Since the cost of culture medium for the production of microbial metabolites is a very important factor in industrial processes, the use of cheaper resources and improving bacterial metabolites’ efficiency by optimizing fermentation conditions are criti- cal. In order to optimize γ-aminobutyric acid (GABA) production, three levels of culture medium (molasses, dairy sludge, and soybean meal), with three bacteria (Lactobacillus brevis PML1, Lactobacillus fermentum 4–17, and Lactobacillus plantarum 1058), in three fermentation times (24, 48, and 72 h) were used. After fermentation, the presence of GABA in the culture medium was investigated by Fourier transform infrared (FTIR), thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC). Also, cytotoxicity, antimicrobial, antioxidant, and biogenic amino acid tests were performed on the fermented extract. The results of statistical design and GABA analysis showed that the optimal treatment included culture medium containing 29.27% dairy sludge, 24.77% molasses, and 10.49% soybean meal for 48 h fermented by L. fermentum 4–17, and under these production conditions, GABA concentration was 359.45 ppm. The highest antimicrobial activity of the fermented extract was against Staphylococcus aureus. The highest level of DPPH radical inhibitory activity was about 78%, and the IC50 level of toxicity on Caco-2 cancer cell line was 41.2 mg/mL. The results showed that none of the studied biogenic amines was detected in culture medium. According to the results, these compounds can be used as an inexpensive substrate to produce valuable medicinal and bioactive compound GABA biologically.

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