Molecular Basis of Alcoholic Aroma Formation Mechanism in Oolong Tea and Jasmine Tea

乌龙茶和茉莉花茶醇香形成机制的分子基础

• 批准号: 07456060
• 负责人: SAKATA Kanzo
• 金额: $ 3.39万
• 依托单位: Shizuoka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07456060/
• 关键词: Oolong tea; Jasmine tea; Tea aroma; Aroma precursor; Primeveroside; Primeverosidase; Camellia sinensis; p-Nitrophenyl beta-primeveroside; 配糖体; β-プリメベロシダーゼ; 酵素合成; β-プリメベロシド; ジャスミン茶; 茉莉花; 香気生成機構; β‐プリメベロシダーゼ; リナロールオキシド; 2‐フェニルエタノール

1) Study on the molecular basis of the alcoholic aroma formation mechanism in oolong tea :We have isolated and identified aroma precursors in most of the alcoholic aroma of oolong tea (Camellia sinensis var.sinensis cv.Shuixian and Maoxie) as glycosides. Most of them were found to be beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides), but the aroma precursors of linalool oxides III and IV (cis-and trans-linalool 3,7-oxides) and (Z)-3-hexenol were exceptionally isolated as 6-O-beta-D-apiofuranosyl-beta-D-glucopyranosides and beta-D-glucopyranoside, respectively.As a preliminary experiment, we have purified a beta-primeverosidase from fresh tea leaves (cv.Yabukita), which showed high substrate specificity toward beta-primeverosides, which are hydrolyzed into primeverose and the corresponding aglycon. As we succeeded in enzymatic synthesis of p-nitrophenyl beta-primeveroside as shown bellow, we tried to purify beta-primeverosidases from fresh tea leaves of green tea (c … More v.Yabukita) and oolong tea (cv.Shuixian). Both enzymes have been shown to be the same enzyme with very similar molecular weight (61KDa by SDS-PAGE analysis). On the basis of the foregoing most of the alcoholic tea aroma such as linalool, geraniol, etc.contributing to the floral aroma of oolong tea have been shown to be generated from their glycosidic aroma precursors (beta-primeverosides) by the action of an endogenous beta-primeverosidase during tea processing.2) Study on the molecular basis of the alcoholic aroma formation mechanism in Jasmine tea : We have isolated, for the first time, aroma precursors of linalool, 2-phenylethanol, and benzyl alcohol as their disaccharide glycosides. We also tried to purify the glycosidases which are concerned with the hydrolysis of the aroma precursors to liberate floral aroma from the acetone powder prepared from flower buds of Jasminum sambac just after flowering. After chromatographic separation we found that three kinds of glycosidases are at least concerned with the hydrolysis of the aroma precursors and these enzymes hydrolyze the disaccharide aroma precursors into aglycon (alcoholic aroma) and disaccharide.3) Enzymatic synthesis of p-nitrophenyl beta-primeveroside (pNP-Pri) : We found that a new enzyme, beta-primeverosidase, is one of the most important glycosidases in the alcoholic aroma formation in tea leaves as well as flowers. We tried to enzymatically synthesize pNP-Pri by transglycosylation reaction. We screened several kinds of commercially available enzymes for transxylosylation activity to find that a few enzymes can transfer a xylose moiety of xylobiose to 6'-position of pNP-beta-D-glucopyranoside. After partial purification of one (Pectinase G) of the enzymes and optimization of the reaction conditions we were able to obtain pNP-beta-primeveroside in an appreciable yield. This useful substrate has let us to effective purify the beta-primeverosidases from tea leaves and flower buds of J.sambac and determine their enzymatic properties. Less

1)乌龙茶醇类香气形成机制的分子基础研究：我们分离并鉴定了乌龙茶（水仙茶和毛蟹茶）醇类香气中的大部分香气前体化合物为苷类化合物。其中大部分是β-樱草苷（6-O-β-D-吡喃木糖基-β-D-吡喃葡萄糖苷），但芳樟醇氧化物III和IV的芳香前体（顺式-和反式-芳樟醇3，7-氧化物）和（Z）-3-己烯醇分别例外地分离为6-O-β-D-阿皮奥呋喃糖基-β-D-吡喃葡萄糖苷和β-D-吡喃葡萄糖苷。我们已经从新鲜茶叶（Cv.Yabukita）中纯化了β-樱草糖苷酶，该酶对β-樱草苷显示出高底物特异性，β-樱草苷被水解成樱草糖和相应的糖苷配基。由于我们成功地酶促合成了对硝基苯基β-樱草苷，如下所示，我们尝试从绿色茶（c ...更多信息 v. Yabukita）和乌龙茶（水仙）。这两种酶已被证明是具有非常相似的分子量（通过SDS-PAGE分析为61 KDa）的相同酶。在此基础上，对乌龙茶花香有贡献的芳樟醇、香叶醇等醇类茶香气，大部分都是由糖苷类香气前体物质产生的。（β-樱草苷）的作用。2）茉莉花茶中醇类香气形成机理的分子基础研究：我们已经分离出，为第一次，芳樟醇，2-苯基乙醇和苯甲醇的香气前体作为其二糖糖苷。我们还试图从刚开花的茉莉花蕾制备的丙酮粉中纯化与水解香气前体物以释放花香有关的糖苷酶。层析分离后发现，三种糖苷酶至少参与了香气前体的水解，并将二糖类香气前体水解为糖苷配基3）对硝基苯基β-樱草苷（pNP-Pri）的酶促合成：我们发现一种新的酶--β-樱草糖苷酶是茶叶和花中醇类香气形成过程中最重要的糖苷酶之一。我们尝试通过转糖基化反应酶促合成pNP-Pri。我们筛选了几种市售酶的转木糖基活性，发现少数酶可以将木二糖的木糖部分转移到pNP-β-D-吡喃葡萄糖苷的6 ′-位。在部分纯化一种酶（果胶酶G）并优化反应条件后，我们能够以可观的产率获得pNP-β-樱草苷。这种有用的底物使我们能够有效地从茶叶和J.sambac的花蕾中纯化β-PrimeveroD酶并测定其酶性质。少

项目成果

Ogawa, K., Moon, J-H,Guo, W., Yagi, A., Watanabe, N.and Sakata, K.: "A study on tea aroma formation mechanism. Alcoholic aroma precursor amounts and glycosidase activity in part of the tea plant." Z.Naturforsch.50C. 493-498 (1995)

Okawa, K.、Moon, J-H、Guo, W.、Yagi, A.、Watanabe, N.和 Sakata, K.：“茶香气形成机制的研究。部分茶中的酒精香气前体含量和糖苷酶活性

Wenfei Guo: "Isolation and characterization of a β-primeverosidase concerned with alcoholic aroma formation in tea leaves." Bioscience Biotechnology and Biochemistry. 60・11. 1810-1814 (1996)

郭文飞：“与茶叶中酒精香气形成有关的 β-primeverosidase 的分离和表征。” 生物科学生物技术和生物化学 60・11（1996）。

J.-H.Moon: "cis-and trans-Linalool 3,7-oxides and methyl salycilate glycosides and (Z)-3-hexenyl β-D-glucopyranoside as aroma precursors from tea leaves for oolong tea." Bioscience Biotechnology and Biochemistry. 60・11. 1815-1819 (1996)

J.-H.Moon：“顺式和反式芳樟醇 3,7-氧化物、水杨酸甲酯糖苷和 (Z)-3-己烯基 β-D-吡喃葡萄糖苷作为乌龙茶茶叶的香气前体。”生物化学 60・11。1815-1819（1996）

坂田完三: "茶のアルコール系香気生成の分子機構" 第38回天然有機化合物討論会講演要旨集. 511-516 (1996)

Kanzo Sakata：“茶中酒精香气产生的分子机制”第 38 届天然有机化合物研讨会摘要 511-516（1996）。

Moon, J-H., Watanabe, N., Iijima, Y., Yagi, A.and Sakata, K.: "cis-and trans-Linalool 3,7-Oxides and Methyl Salicylate Glycosides and (Z)-3-Hexenyl beta-D-Glucopyranoside as Aroma Precursors from Tea Leaves for Oolong Tea." Biosci.Biotech.Biochem.60 (11).

Moon, J-H.、Watanabe, N.、Iijima, Y.、Yagi, A. 和 Sakata, K.：“顺式和反式芳樟醇 3,7-氧化物和水杨酸甲酯糖苷和 (Z)-3-己烯基 beta