In vitro and genetic screening of probiotic properties of lactic acid bacteria isolated from naturally fermented cow-milk and yak-milk products of Sikkim, India

A total of 272 isolates of lactic acid bacteria (LAB) were isolated from 22 samples of naturally fermented milk products of Sikkim in India viz. dahi, soft-variety chhurpi, hard-variety chhurpi, mohi and philu, out of which, 68 LAB isolates were randomly grouped on the basis of phenotypic characteristics, and were identified by 16S rRNA gene sequence analysis. Leuconostoc mesenteroides was the most dominant genus, followed by Leuc. mesenteroides subsp. jonggajibkimchii, Lactococcus lactis subsp. cremoris, Lc. lactis, Lc. lactis subsp. hordniae, Lc. lactis subsp. tructae, Enterococcus faecalis, E. italicus and E. pseudoavium. LAB strains were tested for probiotics attributes by in vitro and genetic screening, based on marker genes. LAB strains showed tolerance to pH 3.0, bile salt, resistance to lysozyme and β-galactosidase activity. Enterococcus faecalis YS4-11 and YS4-14 and Lactococcus lactis subsp. cremoris SC3 showed more than 85% of hydrophobicity. Genes clp L and tdc encoding for low pH tolerance, agu A and Ir1516 encoding for bile tolerance, LBA1446 gene encoding for BSH activity, map A, apf, mub 1 and msa encoding for mucosal binding property were detected. Gene mesY for bacteriocin production was detected only in Leuconostoc spp. Based on the in vitro and genetic screening of probiotic attributes, Leuc. mesenteroides; Leuc. mesenteroides subsp. jonggajibkimchii and Lc. lactis subsp. cremoris were tentatively selected for possible probiotic candidates.