Metabolomics-guided valorization of microbial-enzyme co-fermented mulberry branches and leaves: Flavonoid biotransformation and improved antioxidant capacity

Fermentation represents a sustainable bioprocess for upgrading the utilization value of underutilized agricultural by-products, such as mulberry branches and leaves (MBL). This study applied microbial-enzyme co-fermentation to MBL under simulated industrial conditions, using Lactiplantibacillus plantarum , Saccharomyces cerevisiae , Bacillus subtilis , and a composite enzyme cocktail, and analyzed dynamics in nutrients, bioactive compounds, antioxidant capacity, microbial communities, and flavonoid metabolites. Results showed that fermentation significantly improved nutritional profiles with CP content peaking at day 18 (17.93% relative to 14.30% unfermented MBL, 25.38% increase, p < 0.05) and fiber content reaching the lowest level at day 22 (NDF 49.99% relative to 57.07%, 12.41% reduction; ADF 28.90% relative to 30.45%, 5.09% reduction, p < 0.05). Meanwhile, antioxidant capacity was remarkably elevated, with the hydroxyl radical scavenging rate peaking at day 14 (138.94% increase, p < 0.05). Mechanistically, Lactiplantibacillus rapidly dominated the community and mediated the conversion of flavonoid glycosides to highly active aglycones via glycoside hydrolysis, which directly boosted antioxidant capacity; as fermentation proceeded, these aglycones were further modified to methoxylated flavonoids with improved structural stability. Overall, microbial-enzyme co-fermentation enhanced MBL's nutritional and functional properties. Fermented MBL is a promising high-quality functional feed, alleviating food-feed competition and advancing sustainable circular agricultural bioeconomy.