淡豆豉炮制中γ-氨基丁酸的新发现及其形成机理研究

Semen sojae praeparatum (SSP) is processed by fermenting with black soybean as the key raw material, mulberry leaf and southernwood as the adjuvant. Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the mammalian central nervous system, and it has the similar pesticide effect with SSP. GABA is based on glutamate with glutamic acid decarboxylase (GAD) play a catalytic role. Up to now, there have been no relevant research about GABA of SSP. We have found GABA about 22mg/g in SSP that it was significantly higher than the other known medicinal herbs and fermented products. How so much GABA enriched in SSP? According to the pharmacopoeia of people's republic of china, our group has established the canonical processing technology of SSP, indicated the regularity change of microbial communitythe and unique dominant strains during the fermentation, and dominant microorganism in SSP processing, such as aspergillus, yeast and lactic acid bacteria, can all secrete GAD. We speculate that SSP rich in GABA may be correlated with the complex microbial community. We would like to know the relationship between GABA and glutamate, GAD, pH, related protease in SSP processing. We will also monitor the dynamic change of microbial species and quantity in process with denaturing gel gradient electrophoresis (DGGE), and try to find how microorganism contributed to GABA, to build the foundation of the research about the mechanism of SSP fermentation and the synthesis of GABA.

淡豆豉以黑大豆为主料，桑叶、青蒿为辅料经发酵炮制而成。γ-氨基丁酸（GABA）是中枢神经系统首要抑制性神经递质，有多种功效，并与淡豆豉功效谱相同或超之。GABA由谷氨酸经谷氨酸脱羧酶（GAD）催化生成。迄今未关注淡豆豉GABA。我们首次发现淡豆豉GABA含量为22mg/g远高于已报道中药材、发酵豆制品等。自然发酵炮制的淡豆豉如何高富集GABA？本课题组按《中国药典》已建立规范的淡豆豉炮制工艺，炮制中微生物种类、数量丰富且出现特有优势菌种，曲霉、酵母、乳酸菌等优势菌已报道均分泌GAD。推测淡豆豉高富集GABA与其独特炮制工艺形成的特有微生物菌群密切相关。据此本项目拟研究淡豆豉炮制过程中GABA、谷氨酸、GAD、蛋白酶等指标和微生物菌群的动态变化，明确GABA形成的影响因素、优势菌种及种群间的相互关系在GABA形成中的作用，阐明淡豆豉炮制中微生物调控GABA形成机制，为揭示其炮制机理等奠定基础

淡豆豉以黑大豆为主料，桑叶、青蒿为辅料经发酵炮制而成，被历代本草和历版《中国药典》收录，列入卫生部首批药食两用名单。γ-氨基丁酸（GABA）是哺乳动物中枢神经系统首要抑制性神经递质，有多种功效，并与淡豆豉功效谱相同或超之。本项目首次从微生物角度出发，阐明淡豆豉炮制中GABA形成机制，为揭示淡豆豉炮制机理等奠定基础。.1、参照《中国药典》建立了规范的淡豆豉炮制工艺..2、应用柱前在线衍生HPLC和HPLC-MS法首次测定了淡豆豉炮制中GABA含量动态变化，“再闷”阶段富集了高含量GABA，再闷15天时为6.33 mg/g。.3、从淡豆豉自然炮制中筛选、鉴定出产GABA的微生物，并比较它们的产GABA能力：筛选出产GABA细菌25株，真菌13株，分别为枯草芽孢杆菌、解淀粉芽孢杆菌、克劳氏芽孢杆菌、屎肠球菌、鸟肠球菌、鲑色锁掷酵母菌、米根霉、简青霉、溜曲霉、黄曲霉、黑曲霉、极细支孢霉、桔青霉、白腐菌。细菌产GABA能力较真菌强。.4、对淡豆豉炮制中产GABA微生物在不同pH值和温度下的产酶能力进行了比较，所选的12种微生物产酶最适pH值、温度与淡豆豉自然炮制基本一致，其中真菌有较强的产GAD能力，细菌有较强的产中性蛋白酶能力。.5、对淡豆豉自然炮制中GABA富集的主次因素进行了分析，温度、 pH 值、 GAD、中性及碱性蛋白酶是影响 GABA形成的重要因素。.6、运用PCR-DGGE 技术研究了淡豆豉炮制过程中微生物菌群的动态变化，荧光定量PCR检测了产GABA微生物在淡豆豉炮制过程中的数量变化，分析了微生物种类、数量以及种群间相互关系在GABA形成中的作用。初步认为淡豆豉炮制中细菌种类和数量、霉菌数量、酵母菌数量、米曲霉数量、卵形丝孢酵母数量与GABA含量变化呈负向直线相关，枯草芽孢杆菌数量与GABA含量呈正向直线相关。其余菌种数量与GABA相关系数较小且无统计学差异。枯草芽孢杆菌、鲑色锁掷酵母菌和屎肠球菌3种菌在再闷3天左右存在交互作用，淡豆豉高富集GABA是多菌种共同作用的结果。.7、模拟淡豆豉自然炮制确定了枯草芽孢杆菌纯种发酵高产GABA新工艺。.8、应用CUMS抑郁模型，首次发现淡豆豉能显著改善抑郁小鼠的快感缺失、行为绝望等抑郁症状，具有良好的抗抑郁作用并与GABA含量有关。.9.完成了淡豆豉炮制中各样本纤溶酶活性测定及产纤溶酶菌种的筛选鉴定。