Stage-Resolved Microbial Succession Accompanies Enzymatic and Metabolic Remodelling During the Solid-State Fermentation of Wanying Shenqu

Wanying Shenqu is a traditional solid-state fermented botanical preparation comprising 52 medicinal materials and excipients. However, the stage-resolved relationships among physicochemical changes, hydrolytic enzyme activities, microbial succession, and metabolite remodelling during fermentation remain insufficiently characterised. Physicochemical properties and hydrolytic enzyme activities were monitored throughout the 14-day fermentation. Microbiome and untargeted metabolome profiling were conducted at four stages: D0, D4, D8, and D12. Fermentation involved rapid acidification, transient reducing-sugar accumulation, and moisture loss. Amylase activity increased early, while cellulase and lipase activities peaked on D6 and D10, respectively, and β-glucosidase and protease activities increased later. Pediococcus dominated from D4 onward. Fungal dominance shifted from Paecilomyces to Saccharomycopsis, followed by co-dominance of Saccharomycopsis and Paecilomyces as Aspergillus increased. Of 5031 detected features, 364 metabolites differed among stages. Six Short Time-series Expression Miner (STEM) profiles remained significant after Benjamini–Hochberg correction and comprised 300 metabolites consolidated into four temporal modules. Comparisons among the four omics sampling stages identified three stage-resolved intervals: early substrate mobilisation (D0–D4), metabolic transition (D4–D8), and secondary-metabolite remodelling (D8–D12). Conventional cross-omics analysis identified coordinated microbiome, physicochemical, enzymatic, and metabolic patterns; after stage adjustment and within-family Benjamini–Hochberg false discovery rate correction, no associations remained significant. Together, these results provide a stage-resolved framework and candidate process-monitoring variables for validation across independent batches.