Differential regulatory roles of four distinct microbial types on solid-state fermentation characteristics of Sichuan pepper (Zanthoxylum bungeanum) leaves and distillers’ grains

Sichuan pepper ( Zanthoxylum bungeanum ) leaves (ZBL) and distillers’ grains (DG) are abundant agricultural by-products with high potential for feed utilization, but are limited by high anti-nutritional factors and poor fermentation stability. Anaerobic solid-state fermentation (ASSF) with functional microbes can effectively improve feed safety and nutritional value. However, the functional differentiation and regulatory mechanisms of different microbial strains during single-substrate and mixed-substrate fermentation remain unclear. This study aimed to investigate the effects of four typical strains, including Saccharomyces cerevisiae J, Bacillus subtilis W4, Levilactobacillus brevis RS3, and Lactiplantibacillus plantarum RS4, on fermentation quality, nutritional composition, anti-nutritional factors, bacterial community, and metabolite profiles of ZBL, DG, and their 1:1 mixture. The results showed that substrate type and inoculated strain jointly determined fermentation performance. Inoculation with the four strains effectively reduced pH, promoted lactic acid production, enhanced fiber degradation, and improved dry matter retention and antioxidant activity. Indigenous acid-tolerant microorganisms in DG could inhibit acid-sensitive bacteria in ZBL after mixing, thereby stabilizing fermentation system. Specifically, S. cerevisiae J inhibited aerobic spoilage bacteria and significantly up-regulated phenylpropanoid biosynthesis pathway. B. subtilis W4 promoted flavonoid accumulation and fiber degradation. L. brevis RS3 and L. plantarum RS4 significantly increased lactic acid content and enhanced alkaloid and phenylpropanoid production. Multi-omics analysis revealed that inoculation reshaped bacterial community structure and drove differential enrichment of key metabolites. This study clarified the functional mechanisms of four typical strains in ASSF of ZBL and DG, and confirmed the feasibility of mixed fermentation. The results provide a theoretical basis and technical support for the feed utilization of similar agricultural wastes.