Study On Preparation Of ?-aminobutyric Acid By Biotransformation Of Probiotics

y-Aminobutyric acid(GABA)and foods have active functions to prevent hypertension and depression.High-yielding GABA strains are the key to the preparation and development of GABA foods.As GRAS microorganisms,probiotics have the ability to produce GABA,so it is necessary to screen the probiotic strains of high GABA yield and apply them to the development of GABA functional dairy products.Single factor and response surface methodology were used to optimize GABA preparation conditions of batch fermentation,whole cell transformation and immobilized cell transformation.The High-yielding GABA probiotics strain and direct vat set(DVS)was used to ferment goat milk,the goat yogurt with GABA was obtained and analyzed by metabolomics.The conclusions were as follows.1.In order to screen GABA-producing strains from 60 probiotics,sodium glutamate was used as the substrate,and three response including GABA yield,pH and OD value of culture medium were investigated.The results showed that the transformation ability of each strain was different,57 strains had certain GABA-producing capacity,with the yield between 0.019 g/L and 1.0 g/L.Among them,five strains(SP1,B43,38,355,49)were preliminarily screened out for their good transformation ability,two strains--38 and 49 with high yield of GABA were further obtained and then identified by 16S rDNA.The results of strain identification showed that strains named 38 and 49 were Lactobacillus fermentum and Lactobacillus reuteri.2.Single factor and response surface methodology were used to optimize the GAB A preparing process by batch fermentation of L.fermentum 38 and L.reuteri 49.The optimal GAB A preparing conditions of L.fermentum 38 were obtained as follows:the substrate additive amount was 55 mmol/L,the initial pH was 5.0,the inoculum size was 5.20%,after cultured at a constant temperature of 37? for 73 h,GABA yield was up to 2.767 g/L.The optimal GABA preparing conditions of L.reuteri 49 were as follows:the substrate additive amount was 55 mmol/L,initial pH was 5.0,inoculum size was 4.0%,and after cultured at a constant temperature of 34? for 87 h,GABA yield was up to 2.75 g/L,and the GABA yield was significantly improved compared with that before the optimization.3.The GABA preparing process by whole cell transformation of L.fermentum 38 and L.reuteri 49 were also optimized.The optimum conditions of L.fermentum 38 were as follow:the sodium glutamate concentration of 10.26 g/L,the bacteria concentration of 10 g/L,the pH of 4.80,and the GABA yield was 3.69 g/L In the buffer solution of disodium hydrogen phosphate citric acid,it was transformed at 42? for 15 h The optimal transformation conditions of L.reuteri 49 were:the substrate concentration of 7.28 g/L,bacteria concentration of 10g/L,after conversion at pH 4.6 and 38? for 15 h,the GABA yield was 4.05 g/L.A regression model was established for the preparation of GABA by transformation of these two strains.There was no significant difference between the verified value and the predicted value,which indicated that it was feasible to optimize the preparation of GABA by whole-cell transformation using the response surface method,and the whole-cell transformation method was superior to the batch fermentation method.4.The single-factor and Box-Behnken design were used to optimize the GABA preparing process by immobilized cell transformation of L.fermentum 38 and L.reuteri 49.The results showed that the optimal conditions of L.fermentum 38 at 15 hours were:substrate concentration of 15.24 g/L,cell concentration of 13 g/L,and conversion at 40? in pH 5.0 disodium hydrogen phosphate citrate buffer.Its GABA yield was 2.61 g/L;the optimal transformation conditions for immobilized L.reuteri 49 were:substrate concentration of 15.15 g/L,cell concentration of 15 g/L,conversion at 40? in pH 4.70 disodium hydrogen phosphate citrate buffer.The GABA yield was 2.73 g/L.The immobilized bacterial cells were transformed for a long time.Finally,when L.fermentum 38 transformed for 25 h and L.reuteri 49 transformed for 23 h,the GABA yields were 5.12 g/L and 5.24 g/L,respectively.After seven consecutive transformation tests,the enzyme activity retention rate of immobilized cells reached more than 50%.5.The effects of L.fermentum 38 and L.reuteri 49 on the preparation of GABA fermented goat milk were studied with the DVS used as the control.The results showed that both strains could promote goat milk fermentation and significantly increase GABA content.The content of GABA in goat milk fermentation with the addition of L.fermentum 38 and L.reuteri 49 was 0.32 g/L and 0.25 g/L(The control was 0.06 g/L).Four indicators including GABA content,acidity,pH,and sensory evaluation were investigated to study the storage stability of fermented goat milk.It was found that the content of GABA in fermented milk continued to increase,the acidity increased and the pH decreased,and the sensory evaluation score also occurred reduce with the increase of storage time.Metabolomics differential metabolite analysis of fermented milk found that the GABA content in fermented milk was significantly increased and in the meanwhile,the amino acids and short peptides such as leucine,phenylalanine,vitamin B6,Ile-Lys,Lys-Pro,His-Pro,Leu-Arg were also significantly increased.In this study,L.fermentum 38 and L.reuteri 49 have strong GABA transformation ability.The process of batch fermentation,whole cell transformation and immobilized cell preparation of GABA were compared,and the GABA-containing fermented goat milk was developed,which provides technical reference for the subsequent development of GABA DVS and GABA functional fermented goat milk products.