Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter recognized for its antihypertensive, anxiolytic, neuroprotective, and cognitive-enhancing properties. Functional foods enriched with naturally produced GABA have emerged as promising dietary interventions to modulate the microbiota-gut-brain axis and promote mental well-being. In the current study, a novel GABA-enriched plant-based almond yogurt was developed using the psychobiotic strain Enterococcus faecalis RIT BMS15-3, a human breast milk-derived isolate, as the starter culture. To the best of our knowledge, this is the first report highlighting the use of a human breast milk-derived psychobiotic Enterococcus faecalis RIT BMS15-3 to produce GABA-enriched almond yogurt. The fermentation parameters were systematically optimized to obtain maximum GABA enrichment while ensuring microbial viability and product quality. Unlike previous studies that primarily optimize plant-based yogurts for texture or antioxidant properties, this study focused on maximizing microbial GABA biosynthesis by optimizing L-monosodium glutamate (L-MSG) concentration, fermentation temperature, and fermentation time. The confirmatory experiment resulted in a GABA concentration of 29 mM in the final yogurt developed almond yogurt exhibited high antioxidant activity in DPPH and ABTS assays, with total phenolic and flavonoid contents showing higher activity than those of commercially available dairy and non-dairy yogurt. We optimized the fermentation conditions to improve GABA yield, including L-monosodium glutamate (L-MSG) concentration, fermentation time, and fermentation temperature. The results indicated a GABA yield of 29 mM in the yogurt after optimization. The almond yogurt showed a suitable water-holding capacity, viscosity, and favorable sensory acceptability (overall score: 7.52), as well as functional stability during refrigerated storage. A 21-day storage study showed sustained GABA retention together with maintenance of viable probiotic populations, indicating good functional stability of the developed product. This study emphasizes the feasibility of developing a naturally GABA-enriched, probiotic almond yogurt as a potential next-generation functional food with prospective applications in microbiota-gut-brain axis modulation. Although the observed functional characteristics suggest promise for supporting neurological health, further in vivo and clinical studies are required to establish anti-anxiety and neuroprotective efficacy.
