High-Throughput Selecting And Breeding Lactic Acid Bacteria Producing γ-Aminobutyric Acid

γ-Aminobutyric Acid(GABA) has a variety of physiological activity function, microbial fermentation of GABA in Lactic Acid Bacteria has extensive application prospect. In this paper, in order to quickly and efficiengtly determine the content of GABA, microplate reader is used and the main influencing factors of the method is optimized based on the Berthelot reaction. The optimum condition for the determination of GABA is:0.75mL 0.2mol/L pH10.0 borate buffer,2.5 mL 6% of redistilled phenol,2mL 5.2% available chlorine NaClO solution. In this way, microplate reader can produce higher concentration to detecte GABA, and the precision test is implemented simultaneously. Finally, in order to verify the feasibility of this method,75 mutant strains are fermented by 96-well plates and then are measured by spectrophotometer and microplate reader respectively. The data regression show a significant relationship between two methods, as the correlation is 0.9493. The experimental results show that microplate reader can simply, rapidly and accurately measure the content of GABA. It also lays the foundation for future high-throughput selecting and breeding high-yield GABA from lactic acid bacteria.In this study, the original strain was HX-3-6 and the optimum mutagenesis time was 120s, in order to establish an preliminary screening mechanism, the gradient plate was used to screen mutant strains induced by ARTP. Thepreliminary screening mechanism was that the mutant strains with high productivity may appear in the high concentration of GABA and had a relatively big size. After preliminary screening, the 96-well plateswas used for the further fermentation.In order to verify the correlation between 96-well plates fermation and triangle flask fermation, 50 mutant strains are fermented by 96-well plates and the triangle flask respectively, it was concluded that the correlation coefficient of two methods R2=0.9931, proved that 96-well plates can be used to high-throughput screen the GABA produced by Enterococcus lactic acid bacteria.So, in accordance with the preliminary screening methods, the HX-3-6was induced by ARTP and fermented by 96-well plates. Finally, a mutant strains of L-120-1 was got, the production of GABA can reach 6.322 g/L, which increased 22.49% compared with the GABA production of initial strain HX-3-6.The L-120-1 was proved to have a good genetic stability.In this study, the condition of fermentation on well plates was optimized. First,48-well plates was compared with 96-well plates, which was used for the rest of the fermentation, Then, the single factors of the fermentation condition such as liquid volume in well plates, inoculation amount, carbon source, nitrogen source, L-Glu were determined. Then, on the basis of single factor, the biggest impact factors of the glucose, corn steep liquor, L-Glu and the fermentation time were obtained by the response surface optimization and the key factors of maximum response area were gained by the steepest grade test. Finally, the key factors were further optimizied by the Box-Behnken experiment design.Through response surface optimization, four significant factors--glucose, corn steep liquor, L-Glu and the fermentation time were 8.0g/L,9.2g/L,3.6%,70h. In this condition, the yield of y-aminobutyric acid could reach 12.84g/L, which increased 6.52g/L compared with the yield of the original medium.Then, the fermentation medium and fermentation conditions of GABA produced by Enterococcus lactic acid bacteria on triangle flask was optimized. Firstly, the single factors of the fermentation condition such as liquid volume in flask, inoculation amount, carbon source, nitrogen source, L-Glu were determined. Then, on the basis of single factor, the biggest impact factors of the glucose, corn steep liquor, inoculation amount and the fermentation time were obtained by the response surface optimization and the key factors of maximum response area were gained by the steepest grade test. Finally, the key factors were further optimizied by the Box-Behnken experiment design.Through response surface optimization, four significant factors--glucose, corn steep liquor, inoculation amount and the fermentation time were 22.0g/L, 15.0g/L,19%,48h. In this condition, the yield of y-aminobutyric acid could reach 12.030g/L, which increased 5.602g/L compared with the yield of the original medium.At last, the mutant strain L-120-1 was amplificatory fermented on 3 L fermentor, it was showed that pH and sodium glutamate had a greater influence on the production of GABA, therefore, the pH value of the fermentation process were controlled and the sodium glutamate was added by stages, that the accumulation of GABA can reach 19.813 g/L, which increased 57.8% compared with the fermatiom without any control.