Valorization of the Non-Medicinal Parts of Polygonatum sibiricum and Gentiana scabra Bunge from Liaoning via Solid-State Co-Fermentation: Synergistic Antibacterial Enhancement

The non-medicinal parts of Polygonatum sibiricum (P. sibiricum) and Gentiana scabra (G. scabra) are abundant but underutilized in Liaoning Province, China, creating an environmental burden. Solid-state fermentation (SSF) offers a strategy to enhance their bioactivity, yet triple microbial co-fermentation remains underexplored. This study applied a triple microbiota—featuring Aspergillus niger (A. niger), Bacillus subtilis (B. subtilis), and Saccharomyces cerevisiae (S. cerevisiae)—to ferment the stems and leaves of both plants. Antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was assessed via the Kirby–Bauer test, while Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS)-based non-targeted metabolomics identified differential metabolites and enriched pathways. Co-fermentation significantly increased the inhibition zones to 17.4 ± 0.8 mm for E. coli and 17.7 ± 0.3 mm for S. aureus, a 1.8-fold improvement over the unfermented controls (p < 0.001). Among the 2976 metabolites detected, 1236 were differentially expressed, with Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis highlighting activation of aminoacyl-tRNA biosynthesis, ABC transporter, and phenylalanine–tyrosine–tryptophan pathways. Differential abundance analysis indicated that the aminoacyl-tRNA pathway (DA score > 0.9) is critical for antimicrobial peptide synthesis. Phenylalanine derivatives, including 4-hydroxybenzaldehyde, which increased over 430-fold (Log2 FC = 8.78), contributed to membrane-disruptive antibacterial effects. Mechanistically, A. niger hydrolyzes cellulose to release precursors, B. subtilis synthesizes antimicrobial peptides, and S. cerevisiae enhances metabolite solubility and excretion, collectively boosting antibacterial activity by 80%, suggesting a potent synergistic interaction among the triple microbiota. This cascade mechanism provides a scalable approach for valorizing approximately 55 million tons of traditional Chinese medicine (TCM) waste annually.