Screening Of High Yield ?-Aminobutyric Acid(GABA) Lactic Acid Bacteria And Optimization Of Fermentation Conditions

?-aminobutyric acid(GABA)is an important inhibitory neurotransmitter in the mammalian nervous system,which has many physiological functions,such as calming nerves,improving sleep,regulating blood pressure and enhancing the function of liver and kidney.It is one of the important raw materials of new resource food in the 21st century.At present,the main method of synthesizing GABA is microbial fermentation.In order to obtain food-grade GABA,this study is devoted to selecting high yield GABA Lactic acid bacteria with good biological characteristics and high safety.Then it was mutated and bred by the atmospheric and room temperature plasma(ARTP),and its culture medium and fermentation conditions were optimized.At the same time,the optimum conditions for whole-cell catalytic synthesis of GABA using sodium glutamate(sodium hydrogen glutamate,L-MSG)were explored.Finally,a functional Lactic acid bacteria beverage rich in GABA was developed according to the natural fermentation broth of the strain.The main results were as follows:(1)Eighteen Lactic acid bacteria were screened from traditional fermented dairy products in Aksu area,Xinjiang and Kyrgyzstan,which were belonged to Enterococcus,Lactiplantibacillus and Streptococcus through 16s r RNA molecular biological identification.The results of biological characteristics analysis showed that the 8 strains can tolerate 0.5%bile salt,more than 5%Na Cl and high temperature.The functional characteristics of strains displayed peptonization of milk,the antioxidant,enzyme activity and inhibition of bacteria.The safety analysis showed that all strains had no hemolysis,did not produce nitroreductase and showed different drug sensitivity to 10common antibiotics.No strains did use amino acids to produce biogenic amines,but Z6-4 and Z6-11.(2)A strain of Lactoplantibacillus pentosus Z6 with GABA yield of 1.94 g/L was selected from the tested strains through thin layer chromatography and Berthelot colorimetry.In order to further improve the ability of GABA production,ARTP mutation breeding was carried out with helium as working gas,power supply 120 W,radio frequency power 13 W,spacing 2 mm,gas flow 10 L/min,and treatment time 80 s.Finally,a high-yield GABA mutant Z6-15 with stable genetic characters was obtained,and its GABA yield was 2.84g/L,which was 46.39%higher than before.(3)The medium components and fermentation culture conditions of strain Z6-15 were optimized by single factor test and response surface test design.The results showed that glucose was the best carbon source and peptone and yeast powder were the best nitrogen sources for biomass and GABA production.On this basis,the fermentation and culture conditions were optimized through Box-Behnken's central combination design and response surface analysis,and the best optimal fermentation conditions were as follows:glucose 20 g/L,yeast 10 g/L,peptone11.5 g/L,L-MSG 4.5 g/L,K₂HPO₄·7H₂O 2 g/L,anhydrous sodium acetate 5 g/L,triammonium citrate 2 g/L,Mn SO₄·4H₂O 0.05 g/L,Mg SO₄·7H₂O 0.2 g/L,Tween-80 0.1%,p H 5.5,inoculum4%,dosage 175/500 m L,37?,160 r/min,fermentation time 3 d.Under these conditions,the GABA yield of strain Z6-15 was 3.96 g/L,which was 39.63%higher than before.(4)To explore the ability of whole cell catalytic synthesis of GABA by strain Z6-15,and optimize the conditions of whole cell catalytic synthesis of GABA.The results showed that the optimum reaction conditions were:0.2 M acetic acid sodium acetate buffer system,37?,p H 5.0,V_B6 10 m M,L-MSG 5 g/L,cell concentration 6 g/L(wet weight),and the yield of GABA was1.41 g/L after 24 h reaction.At this time,the molar conversion rate of GABA to L-MSG was51.18%,and the highest production intensity was 0.09 g/(L·h).On the other hand,the effect of ultrasonic crushing treatment on cell catalytic synthesis of GABA was discussed.It was determined that under the optimal conditions of ultrasonic treatment of 40 k Hz and 150 W for 30min,the yield of GABA was 2.58 g/L,and the molar conversion was 93.48%,which was 82.98%and 82.65%higher than that of the untreated group,respectively.Meanwhile,the highest production intensity was 0.15 g/(L·h).(5)The fermentation broth of Lactiplantibacillus pentosus Z6 with high edible safety and higher GABA yield obtained in early was used as the substrate to develop the GABA functional Lactic acid bacteria beverage,and the sensory quality was judged based on the fuzzy mathematics comprehensive evaluation model.The results showed that the best formula of the beverage was as follows:Lactic acid bacteria fermentation broth as the base,6%honey,0.35%calcium carbonate,and 0.01%aspartame.The beverage obtained at this time had a uniform color,sweet and sour,and rich in lactic acid.Aroma,sensory score was the highest 8.52 points.On this basis,agar 0.10%,xanthan gum 0.20%and CMC-Na 0.15%were selected as the composite stabilizers of the beverage.At this time,the centrifugal precipitation rate and stability efficiency of the beverage were 1.96%and 67.81%,respectively.The results of test and analysis showed that the content of GABA in the beverage was 1.98g/L,which was rich in 17 kinds of amino acids such as glutamic acid,glycine and aspartic acid.At the same time,the beverage had good storage at room temperature.