Levilactobacillus brevis CRAI: a novel GABA-producing strain for nutraceutical applications

Gamma-aminobutyric acid (GABA) is a non-proteinogenic amino acid that functions as the principal inhibitory neurotransmitter in the central nervous system and plays a pivotal role in gut–brain axis communication. Reduced GABA levels have been associated with disorders such as irritable bowel syndrome (IBS) and depressive symptoms. Although GABA is employed to mitigate stress and anxiety, conventional production methods are expensive and causes pollution. Certain lactic acid bacteria (LAB) can naturally synthesize GABA during fermentation, offering a more sustainable alternative. In this study different microbial strains were isolated from organic tomatoes. Thirteen LAB strains were isolated and identified by 16S rRNA gene sequencing; PCR screening targeted the gadB gene encoding glutamate decarboxylase. Among the isolates, Levilactobacillus brevis CRAI exhibited the highest production (179 mM GABA). Whole genome sequencing confirmed the absence of virulence and antibiotic resistance genes. Genomic analysis identified two GABA-related genes, gadA and gadB, whose transcription was significantly upregulated in the presence of monosodium glutamate, correlating with enhanced GABA biosynthesis. L. brevis CRAI demonstrated antimicrobial activity, anti-inflammatory effects, and the ability to adhere to intestinal epithelial cells. Fermentation trials using agro-industrial by-products identified tomato pomace as the most suitable substrate that supports L. brevis CRAI growth. Process optimization enabled production of 93 mM GABA on the laboratory scale and 80 mM on the pilot scale. The fermented product was freeze-dried to obtain a GABA-enriched nutraceutical prototype. In vitro gut fermentation models simulating healthy and irritable bowel syndrome (IBS) conditions, following treatment with the strain or GABA-enriched nutraceuticals, showed increased short-chain fatty acid production and partial restoration of microbial balance, including enrichment of beneficial taxa such as Bifidobacteriaceae under dysbiotic conditions. Overall, L. brevis CRAI represents a safe, efficient, GABA-producing probiotic with potential for modulating gut–brain axis dysfunction in IBS.