Functional characterization and safety evaluation of GABA-Enriched <em>Prosopis africana </em>(Okpeye) fermented with <em>Bacillus subtilis</em> AQU under optimized conditions

Gamma-aminobutyric acid (GABA) is a non-protein amino acid synthesized via the α-decarboxylation of L-glutamate. This study explored the production of GABA using Bacillus subtilis AQU isolated from Okpeye and its cytotoxicity effect. Prosopis africana seeds sourced from the Ogege market in Nsukka Local Government Area, Enugu State, Nigeria, were used to produce Okpeye. The fermentation process for Okpeye production was conducted under optimized conditions using Response Surface Methodology (RSM). The isolated B. subtilis AQU were tested for probiotic properties, including low pH tolerance, bile salt tolerance, hydrophobicity, auto aggregation, antimicrobial activity, and cytotoxicity effects. The optimization of GABA production using Central Composite Design (CCD) in Minitab 21.0 software identified the optimal conditions for GABA synthesis: temperature at 40 °C, fermentation time of 177 hours, 5% MSG concentration, and 3% inoculum concentration. Under the optimized conditions, the GABA yield was experimentally validated at 310.03 mg/100 g dry Okpeye, closely matching the predicted value of 313.53 mg/100 g with 83% desirability. The probiotic potential of Bacillus subtilis AQU demonstrated strong tolerance to low pH and bile salts, moderate auto-aggregation capacity, and high cell surface hydrophobicity, collectively suggesting strong colonization potential in the gastrointestinal tract. Cytotoxicity assays performed on L-929 cells indicated that the optimized Okpeye contains B. subtilis AQU, is non-toxic, and maintains above 50% cell viability even at the highest tested concentrations. Findings of the study suggest the potential of utilizing B. subtilis AQU for efficient GABA production, enhancing the nutritional and functional properties of fermented Okpeye.