Development of New Technology for Plant Aroma Emission with New Type of Plant

乌龙茶和红茶的花茶香气已被澄清为由香气前体产生，其中大部分作为β-樱草苷存在于茶叶中，通过β-樱草苷酶在其制造过程中的作用，所谓的发酵过程。近年来，人们发现这些香气形成机制与花、果、酒等中诱人香气的形成有关。基于这些新发现，本论文试图通过从微生物代谢产物中筛选一种新的二糖特异性糖苷酶，并对其进行大量生产和应用，从而建立新型香气释放技术。1）澄清茶β-樱草糖苷酶的底物特异性，通过化学合成和天然来源获得了几种以2-苯基乙醇为糖苷配基的双糖甙类化合物。用β-樱草糖苷酶对其进行水解，以了解其底物特异性。 ...更多信息 城市β-樱草苷酶对β-樱草苷表现出非常高的底物特异性。所有合成的天然二糖糖苷根本不水解。该酶的氨基酸序列分析表明，β-樱草糖苷酶与水解生氰β-D-吡喃葡萄糖苷的β-葡萄糖苷酶有较高的同源性（60%），且存在一定的N-糖基修饰。2）β-樱草苷水解菌的筛选以丁香酚β-樱草苷为唯一碳酱料，从土源微生物中筛选出4株具有β-樱草苷水解活性的菌株。经鉴定，活性最高的菌株为烟曲霉Pr-20。从该菌发酵液中纯化的糖苷酶经SDS-PAGE分析，在47 kDa处显示单一条带，并能水解其它β（1→6）-二糖苷和6-酰化β-D-吡喃葡萄糖苷。3）烟曲霉β-樱草苷酶样二糖苷酶基因的克隆与茶叶β-樱草糖苷酶相同。在酵母和真菌中尝试了该基因的过表达，在真菌中得到了成功的结果。4）通过用二糖苷酶处理来增强内源性植物香气前体的香气的一些试验将二糖苷酶应用于粗茶叶提取物和葡萄汁，并通过GC分析释放的香气。发现许多种类的香气化合物被这种处理大大增强，该酶可用于红茶、红茶酒及其相关行业的增香。少

Floral tea aroma of oolong tea and black tea has been clarified to be generated form aroma precursors, most of which are present as β-primeverosides in tea leaves, by the action of β-primeverosidase during their manufacturing, so called fermentation processes. Recently these aroma formation mechanisms have been found to be concemed with the attractive aroma formation in flower, fruits, wine, etc.Based on these new findings, new type of aroma emission technology was attempted to be established by screening a new disaccharide specific glycosidase among microbial metabolites, its mass production and its application as follows :1) Clarification of substrate specificities of the tea β-primeverosidase, cloning of its cDNA and a trial for its large scale overexpression in E.coli.Several kinds of disaccharide glycosides with 2-phenylethanol as an aglycon were obtained by chemical synthesis or from nature. They were subjected to hydrolysis with the β-primeverosidase to know its substrate specif … More icities. The β-primeverosidase showed very high substrate specificity towards β-primeveroside. All the synthetic unntural disaccharide glycosides were not hydrolyzed at all. Only other natural disaccharide (β 1-6) glycosides were hydrolyzed some extent (0.07-3.0%).The amino acid alignment of the β-primeverosidase obtained by its gene cloning suggested some N-glycosyl modifications and fairly high homology (60%) with that of the β-glucosidase which hydrolyzes cyanogenic β-D-glucopyranosides. The recombinant enzyme easily agglutinated to loose its activity, suggested that the N-glycosyl modifications are necessary for the enzyme stability.2) Screening of microbials with the β-primeveroside hydrolyzing activity Soil-born microbial strains were screened using eugenyl β-primeveroside as a sole carbon sauce to give 4 active strains with the activity. The most active strain was identified as Aspergillus fumigatus Pr-20. The glycosidase purified from the culture medium of the strain showed a single band at 47 kDa in SDS-PAGE analysis and was found to hydrolyze other β (1→6)-disaccharide glycosides as well as 6-acylated β-D-glucopyranoslde.3) Gene cloning of the β-primeverosidase-like diglycosidase from A.fumigatusThe amine acid alignment of the diglycosidase revealed by the gene cloning showed only 14% identity with that of the tea leave β-primeverosidase. The overexpression of the gene was attempted in a yeast and a fungus to give a successful result with a fungus.4) A few trials to enhancing aroma from endogenous plant aroma precursors by treatment with the diglycosidaseThe diglycosidase was applied to crude tea leave extracts and grape juice and liberated aroma was analyzed by GC.Many kinds of aroma compounds were found to be much enhanced by this treatment, suggesting that this new enzyme will be applicable to enhance aroma in black tea and wine production and their related industries. Less