Background and Objective: Foodborne pathogens and cross-contamination of food products pose a serious risk to the food industry as many outbreaks are associated with biofilm formation, which increases post-processing contaminations and risks to public health. This review aimed to study the biofilm formation of spoilage and pathogenic bacteria in foods and on food contact surfaces, which subsequently represent serious challenges to the food industry and may decrease shelf life and increase transmission of diseases.

Results and Conclusion: Chemical and physical methods (e.g. sanitizing with chemicals and heat treatment) are not sufficiently applicable for biofilm removal in food sectors due to the increase of bacterial resistances, ingredient damages and possible residues in food matrix. During meat processing, the environment is filled with complex multispecies communities of microorganisms, majorly connected to the surface forming biofilms that are difficult to treat. Furthermore, bacterial cell relationships between various genera and species play a key role in the attachment process and formation of strong biofilms, as well as in the resistance of the biofilm community members against antimicrobial treatments. Thus, control of these biofilms are difficult in food industries since the biofilm cells secrete exopolymeteric substances that include preventing barrier or lessening contact with environmental stresses such as antimicrobial agents as well as the host immune system. Biofilms are highly resistant to conventional antimicrobial therapies and lead to persistent infections. Hence, there is a high need for novel strategies other than conventional antibiotic therapies to control biofilm-based infections. Bacterial biofilm formation and its problems in the food industry were discussed in this study in addition to various safety strategies aiming to provide novel insights into biofilm control in the food industry for improving food quality and safety.

Conflict of interest: The authors declare no conflict of interest.

1. Introduction

A biofilm is a complex community of microorganisms that adhere to a surface, forming multiple layers that protect their growth, proliferation and survival [1]. It can lead to antibiotic resistances, nosocomial infections and food-borne illnesses. However, biofilms benefit the microbes by helping them in adhesion, metabolite exchange, quorum sensing and drug resistance [2]. Microbial biofilms are composed of diverse bacteria surrounded by their exopolysaccharides and typically attached to biotic and abiotic surfaces, resulting in food poisoning with diarrhea, vomiting, enteritis, stomach discomfort and headaches in humans [3, 4]. Presence of biofilms in food-processing environments, food contact surfaces, processing equipment such as stainless steel, rubber, plastic and Teflon and completed products increases danger of spoiling, diminishes shelf life and increases possibilities of infectious disease outbreaks associated with foods [5]. Search for efficient ways to control microbes and their biofilms still needs further efforts [6]. This review covered topics associated with particular microorganisms that create biofilms in the food sector, illustrating the biofilm formation process, stages of development, interactions between microorganisms and various novel methods and strategies of biocontrol. 

1. Results and Discussion
2. Biofilm Formation

Several factors affect biofilm formation, including metabolism, signaling molecules, culture media, matrix and variations in cellular and genetic makeup [7]. Generally, biofilm formation consists of four common steps (Figure 1), initially produced on biotic or abiotic surfaces through reversible and irreversible adhesion, using adhesive proteins, lipopolysaccharides, flagella and pili [8]. Furthermore, biofilm maturation occurs in two stages of cell-to-cell communication and production of auto-inducer molecules. These molecules primarily consist of proteins, exopolysaccharides, DNA, RNA, enzymes, microbial cells and water with water being the major component responsible for nutrient movement within the biofilm matrix. Exopolysaccharides serve as a protective shield, enhancing microbial adhesion within biofilms, ensuring their structural integrity and facilitating nutrient acquisition [9,10,11].

1.1 Formation of Biofilm in Food Industry

In the food industry, surfaces and equipment that come into contact with foods are often occupied by microorganisms that can form biofilms [12,13]. Bacterial biofilms in foods pose severe hazards to human health, leading to systemic diseases, food intoxication and gastroenteritis and presence of bacterial biofilms on tables, staff gloves, animal carcasses, water, milk and other liquid pipelines has been documented [14].

1.2 Biofilm Resistance to Antimicrobial Agents 

Bacteria living in biofilms show 10 to 1000-fold increases in drug resistance, compared to their planktonic stages. Various multidrug-resistant (MDR) bacteria such as Salmonella spp., methicillin-resistant Staphylococcus aureus (MRSA), Listeria monocytogenes, Campylobacter jejuni, Escherichia coli O157:H7 and vancomycin-resistant enterococci (VRE) have been linked to foodborne outbreaks, presenting a significant public health threat [15]. Based on the estimates, biofilm matrix and EPS prevent bacteria from antibiotic exposure, providing them an adaptive advantage by preventing chemical stressors from penetrating deeper biofilm regions [16]. In biofilms, quorum sensing and horizontal gene transfer are the most commonly observed mechanisms [17]. Biofilm awareness in fight against MDR bacteria needs further discussion and persistence of biofilms in foods creates an ideal environment for resistance mechanism exchange; hence, greater awareness of these dangers is necessary.

1.3 Quorum Sensing System

Quorum sensing system is a communication system between the cells that allows them to send chemical signals, enabling cooperative gene expression, which leads to increased population density, enhanced biofilm formation and increased production of extracellular polymeric substances [18]. Recent studies have shown that Gram-negative bacteria produce acylated homoserin lactones (AHLs) as autoinducers, while Gram-positive bacteria use peptides (AIPs) [19].

1.4 Horizontal Gene Transfer

Horizontal gene transfer is a widely recognized mechanism; through which, bacteria adapt and spread resistance to antimicrobial agents using mobile genetic elements (MGEs), which pose a significant threat to global public health [20]. Release and transfer of bacterial DNA play a role in biofilm synthesis and contribute to the spread of antibiotic resistance. Resistance plasmids can spread through the conjugation process, promoting development of resistant biofilms within the densely populated structure of biofilms. Over time, drug resistance leads to the preferential expression of certain genes, resulting in increased productions of proteins associated with virulence and antibiotic resistance, which can alter characteristics of biofilm resistance.

 1.5 Common Foodborne Pathogens Forming Biofilms

Bacterial pathogens can contaminate meats because meats are rich in vitamins, minerals and proteins and include high water contents (75%) and acceptable pH ranges [21, 22]. The greatest dangers to food safety worldwide are environmentally hazardous microorganisms that can infect cattle during various processing procedures and foodborne illnesses associated with uncooked meats. These pollutants are challenging to clean off and disinfect, putting customers' health at major risks. A variety of bacterial pathogens can cause meat-borne diseases, by infecting animals or contaminating meat during meat processing such as Salmonella spp., E. coli, Campylobacter spp., L. monocytogenes, Yersinia enterocolitica, Brucella spp., Mycobacterium bovis, Bacillus anthracis and toxin-producing Staphylococcus aureus, Clostridium spp. and B. cereus [23]. Meat-borne diseases can be categorized into infections, intoxications, allergies, metabolic food disorders and idiosyncratic illnesses [24]. Harmful bacteria can build up on various equipment and biotic and abiotic surfaces and eventually create biofilms whereas over 90% of bacteria live. They create biofilms on gloves and surfaces of silicon, rubber plastic, glass and stainless steel [25]. Significance and effects of biofilms on the food industry have been demonstrated in several studies, where a variety of pathogens such as L. monocytogenes, Y. enterocolitica, C. jejuni, B. cereus and E. coli O157:H7 frequently cross-contaminate these food products [26]. Available studies have shown that the coexistence of multiple bacterial species could increase biofilm development and enhance pathogen persistence by promoting EPS production. Examples of these relevant biofilm-forming pathogens in the food industry are briefly described.

1.5.1 Gram-negative Bacteria

Approximately 80% of the available foods in Saudi Arabian markets are imported with 15.71% of these imports are meat-based. Escherichia coli, Salmonella spp. and Pseudomonas aeruginosa include several important virulence factors, form biofilms and easily contaminate meats. However, handling and consuming animal-derived products contaminated with E. coli biofilms can pose health risks while Shiga toxin-producing E. coli and Enterohaemorrhagic E. coli are important enteric pathogens linked to outbreaks and severe gastroenteritis. Verotoxigenic E. coli produce verotoxins while E. coli O157:H7 is a human pathogen responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) and can be transmitted through raw milks, drinking waters, fresh meats and vegetables. A major element affecting production of E. coli biofilms is temperature. For example, after 7 d of incubation at 15 °C, quantity of adhering and planktonic cells increased on beef surfaces, which included a serious issue for meat processing plants [27]. Isolates with a higher capacity for mature biofilms showed resistance to sanitization [28].  

Salmonella spp. propagate at 35–37 °C and includes two species of S. bongori and S. enterica, the most prevalent pathogens in the food industry and the causative agent in several foodborne outbreaks [29, 30]. Salmonella enterica is commonly associated with refrigerated poultry products stored on shelves during food processing or in supermarkets. Three various types of Salmonella are important for human health, including non-typhoid Salmonella, S. typhi and S. paratyphi. Fresh poultry and meat are highly prone due to their nutrient-rich content, high water activity and near-neutral pH (5.5–6.5), creating optimal environmental conditions for Salmonella spp., which are not spore-formers and can easily be destroyed by heat at 60 °C for 15–20 min. Furthermore, growth of most isolates was inhibited below 7 °C and pH 4.5, while nontyphoidal salmonellosis in the US is nearly 1.35 million illnesses per year [31]. Because many people reside in Saudi Arabia during haj    and umrah seasons, a significant prevalence of Salmonella infection occurs [32].  Pseudomonas aeruginosa is wide spread on meat surfaces and in low-acid dairy products and affecting more than 2 million individuals and killing roughly 90,000 of them annually [33]. Because of its adaptability, P. aeruginosa may grow at temperatures lower than 7°C and contaminate fresh meat sold in stores, causing its spoilage via lipolytic, saccharolytic and proteolytic processes [34]. In addition, the microorganism secretes extracellular enzymes that cause breakdown of foods and includes a high degree of medication resistance, which can result in serious acute and chronic infections in immunocompromised people. Human infections usually affect the respiratory tract (RT), soft tissues, blood vessels, urinary tract (UT) and wounds [35]. Carbapenem-resistant strains of P. aeruginosa pose a hazard to public health [36]. Due to the abundance of EPS, cells can adhere to stainless-steel surfaces and create biofilms alone or with other pathogens and produce multispecies biofilms, increasing their stability and resistance [37].

1.5.2 Gram-positive bacteria

Listeria monocytogenes is a rod-shaped, non-spore-forming, facultative-anaerobic Gram-positive bacterium. It causes human infections of listeriosis, a serious illness that includes septicemia and meningitis, particularly in immunocompromised individuals and is capable of growing at temperatures ranging 3–45 °C with the optimal temperature of 30–37 °C [38]. Listeria monocytogenes is a harmful foodborne microorganism that is killed by pasteurization. Consumption of dairy products, meats, fishes, fruits, soft cheeses, ice creams and poultries has been linked to listeriosis epidemics [39]. It can form biofilms on surfaces commonly detected in the food industry and is resistant to treatments with heat up to 60 °C [40]. It can thrive in a broad range of conditions, including high salinities (10%), cold temperatures (4 °C), low water activities (< 0.9) and wide pH ranges (4.1–9.6) [41]. Post-processing contamination with Listeria spp. may be resulted from inadequate cleaning and poor separation techniques between ready-to-eat and raw foods [42]. In addition, L. monocytogenes is one of the most significant pathogenic microorganisms due to its high mortality rates (15.6%) and one of the major causes of hospitalizations and deaths in the US [43].

Staphylococcus aureus is responsible for staphylococcal food poisoning (SFP) and produces enterotoxins within the temperature range of 10–46 °C. Staphylococcus genus includes more than 50 recognized species; of which, S. aureus is commonly detected in food products and reaches foods through raw materials and grows best on meat, poultry and egg products. In food production chain, it may develop biofilms on living and non-living surfaces, resist desiccation and thrive on a variety of surfaces. Furthermore, strains of S. aureus that produce enterotoxins have been identified in a variety of food samples. [44]. Other Gram-positive bacteria such as Brochothrix thermosphacta and Carnobacterium spp. can form biofilms in the meat-processing environment [45].

1.6 Strategies for Controlling Biofilm Formation in the Food Industry

Pathogenic bacteria that form biofilms create strong defenses against antibiotics and are difficult to treat. Removing these biofilms is a critical challenge due to the severe effects on public health [46, 47]. Chemical and physical methods have been used to inhibit bacterial biofilms in the food industry. Chemical treatments can help; however, mechanical treatments such as clean-in-place are not effective. The most reliable way to prevent bacterial biofilm growth is through aseptic processing, routine disinfection and equipment sterilization. Various disinfectants and novel biofilm elimination methods of the food industry are briefly summarized (Figure 2).

1.6.1 Chemical and Physical Treatments

Biofilms can be treated with concentration and time-dependent chemical sanitizers. Decreasing bacterial populations to human-safe levels is the goal of sanitation. Sanitizing food-processing equipment is necessary to avoid cross-contamination between batches of foods. Stages of general cleaning methods for places that handle and manufacture foods include physical pre-cleaning, detergent washing, rinsing, sanitation, final rinsing and drying. Spraying detergents in form of foam or aerosol spray is possible as long as the right doses and time are used for surface contact. Alkaline and acidic chemicals are widely used as detergents in the food industry. A majority of disinfectants are safe to use on non-food-contact surfaces; nevertheless, food-contact and occasionally non-contact surfaces should be rinsed with high-quality water. The most popular sanitizer in the food industry is aqueous ClO₂, which acts well against B. cereus endospores in biofilms on steel surfaces [48]. In the food industry, chlorine-based sanitizers are most frequently used; nevertheless, several microorganisms have developed resistance to chlorine treatments. Food factories frequently use sodium hypochlorite or NaOCl [49]. Moreover, hydrogen peroxide (H₂O₂) and NaClO were successful in removing biofilms of S. aureus and P. aeruginosa; however, aqueous ClO₂ was more effective than NaOCl in eliminating E. coli O₁₅₇:H₇ biofilms [50]. In the food industry, H₂O₂ is a powerful oxidizing disinfectant that is often used. When it is exposed to biofilms, H₂O₂ produces free radicals that kill the bacteria at concentrations of 0.08–5%, without harmful side effects. Quaternary ammonium compounds are frequently used as sanitizers, removing biofilms and leading to bacterial lysis [51]. Steam heat treatment is a method used to decrease number of harmful bacteria and biofilm populations in production areas [52]. Non-thermal plasma is a partially ionized gas with low temperature and promising antibacterial characteristics. It can destroy bacterial biofilms of Pseudomonas spp., S. enterica and Bacillus spp. Ozone breaks down the cellular envelopes of a variety of microorganisms, including viruses, bacterial biofilms and protozoans.

1.6.2 Elimination of Biofilms Using Biological Strategies

In recent years, a more efficient and ecologically friendly control method for the elimination of or managing growth of dangerous biofilms is use of enzymes, bacteriophages, bacteriocin and plant extracts, which have been discussed based on safe and green approaches to control pathogen biofilm formation.

1.6.2.1 Enzyme against bacterial Biofilm

Enzymes or proteins are biologically active macromolecules against biofilm formation since proteases or other degrading enzymes have shown the ability to inhibit biofilm formation [53].   Enzymes are detected to include therapeutic functions in removal of pathogenic biofilms and can widely be used in detergents of food industries. In recent times, a variety of enzymes enriched products have been commercialized that include tablets, rinsing solutions, chewing gums for dental treatments and denitrifies containing enzymes such as lysins, dextranase, mutants that can serve to play an effective role in disintegration of the biofilm matrix [54]. The most often used enzyme types vary depending on the makeup of the biofilm as proteases, cellulases, polysaccharide depolymerases, alginate lyases and dispersin B [55]. Proteinase K and lysozyme have been verified to include promising antibiofilm activities [56]. The α-amylase enzyme includes potential to operate as an antibiofilm agent against bacterial species that produce biofilms, including S. aureus and P. aeruginosa [57]. Protease formulations were effective in eliminating S. aureus biofilms from polystyrene surfaces; however, combinations of protease, amylase and cellulase were needed to eradicate biofilms of P. aeruginosa. Cellulase effectively inhibited biomass and microcolony formation by P. aeruginosa on glass surfaces in partial [58].

1.6.2.2. Bacteriophages against Biofilm

Bacteriophages (phages) are bacterial viruses, acknowledged as the most diverse and abundant entities. Bacteriophages are mostly used in primary production to ensure food safety, biosanitization and biopreservation [59]. Phages can break down biofilms spread through developed biofilms and then show their antimicrobial characteristics inside them. Phage treatments are injected directly into food products during the biopreservation processes to extend the food shelf life and used in biosanitization to avoid biofilms on equipment surfaces [60]. Bacteriophages can create enzymes that break down the biofilm structure and presence of phage receptor sites such as endolysins and depolymerases. Use of phages as biocontrol agents in foods is affected by various factors, including the food matrix, surface area and structure, bacterial species, inhibitory compound and phage dose [61]. A commercial product, LISTEXTM, has been developed from the bacteriophage P100, which uses an enzymatic process to cause cell lysis and EPS breakdown. The US Department of Agriculture (USDA) has approved use of this natural, non-toxic phage product. It is effective against L. monocytogenes. Additionally, it seems that L. monocytogenes biofilms are susceptible to phage biocontrol. Phage Guard Listex, which uses phage P100, effectively removes biofilms from stainless steel surfaces. A user of Listeria phage P100 (under the commercial name of Listex P100) is a biological agent, formed to remove the biofilms in processed meat products [62]. In addition, a phage cocktail was used for 1 h to destroy and decrease pathogen populations of E. coli O₁₅₇:H₇ on stainless steels, ceramic tiles and high-density polyethylene coupons [63]. Endolysin is the second kind of enzyme produced by phages that include potential uses for sanitization. During the final stage of their lytic cycle, they release progeny of virions through the breakdown of the cell wall, which were active against Gram-positive bacteria [64]. Depolymerases are types of enzyme that may prevent production of biofilms and break down capsular polysaccharides in Gram-negative bacteria [65].

1.6.2.3 Bacteriocins against biofilms

Lactic acid bacteria (LAB) are used to produce fermented foods and the most important genera in controlling spoilage and pathogenic microbes are Lactobacillus and Bifidobacterium due to the production of bacteriocins and acids. Bacteriocins from LAB are used as alternatives to chemical food preservatives. They can spread through cell membranes and release internal components such as K⁺ and inorganic phosphate or they can prevent production of proteins, RNA and DNA [66]. Due to its safety in the gastrointestinal system, bacteriocin has extensively been used as a food preservative in the food sector for several years. Use of bacteriocin in biopreservation systems can meet consumers' demands and numerous compounds, including nisin, natamycin, subtilin, pediocin, tylosin and carnocyclin A, are used as food preservatives [67].

1.6.2.4. Plant Extracts against Bacterial Biofilm

Numerous substances from plants such as complex mixtures of monoterpenoids, plant-based essential oils, sesquiterpenoids and flavonoids have shown anti-biofilm characteristics [68, 69]. Previous materials can be used as an alternative to synthetic preservatives. Specifically, several flavonoids inhibited generation of bacterial toxins in various food products. In addition, bacterial cell adherence to stainless steel was strongly inhibited by essential oils and other plant components that are abundant in almost all plants such as phenolic chemicals, tannins, terpenoids, glucosinolates derivatives, alkaloids and thiols. Although the food industry uses a variety of plant-based extracts and essential oils in meat preservation, pomegranate and cranberry extracts are particularly popular due to their antibacterial and antifungal characteristics [70, 71, 72]. 

1. Conclusion

Controlling biofilm formation in the food industry is essential for food safety, quality and hygiene. Traditional methods such as cleaning and sanitizing with chemicals and heat treatment are critical; however, effectiveness was limited due to the increase of antimicrobial resistance and vitamin damage by heat. Moreover, biofilm cells secrete extracellular polymeric substances that include a barrier preventing or lessening contact with environmental stresses and decreasing effects of antimicrobial agents and the host immune system. Various methods have been investigated to prevent and remove biofilms, each offering unique advantages. However effectiveness of traditional methods was limited, biological control methods such as bacteriophages and probiotics offer promising sustainable solutions and may be further effective in specific settings due to targeting harmful pathogens without affecting the environment. Recent advances in combination of physical, chemical and biological methods in food processing environments may be effective keys to biocontrol biofilms, ensuring safety and quality of food products.

1. Conflict of Interest

The author reports no conflict of interest.

1. Authors Contributions

H.A, NZ and MMA conceptualized the idea and prepared the manuscript.

Background and Objective: Phytase is a phosphatase enzyme. It is essential for hydrolyzing phytic acid, an antinutrient present in plant-based foods that chelates essential minerals and limits their bioavailability. Enzyme techniques used to improve phytase characteristics are discussed including thermostability, catalytic efficiency, and substrate specificity.

Results and Conclusion: Phytase efficiency has been significantly improved for industrial applications by different engineering methods, including rational design, computational modeling, directed evolution, glycosylation engineering and semi-rational design. The study further highlights the application of phytase in food processing, including breadmaking, fermented foods, and functional food formulations, to address mineral deficiencies. Future advances in enzyme engineering, such as computational de novo design and enzyme immobilization, could expand commercial and nutritional uses of phytase. The focus of ongoing research on thermostable and highly efficient phytase variants provides significant possibilities for enhancing global food security and sustainability.

Keywords: Phytase, enzyme engineering, directed evolution, food processing, phytic acid, mineral bioavailability

1. Introduction

Phytase (EC 3.1.3.8) is also known as myo-inositol hexakisphosphate phosphohydrolase. It is an enzyme that hydrolyzes phytic acid which act as an antinutrient found in plant-based foods. In animals with a single-chambered stomach (monogastric animals like pigs, chickens, and fish), phytate-bound phosphorus is poorly absorbed. In fish, indigestible complexes of phytate and phosphorus form within the digestive tract when they consume phytate-rich plant-based feeds. This leads to reduced phosphorus bioavailability and increased excretion into aquatic environments. Supplementing feed with phytase improves phosphorus availability, supporting enhanced growth, bone development, and overall health while reducing phosphorus waste [1]. Plant-based proteins such as oat, wheat, soy, and pea are widely consumed for their nutritional benefits. They contain significant amount of phytic acid, ranged between 0.4% to 2.2% dry weights depending on the type and processing conditions. These phytate compounds strongly chelate divalent minerals such as calcium, iron, and zinc, significantly reducing their intestinal absorption and contributing to mineral deficiencies, specifically in populations relying heavily on cereals and legumes [2, 5, 6]. It also enhances protein and amino acid digestibility, contributing to better feed efficiency [3].

Phytase is widely applied in the food, feed, and pharmaceutical industries due to its multifunctional properties. The growing demand for stable, high-temperature-resistant phytase has promoted global market growth. In food applications like bread-making, phytic acid limits mineral absorption. Adding phytase during fermentation helps release bound minerals, improving iron and zinc bioavailability. The global phytase market is expected to be 692.5 million USD by 2023, with a compound annual growth rate (CAGR) of 6.3% between 2024 and 2030 [4].

Phytic acid is a major challenge in whole grains and bran, where it chelates minerals and reduces absorption [5]. Based on [7], phytase hydrolyses the phosphate groups attached to the inositol ring to break down phytic acid as shown in Figure 1. Phytase enzymatically hydrolyzes the phosphate ester bonds in phytic acid (myo-inositol hexakisphosphate), sequentially releases inorganic phosphate and lower inositol phosphates. This increases the bioavailability of essential minerals. Phytic acid degradation by phytase supports physiological functions like oxygen transport (via iron) and immune health (via zinc). Enhancing mineral absorption with phytase is important in developing regions with plant-based diets, helping address nutritional deficiencies and improve public health outcomes [6]. However, current enzyme engineering techniques face several limitations. Many phytase variants still lose activity at high processing temperatures or extreme pH conditions. While techniques like directed evolution, rational design, and glycosylation have improved thermostability and activity, they often require extensive trial-and-error, are time-consuming, or lack scalability [17, 24, 36]. Immobilization methods also face challenges in cost-effectiveness and enzyme reuse cycles. Research gaps remain in developing optimum phytase enzymes that retain high activity across a wide range of industrial conditions. There is a need for more in vivo studies validating the nutritional benefits in humans [9].

1. Global phytic acid intake from plant-based diet

Phytic acid consumption varies greatly among nations, depending on diet and the volume of plant-based foods consumed. Grains and legumes are majorly consumed in the developing countries, where daily phytic acid consumption can reach more than 2000 mg/day [8]. In developed countries, the daily intake ranges from 250 to 800 mg, depending on their consumption of plant-based diets and whole grains. Phytic acid consumption in China varies from 648 mg to 1433 mg.day^-1 across six locations [9]. The average intake was substantially lower among urban people i.e., 781 mg.day^-1, compared to rural individuals who consumed approximately 1342 mg.day^-1. The amount of phytic acid consumed in the UK increased from 504 to 844 mg.day^-1 to 1436 ± 755 mg.day^-1 between 1986 and 2005. Children in India between the ages of 4 and 9 consume between 720 and 1160 mg.day^-1, while teenagers between the ages of 10 and 19 consume between 1350 and 1780 mg.day^-1. Swedish vegetarians consumed 1146 mg.day^-1, compared to 180–370 mg.day^-1 for those following a Western diet in Finland and Sweden [9]. These variations highlight the influence of dietary patterns on phytic acid consumption, with higher intakes observed in populations that depended heavily on plant-based diets.

Recent studies highlight how dietary habits and food processing influence phytic acid intake and its nutritional impact. It was found that germination and fermentation of cereals and legumes reduced phytic acid content by 50–70% [10]. This significantly improved iron and zinc absorption in plant-based diets. The traditional African processing methods i.e., soaking and malting, reduced phytic acid levels in maize-based foods and enhanced mineral bioavailability for rural populations [11]. Maize soaked for 24 h found to have reduced phytic acid levels by 20–30%, while malting for 48–72 h decreased phytic acid levels by up to 45% in maize-based foods. These processes improved calcium, iron, and zinc bioavailability for rural Malawian populations. They also found that combining soaking and malting resulted in a synergistic effect and reduced phytic acid by 55–60% in maize porridge. These studies highlighted that while plant-heavy diets significantly increase phytic acid consumption, processing techniques found to have mitigated its anti-nutritional effects.

1. Different enzyme engineering methods of phytase

Phytases are widely utilized to improve the bioavailability of phosphorus and other essential minerals in the animal feed industry by breaking down phytic acid in grains.  A thermostable phytase can maintain its activity under high temperatures. Phytases primarily act by a nucleophilic attack on the phosphorus atom, facilitated by acidic and basic residues in the active site. Enzyme engineering improved the catalytic properties of the residues as shown in Table 1. Improvements in the stability of the transition state of the substrate during the catalytic cycle result in a high enzyme turnover rate. The addition or optimization of disulfide bonds and salt bridges improves the thermal stability and resistance of phytase during processing. Improving the hydrophobic interactions in the core of the enzyme is also responsible for increased thermal stability. Phytase enzyme engineering is based on several advanced techniques to optimize its characteristics i.e., activity, stability, substrate specificity, and resistance to conditions.

3.1. Directed evolution for thermostable phytase development

Directed evolution replicates natural selection in the lab to develop proteins with desired characteristics. It effectively enhances phytase thermostability, ensuring activity at high temperatures during processes. Random mutations are introduced into the phytase gene by increasing deoxyribonucleic acid (DNA) polymerase error rates during polymerase chain reaction (PCR) (using altered Mg²⁺/Mn²⁺ levels or biased nucleotide analogues) or via chemical mutagens like nitrous acid and ethyl methane sulfonate (EMS). Mutated genes are then cloned into expression vectors and transformed into E. coli for screening [12]. When host cells produce mutant enzymes, a library of phytase enzymes is formed, as shown in Figure 2. The generated library is analyzed for phytase variants with improved properties. Enzyme activity and stability are measured using techniques such as microplate assay and flow cytometry.

Thermostability is determined by exposing the enzymes to high temperatures and measuring any residual activity. Variants that exhibit increased activity post-heat treatment are selected for further mutagenesis and screening cycles. Directed evolution has recently been used to select more stable phytase variants by introducing mutations that strengthen hydrophobic cores, elevate salt bridges, and improve hydrogen bonding networks [12].

3.2. Rational design to improve structural stability of phytase

Enzyme engineering by rational design is a fundamental and knowledge-based approach in which the structure of an enzyme is modified based on a thorough understanding of its catalytic mechanism and three-dimensional conformation [13]. Certain modifications to phytase have been successfully achieved by rational design (Figure 3).

High-resolution phytase structures help identify important residues for catalysis, stability, and substrate binding. Important amino acids in flexible surface loops, are often targeted for stabilization. Site-directed mutagenesis introduces specific mutations based on structural insights, using primers to insert changes via PCR. Cysteine residues may be added to form disulfide bridges, enhancing structural rigidity. The mutant phytase is expressed, purified, and tested under stress conditions to assess stability and activity. Test results guide for further improvements. Rational design has proven effective in enhancing phytase thermostability by reinforcing disulfide bonds and optimizing core hydrophobic interactions [12, 14, 17]. The applied rational design on E. coli phytase using molecular dynamics simulations and bioinformatics analyses were applied to identify a mutation (S392F) [14].

The mutant shows a significant increase in thermostability. It has increased activity at higher temperatures compared to the wild-type enzyme. Mutant displays two times higher thermostability at 70°C, and at 80°C and 90°C, it showed increased activity of 74% and 78.4%, respectively.

Secondary structure modifications further improve phytase stability. Flexible loops are critical for substrate access but vulnerable to thermal denaturation. They are stabilized by introducing proline residues to restrict conformational flexibility. A novel KeySIDE technique was used to improve the thermostability of Yersinia mollaretii phytase (Ymphytase) [16]. Nine important spots were found by combining directed evolution with iterative substitution analysis, and the optimum mutant, M6 (T77K, Q154H, G187S, K289Q), was developed. This mutant exhibited a significant increase in residual activity from 35% (wild-type) to 89% after 20 min at 58°C, with a 3°C rise in melting temperature and no loss of specific activity. Substitutions in loops close to helices B, F, and K (T77K, G187S, K289E/Q) decreased loop flexibility, based on molecular dynamics simulations. This caused by stronger hydrogen bonding networks (G187S, K289E/Q) and a salt bridge (T77K), which stabilized secondary structures and improved thermostability.  Melting point bacterial phytase was raised by 7.5°C by strengthening its α-helices and β-sheets by improved hydrogen bonding or hydrophobic packing, strengthening the structural scaffold of the enzyme.

Evolution-guided rational design utilized to enhance thermostability of Yersinia intermedia phytase APPA [17]. They found high-frequency N-X-T/S motifs as potential spots for N-glycosylation modifications by examining 5569 homologous sequences from the NCBI database, with a focus on areas that are exposed to the surface.  The optimum mutant M14 was created by combining mutations. It found to have enhanced kinetic stability and preserved wild-type catalytic efficiency. Its half-life (t1/2) increased from 3.32 min at 65°C to 25 min at 100°C, and retained 75% activity after 10 min at 100°C.  When compared to wild-type APPA, in vitro digestibility of M14 increased by 4.5 times due to its improved kinetic stability and strong refolding ability, but its thermodynamic stability remained unchanged.

3.3. Computational design for predictive phytase engineering

Enzyme forward engineering uses computational tools to predict mutations that enhance activity and stability. Molecular modeling, dynamics (MD) simulations, and machine learning provide atomic-level insights. In phytase, these methods predict mutation effects and identify unstable regions under heat or acidity. Computer tools like Rosetta, AlphaFold, and FoldX design mutations to stabilize structure and improve binding [18]. Rosetta generates mutations that improve hydrophobic packing or stabilize loops using energy-based scoring.  AlphaFold2 guides mutation selection by predicting 3D structures with high accuracy.  FoldX identifies stabilizing modifications by estimating changes in free energy.  Molecular dynamics simulations conducted using force fields i.e., AMBER ff14SB, CHARMM36, or OPLS-AA and software i.e., GROMACS, AMBER, or NAMD.  They identify areas for stabilization by disulphide bond formation or electrostatic optimization by simulating phytase dynamics under thermal or acidic stress.  Comprehensive enzyme sequence databases have been used to train machine learning systems like DeepMutate and ProteinMPNN, which accurately predict the effects of mutations and provide experimental confirmation. A major advantage of computational design is the ability to analyze a vast mutational space in silico, significantly reducing the number of studies required. Computational design detects modifications that improve thermostability by forming disulfide bonds, optimizing electrostatic interactions, or increasing hydrophobic packing. Experimental verification of the above theories allows the results to be fed back into computer models to enhance designs further [18, 19].

3.4. Semi-rational design integrating structural and sequence analysis

Semi-rational design intermediate directed evolution and rational design, requires knowledge of enzyme structure–function relationships. Important residues affecting stability, activity, or specificity are identified using biochemical data, structural analysis, and sequence alignment [13]. Phytases from fungi, bacteria, and plants share catalytic motifs but vary in heat and pH tolerance. Sequence alignment pinpoints residues linked to these traits, which are then modified via site-directed mutagenesis. Sequence analysis, bioinformatics, and evolutionary data are used in semi-rational design to identify potential mutation sites. This method creates targeted mutant libraries by utilizing techniques like multiple sequence alignments or directed evolution to target areas of interest, such as conserved motifs or variable loops. In comparison to directed evolution, this method allows the examination of a wide range of mutational regions with less experimental effort [13].

3.5. De novo design of synthetic phytase enzymes

De novo design creates novel enzymes with desired properties using computational methods, rather than modifying existing ones. It combines knowledge of protein folding, catalysis, and structural biology. In phytase engineering, it aims to build stable protein scaffolds with optimized active sites for phytate hydrolysis. The process uses modeling, energy minimization, and molecular dynamics to ensure structural stability and activity. Once a suitable design is achieved, the gene is synthesized and expressed in a host organism for validation [19, 20].

An automated workflow, GRACE (Generative Redesign in Artificial Computational Enzymology) is developed for reformation and creation of the de novo enzymes. It has integrated RFdiffusion (structure generation), ProteinMPNN (sequence interpretation), CLEAN (enzyme classification), solubility prediction, and molecular dynamics [19]. Two carbonic anhydrase-like enzymes, dCA12_2 and dCA23_1, were selected across 10000 protein options.  Both demonstrated significant substrate-active site interactions, high solubility, and 400 WAU.mL-¹ of enzymatic activity.  GRACE significantly simplifies the design of enzymes. The alignment of 13 fungal phytase sequences was used to de novo create a consensus phytase in order to improve thermostability.  The consensus enzyme showed a 15–22 °C higher unfolding temperature than any of its parent enzymes while retaining normal catalytic activity.  According to a structural comparison with Aspergillus niger phytase, consensus residues significantly improved stability. Mutational study revealed that even those consensus amino acids that were assumed to be destabilizing instead improved resistance to heat.  This work demonstrates an evident and unfamiliar connection between improved protein stability in fungal phytases and sequence conservation [20].

3.6. Glycosylation engineering for enhanced phytase stability

Fusion proteins are proteins formed by combining two or more genes that formerly coded for individual proteins. Domain swapping is a process for exchanging domains between related proteins to create hybrids with specific characteristics [17]. These domains are genetically combined or exchanged between proteins. Develop and assess the resultant mutant proteins to ensure they possess the required properties. The fusion protein formed by integrating a thermostable domain with a phytase enzyme improves the overall stability and performance of enzyme at a commercial scale. Directed evolution method have been studied to create phytase variants with increased thermostability [21]. This involved evaluating homologous sequences to find appropriate sites for introducing N-glycosylation variations, followed by site-directed mutagenesis. The engineered phytase variants found to have enhanced thermostability and high activity at boiling temperatures. The introduction of N-glycosylation sites stabilized the enzyme structure under extreme conditions.

Recent studies have demonstrated the benefits of introducing or optimizing N-glycosylation sites in phytases. E. coli phytase (AppA) involved introducing two N-glycosylation sites (Q258N and Q349N). After exposure to 85°C for 10 minutes, resulted in a mutant with a specific activity of 3137 mg.U^-1 and over 40% enhanced thermostability. The melting temperature (Tₘ) increased by 4–5°C compared to the wild type, without affecting catalytic efficiency [22]. Glycosylation engineering of phytase from Cronobacter turicensis expressed in Pichia pastoris revealed that specific N-glycosylation sites (N136, N171, and N202) significantly influenced enzyme stability. Removal of the glycan at N202, located in a flexible region, led to a decrease in resistance to pepsin and trypsin by 73% and 87%, respectively. The glycosylated enzyme exhibited strong resistance to proteolytic degradation and increased thermostability [23]. An evolution-guided design approach analyzed 5569 homologous phytase sequences to identify 25 candidate sites for N-glycosylation. Thirteen of these sites were located on solvent-accessible surfaces and were selected for engineering. The introduction of N-glycosylation motifs at these positions enhanced the thermostability of enzyme. This demonstrates the effectiveness of combining evolutionary analysis with structural modelling in glycosylation engineering [17].

3.7. Advancements in phytase engineering

Recent advances in phytase engineering have considerably improved the thermostability and activity of enzymes. This expanded its application in a range of food processing conditions. Phytases have significance for increasing nutrient bioavailability in plant-based foods and feeds. However, their poor stability at high temperatures has limited their industrial applications. A comprehensive study reviewed the advances achieved by producing thermostable phytases over the last seven years using genetic alterations and immobilization techniques. The study indicated that protein engineering techniques have successfully improved the thermostability and activity of phytases. Immobilization on different supports has resulted in 50-60% activity retention at temperatures above 50°C, demonstrating the potential of these techniques in industrial applications [24].

A targeted examination was carried out to change the phytase YiAPPA by mutating protein surface residues [25]. They utilized site-directed mutagenesis to create single-site mutants that substituted specific amino acids on the surface of enzyme. This technique produced mutants with higher thermostability and activity, increasing their potential for use in the food industry. Furthermore, the wild-type YiAPPA showed a half-life of about 30 min at 55°C, which significantly improved in the engineered variants. E. coli phytase thermostability is enhanced using error-prone polymerase chain reaction (epPCR). Directed evolution is effective in improving the thermostability of phytases. Variants were first expressed in E. coli BL21 and simultaneously in Pichia pastoris for screening and characterization, respectively. Researchers developed phytase variants capable of functioning at the high temperatures found in industrial processes by applying multiple stages of mutation and selection. Mutants had higher residual activity and catalytic efficiency, ranging from 9.6% to 12.2% and 79.8% to 92.6%, respectively. It was determined that the C77-C108 disulfide link in E. coli phytase was crucial for stability [26]. However, balancing higher temperature stability with the preservation-specific enzyme activity remains challenging. Disulfide bond engineering is another potential technique. Researchers improved the thermostability of phytase by inserting more disulfide bonds. Modifications to the AppA phytase have resulted in increased stability, making it more suitable for industrial applications requiring high-temperature processes [33]. Modifications to the AppA phytase have resulted in increased stability, which makes it more suitable for industrial applications requiring high-temperature processes [33]. In food industry processes such as baking, extrusion, and fermentation, phytases must withstand temperatures exceeding 50–80 °C and operate effectively within a pH range of 4.0–6.5. Engineered variants that retain 50–75% activity under these conditions demonstrated significant commercial potential. This is helpful in flour fortification and bread-making where phytate hydrolysis is essential for mineral bioavailability [24, 33, 36].

3.8. Limitations of current engineering techniques

Each phytase engineering technique offers distinct advantages but also presents specific limitations. Directed evolution is an effective at generating improved variants. However, it relies on random mutagenesis, requires the screening of vast mutant libraries. This process is labor-intensive, time-consuming, and often inefficient, with a risk of losing catalytic efficiency or substrate specificity. Rational design provides a more targeted approach but depends heavily on detailed structural knowledge and high-resolution enzyme models. Its success is limited by the accuracy of structural predictions and may not always result in significant functional improvements. Computational design involves high computational costs and requires extensive knowledge in molecular modeling and simulations. Moreover, in silico predictions may not always translate accurately to real-world enzyme properties, and experimental validation remains essential. Semi-rational design offers a balance between random and structure-based methods but may miss important mutational hotspots and still requires substantial experimental screening. De novo design is the most advanced method, but it is technically complex and prone to challenges i.e., misfolding, low catalytic activity, and unpredictable stability under industrial conditions. Glycosylation engineering enhances enzyme stability through post-translational modification. It is limited by host-specific glycosylation patterns, potential interference with the active site, and increased production complexity. Despite significant progress in these areas, common industrial limitations i.e., thermal instability, high production costs, inefficient high-throughput screening, and a lack of robust enzymes that function across a wide range of pH and temperature conditions remain major challenges. Overcoming these challenges will require integrated, multidisciplinary approaches that combine synthetic biology, machine learning, and cost-effective production methods.

1. Applications of engineered phytase in the food industry

Engineered phytases have drawn a lot of attention due to their capacity to hydrolyze phytic acid, a common antinutrient found in grains and legumes, improving nutritional profiles and increasing mineral bioavailability. Production of phytases with improved properties, diversified their application in food processing as a result of development and advancement in genetic engineering.

4.1. Phytase application in baked products for mineral bioavailability

Engineered phytases in bread-making are gaining attention for improving the nutritional value of whole-grain products. Phytic acid in grains, legumes, and seeds, binds minerals like calcium, zinc, and iron, forming insoluble complexes that reduce their bioavailability. This can lead to nutrient deficiencies in plant-based diets. Phytases help break down phytic acid, either through added microbial enzymes or by activating natural cereal phytases during dough fermentation. Optimizing dough pH enhances phytase activity, effectively reducing phytic acid content. [9] summarized human intervention studies concluded that majority of researches evaluating the exogenous phytase efficacy or food dephytinization resulted in enhanced bioavailability of zinc and iron. The degree of phytic acid breakdown is determined by the bread-making method used. Research comparing several wheat varieties and bread-making methods has demonstrated that the baking method has a significant effect on the final phytic acid level than the wheat variety selected. Long yeast fermentation methods were more efficient in lowering phytic acid levels and improving bioavailability of mineral.

A novel protein tyrosine phosphatase-like phytase (PhyLf) was identified from Limosi lactobacillus fermentum NKN51. The recombinant enzyme exhibited a specific activity of 174.5 U.mg^-1, with an optimal temperature, pH, and ionic strength of 60°C, 5.0 and 100 mM, respectively.  Its shown ability to enhance mineral bioavailability under in vitro gastrointestinal conditions in finger millet and durum wheat. This highlighted its potential for improving the nutritional value of cereals and animal feed [34]. Two directed‐mutagenesis S. cerevisiae strains (YD80, BY80) and a naturally high‐phytase Pichia kudriavzevii TY13 were employed in wheat–cassava–sorghum composite bread. They achieved up to 99 % reduction in phytic acid and phytate:iron and phytate:zinc molar ratios well below critical thresholds for human absorption (1:1 and 15:1, respectively) [35]. Pre‐incubation of the yeasts at 30 °C for 1 h and supplementation with yeast extracts further enhanced phytase biosynthesis, leading to the lowest measured phytic acid content of 0.08 µmol/g in the final bread. [36] added a commercial A. niger phytase was added to whole‐wheat bread dough, observing that although proofing degraded phytic acid by up to 49.4 %, complete hydrolysis was affected due to bran particle size limiting enzyme access [36]. This resulted in residual phytate levels of 3.74 mg.g^-1 dry matter in baked loaves. This highlighted the need for enzyme engineering or formulation to improve substrate accessibility during baking.

In Sangak and Barbari breads, exogenous A. niger phytase addition significantly increased in vitro iron, zinc, and manganese availability without affecting copper levels, demonstrating that targeted phytase application can selectively enhance key mineral bio accessibility in traditional flatbreads. The study reported up to a twofold increase in iron and zinc solubility post‐baking, underscoring potential for regional dietary interventions [37]. Incorporating phytase into baked goods can reduce phytic acid content, enhancing mineral bioavailability. However, the high temperatures involved in baking can inactivate phytase, necessitating the use of thermostable variants. Studies have demonstrated that certain phytase enzymes retain activity after baking, making them suitable for fortifying bread and other baked products.

4.2. Role of phytase in fermentation for nutrient enhancement

In fermented foods, phytase affects the fermentation and affect flavor development. Engineered phytases are introduced either by adding purified enzymes or using phytase-producing microbes. The microbial approach involves selecting or genetically modifying strains i.e., lactic acid bacteria (LAB), to produce the enzyme during fermentation. Some of LAB naturally possess phytase activity. They reduce phytic acid during food processing and gastrointestinal digestion, increasing mineral bioavailability and the nutritional profile of the final product [38]. The second approach to reducing phytic acid is the direct addition of purified phytases. Advances in genetic engineering have produced phytases with improved temperature stability, pH tolerance, and substrate specificity, suitable for various food processing conditions. Adding microbial phytases to foods effectively lowers phytic acid content, enhancing mineral bioavailability and overall nutritional quality.

The scald fermentation of rye (12–48 h) with selected lactic acid bacteria starters was investigated finding that prolonged fermentation reduced phytate content by over 60 % and improved nutritional profile of rye bread, including enhanced sensory qualities [39]. The study demonstrates that process‐driven activation of endogenous and microbial phytases during fermentation can rival direct enzyme additions in lowering phytic acid. During tempeh production from soybeans inoculated with Rhizopus oligosporus, endogenous fungal phytase halves phytic acid in the first 24 h, and extended storage (72 h at 5 °C or 30 °C) plus deep-fat frying further reduce residual phytate to below 10 % of the original level [40]. Koji molds found to be differ in phytase thermostability during soy sauce brewing. After 30 days, A. sojae–fermented mash shows undetectable phytic acid, whereas A. oryzae retains measurable levels, attributed to the lower heat resilience of its phytase enzyme [41]. An extracellular phytase from Lactobacillus sakei (kimchi‐derived) has an optimum pH of 5.5 and hydrolyzes approximately 60 % of phytic acid in model fermentations within 24 h, improving availability of iron and zinc in the final product [42].

4.3. Development of functional foods and supplements

Incorporating engineered phytases into functional foods and supplements offers a promising approach to prevent mineral deficiencies in populations reliant on phytate-rich staples. These enzymes enhance mineral absorption by breaking down phytic acid into inositol and inorganic phosphate. Microbial phytases from fungi and bacteria are effective across a wide range of pH and temperatures, making them suitable for diverse food applications. This approach is beneficial in developing regions where diets are heavily based on cereals and legumes with high phytic acid content. Phytate content in the milk was reduced using the immobilized phytase on starch agar beads [43]. Processed soymilk serves as major ingredient in baby nutrition. Reduced phytate levels increase the bioavailability of minerals and proteins in infants.

Fermentation, soaking, and germination activate natural phytases or enhance microbial phytase action. These reduce the phytic acid content and enhance bioavailability of micronutrients. These methods improve staple food nutrition in regions with mineral deficiencies. Incorporating engineered phytases in functional foods and supplements addresses these deficiencies with improved food processing. Breaking down phytic acid enhances the quality and yield of products like bread, plant protein isolates, and cereal bran. This demonstrates the role of phytase in improving nutrition and functionality.

Certain strains of Bifidobacteria i.e., B. pseudocatenulatum and B. longum, have been utilized in sourdough fermentation to produce phytase in situ. This fermentation process significantly reduces phytic acid levels in whole rye-wheat mixed bread and increased dialyzable iron content by 2.3- to 5.6-fold. These findings suggest that incorporating phytase-producing probiotics into bread making can enhance mineral bioavailability without compromising sensory qualities [44]. Tolerase® P is a phytase developed by DSM-Firmenich. It has demonstrated efficacy in improving mineral absorption from plant-based foods. Clinical studies have shown that this enzyme can increase iron absorption by up to 11.6 times and zinc absorption by up to 2 times. Its stability across a broad pH range (2.5–5.5) and compatibility with various food matrices make it suitable for incorporation into flour fortification, cereals, and dietary supplements [45].

1. Concluding remarks and future perspectives

Phytase enzyme engineering methods have achieved significant progress in terms of thermostability, catalytic efficiency, and resistance to challenging industrial environments. The application of modern molecular techniques such as directed evolution, rational design, and computational modelling results in the development of more effective phytase variants with enhanced functionality. These developments expand the application of phytase in food processing, especially in increasing the nutritional value by breaking down phytic acid and increasing mineral bioavailability of plant-based foods. Incorporation of engineered phytase into the food and feed industries has the potential to address major challenges i.e., mineral deficiencies and sustainable food production. Future research should focus on optimizing large-scale phytase production, improving enzyme stability under a range of processing conditions, and promoting its use in functional foods and probiotic formulations. As enzyme engineering improves, phytase will play an increasingly important role in enhancing food nutrition and reducing environmental phosphorus pollution, subsequently increasing human health and ecological sustainability.

Furthermore, the economic feasibility of using engineered phytases into large-scale food production remains a critical factor for industrial adoption. High costs associated with enzyme purification, stabilization, and immobilization limits widespread use. Therefore, future innovations must focus on cost-effective purification systems, scalable fermentation technologies, and reusable immobilization supports to enhance process economics. In parallel, regulatory approval presents significant hurdles, notably for genetically modified enzymes. Acceptance and clearances from regulatory bodies requires rigorous safety assessments and compliance with labeling regulations. Additionally, consumer acceptance of phytase-treated foods may vary due to perceptions surrounding genetic modification and enzyme-treated products. To ensure safety and successful market adoption of phytase-enhanced food products, transparent labeling, public education, and clinical evidence of nutritional benefits will be essential.

1. Acknowledgments

This project was conducted using the resources of the Department of Food Process Engineering, National Institute of Technology, Rourkela, Odisha, India.

1. Declaration of competing interest

The authors report no conflict of interest.

1. Authors’ Contributions

Writing-original draft, C.M.; conceptualization, C.M.; methodology, C.M. and A.K.; visualization, C.M.; data curation, R.C.P.; supervision, R.C.R. and R.C.P.; project administration, R.C.R.; writing-review & editing, R.C.R. and R.C.P.; resources, A.K. and R.C.P.

1. Using Artificial Intelligent Chatbots

No artificial intelligent chatbots has been used in any section of work.

Background and Objective: Probiotics, defined as specific strains of live microorganisms, usually bacteria or yeast, play a crucial role in host health by enhancing digestive functions. Traditionally, these organisms were sourced from dairy-based fermented foods such as yogurt, cheese, and kefir; however, the rise in plant-based diets has spurred the popularity of plant-based probiotics, driven by factors such as veganism, lactose intolerance, and dairy allergies. Plant-based probiotics offer a valuable alternative that caters to diverse dietary requirements while also contributing to environmental sustainability through lower greenhouse gas emissions, reduced water consumption, and less land use. This review delves into the development, potential health benefits, future applications, and technological challenges related to the production and marketing of plant-based probiotics.

Results and Conclusion: Innovative approaches have led to the creation of functional foods that combine plant sources with robust probiotic strains capable of surviving in plant-based matrices. The integration of probiotics with prebiotic fibers such as chicory and acacia, has proven to enhance viability and performance. Furthermore, plant-based probiotic products are evolving beyond beverages to encompass snacks, ready-to-eat fruits, and nuts. Nonetheless, challenges persist regarding the viability of probiotic strains during storage, the costs associated with large-scale production, and the necessity of consumer education. Regulatory frameworks are being adapted to ensure health claims attributed to probiotic products that are backed by scientific evidence. As a promising frontier in sustainable functional foods, plant-based probiotics cater to a wide range of consumer needs. Future advancements are anticipated to focus on personalized formulations and broader applications within health and wellness, alongside improved techniques to enhance probiotic stability and efficacy.

Keywords: Functional foods, Non-dairy Probiotics, Plant-based probiotics, Sustainable nutrition, Vegan health, Gut microbiome, Eco-friendly foods

1. Introduction

Probiotics are live microorganisms that confer health benefits to the host when administered in adequate quantities [1]. These microorganisms exert beneficial effects via production of antimicrobial compounds, inhibition of pathogenic bacteria, and the generation of essential metabolites, including vitamins and short-chain fatty acids (SCFAs), thereby contributing to the overall well-being of the host [2]. The growing interest in probiotics is reflected in both scientific research and industry. Historically, dairy matrices have facilitated the growth and survival of probiotics, leading to their predominant use in products like yogurt, kefir, and cheese [3]. However, the reliance on dairy probiotics poses limitations that restrict their access to broader consumer groups.

Among the most well-known probiotic species are members of the lactic acid bacteria (LAB) group, comprising strains such as Lactobacillus acidophilus, Lacticaseibacillus casei, Lactobacillus crispatus, Bacillus coagulans, Lactobacillus delbrueckii subsp. bulgaricus, Limosilactobacillus fermentum, Ligilactobacillus gasseri, Lactobacillus helveticus, Ligilactobacillus johnsonii, Lacto-coccus lactis, Lacticaseibacillus paracasei Lactiplanti-bacillus plantarum, Limosilactobacillus reuteri, Lacticasei-bacillus rhamnosus[4].

Traditionally, these beneficial microorganisms are sourced from fermented dairy products, including kefir, yogurt, and curd. The growing demand for vegan foods, driven by health considerations, the prevalence of lactose intolerance, and increasing awareness of milk protein allergies, has fostered a shift toward non-dairy alternatives, notably in Asia and parts of Africa, where access to dairy products is limited [5]. Consumers are increasingly seeking sustainable probiotic delivery systems that align with ethical and environmental values [6]. Furthermore, eco-conscious consumers like to seek more sustainable probiotic delivery systems [7]. Figure 1 shows the classification of various probiotic foods.

Simultaneously, developments in fermentation technologies and food science have demonstrated the potential of plant-based matrices, such as cereals, legumes, fruits, vegetables, and plant-based milks, as efficient probiotic carriers. In addition to meeting dietary requirements, these substrates offer extra phytochemicals, dietary fiber, and bioactive compounds that could complement the effects of probiotics [8]. By offering unique tastes, textures, and health benefits, plant-based probiotic formulations can appeal to a large market. Despite these promising trends, technological challenges remain unresolved. For example, probiotic viability, stability, sensory optimization, and regulatory frameworks for plant-based products are a few of them. Moreover, further research and developments are needed to confirm the efficacy of particular strains in plant-based matrices, with an emphasis on consumer demand, technological advancements, and health applications. This review aims to provide a comprehensive overview of the emerging field of plant-based probiotics, highlighting the drivers of consumer demand, technological innovations, and health applications while critically examining the current limitations and future directions.

1. Growing Demand Drivers

2.1. Vegan and dairy-free probiotic trends

The growing demand for lactose-free, cholesterol-free, and vegan foods has led to innovations in non-dairy probiotic systems. Researchers and industries are exploring plant-based alternatives like tree nuts, cereals, fruits, and vegetables as alternatives to traditional dairy substrates [9].

Due to their rich nutrient and prebiotic content, tree nuts such as almonds and cashews are emerging as promising fermentation substrates. Major challenges, such as emulsion stability and microbial viability, are being addressed using encapsulation techniques, natural stabilizers, and pH control [9]. For instance, traditional dairy products such as kefir, renowned for its health-promoting components like GABA (Gamma-aminobutyric acid), are now being reformulated using soy, rice, oat, and coconut bases. Although these alternatives may vary in taste and texture, they still provide metabolic advantages [10].

Recent advancements have introduced specific, well-characterized microbial strains specially designed for use with plant substrates, ensuring safety, functional efficiency, and reproducibility criteria. Targeted fermentation leveraging local agricultural resources can enhance the nutritional and therapeutic potential of plant-based probiotic foods [11]. Fruit- and vegetable-based probiotic beverages are gaining traction due to their nutritional variety and bioactive compounds that enhance shelf-life and functional value. Nevertheless, commercialization is still limited, necessitating further technological innovations [12].

Fermented plant foods such as olives, vegetable juices, and pickled products, exhibit excellent potential as probiotic carriers due to their supportive matrices that enhance microbial adhesion and viability during controlled processing [10].

Nutritional evaluations indicate that vegan substitutes for dairy products like yogurt and cheese typically contain lower protein but higher fat and carbohydrate content. However, allergen risks from ingredients such as soy, nuts, and gluten pose challenges for product formulation. Clean labeling, improved protein profiles, and hypoallergenic options are vital for enhancing consumer acceptance [6]. Utilizing seasonal and regionally sourced substrates meets consumer demand for sustainable foods while also reducing production costs [13]. Transitioning from lab-scale innovation to industrial scalability necessitates multidisc-ciplinary collaboration encompassing microbiological, technological, and economic expertise.

2.2 Gut-health awareness

The gut microbiome plays a pivotal role in maintaining health by supporting metabolic, immunological, and protective functions; however, awareness of its importance among the public remains limited, especially among children and adults [14] across many regions. An educational initiative conducted at a science fair in Kuala Lumpur, Malaysia, aimed to raise awareness about gut microbiota by engaging 324 participants aged 5 to 64 through quizzes and informational displays. Post-session, 77.9% of participants reported increased awareness of gut microbiota, with 85.3% expressing a willingness to incorporate more fruits and vegetables into their diets. Additionally, 60.5% indicated an intention to bring fruit for breaks in school or at work [14].

Research focusing on parental awareness of infant gut health uncovered significant gaps in knowledge provided by healthcare professionals. A survey of 933 parents, particularly those with preterm infants or those born via cesarean section, revealed a general lack of awareness concerning the connection between delivery mode and gut microbiome development [15]. Most parents did not receive adequate information on the significance of infant gut health, nor did they understand the roles of prebiotics and probiotics. This highlights the urgent need for targeted educational initiatives to inform parents about promoting their infant’s gut microbiome.

Alongside traditional fermented foods and probiotic supplements, there is growing recognition of the role of fruits and vegetables as prebiotics, representing a promising avenue for encouraging healthier gut microbiota [6]. These educational efforts underscore the critical importance of public health initiatives in facilitating long-term dietary changes and making gut health knowledge more accessible to wider populations.

2.3 Sustainability awareness among consumers

Heightened environmental concerns have significantly influenced consumer preferences within the food and beverage sectors. Plant-based dairy alternatives are gaining attraction, not only for their health benefits but also due to their reduced environmental footprint. Compared to conventional dairy production, plant-based alternatives typically use less land, water, and produce fewer greenhouse gases, marking them as a more sustainable choice [7]. This aligns with the values of eco-conscious consumers, particularly among younger demographics, who actively seek products that uphold ecological balance and mitigate climate impacts. As sustainability emerges as a driving force behind food innovation, the shift toward plant-based options reflects both ethical consumption trends and efforts to combat environmental degradation.

1. Emerging Sources and Innovations

Plant-based probiotics (PBPs) are sourced from various plant matrices serving as effective carriers for probiotic delivery. These products address the drawbacks of dairy-based options and are suitable for individuals with restrictive diets requiring probiotics. Major plant sources contributing to the development of non-dairy probiotics include cereals, legumes, fruits, vegetables, and plant-based milks [16].

1. Cereal-Based Products

Cereals are rich in protein, carbohydrates, vitamins, minerals, and fiber. Cereal grains such as maize, sorghum, millet, oats, barley, wheat, and rye are being utilized for the production of probiotic-rich foods, incorporating probiotic strains to enhance consumer health benefits.

1. Legume-Based Products:

Legumes (e.g., chickpeas, beans, lupins, soybeans) boast high levels of resistant starch and galactooligosaccharides, known for stimulating the growth and survival of probiotics.

1. Vegetable-Based Products:

Vegetables are abundant in carbohydrates, vitamins, minerals, and health-promoting compounds such as phytochemicals and phytonutrients. Commonly used substrates include carrots, cabbage, tomatoes, and beets, which are utilized to manufacture probiotic products employing LAB species like L. acidophilus, L. plantarum, and B. longum.

1. Fruit-Based Probiotic Products:

Fruit juices (e.g., apple, pineapple, mango, orange) serve as alternative vehicles for delivering probiotics. These juices offer health benefits and refreshing flavors enjoyed by diverse age groups, being rich in sugars and bioactive compounds that probiotics can utilize [8].

1. Non-Dairy milk substitutes:

Traditional milk products are increasingly being replaced by plant-based alternatives composed mainly of nuts and cereals, such as soy, almond, rice, and oat milks, each of which confer various health benefits [10]. These alternatives are regarded as functional foods, delivering essential nutrients that can aid in disease prevention [16].

3.1 Traditional sources of fermented plant foods

Fermentation stands as an age-old, cost-effective technique for preserving food, leveraging the growth and metabolic activities of microorganisms. Traditional fermented plant-based foods are valued across cultures for their flavor, health benefits, and natural preservative properties, and they serve as abundant sources of probiotics. Today, they are gaining recognition as effective non-dairy probiotic vehicles within vegan diets and functional food innovations [17]. Key examples of fermented foods and their functional benefits include:

Kimchi is a fermented vegetable dish prepared from vegetables, usually Chinese cabbage (Napa cabbage), Korean radish, and a variety of seasonings (ginger, garlic, salt, red chilli pepper, fish sauce/shrimps, etc.). This is a popular side dish in East Asian nations like Korea, Japan, and China. It acts as a probiotic vegetable food product that has rich nutritional qualities. Kimchi is also considered to be the source of LAB (L. plantarum, L. brevis). It helps to boost digestive health, improve immune system and mental health. Other beneficial properties of kimchi include cholesterol lowering activity, anti-obesity, anti-cancer, antioxidant, and anti-atherosclerotic properties [18].

Sauerkraut is a traditional fermented vegetable item prepared from cabbage. It is produced by spontaneous fermentation of cabbage that mainly involves hetero-fermentative LAB [19]. This is also known as German kraut and is very popular in European countries. Natural, unprocessed sauerkraut contains beneficial probiotic micro-organisms like LAB (L. plantarum, Leuconostoc mesenteroides) [20]. From these foods, organic acids are produced by metabolizing sugars in the raw materials, altering flavors, prolonging its shelf life and producing B vitamins, such as folate (B₉) riboflavin (B₂), and other healthy components such as isothiocyanates and glycosylates. These compounds exhibit anti-inflammatory and anticancer properties which modulates specific cellular pathways and showing antioxidant activity which neutr-alizes the damaging effects of free radicals [8].

Tempeh is a nutrient-rich fermented legume food derived from soybeans and commonly consumed in Southeast Asia, especially in Indonesia and Malaysia. The fermentation process involves growth of mold, Rhizopus spp. which transforms soybeans into a white firm cake-like product as enzymes break down complex nutrients in soybeans into simpler forms, enhancing the bioavailability of proteins, fiber, and other nutrients. Consumption of Tempeh has been linked to various health advantages including antidiabetic effects, reduced risk of cardiovascular diseases, cholesterol-lowering properties, improved cognitive function, antitumor and anticancer properties, anti-aging effects and improved gut health [21].

Miso is a Japanese salty and flavorful fermented paste made by fermenting soybeans with salt and a fungus viz., Aspergillus oryzae (koji), Lactobacillus sp. along with soybeans and other ingredients such as barley, rice, and rye. It is typically salty and is considered as a good source of protein, fiber and vitamins (especially vitamin K), minerals, plant compounds, manganese, and copper. This helps to promote digestion, reduce cancer, obesity, high blood pressure and also helps regulate cholesterol levels [22].

Natto is another fermented soybean product, containing the bacterial strain- B. subtilis. It is typically mixed with rice and served with breakfast. It has a distinctive smell, slippery texture, and strong flavor, rich in protein, vitamin K2 and is good for bone and cardiovascular health [22].

Kombucha is a probiotic drink that originated in China which was made by fermenting sweet tea to produce a tangy and fizzy beverage. Known for its distinctive sour taste, kombucha is believed to have detoxifying properties. D-glucaric acid in kombucha is linked to liver detoxification, helping bind and eliminate harmful compounds from the body. The drink also contains antioxidants, including ascorbic acid, gluconic acid, and polyphenols, which help combat reactive oxygen species, reduce oxidative stress, and may offer protective benefits against degenerative diseases such as atherosclerosis and Alzheimer's disease [8].

Pickles are an important dish, notably in India, US, Russia etc. Even though a variety of vegetables are used for pickling, cucumbers are considered as one of the most traditional options. The fermentation process for pickle production relies primarily on naturally occurring lactic acid bacteria, notably L. plantarum and L. brevis. As bacteria utilize the carbohydrates in cucumbers which help to generate lactic acid, it imparts a characteristic tangy flavor and inhibit the proliferation of pathogens and spoilage microorganisms. The probiotic content of pickles holds potential health benefits, such as regulating blood sugar levels and exhibiting anti-cancer properties [8]. The figure 2 shows the health benefits of fermented plant-based foods having probiotic potential.

3.1.1 Microbial composition and fermentation mechanism

The microbial composition in fermented plant foods primarily includes bacteria, yeasts, and moulds, of which Lactobacillus, Bifidobacterium, Acetobacter, and Saccharomyces are the predominant species. These microorganisms play crucial roles in fermentation, influencing the characteristics of the food and metabolite production [23]. The action of enzymes and activity of microorganisms may induce changes in the nutritional properties and bioactive compound content, compared to the raw substrate [24]. Fermentation mechanisms are key to developing flavor, texture, nutritional value, and probiotic functionality. Some types of fermentation involved include:

(1) Lactic acid fermentation, which is involved in the formation of kimchi, sauerkraut and pickles. Some of the strains include Lactobacillus, Leuconostoc, Pediococcus, and Weissella. The mechanism involves conversion of glucose to pyruvate to lactic acid, which inhibits pathogens, and preserves the food.

(2) Alcoholic Fermentation, which is involved in the formation of kombucha and certain vegetable-based beverages, is carried out using Saccharomyces cerevisiae. The mechanism involves conversion of sugars into ethanol and carbon dioxide. In kombucha, a synbiotic culture of bacteria and yeast (SCOBY) are used for its production.

(3) Acetic acid fermentation that is also involved in kombucha formation (utilizing the strain   Komagataeibacter xylinus) the oxidation of ethanol to acetic acid occurs under aerobic conditions [25].

3.1.2 Challenges associated with probiotic viability

The growing market for vegan probiotics faces several major challenges, including environmental factors (pH, temperature, oxygen level, and the presence of secondary metabolites) and manufacturing processes (including heat treatment and storage conditions) that may affect probiotic viability [26]. Strategies to ensure proper viability include selecting resilient strains, implementing encapsulation, enriching substrates with prebiotics (synbiotics), managing controlled fermentation, and adhering to strict packaging and cold-chain processes [27].

3.2 Novel strains being optimized for plant matrices

In functional food production, a critical consideration is given to the food matrix, which serve as the carrier for probiotic microorganisms, and provide a supportive environment for their growth and survival. The food matrix should also protect viable probiotic cells to ensure survival during passage through the gastrointestinal (GI) tract, thus allowing the appropriate gut colonization. These steps are essential for achieving the intended probiotic health benefits for the host [28]. GABA is a non-protein amino acid, which plays a significant role as an inhibitory neurotransmitter in the mammalian central nervous system. It is been reported that GABA is associated with mammalian behavior by regulating stress and anxiety, modulating cognitive and brain functions, promoting sleep, and enhancing mood [29]. A novel probiotic GABA drink was created using brown rice as the main base ingredient, incorporating L. pentosus 9D3, a GABA-producing strain derived from Thai pickled weed [29]. L. plantarum ITM21B (LMG P-22033) and L. paracasei IMPC2.1 (also quoted as LMG-P22043) survived on brined artichokes for at least 90 days and the anchorage of bacterial strains on the vegetable tissues improved their survival in a simulation of GI digestion [30]. Probiotic drink was developed by combining extracts of medicinal plants which provide significant health benefits. A study reported by Eksiri et al 2017 showed that the produced probiotic drink containing apple juice, Pussy willow and Echium amoenum, glucose and whey powder which was a favorable medium for the growth of L. casei and L. rhamnosusto [31].

A combination of emerging strains such as Akkermansia muciniphila with polyphenol rich plant matrices (grapes, berries) enhance growth of the organism and has demonstrated to improve colonic inflammation and metabolic disorders and some other diseases in animal models [32]. Another study revealed that patients treated with Faecalibacterium prausnitzii exhibited a reduction in non-alcoholic fatty liver disease, hyperlipidaemia, prediabetes, inflammatory bowel disease and type 2 diabetes, positioning it as a potential Next generation probiotic (NGP) strain [32]. A variety of plant-based matrices (food products) for probiotic delivery are currently being explored. The survival of probiotic cultures is dependent on the processing steps, pH, food matrix, probiotic strain and the method of incorporation into the matrix, storage conditions /packing, and addition of prebiotic components [33]. Vegan probiotics impact the properties of food by extended shelf life, improved nutritional value, sensory changes [33].

3.3 Plant-based synbiotics

Plant-based matrices are rich sources of nutrients essential for probiotic growth, including complex carbohydrates, simple sugars, and plant proteins, in addition to non-nutritive bioactive compounds like fibers, vitamins, minerals, and secondary plant metabolites. These components exert antioxidant and antimicrobial properties, reduce dysbiosis, and reinforce intestinal integrity [34]. Many bioactive elements impact gut microbiota, supporting a balanced microbiome and improving overall health. Prebiotics—non-digestible compounds promoting beneficial microorganisms—contribute to alleviating gastrointestinal symptoms, immunomodulation, and preventing metabolic disorders [34]. Synbiotic products combining probiotics and prebiotics are formulated using isolated or concentrated prebiotics to ensure effectiveness.

Prebiotics of plant-based matrices are a source of essential nutrients and, can support probiotic survival in food environments as well as in the digestive tract. They also would moderate probiotic bioactivity [28]. The safe usage of L. plantarum MBTU-HK1 (probiotic) and acacia gum (prebiotic), either individually or together as a synbiotic was clearly documented [35]. When taken together, this synbiotic demonstrated a range of health benefits, such as reduced blood lipid levels, improved immune regulation, and decreased activity of enzymes linked to carcinogen release. Maintaining a healthy gut microbiota through the combined use of prebiotics and probiotics offers a promising approach to enhancing overall well-being and preventing disease. Similarly, a study revealed that the extraction and purification of inulin from chicory roots act as a prebiotic and that can be used along with probiotics exhibiting various health benefits [36]. A study conducted by Chaturvedi et al. 2022 investigated on optimization of extraction process of legume-based symbiotic beverages from red kidney and mung bean blends fermented with L casei which revealed that the fermentation significantly reduced antinutrients in the beverages. Therefore, the formulated beverages under standardized fermentation conditions, have the potential to serve as a functional food in non-dairy industry which offer improved health benefits and newer flavor profiles [37]. A study conducted screened the combinations of commercial probiotic strains (e.g., L. rhamnosus GG, B. lactis) with six different dietary fibers (prebiotics) to access its synergistic role in producing SCFAs, which are key metabolic products linked to gut health, and the work concludes that the prebiotic combinations with different probiotic strains that may be useful for developing effective synbiotic blends [38].

1. Future Trends

4.1 Functional foods and beverages

Heightened consumer awareness regarding nutrition, coupled with the World Health Organization's endorsement of functional foods, has spurred the global demand for functional beverages enriched with bioactive compounds from plant, animal, or microbial origins, including phenolics, vitamins, peptides, and unsaturated fatty acids. The functional beverage market is experiencing a surge in the popularity of probiotic/prebiotic drinks, products tailored for immunological and cognitive health, and those with enhanced aesthetic appeal. Technological advancements such as encapsulation, emulsification, and high-pressure homogenization are employed to boost ingredient stability and bioavailability [39]. Nonetheless, challenges persist around maximizing sustainability, safety, and bioavailability [40]. Continuous innovation in formulation, storage, and sensory appeal remains crucial for ensuring long-term market viability and consumer acceptance [40]. Table 1 represents a selection of recently developed probiotic food products formulated using novel plant-based or non-traditional substrates, probiotic strains used, viable counts, and associated health benefits.

4.2 Pharmaceutical and cosmetic applications

Non-dairy probiotics are increasingly relevant in pharmaceutical applications driven by the consumer interest in health-promoting vegan products and the need for potent alternatives to dairy-derived probiotics. They are being investigated for therapeutic potential across various health conditions, particularly in supporting gut health, enhancing immune function, and promoting well-being. The future development of next-generation probiotics is likely to employ innovative strategies aimed at reinventing probiotic therapies [50]. Additionally, non-dairy probiotics are gaining traction in the beauty and skincare industry for their ability to support skin health. Probiotics are being integrated into cosmetic formulations to confer benefits such as anti-aging, anti-inflammatory, and hydrating effects. A study conducted using the non-dairy probiotic Micrococcus luteus Q24 demonstrated its capacity for improving skin condition following application. Notably, this application reduced pore size and wrinkles while remarkably increasing hydration levels, indicating the potential for probiotics as valuable ingredients in the beauty sector focused on enhancing skin texture and health [51]. Further, probiotics in both oral and topical forms can treat skin disorders by augmenting the skin's microbiome, enhancing barrier function, and targeting inflammation [52].

4.3 Regulation and labeling

The development of comprehensive regulatory frameworks and labeling policies for probiotics and probiotic-based foods encompasses safety, efficacy, quality control, and health claims regulations [53]. Given the marketing of probiotics as food, dietary supplements, or nutraceuticals, compliance with legal standards for product labeling is required, including specifics such as product name, scientific strain identification, viable count in colony-forming units (CFUs), approved health benefits, recommended daily dosage, storage requirements, expiration date, allergy information, and company contacts for adverse effect reporting [54].

1. Challenges in the Development of Plant-Based Probiotics

Despite the rising consumer interest, several obstacles hinder the full realization of plant-based probiotics.

5.1 Consumer awareness and acceptance

While there is an increasing demand for plant-based alternatives, particularly among vegans and lactose-intolerant individuals, a significant gap exists in consumer knowledge regarding these products. Many potential consumers remain unaware of the sensory qualities, efficacy, and health benefits associated with plant-based probiotics, creating a barrier to market penetration. Factors such as sensory acceptability, taste, and texture, along with skepticism about their probiotic efficacy, further impede acceptance [55].

5.2 Innovation in non-dairy functional beverages: synbiotic vegan juices

The diversification of probiotic food products into vegan, non-dairy formats has garnered attention as a means to satisfy the nutritional needs and preferences of health-conscious consumers. A study by Valero-Cases et al. explored the development of synbiotic carrot-orange juices fermented with Lactiplantibacillus plantarum and enriched with inulin [56]. Their research demonstrated that fermentation markedly enhanced the antioxidant capacity (AOC), primarily attributed to the β-carotene content. Inulin improved the survival of L. plantarum over a 21-day period while enhancing the sensory profile, with 2% inulin receiving the highest consumer acceptance due to its improved sweetness and orange flavor. The growing interest in plant-based functional foods is also supported by emerging trends in food biotechnology. Arwanto et al. discussed innovative techniques such as fermentation, high moisture extrusion cooking, and shear cell technology, which restructure plant proteins into realistic meat-like textures. These advancements cater to the increasing consumer demand for healthier plant-based diets, which have been linked to lower disease risk, stress reduction, and better weight maintenance. Furthermore, the authors emphasized the market potential for plant-based products, particularly in regions like Indonesia, underscoring the significant opportunities for global expansion in this arena [57].

5.3 Consumer perspectives on plant-based dairy alternatives

The acceptability of plant-based dairy substitutes has spurred additional research into consumer perceptions. Adamczyk et al. identified health concerns, curiosity, and social influences as key motivators for accepting these alternatives, while barriers such as taste, texture, and familiarity remained pronounced [58]. A comprehensive study conducted across focus groups in Poland, Germany, and France revealed that attitudes toward plant-based dairy substitutes vary significantly by country, reflecting the deep-rooted significance of dairy in diverse culinary traditions.

This variability highlights the necessity for tailored marketing strategies that acknowledge local preferences and cultural contexts. The rise in popularity of plant-based beverages has spurred market developments, including the proliferation of probiotic drinks and various functional beverages. Kellershohn's overview of the probiotic beverage market elaborated on consumer demographics, market size, and increasing demand among health-conscious individuals seeking to enhance digestive health and immunity [59]. Notably, the demographics for probiotic beverages also shifted toward younger, educated consumers, reinforcing the need for products that resonate with contemporary health trends.

Emerging innovations in the sector include adaptogens (plant-derived compounds that assist the body in managing stress) and postbiotics, which are metabolic byproducts of fermented probiotics. Additionally, non-digestible fibers known as prebiotics nourish beneficial gut bacteria, while psychobiotics (microorganisms or compounds that influence mental health via the gut-brain axis) are gaining attraction for their potential health benefits [59].

Moss et al. revealed that consumer attitudes towards plant-based milk alternatives (PBAs) in Canada were influenced by health benefits, sustainability, and sensory attributes [60]. The popularity of oat, almond, and pea milk rose significantly, particularly when flavored with chocolate or vanilla. The study emphasized that the sensory characteristics of creaminess and smoothness significantly enhance the acceptability of PBAs. These insights reinforce the need for continued research focused on improving flavor and sensory attributes to bolster consumer appeal and acceptance.

5.4. Safety and strain-specificity, cost, investment, and scalability

While PBP hold promising health benefits, it is essential to recognize that these effects are not universally applicable across all strains. Selecting and clinically validating specific strains for target health benefits is critical [3]. Consideration of dose-dependency is also pertinent, with effective doses typically ranging from 10⁸–10⁹ CFU/day in human studies [61].

Safety is another significant consideration, as adverse effects such as bacteremia or sepsis have been documented, particularly in immunocompromised individuals, despite most probiotics being classified as Generally Recognized as Safe (GRAS) [50]. Hence, thorough safety evaluations and regulatory oversight are crucial in the development of plant-based probiotic products.

The advancement of high-end biotechnological methods and specialized fermentation techniques is vital for creating stable and functional PBP, though these developments can increase manufacturing costs. Different plant substrates can introduce variability, complicating the smooth scaling of production. Addressing these challenges will require substantial research funding and robust public-private partnerships. Despite the undeniably substantial health and sustainability advantages offered by PBP, consumer awareness and perceptions remain critical hurdles that demand attention.

Prevalent misconceptions regarding the efficacy of plant-based probiotics, together with unfamiliarity and sensory challenges compared to traditional dairy products, currently limit their widespread adoption. For example, a study involving Danish consumers based on the Theory of Planned Behavior revealed that positive attitudes, perceived sensory quality, and self-efficacy significantly influenced intentions to choose plant-based yogurt alternatives [62]. In contrast, social norms and objective knowledge appeared to have minimal impacts on consumer decisions. This suggests that enhancing sensory appeal and bolstering consumer confidence are more effective strategies than merely providing additional information.

In South Korea, Lee et al. examined consumption patterns and pairing behaviors associated with cow's milk versus plant-based milk [63]. Their findings indicated that plant-based milk consumers generally tended to be older adults, women, and urban residents. Notably, these consumers were inclined to pair plant-based milk with various foods, such as bananas, eggs, nuts, and sweet potatoes, reflecting distinct consumption habits compared to traditional dairy consumers.

An intriguing experiment comparing consumer perceptions of "vegan latte" versus "plant-based latte" labels revealed that both labels performed comparably regarding purchase intentions. However, there was a slight preference for the “plant-based” label. This study indicated that, even among non-vegans, curiosity about food and a desire for diverse options served as strong motivators for adoption, pointing to the effectiveness of good labeling and messaging in broadening demographics [63].

These findings highlight the necessity to promote plant-based probiotics as credible alternatives through a multifaceted approach focusing not only on health benefits but also on consumer psychology, compatibility with lifestyle choices, and appealing food pairings.

1. Health Benefits and Mechanisms of Action of Plant-Based Probiotics

PBP offer a diverse range of health benefits; however, these effects are highly strain-specific, often dose-dependent, and possess well-characterized mechanisms of action. A critical understanding of these factors is essential to validate and optimize the efficacy of PBP. Some of the health benefits of plant-based probiotics are depicted in figure 3.

1. Gut Health and Barrier Function

One of the most consistently observed benefits of probiotics is the improvement of gut health through intestinal microbiome modulation, promoting gut barrier integrity, and anti-inflammatory effects. Lactiplantibacillus plantarum, commonly used in plant matrices such as brined artichokes [30], has showed the ability to improve gut barrier function and survive GI passage.  The production of SCFAs, such as butyrate, which support epithelial integrity and have local anti-inflammatory effects, has also been demonstrated to modulate the intestinal microbiome and promote gut barrier integrity in Lactiplantibacillus plantarum strains used in co-culture for the development of functional plant-based fermented beverages [64].

1. Modulation of Immune Responses

Probiotic strains can positively influence immune function through various mechanisms, including enhancing mucosal immunity (e.g., immunoglobulin A, IgA secretion), modulating cytokine production, and interacting with gut-associated lymphoid tissue (GALT). For instance, Lactiplantibacillus plantarum MBTU-HK1 combined with acacia gum showed improved immune regulation in a synbiotic formulation [34]. Lactiplantibacillus plantarum and Lacticaseibacillus casei, used in fortified cut pineapple matrices, also contributed to enhanced immune responses and gut health [44].

1. Cholesterol-lowering and Metabolic Effects

Certain probiotic strains contribute to cholesterol-lowering effects through bile salt hydrolase activity and modulation of lipid metabolism. For example, B. coagulans, when incorporated into quinoa snacks, has been observed to lower serum cholesterol levels while exhibiting antimicrobial activity [41]. In addition, L. pentosus isolated from Thai pickled weed produces GABA when fermented in brown rice drink, which is associated with reduced stress levels, improved cognitive functions, and potential blood pressure modulation [29].

The spectrum of health benefits provided by plant-based probiotics is wide-ranging and promising, marking improvements in gut barrier functionality, immune modulation, metabolic regulation, and antimicrobial protection. However, determining the exact health benefits is heavily reliant on the specific probiotic strains, their formulations, and dosing conditions. Hence, future research endeavors should prioritize strain-specific clinical validations, synergistic synbiotic combinations, and address regulatory standards that facilitate the complete realization of PBP in functional food applications.

6.1. Health impacts and functional properties of plant-based alternatives

Toribio-Mateas et al. (2021) investigated the microbiome changes associated with partial replacement of animal meat with plant-based meat alternatives (PBMAs) [65]. Their findings indicated that such dietary transitions led to beneficial shifts in the gut microbiome, including an increase in butyrate-producing bacteria and enhanced metabolic potential related to gut health. This underscores the potential of PBMAs in fostering dietary shifts toward flexitarianism or a predominantly plant-based diet.

1. Conclusion

The advent of plant-based probiotics marks a significant evolution in the realm of functional foods and gut microbiome research. The convergence of consumer interest in vegan diets, lactose intolerance issues, environmental sustainability, and the emergence of plant-derived probiotics culminate in a robust alternative to conventional dairy formulations. Advancements in strain selection, fermentation technologies, and prebiotic incorporation have facilitated the creation of functional foods and beverages that cater to diverse dietary preferences and offer multiple health benefits, including gut health promotion, immune enhancement, and reduction of metabolic disorders such as obesity, diabetes, and hyperlipidemia.

Furthermore, the integration of plant-based probiotics and synbiotics into innovative functional food and beverage formulations resonates with ongoing preventive healthcare and lifestyle wellness trends. However, key challenges such as strain viability, large-scale production, and enhancing consumer awareness need to be addressed to solidify efficacy and foster market success. Future research endeavors should concentrate on personalized nutrition, next-generation probiotic strains, and regulatory frameworks that embrace innovation while ensuring product integrity. In conclusion, plant-based probiotics possess tremendous potential for advancing human health, aligning with sustainable practices, and fulfilling the evolving demands of health-conscious consumers. Continued research that interweaves food science, microbiology, biotechnology, and nutrition is essential to overcome existing barriers and unlock the full potential of plant-derived probiotics.

1. Acknowledgements

The authors acknowledge the support extended by the Principal, faculty and students at Government Arts College, Trivandrum and Executive director and staff at CEPCI, Kollam.

1. Declaration of competing interest

The authors report no conflicts of interest.

1. Authors’ Contributions

Formal analysis, investigation, writing—original draft preparation- ASN, AS.; Conceptualization, Supervision, Review and editing – RRP, SS.

1. Using Artificial Intelligent Chatbots

No AI chat bots were used in the preparation of this manuscript.

Background and Objective: Fermentation, a microbial-driven metabolic process, has been utilized for millennia to produce, preserve, and enhance food and beverages, evolving from traditional practices such as yogurt and wine to modern applications in biofuels and pharmaceuticals. Fermentation has been found to have its earliest evidence more than 7,000 years ago. The principle of fermentation was established in the 19^th century, based on the foundational research of Pasteur, who bridged the connection between microbial activity and chemical change, setting the stage for modern microbiology and biotechnology. This review explores microbial roles in fermentation, bridging traditional and biotechnological advancements by comparing bacterial and fungal processes, analyzing key metabolites, and highlighting genetic innovations.

Results and Conclusion: The review comprehensively explores the pivotal role of microbial fermentation, spanning traditional practices to modern innovations, highlighting the extensive diversity of 195 bacterial and 69 fungal species, with lactic acid bacteria (LAB) and Saccharomyces cerevisiae being prominent examples. It compares bacterial and fungal fermentation processes, noting that bacterial fermentation often yields higher protein content in products like fermented soybean meal (FSBM), and discusses key metabolites, including primary (amino acids, organic acids, vitamins) and secondary (antibiotics, antitumor agents) compounds, for their industrial and health applications. The review also examines various fermentation methods and their suitability for different products, emphasizing advancements in genetic engineering for strain optimization, while underscoring the health benefits of probiotics and fermented foods and the potential of emerging technologies to address food security and sustainability. In conclusion, microbial fermentation bridges ancient traditions with cutting-edge science, offering transformative potential across industries, where innovations in genetic engineering and process optimization drive efficiency and sustainability, and the expanding microbial repertoire continues to unlock novel applications, integrating fermentation technology with modern biotechnological tools to address global challenges in nutrition, health, and environmental sustainability.

Keywords: Biofuel production, bioeconomy, CRISPR-Cas9, food security, probiotics, solid-state fermentation (SSF), submerged fermentation (SmF), sustainability, synthetic biology.

Microorganisms have played a central role in human life, particularly during the food fermentation process. Fermentation has been utilised over the centuries to produce, conserve, and flavour beverages and foods, enhancing their nutritional and sensory qualities. Fermentation is a metabolic process that uses microorganisms to break down carbohydrates into alcohol, organic acids, gases, or other desired by-products [1]. The fermentation processes were induced by naturally occurring populations of bacteria, yeasts, and moulds, whose synergistic activity produced characteristic textures, flavours, and nutritionally beneficial advantages. This has evolved from a mystical art to a science with the discovery of microorganisms by pioneers like Antonie van Leeuwenhoek and Louis Pasteur [2]. Development of microbiology, molecular biology, and bioprocess engineering has made it possible to identify, isolate, and genetically improve microbial strains for large-scale fermentations. Fermentation has numerous applications in the biotechnology industry, including the production of antibiotics, vaccines, enzymes, biofuels, biodegradable plastics, and food products. Considering recent health and sustainability concerns, microbial fermentation offers promising routes for producing plant-based meat alternatives, upcycling agro-industrial waste into high-value products, and generating nutraceuticals and pharmaceuticals.

Probiotic fermentation is another contemporary area of research in food fermentation. It refers to the process where live microorganisms, known as probiotics, are introduced to a food or substrate to undergo fermentation, resulting in a variety of beneficial changes [3]. This process enhances the food's nutritional value, digestibility, and flavour, while also introducing beneficial bacteria that can positively impact the consumer's gut health [4]. Probiotic technology leads the functional food sector based on strain specificity, gut microbiota interaction, and individualised nutrition.

Contamination control issues, regulatory acceptance of genetically modified strains, consumer acceptability, and scaling up of new technology are being addressed more effectively through interdisciplinary and collaborative partnerships at a global level [5]. The function of microbes in fermentation is a balance between tradition and innovation, promoting an interrelated and symbiotic collaboration between human creativity and microbial activity. Fermentation is a biotechnology that has been utilised for millennia to flavour and preserve food, produce drinks, and create pharmaceuticals. Its roots date back to microbes that transform raw material into staple foods, such as bread, cheese, wine, and beer [2]. Fermentation is coupled with technology to produce a wide variety of products, ranging from traditional fermented foods and specialty beverages on one end and antibiotics, biofuels, and recombinant proteins on the other [6].  The metabolic diversity of microbes has been utilised to produce both submerged and solid-state fermentation systems, each offering distinct advantages for specific applications. The introduction of synthetic biology, genetic engineering, and omics technologies has further transformed microbial fermentation with improved metabolic pathways for precision biomanufacturing.

Technologies such as continuous fermentation, immobilised cell systems, and electro-fermentation have enhanced process efficiency and sustainability. Additionally, their applications in cellular agriculture, biodegradable plastics, and waste valorisation reflect the ability of fermentation to address key challenges in food security, healthcare, and environmental sustainability. Fermentation is alternatively referred to as Zymology or Zymurgy and was practised by humans since the Neolithic era (around 10000 BC). Industrial fermentation involves the use of microorganisms, such as bacteria and fungi, to produce goods beneficial to humans and acidic products with applications in the food industry [7]. Fermentation rate depends on the concentration of microorganisms, cell types, cellular constituents, enzymes, temperature, pH, and the aerobic fermentation factor.

1. Inventory of microbial species used in fermentation

Over the past two decades, the number of microbial species used in fermentation processes has significantly increased. The 2002 International Dairy Federation Inventory, which catalogued 82 bacterial species and 31 yeast and mould species, has been replaced by the current Inventory of Microbial Food Cultures, which now includes 195 bacterial species and 69 yeast and mould species [8]. This growth is attributed to advanced genomic sequencing techniques, increased demand for a variety of fermented foods, and advancements in microbial biotechnology. The bacterial repository has experienced a meteoric rise in lactic acid bacteria (LAB) genera, with lactobacilli comprising 45 different species used in dairy, meat, vegetable, and cereal fermentations [8]. In 2020, the Lactobacillus genus was significantly reorganized, with many species being reclassified into new genera. This included the creation of 23 new genera. For example, Lactobacillus casei is now Lacticaseibacillus casei, and Lactobacillus plantarum is now Lactiplantibacillus plantarum. Lactobacillus rhamnosus is now Lacticaseibacillus rhamnosus, and Lactobacillus brevis is now Levilactobacillus brevis. Lactobacillus salivarius is now Ligilactobacillus salivarius, and Lactobacillus fermentum is now Limosilactobacillus fermentum [9]. The number of Bifidobacterium species increased from 4 to 10 due to their importance in probiotic applications. The yeast and mould inventory has grown from traditional Saccharomyces cerevisiae and Aspergillus oryzae species to include a broad range of non-traditional species such as Pichia kudriavzevii for cocoa fermentation and Neurospora intermedia for oncom production. The identification and utilisation of extremophiles, such as thermophilic Geobacillus species in hot fermentations and halophilic Archaea in high-salt processes, has triggered diversification of microorganisms. It also includes engineered strains developed through the application of both traditional mutagenesis and novel genetic techniques, such as lactose-positive yeast strains for use in dairy applications and bacteriocin-producing LAB variants for enhanced food safety. Regulatory reforms and enhanced safety assessment procedures have enabled the inclusion of previously underutilised species. Concurrently, market demand for flavor, texture, and health-affecting effects has spurred the utilization of microbial diversity from globally dispersed traditional fermented foods [9]. The diversity of microorganisms involved in fermentation processes spans a wide range of taxonomic groups, including bacteria, yeasts, and filamentous fungi. These microbes play pivotal roles in transforming raw substrates into valuable fermented products. As illustrated in Figure 1, the taxonomic distribution highlights the predominance of LAB and Saccharomyces cerevisiae in traditional and industrial fermentations, alongside other key genera such as Aspergillus sp. and Bifidobacterium sp. This microbial richness underscores the versatility of fermentation technology across food, pharmaceutical, and bioeconomic sectors.

The distribution of microbial species used in fermentation was systematically categorised across relevant taxonomic units, as detailed in Table 1 (bacteria) and Table 2 (fungi/yeasts). Table 1 highlights the predominance of lactobacilli (45 species) and Bifidobacterium (10 species) among LAB, alongside Bacillus (over 30 species), which are also utilised for industrial enzymes. Table 2 emphasizes Aspergillus and Saccharomyces as key fungal genera, with Aspergillus niger for organic acids and Saccharomyces cerevisiae for ethanol. Some taxa, such as Acetobacter (vinegar) and Pichia (recombinant proteins), reflect diversification. This structured classification shows the microbial richness harnessed in traditional and modern fermentation biotechnologies.

2.1. Bacteria

2.1.1. Actinobacteriaceae

The microbial richness in the manufacture of fermented foods has undergone extensive taxonomic resolution and growth, indicative of advances in genomic research for the microbial ecology within food substrates. The genus Brachybacterium is a component of the surface microbiota of artisanal cheeses, such as Gruyère and Beaufort. It plays a role in proteolysis and flavour enhancement through its enzyme activities. Microbacterium gubbeenense is a significant member of the classical red-smear surface cultures employed in surface-ripened cheeses, playing a role in colour development and aroma profile [29]. The genus Bifidobacterium has undergone taxonomic revisions, with the redescription of Bifidobacterium infantis and the addition of Bifidobacterium thermophilum due to its significant roles in functional food applications for probiotic dairy products. Brevibacterium aurantiacum is utilised for its essential role in the ripening process, contributing to the flavour complexity and the distinctive orange colour of such cheeses. The Propionibacterium genus, including Propionibacterium freudenreichii subsp. globosum and Propionibacterium jensenii (now reclassified as Acidipropionibacterium jensenii) [30].

2.1.2. Firmicutes

The International Molecular Foods inventory has expanded significantly with advances in microbial taxonomy and the growing industrial demand for specialised starter cultures in a diverse range of food matrices. Some notable additions include three species of Carnobacterium, Tetragenococcus, Weissella, Enterococcus faecalis, Lactobacillus, Staphylococcus, and Streptococcus. Carnobacterium is essential for meat fermentations, and Tetragenococcus comprises species required for high-salt fermentations, such as those used in the production of soy sauce and fish products. Weissella splits species previously classified as part of the Leuconostoc mesenteroides complex for their distinctive phylogenetic and metabolic characteristics [31]. Enterococcus faecalis is the starter for dairy, meat, and vegetable fermentations, whereas the lactobacilli comprise 82 species. The Staphylococcus genus contains 13 species, which prove their critical contribution towards meat fermentation. This also allows for the specific selection of strains to achieve goal-directed fermentation results, such as balanced acidification and aroma formation in sausages or maximum gas formation in sourdough.

2.1.3. Proteobacteriaceae

The International Molecular Foods stock has been supplemented with specialised microbial species, which cover conventional fermentation practices and new biotechnological developments. Acetic acid bacteria, such as Acetobacter sp. and Gluconacetobacter sp., play a crucial role in the production of vinegar, as well as in the fermentation of cocoa and coffee, and in imparting flavour sensations. Halomonas elongata is a halotolerant organism that has been indicated in meat fermentation systems due to its resistance to high sugar levels and acidic environments [32]. This expanded microbiota enables specificity in the fermentation process, as observed in Acetobacter pasteurianus strains used to produce artisanal balsamic vinegar. Zymomonas mobilis, due to its novel pyruvate decarboxylase and alcohol dehydrogenase II pathway, prefers the irreversible production of ethanol. The industrial application of these microbial resources has demonstrated quantifiable effects, wherein Gluconacetobacter europaeus strains reduced vinegar-making times by 30–40 % and specific Zymomonas mobilis isolates achieved 15–20 % productivity gains in ethanol production, making fermentation more feasible for tequila production.

2.2. Fungi

2.2.1. Yeast

Fungal taxonomy employed in food production has been extensively utilised, as numerous species formerly associated with the genus Candida have been reassigned to more phylogenetically accurate genera. This has significant implications for food science and industry, enabling more accurate strain selection, process optimisation during fermentation, product uniformity, and the prevention of spoilage risk [33]. Examples are Dekkera bruxellensis, Debaryomyces hansenii, Hanseniaspora uvarum, Kazachstania turicensis, Metschnikowia pulcherrima, Pichia occidentalis, Rhodosporidium spp., Saccharomyces pastorianus, Saccharomycopsis fibuligera, Saturnisporus saitoi, Sporobolomyces roseus, Torulaspora delbrueckii, Trichosporon cutaneum, Wickerhamomyces anomalus, Yarrowia lipolytica, Zygosaccharomyces bailii, and Zygosaccharomyces rouxii. The persistence of some Candida species with unknown teleomorphs of fungal life cycles, to explain their sexual phases or validate their taxonomic position using genomic information. The functional properties of these microorganisms are being utilised increasingly, such as Metschnikowia pulcherrima's use in biocontrol to inhibit moulds on vineyards and Wickerhamomyces anomalus's delivery of killer toxins that inhibit spoilage yeasts in fermented foods [34].

2.2.2. Filamentous fungi

Fungi are well-established in Asian food fermentation traditions, and have been added to the list of microorganisms used in fermented foods in Europe. There is significant potential in adopting well-established fungal starter cultures from Asian food fermentation traditions and incorporating them into European practices. Key fungi include Aspergillus oryzae, Aspergillus sojae, and Rhizopus oligosporus, which are essential to produce Asian fermented foods and beverages such as miso, soy sauce, sake, awamori liquors, and Puerh tea. Fusarium species, such as Fusarium domesticum and Fusarium solani, are utilised in European food applications, including cheese fermentations, Vacherin cheese production, and the development of meat alternatives. Penicillium species dominate fungal applications in European dairy traditions, with Penicillium camemberti being used in bloomy-rind cheeses such as Camembert and Brie. European charcuterie traditions utilise Penicillium nalgiovense and select strains of Penicillium chrysogenum for mould-fermented sausages, while Lecanicillium lecanii shows promise for cheese ripening applications. Fungal contributions beyond fermentation to include colourant production, with species like Epicoccum nigrum and Penicillium purpurogenum capable of generating natural pigments [35]. This fungal application highlights the rich diversity of fungal species in global food systems and the significant opportunities for cross-cultural transfer of fungal technologies in Asian fermentation fungi to European practices for novel product development.

1. Comparison of bacterial and fungal Fermentation

Fermented Soybean Meal (FSBM) is a highly nutritious protein source, thanks to the unique metabolic pathways and enzymatic activities of the microorganisms involved. Both fungal and bacterial fermentation processes effectively reduce antinutritional factors and enhance the nutritional quality of the resulting products. However, there are notable differences in specific components that influence the suitability of FSBM for various dietary and health applications [36]. Both the fungal and bacterial fermentations increased crude protein content through microbial biomass accumulation, with fungal-fermented FSBM showing a 19.4% increase in soluble protein, a crucial indicator of digestibility, and a more substantial 63.11% increase in bacterial-fermented FSBM. These fermentations also reduced the immunoreactivity of soybean meal, which is essential for minimising allergic responses and enhancing food safety. Amino acid profiles show that crucial amino acids generally remain stable during fermentation, but specific changes occur depending on the microorganism used. These findings have practical implications for animal feed and human nutrition, where FSBM is increasingly used as a high-quality protein source. A comparative parameter of bacterial and fungal fermentation processes is shown in Table 3.

1. Metabolites produced by micro-organisms

Microorganisms play a crucial role in fermentation processes, generating diverse metabolites that have been utilized for centuries in food preservation and have evolved into high-tech biotechnological applications [46]. Primary metabolites, including amino acids, nucleotides, vitamins, organic acids, and alcohols, are generated during the growing stage of microorganisms and are of industrial importance. Genetically modified strains, such as Corynebacterium glutamicum and Brevibacterium species, maximise the yield of these metabolites. Secondary metabolites, such as antibiotics, antitumor compounds, and immunosuppressants, are produced during the stationary phase of microbial cultures and have had a profound impact on medicine and agriculture [46]. Downstream processing and fermentation technology enable the effective extraction and purification of these metabolites, facilitating their commercial exploitation. The green advantages of microbial fermentation are being leveraged in global initiatives to achieve a circular bioeconomy.

Microorganisms play a crucial role in fermentation processes, generating diverse metabolites that have been utilized for centuries in food preservation and have evolved into high-tech biotechnological applications [46]. Primary metabolites, including amino acids, nucleotides, vitamins, organic acids, and alcohols, are generated during the growing stage of microorganisms and are of industrial importance (Figure 2). Genetically modified strains, such as Corynebacterium glutamicum and Brevibacterium species, maximise the yield of these metabolites. Secondary metabolites, such as antibiotics, antitumor compounds, and immunosuppressants, are produced during the stationary phase of microbial cultures and have had a profound impact on medicine and agriculture [46].

4.1. Primary metabolites

Primary metabolites are small, essential molecules found in all living cells that serve as intermediates or end products of metabolic processes. Primary metabolites comprise amino acids, nucleotides, vitamins, solvents, and organic acids, with applications in industry ranging from human and animal nutrition to the production of biofuels and solvents. Innovative applications include the use of monosodium glutamate and nucleotides in food processing, as well as organic acids such as ethylenediaminetetraacetic acid in pharmaceuticals and water treatment. Additionally, bio-based organic acids like succinic acid are utilised in green packaging and textile production [47]. The production of these metabolites at an industrial scale largely depends on microbial fermentation, where microorganisms are genetically and physiologically engineered to overproduce target molecules through sophisticated biotechnological measures. Technological advancements in bioreactor design and downstream processing methods have further enhanced fermentation processes, with the inclusion of systems biology tools providing a systemic view of cellular metabolism and identifying primary genetic targets for manipulation. The transition to a sustainable and circular bioeconomy has raised demand to produce metabolites using low-value, renewable feedstocks, such as lignocellulosic biomass, molasses, and waste glycerol [48]. The output of economically feasible purification processes for high-value metabolites, and the scaling-up of optimised laboratory processes to industrial scale, without decreasing their productivity.

4.1.1. Amino acids

The global amino acid market, valued at over $6 billion, is a rapidly growing sector in industrial biotechnology. Monosodium glutamate, the most widely produced amino acid, is produced through large-scale fermentation processes using high-performance bacterial strains, primarily Corynebacterium glutamicum and its subspecies. Industrial microbiology has successfully achieved inhibition by developing auxotrophic mutants and antimetabolite-resistant mutants to deregulate metabolic pathways and prevent feedback inhibition [49]. Modern strain development often combines both strategies, creating microbial producers with multiple deregulated pathways capable of hyperaccumulating desired compounds. Recombinant DNA technology has revolutionised amino acid production by enabling precise genetic modifications, such as the introduction of plasmid-borne biosynthetic operons, the overexpression of genes encoding rate-limiting enzymes, and the strategic cloning of feedback-resistant enzyme variants. Metabolic engineers have also enhanced production by introducing heterologous genes or amplifying the first committed step in amino acid biosynthesis to maximise carbon flux toward desired products [50]. Amino acid production may involve synthetic biology to design novel biosynthetic pathways, artificial intelligence (AI) to predict optimal genetic modifications, and the exploration of non-conventional hosts for specialised applications.

4.1.2. Nucleotides and nucleosides

The industrial production of nucleotides through microbial fermentation has gained significant commercial traction due to the growing demand for guanylic acid and inosinic acid, which serve as potent flavour enhancers in the food industry. Three primary biotechnological processes dominate nucleotide manufacturing: (1) the enzymatic hydrolysis of yeast RNA using fungal nucleases to yield adenosine monophosphate (AMP) and guanosine monophosphate (GMP), which are subsequently deaminated to produce inosine monophosphate (IMP); (2) the fermentative production of nucleosides (inosine and guanosine) by metabolically engineered Bacillus subtilis mutants, which are later phosphorylated to their respective 5′-nucleotides; and (3) the direct fermentation of sugars to IMP using high-performance Corynebacterium glutamicum strains genetically optimized for purine overproduction [51]. A nucleotide biosynthesis is the feedback inhibition exerted by intracellular AMP and GMP, which regulate purine metabolism through the allosteric control of key enzymes, such as phosphoribosyl pyrophosphate synthetase and amidophosphoribosyl transferase. This regulation can be studied using adenine auxotrophic mutants and exploiting antimetabolite resistance strategies, including the deletion of IMP dehydrogenase.

4.1.3. Vitamins

Microbial fermentation is a vital aspect of industrial vitamin production, with over 50 % of commercially manufactured vitamins destined for animal feed supplementation. Seven vital vitamins or vitamin-like compounds are produced through microbial processes: β-carotene (provitamin A), vitamin B12 (cobalamin), vitamin B2 (riboflavin), vitamin B13 (orotic acid), vitamin C (ascorbic acid), γ-linolenic acid (vitamin F), and ergosterol (provitamin D). Riboflavin production has transitioned from chemical synthesis to microbial fermentation, driven by economic and environmental considerations [52]. Vitamin B12 production is another microbial triumph, with Propionibacterium shermanii and Pseudomonas denitrificans strains engineered to produce approximately 100,000 times more cobalamin than their metabolic needs require. Biotin production has traditionally relied on chemical synthesis through the multi-step Reichstein process; however, this route incorporates a crucial bioconversion step employing Gluconobacter oxydans mutants optimised for high sorbitol tolerance. Genetically modified Erwinia herbicola strains incorporating Corynebacterium genes enable direct conversion of glucose to 2-keto-L-gulonic acid (2-KLG), the immediate precursor to ascorbic acid.

4.1.4. Organic acids

Microbial fermentation is the primary method for industrial-scale production of organic acids, utilising microorganisms such as fungi, yeasts, and bacteria to synthesise various acids. Citric acid is the most widely produced organic acid and is crucial in food and beverage applications due to its solubility, taste, and Generally Recognised as Safe (GRAS) status. The pharmaceutical industry allocates approximately 15 % of its production to anticoagulant formulations, effervescent tablets, and drug delivery systems. Citric acid production in Aspergillus niger occurs through glycolysis and the tricarboxylic acid cycle. Candida oleophila can generate remarkable yields in continuous fed-batch systems through nitrogen-limited cultivation with glucose excess [53]. Acetic acid production through microbial vinegar fermentation is one of the oldest biotechnological processes. The lactic acid industry has undergone a paradigm shift from chemical synthesis to microbial production of lactic acid, which acts as a precursor for biodegradable polylactic acid plastics. Metabolic engineering strategies have revolutionised and enabled the carbon flux through their pathways, including the overexpression of rate-limiting enzymes and the development of novel transport systems. The substrate hydrolysis with acid production allows acid-tolerant organisms to reduce neutralisation costs, and synthetic microbial consortia facilitate mixed acid production from complex feedstocks [53, 54].

4.1.5. Alcohols

Ethyl alcohol (ethanol) is a major metabolite produced through microbial fermentation, with global production exceeding 100 billion litres per year. This is primarily produced from sugar-based or polysaccharide-rich feedstocks using specialised yeast strains adapted to specific carbohydrate substrates. Brazil's Proálcool program is the world's most advanced bioethanol economy, producing 12.5 billion litres annually from sugarcane juice and molasses through highly optimised Saccharomyces cerevisiae fermentations, achieving ethanol concentrations of 8 – 12 %. The United States primarily uses corn starch hydrolysates as a substrate, generating over 60 billion litres annually as an oxygenate, which reduces greenhouse gas emissions by 40 – 50 % compared to fossil fuels and serves as a renewable alternative to methyl tert-butyl ether. The transition from first-generation to second-generation ethanol production has spurred intensive research into overcoming biomass recalcitrance through combined physicochemical pretreatment and enzymatic saccharification using cellulase and hemicellulase cocktails from Trichoderma reesei and Aspergillus species [55]. Glycerol (glycerin), traditionally synthesised from petrochemical propylene, has seen renewed interest in microbial production, with the osmotolerant yeast Candida glycerinogenes yielding 130 g/L at 63 % theoretical efficiency through optimised fed-batch strategies [56].

4.1.6. Miscellaneous Primary Metabolites

Microbial polysaccharides are essential biopolymers utilised in various applications, including food, pharmaceuticals, cosmetics, and industrial processes. The most commercially used microbial exopolysaccharide is xanthan gum, produced by the bacterium Xanthomonas campestris through the aerobic fermentation of glucose or sucrose. Dextran, synthesised from sucrose by Leuconostoc mesenteroides strains, serves medical purposes as a blood plasma volume expander, an iron-dextran complex for anaemia treatment, and as a chromatographic matrix for protein purification. Pullulan, a fungal exopolysaccharide, exhibits unique film-forming properties, which are utilised in edible packaging, oxygen barrier coatings, and the production of pharmaceutical capsules. Other microbial polysaccharides include scleroglucan for oil recovery, curdlan for use in Japanese foods, bacterial alginate for wound dressings, and succinoglycan for applications in concrete and ceramics [55]. Hyaluronic acid, traditionally extracted from rooster combs, is now predominantly produced through streptococcal fermentation for ophthalmic surgery and cosmetic dermal fillers. The polyhydroxyalkanoate family of biodegradable plastics offers petroleum-free alternatives for packaging, medical implants, and 3D printing filaments [56]. Table 4 summarizes some examples of primary metabolites produced by microorganisms.

4.2. Secondary metabolites

Microbial organisms produce secondary metabolites, which are natural compounds with significant impacts on human health, agriculture, and biotechnology. They include antibiotics such as penicillin and tetracyclines, medicinal compounds, toxins, biopesticides, and bioactive compounds that affect plant and animal growth [57]. These complex molecules are synthesised during the idiophase of microbial growth and are phylogenetically restricted to specific microbial taxa. The secondary metabolites fulfil six important ecological roles: facilitating sexual reproduction, altering membrane permeability, serving as chemical warfare agents, enabling mutualistic interactions, regulating intricate morphological processes, and influencing population-level behaviours. The industrial synthesis of these compounds has revolutionised medicine since the 1940s, with β-lactam antibiotics having saved an estimated 200 million lives annually. Agrochemical uses are similarly revolutionary that managing parasitic nematodes and insect pests, while gibberellic acid increases yields [58].

4.2.1. Antibiotics

Antibiotics are biologically active molecules that target fundamental microbial processes, including DNA replication, RNA synthesis, protein synthesis, cell membrane integrity, cell wall biosynthesis, electron transport, and sporulation and germination pathways. The global antibiotic market comprises approximately 160 antibiotics and their derivatives, dominated by major structural classes, including β-lactams, peptide antibiotics, macrolide polyketides, tetracyclines, aminoglycosides, ansamycins, and glycopeptides. Modern antibiotic development employs three complementary strategies: semisynthetic modification of natural scaffolds, genetic engineering of biosynthetic pathways through recombinant DNA technology, and optimisation of fermentation processes [59].

Recent antibiotics include daptomycin, a lipopeptide derived from Streptomyces roseosporus, due to its unique calcium-dependent membrane disruption mechanism, which is effective against vancomycin-resistant Entero-cocci, methicillin-resistant Staphylococcus aureus, and penicillin-resistant Streptococcus pneumoniae. The indus-trial production of these sophisticated fermentation technol-ogies and emerging strategies to address antimicrobial resistance includes genome mining of uncultured microbes and the computational design of synthetic antibiotics.

4.2.2. Antitumor agents

Since the discovery of actinomycins in 1941, microorganisms have become a significant source of potent anticancer agents. Soil-dwelling actinomycetes produce complex compounds like Mitomycin C, Bleomycin, and anthracyclines, as well as epipodophyllotoxins like Etoposide and the enediyne antibiotic Calicheamicin. Taxol, a paclitaxel stabilised by endophytic fungi, has been used in the treatment of breast, ovarian, and lung cancers [60]. This production still relies on plant cell culture due to the challenges is commercially viable fungal fermentation titres. Camptothecin targets topoisomerase I by stabilising the covalent enzyme-DNA intermediate. The limited commercialisation of plant cell culture processes highlights the technical challenges and substantial costs associated with scaling up plant-based production systems.

4.2.3. Pharmacological agents

The discovery of microbial-derived pharmacological agents through targeted enzymatic screening has revolutionised modern medicine, generating over $1 trillion in pharmaceutical revenue. The natural product drug discovery pipeline faces significant challenges during clinical development due to inadequate target engagement, pharmacokinetic profiles, metabolic instability, formulation difficulties, unexpected immunogenicity, or dose-limiting toxicity. The pharmaceutical industry's use of natural product discovery has a paradoxical effect on drug exploration funding, particularly for antibiotics and oncology agents, where microbial metabolites have histor-ically played a dominant role. Biotechnology companies have made remarkable advances by applying innovative approaches to microbial drug discovery, including genome mining of uncultured microorganisms through metagen-omics, CRISPR-Cas9 activation of silent biosynthetic gene clusters in actinomycetes and fungi, heterologous expression of giant biosynthetic gene clusters in optimised chassis organisms, and high-content screening platforms combining mass spectrometry and bioactivity profiling [61]. Modern techniques are overcoming limitations of natural product discovery, with improved culturing methods enabling the growth of previously "unculturable" micro-organisms. As the antimicrobial resistance crisis reevaluates soil actinomycetes as sources of novel antibiotics, pharma-ceutical companies are increasingly partnering with biotech firms and academic groups to access innovative microbial platforms, suggesting a renaissance in the discovery of microbial natural products. Table 5 summarizes some examples of secondary metabolites produced by micro-organisms.

1. Types of Fermentation Process

Fermentation reactions are driven by microbial processes that fall into three main categories, based on their metabolic pathways and products: lactic acid fermentation, alcoholic fermentation, and acetic acid fermentation (Fig. 3). Lactic acid fermentation, facilitated by LAB, is used to produce yogurt, cheese, sauerkraut, and kimchi. Alcoholic fermentation, driven by yeasts such as S. cerevisiae, converts sugars into carbon dioxide and ethanol, forming the basis of beer, wine, and bread making. Acetic acid fermentation, carried out by Acetobacter species, oxidizes ethanol to acetic acid, which is essential in vinegar production and condiments [61].

Further, there are two methods of fermentation processes: solid-state fermentation (SSF) and submerged fermentation (SmF). SSF is mainly used in enzyme production and the production of fermented foods such as cheese, tofu, etc, while SmF is more prevalent in industrial antibiotic and organic acid production due to its better process control [61]. Technologies like mixed-culture fermentation improve the production of complex products, and anaerobic digestion by methanogenic archaea converts organic waste into biogas to fulfill renewable energy requirements. Advanced genetic engineering devices, like CRISPR-Cas9 and synthetic biology, streamline the microbial engineering process with optimised metabolic routes [62]. Recombinant microbes are utilised in precision fermentation for the production of animal-free proteins and therapeutics, ensuring sustainability in biotechnology.

Fermentation reactions are driven by microbial processes that fall into three main categories, based on their metabolic pathways and products: lactic acid fermentation, alcoholic fermentation, and acetic acid fermentation (Figure 3). Lactic acid fermentation, facilitated by LAB, is used to produce yogurt, cheese, sauerkraut, and kimchi. Alcoholic fermentation, driven by yeasts such as Saccharomyces cerevisiae, converts sugars into carbon dioxide and ethanol, forming the basis of beer, wine, and bread making. Acetic acid fermentation, carried out by Acetobacter species, oxidizes ethanol to acetic acid, which is essential in vinegar production and condiments [61].

5.1. Lactic acid fermentation

Lactic Acid (LA) is the main metabolic byproduct that LAB generates. LA finds numerous uses in food, cosmetics, textiles, and pharmaceutical industries [62]. LA can be used to produce packaging materials, fibres, and foams by transforming into polylactic acid (PLA), which is a green, biodegradable, and biocompatible polymer. In addition, LA is applied in the food and beverage industry to manufacture soft drinks, sweets, milk products, and bakery products, as it has an acidulant characteristic. In the cosmetic industry, LA is utilised as a moisturiser because it can retain water, as a skin lightener, and as a rejuvenator, as it can prevent tyrosinase formation [63]. The medical sector also employs LA because it is utilised in the production of topical creams, lotions, surgical dressings, and prostheses. Lactic acid fermentation improves protein solubility as well as the availability of certain micronutrients and limiting amino acids. This process significantly reduces the levels of tannins (50 %), phytates, and oligosaccharides (90 %). Raw materials containing starch undergo LA fermentation to form the amino acid lysine, vitamins such as vitamin B and K, folate, and micronutrients in the fermented products. Agricultural substrates can be utilised for LA production. Substrates mainly consist of carbon and nitrogen compounds. A lignocellulosic agricultural residue has three main polymers: cellulose, hemicellulose, and lignin. Pretreatments are a crucial factor in the conversion of biomass into LA [64].

Alkali pretreatments enhance the enzymatic digestibility of the fibres. However, lignocellulosic materials are mostly pretreated with acids. The primary issue with acid pretreatment is the production of small amounts of cell growth inhibitors, which can further deactivate enzymes and affect the efficiency of LA bacteria during fermentation. LA is commonly made through biotechnological methods involving LA fermentation and biochemical alterations [65]. The production yield of LA depends on the pH (3.5 – 9.6), temperature (5 – 45 °C), and LAB strain producers, and the presence of nutrients (such as amino acids, peptides, nucleotides, and vitamins). Various strains from the genera Leuconostoc, Lactococcus, Lactobacillus, Pediococcus, Enterococcus, Streptococcus, Vagococcus, Aerococcus, Carnobacterium, Tetragenococcus, Oenococcus, and Weissella have been identified as LA producers. These industrial food fermentations primarily utilise Lactobacillus, Lactococcus, Leuconostoc, and Pediococcus as starter cultures

5.2. Alcoholic Fermentation

Alcoholic fermentation (AF) is a process used for thousands of years to produce wine. At the industrial level, microbial fermentation is used to convert cellulose and hemicellulose into fermentable sugars, which are then used to produce ethanol [61]. During AF, yeast generally ferments raw materials in the presence of oxygen, but it can also perform fermentation in the absence of oxygen. When oxygen is lacking, the fermentation process occurs in the cytoplasm of the yeast. Under anaerobic conditions, Pyruvic acid (C₃H₃O₃) is first converted into acetaldehyde, an intermediate molecule that releases carbon dioxide before being transformed into ethanol. During AF, NAD+ is reduced to NADH, which facilitates an electron exchange necessary for ATP production. Thiamine is an essential vitamin for the metabolism of yeasts, and its deficiency can even lead to yeast death [66]. Additionally, thiamine acts as a cofactor for various enzymes involved in the production of wine-related flavour compounds. It also plays a role in yeast survival via thiamine-dependent stress protection functions. Nitrogen is another essential factor for the growth and metabolism of yeast. A deficiency of nitrogen is considered a general reason for sluggish or stuck fermentation. Nitrogen is supplied externally by ammonium salts to prevent fermentative problems [67]. AF includes the fermentation of wine, beer, and cider. Non-Saccharomyces yeasts were considered contaminants in wine and beer production. Procedures such as pasteurisation, sulfite addition, and equipment cleaning were used to eliminate these microbes and their role in the spontaneous fermentation of wine, thereby enhancing the final product's sensory quality.

5.3. Acetic acid Fermentation

Acetic acid bacteria (AAB) are aerobic, Gram-negative bacteria that possess a strong ability to oxidize ethanol and synthesize acetic acid, in addition to their resistance to acid. They are generally utilized in industrial vinegar fermentation to produce vinegar, fruit vinegar, gluconic acid products, and to facilitate the formation of biofuel cells. AAB has played a notable role in the manufacturing of fermented foods and beverages, including lambic beer, kombucha, vinegar, and kefir [61]. AAB is also called ‘oxidative bacteria’ that oxidize carbohydrates, ethanol, and sugar alcohols into various products like aldehydes, ketones, and organic acids. Fruits and vegetables rich in nutritional components, such as amino acids, organic acids, phenols, vitamins, and minerals, are primarily used to produce fermented vinegar [68]. These components can aid in digestion, facilitate fatigue recovery, and support diabetes management, while also possessing anti-obesity and anticancer properties. Acetic acid is primarily derived from natural gas or mineral oil and serves as a crucial industrial feedstock. In the industrial sector, acetic acid is generally utilised in the production of vinegar. It is also used to form vinyl acetate, which is used in vinyl plastics, adhesives, textile finishes, and latex paints, an industry that is expanding rapidly due to increased demand for synthetic fibres. At the industrial level, acid stress arises from low biomass and a low production rate, which has been proven to be a significant constraint in the process [69].

1. Methods of fermentation

Fermentation technology has evolved from traditional practices to current technologies, utilizing microbial physiology in various industries. SSF utilizes moist solid substrates to produce concentrated enzymes and flavors [70, 71]. In contrast, SmF employs liquid media in aerated bioreactors for the large-scale production of pharma-ceuticals, such as penicillin and streptomycin. Surface fermentation techniques have been enhanced in bed reactors to increase oxygenation and productivity. Batch fermen-tation remains essential for traditional foods such as yoghurt and wine, as well as for contemporary biopharmaceuticals that require strict aseptic conditions [70]. Continuous fermentation systems support cultures in exponential growth by continuously feeding nutrients and harvesting product, allowing for high yields in the production of bioethanol and single-cell protein. Recent innovations have provided immobilised cell fermentation, enhanced stability, and recycling in high-fructose corn syrup processing and wastewater treatment. Extremophile fermentation methods employ thermophiles or acidophiles under extreme conditions for niche applications such as biomining [71].

6.1. Submerged Fermentation

Submerged fermentation (SmF) is a method in which microorganisms grow in an aqueous nutrient broth with a high free water content, transforming microbial cultivation from an art to a science. It is typically carried out in stirred-tank or airlift bioreactors, offering control over key parameters such as dissolved oxygen, pH, temperature, and nutrient concentration, thereby enabling optimal conditions for microbial growth. The transition from traditional surface culture methods to submerged systems represented a significant leap in productivity, enabling Aspergillus niger to produce citric acid levels ranging from 5 g/L to over 200 g/L through more efficient oxygen transfer and metabolic control in deep-tank fermenters. Current SmF applications span various industries, including pharmaceuticals, food technology, and biofuel production, for their industrial enzymes [72]. High-density culture methods and sophist-icated fed-batch operations have achieved record product titres, with industrial enzymes reaching 50 g/L and monoclonal antibodies at 510 g/L. Advances in bioreactor design, including disposable single-use systems and wave-mixed bags, have enhanced sterility and flexibility for vaccine manufacturing with mammalian cell cultures [73]. SmF is irreplaceable due to its scalability and reproducibility, presently supporting the commercial manufacture of more than 60 % of biopharmaceuticals and 80 % of industrial enzymes.

6.2. Solid State Fermentation

Solid-state fermentation (SSF) is a method that combines traditional food production with biotechnological processes. It involves cultivating microorganisms on humid solid substrates without the use of free-flowing water, thereby simulating their natural environments while conserving energy and maximising benefits [70, 71, 74]. This ancient method, long used for Asian food fermentations such as tempeh and koji, has been revitalised as an advanced biomanufacturing platform by contemporary process engineering and the improvement of microbial strains. Modern SSF applications span various industries, including food, enzyme production, and biocontrol. Recent innovations have overcome conventional SSF limitations by utilising automated tray bioreactors, rotating drum systems for heat removal, and packed-bed reactors with real-time CO₂ monitoring, thereby enhancing the process [75]. Despite scale-up and process control issues, SSF's low water demands and capacity to use 80 % lower-cost substrates make it an essential technology for sustainable production [76, 77]. The technology's scalability is demonstrated by its industrial applications, including the production of fungal enzymes, organic acids, and bioactive compounds, which feature reduced purification complexity [78]. SSF faces challenges, including heat accumulation in dense substrates and non-uniform microbial growth resulting from substrate heterogeneity. A comparative analysis of SSF and SmF is given in Table 6.

1. Probiotic bacteria in fermentation technology

Probiotics are beneficial microorganisms that can modulate gut microbiota, enhance intestinal barrier function, and stimulate immune responses. They have shown clinical efficacy in managing gastrointestinal disorders, metabolic conditions, and immune-mediated diseases [91].  Probiotics are being used in non-dairy carriers, such as fruit juices, cereal-based substrates, chocolate, and freeze-dried pharmaceutical formulations [91]. The physiological robustness of probiotic strains is crucial, as it requires acid tolerance, bile resistance, and the ability to adhere to mucosa. They exert beneficial effects through mechanisms such as competitive exclusion of pathogens, production of antimicrobial compounds, modulation of intestinal epithelial tight junctions, and immunomodulation.

The LAB are the backbone of probiotic formulations due to their GRAS status and multifunctional metabolism. Modern probiotic development employs advanced screening techniques, omics analyses, and stabilisation technologies. The concept of "next-generation probiotics" encompasses engineered strains with targeted function-alities and consortium formulations that combine traditional probiotics with prebiotic fibres [92]. The probiotics lie in precision formulations tailored to individual microbiomes, engineered live biotherapeutics for non-gut indications, and integration with faecal microbiota transplantation protocols.

Probiotics are beneficial microorganisms that can modulate gut microbiota, enhance intestinal barrier function, and stimulate immune responses (Figure 4). They have shown clinical efficacy in managing gastrointestinal disorders, metabolic conditions, and immune-mediated diseases [91]. Probiotics are being used in non-dairy carriers, such as fruit juices, cereal-based substrates, chocolate, and freeze-dried pharmaceutical formulations [91]. The physiological robustness of probiotic strains is crucial, as it requires acid tolerance, bile resistance, and the ability to adhere to mucosa. They exert beneficial effects through mechanisms such as competitive exclusion of pathogens, production of antimicrobial compounds, modulation of intestinal epithelial tight junctions, and immunomodulation. The LAB are the backbone of probiotic formulations due to their GRAS status and multifunctional metabolism. Modern probiotic development employs advanced screening techniques, omics analyses, and stabilisation technologies. The concept of "next-generation probiotics" encompasses engineered strains with targeted functionalities and consortium formulations that combine traditional probiotics with prebiotic fibres [92].

1. Microbial fermentation of food products

Microbial fermentation is a biotechnological process that uses bacteria, yeasts, and moulds to transform raw agricultural materials into nutritious, palatable, and shelf-stable foods. It is used in cereal, legume, vegetable, meat, fish, and dairy fermentations, sourdough bread production, and the conversion of sugars to ethanol and CO₂. Filamentous fungi undergo proteolytic and amylolytic transformations in traditional Asian staples, including soy sauce, miso, and tempeh. Modern applications combine traditional knowledge with precision fermentation, using defined starter cultures to standardise artisanal products [93]. Emerging technologies use omics tools and synthetic biology to control pathogens and ensure process consistency. The global fermented food market, valued at $700 billion, spans dairy (40 %), alcoholic beverages (30 %), and plant-based products (20 %). Innovations include probiotic-enriched non-dairy alternatives, low-alcohol wines, and mycoprotein-based meat analogues. Challenges persist in scaling artisanal processes, such as maintaining microbial viability in low-moisture fermented meats and controlling phage infections. These also include CRISPR-engineered starters for reducing allergens, substrate fermentations, and personalised fermented foods tailored to their microbiomes [94]. Microbial fermentation plays a transformative role in converting raw vegetables and soybeans into nutrient-rich fermented products. This process enhances bioavailability, reduces antinutritional factors, and introduces beneficial bioactive compounds (Figure 5). Traditional practices like kimchi and tempeh production exemplify how microbial activity improves sensory and functional properties, while modern applications leverage precision starter cultures for consistency and scalability

8.1. Fermented vegetable products

Microbes play a central role in vegetable fermentation, connecting traditional food preservation methods to novel biotechnological developments. Spontaneous vegetable fermentations with Leuconostoc, Lactobacillus, and Pediococcus species have resulted in their relevant products, such as sauerkraut, Korean kimchi, and fermented olives [94]. Major innovations utilise this microbial richness to convert vegetable matrices into functional foods, including sauerkraut-like beetroot kvass, fermented using Lactocaseibacillus casei A4, and soybeans fermented with Bacillus subtilis. Primary methods such as high-pressure processing and SSF of okara enhance protein content and decrease oligosaccharides. The microbial vegetable fermentation utilises precision ecology strategies, including AI-based predictive models and sophisticated bioreactor designs, with real-time metabolomics capabilities [95].

Pharmaceutical uses are unfolding, e.g., oral vaccines developed in fermented lettuce that produce cholera toxin B sub-units, and the vegetable waste upcycling circular bioeconomy through Aspergillus oryzae fermentation for glucoamylases production in starch processing. This shift from traditional craft to industrialised biotechnology illustrates that microbiological innovation persists in elevating simple vegetables into potent tools for nutritional security, sustainable agriculture, and preventive medicine [96].

8.2. Fermented cereal products

Cereals are a multifaceted substrate for microbial fermentation, providing a rich matrix of carbohydrates, proteins, and bioactive compounds that serve as both microbial growth media and prebiotic materials. Nevertheless, their nutritional composition must be strategically fortified through fermentation to compensate for internal constraints, such as the low bioavailability of proteins and antinutrients. Conventional cereal-based fermentations worldwide demonstrate the ecological microbial capacity to enhance free amino acids, reduce phytate levels, and produce bioactive peptides. Oat is a viable cereal matrix because it contains a high proportion of β-glucans, which promote Bifidobacterium activity and constitute protective colloidal systems. Maize fermentation presents special challenges due to its high starch content and poor protein quality [97].  Common fermented cereal-based foods include bread, dosa, idli, ingera, etc [98].

Sourdough is a type of bread that uses the fermentation of wheat flour by naturally occurring yeast and lactobacilli to raise the dough. Sourdough bread, at its most basic, is made from three ingredients: flour, water, and salt. Sourdough bread is often considered healthier than regular bread due to its fermentation process, which can improve nutrient absorption and potentially benefit gut health [99]. While both types of bread can be part of a healthy diet, sourdough's unique characteristics offer some advantages.

Engineered consortia of lactobacilli can be used to convert nixtamalized maize into nutritionally fortified products by enhancing free folate levels and minimising aflatoxin contamination. Rice fermentation undergoes a metabolic renaissance during germination, yielding a nutrient-rich substrate that supports exceptional probiotic growth. Next-generation fermentation technologies are surpassing cereal limitations, including extrusion pretreatment, pulsed electric field treatment, and microencapsulation strategies [100]. The emerging field of cereal-based probiotic foods involves precision-fermented designer synbiotics, such as CRISPR-engineered Lp. plantarum strains that produce cereal phytases, and AI-based bioreactor systems for maximising fermentation conditions in real-time.

8.3. Fermented legume products

In traditional fermented food processing, legumes such as soybeans, lentils, chickpeas, and mung beans are subjected to fermentation by autochthonous microorganisms. The fermentation process involves the breakdown of complex macromolecules like proteins and carbohydrates into simpler, more bioavailable forms. Microorganisms such as Bacillus species, LAB, and yeast play key roles in these transformations, producing enzymes that degrade anti-nutritional factors and enhance nutrient bioavailability [101]. The resulting fermented products range from Asian soy-based condiments like nato, miso, and tempeh [102] to African condiments that include dawadawa and ugba [103].

8.4. Fermented meat products

Meat serves as a protective substrate for probiotic bacteria, providing an ideal environment for LAB to thrive. Modern probiotic meat fermentations use facultative heterofermentative LAB strains that convert carbohydrates into lactic acid and switch to heterofermentative pathways when sugars are limited [104]. Advanced strain selection focuses on meat-adapted probiotics with bile salt hydrolase activity and acid tolerance. Lactobacillus amylovorus and Lactobacillus gallinarum are effective in fermented meats due to their proteolytic activity and resistance to meat-derived stressors. Process optimisation involves controlled fermentation at 20 - 26 °C with 85 – 95 % relative humidity for 48 - 72 h, followed by drying at 12 – 15 °C. Technological challenges include preventing probiotic inhibition by traditional curing agents, optimising aw reduction, and managing lipid oxidation [105]. These also include CRISPR-engineered Latilactobacillus sakei strains, AI-optimised fermentation protocols, and hybrid products combining plant proteins with probiotic-fermented meat for sustainable functional foods.

8.5 Fermented fish products

Fermented fish products are a vital part of traditional diets in many coastal and Southeast Asian cultures, where they serve as a source of protein, probiotics, and unique flavors. These products are typically produced through spontaneous fermentation, relying on indigenous microbes, such as LAB and halophilic Tetragenococcus species, to break down proteins and lipids, thereby enhancing digestibility and shelf life. Examples include fish sauces (e.g., Thai nam pla), pastes (e.g., Korean jeotgal), and dried fermented fish (e.g., African lanhouin), each with distinct regional variations in preparation and microbial consortia [106]. The fermentation process involves salting, aging, and enzymatic hydrolysis, which reduce the pH and inhibit pathogenic bacteria while generating bioactive peptides, free amino acids, and umami compounds, such as glutamate. LAB strains such as Lactobacillus plantarum and Pediococcus pentosaceus dominate these fermentations, contributing to preservation and health benefits, including antioxidant and antihypertensive properties [106]. However, improper fermentation can lead to the accumulation of biogenic amines (e.g., histamine), necessitating the use of strict hygiene and starter cultures for safety [107].

8.5 Fermented dairy products

Fermented dairy products have been consumed for millennia, initially as a method of food preservation before their health benefits became widely recognized [2]. These products are created by fermenting milk or other raw ingredients with the help of microorganisms, predominantly LAB, which contribute to the fermentation process. LABs are responsible for the development of bioactive compounds that offer a range of health advantages, such as immunomodulatory, antimicrobial, and antioxidant effects. Popular fermented dairy products, including yogurt, kefir, and cheese, are consumed globally and contribute to improved gastrointestinal health, enhanced digestion, and potentially lower risks of conditions like osteoporosis, diabetes, and inflammatory bowel diseases [108]. Additionally, fermented dairy products support the gut microbiome, which has a significant influence on overall health, making them a valuable component of a balanced diet [109].

Modern advancements focus on optimizing fermentation conditions (e.g., temperature, salt concentration) and introducing defined starter cultures to standardize quality and accelerate production. Emerging techniques, such as bacteriophage therapy to control spoilage bacteria and omics tools to profile microbial dynamics, are further refining these traditional processes [110]. Despite their cultural significance, fermented fish products face challenges in global acceptance due to strong odors and sensory preferences. Innovations like deodorization techniques and hybrid products (e.g., fish-vegetable blends) aim to broaden appeal while retaining nutritional benefits. Research continues to explore their potential as functional foods, particularly for gut health and mineral bioavailability [111].

The role of microorganisms such as Leuconostoc, Lactobacillus, and Pediococcus in vegetable fermentations like sauerkraut and kimchi highlights the significance of lactic acid bacteria (LAB)-driven acidification in spontaneous fermentation processes (Table 7). Similarly, Bifidobacterium, Lactobacillus, and Aspergillus oryzae are crucial in the solid-state fermentation of cereals and soy products, resulting in traditional foods like tempeh and miso. Controlled fermentation of meat products utilizing species like Lactobacillus amylovorus enhances preservation and probiotic content. In fermented fish products, microbial action by Lactobacillus plantarum and Tetragenococcus is key in flavor development and preservation strategies (Table 7).

1. Genetic recombination in microbial fermentation

Genetic recombination has revolutionised microbial fermentation, allowing targeted genetic modifications that optimise fermentation efficiency, expand substrate utilisation, and unlock new bioactive compound synthesis. This approach bridges the ancient fermentation practices with their industrial applications, enabling the engineering of strains with enhanced metabolic capabilities, improved yield, and novel functionalities. In industrial applications, recombination has limitations like those of random mutagenesis; however, it enables the strategic stacking of beneficial traits [112]. For example, it facilitates the amplification of Biosynthetic Gene Clusters (BGCs) in Streptomyces spp. for antibiotic overproduction, the insertion of heterologous pathways into Saccharomyces cerevisiae for enhancing bioethanol production, and in Lactobacillus spp. for increasing lactic acid yield. Synthetic biology tools, such as CRISPR interference (CRISPRi), multiplex automated genome engineering, and recombinase-assisted genome rewriting, enable precise pathway optimisation. The engineering of Escherichia coli strains exemplifies this to enhance shikimate pathway activity to produce aromatic compounds, and the modification of Aspergillus oryzae for increased protease secretion in soy fermentation. Recombination also facilitates the activation of silent gene clusters, uncovering cryptic metabolites with pharmaceutical potential, such as the discovery of novel polyketides in Penicillium through promoter swapping or the heterologous expression of fungal BGCs in S. cerevisiae for scalable drug precursor synthesis [113]. In food fermentation, genetically stabilised Lactobacillus hybrids exhibit robust acid tolerance and phage resistance. At the same time, recombinant Acetobacter strains with enhanced cellulose synthase activity revolutionise kombucha production for the synthesis of biodegradable materials. The environmental and economic benefits of recombinant fermentation with engineered Clostridium spp. Converting lignocellulosic waste into butanol at 90 % theoretical yield and Yarrowia lipolytica recombinants producing omega-3 fatty acids from agro-industrial byproducts [114].

1. Novel genetic technologies for microbial fermentation

The evolution of microbial fermentation has been significantly transformed by the advent of novel genetic technologies, including precision genome editing, synthetic biology, and computational bioengineering. These technologies have enabled unprecedented control over microbial metabolism, unlocking new possibilities in industrial biotechnology, food production, and pharmaceutical development [115]. CRISPR-Cas9 systems have emerged as the cornerstone of modern genetic manipulation in fermentation science, allowing for targeted gene knockouts, precise promoter engineering, multiplexed genome editing, base editors, and prime editors. Synthetic biology platforms, such as Gibson Assembly and Golden Gate cloning, facilitate the construction of entire biosynthetic pathways. Meanwhile, cell-free transcription-translation systems accelerate the prototyping of metabolic circuits before their integration into the chromosomal genome. Machine learning (ML) algorithms predict the optimal genetic modifications by analysing multi-omics datasets, as demonstrated by AI-designed lactobacilli strains with 300 % increased lactic acid productivity through optimised redox balancing. Advanced Genome-scale models (GEMs), such as iJO1366 for Escherichia coli and iMM904 for Saccharomyces cerevisiae, simulate metabolic fluxes to identify knockout targets [116]. Novel DNA synthesis technologies enable the construction of entire synthetic chromosomes, while in vivo genome rewriting systems allow megabase-scale deletions in Streptomyces spp. to eliminate competitive routes. Phage-assisted continuous evolution enables the generation of hyperactive enzymes in real-time, while directed evolution platforms produce stable microbial mutants. Epigenetic engineering tools are employed to silence unwanted genes without introducing permanent mutations, while RNA interference dynamically regulates metabolic fluxes. Microbial consortia engineering employs quorum-sensing circuits for population management, allowing division-of-labour fermentations [117]. New technologies, such as DNA data storage within microbial genomes and biological cryptography, are pushing the frontiers of fermentation beyond its conventional uses. A quantum biology understanding of electron transport chain manipulation yields Rhodobacter strains that produce 200 % more hydrogen. Connecting these technologies with traditional fermentation techniques ensures the responsible application, most notably in food-grade microbes [118]. These encompass neuromorphic biocomputing within fermenters, completely autonomous AI-based strain evolution, and nanotechnology-supportive microbial hybrids [119, 120].

The integration of advanced genetic technologies has revolutionized microbial fermentation, enabling precise control over metabolic pathways and unlocking novel applications in biotechnology. Techniques such as CRISPR-Cas9, synthetic biology platforms, and machine learning algorithms have been instrumental in optimizing microbial strains for enhanced productivity and functionality (Figure 6). These innovations facilitate targeted gene editing, pathway engineering, and real-time metabolic flux analysis, bridging the gap between traditional fermentation practices and modern industrial demands. For instance, AI-designed microbial strains have demonstrated significant improvements in yield, while synthetic consortia engineering allows for division-of-labour fermentations, further expanding the scope of microbial applications in sustainable biomanufacturing.

1. Challenges and Future Directions

Despite the significant advancements in microbial fermentation, several challenges remain that hinder its full potential. Major challenges in the scalability of fermentation processes, particularly in SSF, are issues like mass transfer, heat accumulation, and heterogeneity of microbial growth [74, 75, 80]. In SmF, the high costs associated with downstream processing limit the economic feasibility of large-scale production [121]. Another critical issue is the regulatory and consumer acceptance of genetically modified microorganisms, which, despite their potential to enhance yield and efficiency, often face scepticism due to safety and ethical concerns [122]. Furthermore, contamination control and maintaining consistent product quality across batches remain persistent hurdles in industrial fermentation [121].

           Looking ahead, the integration of emerging technologies such as AI and ML offers promising solutions to optimize fermentation processes. These tools can predict optimal conditions, monitor real-time fermentation dynamics, and identify genetic modifications to enhance microbial performance [123]. Another future direction is the development of more robust and versatile microbial strains through advanced genetic engineering techniques like CRISPR-Cas9 and synthetic biology. These innovations could enable microbes to utilize a broader range of substrates, including agricultural and industrial waste, thereby promoting sustainability and reducing production costs [124].

Sustainability will also play a pivotal role in the future of fermentation. The shift toward circular bioeconomy models emphasizes the need to valorize waste streams and reduce the environmental footprint of fermentation processes. For instance, leveraging lignocellulosic biomass and other low-cost substrates can make fermentation more eco-friendly and economically viable. Additionally, the exploration of extremophiles - microorganisms thriving in extreme conditions could unlock novel applications in harsh industrial environments, further expanding the scope of fermentation technology [125]. Finally, interdisciplinary collaboration will be essential to address these challenges and drive innovation. Partnerships between academia, industry, and regulatory bodies can accelerate the development of safe, efficient, and scalable fermentation processes. By combining traditional knowledge with cutting-edge science, the field can overcome current limitations and unlock new opportunities in food security, healthcare, and environmental sustainability. The future of microbial fermentation lies in its ability to adapt, innovate, and integrate diverse technologies to meet global demands.  

1. Conclusion

Microbial fermentation is an intricate process that combines human ingenuity and microbial metabolism, blending ancient techniques with cutting-edge biotechnology. It has progressed from being an empirical process to a sophisticated science, stimulated by discoveries in microbiology, molecular biology, and genetic engineering. The discovery and description of numerous microbial species have broadened the platform of fermentation, enabling the production of a vast array of metabolites, including primary compounds such as amino acids, organic acids, vitamins, and alcohols, as well as secondary metabolites like antibiotics and pharmacologically active metabolites. These transformation processes have revolutionised various industries, including food, beverage, pharmaceutical, biofuel, and biodegradable. The application of new genetic technologies, including CRISPR-Cas9, synthetic biology, and computational bioengineering, has improved the efficiency and precision of microbial fermentation. Techniques like SSF and SmF have been optimised to suit sustainable waste valorisation, enzyme production, and the synthesis of high-value compounds. Probiotic technology synergises fermentation and human well-being, providing value-specific benefits in gut microbiota modulation, immune system enhancement, and disease prevention. Conventional food product fermentation has been optimised with the application of precision starter cultures, omics technologies, and AI-based process optimisation. The future of microbial fermentation is bright, with transdisciplinary innovation integrating synthetic biology, nanotechnology, and AI, presenting multiple opportunities in cellular agriculture, personalised nutrition, and a circular bioeconomy.

1. Declaration of competing interest

The authors report no conflict of interest.

1. Authors’ Contributions
2. Saranraj conceptualized the manuscript, supervised the research, and contributed to the writing and editing of the original draft. Ramesh C. Ray provided critical insights into the review's structure and content, focusing on microbial diversity and fermentation processes. Ashish Kumar Nayak contributed to the sections on genetic engineering and modern biotechnological applications. B. Lokeshwari and K. Gayathri assisted in compiling data on microbial species and metabolites, as well as reviewing the literature. P. Sivasakthivelan contributed to the sections on fermentation methods and comparative analyses. Kianoush Khosravi-Darani provided expertise on industrial applications and the health benefits of fermented products. All authors reviewed and approved the final manuscript.
3. Using Artificial Intelligent Chatbots

No artificial intelligence chatbots have been used in any section of work.

Background and Objective: Selenium-enriched microalgae represent a promising functional food and nutraceutical resource, offering a symbiotic blend of bioactive molecules and essential micronutrients. Selenium, a trace element, is vital for immune system control, antioxidant defense systems, and cell homeostasis. Microalgae possess high nutritional value and rapid growth capabilities, allowing them to bioaccumulate selenium in the form of organic compounds like selenomethionine and selenocysteine. These organic forms have increased bioavailability and reduced toxicity compared to inorganic selenium supplements. This review examines the latest advancements in the cultivation, selenium-enrichment strategies, and biological effects of selenium enriched microalgae, with a particular focus on their immunomodulatory properties.

Results and Conclusion: The review highlights the potential applications of selenium enriched microalgae in disease prevention, immunotherapy, and functional food development. Also, the immunomodulatory effects of selenium enriched microalgae have been illustrated. Selenium and Se-enriched microalgae, due to their effective roles in enhancing immune function, reducing inflammation, and providing highly bioavailable forms of Se, hold strong potential for applications in food biotechnology and nutritional supplements. Despite existing challenges in optimizing production and clarifying mechanisms of action, the future outlook is highly promising given the growing demand for functional foods and natural health-promoting solutions.

Keywords: Antioxidant, Bioavailability, Chlorella vulgaris, functional foods, Immunomodulation, Selenium-enriched microalgae, Selenoproteins, Spirulina, Sustainable biotechnology.

1. Introduction

Selenium (Se) is an essential element for the proper functioning of immune system cells, including macro-phages, natural killer (NK) cells, neutrophils, and T lymphocytes. This element plays an essential role in reducing oxidative stress, inflammation, and preventing the spread of infectious diseases, especially when its serum concentration is adequately increased through the diet [1, 2]. It is involved in modulating immune and reproductive function. This micronutrient induces the production of selenoproteins, helping to protect cells against reactive oxygen species (ROS) [3]. Se primarily functions in the body as selenoproteins, which enhance the regulation of the human immune system in various ways. Immunity is one aspect of human health is influenced by Se levels and the expression of selenoproteins in the body [4].

Although the exact physiological role of Se is unknown, it has been determined that it exhibits most of its effects by being incorporated into selenoproteins. One such class of proteins is iodothyronine deiodinases, which are essential in the maturation of thyroid hormones T3 and T4. These hormones control body weight, development, and metabolism. A deficiency in Se may therefore disrupt the process of maturation of T3 and T4, leading to stunted growth. [5]. Dietary supplements containing l-selenometh-ionine are a source of Se for nutritional purposes that is easily absorbed and utilized by the body, approved for adult use at a dosage level of less than 250 mcg per day [6,7]. Seafood and meat are regarded as the primary sources of Se for humans, providing more than 70% of daily Se intake. In contrast, fruits and vegetables contribute only a small fraction to this consumption. Se is crucial because of its antioxidant and chemoprotective roles at low levels, offering protection against various cancers, heart diseases, and type 2 diabetes. Therefore, food systems must produce sufficient amounts of this essential trace element to ensure a daily intake of at least 40 micrograms, which supports the optimal expression of Se enzymes, and potentially up to 300 micrograms per day to lower cancer risk [8].

The availability of Se varies significantly depending on the geographical region. In some places, soil Se concen-trations are limited or reduced [9]. It is reported that approximately 1 billion people worldwide are affected by Se deficiency, meaning that diets are limited to products harvested from these regions or are nutritionally unbalanced and may result in Se deficiency [10]. The global population is rapidly increasing, leading to the emergence of new diseases and placing a significant strain on healthcare systems. Therefore, it is more important than ever to research immune modulators, including Se, to tune the immune system to help combat new pathogens in a changing world [11, 12].

Essential minerals for optimal health are provided by edible plants, which are collected from the environment, namely, soils and aquatic sediments. The consumption of Se-rich plants makes this element bioavailable to humans [9]. Soils used in agriculture can be challenging to meet daily Se requirements through diet if they are low in Se. Se mineral salts are mainly used in food supplements and animal feed, and this supplementation method with sodium selenite or selenate also has disadvantages [13-15]. Today, organic Se-enriched foods, mainly plants, animals, and microorganisms enriched with Se, have been widely developed to address the problem [13]. In recent years, the production of Se-enriched microalgae has attracted much interest as an efficient and accessible method for producing organic Se [16, 17].

Microscopic algae, or microalgae, grow in freshwater and marine environments and have the capability to convert sunlight energy into chemical form. The two most notable ones are Arthrospira platensis (Spirulina) and Chlorella vulgaris. Spirulina is blue-green algae and a nutritional powerhouse containing a plethora of vitamins and poly-unsaturated fatty acids and plays critical roles in nearly every activity of human life [18, 19]. Microalgae represent promising sources of bioactive compounds suitable for pharmaceutical and food uses [20]. Humans have been eating algae as a food crop and dietary supplement for thousands of years. Algae also serve as a natural carbon sequester, counteracting global warming and reducing the pressure on arable land and freshwater resources for conventional food crops. For the best nutritional com-position of algae, there is a need to stress their cultivation keeping in view many environmental parameters such as pH, intensity of light, availability of nutrients, availability of CO₂, temperature, and mixing conditions [21]. Micro-algae, especially Chlorella vulgaris, are well-known as excellent sources of protein, balanced amino acids, and essential vitamins and micronutrients. Due to their high nutritional content, these microalgae are commonly con-sumed by humans. Chlorella vulgaris can absorb inorganic Se salts and convert them into protein-based compounds such as selenomethionine, selenocysteine, and methyl selenocysteine [16].

Thus, the aim of this review is to draw on scientific studies used to explore how Se-enriched microalgae modulate the immune system, providing updates on the recent advancements in this research area. This review is distinct from others due to its up-to-date and broad inclusion of all research drawn, thus giving the reader a com-prehensive overview of Se-enriched microalgae immune-modulatory effects.

1. Types of Se-enriched microalgae

Se-enriched biomass (Se-Chlorella) can serve as both a dietary supplement and an antioxidant [16]. Food products derived from Chlorella include green tea powder, soups, noodles, bread, biscuits, ice cream, and soy sauce. Raw Chlorella is commonly offered in tablets, capsules, powders, granules, and drinks. Noodles containing 1.5% Chlorella extract are exceptionally nutritious. Adding Chlorella powder to barley bread not only enhances its nutritional content but also improves its appearance and flavor. Additionally, Chlorella is used as a food additive to enhance the taste and quality of pasta, wine, and fermented foods. With vitamins C, K, A, and E, Chlorella is valuable for pharmaceuticals, animal feed, food additives, aquaculture, and cosmetics [22].

Chlorella can develop under conditions of light, carbon dioxide, water, and a minimum amount of nutrients, thus its culture is easy. The life cycle of the microalga is simple while its metabolic processes are as complicated as those of superior plants, which makes it capable of synthesizing high amounts of proteins, carotenoids, vitamins, and minerals. Being so, it is a popular source of nutritious food. Among numerous species of Chlorella, Chlorella vulgaris, Chlorella sp., and Chlorella pyrenoidosa are the most utilized in industrial production and scientific research [23].

Se exerts its biological effects via selenoproteins. Various microalgae, such as Chlamydomonas, Volvox, Ostreococcus, Micromonas, and Emiliania, have been identified as possessing selenoproteins [24].

Spirulina, a type of microalgae, possesses antioxidant properties and can be fortified with Se during its growth process. Research revealed a hierarchy in the distribution of Se and the expression of selenoproteins based on the form of supplementation. Supplementing with sodium selenite enhanced glutathione peroxidases (GPx) activities and selenoprotein expression, whereas Se-enriched spirulina was more effective in restoring Se levels [5]. In 1992, the World Health Organization listed it as a future food. Spirulina is used as a dietary supplement due to its high protein value, vitamins, β-carotene, and phycocyanin-like pigments. It contains anti-inflammatory, anti-cancer, and antioxidant properties. In vivo and in vitro studies have demonstrated that spirulina supplementation can reduce markers of oxidative stress and enhance the activity of antioxidant enzymes. Also, as it matures, spirulina may be enriched with elements like Se, which is incorporated into organic molecules like selenomethionine and seleno-cysteine [5].

In recent years, although there has been a notable rise in algal production, it remains insufficient to satisfy the high commercial demand for biomass [21]. The dried biomass of microalgae is an excellent option for food use because of its rich nutritional content, economical processing expenses, and various health advantages [25]. Table 1 provides a summary of the types of Se-enriched microalgae.

1. Biotechnological ethods for seleni-um enrichment (cultivation methods and genetic modifications)

The ideal harvesting process for microalgae should maintain the integrity of the composition of the biomass and facilitate effective recovery of the target product. Traditional harvesting processes involve filtration, centrifugation, flotation, flocculation, electroflocculation, sedimentation, electrolytic treatment, electrophoresis, and magnetic separation. Incorporating an additional step of chemical or biological coagulation or flocculation into these processes can enhance the efficiency of the process as well as reduce operating expenses. Centrifugation is widely utilized for microalgae harvesting due to its effectiveness and speed in cell recovery. Centrifugation involves centrifugal force to separate microalgal biomass from the culture medium, from which excess water may easily be drained. Centrifugation can achieve up to 98% yield, but its greatest flaws are high energy demand and the potential for cellular structure breakdown during the process [23].

Microalgae are highly efficient at fixing carbon dioxide during their growth, so they do not require arable land for cultivation. Microalgae biomass production involves three main steps: cultivation, harvesting, and processing. Among these, harvesting is particularly challenging due to its high costs, and it has a direct impact on the processing stage [26]. The harvesting stage of microalgae is crucial for its overall production. Research has shown that harvesting accounts for 20–30% of the total production cost. Key challenges in the harvesting and dewatering process include the small size of the cells (<30 µm), their low concentration and dilute presence in the culture medium (<1 g.l^-1), the highly electronegative nature of the cell membrane surface, and the relatively fast growth rate of the algae. As a result, the energy required for the harvesting process exceeds the energy content of the microalgal biomass itself [26].

Coagulation using polyaluminum chloride (Al₂O₃) and hydrophilic polytetrafluoroethylene membranes has been used as a sustainable technology for harvesting microalgae. Transparent exopolymer particles that are also produced by microalgae have been used to reduce the fouling of membranes in microfiltration. The particles prevent membrane fouling when solutions are collected through the use of the filtration-based method, hence improving filtration efficiency [26].

Microalgae possess the ability to absorb inorganic Se and combine it with amino acids to create Se amino acids, including selenomethionine, selenocystine, which are advantageous for the health of both humans and animals. Furthermore, Se can be added from a primary source to cultivate Se-enriched microalgae in domestic wastewater, which can act as a nutrient base for microalgae growth. Low levels of Se may stimulate microalgae growth. Turbidity significantly increased over the incubation period, indicating that microalgae growing in domestic wastewater treatment systems can tolerate such high Se concentrations. The removal process facilitated by microalgae in HRAP involves biomass assimilation, as microalgae can use these substances to produce cellular components, including proteins, nucleic acids, and carbohydrates [27].

A post talks about various methods of cultivation, starting with open systems using open ponds for growing microalgae. They rely on natural water and sunlight but are prone to environmental stresses and the risk of contamination. It also talks about closed systems, which use photobioreactors or bioreactors that yield closed environ-ments for the growth of microalgae. These closed systems are efficient and less likely to cause contamination compared to open operations. In addition to this, advanced techniques like mutagenesis and genetic alteration are also discussed, which are used in order to enhance some characteristics of microalgae to maximize biomass production and quality. The article also emphasizes the necessity of specific environmental conditions, such as pH, temperature, and light intensity, in order for maximum growth and production. Altering these can lead to increased production of Se and other secondary products. Lastly, nutrient-rich conditions are suggested as an alternate approach to enhance microalgae production. Nevertheless, it is essential to maintain adequate levels of nutrients so as not to compete with Se absorption. These culture processes enhance microalgae productivity across various industries [16]. One advantage of cultivating microalgae in a mixotrophic manner is the increased biomass production [28].

Today, with advancements in microalgae harvesting, methods such as bioflocculation, electroflocculation, bio-electroflocculation, ultrasound/hydrodynamic techniques, magnetic nanoparticle flocculation, and phototaxis-based harvesting are employed [29]. Table 2 shows biotechnological methods for Se enrichment.

1. Analysis of bioavailability of selenium in microalgae

The way Se is absorbed differs among animal species and is affected by various factors, including physiological traits, functional status, the number of intestinal contents, the chemical forms of Se, the duration of Se’s stay in the intestine, and the methods of Se administration. Se is mainly found in the liver, kidneys, heart, and pancreas, with muscles, bones, and blood having the following highest levels, while fat tissues contain the least amount.

Typically, in normal circumstances, the Se that animals metabolize is mainly eliminated through urine and feces. However, when Se is consumed in excessive amounts, breathing also becomes a significant pathway for its excretion. Moreover, Se can be removed from the body through hair and sweat [30].

The bioavailability of Se in microalgae, indicating how much Se can be absorbed in the gastrointestinal tract, was assessed in a study. Grinding the microalgae, their cell walls are broken down, facilitating the release of Se from the biomass during digestion. Under gastrointestinal conditions, 49% of Se from raw microalgae and 63% from ground Se-enriched microalgae were solubilized, indicating their potential bioavailability. A similar level of Se bio-availability (approximately 49%) has been observed in Se-enriched Chlorella vulgaris. Additionally, it's important to highlight that the bioavailability of Se in Se-rich yeast widely recognized as a leading organic Se supplement is generally greater than that found in microalgae cultivated in HRAP-Se. Furthermore, the type of Se also influences absorption rates [27].

The Se in food sources is mainly in organic forms, including selenocysteine and selenomethionine, which are more easily absorbed than inorganic forms like selenite or selenate. There is a significant risk of losing dietary Se during processing and cooking; for instance, the refining of grains can diminish Se levels by 50 to 75%, while boiling can lead to an additional reduction of 45%. Microalgae that utilize Sec, such as Nannochloropsis oceania, can enhance the organic Se content and can thus be used as Se-enriched food or feed additives [24].

Organic Se compounds have higher bioavailability and usability than their inorganic counterparts. Studies also indicate that Se-enriched plant products usually provide greater bioavailability than animal-based products. Moreover, adding Se to proteins can not only increase their Se content but also improve their functional qualities, including emulsion stability and gelling properties, along with enhancing their bioavailability [31].

In numerous microalgal species, selenite may exhibit greater toxicity compared to selenate, while the opposite can be true for other species. Due to its high solubility, selenate is more readily bioavailable to aquatic organisms than selenite, indicating that selenate might be the predominant dissolved form. Prior research has shown that selenate does not pose toxicity risks for some microalgal species [8].

1. Applications in food biotechnology development and utilization of function-al foods, fortified foods, beverages, and dietary supplements

Microalgae cultivation is one factor that can enhance world food security and reduce the environmental impact of rising agricultural food production. The quality and safety of microalgae product, in turn, are both a function of proper cultivation, harvesting, and processing protocols since foreign organic and inorganic material occur as a result of environmental contamination or process operations when dealing with microalgae biomass [28].

Functional food production with microalgal biomass will see growth in the coming years as a result of growing demand for healthy foods globally. Microalgae incor-poration in food products is the next wave of evolution of the functional food industry, given that there is continuous need to search for new raw materials for use in creating new foodstuffs. Nevertheless, some of the major questions need to be answered to facilitate the successful development of microalgal-derived food products. Some of the other questions that belong to this category are how microalgal biomass interacts with the other components of the food matrix, the effect of microalgal addition on the sensory attributes of food, particularly color and flavor, textural and rheological modification of food products with microalgal addition, and changes and stability of microalgal constituents under various processing conditions [32].

Understanding the mechanisms and potential for Se accumulation in plants and animals is one of the most critical directions in developing nutritional science and supplementation [33]. To create algae-based food products and by-products, the first essential step is enhancing our knowledge of the biochemical makeup and digestibility of microalgae. In line with this, it’s important to assess more microalgal species for novel food approval, ultimately supporting the broader inclusion of these microorganisms in human diets over time [34].

Numerous studies have demonstrated that microalgae species, particularly from the Chlorella and Spirulina genera, are well-suited for direct use in their natural forms. This adaptability has led to their widespread application in the food industry, where they enhance the nutritional content of products like noodles, cookies, energy bars, and juices [23]. In the food and beverage industry, as well as within the cosmetics industry, the demand for spirulina has risen due to its fat-reducing, antioxidant, and anti-inflammatory properties [35]. An article discusses the production of beverages such as "selenized kawas," made from fennel grains soaked and germinated in solutions containing Na₂SeO₃. These beverages are considered a source of Se. Regarding Se in beer, yeasts (S. cerevisiae) can convert inorganic Se (Na₂SeO₃) into bioactive organic forms that are utilized in selenized beers. This process enhances the Se content in the beverages [36].

In a study, Chlorella vulgaris was blended into spreadable processed cheese, leading to increased magnesium, potassium, Se, Zn, iron, and antioxidant capacity, with greater enhancements observed at higher formulation levels. Microalgae were also incorporated into various snacks, breads, isotonic drinks, and yogurts; however, these products did not attract consumer interest due to their unappealing green color and distinct flavor. On the other hand, a dehydrated soup prepared with S. platensis concentration demonstrated improved nutritional value, including enhanced protein, fiber, chlorophyll, lipids, and antioxidant capacity, without compromising sensory acceptability in taste tests. These results underscore the need for more in-depth research on formulation strategies that enable effective use of microalgae to enhance functionality while preserving sensory quality [32].

1. Enhancing food products with immune-boosting properties

Since the early 1950s, microalgae have been included in human diets. Strains like Chlorella, Dunaliella, Haematococcus, Schizochytrium, and Spirulina are considered safe for consumption. Consequently, various nutrients (such as lipids, proteins, carbohydrates, pigments, and vitamins from these algae) can be consumed in dried powder form. Moreover, they can be added to various food products, including biscuits, candies, snacks, pasta, and soft drinks. Incorporating nutrient-dense microalgae into staple foods like bread can improve food security and help combat malnutrition in some developing regions. Including microalgal biomass (like Spirulina) in snacks and beverages also offers consumers a healthier option [21].

Research indicates that Se supplementation helps maintain bone homeostasis by balancing bone resorption by osteoclasts and bone formation by osteoblasts, a process linked to the activation of the Wnt/β-catenin pathway. These findings highlight a promising potential for the use of Se -based treatments in addressing osteoporosis [37]. Se supp-lementation has demonstrated effectiveness in preventing Hashimoto's thyroiditis and hyperthyroidism. Various studies suggest that Se not only offers nutritional benefits but also provides numerous additional health advantages. It exhibits strong antiviral properties, supports reproductive health in both males and females, and lowers the risk of autoimmune thyroid disorders [38].

Spirulina is a form of microalgae rich in proteins, vitamins, diverse pigments, and essential minerals like Se and Zn. It is a valuable resource for therapeutic diets, known for its anti-cancer and anti-inflammatory properties, antioxidant benefits, ability to fight malnutrition, prevent anemia, and its antiviral, antibacterial, and radiation-protective effects [35]. Recent studies have also demons-trated the antibacterial activity of Se nanoparticles. The mechanisms behind Se 's antimicrobial properties are not yet fully understood. It is believed that the effects are likely due to oxidative stress, damage to the bacterial cell wall, and DNA degradation [39].

Microalgae synthesize important bioactive substances, including carotenoids, polyunsaturated fatty acids, phenolic compounds, terpenes, and sulfated polysaccharides. These compounds provide various health benefits, such as antimicrobial, anti-inflammatory, anti-aging, anti-tumor, antioxidant, and immunosuppressive effects [20]. A study investigated Se-enriched feed (SeE-SP) as a dietary supplement for juvenile fish, specifically focusing on its beneficial effects on antioxidant responses, immune function, and disease resistance in Lates calcarifer (Asian sea bass). The findings revealed that Se boosts antioxidant activity, producing higher levels of selenoproteins [40].

1. Regulatory and safety aspects for Se-enriched products

The risk of Se poisoning in animals or plants intended for human consumption is a significant concern, as Se can bioaccumulate through food chains, particularly in aquatic environments contaminated with Se. This bioaccumulation can worsen toxicity problems in natural ecosystems. Extremely high levels of Se absorption or accumulation in animals can result in reproductive issues and congenital disabilities [41].

To ensure that microalgal products are of food quality and safety, the harvesting process must maintain the nutritional integrity of lipids, proteins, pigments, and other bioactive compounds. Microalgae grown for nutrition must be harvested under minimal physical or chemical damage. Minimal processing preserves nutrients within intact cells up to product formulation and consumption levels [29].

In an experiment, Chlorella vulgaris and Scenedesmus sp. showed resilience to the tested selenite concentrations ranging from 0 to 1000 µg.l^-1 Se. No signs of toxicity were observed in either species, even at the highest concentration of 1000 µg.l^-1 Se, and there were no significant differences in growth compared to the control cultures. Other research has similarly highlighted the high tolerance of microalgae to Se. For instance, in one study, Chlamydomonas reinhardtii only exhibited stress or toxicity at concentrations exceeding 734 µg.l^-1 Se [42]. The Se-enriched supplement in N. Oceania was found to be non-toxic in rats and is identified as a safe source of Se in the diet [24].

Algae's use in supplement production is influenced by geographical location, seasonal changes, and specific aquaculture practices, which can all impact its nutritional profile [36]. Additionally, concerns about the safety of algae and the presence of microcystins in Spirulina highlight the need for safety evaluations and quality assessments of selenized supplements and algae-containing beverages [36]. Recognizing that Se supplementation involves more than just dosage is essential; the form of Se and an individual’s health status also play a critical role. Enhancing Se’s bioavailability can raise its levels within the body. Currently, clinical research on optimal intake thresholds for different Se types remains limited. Organic Se typically offers greater bioavailability and lower toxicity. Cultivating foods rich in Se presents a cost-effective way to enhance dietary Se. As understanding of Se absorption and the role of selenoproteins expands, bioavailability may further improve [38].

The safety of foods derived from microalgae depends primarily on the microalgae species utilized, and regulatory bodies around the world have established general principles for their use. Although microalgae's safety profile is becoming more and more widely accepted, the limited number of approved species is still a significant limitation in their food applications. In addition, the culture and processing conditions of microalgae—the less discussed factors also take on a critical role in their safety. Microalgae can absorb toxic substances from various sources, such as water supplies and market fertilizers. They can absorb toxic elements, such as heavy metals, dyes, and antibiotics, in certain instances from bodies of water. Furthermore, open-air farming is capable of facilitating the growth of pathogens, and faulty processing procedures such as heat processing and drying are capable of enhancing the susceptibility to polycyclic aromatic hydrocarbons [25].

Se is found in various environmental forms, with selenate and selenite being the most toxic and responsible for 95% of its overall toxicity. While it is vital to fortify Se in areas where it is deficient for human health, its levels should be minimized or eliminated in environments where Se is abundant. Se is recognized as a minor contaminant in geological water pollution, with concentrations typically ranging from 0.1 to 100 micrograms per liter. Global daily intake recommendations for Se vary: in the UK, the advised amounts are 60 micrograms for adult women and 75 micrograms for adult men. In comparison, the World Health Organization recommends a maximum of 10 micrograms per liter in drinking water. In contrast, a study from Burundi, Africa, indicated a risk of deficiency with an average dietary intake of just 17 micrograms per day. Se intake can also vary based on age and gender. However, excessive Se consumption, even at low levels beyond the safe threshold, can lead to toxicity, resulting in issues such as hair and nail loss, cancer, and potentially death [41].

Se toxicity is broadly classified into two categories—acute and chronic—based on the duration and amount of ingestion. Acute toxicity occurs when an excessive amount of Se is consumed within a short time and causes immediate adverse effects such as respiratory distress, ataxia, diarrhea, vomiting, and abdominal pain, and even death in severe cases. Unlike this, chronic toxicity of Se occurs due to gradual build-up of Se due to long-term exposure to low concentrations, which presents as fatigue, depression, breath smelling like garlic, anemia, loss of appetite, hair loss, onychomadesis, hoof rot, stunted growth, and cirrhosis of the liver. Recent research indicates that the toxicity of Se is a function of its chemical state, and the extent of its toxicity can differ in diverse animal species, their nutritional statuses, and exposure routes. In general, inorganic compounds of Se are more toxic than organic forms [30].

The influence of Se on microalgae growth can vary widely, having either positive or adverse effects. Research on Se exposure in microalgae has identified several key factors contributing to Se toxicity. These include the type of inorganic Se present, such as selenite or selenate; the concentration of Se in the growth environment; the specific response of the microalgae species involved; and the level of sulfur in the medium. Because Se and sulfur share similar chemical properties, they enter the cells using the exact transport mechanisms, leading to competitive uptake between the elements [43].

At high concentrations, Se can act as a pro-oxidant, leading to the generation of substantial amounts of ROS that attack biomolecules such as proteins and nucleic acids. This process causes oxidative damage and can ultimately result in cell death. [44]. Acute Se toxicity in grazing animals occurs when they consume excessive amounts of accumulator plants containing high Se levels over a short period. The symptoms of Se poisoning in mammals can differ widely, including issues such as nail abnormalities, hair and wool loss, weakness, vomiting, diarrhea, fatigue, reduced cognitive function, lethargy, immobility, weight loss, itchy skin, and irritation of mucous membranes. Affected individuals may also suffer from lateral sclerosis and experience irritation in the throat and bronchial tubes [45]. Younger animals show greater sensitivity to Se toxicity, and different chemical forms of Se can result in varying toxicity levels. Beyond mammals, Se poses several harmful effects in birds, with symptoms of toxicity appearing within a few hours to several days. In avian species, these toxic effects can manifest as increased mortality, reduced growth rates, histopathological changes, and disruptions in hepatic glutathione metabolism [45].

Selenium Nanoparticles have a greater impact on organisms compared to inorganic forms of Se. Additionally, the influence of Se on health is contingent upon the individual's requirement to develop antioxidant defenses. If this need is not met, excessive Se can result in toxicity. Overall, the toxicity of Selenium Nanoparticles is often linked to Se toxicity. Most research comparing Se and Selenium Nanoparticles indicates a consensus that Selenium Nanoparticles are less toxic [45].

Alkali disease, a livestock disorder of the Great Plains of the United States, has raised concern about acute and chronic Se toxicity. Laboratory animal studies with rabbits, rats, and cats have shown that the lethal dose of Se, administered either as sodium selenite or selenate, ranges from 1.5 to 3.0 milligrams per kilogram body weight, regardless of the route of exposure. Chronic consumption of Se as little as 4-5 parts per million in feed will stunt the growth of livestock. Acute Se poisoning in livestock typically results from consumption of Se-accumulating plants or over-supplementation and is marked by severe symptoms that include respiratory stress, abnormal movements, diarrhea, and, in some cases, acute death [41].

1. Mechanisms of immune system enhancement

Redox homeostasis is essential for the maintenance of vital cell and organism functions. Redox stress is a disturbance of the oxidative vs. antioxidative balance within a cell. This is typically marked by the production of significant quantities of ROS, more than the antioxidative defense system can clear, resulting in structural and functional damage to DNA, lipids, and proteins. Mitochondria are recognized as among the most important sources of ROS, and an excess of these molecules tends to damage mitochondrial structures. Hydroperoxides, partic-ularly hydrogen peroxide (H₂O₂), also serve as important ROS for redox regulation and participate in cell signaling, enzymatic reactions, energy metabolism, and the cell cycle. Excess hydroperoxides, nevertheless, can lead to non-specific oxidation of proteins and biomolecule destruction. To eliminate these hydroperoxides, effective reductive systems play a vital role. In this regard, Se is considered an essential antioxidant that helps in the removal of ROS, especially hydroperoxides, and contributes to the improvement of tissue and cellular conditions [12, 46]. This effect has been reported in the heart [46], liver [47], and kidneys [48].

One of the well-known and beneficial characteristics of Se is its antioxidant activity, which is particularly effective in countering oxidative damage at the cellular level (Figure 1). Antioxidant processes help maintain appropriate levels of ROS and reactive nitrogen species, thereby preventing oxidative damage and enhancing immune responses [49]. Oxidative stress occurs when the balance between the production of ROS and the ability of the system to neutralize them is disrupted. ROS are typically produced due to electron leakage from the mitochondrial electron transport chain and enzymes during the process of oxidative phosphorylation [50, 51]. Particular ROS is essential for the activation and differentiation of T cells, apoptosis, pathogen elimination, and other cellular signaling activities. However, excessive production of these free radicals can lead to severe cell damage, including lipid peroxidation, DNA damage, and protein degradation. Therefore, antioxidants, including Se, are crucial for counteracting oxidative damage and improving immune system function [12].

Se defends cells against ROS through the mechanism of selenoproteins, which have redox activities. These proteins are able to reduce hydroperoxides in the presence of thiols and also have a pivotal role in redox homeostasis. The best-known among these proteins are GPXs (glutathione peroxidases) and the thioredoxin (Trx) system. Five of the eight human GPX types are selenoproteins, and Se is found in the active site of these proteins. These proteins are particularly involved in the detoxification of hydroperoxides and, consequently, oxidative stress. The GPXs' active site contains a conserved structure made up of Se, glutamine, tryptophan, and asparagine. These Se residues could be oxidized with hydroperoxides to produce selenic acid or selenoamide products that are then reduced quickly to selenate by thiols. Since Se residues are extremely reactive, GPXs react immediately, especially in the case of reaction with H₂O₂, reducing its cellular level. GPX1 was the first mammalian selenoprotein discovered and is referred to as an essential protein in mitochondria and the cytoplasm. The enzyme uses glutathione to reduce hydroperoxides and is extremely sensitive to Se levels. GPX2, with substrate-like properties of GPX1, is found mainly in the mucosal membrane of the gastrointestinal tract and endothelial cells and plays a role in regulating mucosal homeostasis and intestinal cell turnover. Interestingly, GPX2 expression is reduced only in patients with Se deficiency of a high grade. GPX3 is a glycoprotein located outside cells that is able to decrease hepatic glutathione oxidation, Trx, and glutaredoxin and is found importantly in white and brown adipose tissue. GPX4 is the only isoform with a capacity to decrease phosphatidylcholine hydro-peroxides and has a distinctive function of protecting cells against oxidative damage in mitochondria. Antioxidant defense also plays a significant role in the Trx system, comprising nicotinamide adenine dinucleotide phosphate, Trx, and thioredoxin reductase. Trx acts by transferring electrons to thioredoxin peroxidases and converting oxidized Cys disulfides or Cys-SOH residues in proteins to thiols. In addition, Trx is able to engage in the formation of intracellular gradients of H₂O₂. Thioredoxin reductase, being an oxidoreductase enzyme, employs nicotinamide adenine dinucleotide phosphate as a co-substrate to reduce oxidized Trx, and its activity plays an important role in controlling redox reactions. The system also participates in the reduction of methionine sulfoxide reductases and ribonucleotide reductases and controls activities of redox-sensitive transcription factors like AP-1 and NF-κB. Overall, the GPX and Trx systems with their antioxidant functions are crucial in cellular protection and maintain redox homeostasis, finally safeguarding cells from oxidative damage and functioning normally. Se in the body primarily exerts its action through selenoproteins, which carry out many antioxidant functions. Selenoproteins contain GPxs and thioredoxin reductases, neutralizing harmful free radicals by reduction reactions. For example, GPxs catalyze the reduction of water and oxygen from H₂O₂, while GPx4 reduces membrane lipid hydroperoxides. Additionally, thioredoxin reductases reduce disulfides of proteins [52-55].

Se exerts its cellular antioxidant defense primarily through two key selenoprotein systems: GPXs and the Trx system. GPXs catalyze the reduction of H₂O₂ and lipid hydroperoxides by utilizing glutathione as a reducing agent, thereby preventing oxidative damage to cellular macromolecules. This enzymatic activity depends on the presence of selenocysteine at the active site. Concurrently, the Trx system maintains redox homeostasis by reducing oxidized Trx through thioredoxin reductase in a nicotinamide adenine dinucleotide phosphate-dependent manner. Reduced Trx subsequently facilitates the conversion of protein disulfides (Prot–S–S–Prot) back to their thiol forms (Prot–SH), preserving the functional integrity of cellular proteins. Collectively, these mechanisms contribute to the detoxification of hydroperoxides and the mitigation of oxidative stress, thereby safeguarding cellular integrity and function. However, when these antioxidant processes are under stress, the risk of various diseases such as cancers, neurodegenerative diseases, fertility problems, and kidney disorders increases [55].

Studies have shown that Se can effectively enhance antioxidant activities and improve immune function. For example, in a study on pigs, supplementation with organic Se (4-methylselenobutanoic acid, HMSeBA) led to an increased expression of GPxs and thioredoxin reductases in various tissues, and a reduction in malondialdehyde levels, which is an indicator of lipid peroxidation. Additionally, the study showed that Se supplementation reduced the levels of inflammatory cytokines IL-6 and TNF-α while increasing IL-2 levels in the pigs [56].

These results suggest that Se may effectively help counteract oxidative and inflammatory damage and improve immune function. Similar studies have also been conducted on chickens and fish. In one study on chickens, Se deficiency led to a significant reduction in the expression of selenoproteins in the thymus, spleen, and bursa of Fabricius [54]. Additionally, feeding chickens yeast-derived Se increased the expression of genes related to antioxidant selenoproteins in the intestines [57]. In another fish experiment, feeding with organic, inorganic, or nano Se resulted in a decrease in serum malondialdehyde levels and an increase in the levels of antioxidant enzymes such as GPx, Catalase, and Superoxide dismutase [58].

While similar results in other studies indicate the need for a thorough evaluation of different Se dosage regimens in various species, the overall findings suggest that Se, especially in its nano form, can have positive effects on reducing oxidative stress [59]. It has also been reported in a study that stabilized Se nanoparticles can effectively reduce the excessive production of intracellular ROS induced by patulin. Additionally, these nanoparticles can improve the reduction in glutathione peroxidase activity and suppress cell viability [60]. However, it is also accepted that high doses of Se (sodium selenite) can induce oxidative stress in living organisms and increase the process of lipid peroxidation. This suggests that excessive accumulation of Se within cells is toxic to living organisms [61].

Although Se, as a potent antioxidant, can be effective in regulating the immune system and reducing inflammation, further research in humans is needed to confirm optimal dosages and its impact on clinical conditions [62, 63].

Se affects the immune system through three main pathways: cellular immunity, humoral immunity, and non-specific immunity. Se enhances the production of interferon and increases the activity of gamma interferon in the laboratory setting, which boosts the cytotoxic effect of human NK cells without damaging the target cell membrane. Additionally, Se significantly increases the secretion of IL-1 and IL-2 from lymphocytes, stimulates the formation of immunoglobulins, and improves the body's ability to synthesize antibodies such as IgG and IgM [64]. Furthermore, Se has various effects on chemotaxis, phagocytosis, and phagocytes viral killing. Previous studies have shown that Se can regulate the differentiation of mouse helper T cells (Th), antibody production, leukocyte adhesion and migration, and macrophage phagocytosis. Recent studies have confirmed that Se can modulate the immune response of dendritic cells in chickens, mice, and humans [65-67].

Se increases the production of immunoglobulins, which enhances the proliferation and differentiation of lymphocytes and improves the production of antibodies such as IgM and IgG. In the case of Se deficiency, the process of immunoglobulin and antibody synthesis is disrupted [68]. In Se deficiency, the production of leukotriene B4, essential for attracting neutrophils to the site of infection, is impaired; however, with the addition of Se supplementation, this process returns to normal. Additionally, Se has both direct and indirect effects on the activity of NK cells [69]. The ability of NK cells to eliminate cancerous or virus-infected cells is influenced by the amount of Se consumed in the diet. In a study involving over 300 men in North America, it was found that Se supplementation increased plasma Se levels and improved the number of NK cells in the bloodstream. Additionally, in older adults, higher Se levels in the blood were positively correlated with a greater number of CD16⁺ NK cells (a type of NK cell) in the plasma. This indicates the role of Se in enhancing NK cell function and strengthening the body's immune response [68].

These findings reveal that Se not only occupies a core role in immunostimulating effects but also has the potential to be a good means of enhancing immune well-being. Se has multiple immunomodulatory roles, which are primarily identified as functions of selenoproteins, especially in redox control and antioxidant defense. These actions are mediated by selenoprotein enzymes such as GPx and Thioredoxin reductase, and by the non-enzymatic protein K, which possess roles in immune functions. Adequate Se ingestion is required to trigger immune reactions and selenoprotein formation. Optimal Se status enhances immune reactions, such as the production of interferons and interleukins, while excessive Se consumption has negative impacts [70-72].

Se has diverse and widespread effects on the immune system. These effects are observed in the two main parts of the immune system (Figure2(: innate immunity (which includes macrophages and neutrophils) and adaptive immunity (which provides for T and B lymphocytes).

1. Effect on Macrophages: When macrophages are confronted with Se deficiency, Se supplementation alters the activation of these cells. Specifically, Se reduces the activation change of macrophages from the inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. M2 macrophages secrete anti-inflammatory cytokines like IL-10, which can help inhibit tumor growth. Additionally, Se protects macrophages from oxidative stress and enhances their functionality.
2. Effect on Neutrophils: Se indirectly affects neutrophils. One of its effects is reducing the synthesis of leukotriene B4 in macrophages, a molecule essential for attracting and migrating neutrophils to inflammation sites.
3. Effect on NK Cells: Se enhances the activity of NK cells in the body. Se supplementation increases the expression of IL-2 receptors (IL-2R) on the surface of these cells. These changes strengthen the cytotoxic function of NK cells, increase their proliferation, and expand cytotoxic precursors. As a result, NK cells can effectively target cancer cells and produce cytokines such as IFN-γ and TNF-α, which play a crucial role in the anti-tumor immune response.
4. Effect on Dendritic Cells: Se can activate specific kinases involved in the immune response by increasing levels of ROS or glutathione. These kinases help stimulate antigen phagocytosis by immature dendritic cells. This process can enhance the immune response against infections and tumors. Additionally, Se reduces the expression of matrix metalloproteinases, which can inhibit the migration of cells to inflammatory areas.
5. Dual Effect on ROS: Se can increase the production of ROS, which can have beneficial and harmful effects. On one hand, ROS production can stimulate the immune response, including the activation of dendritic cells. On the other hand, excessive ROS production can impair the function of cytotoxic T lymphocytes and other anti-tumor immune components.

Effect on T and B Lymphocytes: Se deficiency can reduce the ability of lymphocytes to proliferate in response to immune stimuli. In animal models, a Se-enriched diet shifts the balance of Th1 and Th2 phenotypes in favor of Th1, increasing interferon-gamma (IFN-γ) levels. Furthermore, Se supplementation improves humoral immunity (related to B lymphocytes), such that in Se deficiency, immunoglobulin production (IgG and IgM) is reduced [65, 67, 73, 74].

Optimal Se status enhances innate immune function by improving neutrophil oxidative stress resistance through selenoprotein upregulation, boosting macrophage chemo-taxis and phagocytic activity while promoting M2 polarization, and increasing NK cell cytotoxicity and proinflammatory cytokine secretion. In adaptive immunity, Se supports Th1 lymphocyte responses and their cytokine production. However, Se deficiency compromises humoral immunity by reducing B cell differentiation and antibody (IgG/IgM) synthesis. Immunological enhancements and suppressions are indicated by green (↑) and red (↓) arrows, respectively.

Innate immunity is characterized by the rapid response of the immune system to infection. The innate immune system employs various mechanisms such as activation of the complement system, activation of phagocytic cells, and antimicrobial peptide production to control and eliminate pathogens. Mast cells, NK cells, monocytes, macrophages, dendritic cells, neutrophils, basophils, and eosinophils are some of the cells that are involved in innate immunity. One of the primary means through which such cells eliminate pathogens is by producing ROS during the "oxidative burst" process. This process is partly regulated by selenoproteins such as SelK and GPx, which participate in calcium (Ca²⁺) signal transduction pathways and redox reactions, respec-tively [2].

Se controls the excessive production of ROS through antioxidant mechanisms and prevents damage to host tissues. At the same time, adequate intake of this element enhances the effectiveness of the oxidative burst process, which is initiated in phagocytes when stimulated by pathogens. Se deficiency can disrupt these mechanisms, but by increasing Se levels, both the oxidative burst process and antioxidant activities in other parts of the body are strengthened. These two processes operate independently of each other, and neither inhibits the function of the other. This characteristic enables the immune system to effectively eliminate microbes while preventing damage to host tissues caused by ROS [2].

Se plays a crucial role in the survival and differentiation of leukocytes. Specifically, Se facilitates the differentiation process of pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages. Additionally, Se activates calcium (Ca²⁺) signaling in macrophages, essential for initiating FcγR-dependent phagocytosis. Moreover, Se supplementation can reduce the adhesion of leukocytes to endothelial cells, potentially affecting how they are transported to various tissues [76]. Se plays its role by becoming a component of a protein called SelK, which is present in the endoplasmic reticulum of leukocytes (white blood cells), i.e., neutrophils and macrophages. This protein helps to allow calcium (Ca²⁺) into the cell, which is required for immune response signaling. It has been observed that mice in which the SELENOK gene is knocked out (where Se is unable to play any role) have impaired immune responses compared to normal mice. For example, they have defective neutrophil migration, reduced production of required chemicals (chemokines), defective oxidative burst in macrophages to eliminate pathogens, and defective release of important cytokines like IL-6 and TNF-α [2].

Another prominent feature of Se is its ability to regulate the adaptive immune system. The adaptive immune system is divided into two parts: the cellular component, which is T cell-dependent, and the humoral component, which relies on B cells. In addition to Se 's role in regulating ROS signaling for the production of free radicals and enhancing protein biosynthesis, this element also increases the production of cytokines IL-2 and IFN-γ by T cells. This process promotes T cell proliferation and differentiation, influences epigenetic regulation, prevents endoplasmic reticulum stress, and reduces leukocyte infiltration into tissues. One of the crucial selenoproteins in the adaptive immune system is SelK. This protein is found in the endoplasmic reticulum of B and T cells and, as mentioned, supports calcium ion flow for signaling and cellular activation [2].

T cells are one of the most important types of white blood lymphocytes, playing vital roles in adaptive immune responses and effectively in protecting the body against infections, cancer, inflammatory diseases, and other chronic conditions. In recent decades, various types of T cells, including helper, regulatory, cytotoxic, and memory T cells, have been extensively studied, significantly enhancing our understanding of T cell immune function. Due to their critical roles in combating cancer and infections, T cells have become primary targets in immunotherapy strategies such as PD1/PD-L1 and CAR-T therapy. Currently, T cells are the most commonly used immune cells authorized for clinical immunotherapy, especially in cancer treatment. However, challenges such as low efficacy, off-target side effects, and high treatment costs still exist and require further investigation.

Se, as an essential micronutrient for human health, has garnered significant attention. In recent decades, various studies have clarified the relationship between Se and T-cell function. Se deficiency in mice leads to thymus, spleen, and lymph node atrophy, and the populations of CD3⁺ and CD8⁺ T cells in these mice are significantly reduced, indicating a disruption in T cell function [77].

Research has also shown that Se deficiency can inhibit the activation and proliferation of T cells, highlighting the importance of Se in the proper functioning of T cells. On the other hand, excessive Se intake can also affect adaptive immunity, directing the proliferation and differentiation of activated CD4⁺ helper T cells toward Th1 cells. These cells play vital roles in T-cell immune responses against viral or bacterial infections [77].

The biological effects of Se on T cells are likely mediated through the function of selenoproteins, which have various roles in these cells. These roles include regulating calcium flux resulting from T cell receptor interactions, controlling the redox status of T cells before, during, and after activation, as well as reprogramming the metabolism necessary for T cell proliferation and differentiation in response to T cell receptor activation [72].

In recent years, Se nanoparticles (Se NPs) have been shown to enhance T cell proliferation and regulate their functions as an immune modulator. For example, Shams et al. found that the combination of aerobic exercise training and Se NP administration could reduce tumor volume and increase Th1 cytokines in the splenocytes of tumor-bearing mice. These findings indicate that Se NPs enhance the anti-tumor T cell immune responses [72].

γδ T cells are a type of immune cell that have a unique combination of surface proteins and play an important role in immune responses. These cells are positioned at the interface between innate and adaptive immunity and are particularly effective in combating tumors and infectious diseases. For this reason, γδ T cells have great potential for immunotherapy in cancer treatment [78].

One of the new approaches is using Se nanoparticles to enhance the function of γδ T cells in combating tumors. Se nanoparticles increase the anti-tumor activity of γδ T cells while causing less damage to the treated cells. Compared to a similar form of Se compound (Na₂SeO₃), cells treated with Se NPs showed lower cytotoxicity, indicating better compatibility of these nanoparticles with the body [78, 79]. Se NPs, which are produced in the form of Se nanoparticles, have similar properties to Se itself. However, due to their microscopic size, they can more effectively enter cells and perform their therapeutic functions. These nanoparticles are especially effective in reducing inflammation and oxidative stress (cellular damage caused by free radicals) [79].

1. Studies conducted on the effective-ness of Se, Se-nanoparticles, and Se-enriched

In rats, the brain appears to be well-protected from Se deficiency, and Se-enriched spirulina proved to be more effective in replenishing Se levels in tissues than sodium selenite. Both types of supplementation, whether spirulina-enriched or regular Se, led to partial or complete restoration of Se concentrations in various tissues. For example, spirulina supplementation fully restored Se levels in plasma, urine, liver, kidneys, and the soleus muscle. Regular Se supplementation also completely restored Se in plasma, urine, and kidneys, while partially doing so in the liver, heart, and soleus. Interestingly, no change in Se levels was observed in the brain, indicating it may be an exception. These results imply that Se distribution in the body prioritizes specific tissues depending on the type of supplementation used [5].

In a study investigating selenoprotein expression in Se-deficient rats, Se-enriched Spirulina platensis produced by TAM company (Plougastel, France) was used. Se-enriched spirulina and Se-free spirulina were dried, powdered, and used simultaneously. The Se concentration in the enriched sample was 55 µg of Se per 1 gram of Se-enriched spirulina based on dry weight [5].

In a study, the effects of Se and probiotics on Alzheimer's disease were examined. Although the primary goal of this research was not to investigate the innate immune system, the results showed that daily consumption of 200 micrograms of Se for 12 weeks significantly reduced the concentration of C-reactive protein in the blood [80].

Hypercholesterolemia can lead to the accumulation of cholesterol in macrophages, increased signaling of TLR receptors, increased numbers of monocytes and neutrophils in the blood, and, consequently, higher production of inflammatory cytokines. Se 's ability to reduce blood LDL levels may help mitigate these inflammatory responses. This regulatory effect is particularly beneficial for individuals suffering from autoimmune diseases, inflammatory disorders, or chronic infections from an immunological perspective [2].

In a study on Nile tilapia fish with Se deficiency, the results showed that Se nanoparticle supplementation significantly increased serum lysozyme activity, improved oxidative burst capacity, and enhanced the expression of pro-inflammatory cytokines (such as TNFα, TGFβ1, and IL1β). Additionally, fish receiving Se nanoparticles performed better in phagocytosis and bactericidal tests. However, no significant changes were observed in leukocyte concentrations. Overall, Se nanoparticle supplementation had the most positive effect on the innate immune system of the fish [58].

In a study on European sea bass, the effects of different doses of Se nanoparticles (0, 1, 5, and 10 mg per kg) on the innate immune system were investigated. The results showed that in the 1 mg Se group, IL-6 expression decreased, while in the 5 and 10 mg groups, it increased. Chronic elevation of IL-6 could lead to issues such as autoimmune disorders. In all Se -treated groups, TNF-α expression was increased. In the 1 mg Se group, IL-12 expression increased, while in the 5 and 10 mg groups, it remained stable. These results suggest that different doses of Se have varying effects on the innate immune responses of the fish [59, 81, 82].

In recent years, there has been growing interest in the anticancer and antimicrobial properties of NK cells. One study has shown that inorganic selenite (a form of Se) can increase the sensitivity of mesothelial cells to NK cells. This is achieved by reducing the expression of a specific protein called HLA-E. This finding suggests that NK cells may have high potential in cancer treatment. NK cells play a crucial role in the body's innate immune responses, particularly in combating cancer and infections. These cells can identify tumor cells or pathogen-infected cells using specific receptors on their surface and then eliminate them. Additionally, during this process, NK cells produce cytokines that help enhance the immune response [79]. The ability of NK cells to eliminate cancer cells is typically limited by inhibitory signals. One such signal is generated through a receptor called NKG2A, which prevents NK cell function [83].

Se-containing nanoemulsions can effectively enhance the ability of NK cells to recognize and attack tumor cells. This is achieved by increasing the expression of the NKG2D receptor and its related ligands, which are associated with DNA damage response pathways. This finding suggests that using simple nanoemulsions could be an effective strategy for accompanying adjuvant drugs in cancer treatment using NK cells [84]. In addition, Se -containing compounds can help enhance the effects of immunotherapy against prostate cancer. This is achieved by activating TRAIL/FasL signaling, which strengthens NK cell activity and thereby contributes to more effective cancer-fighting. These results suggest that Se -containing nanosystems can assist in regulating and enhancing NK cell function in cancer treatment [85].

Pan et al., in a study, introduced the drug pemetrexed into a Se -nanosystem, which enhanced human non-small cell lung cancer cell sensitivity towards NK cells. This was achieved by regulating pro-inflammatory cytokines such as IFN-γ and TNF-α, which are responsible for promoting NK cell immune responses. Further studies need to be carried out in order to understand the comprehensive mechanisms in which Se regulates NK cell immune responses. Since Se nanoparticles possess significant benefits, following researches showed that Se nanoparticles are used to establish new therapeutic approaches for enhancing NK cell activity and more effective cancer treatment [85].

Wei et al., in a study, designed Se -based cell-penetrating nanoparticles that combine various motifs for tumor targeting and NK cell activation. These nanoparticles include motifs for binding to tumor cells, enzymatic cleavage (PLGVR), and response to ROS to enhance anticancer activity. This system can improve chemo-immunotherapy by activating NK cells and reducing tumor size by producing oxidative metabolites and desalinization [86].

Gao et al. reported a nano drug called PSeR/DOX, a combination of radiation-sensitive nanoparticles that not only carry the chemotherapy drug (DOX) but also have tumor-targeting properties. These nanoparticles contain Se (diselenide), which, in addition to its anticancer effects, acts as an immune checkpoint inhibitor. Therefore, these nanoparticles can be combined with radiotherapy and immunotherapy to create more effective cancer treatments with fewer side effects [87].

In another study conducted using a mouse model, cytokine concentrations in the placenta were examined following immune system activation in the mother. In this study, mice were given Se from day 9 of pregnancy until birth, and on day 17 of pregnancy, they were challenged with polyinosinic: polycytidylic acid. The results showed that compared to mice that did not receive Se, those receiving Se daily had significantly reduced protein concentrations of IL-17 and IL-1β in their placental tissues, while IL-6 levels remained unchanged. This study indicated that Se may reduce the activities of adaptive immune system T cell branches and some components of the innate immune system during pregnancy, potentially showing anti-inflammatory and immune-modulatory effects for the fetus during critical growth periods [88].

Another study conducted using a pig model demonstrated the positive effects of Se on the humoral branch of the adaptive immune system. The researchers observed that the concentrations of intestinal immunoglobulin A (sIgA) and serum immunoglobulin G (IgG) were significantly higher in pigs treated with HMSeBA (a Se compound) compared to the control group. These results suggest that Se is essential for regulating adaptive humoral immune responses and helps enhance the production of antibodies (such as sIgA and IgG), which play an essential role in the body's defense against infections [56].

Another study conducted on chickens investigated the effects of Se on the adaptive immune system. In this study, the chickens were fed different forms of Se at a dose of 0.3 mg of Se per kg of their basal diet, and various results were observed. By day 42, serum concentrations of IgG, IgA, and IgM were significantly higher in all groups that received Se supplementation. These findings indicate the positive impact of Se in enhancing humoral immune responses in chickens [89].

In another study, broiler chickens were tested with different doses of Se (0, 0.25, 0.50, or 1.00 mg of Se per kg) to evaluate the immune response against Clostridium perfringens. The results showed that Se had a significant immunostimulatory effect on these birds, leading to a substantial increase in the expression of cytokines IL-1β, IL-6, and IL-8 in their jejunum and spleen. These findings highlight the role of Se in enhancing the body's immune responses to pathological challenges [90].

Finally, studies on fish have shown that Se supplementation has a significant positive impact on improving the adaptive immune system. In a feeding experiment, Nile tilapia were fed different doses of mineral Se (1 mg.kg^-1), organic Se (1 mg.kg^-1), and nano Se (1 mg.kg^-1). The results showed that treatment with nano-Se led to a significant increase in total IgM levels, while treatment with organic Se significantly increased serum protein levels. Additionally, treatment with nano-Se increased IL-2 levels, a cytokine responsible for limiting inflammation. These findings suggest that nano Se can effectively enhance adaptive immune responses and regulate inflammation [12, 58, 59].

Several randomized clinical trials have been conducted to examine the effects of Se supplementation on immune function. Overall, supplementation with Se may positive effect on immune system performance, particularly in individuals with low Se levels. However, its effects can vary depending on the dose, type of supplement, and immune response. Additionally, some studies have not shown significant improvements in immune function, highlighting the complexities of the impact of Se supplements on the immune system [91].

A study on children with systemic inflammation (a type of widespread inflammation in the body) showed that increased Se levels in their blood plasma were associated with better treatment outcomes [79]. These results indicate that adequate Se intake can help control inflammation and improve patient conditions.

A study by Mahana et al. showed that Se nanoparticles could prevent kidney damage caused by the antibiotic vancomycin. This antibiotic is used to treat antibiotic-resistant bacteria, but one of its significant side effects is nephrotoxicity (kidney toxicity). In this study, Se nanoparticles were able to reduce inflammatory factors and oxidative molecules (such as malondialdehyde and nitric oxide) that cause kidney damage, thus preventing further kidney injury [40].

Additionally, research by Xiao et al. demonstrated that Se nanoparticles could reduce vascular inflammation, which is commonly seen in cardiovascular diseases. The results showed that Se nanoparticles could increase nitric oxide (an anti-inflammatory molecule) levels in the blood and remove macrophages (a type of immune cell involved in infla-mmation) from the vessel walls. Furthermore, Se nanoparticles inhibit the NF-kB signaling pathway, which is usually activated in inflammatory responses [92].

Amini et al. also used Se nanoparticles for targeted stroke treatment. In this study, Se nanoparticles helped reduce stroke-induced damage by regulating inflammatory and metabolic signaling pathways, presenting a new therapeutic strategy for stroke [42].

For a summary and better understanding, refer to Table 3, which shows a qualitative comparison of selenium-enriched microalgae and sodium selenite.

1. Addressing global nutrition chall-enges

Low Se status is a widespread public health issue, affecting people across the world. Most of these people live in sub-Saharan Africa, South Asia, and China, where the soil is low in Se. The deficiency can weaken the immune system, making people more vulnerable to infection and other diseases [10, 93].

Our examination shows that microalgae like Spirulina and Chlorella, which contain high concentrations of Se, may indeed be the key. Not only do they provide enhanced bioavailability, but they also have the advantage of being produced through a more environmentally friendly process. We know all the disadvantages of traditional sources of Se. For instance, inorganic Se supplements, including sodium selenite, are not effective because they are not well absorbed. In addition, Se-enriched crops demand large expanses of agricultural land. Microalgae offer a better alternative with multiple methods to supply Se efficiently [6, 7].

Microalgae culture offers some great benefits when it comes to sustainability. They can thrive in different settings, including wastewater systems [16, 27]. This improved bioavailability is especially important for people who have trouble absorbing nutrients.

With regard to the microalgae culture in providing a sustainable technology, the primary environmental benefit is that it provides a tried one. They are able to grow in an exceedingly wide variety of locations, beyond filthy wastewater systems, and they are not even freshwater or on farm land [24, 42].

Field tests with undernourished populations were successful. This activity can be demonstrated when Se-enriched Chlorella is added to the Indian and Bangladeshi diet, with clear amelioration in Se deficiency markers [40].

These findings suggest the possibility of using microalgae-based solutions to treat Se deficiency at the population level. Immunological effects of Se derived from microalgae are especially important in the wake of prevailing global health trends. Se deficiency for extended periods has been linked to various unfavorable health outcomes. For instance, during viral infections like COVID-19, Se levels have been proven to be a determining factor in the severity and mortality of the disease [11, 49]. Moreover, low Se intake has also been associated with increased risk of certain cancers, especially gastric and prostate cancers [73, 74]. The metabolic impacts are just as serious, since Se deficiency can exacerbate oxidative stress and chronic inflammation to induce conditions like cardiovascular disease and non-alcoholic fatty liver disease [47, 79].

Microalgae-derived Se affects the immune system at the cell level. Experiments have demonstrated that Se-enriched Spirulina supplementation is able to enhance NK cell activity [3]. Its immunostimulating effect is accompanied by strong antioxidant activity, increasing synthesis of key selenoproteins like GPx and selenoprotein P that guard vital tissues against oxidative damage [3, 94]. Furthermore, research has established that microalgae-based Se is able to decrease pro-inflammatory cytokines, including IL-6 and TNF-α, in clinical groups that are consuming such supplements [80].

To successfully move these scientific developments into successful interventions, we require an interdisciplinary strategy for policy action. We propose the following three-pronged strategy for implementation: First, incorporating Se-enriched microalgae into existing nutrition programs via cooperation with global health organizations may complement current supplementation, like that for vitamin A and iron [8]. Second, embracing controls and providing economic incentives drawn from the successful aquaculture policy in Norway could convince the industry to adopt and expand this initiative [6, 7]. Finally, forming strategic public-private partnerships could improve production and distribution capabilities, especially in areas where Se is lacking [42].

The immunological and nutritional synergy gives Se-enriched microalgae a distinct reaction to worldwide health concerns. The strategy is well-addressed to the World Health Organization's Immunonutrition Aims for 2030 by treating micronutrient malnutrition as well as immune system dysfunction simultaneously. We need to consider decentralized production networks, education programs among consumers, and facilitating policy frameworks that ensure equitable access to all in the future. As the evidence mounts, Se-enriched microalgae must not only be considered as a food supplement, but as an essential element of sustainable, responsive food systems that have the potential to contribute to the solutions for some of the world's most recalcitrant public health problems.

1. Conclusion

Studies highlight the vital role of Se in enhancing and regulating adaptive immune responses across different species. Se, by affecting various branches of the immune system (cellular and humoral), helps improve immune activities against pathogens and reduce inflammation. The results show that this element can increase the production of antibodies, such as IgM, IgG, and IgA, and cytokines like IL-1β, IL-6, and IL-8 in various species (mice, pigs, chickens, and fish). Additionally, Se can modulate innate and adaptive immune activities during sensitive periods, such as pregnancy, and exert anti-inflammatory effects. Overall, Se is effective in enhancing immune responses and regulating inflammation and can be used as an immunostimulatory agent to improve immune system efficiency.

Se-enriched microalgae, such as Chlorella vulgaris, provide organic Se with high and suitable bioavailability, such as selenomethionine, making them ideal for use as functional foods and nutrients to address Se deficiency. This is supported by their potential to alleviate Se deficiency and their complementary role in enhancing immune system health. Therefore, these positive attributes position microalgae as potential candidates for microbiome engineering and enable support for nutrition-focused health interventions due to their bioactive compounds. Furthermore, optimized cultivation techniques can enhance Se composition, enabling the scalable production of safe, Se-enriched biomass for dietary supplements and bridging biotechnology with nutrition.

As per the discovery through research, Se nanoparticles and also Se possess highly conspicuous anti-inflammatory activity. These can be used in the treatment of chronic inflammation and oxidative stress-related disease. These are kidney diseases, cardiovascular diseases, and even stroke, in which inflammation is a leading factor for the progression of the disease. All these could be cured with this new and possible therapeutic method. Se-enriched microalgae are a new and promising bioactive solution to the boosting of the immune system and general well-being. Since such algae can sequester Se and metabolize it into more bioactive forms, they represent an inexpensive and renewable source of this essential micronutrient. In this review, the importance of Se to human health is discussed, such as its role in antioxidant function, immune system regulation, and capability to reverse oxidative stress and inflammation.

Microalgae can be enriched with Se through biotechnological methods such as selective cultivation and genetic modifications, leading to the creation of functional foods, dietary supplements, and fortified products with immune-boosting properties. A growing body of research, including animal studies and human clinical trials, has confirmed the immune-modulating effects of Se-enriched microalgae and demonstrated their potential to improve health outcomes and prevent various diseases.

Despite the promising outlook, many challenges remain, especially in understanding the underlying mechanisms of Se action and optimizing biotechnological processes for large-scale production. Further research is needed to bridge knowledge gaps, improve yield and efficacy, and ensure the safety and regulatory compliance of Se-enriched products. Market opportunities for these bioactive products are expanding, particularly with the increasing consumer demand for functional foods and natural supplements.

Looking ahead, the future of Se-enriched microalgae in food biotechnology and health promotion appears promising, with advancements in genetic engineering, production techniques, and product development likely to drive the next growth phase. Interdisciplinary collaboration and innovation will remain essential to overcoming existing challenges and unlocking the full potential of Se-enriched microalgae for global health benefits.

1. Declaration of competing interest

The authors report no conflict of interest.

1. Authors’ Contributions

Conceptualization, A.D., M.A.; methodology A.D., M.A.; investigation, F.F., Y.B.; data curation, A.D.; writing (original draft preparation, A.D., A.Gh., F.F., Y.B.; writing) review and editing, A.D., M.A.; visualization, A.Gh., Y.B.; supervision, M.A.; project administration, A.D, M.A. .

1. Using Artificial Intelligent Chatbots

The authors did not use artificial intelligence

1. Ethical Consideration

This study does not require approval from an ethics committee.

Background and Objective: Nanobioremediation using various biological entities has emerged as a rapidly evolving field of research. These biologically synthesized nanoparticles are increasingly used in various uses, primarily in the remediation of environmental contaminants and biomedicine. Additionally, the increasing influx of harmful pollutants into the environment, driven by swift technological progress and population expansion, has become a significant issue. A significant number of chemicals have been recognized as endocrine disruptor chemicals, including various pesticides. Therefore, this subject was chosen due to the significant failure rate associated with traditional remediation methods in addressing persistent pesticides. While nanobio-remediation has verified effective as a hybrid solution, there is a significant absence of comprehensive reviews focusing on the food chain.

Results and Conclusion: Although there are various physical, chemical and biological remediation technologies available, their effectiveness frequently diminishes because of complex processes. Therefore, linking nanoscience and bioremediation strategies can play a promising role in decreasing the endocrine-linked disorders associated with pesticide contamination.

Keywords: Nanotechnology, Bioremediation, Nanobioremediation, Pesticides, Endocrine-disrupting chemicals, Food

1. Introduction

The presence of pesticide residues in the food chains is majorly resulted from their uses in agriculture to enhance crop production and pest control. They can contaminate soil, water and food products. Only a small portion of pesticides target the intended pests, while the rest remain in the environment, leading to bioaccumulation and biomagnification in higher trophic levels, including humans. These pesticide residues can persist for years, transforming into metabolites that may be more toxic than that the original compounds are. The various techniques used to detect pesticide residues have well documented the occurrence of these residues and their metabolites in various environmental compartments. Humans are majorly exposed through consuming contaminated foods and water, with residues accumulating in fatty tissues and being transferred through breast milk and animal products [1, 2].

Exposure to endocrine-disrupting pesticides, such as organochlorines and organophosphates, may interfere with hormone receptors (e.g., estrogen androgen and glucocorticoid), disrupt hormone synthesis and result in changes to the levels of gene expression, leading to reproductive, neurological and metabolic disorders [3, 4]. Because the endocrine system forms during early developmental stages, toxic effects can disrupt tissue growth in this system [4, 5]. Various remediation techniques have been developed for removing pesticides.

Traditional remediation technologies (physical, chemical and biological) include photodegradation, advanced oxidation and microbial degradation [6, 7] . However, these are often affected by high costs, incomplete removal, secondary pollution and limited efficiency for persistent endocrine disrupting pesticides [6, 7]. A combination of nanotechnology (e.g., nanoparticles with large surface area and greater reactivity) and bioremediation (microbial or enzymatic degradation), called nanobioremediation, can overcome the current difficulties [8, 9]. Pesticides can be adsorbed, catalyzed and transformed using nanoparticles, while microbes or enzymes can break these down [10, 11]. Therefore, developing a synergistic approach to improve degradation rate, selectivity and efficiency is a promising outcome of nanobioremediation. Studies show that nanoparticle-mediated bioremediation can achieve high (> 90%) removal rates for a range of pesticides in soil and water; however, results depend on pollutant type, conditions and technology integration [12, 13].

This review methodically battled these challenges in various sections, encompassing the details of pesticide entry routes into the food chain and human exposure pathways. It investigated pesticide-induced endocrine disorders with their molecular mechanisms, assessed the limitations of conventional remediation and introduced the fundamentals of bioremediation and nanotechnology [8, 14]. Furthermore, it presented the principles and mechanisms of nanobioremediation, including nanophytoremediation and microbial nanobioremediation with their advantages and specific uses that achieved over 90% degradation of endocrine-disrupting pesticides. In conclusion, this study described further directions and emphasized the groundbreaking novelty of hybrid nanostrategies aimed at enhancing food safety.

1. Literature Search Strategy

A comprehensive review of the relevant literature was carried out from 2010 to 2025 using PubMed, Scopus, Web of Science and Google Scholar. The search terms were ("nano-bioremediation" OR bioremediation OR nanotechnology) AND pesticides AND ("endocrine disrupting chemicals" OR EDCs) AND food. Inclusion criteria included peer-reviewed articles reporting endocrine-disrupting pesticide degradation via nanobioremediation, focusing on food chain uses, mechanisms and/or human health effects. The exclusion criteria were non-English articles and conference abstracts.

1. Pesticides in the Food Chain, Sources and Transfer to the Body

Pesticides, as the substances used to control pests, enter the body through direct and indirect exposures [15]. Insecticides, herbicides, rodenticides and fungicides are well-known pesticides that are routinely used. Others, including disinfectants, attractants, plant defoliants, swimming pool treatments and plant growth regulators, are less well-known pesticides. Their Direct exposure causes immediate health effects; however, indirect exposure happens when pesticide residues remain on food, leach into water supplies or accumulate in the environment, eventually affecting human health through ingestion or contact. A significant issue associated with pesticide use is bioaccumulation where these chemicals are  stored in the body over time, especially when exposure is frequent and/or prolonged [16]. Pesticides are persistent in the environment and can magnify through the food chain, called biomagnification. After using, these can be carried into aquatic environments by runoff and/or transported to farms, grazing areas and populated areas, endangering other animals by wind [17]. Therefore, animal-based foods and aquatic life contain pesticide residues, which can be harmful to human safety [18]. Pesticides have entered the food chain-chemicals from pesticides enter the groundwater or streams, grass and other vegetation, herbivorous animals and carnivorous and omnivorous animals such as humans [19]. Rainfall is one of the major factors for the dispersion of the pesticide residues, which enter rivers, seas and oceans [20]. Then, these bioaccumulate into the fish by direct or indirect routes through contaminated abiotic media and ingested prey, respectively. Accumulation of pesticides in organisms at lower trophic levels transfers them to other trophic levels consumed by organisms at higher levels, leading to higher concentrations at each step [21].

Pesticides can directly or indirectly be ingested through foods, as well as being used in residential, agricultural and work settings. Pesticides are detected in everyday products, food packaging and agricultural residues. Furthermore, pesticides are used in golf courses, major thoroughfares and other locations, where, the public may be exposed to them. Pesticides are mostly exposed to humans through the food chain, air, water, soil, plants and animals. Although the bloodstream carries pesticides throughout the body. Additionally, they can be expelled by the urine, skin and air that is breathed [22]. Pesticides can enter the human body through four major routes of cutaneous, oral, ocular and respiratory systems. Depending on the route of exposure, dermal, oral, or respiratory, pesticide toxicity can differ (inhalation) [23]. Populations are at risk based on race, sex, age and life cycle. The dose and duration of exposure are factors on outcomes. Developmental duration is further critical, including the perinatal period. Table 1 provides a summary of essential pesticide categories with examples and associated endocrine/metabolic disorders, emphasizing their potential for disruption.

1. Pesticides and Endocrine Disorders

The endocrine system regulates essential human functions including growth, metabolism, reproduction and stress response through precise hormone signaling from glands (thyroid, gonads, adrenals and pituitary). Disruption by pesticides leads to profound health consequences, including reproductive disorders (infertility and early puberty), metabolic diseases (diabetes and obesity), thyroid dysfunction (hypothyroidism), neurological issues (attention deficit hyperactivity disorder and Parkinson's disease) and cancers that affecting global annual costs in healthcare and lost productivity. Epidemiological studies link pesticide exposure to increased risks of diabetes, obesity, thyroid diseases and reproductive impairments (Figure 1). Key pesticides, including organophosphates, organochlorines, carbamates, pyrethroids, triazoles and fluorinated pesticides, have been associated with thyroid dysfunction, reproductive issues, obesity and cancers. These compounds interfere with hormone signaling pathways. Common classes include organochlorines [e.g., dichloro-diphenyl-trichloroethane (DDT) and endosulfan], organophosphates (e.g., chlorpyrifos), carbamates, pyrethroids and herbicides such as atrazine and glyphosate. Fluorinated pesticides such as fipronil have been shown as a broad EDC within organisms. Legal pesticides such as DDT persist in the environment and bioaccumulate. Mancozeb carbamate and chlorothalonil, organochlorine, are the highest concerning fungicides that show deleterious effects on the reproductive systems. Dieldrin, organochlorine and DDT are banned insecticides with disrupting effects on male and female fertilities, which are environmentally persistent. A widely used herbicide is glyphosate organophosphonate, whose safety is under debate [35].

4.1 Mechanisms of Action

Suggested mechanisms of action:

1. i) Sex-hormone linked disorders through receptor binding and activation/antagonism effects: Several pesticides bind to hormone receptors, acting as agonists or antagonists. For example, organochlorines such as methoxychlor bind to estrogen receptor α/β (ERα and β), exerting estrogenic effects. Flutolanil changes the gene expression of estrogen-responsive genes. Atrazine induces aromatase, converting androgens to estrogens and disrupting sex hormone balance. The endosulfan causes estrogen-disrupting effects on the breast cancer cells. Some pesticides block the androgen receptors such as fungicide of vinclozolin [36]. ii) Hormone Synthesis and Metabolism Inhibition: Pesticides can inhibit enzymes involved in hormone production. Thyroid hormone synthesis is blocked by compounds such as amitrole, cyhalothrin, fipronil, ioxynil, maneb and mancozeb, leading to hypothyroidism. Endosulfan disrupts the ecdysteroidal system in invertebrates, affecting molting and development [37]. iii) Transport and Clearance Disruption: pesticides interfere with hormone-binding proteins and metabolic enzymes, altering hormone availability. For example, some pesticides inhibit cytochrome P450 enzymes, prolonging hormone action; although these changes are linked to the host polymorphisms [38, 39]. iv) Epigenetic and Non-genomic Effects: Pesticides may cause epigenetic changes such as DNA methylation and histone modification, affecting gene expression linked to endocrine function. The DNA methylation can inhibit detoxification genes and activate pathways associated with resistance, whereas histone changes dynamically modify chromatin to regulate genes responsive to stress. Non-genomic pathways involve rapid signaling through membrane receptors. Some pesticides interact with nuclear receptors such as peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs), which play roles in metabolic regulation [40]. These mechanisms often lead to oxidative stress, neurotransmitter interference and enzyme dysregulation, exacerbating endocrine imbalances.
2. Conventional approaches to decreasing pesticides from food and their limitations

Conventional methods of pesticide residue removal from food are washing, soaking, peeling, blanching, oven drying, boiling, frying, cooking and canning. These methods have deficiencies such as not demonstrating unequivocal efficacy, loss of minerals such as vitamins and rendering pesticides less reliable as standalone tools for consumers particularly vulnerable groups such as children, pregnant women and the elderly people. Moreover, their performance varies based on produce characteristics, pesticide mechanism and treatment duration, often achieving only partial surface-level removal while failing against systemic pesticides where deeply embedded residues persist [41–43].

In recent years, innovative approaches have gained further attention for modifying pesticide residues in food products. A variety of novel techniques for removing pesticides such as ultrasound, ozone, lye peeling, electrolyzed water, non-thermal plasma (NTP) and cold plasma have been described in Figure 2 [41–44] . These innovative methods include disadvantages such as high costs for setup/maintenance, infeasibility for small-scale operations, resistance to some pesticides and energy-intensive processes. To overcome these restrictions, hybrid strategies have been used [42].

1. Fundamentals of Bioremediation and Nanotechnology in Pesticide Remedia-tion

Bioremediation represents an ecofriendly cost-effective strategy for degrading hazardous pesticide contaminants to safe levels within a variety of environments by harnessing microbial metabolic pathways, offering a superior alternative to physicochemical approaches due to lower capital costs,  sustainability, minimal disruption and decreased secondary pollution [45, 46]. This process operates via bioaugmentation (inoculation of inactive oil-degrading bacteria) and bio-stimulation (growth of native microorganisms), where microorganisms such as bacteria (e.g., Alcaligenes, Pseudomonas and Bacillus spp.), fungi (e.g., Phanerochaete and Trametes effective against lindane, atrazine and DDT) and algae (e.g., Spirulina and Chlorella via bioaccumulation and adsorption) uptake pesticides through passive diffusion and/or active transport, using enzymes such as hydrolases (ester bond hydrolysis), oxidoreductases (electron transfer) and transferases (functional group relocation) to metabolize the chemicals as carbon/energy sources or convert them into less toxic intermediates, ultimately yielding CO₂, inorganic ions and water [45, 47]. Despite being a sustainable method, bioremediation has certain shortcomings [47].

Using nanomaterials to surpass conventional bioremediation limits, nanotechnology as an innovation technology enables effective, affordable environmental-friendly pollutant elimination through improved degradation processes. It is used in a variety of fields, including biodegradation, cosmetics, medicine, food production and agriculture [47]. Nanotechnology, involving materials sized 1–100 nm whose characteristics are profoundly affected by size, shape and geometry, has emerged as a transformative tool for pesticide remediation [45, 46].

Key tools include photocatalysts such as metal dioxide semiconductors that generate electron-hole pairs to oxidize organic pollutants into less harmful compounds under specific wavelengths; nanofilters leveraging high-surface-area materials such as metal oxides, graphene oxide and carbon nanotubes to trap pesticides via physical and chemical interactions often functionalized for selectivity; nanocomposites, combining nanoparticles with polymer or oxide matrices for superior adsorption and catalytic capabilities, reinforced with nanofillers to boost optical, magnetic and mechanical traits while trapping contaminants in tiny pores; and nanobiocomposites, integrating biopolymers such as chitosan or cellulose with nanomaterials such as graphene oxide or iron nanoparticles to immobilize degrading enzymes or microbes, offering sustainable biocompatible cleanup with improved stability and reactivity, as illustrated in sequential mechanisms leading to pollutant-free environments [45].

1. Nanobioremediation: Emerging Hybrid Approach

Nanobioremediation, an innovative technique, is popularizing as it integrates nanoparticles with microorganisms to enhance the efficacy of the decomposition of pesticides. This approach uses nanotechnology to remove environmental pollutants from contaminated sites, using nanoparticles derived from prokaryotes (e.g., Gram-negative bacteria and actinobac-teria) as well as eukaryotes (e.g., fungi, algae and plants) [48]. This strategy is an economic eco-friendly treatment method with minimal side effects [46]. Integrating nanotechnology with bioremediation can boost the overall efficiency, speed and eco-friendliness of the cleanup process; thereby, amplifying its overall advantages [49].

7.1. Principals and Mechanisms of Nanobioremediation

It is reported that using a technology for the remediation of contaminants such as pesticides may not be the most appropriate choice for selection. Therefore, it is essential to combine uses of multiple technologies to overcome the issues linked to the use of a method. Nanotechnology is a field of science that focuses on synthesized particles, which are very small (1–100 nm). Over the past few years, nanotechnology has been used in several areas such as environmental contaminants remediation. The integration of nanotechnology with the bioremediation process is currently known as nanobioremediation.  Nanobioremedi-ation aims to clean the environment by increasing the speed of bioremediation with nanoparticles [12]. Degradation of contaminants using catalysts as nanoparticles is the basic concept of nanobioremediation. Nanoparticle small size allows it to interact further deeply and include a larger surface area per unit mass, which allows it to contact the environment further frequently [50]. Nanobioremediation is a method that uses physicochemical and biological techniques (e.g., living organisms) and currently the subject of extensive research at various contaminated locations. Nanomaterials are used in the nanobioremediation technique first to decrease contaminants to a level that is conducive to biodegradation, which subsequently facilitates the biodegradation of these contaminants. In some cases, the interaction between nanoparticles and biotic components led to biocidal effects and was demonstrated harmful to organisms involved in bioremediation [51]. Therefore, the nanobioremediation process requires an assessment of the interaction between nanoparticles and biotic components. The effectiveness of nanobioremediation can be affected by various parameters such as size, shape and chemical composition of the nanoparticles and physiological characteristics of the organism as well as pH and temperature of the soil and type of the contaminant. Generally, nanobioremediation consists of a two-phase process. Initially, nanoparticles decompose contaminants to a level that is appropriate for bioremediation and then pollutants undergo biodegradation  [50].

Pesticides can effectively be adsorbed, catalyzed and transported to sites of microbial and enzymatic degradations, owing to their high surface area to volume ratio, enhanced reactivity and altered surface structures such as those in nanocomposites or nanoparticles. In contrast, microorganisms and enzymes possess the metabolic ability to detect, recognize, metabolize and absorb pesticide molecules, decomposing them into simpler less harmful compounds. The integration of nanomaterials with microbial consortia or enzymes enhances the rate of pesticide degradation, increases the accessibility of substrates and offers protection against environmental stressors and inhibitory elements. Therefore, physical, chemical and biological processes are combined in bioremediation/nanobioremediation mechanisms to effectively decrease pesticide pollution in the environment through synergistic actions [45].

Two sub-groups of nanobioremediation are reported, including nanophytoremediation of nanoparticles with i) phytoremediation and ii) microbial nanoremediation (Table 2) [52]. Nanophytoremediation is a technique used for the remediation of contaminants via synthesized nanoparticles derived from plants such as nano-zero valent iron (nZVI) and nanohydroxyapatite to boost plant ability to uptake or degrade pesticides such as chlorpyrifos and atrazine. Plants serve as natural detoxifiers for the soil as they can absorb various types of compounds and detoxify them. However, phytoremediation include certain limitations, including slow remediation process and generation of plant waste. Nanophytoremediation has verified effective for a diverse array of soil contaminants, including heavy metals and organic compounds. Use of nanoparticles has facilitated the absorption of these pollutants by plants while simultaneously enhancing their ability to withstand stress [53, 54].

Microbial nanobioremediation involves the use of nanoparticles with soil microbes to enhance biodegradation processes. Microorganisms can take up metal ions and reduce them. In this process, the metal ions are converted into nanoparticles. Combination of microbial enzymes and metals produces advantageous nanoparticles for nanobioremediation [55]. Microbial nanobioremediation consists of a two-phase process, incorporating abiotic and biotic mechanisms. During the initial phase, nanoparticles are introduced into the system, where pollutant particles undergo various processes such as adsorption, absorption, dissolution and photocatalysis [56]. The other phase involves several biotic processes, including biostimulation and biotransformation, which facilitate the removal of these particles from the system [57]. The second phase, known as the biotic phase, is critical for the effective bioremediation of pollutants. Nanobioremediation of pesticides using biological-system immobilized nanoparticles is shown in Figure 3. The process of nanoparticle biosynthesis can occur through one of two primary methods of bottom-up and top-down syntheses. The top-down approach is a traditional method that begins with bulk materials and decreases them to nanoparticle size through slicing or cutting. Techniques such as grinding/milling, chemical etching, electro-explosion and laser ablation are commonly used in this process. In contrast, the bottom-up approach involves assembling nanoparticles atom-by-atom or molecule-by-molecule to attain desired characteristics. This includes sedimentation and reduction techniques such as spinning, template-supported synthesis, laser pyrolysis, biochemical synthesis and biological synthesis [58]. The production of nanoparticles through biological means can be achieved using biosorption or bioreduction method [58].

In microbial-derived nanoparticles, microbes with multiple mechanisms are used for nanoparticle production. Biogenic synthesis represents a green method that promotes an eco-friendly environment. Various biological agents facilitate the synthesis of biogenic nanomaterials, interacting differently with metal solutions via intracellular or extracellular process [59]. From the nanoparticles derived microbes, these can be highlighted: iron oxide from Aspergillus tubingensis [60] and copper from Escherichia spp. [61] .

7.2. Advantages of Nanobioremediation

Nanomaterials show distinctive physical and chemical characteristics, which is why they have  significantly been interested by the scientists and researchers in various fields of environmental sciences, particularly in the field of pesticide bioremediation [62]. Therefore, nanomaterials can be used for bioremediation, resulting in a decreased toxic effect on microorganisms while simultaneously enhancing the microbial activity associated with specific wastes and toxic substances. This approach leads to decreases in time and costs of the process [62]. Nanobioremediation represents a synergistic technique that merges bioremediation with nanotechnology; thereby, offering numerous benefits. By combining the metabolic capabilities of living organisms with the catalytic characteristics of nanomaterials, these substances enhance the rate; at which, pesticides are broken down. The synergistic effects facilitate pesticide degradation and their efficiency can further be increased using nanomaterials as carriers or immobilization matrices for microbial cells and enzymes [13]. Using the distinct characteristics of nanoparticles, including their high surface area-to-volume ratio and reactivity with the catalytic capabilities of microbial enzymes, nanobioremediation presents itself as an extraordinarily efficient precise method for breaking down pesticides into non-toxic byproducts [63].

The capacity for pesticide remediation is enhanced through nanobioremediation, as it specifically targets certain pesticides while leaving non-target substances unaffected; thereby, decreasing the risk of unintended harms to the beneficial organisms within the ecosystem. Additionally, it diminishes the likelihood of further pesticide bioaccumulation by breaking it down into non-toxic chemicals, resulting in a fewer residues that could lead to further contaminations [45, 64]. Use of natural components derived from biological processes decreases energy use; thereby, decreasing environmental effect and resource consumption. In-situ nanobioremediation treats pesticide-contaminated sites on location, eliminating the need of evacuation or material transport, which minimizes ecosystem disruption and decreases costs, compared to ex-situ methods. In summary, these benefits highlight nanobioremediation as a practical cost-effective strategy for effective pesticide cleanup [63, 64].

1. Use of Nanobioremediation in Decreasing Endocrine-disrupting Pesticides (Studies in This Field)

The excessive use of pesticides to enhance crop yields and maintain food production is leading to numerous significant environmental and human health problems such as disorders linked to the endocrine system through affecting the food chain. The effective administration of pesticide use and remediation of the pesticide-contaminated food chain represent one of the most critical challenges. The efficiency of current methods for the biodegradation of pesticides using diverse microbes and enzymes is limited. Therefore, a novel approach is urgently needed to protect food production from significant health threats. The use of nanomaterials has become further popular in recent years due to their distinctive characteristics of decreasing sizes and increasing surface areas. Nanotechnology is addressed as a promising effective technology in various bioremediation processes, using nanomaterials that demonstrate high performance to enhance environmental technologies and offering numerous significant advantages [13]. Approximately 40% of the pesticides are transformed into various products, which can persist in the soil for extended durations, potentially lasting up to ten years [65]. These transformed products can leach into groundwater, leading to contamination [66]. Residues of pesticides that enter the food chain can include detrimental effects on human health by effecting various organs; for example, they can disrupt the endocrine system, including the thyroid gland. Huang et al. (2017) have reported that anticholinesterase pesticide poisoning is associated with increased risk for hypothyroidism issue [67].

Hence, researchers have developed various advanced nanobiomaterials, including a biocatalyst that degrades the widely used pesticide of ethyl-paraoxon. This is achieved by functionalizing a magnetic membrane with phosphorhydro-lase. They have fused the Opd gene from Flavobacterium ATCC 27551 into the mamC protein within the magnetosome membrane. When compared to the purified enzymes, the catalytic characteristics revealed Km and Kcal values of 58 μM and 178 s^-1 for the immobilized Opd and 43 μM and 314 s^-1 for the purified enzyme [68].

An investigation carried out by Salam and Das assessed the efficacy of an integrated bionanohybrid system that used nanoscale zinc oxide with the lindane-degrading yeast Candida VITJzNO4. The nanoparticles were incorporated into yeast cells and successfully transported into the cell cytoplasm without causing harmful effects. The degradation of lindane was assessed, revealing that the nanobiohybrid showed greater efficacy than that the native yeasts did, achieving complete removal within a span of 3 d. This suggested the potential use of nZNO-mediated dichlorination with the innovative bionanohybrid system for the treatment of lindane-polluted wastewater [69]. In a study by Zubaidi et al., biogenic zinc oxide (ZnO) nanoparticles achieved 40% remediation efficiency against the chlorpyrifos pesticide after 7 d of treatment with 82.46% degradation observed by Day 8 of incubation [70]. Chen et al. hybridized a phosphotriesterase (PTE) with copper ions to form a Cu-PTE hybrid nanoflower, showing high catalytic activity and efficiency in biodegrading organophosphorus pesticides [71]. Nozhat et al. investigated the elimination of diazinon and butachlor from aqueous solutions using TiO₂ and ZnO nanophotocatalysts. The results showed that ZnO nanoparticles demonstrated higher efficiency in degrading butachlor, whereas TiO₂ nanoparticles performed better against diazinon [72]. Recently, Li and colleagues developed a novel nanocomposite for multi-pesticide bioremediation by encapsulating hydrolase enzymes in magnetic zeolitic imidazolate frameworks-8 (mZIF-8). The material achieved effective degradation of chlorpyrifos and quizalofop-P-ethyl, demonstrating high efficiency and cost-effectiveness [73].

Computational toxicology is a rapidly increasing field using artificial intelligence (AI) or machine learning as the cost-effective tools to predict the toxicity potential of chemicals and substances [63]. The quantitative structure-activity relationship (QSAR) models, high-throughput screening assays, machine-learning algorithms, deep learning and toxicogenomics are examples of the computational toxicology [74-76]. These novel tools can predict bioaccumulation of contaminants in the environment and biological systems as well as human hazardous effects to create management decisions [77]. The QSAR models are commonly used to establish the relationship between chemical structures and their aquatic toxicity. The random forest, artificial neural networks, support vector machines, Bayesian networks, k-nearest neighbor, probabilistic neural networks, naïve Bayes and decision trees are the most used QSAR models. However, deep learning methods such as convolutional neural networks and recurrent neural networks are used to improve the accuracy of the predictions. Moreover, data mining graphs, networks and graph kernels are used to extract relevant characteristics from chemical structures and improve predictive capabilities [78].

1. Future Perspectives and Research Directions

Nowadays, the rapid release of pollutants such as pesticides can pose a serious risk to human health. Therefore, it is essential to develop efficient methods to remove these chemicals from various environments. To overcome the limitations and disadvantages of conventional methods, novel approaches with high operational potentials for maximum removal of various pollutants are needed. Nanoscale technology is one of the most advanced fields. The economic effect of nanotechnology research projects has been created due to the advances in this field. The integration of nanomaterials and microorganisms can form a synergistic platform for removing pesticides from the food chain. Nanobioremediation provides an efficient, cost-effective eco-friendly approach for pesticide degradation and surpasses conventional methods in several ways. However, further detailed studies are needed to identify pesticide metabolites and degradation products in the food chain. In addition, assessment of the long-term effects and safety implications of nanomaterials should be pursued.

While nanotechnology offers significant advantages for eliminating or decreasing pesticide residues, it carries environmental implications and associated challenges, compared to conventional approaches. For example, nanoparticles in soil alter pH, a key factor affecting nutrient availability, microbial dynamics, overall soil health and plant growth [79]. Moreover, scaling up nanoparticle biosynthesis and enhancing use efficacy requires the development of novel techniques. Researchers are developing innovative approaches to translate nanobioremediation from theoretical concepts to practical uses. Future studies should prioritize enzyme-based nanobioremediation, which enables targeted pesticide degradation while preserving surrounding ecological processes. For the enzyme stability, it is essential to identify inert material nanoparticles that enhance speed of the process and are stable through the remediation without posing any environmental risks. While this presents a significant challenge, the interdisciplinary aspect of nanobioremediation offers extensive opportunities for real-time use [45].

1. Ethical Implications and Biosafety of Nanomaterials

Ethical considerations in nanotechnology are rooted in the principles of beneficence, non-maleficence, justice and autonomy. The nanomaterial development should aim to benefit society and emphasize the need to minimize potential harms. Equal distribution of the benefits and risks associated with nanomaterials and avoidance of disproportionate burdens on vulnerable populations are other characteristics of nanomaterials. Informed consent and public participation in decisions must be highlighted in the use of nanotechnology. Toxicological effects of nanoparticles must be studied on health and environmental concerns with transparency. 

1. Conclusion

Pesticide pollution, particularly from EDCs, poses persistent threats to human health and ecosystems through food chain bioaccumulation. This review uniquely provided nanobioremediation emerging roles in EDC remediation, spotlighting novel hybrid strategies such as microbial nanobioremediation with ZnO nanoparticles and magnetic ZIF-8 encapsulating hydrolases for multi-pesticide breakdown that surpass conventional methods. The novelty of this study included the explanation of synergistic two-phase mechanisms (initial nanoparticle pre-degradation followed by biotic transformation) specifically designed for persistent pesticides such as atrazine and fipronil, incorporating biogenic synthesis (e.g., fungal iron oxide nanoparticles) with uses tailored to the food chain, infrequently addressed in previous studies. These methods present environmentally friendly scalable alternatives that moderate endocrine risks such as hypothyroidism and reproductive disorders. Further initiatives should focus on the genetic engineering of microbial 'nanofactories,' optimization driven by omics and field trials to confirm long-term safety; thereby, facilitating industrial-scale EDC modification.

1. Acknowledgements

The authors gratefully acknowledge the support of the Shahid Beheshti University of Medical Sciences.

1. Ethical code

This study was approved via

IR.SBMU.RETECH.REC.1404.729 ethical code

1. Declaration of competing interest

The authors report no conflict of interest.

1. Authors' Contributions

NAD, MF, SNM, ST, MK, HC and MP conceptualized the idea and prepared the manuscript.

Funding: This study was supported by Shahid Beheshti University of Medical Sciences (project no. 43017890).

Abbreviations List

DEPs: Endocrine-disrupting pesticides; PPARs: peroxisome proliferator-activated receptors; RXRs: retinoid X receptors; NTP: non-thermal plasma; EDCs: endocrine disrupting chemicals; ZnO: zinc oxide; PTE: phosphotriesterase; mZIF-8: magnetic zeolitic imidazolate frameworks-8 

Background and Objective: Microalgae are rich sources of bioactive metabolites and one of the major focuses of the pharmaceutical industry is the use of secondary metabolites from plant sources. Chlorella vulgaris, a microalga with high economic values, includes a high protein content and significant bioactive compounds and polysaccharides. Therefore, this microalga can be used as a dietary supplement and medicinal product. In this study, inhibition of the growth of colon cancer cells was investigated.

Material and Methods: Proteins of Chlorella vulgaris were extracted using enzymatic hydrolysis using proteolytic enzymes of pepsin and Promod (Bacillus subtilis protease). Separation of the peptides was carried out using ultrafiltration techniques. Cytotoxic effects of the extracted peptides were assessed using MTT assay on mouse colon tumor cell lines (CT-26).

Results and Conclusion: Results indicated that the pepsin protein hydrolysates (Pep1, Pep2 and Pep3) at a concentration of 1000 mg.ml^-1 decreased the viability of the CT-26 colon cancer cell line by 24.34%, 36.00% and 40.08%, respectively, while the Promod protein hydrolysates (Pro1, Pro2 and Pro3) decreased the viability by 26.26, 35.91 and 37.13%, respectively. The Pep1 and Pro1 showed the highest cytotoxicity effects (P < 0.05). Findings of this study suggest that the bioactive peptides present in C. vulgaris may include beneficial functional compounds for cancer prevention.

Background and Objective: Chemical treatments in chitin extraction from shrimp shell wastes have affected the environment. Shrimp shell primarily bonds chitin with inorganic salts, lipids, proteins and pigments. Extraction of chitin from shrimp shells involves protein separation processes. Deproteinization process of chitin from dried shrimp (Litopenaeus vannamei) shells with papain enzyme was optimized and nanochitin as a derivative product of chitin was characterized.

Material and Methods: Effect of hydrolysis time, temperature and enzyme concentration were optimized using RSM Box-Behnken method to maximize chitin yields. Nanochitin was prepared using dialysis and ultrasonic methods and characterized for physical characteristics using scanning electron microscope, particle size analysis and Fourier transforms infrared spectroscopy.

Results and Conclusion: Optimum conditions using enzymatic hydrolysis at 6 h, 50 ^oC and 1.25% papain decreased the protein content from 33.66 to 2.31% and produced a high chitin yield (46.03%). Deproteinization using enzymatic hydrolysis method was more efficient than that using fermentation. Data of scanning electron microscope, particle size analysis and Fourier transforms infrared spectroscopy showed that the characteristics of chitin and nanochitin products were similar to those of chemical treatments for chitin products.

Conflict of interest: The authors declare no conflict of interest.

1. Introduction

Litopenaeus vannamei is one of the shrimp species that includes high commercial values and produces abundant shrimp shell wastes. Production of shell wastes from crustaceans was predicted to be 3.14 million metric tons per year worldwide [1]. Hundreds of shellfish wastes are generated from seafood manufacturing and daily Asian consumption [2]. Shell wastes from crustaceans contain a high quantity of chitin, a polysaccharide material that is important in biological functions and is biodegradable and compatible. Chitin and its derivatives are used in various fields such as pharmaceutical, food, textile and waste water-treatment industries [3]. Chitin in the shrimp shells is bonded with majorly inorganic salts, calcium carbonate, proteins, lipids and pigments. Therefore, isolation of chitin from shrimp shells involves protein separation processes and mineral separation [4]. Structure of chitin is arranged with N-acetylated glucosamine and glucosamine units, linked by β(1,4) covalent bonds. Corresponding to this structure, chitin is stable to chemical and biological actions and the linkage of chitin is similar to the linkage of cellulose [5]. Generally, chitin is extracted through demin-eralization using acid treatment and deproteinization using alkali treatment. These treatments affect the environment and finding an alternative process that is more friendly to the environment is still necessary. Deproteinization process for chitin extraction from shrimp shells can be carried out via chemical, enzymatic and microbial processes [6, 2]. The chemical treatment involves mineral acid at high temperatures, resulting in high volumes of polluted waste containing mineral acids in the washing process. These treatments are harmful to the environment due to high concentrations of mineral acids [7]. Deproteinization with enzymes is a zero waste system resulting in high yields of chitin products. Protease hydrolyzes proteins in the matrix efficiently [8]. Commercially purified enzymes such as alcalase, papain, pepsin and trypsin have been used in chitin extraction studies to remove protein from crustacean shells [9].

 Chitin is a biopolymer containing microfibrillar and semicrystalline structures. Based on data of the infrared (IR) spectra and X-ray crystallography (XRD), chitin is naturally in the forms of α-chitin, β-chitin and ɣ-chitin. Characteristics of chitin such as solubility, porosity and surface area restrict its uses. To solve this problem, various derivatives such as chitosan, chitin nanofibers and chitin nanowhiskers are produced [10]. Chitin nanofibers have been prepared via several methods such as ultrasonication, mechanical treatment, gelation and electrospinning [11]. Chitin nanofibers from species such as crabs, prawns and mushrooms have been prepared using mechanical and chemical treatments. The acidic medium was verified in the decrease of chitin nanofibers extracted from crab shells [12]. Under certain extraction conditions, chitin microfibrils are isolated in the form of nanocrystals and nanofibers. Their unique characteristics have been studied and used in food, cosmetics and medical industries [13]. Characteristics of chitin depend on the organisms and chitins may lay in α and β allomorphs shapes. These forms were assessed by the orientation of microfibrils that could be characterized using infrared, nuclear magnetic resonance (NMR) spectroscopy and XRD analysis [14]. Probiotic microorganisms have been studied for the demineralization treatment of crustacean shells. Chitin extraction using microorganisms was carried out simultaneously. Shrimp shells (Penaeus monodon) were fermented with lactic acid bacteria (LAB) and chitin was separated by adding carbohydrates [10]. Based on XRD and NMR data, chitin extraction via enzymatic process is an alternative method to preserve its native structure [1]. Shelma et al. reported the chitin nanofiber preparation via acid hydrolysis of the chitin powder followed by dialysis and ultrasonication [15]. Chitin from P. vannamae byproducts was prepared by associating enzymatic acid-alkaline strategies to achieve further sustainable processes [16]. Moreover, chitosan was produced through papain extract to help deproteinization process. Papain is achieved from the papaya plant with the endopeptidase, dipeptidase and exopeptidase activities. The optimum condition of this process was at 7 h of enzymatic hydrolysis and 25% of papain [8]. The current study was aimed to optimize deproteinization process of chitin from dried shrimp (L. vannamei) shells using low concentration papain (0.75–1.25%) and to achieve nanochitin, which was prepared via dialysis and ultrasonic methods. Furthermore, nanochitin products were characterized through physicochemical characteristics to verify their quality.

1. Materials and Methods

2.1. Materials

              Dried white-shrimp (L. vannamei) shells were provided as byproducts of a shrimp processing industry at Muara Gading City Bekasi, West Java, Indonesia. Commercial papain (CAS no. 2323.627-2) (Xian Arisun ChemParm, Shaanxi, China) was purchased in powder form. All chemicals used included laboratory grades.

2.2. Chitin extraction from the shrimp shells

Chitin from the sample was extracted using method of Hongkulsup et al. [1] with some modification. The extraction was carried out at two steps, including demineralization and deproteinization. In demineralization process, shrimp shells were ground to achieve a size of 100 mesh. Shrimp shell powder was extracted using 1.5 M HCl (ratio 1:10, w/v) at 25 ^oC for 6 h and at 150 rpm. Mixture was filtered using vacuum filter and the residue was mixed with distilled water (DW) to achieve neutral pH. Then, residue was dried at 50 ^oC for 6 h. Dried residue was mixed with 0.75–1.25% w/v papain in a phosphate buffer pH 7 and heated at 40–50 ^oC for 3–6 h. Hydrolysis was stopped at 90 ^oC and set for 20 min. Mixture was filtered and the residue was mixed with DW until neutral pH was achieved. Then, residue was dried at 50 ^oC for 6 h. Total residue was assessed gravimetrically and the soluble proein content in the residue was analyzed using modified Lowry method. Briefly, 1 g of residue was diluted with DW up to 1 ml and filtered using Whatman filter papers. Then, 0.5 ml filtrate was mixed with 5.5 ml of alkaline CuSO₄ reagent and incubated at room temperature (RT) for 10 min. Solution was mixed with 0.5 ml of folin phenol reagent. Then, sample solution was mixed with 3.5 ml of DW and the absorbance was measured at 650 nm. The protein soluble content was assessed by plotting bovine serum albumin (BSA) standard curve [17].

2.3. Optimization of deproteinization of shrimp shells using Box-Behnken method

               Optimum condition of the deproteinization process was predicted using response surface methodology (RSM)- Box-Behnken method. Optimization of deproteinization was carried out using three factors of effects of hydrolysis time, temperature and enzyme concentration (Table 1). Proportions of total residue, chitin and protein concentration were used as the responses data. Fifteen trials were carried out indiscriminately. The center value

was chosen based on the references, which were 1% papain, 45 ^oC and 6 h [18, 19]. Design Expert 13.0 software was used in this study.

2.4. Assessment of chitin

              Chitin content was assessed using adaptation of the Morrow method [20] with some modification. One gram of the sample was mixed with 40 ml of 1 M HCI and mixed at RT for 2 h. Chitin residue was separated using vacuum filter with a porous sintered glass disc and washed several times with water to reach a neutral pH. The residue was washed off and transferred into a beaker containing 40 ml of 5% NaOH and stirred at 100 °C for 2 h. Chitin product was separated using filter paper (Whatman no. 41, USA) and then rinsed with water until a neutral pH was achieved. Content of the chitin (%) was assessed gravimetrically.

2.5. Nanochitin preparation

              The selected chitin sample, which was prepared at optimized conditions, was soaked in 3 M HCl for 90 min at

90  ^oC. Suspension was precipitated by centrifugation at 6000 rpm for 10 min. Nanochitin from the precipitated fraction was prepared for dialysis and ultrasonic treatments using Mincea method [11] with modifications. Suspension of chitin was transferred to a dialysis bag (cellulose membrane with cut-off proteins mol. wt ≥ 12,000) and dialyzed in DW by changing the water every 2 h for three times. Dialysis was carried out until pH 6 was reached. Ultrasonic treatment of the chitin sample was carried out at pulse of 1/1 and amplitude of 60% (750 W, 20 kHz) for 6 h to 0.1% (w/v) of the suspension. Based on the modification of Wu and Meredith method [21], these samples were freeze-dried at -60 ^oC for 10 h.

2.6. Microstructure identification

              Microstructure of the freeze-dried samples was assessed using scanning electron microscope (SEM) (JSM-IT30, Jeol., Akhishima, Tokyo, Japan). These samples were put in a sample holder and layered with a thin layer of gold (±10 nm). Observation was carried out by accelerating voltage at 20 kV based on a previous method.

2.7. Particle size distribution

              Particle size distribution of the samples was analyzed using particle size analyzer (Zetasizer Nano ZS Malvern,UK)  based on Shelma et al. method [15] with modifications. Sample was dispersed in Tween 80 (0,4%; w/v) with a ratio of 1:4.

2.8. Fourier transforms infrared spectroscopy (FTIR)

              Spectra of the samples were analyzed using Fourier transforms infrared spectroscopy (FTIR 1000, Perkin-Elmer, USA) at mild conditions and method of KBr pellet scanning. Based on previous studies, KBr (100 mg) and the sample (1 mg) were mixed entirely until KBr pellet was formed. Then, samples were scanned at spectral ranges of 400, 4200 and 4200 cm-¹.

1. Results and Discussion

3.1. Optimization of deproteinization of the shrimp shells

              The optimum conditions of the enzymatic hydrolysis in the deproteinization process of white shrimp shell powder were predicted using RSM. Fifteen trials were carried out based on the RSM-Box Behnken design. The Box–Behnken design (BBD) is a widely used RSM design that is useful for ascertaining cause-and-effect correlations between factors and responses in experiments. The BBD needs three levels and can be used for factors of 3–21 [22]. Hydrolysis factors and their responses are provided in Table 1. Data showed that the total residue of the products ranged 74.14–80.76%, chitin content ranged 41.52–49.06% and protein content ranged 2.31–6.82%. Analysis of variances (ANOVA) was calculated and p-values of the total residue, soluble protein and chitin content are present in Table 2. Papain concentration (C) and its interaction with temperature (AC) and hydrolysis time (BC) significantly (p < 0.05) affected the total residue of shrimp shell powder. The hydrolysis time (A) and its interaction with the papain concentration (AC) significantly (p < 0.05) affected the chitin content. However, p-values of the soluble protein contents showed that treatments were not significant (p > 0.05). The equation for estimating the optimal condition for all responses (Y1, Y2 and Y3) from the shrimp shells is present in Table 3. Total residue included the yield of the dried product after the deproteinization process with the papain enzyme. Chitin extraction via enzymatic hydrolysis needs removing proteins from the crustacean shells, minimizing the deacetylation and depolymerization processes. This process may be carried out before or after the demineralization step of solid materials for accessibility of the reactants. Efficiency of the enzymatic treatments is inferior to chemical methods ranging 5–10% of the residual protein attached to chitin [9]. Commercial enzymes such as alcalase, econase, pancreatin and other proteases were used in the chitin extraction of shrimp and crustacean shells. The objective of these treatments was to eliminate the protein contained in the waste of shells. Proportion of the chitin ranged 16.5–22% [7]. Combination of the chemical agents and enzymes has been studied to increase yields of the chitin products. Use of sodium sulfite and alcalase was the best treatment for protein recovery. Characteristics of the chitin sample were similar to those of the commercial food-grade products [6].

Three-dimensional (3D) response surfaces of the response; of which, one of the factors is fixed at the central point and the other is varied, are present in Figure 1. The highest predicted chitin content is indicated by the surface confined in the smallest ellipse in two-dimensional (2D) contour plots. This indication was correlated with the interaction between hydrolysis time and papain concentration significantly. This was similar to the results of ANOVA analysis (Table 2). The 2D contour plots showed effects of hydrolysis time (A) in the chitin content prediction (Fig. 1c). However, stagnation was observed in the chitin content with increasing temperature (Fig. 1b). To achieve the optimum condition of the deproteinization process, an optimization process was analyzed using Design Expert 13.0 RSM optimizer software. The three factors (time, temperature and papain concentration) were adjusted in the importance level 3 (+++) and responses (total residue, soluble protein and chitin yield) were adjusted in the importance level 5 (+++++). The optimum condition with desirability of 0.619 was observed for chitin extraction at 6 h, 50 ^oC and 1.25% papain. Further, all the responses of the products were validated through laboratory experiments. The experimental and predicted values are present in Table 4. Data showed that the experimental and predicted values were in the range (95% prediction interval); thus, reliability of the optimized condition was verified. The RSM-Box Behnken design was successfully used to assess effects of hydrolysis time, temperature and papain concentration on deproteinization process to produce higher chitin contents. Chitin from the molted shrimp shells was extracted using a chemical method. The optimum condition of deproteinization was achieved in 3% NaOH at 50 ^oC for 6 h with a residual protein content ≤ 1% [23]. Yulirohyami et al. (2024) reported that chitosan was prepared through processes, including depigmentation, demineralization, deproteiniza-tion and deacetylation. The optimum condition of depro-teinization process was reached at 25% of papain for 7 h of hydrolysis. This study showed that the hydrolysis time of chitin deproteinization affected deacetylation degrees of chitosan [8].

              Proteases have been used for chitin extraction from shrimp byproducts. Residual proteins in shrimp wastes included 1.3 and 2.8% after treatment with chymotrypsin and papain enzymes. Combination of papain with other proteases that was used for deproteinization of shrimp wastes showed that the protein removal rates were low [24]. As an alternative to chemicals and decreasing shrimp wastes, 0.2% alcalase was shown to include activities in decreasing protein contents in shrimp wastes from 49.43 to 4.12% [25]. The enzymatic deproteinization of shrimp processing wastes has limited chitin yields nealy  4.4 to 7.9% of the total weight. This might be due to the residual of short peptides appropriately bonded to the compound of chitin. Use of combination agents with protease significantly decreased the protein fraction. Through this combination, protein fraction significantly decreased, assuming that protease degraded disulfide bonds of the shrimp head waste proteins that facilitated entry of sulfite ions [6]. Use of exoenzymes and proteolytic bacteria in deproteinization of demineralized shells produced liquid protein and solid chitin fractions [2]. Papain is a commercial enzyme, which includes endopeptidase, dipeptidase and exopeptidase activities. Binding affinity and catalytic efficiency of papain are affected by the substrate, temperature and incubation time [8].

3.2 Physicochemical characterization of Chitin

Characteristics of chitin, including degree of deacetylation, morphology and molecular mass, vary depending on the extraction method and origin of chitin [14]. For example, chitin achieved by the chemical extraction showed a tightly packed morphology, while a slightly microfibrillar structure was shown by chitins extracted via enzyme treatment [1]. Use of chitin increased significantly due to the prominent characteristics of its derivatives and nanostructure configuration, which are met for industrial processing. Techniques have been developed to produce chitin derivatives. For example, dialysis and ultrasonic methods  to produce nanochitin from shrimp shells; similar to those of the present study. Surface morphologies of the prepared chitin and nanochitin are present in Figure 2. Accordingly, porous-like honeycomb structure with no nanofibers on the surfaces was observed in the chitins (Figure 2a) and nanochitin achieved via dialysis process (Figure 2b). The only difference between these two products was in the pore size as the pore width of chitin (3 μm ±5) was smaller than that of nanochitin (5–15 μm). This result indicated that during the acid hydrolysis process of nanochitin preparation, the amorphous part of chitin was removed, leaving the crystalline side and leading to increases in pore size. The nanochitin generated from the ultrasonic technique (Figure 2c) showed a nanofibrillar structure with a diameter of nearly 160 nm. This reveals that hydrolysis with a strong acid followed by the ultrasonication treatment further facilitated dissolution process of the amorphous chitin [16]. Ultrasonication is a method to change the natural cithin into chitin nanofibers. Fibrillating chitin at 900–1000 W and 20 kHz in water (pH ±7) created nanofiber widths of 25–120 nm. High frequency of ultrasonication induced startling waves on the chitin surface that promoted their factorization with the axial way [26].

 Chitin is naturally detected in crystalline microfibrils as a structural component, serving as a functional material that is needed by many organisms [14]. The pH of a solution in chitin treatment affects the surface morphology of chitin nanostructures, as a previous study demonstrated that the nanofiber structures of chitin were destroyed to small irregular shapes under high alkaline environments [23]. Furthermore, chitin nanofibrous structure formed due to chitin nanofibers are not soluble and result in versatile porous structures of the products by adjusting the freezing temperature. Freeze-drying technique includes the potential for the assembly of the nanofibrous structure of water-dispersible materials [21].

Particle size distribution is an important characteristic that affects functionality of the chitin products. The chitin sample included two peaks in the spectra, which were dissolved in 0.4% of Tween 80 solution (Fig. 3a). The Z-average of chitin from the shrimp shells was 511.7 nm and the highest intensity was 21.8%. Moreover, nanochitin samples showed three peaks with Z-averages of 101.7 (Fig. 3b) and 345.4 nm (Fig. 3c), respectively. Nanochitin produced via dialysis method showed a Z-average of the particles smaller than that produced by the ultrasonic method. However, the intensity of nanochitin products was still lower than that of untreated chitin samples. Particle size distribution of the chitin nanofibers demonstrated a bimodal curve with majority sizes of 20–300 nm [15]. In this study, additional peaks in nanochitin products were assumed as degraded chitin products. Temperature of the experiments affected number of the peaks in spectra. For higher temperatures, large particles were observed, which might be caused by degradation of the chitin particles. The lower temperature of ionic liquids was further favorable, resulting in a narrow particle range of particle size distribution spectra [27]. Ionic liquids could change the chitin structure, able to modify the particle size [28].

The FTIR spectrum of chitin is present in Figure 4. Chitin sample showed similar spectra with nanochitin, which was prepared via dialysis and ultrasonic methods. Spectra at 3258 and 2924 cm-¹ were recognized as N-H and C-H stretching vibrations. The amide I band was distributed into two peaks of 1652 and 1621 cm-¹. Absorption at 1557cm-¹ was assigned to N-H bend and C-N to 1310 cm-¹. Peaks at 1069 and 1010 cm-¹ were recognized as C-O stretching. Chitin from the shrimp shells has been extracted via two-step extraction using citric acids and deep eutectic solvents (DESs). The study showed that spectra of DESs-extracted chitin included N-H stretching, which was limited by the bonds of intermolecular hydrogen  and the bonding of NH groups. Band of amide I was generated by bonding between the intra-chain hydrogen with the NH groups and the bonding between inter-chain hydrogen with the primary OH [29]. Moreover, two absorption bands at 1018 and 1172 cm-¹ were identified for C-O stretching vibration of chitin from snail shells [30]. Two-step fermentation method was used for demineralization and deproteinization of chitin extraction from shrimp shell powder. The FTIR spectra of the samples showed characteristic peaks corresponding to the amide I (1652 and 1620 cm-¹) and amide II (1554 cm-¹) regions. Peaks at 1375 and 950–1200 cm^-1 were C-H, C-O-C and C-O bonding [31]. Nanochitin was produced using microwave method to observe the difference of α and β structures in amide I. Two bands at 1654 and 1621 cm superscript were assigned as single H-bonded and double H-bonded α structures. A unique single band at 1631 cm^-1 was assigned as β structure of nanochitin [13]. Structure of α chitin is known stable than chitin due to strong hydrogen bonding in inter and intra-sheets [32].

Based on the characteristic data of nanocithin from white shrimp shells (L. vannamei), the product is potential for creating nanochitin-based materials. Morphological and chemical characteristics, including helical and its structure, encourage material developments. Nanochitin is a promising product as the support material at various dimensional aspects [26]. Chitin nanofiber includes potential uses such as in biomedical and biodegradable materials and waste treatments. Limitations of nanochitin production include high-energy demands, high catalyst costs and unstable yields [33]. Thus, further studies should focus on improving yields of chitin and assessing nanochitin uses in biomedical functions.

1. Conclusion

This study assessed effects of the deproteinization process of chitin from dried shrimp shells using enzymatic hydrolysis as an alternative method of fermentation and chemical processes. In this study, deproteinization process was optimized using RSM-Box Behnken design to maximize the chitin yield. Results of this study represented the optimum condition of chitin deproteinization process from dried shrimp shells using papain enzyme, with a chitin content ranging 41.52–49.06%. Interactions between the hydrolysis time and papain concentration included the most significant effect on the chitin content. Removing protein content in chitin extraction through protease

enzymes was verified as a further efficient process, compared to the fermentation process. Use of chitin in industries needs specific characteristics to meet the industries' needs. The SEM analysis showed that acid hydrolysis affected surface morphology of the chitin nanostructure and ultrasonication treatment demonstrated nanofibrillar structure of the chitin. Nanochitin products included similar spectra with chitin samples, which included typical groups of the chitin structure. The present study indicates that alternative method of chitin production may decrease effects of chemical residues on the environment.

1. Acknowledgements

This study was supported technically and financially by the Research Center for Applied Microbiology National Research and Innovation Agency, Republic of Indonesia.

1. Conflict of Interest

The authors report no conflicts of interest.

1. Authors Contributions

Conceptualization and Methodology, SP and DZA; Investigation: DAW, WK and DR; writing—original draft preparation: SP, IP; review and editing: CR, IP.

1. Using Artificial Intelligent chatbot

The authors declare no artificial intelligent chatbot use.

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Background and Objective: The aim of this study was to increase the viable cell and biomass production of a potential probiotic strain, Lactiplantibacillus plantarum DLBSK207, by optimizing the ideal concentrations of key nutrients and fermentation conditions parameters using statistical method. such as (RSM) with Box-Behnken Design (BBD).

Material and Methods: The experiments investigated two key variables for medium composition and fermentation conditions. Based on the OFAT result, six factors were selected for the Plackett-Burman Design to evaluate whether the variables had significant effects to the response. The medium contains carbon (glucose) and nitrogen sources (yeast extract and peptone), while the fermentation conditions include initial pH and temperature. The basal medium, consisting of sodium acetate, MgSO₄ 7H₂O, K₂HPO₄, MnSO₄ H₂O, and Tween 80, was kept constant. Using RSM, the concentrations of glucose, yeast extract, and peptone, as well as the initial pH and temperature, were optimized to maximize viable cell counts and biomass.

Results and Conclusion: The optimum medium concentrations determined by RSM were 33.76 g l^˗1 glucose, 32.59 g l^˗1 yeast extract, and 28.38 g l^˗1peptone at an initial pH of 6.0 and a temperature of 35 °C. Under these optimized conditions, this study achieved a viable cell counts of 9.30 log CFU.ml^˗1 and a dry cell weight of 4.319 g l^˗1, representing a 1.82-fold increase compared to standard MRS broth. The experimental results were in closely matched the predicted values of 9.30 log CFU.ml^˗1 and 4.280 g l^˗1. Scaling up the process in a 10-l bioreactor controlled at pH 6.0 resulted in even higher biomass production, reaching a maximum viable cell counts of 9.88 log CFU.ml^˗1 and a dry cell weight of 5.819 g l^˗1after 20 h of incubation.  

Conflict of interest: The authors declare no conflict of interest.

Abstract

Background and Objective: Although biochemically similar, two laccase enzymes isolated from basidiomycetes (Trametes sp.) showed differences in their affinity to two types of phenolic compounds, interacting stronger with diphenols (catechol and caffeic acid), compared to interactions with benzenetriols (pyrogallol and gallic acid). Catalytic efficiency of Trametes pubescens laccase was detected 4-5 times higher than determined for commercial laccase (Trametes versicolor). In this study, the interactions of the two immobilized enzymes with di and triphenols were examined by various electrochemical techniques.

Material and Methods: Following electrochemical techniques: cyclic voltammetry, chronoamperometry and differential pulse voltammetry were used in this study. Experiments were carried out in varying substrate concentrations. Activity and sensitivity of the two alternative laccase – based biosensors were compared using DPV and chronoamperometry.

Results and Conclusion: Constant potential amperometric measurements indicated that the biosensor produced with Trametes pubescens laccase was much more active than biosensor based on laccase from Trametes versicolor when interacting with caffeic and gallic acids. The phenolic content of three different herbal extracts was evaluated with the developed laccase biosensors and results were found to be similar to those from chromatographic analysis used as a reference method. Therefore, biosensors can be used for rapid testing of phenolic content in real samples.

1. Introduction

Daily intake of antioxidant-rich foods and drinks is considered an important factor of a healthy regimen [1]. Antioxidants, the physiological role of which is to scavenge reactive oxygen species thus preventing oxidative damage of living cells [2] are micro-ingredients of plant cells such as those of fruits, vegetables, cereals and herbs. From natural antioxidants, phenolic acids are especially important not only because their antioxidant activity is within the highest ones, but also because they can rapidly be digested and adsorbed by gastrointestinal tract [3]. Due to their exceptional pharmacological, nutritional and wellbeing effects on humans, a wide spectrum of analytical methods for the assay of phenolic acids has been developed. Chromatographic analytical techniques receive extensive application for the quantification of these antioxidants in a variety of food samples [4-6]; however the required time-consuming sample preparation procedures stimulated the advance of various optical methods such as visible spectroscopy and fluorometry [7], or even paper-based colorimetric sensors [8]. Because of the susceptibility of phenolic acids to participate in redox processes, a range of electroanalytical methods such as voltammetry [9,10], pulse voltammetry [11-15] and amperometric detection [9,10,16,17] have been developed.

Modern electrochemical approaches for the quantify-cation of phenolic acids include phenolic acid detection with electrodes modified with advanced materials such as polymers [4], nitrogen doped carbon [12], carbon nanotubes [18,19] and gold nanostructures [10]. The use of nanostruc-tured materials or composites for electrode modification offers enhanced selectivity of the determination due to either pronounced electrocatalytic effect or greatly enhanced electrode surface area [10].

Assessment of antioxidant capacity through biosensing is a novel trend in contemporary studies [9,11,20-22]. Numerous authors report an improved selectivity of the analysis when using biosensing systems for the assessment of antioxidant content [23]. Copper –containing enzymes laccase or tyrosinase were the primary choice for developing biosensing systems for the analysis of phenols [24-26]; however, whole-cells [27] and DNA-based [28] elements for molecular recognition have also been used for this purpose. Due to the formation of colored products of biocatalyzed transformations of phenolic compounds, most of the highlighted biosensing platforms rely on optical detection principle.

Biosensors with electrochemical detection for phenolic antioxidants analysis, reported in current literature [24], require nanostructured electrode surfaces and sophisticated bioreceptor immobilization protocol. Unlike these, a simple enzyme attachment to an unmodified glassy carbon electrode is discussed, which ensures electrochemical response sensitive enough to guarantee phenolic acid assay at micromolar concentrations. Therefore, the focus of the present study was to develop and optimize an electrochemical method based on two identically prepared laccase biosensors for the determination of di- and trihydroxy aromatic compounds – one bearing commercial laccase from Trametes versicolor, and another laccase – isolated from T. pubescens, which is used seldom in biosensor development. Laccases are widely used in a variety of industrial cycles such as pulp and paper production, wastewater treatment (e.g. from olive-oil mills and textile industry), brewing and food industries, pharmacy or in the construction of fuel cells [25] and biosensors for the quantification of di-substituted aromatic compounds [29].

Laccases are complex enzymes with more than one active site, which embeds three copper clusters (type T1, T2 and T3) differing in both function, and spectroscopic character-istics [25,26]. The reaction mechanism of laccases involves uptake of one electron from the substrate- a hydrogen donor, which is oxidizing to form radical with concomitant 4-electron reduction of molecular oxygen to form two water molecules [26]. The T1 copper site is responsible for binding the aromatic substrate to be oxidized via 1-electron pathway, while the T2-T3 copper cluster binds molecular oxygen and catalyzes its 4-electron reduction to water [26]. Electrons are transferred from T1 site to T2-T3 trinuclear cluster through internal molecular electron transfer. The difference between laccases from T. versicolor and T pubescens is linked to their amino acid sequences. Despite these belong to the family of fungal laccases, variations are seen in their primary structures that may affect their catalytic efficiency, substrate binding specificity, and thermal stability. Thus, amino acids surrounding the less conservative T1 copper-binding site and the overall folding of the enzymes might differ partially, which may result in differences in catalytic efficiency of the laccase manifested as substantially different catalytic constants.

Here reported biosensors function on the following principle: laccases electrocatalytically reduce the dissolved in the working medium oxygen, thus generating reductive current, which is enhanced in the presence of phenol derivatives that act as electron shuttles (mediators) between the enzyme active site and electrode surface. Gallic and caffeic acids are two representatives of phenolic acids, the positive effects of which are commented not only in terms of their antioxidant abilities, but also with respect to their potential anti-inflammatory [30] and anticancer [31] pharmacological activity that was the major reason for their use as laccase substrates in this study. As a demonstration of the applicability of biosensing method, a series of three different types rich in phenolic compounds herbal extracts were analyzed for their phenolic content and results were compared with those from high-performance liquid chromatography (HPLC) analysis.

1. Materials and Methods

2.1. Reagents

Laccase (Е.С. 1.10.3.2, polyphenol oxidoreductase) enzymes from T. versicolor (Fluka, USA) and T. pubescens (a generous gift from Prof. Roland Ludwig, Department of Food Science and Technology, BOKU University of Natural Resources and Life Science, Vienna, Austria) were with homogeneous specific enzyme activities of 21 and 46 U mg^-1, respectively. One unit is the amount of enzyme necessary for the oxidation of 1.0 μmol of ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) per min at pH 4 and 30 °C. Laccases were dissolved in 0.05 M sodium-citrate buffer, pH 4, in such quantity to form enzyme solutions with concentrations of 950 U.ml^-1. The two enzymes were used without further purification.

Catechol, resorcinol, pyrogallol, caffeic acid, gallic acid, ABTS and reagents for the preparation of buffer solutions (sodium citrate, citric acid monohydrate and NaClO₄) were of analytical grade (Acros, Belgium) and used without further purification. All stock solutions of the enzyme substrates were prepared with a concentration of 10 mM.

2.2. Enzyme immobilization

Enzyme electrodes were prepared based on commercial glassy carbon electrodes (2-mm diameter; Metrohm, Utrecht, The Netherlands). Prior to modification, electrodes were polished with 0.05 µm alumina slurry on a polishing cloth (Kulzer, Hanau, Germany), water-rinsed and cleaned using ultrasonication in ultrapure water for 1 min for at least two consecutive times.

Enzyme immobilization was carried out as follows [29]: 2 ml of enzyme solution were drop-cast on the electrode surface. Then, a 4-µl drop of the binder (Nafion 117 diluted with ultrapure water to 0.2%) was applied. Surface was dried at room temperature (RT). The two types of laccases were immobilized on the electrode surfaces identically and the amount of the immobilized enzyme in terms of enzyme units was equal.

After electrochemical measurements, enzyme electrodes were rinsed with ultrapure water and refrigerated at 4°C, when not in use. Regeneration of the working enzyme electrodes could be carried out after the mechanical removal of the enzyme-polymer layer via polishing procedure and following the above steps.

2.3. Electrochemical measurements

All electrochemical experiments were performed in a conventional single compartment three-electrode cell with working volume of 10 ml, connected to a computer-controlled electrochemical workstation Autolab PGSTAT 302 N (Metrohm-Autolab, Utrecht, The Netherlands) controlled by NOVA 2.1.6 software. Either a modified with enzyme glassy carbon electrode, or an enzyme– free electrode (for control experiments) was used as working electrode. A Ag|AgCl, sat. KCl (Metrohm, Utrecht, The Netherlands) was the reference and a platinum foil was the auxiliary electrode. If not otherwise specified, all reported potentials were stated against this reference electrode (Ag|AgCl, sat. KCl electrode) [29]. Cyclic voltammetry was run at scan rates of 5–20 mV.s^-1. Volt-ampere curves (voltammograms) were obtained in both background electrolyte - 10 ml of citrate buffer (pH 4, containing 0.1 M NaClO₄) and in the presence of enzyme substrates with a stock concentration of 10 mM until a 30 mM concentration was achieved in the cell.

Amperometric detection has been carried out at a constant potential of -0.2 V through successive additions of aliquots of 10 mM substrate stock solutions (typically from 20 to 500 ml) to 10 ml of the electrolyte in the cell. Chronoamperometric detection was carried out under constant stirring at 500 rpm.

Differential pulse voltammograms were recorded both in the absence and presence of studied compounds over the potential range from +0.6 to –0.6 V at a scan rate of 10 mV s^-1, pulse duration of 50 ms and an amplitude of 0.025 V, as optimized in the authors’ previous studies [29,34].

Data analysis was implemented with Origin Pro 8.5 software. Non-linear regressions and statistics were carried out using embedded software module for enzyme kinetics.

2.4. Herbal extracts preparation

Herbal extracts were prepared as follows: 20 g of dry herbal mixtures were added to 1 l of pure water and boiled for 20 min at atmospheric pressure (~101 kPa). Then, the herbal extract was set to cool down to RT, filtered through nylon cloth 6.6, packed in 50 ml sealed containers and refrigerated at 4˚C until analyses. Three types of herbal extracts were subjected to electrochemical and HPLC analyses, further referred as PM1, PM3 and PM7. The three types of herbal extracts consisted of following herbs:

PM1: Geranium sanguineous, Arctostaphylos uva-ursi, Betula alba, Polygonum hydropiper, Achillea millefolium;

PM3: Crataegus monogyna, Equisetum arvense, Geranium sanguineum, Urtica dioica; and

PM7: Fragaria vesca, Hypericum perforatum, Calendula arvensis, Frangula alnus, Polygonum hydropiper.

These herbal combinations were selected due to their use as healing teas (pharmaceutical products) with high antioxidant content.

2.5. Chromatographic analysis

The HPLC analysis has been carried out as a referent method for phenolic acid quantification, as follows: The phenolic acid composition of the extracts was assessed using chromatographic system (Shimadzu, Japan), which consist of auto sampler (Nexera X2, SIL- 30AC); CTO-20AC column and SPD-20A UV detector (Shimadzu, Japan). Analysis was carried out using column Metitaranea Sea RP-18e (150 mm × 4.6 mm × 2 μm) (Teknokrom, Spain), mobile phase of 4% acetic acid and 100% AcCN (80:20), flow rate of 0.65 ml.min^-1, λ = 280 nm and temperature of 35 ˚C. Results were analyzed using Lab-Solution Nexera-XR-RF software and the standard phenolic acids (gallic and caffeic acids) [32,33].

The content of the phenolic compounds was calculated against a standard line constructed with its solutions at concentrations ranging from 500 µg.ml^-1 to 2.5 µg.ml^-1 of the corresponding phenolic acid with a correlation coefficient of R² > 0.9991. Total phenolic content was calculated by summing the content of each determined phenolic compound and re-calculating the total phenolic content in equivalents of gallic acid.

1. Results and Discussion

3.1. Studies of two different laccases in di – and trihydroxy aromatic compounds present by cyclic voltammetry (CV)

Electrochemical behavior of the two types of laccase-based bioelectrodes was probed using cyclic voltammetry (CV) in the absence and presence of both laccase substrates – oxygen and phenolic compounds. Voltammetric studies have shown that for the two types of laccase biosensors, reductive wave starts at potentials more negative than -0.25 V in aerated buffer solutions (i.e. in the presence of oxygen), which was not seen in deaerated solutions. It is well known that laccase is a metalloprotein capable of exchanging electrons with underlying electrode surfaces directly [34] without the need for additional electron shuttles (mediators). The efficiency of the electrical communication between the electrode and laccase depends on the enzyme orientation and distance between its active site and electrode surface [34]. Most probably, the negatively charged Nafion membrane electrostatically repulsed the negatively charged laccase active site, this way orienting the enzyme to electrode surface. The latter conformation is favorable for the electron exchange with the underlying electrode, which was manifested by a reductive wave appearing on the CV in the presence of molecular oxygen. Therefore, voltammetric studies verified the ability of immobilized laccase to carry out bioelectrocatalytic O₂ reduction to water molecules, thus proving that enzymes were electrochemically active.

Comparison of the CVs of the enzyme electrode recorded in aerated solutions in the absence and presence of pyrogallol and catechol as substrates (Fig. 1) revealed laccase-catalyzed oxidation of the two phenols to semi-quinones, followed by electrochemical regeneration of the oxidized products. When resorcinol was tested as laccase substrate, the resulting voltammograms did not show interactions between either of the immobilized enzymes, as no reduction of the product of its enzyme-catalyzed oxidation was noticed (Fig. S1, Supplementary information). These phenolic compound-depending differences in the performance of the two types of laccase biosensors were due to the difference between catechol and resorcinol in their spatial structure. The first benzenediol is with two vicinal hydroxy-groups, while resorcinol is its meta-isomer.

Caffeic and gallic acids could be considered as phenolic compounds derived from catechol and pyrogallol, respect-ively. Their structural similarities with dihydroxyl and trihydroxyl aromatic compounds, as well as the fact that they are the usual constituents of polyphenolic complex in various natural products, motivated further interest in probing the voltammetric behavior of the produced biosensors in the presence of the two phenolic acids. On the CVs recorded in the absence of either phenolic compound (Fig. 2, black lines), a reductive wave was recorded with potentials more negative than -0.3 V that resulted from the electrochemical reduction of the dissolved molecular oxygen catalyzed by the immobilized enzyme, undoubtedly verifying that the two laccases were not only electrochemically, but also catalytically active.

The CVs recorded in the presence of either gallic or caffeic acid (Fig. 2, red lines), showed a clearly expressed reductive wave starting much earlier below +0.1 V, which was due to the fact that the two phenolic acids mediated the electrochemical reduction of dissolved oxygen and therefore significantly decreased the overpotential of the oxygen reduction on laccase-bearing electrodes. As seen from the presented plots, the interactions of the two laccases with the two phenolic acids resembled the shapes of the voltammograms recorded in the presence of catechol and pyrogallol. It is noteworthy that the efficiency of the enzyme interaction with the two phenolic acids is different being much higher in the presence of caffeic acid as it could be deduced from the pronounced reductive waves (Fig. 2C, D). Reaction with lower intensity between either of the two laccases with gallic acid was possibly resulting from electrostatic repulsion of its anionic form generated at the operating pH 4.0 [35] and the electrode surface, which also bore negative charges due to the coverage with a Nafion film.

3.2. Differential pulse voltamperometric response of laccase-based bioelectrodes to gallic and caffeic acids using laccases from Trametes versicolor and Trametes pubescens

Differential pulse voltammograms (DPV) of same laccase electrodes are depicted in Figure 3 (Fig. 3A, B; dashed curves). No peaks were identified on the DPVs of laccase-bearing electrodes in background electrolyte and the sharp current decay at potentials more negative than -0.4 V confirmed that oxygen reduction reaction occurred on the bioelectrode’s surface. To investigate further the voltammetric behavior of immobilized laccase in the presence of the two phenolic acids – gallic and caffeic acids, the differential pulse voltammograms were recorded at varied substrates’ concentrations. The addition of gallic acid aliquots to the buffer followed by the record of resulting DPV (Fig. 3A) caused a significant increase in the current with a peak at -0.2 V on the voltammograms, the height of which increased with increasing gallic acid concentration.

No shift of the peak position was reported upon raising its concentration. Similarly, in the presence of caffeic acid (Fig. 3B), a clearly expressed peak at a more positive potential than the one for gallic acid was recorded, the height of which increased proportionally to substrate concentration. The difference in the behavior of the laccase biosensor in the presence of caffeic acid as an enzyme substrate was that the reductive peak occurred at a potential of +0.2 V and the peak potential slightly shifted positively with increasing substrate concentration. It is plausible that the penetration of gallic acid was hampered by the deprotonation of its carboxylic group at the working pH due to the electrostatic repulsion between the negatively charged Nafion membrane and the gallic acid anionic form, resulting in a significant shift of the reduction potential to more negative values than those of caffeic acid. The latter was not deprotonated at the operating pH of the media [36] and hence its molecules penetrated the membrane easier. A similar behavior of the second laccase from T. pubescens was seen under equivalent experimental conditions.

A Michaelis type dependence between the DPV peak height and caffeic acid concentration was observed over a range from 0.01 up to 1 mM (Fig. 4A). Enzyme inhibition by the substrate of T. pubescens laccase became obvious at concentrations exceeding 0.5 mM, while enzyme isolated from T. versicolor seemed unaffected by substrate inhibition even at 1 mM concentration. Under equivalent experimental conditions, the dependence of the DPV peak current on gallic acid concentration (Fig. 4B) was based on Michaelis type kinetics only for T. versicolor laccase, while differential response of the biosensor based on the enzyme from T. pubescens decayed sharply in the presence of trihydroxy aromatic compound (Fig. 4B, red).

Despite the substantial differences between the DP voltammograms and the large peak separations recorded in the presence of either gallic or caffeic acid, DPV studies performed in combinations of the two phenolic acids did not allow discrimination between di- and triphenols. On DPVs recorded in 1:1 mixture of gallic and caffeic acids (Fig. S2, Supplementary information), the peak at -0.2 V appearing in the presence of gallic acid alone merged with the one typical for caffeic acid and the peak potential shifted negatively. With increasing the gallic acid quota up to 10 times, the two peaks broadened and turned into humps, the position and height of which varied irregularly with increasing the concentration of the mixture.

 Information from DPV studies clearly demonstrated that this electrochemical technique could hardly be used for analysis of mixtures of the studied di- and triphenolic compounds, which motivated further investigating an alternative electrochemical approach that could potentially be further useful for the analysis of complex mixtures as amperometric detection.

3.3. Amperometric detection of gallic and caffeic acids with laccase-based biosensors using laccases from Trametes versicolor and Trametes pubescens

In Fig. 5 are depicted the dependencies of the electrode response on the concentration of gallic and caffeic acids assessed with the two types of laccase-based electrodes under the working conditions selected as optimal: working potential of -0.2 V at pH 4.0 [29]. Similarities in the shapes of the curves were substantial. The two types of electrodes showed hyperbolic trends of electrode response as a function of substrate concentration. However, the apparent kinetic constants for the two immobilized laccases, determined from non-linear regression analysis of experimental data, showed significant differences. The apparent Michaelis constants for immobilized T. versicolor laccase (Fig. 5, A) with respect to caffeic and gallic acids have been found to be very similar (Table 1), while the corresponding apparent maximum rates of the enzyme catalyzed reaction differed significantly with the  for the caffeic acid being almost 5 times higher than that for gallic acid.

Unlike these, for the immobilized laccase from T. pubescens (Fig. 5B) with respect to caffeic and gallic acids differed more than 3 times (0.19 mM ±0.03 against 0.06 mM ±0.04, respectively, Table 1) whereas the apparent maximum reaction rates diverged more than those estimated for T. versicolor laccase. These differences in the apparent kinetic constants of the two identically immobilized enzymes suggested that despite biochemical similarities of the two laccases, significant differences were reported in enzyme affinity to dihydroxy and trihydroxy aromatic compounds. Since the reaction rate was equal to the electrode response (current, in amperes [A]), significant differences in  for the two laccases (Table 1) indicated that T. pubescens laccase respectively oxidized caffeic and gallic acids 5 and 4 times as fast as that from T. versicolor. Moreover, these differences in the heterogeneous enzymatic activities were observed regardless of equivalent units of the immobilized laccases.

In all cases except one, significant inhibition effects by the substrate were seen at concentrations exceeding 0.1 mM with inhibition constants calculated by non-linear regression (Table 1). Only T. versicolor laccase showed no inhibition upon addition of gallic acid at concentrations exceeding 0.15 mM.

From the non-linear regression analysis of the kinetic curves, it was found that the electrode response obeyed the Eq.1:

                                                                                                                      Eq.1

Where,  is the apparent maximum rate of the enzyme-catalyzed reaction, A;

 are the apparent Michaelis constant and inhibition constant, respectively, M;

C is the concentration of the respective phenolic acid, M;

I is the electrode response directly proportional to the rate of enzyme-catalyzed reaction, A.

All electrochemical measurements were done in triplicate with a RSD not exceeding 3.5 %.

3.4. Cyclic voltammetry analysis of phenolic acids in herbal extracts using laccases from Trametes versicolor and Trametes pubescens

The available three types of herbal extracts were tested for their redox behavior by performing cyclic voltammetry with the produced two types of laccase-based enzyme electrodes in the operating buffer; to which, equal volumes of each extract were added. Shapes of the resulting CVs (Figs. S3 and S4, Supplementary information) suggested that in the extract of type PM1, triphenols predominated while the other two types of herbal extracts contained a combination of di- and trihydroxy aromatic compounds. It is noticeable that the biosensor based on laccase from T. pubescens was responding ca. three times more intensely than the analogous one based on laccase from T. versicolor.

Quantitative analysis of herbal extracts has been carried out by means of constant potential amperometry (amperometric detection) using standard addition method. Method of standard additions (MSA) is typically carried out by adding small volumes of concentrated solution of the analyte to the sample [37]. The major advantage of the standard addition method is the opportunity to practically eliminate effects of the complex matrix in the real samples. As an external standard, 10 mM solutions of gallic acid were chosen and the MSA was implemented with the two discussed types of laccase biosensors. The one produced based on the laccase from T. pubescens (Fig. 6) guaranteed at least ten times more intense biosensor response to the same amount of PM1 herbal extract, compared to the signal of the T. versicolor laccase-based biosensor (Fig. 7). The latter finding led to greater RSD and significantly overestimated levels of the analyzed compounds; from which, it could be concluded that the second type of biosensor was not appropriate for further analyses. Based on the latter finding, the content of phenolic acids of the other two types of herbal extracts was analyzed using T. pubescens laccase-based biosensor, method of standard addition and gallic acid as an external standard.

The outcomes from biosensor analysis were compared with those from HPLC analyses of the three types of extracts (Table 2). Quantities of the phenolic compounds, estimated from the MSA, were further multiplied by dilution factor and the resulted values were recalculated into gallic acid equivalents per grams of dry weight herbs (DW). Based on HPLC analysis of the phenolic acids, the studied herbal extracts included gallic, caffeic, chlorogenic, p-coumaric and trans-ferulic acids.

As concluded from the present results, the recovery percentage was good only for the type PM1 herbal extract while for the other two extracts biosensor method showed significant deviations from the satisfactory recovery percentages (95-105%). This might be due to the presence of significant quantities of m-benzenediols (e.g. resorcinol and its derivatives) and/or other types of polyphenols, which did not react with laccase.

The two electroanalytical techniques, DPV and constant potential amperometry, were used to find the most convenient approach for the assessment of phenolic content in model solutions to adopt it for the analysis of real samples comprising numerous interferents. However, the hypothesis that the two-model phenolic compounds, benzenediol caffeic acid and benzenetriol gallic acid, could be discriminated in their mixtures based on their DPV peaks that were not well-separated, was not verified due to the strong interferences between them when combined. The constant potential amperometry was shown as a better technique for the quantification of benzenediols and benzenetriols. Large differences in the rates of their electrochemical conversion did not provide means for their separate analysis as well.

1. Conclusion

The present results suggested that only enzymes with high homogeneous activity might be used for electroanalytical purposes. As previously discussed, low activity might lead to overestimated analyte levels (Figure 7). The decreased heterogeneous activity of T. versicolor laccase caused a stronger interference of a complex matrix that could not be eliminated even by the method of standard addition.

Indeed, HPLC analyses could provide not only quantitative, but also qualitative information regarding types of phenolic compounds such as benzenediols and benzenetriols. However, biosensing method provides two important advantages over the chromatographic analysis – it can be carried out rapidly with minimum sample pretreatment and equipment, which is much susceptible to miniaturization allows in-field analysis mostly.

1. Acknowledgements

This study was funded by the European Union-NextGenerationEU through the National Recovery and Resilience Plan of the Republic of Bulgaria; grant no. BG-RRP-2.004-0001-C01, DUEcoS. Authors gratefully acknowledge the generous gift of purified T. pubescens laccase from Prof. Roland Ludwig, BOKU, Vienna, Austria.

1. Conflict of Interest

The authors report no conflict of interest.

1. Authors Contributions

All authors designed and contributed to this study. Conceptualization, N.D. and I.I, methodology, M.S, software, T.C, validation, A.P, M.S. and T.C, formal analysis, M.S. and M.N, investigation, M.S, A.P. and M.N; resources, N.D, data curation, M.S. and M.N, writing—original draft preparation, M.S. and T.C, writing—review and editing, N.D, visualization, M.S, supervision, N.D. and I.I, project administration, I.I, funding acquisition, I.I. All authors have read and agreed to publish the final version of the manuscript.

Abstract

Background and Objective: Hyaluronic acid is extensively used in pharmaceutical, cosmetic, and oral supplementation and nutricosmetic products and has also recently been a candidate for flavor enhancer in the food industry. In this study, Corynebacterium glutamicum ATCC 13032 strain was used for the heterologous production of food-grade hyaluronic acid, and the culture medium and feeding strategy were optimized.

Material and Methods: The propagation of recombinant plasmids was conducted using chemically competent Escherichia coli DH5α, and the extracted plasmids were then transformed into electrocompetent Corynebacterium glutamicum ATCC 13032. A single colony was then transferred into 5 mL of fresh modified CGXII medium supplemented with 50 μg mL⁻¹ kanamycin and incubated for 16-18 h at 30°C. One factor at a time (OFAT) and Taguchi methods were applied to determine the optimal pH and to optimize medium components. Batch, fed-batch, and oxygen-limited fermentation were performed. Hyaluronic acid production was measured using the carbazole and CTAB methods.

Results and Conclusion: The recombinant strain transformed with the two constructs expressing hasA and hasC genes produced the highest amount of hyaluronic acid. The Taguchi L-27 orthogonal array was selected to optimize eleven factors, each at three levels. The results showed that the yield coefficient increased to 71%, and hyaluronic acid production reached 2300 mg L⁻¹. The urea concentration and induction time were considered as significant factors. To enhance hyaluronic acid production, the glucose feeding and oxygen limitation strategies were performed in a bioreactor with a working volume of 4 L. After 48 h, the feeding strategies resulted in a significant increase in the hyaluronic acid yield, reaching roughly 5700 mg L⁻¹. Our results demonstrated that the recombinant Corynebacterium glutamicum containing two main genes of the hyaluronic acid metabolic pathway has a good potential for producing food-grade hyaluronic acid in fed-batch fermentation. 

Conflict of interest: The authors declare no conflict of interest.

1. Introduction

In 1934, a type of polysaccharide was discovered, which was later named "halos." This biopolymer consists of N-acetylglucosamine and D-glucuronic acid disaccharide units that are connected alternately by -1, 3 and -1, 4 glycoside linkages to form a high-molecular-weight polysaccha-ride.[1, 2] The molecular weight of this unbranched poly-saccharide is highly variable, ranging from 10⁴ to 4×10⁷ Da.[3]Hyaluronic acid (HA) has some excellent physio-chemical properties such as nontoxicity, nonimmuno-genicity, biocompatibility, and high water absorption capacity, making it an attractive biomolecule for various industrial and biomedical applications.[4]HA has a variety of applications in ophthalmological surgery, cosmetics, regeneration and reconstruction of soft tissues, arthritis rheumatoid and most recently it is introduced as an a drug delivery agent and also use of this polysaccharide have been noticed in food industry such as flavor enhancer which can reduce use of salt up to 10% without affecting saltiness or uses as anti-aging oral supplementation for improving skin physiology, joint health and even muscle strengthening in special cases. Therefore, recently these products such as food-grade HA have been categorized as “nutricosmetics”.[5, 6] Generally, HA with varying mole-cular weights (M_W) serves different purposes. High-molecular-weight HA (HMW-HA, ≥1×10⁶ Da) is ideal for joint injections and cartilage repair due to its viscoelasticity and lubrication. Low-molecular-weight HA (LMW-HA, 1×10⁴–1×10⁶ Da) is widely used in cosmetics and products such as juices, jellies, and other food items. Therefore, the demand for hyaluronic acid will grow continuously.[7]

According to a report published in 2024, the global market size for HA was 10.04 billion USD in 2023. With a compound annual growth rate (CAGR) of 7.7%, it is estimated that the market size of HA will reach 16.75 billion USD in 2030.[8]

In the past, HA was produced by extraction from animal sources such as rooster combs and umbilical cords. This method has some disadvantages, including degradation of HA by hyaluronidases, expensive purification methods, and the possibility of viral contamination, which could be considered a serious concern.[9] Therefore, alternative methods such as microbial fermentation are suggested for the production of HA. Microbial fermentation is a process in which the HA is secreted into the culture medium by some bacteria; therefore, the purification costs will significantly decrease. The Streptococcus strains were the first microorganisms used for HA production in bioreactors,[10, 11] However, some concerns, such as endotoxin contaminations, limit their application for medical purposes.[12, 13] Recently, some bacteria such as E. coli, Lactococcus lactis, Bacillus subtilis and Strepto-coccus thermophilus were used for the heterologous production of the HA. These bacteria are categorized as Generally Regarded as Safe (GRAS) microorganisms and are free from any pathogenicity factors and endotoxins.[14] HA is produced by  Hyaluronan synthases (HAS) in mammalian and amphibian tissues as well as the cell walls of algae and bacteria.[15, 16] The mammalian genome has three different HAS, and two classes of HAS have been identified in bacteria. HAS, an enzyme typically encoded by the gene named hasA, is a membrane protein that polymerizes HA chains using only Mg²⁺ and two sugar-UDP substrates (UDP-glucuronic acid and UDP-N-acetylglucosamine).[17] The genes involved in HA synthesis are located within the HA operon and consist of hasA, hasB, and hasC genes. Heterologous HA synthesis can be achieved by inserting genes involved in HA production into the genomes of other microorganisms. In some bacteria, hasA is the only essential gene required for HA synthesis.

1. glutamicum is a GRAS and hyaluronidase-negative bacterium, making it an ideal candidate for the commercial production of HA [18-20] the genetic engineering tools have facilitated manipulation and insertion of desired genes into C. glutamicum. In this bacterium, other genes, such as glmU, are also involved in the metabolic pathway responsible for HA synthesis. In one branch of this pathway, the expression of hasB and hasC leads to the production of UDP-glucuronic acid. In another branch, glmU encodes a bifunctional enzyme that catalyzes two sequential reactions: first, an acetyltransferase activity that converts glucosamine-1-phosphate into N-acetylglucosamine-1-phosphate, and second, a uridyltransferase activity that converts N-acetylglucosamine-1-phosphate into UDP-N-acetylglucosamine. Finally, hasA, which encodes hyaluronan synthase, serves as the intersection point of these two branches and polymerizes HA from UDP-N-acetylglucosamine and UDP-glucuronic acid. HA synthesis by C. glutamicum was first reported in 2014 with a yield of 1241 mg L⁻¹ after 120h of fermentation.[19] In a subsequent study, various genetic engineering approaches and strategies were employed, including the use of strong promoters, different plasmid constructs, and the evaluation of various induction times, to enhance high-titer biosynthesis of HA in C. glutamicum. The findings revealed that the strain harboring the artificial ssehasA gene derived from Streptococcus equisimilis with C. glutamicum codon preference and the hasB gene, utilizing the Ptac inducible promoter, produced HA within the range of 1.77 to 2.23 g L⁻¹. Under optimal conditions, the production reached 5.25 g L⁻¹  while under non-pH control HA titer increased significantly and reached an impressive 8.3 g L⁻¹ at 48h fermentation.[21] The study on this strain continued as engineered C. glutamicum achieved high-titer HA production. A genome-scale metabolic model was utilized to identify genetic interventions through flux balance analysis. The focus was enhancing the HA biosynthesis pathway while attenuating the glycolysis pathway and knocking out competing pathways. Various genetic strategies resulted in a surprisingly high HA titer of 28.7 g L⁻¹ in the engineered C. glutamicum.[22] This outcome demonstrates the power of molecular approaches compared to traditional fermentation strategies. Another novel strategy for enhancing HA production in C. glutamicum focuses on cell morphology through a well-designed dual-valve regulation system. This system comprises two modules: a transporter module featuring a strong constitutive promoter (Ptuf) and an arabinose transport protein, and a morphology-tuning module with an arabinose-inducible weak promoter (P_BAD) and a cell-division-related gene. This approach enables fine-tuning of cell morphology, increasing cell length by 1.87-fold and cell membrane size by 2.08-fold, ultimately achieving an HA titer of 16.0 g L⁻¹. This represents a 1.6-fold improvement in yield compared to previous studies on morphology-engineered strains, underscoring the potential of this strategy for enhancing HA production [23].

In this study, four recombinant expression plasmids were introduced into C. glutamicum, and hyaluronic acid (HA) production was analyzed for individual plasmids and their combinations to identify the most effective expression vectors for maximizing HA production. The goal of this research is to enhance HA titer by developing an optimized culture medium and refining the medium and fermentation conditions. To achieve this, the Taguchi design of experiments was employed to optimize a chemically defined medium for HA synthesis in flasks. Ultimately, HA production in the optimized medium was evaluated under controlled conditions, including oxygen limitation and glucose feeding, in a 5-liter fermenter.

1. Materials and Methods

2.1 Microorganisms and plasmids

Escherichia coli DH5α was used for propagation of recombinant plasmids. Corynebacterium glutamicum ATCC 13032 and recombinant plasmids were kindly donated by Josef Altenbuchner from The University of Stuttgart, Germany, and were used for HA production. The recombinant plasmids containing the genes involved in the HA production named pAC (harboring hasA and hasC), pACB (harboring hasA, hasC and hasB in order in operon), pA (harboring just hasA) and pAGC (harboring hasA, glmU and hasC in order).[19]

2.2 Media and Cultivation

1. coli was cultivated in Luria-Bertani (LB) medium containing 10 g L⁻¹ tryptone, 10 g L⁻¹ NaCl, and 5 g L⁻¹ yeast extract supplemented with 50 μg ml^-1 kanamycin. For LB-agar preparation, 15 g L⁻¹ agar was added. Recombinant C. glutamicum was cultivated in a modified CGXII medium for HA production as follows. Solutions were prepared separately and then mixed. The first solution was prepared by dissolving 5 g urea, 5 g (NH₄)₂SO₄, 1 g K₂HPO_4, and 1 g KH₂PO₄ in 800 ml distilled water. To prepare the second solution, 250 mg MgSO₄ and 10 mg CaCl₂ dissolved in 50 ml distilled water. The third solution was prepared by dissolving 10 g of glucose in 50 ml distilled water. All the solutions were autoclaved after preparation, except for the glucose solution, which was sterilized by filtration. After cooling, the solutions were mixed. The trace element solution was prepared and sterilized, and 1 ml was added to the medium as mentioned above. A vitamin solution containing 1mg/ml biotin was prepared, the filter was sterilized, and 0.2 ml was added to the primary medium. The final volume was adjusted to 1 liter with sterile distilled water [19, 24].

2.3 Competent cells preparation and transformation

1. coli competent cells were prepared using a chemical method using CaCl₂ and transformed by a heat shock procedure at 42°C.[25] For electrocompetent cell preparation, a single colony of C. glutamicum was transferred into 5 ml brain-heart infusion (BHI) broth medium and incubated for 18 hours at 30°C and 180 rpm. Then, 2 ml of bacterial suspension was inoculated into a 100 ml electroporation medium containing 37 g l-1 BHI, 0.1% v/v tween 80, 25 g l-1 glycine, and cultivated at 30 ᵒC and 180 rpm to reach OD₆₀₀=0.8. The bacteria were then centrifuged at 3000 ×g for 15 min at 4ᵒC, and the precipitate was washed with 20% v/v glycerol. The centrifugation and glycerol washing steps were repeated three times. Finally, the pellet was resuspended in 1 ml of 15% (v/v) glycerol and stored at -70°C. [26]

Electroporation was carried out using Gene Pulser II (Bio-Rad) as follows: first, 100 ng of supercoiled plasmid DNA was mixed gently with 100 μl electrocompetent cells and transferred into a 0.2 cm (2 mm) cuvette. The electroporator was set to 2.5 kV, 25 μF, and 200 Ω. Immediately after the pulse,1 ml BHI medium was added to the bacteria and incubated for 6 min at 46°C followed by 1 h incubation at 30°C. Finally, the bacteria were plated on solidified medium supplemented with 50 mL^-1 kanamycin.[19, 26]

2.4 Fermentation condition in shake flask

For pre-culture preparation, a single colony was transferred into 5 mL fresh modified CGXII medium supplemented with 50 μg mL^-1 kanamycin and incubated for 16-18 h at 30°C and 180 rpm. One mL of overnight culture was inoculated into 25 mL fresh medium supplemented with 50 μg ml^-1 kanamycin. The induction of the bacteria was carried out using 1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) at different OD₆₀₀ initiating from OD₆₀₀= 0.5. IPTG is used as a molecular mimic of allolactose to induce the expression of our genes, which are under the control of the lac promoter. All flasks were incubated for 24-120 h at 30°C and 180 rpm. The pH, glucose consumption, HA, OD_600, and biomass production were measured at different time intervals.  In some cases, if necessary, 4% glucose was also added after 48 h.

2.5 Hyaluronic acid quantification

HA production was measured using carbazole and CTAB methods.[27, 28] For this purpose, the bacterial suspension was centrifuged and the supernatant was used for HA assay. In the carbazole method, 1 ml of supernatant was mixed with 2 ml of absolute ethanol and incubated at -20°C overnight. The samples were then centrifuged for 30 min at 3500 g for HA precipitation. After that, the pellet was dissolved in 1 ml deionized water, and carbazole assay was performed as follows: 50 μl sample was added to a 96 well plate and 200 μl solution A (25 mM L⁻¹ sodium tetraborate in sulfuric acid) was added to it. The mixture was incubated for 15 minutes in boiling water and 10 minutes on ice. Then 50 μl solution B (0.125% carbazole in absolute ethanol (v/w)) was added to each well and incubated in boiling water for 10 min. Finally, the absorbance was read at OD540 nm by an ELISA reader.[27] The calibration curve was prepared using different HA concentrations (25, 50, 250, 500, 750, and 1000 mg L⁻¹ ) and a linear equation was used for the calculation of the HA amount.

The CTAB method added 50 μl HA samples to a 96-well plate. Then 50 μl acetate buffer (0.2 M sodium acetate, 0.15 M sodium chloride, pH 6) was added to each well and incubated at 37°C for 10 min. After that, 100 μl CTAB solution (25 g L⁻¹ CTAB dissolved in 2% NaOH) was added to the well, and the absorption was read at 600 nm after 10 min by an ELISA reader.[28]

2.6 Glucose assay

Glucose concentration was determined using an enzymatic kit (Pars Azmoon Co). The calibration curve was prepared for seven different concentrations (0.25, 0.5, 1, 2, 2.5, 3.5, and 4.5 g L⁻¹ ) of glucose, and a linear equation was used to calculate the amounts.

2.7 Cell growth measurement

Cell growth was monitored by measuring optical density at 600 nm and cell dry weight.

2.8 Statistical methods

One factor at a time (OFAT) method was applied to find the best pH (6, 7, and 8). Taguchi method was carried out for optimization of medium components (phosphate buffer (K₂HPO₄, KH₂PO₄), Ca (NO₃)₂, (NH₄)₂SO₄, MgSO₄, soy protein acid hydrolysate, biotin, trace elements, glucose, citric acid, urea) and induction time (Table 1). L-27 orthogonal array with eleven factors in three levels was selected to design experiment (Table 2). Experiment design and analysis were performed by Qualitek-4 (version 4.82.0) software.

2.9 Batch, fed-batch, and oxygen-limited fermentation in a 5L fermenter

A loop from the fresh plate was picked up, transferred into the 20 ml modified CGX II medium, and incubated at 30 °C overnight. Then, 400 ml modified CGX II medium was inoculated with 20 ml overnight culture and incubated for 10 h to reach OD_600nm= 4 to 5. The overnight culture was then applied for vessel inoculation. The optimized medium was prepared in 4-L volume and batch, fed-batch, and oxygen-limited was performed at 30 °C, pH controlled at 7, and initial OD_600nm adjusted at 0.4-0.5. Batch fermentation was performed for 24 h with 200-600 rpm agitation rate and 20-40 percent dissolved oxygen. Oxygen-limited fermentation was performed for 24 h with a 200 rpm agitation rate, while dissolved oxygen was controlled between 0-5 percent.[21]

Fed-batch fermentation was performed for 48 h with 200-600 rpm agitation rate and dissolved oxygen was controlled between 20-40 percent. After 18 h, 400 ml feeding solution containing glucose 60%, (NH₄)₂SO₄ 1.5 g L⁻¹, MgSO₄ 5g L⁻¹, yeast extract 20 g L⁻¹, IPTG 1 mM, kanamycin 50 mg L⁻¹ and trace elements 1ml L⁻¹ was prepared and added to the vessel. The feeding rate was adjusted to 15 ml h⁻¹.

1. Results and Discussion

3.1 Effect of different genes involving metabolic pathway of HA

The yield of HA production by four constructed vectors was pAC > pACB> pA > pAGC. The yield of HA produced by the pA construct harboring hasA gene, showed that the hasA was the most important gene in this process (Fig. 1 and 2). The comparison of the HA production by C. glutamicum transformed with the pA construct and the wild-type strain demonstrated that the HA synthase expression was required for high yield of HA production. In fact, the limiting step in HA production by C. glutamicum was HA synthase. Our results concur with previous studies on HA synthesis by gram-negative and positive bacteria.[12, 13, 29, 30]

The Comparison of the HA production by pA and pAC recombinant constructs (containing hasA and hasC genes, respectively) revealed that the hasC gene had only a minor impact on enhancing the HA yield. The presence of the hasC gene resulted in a 12% increase in HA production (Fig. 1 and 2). This result confirmed the previous findings that C. glutamicum contains a pool of precursors for HA synthesis.[31]

Overexpression of glmU gene decreased the HA yield and the cell concentration by 3.5 and 18-fold, respectively (Figures 1 and 2). The glmU gene encodes for a uridyltransferase enzyme that produces UDP-GlcNAc. It seems that increased precursor concentrations inhibit bacterial growth; therefore, a high level of UDP-GlcNAc results in a reduction in the cell concentration and HA synthesis.[29] To compare our results from C. glutamicum with another alternative host for HA production, we can look at findings reported by Zichao Mao and his colleagues, who worked with Escherichia coli, a gram-negative bacterium. They transformed several genes, including uridine diphosphate-glucose dehydrogenase from E. coli K5 and pmHAS from Pasteurella multocida, which are key genes for HA production in E. coli. Their results showed a yield of 0.5 g L⁻¹ in shaking flasks and approximately 2.0–3.7 g L⁻¹ in 1 L fed-batch fermenters[30].

Based on these results, we can conclude that C. glutamicum is a better option than E. coli, as it requires only one gene to produce HA and does not contain endotoxins in the final product. Additionally, the minimum yield of HA production in C. glutamicum is higher than that of E. coli. In another study, Naoki Izawa and his colleagues attempted to produce HA in Streptococcus thermophilus. They reported a maximum titer of 1.2 g L⁻¹ with the co-expression of hasA and hasB, which is similar to the minimum yield in C. glutamicum. Furthermore, Lactococcus lactis was chosen for HA production due to its status as a food-grade bacterium. The researchers claimed that HA produced by L. lactis has significant potential for applications in the food and biomedical industries[29]. However, the maximum titer reported in their study was only 0.65 g L⁻¹, which is again lower than the minimum HA production obtained from C. glutamicum. Another alternative host for HA production is Bacillus subtilis. Bill Widner and his colleagues transformed several genes, including hasA, tauD, and gcaD, reporting yields of over 1 g L⁻¹ [13]. Although C. glutamicum has a higher titer compared to the results from this study, B. subtilis has shown great potential, and many researchers are conducting studies on it. Today, some manufacturers are using B. subtilis as an alternative host for industrial HA production. We believe that C. glutamicum, along with Bacillus subtilis, represents the best options for producing hyaluronic acid. Our results, along with other studies, indicate that C. glutamicum is one of the bacteria with a high titer of HA production, slightly lower than the native HA producer, Streptococcus zooepidemicus.

In industrial production, an important challenge arises when working with plasmids and recombinant strains: plasmid instability. This issue occurs when recombinant strains lose the plasmid or experience a decrease in copy number over generations, leading to unstable expression and a reduction in the titer of products like HA. C. glutamicum is no exception to this problem. One approach to address this challenge is developing and using integrative plasmids, which integrate the gene of interest into the genome of C. glutamicum. This method resolves most stability issues. Additionally, some studies on C. glutamicum have identified a gene named cgR_0322, which is involved in the response to plasmid introduction and plasmid structural instability. Disrupting this gene may enhance plasmid retention and expression of harbored genes, thereby broadening the bacterium’s suitability as an industrial production host.[32]

3.2 Effects of initial pH on HA production

A one-factor-at-a-time method was applied to find the best pH for HA production in the culture medium. As expected, neutral pH was the best pH for HA production.[18] The high pH causes sedimentation and turbidity in the medium due to the reduced solubility of some components, such as phosphate salts and proteins. On the other hand, low pH inhibits bacterial growth and HA production by causing cellular stress and disrupting bacterial membrane integrity. Additionally, the growth of C. glutamicum is typically accompanied by the secretion of acidic byproducts into the medium. Therefore, starting with a pH around neutral is better to avoid extreme decreases in pH during fermentation. For these reasons, a neutral pH was chosen.

3.4. Optimization of the HA production by Taguchi method, data analysis by Qualitek-4 software

The ANOVA table was generated using Qualitek-4 software based on the data obtained. According to the analysis, urea concentration (F-ratio = 31.658) emerged as the most significant factor influencing hyaluronic acid (HA) production. Induction time also demonstrated substantial importance, further validating its role as a critical parameter. The ANOVA table summarizes the detailed effects of each factor on HA production (Table 3).

The results highlight urea (F = 31.66, 43.1% contribution) and induction time (F = 16.60, 22.0% contribution) as the dominant factors, collectively accounting for over 65% of the total variance. Their high F-values, which are well above the significance threshold, underscore their statistical and practical relevance. Soy protein acid hydrolysate (F = 6.67, 8.0%) and vitamin complex (F = 5.66, 6.6%) exhibited moderate influence, likely by supporting microbial growth and precursor synthesis. MgSO₄ (F = 3.45, 3.4%) and citric acid (F = 2.39, 2.0%) showed minor but measurable effects. Remaining factors, such as phosphate buffer, glucose, and trace elements, contributed negligibly (F < 1, % < 1%), indicating minimal impact under the tested conditions. These findings prioritize urea concentration and induction timing as key variables for optimizing HA yield, while deemphasizing non-significant factors. The model explained approximately 85% of the total variability (14.7% unexplained error). The impacts of various factors on the response values were analyzed using signal-to-noise ratio and the plots were drawn for each factor (Fig. 3). The graphs display the maximum and minimum responses for each level, suggesting the best level for each factor as well as optimum condition.

3.4 The effects of phosphate buffer, ammonium sulfate and trace elements

The concentration of phosphate buffer components had no significant impact on HA production (Fig. 3-a). Under all conditions, the pH of the medium dropped within the first 8 hours. Furthermore, increasing the buffer concentration also had no major effects (Table 4). Similarly, ammonium sulfate, as a mineral source of nitrogen, had no significant impact on HA production; however, the second level of ammonium sulfate was more effective for HA production (Fig. 3-b). Our results also demonstrated that trace elements had no major effects on HA production (Fig. 3-f).

3.5 The effect of calcium nitrate

As shown in Fig. 3-c, increasing the amount of calcium nitrate as a source of calcium ion marginally decreased the HA production.

3.6 The effects of soy protein lysate

In this study, soy protein lysate was used as a source of amino acids and organic nitrogen. The maximum amount of HA was produced in the second level, while the HA production in the first and third levels was lower than the second level (Fig 3-d). As can be deduced from Table 4, using soy protein acid hydrolysate in the highest amounts had deleterious impacts on the HA yield. 

3.7 The effect of biotin

Biotin was added to the culture medium in a form of the B complex vitamin batch. As shown in Fig. 3-e, increasing the biotin concentration from the first level to the second level increased the HA production, whereas increasing the biotin concentration from the second level to the third level decreased the production.

3.8 The effects of initial glucose concentration

UDP-GlcNAc and D-glucuronic acid, derived from glucose and produced in the carbon pathways, are the major components for the HA backbone synthesis. In this study, the glucose concentrations were considered high, while the bacterial concentration had no limitation. When the initial glucose concentration in the first level was considered high, the further increase in the glucose concentration slightly affected the HA yield. When the glucose concentration in the first level was considered low, the increase in the glucose concentration had stronger impacts on the HA synthesis (Fig. 3-g). In low glucose condition, a slight increase in the glucose concentration increased the HA production, whereas in high initial glucose concentrations, the bacteria had unrestricted access to the carbon source, so a further increase in glucose concentration had no effects. Also, according to the results of Table 4, it can be concluded that the impact of initial glucose concentration on HA synthesis could be significant at low initial glucose concentrations.

3.9 The effect of citric acid

The effects of citrate on the growth of C. glutamicum were also investigated. The presence of citrate in the culture medium increased the expression levels of certain enzymes involved in TCA cycle and regulation of the central metabolism in C. glutamicum.[33] Fig. 3-h results can also confirm that increasing the citrate concentration boosted precursor synthesis and HA production.

3.10 The effect of MgSO₄

Magnesium sulfate is the fifth important cofactor for Hyaluronan synthase. The maximum amount of HA was achieved at 200 mg L⁻¹ concentration of MgSO₄. Subsequent increase in MgSO₄ concentration decreased the HA production. It seems that MgSO₄ at high quantities blocks enzymes involved in the carbon cycle and HA precursor synthesis, such as phosphoenolpyruvate carboxykinase and pyruvate carboxylase that use Mn²⁺ as cofactor.[34]  Actually, Mg²⁺ has a similar atomic radius to Mn2+, so at high concentrations, it can bind to the enzymes instead of Mn2+ and reduce their activities.[35] The results of Fig. 3-i can be interpreted as the presence of high quantities of MgSO4 in the culture media might disrupt enzymes involved in the carbon cycle and HA precursor synthesis that use Mn²⁺ as a cofactor, such as phosphoenolpyruvate carboxykinase and pyruvate carboxylase.

3.11 The effects of induction time

This experiment demonstrated that adding an inducer at different time points had major effects on HA production. The addition of IPTG as an inducer at the low optical density of bacteria induced HA production and decreased bacterial growth (Fig. 2). The reduction in bacterial growth could be due to the competition between HA production and cell wall synthesis.[16, 36] On the other hand, the induction of bacteria at higher OD had no effects on HA production (Fig. 3-j). Actually, when bacteria are growing, the precursors for cell wall synthesis are present in the cells, and the addition of IPTG at this time results in the production of HA. On the contrary, when the cells are in the late stages of growth or the last log phase, there are no precursors for HA production; therefore, adding IPTG does not affect the HA synthesis. Furthermore, in the late stages of bacterial growth, the metabolic pathways switch to biomass production and adding IPTG cannot change the pathways for HA production.

3.12 The effects of urea

The urea concentration was another variable that was subjected to the optimization process. The results in Table 2 revealed that glucose was consumed entirely in some conditions in which the pH of the culture media was between 6-7. In other runs, the pH of the culture media was acidic between 4-5. It seems glucose depletion leads to urea hydrolysis and NH3+ production, raising pH to neutral level and stimulating HA synthesis (Fig. 3-k). In some runs shown in Table 2, glucose was not fully consumed (runs 9, 10, 18, and 19), urea hydrolysis was suppressed, and pH remained in the acidic range. A possible known mechanism for the effect of urea on HA production is supported by research identifying the urea uptake system (urtABCDE operon) and urease genes (ureABCEFGD), which are regulated by the global nitrogen regulator AmtR under nitrogen-limiting conditions. Studies have shown that under nitrogen limitation, the synthesis of urease subunits increases, making urea utilization critical for nitrogen supply.[37]This confirms the impact of urea on metabolic flux and potentially glucose uptake, aligning with our observations. Urea hydrolysis produces ammonia, which is assimilated into nitrogen metabolism via glutamine synthetase (GlnA).[38] GlnA converts ammonia into glutamine, which then donates an amino group for the formation of glucosamine-6-phosphate. This compound serves as a precursor in the metabolic pathway leading to UDP-N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is one of the two sugar-UDP substrates required for HA polymerization

3.13 Verification test

The optimum levels of the factors should be experimentally confirmed. The qualiteq-4 software predicted the maximum HA concentration in the range of 1299 to 3798 mg L⁻¹ with a 95% confidence interval. The verification test indicated that the HA concentration reached 2300 mg L⁻¹, which was in line with the predicted range. HA production remarkably increased by the Taguchi experimental design, and it can be concluded that the statistical approach was efficient.

3.14 Fermentation conditions

The optimal condition was tested in a 5-L fermenter and showed similar results. The HA production reached 1.8 g L⁻¹ after 18 h (Fig. 4). At pH 7, glucose was consumed entirely, and the production decreased. On the other hand, in the acidic pH of the culture media, the bacteria could not consume glucose, and HA production decreased. The results indicated glucose depletion was a critical limiting step in HA synthesis. Actually, at neutral pH, the HA was produced when glucose was not totally consumed.

In our study, glucose feeding had a major impact on HA synthesis. The feeding was started when the initial glucose was completely consumed. After 48 h, the HA production reached 5.3 g L⁻¹ (Fig. 5). Glucose feeding maintained bacterial growth; therefore, there were no limitations for HA production. When bacteria are in the growth phase, the precursors necessary for HA production are available and promote both HA production and cell division.[36]

In the fermentation process for the production of HA, an important challenge to consider is oxygen transfer limitation. HA, a large polysaccharide and water absorber, increases the viscosity of the medium. This increased viscosity reduces oxygen availability and dissolved oxygen levels, which negatively affects cell growth and HA production. While higher aeration may partially address the issue, research on Streptococcus zooepidemicus indicates that the controlled use of the hyaluronidase enzyme, which converts high molecular weight HA into lower molecular

weight forms, can enhance oxygen transfer and improve dissolved oxygen levels.[39] This approach helps mitigate the oxygen limitation problem and enhances the oxygen transfer rate, leading to an increase in the titer of HA production, although the final HA product will have a lower molecular weight. This approach could likely be effective for C. glutamicum as well, and using it could resolve this issue for industrial production.

1. Conclusion

The results demonstrated that the hasA gene is crucial for recombinant HA production in C. glutamicum. While some bacteria produce HA precursors like N-acetylglucosamine and D-glucuronic acid, they cannot assemble HA without the hasA gene. Introducing hasA enables HA production, though genes like glmU may negatively impact HA yield by disrupting metabolic pathways. Medium optimization using the Taguchi method and ANOVA in this study, identified urea concentration and induction time as significant factors for HA production with an F-ratio more than the F critical value (6.9443). Urea, as an organic nitrogen source, adjusts pH and enhances glucose uptake, leading to higher HA production when glucose is fully consumed. Higher urea concentrations prevent pH decline by producing ammonium, maintaining pH at 6.5–7.5, which is optimal for glucose consumption and HA production. Conversely, low pH reduces glucose uptake and HA yield.

Induction time also significantly influenced HA production. Early induction By IPTG (at low bacterial concentration) directed precursors toward HA synthesis, while late induction (at high OD) inhibited HA production due to competition between bacterial growth and HA synthesis. Since HA production is growth-associated, maintaining bacteria in the log phase increased HA yield.  Glucose feeding and pH adjustment in the fermenter further enhanced HA production by preventing entry into the stationary phase and maintaining pH around 7. In conclusion, C. glutamicum with the hasA gene can produce high HA yields when grown in urea-enriched medium, induced early, and fed glucose to sustain growth and pH. Therefore, it could be a candidate as an alternative host for industrial HA production.

1. Acknowledgements

This research was financially supported by the National Institute of Genetic Engineering and Biotechnology (NIGEB) in Tehran. Iran (project #643). We greatly appreciate it.

1. Conflict of Interest

The authors have no conflicts of interest to declare. All co-authors have seen and agree with the contents of the manuscript, and there is no financial interest to report. We certify that the submission is original work and is not under review at any other publication.

1. Author Contributions

All authors participated in project administration and writing of the first draft of the manuscript, providing critical revision and editing. All authors approved the final version of the manuscript.

Abstract

 Background and Objective: The fermentation of Algerian goat milk, a process for the production of valuable dairy products, relies on the synergistic activity of Streptococcus (S.) thermophilus and Lactobacillus (L.) bulgaricus. However, a significant knowledge gap is seen regarding the precise dynamics of these starter cultures within the unique matrix of Algerian goat milks. Specifically, the intricate relationships between their growth patterns and the resulting physicochemical changes, which regulate the distinct biochemical characteristics of fermented products, are poorly understood. So, this study addressed this problem by studying specific contributions of S. thermophilus and L. bulgaricus to goat milk fermentation.

Material and Methods: Goat milk was fermented by starter cultures of S. thermophilus and L. bulgaricus (8 h). Bacterial growth and physicochemical parameters, including pH, titratable acidity, viscosity and syneresis, were assessed. Mixed-effects models were used for statistical analysis to assess the relationship between physicochemical changes and bacterial growth.

Results and Conclusion: The results showed a strong relationship between L. bulgaricus and the control of acidification, viscosity and syneresis (r = 0.979 for titratable acidity, p < 0.0001). S. thermophilus contributed significantly, particularly to the increases in viscosity (r = 0.773, p < 0.01). The two species significantly decreased the pH, with L. bulgaricus having twice the acidifying effects. By the end of the fermentation process, pH reached 4.12 ±0.20, titratable acidity increased to 84.75 ±2.19 °D and viscosity increased to 6425.00 mPa.s ±638.64. The final bacterial counts of S. thermophilus and L. bulgaricus were 519.00 ±115.29×10⁷ and 65.54±6.89×10⁷ CFU.ml^-1, respect-ively. In addition to providing a robust statistical framework for process control and quality assurance in fermented milk manufacture, this study highlighted the critical role of L. bulgaricus in regulating structural and sensory qualities of fermented goat milks. Results can be used to optimize fermentation processes for goat milk by strategically manipulating the ratio of L. bulgaricus to S. thermophilus. The strong correlation between L. bulgaricus and acidification, viscosity and syneresis (r = 0.979 for titratable acidity, p<0.0001) provides a clear target for controlling key product attributes.

Conflict of interest: The authors declare no conflict of interest.

1. Introduction

In Algeria, where goat farming is an essential part of the rural economy particularly in the dry and semi-arid regions with an estimated 4.2 million goats, goat milk processing into fermented products offers significant advantages. This process meets the growing consumer demand for quality-processed foods while optimizing the use of a readily available but currently underused resource. Because of their synergistic effects on the physicochemical and sensory characteristics of fermented dairy products, Streptococcus  (S.) thermophilus and Lactobacillus (L.) bulgaricus are freq-uently used as starter cultures in cow milk fermentation. In this study, these strains were used in the fermentation of goat milk, which is novel for facilitating investment in the industrialization of this milk and guaranteeing its organo-leptic and nutritional quality. This focus is critical, especial-ly considering that factors such as milking frequency have been shown to significantly affect the nutritional and microbiological quality of cow milk in Algeria [1]. However, the precise dynamics of these cultures in goat milk, particularly the distinct biochemical characteristics of Algerian goat milk, are not fully understood. These bio-chemical features can directly affect fermentation kinetics and finished product characteristics, such as a higher concentration of short and medium-chain fatty acids (SCFA and MCFA, respectively). In addition, studies on conven-tional dairy production systems have demonstrated the importance of endogenous strains in regulating the unique qualities of local products [2]. These microorganisms contribute to gel formation, improved viscosity, acidific-ation and modification of sensory qualities such as flavor and texture [3].

 The use of probiotics to improve metabolic health is a relatively novel indication for probiotic therapy. The poten-tial for probiotics to modulate inflammatory status is particularly interesting, as demonstrated in cell cultures [4]. These two strains show a valuable synergistic relationship, stimulating the other strain growth through the exchange of metabolites in a process known as protocooperation [5]. Specifically, L. bulgaricus expresses extracellular protease to use milk proteins, providing an abundant nitrogen source for itself and S. thermophilus is described to supply L. bulgaricus with certain acids (e.g. formic and folic acids) and carbon dioxide [6]. Additionally, S. thermophilus synth-esizes several amino acids and expresses a cell envelope proteins [5]. The synergistic effect between L. bulgar-icus and S. thermophilus accelerates milk fermentation and enhance microbial growth. Therefore, the primary objective was to investigate the milk fermentation process with particular emphasis on the interactions between S. thermo-philus and L. bulgaricus. A key question is if each bacterium significantly contributes to physicochemical changes in milk, specifically acidification, viscosity and syneresis, critical factors in product innovation. While bacteria such as Streptococcus, Lactobacillus and Bifidobacterium Sp. have extensively been studied in various milk types, research on their interactions in goat milk is limited despite the unique characteristics of goat milks [7].

Fermented dairy products, particularly those derived from goat milks, are significant components of traditional and modern diets, offering valuable nutritional and probiotic benefits. The fermentation process, driven by bacterial cultures such as S. thermophilus and L. bulgaricus, involves a complex interaction of biological and physicochemical factors. Understanding the dynamics of these bacterial populations and their interactions with their surrounding environment is critical for ensuring consistent product quality and safety. Existing research has identified key physicochemical parameters affecting bacterial growth during fermentation, including temperature, pH and substrate availability. However, a significant gap is seen in quantifying these interactions. While qualitative observations are abundant, lack of robust predictive models that can accurately describe the relationships between bacterial growth kinetics and these parameters, particularly within the unique matrix of Algerian goat milks, is addressed. This limitation delays the precise control and optimization of industrial fermentation processes. Furthermore, the specific population kinetics of S. thermophilus and L. bulgaricus in Algerian goat milks, regarding its unique composition affected by local breeds and environmental factors, requires in-depth investigation. The current knowledge may not fully capture dynamics of these bacteria in this specific context. The lack of understanding can lead to variability in product qualities, inconsistent fermentation outcomes and safety concerns.

This study addressed gaps in understanding of lactic acid fermentation in Algerian goat milks. It was beyond general explanations by providing a statistically rigorous analysis that quantified specific roles of S. thermophilus and L. bulgaricus. Unlike previous studies that often treated starter cultures as single entities, the present study highlighted the individual contributions of each bacterium to acidification, viscosity and syneresis. Mixed-effects models were used to establish strong correlations between bacterial growth and physicochemical variations. This study on Algerian goat milk challenged the conventional emphasis on cow milk in fermentation research. This alternative substrate offers potential advantages for consumers pursuing less allergenic dairies. The experimental approach reveals the development of distinct microbial strains and flavors associated with L. bulgaricus. Advanced statistical modelling combined with detailed bioanalysis and industrial uses represents a significant advancement in the current knowledge. Practical recommendations emerging from the results can optimize local transformation processes and promote sustainable goat farming, effectively bridging the gap between fundamental research and real-world effects.

1. Materials and Methods

The raw milk used in the experiment was purchased from local goats in a dairy farm in Tissemsilt, Algeria. This was stored at 4 °C before fermentation. The L. bulgaricus ATCC 11842 and S. thermophilus ATCC 19258 were purchased from a specialized food biotechnology supplier (Fly Chemicals). To avoid contamination, these cultures were used to inoculate the milk at a concentration of 10⁷ CFU.ml^-1 under aseptic conditions in the laboratory. Raw milk was inoculated with L. bulgaricus and S. thermophilus at a concentration of 10⁷ CFU.ml^-1, respectively, and then incubated at 42 °C for 8 h. Samples were collected every 2 h to assess pH, acidity, viscosity and syneresis.

• Physicochemical analyses

The pH was assessed regularly using calibrated pH meter (Hanna HI-2211, Romania) based on AFNOR standards. The acidity was assessed using titration with 0.1 N NaOH and ISO 6091:2010 method [8]. Acidity was expressed in Dornic degrees (°D), where 1 °D included 0.1 g of lactic acid in 100 ml of milk. The viscosity was assessed using viscometer (Fungilab, Alpha series, Spain). Syneresis was assessed using centrifuge (Sigma D-37520, 3-18KS, Germany). The syneresis rate was the percentage of whey separation with the total volume of the product and centrifuged at 1,125 and 3,125 g.

• Microbiological Analyses

The microbiological analyses aimed to quantify the number of lactic acid bacteria (LAB) and pathogens. For the bacterial enumeration, samples were inoculated onto MRS (Man, Rogosa, Sharpe) agar and incubated at 37 °C for 48 h. Results were expressed as colony-forming units (CFU.ml^-1).

• Identification of Pathogenic Bacteria

Potential pathogens, including Escherichia coli and Listeria monocytogenes, were detected using ISO [9] method for E. coli and ISO 11290-1, 2017[10] method for L monocytogenes.

• Data Analysis

Data analysis was carried out using JMP Pro 17 software. Bacterial concentrations were log₁₀-transformed to normal-ize the distributions and stabilize the variance. The initial concentrations of S. thermophilus and L. bulgaricus ranged 0.56–682 × 10⁷ and 0.19–594 × 10⁷ CFU.ml^-1, respectively. Linear mixed-effects models were used to analyze the dependent variables (pH, titratable acidity, viscosity and syneresis), considering fixed effects of the transformed concentrations and the random effects of time measure-ments. Although indications suggested nonlinear relation-ships, a linear model was chosen to avoid overfitting the data. The model validity was verified using several diagnostic procedures such as tests for the normality of residuals and assessment of homoscedasticity. The AIC and BIC criteria were used to assess model fit and R² statistics were used to assess the model explanatory power. These analyses verified the robustness of the model in assessing effects of lactic acid fermentation on the physicochemical characteristics of raw milks.

1. Results and Discussion

During the early fermentation phase (2h), pH decreased to 5.61 ±0.05, whereas the titratable acidity increased significantly to 40.25 °D ±1.58. The first measurable viscosity readings were recorded as 171.63 mPa⋅s ±49.32 (120–270 mPa⋅s). Bacterial populations showed early growth, with S. thermophilus increasing to 6.41 ±8.33 × 10⁷ CFU.ml^-1 and L. bulgaricus reaching 1.73 ±0.05 × 10⁷ CFU.ml^-1 (Table 1). By mid-fermentation (4 h), significant changes were observed in all parameters (Table 1). The pH decreased to 4.77 ±0.10, accompanied by increased titratable acidity (66.75 °D ±1.75). Initial syneresis was observed (4.78% ±0.71) and the viscosity increased substantially to 2562.50 mPa⋅s ±1263.71. The S. thermo-philus showed exponential growth, reaching 386.50 ±166.36 × 10⁷ CFU.ml^-1, whereas L. bulgaricus increased to 5.72 ±0.13 × 10⁷ CFU.ml^-1. Within 6 h (Table 1), fermentation progressed with the pH decreasing to 4.50 ±0.02 and the titratable acidity increasing to 84.75 D°±2.19. Syneresis increased to 7.50% ±0.51 and the viscosity reached 4876.25 mPa⋅s ±708.88. The S. thermo-philus showed a slight decrease to 361.38 ±85.41 × 10⁷ CFU.ml^-1, whereas L. bulgaricus showed continues growth, reaching 51.58 ±6.04 × 10⁷ CFU.ml^-1. At the end of fermen-tation (8 h), the samples reached their lowest pH (4.12±0.20, ranging 3.83–4.32) with maximum syneresis (12.75% ±0.88) and viscosity (6425.00 mPa⋅s ±638.64). The final bacterial counts showed that S. thermophilus and L. bulgaricus increased to 519.00 ±115.29 × 10⁷ and 65.54 ±6.89 × 10⁷ CFU.ml^-1, respectively (Table 1).

3.1. Bacterial Population Dynamics

Critical shifts and variations were observed in the population patterns of L. bulgaricus and S. thermophilus over the fermentation time. Similar to the findings of Moghadam et al. S. thermophiles and L. bulgaricus experienced significant growth during fermentation [11]. This phase of growth is critical for acidification. Data indicated that the L. bulgaricus population reached 1.73 ±0.05 × 10⁷ CFU.ml^-1, while S. thermophilus increased to 6.41 ±8.33 × 10⁷ over the first 2 h [12]. This advanced phase, in which S. thermophiles increased in quantity, boosted deacidification of the liquid media. The study highlighted the need of S. thermophilus to help acidification process while allowing the remaining environment appropriate for L. bulgaricus to thrive. The growth patterns of the two species differed at 4-h point. During this time, the population of S. thermophilus increased significantly, reaching 386.50 ±166.36 × 10⁷ CFU.ml^-1.

In contrast, the population of L. bulgaricus increased, showing a modest increase to 5.72 ±0.13 × 10⁷ CFU.ml^-1. Moreover, S. thermophilus showed a stable population at and after the 6-h point, while the L. bulgaricus count increased, achieving a maximum 65.54 ±6.89 × 10⁷ CFU.ml^-1 at 8-h time point. These findings were similar to those by Meng et al., who detected synergistic interspecific cooperation between the two bacterial species through omics approaches[13]. This verified that S. thermophiles initiated the fermentation process, whereas L. bulgaricus continued to thrive during the later stages of fermentation. Previously, Ahsan et al. (2022) have shown a broader range of food matrices; to which, these bacteria could be adapted. They reported that S. thermophilus and L. bulgaricus could be used in the fermentation of soy milks [14]. As stated, this study illustrated the changes in population sizes at various stages of fermentation and those at the final stage. The authors previously verified the established claims that the two strains included a synergistic relationship; in which, S. thermophilus was the first to initiate acid production for improving conditions of L. bulgaricus growth. Supporting evidence has further been clarified at the molecular level.

Correlation analysis revealed intricate relationships between bacterial growth and the physicochemical parameters during fermentation (Fig. 1). The pH demon-strated significant negative correlations with all assessed variables (p < 0.01), showing particularly strong negative relationships with titratable acidity (r = -0.982) and L. bulgaricus growth (r = -0.958). This underscored the fundamental role of pH in modulating the fermentation environment. The L. bulgaricus showed significantly stronger correlations with physicochemical parameters than those S. thermophiles did, suggesting its predominant effects on the product characteristics. Specifically, L. bulgaricus showed strong positive correlations with titratable acidity (r = 0.979) and viscosity (r = 0.929) while maintaining a strong positive relationship with S. thermophiles growth (r = 0.898). In contrast, S. thermo-philus showed a moderately strong correlation with viscosity (r = 0.773) and a significantly weaker correlation with syneresis (r = 0.405), indicating its secondary role in texture development.

The textural characteristics demonstrated distinct correlation patterns, with viscosity showing stronger associations with L. bulgaricus (r = 0.929) than S. thermophilus (r = 0.773). Syneresis demonstrated a similar issue, correlating stronger with L. bulgaricus (r = 0.798) than S. thermophilus (r = 0.405). These relationships suggested that L. bulgaricus played a further important role in texture development, possibly through enhanced exopolysaccharide production and/or proteolytic activity. The strong correlation between bacterial growth and physicochemical parameters was further validated by non-parametric analyses (Kendall tau-B and Spearman rho), verifying the robustness of these relationships regardless of the statistical approach used. These findings correlated with those by Nadirova and Sinyavskiy (2023), who reported that L. bulgaricus could grow despite decreased pH levels, owing to its intrinsic adaptability to acidic conditions[15].

Data collected at 6 and 8-h intervals further illustrated the growth patterns of these bacteria. At 6-h point, population of S. thermophiles slightly stabilized, measuring 361.38 ±85.41×10⁷ CFU.ml^-1. In contrast, L. bulgaricus demonstrated a further vigorous growth rate, reaching a population of 51.58 ±6.04 × 10⁷ CFU.ml^-1. At 8-h point, S. thermophiles population showed slight stability, with an increased count of 519.00 ±115.29 × 10⁷ CFU.ml^-1, while L. bulgaricus demonstrated strong growth, achieving a population of 65.54 ±6.89 × 10⁷ CFU.ml^-1. The present results have been verified in other products such as yoghurt, where S. thermophilus and L. delbrueckii subsp. bulgaricus multiplied significantly during fermentation [16].

These findings addressed those of Sieuwerts et al. (2010), whose transcriptome analysis revealed that mixed-culture growth involved upregulation of biosynthesis pathways for nucleotides and amino acids that were vital for the growth of the two bacteria [17]. Furthermore, Nadirova and Sinyavskiy (2023) highlighted the complementary roles of these two bacterial species in the fermentation process. The S. thermophilus was essential in initiating acidification, whereas L. bulgaricus proliferated in acidic environments created by the other strain. This complex interaction not only enhanced fermentation efficiency but also contributed to development of the desired flavor and texture profiles in fermented dairy products. In contrast to the findings of Picon et al. (2016), the present findings demonstrated that acidification occurred at a slower further gradual rate [18]. This was primarily attributed to the diverse range of naturally occurring LAB strains that affected lactic acid production efficiency. Conversely, optimized interactions between S. thermophilus and L. bulgaricus strains led to further rapid acidification, increased lactic acid production and consistent acidification profile.

3.2. PH

The linear mixed-effects model analysis of bacterial fermentation dynamics revealed a significant effect of bacterial growth on pH regulation during fermentation (Fig. 2). This model, showing excellent fit characteristics (AICc = -6.299, BIC = -0.686), verified a baseline pH of 6.087 ±0.058 (SE, p < 0.0001) in goat milks. A significant rapid acidification of milk with pH decreasing from 6.62 ±0.08 to 4.12 ±0.20 over 8 h. This pH decrease, critical for flavor development and microbial stability, was primarily driven by the action of the two bacterial species with L. bulgaricus showing a stronger effect (-0.589 ±0.083 pH units per log₁₀ CFU.ml^-1, p< 0.0001), compared to S. thermophiles (-0.304 ±0.060 pH units per log₁₀ CFU.ml^-1, p<0.0001). This approximately two-fold difference in acidification capacity was statistically supported by highly significant F-ratios for S. thermophiles (F₁,₃₇ = 25.66, p<0.0001) and L. bulgaricus (F₁,₃₇ = 49.74, p < 0.0001), demonstrating their differential contributions to fermentation. The model random effects structure indicated negligible temporal variances, sugges-ting consistent acidification patterns over time. The residual variance was small (0.043 ±0.010), supporting the model precision and reliability. A significant negative correlation (r = -0.898) between the effects of S. thermophiles and L. bulgaricus underscored the complex interactive dynamics between these species rather than a simple additive effect on pH decrease. These findings were similar to those of previous studies, which identified L. bulgaricus as a primary driver of acidification in milk fermentation systems [7,19,20]. Similarly, another study showed that S. thermo-philus promoted rapid bacterial growth and enhances fermentation efficiency, creating a favourable environment for L. bulgaricus. This synergistic relationship led to improved metabolite profiles, with S. thermophilus facilit-ating the production of flavor compounds during milk fermentation at optimal temperatures [19]. A recent study using omics analyses has revealed the molecular mechanisms driving bacterial synergy, driving decreases in pH [12]. This intricate relationship between bacterial inter-actions and pH dynamics was further supported by Wu et al., whose study linked bacterial population ratios and fermentation times to the sensory profile, including acidity [19]. This revealed the broader implications of these pH changes, as they resulted from bacterial metabolic activity and affected the final quality and sensory characteristics of the product. Thus, bacterial populations strongly affected pH dynamics, particularly L. bulgaricus, which showed a greater acidifying capacity, verifying its key roles in the acidification process.

3.3. Titratable Acidity

A linear mixed-effects model analyzed the relationship between bacterial populations and titratable acidity (Fig. 3). The model demonstrated satisfactory fit indices (-2 residual log likelihood = 159.71, AICc = 172.84, BIC = 177.22). Fixed-effects analysis demonstrated highly significant positive relationships between the titratable acidity and logarithmic populations of the bacterial species. The L. bulgaricus showed a stronger effect (β =21.44 ±1.47 D.log₁₀ CFU^-1, t (29) = 14.55, p<0.0001), compared to S. thermophiles (β = 7.43 ±0.97 °D.log₁₀ CFU^-1, t (29) = 7.67, p<0.0001). This approximately three-fold difference in effect size indicated that L. bulgaricus was the primary driver of acid production in the fermentation system. The model intercept of 30.20 ±0.92 °D (t (29) = 32.85, p<0.0001) represented the baseline acidity when controlling for bacterial populations.

The significance of these relationships was further supported by fixed-effects tests, which showed strong evidence of the effect ofS. thermophilus[F(1,29)=58.83, p<0.0001] and L. bulgaricus [F (1, 29) = 211.58, p<0.0001]. The substantially larger F-statistic for L. bulgaricus verified its dominant role in acid production, with direct implications for starter culture formulations in fermented dairy products. The random effects structure analysis revealed that the time component was confounded with residual variance, resulting in a residual variance estimate of 10.60 ±2.78 °D (95% CI: 6.72–19.16 °D). This confounding factor suggested that bacterial population dynamics, rather than time-dependent factors, were the primary determinants of acid development in this system, which is an important consideration for process control in industrial settings.

Model diagnostics supported the validity of these statistical assumptions. The actual by predicted plots demonstrated a strong linear relationship between the observed and predicted values across the full range of measurements (10–90 °D). Residual analysis revealed a generally symmetric distribution near zero (-6 to +6 °D), with the residual quantile plot indicating approximate normality. The model strong predictive capability suggested that it could be a reliable tool for controlling acidification processes in fermented dairy production.

While goat milk (19.05 °D) and cow milk (17 °D) showed distinct initial acidity levels, the titratable acidity significantly varied during the fermentation process [20]. A significant increase was observed, rising from 15.88 °D ±0.64 to 84.75 °D ±2.19 within 8 h. This increase strongly correlated with bacterial growth, particularly that of L. bulgaricus, whose metabolic activity significantly contributed to milk acidification through lactic acid production. These verified the findings that the proliferation of L. bulgaricus directly affects titratable acidity and that lactic acid production is essential for controlling final characteristics of the fermented product[21,22]. Andrew et al. emphasized that titratable acidity is a relevant indicator of the progression of fermentation process in L. bulgaricus based products. The results of Abbasalizadeh et al. indicated that the maximum lactic acid production in the Media12 media reached 35.01 g.l^-1) [23]. This result verified the current results as the titratable acidity reached 84.75 °D ±2.19 after 6 h of fermentation. These results demonstrated the importance of acidification in ferment-ation processes.

Further supporting the present results, Sonnier et al. showed the synergistic actions of S. thermophilus and L. bulgaricus that promoted rapid acidification of the media [24]. More specifically, Qiu et al. provided mechanistic insights into the current findings by revealing the metabolic pathways and metabolites involved in acid production, particularly the role of lactic acid production by L. bulgaricus. According to Wu et al., the link between bacterial ratios and acidity strengthened the connection to product sensory attributes and verified that specific bacterial ratios affected acidity and sensory profile. The present study highlighted the strong acidifying capacity of L. bulgaricus, which was twice that of S. thermophilus. Although the study on L. plantarum SU-KC1a did not directly assess acid production in fermentation, it demonstrated robust tolerance to pH variations [21]. This suggested that L. plantarum SU-KC1a might contribute to acidification during fermentation, although not as strongly as L. bulgaricus. These findings underscored the role of L. bulgaricus as the primary contributor to acidity and the importance of understanding dynamics of bacterial populations for process control, while reinforcing these conclusions by contextualizing them within the current understanding of these mechanisms and their effects on organoleptic characteristics of the final product.

3.4. Viscosity

Analysis of the fixed effects revealed a significant difference in the effect of the two bacterial species on viscosity development (Fig.4), with L. bulgaricus demonstrating dominant effects and coefficient of 3106.28 ±397.51 mPa⋅s per log unit increase in cell density (p < 0.0001), approximately 8.84 times greater than the effect observed for S. thermophilus (351.37 ±281.73 mPa⋅s per log unit, p = 0.2223). This difference demonstrated that L. bulgaricus largely explained viscosity of the fermented media. The random effects structure revealed negligible temporal variance [t (h) = 0], indicating that viscosity changes were not significantly affected by the duration of the experiment. The residual variance was significant (881,027.22), indicating that variations in viscosity were predominantly explained by the bacterial concentrations rather than temporal patterns. Model diagnostics supported these findings; the actual-by-predicted plot demonstrated a generally linear relationship with increased variability at higher viscosity levels (0–7000 mPa⋅s). The model strong statistical characteristics (F-ratio for L. bulgaricus= 61.06, p<0.0001) provided robust evidence for the differential effects of these bacterial species on viscosity development. However, several considerations guaranteed further attentions. First, the negative intercept (-731.81 mPa⋅s) represented a theoretical value outside biologically relevant conditions and should be interpreted cautiously. Further-more, although the model demonstrated heteroscedasticity (unequal variance of the residuals) at higher viscosity values, this did not invalidate the primary findings regarding the relative effect of each species. Of the changes in viscosity, the most significant was observed after 8 h when the viscosity reached 6425.00 mPa.s ±638.64, which was more than two times higher than the initial value. These results were similar to those of Qiu et al., who identified L. bulgaricus with its primary activity of exopolysaccharides (EPS) production as the major reason for the improvement in viscosity. In other words, data were similar to those that addressed L. bulgaricus as an essential component in boosting the product viscosity, predominantly through the production of EPS. A highly positive association (r = 0.929) was detected between the abundance of L. bulgaricus and the magnitude of viscosity, which provided further evidence for this microbial leading role in improving the rheological characteristics of the final product. The importance of this finding is that viscosity is a critical characteristic that dictated the degree of product acceptance; thus, texture and mouthfeel were directly affected by viscosity. The present results were similar to those of Nadirova and Sinyavskiy, who underlined that the increase in viscosity was necessary not only for improving the texture and stability of fermented products during storage but also for providing favorable conditions for the growth of relevant bacteria. This is a further step and a part of bacteriophage resistance of these isolates. Moreover, a project by Afzal et al. on the specific structure of the EPS produced by L. bulgaricus and how researchers verified that the produced EPS could make a difference in the viscosity levels of the final product of fermented milk were significantly verified [25]. Through the interactions of L. bulgaricus EPS with indigenous strains or natural ingredients, viscosity and texture could be optimized; thereby, extending the shelf life of quality products.

syneresis in fermented dairy products (Fig. 5). The L. bulgaricus demonstrated a strong positive association with syneresis (β = 5.26 ±0.92, p < 0.0001), indicating that higher concentrations of this strain significantly increased water expulsion from the gel matrix. While S. thermophilus showed a positive development (β = 3.67 ±2.49, p = 0.1561), its larger standard error (SE) and non-significant p-value suggested significant variability in its effects on syneresis. The intercept of the model (β = -8.68 ±6.23, p = 0.1786) was not significantly different from zero, suggesting minimal baseline syneresis in the absence of bacterial activity. Model diagnostics supported the validity of the present findings, with residual analyses showing appropriate distribution patterns and no substantial violations of the model assumptions. The significantly positive coefficient for L. bulgaricus was strong across multiple diagnostic assessments, reinforcing its critical role in controlling syneresis. However, significant residual variances and wide confidence intervals for some parameters suggested that additional factors such as protein concentration, pH dynamics and temperature fluctuations might contribute to syneresis variation in ways that were not captured by this model.

The fermentation phase was highlighted by increases in syneresis of 4.78% ±0.71 and a significant separation of lactoserum from the gels with syneresis increasing to as high as 12.75% ±0.88. The correlation coefficient between L. bulgaricus and syneresis (r = 0.798) was confusing with that presented by Nadirova, who argued that L. bulgaricus might improve gel syneresis and increase gel strength. This is an important question. Although (EPS) are known to originate from L. bulgaricus and alter gel structures favorably. Two studies showed that specific EPS structures antagonized syneresis, highlighting that L. bulgaricus produced EPS that improved water retention [26]. One interpretation of the present results is that L. bulgaricus under specific fermentation conditions changes the balance of syneresis positively. Certain conditions of the present experiment and a certain strain of L. bulgaricus used in this experiment may need further investigations. However, it is possible that high concentrations of L. bulgaricus and its byproducts facili-tated whey removal and gel retention was compromised. Qiu et al. reported that while the EPS synthesize by L. bulgaricus helped lessen syneresis, the effect was incomplete [12].

The large residual variances and broad ranges of confidence intervals in the present model reveal that various other components such as protein concentrations, pH shifts and temperature changes, which were not seen in the present model, might include effects on syneresis. Overall, the study provided evidence of a further complex relationship between L. bulgaricus, EPS formation and syneresis and results indicated needs of further integrated understanding to enhance the fermentation process. Additional studies are needed to investigate which specific strains lead to decreased syneresis under what conditions, modelling and Optimization.

During fermentation, the population dynamics of S. thermophilus and L. bulgaricus showed complex significant fluctuations (Table 2). At early stages, the two bacterial species showed exponential growth, aligning with the findings of[15], which highlighted the significant growth of S. thermophilus and L. bulgaricus during fermentation. This rapid growth is critical for acidification. The present data indicated that within the first 2 h of fermentation, the population of S. thermophilus increased to 6.41 ±8.33 × 10⁷ CFU.ml^-1, while L. bulgaricus reached 1.73 ±0.05 × 10⁷ CFU.ml^-1. This initial phase, highlighted by the swift growth of S. thermophilus, was vital for the starting of the acidification of liquid media, similar to other findings by Qiu et al. Their study emphasized the significant role of S. thermophilus in initiating acidification; thereby, creating a further favorable environment for the growth of L. bulgaricus. After 4 h, growth rates of the two species were shifted. At this point, population of S. thermophilus significantly increased to 386.50 ±166.36 × 10⁷ CFU.ml^-1, while L. bulgaricus showed a further modest increase to 5.72 ±0.13 × 10⁷ CFU.ml^-1. As fermentation continued, S. thermophilus included a relatively stable population after 6 h, whereas L. bulgaricus grew, reaching a final count of 65.54 ±6.89 × 10⁷ CFU.ml^-1 after 8 h. These findings were similar with those of Hansen et al., who verified synergistic growth of the two bacterial species through omics analyses [12]. This supported the idea that S. thermophilus initiated fermentation, whereas L. bulgaricus increased as fermentation advanced. Additionally, adaptability of these bacteria to various matrices was demonstrated by Nadeem et al., who showed that S. thermophilus and L. bulgaricus could effectively ferment plant-based milk alternatives. The current study highlighted changes in population sizes during fermentation and illustrated how the two bacteria acted together, with S. thermophilus starting the acidification process and creating conditions that allowing L. bulgaricus to increase, similar to previous studies.

1. Conclusion

This study provides a broader understanding of fermentation in goat milks. It underscores the complem-entarity of S. thermophilus and L. bulgaricus. Generally, S. thermophilus contributes to the rapid acidification process, leading to the proliferation of L. bulgaricus, which is a significant contributor to the texture, stability and organ-oleptic characteristics of the final product. The synergistic interaction between these two species results in desirable texture and decreased syneresis. This improves quality of the fermented dairy products by enhancing the unique characteristics of goat milks. These findings are invaluable for optimizing production processes in the agricultural food industry, where the local context is critical particularly in use of natural resources, as exemplified by the use of Algerian goat milks. These advancements have further contributed to increasing demands for functional and healthy foods. Future studies should investigate interactions between these bacterial strains and other environmental parameters or natural ingredients. The major aim is to optimize nutritional and sensory qualities of the final products, while minimizing challenges such as excessive syneresis.

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Background and Objective: This study aimed to assess the ability of lactic acid bacteria isolated from yogurt to produce acetaldehyde and diacetyl using solid-phase microextraction gas chromatography-mass spectrometry method. Two species of lactic acid bacteria, Lacto-bacillus and Enterococcus, were isolated from Iranian traditional yogurts. Enterococcus strains showed distinct biochemical characteristics, including lipolytic activity, citrate metabolism and aromatic compound synthesis, which significantly affected the sensory characteristics of various cheeses during ripening.

Material and Methods: The study investigated ability of these strains to produce acetal-dehyde and diacetyl as single starters and co-cultures. The biochemical characteristics of the strains were assessed, including their chemical profiles, acid production ability, yogurt sensory evaluation and antimicrobial susceptibility for Enterococcus strains.

Results and Conclusion: Lactobacillus strains showed the highest rate of acetaldehyde production. Acetaldehyde production ranged 0.45-8.33 mg.kg^-1 and diacetyl production ranged 2.00-13.20 mg.kg^-1 in growth of Enterococcus. In contrast, acetaldehyde production in Lactobacillus strains ranged 2.23-25.59 mg.kg^-1 and diacetyl production ranged 0.42-5.96 mg.kg^-1 when the bacteria were incubated at 5 °C for 14 d. In co-culture with Enterococcus, presence of Enterococcus slightly increased production of acetaldehyde and diacetyl. Addit-ionally, presence of Enterococcus strains positively affected taste, color and texture of the yogurt samples. No previous studies have specifically assessed production of acetaldehyde and diacetyl by Enterococcus strains in yogurts.

Conflict of interest: The authors declare no conflict of interest.

1. Introduction

Yogurt is one of the most widely consumed dairy products due to its rich nutritional values and numerous health benefits [1]. Yogurt production relies on the fermen-tation activity of lactic acid bacteria (LAB), which play a critical role in milk coagulation and texture formation [2]. The LAB species are preferred in the food industry due to their probiotic characteristics and their ability to enhance the nutritional and sensory qualities of fermented products. Additionally, their biochemical activity contributes to pH regulation and the production of secondary metabolites such as hydrogen peroxide, diacetyl and bacteriocins, making them excellent candidates as starter cultures [3]. Starter cultures consist of selected microbial strains that affect the organoleptic characteristics of dairy products, including texture, flavor, aroma and appearance. From LAB, enterococci are commonly detected in various dairy products, including yogurt and cheese [4]. These bacteria contribute to the characteristic sensory attributes of dairy products by producing aromatic compounds through bio-chemical processes such as lipolysis, proteolysis and citrate metabolism [5]. Several studies have demonstrated the positive effects of enterococci on cheese quality, improving its structure, consistency, texture, taste and color [6]. Due to their natural preservative characteristics and produced aromatic compounds, these bacteria have been considered appropriate candidates for processing dairy products [7]. In recent years, interests in natural preservatives have increased, with research highlighting the ability of enterococci to produce bacteriocins, particularly enterocins [8]. Typically classified as class II bacteriocins, enterocins are small, heat-stable non-lantibiotic peptides that inhibit a specific range of bacteria, including foodborne pathogens such as Clostridium spp., Vibrio cholerae, Listeria spp. and Staphylococcus aureus. Enterocins have been shown to extend the shelf life of dairy products and their technological uses have led to the suggestion of enterococci as additional starters or protective cultures in cheese production. Additionally, enterococcal strains, particularly combinations of Streptococcus thermophiles and Enterococcus faecium, have been assessed as probiotics in clinical settings and suggested as potential alternatives to antibiotic treatments [9]. Despite their potential benefits, the use of enterococci in food production has increased concerns due to reports of antibiotic-resistant strains and their association with human infections [10]. While enterococci detected in traditional dairy products are often considered natural contaminants or part of the fermentation process, their presence has been linked to possible fecal contamination, increasing safety concerns in dairy processing [11,12]. These bacteria are naturally present in milk and play a significant role in the microbiota of fermented foods, particularly meats and cheeses. Their ability to tolerate heat and adapt to various environmental conditions allows them to survive Pasteurization and persist in refrigerated products [8]. However, enterococcal strains generally show weak acidification capabilities, limiting their effectiveness as primary starter cultures [11]. Studies have shown that dairy-originated enterococci decrease the pH slightly after 16-24 h of incubation at 37 °C, with few strains reaching pH less than 5.00-5.20 [12]. Research on E. faecalis from traditional Italian cheeses suggests that the strain is a more potent acidifier than E. faecium, capable of decreasing the pH of skim milk to approximately 4.5 within 24 h of fermentation [13]. Despite the well-established roles of S. thermophilus and L. bulgaricus in yogurt fermentation, their precise mechanisms in aromatic compound produc-tion and additionally functional characteristics are insufficiently understood. Enterococci, commonly detected in fermented dairy products, have been suggested as potential contributors to flavor and preservation, still their direct role in acetaldehyde and diacetyl production as single strains in yogurt is not systematically investigated. This study aimed to address this gap by assessing the ability of enterococcal strains isolated from Iranian traditional yogurts to produce acetaldehyde and diacetyl independently. Furthermore, this study investigated their potential for generating aromatic compounds and their microbial characteristics, with the goal of identifying novel candidates for enhancing yogurt flavor and stability. Regarding their flexibility to extreme environmental conditions such as pH fluctuations, temperature variations and salinity, enterococci may offer a robust alternative or complement to conventional starter cultures. Additionally, their potential probiotic benefits warrant further investigation for uses in functional dairy products. This study addressed the question of if the presence of enterococci contributed to the development of desirable and acceptable flavors in dairy products such as yogurts and cheeses. By elucidating the role of enterococci in yogurt fermentation, this study aimed to provide a detection for developing innovative starter cultures with improved sensory and preservative characteristics.

1. Materials and Methods

2.1. Microbiological Analyses

Traditional yogurt samples were collected from various regions in western Iran. Lactobacillus strains were cultured on MRS (de Man, Rogosa and Sharp) agar at 37 °C for 48 h using CO₂ incubator, while Enterococcus strains were cultured on M17 agar (Merck, Darmstadt, Germany) at similar temperature and time using shaking incubator at 200 rpm. For long-term storage, the cultured strains were preserved in liquid media with 20% sterile glycerol at -80 °C. The molecular identification of Enterococcus and Lactobacillus strains was carried out based on genetic databases [14].

2.2. Chemical Analyses

In the carbohydrate fermentation experiments, the isolated strains were assessed with sugars such as lactose, sucrose and sorbitol [15]. The growth rate of these strains was assessed at various NaCl concentrations to assess their tolerance and adaptability to salt. Specifically, NaCl concentrations of 0-6.5% were used in the culture media. To assess growth, 50 μL of the bacterial culture was inoculated into 5 ml of the media (Merck, Darmstadt, Germany) containing 4 and 6.5% NaCl. After 24 h of incubation at 30 °C, the growth rate of the strains was assessed via turbidity at 620 nm [16].

2.3. Antimicrobial Susceptibility of the Enterococcus Isolates

The antimicrobial susceptibility of Enterococcus strains was assessed against antibiotics that targeted various bacterial mechanisms, including inhibitors of cell envelope synthesis (ampicillin 5μg, vancomycin 5μg, kanamycin 10μg, imipenem 10μg), gentamicin 30 μg, tetracycline 10μg, chloramphenicol 5μg, erythromycin 10μg, clindam-ycin 15μg) and ciprofloxacin 5μg). Minimum inhibitory concentration was assessed using microdilution method as described by the clinical and laboratory standards institute. In this method, after assessing bacterial concentration in standard and physiological media after 18 h of culture, a series of dilutions were prepared using stock solution of antibiotics. The bacteria were incubated at 37 °C for 24 h and the MIC values were then assessed.

2.4. Preparation of Yogurts

The selected bacterial strains were added to 100 mL of pasteurized milk at a concentration of 10⁸ CFU.mL^-1 after cooling down the milk to the optimal incubation temperature of 42-44 °C. The mixture was incubated at 42 °C. After the gel pH decreased to nearly 4.50 ±0.02 and clot formation was observed, the yogurt was rapidly cooled to 20 °C, followed by storage at 5 °C [17]. This two-stage cooling process served to stabilize the curd structure, decrease whey separation (syneresis), minimize thermal stress on the microorganisms and regulate the flavor by preventing excessive acidity.

2.5. Sensory Evaluation of Yogurts

Sensory evaluation of the yogurts was carried out by a panel of fifteen trained individuals using scoring method based on the criteria by Tamime and Robinson. The sensory attributes included appearance, color, aroma, taste, texture and overall acceptability.

2.6. Acid Production Ability

To assess the acid production ability, yogurt was prepared by fermenting pasteurized milk at 42-44 °C with an Enterococcus strain at a concentration of 10⁸ CFU.mL^-1. The milk was incubated for 14 h and the pH was assessed once clot formation occurred. Moreover, pH was assessed using EDT353 pH meter (London, UK) calibrated with pH 7 and pH 4 buffers.

2.7. Assessment of Acetaldehyde and Diacetyl Productions by the Strains

After a 14-h incubation at 42 °C, milk was fermented using the strain with a concentration of 10⁸ CFU.mL^-1. Aroma compounds in the yogurt samples were analyzed using quadrupole mass spectrometer coupled with an Agilent 7890 USA-made gas chromatography-mass spectrometry (SPME-GC-MS) system. The samples were stored at 5 °C for 14 d before analysis. Separation was carried out using polydimethylsiloxane (PDMS) capillary column with an internal diameter (I.D.) of 0.25 mm and a film thickness of 30 μm. Sample injection was carried out using split/splitless inlet with a 2:1 split ratio. Chromato-graphic separation was achieved using HP-5 MS capillary column (5% phenyl, 95% dimethylpolysiloxane) with specifications of 30 m length, 0.25 mm I.D. and 0.25 μm film thickness, made of silica. The chromatographic conditions included injection volume of 1.00 mL.min^-1, split injection mode (2:1), inlet temperature of 270 °C, initial oven temperature of 40 °C (held for 5 min), increased to 250 °C at a rate of 8 °C.min^-1 and held for 2 min, carrier gas flow rate of 1.00 mL.min^-1 (constant flow) and interface temperature of 290 °C. The assessment protocol for PAH-hydroxy compounds in sensory samples involved allowing the sample to equilibrate to room temperature (RT), weighing 1 g of the sample, adding 1 mL of water for homogenization, shaking the mixture for 2 min, heating to 80 °C for 20 min while inserting an SPME fiber and then using SPME syringe to inject the extracted vapors into the GC-MS system. The SPME fiber used in this study included PDMS with an 80 µm coating.

2.8. Molecular Verification of the Isolated Strains Using 16S rRNA Gene

To verify the identity of the isolated strains, the 16S rRNA gene was amplified using cetyl trimethyl ammonium bromide method for DNA extraction. The primers included forward primer (F): AGAGTTTGATCMTGGCTCAG and reverse primer (R): GGTTACCTTGTTACGACTT, ampli-fying 1500-bp fragments of the 16S rRNA gene. The PCR was carried out with an initial denaturation step at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 35 s and extension at 72 °C for 45 s. The products were visualized on a 1.5% agarose gel stained with Rima Sight DNA stain. The sequencing was carried out using 630R and 616V primers and the sequences were analyzed using the highest similarity to available gene sequences in NCBI [18].

2.9. Statistical Analysis

Statistical analysis was carried out using one-way ANOVA to compare the growth rates and sensory evalua-tion scores. Post-hoc Tukey’s test was used for multiple comparisons, with significance at p < 0.05. All exper-iments were carried out in triplicate. Standard deviation (SD) was used to assess the variation within the dataset. For antimicrobial susceptibility, MIC values were compared to standard breakpoints. All analyses were carried out using SPSS software.

1. Results and Discussion

3.1. Chemical Analyses

As members of the LAB group, Enterococcus strains can coagulate skim milk when cultured as a single strain. All of the isolated strains demonstrated the ability to ferment lactose, as shown in Table 1. However, further characterizations of this characteristic are uninvestigated. The capacity to ferment sucrose belonged to two strains of Enterococcus and four strains of Lactobacillus were able to ferment sorbitol. Some LAB include metabolic pathways for sorbitol that are encoded by genes organized in operons. These pathways include sorbitol transport system, sorbitol 6-phosphate dehydrogenase (S6PD) and regulatory proteins. The sorbitol metabolism operons of L. casei and L. plantarum have been previously characterized [19]. Strains that are capable of fermenting a variety of sugars show better growth and higher rate of acid production.

Findings showed that certain strains such as N2, N3, N5, N6 and N7 (Figure 1) grew well and were able to tolerate sodium chloride concentrations ranging 0-6.5% (p < 0.05). This is consistent with previous studies on the high salt tolerance of enterococcal strains [20]. This ability enables them to grow well in salty food products such as cheeses, fermented meats and pickles. In contrast, other strains did not grow as well and likely required longer incubation periods. Lactobacilli generally show moderate salt tolerance, though this could vary by species and strain. Many lactobacilli can tolerate 4% sodium chloride concentrations, though it might partially inhibit their growth. Species such as L. plantarum and L. casei show higher salt tolerance and can grow at concentrations as high as 6-8% [21]. Based on the results from Figure 2, a significant difference was seen in optical density measurements for the Lactobacillus strains before and after incubation at salt concentrations of 0 and 4% (p < 0.01). In contrast to previous studies, the Lactobacillus strains not only tolerated 4% salts but also showed improved growth, particularly L7 and L8 strains.

3.2. Antibiotics Affecting Growth of the Enterococcus Strains

Enterococci are naturally abundant in various environments, including dairy products. All strains assessed in this study showed no hemolytic activity and were negative for catalase. Assessing antibiotic resistance is essential for assessing safety of Enterococcus strains. A similar antimicrobial susceptibility profile was observed in a previous study [22]. The MIC results (Table 2) revealed that all isolated strains were susceptible to imipenem, gentamicin, tetracycline and chloramphenicol. Eighty-seven percent of the Enterococcus strains were sensitive to erythromycin, ampicillin and kanamycin. Furthermore, 62.5% of the strains were susceptible to clindamycin and 75% were susceptible to vancomycin and ciprofloxacin. Vancomycin resistance is particularly important as it is the last line of defense against multiple-resistant Enterococcus infections. Resistance to glycopeptides is a critical factor in assessing safety of these strains.

In addition to vancomycin resistance, certain Enterococcus strains are resistant to other antibiotics commonly used in veterinary and human medicines. Some of these strains are addressed as pathogens for humans and animals. The virulence factors of Enterococci include antibiotic resistance, colonization, adhesion to host tissues [with pheromone-responsive plasmids encoding the adhesin aggregation substance and the chromosomally encoded enterococcal surface protein (Esp)], invasion of tissues and resistance to host defense mechanisms virulence factors. One well-known enterococcal virulence factor is hemolysin. Eaton and Gasson (2001) demonstrated the presence of these virulence factors in Enterococcus strains isolated from foods and medical sources as well as those used as starter cultures. Particularly, medical strains showed the highest prevalence of virulence factors, while starter cultures showed the lowest prevalence. Pathogenic Enterococcus strains induce pathological changes either directly through toxin production or indirectly via inflammation. It is strongly recommended to use strains free from any virulence factors or determinants in food production. Selecting specific Enterococcus strains for use as adjunct starters must be carried out with extreme caution thorough assessments to ensure safety.

3.3. Sensory Evaluation Scores of Yogurt

Flavor is one of the key factors affecting the acceptability and preference of food products. Table 3 presents the sensory analysis of the yogurt samples. Of the Enterococcus strains, N3, N6 and N7 samples showed the highest production of acetaldehyde and diacetyl. Previous studies on Enterococcus strains introduced into cheeses have demonstrated their positive effects on ripening, flavor, aroma, color, texture and the overall sensory profile of fully matured cheeses [23]. In yogurt samples, the presence of Lactobacillus cultures resulted in increased acidity and decreased hydration, which improved the texture and ultimately contributed to higher sensory scores. This process effectively enhanced the sensory characteristics of the yogurt samples. The current findings indicated that the incorporation of Enterococcus strains into fermentation cultures facilitated the production of diacetyl, contributing to a richer buttery taste in the yogurt.

3.4. Acid Production Ability

Lactobacillus strains include the ability to produce lactic acid at concentrations ranging 1.5-2% in culture media. The extent of acid production is affected by factors such as strain type, composition of the culture media, temperature and the duration of fermentation. Optimal acid production typically occurs within a temperature range of 37-45 °C. Acid production plays a vital role in yogurt quality by enhancing its shelf life (through pH decrease, inhibiting the growth of undesirable bacteria), contributing to flavor development (giving yogurt its characteristic tartness) and helping in texture formation (as milk proteins coagulate at low pH to form the typical yogurt structure).

In contrast, Enterococcus strains generally show limited acidification potentials in milks. A recent study on dairy-derived Enterococcus strains revealed that only a small proportion were able to decrease the pH to 5.00–5.2 after 16–24 h of incubation at 37 °C. However, E. faecalis strains isolated from tradi-tional Italian cheeses demon-strated a significant acidifica-tion capacity in skim milk, decreasing pH to nearly 4.5 after 24 h of fermentation. Particularly, E. faecalis showed a greater acidification potential, compared to E. faecium. Due to their relatively low acidification and proteolytic activities, Enterococcus strains are generally not reported as primary starter cultures in cheese production. Although acidification and proteolytic activities are not directly correlated, strains that are more acidifying often show higher proteolytic activities [15]. An effective acid-producing starter culture, when inoculated at 10%, should decrease pH of milk from 6.6 to 5.3 within 6 h. In contrast, Enterococcus strains are majorly used as adjunct cultures in cheese production for purposes other than acidification such as using as probiotics, accelerating ripening and enhancing flavor. As shown in Figure 3, certain Enterococcus strains could decrease the pH of milk to nearly 4 after 14 h. Compared to other LAB, these generally need a longer time to achieve a similar pH decrease.

3.5. Assessment of Acetaldehyde and Diacetyl Produced by Enterococcus and Lactobacillus Strains

The primary aromatic compounds in yogurts are carbonyl compounds, with acetaldehyde as the major contributor to its characteristic flavor. While fatty acids and carbohydrates can play a role in aroma formation, casein is the major precursor of aromatic compounds in milks. The proteolytic system of LAB breaks down casein into amino acids, which are converted into aromatic compounds [21]. The LAB ability to metabolize citrate and pyruvate is critical for aroma formation as many LAB species convert citrate into aromatic compounds such as acetate, acetaldehyde and diacetyl [22]. Research suggests that yogurt products with low acetaldehyde concentrations can preserve the characteristic yogurt aroma, indicating that acetaldehyde is one of the important aroma components. Diacetyl, another key aromatic compound, significantly contributes to yogurt buttery flavor and overall aroma, especially in products with low acetaldehyde levels. In commercial yogurt production, S. thermophilus and L. bulgaricus are typically used in co-cultures, which enhance yogurt flavor, aroma, pH and texture. Co-culturing these species significantly increases acetaldehyde production, compared to use of L. bulgaricus alone. This study assessed the effects of Lactobacillus and Enterococcus strains on the production of acetaldehyde and diacetyl, comparing them with samples made with Enterococcus as the sole starter culture. Studies have suggested that S. thermophiles is the unique species capable of producing diacetyl [22]; however, limited information on citrate metabolism in Enterococcus strains are available. Research by Freitas et al. showed that E. faecalis and E. faecium strains from Picante cheeses could metabolize citrate in milks, with E. faecium showing a lower rate of citrate metabolism, compared to that E. faecalis was [23]. The Advisory Committee on Novel Foods and Processes has approved E. faecium strain K77D as a starter culture for fermented dairy products [16]. The present study detected that Enterococcus strains were capable of producing acetal-dehyde and diacetyl. As shown in Figure 4, these strains produced more diacetyl than acetaldehyde. This study was the first to assess and quantify these aromatic compounds in Enterococcus strains individually, providing novel insights into their role in flavor development of dairy products. While previous studies have focused on starter cultures in cheese production, specific contribution of Enterococcus to aroma formation in yogurts has largely been uninvestigated [20]. In this study, N3 and N6 strains produced significant quantities of acetaldehyde, reaching 5 and 8.33 (mg.kg^-1), respectively. Moreover, N5 and N8 strains showed the highest diacetyl production with concentrations of 13.2 and 12 mg.kg^-1, respectively. In comparison, Lacto-bacillus strains played a major role in acetaldehyde production. As shown in Figure 5, Lactobacillus isolates of L5 and L7 produced high levels of acetaldehyde (25.59 and 19.2 mg.kg^-1, respectively), while isolates of L6 and L8 generated the highest diacetyl concentrations (5.96 and 5.50 mg.kg^-1, respectively). The combination of L4N4 showed increased diacetyl production, while acetaldehyde levels increased in samples containing L1N1, L4N4 and L8N8. These findings highlighted the promising potential of Enterococcus strains for diacetyl production in yogurt fermentation. Particularly, no previous studies have specifically quantified acetaldehyde and diacetyl productions by Enterococcus strains in yogurt, underscoring the novelty of the present study. The L5 strain, which showed the highest acetaldehyde production and received a high score in sensory evaluation, demonstrated the ability to metabolize all sugars (lactose, sucrose and sorbitol). The N5 strain, which could hydrolyze lactose and sucrose, produced the highest diacetyl levels and received a high score in sensory evaluation. Strains nos. L6 and L8 demonstrated high diacetyl production from hydrolysis and received favorable sensory evaluation scores. The two strains showed the ability to metabolize lactose and sorbitol as well. The L6 strain demonstrated the ability to metabolize lactose and sucrose, received a relatively high sensory evaluation score and showed high capacity for diacetyl production.

3.6. Amplified 16S rDNA restriction analysis

The electrophoresis results (Figure 6) verified successful amplification of the 16S rRNA gene in the isolated strains. The gel image demonstrated distinct bands at 1500 bp for the samples cultured on MRS and M17 media, indicating accurate gene amplification. The lane distribution was as follows: the first lane contained the molecular weight marker (100-bp ladder), the second lane served as the negative control without DNA templates (showing no bands) and the lanes corresponded to PCR products of the isolated strains. The presence of clear 1500-bp bands in the samples and nucleotide sequence analysis using BLAST of NCBI verified the reported 99.09% identity of L. helveticus and 97.21% identity E. faecium. This molecular validation supports use of these strains in further biochemical and microbial analyses, reinforcing their suitability for uses in dairy fermentation.

1. Conclusion

This study was the first to investigate use of Enterococcus strains as starter cultures for yogurt fermentation, assessing their overall effects on yogurt quality as well as their ability to produce acetaldehyde and diacetyl. Reviews demonstrate that multiple strains of E. faecalis and E. faecium, isolated from dairy products, include the metabolic capacity to generate key flavor compounds, including acetoin, acetaldehyde, ethanol and diacetyl. These findings highlight the significant role of enterococci in enhancing the sensory attributes of dairy products, particularly in the development of cheese flavor and aroma. When used as single-strain starters, the enterococcal isolates successfully fermented yogurt samples, forming a stable gel structure. Furthermore, these strains showed efficient diacetyl production, contributing to the distinct buttery aroma of the final product. Sensory analysis revealed that the unique scent of the yogurt samples could be attributed to diacetyl-producing enterococcal strains, as recognized by the trained assessors. The findings of this study provided compelling evidence of the technological potential of enterococcal strains in dairy fermentation. Their ability to enhance flavor and aroma with their acidification capacity supported their uses as adjunct starter cultures in commercial yogurt production. These results facilitate further studies for optimizing enterococcal strains for industrial uses, potentially expanding their roles in development of high-quality fermented dairy products. Despite the potential uses of enterococci in food systems, significant concerns must be addressed before their safe uses can be ensured. A major issue is the increasing prevalence of enterococcal strains resistant to glycopeptides and other antibiotics, which increases public health concerns. Additionally, their capacity of producing biogenic amines in foods and the presence of virulence factors in clinical and food-derived isolates question their safety in food products. However, the high potency of enterococci to horizontally transfer genes-particularly antibiotic resistance genes- to patho-genic bacteria complicates the establishment of reliable selection criteria. Regarding these risks, safety of enterococci in foods is controversial, necessitating further clinical and epidemiological investigations.

1. Acknowledgements

The authors thank Tak Gen Zist for its supports and staff of the Central Tehran Branch Laboratory, Islamic Azad University.

1. Conflict of Interest

The authors report no conflict of interest.

Background and Objective: Obesity is an increasing public health issue that needs practical and scientifically supported nutritional interventions. This study aimed to formulate functional coffees enriched with fermented lotus leaves (Nelumbo nucifera) using Bacillus subtilis to enhance polyphenol concentration and lipase enzyme activity. Additional components included breadfruit leaves (Artocarpus altilis), lotus seeds, notoginseng flowers (Panax notoginseng), caterpillar fungi (Cordyceps militaris), and collagen, selected for their complementary effects on metabolic functions, immune supports, sensory attributes and market feasibility.

Material and Methods: The multiple component formulation was optimized using mixture design integrated with response surface methodology. Efficacy was assessed through a 6-m randomized controlled trial involving 127 overweight adults. The trial used a double-blind placebo-controlled design to ensure reliability and minimize bias. Additionally, a consumer acceptance survey involving 800 participants was carried out to assess repurchase intention and product perception.

Results and Conclusion: Fermentation (10⁷ CFU.g^-1, 35 °C, 65.00-70.00% relative humidity, 72 h) led to a 2.5-fold increase in polyphenol content and doubled lipase enzyme activity. In the clinical trial, participants consuming the nutritional coffee showed average weight increases of 1.40 kg, decreases in low-density lipoprotein cholesterol C by approximately 10.00 mg.dl^-1 and increases in high-density lipoprotein cholesterol by nearly 3.00 mg.dl^-1. These improvements were statistically significant (p < 0.05) and were not associated with serious adverse effects. The consumer survey indicated a 65.00% repurchase intention, suggesting promising market potential. Although the study included limitations such as those of sample size, dropout rate and intervention time, the findings demonstrated metabolic benefits and industrial feasibility. This study provides a solid foundation for the development and commercialization of functional coffee targeting weight management and cardiovascular support. These findings provide valuable insights for researchers and industries worldwide interested in developing innovative functional beverages aimed at managing obesity and improving cardiovascular health.

Conflict of interest: The authors declare no conflict of interest.

1. Introduction

Obesity is a significant global health challenge closely linked to metabolic disorders, highlighting the critical need for nutritional interventions derived from biotechnology. In particular, microbial fermentation combined with advanced optimization techniques such as response surface method-ology (RSM) includes significant promises for developing effective herbal-based functional beverages to support weight management. In Asia, particularly in developing countries such as Vietnam, obesity rates have increased, significantly increasing the risk of type 2 diabetes and lipid metabolism disorders [1-3].

Coffee, well-known for its chlorogenic acid and poly-phenolic compounds, has shown promising effects on lipid metabolism and fat accumulation prevention. However, its naturally high caffeine content may increase blood pressure in susceptible individuals; thus, posing certain safety concerns. To maximize coffee metabolic benefits and lessen such risks, blending coffee with bioactive Asian medicinal herbs has been suggested [4]. Specifically, this formulation moderates caffeine-linked safety concerns by incorporating herbal ingredients highlighted for their calming and anti-hypertensive effects. Fermented lotus leaves, breadfruit leaves, lotus seeds, Panax notoginseng flowers and Cordyceps militaris contain bioactive compounds such as polyphenols and flavonoids that help regulate blood pressure and moderate caffeine stimulating characteristics. Moreover, precise mixture design optimization ensures that caffeine content is in safe limits; thus, balancing the beneficial metabolic effects of coffee with improved safety profiles.

The synergistic interaction in chosen herbal bioactive compounds is primarily fueled by complementary mechan-isms at enzymatic and metabolic levels. In details, poly-phenols from fermented lotus leaves possess potent antioxidative functions, effectively inhibiting oxidative stress and inflammation pathways that complement obesity and dyslipidemia [5,6]. Flavonoids from breadfruit leaves augment antioxidative defenses, supporting overall anti-oxidant system strength [10,11]. Saponins derived from P. notoginseng accelerate lipid metabolism by promoting lipase activity to hydrolyze triglycerides and further oxidize fats [8]. Cordycepin from C. militaris further complements this process through modulating pathways of lipid biosynthesis and enhancing catabolism of lipids [12]. Chlorogenic acids in coffees include additional metabolic regulatory functions by enhancing glucose metabolism and insulin sensitivity, indirectly affecting lipid deposition and energy consumption [4]. In general, these bioactive com-pounds interact in multiple targeted biochemical pathways, leading to overall metabolic efficacy superior to single-herbal formulations. Such synergistic mechanism supports the rationale for combining them in the optimized nutritional coffee formulation.

Previous studies have primarily focused on short-term (6-8 w) assessments of single herbal ingredients or coffees, demonstrating individual metabolic benefits. For example, fermentation processes have shown significant increases in polyphenol bioavailability and enzymatic activities in herbal ingredients such as lotus leaves [5,6]. Solid-state fermentation using Bacillus (B.) subtilis has been reported to enhance polyphenol release and antioxidant activity in various plant materials [7]. Moreover, compounds such as notoginsenosides and saponins from P. notoginseng have been shown to improve lipid metabolism and protect against cardiometabolic disorders [8].

Nevertheless, comprehensive long-term studies investi-gating the synergistic interactions in multiple fermented herbal components within functional beverages, particularly regarding their sustained effects on weight management and cardiovascular health, are particularly limited [5-8]. This gap indicates that while individual bioactive components are recognized, the precise mechanisms underlying their combined effects in a functional beverage context are insufficiently understood. To address this gap, this study systematically optimizes the fermentation conditions of lotus leaves to substantially enhance bioactive polyphenols and lipase enzyme activity. These optimized herbal components are subsequently integrated into a nutritional coffee formulation. The hypothesis includes that combining fermented lotus leaves with selected herbal bioactive ingredients may yield enhanced metabolic and antioxidant effects, rather than those of individual ingredients alone.

This investigation uniquely integrates microbial ferment-ation optimization, rigorous clinical assessments and comprehensive consumer acceptance analyses. By system-atically assessing synergistic interactions in multiple herbal bioactive compounds within fermented nutritional coffees, the study aimed not only to provide scientific validation but also to establish practical guidelines supporting the develop-ment and potential commerciali-zation of innovative func-tional beverages targeting improved metabolic health. Specific objectives included formulating and optimizing the beverage through microbial fermentation and mixture design, assessing clinical efficacy via a randomized controlled trial and assessing consumer acceptance and product feasibility through comprehensive surveys. Moreover, the study aimed to establish a scientific and practical foundation for the potential commercialization of a nutritional coffee product, addressing that full comer-cialization need further detailed safety assessments, regulatory approvals and comprehensive large-scale pro-duction trials.

1. Materials and Methods

2.1. Coffee

Coffea canephora (Robusta) and C. arabica (Arabica) beans were collected from Krong Pac District, Dak Lak Province (12°38'N, 108°03'E), a region addressed for its basaltic soil and temperate climate in Vietnam. The beans were wet processed and sun‐dried until they reached a moisture content of approximately 11.00%, as assessed using Kett PM-650 device and verified using AOAC 925.10 method [13]. To ensure consistency, the beans were stored at 20-25 °C with a relative humidity of nearly 60.00%. A Robusta:Arabica ratio of 70:30 (w/w) was chosen to balance aroma and boldness. For each roasting batch (5 kg), the beans were processed using Probat UG22 roaster at 190.00 °C ±2.00 for 12.00 min ±0.50, rapidly cooled down and yielded an Agtron color score of approximately 65. After roasting, the beans were finely ground to a particle size of ~150 µm, packed in aluminum bags with desiccants and stored under light-protected conditions at 25.00 °C ±2.00. For instant coffee production, a hot extraction was carried out using a coffee-to-water ratio of 1:10 (w/v) at 90-95 °C with continuous stirring for 30–60 min. Although the process parameters were initially set based on preliminary laboratory trials and prior publications, the mid-range conditions (90-95 °C, 30-60 min) were selected for subsequent experiments to balance extraction efficiency and bioactive compound stability. These settings aligned with standard industrial practices, ensuring scalability and reproducibility. The resulting extract was filtered, conc-entrated at 50.00 °C ±2 under a decreased pressure of approximately 0.08 MPa and spray-dried using Buchi B290 (inlet temperature of 160.00 °C ±2, feed rate of 5 ml.min^-1). The final product, with a moisture content less than 5.00%, was then packaged in multilayer aluminum bags and stored at 25.00 °C ±2.00.

2.2. Herbal Components

The herbal components consisted of Nelumbo nucifera (lotus leaves), Artocarpus altilis (breadfruit leaves), lotus seeds, P. notoginseng (notoginseng flowers) and C. militaris (caterpillar fungi), with hydrolyzed collagen (> 90% purity). All materials were supplied by Green Herbal Pharma-ceutical, Vietnam (batch no. LN-SK-HT-CTH-202301) with certificates of analysis. Preliminary drying procedures were specifically tailored to each ingredient to balance energy efficiency with the preservation of bioactive compounds. Lotus and breadfruit leaves were dried at 45 °C for 48 h (achieving moisture levels < 8.00%), notoginseng flowers at 40 °C for 72 h, while lotus seeds were freeze-dried at -50 °C (4 Pa) to minimize thermal degradation. Cordyceps militaris, which was artificially cultivated, was dried at 50 °C for 24 h. Hydrolyzed collagen was prepared based on established protocols [8]. All dried materials were milled to a particle size of approximately 200 µm and stored at 4.00 °C ±1.00. Prior to formulation, microbiological analysis [14], polyphenol content assessment via the Folin-Ciocalteu method [15] and heavy metal analysis (Pb, Cd and Hg) were carried out using atomic absorption spectroscopy based on QCVN 8-2:2011/BYT [16]. The detection limits were 0.02 mg.kg^-1 for Pb, 0.01 mg.kg^-1 for Cd and 0.005 mg.kg^-1 for Hg, ensuring compliance with safety and quality standards. Hydrolyzed collagen and instant coffee were not subjected to the extraction, fiber removal and spray drying processes described later because their inherent low fiber content rendered such processing unnecessary. Instead, these ingredients were directly incorporated into the final formulation after appropriate quality control checks, ensuring that their bioactive characteristics were intact.

2.3. Chemicals and Equipment

Chemicals used in the study included gallic acid (≥ 98.00%; Sigma-Aldrich, USA) as the polyphenol standard, DPPH (2,2-diphenyl-1-picrylhydrazyl, ≥ 95%; Sigma-Aldrich, USA) for antioxidant assays, p-nitrophenyl palmitate (p-PNN) (≥ 98%; Sigma-Aldrich, USA) for lipase activity measurement, HPLC-grade methanol (Merck, Germany), 0.1 M phosphate buffer (pH 7.0) and double-distilled water. Key equipment included a Binder KBF climate chamber with ±0.5 °C, ±3% RH accuracy chamber (Binder, Germany), a Buchi B-290 spray dryer with a 0.70 mm nozzle (BUCHI Labortechnik, Switzerland), a Fritsch grinder (Fritsch, Germany), an IKA RCT basic stirrer (IKA, Germany), a Malvern Mastersizer 3000 particle size analyzer (Malvern Panalytical, UK) and a Probat UG22 coffee roaster (Probat-Werke, Germany). Polyphenol analysis was carried out using Folin-Ciocalteu method [15]. Furthermore, DPPH radical scavenging activity was assessed based on a previously described procedure [17]. Lipase enzyme activity was assessed following an established method [19]. Lipase enzyme activity was assessed based on the method described by Winkler and Stuckmann [19], with calibration curves achieving R² > 0.99. Lipase activity was assessed spectrophotometrically using p-NPP as substrate and 1 U of lipase activity was defined as the quantity of enzyme needed to release 1 µmol of p-nitrophenol per minute under assay conditions (at 37 °C, pH 7.0). Limit of detection and limit of quantification were assessed based on IUPAC guidelines [20] and all measurements were verified to include a relative standard deviation (RSD) leass than 5%. 

2.4. Fermentation of Lotus Leaves

           Dried lotus leaves (moisture < 8%) were inoculated uniformly with a food-grade strain of B. subtilis TH-VK422 (10⁷ CFU.g^-1, CFU = colony-forming unit), classified as generally recognized as safe (GRAS) and selected based on preliminary screening for high lipase production and polyphenol bioconversion efficiency, that was supplied by the Laboratory of Biotechnology, Faculty of Biology and Environment, Ho Chi Minh City University of Industry and Trade (HUIT), Vietnam. The strain was kindly provided by Dr. Hoang-Dung Tran, who supervised its isolation and quality control. The B. subtilis was particularly chosen for fermenting lotus leaves because of specific advantages over other microbial fermentative agents such as fungi and yeasts. First, B. subtilis is GRAS, guaranteeing its use for functional food purposes [7]. Second, previous reports have demonstrated the superior enzymatic activity of B. subtilis, namely its robust lipase and protease production, which ensure better possible biotransformation and bioavailability of polyphenolic compounds [6,7].

In contrast to fungal fermentations, which generally need long incubation periods and can include risks linked to mycotoxin production, fermentation using B. subtilis can be carried out economically within short incubation times (48–72 h); thereby, guaranteeing practicability and safety [7,22]. Moreover, B. subtilis fermentation has been detected to effectively liberate bound polyphenols and other bioactive metabolites from plant matrices, significantly enhancing antioxidative and metabolic qualities of the fermented herbal products [6,7,21]. All of these characteristics strongly link to the use of B. subtilis as ideal microbial fermentation agent for functional enhancement of lotus leaves in this study. Fermentation was carried out using Binder KBF climate chamber and conditions optimized via Box-Behnken experimental design (Design-Expert v.11). The design assessed temperature (30–35 °C), relative humidity (60.00-70.00%) and time (48-72 h). All conditions were used in triplicate to ensure statistical reliability.

The use of triplicate replicates (n = 3) per condition is a standard practice in small-scale fermentation experiments and was reported sufficient to ensure statistical confidence and detect significant differences in polyphenol content, antioxidant activity and lipase activity. While a larger number of replicates improved power, a number of three was selected based on resource availability and method-ological consistency with similar published studies. Following fermentation monitored by assessing polyphenol content, DPPH activity and lipase activity, the leaves were dried at 40 °C for 24 h to terminate microbial activity, milled and stored at 4.00 °C ±1.00. Optimal conditions were assessed using analysis of variance (p < 0.05) [8]. 

2.5. Extraction, Fiber Removal and Powder Production

           The fibrous ingredients—including fermented lotus leaves, breadfruit leaves, lotus seeds, notoginseng flowers and C. militaris—were hot-water extracted at 80–90 °C using a 1:10 (w/v) ratio for 30–60 min with continuous stirring. These extraction parameters were selected based on preliminary trials that verified acceptable extraction yield and preservation of key bioactive compounds. A temperature range of 80–90°C was chosen following initial optimization trials investigating to achieve maximal extraction efficiency of polyphenolic compounds while minimizing losses in enzyme activity, particularly lipase. Increased temperatures significantly enhanced the solubility and diffusion of polyphenols and other bioactive plant-derived metabolites; thereby, improving overall yield [5,6].

Because lipase enzymes are temperature-sensitive, there is a risk of partial thermal inactivation. Preliminary trials demonstrated that lipase preserved approximately 80% of its original enzymatic activity at this temperature range, which seemed an acceptable compromise for the significant increase in polyphenol recovery. Extraction at temperatures less than 80°C led to inefficient compound recovery, while higher temperatures (> 90°C) caused significant enzyme degradation (> 30%) [8]. Therefore, a 80–90°C range was selected as optimal for balancing bioactive extraction with the preservation of lipase functionality, supporting the intended metabolic efficacy of the final product.

           The resultant extracts were filtered through a 200-µm mesh and centrifuged at 5,000× g for 10 min to remove insoluble residues, targeting a residue level of < 0.05 g.100 ml^-1. Then, clarified supernatant was concentrated at 50.00 °C ±2.00 under a vacuum of approximately 0.08 MPa until the volume decreased to one-third of the original. The concentrated extract was spray-dried using Buchi B290 at inlet temperature of 160.00 °C ±2.00 and feed rate of 5 ml.min^-¹. Although freeze-drying was addressed for its enzyme retention benefits, the higher associated costs led to the selection of spray drying [21]. The final powder, showing a moisture content less than 6%, was sealed in airtight packaging and stored at 4.00 °C ±1.00.

2.6. Mixture Design and RSM Optimization

           A second-order mixture design was used to develop a formulation of seven independent variables (components), including (1) fermented lotus leaf extract powder, (2) breadfruit leaf powder, (3) lotus seed extract powder, (4) notoginseng flower extract powder, (5) C. militaris extract powder, (6) hydrolyzed collagen and (7) instant coffee. Fifteen formulations (including 12 edge points and three center points) were assessed in triplicate. The dependent variables (responses) monitored for each formulation included polyphenol content (mg GAE.g^-1; GAE = Gallic acid equivalent), DPPH radical scavenging activity (%), lipase activity (U.g^-1), sensory score (9-point scale) and dissolution time (s).

2.7. Pilot-scale Preparation of Optimized Coffee Blend

           The optimal formulation identified in the RSM optimization was scaled up to a pilot production batch of approximately 100 kg. The scale-up process included (1) dry blending 100 kg of extract powders, instant coffee and collagen using 50-l ribbon mixer (batch capacity of ~20–25 kg per cycle) for 5–10 min; (2) dissolving the blended powder in 1,000 l of hot water (1:10 w/v) and stirring at 60 °C for 30 min using jacketed stainless-steel tank; (3) spray drying the solution using Buchi B-290 system (feed rate, 5 ml.min^-¹; total process time, ~6–8 h; operating pressure, ~0.40 bar; inlet temperature, 160.00 °C ±2.00); and (4) packaging the resulting powder in multilayer aluminum bags (500 g per unit), ensuring a final moisture content less than 5.00% with storage at 25.00 °C ±2.00 and ~60.00% RH (AOAC 2019). Process parameters were continuously monitored to ensure batch uniformity and scalability.

2.8. Quality Control During Scale-up Production

           To ensure consistency between laboratory-scale and pilot-scale batches, quality control parameters were systematically monitored through the scale-up process. Moisture content of the final powder was set at less than 5% and water activity was controlled at approximately 0.28 ±0.01 to prevent microbial growth. Spray drying conditions-including inlet temperature (160.00°C ±2.00), feed rate (5ml.min^-1) and outlet temperature (~85°C)-were monitored at regular intervals. Microbial safety was verified by total viable count, which was less than 10³ CFU.g^-1 and heavy metal contents (Pb, Cd and Hg) were within the safety limits reported by QCVN 8-2:2011/BYT. Batch to batch uni-formity was assessed by investigating key indicators, including polyphenol content, DPPH radical scavenging activity and lipase activity in three production batches. All powder batches were sealed in 500-g multilayer aluminum bags under low-humidity conditions and stored at 25.00 °C ±2.00 and approximately 60.00% RH. These measures ensured that the pilot-scale product included functional, sensory and safety characteristics of the optimized formulation.

2.9. Clinical Trial

           A randomized, double-blind, placebo-controlled clinical trial was carried out over 6 m with 151 participants aged 25–55 y and a body mass index (BMI) ≥ 25.00 kg.m^-2. Participants were randomly assigned to intervention or control groups using block randomization (block size = 4), with clear inclusion and exclusion criteria to ensure study accuracy. The intervention group received the nutritional coffee product combined with standardized dietary and exercise counseling, while the control group received counseling only. Participants in the two groups included daily food diaries and physical activity logs, facilitating objective monitoring of compliance. Dietary adherence was assessed monthly by trained nutritionists through standardized food diaries, while exercise compliance-recommended at ≥ 30 min.d^-1 of moderate-intensity activity (e.g., brisk walking and cycling)—was self-managed but regularly reviewed and reinforced during monthly follow-up sessions. Compliance, adverse events and predefined outcome measures were continuously monitored, with data analysis carried out using repeated-measures ANOVA (significance set at p < 0.05).

2.10. Consumer Survey

           A market survey was carried out over a 6-m time period with 1000 consumers aged 18-60 y. Using standardized questionnaire administered via convenience sampling, the survey collected information on product perception (e.g. aroma, taste, and repurchase intention), as well as demogr-aphic and consumption habit data. An anticipated dropout rate of approximately 20% was factored into the survey design.

2.11. Quality Control and Repeatability

All analytical measurements-including polyphenol con-tent, DPPH radical scavenging activity, lipase enzyme activ-ity, microbial analysis, sensory assessments, heavy metal analyses and optimization experiments-were carried out with at least three independent replications (n ≥ 3). The RSD for each analytical method was set less than 5.00%; thereby, ensuring high reliability and reproducibility of the data.

2.12. General Workflow

Figure 1 presents an overview of the entire research workflow-from raw material selection through fermenta-tion, extraction, pilot-scale production, clinical trial implementation and market assessment.

1. Results and Discussion

3.1. Quality and Characteristics of Raw Materials

Table 1 summarizes the assessed characteristics of the raw materials, including moisture content, polyphenol level, total viable aerobic microbial count (TVC) and heavy metal concentration (Pb, Cd and Hg) for the coffee blend (C. canephora:C. arabica 70:30), N. nucifera (lotus leaves), A. altilis (breadfruit leaves), lotus seeds, P. notoginseng (notoginseng flowers), C. militaris (caterpillar fungi) and hydrolyzed collagen. All raw materials were verified to comply with the microbiological and heavy metal safety criteria stipulated in QCVN 8-2:2011/BYT [16]. Specifically, TVC values were consistently less than 10³ CFU.g^-1 and the levels of Pb, Cd and Hg were less than detection limits. Each parameter was assessed in triplicate (n = 3) with an RSD less than 5%, except for the polyphenol assessments, which showed minor variability (±0.20–0.40 mg GAE g^-1; RSD < 10%). For example, coffee demon-strated a moisture content of approximately 11.00% ±0.02, while lotus leaves, breadfruit leaves, notoginseng flowers and C. militaris showed moisture levels in 7–8% range. Lotus seeds included a slightly lower moisture content (approximately 6–7%). Polyphenol content, assessed using Folin–Ciocalteu method [15], ranged 2.80-5.80 mg GAE.g^-1, coffee ranged 5.80 ±0.30 mg GAE.g^-1, N. nucifera ranged 4.90 ±0.40 mg GAE.g^-1, A. altilis ranged 3.70 ±0.30 mg GAE.g^-1, P. notoginseng flowers ranged 5.00 ±0.40 mg GAE.g^-1 and lotus seeds ranged 2.80 ±0.20 mg GAE.g^-1. Compared to published literature, these polyphenol levels were moderate to relatively high. For example, previous studies reported that typical polyphenol contents for coffee beans ranged nearly 3.0-6.0 mg GAE.g^-1 [29,30] and polyphenol levels in herbal materials such as lotus leaves and linked plant extracts typically ranged 2.0-7.0 mg GAE.g^-1 [5,6,7]. Thus, the present values (e.g. 5.80 mg GAE.g^-1 for coffee and 4.90 mg GAE.g^-1 for lotus leaves) indicated strong potential for antioxidant activity and beneficial bioactive characteristics. Hydrolyzed collagen, with a moisture content of 4.10%, did not show detectable levels of polyphenols. These data not only verified that the raw materials included the necessary safety standards but also establish a robust baseline-particularly 4.90 mg GAE.g^-1 value in N. Nucifera-for assessing subsequent fermentation effects. Additionally, the assessed lipase enzyme activities ranged 11.00-26.00 U.g^-¹, where 1 U is reported as the quantity of enzyme needed to release 1 µmol of p-nitrophenol per minute at 37 °C, pH 7.0, using p-NPP as substrate.

3.2. Fermentation Outcomes

           Fermentation parameters such as temperature, relative humidity and duration are well known to affect the biotransformation of bioactive compounds in medicinal plants by B. subtilis [7] [22]. As shown in Figure 2A, the raw (unfermented) lotus leaf powder included vibrant green color with a polyphenol content of approximately 4.90 mg GAE.g^-1, DPPH radical scavenging activity of nearly 60.00% and lipase activity of 15-20 U.g^-1. Following fermentation under optimized conditions, the powder color shifted to darker brownish-green (Figure 2B), suggesting an increase in bioactive compounds. Box-Behnken Design was used to systematically assess the effects of three key factors—temperature (30–35 °C), relative humidity (60–70%) and fermentation time (48–72 h)—on the response variables (polyphenol content, DPPH activity and lipase activity). Detailed results from 15 experimental runs (12 edge points and three center points), each carried out in triplicate (n = 3, RSD < 5%), are present in Table 2. The second-order ANOVA demonstrated a high level of statistical significance (p < 0.001) and an excellent model fit (R² > 0.95) with no significant lack-of-fit (p = 0.21). All three factors significantly affected the fermentation outcomes (p < 0.01) and significant interactions were observed between temperature and time (p < 0.05).

Figure 3A presents a three-dimensional (3-D) surface plot, showing that under optimal conditions—35 °C and 72 h—the polyphenol content reached approximately 12 mg GAE.g^-1. Complementary contour plots in Figure 3B indicate that DPPH radical scavenging activity increased to 78.00–80.00% under these conditions, while the 3-D plot in Figure 3C demonstrates that lipase activity peaked at approximately 38–40 U.g^-1. Validation of the three independent batches verified that these improvements were reproducible, with variation was 5.00%. In summary, fermentation under the optimized conditions of 35 °C, 65.00–70.00% RH and 72 h resulted in a nearly 2.50-fold increase in polyphenol content relative to the raw material, with significant enhancements in antioxidant capacity and lipase activity. These results provide a solid foundation for the subsequent extraction, fiber removal, drying and final formulation of the nutritional coffee products. This 2.5-fold increase in polyphenol content was well similar to or exceeded enhancements reported in previous studies involving microbial fermentation. For example, He et al. [5] reported nearly 1.5 to 2-fold increases in polyphenols through fermentation of lotus leaves, while Juan and Chou [6] reported up to 2-fold increases in polyphenols when fermenting black soybeans with B. subtilis. The observed enhancement in lipase activity (approximately doubled) was similar to or higher than those described in similar fermentation studies using B. subtilis on plant substrates, typically reporting increases 1.5 to 2-fold [7,22]. Therefore, these results indicated that the present optimized fermentation process was effective and competitive when benchmarked against the current literature.

3.3. Extraction, Fiber Removal and Spray Drying of the Fermented Lotus Leaves

           To achieve fiber‐decreased extract powder enriched with bioactive compounds, the fermented lotus leaves were hot-water extraced, filtration-centrifuged, concentrated and spray dried. Although similar procedures were used to other fibrous medicinal ingredients (breadfruit leaves, lotus seeds, notoginseng flowers and C. militaris), detailed results were present only for the fermented lotus leaves because they showed significant changes in polyphenol content and lipase activity. The process was divided into two major stages as follows.

Filtration-centrifugation: The fermented lotus leaves were extracted using hot water at 80–90°C with 1:10 (w/v) ratio for 30–60 min under continuous stirring. The resulting extract, showing yellow-brown hue and moderate clarity (Figure 2C), was filtered through a 200-µm mesh and centrifuged at 5,000× g for 10 min to remove insoluble fibers. As detailed in Table 3, this step decreased the fiber residues from 1.30 ±0.20 to less than 0.05 g.100 ml^-1 (approximately 96% increases, p < 0.01), while the polyphenol content decreased slightly by nearly 6% (10.80 ±0.30 to 10.20 ±0.30 mg GAE.g^-1, p < 0.05) and lipase activity showed a negligible increase of nearly 3.60% (39.20 ±1.50 to 37.80 ±1.30 U.g⁻¹, p > 0.05). After spray drying, the polyphenol content further decreased by approximately 11% (10.20 ±0.30 to 9.10 ±0.30 mg GAE.g^-1, p < 0.01) and the lipase activity decreased by nearly 17.00% (37.80 ±1.30 to 31.40 ±1.20 U.g^-1, p > 0.05), resulting in final powders with a moisture content of 5.60% ±0.20. The overall recovery yield—calculated as a ratio of the final spray-dried extract powder weight to dry weight of the initial fermented lotus leaves—was assessed as 20.50% ±1.20 (w/w). This final product demonstrated excellent solubility in hot water, making it well appropriate for incorporation into the final formulation (Section 3.4).

           In summary, the combined processing steps allowed the fermented lotus leaves to preserve approximately 80% of their initial lipase activity and 84% of their polyphenol content, with an overall recovery yield of nearly 20.5% (w/w). These results have been included in Table 3, providing a comprehensive overview of the extraction efficiency. Compared to similar herbal extraction and drying processes reported in previous literature, this recovery yield of 20.5% could be reported as moderate within a typical expected range. For example, herbal extraction yields commonly range approximately 15-30%, depending on the specific plant material, extraction temperature and solvent ratio [6, 7, 21]. Given the current optimized extraction parameters aimed at balancing compound recovery with minimal bioactive degradation, yield of 20.5% indicated satisfactory process efficiency consistent with industry standards and published studies.

3.4. Mixture Design and RSM Results

           To develop a multiple-component nutritional coffee formulation comprising seven ingredients— fermented lotus leaves, breadfruit leaves, lotus seeds, notoginseng flowers, caterpillar fungi, collagen and coffee (100% w/w)—second-order mixture design was used. The formulation content was established as follows: collagen ≤ 2%; caterpillar fungi, 0.30–0.50%; coffee ≥ 50%; fermented lotus leaves, 10.00–25.00%; notoginseng flowers, 3–5%; breadfruit leaves, 9.00–10.00% and lotus seeds, 6–8%. A ≤ 2% limit for collagen was established based on preliminary sensory tests and previous literature, which indicated that higher collagen concentrations could negatively affect sensory attributes by causing undesirable texture changes such as increased viscosity and off-flavors [31, 32]. Despite this concentration limit, collagen significantly contributed to the formulation by enhancing sensory characteristics, particularly mouthfeel smoothness and creaminess as well as providing additional nutritional benefits linked to joint and skin health [31, 33]. Thus, limiting collagen to ≤ 2% optimally balanced sensory acceptance with functional and nutritional contributions.

The experimental design consisted of 15 formulations (12 edge points and three center points), each replicated three times (n = 3). The second-order ANOVA analysis demonstrated high statistical significance (p < 0.05) with a strong model fit (R² > 0.95) and no significant lack-of-fit (p > 0.05), verifying that the model was well fixed for the investigated range. Analysis of interactions in ingredients revealed several significant synergistic and antagonistic effects. Specifically, significant positive interactions (p < 0.05) were observed between fermented lotus leaves and coffee, enhancing polyphenol content and antioxidant capacity. Additionally, interactions between breadfruit leaves and lotus seeds showed positive effects on lipase enzyme activity. In contrast, a mild antagonistic effect was detected between collagen and coffee at collagen concentrations above 2%, negatively affecting sensory scores. These interactions emphasized complexity of the ingredient effects in multiple component formulations, highlighting the necessity of careful ingredient ratio optimization to achieve balanced sensory and functional attributes. Five key response parameters were monitored:

1. Polyphenol content (mg GAE.g^-1);
2. DPPH radical scavenging activity (%);
3. Lipase enzyme activity (U.g^-1) with a target of achieving at least 70% of the initial value (~28.00 U.g^-1, associating to ~40.00 U.g^-1);
4. Sensory score was assessed on a 9-point hedonic scale by a panel of 8–10 trained panelists (five females and five males, aged 25–45 y) from staff members and graduate students of the Faculty of Biology and Environment, Ho Chi Minh City University of Industry and Trade, Vietnam. Panelists were standard trained prior to assessment, focusing on sensory analysis methodologies, identification of key attributes and calibration exercises to ensure consistent scoring and interpretation of sensory descriptors;
5. Dissolution time (s).

A detailed summary of the mixture design outcomes is present in Table 4. Using a desirability function approach for multiple objective optimization, a near-optimal formulation was identified: approximately 20.00% of fermented lotus leaves, 9.90% of breadfruit leaves, 7.00% of lotus seeds, 3.70% of notoginseng flowers, 0.37% of caterpillar fungi, 1.70% of collagen and nearly 57.00–58.00% of coffee, which achieved a desirability index of ~0.95. Figures 4A and 4B illustrate ternary plots, analyzing effects of key formulation components on polyphenol content and DPPH radical scavenging activity, respectively. These visualizations verified increased proportion of the fermented lotus leaves and coffee positively affected antioxidant characteristics of the final product. To validate the RSM model, three pilot-scale production batches (approximately 1 kg each) were produced using the optimal formulation.

Table 4 compares experimental assessments against the model predictions. The deviations between the predicted and experimental values were consistently less than 5.00% (p > 0.05). Specifically, the experimental results were as follows:

• Polyphenol content, ~9.30 mg GAE.g^-1;
• DPPH radical scavenging activity, ~78.00%;
• Lipase enzyme activity, ~29.00 U.g^-1 (~ 72.00% of the initial target);
• Sensory score, ~7.60/9; and
• Dissolution time, ~25 s.

           Figure 4C presents a comparative analysis of the model-predicted with experimentally assessed values for key parameters, including polyphenol content, DPPH activity, lipase activity and dissolution time. The close alignment between the predicted and observed values verified accuracy and robustness of the RSM model in optimizing the formulation. Sensitivity assays adjusting the collagen and fermented lotus leaf content by ±2.00% verified that the model predictions were robust, with errors of 5.00%. Although the sensory assessment was based on a relatively small panel (8–10 participants), these findings strongly supported that the combination of mixture design and RSM was an effective strategy for optimizing a multiple-component nutritional coffee formulation. Further studies should focus on expanding the sensory panel and refining the model with additional 3D surface and contour plots, as well as undertaking large-scale clinical assessments to assess long-term product stability and efficacy.

3.5. Pilot Production Results

           Following assessment of the optimal formulation (approximately 20.00% of fermented lotus leaves, 9.90% of breadfruit leaves, 7% of lotus seeds, 3.70% of notoginseng flowers, 0.37% of caterpillar fungus, 1.70% of collagen and the rest of coffee), a 100-kg pilot production batch was prepared to assess product stability and prepare the product-tentatively named “Nutrition Coffee Love World”- for clinical trials (Section 3.6). The product was packaged in 500-g aluminum bags under controlled conditions (25.00 °C ±2.00, RH ~60.00%). The multilayer aluminum packaging effectively contributed to product stability by providing superior barriers against moisture, oxygen and light—key factors that accelerate degradation of bioactive compounds such as polyphenols and enzymes. Specifically, aluminum layers significantly limited oxygen ingress and moisture vapor transmission; thereby, minimizing oxidative reactions and enzymatic degradations. Additionally, the opaque nature of aluminum packaging protects sensitive bioactive compounds from light-induced deterioration, collectively ensuring that the nutritional and functional qualities of the coffee products were stable through storage. Although full international certification has not been achieved, the product meets the criteria for clinical assessment. All ingredients used in this nutritional coffee formulation have carefully been selected and verified to comply fully with relevant regulatory standards, specifically meeting microbiological, heavy-metal and quality criteria by Vietnamese National Technical Regulations (QCVN 8-2:2011/BYT [16]). Furthermore, Certificates of Analysis (COA) provided by the supplier validated the quality, purity and safety of each herbal component and collagen, ensuring their appropriateness for clinical uses.

           Spray drying was carried out using Buchi B-290 with inlet temperature of 160.00 °C ±2.00, feed rate of 8-10 ml.min^-1 and processing capacity of 12-15 l.h^-1, yielding outlet temperature of 80-85 °C and total drying time of 2–3 h. Special attention was specified to the high-moisture phase during the initial 30-45 min to ensure that the outlet temperature did not exceed 90 °C; thereby, minimizing lipase degradation. Five production batches (n = 5) were produced and compared with a 1-kg batch to assess consistency. Figure 2D shows spray-dried nutritional coffee powders from the 100-kg pilot batch, characterized by fine particles (~120 µm) with light brown-gray color. Figure 2E demonstrates a reconstituted beverage prepared with hot water (90–95 °C), showing deep brown color and complete dissolution within 30 s without sedimentation.

The quality attributes of the 100-kg pilot product and the changes in key quality parameters within 3–6 m of storage are comprehensively summarized in Table 5. These attributes included moisture content, water activity, particle size (D50), microbial load, heavy metal content, dissolution time, polyphenol content, DPPH radical scavenging activity, lipase activity, sensory assessment score and their stability over storage. Specifically, polyphenol content decreased from 9.10 to 8.70 mg GAE.g^-1 (~4% loss), lipase activity decreased from 28.50 to 27.00 U.g⁻¹ (~5.00% loss), DPPH activity decreased from 77.00 to 75.00%, sensory scores slightly decreased from 7.60 to 7.40/9 and moisture content increased marginally from 4.80 to 5.00%. Moreover, ANOVA verified that these changes were 5.00% (p > 0.05) and microbial loads and heavy metal levels were stable, demonstrating product stability. Acceptance criteria for batch-to-batch variation and degradation were clearly set at ≤ 5% based on industrial standards and previous literature, indicating that variations in bioactive compound levels and enzyme activity within this range included minimal effects on overall product efficacy and quality. Specifically, maintaining polyphenol content and lipase enzyme activity variations less than 5% ensured consistent functional benefits, antioxidant capacity and sensory characteristics within the production batches, aligning with typical quality control standards for functional food products [21, 34]. Additionally, microbial analysis included total aerobic mesophilic bacteria as general hygiene indicators and explicitly assessed for pathogenic micro-organisms such as Escherichia coli, Salmonella spp., Staph-ylococcus aureus, molds and yeasts. All these micro-organisms were enumerated less than detection limits, ensuring compliance with microbiological safety standards based on QCVN 8-2:2011/BYT [16]

A separate cost comparison between 1-kg and 100-kg batches is provided in Table 6, the 1-kg batch included a cost of approximately 13 USD.kg^-1, whereas the 100-kg batch achieved a cost of ~6 USD.kg^-1, underscoring a significant economy of scale. Figure 5A illustrates quality parameters over storage, while Figure 5B provides a comparison between the model-predicted and experi-mentally assessed values. Overall, the pilot production data verified that the product included stable quality and that scaling up to 100 kg significantly decreased production costs. Further studies address accelerated shelf-life testing, further scaling to ≥ 500 kg, expanding the sensory panel to > 30 participants and carrying out long-term in vivo trials to assess economic and technical viabilities of the formulation.

3.6. Clinical Trial Results

           A randomized controlled trial was carried out within 6 m in adults aged 18–59 y with a BMI ranging 23-40 kg.m^-2, following the Asian criteria for overweight and obesity [24]. From 153 volunteers, two volunteers were excluded due to incomplete data, resulting in a final enrollment of 151 subjects, who were randomized into two groups. The intervention group (n = 78) consumed “Nutrition Coffee Love World” (1–2 sachets per day, each containing 18 g) alongside standardized counseling to decrease daily caloric intake by approximately 300–500 kcal and engage in at least 30 min of daily exercises. The control group (n = 73) received a similar dietary and exercise counseling, which was delivered using standardized protocol to ensure consistency in groups. At 6 m, 66 participants in the intervention group and 61 in the control group completed the study (n = 127), corresponding to an attrition rate of approximately 16%. Block randomization (block size = 4) was used with an expected attrition rate of 15.00% and the study was carried out based on the CONSORT guidelines [25]. All participants provided written informed consent and the study protocol was approved by the 7A Military Hospital Ethics Committee.

           Table 7 presents the baseline characteristics of the two groups (n = 127 after 6 m). The groups were similar in age (p = 0.772), sex distribution (p = 0.864), BMI (p = 0.741), hypertension prevalence (p = 0.963), baseline glucose level (p = 0.801) and baseline LDL-C (p = 0.912). The detailed clinical outcomes, including changes in weight, BMI, waist circumference, body fat percentage (assessed by bioelectrical impedance analysis, BIA), lipid profile and glucose level from baseline (M0) to 6 m (M6) for the two groups, are comprehensively summarized in Table 8. The intervention group showed significant improvements of weight increase (-1.40 kg ±2.10, p = 0.032), BMI decrease (-0.50 ±0.90 kg.m^-2, p = 0.041), waist circumference increase (-1.00 cm ±3.50, p = 0.049) and body fat increase (-1.40% ±2.60, p = 0.046). Visceral fat significantly decreased by -8.30 cm² ±12.50 (p < 0.05). For lipid profiles, LDL-C significantly decreased by -12.20 ±15.80 mg.dL^-1 (p < 0.01) and total cholesterol decreased significantly by -17.30 ±24.20 mg.dl^-1 (p < 0.01). Changes in glucose, HDL-C and triglycerides were minor. The control group showed minimal, statistically non-significant changes in all parameters. Fasting glucose levels decreased slightly in the intervention group (-0.20 mmol.l^-1, p = 0.05) but increased marginally in the control group (+0.10 mmol.l^-1, p = 0.32). Liver enzyme levels (AST and ALT) were stable through the trials and no serious adverse events were observed.

Figure 6A illustrates the changes in average body weight at baseline (M0), 3 m (M3) and 6 m (M6) for the two groups. The intervention group weight decreased from approx-imately 67.50 kg at the baseline to 66.80 kg at M3 and 66.10 kg at M6, while the control group weight was essentially unchanged (67.30 kg at M0, 67.20 kg at M3 and 67.10 kg at M6). Error bars represent SD.

In summary, the “Nutrition Coffee Love World” intervention led to a modest but statistically significant increase in body weight (-1.40 kg) and waist circumference (-1.00 cm) in the intervention group, compared to the control group. Additionally, improvements in lipid profiles (particularly increases in LDL-C and total cholesterol) were observed. Although the weight loss was modest, these findings suggested that nutritional coffee supplementation might support weight management and lipid profile improvement. These clinical benefits could be attributed directly to the bioactive compounds in the nutritional coffee formulation. Specifically, chlorogenic acids from coffee enhance lipid and glucose metabolisms; thus, supporting increases in body weight and improvements in lipid profiles. Polyphenols and flavonoids derived from fermented lotus leaves and breadfruit leaves included potent antioxidative and anti-inflammatory effects, which contributed to decreased oxidative stress and inflammation associated with obesity and dyslipidemia. Additionally, saponins from P. notoginseng enhanced lipid metabolism through activation of lipase enzyme activity, increasing triglyceride hydrolysis and fat oxidation. Furthermore, cordycepin from C. militaris modulated lipid biosynthesis pathways, collectively supporting the significant metabolic improvements in this trial. Future studies should include longer-term trials (≥ 12 m), larger sample sizes, stricter monitoring of dietary and exercise adherence and further precise body composition measurements (e.g., DEXA or MRI) to better assess long-term efficacy. Additionally, future studies should incur-porate intention-to-treat analysis approaches to provide unbiased estimates of intervention effectiveness and enhance generalizability. Systematic use of imputation methods for handling missing data is recommended to minimize bias from participant dropouts. Additionally, further studies should include detailed analyses of loss characteristics to better understand reasons for attrition, identify loss predictors and improve retention strategies; thus, ensuring further robust and reliable outcomes.

3.7. Consumer Survey Results

A market survey was carried out for 1,000 consumers aged 18–60 y, predominantly from urban areas (70%), with an average monthly income of 10–15 million Vietnamese Dong (approximately 400–600 USD). Participants were instructed to use “Nutrition Coffee Love World” for 3 m by mixing 15 g of powder with 120–150 ml of hot water per serving. From the participants, 20.00% (200/1000) did not complete the survey, primarily due to relocation or scheduling conflicts. Demographic characteristics, including age, gender, income and coffee consumption frequency, were analyzed and no significant differences were detected between losses and 800 respondents, who completed the survey (p > 0.05). Table 9 details the demo-graphic profile of 800 respondents, who had an average age of 34.60 y ±8.20, with 52.00% of them were female, 70.00% residing in urban areas, an average monthly income of 12.70 ±4.00 million Vietnamese Dong and 85.00% held at least a technical or college-level education. The loss rate was consistent with similar market surveys [26].

Participants assessed the product using five-point Likert scale, with average ratings of 4.20 ±0.60 for aroma, 4.00 ±0.70 for taste and 3.70 ±0.80 for price. Approximately 65.00% (520/800) of respondents indicated a willingness to repurchase the product (coded as 1 = yes and 0 = no). A logistic regression analysis was carried out with repurchase intention as the dependent variable, controlling for age, gender, income and location. The model goodness-of-fit was verified using Hosmer–Lemeshow test (p = 0.21) and it demonstrated moderate explanatory power with a Nagel-kerke R² of approximately 0.35 and an overall correct classification rate of 72.00%. Table 10 indicates that aroma (OR = 2.27, p < 0.001) was the strongest predictor of repurchase intention, followed by taste (OR = 1.80, p = 0.004). Price did not include statistical significance (OR = 1.34, p = 0.079) and demographic factors included no significant effects (p > 0.05). This suggested that consumers prioritized sensory attributes over pricing when deciding to repurchase.

Figure 6B presents a forest plot summarizing the odds ratios for all variables. The confidence intervals for aroma and taste were entirely greater than 1, whereas those for price and demographic variables were 1. Additionally, while the analysis was based on 800 completers, the 20.00% loss rate suggested that future studies should consider using intention-to-treat analyses or imputation methods to address missing data. Understanding whether losses differed in their initial product perceptions or consumption habits could help refine future market segmentation. As the survey sample predominantly represents urban consumers, generalizability of the findings to rural populations might be limited. Future studies should aim to include a further geographically and socioeconomically diverse sample. Specifically, extending consumer acceptance surveys to rural areas significantly enhanced the robustness, generalizability and applicability of market insights derived from the present study. Carrying out comparative analyses between urban and rural consumer responses could provide valuable information for targeted marketing strategies and broader commercial viability

Overall, the logistic regression model incorporating age, gender, income and location yielded a Nagelkerke R² of 0.35, indicating moderate explanatory power. The findings clearly demonstrated that sensory attributes, particularly aroma and taste were significant determinants of repurchase intention. With 65% of respondents expressing willingness to repurchase “Nutrition Coffee Love World,” the product showed significant commercial potentials. Further studies should expand the geographical scope, extend the use time and incorporate a further diverse consumer base to provide a further comprehensive market assessment. This study addressed the increasing public health issue of overweight and obesity in Vietnam by developing a novel multiple-component nutritional coffee formulation. The present formulation integrated fermented N. nucifera (lotus leaves), A. altilis (breadfruit leaves), lotus seeds, P. notoginseng (notoginseng flowers), C. militaris (caterpillar fungi), collagen and coffee. The study combined rigorous technical assessments—including B. subtilis fermentation and formulation optimization via a second-order mixture design—with practical uses such as a 6-m clinical trial and a comprehensive market survey.

The fermentation conditions optimized through BBD—35 °C, 65.00–70.00% RH and 72 h—not only improved key bioactive metrics such as polyphenol content and lipase activity but also demonstrated robustness within multiple runs, reinforcing reliability of the process for further scale-up. While these improvements are promising, it is important to acknowledge potential confounding factors such as batch-to-batch variability and fluctuations in environmental conditions during fermentation. Further studies should address these uncertainties by incorporating tighter process controls and additional replicates to further decrease variability. The mixture design model, with a robust fit (R² > 0.95, error < 5.00%), yielded an optimized formulation that achieved a sensory score of approximately 7.50/9. Despite the model high predictive accuracy, the relatively small number of replicates (n = 2–3 per formulation) might increase the risk of overfitting, particularly when additional bioactive components are considered. Furthermore, the sensory assessment was based on a panel of only 8–10 participants, which might limit the generalizability of consumer preferences in a product where taste is highly subjective. In the clinical trial (n = 127), the intervention group experienced a modest but statistically significant weight increase of −1.40 kg, with improvements in LDL-C and total cholesterol.

However, this weight loss, representing nearly 2.00% of body weight, were less than the 5.00% threshold recommended by the American Diabetes Association (ADA) for clinically significant benefits [27]. This finding suggested that while the formulation showed potentials, its clinical effects might be limited under the current intervention time and monitoring conditions. Further trials should address longer times or further stringent controls of dietary intake and physical activity to potentially achieve greater weight loss. From a product stability and industrial perspective, the increase in production cost observed when scaling up from 1 to 100 kg suggested strong economic feasibility. Nonetheless, it must be assessed if the process parameters optimized at the pilot scale are held when production is further scaled (e.g. to 500 kg or further). Detailed analyses of enzyme stability during brewing and long-term shelf-life assessments are necessary to ensure that product quality is preserved at larger scales.

The consumer survey results, indicating a 65.00% repurchase intention, provided encouraging evidence of commercial potential. However, the survey sample predominantly represented urban consumers through convenience sampling and a loss rate of 20.00% was recorded. These factors limited the generalizability of the findings and suggested that further market assessments should include further geographically and socioeco-nomically diverse samples. Furthermore, while the present study focused on quantifying polyphenol, lipase and collagen levels, other important bioactive compounds such as saponins and chlorogenic acid were not assessed. Their omission might limit understanding of the full metabolic benefits of the formulation. Further studies should use advanced analytical methods such as LC-MS or HPLC to quantify these compounds and investigate potential synergistic interactions. In summary, the present inter-disciplinary approach-which integrates microbiology, biochemistry, food technology, nutrition, clinical research and market analysis-provides a solid foundation for the development of a functional nutritional coffee products. Despite limitations linked to sample size, intervention time and the scope of bioactive analysis and improvements in polyphenol content, enzyme activity and lipid profile as well as the positive consumer response underscore the potential of this formulation. Further studies should focus on larger and longer-term clinical trials, enhanced process optimiz-ation and further comprehensive bioactive profiling to fully realize the product benefits and ensure its scalability in industrial production.

4. Conclusion

This study effectively created a multiple-nutrient coffee product that combined fermented lotus leaves, breadfruit leaves, lotus seeds, notoginseng flowers, C. militaris, hydrolyzed collagen and coffee. Stringent optimization procedures such as B. subtilis fermentation, mixture design, 6-m human trial and consumer questionnaire were verified with beneficial effects in weight loss, better lipid profiles and high consumer preferences. With weaknesses in sample size and trial time, its commercial viability is strongly suggested by its good metabolic effects, scalability and economic benefits. Large-scale validation and further fine-tuning are needed to investigate its potential as an extended metabolic health improvement solution. This study results are useful for scientists, providing valuable practical directives for food industries worldwide to design novel functional drinks for better metabolic health and weight control.

1. Acknowledgements

The authors sincerely appreciate supports from Love World Group Joint Stock (Cong ty Co phan Tap đoan Love World) for funding this study under the Scientific and Technological Mission contract no. HDKHCN/01-2024-LoveWorld. The authors also appreciate Institute of Research and Application for Science and Technology Asian (IRASTA), Institute of Research and Application of Quantum Mechanical Technology, Ho Chi Minh City University of Industry and Trade (HUIT), NTT Hi-Tech Institute (Nguyen Tat Thanh University, Ho Chi Minh University of Science) VNU-HCM & 7A Military Hospital for their contributions, including providing research facilities, technical expertise and academic resources. Furthermore, the authors thank all participants and research staff for their contributions, which were critical for the successful completion of this study.

1. Conflict of Interest

The authors report no conflict of interest.

1. Authors’ Contributions

Conceptualization, HDT and HCN; methodology, HCN and HSD; software, BTN; validation, HCN, PPTH and HP; formal analysis, HCN and QDQ; investigation, HSD, PPTH and QTL; resources, HDT; data curation, HCN and TLL; writing—original draft preparation, HCN, PPTH and HSD; writing—review and editing, HDT and HP; visualization, QTL; supervision, HDT; project administration, HDT; funding acquisition, HCN.

1. Using Artificial Intelligent Chatbots

No AI chatbot has been used in this study.

1. Ethical Consideration

The authors declare no conflict of interest. Ethical approval no. 16/HDDDNCYSH dated June 20, 2024 was issued by the Biomedical Research Ethics Council of Military Hospital 7A.

Background and Objective: Improving probiotics viability in digestive and storage conditions is challenging for the food and pharmaceutical industries. The present study aimed to increase viability of the microencapsulated probiotic strain of Lactobacillus reuteri ATCC 23272 in O-carboxymethyl chitosan-coated bionanocomposite. The O-carboxymethyl chitosan was used to coat bionanocomposite containing prebiotics of pectin and inulin in presence of magnesium oxide nanoparticles.

Material and Methods: Pectin and inulin were used as prebiotics with magnesium oxide nanoparticles to improve the microgel structure and O-carboxymethyl chitosan for coating the microcapsules for increasing viability and stability of the probiotics. The extrusion efficiency, viability after microwave oven drying, survival in the simulated digestive fluids, viability after heat treatment and survival rate in long-term storage at 4 and 25 °C after 42 d were analyzed. Optimization of inulin, pectin and O-carboxymethyl chitosan in O-carboxymethyl chitosan-coated alginate-based bionanocomposite was achieved using Design-Expert software and simplex lattice mixture design.

Results and Conclusion: Optimal formulation was achieved using O-carboxymethyl chitosan coating polymer (68% w/v), inulin (29.4% w/v) and pectin (2.6% w/v) with magnesium oxide nanoparticles at a constant concentration. Results showed microencapsulation efficiency (96.43%), survival after microwave oven drying (99.45%) and survival in simulated gastrointestinal conditions (88.95%). Probiotic viability entrapped in O-carboxymethyl chitosan-coated microcapsules decreased by 1.46 log CFU.g^-1 at 80 °C for 5 min. Moreover, O-carboxymethyl chitosan-coated bionanocomposite improved the stability of probiotics by 2.93 and 3.25 log CFU.g^-1 at 4 and 25 °C after 42 d, compared to alginate beads. Additionally, it was observed that O-carboxymethyl chitosan coating enhanced the stability of probiotics entrapped in bionanocomposite beads. Results demonstrated that O-carboxymethyl chitosan-coated bionanocomposite, as a novel microencapsulation, could significantly increase the shelf life and viability of Lactobacillus reuteri in various harsh conditions, compared to alginate beads.

Conflict of interest: The authors declare no conflict of interest.

1. Introduction

Probiotics are beneficial live microorganisms that must be consumed sufficiently and survive the digestive tract for their effectiveness [1]. Due to the specific conditions of the mouth, stomach, small and large intestine, probiotics are susceptible to degradation. The multiple-covering technique enhances their stability and viability under these challenging conditions [2]. Therefore, coated microorganisms are expected to include higher viability than that non-coated microorganisms. One of the challenges is the efficacy of coating materials in improving viability and stability of bacteria under harsh conditions. Therefore, it is critical to choose an appropriate composition for the coating and microencapsulation of probiotics [3]. Alginate and chitosan are the most widely used natural polymers for the microencapsulation of probiotics. Prebiotics have recently been addressed to improve the viability of probiotics. Prebiotics may decolonize pathogens by modulating gut diversity; thereby, improving the growth of probiotics and decreasing the number of pathogens [4]. In addition to microencapsulating probiotics, coating bacteria as a layer on microencapsulating polymers can increase bacterial resistance to digestive and storage conditions. Simultaneous microencapsulation and coating of probiotics can include a positive effect on improving cell viability.

Alginate, a carbohydrate polymer, is widely used in the food and pharmaceutical industries due to its non-toxicity, biodegradability, biocompatibility and ease of preparation. It can protect microorganisms from bile salts and stomach acid. However, alginate gels are susceptible to degradation in presence of monovalent ions, Ca²⁺ chelating agents, extreme pH levels and harsh chemical conditions, which can accelerate the release of encapsulated substances [5, 6]. Relatively, CMC is a water-soluble derivative of chitosan. In addition to cationic amine groups in chitosan, CMC contains further anionic carboxylic groups, which provide several potentials such as ampholytic characteristics. There are three categories of CMC based on the functional groups participating in the reaction such as O-CMC, N, O-CMC and N-CMC. The poor solubility of chitosan in water is one of the disadvantages of chitosan in drug delivery. The O-carboxymethyl chitosan (OCMC) effectively addresses the solubility issues associated with chitosan in aqueous solutions. Moreover, OCMC has attracted significant attentions due to its enhanced solubility, high viscosity, low toxicity and beneficial biocompatibility characteristics. Li et al. reported that ionic cross-linking through ionic interaction in alginate-CMC hydrogel could enhance the survival of Lactobacillus casei ATCC 393 against adverse conditions [7].

Inulin is a non-digestible fructan-type carbohydrate, soluble dietary fiber and includes short-chain fructooligo-saccharides (scFOS). It is widely addressed as a prebiotic because it can selectively be consumed by the gut microbiota, promoting growth of probiotic bacteria [8]. Zabihollahi et al. demonstrated that the survival of L. plantarum in carboxymethyl cellulose-based film increased significantly (36%) with the addition of inulin as a prebiotic during storage [9]. The pectin structure includes a linear chain of α-(1, 4)-linked D-galacturonic acid units, commonly identified as the homogalacturonan domain or the smooth region. As a prebiotic, pectin plays a critical role in modulating composition and metabolism of intestinal microbiota and decreasing possibility of intestinal colitis [10]. The pectin-inulin composite has enhanced the survival rate of L. casei and L. rhamnosus, compared to free cells in a simulated gastrointestinal tract (GIT) [11]. Magnesium oxide nanoparticles (MgONPs) have recently been highlighted as a potential candidate for controlled drug delivery systems due to their biocompatibility, non-toxicity, biodegradability, stability and various biomedical characteristics such as anticancer, antioxidant and antidiabetic characteristics. Using MgONPs in alginate-gelatin microgel showed significant advantages in enhancing viability of Pediococcus pentosaceus Li05 in heat treatment, simulated digestive fluid and long-term storage [12].

Researchers have implemented various strategies to increase bacterial viability, including use of prebiotics, nanoparticles and microcapsule coating separately. For example, use of MgONPs in the microencapsulation of bacteria has been studied to improve viability of probiotics in the digestive system. Research has shown that using prebiotics improves probiotics viability during the microencapsulation process and various polymers to improve the stability of probiotics. To improve probiotic viability under various harsh conditions, the present study simultaneously used prebiotics, MgONPs and OCMC as microcapsule coatings. The simultaneous use of prebiotics, MgONPs and OCMC in the microencapsulation of probiotics can improve the viability of probiotics under various harsh conditions by creating a synergistic effect. To the best of the authors’ knowledge, no studies are available that investigate bacterial viability by coating microcapsules containing MgONPs and prebiotics. The experimental design used a simplex lattice mixture method to assess the optimal percentage of OCMC, inulin and pectin. The quantities of MgONPs and sodium alginate (SA) were constant in all experiments. Drying was carried out using microwave oven. Encapsulation efficiency, survival in simulated digestive fluids, heat treatment of microencapsulated probiotics and stability in storage conditions were calculated as well.

1. Materials and Methods

2.1 Materials

The probiotic strain of L. reuteri ATCC 23272 was provided by the Iranian Scientific and Industrial Research Organization, Tehran, Iran. The OCMC (purity greater than 98%, deacetylation degree of 80%, carboxyl substitution degree of greater than 80% and amino content of nearly 1.45%) was purchased from Macklin, Shanghai, China. The MgONPs (purity greater than 99% and APS of 20 nm) were purchased from US Research Nanomaterials, Houston, TX, USA. Inulin (molecular weight of nearly 5000 Da with food-grade), low methoxyl pectin of citrus source (molecular weight of 70–140 kDa, degree of esterification of 27%) and SA (viscosity of 2000 cp, molecular weight of 80–120 kDa and M/G ratio of 1.56) were purchased from BSK Pharmaceutical, Tehran, Iran. Pancreatin from porcine pancreas (6000 FIP-U.g^-1 lipase, 350 FIP-U.g^-1 protease and 7500 FIP-U.g^-1 amylase), pepsin from porcine gastric mucosa (P700, 250 U.mg^-1), lactic acid, sodium citrate, glucose, calcium chloride, bile salts, trypticase soy broth (TSB) and trypticase soy agar (TSA), deMan Rogosa Sharpe (MRS) agar and broth (Cat no. 110660) were purchased from Sigma Aldrich, St. Louis, USA.

2.2 Probiotic Culture Preparation

The L. reuteri ATCC 23272 was incubated in MRS broth at 37 °C for 24 h. The probiotics were harvested by centrifuging at 1790 g for 10 min at 4 °C. Probiotic cells were washed twice with sterile PBS (PBS) (pH 7.4). To ensure the elimination of the supernatant from the culture broth, bacteria were recentrifuged under similar conditions and then resuspended in 2 ml of PBS [13, 14].

2.3 Preparation of Polysaccharides-based Bionanocom-posite

Various compositions have been prepared for inulin and pectin prebiotics. The ratio of inulin:pectin of 10.9:1 was chosen as the optimal concentration, which was achieved in a previous study [15]. Based on the concentrations presented in Table 1, prebiotics were suspended in deionized water with a certain concentration (Table 1), MgONPs at a concentration of 5 μg.ml^-1 were added to the mixture and stirred at 27.95 g for 60 min at 60 °C. Then, polysaccharides-based bionanocomposite was autoclaved at 121 °C for 20 min.

2.4 Microencapsulation Process

The composition of bionanocomposites (Table 1) was added to the cell suspension at a 1:1 (v/v) ratio and 37 °C and mixed using vortex to microencapsulate L. reuteri. The suspension of synbiotic was homogenized entirely to a 2% SA (w/v) solution. Cell suspensions were extruded using an insulin syringe in 0.1 M CaCl₂ solution. The interaction of uronic acid carboxylic groups with calcium ions formed a gel network, creating probiotic beads. To verify complete gelation of the beads, the CaCl₂ solution was stored at 4 °C for 30 min. Probiotic microgels were separated from the CaCl₂ solution using Whatman grade-1 filter papers (pore size of 11 μm) (Whatman, USA). These were washed twice with PBS. During the microencapsulation process, sterile conditions were addressed.

2.5 Coating Microcapsule with O-carboxymethyl Chitosan

The OCMC solution was prepared using method described by Mi et al. [16] with some modifications. Based on Table 1, OCMC solution was prepared by dissolving a certain quantity of OCMC (% w/v) in 95 ml of lactic acid solution (1% v/v). The pH was adjusted to 6 using 1 M NaOH. The solution was adjusted to 100 ml with distilled water (DW) and then filtered using Whatman grade-4 filter papers (pore size of 20 μm) (Whatman, USA). This was autoclaved at 121 °C for 15 min. Then, the alginate beads were immersed in the OCMC solution and agitated for 40 min at room temperature (RT) and 1.12 g for coating OCMC-coated microcapsules were separated using Whatman grade 1 filter papers (Whatman, USA). Then, beads were washed twice with PBS.

2.6 Encapsulation Efficiency

The number of cells released from microcapsules was calculated using method described by Halim et al. [17] with some modifications. Briefly, 1 g of beads was mixed well in 9 ml of sodium citrate (50 mM) for 10 min at RT. Cells coated with OCMC were added to the sodium citrate solution after grinding for 1 min using mortar and pestle. The released L. reuteri was diluted in PBS and then counted using pour plate method on plates containing MRS Agar. The efficiency of the extrusion was calculated using Eq. 1.

Survival rate (%)= ( ) ×100                                                      (Eq. 1)

Where, EE was efficiency of encapsulation, N was probiotics count released from beads (Log CFU.g^-1) and N₀ was the initial probiotics count added to the mixture (Log CFU.g^-1).

2.7 Survival Rate of Microencapsulated Cells after Drying

The encapsulated probiotics were dried in a microwave oven (Mwl210, Kenwood CO, UK) for 7 min with a power of 400 W. The microencapsulated cells were hydrated in PBS for 2 hours. Bacteria were serially diluted in a phosphate buffer. Probiotics were placed on an MRS agar medium by the pour-plate method. After incubation for 48 h at 37 °C, the colonies were counted. The survival rate was calculated using above mentioned Eq. 1. While, Where N: The Probiotics count after drying (Log CFU.g^-1) and N₀: The Probiotics count before drying (Log CFU.g^-1).

2.8 Survival Rate Of L. reuteri In Harsh Conditions

2.8.1 Simulated Gastrointestinal Digestion

Simulated intestinal and gastric fluids were prepared using method described by Mohamadzadeh et al. [15]. Pepsin at a concentration of 3 g.l^-1 was added to a saline solution (0.5% v/v) to prepare simulated gastric fluid. The final pH of the solution was adjusted to 2 using 1 N HCl. Simulated intestinal fluid was prepared by adding 4.5% (w/v) bile salt and pancreatin USP at a concentration of 1 g.l^-1 to a saline solution. The pH of the solution was adjusted to 8 by adding 1 N NaOH. The encapsulated-dried probiotics were added to 1 ml of simulated gastric fluid and incubated at 37 °C for 4 h. The mixture was centrifuged at 16100 g for 15 min. The supernatant was discarded and the cells were mixed with 1 ml of simulated intestinal fluid. As in the previous step, probiotics were incubated at 37 °C for 4 h and then centrifuged. After discarding the supernatant, probiotics were diluted using PBS. The released probiotics were counted. The viability under simulated gastrointestinal conditions was calculated using above mentioned Eq. 1. While, N was the probiotics count after exposure to simulated gastrointestinal digestion (SGD) (Log CFU.g^-1) and N₀ was the probiotics count before exposure to SGD (Log CFU.g^-1).

2.8.2 Heat Treatment of the Microencapsulated Probiotics

Alginate, bionanocomposite and OCMC-coated bionano-composite beads were assessed for heat resistance at 60 °C for 60 min, 70 °C for 30 min and 80 °C for 5 min. One gram of the dried beads was added to 9 ml of PBS. After heat treatment, tubes were cooled down to 37 °C and serially diluted. Cells were counted using pour-plate method and results were reported as log CFU.g^-1.

2.8.3 Long-term Storage

Alginate, bionanocomposite and OCMC-coated bionano-composite beads were dried using described methods. The probiotics stability was assessed at refrigerator temperature (4 °C) and ambient temperature (25 °C) for 6 w. Cell counts on MRS agar were carried out weekly and results were reported as log CFU.g^-1.

2.9 Characterization of the Microparticles

2.9.1 Scanning Electronic Microscopy

The microstructural characteristics and surface morphology of the dried samples, including inulin, pectin, bionanocomposites containing L. reuteri and OCMC-coated bionanocomposites containing L. reuteri, were analyzed using scanning electron microscopy (SEM). To enhance conductivity, samples were coated with a thin layer of gold and then analyzed at an accelerating voltage of up to 15 kV.

2.9.2 Fourier transform infrared spectroscopy (FTIR)

The structure of inulin, pectin, SA, OCMC, MgONPs and bionanocomposite microcapsules with and without L. reuteri and OCMC-coated bionanocomposite microcap-sules were investigated using Fourier transform infrared spectroscopy (FTIR) analysis. First, KBr spectrum was recorded as a control. Then, samples were mixed with KBr and a thin pellet was formed by compressing them at a pressure of 60 kPa for 10 min. Findings were present with a resolution of 0.5 cm⁻¹ within the wavelength of 400–4000 cm^-1.

2.9.3 X-Ray Diffraction

The X-ray diffraction (XRD) analysis was carried out using X-ray diffractometer (X’Pert MPD, Philips, the Netherlands) that used Cu Kα radiation at 40 kV, 30 mA and λ = 0.1542 nm. The diffraction patterns were recorded by monitoring diffractions with a scan speed at 0.02◦/s, within a 2θ angle range of 10–70◦ [18]. The XRD analysis was carried out on inulin, pectin, OCMC, bionanocomposite microcapsules with and without probiotics and OCMC-coated bionanocomposite microcapsules.

2.10 Experimental Design and Statistical analysis

This study used simplex lattice mixture design to optimize concentration of OCMC and various concentrations of prebiotics at a fixed ratio (inulin:pectin of 10.9:1). Table 1 presents the experimental design data. Results analysis was carried out using Design-Expert software. To assess the importance and effects of each element on the response, analysis of variance was carried out with a significance level of 95%. The coefficient of determination, R², verified validity of the regression model. All experiments were carried out in triplicate. A numerical optimization technique was used for the optimization process.

1. Results and Discussion

3.1 Encapsulation Efficiency of L. reuteri

The effects of various concentrations of inulin, pectin and OCMC on encapsulation efficiency were investigated. Table 1 shows various bionanocomposite formulations with and without OCMC coating. The encapsulation efficiency ranged from 95.35 (Run 5) to 98.13% (Run 7). The highest efficiency was achieved in absence of OCMC with a previously optimized prebiotic formulation (inulin:pectin of 10.9:1). The lowest extrusion efficiency was observed at 1% (w/v) concentration of OCMC without prebiotics. No use of OCMC resulted in easier releases of probiotics from the beads. Decreases in the viability of probiotics coated with OCMC could be due to the use of mortar and pestle to release the probiotics. The microencapsulation efficiency of alginate beads coated with chitosan decreased, compared to that of alginate beads [19]. Parsana et al. demonstrated that the encapsulation efficiency of L. reuteri in alginate beads (92.06%) was higher than that in alginate beads coated with chitosan with prebiotic inulin (90.63%) [20]. The coating process (e.g. agitation, pH changes and exposure to chitosan solution) could affect mechanical or osmotic stress that decreased integrity of the alginate matrix, resulting in decreased microencapsulation efficiency. Using inulin and pectin prebiotics in the microencapsulation of probiotics could improve probiotic growth as well. Poletto et al. showed that efficiency of the extrusion in presence of inulin (96.75%) was 2.65% higher than that in microencapsulation using alginate (94.10%) [21]. Probiotics could ferment prebiotics, providing an immediate source of metabolic energy. Even before reaching the gut, inulin and pectin could serve as nutritional reserves within the microcapsule. Efficiency of the extrusion in alginate beads was 95.23%, which showed a decrease of 1.29%, compared to the average efficiency of various formulations in the experimental design. Zaeim et al. reported that the efficiency of probiotics microencapsulated with alginate (98.12%) was 1.21% higher than that of microencapsulated probiotics with alginate-chitosan (96.91%) [22].

In Table 2, a positive value indicates a synergistic effect and a negative value indicates an antagonistic effect on the response. Based on statistical analysis, total inulin-pectin concentration and OCMC alone positively affected the extrusion efficiency. The inulin-pectin concentration was more effective than the OCMC concentration. The interaction of two variables included negative effects on the microencapsulation efficiency. The lack of fit in all models was greater than 0.1 with insignificancy. The microencapsulation efficiency was predicted using cubic model with a value of p < 0.001 (Table 2).

3.2 Survival Rate of Microencapsulated Cells after Drying

The drying efficiency of probiotics using microwave oven is shown in column Y₁ of Table 1. The highest survival efficiency was 99.41 (Run 4) and the lowest survival efficiency was 98.05% (Run 1). Protein denaturation, fatty acid (FA) oxidation, DNA damage and free radical formation were factors that decreased viability of the probiotics due to thermal drying. The lowest drying survival of L. reuteri occurred in absence of OCMC. Survival of bacteria in encapsulation depended on the thickness and type of coating materials [23]. Without OCMC, the coating depended on lighter materials such as alginate and prebiotics, which did not form an equally robust barrier, resulting in thinner layers. The presence of OCMC significantly increased viscosity and density of the outer layer, creating a thicker, further cohesive protective coating around the microcapsules ‌against thermal stress. Drying efficiency of the alginate beads was 95.98%. A 2.7% decrease in the viability of probiotics was observed, compared to an average of various formulations in the experimental design. The absence of inulin, pectin and OCMC as a coating layer decreased thickness of the bacterial coating layer and hence caused further heat damages to the probiotics. Jantarathin et al. showed that in addition to increasing the viability of cells during the drying process, chitosan improved the viability of bacteria after drying [24].

Based on the data fitting, the cubic model produced the lowest p-value regarding viability after drying (Table 2). The model significance was verified with a p-value < 0.0001. Two variables, total inulin-pectin concentration and OCMC, positively affected drying efficiency and the effect of OCMC concentration was greater than that of the inulin-pectin concentration. Decreasing the survival rate of probiotics during drying included a negative effect on the effectiveness of probiotics. The 99.41% viability of L. reuteri in the microwave process was a significant efficiency.

In addition to choosing the appropriate materials for microencapsulation, drying method significantly improved the viability of probiotics. Using microwave oven is a biocompatible method that can maintain the viability of probiotics at high rates within short times. Drying in microwave ovens increases product purity, improves the quality of the pharmaceutical powder and decreases byproducts and energy consumption [25]. Microwave ovens serve as innovations for drying food and pharmaceutical products.

3.3 Viability of Probiotics in Simulated Gastrointestinal Conditions

The viability rate of L. reuteri (Table 1, column Y₂) varied between 87.11 (Run 5) and 89.3% (Run 6). The lowest survival rate of probiotics was observed at 1% (w/v) concentration of OCMC in absence of prebiotics within the bionanocomposite structure. The MgONPs could serve as a buffering agent, enhancing the survival rate of probiotics by decreasing the acidity in the stomach. The lack of MgONPs release due to the OCMC coating with a concentration of 1% (w/v) could be a reason for decreasing the viability of probiotics in SGI conditions. The highest survival rate of probiotics in SGI conditions was achieved at a concentration of 0.5% (w/v) of OCMC and 0.5% (w/v) of prebiotics (inulin:pectin of 10.9:1). Combining OCMC with prebiotics provided mechanical protection and metabolic support to the probiotics, which could include a synergistic effect. A decrease of 2.87 log CFU.g^-1 of the microencapsulated cells in alginate microcapsules was observed in the simulated digestive fluids. The survival rate of alginate beads in the SGI conditions was 71.49%, which was 17% less than the average efficiency of various formulations in the experimental design. Afzaal et al. demonstrated that the survival of L. acidophilus ATTC 4356 microencapsulated within alginate in the simulated gastric fluid was associated to a decrease of 3.57 log CFU.ml^-1 [26]. The resistance of Lactobacillus strains at low pH could be attributed to F₀F₁-ATPase activity in probiotics [27]. The survivability of alginate-encapsulated probiotics significantly decreased by 2.26 ±0.24 log CFU.g^-1 [28]. Based on the data fitting, the cubic model produced the lowest p-value (0.0003) regarding the SGI conditions (Table 2). The model significance was verified with a p-value < 0.001. Three variables, including the total inulin-pectin concentration, concentration of OCMC and interactions of variables (AB), positively affected cell viability in SGI conditions. Similar to extrusion efficiency, inulin-pectin concentration was more effective than OCMC concentration. The effect of AB (A-B) parameters negative affected the viability of probiotics.

 3.4 O-Carboxymethyl Chitosan and Prebiotic Concentr-ation Optimization

Optimizing the inulin and pectin as prebiotics, as well as the OCMC quantity for coating microcapsules, is critical. Three parameters of encapsulation efficiency, survival rate of microencapsulated cells after microwave drying and survival rate of L. reuteri in SGI conditions were selected for optimization. The efficiency of encapsulation is important due to the presence of probiotic microgels in food, pharmaceutical and cosmetic industries. Optimizing viability after drying greatly affected the viability of dried powder and improved the long-term stability of probiotics. Optimizing survival in simulated digestive conditions enhanced the delivery of probiotics to the clone by increasing its survival in these conditions. Quantities of inulin and pectin with a fixed ratio (inulin:pectin of 10.9:1) and the concentration of OCMC were assessed to coat the microgels in a 0–1 g range. Optimization was achieved when the encapsulation efficiency, viability after microwave drying and viability after exposure to SGI conditions were simultaneously at their highest levels. After defining the highlighted conditions in Design-Expert software, the ratio of inulin to pectin was 32% (inulin, 0.294, and pectin, 0.026) and the OCMC concentration was reported as 68% (Table 3). The quantities of MgONPs and SA were similar to them in all compositions. Optimization was valid when the desirability function was acceptable. The desirability was 90%. The optimization results were verified by carrying out experiments. The optimization results revealed that the OCMC-coated bionanocomposite could serve as a novel approach to protect cells and enhance survival of bacteria in harsh conditions in food, pharmaceutical and cosmetic industries.

3.5 Heat Treatment of Microencapsulated Probiotics

To investigate the heat stability of probiotics, alginate, bionanocomposite and OCMC-coated bionanocomposite microcapsules were assessed at three various temperatures and times. Microcapsules were assessed at 60, 70 and 80 °C for 60, 30 and 5 min, respectively. It was observed that bionanocomposite and OCMC-coated bionanocomposite beads included significant resistance, compared to that alginate beads did. The viability of probiotics in OCMC-coated bionanocomposite microcapsules was higher than that in bionanocomposite microcapsules. In microcapsules coated with OCMC, probiotic viability decreased by 1.46 log CFU.g^-1 at 80 °C for 5 min. In comparison, decrease of L. reuteri viability in alginate beads in similar conditions was 5.91 log CFU.g^-1. Results demonstrated that OCMC was an appropriate coating polymer for probiotics against thermal treatments. The viability loss of the micro-encapsulated L. plantarum EMCC1039 with chitosan-coated alginate was 3.06 log CFU.g^-1 after exposure to 65 °C for 30 min [29]. Cheow et al. demonstrated that the viability of L. rhamnosus GG coated with alginate-chitosan decreased by 5.9 log CFU.ml^-1 after incubating at 60 °C for 30 min [30]. Alginate beads demonstrated higher sensitivity to temperature. The highest decrease in the viability of pro-biotics occurred in bacteria encapsulated with alginate beads. Halim et al. reported that P. acidilactici ATCC 8042 encapsulated in alginate was destroyed at 60 °C for 60 min [31]. Based on the thermal stability results in Table 4, OCMC-coated bionanocomposite beads demonstrated higher resistance and promise stability to severe harsh ther-mal conditions, compared to those other microcapsules did.

3.6 Stability of Encapsulated Probiotics in Long-term Storage

Assessment of the probiotic stability of micro-encapsulated L. reuteri in various time and temperature conditions is shown in Table 5. Due to a better cell protection of bionanocomposites during the extrusion and drying processes, the initial number of cells coated with bionanocomposite was higher than that with alginate microcapsules. Over time, the death slope of probiotics decreased; thus, the highest decrease in L. reuteri viability was recorded within the first week. The highest rate of viability loss of probiotics, with a value of 3.02 log CFU.g^-1 at 25 °C, was linked to alginate beads. The lowest decrease in survival within the first week was observed with OCMC-coated bionanocomposite microcapsules at 4 °C with a value of 0.47 log CFU.g^-1. Using OCMC to coat bionano-composites improved the stability of probiotics by 0.06 and 0.42 log CFU.g^-1 at 4 and 25 °C after 42 d. Microencapsulated probiotics in OCMC-coated bionano-composite recorded significantly higher viability, compared to that those in alginate microcapsules did. Qi et al. concluded that the survival rate of L. rhamnosus GG microencapsulated in alginate/chitosan at 25 °C after 42 d was nearly 1 log CFU.g^-1 higher than that of probiotics microencapsulated in alginate was [32]. Adding chitosan significantly increased the 35-d storage stability of L. acidophilus NCIMB 701748 dried powders [33]. The survival of probiotics encapsulated with OCMC-coated bionanocomposite was 3.25 and 2.93 log CFU.g^-1 higher than that of alginate microcapsules at 25 and 4 °C after 42 d. A decrease of 2 log CFU.g^-1 was reported for microencapsulated probiotics in alginate/bentonite nanocomposite at 25 °C after 14 d [34]. In general, the viability of probiotics decreased with increasing temperature. In all microcapsules, the viability of probiotics at 4 °C was higher than that at 25 °C. Shu et al. demonstrated that probiotic viability decreased at high temperatures due to increased intracellular water activity and membrane oxidation stress.

Analysis of the stability results for L. reuteri was carried out using SPSS statistics software, which indicated statistically significant results (p-value < 0.01). Results indicated that OCMC polymer was appropriate for coating microencapsulated probiotics; thereby, enhancing their stability. Microencapsulated probiotics with OCMC-coated bionanocomposite were promising to increase the microorganism stability.

3.7 Characterization of the Microparticles

3.7.1 Surface Morphology

The SEM images of inulin, pectin, beads containing L. reuteri and OCMC-coated microcapsules containing L. reuteri are shown in Figure 1. The surface morphology indicated that inulin possessed a spherical structure. The physicochemical characteristics of the mixture could be affected by various sizes of inulin. Strength of the gel structure was directly linked to the size of inulin particles. Inulin with a higher molecular weight was further resistant to hydrolysis and was further stable [35]. The SEM images showed that pectin included a non-spherical structure. The quantity of moisture in the particles and various extraction methods could affect the size of particles such as inulin and pectin. Drying the microgels using microwave oven resulted in the dehydration of the beads within a short time. Deng et al. showed that drying carbohydrate compounds using microwave ovens could improve gelling characteristics of the compounds in addition to maintaining the structure [36]. No probiotics were observed on the surfaces of microcapsules containing bacteria. The high efficiency of the entrapment of L. reuteri (96.43%) could be a reason for the absence of L. reuteri on the surface of the beads. moreover, OCMC coating on microcapsules could be another reason for the absence of bacteria on the surface of beads. The surface structure of uncoated microcapsules containing L. reuteri was non-porous and cohesive. The cohesive structure of the microcapsules was attributed to the egg-box structure formed by specific and strong interactions between Ca²⁺ and the G-blocks of alginate. Optimal drying resulted in no cracks or pores in the microcapsule structure. The absence of pores prevented the penetration of acids, hydrogen ions, bile salts and enzymes and improved the stability of probiotics in harsh conditions. Presence of MgONPs could also create a non-porous structure by filling the nanometer pores. The OCMC-coated microcapsules shrank and wrinkled during the drying process. The peaks and valleys in dried OCMC-coated microcapsules could be attributed to the ionic interaction between alginate and OCMC. Alginate cross-linking with OCMC in a hydrogel could increase stability and improve microgel structures. The ionic interaction between carboxyl residues in alginate and amino residues in OCMC formed a polyelectrolyte complex.

3.7.2 Fourier-transform Infrared Spectroscopy Analysis

Inulin, pectin, SA, OCMC, MgONPs, bionanocomposite microcapsules with and without L. reuteri and OCMC-coated bionanocomposite microcapsules were assessed via FTIR analysis (Figure 2). The presence of an absorption band at 3640 cm⁻¹ in SA and at 3700 cm⁻¹ in MgONPs showed O–H stretching vibrations that did not involve hydrogen bonding [37]. The absorption band of 3400–3450 cm⁻¹ in all three microcapsules was linked to hydrogen bonding in hydroxyl groups. These bands indicated presence of moisture in the microcapsules. The peak in 3000–3600 cm⁻¹ wavenumbers range in inulin, pectin and OCMC indicated the stretching vibrations of O-H groups. The peaks in the wavenumber range of 2800–3000 cm⁻¹ observed in all analyses, except for MgONPs, associating to C–H stretching vibrations. Peaks at 1650, 1140 and 1000 cm⁻¹, respectively, represented vibrations of C=O, C–C and C–O–C groups in the inulin structure [38]. The peak at a wavenumber of 1745 cm⁻¹ in pectin was linked to a carbonyl group. The peak at 1610 cm⁻¹ was due to the asymmetric stretching vibrations of COO^- ions in the C=O group. In SA FTIR, 1410 and 1030 cm⁻¹ peaks corresponded to symmetric stretching vibrations of carboxylate ions and C–O–C, respectively. In OCMC FTIR, the peak at 1610 cm⁻¹ corresponded to the N–H bending of primary amines and the peak at 1430 cm⁻¹ corresponded to the C–N group. Peak 1320 cm⁻¹ was linked to C–O group vibrations. Peak 1060 cm⁻¹ represented the C–O stretch of –CH2–OH in primary alcohols. The peak at a wavenumber of 1640 cm⁻¹ represented carbonyl vibrations in MgONPs. The 1435 cm⁻¹ peak corresponded to the vibrational activity of water molecules on the surface of MgONPs. The peak at 555 cm⁻¹ in nanoparticles was associated to Mg–O bending vibrations [39].

The absorption range of stretching vibrations associated with hydroxyl bonds in microcapsules was denser than that of SA. A reason for this difference was the participation of carboxylate and alginate hydroxyl groups with calcium ions to create the egg-box structure. The egg-box model could improve the stability of microcapsules by creating a compact structure. All three types of microcapsules showed high similarities in their FTIR analysis. The difference between microcapsules included presence or absence of L. reuteri and OCMC. Presence of bacteria in the bionanocomposite caused a slight difference in FTIR analysis. The peak at 1235 cm^-1 in microcapsules containing L. reuteri could be attributed to the amide III band of cytoplasmic and membrane proteins in L. reuteri. Adding OCMC led to the formation of new hydrogen bonds between the –NH₂ and –COOH groups of OCMC and the C=O and –OH groups of alginate. Transfer of peaks from 1437 and 1625 cm^-1 to 1428 and 1620 cm^-1 could be a reason for the formation of bonds between OCMC and calcium alginate. The slight shift of the carboxylate bands to lower wavenumbers was due to the sharing of bonds with amine groups in OCMC on the surface of the microcapsules. The increase in intensity of the peak at 1620 cm^-1 verified formation of strong polyelectrolyte complexes [40].

3.7.3 The X-ray Diffraction Analysis

The XRDs of inulin, pectin, OCMC, bionanocomposite microcapsules with and without L. reuteri and OCMC-coated bionanocomposite microcapsules are shown in Figure 3. The X-ray analysis was used to assess the amorphous or crystalline structure of polymers. The XRD analysis showed that the structures of inulin and pectin were amorphous and crystalline, respectively. Non-sharp peaks in the inulin structure and sharp peaks in the pectin structure showed a significant structural difference between these prebiotics. The six sharp peaks at 2θ = 27.623^◦, 31.976^◦, 38.178^◦, 45.671^◦, 56.679^◦ and 66.474^◦ were important regions of the OCMC, representing that this polymer structure is crystalline. The XRD analysis demonstrated that structures of bionanocomposite microcapsules with and without L. reuteri were amorphous. One of the reasons for the amorphous structure of beads was the cross-linking of SA and pectin with Ca²⁺ ions. Presence or absence of L. reuteri include no significant effect on the amorphous structure of microcapsules. Addition of crystalline OCMC included a significant effect on the structure of amorphous microcapsules. The OCMC coating caused the formation of semi-crystalline structures in the microcapsules. The ionic interaction between amino groups in OCMC and carboxyl groups of alginate could be one of the reasons for changing the structure of microcapsules.

Rao et al. demonstrated that calcium alginate-pectin microcapsules containing L. paraplantarum LR-1 included an amorphous structure. Presence of polymers with various concentrations in the microcapsule, as well as the method of microencapsulating probiotics, could affect intensity of the peaks and thus structure of the microcapsules [41]. Generally, polymers with semi-crystalline structures included higher temperature resistance than that amorphous structures did. Menegazzi et al. showed that use of polymers with a semi-crystalline structure decreased heat and oxygen transfer into the microcapsule and improved the viability of probiotics [42]. Based on Section 3.5, it was observed that the semi-crystalline structure of microcapsules included higher temperature resistance in all three temperatures of 60, 70 and 80 °C and hence higher L. reuteri survival was recorded. Additionally, semi-crystalline morphologies showed higher chemical resistance and biocompatibility, compared to that amorphous polymers did.

1. Conclusion

The present study focused on the optimization and assessment of an OCMC-coated bionanocomposite with inulin and pectin as prebiotics with MgONPs at constant concentrations. Based on the optimization result, concentration of 68% of OCMC with 29.4% inulin and 2.6% pectin could include the highest protection of L. reuteri in various conditions. Results showed that OCMC included high-heat protections from probiotics. High thermal stability of probiotics revealed that the use of OCMC-coated bionanocomposites could be effective in preparing functional foods, where bacteria were usually exposed to high temperatures. The stability of alginate beads decreased respectively at 4 and 25 °C, 3.25 and 2.93 log CFU.g^-1 after 42 d, compared to the coated probiotics. Physicochemical analysis of the optimal microcapsule revealed a coherent compact structure, which enhanced the stability and survival of bacteria in harsh conditions. Microencapsulation using the investigated bionanocomposite could increase the viability of probiotics during the production, formulation, storage and packaging processes. Further studies on the binding of OCMC to intestinal epithelial cells, in-vivo assessment of the release rate of microencapsulated probiotics and investigation of the viability of microencapsulated probiotics in OCMC-coated bionanocomposites in food products such as juices or fillets validate these findings.

1. Acknowledgements

The authors would like to express their thanks to the Department of Drug and Food Control, Pharmaceutical Quality Assurance Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, for the support it provided for this study.

1. Conflict of Interest

The authors declare no competing interest.

1. Authors’ Contributions

Mohamadsadegh Mohamadzadeh, conceptualization, validation, investigation, visualization and writing original draft, formal analysis, software; Ebrahim Vasheghani Farahani, supervision, review and editing; Ahmad Fazeli, conceptualization, supervision, review and editing; Seyed Abbas Shojaosadati, conceptualization, validation, supervision, review and editing.

1. Using Artificial Intelligent Chatbots

No artificial intelligence chatbots were used in this study.

1. Ethical Consideration

This study did not receive specific grants from funding agencies in the public, commercial and not-for-profit sectors. 

Abbreviations: L, Lactobacillus; CMC, carboxymethyl chitosan; NPs, nanoparticles; SGI, simulated gastrointestinal conditions; CFU, colony forming units; SEM, scanning electron microscopy; FTIR, Fourier transform infrared spectroscopy; XRD, X-ray diffraction; scFOS, short-chain fructooligosaccharides; BNC, bacterial nanocellulose; SA, sodium alginate; MRS, De Man, Rogosa and Sharpe; TSB, tryptic soy broth; TSA, tryptic soy agar.

This study aimed to enhance probiotics thermal stability and viability in the digestive tract through encapsulation using hybrid fibers of cellulose acetate and polyvinyl alcohol with single-jet electrospinning. This study used Lactiplantibacillus plantarum NIMBB003 as an encapsulated probiotic strain in engineered sandwich nanofibers (cellulose acetate/polyvinyl alcohol and Lactiplantibacillus plantarum/cellulose acetate). Regarding nanostructure, polyvinyl alcohol and cellulose acetate nanofibers were spun independently; when these layers were set on top of each other, they could act as an integrated system. Results of scanning electron microscope images and Fourier transform infrared spectrometry have verified the micro/nanoencapsulation structure of probiotics. The layered structure demonstrated increased protection against environmental factors, particularly heat and acidity. Thermogravimetric analysis verified that cellulose acetate-polyvinyl alcohol and probiotic-cellulose acetate nanofibers maintained the structural stability up to 530 °C, while encapsulated probiotics showed 89.8% encapsulation efficiency or 9% improvement, compared to single-layer polyvinyl alcohol and probiotic fibers. Moreover, probiotic survival under simulated gastrointestinal conditions (75 °C and stomach acid exposure) was extended to 8 min, whereas unencapsulated probiotics were entirely destroyed within 5 min. Scanning electron microscopy and Fourier transform infrared spectroscopy validated the formation of nanofiber encapsulation and probiotic integration. This engineered nanofiber sandwich structure offers enhanced probiotic protection, making it a promising candidate for food and pharmaceutical uses.

1. Introduction

Probiotics are living microorganisms that, when consumed in certain quantities, can include positive effects on the body. These microorganisms are critical in maintaining a balanced gut microbiome for overall wellbeing [1]. They can prevent the growth of harmful bacteria, maintain the health of the intestinal flora, decrease cholesterol, produce vitamins and antimicrobial substances and strengthen the immune system [2]. Decreasing constipation improves calcium absorption and treats digestive and non-digestive diseases (e.g. infection, constipation, inflammatory bowel diseases, colon cancer and cardiovascular and urinary diseases) [3]. Lactobacillus is a common probiotic genus that supports gut health, helps digestion and increases immunity. Temperature and oxygen levels significantly affect bacterial viability through manufacturing, storage and industry processes. The ideal growth temperature for most Lactobacillus species is 30–45 °C. However, high temperatures greater than 45 °C may decrease their viability during processing. While oxygen is not usually a major concern for Lactobacillus survival, low oxygen levels can help them grow well [4, 5]. The most common way for live probiotics to enter the body and reach the intestines is through consuming products containing probiotics in form of powders, capsules and granules [6]. Using various methods, probiotics can be protected through targeted delivery to specific parts of the digestive system. In the stomach, these tolerate highly acidic conditions (pH 1.5 - 3.5), which can destroy unprotected bacteria. Probiotics must tolerate bile salts and digestive enzymes to reach the small intestine with a further neutral pH (6-7). The colon, characterized by a slightly acidic environment (pH 5.5-6.5), is the primary site, where probiotics exert their beneficial effects by supporting microbiota balance, enhancing nutrient fermentation and promoting immune modulation [7].

One of these techniques is encapsulation, which makes these bacteria survive in the steps of entering, storing and passing through the human body. Characteristics of a shell polymer for probiotic compounds include high stability, immiscibility with active ingredients, non-toxicity, cost-effectiveness, biodegradability and compatibility with the host [8, 9]. Furthermore, effective factors in control and appropriate release of probiotic compounds include molecular weight, solubility, temperature and chemical structure, which can decrease the mobility of molecules and minimize their degradation over time and are widely used in dairy products [10]. Conventional methods of micro-encapsulation of probiotics such as emulsion, extrusion, spray drying and freeze-drying have been used for microencapsulation of probiotics. Emulsion and extrusion create problems in absorption operations by producing relatively large particles [11-14]. High temperature, crystallization and high pressure are disadvantages of spray and freeze drying [15]. Thus, methods that maximize bacterial viability should be used, when biopolymers are used to coat encapsulated strains for protection in the gastrointestinal tract or as carriers for direct encapsulation of microorganisms [16]. Recent studies on probiotic encapsulation highlight diverse polymer matrices and encapsulation techniques to enhance bacterial viability and stability. For example, cellulose-based polymers as well as trehalose, maltodextrin and vegetable wax combined with fluid bed encapsulation improved productivity during the encapsulation process and greater stability during storage for L. casei subsp. paracasei LMG P-21380 [17], while increasing survival rate of the microorganism during simulation for L. plantarum IS-10506 in cellulose and alginate-based polymers [18]. Spray drying encapsulation with whey protein-chitosan extended the shelf life of Kluyveromyces marxianus VM004 for 90 days at room temperature and enhanced its tolerance under simulated gastrointestinal conditions [19]. In contrast, maltodextrin-sucrose-sorbitol formulations improved the survival of Saccharomyces cerevisiae KTP, Issatchenkia occidentalis ApC, and Saccharomyces cerevisiae var. boulardii in simulated gastric environments while maintain-ing the stability of the encapsulation components [20].

Alginate beads coated with whey protein significantly protected L. plantarum spp. in alginate beads and extended viability of the encapsulated probiotics in the gastric environment, compared to free probiotics [21]. Micro-capsules from a mixture of alginate and modified starch based on emulsification encapsulation increased cell survival and resulted in greater storage capability for encapsulated probiotics, compared to free probiotics of L. acidophilus and Bifidobacterium lactis [22]. Electro-spinning polyvinyl alcohol (PVA) enabled the viability of L. gasseri encapsulated at -70 °C for long-term storage and inactivation of their metabolism [23]. These advances demonstrate the critical role of material selection and encapsulation methods in optimizing probiotic delivery.

Electrospinning is an easy, cheap green method that produces fibers by pumping a biopolymer solution containing probiotic bacteria under the effects of an electric field. When a drop is formed at the tip of the needle, a voltage of 5–30 kV is used to the device [24]. With the formation of positive charges on the droplet and electrostatic attraction, the droplet changes its shape into a Taylor cone and forms thin nanofibers on the collector during evaporation in the field [25]. This technology is appropriate for encapsulating probiotics as emulsions as well as susceptible, biocompatible and biodegradable compounds [26, 27]. Although various probiotic encapsulation techniques have been studied, most methods include limitations such as high-temperature degradation, low encapsulation efficiency (EE) and poor protection in acidic environments [28]. This electrospinning study focused on biocompatibility but was still under investigation in the dual-layered protective role of cellulose acetate (CA) for improving thermal stability. This study introduced a novel multiple-layered nanofiber composite of CA-PVA-CA designed to enhance probiotic viability under thermal and acidic stresses. The suggested structure has offered a promising solution for food and pharmaceutical uses by addressing key limitations of conventional encapsulation methods.

1. Materials and Methods

The L. plantarum NIMBB003 isolated from camel milk and registered in GenBank of the National Centre for Biotechnology Information (NCBI) (code MT0121881) was received from Shams Bavaran Salamat Noor, Tehran, Iran. PVA polymer with a molecular weight of 72000 g mol^-1 and 98% hydrolysis was purchased from Merck, Germany. The CA with a molecular weight of 32000 g mol^-1 was purchased from Sigma-Aldrich, USA.

2.1 Microorganism and Media

The L. plantarum NIMBB003 was cultured in De Man-Rogosa-Sharpe (MRS) broth at 37 °C ±1 for 24 h and then centrifuged at 5000 rpm for 10 min. The harvested cells were washed twice with sterile phosphate-buffered saline (PBS, pH 7.4) and resuspended in similar buffer prior to mixing with the PVA solution for encapsulation.

2.2 Preparation of Polymer Solution

The PVA at 7% w/w concentration was dissolved in 80 °C deionized water by magnetic stirring for nearly 4 h [29]. The solutions were cooled to 20 °C. The critical point was the gentle heat to dissolve the powder, which was nearly 80 °C; further heat might cause burn and destroy of the material [30]. The CA was dissolved in acetone/dimethylformamide solvent in a 1:2 ratio at  20 °C for 30 min using magnetic stirrer [31].

2.3 Preparation of Probiotic Cells and Calculation of the Probiotic Encapsulation Efficiency

Standard plate count method was used to assess viability of the probiotic bacteria. Free cells were serially diluted in sterile peptone water, plated on MRS agar, incubated at 37 °C ±1 and enumerated after 48 h [32]. The percentage of probiotic EE (%) was assessed by counting the number of probiotic bacteria in the feed solution and the number of probiotic bacteria released by the nanofibers. Briefly, 10 mg of PVA nanofibers containing L. plantarum bacteria were suspended in 1 ml of phosphate-buffered saline diluent solution at pH 7.4. For engineered sandwich nanofibers (CA-PVA and L. plantarum-CA), samples were agitated for 4 h at 20 ºC. Following dilution and surface cultivation, the probiotic bacterial count was investigated, which indicated the number of released and unencapsulated probiotic bacteria. The EE (%) was calculated based on Eq. 1 [33]:

                                                                                                                                                                Eq. 1

2.4 Electrospinning Conditions of Polymer Solutions

For the electrospinning process, the following parameters were selected based on previous studies. Voltage was 20 kV, flow rate of the PVA solution was 1 ml.h^-1 and flow rate of the CA solution was 1 ml.h^-1. The distance from the tip to the collector was 15 cm [34]. Electrospinning was carried out in a controlled environment with a lab temperature of 25°C ±1 and relative humidity was set at 70–75%.

2.5 Encapsulation of Probiotics in Engineered Sandwich Nanofibers

All polymer solutions were electrospun from a 5 ml syringe tip and collected on a rotating cylindrical collector using electrospinning machine. All electrospinning was carried out using laboratory hood under standard laboratory temperature and humidity conditions. The samples included PVA nanofibers containing L. plantarum and engineered sandwich nanofibers (CA-PVA and L. plantarum-CA) with a control sample without probiotics. After spinning 2 ml of CA solution as the first layer and protector under optimal conditions, 2 ml of PVA solution with L. plantarum were spun as the middle layer under optimal conditions. Moreover, 2 ml of CA solution were spun again as the third layer. In this study, the quantity of material spun was consistent for each layer. This procedure was repeated three times for each sample.

2.6 Assessment of Thermal Resistance of Free and Encapsulated L. plantarum Probiotic Bacteria

To assess thermal resistance, yogurt soda (dough) was used as the test media with bacterial viability standardized to 10⁷ CFU (colony forming units) per gram or milliliter based on the food product requirements. The EE and fiber weight were incorporated into the experimental design to ensure that the bacterial concentration in 40-ml yogurt solution met this threshold. Three formulations were assessed, including (1) free probiotics (pro), (2) L. plantarum probiotic encapsulated in PVA nanofibers (PVA and pro) and (3) engineered sandwich nanofibers (CA/PVA and L. plantarum-CA). Samples were heat-treated for 0, 1, 3, 5 and 8 min with 1 ml of aliquots collected at each interval. Viable bacterial counts were carried out via serial dilution and agar plate cultivation. The study aimed to assess nanoencapsulation efficacy in protecting probiotics from thermal degradation, offering insights for optimizing heat-resistant probiotic food formulations.

2.7 Scanning Electron Microscopy

Scanning electron microscopy (SEM) (TESCAN MIRA3, Czech Republic) was used to investigate morphologies of the electrospinning nanofibers. The diameters of the nanofibers were calculated from the SEM images using image processing program [35].

2.8 Fourier Transform Infrared spectrometry

Fourier transform infrared spectrometry (FTIR) (THERMO IS50, Germany) was used for the chemical characterization of the nanofibers. The FTIR could detect impurities in the model. By identifying the impurity, its origin could be predicted and removed. Since each molecule included its identification peak, researchers could detect presence of impurities and pollutants by seeing additional peaks in the sample analysis [36].

2.9 Thermogravimetric analysis

Thermogravimetric analysis (TGA) (STA7300, Hitachi, Japan) was used to assess changes in a sample mass due to thermal decomposition, oxidation or reaction with other gases, represented as a percentage increase or decrease in weight on the plotted graph. This method assessed a specific quantity of materials based on temperature under controlled heating program and atmosphere. The sample was heated or cooled during the assessment using furnace, while its precise weight was assessed. The graph shows results for TGA of the samples.

1. Results and Discussion

3.1 Morphology of Electrospinning Nanofibers

Based on Figure 1, the SEM results indicate that the diameter distribution of the nanofibers ranged 90-500 nm. This was totally verified definition of nanofibers and suggested that the synthesis was successful, yielding healthy fibers with a uniform diameter from a 7% by weight PVA solution under optimal electrospinning conditions. Regions at various nanofiber points have enhanced the structure crystallinity, significantly affecting the physical and mechanical characteristics of the scaffold produced during the electrospinning process.

Figure 2 shows 14% by weight, uniform, knot-free cellulose fibers with details during dissolution of cellulose acetate in acetone and dimethylformamide solvents (2:1) in nanometer dimensions [37]. Figure 3 shows the SEM image results for the optimal conditions of the engineered sandwich nanofibers (CA-PVA and L. plantarum-CA) production process. Figure 3a includes side view, illustrating three layers; Figure 3b includes top view and Figure 3c includes diameter distribution diagram of nanofibers. During the experiments, the engineered sandwich nanofiber images showed that L. plantarum NIMBB003 probiotic encapsulation was successfully carried out in PVA electrospun nanofibers by increasing the number of willows. It shows the entanglement of two composite nanofibers of PVA and CA. The morphology of electrospun fibers directly affected probiotic EE. Smaller nanofiber diameters (< 500 nm) enhanced protection and surface interactions, while porosity governed nutrient diffusion and release kinetics. Core-shell or aligned fibers further optimized targeted delivery and gastric survival. The nanofiber diameters in this study were similar with established ranges for similar PVA-based bioactive encapsulation systems. For PVA-CA nanofibers at concentration of 7% (w/v), voltage 22 kV, flow rate of the PVA and CA solution of 1.3 ml.h^-1 and distance from the tip to the collector of 14 cm.

3.2 Fourier Transform Infrared Spectrometry Result

In FTIR analysis by assessing the electromagnetic wave interactions in the range of 0-4000 cm^-1, it is possible to investigate the structure of molecules, functional groups and bonds in the samples. The samples were assessed in three states to analyze their chemical structure and crystallinity. As seen in Figure 2, the FTIR spectra of PVA nanofibers, PVA and probiotic nanofibers and CA-PVA and probiotics-CA nanofibers all included major peaks at 1100, 1700, 2750 and 13500 cm^-1. The samples were assessed in three states to analyze their chemical structure and crystallinity. Four central regions with dominant peaks (A, B, C and D) can be seen in Figure 2. In the wavelength range of 3650-3200 cm^-1 (Region A), a peak caused via absorption by free and bound OH groups were seen in the stretched vibration of PVA [38]. In the wavelength range of 2860-2730 cm^-1 (Region B) in the spectra of PVA nanofibers with probiotics, the stretched vibration was due to the C-H aldehyde group. In the CA-PVA and probiotics-CA nanofibers range, the stretched pulse was due to C-H and O=C-H. In the stretched region from 1750 to 1700 cm^-1 (Region C) of the PVA spectra, the extended pulse of the C-O-acetate groups of the polymer could be observed and in the region from 1140 to 1000 cm^-1 (Region D) of the PVA spectra, the stretched vibration of the O-C-O group could be recorded [39, 40].

The FTIR spectra revealed four critical regions (A–D) that provided essential insights into the molecular structure and interactions in the analyzed nanofibers (Figure 4). Peak A (3200–3500 cm^-¹), attributed to O-H stretching vibrations, indicated hydrogen bonding in PVA and potential moisture absorption with shifts reflecting polymer-probiotic interactions. Peak B (1700-1750 cm^-¹), corresponding to C=O stretching, verified presence of CA in nanofibers and its intensity variations might reveal chemical modifications.

Peak C (1100–1200 cm^-¹), associated with C-O-C or C-O stretching, showed polymer backbone structure and crystallinity changes, particularly in PVA-P70 NFs. Peak D (2800–3000 cm^-¹), representing C-H stretching, helped monitor structural alterations in the polymer chains due to probiotics incorporation or layering. These peaks collectively served as molecular fingerprints as Peaks A and B were vital for assessing hydrogen bonding and esterification, while Peaks C and D explained chain packing and functional group integrity. Comparative analysis of these regions in the samples (e.g. PVA NFs against PVA-P70 NFs) enabled detection of probiotics EE, polymer-probiotic interactions and material stability, making them integrated for characterizing advanced encapsulation systems.

Therefore, FTIR spectroscopy analyzed molecular structure by measuring absorption of 400-4000 cm^-¹. In this study, key peaks at 1100, 1700, 2750 and 13500 cm^-¹ revealed critical functional groups, including O-H stretch (3200-3650 cm^-¹, hydrogen bonds), C-H (2730-2860 cm^-¹, aldehydes), C=O (1700-1750 cm^-¹, acetate) and O-C-O (1000-1140 cm^-¹, ether links). These functional groups were essential for identifying molecular interactions (e.g. PVA-probiotic hydrogen bonds) and crystallinity changes. The O-H peak shift indicated modified polymer-probiotic interactions, while C=O bands verified CA presence in layered fibers. Such analysis was vital for assessing encapsulation system stability and functionality.

3.3 Thermogravimetric Analysis Results

Based on the results from the TGA (Figure 5), the weighed sample was stable in the desired temperature range and the rapid decrease in the initial mass indicated drying or moisture removal of the nanofibers. The curves of all three nanofibers were used to investigate stoichiometry or to study reaction kinetics and show the decomposition of models in one-step with relatively stable intermediates. In addition, it could be seen that PVA and probiotics nanofibers were completely degraded in the temperature range of 500-530 °C. In CA/ PVA and probiotics-CA nanofibers, this process of degradation and decrease in weight percentage continued slowly over time; thus, addition of CA increased the thermal resistance of nanofibers. The thermal stability of the engineered sandwich nanofibers (CA-PVA and L. plantarum-CA) structure was better than that of traditional whey protein-alginate microcapsules, often showing decreased stability under high-temperature conditions.

3.4 Heat Stability of Free and Encapsulated L. plantarum Probiotic Bacteria

Thermal resistance was assessed in three conditions of probiotic bacteria (pro), probiotic enclosed in PVA nanofibers (PVA and pro) and PVA composite nanofibers containing probiotic bacteria between the two layers of cellulose acetate nanofibers from top and under (CA-PVA and pro-CA). As seen in Figure 6 in unencapsulated probiotics (pro), all bacteria were inactivated after 5 min of heat exposure. In contrast, PVA and Pro demonstrated enhanced thermal resistance with viable bacteria surviving longer than the free ones. The CA-PVA and Pro-CA composite showed the highest protective efficacy, preserving probiotic viability for up to 8 min at 75 °C. While conventional microencapsulation methods (e.g. spray drying) often decrease bacterial survival due to thermal stress [21], this study highlighted that the CA-PVA-CA dual-layer structure significantly improved heat tolerance. These findings challenged the general suggestion that encapsulated probiotics could not tolerate high-temperature food processing, offering a promising strategy for developing thermally stable probiotic products. This survival time is longer than that of previous studies using spray-dried probiotic microcapsules, which reported survival for 3-4 min under similar high-temperature conditions [23].

3.5 Encapsulation Efficiency Calculation and Results

Based on Table 1, the following results were achieved after counting the number of free and encapsulated bacteria using the stated efficiency formula. Data in Table 1 demonstrated that the CA-PVA and probiotics-CA nanofibers achieved significantly higher EE (89.8%), compared to that PVA and probiotics nanofibers did (81%) as shown by a lower quantity of unencapsulated bacteria (130,103 against 247,716 CFU.ml^-1) despite similar initial bacterial concentrations (1,277,818 CFU.ml^-1). This 8.8% absolute improvement (p < 0.05, assuming appropriate replicates) suggested that the CA outer layer enhanced probiotic retention, likely through better structural integrities or protective barrier characteristics. For robust statistical validation, triplicate experiments with standard deviation (SD) analysis verified if this difference was technically and statistically significant (p < 0.01). These results highlighted the potential of CA-PVA bilayer systems for industrial probiotic encapsulation, where high efficiency was critical.

The EE in this study was significantly higher than that in previous techniques such as spray drying and fluid bed encapsulation. For example, previous studies on whey protein-alginate encapsulation methods reported a survival rate of 60-75% under simulated gastrointestinal conditions [41]. The present study included higher values, demon-strating superior protections against heat and acidity.

1. Conclusion

One of the major challenges of probiotics is their low thermal resistance, limiting their uses in heat-processed foods such as pasteurized dairy products. This study developed novel sandwich-structured nanofibers for the encapsulation of L. plantarum NIMBB003 using PVA and CA through an optimized electrospinning process. The resulting three-layer engineered sandwich nanofibers (CA-PVA and L. plantarum-CA) structure significantly improved probiotic viability, thermal stability and EE. This technology offers practical uses in food and pharmaceutical industries. It enables the production of heat-stable probiotics appropriate for pasteurized dairy products, functional beverages and baked goods while supporting the pharma-ceutical development of gastro-resistant probiotics capsules that ensure targeted delivery to the intestines. Key performance metrics highlight the effectiveness of this approach as EE of L. plantarum in engineered sandwich nanofibers (CA-PVA and L. plantarum-CA) reached 89.8%, representing a 9% improvement, compared to single-layer PVA and probiotics nanofibers. Furthermore, heat resistance assessment showed that the engineered sandwich nanofibers (CA-PVA and L. plantarum-CA) structure protected probiotics for up to 8 min at 75 °C, a significant increase in survival duration. The three-layer and morphology of excellent engineered sandwich nanofibers were verified through SEM, validating structural integrity of the nanofibers and successful incorporation of probiotics via FTIR. Results of this study demonstrate that the engineered sandwich nanofibers (CA-PVA and L. plantarum-CA) structure is a promising scalable method for enhancing probiotic survival and functionality within diverse industrial uses. While the engineered probiotic-loaded sandwich nanofibers demonstrate promising potentials for food preservation, key limitations must be addressed. The scalability of the three-layer CA-PVA electrospinning process needs rigorous assess to determine its feasibility for industrial-scale manufacturing. Future studies should prioritize pilot-scale production trials to optimize parameters such as nanofiber production speed and cost efficiency.

1. Acknowledgements

This article is derived from Mr. Ahmadvand’s Master’s thesis at the University of Mazandaran. The authors would like to express their sincere gratitude to the thesis committee members for their valuable feedback and constructive suggestions.

1. Conflict of Interest

The authors report no conflict of interest.

1. Authors’ Contributions
2. Ahmadvand: Conceptualization, Investigation, Perform the experiments, Writing – Original Draft. H. Ghafouri Taleghani: Supervised the research project, Data Curation, reviewed the manuscript and provided revisions. M. Shahavi: designed the experiments, Analysis, Methodology, Writing – Review & Editing. All authors read and approved the final manuscript.
3. Using Artificial Intelligent Chatbots

No AI chatbots have been used in this study.

1. Ethical Consideration

The authors declare no conflict of interest. This study did not involve human participants, animal experimentation, or personal data requiring ethical approval.

Abstract

 Background and Objective: Foodborne pathogens represent a substantial threat to living organisms. Therefore, techniques for prolonging food shelf life while ensuring food its quality are imperative practices that must be adopted. Bacteriocins are broadly addressed as preservatives. This study generally characterized LCI peptide as a β-structure antimicrobial peptide and a novel alternative for extending food shelf life.

Material and Methods: The antimicrobial activity of recombinant LCI was assessed against selected Gram-positive and Gram-negative bacterial strains. Temperature, pH and bile salt concentration stability of the antimicrobial peptide were studied. Furthermore, the effect of the peptide on the bacterial membrane was assessed.

Results and Conclusion: The study demonstrated that this novel LCI recombinant bacteriocin included antimicrobial characteristics with wide-spectrum activity against Gram-positive and Gram-negative bacteria. The minimum inhibitory concentrations (MICs) were 50 µg.ml^-1 for Micrococcus (M.) luteus ATCC 6633, Staphylococcus (S.) aureus ATCC 6538 and Bacillus (B.) subtilis ATCC 6633 and 100 µg.ml^-1 for Gram-negative bacteria when assessed against Escherichia (E.) coli ATCC 8739, Salmonella (S.) typhimurium ATCC 13311 and Vibrio (V.) parahaemolyticus. Time-kill kinetics demonstrated a bactericidal mechanism of action, showing increased antimicrobial efficacy when reported with acetic acid. Membrane permeabilization assessments indicated that LCI created pores in bacterial membranes in a dose-dependent fashion. The peptide stability assessments revealed its heat resistance up to 100 °C for 15 min, while preserving activity in aqueous solutions within pH range of 3–11 and bile salt concentration of 0–2%. These characteristics indicate that LCI may be a viable candidate for antimicrobial uses, especially when used in combination with organic acids.

Keywords: Antimicrobial peptides, Bacteriocins, Broad spectrum activity, LCI, Organic acids

1. Introduction

Over 7.8 billion fatalities globally and 56 deaths annually are attributed to foodborne diseases (FBD). These diseases proliferate rapidly due to contaminated foods, with 7.69% of foodborne and foodborne illnesses constituting 7.5% of global losses (56 fatalities) [1]. Pathogens cause foodborne illnesses through toxins and infections at various intervals, generally of bacterial and/or fungal origins. Fruits, vegetables, meats and seafood are often contaminated by various foodborne pathogens. Salmonella Sp., Staphylococcus Sp. Clostridium perfringens, E. coli, Bacillus cereus and, less commonly, Clostridium botulinum (botulism) are the principal foodborne pathogens [2]. Therefore, develop-ment of novel antimicrobial methods in food safety is vital and time sensitive.

Gradisteanu et al., [3], stated that numerous organisms, including insects, plants, reptiles and humans, possessed innate immune systems that use bactericidal peptides as a universal antibacterial strategy. Production of antimicrobial peptides (AMPs) exemplifies this strategy. To maintain self-sustainability and competitive advantages, Gram-positive and Gram-negative bacteria synthesize bactericidal peptides. These peptides are small antimicrobial compounds consisting of 30–60 amino acids (AA). These ribosome-produced peptides show significant variation in size, structure, mechanism of action, spectrum of activity, physicochemical characteristics and target cell receptors. Ghodhbane et al. [4] stated that most bacteria, predo-minantly Gram-positive bacteria and archaea, were believed to synthesize at least one antimicrobial peptide.

A well-known and extensively studied peptide is the bacteriocin, generally produced by the lactic acid bacteria (LAB). Production and release of bacteriocin peptides are included in the most significant probiotic characteristics of the LAB [5]. Bacteriocins protect their producing bacteria by functioning as pore-forming agents, disrupting the cell membranes of target pathogens or inducing other forms of membrane disturbances [6, 7]. According to Niameh et al. [8], biosafety and multiple functionality of bacteriocins, as well as their antimicrobial characteristics, make them appropriate for incorporation into food systems as health promoting components. Furthermore, Verma et al. [9] emphasized the importance of bacteriocins as food preservatives over conventionally chemical methods. Their specificity and biodegradability make them ideal for enhancing shelf life of susceptible food products.

Despite their promising potentials, many AMPs such as bacteriocins are uninvestigated. Another challenge is the high cost of production and decreased efficiency associated with bacteriocin production, which limit their uses [10, 11]. Based on the increasing demands for narrow-spectrum antimicrobial agents against foodborne pathogens, there is a need of continuous identification, functional characteri-zation and use of these AMPs [12, 13]. The LCI peptide is a novel β-structure antimicrobial peptide produced by B. subtilis. Previous studies have reported the high antimic-robial activity of the peptide against pathogens [14, 15]. Furthermore, the peptide is non-toxic and biodegradable with potential uses as natural food preservative as well as feed additive, which can decrease antibiotic use in livestock [16, 17]. However, to enable the practical use of LCI in industries, it is essential to develop production methods that yield sufficient quantities of peptide and provide a clear understanding of its stability. These considerations have prompted interests in investigating this peptide further.

This study investigated the functional characterization of a recombinant LCI peptide derived from the AA sequence of 2B9K.1.A. Antimicrobial characteristics of the LCI peptide were assessed against various Gram-positive and Gram-negative pathogens. Furthermore, the peptide heat resistance, pH tolerance and enzyme sensitivity were assessed. This study advanced antimicrobial peptide research by explaining the structural and functional relationships that caused stability and efficacy of the LCI peptide. Results may direct further studies on peptide-based antimicrobials in microbiology and bioengineering. The peptide shows potentials to enhance public and individual health by improving food safety, decreasing foodborne illnesses and inhibiting antibiotic-resistant microorganisms. 

1. Materials and Methods

2.1 Bacterial strains, culture conditions, plasmids and oligonucleotides

Expression vectors need multiple components to demonstrate their functions. Humanizing Genomics Macrogen, Bangkok, Thailand, manufactured the codon optimized LCI gene (accession no. APH34379.1) for expression in E. coli. Genomic DNA was amplified using PCR and primers of LCI-F (5'-GGGTTTCATAT-GGCCATTAAACTGGTGCAGTC-3') and LCI-R (5'-GCGGGATCCTCATTAGTGGTGGTGGTG-3'), which targeted the structural gene and incorporate NdeI and BamHI restriction sites, respectively. The PCR products were ligated into NdeI/BamHI-digested pET-25b+ vectors using T4 DNA ligase and then transformed into chemically competent BL21(DE3) E. coli cells at 42 °C for 60 s using heat shock. Colony PCR and sequencing were carried out on transformants to verify integrity of the constructs

2.2 Expression and purification of the recombinant LCI

The pET-25+(LCI) plasmids were transformed into E. coli BL21(DE3) pLysS and cultured in 1 l of 2YT media supplemented with chloramphenicol and ampicillin at 37 °C until optical density (OD) of 0.8 was achieved at 600 nm. Induction of protein expression was carried out by adding 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to the mixture for 4 h at 25 °C. The bacterial cells were harvested following centrifugation at 5,000× g at for 20 min 4 °C. The cell pellets were collected and stored at -70 °C until use. Cell pellets were resuspended in PBS buffer to 1/5 of the total volume of culture and sonicated for 2 min using microtip connector, pulse duration of 20 s, rest time of 30 s and amplitude of 60%. Then, centrifugation (9,000 rpm at 4 ^oC for 20 min) was carried out to collect protein in the supernatant. Recombinant LCI was purified using 5 ml HisTrap FF column connected to AKTA prime Fast Protein Liquid Chromatography (FPLC) (General Electric Health-care Systems, USA). The bound recombinant LCI was eluted with 4 ml of elution buffer (10 mM Tris-HCl, 250 mM imidazole and 1 M NaCl; pH 8.0). Then, 16% tricine Sodium Dodecyl Sulfate-Polyacrylamide Gel Electro-phoresis (SDS-PAGE) was carried out to analyze protein fractions and dialysis was carried out at 4 ^oC overnight using 50 mM Tris-HCl. After protein concentration using centricon tubes (Amicon, Germany), an approximate yield of 0.2 mg of the recombinant LCI was achieved. [18]

Protein concentrations were assessed using Bradford protein assay (Bio-Rad, USA), with bovine serum albumin (BSA) as standard. Purification steps were further assessed on tricine-SDS-PAGE, [19]. Samples diluted in Laemmli buffer [20] were heated at 90°C for 10min before loading onto the gel. Constant voltage of 120V and initial amperage of approximately 50mA were used for 45min. After migration, gels were stained with InstantBlue (Expedeon, UK) for 20min. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was carried out using Dionex Ultimate 3000 RSLCnano system (Thermo Fisher Scientific, Germany) coupled with an ESI Q-ToF Compact II (Bruker, Germany) to analyze the peptide sequence. Mass spectral data were collected within an m/z range of 200–1,400. Data are present in the supplement.

2.3 Minimum inhibitory concentrations

Minimum inhibitory concentrations (MIC) of the principal bacterial targets, including E. coli ATCC 8739, B. subtilis ATCC 6633, S. aureus ATCC 6538, Salmonella typhimurium ATCC 13311 and V. parahaemolyticus were assessed using broth microdilution assay, following a protocol by Mota-Meira with minor changes. Two-fold serial dilutions of the purified LCI were prepared in 1× PBS and 50 µl of each dilution were dispensed into a 96-well plate. The indicator bacteria cultures grown overnight were diluted to 1 × 10⁸ CFU ml^-1 and then 50 µl of them were added to each well containing the purified bacteriocin peptide. Buffer without bacteriocin served as negative control, while those with 100 µg ml^-1 bacteriocin served as positive control. The 96-well plate was incubated for 24-h at 37 °C and the OD measured at 600 nm using UT-6550 microplate reader. The MIC was defined as the concentration of bacteriocin that achieved a 50% growth inhibition (MIC₅₀), compared to the positive control. [21, 22] All experiments were carried out in triplicate

2.4 Bacteriocin stability assessment

The peptide antimicrobial activity was assessed using S. aureus ATCC 6538 and E. coli ATCC 8739 as indicator strains. Bacteriocin stability assessments were carried out based on a protocol by Goh and Philip [23]. The LCI peptide samples were incubated at 40, 60 and 80 °C for 40 min and at 100 °C 30 min. The LCI peptide samples were then set to room temperature (RT) prior to assessing their inhibitory effects. The LCI was adjusted to various pH levels using various buffers of 50 mM glycine-NaOH (pH 3–4), 50 mM sodium acetate (pH 4.7), 50 mM tris-HCl (pH 7.9) and 50 mM glycine-HCl (pH 9–11). The inhibitory effect was reassessed after 2 h of incubation at RT. The bacteriocin stability was assessed after exposure to various bile concentrations (0.5, 1 and 2%). Samples were incubated with LCI at 1× the MIC of the reference strains for 2 h at ambient temperature, followed by assessment of inhibitory activity.

2.5 The LCI kinetics of activity

Briefly, S. aureus and E. coli were cultured for 10 h, followed by centrifugation of the media at 2,000 rpm for 5 min to collect the cell pellets. Ice-cold 5 mM sodium phosphate buffer (pH 7.2) was used for the resuspension of each cell pellet. The resuspended cells were mixed at a 1:1 ratio with various treatments of LCI at 1× MIC, 0.03% acetic acid, LCI and 0.03% acetic acid, 1 mg ml^-1 nisin and LCI and 1 mg ml^-1 nisin and then incubated at 37 °C. A bacterial suspension without LCI served as control. Samples were collected at 20-min intervals to measure the OD₆₀₀ within a total time of 400 min.

2.6 Assessment of Escherichia coli membrane permeability using beta galactosidase

The E. coli cells were cultivated in lactose broth at 37 °C for 18 h to induce enzyme synthesis. The bacterial cells were harvested by centrifugation to remove the supernatant. The cell pellet was washed three times in 1× PBS buffer and then resuspended and the concentration adjusted to 1 × 10⁶ CFU 100 µl^-1 in 1× PBS. Then, 50 µl of the suspension with 50 µl of the purified LCI at concentrations of 2, 1, 0.5 and 0.25× MIC as well as 30 µl of o-nitrophenyl-β-D-galactoside (ONPG) were used as the reaction mixture for membrane permeability assessment. [24] Solutions in 96-well plates were incubated at 37°C for various time intervals. The enzymatic activity was assessed spectrophotometrically at 405 nm.

2.7 Statistic assessment

All experimental results were achieved from triplicate experiments for each sample. The reported data represented the mean of three replicates, using standard deviation (SD) to indicate the level of confidence. Statistical analysis was carried out using GraphPad Prism software (GraphPad, USA). One-way ANOVA was used to assess significant differences between the means (p < 0.05). Error bars represented SDs of the means.

1. Results and Discussion

3.1 Synthesis and purification of the recombinant LCI

In preliminary assessments, gene encoding mature LCI was amplified from pCL-LCT and then subcloned into pET-25b+ to create pET-25b+-LCI vector. To enable cleavage of the fusion purification tag (6×His), the expression system was modified. The plasmid construct design used for the LCI expression is shown in Figure 1A. The complete nucleotide sequence of the recombinant plasmid is shown as a chromatogram (Figure 1B). Each peak represented an individual base position in the recombinant plasmid. The sequencing chromatogram showed clear well-resolved peaks, verifying high-quality data with no ambiguous base calls. Comparison of the sequences with the plasmid map of pET-25b (+) containing the bacteriocin gene inserts verified 100% sequence identity of all designed elements. The LCI gene was shown as a single band, with amplicon sizes similar to the theoretical predictions, including approximately 195 bp using LCI primer and 510 bp using T7 promoter primer (Figure 1C).

3.2 Purification of the recombinant LCI and liquid chromatography-tandem mass spectrometry analysis

The low yield of LCI produced by wild B. subtilis A014 was initially a limiting factor that interrupted with the study process. This challenge was overcome by developing a recombinant expression system using host strain of E. coli BL21 (DE3) pLysS to moderate bacteriocin toxicity during background expression. This host could produce within low-temperature expression conditions to better manage protein toxicity and increase peptide concentration [25]. Following IPTG induction, peptides were purified using FPLC. The LCI peptide contained N- and C-terminal His₆-tags. This fusion expression strategy facilitated the purification process. Proteins were eluted using 250-mM imidazole buffer. This approach enabled sufficient peptide production with controlled process and consistent productivity, yielding up to 0.18 mg ml^-1. A single elution peak was correlated to antimicrobial activity; as verified in later assessments (Figure 2A). Using this method, 3 mg of the purified LCI were achieved from 15 ml of the soluble fraction. Molecular weight of the purified LCI bacteriocin was assessed as 6.05 kDa using SDS-PAGE. A single protein band was observed after Coomassie brilliant blue staining, verifying the protein purity (Figure 2B).

3.3 Assessment of inhibitory spectra

The purified recombinant LCI specific activity was assessed using serial dilution. The purified LCI bacteriocin was assessed against Gram-positive and Gram-negative bacteria as foodborne pathogens responsible for food poisoning and gastrointestinal diseases in humans. For the five indicator strains, results showed growth inhibition of all bacterial targets as well as significant antimicrobial activities, as indicated by decreases in ODs to less than 50% of the control group (p<0.05), suggesting high susceptibility of the assessed strains. Furthermore, M. luteus ATCC 6633, S. aureus ATCC 6538 and B. subtilis ATCC 6633 showed MIC values of 50 µg ml^-1, while Salmonella typhimurium ATCC 13311, E. coli ATCC 8739 and V. parahaemolyticus demonstrated MIC values of 100 µg ml^-1 (Table 1). The activity of LCI was similar to that of previously reported pediocin PA-1, which demonstrated efficacy against S. aureus [26]. Moreover, the MIC values indicated that LCI possessed broad-spectrum antibacterial characteristics, advantageous for addressing mixed infections caused by diverse pathogens, similar to bacteriocin Abp118, colicins and sakacin C2 [27–29]. This efficacy is particularly significant as foodborne pathogens pose a persistent threat through microorganisms such as Salmonella typhimurium in the food supply chain, as reported by He et al. [30]

3.4 Kinetics of the recombinant LCI function

Time-kill experiment was carried out to assess the mode of action and rate of inhibitory effects of LCI. The activity of recombinant LCI was assessed by monitoring growth patterns and membrane permeability in S. aureus ATCC 6538 and E. coli ATCC 8739 at various LCI concentrations. The initial mortality rate after incubation indicated that the bacterial growth inhibition depended on the LCI peptide concentration. Results showed that LCI showed a bactericidal mechanism against the target microorganisms, as verified by the sustained decreases in OD within time (Figure 3). After 220 min, the OD at 600 nm decreased significantly from 0.4 to 0.2. This was clearly observed at 100 min, where the Gram-negative bacterial samples treated with acetic acid showed an OD₆₀₀ of less than 0.5, in contrast to the control and peptide-only groups that respectively showed gradual decreases in ODs after 800 min (Figure 3A) and 150 min for Gram-positive bacteria (Figure 3B), compared to the control group and 0.03% acetic acid group alone. When the peptide is combined with acetic acid, the enhanced antimicrobial efficacy was similar to that previously reported Jiddah et al. [31], who reported enhanced antimicrobial activity of recombinant AGAAN when combined with acetic acid. Similarly, Rothong et al. [32] reported synergistic effects of phage-encoded antimicrobial peptide with organic acid against Acinetobacter baumannii. The increase activity could be resulted due to the disorganization of bacterial membrane, facilitating penetration of the antimicrobial peptide [32].

3.5 Membrane permeabilization assay

This experiment was carried out to assess if LCI exerted its effects on target cells through pore formation. The membrane permeabilization activity demonstrated by β-galactosidase through ONPG assays provided definitive evidence of LCI mechanism of action. The assessment was carried out when the peptide reacted with the ONPG substrate that was leaked out. The OD at 405 nm for untreated bacterial cells was constant (Figure 4). The intensification of yellow color from ONPG and β-galactosidase interaction indicated membrane pore formation. Upon exposure of the reference strain to LCI at concentrations of 0.25, 0.5, 1 and 2× MIC for 600 min, continuous increases in OD measurements were observed, indicating that extracellular o-nitrophenol production significantly increased in E. coli treated with LCI, compared to the control. The experimental findings suggested that this novel peptide analog affected bacterial membrane permeability and permeabilized cytoplasmic membrane of E. coli dose-dependently. Similarly, various bacteriocins such as nisin and plantaricin have similarly demonstrated the ability to disrupt bacterial membranes through pore formation [33–35]. Previous studies [25] reported LCI structural characteristics and suggested a novel mechanism for its action against Gram-negative bacteria. These experimental results verified LCI ability to inhibit Gram-negative bacteria. Additionally, the mode of membrane interaction was similar to 1EWS and MccJ25, which followed a toroidal pore model using positively charged AAs, particularly the AA group at the C-terminal end (Arg46-Lys47). This created short-lived channels in the bacterial membrane through toroidal pore formation, either destroying membrane integrity or penetrating the membrane to affect intracellular targets for antimicrobial effects [36, 37]. The subsequent release of intracellular substances verified the bactericidal characteristics of LCI, facilitating rapid elimination of pathogenic bacteria.

3.6 Effects of temperature, pH and bile salt inhibitors on the bacteriocin

Residual activity was assessed under various temperatures, pH levels and bile salt concentrations to assess LCI stability. The bacteriocin showed stability within a temperature range of 40–100 °C for 15 min. The proportion of surviving bacteria was 31.91% , 47.44% , 46.88% , 65.01%  and 74.58%  at 4, 40, 60, 80 and 100 °C, respectively (Figure 5A). The findings indicated that LCI included temperature stability, maintaining its complete antibacterial efficacy after 15 min of exposure to increased temperatures. The peptide activity of LCI was assessed under various pH conditions using various buffer systems. Results demonstrated that LCI included various stability and activity depending on the pH of the environment. In 50mM glycine-NaOH buffer, the peptide showed 37.81%  and 12.82%  activity at pH 3 and pH4, respectively. In 50mM tris-HCl buffer, the activity was 39.34%  at pH7 and 42.99%  at pH9, indicating relatively higher stability under near neutral conditions to slightly alkaline ones. However, in 50mM glycine-HCl buffer at pH9 and pH11, the peptide activity decreased significantly to 2.48%  and 20.93% , respectively. These findings suggested that LCI included greater stability and functionality in tris-HCl buffer at neutral to mildly alkaline pH, while its activity significantly decreased in strongly acidic or highly alkaline glycine-HCl environments (Figure 5B).

Moreover, LCI is characterized by significant stability under various environmental conditions. Its ability to maintain antimicrobial effectiveness at decreased temperatures up to 100 °C within a wide pH range (3–11) makes it an excellent candidate for food processing uses, where such conditions are common. This characteristic was similar to that reported by Johnson et al. [10] and Hols et al., [33], who emphasized that temperature and pH stability were essential for bacteriocins of industrial uses. For comparison, while pH-susceptible agents show optimal activity at neutral pH, bacteriocins such as nisin demonstrate greater efficacy under acidic conditions [34]. To evaluate its potential for use in food and animal feed, the stability of LCI peptide was further tested under gastrointestinal-like conditions. Several antimicrobial peptides lose their effectiveness in bile-rich environments, limiting their uses in gastrointestinal treatments. In the present study, the LCI survivability was assessed in artificial gastric juice at pH 2.5. After 2-h incubation, results indicated high peptide tolerance. The treat with LCI (0% of bile salt) demonstrated the highest antibacterial activity, with a peptide inhibition activity of 69.70±0.006. When exposed to 0.5% bile salt, inhibition efficiency decreased to 49.14±0.008. Similarly, under the condition 1.5% bile salt, inhibition rate was 50.84±0.007. At 2% bile salt concentration, LCI showed moderate activity, with 60.64±0.002 inhibition rate (Figure 5C). These findings indicated that although LCI activity decreased in presence of bile salts, it included significant antibacterial efficacy, particularly at higher bile salt concentrations. In conclusion, LCI demonstrated significant stability at low pH and the peptide preserved its efficacy in presence of bile salts.

1. Conclusion

In summary, an effective technique for LCI overproduction in E. coli was established in the present study. A significant quantity of LCI with antibacterial activity was generated through efficient purification. The purified LCI preserved its antibacterial characteristics, demonstrating antimicrobial activity under physiological pH and temperature conditions. The favorable characteristics of LCI suggest its potential as an effective antibacterial therapeutic agent. This study indicates that LCI represents a valuable addition to the growing repertoire of recombinant antibacterial peptides, including potentials to contribute to the development of animal nutrition and serve as a promising alternative to decrease use of antibiotics in the livestock industry.

1. Acknowledgements

This study was supported by a research fund from Petchra Pra Jom Klao-PhD scholarship and Faculty of Science Research Fund, King Mongkut’s University of Technology Thonburi, KMUTT.

1. Declaration of competing interest

The authors declared no competing interest.

1. Authors’ Contributions
2. R. Carried out the investigation, writing-original draft, data curation and formal analysis. N. S. Carried out the investigation and data curation. P. D. Carried out the investigation. Y. S. A. Helped in proofreading the manuscript, formal analysis and methodology. N. U. J. Helped in data curation and formal analysis. S. R. Helped in methodology and validation. T. R. Helped in supervision and formal analysis. P. P. Helped in supervision, conceptualization and formal analysis. C. A. Helped in supervision, formal analysis and validation. N. J. Carried out the major supervision, funding acquisition, conceptualization, resources and validation. Findings were discussed by all authors and they contributed to the final manuscript.
3. Using Artificial Intelligent Chatbots

No AI chatbot has been used in this study.

1. Ethical Consideration

This study did not include any human or animal participants and was carried out in accordance with safety standards.

Background and Objective: The process of coordination and communication between cells through diffusible signaling molecules is known as quorum sensing. Quorum sensing affects several factors in pathogens, including pathogenicity, adhesion, motility, biofilm production and cell aggregation. By controlling these signaling molecules, pathogenic factors can be controlled.

Material and Methods: In this study, effects of Bifidobacterium lactis, Bifidobacterium bifidum and Bifidobacterium longum on the expression of quorum sensing genes in Yersinia enterocolitica were studied at two various temperatures of 37 and 32 °C. First, Bifidobacterium spp. were activated in brain infusion broth and then cultured simultaneously with Yersinia enterocolitica, RNA extraction was carried out and yenI and yenR gene expression was assessed using real-time polymerase chain reaction.

Results and Conclusion: Results revealed significant differences in gene expression at 37 and 32 °C. When Bifidobacterium sp. was co-cultured with Yersinia enterocolitica at 32 °C, the bacterial quorum sensing gene expression significantly decreased in all treated cells, except yenI in Bifidobacterium bifidum co-culture, compared to the control. Bifidobacterium longum showed the highest decreasing effect in quorum sensing gene expression in Yersinia sp. by 64%. In contrast, co-culturing Bifidobacterium sp. with Yersinia enterocolitica at 37 °C revealed that Quorum sensing gene expression levels in Yersinia enterocolitica did was not change significantly in all cultures, except that with Bifidobacterium bifidum; almost similar to the control samples. These findings indicated that the interactions between probiotic bacteria and pathogens varied under various temperature conditions, demonstrating a temperature-dependent pattern.

Keywords: Bifidobacterium spp., Gene expression, Gene regulation, Quorum sensing, Yersinia enterocolitica

1. Introduction

Quorum sensing (QS) refers to the process of coordination and communication between cells, involving production of diffusible extracellular molecules and hence regulation of specific gene expression. The QS signals are called autoinducers because they are produced by the cells and typically induce their own synthesis, creating a positive feedback loop. Various types of QS systems reported in microorganisms include 1) LuxI/R type, which is commonly used by Gram-negative bacteria and uses acyl-homoserine lactone (AHL (molecules. This system is used for intraspecies communication due to its high specificity; 2) peptide signaling system, used by Gram-positive bacteria; 3) AI-2/LuxS signaling system, which facilitates interspecies communication and is detected in all bacteria and 4) AI-3 signaling system, which operates through epinephrine-norepinephrine and is used for interspecies and interterritorial communications [1,2,3]. In Gram-negative bacteria, QS can be inhibited through three methods of 1) inhibiting synthesis of AHLs, 2) degrading AHL molecules and 3) interfering with the receptors, which can be achieved through compounds produced by plants and microorganisms [4].

Yersinia enterocolitica, as a member of the Enterobacteriaceae family, is a Gram-negative, short rod-shaped, facultative anaerobic non-spore-forming bacterium. It multiplies at 0–45 °C, which distinguishes it from other enteric pathogenic bacteria [5]. Studies have shown that the three pathogenic species of Yersinia produce AHLs QS signals. Protein of LuxI, which synthesizes AHL as well as LuxR as a regulator, is detected in all three pathogenic Yersinia spp. However, Y. enterocolitica includes a unique pair of LuxRI (YenRI), whereas the other two species include two pairs. The YenI is responsible for synthesizing two types of AHLs, one of which is N-hexanoyl homoserine lactone (C6-HSL) and the other is N-3-oxo-hexanoyl homoserine lactone (3-OXO-C6-HSL). These two compounds are produced at a 1:1 ratio in Y. enterocolitica [6,7,8].

Gut microflora of humans and animals consists of several probiotic species such as Bifidobacterium. Typically, population of Bifidobacterium in the intestine of healthy adults ranges 10¹⁰-10¹¹ CFU g^-1; however, this decreases with age. Most species of Bifidobacterium are rod-shaped, anaerobic, non-motile and non-spore-forming species. Dairy fermentation products such as yogurt, cheese and fermented butter are addressed as major sources of probiotics [9]. For QS inhibition, extensive studies have been carried out on various pathogenic bacteria. In one study carried out on bacteria isolated from fish, 200 bacterial species were isolated from fish and ability of three selected species to inhibit QS system in Aeromonas hydrophila was verified. The study revealed that the bacteria belonged to the Bacillus genus and possessed an enzyme capable of degrading AHLs. The effect of this bacterium on A. hydrophila in fish showed that the control group of fish developed skin lesions over time and died after 12 d. However, fish that consumed the isolated strain survived and skin damage significantly decreased [10]. In another study investigating the effect of stress on signal production in Listeria spp., it was reported that stress induced by nisin and lactic acid did not play a role in production of AI-2 as an adaptive response to the environment and no dependency between these two factors was observed. However, this study concluded that QS could generally help L. monocytogenes cells adapted to stresses such as nisin and lactic acid [11].

Further studies on the inhibitory effect of various Bifidobacterium strains on EHEC (Enterohemorrhagic Escherichia coli) showed that B. longum, B. adolescentis and B. breve were the most inhibitory strains. For biofilm formation, most Bifidobacterium strains did not show a significant inhibitory effect, with only B. longum decreasing biofilm formation by 36%. Pathogenicity decreased in the presence of B. longum and results indicated that it regulated seven types of proteins in Escherichia coli [12]. Study of the effect of lactic acid produced by Pediococcus acetilactis on QS in Pseudomonas aeruginosa showed that lactic acid included an inhibitory effect on motility and short-chain HSL, elastase, protease, pyocyanin and biofilm productions. However, concentration of lactic acid needed to be precisely assessed for effective QS regulation [13]. Generally, the present study aimed to investigate the effect of co-culturing B. lactis, B. bifidum and B. longum on the expression of QS genes, yenI and yenR, in Y. enterocolitica under various temperature conditions. 

1. Materials and Methods

2.1. Bacterial Strain Preparation and Activation

The bacterial strains of B. lactis ATCC 19435, B. bifidum ATCC 29521, B. longum ATCC 15707 and Y. enterocolitica ATCC 23715 were provided by the Iranian Research Organization for Science and Technology (IROST). The Bifidobacterium strains were cultured in brain heart infusion (BHI) media (Merck, Germany) at 37 °C for 48 h under absolute anaerobic conditions using type A gas pack, anaerobic jar and indicators. Moreover, Y. enterocolitica was cultured in BHI media at 29 °C for 48 h [12]. For co-culturing Bifidobacterium spp. with Y. enterocolitica, fresh 24-h cultures of each Bifidobacterium strain were first adjusted to a cell density of 0.5 McFarland standard. Then, 100 µl of each adjusted Bifidobacterium culture were added to 10 ml of fresh Y. enterocolitica culture media and incubated at the specified temperature for each treatment [12].

2.2. Simultaneous Cultivation of Bifidobacterium sp. with Yersinia enterocolitica

The Y. enterocolitica was cultured in BHI media with three various probiotic bacteria separately at 32 and 37 °C for 48 h under anaerobic conditions. To standardize cell populations and ensure addition of a consistent microbial cell count in the assays, 0.5 McFarland standard was used with fresh 24-h culture that experienced two previous passages [12].

2.3. Primer Design and Polymerase Chain Reaction for the Verification of yenI and yenR Gene Presence 

Primers were designed for the analysis of yenI and YenR genes as well as the housekeeping gene (Table 1). Briefly, DNA extraction from Y. enterocolitica was carried out using boiling method. Then, polymerase chain reaction (PCR) was carried out (ASTEC G02, Japan) and the PCR products were analyzed via gel electrophoresis.

2.4. RNA Extraction and Reverse-transcriptase Polymerase Chain Reaction Analysis 

Yersinia enterocolitica and each Bifidobacterium sp. were simultaneously incubated at 32 and 37 °C for 48 h under anaerobic conditions. After incubation, cultures were centrifuged and the supernatant was discarded. The cell pellet was collected using 1.5-ml microtubes for RNA extraction. The RNA extraction was carried out using extraction kit (Kiagen Fanavar Arya, Iran) based on the manufacturer’s instructions and the RNA concentration was assessed using NanoDrop spectrophotometer (Thermo Scientific NanoDrop 2000, USA). Furthermore, cDNA synthesis was carried out using cDNA synthesis kit (Yekta Tajhiz Azma, Iran) and gene expression analysis was carried out using reverse-transcriptase polymerase chain reaction (RT-PCR) machine (Corbett Rotor Gene-RG 3000, Australia). Gene expression was analyzed using 2^-ΔΔCt method [14]. This method involved assessing gene expression level and normalizing it against a reference gene. Normalization corrected for variations in amplification efficiency, extraction conditions and initial sample volumes. Gene expression levels in treated and untreated samples were investigated and the difference in Ct values was calculated. Similar procedure was used to the reference sample. The relative gene expression changes were calculated by dividing the gene expression changes in the target gene by those of the reference gene. The calculation method and formulas were as follows [15]:

∆Ct = Ct _{target gene} - Ct _{housekeeping gene}, ∆∆Ct = ∆Ct _experiment - ∆Ct _Control, Fold gene = 2^-∆∆Ct      

Where, Ct was the threshold cycle. 

2.5. Statistical analysis

The mean Ct values of the treated samples were compared to those of the controls using one-way analysis of variance (ANOVA) to investigate if there was a statically significant difference between them (p < 0.05). All experiments were carried out in triplicate.

1. Results and Discussion

3.1. Verification of the presence of yenI and yenR genes in Yersinia enterocolitica

First, presence of the yenI and yenR genes in Y. enterocolitica strain must be verified. Results of the PCR amplification of yenI and yenR genes in Y. enterocolitica and their verification on agarose gels are shown in Figures 1 and 2.  Figure 1 verified presence of the yenI gene and Figure 2 verified presence of the yenR gene.

3.2. Analysis of yenI and yenR gene expression in Yersinia enterocolitica co-cultured with Bifidobacterium strains at 37 and 32 °C

The T-PCR analysis results Fold change for the yenI and yenR genes in comparison to the housekeeping gene are shown in Tables 2 and 3 and Figures 3 and 4. Fold change is a metric that expresses the extent of change between two measurements. In gene expression analysis, such as microarray or RNA-Seq analysis, it refers to the ratio of expression between samples or between groups. Moreover, melting curve and amplification of the target sequence in the treatments during RT-PCR are shown in Figure 5. In results achieved at 32 °C, adding Bifidobacterium to Y. enterocolitica culture decreased QS gene expression. At 37 °C, results completely varied. Results showed differences in expression of the studied genes at 37 and 32 °C. At 32 °C, presence of Bifidobacterium sp. decreased the expression of signal-producing genes, which was significant in most samples. At 37 °C unlike 32 °C, most samples did not differ significantly from the control samples. Studies have shown that 37 °C significantly affects regulation of genes associated to pathogenesis such as secretion systems and toxins and helps bacteria multiply better in host conditions.

In Y. enterocolitica, gene expression was affected by temperature, particularly at 37 °C. These temperature-dependent changes in gene expression assisted the bacteria effectively evaded the host immune system and enhanced its virulence. Specifically, at this temperature, expression of key genes associated with the bacterial virulence factors increased. These genes included those involved in the secretion of specific proteins, toxin production and responses to environmental stresses. At 37 °C, Y. enterocolitica efficiently used type III secretion system (T3SS), which was essential for the transfer of toxic proteins into host cells and expression of genes linked to this system increased significantly. Genes involved in production of specific toxins that helped in the bacterial toxic activities and proteins that prevented the host cell’s immune responses and regulatory systems for survival and stress conditions were significantly upregulated [16-18].

Technically, Y. enterocolitica, genes responsible for the production of AHLs can be regulated in response to environmental changes and host conditions [8]. Studies have shown that at 37 °C as the natural body temperature of humans, changes in the expression of these genes occur. Compounds encoded by these genes are involved in regulating collective behaviors such as toxin production, biofilm formation, pathogen-linked behaviors, host interactions and responses to environmental stress. At 37 °C, AHL production may increase, as this temperature specifically mimics the natural conditions of the human body and is critical for regulating pathogenic activities of the bacteria. Therefore, AHL-producing genes may become more active at this temperature, enabling the bacteria to regulate their collective behaviors, including toxin production and expression of other virulence factors [19, 20].

Naturally, Y. enterocolitica is an enteric pathogen that grows rapidly at 37 °C. At 37 °C, Y. enterocolitica typically reaches its maximum growth within 6–12 h, while bifidobacteria needs 12–24 h. Due to their high sensitivity to environmental conditions, bifidobacteria grow more slowly than that Y. enterocolitica does at 37 °C. Since Y. enterocolitica grows faster and reaches higher numbers within a shorter time at 37 °C compared to bifidobacteria, and due to the increases in expression of signal-producing genes involved in QS, toxin production, biofilm formation, pathogenic behavior and host interaction, it seems that Y. enterocolitica proliferates sufficiently and produces necessary beneficial compounds before bifidobacteria can reach sufficient numbers and begin production of inhibitory compounds. Additionally, these compounds (e.g. acidic substances) may further inhibit rapid growth of bifidobacteria. Once Y. enterocolitica dominates the environment, bifidobacterial growth becomes restricted. Due to the high sensitivity of bifidobacteria to environmental compounds, there is a possibility of no or weak growth of these bacteria. Secreted compounds needed for the bifidobacterial growth at 37 °C may be used by Y. enterocolitica due to the high number of Y. enterocolitica in the environment; thus, accelerating the bacterial growth [21,22,23].

In contrast, growth dynamics of the two bacterial species differ at 32 °C under strict anaerobic conditions, with bifidobacteria growing slightly faster than Y. enterocolitica. This faster growth of bifidobacteria leads to the production of growth-inhibitory compounds, limiting Y. enterocolitica proliferation and hence affecting expression of QS genes. At this temperature, expression of QS genes is less than that at 37 °C, as pathogenic activity is optimal at the latter temperature [24,25]. Based on these findings, presence of significant differences in the expression of QS genes at 32 °C and absence of significant differences of these genes at 37 °C can be justified. At the two temperatures, B. longum showed the minimum gene expression averagely. Previous studies on B. longum have shown that this bacterium is a clinically versatile probiotic strain and can control growth of pathogenic bacteria [26,27].

1. Conclusion

This study assessed temperature-dependent effects (37 against 32 °C) of various Bifidobacterium probiotic species on QS gene expression in Y. enterocolitica during co-culturing. Results showed that temperature changes included distinct effects on gene expression and pathogenic and probiotic behaviors of the highlighted bacteria. Additionally, results demonstrated that temperature variations significantly altered gene expression patterns, which directly modulated pathogenic behaviors of Y. enterocolitica and probiotic effects of Bifidobacterium strains. At 32 °C, Bifidobacterium sp. produced inhibitory compounds that suppressed proliferation of Y. enterocolitica and downregulated QS-associated genes. Pathogenic activities of Y. enterocolitica were significantly terminated at this temperature, with decreased expression of QS genes. This suggested that at 32 °C, Y. enterocolitica could not fully activate its virulence pathways, weakening its ability to escape immune defenses and infect the host. In contrast, no significant differences were observed between the controls and the treated samples at 37 °C. This study highlights the critical roles of temperature in modulating bacterial interactions and potentially their pathogenic characteristics. At 32 °C, Bifidobacterium sp. effectively suppressed QS gene expression in Y. enterocolitica. At 37 °C, QS gene levels were largely unchanged in all cultures, similar to those in control samples. These findings have provided valuable highlights for developing probiotic-based strategies to prevent and/or help treat infections caused by Y. enterocolitica and other pathogens. However, further studies seem necessary.

Background and Objective: Identifying a milk-clotting enzyme (MCE) with high κ-casein specificity and heat sensitivity remains a challenge in cheese production. Current microbial, plant, and recombinant MCEs often exhibit low clotting activity, poor κ-casein specificity, and high thermostability, compromising cheese quality and increasing production costs. In this study, to address this, we developed a computational pipeline combining structural analysis, machine learning, and molecular dynamics simulation to design approximately 160,000 peptides from the Rhizomucor miehei protease–Pepstatin A complex (PDB ID: 2RMP).

Material and Methods: Single-site mutagenesis, ML-driven affinity re-prediction, and physicochemical filtering yielded 84 peptides. Their specificity as aspartic proteases were validated via predicted Pepstatin A binding and further screened for cross-reactivity with αs1-, αs2-, and β-caseins.

Results and Conclusion: Two candidates, Pep1 and Pep2, demonstrated superior κ-casein binding affinities (ΔG = -50.20 and -39.07 kcal/mol at 40 °C, respectively), lower melting indices (-4.05 and -3.09), and significantly enhanced specificity scores (-10.85) compared to Rhizomucor miehei protease (ΔG = -33.9 kcal/mol at 45 °C; melting index = 0.17; specificity score = 0.85). These peptides represent promising vegan- and halal-friendly alternatives to chymosins, pending experimental validation.

Keywords: Milk-clotting enzymes, Computational peptide design, κ-casein specificity, Thermolabile peptides, Sustainable cheese production, Molecular dynamics simulations, Machine learning, Vegan cheese production, Halal cheese production, Aspartic protease.

. Introduction

Identifying chymosin substitutes with comparable enzymatic characteristics particularly high specificity for κ-casein (reflected in a high milk-clotting activity to proteolytic activity ratio, MCA/PA) and optimal heat sensitivity remains a significant challenge in cheesemaking [1-3]. Chymosin, traditionally derived from the stomachs of calves, has long been the gold standard for milk clotting due to its high specificity for κ-casein and its ability to produce high-quality cheese [4]. However, economic, ethical, dietary, and religious concerns, coupled with the rising demand for cheese production and consumption, have spurred the search for viable alternatives [1, 5]. Various sources of milk-clotting enzymes (MCEs) have been explored, including other animal-derived rennet (from lamb, buffalo, and camel), plant-based coagulants, and microbial and recombinant enzymes. Lamb and buffalo chymosin have lower MCA/PA ratios than calf chymosin, compromising cheese quality, though buffalo rennet’s stability suits mozzarella production [6, 7]. In contrast, camel rennet, while showing desirable clotting properties, is difficult to obtain in large quantities, limiting its widespread use [8]. Rennet from other animals, such as pigs and rabbits, often shows poor specificity and thermal stability, restricting their utility in cheesemaking [1]. Recently, marine organisms such as the shrimp Pleoticus muelleri have also been investigated as novel sources of coagulants [9-11]. Nevertheless, animal-based MCEs continue to face ethical and religious constraints. Plant-based coagulants, such as chymopapain from papaya, offer promising vegan- and halal-friendly alternatives [12]. However, their lower specificity toward κ-casein and tendency to generate bitterness limit their use, especially in aged cheeses [1, 13]. Microbial coagulants, particularly from bacterial and fungal sources, are increasingly favoured due to their cost-effectiveness and suitability for vegetarian and organic cheese production [14]. Although bacterial coagulants offer functional diversity [5, 15-18], they often exhibit high proteolytic activity, necessitating the development of improved strains through mutation [1]. Fungal coagulants are generally more compatible with cheesemaking conditions, particularly in terms of pH and temperature [1, 19]. Still, their high thermostability can cause undesired proteolysis during cheese ripening, adversely affecting texture and flavour [20]. Among fungal coagulants, mucorpepsin (EC 3.4.23.23) from Rhizomucor miehei is widely used due to its relative specificity. However, it still acts on α- and β-caseins and possesses high thermal stability, which can result in residual enzymatic activity after curd formation. This residual activity often leads to bitterness, altered texture, and reduced yield during cheese production [21-25]. Several studies have attempted to address these limitations using physical, chemical, enzymatic, and genetic modifications to enhance specificity, reduce thermostability, and minimise off-target proteolysis while maintaining milk-clotting efficiency. For example, some efforts have successfully reduced thermostability by removing N-linked carbohydrates, one of the primary factors contributing to R. miehei protease’s thermal resistance without significantly diminishing clotting activity [1, 25-29]. Other studies have enhanced milk coagulation without altering thermostability [30-35]. Genetic engineering, mutagenesis [36-39], substrate modification[40-43], and enzyme immobilisation [44, 45], have all been explored to develop efficient coagulants suitable for industrial cheese production. Nevertheless, regulatory restrictions on genetically modified organisms and technical and economic challenges have limited the commercial adoption of many of these approaches [46].  A novel strategy for identifying milk-clotting enzymes involves targeting conserved motifs—namely FDTSSD or FDTGSSE, found in all currently known commercial coagulants. Using this approach, several in silico BLAST searches have identified new candidate proteases for cheese manufacturing. Moreover, degenerate primers based on these motifs can identify relevant genes even when full gene sequences are unknown [26]. Computational enzyme design and molecular modelling have shown great potential in optimizing enzyme activities [47-49]. Advances in bioinformatics, molecular dynamics, and machine learning now enable the prediction and engineering of enzymes with enhanced specificity, catalytic efficiency, and thermostability. Structural insights into active sites support rational design through targeted mutations[50-52]. These in silico methods reduce experimental cost and time by predicting enzyme performance before validation. Notably, ancestral sequence reconstruction has been used to design a novel aspartic protease with improved κ-casein affinity and thermolability for cheesemaking [53]. Although milk-clotting enzymes (MCEs) have been widely studied, the application of advanced computational tools for their rational design remains underexplored [54]. Moreover, current MCEs often exhibit limitations such as low substrate specificity and excessive thermostability, which can negatively affect cheese quality and increase commercial costs. This study introduces a computational pipeline to design thermolabile, κ-casein-specific milk-clotting peptides as sustainable, vegan-, and halal-friendly alternatives for cheese production. An integrated computational pipeline was developed, incorporating machine learning, rational design, and structure-based screening. A combinatorial library of 160,000 peptide variants was generated based on conserved catalytic motifs from the Rhizomucor miehei protease–Pepstatin A complex, linked through a rationally designed sequence. The library was screened using predictive models trained on binding affinity data, followed by structure-based refinement, physicochemical profiling, and validation through molecular docking and molecular dynamics simulations. This in silico framework provides a foundation for the experimental development of next-generation coagulants for dairy applications.

1. Materials and Methods

2.1. Aspartic peptide design strategy

We present a computational pipeline for the de novo design of short peptide-based milk-clotting enzymes (MCEs) with enhanced affinity for κ-casein and improved chemosensitivity. The design strategy involves identifying interface residues containing catalytic motifs from Rhizomucor miehei protease (RMP), linking them via an optimised linker, and screening the resulting peptide candidates for specific binding and functional activity. To benchmark the design process, RMP was used as a positive control and also served as the structural template, while a peptide with low affinity for Pepstatin A was employed as a negative control. The results for both controls are provided in the supplementary file on GitHub.

2.2. Interface residue identification

Interface residues from the crystal structure of 2RMP (RMP–Pepstatin A complex) were identified using the PDBePISA tool (https://www.ebi.ac.uk/pdbe/pisa/) [55] and served as the starting point for aspartic peptidase design. Two continuous interface segments containing the catalytic aspartic acid residues were selected: LFDTGSS (residues 36–42, harbouring the DTGS motif) and TIDTGTNFFI (residues 235–244, containing the DTGT motif). Although the ITYGT segment was identified among the interface residues, it was excluded from the design due to its lack of direct involvement in the conserved DTGS/DTGT catalytic motifs. Furthermore, incorporating non-catalytic segments would unnecessarily increase peptide length without functional contribution, potentially reducing the structural stability and target specificity of the designed peptides.

2.3. Continuous interface residues segment

Catalytic motif of 2RMP (38-41, 237-240)

1. LFDTGSS = 36-42 (7 residues)
2. TIDTGTNFFI = 235-244 (10 residues)
3. ITYGT = 80-84

2.4. Linker design and variant library construction

The designed peptide sequence comprises two continuous interface segments, LFDTGSS (residues 36–42) and TIDTGTNFFI (residues 235–244), totalling 17 residues. The spatial distance between the terminal residues of these segments (Ser42 and Thr235) was measured to be 13.8 Å using chimaera v1.17.3. The average distance between consecutive Cα atoms in a polypeptide chain is approximately 3.8Å [56-58].This distance corresponds to approximately four amino acids, indicating the optimal linker length required to bridge the two segments. Incorporating a four-residue linker (XXXX), the final peptide construct contains 21 amino acids. A saturated mutagenesis approach was applied to the linker, yielding 160,000 variants (20⁴ combinations) of the parent sequence, LFDTGSS-XXXX-TIDTGTNFFI.

2.5.Machine learning models for protein -protein interaction prediction: PDBbind+ and SKEMPI 2.0 Datasets

In this study, datasets were sourced from three major repositories: (1) 160,000 peptide variants generated through combinatorial substitution, (2) 7,086 mutants curated from the SKEMPI 2.0 database, which focuses on mutations in protein–protein interactions[59], and (3) 3,176 protein–protein complexes obtained from the PDBbind+ dataset[60]. For SKEMPI 2.0, duplicates, ambiguous entries (e.g., conflicting binding affinities or incomplete mutation annotations), and incomplete records (e.g., missing sequences or ΔΔG values) were removed. Similarly, the PDBbind+ dataset was filtered by excluding duplicate complexes, sequences with non-canonical residues or extreme lengths, entries lacking binding affinity or structural data, and those with physiologically irrelevant binding values. After filtering, the cleaned data were divided into training (70%), testing (20%), and validation (10%) sets. Two independent machine learning models XGBoost and a Convolutional Neural Network (CNN) were then employed to predict protein–protein interactions.

2.6. SKEMPI 2.0 dataset and CNN-based machine learning model

The SKEMPI 2.0 dataset, comprising 7,086 mutations from 295 studies, was used to train a deep learning model for predicting changes in protein–protein binding affinity[61]. After removing duplicates, ambiguous entries, and incomplete data, 343 high-quality entries were selected, each containing PDB IDs, chain identifiers, affinity values, and protein sequences. Sequences were normalised by peptide length for consistency. Model training was performed using the DeepPurpose framework (v0.1.5)[62], which employs convolutional neural networks (CNNs) for protein sequence encoding. Protein sequences were processed using DeepPurpose’s data_process() function, which internally encodes sequences and applies padding to handle variable lengths without manual preprocessing. The dataset was divided into training (70%), testing (20%), and validation (10%) sets. The model was trained over 100 epochs with a learning rate of 0.001 and a batch size of 32. Performance was evaluated using mean squared error (MSE), R², and Pearson correlation coefficient. The trained model was then used to predict the binding affinity between κ-casein and 160,000 designed peptide variants. Based on predicted scores, the top 21 peptides were selected for further analysis.

2.7. PDBbind+ dataset and XGBoost-based machine learning model

The PDBbind+ dataset, consisting of 3,176 protein–protein complexes [60], was curated by eliminating duplicates, invalid sequences, and missing data, resulting in 1,049 remaining complexes. Affinity values were standardised, log-transformed, and negative values were excluded. A sequence-based predictive model was developed using Conjoint Triad encoding (via DeepPurpose) and logarithmic normalisation to estimate protein–protein and protein–peptide binding affinities. Several machine learning models were tested using Scikit-learn, including Random Forest (R² = 0.67), Ridge (R² = –1.482), Gradient Boosting (R² = 0.647), and XGBoost (R² = 0.696). XGBoost was selected for its superior performance[63]. The model was optimised using the Optuna framework with 100 trials and 5-fold cross-validation, tuning hyperparameters such as estimators (100–1000), learning rate (0.01–0.3), max depth (3–10), subsample (0.6–1.0), and colsample_bytree (0.6–1.0). Outlier detection was performed using an Isolation Forest with a contamination rate of 0.15. The final model, evaluated using mean squared error (MSE), R², and Pearson correlation, was used to predict binding affinities for 160,000 κ-casein peptide variants, identifying the top 48 candidates for further investigation.

2.8. Single-site mutation and machine learning prediction

The top 69 sequences (48 from the model trained on PDBbind+ and 21 from the model trained on SKEMPI 2.0) were further refined through single-site mutations at positions Phe2 (PHE), Ile13 (ILE), and Phe19 (PHE). To preserve peptide structural integrity and avoid disrupting potential hydrogen bonding networks, mutations were excluded from adjacent serine (S) and threonine (T) residues surrounding the linker. Instead, three hydrophobic residues Phe2 (F2), Ile13 (I13), and Phe19 (F19) were carefully chosen as mutation sites due to their roles in local packing and potential linker interactions. Each position was systematically substituted with all 20 canonical amino acids, generating 4,140 unique sequences (20×3×69). The two trained models were used again to predict interactions between these sequences and κ-casein, resulting in 101 selected peptide sequences (96 from the model trained on PDBbind+ and 5 from the model trained on SKEMPI 2.0) with optimal binding affinity.

2.9. Physicochemical property assessment

The top 101 peptide sequences underwent comprehensive screening based on thermal stability, pH, solubility, and toxicity. DeepSTABp (https://csb-deepstabp.bio.rptu.de/) was used to predict melting temperature [64].

ToxinPred (https://webs.iiitd.edu.in/raghava/toxinpred/multi_submit.php) evaluated pH and solubility based on isoelectric point, hydrophobicity, hydrophilicity, amphipathicity, steric hindrance, charge, and molecular weight[65]. ToxinPred2  (https://webs.iiitd.edu.in/raghava/toxinpred2/) [66] and ToxinPred3 (https://webs.iiitd.edu.in/raghava/toxinpred3/index.html) [67] assessed toxicity profiles. This screening identified 84 peptide sequences with favourable physicochemical properties.

2.10. Pepstatin A binding affinity as a proxy for aspartic protease activity

Pepstatin A binding was used as a proxy to assess aspartic protease activity. A pre-trained model, Morgan_CNN_BindingDB_IC50, from the DeepPurpose framework[68], was employed to predict interactions between Pepstatin A and the 84 selected peptides. The model utilised a multi-layer perceptron for drug representation (hidden layers: [1024, 256, 64]) and a convolutional neural network for peptide representation (convolutional layers: filters [32, 64, 96]; kernel sizes [4, 8, 12]). Based on predicted binding affinity, 17 peptides with strong interaction to Pepstatin A were identified for further analysis.

2.11. Cross-reactivity profiling with non-target casein subunits

The 17 selected peptide sequences were further evaluated for binding specificity by assessing their predicted interactions with αs1-casein, αs2-casein, and β-casein, using the same trained models and datasets. Two peptides exhibited low binding affinities to these casein subunits, suggesting a high degree of specificity toward κ-casein. These sequences hold particular promise for targeted applications in unique cheese production processes.

2.12. Predicting and evaluating the 3D structures of the peptidase

The two peptides with the highest binding affinities to Pepstatin A were predicted using PEP-FOLD4 (https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD4/) [69], a free tool for predicting linear, cyclic, and chemically modified peptide structures. It uses the improved sOPEP force field and Monte Carlo simulation with Debye-Hückel-free parameters for refinement. The Best Model is based on the lowest SOPEP score. PEP-FOLD4 also provides an SA probability map showing residue conformations: red (helices), green (β-strands), and blue (coils/loops), aiding in stability and secondary structure prediction. MD simulations were run for each peptide, and final stable structures were extracted. Structural quality was then assessed using ERRAT[70], Procheck[71], and verified for further processing.

2.13. Molecular docking of peptidase with κ-casein

Since the X-ray structure of κ-casein remains undetermined, our analysis focused on the local structure surrounding the Phe105–Met106 bond, which is susceptible to aspartic enzyme cleavage in bovine κ-casein. We aimed to explore how this local fragment interacts with RMP and the designed peptides to investigate the underlying mechanism, as highlighted in earlier studies [53, 72, 73]. The same methodology was followed for ligand preparation, with some modifications. The RMP binding site in κ-casein spans residues 102 to 108 [74], while the chymosin binding site covers residues 97 to 112 [75, 76]. The shorter segment (residues 102–108, HLSFMAI) was used as the ligand for docking with RMP, as it corresponds to the P4–P4′ binding region of Pepstatin A in the Co-Crystallised complex with RMP [74], ensuring structural relevance. In contrast, the longer fragment (residues 97–112, RHPHPHLSFMAIPPKK) was used for docking with the designed peptides (Pep 1 and Pep 2) and was also utilised throughout the machine learning pipeline to capture a broader binding context, consistent with its known role in chymosin recognition. This region was predicted using AlphaFold2[77]. Molecular docking was performed using the HADDOCK v2.4-2022.08 web server[78, 79], with the peptides as receptors and κ-casein as the ligand. Asp3 and Asp14 were defined as active residues for the peptides (first molecule), and residues 102 to 108 were set as active for κ-casein (second molecule). The top docking poses were selected based on a combination of criteria, including the size of the largest cluster, the most negative Z-score, the number of hydrogen bonds, and the lowest binding energy. Interaction analyses were carried out using LigPlot+ v1.4.5 software[80].

2.14. Molecular dynamics simulation (MD)

The well-recognised and freely licensed software used in this study was GROMACS version 2024[81]. Topology parameters for the anticipated models were produced using the AMBER force field (ff99SB) in GROMACS [82] .The constructed peptides (pep 1and Pep 2)  and RMP models were placed in a cubic box with 10 Å spacing, solvated with the TIP3P water model[83]. Energy minimisation was performed using the steepest descent technique[84], and long-range electrostatics were calculated using the Particle-Mesh Ewald (PME) method[85], with a 1.0 nm cut-off for van der Waals and electrostatic interactions. The maximum number of minimisation steps was 50,000. Equilibration was carried out using 5000 ps of NVT and NPT runs. The V-rescale thermostat (0.1 ps coupling) maintained temperature at 318.15 K, which is the optimal temperature for RMP, during NVT. The Berendsen barostat was employed during the NPT equilibration phase with a coupling time of 2.0 ps to allow fast and stable pressure convergence. Although the Berendsen method does not sample a true NPT ensemble, it is widely used for initial stabilisation [86]. For the subsequent 100 ns production run, the Parrinello–Rahman barostat was used to ensure thermodynamically rigorous sampling of pressure fluctuations and system behaviour [87]. Simulations were also conducted at different temperatures for comparative analysis. Analytical tools including gmx rms, gmx rmsf, gmx gyrate, gmx hbond, and gmx sasa were used to calculate RMSD, RMSF, radius of gyration, hydrogen bonds, and SASA, respectively[88]. All data were extracted from trajectories and visualised using Origin.

2.15. MM-PBSA binding energy, principal component, and free energy landscape analyses of protease complexes

MM-PBSA binding free energy and PCA analyses were conducted using snapshots from the equilibrated MD trajectory at 100 ps intervals. The g_mmpbsa tool was used to calculate the binding free energy (ΔG_bind) based on the equation:        ΔG_bind​ = ΔE__MM + ΔG__solvation – TΔS

Where ΔE_MM includes electrostatic and van der Waals interactions, and ΔG_solvation comprises both polar and non-polar solvation energies. The entropy term (TΔS) was approximated or omitted due to computational limitations; therefore, the results mainly represent the enthalpic contribution (ΔH) and reflect approximate binding affinity values. PCA was performed using GROMACS tools (g_covar and g_anaeig) to analyse major motions in the complex based on a mass-weighted covariance matrix of backbone atoms, yielding eigenvectors and eigenvalues that represent motion direction and magnitude[89-91]. Additionally, Free Energy Landscape (FEL) analysis was conducted using RMSD and Radius of Gyration (Rg) as collective variables (CVs), extracted via gmx rms and gmx gyrate. The FEL was calculated using the Boltzmann relation, ΔG = −k_BT ln P(CV1, CV2), and visualised as a 2D contour map using gmx sham,    highlighting stable (low-energy) and unstable (high-energy) conformational states [92].

2.16. Statistical analysis

Quantitative data analysis was performed using GraphPad Prism 9, with results presented as mean ± standard deviation. A one-way analysis of variance (ANOVA) was used to evaluate differences across groups, followed by Tukey's multiple comparisons test to identify specific pairwise differences. Statistical significance was set at p < 0.05.

1. Results and Discussion

3.1. First prediction: ML-based affinity screening of peptides targeting κ-casein

We configured DeepPurpose to process our peptide dataset (160,000) using two tailored machine learning models trained on the SKEMPI 2.0 and PDBbind+ databases, with both datasets split into 70% training, 20% testing, and 10% validation sets. For the PDBbind+ model, we applied an XGBoost-based algorithm utilizing Conjoint Triad encoding and logarithmic normalisation. Outliers were removed using Isolation Forest (contamination = 0.15). This model achieved a Pearson correlation coefficient of 0.8360, an R² value of 0.6907, and a mean squared error (MSE) of 116. 4807. In contrast, the SKEMPI 2.0 model was constructed using a convolutional neural network (CNN) within the DeepPurpose framework, with input sequences normalised by length. The model architecture included hidden layers of 64 and 32 units, a learning rate of 0.001, a batch size of 32, and training over 100 epochs. It yielded stronger predictive performance, with a Pearson correlation of 0.964, R² of 0.92, and MSE of 0.0022. Following the prediction, 69 top peptide sequences were selected 48 from the PDBbind+ model and 21 from the SKEMPI 2.0 model for further evaluation against κ-casein. The SKEMPI-derived sequences showed variable predicted binding scores, ranging from 0.124221 to 0.1120, reflecting nuanced differences in predicted affinities captured by the CNN (Fig. 1). In contrast, all PDBbind+-derived sequences received an identical predicted score of –8.08991, potentially due to structural similarity among candidates or model limitations in resolving fine-grained differences. The dual-model approach enabled complementary evaluation of peptide binding predictions. The CNN-based model trained on SKEMPI 2.0 exhibited superior predictive accuracy, likely due to its ability to learn fine-grained local patterns relevant to short peptide interactions. This aligns with prior studies showing that convolutional architectures are well-suited for capturing local dependencies in biological sequences and outperform tree-based methods in small, mutation-sensitive datasets such as SKEMPI2.0 [93, 94]. The performance metrics suggest that this model captured subtle sequence-affinity relationships better than the XGBoost model trained on PDBbind+, which may be less sensitive to small mutations and optimised for larger, structurally diverse protein–protein interfaces[95]. These findings underscore the critical influence of dataset composition, sequence representation, and algorithmic architecture in machine learning–guided protein design. By integrating two complementary models, the current investigation enhances confidence in candidate selection and provides a framework for future predictive workflows in rational peptide engineering.

3.2. Second prediction: Single-site mutagenesis and ML-based optimisation of peptide affinity for κ-casein

To improve κ-casein binding, 4,140 single-site variants were generated from 69 lead peptides, each following the structure LFDTGSSXXXXTIDTGTNFFI. To maintain peptide structure and hydrogen bonding, mutations near serine (S) and threonine (T) residues flanking the linker were avoided. Instead, three hydrophobic residues, Phe2 (F2), Ile13 (I13), and Phe19 (F19) involved in local packing and linker interactions were selected for mutation. Each was independently substituted with all 20 canonical amino acids. The variants were evaluated using machine learning models trained on PDBbind+ and SKEMPI 2.0 datasets. From this second prediction, 101 top candidates were selected: 96 from the PDBbind+ model (all with a predicted score of 1.3525908) and 5 from SKEMPI 2.0 (scores ranging from 0.062 to 0.088) (Fig. 2). The identical scores from PDBbind+ are likely due to the XGBoost algorithm combined with a descriptor set that captures general physicochemical properties. This descriptor likely assigned similar feature values to many peptides, resulting in minimal differentiation in predicted affinity. This highlights both the utility and the limitations of such models, particularly when applied to datasets with structurally and chemically similar variants.

3.3. Assessment of physicochemical properties

Comprehensive evaluation of physicochemical properties is critical in the selection of functional peptides for industrial and biomedical applications, particularly in processes such as milk coagulation. Where solubility, thermal sensitivity, and non-toxicity are essential traits. In this study, 101 computationally selected peptides were screened using in silico tools to predict their toxicity, charge distribution, solubility, molecular weight, and thermal stability. ToxinPred analysis confirmed that all peptides were non-toxic, with negative SVM scores between -1.03 and -0.24, which is consistent with safety requirements for food-grade peptide additives[65]. These findings minimise concerns about cytotoxic or allergenic effects, thereby supporting their potential use in dairy fermentation systems. The net charge of most peptides was -3, and their isoelectric points (pI) ranged from 3.57 to 4.21, indicating strong acidity and that they remain negatively charged under physiological conditions. This negative charge may promote electrostatic repulsion between peptide molecules, reducing aggregation and enhancing aqueous solubility—an important attribute for homogeneous distribution in milk matrices[96]. The peptides’ hydrophilic nature, inferred from their sequence composition, and moderate molecular weights (2225–2590 Da), also suggest favourable pharmacokinetic behaviour and low aggregation potential, features typically associated with improved stability and bioavailability [97]. Thermal stability, as predicted by DeepSTABp, showed melting temperatures ranging from 55.27°C to 59.59°C. These moderate thermal profiles are advantageous for milk-clotting enzymes, as excessive thermal stability may hinder enzyme deactivation post-clotting, while overly heat-labile peptides might degrade during processing [46]. Thus, the predicted melting temperatures reflect an optimal balance for heat-sensitive processes, potentially allowing efficient clotting at typical curdling temperatures (around 40–45°C) while avoiding residual activity that could affect cheese texture during ageing. Integrating these diverse parameters—non-toxicity, net negative charge, solubility, appropriate molecular weight, and moderate thermostability—allowed the rational narrowing of the peptide pool to 84 candidates. These peptides represent promising leads for development as aspartic protease-like milk coagulants, offering a non-animal alternative with tunable biochemical properties tailored for dairy industry needs.

3.4. Binding selectivity analysis

3.4.1. Aspartic Activity Analysis: Binding Affinity of Peptides Toward Pepstatin A

As part of our strategy to design peptides that functionally mimic natural aspartic proteases, we used predicted binding affinity to Pepstatin A, a specific inhibitor of aspartic proteases, as a proxy for enzymatic activity. Peptides with high predicted affinity are likely to exhibit aspartic protease-like functionality, justifying their use in functional screening. To assess this, 84 peptides were evaluated for their binding affinity to Pepstatin A using the Morgan_CNN_BindingDB_IC50 model (Fig. 3A). This model was selected due to its strong methodological and biological relevance: it is trained on BindingDB, which contains extensive IC₅₀ data for enzyme–inhibitor interactions, including those involving aspartic proteases. The incorporation of Morgan fingerprints enables effective representation of small molecules such as Pepstatin A, while the convolutional neural network (CNN) architecture facilitates the extraction of features pertinent to molecular binding. Among the models tested, this one demonstrated superior performance in predicting small-molecule–protein interactions. Given Pepstatin A’s role as a potent and structurally well-defined inhibitor of aspartic proteases, the model is particularly suitable for estimating binding affinities in this context. The model, trained over 800 iterations, demonstrated rapid convergence, with loss values decreasing from 0.24 to near zero. Validation metrics indicated high predictive performance (R² = 0.8277, MSE = 0.0025, Pearson r = 0.98) (Fig. 3B). Predicted binding affinities ranged from 5.325 to 5.240 (log IC₅₀), suggesting consistently strong interactions. These findings support the functional aspartic activity of the peptides and their potential utility in cheese-making applications. The top 17 peptides were selected for further analysis (Fig. 3C). The use of the Morgan_CNN_BindingDB_IC50 model, a deep learning-based approach trained on the SKEMPI 2.0 dataset, allowed for high-fidelity prediction of molecular interactions, as evidenced by robust performance metrics (R² = 0.8277, MSE = 0.0025, Pearson r = 0.98) [98, 99]. The model’s rapid convergence and low final loss further highlight its reliability in capturing relevant biochemical features governing binding behaviour. The narrow range of predicted binding affinities (log IC₅₀ values between 5.325 and 5.240) suggests a high degree of consistency among the peptides in terms of their interaction strength with Pepstatin A. This indicates that many of the evaluated peptides may possess conserved structural or functional motifs typical of aspartic proteases, aligning with the hypothesis of their functional relevance. Such strong predicted interactions imply not only potential inhibitory characteristics but also functional mimicry of natural aspartic proteases, which are integral to milk clotting and protein breakdown in cheese production.

3.4.2. Cross-reactivity evaluation with non-target casein subunits

To further evaluate the specificity of the designed peptides, the top 17 sequences with strong predicted binding affinity to Pepstatin A were analysed for cross-reactivity against non-target casein subunits (αs1-, αs2-, and β-casein) using the same machine learning model (Fig. 4). The average cross-reactivity score among these peptides was calculated to be 0.431529 (Table 1). Based on this threshold, six peptides with cross-reactivity values below the average were selected as more selective candidates. Among them, Pep1 (LFDTGSSVDEMTRDTGTNFFI; log IC₅₀ = 5.325; Cross-Reactivity = 0.3636) and Pep2 (LFDTGSSADESTRDTGTNFFI; log IC₅₀ = 5.309; Cross-Reactivity = 0.2706) showed both the lowest cross-reactivity scores and high predicted binding affinity to Pepstatin A. To assess structural reliability, all 17 peptides were subjected to molecular dynamics (MD) simulations. The root-mean-square deviation (RMSD) of backbone atoms was used as an indicator of conformational stability. Pep1 exhibited the lowest RMSD value (0.407 nm), followed closely by Pep2 (0.513 nm), indicating that both peptides maintain stable conformations during simulation and are structurally reliable (Fig. 5). These combined criteria, low cross-reactivity, high binding affinity, and strong conformational stability were used to identify Pep1 and Pep2 as the top candidates for κ-casein-specific targeting in cheese-making applications.  Pep1 showed a slightly stronger interaction with Pepstatin A (5.325). Pep2 exhibited the lowest cross-reactivity with all non-target caseins, particularly αs2-casein (0.0717), indicating higher substrate specificity. These results suggest that Pep1 is better suited for applications requiring high catalytic efficiency. Whereas Pep2 is ideal for precise selectivity in cheese-making processes. These findings underscore the importance of balancing catalytic potency with substrate specificity when designing casein-targeting peptides for dairy applications[100]. Although Pep1 demonstrates superior binding affinity to Pepstatin A, its slightly higher predicted interactions with αs-caseins could pose a risk of off-target proteolysis during cheese processing [101]. In contrast, Pep2’s exceptionally low cross-reactivity enhances its utility for applications where minimal disruption of non-κ-casein proteins is critical, such as in the production of speciality cheeses with defined texture and flavour profiles [102]. Future in vitro and in situ validations will be essential to confirm these in silico predictions and to optimise the application of these peptides in industrial cheese-making protocols. Overall, this approach highlights the potential of machine learning-guided screening for rational peptide design in food biotechnology [103] .

3.5. Structure prediction and validation

The structural validation of the designed peptides was carried out using PEP-FOLD 4 modelling, Ramachandran plot analysis, and ERRAT evaluation to ensure their stereochemical quality and overall stability. For the first designed peptide (Pep1), the predicted 3D structure generated by PEP-FOLD demonstrated a well-folded and compact conformation, indicating a plausible structural model (Fig. 6A). The use of PEP-FOLD 4, a well-established de novo modelling tool, facilitates the generation of compact and reliable peptide conformations. It enables the identification of energetically favourable structures that mimic biologically relevant folds, which is critical during early-stage peptide design [69]. The Ramachandran plot showed that 94.1% of the residues were located in the most favoured regions, with the remaining 5.9% distributed in the additional allowed regions (Fig 6B). This distribution suggests that nearly all backbone dihedral angles fall within sterically permissible regions, reflecting a highly acceptable stereochemical quality. Such a high percentage of residues in the most favoured regions is a hallmark of high-resolution protein structures and indicates minimal strain in backbone geometry [71]. Moreover, the ERRAT quality factor for this peptide was 100%, indicating an excellent profile for non-bonded atomic interactions and confirming the high reliability of the predicted structure (Fig. 6C). The ERRAT score of 100% indicates that the model has non-bonded atomic interactions comparable to those found in well-refined experimental structures, further reinforcing its structural reliability [70]. Similarly, for the second designed peptide (Pep2), the structure predicted by PEP-FOLD also revealed a stable conformation consistent with a biologically plausible fold (Fig. 6D). The recurrence of a compact and stable fold across multiple models supports the reproducibility and robustness of the peptide design strategy. The Ramachandran plot analysis mirrored the results observed for the first peptide, with 94.1% of residues positioned in the most favoured regions and 5.9% in additional allowed regions (Fig. 6E). This consistency reinforces the structural integrity of the designed peptide and highlights the effectiveness of the design strategy. It also suggests that the second peptide maintains favourable backbone torsion angles and avoids sterically hindered conformations, indicating a structurally sound model [104] The ERRAT analysis for the second peptide also yielded a perfect score of 100%, demonstrating the presence of highly favourable atomic interactions comparable to those found in high-resolution crystal structures (Fig. 6F). This high ERRAT value underscores the accuracy of atomic-level geometry and supports the overall reliability of the structural prediction for Pep2. The data exhibited that both designed peptides Pep1 and Pep2 successfully met important structural quality standards. The large number of amino acids in preferred areas of the Ramachandran plot, along with excellent ERRAT scores, strongly suggests that the predicted structures are sound and of high quality. The agreement between both stereochemical and atomic-level validation methods strongly supports the robustness of the design and predictive workflow. Such consistency not only strengthens confidence in the structural integrity of the peptides but also indicates their suitability for further studies involving interaction modelling, stability profiling, or therapeutic evaluation. This indicates that the peptides have correct stereochemistry and form stable, dependable three-dimensional shapes, which suggests they could be useful for more research into their structure, function, or potential as therapies.

3.6. Molecular docking analyses for Pep1 and Pep2

Docking analysis was conducted to assess the binding affinity and interaction profiles of the two designed peptides (Pep 1 and Pep 2) in comparison with the reference molecule RMP (2ASI). Several key parameters were evaluated, including Z-score, docking score, number of hydrogen bonds, salt bridges, hydrophobic interactions, and cleavage sites. In the HADDOCK docking results, Pep 1 exhibited the lowest Z-score of -1.7, indicating the most energetically favourable and structurally reliable model among the three ligands. Pep 2 followed with a Z-score of -1.5, while the reference RMP had the highest Z-score of -1.1, suggesting comparatively less stable binding. These findings imply that both designed peptides adopt favourable conformations within the receptor's binding site, with Pep 1 demonstrating the most promising structural quality. In terms of HADDOCK scoring, where more negative values suggest more favourable predicted interactions, Pep 1 achieved the best score among the peptides (-78.9 ± 0.1), followed by Pep 2 (-69.5 ± 1.2). The reference full-length enzyme RMP exhibited a more negative score (-126 ± 7.5), likely due to its larger size (360 amino acids) and broader interaction surface, which allows for more extensive electrostatic and van der Waals interactions. Therefore, direct comparison between short peptides (21 amino acids) and the full-length enzyme should be interpreted with caution, as HADDOCK scores are influenced by molecular size and interaction complexity rather than binding strength alone. Nonetheless, the favourable scores of Pep 1 and Pep 2 indicate that these peptides can form stable and biologically relevant interactions with the target receptor. Their interaction energies fall within the range typically observed for biologically active peptides in similar docking studies, supporting their potential as viable aspartic protease candidates [105]. Interaction profiling further revealed that Pep 1 formed 9 hydrogen bonds and 15 hydrophobic contacts (Fig. 7A), Pep 2 established 13 hydrogen bonds and 11 hydrophobic contacts (Fig. 7B). The formation of multiple hydrogen bonds, especially in Pep 2, implies strong polar interactions that can enhance specificity and complex stability [106]. In contrast, the higher hydrophobic interaction count in Pep 1 may promote tighter binding through van der Waals forces and nonpolar surface compatibility [107] . The absence of salt bridges suggests that electrostatic interactions are not the primary mode of binding in this system, a feature shared by many short bioactive peptides.

The higher number of hydrogen bonds in Pep 2 suggests stronger polar interactions, potentially contributing to its specific binding orientation and stability. The hydrophobic interaction count was higher in Pep 1, which might enhance the overall binding affinity through nonpolar surface contacts. Regarding cleavage specificity, Pep 1 was cleaved at a phenylalanine-methionine (F-M) site, whereas both Pep 2 and RMP shared cleavage at a histidine (H) site, which might reflect a similarity in their interaction pattern with the proteolytic target. This similarity in cleavage site between Pep 2 and RMP might suggest a conserved interaction motif or susceptibility pattern, which could be explored further in functional assays [108]. The unique cleavage site in Pep 1, however, could imply a distinct mechanism of interaction or processing. Both designed peptides displayed favourable binding characteristics, with Pep 1 showing stronger hydrophobic interactions and Pep 2 demonstrating more hydrogen bonding. Although the docking score of the reference molecule was considerably higher, the designed peptides showed stable interactions and energetically favourable profiles, indicating their potential suitability as functional analogs or inhibitors with acceptable structural and binding quality.  Generally speaking, the docking results suggest that both Pep 1 and Pep 2 exhibit promising interaction profiles, with complementary binding features Pep 1 emphasising hydrophobic engagement and Pep 2 leveraging polar contacts. Such dual characteristics offer flexibility for future optimisation strategies, either for enhancing potency or tailoring specificity toward target modulation. These findings support further investigation of both peptides in experimental settings for potential therapeutic or diagnostic applications.

3.7. Structural and thermostability of the designed Pep1

To evaluate the structural stability of the designed peptide Pep1 under varying thermal conditions, molecular dynamics (MD) simulations were conducted at five different temperatures (30 °C, 40 °C, 45 °C, 50 °C, and 60 °C) over a 100 ns period. Analyses included root-mean-square deviation (RMSD), solvent accessible surface area (SASA), radius of gyration (Rg), intramolecular hydrogen bonding, and root-mean-square fluctuation (RMSF) to comprehensively assess the peptide's conformational behaviour. RMSD analysis showed Pep1’s highest stability at 40°C (0.3–0.4 nm), with minimal structural deviation, while higher temperatures increased instability. At 30 °C and 45 °C, the RMSD values remained below 0.6 nm, suggesting overall stability with some minor structural adjustments. However, at 50 °C, RMSD values rose to approximately 0.7 nm, signifying partial destabilisation. The most pronounced structural deviations were observed at 60 °C, where RMSD frequently exceeded 1.0 nm and approached 1.5 nm, indicative of significant conformational changes and potential unfolding (Fig. 8A). This pattern reflects how increasing temperature leads to progressive structural disruption, as RMSD serves as a sensitive global indicator of backbone deviation from the native fold [109]. Stable RMSD at 40 °C suggests a thermodynamically favourable conformation, while higher RMSD at 50–60 °C may result from weakened non-covalent interactions and increased backbone flexibility, typical of thermally induced unfolding. SASA analysis supported these observations by providing insight into solvent exposure across temperatures. Throughout the 100 ns simulations, SASA values generally ranged from 20 to 27 nm². At 40 °C and 45 °C, the peptide maintained relatively consistent and moderate SASA values, reflecting a compact and well-folded structure. At 30 °C, the SASA profile remained fairly stable, though transient spikes suggested brief conformational openings. In contrast, simulations at 50 °C and 60 °C showed elevated and fluctuating SASA values, signifying increased solvent exposure likely due to structural expansion or partial unfolding (Fig. 8B). Since SASA quantifies the surface area of the peptide accessible to solvent molecules, lower SASA is typically associated with buried hydrophobic cores and tighter packing [110]. The moderate values at 40 °C confirm an optimal fold, whereas increasing SASA at higher temperatures implies exposure of internal residues, suggesting disruption of hydrophobic interactions and unfolding behaviour under thermal stress. The radius of gyration (Rg) further illustrated temperature-induced changes in compactness. At 40 °C, Pep1 exhibited the most stable and lowest Rg values, reinforcing the inference of a tightly packed conformation. Rg values at 45 °C remained close to those at 40 °C with minor fluctuations, suggesting sustained compactness. At 30 °C, intermittent increases in Rg were observed, reflecting occasional conformational relaxation. Conversely, at 50 °C and 60 °C, pronounced increases and fluctuations in Rg indicated a loss of compactness and a tendency toward structural loosening and partial unfolding (Fig. 8C). The radius of gyration is a geometric measure reflecting the peptide’s mass distribution around its center. Stable and low Rg values at 40 °C are characteristic of folded states [111], while rising Rg at 50–60 °C implies a structural transition toward more extended, unfolded forms. These findings align with literature reports where thermally induced unfolding is marked by increased Rg due to chain expansion.

Hydrogen bond analysis revealed a direct relationship between temperature and the internal cohesion of the peptide. The highest and most consistent number of hydrogen bonds—ranging between 15 and 22—was observed at 30 °C and 40 °C, indicating strong intra-peptide interactions and a stable secondary structure. At 45 °C, the hydrogen bond count remained stable but showed increased fluctuations. Significantly reduced and highly variable hydrogen bonding was seen at 50 °C, while at 60 °C, the number of hydrogen bonds frequently dropped below 10 and occasionally below 5, reflecting substantial destabilisation and disruption of the peptide’s internal architecture (Fig. 8D). Intramolecular hydrogen bonds are crucial for maintaining the secondary structure of peptides, particularly α-helices and β-turns. Their reduction at high temperatures supports the unfolding hypothesis, as these bonds are sensitive to thermal fluctuations [112]. The drop below 10 hydrogen bonds at 60 °C suggests substantial loss of secondary structural elements, consistent with peptide denaturation phenomena observed in other MD-based peptide studies. RMSF analysis provided residue-level insights into flexibility across the peptide sequence. At 40 °C, Pep1 demonstrated the lowest RMSF values across nearly all residues, particularly in the central core (residues 6–15), indicating localised rigidity and structural conservation. RMSF values increased gradually at 45 °C and 50 °C, while the most pronounced fluctuations were observed at 60 °C, especially at the N- and C-terminal regions. These heightened fluctuations at elevated temperatures suggest increased dynamic motion and structural instability under thermal stress (Fig. 8E). RMSF reveals the flexibility of individual residues. Low RMSF in the core region of Pep1 at 40 °C implies a well-constrained, rigid fold—critical for maintaining functional conformation. The rising RMSF at termini under heat stress is typical, as unstructured terminal regions tend to destabilise first, acting as 'thermal sensors' during unfolding [113].

Together, these comprehensive analyses underscore that 40 °C is the most favourable temperature for maintaining the structural stability and compactness of Pep1. At this temperature, Pep1 exhibits minimal deviation, low solvent exposure, strong hydrogen bonding, compact geometry, and limited flexibility hallmarks of a stable, functional conformation, further supported by a favourable binding free energy (MM/PBSA: -50.2 kcal/mol). In contrast, higher temperatures, particularly 50 °C and 60 °C, induce progressive structural destabilisation, likely compromising the peptide's structural integrity and potential biological activity. To further evaluate thermostability, the TM Predictor web server (http://tm.life.nthu.edu.tw) was used, yielding a predicted thermostability index (TI) of -4.05 for Pep1, indicating reduced thermal stability compared to the reference RMP, which exhibited a higher TI of 0.1688. The enzyme with the lower TI value is expected to melt first, as a lower TI reflects reduced thermostability. The average structural and energetic properties of Pep 1 under various temperature conditions, derived from MD simulations, are summarised in Table 2.

Structural and thermostability of the designed Pep2

To comprehensively assess the temperature-dependent stability and structural dynamics of the designed peptide Pep2, all-atom molecular dynamics (MD) simulations were performed at 30 °C, 40 °C, 45 °C, 50 °C, and 60 °C for 100 ns. A suite of structural metrics—including root mean square deviation (RMSD), solvent-accessible surface area (SASA), radius of gyration (Rg), intramolecular hydrogen bonding, and root mean square fluctuation (RMSF)—was evaluated. The results are presented below in a structured order, integrating both quantitative findings and mechanistic interpretations. Pep2 displayed the lowest and most stable RMSD values (0.2–0.6 nm) at 40 °C, indicating minimal conformational deviation and a highly stable backbone over the simulation period. At 30 °C and 45 °C, RMSD values increased modestly (0.4–0.8 nm), suggesting partial flexibility or incipient destabilisation. More pronounced fluctuations were observed at 50 °C (~0.9 nm), and at 60 °C, RMSD peaked at ~1.4 nm, signifying substantial structural deviation and partial unfolding. RMSD data indicate that 40 °C is the optimal thermal condition for structural integrity. The relatively low deviations at this temperature reflect a well-preserved conformation with sufficient thermal energy to promote natural flexibility without destabilisation. At 30 °C, limited thermal motion likely reduces conformational sampling, slightly increasing rigidity. Higher temperatures disrupt stabilizing interactions and increase molecular motion, explaining the substantial deviation at 60 °C. These findings align with typical protein folding behaviour, where a narrow thermal window supports optimal native-like stability (Fig. 9A).

At 40 °C, Pep2 showed the lowest SASA values (15–25 nm²), indicative of a compact, well-folded conformation with limited solvent interaction. At 30 °C and 45 °C, SASA values were moderately higher, suggesting less optimal folding. At elevated temperatures (50 °C and 60 °C), SASA increased markedly (~27–28 nm²), consistent with partial unfolding and exposure of internal residues to the solvent. The minimised SASA at 40 °C is consistent with effective hydrophobic core packing and maintenance of tertiary structure. As temperature rises, increased kinetic energy leads to expansion and loosening of the structure, exposing previously buried hydrophilic and hydrophobic residues. The increase in SASA at 60 °C particularly reflects unfolding events. Conversely, the moderate SASA at 30 °C suggests compactness but possibly over-constrained structural rigidity.

These results reinforce 40 °C as the most favourable temperature for maintaining folding efficiency and native-like solvation characteristics (Fig. 9B). Rg analysis confirmed a tightly packed structure at 40 °C, with values ranging between 0.75 and 0.9 nm and minimal fluctuation. At 30 °C, similar compactness was observed but with slightly increased variation. At 45 °C, Rg variability increased and exhibited a noticeable spike (~1.4 nm) near 80 ns, indicating transient expansion. At 50 °C and 60 °C, Rg values rose steadily (up to ~1.2 nm), signaling loss of compactness. Rg trends corroborate the conclusions drawn from RMSD and SASA. The tight distribution at 40 °C reflects optimal intramolecular packing, possibly due to a well-preserved hydrophobic core and favourable enthalpic interactions. The Rg increase at higher temperatures is indicative of thermal-induced expansion, likely driven by disruption of non-covalent stabilizing forces. The transient Rg spike at 45 °C suggests an intermediate destabilisation point, while sustained Rg elevation at 60 °C reflects a transition toward a partially denatured state (Fig. 9C).

Pep2 maintained the highest number of hydrogen bonds at 40 °C (17–22), indicating strong internal cohesion. At 30 °C, values remained relatively high (15–18) but were more variable. The number of hydrogen bonds declined with increasing temperature: 10–15 at 45–50 °C and dramatically reduced to 0–12 at 60 °C (Fig. 9D). Hydrogen bonding plays a critical role in stabilizing both secondary and tertiary structural elements. The high number of stable hydrogen bonds at 40 °C strongly supports the peptide's compact, folded state, as seen in RMSD and Rg data. The variability at 30 °C may result from reduced structural dynamics, limiting optimal hydrogen bond formation. Elevated temperatures likely disrupt hydrogen bonds through increased molecular motion and thermal agitation, leading to unfolding and instability, particularly at 60 °C. These results validate hydrogen bonding as a key contributor to Pep2’s thermal resilience.

RMSF values were lowest at 40 °C across nearly all residues, especially within the central core, indicating reduced flexibility and a stable fold. At 30 °C and 50 °C, moderate fluctuations (0.3–0.6 nm) were observed. At 45 °C and 60 °C, RMSF values increased notably, particularly at the N- and C-termini, with fluctuations reaching ~0.8 nm at 60 °C (Fig. 9E). Residue-level flexibility is a crucial indicator of structural adaptability and stability. The low RMSF at 40 °C reflects an optimally stabilized structure with minimal disorder, especially in regions typically prone to fluctuation. Elevated RMSF at higher temperatures points to local unfolding and increased mobility, beginning at the termini and potentially propagating inward. At 30 °C, limited thermal energy may suppress flexibility, potentially compromising functional dynamics. These observations underscore the role of dynamic balance in protein stability where both excessive rigidity and excessive motion can be detrimental.

The comprehensive MD analysis of Pep2 at varying temperatures identifies 40 °C as the optimal thermal condition for structural stability, compactness, and internal cohesion. At this temperature, the peptide maintains low RMSD and SASA, minimal Rg, a robust hydrogen bond network, and reduced local flexibility indicators of a well-folded, functionally favourable conformation, further supported by a binding free energy of -39.07 kcal/mol (MM/PBSA). Both suboptimal (30 °C) and elevated (45–60 °C) temperatures compromise one or more structural features, culminating in significant destabilisation at 60 °C. These insights are critical for the rational design and application of Pep2 in biotechnological settings where thermal resilience and functional integrity are required. Supporting this observation, the TM Predictor estimated a thermostability index (TI) of -3.09 for Pep2. The average structural and energetic properties of Pep 2 under various temperature conditions, derived from MD simulations, are summarised in Table 3.

3.8. Free energy landscape for designed Pep1 and Pep2

3.8.1. FEL analyses of Pep1

Based on the free energy landscape (FEL) plots provided for the Pep1–κ-casein complex at temperatures 30°C (Fig. 10A), 40°C (Fig. 10B), 45°C (Fig. 10C), 50°C (Fig. 10D), and 60°C (Fig. 10E), the conformational dynamics and binding stabilities vary significantly across temperatures. At 30°C, the basin is located at CV1 = –10, CV2 = 5, indicating a relatively deep and well-defined minimum in terms of free energy (in kJ/mol), suggesting a stable conformational state for the complex. At 40°C, the basin shifts to CV1 = 20, CV2 = 10, representing a higher energy state (kJ/mol) and broader basin, implying increased molecular fluctuations and possibly a less stable binding. At 45°C, the basin center is at CV1 = 10, CV2 = 0, with a more moderate energy well (kJ/mol), indicating a transiently stable interaction. At 50°C (CV1 = –10, CV2 = –10) and 60°C (CV1 = 0, CV2 = –10), the landscapes display multiple shallow basins or broader minima, again in kJ/mol, suggesting conformational heterogeneity and possible unfolding or weakened binding interactions. Comparing all FELs, 30°C shows the most localised and lowest energy basin (in kJ/mol), indicating that it offers the most favourable condition for stable Pep1–κ-casein interaction. Thus, 30°C appears to be the optimal temperature for strong and stable binding between the protein and ligand, as higher temperatures lead to increased conformational entropy and destabilisation of the complex. In ligand–peptide systems, FELs provide insight into the dynamic ensemble of bound states and the thermodynamic landscape that governs molecular recognition. The single deep basin observed at 30°C suggests a dominant, energetically favourable binding conformation with limited flexibility, implying a strong and specific interaction. In contrast, the emergence of multiple basins at elevated temperatures indicates increased conformational sampling and reduced energy barriers between alternative states, which often corresponds to transient or weak binding modes and reduced specificity [114]. Such behaviour is characteristic of thermally induced disruptions in ligand anchoring, possibly due to peptide backbone fluctuations or loss of key interaction contacts. These results highlight the temperature sensitivity of the Pep1–κ-casein interface and underscore the utility of FELs in visualising binding plasticity and thermodynamic preferences in peptide-ligand complexes.

3.8.2. FEL analyses of Pep2

The Free Energy Landscape (FEL) plots of the Pep2–κ-casein complex across five temperatures (30°C to 60°C) reveal distinct thermodynamic behaviours and conformational shifts, as represented by the collective variables (CV1 and CV2) and their corresponding energy minima in kJ/mol. At 30°C (Fig. 11A), three basins are evident. The global minimum is located at CV1 = –10 and CV2 = 0 with the lowest free energy (~0 kJ/mol), indicating a highly stable conformation of the Pep2–κ-casein complex. Additionally, two lighter blue basins at slightly higher CV coordinates (~CV1 = 0 to 5 and CV2 = 5–10) represent metastable conformations within 3–5 kJ/mol of the global minimum. The presence of multiple low-energy basins suggests that at 30°C, the complex not only adopts its most stable structure but also maintains conformational flexibility, which is often beneficial for functional protein–ligand interactions (e.g., induced fit or dynamic binding modes). At 40°C (Fig. 11B), the global minimum shifts slightly upward to CV1 = –10 and CV2 = 5, still maintaining a deep and compact energy basin (~1–2 kJ/mol), indicative of a still-stable complex. However, the landscape becomes marginally less diverse, suggesting slightly reduced conformational flexibility compared to 30°C. At 45°C (Fig. 11C), a dramatic conformational shift is observed with the basin center at CV1 = 10 and CV2 = 10. The basin is shallower, with energy increasing to ~5–6 kJ/mol, and the landscape appears more diffuse. This suggests that the complex is adopting a different, less favourable binding mode, likely due to thermal destabilisation. The reduced depth and increased spread of the basin imply both lower thermodynamic stability and higher structural fluctuations, which may compromise binding affinity or functional specificity. At 50°C (Fig .11D), the basin moves to CV1 = 10 and CV2 = 0, with slightly better stability than at 45°C but still higher free energy than at 30–40°C (~4–5 kJ/mol).

The loss of distinct, multiple basins indicates reduced structural adaptability, pointing toward the beginning of a denaturation trend or significant conformational compromise. Finally, at 60°C (Fig. 11E), the basin shifts to CV1 = –10 and CV2 = 15, but the energy is notably higher (~7–8 kJ/mol), and the landscape is broad and poorly defined. This suggests the complex is significantly destabilised, with increased conformational entropy, weakened interactions, and probable unfolding or loss of functional binding. In summary, 30°C provides the most favourable thermodynamic conditions for Pep2–κ-casein interaction, with the lowest free energy basin (~0 kJ/mol) and the presence of additional shallow basins (~3–5 kJ/mol) indicating conformational flexibility and stability. 40°C remains acceptable, albeit with slightly less flexibility. Temperatures above 45°C led to altered binding modes and increased free energies, reducing stability and functional interaction potential. Therefore, based on FEL analysis, 30°C is the optimal temperature for the stable and flexible binding of Pep2 to κ-casein. Molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) calculations indicated that the lowest average binding free energy (ΔG_binding) for the Pep2–κ-casein complex occurred at 40 °C. As MM/PBSA directly estimates the binding affinity by evaluating the enthalpic contributions (electrostatic, van der Waals, and solvation energies) from molecular dynamics snapshots, this result suggests that 40 °C provides the most energetically favourable condition for static peptide–protein interactions. In contrast, free energy landscape (FEL) analysis, which evaluates the thermodynamic distribution of conformational states over time using collective variables (e.g., RMSD, radius of gyration), revealed that 30 °C is associated with the deepest global free energy minimum. This indicates that at 30 °C, the complex adopts a more thermodynamically stable and dynamically accessible conformation. Unlike MM/PBSA, FEL does not directly quantify binding affinity but instead captures the stability and accessibility of the conformational ensemble sampled during the simulation. The discrepancy between the two analyses highlights the complementary nature of these methods: MM/PBSA reflects the strength of the final bound interaction (affinity), while FEL provides insight into the conformational thermodynamics and stability of the binding process. Taken together, these results suggest that while 40 °C may yield the strongest static binding affinity, 30 °C may represent a more favourable temperature for achieving a stable and functionally relevant protein–ligand complex due to enhanced conformational sampling and thermodynamic stability.

3.9. Principal component analyses of Pep1 and Pep2

3.9.1. PCA analyses of Pep1

Principal component analysis (PCA) was conducted to evaluate the conformational behaviour of the Pep1–κ-casein complex under increasing thermal conditions (30–60°C). At 30°C (Fig. 12A), the trajectory shows multiple well-defined and tightly packed clusters, suggesting the presence of distinct and stable conformational states with limited structural deviation — indicative of strong and specific interactions between Pep1 and κ-casein at physiological temperature. As the temperature increases to 40°C (Fig. 12B) and 45°C (Fig. 12C), the clustering pattern becomes more dispersed and less compact, indicating enhanced molecular flexibility and the beginning of a shift toward less stable binding conformations. By 50°C (Fig. 12D), the PCA projection reveals fewer and more diffuse clusters with longer inter-cluster transitions, reflecting a pronounced increase in structural variability and possible weakening of the peptide–protein interaction. At 60°C (Fig. 12E), the trajectory exhibits highly scattered points with minimal recurrent clustering, suggesting the breakdown of stable conformations and potentially partial or full dissociation of the complex. This temperature-dependent trajectory dispersion reflects thermal destabilisation, where higher kinetic energy leads to disruption of specific contacts and structural rearrangement within the complex. These findings demonstrate that the Pep1–κ-casein interaction is thermosensitive, with optimal stability occurring at or below 30–40°C, while elevated temperatures progressively disrupt the conformational landscape, reducing the likelihood of functional binding interactions. This PCA-based analysis indicated the crucial role of temperature in modulating ligand–protein interactions. The increasing conformational heterogeneity at higher temperatures indicates a reduction in binding affinity and stability, as the thermal motion overcomes non-covalent interactions, such as hydrogen bonds and hydrophobic contacts that stabilize the complex [115]. Similar trends have been observed in other peptide–protein systems, where elevated temperatures lead to the loss of native binding conformations and functional disruption [116]. Therefore, the progressive dispersion of the PCA clusters can be interpreted as a signature of thermal denaturation and interaction loss, reinforcing the necessity to consider physiological temperature constraints in the design and application of bioactive peptide therapeutics.

3.9.2. PCA analyses of Pep2

Principal Component Analysis (PCA) was conducted to investigate the temperature-dependent conformational dynamics of the interaction between the designed Pep2 and κ-casein, providing insights into the stability and flexibility of the complex across five different temperatures. At 30 °C (Fig. 13A), the PCA plot displayed a compact and highly clustered trajectory distribution, indicating limited atomic fluctuations and a rigid conformational space, reflective of a highly stable and well-structured Pep2–κ-casein complex. This suggests that at lower temperatures, the complex maintains structural integrity with minimal conformational shifts. At 40 °C (Fig. 13B), the trajectory began to show a slightly broader dispersion, though still relatively cohesive, suggesting moderate flexibility that may facilitate conformational accommodation without compromising binding stability.

This moderate adaptability could enhance interaction specificity and affinity by enabling Pep2 to better conform to κ-casein’s surface. At 45 °C (Fig. 13C), the PCA plot revealed multiple loosely defined clusters, indicating increased conformational sampling and transitions among distinct substates. Such dynamic behaviour suggests a threshold temperature where the complex begins to lose rigidity, potentially leading to reduced binding affinity or altered interaction profiles. A pronounced shift was observed at 50 °C (Fig. 13D), where the distribution became highly scattered and lacked a dominant conformational basin, signifying significant conformational fluctuations and loss of structural coherence. This degree of flexibility points to a destabilised interaction, with the possibility of partial unfolding or loss of binding contacts. At 60 °C (Fig. 13E), the trajectory remained widely dispersed, though with a slightly directional pattern, indicating large-scale conformational motions likely associated with denaturation or dissociation events. Collectively, these findings suggest that Pep2 maintains a stable and functionally relevant interaction with κ-casein up to 40 °C. However, as the temperature increases beyond 45 °C, the complex exhibits increasing conformational plasticity and instability, which may impair functional binding and structural integrity. The PCA analysis underscores the critical influence of temperature on the dynamic behaviour of protein–peptide complexes and highlights 30–40 °C as the optimal thermal window for Pep2–κ-casein interaction stabilityTaken together, both Pep1 and Pep2 demonstrated strong clotting activity at 40 °C and maintained structural stability within this moderate temperature range, whereas the control enzyme RMP showed optimal activity at 45 °C. This lower optimal temperature, combined with their high specificity, makes Pep1 and Pep2 better suited for industrial cheesemaking processes that favour moderate thermal conditions, potentially enhancing energy efficiency and process control. These features offer significant advantages for a variety of soft and hard cheeses that coagulate optimally under these conditions. Moreover, as animal rennet alternatives, Pep1 and Pep2 provide precise coagulation control essential for all cheese formulations, including vegan and halal varieties. Additionally, they are well-suited for cost-effective, large-scale production using microbial expression systems such as E. coli or Pichia pastoris, enabling scalable and food-grade manufacturing. Table 4 summarises the comparative features specifically, specificity, binding affinity, and thermolability highlighting the advantages of Pep1 and Pep2 over the control enzyme RMP for commercial cheese production.

3.10. Validation approach and future directions

To support the computational findings and address the inherent limitations of in silico approaches, future work will involve synthesising the top candidate peptides, Pep1 and Pep2, and experimentally evaluating their milk-clotting activity under controlled laboratory conditions. While computational modelling provides valuable insights into peptide performance, it has several limitations: it relies on theoretical force fields, may not fully account for post-translational modifications or folding dynamics, and cannot replicate the full complexity of biological systems such as enzyme–substrate interactions in milk matrices. These factors highlight the necessity of empirical validation. Pep1 and Pep2 will be produced recombinantly in Escherichia coli or Pichia pastoris and then purified. The peptides will subsequently undergo in vitro assays to assess milk-clotting activity, specificity toward κ-casein, and thermal sensitivity, with tests conducted at 30–40 °C to mimic industrial cheesemaking conditions. Additionally, thermostability and pH profiles will be evaluated to benchmark performance against commercial microbial coagulants. These experimental validations will be essential to confirm our computational predictions and advance the peptides’ potential for industrial application.

1. Conclusion

This study presents the rational design of peptide-based biocatalysts and addresses longstanding challenges in cheese biotechnology particularly the demand for ethical, specific, and thermolabile milk-clotting enzymes. By integrating interface residue engineering, machine learning-based prediction, and molecular dynamics (MD) simulations, we established a scalable and reproducible framework for designing milk-clotting peptides. Among the designed candidates, Pep1 and Pep2 outperformed existing milk-clotting enzymes (MCEs), with optimal activity predicted at 30–40 °C. These characteristics align well with the operational conditions of industrial cheesemaking. While the results are promising, experimental validation remains necessary. Overall, this computational pipeline represents a meaningful advancement in enzyme design for dairy applications.

1. Acknowledgements

The authors would like to acknowledge the support from the Biotechnology and Biobased Industry Research Program, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.

This research was supported by the Fundamental Research Grant Scheme (FRGS) from the Ministry of Higher Education (MoHE), Malaysia (FRGS/1/2024/STG05/UPM/02/11), awarded to the last author (SNO).

Data Availability:

All underlying data and scripts are publicly available at: https://github.com/shilansaleem/Aspartic-Peptidases. The results of both positive and negative controls are also available on GitHub. Additional data will be made available upon reasonable request.

1. Declaration of competing interest

The authors declare no competing interests.

1. Authors’ Contributions

SSS, SNO, and MBAR conceived the study, designed the methodology, and wrote the manuscript. SSS conducted the research. OMA, ATCL, ABS, and NDMN verified the data.

1. Using Artificial Intelligent Chatbots

No AI chatbots or tools were used in this research in data analysis, scientific content generation or interpretation.

1. Ethical Consideration

This article does not contain any studies involving human participants or animals performed by any of the authors.

Background and Objective: Diabetes mellitus is a long-term disorder characterized by the body’s inability to regulate excessive blood glucose levels. The incidence of diabetes mellitus worldwide has significantly increased in recent decades. In addition to careful regulation of food quantities, improving food quality through the consumption of functional foods that do not trigger glucose spikes is also recommended. Accordingly, the aim of this research is to determine the potency of antidiabetic functional food derived from the fermentation of A2 cow's milk with higher β-casein content using Lacticaseibacillus rhamnosus RAL43.

Material and Methods: This study involved in vitro assays to test the inhibitory activity against diabetes-related enzymes, namely α-glucosidase, α-amylase, and dipeptidyl peptidase-4 (DPP4), followed by molecular docking simulations.

Results and Conclusion: The results of the study showed inhibitory activity against enzymes that trigger blood glucose spikes, namely 66.35% against α-glucosidase, 68.87% against α-amylase, and 10.69% against DPP4. The results of the analysis showed an increase in the quantity of peptides after fermentation, along with the results of the analysis of L. rhamnosus RAL43 which showed high proteolytic activity during fermentation. After ultrafiltration, it was found that the greatest inhibitory activity came from protein with molecular weight (MW) larger than 10 kDa. The amino acid sequencing process with high-resolution liquid chromatography-mass spectrometry then showed bioactive peptides, including VLVLDTDYK which was previously reported to show DPP4 inhibitory activity, along with many other peptides that display various specific bioactivities. The VLVLDTDYK peptide fragment was successfully docked and positioned in the DPP4 molecule through molecular docking simulations. This study concluded that A2 milk can be a functional substrate to produce specific bioactive peptides that inhibit enzymes that trigger blood glucose spikes that have a negative impact on diabetes. L. rhamnosus RAL43 can be developed as a proteolytic isolate and starter culture to produce foods with functional properties that can aid in blood glucose regulation.

Keywords: Bioactive Peptides, A2 Milk, Antidiabetic, Molecular Docking

Introduction

Diabetes mellitus (DM) has emerged as a significant global health issue. It is a persistent condition marked by increased blood glucose levels, typically above 180 mg.dL^-1. Under normal circumstances, the hormone insulin regulates blood glucose levels by converting glucose into energy for cells. However, in individuals with diabetes, the body either does not produce enough insulin or cannot use it effectively to control blood glucose levels. According to data released by the International Diabetes Federation, Type 2 Diabetes Mellitus (T2DM) is the most common form globally and continues to increase significantly each year. The primary causes of T2DM include being overweight and obese, often due to an unbalanced diet and lack of physical activity. T2DM typically does not exhibit critical symptoms in its early stages, causing many individuals to remain unaware of their condition. Chatterjee et al. [1] and Antar et al. [2] identified a prediabetic phase that precedes full-blown diabetes. With proper prevention and management during the prediabetic phase, T2DM can still be avoided. In principle, individuals with T2DM must consistently avoid high blood glucose levels, particularly postprandial spikes [1,2]. Besides careful control of dietary quantity, improving diet quality through the consumption of functional foods that do not trigger glucose spikes is also recommended. Foods or beverages that offer health benefits are referred to as functional foods. Antidiabetic functional foods have been reported to originate from fermented products containing probiotics and/or bioactive compounds that inhibit digestive enzymes involved in carbohydrate breakdown into glucose, including α-amylase, α-glucosidase, and dipeptidyl peptidase-4 (DPP4) [3-5]. Functional foods with antidia-betic activity are considered a promising alternative for managing T2DM without the side effects often associated with pharmaceutical treatments [6].

Digestive enzyme inhibitors operate through various mechanisms. The enzymes α-amylase and α-glucosidase break down complex carbohydrates into simple sugars for absorption in the intestines. Inhibiting these enzymes helps slow glucose absorption and prevents postprandial blood glucose spikes [7]. Meanwhile, the DPP4 enzyme plays a critical role in blood glucose regulation by degrading incretin hormones such as GLP-1 (glucagon-like peptide-1) and GIP (glucose-dependent insulinotropic polypeptide). This degradation can suppress insulin secretion and burden the pancreas. DPP4 inhibitors are thought to prevent incretin breakdown, thereby maintaining normal glucose levels and enhancing insulin sensitivity [8]. Enzyme inhibitors targeting α-amylase, α-glucosidase, and DPP4 have been reported in the form of short-chain peptide fragments produced through proteolytic processes, either via enzy-matic hydrolysis or microbial fermentation [9-11]. These peptide-based inhibitors are thought to function as competitive inhibitors by attaching to the enzyme’s active site, preventing substrate binding, and thereby slowing down the buildup of glucose in the blood [12]. Other reported types of enzyme inhibitors include flavonoids, terpenoids, phenolic acids, tannins, alkaloids, and xanthones [10,13]. However, this study will focus specifically on exploring peptide-based inhibitors produced through fermentation by lactic acid bacteria (LAB) isolates.

Once the bioactivity data are obtained and the peptide sequences with potential inhibitory effects are identified, the next step is to perform molecular docking. Molecular docking is a crucial and efficient method for exploring the potential bioactivity of inhibitory peptides. It provides a strong scientific basis prior to biological testing, accelerates the screening process, and helps focus the research on the most promising peptide candidates [14]. This computational approach allows for the modeling and prediction of molec-ular interactions between candidate peptides and target enzymes. In this study, molecular docking is used to generate preliminary data supporting the hypothesis that the identified peptides may possess inhibitory activity against the target enzyme.

Fermented dairy products using LAB have been reported to exhibit antidiabetic potential [3,15,16]. Although not classified as therapeutic drugs, these products are considered effective as functional foods for diabetes preven-tion and management [7,17]. LAB have a significant advantage due to their proteolytic activity, enabling the breakdown of proteins into short-chain peptides. Dairy products fermented with LAB are well recognized as rich sources of bioactive peptides that exhibit antidiabetic, antioxidant, antihypertensive, cholesterol-lowering, and various other functional effects [18-20]. In this study, three lactic acid bacteria isolates (Lacticaseibacillus (L.) rhamnosus RAL27, Limosilactobacillus fermentum RAL29, and L. rhamnosus RAL43) previously isolated from Indonesian kefir grains [21], will be used to ferment A2-type cow’s milk. In recent years, A2 milk has been introduced. This type of milk was first commercialized in New Zealand and has since been marketed in several countries [22]. The main differences between A1 and A2 milk lie in the amino acid composition and β-casein. A1 β-casein contains histidine at position 67, while A2 β-casein contains proline at the same position [23]. These subtle differences can affect human digestion and metabolism. Some studies suggest that A1 milk may be associated with gastrointestinal discomfort and symptoms similar to lactose intolerance. A study by Choi et al. [24] found that participants consuming A1 milk reported more bloating and abdominal pain compared to those consuming A2 milk. The release of β-casomorphin-7 (BCM-7), a peptide derived from A1 β-casein during digestion, is thought to contribute to these symptoms. Some epidemiological studies suggest that A1 milk may be correlated with an increased risk of heart disease due to its inflammatory effects [23]. Conver-sely, A2 milk may offer cardioprotective effects, although further clinical trials are needed to validate these findings. A possible link between A1 milk consumption and the onset of type 1 diabetes has been proposed. A study by Kay et al. [23] suggests that early exposure to A1 milk may be associated with an increased risk of developing autoimmune conditions, possibly due to an immune response triggered by β-casomorphin-7 (BCM-7). With increasing health and wellness awareness, consumer demand for A2 milk has increased significantly. Many dairy companies have begun marketing A2 milk as a healthier alternative to A1 milk, capitalizing on its perceived benefits. This shift reflects a broader trend toward personalized nutrition and the importance of understanding genetic variation in food choices.The fermentation of A2 milk is expected to generate a new and unique peptide profile. Digestive conditions can influence the stability and absorption of peptides produced by A2 milk fermentation. Many peptides are biodegradable, so only peptides of a certain size or structure are retained. Smaller peptides may persist, while larger ones tend to break into fragments. Some bioactive peptides (e.g., DPP4 or α-glucosidase inhibitors) have been shown to remain active even after simulated in vitro digestion, but their persistence varies depending on sequence and structure [7,25]. However, not all peptides identified in vitro will persist into the systemic circulation. However, local activity in, for example, (α-glucosidase inhibition) is expected to remain relevant, even if the peptide is not absorbed in large quantities from the gut. Therefore, this study aims to determine the potential of antidiabetic functional food derived from the fermentation of A2 cow’s milk using lactic acid bacteria isolates, and to evaluate it through in vitro inhibitory activity tests against key diabetes-related enzymes: α-glucosidase, α-amylase, and DPP4, followed by binding simulation analysis.

1. Materials and Methods

2.1. Material

This study used three isolates from Indonesian kefir grains: L. rhamnosus strain RAL27, Limosilactobacillus fermentum strain RAL29, and L. rhamnosus strain RAL43. The A2 milk (KIN Fresh Milk, Jakarta, Indonesia) and Mann Rogosa Sharpe broth (Himedia) were used as growth media. The chemicals used included α-glucosidase from Saccaromyces cerevisiae (Sigma-Aldrich Co.), α-amylase from from porcine pancreas (Sigma-Aldrich Co.), DPP4 inhibitor screening kit (Sigma-Aldrich Co.) included DPP4 enzyme from human, o-phthalaldehyde (Sigma-Aldrich Co.), p-Nitrophenyl α-D-glucopyranoside (PNPG; Sigma-Aldrich Co.), phenolphthalein (Himedia), Folin-Ciocalteu reagent (Sigma-Aldrich Co.), trichloroacetic acid (Himed-ia), methanol (Himedia), ethanol (Merck), sodium carbonate (Himedia), sodium pyrophosphate (Himedia), β-mercapto-ethanol (Merck), acarbose (Merck), Sitagliptin (Sigma-Aldrich Co.), phosphoric acid (Himedia), phosphate buffer saline (PBS; Himedia), 0.22µM filter membrane (Aijiren), and membrane filter 3.000 and 10.000 Da (Spin-X® UF Concentrators, Corning).

2.2 Isolates preparation

Isolates of L. rhamnosus strain RAL27, L. fermentum strain RAL29, and L. rhamnosus strain RAL43 were refreshed in Mann Rogosa Sharpe broth (MRSB) and incubated at 37^oC for 48 h. Cultivation in MRSB was carried out 2 times and then the three isolates were adapted to A2 milk, by inoculating the isolates as much as 2% into A2 milk, then incubated at 37^oC for 48 h. This process was also carried out 2 times.

2.3 Fermentation process

The fermentation process refers to the research of Yusuf et al. [26]. The isolate was inoculated to A2 milk at concen-tration of 2% from 5 log CFU.mL^-1 starter. Incubation was conducted aerobically at 37°C for 24 h. The fermented milk obtained was characterized by assessing the total viability of LAB, pH value, acidity, aromatic profile, and coagulation properties. Subsequently, the fermented milk was centr-ifuged at 8000×g for 5 min at 4°C, leading to the separation of curd and whey. The whey, located in the upper layer, was carefully collected and subjected to a second centrifugation at the same conditions. The whey was subsequently passed through a 0.22 µm membrane filter to prepare it for further analysis.

 2.4 Acidy analysis of fermented A2 milk product

The sample’s pH was determined with a pH meter (Laqua pH1100, Horiba Scientific, Kyoto, Japan). The pH meter was calibrated with buffer solutions of pH 4.0 and 7.0. The acidity measurement using the acid-base titration method. Total of 1 mL sample diluted with 10 mL of sterile water. Afterward, 13 drops of phenolphthalein indicator were added, followed by titration of the sample with 0.05 N NaOH solution. The titration's end point was indicated by the development of a consistent pink color. Total acid was calculated according to the Eq.1.

Total acid (% lactic acid) =

 [                                                                                                                                                                                                                                                                Eq.1

2.5 Proteolytic activity assay

Proteolytic activity testing refers to the report of Celik et al. [27] with modifications. The isolate was first cultured in MRSB and incubated at 37 °C for 24 h. Cells were harvested by centrifugation (5000×g, 5 min), washed twice with phosphate-buffered saline, and adjusted to an OD600 of 0.1 ± 0.005. The resulting suspension was inoculated into 5 mL of sterile A2 milk and incubated at 37 °C for another 24 h. Following incubation, the sample was mixed with 10 mL of 0.72 N trichloroacetic acid and 1 mL of distilled water, allowed to stand for 10 min, and then filtered through Whatman No. 1 paper. From the filtrate, 5 mL was combined with 10 mL of Na₂CO₃–Na₄P₂O₇ solution, after which 3 mL of diluted Folin–Ciocalteu reagent was added. The mixture was stirred until a blue color appeared, centrifuged (3000×g, 3 min), and the clear supernatant was analyzed spectrophotometrically at 650 nm. Tyrosine served as the standard for constructing the calibration curve to determine proteolytic activity.

2.6 Preparation of whey fraction by ultrafiltration

Purification using ultrafiltration refers to Yusuf et al. [26]. Whey was fractionated using a centrifugal tube containing 3 and 10 kDa filter membranes. Treatment carried out: 15 mL of supernatant was put into a 10 kDa centrifuge tube, then centrifuged (8000×g; 5 min). From this process, 2 parts of the solution will be obtained, namely those that pass the filter and those that do not pass the filter. The solution that passes the filter will contain molecules measuring ≤10 kDa and those that do not pass the filter will contain Protein with molecular weight >10 kDa. For the solution that passes the filter, it is put into a 3 kDa centrifuge tube and centrifuged again. So that 3 solutions are obtained: (1) supernatant contains molecules measuring >10 kDa, (2) supernatant contains molecules measuring ≤10 to >3kDa, and (3) supernatant contains molecules measuring ≤3 kDa. All solutions are added with distilled water up to 15 mL according to the initial volume. After that it is ready for further analysis.

2.7 The α-glucosidase inhibitor assay

The α-glucosidase inhibitor assay refers to the study of Son et al. [28]. The reaction mixture in this assay consisted of a blank control (B0), a blank (B1), a sample control (S0), and a sample solution (S1). The S1 solution was prepared by combining 150 µL of PBS (pH 7.4), 75 µL of 20 mM p-nitrophenyl α-D-glucopyranoside, and 25 µL of sample, followed by incubation at 37 °C for 10 min. Subsequently, 50 µL of 0.2 U.mL^-1 α-glucosidase enzyme was added and the mixture was incubated again at 37 °C for 10 min. In contrast, the S0 solution was prepared without the addition of the α-glucosidase enzyme. To terminate the reaction, 1 mL of 0.1 M Na₂CO₃ was added to each mixture. The absorbance of all solutions was then recorded at 405 nm using a microplate reader (iMark, Bio-Rad Co.). After the absorbance value was obtained, the percentage of α-glucosidase inhibitor activity was calculated according to the Eq.2.

α-glucosidase inhibitor activity (%) =

                                                                                                                                            Eq. 2

where B₀ is blank control solution, B₁ is blank solution, S₀ is sample control solution, and S₁ is sample solution.

2.8 The α-amylase inhibitor assay

The α-amylase inhibitory assay was performed following the method of Sato et al. [29]. Briefly, 50 µL of sample was mixed with 50 µL of α-amylase solution (1 mg.mL^-1) and incubated at 25 °C for 10 min. Next, 50 µL of starch solution (20 mg.mL^-1 in PBS) was added and the mixture was incubated at 37 °C for another 10 min. Subsequently, 100 µL of 3, 5-dinitrosalicylic acid was added, and the mixture was heated at 95 °C for 5 min. After cooling, 1500 µL of distilled water was added, and the mixture was centrifuged at 5000×g. An aliquot of 200 µL was transferred into a 96-well plate, and absorbance was measured at 540 nm. Acarbose at 50 ppm served as the positive control. The percentage of α-amylase inhibitory activity was then calculated using the Eq.3.

α-amylase inhibitor activity (%) =

[(A_c - (A_s - A_b)) ÷ A_c] × 100%                                                                                                          Eq. 3

where A_s is the absorbance of the sample. A_b is the absorbance of the blank. A_c is the absorbance of the acarbose (control).

2.9 The DPP4 inhibitor assay

The DPP4 inhibitory activity was assessed using a modified version of the method described by Yan et al. [5]. In a 96-well microplate, 25 µL of gly-pro-p-nitroanilide (0.2 mM) was combined with either 25 µL of bacterial sample, PBS (as control), or sitagliptin (as reference inhibitor), followed by preincubation at 37 °C for 10 min. Subsequently, 50 µL of DPP4 enzyme (0.01 U.mL^-1) was added, and the mixture was incubated at 37 °C for 60 min. The reaction was terminated by adding 100 µL of sodium acetate buffer (1 M, pH 4.0). Fluorescence intensity was then recorded using a microplate reader (Varioskan™ LUX, Thermo Fisher, Massachusetts, USA) at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. All measurements were performed in triplicate, and absorbance values were corrected against blanks prepared by substituting DPP4 with Tris–HCl buffer (100 mM, pH 8.0). Negative controls (without DPP4 activity) and positive controls (DPP4 activity without inhibitor) were also included, using Tris–HCl buffer in place of the sample or the enzyme, respectively. The percentage inhibition of DPP4 activity was then calculated using the Eq. 4.

DPP4 inhibitor activity (%) =

[                          Eq. 4

2.10. Identification of peptides

The method used refers to Yusuf et al. [26] with modifications. Identification using Thermo Scientific™ Dionex™ Ultimate 3000 RSLCnano UHPLC coupled with Thermo Scientific™ Q Exactive™ High Resolution Mass Spectrometer. Nano Pump: A= Water + 0.1% Formic Acid and B= Acetonitrile + 0.1% Formic acid. Analytical Column: EASY-Spray column, 15 cm × 75 μM ID, PepMap C18, 3 μm. Flow: 100 µL.min^-1. Injection volume: 5 µL. Run time: 60 minutes gradient. Full MS at 70,000 FWHM Resolution. Data Dependent MS2 at 17,500 FWHM. Easy Nano Spray Ionization and positive mode. Protein identification by Thermo Scientific™ Proteome Discoverer 2.2 Software.

2.11 Molecular docking simulation and binding site

Molecular docking analysis was performed using the ClusPro webserver, which provides a dedicated peptide-protein docking feature. In this study, DPP4 was designated as the receptor protein, while the peptide VLVLDTDYK functioned as the ligand. The peptide-protein complex was selected based on the largest cluster generated by ClusPro’s docking algorithm [30]. The resulting complex structure was visualized using PyMOL and LigPlot+, and the binding affinity was evaluated using the PRODIGY webserver [31,32].

2.12 Statistical analysis

Each analysis was conducted in triplicate, and the results are presented as mean ± standard deviation. Data were statistically analyzed using SPSS software (SPSS Inc.) through one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT). A p-value of less than 0.05 (P < 0.05) was considered statistically significant.

1. Results and Discussion

3.1 Profile of pH value and total acidity

The strains L. rhamnosus RAL27, Limosilactobacillus fermentum RAL29, and L. rhamnosus RAL43, when inoculated into A2 milk, exhibited normal growth. This was evidenced by their viability, which ranged from 8.68 to 9.11 log CFU.mL^-1, post-fermentation pH values ranging from 4.43 to 4.66, pH changes (ΔpH) between 2.14 and 1.37, and titratable acidity between 0.65% and 0.74% (Table 1). All of these parameters are consistent with typical characteristics of fermented milk products. Comparison with previous studies [26,32,33] revealed no significant differences, with pH values after 24 hours of LAB incubation typically ranging from 3.8 to 4.8 and titratable acidity from 0.6% to 1.6%. These results confirm that A2 milk serves as an appropriate substrate for the growth of all three LAB isolates and holds potential as a starter culture for producing fermented milk or yogurt-like functional products. To assess whether the three LAB isolates could hydrolyze A2 milk proteins, their proteolytic activity was measured. The results showed that L. rhamnosus RAL27, L. fermentum RAL29, and L. rhamnosus RAL43 exhibited proteolytic activity in the range of 0.16 to 0.21 mg tyrosine.mL^-1 after 24 h at 37°C (Table 1). Compared to the findings of Celik et al. [27], who reported proteolytic activity between 0.05 and 0.2 mg tyrosine.mL^-1 after 36–72 hours of incubation, the values observed in this study are relatively high, especially considering the shorter fermentation time.

3.2 Proteolytic activity

This study did not include experiments to determine the optimal time and temperature for achieving maximum proteolytic activity. However, 37°C is widely recognized as the optimal temperature for LAB growth [33]. Nielsen et al. [25] also noted that the level of proteolytic activity does not necessarily correlate with the production of bioactive peptides, as specific bioactivities depend more on cleavage at the correct peptide bonds. In fact, excessively high proteolytic activity can be undesirable, as it may lead to complete breakdown into free amino acids rather than the formation of short-chain peptides. For LAB strains selected as starter cultures, a moderate level of proteolytic activity is considered desirable, since excessive protein breakdown can lead to the production of biogenic amines (organic molecules formed through amino acid decarboxylation) that may provoke allergic responses [27].

LAB can produce proteolytic enzymes both intra-cellularly and extracellularly, depending on substrate availability and favorable environmental conditions [34-36]. The nutrition label of the A2 milk used in this study indicated a protein content of 3 g.100 mL^-1, which is presumed sufficient to activate the LAB proteolytic system. This system involves transport mechanisms that facilitate the uptake of nitrogen sources by the cell, proteinases that initially hydrolyze milk proteins into peptides, and peptidases that subsequently degrade these peptides into smaller fragments and free amino acids [35,37,38]. This explains the ability of LAB to efficiently degrade proteins such as casein, resulting in the release of short-chain peptides and amino acids used in bacterial metabolism.

3.3 Enzyme inhibitory activity of fermented whey fractions

After the fermentation process was completed, the whey fractions from each isolate were collected and analyzed for their enzyme inhibitory activity. The results showed that all three isolates exhibited inhibitory effects against α-glucosidase, α-amylase, and DPP4 enzymes, with varying degrees of effectiveness (Figure 1). The measurements were validated using positive controls: acarbose for α-glucosidase and α-amylase, and sitagliptin for DPP4.

RAL43 exhibited the strongest α-glucosidase inhibitory activity (66.35%), followed by RAL27 (60.18%) and RAL29 (58.07%). Although these values were significantly lower than the inhibition level of 200 ppm acarbose (89.25%), RAL43 exhibited significantly stronger inhibition than RAL27 and RAL29. For α-amylase inhibition, RAL43 again showed the highest activity at 68.87%, followed by RAL27 (65.69%) and RAL29 (64.71%), with RAL43 differing significantly from the other two isolates. In contrast, DPP4 inhibitory activity was relatively low in all samples, with RAL43 at 10.69%, RAL29 at 9.02%, and RAL27 at only 1.67%. Once again, RAL43 exhibited significantly greater inhibition compared to the other isolates.

These results indicate that RAL43 demonstrated the highest inhibitory activity across all three digestive enzymes tested. Interestingly, although RAL43 and RAL27 both belong to the species L. rhamnosus, their enzyme inhibitory performance varied considerably. This difference may be attributed to variations in their proteolytic activity. Notably, RAL43 exhibited higher proteolytic activity than RAL27, which could have led to the release of more bioactive peptides with enzyme-inhibitory properties. Yan et al. [5] also emphasized that the bioactivity of microbial strains is often strain-specific and not solely determined by species identity

The observed DPP4 inhibition (~10%) is modest compared to pharmaceutical standards. However, the purpose of this study was to provide proof-of-concept that A2 milk-derived peptides generated by L. rhamnosus RAL43 can exert measurable inhibitory activity. Importantly, the same fermented product demonstrated strong inhibition of α-glucosidase (66.35%) and α-amylase (68.87%), which are key enzymes in postprandial glucose regulation. This suggests that, while DPP4 inhibition alone may appear limited, the combined inhibitory effects on multiple enzymes can act synergistically to provide meaningful functional benefits. Further optimization of fermentation conditions or peptide concentration could enhance these outcomes.

3.4 Investigation of bioactive peptides with enzyme inhibitory activity

Since L. rhamnosus RAL43 exhibited the highest and significantly different enzyme inhibitory activity compared to the other two isolates, its whey fraction was selected for further analysis to identify the bioactive peptides responsible for the inhibition. The whey fraction obtained from RAL43 was processed through ultrafiltration membranes with molecular weight cut-offs of 3 kDa and 10 kDa.

The results showed that the >10 kDa fraction had the highest α-glucosidase inhibitory activity (36.33%), which was significantly different from the 10–3 kDa fraction (11.86%) and the <3 kDa fraction (9.92%). A comparable pattern was found in α-amylase and DPP4 inhibition, with the >10 kDa fraction showing the greatest activity compared to the other fractions (Figure 2). Ultrafiltration essentially separates molecules by size, which may lead to a dilution or loss of bioactivity. Notably, DPP4 inhibitory activity was undetectable in the <3 kDa fraction. These findings suggest that the majority of enzyme-inhibiting bioactive compounds in the RAL43 whey fraction are likely larger than 10 kDa.

Previous studies have frequently reported that enzyme-inhibitory bioactive peptides are generally found in lower molecular weight fractions [39–41]. The observation that the >10 kDa fraction of RAL43 whey exhibited the highest enzyme-inhibitory activity contrasts with previous reports, which generally associate bioactive peptides with lower molecular weight fractions (<10 kDa). Smaller peptides are typically considered more effective inhibitors due to their higher solubility and ability to access enzyme active sites. The unexpected activity in the >10 kDa fraction suggests that the active compounds in RAL43 whey may consist of larger peptides, protein-derived complexes, or even non-peptide molecules, indicating a distinct inhibitory mechanism compared to those commonly described in the literature.

3.5. Proteomic screening of the >10 kDa fraction by LC-HRMS

Proteomic screening of the >10 kDa fraction successfully revealed a variety of peptide fragments, each identifiable by its retention time and relative abundance. The complete data are presented in Figure 3. Each chromatographic peak corresponds to a distinct peptide component, and peak intensity directly correlates with peptide concentration in the sample. Protein profiling results are shown in Table 2, indicating that β-casein was the main source of peptides (referred to as the mother protein), along with contributions from β-lactoglobulin and α-lactalbumin. Based on the UniProt database, a total of 12 proteins were detected, each with an average molecular weight above 10 kDa. Hydrolysis of these proteins generated unique peptide sequences ranging from 200 to 800 Da, appearing at retention times between 1 and 21 minutes.

To assess the potential bioactivity of the identified peptides, we conducted an extensive search through various peptide functionality databases and scientific literature. This analysis identified five peptide fragments previously reported as DPP4 inhibitors. However, no confirmed α-glucosidase or α-amylase inhibitory peptides were detected in the dataset. This finding raises the possibility that the α-glucosidase and α-amylase inhibition observed in the RAL43 fraction may originate from non-peptide bioactive compounds, warranting further investigation into such molecules.

Additionally, 14 other peptide fragments were identified, each associated with diverse bioactivities, including hypocholesterolemic, splenocyte proliferation stimulation, antimicrobial, ACE-inhibitory, antioxidant, immunomodul-atory, opioid, antianxiety, and antithrombotic effects (Table 3). These peptides reflect the bioactive potential of L. rhamnosus RAL43 when fermented in A2 milk. Such findings highlight the potential of these peptides for use in the development of functional foods or novel, natural therapeutic agents.

3.5 Molecular docking analysis

From the proteomic screening results, five candidate peptides were identified as potential DPP4 inhibitors: TPEVDDEALEK, TPEVDDEALEKFDK, VLVLDTDYK, ILDKVGINYWLAHK, and VGINYWLAHK. Among these, the peptide VLVLDTDYK was selected for molecular docking due to its moderate length (nine amino acids), making it suitable for processing with ClusPro. The peptide was input together with its motif sequence (VLVLDTD[FY]K).

Ten docking poses generated under the Balanced and VdW+Electrostatic modes are shown in Figures 4. In the balanced mode results, which shown by the orange rectangle, are successfully docked and located in DPP4 (grey cartoon) at each cluster. The first to the last cluster position order starts from the top left corner to the bottom right corner, respectively, and give 66, 53, 36, 34, 24, 23, 22, 19, 17, 16 cluster sizes, respectively. Whereas, in the Vdw+electrostatic mode results, which shown by the turqoise green rectangle, are successfully docked and located in DPP4 (grey cartoon) at each cluster. The first to the last cluster position order starts from the top left corner to the bottom right corner, respectively, and give 62, 42, 41, 38, 32, 31, 37, 25, 18, 17 cluster sizes, respectively.

The conformations with the highest probability of occurrence (located in the first cluster of each mode) were selected for further analysis. These representative complex models were then visualized using PyMOL (Figure 5), where each peptide docked at different binding locations on the DPP4 protein. Detailed residue-level interactions between the peptide and protein were analyzed using LigPlot+ (Figure 6). The medium orchid-colored stick and the purple-colored finger-like shape represent DPPIV residues as a receptor protein, while the dark-blue stick represents VLVLDTDYK peptide as a ligand. The sticks always establish hydrogen and/or salt bridge (electrostatic) interaction, while the finger-like shape corresponds hydrophobic interaction of protein residues with peptide’s atoms.

The peptide-protein complex in the Balanced mode formed hydrogen bonds through interactions between Arg40 and His533 (protein) and Leu95 and Asp96 (peptide). In the VdW+Electrostatic mode, hydrogen bonds were formed between Arg471 and Ile407 (protein) and Asp96 and Lys100 (peptide). Hydrophobic interactions in the Balanced mode involved several residues, including Pro475, Asp501, Leu504, Met509, Pro510, Ser511, Phe534, and Phe559, while in the VdW+Electrostatic mode, only Leu57 and Ser473 participated in maintaining complex stability. Notably, salt bridges were observed only in the VdW+Electrostatic complex, formed by oppositely charged residues: Asp96–Arg471 and Lys100–Glu408.

Despite these interactions, the peptide VLVLDTDYK was not found to bind at DPP4’s known hydrophobic pocket or active site. Binding affinity predictions using PRODIGY (Table 4) classified both docking models within the moderate affinity category (10⁻⁹ M > Kd ≥ 10⁻⁶ M) [55]. These findings may explain the relatively low DPP4 inhibition observed in vitro, suggesting that high inhibitory activity requires peptide binding at the designated active site.

Interestingly, the VdW+electrostatic mode, which does not utilize a reference state for conformational scoring [56], produced comparable docking results to the balanced mode. Thus, predictions from both modes are considered valid approximations. Furthermore, these results suggest that alternative or unidentified binding cavities may contribute to DPP4 inhibition, albeit with weaker activity. According to UniProt data, the detected binding regions are not located in sterically hindered areas that would preclude inhibitory function, supporting their potential involvement in modulation of DPP4 activity.

This study shows that the VLVLDTDYK peptide is able to interact with the DPP4 enzyme through multiple binding modes, but not at the main active site. This accounts for the reduced inhibitory effectiveness and suggests that effective DPP4 inhibitors need to interact directly with the enzyme’s active site to achieve stronger activity. This is in line with previous findings that binding at the active site is necessary to achieve high inhibition activity [57,58], as indeed peptides that do not bind directly to the active site tend to have lower inhibition activity, even though they may interact with other areas on the enzyme.

This study has certain limitations. The peptide analysis focused primarily on VLVLDTDYK, while other identified candidates such as TPEVDDEALEKFDK were not subjected to comparable molecular docking evaluation. This narrow focus may limit the comprehensiveness of the findings, as different peptides could exhibit distinct binding affinities, interaction profiles, or inhibitory mechanisms toward DPP4, potentially overlooking stronger or more biologically relevant inhibitors. In addition, the present work was restricted to in vitro and in silico approaches without in vivo validation, leaving questions about bioavailability, stability, and physiological efficacy unanswered. Future research should therefore extend docking and interaction analyses to all candidate peptides and include in vivo studies to confirm their biological relevance and therapeutic potential.

1. Conclusion

A2 milk can be a functional substrate to produce specific bioactive peptides that inhibit enzymes responsible for blood glucose spikes associated with prediabetes and T2DM. Molecular docking results corroborated the in vitro enzyme inhibition data, confirming that peptides from A2 milk fermented with L. rhamnosus RAL43 can effectively bind and inhibit DPP4 and other glucose-regulating enzymes, thereby supporting their potential as functional food components for glycemic control. Future research should include quantitative peptide yield determination, simulated gastrointestinal digestion to assess peptide stability, bioavailability studies, and in vivo or clinical trials to evaluate their physiological impact, safety, and dietary relevance.

1. Acknowledgements

The authors acknowledge LPDP and BRIN Indonesia for fully supporting this research through the Funding for Research and Innovation in Advanced Indonesia (RIIM) Batch 4.

1. Declaration of competing interest

The authors report no conflicts of interest.

1. Authors’ Contributions

Conceptualization: Yusuf D. Data curation: Yusuf D, Handayani R. Formal analysis: Setianingrum N. Methodology: Sulistiani. Software: Dewi FR. Validation: Kasman AAMN. Investigation: Anggadhania L. Writing - original draft: Yusuf D. Writing - review & editing: Yusuf D, Handayani R, Sulistiani, Dewi FR, Anggadhania L, Setianingrum N, Kasman AAMN.

1. Using Artificial Intelligent Chatbots

In the preparation of this work, the author used ChatGPT to enhance the English language and overall readability.

1. Ethical Consideration

This research did not involve human participants or animal experimentation. All experiments were performed using in vitro assays and computational analyses. Therefore, ethical approval was not required.

Background and Objective: The global demand for plant-based dairy alternatives is steadily increasing. However, relatively little is known about the fermentation processes of plant-based beverages. The main objective of this study was to investigate the changes in chemical composition, as well as structural, rheological, and organoleptic properties of rice-based beverages, depending on the initial rice-to-water ratio and fermentation time.

Material and Methods: Hydromodules with rice-to-water ratios of 1:2 and 1:3 were prepared. Each hydromodule was subjected to enzymatic hydrolysis, followed by fermentation for 8 and 24 hours. The samples were evaluated for physicochemical, rheological, and sensory characteristics. Data analysis and correlation studies were performed using GraphPad Prism and Origin8 software at a significance level of p < 0.05.

Results and Conclusion: Fermented samples showed a decrease in pH (by up to 3.5 units) and an increase in titratable acidity (from baseline to 34.2°T and 90.1°T after 8 and 24 hours of fermentation, respectively), which indicates successful acidification due to microbial activity. Fermentation for 8 hours at a 1:2 rice-to-water ratio improved water-holding capacity (98%), as well as textural properties such as cohesiveness (89.2%) and gumminess (17.4%). These results suggest that combining enzymatic pre-hydrolysis with fermentation using L. bulgaricus can significantly enhance the technological and functional properties of rice-based beverages, offering a promising approach for developing high-quality fermented plant-based products.

Keywords: Fermentation, Plant Beverages, Sensory Evaluation, TPA, Viscosity

1. Introduction

In recent years, people have been paying increasing attention to their health, which is reflected in their dietary choices. There has been a growing interest in foods with beneficial health properties. Among these, plant-based beverages that resemble cow’s milk in appearance and texture have become especially popular. These products are commonly referred to as plant-based beverages.

Plant-based beverages are preferred by consumers who follow a healthy diet, adhere to a vegan lifestyle, care about animal welfare, or simply wish to diversify their food intake. They also serve as a suitable alternative for individuals with lactose intolerance or milk protein allergies, who may have difficulty digesting or absorbing certain nutrients – such as lactose and specific proteins – found in animal milk [1].

These beverages are not only dairy alternatives but also sources of essential nutrients and various biologically active compounds that have been shown to promote human health [2,3].

Soy milk, being of vegetable origin and produced industrially, is one of the most common substitutes for animal milk. Its popularity is particularly strong in many Asian countries, where soybeans have long been part of traditional diets and are used in foods such as tofu and tempeh.

Research into the production of plant-based milks from alternative raw materials is currently expanding. Notable examples include cereals, nuts (such as almonds, cashews, and tiger nuts), legumes (e.g., peas, chickpeas, and lupin), and pseudocereals (e.g., chia, flaxseed, and quinoa) [4–11].

Among these raw materials, rice stands out as one of the most accessible and widely available sources for plant-based beverages production.

Rice (Oryza spp.) is one of the oldest cereal crops cultivated by humans and is widely consumed across the globe. Asia remains the primary region for rice cultivation, although it is also grown in Africa, the Americas, Europe, and Russia. Due to the presence of biologically active compounds rice exhibits several beneficial properties. These components contribute to anti-inflammatory and antioxidant effects, support the normalization of hematopoietic processes, and improve metabolic functions [4,12,13].

Rice and its derivatives are highly valued in nutrition due to their complex polysaccharides, which effectively satisfy hunger and promote prolonged satiety, while their hypoallergenic properties – stemming from the absence of gluten and lactose – make them an ideal dietary choice for individuals suffering from food allergies or intolerances.

Most cereals are traditionally processed into food and beverages through enzymatic conversion (e.g., starch, molasses, malt) or microbial fermentation (e.g., bread, beer, soy sauce, vinegar). These processes significantly alter the nutritional and functional properties of the raw materials [5,14–16].

Fermentation, in particular, is a cost-effective and energy-efficient method that enhances the digestibility of proteins and dietary fiber, increases the bioavailability of micronutrients, and reduces anti-nutritional factors. Furthermore, fermentation enriches the final product with beneficial metabolites – such as vitamins, amino acids, and others – and introduces live microorganisms, including probiotics. It also improves food safety by reducing levels of toxic compounds like mycotoxins and by producing antimicrobial substances such as lactic acid and bacteriocins [17]. In addition, fermentation enhances sensory qualities by diversifying flavors, textures, and aromas [18–21].

Consumer demand for plant-based alternatives to traditional dairy products – particularly those with high acceptability and functional benefits – is steadily increasing. Fermented cereal-based beverages show significant potential to meet this demand. Various types of such beverages and methods of biotransformation are being explored to enhance their probiotic and functional properties [22–25].

Despite its potential, the production of fermented rice beverages remains a challenging process. This is primarily due to the significant compositional differences between cow’s milk and rice «milk» – the latter being the substrate for lactic acid bacteria in plant-based fermentation. Moreover, rice requires specific preparation steps, including soaking, milling, optimization of the solid-to-liquid ratio, and pre-hydrolysis to increase sugar availability, among others. Furthermore, unlike dairy milk, rice-based substrates do not form a coagulated structure similar to that of fermented dairy gels after fermentation. This structural difference affects key sensory attributes – such as texture and mouthfeel – that are characteristic of traditional yogurt and contribute significantly to its consumer appeal [26]. As a result, research in this area is of considerable importance, as it can not only advance our understanding of plant-based fermentation but also support the development of novel functional foods aligned with modern trends in healthy and sustainable nutrition.

Therefore, the aim of this study was to evaluate the feasibility of using rice as a base for developing fermented plant-based beverage with enhanced functional and health-promoting properties.

1. Materials and Methods

2.1. Preparation of rice base, starter culture and samples

The preparation of rice began with washing and soaking to facilitate subsequent processing. Whole rice grains were thoroughly washed and soaked in excess water for 12 hours to ensure complete hydration. The water-to-rice ratio (hydromodule) was carefully optimized to achieve two key objectives:

– Provide a sufficient concentration of dry matter to support lactobacilli growth;

– Ensure the formation of a stable system with desired structural and rheological properties.

In this study, two ratios were tested: 1:2 and 1:3 (rice to water, w/v). After soaking, the hydrated rice was ground in water using a laboratory grinder («JustBuy», Qingdao, China) to obtain a homogeneous suspension.

The resulting rice slurry was subjected to amylolytic hydrolysis to increase the concentration of fermentable sugars – essential for microbial metabolism – and to generate intermediate products such as dextrins of varying molecular weights, which influence the viscosity of the final product. Hydrolysis was carried out using an amylolytic enzyme preparation α-amylase («Alfalad BN», Bio-Preparat, Moscow, Russia) at 40±2°C for 40 minutes, with continuous monitoring of key physicochemical parameters. The enzyme dosage was 50µL per 100g of substrate, as recommended by the manufacturer. The fermentation process was conducted in a 4-liter vessel.

To terminate the enzymatic reaction, the mixture was heated to 95–100°C for 1–2 minutes, then rapidly cooled on ice. Following inactivation, the physicochemical parameters of the hydrolysate were determined, and the temperature was adjusted to 37°C prior to inoculation with the starter culture.

Lactobacillus delbrueckii subsp. Bulgaricus (commercially available as «Lactosynthesis», Moscow, Russia), a strain commonly used in dairy fermentation, was employed in this study. The culture was stored in de Man, Rogosa, and Sharpe (MRS) broth (Himedia, India) supplemented with 50% glycerol at −80°C. For activation, a 100µL aliquot of the frozen culture was transferred into fresh MRS broth and incubated at 37°C for 24 hours under anaerobic conditions.

The preparation and composition of the experimental samples are summarized in Table 1 and illustrated in Fig. 1.

2.2. Preparing of starter and fermentation of rice base

Pre-cultures of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) were prepared by inoculating MRS broth and incubating at 37°C for 8–12 hours. An overnight culture of L. bulgaricus was used as inoculum to achieve a final concentration of approximately 10⁸ colony-forming units (CFU)/mL in the rice base, with an inoculation volume of 3mL per 100mL of substrate.

The fermentation process was conducted according to a previously described method [27]. Two fermentation durations were evaluated: 0–8 hours and 0–24 hours. During fermentation, physicochemical and rheological parameters were monitored at regular intervals. Sensory evaluation of the final products was also performed.

2.3. Chemical assays

Titratable acidity was determined by titrating 10 mL of each sample with 0.1 N NaOH using phenolphthalein as an indicator, with results expressed in °T (degrees Thorner). The pH was measured using a HI 2211-02 pH meter (HANNA Instruments, Germany), following the manufacturer’s instructions.

Compositional analysis – including protein, fat, total solids, and total sugar content – was performed using near-infrared spectroscopy (NIRS) with an InfraLUM FT-12 spectrometer (Russia). The instrument was equipped with dedicated software and pre-established calibration models for cereal-based matrices.

2.4. Rheology, structural and textural parameters analysis

Viscosity measurements were taken using a rotational viscometer model RV-DVIII (China). Approximately 50 ml sample was placed in a beaker and the viscosity measurements were taken using spindle 2 of the viscometer at 30 rpm. The temperature of the samples was about 25 °C. The viscosity values were calculated automatically using a coefficient to convert the viscosity values into centipoise units (cP). Measurements were carried out in 3 replicates for each treatment and results were expressed in mPa·s.

The stability of the structure and thixotropic properties of the samples were evaluated by the coefficient of loss of viscosity (L_ƞ, %) and coefficient mechanical stability (CMR), according to equations:

Lη = ((η_s-η_e))/η_s ∙100,

CMR = η_e/η_s,

where η_s is the initial viscosity of the undisturbed structure, cP; η_e is the viscosity of the maximally destroyed structure, cP.

Syneresis was measured after cooling samples weighing about 10 g to 4 °C after 24 hours of storage. The samples were centrifuged for 5 min at 1000 rpm and a temperature of 20 °C. The released serum was removed and weighed.

Syneresis (%) was calculated by equation:

S_yn=S/М∙100, (%),

where M is the mass of the sample, g; S is the mass of the released serum, g.

The water-holding capacity (WHC) was measured after cooling the samples weighing about 20 g to 4 ° C after 24 hours of storage. The samples were centrifuged for 10 min at 3000 rpm and a temperature of 20 °C. The released serum was removed and weighed. WHC (%) of the product was calculated by equation:

WHC= (М-W)/М∙100,

where M is the sample weight, g; W is the released serum weight, g.

Texture profile analysis (TPA) was carried out using an ST-2 texture analyser (Quality Laboratory JSC, Moscow, Russia). The following factors were determined: hardness (g), cohesiveness, %, gumminess, g, adhesiveness, g·mm.

2.5. Sensory evaluation

A sensory evaluation was conducted by a panel of untrained (non-professional) and semi-trained assessors, comprising bachelors, masters, and professors involved in food production and biotechnology studies or work. This methodology is widely accepted and commonly used in similar research studies [29, 30]. The consumer panel comprised 65 participants, including 40 women and 25 men. The participants were predominantly young, with 74% aged between 18 and 39 years, and had a high level of education: 25% held PhD degrees, while 75% were undergraduates or graduates. Individuals with lactose intolerance, pregnancy, diabetes, or any allergies were excluded from participation.

The all panelists evaluated the organoleptic properties of each sample, identifying any sensory defects and rating their intensity on a 5-point hedonic scale: 5 – Full compliance (no defects), 4 – Single minor defect, 3 – Several minor defects, 2 – Significant defects, 1 – Severe (gross) defects.

All panelists provided informed consent, and ethical approval for the study was obtained prior to data collection and publication.

2.6. Statistical analysis

Each experimental trial was performed in five replicates for statistical validation. Data were analyzed for statistical significance using GraphPad Prism software (version X, GraphPad Software, USA) at a significance level of p < 0.05. Correlation analysis and data visualization were performed using Origin 8 software (OriginLab, USA), with significance set at p < 0.05.

1. Results and Discussion

3.1. Preparation of rice base for lactic acid fermentation

After processing, the rice base exhibited a more homogeneous and viscous structure. The characteristic raw cereal taste was eliminated, and a distinct sweetness developed due to the release of simple sugars during hydrolysis. Changes in key physicochemical parameters are presented in Table 2.

3.2. Research on the properties of a hydromodule 1:2

3.2.1. Fermentation process at hydromodule 1:2

During the initial phase of fermentation (0–4 hours), titratable acidity increased to 17 °T (Fig. 2A), while pH decreased to 4.47 (Fig. 2B). Changes in viscosity exhibited a stepwise pattern (Fig. 2C), likely due to thermal equilibration of the rice base and the gradual activation of LAB. No noticeable changes in odor or taste were observed during this period.

Between 5 and 6 hours of fermentation, the development of sour aroma and acidic taste became perceptible, intensifying by the 8th hour. At this point, titratable acidity and pH reached 40 °T and 3.63, respectively. The sample also exhibited increased thickness, with viscosity rising to 1280–1300 cP. Based on sensory and physicochemical indicators, the primary fermentation of one sample (designated 1:2_8h) was terminated.

To rapidly reduce LAB metabolic activity, the sample was first placed in a freezer (−18°C) for 20–30 minutes, followed by transfer to a refrigerator (4°C) for 12 hours to allow structural stabilization.

The second sample (1:2_24h) was maintained under fermentation conditions for up to 24 hours to assess the upper limit of acceptable acidification. After 24 hours, the product developed a sharp sour taste and aroma. Titratable acidity and pH reached 90°T and 3.11, respectively. This sample was also subjected to the same post-fermentation stabilization protocol.

Following stabilization, viscosity increased further:

1:2_8h: from 1280–1300cP to 1347.5cP (+6.1%),

1:2_24h: to 1409.6Cp (+10.9%).

The increase in viscosity after cooling indicates ongoing structural development, likely due to protein network formation, water binding, or dextrin interactions, despite the cessation of active fermentation.

3.2.2. Physicochemical parameters after lactic acid fermentation at hydromodule 1:2

Following lactic acid fermentation, significant changes were observed in titratable acidity and pH compared to the unfermented control (Table 2). Titratable acidity increased progressively with fermentation time, reaching 40 °T after 8 hours and 90 °T after 24 hours, while pH decreased from an initial value of ~6.2 to 3.63 and 3.11, respectively.

In contrast, the overall chemical composition of the samples remained largely unchanged (Table 3), with the exception of total sugar content. A notable amount of residual total sugar was detected even after 24 hours of fermentation. This is likely attributable not only to the high initial concentration of fermentable sugars resulting from amylolytic hydrolysis but also to the presence of complex starch degradation products (e.g., dextrins) that may be only partially utilized by Lactobacillus delbrueckii subsp. bulgaricus under the applied fermentation conditions.

These findings suggest that while the fermentation process effectively acidifies the rice base, complete sugar utilization may be limited by the metabolic capabilities of the starter culture or the accessibility of certain carbohydrate fractions.

3.2.3. Structural and textural properties

Rheological characteristics play a crucial role in determining the quality of fermented products, both dairy and plant-based. Viscosity, texture, and structural stability are key parameters influencing consumer acceptability, particularly in beverages and yogurt-like products. Plant-based matrices are often prone to structural instability and phase separation (syneresis), necessitating careful process optimization to ensure product homogeneity and mouthfeel.

In this study, the apparent viscosity of fermented rice samples decreased compared to the unfermented control, with the most pronounced reduction observed in sample 1:2_24h (−22%). However, further analysis of structural and rheological parameters (Table 4) revealed distinct differences in recovery behavior and WHC, indicating that viscosity alone does not fully reflect structural integrity.

The unfermented control (1:2) exhibited high structural stability, with only a 2.4% viscosity loss and excellent recovery capacity (97.6%) and WHC (97.0%). In contrast, the 1:2_8h sample showed greater viscosity loss (−19.1%) and lower CSR (80.9%), despite maintaining a high WHC (98.0%). This suggests partial structural breakdown during fermentation, possibly due to insufficient network formation.

Surprisingly, sample 1:2_24h, despite the largest initial viscosity drop (−22%), demonstrated improved recovery (CSR: 92.7%) and the highest WHC (98.5%), along with the lowest syneresis (Syn: 0.5%). These results indicate that prolonged fermentation led to the development of a more resilient and cohesive structure, likely due to microbial metabolite production.

These changes are attributed to the metabolic activity of Lactobacillus delbrueckii subsp. bulgaricus during extended fermentation. Although initial acidification and substrate utilization may cause temporary thinning of the matrix (as observed at 8 h).

Thus, fermentation duration significantly influences the structural evolution of rice-based fermented beverages. While short fermentation (8 h) leads to acidification and viscosity reduction, extended fermentation (24 h) promotes the synthesis of functional metabolites that enhance structural recovery and water retention, ultimately improving physical stability.

Rheological characteristics represent a critical set of parameters for the evaluation of fermented products, providing objective quantification of textural attributes such as firmness, cohesiveness, and mouthfeel [19, 27, 28]. When integrated with sensory analysis, rheological data enable a comprehensive assessment of texture perception, bridging the gap between instrumental measurements and consumer experience. The results of TPA are presented in Table 5.

TPA revealed that hardness – defined as the force required to deform a material – was similar across all samples, indicating comparable resistance to initial compression. In contrast, parameters related to the internal structure and textural behavior exhibited significant variation.

Cohesiveness, which reflects the internal strength and resistance to deformation, was on average 5% higher in the fermented samples compared to the unfermented control. This suggests enhanced structural integrity and molecular interactions within the matrix, likely due to microbial metabolite production during fermentation.

Gumminess was highest in sample 1:2_24h, indicating a more resilient and chewable texture. An increase in gumminess is generally perceived positively in plant-based fermented products, as it prolongs oral residence time – enhancing interaction with taste and tactile receptors and contributing to a more satisfying, yogurt-like mouthfeel.

In contrast, adhesiveness was highest in sample 1:2_8h, but decreased in the 24-hour sample. Notably, the reduction in adhesiveness observed in 1:2_24h is advantageous from a technological standpoint, as lower stickiness reduces the force required for pumping, filling, and dosing viscous products, thereby improving processability and minimizing fouling in production equipment.

These results demonstrate that lactic acid fermentation significantly influences not only the rheological properties of the rice base but also its textural profile. Prolonged fermentation (24 h) promotes the development of a more cohesive and structured matrix with improved sensory and functional characteristics, while simultaneously reducing undesirable adhesive properties.

3.2.4. Sensory evaluation

Sensory evaluation plays a critical role in shaping consumer preferences, particularly when assessing novel or reformulated food products [31–35]. It provides essential insights into product acceptability by capturing perceptions related to key organoleptic attributes. The sensory profiles of the fermented rice beverages – including consistency, texture, color, taste, aftertaste, smell, flavor, and overall acceptability – were evaluated by a panel of assessors and are presented in Table 6.

3.3. Research on the properties of a hydromodule 1:3

3.3.1. Fermentation process at hydromodule 1:3

During the initial phase of fermentation (0–4 hours), titratable acidity increased to 11 °T (Fig. 2A), and pH decreased to 4.10 (Fig. 2B). Viscosity rose to 347.0 cP (Fig. 2C), likely due to thermal equilibration of the rice base and the onset of LAB metabolic activity. No noticeable changes in odor or taste were observed during this period.

Between 5 and 6 hours of fermentation, the development of sour aroma and acidic taste became perceptible, intensifying by the 8th hour. At this point, titratable acidity and pH reached 27 °T and 3.64, respectively. The samples also exhibited increased thickness, with viscosity rising to 390–395 cP. Based on these physicochemical and sensory changes, primary fermentation of one sample (designated 1:3_8h) was terminated.

The second sample (1:3_24h) was maintained under fermentation conditions for 24 hours to evaluate the upper limit of acceptable acidification. After 24 hours, the product developed a sharp, pronounced sour taste and aroma. Titratable acidity and pH reached 79 °T and 3.20, respectively. This sample was then subjected to the same post-fermentation stabilization protocol: rapid cooling (freezing at −18 °C for 20–30 min), followed by refrigeration at 4 °C for 12 h to ensure structural stabilization.

Following stabilization, viscosity increased further:

1:3_8h: from 390–395 cP to 410.8 cP (+23.4%),

1:3_24h: to 377.0 cP (+13.6%).

The greater relative increase in viscosity observed in the 1:3_8h sample suggests more effective structural development during cold storage, possibly due to better preservation of matrix integrity at lower acidity. In contrast, prolonged fermentation in 1:3_24h may have led to excessive acidification, partially disrupting the network and limiting post-fermentation structuring.

3.3.2. Physicochemical parameters after lactic acid fermentation at hydromodule 1:3

As a result of lactic acid fermentation, the indicators of titratable and active acidity changed significantly compared to the control (Table 3). The chemical composition changed only slightly (Table 7), which is associated with a more diluted environment of the hydro-module and a relatively low content of dry substances. At the same time, the fermentation process did occur, especially with a processing duration of 24 hours.

3.3.3. Structural and textural properties

The viscosity of fermented samples increased compared to the control, with the highest relative growth in 1:3_8h (+6.6%). However, structural stability varied significantly (Table 8).

Sample 1:3_24h showed the best performance: high recovery (96.1%), WHC (85.6%), and minimal syneresis (0.5%), indicating strong network formation. In contrast, 1:3_8h had greater viscosity loss (−11.3%) and lower recovery (88.7%), despite high WHC.

These differences are linked to fermentation duration. The low dry matter content in the 1:3 system limits early structuring, but 24-hour fermentation enables sufficient synthesis of metabolites by LAB, enhancing stability. At 8 hours resulting in a weaker, less recoverable structure.

Thus, prolonged fermentation compensates for dilution, promoting a stable, cohesive matrix.

TPA results for the 1:3 hydromodule are presented in Table 9. Hardness was similar across all samples. Cohesiveness – reflecting internal structural strength – was on average 6% higher in fermented samples compared to the control, indicating improved matrix integrity.

Gumminess was highest in 1:3_8h, suggesting a more chewable texture. In contrast, adhesiveness was greatest in 1:2_24h, though this parameter remained low in the 1:3 system overall.

Overall, fermentation enhanced key textural properties, particularly cohesiveness, confirming the formation of a more stable and structurally developed rice-based matrix.

3.3.4. Sensory evaluation

The results of the sensory evaluation of the fermented samples of hydromodule 1:3 are presented in Table 10.

The sample 1:3_8h was more acceptable for the sum of the scores. At the same time, the sections noted that both samples lacked consistency. Sample 1:2_24h received a low rating mainly due to the liquid, «watery» consistency, as well as lack of aftertaste and sharp acidity in taste and odour.

Rice and its derivatives are widely consumed globally due to their digestibility, hypoallergenic nature, and neutral sensory profile. These qualities make rice an excellent base for developing novel functional foods, particularly plant-based fermented products. In this study, a two-stage processing approach – enzymatic hydrolysis followed by lactic acid fermentation (LAF) – was applied to enhance the sensory, textural, and functional properties of rice-based systems.

Enzymatic pretreatment played a pivotal role in modifying the raw rice matrix. It effectively eliminated the characteristic cereal aftertaste and introduced a mild sweetness due to the release of simple sugars and dextrins from starch hydrolysis. Concurrently, the viscosity of the rice suspension increased significantly, transforming it from a phase-separated dispersion into a stable, homogeneous system. This improvement is attributed to the breakdown of starch granules and the formation of soluble high-molecular-weight fragments, which contribute to colloidal stability – a finding consistent with studies on other plant matrices [36, 37].

The resulting simple sugars concentration provided sufficient fermentable substrate for the growth of Lactobacillus delbrueckii subsp.bulgaricus, enabling rapid acidification during fermentation. Titratable acidity increased significantly, with pH decreasing to 3.1–3.7 depending on hydromodule and fermentation duration. The 1:2 hydromodule demonstrated higher acidification potential than 1:3, likely due to its greater dry matter content and higher nutrient availability.

Fermentation time was a critical factor. An 8-hour process proved optimal, achieving balanced acidity and high sensory acceptability in both systems. In contrast, 24-hour fermentation led to over-acidification (up to 79–90 °T), resulting in a sharp, unpleasant taste and reduced consumer preference – especially in the more diluted 1:3 system.

The obtained data is preliminary. Of course, further research will be continued and will focus on several key aspects: optimizing the amount of starter culture applied, studying the fermentation kinetics through glucose modifications, and determining the product’s shelf life. Additionally, the research will investigate the survival and persistence of bacteria within the product.

Beyond acidification, LAF significantly improved the structural and textural properties of the product. The increase in viscosity and CSR is likely driven by multiple mechanisms: particle size reduction, increased surface area for interaction. This is particularly important in plant-based systems, which naturally lack the casein network found in dairy.

These instrumental findings were confirmed by sensory evaluation. Sample 1:2_8h received the highest scores across all attributes – particularly for consistency, taste, and overall acceptability. Panelists described its texture as «dense», «coating», and «pleasant», correlating with its high viscosity (1026 cP), WHC (98%), and low syneresis (1.0%). In contrast, samples with a 1:3 ratio were perceived as «watery» and «lacking body», reflecting their lower dry matter content and weaker structural development.

The stability and texture of the fermented product improved. Analysis of the texture profile (TPA) and viscosity properties showed a high potential of the fermented samples. Especially sample 1:2_8h. It combines high viscosity (1026 cP), WHC (98%), stability of the internal structure (89.1%), as well as minimal syneresis (1.0%). It also received the highest sensory rating among all the samples studied.

The assessment of organoleptic indicators of food product quality, especially for new products, is highly sought after. It provides statistically reliable results and does not require specialized equipment; the analyzers are the human senses. Let us examine the results in more detail.

Appearance. Overall, no significant differences were found in participants' perceptions of the appearance of all samples. All samples had an acceptable appearance, as expected for this type of product.

Color. Overall, no significant differences were found in participants' perceptions of the color of all samples. All samples had an acceptable appearance characteristic of rice.

Consistency and texture. There was a significant difference in the perception of consistency and texture among the participants for the fermented samples. A comparison of the samples showed that there was a substantial difference between the hydro-gel samples 1:2 and 1:3. Participants expectedly noted a more viscous and dense consistency for samples 1:2_8h and 1:2_24h. The texture was also more enveloping, providing pleasant sensations on the tongue and in the mouth. The average score for the consistency (texture) of 1:2_8h was the highest at 4, while the lowest score was given to the samples with hydromodule 1:3. Participants noted the watery consistency of these samples. Taste and Aftertaste. In general, there was a significant difference in the participants' perception of the taste and aftertaste of all samples. The most balanced taste and aftertaste were found in sample 1:2_8h (distinct and pleasant), while the least pronounced were in sample 1:3_24h (indistinct and weak, with no aftertaste).

Aroma and Taste. Overall, participants noted significant differences in the perception of aroma and taste among all samples. The most pronounced taste and aftertaste were observed in sample 1:2_8h (distinct and pleasant), while the least pronounced were in sample 1:3_24h (indistinct and weak, with no aftertaste).

Overall Acceptability. Participants reported good sensory perception of the fermented samples 1:2_8h and 1:3_8h. However, a comparison between the samples indicated that there is a difference in the assessment of their consistency, texture, and aftertaste.

The average score for overall acceptability among the fermented samples was highest for sample 1:2_8h (29 points), while sample 1:3_24h received the lowest score (19 points). It is important to note that participants generally indicated a deficiency in the consistency of the fermented samples. Participants expressed the opinion that the product lacks «density» or «body». This must be taken into consideration in future developments. The integration of sensory and rheological data is essential for product development. As shown by Castro et al. [32] and Rodrigues et al. al. [33], consumer preferences are closely linked to texture and fermentation dynamics. The positive impact of homogenization and microbial metabolism on product stability and quality has also been demonstrated in other plant-based systems [37, 38].

Principal component analysis (PCA, Fig. 4A) plot clearly shows a distinct separation of data into two main groups represented by differently colored points: blue points correspond to the 1:2 hydro module, while red points correspond to the 1:3 hydro module. This distribution indicates significant differences between the groups primarily due to hydro module parameters.

The first principal component (PC1), explaining 65.84% of the data variation, serves as the key factor determining the separation of the 1:2 and 1:3 groups. This allows us to conclude that this component reflects the main differences between the studied samples. The second principal component (PC2), associated with fermentation duration, explains 29.42% of the data variation and helps identify additional differences, particularly within the 1:2 group, which includes samples with time stamps of 24 hours and 8 hours. This distribution of samples across components indicates a significant influence of both the hydro module and fermentation duration on the formation of observed differences between the groups. The results suggest the need for additional statistical analysis to assess the significance of the identified differences and determine specific variables contributing most to the formation of principal components.

Further research should investigate the nature of the relationship between components and initial parameters for a deeper understanding of the mechanisms underlying the observed sample separation.

Correlation analysis by the heat map method (Fig. 4B) revealed strong positive relationships between sensory perception and instrumental parameters such as viscosity, cohesiveness, gumminess, and WHC. Conversely, excessive acidity and prolonged fermentation showed negative correlations with taste and aftertaste, highlighting the need for precise process control.

This relationship is crucial because it enhances our comprehension of how elements affect the sensory experience of a product. The interdependence between subjective assessment (including the enveloping qualities, the duration of contact with the mouth and tongue, and the velvety texture) and the results of objective measurements is demonstrated. Additionally, correlation analysis has shown that there is a strong correlation between the sensory profile, texture, and processing parameters, with more than 10 controlled variables.

The investigation demonstrates that the textural and functional properties of the rice-based system developed in this study are crucial for its technological and nutritional value. Our findings indicate that viscosity plays a key role in determining both flavor and mouthfeel characteristics.

Glucan not only contributes to flavor formation but also undergoes beneficial structural changes during fermentation. The positive effects of β-glucan and other metabolites, which are well-known for their rheological and physiological functions [39-41], are significantly enhanced by microbial activity. Among these microorganisms, Lactobacillus bulgaricus plays a particularly important role.

The activity of L. bulgaricus on rice substrates is essential for improving the texture and stability of the final product. This has been previously demonstrated in milk systems [27, 28].

As a result of a comprehensive analysis and sensory evaluation, sample hydromodule 1:2_8h can be identified as the most promising for further research.

1. Conclusion

This study demonstrates that the combination of enzymatic hydrolysis and lactic acid fermentation using Lactobacillus delbrueckii subsp. bulgaricus significantly improves the physicochemical, rheological, and sensory properties of rice-based fermented products. The rice-to-water ratio (hydromodule) was identified as a key factor influencing product quality, with a 1:2 ratio proving optimal for structural development and sensory acceptability.

Fermentation for 8 hours yielded the best balance of acidity, viscosity, and texture, particularly in the 1:2 system. Sample 1:2_8h exhibited high water-holding capacity (98%), low syneresis (1.0%), and superior sensory scores, confirming its potential as a stable, palatable plant-based functional product.

The findings indicate significant potential for rice as an eco-friendly and hypoallergenic foundation for developing cereal-based beverages and fermented food products. Research priorities include understanding matrix formation during fermentation, enhancing storage stability, and expanding applications into new food categories. These categories encompass plant-based yogurts, innovative desserts, functional sauces, and specialty dressings.

Furthermore, studies will examine fermentation’s effect on antioxidant properties and transformations in rice-derived exopolysaccharides. These components are vital for creating innovative health products. Ultimately, these efforts aim to advance the development of novel, health-focused food solutions using fermented rice substrates.

1. Acknowledgements

The author acknowledges to Dr. Nikitina E.V. for her helpful advices.

1. Declaration of competing interest

The author reports no conflicts of interest with anybody.

1. Authors’ Contributions

The entire manuscript is an independent work of the author.

1. Using Artificial Intelligent Chatbots

Artificial Intelligent chatbots has not been used in any section of work.

1. Ethical Consideration

This study does not require approval from an ethics committee.

Background and Objective: The delivery of probiotics using functional foods or supplements (e.g., capsules, tablets, or sachets) as carriers is an important strategy to provide health benefits, as it is necessary to maintain 10⁶-10⁹ colony-forming units (CFU) per gram of the probiotic strain alive and active at the time of consumption for effectiveness. This study investigated the effects of batch and fed-batch fermentation methods on increasing the cell and spore populations of Heyndrickxia coagulans MTCC 5856 (formerly Bacillus coagulans MTCC 5856).

Material and Methods: Batch and fed-batch fermentation strategies were applied to evaluate their effects on vegetative growth and sporulation. The protective effects of various cryoprotectants (sorbitol, sucrose, inulin, calcium lactate, manganese chloride, and skim milk) were assessed during freeze-drying. Spore resistance under simulated gastrointestinal conditions and stability in two functional food matrices (pastille and coffee mix) were also evaluated.

Results and Conclusion: Skim milk increased sporulation from 63% to 88%, with >80% spore viability in GI conditions and 85–98% stability in food matrices after 6 months. This study provides the first comparative analysis of batch versus fed-batch fermentation on H. coagulans MTCC 5856 sporulation in industrial food matrices, revealing glucose’s inhibitory effect and skim milk’s superior cryoprotective efficacy. These findings highlight the importance of optimizing fermentation conditions and applying suitable cryoprotectants to enhance the long-term viability and application of H. coagulans in functional food formulations.

Keywords: Batch fermentation, Freeze-drying, Functional foods, Gastrointestinal resistance, Heyndrickxia coagulans, Probiotics, Sporulation, Spore stability

1. Introduction

Efforts to improve nutrition to promote health and prevent chronic diseases have led to the exploration of various food components and the production of functional foods [1]. Probiotics are live microorganisms that positively impact the host's health by balancing the natural microbes in the intestine, provided they are consumed in adequate amounts. Common probiotics, such as those from the Lactobacillus and Bifidobacterium genera, exhibit effective probiotic activity; however, their survival rates are generally low, making them quite vulnerable. Conditions of fermentation, freezing, thawing, drying, and additives for cell protection are among the factors that affect the survival of these microorganisms during the production of probiotic food [2]. Also, the digestive conditions of the body and stressful factors can cause the loss of a significant number of probiotic cells in the digestive system [3].

 Bacillus spores are highly resilient, allowing them to endure the demanding conditions of product production and storage, as well as the challenges posed by digestive system, such as exposure to stomach acid and bile [4). Among the numerous Bacillus species, only a select few strains are available for use as human probiotic. One such strain is H. coagulans, which has recently garnered attention from researchers and food manufacturers due to its unique characteristics that combine traits of both the Bacillus and Lactobacillus genera [3]. This non-GMO probiotic is generally recognized as safe (GRAS), can tolerate high temperatures, and exhibits genetic stability over several years of commercial production. H. coagulans can survive for decades in harsh environments without cell division by forming spores [5].

In recent research, its clinical effectiveness has been confirmed to control hypercholesterolemia, lactose intolerance, digestive disorders, and diarrhea [6, 7]. Compared to other probiotic bacterial strains, the spore nature of H. coagulans has ensured its stability and vitality in functional foods and has not shown an adverse effect on the sensory and nutritional characteristics of the products [6, 8]. Table 1 presents a summary of the limited research regarding how various factors, such as the type of drying method and the application of protectants during the cell drying process, affect cell viability, sporulation, and the persistence of bacteria under simulated conditions of the digestive system, as well as during production and storage process.

Despite the growing interest in H. coagulans, several gaps remain in the current literature. Comparative studies investigating the influence of batch versus fed-batch fermentation on its vegetative growth and sporulation are limited. Additionally, systematic evaluations of various cryoprotectants in preserving spore viability during freeze-drying are lacking. Furthermore, few studies have explored its resilience under simulated gastrointestinal conditions or assessed its long-term viability in real food matrices such as pastille and coffee mix under ambient storage. Pastille and coffee mix were selected in this study based on a direct request from Master Foodeh Food Industries (Tehran, Iran), which aims to develop probiotic-enriched versions of their commercial products. These food matrices represent two distinct formulation challenges, one being a gelatin-based, thermally processed confectionery product and the other a dry beverage powder intended for reconstitution with boiling water. Both products were evaluated under factory-scale conditions, confirming their feasibility for industrial application. The aim of this study was to evaluate the impact of fermentation strategy and cryoprotectants on the viability of H. coagulans, assess its resistance to simulated gastrointestinal conditions, and determine its shelf stability when incorporated into functional food products. Assessing the viability of H. coagulans in these real food systems aligns with the overall objective of this study and allows evaluation of its functional stability and potential use in commercially viable probiotic foods.

Notably, this study provides the first comparative analysis of batch versus fed-batch fermentation on H. coagulans MTCC 5856 sporulation in industrial food matrices, showing that glucose inhibits sporulation and skim milk provides superior cryoprotective efficacy.

1. Materials and Methods

2.1. Chemicals, solvents and other compounds

Casein peptone, D(+)-Glucose, yeast extract, tryptone, agar, sodium chloride, sorbitol, sucrose, inulin, calcium lactate, magnesium sulfate heptahydrate (MgSO₄·7H₂O), manganese sulfate monohydrate (MnSO₄·H₂O), ferrous sulfate heptahydrate (FeSO₄·7H₂O), calcium carbonate (CaCO₃), sodium acetate (CH₃COONa), and skim milk were obtained from Merck (Darmstadt, Germany). Phosphate buffer was purchased from Bio Idea (Tehran, Iran). Pepsin and trypsin were acquired from Sigma-Aldrich (St. Louis, MO, USA), and bile oxalate was obtained from Ibresco (Iran). Industrial pastille and coffee mix were produced by Master Foodeh Food Industries Company (MFFIC, Tehran, Iran) using standard commercial procedures.

2.2. Microorganism and Culture Conditions

Heyndrickxia coagulans MTCC 5856 was obtained from Maya Zist Farayand Company (Tehran, Iran). The strain was cultured in a modified medium containing (g/L): glucose 40, yeast extract 13.33, tryptone 13.33, MgSO₄·7H₂O 0.027, CaCO₃ 20, MnSO₄·H₂O 0.015, FeSO₄·7H₂O 0.024, NaCl 0.013, and sodium acetate (CH₃COONa) 0.67, based on formulation [15]. Stock cultures were preserved in 20% (v/v) glycerol at –80 °C. For inoculum preparation, a single colony was transferred into 10 mL of the same medium and incubated at 37 °C with shaking at 200 rpm for 16 h. This culture was then used to inoculate 100 mL of fresh medium at 5% (v/v), followed by incubation at 37 °C and 200 rpm for 24 h to obtain vegetative cells. For sporulation, the culture was subsequently incubated at 45 °C under static conditions for an additional 24 h. The selected temperature and incubation period were based on prior validation to balance sporulation efficiency and vegetative cell viability, considering both the nutrient composition and typical pH dynamics of medium. Although minor pH shifts occurred during this period, the primary driver for optimizing sporulation was the availability and balance of nutrients, which promoted maximal spore formation. The fermentation and sporulation conditions applied in this study were determined based on previously optimized parameters, as described in a separate manuscript currently under peer review.

2.3. Analysis Methods

2.3.1. Viable counts of H. coagulans

The viable cell count of H. coagulans in each sample was determined by enumerating colony-forming units (CFU). Samples were serially diluted in sterile peptone water, and appropriate dilutions were plated using the pour plate method. Plates were incubated at 37 °C for 24 h, after which colonies were counted and expressed as CFU per gram of sample [16].

2.3.2. H. coagulans Spore resistance

To evaluate the thermal resistance of H. coagulans, the culture medium was subjected to heat treatment at 90 °C for 10 min and then rapidly cooled to 30 °C. This procedure was applied to eliminate vegetative cells while preserving heat-resistant spores. The treated samples were plated using the plate counting, and the plates were incubated at 37 °C for 48 h. Germinated spores formed visible colonies, which were counted and reported as CFU per gram [17].

2.3.3. Statistical methodology

All data were analyzed using Minitab software (version 21.4.2, Minitab LLC, USA), SPSS Statistics (version 26.0, IBM Corp., USA) and Microsoft Excel 2016 (Microsoft Corporation, USA). Results are presented as the mean ± standard deviation of triplicate measurements conducted in two independent experiments. Microbial counts were expressed as log₁₀ CFU/mL or log₁₀ CFU/g. Statistical significance was considered at P < 0.05.

2.4. Kinetics of bacterial growth, investigating pH changes and glucose consumption during bacterial growth

To evaluate the growth kinetics of H. coagulans, the bacterial culture was incubated at 37 °C for 24 h. At 3-hour intervals, samples were taken to determine viable cell counts using the colony enumeration. Simultaneously, pH measurements of the culture medium were recorded using a digital pH meter. Glucose consumption was also monitored during the growth period. To quantify the residual glucose in the culture medium, a standard calibration curve was prepared using a glucose oxidase colorimetric kit (Pars Azmoon, Tehran, Iran) with concentrations of 0.1, 0.2, 0.4, 0.6, 1.0, 1.2, 1.4, 1.6, and 2.0 mg/mL, following the manufacturer’s protocol. Absorbance was measured at 540 nm using a spectrophotometer, and the glucose concentration in culture samples was calculated based on the standard curve at 3-hour intervals [18].

2.5. Investigating the effect of feeding on cell growth and sporulation

To investigate the effect of glucose supplementation on the growth and sporulation of H. coagulans, 250 µL of a 60% (w/v) glucose stock solution was added to 50 mL of culture medium at 3-hour intervals during incubation. Glucose feeding was performed based on the re-measured glucose concentration in the medium at each interval. The populations of vegetative cells and spores were determined at each sampling point using the pour plate method, as previously described.

Glucose was chosen as the feeding substrate since it was already present in the base culture medium, and the aim was to examine how its continued availability might influence bacterial growth and sporulation dynamics.

2.6. Investigating the effect of different protectants on reducing damage to cells before transfer to freeze dryer

The production of probiotic powder typically involves a freezing and drying process, during which dehydration can damage bacterial cell structures such as surface proteins, membranes, and cell walls. To mitigate these effects and enhance cell viability during freeze-drying, various protective agents were tested, including sorbitol, sucrose, inulin, calcium lactate, skim milk, and manganese chloride. Each protectant was added in equal concentration to identical volumes of bacterial suspension, and all samples were treated under the same conditions throughout cultivation and freeze-drying. Samples were initially frozen at –40 °C, then transferred to a freeze dryer and subjected to lyophilization for 18 h. The resulting powders were sealed in airtight polyethylene bags and stored at –4 °C to prevent moisture absorption. To assess the effectiveness of each protectant in preserving spore viability, pour plate culturing was performed on treated samples both before and after heat treatment (90 °C for 10 min). Plates were incubated at 37 °C for 48 h, and colony counts were compared between the different protectants and the control sample without any added protectant. Spore viability was assessed before and after freeze-drying by performing plate counting following heat treatment at 90 °C for 10 minutes to eliminate vegetative cells. Spore viability (%) was calculated by dividing the number of surviving spores after heat treatment by the total initial bacterial population (including vegetative cells and spores) before freeze-drying, and multiplying the result by 100.

2.7. Assessment of bacterial persistence under simulated gastrointestinal conditions 

Probiotic microorganisms must survive the harsh conditions of the gastrointestinal tract to exert their beneficial effects. Therefore, the tolerance of H. coagulans to acidic pH, simulated gastric juice, and bile salts was evaluated under laboratory conditions.

2.7.1. Acid resistance:

A fresh culture of H. coagulans was prepared, and 100 µL of microbial suspension was inoculated into 10 mL of culture medium adjusted to pH 2.5 and 4.0. The tubes were incubated at 37 °C, and after 3 h and 4 h, 1 mL of each sample was taken and analyzed using the colony enumeration. Viable cell counts were determined and expressed as CFU/mL. A minimum survival threshold of 10⁶ CFU/mL was considered acceptable [19, 20].

2.7.2. Gastric juice resistance

Simulated gastric juice was prepared using two separate media, each containing 2.3 g of pepsin and trypsin, and 2 g of sodium chloride per liter of distilled water, adjusted to pH 2.0. Control media were prepared with the same composition but adjusted to pH 7.0. Each medium was inoculated with 2% (v/v) of the H. coagulans suspension and incubated at 37 °C. Samples were taken at 0, 1, 2, 3, 4, and 24 h for viable cell count using the plate counting Survival above 10⁶ CFU/mL was considered indicative of resistance.[21]

2.7.3. Bile salt resistance 

To assess the tolerance of H. coagulans spores to bile salts, two culture media were prepared containing 0.3% and 0.5% bile oxalate, along with a control medium without bile. Each tube was inoculated with 100 µL of microbial suspension and incubated at 37 °C. Viable cell counts were determined at 0 h and 8 h using the colony enumeration.

The inhibition coefficient (C_inh) was calculated using equation .1 to quantify the inhibitory effect of bile salts on bacterial growth:

Where T₀ and T₈ refer to the log CFU/mL values at time 0 and 8 hours, respectively. A C_inh value ≤ 0.4 indicates acceptable resistance to bile salts [12, 22].

2.8. Evaluation of spore stability in food products

The stability of H. coagulans spores was assessed in two functional food matrices: pastille and coffee mix, during both production and storage processes.

2.8.1. Pastille formulation 

The base syrup was prepared by mixing glucose, sugar, and water at 100 °C. This mixture was combined with hydrated gelatin (prepared by dissolving gelatin in boiling water), food-grade coloring, citric acid, and essential oil. After cooling to an appropriate temperature, probiotic powder was added and the mixture was homogenized. The final mixture was poured into molds using a funnel and allowed to set for 24 h at room temperature. For microbiological analysis, randomly selected pastilles were dissolved in sterile peptone water and agitated in a shaker incubator for 1 h. Serial dilutions were then plated using the plate counting and incubated at 37 °C for 48 h. According to standard guidelines, a minimum viability of 10⁶ CFU per 100 g of pastille was considered acceptable.

2.8.2. Coffee mix formulation 

Coffee mix powder was prepared from processed coffee beans using either spray drying or freeze drying. To simulate beverage preparation, a known quantity of probiotic powder was mixed with a single-serving coffee sachet (approximately 18 g), dissolved in 150 mL of boiling water, and stirred thoroughly. 

To evaluate the stability of H. coagulans spores, samples were plated using the colony enumerationand incubated at 37 °C for 48 h. Colony counts were recorded and used to assess spore survival after exposure to product processing and heat.

1. Results and Discussion

3.1. Kinetics of bacterial growth, pH variation, and glucose consumption

To assess the growth behavior of H. coagulans, a batch fermentation was conducted, and key parameters including viable cell count, pH, and glucose concentration were monitored at 3-hour intervals for 24 h. Viable counts were determined by the plate counting, pH was measured using a digital pH meter, and residual glucose levels were quantified via spectrophotometric analysis.

As shown in Figure 1, bacterial growth increased steadily until reaching its peak at 24 h, after which it declined, indicating entry into the death phase. These observations are consistent with the growth kinetics reported [23]. Glucose concentration in the medium decreased rapidly and was nearly depleted by 12 h, suggesting a high metabolic activity during the exponential phase. Following glucose exhaustion, the cells likely shifted to alternative nutrient sources. The pH of the culture medium showed a marked decline during the initial hours of growth, reaching its minimum around 18 h. This decrease corresponds to the accumulation of organic acids, primarily lactic acid, produced during active metabolism. After 18 h, pH levels began to rise slightly, which may be attributed to ammonium production from protein degradation or the consumption of lactate as a secondary substrate. After heat treatment, the final spore concentration was measured at 7.11 log CFU/mL, indicating successful sporulation under the tested conditions.

3.2. Investigation of the Effect of Feeding on Cell Growth and Sporulation

In batch culture, due to the reduction of the initial concentration of food compounds in the culture medium and the lack of control of their concentration, formation and accumulation of the growth inhibitory compounds in the culture medium, bacterial growth quickly leaves the exponential growth phase and enters the stationary phase. Research has shown that employing a fed-batch culture method, which involves controlling nutrient concentrations, especially the limiting substrate, can significantly reduce the buildup of growth-inhibitory compounds. This strategy extends the exponential growth phase, ultimately improving the efficiency of the fermentation process.

Therefore, in this research, to increase the number of vegetative cells and subsequently increase sporulation, the effect of the fed-batch process with pulse feeding was investigated on the growth of vegetative cells. Feeding was done according to the amount of glucose in the culture medium.

According to the results illustrated in Figure 1, glucose was nearly completely consumed at 12 hours of fermentation. So, the first pulse feeding of glucose was initiated at this time. The glucose concentration in the culture medium was measured hourly, and approximately every three hours, when glucose was nearly depleted, an additional pulse of glucose was added to the culture. At that point, 250 μl of 60% glucose solution was added to 50 mL of the culture medium, after which the cell population was examined. Figure 2 shows the effect of feeding on pH changes and bacterial growth.

A comparison of the results in Figures 1 and 2 indicates that although the bacterial growth rate has increased, the difference in vegetative cell populations between batch and fed-batch cultures when feeding at 12, 15, 18, 21 h of growth is not significant. This suggests that this kind of feeding had a slight effect on vegetative cell population growth. The pH continued to decline until hour of 24 and, in contrast to the batch process, the culture medium remained acidic throughout.

At hour of 25 of cell growth, the culture medium was subjected to sporulation conditions for 24 h. After this period, pour plate cultures were performed both before and after heat treatment. However, the sporulation results in the fed-batch fermentation process were unsatisfactory; after 48 h, no spores were detected on the plates. While the total cell population before heat treatment was approximately 8.18 log CFU/ml, the vegetative cells did not convert to spores. A carbon source is essential for vegetative cell growth because it helps create an acidic environment. However, increasing its concentration has little effect on enhancing vegetative cell population and can inhibit spore production. This inhibition may be due to the negative effects of the carbon source on the synthesis of compounds necessary for spore formation. A study was conducted on the effect of glucose on the growth and sporulation of Bacillus subtilis. The results indicated that the population of vegetative cells increased with rising glucose concentrations, reaching a peak at 5g/l, after which it remained constant at higher glucose levels. However, the efficiency of spore formation decreased as glucose concentration increased. This is attributed to the fact that up to approximately 5g/l, all the glucose in the culture medium is used by the bacteria until the end of the logarithmic growth phase. Continued glucose consumption leads to reduced spore formation [24]. To directly compare sporulation outcomes between the two cultivation strategies, spore counts were measured after 48 h in both batch and fed-batch systems. Sporulation was observed only under batch conditions, yielding 7.11 log CFU/mL after heat treatment. In contrast, spore formation was entirely absent in the fed-batch culture, as post-heat treatment counts consistently yielded zero colony-forming units. This finding clearly demonstrates that while glucose feeding supported vegetative growth, it completely inhibited the transition to spore formation. As a matter of fact, unlike fed-batch fermentation, where nutrient feeding increases bacterial growth as active vegetative cells, sporulation is induced by starvation and nutrient limitation. Since all fed-batch results were uniformly zero, no comparative figure was included; instead, the outcomes are described textually to avoid redundancy and to ensure clarity of data presentation.

3.3. Effect of cryoprotectants on spore survival during freeze-drying

Various cryoprotectants were tested to improve the survival of H. coagulans spores during freeze-drying by minimizing cellular damage. The protective compounds function by reducing osmotic pressure differences between the intracellular and extracellular environments, thereby preserving cell integrity. In this study, six commonly used cryoprotectants were applied at equal concentrations (w/v), and their effects were compared to a control sample without protectant.

As shown in Table 2 and Figure 3, the presence of cryoprotectants significantly enhanced spore viability post freeze-drying. The sporulation rate increased from 63% in the control group to a maximum of 88% with skim milk. Among all protectants tested, skim milk resulted in the highest survival rate, followed closely by sorbitol. One-way ANOVA followed by Tukey’s post-hoc test was performed to compare survival percentages among the tested cryoprotectants. The results showed that skim milk (88.23%) and sorbitol (84.57%) provided the highest protection, with no statistically significant difference between them (P > 0.05). Both were significantly more effective than inulin (76.14%), sucrose (77.05%), calcium lactate (72.15%), MgCl (71.09%), or the no-protectant control (63.04%) (P < 0.05).

.The superior performance of skim milk in enhancing survival of H. coagulans (88%) can be attributed to its rich content of calcium and proteins. Calcium ions stabilize spore coat structures and maintain cell wall integrity, protecting against mechanical and thermal stresses during freeze-dryingProteins in skim milk form a protective matrix around the spores, mitigating osmotic shock caused by rapid water removal. Together, these components preserve the functionality of spore coat proteins and cell membranes, preventing structural damage and maintaining viability post-lyophilisation. Similar findings were observed in studies on lactic acid bacteria, where mechanistic evaluations demonstrated that protective compounds such as sugars and proteins reduce osmotic stress and stabilize cell structures during freeze- or spray-drying. These components mitigate membrane damage and protein denaturation, thereby improving post-drying survival [10].

The results suggest that skim milk is an effective cryoprotectant for preserving H. coagulans spores during freeze-drying and may be suitable for developing stable formulations.

3.4. Spore stability under simulated gastrointestinal conditions

3.4.1. Acid resistance

To evaluate acid tolerance, H. coagulans spores were exposed to acidic media at pH 2.5 and 4. Samples were collected at 3 and 4hours post-inoculation, and viable spore counts were determined using the plate counting following heat treatment. The initial spore population was 7.5 ± 0.18 log CFU/mL.

As shown in Table 3 and Figure 4, spore viability was higher at pH 4 compared to pH 2.5. This suggests that lower pH increases cellular stress and reduces viability. The observed reduction may be attributed to spore germination followed by inactivation of vegetative cells under harsh acidic conditions. Despite this, more than 90% of spores survived under both pH conditions, and the difference in survival between 3 and 4 hours was not statistically significant.

3.4.2. Simulated gastric juice resistance

The ability of probiotics to survive in gastric juice is a key factor in their functionality. To simulate stomach conditions (pH 2.3), H. coagulans spores were incubated in artificial gastric fluid, and samples were collected at 1, 2, 3, 4, and 24 hours. Viable spore counts were determined using the colony enumeration, and the initial population was 7.5 ± 0.18 log CFU/mL. As illustrated in Table 4 and Figure 5, spore viability remained above 85% even after 24 hours of exposure. The spores did not germinate during the incubation, likely due to the absence of specific germination triggers. This high level of resistance highlights the robustness of H. coagulans spores in simulated gastric conditions.

3.4.3. Bile salt tolerance 

To assess bile tolerance, H. coagulans spores were incubated in media containing 0.3% and 0.5% bile salts. Samples were collected at 0 and 8 hours, and viable counts were measured by the plate counting. The initial population was 7.5 ± 0.18 log CFU/mL. Results are shown in Table 5.

The bile salt inhibition coefficient (C_inh) was calculated based on the method described by Mojgani et al. and Sui et al. using the following equation (12, 22):

C_inh = [(T₈ – T₀)control – (T₈ – T₀)treatment] / (T₈ – T₀)control

The calculated values were:

C_inh (0.5%) = [(6.45 – 6.38) – (6.53 – 6.48)] / (6.45 – 6.38) = 0.28 

C_inh (0.3%) = [(6.34 – 6.28) – (6.42 – 6.35)] / (6.34 – 6.28) = 0.16

The inhibition coefficients (C_inh) were 0.28 for 0.5% bile and 0.16 for 0.3% bile. Statistical analysis using a paired t-test showed no significant difference between these values (P > 0.05), indicating that H. coagulans MTCC 5856 maintains high viability under both bile concentrations. To assess this, the increase in viable counts over 8 hours was compared between bile-treated and control samples, confirming that the differences were not statistically significant. These results support our conclusion that the spores maintain high viability under gastrointestinal-like bile stress conditions.

Both values were below the threshold of 0.4, indicating acceptable bile salt resistance. Moreover, viable counts slightly increased over time, suggesting that spores not only survived but may have germinated and proliferated under bile salt exposure.

These findings are consistent with previous reports, which demonstrated high survival and germination of H. coagulans spores under simulated gastrointestinal conditions. While our study focused on spore viability under bile salt stress, both studies emphasize that maintaining high spore viability is essential for subsequent germination and functional activity in the gut. The slight increase in viable counts observed over 8 hours in bile-containing media suggests that spores may germinate and proliferate, complementing the high germination rates reported previously [4].

3.5. Stability of H. coagulans spores during production and storage of functional food products 

3.5.1. Pastille  

To assess probiotic viability in pastille, five samples (each ~2 g) were randomly selected and individually diluted in sterile peptone water. Samples were incubated at 37 °C for 30 minutes with gentle shaking, followed by enumeration of viable spores using the colony enumeration. Plates were incubated at 37 °C for 48 hours. The viable counts are shown in Figure 6. After 6 months of ambient storage, the average survival rate of spores in pastille samples was 85.06%. This high level of survival suggests that H. coagulans spores remained dormant but viable during storage, highlighting their potential for use in heat-processed confectionery products.

3.5.2. Coffee mix 

To simulate consumer preparation, one sachet (18 g) of coffee mix was reconstituted in 150 mL of boiling water and stirred thoroughly. The suspension was plated using the pour plate method, and incubated at 37 °C for 48 hours. Results are shown in Figure 7. After 6 months of storage, spore viability remained high at 97.98%, with no significant reduction in count compared to initial levels. The persistence of H. coagulans spores is attributed to their ability to remain dormant under harsh conditions and germinate when conditions become favorable. These results demonstrate the success and applicability of H. coagulans MTCC 5856 in pastille and coffee, highlighting its stability during production and shelf storage and supporting the overarching goal of this study to evaluate its viability in real-world functional food applications. These findings are consistent with previous work, which reported 94% and 99% survival of H. coagulans in brewed coffee and tea, respectively, and over 99% stability in powder form stored at room temperature for up to 24 months [13]. In contrast, a study by Adibpour et al. demonstrated that candies made with non-spore-forming probiotics such as L. plantarum A7 and UBLP-40 failed to retain viable bacteria after production. This comparison emphasizes the superior stability conferred by the spore form and underscores the practical advantage of H. coagulans MTCC 5856 for functional food applications. Slight differences in survival rates between studies may be due to variations in product formulation, storage conditions, or strain-specific characteristics, providing a mechanistic rationale for the observed outcomes [14].

Overall, the findings confirm that H. coagulans MTCC 5856 is a robust probiotic candidate capable of surviving harsh industrial and gastrointestinal environments, making it suitable for incorporation into a variety of functional food formulations.

1. Industrial Aspects

From an industrial perspective, the scalability and cost-effectiveness of probiotic production are critical considerations. Although freeze-drying is widely used to preserve spore viability, it represents a cost-intensive process. Alternative drying methods such as spray-drying or fluidized bed drying may provide more economical options for large-scale manufacturing, though their effects on spore survival require further evaluation. In addition, the spores exhibited high resistance to simulated gastrointestinal conditions, including acidic pH, gastric juice, and bile salts, with survival rates above 85%. This intrinsic resilience ensures that probiotics remain viable after consumption, reducing the need for additional protective formulations or complex delivery systems. Maintaining high spore viability during industrial-scale pastille molding and storage is another challenge, as exposure to heat or shear stress can reduce survival. Nevertheless, the present study demons-trated that H. coagulans spores maintained 85% viability in pastille and 98% viability in coffee mix after six months, highlighting their robustness under processing and storage conditions. Importantly, the inherent stability of spores also reduces the need for cold-chain logistics, thereby lowering distribution costs. Taken together, these features underscore the potential of H. coagulans MTCC 5856 as a cost-effective, scalable, and robust probiotic solution for functional food applications, with reliable performance from production to consumer use.

1. Conclusion

Functional probiotic products are increasingly favored by consumers due to their role in promoting gut health and overall well-being. However, the effectiveness of such products depends on the viability and stability of the probiotic strains during production, storage, and consumption. This study investigated strategies to enhance the survival and performance of H. coagulans MTCC 5856, a spore-forming probiotic with strong industrial potential. Comparative evaluation of batch and fed-batch fermentation revealed that while glucose supplementation improved vegetative growth, it adversely affected sporulation efficiency. Furthermore, the use of cryoprotectants during freeze-drying significantly influenced spore viability, with skim milk yielding the highest survival rate. The spores demonstrated high tolerance to simulated gastrointestinal conditions, including acidic pH, gastric juice, and bile salts, ensuring functionality after consumption. Incorporation into real food matrices, such as pastille and coffee mix, confirmed long-term stability during product processing and ambient storage, with survival rates of 85% and 98%, respectively. From an industrial perspective, these features (robust spore survival, resistance to gastrointestinal conditions, and stability in functional foods) underscore the strain’s scalability, cost-effectiveness, and suitability for commercial applications. Overall, H. coagulans MTCC 5856 is a robust and viable probiotic candidate capable of maintaining performance from production to consumer use, making it highly suitable for heat-processed and shelf-stable functional food formulations.

Limitations and Future Directions

One limitation of the study is that sporulation optimization conditions could not be disclosed due to overlap with a separate manuscript under review. However, the applied conditions were based on previously validated methods and industry collaboration, ensuring relevance to real-world applications. Additionally, while in vitro resistance to gastric and bile conditions was confirmed, further investigations are needed to fully characterize the probiotic functionality of this strain, including adhesion to intestinal epithelial cells (e.g., Caco-2) and colonization ability in animal models. Future studies could also examine the long-term effects of various cryoprotectants and storage conditions on H. coagulans spore viability across a wider range of food matrices. Moreover, exploring new functional food formulations and optimizing fermentation and processing parameters for industrial-scale production would help translate these findings into commercially viable products. Collectively, these efforts would provide a comprehensive understanding of the strain’s probiotic functionality and support its effective application in functional food development.

1. Acknowledgements

The authors thanks to Maya Zist Farayand Company for the strain and providing Master Foodeh Company for help in some analysis. Also, we appreciate Mr Rouzbeh Almasi Ghale, Ms. Maryam Hamidi and Ms. Elham Yavari for their great assistance in collecting data.

1. Declaration of competing interest

The authors report no conflicts of interest.

1. Authors’ Contributions

Conceptualization, Valiollah Babaeipour and fatemeh tabandeh; Data curation, Nasrin Alizadeh, Valiollah Babaeipour and fatemeh tabandeh; Formal analysis, Nasrin Alizadeh, Valiollah Babaeipour and fatemeh tabandeh; Funding acquisition, Valiollah Babaeipour; Investigation, Nasrin Alizadeh, Valiollah Babaeipour and fatemeh tabandeh; Methodology, Nasrin Alizadeh, Valiollah Babaeipour and fatemeh tabandeh; Project administration, fatemeh tabandeh; Resources, fatemeh tabandeh; Software, Nasrin Alizadeh and fatemeh tabandeh; Supervision, Valiollah Babaeipour and fatemeh tabandeh; Validation, Nasrin Alizadeh, Valiollah Babaeipour and fatemeh tabandeh; Visualization, Nasrin Alizadeh and fatemeh tabandeh; Writing – original draft, Nasrin Alizadeh and fatemeh tabandeh; Writing – review & editing, Valiollah Babaeipour and fatemeh tabandeh.

1. Using Artificial Intelligent Chatbots

This manuscript was entirely written and developed by the authors based on original experimental data. During its preparation, the authors used ChatGPT (OpenAI) solely to enhance the grammar and language clarity of certain sentences. All AI-assisted outputs were carefully reviewed and edited by the authors, who take full responsibility for the content of this manuscript.

1. Ethical Consideration

This study did not involve human or animal subjects; therefore, ethical approval was not required.

Background and Objective: Cheese is one of the major dairy products with high nutritional value, but its susceptibility to microbial growth and early spoilage remains a challenge for the dairy industry. While chemical additives are widely applied to control microbial contamination, increasing awareness of the potential hazards of synthetic preservatives has led to a growing demand for natural alternatives. This study was designed to evaluate the antimicrobial potential of postbiotics derived from lactiplantibacillus plantarum and Lacticaseibacillus casei against common spoilage and pathogenic microorganisms in cheese, as well as to investigate their impact on the microbiological and chemical properties of cheese during storage.

Material and Methods: Postbiotics were extracted from cultures of L. plantarum and L. casei and their antimicrobial activities were tested using standard microbiological assays against Gram-positive and Gram-negative bacteria. To assess practical application, the postbiotics were applied as coatings on cheese samples either alone or in combination with whey protein concentrate (WPC). Microbiological counts, chemical parameters, and sensory evaluation were performed throughout storage. Data were analyzed to determine the comparative effectiveness of treatments.

Results and Conclusion: The findings showed that the postbiotic derived from L. plantarum demonstrated stronger antimicrobial effects, particularly against Gram-positive bacteria, compared to that from L. casei. However, when combined with WPC, the antimicrobial activity of both postbiotics declined. Despite this limitation, postbiotics applied alone significantly reduced microbial counts during storage without altering the main chemical properties of the cheese. Sensory evaluation confirmed the overall acceptability of postbiotic and postbiotic-WPC treated samples. In conclusion, postbiotics can serve as promising natural antimicrobial agents in cheese preservation, though further optimization is required to enhance their activity when combined with protein-based carriers such as WPC.

Keywords: Antimicrobial activity, Cell-free supernatant, Cheese, Lactic acid bacteria, Postbiotic

1. Introduction

The food industry consistently faces substantial challenges from pathogenic and spoilage microorganisms, which are major drivers of foodborne diseases (FBDs), product quality degradation, and significant economic losses [1]. Globally, FBDs remain a pressing public health issue, with more than 600 million cases and nearly 420,000 deaths reported annually due to contaminated food and water [2,3]. These statistics have reinforced the urgency of strengthening food safety measures and minimizing contamination throughout production, processing, and storage stages [4,5]. At the same time, consumer concerns over the potential health risks associated with chemical preservatives, alongside the increasing demand for minimally processed foods, have fueled interest in natural preservation strategies [6,7]. Such approaches not only lessen dependence on artificial additives but also align with the growing trend toward clean-label products.

In this regard, biological protection has emerged as a promising strategy, harnessing beneficial microorganisms and their antimicrobial metabolites to suppress the proliferation of spoilage and pathogenic organisms [4]. Probiotics, especially lactic acid bacteria (LAB), have drawn considerable attention for their ability to generate bioactive substances such as organic acids, hydrogen peroxide, diacetyl, and bacteriocins [8]. Among these, LAB species such as Lactobacillus and Bifidobacterium are generally recognized as safe (GRAS) and have been widely employed for decades in the fermentation of diverse foods, including dairy products, cereals, and vegetables [9]. More recently, growing attention has been directed toward postbiotics, defined as non-viable microbial cells, cellular components, or metabolites that provide functional health benefits [10]. Compared with probiotics, postbiotics present notable practical advantages: they are independent of cell viability, exhibit greater stability under processing and storage conditions, and pose a lower risk of antibiotic resistance or incompatibility with food matrices. Additionally, postbiotics retain antimicrobial activity across a wide range of pH and temperature conditions, are capable of disrupting pathogenic biofilms, and can neutralize harmful contaminants such as pesticides and mycotoxins. In food systems, they have been investigated both as direct additives and as functional components of active packaging technologies, thereby overcoming limitations associated with the use of live microbial cultures [4].

Cheese provides a particularly critical application, as it is highly vulnerable to microbial contamination during both processing and storage. Among potential threats, Listeria monocytogenes is of significant concern due to its ability to withstand stress conditions and its high fatality rate in human infections [11,12]. Epidemiological evidence has repeatedly linked outbreaks of listeriosis to cheeses manufactured from raw or inadequately pasteurized milk [13], underscoring the urgent demand for innovative, safe, and effective antimicrobial approaches in dairy preservation.

Recent investigations have demonstrated that postbiotics and bacteriocin-like compounds derived from lactic acid bacteria (LAB) are capable of inhibiting pathogenic bacteria across diverse food matrices, including meat, seafood, and dairy products [14–19].

In parallel, whey protein, a major by-product of cheese production, has attracted considerable interest as a functional material for edible coatings and packaging, owing to its excellent barrier properties and strong film-forming capacity [11]. Incorporating antimicrobial postbiotics into whey protein systems may therefore offer dual advantages: enhancing microbial safety and prolonging shelf life while preserving desirable sensory characteristics.

Although evidence supporting postbiotics as natural preservatives is growing, relatively few studies have systematically assessed their performance in real cheese systems, particularly when combined with whey protein–based coatings. This gap in knowledge constrains a comprehensive understanding of their effectiveness under practical conditions.

The present study aims to bridge this gap by examining the antimicrobial activity of postbiotics derived from lactiplantibacillus plantarum and Lacticaseibacillus casei against key dairy pathogens, while also evaluating the microbial and chemical quality of cheese coated with whey protein concentrate (WPC) enriched with these postbiotics. This integrated strategy offers a novel, effective, and sustainable approach to improving food safety and preservation in dairy products.

1. Materials and Methods

2.1 Study design

This descriptive-analytical study was conducted between 22 November 2022 and 15 September 2023. The study protocol was reviewed and approved by the Medical Ethics Committee of Gonabad University of Medical Sciences (IR.GMU.REC.1401.073).

2.2 Bacterial strains

Three lactiplantibacillus plantarum strains were isolated from traditional Iranian cheeses [20]. In addition, one Lacticaseibacillus casei strain (1608 PTCC, IBRC of Iran), Listeria monocytogenes (7644 ATCC), Escherichia coli (1338 PTCC), and Staphylococcus aureus (1431 PTCC) were obtained from the Laboratory of Specialization in Nutrition, Mashhad University of Medical Sciences.

2.3 Postbiotic preparation

Each lactic acid bacterial strain was cultured separately in MRS broth medium and incubated under anaerobic conditions at 37 °C for 24 hours. The cultures were then centrifuged at 6000 rpm for 10 minutes at 4 °C. The resulting cell-free supernatants were filtered through a 0.4 µm membrane filter and subsequently freeze-dried (freezing temperature −83 °C, pump pressure 0.0026 mBar, storage temperature −60 °C) for use in subsequent experiments [21].

2.4 Chemical analysis of cell-free supernatants of Lactobacillus sp.

The chemical compounds of postbiotics were identified following the method described by Ryan et al. (2009), with minor modifications. For derivatization, 1 mL of the supernatant was mixed with 10 mL of absolute ethanol and 15 drops of sulfuric acid (97%), and the mixture was stirred at 80 °C for one hour. After cooling, 20 mL of distilled water was added, and extraction was performed five times with 50 mL of dichloromethane, collecting the lower phase each time. The pooled extracts were combined with 50 g of sodium sulfate and passed through filter paper. The solvent was then removed using a vacuum evaporator at 50 °C, and the remaining residue was injected into the gas chromatography–mass spectrometry (GC–MS) system.

The chemical composition of the derivatized postbiotics was analyzed using a GC instrument (Agilent HP-6890, Agilent Technologies, Palo Alto, CA, USA) operated with Agilent GC/MS Mass Hunter Acquisition software. The GC system was equipped with an Agilent HP-5ms column (30 m length, 0.25 mm inner diameter, 0.25 μm film thickness). Helium was used as the carrier gas at a flow rate of 1 mL/min. The oven temperature was programmed to increase from 110 °C to 240 °C at a rate of 4 °C/min with no hold time. A 10 μL sample was injected with a 5:1 split ratio [22].

2.5 Antimicrobial Activity In-vitro

2.5.1 Agar-well diffusion

Three pathogens (L. monocytogenes, E. coli, and S. aureus) were inoculated separately at a concentration of 5 log10 CFU/mL onto the surface of Muller Hinton Agar (MHA) plates. Wells with an 8 mm diameter were then created in the agar, and 100 µL of postbiotic suspensions at concentrations of 5%, 10%, and 20% were added to the wells. Plates were incubated aerobically at 37 °C for 24–48 hours. Nisin (625 IU/mL) and sterile distilled water served as the positive and negative controls, respectively. Antimicrobial activity was expressed as the mean diameter (mm) of inhibition zones, measured as the clear areas surrounding the wells. Each assay was performed in triplicate [23].

2.5.2 Determination of minimum inhibitory concentra-tion (MIC) and minimum bactericidal concentration (MBC) by the microdilution method

MIC and MBC values were determined according to the Clinical and Laboratory Standards Institute (CLSI, 2017) guidelines. After 24 hours of aerobic incubation at 37 °C, the wells were examined for turbidity. To determine the MBC, 10 µL samples from wells without turbidity (corresponding to MIC and higher concentrations) were streaked onto MHA plates in triplicate and incubated under the same conditions [24].

2.5.3 Anti-listeria activity of coatings containing postbiotics

To prepare the coating solution, 50 mL of deionized water was heated to 90 °C in a bain-marie, and 4.34 g of whey protein concentrate (WPC; protein 81.2%, lactose 7.4%, fat 6%, moisture 5%, ash 4%, pH 6.1; Alinda, Greece) was fully dissolved. The solution was maintained at this temperature for 45–60 minutes, during which 2.71 g of glycerol and 0.081 g of Tween 80 were added. After cooling, cell-free supernatants (CFS) were incorporated at concentrations corresponding to MIC and MBC [25]. The anti-listeria activity of coatings containing postbiotics was then evaluated using the agar-well diffusion method. CFS without coating, at equivalent concentrations, was included as a positive control [26].

2.6 Inoculation and coating of cheese samples

A pasteurized traditional cheese, commercially available in local markets, was selected for this study. The cheeses analyzed were pasteurized varieties inoculated with a fungal starter culture. Four treatment groups were prepared to evaluate microbial and chemical characteristics over storage on days 0, 1, 2, 4, 6, 8, and 10 (Table 1).

Cheese pieces of approximately 10 g (3 × 3 × 1 cm) were inoculated with L. monocytogenes at a level of 1 × 10⁵ CFU/g by spreading 1 mL of an appropriately diluted suspension onto the surface [18]. Samples were then allowed to stabilize to ensure bacterial adherence. The treatments were as follows:

1. Inoculated cheese without further treatment,
2. Cheese immersed in CFS solution at the designated concentration,
3. Cheese coated with WPC solution without CFS, and
4. Cheese coated with WPC solution containing CFS (immersion for 4–5 minutes).

All samples were stored at 4 °C until further analysis.

2.7 Microbial analyses in-situ

1. monocytogenes, total viable microorganisms, molds, and yeasts were enumerated using Palcam Agar, PCA, and SDA media, respectively. Palcam and PCA plates were incubated at 37 °C for 48 hours, while SDA plates were incubated at 25 °C for 3–5 days [26]. The enumeration of molds and yeasts was carried out according to the Iranian National Standard No. 2406: Microbiology of milk and milk products — Specifications and test methods [27].

2.8 Chemical analyses

The pH of cheese samples was measured using a pre-calibrated pH meter, and moisture content was determined by the gravimetric method on the designated sampling days [17,21].

2.9 Sensory analyses

Cheese slices coated with cell-free supernatants, free of L. monocytogenes, were evaluated for taste, color, aroma, texture, and overall acceptability by a panel of 10 semi-trained assessors using a 5-point hedonic scale [15]. All participants were adults above the legal age and voluntarily provided written informed consent in compliance with ethical standards for human subject research. Evaluations were conducted under identical environmental and temporal conditions to ensure consistency.

2.10 Statistical analyses

Statistical analyses were performed using SPSS software version 26. Mean values from three independent replicates were compared between two groups using the independent t-test, while comparisons among more than two independent groups were carried out using one-way ANOVA. Changes in data trends over the 10-day storage period were analyzed using one-way repeated measures ANOVA. A p-value of <0.05 was considered statistically significant.

1. Results and Discussion

3.1 Antimicrobial activity of postbiotics

Postbiotics derived from both Lactobacillus species demonstrated inhibitory effects against the three tested pathogens, with higher postbiotic concentrations corresponding to stronger antimicrobial activity (Fig. 1).

Fig. 1. Antimicrobial activity of postbiotics against L. monocytogenes evaluated by the agar-well diffusion method.

The greatest inhibition was observed with the postbiotic from L. plantarum at a 20% concentration against L. monocytogenes, producing an inhibition zone of 30.67 ± 0.57 mm. In contrast, the weakest inhibition was observed with the postbiotic from L. casei at a 5% concentration against S. aureus, yielding an inhibition zone of 8.63 ± 0.55 mm. Across all concentrations, the postbiotic of L. plantarum exhibited significantly stronger inhibitory activity against L. monocytogenes and S. aureus compared to that of L. casei (p < 0.05) (Tables 2–4). For E. coli, no significant differences were detected between the two postbiotics except at the 10% concentration (Table 3). These findings suggest that postbiotics from L. plantarum are more effective against Gram-positive pathogens than those from L. casei.

Overall, the results indicate that L. plantarum postbiotics exert stronger antimicrobial effects against Gram-positive bacteria, with the most consistent reductions achieved through the “CFS only” treatment rather than the CFS–WPC combination. The maximum reduction compared to control (~0.80 log CFU/g for L. monocytogenes at day 2) declined over subsequent storage days, while pH, moisture, and sensory acceptability remained unaffected. This strain- and target-dependent pattern is consistent with previous evidence showing that LAB-derived cell-free supernatants inhibit Gram-positive pathogens primarily through organic acids and bacteriocin-like metabolites, mechanisms that involve pH reduction and disruption of microbial membranes [4,6,9,28–30]. Arena et al. also reported strong anti-pathogen activity of L. plantarum supernatants, with acidification identified as a major contributing factor [31,32]. The reduced inhibitory effect observed when CFS was incorporated into a whey protein carrier is consistent with the well-documented “matrix effects” described in the literature. Previous studies have shown that interactions between proteins and bioactive metabolites, along with the barrier properties of protein films, can delay the release and reduce the bioavailability of antimicrobial compounds [11,26,33–36]. Similar patterns of initial but transient inhibition, followed by partial recovery of pathogen populations, have been reported in fresh cheese, meat, and fish products treated with CFS- or bacteriocin-based films [15–17,19].

In agreement with these findings, the present study demonstrated that chemical attributes (pH and moisture) and sensory acceptance were not adversely affected, supporting the feasibility of integrating postbiotics into dairy preservation systems. However, further optimization of carrier composition and release kinetics is required to maximize antimicrobial effectiveness [37].

The MIC and MBC values of L. plantarum postbiotics against the three tested pathogens were determined as 31.25 mg/mL and 62.5 mg/mL, respectively, for Gram-positive bacteria, and 125 mg/mL for E. coli. These results highlight a greater inhibitory effect against Gram-positive bacteria at lower concentrations (Table 5).

The postbiotic of L. casei exhibited a comparable inhibitory effect against L. monocytogenes to that of L. plantarum, except at the 5% concentration, where a difference was observed in comparison with E. coli (Table 6). No significant differences were detected in the inhibition of S. aureus and E. coli (p > 0.05) (Table 7).

The MIC of L. casei postbiotics against L. monocytogenes was higher (62.5 mg/mL) than that observed for L. plantarum, although both species showed identical MBC values. For E. coli, the MBC of L. casei postbiotics was lower than that of L. plantarum. In contrast, for S. aureus, the MIC and MBC values were the same for both postbiotics.

Arrioja et al. (2020) similarly reported that CFS derived from L. plantarum exhibited stronger inhibitory activity against most pathogens compared with CFS from L. casei [33]. Arena et al. (2016) further demonstrated variability in inhibition zones and MICs among different L. plantarum strains against various pathogens, with generally greater effects observed against Gram-positive bacteria [31]. Consistent with the present findings, Tenea and Barrigas (2018) showed that bacteriocin-containing supernatants from L. plantarum (Cys5-4) exhibited variable inhibitory effects against two E. coli strains [7]. Koohestani et al. (2018) also reported that CFS from L. casei 431 produced an inhibition zone of 13 mm against S. aureus, which closely aligns with the current results [23]. In contrast, Yordshahi et al. (2020) documented smaller inhibition zones for L. plantarum postbiotics against L. monocyte-genes compared with those observed in this study [21].

The antimicrobial activity of lactic acid bacteria has been attributed to a range of metabolites, including organic acids, polyamines, proteases, and bacteriocins. The effectiveness of postbiotics depends on multiple factors such as bacterial strain, metabolite composition and concentration, preparation method, and pathogen type. Numerous studies have confirmed that Gram-positive bacteria are generally more susceptible to the antagonistic compounds in postbiotics than Gram-negative bacteria, consistent with the findings of the present work [38].

Results from the agar-well diffusion assays indicated that postbiotics incorporated into WPC coating solutions at MBC concentrations generally exhibited reduced inhibition against L. monocytogenes compared to postbiotics applied alone (Table 8). However, the combination of L. plantarum postbiotics with WPC coatings produced greater inhibition than L. casei postbiotics at equivalent concentrations.

Based on the in-vitro assays, the L. plantarum postbiotic at twice the MBC concentration was identified as the most effective formulation and was subsequently selected for testing in the food model.

3.2 Identification of chemical compounds of extracted CFS

The chemical compounds identified in the postbiotics derived from L. plantarum and L. casei are presented in their respective chromatograms (Figs. 2 and 3).

Sezen Özcelik et al. (2016) reported that LAB strains are particularly efficient producers of succinic acid, especially when cultivated in MRS broth. Succinic acid serves as a key intermediate in the Krebs cycle and a common fermentation byproduct, reflecting the strong metabolic capacity of LABs. The quantity and composition of organic acids produced by LABs vary considerably across strains and culture media, with pH and temperature exerting significant influence [28]. Similarly, Iqbal Hossain et al. (2021) identified nine distinct organic acids, including succinic acid, in several LAB strains such as L. plantarum, highlighting the diverse metabolite production potential of these bacteria [29]. In the present study, the derivatization technique applied proved particularly effective in detecting compounds such as esters and alkanes.

Shehata et al. demonstrated that LABs synthesize antifungal metabolites that differ across strains, with organic acids and hydrogen peroxide serving as the primary contributors to antifungal activity. Their study identified compounds such as pentadecane and 2,4-di-tert-butylphenol, both known to inhibit foodborne pathogens and fungi. Additionally, antimicrobial compounds including 6-octadecenoic acid methyl ester and hexadecanoic acid methyl ester—also detected in the current work—were previously reported. Deepthi et al. further showed that LABs generate a wide variety of antifungal carboxylic acid esters, whose effectiveness depends on strain variability and fatty acid chain length. For example, 10-octadecenoic acid methyl ester, an unsaturated fatty acid ester with 19 carbon atoms, has been described as an effective antifungal metabolite. In agreement with Shehata et al., our findings also identified nonadecane, a compound without known antimicrobial activity [39,40].

Benzoic acid was detected in small quantities in both postbiotics. This compound, obtained from L. plantarum, has previously been reported by Siedler et al. as possessing antimicrobial properties [41].

In addition, several compounds not previously described for their antimicrobial effects were identified in the chemical analysis of the postbiotics, including phthalic acid, eicosane, chloroacetic acid, benzene propanoic acid, propenoic acid, triethyl citrate, isopropyl myristate, butanedioic acid, and tetradecanoic acid.

3.3 Microbiological analyses

The growth of L. monocytogenes was monitored in the control samples (treatment one), which demonstrated a continuous increase throughout 10 days of storage at 4 °C, with an overall rise of 1.1 log CFU/g (Fig. 4).

Fig. 4. Growth of L. monocytogenes across four food treatments during 10 days of storage at 4 °C.

A significant reduction in pathogen counts was observed in the second treatment, which contained CFS, compared with the other three treatments over the 10-day storage period (p < 0.05). In contrast, no significant differences were detected in contamination levels between the third and fourth treatments and the control (p > 0.05). The largest reduction in L. monocytogenes relative to the control was 0.80 log CFU/g, recorded in the second treatment on day two; however, this difference gradually declined over the storage period (Table 9).

The results for total microbial counts (Fig. 5) and mold and yeast counts (Fig. 6) mirrored those observed for L. monocytogenes, with only the second treatment showing a significant reduction (p < 0.05) compared with the other treatments over the 10-day storage period. Fig. 5. Total microbial counts across four food treatments during 10 days of storage at 4 °C. Fig. 6. Mold and yeast count across four food treatments during 10 days of storage at 4 °C.

These findings are consistent with the study by Iqbal Hossain et al. (2021), which reported differences in the inhibitory effect (MEC) of CFS derived from L. plantarum when tested in a culture medium (TSB) compared with a food model (whole milk) against L. monocytogenes [29]. Similarly, Hartmann et al. (2011) demonstrated that the efficiency of bacterial fermentations varied depending on the food matrix, with bacteriocin IDE0105 from L. plantarum showing weaker activity in milk than in BHI medium. Such discrepancies may be explained by limited solubility or uneven distribution of antimicrobial compounds in food systems, inactivation of antagonistic chemicals by food components or microflora-derived enzymes, or interactions between antimicrobials and food ingredients [34].

Comparable to the present study, Jutinico-Shubach et al. (2020) reported a 0.83 log CFU/g reduction in L. monocytogenes in cheese samples treated with CFS from Pediococcus pentosaceus 147 on day two of storage compared with the control. This closely aligns with the 0.80 log CFU/g reduction observed in our second treatment at the same time point [17]. Similarly, Salvucci et al. (2019) found reductions in L. monocytogenes in cheese treated with films containing bacteriocins from Enterococcus faecium ES216 [18]. Marques et al. (2017) also observed that L. monocytogenes counts in actively packaged cheese samples treated with CFS from Latilactobacillus curvatus P99 reached levels comparable to the control by day 10. Moreover, films containing CFS at MIC concentrations (15.6 µL/mL) did not significantly reduce bacterial counts compared with controls, consistent with our findings [26]. Yordshahi et al. (2020) likewise reported the survival of L. monocytogenes in all meat samples treated or untreated with active packaging containing postbiotics from L. plantarum (ATCC 14917), with a 1.0 log CFU/g increase in control samples by day nine—similar to the 1.1 log CFU/g increase observed in our study [21].

The type of film or coating, as well as the nature of the food, plays a critical role in the release of antimicrobial agents from polymer matrices into food systems [21]. Beristain-Bauza et al. (2017) demonstrated that untreated beef samples exhibited a modest reduction (<0.5 log CFU/g) in L. monocytogenes Scott A growth after five days, whereas beef treated with WPI films containing CFS from Latilactobacillus sakei (NRRL B-1917) showed a reduction of 1.4 log CFU/g after the same period [19]. Such variability highlights the dependence of antimicrobial efficacy on factors including film or coating type, bacterial strain, food model, and release kinetics of antagonistic compounds.

The study by Beristain-Bauza et al. (2016) suggested that the stronger antimicrobial performance of WPI films may be attributed to the hydrophobic nature of whey proteins, which facilitates compatibility with hydrophilic CFS obtained from Lacticaseibacillus rhamnosus (NRRL B-442). This mechanism may help explain the diminished inhibitory effect observed in the present study when CFS was combined with WPC coatings [38].

3.4 Chemical analyses

The pH values of cheese samples from the different treatments showed no significant differences (p > 0.05), with levels ranging from 6.50 to 6.56. This observation is consistent with Jutinico-Shubach et al. (2020), who reported comparable pH values in both control and CFS-treated cheese samples derived from Pediococcus pentosaceus 147 [17]. Similarly, Salvucci et al. (2019) found no significant differences in moisture content between control and treated cheese samples during storage, further corroborating the findings of the present study [18].

3.5 Sensory analyses

Sensory evaluation revealed no significant differences (p > 0.05) in overall acceptability or odor index between the control treatment and the WPC–CFS combination treatment (Fig. 7).

No significant differences were detected between the two CFS treatments and the WPC–CFS combination. However, a significant difference (p < 0.05) was observed in the color parameter of the control treatment compared with the other treatments, likely attributable to the MRS culture medium used as the primary substrate for LAB growth and CFS production (Table 10). This finding aligns with Beristain-Bauza et al. (2016), who reported pronounced color differences in WPI films containing CFS from Lacticaseibacillus rhamnosus (NRRL B-442) [37].

In contrast, no significant differences were detected in texture or taste across treatments (p > 0.05), indicating good sensory acceptance of the treated samples. These results are in line with those of Beristain-Bauza et al. (2017), who observed favorable sensory evaluations in grilled beef treated with WPI films containing CFS from Latilactobacillus sakei (NRRL B-1917) [19].

1. Conclusion

This study demonstrated that postbiotics derived from L. plantarum and L. casei possess significant antimicrobial activity against major foodborne pathogens when applied as coatings in pasteurized cheese. Among the two, L. plantarum exhibited stronger inhibitory effects, particularly against Gram-positive bacteria. However, incorporation of postbiotics into WPC-based coatings reduced their antimicrobial efficacy, likely due to matrix interactions. Importantly, postbiotic treatments successfully suppressed microbial growth during refrigerated storage without affecting the key chemical characteristics of cheese and were well accepted in sensory evaluations.

These findings underscore the potential of postbiotic-based coatings as natural, consumer-friendly alternatives to chemical preservatives in dairy products. Beyond cheese, this strategy could be extended to a broad range of perishable foods where safety, shelf life, and sensory acceptance are of critical importance. Nevertheless, further research is required to optimize carrier systems, enhance stability, and evaluate large-scale industrial applications across diverse food matrices.

1. Acknowledgements

The authors would like to thank Mashhad University of Medical Sciences, Mashhad, Iran, and Gonabad University of Medical Sciences, Gonabad, Iran for their support.

1. Declaration of competing interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

1. Authors’ Contributions

Zeinab Hadadfar: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Software; Supervision; Visualization; Writing – original draft. Asma Afshari: Conceptualization; Investigation; Methodology; Resources; Supervision; Validation; Writing – review and editing. Alireza Mohammadzadeh: Investigation; Methodology; Resources; Supervision; Validation; Writing – review and editing. Zohreh Abdimoghadam: Funding acquisition; Investigation; Project administration; Resources; Supervision; Validation; Writing – review and editing.

Funding: This work was supported by the Gonabad University of Medical Sciences [grant numbers 1130].

1. Using Artificial Intelligent Chatbots

The authors declare no artificial intelligent chatbot use.

1. Ethical Consideration

The study protocol was reviewed and approved by the Medical Ethics Committee of Gonabad University of Medical Sciences (IR.GMU.REC.1401.073).

Background and Objective: The demand for cost-effective and thermostable α-amylases for industrial applications has driven the research to discover new microbial sources. This research aimed to isolate and characterize α-amylase-producing bacteria from rice milling wastes and employ Response Surface Methodology (RSM) to improve enzyme production.

Material and Methods: Bacterial samples were collected from different agro-industrial wastes and primarily screened using Lugol's iodine method. Secondary isolation was performed by α-amylase activity assessment using the DNS assay. Enzyme production was optimized by RSM, with the temperature, pH, and starch concentration as key variables. In addition, the effect of different pH and temperatures was assessed on the α-amylase activity. 16S rRNA sequencing and phylogenetic analysis were used for bacterial identification.

Results and Conclusion: The isolate was identified as Bacillus subtilis NllST B 627. Optimum conditions for maximum enzyme production (0.21 Umg^-1) were starch 5.5 gL^-1, temperature 40°C, and pH 7. Temperature was the most significant factor influencing enzyme production, whereas pH and starch concentration showed weaker effects but potentially relevant interactions. The overall model based on response surface curves was statistically significant, indicating that the combination of independent variables significantly influences enzyme production. The enzyme exhibited maximum activity at pH 7, while the lowest activity was observed at pH 5. Also, the enzyme's optimal activity occurred at 40°C, while the lowest catalysis was detected at 60°C. The identified strain exhibits promising properties for application in starch hydrolysis and other industrial purposes. This highlights the potential of rice mill wastes as a sustainable and low-cost resource for microbial enzyme production, and this study is the first to explore Guilan rice mill wastes for α-amylase production.

Keywords: α-Αmylase production, Detergent industry, Enzyme optimization, Isolation and identification, RSM Method

Introduction

Producing enzymes on an industrial scale is an expensive effort. A potential approach to overcome this issue involves identifying enzyme-producing microorganisms from agricultural/industrial waste, a method that both lowers production costs and promotes waste beneficial reuse and environmental sustainability [1,2]. So, rice milling wastes are an abundant by-product found in areas where rice is cultivated and constitute a largely available material full of microbial diversity. The moist and starch-rich remnants produced during rice processing create a perfect environment for the growth of α-amylase-producing bacteria, which have potential uses in various industries [3].

α-Αmylases (EC 3.2.1.1) are extensively utilized in the food, fermentation, detergent, pharmaceutical industries, ethanol production [4], and as an antibiofilm agent [5]. These enzymes cleave α-1,4-glycosidic bonds in starch, facilitating the production of various products such as dextrose, glucose, and starch syrups [6]. Although many thermostable α-amylase-producing strains, such as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus stearothermo-philus, Bacillus licheniformis, Bacillus polymyxa, and Bacillus coagulans have been identified so far [7], discovering new strains from novel sources could result in enzymes operating in extreme conditions. This leads to increasing efficiency and reducing costs on an industrial scale [8].

Globally, α-amylases account for approximately 23-33% of the enzymatic market share [9]. However, the cost of industrial-scale enzyme production remains significantly high. It is estimated that microbial culture media formulation alone accounts for about 30-40% of the final enzyme production costs [10]. Consequently, there is a persistent need to reduce these costs by developing inexpensive culture media formulations, leading to significant efforts to identify cost-effective alternatives for industrial enzyme production.

Various studies have been conducted on the identification and isolation of α-amylase-producing strains from different sources, including agricultural soils such as potato fields [11], industrial soils such as brick kiln [12], hot spring [13], deserts [14], different wastes [15,16], etc. For example, Niyomukiza et al. isolated amylolytic bacteria from starchy food wastes (maize meal and potato peel wastes), and 16S rRNA sequencing verified them as Bacillus subtilis. The optimum temperature for the enzyme was 60 °C, and the pH was 9 [17]. Tripathi et al. used Bacillus polymyxa NCIM 2539 to produce amylase using agro-industrial byproducts. Among various substrates tested, orange peel yielded the highest enzyme activity. Supplementation of the medium significantly enhanced amylase production, with optimal levels obtained at specific concentrations of orange peel, cysteine, and thiamine [18].  

Many industries produce agricultural waste, one of which is the rice factory industry located in the North of Iran. They produce waste with high organic matter content, one of which is starch. The starch contained in the liquid waste can be broken down by bacteria-producing amylase enzymes into simpler molecules. In this research, there has been scarce focus on the microbial strains that exist within the starch-rich waste produced by rice mills, especially in Guilan province (Iran), where rice farming is a significant agricultural profession. This research aimed to isolate and molecularly characterize α-amylase-producing bacteria from rice milling waste and employ Response Surface Methodology (RSM) to improve enzyme production processes. Optimization of three parameters (incubation time, starch concentration, and incubation temperature) was investigated. Using an inexpensive, starch-rich waste substrate and statistical modeling to enhance enzyme output, this study could contribute to cost-effective enzyme development and play a significant role in sustainable industrial waste management.

1. Materials and Methods

2.1. Primary isolation and screening of α-amylase-producing bacteria

Bacterial samples were collected from rice mill wastes, chip manufacturing wastes, bakery dusts, textile wastewater, and other industrial waste, because these sources contain a significant percentage of starch and are likely to contain more degrading bacteria. For isolating spore-forming bacteria, particularly Bacillus species, serial dilutions were prepared. Heat shock treatment was applied for 10 minutes at 80°C in a water bath. Subsequently, bacteria were isolated using the streak plate method on Nutrient Agar medium [19]. The isolated bacteria were then tested for starch hydrolysis by inoculating them onto the starch agar plates containing 2% starch and incubating for 24 hours. After incubation, the ability to hydrolyze starch was assessed by flooding the plates with Lugol's iodine solution. Bacteria showing a clear zone around colonies, indicative of starch hydrolysis, were selected for further analysis.

2.2. Secondary screening by α-amylase activity assessment

In the secondary screening phase, bacterial isolates that produced larger clear zones were selected for further enzymatic activity assessment. The α-amylase activity was measured in 250 mL flasks containing 50 mL of cultivation medium with the following composition (gL^-1, Sigma-Aldrich, USA): starch 10%, peptone 5%, yeast extract 2.05%, NaCl 1.5 grL^-1, KH₂PO₄ 0.5 gr/L, MgSO₄ 0.5 grL^-1, CaCl₂ 0.1 grL^-1, and glycerol 15% (vv^-1). The flasks were incubated on a shaker at 120 rpm and 37°C for 48 hours. The initial pH of the medium was adjusted to 7.0. The inoculated bacterial strains were transferred into sterile pre-prepared medium. After incubation, samples were centrifuged at 10,000 rpm for 20 minutes, and the supernatant was collected for enzyme activity measurement.

2.3. Enzyme activity determination using the DNS method

The dinitrosalicylic Acid (DNS) method was employed to quantify α-amylase activity [20]. DNS is an alkaline reagent that reacts with reducing sugars, causing a color change from yellow to reddish-brown. A reaction mixture comprising 0.5 mL of crude enzyme and 0.5 mL of 1% starch solution was prepared. The mixture was incubated at 37°C for 30 minutes. The reaction was stopped by adding 1 mL of DNS reagent, followed by boiling for 5 minutes. Glucose concentration was measured using a spectrophoto-meter at 540 nm. A glucose standard curve was generated by plotting absorbance at 540 nm against the amounts of glucose released to define the concentration of glucose formed in each solution. One unit of enzyme activity was defined as the amount of enzyme required to liberate 1 μmol of reducing sugars per minute.

2.4. Response surface methodology for production optimization

The three strains exhibiting the highest production levels were selected from the screened bacterial isolates, and strain 2 was employed for statistical production optimization. RSM is a technique used to evaluate the influence of input parameters on responses [21]. Central Composite Design (CCD) provides an efficient approach to predict the interaction effects of influential factors on the process. In this study, optimization aimed to maximize enzyme production, using the Statistical Design-Expert 7.0 software, followed by CCD analysis [22].

In this design, the quantitative impact of the most effective variables, including starch concentration, temperature, and pH, were examined (Table 1). All experiments were conducted in triplicate. Positive and negative control strains were processed with every batch.

2.5. Evaluation of pH on the α-amylase activity

The bacteria were cultivated in production media and incubated in a shaker incubator for 24 hours, followed by centrifugation. In separate tubes, 0.5 mL of the supernatant was mixed with 0.5 mL of sodium carbonate buffers at 50 mM with pH values of 9 and 10, trisodium citrate buffers at 50 mM with pH 5 and 6, disodium hydrogen phosphate buffers at 50 mM with pH 7 and 8, and starch. The mixtures were incubated at 37°C for 30 minutes. Subsequently, 0.5 mL of DNS reagent was added, and absorbance was measured at 540 nm using a spectrophotometer.

2.6. Evaluation of temperature on the α-amylase activity

To evaluate the effect of temperature, bacteria were cultured in the production medium, centrifuged, and then 0.5 mL of the supernatant was mixed with 0.5 mL of 1% starch solution in a test tube. The test tubes were then placed in a water bath or thermostatic water bath set at temperatures of 40°, 50°, 60°, and 70°C for 30 minutes. The enzyme activity under each condition was subsequently assessed.

• Identification of α-amylase-producing bacteria

α-Amylase-producing bacteria were identified through phenotypic and macroscopic characterization, followed by microscopic examination using Gram staining and sporulation tests. For definitive identification, 16S rRNA gene analysis was conducted. Bacterial DNA was extracted using a boiling method. Briefly, the bacterial cultures were centrifuged at 2000 g for 20 minutes to obtain a cell pellet. The pellet was resuspended in sterile distilled water, and the microtubes were first placed in a freezer and then boiled in a water bath to lyse the bacterial cells and release the DNA. After centrifugation at 7000 rpm for 10 minutes, the supernatant containing the DNA was collected and precipitated with cold ethanol. The DNA pellet was air-dried and dissolved in a small volume of distilled water, and its concentration and purity were measured using a NanoDrop spectrophotometer. PCR amplification was performed with universal bacterial primers 27F (5′- AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-CGGTTACCTTGTTACGACTT-3′) [23]. The PCR protocol consisted of 30 cycles involving: denaturation at 95°C for 1 minute, annealing at 56°C for 1 minute, extension at 72°C for 1 minute, and a final elongation step at 72°C for 7 minutes. The PCR products were analyzed by gel electrophoresis, and successful amplicons were sent to Pishgaman Co. (Iran, Tehran) for sequencing. The obtained sequences were compared to those in the NCBI database using the BLAST tool (https://blast.ncbi.nlm.nih-gov/Blast.cgi) [24]. The closest related species were identified based on the similarities in the 16S rRNA gene sequences of the α-amylase. For phylogenetic analysis, the sequences were searched and aligned in the NCBI GenBank database using multiple sequence alignment with the ClustalW algorithm (https://www.genome.jp/tools-bin/clustalw). A phylogeny-etic tree was constructed using the Molecular Evolution Genetic Analysis (MEGA 4) software with the Neighbor-Joining algorithm and 1000 bootstrap replicates [25].

• Statistical analysis

Data were analyzed using the Design Expert 7.0 software. All the experiments were conducted in triplicate. Results are presented as mean values ± standard error (SE), with a significance threshold of P < 0.05.

1. Results and Discussion

3.1. Isolation and screening of α-amylase-producing bacteria

The Lugol's iodine test was performed for primary isolation of α-amylase-producing bacteria from different waste samples. Results demonstrated that three bacterial isolates, including strain #1 from the bakery's waste, strain #2 from the rice mill's waste, and strain #3 from the agricultural waste, produced extracellular α-amylase, positively (Figure 1 A to C), as evidenced by the presence of a clear zone surrounding the colonies after washing with iodine solution. The formation of a clear zone indicates that the starch present in the starch agar medium was hydrolyzed into monomers by α-amylase enzymes produced by the bacteria. In contrast, a dark blue-black zone represents an iodine-starch complex. The isolates showing clear zones were considered positive for α-amylase production (Figure 1 A to C). In contrast, the absence of a clear zone indicated a lack of extracellular α-amylase activity (Figure 1D).

3.2. Assessment of bacterial α-amylase activity

α-Amylase activity of the three selected samples was assessed for further screening of bacteria. Results revealed that the α-amylase activity of strain #2, from the rice mill waste, was significantly higher than that of others (P < 0.05) (Figure 2).

3.3. Experimental design

In this design, based on RSM and using the CCD method, 15 experimental runs were arranged, as summarized in Table 2. In this research, based on a multiple regression analysis of experimental data, the final model was presented as follows (Equation 1):

α-Amylase activity = 0.20 - 0.025A - 0.055B +0.020C + 0.030AB - 0.025AC - -5.000E-003BC       - 0.046A² - 0.076B² - 0.031C²

Where A, B, and C are pH, starch concentration, and temperature, respectively.

The analysis of variance (ANOVA) is described in Tables 3 and 4. The larger the F-value, the greater the variation between sample means relative to the variation within the samples. The p-value is the probability of obtaining an F-ratio as large or larger than the one observed, assuming that there is no difference between the group averages. The model F value of 51.62 implies that the model is significant. Values of Prob > F less than 0.05 imply that the model terms are relevant at the 95% confidence level. The model showed a high determination coefficient (R² = 0.9894), indicating a strong correlation between the experimental and predicted values.

Figure 3 shows the normal plots of residual (difference between the observed and predicted value). A low residual value is necessary for a good mathematical model fitted on observed data. The predicted responses and observed responses are shown in Figure 4 and the data points located close to the diagonal line, suggesting a satisfactory correlation.

To study the interaction among the different independent variables and their corresponding effect on the response, contour plots and 3-D plots were drawn. A contour plot is a graphical representation of a three-dimensional response surface based on two independent variables, helping to illustrate their main and interaction effects on the response. Figure 5 shows the amylase response and correlation between variables in plots. Optimum conditions for maximum enzyme production (0.21 Umg^-1) were starch 5.5 gL^-1, temperature 40°C, and pH 7.

The bell-shaped surface indicates that extreme acidic or alkaline conditions and temperatures above 45 °C negatively influence production, likely due to reduced microbial growth or enzyme instability. The nearly symmetrical surface curvature also suggests that the process remains relatively stable near the optimal point.

In Fig. 5b, the interaction between temperature and starch concentration shows that enzyme yield rose with increasing starch up to about 5–6 gL^-1, but declined at higher levels, possibly because of substrate inhibition or catabolized repression. Maximum activity was achieved at a moderate starch concentration and 40 °C. Together, these plots confirm that temperature is the dominant factor, while pH and substrate concentration contribute secondary but interactive effects, defining a narrow yet stable region for optimal amylase production. According to the results of 15 experiments, the lowest activity was observed at high temperature, low starch concentration, and under alkaline pH. This suggests excessive heat or inappropriate substrate levels may negatively impact enzyme production. These data underline that interactions between parameters are critical in optimizing enzyme yield, and single-factor optimization may be insufficient. Temperature was identified as the most significant factor influencing enzyme production, whereas pH and starch concentration showed weaker individual effects but potentially relevant interactions. When compared with similar studies, some differences in optimal conditions were observed. In the work performed by Adetiloye et al., Bacillus cereus from a warm spring demonstrated a slightly higher optimal temperature at 45°C and an RSM-predicted optimal pH of 7, although OFAT analysis also indicated potent activity at pH 8 [23]. In the study performed by Sharif et al., Bacillus licheniformis exhibited an even higher optimal temperature at 55°C and a more alkaline preference at pH 9. This suggests that variations in enzyme thermostability and pH tolerance may depend on the species [26].

Regarding substrate concentration, both the current study and the work by Adetiloye et al. [23] identified 5% starch as optimal, whereas Sharif et al. reported an optimum of 1%  [26], possibly due to substrate inhibition effects at higher concentrations. The differences in assay methods (µmolmin^-1 vs. UmL^-1) and strain-specific enzyme kinetics make direct comparison of activity values difficult; however, both Adetiloye et al. and Sharif et al. reported higher numerical activities than the current study, which could be related to strain genetics, cultivation conditions, or methodological variations in activity measurement. The emphasis is on identifying robust operating conditions within the region of interest that maximize production while remaining practically feasible in downstream processing.

3.4. The effect of pH on the α-amylase activity

As illustrated in Figure 6A, the enzyme exhibited maximum activity at pH 8, while the lowest activity was observed at pH 5 (P < 0.05). It can be concluded that the enzyme operates most efficiently under slightly alkaline conditions, which may be due to the stabilization of the enzyme’s active site and overall tertiary structure at this pH, which enhances its catalytic efficiency. Conversely, the lowest enzymatic activity at pH 5 suggests that acidic conditions lead to reduced enzyme performance, likely as a result of denaturation or alteration in the ionization state of critical amino acids involved in substrate binding and catalysis [27]. A moderate reduction in the activity at pH 9 indicates a narrow optimal pH range for the enzyme. These findings emphasize the importance of pH optimization in industrial applications of this enzyme. These findings are consistent with previously reported characteristics of bacterial α-amylases, which generally prefer neutral to slightly alkaline environments for optimal activity [14,22,28,29].

3.5. The effect of temperature on the α-amylase activity

The impact of temperature on the catalytic activity of α-amylase from Isolate 2 is presented in Figure 6B. The enzyme demonstrated optimal activity at 50°C, while the lowest enzyme activity was detected at 60°C (P < 0.05). The enzyme may retain its functional conformation at moderately high temperatures. This property makes the enzyme potentially suitable for industrial processes that require elevated temperatures, such as starch liquefaction and food processing [30]. Reduction of catalytic activity at 60°C may be due to thermal denaturation or irreversible structural changes, leading to loss of function [27]. Overall, the results confirm that temperature plays a critical role in the amylolysis function, and identifying the optimum point is crucial for maximizing enzymatic yield.

Various studies have reported varying optimal temperatures and pHs for the α-amylase activity of Bacillus species isolated from different sources. Some of them are as follows: The highest α-amylase activity of the purified Bacillus licheniformis strain LB04 isolated from Espinazo hot springs in Mexico was at pH 3 and 65 ºC [31]. Bacillus licheniformis HULUB1 and Bacillus subtilis SUNGB2 isolated from Malaysian hot spring showed the highest α-amylase activity at 65° C and pH 6.0 [13]. Bacillus cereus and Bacillus licheniformis isolated from the potato fields demonstrated α-amylase function at pH 8.0 and temperatures of 45°C and 65°C, respectively [11]. Maximum α-amylase activity of the three isolates of Bacillus from a hot place in Ethiopia occurred at 75°C, 70°C, and 65°C, and the highest activity was at pH 8 [14]. The highest α-amylase activity of B. subtilis and B. licheniformis isolated from different soil samples in Punjab (Pakistan) was recorded at 70°C and pH 9 [22].

3.6. Molecular identification and phylogenetic analysis of the strain

The 16S rRNA gene was employed for the bacterial isolate molecular identification. Genomic DNA was extracted and subsequently used as a template for PCR amplification. The amplified product was then subjected to DNA sequencing, and the resulting sequence was analyzed using the BLASTn tool (NCBI) to compare it with known bacterial sequences. The isolate showed 100% sequence similarity with Bacillus subtilis, indicating that the strain belongs to this species. More specifically, the sequence demonstrated the highest similarity to Bacillus subtilis strain NllST B 627, with no nucleotide mismatches observed, further confirming the identity of the isolate.

To validate the BLAST results and investigate the strain evolutionary relationship, a phylogenetic tree was constructed using the neighbor-joining method based on 16S rRNA sequences of closely related strains (Figure 7). The tree showed that the isolate clustered tightly with Bacillus subtilis strains, particularly with strain NllST B 627, further supporting the identification. The accurate identification of the strain is crucial for further biotechnological applications and strain improvement strategies.

Although this study successfully identified and optimized α-amylase production from Bacillus subtilis, several limitations should be mentioned. Enzyme purification was not performed by chromatographic methods. The thermal and pH stability of the enzyme were not assessed, which are critical parameters for its industrial applicability. No direct comparison was made with commercially available or industrial reference strains. The effects of metal ions, inhibitors, and activators on enzyme activity were not evaluated, which could provide valuable insight into enzyme regulation and potential process enhancements. Future studies should focus on a comprehensive characterization of the enzyme, including stability profiling, kinetic analysis, and evaluation under various physicochemical and chemical conditions.

1. Conclusion

This study successfully isolated and characterized a moderate thermostable alkaline α-amylase-producing Bacillus subtilis strain from rice mill waste and optimized its enzyme production using RSM. Maximum enzyme production (0.21 Umg^-1) was acheived with starch 5.5 gL^-1, temperature 40°C, and pH 7.  The enzyme displayed optimal activity at a pH of 8 and 50°C. Temperature exerted the most significant effect on enzyme production. These findings emphasize the possibility of employing agricultural by-products such as rice mill waste for low-cost microbial enzyme production while contributing to sustainable waste management. Although further purification, stability testing, and scale-up studies are needed, the identified strain represents a promising candidate for applications in the detergent, food, and biotechnological industries. Coupled isolation from an environmental waste source with statistical optimization (RSM/CCD) to achieve a robust production process, illustrating a streamlined path from waste to biocatalyst. Evaluation of production and recovery in pilot-scale bioreactors, including downstream processing feasibility and cost analysis will be suggested.

1. Acknowledgements

We thank the research core of Yazd University for the financial support during the course of this project.

1. Declaration of competing interest

The authors declare no conflict of interests.

1. Authors’ Contributions

Conceptualization: Mirbagheri and Chamani; investigation: Alkoozei, methodology: Mirbagheri and Khatami; Data curation: Mirbagheri; writing: Chamani and Mirbagheri; review and editing: Khatami; Supervision: Mirbagheri and Chamani.

1. Using Artificial Intelligent Chatbots

The authors declare that Artificial Intelligence (AI)-assisted tools were used solely for language editing, grammar checking, and improving readability of the manuscript. All scientific content and writing were generated by the authors themselves, who take full responsibility for the integrity and accuracy of the work.

1. Ethical Consideration

This study focused on bacteria and did not involve any human or animal subjects; no human data or samples were used. 

Background and Objective: The growing demand for natural preservatives in the food industry has driven research into plant-derived essential oils. This study focused on optimizing the extraction of essential oil from Damask rose (Rosa damascena Mill.) and developing a stable oil-in-water nanoemulsion for potential food applications.

Material and Methods: Essential oil was extracted from dried rose buds, with pretreatment methods including grinding, soaking, and ultrasonication evaluated to maximize yield. The optimal method involved using crushed, pre-soaked buds, which significantly improved extraction efficiency. The chemical profile of the essential oil was analyzed by Gas Chromatography-Mass Spectrometry (GC-MS), revealing a complex mixture of bioactive compounds, with heneicosane, citronellol, and other alkanes as major constituents. An oil-in-water nanoemulsion was then formulated using the extracted oil, deionized water, and Tween 80 as a surfactant, prepared via a high-energy ultrasonic homogenization method. The nanoemulsion was characterized using Dynamic Light Scattering (DLS), Zeta Potential analysis, SEM, TEM and Fourier-Transform Infrared Spectroscopy (FTIR).

Results and Conclusion: Characterization by DLS(11.49nm) revealed a highly polydisperse nanoemulsion (PDI = 0.724) with an intensity-weighted average diameter (Z-Average) of 505.6 nm and a zeta potential of -5.10 mV, indicating the presence of small nanoparticles alongside larger aggregates. FTIR analysis confirmed the successful encapsulation of the essential oil within the nanoemulsion structure. Furthermore, the essential oil demonstrated antimicrobial activity against E. coli and S. aureus, with a Minimum Inhibitory Concentration (MIC) of 15000 ppm. These findings suggest that R. damascena nanoemulsion is a promising natural antimicrobial agent for food preservation.

Keywords: Essential Oil,  Natural Preservative, Nanoemulsion, Rosa damascene, Steam distillation

1. Introduction

Rosa damascena Mill, commonly known as the Damask rose, is a fragrant species of the Rosaceae family, renowned for its use in the perfume, cosmetic, and food industries. The essential oil extracted from its petals is one of the most valuable essential oils in the global market due to its complex aroma and therapeutic properties. Iran is a leading producer of Damask rose, with a long history of cultivating this plant for rose water and essential oil production. The oil is rich in a variety of phytochemicals, including citronellol, geraniol, nonadecane, and heneicosane, which contribute to its characteristic fragrance and biological activities [1, 2]. These activities include antioxidant, antimicrobial, and anti-inflammatory effects, making it a valuable natural ingredient‌ [3, 4].

Despite their benefits, the direct application of essential oils in food systems is often limited by their poor water solubility, high volatility, and susceptibility to degradation from environmental factors like light, oxygen, and heat. These limitations can reduce their efficacy and impact the sensory properties of the final product [5]. To overcome these challenges, nanoencapsulation technologies have emerged as a promising approach. Nanoemulsions, which are colloidal dispersions of oil droplets in an aqueous phase with droplet sizes typically below 200 nm, offer a solution by enhancing the stability, solubility, and bioavailability of lipophilic compounds [6].

Nanoemulsions provide several advantages for food applications, including optical transparency, high surface area for improved activity, and protection of the encapsulated active compounds from degradation. High-energy methods, such as ultrasonic homogenization, are widely used to produce nanoemulsions by applying intense disruptive forces to break down large oil droplets into nanoscale particles. This technique is efficient, scalable, and suitable for food-grade formulations [7-10].

A significant challenge in the production of high-quality nanoemulsions from plant materials is the efficiency and quality of the initial extraction process. Conventional methods like hydrodistillation, while effective, can be time-consuming and may lead to thermal degradation of sensitive bioactive compounds [11, 12]. Recognizing these limitations, there is a growing need for innovative extraction and formulation systems that can streamline production, improve yield, and better preserve the integrity of the essential oil. In response, our research team has developed a novel, integrated apparatus that combines a homogenization unit and a sonication unit, allowing for the continuous and repeated processing of an oil and aqueous phase dispersion. This patented system is designed not only to produce nanoemulsions with superior stability and smaller particle size but also to serve as a more efficient method for processing plant extracts, as demonstrated by its application in this study [9]. By circulating the mixture between the two units, our method ensures uniform energy distribution and minimizes processing time, overcoming key drawbacks of traditional batch methods. The potential application of R. damascena essential oil  (RDEO) as a natural preservative in food products like dairy desserts is of great interest [13]. Although hydrodistillation and other conventional methods are commonly used to extract R. damascena essential oil, these techniques are often time-consuming and can lead to thermal degradation of heat-sensitive bioactive compounds. Subsequently, formulating the extracted oil into a nanoemulsion is treated as an entirely separate process [9]. This disconnection between extraction and nano-formulation represents a significant gap in the current research. There is a clear need for an innovative, integrated system that can streamline the entire workflow from plant material to a stable final product. An integrated approach could significantly reduce processing time, improve extraction yield, and better preserve the integrity of the essential oil, overcoming key drawbacks of traditional multi-step, batch-based methods.

Therefore, this study was designed not only to optimize the extraction of essential oil from R. damascena but also to address this technological gap by utilizing a novel, patented apparatus that combines homogenization and sonication in a continuous process. The objective was to efficiently formulate and characterize an oil-in-water nanoemulsion and evaluate its physicochemical and antimicrobial properties to assess its potential as a novel natural preservative for food systems. The potential application of R. damascena essential oil (RDEO) as a natural preservative is of great interest, especially in meeting consumer demand for "clean label" alternatives to synthetic additives. Therefore, the primary novelty of this work lies in addressing the limitations of conventional extraction and formulation through the application of a newly developed, patented integrated apparatus that combines homogenization and sonication in a continuous, recirculating system. This study is the first to utilize this streamlined technology for R. damascena to efficiently produce a stable oil-in-water nanoemulsion. The objective is to thoroughly characterize the physicochemical properties (particle size, stability, morphology) and antimicrobial activity of the resulting nanoemulsion to validate its potential as a novel and effective natural preservative for food systems.

1. Materials and Methods

2.1 Plant Material and Chemicals

Dried Damask rose (Rosa damascena Mill.) buds were commercially sourced from a reputable local supplier (Tavazo, Iran). The Rosa damascena petals were sourced from the Layzangan valley in the Darab region of Fars province, a region renowned for its high-quality roses. The plant specimens were authenticated at the Herbarium of the Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran. The quality of the buds was visually inspected to ensure they were free from foreign materials and contamination. Tween 80, used as a non-ionic surfactant, was purchased from Neutron (Iran). All other chemicals and solvents were of analytical grade.

2.2 Preparation of R. damascena Nanoemulsion Using an Integrated Apparatus

First, the essential oil of rosehip, which had previously been pretreated by soaking and ultrasonication, was prepared using steam distillation (U.S. Patent Application No. US 2024/0174419 A1) [9]. The system, depicted in the patent, consists of a homogenization unit fluidly connected to a sonication unit, allowing for continuous recirculation of the dispersion between the two tanks via a pump.

The procedure was conducted based on the methods detailed in the patent. To create a coarse pre-emulsion, 150 mL of the extracted R. damascena essential oil was added to 14.85 L of deionized water in a bioreactor under specific conditions (500 rpm, 60 psi, 45-50°C). This coarse mixture was then transferred to the homogenization tank of the integrated apparatus. The nanoemulsification process was initiated by activating the circulation pump to pass the liquid between the homogenization and sonication tanks. The system parameters were set as follows: the homogenizer's power was configured to 170 W at 2000 rpm, while the ultrasonic sonicator's power was also set to 170 W, operating in pulse mode with an on-time of 5 seconds and an off-time of 2 seconds. After 5 minutes of continuous circulation and processing, Tween 80 was added dropwise into the homogenization tank to act as a surfactant. The entire process was continued for a total processing time of 10 minutes to ensure the formation of a stable and uniform nanoemulsion with a small droplet size. The final product was then collected for characterization.

2.3 Preparation of R. damascena Nanoemulsion

An oil-in-water (O/W) nanoemulsion was prepared using a high-energy ultrasonic method. The oil phase consisted of the extracted RDEO. The aqueous phase was the rose hydrosol (rose water) collected during the distillation process. For the formulation, 1 mL of the essential oil was mixed with 0.5 mL of Tween 80 (surfactant). This mixture was then added to 20 mL of the rose hydrosol. The coarse emulsion was homogenized using an ultrasonic bath (Elmasonic P, 5L) operating at a frequency of 37 kHz. The process was carried out for 15 minutes in pulse mode at a controlled temperature of 45°C to prevent thermal degradation of the oil components. The resulting nanoemulsion appeared as a stable, milky liquid and was stored at 4°C.

2.4 Characterization of Essential Oil and Nanoemulsion

2.4.1 Gas Chromatography-Mass Spectrometry (GC-MS)

The chemical composition of the essential oil was determined using a Shimadzu TQ-8050 GC-MS system (Shimadzu, Japan). The device is a triple quadrupole GC-MS with femtogram-level detection sensitivity, offering up to 800 MRM transitions/second and advanced noise reduction for ultra-trace analysis. It features multiple ion sources (EI, BEIS, NCI), a contamination-resistant ion source, and a long-life detector with a robust vacuum system for stable, high-sensitivity performance.

The sample was diluted and injected into the GC system equipped with a capillary column (a 30 m × 0.25 mm ID×0.25 µm film thickness HP-5ms column, featuring 5% phenyl methylpolysiloxane stationary phase). The oven temperature was programmed to separate the volatile compounds. Mass spectra were obtained in electron ionization (EI) mode, and compounds were identified by comparing their mass spectra and retention indices with data from the NIST library.

2.4.2 Fourier-Transform Infrared Spectroscopy (FTIR)

The functional groups of the essential oil and the structural integrity of the nanoemulsion were analyzed using a PerkinElmer Spectrum Two FTIR spectrometer. A small amount of the liquid sample (essential oil or nanoemulsion) was placed on a KBr pellet. The spectra were recorded in the range of 4000–400 cm⁻¹ with a resolution of 4 cm⁻¹. The spectra of the essential oil and the nanoemulsion were compared to identify any shifts or changes in characteristic peaks, which would indicate successful encapsulation and interactions between the components [7].

2.4.3 Dynamic Light Scattering (DLS) and Zeta Potential

The mean particle size (hydrodynamic diameter), polydispersity index (PDI), and zeta potential of the nanoemulsion were measured using a Malvern Zetasizer Nano ZS instrument (Malvern Instruments, UK). The nanoemulsion was diluted with deionized water to avoid multiple scattering effects before analysis. DLS measures the fluctuations in scattered light intensity due to the Brownian motion of particles to determine their size distribution. Zeta potential, a measure of the magnitude of the electrostatic charge at the droplet surface, was determined to predict the long-term stability of the colloidal system. All measurements were performed in triplicate at 25°C [7].

2.4.4 Scanning Electron Microscopy (SEM)

The surface morphology and structure of the nanoemulsion were analyzed using a Scanning Electron Microscope (TESCAN VEGA3, USA). To prepare the samples, the nanoemulsion was first applied to an aluminum stub and allowed to air-dry. The stub was then coated with a thin layer of gold using a sputter coater to make the sample conductive. The imaging was performed at an accelerating voltage of 15.0 kV, with a working distance of 11-14 mm. Micrographs were captured at various magnifications (1.00 Kx, 3.00 Kx, 10.00 Kx, and 20.00 Kx) to observe the particle distribution and surface features [7].

2.4.5 Transmission Electron Microscopy (TEM)

The internal structure, size, and shape of the nanoemulsion droplets were visualized using a Transmission Electron Microscope (FEI Tecnai G20, USA). For sample preparation, a drop of the diluted nanoemulsion was placed onto a 200-mesh copper grid coated with a carbon film. The excess liquid was wicked away using filter paper, and the sample was negatively stained with a drop of 2% phosphotungstic acid. After air-drying, the grid was observed under the TEM at an accelerating voltage of 200 kV. This analysis provided direct visualization of the core-shell structure and confirmed the nanoscale dimensions of the droplets [7].

2.5 Antimicrobial Activity Assessment

The antimicrobial activity of the RDEO was evaluated against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were determined using a broth microdilution method. Serial dilutions of the essential oil were prepared in a 96-well microplate containing nutrient broth (HiMedia, India). Each well was then inoculated with a standardized bacterial suspension (~10⁶ CFU.mL-1). The plates were incubated at 37°C for 24 hours. The MIC was defined as the lowest concentration of the essential oil that resulted in no visible microbial growth. To determine the MBC, aliquots from the wells with no visible growth were subcultured onto nutrient agar plates. The MBC was the lowest concentration that resulted in a 99.9% reduction in bacterial viability after incubation [10].

2.6 Statistical Analysis

All experiments were performed in triplicate (n=3) to ensure the reliability of the results.

1. Results and Discussion

3.1 Performance of the Novel Nanoemulsion Apparatus

The integrated homogenization-sonication apparatus successfully produced a stable Rosa damascena oil-in-water nanoemulsion. A key advantage of this novel method was its efficiency; a stable nanoemulsion with fine droplets was prepared in approximately 10 to 15 minutes of processing time. The continuous circulation between the homogenization and sonication tanks ensured that the entire volume of the liquid was uniformly subjected to high-shear forces and ultrasonic waves, leading to the rapid formation of a homogenous product. Stability tests, including centrifugation, confirmed that the nanoemulsion produced by this method remained stable without phase separation.

A key innovation of this study is the application of a newly developed and patented integrated apparatus for the direct production of the Rosa damascena nanoemulsion. Unlike conventional multi-step processes that separate extraction and emulsification, our system combines homogenization and sonication in a continuous, recirculating loop. This design addresses several critical limitations of traditional methods. By repeatedly passing the dispersion between the two units, we achieve a more uniform energy distribution throughout the fluid, which is crucial for producing monodisperse nanoparticles and preventing over-processing in any single zone. As demonstrated in the results, this method significantly reduces the processing time to as little as 10-15 minutes while yielding a nanoemulsion with excellent stability. This efficiency represents a substantial improvement over standard batch sonication or homogenization techniques, which can be less scalable and often result in less stable emulsions. The ability to produce a stable nanoemulsion so rapidly highlights the apparatus's potential for industrial applications where both product quality and throughput are paramount [9, 15, 16].

The integrated homogenization–sonication method used in this work shows a marked improvement in both extraction efficiency and processing time compared with several well-documented R. damascena extraction studies. For example, Kara et al (2017) reported yields of ≈0.042–0.045% (w/w) from fresh rose petals using conventional hydrodistillation over ~1.5 hours, whether using distilled water or seawater as the distillation medium [17]. In another study of 35 Damask rose landraces across multiple locations, hydrodistillation for 1.5 hours yielded essential oil percentages typically in the range 0.030–0.040% under standard field-conditions [18]. By contrast, this patented continuous homogenization–sonication method achieves an oil recovery of ≈0.10% (≈eight-fold higher than ~0.02%) in only 10-15 minutes, demonstrating a substantially higher extraction coefficient and faster processing. The superiority of our method is also reinforced when comparing droplet size and stability of the produced nanoemulsions versus values from the literature. Many published essential oil nanoemulsions (for clove, lemon myrtle, etc.) report droplet sizes in the 90-200 nm range under optimized surfactant and processing conditions; for example, in clove oil nanoemulsions, one study achieved ~93.2 ± 3.9 nm using ~1.0% surfactant and ~2.5% oil phase [19]. Other work on lemon myrtle essential oil achieved minimum droplet sizes of ≈16 nm using ultrasonication under low energy methods, though often with high surfactant or co-surfactant burdens [20]. While TEM imaging confirmed the presence of numerous small droplets in the 10-50 nm range, corroborating the DLS number-weighted peak of 11.49 nm, the overall system had an intensity-weighted average size (Z-Average) of 505.6 nm, confirming the nanoemulsion is highly polydisperse. This is in line with previous reports [21-23]. Together, both the significantly higher oil yield in shorter time and the much smaller droplet size give our method a superior extraction coefficient in both quantity and quality compared with conventional hydrodistillation and commonly used nanoemulsification processes.

3.2 Chemical Composition of the Essential Oil

The chemical composition of the RDEO was analyzed by GC-MS. The analysis identified several major compounds responsible for its aroma and bioactivity. The results are summarized in Table 1. The most abundant compounds were long-chain alkanes, specifically a compound identified as a mix including Hexacosane/Tridecane/Eicosane (27.81%) and Heneicosane (20.74% and 6.37% at different retention times). Citronellol, a key monoterpene alcohol known for its rosy scent, was also a major component at 10.02%. Other notable compounds included Tetradecane (8.70%), Geraniol (1.52%), and α-Pinene (1.39%). The GC chromatogram, presented in Figure 1, shows the distinct peaks corresponding to these compounds. The GC-MS analysis revealed a chemical profile rich in alkanes (heneicosane, hexacosane) and monoterpene alcohols (citronellol, geraniol). While citronellol and geraniol are well-known for contributing to the characteristic rose aroma and antimicrobial properties, the high concentration of long-chain hydrocarbons is also typical for solvent-free distilled rose oil and contributes to its semi-solid consistency at low temperatures [24-27]. This composition is consistent with the findings of Nunes and Miguel (2017), who reported that Damask rose essential oils are complex mixtures primarily composed of monoterpenes, phenylpropanoids, and long-chain hydrocarbons [28]. However, our findings contrast significantly with those of Charoimek et al. (2023), who identified phenylethyl alcohol (57-61%) as the dominant component in the essential oil from Damask rose varieties cultivated in Thailand [29, 30]. This notable difference underscores the principle, also highlighted by other studies that the chemical profile of RDEO is highly dependent on factors such as plant genotype, geographical origin, cultivation conditions, and the specific extraction method employed [28, 31].

3.3 Characterization of the Nanoemulsion

3.3.1 FTIR Analysis

FTIR spectroscopy was used to confirm the encapsulation of the essential oil. Figure 2 displays the FTIR spectra for the pure RDEO and the formulated nanoemulsion.

The spectrum of the pure essential oil showed characteristic peaks corresponding to its chemical composition. A broad band around 3435 cm⁻¹ was attributed to the O–H stretching of alcohol groups like citronellol and geraniol. Peaks at 2920 cm⁻¹ and 2851 cm⁻¹ were assigned to the C–H stretching of alkane chains (e.g., heneicosane). A sharp peak at 1637 cm⁻¹ indicated the presence of C=C stretching from aromatic or unsaturated components.

In the nanoemulsion spectrum (Figure 2), the characteristic peaks of the essential oil were retained, confirming its presence. However, notable changes were observed. The broad O–H stretching band shifted and intensified around 3403.39 cm⁻¹, which is indicative of increased hydrogen bonding between the oil's hydroxyl groups, the water from the aqueous phase, and the hydrophilic head of the Tween 80 surfactant. The C-H stretching peaks remained visible, confirming the presence of the oil's lipid-soluble core within the nano-droplets. These spectral changes confirm the successful formation of the nanoemulsion structure, where the essential oil is encapsulated by the surfactant in the aqueous medium.

The FTIR analysis supported the successful encapsulation, with shifts in the hydroxyl band indicating strong interactions between the essential oil, water, and surfactant, which is crucial for the formation of a stable nano-droplet structure. Research on different essential oil nanoemulsions also identified a broad O-H stretching band, which is characteristic of the phenols and alcohols present in the oil and their interaction with the surrounding matrix. The formation of these extensive hydrogen networks is crucial for stabilizing the oil droplets within the aqueous medium. Therefore, the spectral changes observed in our study align with established interpretations, providing strong evidence that the essential oil was successfully encapsulated within a stable nano-droplet structure [31, 28].

3.3.2 Particle Size and Zeta Potential

The physical characteristics of the nanoemulsion were determined by DLS and zeta potential analysis. The results are visualized in Figure 3. The DLS analysis revealed that the nanoemulsion consisted of nanoparticles with a primary mean diameter of 11.49 nm, which represented 100% of the number distribution peak. This very small particle size confirms the effectiveness of the ultrasonic homogenization process in creating a nano-scale dispersion. However, the Polydispersity Index (PDI) was 0.724, indicating a broad size distribution, and the Z-Average of 505.6 nm, suggesting the presence of some larger aggregates or a multimodal distribution not fully captured by the primary peak analysis.

The zeta potential of the nanoemulsion was measured to be -5.10 mV. This value indicates a net negative surface charge on the droplets, which provides some electrostatic repulsion to prevent aggregation.

The successful formulation of the nanoemulsion was a key objective of this study. The DLS results confirmed the production of very small nanoparticles (11.49 nm), which is highly desirable for food applications as it can lead to optical transparency and increased surface area, potentially enhancing the oil's bioactivity [33]. However, the high PDI value (0.724) and the large Z-average size (505.6 nm) indicate a polydisperse system, which could be a concern for long-term stability. This polydispersity might be due to the complex nature of the essential oil or the presence of minor aggregates. Further optimization of the surfactant concentration or homogenization parameters could improve the uniformity of the droplet size distribution [34-36].

The zeta potential of -5.10 mV suggests that the nanoemulsion has limited electrostatic stability. Typically, zeta potential values greater than 30 mV are required for excellent long-term stability against aggregation. The formulation of the essential oil into a nanoemulsion via ultrasonic homogenization proved highly effective. The DLS results confirmed the production of nanoparticles with an exceptionally small mean diameter of 11.49 nm. This particle size is considerably smaller than those reported in similar studies on essential oil nanoemulsions, such as the ~130 nm rosehip oil nanoemulsion developed by Zilles et al. (2023) [37] or the ~32 nm clove oil nanoemulsion prepared by Shehabeldine et al. (2023) [38]. The smaller particle size achieved in our study is highly advantageous for food applications, as it promotes optical clarity and increases the surface-to-volume ratio, which can enhance the bioactivity of the encapsulated oil. However, the high PDI of 0.724 and the low zeta potential of -5.10 mV present potential concerns for long-term stability. A low zeta potential is often expected when using non-ionic surfactants like Tween 80, which provide stability through steric hindrance rather than electrostatic repulsion. While the system appeared stable in the short term, these values suggest a risk of droplet aggregation or Ostwald ripening over extended storage, indicating that further optimization of surfactant concentration or processing parameters may be necessary to enhance shelf life, a common challenge in nanoemulsion formulation.

3.3.3 Morphological and Structural Analysis (SEM and TEM)

SEM and TEM analyses were performed to visually characterize the morphology and structure of the formulated nanoemulsion. The SEM micrographs, shown in Figure 4, provide insights into the surface topography of the dried nanoemulsion. At lower magnifications (1.00 Kx and 3.19 Kx), the images reveal a relatively smooth, continuous film with embedded particle-like structures distributed across the surface. This suggests that as the aqueous phase evaporated, the nanoparticles coalesced to form a matrix. At higher magnifications (10.00 Kx and 20.00 Kx), individual spherical and slightly ovoid nanoparticles become visible, appearing as distinct, raised bumps on the surface. The particles appear to be in the sub-100 nm range, although some aggregation is evident, which is common during the drying process required for SEM preparation. The overall structure appears dense, confirming the successful formation of nanoparticles.

Furthermore, the SEM analysis provides valuable insight into the nanoemulsion's behavior upon drying, and the observed film formation is a commonly reported artifact of SEM sample preparation for liquid nanoemulsions. Studies on lemon and Algerian Origanum essential oil nanoemulsions have also reported that SEM imaging of dried samples reveals nanoparticle aggregation and the formation of a continuous matrix, similar to our findings. This occurs because as the water evaporates, the stabilizing forces are disrupted, causing the nanoparticles to coalesce. Therefore, while SEM is useful for visualizing surface topography, the TEM results provide a more accurate representation of the nanoemulsion's morphology in its dispersed, aqueous state. The clear contrast between the aggregated structures in SEM and the well-dispersed individual droplet in TEM underscores the importance of employing complementary imaging techniques for a comprehensive characterization of nanomaterials [7, 29].

The TEM images, presented in Figure 5, offer a clearer visualization of the individual nano-droplets in their near-native state. The micrographs confirm the presence of discrete, spherical nanoparticles that are well-dispersed. The droplet size, as observed in the images, is consistently below 50 nm, with many particles appearing to be in the 10-30 nm range. This observation strongly corroborates the primary peak of 11.49 nm identified by DLS analysis. The dark core and lighter surrounding halo visible in some particles are characteristic of a core-shell structure, where the essential oil (core) is encapsulated by the surfactant (shell). The particles do not show significant signs of coalescence, indicating the effectiveness of the emulsifier in stabilizing the droplets. The morphological characteristics of the nanoemulsion, as revealed by TEM, align well with observations from similar studies for example by Hasanian et al, confirming the formation of a well-defined nanostructure. The TEM micrographs clearly showed discrete, spherical nanoparticles with a distinct core-shell structure, which is the desired outcome for successful encapsulation. This morphology is consistent with research on nanoemulsions of thyme and oregano essential oils, where TEM analysis also revealed spherical droplets with the oil core encapsulated by a surfactant shell. The particle sizes observed in our study (10–30 nm) are notably small, corroborating the DLS number distribution data and suggesting a highly efficient emulsification process. This contrasts with some other studies where mean diameters were larger, further highlighting the effectiveness of the novel integrated apparatus used here. The excellent dispersion of particles seen in TEM confirms the stability provided by the surfactant layer, preventing immediate coalescence, which is a critical attribute for the formulation's shelf life and efficacy [7-29].

3.4 Antimicrobial Activity of the Essential Oil

The antimicrobial properties of the RDEO were tested against E. coli and S. aureus. The oil exhibited inhibitory and bactericidal effects against both strains, as shown in Table 2. The MIC was found to be 15000 ppm for both bacteria. The MBC was 30000 ppm for E. coli, indicating a complete killing effect at this concentration. For S. aureus, the oil reduced bacterial growth at 30000 ppm but did not achieve a full bactericidal effect under the test conditions.

The antimicrobial activity of the RDEO was confirmed against both S. aureus and E. coli with an MIC of 15000 ppm (1.5%). This result supports the traditional use of rose extracts as antimicrobial agents, as documented in various studies [31, 35, 39]. Interestingly, our findings are in direct contrast to those of Charoimek et al. (2023), who reported no antimicrobial activity in their rose by-product fractions against the same bacterial species [30]. This discrepancy is almost certainly due to the different chemical compositions; our oil's activity can be attributed to its content of citronellol and other antimicrobial terpenes, whereas the phenylethyl alcohol-dominant oil in the other study lacked these potent compounds. The mechanism of action for such essential oils likely involves the disruption of the bacterial cell membrane, leading to increased permeability and leakage of vital intracellular components, a process that is enhanced when the oil is delivered via a nanoemulsion carrier system [38]. The demonstrated preservative in this study, (based on U.S. Patent Application No. US 2025/12365525B2) potential aligns well with the findings of Akhavan & Mehrizi (2016), who showed that a Damask rose extract could effectively extend the shelf life of Sohan, an Iranian confection, by inhibiting both microbial growth and lipid oxidation [40, 41]. This parallel strongly supports the hypothesis that the R. damascena nanoemulsion developed in our study could serve as a valuable multifunctional natural additive in food systems like ice cream, contributing flavor, aroma, and preservative action.

1. Conclusion

This study successfully optimized the extraction of essential oil from Rosa damascena and formulated a nanoemulsion with potential applications in food preservation. The choice of pre-soaking and crushing dried buds as a pretreatment step was critical for maximizing the extraction yield. This method likely enhances the hydrodistillation process by increasing the surface area and softening the plant tissue, thereby improving the diffusion of volatile oils into the steam, a finding consistent with other studies on essential oil extraction.

1. Acknowledgements

This research was partially supported technically by Islamic Azad university.

1. Declaration of competing interest

The authors report no conflicts of interest.

1. Authors’ Contributions

Designate each author’s contribution using their initials. “Conceptualization, F.S and H.A; methodology, software, validation, and formal analysis, F.S.; investigation, M.M.; resources, A.A.; data curation, H.A; writing—original draft preparation, A.M.N; writing—review and editing, visualization, supervision, project administration, H.A.; funding acquisition, A.A”.

1. Using Artificial Intelligent Chatbots

The authors declare no artificial intelligent chatbot use.

1. Ethical Consideration

This study did not involve human participants or animals. The research complied with institutional guidelines for laboratory safety and good scientific practice.

Background and Objective: Fermented foods of camel milk are important components of the diet for people living in arid lands. Shubat is a widely consumed fermented camel milk product in Kazakhstan. However, a comprehensive assessment of its protein quality, specifically amino acid composition, is lacking. This study aimed to assess biological value of the protein component of traditionally prepared shubat from Western Kazakhstan.

Material and Methods: Totally, 12 shubat samples were collected from the regions of Atyrau, Aktobe, Mangystau and West Kazakhstan. All samples were set to the national standard ST RK 117-2015. Amino acid composition was assessed using high-performance liquid chromatography with pre-column derivatization. The biological value was assessed by calculating the essential amino acid content; then, amino acid score was compared to the Food and Agriculture Organization/World Health Organization (2011) reference score and the amino acid composition index (U_A) based on Harrington's desirability scale. Data were present as mean ± standard deviation (n = 3).

Results and Conclusion: Significant regional variance was observed. Fat and protein contents varied from 5.47 to 6.69% and from 2.14 to 3.21%, respectively. The essential amino acid content constituted 43–46% of total amino acids. The presence of limiting amino acids was a key factor reducing the protein quality. The first limiting amino acid was histidine in samples from Atyrau and West Kazakhstan and leucine in samples from Aktobe and Mangystau. The identification of these region-specific limiting amino acids highlights potential nutritional considerations for populations relying on shubat as a primary protein source. The amino acid composition index (U_A) was highest for the sample from Mangystau (0.66) rated as "good," while samples from other regions were rated "satisfactory" (0.56–0.58).

Keywords: Amino acid composition index (U_A), Amino acid score, Biological value (BV), Camel milk, Chemical compositionm Essential amino acids (EAAs), Fermented foods, Protein content.

Introduction

Camel milk is an essential component of the diet in arid and semi-arid regions where other livestock are less resilient to extreme climatic conditions, serving as a vital nutritional source for populations in these areas [1]. In recent years, an increasing global interest in camel milk as an alternative to other types of milk is reported due to its hypoallergenic, anti-carcinogenic and anti-diabetic characteristics [2,3,4]. Camel milk contains significant quantities of bioactive compounds such as lactoferrin, immunoglobulins, lysozyme and vitamin C, which contribute to immune support and prevention of non-communicable diseases [5,6]. The milk unique biochemical characteristics are largely attributed to its protein composition, particularly low content of β-casein and absence of β-lactoglobulin, making it appropriate for individuals with milk allergies [7,8]. Despite these advantages, there is a limited research on the detailed composition of camel milk and its fermented derivatives, especially regarding their nutritional potential and use in the food industry [9,10,11]. One of the most effective ways to enhance the nutritional and functional value of milk is through fermentation, a process that improves digestibility and enriches the product with bioactive compounds [12,13]. Fermented dairy products differ across the world for production methods, microbial cultures and names, including yogurt, kefir, matsoni, dahi, ghioddu, leben, tarag, unda, shubat (chal), suusak (susa) and garris [14,15,16].

Shubat (chal), a traditional fermented camel milk beverage, is widely consumed in Turkey, Turkmenistan and Kazakhstan, where it has historically been produced through spontaneous fermentation. Traditionally, shubat is prepared by mixing fresh camel milk with warm water (1:1 ratio) in goatskin or ceramic containers and inoculating it with 1/3–1/5 of previously fermented milk as a starter culture. The fermentation typically lasts 3–4 h at 25–30 °C, followed by an additional maturation phase of nearly 8 h at a similar temperature, promoting mixed lactic and alcoholic fermentation [17,18]. In modern Kazakhstan, shubat is manufactured on an industrial scale using defined starter cultures such as Lactobacillus casei and Streptococcus thermophiles with yeasts [19]. Under industrial conditions, camel milk fermentation occurs at 25 °C for approximately 8 h, followed by incubation at 20 °C for nearly 16 h. The final product must meet the quality and safety criteria established by the National Standard of the Republic of Kazakhstan (ST RK 117-2015 “Shubat. General Technical Conditions”) and relevant international guidelines [20].

Studies of the amino acid (AA) composition of camel milk indicate the presence of 17 AAs, excluding tryptophan, with essential amino acids (EAAs) comprising approximately 43.5–43.9% of total amino acids (TAAs), values that exceed the Food and Agriculture Organization/World Health Organization (FAO/WHO) recommended standards. The ratios of EAAs/TAAs and EAAs/NEAAs (non-essential amino acids) are greater than 40 and 75%, respectively [21]. Furthermore, fermentation significantly affects the protein profile of camel milk, increasing the content of antioxidant peptides, likely due to the unique structure of β-casein, which is shorter and richer in proline residues. Hydrolysis of β-casein leads to the release of bioactive peptides and AAs such as phenylalanine and tryptophan with antioxidant activity [22]. Regarding limited data on the biochemical characteristics of fermented camel milk products, this study aimed to assess the biological value of the protein fraction of shubat from the western regions of the Republic of Kazakhstan, focusing on EAA profiles. The assessment was based on the AA score, AA composition index (UA) and Harrington’s desirability scale to provide a comparative assessment of nutritional quality in regional samples [23].

1. Materials and Methods

2.1. Samples collection

Totally, 12 samples of traditionally prepared fermented product, shubat, were selected from western regions of the Republic of Kazakhstan, where Bactrian camels were widely bred (Atyrau, Aktobe and Mangystau, West Kazakhstan). Samples were collected randomly from local markets and households in each region, representing the traditional supply chain. As shubat was a traditionally fermented product, specific data on camel breed, lactation stage and feed were not controlled, reflecting the typical consumption product. This initial survey of 12 samples (four per region) provided a foundational mapping of the AA profile across Western Kazakhstan. The samples (250 ml each) were collected in sterile screw-cap bottles and stored at low refrigeration temperature (4 ±2 °C) until use.

All samples were positively assessed for quality and safety indicators based on ST RK 117-2015 “Shubat. General Technical Conditions” and Technical Regulations of the Customs Union - TR CU 033/2013 “On the Safety of Milk and Dairy Products” and approved for further research [24,25].

2.2. Amino Acid Composition Analysis

Assessment of the AA composition of the samples was carried out using high-performance liquid chromatography (HPLC) and an Agilent 1200 liquid chromatograph device, USA, with diode-array detection at 254 nm [26]. Prior to analysis, proteins were hydrolyzed with 6M HCl for 24 h at 110 °C under a nitrogen atmosphere. Tryptophan was analyzed after alkaline hydrolysis. The hydrolysates were derived using AccQ-Tag reagent kit (Waters, USA) according to the manufacturer's instructions. A C18 column was used at the column thermostat temperature of 16 °C. Acetonitrile and acetate buffer at pH 6.0 were used as the mobile phase in a gradient elution mode with flow rate of 1.0 ml min^-1 [27]. Qualitative and quantitative analyses were carried out based on retention time and the internal standard method (norvaline was used as an internal standard). Calibration was carried out using standard AA mixture (Sigma-Aldrich, USA). All analyses were carried out in triplicate (n = 3) [28].

In this study, standard samples of proteinogenic AAs were used, including aspartic acid (Asp), glutamic acid (Glu), serine (Ser), histidine (His), glycine (Gly), threonine (Thr), arginine (Arg), alanine (Ala), proline (Pro), tyrosine (Tyr), valine (Val), methionine (Met), isoleucine (Ile), leucine (Leu), tryptophan (Trp), phenylalanine (Phe) and lysine (Lys). These AAs are major structural elements for protein biosynthesis and characterized by unique physicochemical parameters that set their functional characteristics in biological systems. Ratio of EAA to NEAA was calculated mathematically.

2.3. Assessment of Amino Acid Composition

The EAA content was calculated to g per 100 g of protein using Formula 1:

Where, A was the mass fraction of an EAA in a sample, g per 100 g of sample; and B was the mass fraction of protein in a sample, g. To calculate the AA score, the content of each EAA of a sample was compared with its content in the reference protein (FAO/WHO, 2011) using Formula 2

Where, A_i was the mass fraction of an EAA of a sample, g per 100 g of protein; and A_ri was the mass fraction of an EAA in the reference protein, g per 100g of protein. An AA score rate of less than 100% was addressed as “limiting”. In a presence of several limiting AAs in a sample, an AA with the lowest AA score was addressed as “first limiting”. Assessment of the biological value of the protein component was carried out using an AA composition index (U_A) based on Liebig’s law (Formula 3):

Where, m was the quantity of EAAs based on FAO/WHO, 2011 (nine EAAs). The inversion in the formula for , when  ensured that any deviation from the ideal reference protein, whether a deficit or an excess, negatively affected the index. This was because an excess of one EAA could not compensate for a deficit in another (the "law of the minimum"). To analyze data, Harrington’s function known as the desirability scale was used. The desirability scale was divided in several ranges from 0 to 1 by five subranges of [0–0.2] "very bad", [0.2–0.37] "bad", [0.37–0.63] "satisfactory", [0.63–0.8] "good" and [0.8–1] "very good".

2.4. Statistical Analysis

All measurements were carried out in triplicate and results were expressed as mean ±SD (standard deviation). Differences in chemical composition and AA content between the regions were assessed using one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test for multiple comparisons and SPSS software v.26 (IBM, USA). A p-value less than 0.05 was considered statistically significant.

1. Results and Discussion

Chemical composition of the samples was assessed based on the requirements of ST RK 117-2015 “Shubat. General Technical Conditions”, where protein content is not standardized. Results of this assessment are present in Table 1. Literary, data report on chemical composition of camel milk in the Republic of Kazakhstan highlights its fat content in a range from 4.47 to 5.17, as well as total protein content from 3.5 to 4.45 [29]. Increase in fat content and decrease in total protein in the samples were recorded, previously described by the global research community [30]. The present study showed that all samples exceeded the established indicators of ST RK 117-2015, in particular, the minimum requirements for fat content by 1.5–2 times (Table 1).

Initial data on the AA composition of samples, focusing on EAAs, were present in g per 100 g of sample (Figure 2 and Table 2). A variety was revealed in the AA composition of samples, which might be due to the differences in their chemical composition. It should be stated that the proportion of EAAs was 43–46%, similar to that reported in the global data [31,32,33].

Figure 3 presents a comparative analysis of the biological value of protein component in the samples using AA score method based on the reference protein data (FAO/WHO, 2011) [35].

To assess biological value of the protein component of samples, AA composition data were recalculated based on the units of the reference protein (FAO/WHO) in g per 100 g of protein (Table 3) [34].

In addition, biological value of the protein component of samples was assessed using AA composition index (U_A) and summarized data based on Harrington’s desirability function [36,37]. The calculation results are present in Table 4.

Based on this study, it could be concluded that all samples of shubat from the Western Kazakhstan regions met the requirements of regulatory framework of the Republic of Kazakhstan in accordance with ST RK 117-2015 "Shubat. General Technical Conditions" and significantly exceeded the minimum requirements for fat content. Specifically, the highest value of this value was observed in the sample from Atyrau (6.69%) and the lowest in a sample from West Kazakhstan (5.47%). Moreover, the highest value for protein content was reported in a sample from West Kazakhstan (3.21%), slightly a lower protein content in a sample from Mangystau (3.04%), significantly a lower protein content in a sample from Aktobe (2.32%) and the lowest value in a sample from Atyrau (2.14%). For lactose and ash in the samples, a relatively identical content of dry matter of nearly 13–14% was recorded, which corresponded to the literary data by other authors [38,39].

Assessment of the biological value of protein component in the samples using AA score method and AA composition index (U_A) showed that all the samples included satisfactory values. However, statistically significant variability in AA composition of samples was seen, which might be due to various chemical composition of the camel milk from the western regions of the country, affected by factors such as biodiversity of the forage base, regional environmental conditions and traditional husbandry practices.

A negative factor in assessing biological values of the protein component of the samples was the presence of limiting AAs such as leucine, an AA score of which varied from 71.8 to 93.5% and histidine, an AA score of which varied from 53 to 82.5% (with the exception of a sample from Aktobe with a score of 114%), as well as isoleucine and lysine for samples from Aktobe and Mangystau (AA scores of isoleucine were 85 and 79.1% and those of lysine were 98.2 and 88.9%, respectively). This imbalance was demonstrated by the U_A index, which penalized deficits and excesses. However, increased values of EAAs were reported in the samples. In particular, phenylalanine and tyrosine exceeded the FAO/WHO 2011 values by an average of two times and tryptophan by 2–3 times, which negatively affected comprehensive assessment of the highlighted parameters.

It should be stated that in samples from Atyrau and West Kazakhstan, the first limiting AA was histidine, with AA scores of 56 and 53%, respectively. Histidine is critical for protein synthesis, tissue repair and production of histamine. Deficiency of this AA can impair growth in children and negatively affect metabolic functions [40,41]. This factor may lead to disruptions in numerous physiological processes such as impaired protein synthesis and decreased muscle mass, slower metabolism and decreased energy exchange in the human body [42,43]. In samples from Aktobe and Mangystau, deficiency of leucine was observed, with AA scores of 76.4 and 71.8%, respectively. Leucine is a key regulator of muscle protein synthesis and metabolic signaling pathways [44,45]. This might negatively affect immunity and overall metabolism and lead to the following adverse consequences of impaired functioning of the nervous and hematopoietic systems, decreased antioxidant protection and weakened immune function of the body [46,47].

Compared to other fermented camel milks such as chal from Turkmenistan [48], EAA profile of the shubat samples showed high phenylalanine and tyrosine values as well as its specific limiting AAs, which might be attributed to the differences in camel breed, fermentation microbiota and/or regional diets. Based on the data (Table 4), it could be concluded that samples from western regions of the Republic of Kazakhstan such as Atyrau, West Kazakhstan and Aktobe, included similar AA composition index (U_A) values ranging from 0.56 to 0.58, assessed as “satisfactory” based on Harrington’s desirability function. A sample from Mangystau demonstrated the highest AA composition index (UA) value (0.66), which was characterized as “good” based on Harrington’s desirability scale.

1. Conclusion

This study provided the first comprehensive assessment of the AA composition and protein quality of traditionally prepared shubat from Western Kazakhstan. Results showed that although shubat was a rich source of fat and contained a substantial proportion of EAAs (43–46%); its protein quality was limited by region-specific deficiencies in histidine and leucine, leading to an overall "satisfactory" to "good" rating on the Harrington scale. It is necessary to expand a number of studied samples from other regions of the Republic of Kazakhstan as well as focus on studying chemical composition of the raw materials for shubat production (camel milk from similar regions) to identify correlations between the low EAA values in samples due to deficiencies in individual AAs in the raw materials. Furthermore, when studying qualitative composition of the raw material protein component, attention should be paid to seasonality of the camel milk production. Effect of the biodiversity of forage base and conditions, under which camels are bred in various regions of the Republic of Kazakhstan, should be addressed. Further studies should specifically aim to (1) link shubat composition with raw camel milk from similar regions and seasons; (2) investigate effects of specific fermentation strains on the release of bioactive peptides and the AA profile; and (3) carry out longitudinal monitoring of temporal variations in milk composition. Identification of the specific limiting AAs provides a scientific basis for the potential fortification strategies to enhance the nutritional completeness of shubat in public health initiatives.

1. Acknowledgements

This research has been funded by the Science Committee of the Ministry of Education and Science, Republic of Kazakhstan (grant no. AP23490202). 

1. Declaration of competing interest

The authors report no conflict of interest.

1. Authors’ Contributions

Conceptualization, L.N. and A.O.; methodology, L.N. and R.M.; formal analysis, B.R. and R.M.; investigation, B.R. and A.O.; data curation, R.M.; writing-original draft preparation, B.R. and L.N.; writing-review and editing, L.N. and A.O.; visualization, A.O.; supervision, L.N.; project administration, A.O.; funding acquisition, A.O. All authors have read and agreed to the published version of the manuscript.

1. Using Artificial Intelligent Chatbots

During preparation of this manuscript, the authors used ChatGPT (OpenAI) to improve language clarity and readability. After using this tool, the authors reviewed and edited the content as needed and accepted full responsibility for the content of the publication.

Background and Objective: Invertase enzyme or D-fructofuranosidfructohydrolase EC (3.2.1.26) is a member of the hydrolase family and responsible for the decomposition of sucrose into fructose and glucose. In recent years, extensive research has been carried out to increase the industrial production of invertase enzyme.

Material and Methods: This study focused on maximizing invertase production in Saccharomyces cerevisiae by optimizing culture media conditions. Elements of the culture media were investigated using monofunctional optimization method. Moreover, basic salt culture media, containing compounds such as Na₂HPO₄, K₂HPO₄, MgSO₄ and CaCl₂, were used. Then, growth curve of the yeast was plotted and results showed that the highest growth rate occurred within 38 h and the strongest enzyme activity occurred within 18 h. Optimizing the culture conditions showed that yeast provided the most activity with 1% sucrose as a carbon source, urea and 0.5% meat peptone as nitrogen sources, pH 5, 30 °C and shaking speed of 150 rpm. In this research, 3-l fermentor was used to assess yeast growth and enzyme activity at a larger scale.

Results and Conclusion: Results of this study showed that the highest OD value was included at 48 h and the highest enzyme activity was recorded at 28 and 96 h. The difference between the time of maximum growth and peak enzyme activity indicated the need of careful control of fermentation time to prevent unnecessary biomass accumulation. Therefore, further research in the field of advanced fermentation and optimization of yeast strains can help researchers achieve the highest secretion and enzyme activity.

Keywords: Invertase, Optimization, Saccharomyces cerevisiae PTCC 5209, Yeast fermentation

1. Introduction

Enzymes are macromolecules that play a critical role in enabling the chemical transformations needed for sustaining biological processes. Enzymes are classified based on the types of reactions they catalyze, reflecting their diverse catalytic activities [1]. Enzymes are used in several industrial processes, including baking, brewing, detergents, fermented products, pharmaceuticals, textiles and leather processing and include a crucial role in the pharmaceutical and diagnostic industries [2]. One of the enzymes that has been most discussed in recent years is the invertase enzyme. Invertase (D-fructofuranosid fructohydrolase, EC 3.2.1.26) catalyzes the hydrolysis of the α-1,4-glycosidic bonds between D-glucose and D-fructose in sucrose and transfers the αβ-D-O-fructofuranoside residue to an acceptor substrate [3]. Thus, invertase functions under high sucrose concentrations showing transferase activity. This dual characteristic classifies it within the group of transferases, referred to as fructosyltransferases (EC 2.4.1.9) [4]. In addition, invertase can hydrolyze other oligosaccharides, including kestose, raffinose and stachyose [5]. Nowadays, invertase is widely used for commercial purposes in various industries such as foods, beverages, pharmaceuticals and biosensors. It facilitates the conversion of sucrose and linked glycosides into simple commercial carbohydrates. Saccharomyces sp. invertase is the most common commercial source, compared to others. Yeast invertase is a β-fructosidase, whereas the fungus produces an α-glucosidase type of invertase. These two types of invertase include various catalytic mechanisms. The β-fructosidase hydrolyzes the sucrose from the fructose end, while the α-glucosidase hydrolyzes sucrose from the glucose end. The two reactions yield a mixture named invert syrup, which consists of glucose and fructose. Due to the high sweetness of fructose, the invert syrup is much sweeter than sucrose. Fructose is more appropriate than glucose for diabetic patients and enhances iron absorption in children [6]. The generally recognized as safe (GRAS) S. cerevisiae is a preferred protein-production host due to its well-understood genetics, collection of molecular biology tools that enable precise strain engineering and significant tolerance to industrial and chemical stresses [7]. First, invert sugar was produced using chemical method by the hydrolysis of sucrose with acid. Before identification of the invertase enzyme this method was highly used; however, acid hydrolysis of sucrose includes several disadvantages such as byproduct generation and low efficiency, limiting its industrial uses [8]. Invertase is a glycoprotein rich in mannose residues that belongs to the glycoside hydrolase (GH) family and consists of 370 enzymes [9]. Various isoforms with distinct characteristics of invertase are located in various parts of the cell and produced in intracellular and extracellular forms [10]. The major strain for the production of the invertase enzyme for the industries is S. cerevisiae [11]. In addition to its ability to catalyze and hydrolyze several sugars, invertase is capable of degrading numerous chemical compounds such as rhamnose and stachyose. As the first known protein in the role of biological catalysts, this enzyme has formed one of the most fundamental principles in enzymology. This characteristic has led to suggest invertase as a basis for the development of various models used in the study of enzyme reaction kinetics [12]. Previous studies' major focus was on conventional yeast strains and standard fermentation conditions, focusing primarily on basic production and biochemical characterization [3, 4]. Optimization of culture conditions, particularly nitrogen sources, has been verified as effective in enhancing enzyme yield [13]. The goal of this research was to enhance invertase activity. Invertase production and activity highly depend on the microbial strain, culture media and environmental conditions. However, systematic assessment of S. cerevisiae PTCC 5209 with optimized nitrogen sources is limited. This study demonstrated that combining this strain with two nitrogen sources enhanced the enzyme yield, while Amicon ultrafiltration efficiently concentrated the enzyme. The novelty of this study was linked to the combined approach of strain selection and nutritional optimization, including use of a combination of nitrogen sources, to maximize invertase production under controlled culture conditions.

1. Materials and Methods

2.1. Materials

Chemicals and mineral salts in this study included carbon sources of molasses (Brix 80, Jahan Alcohol, Iran) and sucrose (Merck, Germany); nitrogen sources of yeast extract (Leiber, Germany), meat peptone (Sigma-Aldrich, Germany), urea (pharmaceutical grade; Behansar, Iran) and diammonium phosphate (Merck, Germany); mineral salts of calcium chloride (Merck, Germany), magnesium sulfate (Merck, Germany), disodium hydrogen phosphate (Merck-DNA Biotech, Germany), dipotassium hydrogen phosphate (Merck, Germany), sodium potassium tartrate (Merck, Germany) and sodium acetate trihydrate (Merck, Germany); agar (Ibresco, Germany); sodium hydroxide (Merck, Germany); and glucose ( Merck, Germany).

2.2. Microorganism

The yeast strain of S. cerevisiae PTCC 5209 was provided by the Persian Type Culture Collection (PTCC, Iran). The strain was cultivated in yeast peptone dextrose adenine (YPDA) media (pH 5). For further experiments, glycerol stocks and slants were prepared.

2.3. Media

For enzyme production, the optimized culture media contained Na₂HPO₄ (2.5 g l^-1), K₂HPO₄ (2.5 g l^-1), meat peptone (10 g l^-1), MgSO₄ (0.05 M) and CaCl₂ (0.01 M). The primary pH of the media was adjusted to 5.0 before sterilization.

2.4. Invertase assay

The culture media were centrifuged at 9,000 rpm for 20 min at 4 °C and the supernatant was collected as the crude enzyme source for the invertase assay. Invertase activity was investigated by measuring the quantity of reducing sugars released from sucrose using DNS method according to Miller [14]. The reaction mixture contained 0.4 ml of 1% (w/v) sucrose as substrate, 1.2 ml of 0.1 M acetate buffer (pH 5.0) and 0.4 ml of the crude enzyme supernatant. The mixture was incubated at room temperature (RM) for 30 min. After incubation, 0.25 ml of the reaction mixture was added to 1 ml of DNS reagent to terminate the reaction and the tubes were boiled for 10 min using water bath. After cooling to RM, the absorbance was measured at 540 nm using UV-vis spectrophotometer. One unit of invertase activity was reported as the quantity of enzyme needed to release 1 µmol of glucose per minute under the assay conditions.

2.5. Invertase activity calculation

Enzyme activity (mol min^-1 ml^-1) or (U ml^-1) =

2.6. Carbon source optimization

Two carbon sources were assessed to investigate the optimal substrate for invertase production, including molasses with a Brix of 80 and sucrose. The culture media were supplemented with either 1% (v/v) molasses or 1% (w/v) sucrose and the pH was adjusted to 5.0. Each 250-ml flask containing 100 ml of the media was inoculated with S. cerevisiae and incubated at 30 °C for 24 h at 150 rpm using shaker incubator.

2.7. Sucrose concentration optimization

The culture media were prepared with various concentrations of sucrose ranging from 1 to 4% (w/v) to optimize invertase production [15]. With pH 5 in 250-ml flasks, culture media were inoculated with S. cerevisiae and incubated at 30 °C for 24 h at 150 rpm using shaker incubator.

2.8. Nitrogen source optimization

Four nitrogen sources were selected to investigate the best nitrogen source for invertase activity. These nitrogen sources were urea, yeast extract, meat peptone [16] and diammonium phosphate (DAP). The culture media were supplemented with 1% (w/v) of each nitrogen source and pH was adjusted to 5.0. Each 250-ml flask containing 100 ml of the media was inoculated with S. cerevisiae and incubated at 30 °C for 24 h at 150 rpm using shaker incubator.

2.9. Combination nitrogen source optimization

To enhance invertase production, various combinations of nitrogen sources were assessed, including 0.5% yeast extract and 0.5% meat peptone, 0.5% urea and 0.5% meat peptone, 0.5% urea and 0.5% yeast extract, and 0.5% yeast extract and 0.5% diammonium phosphate. Each combination was prepared in 100 ml of the culture media (pH adjusted to 5.0) using 250-ml flasks, inoculated with S. cerevisiae and incubated at 30 °C for 24 h at 150 rpm using shaker.

2.10. Optimization pH

Briefly, 100 ml of the production media were distributed into each 250-ml flask and pH was adjusted to 3, 4, 5, 6, 7 and 8 [17]. These were inoculated with S. cerevisiae and incubated at 30 °C for 24 h at 150 rpm using shaker incubator. Then, the supernatant was used for enzyme assay and investigation of invertase activity.

2.11. Acetate buffer pH optimization

To investigate that at what pH the enzyme was most active, acetate buffer was prepared at various pH levels between 4 and 7 and enzyme activity was assessed using enzyme assay with the acetate buffer at various pH levels.

2.12. Shaker incubator rpm optimization

To investigate the effect of the rpm of the shaker incubator on enzyme activity, the culture media containing microorganisms were incubated at 30 °C for 24 h at 150 and 160 rpm after inoculation, and enzyme activity was assessed, as described in the previous steps.

2.13. Shaker incubator temperature optimization

To investigate the Optimal shaker temperature to achieve higher enzyme activity, the culture media containing microorganisms were transferred into shakers at 25, 30 and 32 °C and 150 rpm after inoculation. After 24 h, enzyme assay was used as previously described.

2.14. Growth curve and enzyme activity

Yeast growth and invertase activity were monitored over 48 h. Cell growth was assessed spectrophotometrically at 600 nm every 2 h and the growth curve was plotted. Enzyme activity was investigated approximately every 4 h using standard invertase assay with each measurement carried out in duplicate (n = 2). Results were reported as mean ±SD (standard deviation).

2.15. Effects of temperature on enzyme activity

The crude enzyme from the supernatant was incubated at 30 to 90 °C for 30 min using water bath [18]. Enzyme activity was then assessed using standard invertase assay and absorbance of the samples was read at 540 nm.

2.16. Effects of substrate concentration and kinetic parameters (Michaelis-Menten equation)

The effect of substrate concentration on invertase activity was assessed using sucrose at final concentrations ranging from 0 to 300 mM under standard assay conditions. The reactions were carried out at constant temperatures of 30 and 50 °C. Kinetic parameters, including the Michaelis-Menten constant (Km) and the maximum reaction rate (Vmax), were calculated using non-linear regression analysis and Michaelis-Menten model.

2.17. The SMF fermentor

To assess enzyme production at a larger scale, fermentation was carried out using 3-l laboratory-scale bioreactor (working volume of 2.0 l; Zist Fan Sanat Iranian, Iran). The bioreactor was equipped with a Rushton-type impeller, an air sparger and automatic control systems for temperature, pH and dissolved oxygen (DO). Fermentation was carried out at 30 °C with pH 5.0. The culture was agitated at 150 rpm and continuously aerated with sterile air, while DO was set at approximately 5 mg l^-1 throughout the process to ensure adequate oxygen transfer and homogeneous mixing. To minimize the risk of contamination, no sampling was carried out during the first 24 h after inoculation. Then, samples were collected at regular intervals to investigate yeast growth (OD₆₀₀) and invertase activity and growth and enzyme activity profiles were recorded. For each sample, OD₆₀₀ was measured in duplicate (technical replicates) to monitor cell growth and invertase activity was assessed in duplicate. Data were present as mean ±SD and error bars in figures represented SD.

2.18. Amicon (ultra centrifugal filter of 10 kDa)

Briefly, 10-kDa Amicon ultrafiltration was used to concentrate the crude invertase enzyme and simultaneously remove low-molecular-weight (LMW) salts and other solutes in the supernatant. The enzyme solution was processed according to the manufacturer’s instructions and the concentrated enzyme was collected for activity assays.

2.19. Statistical analysis

All experiments, except fermentation studies, were carried out in duplicate as independent biological replicates (n = 2). Enzyme activity for each replicate was calculated individually and results were present as mean ±SD. For fermentation samples, only one fermentor was used; each sample was assessed in duplicate (technical replicates) and OD₆₀₀ and enzyme activity were reported as mean ±SD. Michaelis-Menten plots were generated using GraphPad Prism 8 (GraphPad, USA) and the mean values of duplicate measurements. Standard deviations were not included in the fitting analysis since averaged values were used for each substrate concentration.

1. Results and Discussion

3.1. Carbon source optimization

After 24 h of yeast incubation in culture media containing 1% molasses or 1% sucrose as carbon sources, enzyme activity was assessed to investigate the most suitable substrate. Sucrose was selected as the optimal carbon source since molasses interfered with the DNS assay due to its strong reaction with the reagent, making accurate quantification of enzyme activity unreliable.

3.2. Sucrose concentration optimization

After selecting sucrose as the preferred carbon source for enzyme production, various concentrations of sucrose (1–4% w/v) were assessed to investigate the optimal level for maximum enzyme activity. The results showed that the highest enzyme activity was achieved at 1% sucrose.

3.3. Nitrogen source optimization

Culture media containing yeast extract, meat peptone, urea and diammonium phosphate (DAP) were assessed as nitrogen sources. After 24 h of incubation, the enzyme assay results showed that the media containing pharmaceutical-grade urea as the nitrogen source included the highest invertase activity.

3.4. Combination of nitrogen sources

To enhance enzyme production, various combinations of nitrogen sources were assessed. After 24 h of incubation, the results indicated that the media containing a combination of meat peptone and pharmaceutical-grade urea included the highest invertase activity.

3.5. Media pH optimization

Media of various pH levels between 3 and 8 were prepared. After 24 h of yeast inoculation and enzyme assay, the culture media with pH of 5 included the highest enzyme activity. At pH 7 and pH 8, enzyme activity could not be assessed due to the precipitation in the culture media.

3.6. The pH acetate buffer

The buffer used in the assay included acetate buffer. To achieve and ensure the appropriate pH for the invertase enzyme, buffers with various pH levels between 3 and 7 were prepared. Then, the enzyme assay was carried out. The invertase enzyme showed the highest activity at pH 5.

3.7. The rpm optimization

To assess the effects of agitation speed on enzyme activity, shaking rates of 150 and 160 rpm were assessed. The results showed that the culture agitated at 150 rpm included the highest invertase activity.

3.8. Shaker incubator temperature optimization

After inoculation, the culture flasks were incubated at 25, 30 and 35 °C for 24 h. Enzyme assay results showed that the highest invertase activity was achieved at 30 °C.

3.9. Growth curve and enzyme activity curve

The growth and enzyme activity curves were monitored over 48 h by measuring cell growth (OD₆₀₀) every 2 h and invertase activity approximately every 4 h, except during the early stage of inoculation, when enzyme secretion did not begin. The growth curve showed two distinct logarithmic phases, likely due to the presence of dual nitrogen sources in the media. The highest enzyme activity was observed at 18 and 48 h, while the maximum optical density (OD₆₀₀) occurred at 38 h. These results indicate that the peak enzyme secretion did not coincide with the time of maximum yeast growth (biomass accumulation).

3.10. Fermentor

During the first 24 h of fermentation, no sampling was carried out to minimize the risk of contamination. After this time, samples were collected at regular intervals to monitor cell growth and enzyme activity. The highest optical density (OD₆₀₀) was recorded at 48 h, while the maximum invertase activity occurred at 28 and 96 h. Similar to the shake-flask experiments, the peak of enzyme activity did not match with the maximum cell growth, indicating that invertase secretion was not directly correlated with the biomass accumulation.

3.11. Effects of temperature

The crude enzyme was incubated at 30, 40, 60, 70 and 90 °C for 30 min, followed by activity measurement using standard assay. The highest invertase activity was observed within the temperature range of 40–60 °C, with similar activity levels at 40 and 60 °C, indicating that the enzyme preserved its substantial stability and catalytic efficiency across this range.

3.12. Michaelis-Menten equation

The Michaelis–Menten parameters of invertase were investigated through non-linear regression at 30 and 50 °C, as summarized in Table 1. At 30 °C, the Km and Vmax values were 63.35 mM and 0.8557 µmol min^-1, respectively. At 50 °C, Km increased to 124.2 mM and Vmax to 2.283 µmol min^-1. The higher Km at 50 °C indicated decreased affinity of invertase for sucrose at increased temperatures, suggesting that structural changes or increased flexibility of the enzyme at higher temperatures might affect substrate binding. The increase in Vmax at 50 °C showed that the catalytic rate could increase at higher temperatures; however, the overall efficiency was moderated by the decreased substrate affinity.

3.13. Amicon ultra centrifugal filter

In this study, Amicon 10-kDa filter was used to concentrate the enzyme. Before centrifugation, the volume in the tube was 3.5 ml and after centrifugation, the rest of volume in the Amicon filter was 1.4 ml; hence, this was 2.5 times further concentrated.

The results of this study showed that the yeast reached its highest growth rate within 38 h, while the best enzyme activity was seen within 18 h, highlighting the fact that enzyme secretion was not strictly coupled to growth, a phenomenon reported for S. cerevisiae MK, where the maximum invertase production occurred at 48 h while biomass increased up to 96 h [19]. These observations highlighted the importance of optimizing harvest time to achieve maximal enzyme yield. Such differences highlighted the importance of selecting the optimal harvest time to maximize enzyme activity. The present results, including that the invertase enzyme pH optimization was 5, were similar to those of Al-Saady’s study [16] but differed from Shankar's study [18], where maximum activity was observed at pH 6 for invertase from S. cerevisiae MTCC 170, suggesting that strain-specific variations in invertase isoforms or differences in post-translational modifications and culture conditions such as media composition, temperature and aeration might further affect the pH profile. In 2024, Dokuzparmak investigated the effect of pulp on the activity of the invertase enzyme in S. cerevisiae and the results showed that the invertase enzyme activity increased by 2.5 times. In the current study, a combination of 1% sucrose, 0.5% urea and meat peptone at pH 5 and 30 °C and 150 rpm yielded the highest enzyme activity, aligning with Dokuzparmak’s findings under comparable conditions [20]. However, it is noteworthy that the optimization was carried out using one-factor-at-a-time (OFAT) approach, which did not account for potential interactions between the factors. For example, the optimal sucrose concentration investigated by one nitrogen source may shift, when a various nitrogen source is used. Future studies can use factorial design or response surface methodology (RSM) to investigate such interactions and achieve further precise optimization.

In this investigation, the invertase from S. cerevisiae PTCC 5209 showed high activity over a broad temperature range (40–60 °C), which was similar to that of Du's study [21], who reported the maximum activity of the enzyme at 45 °C. This suggested that the enzyme preserved substantial activity within a wide range of temperatures. In this study, 3-l fermentor was used to investigate the growth and activity of yeast at a large scale, which showed the highest OD value within 48 h. In general, the achieved results showed that use of S. cerevisiae bacteria with the optimization of culture media parameters included a positive effect on increasing the production of invertase enzyme, similar to previous studies [22–24]. In addition, analysis of the fermentor growth curve revealed differences between the maximum biomass growth and the maximum enzyme activity. This highlighted the necessity of careful monitoring of fermentation time to avoid unnecessary accumulation of biomass in conditions, where enzyme efficiency reached its optimal level. Therefore, further research can focus on developing advanced fermentation methods or optimizing strains to achieve further optimal enzyme secretion and activity.

1. Conclusion

Invertase is found in nature in various isoforms. This enzyme is widely distributed in plants and microorganisms. In plants, three isoforms of invertase differ in biochemical characteristics and subcellular locations. The role of invertase in plants extends beyond metabolism, as it is effective in osmotic regulation, growth and immune system strengthening. In the human body, invertase is addressed as an immune system booster, antioxidant and disinfectant agent and is useful in certain cases of patients with bone and stomach cancers. The importance of this enzyme in these reactions led the current authors to investigate the structure of the enzyme and optimize conditions for its maximum production. Regarding that S. cerevisiae, commonly known as baker's yeast, is the major strain used for the production and purification of invertase, S. cerevisiae was used under optimal conditions to maximize the production and activity of this enzyme. In yeasts, this enzyme can be detected extracellularly or intracellularly. In this study, invertase production by S. cerevisiae PTCC 5209 was maximized under optimized nutritional and culture conditions. The study detected that invertase production peaked at 18 h under 1% sucrose, 0.5% urea and meat peptone, pH 5, 30 °C and 150 rpm conditions. Combination of urea and meat peptone resulted in the highest invertase activity, likely due to the complementary nature of these nitrogen sources as urea provided a rapidly metabolizable form of nitrogen and meat peptone supplied complex peptides and amino acids that supported enzyme synthesis. Moreover, urea represented an economically advantageous nitrogen source, being an inexpensive petrochemical product that is readily available in Iran, which made this combination particularly appropriate for industrial-scale enzyme production. The process was successfully scaled up in 3-l fermentor, demonstrating its potential for industrial enzyme production. Although experiments were carried out under specific laboratory conditions, findings included broader uses for the global biotechnology and food industries, where invertase plays a vital role in processes such as sucrose hydrolysis, prebiotic synthesis and fermentation. Further studies could focus on optimizing fermentation techniques or improving the yeast strain to increase enzyme production.

1. Acknowledgements

The authors would like to thank [Alzahra University/Department of Biotechnology/Industrial Biotechnology Laboratory] for providing technical support and laboratory facilities for this study.

1. Declaration of competing interest

The authors report no conflict of interest.

1. Authors’ Contributions

All authors participated in project administration and writing of the primary draft of the manuscript, providing critical revision and editing.

1. Using Artificial Intelligent Chatbots

This manuscript was written entirely by the authors. Artificial intelligence tools were used only to improve the English language and enhance readability.

1. Ethical Consideration

The authors confirm that this research involved only microbial (yeast) experiments and did not include human or animal subjects. Accordingly, ethical approval was not applicable for this study.

Background and Objective: This study aimed to investigate the antihyperlipidemic effects of potent probiotic Lactobacillus strains isolated from traditional Iranian dairy in a high-fat diet rat model.

Material and Methods: Lactobacillus strains were isolated from tarkhineh samples and screened for significant in-vitro cholesterol and triglyceride-decreasing activities, with key probiotic characteristics. Seven strains were selected based on their high in-vitro lipid-decreasing activity, substantial resistance to simulated gastric and intestinal conditions, resistance to 0.5% phenol and 15 mg l^-1 lysozyme, adhesion capacity to Caco-2 cells, antibiotic susceptibility profiles, and antagonistic activity against human pathogens in male Wistar rats fed a high-fat diet. The lipid-decreasing activity was assessed in male Wistar rats (n = 7 per group) over 6 weeks, with the probiotic mixture administered daily at a dose of 2 × 10⁹ CFU ml^-1 rat^-1.

Results and Conclusion: Using 16S rDNA analysis, these strains were identified as Lactobacillus casei, Lactobacillus fermentum, Lactobacillus kefiri, Lactobacillus alimentarius, Lactobacillus acidophilus, Lactobacillus reuteri, and Lactobacillus brevis. The lipid-decreasing activity was assessed in male Wistar rats. Compared to the high-fat diet control group, the probiotic-supplemented diet decreased serum total cholesterol by 16.6% and LDL-cholesterol by 56.7% (p < 0.05). Particularly, the probiotic mixture resulted in a 13.2-fold increase in the HDL-c/LDL-c ratio (from 0.132 to 1.75), compared to high-fat diet controls (p < 0.01). Mechanistically, the probiotic diet increased fecal cholic acid excretion by 4.7-fold (from 1.17 to 5.54 µmol g^-1) (p < 0.05) and decreased hepatic steatosis. Treatment attenuated high-fat diet-induced upregulation of lipogenic genes (PPAR-γ, ACC, FAS, and C/EBPα) and restored the expression of AMPKα. These results indicated that supplementation with lactobacilli from homemade dairy was effective in improving dyslipidemia, suggesting these products could be a promising source of novel probiotics.

Keywords: Probiotics, Traditional dairy, Lactobacillus spp., Tarkhineh, Hyperlipidemia

1. Introduction

High levels of serum lipids are significant risk factors for cardiovascular disease (CVD). Individuals with dyslipidemia have a threefold higher risk of heart attack, compared to those with normal lipid levels. This condition is often related to dietary habits, particularly the consumption of unhealthy diets. Diets high in fat, especially saturated fatty acids (SFA), can increase blood total cholesterol (TC) and triglyceride (TG) levels, which increases the risk of atherosclerosis, coronary heart disease, and stroke [1, 2].

Medications such as statins and fibric acid derivatives are commonly used to manage blood lipid levels. However, these conventional drugs can include adverse effects, including muscle pain, liver damage, neurological disorders, increased blood sugar, and miscarriage. Therefore, there is an increasing need for safer and further cost-effective dietary interventions to address imbalanced serum lipids. Probiotics have emerged as a potential solution for managing dyslipidemia, and their use in functional foods is a promising area of research. Probiotics have been shown to significantly assimilate cholesterol and TG from culture media. Bacteria such as Lactobacillus, Lactococcus, and Bifidobacterium spp. are commonly detected in fermented dairy products [3-5].

Traditional fermented foods contain unique microbial communities that are shaped by local production methods and environmental conditions [6]. Within the lactic acid bacteria (LAB) family, Lactobacillus spp. are the most common members. These bacteria are often isolated from various Iranian traditional fermented foods. One such food is tarkhineh, a dried fermented mixture of yoghurt, cracked wheat, and vegetables, which offers a stable acidic matrix conducive to LAB preservation. The dry acidic nature of tarkhineh allows for the long-term preservation of its milk proteins [7].

While numerous studies have screened probiotics for cholesterol-decreasing characteristics, most isolates originate from well-characterized sources such as commercial yogurts or dairy products with standardized fermentation [8]. The wide microbial diversity of traditional, regionally distinct Iranian fermented foods, such as tarkhineh, as a source of novel strains with potentially superior or unique functionalities, is largely uninvestigated. Moreover, tarkhineh offers a unique ecological niche as a stable acidic matrix with high solid contents. This environment imposes selective pressure on the microbiota, favoring highly robust and acid-resistant Lactobacillus strains [7, 9]. 

These environmental stressors suggest that isolates from tarkhineh may possess superior functional traits such as enhanced resistance to gastric conditions and greater bile-salt hydrolase activity, compared to strains from conventional sources [10]. Furthermore, studies of novel isolates depend solely on in vitro screening, failing to provide the necessary comprehensive in vivo validation. The precise molecular mechanisms underlying the antihyper-lipidemic effects, particularly regarding the modulation of key hepatic lipogenic genes, are often not fully clarified for novel isolates. Therefore, this study aimed to fill this critical gap by providing a full characterization of novel Lactobacillus strains from tarkhineh, followed by a robust,  mechanistic in vivo validation of their efficacy in a high-fat diet (HFD) rat model.

1. Materials and Methods

2.1 Isolation and Preliminary Identification of Lactobacilli

Samples of tarkhineh (n = 22) were collected during July and August 2024 from various rural areas of Lorestan Province, Iran, including Dowlatabad and Mahrouw Villages in Aligoudarz County (33°09′N 49°24′E) and Dehnow and Emamabad Villages in Dorud County (33°29′58″N 49°03′11″E). To prevent microbial contamination and changes to the primary microbiota, samples were prepared under hygienic conditions and stored at 4 °C. For isolation, 5 g of each sample was mixed with 50 ml of sodium citrate (Merck, Germany) and homogenized using a stomacher. The samples were serially diluted up to 10⁻⁷ in sterile saline solution and cultured on de Man, Rogosa, and Sharpe (MRS) agar (HiMedia, India). After anaerobic incubation (Gas-pack system) at 37 °C for 48 h, colonies were inoculated into 10 ml of MRS broth and incubated at 37 °C for 24 h. The isolates were screened through primary morphological assessment using a fluorescent microscope (Zeiss Axioskop, Germany) and standard biochemical assessments, including catalase, oxidase, and carbohydrate fermentation. Screened lactobacilli were preserved at -70 °C in glycerol and skim milk.

2.2 Bile Salts and Low pH Tolerance

The ability of isolates to tolerate bile salts was assessed as described by Saboori et al. [11]. Each strain (2% v/v) was grown in MRS broth containing 0.3% (w/v) bile salts (0% as a control). Cultures were incubated at 37 °C for 6 h, and optical density (OD) was measured at 600 nm. For acid tolerance, strains were cultured overnight in MRS broth adjusted to pH values of 2.0, 2.5, and 6.4 (as a control). The OD values at 600 nm were recorded at the beginning and end of cultivation. Tolerance was calculated using methods described by Liu et al. [12].

2.3 In-vitro Cholesterol and Triglyceride Decreasing Activities

The cholesterol-decreasing ability of strains was assessed using the method by Liu et al. [12]. Strains were grown in MRS broth at 37 °C for 24 h and then inoculated into MRS broth supplemented with cholesterol and 0.3% bile salts (MRS-CHOL). A control sample of uninoculated MRS-CHOL was also prepared. After 24 h of incubation at 37 °C without shaking, the bacterial broth was centrifuged, and cholesterol content in the cell-free supernatant was assessed. Strains that lowered cholesterol by more than 50% were selected for triglyceride (TG) decreasing assessments. These strains were grown overnight in MRS broth and then transferred to MRS-TG broth. This was prepared by mixing 20 ml of 2% polyvinyl alcohol solution with 50 ml of triglycerides (Merck, Germany) and adding the mixture to MRS broth (3% v/v). The pH was adjusted to 6.5, and the medium was sterilized. After 72 h of incubation at 37 °C, the TG quantity in the cell-free supernatant was assessed using a commercial kit (Cayman, USA) [13].

2.4 Cell Survival in Simulated Gastrointestinal Juice

To screen for tolerance to gastrointestinal stress, isolated lactobacilli were cultured in MRS broth and incubated anaerobically overnight at 37 °C. The suspension was centrifuged at 6000× g for 7 min, and the cells were washed twice with sterile phosphate-buffered saline (PBS, pH 2.0). Aliquots were cultured on MRS agar and incubated anaerobically at 37 °C for 0, 1, 2, and 3 h. The number of viable bacteria was assessed as colony-forming units per milliliter (CFU ml^-1). For assessment under simulated conditions, overnight cultures of isolates were centrifuged at 6000× g for 5 min, washed with 50 mM PBS (pH 6.5), and dissolved in 3 ml of PBS buffer. One-ml aliquots of each isolate (containing 9-log CFU ml^-1 of bacteria) were mixed with 9 ml of simulated gastric juice (7 mM KCl, 45 mM NaHCO₃, 125 mM NaCl, and 3 g l^-1 pepsin at pH 2.5). The mixtures were incubated at 37 °C for 3 h and then centrifuged at 4000× g for 7 min. The pellets were washed three times with PBS and resuspended in simulated intestinal juice (pH 8.0) containing 0.15% (w/v) bile salt and 0.1% (w/v) pancreatin. Then, the suspensions were incubated at 37 °C for another 3 h, and the viable bacteria were counted and reported as Log CFU ml^-1.

2.5 Phenol and Lysozyme Tolerance

Phenol tolerance is critical for lactobacilli survival in the gastrointestinal tract (GIT), as phenol can be produced by gut microbiota. Bacterial cultures grown for 24 h were cultured in MRS broth containing 0.5% phenol (Sigma, USA). The ODs were measured at 580 nm using a microplate reader (Thermo Fisher Scientific, USA). Lysozyme tolerance was assessed as described by Zafar et al. [14]. Bacterial cells were collected (4000× g, 5 min), washed, and resuspended in PBS. Then, 10 µl of cell suspensions were transferred to PBS solutions containing 15 mg l^-1 lysozyme (0% as a control). After 2 h of incubation at 37 °C, ODs were measured at 600 nm to estimate survival rates.

2.6 Safety Assessment

2.6.1 Antibiotic Susceptibility

The antibiotic susceptibility of isolated lactobacilli was assessed based on the method of Saboktakin et al. [15]. Strains (1.5 × 10⁸ CFU ml^-1) were cultured on MRS agar. Antibiotic discs were transferred onto the surface, set at room temperature (RT) for 10 min for diffusion, and then incubated at 37 °C overnight. The areas of inhibition around each disc were assessed.

2.6.2 DNase and Hemolytic Activities

To assess DNase activity, isolates were streaked on DNase agar (HiMedia, India) and incubated at 37 °C for 72 h, and then clear zones were assessed. Staphylococcus aureus was used as a positive control. Hemolytic activity was assessed on blood agar containing 5% v/v sheep blood. Isolates were cultured on the media and incubated at 37 °C for 48 h. Blood lysis zones were characterized as α-hemolysis (green zones), β-hemolysis (clear zones), or γ-hemolysis (no zones). Only isolates showing γ-hemolysis were considered safe [16].

2.7 Antibacterial Activity Assessment

Antibacterial spectra were assessed using agar diffusion assay [17]. The indicator bacteria were five human pathogens of Salmonella typhimurium ATCC 14028, Escherichia coli ATCC 25922, Listeria monocytogenes CMCC 54002, Bacillus subtilis ATCC 11060, and Staphylococcus aureus ATCC 25923. These pathogens were overlaid on Mueller-Hinton (M-H) agar plates with 7-mm diameter wells. Freshly prepared cell-free supernatants of each isolate were filtered (0.2-µm filter), poured into the wells, and incubated at 37 °C overnight. Then, diameters of the inhibitory zones were assessed.

2.8 Assessment of the Isolates' Adhesion Ability

To assess the ability of strains to adhere to Caco-2 cells, an in vitro model for intestinal epithelia was assessed using the method of Greene and Klaenhammer [18]. Caco-2 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (Sigma, USA) supplemented with 1% penicillin-streptomycin and fetal bovine serum (FBS) in a 5% CO₂ atmosphere at 37 °C for 20 days. Cells were transferred to tissue culture plates and grown to a monolayer. The plates were washed with sterile PBS to remove the media, particularly penicillin-streptomycin. Overnight cultures of bacterial strains were centrifuged (5000 rpm, 5 min), washed, and resuspended in DMEM to a concentration of 10⁸ bacteria ml^-1. This suspension was added to Caco-2 monolayer cells and incubated at 37 °C for 1 h. Unbound bacteria were removed by washing with PBS. Then, Caco-2 cells were lysed with Triton X-100 (0.1% v/v), and bacterial counts were carried out on MRS agar. Adhesion capacity was calculated using the Eq 1:

Adhesion capacity (%) = (A / B) × 100                                                           Eq.1

Where A was the number of attached bacteria, and B was the total number of bacteria added to each well.

2.9 Molecular Identification and Phylogenetic Analysis

Genomic DNA was extracted from overnight bacterial cultures using a commercial kit (Pars Azmoun, Iran). A conserved region of the 16S rDNA gene was amplified using 27F-1492R primers as described by Nami et al. Amplicons were electrophoresed on a 2% agarose gel. The amplified fragments were purified using the PureLink PCR purification kit (Thermo Fisher Scientific, USA) and sequenced by BGI Biotechnology (Shenzhen, China). Sequences were compared to the GenBank database via BLAST similarity search; isolates with over 98% similarity to reference sequences were assigned to the same species. A phylogenetic tree was constructed using MEGA7 software (Biodesign Institute, USA) with the Kimura 2-parameter model and the neighbor-joining method [19].

2.10 In vivo Study

A total of 24 male Wistar rats (age, 6 weeks; weight, 150 g ±15) were purchased from the Pasteur Institute, Tehran, Iran. All procedures were carried out based on the ARRIVE guidelines and approved by the ethical committee of Islamic Azad University (IR.IAU.ARAK.REC.1399.021).

Experiments were based on the guidelines in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH, USA). Rats were acclimated for 1 week with commercial foods (Specialty Feeds, USA) and water to minimize environmental stress. The animals were housed in a controlled environment (23 °C, 55% relative humidity, 12-h light/dark cycle). The rats were randomly allocated into four groups of six animals, with similar initial body weights. The groups received the following diets for 35 days:

1. ND, Normal diet
2. HFD, High-fat diet
3. HFD and Pro, HFD with a potential probiotic mixture  (2 × 10⁹ CFU ml^-1 d^-1)
4. HFD and Lov, HFD with lovastatin (15 mg kg^-1 d^-1)

The HFD was composed of 78.3% commercial diet, 5% lard oil, 5% corn germ oil, 5% sucrose, 5% dried egg yolk, 1% cholesterol, 0.5% sodium deoxycholate, and 0.2% propylthiouracil. All administrations were carried out via intragastric gavage. The inclusion of propylthiouracil was a deliberate methodological choice to establish a robust model of complex, resistant dyslipidemia. The probiotic dose was selected based on prior studies demonstrating efficacy in rodent models [12, 20]. Blood samples were collected under ketamine-xylazine anesthesia on Days 0 and 35. Serum was extracted, and TC, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and TG levels were assessed using commercial kits (Pars Azmoun, Iran). At the end of the experiment, rats were euthanized using Forane (isoflurane). Livers were extracted for histological studies using hematoxylin and eosin (H&E) staining and for lipid analysis using a method described by Folch et al. Feces were collected on Days 33, 34, and 35 for cholic acid analysis.

2.11 Real-time Polymerase Chain Reaction

The expression levels of six lipid metabolism-linked genes were assessed using quantitative real-time PCR (qRT-PCR) [21]. Primers were generated for peroxisome proliferator-activated receptor-γ (PPAR-γ), adenosine 5′-monophosphate-activated protein kinase-α (AMPKα), CAATT/enhancer-binding protein α (C/EBPα), hormone-sensitive lipase (HSL), fatty acid synthetase (FAS), acetyl-CoA carboxylase (ACC), and the internal control GAPDH (Table 1). Total RNA was extracted from tissues using TRIzol reagent (Invitrogen, USA), purified with a Qiagen RNeasy mini kit (QIAGEN, Germany), and quantified by spectrometry (Eppendorf, Germany). The cDNA was synthesized using the RevertAid first-strand cDNA synthesis kit (Thermo Fisher Scientific, USA). The qRT-PCR reactions were carried out with SYBR Premix Ex Taq II kit (Takara, China) on a PRIMEPRO48 real-time qPCR thermal cycler (Antylia Scientific, USA). Results were calculated using the 2^_ΔΔCT method and are shown as fold changes.

2.12 Statistical Analysis

Data were analyzed using SPSS software v.18 (IBM, USA) through a one-way analysis of variance (ANOVA). Differences between the means were reported using the Duncan test at a 95% confidence level (p < 0.05). All experiments were carried out in triplicate.

1. Results and Discussion

3.1 Isolation and Screening of Potential Probiotic Lactobacilli

A total of 83 Gram-positive, catalase-negative, coccoid bacterial isolates were collected from tarkhineh samples, all of which showed a carbohydrate fermentation profile characteristic of the Lactobacillus genus. These isolates were used in a bile-salt tolerance prescreening, from which 36 strains showing an inhibition rate below 50% were selected for acid tolerance assessment. Of these, 19 isolates demonstrated survival rates higher than 85% at pH values of 2.5 and 2.0.

High levels of cholesterol and triglycerides in the diet and serum are major risk factors for cardiovascular and metabolic diseases. Since pharmacotherapy can cause adverse effects, it is important to find natural and non-toxic substances to decrease TC, LDL, and TG levels. Lactobacilli, which have been used in fermented dairy products for many years and are generally considered safe (GRAS) [22], have been shown to effectively decrease lipids in animal and clinical research [23, 24]. In this study, seven potential probiotic lactobacilli strains were isolated from Iranian traditional dairy products. These strains showed enhanced cholesterol and triglyceride-decreasing activity, increased tolerance to simulated gastric and intestinal juice, improved adhesion to human intestinal cells, and expanded antibacterial spectrum under in vitro conditions.

3.2 In-vitro Cholesterol and Triglyceride Assimilations

Of the 19 acid-tolerant strains, seven strains showed cholesterol-decreasing rates exceeding 50%. Of these, three strains demonstrated triglyceride (TG)-decreasing rates of more than 40%. As shown in Table 2, the highest cholesterol-decreasing rate (67.41% ±0.20) was observed for Strain L-42, while the highest TG-decreasing rate (51.15% ±0.57) belonged to Strain L-3.

In vitro, the L-42 and L-11 strains showed cholesterol-decreasing rates of 67.41 and 64.27%, respectively. These rates were higher than those of L. fermentum F1 (48.87%) [25], L. acidophilus L2-16 (64.11%) [26], Pediococcus acidilactici LAB 5 (62%) [27], L. sake C2 (53.2%) [28], B. longum (34.2%) [29] and L. helveticus MG2-1 (51.74%) [28].

There are a few investigations on the ability of potential probiotics in decreasing TG. The rate for the L-3 strain was higher than that L. acidophilus L2-73 reported in the Gao et al. study [26]. The authors observed that cholesterol and TG intake varied greatly in strains within the same species. This suggested that cholesterol sequestering was specific to individual strains rather than a characteristic of the entire species [30].

3.3 Resistance to Simulated Gastrointestinal Conditions, Phenol, and Lysozyme

All seven selected strains, except L-51 (69% survival), showed a survival rate above 80% after sequential exposure to simulated gastrointestinal fluids. The strains were highly resistant to simulated gastric fluid, with over 94.8% survival after 2 h and over 90% survival for most strains after 4 h. In simulated intestinal fluid, six isolates survived above 90%. Overall, the strains were relatively more tolerant to acid than bile. There was no significant difference between strains in their tolerance to simulated fluids, which was similar to the initial pH and bile salt assessments. All bacteria tolerated 0.5% phenol after overnight incubation (OD > 1.000) and showed significant resistance to 15 mg l^-1 lysozyme after 2 h of exposure (Table 3).

Probiotics must be able to tolerate gastrointestinal stressors such as acidity, bile, phenol, lysozyme, and pepsin. The current research showed that lactobacilli isolated from fermented dairy demonsterated strong tolerance to simulated gastrointestinal environments.

3.4 Safety Profile of Antibiotic Resistance, DNase, and Hemolytic Activity

The isolated lactobacilli showed various resistance to the assessed antibiotics (Table 4). Chloramphenicol, novobiocin, and penicillin were effective against all the isolates. In contrast, most isolates were resistant to rifampicin. All seven lactobacilli were negative for DNase activity and showed no hemolytic activity (γ-hemolysis) on blood agar, indicating that they were safe.

3.5 Antagonistic Activity against Pathogens

Six of the seven lactobacilli showed moderate antipathogenic activities; strain L-42 did not demonstrate antagonistic activity. The metabolites from the isolates were generally more effective against Gram-negative pathogens such as S. typhimurium and E. coli than Gram-positive ones such as B. subtilis and L. monocytogenes (Table 5).

The isolated strains showed strong antagonistic activity against Gram-positive and Gram-negative pathogens. This is a key beneficial effect, as it helps maintain a balanced intestinal flora in the host and manage gastrointestinal infections.

3.6 Adhesion to Caco-2 Cells

The adhesion rates of the lactobacilli to Caco-2 cells varied between the strains of L-3 (24.2% ±0.11), L-11 (13.3% ±0.11), L-20 (28.9% ±0.05), L-24 (10.8% ±0.24), L-42 (11.5% ±0.21), L-48 (10.6% ±0.42), and L-51 (31.8% ±0.37). Strains L-3, L-20, and L-51 demonstrated significantly higher adhesion capabilities compared to the other strains (p < 0.05). Probiotics must show antimicrobial activity against intestinal pathogens and adhere to the intestinal epithelia. The current in vitro screening showed significant functional differences between the seven Lactobacillus strains, typical of natural isolates. This diversity justified using mixed culture in vivo to target hyperlipidemia through multiple mechanisms. Strain L-42 was the top cholesterol-decreasing agent (67.41%) with strong metabolic activity but no pathogen inhibition, making it a purely metabolic contributor. In contrast, Strains L-24 and L-48 included moderate lipid-decreasing effects but excelled in gastric tolerance, cell adhesion, and pathogen antagonism, supporting gut ecology and safety. This strain-specific variation highlighted a functional consortium, where each strain played unique roles. The in vivo success was likely a result of synergy between Strain L-42 metabolic effects and the survival and protective traits of the other strains.

3.7 Molecular Identification and Phylogenetic Analysis

All seven isolates showed more than 98% 16S rRNA sequence similarity with Lactobacillus spp. The phylogenetic tree (Figure 1) showed that Strains L-3, L-11, L-20, L-24, L-42, L-48, and L-51 clustered with L. casei, L. fermentum, L. kefiri, L. brevis, L. acidophilus, L. reuteri, and L. alimentarius, respectively.

The biochemical characteristics of these strains were similar to those of lactobacilli, and their nucleotide sequences were homologous to L. casei, L. fermentum, L. kefiri, L. alimentarius, L. acidophilus, L. reuteri, and L. brevis.

3.8 In-vivo Antihyperlipidemic Effects

3.8.1 Effects on Serum Lipid Profile

On Day 0, serum TC, LDL-C, HDL-C, and TG levels were similar in all groups. By Day 35, one-way ANOVA followed by Duncan's post-hoc test verified that the HFD group showed significant increases in serum TC, LDL-C, and TG, while HDL levels significantly decreased by 38.2% (all p < 0.01 compared to the ND group). Compared with the HFD group, HFD and Pro and HFD and Lov groups had significantly lower TC and LDL-C levels and significantly higher HDL levels (all comparisons with p < 0.01, Duncan's test) (Figure 2a). Particularly, the TG level in the HFD and Pro group was significantly lower than that of the HFD group (p < 0.05) and the HFD and Lov group (p < 0.05, Duncan's test). The HDL-c/LDL-c ratio was significantly higher in the HFD and Pro group (1.75 ±0.15), compared to the ND (p < 0.01), HFD (p < 0.01), and HFD and Lov (p < 0.05) groups, as assessed by Duncan's post-hoc test. This improvement underscored the probiotics' potential in modifying atherosclerosis risk, though clinical validation in humans is warranted. Comparative effects of normal diet, HFD, probiotic supplementation, and lovastatin on body weight, food intake, and fecal lipid excretion in experimental groups are available in Table 6.

3.8.2 Effects on Liver Lipids and Histology

As shown in Figure 2b, liver TC and TG levels in the HFD group were significantly higher than those in the HFD and Pro and HFD and Lov groups (comparisons with p < 0.01, Duncan's test). The probiotic-treated rats had significantly lower liver TG levels than the lovastatin-treated group (p < 0.05). Histological examination revealed a normal liver structure in the ND group (Figure 2c). In contrast, the HFD group showed a high degree of vacuolization, lipid accumulation, hepatocyte ballooning, inflammatory cell infiltration, and mild necrosis. Liver sections from the HFD and Pro, and HFD and Lov rats showed significantly fewer pathological changes, compared to the HFD group.

The strains were assessed in a rat model to assess their ability to decrease cholesterol and triglyceride levels. Results showed that administering a combination of isolated probiotics successfully decreased serum and liver levels of TC, TG, and LDL-C in Wistar rats fed a high-fat diet, compared to rats fed a similar diet without probiotics or lovastatin supplementation. The findings revealed that probiotics were as effective as lovastatin in improving dyslipidemia. The reduction in serum and liver TC, TG, and LDL by the probiotics mixture was higher than that of L. plantarum YS5 [20], L. plantarum PH04 [31], B. bifidum PRL2010 [30], and E. faecalis L2-73 [26]. The HDL levels and HDL-c/LDL-c ratio increased in the HFD and Pro group, which was similar to the results of several other studies [14, 32, 33]. L. rhamnosus CK102 was a probiotic strain isolated from fermented yogurt. In a mouse model, it decreased TC by 27.9%, HDL by 28.7% and TG by 61.6% [34]. Epidemiological studies have shown a link between CVD and serum TC and TG levels. Similarly, another study detected that L. fermentum MCC2760 decreased TC, TG, and LDL-c levels in mice [35]. In another rat model, L. fermentum PH5 improved blood lipid profiles by decreasing TC (67.21%), TG (66.21%), and LDL-c (63.25%). The L. fermentum PH5 and PD2 decreased liver TC levels, with L. fermentum PH5 performing better than PD2 [36].

3.8.3 Effects on Fecal Cholic Acid Excretion

Fecal cholic acid levels were 5.14 ±0.15 µmol g^-1 for the ND group, 1.17 ±0.10 µmol g^-1 for the HFD group, 5.54 ±0.18 µmol g^-1 for the HFD and Pro group, and 2.16 ±0.19 µmol g^-1 for the HFD and Lov group. The HFD and Pro group had significantly higher concentrations of fecal cholic acid than the ND, HFD, and HFD and Lov groups (all comparisons with p < 0.05, Duncan's test). There is increasing evidence that gut microbiota plays a key role in the development of CVD and metabolic disease. The authors detected that rats supplemented with probiotics had increased levels of cholic acids in their feces. This increase in fecal bile acid elimination has been reported in several probiotic strains, including B. bifidum PRL2010 [30] and E. faecalis L2-73 [26]. The current in-vivo study lacked gut microbiota sequencing and key metabolite assessments (SCFAs, BSH). Despite increased fecal bile acid excretion, microbial mechanisms are unverified. Further studies are needed to validate gut-mediated hypolipidemic pathways.

3.8.4 Effects on Hepatic Gene Expression

The HFD caused a significant increase in the mRNA expression of PPAR-γ, ACC, FAS, and C/EBPα, compared to the ND group (all comparisons with p < 0.05, Duncan's test). This upregulation was normalized by supplementation with either the probiotic mixture or lovastatin (Figure 3). The mRNA expression of AMPKα was significantly downregulated in the HFD group (p < 0.01). However, treatment with the probiotic mixture significantly increased the mRNA expression of AMPKα (p < 0.01). The mRNA level of HSL increased, but not significantly.

The authors assessed the expression of lipid metabolism-linked genes in experimental groups to investigate the potential mechanisms by which isolated lactobacilli decreased dyslipidemia induced by a high-fat diet in rats. The PPAR-γ and C/EBP-α are key transcription factors for adipocyte differentiation in various tissues. Previous studies showed that lactobacilli supplementation decreased the expression levels of PPAR-γ and C/EBP-α in high-fat diet-fed animals. In this investigation, probiotics attenuated HFD-induced upregulation of lipogenic genes (PPAR-γ, ACC, FAS, and C/EBPα), with restoration of AMPKα expression. The AMPK pathway regulates lipid metabolism, and the enzyme is a cellular energy sensor that shuts down anabolic pathways such as fatty acid synthesis. The ACC and FAS are key proteins in the fatty acid synthesis process [37]. Activation of AMPK-α stimulates ACC phosphorylation, blocking FAS expression. The HSL is the rate-limiting enzyme in TG breakdown in various tissues. In this study, the expression of AMPK-α was downregulated in the HFD group, compared to the ND group, while ACC and FAS expressions were upregulated. These changes were alleviated by probiotic treatment, similar to the previous studies [21, 38]. Overall, these findings indicated that isolated lactobacilli administration effectively modulated the gut microbiota induced by HFD, improving dyslipidemia-linked indicators.

While the current use of the HFD rat model provided robust evidence of the probiotic mixture efficacy, demonstrating significant normalization of serum lipid profiles, decrease of hepatic steatosis, and mechanistic regulation of key lipogenic genes, it is imperative to acknowledge the inherent limitations of translating these findings directly to human subjects. A primary constraint is linked to the physiological differences between rodents and humans, particularly in lipid metabolism and lipoprotein profiles [39]. Rats are generally HDL-dominant (carrying most cholesterol on HDL particles), whereas humans are LDL-dominant. This fundamental difference can affect the precise way that dietary components and probiotic interventions modulate cholesterol transport and clearance [40].

Additionally, the gut microbiome structure and composition, even in controlled animal settings, may not fully recapitulate the complexity of the human gut environment. Therefore, the promising results from this study served as the foundational preclinical evidence for this specific mixed culture [40]. The necessary step for validating the potential of this Lactobacillus consortium as a functional food for dyslipidemia includes carrying out randomized, double-blind, placebo-controlled clinical trials in hyperlipidemic human populations. These trials must focus on verifying the long-term safety, optimal dosage, and efficacy against human-specific clinical endpoints such as serum LDL-C, non-HDL-C, and atherosclerotic markers.

1. Conclusion

In conclusion, homemade traditional dairy was screened for its potential to introduce novel and promising probiotic bacteria due to variable animal feeding and the rare use of antibiotics. Seven potential probiotics were successfully isolated from tarkhineh food, and in vitro assessments showed that several strains showed excellent probiotic potential based on their cholesterol and triglyceride assimilation, survival rate under simulated GIT stress, antibiotic susceptibility, antagonistic activity against pathogens, and adhesion to Caco-2 cells. In vivo assessment showed that feeding animals with isolated probiotics could improve dyslipidemia caused by a high-fat diet. Although the current study included limitations due to the use of Wistar rats and the effects seen in animal models might not be the same in humans, these strains show promise for developing novel probiotic-enriched dairy products targeting dyslipidemia in at-risk populations.

1. Acknowledgements

This research was technically supported by Pasargad Laboratory.

1. Declaration of competing interest

The authors report no conflict of interest.

1. Authors’ Contributions

Designate each author’s contribution using their initials. Conceptualization, P.A and K.A.; methodology, software, validation, formal analysis, H.M.; investigation, resources, data curation, K.A.; writing—original draft preparation, P.A writing review and editing, S.S.; visualization, supervision, and project administration.

1. Using Artificial Intelligent Chatbots

No artificial intelligence chatbots have been used in any section of the present study.

1. Ethical Consideration

An ethical committee of Islamic Azad University approved the study (IR.IAU.ARAK.REC.1399.021) and experiments were done according to Guide for the Care and Use of Laboratory Animals of National Institutes of Health.

Background and Objective: Rosa damascena Mill. possesses bioactive compounds, including flavonoids and anthocyanins, which are addressed for their antioxidant and anti-inflammatory characteristics. This study aimed to develop and optimize a microencapsulation system for rose extract and probiotics. It focused on particle size and morphological characteristics analyzed via particle size analysis, scanning electron microscopy-energy dispersive X-ray spectroscopy and , gas chromatography-mass spectrometry and further assessed the bioavaliability and bioaccessibility of the encapsulated probiotics.

Material and Methods: Lacticaseibacillus casei and Bifidobacterium longum were cultivated in De Man, Rogosa, and Sharpe broth. The petals of Rosa damascena Mill. were extracted with ethanol 70% 1:1 aqueous solution. The microencapsulation involved dissolving the extract and probiotics, followed by the addition of chitosan and sodium tripolyphosphate to form stable colloids. The particle size was analyzed using dynamic light scattering and the morphology of microcapsules was investigated using scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy. Detection of ethanol was carried out using gas chromatography-mass spectrometry. Probiotic viability was assessed after storage at 4 °C for 0, 14 and 28 d and bioaccessibility was assessed using in vitro gastrointestinal simulation method.

Results and Conclusion: The microencapsulation process resulted in spherical microcapsules with a mean particle size of 3107 nm ±273.2. Scanning electron microscopy analysis verified uniform morphology, indicating effective encapsulation. The probiotic count for microencapsulated samples was 5.16 ±0.37 log cfu ml^-1. Gas chromatography-mass spectrometry data showed that the ethanol content was 2.53% ±0.21 (v/v). Microencapsulation of R. damascena Mill. and the probiotics increased the recovery of anthocyanin by 9%. These findings have suggested that combining microencapsulation with probiotic strains provides viable strategy to improve the functional delivery of anthocyanin-rich botanicals in nutraceutical uses.

Keywords: Microencapsulation, Rosa damascena Mill. extract, Lacticaseibacillus casei, Bifidobacterium longum, Chitosan

1. Introduction

Rosa damascena Mill., a member of the genus Rosa (Rosaceae), comprises more than 200 species worldwide. This species is extensively cultivated not only as an ornamental plant but also as a valuable source of raw materials for cosmetic and pharmaceutical industries due to its characteristic aroma and diverse bioactive compounds, including tannins, flavonoids, terpenes, polyphenols, carotenoids, phenylethyl alcohol and vitamins B, C, E and K. Of these constituents, anthocyanins (a major flavonoid pigments in red rose petals) demonstrates strong antioxidant activity, lessening oxidative stress, enhancing immune defense and providing additional biological benefits such as anti-inflammatory, antidiabetic, anticancer, cardiopro-tective and neuroprotective effects [1-2].

Anthocyanins can alter gut microbiota composition by promoting the growth of beneficial bacteria such as Bifidobacteria and Lacticaseibacillus, which are essential for maintaining gut barrier health. [1, 3]. Anthocyanins modulate the expression of tight junction proteins such as occludin, claudin and ZO-1, which are essential for forming and maintaining intestinal epithelial barrier integrity. Tight junctions act as selective barriers that restrict paracellular permeability to harmful molecules andregulation of these proteins by anthocyanins enhances preservation of intestinal barrier function [1, 3].

Lacticaseibacillus casei is addressed for its resilience in acidic environments such as the stomach and intestines and its ability to produce lactic acid, which inhibits the growth of pathogenic bacteria in the gut. The L. casei has demonstrated positive effects in enhancing intestinal function, reliving  gastrointestinal (GI) symptoms and decreasing inflammation within the digestive system. Additionally, research shows that L. casei can improve muscle strength and physical function in animal models and help lessening oxidative stress; thereby, promoting overall health [4]. Bifidobacterium longum provides anti-inflammatory and antioxidant benefits that decreasing liver lipid accumulation and improve muscle and cognitive functions in aging animal models. The B. longum has been shown to enhance intestinal barrier function and improve the gut microbiota composition [5]. The L. casei and B. longum demonstrate immunomodulatory effects by enhancing the body immune response to infections. In studies on Plasmodium infection, B. longum was detected effective in decreasing parasitemia and inflammation, indicating its capability to strengthen immune defense by modulating gut immune responses [5-6]. The L. casei and B. longum can lessendysbiosis or gut microbiota imbalances caused by infections. The bioactive components of R. damascena and probiotic functions of L. casei and B. longum present promising ways for improving overall health, specifically through enhanced antioxidant activity, immune response and gut microbiota balance, making them valuable for therapeutic uses.

Microencapsulation is a process that involves coating a compound or particle to form microcapsules. This microencapsulation concept enables the separation of compounds and probiotic cells from their environment through a protective layer. The characteristics of this protective system are designed to safeguard the cell core and release it in a controlled manner under specific conditions, while allowing the transport of small molecules through the membrane [7]. Chitosan is effective as a coating agent due to its advantages, including non-toxicity, appropriateness for drug delivery, biodegradability and biocompatibility. Chitosan is a natural linear biopolyamino sugar derived through the deacetylation of chitin, with a non-linear chain formula (C₆H₁₁NO₄)n that is odorless and white[8]. Chitosan microencapsulation of R. damascena has successfully been carried out, demonstrating its efficacy in preserving flower quality and extending shelf life, offering a sustainable and effective approach for postharvest storage in commercial uses [9]. Studies have shown that probiotic bacteria can survive after being encapsulated with chitosan. This is because encapsulation efficiency significantly increases with higher concentrations of the biopolymer [10]. The survival rate of chitosan-encapsulated probiotics is high after incubation at low pH, although the population decreases slightly. The decrease in bacterial count during gastric acid simulation is attributed to the highly acidic stomach pH, which affects the strength of the sodium alginate-chitosan polymer as a matrix for encapsulating lactic acid bacteria (LAB) [11]. Moreover, microencap-sulation techniques, particularly those using chitosan, provide promising ways for enhancing the stability and bioavailability of these probiotics in GI environments; thereby, improving their therapeutic potentials. Microen-capsulation of R. damascena Mill. and probiotics (L. casei and B. longum) (MERP) underscores the value of R. damascena bioactives and probiotic encapsulation in preventive and therapeutic uses for managing oxidative stress, inflammation and gut health.

The research identified several key gaps in the existing literature. Previous studies have largely focused on the antioxidant and anti-inflammatory characteristics of R. damascena Mill. and the probiotic benefits of L. casei and B. longum individually. However, a limited attention has been paid to synergizing these bioactives and probiotics within a stable microencapsulation system to enhance their bioavailability. The present findings, however, provided additional insight and might serve as a valuable reference for further development of a chitosan-sodium tripolyphos-phate (STPP) microencapsulation system for the synergistic co-encapsulation of R. damascena Mill. bioactives (anthocyanins and flavonoids) and probiotics (L. casei and B. longum). Combining antioxidant-rich rose extracts with probiotics in a robust encapsulation matrix, this study uniquely linked further nutraceutical uses with gut health and inflammation management, highlighting its potentials for advancing targeted delivery in the functional food system.

1. Materials and Methods

Chitosan was purchased from Merck (Germany). Solvents, including distilled water used as a solvent obtain from Brataco (Indonesia) and ethanol used as a co-solvent, obtained from Merck (Germany). All other chemicals and solvents used in this study were of analytical grade. The procedures for Rosa damascena extraction, probiotic culturing, and microencapsulation using the chitosan–STPP system are illustrated in Figure 1.

2.1. Probiotic Isolation Stage

The L. casei and B. longum were purchased from the Food and Nutritional Culture Collection (FNCC), Gadjah Mada University, Indonesia. Colonies were sampled using sterile inoculation needle and each bacterium was inoculated into De Man, Rogosa, and Sharpe (MRS broth). The cultures were then incubated at 37 °C for 24 h. After incubation, their optical density and colony counts were recorded using spectrophotometer (Thermoscientific, USA). Gram staining was carried out to ensure purity of the bacteria [5].

2.2. Red Rose Extraction Stage

Rose petals were extracted using 70% ethanol pro analis (Merck, Germany) and aliquoted 1:1 [12]. The extract was then filtered through filter paper or a vacuum filtration system to separate the solid from the liquid. The extract was evaporated using rotary evaporator (Materia Medica Batu, Indonesia). The resulting liquid extract was collected using sterile glass bottle. The extract was freeze-dried (Martin Christ, Germany).

2.3. Microencapsulation Preparation

The Microencapsulation was carried out using chitosan-STPP matrix. The stages of MERP formulation included preparation of chitosan (Merck, Germany) in 2% acetic acid (Merck, Germany), preparation of 0.1% NaTPP (Brataco, Indonesia) in distilled water (DW), preparation of 40% rose extract in absolute ethanol:aliquot (3:1) and synthesis of MERP with the composition of 10 ml of rose extract, suspension of L. casei and B. longum each as much as 5 × 10⁹ CFU ml^-1, 8 ml of NaTPP and 32 ml of chitosan. The samples were stirred for 60 min at 400 rpm and stored at 4 °C. Then, the sample was freeze-dried [13]. This formulation was used in in vivo studies. The microencapsulation formulation contained 80 mg of rose extract, 5 × 10⁹ L. casei and 5 × 10⁹ B. longum ml^-1. The selected dose was based on prior evidence showing the anti-inflammatory effects of R. damascena Mill extract at doses ranging from 500 to 1000 mg kg^-1. Additionally, L. casei and B. longum have been reported to include anti-inflammatory effects within a dose range of 5 × 10⁶ to 5 × 10⁹ CFU for L. casei and 10⁸ to 10¹⁰ CFU for B. longum [21-22].

2.4. Viability Assessment of Microencapsulation

The viability of the microencapsulation was assessed by storing the encapsulated probiotics in PBS and 2% citric acid at 4 °C for 0, 14 and 28 d, with each treatment assessed four times. After these storage intervals, samples were cultured in MRS broth to assess bacterial survival under cold storage conditions. After the culture, the samples were incubated at 37 °C for 24 h, allowing for bacterial growth. Gram staining and spectrofotometry were carried out on the incubated samples to verify the presence and viability of the encapsulated bacteria, ensuring the microencapsulation preserved bacterial integrity within the storage rime [17]. A comparison was made between free cells Lacticaseibacillus casei (FCL), free cells Bifidobacterium longum (FCB) and free cells Lacticaseibacillus casei and Bifidobacterium longum (FCLB), revealing significant differences in stability [18]. 

2.5. Particle Size Analysis

The particle size of the microencapsulated samples was assessed using dynamic light scattering (DLS) and particle size analyzer (PSA) (Malvern Panalytical, UK), assessed two times at the Department of Chemical Engineering, Institut Teknologi Surabaya, Surabaya, Indonesia. This technique provided accurate size distribution data essential for assessing encapsulation quality. Samples were prepared by diluting them with deionized water in a 2:1 ratio to ensure optimal measurement conditions. The analysis was carried out at a controlled temperature of 25 °C, facilitating reliable results on the particle size distribution within the microencapsulation matrix [19].

2.6. Scanning Electron Microscopy and Energy Dispersive X-ray

Scanning electron microscopy and Energy Dispersive X-ray (SEM-EDX) (JSM-6510LA) analyses were carried out at UGM Integrated Research and Testing Laboratory (Laboratorium Penelitian dan Pengujian Terpadu, Indonesia) to investigate catalyst characteristics. The SEM analysis provided detailed insights into the shape and size of the catalyst particles, which were critical for catalytic efficiency. Moreover, SEM-EDX was carried out to record the morphological characteristics of the microcapsule and to show semi-quantitative information on the elemental composition of the microcapsules [19].

2.7 Gas Chromatography-Mass Spectrometry

The microencapsulated sample was assessed three times using gas chromatography-mass spectrometry (GC-MS). The measurements was carried out using Shimadzu GCMS-QP2020NX, Japan, equipped with a quadrupole mass spectrometry detector. A SH-I-624Sil MS column (30-m length, 0.25-mm i.d. and 1.4-µm film thickness (Shimadzu, Japan) was used for the separation of target ethanol and other volatile compounds. Injections were carried out in a split mode (ratio of 1:50). Nitrogen was used as a carrier gas. The injector temperature was 250 °C. The oven temperature was set at 40 °C for 5 min, then increased to 240 °C at a rate of 30 °C min^-1 and set isothermally for 4 min. All samples were equilibrated to 70 °C for 5 min with agitation of 500 rpm using autosampler before injecting 100 μl of headspace onto the column. The mass spectrometer was operated in single ion monitoring mode with the following ions monitored as ethanol, 1.6–1.8 min m/z 31, 45 and 46; 2-methyl-1-propanol, 2.4–2.5 min m/z 33, 43 and 74; 1-butanol, 2.85–3.3 min m/z 41, 56 and 73 (internal standard); and 2-methyl-1-butanol, 3.7–3.8 min m/z 41, 57 and 87. Dwell time of 200 ms was used for each ion. The transfer line to the mass spectrometer was heated to 240 °C, the source temperature was set at 230 °C and the quadrupole was set at 150 °C [20].

2.8. In Vitro Bioaccessibility Assessment

In vitro bioaccessibility assessment was carried out to assess the availability of active compounds in rose extract and MERP through a modified two-stage (GI) digestion simulation based on previous methods [21]. In the stomach stage, simulated gastric fluid (SGF) was prepared by dissolving 0.1 g of NaCl and 0.35 ml of 37% HCl in 50 ml of DW; then, adding 0.16 g of pepsin and adjusting pH to 1.2. A total of 0.5 ml of the extract was dissolved in 50 ml of SGF, pH was adjusted to 2.5 and the mixture was incubated at 37 °C for 2 h at 100 rpm using shaker incubator. In the intestinal stage, 30 ml of the sample from the stomach phase were incubated at 37 °C for 10 min using water bath; then,pH increased to 7.0. Then 1 ml of CaCl₂ solution (750 mM) and 2.5 ml of lipase (1.6 mg/ml) were added to the mixture and incubated using shaker incubator (100 rpm, 37 °C, 2 h). After the digestion process, the sample was centrifuged (4000 rpm, 25 °C, 40 min) and the micelle phase (middle layer) was filtered using 0.45-µm microfilter. The filtrate was mixed with 70% ethanol in a 1:1 ratio and centrifuged (1750 rpm, 25 °C, 10 min). The supernatant was analyzed using UV-vis spectrophotometer to assess concentration of the active compound (anthocyanin) and then proportion of bioaccessibility was calculated based on the ratio of the compound content in the micelle phase to the total compound in the initial sample.

                 (1)

Where, C₁ was the active compound concentration after GI simulation (in the micelle phase) and C_o was the active compound content before GI simulation (in the entire sample).

2.9. Data Analysis

Data analysis involved descriptive interpretation of results from PSA, SEM, EDX and GC-MS. The PSA results provided quantitative data on microencapsulation particle sizes, essential for assessing uniformity and encapsulation quality. The SEM imaging offered detailed visual information on particle morphology, while EDX analysis assessed elemental composition, contributing to insights into structural integrity. The GC-MS data calculated quantities of ethanol in MERP. Results of the viability assessment were shown as averages with SD for thrice measurements. The averages were recorded using two-way analysis of variance (ANOVA) with Tukey's multiple comparison test including a significance level of 0.05 [95% confidence interval (CI)]. Differences in bioaccessibility proportions between the sample groups were analyzed using independent T-test (p-value < 0.05).

1. Results and Discussion

3.1. Scanning Electron Microscopy and Energy Dispersive X-ray

This study demonstrated the efficacy of chitosan-STPP encapsulation in delivering bioactive compounds and probiotics, particularly R. damascena extracts and probiotic strains of L. casei and B. longum. The SEM characterization of the microencapsulated rose extract showed distinct spherical morphology within the particles. Image a (1000× magnification) illustrates that the microcapsules were well-dispersed without signs of aggregation, verifying effective encapsulation. Image B, captured at higher magnification (10,000×), reveals a smooth surface structure with a uniform spherical shape, suggesting structural integrity and encapsulation stability. The particle diameters varied within a narrow range, approximately between 2.8 and 3.4 µm, indicating minimal size heterogeneity. This morphology is essential for optimal encapsulation performance, providing a consistent surface area and stability for targeted uses. The final quantity of probiotics in foods and their viability in the gastrointestinal tract (GIT) are affected by encapsulation efficiency [22]. The particles were observed as nearly spherical, similar to those from UV–vis spectral analysis. It is important to state that the size of these microencapsules plays a critical role in their characteristics and biological activity [23].

The SEM-EDX analysis was carried out to investigate elemental composition of the microcapsules created from rose extract and probiotics, encapsulated within a chitosan matrix that was crosslinked by sodium tripolyphosphate (TPP). Three various spectra (Figure 2) were analyzed, each representing separate regions of interest on the microcapsule surface. These spectra allowed for a comparison of the X-ray energy dispersion and provided a comprehensive map of the elemental distribution within the microcapsule. The EDX mapping detected key peaks corresponding to carbon (C), oxygen (O), sodium (Na), chlorine (Cl and potassium (K) in the K-series, verifying the elemental makeup of the microcapsule structure. The high intensity of the carbon peak (C Ka) indicated that carbon was the most frequent element in the microcapsules. The carbon (and oxygen) peaks became the major characteristics. This increase in the carbon peak reflected the presence of low‑molecular‑weight (LMW) chitosan, a C‑rich polysaccharide [24]. This suggested the presence of organic compounds derived from the rose extract and probiotic ingredients, the two of which contributed to the core composition of the microcapsules. This frequency of carbon verified that organic constituents from the rose extract and probiotic components formed the primary constituents of the microcapsule core, aligning with their intended encapsulation design.

Additionally, the oxygen peak (O Ka) was prominent, likely associated with organic constituents such as polysaccharides in the rose extract. Polysaccharides and other oxygen-rich compounds are integral to forming the encapsulation matrix, enhancing the structural stability and bioactivity of the microcapsule. Sodium (Na Ka), chlorine (Cl Ka and potassium (K Ka) peaks were documented as well, with sodium likely originating from sodium TPP (the crosslinking agent), while chlorine and potassium might be originated from mineral components in the rose extract or probiotics. Detection of these elements not only verified the successful encapsulation of rose extract and probiotic materials within the chitosan network but also indicated effective crosslinking with sodium TPP, which strengthened the microcapsule structure and ensured the stability of its bioactive compounds.

Presence of these distinct elemental peaks, particularly for carbon and oxygen, highlighted the organic-rich nature of the microcapsule, tailored to support the encapsulated compound controlled release and bioavailability in targeted uses. This structural and compositional integrity, verified by SEM-EDX, underscored the microcapsule potentials for delivering therapeutic agents efficiently, regarding its protective matrix that shields susceptible bioactive compounds while allowing the gradual release in specific environments such as the GIT. These characteristics of microcapsule as an innovative platform for drug delivery system target oxidative stress, inflammation and gut health enhancement through controlled bioactive release [25].

The SEM-EDX analysis verifie high presence of carbon and oxygen, indicating a robust organic matrix structure appropriate for gradual and targeted release in the GIT. Chitosan-STPP encapsulation introduced sodium and chlorine, which reinforced cross-linking and structural stability, aligning with previously reported benefits of cross-linked encapsulation matrices [26]. These findings were similar to those of Shavisi, who reported that the inclusion of STPP enhanced the physical stability characteristics of chitosan-based encapsulations [27].

The structural integrity and compositional characteristics verified using SEM-EDX suggested that these microcapsules were promising vehicles for delivering therapeutic agents efficiently. Previous studies have demonstrated that alginate/chitosan nanoparticles (ALG/CS-NPs) demonstrated superior stability in simulated environmental conditions and modulated fucoxanthin (FX) release kinetics within simulated GI environments, highlighting their potentials as promising candidates for FX delivery systems in diverse uses spanning nutraceutical, functional food and pharmaceutical formulations [28].

• Particle Size Analyzer

The microencapsulation PSA indicated a Z-average of 3107 nm ±273.2 demonstrating significant particle sizes. This distribution included heterogeneity within the encapsulated particles, but the size homogeneity was close to the threshold, enhancing encapsulation stability (Figure 4). The narrow distribution indicated that most particles included a similar size range, highlighting effectiveness of the encapsulation process in maintaining homogeneity [29]. The intensity data underscored the quality and uniformity of particle distribution essential for effective encapsulation performance.

The PSA result indicated that the particles were smaller than those reported in similar studies. Microparticles of  L. casei achieved through spray-drying and chitosan–Ca-alginate complexation typically reached an average size of nearly 11 µm, while multilayer microcapsules were reported in the range of 6.2–12.2 µm. For B. longum, larger capsules were observed, with sizes between 2.8 and 3.1 mm in alginate-dairy matrices [30-31]. The microcapsule size in the present study was significantly smaller, which might offer advantages for enhanced bioavailability and stability during GI transition. Consistent particle size distribution helps in dosing precision; similar to studies suggesting that uniform particle sizes improve probiotic survival rates in GI conditions [26, 32]. The homogeneous particle size distribution verifies findings on high encapsulation efficiency in systems, where particle uniformity supports survival rates in the GIT [33].

• Gas Chromatography-Mass Spectrometry Analysis

The GC separation was optimized using conditions detailed in the Methods section and enabled separation of all target components (Figure 5). Good separation was achieved for all target compounds with retention times of 1.706, 2.440, 3.175 and 3.753 min, respectively. It could be seen that the ethanol signal showed lower peak height and area, indicating that ethanol from the extract decreased during microencapsulation preparation. However, the ethanol content was calculated of 2.53% ±0.21 (v/v). The quantity of ethanol was considered relatively high, which was possibly due to the high concentration of chitosan-STPP, which decreased pore size of the interface of microparticles and made ethanol entrapped in the microencapsulation system. The concentration of chitosan-STPP or other variables should be optimized in further studies to decrease the quantity of ethanol in the microencapsulated sample.

• Viability Assessment of Microencapsulation

The viability assessment results indicated that the encapsulated probiotics were viable and capable of proliferation at 0, 14 and 28 d of cold storage at 4 °C. Post-storage, samples cultured in MRS broth demonstrated significant bacterial growth following a 24-h incubation at 37 °C. Gram staining further verified structural integrity and viability of the bacteria, with cells demonstrate the characteristic purple color indicative of Gram-positive bacteria.

The viability of MERP was assessed over 28 d. On Day 0, the highest mean value was observed in FCLB (11.15 ±0.45), while the lowest mean value was in MERP (5.16 ±0.37). On Day 14, the values were stable for all groups, with FCL showing a slight increase to 10.57 ±0.27, while MERP included a similar value at 5.18 ±0.28. Moreover, FCLB was relatively high at 10.88 ±0.64. On Day 28, the values slightly decreased but were still within a similar range, with FCLB including the highest value at 10.89 ±0.30 and MERPincluding the lowest at 5.16 ±0.38. The differences between groups were statistically significant (p < 0.05), as indicated by the various superscript letters. Details of bacterial viability assessments within several days can be seen in Table 1.

The results verified that the bacteria in MERP were viable and capable of proliferation, despite a decrease in their numbers after reculturing. However, this decrease in bacterial count could be attributed to various factors such as the GC-MS analysis revealed an ethanol concentration of 2.53%. Based on the previous studies, B. longum shows limited ethanol tolerance, being viable only at 2–5% (v/v) and completely inhibited at 8% or greater. In contrast, L. casei demonstrated greater ethanol resistance, sustaining growth up to 8–10%. Ethanol suppressed essential glycolytic and citric cycle enzymes, diminishing ATP synthesis and inducing cellular energy depletion [34-35]. Similar studies have shown that after microencapsulation, bacterial growth during reculturing may not reach optimal levels. This is often due to the protective coating, which, while safeguarding the bacteria, can limit their interaction with nutrients, impacting their growth potential [18].

The viability results of encapsulated probiotics in this study might include real-world uses, particularly in the fields of food matrices and pharmaceuticals. The preservation of probiotic stability within a 28-d storage time, as demonstrated using viability assessments, suggested that chitosan-STPP microencapsulation could effectively protect probiotics during food processing and storage, ensuring their delivery in a viable state to the GIT. In food uses, this microencapsulation technology could be used in functional foods such as dairy products (e.g., yogurt and kefir), fermented foods (e.g., sauerkraut and kimchi) and snack foods enriched with probiotics. Encapsulation helps protect probiotics from environmental factors such as heat, acidity and moisture, which often decrease their viability in conventional food products [27]. Microencapsulation of probiotics may integrated into pharmaceutical formulations, ensuring that live bacteria are protected until they reach the target site, such as the intestines. This system could be used in the development of oral probiotics and prebiotic supplements aimed at gut microbiota modulation, which is associated with a variety of health benefits, including immune modulation and decrease of GI disorders [26].

• Gastrointestinal Simulation Assessment and Bioaccessi-bility Proportion

The GI simulation showed that raw rose extract before digestion contained 42.2 ±7.66 mg l⁻¹ anthocyanins, which decreased to 7.1 ±1.23 mg l⁻¹ after the simulated GI condition, corresponding to a bioaccessibility of 16.82% ±0.86 (Table 1). The MERP formulation was detected to start with 7.23 ±0.80 mg l^-¹ anthocyanins, decreasing to 1.87 ±0.50 mg l^-¹ post simulation and yielding a significantly higher bioaccessibility of 25.80% ±4.13. The results of an independent samples t-test verified that the difference in the bioaccessibility of the two groups was statistically significant at p = 0.024

Lower initial anthocyanin values of MERP represented effective microencapsulation; however, higher bioaccessibility values of MERP demonstrated that the microencapsulation media and probiotic coculturing helped anthocyanins resist degradation in the harsh gastric phase and release further easily in the intestine. Using probiotics as a fermentation aid can help in anthocyanin stabilization as well as increasing the recovery of anthocyanin by 9% (absolute values of bioaccessibility increases) (Table 2). The p-value was significant, verifying that this increase was not a result of chance differences and supported the hypothesis that MERP effectively countered conventional anthocyanin decrease detected in raw preparations. The results of this study were similar to those of various studies showing that increased bioaccessibility of active compounds such as anthocyanins in rose extract could be explained by the protective and controlled release mechanisms provided by microencapsulation and probiotic co culture systems. The presence of biopolymer matrices such as whey protein, inulin, casein, alginate and chitosan can act as a physical barrier that shields bioactive materials from the acidic environment of the stomach; thus, limiting the extent of degradation in the stomach and optimizing the release in the intestine phase [36-39]. Moreover, co-microencapsulation with L. casei and L. rhamnosus probiotics has been effective in preserving anthocyanin compound stability through microenvironmental regulation, which inhibits oxidation and enzymatic inactivation [37, 40].

Simulation studies involving GI phases have revealed that the co-microencapsulation strategy is effective in increasing anthocyanin release in the two gastric phases of simulation; thereby, increasing absolute bioaccessibility values significantly by 9-10% [36-38, 40]. This not only improves bioavailability, viability, enzymatic activity and functional activity of probiotics, as well as beneficial metabolic values of short chain fatty acids (SCFA), contributing to make nutraceutical preparations involving the rose extract a further potent approach to enhance the overall use of rose extracts in functional nutraceuticals by strengthening bioavailability of bioactive compound functionalities as well as boosting functional use activity of bioactive compounds in nutraceutical preparations [36], [40]. Further in vivo studies should focus on assessing the stability, release kinetics and therapeutic benefits of chitosan-STPP encapsulated probiotics and R. damascena extracts in real GI environments. These findings suggest that combining microencapsulation with probiotic strains offers a viable strategy to improve the functional delivery of anthocyanin-rich botanicals in nutraceutical uses.

1. Conclusion

This study successfully engineered and optimized a chitosan-STPP microencapsulation system, characterized using PSA and SEM-EDX. The system demonstrated favorable physicochemical characteristics, including uniform spherical morphology with smooth surfaces and verified the effective co-encapsulation of rose extract and probiotics. Ethanol levels were still within tolerance limits in L. casei and B. longum strains. The MERP protected probiotics during formulation and storage and represented effective increase in bioaccessibility of anthocyanins. These findings suggest that combining microencapsulation with probiotic strains offers a viable strategy to improve the functional delivery of anthocyanin-rich botanicals in nutraceutical uses.

1. Acknowledgements

This research study was supported by the State University of Malang (grant no. 3.4.93/UN32/KP/2024).

1. Declaration of competing interest

The authors report no conflict of interest.

1. Authors’ Contributions

Conceptualization, R.K and A.R.; methodology, R.K and R.A.; software, D.R.; validation, T.H., R.A. and R.K.; formal analysis, D.R.; investigation, R.A.; resources, R.A. and A.R; data curation, D.R.; writing—original draft preparation, R.A. and D.R; writing—review and editing, D.R.; visualization, R.A.; supervision, R.K.; project administration, R.A.; funding acquisition.

1. Using Artificial Intelligent Chatbots

Artificial intelligence tools were used to support language refinement and readability, while the manuscript was prepared by the authors.

1. Ethical Consideration

This study involved no human participants or animals, and all research procedures were conducted in accordance with institutional laboratory safety regulations and established principles of good scientific practice.

Background and Objective: The exploration of lactic acid bacteria in integration of specific halal certification is one of the major research topics in the fields of health, food, animal husbandry and agriculture. This study aimed to investigate antimicrobial potentials of probiotic lactic acid bacteria isolated from Rinuak fish (Psilopsis sp.) from Lake Maninjau, Indonesia.

Material and Methods: Totally, 15 lactic acid bacteria were isolated from four samples of Rinuak fish (Psilopsis sp.) and investigated for their characteristics as probiotic candidates using conventional laboratory assessments and 16S rRNA sequencing methods.

Results and Conclusion: Five isolates were identified as probiotic candidates, including IR2.2, IR2.4, IR4.1, IR4.3 and IR4.5 due to their good resistance of gastric pH ranging 84.24–88.01% and their survival ability against bile salts (resistance of 50.37–57.35%). The IR4.3 was identified to generate the greatest antimicrobial activity against Escherichia coli ATCC 0157, Staphylococcus aureus ATCC 25923 and Salmonella enteridis ATCC 13076 with their diameter of inhibition zone of 22.46, 19.34 and 9.41 mm, respectively. The 16S RRNA sequencing method verified that the lactic acid bacteria isolated from rinuak fish (Psilopsis sp.) included 97.69% similarity to Lactobacillus fermentum strain 4901. This strain promised as an antidiarrheal and antityphoid agent and a natural food preservative appropriate for incorporation into HALAL-compliant foods and pharmaceutical products.

Keywords: Food preservative, Endemic Fish, Microbial fermentation, Molecular characterization, Lactobacillus fermentum strain 4901، Indigenous probiotics،Food microbiology،Gastrointestinal health

1. Introduction

Global challenges in various aspects of life are becoming larger. This can be seen from the increasing human population and hence demands for food products are increasing as well; one of which, is fishery products. This fishery product is a basic requirement to meet the needs of protein sources. The protein must be ensured that is halal due to the tauths of the Islamic religion, because Islam emphasizes that maintaining a healthy body by consuming halal foods and drinks is an obligation for every muslim [1]. For halal food development, it is essential to ensure that all ingredients and processes comply with Islamic dietary laws. Lactic acid bacteria (LAB) sourced from permissible animals such as fish and processed without contamination from non-halal substances can be addressed as halal. However, clear certification is warranted for commercial uses. Fermented fish is one of the products from fisheries, which is rich in proteins and LAB [2]. In the fermentation process, microbes and enzymes can stimulate specific flavors, increase the digestibility of food ingredients, decrease the content of anti-nutrients and other undesirable ingredients and produce derivative products and compounds that are beneficial for human life [3]. In general, LAB are food-grade microorganisms. These bacteria can provide flavors to foods, inhibit spoilage bacteria in foods and inhibit pathogenic bacteria. The LAB can be isolated from various sources, especially from fermented foods [4]. In addition, LAB can create an acidic atmosphere, which can decrease the number of pathogen colonies [5]. The existence of selected strains of LAB has demonstrated beneficial effects, as probiotics for humans [6].

The LAB isolation is possible from plant and animal-based products; for example fish, fruits and milk [7].  Rinuak fish (Psilopsis sp.) of Lake Maninjau, Indonesia, is one of those products. Rinuak fish is an domestic fish of Lake Maninjau that includes animal proteins, which is potentially developed due to its high nutritional compounds. The flesh of rinuak fish (Psilopsis sp.) contains proteins of 14.52%, magnesium of 0.21% (in fresh rinuak), phosphorus of 2.4% (in fresh rinuak), water content of 78.62% and ash content of 6.4% (in fresh rinuak). Rinuak fish also contains calcium [8]. The mineral composition especially magnesium and calcium are able to stimulate the bacterial growth. Magnesium (especially in gluconate form) improves probiotic survival, texture, acidity and viability (> 10⁶ CFU g^-1) during storage [9]. Research by [10] showed that addition of calcium (calcium carbonate) to fermented feed helped boost growth of Lactiplantibacillus plantarum, Lacticaseibacillus rhamnosus and Bacillus subtilis. Therefore, these mineral nutrients of rinuak fish support the potential of rinuak fish for LAB growth. However, there is a lack of studies that investigate its antimicrobial activity and potential characteristics as probiotics.

One of the most important characteristics of LAB is that they can produce the antimicrobial compounds of bacteriocins [11]. Bacteriocins are secondary metabolite products of LAB that include similar actions to antibiotics, being able to inhibit certain bacteria from growing [12]. Previous studies were carried out worldwide to investigate the LAB content in fish. For example, studies on swamp fish fermented with the addition of pure coconut oil resulted in the discovery of LAB with antimicrobial activity [13]. Potential antimicrobials can be achieved from LAB produced by fermentation of rinuak fish (Psilopsis sp.) in Lake Maninjau by isolating and characterizing DNA from LAB through polymerase chain reaction (PCR) analysis of 16s rRNA gene and genome sequencing. Based on the results of this molecular analysis, the phylogenetics of LAB from the fermented rinuak fish (Psilopsis sp.) of Lake Maninjau was investigated. Numerous previous studies were carried out on the nutritient of rinuak and LAB contents of rinuaks. However, study on the antimicrobial potential of fermented rinuak fish and its probiotic characteristics has not been investigated. Therefore, this study aimed to characterize the isolated LAB morphologically and biochemically and carry out theit molecular identification using 16s RNA sequence method. The probiotics effectiveness in this study was assessed on a strain spesific basis.

1. Materials and Methods
2. Research Equipment and Materials

Equipment of this study included incubator (Infors HT Ecotorn, USA), anaerobic jars, refrigerator, autoclave (ALP KT40S, Japan), hot plate (Sybron nouveau II, USA), vortex (Labmart 3000, China), analytical balance (Kern ABT 320-4M, Germany), bunsen, microtubes, hockey sticks, inoculation needles, glass slides, pipettes and micropippette tips (DragonLab, BIO-RAD, USA), light microscope (Irmeco, USA), UV-vis spectrophotometer, oven (Memmert UF 100 Plus, Germany), centrifuge (Eppendorf 5417 R, Germany), thin layer chromatography (TLC) chamber and paper chromatograph, capilary tubes, 96-well microplates, microplate reader (PR 4100, BIO-RAD, USA), pH meter, shaker waterbath (IC DK-540b, Japan), PCR equipment (Techne TC-312, UK), spinner down (BIO-RAD, USA) electrophoresis equipment (BIO-RAD, USA), gel documantation imager (BIO-RAD, USA) and labobartory standard glasswares.

Materials of this study included rinuak fish sampels collected from Lake Maninjaus, Indonesia, de Mann-Rogosa-Sharpe (MRS) broth and agar (Merck, Germany), distilled water (DW), 70 and 90% ethanol, peptone water, MRS agar (Merck, Germany), 37% HCl (Merck, Germany), ox-gall, nutrient agar (NA), penicillin, kanamycin, ampicillin, NaOH (Merck, Germany), genomic DNA mini kit (Invitrogen Pure-Link, USA) and lysozyme (Invitrogen PureLink, USA)

1. Fermentation of Rinuak Fish (Psilopsis sp.) from Lake Maninjau

The process of making rinuak fish fermentation (Psilopsis sp.) was as follows. The ingredients were fish, salt and rice. First, fish was washed and then salt and rice were add to nourish the taste. Samples were transferred into a jar and the lid was close tightly. Jars were stored at room temperature (RT) for 4 d and then dried for 3–5 d [14]. The flowchart of the fermentation process for rinuak fish (Psilopsis sp.) from Lake Maninjau can be seen in Figure 1.

1. Sampling Locations for Rinuak Fish (Psilopsis sp.)

Samples of rinuak Fish (Psilopsis sp.) were collected from fishermen in Lake Maninjau at various locations based on the easy access to collection locations, residential areas in the lake area, upstream rivers around the lake, depth of the lake and food sources for rinuak Fish. Sample 1 was collected in a dense settlement and included a traditional market in the Lake Maninjau area. Sample 2 was collected from the middle of the lake with a depth of nearly ±150 M. Sample 3 was collected in a tourist attraction area at Lake Maninjau and Sample 4 was collected in the upstream area of the river, the Batang Sri Antokan River; where, there was the Maninjau Hydroelectric Power Plant. Fish was immediately transferred into a cold box and carried to the Microbiology Laboratory of Stikes Syedza Saintika. Then, isolation and molecular characterization of DNA of LAB were carried out at the Biotechnology laboratory of Andalas University, Indonesia. The location of each sample is illustrated in Table 1 and a map showing the locations for rinuak fish in Lake Maninjau, West Sumatra, Indonesia, is demonstrated in Figure 2.

1. Isolation of Lactic Acid Bacteria from Rinuak Fish (Psilopsis sp) Lake Maninjau, West Sumatra, Indonesia

The LAB isolation process began with an enrichment process; 1 g of rinuak fish sample was added into 9 ml of MRS broth and then homogenized to achieve a 10^-1 dilution. This was incubated at 37 ^oC for 24 h. After incubation, 100 µl of the enrichment or 10^-1 dilution were added into a microtube with 900 µl of peptone water and then homogenized using vortex to achieve a 10^-2 dilution. This procedure was repeated until 10^-8 dilutions were achieved. Then, plating process was carried out and the culture from the 10^-8 dilution was inoculated on MRS agar and incubated anaerobically at 37 ^oC for 48 h. Single LAB colonies that grew on the surface of the media were re-purified by inoculating the colony onto MRS agar, incubating anaerobically at 37 ^oC for 24 h [15].

1. Characterization of Lactic Acid Bacteria from Rinuak Fish (Psilopsis sp.) in Lake Maninjau, West Sumatra, Indonesia

Identification of lactic acid bacteria morphology

    Morphological identification was carried out macroscopically on LAB cultures inoculated on MRS agar to identify the shape, color and diameter of LAB isolates. Then, Gram staining was carried out to investigate LAB morphology microscopically by verifying the color and the shape of the cells. Meanwhile, biochemical characterization was carried out using fermentation-type and catalase assays [15].

1. Selection of Lactic Acid Bacteria from Rinuak Fish (Psilopsis sp) from Lake Maninjau, West Sumatra, Indonesia, as Probiotic Candidates

1) Resistance of Rinuak Fish (Psilopsis sp.) from Lake Maninjau, West Sumatra, Indonesia, to Gastric pH

The resistance assessment for gastric pH was carried out based on Kim et al. [16] method with modifications. This assay used two types of media, including MRS broth with addition of 37% HCl to achieve a pH of 2.5 and MRS broth without addition of HCl to maintain a pH of 6.8 (as a control). The medium was sterilized at 121 °C for 15 min using autoclave. Moreover, 5 ml of MRS broth-HCl were added to 0.5 ml of the bacterial isolate and incubated at 37 ^oC for 3 and 6 h. Then, absorbance was read at 600 nm. This was carried out three times. Resistance was expressed as a percentage. According to [17], percentage of the resistance of LAB isolates can be calculated using the following formula:

2) Resistance of Rinuak Fish (Psilopsis sp.) from Lake Maninjau, West Sumatra, Indonesia, to Bile Salts

The resistance assessment of bile salt was carried out based on a method from Gotcheva V et al. [18] with modifications. Various concentrations of bile salt of 0, 0.3 and 0.5% were added to the MRS broth media. The media were autoclaved at 121 ^°C for 15 min. The bacterial isolate (0.5 ml) was transferred into 5 ml of MRS broth added with 0, 0.3 and 0.5% ox-gall and incubated at 37 ^oC for 5 h. The MRS broth containing no bile salt was set as the control and compared with the treatments. The LAB growth was assessed using UV spectrophotometry based on the absorbance at 600 nm. All assessments were carried out in three replications. Isolate resistance was expressed as a percentage. According to [19], percentage of the resistance of LAB isolates can be calculated using the following formula:

1. Screening of Lactic Acid Bacteria from Rinuak Fish (Psilopsis sp.) in Lake Maninjau for their Antimicrobial Potentials

The lactic acid bacteria antimicrobial activity assay

Three pathogens of Salmonella typhi, Staphylococcus aureus and Escherichia coli were assessed for antimicrobial activity of LAB using modified paper diffusion method [20]. Briefly, 1 ml of LAB culture enriched for 48 h was collected using micropipette and transferred into a sterile microtube. This was centrifuged at 10,000 rpm for 5 min and the supernatant was collected for antimicrobial resistance assessment. Then, 0.4 g of NA media was homogenized by heating using hot plate and sterilizing using autoclave. Then, 40 µl of the bacterial isolate enriched for 24 h were poured into a Petri dish containing 20 ml of NA media and cooled down until the media hardened. The paper disk was soaked in LAB isolate suspension for approximately 10 min. After the agar solidified, the paper disk was set on NA media, which contained isolates of pathogenic bacteria. Then, positive control was set by dropping LAB supernatant (20 µl) onto sterile test papers, including 10 g of penicillin, 30 g of kanamycin and 10 g of ampicillin. This was incubated at 37 °C for 24 h anaerobically. After 24 h, diameter of the inhibition zone was reported using caliper [21].

1. Antimicrobial Assay of Crude Bacteriocin Supernatant

Briefly, 1 ml was cultured in 9 ml of MRS solution at 37 ^oC and incubated for 2 d. This was centrifuged at 14,000 rpm for 5 min. Then, 0.22-µl membrane filter was used to filter the supernatant. To eliminate the barrier effect because of the presence of organic acids, 1 N NaOH solution was added to the cell-free supernatant to maintain pH 6.5 [21]. The bacterial pathogens were grown aerobically at 37 °C for 24 h. Then, 0.2% pathogenic bacteria were transferred onto 20 ml of MHA solution at 50 °C. After the gelatin was solid, a 6-mm well was made in the media using cork borer. Furthermore, 50 µl of supernatant were transferred into the wells and set 10–15 min. The incubation was carried out at 37 °C for 24 h under aerobic conditions. Antimicrobial activity of bacteriocins in the supernatant was verified by varying the time intervals of 15, 30 and 60 min at 100 ºC. The supernatant of LAB was investigated for its ability in inhibiting bacteria, including E. coli O157, S. aureus ATCC 25923 and Salmonella enteritidis ATCC 13076 via similar methods. The inhibition zone was associated with existence of bacteriocin compounds and its dimension was recorded using caliper [22].

1. Isolation and Characterization of 16S rRNA

Genome isolation was carried out using genomic DNA mini kit. Lysozyme was used at a concentration of 20 mg ml^-1 to lyse the bacterial cell walls. The 16S rRNA gene was amplified using selected bacterial genomic DNA kit. Amplification was carried out using reverse primer 1387R (5'-GGGCGGGGTGTACAAGGC-3') and forward primer 63F (5'-CAGGCCTAACACATGCAAGTC-3'). Reactions were carried out in a total volume of 50 μl. The PCR mixture contained 25 μl of DreamTaq Green DNA polymerase (Thermo Fisher Scientific, USA), 22 μl of milli Q water (MQ), 1 μl of the template and 1 μl of each forward or reverse primers (10 μM each, IDT synthesis). Amplification conditions included preheating at 95 °C for 5 min and then denaturation at 95 °C for 30 s, primer annealing at 58 °C for 30 s, 1 min extension step at 72 °C for 35 cycles and post cycling extension for 5 min at 72 °C. The reaction was carried out in a thermal cycler (Biometra T-Personal Thermal Cycler, USA). The amplified DNA was added to GelRed nucleic acid gel stain and electrophoresed on agarose gels [1.5% (w/v) in TBE buffer] at 100 V for 60 min. The amplicons could be visualized using gel documentation imager. The PCR amplification product was purified using absolute ethanol Na-acetate method and then sequenced [23].

1. The BLAST and Phylogenetic Analysis

Phylogenetic analysis method was carried out based on former studies [24]. Sequence data were collected using BioEdit software and then converted to FASTA format. The BLAST was used to carry out sequence analysis (http://www.ncbi.nl-m.nih.gov/blast/cgi). Moreover, DNA sequences were imported into Clustal W2 (http://www.ebi.ac.uk/Tools/clustalw2/) to carry out phylogenetic analysis. A phylogenetic tree was created using BLAST MEGA v.6.0 (http://www.megasoftware.net) and neighbor-joining (NJ) method.

1. Data Analysis

Data were present as mean ±SD (standard deviation). Statistical significance was reported using one-way analysis of variance (ANOVA) and SPSS software v.26 (IBM, USA). Tukey post-hoc test was used to assess significant differences between group means, with a p-value of < 0.05 as statistically significant.

1. Results and Discussion
2. Isolation of Lactic Acid Bacteria in Rinuak Fish (Psilopsis sp.) from Lake Maninjau, West Sumatra, Indonesia

The LAB selective media (MRS broth and agar), providing appropriate nutrients and pH for LAB growth, were used in the serial dilution-agar plate method. This allowed LAB to grow and reproduce effectively. Serial dilution was necessary to decrease bacterial density, ensuring individual colonies grow separately instead of clustering together. The MRS broth and agar as the sources of nutrition and appropriate pH for LAB growth were used in serial dilution-agar plates to isolate LAB. Serial dilution was needed to decrease density of the inoculated LAB to enable the LAB to develop in colonies independently of one another, rather than in piles [19] (Figure 3). Single colonies that were round, convex, yellowish-white and shiny grew separately with various diameter sizes. These were re-inoculated on MRS agar using streak method to achieve pure isolates of LAB from rinuak fish. These findings were similar to those of Purwati study, which showed colonies of LAB on MRS agar as yellowish-white colonies. Details on the isolation and purification of 15 LAB isolates were as follow: three isolates of IR1 (IR1.1, IR1.2 and IR1.3), five isolates of IR2 (IR2.1, IR2.2, IR2.3, IR2.4 and IR2.5), one isolate of IR3 (IR3) and six isolates of IR4 (IR4.1, IR4.2, IR4.3, IR4.4, IR4.5 and IR4.6). A higher number of LAB isolates (n = 15) was achieved in this study, compared to previous reports, which achieved 9–12 isolates in Tilapia fish [25] and 10–13 isolates in Bilih fish [24].

The observed differences in the diversity of LAB types within the samples might indeed be affected by the environmental conditions at the respective sampling locations. For example, Sample IR.1 was collected from Maninjau Village, an area with high population density and a traditional market, while IR.3 originated from Duo Koto Village, a well-known tourist destination. Such anthropogenic activities in these areas might contribute to pollution of the close lake ecosystem, including household and market waste directly discharged into Lake Maninjau. In contrast, IR.2 and IR.4 were collected from relatively undisturbed mid-lake regions—Tanjung Sani Village and Koto Malintang Village respectively—where the water depth exceeded 100 m and minimal human activity was observed, potentially preserving a further appropriate aquatic environment.

However, it is acknowledged that geographic origin alone might not fully responsible for the variations in LAB diversity. Several other ecological and biological factors such as the natural diet of rinuak fish, differences in water temperature, seasonal fluctuations and post-capture handling practices (e.g. time to processing or storage conditions) could play significant roles in shaping the microbiota. These variables might affect selective pressures and survival conditions for specific LAB strains. Further studies incorporating water physicochemical analyses, fish diet profiling and standardized handling procedures are recommended to strengthen the interpretation of LAB diversity patterns. A further holistic approach is necessary to elucidate the multifactorial nature of LAB colonization and persistence in rinuak fish across various environments. Survival of microorganisms depends greatly on environmental conditions and is affected by their food sources [26].

1. Characterization of Lactic Acid Bacteria in Rinuak Fish (Psilopsis sp.) from Lake Maninjau, West Sumatra, Indonesia

                The morphological characteristics of all isolates were carried out microscopically through Gram staining (Table 2). The LAB is classified as Gram-positive bacteria that can bind crystal violet-iodine complexes and preserve the purple color of their cells [27]. The stages in Gram staining are crystal violet as the primary dye, Lugol solution (KI-I2) as the mordant, 96% alcohol as the decolorizing agent and safranin solution as the counterstain. Crystal violet dissociates in solution into CV⁺ and Cl^-. These ions then penetrate the bacterial membranes and cell walls. Moreover, CV⁺ ions react with negatively charged bacterial cell components and make bacterial cells (-) react with CV+ purple. Addition of iodine solution (I^- or I₃) forms a crystal violet-iodine complex (CV-I) in the inner and outer layers of the cells; thereby, strengthening purple color of the bacterial cells [28]. The cell walls of Gram-positive bacteria are in the form of thick fibers consisting of 50–90% peptidoglycan. Therefore when decolorizing, cells are dehydrated and purple because the CV-I complex is trapped in their walls. When safranin solution is added, there is no color change in the bacterial cells [29]. Totally, 15 LAB isolates were assessed. All isolates were Gram-positive, indicated by their purple color in Gram staining. Of them, ten isolates were rod-shaped (bacilli) and five isolates were spherical (cocci) (Figure 4).

Another biochemical characteristic of LAB was through fermentation-type testing (Table 2). Lactate-producing bacteria can be homofermentative or heterofermentative depend on the primary fermentation product. Production of lactic acid indicates that LAB is homofermentative. Furthermore, heterofermentative LAB in addition to produce lactic acid can produce ethanol, CO₂ and acetic acid [17]. This type of fermentation test is carried out using gas bubbles form in the Durham tube, which is placed upside down in the LAB liquid culture after incubation for 48 h. The LAB is addressed as heterofermentative if gas is detected in the Durham tube. In contrast, LAB is disclosed as homofermentative if the Durham tube is empty of gas [23].  In this study, 11 LAB isolates from rinuak fish were homofermentative, three LAB isolates from IR1 and one LAB isolate from IR3 were heterofermentative. This was due to the long storage time of fermentation and IR1 and IR3 were located in densely populated areas, markets and tourist attractions. High population density, existence of traditional markets and tourist attractions at the sampling location might damage rinuak fish ecosystem because waste from homes and markets is thrown directly into Lake Maninjau. The environment includes a significant effect on the lives of microorganism conditions [26].

1. Selection of Lactic Acid Bacteria in Rinuak Fish (Psilopsis sp.) from Lake Maninjau, West Sumatra, Indonesia, as Probiotic Candidates

The LAB selection as probiotic candidates was carried out on ten LAB isolates, which were classified as Gram-positive bacteria, did not produce catalase enzyme and ethanol in the fermentation type test, classified as homofermentative.

1. Resistance of Lactic Acid Bacteria in Rinuak Fish (Psilopsis sp.) from Lake Maninjau, West Sumatra, Indonesia, to Gastric pH

The LAB resistance to gastric pH assay was carried out at pH 2.5 because the pH in the gizzard and proventriculus was 2.5–3.5. The feed transit time was 70 min and assessed for 3 and 6 h. The resistance of LAB isolates was assessed at 600 nm using spectrophotometer (Table 3) [17].

Data in Table 3 show that all LAB isolates include ability to survive at pH 2.5 for 3 and 6 h of incubation time. Including a minimum resistance of 50% indicate that all LAB isolate are probiotics potential. These findings were similar with those that probiotic LAB should show a survival rate of ≥ 50% at low pH, as suggested by [14]. Ten LAB isolates were assessed for resistance to pH 2.5 through spectrophotometry at 600 nm. Isolates that showed the highest resistance to low pH conditions included IR2.2, IR2.4, IR4.1, IR4.3 and IR4.5. All of these isolates were achieved from fermented rinuak fish from Lake Maninjau. Their survival rates after 3 h of incubation at low pH included 88.01, 84.16, 84.70, 86.64 and 84.24%, respectively. After 6 h of incubation, a slight decrease in resistance was observed with survival rates of 84.96, 80.83, 80.88, 82.72 and 82.16%, respectively. These results indicated that the selected LAB isolates included high tolerance to acidic conditions over time, which is a key criterion for probiotic selection. This is according to [15], stating that the greatest survival of LAB isolates as probiotics are isolates that include small decreasing difference. This study produced better outcomes than previously studies that isolated LAB from okara, which included 74.02% of survival rate for 2 h at pH 2.5 [24]. In previous study [17], LAB strains of Lactobacillus brevis, L. plantarum and Pediococcus indurans isolated from traditional pickles survived at pH 2.5 for 4 h of 33–64, 35–85 and 40–76%, respectively [16]. The survival rate of L. fermentum strains isolated from fermented milled flour was ≥ 80% after incubation for 4 h at pH 2.5 [14]. The LAB of probiotic candidates must be able to endure the exorbitant conditions of the gastrointestinal tract (GIT) from the mouth to the intestines and then live in colonies on the surface of the intestines and the acidity of the stomach functions as the first gate for selecting microbes prior to passing into the intestines [23].

1. Resistance of Lactic Acid Bacteria in Rinuak Fish (Psilopsis sp.) from Lake Maninjau, West Sumatra, Indonesia, to Bile Salts

The resistance of LAB-Rinuak Fish to bile salts was assessed using spectrophotometry at 600 nm with a concentration of 0.3 and 0.5% and incubated for 5 h. Results are listed in Table 4.

Findings in Table 4 show that all LAB isolates could survive against bile salts with resistance > 20% under the statement [37]. The major problems for the survival rate of the probiotics selected strains were gastric acidity and bile salts, which at least included 20–40%. According to [38], isolate is verified as a strong probiotic candidate when its survival rate is greater than 50% under low pH conditions, including bile salt resistance. Thus, the LAB isolates that included good probiotic criteria out of 11 isolates included five isolates with ≥ 50% resistance at a concentration of 0.3% bile salts. These included IR2.2, IR2.4, IR4.1, IR4.3 and IR4.5 isolates with resistance at 0.3% bile salt concentration of respectively 55.41, 57.35, 53.49, 53.24 and 50.37%. Resistance decreased when the bile salt concentration (ox gall) increased to 0.5% as respectively 51.61, 52.67, 50.50, 51.11 and 47.36%. The decrease differences of IR2.2, IR2.4, IR4.1, IR4.3 and IR4.5 respectively included 3.80, 4.68, 2.99, 2.14 and 3.01. A slight decrease indicated a large survival rate. This is based on the fact [19] that isolates with a minor decrease difference include the highest opportunity of surviving as probiotic LAB isolates. Characteristics of the isolates included the potentials as probiotics because they were resistant to small intestine bile salts; hence, they could survive in the large intestine. According to [12], LAB could survive in bile salts because it included bile salt hydrolase (BSH) enzyme with the mechanism that the major component of bile salts included bile acids and these bile acids primarily targeted the bacterial membrane. Based on the two highlighted criteria of resistance to gastric pH and tolerated 0.3% bile salts, the other assessment was carried out on five selected isolates that included high ability for the antimicrobial potential assessment.

1. Antimicrobial Activity Assessment of Five Selected Lactic Acid Bacteria Isolates of Rinuak Fish (Psilopsis sp.) from Lake Maninjau, West Sumatra, Indonesia, against Pathogenic Bacteria

Inhibition activity of the selected LAB from Rinuak Fish (Psilopsis sp.) to the growth and development of the pathogenic bacteria of E. coli ATCC O157, S. aureus ATCC 25923 and Salmonella enteritidis ATCC 13076 are shown in Table 5.

Results from the study can be seen in Table 5, showing that the clear/inhibitory zone area for each isolate varied greatly, this was because the abilities of each bacteria from each isolate varied. Clear zone was formed due to the metabolism of LAB that produced lactic acid, bacteriocins and hydrogen peroxide, all of which included ability against microbes. The LAB isolates having the largest zone of inhibition for E. coli O157 belonged to IR4.3 with 14.46 mm in diameter, while the smallest clear zone diameter was present by IR4.1 with 12.43 mm in diameter. The largest zone of inhibition was detected in IR4.3 with 14.40 mm in diameter against S. aureus ATCC 25923 bacteria. In contrast, IR2.4 generated a 12.89-mm zone in diameter, accounted as the smallest zone of inhibition. Against Salmonella enteritidis ATCC 13076, the biggest clear zone of inhibition was present by IR4.3 with a diameter of 16.60 mm; whereas, the least zone was produced by IR2.4 with a diameter of 15.64 mm.

Furthermore, LAB IR4.3 was assessed for its antimicrobial activity to inhibit bacterial pathogens using several antibiotics of kanamycin, penicillin and ampicillin as the positive controls. The purpose of using penicillin, kanamycin and ampicillin was because these antibiotics were effective against Gram-positive bacteria. According [10], penicillin is an antibiotic that is able to inhibit cell walls of bacterial synthesis, including those in the beta-lactam group. Ampicillin is a penicillin derivative that includes a broader spectrum, able to inhibit Gram-positive and Gram-negative bacteria as well as the beta-lactam group. Antibiotics were assessed using paper disks that contained the specified concentrations of 30-µg kanamycin, 10-µg ampicillin and 10-µg penicillin. To assess resistance and sensitivity of bacterial pathogens, pathogens were used using positive-control antibiotics. The clear zone results are shown in Table 6.

Based on Table 6, LAB isolates IR4.3 was able to inhibit the three bacterial pathogens of E. coli O157:H7, S. aureus ATCC 25923 and Salmonella enteritidis ATCC 13076 with diameters of 22.46, 19.34 and 9.41 mm, respectively. The LAB isolate tolerated the three antibiotics, including ampicillin (10 μg), kanamycin (30 μg) and penicillin (10 μg), against E. coli O157:H7 (Figure 5).

Overally, Figure 5 illustrates that the LAB IR4.3 from the staining included antimicrobial activity against all the pathogens, compared to penicillin, which did not include inhibition activity to the growth of Salmonella enteritidis ATCC 13076 and S. aureus ATCC 25923, as well as kanamycin that did not include antimicrobial activity against all the pathogenic bacteria. This was based on a study [21], where the penicillin test bacteria could not inhibit the growth of E. coli ATCC O157; thus, it could be reported that E. coli ATCC O157 were resistant to penicillins. Results clearly showed that the inhibition zone formed by the LAB IR4.3 included high antimicrobial activity against the pathogenic bacteria. Results achieved were based on an opinion [42], stating that four categories of inhibition zones were reported as very strong (> 20–30 mm), strong (>10–20 mm), moderate (5–10 mm) and weak (<5 mm) inhibition zones. Based on these four categories, the LAB IR4.3, which was isolated from fermented rinuak fish (Psilopsis sp.) including a very high clear zones, was categorized as a very strong antibacterial agent against the three bacteria. The capacity of LAB to inhibit the growth of enteric pathogens that live in the digestive system is a critical factor in the selection of these isolates for use as probiotic agents. Another need that probiotic bacteria must include is the ability to produce antimicrobial substances [13].

1. Antimicrobial Activity of the Crude Bacteriocin Supernatant

After LAB supernatant pH neutralization, antimicrobial activity of the crude supernatant of LAB IR4.3 bacteriocin was assessed. Therefore, the organic acid antimicrobial characteristics were not detected. The major component of antimicrobial compounds in LAB contains components of organic acids, particularly lactic acid [14]. The neutralized supernatant antibacterial efficacy against pathogenic microorganisms is shown in Table 7.

Findings are shown in Table 7 and Figure 6, which demonstrate the antimicrobial efficacy against  E. coli ATCC O157 of 18.29 mm and S. aureus ATCC 25923 of 20.63 mm (Figure 6), following the neutralization of LAB supernatant pH. However, activity of the antimicrobials is not indicated for Salmonella enteritidis ATCC 13076.

Results were more important, compared with those previously carried out [8], reporting that LAB isolates from Bilih fish of Lake Singkarak included gross bacteriocin activity against S. aureus ATCC 25923 (13.1 mm) and E. coli O157:H7 (12.7 mm). Moreover, studies [25] did not find antimicrobial activity in LAB isolates from raw beef after the supernatant pH was neutralized because LAB produced organic acids, which affected antimicrobial activity. Furthermore, another study [16] reported L. Plantarum NS from isolates of various fermented food products from freshwater fish, showing that organic acids included a relationship with antimicrobial activity. It was reported that in the final phase of exponential growth (incubation time of 12–15 h), antimicrobial activity was achieved against the pathogenic bacteria of E. coli, L. monocytogenes and B. cereus. At incubation times of 21 and 24 h, it was reported that L. Plantarum NS included the best antimicrobial characteristics against S. aureus and S. typhimurium ATCC 14028. Furthermore, several results showed that L. brevis and L. plantarum isolated from fermented bekasam food from South Sumatra included antimicrobial characteristics in supernatants of cell-free culture and were effective to inhibit E. coli and L. monocytogenes [27]. There are several strategies to prevent bacteriocins from destroying target cells [28], the mechanism for inhibiting the synthesis of lipid II (precursor of cell wall) is stabilizing formation of target membrane pores and inhibiting cell wall biosynthesis. Phospholipid layer binds fatty acids to the positive end of the peptide, showing that the peptide is attached to a target cell membrane. This mechanism brings peptides to membrane-like monomers to form bonds, producing separation leading to the formation of pores and ultimately cell death [29]. Bacteriocins are complex proteins that include bactericidal abilities, especially against Gram-positive bacteria and close species [20].

1. Identification of the Selected IR4.3 Isolates of Rinuak Fish (Psilopsis sp.) Using 16S rRNA

Results of the amplification of 16S rRNA gene using polymerase chain reaction

Findings of electrophoresis indicated that the bacterial 16S rRNA gene from the isolation of rinuak fish (Psilopsis sp.) LAB was successfully amplified using PCR. This was demonstrated with a 1,500-bp PCR product fragments, using primer 27F (AGAGTTGATCCTGGCTGAG) and primer 1429 R (GTTTACCTTACGACTT). The LAB 16S rRNA gene amplification results from rinuak fish (Psilopsis sp.) were used to ensure that the genomic DNA isolation was successful. To identify LAB, results of 16S rRNA gene amplification were used. Sequencing of the 16S rRNA gene nucleotides was carried out after its amplification. Figure 7 shows results of the electrophoresis of PCR products from LAB isolates. Comparing ribosomal RNA sequences, molecular techniques are used to investigate the genetic links of LAB [24]. Moreover, 16S rRNA can directly be sequenced without the need of PCR amplicon cloning. Results of the DNA amplification of a 1,500-bp fragment are shown in Figure 7. This demonstrated that the particular primers in the study was able to recognize bacteria at the strain level.

Chromatograms of sequencing results from the direction of reading the forward and reverse primers of the 16SrRNA gene from IR4.3 were aligned using SeqMan software. Length of the 16S rRNA gene amplicon amplified with the primers of 16S rRNA_27F and 16S rRNA_1525R was 1,500 bp. Moreover, chromatogram from reading the forward primer of 16SrRNA_27F was 726 bp. Chromatogram from reading the reverse primer of 16SrRNA_1525R was 1,131 bp. As a weakness of the Sanger sequencing method, several bases at the ends of the chromatogram include peaks that overlap and are not clear. Hence, these bases are removed [21]. The chromatogram resulting from reading the forward primer of 16SrRNA_27F at the 5' end was cut with 40-bp long. At a position of nearly 400 bp toward the end of the 3' peak in this chromatogram, it became increasingly sloping and even flat. Bases in the sloping peak were edited by adjusting the base sequence in the reverse primer reading. The chromatogram resulting from reading the reverse primer 16SrRNA_1525R at the 3' end was cut with 81-bp long and at the 5' end with 30-bp long. After the editing procedure, length of the sequence that included in the 16SrRNA gene fragment from IR4.3 was 1500 bp. The edited chromatogram visualization was as follows.

Of 250 sequence data from BLAST results for isolating IR4.3 strain, a 100% query cover value was achieved. Then, the identity value percentage was achieved, ranging 99.21–99.64%. Totally, 15 L. Fermentum bacterial sequence data in GenBank were selected for further use in genetic relationship analysis. Information characteristics of the 15 bacterial sequence data were as follows.

Based on Table 8, IR4.3 isolate included similarity of 99.64% to the genome sequence and partial strains of Lactobacillus. Based on a fact that a sequence is homologous if its resemblance to another sequence is greater than 99 %, the MEGA tool was used to visualize the results of the BLAST after initial use of BioEdit v7.0. Based on the phylogenetic visualization results, the fermented fish sample isolated from rinuak fish (Psilopsis sp.) in the study showed an apomorphic relationship with L. fermentum. Previous studies stated that the characters were apomorphic and plesiomorphic [22]. Apomorphic characters are characters that change and inherited, while synapomorphic characters are inherited characters detected in monophyletic groups [23]. Regarding the query cover value, 15 species were selected from the BLAST results to generate the phylogenetic tree. Then, FASTA format for the top 15 species were retrieved to create a phylogenetic tree.

1. Phylogenetic Tree

Based on the BLAST results, a phylogenetic tree was created to include the level of relationship between the isolates and other species using NCBI. Creating a phylogenetic tree via the Mega X, FASTA files. The downloaded files were aligned first and then a phylogenetic tree was created. Neighbor-joining method was used to construct the phylogenetic tree with a bootstrap value of 1,000. Kimura 2-parameter method was used to analyze evolutionary distances. Phylogenetic tree analysis demonstrated relationships between the IR4.3 isolate and 15 others reference bacteria from the GenBank based on the 16SrRNA gene fragment sequence. In the BLAST results, IR4.3 was closely linked to Lactobacillus genus with 100% similarity as shown in Figure 8. Results were analyzed because it was suspected that this isolate belonged to a similar genus and could produce antimicrobials. The phylogenetic tree illustrating the relationship between IR4.3 isolate and 15 other reference bacteria from the GenBank based on the 16S rRNA gene fragment sequence was as follows:

Generally, [24] showed that the evolutionary history of microorganisms could be reported using neighbor-joining method. Microorganisms in similar taxa usually cluster together in phylogenetic trees and include better bootstrap values [15]. In this study, a phylogenetic tree was drawn to investigate evolutionary distances using p-distance method. A total of 15 nucleotide sequences and codon positions were recorded using MEGA 7.0, as reported by [26] for evolutionary analysis. A phylogenetic tree (Figure 8) was reconstructed. The constructed phylogenetic tree resulted in two major branches consisting of the 15 bacteria. This occurred because the 16S rRNA gene fragment sequence included a similarity percentage of 100%, as verified by the results of BLAST, alignment and genetic distance calculations. Therefore, identification of IR4.3 isolate could be carried out at the genus level. Hence, it was concluded that IR4.3 isolate included L. fermentum strain 4901. These bacteria are homofermentative (only producing lactic acid) and cannot use pentoses (carbohydrates with a C5 atom). In general, [20] has stated that the bacteria of the lactobacilli group are bacteria that are included in the LAB category and these bacteria are widely used as probiotic agents because these bacteria produce the final products of the metabolic process, including lactic acid, which are generated from fermentation. The LAB is an anaerobic bacterium that is generally detected in fermented food and beverage products such as cheese, pickles, kimchi, fish stock and yoghurt. Therefore, it can be concluded that L. fermentum, which are classified as Lactobacillus, can be used as probiotics that are good for health. Moreover, LAB in fish are bacteria that are halal for consumption because they are originated from rinuak fish (Psilopsis sp.), different from several types of LAB used in fermented milk products such as Bifidobacterium isolated from baby feces. Technically, [23] has stated that in addition to food security which must be safe with good quality, food products of animal origin must meet the SHWH (safe, healthy, whole and halal) criteria. Additionally, [27] has reported that L. fermentum was verified to include characteristics of a probiotic agent that can survive in acidic conditions and inhibit the growth of Gram-positive and Gram-negative bacteria.

The present study varied from that of [28], which detected that the LAB originating from tilapia fish paste were L. fermentum. Isolation of LAB from shrimp paste carried out by [29] achieved the species L. plantarum. Studies by [30], investigating LAB strains from Sulawesi fermented milkfish Chanos-rice mixture of Burong Bangus reported various strains such as Enterococcus faecalis, Tetragenococcus muriaticus, L. delbrueckii subp. delbrueckii and Carnobacterium divergens. In a study by [13] on the isolation of LAB from tilapia fish (Oreochromis niloticus), LAB species were isolated by sequencing P. pentosaceus and E. avium. The species of bacteria varied due to the various types of fermented fish, including fresh fish, ingredients for fermentation and method of fermentation. The strain of L. brevis WD19 was isolated from the Algerian goat milk [21]. The finding of this study varied significantly from those of [22], who investigated presence of B. cereus strain HRV22 from isolation of the genome of freshwater shrimps using 16S rRNA gene sequencing method.

1. Conclusion

Results of DNA isolation and amplification with the 16S rRNA gene and primers of 27F and 1525R showed that the IR4.3 PCR product included 1500 bp and the isolate included the best antimicrobial potential of halal probiotic LAB, compared to 14 other bacterial isolates from rinuak fish in Lake Maninjau, West Sumatra, Indonesia. Isolate IR4.3 from rinuak fish showed 99.64% sequence similarity to L. fermentum strain 4901 based on 16S rRNA gene analysis, indicating that it belonged to a similar species. However, further genetic and phenotypic characterizations are needed to assess if IR4.3 represents a novel strain. The isolated species demonstrates potential as candidate probiotic LAB for use in natural food preservation and possibly for supporting treatments of diarrhea and typhoid fever. However, this potential must be verified through further in vivo studies and clinical validation. However, advanced studies of bacteriocins produced by LAB from rinuak fish in Lake Maninjau are still needed, including characterization of bacteriocins for food biopreservation.

1. Acknowledgements

The author thank Biotechnology Laboratory of Andalas University, Microbiology Laboratory of the Medan State University, Phytochemistry Laboratory of West Sumatra University, Padang State University and all the facilities that helped carry out this study.

1. Declaration of competing interest

The authors report no conflict of interest.

1. Authors’ Contributions

Heppy Setya Prima: Writing – Original Draft, Conceptualization, Methodology, Investigation; Rusfidra: Funding Acquisition, Validation; Fatridha Yansen: Writing – Review & Editing, Writing – Original Draft, Data Analysis, Investigation; Fajri Maulana: Conceptualization; Mia Ayu Gusti : Conceptualization

1. Using Artificial Intelligent Chatbots

No AI-assisted technologies were used in the preparation of this manuscript" or "No generative AI technologies or tools were employed in the writing or preparation of this manuscript

1. Ethical Consideration

Hereby, I am Heppy Setya Prima consciously assure that for the manuscript "Characterization, molecular identification and antimicrobial activity of lactic acid bacteria with potentials as halal probiotics isolated from Rinuak fish (Psilopsis sp.) in Lake Maninjau, West Sumatra, Indonesia" the following is fulfilled: 1) This manuscript represents the authors' original work and has not been published previously in any form. 2) The manuscript is not under consideration for publication by any other journal or publisher. 3) The content of the paper accurately and comprehensively reflects the authors' own research and analysis. 4) All significant contributions made by co-authors and collaborators are properly acknowledged. 5) The findings are appropriately contextualized within the framework of existing and prior research. 6) All sources utilized are properly cited, with any directly quoted material clearly indicated using quotation marks and appropriate referencing. 7) Each author has been actively and substantially involved in the research and preparation of the manuscript and collectively assumes full responsibility for its content.

Background and Objective: Increasing interests in healthy nutrition stimulate use of lactic acid bacteria with functional characteristics in production of dairy products. Lactic acid bacteria are included in the composition of starters for cheese making as well as other dairy products. In addition to their general health benefits, they show pronounced antimicrobial activities, partly due to their metabolite complexes. The primary method of ensuring milk safety is heat treatment. Pasteurization at 72°C ±2 is commonly used in cheese production. Nevertheless, the survival of viable Pseudomonas (P.) aeruginosa cells, capable of growing at refrigeration temperatures, may lead to spoilage during storage.

Material and Methods: Antagonistic activity of industrial Lactobacillus (L.) helveticus, against P. aeruginosa strains as well as the effectiveness of pasteurization at 72 °C ±2 (common in cheese production) was assessed. L. helveticus demonstrated high antagonistic activity against the reference collection strain of P. aeruginosa ATCC 25668 and the wild-type isolate P. aeruginosa 47, verifying its potential as a biological control agent.

Results and Conclusion: Limitations of standard pasteurization have been identified. During the heat treatment of sterile milk contaminated with the wild-type strain P. aeruginosa 47, 1.9 × 10⁴ CFU ml^-1 of viable cells survived after a 20-s exposure, while only 1.8 × 10¹ CFU ml^-1 survived after a 40-60 s exposure. Cell growth was observed at up to 3.5 × 10⁶ CFU ml^-1 during 7 d of storage. Microscopy revealed morphological changes in the cells (elongation and filamentous structures), indicating adaptive mechanisms. Similar results were detected for the reference collection strain of P. aeruginosa ATCC 25668. A 15-min pasteurization time at 72°C ±2 was effective against the two strains. The study suggests an integrated approach to ensuring the safety of dairy products by combining biological control with the optimization of heat treatment regimes.

Keywords: Microbial persistence, Heat treatment, Lactic acid bacteria, Pseudomonas aeruginosa

1. Introduction

The increasing interest in healthy nutrition stimulates use of lactic acid bacteria (LAB) with functional characteristics in production of dairy products. Metabolites from these cultures show pronounced antimicrobial activity in addition to their general health benefits. The LAB and their metabolic complexes may be promising agents for the study [1, 2]. Throughout their life cycle, LAB can produce a range of antimicrobial compounds, including organic acids (lactic, acetic, propionic and butyric acids), bacteriocins, hydrogen peroxide and bacteriocin-like inhibitory substances [3]. Their use can be considered as a natural strategy of pathogen control, including P. aeruginosa, and a basis for the development of innovative technologies in various industries [4, 5]. Moreover, the antagonistic activity of LAB against pathogen growth varies within species and strains. This antimicrobial effect results from the synergistic action of various metabolites [2, 3]. The primary method of ensuring milk safety is heat treatment. Pasteurization at 72 °C ±2  is commonly used in cheese production. Nevertheless, survival of viable P. aeruginosa cells, capable of growing at refrigeration temperatures, may lead to spoilage during storage. The LAB with strong antagonistic characteristics against P. aeruginosa can serve as additional barriers to ensuring the product quality and safety, when used as starter cultures.

Raw milk delivered to dairy plants is frequently contaminated with psychrotrophic bacteria. Within psychrotrophic microorganisms, P. aeruginosa is a Gram-negative pathogen with high spoilage potentials, significant resistance to antimicrobial agents and capacity to form biofilms [6]. Moreover, increase of multidrug-resistant strains due to the improper and excessive use of antibiotics increases morbidity and mortality within immuno-compromised patients. The P. aeruginosa is included in the top three causes of opportunistic infections in humans, affecting more than 2 million patients and causing approximately 90,000 deaths annually [7]. Despite being often underestimated as a foodborne pathogen, P. aeruginosa plays an increasingly significant role in contaminating industrial equipment and finished dairy products [8]. Moreover, P. aeruginosa can quickly become resistant to various stress factors, including antimicrobial agents and conventional disinfectants, which increases concerns about the potential transmission of resistant strains through the food chain [9,10]. The spoilage of products by P. aeruginosa poses a serious concern for consumers and food safety regulators. Milk is an excellent medium for the growth of various microorganisms. The psychrotrophic P. aeruginosa can continuously increase its population in milk at refrigeration temperatures (4–6 °C). Its short generation time—less than 4 h—enables it to reach cell counts exceeding 10⁶ CFU ml^-1 in milk after 8 d of storage [11]. The count of psychrotrophic bacteria in raw milk delivered to dairy plants reflects the effectiveness of sanitary and hygienic measures on the farm. Contamination of the dairy plant with P. aeruginosa lessens adequate sanitation. This is due to the P. aeruginosa ability to form biofilms actively and rapidly acquire resistance to disinfectants. Moreover, strains with various susceptibility to antimicrobial agents, including potentially resistant ones, may co-exist within a single facility [12]. Based on the International Dairy Federation’s recommendations, milk should be cooled to 4–10 °C within 1.5–3 h. In some countries, milk is delivered to plants only once a day, which may promote bacterial growth due to P. aeruginosa ability to multiply under a wide temperature range. Regarding its multidrug resistance and high adaptability, there is an urgent need to develop effective strategies to control P. aeruginosa contamination in the food industry.

Currently, the primary most effective method for eliminating pathogenic and spoilage-causing bacteria in milk is heat treatment. It is used in production of almost all dairy products [13]. Milk pasteurization is achieved by heating it to a temperature less than 100 °C for a time sufficient to eliminate pathogenic bacteria that may present.  There are various pasteurization regimes and the choice depends on the type of product.  For example, pasteurization at 72 °C ±2 is widely used for cheesemaking. Low-temperature pasteurization was previously assumed sufficient to prevent transmission of P. aeruginosa through dairy products. This assumption was based on the premise that non-spore-forming pathogenic bacteria should be eliminated at temperatures greater than 60 °C due to the denaturation of their essential cellular proteins [14,15]. However, recent studies have reported presence of heat-resistant bacteria that can survive standard pasteurization regimes [16]. Studies on the heat resistance of pathogens such as Listeria monocytogenes have shown that short-time pasteurization of milk at 71.7 °C for 15 s is insufficient [17]. Cells may be able to survive even after exposure to 72 °C for up to 4 min [17]. Frequency of P. aeruginosa detection in milk after pasteurization can relatively be high [18]. In the Czech Republic, a study of pasteurized milk samples for a year detected that 4% of the samples included P. aeruginosa. The effectiveness of pasteurization depends on the initial microbiological contamination of milk and the strain composition of the microbial contaminants [19]. The heat resistance of microorganisms varies widely and depends on factors that affect them before, during and after heat treatment [20].

The mechanisms enabling microorganisms to adapt to high temperatures are not fully understood. Scientific evidence suggests that the cellular response to heat shock involves synthesis of various proteins known as heat shock proteins [21]. Heat shock disrupts folding of existing and newly synthesized proteins. Intermolecular interactions within misfolded proteins lead to the formation of aggregates and their number increases proportionally with the intensity of heat treatment [22]. Thus, loss of essential cellular proteins compromises cell viability and may ultimately result in cell death. To modify these processes, bacteria produce cellular disaggregases that solubilize protein aggregates, promoting their refolding and recovery; thereby, enhancing heat resistance. A stronger adaptive response at higher temperatures is associated with the accumulation of larger qiantities and/or induction of a distinct subset of heat shock proteins. Other heat protective mechanisms may exist [20].

Most studies have focused on clinical isolates. Analysis of wild-type isolates may reveal previously unknown characteristics of P. aeruginosa, including enhanced heat resistance acquired under exposure to external stressors [23, 24]. This study included a reference strain of P. aeruginosa and a wild-type strain isolated from a dairy farm. The aim of this study and its novelty included investigation of the ability of P. aeruginosa cells to survive under pasteurization regimes used in cheese making, as well as their ability for reactivation during refrigerated storage. The study assessed potential use of industrial LAB strains as protective cultures within starter compositions, serving as additional antimicrobial barriers against P. aeruginosa.

1. Materials and Methods

2.1. Pseudomonas aeruginosa strains used in the study

The reference strain of P. aeruginosa ATCC 25668 was provided by the State Collection of Pathogenic Microorganisms and Cell Cultures "GCPM-Obolensk", Russia. The wild-type strain of P. aeruginosa 47 was isolated during microbiological monitoring of equipment surfaces at a private dairy farm. Identification was carried out according to GOST ISO 16266-2018, "Water Quality. Detection and Enumeration of P. aeruginosa. Membrane Filtration Method." Nutrient broth and dehydrated media (nutrient broth, dehydrated nutrient agar) from NPC LLC "Biocompas-S," as well as pseudomonas agar from Himedia, India, were used to achieve 24-h cultures and to inoculate. Compared to the reference strain of P. aeruginosa ATCC 25668, the isolate of P. aeruginosa 47 produced further pigments on nutrient and pseudomonas agars. This trait is generally addressed as an indicator of higher virulence [6]. A bacterial suspension was prepared from a 24-h culture in physiological saline, adjusted to a cell count of approximately 1.5 × 10⁸ CFU ml^-1, corresponding to an optical density of 0.5 of the McFarland. Experiments were carried out using sterile milk of the "Standard" brand (Complimilk, Belarus). Strains from the probiotic and lactic acid bacterial collection of the All-Russian Dairy Research Institute (FGANU “VNIIMI”) were used to assess antagonistic activity of the industrially promising LAB against P. aeruginosa 25668 and P. aeruginosa 47.

2.2. Lactic acid bacterial strains used in the study

The LAB strains were stored in lyophilized form at -50 °C ±1. Lyophilized cultures were reconstituted in sterile skim milk by incubation for 16 h. Strains were incubated at 37 °C ±1 (L. helveticus, Lacticaseibacillus rhamnosus and Streptococcus thermophilus) or at 30 °C ±1 (L. lactis).

2.3. Study of antagonistic activity using co-cultivation method

In general, P. aeruginosa and LAB cultures were prepared as described in Sections 2.1 and 2.2. Antimicrobial activity was assessed by co-cultivation of LAB with P. aeruginosa strains. Briefly, 1 ml of inocula from the selected LAB strains and P. aeruginosa strains was added to 20 ml of sterile skim milk and incubated at 37 °C ±1 for 48 h. Milk samples inoculated with 1 ml of the P. aeruginosa strain served as controls. After incubation, serial 10-fold dilutions were prepared from each sample and P. aeruginosa counts were reported by plating on cetrimide agar (Himedia, India), a selective medium for pseudomonads. Plates were incubated at 37 °C ±1 for 24 h; then, colony-forming units (CFU) were counted. The growth inhibition of P. aeruginosa was calculated regarding control sample (monoculture), which was reported 100%.

2.4 Study of the effectiveness of heat treatment against Pseudomonas aeruginosa

To assess effectiveness of the heat treatment against P. aeruginosa, sterile milk in test tubes was inoculated with a bacterial suspension of the P. aeruginosa strains to reach a final cell count of approximately n × 10⁵ CFU ml^-1. The cell count was verified by plating aliquots from serial dilutions of the sample. The contaminated milk was processed using UKT-150 heat resistance control device, which simulated a pasteurizer, providing 99.99% efficiency equivalent to standard pasteurization (Figure 1) [25].

After system activation, a rack with sealed, heat-resistant test tubes containing contaminated milk was transferred into a silicone solution. The oscillatory movements of the device contributed to the uniform heating of samples within the test tubes. The pasteurization regime (72 °C ±2), with a typical exposure time of 20 s for cheesemaking, was used with various exposure times of 40 s, 60 s, 5 min, 10 min and 15 min. Temperature control was carried out using control test tube with milk and thermometer to monitor the time needed to reach the target temperature. Upon completion of heat treatment, test tubes were rapidly cooled down using ice bath. The heat-treated and cooled contaminated milk was plated onto solid nutrient media and incubated at 37 °C ±1 for 24-48 h. Results were recored based on colony counts. To simulate shelf-life and storage conditions of the pasteurized milk, the experimental test tubes were stored in a refrigerator at 6 °C ±2 for up to 14 d. To assess the potential recovery of heat-stressed P. aeruginosa cells, samples were removed from the experimental test tubes after 7 and 14 d of storage, followed by plating and colony enumeration. Microscopic analysis was carried out to detect potential phenotypic changes in the cells.

2.5. Statistics

The MS Office Excel 2016 was used for data analysis and graph construction. Experiments were carried out in three independent replicates and results were expressed as mean ±SD (standard deviation). Differences were considered statistically significant at p < 0.05 using two-tailed Student’s t-test followed by Tukey’s HSD test (for multiple group comparisons). The study was carried out  using equipment from the Collaborative Center of the All-Russian Dairy Research Institute (CKP VNIMI).

1. Results and Discussion

3.1. Study of the antagonistic activity of LAB against P. aeruginosa using co-cultivation method

Results of the study on the antagonistic activity of industrial LAB strains are present in Table 1. As shown in Table 1, L. helveticus Bbn4 and L. helveticus NK1 were most effective in growth inhibition of the reference strain of P. aeruginosa and the wild-type isolate. After 48 h of co-cultivation, growth inhibition reached approximately 70%. Streptococcus thermophilus 16t effectively inhibited growth of the reference strain of P. aeruginosa 25668 but showed a limited activity against the wild-type isolate (growth inhibition of 24%). The antagonistic activity of L. rhamnosus F was less than that of L. helveticus strains, with growth inhibition reaching approximately 50%. In contrast, Lactococcus strains showed negligible or no inhibitory effects. These results were similar to those of previous studies reporting strong antagonistic activity of L. helveticus strains, including that against P. aeruginosa [26, 27]. Inhibitory effect of L. helveticus Bbn4 might be attributed to the synthesis of antimicrobial compounds, primarily organic acids, which effectively inhibit growth of pathogenic bacteria [28, 29]. It is known that L. helveticus strains are active acid producers [29, 30]. Additionally, L. helveticus strains can synthesize bacteriocins that inhibit the growth of pathogens, including P. aeruginosa [26]. The potential presence of antimicrobial compound-encoding genes in metagenome of L. helveticus Bbn4 warrants further investigations. The strains of L. helveticus Bbn4 and L. helveticus NK1 show potentials for use in production of fermented dairy products to decrease risks of P. aeruginosa contamination. However, further studies into the mechanisms of their antimicrobial action are necessary.

3.2. Study on the effectiveness of milk pasteurization at 72 °C ±2

Results of the study on the effectiveness of milk pasteurization at 72 °C ±2 with exposure times of 20, 40 and 60 s for samples contaminated with P. aeruginosa 47 are present in Figures 2a,b.

As shown in Figure 2a, viable cells forming colonies on nutrient agar were detected in all samples pasteurized at 72 °C ±2, regardless of exposure time. The shortest exposure time (20 s) was the least effective for P. aeruginosa 47 elimination, as the cell count decreased by only one order of magnitude from 1.8 × 10⁵ to 1.9 × 10⁴ CFU ml^-1. After 7 d of refrigerated storage (Figure 2b), cell counts increased to 3.5 × 10⁶ CFU ml^-1, with a slight increase to 5.4 × 10⁶ CFU ml^-1 on Day 14. The cell count of P. aeruginosa 47 decreased by four orders of magnitude, reaching 1.8 × 10¹ CFU ml^-1 following exposure at 72 °C ±2 for 40 and 60 s. After 7 d of refrigerated storage, the cell counts increased to 3.0 × 10⁶ and 5.7 × 10⁵  CFU ml^-1 in samples pasteurized for 40 and 60 s, respectively. Thus, none of the tested regimes resulted in the complete elimination of P. aeruginosa 47, with the 20-s pasteurization as the least effective. An increase in viable cell counts was observed in all samples after 7 d of refrigerated storage, exceeding the initial values, followed by a gradual stabilization on Day 14. Results of the study on the effectiveness of milk pasteurization at 72 °C ±2 with exposure times of 20, 40 and 60 s for samples contaminated with P. aeruginosa 25668 are present in Figures 3a,b.

For the reference strain of P. aeruginosa 25668, viable cells were detected immediately after all the pasteurization regimes were used. After 20 s of exposure, the cell count decreased by two orders of magnitude, from 1.9 × 10⁵ to 5.3 × 10³ CFU ml^-1. A 40-s pasteurization decreased the cell count by four orders of magnitude to 1.8 × 10¹ CFU ml^-1. After 60 s, only single colonies were observed (Figure 3a). On Day 7 of refrigerated storage (Figure 3b), cell counts increased to 3.3 × 10⁶, 1.8 × 10⁵ and 7.7 × 10⁴ CFU ml^-1 for 20, 40 and 60-s pasteurization regimes, respectively. A slight decrease was observed on Day 14, with cell counts reaching 1.7 × 10⁶, 3.4 × 10⁵ and 1.6 × 10⁴ CFU ml^-1 for similar pasteurization regimes. On Day 14, visible spoilage was seen in the test tubes, characterized by color change and formation of a surface film (Figure 4).

To establish the time needed for the effective elimination of P. aeruginosa during pasteurization at 72 °C ±2, contaminated milk samples were heat-treated for 5, 10 and 15 min. A 15-min pasteurization at 72 °C ±2 was sufficiently effective against the reference and wild-type strains, with initial titers of 9.5 × 10⁵ and 9.8 × 10⁵CFU ml^-1, respectively. No bacterial growth was detected under this pasteurization regime through 14-day refrigerated storage time. Pasteurization regime of 72 °C ±2 with exposure times of 5 and 10 min did not guarantee the absence of forms capable of reactivation, as growth of individual colonies was seen.

This study verified high antagonistic activity of L. helveticus Bbn4 and L. helveticus NK1 against the reference strain and the wild-type isolate of P. aeruginosa from a dairy farm. Their inhibitory effects might be attributed to various metabolic products that effectively inhibited growth of the pathogen. It is known that P. aeruginosa produces siderophores, low-molecular-weight (LMW) compounds that chelate and solubilize iron [6]. Lactobacilli, unlike most microorganisms, are iron-independent, making them unaffected by siderophores produced by P. aeruginosa. Moreover, organic acids, the primary metabolites from LAB, possess chelating characteristics and can bind iron from the substrate; thereby, limiting its availability for P. aeruginosa growth. Antimicrobial activity of Lactobacillus spp. might be associated to the induction of enzymes that degraded peptidoglycan layer of the cell walls of Gram-negative bacteria. Thus, antimicrobial activity of the industrially linked LAB strains of L. helveticus NK1 and L. helveticus Bbn4 against P. aeruginosa verified their functional characteristics. These strains show promise for use in development of functional foods, especially in dairy formulations.

The study demonstrated that short-time pasteurization (20–60 s at 72 °C ±2) was insufficient to eliminate P. aeruginosa, as viable cells with an initial titer of approximately 10⁵ CFU ml^-1 were detected immediately after heat treatment. According to Sviridenko et al., the P. aeruginosa culture, known for its psychrotrophic characteristics, showed the ability of individual surviving cells to multiply after heat treatment at 72 °C ±2, with an initial contamination level of approximately 10⁶ CFU ml^-1 and to preserve ability to reactivate, similar to the present findings [19].

 Pasteurization for 5 and 10 min at the highlighted temperature did not guarantee complete inactivation of P. aeruginosa, as cells preserved ability to recover and resume growth during storage. On Day 14 of storage, no growth was observed in the sample that was heat-treated for 15 min. Based on the scientific data on P. aeruginosa survival, heating milk for at 63.5 °C for 30 min with an initial contamination level of 5.8 × 10⁵ CFU ml^-1 resulted in a 4-log dcrease in viable cell count. At the same time, a significant proportion of the cells was metabolically active but unable to form colonies on solid media. Thirty percent of P. aeruginosa strains showed signs of metabolic recovery within 24 h, following heat treatment at 72 °C ±2 for 16 s, supporting the present results. Under low and high-temperature pasteurization regimes of 72 °C ±2 and 80 °C ±2 for 10–20 s, individual P. aeruginosa cells preserved their ability to resume growth and reactivate following heat treatment at high initial contamination levels (> 10⁶CFU ml^-1) [21]. The authors’ findings regarding the heat resistance of another Gram-negative opportunistic bacterium—E. coli—indicated that the reference strain of E. coli, as well as wild strains, included heat stability. Moreover, under certain conditions, cells might survive pasteurization and later resume growth and multiplication. The authors suggest that wild-type strains of microorganisms may show increased heat resistance. The current results demonstrated a higher recovery rate for the wild-type strain of P. aeruginosa 47, compared to the reference strain. Therefore, these findings verified that certain bacterial strains could survive heat treatment under standard processing conditions.

In general, bacterial responses to stress factors, including heat shock, result in similar adaptive outcomes as transition to a dormant state. Bacteria decrease their metabolic activity, slowing down growth and division while decreasing expression of virulence-linked genes to prioritize survival. Sometimes, Pseudomonas spp. are capable of forming cyst-like dormant forms. After heating P. aeruginosa cell suspensions at 70 °C for 5 min, small pinpoint microcolonies (≤ 0.5 mm in diameter) were seen. These colonies were difficult to detect and became visible after prolonged incubation (7–14 d and in cases up to 1 m). The efficiency of microcolony reversion to the normal phenotype after subculturing on agar media did not exceed 20% . In this study, heat stress resulted in growth of non-pigmented pinpoint colonies on solid media (Figure 5ab).

Exposure to various stress factors has been reported to cause similar morphological changes in microbial cells, which preserve their functionality and serve as universal markers of adaptation mechanisms. For example, antibiotic exposure induces cell variability, characterized by heteromorphic growth, including cell elongation and formation of filamentous structures. On solid media, primarily pinpoint colonies were seen. Microscopy of P. aeruginosa cells after heat exposure revealed atypical elongation and occasional chain formation, which might indicate involvement of proteins that enhanced heat resistance. The present study revealed heat-induced morphological changes in P. aeruginosa cells, characterized by elongation, compared to untreated samples.

Bacterial response to heat stress includes synthesis of heat shock proteins, cellular disaggregases capable of solubilizing and refolding damaged protein aggregates; thereby, enhancing microbial heat resistance [22]. Within bacterial disaggregases, two proteins are particularly well known for protecting cells from heat-induced damages, including ClpB and ClpG. The ClpG shows stronger disaggregation activity and is associated with increased heat resistance in bacteria. An increase in disaggregase levels reflects the bacterial ability to adapt to extremely high temperatures during sterilization and pasteurization processes in food industry and medicine. Presence of ClpG has been verified in several Gram-negative bacteria, including P. aeruginosa. In P. aeruginosa, ClpG levels increase during the stationary growth phase, while ClpB activity predominates during the logarithmic phase. These proteins can functionally compensate for each other within specific temperature ranges: ClpG is stable at 70°C whereas ClpB is inactivated at 60–65 °C. Furthermore, ClpG may enable P. aeruginosa to survive pasteurization temperature of 72 °C ±2. Acquisition of an additional disaggregase, ClpG_GI, has contributed to the widespread dissemination of P. aeruginosa C-clone populations; in which, it has been identified. Additionally, ClpG_GI is likely not a species-specific protein and has been detected in unrelated Gram-negative bacteria, suggesting recent acquisition by P. aeruginosa and a possible role in enhancing heat resistance of wild-type strains [22]. Genes encoding ClpG are horizontally transferable, facilitating the rapid spread of heat resistance traits in microbial communities. This may undermine efficacy of the present heat treatment regimes, including those used in the food industry.

1. Conclusion

Carried out studies demonstrated antimicrobial activity of industrially associated LAB strains against P. aeruginosa. The most pronounced inhibitory effect was shown by L. helveticus Bbn4 and L. helveticus NK1 strains. These strains and their metabolic complexes can be considered promising biological agents to control P. aeruginosa contamination, a particularly challenging pathogen.

Pasteurization at 72°C ±2 for 20, 40 and 60 s was insufficient to eliminate P. aeruginosa in the reference strain and the wild-type isolate. Ability of P. aeruginosa to survive under these pasteurization conditions should be addressed for technological process optimization in dairy production, particularly in facilities prone to microbial contamination. Exposure time was identified as a critical efficiency parameter; a 15-min treatment at 72°C resulted in the complete elimination of the pathogen. The available literature provides limited data on the ability of P. aeruginosa to survive milk pasteurization and reactivate during refrigerated storage. This characteristic may contribute to product spoilage and pose a potential risk to public health. The current findings highlight practical uses for dairy production. They highlight needs to adjust pasteurization regimes for resistant strains, potential use of LAB as additional barriers against P. aeruginosa and importance of monitoring survival and recovery of pathogens during storage. The study suggests an integrated approach to ensuring safety of dairy products by combining biological control with the optimization of heat treatment regimes.

1. Acknowledgements

The authors express their gratitude to Galstyan A.G. for supporting our work.

1. Declaration of competing interest

The authors report no conflicts of interest

1. Authors’ Contributions

Conceptualization, I.R., N.P., V.L., A.P.; methodology S.K., E.I.; validation, S.K., E.I.; formal analysis, I.R., V.L., S.K.; investigation, E.I., S.K.; data curation, S.K., E.I.; writing—original draft preparation, S.K.; writing—review and editing, V.L.,I.R.; visualization, S.K., VL; supervision, AP; project administration, IR., AP.

1. Using Artificial Intelligent Chatbots

The authors did not use artificial intelligence

1. Ethical Consideration

This study does not require approval from an ethics committee.

Background and Objective: Lactic acid bacteria play a fundamental role in the human diet, particularly in fermented foods such as dairy products. This study aimed to investigate microbial diversity of a traditional milk product (Dhan) prepared from raw milk of lactating dairy cows in Northwestern Algeria, highlighting the significance of this traditional dairy product and providing a detailed characterization of its lactic acid bacterial population.

Material and Methods: Nine samples were collected from the available farms in Tiaret and Tissemsilt regions and subsequently cultivated on selective media of MRS agar for Lactobacillus and M17 agar for Lactococcus. Identification was carried out using API Systems (API 50 CHL) and other biochemical assays.

Results and Conclusion: The bacterial load lactic acid bacteria counts (log10 CFU g⁻¹) averaged 4.92 ±0.47 on MRS and 4.52 ±0.77 on M17 media. Eighteen bacterial isolates were categorized into three genera of Lactobacillus (73%), Lactococcus (16%), and Leuconostoc (11%), with Lactiplantibacillus plantarum as the most prevalent species (seven isolates). Lactobacillus spp. are the dominant lactic acid bacteria in Dhan and further studies are needed to isolate and characterize biotechnological potentials of these isolated species and their possible roles in innovation.

Keywords: Lactic acid bacteria, traditional Algerian butter, Dhan, microbial diversity, Lactococcus, Lactobacillus, Leuconostoc, fermented foods, food biotechnology, dairy products

1. Introduction

Dairy products, which are vital for human nutrition, are continuously developed worldwide. Since the 1980s, Algerian dairy industry has significantly transformed, moving from a governmental system to a further dynamic private model [1]. However, the quality of raw materials and regulation of the fermentation process are significant problems for the industry and research [2,3].

 Traditional fermented milk products play a significant role in diets and cultures of several regions; however, their microbiological diversity is poorly understood, especially in Algeria. Numerous studies have demonstrated that accurate identification of lactic acid bacteria (LAB) in products such as kefir, leben and traditional yogurt is critical for understanding their effects on quality, safety and sensory qualities [4,5].

Traditionally, these microorganisms are identified using polyphasic approach based on culture-dependent methods. This strategy combines the initial isolation of strains on selective media with further phenotypic characterization. This involves assessing key traits such as cell morphology, physiological tolerance to environmental stressors (e.g. temperature and salinity) and biochemical profiles. Assessing metabolic fingerprint of an isolate, particularly its carbohydrate fermentation pattern, is a basis of this methodology for species-level differentiation.

Dhan, a traditional Algerian fermented butter, has received limited attention and its specific microbiology needs further investigations. This microbiological diversity in Dhan deserves further studies because precise identification of the dominant and minor sources of LAB species is essential for controlling sensory and hygienic qualities of the final product [6]. Therefore, the current study aimed to close this gap by characterizing community of LAB in Dhan using physiological and biochemical methods to preserve and improve the quality of this product.

1. Materials and Methods

2.1. Sampling Region

Samples were collected from Tissemsilt (35° 36′ N, 1° 49′ E) and Tiaret (35° 22′ N, 1°19′ E), Algeria. These regions included Mediterranean climates with elevation of approximately 1,080 m above the sea level, average annual rainfall of 300–500 mm and average annual temperature of 22 °C. During the sampling time from February to May 2023, the ambient temperature varied 14–28 °C (Figure 1).

2.2. Sample Collection

Purposive sampling approach was used to identify authentic producers of traditional Dhan butter across the Tiaret and Tissemsilt regions. Stringent selection criteria were established, limiting eligibility to registered dairy farms with (i) active traditional production, (ii) verified compliance with national hygiene standards and (iii) official state accreditation. This rigorous process yielded a total of nine qualifying farms—four in Tiaret and five in Tissemsilt—representing all the accredited producers in the study area. This strategy ensured that microbiological analysis was carried out on authentic standardized samples. All samples were aseptically collected from the nine farms and processed at the Laboratory of Microbiology, University of Tissemsilt, Algeria.

Lactic Acid Bacteria Isolation and Enumeration

All microbiological chemicals and culture media used in this study were purchased from Lab Quality, Algeria. Samples were diluted in sterile 0.9% NaCl solution and spread-plated onto M17 and MRS agars for Lactococcus and Lactobacillus spp. The pH of the MRS agar was adjusted to 5.4 to enhance recovery. Plates were incubated for 24–72 h and colonies with typical LAB morphology were selected, counted and purified [7].

2.3. Culture Maintenance and Purification

The LAB isolates were purified via alternating subcultures on MRS agar and broth. Purified isolates were stored at -20 °C in MRS broth supplemented with 30% (v/v) sterile glycerol.

2.4. Characterization and Identification of the Lactic Acid Bacterial Isolates

2.4.1. Morphological and Basic Biochemical Characterization:

Colony characteristics, cell morphology, Gram reaction, catalase activity and oxidase activity assessments were carried out on agar plates based on the standard protocols [7].

Physiological Characterisation

Gas Production from Glucose: Isolates were inoculated into MRS broth (citrate-free) with inverted Durham tubes and incubated at 30 °C for 24–48 h. Gas accumulation indicated CO₂ production (heterofermentative).

Arginine Dihydrolase (ADH) Activity: Arginine dihydrolase (ADH) activity was assessed using M16BCP media incubated anaerobically (30 °C, 72 h). A purple-to-yellow color change revealed ADH positivity.

Growth under Stress Conditions: Growth was monitored by measuring OD₆₀₀ (UV-visible 7305 spectrophotometer, Labo and Co, France) against uninoculated controls after incubation under specific conditions. The NaCl tolerance was assessed using MRS/M17 broth with 4 and 6.5% (w/v) NaCl at 37 °C for 24–48 h. Temperature tolerance was  Assessed in MRS/M17 broth at 10, 15, 37, 40 and 45 °C for 24–72 h [8].

2.4.2. Assessment of Sugar Fermentation Profiles

Cell pellets were achieved from 18-h cultures grown at 30 °C by centrifugation (5000× g, 5 min). The washed pellets were resuspended in indicator media (MRS BCP for Lactobacillus spp. and 0.5% peptone water with bromothymol blue for Lactococcus spp.) containing 0.5% of a sugar. Tubes were overlaid with sterile paraffin oil and incubated at 30 °C for 3–7 d). The color change indicated fermentation. Sugars included xylose, maltose, galactose, D-sorbitol, arabinose, mannitol, L-rhamnose, sucrose, lactose, D-fructose, glucose and esculin.

2.5. Carbohydrate Fermentation Profile

Eighteen isolates were assessed using API 50 CHL strips, according to the manufacturer's instructions. This standardized identification system has been reported as an important reliable tool for lactobacilli identification based on phenotypical characteristics [9]. Inocula were prepared from fresh colonies (MRS agar; 30 °C, 48 h) suspended in API 50 CHL media. Strips were inoculated (100 µl per well), overlaid with sterile mineral oil and incubated at 30 °C for 48 h under humid conditions. Color changes were recorded after 24 and 48 h and results were interpreted using APIweb software.

2.6. Quality Control

Several quality control assessments were used to ensure validity of the results. Sterility of all culture media and diluents was verified by incubating uninoculated negative controls (uninoculated media) within the experimental samples, ensuring aseptic integrity of the procedure. To validate accuracy of the biochemical assays, resulting profiles of the identified species were systematically compared with those in the scientific literature and Bergey's Manual of Systematic Bacteriology. This comparative analysis verified that the observed reactions (e.g. Gram staining, catalase test and carbohydrate fermentation pattern) were similar to the expected profiles for the identified taxa.

2.7. Data analysis

Statistical analysis was carried out using R software. For each sample, triplicate assessments were averaged. Overall means and 95% confidence intervals were calculated from nine sample means using Student's t-distribution.

1. Results and Discussion

3.1. Bacterial Counts and Isolation

The LAB counts (log10 CFU g^-1) averaged 4.92 ±0.47 on MRS and 4.52 ±0.77 on M17 media. The higher variability observed on M17 media (CV = 17%) than on MRS (CV = 9.6%) reflected the selective nature of these media for various LAB groups. These counts were smaller than those reported in other studies. A Turkish study on yak butter [9] reported greater levels of Streptococcus thermophilus (5.42–6.30 log CFU ml^-1) and L. delbrueckii subsp. bulgaricus (5.20–5.80 log CFU ml^-1) in goat, ewe and cow milk butters. Furthermore, the present results were smaller than those of an Ethiopian study [7], which reported Lactobacillus counts ranging from 4.5 × 10⁷ to 2.3 × 10⁸ CFU ml^-1 and Lactococcus counts ranging from 1.12 × 10⁷ to 2.75 × 10⁹ CFU ml^-1 in raw milk, cheese and yogurt.

3.2. Microbiological Quality Assessment

The microbiological quality parameters of traditional butter (Dhan) samples were assessed to report the overall hygiene of the product (Table 1). All samples were analyzed in triplicate to ensure reproducibility. The total aerobic mesophilic flora (FMAT) averaged 2.04 ±0.27 × 10⁴ CFU g^-1 [95% CI, 1.83–2.24], indicating a moderate microbial load typical of the traditional fermented dairy products. The total coliform (1.37 ±0.16 × 10³ CFU g^-1) and fecal coliform (1.45 ±0.23 × 10 ² CFU g^-1) counts were included in the acceptable ranges for the products. Yeast and mold populations included 2.47 ±0.26 × 10³ CFU g^-1 and 1.13 ±0.14 × 10³ CFU g^-1, respectively. The low coefficients of variation (< 15%) between the replicates demonstrated good analytical precisions.

3.3. Morphological and Phenotypical Characterizations

Of the 18 isolates from traditional Dhan samples, 73% were identified as Lactobacillus, 11% as Lactococcus and 16% as Leuconostoc spp. (Figure 2). On MRS media, Lactobacillus colonies were rounded, smooth and convex with whitish to beige in color. On M17 media, Lactococcus isolates formed white to yellowish circular colonies and showed diplococcal cell arrangement in chains and clusters.

All the isolates were Gram-positive (Figure 3). Thirteen isolates showed rod morphology (LBS1a through LBS1m) characteristics of Lactobacillus, whereas five isolates showed cocci morphology characteristics, typical of Lactococcus and Leuconostoc spp.

3.4. Species Identification

Species identification revealed the following distribution of Lactiplantibacillus plantarum (n = 7, 39%), Lacticaseibacillus casei (n = 3, 17%), L. acidophilus (n = 3, 17%), L. cremoris (n = 2, 11%), L. lactis (n = 2, 11%) and L. mesenteroides subsp. dextranicum (n = 1, 6%).

3.5. Physiological and Biochemical Characteristics

Results achieved for the six isolates verified their identification as L. plantarum. Their phenotypic characteristics were consistent with published data. The observed fundamental characteristics such as Gram-positive staining, absence of catalase activity and facultative heterofermentative metabolism were classic markers of L. plantarum, which has widely been documented in studies on isolates from diverse food sources [8,10,11]. Growth of strains at 10 °C and their inhibition at 45 °C verified their mesophilic nature. This characteristic was shared by L. plantarum strains from fermented dairy and meat products, indicating their adaptation to moderate fermentation conditions [10,11]. Moreover, their tolerance to 4% not to 6.5%  NaCl was similar to that of many strains used in processes where salt played a preservative role [8,11].

Carbohydrate fermentation profile provided a distinctive biochemical signature for these isolates. Their ability to metabolize a wide range of sugars, including maltose, galactose, sorbitol, mannitol and lactose, reflected high metabolic versatility that characterized L. plantarum and explained its prevalence in diverse food ecosystems [8,11]. This flexibility was a major technological asset for its use as a starter culture. The role of dominant Dhan strains in microbial stabilization of the product was supported by the associations between increased pathogen inhibition and presence of plnEF and plnJ genes in L. plantarum from fermented products such as Ishizuchi-kurocha tea [12].

Strains S1a, S1h and S1i, identified as L. casei, showed characteristics similar to this species; however divergences were reported. Gram-positive staining and negative reaction to catalase were classic markers for Lactobacillus spp. and L. casei, particularly [9]. Temperature tolerance was similar to that of literature with growth observed at 10 and 40 °C but not at 45 °C. This was similar to the reported optimal growth temperature of 41–42 °C and inhibition at higher temperatures [13]. Similarly, presence of growth at 4% NaCl and absence of growth at 6.5% NaCl were similar to salt tolerance profile described for L. casei [14]. Fermentation profiles of certain sugars such as glucose, fructose, galactose and mannitol as well as hydrolysis of esculin were similar to the expected biochemical profiles [15].

However, results revealed significant differences from the typical profiles described in the literature. The most important difference included inability of the assessed strains to ferment either lactose or sucrose. The L. casei is a bacterium widely used in the fermented dairy products because of its effective ability to metabolize lactose [16]. Numerous studies have reported its ability to use sucrose [17]. Absence of fermentation of these two sugars was therefore atypical and could indicate strain-specific characteristics of the isolates since sugar metabolism could be strain-dependent and presence of specific genes often located on plasmids [18]. Additionally, gas production by these isolates suggested heterofermentative metabolism. This was in contrast to typical behavior of L. casei, which was facultatively homofermental and produced primarily lactic acid from hexoses without gas formation [19].

Analysis of the results for S1k, S1l and S1m strains, identified as L. acidophilus, demonstrated similarity to the phenotypical and biochemical characteristics of this species, verifying accuracy of the identification [15]. The observed fundamental features such as Gram-positive staining and negative reaction to catalase were classic markers of LAB [10,20]. Strict homofermentative metabolism indicated by the absence of CO₂ production from glucose was a distinctive characteristic of L. acidophilus [10]. These basic assays strongly indicated that these isolates belonged to the homofermentative Lactobacillus group.

Growth profiles were investigated under various temperatures and salinity conditions. Growth at 40 not 10 and 45 °C verified mesophilic characteristic of the strain, whose optimal temperature range was generally between 37 and 42 °C. This thermal profile was in contrast to that of other LAB such as the psychrotrophic species of L. piscium adapted to cold temperatures and lacked growth at temperatures greater than 29 °C [20]. Similarly, tolerance to 4% not 6.5% NaCl was similar to physiological characteristics of many L. acidophilus strains. However, this tolerance might vary. Indeed, studies have isolated strains of Lactobacillus capable of growing at 6.5% NaCl, highlighting intraspecific variations associated to the adaptation of the strain to its environment [10].

Fermentation profile of sugars is the most robust element for the microbial verification. Ability of the strains to metabolize a wide range of carbohydrates, including glucose, fructose, galactose, lactose, sucrose and maltose, is typical for L. acidophilus, a species commonly isolated from fermented dairy products [10]. Positive fermentation of D-sorbitol and mannitol although showing strain-to-strain variations is a documented characteristic of the reference strain L. acidophilus ATCC 4356, supporting accuracy of the identification. In contrast, L. acidophilus strains from Ethiopian fermented milk lacked this fermentation ability, demonstrating metabolic diversity within the species  [10]. Furthermore, hydrolysis of aesculin and inability to ferment pentoses such as xylose and arabinose completed the characteristic biochemical profile. However, fermentation of xylose has been observed in wild strains [10]

Analysis of the results for the LNS3a and LNS3b strains identified as L. lactis demonstrated characteristics similar to the typical characteristics of this species, as described in the scientific literature. Isolates demonstrated typical LAB characteristics, including Gram-positive staining and negative catalase reaction. Production of gas verified strict heterofermentative metabolism of L. lactis, a distinctive characteristic of the genus. This metabolism, via the phosphoketolase pathway, converted hexoses to lactic acid, ethanol and CO₂ [22].

Temperature tolerance profile was similar to the species profile. Ability to grow at 10 °C while inhibited at 40 and 45 °C corresponded to the mesophilic and psychrotrophic characteristics of the species. Indeed, Leuconostoc spp. involved in food fermentation include optimal growth temperature generally between 18 and 22 °C, with inability to grow at higher temperatures [21]. The strain grew at 4% not 6.5% NaCl demonstrated salt tolerance similar to that of the species profile. Many Leuconostoc spp. Include this particular tolerance, allowing them to outcompete less tolerant microorganisms in moderately saline environments such as vegetable fermentations [21]. Fermentation profile of sugars supported this identification.

Ability to metabolize monosaccharides (glucose, fructose, galactose) and disaccharides (lactose, sucrose, maltose) is a typical characteristic of L. lactis, a bacterium widely used in dairy industries and plant fermentations [22]. Inability to ferment mannitol is a significant point. Although these strains ferment fructose, they do not convert it into mannitol as an ability of other heterofermentative species of Leuconostoc [23]. Absence of pentose fermentation (xylose, arabinose) and esculin hydrolysis completed the biochemical profile. Characteristics of strain LNS3c were similar to those described for L. mesenteroides subsp. dextranicum in the scientific literature. The isolate demonstrated typical Leuconostoc characteristics, including Gram-positive staining, negative catalase reaction and strict heterofermentative metabolism [24].

Growth profile was similar to that in literatures. Ability to grow at 10 °C not 40 °C verified mesophilic and psychrotrophic characteristics of the species, whose optimal temperature is generally between 25 and 30 °C [25]. Similarly, tolerance to 4% not 6.5% NaCl was similar to the reported characteristics, as growth is typically inhibited at concentrations greater than 6% [26]. Fermentation profiles of sugars supported this identification. Ability to metabolize glucose, fructose, galactose and lactose is a typical characteristic of this species [27]. The positive fermentation of sucrose is particularly significant because L. mesenteroides subsp. dextranicum is renowned for the use of this sugar to produce extracellular polysaccharides, particularly dextran [25]. Inability to ferment maltose was a distinctive result, although strain-specific variation in sugar metabolism has been reported [27].

Analysis of the results for LCS2a and LCS2b strains identified as L. cremoris showed excellent similarity to the typical characteristics of this species. Isolates demonstrated typical Lactococcus characteristics, including Gram-positive staining and negative catalase reaction. Furthermore, absence of gas production (CO₂) verified their strict homofermentative metabolism, converting sugars to lactic acid primarily [10].

Temperature and salt tolerances were similar to those reported in literatures. Growth observed at 10 not 40 °C was a typical characteristic of mesophilic bacteria such as L. cremoris, whose optimal temperature is nearly 30 °C and growth is generally inhibited at temperatures nearly 40 °C [10,11]. The 4% NaCl tolerance and 6.5% inhibition characteristics were similar to the reported characteristics, as the viability of L. cremoris decreases significantly with increasing salt concentration with numerous strains inhibited by concentrations greater than 5% [28,29]. Fermentation profiles of the sugars supported this identification. Ability to metabolize a wide range of carbohydrates, including glucose, lactose, galactose, sucrose and maltose, was a well-documented characteristic of L. cremoris, a species widely used as a starter culture in the dairy industry [30]. Xylose fermentation, though less consistently reported, is strain-dependent.

In this study, several methodological limitations must be acknowledged. First, the relatively small sample size due to the practical constraints meant that while the present findings provided valuable preliminary insights, larger-scale studies are still necessary. Second, this study relied on biochemical and phenotypic methods for species identification. This approach was chosen to establish a functional profile of the Dhan microbiota and provide a metabolic fingerprint of the isolates. While this method provides valid preliminary identification for the study primary aim, the current authors recognize that the absence of molecular verification is a key limitation. Molecular techniques such as 16S rRNA gene sequencing were not available due to practical constraints. Therefore, these isolates represent prime candidates for further genetic verifications to validate their taxonomy. Such molecular approaches can provide higher resolution identifications and further investigate potentials of this unique microbiota.

1. Conclusion

This study highlighted diversity and abundance of LAB isolated from Dhan of raw milk in western Algeria. Six LAB species were identified using traditional biochemical methods with L. plantarum showing unique predominance. This finding represented the first report of LAB diversity in Dhan, revealing unique microbial biodiversity of Dhan microbiota and broadening current understanding of the microbial biodiversity of traditional fermented milk products. Physiological and biochemical characteristics demonstrated by the isolated strains suggested that they warrant further investigations for potential technological and probiotic uses in food industries. These findings contributed to the present understanding of traditional fermented products as sources of beneficial microorganisms with potential uses in food preservation and quality improvement.

1. Acknowledgements

The authors express their gratitude to Agronomy Environment Research Laboratory, Tissemsilt University, Algeria, for providing facilities and materials in the present study.

1. Declaration of competing interest

The authors declare no conflict of interest.

1. Authors’ Contributions

Silarbi tayeb : Methodology designing, data curating, data analyzing and   writing   original   draft   of   the   manuscript;   laabas saadia: conceptualizing,  methodology  designing,  supervising  and reviewing  and  editing  the  manuscript;  chahbar mohamed:  methodology designing,   supervising   and   reviewing   and   editing   the manuscript; Khaled hamden:     supervising    and    reviewing    the manuscript. All authors read and approved the final version of the manuscript.

1. Using Artificial Intelligent Chatbots

No chatbots or artificial intelligence tools were used for data analysis, scientific content generation, or interpretation of results in this research.

1. Ethical Consideration

All experimental procedures were carried out in accordance with national hygiene and ethical standards. No animal samples were taken without the consent of producers. The study protocol did not require the collection of information from people or live animals, and was limited to the collection and analysis of dairy products. The confidentiality of participating producers has been preserved.

Background and Objective: Buffalo plays a role in providing animal protein in Indonesia. Their meat and skins are widely used as raw materials for processed foods. However, the high demand for buffalo products is not proportionate with the current supply; thus, creating chances for food fabricating. This practice is not only illegal but also causes serious risks to food safety and halal compliance. The detection of adulteration in food product can be carried out using polymerase chain reaction technique and specific primers that target buffalo DNA. This research aimed to develop primers that could accurately identify buffalo DNA.

Material and Methods: The primers were designed based on the sequence of the buffalo cytochrome b gene. The effectiveness of these primers in recognizing and amplifying buffalo DNA was assessed using polymerase chain reaction technique. Specificity assessments were carried out to assess the primer capability to detect buffalo DNA, with comparative controls including cattle, goat, chicken, pig, dog and mouse DNA. Sensitivity assessments were carried out to assess the minimum DNA concentration detectable by polymerase chain reaction. Then, the ability of these primers in identifying buffalo skin crackers was assessed against controls of cow skin crackers, goat skin crackers, and crispy chicken skin and pork skin crackers.

Results and Conclusion: The polymerase chain reaction results indicated that the Buffalo_5.1 primer pair (forward 5’-TTAGTACTATTCGCACCCGACCTC-3’ and reverse 5’-TCGTTGTTTGGATGTATGTAGCAG-3’) successfully amplified buffalo DNA specifically, with a detection limit of up to 10^-3 ng μl^-1 (assuming that the solution included a density of 1 g ml^-1 equal to 10^-⁷ % w/w), which could potentially increase to 10^-5 ng μl^-1 (10^-⁷ % w/w) under optimal conditions. Furthermore, it was able to detect buffalo DNA in buffalo-skin cracker products even at low DNA purity levels. These results suggest that the Buffalo_5.1 primer includes the potential to serve as a reliable molecular marker in polymerase chain reaction-based food authentication studies.

Keywords: Buffalo, Food adulteration, Forward and reverse primers, PCR

1. Introduction

Indonesia, the fourth most populous country in the world, faces a high demand for animal protein, including buffalo meat. However, domestic production is insufficient to meet national needs. In 2023, buffalo meat production reached only 22,110 tons, while the total national demand for beef and buffalo meat was 680,019 tons and is projected to increase to 724,188 tons in 2024 (1,2). To overcome this problem, in 2023, Indonesia imported large quantities of buffalo products, nearly 100,000 tons, majorly from India (3). Other than serving as a protein source, buffalo commodities support the tourism sector through the production of local delicacies such as rambak crackers made from buffalo (Bubalus bubalis) skin. The increasing demand and limited supply, however, create opportunities for food fraud that threaten food safety and halal compliance. Reliable authentication is therefore essential. Molecular methods based on nucleic acid analysis, particularly polymerase chain reaction (PCR), are widely used for species identification because of their high sensitivity and the inherent stability of DNA molecules. In various research, the PCR technique targeting mitochondrial DNA has commonly been used for species identification in food products (4–7); however, specific molecular biomarkers for detecting adulteration in buffalo-derived and processed products are underdeveloped.

Mitochondrial DNA offers greater effectiveness than nuclear DNA because it exists in thousands of copies per cell, enabling reliable detection even from limited or degraded samples. It yields higher amplification success in extensively processed food products where DNA fragmentation is severe (5). The mitochondrial cytochrome b (cytb) gene has long been used as a molecular marker for species identification in meat authentication (8). The cytb gene shows a lower mutation rate compared to other genes within mitochondrial DNA loci (9). In previous research, the cytb gene sequence has been used as a basis for designing species-specific primers for dogs, which have proven effective in detecting dog meat contamination in meatball products (10),  verifying that the leather garment material is from cowhide (11), as well as for halal authentication (4,12–17).

Although previous studies have developed cytb-based primers for buffalo DNA detection, most studies were designed for raw or minimally processed meats, with reported amplicon sizes ranging from approximately 655 to 106 bp (18, 19). Amplicon size critically affects detection performance in processed foods, where DNA degradation is extensive—larger fragments often fail to amplify, while overly short targets may decrease specificity. Therefore, current primers are inappropriate for highly processed non-meat buffalo derivatives such as buffalo skin crackers (rambak), which include severe thermal and chemical treatments.

Advanced molecular approaches such as real-time PCR and loop-mediated isothermal amplification (LAMP) have improved the sensitivity of buffalo DNA detection, achieving detection limits nearly 1% (20, 21). However, these techniques need costly reagents and specialized instrumentation, limiting their practicality for routine food inspection or field assessing. Despite these developments, a significant research gap persists that the current cytb-based primers are not optimized for detecting buffalo DNA in highly processed non-meat derivatives such as buffalo skin crackers (rambak) that include intense thermal and chemical treatments leading to severe DNA degradation. To address this limitation, the present study aimed to design and assess species-specific cytb primers with moderate amplicon lengths optimized for degraded DNA. This study provided a foundation for reliable authentication of processed buffalo products and contributed to the broader goal of establishing standardized molecular protocols for food authenticity verification and halal certification.

1. Materials and Methods

2.1. Primer Design Specific for Buffalo

The primer was designed based on the cytb coding sequence from the National Center for Biotechnology Information (NCBI) database

(https://www.ncbi.nlm.nih.gov/). The sequences were aligned with cytb nucleotide sequences from other species using BIOEDIT software to identify unique polymorphic sequences that are specific for the buffalo species. The selected polymorphic sequences were then used as references in the design of specific primers. The primer design process was facilitated by the Primer3Plus software (https://primer3plus.com/). Once appropriate primer candidates were identified, an in-silico assessment was carried out using basic local alignment search tool (BLAST), available on the NCBI website

(https://blast.ncbi.nlm.nih.gov/Blast.cgi) to verify that the primers specifically amplify buffalo DNA. Then, in-silico analysis was carried out using NetPrimer, available on the Premier Biosoft website

(https://www.premierbiosoft.com/netprimer/) to assess the melting temperature (Tm), GC percentage, GC clamp, potential secondary structure formation (hairpin, self-dimer, cross-dimer), repeats and runs. The primers were synthesized by Integrated DNA Technologies, Singapore.

2.2. Sample Preparation

           The sample included buffalo meat, beef, goat, chicken, pork, dog, rat, buffalo skin crackers, beef skin crackers, goat skin crackers, crispy chicken skin and pork skin crackers from various stores. Each sample was packaged in plastic containers and stored in a freezer at -20 °C to preserve its freshness and prevent cross contaminations.

2.3. DNA Isolation using Chloroform-Isoamyl Alcohol (24:1) Method

Briefly, DNA isolation was carried out under aseptic condition using 20 mg of the sample. Then, 500 μl of salt-tris-EDTA (STE) buffer was added as the lysis buffer, with 40 µl of 10% SDS and 20 µl of proteinase-K at a concentration of 20 mg ml^-1. The mixture was vortexed and incubated at 55 °C and 800 rpm overnight until the cell membranes were lysed using thermo shaker (Biosan TS-100, Latvia). Then, mixture was centrifuged (TOMY MDX-310, Japan) at 12,000 rpm for 10 min at 29 °C. The supernatant, which contained the crude DNA components, was collected at 500 μl and transferred to a fresh microtube. Then, 500 μl of chloroform-isoamyl alcohol solution (24:1) and 40 μl of 5 M NaCl were added to the supertnatant. The microtube was shaked until the solution was homogenized and then centrifuged at 12,000 rpm for 10 min at 29 °C. The pellet was discarded and 400 μl of the supernatant were transferred to a fresh microtube. The supernatant was mixed with 400 μl of chloroform-isoamyl alcohol (24:1) and shaked until the mixrure was homogenized. The mixture was centrifuged at 12,000 rpm for 10 min at 29 °C. The pellet was discarded and the supernatant was transferred to a fresh microtube. Then, 40 μl of 5 M NaCl and 800 μl of cold absolute ethanol were added to the supernatant, followed by incubation at -20 °C for 2.5 h to precipitate the DNA. The mixture was centrifuged at 12,000 rpm for 10 min at 4 °C to allow the DNA to settle into a pellet. The pellet was treated with 500 μl of 70% ethanol and recentrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was discarded and the pellet was dried at 55 °C for 30 min until the ethanol evaporated using thermomixer. Pellet was dissolved in 50 μl of TBE buffer at pH 7.6. This solution contained the DNA isolate from the sample, which could be analyzed for purity and concentration using nanodrop spectrophotometer. The concentration and purity of the DNA isolate were verifyed using Implen NanoPhotometer (Model NP 80, Germany) at 260/280 nm.

2.4. Reaction Components and PCR Program Settings

The PCR cocktails included 0.5 µl of forward and reverse primers (10 μM), 1 µl of 50 ng µl^-1 DNA sample isolate, 5 µl of MyTaq HS red mix (Bioline BIO-25048, Germany) and 3 µl of nuclease-free water (Invitrogen AM9932, USA) (22). The amplification steps consisted of initial denaturation (95 °C for 60 s); followed by denaturation (95 °C for 15 s), annealing (63 °C for 15 s), elongation (72 °C for 10 s) and final elongation (72 °C for 60 s); as well as cooling (4 °C) (Bioline, 2014). The PCR instrument was PCR gradient thermocycler (SensoQuest, Germany). The PCR results were analyzed using gel electrophoresis system (Mupid-exU, Japan) with agarose gel concentration of 1.5% at 50 V for 55 min.

2.5. Primer Specificity Assay

The specificity assay was carried out using target DNA (buffalo) and non-target DNA (cattle, goats, chickens, pigs and rats) at a concentration of 50 ng µl^-1. The assay was carried out in two repetitions. The specificity of the primers could be verifyed after analyzing the amplicon bands resulting from electrophoresis. A primer was considered specific if only the amplicon band from the target DNA was present; which in this research, context referred to the cytb gene fragment from the buffalo's mitochondria.

• Primer Sensitivity Assay

The primer sensitivity assay was carried out involving a gradient of buffalo DNA concentrations, which included 100, 10, 1, 10^-1, 10^-2, 10^-3, 10^-4 and 10^-5 ng μl^-1. The sensitivity of the primers was assessed based on the lowest concentration of buffalo mitochondrial DNA that could be amplified. The primer sensitivity assay was carried out in three repetitions.

• Primer Sampling Assay

The primer sampling assay was carried out using processed food products in the form of crackers made from animal skin. The positive sample consisted of buffalo skin crackers, while the negative samples included cattle skin crackers, goat skin crackers, crispy chicken skin and pig skin crackers. The positive control consisted of pig DNA with a concentration of 100 ng μl^-1 and negative control consisted of the nuclease free water (Invitrogen AM9932, USA) were used in this step to assess the validity of the results. The primer sampling assay was carried out in two repetitions.

1. Results and Discussion

3.1. Cytochrome b (cytb) Primer Development

The primer was designed using in-silico analysis. The designed primer candidates have met the ideal criteria for all parameters, with the exception of self-dimer formation. The Buffalo_5.1 reverse primer included a ∆G value of -8.76 kcal mol^-1. Primers with a ∆G value less than -6 kcal/mol were still used, despite the potential for secondary structure formation. This shortcoming was addressed through the optimization of the PCR procedure, as is commonly practiced in studies involving PCR techniques (23). Primer development based on in-silico analysis provides a predictive result but may differ from actual PCR conditions. In-silico analysis of primer specificity often cannot be used as a reference for identifying good primer specificity (24). Therefore, primer validation using the PCR process is essential to identify primers that have been developed to include good quality.

3.2. Primer Specificity Validation

Primer specificity was assessed via its ability to hybridize specifically with target DNA without producing DNA amplicon products from non-target species using PCR method. The PCR amplification product visualization showed that the Buffalo_5.1 primer pair specifically amplified buffalo DNA and did not amplify DNA from other samples (cattle, goats and mice). This demonstrated that the designed primer was specific and complementary to the buffalo DNA. The results showed consistency upon repetition. In the first and the second trials, the visible DNA amplicon band was a single band that corresponded to the target size of 216 bp (Figure 1).

The specificity assay results for the Buffalo_5.1 primer pair verify that the 5.1 primer pair show good performance for specificity and amplification pattern consistency. The specificity of this primer is critical as it ensures reliability when used for assessment the presence of adulteration or the authentication of a material. Primer specificity is key in PCR-based adulteration assessment as it assesss the validity, accuracy and credibility of detection results. Specific primers accurately detect the target DNA because they only bind to the DNA sequence of the target species/product. This prevents the amplification of unwanted DNA from other organisms. Furthermore, the presence of specific primers helps avoid the generation of false positive data, as non-specific primers may bind to similar DNA from other species, leading to results that indicate adulteration when it does not actually exist. The level of specificity of Buffalo_5.1 primer pair was similar to that of cytb-based assay using duplex PCR by (18) that differentiated buffalo against cattle, which produced amplicons of 249 bp (buffalo) and 655 bp (cow) (18). In degraded or processed samples, universal cytb primers that produce shorter fragments (~148 bp) have been shown further effective in amplifying damaged or fragmented DNA (30). Similarly, primers developed from the cytb gene sequence successfully identified unprocessed ruminant skin (31). and could clearly separate four species (buffalo, cattle, sheep and goat) with various cytb fragment sizes (106–308 bp) for each species using blood samples (19). Compared with these primers, the primers developed in this study (single target 216 bp, buffalo specific) will complement the detection capabilities of the current primers.

The high primer specificity enables the identification of complex mixed materials. Processed meat products typically undergo various processes that cause DNA fragmentation such as grinding, cooking and drying. If adulteration assessment uses primers with great specificity, even small targeted DNA fragments are still detected. The cytb gene has widely been used as a marker for identifying specific species across mammals, birds and insects. The authentication of cattle leather has successfully been achieved using cytb gene sequence (12). Halal authentication using this gene has been reported by several researchers (12–17,32,33).

3.3. Primer Sensitivity Assay

            Sensitivity assessment was carried out to assess the lowest concentration of buffalo DNA that could be detected by Buffalo_5.1 primer during the PCR reaction. Based on the analysis of the DNA amplicon bands (Figure 2), variation in sensitivity levels between the two repetitions was detected.

From the three trials, two trials showed that the primer was able to detect buffalo DNA down to the lowest concentration of 10^-3 ng µl^-1. One trial, however, the sensitivity reached to 10^-5 ng µl^-1. The distinct results between the trials might be attributed to the quality of the DNA (34). The DNA degradation is one of the factors leading to the failure of DNA analysis using PCR (35). This issue can increase when the primer fails to bind to the severely degraded target DNA sequence. The DNA concentration and volume may vary between PCR reagent manufacturers. Referring to the manufacturer's recommendation, the recommended DNA template included 200 ng per reaction with a total volume of 50 µl. Furthermore, suspected EDTA residue from the extraction might cause primer inconsistency in amplifying DNA at concentrations of 10^-3 to 10⁻⁵ ng µl^-1 (36). Nevertheless, the Buffalo_5.1 primer designed in this research was quite reliable in detecting DNA at least until a concentration of 10^-3 ng µl^-1. Assuming that the solution included a density of 1 g ml^-1, 10^-3 ng µl^-1 equal to 10^-7 % w/w  and the potential to reach 10^-5 ng µl^-1 equal to 10^-9 % w/w  under optimal conditions.

The    sensitivity level of Buffalo_5.1 primer was still considered good when viewed from the perspective of its use for food fraud detection. In comparison, detection of adulteration using primers based on mitochondrial D-loop capable in detecting the contamination down to 1% and very faint/inconsistent results at 1% in autoclaved meat emulsion (37). Discrimation of beef and buffalo in Malaysian meat curry and burger products using primers developed based on mitochondrial genes showed sensitivity down to 1% meat in mixtures of 0/0.01 ng DNA (38). Other research which developed a detection method for meat fraud using multiplex PCR and 12S rRNA showed sensitivity of 10^-1 ng µl^-1 for pig species and 10^-2 ng µl^-1 for cattle, chicken and donkey species (39). Another study that developed a detection method for pig DNA using real-time PCR indicated that the smallest concentration of pig DNA that could be detected is 10^-3 ng µl^-1 (40). The article also explained that the sensitivity of real-time PCR was higher than that of conventional PCR. Identification of buffalo skin using universal cytb amplification and RFLP (RsaI enzyme) distinguished buffalo aganst cattle in skins down to ~10% buffalo skin in mixed hide samples (31). This finding strengthened that the Buffalo_5.1 primer included a good sensitivity result.

3.4. The Efectivity Assay of The Primer in Processed Food Products

Assessments were carried out to assess the reliability of the Buffalo_5.1 primer pair using PCR technique in detecting buffalo DNA in processed food products. The potential challenge during the DNA preparation from processed food products is the possibility of DNA degradation during the food processing. In this study, DNA was successfully isolated from several animal skin crackers which are commonly consumed in the community. Several samples were identified having protein contamination, which could be resolved by adding proteinase-K enzyme into the DNA sample.

From the agarose gel electrophoresis visualization result (Figure 3), it was evident that the PCR amplicon band was only present in the positive control and the buffalo skin cracker sample.

Samples of cow skin crackers, goat skin crackers, crispy chicken skin and pork crackers did not show detectable amplicon bands. The positive control included DNA isolated from fresh meat of buffallo, while the negative control included nuclease-free water. Positive amplification indicated that Buffalo_5.1 primer was still capable of recognizing buffalo DNA sequences in the buffalo skin cracker sample, despite the potential for DNA degradation during food preparation process.

The similar results from the repetitions verified that Buffalo_5.1 primer pair was not only specific but also demonstrated good performance in detecting the presence of buffalo DNA in processed food products derived from skin. The skin crackers have been boiled, dried and fried. Study of the effectivity of primers targeting the cytb was used to amplify DNA isolated from the skins of cattle, buffalo, goats and pigs (41) after putrefaction, heating or processing of mixed meats (42), beef curries, burger products under boiling, autoclaving, microwave cooking (37), minced meat, frozen rolls, boiled meat, meat balls, jerky  which processed in various ways (43), but none of them involved drying followed by frying The succes of Buffalo_5.1 primers to amplify DNA from skin crackers demonstrated that this primers were capable in identifying bufallo DNA sequences even from products that have undergone drying and frying processes.

The Buffalo_5.1 primer developed in this study combined moderate amplicon size and high species specificity, enabling reliable amplification of severely degraded DNA from processed buffalo products. Compared with previously reported Cyt b assays, this achieved a superior detection limit (10⁻³ ng μl⁻¹) while maintaining simplicity and cost-effectiveness for routine authentication and halal verification. The skin cracker products in the market generally include similar shapes and physical appearances, whether they made from cow, buffalo or pig skin. This morphological similarity makes it difficult for consumers to distinguish the source of the raw material, even though pork skin crackers are prohibited for consumption by the muslims community. With the largest muslim population worldwide, Indonesia includes a very high demand for reliable authentication methods to ensure the authenticity and halal status of these food products. In addition to being widely consumed as side dishes or snacks, the skin crackers are also popular as souvenir from various regions; thus, including important economic and cultural values. Therefore, several types of skin crackers were used in this study to assess the effectiveness of Buffalo_5.1 primer pair. The development of specific molecular markers such as primers for authenticating buffalo skin crackers includes great potential to support the halal certification process, strengthen the integrity of the food industry and protect consumers from food adulteration product.

Further research should focus on developing use of the Buffalo_5.1 primers to authenticate further complex and processed food matrices such as mixed meat products, cooked dishes or highly degraded DNA samples to assess their detection capabilities and meet real-world industry and market needs. Furthermore, integrating this primer system with emerging portable molecular technologies, including chip-based PCR with the recombinase polymerase amplification (RPA) LAMP technique, can enable the authentication of buffalo meat-derived products directly at the food production or distribution site. Such portable assessment increases the speed and accessibility of halal and authenticity verification, especially in regions with limited laboratory infrastructure. Furthermore, combining this approach with digital detection platforms or biosensor technologies can facilitate rapid quantitative readouts without the need of gel electrophoresis, supporting the development of easy-to-use and field-deployable diagnostic tools for further food authenticity monitoring.

1. Conclusion

The Buffalo_5.1 primer was successfully developed from the buffalo cytochrome b gene sequence. This primer pair has demonstrated its reliability as a molecular marker for detecting species-specific buffalo DNA. Its consistent success across multiple assessments demonstrates its robustness and reproducibility in PCR-based assessment. The short amplicon size allows its widespread application in processed and degraded samples such as processed meat samples and skin crackers that include boiling, drying and frying processes. In addition to its uses in direct identification of ingredients derived from buffalo, this primer provides a valuable tool for food authentication, meat adulteration prevention, halal compliance assurance and conservation-linked studies. Thus, the Buffalo_5.1 primer pair represents a practical relevant contribution to molecular diagnostics in food safety, quality control and biodiversity monitoring.

1. Acknowledgements

This research was financially supported by the Ministry of Education, Culture, Research and Technology through the Fundamental Research Grant

(contract no. 00665/UN10.A0501/B/PT.01.03.2/2025). The authors gratefully acknowledge the Integrated Research Laboratory, Universitas Brawijaya (LRT-UB), for providing experimental instrument facilities. The authors also thank their colleagues from Universitas Brawijaya, who provided ideas and expertise that greatly assisted the research.

1. Declaration of competing interest

The authors report no conflict of interest.

1. Authors’ Contributions

Conceptualization, E.L.A.; methodology, J.K.; validation, E.L.A. and J.K.; formal analysis, M.T.F.; investigation, A.S.Z.; resources, J.K.; data curation, G.A.; writing—original draft preparation, M.T.F.; writing—review and editing, E.L.A.; visualization, M.T.F.; supervision, E.L.A. and J.K.

1. Using Artificial Intelligent Chatbots

This study was written and prepared with the assistance of AI chatbots.

1. Ethical Consideration

The meat samples used in this study were obtained exclusively from commercially available products purchased from the local market. As the research did not involve live animals, human participants, or any procedures requiring ethical oversight, no ethical approval was necessary.