Study On Influence Factors And Metabolic Pathways Of Gamma-aminobutyric Acid In Lactobacillus Plantarum

Lactobacillus plantarum is one of the most important probiotics with excellent fermented properties and various health-promoting functions, which are widely applied in dairy products, especially in fermented dairy products. gamma-Aminobutyric acid(GABA) is an important inhibitory neurotransmitter in central nervous system of mammal. It has several well-known physiological functions, such as induction of hypotension, tranquilizing and allaying excitement, reinforcement excreting of insulin, protection of liver and kidney, preventing obesity, and so on.In the present study, firstly, single factor experiments were performed to explore factors that influence Lb. plantarum ATCC14917 to produce GABA, and then orthogonal experiment design was used to determine the significance level of the influence factors above. Secondly, fermentation system leading to significant increase GABA production was structured by taking LMSG as the single inducing factor and chemical defined medium(CDM) as fermented substrat. Finally, next-generation high throught put sequencing technology was applied to perform transcriptome analysis of Lb. plantarum ATCC14917 at the specific time point at which appears the most significantly different GABA production between L-MSG induction group and L-MSG free group. Moreover, gene quantitative analysis, gene function annotation, and metabolic pathway enrichment analysis were also carried out to probe the GABA metabolic pathways inducing by LMSG of Lb. plantarum ATCC14917.Results of the present study through the above experiments were presented as following: 1. Concentration of L-MSG, concentration of PLP, initial fermented p H value, and fermented temperature can significantly influence GABA production, but the concentration of Ca2+ exerted little effect. The result of variance significance analysis and range analysis suggested that the significant degree of these factors was p H value ? fermented temperature ? L-MSG concentration ? PLP concentration ? Ca2+ concentration. 2. The fermentation system resulting in different GABA production was showed below: the initial fermented p H value was 5.5, fermented temperature was 37 ℃, concentration of LMSG was 100 m M, inoculation amount was 3%(v/v), medium volume was 2 L. In this fermented condition, the GABA production was 721.35 m M in L-MSG induction group at 36 h, and this yield was 7.77 folds of that in L-MSG free group. 3. 87 significant difference genes induced by L-MSG were detected by high throught put transcriptome sequencing, such as phosphotransferase encoding gene pts, pyruvatedehydrogenase encoding gene pdh, glutamate decarboxylase encoding gene gad B, GABA transferase encoding gene gad T, and succinic semialdehyde dehydrogenase encoding gene gab D, etc., and among these genes, up-regulated genes were 69, and down-regulated were 18. Functions of these genes were closely related to carbohydrate transmembrane transporter activity, sugar- phosphotransferase system and transferase activity, and meanwhile, these genes also participate in various metabolic pathways, including pyruvate metabolism, pentose phosphate pathway(HMP), fatty acid synthesis, amino acid metabolism. 4. The potential molecular mechanism of Lb. plantarum ATCC14917 producing GABA induced by L-MSG was predicted by genes functional annotations and pathway enrichment analysis. ① L-MSG could induce gad B to up-regulate, which facilitated GABA synthesis; ② L-MSG could induce gab T to down-regulate, which inhibited the degradation of GABA and contributed to GABA accumulation; ③ L-MSG could induce glucose to translate into succinic acid through glycolysis and pyruvate metabolism, and the succinic acid could also inhibit the catabolic pathways of GABA; ④ L-MSG could activate the synthesis of fatty acid, which increased the membrane permeability, contributed to substrata absorption, and facilitated to cell growth and metabolism. In this study, the influencing factors for GABA production of Lb. plantarum ATCC14917 were analyzed, and the molecular mechanisms of GABA synthesis and GABA metabolic pathways were predicted by the next-generation transcriptome sequencing. This research laid an important theoretical foundation for control GABA fermentation by Lb. plantarum in the molecular level, and provide a good research idea and approach for related work.