- Article
18 Pages
This study investigated the regulatory effects of Bacillus cereus S1 combined with cinnamaldehyde on microbial community dynamics, forage nutritional quality, and virulence-gene-associated molecular risks during the aerobic storage of alfalfa hay with 30% moisture content. Storage experiments were performed aerobically at ambient temperature (25–30 °C). Three treatments were established: a blank control (CK), single-strain inoculation with B. cereus S1 (BS), and the combined application of B. cereus S1 plus cinnamaldehyde (BC). Sampling was conducted on days 0, 8, and 32. Metagenomic sequencing was performed, followed by joint functional annotation using the Carbohydrate-Active EnZymes (CAZy) and Pathogen–Host Interaction (PHI) databases to decipher how the BC treatment modulated microbial succession, nutritional traits, and molecular-level biosafety risks. At day 32 of storage, spoilage-related genera, including Aspergillus, Acinetobacter, and Oceanobacillus, proliferated extensively in the CK group. Both BS and BC suppressed the massive proliferation of undesirable microbes and stabilized community structure, with BC exhibiting the best regulatory performance. The BC treatment drove a clear community succession, where the dominant genus shifted from Enterococcus toward the inoculated functional strain Bacillus cereus S1. Functional gene profiling revealed continuous enrichment of AA10 family lytic polysaccharide monooxygenases (LPMOs)—key cellulose-degrading enzymes—in the BC group, accompanied by significant reductions in neutral detergent fiber (NDF) and acid detergent fiber (ADF). Meanwhile, accumulation of lethal and increased-virulence PHI genes was markedly restrained. The relative abundance of Aspergillus was positively correlated with high-risk PHI genes (r = 0.556, p < 0.01). On day 32, the BC group achieved a dry matter preservation rate of 94.22%, crude protein (CP) content of 28.91%, and an in vitro dry matter digestibility of 90.88%. This work represents the first mechanistic investigation of the combined Bacillus-cinnamaldehyde system specifically for 30%-moisture aerobic-stored alfalfa hay. Our results demonstrate that this combined treatment reshapes microbial communities, constrains toxigenic fungal proliferation, improves forage quality, and mitigates molecular-level biosafety risks. It provides mechanistic insights for developing green bioregulators for high-moisture forage storage.
Agronomy
1 November 2026





