In vitro identification of GABA-producing psychobiotic candidates from human breast milk

Human breastmilk is a rich source of beneficial microbiota and bioactive compounds, yet its potential as a reservoir of psychobiotics remains underexplored. In this study, breastmilk samples were aseptically collected from healthy lactating mothers and cultured using MRS media. A total of 188 bacterial isolates were screened for gamma-aminobutyric acid (GABA) production using monosodium glutamate (MSG) as a substrate, with quantification by High-Performance Liquid Chromatography (HPLC). Several isolates, mainly Gram-positive cocci of the Enterococcus genus, showed GABA production ranging from 0.3343 to 0.8471 g/L. The most potent isolate identified as Enterococcus faecalis RIT BM S15-3 through 16S rRNA sequencing, exhibited notable resistance to gastrointestinal conditions and antimicrobial activity. Further, fermentative GABA production was optimized by adjusting pH, temperature, and MSG concentration. These findings highlight breastmilk as a promising source of GABA-producing bacteria with potential psychobiotic relevance. However, comprehensive strain-specific safety and in vivo evaluations are required before considering applications in functional foods or microbiota-based strategies targeting the gut–brain axis.