Comparative analysis of the adhesion capacity of lactic acid bacteria and bifidobacteria isolated from traditional dairy products using a Сaco-2/HT29-MTX co-culture modelAbstract
The aim of the present study was to evaluate the adhesive properties of lactic acid bacteria and bifidobacterial strains isolated from traditional fermented dairy products of Kazakhstan using an in vitro model of the human intestinal epithelium (Сaco-2/HT29-MTX). The investigated strains were isolated from shubat, koumiss, kurt, and raw camel milk. A total of 11 bacterial strains were included in the study, comprising 7 strains of lactic acid bacteria (genera Lactobacillus and Lactococcus) and four strains of Bifidobacterium crudilactis isolated from shubat (fermented camel milk), koumiss (fermented mare’s milk), kurt (dried fermented dairy product), and raw camel milk. Microbial identification was performed by 16S rRNA gene sequencing, revealing sequence similarity levels ranging from 99.71% to 100.0%. Adhesion activity was assessed based on optical density measurements and adhesion index values. The results demonstrated substantial differences in adhesion capacity among the lactic acid bacterial strains. The highest adhesion activity was observed for Lactobacillus crustorum 8-9M and Lactococcus lactis subsp. lactis 7-8M, whereas the lowest was detected in Lactobacillus pontis 6-12M. Among the bifidobacteria, Bifidobacterium crudilactis 7-5C exhibited the greatest adhesion capacity, while strain 7-6C showed no detectable adhesion activity. The obtained results indicate pronounced strain-specificity of the adhesive properties of the investigated microorganisms and allow the identification of promising cultures with high probiotic potential.
Keywords:
lactic acid bacteria; Bifidobacterium crudilactis; adhesion; probiotics; fermented dairy products; in vitro
Resumo
O objetivo do presente estudo foi avaliar as propriedades de adesão de cepas de bactérias ácido-láticas e bifidobactérias isoladas de produtos lácteos fermentados tradicionais do Cazaquistão, utilizando um modelo in vitro de epitélio intestinal humano (Caco-2/HT29-MTX). As cepas investigadas foram isoladas de shubat, koumiss, kurt e leite de camela cru. Um total de 11 cepas bacterianas foi incluído no estudo, compreendendo 7 cepas de bactérias ácido-láticas (gêneros Lactobacillus e Lactococcus) e quatro cepas de Bifidobacterium crudilactis isoladas de shubat (leite de camela fermentado), koumiss (leite de égua fermentado), kurt (produto lácteo fermentado seco) e leite de camela cru. A identificação microbiana foi realizada por meio do sequenciamento do gene 16S rRNA, revelando níveis de similaridade das sequências variando de 99,71% a 100,0%. A atividade de adesão foi avaliada com base em medições de densidade óptica e valores do índice de adesão. Os resultados demonstraram diferenças substanciais na capacidade de adesão entre as cepas de bactérias ácido-láticas. A maior atividade de adesão foi observada para Lactobacillus crustorum 8-9M e Lactococcus lactis subsp. lactis 7-8M, enquanto a menor foi detectada em Lactobacillus pontis 6-12M. Entre as bifidobactérias, Bifidobacterium crudilactis 7-5C exibiu a maior capacidade de adesão, ao passo que a cepa 7-6C não apresentou atividade de adesão detectável. Os resultados obtidos indicam uma acentuada especificidade de cepa nas propriedades de adesão dos microrganismos investigados e permitem a identificação de culturas promissoras com alto potencial probiótico.
Palavras-chave:
bactérias ácido-láticas; Bifidobacterium crudilactis; adesão; probióticos; produtos lácteos fermentados; in vitro
1. Introduction
In recent years, there has been growing interest in probiotic microorganisms isolated from traditional fermented dairy products, which are considered promising sources of functionally active strains for the food and biotechnology industries. Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (Hill et al., 2014, p. 507). Their functional effects are associated with modulation of the intestinal microbiota, enhancement of intestinal barrier function, and regulation of immune responses (Sanders et al., 2019).
One of the key criteria for the selection of probiotic cultures is their ability to survive passage through the gastrointestinal tract and adhere to intestinal epithelial cells. Adhesion promotes temporary colonization of the mucosal surface, contributes to the competitive exclusion of pathogenic microorganisms, and enhances interactions with the host immune system (Tuomola and Salminen, 1998; Monteagudo-Mera et al., 2019). Adhesion capacity has been shown to be highly strain-specific and is determined by structural characteristics of the bacterial cell surface, including pili, exopolysaccharides, and mucus-binding proteins (Kankainen et al., 2009; Lebeer et al., 2012).
In vitro models based on human intestinal cell lines such as Caco-2 and HT29-MTX are widely used to evaluate the adhesion and interaction of probiotic microorganisms with the intestinal epithelium. Caco-2 cells differentiate into enterocyte-like cells, whereas HT29-MTX cells are capable of mucus production, thereby enabling the simulation of different components of the intestinal barrier. The use of co-cultures of these cell lines increases the physiological relevance of the model and allows more accurate assessment of microbial adhesion properties (Gopal et al., 2001; Jose et al., 2017).
Traditional fermented dairy products of Kazakhstan, such as shubat, koumiss, and kurt, represent unique microbial ecosystems formed under specific climatic and technological conditions. These products are characterized by high diversity of lactic acid bacteria and bifidobacteria adapted to extreme environmental conditions and possessing potentially valuable probiotic properties (Jans et al., 2012; Akhmetsadykova et al., 2015). Particular attention has been paid to camel milk and its fermented products because of their unique chemical composition and their potential as sources of novel functionally active strains (Shori, 2017).
Koumiss is a fermented dairy product traditionally prepared from mare’s milk. This beverage continues to play an important role in the diet of the Central Asian steppe populations of Hunno-Bulgar, Turkic, and Mongolian origin, including Kazakhs, Bashkirs, Kalmyks, Kyrgyz, Mongolians, and Yakuts.
Traditional artisanal production of koumiss is based on inoculation of raw mare’s milk with a starter culture obtained from a previously prepared batch of koumiss. Currently, koumiss is often produced from pasteurized mare’s milk supplemented with approximately 30% of a previous batch of the product, followed by fermentation at 26-28 °C for 2-4 h with periodic stirring. After fermentation, koumiss is bottled, sealed, and stored at 4-6 °C for 1-3 days. The main fermentation products are lactic acid, ethanol, and carbon dioxide.
Shubat is a fermented camel milk beverage that constitutes an important component of the daily diet of many people in Kazakhstan. Traditionally, shubat has been considered beneficial in the management of diseases such as asthma, tuberculosis, hepatitis, diabetes, and psoriasis. The preparation process is relatively simple and, similarly to koumiss production, is based on the backslopping method. Fresh camel milk is poured into a wooden container, inoculated with previously prepared shubat, sealed, and left to ferment.
Kurt is a traditional Kazakh dairy product prepared from pasteurized cow’s, sheep’s, or goat’s milk. The product is manufactured from skimmed milk, which is pasteurized at 80-85 °C for 10-20 s, cooled to 32-34 °C, and inoculated with starter culture. The starter culture, consisting of Lactococcus, Streptococcus thermophilus, and Lactobacillus delbrueckii subsp. bulgaricus, is added at a concentration of 5%. The milk is incubated until the acidity reaches 75-76 °T (0.67-0.68% titratable acidity).
Despite the growing interest in probiotic microorganisms, data regarding the adhesive properties of strains isolated from traditional Kazakh dairy products remain limited. In particular, comparative characteristics of lactic acid bacteria and bifidobacteria isolated from shubat, koumiss, kurt, and camel milk under standardized intestinal epithelial models have not been sufficiently investigated. The lack of such data restricts the selection of promising strains for the development of functional foods and probiotic cultures.
Therefore, the aim of the present study was to evaluate the adhesion capacity of lactic acid bacteria and bifidobacterial strains isolated from traditional fermented dairy products of Kazakhstan using in vitro models of the human intestinal epithelium (Caco-2 and HT29-MTX). The obtained results will facilitate the identification of strains with high probiotic potential and provide a scientific basis for the development of novel functional products derived from regional raw materials.
2. Materials and Methods
A total of 11 bacterial strains were isolated and investigated, including 7 strains of lactic acid bacteria (Lactobacillus and Lactococcus) and four strains of Bifidobacterium crudilactis isolated from traditional fermented dairy products of Kazakhstan (shubat, koumiss, kurt, and camel milk).
2.1. Sampling and characterization of dairy products
Samples of traditional fermented dairy products, including shubat (fermented camel milk), koumiss (fermented mare’s milk), and kurt (dried fermented dairy product), as well as raw camel milk, were collected from private farms located in the Almaty and Kyzylorda regions of the Republic of Kazakhstan.
Sampling was performed under aseptic conditions using sterile containers. Samples were transported under refrigerated conditions at 4 ± 1 °C and analyzed within 24 h after collection. The pH values were measured immediately upon arrival using a calibrated pH meter (Mettler Toledo, Switzerland). Prior to microbiological analysis, samples were homogenized.
2.2. Isolation and cultivation of lactic acid bacteria and bifidobacteria
Traditional fermented dairy products and raw camel milk were used as sources for the isolation of microorganisms. The camel milk sample was collected from a clinically healthy Bactrian camel (Camelus bactrianus) at a farm in the Almaty region by manual milking into sterile hermetically sealed bottles. Detailed information regarding the investigated strains, including the isolation source and geographical origin, is presented in Table 1. Following pH determination, samples were aseptically transferred into sterile tubes, transported under refrigerated conditions, and analyzed in the microbiology laboratory.
Samples of traditional fermented dairy products (shubat, koumiss, kurt, and camel milk) obtained from different regions of Kazakhstan were used as sources for the isolation of lactic acid bacteria (LAB) and bifidobacteria.
For microbiological analysis, 1 g (or 1 mL) of each sample was suspended in 9 mL of sterile physiological saline solution (0.85% NaCl), followed by preparation of tenfold serial dilutions. Plating was performed in triplicate on selective culture media.
Lactic acid bacteria were cultivated on MRS agar (de Man, Rogosa and Sharpe; Difco™, USA) under anaerobic conditions at 37 °C for 48-72 h using the GasPak™ system (BD Biosciences, USA).
Bifidobacteria were isolated on Wilkins-Chalgren agar (Oxoid, UK) supplemented with soy peptone (5 g/L), L-cysteine-HCl (0.5 g/L), Tween 80 (1 mL/L), mupirocin (100 mg/L), and glacial acetic acid (1 mL/L). Incubation was performed under anaerobic conditions at 37 °C for 72 h.
The selective media were chosen based on their ability to support the preferential growth of the corresponding microbial groups: MRS agar for lactic acid bacteria and modified Wilkins-Chalgren agar for bifidobacteria.
Selected colonies were purified by repeated streaking until pure cultures were obtained. Pure cultures were stored at −80 °C in MRS broth supplemented with 20% (v/v) glycerol until further use.
Identification of the isolated strains was performed based on 16S rRNA gene sequence analysis (or by MALDI-TOF mass spectrometry), which enabled taxonomic classification to the species level.
As a result of the identification procedure, the strains presented in Table 1, including representatives of the genera Lactobacillus, Lactococcus, and Bifidobacterium isolated from traditional fermented dairy products of Kazakhstan, were characterized.
2.3. Molecular identification of strains
Genomic DNA was extracted from pure bacterial cultures using the Genomic DNA Mini Kit (Applied Biosystems, USA) according to the manufacturer’s instructions.
A nearly complete fragment of the 16S rRNA gene was amplified using universal primers under previously described PCR conditions. Amplification was carried out using a Biometra T Professional Gradient Thermocycler (Germany). The obtained PCR products were verified by electrophoresis in 1.5% agarose gel (110 V, 30 min) followed by visualization under UV light.
Amplified fragments were purified using a PCR purification kit (Qiagen, Germany) and submitted for sequencing to a specialized sequencing company (GATC Biotech, Germany).
Sequence assembly and editing were performed using BioEdit software (version 7.2.5). Strain identification was conducted by comparison of the obtained sequences with the GenBank (NCBI) and EzTaxon databases using the BLAST algorithm. Sequence similarity values ≥99% were considered sufficient for species-level identification.
The obtained sequences were deposited in the GenBank database using the BankIt submission system (Omarova et al., 2016).
2.4. Cultivation of cell lines
Human intestinal epithelial cell lines Caco-2 (ATCC HTB-37) and HT29-MTX were used for adhesion assays. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin.
Cell cultivation was performed at 37 °C in a humidified atmosphere containing 5% CO2. The culture medium was replaced every 2-3 days. For differentiation, Caco-2 cells were incubated for 14 ± 1 days after reaching confluence.
For co-culture experiments, Caco-2 and HT29-MTX cells were seeded at a ratio of 90:10 in 24-well plates and cultivated until formation of a differentiated monolayer.
2.5. Preparation of bacterial suspensions
Bacterial strains were cultivated in MRS broth at 37 °C for 24 h under anaerobic conditions. Cells were harvested by centrifugation at 2000 × g for 10 min, washed twice with sterile phosphate-buffered saline (PBS, pH 7.0), and resuspended in PBS. Cell concentrations were adjusted to approximately 1 × 108 CFU/mL by monitoring optical density at 420 nm using an Infinite M200 microplate reader (Tecan, Austria).
2.6. Determination of adhesion capacity
The adhesive properties of the strains were evaluated using a Caco-2/HT29-MTX co-culture model. Prior to the experiment, cell monolayers were washed twice with PBS to remove antibiotics.
Each well received 100 μL of bacterial suspension (~108 CFU/mL) and 100 μL of either milk hydrolysate solution (5%) or PBS (control). All experiments were performed in triplicate.
Incubation was carried out at 37 °C in an atmosphere containing 5% CO2 for 1-2 h. After incubation, cells were washed three times with PBS to remove non-adherent bacteria.
Adherent bacteria were released by treatment with 0.1% Triton X-100 for 3 min. The resulting suspensions were serially diluted and plated on MRS agar. Colony counts were determined after incubation at 37 °C for 48 h.
Adhesion was expressed as the percentage of the total number of bacteria initially added. Lactobacillus rhamnosus GG was used as a positive control strain.
2.7. Statistical analysis
Statistical analysis was performed using both parametric and non-parametric methods. Normality of distribution and homogeneity of variances were assessed using standard statistical tests. When assumptions were met, one-way analysis of variance (ANOVA) was applied, whereas Welch’s ANOVA was used in cases of unequal variances. In the absence of normal distribution, the non-parametric Kruskal-Wallis test followed by Dunn’s post hoc test was employed. Differences were considered statistically significant at p < 0.05.
3. Results and Discussion
3.1. Identification of strains
Based on 16S rRNA gene sequencing, the investigated microorganisms were identified with a high degree of confidence. The similarity of nucleotide sequences to those deposited in the GenBank database ranged from 99.71% to 100.0%, allowing unambiguous assignment of the isolates to the corresponding species.
Specifically, the strains were identified as Lactobacillus helveticus, Lactobacillus pontis, Lactobacillus kefiranofaciens, Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus crustorum, as well as Bifidobacterium crudilactis and representatives of the genus Lactococcus.
3.2. Adhesive properties of lactic acid bacteria
The results of the evaluation of the adhesion capacity of lactic acid bacteria are presented in Table 2.
Adhesive properties of lactic acid bacterial strains using the Сaco-2/HT29-MTX cell model (in vitro).
The investigated LAB strains demonstrated substantial differences in adhesion capacity to Сaco-2/HT29-MTX cells. The highest adhesion activity was observed for Lactobacillus brevis 8-6M (37.09 ± 2.54), followed by Lactobacillus crustorum 8-9M (24.82 ± 6.80) and Lactobacillus paracasei 8-2M (19.89 ± 5.58).
Intermediate adhesion values were recorded for Lactobacillus kefiranofaciens 6-2M (18.39 ± 2.70) and Lactococcus lactis subsp. lactis 7-1M (18.29 ± 6.34), whereas the lowest adhesion level was observed for Lactobacillus helveticus 5-2M (7.27 ± 1.62).
Certain strains, including Lactobacillus helveticus 5-2M and Lactobacillus pontis 6-12M, did not demonstrate pronounced adhesive activity. The graphical representation of the adhesive properties of lactic acid bacterial strains using the Сaco-2/HT29-MTX cell model (in vitro) is presented in Figure 1. The high adhesion capacity of Lactobacillus brevis 8-6M may be associated with the presence of surface-associated proteins and exopolysaccharides involved in interactions with epithelial cells. These characteristics are considered key determinants of probiotic potential, contributing to intestinal colonization and competitive exclusion of pathogenic microbiota.
Adhesive properties of lactic acid bacterial strains using the Сaco-2/HT29-MTX cell model (in vitro).
The graphical representation confirms the considerable variability of adhesive properties among the investigated strains and highlights the most active cultures.
3.3. Adhesive properties of bifidobacteria
The results of the adhesion assay for Bifidobacterium crudilactis strains are presented in Table 3.
Adhesive properties of Bifidobacterium crudilactis strains using the Сaco-2/HT29-MTX cell model (in vitro).
The investigated bifidobacterial strains exhibited substantial differences in their adhesion capacity to Сaco-2/HT29-MTX cells. The highest adhesion values were observed for Bifidobacterium crudilactis 7-5C (76.23 ± 1.59%) and B. crudilactis 7-2C (73.68 ± 1.26%), indicating a pronounced adhesive potential of these isolates.
A low level of adhesion was detected for B. crudilactis 7-1C (6.19 ± 0.70%), suggesting a limited capacity for interaction with epithelial cells.
The strain B. crudilactis 7-6C did not demonstrate detectable adhesion activity, as all measured values remained below the detection limit under the experimental conditions.
The obtained results confirm the pronounced strain-specificity of the adhesive properties of bifidobacteria.
The adhesion properties of Bifidobacterium crudilactis strains using the Сaco-2/HT29-MTX cell model (in vitro) are presented in Figure 2.
Adhesive properties of Bifidobacterium crudilactis strains using the Сaco-2/HT29-MTX cell model (in vitro).
The graphical representation clearly demonstrates the differences in adhesion activity among the bifidobacterial strains.
The statistical significance of differences between groups was evaluated separately using one-way analysis of variance (ANOVA), Welch’s ANOVA, and the non-parametric Kruskal-Wallis test.
The obtained results demonstrate pronounced strain-specificity of the adhesive properties of the investigated lactic acid bacteria and bifidobacteria, which is consistent with current concepts describing probiotics as a functionally heterogeneous group of microorganisms. Adhesion to the intestinal epithelium is considered one of the key factors determining probiotic potential, including the ability to colonize the intestinal mucosa, competitively exclude pathogens, and interact with the host immune system (Tuo et al., 2018; Rocha-Mendoza et al., 2020).
In the present study, LAB strains isolated from traditional fermented dairy products of Kazakhstan exhibited a broad range of adhesion activity. The highest adhesion values were observed for Lactobacillus brevis 8-6M and Lactobacillus crustorum 8-9M, whereas some strains, such as Lactobacillus helveticus 5-2M, displayed minimal adhesion capacity. Similar variability has previously been reported for Lactobacillus plantarum strains, where even closely related isolates demonstrated substantial differences in adhesion properties (Jiang et al., 2016; Huang et al., 2013; Zheng et al., 2013).
Mechanistically, high adhesion capacity may be associated with structural features of the bacterial cell surface. It has been demonstrated that the adhesive properties of LAB depend on the presence of surface-associated proteins, lipoteichoic acids, and the ability to undergo auto-aggregation (Kotzamanidis et al., 2010; Collado et al., 2008). In addition, exopolysaccharides (EPS) play an important role by facilitating stable interactions with epithelial cells and protecting bacteria from adverse environmental conditions (Živković et al., 2016; Castro-Bravo et al., 2018).
Particular attention should be paid to the influence of the milk matrix on bacterial adhesion. Rocha-Mendoza et al. (2020) demonstrated that milk components, particularly phospholipids, may enhance LAB adhesion to Сaco-2/HT29-MTX cells, which is consistent with our findings for strains isolated from shubat and koumiss. These observations support the hypothesis that fermented dairy products represent a natural ecological niche for the selection of strains with enhanced probiotic potential.
These findings are also consistent with the results reported by Omarova et al. (2016) who demonstrated that traditional fermented dairy products of Kazakhstan constitute an important source of diverse probiotic microorganisms, including lactic acid bacteria and bifidobacteria.
The results obtained for bifidobacteria also revealed pronounced strain-specificity. Bifidobacterium crudilactis 7-5C exhibited the highest adhesion activity, whereas strain 7-6C completely lacked detectable adhesion capacity. Similar differences have been described previously and are associated with genetically determined characteristics of the bacterial cell surface and the presence of specific adhesion-related factors (O’Callaghan and van Sinderen, 2016).
Experimental evidence indicates that bifidobacterial exopolysaccharides play a crucial role in host interaction by contributing both to adhesion and immunomodulatory activity (Fanning et al., 2012; Castro-Bravo et al., 2018). The absence of adhesion observed for strain 7-6C may therefore be associated with alterations in cell surface structure or the absence of relevant adhesion-associated genes.
The use of the Сaco-2/HT29-MTX model in the present study is methodologically justified, as these cell lines mimic small intestinal enterocytes and are widely applied for the evaluation of probiotic adhesion properties (Duary et al., 2011). Furthermore, adhesion levels to Caco-2 cells have been shown to correlate with the probiotic potential of bacterial strains (Zawistowska-Rojek et al., 2022).
It should also be noted that bacterial adhesion properties may vary depending on external factors, including cultivation conditions and the physiological state of the cells. In particular, damage to the cellular membrane significantly reduces bacterial adhesion capacity, as demonstrated by Kiepś et al. (2023). Similarly, the growth phase of the culture substantially influences the expression of adhesion-associated factors (Deepika et al., 2009).
Recent studies further indicate that probiotic adhesion is associated not only with colonization ability but also with enhancement of intestinal barrier function. For example, Lactobacillus plantarum has been shown to strengthen the epithelial barrier and modulate the intestinal microbiota (Wang et al., 2018). Similar effects may also be characteristic of the highly adhesive strains investigated in the present study.
Novel approaches for studying microbial adhesion, including intestinal organoid models, have confirmed that highly adhesive strains possess increased colonization and functional potential (Jo et al., 2025), thereby opening new perspectives for more accurate evaluation of probiotic properties in future studies.
Overall, the obtained results confirm that adhesion is a multifactorial and highly strain-specific characteristic. Among the lactic acid bacteria, Lactobacillus brevis 8-6M demonstrated the highest adhesion activity, whereas Lactobacillus crustorum 8-9M and Lactococcus lactis subsp. lactis 7-1M exhibited moderate adhesion levels. Among the bifidobacteria, the most pronounced adhesive properties were observed for Bifidobacterium crudilactis 7-5C.
The highly adhesive strains identified in the present study, particularly Lactobacillus brevis 8-6M and Bifidobacterium crudilactis 7-5C, are of considerable interest as potential probiotic cultures for the development of functional dairy products.
4. Conclusions
The adhesion capacity of the investigated lactic acid bacteria and bifidobacteria exhibited pronounced strain-specificity. Using the Сaco-2/HT29-MTX cell model, Lactobacillus brevis 8-6M demonstrated the highest adhesion activity among the lactic acid bacteria (37.09 ± 2.54%), whereas Lactobacillus crustorum 8-9M and Lactococcus lactis subsp. lactis 7-1M showed moderate adhesion levels.
Among the bifidobacteria, the highest adhesion values were observed for Bifidobacterium crudilactis 7-5C (76.23 ± 1.59%) and 7-2C (73.68 ± 1.26%), substantially exceeding those of the lactic acid bacterial strains. In contrast, B. crudilactis 7-6C did not exhibit detectable adhesion activity.
The obtained results indicate substantial variability in adhesive properties even among strains belonging to the same species and confirm the necessity of individual evaluation of probiotic potential at the strain level. Highly adhesive cultures, particularly Lactobacillus brevis 8-6M and Bifidobacterium crudilactis 7-5C, are of greatest practical interest as promising candidates for the development of functional dairy products. The high adhesion capacity of these strains may be associated with the presence of surface-associated proteins and exopolysaccharides that mediate interactions with epithelial cells, representing key determinants of colonization potential and competitive exclusion of pathogenic microorganisms.
Acknowledgements
This work was supported by the Ministry of Science and Higher Education of the Republic of Kazakhstan [grant number AP22685499] within the framework of grant funding for research by young scientists under the “Zhas Galym” project for 2024–2026, titled “Potential application of probiotic lactic acid bacteria exopolysaccharides in food biotechnology”.
Data Availability Statement
The entire set of data that supports the results of this study were published in the article itself
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