Multi-Parameter In Vitro and Genomic Characterization of Bovine-Derived Bacteria with Probiotic Potential

Raw milk and bovine feces represent important sources of microorganisms with probiotic potential. This study aimed to identify potential probiotic candidates from bovine-derived samples and to comprehensively characterize their functional and safety-related features by integrating multi-parameter in vitro evaluation with whole-genome analysis. Acid-producing bacteria were isolated from 12 raw milk and 15 bovine fecal samples using bromocresol green agar. Following PCR-based exclusion of Escherichia coli and RAPD-PCR dereplication, the isolates were evaluated for acid and bile tolerance, survival under simulated gastrointestinal conditions, and autoaggregation, leading to the selection of five candidate strains: Lactiplantibacillus plantarum MX13, Lacticaseibacillus paracasei MX165, Enterococcus faecalis MX179, Enterococcus faecium MX125, and Enterococcus durans FX198. Further in vitro characterization revealed marked strain-specific differences in gastrointestinal stress tolerance, autoaggregation, cell-surface hydrophobicity, and antibacterial activity, with the two lactobacilli displaying broader antibacterial spectra than the Enterococcus strains. Multivariate analysis further revealed distinct overall phenotypic profiles among the candidates, with MX13 exhibiting a relatively balanced functional profile across the traits evaluated. Whole-genome analysis revealed clear differences between MX13 and MX125 in antimicrobial resistance-associated, putative virulence-associated, and other functional genomic features, providing additional information for candidate prioritization. Overall, this study identified and characterized five bovine-derived potential probiotic candidates with distinct functional properties through integrated phenotypic and genomic evaluation, providing a basis for their further functional validation and application-oriented assessment.