Antibacterial activity of soy milk fermented by kefir grain against a number of pathogenic bacteria

Introduction: Probiotics are live microorganisms that have beneficial effects on human health when consumed in sufficient quantities (Fuller 1989). Kefir beverage is a natural complex probiotic and one of the oldest fermented milk products. This microbial symbiotic is obtained by fermenting milk with kefir grains. Consumption of kefir is effective in promoting health and increases the body's immune system, balances blood pressure, treats digestive diseases and reduces serum cholesterol levels, and also has antibacterial, antifungal and antitumor activities (Farnworth 2005). The microflora of kefir grains contains a group of specific microbes that exist in a complex symbiotic relationship, including species of yeasts, lactic acid bacteria (Lactobacillus and Lactococcus), and acetic acid bacteria (Garbers 2004). Kefir is a complex probiotic, from a microbial symbiotic that is obtained from milk fermentation by kefir grains, and its consumption is effective in promoting health. The main raw material for making kefir beverage is cow's milk. For various reasons, some people do not consume cow's (animal) milk: they may be vegetarians or have health problems such as lactose intolerance or a casein allergy (Bau et al., 2015). The purpose of this study is to investigate the antibacterial activity of kefir samples prepared with soy milk against 10 pathogenic bacteria.
Material and methods: The regeneration and fermentation process of grains was done with successive subcultures in milk according to the method presented by Ajam and Koohsari (2020). Activated kefir grains were added to soy milk (with 2% and without sucrose) and the fermentation process was performed at 25°C and 37°C. After fermentation, kefir grains were separated from kefir extract and the antibacterial activity of kefir extract against 10 pathogenic bacteria including Staphylococcus aureus, Bacillus cereus, Shigella dysenteriae, Enterococcus faecalis, Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli (native isolate and standard strain) and Klebsiella pneumoniae (native isolate and standard strain), was evaluated based on diffusion in agar and by the well method. For this purpose, the method presented by Weinstein et al., 2018 was used. Regarding sensory tests, in order to compare with kefir samples prepared from soy milk, kefir samples with cow's milk were also prepared, so that a comparison can be made between these samples. In order to study this comparison, indicators of overall appearance, color, fragrance, taste and mouthfeel and total score were measured.
Results and discussion: The results of the effect of fermentation temperature (25°C and 37°C) and the presence of sucrose in kefir samples prepared with soy milk on the antibacterial activity against the tested bacteria, except for the gram-positive bacteria E. faecalis and S. aureus, indicate a significant difference between treatments (P>0.05). The presence of sucrose and fermentation temperature had a significant effect on antibacterial activity against all tested bacteria except E. faecalis and S. aureus. And kefir samples prepared with soy milk containing sucrose showed more antibacterial activity. The highest antibacterial activity of kefir samples prepared with soy milk against P. aeruginosa was related to kefir samples prepared with soy milk containing sucrose at fermentation temperatures of 25°C and 37°C with mean of diameter of inhibition zone of 20 mm. The highest antibacterial activity of the kefir samples prepared with soy milk against E. faecalis is related to the samples prepared with soy milk without sucrose and at the fermentation temperature of 25°C with mean of diameter of inhibition zone of 18.5 mm and the highest antibacterial activity of the samples against S. aureus was related to the samples of soy milk without sucrose at the fermentation temperature of 37°C with mean of diameter of inhibition zone of 16 mm. The highest antibacterial activity against S. dysenteriae and S. typhimurium was observed in kefir samples prepared with soy milk containing sucrose at fermentation temperatures of 25°C and 37°C. The highest antibacterial activity of kefir samples prepared with soy milk against the native isolate and standard strain of K. pneumoniae was related to kefir samples prepared with soy milk containing sucrose at fermentation temperatures of 37 with mean of diameter of inhibition zone of 21 and 18 mm, respectively. Compared to other tested bacteria, E. coli and especially the standard strain of this bacteria, showed more resistance than kefir samples prepared with kefir soy milk. In this regard, B. cereus showed resistance to all treatments and no antibacterial activity was seen against this spore bearing bacteria bacterium. Based on the results of sensory tests, no significant difference was observed between the treatments in any of the indicators (P<0.05), but nevertheless, the sensory evaluations indicated low sensory acceptability of kefir samples prepared with soy milk, so compared to kefir samples obtained from cow's milk, it obtained a lower overall score. Antibacterial activity of fermented beverage is related to the compounds present in this beverage. It has also been reported that the compounds in kefir drink are affected by the type of substrate or milk (soy milk in the present study), fermentation conditions, kefir grain origin and storage conditions (Rosa et al., 2017). Soy milk has different carbohydrates, the most important of which are sucrose, raffinose and stachyose, while lactose is the main carbohydrate in cow's milk (Pinthong et al., 1980). Among the mechanisms of antibacterial activity of kefir extracts, we can mention acidic pH, as a result of lactose fermentation or alternative substrates, increasing the content of organic acids, such as lactic acid, acetic acid, etc., by microorganisms in kefir grains (Kourkoutas et al., 2007). Addition of carbohydrates (including sucrose) improves the ability of kefir grain microorganisms to produce lactic acid in soy milk. In addition to the role of organic acids, the antimicrobial activity of kefir beverage is related to the production of peptides (bacteriocins), carbon dioxide, hydrogen peroxide, ethanol and diacetyl (Oliveira Leite et al., 2013). In addition to these compounds, the exopolysaccharide produced by kefir grains called kefiran also has antibacterial activities (Wang et al., 2008 and Prado et al., 2015).
Conclusion: The results indicated significant antibacterial activity of kefir samples prepared with soy milk against the tested bacteria except for B. cereus and the standard strain of E. coli. The remarkable effect of kefir samples prepared with soy milk against Gram-negative bacteria such as P. aeruginosa, K. pneumoniae, both native isolates and standard strains, and S. dysenteriae was very interesting.