【作者】 刘坤；

【导师】 张正竹； 俞杰；

【作者基本信息】 安徽农业大学 ， 茶学， 2023， 博士

【摘要】 茶叶中的糖苷类香气物质(Glycosidically Bound Volatiles,以下简称GBVs)在茶叶香气的形成中扮演着关键角色。这些物质通过水解反应,来释放出挥发性香气化合物,从而参与了茶叶香气的构建。挥发性芳香物质的含量和组成对于茶树的抗逆性和茶叶的香气品质具有显著的影响。然而,传统的糖苷定量分析方法存在一些挑战,比如时间消耗长、效率低下以及分析效果不尽如人意等诸多问题。这些局限因素不仅制约了对糖苷类香气物质的深入研究,同时也妨碍了对茶叶香气形成机理更为深入的探讨。首先,通过化学合成的方法成功合成了茶叶中的七种糖苷类香气前体物。建立了一种基于超高效液相色谱的全新糖苷检测方法。成功测定了五种不同茶树品种的糖苷类香气前体的含量,能够更深入地从茶鲜叶的角度分析不同品种茶叶的适制性。此外,为了揭示糖苷类香气前体物质在红茶和绿茶加工过程香气形成中的贡献,分别研究了在红茶和绿茶加工过程中糖苷类香气物质含量的变化情况,并取得了以下结果:1.使用3,5-二取代水杨醛和天然氨基酸在乙醇作溶剂条件下一步合成了一类易于制备、结构稳定的阴离子手性钴(Ⅲ)配合物钠盐,该类配合物中心配位金属不会与底物分子发生直接或间接的相互作用,仅在反应中作为手性源进行立体选择性控制。此外,配体基团的空间大位阻使它成为一类弱配位阴离子,使碱金属钠离子可作为路易斯酸。这类催化剂可以做为路易斯酸催化多种不对称反应,受到烯糖法合成糖苷的启发,将这类催化剂应用于路易斯酸催化的糖苷合成中。并成功的以中等对映选择性(66%ee)和高立体选择性(α/β=9/1)得到了两种2-卤代糖苷化合物。在后续研究中,用这类催化剂成功实现了高对映选择性分子内溴胺化反应(87%ee),和高对映选择性多组分Post-Ugi反应(98%ee)。2.利用三氯乙酰亚胺酯法,并利用不同的保护基策略,配基为芳香醇(苯甲醇,苯乙醇)时采用了苯甲酰基保护的糖基供体,而配基为脂肪醇(顺式-3-己烯醇,香叶醇)时,采用了乙酰基保护的糖基供体,以高立体选择性和良好收率成功合成了茶叶中的苯甲醇葡萄糖苷和樱草糖苷、苯乙醇葡萄糖苷和樱草糖苷、顺-3-己烯醇葡萄糖苷、香叶醇葡萄糖苷和对硝基苯酚樱草糖苷等七种香气前体物质。3.利用合成的糖苷类化合物作为标准物质,建立了一种采用超高效液相色谱的直接分析方法。这一方法具有多重优点,包括短时间内(25分钟内)完成对六种糖苷的定性和定量分析,样品要求较少(5.0 mg),前处理流程相对简单,以及高效性。此外,观察到各种糖苷在浓度范围为5-100μM之间具有出色的线性关系(R~2>99%),而检测限范围为0.21至3.79μM。这一方法为糖苷分析提供了更为灵活的选择,有望在研究和实际应用中得到广泛应用。4.首次利用合成的对硝基苯酚-β-樱草糖苷检测茶叶中的β-樱草糖苷酶活性。并且通过比色法建立了β-樱草糖苷酶的酶活性测定方法。分析了不同茶树品种不同叶位上β-樱草糖苷酶活性的变化,结果显示,在大多数茶树品种的不同叶位上,嫩叶中β-樱草糖苷酶活性较高。这些结果表明了β-樱草糖苷酶活性在茶叶不同叶位和不同茶树品种之间存在显著的差异。5.分析了红茶、绿茶加工过程中糖苷含量变化,以及在不同茶树品种中的含量差异。在红茶加工过程中,樱草糖苷含量在揉捻过程中下降比较明显。表明,在红茶制备中樱草糖苷受到樱草糖苷酶的水解作用,从而释放出挥发性香气物质,因此红茶香气形成的关键因素是樱草糖苷。在绿茶制备过程中,观察到樱草糖苷和葡萄糖苷的含量都呈上升趋势。发现最终制成的绿茶香气与鲜叶中的樱草糖苷浓度之间存在显著的相关性。强调了樱草糖苷在茶叶制备中的关键作用。另外在五种不同品种的茶鲜叶中,观测到黄山早芽的糖苷含量整体都高于其他品种,也为黄山早芽适制红茶和绿茶做出有力证明。然而,需要指出的是,利用糖苷类物质的含量来预测不同品种茶叶的香气潜力等相关研究仍需要进一步深入探讨。这一领域的研究将有助于更好地理解不同茶叶原料的香气特性,进一步提高茶叶的品质和调控香气的能力。更多还原

【Abstract】 Glycosidically Bound Volatiles(GBVs)in tea leaves play a crucial role in the formation of tea aroma.These compounds release volatile aromatic compounds through hydrolysis,contributing to the aroma profile of tea.The content and composition of these volatile aromatics significantly affect the tea plant’s stress resistance and the aromatic quality of the tea leaves.However,traditional quantitative analysis methods for glycosides face challenges such as time consumption,low efficiency,and unsatisfactory analytical results.These limitations not only impede in-depth research into glycosidic aromatic substances but also hinder a more thorough understanding of the mechanisms behind tea aroma formation.Initially,we successfully synthesized seven types of glycosidic aroma precursors found in tea leaves using chemical synthesis and established a novel glycoside detection method based on Ultra-High Performance Liquid Chromatography(UPLC).We quantified the glycosidic aroma precursors in five different tea plant varieties,enabling a more comprehensive analysis of the suitability of different tea varieties from the perspective of fresh tea leaves.Additionally,to uncover the contribution of glycosidic aroma precursors in the aroma formation during the processing of black and green tea,we studied the changes in the content of these substances during their processing,achieving the following results:1.Using 3,5-disubstituted salicylaldehyde and natural amino acids,we synthesized a class of easily prepared,structurally stable anionic chiral cobalt(Ⅲ)complex sodium salts in ethanol.These complexes,where the central coordinating metal does not interact directly or indirectly with the substrate molecules,only serve as a chiral source for stereoselective control in reactions.Moreover,the large steric hindrance of the ligand groups renders them weakly coordinating anions,allowing the sodium ions to act as Lewis acids.Inspired by the alkene glycosylation method,we applied these catalysts in Lewis acid-catalyzed glycosylation,successfully obtaining two types of 2-halogenated glycoside compounds with moderate enantioselectivity(66%ee)and high stereoselectivity(α/β=9/1).In subsequent studies,these catalysts achieved high enantioselective intramolecular bromoamination(87%ee)and enantioselective multi-component Post-Ugi reactions(98%ee).2.Employing the trichloroacetimidate method and using different protecting group strategies,we synthesized several aroma precursors found in tea leaves.For aromatic alcohol substrates(benzyl alcohol,phenethyl alcohol),glycosyl donors protected with benzoyl groups were used,while for aliphatic alcohols(cis-3-hexenol,linalool),glycosyl donors protected with acetyl groups were employed.This approach successfully synthesized benzyl glucoside and primveroside,phenethyl glucoside and primveroside,cis-3-hexenyl glucoside,linalool glucoside,and p-nitrophenol primveroside in tea leaves with high stereoselectivity and good yield.3.Utilizing the synthesized glycosidic compounds as standard substances,we established a direct analysis method using Ultra-High Performance Liquid Chromatography.This method offers multiple advantages,including the ability to complete qualitative and quantitative analysis of six glycosides within a short time frame(within 25 minutes),requiring a small sample amount(5.0 mg),a relatively simple preprocessing workflow,and high efficiency.Additionally,we observed an excellent linear relationship for various glycosides within a concentration range of 5-100μM(R~2>99%),with detection limits ranging from 0.21 to 3.79μM.This method provides a more flexible option for glycoside analysis and is expected to find widespread application in research and practical use.4.For the first time,we used the synthesized p-nitrophenol-β-primeveroside to detectβ-primeverosidase activity in tea leaves and established a colorimetric method for measuring the enzyme’s activity.The analysis ofβ-primeverosidase activity in different tea tree varieties and leaf positions showed that tender leaves in most tea tree varieties have higherβ-primeverosidase activity.These results indicate significant variations inβ-primeverosidase activity among different leaf positions and tea tree varieties.5.We analyzed the changes in glycoside content during the processing of black and green tea,as well as the content differences among different tea tree varieties.During black tea processing,a noticeable decline in primeveroside content was observed,particularly during the rolling stage,indicating that primeveroside undergoes hydrolysis by primeverosidase,releasing volatile aromatic substances.Therefore,primeveroside is a key factor in the formation.However,it should be noted that further in-depth exploration is required for research related to the utilization of gentiobiosides in predicting the aroma potential of different tea varieties,among other related aspects.Research in this domain holds the promise of enhancing our understanding of the aromatic characteristics of diverse tea raw materials and,subsequently,improving the quality and capacity for aroma regulation in tea production.更多还原