Optimization Of Fermentation Conditions Of ?-aminobutyric Acid Produced By Lactobacillus Plantarum And Genome Analysis

Gamma-aminobutyric acid(GABA)is a non-protein amino acid produced by Lglutamic acid catalyzed by glutamate decarboxylase(GAD),and distributed widely in microorganisms,plants,and animals.In the case of human health benefits,GABA is an inhibitory neurotransmitter in the human brain.Moreover,GABA also plays a vital role in physiological functions,such as lowering blood pressure,antidepressant,diuretic effect,and calming effect.In addition,GABA can produce from plants,animals,and microbe,but GABA content in animals and plants is very low,and it is difficult to extract.Therefore,microorganisms are one of the best choices for GABA production because microorganisms can rapidly grow and friendly to humans.Lactic acid bacteria(LAB)are food-grade microorganisms,and also the main GABA producers to directly produce GABA-enrich foods.There are many GABAproducing species,such as Lactobacillus species,Streptococcus species,and Enterococcus.It is particularly important to identify strains to determine their genetic characteristics.In this study,we study the subsequent screening via isolation techniques and in order to identify high GABA-producing strains.In this project,68 isolatedwere identified by 16 S r RNA and specific species PCR amplification technology,including11 strains of S.thermophilus,21 strains of L.plantarum and 36 strains belong to the L.paracasei.For examination,the ability of GABA-producing L.plantarum,S.thermophilus,and L.paracasei strains via the presence of gad gene.The results showed that S.thermophilus and L.paracasei did not contain gad gene,while L.plantarum contained GAD gene.This study used a combination of paper chromatography and HPLC to analyze the amount of GABA.Finally,the strain that produces high GABA is L.plantarum LP-F1.The results showed that cultural conditions and the development of high-GABA producing strains through mutagenesis selection and breeding are the main important role in improving GABA production.The optimization of GABA production such as p H value,temperature,MSG content and PLP content were optimized.Comprehensive optimization results analysis found that p H-adjusted,temperature,MSG content,PLP content can affect the GABA production of the strain.The highest GABA yield of L.plantarum LP-F1 reached 1065 mg/L with 2% of MSG supplemented,initial p H adjusted is 6.5,the optimal temperature is 42 ?,and the optimal PLP concentration is50 ?M.Compared with before optimization(707.70 mg / L),the GABA yield increasedby 50.48%.At the same time,the co-cultured between two species of GABA-producing LAB effect to increase GABA production.In this study,we use L.plantarum and S.thermophilus for co-cultured and optimization of the inoculation ratio.The results showed that the symbiosis had no direct effect on the GABA yield.On the other hand,the whole-genome sequencing of high GABA-producing L.plantarum LP-F1 genome was carried out,and the GABA synthesis pathway of this strain was analyzed.At the same time,the metabolic pathways of folic acid,riboflavin,and glutamate were analyzed via the bioinformatics analysis method,and the comparative genomic analysis was conducted by 15 strains including L.plantarum WCFS1,JDM1,ST-III,and J26 by searched from the NCBI database.It aims to fully understand the characteristics of L.plantarum LP-F1 and the experimental foundation for its future industrial application.The analysis found that L.plantarum LP-F1 contains a glutamate decarboxylase gene gad and most of the key genes of glutamate metabolism,so it has the biosynthesis ability of GABA.The bioinformatics analysis result is consistent with the results of GABA evaluation of strain.Furthermore,it contains the complete riboflavin metabolism and folate metabolism related key genes.Therefore,it was speculated that this strain can synthesize riboflavin and folic acid.Comparative analysis between L.plantarum LP-F1 and reference strain,all reference strains contain the gad gene,so the reference strains can synthesize GABA.L.plantarum 16,ST-III,and CAUH2 strains have key genes for the complete synthesis of folic acid and riboflavin,so the above strains can synthesize folate and riboflavin.However,other reference strains lack related genes or contain pseudogenes,so they cannot synthesize folic acid and riboflavin at the same time.