【作者】 李海星；

【导师】 曹郁生；

【作者基本信息】 南昌大学 ， 食品科学与工程， 2012， 博士

【摘要】 γ-氨基丁酸是一种非蛋白质组成的天然氨基酸,广泛存在于自然界。γ-氨基丁酸是哺乳动物中枢神经系统的一种主要抑制性递质,具有多种生理功能,可作为生物活性因子应用于食品、医药和饲料工业。我国己批准γ-氨基丁酸作为新资源食品用于食品生产加工。本论文就高产γ-氨基丁酸乳酸菌的分离鉴定,Y-氨基丁酸的检测方法,发酵工艺的建立优化,产物的分离纯化,以及关键基因分析进行了较深入的研究,主要结果如下。1.建立了预染纸色谱方法用于氨基酸的高通量定性分析,预染纸色谱-分光光度法用于γ-氨基丁酸的高通量定量。预染纸色谱的操作方法为：滤纸在含茚三酮的展开剂(正丁醇：冰乙酸：水=5：3：2,v/v/v)中展开后,直接加热显色。预染纸色谱能将谷氨酸和γ-氨基丁酸完全分开,氨基酸斑点致密,避免了传统纸色谱的拖尾和重叠现象。优化了影响γ-氨基丁酸与茚三酮显色的重要因素如茚三酮浓度、显色温度、显色时间和Cu2+浓度等,使显色反应完全、生色基团稳定。优化上述因素后,将预染纸色谱偶联分光光度法用于γ-氨基丁酸的定量,实验条件为：准确吸取2μL样品点样,色谱纸在含1.2%茚三酮的展开剂中展开后,70℃显色80min,将γ-氨基丁酸-茚三酮斑点从色谱纸上剪下,置于5mL洗脱液(75%乙醇：0.6%CuS04·5H20=38：2,v/v)中,40℃50rpm震荡洗脱60min,测定洗脱液在512nm处的光吸收值。结果表明,方法在0.50～20.00g/L的浓度范围内线性关系良好(R2=0.998),相对标准偏差RSD<2.64%,回收率102.7～103.9%。利用预染纸色谱-分光光度法和HPLC同时测定样品的GABA浓度,两种方法的测定结果无显著性差异,表明所建立的方法可用于Y-氨基丁酸的定量。2.从泡菜中分离到高产γ-氨基丁酸的短乳杆菌NCL912。利用纸色谱初筛、HPLC复筛,从1000余株分离自泡菜样品的乳酸菌中筛选到23株产γ-氨基丁酸的疑是细菌,其中菌株NCL912的产量最高(15.37g/L)。经LC-MS测定,NCL912转化产物的分子量与γ-氨基丁酸标准品完全一致。上述结果表明,NCL912确实能转化谷氨酸,且转化产物为Y-氨基丁酸。根据表型特征、生理生化特性和16SrDNA全序列比对,鉴定NCL912为短乳杆菌(Lactobacillus brevis)。该菌现保藏于中国典型培养物保藏中心,保藏编号为CCTCCM208054(=NCL912)。3.设计并优化了Lactobacillus brevis CCTCCM208054产Y-氨基丁酸的发酵培养基。用单次单因子方法筛选到影响γ-氨基丁酸合成的四个重要因子为葡萄糖、大豆蛋白胨、Tween-80和MnSO4·4H2O,并确定了各重要因子的合适浓度范围。然后利用响应面方法对重要因子进行分析,确定了四个因子的最佳水平,分别为55.25g/L、30.25g/L、1.38mL/L和0.0061g/L。在各因子的最佳水平条件下,模型预测γ-氨基丁酸产量为36.06g/L,验证实验中实测值为35.66g/L,二者基本一致。优化发酵培养基后,CCTCCM208054的γ-氨基丁酸产量比优化前提高了130%。4.考察了5’-磷酸吡哆醛、温度、pH和初始谷氨酸钠浓度对CCTCCM208054生长和合成γ-氨基丁酸的影响。5’-磷酸吡哆醛对细胞生长和γ-氨基丁酸合成没有影响；温度、pH和初始谷氨酸钠浓度则有重要影响,最佳水平分别为30～35℃、5.0和0.25～0.50M。在此基础上,建立了CCTCCM208054合成γ-氨基丁酸的补料分批发酵工艺如下。种子培养基(g/L)：葡萄糖50,大豆蛋白胨25,MnSO4·4H2O0.01,Tween-802mL/L,MSG0.5M,pH5.0；发酵培养基除葡萄糖为35g/L、MSG为0.4M外,其余成分与种子培养基相同；CCTCCM208054在种子培养基中32℃培养约10h(A600=4.0～6.0)作为种子液；补料分批发酵的具体参数为：发酵培养基3L,接种量10%(v/v),培养温度32℃,搅拌速度100rpm,发酵周期48h,12h和24h时分别补入280g和112gMSG,整个过程用5M H28O4控制pH5.0。发酵48h时,发酵液中γ-氨基丁酸浓度达到102.78±5.30g/L,无谷氨酸钠和葡萄糖残留。5.通过离心、活性炭脱色、70%乙醇脱盐、离子交换色谱精制和乙醇结晶的方法,从发酵液中分离纯化γ-氨基丁酸。产品回收率约为50%；产品纯度达98.66±2.36%。6.克隆到了CCTCCM208054的gadA及其侧翼序列,从上游至下游依次为：乙酰转移酶基因(act)、PgadR、gadR、Pgad、gadC、gadA和谷氨酰-tRNA合成酶基因(gts),与Lactobacillus brevis ATCC367中相应的基因结构类似,但ATCC367的PgadR上游为NADPH-醌还原酶相关的锌依赖氧化还原酶基因。CCTCCM208054的gadR、gadC、gadA与ATCC367的同源性分别为66%、79%、79%,编码蛋白的同源性分别为66%、91%、91%；CCTCCM208054的act与gadR的间隔区、gadR与gadC的间隔区、gadC与gadA的间隔区长度分别为278、210、59bp,而ATCC367相应的间隔区长度分别为270、193、55bp；两株菌的上述间隔区的同源性分别为43%、58%、62%。CCTCCM208054的gadA和gadC的间隔区或其附近序列中不存在任何转录信号,表明gadCA可能形成操纵子结构。不能直接扩增到CCTCCM208054的gadB,但扩增到了其醛酮还原酶基因(akr)(ATCC367的akr距gadB仅1003bp),往CCTCCM208054的akr下游步移了3027bp也未发现gadB,提示CCTCCM208054中可能没有gadB。7.利用实时荧光定量PCR研究了添加与不添加MSG的条件下,CCTCCM208054的gadA、gadC和gadR在不同培养时期的转录情况。MSG可以诱导三个基因表达；gadC和gadA的转录量一致；与Lc. Lactis的gadR为组成型表达不同,CCTCCM208054的gadR和gadCA同步转录,且转录量大幅高于gadA和gadC,因时期不同为后者的14～156倍。上述结果表明,CCTCCM208054可能通过以下三个方面实现了高产GABA:(1)可能通过优化GAD系统的功能基因和调控序列,提高了gadCA的转录量以及GadA和GadC的活性；(2)可能通过gadCA组成操纵子结构实现了gadC和gadA同步表达,使脱羧反应和氨基酸运送相协调；(3)可能通过gadR的高表达保障了gadCA正常转录。更多还原

【Abstract】 Gamma-aminobutyric acid (GABA) is a naturally non-protein acid that is widely distributed in nature. GABA acts in animals as a major inhibitory neurotransmitter, and has several important physiological functions and the potential as a bioactive component in foods, pharmaceuticals and feeds. GABA has beeen approved as a new resource food for food processing in China. This dissertation was focused on the bioconversion of GABA with lactic acid bacteria (LAB), including the isolation and identification of a high-yielding GABA producer; the methods for the analysis of GABA; the establishment and optimization of fermentation process for GABA production; the isolation and purification of GABA; and the analysis of GAD system key genes in CCTCCM208054. The main results are as follows.1. Pre-staining paper chromatography was developed for qualitative analysis of amino acids. Pre-staining paper chromatography-vis-spectrophotometry was developed for quantitative analysis of GABA. Operating conditions for pre-staining paper chromatography were as follows:the chromatography paper was developed in a developing solvent (n-butanol:acetic acid:water=5:3:2, v/v/v) containing ninhydrin, then the paper was directly dried for color yield. Glutamate and GABA were completely separated with condense spots in the pre-staining paper chromatography while the spots in classical method were partially overlapped and with long tails. The effects of ninhydrin concentration, color temperature and time on the color yield in the ninhydrin reaction, and the effect of Cu2+concentration on the stability of GABA-ninhydrin compound were optimized. The optimized pre-staining paper chromatography coupled with vis-spectrophotometry could be applied to gamma-aminobutyric acid quantification as follows. Appropriate2μL of samples were spotted onto the chromatography paper. The paper was developed at30℃with n-butanol-acetic acid-water (5:3:2) containing1.2%of ninhydrin. After development, the paper was directly heated for color yield at70℃for80min. Then the GABA-ninhydrin spots were cut out from the paper and were extracted with5.0mL eluent (75%ethanol:0.6%CuSO4·5H2O=38:2,v/v) at40℃50rpm for60min. The absorptions were read in a UV-vis spectrophotometer at512nm. The results indicated that the linear range of the developed method was from0.5to20.0mg/mL. Furthermore, an excellent correlation coefficient was observed with an R2=0.998. The method is accurate (RSD<2.64%), and has good recoveries (102.7-103.9%). The concentrations of GABA determined by the current method and HPLC were quite close.2. A high GABA-producing strain, Lactobacillus brevis NCL912was isolated from Chinese traditional paocai. More than1000strains of lactic acid bacteria from paocai samples were screened by the ability in production of GABA, analysed with paper chromatography and HPLC, and23suspicious bacteria were obtained. Among them, strain NCL912exhibited the highest conversion ability (15.37g/L). The molecular weight of the suspicious product was almost the same to that of GABA standard determined by LC-MS. It showed that strain NCL912could indeed convert glutamate to GABA. Strain NCL912was identified as Lactobacillus brevis according to its phenotye, physiological and biochemical characteristics and full16S rNDA sequence alignment. This strain has been deposited in China Center for Type Culture Collection with the accession number CCTCCM208054(=NCL912).3. Fermentation medium for the production of GABA by CCTCCM208054was optimized. Single-dimensional search method was first adopted to select the key factors that impact the GABA production to preliminarily determine the suitable concentration ranges of the key factors. Then response surface methodology was applied to analyze the optimal contents of the key factors glucose, soya peptone, Tween-80and MnSO4·4H2O, and their optimal levels were55.25g/L,30.25g/L,1.38mL/L and0.0061g/L, respectively. The production of GABA was predicted as36.06g/L under the optimized conditions with this model. While the measured GABA content was35.66g/L in the verification test, which was basically identical with the predicted value. GABA production of CCTCCM208054in optimized medium was130%higher than that in the initial medium.4. The impacts of pyridoxal-5’-phosphate, pH, temperature and initial glutamate concentration on the GABA production and cell growth of CCTCCM208054were investigated. Pyridoxal-5’-phosphate did not affect the cell growth and GABA production of CCTCCM208054. Temperature, pH and initial glutamate concentration had significant effects on the cell growth and GABA production of CCTCCM208054. The optimal temperature, pH and initial glutamate concentration were30～35℃,5.0and0.25～0.50M. According to the data obtained in the above, a fed-batch fermentation process was developed to produce GABA as follows.The seed medium was composed of (g/L):glucose,50; soya peptone,25; MnSO4·4H2O,0.01; L-glutamate,0.15M; and Tween80,2mL/L; pH5.0. The fermentation medium was the same to the seed medium except for glucose50g/L and glutamate0.4M. CCTCCM208054was cultured in the seed medium at32℃for about10h till A600between4.0and6.0and then used for seed culture inoculation. The specific fed-batch fermentation parameters were:fermentation medium3L, seed culture300mL, temperature32℃, stirring speed100rpm, fermentation period48h,280g and112g glutamate were supplemented into the bioreactor at12h and24h, respectively. pH value of the fermentation broth was controlled at5.0with5M H2SO4during the whole process. The GABA concentration reached102.78±5.30g/L while no glutamate and glucose remained at48h.5. Centrifugation, activated carbon decoloration,70%ethanol desalination, refining by ion exchange chromatography and crystallization from ethanol were successively conducted for the purification GABA from the fermented broth. The recovery rate for the whole purification process was about50%. The purified product displayed a single spot in TLC chromatogram. Its purity reached98.66±2.36%through HPLC determination.6. gadA and its flanking region in CCTCCM208054was cloned. Gene order of the cloned region is (from upstream to downstream):acetyltransferase gene(act), PgadR, gadR, Pgad, gadC, gadA and glutamyl-tRNA synthetase gene (gts). This is almost the same to that in Lactobacillus brevis ATCC367. However, NADPH:quinone reductase related Zn-dependent oxidoreductase gene is located immediately upstream of PgadR in ATCC367. The homologous coefficients of gadR, gadC and gadA in CCTCCM208054with those in ATCC367are66%,79%and79%, respectively; and those of the encoded proteins are66%,91%and91%, respectively. Intergenic regions of between act and gadR, gadR and gadC, and gadC and gadA are278,210and59bp, respectively in CCTCCM208054, while they are270,193, and55bp, respectively in ATCC367. In the two microbes, the homologous coefficients of the above intergenic regions are43%,58%and62%, respectively. No possible transcription signals could be identified in or near the59bp intergenic region between gadA and gadC. We could not clone gadB by using primer pairs for direct amplification it. We amplified aldo-keto reductase gene (akr) and walked into downstream of akr for3027bp but did not find gadB although it locates downstream only1003bp of akr in ATCC367. This suggests that CCTCCM208054maybe contain no gadB.7. Real-time fluorescence quantitative PCR was applied to analyze the transcriptional levels of gadA, gadC and gadR in CCTCCM208054during the fermentation process in the fermentation media supplemented with or without glutamate. The results showed that glutamate induced their expression. The transcriptional level of gadC is identical to that of gadA. Different from constitutive transcription of gadR in Lactococcus lactis, transcription of gadR in CCTCCM208054is synchronous with gadCA, and its transcriptional level is14-156times of that of gadCA. Sequence analysis and quantitative PCR results suggested that gadCA maybe form an operon structure. The high GABA-producing ability of CCTCCM208054maybe derived from three reasons:first, transcriptional level of gadCA and activity of the enzymes maybe enhanced via optimizing functional gene and regulatory sequences of GAD system; second, the synchronous expression of gadC and gadA via forming an operon is conducive to coordinating the decarboxylation and antiport; third, high expression of gadR guarantees normal transcription of gadCA.更多还原