Potential probiotic strains from milk and water kefir grains in Singapore—use for defense against enteric bacterial pathogens

Abstract

Kefir grains consist complex symbiotic mixtures of bacteria and yeasts, and are known to impart numerous health-boosting properties to milk and water kefir beverages. Accordingly, studies have been conducted to investigate the microbiota communities in kefir grains, as well as the possibility of deriving useful probiotic strains from them. This study began with milk and water kefir grains collected from a Singapore-based homebrewer, and a total of 158 microbial strains, representing 6 fungal and 17 bacterial species, were isolated from these. Isolated genera include Lactobacillus, Liquorilactobacillus, Lacticaseibacillus, Lentilactobacillus, Leuconostoc, Lactococcus, Acetobacter, Gluconobacter, Oenococcus, Clostridium, Zymomonas, Saccharomyces, Kluyveromyces, Pichia, Lachancea, Candida and Brettanomyces. Next, a funnel approach, involving numerous phenotypic and genomic screening assays, was applied to identify kefir-derived microbial strains with highest probiotic potential. Particular focus was placed on examining the antipathogenic properties of kefir isolates towards enteric pathogens, which pose considerable global health burden. Enteric pathogens tested include from the genera Bacillus, Salmonella, Vibrio, Clostridium, Klebsiella, Escherichia and Staphylococcus. Well diffusion assays were conducted to determine the propensity of kefir isolates to inhibit growth of enteric pathogens, and a competitive adhesion/exclusion assay was used to determine the ability of kefir isolates to out-compete or exclude attachment of enteric pathogens to Caco-2 cells. Seven bacterial strains of species Lentilactobacillus hilgardii, Lacticaseibacillus paracasei, Liquorilactobacillus satsumensis, Lactobacillus helveticus and Lentilactobacillus kefiri, were ultimately identified as potential probiotics. Desirable probiotic characteristics, including good survival in acid and bile environments, bile salt hydrolase activity, antioxidant activity, non-cytotoxicity and high adhesion to Caco-2 cells, lack of virulence or antimicrobial resistance genes and presence of vitamins and GABA synthesis genes, were identified in these kefir isolates. Overall, probiotic candidates derived in this study are novel strains which can add diversity to the existing probiotics repertoire, and may also provide consumers with alternative product formats to attain the health benefits of kefir.