Valorization of mushroom byproducts by co-fermentation with soybeans for a functional Douchi development

This work explores the valorization of Edible Mushroom By-Products (EMBP) in Douchi production. We first focused on optimizing the critical koji-making stage, utilizing a mixed culture of Aspergillus oryzae and Mucor racemosus to maximize enzymatic activity. Through response surface methodology, we identified optimal conditions, specifically a 1:1 strain ratio, 6% inoculation, and 21% EMBP supplementation for fermenting over 63 hours. These parameters significantly enhanced protease and β-glucosidase activities, facilitating the biotransformation of isoflavone glycosides into bioactive aglycones like daidzein and genistein, thereby boosting antioxidant capacity compared to traditional soybean-only Douchi. Building on this, we further employed untargeted UPLC-MS/MS metabolomics to map non-volatile metabolites throughout the entire fermentation and 30-day post-fermentation process. Identifying 695 metabolites, the analysis revealed that the most profound metabolic shifts occur during koji-making and the first five days of post-fermentation. Differential analysis highlighted flavonoids and amino acids as primary drivers of metabolic variance, while correlation data linked the escalating antioxidant potential to specific accumulations of aglycones, free fatty acids, phenolic acids, and group A saponins. Collectively, our work demonstrates that co-fermenting soybeans with mushroom waste effectively enhances the nutritional profile, flavor precursors, and functional bioactivity of Douchi, offering a sustainable solution for agricultural by-product utilization.