Evaluation of probiotic and antimicrobial properties of Levilactobacillus brevis NKN55 isolated from local yogurt

Introduction: Probiotics are living microorganisms that, when consumed in food, provide health benefits to the host. In the past, probiotics were used as substances consumed by microorganisms that stimulate the growth of other microorganisms. The composition of probiotics is defined as microbial supplements that create a positive effect by modifying the composition of the microbial flora (Joyande et al. 2021). Among the effects of probiotics on human health, we can mention the improvement of milk digestibility in people with lactose intolerance, the enhancement of the immune system through antimicrobial peptide production, the synthesis of group B vitamins, the boost in the body's immunity, and the prevention of cell carcinogenesis (Fallah et al. 2019). Due to the conversion of fermentable sugars into organic acids, ethanol, and other metabolites with antimicrobial potential, these microorganisms create unfavorable conditions for the growth of potentially pathogenic microorganisms or agents of spoilage (Alizadeh Behbahani & Noshad. 2021). Native strains of lactic acid bacteria have found special importance in the dairy industry because these strains, in addition to being compatible with the conditions of the region, possess a unique ability to produce the desired taste and aroma in various types of fermented products. Additionally, these strains exhibit characteristics such as inherent resistance to destructive phages and antimicrobial effects (Rokhtabnak et al. 2015). The aim of this research was to investigate the functional potential and antimicrobial activity of the Levilactobacillus brevisNKN55 strain isolated from local yogurt in Tashan (Behbahan). If it exhibits desirable functional and antimicrobial properties, it can be utilized in the production of dairy products either as a complementary culture or as a natural preservative.

Materials and Methods: Firstly, the strain was isolated and identified using molecular methods. Subsequently, the strain was evaluated for probiotic properties such as acid resistance (pH 2.5, 3.5, and 4.5), hydrophobicity, and bile resistance (0.3, 0.5, and 0.7). Cholesterol absorption was also assessed. Additionally, the strain underwent evaluation for biogenic amine production, hemolytic and DNase properties. The antioxidant property of the isolated strain (measured using DPPH and ABTS assays) was determined, and its antimicrobial activity against 6 key pathogens (Escherichia coli, Bacillus cereus, Salmonella typhimurium, Klebsiella aerogenes, Staphylococcus aureus, and Listeria monocytogenes) was investigated using disc diffusion and agar well diffusion methods. Furthermore, adhesion potential to Caco-2 cells, anti-adhesion properties, auto-aggregation capacity, and co-aggregation of the strain were also evaluated.
Results and discussion: The maximum reduction in strain viability is associated with a shelf life of 3 hours at pH 2.5. As the pH decreased from 4 to 2, a significant decrease in the number of viable cells was observed, dropping from 7.8 to 6.90 Log CFU/mL. In this study, the growth of the strain decreased by 0.7% with an increase in bile salt percentage. The hydrophobicity of the strain was 58.4±0.40%. For the investigated strain, DNase, biogenic amine production, and hemolytic activity were negative. The cholesterol absorption rate was 10.39, while DPPH and ABTS free radical capacity were 33.46 and 38.5, respectively. The auto-aggregation potential was 33.8, and Co-aggregation was 21.45. Adhesion potential to Caco-2 cell was 10.50, and anti-adhesion potential against K. aerogenes was 38.90 in competition, 31.20 in ability, and 19.8 in displacement. One of the important characteristics of lactic acid bacteria, crucial in their role in the food industry, is resistance to exposure to acidic conditions in products such as yogurt or buttermilk. For this purpose, the survival of these bacteria was tested at pH 2.5, 3.5, and 4.5. The results showed that the Lev. brevisNKN55 strain has a favorable shelf life under the investigated conditions.

To produce beneficial effects in the body, probiotics must be able to grow in the stomach and intestines and have the ability to live there. For this purpose, they must have the necessary resistance to face hydrochloric acid in the stomach and bile salts in the intestine (Alizadeh Behbahani et al. 2020). The results showed that Lev. brevis NKN55 has good resistance to different concentrations of bile salts. In this study, the growth of the tested strain was inhibited by 0.7% with an increase in the percentage of bile salt. However, the growth rate depends on the concentration of bile salts. These results are similar to other studies that have shown lactobacilli can survive in high bile levels. Surface hydrophobicity can be used as a primary way to identify probiotic bacteria with adhesion properties and suitable characteristics for commercial purposes (Vasechi et al. 2020). The presence of hydrophobic molecules on the cell surface, such as surface proteins, cell wall proteins, cytoplasmic membrane proteins, and lipids, increases the cell's hydrophobicity. There is always concern about the commercial use of these bacteria because it is possible to transfer this gene to pathogenic bacteria and create resistance against them. The significant sensitivity of the isolated strain to multiple antibiotics indicates that this strain may not possess genes that cause antibiotic resistance. One critical aspect of probiotic bacteria that should be considered during evaluation is their antibacterial effect, attributed to metabolites such as organic acids, hydrogen peroxide, diacetyl, ethanol, phenols, and protein compounds that inhibit growth (Barzegar et al. 2020). The most common antimicrobial compounds reported to be produced by probiotic bacteria include bacteriocins, hydrogen peroxide, and organic acids (especially lactic and acetic acids). Auto-aggregation is directly related to the adhesion potential of probiotic bacteria, while aggregation has a close interaction with pathogens (Patel et al., 2011). The auto-aggregation property helps bacteria adhere to intestinal cells and mucosal surfaces. Cell aggregation may enable bacteria to form a barrier that prevents colonization and biofilm formation by pathogenic bacteria. The ability of bacterial cells to attach to intestinal mucosa is called adhesion (Jena et al. 2013). In case of damage to the epithelial tissue, the probability of bacterial cell adhesion decreases. Probiotic bacteria are used to treat damage to the digestive system and replace its lost flora. By correcting the microbial balance inside the intestine, damaged tissue is improved, and the ability of microorganisms to bind to the surface of intestinal cells increases (Fontana et al., 2013).

Conclusion: The process of collecting and identifying native strains from fermentation products in any part of the country can cause Preservation of microbial and genetic reserves and provides useful information for scientific and commercial applications, especially in the field of dairy industries and the discussion of probiotics and functional foods. In this study, Lev. brevisNKN55 strain isolated from Behbahan yogurt was evaluated for its probiotic and antimicrobial potential. The results showed that this strain has a high ability to inhibit pathogenic bacteria. This bacterium tolerates different concentrations of bile salts well. It also has the ability to survive in acidic conditions. This bacterium tolerates different concentrations of bile salts well. It also has the ability to survive in acidic conditions. The studied strain was sensitive to common antibiotics and showed acceptable adhesion, hydrophobicity, auto-aggregation and accumulation. According to the results, it is suggested to use this strain as a probiotic supplement in fermentation cultures or as a co-culture in the production process of fermented food products after conducting more confirmatory tests. The results showed that this strain has a high ability to inhibit pathogenic bacteria. This bacterium tolerates different concentrations of bile salts well. It also has the ability to survive in acidic conditions. This bacterium tolerates different concentrations of bile salts well. It also has the ability to survive in acidic conditions. The studied strain was sensitive to common antibiotics and showed acceptable adhesion, hydrophobicity, auto-aggregation and accumulation. According to the results, it is suggested to use this strain as a probiotic supplement in fermentation cultures or as a co-culture in the production process of fermented food products after conducting more confirmatory tests. The results showed that this strain has a high ability to inhibit pathogenic bacteria. This bacterium tolerates different concentrations of bile salts well. It also has the ability to survive in acidic conditions. This bacterium tolerates different concentrations of bile salts well. It also has the ability to survive in acidic conditions. The studied strain was sensitive to common antibiotics and showed acceptable adhesion, hydrophobicity, auto-aggregation and accumulation. According to the results, it is suggested to use this strain as a probiotic supplement in fermentation cultures or as a co-culture in the production process of fermented food products after conducting more confirmatory tests.The results showed that this strain has a high ability to inhibit pathogenic bacteria. This bacterium tolerates different concentrations of bile salts well. It also has the ability to survive in acidic conditions. This bacterium tolerates different concentrations of bile salts well. It also has the ability to survive in acidic conditions. The studied strain was sensitive to common antibiotics and showed acceptable adhesion, hydrophobicity, auto-aggregation and accumulation. According to the results, it is suggested to use this strain as a probiotic supplement in fermentation cultures or as a co-culture in the production process of fermented food products after conducting more confirmatory tests.