Integrated analysis of microbiome and metabolomics: mechanism of different pre-fermented juice regulating protein degradation during mulberry silage

Mulberry ( L.) is a valuable woody protein feed that alleviates protein feed shortages, yet its silage production faces key challenges including high moisture content, scarce native lactic acid bacteria, and severe protein degradation. This study innovatively adopted alternative raw materials to prepare pre-fermented juices, with grape pomace pre-fermented juice (treatment G) and red clover pre-fermented juice (treatment R) compared against mulberry pre-fermented juice (treatment M) and an untreated control (CK). Combined with bacterial community and metabolomic analyses, we evaluated the effects of these treatments on mulberry silage quality over a 60-day ensiling period. Results showed that treatment R delivered the best performance: it enriched , upregulated isoflavonoids including formononetin, glycitein and biochanin A, reduced silage pH to 3.99, increased lactic acid content, and inhibited protein degradation by suppressing protease activities. Treatment M promoted the growth of and the accumulation of beneficial metabolites such as 3,4-dihydroxybenzaldehyde, while treatment G showed moderate effects. All pre-fermented juices improved silage fermentation quality and nutrient retention. This study clarifies the microbial-metabolite regulatory mechanisms underlying mulberry ensiling, provides a low-cost and eco-friendly silage production technology, and promotes the sustainable utilization of mulberry in ruminant production.