Probiotic Properties and Optimization of Gamma-Aminobutyric Acid Production by Lactiplantibacillus plantarum FBT215

Abstract

Gamma-aminobutyric acid (GABA) improves various physiological illnesses, including diabetes, hypertension, depression, memory lapse, and insomnia in humans. Therefore, interest in the commercial production of GABA is steadily increasing. Lactic acid bacteria (LAB) have widely been reported as a GABA producer and are safe for human consumption. In this study, GABA-producing LAB were preliminarily identified and quantified via GABase assay. The acid and bile tolerance of the L. plantarum FBT215 strain were evaluated. The one-factor-at-a-time (OFAT) strategy was applied to determine the optimal conditions for GABA production using HPLC. Response surface methodology (RSM) with Box-Behnken design was used to predict the optimum GABA production. The strain FBT215 was shown to be acid and bile tolerant. The optimization of GABA production via the OFAT strategy resulted in an average GABA concentration of 1688.65 ± 14.29 μg/ml, while it was 1812.16 ± 23.16 μg/ml when RSM was applied. In conclusion, this study provides the optimum culture conditions for GABA production by the strain FBT215 and indicates that L. plantarum FBT215 is potentially promising for commercial functional probiotics with health claims.

Keywords: Gamma-aminobutyric acid; Lactiplantibacillus plantarum; one-factor-at-a-time strategy; optimization; probiotic properties; response surface methodology.

Fig. 1. The amount of GABA produced by LAB isolates cultured in modified-MRS broth supplemented with 50 mM MSG at 37°C for 48 h.

The GABA concentration was quantified using the GABase assay. GABA, gammaaminobutyric acid; LAB, lactic acid bacteria; MSG, monosodium glutamate.

Fig. 2. Probiotic properties of Lactiplantibacillus plantarum FBT215.

(A) Acid tolerance of L. plantarum FBT215 at pH 2.5, 3.0, and 6.0 (control). The cell viability was evaluated every hour for 2 h on an MRS agar plate. (B) Bile tolerance of L. plantarum FBT215. The cell viability was calculated every 3 h for 6 h on an MRS agar plate. (C) Gel electrophoresis of sortase A coding gene. The amplicon was visualized using 1.2% (w/v) agarose gel. Lane M, molecular mass marker (Thermo Fisher Scientific, SM0311); 1, sortase A amplicon.

Fig. 3. The concentration of GABA produced by Lactiplantibacillus plantarum FBT215 in modified-MRS broth.

(A) The optimal temperature was determined by evaluating the GABA production at 25°C, 30°C, 37°C, 40°C, and 45°C in MRS broth supplemented with 50 mM MSG. (B) The optimal pH was determined by investigating the GABA production, at different pH conditions (pH 3.5-9.5), at 37°C in MRS broth supplemented with 50 mM MSG (C). The optimal incubation time was investigated for 96 h in MRS broth supplemented with 50 mM MSG at 37°C. (D) The optimum carbon source among nine different carbon sources (glucose, fructose, sucrose, lactose, galactose, xylose, mannose, mannitol, and lactulose) was determined by culturing in MRS broth supplemented with 50 mM MSG, at 37°C and pH 7.5, for 72 h. (E) The optimal fructose concentration in the range of 1−5% was investigated by culturing in MRS broth supplemented with 50 mM MSG, at 37°C and pH 7.5, for 72 h. (F) The optimum nitrogen source among seven different nitrogen sources (control, peptone, tryptone, soytone, proteose-peptone No. 3, malt extract, and beef extract) was determined by culturing in MRS broth supplemented with 50 mM MSG, at 37°C and pH 7.5, for 72 h. (G) The optimal tryptone concentration in the range of 1−5% was determined by culturing in MRS broth supplemented with 50 mM MSG, at 37°C and pH 7.5, for 72 h. (H) The optimal fructose-containing polymers among three different sources (Fructooligosaccharides, raffinose, and inulin) were determined by culturing in MRS broth supplemented with 50 mM MSG, at 37°C and pH 7.5, for 72 h. (I) The effect of MSG supplementation was evaluated in the range of 25-250 mM by culturing in MRS broth at 37°C and pH 7.5 for 72 h. (J) The effect of PLP supplementation was evaluated in the range of 0–50 mM by culturing in MRS broth supplemented with 50 mM MSG, at 37°C and pH 7.5, for 72 h. MSG, monosodium glutamate; PLP, pyridoxal 5’-phosphate.

Fig. 4. Three-dimensional response surface plots illustrating the effect of each variable on GABA production by L. plantarum FBT215.

(A) GABA concentration by the interaction between fructose concentration and tryptone concentration. (B) GABA concentration by the interaction between fructose concentration and initial pH. (C) GABA concentration by the interaction between tryptone concentration and initial pH.