Screening Of Lactic Acid Bacteria Producing ?-aminobutyric Acid And Optimization Of Culture Conditions

?-Aminobutyric acid(GABA)is widely present in mammals.Because of its various physiological functions,GABA is added as an active substance in various foods.Screening lactic acid bacteria with high GABA production from nature has become a research hotspot in recent years.After primary screening,low temperature acclimation and rescreening of fermentation characteristics,5 strains of lactic acid bacteria with normal growth at 20?,excellent fermentation characteristics and good organic acid production and antibacterial ability were obtained from traditional fermented meat products in pastoral areas.The morphological observation and molecular biological identification of 5 strains of lactic acid bacteria were carried out.The 5 strains of lactic acid bacteria were identified as Lactobacillus plantarum,numbered CM25,CM41,CM44,CM46 and CM48.High performance liquid chromatography(HPLC)was used to determine the GABA production ability of 5 lactic acid bacteria.The results showed that the average GABA production of 5 lactic acid bacteria was 0.818±0.15 g/L in MRS medium containing L-glutamic acid for 24h,and the highest GABA production was 1.21±0.10g/L in strain CM25.Subsequently,the glutamate decarboxylase(GAD)gene of the strain was sequenced and the expressed proteins were analyzed by bioinformatics.The results showed that the molecular formula of the protein was C₁₉₇₄H₂₉₉₂N₅₂₄O₅₆₂S₂₀,and the molecular weight was 43697.85,which belonged to the aspartate aminotransferase(AAT)superfamily(fold I)pyridoxal phosphate(PLP)dependent enzyme.With CM25 as the test strain,the culture conditions and medium components were optimized by single factor test and GAD gene expression verification.The optimal conditions were obtained as follows:initial pH 5.20,inoculation amount 5.2%,culture temperature 30?,substrate L-glutamate addition amount 20g/L,PLP 0.4g/L,Mg²⁺0.6mg/L,Ca²⁺0.65mg/L,and Mn²⁺0.8mg/L.The GABA yield of CM25 was 2.68±0.06g/L,which was 121.49%higher than that of 1.21±0.10g/L before optimization.The relative expression of GAD gene was 8.43±0.07,which was 8.43 times higher than 1.00±0.00before optimization.