This study aimed to investigate the Gamma-aminobutyric acid (GABA) production potential of Levilactobacillus brevis PML1 using an industrial waste-based culture medium. The optimization of GABA production was carried out in Spent Coffee Grounds (SCG) medium employing Response Surface Methodology (RSM). A Central Composite Design (CCD) was employed, revealing that the optimal SCG treatment conditions were as follows: 6% H2SO4 concentration, 120 min incubation, 0.14 mL Accellerase-1000 enzyme. High-performance chromatography analysis demonstrated a GABA yield of 492 mg/L after 48 h at 37 °C when supplemented with 3% monosodium glutamate. The antimicrobial activity assessment exhibited sensitivity towards Staphylococcus aureus among tested pathogenic bacteria. Scanning electron microscopy elucidated the antibacterial mechanism on Escherichia coli and S. aureus as representative strains. Furthermore, radical inhibitory activities were observed through DPPH assay recording a maximum inhibition rate of 13.15%, while FRAP assay indicated an antioxidant capacity of up to 0.419 mmol/L. Also, L. brevis demonstrated 53.40% cholesterol removal capability. Cytotoxic effects on human colon cancer cell lines Caco-2 were examined using MTT assay, yielding an IC50 value of approximately 3.25 g/mL for the extracts. In conclusion, this study highlights that SCG can serve as an affordable culture medium for efficient metabolite production.

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