Evaluation of the techno-functional properties of lactobacilli strains originated from Bos indicus and Bubalus bubalis calves for probiotic potential.

The current study was undertaken to isolate, characterize, and cluster lactobacilli strains of indigenous cattle (Bos indicus) and buffalo (Bubalus bubalis) calf origin to develop a species-specific multistrain potential probiotic adjunct. Bacterial strains were isolated from the feces of Bos indicus and Bubalus bubalis calves of different breeds, i.e., Sahiwal (SC), Tharparker (TP), and Gir (GC) cattle and Murrah buffalo (MB). The fecal samples were aseptically taken from five healthy calves (5-10 days old) of each breed (a total of 20 animals). Accordingly, 105 bacteria were isolated, 52 of which were catalase negative, Gram-positive, and vancomycin resistant. These isolates were presumed to be Lactobacillus species, and genus-specific PCR was used to confirm their identification. Among the 52 isolates, 22 strains demonstrated greater acid tolerance, while 29 isolates showed superior bile tolerance, with survivability greater than 90%. Fifteen isolates capable of tolerating both acid and bile were further selected, and their identities were confirmed via 16S rRNA sequencing. Cluster analysis of the phylogenetic tree revealed three different species: Ligilactobacillus salivarius (8), Ligilactobacillus agilis (1), and Limosilactobacillus reuteri (6). All lactobacilli strains showed coaggregation with Escherichia coli ATCC25922 and Salmonella arizonae ATCC13314. All the isolates were found non-hemolytic and non-mucinolytic. Principal component analysis (PCA) revealed that L. reuteri GC09, L. salivarius GC12, L. agilis MB08, L. salivarius MB14, L. reuteri SC01, and L. reuteri MB11 possessed the most desirable attributes of probiotics. Based on these findings, these strains could be used to develop multi-strain probiotic adjunct for calves.