茶叶微生物固态发酵中咖啡碱降解途径初探

为探究微生物作用下咖啡碱的降解产物与途径,将普洱茶发酵中筛选鉴定的Aspergillus sydowii NRRL250（聚多曲霉）、Aspergillus pallidofulvus NRRL4789、Aspergillus sesamicola CBS137324和Penicillium mangini CBS253.31等优势菌株分别接种至晒青毛茶进行单菌种固态发酵,并采用高效液相色谱（HPLC）测定咖啡碱、可可碱、茶碱的含量,探究微生物对咖啡碱代谢的影响;另外,基于UHPLC-QTOF-MS代谢组学技术,以灭菌处理组（ST组）和原料组（RM组）为对照,对聚多曲霉接种发酵样进行代谢组学分析。结果表明,A. pallidofulvus NRRL4789、A. sesamicola CBS137324和Penicillium mangini CBS253.31等优势菌株对咖啡碱等嘌呤类碱代谢均无显著影响,而在聚多曲霉接种发酵中,咖啡碱含量显著下降（P<0.05）,降幅达83.89%;茶碱含量显著增加（P<0.05）,发酵末期含量为(25.03±1.17) mg·g^-1;而可可碱保持基本稳定。由此可知,聚多曲霉对咖啡碱降解代谢有显著影响。采用UHPLC-QTOF-MS方法检出茶碱、3-甲基黄嘌呤、1,7-二甲基黄嘌呤等9种与咖啡碱降解相关的代谢物。在聚多曲霉作用下,茶碱、3-甲基黄嘌呤、1,7-二甲基黄嘌呤、7-甲基黄嘌呤含量显著提高（P<0.05）。茶碱、3-甲基黄嘌呤、1,7-二甲基黄嘌呤和1-甲基黄嘌呤与咖啡碱及其相关代谢物的N-脱甲基化途径相关。1,7-二甲基尿酸、1-甲基尿酸与咖啡碱相关代谢物的氧化途径相关。由此可知,聚多曲霉为降解普洱茶咖啡碱的优势菌株,且具有将咖啡碱转化为茶碱的潜在能力;在咖啡碱降解代谢过程中,存在聚多曲霉作用下的N-脱甲基化和氧化,并以N-脱甲基化为主。     

普洱茶渥堆发酵中可降解咖啡碱真菌菌株的筛选和鉴定

为了在普洱茶渥堆发酵中筛选出可降解咖啡碱的菌株,采用咖啡碱液体筛选培养基从不同阶段普洱茶发酵样中筛选目标菌株,结合菌落特征、分生孢子结构以及18 S r DNA序列对目标菌株进行鉴定。并将目标菌株接种至茶叶内进行单菌种发酵,通过高效液相色谱法(HPLC)测定咖啡碱、茶碱、可可碱、3-甲基黄嘌呤等嘌呤碱含量。结果表明,在普洱茶发酵样中筛选出一株有效降解咖啡碱的菌株,此菌株在48 h内对咖啡碱的降解率为87.7%。经鉴定为聚多曲霉(Aspergillus sydowii),序列相似性为99.8%。在聚多曲霉茶叶单菌种发酵中,咖啡碱含量急剧下降,在发酵结束时仅占干物质重的(0.414±0.077)%;茶碱质量分数大幅度上升,由最初的不足0.1%上升至(2.129±0.246)%,成为主要的嘌呤碱;可可碱含量无显著变化(P0.05);3-甲基黄嘌呤有大幅度增加,增幅达130%。本文首次从发酵茶叶中筛选出可降解咖啡碱的菌株,探究了微生物发酵中咖啡碱的转化产物,为低咖啡碱普洱茶的开发提供了菌种。     

茶树咖啡碱合成酶CRISPR/Cas9基因组编辑载体的构建

CRISPR/Cas9技术是一门新兴的基因组定点编辑技术,具有操作简单、高效的优点,可轻松实现对目标基因的敲除、替换和定点突变等操作。该技术刚诞生,就受到了全球生命科学领域研究者的关注,不到3年的时间就已经成功应用于多种动、植物当中。然而CRISPR/Cas9技术在茶树中的应用面临载体构建问题,本文以茶树咖啡碱合成酶为例,联合采用常规PCR、Overlapping PCR和Golden Gate Cloning技术,构建了包含茶树咖啡碱合成酶双靶点的CRISPR/Cas9基因编辑载体,为CRISPR/Cas9介导的基因组编辑技术在茶树中的应用奠定了坚实基础。     

Inheritance studies of caffeine and theobromine content of Mate (Ilex paraguariensis) in Misiones, Argentina

In order to start a genetic improvement program for quality in the Mate crop (Ilex paraguariensis), it was attempted to estimate the actual genetic parameters of caffeine, theobromine and related quality traits, using available progeny tests in Misiones, Argentina. Using cluster analysis, eight groups of similar characteristics could be identified, and individual within full-sib family selection for quality was performed. Additive effects were strong for caffeine but weak for theobromine. Co-heritability between caffeine and theobromine was positive, suggesting that additive genetic correlations and dominance correlations are concurring. The strong influence of paternal progenitors regarding caffeine needs more research. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biochemical diversity in the genus Coffea L.: chlorogenic acids,caffeine and mozambioside contents

Summary The extent and nature of biochemical diversity in green coffee beans was established by a large sampling of genus Coffea, representing nine species from the Guineo-Congolian region, nine species from east Africa and seven species from Madagascar. HPLC analyses were used to determine the contents of caffeine, chlorogenic acids and mozambioside. Data processed by principal component analyses showed the existence of two metabolic pathways. One leads to the synthesis of small quantities of chlorogenic acids (< 2.5% dmb) and caffeine ( < 0.3 % dmb) while the other leads to large concentrations of chlorogenic acids (> 4.5% dmb) and caffeine (> 0.4% dmb). Their distribution in the genus Coffea is discussed in relation to the biogeographic origin of the plant material.     

用超临界CO2脱除绿茶浓缩液中咖啡碱的工艺研究

对绿茶浓缩液中咖啡碱的超临界CO₂萃取工艺进行了研究，通过4因素正交试验探讨了浓缩液的浓度、操作压强、操作温度、萃取时间对咖啡碱脱除率的影响。结果表明，超临界CO₂萃取技术可以有效地脱除绿茶浓缩液中大部分的咖啡碱，在此基础上完成了用超临界CO₂脱除绿茶浓缩液中咖啡碱的连续作业试验，从而获得了加工脱咖啡碱绿茶浓缩液或速溶绿茶的新工艺。     

Effects of dietary caffeine on growth,body composition,somatic indexes,and cerebral distribution of acetyl‐cholinesterase and nitric oxide synthase in gilthead sea bream (Sparus aurata), reared in winter temperature

This study aims at examining the effect of caffeine administration on growth, feed efficiency, and consumption of sea bream (Sparus aurata), reared in winter temperatures. Moreover, it is questioned whether caffeine has a central action in the brain and its effects are partly mediated via central brain mechanisms. For this, we studied the influences of caffeine treatment on the cerebral pattern of the cholinergic neurotransmission and the novel neuromodulator nitric oxide (NO), by means of acetyl‐cholinesterase (AchE) and nitric oxide synthase (NOS) histochemistry. Five different diets containing 0.0, 0.1, 1.0, 2.0 and 5.0 g caffeine kg^?1 of diet were administrated to five groups of fish. Caffeine adversely affected sea‐bream growth at a concentration higher than 1 g kg^?1 diet and increased feed conversion ratio in the treatments of 2 and 5 g kg^?1 (P < 0.05). The daily consumption of feeds was similar to all groups, indicating that caffeine did not influence diet palatability. AChE‐ and NADPH‐diaphorase histochemistry showed densely labeled cells and fibers mainly in dorsal telencephalon, preoptic, pretectal, hypothalamic areas, optic tectum, reticular formation, cerebellum and motor nuclei. When compared with matched caffeine‐treated animals, no differences in the histochemical pattern and cell densities of cerebral AChE and NADPH‐diaphorase were found.     

茶树咖啡碱合成酶基因cDNA在烟草中的表达

为了在烟草中合成咖啡碱,将咖啡碱合成酶(Tea caffeine synthase,TCS)基因cDNA全序列克隆到双元载体pBI121中,通过农杆菌叶盘转化法将TCS基因成功转入烟草,结果得到72株转基因烟草植株。对其中3株转基因植株进行PCR检测和PCR-Southern检测,证实TCS基因已整合到基因组中;RT-PCR和Northern分析显示,TCS在这些植株中均能正常转录。用从转基因植株中提取的粗酶液进行体外酶促反应,能催化7-甲基黄嘌呤和可可碱向咖啡碱的转变,由此表明,转基因烟草植株中能产生具有正常生物学活性的TCS,但从3株转基因烟草中均未检测到咖啡碱。     

咖啡因Na_2─EDTA对不同辐射敏感性作物质膜过氧化的维护效应

本文研究了咖啡因和Ｎａ２─ＥＤＴＡ辐照后处理对大豆和油菜７日龄幼苗质膜过氧化水平的生物学效应。结果表明，随着辐射剂量的增加，细胞膜透性、丙二醛、脯氨酸含量和过氧化物酶活性增大，而超氧化物歧化酶和过氧化氢酶则先增大而后降低。辐照后，大豆幼苗质膜过氧化程度高于油菜。咖啡因、ＥＤＴＡ后处理降低了两种作物的细胞膜过氧化水平和丙二醛含量，提高了脯氨酸含量和超氧化物歧化酶，过氧化氢酶和过氧化物酶的活性，减轻了辐射对细胞膜产生的损伤。咖啡因和ＥＤＴＡ后处理对油菜质膜的保护作用强于大豆，ＥＤＴＡ的作用效应强于咖啡因，咖啡因与ＥＤＴＡ复合作用协同效应不明显。     

嫁接对茶树新梢化学成分的影响

以云南大叶种为砧木，乌牛早和浙农113为接穗进行嫁接试验，并与同期移栽的乌牛早和浙农113进行化学成分分析比较。结果表明，嫁接对该2品种的化学成分含量的明显影响，氨基酸和咖啡因含量增加；茶多酚和主要儿茶类素含量减少。