Mixed Substrate Mediated P H Self-buffering Fermentation Promotes Gaba Production By Lactobacillus Brevis And Establishment Of Related Analytic Methods

Gamma-aminobutyric acid（GABA）is a natural non-protein four-carbon amino acid with many probiotic functions,which was widely applied in food,medicine,feed and other industries,as well as a new resource food by the Ministry of Health in 2009.Given the high efficiency of GABA synthesis by fermentation and it is generally recognized as safe for lactic acid bacteria,the production of GABA by lactic acid bacteria has become a future development trend.In this paper,a sensitivity intensified ninhydrin-amino acid chromogenic system（SINACS）mediated by ethanol-ethyl acetate was established to monitor the fermentation process of GABA.Then,the mixed substrate mediated p H self-buffering fermentation promotes GABA production based on Lactobacillus brevis CD0817 was constructed,and the mechanism of GABA production was explored.Finally,an efficient and universal genome walking method for obtaining unknown sequences is proposed based on partially overlapping primers.The main results of this paper are as follows.（1）Sensitivity intensified ninhydrin-amino acid chromogenic system was constructed by single factor experiment which based on the substrate of glutamic acid and GABA,successively optimizing the ninhydrin-amino acid reaction parameters such as ethanol concentration,ethyl acetate concentration,and their volume ratio,reaction temperature,and the concentration and p H of sodium acetate buffer to obtain the optimal reaction medium and chromogenic conditions.The optimized SINACS consist of the following components:40%ethanol,25%ethyl acetate,35μL 0.2 M sodium acetate buffer（p H 5.0,final concentration 2.3 m M）and 1%ninhydrin solution.The optimal chromogenic conditions were as follows:0.1 m L of the sample was added to 2.9 m L of SINACS,and incubated at 70℃ for 30 min,cooling at room temperature for 3 min,then determining the A₅₇₀.The results showed that A₅₇₀ was stable for at least 120 min,and the sensitivity of glutamic acid and GABA were increased by 15 and 35 times,respectively.The synthesis of GABA during fermentation by L.brevis CD0817 was monitored by SINACS and HPLC these two methods,have identical synthesis trends,which could be employed for the biological processes such as amino acid-producing microorganism breeding,fermentation process optimization and monitoring.（2）Using glutamic acid-monosodium glutamate as the mixed substrate,a single factor 250 m L-shaking flask test was carried out to optimize the fermentation parameters affecting the synthesis of GABA by L.brevis CD0817.The fermentation parameters including the concentration of L-monosodium glutamate,glucose,yeast extract,MnSO₄·H₂O,and Tween-80,as well as fermentation temperature were optimized on the following shaking flask fermentation conditions:10%（v/v）of the inoculum volume,incubation at 30℃ for 48 h.According to the optimization results,the reconstituted fermentation medium（g/L）:L-monosodium glutamate 33.8,Glucose 5,Yeast Extract FM408 35,MnSO₄·H₂O 0.05,Tween-80 1,and 800 g/L of the glutamic acid was added to the fermentation broth before inoculation.Compared with single glutamic acid fermentation,mixed-substrate fermentation improved GABA synthesis efficiency by promoting early cell growth.（3）A high-efficiency mixed-substrate biotransformation system according to the above optimized shaking flask parameters was verified in a 10-L fermenter,fermentation conditions were as follows:seed liquid inoculum volume 10%（v/v）,fermentation medium 4 L,fermentation temperature 30°C,stirring speed 100 rpm,glutamic acid 3000 g,the GABA yield reached 353.1±8.3 g/L after 48 h fermentation,compared with single glutamic acid fermentation,it increased by 9.1%,and the substrate conversion rate reached 99.8%.（4）Wristwatch PCR（WW-PCR）,a genome walking method based on partially overlapping primers,was established to retrieve unknown sequences flanking the known genomes to facilitate the subsequent analysis of the mechanism of high-yield GABA fermentation with mixed substrates.Three wristwatch primers feature 5’-end（12 nt）and 3’-end（3 nt）overlap,as well as a heterologous interval（10 nt）,that is,any wristwatch primer can be annealed to the complementary site of another wristwatch primer in a low-stringency cycle to form a wristwatch-like structure,and prime DNA elongation.Each WW-PCR set is comprised composed of three nested（primary,secondary,and tertiary）PCRs individually performed by three WWPs.The WWP is annealed arbitrarily anneal to somewhere on the genome in the one low-stringency cycle of the primary PCR,or directionally to the previous WWP site in one reduced-stringency cycle of the secondary/tertiary PCR,producing a pool of single-stranded DNAs（ss DNAs）.A target ss DNA incorporates a gene-specific primer（GSP）complementary at the 3’-end and the WWP at the 5’-end and thus,can be exponentially amplified in the next high-stringency cycles.Nevertheless,a non-target ss DNA cannot be amplified as it lacks a perfect binding site for any primers,which can effectively enrich the target products and eliminate non-target products after three consecutive rounds of PCR.WW PCR charismatic high versatility and advanced specificity,as an attractive alternative to the existing genome stepping method,gap filling can be used in chromosome sequencing to obtain a complete genome sequence.