Multifactorial Neuroprotective Potential of γ-Aminobutyric acid-Producing Potential Probiotics Against Stress Induced Neuronal Cell Lines.

Gamma-aminobutyric acid (GABA) is associated with depressive-like behaviours and influences the gut-brain axis through vagal nerve-dependent mechanisms. Studies indicate that depression can be reversed with GABA administration, but the neuroprotective potential of GABA-producing bacteria remains underexplored. Subtractive screening identified five potential GABA-producing strains out of 95 distinct isolates, which were subsequently confirmed using thin-layer chromatography, NMR, and mass spectrometry. All five strains survived in acidic conditions, with survivability rates of 72-84%, and in 0.3% bile, with survivability rates of 93-96%. The selected strains showed higher hydrophobicity to chloroform, possessed free radical scavenging activities, and were capable of forming stronger biofilms. This study identifies Limosilactobacillus fermentum PB02 and Levilactobacillus brevis MM as potential GABA-producing probiotics. The neuroprotective effects of GABA-containing cell-free supernatant (CFS) were tested in stress-challenged Human Neuroblastoma SH-SY5Y cell lines. The CFS supplementation increased cell viability after exposure to high glucose and lipopolysaccharide, and improved viability in a glutamate-induced excitotoxicity assay, indicating the multifactorial in vitro neuroprotective potential. This study enhanced the viability of cells by 20-25% viability in cells challenged with 250mM glucose and 1 µg/mL LPS, and in the glutamate-mediated excitotoxicity assay. Additionally, L. fermentum PB02 and L. brevis MM emerged as promising psychobiotic candidates with neuroprotective effects, as indicated by Principal Component Analysis and weighted scoring matrix analysis. By integrating clustering analysis, we demonstrate that isolated strains can be promising candidates showing neuroprotective potential using in vitro methods. Further in vivo validation will be required to confirm these effects.