Sequential fermentation with Saccharomyces cerevisiae, Levilactobacillus brevis and Acetobacter pasteurianus for GABA-enriched low-alcohol functional beverages: Optimization and synergistic formation mechanism of GABA

To address the issues of low γ-aminobutyric acid (GABA) synthesis efficiency and excessive ethanol residue in the preparation of cereal-based functional fermented beverages using single-strain fermentation, we utilized a composite fermentation substrate of Cordyceps militaris and germinated brown rice to establish a three-strain sequential fermentation strategy. We screened three functional strains compatible with the composite substrate: Saccharomyces (S.) cerevisiae Fresco, Levilactobacillus (L.) brevis GH1, and Acetobacter (A.) pasteurianus CICC20001. The optimal sequential fermentation process was optimized: first inoculating S. cerevisiae and fermenting at 37°C for 24 h, then cofermenting with L. brevis at 34.4°C for 72 h, and finally inoculating A. pasteurianus and fermenting at 30°C for 48 h. Under these conditions, GABA production reached 24.29 g/L, ethanol content decreased to 4.86 g/L, organic acid components in the beverage were optimized, and flavour quality significantly improved. Additionally, by controlling the fermentation pH using lactic acid, GABA production was further increased to 30.03 g/L. During the co-fermentation phase, S. cerevisiae metabolizes acids to reduce the microenvironmental pH, thereby activating glutamate decarboxylase (GAD) and utilizing nucleotide metabolism to provide precursor substances for the synthesis of pyridoxal phosphate (PLP) coenzymes. L. brevis maintained nitrogen balance through glutamine and γ-glutamyl dipeptide metabolism, stabilizing GAD activity and promoting GABA synthesis. Our results clarify the optimal strategy for three-strain sequential fermentation, elucidate the GABA enrichment mechanisms, and develop a novel low-alcohol, GABA-rich, medicinal and edible cereal-based fermented beverage.