Editorial


Exploration and Understanding of Beneficial Properties of Lactic Acid Bacteria: 10 years of experience in Applied Food Biotechnology

Svetoslav Dimitrov Todorov, Richard Weeks, Kianoush Khosravi-Darani, Michail Leonidas Chikindas

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e1
https://doi.org/10.22037/afb.v11i1.43665

The scientific community is currently facing a more than exponential increase of knowledge in all areas and disciplines. In the last 10 years, the contribution of the journal Applied Food Biotechnology was eminent in distributing that knowledge by providing a tribune for researchers from different countries all over the world to share their ideas, observations, and hypotheses. With a focus on different aspects of applied and fundamental biological sciences, the journal Applied Food Biotechnology was established as a reference of the Iranian scientific community. In the last 10 years, the journal has been covering several topics related to exploring the beneficial properties of lactic acid bacteria (LAB) and their role in modern food technologies. LAB are already proven as a realization of Hippocrates' vision for the potential role of food in human and other animals' health. However, what will be the next frontier? What are the challenges in understanding the interactions between microbiota and host microorganisms? What will be the novel analytical tools, facilitating a new era of probiotic research? These were only a few research topics presented and discussed in Applied Food Biotechnology in the last decade. This editorial overview aims to celebrate the scientific contribution of Applied Food Biotechnology in the area of research associated with the beneficial properties of LAB, summarizing some of the studies published in the journal.

In this editorial article, nationality of the authors from establishment of Applied Food Biotechnology from 2014 to the present time has been overviewed. The editorial board, especially chief editor, wish to make a broad global audience to spread knowledge of food biotechnology via publication of outstanding articles in this journal. At the beginning of the activity of journal office, a limited number of non-Iranian authors submitted their manuscripts to this journal; however immediately after the publication of the first issue, number of the foreign authors increased further, while they showed their satisfaction with the acceleration in peer-review processes of their manuscripts. From the published articles, probiotics has been the major scope; therefore, screening of the beneficial probiotics from various natural sources and their uses in prevention of diseases have been introduced by various authors. Thus, the most interesting findings of the authors have been introduced; through which, readers are further adapted to the journal priorities and preferences in probiotics and postbiotics. It is believed that invitation of prestigious authors and carrying out rapid peer-review processes are a key success to achieve high article citations and authors’ satisfactions.

Plants Extract and Essential Oil as Natural Preservatives in Foods: One-Decade Editorial Experiences

Khadijeh Khoshtinat, Zahra Beig Mohammadi

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e13
https://doi.org/10.22037/afb.v11i1.44605

The researchers are fronting the increasing of knowledge in extraction, application, and also antioxidant and antimicrobial properties of plant extracts and essential oils. In recent decade, the journal "Applied Food Biotechnology" has been established a channel for scientists all around the world to share their own hypotheses, results, and conclusions. As a peer-reviewed multi-disciplinary biotechnological publication, it covers several scopes which one important one is food microbiology. In this context, the journal has published several reports on food application of plant extracts and essential oils. The aim of this text is to determine the main categories of published articles in this context in the Journal of "Applied Food Biotechnology" and so on by editors. It seems that research tend to show the effective function of essential oils, as well as comparison of free and encapsulated forms as antimicrobial and antioxidant agents in food. With the aim of holding the potential to alleviate certain complexities, enhance yield, and simplify the isolation process of bioactive metabolites or their individual components, research has played a significant role in reducing production cost of essential oil and herbal extract.

Original Article


Design and Synthesis of a New Anticancer and Antimicrobial Nanocomposite by Microalgae Based on an Up-down Approach

Marjan Rajabi, Mahdi Rahaie, Hossein Sabahi

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e15
https://doi.org/10.22037/afb.v11i1.43923

 

Abstract

 

Background and Objective: Use of natural ingredients is a safe efficient approach to overcome various diseases. Encapsulated ginger extract has shown improved physicochemical characterizations, compared with that, the ginger extract has. In this study, a natural system integrated with ginger bioactive compounds (6-gingerol) and green microalgae of Chlorella vulgaris was reported to increase bioactive compounds medicinal effectiveness and introduce a novel food supplement.

Material and Methods: First, nanoparticles of microalgae were produced using ball-milling technique. Ethanolic ginger extract, loaded on microalga nanoparticles, was investigated at various pH values (2-7.4) to effectively release the active agents. Various analytical techniques (e.g., Fourier transform infrared, thermogravimetric analyses) were used to characterize the nanocomposite and investigate its anticancer and antimicrobial effects.

Results and Conclusion: Dynamic light scattering showed a medium size of 20.9 nm for the microalga nanoparticles. The release assay of ginger polyphenols showed a releasing process controlled by the pH. Fourier transform infrared, thermogravimetric analysis and differential thermal analysis revealed adsorption of ginger extract on nano Chlorella vulgaris surface. Moreover, 2,2-diphenyl-picrylhydrazyl bioassay results on the nanocomposite (GE@nano C.v) verified its significant antioxidant, antibacterial and anticancer activities. The nanocomposite has the minimum inhibitory effect on human breast adenocarcinoma cells and bacterial growth at 1 and 6.25 mg ml-1 concentrations, respectively. In brief, adsorption of ginger extract on the microalga nanoparticle surfaces enhanced physical and chemical characteristics of the ginger extract, compared to its free form. Bioactive compounds in Chlorella vulgaris and ginger extract strengthen their reported activities. Furthermore, microalgal nanoparticles could act as a safe carrier for the controlled release of 6-gingerol in addition to their nutraceutical characteristics. 

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

 

Article Information

 

Article history:

- Received

19 Nov 2023

- Revised

17 Jan 2024

- Accepted

17 Feb 2024

 

Keywords:

▪ Antitumor

▪ Food supplement

▪ Ginger

▪ Microalgae

▪ Natural nanomedicine

 

*Corresponding author:

 

Mahdi Rahaie *

Department of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran, Tehran 14399-57131, Iran.

 

Tel: +98-21-86093408

 

E-mail:

mrahaie@ut.ac.ir  

 

How to cite this article

Rajabi M, Rahaie M, Sabahi H. Design and Synthesis of a New Anticancer and Antimicrobial Nanocomposite by Microalgae Based on an Up-down Approach. Appl Food Biotechnol. 2024; 11 (1): e15. http://dx.doi.org/10.22037/afb.v11i1.43923

 

  1. Introduction

 

By decreasing consumption of essential nutrition, various chronic diseases emerge. This dilemma needs a reliable effective solution to prevent future malnutrition illnesses. Nowadays, plant and algal-derived supplies as functional foods are attracting public attentions due to healthful bioactive components and their therapeutic effects [1,2]. One of the wide subgroups of algae includes microalgae. Algal biomass includes diverse health-beneficial bioactive compounds such as fibers, carotenoids, polysaccharides, polyphenols and peptides that are resulted from various metabolic pathways and effective on cardiovascular diseases (CVDs), types of cancers, atherosclerosis, neurodegenerative diseases, obesity, gut health, bone health, inflammation, type II diabetes and antioxidant and antiviral activities [3,4]. In developing seaweeds and microalgal foods, aspects such as consumer awareness and demands, bioactive compound bioavailability and stability, cost-effectiveness and life durability needs further attentions. Moreover, only a few species of microalgae are approved for the human consumption as foods due to strict food safety regulations. These include Arthrospira platensis (spirulina), Chlorella spp., Aphanizomenon flosaquae, Schizochytrium spp., Scenedesmus spp., Dunaliella salina, Tetraselmis chuii, Haematococcus pluvialis and Porphyridium purpureum as resources of human nutrition within the microalgal species [5,6]. It has been reported that bioactive agents are critical for better health conditions. Therefore, plant phytochemicals have been selected as they include generally favorable characteristics such as less toxicity and well bearing by normal body cells. Frequently assessed bioactive compounds in plant extracts include curcumin, gingerol, β-carotene, quercetin and linamarin, which are developed as anticancer drugs. These compounds are rich in active agents such as phenols, alkaloids, flavonoids and tannins, which include high anti-inflammatory, antioxidant, antimicrobial, antica-ncer and antiaging activities [7,8].

One of the active agents of the ginger (Zingiber officinale) rhizome is found in the polyphenol group of 6-gingerol, which is popular for the protection of several cancers. The 6-gingerol mechanism includes interfering with a number of cell signaling pathways that affect balances between the cell proliferation and apoptosis [9]. These lead to effective approaches majorly for liver carcinoma and breast cancer. In addition, antiviral, antimicrobial, antihyperglycemic, antilipidemic, cardioprotective and immunomodulatory activities have been shown. However, 6-gingerol disadvantages such as pH and oxygen sensitivities, temperature lability, light instability and poor aqueous solubility limit its potential administrations. Thus, novel approaches for efficient delivery of 6-gingerol in a targeted controlled manner is critically important [10]. Drug delivery paths have been improved with developing of nanotechnology science. Appropriate drug-delivery nanosystems are used to enhance drug stability, specificity and durability in human blood circulatory system. Various drug vehicles have been introduced to improve drug efficiency and therapeutic effects. Some nanotechnology systems used for targeted drug delivery include dendrimers, micelles, carbon nanotubes, nanoparticles and liposomes, which are extensively used in various industries such as cosmetic, food, medicine, health, energy, electronics and environment industries [11,12]. Additionally, nanote-chnology has provided a path; in which, well-qualified and practical forms of foods with nutrient bioavailability can be produced. Moreover, most of the recent studies is dedicated to crop and food processing developments through nanotechnology [13].

One of the nanotechnology approaches in food technology includes a top-down approach that uses nanostructures from bulk materials by size decreasing via milling, nanolith-ography or accurate-engineering techniques. In contrast to nanostructures with a large surface area-to-volume ratio, this nano-approach induces a higher activity and enhances performance. Factors such as non-specificity, low stability and aggregation can delay these nanotechnologies' uses because of decreased functions. To extend stability of nanosize structures, associating a host material (as a matrix or support) can be an alternative way to overcome the former issues [13-15]. Ginger is an old additive and traditional medicine; previous studies on using ginger extract for therapeutic approaches have been less effective, compared with the loaded ones in a carrier due to less stability and low solubility. Therapeutic characteristics of this natural medicine are limited as previously stated. Moreover, Chlorella (C.) vulgaris is approved by the Food and Drug Administration (FDA) with nutritional and medicinal characteristics and potential of uses as a natural carrier. To enhance physiochemical characteristics of ginger extract, containing health-promoting bioactive compounds, as well as natural ingredient demands in food nutraceutical and medical industries, a nanocomposite was designed that was a novel approach to overcome disadvantages of using ginger extract alone. In this study, a nano-microalga was synthetized using up-down path as a carrier for ginger extract. Moreover, composite was assessed using several bioassays.

  1. Materials and Methods

2.1. Materials

Ginger rhizome was purchased from a local market in Tehran, Iran. Moreover, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, Gallic acid and sodium carbonate were purchased from Sigma-Aldrich, USA. Methanol (purity>99%) and ethanol (purity≥99.7%) were provided by Merck, Germany. Fresh C. vulgaris microalgae was provided by the Faculty of New Sciences and Technologies, University of Tehran, and cultured before use. All the chemicals included analytical grades and deionized water was used to prepare solutions.

2.2. Microalgae culture

Briefly, BG-11 liquid media were used to propagate C. vulgaris. Microorganism was inoculated at 10% using 500-ml flasks of 200 ml of BG-11. Culture flasks were incubated using rotary incubator at 25 ℃ ±2 and 100 rpm. Cells were harvested by centrifugation (Awel, model MF 20-R, France) at 8000 rpm for 10 min at 4 ℃. These were freeze-dried (Operon, model FDB 5503, South Korea) [16].

2.3. Ginger extraction

Ginger rhizomes were dried at room temperature (RT) and grounded using blender and then the fine powder was stored at -20 ℃ for further use. Extraction method (maceration technique) reported by Ali, et al. was used with modifications [17]. Generally, 5 g of the ginger powder were transferred into a 50-ml tube and extracted with 25 ml of 70% ethanol at 26 ℃ for 72 h using shaker-incubator. The hydroalcoholic extract was filtered through Whatman filter papers (Whatman, UK) to separate from the solid phase. Concentrated extract, achieved using rotary evaporator and freeze-drier, was stored at 4 °C until use.

 

2.4. Characterization of the ginger extract

2.4.1. Total phenol content of the ginger extract

To investigate total phenolic content of the extract, Folin–Ciocalteu method was used [18]. Gallic acid calibration curve was plotted using mixture of ethanolic solution of Gallic acid (1 ml) and Folin–Ciocalteu reagent (200 µl, 10× diluted) and sodium carbonate (160 µl, 0.7 M). Absorbance ratio of the solution was read using UV-Vis spectrophotometer (Thermo, WPA, Germany) at 760 nm. To assess the total phenolic content, ginger extract was mixed with the highlighted reagent and the absorbance ratio was measured with three replicates. Equation 1 was used for the calculation of total phenolic compounds as follows.

T = C ^ V/M                                                                                          Eq. 1

Where, T was the total phenolic content [mg g-1 sample extract in Gallic acid equivalents; C was the concentration of Gallic acid established from the calibration curve (mg ml-1 )]; V was volume of the extract (ml); and M was mass of the sample extract (g) [19]. Results were expressed as µg of ginger extract ml-1 of supernatant based on the calibration equation. Calibration equation of the ginger polyphenols (6-gingerol) was achieved via UV [absorbance value = 0.0507 (ginger polyphenols concentration in mg ml-1) - 0.0636 (R2 = 0.991)].

2.4.2. High-performance liquid chromatography analysis

Waters liquid chromatography apparatus, including a separation module (Waters 2695, USA) and a photodiode array detector (PDA) (Waters 996, USA) was used for high-performance liquid chromatography (HPLC) analysis. Data acquisition and integration were carried out using Millennium 32 software. Injection was carried out using auto-sampler injector. Chromatographic assay was carried out on a 15 cm × 4.6 mm with pre-column, Eurospher 100-5 C18 analytical column provided by Waters (sunfire) reversed-phase matrix (5 μm) (Waters, USA) and eluting was carried out in a gradient system with ACN as the organic phase (Solvent A) and distilled water (DW) (Solvent B) with a flow-rate of 1 ml min-1. Injection volume was 20 µl and temperature was set at 25 °C (run time, 40 min and columns size, 2.1 mm) [20].

2.5. Size decreases in nanoscale, dynamic light scattering and zeta potential

Nano-microalgae powder with nanometer dimensions was produced using ball-milling technique (600 rpm, 6 h). Ball-milling process was carried out at 27 ℃, to avoid excessive heating. Temperature was controlled using air-cooling system. After the process, samples were transferred into a closed container to prevent moisture. The most common technology for assessing particle sizes based on particle-light interactions is dynamic light scattering (DLS) technique. Assessment of the nano C.v size was carried out using particle size analyzer (Horiba, SZ100 model, Japan). A critically physical parameter to identify surface charges of a particle (microalgae, GE, nano C.v and GE@nano C.v) in suspensions, which could anticipate interactions, is zeta potential analysis (ζ). Technically, ions around the particles dispersed in the fluid regulate charges of the particle surface layer. In this study, zeta potential was assessed using Smoluchowski formula (Eq. 2) as follows.

Ζ = µ                                                                                                     Eq. 2

Where, η was viscosity of the media, ε was the permittivity and μ was the electrophoretic mobility [21]. In this study, zeta potential of the samples (ginger extract, micro C. vulgaris cell, nano C. vulgaris, GE@micro C.v and GE@nano C.v) homogenized in deionized water via ultrasound technique was assessed (Malvern, model ZEN 3600, UK).

2.6. Adsorption experiment

Ethanolic ginger extract was adsorbed onto C. vulgaris surface as a function of stirring time and ginger extract dosage. A suspension of 5 mg ml-1 nano C.v and 0.5 mg ml-1 ginger extract was stirred for 1, 2, 4, 6, 8, 10 and 12 h and then centrifuged (5000 rpm, 15 min). The harvested GE@ nano C.v (ginger extract on nanoparticles of C. vulgaris) was freeze-dried. As the adsorption yield curve (%) reached a plateau pattern after 1 h, this time was selected as the stirred time. Various quantities of GE (0.1, 0.2, 0.4, 0.6, 0.8 and 1 mg ml-1) were suspended in flasks containing 5 mg ml-1 nano C.v (dissolved in 70% ethanol), stirred for 1 h and then centrifuged (5000 rpm, 15 min). The harvested GE@nano C.v was freeze-dried. Polyphenol assessment (especially 6-Gingerol) of ginger extract adsorbed onto nano C.v was carried out as follows: 1 ml of the solution was collected and centrifuged (5000 rpm, 15 min). Supernatant was separated from the sediment and re-centrifuged (12,000 rpm, 10 min). Concentration of the GE was calculated at 760 nm, using UV-visible spectroscopy (Thermo, WPA, Germany) [16]. Encapsulation efficiency (EE%) and encapsulation yield (EY%) were calculated using Eqs. 3 and 4:

Encapsulation efficiency (%) =  × 100                                                Eq. 3

Encapsulation yield (%) =  × 100                                                Eq. 4

2.7. Verification of adsorption

To verify adsorption of ginger extract on nano Alg surface, Fourier transform-infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and differential thermogravimetric analysis (DTA) were carried out.

2.7.1. Thermogravimetric analysis

The TGA was carried out at a heating rate of 10 ℃ min-1 from RT to 600 ℃ with the inert nitrogen gas of 50 ml min-1 using thermal gravimetric analyzer (TGA/DSC/1, Mettler Toledo, Singapore).

2.7.2. Differential thermogravimetric analysis

The DTA analysis of the GE, nano C.v and nano Alg/GE was carried out to assess physical state of the extract in this carrier and possibility of the interactions between the GE and the microalga.

2.7.3. Fourier transform-infrared spectroscopy

Chemical characteristics of the material were investigated using FTIR spectroscopy (Perkin-Elmer Frontier, USA) using KBr disks in the range of 400–4000 min-1.

2.7.4. Antioxidant assay (total antioxidant capacity)

Photochemical stabilities of the nanocomposite (GE@ nano C.v) and ginger extract were assessed via a procedure described by Paramera et al. [22] with minor modifications. Free GE (200 mg) and a quantity of each nanocomposite (containing 200 mg of ginger polyphenols) were exposed to sunlight for a month using enclosed glass Petri dishes. Time of the light exposure included 12 h day-1 with a total of 720 h. The average daily temperature within the month was 25 ℃. After exposure for 7, 14 and 30 d, samples were collected and bioactivities of their 6-gingerol were assessed as follows: antioxidant activity of dispersed GE and nanocomposite (GE@nano C.v) were assessed using DPPH free radical scavenging method [23]. Inhibition of DPPH radicals by the samples was calculated using Eq. 5 as follows.

DPPH inhibition (%) =  × 100              Eq. 5

Where, A control was the absorbance spectrum without GE and A sample was the absorbance of ginger polyphenol nanocomposite.

2.8. In vitro assays

2.8.1. Antibacterial assay

Staphylococcus aureus ATCC 33591, Pseudomonas aeruginosa ATCC 9027, Salmonella enterica ATCC 9270 and Escherichia coli ATCC 10536 were selected for the antibacterial susceptibility assay. Minimal inhibitory concentrations (MICs) of the ginger extract and nanocomposite were assessed using microtube dilution assay as described by Acharya and European Committee for the highlighted bacterial strains. Ethanolic extract concentrations of 2.5, 1.25 and 0.625 mg ml-1 and nanocomposite concentrations of 1-4 mg ml-1 (consisting of ginger extract) were prepared using serial dilution in LB broth and 0.5 McFarland standard (108 CFU ml−1) of the bacterial suspensions was added to each tube. All tubes were incubated at 30 °C for 14 h at 100 rpm. Absorption of each sample was measured at 600 nm using UV-vis spectroscopy (Thermo, WPA, Germany). The lowest extract concentration that inhibited bacterial growth was recorded as MIC (in three independent experiments) [24]. Inhibition of the bacterial growth was calculated using Eq. 6 as follows.

Antibacterial activity (%) =  × 100                                                                                                                         Eq. 6

Where, Apositivecontrol was the absorbance spectrum without samples (ginger extract and nanocomposite) and Asample was the absorbance of bacteria with certain concentrations of ginger extract and nanocomposite. Culture media was used as blank.

2.8.2. Cytotoxicity assay

In this study, human breast adenocarcinoma MCF-7 cell line was cultured using T-25 cell culture flasks containing Hi-Gluta XL Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), streptomycin (100 µg ml-1) and penicillin (100 U ml-1). Cells were propagated using cell culture incubator supplied with 5% CO2 at 37 ℃. cells were seeded overnight using 96-well cell culture plates and RPMI media suspension of three treatments (ginger extract, nano C.v and GE@nano C.v) was added to the wells at a confluence of 70% to avoid possible interferes of DMEM media with the samples (200 µl total volume). The control wells included no agents. Experiments were carried out with three replicates. Effects of each treatment on cancer cell growth were assessed after 24, 48 and 72 h, separately. After treatment of cells with various concentrations of the samples (0.5, 0.75 and 1 µg ml-1) in each well for 24, 48 and 72 h, cells were rinsed with 1× PBS buffer and incubated with 100 µl of 0.5 mg ml-1 MTT at 37 ℃. After 4 h of incubation, 100 µl of DMSO were added to dissolve the dark-blue crystals of formazan (MTT metabolites) and incubated for 30 min at 37 ℃. Then, absorbance of the reduced MTT was measured at 560 nm using plate reader device (Convergys ELR 96×, Germany) [25].

2.9. Statistical analysis

Data represent mean ±SD (standard deviations) of three replicates. The mean comparisons were carried out using one-way variance analysis (ANOVA). Antioxidant activity data of nanocomposite and ginger extract were analyzed in a completely randomized block design with three replicates and three treatments (exposure to sunlight for 0, 14, 30 and 60 d). Duncan multiple range test (p<0.05) was used to assess significance of the difference within the treatment means. Data for each analysis were represented as the mean ±SE (standard error) of the mean.

  1. Results and Discussion

3.1          High-performance liquid chromatography analysis

The HPLC analysis was carried out to quantify 6-gingerol as a bioactive compound (polyphenol) with therapeutic characteristics as well as antioxidant activities. Chromatograms in Fig. 1b show spectra associated to ginger extract as well as the standard spectra of 6-gingerol (Fig. 1a). Peaks linked to 6-gingerol at 14.38 min were clearly present in the two spectra, showing 6-gingerol in the ginger extract. Using data, standard curve of 6-gingerol was first plotted and then quantity of 6-gingerol in the ginger extract was calculated as 96 mg g-1 DW (dry weight) extract (Fig. 1 c). This value was higher than that by Yamprasert et al. (2020), where concentration of 6-gingerol in the extract was calculated as 71.13 mg g-1 using soaking method [26]. Compared to the method by Simonati et al. (2009) that calculated 170 mg g-1, the former value was lesser, which could be due to the use of supercritical fluid[1] extraction technique and high pressure for extraction in that study [27].

3.2. Release assay

Release assessment of 6-gingerol in the ginger extract was carried out based on the absorbance at 760 nm in the simulated environment of body conditions with pH 7.4 using Folin-Ciocalteu reagent at various times. Since extract was loaded on nanoparticles of the microalgae, further releases were expected at the beginning. At t = 1 h in the acidic conditions similar to the stomach with pH 2, the release rate was 16% and at pH 7.4, the rate was 14%. Up to 72 h, the release rate was constant with mild changes and reached 18% (Fig. 2). Complete release of the ginger extract was not observed at no pH conditions. However, release of the extract in acidic conditions of the stomach was higher, which was due to the effects of acidic pH on interactions between the algal nanoparticles and the extract as well as the surface load. According to Zarei et al., release rates of gingerol loaded in pegylated and non-pegylated nanoliposome respectively were 3.1 and 4.8% within 48 h [28]. Release rate in report of Jafari et al. was nearly 39% within 2 h and 59% within 4 h [16]. In another study by Shateri et al., release comparison of two synthetic systems (BCE-Spirulina and BCE-nanosized Spirulina) was investigated in physiological (pH 7.4) and acidic (pH 1.2) conditions within 96 h. Release rates of the extract were respectively calculated as 50 and 40% within the first 12 h. Based on the results, release of extract from the nanocomposite was faster and higher at the two pH conditions because of loading further extracts on the Spirulina surface, compared with the microalgal whole cell. The plateau curve was seen after 48 h, which reached 50% in acidic pH [29].

3.3. Dynamic light scattering and zeta potential assessments

To assess sizes of the ground C. vulgaris nanoparticles, an appropriate quantity of the microalgal nanoparticles was first dissolved in DW and sizes of the particles were assessed after sonication for 15 min. Sizes of Chlorella nanoparticles included 20.9 nm. Sharp peak and high height of the peak ndicated the high number of the particles (90%) in this size range (Fig. 3). Furthermore, C. vulgaris included three growth phases of log, stationary and lag phases. In the early log phase, a fragile unilamellar layer with 2-nm thickness could be demonstrated. Through maturation (log phase), cell wall thickness gradually increased to 17-20 nm, forming a microfibrillar layer of glucosamine. Due to the variation of cell wall rigidity of C. vulgaris within the growth phase, appropriate digestion and absorption of the valuable nutritional substances were limited [30]. Therefore, nano-sized microalgae were used not only to overcome problems of the ginger extract adsorption onto the Chlorella surface, but also to enhance drug loading efficiency of the synthetic nanocomposite.

Naturally, surface groups, extracellular products and cell structures affect electric characteristics of cells. In physiological pH, zeta potentials of the ginger extract (GE), C. vulgaris (microalgae), nanoparticles of C. vulgaris (nano C.v), microcomposite (GE@micro C.v) and nanocomposite (GE@nano C.v) included -22.8, -16.8, -27.2, -4.3 and -9.3 mV, respectively (Fig. 4). Based on the results, the lowest quantity of the extract could be loaded on the particles of C. vulgaris, this occurred due to the negative surface charge and repulsion in loading process. The whole cell of C. vulgaris included a negative surface charge of -16.8 mV; similar to that of Hao, et al., which linked to cell wall compositions of majorly cellulose, hydroxyl(-OH), carboxyl(-COOH) and aldehyde(-CHO) groups with negative charges [31]. Surface charge of the nanocomposite was -9.35 mV, becoming further positive and closer to the charge of the extract that indicating better adsorptions of the ginger extract on the microalgal nanoparticles. Zeta potential of the synthesized nanocomposite could be addressed as the reason for the low adsorption of ginger on the microalgal surfaces. Results showed that by shrinking the size of C. vulgaris particles from micrometers to 20 nm (less than 100 nm) with effects on physical characteristics, its surface charge became further negative and closer to -30 mV; thus, its stability increased due to the increases in the ratio of surface to volume [32]. Moreover, surface charge of the ginger extract was -22.8 mV, similar to that reported by Min Ho et al. This was because of the presence of carbohydrates and numerous anion sites, lipids and polyphenols due to possible groups in the extract [33].

3.4. Fourier transform-infrared spectroscopy analysis

Figure 5 shows FTIR graphs of ginger extract, microalgae and nanocomposite (GE@nano C.v), to indicate chemical groups in each treatment individually and characterize their interactions in synthetized nanocomposite.

 

 

A

 

 

 

B

 

 

 

C

 

 

 

Figure 1. High-performance liquid chromatography peaks corresponding to 6-gingerol standard (260 ppm) (a), ginger extract (b) and 6-gingerol content of the ginger extract (c).

 

 

Figure 2. Release rate curves of the ginger extract from nanocomposite at two acidic (red line) and neutral (black line) pH values.

Figure 3. The size measurement of Chlorella vulgaris particles after milling by DLS technique. Most of the particles' sizes are in the average of 20.9 nm.

 

Figure 4. Surface charges (zeta potentials) of the four samples. GE (orange), Chlorella vulgaris whole cell (green), Chlorella vulgaris nanoparticles (green pattern), microcomposite (blue) and nanocomposite (light blue pattern).

 

 

3.4.1. The ginger extract

 

The strong band located in the area of 3527.24 was linked to alcohol groups (O-H) and the presence of intramolecular and intermolecular hydrogen bonds; which could be associated to carbohydrates and polyphenols in the extract. The peak in the area of 2916.69 showed asymmetric stretching vibration of the C-H group and possibly the carboxyl group [34]. In addition, FTIR spectrum of the ginger included a relatively strong peak at 1606.5 , which was in the range of 1600–1609 and linked to the ring of polyphenols. The absorption spectrum in 1515.8 belonged to the nitro-aromatic groups of ginger extract. The broadening of the peak in the area of 1500–1800 indicated double and amide bonds. Moreover, C=O stretching bonds and N-H bending bonds when showing a broad peak in the range of 1516-1863.86 centered on the 1638  area. Presence of a 1268.13 was associated to the vibrations of alkyl ether and ester groups, demonstrating presence of triglycerides in the extract [35]. Furthermore, peak of 1040.10  included bending vibrations of the C-C bond of cellulose extract [33]. Absorption bands in the area of 924.45 were linked to carbon-carbon double bonds [36].

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 5. Fourier transform-infrared spectroscopy spectra of the ginger extract (GE, orange), nanocomposite (GE@nano C.v, blue) and nano Chlorella vulgaris (nano C.v, green).

 

 

3.4.2. Microalgae

Spectrum of C. vulgaris microalgae showed several peaks in various areas. The band of 3316.01  corresponded to the O-H stretching group, revealing presence of a strong alcohol group. Area of 1659.46  showed a strong band, sign of the presence of the first type of amide group within the proteins. Band of 2927.12 demonstrated presence of lipids in microalgae [37]. Band in the area of 2855.44 showed the bending CH2 group linked to carbohydrates and lipids. Band in the area of 1738.96 cm-1 highlighted ester bond of the carbonyl group. Peak in the area of 1547.97 belonged to the proteins and C=O stretching group. Presence of peaks in the areas of 1406.05 and 1242.77 was linked to the carbon-carbon bending group and the carboxylic acid group of microalgae, respectively. Peak of 1072–1099  was associated to -O-C group of carbohydrates, nucleic acids and other phosphate-containing compounds. Moreover, peak of 980–1072 belonged to the group (C-O-C) of polysaccharides [16,38].

3.4.3 Nanocomposite

Nanocomposite spectrum was located between the FTIR spectra of extract and microalga with further similarity to the extract, which could be attributed to loading of the extract on the microalgae. In FTIR spectra of the nanocomposite, bands were removed, replaced or decreased in depth. Band in the range of 3500–3300  of the spectra of the extract and microalga nanoparticles shifted to the left (3667.23 ) in the spectra of the nanocomposite, revealing connections of the extract with microalga. Lack of 3527 peak of the extract could be due to the interactions of O-H group of phenol with microalga. In contrast, 2927 peak of Chlorella became wider in the present nanocomposite, which was possibly due to the interactions of microalga with the extract. The microalga peak shifted from 1738 to 1728.65 and the extract from 1515 to 1534.94 in the nanocomposite could be due to the interactions of microalgal proteins with the extract through C=O and N-H groups, respectively. Peak in 1047  area of the extract could be seen in the spectra of the nanocomposite. Peaks between 924 and 564 cm-1 in the extract and peaks between 1242 and 582 cm-1 of Chlorella in the nanocomposite were removed, demonstrating interactions between the extract and microalga and the formation of C-C and C-O single bonds.

3.5. Thermogravimetric analysis assay

Investigating thermal stability (Fig. 6), graph of microalga showed its three-stage mass losses due to the pyrolysis process (25-600 ℃), which ultimately led to 70% decreases in the mass of microalga. Biomass's type and composition could change the TGA curve. In the first stage, mass losses occurred due to dehydration [16]. Nearly 4.65% of the microalga mass decreased as water evaporation up to 117 °C occurred (moisture loss). Small changes in the slope of the graph at 117-201 °C could be due to the beginning of the decomposition of compounds such as hemicellulose and carbohydrates. By increasing the temperature to values higher than 117 ℃, the second stage of biomass degradation began with a sharp slope (major zone).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 6. Thermogravimetric analysis of the three samples: nano C.v (green), nanocomposite (blue) and ginger extract (orange).

 

 

Hence in the temperature range of 201–379 °C, nano C.v biomass quickly decreased by 46%, which could be caused by decomposition of the components. Volatile compounds such as organic materials, proteins, lipids, carbohydrates and cellulose and at higher temperatures (up to 379 °C), lignin, carbon materials and minerals (nearly 12% wt), were decomposed slowly [16,39,40]. With a low slope of the graph (third stage of 400–600 ℃), all volatile components of nano C.v were removed and non-volatile components were destroyed left as amorphous carbon forms [39]. In ginger extract graph, the mass curve decreased gradually. At 100 ℃, the first peak corresponded to the water loss (6.46%) [41]. The second weight loss occurred at 142.8–172.4 ℃, which represented decomposition of several aromatic compounds [42]. In the third stage (172.4-241.6 ℃), organic compound degradation occurred in decomposition of organic materials [43]. The last and the major step of mass loss occurred at 241.6-600 ℃ (36.6%) due to organic compound oxidation, lignin and cellulose degradation and complete decomposition [43]. The second and the third stages of GE degradation occurred at 142–241 ºC, while this range for GE@nano C.v was 210-300 ºC, meaning that synthesize of nanocomposite (GE@nano C.v) led to thermal stability of the ginger extract up to 300 ℃. In contrast, onset of the destruction temperature of microalgae was at 200 ℃. In nanocomposite graph, degradation temperature began at 150 ℃, showing that when the extract was loaded on C. vulgaris, degradation began earlier. As previously reported by Shetta et al. [44], green tea extract on the surface of chitosan nanoparticles was destroyed faster. This higher thermal stability of GE after adsorption on nano C.v is similar to that of Jafari et al. study, which demonstrated that thermal stability of curcumin was improved after its encapsulation into C. vulgaris [16]. Therefore, thermal degradation of organic compounds (the major step) in the nanocomposite occurred at higher temperatures, compared to those in ginger extract and nanomicroalga (two degradation steps). This could address improved intramolecular interactions between the ginger extract chemical groups and nanomicroalga that suggested a natural nanocomposite with enhanced thermal characteristics.

To calculate the quantity of ginger extract adsorbed on C. vulgaris surface, Eq. 7 by Shateri et al. [29] was used as follows.

Calculated mass residue =    Eq. 7

Where, x was the load quantity (%). Based on the formula, quantity of the extract loaded on the microalga was 31.64%. Table 2 shows weight decompositions (%) of the three samples.

3.6. Differential thermal analysis

The DTA curve shows thermal difference analysis. The technique calculated differences between the sample temperature and the reference temperature. The DTA graph shows a better temperature difference, compared with the TGA curves [49]. As shown in Fig. 7, DTA curves showed degradation peaks of nano CV, GE and GE@nano CV composite. In nano C.v, a calorific peak was seen at 100 °C, revealing dehydration and water loss. A strong second peak and a weak peak at 200 °C specify thermal decomposition of various Chlorella compounds and beginning of their degradation.

 

 

 

Table 1. Comparison of the current study with similar studies using various drug carriers and their effects.

Carrier

Cargo

Loading (%)

Positive effects

Reference

Spirulina platensis

Doxorubicin

85

+ Anticancer efficacy on lung metastasis

Biodegradable

Improved fluorescence imaging

[45]

Spirulina platensis

Black cumin

81.94

+ Antibacterial activities

+ Antioxidant activities

+ Anticancer activities

+ Thermal stability

[29]

Chlorella vulgaris

Curcumin

55

+ Thermal stability

+ Photostability

[16]

Chlorella pyrenoidosa cells

Lycopene

96.31

+ Stability

+ Antioxidant activity

[46]

Polymeric micelles (TPGS/PEG-PCL)

6- Gingerol

79.68

+ Solubility of 6-gingerol

+ Oral bioavailability

+ Improved brain distribution

[47]

Nanostructured lipid carriers

6- Gingerol

76.71

Higher drug concentrations in serum

+ Solubility of 6-gingerol

+ Oral bioavailability

[48]

Chlorella vulgaris

Ginger extract

31.64

+ Antibacterial effect

+ Antioxidant activities

+ Anticancer activities

+ Thermal stability

This work

 

Table 2. Thermogravimetric analysis assay with weight decreases as the temperature changes for the three samples (nanocomposite, nano C.v and GE).

Samples

Temperature range (℃)

Weight loss (%)

Nanocomposite

25.00-157.88

5.77

 

157.88-600.70

63.43

Nano Chlorella vulgaris

24.50-117.31

5.46

 

117.31-201.39

4.90

 

201.39-379.55

46.65

 

379.55-600.77

12.94

Ginger extract

24.60-142.8

6.46

 

142.8-172.4

6.39

 

172.4-241.62

18.26

 

241.62-600

36.69

 

 

 The fourth strong heating peak was observed at 300 °C, which included almost destruction of the microalga [39]. In DTA curve of GE, three strong peaks were seen in the temperature range of 100-200 °C (respectively from left to right: endothermic, endothermic and exothermic); which belonged to dehydration, destruction and decomposition of the major compounds in the ginger extract. Other exothermic and endothermic peaks were seen in the range of 200-300 °C. Residual and volatile compounds and carbohydrate depolymerization of the extract were almost completely decomposed, based on the studies by Kuk et al. [50].

Diagrams of DTA of GE@nano C.v were similar to diagrams of nano C.v, showing low loads of the extract. The two peaks of nano C.v degradation at 200 and 300 °C decreased to one peak at 250 °C. This major change in the decomposition peak of nano C.v showed that GE absorption on microalga nanoparticles was so strong that caused major changes in the rate and point of its decomposition. Diagram of the nanocomposite is plotted to the left, compared to that of the extract. Interactions between the extract and the microalga decreased thermal resistance in the range of 200-300 °C, which could be caused by weak interactions. In contrast, heat flow of the microalgae and its mild changes could be due to the low superficial loading of the extract on surface of the microalga as well as weak connections caused by it. It could be interpreted at a heating rate of 10 min/℃ in the first stage of the heating process with a peak at 55 °C with a molecular weight (MW) of 0.09 and a second peak of heat removal at 114 °C with an MW of 0.05 possibly due to the water loss. Thermal decomposition in the second stage was exothermic with two weak peaks at 236 °C and a strong peak at 297 °C with MWs of 0.2 and 0.35. This is the maximum heat loss due to protein decomposition at the top of the peak and carbohydrates, where bonds included O-O, N-O, C-N, C-C, C-O, N-H, C-H, N=N, H=H, O-H, O=O, C=C, C=N and C=O.

 

 

Figure 7. Differential thermogravimetric analysis diagram of nano C.v, ginger extract and nanocomposite.

 

 

Following the thermal decomposition curve, gasification occurred in the third stage with a weak peak at 370 °C and MW of 0.1. These results were similar to the results of Wang et al. [40]. In the third stage, only non-condensable gases such as CO, CO2, H2 and CH4 were released with a little weight loss. This might reflect that the necessary heat was directly proportional to the increased temperature, as almost all the volatiles in the microalga were released in the thermal decomposition zone. This could be understood due to the soft structure of microalga with a relatively low lignin concentration (7.33-9.55% wt), thus volatile substances were easily separated from the tissues. Compared to hardwoods with high lignin contents (25%) and complex textures, thermal decomposition of hardwoods over 600 °C still produced non-condensable gases, explaining its relatively high weight losses.

3.7. The 2,2-diphenyl-1-picrylhydrazyl  test

Ginger increases blood plasma antioxidant capacity and decreases lipid peroxidation and renal nephropathy in rats. Gunathilake et al. detected that 6-gingerol in ginger extract removed peroxide radicals and hence could be used as a natural food additive and a substitute for artificial antioxidants [51]. Antioxidant activities of the ginger extract and nanocomposite (GE@nano C.v, loaded with 0.5 mg mg ml-1 GE) were investigated for one month. As shown in Fig.8, antioxidant activity of the nanocomposite on the first day was nearly 60% at a concentration of 1.5 mg ml-1. This increased by ~2% within one month as a result of antioxidant characteristics of Chlorella, when GE adsorbed on the surface within the first week and then made the antioxidant agents of C. vulgaris available. Karaman et al. detected that the higher activity of inhibiting free radicals for the sample encapsulated in yeast cells was associate to the higher anti-radical contents of the yeast cells [52].

In a study by Ghasemzadeh et al., antioxidant activity of the methanol extract of ginger was reported as 51-58% at a concentration of 40 µg ml-1[53], possibly due to differences in the extraction method and various bioactive contents. In a study of Stoyanova et al., antioxidant activity of the ginger extract at 20 µg ml-1 was calculated as 90% [54]. In the study of Abdel Karim et al., C. vulgaris showed a 50% antioxidant activity with a concentration of 1.95 mg ml-1 due to the presence of plant chemicals (e.g., phenols, flavonoids, etc.) [55].

3.8. Antibacterial assay

Antibiotics are the compounds, which are used for human, animal and aquaculture treatments. Recently, their residues and degraded products in environment have included potential risks and toxicity worldwide. Antimicrobial activity against pathogenic and food-spoiling microorganisms is due to the bioactive compounds. Phenolic compounds such as gingerol and shogaol and their relationships majorly with other compounds such as β-sesquiphalendrene, cis-caryophyllene, zingiberene and α-farnesin are responsible for their antimicrobial activities in ginger essential oil and extract [56]. Hydrophobic residues of the ginger extract may interact with the lipophilic part of the cell membrane, disrupting their membrane integrity and function (e.g., electron transfer, nutrient absorption, protein and nucleic acid synthesis and enzymatic activity). In this study, results of the MIC assay showed that the ginger extract (Fig. 9a) and nanocomposite (Fig. 9b) included antibacterial charact-ristics and rates of their inhibition depended on their doses. By decreasing concentrations of the extract and nanocomposite, their antibacterial activities decreases. Concentrations of 1, 2 and 4 mg ml-1 of the extract and 6.25, 12.5 and 25 mg ml-1 of the nanocomposite were investigated.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 8. Antioxidant diagrams of the ginger extract (GE) and nanocomposite at concentrations of 0.5 and 1.5 mg ml-1, respectively. All data are expressed as the mean ±SEM. Statistical analysis was carried out using ANOVA (letters, p < 0.05). Charts with similar letters include no significant differences.

 

 

 

 Results of the antibacterial assay showed that absorption of the extract on the nanoparticle significantly decreased its antibiotic characteristics, which could be linked to the slow releases of the extract active compounds from the surface of the nanoparticles; however, this could be active for a longer time. Antibacterial activity of the ginger extract was due to its polyphenol compounds, especially zingibrene, gingerol and terpenoids [57]. Based on the study of Hussein et al., E. coli and S. aureus were susceptible to the active compounds of C. vulgaris at a high concentration of 100 mg ml-1. This could be due to the fatty acid contents, bioactive compounds, effects on cell cycle and protein and DNA syntheses or hydrophobic interactions that ultimately lead to cell leakage and death [58]. Inhibitory effects of the ginger extract on S. aureus, Pseudomonas spp. and E. coli was estimated as 90%. Inhibitory effects of nanocomposite on Salmonella spp. was higher than 60% and its effects on Pseudomonas spp. reached the maximum, increasing by ~6% for S. aureus to 50%. These differences could occur due to the structural differences in these bacteria [59].

3.9. Cytotoxicity assay

Ginger compounds (6-gingerol and its derivatives) have been shown to decrease hazards of several diseases, majorly in the gastrointestinal tract (GIT) and cancers such as carcinogenesis in the skin and breasts [60]. To assess cell viability of the breast cancer cell line (MCF-7) for their ability to decrease tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide], various concentr-ations of GE, nano C.v and GE@nano C.v within 24, 48 and 72 h were investigated (graphs of 24 and 48-h assays not shown). Results showed outstanding cancerous-cell inhibitions by the nanocomposite (GE@nano C.v), compared to ginger extract and nano C.v. Based on the growth inhibition chart of the MTT assay in Fig. 10,

growth inhibitions of 67, 73 and 86%, were respectively reported after 24 h of incubation (37 ℃, 98% humidity, 5% CO2), with ginger extract at concentrations of 0.5, 0.75 and 1 mg ml-1,. After 48 h, this value reached 28, 46 and 84%, respectively, which indicated dose and time-dependent cytotoxicity of the three treatments, as the more concentration, the higher growth inhibition. Cell viabilities were inhibited by the ginger extract treatment during 48 h. After 72 h of adding GE, cytotoxicity effects on the breast cancer cells for the concentrations of 0.5, 0.75 and 1 mg ml-1 were 47, 48 and 47%, respectively. It was demonstrated that anticancer activity of the ginger extract decreased within 72 h. Various studies have shown effectiveness of the ginger extract on tumors. However, the extract not only includes therapeutic characteristics (due to 6, 8 and 10-gingerol and 6, 8 and 10-shogaol agents) but also decreases vomiting and nausea of the patients after chemotherapy [61]. In a study by Mohammed et al., further gingerol concentrations caused higher effects on cancer cell growth inhibition as 0.1 mg ml-1 gingerol included significant cytotoxicity for 24 h and 0.4 mg ml-1 gingerol showed 80% inhibition on MCF-7 cell line [62].

As previously stated, microalgae include bioactive compounds such as phytochemicals and carotenoids (lutein in C. vulgaris) with verified anticancer activities [63,64]. Cytotoxicity rates included 82, 75 and 76% after 24 h of treatment with nano C.v at concentrations of 0.5, 0.75 and 1 mg ml-1 that reached to 96, 77 and 67% after 48 h, respectively.

 

A

B

 

 

Figure 9. Graphs of the average inhibitory proportions of the ginger extract (GE) (a) and nanocomposite (b) on four bacterial strains of Salmonella typhi (black), Staphylococcus aureus (gray dotted black pattern), Pseudomonas aeruginosa (light gray-black pattern) and Escherichia coli (gray-black pattern) using MIC assay in three various concentrations with controls. Significant differences (mean ±SE) of the effects of concentrations on the bacteria is illustrated with letters at p <0.05.

 

 

 

 Growth rates of the cancer cells at 0.5, 0.75 and 1 mg
ml-1 respectively included 62, 62 and 67% after 72 h. This showed mild increases at 72 h, which could be due to the limited anticancer compounds in the cell wall of C. vulgaris microalga after grinding. Anticancer activities of the nanocomposite included 63, 63 and 71% after 24 h at 0.5, 0.75 and 1 mg ml-1, respectively. After 48 h from the addition of nanocomposite, these values included 74, 71 and 59% for the concentrations of 0.5, 0.75 and 1 mg ml-1 with significant changes, compared to those after 24 h, respectively. After 72 h at the concentrations of 0.5, 0.75 and 1 mg ml-1, growth rates of the cancer cells included 51, 54 and 53%, respectively. The maximum inhibitory effect could be seen at 1 mg ml-1 concentration of the nanocomposite due to the increases of ginger extract concentration adsorbed on nano C. vulgaris.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 10. Cell viabilities of the cancer cells (MCF-7 cells) after incubation in 0.5, 0.75 and 1 mg ml-1 ginger extract, nanoparticles of Chlorella vulgaris and nanocomposite (GE@nano C.v) with different significant values at p<0.05.

 

 

 

Anticancer activity of the ginger extract alone decreased after 72 h. However, activity of the nanocomposite increased and was set constantly, demonstrating preservation of the activity of ginger extract adsorbed on the surface of microalga. This is a significant advantage for the use of nano C.v as a carrier for GE.

  1. Conclusion

In this study, the synthesized nanocomposite included promising characteristics, which could be used in medicinal and nutritional industries. The HPLC assay showed bioactive compounds, especially 6-gingerol, in the extract (0.48 mg in 5 mg total extract). Furthermore, FTIR, TGA and DTA results verified adsorption of ginger extract on nano Chlorella surface. Due to intramolecular interactions of ginger with Chlorella chemical groups, improved thermal stability in range of 200-300 ℃ was observed. In addition, nanocomposite included a mild improved antioxidative activity within a month with 1.5 mg ml-1 concentration. Antimicrobial and anticancer assays indicated good effects on microbial and breast-carcinoma cell-line (MCF-7) growth at concentrations of 6.25 and 1 mg ml-1, respectively. In conclusion, combination of ginger and C. vulgaris could improve further antioxidant, antimicrobial and antitumor effects of ginger extract, compared to ginger extract alone. Further studies are needed to focus on novel methods for increasing adsorption of ginger extract on C. vulgaris cell wall to enhance nanocomposite stability.

  1. Acknowledgements

The authors acknowledge Prof. Ali Reza Zomorodipour from NIGEB, Iran, for his support to provide research instruments of cell culture. In addition, authors acknowledge the instrumental supports of the University of Tehran.

  1. Conflict of Interest

The authors declare no conflict of interest.

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Research Article

Applied Food Biotechnology, 2024, 11(1):e15

Journal homepage: www.journals.sbmu.ac.ir/afb

pISSN: 2345-5357

eISSN: 2423-4214

طراحی و ساخت  یک نانوکامپوزیت جدید ضد سرطان و ضد میکروب با استفاده از ریزجلبک‌ و مبتنی بر یک رویکرد بالا به پایین

مرجان رجبی، مهدی رهایی*، حسین صباحی

گروه مهندسی علوم زیستی، دانشکده علوم و فناوری های نوین، دانشگاه تهران، تهران، ایران

تاریخچه مقاله

دریافت 19 نوامبر 3202

داوری 17 ژانویه 2024

پذیرش 17 فوریه 2024

 

چکیده

سابقه و هدف: استفاده از مواد طبیعی روش کارآمد و ایمنی برای غلبه بر بیماری­های گوناگون است. نشان داده شده است که ویژگی­های فیزیکوشیمیایی عصاره زنجبیل ریزپوشانی شده در مقایسه با عصاره آزاد، بهبود یافته­ است. این مطالعه، یک سامانه طبیعی حاوی ترکیبات زیست فعال زنجبیل (6 -جینجرول) و ریزجلبک سبز کلرلا ولگاریس، ترکیبات زیست فعال با اثربخشی دارویی بیشتر و مکمل غذایی جدید را معرفی می­کند.

مواد و روش ها: ابتدا، نانوذرات ریزجلبک با روش آسیاب گلوله­ای یا گوی­آس[2] تولید شدند. عصاره اتانولی زنجبیل، بارگذاری شده بر روی نانوذرات ریز جلبک، در pH  های گوناگون (4/7-2) مورد بررسی قرار گرفت تا عوامل فعال رهایش موثری داشته باشند. روش­های تحلیلی گوناگونی (به­عنوان مثال، تبدیل فوریه مادون قرمز[3]، تجزیه و تحلیل گرما وزن­سنجی[4]) برای توصیف نانوکامپوزیت و بررسی اثرات ضد سرطانی و ضد میکروبی آن استفاده شد.

یافته‌ها و نتیجه­گیری: اندازه نانوذرات ریزجلبک با روش پراکندگی نور دینامیک به­طور متوسط 9/20 نانومتر تعیین شد. بررسی رهایش پلی­فنول­های زنجبیل نشان داد pH فرآیند رهایش را کنترل می­کند. تبدیل فوریه مادون قرمز، تجزیه و تحلیل گرما وزن­سنجی و آنالیز حرارتی افتراقی[5]، جذب عصاره زنجبیل بر سطح نانو کلرلا ولگاریس نشان داد. علاوه بر این، نتایج تعیین مقدار زیستی[6] 2،2-دی فنیل-پیکریل هیدرازیل بر روی نانوکامپوزیت (GE@nano C.v) فعالیت­های قابل­توجه ضداکسایشی، ضد باکتریایی و ضد سرطانی آن را تأیید کرد. این نانوکامپوزیت به­ترتیب در غلظت­های 1 و 25/6 میلی­گرم بر میلی­لیتر، کمترین اثر بازدارندگی را بر روی سلول­های آدنوکارسینومای پستان انسان و رشد باکتری­ها دارد. به­طور خلاصه، جذب عصاره زنجبیل بر سطوح نانوذرات ریز جلبک، ویژگی‌های فیزیکی و شیمیایی عصاره زنجبیل را در مقایسه با فرم آزاد آن افزایش داد. ترکیبات زیست فعال موجود در کلرلا ولگاریس و عصاره زنجبیل فعالیت­های آنها را تقویت می­کند. علاوه بر این، نانوذرات ریزجلبک می‌توانند علاوه بر ویژگی‌های تغذیه‌ای، به عنوان یک حامل امن برای رهایش کنترل‌شده 6-جینجرول عمل کنند.

تعارض منافع: نویسندگان اعلام میکنند که هیچ نوع تعارض منافعی مرتبط با انتشار این مقاله ندارند.

واژگان کلیدی

▪ ضدتومور

▪  مکمل غذایی

▪ زنجبیل

▪ ریزجلبک

▪ نانو داروی طبیعی

 

*نویسنده مسئول

مهدی رهایی

گروه مهندسی علوم زیستی، دانشکده علوم و فناوری های نوین، دانشگاه تهران، تهران، ایران

تلفن: 811118583-62+

پست الکترونیک:

mrahaie@ut.ac.ir  

 

 

 

[1] SC-CO2

[2] Ball-milling آسیابی که در آن از گلوله‏های فولادی یا سرامیکی برای خرد و نرم کردن مواد غیرفلزی استفاده کنند

[3] Fourier transform infrared (FT IR)

[4] Thermogravimetric   روشی تحلیلی بر مبنای اندازه‏گیری تغییرات وزن ترکیب یا آمیزه با افزایش دما 

[5] Differential thermal analysis

[6] Bioassay   تعيين قدرت يا غلظت يك مادة دارويي ازطريق سنجش اثرات آن بر بافت‌هاي زنده

 

Antibacterial Activity of Lactiplantibacillus Strains Isolated from Commercial Yogurt against Foodborne Pathogenic Bacteria

Mona Othman Ibrahim Albureikan, Rawan Hassan Alshahrani

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e3
https://doi.org/10.22037/afb.v11i1.43275

Abstract

 Background and Objective: Lactic acid bacteria are well known as beneficial microorganisms and most of them are probiotic distributed widely, especially in fermented dairy products e.g. yogurt. This study aimed to isolate, characterize and assess antimicrobial effects of lactic acid bacteria producing bacteriocin-like inhibitory substances against foodborne pathogenic bacteria.

Material and Methods: In the present study, 17 lactic acid bacteria strains were isolated from 10 commercial yogurt samples and the antibacterial effects of lactic acid bacterial cell culture, cell-free supernatant and neutralized cell-free supernatant was assessed against standard foodborne pathogenic bacteria of Escherichia coli, Listeria monocytogenes, Klebsiella pneumonia and Salmonella typhimurium using agar well diffusion assay. Although various treatments were used, most of the lactic acid bacterial isolates showed antimicrobial activity against the foodborne pathogenic bacteria. Moreover, Lactiplantibacillus pentosus (SY1), Lacticaseibacillus rhamnosus (SY5), Lactiplantibacillus plantarum (SY8) and Lactiplantibacillus plantarum (SY9) showed significantly the best antimicrobial activity against the foodborne pathogens and thus were further identified using 16S rRNA gene molecular method.

Results and Conclusion: Results showed that four isolates could produce bacteriocin-like inhibitory substances, which was significantly effective to inhibit growth of the pathogens. Primary screening for antimicrobial activity showed that 10 lactic acid bacterial strains inhibited Escherichia coli. The results revealed that Listeria monocytogenes and Salmonella typhimurium was inhibited by six and one lactic acid bacterial isolates. Moreover, results showed that Klebsiella pneumoniae was not affected by the isolates or treatment methods. It is concluded that a bacteriocin-like inhibitory substance of lactic acid bacterial isolates was effective; hence, it could be used as a natural food additive to prevent foodborne infections and improve the food quality.

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

Development of Low-Lactose Probiotic Yogurt Drinks with Lactiplanti-bacillus plantarum subsp. plantarum Dad-13: Physicochemical and Sensory Characteristics

Rendra Lebdoyono, Tyas Utami, Endang Sutriswati Rahayu, Dian Anggraini Suroto

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e6
https://doi.org/10.22037/afb.v11i1.41903

Background and Objective: Lactose intolerance is a prevalent clinical syndrome due to consumption dairy products. Development of low-lactose products may help consumers to receive nutrition of dairy products without negative health effects. The aim of this study was to investigate survival, physicochemical and sensory characteristics of low-lactose yogurt drinks produced with probiotic Lactiplantibacillus plantarum subsp. plantarum Dad-13 during cold storage.

Material and Methods: Low-lactose milk was inoculated with probiotics in fermentation process of yogurt at 37, 39 and 42 °C while the growth of lactic acid bacteria and pH were monitored. Prepared yogurt drink was stored at 4 °C for 35 days and characterized for microbial and physicochemical aspects.

Results and Conclusion: Lactiplantibacillus plantarum subsp. plantarum Dad-13 showed high count at all the three temperature and reached to 7.64-8.96 log CFU.ml-1 at 37 °C within 36 h when cultured with yogurt. During the 35-d storage, cell viability of Lactiplantibacillus plantarum Dad-13 was 7.79 log CFU.ml-1. Furthermore, sensory assessment significantly increased (p≤0.05) for odor, viscosity, taste and total acceptability, compared to the non-probiotic yogurt.

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

Ganoderic Acid Production via Aerial Co-cultivation of Ganoderma lucidum with Bacillus subtilis and Aspergillus niger Using Bubble Column Bioreactor

Soheil Kianirad, Dana Shakiba, Ashrafalsadat Hatamian, Zahra-Beagom Mokhtari-Hosseini, Hale Alvandi, Elham Ansari, Bahman Ebrahimi Hosseinzadeh

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e7
https://doi.org/10.22037/afb.v11i1.43684

Abstract

Background and Objective: Ganoderma lucidum, with its medicinal characteristics, is one of the most beneficial fungi in traditional Asian medicine. This fungus low efficiency of ganoderic acid production has limited its use as a valuable secondary metabolite. Environmental stresses and elicitors such as microbial volatile organic compounds in co-cultures can increase ganoderic acid production. To investigate effects of variables of co-culture time and volume on Ganoderma lucidum growth and ganoderic acid production, Bacillus subtilis and Aspergillus niger were aerially co-cultured with Ganoderma lucidum.

Material and Methods: To investigate fungus growth and production of ganoderic acid using bubble column bioreactor, effects of independent variables of temperature, initial inoculation, length-to-diameter ratio (L: D) and aeration were investigated using Taguchi method. Then, effects of co-culture of Ganoderma lucidum with Bacillus subtilis and Aspergillus niger under optimum conditions were investigated.

Results and Conclusion: Optimizing effects of co-culture time and volume variables led to 2.9-fold increases in production of ganoderic acid, compared to the control sample. Optimization of biomass production in the bioreactor showed that biomass production increased significantly by increasing the initial inoculation percentage and temperature. These two variables significantly affected ganoderic acid production and its optimum production point was 10% of initial inoculation, temperature of 25.6 °C, L: D of 4:8 and aeration rate of 0.64 vvm. Gas holdup investigation for air-water and air-fermentation media systems showed that the presence of suspended solids and aeration rate affected gas holdup. Microbial volatile organic compounds in co-culture of microorganisms can increase ganoderic acid production by Ganoderma lucidum.

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

Effects of Organic Solvents on Acceptor Reactions for Oligosaccharide Synthesis Catalyzed by Glucansucrase URE 13-300

Stanimira Angelova, Tonka Vasileva, Veselin Bivolarski, Ilia Iliev

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e9
https://doi.org/10.22037/afb.v11i1.43668

 

Abstract

 

Background and Objective: Glucansucrases from GH70 family are effective transglucosylases, able to use non-carbohydrate acceptors. Glycosylation of flavonoids or terpenoids increases their water-solubility and bioavailability. Enzymatic glycosylation by glucansucrases can be improved by addition of organic solvents to the reaction media. Thus, the aim of the study was to assess effects of menthol, carvacrol and thymol solubilized in organic solvents on the activity of glucansucrase URE 13-300 and transferase reaction.

Material and Methods: Several organic solvents were assessed for their effects on glucansucrase activity using DNS method. Kinetic parameters in presence of the most appropriate solvents were evaluated as well. Thymol, carvacrol and menthol were solubilized in DMSO and their effects on the enzyme activity was assessed. Dynamic of oligosaccharides synthesis in aqueous-organic media was investigated using high-performance liquid chromatography.

Results and Conclusion: Maltose-derived oligosaccharides synthesized by glucansucrase URE 13-300 showed degrees of polymerization from 3 to 6 in presence of organic solvents, as well as in presence of buffer alone. Their concentrations did not differ significantly in each of the reactions in aqueous-organic media. Furthermore, kinetic parameters showed adjacent Km values with 5% solvents compared to the control reaction in buffer. These findings revealed that the overall synthesis of glucooligosaccharides was not altered by the organic solvents, nevertheless they changed the product distribution throughout the transferase reactions. These moderate effects of the selected organic solvents were important requirement for the glycosylation of biologically active compounds for use in the food industry.

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

In-vitro Assessment of Antifungal and Antioxidant Activities of Olive Leaves and Fruits at Various Extraction Conditions

Jalal Hassan, Aghil Sharifzadeh, Sogand Moghadam, Hamid Hajigholamreza, Gholamreza Shams, Amirali Aghamohammadi, Kiandokht Ghanati

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e10
https://doi.org/10.22037/afb.v11i1.43655

Abstract

 

Background and Objective: Nowadays, there is a growing interest for use of plant-based products such as extracts in various industrial sectors. Therefore, optimization of conditions for ideal extraction of bioactive compounds is highly important. Olive leaves and fruits include biophenols, which can be used as natural antimicrobial and antioxidant agents. Therefore, extraction of these bioactive compounds can create value-added products, which can be used as natural preservatives in food industries. The aim of this study was to investigate effects of various extraction parameters (type of solvent, solvent volume, temperature, time and pH) on in-vitro antioxidant and antifungal activities of Iranian olive leaf and fruit extracts against five Candida species.

Material and Methods: Olive fruit and leaf extracts were achieved using maceration method at various extraction conditions. Antioxidant activity of the prepared extracts was assessed by cupric reducing antioxidant capacity method. The phenolic profile in olive leaf extract was assessed using high performance liquid chromatography. Antifungal activity of the olive leaf extract was assessed using disk diffusion method and minimum inhibitory concentration and minimum fungicidal concentration values.

Results and Conclusion: Results showed that the highest antioxidant activity was recorded in olive leaf extract prepared by 100 ml of 96% ethanol at pH 7 and 80 °C for 6 h. Moreover, HPLC analysis of the ethanolic olive leaf extract showed that oleuropein was the major compound of the extract. Antioxidant activity of the olive leaf extract was higher than that of the fruit extract in various conditions. Regarding antifungal activity, the olive leaf extract showed a higher activity, compared to olive fruit extract at all concentrations. In olive leaf extract, the highest (62.5 µg ml-1) and the lowest minimum fungicidal concentration (15.6 µg.ml-1) values were reported for Candida tropicalis and Candida albicans, respectively. The minimum fungicidal concentration of the olive leaf extract was 250 µg ml-1 for Candida albicans, Candida parapsilosis, Candida glabrata and Candida krusei and 500 µg ml-1 for Candida tropicalis. It can be concluded that olive leaf extract is a source of antioxidant and antifungal substances with potential uses in food industries.

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

Development of a Cost-Effective Culture Medium for the Bacterial Cellulose Production Using Food Industry Wastes

Maryam Nasresfahani, Valiollah Babaeipour, Mohammad Imani

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e11
https://doi.org/10.22037/afb.v11i1.43875

Abstract

 

Background and Objective: Use of bacterial cellulose has been interested in various industries, especially medical and pharmaceutical industries, due to its unique characteristics, compared to plant cellulose. However, bacterial cellulose production costs have limited its industrial uses, compared to plant cellulose. Decreasing costs of the culture media is one of the effective parameters for the industrial production of bacterial cellulose. This is the first report on combination of vinasse and glucose syrup as a bacterial cellulose culture medium.

Material and Methods: Two inexpensive culture media were developed for high-level production of bacterial cellulose based on food industrial wastes of corn steep liquor-glucose and vinasse-glucose syrups. Concentrations of glucose syrup and corn steep liquor as a culture medium and concentrations of vinasse and glucose syrup as another culture medium were optimized using response surface method with central composite design to maximize bacterial cellulose production yields.

Results and Conclusion: Under the optimal conditions after seven days, 14.8 and 13.3 g.l-1 dry bacterial cellulose were achieved in corn steep liquor-glucose syrup and vinasse-glucose syrup respectively. Yield of produced bacterial cellulose from these two cost-effective culture media was one of the highest values reported for bacterial cellulose. Furthermore, the produced bacterial cellulose was characterized using Fourier-transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy.

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

Effects of Critical Storage Temperatures on Microbiological, Physico-chemical and Sensory Indicators of Sweetened Condensed Milk

Aleksandr Kruchinin, Elena Yurova, Bolshakova Ekaterina , Svetlana Turovskaya , Elena Illarionova, Irina Barkovskaya, Victoria Leonova

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e12
https://doi.org/10.22037/afb.v11i1.44478

Background and Objective: Principles of osmo and thermoanabiosis are used to produce sweetened condensed milks. Regarding their extended shelf lives, there are demands for their export to countries with various climates. However, high-positive and low-negative ambient temperatures during sweetened condensed milks transportation can affect their quality. Hence, it is important to study effects of critical storage temperatures on microbiological, physicochemical and sensory indicators of sweetened condensed milks.

Material and Methods: This investigation included a comprehensive study of the physicochemical, microbiological and sensory characteristics of sweetened condensed milks after storage under conditions involving multiple-stage and single-stage temperature changes within various ranges (from 5 to 50 °C; from 5 to -50 °C, from 50 to -50 °C and reverse cycles.).

Results and Conclusion: Analysis of samples subjected to cyclic changes, including multiple-stage heating for 9 d followed by multiple-stage cooling for 11 d, revealed that only viscosity changed relative to the control samples. In the reverse similar cycle (cooling to heating), formation of destabilized fat was observed. Moreover, changes of cycles and subsequent storage of the samples for 6 m led to increased viscosity, compared to control samples. It was established that single-stage freezing with a 14-d storage did not critically affect its quality. In contrast, rapid heating of the sweetened condensed milk up to 50 °C and storage under such critical conditions outside a cooled storage area were unacceptable. Further storage of samples subjected to cycles of single-stage freezing and heating for 6 m demonstrated a complete non-compliance with control samples for all parameters. Thus, sweetened condensed milk can be subjected to single-stage freezing to -50 °C and storage for 14 d, as well as multiple-stage cooling/freezing to -50 °C and multiple-stage heating to 50 °C following by cooling to 5 °C without loss of quality and safety during 6 m.

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

Effects of Adding Non-viable Lacticaseibacillus casei and Lactobacillus acidophilus on Physicochemical, Microbial, Chemical and Sensory Attributes of Probiotic Doogh

Saba Kamalledin moghadam, Mahdi farhoodi, vahid Mofid, Aziz Homayouni-Rad, Amir-Mohammad Mortazavian-Farsani, Ali Milani

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e14
https://doi.org/10.22037/afb.v11i1.44105

 

Background and Objective: Inactivated probiotics provide various health and technological benefits, making them appropriate for the production of functional dairy products. The aim of this study was to investigate effects of adding nonviable probiotics (Lactobacillus acidophilus LA-5 and Lacticaseibacillus casei 431) to doogh (a typical Iranian fermented milk drink).

Material and Methods: Probiotics were inactivated by heat or sonication and added to the samples before or after fermentation. Various parameters such as pH, titratable acidity, redox potential, antioxidant capacity, color, viscosity, and phase separation, viability of traditional starter bacteria and probiotics and sensory characteristics were assessed during fermentation and refrigerated storage at 5 °C.

Results and Conclusion: Sonicated probiotic-containing treatments included the highest pH decrease rate (0.011 pH min-1) during fermentation, as well as the highest antioxidant capacity (16.45%) and viscosity (35.15 mPa.s), while heat-inactivated probiotic- containing treatments included the lowest viscosity (17.60 mPa.s). Treatments with viable probiotics reasonably included the highest post-acidification rate during storage (4.14 °D d-1), compared to those containing nonviable cells, as well as the minimum phase separation rate. The b* and L* values of color did not differ significantly within treatments, but the highest a* value was observed in the treatments with sonication. The highest populations of Lactobacillus delbrueckii ssp. bulgaricus (log 11,891 cfu ml-1) and Streptococcus thermophilus (log 14,977 cfu ml-1) at the end of the storage were observed in treatments with heated probiotics (compared to viable probiotics) and treatments with sonicated probiotics, respectively. In addition, Lactobacillus acidophilus was more susceptible than Lacticaseibacillus case and included lower viability. Taste, mouth feeling and total acceptance of all samples did not differ significantly within treatments. The present study suggests that inactivated probiotics can successfully be used for the production of fermented milk beverages with appropriate sensory characteristics and higher antioxidant capacity, compared to the control group.

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

 

  1. Introduction

 

Recently, it has been suggested that probiotics, viable or non-viable, are bacterial cells that include positive effects on human health. By this general definition, probiotics are divided into two categories of viable and non-viable probiotics [1, 2]. The idea of using non-viable probiotics in food industries is originated from the fact that probiotic bacteria are susceptible to environmental conditions during passage through the gastrointestinal tract (GIT), include limited stability over a wide range of pH and temperature, include a shorter shelf-life and need refrigerated storage. Therefore, their use in various industries is further technologically and economically feasible [3-6]. Additionally, it has been verified that non-viable probiotics include beneficial effects for humans such as immunostimulating activity [7], cholesterol decrease [8], anticancer characteristics [9], healing gastrointestinal disorders [10] and suppression of pro-inflammatory cytokine production [11]. There are several available methods to inactivate probiotics, including heat treatment, ultraviolet (UV) irradiation, irradiation, sonication (ultrasound), high pressure, ionizing radiation, pulsed electric field (PEF), supercritical CO2, drying and changes in pH [8,12]. Sonication and heating are the most commonly used methods for inactivating probiotics, majorly because they are cost-effective and time-efficient. Ultrasound at frequencies of 20–40 kHz can be lethal to microorganisms by creating acoustic cavities on their cell membrane (CM), leading to the release of their contents [13]. In contrast, during heating, intracellular contents are not released.

Doogh is a fermented beverage whose major ingredients include yogurt, water, salt and flavoring agents [14]. However, studies on adding non-viable probiotics to fermented foods are limited. Parvayi et al. studied effects of inactivated Lactobacillus acidophilus ATCC SD 5221 and Bifidobacterium lactis BB-12 on yogurt characteristics and reported that incorporation of heat-inactivated probiotics to yogurts included less technological challenges and could be deliberated as an appropriate alternative for probiotics in functional yogurts [15]. Overall, there is still a research gap in the development and commercialization of inactivated probiotic dairy products in food industries. While interests in probiotics and prebiotics are increasing, inactivated probiotics have not received much attention for product development and market availability. In addition, knowledge on specific inactivated probiotic compounds in dairy products and their potential effects on human health is limited. Further research are needed to identify and characterize these compounds and assess their potential health benefits and uses in functional foods [16,17]. Moreover, there is a lack of standardized methods for the production and quality control of inactivated probiotic dairy products, which limits their widespread commercialization. Research in this area is essential to establish industry standards and guidelines for the production and commercialization of inactivated probiotic dairy products. The aim of this study was to assess effects of adding non-viable forms of Lacticaseibacillus casei 431 and lactobacillus acidophilus LA-5 probiotics inactivated by heating or sonication on the quality characteristics of doogh, a traditional fermented milk beverage from Iran. Probiotics were added before or after the milk fermentation processes.

  1. Materials and Methods
    • Materials

Skim milk powder was purchased from Pegah, Tehran, Iran. Starter culture included Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus (YF-3331) and the probiotics (Lacticaseibacillus casei 431 and Lactobacillus acidophilus LA-5) were provided by Chr. Hansen, Copenhagen, Denmark. De Man-Rogosa-Sharpe (MRS) agar and M17 agar were purchased from Quelab, Montreal, Canada, and salt from a local market.

  • Preparation of nonviable probiotics

Probiotic suspension was subjected to thermal inactivation by heating at 121 °C for 15 min [18].

To achieve ultrasound inactivation, probiotic suspension was exposed to ultrasound waves at a frequency of 250 kHz for 25 min [19].

  • Preparation of doogh

To prepare doogh, skim milk powder was reconstituted and diluted to a total solid content of 3.5%. Mixture was heated to 90 °C and set for 15 min before cooling down to 45 °C. Probiotics in viable or nonviable form were added before heat treatment (B) or after fermentation (A). Mixture was incubated at 42 °C until the pH reached 4.5, cooled down to 5 °C and stored in refrigerator for 28 d, as presented in Fig. 1.

  • Assessment of pH, redox potential and titratable acidity

The pH, RP (redox potential) and titratable acidity of the doogh samples were checked every 30 min during fermentation. After fermentation, doogh samples were cooled and stored in refrigerator for 28 d, during which, pH, RP (redox potential) and titratable acidity were assessed every 7 d to monitor the shelf life. The pH and RP were assessed using pH meter at room temperature (RM). Titratable acidity was assessed by titrating with 0.1 M NaOH solution and 0.5% phenolphthalein indicator [20]. Increase in acidity, decrease in pH value (pH value min-1) and increase in redox potential (mV min-1) were calculated using Eqs. 1, 2 and 3:

Figure 1. Study design of the present study.

 

  • Serum separation analysis

After cooling down, samples were stored in 10 ml vials and incubated at 5 ºC to assess serum separation. During the shelf-life period, height of the supernatant was assessed every 7 d to assess degrees of serum separation that were expressed as proportions using the following Eq. 4 [21]:

 

                Eq. 4

  • Rheological assessment

Rheological assessments were carried out using Brookfield viscometer at refrigerator temperature, one day after the samples were prepared [22]. Briefly, no. 2 cylindrical spindle and spindle speeds of 0.3, 0.6, 1.5, 3, 6, 12, 30 and 60 rpm were used during 90 s if the torque to rotate the spindle in the samples was between the 15.0 and 85.0% of the maximum torque.

  • Assessment of antioxidant capacity

To assess antioxidant capacity of the samples, a method was used based on the ability of antioxidants to scavenge the stable radical DPPH (1,1-diphenyl2-picrylhydrazyl). This method was described by Farahmandfar et al. essentially, sample ability to reduce the concentration of DPPH was assessed by measuring absorbance of the solution before and after exposure to the samples [23]. Antioxidant capacity of all samples and inactivated bacterial suspension were assessed on two occasions. The first assessment was carried out on the day of production, while the second assessment was carried out on Day 28 of the shelf-life.

       Eq. 5

  • Color assessment

Color characteristics of doogh were assessed using Hunter Lab Color Flex EZ explained by Milovanovic et al. [24]. Color parameters were L* (brightness, white = 100, black = 0), a* (+, red; -, green) and b* (+, yellow; -, blue).

  • Bacterial enumeration

Pour plate method was used to count numbers of L. delbrueckii subsp. bulgaricus, S. thermophilus and L. casei [25]. The L. bulgaricus, starter bacteria of doogh, was cultured in MRS-bile agar at 42 ºC for 72 h under anaerobic conditions using Gas Pac system. Enumeration of S. thermophilus was carried out using M17 agar at 37 ºC for 24 h under aerobic conditions [26]. Lactobacillus acidophilus LA-5 and L casei were cultured in MRS agar with added bile (0.15% w w-1) to prepare selective media of probiotic enumeration at 37 ºC for 72 h under aerobic conditions [27, 28]. The initial counts of L. acidophilus and L. casei were 107 CFU ml-1. To calculate the viability proportion index, final cell population of the microorganisms was divided into the initial cell population based on the Eq. 6 [25].

                                                           Eq. 6

  • Sensory evaluation

Taste, mouth feel and overall acceptance of doogh were assessed using 5-point hedonic scale rating test (with 5 excellent, 4 good, 3 acceptable, 2 bad and 1 very bad) [29]. Twenty consumers assessed the sensory attributes of doogh samples after the first day of preparation.

  • Statistical analysis

All experiments were carried out in triplicate and expressed as mean ±SD (standard deviation) (n = 3). Data were analyzed using univariate analysis of variance (Tukey test) AND SPSS statistical software v.26 (SPSS, Chicago, USA). Generally, p < 0.05 was addressed as the significance threshold.

  1. Results and Discussion
  • Assessments of pH, redox potential and titratable acidity

During milk fermentation, growth of starter bacteria leads to the conversion of lactose into various compounds such as lactic acid, acetate, formate, acetaldehyde and ethanol. This process results in lactic acid production, causing decreases in pH and increases in redox potential and titratable acidity [30]. Figure 2 illustrates changes in pH, redox potential and titratable acidity during the fermentation process. The initial pH of milk at the beginning of fermentation was 6.8, dropping to 4.5 by the end of fermentation. As shown in Fig. 2, and Table 1 fermentation process included three distinct phases of lag, log and constant phases. During the first 30 min, the lag phase, no significant changes were seen, possibly due to the adaptation of the starter bacteria and buffering characteristics of milk [31]. The fastest decrease in pH and increase in redox potential were observed in sample with ultrasound-inactived L. casei. This might be attributed to the ultrasound treatment, which caused puncturing of the membrane of the probiotics, resulting in the release of their cell contents into doogh [5,13]. Feeding the starter bacteria resulted in decreases in the rate of pH and pH of BUC reached 4.5 as the fastest rate (after 210 min). However, BUA included the highest titratable acidity, indicating that the type of probiotic bacteria included major effects on the rate of pH drop and acidity increase. Similarly, Tian and colleagues (2017) reported that the type of bacteria included effects on the quantification of organic acids [32]. In addition, postbiotics produced from L. acidophilus LA-5, L. casei 431 and L. salivarius included 62 vrious components, including alcohols, terpenes, norisoprenoids, acids, ketones and esters [33]. Hence, these compounds were available in the environment and might improve the fermentation stage.

Based on Fig. 2, BHC included similar rates of pH decrease and increase in redox potential through the fermentation process as the sample without probiotics. However, BHA showed the lowest rate of acid increase at the end of the fermentation, indicating that the starter bacteria alone were responsible for lactic acid production and the intact cells of the probiotic bacteria included no significant effects on acid production. Furthermore, heat-inactivated L acidophilus demonstrated the antibacterial activity [34]. Samples containing live probiotics needed longer times (240 min) to reach pH 4.5. This finding was similar to the finding of Parvayi (2021), who reported that live probiotic samples needed longer times to reach pH 4.5, compared to paraprobiotic samples [15]. Based on a study by Vinderola et al. (2002), adding L. casei and L. acidophilus to the media with the starter bacteria included negative effects on the growth of the starter bacteria, resulting in decreases in lactic acid production [35].

Statistical analysis showed no significant differences in redox potential between various types of bacteria (p>0.05). However, L. acidophilus resulted in further decreases in pH and increases in titratable acidity during the storage, compared to that L. casei did (p<0.05) as represented in Table 2. These results suggested that the selection of probiotic bacteria should carefully be considered based on the specific goals of the fermentation process [36]. Throughout the storage, the highest level of titratable acidity was seen in sample containing live probiotics of L. acidophilus (181AD∘), which could be attributed to the ongoing acid production by the live probiotics at the refrigerated storage. In contrast, BUC sample included the lowest acidity (117A), suggesting that the addition of probiotics after the fermentation process could lead to uncontrolled increases in acidity and continued fermentation during cold storage [15]. Moreover, samples containing sonicated and live probiotics included the maximum and the minimum RP increasing rates because of producing the minimum and the maximum lactic acid quantities during storage (p<0.05).

  • Serum separation

The study detected that the activity of starter bacteria and their ability to generate acids included significant effects on the separation of serum in the samples [37]. Data of Table 3 show increases in serum separation values for all samples during the storage. The initial and the final separation rates of BUC were the highest (32.4%), suggesting that the released intracellular contents were heavier than the whole bacterial cells, causing further sedimentations. In addition, Samples containing live probiotics included smaller serum separation ratios at the end of storage, indicating that they frequently produced lactic acid and their pH was further different from the isoelectric pH [38].

Relatively, Amani et al. reported effects of the activity of starters during storage due to their protein hydrolyzing characteristics on phase separation [37]. In addition, L. casei was reported to include lower serum separation ratios than that L. acidophilus did (p<0.05). This suggested that the type of bacteria in the samples played important roles in the serum separation rate because various strains of probiotic bacteria included various abilities to ferment and break down organic compounds and producing exo-endo polysaccharides as discussed in viscosity section [39]. However, np statistically significant differences were detected between the sequences of probiotic additions (p> 0.05).

  • Viscosity

Naturally, acidification and lowering of pH during fermentation cause milk casein proteins to clump, affecting viscosity of the final products. Figure 3 shows assessed viscosity of the samples. Sonicated probiotic-containing treatments (BUC and BUA) included the highest viscosity (3.083 ±0.6 and 3.515 ±0.5, respectively). Additionally, addition of live probiotics during fermentation led to increased viscosity, compared to samples without probiotics. It was reported that the release of exopolysaccharides and intracellular polysaccharides from the probiotics significantly increased viscosity [40, 41]. Exopolysaccharides secreted by Lactobacillus spp. during their growth affect viscosity of dairy products [42]. Moreover, "intracellular polysaccharides" are polysaccharides that accumulate within cells. The intracellular biosynthetic process involves transferring sugar residues into the cell, converting them into various monomeric units, partially polymerizing them and attaching them to isoprenoid lipid carriers [43]. Viscosity of heat-inactivated treatments was similar to that of control treatment, possibly because intact cells of probiotic bacteria did not release biopolysaccharides into doogh samples. Furthermore, type of bacteria significantly affected the viscosity (p<0.05). It was previously reported that variations in the viscosity values could be affected by characteristics of the probiotics cultures as well as adaptability of the bacteria [44].

  • Antioxidant activity assessment

The DPPH radical scavenging method, widely used to assess antioxidant activities, is simple, rapid, sensitive and reproducible compared to other methods [45]. Figure 4 shows antioxidant capacities of the samples on Days 1 and 28. Antioxidant capacity of the samples decreased significantly during the storage due to inappropriate sealing, oxygen entry into the samples and uncontrolled bacterial activity. Sonicated probiotic-containing treatments increased the antioxidant capacity, as the intracellular content of lactic acid bacteria (LAB) demonstrated greater antioxidant characteristics than that the whole cell or the extracellular metabolites did [46,47]. Antioxidant activity of the intracellular contents of LAB was linked to the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), nicotinamide adeninedinucleotide (NADH)-oxidase, NADH-peroxide and glutathione (GSH) enzymes [48]. In addition, use of live or heat-inactivated probiotics did not result in significant differences (p>0.05). This was due to the cell contents that were not released. Relatively, lyophilized cells of Lactococcus lactis subsp. cremorishave included the highest antioxidant capacity, compared to those the heat-killed and intact cells did [49].

In contrast, use of L. acidophilus rather than L. casei significantly increased the antioxidant capacity (p<0.01). Amdekar et al. assessed antioxidant and anti-inflammatory potentials of L. casei and L. acidophilus in in-vitro models of arthritis. Results indicated that arthritic rats treated with L. acidophilus included higher glutathione peroxidase and decreased glutathione concentration, compared to that arthritic rats treated with L. casei did [50]. Additionally, adding probiotics before fermentation improved the antioxidant capacity of doogh samples (p>0.05).

  • Color analysis

 

Color is a critical characteristic in assessing quality of products such as yogurts and doughs. The L* parameter indicates lightness or darkness of the color, the a* parameter shows redness or greenness of the color and the b* parameter represents yellow or blueness of the color [51]. The color values are shown in Fig. 5. Integration of probiotics inactivated using ultrasound resulted in increases in a* value, indicating release of probiotic contents into the doogh sample (p<0.05) and showing that green pigment substances such as thiamine were present in intracellular probiotics [52]. However, no significant differences were reported between the paraprobiotics and probiotics in a* value (p>0.05). Additionally, no significant differences were demonstrated between the sequential additions of probiotics in a* value (p > 0.05). Type of the probiotics in doogh samples included significant effects on a* value (p < 0.05). It has previously been suggested that various types of bacteria with special characteristics can affect color of the products [53]. In L* and b* values, no differences were observed within the addition of active/inactivated probiotics into doogh samples (p>0.05). Furthermore, types of probiotic bacteria (L. casei or L. acidophilus) and probiotic adding sequences did not include significant effects on L* and b* values (p > 0.05).

  • Viable counts of the starter bacteria and probiotics

The L. bulgaricus and S. thermophilus are critical for acidification and production of doogh [54]. Survival of the starter and probiotic bacteria in yogurts depends on various factors such as the specific strains, interactions between the species, chemical compositions of the yogurts, the culture conditions, production of hydrogen peroxide during bacterial metabolism, final acidity of the yogurts, rates of lactic and acetic acids, nutrient availability and the storage temperature [46]. Table 4 shows the number of L. delbrueckii subsp. bulgaricus and S. thermophilus for all samples during the storage. The BUA included the highest number of L. delbrueckii subsp. bulgaricus on the first and the last days of enumeration (logs 11.89 and 10.91 cfu ml-1, respectively), while BC included the lowest number (logs 11.35 and 9.68 cfu ml-1, respectively). Additionally, BA and BUA included the lowest and the highest L. delbrueckii subsp. bulgaricus counts at the end of storage (logs 7.84 and 10.91 cfu ml-1, respectively). Type of probiotics (viable or non-viable) in doogh samples included significant effects on the viability of L. delbrueckii subsp. bulgaricus (p<0.05) through competitive interactions, metabolic activities, cell-cell interactions and protective effects. However, a study by Parvayi et. al (2021) showed that addition of viable or non-viable probiotics did not affect L. delbrueckii subsp. bulgaricus when used as the starter bacteria [15].

As a statistical result, significant differences were observed between using L. casei and L. acidophilus probiotic bacteria (p<0.05). Selection of probiotic strains such as L. casei and L. acidophilus in doogh could affect viability of L. delbrueckii subsp. bulgaricus through species-specific interactions, metabolic compatibility, competition for resources, synergistic or antagonistic effects and stability of the microbial community. In addition, inhibition of L. delbrueckii subsp. bulgaricus growth by L. acidophilus has previously been reported. Vinderola reported L. casei strains did not include effects on the growth of L. delbrueckii subsp. bulgaricus [35]. Furthermore, no statistical differences were reported between the sequences of adding probiotics (before or after fermentation) into doogh samples (p>0.05). In contrast, viability of S. thermophilus decreased significantly (p<0.05) during the storage. On the first day of storage, BUC included the highest number of S. thermophilus (log 14.67 cfu ml-1), whereas BA included the lowest number of S. thermophilus (log 13.54 cfu ml-1). In addition, AA and AUC included the lowest and the highest S. thermophilus counts at the end of storage (logs 10.11 and 12.85 cfu ml-1, respectively). The highest rate of decrease in S. thermophilus viability was associated to AA (0.73), while AUA included the lowest rate of decrease in S. thermophilus viability (0.88). Addition of non-viable probiotics caused significant differences in the bacterial population, compared with that addition of live probiotics did (p<0.05). This could be due to the indirect antagonistic effects of live probiotics [4, 35].

Doogh samples with inactivated probiotic cells showed significantly higher starter proliferation, compared to those treated with probiotic bacteria due to the cell wall structure of L. acidophilus and L. casei in their ruptured cells (p<0.05). Naturally, cell wall majorly consists of teichoic acids, cell structural protein (S-layer), peptidoglycan and polysaccharides [10]. Additionally, LAB intracellular contents include GABA, B-vitamin complex, polysaccharides, biopeptides, polysaccharides and lipoteichoic acids [4,47,55]. Therefore, fermentation in the environment is strengthened when the intracellular contents are released into doogh. It is possible that choosing the right time for inoculation can significantly affect growth of starter bacteria. Results showed significant differences (p < 0.01) between adding probiotics before or after the fermentation process, affecting viability and activity of the starter bacteria due to its adaptation to the culture medium during fermentation and storage. Moreover, it was reported that L. acidophilus included stronger growth inhibitory effects on S. thermophilus than that L. casei did (p <0.01). In a study by Vinderola et. al (2002), L. casei strains inhibited growth of S. thermophilus while L. acidophilus did not affect the growth of S. thermophilus [35]. Sample inoculated with live probiotics before fermentation included the lowest count of S. thermophilus due to the potential antagonism effects of the probiotics (p<0.01).

Generally, live probiotics included side effects on the growth and viability of the starter bacteria. For nonviable probiotic cells, these inhibitory effects are rarely observed and can surprisingly promote starter bacterial activity by providing various nutritious (e.g., amino acids, minerals, B vitamins and saccharides) and growth stimulatory elements [56]. Moreover, live probiotic bacteria include side effects on the starter bacterial growth because of their antimicrobial secretion and competition. Probiotic bacteria included more inhibitory effects on LAB than that LAB did when probiotics were not present [35]. For inactivated probiotics, there are no severe competitions for nutrition between the starter bacteria that may nourish them and enhance their growth due to the release of cytoplasmic contents. It is noteworthy that addition of live probiotics to media before fermentation increased the number of probiotic cells during storage due to better adaptation (p < 0.05). Furthermore, L. acidophilus was more susceptible than L. casei and statistically significant differences were recorded in viability of the probiotic bacteria (p < 0.05). Therefore, it can be concluded that selection of an appropriately adaptable strain may play critical roles in preserving viability of probiotics during the shelf life of the products [36].

  • Sensory evaluation

Sensory attributes play key roles in attracting consumers. Daily probiotic products may include distinct tastes that are not be accepted by the consumers [57]. Therefore, studies have focused to improve consumer acceptance of probiotic beverages. In this study, taste, mouthfeel and overall acceptability of sensory aspects were assessed on the first day of fermentation (Figure 6). The AHC included the highest score (4.1/5) for taste, while AC included the lowest score (2.6/5) due to uncontrolled lactic acid formation. Nevertheless, no significant differences were seen for taste, mouthfeel and total acceptance of doogh samples (p > 0.05). A study demonstrated that probiotic beverages containing L. casei included high acceptance, compared with beverages containing L. acidophilus due to desirable flavors [58].

  1. Conclusion

These non-viable probiotics have been shown to eleminate technological limitations by enhancing rates of titratable acidity and fermentation, texture, viability of starter bacteria including S. thermophilus and L. bulgaricus and decreasing post-acidification rate as well as potentially improving gut health and immunity by increasing antioxidant capacity of doogh samples. Further studies are needed to fully understand mechanisms of these effects and optimize formulation of non-viable probiotics in fermented milk products. Overall, findings suggest that incorporating non-viable probiotics into fermented milks can be a valuable strategy for enhancing functional characteristics of dairy products.

  1. Acknowledgements

Please write one paragraph

  1. Conflict of Interest

The authors report no conflicts of interest.

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Production of Nanocomposite Silver Packaging using Solution Blending Method for the Supplement of Antibacterial Coating

Zahra Taati Jafroudi, Hamed Ahari, Nekisa Sohrabi Haghdoost, Shahrokh Shabani

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e16
https://doi.org/10.22037/afb.v11i1.43876

 

Background and Objective: The objective of this study was to assess antimicrobial effects of silver nanoparticles on Gram-positive and Gram-negative bacteria that used in preparing silver nanocomposite with the antibacterial characteristics using solution method. Moreover, the aim of the current study was to produce antimicrobial silver nanocomposites for food coating with their effects on a wide range of bacteria.

Material and Methods: To assess antibacterial characteristics of silver nanoparticles, several steps were carried out. First, nanoparticles were synthesized through a chemical reduction method using NaBH4 and then analyzed using x radiation diffraction, ultraviolet and visible spectroscopic analysis, dynamic light scattering and scanning electron microscopy nanometric assays. Then, Staphylococcus aureus and Escherichia coli were used as Gram-positive and Gram-negative bacterial indicators. Minimum inhibitory concentration, minimum bactericidal concentration and inhibition zone levels were measured. Nanocomposite was produced using solution blending method and its antibacterial characteristics were assessed using inhibition zone method.

Results and Conclusion: Results indicated that silver nanoparticles with 20 and 50 µg.l-1 concentrations included inhibitory effects on Staphylococcus aureus and Escherichia coli, respectively. Furthermore, concentrations of 40 to 60 mg.l-1 included lethal effects on Staphylococcus aureus and Escherichia coli, respectively. Based on the results, the highest antibacterial effects were observed on Gram-positive Staphylococcus aureus. In inhibition zone assays, a 3-5 mm zone was seen around the silver nanoparticle discs in cultures of the microorganisms. In the inhibition zone assay of the produced nanocomposites, the zone was expected regarding the concentrations. Results were calculated in three repetitions and the value estimated through ANOVA was significant when p<0.0001. It has been concluded that silver nanoparticles are useful in Gram-positive and Gram-negative bacteria for the inhibition and destruction. Moreover, it has been verified that using the method includes great effects on antibacterial characteristics of the nanocomposites.

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

 

 

*Corresponding authors:

 

Hamed Ahari *

Food Biotechnology, Tehran Science and Research Branch, Islamic Azad University, Tehran, Iran

 

Tel: +98-9121872334

 

E-mail:

dr.h.ahari@gmail.com

 

 

  1. Introduction

 

In recent years, use of metal nanoparticleshas increased due to the resistance of pathogenic microorganisms (bacteria, fungus and viruses) against conventional antimicrobials. General concerns on the safety and quality of foods, particularly marine foods, during storage and stocking have led the microbial growth control a fundamental part of the distribution and storage chain of such products [1-4]. Based on various studies and assessment of silver nanoparticle function mechanism against microorganisms, their use as antimicrobial agents, especially in the food and medical industries, can be one of the novel solutions for conquering problems caused by the pathogenic microorganisms. Food products are infected by various microbial agents during production processesss. Infections may occur in formulation of ingredients, using chemical additives and posing high pressure and flash pasteurization. Harmful materials likely enter food formulation during this process, or chemical reactions may occur with dangerous outcomes to humans.

If food products are in contact with contaminated surfaces or surfaces with metal ions during the production processes, this can endanger consumers’ health. Therefore, use of proper antimicrobial packaging based on bio-polymers is nowadays highly interested due to being biodegradable, lack of collection of various synthesized materials in the natural ecosystem and improvement of mechanical and viscoelastic characteristics as well as appropriate antimicrobial characteristics [5]. Liao et al. studied the antibacterial activity and action mechanisms of silver nanoparticles (AgNPs) against Pseudomonas aeruginosa resistant to several medicines. In that study, use of morphological changes and assessment of active oxygen and activity of enzymes in the bacteria when exposed to AgNPs as well as reporting minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) showed the potential antibacterial effects of AgNPs on the bacteria [6]. Jo et al. investigated antibacterial characteristics of polyethylene and polypropylene nanocomposite films using AgNPs. First, nanocomposites were prepared using melting method and extruder. Based on the results, these nanocomposites included a 99.9% destructive effect on Staphylococcus aureus and Escherichia coli bacteria. This result showed well that using these nanocomposites could be effective and efficient in food packaging [7]. Furthermore, researchers synthesized degradable films with mixed clay and polyvinyl alcohol (PVA). They used these films against essential food pathogens such as Salmonella typhimurium and Staphylococcus aureus. Their results could reflect high antimicrobial effects of these nanoparticles, their mechanical characteristics and appropriate flexibility caused by PVA, as well as biodegradability of these films, which were verified through burring assaessment of them in the ground. To show efficiency of the highlighted packaging bags, shelf life of the chicken sausage samples was compared with that of regular polyethylene bags. Results showed enhancements in shelf life by decreasing microbial loads [7].

Mathew et al. synthesized nanocomposites, combining clay and biodegradable PVA. Based on their findings, the combined nanocomposite films included sufficient antimicrobial characteristics against food pathogens such as S. typhimurium and S. aureus and higher mechanical characteristics such as resistance against water and light transmission, compared to control films. The soil burial assay revealed that the nanocomposites degraded within 110 d and hence were considered biodegradable. Then, nanocomposite combined films were included in the bags used for keeping chicken sausages, which resulted in decreases in microbial loads compared to the control polyethylene bags and were much more effective in increasing shelf life of the chickens [8]. Findings from their study were similar with those of the the current study.

Liao et al. studied antibacterial characteristics and mechanisms of AgNPs against P. aeruginosa resistant to drugs. In this study, antimicrobial effects of AgNPs on resistant clinical isolates against P. aeruginosa with MIC and MBC were investigated. Morphological changes were observed in P. aeruginosa resistant against drugs under transmission electron microscopy (TEM). Distinct protein highlighted in the proteomics approach was studied quantitatively and production of reactive oxygen species was assessed using 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA) coloring. Activity of superoxide dismutase (SOD), catalase and peroxidase was chemically assessed and apoptosis effects were studied through flow cytometry. Findings revealed that AgNPs included strong inhibitory effects on P. aeruginosa resistant against the antimicrobials with MIC of 1.406-5.625 mg.ml-1 and MBC of 2.81-5.62 mg.ml-1. Results of TEM revealed that AgNPs could penetrate resistant bacteria and disrupt their structure. Furthermore, quasi-apoptosis in bacteria affected by AgNPs was significantly higher. General findings and the estimated p-value (p<0.01) revealed strong antibacterial effects of AgNPs on multiresistant P. aeruginosa [9]. Active packaging incorporating AgNPs becomes popular due to its efficacy in combating foodborne pathogens. This technology uses AgNPs directly embedded in the packaging materials or adsorbed as ions, offering a safe effective antimicrobial shield. Recent approvals by the European Food Safety Authority (EFSA) for specific silver compounds further facilitates broader implementations [10].

This study investigated antibacterial effects of AgNPs on Gram-positive and Gram-negative bacteria and produced silver nanocomposites with appropriate antibacterial characteristics using solution blending. As previously stated, the present experimental study investigated use of AgNPs synthesized via chemical resuscitation method by assessing their MIC, MBC and inhibition zone against S. aureus, E.coli and Candida albicans, leading to decreases of food spoilage and enhancement of food shelf life. Moreover, their antimicrobial effects were studied as alternatives to antimicrobials.  The aim of this study was to investigate antibacterial effects of AgNPs on Gram-negative and Gram-positive bacteria and produce silver nanocomposites with appropriate antibacterial characteristics using solution blending method.

  1. Materials and Methods

2.1. Synthesis and characterization of silver nanoparticles

The AgNPs were synthesized using chemical reduction method with sodium borohydride (NaBH4). Dynamic light scattering (DLS) verified the particle sizes within the desired range of 25-40 nm. The X-ray diffraction (XRD) analysis revealed crystal structure of the materials, while UV-VIS spectroscopy provided information on nanoparticle size and homogeneity. Additionally, scanning electron microscopy (SEM) visualized morphology of the synthesized nanoparticles.

2.2. Antimicrobial activity assessment

Culture media and autoclaves were sterilized. Each experimental tube included 5 ml of culture media, 100 mg of bacteria/fungi and calculated concentrations of AgNPs. Triplicate experiments were carried out.

2.3. Minimum inhibitory concentration and minimum bactericidal concentration assessments

Microdilution assay in gamma tubes was used. Nutrient broth was used for S. aureus and E. coli and Sabouraud dextrose (SD) broth for C. albicans. Standardized inocula (100 μl) were added to the broths and incubated at 37 °C for 24 h. The MIC was assessed as the lowest concentration inhibiting visible growth. The MBC included plating 100-μl aliquots onto agar media (nutrient agar for bacteria and SD agar for fungi) and incubating at 37 °C for 24 h. Moreover, MBC was defined as the lowest concentration demonstrating no microbial growth or less than three colonies (99-99.5% killing).

2.4. Nanoparticle synthesis method

First, sodium borohydride was dissolved in water (ice bath) and mixed with polyvinylpyrrolidone (PVP). Silver nitrate solution was then added to the mixture, which changed the color from yellow to orange, brown and then black. This was then stirred quickly (~1500 rpm) at 50–60º C, which created clods. Drops of silver nitrate (0.001 M) were added to sodium borohydride (0.002 M) set in the ice bath, which changed color of the final product to yellow. This color became darker over time. To increase stability of the product, 1% PVP was added to the solution, which changed color of the product to pale orange-red. Concentration of the colloidal nanosilver was 6 mg.ml-1 and based on the UV-VIS assay, size of the particle was 25–40 nm. The quantity of PVP used for increasing stability was 3 mg.ml-1.

2.5. Nanocomposite production through solution blending

In brief, 500 ml of the polymer PVA were divided into five beakers with volume of 0.28, 0.42, 0.83, 1.67 and 2.5 ml. Then, AgNPs were added to 12.5 25, 50, 100 and 150 ml with a concentration of 6 mg.ml-1. These were stirred on a stirrer heater at 50 ºC for 24 h until volume of the solution reached 20 ml. The final product was poured into a Petri dish and set in the oven for 24 h to dry. Then, the final composite with similar thickness and appropriate level of flexibility was ready.

2.6. Analysis of the size of nanoparticles using dynamic light scattering method

In general, DLS is a technique used to assess particle sizes in solutions and suspensions. In this method, specialized devices analyze the motion of particles while they are suspended in a liquid. It provides a rapid and non-destructive way to assess particle sizes, ranging from nano to micrometers. For example, researchers transferred nanosilver colloids into the DLS device cell. The subsequent analysis estimated the particle size. Specifically, 5 ml of nanosilver colloid were analyzed at 25 °C with a laser strength of 60%.

2.7. UV-VIS analysis

Characteristics of photons on samples and measuring the rate of passage or absorption (rate of adsorption or reflectance of light) in various wavelengths ranging 200-1100 nm. Results of the assay were presented in a typical surface absorption plasmon at 420 nm  achieved from the AgNPs.

2.8. Scanning electron microscopy analysis

The SEM is an exceptionally well-suited method for the study of nanoparticle structure and it depicts the size of AgNPs in ranges from 10 to 100 nm. No agglomeration was seen in nanoparticles, showing stabilization of the nanoparticles.

  1. Results and Discussion

3.1. Analysis of the nanoparticle characteristics

The DLS results from Fig. 1 (a, b, c) revealed essential information on AgNPs. Based on the figure, AgNPs demonstrated the following characteristics. Number distribution, approximately 41% of the particles within specific size ranges; volume distribution, nearly 48% of the particles contributing to the overall volume; intensity distribution, significantly 92% of the scattered light intensity originating from the specific particle sizes.

Additionally, Fig. 2 shows a SEM image of AgNPs synthesized through chemical reduction. These nanoparticles exhibited spherical shapes and included a size range of approximately 25 to 40 nm.

Furthermore, Fig. 3 presents the UV-VIS diagram, providing further characterizations of the AgNPs. To assess structural characteristics of the nanocomposite films, XRD analysis was used. Nanosilver samples were irradiated with Cu-Kα radiation (λ = 54.1 Å) using X-ray spectrometer operating at 40 kV and 30 mA. The resulting XRD pattern provided valuable information on the crystalline phases and crystallographic orientation within the nanocomposite films (Fig. 4).

 

 

 

 

  1. Nanoparticles with a size of 145.8 nm

Figure 1. Dynamic light scattering diagram of the produced silver nanoparticles with various sizes: a, 121.9, b, 145.8 and c, 150.3

 

 

 

 

 

Figure 2. Scanning electron microscopy of the silver nanoparticle synthesis using NaBH4

 

Figure 3. The UV-VIS diagram of the produced silver nanoparticles

 

 

 

 

 

 

Figure 4. The X-ray diffraction diagram of the produced silver nanoparticles

 

 

 

3.2. Minimum inhibitory concentration and minimum bactericidal concentration assessments

The MIC and MBC of the biosynthesized nanoparticles were assessed against various pathogens. Nanoparticles showed potential antibacterial activities against E. coli (MIC, 50 µg.ml-1 and MBC, 70 µg.ml-1) and S. aureus (MIC, 25 µg.ml-1 and MBC, 45 µg.ml-1). However, nanoparticles demonstrated weaker antifungal activities against C. albicans (MIC, 350 µg.ml-1 and MBC, 380 µg.ml-1). Results suggested potentials of these nanoparticles as broad-spectrum antimicrobials. Although further optimizations may be necessary for the enhanced antifungal efficacies (Fig. 5).

3.3. Antimicrobial susceptibility assay for the assessment of inhibition zone diameters (disk diffusion)

To assess antibacterial and antifungal activities of the AgNPs, inhibition zone assay was used via diffusion disks impregnated with various concentrations (200 and 6000 µg.ml⁻¹). Three microorganisms were assessed, including S. aureus, E. coli and C. albicans. Isolated bacterial colonies of S. aureus and E. coli were suspended in sterile serum, creating a homogenous solution. This solution was then streaked onto agar plates using sterile swabs. Blank disks loaded with either AgNPs or control antibiotics (amikacin for bacteria and itraconazole for fungi) were transferred onto the inoculated plates. Following incubation at 37 °C for 24 h, diameters of the resulting inhibition zones around the disks were measured using caliper. For the nanocomposite disks, another experiment was carried out, where blank disks were punched out and loaded with various nanoparticle concentrations. These disks were then added to the bacterial cultures and the inhibition zones were measured as described. Results of this study provided information on the potentials of the AgNPs as antimicrobial agents against various pathogens (Table 2).

Findings of MBC and MIC assays verified that by prohibiting microorganisms, AgNPs could increase the shelf life of foods (Table 1). Results revealed the higher effects of AgNPs on Gram-positive S. aureus, compared to Gram-negative E. coli. According to Abbaszadegan et al., the major reason for differences in antibacterial effects of Gram-positive and Gram-negative bacteria included the quantity of peptidoglycan in the bacteria cell wall. Ggram-positive strains included further peptidoglycans in their cell walls, compared to those Gram-negative strains did, allowing AgNPs to include extended inhibition zones for these strains.

This study demonstrated effectiveness of AgNPs in extending food shelf life by inhibiting microbial growth. Studies, including those by Abbaszadegan et al. and Eslami et al., highlighted the nanoparticle efficacy against various bacteria, with Gram-positive strains such as S. aureus exhibiting a greater susceptibility, compared to that Gram-negative E. coli doing. This difference was attributed to the thicker peptidoglycan layer in Gram-positive bacterial cell walls, offering a larger target for AgNPs.

Researchers investigated the potential of AgNPs to combat microbes in various settings, including food preservation. In a study by Eslami et al. (2016), effectiveness of AgNPs in preserving saffron was investigated. Various concentrations of nanoparticles were incorporated into packaging materials and the microbial loads on the saffron were monitored over time.

 

Figure 5. Statistical findings of the minimum inhibitory concentration assay using ANOVA

 

Table 1. Estimated nanosilver concentrations for MBC, MIC and MFC

 

Table 2. Results of the inhibition zone assay for silver nanoparticles and nanocomposites

 

 

Results showed significant decreases in microbial growth, particularly at higher nanoparticle concentrations, highlighting the potential of this technology for extending the shelf life of food products [10]. Antibacterial activity of the AgNPs against hospital-acquired antibiotic-resistant strains of P. aeruginosa was assessed by Salomoni et al. [11]. Commercial nanoparticles effectively inhibited bacterial growth at specific concentrations, suggesting their potentials as tools to combat challenging infections. Further studies such as that by Alsharqi et al. deeper investigated mechanisms; by which, AgNPs exert their antimicrobial effects. Their in-vitro experiments demonstrated that nanoparticles interacted with bacterial cell membranes, ultimately suppressing bacterial growth. Significantly, this effect was observed against Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria, although various degrees of susceptibility were observed [12]. Similar to the current findings, these studies collectively present encouraging evidence for the use of AgNPs as a novel antimicrobial strategy. Further studies are needed to assess their potential uses and safety profiles.

The SEM images of the treated bacteria cells revealed significant morphologic changes in the cell membranes after processing with AgNPs. Results indicated strong antibacterial reactivities in AgNPs that could inactivate harmful and pathogenic microorganisms [12]; similar to results of the present study.  Previous findings showed that AgNPs directly attacked the cell membrane of bacteria, causing significant morphological changes [12]. This verified strong antibacterial activities of these nanoparticles, further supporting their potentials to combat harmful microorganisms.

Yan et al. [13] investigated broadly mechanisms of action using proteomics approaches, revealing 59 proteins affected by silver interactions. Interestingly, silver interacted with several membrane proteins and triggered production of ROS within the bacteria. This ROS production ultimately damaged the cell membrane, leading to bacterial death. These findings were perfectly similar to findings from the current study and other studies, highlighting the potential antimicrobial roles of AgNPs. Additionally, Pooyamanesh et al. [14] successfully incorporated AgNPs into food packaging materials, demonstrating their effectiveness against various foodborne bacteria such as E. coli and S. aureus. This evidence strongly suggests that AgNPs include tremendous potentials in combating harmful bacteria, opening doors for novel uses in food preservation. However, further studies are necessary to fully understand their safety and optimize their effectiveness for various uses.

  1. Conclusion

This study has validated potentials of AgNPs and their nanocomposites for antimicrobial uses in food packaging. Findings from MIC, MBC and inhibition zone assays consistently have demonstrated their effectiveness against various bacteria, especially Gram-positive strains. These provide direct benefits for food preservation, extending shelf life while eliminating needs of harmful chemical additives. Integrating AgNPs into food packaging offers more than a chemical-free alternative; it presents a multifaceted solution with far-reaching benefits. First, it develops organic food production by effectively combating bacteria without conventional preservatives, fostering trust and enhancing food quality. Second, their significant antibacterial ability originates from their high surface areas and positive charges, disrupting the bacterial membranes and significantly extending food shelf lives. Third, the chemical resuscitation method allows for precise control of nanoparticle sizes, tailoring their interaction with specific bacteria for optimized performance. Fourth, the suggested solution blending method improves cost-effectiveness, making this innovative technology readily accessible. While further studies are critical to understand long-term effects and ensure responsible implementation, these diverse advantages offer AgNPs as a promising solution for the challenges of food preservation. While Gram-positive bacteria have demonstrated greater susceptibilities due to their cell wall structures, effectiveness of AgNPs even at authorized low concentrations and their minimal risks of release into foods further highlight their potentials as safe sustainable alternatives to the available antimicrobial agents. This study pioneers further investigations and optimization of silver nanoparticle-based food packaging solutions, offering a promising path towards enhanced food safety and decreased environmental adverse effects. However, it is important to acknowledge needs of continuous studies to comprehensively understand potential long-term effects of the current technology.

  1. Acknowledgements

Special thanks to the Nano Research Laboratory (Ultrasonic Section), Science and Research Branch of Islamic Azad University.

  1. Conflict of Interest

The authors declare no conflict of interest.

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Investigating Untapped Potentials: Velvet Beans as Novel Prebiotic Sources and Their Effects on Gut Microbiota and Short-Chain Fatty Acid Level

Amalia Eka Puspita, Artini Pangastuti, Shanti Listyawati, Siti Lusi Arum Sari

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e17
https://doi.org/10.22037/afb.v11i1.44643

 

Abstract

 

Background and Objective: Prebiotics are non-digestible carbohydrates that selectively facilitate growth of beneficial microorganisms in the gut. Legumes naturally contain carbohydrates with potentials as prebiotic sources. However, numerous legume species remain uninvestigated in this context. The aim of this study was to identify such uninvestigated legumes as potential sources of prebiotics.

Material and Methods: Nine legume samples collected from Central Java and East Java, Indonesia, were extracted using maceration method. Digestion with HCl buffer and α-amylase followed by analysis with dinitrosalicylic acid and phenol-sulfuric acid methods assessed quantities of non-digestible carbohydrates. Three legumes with the highest non-digestible carbohydrates levels were assessed in vitro to investigate their abilities to promote the probiotics growth. Then, the most promising extract was assessed on mice to assess its effects on short-chain fatty acid levels using GC-2010 Plus and gut microbiota composition using metagenomic 16S rRNA markers.

Results and Conclusion: From the nine legumes assessed, bambara groundnut (23,51 mg.g-1), velvet beans (22.36 mg.g-1) and chickpeas (12.1 mg.g-1) included the highest non-digestible carbohydrates levels. Velvet beans showed a greater ability to stimulate growth of Lactobacillus plantarum and Bifidobacterium bifidum, compared to bambara groundnut and chickpeas. Administration of velvet beans to mice increased short-chain fatty acid levels in forms of acetate (12.6 mM) and propionate (3.28 mM). Significantly, velvet beans could modify composition of the gut bacteria by increasing diversity, decreasing dominance levels, increasing abundance of Bacteroides, Helicobacter, Mucispirillum, Bifidobacterium and Lawsonia genera and decreasing abundance of Lachnospiraceae NK4A136 group, Blautia and Lachnoclostridium species.

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

  1. Introduction

 

Prebiotics consist of non-digestible carbohydrates (NDCs) and are resistant to stomach acid and digestive enzymes; therefore, they selectively promote growth of beneficial bacteria in the large intestine [1,2]This fermentation process by gut bacteria produces short-chain fatty acids (SCFAs) such as acetate, propionate and butyrate, which can lower pH of the gastrointestinal tract (GIT) and affect gut microbiota compositions [3]. Decreases in pH can decrease number of pathogenic microorganisms and increase growth of beneficial microorganisms, which is linked to the tolerance of the latter microorganisms to acidic conditions. Previous studies have shown that specific prebiotics such as inulin can promote growth of beneficial gut bacteria such as Lactobacillus and Bifidobacterium. [4,5]. Similar effects were observed with fructo-oligosaccharides, increasing Bifidobacterium while decreasing harmful bacteria [6].

Legumes are renowned for their nutritional values, particularly as protein sources. However, they contain NDCs in the form of oligosaccharides and polysaccharides, which include potentials as prebiotics [7]. These components pass through the small intestine undigested and reach the large intestine, where they can promote growth of beneficial gut bacteria [8]. Studies have demonstrated prebiotic effects of common legumes such as cowpeas and black beans, including decreased pH levels, increased growth of Bifidobacterium and Lactobacillus and increased SCFA levels [9,10] however, a vast majority of legume species are grouped under the minor category, remaining virtually uninvestigated. Much legumes are consumed only by local communities in Java, Indonesia. Additionally, food industries ignore legumes due to a limited knowledge of their compositions and potential benefits. Investigating prebiotic potentials of the minor legumes presents a compelling opportunity to enhance their economic values.

No reports have been published on the prebiotic potentials of specific minor legume varieties used in the current study. While most studies on prebiotics have focused on familiar major legumes, the current study investigated the lesser-known varieties. Significantly, one minor legume, Mucuna pruriens (velvet bean), has shown promises to combat obesity in mice, but its prebiotic potentials must be investigated [11]. In the current study, minor legume samples were selected based on their NDCs content and ability to promote growth of common gut bacteria of Bifidobacterium bifidum and Lactobacillus plantarum [12,13]. Legumes with the most promising prebiotic potentials were further assessed in mice to assess their effects on SCFA levels and gut microbiota compositions. This study targeted caecum, the major fermentation site in mice (pH 4.4-4.6), and used metagenomic analysis of 16S rRNA gene sequences to characterize the microbial communities. The aim of this study was to investigate novel potential prebiotic sources from several assessed legume candidates.

  1. Materials and Methods

2.1. Selection of Legumes

A total of nine types of legumes that were not investigated as prebiotics were species grown and consumed by local people in Java, Indonesia. The minor legumes, including velvet beans (M. pruriens), bambara groundnut (Vigna subterranea), chickpea (Phaseolus vulgaris), calopo beans (Calopogonium mucunoides), snow pea (Pisum sativum var. saccharatum), winged beans (Psophocarpus tetragonolobus), sword beans (Canavalia ensiformis), red beans or senerek beans (P. vulgaris) and turi beans (Sesbania grandiflora).

2.2 Extraction of Carbohydrates

The prebiotic components in the legume samples were extracted using maceration method. Ethanol, a polar solvent known for its attraction to polar carbohydrates, was chosen as the extraction solvent. Then, 70% ethanol (v v-1) was used for maceration with a 1:10 sample:solvent ratio [14,15]. The mixture was set for 4 d and then filtered and the solvent was evaporated at 60 °C using rotary evaporator.

 

 

2.3. Assessment of non-digestible carbohydrate contents

Carbohydrate resistance of nine minor legume extracts was assessed using simulated stomach acid and digestive enzymes. Each extract was prepared as a 10% stock solution (w v-1) in distilled water (DW). Resistance was assessed using acidic and enzymatic digestions. Acidic digestion involved incubating 200 µl of 1% extract solution (v v-1) with 200 µl of HCl buffer (pH 1) at 37 °C for 4 h (16). The reaction was stopped with 1 N NaOH. Enzymatic digestion was followed by further incubation of 200 µl of acid-digested solutions with 200 µl of α-amylase enzyme (2 U.ml-1) in sodium phosphate buffer at 37 °C for 6 h. Heating at 80 °C for 10 min stopped the reaction. Each digestion was carried out in triplicate [15].

   The NDCs content was assessed using dinitrosalicylic acid (DNS) method for reducing sugars before digestion and the phenol sulfuric acid method for total sugars after acid-enzymatic digestion. Acid and enzyme analyses were carried out respectively to simulate digestion in the stomach and small intestine. The DNS method involved preparation of a reagent by dissolving 10 g of 3,5-dinitrosalicylic acid, 2 g of phenol, 0.5 g of sodium sulfite and 10 g of sodium hydroxide in 1 l of DW. Assay involved adding 100 µl of 1% legume extract solution (v v-1) and 100 µl of DNS reagent, followed by vortexing for 30 s, heating at 95 ℃ for 10 min (until color change), adding 33 µl of 40% sodium potassium tartrate
(w v-1) and diluting 10× with DW. Absorbance was measured at 540 nm using ELISA reader [17] For the phenol sulfuric acid method, 50 µl of digested solution were mixed with 50 µl of phenol solution and vortexed for 30 s; followed by mixing with 250 µl of H2SO4 (sample:phenol:H2SO4 ratio of 1:1:5), vortexing for 30 s, diluting 10× with DW and measuring the absorbance at 490 nm using ELISA reader (18). The NDC content in the extracts was calculated using Eq. 1 (15):

 

                                                   Eq.1

2.4. Probiotics Growth Stimulation

This study used the probiotic B. bifidum and L. plantarum. Bacterial growth was estimated using standard growth curve based on optical density at 600 nm and colony counts (CFU.ml-1)19. Bifidobacterium bifidum was incubated anaerobically at 37 °C, while L. plantarum was incubated aerobically at 37 °C using shaker incubators. The two bacteria were first propagated on MRS agar media for 24 h at 37 °C before inoculation into MRS broth media. After 24 h, inoculum was used to count cells and colonies. The OD 600 measurement at each dilution (100-10-10) estimated the number of cells. Colony counts for dilutions of 10-7, 10-8 and 10-9 were carried out using total plate count (TPC) method with triplicate plating.

Bifidobacterium bifidum and L. plantarum were propa-gated in each modified MRS broth growth medium. The MRS broth was prepared using 10 g of peptone, 4 g of yeast extract, 8 g of meat extract, 20 g of carbon sources (glucose, inulin and three types of legume extracts with the highest NDCs), 1 g of Tween 80, 2 g of potassium phosphate dibasic (K2HPO4), 5 g sodium acetate (CH3COONa.3H2O), 2 g of tri-ammonium citrate (C6H17N3O7), 0,2 g magnesium sulfate (MgSO4·7H2O) and 0,04 g manganese (II) sulfate (MnSO4·H2O), which were dissolved in DW up to 1 l [20]. The OD 600 was measured at 0, 24 and 48 h using ELISA reader. Then, number of the bacterial colonies was estimated from the standard curve.

2.5. Experiments on Animal Models

Promising prebiotic function legumes, based on their NDC levels and probiotic-stimulating abilities, were assessed in mice to investigate their effects on SCFA levels and gut microbiota compositions. Fifteen mice were equally divided into three groups of a negative control group fed with a standard diet, a positive control group fed with a standard diet supplemented with 5% inulin (w w-1) and a treatment group fed with a standard diet supplemented with 5% of the selected legume extract (w w-1). Each mice was housed individually, fed at 5 g per min rate with ad libitum water. Body weight and food intake of each mice were assessed daily for 28 d. After 28 d, mice were euthanized and their cecum were collected for further analysis.

2.6. Assessment of Short-Chain Fatty Acid Levels

   Analysis of SCFA levels in caecum contents was carried out using gas chromatography (Shimadzu GC-2010 Plus, Japan). First, 0.15 g of the sample was mixed with 1 ml of DW and centrifuged at 1008 × g for 10 min. Then, supernatant was analyzed using GC-202 Plus, Japan, provided by the Food and Agricultural Product Technology Testing Laboratory, Faculty of Agricultural Technology, UGM, Indonesia [21].

2.7. Metagenomic Analysis

   Genomic DNA was extracted from the caecum using ZymoBIOMICS DNA mini kit, following the manufacturer’s instructions. The extracted DNA concentration was assessed using BioPhotometer Plus (Eppendorf, Germany). Poly-merase chain reaction (PCR) amplified the V3 and V4 regions of the 16S rRNA gene using primers of 341F (CCTACGGGRGGCAGCAG) and 806R (GGACTACC-AGGGTTTCTA) [22]. Then, DNA sequencing was carried out using next-generation sequencing (NGS) method and MGISeq platform. All PCR and NGS studies were carried out at PT. Genetic Sciences Jakarta, Indonesia.

2.8. Data Analysis

Data were analyzed using SPSS software v.24.0 and one-way ANOVA with a significance level of α = 0.05. For significant differences, Duncan's multiple range test (DMRT) was used for post-hoc analysis. FASTA-formatted sequence data were analyzed with UPARSE v7.0.1001 to group sequences into OTUs (operational taxonomic units). Sequences with ≥97% similarity were assigned to the same OTU. Taxonomic profiling was carried out using QIIME v.1.7.0 and SILVA database. Multiple sequence alignment was carried out using MUSCLE v.3.8.31. Then, OTUs with abundance less than 0.005% were removed. Normalized OTU abundance was used to assess alpha and beta diversities. Alpha diversity analysis was carried out using QIIME v.1.7.0 and visualized using R v.2.15.3. This captured the within-habitat bacterial diversity, including the number of OTUs, Shannon-Wiener diversity index and Simpson index. Beta diversity, reflecting bacterial diversity between the habitats, was estimated using PCoA-based index calculated using FactoMineR, ggplot2 and R v.2.15.3.

  1. Results and Discussion

3.1. Quantity of Non-digestible Carbohydrates in Legumes

To qualify a compound as a prebiotic agent, its carbohydrates must resist digestion in the stomach and small intestine. Table 1 shows that velvet beans and bambara groundnut included significantly higher quantities of NDC, compared to other beans (p<0.05). Chickpeas demonstrated a relatively high quantities of NDC, compared to other legumes. In addition to the high quantity of NDCs, the low hydrolysis proportions of velvet beans (4.57%), bambara groundnut (8%) and chickpea (24%) indicated that most of the carbohydrates in these three beans were resistant to acid and enzyme digestions.

These results suggested that these three beans could reach the large intestine for selective fermentation by the beneficial bacteria. Based on the results, these three beans were further assessed in vitro to assess their abilities to stimulate beneficial bacteria.

Velvet beans (M. pruriens) are beans with high carbohydrates contents. Previous studies reported that raw velvet bean seeds contained up to 49.22% of carbohydrates [23] Total dietary fiber content of the velvet beans is known to reach 86.6 mg.g-1, which is higher than that of Canavalia gladiata and Vigna unguiculata [24]. Bambara groundnuts (V. subterranean) are known as food sources with high carbohydrate contents, reaching up to 64.4%. Previous studies have shown that most of the carbohydrates contained in bambara groundnut are dominated by oligosaccharides and polysaccharides [25]. Chickpeas (P. vulgaris) are known as sources of carbohydrates that consist of starch, fibers and oligosaccharides. Previous studies have reported that the total polysaccharide contents in chickpeas reach to 300-370 mg.g-1, while the indigestible carbohydrate contents in form of oligosaccharides reach to 41.8-85.3 mg.g-1 [26].

3.2. Propagation of Probiotics on Various Carbon Sources

The NDCs are qualified as prebiotics if they selectively encourage propagation of beneficial bacteria in the large intestine. This study investigated several prebiotic candidates known for their acid and enzymatic resistances, based on their high quantity of NDCs. The candidates included velvet beans, bambara groundnuts and chickpeas. The study assessed their abilities to stimulate propagation of the representative probiotics of B. bifidum and L. plantarum, commonly detected in GIT of humans and rodents [27,13]. Bacteria were cultured in MRS broth media with various carbon sources. Each medium contained 2% extracts (w v-1) of velvet beans (MRS-VB), bambara groundnuts (MRS-BR) and chickpeas (MRS-CP). Propagation of B. bifidum and L. plantarum on these sources was compared with those on cultures using 2% of prebiotic inulin (w v-1) (MRS-INU), 2% of glucose (w.v-1) (MRS+) and no carbon sources (MRS-). Figure 1a,b showed that the growth of B. bifidum and L. plantarum in MRS- media did not show significant increases after incubation for 48 h. Carbon source in the media is a substrate that is utilized by bacteria to form amino acids and other cell components, making it important for the multiplication of bacterial cells [28].

Figure 1a reveals diverse growth patterns for B. bifidum in various MRS media. Significantly, MRS-VB and MRS-CP media stimulated B. bifidum propagation after 24 h, compared to MRS-BR. However, the number of B. bifidum colonies in MRS-VB and MRS-CP media was lower than that in MRS-INU at 24 h (p<0.05). At 48 h, B. bifidum in media with the prebiotic candidate (MRS-VB, MRS-BR and MRS-CP) included a lower number than that it did in MRS+ and MRS-INU media (p<0.05). These results indicated that the three prebiotic candidates were not able to stimulate propagation of B. bifidum as well as inulin prebiotics. However, B. bifidum in MRS+ media included a better propagation rate, compared to that in MRS-INU. Despite its positive effects, glucose failed to control harmful bacteria such as Escherichia coli and Salmonella spp. This suggests that specific media and prebiotics might be needed to support particular beneficial bacteria while limiting harmful ones [29].

Figure 1b reveals that L. plantarum in MRS-VB, MRS-BR and MRS-CP media included a lower number of colonies at 24 h than that it did MRS-INU (p<0.05). At 48 h, L. plantarum in MRS-VB media included the highest number of colonies, compared to that it did in MRS-VB, MRS-CP and MRS-INU media. A carbon source of 2% velvet bean extract could stimulate propagation of L. plantarum to reach to 99.83 ±1.45 × 107 CFU.ml-1, which was significantly higher than inulin prebiotics and the other two prebiotic candidates (p<0.05). These results indicated that velvet beans included a better ability to stimulate L. plantarum during 48 h of incubation, compared to that inulin prebiotics did.

Proliferative Index was reported from log cell number at 48 h minus log cell number at 0 h. Table 2 shows proliferative index of the probiotic bacteria during 48 h of incubation in media containing various prebiotic carbon sources. Significantly, B. bifidum in MRS-VB media included a higher proliferative index and was significantly different, compared to that it did in MRS-BR and MRS-CP (p<0.05). However, the proliferative index of B. bifidum in MRS-VB media included a lower value, compared to that it did in MRS-INU and MRS+ (p<0.05). These results indicated that B. bifidum utilized inulin and glucose better than that the prebiotics in velvet beans did. These results were similar to previous results, which stated that propagation of B. bifidum could only be stimulated by inulin [30]. This was because B. bifidum could not utilize oligosaccharides such as fructooligosaccharides (FOS) and galactooligosaccharides (GOS) in legumes. Previous studies have reported that FOS-type prebiotics could stimulate propagation of all Bifidobacterium spp., except B. bifidum [30]. This was because B. bifidum did not include genes encoding β-fructofuranosidase enzyme, functioning to hydrolyze FOS [31].

   The proliferative index of L. plantarum (Table 2) in MRS-VB media showed the highest value, compared to that the other groups did (p<0.05). These results indicated that L. plantarum could better utilize carbohydrates in velvet beans better, compared to that inulin prebiotics and the other two prebiotic candidates did. Velvet beans (M. pruriens) are known to contain FOS prebiotics, which consist of glucose and fructose units linked by β(2-1) glycosidic bonds [32,33]. Previous studies have shown that L. plantarum can utilize FOS as a selective carbon source because it includes β-fructofuranosidase enzyme, which plays roles in FOS degradation [34,33]. Nucleotide sequence of the L. plantarum genome has demonstrated presence of the sacA gene, which expresses an enzyme that can hydrolyze FOS internally. Unlike extracellular enzymes in L. pentosus and L. paracasei, intracellular enzymes in L. plantarum can maximally utilize FOS prebiotics because there are no hydrolysis products that are consumed by other bacterial species in the large intestine. This reveals that L. plantarum utilizes FOS to compete with other microorganisms in the large intestine. Fermentation of FOS by L. plantarum produces secondary metabolites that inhibit propagation of pathogenic bacteria such as E. coli that produce β-glucuronidase enzyme [34].

3.3. Assessment of the prebiotic effects with prebiotic index

A prebiotic index value greater than 1 (>1) shows that a carbohydrate includes positive effects on propagation of the probiotic bacteria. Figure 2 shows that the prebiotic index of velvet beans and inulin in L. plantarum culture includes values greater than 1 (>1). Velvet beans included the highest value, compared to that inulin and other prebiotic candidates did (p<0.05). In B. bifidum culture, the three legume candidates included a lower proliferative index, compared to that inulin prebiotics did (p<0.05). These results demonstrated that prebiotics in velvet beans included good abilities to stimulate L. plantarum, compared to that the inulin did. Moreover, propagation of B. bifidum could only be stimulated by the inulin prebiotics.

3.4. Experiments on Animal Models: Changes in Body Weight and Food Consumption

Identified as promising prebiotic candidates, velvet beans were assessed in vivo using mice models. Mice in all three groups included similar body weights at baseline (Day 0) (Table 3). On Day 7, all groups gained weight with the treatment group (13.3%) showed the highest increase (not statistically significant), compared to the negative (12,3%) and positive controls (12,67%). By Days 21 and 28, all groups gained weight steadily with no significant differences between them (Table 4). However, all groups consumed more than 78% of the provided foods, demonstrating that prebiotics reached digestive system of all mice. Although significantly indifferent, treatment and positive control groups included lower body weights than that the negative control group did. A relatively high feed consumption in the treatment group did not cause significant increases in body weight of the mice. Previous studies have reported that velvet beans include anti-obesity effects by decreasing body weight in obese groups [11].

3.5. Short-chain fatty acid levels in animal models

Anaerobic bacteria primarily produce SCFA such as acetic, propionic and butyric acids via fermenting NDCs [35]. Significantly, positive control and treatment groups demonstrated higher levels of these SCFAs, compared to the negative control (Table 5). Levels of acetic and propionic acid in the treatment group significantly higher than those in negative control group (p<0.05), but significantly lower than those in positive control group (p<0.05). Levels of butyric acid in treatment and negative control groups were significantly lower than those in positive control group (p<0.05). This suggests that velvet beans supported propagation of the bacteria that produced these SCFAs, potentially leading to shifts in gut microbiome metabolism and SCFA production with benefits to gut health.

 Previous studies have reported that feeding germinated velvet beans can increase SCFA production in the cecum of broilers [36].

3.6. Diversity of the Gut Microbiota in Mice

   Structure of the microbiota community in each group was assessed using various alpha diversity indices (Table 5). Figure 3 shows a flattened rarefaction curve indicating that all OTUs in the three groups were detected.

Based on Table 5, Shannon index in the treatment group with velvet bean supplementation showed a lower value compared to the negative control (standard diet), but higher compared to the positive control (inulin supplementation). The Shannon index (H') is positively correlated with the diversity and evenness of bacterial species in a bacterial community [37]. These results revealed that species diversity and evenness in the treatment group were higher than the positive control but lower than the negative control. This was similar to the higher number of OTUs (species richness) in the treatment group compared to the positive control, but lower compared to the negative control. In addition to Shannon index, Simpson index was used as an indicator to estimate the diversity of gut microbiota in mice. A high Simpson index value demonstrates that the diversity of bacteria in a community is low. Furthermore, a higher Simpson index value indicates greater dominance of a particular bacterial species in the community [38]. Based on Table 5, it was detected that the Simpson index in the treatment group included a lower value, compared to the positive and negative control groups. These results showed that species diversity in the treatment group was higher than that in the controls. Moreover, a low Simpson index in the treatment group demonstrated a low dominance level, hence, it could be assumed that no community balances existed in the mouse gut bacteria. Bacterial community balance can create a positive ecosystem that includes good resistances to pathogens and improves digestive track health of the host [38].

Diversity between the groups (beta diversity) was analyzed using principal coordinates analysis (PCoA). Figure 4 shows the PCoA analysis, revealing distinct variations in the bacterial communities within the groups. Distances between the points that represent positive control and treatment groups on the PCoA plot show a closer proximity, indicating that the bacterial composition of these two groups included a greater similarity, compared to the negative control group. This verified that the bacterial communities in positive control and treatment groups were more similar to each other than the negative control.

3.7. Abundance and Composition of the Gut Microbiota in Mice

  Metagenomic analysis of the bacterial communities in mice cecal samples (Figures 5A) revealed that the Firmicutes and Bacteroidetes phyla included the highest relative abundance in all three groups. This finding is similar to that of previous studies, which showed that Firmicutes and Bacteroidetes were the dominant phyla in the gut of healthy individuals [41]. Abundance of Firmicutes in the treatment group (45%) was relatively lower than that in the positive (64%) and negative (62%) control groups. However, abundance of Bacteroidetes did not show significant differences between the three groups (20,23 and 21%). The Firmicutes/Bacteroidetes (F/B) ratio indicates homeostasis of the GIT. Increases or decreases in the F/B ratio reveal dysbiosis, which is associated to metabolic and inflammatory disorders. Increased F/B ratios are associated to obesity, which is characterized by increased abundances of the Firmicutes phylum [42]. Previous studies have reported that obese individuals received high-fiber diets for 1 y had decreased Firmicutes abundances [43]. Similar studies have reported that African children who consumed high-fiber diets had lower Firmicutes abundances than that children who consumed fast food [44] The current study revealed that the F/B ratio in the treatment group was lower than that in positive and negative control groups. This suggests that a 5% supplementation of velvet bean extract includes anti-obesity characteristics in mice. In addition to the dominant phyla, treatment group demonstrated enrichments in Proteobacteria (2%) and Actinobacteria (0.8%), compared to the control groups (respectively 0.8-4 and 0.5-1%).

 Significantly, Desulfobacterota, Deferribacterota and Campylobacerota showed significant enrichments in the treatment group (14, 12, 8%), compared to controls (respectively 4-7, 3, 2-5%).These findings highlight potential shifts in gut microbial composition following the interventions.

Figure 5B reveals that Lachnospiraceae dominated gut microbiota in all groups. However, the treatment group (21%) showed significantly lower abundance of Lachnospiraceae, compared to positive control (39%) and negative control (40%). While genera within this family produce beneficial metabolites, the high abundance was linked to glucose metabolism disorders and inflammatory bowel disease (IBS) [45,46].

Interestingly, Helicobacteraceae (8%), Deferribacteraceae (12%) and Desulfovibrionaceae (14%) were significantly enriched in the treatment group, compared to positive and negative control groups (2-4 and 3-7%, respectively). Significantly, Lactobacillaceae (7–11%) was abundant within all groups (Figure 5C).

Figure 6C shows genera that belonged to the top 10 highest abundances in all three groups. The Lachnospiraceae NK4A136 group, Blautia and Lachnoclostridium were the most identified genera from the Lachnospiraceae family. The Lachnospiraceae NK4A136 group included a lower relative abundance in the treatment (3%) and positive control (2%) groups, compared to the negative control group (18%). The Lachnospiraceae NK4A136 group include anaerobic bacteria that could produce SCFAs through the fermentation of polysaccharides, making them beneficial bacteria. However, previous studies reported that the negative control group included higher abundance of Lachnospiraceae NK4A136 group, compared to the group fed prebiotic tape ketan diets. The study showed that the Lachnospiraceae NK4A136 group was more abundant in stressed mice and its abundance decreased after feeding with prebiotic tape ketan diets [47].

Blautia genus, belonging to the Lachnospiraceae family, exhibited a diminished relative prevalence in the treatment (6%) and negative control (5%) groups in contrast to the positive control group (12%). Blautia is addressed for its involvement in synthesis of SCFAs, particularly propionate and butyrate. Similarly, Lachnoclostridium genus demonstrated a comparatively decreased prevalence in the treatment (2%) and negative control (3%) groups, compared to the positive control group (11%). Lachnoclostridium is highlighted for its anti-inflammatory characteristics and production of butyrate (48, 49). The higher propionate and butyrate levels in the positive control group (Table 5) support the current findings, as these levels were significantly different from that of the treatment and negative control groups.

Based on Figure 6C, Helicobacter genus from Helicobacteraceae included a higher relative abundance in the treatment group (8%), compared to the positive (2%) and negative (5%) control groups. Several Helicobacter spp. were detected in the treatment group, including H. typhlonius, H. bilis, H. japonicus, H. apodemus, H. rodentium and H. ganmani. Although most Helicobacter spp. in the GIT are potential pathogens, studies have investigated interactionS of Helicobacter spp. with other microbiota members that create positive effects. Helicobacter in the mouse GIT can enhance resistance to infectious diseases caused by Citrobacter rodentium and prevent infection without adaptive immunity [50]. Previous studies reported that H. bilis colonization in the cecum could induce specific immune responses in form of immunoglobulin G (IgG) activation, which could protect mucosal layers from Mucispirillum schaedleri infections [51].

Mucispirillum, part of the Deferribacteraceae family, showed higher abundance in treatment group (12%), highlighting potential interactions with the velvet bean interventions, compared to the control groups (3%). Identified species from this genus included M. schaedleri, which is known to invade the cecal mucosal layers and cause intestinal inflammations [52]. In addition to its negative effects, Mucispirillum is known to interact with the mucus and create a positive environment for the bacterial propagation in GIT. Previous studies detected that M. schaedleri, which colonized the mucosal layers played positive roles in protecting the host's GIT by forming a mucus barrier against the pathogen infections [53]. The studies showed that mice infected with Salmonella typhimurium that were previously colonized with M. schaedleri experienced significant decreases in intestinal inflammation, compared to the controls. Generally, M. schaedleri is antagonistic to S. typhimurium and can therefore inhibit virulence factors, tissue invasion and inflammation [52].

   Bacteroides spp. in the treatment (8%) and negative control (12%) groups included higher abundances, compared to the positive control group (5%) (Figure 6C). Bacteroides spp. produce SCFAs that can be absorbed by the intestine and include positive effects on the hosts [53]. Studies have shown that Bacteroides spp. are low in abundance in people with diarrhea, indicating that high abundances of Bacteroides spp. are positively correlated with digestive health [54]. Identified species from the genus Bacteroides included B. uniformis, B. acidifaciens and B. vulgatus. Techniqually, B. uniformis has been identified as a potential therapeutic agent for obesity due to its demonstrated capacity to restrict weight gain and increase butyrate concentrations within the intestinal tract [55]. Detected broadly in high-fiber diets, commensal gut bacteria of B. acidifaciens offer multiple health benefits. These bacteria help prevent obesity and improve insulin sensitivity, potentially decreasing risks of type 2 diabetes mellitus [56]. Known for its enzyme production, B. vulgatus degrades complex polysaccharides into SCFAs, offering gut health benefits [57]. This versatile bacterium possesses the ability to fight against pathogenic infections associated with inflammatory bowel disease (IBD) [58]. . Additionally, B. vulgatus serves as an anti-obesity agent and helps hyperlipidemia treatment, expanding its health effects [59].

   The relative abundance of Lactobacillus was lower in the treatment group (3%), compared to the positive and negative control groups (5% each). Lactobacillus is a genus known as probiotic bacteria widely used in the production of functional foods. However, abundance of Lactobacillus spp. is relatively low in the GIT [60]. Although Lactobacillus spp. include positive roles in host health as studies have reported that Lactobacillus spp. with high abundance are detected in groups with digestive infection diseases, IBD, diarrhea and IBS [61]. A majority of species detected in the treatment and negative control groups were L. intestinalis, while the species detected in the positive control group were L. intestinalis and L. hominis. The L. intestinalis, recently recognized as a probiotic agent, offers protective and homeostatic benefits for GIT. Studies have shown that the microorganism modulates the immune system to combat colitis infections in mice [62]. In addition to Lactobacillus, Ligilactobacillus genus was detected to include a higher relative abundance in the treatment group (3%) than the positive control (1%). Species of the Ligilactobacillus genus that have been investigated as probiotics include L. salivarius [63]. However, this study was unable to identify Ligilactobacillus to the species level.

   The relative abundance of Bifidobacterium spp. in the treatment (0.2%) and positive control (0,3%) groups was higher than that in the negative control group (0.04%). Similar to the established prebiotic inulin, supplementing mice with velvet beans significantly increased the presence of Bifidobacterium spp. in their caecum. This genus largely includes probiotic species. Bifidobacterium genus detected in the positive control group was dominated by B. animalis, while the treatment group was dominated by B. pseudolongum. The B. animalis, a probiotic species, modulates gut microbiota composition for anti-obesity effects and protects GIT by enhancing intestinal barrier functions [64]. Studies have shown that the microorganism improves insulin sensitivity and restores blood glucose homeostasis in diabetic mice [65]. Furthermore, B. animalis produces beneficial organic acids such as acetic acid, linoleic acid and DHA [66]. Well-known for its anti-tumor characteristics, B. pseudolongum as a probiotic that combats non-alcoholic fatty liver disease (NAFLD) boosts intestinal barrier functions and restores healthy gut microbiota compositions [67,68].

  1. Conclusion

This study assessed several legumes with potentials of development as prebiotic sources or functional foods. Bambara groundnuts, velvet beans and chickpeas passed the initial selection due to their high quantities of NDCs as a prebiotic characteristic. Velvet beans showed a better ability to stimulate B. bifidum and L. plantarum probiotics that were assessed in vitro, compared to bambara groundnuts and chickpeas. Thus, these passed the second selection. The abundance of Lactobacillus spp. in the velvet bean group (3%) was lower than that in the two controls (5%). Presence of prebiotic characteristics in velvet beans was validated by the increased levels of SCFAs in the cecum of mice as a result of NDC fermentation. Supplementation of velvet beans in mice can alter gut microbiota compositions. This alteration involves balancing the bacterial community, which is positively correlated with enhanced host defense systems.

  1. Acknowledgements

This study was supported financially by Sebelas Maret University.

  1. Conflict of Interest

This study was supported financially by Sebelas Maret University.

  1. Authors Contributions

Conceptualization and design of the experiments, A.P and S.L.A.S; methodology, A.P and A.E.P; carry out of the experiments, A.E.P; analysis of data, A.E.P; contribution to reagents/materials/analysis tools, A.P; original draft preparation, A.E.P; review and edition, A.P. and S.L.

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Background and Objective: Chocolate is a trendy food consumed by various age groups. It has been hypothesized that shocolate can become a significant functional product by incorporating probiotics into it. In this study, chocolate was used as a food matrix to transfer probiotic microorganisms to it. Bitter chocolate was chosen due to its preference by the consumers. Therefore, free and microencapsulated probiotic cultures were prepared.

Material and Methods: Lactiplantibacillus plantarum was used as the probiotic microorganism and calcium-alginate gel capsule was used for microencapsulation. The number of microorganisms and sensory characteristics of free, microencapsulated probiotic culture and culture-free bitter chocolates were assessed after 60 d of storage at 18 °C.

Results and Conclusion: Based on the results, count of the microorganisms in probiotic chocolate was 5.8×107 CFU g-1 on Day 0, while it decreased to 1.7×107 CFU g-1 on Day 60. Although decreases were seen in the level of probiotics, it has been shown that shocolates included sufficient counts of probiotics to be reported as probiotic chocolates. The microbial count of probiotics in microencapsulated probiotic chocolate (2.1×107 CFU g-1 on Day 0) decreased significantly to 2.4×105 CFU g-1 on Day 60. The highest microbial count was observed in samples containing free probiotic cultures after 60 d. However the microbial count did not decrease significantly in samples containing free cultures, 2-log decreases were observed in microencapsulated cultures. Thus, chocolate can be used as matrix for the probiotics. For sensory analysis, sample containing free culture was the most preferred after 60 days of storage regarding the overall acceptability.

Conflict of interest: The authors declare no conflict of interest

1. Introduction

Consumption of functional products, including probiotic foods, has increased worldwide. Pursuit of healthy eating and lifestyle has affected humanity in recent years, leading to health-conscious individuals to turn to functional foods. Probiotics, live microorganisms with positive health effects when consumed appropriately, are incorporated into foods, enhancing nutritional and technological characteris-tics of the foods. These functional probiotic foods promote intestinal health by increasing beneficial microorganisms, preventing diarrhea and inhibiting harmful pathogen colonization. Other benefits include lowering blood cholesterol, strengthening the immune system and neutr-alizing cancer-causing compounds. Addition of probiotics to foods is critical for the human health [1-4]. Lactiplanti-bacillus plantarum (L. plantarum) is a microorganism belonging to the probiotic microorganism group [5-7]. Addition of probiotic microorganisms to food products can lead to decreases in the number of probiotic cultures due to the stressful environments. To minimize these barriers, techniques such as the selection of bacterial strains, regulation of food processing processes and microencap-sulation have been developed and used to protect probiotic bacteria. Microencapsulation is the process of entrapping microorganisms with appropriate carrier support materials. This creates a film layer around the cells, protecting the cell viability against the barriers. The most studied technique in this method includes extrusion coating based on the forming calcium-alginate gel capsules [7].

Alginate is used in the microencapsulation method due to several advantages. These include being non-toxic to the body, having characteristics that easily encapsulate bacteria, being safe for foods, being inexpensive and being soluble in the intestines. Size and shape of the beads formed in the microencapsulation method depend on the diameter of the needle; through which, alginate is transferred, density of the alginate and distance; to which, the alginate is transferred [8-12]. Probiotic foods include approximately 60-70% of the functional food market. Although a majority of the probiotic products are yogurts and fermented dairy products, production of non-dairy probiotic products such as chocolates has increased in recent years [10,13]. Cocoa butter, sugar and cocoa particles include basic components of the chocolates. Researchers have reported that chocolate has characteristics that can carry probiotic microorganisms and tolerate adverse effects of the gastrointestinal system [14,15]. It has been reported that Bifidobacterium lactis, Lactobacillus (L.) acidophilus, L. paracasei, L. casei and L. rhamnosus probiotics have successfully been used in production of chocolate and cocoa desserts. Based on a similar study results, no difference was found in sensory characteristics and the products could be used as carrier matrices for the probiotics, comparing probiotic-added products with control samples [16].

The primary aim of the present study was to decrease digestive problems caused by the changes in the intestinal microbiota due to the changes in the current diet systems and frequent uses of fast ready-to-eat meals. Reactions can develop against the probiotic isolates, especially in childhood. The target includes development of intestinal microbiota by adding probiotic cultures to foods such as chocolates, which are loved by the people of all age groups. So, aim of this study was to add L. plantarum probiotics (microencapsulated and free) to bitter chocolate samples that provided them functional characteristics. A probiotic strain, which was a food supplement in capsule form, was used in the study. This was a novel approach for the chocolate matrix. In addition, temperature value assessed as the storage temperature in functional chocolate experiments was a temperature value that was not used previously. Changes in the number of microorganisms in chocolates turned into functional products at the end of the storage and their sensory characteristics were assessed by trained panelists. Another aim included assessment of the matrix characteristics of chocolates in carrying probiotic cultures.

2. Materials and Methods

Bitter chocolate was purchased from Solen Cikolata Gida Sanayi, Gaziantep, Türkiye. The probiotic microorganism used included L. plantarum 299v. This microorganism (later named L. plantarum) was isolated and used from a commercially available probiotic food supplement (Probest, Abdi Ibrahim Ilac Sanayi ve Ticaret Anonim Sirketi, Istanbul, Türkiye). McFarland Unit Cell Densitometer (Biosan SIA, Latvia) is used to measure cell concentrations. All the chemicals were purchased from Merck, Germany.

2.1. Chocolate

Chocolate production includes several stages. In the final stage, semi-finished chocolate is tempered, molded and packaged. In the tempering process, semi-finished chocolate is melted at 50 °C and then cooled down to 28 °C. This process is repeated 4-5 times. As a result of the process, chocolate is poured into molds. With the tempering process, chocolate becomes much smoother and shiny. Semi-finished chocolate was used in the current study and no tempering process was used to assess the matrix formation characteristics of chocolates for the probiotic microorgan-isms. Chocolate production was designed in three various ways. The first sample included normal chocolate without microbial culture, the second sample contained free-form probiotic microorganism culture (L. plantarum) and the third sample included microencapsulated probiotic L. plant-arum culture (Figure 1). In preparation of the chocolate, semi-finished chocolate was melted at 50 °C using water bath and then transferred to sterile molds [17]. Molded chocolate was cooled down at room temperature and set to harden. Chocolate was stored at 18 °C until analysis. Characteristics of bitter chocolate used as semi-finished product in the study are listed in Table 1.

2.2. Addition of Probiotic Microorganisms to Chocolates

In this study, L. plantarum probiotics were used. The L. plantarum was cultured in MRS media at 37°C for 24 h. To quickly assess the probiotic culture for the addition to chocolates, bacterial count was set to 1 McFarland (approximately 3.0×108 CFU ml-1). In assessment of the exact number of the bacteria, necessary dilutions were prepared and the number of microorganisms was assessed using spreading method. Probiotic and microencapsulated cultures were added to the chocolates and the chocolates were then transferred to sterile packages. The mixture was allowed to cool down at RT until the desired hardness was achieved. The chocolate packages were sealed and stored at 18 °C using incubator. Number of microorganisms was assessed on Days 0, 30 and 60 of storage and sensory analyses were carried out [18,19]. All studies were carried out in two parallels and three replications.

2.3. Microencapsulation of Probiotic Microorganisms

Extrusion technique was used for the microencaps-ulation of probiotic microorganisms. During preparation of the probiotic cultures, cultures were centrifuged at 3000 g after incubation in MRS media at 37 °C for 24 h). After discarding the supernatant, cultures were dissolved and concentration of the microorganisms increased using 1 McFarland (approximately 3.0×108 CFU m-l). Microorgan-isms were added into a previously prepared 1% sterile sodium alginate solution using syringe. The homogenized alginate with bacterial mixture was transferred to a 1% (w v-1) sterile CaCl2 solution [18]. The alginate with the bacterial mixture was homogenized in CaCl2 solution using magnetic stirrer and then solidified. Beads were filtered using Whatman no. 4 filter papers and transferred to sterile Petri dishes [20]. During chocolate production, microencap-sulated culture from these Petri dishes was homogeneously added to the chocolates [21].

2.4. Assessment of the Number of Probiotic Microorganisms

The L. plantarum probiotic was homogenized using sterile dilutions containing 0.85% (w v-1) salt and 0.1% (w v-1) peptone. Briefly, 1 ml of the homogenized sample was diluted with dilution fluid. Diluted samples were spread plated on de Man, Rogosa and Sharpe (MRS) agar and incubated at 37 °C for 48 h [22]. After incubation, number of the microorganisms was assessed. To assess number of the microencapsulated L. plantarum, sterile sodium citrate buffer was added to the sample and homogenized for 15 min using magnetic stirrer. Then, 1 ml of the homogenized chocolate was used to prepare sufficient dilutions [21]. Number of the microorganisms was assessed using sprea-ding method. After incubation at 37 °C for 48 h, number of the microorganisms was assessed. All studies were carried out in two parallels and three replications.

2.5. Sensory Analysis of Probiotic Chocolates

For sensory analysis, a panel of ten people was selected and trained. Samples were assessed for appearance (smoothness-appearance, brightness and blooming), aroma (cocoa flavor and off-flavor), taste (cocoa taste and sweetness), texture during the first bite (hardness and brittleness) and chewing (strength, smoothness-texture, melting level, stickiness, spread and mouth covering), color (brown color) and overall acceptability. Participants rated each characteristic on a scale of 1-9. Numbers representing the scale were established as 9, extremely like; 5, neither like nor dislike and 1, extremely dislike [17].

2.6. Statistical analysis of Probiotic Chocolates

Findings of this study were reported as means of triplicate data with standard deviations (SD). Analysis of data was carried out using ANOVA. Significant differences (p<0.05) in individual means were assessed using Tukey’s HSD test. The SPSS software v.22.0 (IBM, Chicago, USA) was used to analyze the results. Results were expressed as means ±SD.

3. Results and Discussion

In the study, number of the probiotic microorganisms in each chocolate (chocolate without probiotics, probiotic chocolate and microencapsulated chocolate) after storage was assessed and sensory characteristics of the chocolates were analyzed.

3.1. Microbiological Analysis

With the microbial analysis results, potential of the bitter chocolate as a matrix for L. plantarum was assessed. Results of the microbiological analysis are present in Table 2. Based on Table 2, it was assessed that chocolate without added probiotics did not contain microorganisms on Days 0, 30 and 90. Chocolate is generally not a product; in which, microorganisms can multiply. Moreover, it was assessed that the microorganism counts of chocolate with probiotics were 5.8×107 CFU g-1 on Day 0, 2.5×107 CFU g-1 on Day 30 and 1.7×107 CFU g-1 on Day 60. It was shown that number of the microorganisms did not change significantly (p<0.05) after storing probiotic chocolate for 60 d. It was concluded that chocolate could be used as a matrix for probiotics as the number of microorganisms was 1.7×107 CFU g-1 on Day 60. When free form of L. plantarum was added to chocolates, no significant changes (p<0.05) were observed in the number of microorganisms for 60 d. Although addition of high quantities of the culture was initially carried out to achieve a high culture count in the final products, high quantities of the free culture could adversely affect taste of the chocolate. For the probiotic microorganisms to include beneficial effects on human health, probiotics should be present in foods at a level of 106-107 CFU g-1(or ml-1) [23,24]. Regarding the results, it has been demonstrated that the measured free probiotic culture was included in these values with beneficial effects. Several studies support these results. A study reported bitter chocolate as an appropriate matrix for the transport of B. breve NCIM5671 strain [25]. Another study stated that the number of probiotic bacteria added to chocolates was constant [26].

Based on Table 1, 2.1×107 CFU g-1 microorganisms were inoculated into the chocolate in production of microen-capsulated chocolates. Number of the microorganisms was 6.0×105 CFU g-1 on Day 30 and 2.4×105 CFU g-1 on Day 60. Furthermore, number of the microorganisms decreased significantly (p<0.05) at the end of Day 30. Number of the microorganisms did not change significantly (p<0.05) within 30 and 60 d of storage. In a study by Erginkaya et al., [17], it was stated that bitter chocolates stored at 4 and 25 °C for 60 d showed differences in physical characteristics due to storage at various temperatures. It was also stated that a storage temperature of 25 °C caused significant decreases in the microbial count. In the present study, a storage temperature of 18 °C was set, which was within the temperature range in the highlighted study and the commercial storage temperature for chocolate. Studies showed that storage temperature caused decreases in microbial counts at 4 °C and probiotic cell concentration decreased by 1-2 log CFU g-1. At 25 °C, microbial counts decreased by 4-7 log CFU g-1 [14,18,27]. Based on the results, it was clear that chocolate could be used as a matrix to include probiotics. Several studies support this conclusion [14-17].

The global market for probiotic foods, supplements and probiotic foods is growing significantly. Therefore, cell encapsulation is emerging as an alternative for the incorpo-ration of probiotics into various food matrices. [28]. In the present study, alginate concentration used for the capsule formation was set at 1% (w v-1); thus, avoiding adverse effects on the appearance of chocolates. However, capsules formed within the gel concentrations did not sufficiently protect the probiotic cultures. In addition to the studies supporting these results, there are studies indicating that the microencapsulation process is an effective method for protecting probiotics. In a study, although starch-alginate capsules included the highest survival rate within the encapsulated cells, less than 1 log CFU g-1 of loss occurred [29,30].

In another study, probiotic strain of Limosilactobacillus reuteri DSM 17938 and non-probiotic strain of L. plantarum 48M were microencapsulated in alginate matrix using emulsion technique. Survival of microorganisms in microcapsules was assessed against gastric conditions and heat stress. Results showed that the microencapsulation process increased viability of L. plantarum 48M cells following exposure to gastric conditions, resulting in similar survival to L. reuteri DSM 17938. Additionally, it was stated that microencapsulation could not protect L. reuteri DSM 17938 and Lactiplantabacillus plantarum 48M cells when exposed to heat treatment [31]. There are studies indicating that microencapsulation is effective in protecting probiotics against adverse environmental conditions [5,6]. The fact that the microencapsulation process cannot protect microorganisms exposed to heat in the results shows that decreases in the number of microorganisms as a result of the microencapsulation process may be a normal result. Studies have demonstrated that chocolate can be used to transport probiotics without the need of microencapsulation process [18,32-35]. İn contrast, there are several studies indicating that the use of probiotics in foods via micro-encapsulation method is more effective than the use of free probiotics [11,13,23,30,36-40]. Chocolate enriched with encapsulated probiotics (probiotic-chocolate) and control (chocolate with non-encapsulated probiotics) were stored under aseptic conditions at 25 and 4 °C for 120 d. Relatively, when the protective effects of probiotic chocolates were compared, encapsulated chocolates includ-ed further probiotics (at least 2.0 log) after 120 d, compared to non-encapsulated probiotics. This verified that the encapsulates were protective under the two storage conditions [41]. Despite the encapsulation process, probiot-ics could be affected by stress conditions and the microencapsulation process could be affected by several parameters. These parameters were assessed as the size of microcapsules [29, 42], encapsulation method and coating materials as well as the concentration [9,18,24,28,37,40,43]. In production of capsules, encapsulation is based on alginate materials and addition of prebiotics to alginate increases protection of the capsules [28,29,40]. Studies have shown that the survival rates of probiotics depend on the type of probiotic, type of chocolate, storage temperature and time [18,32,43,44].

In a study by Sedefoglu et al. [7], ice cream samples were encapsulated with alginate to assess bacterial counts. They concluded that encapsulation did not provide additional protective effects to the probiotics. In cases; where the storage duration exceeded 120 d, microorganisms in microencapsulated samples were still viable. In a study, encapsulated L. plantarum 564 and commercial probiotic L. plantarum 299v were added in the production of bitter chocolates and it was seen that the probiotic bacteria survived very well after the production and during storage [45]. Although effects of alginate encapsulation on the survival of lactic acid bacteria in the food matrix has been studied, uniformity in the encapsulation procedure has not been identified in studies yet. Studies vary in capsule size, alginate concentration, calcium chloride concentration, hardening time of the capsules in calcium chloride and the initial number of the cultures, leading to differences in the survival of encapsulated bacteria. In summary, a higher number of microorganisms were detected in chocolates produced using free probiotic culture than in those with added probiotic cultures through microencapsulation. Additionally, it has been reported that chocolate can be used as a matrix to include probiotics.

3.2. Sensory Analysis Results

Sensory analysis results of the probiotic bitter chocolate samples during storage are provided in Tables 3 and 4. Description of the characteristics is as follows. Smoothness-appearance, smooth appearance of the product’s surface without lumps of grits; brightness, intensity of light reflection in the product, opposite of opaque; bloom, white-gray layer of the visible lipid/sugar crystals on the surface; cocoa aroma, aroma of the cocoa powder (from none to very); off-flavor, unpleasant and unwanted aroma in the product; cocoa taste, intensity of the taste of cocoa in the product; sweetness, taste quality most often associated with sucrose (none to very); hardness, force needed to cut the food using central incisor teeth; brittleness: breakage level of the piece of chocolate in first bite; strength, force needed to compress samples between tongue and palate; smoothness-texture, levels of even and consistent continuity of the product in mouth; melting level, time needed to melt half of the sample while chewing (slow to fast); stickiness, level of stickiness to molar teeth; spread, level of covering surface of the mouth; mouth covering, the after-feel film, which covers the mouth surface; brown color, light brown to dark brown; general acceptability, acceptability of the product in general with all of its sensory characteristics [7].

As seen in Table 3, no significant level (p<0.05) was detected in sensory characteristics of the chocolates without probiotics, probiotic chocolates or microencapsulated chocolates on Days 0 and 30. Sensory characteristics of the chocolate samples on Day 60 showed significant differences (p<0.05). From daily parameters, the highest smoothness value was assessed in the microencapsulated chocolate sample. This was due to the structure of the chocolate changing after a certain time. It was assessed that the brightness, blooming, cocoa flavor, off-flavor, cocoa taste [similar importance as probiotic chocolate (p<0.05)], hardness, stickiness and brown color parameters of the chocolates without probiotics were significant (p<0.05).

It was also stated that cocoa taste, sweetness, brittleness, strength, melting level, spread and brown color [similar importance as chocolate without probiotic (p<0.05)] were significant as well (p<0.05) in probiotic chocolates. In microencapsulated probiotic chocolate, smoothness-appearance, strength [similar importance as probiotic addition (p<0.05)], smoothness-texture and mouth covering characteristics were statistically significant (p<0.05). Various characteristics of chocolates were assessed through sensory analyses. On Day 60, it was reported that eight sensory parameters of the chocolates without probiotics, eight sensory parameters of the chocolates with probiotics and four sensory parameters of the microencapsulated probiotic chocolates were significant (p<0.05). It has been assessed that chocolates with probiotics were different significantly (p<0.05) from chocolate with microencapsul-ation and chocolate without probiotics addition in general acceptability. This is one of the important characteristics in sensory analyses. At the end of 60 d of storage, the most favored chocolate was that containing free culture, followed by the microencapsulated chocolate. Graphical represen-tation of the results is provided between Figures 2 and 3. Table 4 presents sensory analysis results of the probiotic bitter chocolate samples over the storage, based on the added cultures. Based on Table 4, sensory analysis results on Days 0, 30 and 60 were provided based on the added cultures. In chocolate samples without probiotics, the brittleness parameter included a significant level (p<0.05) on Day 0, whereas this characteristic decreased on Days 30 and 60. Regarding sensory parameters of the probiotic chocolate, hardness showed significant decreases (p<0.05) over time. In general acceptability parameter, the highest value was reached after 60 d. It was recorded that cocoa taste and hardness parameters of the chocolate containing microencapsulated probiotics decreased significantly on Day 60. Based on the general acceptability results, chocolate containing probiotics was acceptable at a significant level (p<0.05) depending on the 60-d storage. Studies indicate that the addition of probiotics to affect functional characteristics of the chocolates does not make changes in sensory characteristics [16,18,32-36,45]. In a study, sensory and physiological characteristics such as color, texture, rheology and melting profile of bitter chocolates with added probiotic cultures and control bitter chocolate samples without probiotics were compared. Results showed that physiological characteristics of the chocolate containing probiotics and control chocolate did not differ significantly [25].

Other studies reported that chocolates with microencap-sulated probiotics received similar scores in sensory evaluations, compared to those containing free probiotics [18,35]. In another study, it was assessed that microcapsules containing bitter chocolate included no differences with two commercially available chocolates based on the hedonic sensory assay [26]. Although it was stated that adding probiotics to chocolate varieties did not include negative effects based on the sensory assessment results; however, the study reported significant decreases in the general liking scores of all chocolate samples after 60 d of storage [18]. In another study, chocolates were investigated for aw, pH, surface color and morphology, hardness, microbiological quality, sensory acceptance and probiotic viability. After 120 d of storage at 25 °C, probiotic populations in chocolate decreased by a maximum of 1.4 log due to the stress environment. Based on these results and a good acceptance of all samples by the panelists, semisweet chocolate can be addressed as a good matrix for probiotics [33].

4. Conclusion

This study concludes that chocolate is an appropriate food for carrying probiotic cultures. Additionally, importance of chocolates containing probiotic cultures emerges due to their popularity within children and difficulty in supplementing children foods with probiotic cultures. By adding probiotic cultures to chocolates, functional foods can be prepared. The fact that chocolates containing free culture did not show decreases in the number of microorganisms indicates that cultures can be added without the microencapsulation process. No changes in sensory characteristics were observed after adding probiotic cultures to the chocolates and their general acceptability values were higher than those of other samples. High values were achieved in microbial and sensory analysis results by adding free probiotic cultures to the chocolates without microencapsulation. This eliminates needs of further labors, times and costs for the microencapsulation process. Thus, chocolates can be added with functional characteristics using added free probiotics. Several parameters were effective in low detection of probiotics as a result of the microencapsulation process. Microencapsulation method used in the present study included container material, CaCl2 volume and alginate section, where various microencapsulation blockers were stored. As a result of the study, it is concluded that chocolate can be used as a matrix in distribution and formation of probiotic cultures. The current study suggests further studies to carry out. These studies can involve various probiotics, adjusted storage temperatures and durations and modified parameters in microencapsulation processes

5. Acknowledgements

The author expresses his deepest gratitude to Kilis 7 Aralık University, BAP Unit, for its support provided under the current project of 21-13392.

6. Conflict of Interest

The author declares no conflict of interest.

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Assessment of the Physicochemical, Antioxidant, in-vitro Anti-diabetic and Nutritional Characteristics of Pigeon Pea Protein Hydrolysates

Jyoti Mukherjee, Nagaveni Shivshetty, Venkata Giridhar Poosarla

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e19
https://doi.org/10.22037/afb.v11i1.44371

Background and Objective: Cajanus cajan (pigeon pea) seeds include special characteristics that can serve as alternative vegan protein sources. The aim of this study was to investigate bioactive peptides in the pigeon pea using economically feasible method of acid and enzymatic hydrolysis.

Material and Methods: In this study, pigeon pea was subjected to hydrolysis by two methods of acid and enzymatic hydrolysis. The generated hydrolysates were characterized by result analysis of the protein content and yield, degree of hydrolysis, anti-nutritional profile, Fourier transform infrared spectroscopy, antioxidant assay of 2,2-diphenyl-1-picryl hydrazyl, hydroxyl radical scavenging assay, metal chelating ion assay and reducing power. Moreover, antidiabetic effects were assessed using α-amylase inhibition assay.

Results and Conclusion: Pigeon pea was digested by acid (pH 4) and enzyme hydrolysis, further subjected to membrane filtration to achieve peptide fractions with bioactive characteristics. The hydrolyzed pigeon pea showed good increased protein contents and degree of hydrolysis, compared with the control. Degree of hydrolysis were 62.7% for acid, 68.42% for enzyme and 34.32% for unhydrolyzed proportion. Hydrolyzed samples included Fourier transform infrared  peaks at 3500–4000 cm-1, showing amides I and II. The resulting peptides after the hydrolysis showed a higher range in acid hydrolysis (250–20 kDa), whereas the EH fractions showed a very low molecular weight of less than 15 kDa. Peptides produced by AH demonstrated considerable bioactive characteristics, compared to EH antioxidant and anti-diabetic characteristics against the standards. This study highlights production of pigeon pea protein hydrolysates using two methods of traditional (acid) and modern (enzymatic), showing that acid hydrolysate can be a cheap economical method for generating protein hydrolysate with good bioactive characteristics.

 1. Introduction

Recently, high demand for plant proteins have been reported due to production of plant-based meat, shifting consumer preferences and increased awareness of their role in health and fitness. Legume seeds include a critical place in the human diet worldwide as a rich source of proteins. Legumes are classically called the poor man’s meat when animal proteins are limited or when poverty, spiritual or holy preferences prevent consumption of meat. One of the most essential dietary legumes is Cajanus cajan, commonly known as pigeon pea and red gram in English [1, 2]. Production statistics of pigeon pea reveal that India contributes to nearly 90% of the global production. Despite having a high nutritional profile, pigeon pea is underused and received little attention from research and development to unlock its potential uses in food industries. Cajanus cajan is a significant source of proteins, vitamins and minerals rich in essential amino acids (EAA), with large quantities of lysine, which is often a limiting factor in plant-based proteins within the dietary legumes playing critical roles in human nutrition. Numerous studies on pigeon pea have revealed significant findings such as antioxidant, anti-hypertensive, anti-diabetic, anti-carcinogenic, anti-coagulant, anti-inflammatory and certain satiety effects. It is the most appropriate alternative for individuals with allergies or sensitivities to other popular sources such as soy, dairy, or wheat. It offers a hypoallergenic option for incorporating proteins into various food systems. Pulse proteins are a rich source of potential bioactive sites with the help of hydrolysis, autolysis, gastrointestinal digestion and fermentation. Complex protein in pigeon pea when subjected to artificial hydrolysis, natural gastrointestinal digestion or fermentation hydrolysis forms peptides with good bioactive characteristics that can be used as functional foods, benefiting human health [1].

Multiple processes have been used for deriving protein hydrolysates, chemical and modified, of which, enzymatic protein hydrolysis is the most common process. Currently, most proteins are hydrolysed using proteolytic enzymes at the ideal temperature and pH. These often target particular peptide bonds, resulting in the release of AAs and peptides of various sizes [3]. In chemical methods, acid/alkaline hydrolysis are a conventional method. In chemical method, it is seen that mostly non-essential amino acids (asparagine, glutamine, cysteine, and tryptophan) are destroyed that are difficult to recover by acidic hydrolysis; hence, neutralization with a base (hydroxide) is recommended after heating. The hydrolysed protein is further subjected to membrane filtration or purification method [4]. The major problem of enzymatic hydrolysis is its expensive costs as well as presence of enzyme inhibitors in raw materials. The need of careful optimization and handling is essential, which can lead to enzyme denaturation or inactivation, resulting in incomplete hydrolysis with lower yields. An alternate economical method that can be used for protein hydrolysis includes acid hydrolysis. Studies have shown that essential amino acids (EAAs) such as aspartic acid, glutamic acid, proline, glycine, alanine, leucine, phenylalanine, histidine, and arginine can be achieved by acid hydrolysis (AH) [4]. Therefore, the aim of this study was to investigate the best cheap method for the production of pigeon pea protein hydrolysates via AH or enzymatic hydrolysis (EH) with good bioactive characteristics.

Bioactive peptides are addressed as nutraceuticals with health advantages associated with illness prevention or therapy. Studies on the antioxidant characteristics of crude protein hydrolysates have been carried out by several researchers [5–8]. Nutritional characteristics of pigeon pea have been associated with decrease in occurrence of various cancers, HDL cholesterol, type-2 diabetes, and heart diseases. These include compounds such as protease inhibitors, non-antinutritional components and angiotensin I-converting enzyme (ACE) inhibitor with possible beneficial characteristics [5,9,10]. Studies have shown that foods packed with antioxidants provide functional health benefits by acting as exogenous sources of antioxidants to neutralize oxidants. Recently, foods rich in protein-derived peptides, typically achieved through the hydrolysis of food proteins, have been assessed for potential therapeutic functions in preventing cellular damages from oxidative stress that promotes human health. These pulse proteins are the most investigated naturally occurring alternatives to synthetic antioxidants and antidiabetic characteristics. Pulse proteins have become popular due to their availability, accessibility, affordability and simpler derivative [11]. Furthermore, the novelty of this study is to highlight advantages, disadvantages and cost-effectiveness of various methods for producing superior bioactive peptides from pigeon pea. Research on the specific effects of AH on pulse hydrolysates is in its early stages, however presenting an exciting opportunity to tailor their functional characteristics.

2. Materials and Methods

Unpolished pigeon pea seeds were purchased from a local market in Karnataka, India. Olive oil was purchased from a local supermarket in Vishakhapatnam, India. All the chemicals and reagents were in analytical grade  from Sigma, Germany, Merck, Germany, and Himedia, India, and provided by a local vendor.

2.1. Preparation of the protein hydrolysate

Sample preparation was carried out based on a method described previously [12]. Seeds purchased were washed, sun-dried and powdered into fine meal using 40-mesh sieve. Roasted seeds were subjected to fat extraction process for nearly 6 h using an ethanol extraction-based system. Mixture was further subjected to distillation to achieve the final fat-free product for hydrolysis. For acid hydrolysis, hydrolysates were prepared using 6 M of HCl and were then added to defatted pigeon pea flour. Extracted samples were  with 6 M hydrochloric acid per mL for various time constraints under high pressure at 121 PSI for 3–4 h. Precipitated protein at isoelectric pH was removed from the suspension by centrifugation at 12298 g for nearly 20–30 min, adjusted to pH 7.0 with 0.1 M NaOH, lyophilized and stored at 4 °C [13]. For EH, pigeon pea sample was hydrolysed enzymatically using protease pigeon pea and fat-free suspensions were prepared using 100 g of the samples that was brought to a volume of 1 L with 0.10 M phosphate buffer (pH 7–7.4 under optimum conditions with the enzyme). The enzyme complex was added for each of the experiments at room temperature (RT), based on the enzyme/substrate (E/S) ratios (m/m) (0.1, 0.3, 0.5, 0.7, and 1). The reaction mixture was transferred into a bioreactor with a capacity of 20 L. Ranges used to assess variables of pH, temperature, and time included 1–9, 50–70 °C, and 100–180 min, respectively. Separation of the solid from the supernatant was carried out for each sample when the enzymatic activity ended via heating at 90 °C for 20 min. After decanting the samples, supernatant was centrifuged at 2490 g. Further selective filtration was carried out using 0.4-µm membrane filtration and the supernatant was freeze-dried and stored at 4 °C until further use [10,11,14].

2.2 Proximate composition of pigeon pea

Pigeon pea seeds (unhydrolysed, defatted, and hydrolysate) were analyzed for crude fiber, ash, and relative humidity using standardized methods by the Association of Official Analytical Chemists (AOAC) [15]. Crude fat was assessed using Soxhlet solvent extraction system [13]. Crude fat was calculated using the Eq. 1:

                                  Eq. 1

Where, W1 was the empty thimble, W2 was weight of the sample and thimble and P was weight of the sample.

2.3. Protein content assessment

Kjeldahl protocol was used as described previously [16]. Briefly, 200 mg of the samples were used in the analysis. Kjeldahl method was used to assess the protein content based on the protocol of AOAC IS 7219 [15]. The assessed nitrogen content was multiplied by 6.25 (the nitrogen-to-protein conversion factor for legumes) to achieve the final protein concentration of the samples [13] using Eq. 2:

                                                                                       Eq. 2

Where, a was the volume in 0.5 N acid assessed for distillation, b was the volume in 0.1 N base used for back titrating a, c was the volume in 0.5 N acid used for blank distillation a, and d was the volume in 0.1 N alkali used for back titrating c.

2.4. Degree of hydrolysis

The O-phthalaldehyde assay (OPA) procedure was used for assessing degree of hydrolysis (DH) as described by Nielsen [13]. The OPA was a sensitive technique widely used for pulse proteins [14] and DH was calculated using Eq. 3:

                                       Eq. 3

Where, V was the sample volume (0.1 lL), X was the sample weight (0.125 g), P was the soluble protein content (90%) of the sample and serine-NH2 was in meqv serine-NH2·g-1 protein (Eq. 4).

                                                             Eq. 4

Where, α and β were respectively the constants of 1.00 and 0.40 for the raw materials that were not assessed. The DH was calculated using Eq. 5

                                                      Eq. 5

Where, htot was the total number of peptide bonds per protein equivalent.

2.5. Protein yield assessment

The yield of protein pigeon pea samples (defatted, AH, and EH) was assessed as per modifications and was calculated using Eq. 6:

                                      Eq. 6

2.6. Assessment of anti-nutritional factors

Anti-nutritional factors of tannic acid, total phenol content, phytic acid, and trypsin inhibition were carried out as previously described [15].

2.6.1. Total phenolic content assessment

The total phenol content of various samples was assessed quantitatively using Folin-Ciocalteu method; in which, gallic acid was taken as standard and the absorbance was measured at 760 nm [15].

2.6.2. Tannins assessment

Presence of tannins was assessed using Folin-Denis reagent spectrophotometric method at 700 nm [15].

2.6.3. Phytic acid content assessment

Phytic acid content of the samples was assessed spectrophotometrically at 480 nm, where Fe (NO3)3 was used as standard [15].

2.6.4. Trypsin inhibition assessment

Trypsin inhibition activity was assessed indirectly by inhibiting activity of the synthetic substrate [Nα-benzoyl-D,L-arginine p-nitroanilife hydrochloride (BAPNA)], which was subjected to hydrolysis by trypsin to produce yellow colored p-nitroanilide at 400 nm [17].

2.6.5. Saponin assessment

Saponins were assessed spectrophotometrically at 430 nm using saponin (0–40 µg) as standard [17].

2.7. Fourier transform infrared spectroscopy

The Fourier transform infrared (FTIR) spectroscopy spectra of pigeon pea AH were recorded using ALPHA-II FTIR spectrometer (Bruker Optics, Germany) fitted with an ATR (attenuated total reflectance) sampling device containing diamond crystals. The absorbance spectra were 4000–400 cm-1 (with a standard KBr beam splitter) at a spectral resolution of 4 cm-1 with 16 scans co-added and averaged. Additionally, 1–2 g of the sample were finely powdered for the scan. Data were translated into transmittance units [18].

2.8. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of unhydrolyzed and hydrolyzed samples was carried out using 5% stacking gel and 15% separating gel using Biobase gel system (Shandong, China) at 100 V. Gels were fixed in Coomassie brilliant blue (CBB) under RT and destained to visualize bands [18].

2.9. Amino acid composition

 The pigeon pea fat-free unhydrolysed control and protein hydrolysates (AH and EH) were digested with 6 M of HCl for 24 h. Then, AA composition was assessed using HPLC 1260 Infinity Agilent system, USA, based on a method previously described [19]. Cysteine and methionine contents were assessed post performic acid oxidation as described previously [20] and the tryptophan content was assessed as described by Li et al. [8].

2.10. Assessment of functional characteristics

2.10.1. Water and oil absorption capacities

Water absorption capacities (WAC) of the protein was assessed [17, 18] and then calculated using Eq. 7:

                                    Eq. 7

Where, W was the protein weight, V1 was quantity of the distilled water (DW) and V2 was volume of the water. For oil absorption capacities (OAC), 0.5 g of the samples were mixed with 5 mL of the appropriate vegetable oil and vortexed for 5 min. Slurry was centrifuged at 1968 g for 30 min and then weight of the adsorbed oil was assessed. The OAC was calculated using Eq. 8:

                                                 Eq. 8

Where, W was weight of the protein sample, W1 was weight of the oil and W2 was quantity of the free oil.

2.10.2.    Emulsifying activity index (EAI)

Emulsifying ability of the samples (unhydrolyzed, defatted and hydrolyzed) was carried out based on a method with modifications [19]. The EAI was calculated using Eq. 9:

                                      Eq. 9

Where, A0 was the absorption at 500 nm, φ was volume of the oil fraction, and C was protein concentartion of the sample.

2.10.3.    Emulsifying stability index

For emulsifying stability index (ESI), samples at a protein concentration of 1% were mixed with 9 g of vegetable oil (e.g. olive oil) and then homogenized for 5 min using ultrasonic cell crusher noise isolating chamber [19]. Two aliquots were pipetted at 0 and 10 min and further diluted with 5 ml of 0.1% SDS solution. Absorbance of the solution was recorded at 500 nm (in min) and was calculated using Eq. 10:

                                                   Eq. 10

Where, A0 was the absorbance at 0 min., A10 was the absorbance at 10 min, ∆t was the time difference of 10 min.

2.10.4 Foaming characteristics

Foaming characteristics were assessed, including foaming capacity (FC) and foaming stability (FS). Briefly, 200 mL of the sample solutions (unhydrolysed, defatted, and hydrolysates) with 0.5% protein concentration at various ranges of pH (2–10) were homogenized for 10 min using ultrasonic cell crusher noise isolating chamber and φ 6 probes to induce air at RT [20]. Generally, FC and FS were calculated using Eqs. 11 and 12:

                        Eq. 11

                         Eq. 12

Where, A was the volume before whipping (mL), B was the volume of foam at 0 min after whipping (mL) and C was the volume of foam at 20 min after whipping (mL).

2.11. Antioxidant characteristics

2.11.1 DPPH radical scavenging activity

For DPPH radical scavenging activity (DRSA), antioxidant activity was assessed using samples with a concentration of 1 mg·mL-1 that were mixed in water; 0.1 mg of DPPH-1:1 (v:v) was dissolved in 99.5% methanol [14]. Potential of the radical scavenging activity was assessed using absorbance at 517 nm and Shimadzu UV-1800, Nakagyo-Ku, Japan. In general, DPPH radical scavenging activity was calculated based on Eq. 13:

                   Eq. 13

Where, Asample was absorbance of the sample and Acontrol was absorbance of the control.

2.11.2. Hydroxyl radical scavenging activity

For hydroxyl radical scavenging activity (HRSA), nearly 100 μl of each sample with concentrations of 20–200 g were collected and mixed with 100 μl of ferrous sulphate (3 mM) and 100 μl of 1,10-phenanthroline (3 mM; dissolved in 0.1 M phosphate buffer; pH 7.4). Then, 100 μl of 0.01% hydrogen peroxide were added to the mixture to initiate the reaction. Mixture was incubated at 37 oC for 1 h and absorbance was measured at 536 nm using Shimadzu UV-1800 spectrophotometer, Nakagyo-Ku, Kyoto, Japan [14]. Equation 14 was used to assess hydroxyl radical scavenging capacity.

                           Eq. 14

Where, Asample was absorbance of the sample, Acontrol was absorbance of the control and Ablank was absorbance of the blank.

2.11.3. Metal ion-chelating assay

For metal ion-chelating assay (MCA), samples (100 μg) were mixed with 250 μl of 100 mM Na acetate buffer (pH 4.9) and 30 μl of FeCl2 (0.01%, w v-1). Ferrozine (12.5 μl, 40 mM) was added to the mixture after incubation at RT for 30 min. Generally, EDTA was used as positive control. Binding of Fe (II) ions to ferrozine generated a colored complex that was measured at 562 nm using Shimadzu UV-1800, Nakagyo-Ku, Kyoto, Japan [14,21,22]. The ferrous ion chelating ability was calculated using Eq. 15:

                                                                                Eq. 15

Where, Acontrol was absorbance of the control and Asample was absorbance of the sample. Reduced glutathione (GSH) was assessed (1 mg·ml-1) concurrently with the samples as positive control for all the antioxidant activity assays (DRSA, HRSA, and MCA).

2.11.4.    Assessment of reducing power

Reducing power of the protein was assessed based on a modified method [8]. Protein hydrolysate solutions with various concentrations (1, 5, 10 and 15 mg·ml-1) were prepared. Then, 1 ml of the vortexed sample was added to 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. Reaction mixture was rapidly vortexed and incubated at 50 °C for 20 min. Then, 2.5 ml of 10% TCA were added to the mixture and centrifuged at 12298 g for 10 min. Supernatant of 2.5 ml was mixed with 200 µl of deionized water and 40 µl of 0.1% FeCl3. This was set to react for 10 min and absorbance was measured at 700 nm using Shimadzu UV-1800, Nakagyo-Ku, Japan.

2.12. Assessment of anti-diabetic characteristic using α-amylase inhibition activity

Inhibition of α-amylase activity was assessed using a protocol with slight modifications [14]. Briefly, 6 mM of NaCl and 100 μl of 20 ml l-1 sodium phosphate buffer (pH 6.9) were mixed with 100 μl aliquot of the sample containing 1 mg ml -1 of α-amylase solution. Further, 100 μl of 1% starch solution in 20 mM sodium phosphate buffer (pH 6.9, 6 mM) were mixed with the sample and incubated. Reaction was terminated by the addition of 200 μl of dinitrosalicylic acid and incubated at 100 °C for 5 min using boiling water bath. The reaction mixture was cooled down to RT, followed by the addition of 3 ml of double DW. Absorbance of the samples, Control 1 and Control 2 was measured at 540 nm. A standard synthetic drug (Glyciphage) was used to compare the values. Inhibition proportion of α-amylase activity was calculated using Eq. 16:

       Eq. 16

Where, Control 1 represented mixture of starch solution, protein sample excluding α-amylase enzyme and Control 2 represented mixture of starch solution, α-amylase enzyme excluding protein sample.

2.13. Statistical analysis

Analyses were carried out in three independent replications, with the outcomes subjected to one-way variance analysis. Statistically significant differences (p ≤ 0.05) between the mean values were assessed using Tukey test and Origin Pro software v.8.1.

3.Results and Discussion

3.1. Proximate composition of pigeon pea

Crude fiber contents of unhydrolysed, defatted, acid, and enzyme hydrolysed pigeon pea seed samples were 7.56% ±0.96, 7.45% ±0.90, 7.10% ±0.80 and 6.95% ±0.80, respectively (Table 1). There was a little difference within the samples. The total ash contents were 3.76% ±0.46, 3.45% ±0.60, 3.97% ±0.70, and 4.05% ±0.60 for the highlighted samples, respectively.

For crude fat content, decreases in fat concentration were reported with a decreasing order from unhydrolysed to hydrolysate samples due to the extraction of defatted seeds using Soxhlet ethanol-based extraction, leading to the removal of fat in outer and inner pods of the seeds, which resulted in 4.45% ±0.50, 2.78% ±0.40, 2.18% ±0.50, and 1.95% ±0.50, respectively. Moisture contents of the samples were respectively 85.63% ±0.50, 78.2% ±0.50, 71.5% ±0.50, and 69% ±0.50 for unhydrolyzed, defatted and hydrolyzed samples [20].

3.2. Protein content

Protein content (PC) of the pigeon peas showed significant increase in hydrolysed seeds, compared to unhydrolysed (UH) seeds. Moreover, EH included a protein content of 24.05% ±0.5; AH included a protein value of 22.05% ±0.5 and UH included a protein content of 20.07% ±0.80. Significant increases in the total protein content of EH were recorded, compared with those of AH and UH (Table 2).

Technically, standard Kjeldahl method measures total protein content on a dry weight basis. From the previously reported studies, it is understood that the protein content changes when subjected to hydrolysis; however, there are mild changes in the protein content, depending on the used methods. Values reported in the current study indicated that they were within a similar range of 21–28%. With the help of hydrolysis, germination and soaking in improved varieties, significant increases were reported in protein contents [17, 20]. The protein bioavailability increased due to the hydrolysis achieved in a shorter time. The hydrolysates solid-liquid extraction method was a further efficient economically acceptable process for producing fish protein hydrolysates. Due to increased DH, it could hydrolyze proteins in a shorter time than enzymatic hydrolysis could [2, 8].

3.3. Degree of hydrolysis

Table 3 shows that the enzymatically produced hydrolysates included 68.42% ±0.48, better than unhydrolysed samples with 34.32% ±0.57  DH when treated with acids [6, 23]. Increased rate of hydrolysis during the later stages of hydrolysis might be because of increase in cleaved peptides that were unfolded and exposed further to AH surface area, allowing for easier proteolytic cleavages. In this study, it was observed that the extractable protein affected degrees of hydrolysis, which was similarly observed in a study by Abbe et al., 2022 [2]. The DH for EH were detected as 68.42 ±0.48. Annihilation of the protein natural secondary structure and cleavage of peptide fragments from the larger UH protein structure increased the protein solubility, indicating sharp increases in DH. Additionally, increased hydrolysis might result in further hydrophobic AAs unfolding, which enhanced surface hydrophobicity. Increased surface hydrophobicity could help the formulation textural probiotic encapsulation [24]. The EH and AH included better DH and higher protein content but EH included defined limitations for its effects on peptide characteristics such as hydrolysate, length of peptide, enzyme-substrate ratio, specificity of enzyme, hydrolysis time, molecular weight and bioactivity, as well as AA composition. Additionally, pre-treatment was needed to enhance hydrolysis as the cleavage sites were exposed with restrictions to higher exposure to heat, resulting in AA structures were deformed. Table 4 shows resulting barriers in the industrial sector due to it cost-ineffectiveness [25].

The study economic viability was assessed using a range of economic measures, one of the most important of which was the cost of manufacturing, sample preparation and total running expenses. The total operating costs was 3.65 USD/kg for AH and 52.48 USD/kg for EH, respectively (Table 4). With its competitive overall operative costs and good protein recovery yields, AH clearly was as the most economically viable agent.

3.4. Anti-nutritional factors

Tannic acid equivalents (mg·g-1) were assessed as 7.07, 6.46, 5.41 and 4.8 in unhydrolyzed, defatted and hydrolysed pigeon peas, respectively (Figure 1A). A study reported the range of tannin in unprocessed pigeon pea as 4.3–11.4
mg·g-1 [26]. Tannin-protein complexes have been suggested to cause decreased AA availability, increased fecal nitrogen, inadequate protein digestibility and decreased iron bioavailability. These complexes might not be separated, which caused them as eliminated with the wastes [27]. Hydrolysates effectively eliminated tannins from the protein complex and improved nutritional values of the hydrolysates. The total phenolic content was assessed as 1.88 mg 100 g-1, 1.504 mg·100 g-1, 1.45 mg·100 g-1 and 1.376 mg·100 g-1 for unhydrolyzed, defatted, acid and enzymatic hydrolysates, respectively (Figure 1A). Prior investigations reported 1.6 mg·100 g-1, which verified the reported values [26]. After hydrolysis (acid or enzyme), the OH group decreased significantly and enhanced bioavailability of the macronutrients, as revealed in similar studies where heat and acid treatments decreased quantity of the free form as well as bound phenolic compounds that even leached out in water, ultimately making dormant sites of the proteins active [28]. Phytic acid levels were assessed as 7.13, 6.175, 5.415 and 4.7 mg·g-1 for unhydrolysed, defatted, acid and enzymatic hydrolyzed forms, respectively (Figure 1A). A study reported that the phytic acid content of pigeon pea was assessed from 4.87 to 6.54 mg·g-1, similar to the current findings [26]. Based on studies, total free (unbound) mineral contents increased after the whole wheat bread was autoclaved and microwaved simultaneously and the quantity of phytic acid decreased [29].

Saponins belong to a complex class of naturally occurring triterpenes or steroidal glycosides, detected in numerous types of plants, including oil seeds and pulses. Since saponins change permeability of the cell walls, they may result in adverse effects when consumed. The small intestine cells are bound by saponin, which alters how nutrients are absorbed through the intestinal membrane [1]. Saponins were assessed to be 9 mg.g-1 for unhydrolyzed and 11.5 mg.g-1 for the enzymatic hydrolysates. Similar values were reported in previous studies by Sekhon et al. [26]. Saponins include positive correlations with HRSA. This study highlighted that similarly hydrolysates included higher values than those unhydrolysates did. Saponins are well known for their capacity to yield foam that is exceptionally stable [3]. Trypsin inhibition activity of the unhydrolyzed samples was lower than that of hydrolysed samples (Figure 1B) [30]. After hydrolysis of H-bonds, trypsin-protein complexes were undigested, leading to decreases in inhibitors. These studies justifies relevance of the assessment of inhibitors as they delay the metabolism; hence, it must be addressed while consuming legumes as the source proteins. It is reported that trypsin-protein complexes are undigested after the lysis of H-bonds and decreases in inhibitors [21]. Studies on trypsin protease inhibitors (tannins, phytates, trypsin inhibitors and goitrogens) have shown their interference with the digestion processes and production of pancreatic hypertrophy or hyperplasia, inhibiting growth of the epithelial cells that lining mucosa and thus including anti-nutritive effects. Various methods have been used in food processes to address effects of these food anti-nutrients, including milling, soaking, germination, autoclave and microwave treatments and fermentation [31].

3.5. Fourier transform infrared spectrum analysis

Secondary structure of the proteins was assessed using FTIR spectroscopy [30]. Figure 2A shows that spectral characteristics of the globular complex (GC) proteins in unhydrolyzed samples were the Amide-A band at approximately 3274.31 cm-1 (NH stretching), the Amide-B band at approximately 2854.99 cm-1 (C-H symmetric stretching modes of methyl), the amide-I band at 1743 cm-1 (C=O carbonyl stretching), the amide-II band at 1634 cm-1 (C=C aromatic stretch) and N–H bending at 1540 cm-1 and the aromatic C-H in plane bend at 1074.88 cm-1. These findings were consistent with the previous studies [17, 31]. The AH spectra showed characteristic peaks in the range of 3273–2874.17 cm-1, indicating that respectively Amide A (N-H and C-H stretching) and Amide B (methyl C-H stretching) bands brought on by the bending resonances of the intra and intermolecular hydrogen bonds (Figure 2B). Peaks at 3231.46, 3065.45, 2958.78 and 2875.78 cm-1 showed that Amides A and B were visible in the IR spectra of EH (Figure 2C). The aromatic C-H out of the peaks were visible at 666.10 and 1398.04 cm-1 indicating N-O aliphatic stretch. Moreover, peak of 1451.70 cm-1 showed visible peaks of C-H asymmetric bend, and 1579.13 cm-1 showed a band of the NH bend (Figure 2C). The connection between functional groups of C-O and N-H forms a helical structure [32]. 

The conventional protein bands of Amide I (1600–1700 cm-1), Amide II (1500–1580 cm-1) and amide III (1200–1400 cm-1) showed visible bands of Amides A and B. A previous study showed that the characteristic protein bands were linked to specific stretching and bending vibrations of the protein backbones. The α-helix (1650–1658 cm-1) and β-sheet (1638–1687 cm-1) were the primary contributors to Amide I, while Amide II was mostly caused by N-H bending vibrations (60%) connected to C-N stretching vibrations (40%), representing dominance of the protein contents [33]. Therefore, peaks (1300, 1650 and 1688 cm-1) investigated in the hydrolysate spectra were similar to α-helix and β-sheet [4, 17].

3.6. Protein Profile Analysis (SDS-PAGE)

In vertical gel electrophoresis, proteins are separated based on their molecular weights. In this study, acid and enzymatically hydrolyzed pigeon pea samples were subjected to SDS-PAGE (15%) to assess molecular weights of the hydrolysates. Protein marker (Lane 1) ranged 245–11 kDa which included various molecular weights, followed by acid samples in Lane 2, unhydrolysed samples in Lanes 3 and 4 and enzymatic hydrolysates in Lane 4. Acid hydrolysed samples showed distinct bands in the range of 250–20 kDa (Lane 2), which were suggested as albumins and globulins and the difference in molecular weights showed the extraction procedure, making them high-molecular weight (HMW) peptides. The control unhydrolyzed protein demonstrated intense bands at 135–100 kDa (Lanes 3 and 4). The SDS-PAGE analysis of the EH revealed generation of low-molecular weight (LMW) peptides with a mass range of less than 15 kDa (Figure 3, Lane 5).

Figure 3 illustrates differences in bands affected by hydrolysis differently. Thus, it could be assessed that LMWs were produced by breaking the complex protein structure and peptide bonds by use of AH conditions while enzymatically hydrolysed samples included the presence of LMW peptides, indicating complete hydrolysis of the pigeon peas. A similar LMW band was seen in casein and flaxseed hydrolysate studies [10, 11].

3.7. Amino acid composition

The AA compositions of pigeon pea unhydrolysed (UH), acid (AH), and enzymatic hydrolysates (EH) are shown in Table 5.

The current composition supports protease ability to specifically cleave proteins at peptide bonds that hydrophobic AAs contribute to, increasing quantity of the linked AAs [16]. The pigeon pea unhydrolysed AA profile showed that the legume was rich in proteins and included significant quantities of EAAs that were better than the needs of the World Health Organization/Food and Agriculture Organization/United Nations University (FAO/WHO/UNU) Expert Consultation [10]. Findings from this study revealed greater concentrations of hydrophobic AAs (38.4 and 37.88%), EAAs (39.68, 42.26%), sulphur containing AAs (2.52 and 1.29%) and aromatic AAs (9.89 and 11.69%) for unhydrolyzed and enzymatic hydrolyzed samples, respectively. These were

similar to previous findings by Nwachukwu et al. for the flaxseed protein and thermoase hydrolysates (8.62 and 9.03% of aromatics; 34.55 and 35.72% of hydrophobics) [34].

Comparison of AA profiles of the protein hydrolysates derived by the isoelectric precipitation method to native protein (unhydrolysed pigeon pea) revealed a little detectable variation, suggesting that the protein composition was largely unaffected by the extraction process [30]. Olagunju et al. [14] detected that use of distinct proteases during the hydrolysis of pigeon pea protein produced similar findings. The hydrolysates included higher concentrations of particular AAs, compared to those the non-hydrolyzed protein did, including glutamic acid, histidine, leucine, isoleucine, phenylalanine, arginine, tyrosine, and tryptophan. Glutamic and aspartic acids were the most frequent AAs in AH and EH, whereas levels of tryptophan and methionine were below the recommended levels. Branching-chain amino acids (BCAAs) and a majority of hydrophobic AAs (glycine, alanine, valine, leucine, isoleucine, proline, and phenylalanine) increased upon enzymatic degradation using protease enzymes.

3.8. Functional characteristics of bioactive peptides

3.8.1. Water and oil absorption capacities

Technically, WAC and OAC are pH dependent and detrimental parts of this study. The WAC values of the hydrolysates were higher than those of unhydrolysed samples. At pH 4, sharp decreases were seen in WAC and OAC values due to the pH near the isoelectric point of the proteins (Figure 4A). Similar findings were reported by the literatures, showing that peptides typically included higher water-holding capacities than those of their associated flour forms. This finding was similar to another finding and likely resulted from changes in protein conformation that exposed further water-binding sites, increasing protein polarity, electric charge and proportion of the proteins bound in water [4, 17]. The OAC value is important as an estimate of oil ability to absorb proteins, which can indicate how hydrophobic a protein is. The non-protein components in flours, including starch granules and lipids, may partially function as a barrier to water penetration, fractions with higher protein contents with fibers include greater potentials to hold water. Due to their lower lipid contents and smaller particle sizes, lentil protein peptides included better water-holding capacities than those the lentil flours did.

The OAC of pigeon pea unhydrolysed seeds was lower than that of the enzymatic hydrolysate with significant general decreases at pH 4 (Figure 4A). Compared with other spectra of legumes, these values were at average levels, as reported in a study of protein hydrolysates with higher surface hydrophobicity and better surfactant characteristics. However, denaturation of the globular proteins might expose hydrophobic regions. It is believed that OAC is created by the binding of non-polar side groups of proteins, leading to oil entrapment. The OAC is therefore affected by the quantity of hydrophobic AAs that are exposed, as well as the quantity of hydrophobic AAs in the proteins that can visibly be demonstrated in the AA profile (Table 5 ) for the unhydrolysed and hydrolysed samples [3, 20, 35]. Based on a study, globular proteins with further hydrophilic and polar AA residues on the surface included a higher WAC and proteins with further hydrophobic and non-polar AA residues might include a higher OAC [36]. Plant-based proteins with good water and oil absorption characteristics can create plant-based protein analogues and emulsions, which are beneficial in food industries.

3.8.2. Emulsifying capacity

The EAI (Figure 4B) and ESI (Figure 4C) values of the hydrolysed samples were higher than those of unhydrolyzed and defatted samples, which was observed with pH as a critical factor. The lowest EAI and ESI values were reported at pH 4, which was near the isoelectric point of soy and whey proteins and included a lesser solubility based on Figures 4B and 4C, respectively. This indicated that pH substantially affected emulsifying characteristics of the peptides with and without hydrolysis. Stability of the emulsion increased by pH levels that were below the isoelectric point. The maximum EAI and ESI values for the emulsions at pH 8 are shown in Figures 4B and 4C [19]. It is shown that the hydrophilic/hydrophobic AA composition and equilibrium development of the interfacial film, as well as the solubility and flexibility of the protein molecules, are interrelated to differences in EAI and ES for various proteins [37]. Protein isolates with higher EAI and ES additionally included further hydrophilic AA residues and were further soluble (Figures 4B and 4C). Amphiphilicity of the peptides includes major effects on the emulsifying abilities of hydrolysates. Based on multiple studies, hydrolyzed peptides enhance hydrolysates abilities to emulsify due to their potentials to unfold at the oil/water interface [37]. Additionally, they are more likely to include residues that interact with the aqueous phase and the oil droplets, respectively, whether they are hydrophobic or hydrophilic [2, 14]. Due to steric effects, this interaction makes the emulsion further stable [38]. The AH offers further potentials for food emulsion uses, including yoghurt, mayonnaise and ice cream since EAI and ESI are essential indices for food emulsion use and quality control. Moreover, higher molecular weight of the protein hydrolysates may result in higher emulsion capacities of AH.

3.8.3. Foaming characteristics

Proteins in dispersions decreased surface tension at the water-air interface, resulting in the ability to foam. The hydrolysates half-life could be changed by the preservation of the viscoelastic adsorbed layer, suggesting that FS might necessitate addition of functionality improvers to the hydrolysate. It is well known that globular proteins frequently produce adsorbed layers that are elastic with enhanced viscosity and hence support stability of the foam [39]. Foaming capacity and stability are pH dependent. The FC decreased at pH 4 and reached its maximum value at pH 6 (decreased in alkaline conditions). The FC was inversely proportional to pH. At pH 6, net charge increased, which led to increases in foaming capacities. At pH 4, FC achieved a lower solubility at its isoelectric point. Increased concentrations of amphiphilic peptides contributed to increased FC (Figure 4D). Moreover, FS depended on protein-protein interactions with the matrix [20]. An extensive intermolecular network (protein-protein interactions) preserved by proteins maintained their tertiary structures at the interface, resulting in strong films and further stable foams.

Protein LMWs and other structural characteristics helped generate foams further quickly. However, they might not be effective for creating protein-protein interactions that resulted in stable foams (Figure 4D). These reports help explain why the unhydrolysed control pigeon pea included a small foam ability (expansion of foam) [37]. Large concentrations of hydrophobic AAs could be the reason for decreased FC values. Stability of the foam formation was affected by the strength of protein films and permeability for gases [18]. Several peptides with various hydrophobicity, charge balances and conformations from the native molecule are created during the digestion of proteins. These peptides are more flexible due to their lower molecular weight; hence, a stable interfacial layer is seen and the rate of diffusion to the interface increases, improving the foamability characteristics. For a protein to foam effectively (e.g. including high foamability), it should adsorb quickly during the transient stage of foam formation. These findings revealed increases in surface activity, most likely due to the originally larger surfaces created by partial proteolysis that allowed for the incorporation of further air [39].

3.9. Antioxidant activities of peptides

Several chemical or biological assays are needed to assess antioxidant mechanism and quantify antioxidant activity of the legume extracts [40]. Although this in-vitro study is fascinating from a strictly predictive viewpoint based on chemical processes, it might sometimes correspond to in-vivo systems. The MCA and HRSA are based on electron transfer reactions (ET) and hydrogen atom transfer reactions (HAT), respectively; DPPH is a hybrid HAT and ET-based assay [7].

3.9.1. The DPPH radical scavenging activity

Due to the method simplicity, efficacy and credibility, DRSA is often used to assess antioxidant capacity of foods, peptides and biological samples. In the present study, DRSA in pigeon pea samples was 38.60, 42.45, 74.25 and 80% for unhydrolysed, defatted, acid and enzymatic hydrolysates, respectively (Figure 5A). Results were almost similar to those of literature. The DRSA of hydrolysates was able to achieve a similar efficiency to that of GSH, which is the predominant intracellular antioxidant that helps in keeping redox equilibrium in the body and as well as modulating cell viability. Other studies have reported similar increase in antioxidant activity with increase in DH. Increased hydrolysis led to further hydrophobic side chains exposed, improving DRSA. Moreover, positive correlations were recorded between the phenolic content and DRSA, which justified the oxidative stress decreases on cellular levels. Relatively, as phenolic substances or their respective levels of hydroxylation increase, their abilities to scavenge DPPH radicals increase as well. Free radicals combine with cellular components such as DNAs, proteins and cell membranes; hence, these metabolites (primary and secondary) solve the problem of oxidative stress by neutralizing and disguising them as harmless agents through biological processes [10, 40].

3.9.2. Hydroxyl radical scavenging activity

Technically, HRSA assesses scavenging ability of peptides when hydroxyl radicals are generated due to the reaction between H2O2 and metal ions such as Fe2+ ion form. Hydroxyl ion includes short lifespan and highly reactive species that degrades macromolecule proteins, DNAs and lipids. Hydroxyl ion is addressed as a responsible factor for initiating lipid oxidation processes, causing cell damages that result in ageing and other chronic illnesses. Scavenging abilities of pigeon pea unhydrolyzed, defatted, acid, and enzymatic hydrolyzed samples were 38.55, 40.2, 45.15, and 50%, respectively (Figure 5A). In the current study, HRSA showed similar results to those of DPPH and phenolic contents did in the samples. Total length of the peptides, makeup and sequencing of the AAs and other variables alter antioxidant activity of protein hydrolysates. It can be concluded from the results that hydrolysis enhanced the hydroxyl radical scavenging capacity of pigeon pea proteins; similar to that observed in the enzymatic hydrolysis of pigeon pea using various enzymes [23].

3.9.3. Metal ion-chelating assay

Transition metal Fe2+ stimulates production of OH- and superoxide radicals (O2), triggering oxidative chain reactions. Chelating chemicals regulate the oxidative chain reactions caused by the radicals and decrease quantity of transition metals available in organic materials. The Fe2+ Scavenging ability of the hydrolysed samples was similar to that of GSH and significantly better than that of unhydrolysed samples [8]. The current study indicated that metal chelating abilities were high; similar to other studies that verified hydrolysate antioxidant abilities could inhibit oxidant reactions and help in prolonged storage. Increased hydrolysed peptides or polar AA concentrations might be associated with increased Fe2+ chelating activity within hydrolysis. Protein hydrolysate ability to chelate metal ions is generally reported responsible for their abilities to bind with ions. It has been stated that histidine-containing peptides include metal-chelating activities via their imidazole rings. Another significant reason for antioxidant activity included ability to bind transition metals because transition metal ions such as Fe2+ and Cu2+ naturally stimulate creation of reactive oxygen species (ROS) such as hydroxyl radical (OH), triggering oxidation of fatty acids [23].

3.9.4. Reducing power

The reducing power of a compound is studied to assess its antioxidant potentials. Figure 5B demonstrates the reducing power reached by acid-base hydrolysis for 5 h at various doses (1–15 mg·mL-1). Hydrolysates included the highest reducing power, showing that these hydrolysates in EH followed by AH included further bioactive peptides that could scavenge free radicals. The activity assessed by UH was the lowest. Reducing power of the samples increased with a higher absorbance value. When reducing substances were present, the Fe3+/ferricyanide complex was reduced to the ferrous form (Fe2+) through electron donation. Briefly, quantity of Fe2+ was measured at 700 nm [7]. Variation in reducing power might be a significant characteristic, indicating changes in AA composition after hydrolysis. Information suggested that hydrolysates might help free radicals by donating one electron [8].

3.9.5. Anti-diabetic assay

In general, α-amylase enzyme in hydrolysis of glycosides is responsible for the breakdown of complex carbohydrates into oligosaccharides and glucose for a better uptake. Hence, inhibition of such an enzyme is helpful in decreasing breakdown of complexes to simpler levels, making it a better strategy to manage diabetes. For this reason, naturally occurring bioactive substances in foods that are helpful in treatment of diabetes have widely been reported. Figure 6 shows that various samples included anti-diabetic activities. The synthetic drug glyciphage showed the highest inhibition rate (56%), whereas hydrolysates EH (44%) and AH (38%) showed the best inhibitory effects, compared with the activity of UH (18%) and DF (22%). Low activities of the UH and DF might be due to weaker bond interactions, including electronic and hydrophobic bonds with lesser bond availability to bind with polysaccharides and dietary fibers, ultimately leading to lesser inhibition. After hydrolysis, synergistic activities between the peptides and inhibition were recorded. Results

were similar to those of various hydrolysates. Although studies have shown certain differences in inhibition due to the sources of raw materials, filtration process and type of hydrolysis, inhibiting activity of α-amylase may lessen the likelihood of hyperglycemia in the body [14].

4. Conclusion

This study highlights synthesis of functional proteins with comparative findings of the various methods. Effectiveness of pigeon pea as a potential alternative protein source was the major finding of this study as well as assessment of the peptide functional, antioxidant and anti-diabetic characteristics. This study has demonstrated use of acid and enzymatic hydrolysis methods for the extraction of pigeon pea protein hydrolysates to subdue anti-nutritional factors that delay efficacy of metabolism. This approach can be used as an excellent alternative to traditional proteins with a higher concentration of bioactive peptides. It has been observed that the bioactive peptides include beneficially functional qualities such as helping in developments of emulsions, gels and suspensions, making them ideal choices for potentially commercial protein powders. This study provides numerous novel possibilities for research on plant-based protein sources and pigeon pea can be used as a vegan source while preserving the protein texture, flavor and nutritional profile. These help in treating various chronic diseases and metabolic disorders; hence, contributing significantly to health and food industries. The present study addresses usethe use of cheaper vegan protein sources in human diets.

5. Acknowledgements

None.

6.Conflict of Interest

The authors declare no potential conflict/competing interest.

7. Authors Contributions

Conceptualization, N.S.; methodology, J.M.; software, J.M.; validation, N.S..; formal analysis, J.M.; investigation, J.M.; resources, N.S.; data curation, writing—original draft preparation, J.M..; writing— review and editing, N.S. and V.G.P.; visualization, N.S.; supervision, N.S.

 

Synergistic effects of recombinant AGAAN antimicrobial peptide with organic acid against foodborne pathogens attached to chicken meat

Nafiu Usman Jiddah , Ya'u Sabo Ajingi, Neeranuch Rukying, Triwit Rattanarojpong, Worapot Suntornsuk, Patthra Pason, Nujarin Jongruja

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e21
https://doi.org/10.22037/afb.v11i1.44981

Background and Objective: Fresh chicken meat includes the capacity to contain foodborne pathogens. A previous study has demonstrated efficacy of recombinant AGAAN antimicrobial peptide against various bacterial strains. In general, AGAAN is a newly discovered antimic-robial peptide with a unique cationic alpha-helical structure. The peptide is originated from the skin secretions of Agalychnis annae. This peptide showed a significant affinity towards the negatively-charged microbial lipid bilayer, as previously demonstrated by the experimental and in-silico analyses. However, the major concerns include high production costs, limited expression, laborious process and potential toxicity associated with concentrated peptides. In this research, the synergistic effects with organic acid were addressed to decrease these problems while preserving its bactericidal activity.

Material and Methods: Recombinant AGAAN and organic acids were assessed on Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 8739. This was carried out by assessing minimum inhibitory concentration and fractional inhibitory concentration. In addition, effects of the combination on bacterial membrane integrity by carrying out beta-gala-ctosidase assessment. Additionally, the potential efficacy of this combination in preserving poultry meat was investigated.

Results and Conclusion: Minimum inhibitory concentration of the recombinant AGAAN against the two bacterial strains was 0.15 mg.ml-1. In contrast, the minimum inhibitory concentration of acetic acid against Staphylococcus aureus and Escherichia coli were 0.2 and 0.25% v v-1, respectively. The combination demonstrated significant synergy, as evidenced by fractional inhibitory indices of 0.375 against the two foodborne pathogens. Based on the study, the combination effectively inhibited proliferation of these disease-causing microorganisms that led to foodborne illnesses within 300 min. Presence of intracellular beta-galactosidase indicated that the combination of factors has caused damages to the cell membrane, resulting in its compromised integrity. Red blood cells exposed to various concentrations of recombinant AGAAN and acetic acid did not result in hemolysis. Results showed significant differences (p < 0.05) in all the experiments on meat samples that received treatments with recombinant AGAAN and acetic acid. The current study detected that a combination of recombinant AGAAN antimicrobial peptide with organic acid could effectively inhibit growth of pathogens at lower concentrations. Data presented in this study can help food industries develop further efficient cost-effective antimicrobial uses.

  1. Introduction

 

Prevalence of foodborne diseases has emerged as a significant global health concern. The World Health Organization (WHO) report indicates that nearly 600 million cases of foodborne illnesses occur annually due to the consumption of food substances contaminated with microorganisms and chemicals [1]. Food contamination and increases in the risk of foodborne diseases are caused by pathogenic microorganisms [2]. Meat and meat products are important sources of nutrients for humans due to their high protein composition and other essential nutrients [3].  However, these foods provide appropriate environments for the growth of foodborne microbes due to their high water content and nutrients, [4]. A significant number of studies have shown that Staphylococcus aureus and Escherichia coli are associated with meat contamination [5-7]. The S. aureus is a facultative anaerobic, Gram-positive non-spore-forming bacterium [8]. It is a major problem in foodborne illnesses [9]. The S. aureus infections cause significant morbidity and mortality in developing and developed countries [10]. Similarly, E. coli is a non-spore-forming bacterium and the major cause of foodborne diseases in Gram-negative bacteria. Disease-causing strains of E. coli can infect the stomach, leading to serious abdominal symptoms [11]. Previous studies have primarily concentrated on spore-forming microorganisms, thereby overlooking non-spore-forming ones such as E. coli and S. aureus. Based on their contribution to foodborne illnesses, it is important to develop a cost-effective user-friendly approach to slow their rapid proliferation in food products.

Organic acids have been used as antimicrobial agents to inhibit foodborne pathogenic bacterial growth in chicken meats during processing [12]. Due to the potential resistance development by microorganisms, there are needs of drug alternatives that can efficiently kill resistant bacteria and enhance preservation [13]. Antimicrobial peptides (AMP) are produced by living organisms and include critical functions in protecting hosts against infections [14,15]. Likelihood of microbes exhibiting resistance to AMP is exceedingly low because of their wide range of mechanisms of action. Multiple studies have emphasized potential of AMP as a viable option for preventing meat spoilage and foodborne diseases [16-19]. In a previous investigation by the current authors, recombinant AGAAN (rAGAAN) effectively was cloned, expressed and analytically characterized [20]. Technically, AGAAN is a novel antimi-crobial peptide with a cationic α-helical structure from the skin secretions of the blue-sided frogs. The rAGAAN is stable at various temperatures and pH and destroys a wide range of bacteria [20]. A hemolytic assay has shown that the peptide is relatively non-toxic to mammalian red blood cells (RBCs). Combination of these characteristics with its rapid killing kinetics demonstrates that rAGAAN includes the potential as an effective food preservative against foodborne pathogens. Nevertheless, major issues include exorbitant production expenses, labor-intensive procedures and potential toxicity of using high concentrations of peptides.

Combining two or more AMPs may boost antimicrobial activity at lower doses [21]. The present study assessed pairwise combinations of the rAGAAN with formic and acetic acids against E. coli and S. aureus. Selection of these two organic acids was based on their high effectiveness against the highlighted bacterial strains. In addition, FAO/WHO Expert Committee on Food Additives has classified acetic and formic acids as generally regarded as safe. The former chemical was assigned to an unrestricted group acceptance daily intake (ADI), while the latter was assigned to an ADI range of 0–3 mg.kg-1 [22]. Combination of rAGAAN and these organic acids could decrease the concentration while preserving their potentially bactericidal activity. Differences in their mechanisms of action necess-itate assessment of synergy in membrane permeation and kinetics of inactivation. This study could provide an additional option for poultry industries to protect chicken meats from pathogens.

  1. Materials and Methods

2.1 Bacterial strains

Department of Microbiology at King Mongkut's University of Technology Thonburi in Bangkok, Thailand, supplied the foodborne pathogenic strains of E. coli ATCC 8739 and S. aureus ATCC 6538.

2.2 Recombinant AGAAN peptide expression and purification

The rAGAAN was produced based on the method of Ajingi et al., [20]. Briefly, the recombinant plasmid (pET-AGAAN) was transformed into E. coli BL21 (DE 3) competent cells. A colony of the competent cells with recombinant plasmids was inoculated into Luria-Bertani (LB) broth supplemented with chloramphenicol and ampicillin and grown at 37 °C and 200 rpm overnight. Then, 1% v v-1 from the overnight culture was introduced into a fresh 1-l LB broth supplemented with chloramphenicol and ampicillin as well as 1% w v-1 glucose. Culture was grown to an optical density (OD 600 nm) range of 0.4–0.6 at 37 °C and 200 rpm. Then, rAGAAN was expressed through induction with isopropyl β-D-1-thiogalactopyranoside at a concentration of 500 mM. Culture was grown at 16 °C for 18 h at 150 rpm. Cells were collected through centrifugation at 6,120× g for 30 min at 4 °C. Then, cells were suspended in 10 ml of buffer solution (10 mM Tris-HCl, 1 M NaCl; pH 8.0). These were subjected to sonication at an amplitude of 60% for 2 min, repeated for five cycles to induce cell disruption. Supernatant was purified after sonication and centrifugation at 6,120× g for 25 min at 4 ℃ using HisTrap FF column linked to the FPLC system. The column was pre-equilibrated with binding buffer (10 mM Tris-HCl, 1 M NaCl; pH 8.0). Elution of the bound peptide was carried out using buffer B (10 mM Tris-HCl, 1 M NaCl, 250 mM imid-azole; pH 8.0). Then, dialysis was carried out overnight at 4 °C using 50 mM Tris-HCl solution. Then, peptide was concentrated using 3-kDa centricon centrifugal filter tubes (Amicon, Germany). Concentration of the rAGAAN was measured using Bradford protein assay and its purity was assessed using 16% tricine-sodium dodecyl sulfate–polyacrylamide gel electrophoresis (tricine-SDS-PAGE).

2.3 rAGAAN and organic acid preparation

The rAGAAN was formulated in milligrams per milliliter (mg.ml-1). It was dissolved in 1× phosphate-buffered saline (PBS), whereas the organic acids were formulated in percentages (% v v-1) by dissolving in distilled water (DW).

2.4 Culture preparation

A volume of 20 μl of microbial stock, previously stored at -80 ◦C, were plated on LB agar. The resulting culture was incubated at 37 oC for 18 h. Then, subculture process was carried out for each strain under identical conditions to preserve integrity and purity of the cells. On the next day, a suspension was generated by transferring isolated colonies into sterilized 10-ml LB media. The bacterial strains were cultured until they reached an OD of 108 cfu.ml-1. This measurement was achieved at 600 nm using spectrophot-ometer (U-2900UV/VIS Hitachi Tokyo, Japan). Concen-tration was modified to 105 cfu.ml-1 using sterile LB broth.

2.5 Minimum inhibitory concentration assessment

Briefly, 50 μl of the inoculated sample were administered into each well of the 96-well plates. Then, aliquots of 50 μl were dispensed into the wells, containing rAGAAN and organic acids at various concentrations. The 96-well plates with the lids closed were incubated at 37 °C for 18 h. Results were analyzed at 600 nm using microplate reader (BioTek, synergy H1, Winooski, USA). Control contained 100 μl of the bacterial inoculum. The MIC values included the lowest concentrations of the antimicrobial agents that cause bacterial growth inhibition.

2.6 Synergistic effects of rAGAAN with acetic and formic acids

Combination effects of rAGAAN with organic acids against the bacterial strains were assessed using checkerboard method. Briefly, 18-h cultures in LB broth were used to inoculate fresh LB broth to achieve a cell density of approximately 105 cfu.ml-1. Generally, 50 μl of the inoculated sample were added into 96-well microplates. Then, rAGAAN and organic acids were transferred into the 96-well microplates with increasing concentrations arran-ged in columns and rows, respectively. The organic acids were mixed with rAGAAN separately to assess their combinatorial effects on pathogenic bacteria. The purpose was to decrease the effective concentration of rAGAAN while preserving its antimicrobial activity. Assessment of the synergistic interactions involved the summation of the fractional inhibitory concentration indices (FICI) as Eq. 1 [23].

                                                                                                 Eq. 1

where, FICI ≤ 0.5 indicated synergistic relationships between the rAGAAN and organic acids that increased the antimicrobial activity, FICI > 0.5–4.0 was indifferent and FICI > 4.0 was antagonistic.

2.7 Kinetics of inactivation

The OD of bacterial strain was measured to assess the rate of inactivation when treated with rAGAAN, acetic acids or their combination. Bacterial culture, diluted in LB broth to a concentration of approximately 105 cfu.ml-1, was added to 96 well plates. The rAGAAN and acetic acid were added at their minimum inhibitory concentration (MIC) levels, individually and in combination with their fractional inhibitory concentration (FICI) at 1×, 2× and 3× to separate wells. The 96-well plate was incubated at 37 °C. The procedure entailed monitoring the rate of inactivation for various bacterial strains by measuring the OD at consistent intervals of 1 h for 5 h. The OD was measured using spectrophotometer set at 600 nm and microplate reader (BioTek, synergy H1, Winooski, USA).

2.8 β-Galactosidase assay

The β-galactosidase assay was carried out to assess effects of the rAGAAN and acetic acid or their combination on membranes of the bacteria. First, E. coli was inoculated into lactose broth and incubated at 37 °C for 18 h to stimulate β-galactosidase production. The bacterial cells were centrifuged and the pellet was washed thrice with 1× PBS. Then, the bacterial concentration was modified to roughly 105 cfu.ml-1 in 1× PBS solution. Moreover, 50 μl of purified rAGAAN, acetic acid and their combination at 1× FICI, 2× FICI and 3× FICI were added into wells of a microplate containing 50 μl of E. coli cell suspension. A volume of 30 μl of O-nitrophenyl-β-D-galactoside (ONPG) were added into every well of the microplate. The microplate was incubated at 37 °C and activity was assessed by measuring the spectrophotometric absorbance at 405 nm and various time intervals.

2.9 Hemolysis assay

The RBC lytic assay was carried out based on a procedure by Taniguchi et al. [24] with minor adjustments. The RBCs were washed thrice in 1× PBS and centrifuged at 14,530× g for 10 min. Pellet was dissolved in 1× PBS to achieve a concentration of 4%. Generally, 500 μl of blood were mixed with 500 μl of rAGAAN and acetic acid, indivi-dually and in various combinations (1×FICI, 2×FICI, and 3×FICI). The positive control included a solution containing 0.1% TritonX-100, while the negative control included a solution containing 1× PBS. Solution was incubited in microtubes at 37°C for 1 h and centrifugation was carried out at 14,530× g for 5 min. Then, 100 μl of the supernatant were extracted from each microtube and transferred to each well of 96-well plate. Assessment of hemoglobin release was carried out by measuring the absorbance at 540 nm.

2.10 rAGAAN-acetic acid against chicken meat spoilage

Antimicrobial efficacy of the rAGAAN and acetic acid combination was assessed using a methodology described by Ajingi et al. [25], with minor adjustments. In brief, fresh chicken meat was purchased from a local market and immediately transferred to the laboratory. Meat was divided into approximately 10-g specimens and washed thoroughly. Specimens were transferred into a laminar flow hood and 100 µl of 105 cfu of E. coli were divided to five separate locations. Sample was set for 1 h to promote appropriate attachment of the bacterial strains. Then, meat sample was submerged into 200-ml solution of rAGAAN/acetic acid for 1 h. Furthermore, sample was extracted, transferred into a plastic bag and incubated at 37 oC for 3 d. The chicken meat sample was transferred into a plastic bag with solution consisting of 0.1% peptone water. Sample was mechanically pulverized using stomacher to enhance liberation of the bacterial cells. Following the process of serial dilution, a 100 µl of sample were transferred onto an LB-agar plate. Number of colonies on the plate was counted at intervals of 0, 1, 2 and 3 d. Control group was administered with DW.

2.11 Statistical analysis

Results were present as mean ±SD (standard deviation) of three replicates. Statistical distinction was assessed using one-way analysis of variance (ANOVA) with Duncan’s multiple-range test. Differences with p < 0.05 were regarded as statistically significant.

  1. Results and Discussion

3.1 Minimum inhibitory concentration

The MICs of rAGAAN and organic acids against S. aureus and E. coli are present in Table 1. The MIC of rAGAAN against S. aureus and E. coli was assessed as 0.15 mg.ml-1. The organic acids inhibited proliferation of the pathogenic bacteria at various concentrations expressed as proportions (%).

Acetic acid demonstrated inhibitory effects on the growth of S. aureus at 0.2% v v-1 and on the growth of pathogenic E. coli at 0.25% v v-1. Formic acid inhibited growth of S. aureus at 0.25% v v-1 and growth of E. coli at 0.2% v v-1. The findings for acetic acid were similar to those against eleven mastitis pathogens in dairy cows with MIC values ranging of 0.125–0.25% v v-1 [26]. Similarly, Fraise et al. [27] reported antimicrobial activity of acetic acid against Pseudomonas aeruginosa and S. aureus at 0.166 and 0.312% v v-1, respectively. Manuel et al. [28] detected that formic acid at a concentration of 0.06% v v-1 exhibited antimicrobial effects against E. coli. Variations in their effectiveness against the microorganisms might be attributed to their chemical compositions. Methyl group (CH3) in acetic acid donated electron density to O-H bond, resulting in increased difficulties in removing the hydrogen atom. Consequently, acetic acid was weaker than the formic acid. Weak acids included a higher ability to pass through bacterial membranes, compared to strong acids due to the balances between their ionized and non-ionized states. The non-ionized form could easily diffuse through hydrophobic membranes. As a result, they provided proton gradients needed for ATP synthesis to collapse. This occurred because free anions such as acetate in this situation combined with periplasmic protons that were pumped out by the electron transport chain. Then, anions transported the protons back across the membrane without F1Fo ATP synthase [29].

3.2 Synergistic effects of rAGAAN with organic acids

The inhibitory concentration index (FICI), demons-trating combined effects of rAGAAN and organic acids, is present in Table 2. The compound rAGAAN demonstrated synergistic effects against S. aureus and E. coli when combined with organic acids. Results showed that the synergistic effects were strongest when using acetic acid for the two bacterial strains, compared to when using formic acid. The FICI values for the combination of rAGAAN with acetic acid were assessed as 0.375 (p < 0.5) for S. aureus and E. coli. The FICI values for the combination of rAGAAN with formic acid were assessed as 0.375 for S. aureus and 0.5 for E. coli.

 

 

 

 

Results indicated that sub-MICs of the antimicrobials were needed to effectively terminate the bacterial growth. Combination of rAGAAN and acetic acid resulted in a 25% decrease in the concentration of each antimicrobial, compared to their MICs. Synergism can occur when two various antibacterial agents, each with non-overlapping mechanisms of action, are combined with each other [30]. Therefore, the authors suggest that the antimicrobial effects could be strengthened using synergistic effects of combined organic acid with rAGAAN. While the precise process; by which, combination of rAGAAN with organic acid created synergistic effects is still unknown, studies have demonstrated that the cell membrane of bacteria is a shared target for the antibacterial effects of various antimicrobial peptides. Additionally, these peptides include an affinity for bacterial cellular components, including DNA [31]. In contrast, it is suggested that organic acids can delay absorption of nutrients and disrupt flow of electrons, leading to decreases in ATP production [32]. This various mechanism of action enables swift eradication of bacteria.

3.3 Kinetics of inactivation

Acetic acid was chosen for the study because it included stronger antimicrobial effects than that formic acid with rAGAAN did. Growth inhibition kinetics of rAGAAN, acetic acid and their combination on the logarithmic phase of the pathogenic bacteria are illustrated in Figure 1. When the peptide rAGAAN was mixed with acetic acid at the FIC, there was no noticeable alteration in OD for either of the bacterial strains during 5 h. This indicated that the bacterial growth was entirely suppressed. The combination demonstrated significant inhibitory effects, greater than that of the individual antimicrobial agent and control group. The combination exhibited the capacity to inhibit proliferation of S. aureus ATCC 6538 and E. coli ATCC 8739 at various concentrations within a few hours of exposure. Upon analyzing each treatment individually, it became clear that progressive decreases occured in OD measurements as time progressed. Nevertheless, use of rAGAAN with acetic acid led to further pronounced decreases in the turbidity level of the culture.

3.4 β-Galactosidase assay

To clarify the mechanism; by which, the combination acted, membrane permeability assay was carried out. This experiment used E. coli that was cultured in media containing lactose broth, which stimulated the synthesis of β-galactosidase. The β-galactosidase is an endogenous enzyme synthesized by the lac operon in bacteria. Release of this enzyme depends on disruption of the cell membrane. Release of the β-galactosidase enzyme from the disrupted cytoplasmic membrane was detected within 10 min of incubation with rAGAAN alone. In addition, cell membrane was destabilized by a combination of rAGAAN with acetic acid at 1× FICI, 2× FICI and 3× FICI, as shown in Figure 2.

Findings showed that the presence of rAGAAN, independently and in combination with acetic acid, could result in permeability of the cell membrane of E. coli. How-ever, acetic acid alone did not demonstrate effects on perme-ability of the membrane. Increasing OD measurements over time were directly linked to the rate of O-nitrophenol prod-uction from the breakdown of ONPG. The current results were similar to those of Yuan et al. [33], who observed increases in OD due to the degradation of ONPG when Larimichthys crocea myosin heavy chain protein-derived peptide was combined with low-intensity ultra-sound. Cell membrane disruption might lead to releases of intracellular components [34].

3.5 Hemolysis assay

Potential cytotoxicity of the chemical combination on human RBCs was assessed. Results of the hemolytic assay are present in Figure 3. The experimental group supernatant included clarity and transparency, which sharply contrasted to that the positive control group supernatant did. Further-more, no substantial fluctuation was recorded in absorbance of the experimental groups. This demonstrated that combin-ation of acetic acid and rAGAAN did not include any adverse effects on RBCs. However, absorbance of acetic acid alone resulted in approximately 50% hemolysis of the RBCs presented at high concentrations. Results suggested that the two antibacterial agents could effectively be combined as harmless food additives to combat pathogens.

 

3.6 rAGAAN-acetic acid against chicken meat spoilage

Results of the experiment are shown in Table 3 and Figure 4. Use of rAGAAN and acetic acid, either separately or in combination, led to significant suppressions of the E. coli growth. Inhibition was observed at various durations of exposure, in contrast to the control group (Table 3). At the beginning of the experiment (Day 0) after exposure to DW for 1 h, the control group showed decreases in the cell population to a value of 6.06 log 10 cfu.g-1. Significant decreases (p = 0.05) were observed in the bacterial count after individual treatment with rAGAAN and acetic acid (4.87 and 5.01 cfu.g-1, respectively). Similarly, when the combination therapy was administered at concentrations of 1×, 2× and 3×, the resulting bacterial counts were 4.85, 4.82 and 4.81 cfu.g-1, respectively. Significant differences in decreases were reported when comparing the control group to the groups that received rAGAAN treatment alone and the groups that received combination treatments at a three-fold fractional inhibitory concentration index as the duration of treatment increased. On Day 1 of the experiment, statistically significant decreases were seen in values of the microorganisms. More precisely, rAGAAN alone and its combination resulted in decreases of 1.39 and 1.40 log 10 cfu.g-1, respectively.

 

On Day 2, the 3× FICI combination group showed statistically significant decreases of 1.53 log 10 cfu.g-1, compared to the control group (p = 0.05). Furthermore, significant decreases of 1.63 log 10 cfu.g-1 were reported on Day 3, compared to the control group. Results of this study demonstrated that use of rAGAAN and acetic acid included combined antibacterial effects on E. coli in meat samples stored at 37 °C for 3 d. The synergistic effect was detected as superior to the antibacterial effects of rAGAAN when used separately. Results supported changes in the color of the meat. The transformation is documented through the alteration in color of the meat (Figure 4). The control group demonstrated a significant color change and included an unpleasant smell. This might be attributed to the prolif-eration of microbial organisms. Katiyo et al. [35] reported strong correlations between the odor and growth of microorganisms in chicken legs. The putrid smell of spoiled meat might be due to the existence of various compounds such as sulfur compounds, carbonyls, ketones, diamines and alcohols [36].

  1. Conclusion

This study provided evidence on the synergistic effects of combining rAGAAN antimicrobial peptides with organic acids to inhibit growth of two prevalent foodborne pathogens of S. aureus and E. coli. Significantly, this integr-ated approach created inhibitory effects at decreased con-centrations, compared to separate uses of these substances. The study revealed that rAGAAN included the highest beneficial synergy when combined with acetic acid. Simultaneous administration of rAGAAN and acetic acid caused disruption of the bacterial cell membrane without causing detectable harms to mammalian RBCs. Further-more, implementation of multiple treatments led to decre-ases in the presence of microorganisms in chicken meats after 3 d, compared to the group with no treatments. The study results can be used as secure economically feasible alternatives for the preservation of chicken meats. While the current study assessed two pathogens, further studies are necessary to investigate effects of the chemical combi-nations on a wider range of microorganisms as well as assessing precise mechanisms of the synergistic intera-ctions.

  1. Acknowledgements

This study was supported by research funds from Petchra Pra Jom Klao Research Scholarship (contract no. 74/2563) and Faculty of Science, King Mongkut’s University of Technology Thonburi.

  1. Conflict of Interest

The authors declare no conflict of interest.

  1. Authors Contributions

Investigation, writing-original draft, data curation and formal analysis, N.U.J.; formal analysis, validation, Y.S.A.; Data curation, formal analysis, N.R.; project administration and supervision, T.R.; methodology, supervision, W.S.; project administration and supervision, P.P.; supervision, conceptualization, funding acquisition, resources, methodology, N.J.

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Background and Objective: Komak beans include high nutritional values, making them promising raw materials for alternative food sources such as tempe. Because the beans are hard, they need soaking in water for 72 h with the water change every 12 h. Soybeans only need soaking for 24 h without changing the water during tempe processing. In this study, pressure cooking was used for Komak beans prior to soaking and a starter culture of Lactiplantibacillus plantarum subsp. plantarum WGK4 was added to the soaking water to decrease the quantity of water soaking and soaking time.

Material and Methods: Komak beans were pretreated by pressure-cooking for various times and texture and anti-nutritional factors were assessed. The selected pressure-cooked Komak beans were soaked in water and inoculated with Lactiplantibacillus plantarum subsp. plantarum WGK4. The pH, titratable acidity, soluble protein and minerals were assessed in the soaked water and Komak beans. The soaking water was assessed for viable lactic acid bacteria and anti-nutritional and volatile compounds were assessed in the soaked Komak beans. Mold fermentation was carried out by adding 0.2% (w.w-1) tempe starter culture to the drained Komak beans and incubating for 48 h at room temperature. 

Results and Conclusion: Dehulled Komak beans that were pressure-cooked for 15 min included a hardness value of 34.47 N, which was close to the hardness of boiled soybeans in traditional tempe preparing. Pressure-cooking Komak beans significantly decreased anti-nutritional factors. Addition of Lactiplantibacillus plantarum subsp. plantarum WGK4 during the 24-h soaking step decreased pH of Komak bean from 6.7 to 4.5. Decreases in tannin concentration was observed. Volatile compounds responsible for the beany flavor were not detected in the Komak beans at the end of the soaking. Pressure-cooking and addition of Lactiplantibacillus plantarum subsp. plantarum WGK4 significantly shortened the soaking time and decreased water needed for Komak Tempe processing. This process provides tempe as an affordable plant-based protein alternative.

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

 

  1. Introduction

 

Komak bean (Lablab purpureus (L.) Sweet) is a legume that is widely cultivated in arid areas such as Lombok Island, West Nusa Tenggara, and Indonesia [1]. Commonly cultivated as a backyard crop, intercrop or monoculture crop, Komak can be harvested four months after planting [2], allowing for year-round use and ensuring supply availab-ility. Komak beans have traditionally been consumed as vegetables and snacks. These beans are valued for their nutritional composition, characterized by moderate protein, high carbohydrate and low fat contents [3]. Availability and rich nutritional contents of these beans make them promising alternative food sources, especially as plant-based protein options such as tempe. Tempe, a traditional Indonesian fermented food, is processed by fermentation of soybeans with Rhizopus spp.

Soybeans are typically used as ingredients in tempe production. However, other beans such as jack beans [4], velvet beans [5] and other common beans [6] can be used. Time needed to prepare tempe varies, especially in the soaking step, depending on characteristics of the beans. Soybeans only need 24 h of soaking to soften the beans in tempe processing [7] but jack beans need 48 h of soaking with water changing every 12 h [4] and velvet beans need a 96-h soaking time with water changing every 12 h [5]. A preliminary study showed that Komak beans could be used as raw materials for Tempe; however, they need 72 h of soaking, with water changing every 12 h. Komak beans are hard with larger bean size, compared to soybeans [7]; however, Komak bean size is smaller than jack [8] and velvet [9] bean sizes. Therefore, Komak Tempe-preparing needs a longer soaking time and consumes six times more water quantity. Tempe production needs a large quantity of water for soaking, changing water and boiling, which hence generates wastewater containing components that can pollute the environment. Therefore, it is necessary to shorten soaking time and decrease water use while creating conditions appropriate for mold fermentation.

Studies have used pressure-cooking methods to speed up softening of beans during soaking. High pressure and uniform heat distribution improved softening of the beans. Time needed for softening can vary depending on size and hardness of the beans [10]. For example, pressure-cooking for 15 min at 110 °C effectively softens common beans [11] and a similar time at 120 °C is appropriate for barlotto beans, chickpeas and kidney beans [12]. Pressure-cooked Komak beans may decrease soaking time and provide a soft texture appropriate for acid fermentation. In soaking, natural acidification occurs due to spontaneous lactic acid bacteria (LAB), decreasing pH of the beans to 4–5, which can inhibit the growth of contaminant microorganisms and pathogenic bacteria [13].

The LAB are addressed for their significant roles in bean soaking of Tempe production. Studies by [14,15] have identified LAB at various stages of soybean tempe production. Moreover, LAB strains isolated at various stages of Tempe processing exhibit antimicrobial activities [16]. The LAB decrease anti-nutritional factors such as phytic acid, tannin [17] and beany flavor [18]. In studies, LAB were added when beans were soaking. These bacteria include Lactobacillusplantarum DSM 20174 in common beans [6] and Lactobacillus fermentum HPBD2 in soybeans [19]. This addition could decrease the pH value, shorten the soaking time and inhibit unwanted bacteria such as Enterobacteriaceae. The LAB of Lactiplantibacillus plantarum subsp. plantarum WGK4 isolated from the soaking water of legumes can grow and produce acids in jack bean [15] and black soy [20] milks. Research regarding the combination of pressure-cooking and LAB addition during the soaking stage prior to mold fermentation have not been carried out. Therefore, the major aim of this study was to investigate effects of pressure-cooking time of Komak beans prior to soaking and addition of Lactiplantibacillus  plantarum subsp. plantarum WGK4 starter culture to soaking water in Tempe processing on decrease of soaking-water quantity and soaking time.

  1. Materials and Methods

2.1. Materials

Cream-colored Komak jamak putek (Lablab purpureus (L.) Sweet) was received from farmers in the North Lombok District of Lombok Island, Indonesia. Samples were collected during a dry season (July–September), 2020. The L. plantarum subsp. plantarum WGK4 (WGK4) was isolated from water soaked in red lima beans in Tempe production [15],  Biotechnology Laboratory, Department of Food and Agricultural Product Technology, Faculty of Agricultural Technology, Universitas Gadjah Mada, Yogyakarta, Indonesia. A commercial Tempe starter (containing R. oligosporus) (Raprima, Bandung, Indonesia) was purchased from a local market in Yogyakarta, Indonesia. De Mann, Rogosa and Sharpe (MRS) media were supplied by Merck, Darmstadt, Germany. All chemical reagents were purchased from Sigma-Aldrich, USA.

2.2. Preparation of Starter Culture

The starter culture was prepared as previously described by Yudianti et al. [15]. Culture stock was stored at -20 °C in a sterile solution containing 1:1 ratio of 20% w.v-1 sucrose and 10% w.v-1 skim milk. Working culture was prepared by incubating the culture in MRS broth at 37 °C for 24 h, then inoculating it into MRS deep tube agar. This was incubated at 37 °C for 24 h and stored at 4 °C. Starter culture was activated by inoculation of the working culture into MRS broth and incubation at 37 °C for 24 h twice. Cell pellets were harvested by centrifugation at 2000 rpm for 10 min (Thermo-Fischer Scientific, Germany) and washing twice with sterile saline water. The LAB starter culture was quantified and recorded as CFU.ml-1.

2.3. Preparation of Komak Beans and Pressure-cooking Treatments

Komak beans were peeled using peeler machine (Yamamoto SY 150, Medan, Indonesia). Beans were rinsed and soaked in distilled water (DW) (1:5 w.v-1) for 6 h at ambient temperature. After discarding the soaking water, beans were washed with DW. Beans were transferred into an Erlenmeyer flask and filled with DW (1:2 w.v-1). Then, flask was subjected to pressure-cooking at 110 °C for 10, 15 and 20 min using portable pressure cooker (All American, Wisconsin, and USA). Raw, soaked dehulled and pressure-cooked soaked beans were assessed for hardness. These samples were freeze-dried (Labconco, Kansas City, USA) and transferred into a freezer at -20 °C until analysis of phytic acid, trypsin inhibitor (TI), tannin and volatile compounds.

2.4. Acid Fermentation of Komak Beans with Addition of Lactiplantibacillus plantarum subsp. plantarum WGK4

A starter culture of WGK4 was added to the selected pressure-cooked Komak beans with the soaking water. The primary LAB concentration was 10⁶ CFU.ml-1. Acid ferme-ntation was carried out at room temperature (RT) for 24 h. Soaked pressure-cooked Komak beans without addition of LAB starter culture were used as control. Every 2 h, samples of soaking water and soaked Komak beans were collected and assessed for pH and titratable acidity. Soaking water sample was assessed for viable cells as well. When pH of the Komak beans reached to 5.0 and 4.5, soaking water was analyzed for soluble proteins and minerals: potassium, magnesium, phosphorus, iron and calcium. Soaked beans were freeze-dried and stored at -20 °C until analysis of soluble proteins, minerals, anti-nutritional compounds and volatile compounds.

2.5. Komak Tempe Fermentation

Komak beans were added to a starter culture (WGK4) and soaked to achieve pH appropriate for mold fermen-tation. Beans were drained and inoculated with a comercial Tempe starter (0.2% w.w-1). Tempe fermentation was carried out for 48 h at RT using perforated plastic bags. Komak Tempe was freeze-dried and stored at -20 °C until analysis of soluble protein, phytic acid, TI and tannin.

2.6. Hardness Assessment

Hardness of the raw, soaked dehulled and pressure-cooked beans was assessed using universal testing machine (Z0.5, Zwick/Roell, and Germany) [9]. Assessment was carried out at a speed of 10 mm.min-1 with pre-load of 0.01 N. Hardness was assessed as the compression force used to deform the beans. Force was assessed in Newtons (N).

2.7. Viable Lactic Acid Bacteria Count

Viable LAB count was assessed based on a procedure described by Yudianti et al. [15]. Viable cells in soaking water were assessed using serial dilution and pour-plate methods as well as MRS agar added with CaCO3 and incubated at 37 °C for 48 h. After incubation, colonies on plates were calculated and reported as log CFU.mL-1.

2.8. pH and Titratable Acidity

The pH of the soaking water and beans was assessed using pH meter (FiveEasy F20, Mettler-Toledo, Switzer-land). Approximately 5 g of the crushed bean sample were mixed with DW (1:1). Titratable acidity analysis was carried out by titration of 5 ml of soaking water or 5 g of crushed bean mixed with DW (1:1) and 0.1 N NaOH using phenolphthalein (PP) as indicator [21].

2.9. Analysis of Minerals Contents

Mineral contents (K, Mg, P, Fe and Ca) were assessed using ICP-OES (Agilent Technologies 700 Series ICP-OES, Santa Clara, CA, USA) and assessed based on AOAC 2011.14 [21]. Nearly 0.5 ml of soaking water and 0.5 g of freeze-dried sample were digested and mineralized by adding 10 ml of concentrated HNO3, followed by 15 min of heating at 150 °C using microwave oven. Digested sample was set to reach RT and then an internal standard containing 100 mg.l-1 yttrium was added to the sample, followed by addition of DW to reach 50 ml of the final solution. This was filtered before ICP-OES analysis, using filter papers.

2.10. Analysis of Soluble Protein

Soluble protein assessment in soaking water and soaked Komak beans was carried out using Lowry method [22]. Freeze-dried sample (1 g) was added to 10 ml of DW and mixed for 30 min using water bath shaker. After centrifuge-ation at 3000 rpm for 15 min, supernatant (1 ml) and soaking water (1 ml) were mixed with 0.9 ml of Lowry A. Then, mixture was incubated using water bath shaker. Incubation time included 10 min at 50 °C. Cooled mixture was added with 0.1 ml of Lowry B and left for 10 min at RT. Then, 3 ml of Lowry C were added to the mixture and incubated at 50 °C for 10 min using water bath shaker. Mixture was cooled down to RT and absorbance was recorded at 650 nm immediately. Bovine serum albumin was prepared for the standard curve plotting.

2.11. Analysis of Phytic Acid 

Phytic acid content was assessed using a method described by Fitriani et al. [23]. Freeze-dried sample (0.1 g) was mixed with 20 ml of 0.5 M HNO₃ and incubated for 4 h at RT using water bath shaker. Then, mixture was filtered and 1 ml of the resulting extract was mixed with 1 ml of 0.005 M FeCl₃.6H₂O and 0.4 ml of DW followed by heating for 20 min in boiling water (100 °C). After cooling down, mixture was mixed with 5 ml of N-amyl alcohol and 0.1 ml of 0.1 M ammonium thiocyanate and centrifuged at 3000 rpm for 10 min. Then, absorbance was assessed at 495 nm. To generate phytic acid standard curve, a mixture of Na-phytate and HNO₃ solution was used.

2.12. Analysis of Tannin

Tannin concentration was assessed using a procedure described by Fitriani et al. [23]. A freeze-dried sample of 0.31 g was mixed with 62.5 ml of DW and boiled at 100 °C for 2 h. After cooling down, solution was filtered through Whatman filter papers no. 1. Then, 0.5 ml of Folin-Ciocalteu reagent and 2 ml of 20% Na₂CO₃ were added to 1 ml solution. Mixture was incubated at RT for 30 min. Absorbance was read at 748 nm.

2.13. Analysis of Trypsin Inhibitor

The TI was analyzed as previously described by Fitriani et al. [23]. The TI analysis was carried out by preparing TI extract, a substrate (BAPNA solution) and trypsin solution. The TI extract was prepared by dissolving 1 g of freeze-dried sample in 50 ml of 0.01 M NaOH. Mixture was agitated for 3 h at RT using water bath shaker, followed by centrifugation at 3500 rpm for 10 min. Substrate was prepared by dissolving 40 mg of BAPNA in 100 ml of 0.05 M tris-buffer (pH 8.2) in 1 ml of DMSO and stored at 37 °C. Substrate was prepared freshly before assessment. Trypsin solution was prepared by dissolving 4 mg of trypsin in 200 ml of 0.001 M HCl and stored at 4 °C. Generally, TI analysis initiated with the preparation of the control and sample solutions. Control solution was prepared by mixing 2 ml of DW, 5 ml of BAPNA and 2 ml of trypsin solution. Sample solution was prepared using a similar procedure but with addition of the extract. Solutions were incubated at 37 °C for 10 min using water bath shaker. Following incubation, 1 ml of 30% acetic acid was added to terminate the reaction and the mixture was centrifuged for 10 min. Absorbance measurement was carried out at 410 nm using UV/visible spectrophotometer. The TI formula was as Eq. 1:

                                                                                           Eq. 1

2.14. Analysis of Volatile Compounds

Headspace SPME/GC-MS was used to analyze volatile compounds of freeze-dried samples released in the headspace. Briefly, 5 g of freeze-dried sample were transferred into a headspace vial (22 ml) with polytetrafluorethylene-silicone septa. A divinylbenzene/ carboxen/polydimethylsiloxane (DVB/ CAR/PDMS) fiber was inserted into the vial and incubated at 80 °C for 45 min using water bath. Fiber was immediately injected into the injector port of the GC-MS for 5 min of desorption at 250 °C. Agilent 7890A GC and Agilent 5975C XL EI/CI MS (Santa Clara, CA, USA) were the instruments of analysis. The capillary column included DB-Wax (30 × 250 × 0.25 m). The oven temperature was initially set to 40 °C, increased by 5 °C.min-1 to 120 °C, then increased by 9 °C.min-1 to 240 °C and set for 5 min. Helium was used as carrier gas at a flow rate of 1 ml.min-1. The quadrupole and ion source temperatures were set at 150 and 250 °C, respectively. The volatile compounds were identified based on their mass spectra and the NIST 14.0 database. The linear index was calculated using retention data from the standard alkane series (C9-C31). Relative peak area (%) was estimated by comparing the peak area of each compound with the peak area of all compounds [24].

2.15. Statistical Analysis

A completely randomized experimental design with three replications was used in this study. Data were analyzed using one-way ANOVA followed by Duncan's multiple range test when significant differences were present (p < 0.05). Software used for the analysis was SPSS software v.25 (SPSS, Chicago, USA). Results are presen as mean and standard deviation (SD).

  1. Results and Discussion

3.1. Pressure cooking treatment

Komak beans were pressure cooked for various quantity of time and the hardness and anti-nutritional contents were assessed. This study demonstrated that pressure-cooking treatment significantly affected hardness (p < 0.05). Findings showed that raw Komak beans included the highest hardness (341.93 ± 0.86 N). Dehulling followed by soaking for 6 h (RBPS) significantly decreased the hardness to 84.29 N ±0.73 (Table 1). In soaking, dehulled beans absorbed water, decreasing hardness and increasing swelling of the beans, which might decrease the cooking time [10].

Pressure-cooking of the beans further decreased their hardness. The longer the pressure-cooking time, the softer the Komak beans. This softening was associated to structural changes in the beans. Breakdown of cell walls in the beans resulted in the release of pectin and protein middle lamella, which contributed to binding and firmness of the beans [11]. Dehulled Komak beans that were pressure-cooked for 15 min included a hardness value of 34.47 N ±0.51, which was similar to the value of the hardness of boiled soybeans from traditional Tempe (34 N).

Phytic acid, trypsin inhibitor and tannin content in raw Komak beans were 11.77 mg.g-1 ±0.97, 2.14 TIU.mg-1 ±0.02 and 11.94 mg.g-1 ±0.23, respectively (Table 2). Dehulling and soaking of komak beans significantly decreased the trypsin inhibitor and tannin content (p < 0.05) but did not significantly decrease the phytic acid content (p > 0.05). Pressure-cooking treatment significantly decreased the anti-nutrient factors in this study. Phytic acid concentration of the pressure-cooked dehulled komak beans for 10, 15 and 20 min decreased by 47.1-51%, compared to dehulled beans (RBPS). Phytic acid could bind to minerals such as calcium, copper, zinc and iron, delaying their absorption.

It has been reported that soaking and pressure-cooking of soaked kidney beans decreased phytic acid contents by 19 and 62%, respectively [25]. The higher phytic acid decrease in kidney beans could be attributed to longer soaking time and higher temperature during pressure-cooking. Soaking time for kidney beans was 12 h and pressure-cooking temperature and time were 121 °C and 30 min, respectively [25]. Trypsin inhibitor content of Komak beans was 2.14 TIU.mg-1 ±0.02 and trypsin inhibitor loss during soaking was 3.7%. However, a high decrease rate in trypsin inhibitor was detected in the pressure-cooking treatment (91.1-93.9%). High temperature and pressure could disrupt covalent and non-covalent bonds of proteins, resulting in inactivation of trypsin inhibitor activity [26]. Compared to raw beans, dehulled soaked beans decreased the tannin content by 7.8%, whereas pressure-cooked beans decreased the tannin content by 41.6-64.8% for 10-20 min. Decreases in tannin concentration during the soaking process might be caused by leaking of its compounds into the soaking water, while the pressure-cooking process might decrease the tannin content by thermal degradation of these compounds [27]. Decreases in tannin concentration due to pressure cooking of presoaked and soaked kidney beans and cowpeas have been reported as well [25,27]. This suggests that pressure-cooking Komak beans for 15 min can decrease the hardness similar to that of soaked soybeans and significantly decrease phytic acid, trypsin inhibitor and tannin contents. The pressure-cooking method can be used to soften beans and decrease anti-nutrient substances prior to the soaking step.   

3.2. Acid Fermentation of Komak Beans with Addition of Lactiplantibacillus plantarum subsp. plantarum WGK4

3.2.1. Viable cells, pH and Total Titratable Acid of Soaking Water and Komak Beans

Acid fermentation of Komak beans by pressure-cooking treatment for 15 min was carried out by the addition of WGK4 starter culture, followed by 24 h of incubation at RT. The soaking water used included water from the pressure-cooking step. Acid fermentation without starter culture addition was used as control (Figure 1A). No viable LAB were present during the soaking time. A possible explanation for this might be that natural LAB from the raw materials and soaking water were killed by heating during the pressure-cooking. Thus, titratable acidity of the soaking water and Komak beans in the control did not increase and pH of soaking water and Komak beans was approximately similar during soaking.

Acid fermentation supplemented with WGK4 starter culture (Figure 1B) showed increased LAB growth from 6.2 to 9.5 log CFU.ml-1 by the end of the soaking time. Viable LAB count increased significantly for 12 h and then was relatively constant until the completion of soaking. The LAB needed carbon and nitrogen sources, vitamins and minerals for their growth and metabolic activities [28]. Fermentable sugars in soaked beans and soaking water were used by LAB and metabolized to compounds, especially lactic acid, increasing titratable acidity and decreasing pH of the soaked beans and soaking water. Sucrose was the most common sugar in Komak beans.

The beans also contained stachyose (2.77%) and raffinose (0.45%) [29]. The WGK4 has been reported to use fructose, glucose, sucrose and raffinose for growth [15, 20]. According to [30], L. plantarum C6 used stachyose and raffinose to grow through the production of α-galactosidase. 

In this study, WGK4 showed a good growth rate during fermentation of the soaked Komak bean. After 24 h of ferm-entation, growth of WGK4 reached to 9.5 log CFU.ml-1. In addition, black soymilk fermentation using WGK4 at 37 °C increased the cell count from 6.4 to 9.1 log CFU.ml-1 [20]. This indicated that WGK4 could use nutrients in black soymilk for its growth. Kitum et al. [17] detected that addition of L. plantarum BFE 5092 to soaked kidney beans increased the cell count by 2 log cycles from 6.5 to 8.5 log CFU.ml-1 following fermentation for 24 h. The WGK4 growth during acid fermentation of Komak beans increased by more than 3 log cycles. It is possible that during pressure-cooking prior to soaking, nutrients from the Komak beans needed for the growth of WGK4 were released into the soaking water. Tables 3 and 4 show increases in soluble protein and minerals in the soaking water after pressure-cooking.

3.2.2. Soluble Protein and Mineral of Soaking Water and Komak Beans

After pressure-cooking treatment for 15 min, protein contents in water and Komak beans were 0.28% ±0.03 and 4.16% ±0.05, respectively (Table 3). Pressure-cooking increased the soluble protein of Komak beans, compared to raw beans. Total and soluble s of the Komak beans were 26 and 1.67%, respectively. Water used for the pressure-cooking was used for acid fermentation without changes or addition of fresh water.

During soaking, soluble protein in the Komak beans decreased, while soluble protein in the soaked water, either soaking without or with the addition of starter culture WGK4, increased. Decreases in soluble protein in the Komak beans without LAB addition could be due to the protein leaking from the beans to the soaking water. Addition of WGK4 lowered the soluble protein in soaking water and beans. During soaking, LAB used nutrients, including soluble proteins, for their growth and metabolic activity. These bacteria could produce proteolytic enzymes such as proteases and peptidases, which provided nitrogen sources for their growth and metabolic activity. Meng et al. [31] showed that soluble proteins in bean milk (soy, peanut and chickpea) decreased after fermentation with Lactobaci-llus fermentum GD01. The WGK4 grew well in black soymilk containing amino acids such as leucine, valine, glutamic acid, cysteine, arginine, methionine and histidine [20]. Raw Komak beans contain 18 various amino acids, including arginine, glutamic acid, leucine, valine and cysteine. These essential amino acids (EAAs) are needed by LAB, especially L. plantarum [32]. Soluble proteins in soaking water and Komak beans might contain these EAAs.

Minerals are micronutrient compounds detected in legumes. Komak beans are rich in potassium, magnesium, phosphorus, iron and calcium [2]. Raw beans contain 1686.95 mg of potassium, 150.26 mg of magnesium, 358.05 mg of phosphorus, 4.71 mg of iron and 123.07 mg of calcium per 100 g. After pressure-cooking, mineral content of the Komak beans decreased. Potassium, magnesium, phosphorus, iron and calcium concentrations in pressure-cooked Komak beans were nearly one-third to two-thirds lower than those in raw beans. The pressure-cooking process, which uses water as a medium, could cause minerals to leak into cooking water [33]. Mineral contents in Komak beans and soaking water after pressure-cooking soaked without and with WGK4 are shown in Table 4. The mineral mostly lost in the Komak beans was potassium. According to Damodaran et al. [34], potassium is present in foods as a free ion. Martinez-Pineda et al. [35] reported that pressure-cooking soaked chickpeas decreased potassium and phosphorus contents by 88.9 and 67.2% with final values of 95 and 104.6 mg per 100 g, respectively. Pressure-cooking was carried out at 118 °C for 40 min. The pressure-cooking process led to the loss of the mineral concentration of the beans. After pressure-cooking, Komak beans were soaked without and with addition of WGK4.

The current study reported that soaking Komak beans decreased the mineral content. Soaking with addition of WGK4 decreased the potassium content by more than 50%. Decreases in minerals of the Komak beans resulted in increases in minerals in the soaking water. The mineral loss during soaking might be due to the release of minerals into the soaking water [36]. They reported that soaking white faba beans for 24 h decreased iron and phosphorus by 28 and 16%, respectively. Iron content of the cowpeas decreased by 14% after soaking for 24 h [37]. However, concentration of the minerals in soaking water with the addition of WGK4 was much lower than that without the addition of WGK4 (Table 4). Increasing the soaking time with WGK4 significantly decreased minerals in Komak beans and soaking water. Technically, LAB need minerals in their growth media because they are unable to synthesize these essential nutrients. Therefore, media containing minerals must be provided. Soaking water provides minerals used by LAB for growth and metabolic activity. Examples of the minerals needed by LAB are Fe, Mg, Mn and Zn and their quantities vary depending on the microbial strain [38]. As reported by Leksono et al. [20], WGK4 LAB grew well in black soymilk containing minerals including iron, zinc, magnesium and manganese. Minerals were used to grow L. acidophilus KLDS 1.0738. During 14 h of fermentation, L. acidophilus KLDS 1.0738 utilized iron, magnesium and potassium at rates of 83.5, 27.2 and 9.6%, respectively [28]. This finding indicated that nutrients in the media could support growth of WGK4.

3.2.3. Anti-nutrient Factors of Komak Beans

Raw Komak beans contained 11.77 mg.g-1 ±0.97 phytic acid, 2.14 mg.g-1 ±0.02 trypsin inhibitor and 11.94 mg.g-1 ±0.23 tannin (Table 2). Pressure-cooking for 15 min decree-sed more than 50% of these anti-nutrient factors to 5.77 mg.g-1 ±0.34, 0.16 mg.g-1 ±0.03 and 4.56 mg.g-1 ±0.58 for phytic acid, trypsin inhibitor and tannin, respectively. Soaking the Komak beans without WGK4 addition did not significantly decrease contents of the anti-nutrient factors, except for tannin which decreased by more than 50% after 24 h of soaking (Table 5). Soaked Komak beans with the addition of WGK4 significantly decreased the anti-nutrient factors, compared to pressure-cooked beans. Decreases in phytic acid contents throughout fermentation might be associated to the phytase enzyme activity of beans and microbial fermentation [17]. Previous studies have shown that LAB fermentation can decrease the phytic acid contents of materials [17, 39]. For example, fermentation of kidney beans and faba bean flour for 24 h, using L. plantarum BFE5092 and L.plantarum E-78076, resulted in 28 (5.02 to 3.61 mg.g-1) and 14% (9.7 to 8.9 mg.g-1) decreases in phytic acid, respectively. Pressure-cooking followed by soaking with addition of WGK4 significantly eliminated phytic acid in the Komak beans. Although phytic acid is an anti-nutritional component, low levels of phytic acid are beneficial for health such as prevention of diabetes [40]. Consumption of phytic acid is safe and can be consumed up to 4500 mg per day [41].   

As shown in Table 5, trypsin inhibitor content was unaffected by addition of LAB to Komak beans. It showed relatively consistent values ranging 0.13-0.15 TIU.mg-1. Chandra-Hioe et al. [42] reported no significant differences in the content of trypsin inhibitor of chickpeas and faba bean flour fermented by LAB for 16 h. Addition of LAB significantly decreased tannin content of the Komak beans, with tannin content ranging 1.16-1.45 mg.g-1. Tannin content in soaked kidney beans fermented with L. plantarum BFE 5092 for 24 h decreased by 7% (3.07 to 2.83 mg.g-1) [17]. Decreases in tannin in Komak beans fermented with WGK4 could be linked to the ability of LAB to hydrolyze the tannin complex. Tannin content in Komak beans could be decreased by pressure-cooking followed by the addition of WGK4 during soaking. Tannin is safe to consume and consumption less than 1.5-2.5 g per day shows no side effects [43].

3.2.4. Volatile Compounds of Komak Beans

Volatile compounds in raw Komak beans were identified as aldehydes, alcohols, phenols, hydrocarbons, ketones, pyrazines, acids, esters, terpenes, furans and naphthalene (Table 6). After pressure-cooking for 15 min, concentrations of the volatile compounds, except aldehydes, mostly decreased. Further decreases in volatile compound concentrations were detected when Komak beans were soaked with or without addition of WGK4. Soaking Komak beans for 16 h with addition of WGK4 were selected since it achieved an appropriate pH value for mold fermentation. Similar to other legumes, Komak beans include a distinctive aroma that can affect consumer perception and hence limit their use. This distinct aroma is associated with an unpleasant odor, also called beany flavor. Volatile compounds contributing to the beany flavor have been described as beany, green and earthy [44, 45], derived from volatile aldehydes such as hexanal, nonanal and (E, E)-2, 4-nonadienal; alcohols such as 1-hexanol and 1-octen-3-ol; ketones such as 3-octen-2-one; and furan such as 2-pentyl furan [18, 44, 46]. In Komak beans, volatile compounds of the aldehyde group associated with beany flavor include nonanal and (E, E)-2, 4-nonadienal. Nonanal concentration of the beans soaked with addition of WGK4 was significantly lower than that of the pressure-cooking treatment. Nonanal and (E, E)-2, 4-nonadienal was detected at the beginning of soaking because of pressure-cooking. Increases in aldehyde compounds in yellow and grey peas (Pisum sativum) were reported due to enzymatic and non-enzymatic oxidation processes during heating [47]. Moreover, (E, E)-2, 4-nonadienal was undetected in Komak beans fermented with WGK4. According to Pei et al. [46], pea flour fermentation with L. rhamnosus L08 for 6 h decreased this compound to undetectable levels.

After soaking Komak beans with addition of WGK4 for 16 h, 1-octen-3-ol, an alcohol compound and 2-pentyl-furan, contributing to the beany flavor, were not detected. It might be linked to the enzymatic activity of these LAB. Significant decreases in 1-octen-3-ol were observed in L. plantarum X7021 soymilk fermentation. These decreases might be attributed to the strong oxidoreductase system of these LAB [48]. This result is similar to that of Saadoun et al. [49] in okara fermentation using L. acidophilus 8151, P. acidilactici 3992 and L. rhamnosus 1473. Furthermore, decreases in 2-pentyl furan by LAB fermentation were demonstrated by Saadoun et al. [49]. In addition to disappearance of volatile compounds contributing to the beany flavor, several pleasant odors were detected in Komak beans during 16-h soaking with addition of WGK4. These volatile compounds included 3-carene (citrus), D-limonene (citrus) and cis-β-farnesene (citrus green). This indicated that addition of WGK4 in the soaking step not only decreased pH of the Komak beans to desirable levels, but also decreased the beany flavor and tannin levels and produced volatile compounds responsible for the pleasant flavors.

3.3. Komak Tempe with Soaking in Lactiplanti-bacillus plantarum subsp. plantarum WGK4

Pressure-cooking followed by soaking Komak beans for 16 h with addition of WGK4 shortened the soaking time and decreased water requirements to one-sixth, compared to traditional Komak Tempe production. Tempe production needs a large quantity of water for soaking, water changes and boiling, which subsequently generates wastewater containing components that can pollute the environment. In the pre-mold fermentation process of jack bean Tempe production, beans with a hard texture were soaked for 24 h, boiled for 30 min and then soaked for 24 h. Then, beans were peeled and sliced into 4–6 pieces and soaked for 48 h. Soaking water was changed every 12 h. Sliced beans were boiled for 30 min. This process needed a large quantity of water and a soaking time of 96 h [4]. In this study, the soaking time was 16 h, which included a faster method. Studies by [5] on velvet beans revealed that the beans were soaked for 24 h, boiled for 30 min, peeled, sliced, soaked for 96 h with water changes every 12 h and boiled again for 30 min. This process needed eight times more water and included a longer time. Therefore, method used in this study was shorter and involved a fewer steps than that other processing methods did.

A 48-h fungal fermentation of the Komak beans formed compact mycelia, white cake-form products (data not shown), containing 6.23% ±0.55 soluble protein and anti-nutrient factors of 3.46 mg.g-1 ±0.21 phytic acid, 0.13 TIU.mg-1 ±0.03 trypsin inhibitor and 6.69 mg.g-1 ±0.26 tannin. Mold fermentation increased the soluble protein of the Komak Tempe by 59.8%. Raw Komak beans included a protein solubility value of 1.67%. Increases in soluble protein during Tempe fermentation were caused by the presence of protease enzymes produced by the molds throughout the fermentation [50]. Proteolytic enzyme activity is considered as the major factor in protein hydrolysis during Tempe fermentation, causing releases of peptides and free amino acids, thereby increasing dissolved nitrogen. Protein solubility in soybean, chickpea, pea and horse bean Tempe respectively increased by 66.4, 62.7, 62.3 and 60.7%, compared to raw beans [51]. Increases in soluble protein during Tempe fermentation could include positive effects on the nutritional quality of Tempe. As the protein in beans is broken down into simpler forms during ferment-ation, protein solubility increases, making it easier for the body to absorb the protein.

The anti-nutritional components of Komak Tempe resulting from the addition of WGK4 during soaking are present in Table 7. Phytic acid in Komak Tempe decreased from 4.96 to 3.46 mg.g-1 compared to beans prior to Tempe fermentation (beans soaked with WGK4) (Tables 5 and7). Fermentation of Rhizopus oligosporus generates phytase enzymes that hydrolyze phytic acid present in beans into organic phosphate and inositol [52]. Decreases in phytic acid were reported in soybean Tempe from 32.30 to 28.13 mg.g-1 and kidney bean Tempe from 3.90 to 3.53 mg.g-1, compared to cooked beans from dehulled soaked beans [53]. Phytic acid content observed in the Komak Tempe was lower than that in soybean and kidney bean Tempe.

 Trypsin inhibitor in the fermented Komak Tempe did not decrease. Trypsin inhibitor in the Komak Tempe was 0.13 TIU.mg-1. According to [6], thermal use (cooking and autoclaving) is more effective in eliminating trypsin inhibitors than the activity of microorganisms alone. For tannin contents, Tempe fermentation increased tannin contents of Komak Tempe, compared to the beans prior to Tempe fermentation (Tables 5 and 7). Tannin content of the Komak Tempe was 6.69 mg.g-1. This increase might be due to the enzymatic hydrolysis of condensed tannins [52]. Increases in tannin content of the velvet bean Tempe have been reported by Ezegbe et al. [54]. Tannin content of soybean Tempe was 7.6 mg.g-1 [52]. Although soybean Tempe contains anti-nutritional components such as phytic acid and tannin, it is widely consumed by Indonesian people, especially the Javanese. Therefore, Komak Tempe can safely be consumed as an alternative to soybean Tempe. Based on the findings of this study, addition of WGK4 during acid fermentation of softened Komak beans can be used for the Tempe production, resulting in Komak Tempes that are safe for consumption.

  1. Conclusion

Dehulled Komak beans pressure cooked for 15 min included a hardness value of 34.47 N. This value was close to the hardness value of boiled soybeans from traditional Tempe preparing. Pressure-cooking of Komak beans significantly decreased their anti-nutritional components, including phytic acid, trypsin inhibitor and tannin. Addition of L. plantarum subsp. plantarum WGK4 during the soaking step for 24 h decreased pH of the beans from 6.7 to 4.5.

These LAB use nutrients in the media such as soluble proteins and mineral elements for growth and metabolism. At the end of the soaking period, tannin level significantly decreased and the volatile compounds associated with the beany flavor were no longer available in the Komak beans. Therefore, L. plantarum subsp. plantarum WGK4 can potentially be used as a starter culture for acid fermentation in Tempe processing. Pressure-cooking and addition of these LAB significantly decreased the soaking time and water needed for Komak Tempe processing. These findings can highly help Tempe processing in resource-rich regions, as they shorten the processing time and save water. However, it is necessary to provide LAB culture starters in further applicable powdered forms.

  1. Acknowledgements

The authors thank the Ministry of Education and Culture of Indonesia for awarding Domestic Postgraduate Education Scholarships (no. B/67/D.D3/KD.02.00/2019). This study was carried out with independent funding.

  1. Conflict of Interest

The authors report no conflicts of interest.

  1. Authors Contributions

Conceptualization, T.U. and E.S.R.; methodology, W.W.; software, W.W.; validation, T.U., E.S.R. and W.S.; formal analysis, W.W.; investigation, W.W.; resources, T.U.; data curation, T.U., E.S.R. and W.S.; writing-original draft, W.W.; writing-review and editing, T.U., E.S.R. and W.S.; visualization, W.W.; supervision, T.U.; project administration, W.W.; funding acquisition, T.U.

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Review Article


Emerging Studies on Zataria multiflora Boiss L.: Pioneering the Antimicrobial and Antifungal Characteristics–A Systematic Review

Zahra Pilevar, Kristin Haglund, Vahid Ranaei, Mansoureh Taghizadeh, Nasim Maghboli Balasjin, Hedayat Hosseini

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e20
https://doi.org/10.22037/afb.v11i1.44252

Background and Objective: Zataria multiflora Boiss L., a medicinal herb, is addressed for its diverse biological characteristics, including antibacterial, antiviral, antifungal, antioxid-ant and pain-relieving characteristics. However, specific mechanisms and compounds responsible for these effects are still under investigation, particularly for their comparative efficacies. The aim of this study was to bridge this knowledge gaps by providing a focused novel analysis of the chemical composition and antimicrobial and antifungal effects of Zataria multiflora Boiss, highlighting its potential health benefits and therapeutic uses.

Material and Methods: This review was carried out following standard protocols for systematic analyses. Comprehensive literature searches were carried out in multiple databases, including PubMed, EMBASE, CINAHL, Cochrane Library and Web of Science. Key search terms included "Zataria multiflora Boiss", "antibacterial", "antifungal", "chemical composition" and "biological activities". The review time period included 2003 to 2023 with 71 relevant articles selected based on predetermined inclusion and exclusion criteria. This approach ensured adherence to the journal formatting standards for systematic reviews.

Results and Conclusion: The present analysis highlighted thymol and carvacrol as the primary compounds of interest in Zataria multiflora Boiss L. linked to its most potent antimicrobial and antifungal effects. Additionally, it was discovered that the antifungal characteristics of this herb were particularly pronounced, surpassing its other biological activities. However, the review included a limited evidence regarding the plant sedative and muscle relaxant characteristics, which fell outside the primary scope of this study on antimicrobial and antifungal effects.

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

1.Introduction

Use of medicinal plants and herbal medicines is still significant despite advances in synthetic drug develop-ment. In some countries, these natural remedies are critical parts of the healthcare system, often surpassing the trade of chemical drugs [1–3]. The World Health Organization (WHO) "Health for all by the year 2000" program emphas-ized importance of traditional medicine, leading to increased scientific and commercial interests in this field. Iran, with its diverse climate, is particularly addressed for its wealth of medicinal plants [4–7]. Shirazi thyme or Zataria (Z.) multiflora [8] is a significant example that is majorly detected in Iran, Pakistan and Afghanistan. This plant, as a part of the Lamiaceae family, grows up to 90-cm tall and is characterized by ovate circular leaves, dense tubercular mottling and white, hairy rounded buds in the leaf axils [9]. It is well-known for its culinary and medic-inal uses, particularly for its antiseptic, analgesic, anti-parasitic and antidiarrheal characteristics [10, 11]. Modern pharmacology verifies its therapeutic effects, including pain relief, spasm decrease and antiinflammatory effects. The Z. multiflora is used in various medicinal forms e.g. syrup and cream to treat a wide range of medical conditions, like inflammatory bowel disease (IBD), vagin-itis, dental pain, oral infections, respiratory and digestive diseases, pain, fever as well as common colds [9, 12–15].

The Z. multiflora essential oil (ZMEO) has been approved by the United States Food and Drug Administration (US FDA). Naturally, ZMEO contains phenolic oxygenated monoterpene compounds (carvacol, linalool and thymol) that act as free radical scavengers. Phenolic compounds are addressed as plant secondary metabolites, which are formed by the connection of an aromatic nucleus to one or more hydroxyl groups. These compounds are abundantly distributed in all parts of the Z. multiflora and include significant antioxidant activities. The antioxidant activity can be attributed to their redox characteristics and chemical structures [8, 17, 18]. A standard method for analysis of Z. multiflora secondary metabolites is gas chromatography (GC) coupled with mass spectrometry. Recently, methods such as mid-infrared spectroscopy, near-infrared spectroscopy and Raman spectroscopy have been used as chemical fingerprinting methods to analyze various secondary metabolites of plants [19]. Phenolic acids, poly-phenols and flavonoids are important compounds that include a wide range of biological activities. Their antioxidant activities are due to their polyphenolic natures. Their medicinal use includes a long history and are marketed as antispasmodic and anti-inflammatory drugs [20–22]. The study of yield efficiency and chemical compositions of Z. multiflora flowering branches essential oil (EO) showed that the highest and the lowest efficiencies of EO were associated to Zarghan (4%) and Sivand (2.91 %), respectively.

Increasing global interests in medicinal plants, reinforced by WHO emphasis on traditional medicine, scores relevance of investigating less used natural remedies such as Z. multiflora [8]. Studies have shown medical uses of Z. multiflora such as mouthwash for the treatment of aphthous stomatitis [9], IBD and vaginitis [23]. Originating from regions such as Iran, Pakistan and Afghanistan, Z. multiflora has long been recognized for its culinary and medicinal uses. While its antiseptic, analgesic, antiparasitic and antidiarrheal characteristics are well-documented, recent advancements in pharmacology include further validations of its broad therapeutic potentials. Therefore, the aim of this systematic review was to investigate less-studied dimensions of Z. multiflora, particularly focusing on its phenolic compounds such as carvacol, linalool and thymol, which are critical for their antioxidant activities. While the FDA has acknowledged benefits of ZMEO, significant scopes for the comparative studies on this EO with those of other medicinal plants and with investigation of its novel clinical uses are still available. The aim of the present study was to bridge this gap by providing a comprehensive analysis of Z. multiflora chemical composition and its various effects, especially in antimicrobial and antifungal fields. Furthermore, this review addressed phytochemical diversity of Z. multiflora, considering variations in its EO composition from various habitats. Such insights are important for cultivating Z. multiflora for industrial, food, pharmaceutical and cosmetic means. By investigating these novel aspects, this study developed understanding of Z. multiflora roles in healthy living, especially in modern medicine and therapy.

2. Materials and Methods

2.1. Search strategy and study selection

Published research articles of 2003–2023 (last 20 years) in English were analyzed using relevant terms in Google Scholar, PubMed, EMBASE CINAHL, Cochrane Library and Web of Science databases. Search was carried out using the following keywords of "antibacterial activity", "anti-fungal", "chemical”, "Zataria multiflora", "biological act-ivities", "chemical composition", "Zataria multiflora Boiss", "antioxidant activity", "antioxidant" and "extract". Combinations of these words with the operators "and" and "or" were searched as well.

2.2. Inclusion and exclusion criteria

Inclusion criteria included studies; in which, Z. multiflora Boiss L. and parameters associated to the extract and EO of this plant were assessed without limitations. Studies; in which, Z. multiflora Boiss L. was examined along with other plant species with no restrictions in terms of chemical characteristics included were investigated as well. Interventional studies exclusively assessing anti-fungal characteristics of Z. multiflora Boiss L. were selected for the present study. Exclusion criteria were extended for the studies involving plants other than Z. multiflora Boiss L. and those that did not primarily investigate antifungal attributes of Z. multiflora. Articles with no full texts were excluded from the study, as well as review articles, descriptive studies and manuscripts that were invalid, unassociated or did not meet the specific criteria of focusing on the antifungal aspects of Z. multiflora Boiss L.

2.3. Articles and screening selection protocol

The primary search yielded 2912 citations. Removal of 941 duplicates resulted in 1971 citations. Titles and abstracts of 1971 articles were screened. Furthermore, 839 articles that did not meet inclusion criteria or were inappropriate due to indirect links with the subject were excluded. The rest of 1132 full-text articles were reviewed. of these articles, 1061 were excluded and 71 relevant studies were selected based on their relationships with the article goals, inclusion and exclusion criteria and their qualities. Method of analysis and interpretation included determining the study purpose and collecting findings based on preferred reporting items for the systematic review (PRISMA) (Figure 1). Quality of the final articles was assessed separately by an evaluating researcher with experience in systematic reviews and biological topics.

  1. Results and Discussion
3.1. Selection of articles

In the primary search, 2912 records were identified. Totally, 941 articles were excluded due to duplication. By assessing texts of the articles based on the inclusion and exclusion criteria and topic relevance, a total of 71 articles were included in this review (Figure 2). Moreover, 48 articles assessed for antimicrobial and antifungal effects or pharmacological activities and 20 articles assessed for antimicrobial and antifungal effects. Antimicrobial and antifungal effects referred to the highlighted effects on patients’ symptoms or outcomes. Data regarding chemical composition were collected and reported as follows.

3.2. Comprehensive pharmacological analysis of Zataria multiflora Boiss L.

Table 1 provides a detailed analysis of the pharma-cological characteristics of Z. multiflora Boiss L. from 48 scholarly articles. This table extensively docum-ents the plant robust antifungal capabilities, with 25 studies corro-borating this attribute. Furthermore, it highlights the plant significant antibacterial characteristics, as identified in nine studies. The present study further includes investigation of Z. multiflora potentials in anti-cancer and antioxidant uses, each detailed in five studies. The table uniquely highlights studies assessing combined anti-bacterial and antioxidant effects of the plant, thereby enriching knowledge of its multifaceted pharmacological uses. This in-depth compilation of research scores Z. multiflora Boiss L. broad therapeutic potentials, illustrating its various uses in modern medicine. Results from studies on Z. multiflora Boiss L. can be translated into practical uses in medicine and therapeutics in various ways including antifungal and antibacterial treatments, cancer therapies and antioxidant benefits. The verified antifungal characteristics can lead to development of novel antifungal drugs, especially for drug-resistant fungal infections. Its anti-bacterial effects suggest potential uses in treating bacterial infections, possibly as an alternative to traditional antibiotics. Anti-cancer findings may contribute to novel approaches in cancer treatment, possibly as complementary therapies with conventional treatments. Antioxidant characteristics indicate uses of the plant in combating oxidative stress-related diseases, potentially as a dietary supplement. These potential uses demonstrate the therapeutic versatility of Z. multiflora, offering promising capabilities for future pharmaceutical developments. It is noteworthy that geographic origin and research methodology significantly affect results of studies on Z. multiflora Boiss L. Chemical com-position of plants can vary based on soil type, climate and other environ-mental factors. Studies from various regions may report variations in the concentration of active compounds, affecting their pharmacological efficacies. Moreover, selection of the extraction methods, experimental models and assay techniques can lead to various outcomes. For example, in-vitro studies may show various levels of efficacy, compared to those in-vivo studies may due to the complexity of biological systems. The reviewed studies show a wide range of pharmacological activities for Z. multiflora Boiss L., including antifungal, antibacterial, anti-cancer and antioxidant activities. This addresses potentials of this plant for various therapeutic uses.

3.3. Diverse pharmacological effects of Zataria multiflora

The Z. multiflora, known for its antimicrobial and antifungal characteristics, has extensively been studied for various pharmacological effects. A comprehensive review of 20 articles has revealed a wide range of therapeutic potentials. These include relaxant effects in ten studies, which have shown significant improvements in conditions such as asthma and premenstrual syndrome. Three studies highlight its anti-parasitic efficacy, demonstrating effecti-veness against malaria vectors and Leishmania spp. Anal-gesic characteristics have been verified in two studies, showing decreased pain in conditions such as irritable bowel syndrome (IBS) and postpartum pain. Similarly, two articles have reported its anti-inflammatory effects, indicat-ing decreases in inflammatory cytokines and factors. Furthermore, a significant study has investigated its potential in nerve repair, particularly in a rat model of Alzheimer's disease, showing its neuroprotective charac-teristics. These diverse findings highlight the multifaceted pharmacological uses of Z. multiflora (Table 2).

3.4. Antifungal activity

Antifungal effects of thyme extract and EO have been reported in various studies [24–27, 29–33]. Various studies have shown that Candida albicans is one of the most important causes of oral thrush and vaginal yeast infection and is strongly affected by the Z. multiflora extract and EO [24, 26, 32–34, 42–45, 48]. In 2015, Avaei et al. identified a total of 43 Z. multiflora compounds, whose major components included thymol, carvacrol, p-cymene, γ-terpinene and α-pinene. Other components make up less than 19.81% of the oil. Results of the antimicrobial analysis showed that Bacillus cereus was more resistant than the other two bacteria. Of the yeasts, Saccharomyces cervicii was more resistant than C. utilis. From the fungal species, growth of Penicillium digitatum and Aspergillus niger was inhibited by a similar oil concentration. Results of the present study showed that ZMEO included significant antimicrobial activity [30]. In a study, the lowest inhibitory concentration of EOs of 0.007–0.5 μg/ml was achieved [40]. In another study, it was detected that the antifungal effects of ethanolic and methanolic Z. multiflora extracts were significantly higher than its aqueous extract [47]. Further clinical studies in this field can help better understand antifungal characteristics of this plant.

3.5. Antibacterial activity

Various studies have shown effects of Z. multiflora extract and EO on various pathogenic bacteria [50, 51]. Results showed that the extract affected Gram-positive and Gram-negative bacteria [23, 49, 52]. Assessing the anti-bacterial and antioxidant characteristics of ZEO and Rhus coriaria L. hydroalcoholic extracts, Mojaddar Langroodi et al. showed that sumac extract included a stronger antioxidant activity than that the ZEO did. Based on antibacterial activity results, ZEO was more potent than sumac extract [67, 91]. Mansour et al. investigated antibacterial effects and physicochemical characteristics of ZMEO. Results showed that ZMEO was effective on pathogenic bacteria, especially Staphylococcus aureus. Investigating physicochemical characteristics such as effects of pH, temperature, detergents and enzymes on ZMEO activity showed that EO was completely stable against temperature and very stable in a wide range of pH. Antibacterial activity of EO is insensitive to all types of protein-denaturing detergents (e.g. Tween 80, Tween 20 and Triton 100) and enzymes (e.g. proteinase K, trypsin, lipase and lysozyme). Therefore, potential use of ZMEO is suggested. However, further studies including purification, mass spectrometry, nuclear magnetic resonance (NMR) and toxicity assessment are needed to verify this suggestion [92, 93].

3.6. Antioxidant and antitumor activities

Appropriate anti-cancer effects of Z. multiflora extract and EO were reported in five studies [57–61]. In several studies, it was detected that this plant could increase cell death in colon carcinoma, cervical cancer and breast cancer and this increase indicated toxicity caused by the plant extract [59–61]. In 2022, Saffari et al. investigated chem-ical composition, bioactive functional groups, antioxidant ability and cytotoxicity of Shirazi-thyme EO on HT29 cell line. Results showed that with increases in Shirazi-thyme EO concentration, effects on HT29 cell line increased and its survival rate decreased. Based on antioxidant power results, phenol and flavonoid of ZMEO, it is possible to use Z. multiflora as a natural preservative in the food industry [94].

3.7. Anti-inflammatory and analgesic activities

Based on the studies, blood inflammatory factor levels in the Zataria group were significantly improved [88, 89]. In addition, anti-inflammatory and analgesic effects of this plant have been observed in interventional studies. Ghorani et al. demonstrated that patients improved significantly in their blood inflammatory factors after two months of treat-ment [88]. Another study showed that gastrointestinal patients consuming Shirazi thyme experienced less pains [86]. These have verified anti-inflammatory and analgesic activities of Z. multiflora.

3.8. Antiparasitic activity

Antiparasitic activity of plants has been identified during various investigations [83–85]. Based on a study, ZMEO could be effective against Anopheles mosquitos [84]. In another study, antiretroviral, antimalarial and anti-inflammatory roles of Betulinic (one of the important compounds of ablution) were reported [84, 95] in a study, nanoparticles containing extracts of several plants, including Shirazi thyme, inhibited major malaria vectors [83]. These findings show the importance of thyme as a complementary anti-parasitic treatment.

3.9. Relaxant effects

Various therapeutic effects of Z. multiflora such as bronchial dilation and decreases in lung inflammation, common colds and women's disorders have been reported [72, 73]. Findings have shown that Z. multiflora includes good relaxing effects on smooth muscles. Relaxation can be therapeutically important, especially in respiratory obstructive disorders, high blood-pressure vasodilation and digestive disorders [75–79]. Possible mechanisms of Z. multiflora and its component relaxant effects (majorly carvacrol) on smooth muscles, including inhibitory effects on histamine (H1) and muscarinic receptors, blocking effects on calcium channels and stimulating effects on beta-adrenergic receptors, have been verified [96, 97]. Based on the findings, relaxant effect is one of the most important characteristics of carvacrol and thymol compounds, which are abundantly detected in this plant [98]. Several studies have verified these effects, indicating potential roles for this herb in smooth-muscle relaxation.

3.10. Chemical composition

Table 3 comprehensively details chemical composition of Z. multiflora Boiss, as identified in various research studies within the last two decades. This table highlights that Z. multiflora Boiss is predominantly comprised of monoterpenes. Significantly, carvacrol is the predominant compound, with its concentration ranging 26.69–76.18% in 13 distinct studies. Another compound is closely thymol, with its concentration varying 19.89-71.40%. Other significant compounds such as p-cymene, γ-terpinene, linalool and myrcene have been reported. Presence of fatty acids, particularly β-sitosterol and Stigmasterol, has been addressed in two studies. Flavonoids, including 6-hydroxyluteolin, apigenin and luteolin were identified, each in two separate articles. This table provides an in-depth insight at the phytochemical diversity of Z. multiflora Boiss, underlining its potentials for various biological and therapeutic uses.

3.11. Chemical composition

The most important compounds in this plant are monoterpenes with nearly 70% reported [54, 102]. Two researches have reported carvacrol, which is a type of monoterpene as the major compound of this plant extract [106, 107]. Thymol has been identified as the major compound in several research [101]. Various results have been reported regarding quantities of carvacrol and thymol. In a semi-experimental study, it was detected that 61% of the content included carvacrol and 25% included thymol [104]. In another similar study, it was detected that nearly 73% of thymol were present in fresh plants and nearly 63% of carvacrol were present in dried plants [105]. These two important substances in Z. multiflora plant include high anti-carcinogenic roles and environmental factors and plant stresses, stage of plant growth; geographical area is another important factor that affects their size [75, 108, 109]. Another major compound is p-cymene [110]. Based on a study, Z. multiflora contains flavonoids such as apigenin, luteolin and 6-hydroxyluteolin [111].

In a current systematic review, the most detected compound was carvacrol, which was reported as 26.69–76.18% followed by thymol as 19.89–71.40% in 13 studies. Based on the findings, thymol and carvacrol are phenolic monoterpenes that include positive effects on cellular functions and cellular and humoral immune responses [109, 112]. Older findings have shown that Z. multiflora contains small quantities of tannin, resin and saponins. Alkaloid compounds have not been observed in this plant [113]. Thyme contains fatty acids such as behenic acid, lignoceric acid, cerotic acid and montanic acid. Based on other findings, triterpenes such as hydroxycinnamic acids, rosmarinic acid, betulin, betulinic acid and oleanolic acid and fatty acids such as β-sitosterol and stigmasterol are detected in Z. multiflora extracts [99, 111]. In 2017, Majdi et al. reported that thyme played key roles in biosynthesis of antimicrobial and antifungal activities in addition to abiotic stimuli and growth and spatial factors. Hence, optimization of these factors can be addressed as a useful strategy to achieve high yields of valuable compounds in T. vulgare or other closely-related plant species [114].

Findings by Karimi et al. have shown that the constit-uent content of oil and hydroalcoholic extracts is correlated with various geographical locations, weather and demo-graphic factors [103]. Increasing effective compounds in this plant can help improve its effectiveness.

  1. Conclusion

In all the studies, the most important compounds of Z. multiflora were carvacrol and thymol. The Z. multiflora extract and EO included significant antifungal, antioxidant, antibacterial, antiparasitic, anti-inflammatory and pain-relieving effects. Antifungal activity of this plant was more significant, compared to other biological activities of the plant. In addition, sedative effects of Z. multiflora and its compounds included relatively strong relaxing effects on various types of smooth muscles. Due to the extraordinary characteristics of this plant, further clinical and laboratory studies are needed to clarify its clinical uses, possible side effects and effective concentrations.

  1. Acknowledgements

Authors appreciate the support provided by Shahid Beheshti University of Medical Sciences through project NO. 5- 43005777 for this study.

  1. Conflict of Interest

The authors declare no conflict of interests.

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https://doi.org/10.1111/j.1755-5949.2011.00237.x

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Short Communication


Effects of Pasteurization on Antihyperglycemic and Chemical Parameter of Xoconostle (Stenocereus stellatus) Juice

Jose Alberto Mendoza-Espinoza, Sindu Irais Gomez-Covarrubias, Edgar Sierra Palacios, Erika Alvarez-Ramirez, Rayn Clarenc Aarland, Fernado Rivera-Cabrera, Rocio Gomez-Cansino, Patricia Bustamante-Camilo, Fernando Diaz de Leon-Sanchez

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e4
https://doi.org/10.22037/afb.v11i1.43252

Background and Objective: The antihyperglycemic effect is associated with the pre-hispanic fruit xoconostle or tunillo (Stenocereus stellatus, Pfeiffer and Riccobono). This fruit includes in various varieties, distinguished by color. Xoconostle fruits are highly perishable. Therefore, the aim of this study was to assess antihyperglycemic effects of xoconostle juice before (fresh) and after pasteurization. The study focused on the white and red varieties of xoconostle.

Material and Methods: In this study, the method involved collecting juice from xoconostle fruits, followed by pasteurization. Chemical, physical and microbial parameters were assessed for the juice and the ability to decrease capillary glucose levels (antihyperglycemic effect) was assessed in male Wistar rats.

Results and Conclusion: Pasteurization process led to decreases in total phenolic content of the red variety of xoconostle fruit, while the white variety showed increases in malic acid content. Despite these changes, fresh and pasteurized juices of the two varieties showed lower blood glucose levels, compared to the control group. Red variety demonstrated a stronger antihyperglycemic effect. In conclusion, pasteurization did not affect pharmacological effects of xoconostle juice, making it a viable preservation method without compromising the antihyperglycemic charac-teristics. Results of this research suggest a conservation method which preserve the antihyperglycemic effects while extending its shelf life.

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

Leptospirosis in Slaughterhouse Personnel: A Seroepidemiologic Study Using Microscopic Agglutination Test

Ali Hokmi, Seyyed Saeed Eshraghi, Abbas Rahimi Foroushani, Gholamreza Abdollahpour, Ramin Mazaheri Nezhad Fard

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e5
https://doi.org/10.22037/afb.v11i1.43527

Background and Objective: Meat can be contaminated by Leptospira species. This bacterial pathogen causes severe leptospirosis disease in humans and animals. The major aims of this study were to assess seroepidemiological prevalence of leptospirosis in employees of a slaughterhouse in Guilan Province, Iran, using microscopic agglutination test and further investigate the positive samples using nested polymerase chain reaction method.

Material and Methods: In this study, 150 employees of a slaughterhouse in Guilan Province, Iran, were participated after completing written consents and personal questionnaires. This sample size was calculated based on the mean prevalence of the pathogen in the region. After assessing sera of the participants for Leptospira antibody using microscopic agglutination test, urine samples were collected from the positive participant for further assessments using nested polymerase chain reaction.

Results and Conclusion: Based on the results, microscopic agglutination test was positive for 10.7% of the participants. However, Nested-PCR was negative for the positive microscopic agglutination tests on sera collected from the participants with antibodies against Leptospira antigens. The current results demonstrate that Leptospira can occur in asymptomatic humans in slaughterhouses and highlight the high potential of the disease transmission to humans in the province. Therefore, further extended control and prevention measures for slaughterhouse workers are recommended to guarantee the food safety.

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

Black Grass Jelly Encapsulated Lactobacillus plantarum Mar8 in Honey and D-Allulose Beverage Enriched with Mangosteen Pericarp Extract

Titin Yulinery, Novik Nurhidayat, Nilam Fadmaulidha Wulandari, Sri widawati, Suliasih Suliasih, Lusianawati widjaja

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e8
https://doi.org/10.22037/afb.v11i1.43264

Abstract

Background and Objective:

Black grass jelly served in sweet syrup is one of the Chinese and East and Southeast Asian traditional beverages. An innovative enrichment can make it a better functional food. This study innovatively enriched the black-jelly food with formulas of probiotic Lactobacillus plantarum Mar8, honey, D-allulose and mangosteen pericarp extract. The probiotic viability, antioxidant and hypoglycemic potential were investigated as well.

Material and Methods: Ready-to-drink functional beverages included mangosteen pericarp extract varied in concentrations of 0.1, 0.2 and 0.4 mg ml-1, D-allulose in honey and encapsulated probiotic Lactobacillus plantarum Mar8 in black grass jelly containing konjac and carrageenan. The probiotic viability, antioxidant activity and hypoglycemic potential were the selective parameters for the functional beverage formulas. The viability of probiotic Lactobacillus plantarum Mar8 was assessed using total plate count method. Antioxidant activity was assessed based on the reaction of 2,2-Diphenyl-1-picrylhydrazyl radical scavenging. Hypoglycemic potential was investigated by counting petite yeast cells after treating with black grass jelly formulas. Significant differences were reported using one-way analysis of variance and Duncan's test. Statistically significance included p-values≤0.05.

Results and Conclusion: The probiotic Lactobacillus plantarum Mar8 encapsulated in black grass jelly survived well in the honey, D-allulose and mangosteen pericarp extract formulated beverages. Honey supported the probiotic viability better, producing further antioxidants and high potentials in hypoglycemia than that those of other formulas did. Mangosteen pericarp extract enriched the functionality of the black grass jelly probiotic beverages. However, further studies are needed to assess favorability and stability of this functional food.

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

Food Storage, Processing and Genetic Stability Studies of Bacillus (Heyndrickxia) coagulans BCP92 (MTCC 25460)

Sohel S Shaikh, Chinmayi Joshi, Farhana Malek, Anis Malik, Manoj Gandhi

Applied Food Biotechnology, Vol. 11 No. 1 (2024), 18 November 2023, Page e22
https://doi.org/10.22037/afb.v11i1.44919

Background and Objective: Bacillus coagulans are spore-forming probiotics that provide health benefits when consumed in adequate amounts. Therefore, they can be added to functional foods to enhance their nutritional values. The aim of the present study was to investigate stability of Bacillus coagulans BCP92 in various functional foods during food processing and storage conditions as well as genetic stability study of the strain using DNA fingerprinting method.

Material and Methods: Bacillus coagulans BCP92 was incorporated into a range of functional foods and beverages such as instant coffee, tea, sweet corn soups, oatmeal, upma, gummies, brownies, ice creams, non-alcoholic beverages, chocolates, peanut butter and shrikhand. Viability of the bacteria was assessed using pour plate method under various processing and storage conditions. Genetic stability of B. coagulans was assessed using DNA fingerprinting.

Results and Conclusion: The viability was shown in food processing conditions of teas (99.97%), coffees (99.45%), sweet corn soups (99.36%), oatmeal (98.81%), upma (99.57%), gummies (99.67%) and brownies (98.14%). In food-storage conditions, relative viability was as follows: fruit juices (98.91%), lassi (98.72%), energy drinks (98.70%), cold coffees (99.29%), milk chocolates (99.87%), white chocolates (100.13%), dark chocolates (99.20%), shrikhand (99.04%), ice creams (99.45%), and peanut butters (98.32%). Furthermore, DNA fingerprinting showed genetic stability of the probiotic B. coagulans BCP92. In conclusion, B. coagulans BCP92 has shown good viability in various food processing and storage conditions. Moreover, it is genetically stable, thus making it a good candidate for addition to functional foods.

  1. Introduction

 

The potential health benefits of probiotics, which involve improving gut microflora, have been a topic of scientific interests for many years. However, it has only recently begun to receive scientific assessments [1]. Probiotics are living microorganisms that confer health benefits to the host when consumed in sufficient quantities [2]. Several studies have revealed that consumption of probiotics decreases risks of antibiotic-associated diarrhoea [3], symptoms of irritable bowel syndrome (IBS) [4], risks of lactose intolerance [5] and constipation [6] and risks of carcinogens and helps in decreases of obesity and enhancement of immune responses and decreases of cholesterol levels [7]. To confer the specific health benefits of incorporated probiotics in food products, the recommended adequate levels of probiotics (106–107 CFU.ml-1) should be provided in the final products [8]. Preserving viability of the probiotic cultures in foods until the end of shelf life is an important criterion for providing effective probiotic food products [9]. It has been observed that the viability of most probiotic bacteria is lost during processing and storage conditions. Furthermore, only a limited number of bacteria can survive harsh conditions of the gastrointestinal tract (GIT) [10]. An effective way to deliver probiotic bacteria includes incorporation of them into food products, making it easier for the consumers to maintain their gut health, considering that many people choose probiotic food products instead of probiotic capsules and pills [11].

Awareness of the importance of maintaining gut health has led to great increases in demands for probiotic foods. Probiotics are either used as starter cultures in combination with traditional starters or alone and incorporated into dairy products, where many functional characterisations are improved by the addition of probiotics. However, there are several challenges linked to function and stability of the probiotics in dairy products [12]. It is generally accepted that probiotic products should include a minimum concentration of 106–107 CFU.g-1 or CFU.ml-1 and a total concentration should be 108–109 CFU.g-1 consumed daily to exert the probiotic effects [13,14]. Numerous probiotic foods include dairy products such as ice creams, fermented milks, frozen desserts, yoghurts, cheeses, milk powders and cheesecakes, [12,15,16], as well as non-dairy products such as oat drinks, commercial fruit juices, soya milks [17–19]. However, spore-based Bacillus-based probiotics have shown higher survival rates than those others have. In a study by Hashemi et al. [20], it was observed that survival rates of the samples with B. coagulans were higher than those with Lactobacillus acidophilus. Similarly, Soares et al. detected that the Bacillus strains, which included probiotic characteristics, showed a greater viability than that the probiotic strains of Bifidobacterium and Lactobacillus did [21]. Stability of probiotics is always a concern during the storage and processing conditions. The present study focused on the stability assessment of Bacillus coagulans BCP92 under various food processing and storage conditions to assess its potential as a probiotic addictive for enhancing the nutritional values of food products. Furthermore, assessment of genetic stability of the strain was carried out using DNA fingerprinting method.

  1. Materials and Methods

2.1 Microbial culture

Bacterial spores of Bacillus coagulans BCP92 (MTCC 25460) used in this study were produced at Pellucid Lifesciences, India. Concentration of the prepared B. coagulans spores was 150 billion CFU.g-1 (11.146 log CFU.g-1). Standard pour plate technique was used to assess the total viable bacterial count. The B. coagulans spores were thoroughly mixed in the food product and incubated at 75 °C for 30 min using water bath, followed by rapid cooling down to below 45 °C. Suspension was serially diluted in sterile peptone water. An appropriate quantity of diluents was poured into a sterile petri plate and mixed with molten glucose yeast extract BC agar (Hi media M2102, India) in triplicate. Plates were incubated at 40 °C for 48–72 h. The mean of these viable counts was expressed as log10 CFU.g-1. All the Chemicals and reagents were purchased from Merck, Germany, and the microbiological media were purchased from Hi Media, India.

2.2 Assessment of the stability of Bacillus coagulans BCP92 under food processing conditions

The B. coagulans BCP92 was added to a variety of food products and beverages such as instant coffees, teas, sweet corn soup powders, oatmeal, upma, cornflakes, gummies and brownies to assess its stability under certain processing conditions. The B. coagulans BCP92 stability under food processing conditions was studied by comparing the initial count with that after the processing conditions.

2.2.1 Instant coffees and teas

Instant coffee (7.5 g) and tea (1.9 g) powders were mixed with 14 mg each of B. coagulans powder. The prepared mixtures were dissolved in 100 ml of hot water (80–85 °C). Samples from tea and coffee were collected at 0 and 30 min and viable counts of B. coagulans BCP92 were analysed using standard pour plate method.

2.2.2 Sweet-corn soup powders and oatmeal

Briefly, B. coagulans BCP92 (14 mg) was mixed uniformly with sweet-corn soup powder (10 g.serving-1) and oatmeal (100 g.serving-1) separately. Mixture was cooked in 150 ml hot water (80–85 °C).  Cooked samples were collected at 0 and 30 min to analyse the viable cell count of B. coagulans BCP92 using pour plate method.

2.2.3 Upma and cornflakes

Ready-to-eat breakfast upma and cornflakes were purchased from a local market. Upma (50 g·serving-1) was added into warm water (80–85 °C) and cooked for 2 min. Then, 14 mg of B. coagulans BCP92 were added to the mixture, stirred well and cooked for 2 min. Pour plate method was used to assess viable count of B. coagulans BCP92 in samples of 0 and 30 min. One serve of 100-g cornflakes was mixed with 150 ml of hot milk and cooked for 5 min. Then, 14 mg B. coagulans BCP92 were added to the mixture and set for 5 min. Samples were collected at 0 min and 30 min to assess the viable count of B. coagulans BCP92 using pour plate method.

2.2.4 Gummies

All the requirements for gummies such as sugar, flavour, sodium citrate, citric acid, corn syrup, water and pectin were mixed for a batch of gummies for 1 kg material to investigate the bacterial survival under processing conditions. All the ingredients of gummies were mixed and heated to dissolve all the contents. Once all the ingredients dissolved, they were mixed with B. coagulans BCP92 (14 mg.3 g-1) at not greater than 85–90 °C in the final mixture before it solidifies. Viable count of B. coagulans BCP92 was measured in gummies and the molten sample once mixed using pour plate method.

2.2.5 Brownies

All the necessary ingredients for preparing brownies such as flour, salt, cocoa powder, eggs, brown sugar, vanilla essence, vegetable oil and butter were mixed thoroughly to prepare the batter, then B. coagulans BCP92 (14 mg.30 g·serving-1) was added to the batter and baked at 175 °C for 22–25 min. Viable count of B. coagulans BCP92 was calculated in the brownies before and after baking using pour plate method.

2.3 Storage stability of Bacillus coagulans BCP92 in various food matrices

Stability of B. coagulans BCP92 was assessed under standard food storage conditions for ice creams (-20 °C), peanut butters (22–25 °C), non-alcoholic beverages, chocolates and shrikhand (4 °C) based on the ICH Guideline Q1A(R2) [22].

2.3.1 Ice creams

A batch of ice cream was homogenously mixed with B. coagulans BCP92 (14 mg.100 ml serving-1) and 100 ml of ice cream were dispensed in a sterile ice cream cup. The ice cream was stored at -20 °C for 6 m. The primary bacterial count of the B. coagulans BCP92 was carried out immediately after mixing of B. coagulans BCP92. Samples were collected monthly for enumeration up to 6 m of storage. Pour-plate technique in triplicates was used to carry out the bacterial count.

2.3.2 Non-alcoholic beverages

Four various types of commercial beverages of fruit juices, energy drinks, lassi and cold coffees were purchased from a local market. The B. coagulans BCP92 (14 mg.serving-1) was inoculated into four sterile flasks containing fruit juices (100 ml), energy drinks (250 ml), cold coffees (200 ml) and lassi (180 ml). All flasks were sealed and stored at 4 °C. Viability of B. coagulans BCP92 in all beverages was assessed using pour plate method. Stored samples were collected for analysis on Days 0, 30, 60, 90, 120 and 180. All analyses were carried out with three replicates.

2.3.3 Chocolates

Three types of chocolates were purchased from a local market, including white, dark and milk chocolates. Chocolate bars were melted by heating at 45–50 °C. Then, B. coagulans BCP92 (14 mg.100 ml serving-1) powder was thoroughly mixed with the molten chocolates. The mixture of chocolate and probiotics was stored at 4 °C. Viability of B. coagulans BCP92 in chocolates was assessed using pour plate method. Stored samples were collected for analysis on Days 0, 30, 60, 90, 120 and 180. All analyses were carried out with three replicates. 

2.3.4 Peanut butter

Peanut butter was purchased from the local market. Samples (32 g·serving-1) were inoculated with B. coagulans BCP92 (14 mg·serving-1). Peanut butter and probiotic powder were mixed uniformly and stored at room temperature (RT). Then, B. coagulans BCP92 viability was assessed after 0, 30, 60, 90, 120 and 180 days of storage.

2.3.5 Shrikhand

Shrikhand was purchased from a local market, added into probiotic B. coagulans BCP92 (14 mg.100 g.serving-1) and stored at 4 °C. Stored samples were collected for analysis on Days 0, 30, 60, 90, 120 and 180. All analyses were carried out with three replicates. 

2.4 Genetic stability of Bacillus coagulans BCP92

For the comparisons from two various stages of the production process, primary cultures in the form of VIAL and final product batch samples were used. Generally, DNA was isolated from each sample. Quality of the DNA was assessed on 1.0% agarose gel and a single band of high-molecular weight DNA was observed. Moreover, DNA fingerprinting of the cultures was carried out using rep-PCR method and MSP-PCR fingerprinting. Two types of rep-PCR fingerprinting were applied [23] using BOX (50-CTACGGCAAGGCGACGCTGACG-30) and (GTG)5 primers (5-GTGGTGGTGGTGGTG-3) [24]. Briefly, 20 ul of PCR amplicons were separated on 2% agarose gel and banding patterns were analyzed using Gel-analyzer software. The dendrogram was plotted using unweighted pair-group method and arithmetic averages with correlation levels expressed as proportions of the Pearson correlation coefficient.

2.5 Statistical analysis

Viable count of B. coagulans BCP92 was expressed as log10 CFU.serving-1 in food processing conditions and log10 CFU g-1.ml-1 in food storage conditions. All analyses were carried out with three replicates. Results included averages of the three independent determinations. Differences between the two values were calculated using student’s t-test. Level of the significance for all statistical tests was p < 0.05.

  1. Results and Discussion

3.1 Stability of Bacillus coagulans BCP92 in food processing conditions 

Stability of B. coagulans BCP92 was studied by measuring viability of the bacteria in various food matrices under certain processing conditions (Figure 1). The primary viable count of B. coagulans BCP92 in tea was 9.31 ±0.02 log10 CFU serving-1 and in coffee was 9.38 ±0.02 log10 CFU serving-1. After 30 min, these values were 9.30 ±0.05 and 9.33 ±0.02 log10 CFU serving-1, respectively. The B. coagulans preserved 99.97% of its viability in tea. In instant coffee, viability of B. coagulans after processing was preserved up to 99.45%. Viability of B. coagulans BCP92 was assessed in sweet corn soup and oatmeal during processing. The B. coagulans was incorporated into corn soup powder and oatmeal by adding hot water. In soup preparation, the primary B. coagulans of 9.38 ±0.03 log10 CFU serving-1 preserved a 99.36% viable count after 30 min. The oatmeal primary concentration was 9.25 ±0.03 log10 CFU serving-1 and after 30 min, it preserved 98.81% viability (Figure 1).

Upma primary concentration was 9.20 ±0.05 log10 CFU serving-1 and after 30 min, it preserved 99.57% viability. The viability studies on cornflakes showed a primary count of 9.22 ±0.02 log10 CFU.serving-1, and after 30 min, it preserved 99.78% of viability (Figure 1). Viability was studied in gummies as well. For gummies before and after processing, they showed 99.67% of relative viability per gummy. The viable count of B. coagulans BCP92 spores in gummy processing conditions showed slight non-significant  decreases in count (Figure 1). Viability studies in brownies showed that the primary concentration of probiotics in each brownie was 9.22 log10 CFU serving-1. After heating, this showed a 98.14% relative survival rate (Figure 1). Studies have shown use of probiotics in functional foods. Polo et al. [25] reported use of B. coagulans in herbal teas. Majeed et al. [26] reported viability of B. coagulans up to 2 y of shelf life when stored with tea and coffee powders. Kahraman et al. [27] and Miranda et al. [28] studied B. coagulans stability in gummies and showed B. coagulans survivals during production and processing. Majeed et al. [29] reported stability of B. coagulans in various food matrices such as hot fudge toppings, chocolate fudges (97.23%) and peanut butters (99.6%) with viability over 95% as well as its viability in apple juices (99.3%) of baked products. Almada et al. [30] showed that eight strains of Bacillus in various baking, cooking and drying processes affected γ of the Bacillus strains; of which, B. coagulans reported higher resistance. Foods containing spore probiotics are becoming popular due to their resistance to heat processes, low water activity, acidic pH and heat stability [31]. In this study, B. coagulans BCP92 showed high viability in various foods during food processing conditions, with viabilities ranging 98.14–99.97% in food products such as teas, coffees, sweet corn soups, oatmeal, upma, gummies and brownies.

3.2 Storage stability of Bacillus coagulans BCP92 in various food storage conditions

Incorporation of B. coagulans BCP92 into a food product was studied to assess viability and stability of B. coagulans BCP92 during storage and its possible use as a food ingredient (Table 1). The relative viability of B. coagulans in ice creams was 99.41%, The primary viable count of B. coagulans in ice creams was 7.32 ±0.04 log10 CFU ml-1 and the final count was 7.28 ±0.06 log10 CFU ml-1 over 6 m of storage (Table 1). Studies show use of ice creams as vehicles for probiotics. Due to exposure of the cells to various stress factors associated with formulation, overrun, melting and storage, losses in viability occur [9]. Fruit juices, energy drinks and cold coffees showed primary counts of 7.34 ±0.01, 6.92 ±0.01 and 7.00 ±0.03 log10 CFU ml-1, respectively. After storage up to 6 m, the preserved viability rates were up to 98.87, 98.74 and 99.23%, respectively (Table 1). Viability of B. coagulans was assessed in various types of chocolates. Results revealed that B. coagulans BCP92 preserved its high viability throughout the entire 180-d storage time. Viability of B. coagulans in milk, white and dark chocolates were 99.99, 100 and 99.16%, respectively (Table 1)

Stability studies in shrikhand and lassi showed consistency of B. coagulans BCP92 as the primary concentration of B. coagulans was 7.32 ±0.08 and 7.03 ±0.02 log10 CFU ml-1 and after the study, it preserved its 99.08 and 98.80% relative viabilities, respectively (Table 1). Stability studies in peanut butters demonstrated a good viability of 98.40% from primary 7.76 ±0.02 log10 CFU ml-1 per serving after 6 m of storage (Table 1).

Various studies report stability of non-spore-forming probiotics, showing survival of probiotics during storage [32,33]. However, probiotic Bacillus strain showed a higher survival rate [20, 21] than Lactobacillus and Bifidobacterium during storage under GIT conditions when studied in cheeses, pasteurized orange juices and breads [21]. A study with L. acidophilus and B. coagulans in ice creams stored at -18 °C for 90 d showed a higher survival rate in  B. coagulans than L. acidophilus [20]. Marcial-Coba et al. [33] microencapsulated Akkermansia muciniphila and L. casei in dark chocolates. In another study, Cielecka-Piontek et al. [34] demonstrated stability of B. animalis subsp. Lactis, Saccharomyces boulardii and B. coagulans GBI-30, 6086 in chocolates. Similar results were reported by Silva et al. [35] in L. acidophilus LA3 and B. animalis subsp. lactis BLC1, showing the highest viabilities of approximately 7.7 and 7.3 log CFU.g-1 in semisweet chocolates, respectively. Lavrentev et al. [36] showed use of B. coagulans as a starter culture and its viability for 60 d and reported satisfactory results for stability and quality characteristics of the product. Maity et al. reported the B. coagulans stability in various food matrices under processing conditions, including lemon iced teas (99.46%), green teas (98.48%), masala teas (98.96%), lemon teas (99.59%), instant coffees (99.79%), upma (99.89%), corn soups (99.79%) and noodles (99.68%) [37]. The B. coagulans BCP92 preserved its stability over 6 m in foods such as fruit juices, lassi, energy drinks, cold coffees, chocolates, shrikhand, ice creams and peanut butters with relative viabilities ranging 98.32–100.13%. In the present study, non-significant decreases were observed in all the food matrices under food processing and storage conditions, showing the versatile nature of B. coagulans BCP92.

3.3 Genetic stability

Samples of “VIAL” (primary sample) and “final product” were provided for the study. Generally, DNA was extracted and DNA fingerprinting was carried out using BOX primers sets, followed by PCR analysis and dendrogram plotting. Genotype of each strain could be differentiated by the distribution of PCR bands and the two samples were closely linked to each other based on DNA fingerprinting patterns in the experiments (Figures 2 and 3). Genomic safety and probiotics attributes showed that B. coagulans BCP92 was safe [38]. Genomic fingerprinting also showed genetic stability of B. coagulans BCP92 in the production cycle; in which, it showed the genetic stability in primary and final samples of production. Majeed et al. reported genetic stability of three various sample batches [24]. Genetic stability studies using DNA fingerprinting verified stability of B. coagulans BCP92.

  1. Conclusion

The present study reported stability of B. coagulans BCP92 in various food matrices under processing and storage conditions. The B. coagulans BCP92 tolerated the low pH of juices, low-temperature storage and heating during food processing conditions. These findings focused on the potential of these food products as carrier vehicles for the delivery and stability of spore-forming probiotics. The B. coagulans BCP92 was also genetically stable in the production process, which was a positive indication of genetic stability of culture. Hence, this finding suggests use of spore-forming probiotic B. coagulans BCP92 in functional foods for gut health improvement and gastrointestinal disorders. Further studies can be carried out on incorporating B. coagulans BCP92 in food products and assessing them on human subjects to gauge their effects on human health. Additional studies on food supplemented with B. coagulans BCP92 can help deeper understanding of its potential benefits for human health and sensory profiling, ultimately leading to advancements in the field of nutrition and wellness.

 

  1. Conflict of Interest

The authors report no conflict of interest.

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Background and Objective: Various products on the market contain probiotics such as lactic acid bacteria, which are promoted with a wide range of benefits. Functionality of these products is linked to the specific strains, bacterial species and viable cell counts. This study aimed to assess conformity of targeted lactic acid bacterial species and viable cell counts in commercially available probiotic products with their labeling, ensuring efficacy of the products.

Material and Methods: Multiplex polymerase chain reaction technique was developed using specific primers to effectively differentiate lactic acid bacteria in probiotic products. Therefore, strains used in the products were targeted and relevant nucleotide sequence data were searched to select two sets of polymerase chain reaction primer pairs of L. pla-F/R and L. para-F/R, targeting 16S ribosomal RNA genes, and L .pen-F/R, targeting recA genes.

Results and Conclusion: The individual primer sets produced the expected target products that matched the labeling for the tested strains of Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillus pentosus. Then, specificity assessing was carried out using multiplex primer sets for single strains, pairwise combinations and triple combinations of lactic acid bacteria. After verifying specificity for all the three strains under similar polymerase chain reaction conditions, sensitivity of the multiplex polymerase chain reaction was investigated by assessing various dilutions of the three lactic acid bacterial strains and commercially available probiotic products. These findings demonstrated potential uses of multiplex polymerase chain reaction in lactic acid bacterial detection techniques. In conclusion, specific primer sets can be used in multiplex polymerase chain reaction to rapidly and effectively detect lactic acid bacterial strains in commercial products.

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

Lactic acid bacteria (LAB) are addressed for their beneficial effects on the human gastrointestinal tract, enhan-cing overall immune health. There are recent interests in development of functional LAB products. Demands for the probiotic functional foods are rapidly increasing due to the increased consumer awareness of food effects on health [1]. Researchers have assessed potential uses of probiotics in dairy and nondairy products and their viabilities during storage [2]. They have concluded that the final product should contain a minimum of 106-107 viable cells per serving to benefit consumer health [2]. Their studies have shown that mislabeling of probiotic species is common in commercial products [3]. Lack of appropriate identification of the strains and false efficacy claims have led to confusion. Probiotic products available in the market often use mixed strains. Thus, there are needs to monitor conformities of the labeled bacterial species and viable cell counts.

Polymerase chain reaction (PCR) technique has success-fully been used to detect and differentiate viruses and bacteria in various foods [4]. Rapid and reliable nature of PCR provides a valuable tool for distinguishing closely related species within two groups of lactobacilli [5]. This technique has been used to rapidly identify Lactobacillus plantarum in kimchi [6]. Oligonucleotide primers have been developed from sequences between the 16S and 23S rRNA genes, enabling identification of various lactobacilli strains in dairy products and probiotics using PCR [7]. Further-more, strain-specific PCR can be used for the rapid identi-fication of lactobacilli isolated from food samples [8] and specific identification of ten common lactobacilli and bifi-dobacteria strains in fermented milks [9].

In this study, multiplex PCR method was developed to investigate applicability of molecular detection techniques for LAB using three sets of specific primers sourced from the literature. Moreover, 16S rRNA gene sequences were targeted to explore molecular LAB detection. By amplifying 16S rRNA and recA gene sequences through PCR, this study simultaneously detected three LAB strains as well as mixed LAB strains in the products. Sensitivity of detection was assessed to establish a simple, reliable rapid method appropriate for the effective identification of LAB strains in probiotic products.

  1. Materials and Methods

2.1 Bacterial strains and culture conditions

The LAB strains were stored in a -80 °C freezer. Before the experiments, strains were activated twice using lactobacilli MRS broth (Difco, Detroit, MI, USA) supplemented with 0.05% (w/w) L-cysteine (Merck, Taipei, Taiwan). Strains were cultured under optimal growth conditions at 37 °C for 24 h. Reference strains were provided by the Bioresources Collection and Research Center (BCRC, Hsin-Chu, Tai-wan). The L. pentosus BCRC 17972 and 17973, L. plantar-um F7-1 and L. paracasei BCRC 12193, 12188, 12248 and 17002 were used in this study. Seven strains were used in the current study as well. The commercial probiotic product was purchased from Li-Fong, Tainan, Taiwan, for PCR detection. Each sachet of the bacterial powder contained a high level of viable probiotic cells with 5.0 × 1010 CFU.g-1. Specific strains included in the product were L. plantarum LP112, L. paracasei LPC188 and L. pentosus LPE588. ThIS study was carried out at Testing and Analysis Center for Food and Cosmetics, HungKuang University, Taichung City, Taiwan.

2.2 Genomic DNA preparation and polymerase chain reaction primers

Total chromosomal DNA of the LAB cells was extracted using Blood and Tissue Genomic DNA Extraction Miniprep System (Viogene, Taipei, Taiwan) based on the manuf-acturer’s instructions. Specific primer sequences for the LAB detection are shown in Table 1. Experiments were repeated thrice [10-12].

2.3 Polymerase chain reaction amplification

Method was carried out according to [13]. For each PCR cycle, denaturation, annealing and extension were carried out at 94 °C for 60 s, 57 °C for 60 s and 72 °C for 120 s, respectively. Final extension was carried out at 72 °C for 5 min.

2.4 Sensitivity of the polymerase chain reaction assay

A 24-h culture of the LAB strain was serially diluted 10-fold with sterile water. Purification of DNA was carried out as described in Section 2.2 [14].

2.5 Polymerase chain reaction detection in the commercial probiotic product

The probiotic product was purchased from Li-Fong, Tainan, Taiwan. After diluting the product to 108–106, 1 ml of the diluted sample was collected and DNA extraction was carried out. Then, 2 μl of the extracted DNA was used for multiplex PCR. Experiments were repeated thrice.

  1. Results and Discussion

3.1 Multiplex Polymerase chain reaction

Figure 1 shows gel electrophoresis results of the multiplex PCR for DNA detection of individual LAB strains. Results demonstrated specificity of the three primer sets for their respective target genes in each strain. Small interferences were seen for L. plantarum F7-4 with no effects on amplification of other strains. Figure 1 shows gel electrophoresis results of multiplex PCR for DNA detection of two LAB strains. Results demonstrated that L.pla-F/R, L.pen-F/R and L.para-F/R primer sets could accurately amplify DNA from combination of two LAB strains. No nonspecific products were observed. Thus, 57°C was determined as the optimal annealing temperature for successful primer binding and DNA polymerase activity.

Gel electrophoresis results validated effectiveness and specificity of the multiplex PCR for identifying and discriminating various LAB strains. These findings demonstrated applicability of the developed primer sets and verified their suitability for use in multiplex PCR. The optimized annealing temperature ensured robust amplifica-tion without nonspecific amplification products.

3.2 Sensitivity assessment of the lactic acid bacterial strains using multiplex polymerase chain reaction

In Figure 2A, DNA extracted directly from the mixed cultures of three LAB strains (109, 108 and 107 cfu ml-1) are shown. At a concentration of 106 cfu ml-1, only L. paracasei (BCRC 12188) demonstrated amplifications in multiplex PCR, indicating that detection sensitivity of L. plantarum and L. pentosus was limited to 107 cfu ml-1. Figure 2B shows mixed cultures of the three LAB strains at similar concen-trations after preculture in MRS broth (37 °C, 24 h). Multi-plex PCR detected all the three strains at a sensitivity of 106 cfu ml-1, suggesting that L. plantarum and L. pentosus proliferated and could be detected. Figure 2 shows detection limits and proliferation capabilities of the three LAB strains using multiplex PCR. Results indicated that preculture of the mixed cultures in MRS broth improves detection sensitivity, enabling accurate identification of L. plantarum and L. pentosus strains at lower concentrations.

3.3 Preculture and mixed various concentrations of lactic acid bacterial strains using multiplex polymerase chain reaction

Figure 3A shows gel electrophoresis results of the multiplex PCR carried out on DNA samples extracted from a mixture of L. plantarum (109 cfu ml-1) with two other LAB strains after preculture for 24 h. In Lanes 5–8, amplification products of L. pentosus and L. paracasei diluted to 106 cfu ml-1 were less expressed. The PCR amplification products of 106 cfu ml-1 DNA could be observed. The L. pentosus BCRC 17973 demonstrated a bacterial count of ~109 cfu ml-1 after 24 h of cultivation, whereas L. paracasei BCRC 12188 showed increased bacterial count, suggesting that preculture could enhance detection rate of low-concen-tration bacterial strains. Figure 3B shows gel electrophoresis results of the multiplex PCR on DNA extracted from a mixture of L. plantarum (108 cfu ml-1) with two other LAB strains after preculture for 24 h. The PCR products in Lanes 1–16 indicated that all the three sets of species-specific primer pairs yielded the expected PCR products for various concentrations of the bacterial suspensions after 24 h of preculture. Figure 3 shows effectiveness of the multiplex PCR in detecting L. plantarum at various concentrations in a mixed culture with other LAB strains. Results highlighted effects of preculture on enhancing assay detection rate and sensitivity.

3.4 Polymerase chain reaction detection of the commercial probiotic product

Figure 4 demonstrates results of the multiplex PCR. Whether directly detected or precultured, the three LAB strains provided DNA amplification products from 107 to 109 cfu ml-1. It was suggested that 107 cfu ml-1 was the detection limit of the product (not detected when diluted to 106 cfu ml-1).

The PCR-based species identification is a critical highly valuable tool for detecting and identifying bacteria. It offers advantages, including time efficiency and reliability in microbial identification. Compared to traditional methods such as culture-based techniques, PCR can provide results in a relatively short time. It eliminates the need of time-consuming cultivation of bacteria, allowing for further rapid identification and subsequent decision-making processes. In industrial uses, species identification is critical in selecting and developing bacterial strains appropriate for specific purposes.

Whether in food, agriculture or biotechnology industries, PCR-based species identification enables researchers to screen and select the most appropriate bacterial species for desired characteristics and functions.

The 16S rRNA gene in the ribosomal RNA of prokaryotes is the best molecular marker for bacterial evolutionary analysis. This is due to several gene characteristics, including its presence across various species, abundance, sufficient sequence length and presence of conserved and variable loci. The 16S rRNA gene is widely used in identifying lactobacilli and a commonly rapid technique for bacterial classification and identification in dairy products. Caro et al. reported that partial sequencing of 16S rRNA genes is often used for lactobacilli identification [15]. The RecA protein is a DNA recombinase that plays critical roles in DNA repair and recombination processes of bacteria. It is encoded by the recA gene in the genomes of various prokaryotic microorganisms. The recA gene and its corresponding protein have extensively been studied and used in various research, including evolutionary analysis, phylogenetic studies and identification of bacterial strains. Conserved nature and functional importance of the RecA protein make it a valuable molecular marker for understanding genetic relatedness and evolutionary relationships within bacteria. By comparing the recA gene sequences of various bacterial strains, researchers can have insights into their genetic diversity, evolutionary history and phylogenetic classification.

Lu et al. developed a multiplex PCR method that could effectively be used in key vaginal microbiota evaluation in women with bacterial vaginosis [16]. You et al. used multiplex PCR to detect six species of L. acidophilus group [17]. Petri et al. used multiplex PCR for the rapid identification of wine-associated LAB [18]. Settanni et al. introduced a method and reported its state-of-the-art uses for microbial identification in foods and beverages [19]. Sciancalepore et al. described use of a simple, low-cost, rapid sensitive method based on droplet-based multiplex PCR directly on food matrices for the simultaneous detection of bacterial genes involved in biogenic amine synthesis [20]. Specific primers were designed based on similar sequences and multiplex PCR was optimized for the simultaneous identification of L. plantarum, L. pentosus and L. paraplantarum [21]. Sul et al. developed a multiplex PCR to detect Lactobacillus and Bifidobacterium spp. in commercial probiotic products [13].

Previously, Gram-negative broth enrichment was used in studies, followed by immunomagnetic separation multiplex PCR to enable the simultaneous detection of Salmonella spp. and enterohemorrhagic Escherichia coli in food samples, irrespective of their significant discrepancy in cell counts [22]. A PCR primer set derived from the seque-nce in 16S to 23S internal transcribed spacer (ITS) region was also developed for the specific detection of B. adoles-centis in probiotics. This primer set included potentials for inspecting dairy food and environmental samples [14]. To ensure food safety during direct vat inoculation of Paocai, a propidium monoazide-based quantitative PCR method was developed to quantify L. plantarum NCU116 fermentation starter, as well as Saccharomyces spp. and potentially present pathogenic bacteria [23]. Plate counting was carried out and demonstrated similar results to quantitative PCR analysis, indicating appropriateness and effectiveness of absolute quantitative PCR for rapidly detecting microbial composition in the Paocai system [23]. In a recent study, surveillance of Lactobacillus bacteremia was carried out using biochemical and conventional-PCR assays. However, these methods could not provide target quantification and might lead to false-positive results [24]. To address this limitation, a L. rhamnosus-specific quantitative PCR assay was developed. This assay delivers accurate and reproduci-ble results, leveraging specificity of a TaqMan probe, targ-eting unique 16S rDNA sequences of L. rhamnosus [24].

  1. Conclusion

In summary, the three sets of PCR primer combinations, targeting various LAB strains, demonstrated specificity and generated the expected amplicons during PCR amplifica-tion. Use of multiplex PCR in LAB genomic detection showed potentials and was appropriate for detecting various species in food products. Multiplex PCR decreased experimental cost and time, eliminating the need of time-consuming sequencing processes. Therefore, this method is expected to contribute to the reliability of probiotic labeling systems by facilitating strain identification. This molecular technique offers a valuable tool for quality control, product development and microbial monitoring of probiotic strains in various fields.

  1. Conflict of Interest

The authors report no conflict of interest.

  1. Authors Contributions

Conceptualization, TCC; methodology, LZY; data curation, LZY; writing, TCC.

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