Mutagenesis Breeding Of Enterococcus Faecalis Producing Gaba And Optimization Of Fermentation Conditions

Gamma-aminobutyric acid(GABA)is a natural non-protein amino acid that is widely distributed among various animals,plants and microorganisms.GABA has important physiological functions such as lowering blood sugar,lowering blood pressure,and anti-depression.At present,it is mainly used as a functional food and has a broad prospect in the market.In this study,in order to improve the screening efficiency of mutant strains of GABA after mutagenesis,a paper chromatography-microplate reader method was established first to determine the yield of y-aminobutyric acid in microplate fermentation broth.Then,the high-yield mutant EM05 of GABA was obtained by seven rounds of ARTP mutagenesis and two rounds of adaptive evolution by microbial microdroplet culture system(MMC).Next,by analyzing the fermentation curve and kinetic parameters at different rotational speeds,a two-stage rotational speed regulation strategy was proposed,and the fermentation conditions that affected this strategy were optimized.Finally,the fermentation medium was optimized by single-factor experiment and orthogonal design to increase the fermentation production of the high-yield strainEM05.The main research contents and results are as follows:(1)Determination of GABA in fermentation broth by paper chromatography-microplate reader.Important factors that affected the performance of paper chromatography-microplate reader method were optimized,and a corresponding spectrophotometric method was established.The results showed that the optimized chromatography solution contained 8 g/L ninhydrin,and n-butanol,glacial acetic acid and water were in a volume ratio of 2:1:1.Moreover,the optimized eluent had 75%(v/v)ethanol,and the volume ratio of ethanol to 6 g/L pentahydrate copper sulfate was 39:1.It was found that the absorbance and GABA concentrations showed a good linearity(R2=0.9990)within 1?7 g/L.The average recovery rate of the method was 98.249%,and the RSD was 2.6255%.(2)Mutagenesis breeding of Enterococcus faecalis producing GABA and adaptive evolution.Enterococcus faecalis which producing GABA were mutated by the novel ARTP breeding technique,and MMC was used to adaptively evolve the mutant strains that after primary screening of plate resistance and the re-screening of well-plates.The results showed that after seven rounds of ARTP iterative mutagenesis,resistance plate preliminary screening,and well plate re-screening,the mutant strain En1203 was finally obtained with a yield of 7.246 g/L,which was an increase of 104.7%compared with the original strain L-120-1.The En1203 mutant was subjected to environmental stress with different concentrations of the substrate MSG.After two adaptive evolution experiments by MMC,a high-yielding strain of EM05 was obtained.At a substrate MSG concentration of 100 g/L,the GABA yield of EM05reached 28.28 g/L.After five consecutive generations of genetic stability investigations,the high-yield strain of EM05 has less fluctuations in GABA-producing ability and has better genetic performance.(3)Effects of two-stage rotational speed control on GABA production by Enterococcus faecalis.By analyzing the fermentation curve and kinetic parameters of En1202 at different rotational speeds(0 and 100 r/min),a two-stage rotational speed regulation strategy was proposed,and the fermentation conditions that affected this strategy were optimized.The results showed that the yield at 72 h and 0 r/min was the highest,and the biomass at 100 r/min in stable periods was the highest.The optimized fermentation conditions were as follows:the liquid volume was 100 mL,rotational speed was 150 r/min at the first stage of fermentation(0-12h),then switched to 0 r/min at the second stage(12-72h).On this basis,the production of GABA reached(6.713 ± 0.135)g/L,which was 12.2%and 60.1%higher than that before optimization at 0 and 100 r/min,respectively.(4)Optimization of fermentation medium for Enterococcus faecalis producing GABA.The single-factor experiment and orthogonal design were used to optimize the concentration ratio of the mixed substrates(L-glutamic acid and sodium L-glutamate),the concentration of carbon and nitrogen sources,the concentration of sodium succinate,and finally validated in a 3L bioreactor.The results showed that under the optimal fermentation medium(glucose 15 g/L,tryptone 25 g/L,sodium succinate 8 g/L,L-glutamic acid 70 g/L,MSG 30 g/L),the yield of GABA synthesized by EM05 was 60.658 g/L,which is 114.49%higher than before optimization(the substrate addition amount of MSG is 100 g/L).Finally,after verification by a 3L bioreactor,the yield of GABA synthesized by EM05 can reach 64.233 g/L.