Substrate Sustained Release-based High Efficacy Biosynthesis Of GABA By Lactobacillus Brevis NCL912

Gamma-aminobutyric acid?GABA?is a major inhibitory neurotransmitter in the mammalian central nervous system.GABA possesses numerous well-known physiological properties such as hypotensive,diuretic and diabetes reversal effect.Thus,much attention has been paid to the large-scale bioconversion of GABA by lactic acid bacteria.Previously we established an efficient fed-batch fermentation process for Lactobacillus brevis NCL912 producing gamma-aminobutyric acid from monosodium L-glutamate,of which GABA production was 104 g/L after fermenting for 48 h and it can hardly be improved.L-glutamic acid is an acdic amino acid and has low solubility?6 g/L?,which confers L-glutamic acid the properties of sustained release and pH buffering.Therefore,L-glutamic acid may have the advantage of needing to only be added one time prior to cultivation due to its sustained release.In addition,extra inorganic acid would not be required.These key characteristics of L-glutamic acid may allow efficient GABA production by reducing the inhibition of extra by-products.In this study,the formulation of the culture medium was re-screened and re-optimized for the synthesis of GABA by L.brevis NCL912.This dissertation was focused on the quantitative method of?-aminobutyric acid,the optimization of fermentation medium when using L-glutamic acid as the substrate and batch fermentation in a 10 L fermenter.The main experimental results of this study are as follows:1,Cu²⁺free elution pre-staining paper chromatography-vis-spectrophotometry was established for quatitative analysis of gamma-aminobutyric acid,which facilitates the follow-up experients.Different concentrations of ethanol solution were screened with elution time and stability of the eluent as indicators.It turned out that elution rate is fastest and the stability is the best when the ethanol concentration is75%.75%ethanol solution could completely elute the GABA-ninhydrin compound from paper within 25 min and the A570 value of eluent showed almost the same stability being maintained for at least 150 min.HPLC was employed as a reference method to validate the current method and no significant difference existed between the GABA contents determined by these two methods?p>0.05?.The calibration curve was linear over the selected GABA concentrations?0.25-10 g/L?with the linear equation being y=0.046x+0.0106?R²=0.9936?,where y means A570 value and x stands for GABA concentration.Satisfying recoveries ranging from 96.95%to102.9%were obtained,RSD was ranging from 0.82-1.72%,indicating that the developed approach could be an alternative of the existing methods for determination of gamma-aminobutyric acid.2,L-glutamic acid was proposed as a substrate and the key components of the fermentation medium affecting GABA synthesis were re-screened and re-optimized to enhance GABA production from L.brevis NCL912.The initial fermentation medium was composed of 50 g/L glucose,25 g/L yeast extract,0.1 g/L MnSO4·H2O,220 g/L glutamic acid,and 2 g/L Tween-80.The fermentation conditions were:inoculum volume,10%?v/v?;incubation temperature,32°C;and fermentation period,48 h.It turned out that carbon source?glucose?,nitrogen source?yeast extract powder FM408?,Tween-80 and Mn²⁺were the four key components that affected GABA production.The optimized levels of the four key components in the fermentation medium optimizing by single-factor experiments were 25 g/L glucose,25 g/L yeast extract FM408,0.15 mM MnSO4·H2O,and 2 g/L Tween-80.3,The fermentation process of substrate sustained-release was established basing on flask-shaking optimized parameters.The batch fermentation conditions were:fermentation medium load,5 L;inoculum volume,10%?v/v?;incubation temperature,32°C;agitation speed,100 rpm;L-glutamic acid,295 g/L.After 48 h of fermentation,the final GABA concentration increased up to 205.8±8.01 g/L,which was approximately double the yield obtained from the original fermentation process using monosodium L-glutamate.