武夷岩茶炭焙工艺对肉桂乌龙茶挥发性组分的影响

以武夷岩茶当家茶树品种肉桂(Camellia sinensis ‘Rougui’)鲜叶制成的乌龙茶为试材,基于顶空固相微萃取法(HS-SPME)结合气相色谱-质谱联用技术(GC-MS),探讨武夷岩茶炭焙工艺对肉桂乌龙茶挥发性组分的影响。实验中共检测到样本挥发性成分443个,其中包括96个杂环化合物、81个酯类化合物、31个萜类化合物、42个芳香烃类化合物、55个酮类化合物、24个其他烃类化合物、35个醇类化合物、24个醛类化合物、15个酚类化合物、14个胺类化合物、10个酸类化合物、5个含氮化合物、3个含硫化合物等。经主成分分析及聚类分析显示,焙火工艺是影响乌龙茶挥发性成分含量的重要影响因子。随炭焙程度增加,不同类别物质中的多数挥发性成分含量随之显著升高,且以美拉德反应产物等具有茶叶烘炒香的吡嗪类、糠醛类衍生物、吡咯类等化合物最具代表性;同时,部分含量丰富且具有乌龙茶特殊花果香气的醇类、萜烯类物质如香叶醇、反式-橙花叔醇、植物醇、α-法尼烯等,及具清新花香的吲哚含量显著降低。然而,大多数挥发性差异代谢物随炭焙程度增加相对含量显著升高,并不代表茶叶中芳香物质总量增加。研究表明,乌龙茶精制烘焙过程中,香气物质的积累主要来自热作用下的美拉德反应及非酶促的降解和氧化,如黄酮苷类物质水解。     

Cloning of beta-primeverosidase from tea leaves,a key enzyme in tea aroma formation

A beta-primeverosidase from tea (Camellia sinensis) plants is a unique disaccharide-specific glycosidase, which hydrolyzes aroma precursors of beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides) to liberate various aroma compounds, and the enzyme is deeply concerned with the floral aroma formation in oolong tea and black tea during the manufacturing process. The beta-primeverosidase was purified from fresh leaves of a cultivar for green tea (C. sinensis var sinensis cv Yabukita), and its partial amino acid sequences were determined. The beta-primeverosidase cDNA has been isolated from a cDNA library of cv Yabukita using degenerate oligonucleotide primers. The cDNA insert encodes a polypeptide consisting of an N-terminal signal peptide of 28 amino acid residues and a 479-amino acid mature protein. The beta-primeverosidase protein sequence was 50% to 60% identical to beta-glucosidases from various plants and was classified in a family 1 glycosyl hydrolase. The mature form of the beta-primeverosidase expressed in Escherichia coli was able to hydrolyze beta-primeverosides to liberate a primeverose unit and aglycons, but did not act on 2-phenylethyl beta-D-glucopyranoside. These results indicate that the beta-primeverosidase selectively recognizes the beta-primeverosides as substrates and specifically hydrolyzes the beta-glycosidic bond between the disaccharide and the aglycons. The stereochemistry for enzymatic hydrolysis of 2-phenylethyl beta-primeveroside by the beta-primeverosidase was followed by (1)H-nuclear magnetic resonance spectroscopy, revealing that the enzyme hydrolyzes the beta-primeveroside by a retaining mechanism. The roles of the beta-primeverosidase in the defense mechanism in tea plants and the floral aroma formation during tea manufacturing process are also discussed.     

Production of volatiles by degradation of lipids during manufacture of black tea

During manufacture of black tea, lipids are degraded to volatile constituents. Cis-3-hexenal was present in appreciable amounts in the various parts of fresh shoots and decreased in the second leaves during manufacture. There was a simultaneous increase in trans-2-hexenal. Linalol and methyl salicylate also increased appreciably during rolling and fermentation. Most of the volatiles were lost during the firing process. The above trend was borne out by the ‘potential’ of the leaves for the production of volatiles as indicated by the increased amounts of volatiles produced by homogenizing the tissue in water against controls homogenized in 0.1 N acid. The C₆-aldehydes present in the headspace of withered shoots increased significantly following mechanical damage. The major fatty acids of the lipids in the various parts of the shoots were linolenic, linoleic, palmitic, oleic and stearic acids. The ratio of linoleic to linolenic acid in the stems was much higher than that of the leaves or buds and this was reflected in its higher 'potential for formation of hexanal. During withering and rolling of the second leaves, the unsaturated fatty acids showed substantial losses compared with the saturated acids. It is suggested that the enzymic breakdown of membrane lipids initiate the formation of volatile carbonyl compounds which are partly responsible for the flavour of black tea.     

Volatile and Non-volatile Forms of Aroma Compounds in Tea Leaves and Their Changes due to Injury

Fresh tea leaves were homogenized in a chloroform-methanol mixture (1:1, v/v), and separated into chloroform-soluble and methanol-water-soluble fractions after addition of water. From the chloroform-soluble fraction, the volatile forms of the aroma compounds were obtained. The non-volatile forms of the aroma compounds were associated with the methanol-water-soluble fraction, and were converted to volatile forms by hydrolysis with dilute acid.

The amount of the aroma compounds in the free form, such as cis-3-pentenol, hexanol, cis-3-hexenol, trans-2-hexenol, linalool, linalool oxide (cis, 5-membered), linalool oxide (trans, 5-membered), linalool oxide (trans, 6-rnembered), linalool oxide (cis, 6-membered), nerol, geraniol, phenylmethanol, and 2-phenylethanol, markedly increased during black tea manufacture. However, those in the bound form, showed a slight decrease during the manufacture. The increases in the former were also brought about by maceration, or treatment of the tea leaves with monoiodoacetate or malonate.     

不同香型茶树鲜叶挥发性组分与β-葡萄糖苷酶的相关性分析

为了明确不同香型茶树(Camellia sinensis)品种鲜叶挥发性物质的组成、含量、β-葡萄糖苷酶活性及其基因差异表达特征,以黄观音、0318E、福云6号、0213-2和C19五个四年生茶树品种(系)为实验材料,使用GC-MS测定其鲜叶挥发性物质的组成及含量,并对β-葡萄糖苷酶活性进行测定,同时克隆β-葡萄糖苷酶基因,分析其在不同品种中的表达差异。结果表明,黄观音和0318E的鲜叶中,萜烯类与衍生物种类及含量都高于普通香型茶树鲜叶(福云6号、0213-2和C19);鲜叶中β-葡萄糖苷酶活性以黄观音(7.8 U·g–1)和0318E(7.3 U·g–1)最高,福云6号和C19次之,0213-2(6.1 U·g–1)最低。β-葡萄糖苷酶基因在黄观音和0318E中表达最强,显著高于C19、福云6号和0213-2,在0213-2中的表达量最低。     

Influence of Fungal Fermentation on the Development of Volatile Compounds in the Puer Tea Manufacturing Process

Puer tea is a unique Chinese fermented tea with natural flora manufactured in Yunnan Province of China. Very complex changes take place to form special quality and flavor characteristics in Puer tea due to the coordination of microbial metabolic action and natural oxidation. This paper investigates the isolation and identification of fungi responsible for the fermentation and the development of main volatile compounds of Puer tea during the fermentation process by means of GC/MS. Aldehydes and ketones in parched green tea (raw material) with low boiling‐points decreased significantly, while the amount of terpene alcohols – such as linalool and linalool oxides, methoxybenzene and derivatives, and indole – increased remarkably from the parched green tea to the Puer tea product after the manufacturing process. Degradation caused by heat and microbial growth at the piling stage likely played a key role in the generation of these compounds that contributed to the aromatic characteristics of Puer tea. It is hypothesized that the fungus Aspergillus niger plays a decisive role in the development of the volatile compounds.     

Productivity and biochemical properties of green tea in response to full-length and functional fragments of HpaG Xooc, a harpin protein from the bacterial rice leaf streak pathogen Xanthomonas oryzae pv. oryzicola

Harpin proteins from plant pathogenic bacteria can stimulate hypersensitive cell death (HCD), drought tolerance, defence responses against pathogens and insects in plants, as well as enhance plant growth. Recently, we identified nine functional fragments of HpaG;Xooc, a harpin protein from Xanthomonas oryzae pv.oryzicola, the pathogen that causes bacterial leaf streak in rice. Fragments HpaG;1-94'HpaG;10-42, and HpaG;62-138, which contain the HpaG;Xooc regions of the amino acid sequence as indicated by the number spans, exceed the parent protein in promoting growth, pathogen defence and HCD in plants. Here we report improved productivity and biochemical properties of green tea (Camellia sinensis) in response to the fragments tested in comparison with HpaG;Xooc and an inactive protein control. Field tests suggested that the four proteins markedly increased the growth and yield of green tea, and increased the leaf content of tea catechols, a group of compounds that have relevance in the prevention and treatment of human diseases. In particular, HpaG;1-94 was more active than HpaG;Xooc in expediting the growth of juvenile buds and leaves used as green tea material and increased the catechol content of processed teas. When tea shrubs were treated with HpaH;Xooc and HpaG;1-94 compared with a control, green tea yields were over 55% and 39% greater, and leaf catechols were increased by more than 64% and 72%, respectively. The expression of three homologues of the expansin genes, which regulate plant cell growth, and the CsCHS gene encoding a tea chalcone synthase, which critically regulates the biosynthesis of catechols, were induced in germinal leaves of tea plants following treatment with HpaG;1-94 or HpaG;Xooc. Higher levels of gene expression were induced by the application of HpaG;1-94 than HpaG;Xooc. Our results suggest that the harpin protein, especially the functional fragment HpaG;1-94, can be used to effectively increase the yield and improve the biochemical properties of green tea, a drink with medicinal properties.     

Substrate specificity of beta-primeverosidase, a key enzyme in aroma formation during oolong tea and black tea manufacturing

We synthesized nine kinds of diglycosides and a monoglycoside of 2-phenylethanol to investigate the substrate specificity of the purified beta-primeverosidase from fresh leaves of a tea cultivar (Camellia sinensis var. sinensis cv. Yabukita) in comparison with the apparent substrate specificity of the crude enzyme extract from tea leaves. The crude enzyme extract mainly showed beta-primeverosidase activity, although monoglycosidases activity was present to some extent. The purified beta-primeverosidase showed very narrow substrate specificity with respect to the glycon moiety, and especially prominent specificity for the beta-primeverosyl (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosyl) moiety. The enzymes hydrolyzed naturally occurring diglycosides such as beta-primeveroside, beta-vicianoside, beta-acuminoside, beta-gentiobioside and 6-O-alpha-L-arabinofuranosyl-beta-D-glucopyranoside, but were unable to hydrolyze synthetic unnatural diglycosides. The purified enzyme was inactive toward 2-phenylethyl beta-D-glucopyranoside. The enzyme hydrolyzed each of the diglycosides into the corresponding disaccharide and 2-phenylethanol. These results indicate the beta-primeverosidase, a diglycosidase, to be a key enzyme involved in aroma formation during the tea manufacturing process.     

Characterization of novel small RNAs from tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> L.)

Small RNAs play important roles in plant development, metabolism, signal transduction and responses to biotic and abiotic stresses by affecting gene expression. Tea (Camellia sinensis L.) is an important commercial crop in the world. To understand the regulatory mechanisms involving small RNAs in tea metabolism, we constructed a small RNA (sRNA) library from its tea drink manufacturing tissue part i.e. topmost two leaves and a bud. For the first time, we isolated and cloned six novel small RNAs candidates from tea. These were predicted to target 67 genes responsible for various important plant functions. Isolated small RNAs were validated through expression analysis in young leaf and old leaf during non-dormant and dormant growth phases of tea. Results suggest the probable role of isolated small RNAs in development and seasonal variations of tea.     

茶树硝酸盐转运蛋白基因的克隆和表达分析

硝酸盐转运蛋白（NRT）是植物吸收和利用硝态氮的一种关键蛋白。运用RACE技术从茶树中扩增出NRT基因的cDNA,并利用实时荧光定量PCR检测了CsNRT基因在不同茶树器官与品种之间的差异表达。结果表明：CsNRT基因的cDNA全长2 061 bp,开放阅读框为1 818 bp,编码含由605个氨基酸组成的蛋白质,GenBank登录号为KJ160503,属于NRT2基因家族。CsNRT为组成型基因,对不同处理的水培茶苗进行定量表达分析显示,该基因在根、茎、叶中都有表达,其中在根部的表达水平最高,1.0 mmol·L-1的NO3-可诱导其表达量上升7.53倍。不同茶树品种中CsNRT基因的表达也有较大差异,‘龙井长叶’和‘凫早2号’的表达量较高,前者强烈响应0.5和1.0 mmol·L-1 NO3-的诱导,后者的响应浓度为1.0和2.0mmol·L-1,而‘舒茶早’在各浓度下的表达差异不明显。     

茶树CsCCD基因家族全基因组鉴定及乌龙茶LED补光晾青下表达分析

类胡萝卜素裂解双加氧酶(carotenoid cleavage dioxygenase, CCD)家族成员能催化类胡萝卜素裂解生成挥发性芳香物质并参与植物激素的合成。为探究茶树CsCCD基因家族成员生物学功能及基因表达模式,采用生物信息学手段进行了茶树全基因组CsCCD基因家族成员的鉴定,预测分析了其基因结构、保守基序、染色体定位、蛋白的理化性质、进化特性、互作网络、启动子顺式作用元件,并通过RT-qPCR测定了茶树不同叶位、乌龙茶加工过程中光照处理下CsCCD的相对表达量。共鉴定出11个茶树CsCCD基因家族成员,含有1–11个外显子、0–10个内含子不等;平均氨基酸个数为519 aa,平均分子质量为57 643.35 Da;聚类分析显示,CCD1、CCD4、CCD7、CCD8和NCED5个亚族各自聚成一类。茶树CsCCD基因家族主要含有胁迫响应元件、激素响应元件、光响应元件与多因素响应元件,且以光响应元件最多(142个)。进一步对茶树CsCCD基因在茶树不同叶位及乌龙茶加工过程中LED补光晾青过程的表达模式分析发现,CsCCD1、CsCCD4在成熟叶中表达量高于嫩叶及嫩茎,且随做青...     

Preliminary Studies on Flavour Volatiles in Qimen Black Tea

Three kinds of flavor enriched black tea in the world are produced from Qimen in China, Darjeeling in India and Uva in Sri Lanka. The results of analysis from the Qimen black tea showed that there are more than twenty flavour volatiles. The essential aromatic compounds are geraniol, 2,6-di-butul-l,4-benzoquinone, 2,6-di-t-butyl-4-methyl phenol, benzenemethanol, benzenethanol, linalool and linalool oxides. In Qimen black tea the aroma acquired is produced during the processing aside from the original compounds of fresh leaves.     

茶叶叶绿体的嗜锇颗粒含量和适制茶类的关系

应用电子显微镜技术,从细胞亚显微水平研究了81个茶叶品种(或品系)的叶绿体内嗜锇颗粒的分布情况及其适制茶类的关系。结果表明,适制乌龙茶的茶树品种嗜锇颗粒含量都比较多,而有特殊香气的凌云白毛茶嗜锇颗粒含量高达100—46(平均值58),认为嗜锇颗粒含量与成茶香气有关,研究不同茶树品种的嗜锇颗粒含量可为茶树品种的成茶适制性和育成高香型品种的亲本选择提供参考依据。     

Chemical Compositions and Antioxidant Activity of Essential Oil from Green Tea Produced from Camellia taliensis (Theaceae) in Yuanjiang,Southwestern China

Camellia taliensis belonging to Camellia sect. Thea (Theaceae) is distributed from the western and southwestern areas of Yunnan Province, China to the north of Myanmar. Known as the “wild” tea plant, it has been commonly used for making tea by the local people of its growing area. It is the first investigation of the volatile constituents of the fresh tender leaves of C.taliensis and green teas produced from its tender and older leaves. The volatile constituents were obtained by hydrodistillation and analyzed by GC and GC MS. Ninety one compounds were identified. The results showed that the main compositions of volatile oil of the fresh tender leaves were hexadecanoic acid (30.52%), linoleic acid (19.82%), phytol (8.75%), and geraniol (2.54%), while monoterpenoids (58.51%) composing of linalool (28.43%), hotrienol (1.13%), α terpineol (11.68%), nerol (4.92%) and geraniol (12.34%) were the major volatile components of its green tea product. From the fresh leaves to the green tea products, 28 aroma components were formed. Among then, the content of (Z, Z, Z) 9, 12, 15 octadecatrien 1 ol (peak 77) was up to 1.21% (from tender leaves) and 11.2% (from older leaves), respectively. The DPPH and ABTS+ radical scavenging assays demonstrated a moderate activity of essential oil from the three essential oils of C.taliensis.     

我国西南元江大理茶的挥发性成分及其抗氧化活性(英文)

大理茶 ( Camellia taliensis) 为山茶科山茶属茶组植物,主要分布于云南横断山脉澜沧江至伊洛瓦底江流域,即从云南的西部及西南部至缅甸北部。在其分布区,大理茶亦被称为野生大茶树,常用于加工制作茶叶。采用水蒸气蒸馏法、GC 及 GC/MS 联用技术,首次对大理茶的鲜幼叶和鲜幼叶及老叶分别制成的绿茶中的挥发性成分进行提取和分析,共鉴定出 91 个化合物。研究结果表明,大理茶鲜幼叶的主要香气成分为棕榈酸 ( 30. 52%) ,亚油酸 ( 19. 82%) ,植醇 ( 8. 75%) 和亚麻酸乙酯 ( 2. 54%) 等有机酸及其酯和二萜类,而制成绿茶后,其主要香气成分则为芳樟醇 ( 28. 43%) ,脱氢芳樟醇 ( 1. 13%) ,α-松油醇( 11. 68%) ,橙花醇 ( 4. 92%) 和香叶醇 ( 12. 34%) 等单萜醇类成分。从大理茶鲜叶到由其制成的绿茶,香气成分发生了较大变化,形成了 28 种原鲜叶中未检测到的香气成分,其中,( Z,Z,Z) -9,12,15-十八烷三烯-1-醇的含量分别达到 1. 21% ( 幼叶绿茶) 和 11. 2% ( 老叶绿茶) ,是大理茶制作的绿茶的特征香气成分。DPPH 和 ABTS+自由基清除实验结果显示大理茶鲜叶及其制成的绿茶的挥发性成分均具有一定的抗氧化活性,但均弱于茶多酚的抗氧化活性。     

甜橙SPL基因家族的鉴定及其在成花诱导中的表达分析北大核心CSCD

SQUAMOSA promoter binding protein-like(SPL)基因家族在植物成花转变与花器发育中起重要调控作用。该研究基于甜橙(Citrus sinensis)基因组数据,对甜橙SPL家族成员及其启动子区进行生物信息学分析,并采用qRT-PCR检测其在甜橙不同花期、不同品种花芽和叶片的表达特异性,为深入探讨CsSPLs基因在甜橙成花诱导中的功能奠定基础。结果表明:(1)共鉴定出15个含有SBP保守结构域的甜橙SPL基因,分别命名为CsSPL1~CsSPL15,且分布在6条染色体上。(2)CsSPL1~CsSPL15基因编码的氨基酸长度为130~1075 aa,分子量为14.73~118.75 kD;系统发育分析将15个CsSPLs分成8个亚族,除CsSPL4外,其他CsSPLs均与拟南芥SPL家族成员聚类。(3)顺式作用元件分析表明,CsSPLs启动子中含有大量光响应元件,推测CsSPLs可能在甜橙响应光调控过程中发挥重要作用。(4)转录组数据分析显示,CsSPLs在甜橙愈伤组织、花、叶片和果实中均有表达,其中CsSPL3、CsSPL4、CsSPL7、CsSPL8、CsSPL12、CsSPL13、CsSPL14和CsSPL15在花和叶片中较高表达。(5)qRT-PCR检测显示,CsSPLs在甜橙不同花期、不同品种花芽和叶片组织中的表达均有显著上调趋势,且采样各时期下早花品种‘赣南早’花芽和叶片中CsSPLs的表达量均高于对照品种‘纽荷尔’。研究认为,甜橙成花组织内CsSPLs基因的高表达是其花期提前的主要因素。     

叶绿体基因编码蛋白质在水稻叶片生长过程中的表达研究

叶绿体是绿色植物把光能转化为化学能的重要细胞器.目前,多种植物的叶绿体基因组序列已经获得,对叶绿体内发生的各种生物学过程人们也已经有相当深入的了解,但对叶绿体基因编码蛋白质的表达还所知甚少.用蛋白质印迹实验系统地检测了15个叶绿体基因编码蛋白质在水稻叶片不同生长时期的表达.其中7个与光合作用相关蛋白质的表达具有相似的模式,其表达量随叶片生长而增加,一般在孕穗期、开花期达到最高峰,在成熟期叶片下降,这种模式与水稻生长对光合作用的需求有明显相关性.4个与DNA复制相关的RNA聚合酶在苗期叶片中表达量达到最高,说明这些聚合酶在较早时期发挥作用.4个NADH脱氢酶蛋白质的表达呈2种不同的模式,其中亚基2和4在种子萌发后的早期叶片中就达到最高峰,亚基5和7的最高峰出现在中后期,反映了它们之间功能上的不同.实验结果直观且相对定量地揭示了叶绿体编码蛋白质的表达与叶片生长之间的关联关系,为深入了解其功能提供了重要的线索.     

茶丽纹象甲为害诱导的茶树防御反应分析

基于转录组测序,获得了茶树新梢叶片被茶丽纹象甲为害前后的差异基因表达谱,并从信号监测与转导、转录因子、次生代谢物生成等方面分析了相关基因的表达变化情况,为进一步研究茶丽纹象甲为害诱导的茶树防御机制奠定基础。结果表明:(1)茶丽纹象甲为害茶树新梢叶片后共检测到显著上调表达基因309个,显著下调表达基因297个,这些显著差异表达基因共分成23类。(2)检测到信号监测与转导过程中合计17个单基因上调表达2.0～2.8倍,包括富含亮氨酸重复序列的类受体蛋白激酶基因、促分裂原活化蛋白激酶级联信号系统蛋白激酶基因、茉莉酸信号通路基因、钙离子信号基因、水杨酸代谢通路基因;检测到21个转录因子单基因上调表达1.8～2.8倍,包括bHLH、WRKY、bZIP、MYB、UAF等转录因子;检测到苯丙类、类黄酮及萜类物质代谢过程中合计8个单基因上调表达2.1～2.5倍,包括苯丙酮酸互变异构酶基因、肉桂酰辅酶A还原酶基因、二氢黄酮醇还原酶基因、花色素还原酶基因、法尼基二磷酸合成酶基因、法尼醇脱氢酶基因。研究认为,茶丽纹象甲为害诱导信号转导、转录因子及次生代谢过程中的基因增量表达以机械损伤作用为主,昆虫口腔分泌物对这些基因的诱导表达具有协同促进作用。     

我国西南元江大理茶的挥发性成分及其抗氧化活性

大理茶(Camellia taliensis)为山茶科山茶属茶组植物,主要分布于云南横断山脉澜沧江至伊洛瓦底江流域,即从云南的西部及西南部至缅甸北部.在其分布区,大理茶亦被称为野生大茶树,常用于加工制作茶叶.采用水蒸气蒸馏法、GC及GC/MS联用技术,首次对大理茶的鲜幼叶和鲜幼叶及老叶分别制成的绿茶中的挥发性成分进行提取和分析,共鉴定出91个化合物.研究结果表明,大理茶鲜幼叶的主要香气成分为棕榈酸(30.52％),亚油酸(19.82％),植醇(8.75％)和亚麻酸乙酯(2.54％)等有机酸及其酯和二萜类,而制成绿茶后,其主要香气成分则为芳樟醇(28.43％),脱氢芳樟醇(1.13％),α-松油醇(11.68％),橙花醇(4.92％)和香叶醇(12.34％)等单萜醇类成分.从大理茶鲜叶到由其制成的绿茶,香气成分发生了较大变化,形成了28种原鲜叶中未检测到的香气成分,其中,(Z,Z,Z)-9,12,15-十八烷三烯-1-醇的含量分别达到1.21％(幼叶绿茶)和11.2％(老叶绿茶),是大理茶制作的绿茶的特征香气成分.DPPH和ABTS+自由基清除实验结果显示大理茶鲜叶及其制成的绿茶的挥发性成分均具有一定的抗氧化活性,但均弱于茶多酚的抗氧化活性.