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A total of 50 lactic acid bacteria (LAB) isolates from Iranian traditional dairy products (Motal and Lighvan cheeses, and artisanal yogurt) were screened for gamma-aminobutyric acid (GABA) production. Firstly, a rapid colorimetric test was performed to evaluate the glutamate decarboxylase (GAD) activity among the LAB isolates examined. Thin layer chromatography (TLC) was then performed on selected strains to identify isolates with high/moderate GABA producing capacity, and a GABase micro-titer plate assay was employed to quantify GABA. Finally, two Lactococcus (Lac.) lactis strains were selected for GABA production optimization via Response Surface Methodology (RSM) following Central Composite Design (CCD). Forty-one out of the 50 isolates showed GAD activity according to the colorimetric assay. Eight isolates displayed strong GAD activity, while nine showed no activity; low to moderate GAD activity was scored for all other isolates. GABA production was confirmed by TLC in all isolates with high GAD activity and in four selected among isoaltes with moderate activity. Among the Lactococcus strains tested, Lac. lactis 311 and Lac. lactis 491 were the strongest GABA producers with amounts of 3.3 and 1.26 mM, respectively. These two strains were subjected to GABA production optimization applying RSM and CCD on three key variables: Monosodium glutamate concentration (MSG) (between 25 and 150 mM), incubation temperature (between 25 and 37 °C), and pH (between 4.0 and 5.0). Optimal conditions for GABA production by Lac. lactis 311 and Lac. lactis 491 of temperature, pH and MSG concentration were, respectively, 35.4 and 30 °C, pH 4.5 and 4.6, and MSG concentration of 89 and 147.4 mM, respectively. Under the above conditions, the amount of GABA produced by Lac. lactis 311 and Lac. lactis 491 was 0.395 and 0.179 mg/mL, respectively. These strains and the optimal culture conditions determined in this study could be used for the biotechnological production of GABA or applied in food fermentations for the development of naturally GABA-enriched foods.

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