In Vitro Screening of Cattle-Derived Lactic Acid Bacteria Fermented Raw Milk for Probiotic Properties and Fecal Fermentation: Preliminary Insights into Potential Mechanisms Against Calf Diarrhea

In this study, three bovine-derived lactic acid bacteria (LAB) strains were isolated from feces of healthy Simmental calves: Lacticaseibacillus paracasei (Lp), Enterococcus hirae (Eh), and Enterococcus faecalis (Ef). Bacterial suspensions were inoculated into pasteurized milk at a 2% inoculation ratio and anaerobically fermented at 37 °C for 24 h to obtain three kinds of LAB-fermented milks: Lacticaseibacillus paracasei fermented milk (LpM), Enterococcus hirae fermented milk (EhM), and Enterococcus faecalis fermented milk (EfM). Their probiotic potential was comprehensively evaluated through a series of in vitro assays, and in vitro colonic anaerobic fermentation was conducted using fecal inoculum from four Simmental calves (three biological replicates per group) to investigate the regulatory effects of these strains on the colonic microbiota and short-chain fatty acids (SCFAs). The three strains exhibited similar growth trends during raw milk fermentation. Compared with Eh and Ef, Lp showed significantly greater utilization of galactooligosaccharides (GOS) and lactooligosaccharides (LOS) (p < 0.01), whereas no significant difference in proteolytic activity was observed among the three strains (p = 0.192). LpM exhibited significantly higher water-holding capacity and cell-surface hydrophobicity than EhM and EfM (p < 0.01), along with a higher auto-aggregation rate (p < 0.01). Conversely, Eh and Ef displayed stronger co-aggregation with Staphylococcus aureus, Escherichia coli, and Salmonella, as well as greater antibacterial activity, with Ef being the most effective (p < 0.01). All three strains were highly sensitive to kanamycin, streptomycin, tetracycline, and doxycycline, whereas Eh and Ef showed intermediate sensitivity or resistance to several antibiotics. After fermentation, LpM and EhM retained IgG more effectively, while EfM consumed the least lactose. EhM produced the most acetic acid, LpM was dominant in propionic and butyric acid production, and both LpM and EhM generated more lactic acid than EfM (p < 0.01). The fermented milks displayed distinct 2,2-Diphenyl-1-picrylhydrazyl(DPPH) and hydroxyl-radical-scavenging capacities; LpM possessed the strongest antioxidant activity, followed by EhM, while EfM showed the weakest antioxidant performance (p < 0.01). During in vitro fermentation, both LpM and EhM produced more SCFAs than EfM. Through 16S rRNA gene sequencing it was revealed that LpM increased the abundance of Butyricicoccus pullicaecorum, Lactococcus, Streptococcaceae, and Lachnospiraceae. Although Eh and Ef could enrich genera such as Bacillus to inhibit diarrhea-associated pathogens, they also coincided with an increase in opportunistic pathogens including Escherichia-Shigella, Klebsiella, and Enterococcus. Therefore, Lp represents a promising candidate strain of bovine-derived lactic acid bacteria for fermented milk aimed at preventing calf diarrhea. Notably, Eh and Ef also enriched opportunistic pathogens, and further safety assessment is required before practical application.