Screening Of GABA-Producing Lactobacillus Sp. And Optimization Of The Fermentation Conditions

γ-Aminobutyrc acid (GABA) is a non-protein amino acid that is widely occuredin nature. GABA is a major inhibitory neurotransmitter in the central nervous systerm.GABA has very important physiological functions, such as neurotransmissionmediator, depression of hypertension, diuretic effects, and tranquilizer effects etc. Itmakes GABA an important functional factor in food and medicine. In the presentstudy, a strain of Lactobacillus sp. that produce GABA was screened, and itsfermentation process were studied. The main results were as follows:1. A HPLC method for analysis GABA in the fermenting liquor was established.The GABA was extracted from fermented broth, and derivatized to OPA-GABA withOPA; the latter was subsequently separated with C₁₈separation column and detectedwith UV absorbance detector at 338 nm. Under the conditions described above, theHPLC separation gave a well-resolved and symmetrical OPA-GABA peak. Theretention times was 6.63 min and reproducible. Peak area values showed goodreproducibility with relative standard deviation (RSD) values at 1.7725ï¼…. Thecalibration curve for OPA-GABA was calculated by linear least-squares squaresregression. The regression equation wasy=2.2203x-0.0428(R²=0.9996) when theinitial GABA concentration in the range 0～3.000 g/L. The result indicated that themethod described was specific and useful for the determination of GABA in thefermenting liquor.2. A GABA-producing strain of Lactobacillus sp. L139 was isolated andscreened from the traditional .fermented food. 4.35 g/L of GABA with a substrateconversion of 78.94ï¼…was produced by the initial strain L139 after fermentation for72 hours under the conditions without being optimized.3. The culture medium and conditions of L139 for GABA production wereinvestigated and optimized with one-factor-at-a-time experimental design andresponse surface methodology. The MRS-based fermentation medium forγ-aminobutyric acid (GABA) production was optimized using SAS softwarecombined with the Plackett-Burman design and response surface analysis method. Firstly, Plackett-Burman design was used to evaluate the influence of related factors.The results showed that glucose, C/N ratio and MnSO₄·H₂O were the main affectingfactors while the other additives had no significant effect on the GABA production.Then, the Box-Behnken design and response surface analysis were used to determinethe optimal levels of the main factors. The results indicated that the optimalcomponents of medium for GABA production was as follows: glucose,25.4 g/L; yeastextract,6.2g/L;. peptone,6.2g/L; Tween 80,2mL/L; ethonal, 4mL/L; MgSO₄·7H₂O,0.2g/L; MnSO₄·H₂O,0.045 g/L; L-MSG,12.5 g/L. Based on the above optimumcondition, the effects of temperature, pH, oxygen supply and the concentrations ofMSG were investigated. As a result, the optimal concentrations of MSG, pH,temperature was 20 g/L, 5.0, 34℃respectively. The yield of GABA afteroptimization reached 10.21 g/L that was increased by 1.35-fold in comparison tonon-optimization, The conversion, rate was 92.64ï¼….4. According to the optimal conditions, the batch fermentation was carried out in5 L fermentor to demonstrate the result of optimization. The substrate was mostlytransformed to GABA after fermentation for 80 hours; the yield of GABA was 9.86g/L and the conversion rate of substrate was 89.46ï¼….5. Based on the optimal conditions, the fed-batch fermentation was performedwith strain L139 in 5. L fermentor. When the fermentation was conducted withagitation at 100 r/min and without air addition for nearly 40 hours, the glucose andL-MSG were added into the medium. The addition of glucose and L-MSG couldresult in accumulation of GABA to reach as high as 32 g/L with a substrateconversion of 72.59ï¼…after fermentation for 120 hours.