Time-Series Metabolomics Reveals a Three-Stage Metabolic Relay Driving Antibacterial Enhancement in Co-Fermented Polygonatum sibiricum and Gentiana scabra Byproducts

Abundant non-medicinal byproducts of Polygonatum sibiricum and Gentiana scabra are severely underutilized, resulting in resource waste and environmental burden. A previous study confirmed that triple-microbial co-fermentation enhances their antibacterial activity, yet the temporal metabolic mechanism and optimal process parameters remain unclear due to endpoint-only metabolomics limitations. This study aimed to optimize the staged solid-state fermentation (SSF) system for maximum antibacterial activity, verify the triple-microbial consortium’s synergistic enhancement effect, and elucidate the dynamic metabolic mechanism via time-series metabolomics. A staged SSF strategy was established: Aspergillus niger monoculture (0–48 h) followed by Bacillus subtilis and Saccharomyces cerevisiae co-culture (48–72 h). Key parameters were optimized via single-factor experiments and a Box–Behnken design. Under optimal conditions, inhibition zones against Staphylococcus aureus and Escherichia coli reached 20.8 ± 0.3 mm and 17.6 ± 0.2 mm, respectively, with a 17.5% increase in S. aureus inhibition and markedly improved E. coli inter-batch consistency. Time-series untargeted LC-MS/MS metabolomics (2681 identified metabolites) revealed a three-stage metabolic relay model driving antibacterial enhancement: 0–48 h shikimate pathway activation for phenolic precursor accumulation; 48–60 h dipeptide conversion and ABC transporter enrichment initiating antibacterial synthesis; 60–72 h metabolic flux redirected to indole alkaloid biosynthesis for complex antibacterial compound accumulation. This work provides a mechanistic paradigm for the high-value valorization of herbal byproducts, with applications in natural antibacterial agents and functional feed additives.