This study aimed to investigate the potential of Bacillus coagulans (BC) as an inoculant in alfalfa silage fermentation. Fresh alfalfa was harvested at a dry matter (DM) content of 329.60 g/kg fresh weight (FW), and inoculated without (CON) or with BC (1 × 106 CFU/g FW), Lactobacillus plantarum (LP, 1 × 106 CFU/g FW), and their combinations (LP+BC, 1 × 106 CFU/g FW, respectively). Samples were taken at 3, 7, 14, 30, and 60 d, with three replicates for each. The prolonged ensiling period resulted in a decrease in pH values and an increase in lactic acid (LA) concentrations in alfalfa silages. After 60 d of fermentation, the application of BC and LP decreased the pH values and increased LA concentrations in treated silages, especially when their combination was applied. Application of BC preserved more water-soluble carbohydrates (WSC), and further application of BC increased WSC in LP+BC-treated silage compared to LP-treated silage. There was no significant difference in the crude protein (CP) content between the CON and treated silages, however, the BC and LP treatments reduced the ammonia nitrogen (NH3-N) concentration, especially when their combination was applied. Additionally, the BC and LP-treated silages had lower neutral detergent fiber (NDF) and acid detergent fiber (ADF) when compared to the CON silage (p < 0.001). Inoculants also increased Lactobacillus abundance and decreased Enterococcus abundance after 60 d of fermentation. Spearman's rank correlation analysis revealed a positive correlation between LA concentration and Lactobacillus abundance. It was noteworthy that LP, BC, and their combination increased the relative abundances of carbohydrate metabolism, energy metabolism, cofactors, and vitamin metabolism, decreasing the relative abundances of amino acid metabolism and drug resistance: antimicrobial. Therefore, the inclusion of BC increased the fermentation quality of alfalfa silage, with the optimal combination being LP+BC. According to the findings, BC could be considered a viable bioresource for improving fermentation quality.
Keywords: Bacillus coagulans; alfalfa; fermentation composition; microbial diversity.