黑曲霉产生的羧酸酯水解酶在普洱茶后发酵中作用研究

The unknown of the roles of key enzymes of dominant fungi seriously restricts the study on quality formation of Pu-erh tea. In this project, the roles of 14 Carboxylic ester hydrolases (CEHs) including Tannases and Lipases produced by Aspergillus niger in the post-fermentation of Pu-erh tea will be studied on the basis of that these CHEs have been identified in our previous research. These enzymes will be obtained through gene clone, His-tag expression and purification using Ni-NTA chromatography. The roles of Tannases in tea phenolic metabolism will be studied through in vitro analysis of these purified enzymes with substrates of the standards of tea polyphenols and extraction of Sun-dried green tea, and the compare metabolomics analysis of compound compositions in samples fermented by adding enzymes and control. The effects of Lipase to metabolism of tea aroma components will be investigated by comparison of tea aroma components using gas chromatography-mass spectrometry in enzymatic model, samples fermented by adding enzymes and control. The bioactivity and sensory quality of reaction products and fermented samples will be investigated by search of database, sensory evaluation and antioxidant activity tests. Taken together, the roles of CEHs in the post-fermentation of Pu-erh tea will be studied through the survey of effects to metabolism of tea phenol and aroma components, to bioactivity and sensory quality. The roles of key enzymes of dominant fungi in the post-fermentation of Pu-erh tea will be revealed, and novel knowledge for the quality formation of Pu-erh tea will be provided, through the development of this project. The technical system integration of the key links including of microbe, enzymes, chemical compounds and quality will be established and technical supports will be provided for further study on other teas, microorganisms and enzymes. And Tannase and Lipase of A. niger will be obtained, which will provide the basis for the application of these enzymes.

优势菌关键酶在后发酵中作用不明，严重制约普洱茶品质形成基础研究。在已发现黑曲霉产生的单宁酶、脂肪酶等14个羧酸酯水解酶的基础上，本项目拟研究其在普洱茶后发酵中的作用。拟通过基因克隆、酵母表达、镍柱纯化制备酶。通过标品、晒青提取物为底物的体外酶促研究，加酶发酵与对照样品的比较代谢组研究，探究单宁酶在普洱茶酚类物质代谢中的作用。应用气质联用比较体外酶促、加酶发酵和对照样品的香气物质，研究脂肪酶在茶叶香气物质代谢中的作用。通过数据库、活性测定和感官审评，研究酶催化产物与发酵样品的生物活性及感官品质。从对酚类和香气物质代谢、生物活性及感官品质的作用入手研究酶在后发酵中的作用。项目将阐明优势菌关键酶在后发酵中的作用，获得普洱茶品质形成新知识；将建立贯通微生物、酶、化学成分、生物活性与感官品质各关键环节的技术体系，为研究其它茶、微生物和酶提供技术支撑；将获得黑曲霉的单宁酶和脂肪酶等具有应用前景的酶。

为解决普洱茶发酵优势菌关键酶的作用不明，这一普洱茶发酵机理研究的关键科学问题，本项目开展了8项研究。项目主要研究与结果包括：（1）建立了一种同时测定茶叶20种特征物质的HPLC方法，为研究酶的作用提供了基础；（2）开展了普洱茶发酵系统微生物组的多维宏组学研究，鉴定了微生物产生的羧酸酯水解酶，揭示了单宁酶等催化的酚酸酯等物质代谢；（3）应用单菌发酵、蛋白组学和代谢组学技术，揭示了普洱茶发酵优势曲霉黑曲霉（Aspergillus niger）、溜曲霉（Aspergillus tamarii）和烟曲霉（Aspergillus fumigatus）在茶叶发酵中产生的羧酸酯水解酶，明确了其催化的酚酸酯类物质代谢；（4）应用基因工程方法制备得到了黑曲霉的8个单宁酶和1个脂肪酶；（5）开展了单宁酶催化茶叶物质研究，揭示了单宁酶对茶叶酚酸酯类物质的代谢；（6）应用脂质组学技术揭示了普洱茶发酵中脂类物质的代谢，鉴定到752个脂类物质，阐明了亚油酸等重要香气物质或前体在发酵中的变化；（7）揭示了脂肪酶催化茶叶脂类物质的变化，关联分析发现酶催化导致的部分变化与发酵中的变化相同，表明发酵中黑曲霉脂肪酶参与了部分茶叶脂类物质代谢。通过项目研究明确了单宁酶在发酵中的作用为水解酚酸酯类物质，降低其含量，增加对应的酚酸含量，进而降低茶叶的涩度，增加醇厚度，同时增加发酵阶段样品的酸味；明确脂肪酶的主要作用是水解脂类物质，造成发酵中脂类物质剧烈变化，但其对香气滋味的作用有待进一步研究。项目阐明普洱茶发酵优势菌（黑曲霉）关键酶（单宁酶和脂肪酶）在发酵中的作用，获得了普洱茶发酵机理的新知识，建立的微生物组学、基因克隆表达平台，为研究其它茶、微生物或酶提供了支撑。项目研究发表论文18篇，其中SCI收录论文5篇。培养硕士研究生2人。