Background and Objective: Staphylococcus aureus is a significant foodborne and zoonotic pathogen. This study aimed to enhance the anti-Staphylococcus aureus activity of egg-white lysozyme through heat treatment and synergistic combinations with natural antimicrobials.

Material and Methods: The lysozyme was purified from egg white via ammonium sulfate precipitation and cation-exchange chromatography, yielding a homogeneous protein. Anti-Staphylococcus aureus activity of native lysozyme, heat-treated lysozyme and its combination with ferulic acid or Mycobacterium smegmatis acyltransferase was assessed, respectively. All experiments were carried out in triplicate and statistical analysis was carried out using SPSS software.

Results and Conclusion: The specific activity of lysozyme to Micrococcus lysodeikticus was 27,407.4 U.mg^-1. The lysozyme IC₅₀ against Staphylococcus aureus was 300.8 µg.ml^-1, with transmission electron microscopy verifying bacteriolytic action. Heat treatment under optimized conditions (90°C, 15 min, pH 6.2) significantly enhanced the lysozyme antibacterial activity by 35.1%, which was correlated with structural changes evidenced by circular dichroism spectroscopy. Furthermore, synergistic effects were observed when heat-treated lysozyme was combined with ferulic acid or Mycobacterium smegmatis acyltransferase (MsAcT), leading to prolonged inhibition and decreased viable bacterial counts. The findings of this research demonstrated that structural modifications and combinatorial strategies could effectively improve the efficacy and application potential of lysozyme as a natural antimicrobial agent in food safety.

Keywords: Egg-white lysozyme, Ferulic acid, Heat-induced fibrillar aggregates, Mycobacterium smegmatis acyltransferase, Staphylococcus aureus

1. Introduction

Staphylococcus aureus is a Gram-positive bacterium widely spread in nature and a common foodborne pathogen. The enterotoxins (SEs) of the bacteria show significant heat resistance, rendering them difficult to completely eliminate through conventional cooking methods and posing a significant risk of food poisoning. Furthermore, S. aureus facilitates cross-infection between humans and animals via the food chain through contamination of animal feed, subsequent infection of animals and transmission to humans, establishing it as an important zoonotic pathogen. The S. aureus has been detected in various animal species and a wide range of food products, with its prevalence continuously increasing on a global scale. Therefore, S. aureus is still a high-priority target in food safety monitoring [1, 2].

To combat S. aureus contamination in food processing, diverse biological control strategies have been investigated. These include inhibition using lactic acid bacteria (LAB) probiotics [3], Lysostaphin [4], bacteriophages [5,6], essential oils (EO) [5] and lysozyme [7,8]. From these, egg-white lysozyme has widely been used due to its high catalytic activity, simple preparation and cost-effectiveness [7,9]. It inhibits S. aureus through two primary mechanisms of (1) lytic mechanism as lysozyme hydrolyzses β-1, 4-glycosidic bonds to peptidoglycan, which causes cell wall damage, induces cell lysis and results in bactericidal activity; and (2) the non-lytic mechanism, where under denaturing conditions, it suppresses growth through inherent protein characteristics such as hydrophobicity and cationic effects [8,10]. While the antimicrobial characteristics of native egg-white lysozyme are well-documented, its efficacy under common food processing conditions, particularly those involving heat, needs further investigation. Moreover, strategies to enhance its activity, especially in a heat-treated state, through combination with other natural antimicrobial agents are still under-investigation. Under various reaction conditions, the interactions between other natural antibacterial agents and egg-white lysozyme can lead to various effects on its antimicrobial activity. For example, in an alkaline solution, theaflavin covalently binds to egg-white lysozyme, resulting in significant decrease of its antibacterial activity [11]. However, in an amyloid fibril hydrogel, the interaction between epigallocatechin gallate (EGCG) and egg-white lysozyme significantly broadens the antibacterial spectrum of egg-white lysozyme [12]. Therefore, an in-depth investigation into the interaction conditions between natural antibacterial factors and egg-white lysozyme is greatly important for enhancing the antibacterial efficiency of egg-white lysozyme.

This research aimed to isolate and purify egg-white lysozyme using chromatography and to assess the effect of heat treatment on its anti-S. aureus activity. Furthermore, the study prepared a synergistic combination of heat-treated lysozyme with ferulic acid and acyltransferase to develop an enhanced strategy for suppressing S. aureus growth. The experimental results demonstrated that heat-induced structural modification (fibrillar aggregation) enhanced lysozyme antimicrobial mechanism beyond native peptidoglycan hydrolysis. Moreover, it has first been reported that a synergistic combination of heat-treated lysozyme with ferulic acid and MsAcT against S. aureus significantly improves the efficacy and time of inhibition.

1. Materials and Methods

2.1. Strains, biochemical reagents and chemical reagents

Micrococcus lysodeikticus CGMCC 1.4547 and S. aureus CGMCC 1.282 were purchased from China General Microbiological Culture Collection Center, China. Bovine serum albumin (BSA) and standard protein molecular weight marker were purchased from Takara Biomedical Technology, China. Moreover, CM-Sepharose fast flow chromatography column was purchased from GE Healthcare, China. Ferulic acid, caffeic acid, gallic acid and N-acetylglucosamine (NAG) included analytical grade unless otherwise specified and purchased from Shanghai Macklin Biochemical Technology, China.

2.2. Purification of egg-white lysozyme

Egg white was initially diluted by 50-fold using 50 mmol.l^-1 pH8.0 tris-HCl buffer and the resulting protein solution was ultrasonicated for 10 min. The supernatant was collected by centrifugation at 12,000 rpm for 5 min at 4 °C. Solid ammonium sulfate was added to the supernatant to achieve 40% (w/v) saturation and the mixture was set to precipitate for 2 h at 4 °C. After centrifugation, the precipitate was removed and the collected supernatant was further loaded onto a CM-Sepharose fast flow column (5 × 20 cm) that was pre-equilibrated with 50 mmol.l^-1 tris-HCl buffer (pH 8.5). The lysozyme was eluted with a 5-fold column volume of 50 mmol.l^-1 tris-HCl buffer (pH 8.5) with increasing concentrations of NaCl (0.1 and 0.5 mol.l^-1) at a flow rate of 20 ml.h^-1, respectively. The active fractions were pooled. Protein concentration was assessed using Bradford method with BSA as a standard. The homogeneity of the purified egg-white lysozyme was assessed using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

2.3. Activity assay of egg-white lysozyme

The cell lysis activity of lysozyme from egg white to M. lysodeikticus was quantitatively assessed using spectrophotometeric assay method, which was described by the National Standard of the People's Republic of China (GB/T 30990-2014, Determination of Lysozyme Acyivity) as well as Naveed et al. [13]. Briefly, after incubation overnight, M. lysodeikticus was transferred into the fresh LB liquid media at 1% (v/v) and incubated at 37 °C for 2 h at 220 rpm. The cells were harvested by centrifugation and resuspended in 50 mmol.l^-1 Na₂HPO₄-Na₂HPO₄ buffer (pH 6.2) to a final concentration of 5 × 10⁵ CFU.ml^-1. The reaction system contained 2.5 ml of cell suspension and 0.5 ml of lysozyme solution, while the control system received 0.5 ml of inactivated lysozyme solution. The reaction was carried out at 50 mmol.l^-1 Na₂HPO₄-Na₂HPO₄ buffer (pH 6.2) for 5 min at 25 °C and the absorbance of the reaction system was continuously monitored at 450 nm. One unit of lysozyme activity (U) was defined as the quantity of enzyme necessary to decrease OD₄₅₀ by 0.001 per minute under the standard assay condition.

2.4. Anti-Staphylococcus aureus activity of egg-white lysozyme

Briefly, S. aureus in the logarithmic growth phase was used as an indicator microorganism to assess antibacterial activity of the purified lysozyme. To set the growth curve, overnight-cultured S. aureus was subcultured into the fresh LB liquid media at a 1% (v/v) inoculum and incubated at 37 °C for 14 h at 220 rpm. Samples were collected every 30 min to measure the optical density (OD) of the culture broth at 600 nm. The growth curve was plotted with OD₆₀₀ on the y-axis and incubation time on the x-axis.

The antibacterial assessments were carried out using a method described by Carrillo et al. [14] with slight modification. Following 2 h of subculture, S. aureus suspensions were adjusted to 5 × 10⁵ CFU.ml^-1 through serial dilution in LB liquid media. Aliquots of S. aureus suspensions were mixed with equal volumes of the purified lysozyme at various concentrations and incubated at 37 °C for 6 h at 220 rpm. The OD₆₀₀ of the mixture was recorded after a 6-h subculture. The control group used 50 mmol.l^-1 Na₂HPO₄-Na₂HPO₄ filter-sterilized buffer (pH 6.2) instead of lysozyme. Antibacterial activity of the purified lysozyme to S. aureus was assessed using the antibacterial ratio. Antibacterial ratio was calculated using the following equation of R = (A - B) ÷ A × 100%; in which, R was the antibacterial ratio (%); A was the OD₆₀₀ value of the control group; and B was the OD₆₀₀ value of the experimental group. The antibacterial effect curve was plotted with the antibacterial ratio on the y-axis and the logarithmic of the purified lysozyme concentration on the x-axis. The IC₅₀ was defined as the purified lysozyme concentration, which resulted in a 50% decrease in the level of the antibacterial ratio, compared with untreated groups after a 6-h treatment. The IC₅₀ value was calculated using GraphPad Prism software. The cell morphology of S. aureus after a 6-h lysozyme treatment (IC₅₀ concentration) was reported using transmission electron microscopy (TEM).

2.5. Effects of heat treatment on anti-Staphylococcus aureus activity of the purified egg-white lysozyme

To assess the anti-S. aureus effects of the heat-treated egg-white lysozyme, a specific concentration of the purified lysozyme solution was incubated at various temperatures for a set duration before assessing its anti-S. aureus activity using water bath. In this study, three key parameters were primarily investigated, including heat treatment temperature, time and initial lysozyme concentration. The temperatures were set at 37, 50, 60, 70, 80, 90 and 100 °C, respectively. The treatment times were set at 5, 10, 15, 60, 120 and 240 min, respectively. The initial lysozyme concentration were set at 601.6, 1000, 2000, 4000 and 6000 μg.ml^-1, respectively. The three highlighted factors were optimized using one-factor-at-a-time method. After heat treatment, equal volumes of the lysozyme solution and S. aureus suspension were thoroughly mixed and co-cultured at 37 °C for 6 h at 220 rpm. The antibacterial ratio was then quantified. Untreated lysozyme was used as control group.

To assess the effect of heat treatment on lysozyme molecular structure, circular dichroism (CD) spectroscopy was used to analyze changes in its secondary structural components before and after thermal exposure. The CD spectra were assessed using Jasco J-1500 spectropolarimeter (Jasco, Japan) with a 1-mm cell in the far-UV region from 190 to 300 nm. The concentration of lysozyme was 0.1 mg.ml^-1 in 5 mmol.l^-1 phosphate buffer (pH 6.2).

2.6. Inhibitory effects of phenolic acids combined with heat-treated egg-white lysozyme on Staphylococcus aureus

2.6.1. Compatibility screening of various phenolic acids with egg-white lysozyme

In this experiment, the inhibitory effects of egg-white lysozyme combined with three phenolic acids (ferulic acid, caffeic acid and gallic acid) at various concentrations on S. aureus growth were assessed, respectively. The final concentrations of each phenolic acid were 200, 400, 800 and 1000 μg.ml^-1, while the final concentration of egg-white lysozyme was set at 300.8 μg.ml^-1. The anti-S. aureus activity analysis and antibacterial ratio calculation methods were based on those described in Section 1.4. For the control group, phenolic acids or lysozyme were replaced with 50 mmol.l^-1 Na₂HPO₄-Na₂HPO₄ buffer solution (pH 6.2).

2.6.2. Effects of the heat-treated lysozyme and ferulic acid combination on Staphylococcus aureus growth

The method for assessing the growth curves of S. aureus was based on Section 1.4. The heat treatment procedure for egg-white lysozyme was carried out according to Section 1.5, with the final concentration of heat-treated lysozyme adjusted to 300.8 μg.ml^-1. For the control group, ferulic acid or lysozyme were substituted with 50 mmol.l^-1 Na₂HPO₄-Na₂HPO₄ buffer solution (pH 6.2).

2.7. Numberized subsection inhibitory effects of Mycobacterium smegmatis acyltransferase combined with heat-treated egg-white lysozyme on Staphylococcus aureus growth

The mechanism by which, M. smegmatis acyltransferase (MsAcT) combined with heat-treated egg white lysozyme inhibited the growth of S. aureus is illustrated in Fig. 6.1. The preparation of the purified MsAcT was based on the methods described by Jia et al. [15]. Briefly, recombinant Escherichia coli BL21(DE3) strain was inoculated to Luria-Bertani (LB) broth and grown at 30 °C. The IPTG was added to culture broth to the final concentration of 1 mmol.l^-1, when the OD₆₀₀ reached 0.6–0.8. After 14 h interval, cell pellet was collected, resuspended using loading buffer (20 mmol.l^-1 pH 7.4 Na₂HPO₄-NaH₂PO₄, 20 mmol.l^-1 imidazole and 500 mmol.l^-1 NaCl) and then lysed using sonication. The supernatant from the cell lysate was collected and directly loaded on the HisTrap HP affinity chromatography column pre-equilibrated with loading buffer, respectively. Recombinant protein was eluted with a linear gradient of 20 ml of 20–500 mmol.l^-1 imidazole in the buffer with a flow rate of 0.8 ml.min^-1. The fractions with pure MsAcT were pooled and dialyzed against 20 mmol.l^-1 Na₂HPO₄-NaH₂PO₄ (pH 6.2) buffer overnight at 4 ^◦C. The hydrolysis activity of MsAcT to NAG was assessed according to Jiang et al. [16] and Muzzarelli and Rocchetti [17]. In brief, the reaction mixture contained 20 μg.ml^-1 NAG in 20 mmol.l^-1 Na₂HPO₄-Na₂HPO₄ buffer (pH 6.2). Appropriately diluted MsAcT was added into the reaction mixture to create a linear dependence of the reaction rate to protein concentration. The reaction was carried out at 37 ◦C and the kinetics was detected for 3 h at 202 nm.

The specific procedure for inhibiting S. aureus growth using MsAcT combined with heat-treated egg-white lysozyme was carried out as follows: MsAcT, heat-treated egg white lysozyme and buffer solutions were sterilized using 0.22-μm filters. Then, 200 μl of appropriately diluted log-phase S. aureus cells were mixed with 200 μl of 1 mg.ml^-1 MsAcT solution, while the control group received 200 μl of 50 mmol.l^-1 NaH₂PO₄-Na₂HPO₄ solution (pH 6.2). After incubation at 37 °C for 3 h, 200 μl of the heat-treated egg-white lysozyme (final concentration of 300.8 μg.ml^-1) were added to the experimental and control groups and mixed thoroughly, followed by incubation at 37 °C for 3 h. The samples were then centrifuged at 12,000 rpm for 3 min to separate the supernatant and the bacterial cell pellet. The protein concentration in the supernatant was measured to calculate the increase in protein, while the number of viable bacteria in the pellet was assessed according to the National Food Safety Standard of China (GB4789.2-2022, Food Microbiological Examination: Determination of Total Bacterial Count).

1. Results and Discussion

3.1. Purification and characterization of egg-white lysozyme

The purification of egg-white lysozyme was achieved through ammonium sulfate precipitation and CM-Sepharose fast flow column chromatography, resulting in 8.7-fold purification and yield of 41.0% (Table 1). Cation exchange chromatography verified particularly effective, accounting for 5.8-fold increase in specific activity. The purified enzyme was verified as homogenous using SDS-PAGE, showing a single band at 14.3 kDa (Fig. 1, Lane 6), which was similar to the molecular mass of egg-white lysozyme [18]. Its specific activity was assessed as 27,407.4 U.mg^-1 using M. lysodeikticus cells as substrate. This value was less than that reported by Chen et al. [18], a discrepancy likely attributable to the use of whole cells in this assay instead of the isolated cell walls used in the highlighted study.

A gradual loss of total activity was observed throughout purification. Particularly, lysozyme was detected in pellets at a relatively low ammonium sulfate saturation (40%), suggesting the potential formation of insoluble aggregates under these conditions, as previously documented [19, 20]. For practical uses, it is critical to state that the growth state of M. lysodeikticus, affected by culture conditions and equipment, significantly affects the assessed specific activity [11, 13, 21]. Therefore, standardizing the substrate by setting the growth curve and harvesting log-phase cells under local laboratory conditions is essential for accurate activity assessment. In this study, the buffer solution for the assessment of the egg-white lysozyme activity according to the National Standard of the People's Republic of China (GB/T 30990-2014) requirements included 50 mmol.l^-1 Na₂HPO₄-NaH₂PO₄ buffer (pH 6.2). In practical fields, particularly in the food processing industry, buffer solutions with pH 6.2 are rarely used.

Therefore, it is essential to assess optimal pH and temperature under the specific conditions necessary for the target uses, when assessing using effectiveness of egg-white lysozyme.

3.2. Anti-Staphylococcus aureus activity and mechanism of the purified egg-white lysozyme

The growth curve of S. aureus was characterized, identifying a log phase from 1 to 8 h (Fig. 2A), similar to the previous reports [22]. Cells from the exponential phase (2 h) were used for the assays. The IC₅₀ of native lysozyme against S. aureus was 300.8 μg.ml^-1 (Fig. 2B). The antibacterial efficacy of lysozyme is affected by the composition and sequence of bacterial cell wall peptidoglycan, as well as the physiological state of the enzyme [23, 24]. Lysozyme fights microbes through bacteriostatic, bactericidal and bacteriolytic mechanisms [25]. The transmission electron microscopy (TEM) images provided direct evidence of the bacteriolytic action, showing damaged S. aureus cell walls with distinct light/dark contrast and the collapse of cells, leading to leakage of intracellular contents (Fig. 2C, arrows). Despite this significant effect, the inhibitory activity of the native lysozyme is difficult to sustain over extended times (Figs. 2A, 5A), highlighting a limitation for its use as a standalone antimicrobial agent.

3.3. Enhancement of anti-Staphylococcus aureus activity using heat treatment and structural changes

Heat treatment under optimized conditions (600 μg.ml^-1, 90°C, 15 min, pH 6.2) enhanced the anti-S. aureus activity of lysozyme by 35.1%, achieving 82.5% inhibition compared to the native enzyme (Fig. 3A–C). This increase in activity after thermal denaturation was similar to that against other microbes such as SARS-CoV-2 and Bacillus subtilis [26, 27, 28]. Structural analysis revealed the reason behind this enhancement as a significant rearrangement of secondary structure occurred, with α-helix content decreasing from 35.59 to 23.60% and β-sheet, β-turn and random coil structures increasing (Fig. 3D, Table 2). This unfolding and proliferation of β-sheets drived the formation of fibrillar aggregates [30,29], which were postulated to perforate microbial membranes, a mechanism distinct from the native enzyme peptidoglycan hydrolysis [27]. This suggested that structural modification wa a viable strategy to improve the efficacy of lysozyme.

3.4. Synergistic anti-Staphylococcus aureus effects of heat-treated egg-white lysozyme and phenolic acids

From the phenolic acids (ferulic, caffeic and gallic acids), ferulic acid showed the highest inhibition (52.2%) at 200 μg.ml^-1, though differences diminished at higher concentrations (1000 μg.ml^-1), where all acids reached ~99% inhibition (Fig. 4A–C). Ferulic acid is reported to inhibit S. aureus by suppressing tetK and MsrA efflux pumps on the bacterial membrane [31,32].

A combination of 400 μg.ml^-1 ferulic acid with native lysozyme (300.8 μg.ml^-1) showed a synergistic effect, increasing inhibition by respectively 12.3 and 29%, compared to either compound alone (Fig. 4A). This synergy was further increased, when ferulic acid was combined with heat-treated lysozyme, reducing bacterial biomass (OD₆₀₀) by additional 18.7%, compared to the combination with native enzyme (Fig. 5A). The optimal protocol involved sequential addition of heat-treated lysozyme added at time zero, followed by ferulic acid after 6 h. This not only delayed the entry into the log phase by an additional hour but also decreased final biomass by 48.8% (Fig. 5B). This demonstrated that combining lysozyme with other antibacterial compounds, particularly after structural modification, could significantly enhance and prolong its inhibitory effect, potentially broadening its antibacterial spectrum [12,33]. However, when designing such combinations, concentration and addition sequence of egg-white lysozyme and phenolic acid, biocompatibility, reaction condition and differences in the mechanisms of action must carefully be addressed. Otherwise, adverse effects may occur. For example, in a solution with pH 9.0, phenolic acids (e.g., theaflavin) form covalent bonds with egg-white lysozyme, resulting in a 20% decrease in the antibacterial activity of egg-white lysozyme [12,33].

3.5. Synergistic inhibition using MsAcT and heat-treated lysozyme

The enzyme MsAcT hydrolyzes acyl groups on peptidoglycan structural units, theoretically facilitating subsequent hydrolysis by lysozyme (Fig. 6A). Experimental data verified this synergy. Treatment with heat-treated lysozyme alone increased the extracellular protein concentration by 0.115 mg.ml^-1, indicating cell lysis. When combined with MsAcT, the protein release increased further substantially by 0.208 mg.ml^-1 (Fig. 6B). This was verified by colony counts, which showed a decrease in viable cells from 1.69 × 10⁸ CFU.ml^-1 (lysozyme alone) to 1.41 × 10⁸ CFU.ml^-1 (combination treatment) (Fig. 6C). This is a significant finding, as bacterial deacylation of peptidoglycan is typically a resistance mechanism that decreases hydrophobic interaction with lysozyme [10]. However, the present results demonstrated that external enzymatic deacylation by MsAcT could instead sensitize cells to lysozyme. It is challenging to explain this novel result at the molecular level. Since the lysozyme used in this research was achieved through separation and purification from egg-white and its 3-dimensional (3D) structure has not been resolved, molecular docking techniques cannot be used to investigate the differential interactions between NAM/NAG and deacetylated NAM/NAG with the lysozyme. Additionally, MsAcT is a promiscuous biocatalyst possessing multiple catalytic activities such as hydrolytic activity and perhydrolysis activity [15]. Further in-depth investigation is needed to assess which catalytic activity dominates during its synergistic action with egg-white lysozyme in inhibiting S. aureus.

When egg-white lysozyme acts synergistically with other natural antibacterial agents to inhibit the growth of S. aureus, biocompatibility is one of the primary key parameters. Environmental conditions such as buffer pH [10,24], ionic strength [10,20,24] and temperature [14,27] of the egg-white lysozyme solution can alter its conformational state to various degrees; thereby, decreasing its antibacterial activity. Polyphenolic compounds may modify and disrupt the conformation of egg-white lysozyme, diminishing or eliminating its antibacterial activity [11, 12, 21, 31]. When other antibacterial proteins (e.g., proteases) act synergistically with egg-white lysozyme to inhibit the growth of S. aureus, the degradation of lysozyme by proteases can directly lead to its inactivation [14]. This study investigated the synergistic inhibitory effect of egg-white lysozyme and ferulic acid on growth of S. aureus under a sequential interaction mechanism, where two antibacterial agents were sequentially added to the reaction system to inhibit the bacterial growth. The experimental results indicated that the sequential interaction mechanism could partially overcome the issue of biocompatibility and maximize the antibacterial efficacy [Fig. 5B].

With the public in-depth understanding of the bacterial resistance mechanisms, growing awareness of environmental hazards caused by antibiotic misuse and heightened self-health consciousness, prohibition of antibiotics in animal feed and breeding processes has become a global interest. Therefore, it is vital to actively search for various alternatives to antibiotics [34, 35, 36]. From various antibiotic alternatives, egg-white lysozyme is a promising candidate due to its excellent biosafety. This study demonstrated that structural modification through heat treatment and strategic combination with other antimicrobials such as ferulic acid or enzymes such as MsAcT could significantly enhance its efficacy and time of action against S. aureus. Further studies should focus on clarifying the detailed mechanisms of these synergistic interactions and investigating other modification strategies such as glycosylation and lipophilization [10,24] to fully realize the potential of lysozyme as an effective antibiotic alternative.

1. Conclusion

In conclusion, this study successfully purified and characterized egg-white lysozyme and demonstrated that its anti-S. aureus activity could significantly be enhanced through thermal denaturation and synergistic combinations. Heat treatment induced structural reorganization of lysozyme, increasing its β-sheet content and enabling membrane-perforating antibacterial mechanisms distinct from its native lytic activity. Furthermore, combining heat-treated lysozyme with ferulic acid or MsAcT resulted in the synergistic effects, delaying bacterial growth and improving bacteriolytic efficacy. The results highlighted the potential of modified lysozyme as part of integrated natural preservation systems to improve food safety. Further studies should focus on explaining detailed mechanisms of synergy and investigating other modification methods to further broaden the applicability and effectiveness of lysozyme-based antimicrobial interventions. To better promote this process in the field of food processing, follow-up research should thoroughly investigate its use effects, affecting factors and long-term stability in real food matrices (e.g., meat and dairy products). Additionally, a comparison of production costs and profitability with other antibacterial processes is necessary.

1. Acknowledgements

This research was supported financially by the National Natural Science Foundation of China (nos. 31370802 and 31870787) and Fujian Province Natural Science Foundation (no. 2021J01169).

1. Declaration of competing interest

The authors declare no conflict of interest.

1. Authors’ Contributions

Conceptualization, W.H. and X.X.; methodology, W.H.; software, Q.W.; validation, L.J., .J.W. and Q.W.; formal analysis, W.H. and S.Z.; investigation, X.X.; resources, J.W.; data curation, L.J.; writing—original draft preparation, W.Y.; writing—review and editing, S.Z.; supervision, S.Z.; project administration, H.J.; funding acquisition, S.Z.

1. Using Artificial Intelligent Chatbots

No artificial intelligent chatbots have been used in sections of this manuscript.

1. Ethical Consideration

The subjects of this study are egg white lysozyme and Staphylococcus aureus. As no animal or human trials are involved, no ethical review documentation is required.

Background and Objective: Escherichia coli O157:H7, a major foodborne bacterial pathogen, is commonly detected in the intestines of healthy cattle, deer, goats and sheep. It can readily contaminate veal, ground beef and hamburgers, causing a range of human diseases, including mild diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome and thrombotic thrombocytopenic purpura. Various treatment strategies are used to reduce E. coli O157:H7 contamination, ranging from washing to chemical and physical food decontamination. The increase of antibiotic-resistant bacteria has hence increased interest in lytic bacteriophages as biocontrol agents. This study investigated the use of bacteriophages to control E. coli O157:H7 growth in experimentally contaminated meat.

Material and Methods: Six E. coli O157:H7 specific bacteriophages were isolated from sewage, purified via plaque assay and assessed individually and in cocktails for lytic activity at various multiplicity of infection rates. Temperature-dependent effects on adhesion and lytic efficiency were assessed at refrigeration and ambient conditions. Phages were further used in commercial polyethylene food wraps to assess realistic surface delivery performance on inoculated meat samples and long-term phage stability was monitored during storage at 4 °C.

Results and Conclusion: Single-phage treatments achieved up to 100% bacterial elimination at higher multiplicities of infection, while optimized phage cocktails completely eradicated bacteria. Temperature affected bacterial recovery, with refrigeration decreasing bacterial adhesion and slightly slowing phage-mediated lysis. Importantly, this study has introduced an innovative practical approach using phages onto commonly used commercial polyethylene plastic wraps, demonstrating 99 and 93% reductions when phage‑treated wraps were used after 20 min and 24 h storages, respectively. Long-term storage of phage tf1 at 4 °C for seven months demonstrated stable plaque-forming ability, verifying durability for practical uses. These results highlight bacteriophages as effective, safe and scalable interventions to control E. coli O157:H7 in meat products, providing a practical strategy to enhance food safety, particularly for pathogens with extremely low infectious doses.

Keywords: Bacteriophage biocontrol, Escherichia coli O157:H7, Food safety intervention, Meat contamination, Phage cocktail therapy, Polyethylene wrap application

1. Introduction

Escherichia coli O157:H7 was identified in 1983 and has since been addressed as a major foodborne bacterial pathogen affecting humans [1, 2]. It is commonly detected in the intestines of healthy cattle, deer, goats and sheep and can therefore readily contaminate veal, ground beef and hamburgers [3]. The E. coli O157:H7 produces Shiga toxins and is classified as an enteric bacterium responsible for diarrhea and dysentery, also known as enterohemorrhagic E. coli (EHEC) [4]. It causes a range of human diseases, including mild diarrhea, hemorrhagic colitis (HC), hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) [5]. Although other foodborne pathogens such as Listeria, Salmonella enterica Serovar Enteritidis and Shigella species have been isolated from meats, E. coli O157:H7 is particularly concerning due to its extremely low infectious dose, as few as 10 cells, and its ease of transmission during slaughter and meat processing [6]. The microorganism can spread directly from person to person, especially in child day-care settings and from animals to humans [7].

Globally, E. coli infections are estimated to cause 2.8 million cases annually, with approximately 63,000 cases of HC reported each year in the United States alone. Children are particularly susceptible to severe complications such as HUS, a leading cause of acute renal failure (ARF) [8]. Numerous studies have investigated the prevalence of E. coli O157:H7 in food products and the bacterium has been isolated from a variety of plant and animal-derived foods within several countries. Contamination is frequently associated with meat products due to exposure to intestinal contents during slaughter and processing. However, contamination has been reported in raw fruits and vegetables, often linked to the use of untreated animal waste or contaminated irrigation water. Reported prevalence rates vary by country and food type, but the detection of the pathogen in ready-to-eat foods highlights the risk of foodborne illness outbreaks [9]. From 2003 to 2012, 390 outbreaks of Shiga toxin-producing E. coli O157 in the United States led to 4,928 diseases, 1,272 hospitalizations and 33 deaths, with cattle serving as the primary reservoir and ground beef as the most common route of transmission. Undercooked ground beef and other non-intact cuts pose a high risk, making it critical to decrease contamination during beef production, processing and storage and to protect consumers through appropriate handling and cooking of raw beef products [10].

Despite the urgent need to control E. coli O157:H7 contamination, current treatment methods vary in effectiveness and present challenges. To effectively decrease or eradicate E. coli O157:H7 contamination, a variety of treatment methods are currently used, ranging from simple washing of foods to chemical (peroxyacetic acid, chlorine dioxide, sodium hypochlorite and organic acids) or physical (irradiation, pasteurization, high-pressure processing and pulsed electric fields) decontamination techniques. These methods vary in their efficacy, costs and effects on the sensory quality and visual appeal of foods. However, their practical use is limited because several of these treatments can modify texture, flavor and nutritional attributes and some also increase concerns associated to residue safety and toxicity. Furthermore, they are not universally usable within all food types, which limits their feasibility and consistency in real processing environments [11]. Although antibiotics are used as effective chemical agents for removing these bacteria, increasing reports of antibiotic-resistant bacteria suggest that an alternative effective method is needed [12]. The growing threat of multidrug-resistant bacterial pathogens necessitates urgent investigation of alternatives to traditional antibiotics [13].

Regarding the limitations of current treatments, bacteriophage therapy, first developed by Felix d’Herelle and historically overshadowed by antibiotics, has re-emerged as a promising alternative to traditional antibiotics. This is due to its specificity, ability to target biofilms, minimal side effects and potentials to decrease antibiotic resistance development [14]. In food safety, bacteriophages offer unique advantages because of their narrow host range, which enables them to eliminate pathogenic E. coli strains without harming beneficial microbiota [15]. Coliphages, in particular, are abundant in environments such as sewages, animal wastes and water sources, making them accessible for isolation and use against foodborne pathogens [16]. One challenge to their use is the possible emergence of phage-resistant bacteria; however, this can be addressed through the use of phage cocktails composed of multiple phages with complementary host specificities. Such formulations not only broaden the range of susceptible bacterial targets but also decrease the likelihood of resistance development. Moreover, phages include the ability to evolve in bacteria, allowing them to overcome resistance mechanisms that limit the effectiveness of antibiotics. These characteristics have provided a way for regulatory acceptance of several phage-based products in the food industry, particularly for the control of E. coli O157:H7 in beef and fresh products, highlighting their potential as sustainable and innovative tools to enhance food safety [17].

Bacteriophages have been recognized as safe for use in food production and are classified as generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (FDA). While phages targeting E. coli O157:H7 such as EcoShield and SecureShield E1 have received GRAS approval for use in beef and other food products, similar approvals exist for phages against other foodborne pathogens. The GRAS phages are valued for their specificity, safety for human consumption, minimal effects on the food sensory characteristics and ability to decrease pathogen levels without contributing to antimicrobial resistance [18]. This research investigated the use of bacteriophages as biocontrol agents to control the propagation of artificially contaminated E. coli O157:H7 in meat samples.

1. Materials and Methods

2.1. Bacteria cultivation

Lyophilized E. coli O157:H7 PTCC 1860 was provided by the Persian Type Culture Collection (PTCC), Iran. The strain was cultured on tryptic soy agar plates at 37 °C for 16–18 h. For storage, bacterial cultures were suspended in tryptic soy broth (TSB) with 15% glycerol and stored at -80 °C. Bacterial counts were determined as colony-forming units (CFU) defined as viable bacterial cell capable of forming a visible colony on solid media.

2.2. Isolation and purification of bacteriophages

Bacteriophages were isolated from environmental water, sewage and slaughterhouse sewage samples. Briefly, 100 mL of sewage samples were collected in sterile bottles and transported to the laboratory. To enrich bacteriophages, samples were mixed with 200 mL of TSB media and inoculated with the E. coli host and then incubated overnight at 37 °C. Chloroform was added to the mixture for 10 min to lyse the bacterial cells, followed by centrifugation to remove debris. The resulting supernatants, containing potential bacteriophages, were assessed against host bacteria using double-layer agar method [19]. After overnight incubation at 37 °C, plates were investigated for plaques. Single plaques were isolated and subjected to repeated culture using double-layer agar technique to purify individual bacteriophages. Isolated bacteriophages were stored in SM buffer at 4 °C. Phage titers were expressed as plaque-forming units (PFU), where one PFU corresponded to an infectious phage particle that produced a visible plaque on a bacterial lawn.

2.3. Contaminated-meat experiments

Fresh beef was purchased from a local butchery and cut into similar pieces. Their sterility was assessed before use. For experimental contamination, E. coli O157:H7 was cultured overnight and a bacterial suspension was prepared at a concentration of 10⁷ CFU.mL^-1. Meat samples were immersed in the suspension and agitated for 10 min to allow bacterial adhesion. The multiplicity of infection (MOI) was reported as the ratio of phage particles (PFU) to bacterial cells (CFU).

2.3.1. Single Phage Treatment

Meat samples were divided into three groups and suspended in sterile water, including untreated meat (control), meat treated with E. coli O157:H7 at 10⁷ CFU.mL^-1 (bacteria only), meat contaminated with E. coli O157:H7 (10⁷ CFU.mL^-1) and subsequently treated with a specific bacteriophage at 10⁸ PFU.mL^-1 (phage-treated). All samples were agitated for 20 min; after which, aliquots were serially diluted (10-¹, 10-² and 10-³) for plating on eosin methylene blue (EMB) agar. Sampling and culturing were repeated after 60 min to assess bacterial decrease over time. The entire procedure was carried out at room temperature (RT) and 4 °C to assess the effect of temperature on phage efficacy.

2.3.2. Phage Cocktail Treatment

A mixture of three bacteriophages (TF1, TF2 and SC) was used in meat samples inoculated with E. coli O157:H7 (10⁷ CFU.mL^-1) at a total phage concentration of 10⁸ PFU.mL^-1. To further assess the cocktail performance, an additional trial was carried out; in which, each phage was used individually at 10⁸ PFU.mL^-1.

2.4. Contaminated ground beef experiments

Ground beef was prepared from the meat under sterile laboratory conditions and divided into three portions of equal weight. One portion was untreated as a control. The second portion was inoculated with E. coli O157:H7 at a concentration of 5 × 10⁸ CFU.mL^-1 without further treatment. The third portion was similarly contaminated with the bacteria, agitated for 20 min to ensure homogenized distribution and then treated with a bacteriophage suspension at 10⁸ PFU.mL^-1. These experiments were carried out at RT and 4 °C.

2.5. Plastic wrap treatment with bacteriophages

Beef samples were covered with plastic wrap under various conditions. The wrap included commercial food-grade low-density polyethylene available in the market. In the control setup, uncontaminated beef was wrapped with untreated plastic. A second setup involved contaminated beef wrapped with plastic that did not contain bacteriophages. In the phage-treatment setup, plastic wrap was pretreated with a suspension containing 10⁹ PFU.mL^-1 bacteriophages by spraying and storing at 4 °C for 20 min and then used in contaminated beef for 30 min at 4 °C. To further assess the protective effect of wraps over time, additional plastic sheets were incubated at 4 °C for 24 h after sprayed with the phage suspension followed by covering beef samples for 30 min.

2.6. Statistical analysis

All experiments were carried out in three replicates (n = 3). Quantitative data were presented as mean ± SD (standard deviation). Error bars in figures represented SD of the replicates.

1. Results and Discussion

Four bacteriophages were isolated from municipal sewage and two others from slaughterhouse wastewater. These were designated as tf1-tf4 and sc-sg, respectively. Of the six bacteriophages isolated, tf1 was chosen for further individual assessments based on its distinct plaque morphology. This produced large, clear and well-defined plaques, suggesting high lytic efficiency and strong bactericidal potential against E. coli O157:H7. In contaminated beef experiments, when 10⁶ PFU.mL^-1 of tf1 bacteriophage was used at RT against 10⁷ CFU.mL^-1 of E. coli O157:H7, 65% ±0.5  of bacteria were removed after 20 min (MOI = 0.1) at RT. Increasing phage dose up to 10⁹ PFU.mL^-1 led to 99% ±0.4  bacterial elimination (MOI = 100), as shown in Figure 1.

Exposure with phage for 60 min at RT, addition of 10⁷ PFU.mL^-1 of phage to 10⁶ CFU.mL^-1 of bacteria resulted in nearly 93.5% ±0.4 elimination (MOI = 10). However, bacteria were more effectively removed, when treated by 10⁸ PFU.mL^-1 phage even up to 99% ±0.2 (MOI = 100). Elimination rate was 100%, when 10⁹ PFU.mL^-1 of phage was added to 10⁷ CFU.mL^-1 of bacteria (MOI = 1000), as shown in Figure 2. Bacterial decreases are shown at the second y axis (right y axis).

For assessing the effect of phage cocktail, including tf1, tf2 and sc, cocktails with two various formulas were assessed. Meat containing 10⁷ CFU.mL^-1 bacteria was exposed to a dose of 10⁸ PFU.mL^-1 cocktail phages and showed nearly 99% ±0.1 of bacteria being lysed after 20 min and 99.5% ±0.1 after 60 min. When a cocktail included 10⁸ PFU.mL^-1 separately, bacterial removal was 100%; as shown in Figure 3. Comparison of performance between single-phage (Figure 1-2) and cocktail treatments (Figure 3) indicated that while tf1 alone achieved strong bacterial decrease at higher MOIs, the cocktails demonstrated enhanced efficacy and reliability in reaching levels below infectious (< 100 CFU) and ingestion (< 10 CFU) thresholds. The optimized cocktail not only maintained 99% bacterial elimination but achieved complete eradication when used at equivalent individual phage titers, demonstrating superior robustness in ensuring bacterial counts fall below clinically relevant exposure limits.

In ground beef experiments, meat samples were inoculated with E. coli O157:H7 at an initial concentration of 10⁷ CFU.mL^-1 and then treated with 10⁸ PFU.mL^-1 of bacteriophages for either 20 or 60 min at 25 and 4 °C. Bacterial recovery from the meat was affected by temperature, as adhesion decreased at refrigeration temperature, compared to ambient conditions, consistent with the lower metabolic activity and decreased surface interactions of bacteria at colder environments. After 20 min of phage exposure, bacterial counts decreased by 96.4% ±0.1 at 25 °C and 82.0% ±0.4 at 4 °C (Figure 4). When exposure time was extended to 60 min, decrease rates increased to 99.95% ±0.05 and 87.0% ±0.5 at 25 and 4 °C, respectively, indicating that phage-host interactions were kinetically dependent, with higher temperatures favoring further efficient adsorption and subsequent lysis. These findings suggested that rapid refrigeration of meat could decrease infection risk by limiting bacterial attachment, even though phage-mediated lysis occurred further slowly at lower temperatures

Contaminated meat samples were covered for 30 min with plastic wraps coated with 10⁹ PFU.mL^-1 phage for 20 min and 24 h. significant viable cell decreases, at the two conditions were observed with the greatest decrease in the 20 min-treated plastic wrap, compared to controls without phages. This included 99.0% ±0.9, compared to 93% ±2 in 24 h-treated group; as shown in Figure 5.

To assess the long-term stability of phage tf1, its plaque-forming ability was assessed over a 7 m storage at 4 °C. At a 10⁻⁷ dilution, plaque counts were consistent within all measured time points, showing no appreciable decreases in infectivity (Figure 6). The average plaque count calculated from all sampling intervals was stable, further verifying the persistence of active phage particles during prolonged storage. Minor deviations observed between the months were included within the expected range of experimental variability, which might be seen due to the counting precision or inherent assay fluctuations. Overall, these results indicated that phage tf1 preserved its infective capacity and structural integrity for at least 7 m under refrigerated conditions, highlighting its strong potential for use in long-term biocontrol uses.

The EHEC are pathogenic E. coli strains that produce Shiga toxins and cause HC and its life-threatening sequelae of HUS [20]. Since identification of serotype O157:H7 in 1983, many outbreaks associated with EHEC have been reported in the United States and E. coli O157:H7 has become one of the most important foodborne pathogens [21]. It causes severe human disease worldwide. It seems important to eliminate this pathogen in appropriate way [5]. Antibiotics were always an option; however, with increasing antibiotic-resistant strains due to use of antibiotics in cow feed, an alternative treatment is needed [22].

In a study, the overall prevalence of Shiga toxin-producing E. coli in raw beef samples collected from an industrial slaughterhouse in Hamadan, Iran, was 10.4%, with one isolate (12.5%) belonged to the O157 serogroup and the rest isolates of 87.5% classified as non-O157. The prevalence of O157 (1.3%) was similar to that reported in Iran (e.g., 1.35% in Mashhad), greater than those reported from the EU (0.3%), the U.S. (0.8%) and Brazil (1%) and less than those reported in Nigeria (2.2%) and Australia (1.7%). The study verified cattle as a reservoir for Shiga toxin-producing E. coli and highlighted seasonal and animal-associated factors such as spring sampling and the use of feeder cattle, which might contribute to the prevalence [23]. Additionally, they reported that 5.3% of raw poultry meat samples were contaminated with E. coli O157 strains. Out of 257 samples, 36% of the samples contained E. coli isolates, with 38.7% of the isolates identified as Shiga toxin-producing E. coli carrying virulence genes such as stx1, stx2 and eae. The isolates showed significant genetic diversity and high resistance to antibiotics, including nalidixic acid, tetracycline, ampicillin and trimethoprim-sulfamethoxazole [24].

Since discovery of bacteriophages, they included therapeutic potential as natural enemies of bacteria and phase-based elimination of bacteria in agriculture and food industry was started [25]. Therefore, bacteriophages have been reported as good antibiotic alternatives. They provide a highly specific, environmental-friendly effective means of controlling bacterial pathogens in agriculture; thereby, contributing to efforts against the increase of antibiotic resistance in the food sector [26].

This study aimed isolation of effective bacteriophages for biocontrol of E. coli O157:H7 in beef and ground beef. Six phages were isolated and assessed in suspension and spray methods on beef and beef ground samples at RT and 4 °C. The use of the submersion method to assess bacterial attachment to meat showed less bacterial recovery at 4 °C, compared to 25 °C, verifying that adhesion and phage inactivation efficiency were modulated by temperature, likely due to differences in bacterial physiology and phage adsorption kinetics. These findings highlighted the importance of environmental conditions and exposure time in optimizing phage-based biocontrol strategies in meat systems. Furthermore, the effect of various MOIs were assessed after incubation times of 20 and 60 min. The results showed that the phage treatment eliminated 82 to 100% of E. coli O157:H7. Phage cocktails included a better elimination rate. In a similar study, a cocktail of all three phages was assessed for their ability to lyse the bacterium in vivo and in vitro. The phage cocktail totally eradicated E. coli O157:H7 from beef surfaces in seven out of nine experiments. They have isolated phages from bovine farmyard slurry samples. The study achieved superior results when using phage cocktails similar to those used in this study, suggesting that a combination of multiple phages could enhance the effectiveness against the target bacteria. Another bacteriophage cocktail of three lytic phages for E. coli O157:H7 was investigated for its ability to decrease experimental contamination of hard surfaces, tomato, spinach, broccoli and ground beef by three bacteria. The observed decreases ranged from 94 to 100%. This suggested that naturally occurring bacteriophages might be useful for decreasing contamination of various hard surfaces, fruits, vegetables and ground beef by E. coli O157:H7 [10]. The E. coli O157:H7 is addressed for its extremely low ingestion (number of viable microorganisms that must be consumed via contaminated food or water orally to establish infection in a susceptible host) and infectious dose (the smallest number of viable microorganisms needed to cause infection in a host). Epidemiological evidence indicates that ingestion of fewer than nine viable cells can cause infection, with most reports estimating an infectious dose of fewer than 100 CFU [27]. This exceptionally low threshold highlights the importance of complete bacterial elimination in any antimicrobial intervention [28]. In the present study, the initial bacteriophage cocktail achieved a 99.95% decrease in E. coli O157:H7 counts, creating approximately 730 CFU. In contrast, the optimized cocktail achieved complete eradication (no detectable CFU). Regarding that single-digit cell numbers can lead to infection, the ability to decrease bacterial levels to less than the detection limit represents a critical improvement in ensuring microbiological safety.

Extending previous research on phage-based biocontrol strategies, the current study introduced a novel approach by spraying bacteriophage preparations onto plastic wrap used in cover meat containers, demonstrating that phage could effectively decrease E. coli O157:H7 contamination. This study assessed the stability of bacteriophage-coated plastic wrap under refrigeration. Wraps stored for 20 min or 24 h before contact with E. coli O157:H7 significantly decreased bacterial counts, with 99 and 93% decreases, respectively. These results indicated that phages were active on the wrap for at least 24 h, though shorter storage preserved slightly higher lytic activity. While phage uses in food safety such as sprays, edible films and integration into packaging have been reported in the literature [29, 30], the use of phage-coated plastic wrap directly covering meat surfaces is novel. This method offers a practical potentially-scalable strategy for controlling pathogenic contamination, particularly for pathogens such as E. coli O157:H7 with very low infectious doses, using a simple, economic readily available packaging material. The results showed that brief exposure to phage-coated surfaces could substantially decrease bacterial load, highlighting its relevance for enhancing food safety in processing and retail settings.

Previous studies have investigated the use of bacteriophages as natural antimicrobial agents in active or edible packaging by incorporating phages into biopolymer films or coatings [31]. Several studies have used phages within chitosan and PVA matrices for suppressing E. coli on food surfaces, demonstrating decreases in bacterial load on beef, poultry and other substrates [32-34]. These film or coating-based approaches, while effective, often need specialized formulation and fabrication and may face challenges such as controlled phage release, stability during storage and ensured sufficient contact with the food surface. In contrast, the present approach, spraying phage preparations directly onto standard commercial plastic wrap covering meat containers, eliminates the need for film casting or coating, enabling immediate phage contact with contaminated surfaces. This method can be rapidly implemented within existing packaging workflows without modifying materials or supply chains and the large decreases observed in these experiments suggest that sprayed phages may achieve antimicrobial effects on phage-embedded films while offering greater operational simplicity and flexibility.

Monitoring the viability of the tf1 phage over 7 m demonstrated long-term stability, with plaque numbers showing a slight increase during storage. This modest increase might reflect the gradual disaggregation of phage particles over time and further verifies that the phage preserves its stability and plaque-forming ability under standard storage conditions.

Bacteriophages can be limited by their narrow host range, complexity of formulating effective cocktails and decreased diffusion and activity within dense food matrices such as meats. Their stability is affected by environmental conditions and bacterial resistance may increase through receptor modifications. Additionally, regulatory restrictions and consumer perception challenges are difficulties to widespread adoption in food systems. In contrast to FDA, the European Food Safety Authority (EFSA) provides a further restrictive stance and bacteriophages are not currently approved as food additives or antimicrobials within the EU due to unresolved concerns regarding genetic stability, host-range specificity and safety classification [35]. This regulatory disparity highlights the need of continued investigation into phage safety, efficacy under practical food-processing conditions and standardized assessment frameworks to support future approval.

1. Conclusion

Use of bacteriophages is an effective strategy for controlling pathogenic E. coli O157:H7 in the beef and ground beef industry. Suspension and spraying methods demonstrated promising results in decreasing bacterial contamination. The ability to target dangerous pathogens with phages that are relatively easy to isolate highlights their potential as a practical and safe biocontrol tool. Widespread use of bacteriophages could enhance food safety by decreasing pathogenic E. coli contamination and should further be investigated for integration into standard food processing protocols, including their incorporation into sanitation procedures, formulation optimization and development of practical delivery methods appropriate for industrial use. The findings of this study represent an early-stage investigation of phage use in beef packaging systems. While the decreases are highly encouraging, continued research under diverse storage conditions, product types and scaling environments is needed before full industrial adoption. These preliminary results support further development of phage-based interventions as a promising addition to meat safety strategies.

1. Acknowledgements

The authors gratefully acknowledge the Microbiology Laboratory staff at Tabriz University, Ms. Fatemeh Khodayi and laboratory colleagues for their technical support and assistance throughout this study.

1. Declaration of competing interest

The authors declare no conflict of interest associated to this study.

1. Authors’ Contributions

Investigation, methodology, data curation, HMG; original draft preparation, editing and review, analysis, visualization, RR; supervision, conceptualization, FSH, supervision, conceptualization, methodology, formal analysis, review, resources, GZ.

1. Using Artificial Intelligent Chatbots

No AI assistance was used.

1. Ethical Consideration

No ethical approval was required for the conduct of this study

Abstract

Background and Objective: Probiotic whey-apple juice fermented beverages are becoming popular for their health benefits and incorporating bioactive ingredients such as licorice extract and caffeine enhances their functionality and consumer appeal. This study aimed to assess the physicochemical, microbial and sensory characteristics of a whey-apple juice based probiotic fermented beverage enriched with licorice extract and caffeine during storage.

Material and Methods: A probiotic beverage was formulated with various licorice extract (0–0.6%) and caffeine (60–100 mg.100 ml^-1) levels and stored for 1, 15 and 30 d. This was analyzed for pH, total soluble solids, viscosity, color, phenolic content, antioxidant activity, probiotic viability and sensory traits. A three-factor factorial design with statistical analysis was used for all experiments.

Results and Conclusion: Higher licorice extract and caffeine levels significantly increased Lactobacillus plantarum viability (from 6.27 to 7.79 CFU.ml^-1), phenolic content (from 125.87 to 164.16 mg GAE·g⁻¹), antioxidant activity (IC₅₀: from 145.46 to 112.26 μg.mg^-1) and viscosity (from 142.62 to 168.36 mPa.s^-1) while decreasing pH (from 4.26 to 4.17) and lightness (from 53.74 to 46.1) (p<0.05). The two additives altered color values, with a significant increase in redness only for licorice extract. The sample with 0.3% licorice extract and 80 mg caffeine included the highest sensory acceptability. Licorice extract and caffeine can improve the functional, physicochemical and sensory qualities of probiotic beverages during storage.

Keywords: Antioxidants, Caffeine, Fermented beverage, Glycyrrhiza, Lactobacillus plantarum, Probiotics

1. Introduction

Whey, a by-product of global cheese and casein production, represents environmental pollutant and valuable nutritional resource due to its high production volume and organic load. Rich in essential amino acids, peptides and proteins, whey offers numerous health benefits, including antihypertensive, antioxidant, anti-inflammatory and antimicrobial effects. Efficient use of whey supports sustainable waste management and creation of value-added functional foods [1-3]. Apple juice is a nutrient-rich beverage containing natural sugars, vitamins particularly vitamin C, minerals such as potassium and antioxidants such as polyphenols. Combining whey protein with apple juice not only improves the beverage taste but also enhances its nutritional profile by adding natural sugars, vitamins and antioxidants. Additionally, whey serves as an excellent base for probiotic beverages, promoting gut health and overall wellness [4, 5].

Licorice (Glycyrrhiza uralensis) is a widely used medicinal herb characterized by a diverse array of bioactive compounds, including prominent flavonoids such as liquiritigenin and isoliquiritigenin. Liquiritigenin, a primary constituent, shows multiple pharmacological activities encompassing antioxidant, anti-inflammatory, antibacterial, antidepressant and anxiolytic effects. Caffeine, the principal active ingredient in energy beverages, acts as a central nervous system (CNS) stimulant, enhancing alertness and cognitive function. The incorporation of licorice extract and caffeine significantly augments the functional attributes of the beverage. Collectively, these ingredients exert a synergistic effect, enhancing health-promoting potential and sensory qualities of the formulation [6-8].

Previous studies have documented the development and quality assessment of apple-whey based herbal functional ready-to-serve beverages [4,9]. For example, Sharma et al. [4] formulated a low-calorie beverage by mixing 75% apple juice with 25% whey protein, achieving the desired protein content. Additionally, kombucha beverages incorporating licorice and ginger have demonstrated strong antioxidant capacities and increased phenolic content, enhancing their functional characteristics [6]. Moreover, a non-alcoholic mixed beverage, containing coconut water, cashew apple juice and caffeine, has successfully been developed [10]. Despite the growing interest in functional probiotic beverages, no study has investigated the integration of probiotic cultures into whey-apple juice systems enriched with licorice extract and caffeine. This gap creates unresolved questions regarding microbial stability, physicochemical characteristics, sensory acceptance and health-promoting potential. Conventional formulations often suffer from decreased probiotic viability due to acidic fruit components and lack of natural synergistic stabilizers. The present study addressed this challenge by developing a novel probiotic apple-whey beverage fortified with licorice extract (0, 0.3 and 0.6%) and caffeine (60, 80 and 100 mg). This innovative combination affected licorice polyphenols and caffeine stabilizing effects to enhance probiotic viability, while assessing physicochemical, microbial and sensory attributes during storage.

1. Materials and Methods

2.1. Materials

Caffeine powder and carboxymethylcellulose (CMC) were purchased from Merck, Germany and licorice root was purchased from Shirin Darou, Shiraz, Iran. Lactobacillus plantarum (L. plantarum) PTCC 1058 cultures were provided by the Iranian Research Organization for Science and Technology (IROST, Tehran, Iran). The lyophilized strain (≥ 10⁸ CFU.ml^-1) was propagated in MRS broth for 24 h at 37 °C using CO₂ incubator (Memmert, Munich, Germany). Man, Rogosa and Sharpe (MRS) agar and broth were supplied by Merck, Darmstadt, Germany. All reagents in this study included analytical grade and were purchased from Merck, Germany.

2.2. Licorice Extract Processing

Licorice root was washed, dried and ground into powder, which was then extracted with 70% ethanol through a 72-h shaking process. The extract was filtered to remove residues, concentrated using rotary evaporation and dried to achieve the final extract [11].

2.3. Apple Juice Processing and Conservation

The apple juice was prepared by thoroughly washing and sorting the fruits, followed by cutting and grating. Apple juice was achieved using electric juicer and then passed through a muslin filter. The extracted juice was pasteurized in glass containers via heating and stored at room temperature (RM) until further use [9].

2.4. Whey Preparation from Cow Milk

Fresh cow milk was processed using method of Sharma et al. [9]. The milk was heated to 82 °C for 10 min, then cooled to 70 °C and acidified with 2% citric acid under continuous stirring to induce casein coagulation. The clotted mixture was passed through a double layer of muslin cloth and the achieved whey was centrifuged at 5000 rpm for 10 min to eliminate residual fat prior to its use in product formulation.

2.5. Production of Probiotic Beverage

Apple juice and whey were heated to 70 °C for 15 min and filtered and then mixed at 60:40 ratios. Carboxymethyl cellulose (0.5 g per 100 ml), caffeine (60, 80 and 100 mg per 100 ml) and licorice extract powder (0, 0.3 and 0.6%) were added to the mixture. The mixture was cooled to 37 °C and inoculated with a fresh L. plantarum probiotic culture at an initial level of approximately 10⁹CFU.ml^-1. Fermentation was carried out at 37 °C until the beverage reached a target pH of 4.5. The final pH after fermentation was assessed at 4.3 ±0.1. After fermentation, samples were stored at 4 °C. Experiments were carried out on Days 1, 15 and 30 to monitor physicochemical characteristics, microbial viability and sensory attributes of the beverage [12].

2.6. Physicochemical Analysis of Probiotic Beverage

During the storage time, probiotic beverage samples were analyzed for pH, total soluble solids (TSS) and viscosity. The pH and TSS were assessed using digital pH meter (AZ 86502, Taiwan) and digital refractometer (RX-7000α, India), respectively. The apparent viscosity was assessed using Brookfield viscometer (DV II + LV, Brookfield, Middleboro, MA, USA) equipped with an LV2 spindle. Viscosity was expressed in mPa_·s^-1 and assessed at 20 °C within fourteen spindle speeds ranging 1.5-2000 rpm; the primary reported values corresponded to 60 rpm. Assessments were carried out in triplicate to enhance reproducibility [9].

2.7. Color Assessment of Probiotic Beverage

The color characteristics of the probiotic beverage were assessed using HunterLab UltraScanvis US-Vis 1,310 device, USA, which assessed L*, a* and b* color values. The lightness parameter (L*) ranged from 0, representing black, to 100, representing white, a* scale assessed color on a spectrum from +127 (red) to -128 (green), while the b* scale ranged from +127 (yellow) to -128 (blue) [13].

2.8. Assessment of Phenolic Levels and Antioxidant Potential

Probiotic beverage samples were centrifuged and filtered prior to analyzing total phenolic content (TPC) using Folin-Ciocalteu method. The TPC was assessed using Folin-Ciocalteu method. A calibration curve was prepared with Gallic acid standards (0–200 mg.l^-1) and results were expressed as Gallic acid equivalents (GAE) per liter. Each assessment was carried out in triplicate. Antioxidant activity was assessed using DPPH radical scavenging assay. Sample extracts at various concentrations were reacted with 0.1 mM DPPH solution and absorbance was assessed at 517 nm after 30 min incubation in dark. The inhibition percentage was calculated and IC₅₀ values were achieved from a dose-response curve generated by plotting inhibition percentages against sample concentrations. All assessments were carried out in triplicate to ensure reproducibility [9].

2.9. Assessment of Probiotic Bacterial Survival

For microbiological assessment, 10 ml of the sample was mixed with 90 ml of sterile saline to achieve a 10^-1 dilution, followed by preparation of successive serial dilutions. Enumeration of L. plantarum was carried out by spreading the diluted samples onto MRS agar supplemented with vancomycin and incubating at 37 °C for 72 h using CO₂ incubator. The results were reported as logarithmic colony-forming units (Log CFU) per milliliter [14-15].

2.10. Sensory Analysis

A sensory analysis was carried out with a panel of 12 trained participants (six men and six women, aged 20–30 y) using five‑point hedonic scale ranging from 1 (“extremely dislike”) to 5 (“extremely like”). The panelists assessed the probiotic beverage samples for color, flavor, texture and overall acceptability during a 30‑d storage time. For each session, 20-ml portions of the beverage were present on coded plates, set at 4°C ±1 and panel members rinsed their mouths with water between the assessments [16].

2.11. Statistical Analysis

All experimental trials were carried out using completely randomized factorial design with three variables of caffeine concentration, licorice powder level and storage time to assess their individual and combined effects on the physicochemical, microbiological and sensory characteris-tics of the probiotic drink. Data analysis was carried out using SPSS software v.22 and significant variations within the treatments were identified using Duncan’s multiple range test at a 95% confidence threshold (p < 0.05).

1. Results and Discussion

3.1. The pH and Probiotic Viability Assessment

Figure (1a, 1b) shows the pH levels and L. plantarum viability in probiotic beverages formulated with various quantities of licorice extract powder and caffeine through storage. The pH values of all samples ranged 4.13-4.32, which set within the acceptable range for apple juice (3.2–4.2) according to the Iranian National Standardization Organization (INSO) (ISIRI 14345, 2011), though the use of whey increased the highest pH. The ANOVA revealed significant major effects of licorice (p < 0.001) and caffeine (p < 0.001) on pH, as well as a significant interaction between the licorice and caffeine (p < 0.001). Similarly, L. plantarum viability was significantly affected by licorice (p < 0.001) and caffeine (p < 0.001), with a strong interaction effect (p < 0.001). The two parameters decreased significantly over the storage time (p < 0.05).

These results verified that licorice and caffeine acted synergistically to enhance probiotic viability while decreasing pH. Additionally, caffeine and associated phenolic compounds affected L. plantarum metabolic pathways through enzymatic activity, potentially increasing bacterial viability.

The observed decrease in pH with higher licorice and caffeine concentrations reflected intensified fermentation by L. plantarum, resulting in greater production of organic acids such as lactic acid that further promoted probiotic function. Caffeine positive effect might be linked to its potential degradation by these bacteria into methylxanthines, which could play an important role in cultivating beneficial gut microbiota such as Lactobacillus species. Methylxanthines might stimulate lactic acid bacteria (LAB) through nutritional enhancement, serving as possible carbon sources; thereby, creating a further favorable environment for bacterial proliferation. This combination of biochemical interactions suggested the potential beneficial effects of licorice extract and caffeine on probiotic viability and activity [14,17].Over the storage time, a significant decrease in L. plantarum viability and pH was typical for probiotic beverages. As nutrients decreased and metabolic waste accumulated, probiotic cell numbers decreased. Acid production might decrease or stabilize over time, affecting pH measurements. The initial pH decrease was linked to active bacterial metabolism during early storage, but extended time led to lower bacterial activity and viability; thereby, decreasing the two parameters significantly (p < 0.05). This decrease highlighted the importance of optimizing storage conditions and duration for maintaining probiotic efficacy in commercial products [18]. Similarly, a study on Iranian ultrafiltered white probiotic cheese containing caffeine detected that the addition of caffeine improved the survival of L. fermentum [19]. Tsirulnichenko and Kretova [14] reported that licorice root was a valuable source of probiotic-supporting substances, particularly fructans. They identified that a 1% concentration of licorice root extract represented the minimum effective dose needed to significantly stimulate the growth of probiotic microorganisms. In addition, Aleman et al. [20] studied the effects of functional ingredients on yogurt and detected that adding licorice root improved the viability of L. bulgaricus during storage.

3.2. Total soluble solids and Viscosity Assessment

Figure (2a, 2b) illustrate the TSS and viscosity of probiotic beverages formulated with varying concentrations of licorice extract powder and caffeine during storage. The TSS values of the samples ranged 10.3-11.85 °Brix, while viscosity values varied 141.21-171.43 mPa.s^-1. The ANOVA results showed that caffeine concentration included no significant effect on TSS (p = 0.20) or viscosity (p = 0.11). Licorice extract concentration included a non-significant effect on TSS (p = 0.42), but significantly increased viscosity (p=0.004). Storage time significantly affected TSS (p = 0.002) and viscosity (p = 0.001). Importantly, the interaction between licorice and caffeine was not significant for TSS (p = 0.292) but was significant for viscosity (p = 0.018), indicating that higher licorice levels amplified the thickening effect of caffeine. These findings suggested that while variations in caffeine concentration might affect TSS and viscosity, the absence of statistical significance was likely attributed to the relatively low caffeine levels used in the formulation. The statistically significant increase (p < 0.05) in the viscosity of the probiotic beverage with increasing licorice extract concentration could be linked to several factors inherent to the licorice composition. Licorice extract is rich in soluble solids and crude fibers, which act as natural thickeners, enhancing the beverage viscosity. Additionally, these components improve the water-holding capacity, contributing to a thicker further stable texture [20]. The significant increase in TSS and viscosity of samples during storage could be attributed to several factors. Over time, the evaporation of water leads to a concentration effect, increasing TSS, which can contribute to higher viscosity. Additionally, licorice extract contains carbohydrates that may interact with other components in the beverage matrix, enhancing thickening and water retention during storage.

The presence of caffeine and bioactive compounds from licorice affects microbial activity and biochemical reactions during fermentation, potentially increasing the production of organic acids and other metabolites that modify the beverage rheological characteristics [17]. De Carvalho et al. [7], in their study on developing a mixed beverage made from coconut water and cashew apple juice with caffeine, detected that the TSS content of their samples ranged 10.6-11.6, closely similar to the current findings. Similarly, Mohamed et al. [22] reported that formulations containing licorice extract showed significantly greater viscosity than the control samples, suggesting that licorice extract enhanced the thickness and texture of health drinks. Additionally, Khoshdouni et al. [23] reported a slight increase in TSS values over time, corresponding to the results achieved in the current study.

3.3. Total Phenolic Content and Antioxidant Activity (IC₅₀)

Fig. (3a, 3b) show the effect of licorice extract powder and caffeine on TPC (mg GAE·g⁻¹) and antioxidant activity (IC₅₀, µg·mL⁻¹) of probiotic beverages during storage. The TPC values ranged 116.12-171.53 mg GAE·g⁻¹, while IC₅₀ values varied 108.25-156.74 µg·ml⁻¹ within the treatments and storage times. Moreover, ANOVA results indicated that the licorice extract concentration significantly increased TPC (p < 0.001) and antioxidant activity (p< 0.001). Caffeine concentration included a non-significant effect on TPC (p = 0.27), but significantly improved antioxidant activity as assessed by IC₅₀ (p < 0.001). Importantly, the interaction between licorice and caffeine was significant for TPC (p= 0.031) and IC₅₀ (p = 0.024), indicating that higher licorice levels amplified the antioxidant-enhancing effect of caffeine. The TPC and antioxidant activity decreased significantly over the storage time (TPC, p < 0.001; IC₅₀, p < 0.001), reflecting the gradual degradation of phenolic compounds and decreased bioactivity during prolonged storage.

Licorice extract contains abundant phenolic compounds and flavonoids, which confer strong antioxidant potential. From its significant constituents are phenylflavonoids such as dehydroglyasperin C (DGC), recognized for their effectiveness in neutralizing free radicals and safeguarding cells against oxidative injuries. The antioxidant activity of licorice is primarily attributed to its ability to quench reactive oxygen species, limit lipid peroxidation and enhance the function of antioxidant enzymes. These mechanisms highlight licorice reported health-promoting characteristics, including anti-inflammatory and anti-aging effects and highlight its value as a natural ingredient for improving the antioxidant profile of foods and beverages. Collectively, the diverse bioactive molecules in licorice provide substantial protection against oxidative stress and may help decrease the risk of associated disorders [24]. Incorporation of caffeine into probiotic beverage formulations has been shown to progressively increase antioxidant activity. Caffeine shows antioxidant capacity through radical scavenging and moderation of oxidative stress. Within a probiotic system, caffeine can act synergistically with other bioactive compounds; thereby, enhancing the overall ability of the beverage to counteract reactive oxygen species. This gradual improvement is likely associated with interactions between caffeine, probiotic microorganisms and phenolic constituents, ultimately strengthening the functional attributes of the product over time [25]. A significant decrease in phenolic content and antioxidant activity during storage (p < 0.05) is commonly observed in probiotic beverages. Throughout storage, factors such as exposure to oxygen and light, fluctuations in temperature and changes in pH contribute to the breakdown of phenolic compounds. Additionally, biochemical reactions and interactions between phenolics and other components within the beverage can further destabilize these antioxidants, resulting in decreased antioxidant effectiveness over time [26]. Quintana et al. [27] reported that the TPC in licorice root extract ranged 48.47-180.06 mg GAE.g^-1 while assessing its antioxidant activity. Similarly, Vieira et al. [25] demonstrated that caffeine effectively scavenged hydroxyl radicals and investigated the mechanisms behind its antioxidant potential. Fatima et al. [28] observed that antioxidant activity and free radical scavenging capacity gradually decreased during the storage of functional fruit and vegetable-based drinks containing herbal and spice extracts. Although licorice and caffeine significantly enhanced the phenolic profile and antioxidant activity of probiotic beverages, limitations should be addressed. The storage time was relatively short (30 d), which restricted conclusions on long-term stability; future studies should extend to 60 d to provide further comprehensive shelf-life data. Quantitatively, the TPC values observed in this study (116–171 mg GAE·g⁻¹) were within or slightly higher than the values reported by Quintana et al. (48–180 mg GAE·g⁻¹), highlighting the strong contribution of licorice extract. Importantly, published studies on probiotic beverages containing caffeine were limited, underscoring the novelty of this study and the need of further investigations in this area.

3.4. Color Assessment of the Probiotic Beverage

Figure (4a, 4b, 4c) shows the changes in the L*, b* and a* color indices of the probiotic beverage containing licorice extract and caffeine over the storage time. The L* values ranged 44.61-56.45, the a* values ranged 14.42-25.76 and the b* values ranged 17.23-26.4 within the treatments and storage times. The ANOVA results indicated that increasing licorice extract and caffeine concentrations significantly decreased L* (p = 0.012) and significantly increased b* (p = 0.008). The two factors increased a* values, but this increase was significant only for licorice extract (p = 0.021). Storage time significantly affected all three indices (p < 0.001). Importantly, the interaction between licorice and caffeine was significant for L (p = 0.033,) and b* (p = 0.027), but not for a* (p = 0.29). These findings verified that higher licorice levels amplified the color‑modifying effect of caffeine, leading to darker and further saturated.hues during the storage.

This decrease in lightness could be linked to the inherent color compounds present in licorice extract and caffeine such as polyphenols and alkaloids, which created a deeper color. Additionally, interactions between these compounds and the beverage matrix during storage might enhance pigment formation or aggregation, further decreasing brightness. The significant change at p < 0.05 verified that this effect was statistically robust, suggesting a clear effect of these ingredients on the visual appearance and potentially consumer perception of the product [29-30]. Increasing the quantities of licorice extract and caffeine causing significant increase in the b* (yellow-blue) value could be explained by the yellowish pigments naturally present in licorice, primarily flavonoids such as liquiritin and glabridin, which contributed to its characteristic yellow color. Studies indicate that licorice flavonoid content is responsible for this yellow hue, which can intensify as concentration increases, thus increasing the b* value towards yellow tones [31]. The significant increase in a* (red-green) values for licorice extract might be attributed to the presence of reddish-brown compounds such as melanoidins, which were formed during processing or storage through Maillard reactions. These polymeric brown pigments accumulated with heat treatment, contributing to darkening and shift towards red color. Additionally, heat treatment could increase the TPC and antioxidant activity of licorice extracts, affecting their color characteristics [32-33]. Kang et al. [31] demonstrated that during storage, licorice extract created the formation of brown melanoidin pigments, resulting from Maillard reaction between sugars and amino acids. Similarly, Ardalanian and Fadaei [33] observed that adding ginseng extract to doogh samples caused increases in a* and b* color values. In an associated study, Jooyandeh and Alizadeh Behbahani [32] reported that the inclusion of pepper extract in functional yogurt led to a decrease in L* (lightness) with increases in a* (red-green) and b* (yellow-blue) values.

3.5. Sensory Assessment of Probiotic Beverage

Figure (5) presents the sensory evaluation outcomes of probiotic beverage samples assessed by the panel on Day 30 of storage, considering attributes such as taste, aroma, color, texture and overall acceptability. Statistical analysis indicated that variations in licorice extract and caffeine levels significantly affected taste scores (p < 0.05). The combination of 0.3% licorice extract with 80 mg of caffeine improved taste perception, whereas higher concentrations resulted in decreased ratings. Caffeine content showed no significant effect on aroma or color, while 0.3% licorice extract enhanced color scores (p < 0.05). Texture was favorably affected by 0.6% licorice extract, with no significant effect from caffeine. The sample containing 0.3% licorice extract and 80 mg of caffeine achieved the highest overall acceptability.

Similarly, Azami et al. [34] developed a dairy beverage with licorice extract and cocoa powder and detected that moderate licorice levels improved taste and aroma, but excessive quantities led to decreased acceptability due to bitterness and off-flavors. Mahmoudi et al. [35] produced a functional non-dairy probiotic beverage containing fermented jujube extract, reporting that addition of extract enhanced aroma and taste, contributing to higher acceptability scores. Similarly, Ghosi Hoojaghan et al. [36] studied the effect of fennel extract in doogh, finding that low concentrations included insignificant effects on flavor, but higher concentrations decreased flavor scores.

1. Conclusion

This research formulated a functional probiotic drink enriched with licorice extract and caffeine, which showed improvements in probiotic survival, antioxidant potential and sensory attributes. Licorice extract significantly enhanced phenolic content, viscosity and antioxidant capacity, whereas caffeine contributed to antioxidant activity and probiotic viability. Appropriate concentrations of these components optimized flavor, appearance, texture and overall consumer acceptance. Overall, the results indicate that licorice extract and caffeine act synergistically to strengthen the health-promoting characteristics and sensory quality of probiotic beverages, highlighting their promise as valuable functional ingredients.

1. Declaration

5.1. Acknowledgements

Contribution of Islamic Azad University, Iran, Tehran, is deeply appreciated.

5.2. Declaration of competing interest

The authors declare no conflict of interest or finance.

5.3. Authors’ Contributions

Aydin Ali Bigelow: Writing original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Funding acquisition. Marjaneh Sedaghati: Writing–review and editing, Supervision, Methodology, Resources, Projected administration, Conceptualization. Mozhgan Emtyazjoo: Supervision, Resources, Projected administration, Conceptualization.

5.4. Using Artificial Intelligent Chatbots

AI chatbot was used only for grammar correction.

5.5. Ethical Consideration

No ethical approval was required for the conduct of this study.

Background and Objective: The metabolomic profile represents the totality of all low-molecular-weight metabolites. It reflects the physiological state of micro-organisms and their potential for biotechnological uses. The aim of this study was to analyze the metabolomic profile of Lactococcus strains, which allowed the researchers to study the characteristics of these microorganisms. As a result, principles for designing lactic acid bacteria consortia with pronounced production-significant chara-cteristics for the creation of next-generation fermented milk products can be developed.

Material and Methods: The study microbes were Lactococcus strains. The content of organic and amino acids in the experimental samples was assessed by capillary electrophoresis. The contents of vitamins, mono- and disaccharides were assessed using high-performance liquid chromatography.

Results and Conclusion: The metabolomic profiles of 16 industrially associated Lactococcus strains were analyzed. All strains produced lactic acid, with the highest content observed in Strain Ll1, at 8677.8 mg·kg^-1. The experimental samples showed a significant increase in methionine content, with the highest value of 311.2 mg·(100 g)^-1 in Strain Ll3 and cystine content, with the highest value of 45.6 mg·(100 g)^-1 in Strain Ld1. Strain Lc8 showed the ability to synthesize methionine and cystine, which might be promising in cheese production, since these sulfur-containing amino acids contribute to the aroma and flavor of fermented milk products. This strain produced formic, succinic and acetic acids and could catabolize citric acid and galactose. The Strains Ld1, Ld2 and Ld3 showed galactose accumulation, indicating a lack of enzymatic activity for the oxidation of this monosaccharide. Literature data on variability in vitamin synthesis levels between the strains have been verified. Thus, only Strain Ld2 synthesized vitamin B2, the content of which significantly exceeded the control and reached 103.18 ±2.06 μg·(100 g)^-1. Five strains were capable of synthesizing vitamin B6 up to 247.69 ±5.45 μg·(100 g)^-1. The vitamin B9 content increased, compared to the control, in eight samples, with maximum values of 69.59 ±1.46 μg·(100 g)^-1 for Strains Ll3. The dataset on lactococci metabolomic profiles can serve as a basis for the further development of methodological systems, mathematical models and algorithms for selecting strains for the creation of consortia and the production of products, including those with functional characteristics. The results can serve as a basis for the design of consortia of lactic acid bacteria in biotechnological approaches to produce novel fermented milk products with specific characteristics, including functional ones, which include positive effects on human organs when consumed regularly.

Keywords: Lactic acid bacteria, Lactococcus, Functional characteristics, Metabolomic profiles, Metabolites

1. Introduction

Fermented dairy products contain various metabolites produced by starter cultures that offer health benefits to the human body. These include organic acids, exopolysacchar-ides, vitamins and nitrogenous compounds such as peptides and amino acids. Metabolites are formed as a result of the vital activity of lactic acid bacteria (LAB), which ferment lactose into lactic acid, hydrolyze proteins and lipids to peptides, amino acids and free fatty acids. Some metabolites can regulate human metabolism, decrease inflammation or include antimicrobial effects [1]. Lactococcus bacteria (Streptococcaceae family) are among industrially asso-ciated bacteria in the dairy industry. They are Gram-positive, facultative anaerobic catalase-negative cocci [2]. Their primary role in fermentation processes includes efficient conversion of lactose into lactic acid, thereby increasing medium acidity, which enhances antimicrobial characteristics and extends the shelf life of the final product [3]. Lactose metabolism occurs via the pentose phosphate pathway and/or the Embden-Meyerhof-Parnas pathway (glycolysis) through the action of the enzyme β-galactosidase, which hydrolyzes lactose into glucose and galactose. Glucose enters glycolysis, forming pyruvate, which is subsequently reduced to L-lactic acid by lactate dehydrogenase. Another major route of lactose catabolism is the pentose phosphate pathway, in which lactose is phosphorylated to lactose-6-phosphate during transport and then cleaved into glucose and galactose-6-phosphate by phospho-β-galactosidase. Then, galactose-6-phosphate is converted into tagetose-6-phosphate by isomerase, which turns into glyceraldehyde-3-phosphate and dihydroxyace-tone phosphate. Glyceraldehyde-3-phosphate enters glycol-ysis and leads to pyruvate and L-lactic acid formation [4]. The accumulation of L-lactic acid leads to a decrease in the pH of the medium, which determines the intensity and pathway of lactic acid fermentation. Other key factors affecting this process are the acidification rate and the presence of oxygen and carbon dioxide in the medium. Within the Genus Lactococcus, L. lactis subsp. lactis stands out for its higher rate of lactose utilization and the ability to ferment maltose and trehalose [2].

Moreover, acid production is indirectly linked to exopolysaccharide (EPS) production. The EPS are high-molecular-weight carbohydrate polymers (either homo or heteropolysaccharides) secreted by microorganisms into the extracellular environment. Their precursors are intermediate metabolites of glycolysis and the pentose phosphate pathway glucose-6-phosphate, fructose-6-phosphate, uridine diphosphate glucose and other nucleotide sugars. Since these intermediates serve as precursors for lactic acid, a competitive relationship for carbohydrate substrates occurs [5]. The highest activity of the enzymatic complex of EPS synthesis is achieved at lower acidity levels during lactate biosynthesis (pH 5.0–5.5) [6, 7]. The production of EPS leads to the formation of a slimy layer surrounding the cell, which forms a colloidal aggregate in the form of an amorphous substance with little or no cell adhesion or a cohesive capsule [8]. Acid formation and then EPS production by Lactococcus spp. contribute to the texture of dairy fermented products, resulting in a smooth, creamy, homogeneous consistency with possibly slight ropiness.

Cow milk contains 0.4–0.5 mg·(100 g)^-1 of potassium and sodium citrates. Among Lactococcus species, only L. lactis subsp. lactis var. Diacetylactis is capable of citrate metabolism. End products of citrate metabolism include diacetyl, acetoin, 2,3-butanediol, acetaldehyde, ethanol and lactic acid. The induction of the citrate permease catalytic enzyme is caused by the presence of lactic acid rather than citrate, which is synthesized from glucose and citrate under acidic pH conditions [9]. Thus, acid formation affects citrate metabolism. The most valuable metabolic products in this biosynthesis are diacetyl and carbon dioxide, as they improve the texture and flavor of fermented dairy products.

Lactococcus species include a limited ability to synthesize amino acids de novo from inorganic carbon sources and are therefore auxotrophic. However, fermented dairy products can be enriched with amino acids through the proteolysis of milk caseins [10]. Extracellular proteinases of Lactococcus hydrolyze caseins into oligopeptides, which are then transported into the cell and cleaved by intracellular peptidases into amino acids [11]. The activity of proteolytic enzymes and transport systems depends on the availability of nitrogen, acidity and the content of free amino acids and is controlled by regulatory systems. Particularly, L. lactis subsp. cremoris cleaves β-casein with the similar protease specificity as L. lactis subsp. lactis, but has less active proteolysis [12]. Amino acids are used for cellular biosynthesis and as precursors of biologically active compounds. Several amino acids are degraded to volatile compounds (aldehydes, ketones and alcohols), which can affect the sensory characteristics of fermented dairy products [12, 13]. Partial hydrolysis of milk proteins by lactococci, with lactose reduction, enhances digestibility in the human gastrointestinal tract (GIT) and may decrease the risk of intolerance, compared to raw milk.

Amino acids play a critical role in the synthesis of the bacteriocin of nisin (a polycyclic peptide composed of 34 amino acid residues, including the non-proteinogenic amino acid lanthionine) which is active against a broad spectrum of Gram-positive bacteria [14]. Studies have shown that the presence of amino acids such as alanine, isoleucine, serine, glutamic acid, tyrosine and tryptophan in the culture medium enhances bactericidal activity against strains of Escherichia coli and Staphylococcus aureus [14, 15]. Regarding vitamin biosynthesis, Lactococcus species are largely auxotrophic due to their adaptation to nutrient-rich environments such as milk, where several vitamins are readily available. Nevertheless, certain strains have the ability to synthesize B-group vitamins, such as folates (B9) and riboflavin (B2) [16]. Folate production levels vary significantly between the strains. Efforts to enhance B9 biosynthesis in LAB have involved optimized cultivation conditions and genetic modification through strain selection and metabolic engineering [17]. The ability of Lactococcus strains to produce riboflavin is limited and highly strain-dependent. Some strains function as “riboflavin producers” and are used to enrich dairy products with improved nutritional value, as well as imparting a yellowish color to whey and cheeses [18, 19].

The metabolomic profile reflects the key characteristics of the microorganism that determine its technological potential in dairy production. Industrially associated characteristics of strains include technological significance in the production of dairy products (e.g., fermentation time and viscosity) Understanding the genetic and regulatory mechanisms underlying metabolite biosynthesis pathways in Lactococcus species enables the targeted selection of starter cultures and development of microbial consortia, which is critical for creating novel biotechnological products. The aim of this study was to analyze the metabolomic profile of Lactococcus strains, which allowed the researchers to study the characteristics of these microorganisms. As a result, principles for designing LAB consortia with pronounced production-significant charact-eristics for the creation of next-generation fermented milk products can be developed.

1. Materials and Methods

2.1. Experimental samples

The microorganisms of this study were Lactococcus strains from the collection of the All-Russian Research Institute of the Dairy Industry (“VNIMI”), selected based on an evaluation of the most important characteristics for dairy processing, including the viability after long-term storage, acidification rate, titratable acidity and pH, apparent viscosity and sensory characteristics. The selected strains included four strains of L. lactis subsp. lactis biovar diacetylactis (Ld1, Ld2, Ld3 and Ld4), eight strains of L. lactis subsp. cremoris (Lc1, Lc2, Lc3, Lc4, Lc5, Lc6, Lc7 and Lc8) and four strains of L. lactis subsp. lactis (Ll1, Ll2, Ll3 and Ll4). Before use, the strain was preserved at -80 °C under glycerol. The strain was then cultured in milk at 30 °C. A second-generation culture was used. The initial cell concentration was 10⁷ CFU·ml^-1. To obtain experimental samples, sterile skimmed milk (the "Standard" brand Complimilk, Belarus) was fermented with the test strain at a concentration of 5% and incubated at (30 °C ±2) until a clot formed (рН 4.5 ±0.2). The control was sterile skim milk. The milk was prepared by dissolving 90 g of dry skim milk in 1000 ml of tap water and sterilized by autoclaving using N-Bioteck sterilizer, Korea, at 121°C for 3 min. The initial pH was 6.8 and was set in a thermostat with the experimental samples at 30 °C. A limitation of this study was the number of strains (40). The study was limited to assessing the characteristics of representatives of a genus, lactococci, the most common type of LAB used in food production. The studied strains were isolated from homemade fermented products and natural sources in a temperate climate zone. Moreover, the study did not include genetically modified strains.

2.2. Assessment of organic and amino acids

The content of organic acids in the experimental samples was assessed using capillary electrophoresis and "Kapel 205" system, equipped with a spectrophotometric detector and a quartz capillary (75-µm inner diameter, 60-cm total length). Samples were pre-diluted with distilled water. The buffer electrolyte was prepared using benzoic acid, diethanolamine, cetyltrimethylammonium bromide and Trilon B. Separation was carried out at 20 kV with UV detection at 254 nm. Electropherograms were processed using "Elforan" software.

For the assessment of amino acid composition, the samples underwent acid hydrolysis and, alkaline hydrolysis for tryptophan, to convert protein-bound amino acids into free forms. All amino acids, except tryptophan, were derivatized into phenylisothiocarbamyl derivatives and quantified via capillary electrophoresis. Tryptophan was assessed directly without derivatization using borate buffer, +25 kV voltage and UV detection at 219 nm. Glutamic acid, aspartic acid and cystine were analyzed in phosphate buffer with β-cyclodextrin under +25 kV voltage, 50 mbar pressure and UV detection at 254 nm. The rest of amino acids (arginine, lysine, tyrosine, phenylalanine, histidine, leucine and isoleucine, methionine, valine, hydroxyproline, proline, threonine, serine, alanine and glycine) were assessed using similar method without pressure. All electropherograms were processed using "Elforan" software. For such amino acids as tryptophan, glutamic acid and glutamine, and aspartic acid and asparagine, the associated error was ±20%; for arginine, tyrosine, phenylalanine, histidine and methionine, the associated error was ±23%; for lysine, leucine and isoleucine, valine, proline, threonine, serine, alanine and glycine, the associated error was ±18%; for cystine and cysteine, the associated error was ±24%; and for organic acids, the associated error was ±20%.

2.3. Assessment of mono and disaccharide contents

The contents of mono and disaccharides in the experimental samples were assessed using high-perfor-mance liquid chromatography (HPLC) in accordance with GOST 54760-2011, “Component milk products and infant milk products. Assessment of mono and di-sugars mass concentration using HPLC method.” The analysis was carried out using MAESTRO liquid chromatograph (INTER-LAB, Russia), with a Zorbax carbohydrate analysis column (5 μm, 4.6 × 250 mm; Agilent Technologies, USA), CAU-X-320 electronic analytical balance (CAS, Korea), variable-volume single-channel pipette (100–1000 μl; BIOHIT, Finland; Sartorius Biohit Liquid Handling, Germany) and Sigma 1-14 microcentrifuge (Sigma Laborz-entrifugen, Germany). The associated measurement error was ±12%.

2.4 Assessment of vitamins

Vitamin C was assessed using Agilent 1260 chromato-graphy system equipped with a gradient 4-channel pump, a diode array detector, a column thermostat and an automatic sample delivery system. The associated measurement error was ±34%. The analyte was extracted by diluting the sample aliquot 1:2 with an extracting solution (3% metaphosphoric acid). It was vortexed and centrifuged at 10.850× g. The supernatant was filtered through a 0.22-μm filter. The resulting filtrate was used for analysis. Separation of components was carried out by reversed-phase chromate-graphy on an Agilent Extend-C18 4.6*250 mm column. Vitamin C was eluted under isocratic conditions using mobile phase of 30 mM phosphate buffer, pH 2.6, at a flow rate of 1 ml·min^-1. Detection was carried out using diode array detector at 254 nm.

B-group vitamins were assessed using HPLC with an Agilent 1260 Infinity II HPLC device combined with an Ultivo Triple Quad LC/MS mod. 6465, Singapore, Agilent Technologies and an Agilent 1260 Infinity II HPLC device with a diode array detector G7115A. Chromatographic separation was carried out using Agilent InfinityLab 120 Poroshell 120 Phenyl-Hexyl column (3.0 × 100 mm, 2.7 μm). For sample preparation, 1 g of the sample was mixed with 4 cm³ of deionized water, vortexed and then 5 cm³ of acetonitrile and 0.1 g of ascorbic acid were added to the mixture. The mixture was re-vortexed, ultrasonicated for 30 min and centrifuged at 3.650× g for 10 min. The sample was stored at -4 to -6 °C and filtered through a 0.22-μm filter.

2.5. Statistics

The reliability of data was verified through the execution of experiments in at least three independent replicates. Furthermore, MS Office Excel 2016 was used for data analysis and graph creation. Experimental data were presented in“mean value ± measurement error (relative)”. The data on vitamins were present as mean ± standard deviation. To calculate the significance of differences, ANOVA statistical method was used, with the accepted limitation of statistical significance of differences between options at p ≤ 0,05. The study was carried out using equipment of the Collaborative Center of the All-Russian Dairy Research Institute (CKP “VNIMI”).

1. Results and Discussion

3.1. Assessment of amino acids

Amino acids are promising metabolites for biotech-nological uses. These compounds are used as artificial sweeteners, flavoring agents and feed additives and for pharmaceutical purposes [19, 20]. During the production of fermented dairy products, specific amino acids generated via casein proteolysis are responsible for the formation of thiols, alcohols, esters and aldehydes, contributing to a broad range of flavors [20]. Sulfur-containing amino acids play a critical role in the development of sensory charac-teristics, as well as in the antioxidant activity of fermented dairy products [21]. In the experimental samples containing the industrially associated Lactococcus strains, the amino acids were assessed. Compared with the control (milk), a significant increase in the content of methionine and cystine was detected in the experimental samples. The results are presented in Figure 1.

A significant increase in amino acid content relative to the control was observed for methionine (Figure 1A) in Strains Lc2, Lc6, Lc8, Lc4, Ll2, Ll3, Ll4, with the highest content in Strain Ll3 at 311.2 mg·(100 g)^-1. The sulfur-containing amino acid methionine plays a role in the initiation of transcription and is critical in various methyltransferase reactions. Aromatic compounds such as 3-methylbutanal, methanethiol, dimethyl sulfide, 2-methylpropanol and dimethyl trisulfide are synthesized from methionine [22]. Among these, methanethiol is particularly important for its contribution to the desirable flavor of cheddar cheese, especially in combination with diacetyl and butyric acid [23]. Increase in cystine content (Figure 1B) was observed in Strains Ld1, Ld2 and Lc8, with the highest content in Strain Ld1 [45.6 mg·(100 g)^-1], compared to the control. Cystine is the oxidized form of cysteine, which serves as a key metabolite for synthesizing most sulfur-containing cellular compounds. Cysteine is involved in protein folding, assembly and stability through the formation of disulfide bonds. Additionally, cysteine-containing proteins such as thioredoxin and glutathione play essential roles in protecting cells against oxidative stress [24]. The strain with the conditional designation (Lc8) is capable of synthesizing methionine and cystine. The levels of other amino acids were similar to or less than those of the control, suggesting their active utilization by microorganisms during growth.

3.2. Assessment of organic acid content

One of the fundamental criteria for selecting Lactococcus strains for starter culture consortia is their ability to produce acid. Organic acids such as acetic, lactic, citric, succinic and propionic acids are produced by LAB during carbohydrate metabolism. As intermediates in metabolic pathways, these acids enhance the taste of products and extend shelf life through their antimicrobial and antioxidant characteristics; thereby, increasing consumer value [25,26]. The results of assessment of the content of organic acids are presented in Figure 2.

All the samples showed a significant increase in lactic acid content, compared to the control (Figure 2). The highest levels were observed in Strains Ld1 and Ll1, reaching 8116.5 and 8677.8 mg·kg^-1, respectively. Lactic acid synthesis indicates the β-galactosidase enzymatic activity of these microorganisms. Lactic acid in fermented milk products acts as an acidity regulator and preservative. When ingested, lactic acid accelerates metabolism, decreases the number of pathogenic bacteria and also has antioxidant characteristics.

Only trace quantities of citric acid were detected in Samples Lc1, Lc3 and Lc5, which indicated the presence of a citrate pathway for the breakdown of this substrate. The consumption of citric acid depends on the presence of genes encoding the enzymes citrate permease and citrate lyase, which ensure the transport of citrate into the cell and its catabolism into oxaloacetate [27]. As a result of breakdown by lactococci, gas formation occurs in dairy products. In the production of certain products such as cheeses, this is addressed as a positive phenomenon, leading to the formation of "eyes." However, if its presence is assessed, this can lead to product defects. The breakdown of citric acid by these microorganisms leads to the formation of acetate, lactic acid, diacetyl and acetoin. Diacetyl and acetoin create flavor and aroma in food products, which is promising for the production of starters for butter, sour cream, yogurt and certain types of cheese.

Significantly, formic acid was detected in Strains Lc1, Lc2, Lc4, Lc6, Lc7, Lc8, Ll2, Ll3 and Ll4. In Lactococcus, formic acid is produced primarily by the pyruvate-formate lyase enzyme, which catalyzes the breakdown of excess pyruvate into acetyl-CoA and formate, which is the initial step in the formation of mixed-acid products. In the L. lactis genome, the genes responsible for this pathway are pfl, encoding pyruvate-formate lyase and pflA, a formate activator. Formate is formed with acetyl-CoA, which is consumed by the cell for energy needs. Formate itself can be excreted from the cell to maintain pH and redox balance [28]. In fermented dairy products, formic acid has little effect on the taste of the product, but it can serve as a preservative to preserve nutrients and it also has a bactericidal effect against pathogens.

Succinic acid was detected in Strains Lc4, Lc8, Ll3 and Ll4, with contents of 83.5, 89.9, 74.9 and 63.3 mg·kg^-1, respectively. This acid is synthesized via heterofermentative lactic acid fermentation alongside lactic acid. Succinic acid is formed upon activation of the reductive branches of the tricarboxylic acid cycle. In Lactococcus strains, the complete tricarboxylic acid cycle is inactive, but succinate can be synthesized through carboxylation of C3–C4 units as pyruvate or phosphoenolpyruvate is carboxylated to oxalo-acetate by pyruvate carboxylase or phosphoenolpyruvate carboxy-lase and then oxaloacetate is sequentially reduced to malate and ultimately converted to succinate [29]. In the human body, succinic acid promotes the synthesis of propionic and butyric acids in microbiota.

Acetic acid production was observed in many strains, with the highest contents recorded in Strain Lc4 and Lc6 at 1148.7 and 1129.8 mg·kg^-1, respectively. Acetate is formed during mixed fermentation from acetyl-CoA by phospho-acetyltransferase and acetate kinase. This results in the generation of energy in the cell in the form of ATP [30]. Acetic acid enhances the sensory characteristics of the fermented dairy products and contributes to microbial safety and shelf-life extension due to its antimicrobial activity.

3.3. Assessment of mono and disaccharide contents

Lactose, the primary carbohydrate in milk, is a disaccharide composed of glucose and galactose residues, hydrolyzed by lactase and β-galactosidase. Lactose metabolism in Lactococcus differs from that of other LAB by allowing simultaneous catabolism of glucose and galactose [9]. Galactose participates in the formation of glycoproteins and glycolipids, which are essential for cell-cell communication. However, its presence in food poses risks for individuals with galactosemia.

Another important disaccharide is lactulose, formed from lactose during high-temperature treatment of milk through isomerization involving hydrogen transfer from the second to the first carbon atom in the glucose residue. Lactulose content in milk serves as an indicator of thermal processing [31]. It is composed of D-fructose and D-galactose linked via a β-1,4-glycosidic bond and shows prebiotic effects at low concentrations. Previous research [32] demonstrated that the presence of lactulose improves the viability of starter cultures during storage, freezing and freeze-drying.

The analysis of monosaccharides and disaccharides (Figure 3) revealed a slight decrease in lactose content, compared to milk in all experimental samples. Particularly, lactulose was detected in all samples except Strains Ld1 and Ld4, including control. A decrease in lactulose content was observed in Samples Ld2, Ld3 and Ll1, compared to the control. The presence of lactulose can enhance consistency, increase starter culture viability and extend the shelf life of fermented dairy products. Galactose was detected in Strains Ld1, Ld2 and Ld3, with contents of 0.295%, 0.605% and 0.354% by mass, respectively. This indicated that the other strains—Lc1 through Lc8, and Ll1 through Ll4 and Ld4—were capable of galactose utilization. After transport into the microorganism, lactose contained in milk is hydrolyzed by β-galactosidase to glucose and galactose. Galactose can either be released by cells into the environment or undergo further metabolism into glucose-1-phosphate by four enzymes of galactokinase (GalK), hexose-1-phosphate uridine transferase (GalT), UDP-glucose-4-epimerase (GalE) and aldose-1-epimerase (GalM). These enzymes make up the Leloir pathway, whose genes can be present either alone (gal genes) or in combination with lactose metabolism genes (lac genes), making the final fermented products safer for individuals with galactosemia.

3.4. Assessment of vitamin contents

Based on the results of amino acid analysis, nine strains that showed a significant increase in methionine and cystine contents in samples were selected, compared to the control (Lc2, Lc4, Lc6, Lc8, Ll2, Ll3, Ll4, Ld1 and Ld2). These strains are of interest for possible inclusion in consortia for the development of products with specific functional characteristics. The selected samples were additionally assessed for vitamin C and vitamin B group (B2, B6, B7 and B9) contents. The results of vitamin C assessment in the samples are presented in Table 1.

As the studies have shown, the content of vitamin C in the samples did not exceed the control levels. The results of the assessment of B vitamins in the samples are presented in Table 2.

A number of foods, including milk, contain vitamin B2 (riboflavin). It is a key enzyme in redox reactions and essential for hemoglobin synthesis, maintaining the normal condition of the GIT mucosa and supporting nervous system function [33]. The ability of lactococci to synthesize riboflavin is limited, but some strains produce the compound in significant quantities [18]. In this study, only Strain Ld2 showed an increased level of vitamin B2 [103.18 μg·(100 g)^-1], compared to the control [79.97 μg·(100 g)^-1]. In the other samples, the content of riboflavin was at the control level or decreased.

Vitamin B6 plays a critical role in the normal functioning of the human body systems, including the digestive, cardiovascular and immune systems. It participates in hundreds of biochemical reactions, affecting metabolism. It is involved in the synthesis of neurotransmitters such as serotonin and dopamine, improves cognitive functions and promotes a positive mood [34]. It affects collagen synthesis and regulates hormonal balance in women. In the samples of industrially associated Lactococcus strains Lc6, Ll4, Ll3, Ll8 and Ll2, a significant content of vitamin B6 was detected, exceeding the control by 3–5 times. The vitamin B7 content in all samples was lower than that in the control.

Analysis of vitamin B9 content showed a significant increase in eight out of nine samples, ranging from 10.02 to 69.59 μg·(100 g)^-1, compared to 4.37 μg·(100 g)^-1 in the control group. Significantly, the Lc2 strain contained less than 1 μg·(100 g)^-1 of vitamin B9. Folic acid (vitamin B9) is essential for appropriate cell growth and division, acting as a coenzyme in nucleic acid synthesis. Folic acid deficiency can lead to serious fetal development disorders in pregnant women [35]. Vitamin B9 plays a critical role in the conversion of homocysteine, a substance associated with an increased risk of cardiovascular disease, into methionine. This compound is necessary for the synthesis of several neurotransmitters, prevents neurological damage, improves cognitive function and decreases the risk of certain types of cancer (breast, cervical and intestinal cancers) [36]. Receiving vitamins through foods is more physiologically beneficial for the human body than introducing chemically synthesized preparations. A comparative assessment of lactococci from the VNIMI collection, stored since 1960, was carried out for the regenerative capacity of LAB strains, as well as for the production-significant characteristics of these microorganisms such as fermentation activity, active and titratable acidity, viscosity and organoleptic indicators. Of the 40 Lactococcus strains, 39 showed metabolic activity regardless of storage time [37].

1. Conclusion

The study included metabolomic profiling of industrially associated Lactococcus strains based on the analysis of amino acids, organic acids, monosaccharides, disaccharides and vitamins. All strains produced lactic acid, with the highest content observed in Strains Ld1 and Ll1, at 8116.5 and 8677.8 mg·kg^-1, respectively. The experimental samples showed a significant increase in the content of methionine, with the highest content in Strain Ll3 and cystine in Strain Ld1. The content of other amino acids was similar to the control level or decreased. Strain Lc8 showed the ability to synthesize methionine and cystine, which may be promising in cheese production, since these sulfur-containing amino acids contribute to the aroma and flavor of fermented milk products. This strain produced formic, succinic and acetic acids and could catabolize citric acid and galactose. The Strains Ld1, Ld2 and Ld3 showed galactose accumulation, indicating lack of enzymatic activity for the oxidation of this monosaccharide. Literature data on significant variability in vitamin synthesis levels between the strains have been verified [18,20]. Thus, only Strain Ld2 synthesized vitamin B2, the quantity of which significantly exceeded that in the control and reached 103.18 ±2.06 μg·(100 g)^-1. Five strains were capable of synthesizing vitamin B6 up to 247.69 ±5.45 μg·(100 g)^-1. The vitamin B9 content increased in eight of the nine samples, with maximum values of 69.59 ±1.46 and 51.16 ±1.28 μg·(100 g)^-1 for the strains Ll3 and Ll4, compared to the control. For further formation of consortia, strains Lc1, Lc8, Ll2, Ll3, Ld1 and Ld2 were selected. The results can serve as a basis for the design of consortia of LAB in biotechnological approaches to produce novel fermented milk products with specific characteristics, including functional ones.

1. Acknowledgements

The authors would like to thank Elena Yurova and Nikolai Zhizhin for their assistance.

1. Declaration of competing interest

The authors report no conflict of interest (If authors have any kind of interest mention please clearly).

1. Authors’ Contributions

Conceptualization, I.R., N.P., V.L., V.S., S.K., V.M.; methodology S.K.; validation, S.K., V.S.; formal analysis, I.R., V.M., S.K..; investigation, V.S., S.K.; data curation, S.K., V.S; writing—original draft preparation, V.S.; writing—review and editing, V.L., I.R., V.S., S.K.; visualization, V.S.; supervision, I.R. N.P.; project administration, I.R, N.P.

1. Using Artificial Intelligent Chatbots

No using Artificial Intelligent Chatbots

1. Ethical Consideration

The ethics committee's opinion is not required in this work