**Background:** Concentrate-based diets for beef cattle increase weight gain but are associated with reduced microbial diversity, ruminal acidosis, liver abscesses, and endotoxins, often treated with antimicrobials. Due to concerns over antimicrobial resistance, alternatives such as prebiotics and probiotics are needed. α-Mannooligosaccharides (MOS) from yeast cell walls (e.g., Bio-Mos®) are widely studied prebiotics. Bacteroides thetaiotaomicron (B. theta) is a dominant rumen phylum member known for polysaccharide utilization loci (PULs) that enable complex carbohydrate metabolism, including yeast mannan (YM). This study aimed to assess the impact of Bio-Mos® and two B. theta strains (MD33 and MD40) on rumen microbiome composition and fermentation.
**Methods:** For the FACS experiment, rumen fluid from one cannulated cow on a barley silage diet was incubated with or without 2% Bio-Mos® for 48 h, then incubated with fluorescently labeled YM (FLA-YM). Cells interacting with FLA-YM were sorted by FACS and identified by 16S rRNA sequencing (V4 region, Illumina MiSeq). For ex vivo experiments, three cannulated Angus X Hereford crossbred cows were fed alfalfa hay with or without 1% Bio-Mos® (20 g/day) for one week each. Rumen fluid was collected, pooled, and used to inoculate batch cultures containing feed ±2% Bio-Mos®. Cultures were inoculated with PBS (control), MD33, or MD40 (1.4–1.9 × 10^9 CFU/ml). Gas production was measured at 0, 3, 6, 9, 12, 24, and 48 h. VFAs, ammonia, and 16S rRNA sequencing were analyzed at 0, 3, 6, 12, 24, and 48 h. Glycomics analysis of indigestible polysaccharide residues was performed at 48 h.
**Key Results:** FACS analysis showed 6.87% of cells in the control community and 7.67% in the Bio-Mos®-treated community displayed FLA-YM uptake. Sorted communities had significantly lower alpha-diversity than unsorted samples (p < 0.001). ANOSIM showed significant differences between sorted and unsorted communities (R: 0.822; p < 0.001). Bio-Mos® supplementation increased species richness (p = 0.077) and significantly altered community composition (p < 0.001), with increased Bacteroidetes and decreased Clostridiales. Bio-Mos® resulted in significantly lower gas production at all time points (p < 0.05). Total VFA concentrations were significantly lower in Bio-Mos® samples at 0, 3, 6, and 48 h. Butyric acid and valeric acid were significantly lower in Bio-Mos® samples at all time points, while C2/C3 ratio was significantly higher. Ammonia production was significantly lower in Bio-Mos® samples at all time points except 24 h. Probiotic effects of MD33 and MD40 were modest alone, with most significant differences at 48 h. When combined with Bio-Mos®, MD33 significantly increased gas production at all time points (p < 0.05), while MD40 increased gas only at 3 and 6 h. MD33 with Bio-Mos® resulted in significantly lower total VFA, propionic acid, butyric acid, isobutyric acid, isovaleric acid, valeric acid, and caproic acid at 48 h. MD40 with Bio-Mos® resulted in significantly lower propionic acid, butyric acid, isobutyric acid, and caproic acid, and significantly higher C2/C3 at 48 h. Glycomics analysis revealed changes in monosaccharide and glycosidic linkage composition, with Bio-Mos® treatment increasing fucose, arabinose, mannose, galactose, glucose, and glucosamine, while decreasing rhamnose.
**Clinical Implications:** Bio-Mos® supplementation altered rumen fermentation by decreasing gas, total VFA, and ammonia production, which may help prevent ruminal acidosis in cattle fed high-grain diets by reducing fermentation efficiency and organic acid buildup. The combination of Bio-Mos® with B. theta strains showed some synbiotic effects, particularly for MD33, but the effects on overall community composition and VFA production were modest. The study demonstrates that FLA-PS combined with FACS and 16S sequencing can identify YM-interacting bacteria, but the clinical significance of the synbiotic approach requires further research with optimized dosages and longer treatment periods.