A new strategy for the fermentation of Massa Medicata Fermentata by combining multiple strains of fermentation and their fermentation mechanisms.

To address the quality instability of the traditional fermentation process of Massa Medicata Fermentata (LSQ), we designed an innovation strategy for dual-strain co-fermentation LSQ. Rhizopus arrhizus, Bacillus velezensis, Bacillus subtills, and Bacillus cereus were selected as the fermentation strains for the LSQ. After dual-strain co-fermentation, its pro-digestive enzymes and anti-inflammatory activities were significantly enhanced. Particularly, R. arrhizus/B. subtills fermentation group showed the prominent promotion of the enzymatic activities of amyloglucosidase, cellulase and trypsin, with AC200 values < 1.00 and Max fold increase values of 27.39 ± 0.22, 25.39 ± 0.87 and 48.07 ± 1.84, respectively, and anti-inflammatory activity with an IC₅₀ value of 2.35 ± 0.18 mg/mL. Based on the correlation analysis of differential metabolic profiles and activities, the key pharmacodynamic metabolites were analyzed and validated, such as levomycetin succinatea, β-citrylglutamate, D-glucosaminic acid, nikkomycin and fucose 1-phosphate. Among them, D-glucosaminic acid was positively correlated with the promoting activity of amyloglucosidase, cellulose, enzyme trypsin, pepsin, and the inhibitory activity of NO production, and fucose 1-phosphate and nikkomycin had the prominently positive correlation with the promoting activity of pepsin (p < 0.01). In addition, the docking scores between them and digestive enzyme proteins were all < - 5. A new strategy involving the dual-strain fermentation of LSQ was investigated, and clarified the LSQ fermentation strain-constituent-pharmacological activity correlations, which provides a valuable reference for delving into the LSQ fermentation mechanism.