Enzyme-Assisted Bacteria–Yeast Co-Fermentation of Ganoderma lucidum: Metabolomic Reprogramming, Sensory Evolution, and Sleep-Promoting Effects

To enhance the extraction efficiency and bioactivity of Ganoderma lucidum, this study developed a novel enzyme-assisted bacteria–yeast co-fermentation system utilizing Lactobacillus acidophilus and Saccharomyces cerevisiae. Following optimization using response surface methodology, the maximum yields of polysaccharides and triterpenoids reached 2.07% and 3.55%, respectively, substantially exceeding those obtained by direct extraction. Structural characterization revealed that the integrated process induced substantial changes in the polysaccharides, including increased homogeneity, mannose enrichment, and a more porous microstructure. Furthermore, untargeted metabolomics demonstrated that co-fermentation significantly altered the small-molecule profile, characterized by the differential regulation of specific triterpenoids and the enrichment of peptides and amino acids. These metabolic shifts were associated with the evolution of the sensory profile, particularly increased taste richness. In vivo evaluations using a mouse model of PCPA-induced insomnia revealed that the fermented extract exhibited greater sleep-promoting activity than the direct extract. The fermented extract increased hypothalamic 5-HT and GABA levels and was accompanied by modulated gut microbiota composition by increasing the abundance of Lactobacillus and reducing that of Prevotella. These findings suggest that the combined enzymatic-fermentation strategy alleviates insomnia, potentially involving modulation of the microbiota–gut–brain axis, offering preliminary insights into the potential application of Ganoderma lucidum in functional foods.