【作者】 陈海军；

【导师】 林亲录；

【作者基本信息】 湖南农业大学 ， 农产品加工及贮藏工程， 2007， 硕士

【摘要】 γ-氨基丁酸(γ-amino butyric acid，GABA)是一种广泛分布于动植物体内的非蛋白质氨基酸，它对人体具有多种生理功能，γ-氨基丁酸是哺乳动物中神经系统的主要抑制性递质，介导了40％以上的抑制性神经传导，参与脑循环生理活动，具有抗心律失常功能，具有降血压、抗惊厥、镇痛、改善脑机能、精神安定、促进长期记忆等功能，GABA可作为少年儿童智力的营养补充剂和促进剂。促进生长激素分泌，肾功能活化，肝功能活化，抗疲劳等生理功能。在国外利用乳酸菌发酵制作的富含GABA的食品配料，均可直接作为或者用于抗高血压、增进脑机能及肝功能的功能食品。在我国，有关利用生物技术生产GABA的研究，主要集中在一些高校和科研院所。但其研究成果中产GABA能力都不高，也很少能应用于工业化生产。筛选具有高活力GAD的乳酸菌菌株以及探索实用的乳酸菌生产GABA的途径和高效的提取纯化工艺将具有广泛的经济效益和社会效益。本论文主要包括了以下几方面的研究：(1)高产GABA乳酸菌的筛选采用梯度稀释法从加有质量分数为1％的米糠的自然发酵泡菜中分离筛选出产GABA的菌株，并对其进行鉴定，测定其发酵产GABA的产量，发现菌株B2产量最高，可达1.2mg／mL。(2)乳酸菌发酵液中GABA的定性与定量检测方法的研究本文通过对纸层析法、比色法等定性与定量测定GABA的方法。在需要精确测定GABA时，本文研究了PITC柱前衍生-紫外检测GABA的HPLC法。确定了定性检测GABA的纸层析方法：展开剂的配方为正丁醇：醋酸：水=4：1：3，内含0.4％的茚三酮；显色温度和时间为90℃、10min。研究了PITC柱前衍生-紫外检测GABA的HPLC法测定乳酸菌发酵液中的GABA，此法检出限为5mg／L，RSD为1.2％，回收率为101.4％。(3)乳酸菌GAD活性的研究以实验室筛选所得的乳酸菌B2作为出发菌株，对其酶学性质进行研究，即酶的热稳定性、pH稳定性及温度、pH、底物浓度、酶浓度与酶活性的关系，并考察了几种金属离子，酶的激活剂，抑制剂对酶的影响。确定该脱羧酶作用的合适条件。为进一步对乳酸菌发酵条件优化指明了方向。(4)乳酸菌发酵条件的优化主要以试验中所筛选的产GABA的乳酸菌菌株作为出发菌株，对其发酵产GABA条件进行了摸索和探讨，主要从培养基的成分、配比、pH值、发酵温度、发酵周期等方面进行了试验。试验结果表明，最佳发酵培养基组成为：复合碳源质量分数为2％，其中乳糖与谷氨酸钠的质量比为1：1；复合氮源蛋白胨与酵母膏的质量比为1：1；碳源与氮源比为1：1。通过发酵条件优化可知，培养基的初始pH为7.0，培养温度为33℃，谷氨酸钠的添加量为80 g／L，PLP的加入量为50umoL／L，为最适于GABA的生成条件，并确定最佳发酵周期为3d。在最佳发酵条件下，菌株B2产GABA量可达4.2mg／mL。更多还原

【Abstract】 Theγ-amino butyric acid (GABA), is a kind of non- protein amino acid which widelyexists in the body of vegetables and animals. It has various physiological functions tothe human body, GABA is a main depressor of the mammal nervous system, lying tolead 40% to restrain nerve conduction above, participating in the physiologicalactivities of the brain circulation, having anti-out of gear rhythm of the heart todisorder function, having antihypertensive, the anti- convulsion, ease pain,improving the function of the brain, spirit stabilize, promote to remember over a longperiod of time, the GABA has become a young child intellectual nourishmentsupplement and accelerant Promote the exudation of growth hormone, the activationof kidney function, the activation of liver function, physiology function of theanti-tired etc.It is reported tha : food with the enrichment GABA which makes use oflactobacillus to ferment a creation goes together with to anticipate, all can be thefunction food which uses for anti- high blood pressure and promotes brain functionand liver function directly. In our country, the study on which makes use of theproduction GABA of the biotechnology also have in recent years, main concentrationin some high schools and research studio.But it produces GABA abilities all verygenerally, also can seldom be applied in industrialization to produce. Searching thelactobacillus germ of high vitality GAD stub and investigating practical lactobacillusto produce GABA path will have extensive economic benefit and social significationwith withdrawing a pure chemical engineering skill efficiently. This thesis mainlyseveral aspects fermented to produce GABA to carry on a study to the lactobacillusfrom the following:(1) The screening of a Lactobacillus with high production of GABAAdopt a steps degree a dilution method separation sieving to produce GABAgerm stub from add to have a quality score to ferment pickles for 1% natures, and as to it’s carry on authenticating, measures it to ferment to produce GABA yield,discover the germ stub B2 yield topmost, can reach to 1.2 mg/mL.(2) The study on the method for measuring GABA in the liquid of fermentationIn this paper, paper chromatography method colorimetric method high pressureliquid chromatography were studied on qualitative analysis and quantitative techniqueof GABA which fermented by lactobacillus .While needing a precision measurementGABA, this text studied PITC pillar developed before- the HPLC method of theultraviolet examination carded on the accurate quantitative analysis. Made surepaper chromatography method to qualitative analysis GABA .method:Theformulation which launches is a positive n-butyl alcohol:Acetic acid:Water=4:1:3,conta 0.4% ninhydrin; the temperature and time of the colordevelopment is 90℃10min respectively, the HPLC method of PITC pillar-purpleexamine the measurement lactobacillus of the GABA in the liquid outside, thismethod checks to limit for the 5 mg/L, RSD is 1.2%, recovery rate is 101.4%.(3)The research of Activities of the lactobacillus GADUse the laboratory Lactobacillus B2 gained by sieving B2 as to set out germstub, as to it’s the enzyme learn a property to carry on a research, namely hotstability, pH stability and, the relation of temperature, pH The density of substrateenzyme density and enzyme activity, and investigated a few metals, the enzymeactivates, depressor influences to the enzyme.The assurance should take off thesuitable condition of the function of GAD.For further fermenting a condition to thelactobacillus excellent turn it to understand. Giving a direction to the study ofoptimization of the condition of lactobacillus fermentation.(4) Optimization of the condition of lactobacillus fermentationUse the lactobacillus which can produce GABA searched from the frontexperiment as the set out bacteria do some study on it’s fermented condition abouthow to produce GABA, mainly from culture medium of composition, go togetherwith a ratio, pH value, fermentation temperature, fermentation period etc. to carryon experiment. the study showed that the best culture medium is that best fermentculture medium to constitute for:Compound carbon source quality score is 2%, (among them, the quality of lactose and the valley ammonia sour sodium compare to1:1), reunite the nitrogen source peptone and the quality of leaven cream to compareto 1:1, total carbon nitrogen compare is 1:1.Pass to optimize fermentation condition,the result is, The beginning pH 7.0,temperature 33℃, sodium glutamate 80 g/L, thePLP is 50umoL/L, and make sure the best fermentation period is 3 days. under theoptimist fermentation condition, the GABA yield of B2 can reach to 4.2mg/mL.更多还原