【作者】 张佳；

【导师】 阮建云；

【作者基本信息】 中国农业科学院 ， 茶学， 2010， 硕士

【摘要】 氨基酸是生物体代谢过程中起重要作用的物质,在许多生理机能方面都起着不可替代的作用。茶氨酸是茶叶中所含有的一种特殊非蛋白质的氨基酸,除此之外,茶叶中主要的游离氨基酸还有谷氨酸、谷氨酰胺、丙氨酸、丝氨酸、天冬氨酸等。并且氨基酸是构成茶叶滋味的重要组成部分,其含量的多少会直接影响茶叶的品质。对于茶叶氨基酸检测已有大量研究,目前主要以仪器分析为主,而应用气相色谱-质谱方法检测茶叶中游离氨基酸尚未见报道。本文研究主要分为两部分:第一部分是应用衍生试剂N-(特丁基二甲基硅烷)-N-甲基三氟乙酰胺(≥95%;MTBSTFA)含叔丁基二甲基氯硅烷(1%;TBDMSCl)衍生、气相色谱和气相质谱联用仪测定茶叶中的游离氨基酸方法。比较了衍生温度、时间、酸度及辅助试剂等对衍生效率的影响,提出以正缬氨酸为内标,乙腈为辅助衍生试剂,衍生温度110℃,衍生时间30 min,可以定量检测茶叶中主要的游离氨基酸。茶氨酸产生三个衍生峰,经质谱鉴定第一个峰(按出峰时间)为环状衍生峰,第二个峰为一个N端没有衍生,第三个峰为完全衍生峰。各氨基酸在10～1000nmol范围内线性良好(相关系数均大于0.99),对茶叶样品测定结果与氨基酸自动分析仪基本一致,添加回收表明茶氨酸、谷氨酸、谷氨酰胺、丝氨酸、天冬氨酸等的回收率均85%～108%。结果表明,本文建立的GC-MS及GC-FID方法操作比较简便、准确,重现性好,适用于茶叶中主要游离氨基酸的分离检测。第二部分是利用气相及气相色谱-质谱联用(GC-MS)检测稳定性同位素15N标记不同氮源吸收的茶叶中主要游离氨基酸(茶氨酸、丙氨酸、谷氨酸、谷氨酰胺等),初步建立了检测方法。稳定同位素示踪技术应用较广,但是利用稳定同位素研究茶叶中主要游离氨基酸特别是茶氨酸合成与代谢方面尚无报道。应用稳定同位素方法比常规的氮代谢平衡技术以及单纯测定体内氨基酸谱的方法更优越,可以动态的观察外源氮进入蛋白质、氨基酸代谢过程,深入了解它们的代谢动态变化。通过MTBSTFA衍生后M-57特征离子峰分析,用纯度>99%的丙氨酸、谷氨酸及谷氨酰胺与普通氨基酸进行0、20%、40%、60%、80%、100%比例混合,结果符合检测要求。而实验中检测标记的茶氨酸情况比较复杂,共有十种情况,分为标记一个氮和标记两个氮两大类,并分别进行了分析。同时选取温室吸收48h后的根与茎叶与对照进行比较,茶氨酸的标记不明显,而谷氨酸、谷氨酰胺及丙氨酸等都有不同程度的差异。此方法的建立为代谢吸收实验提供研究基础。更多还原

【Abstract】 Free amino acids are important metabolites and contributor to tea quality. L-Theanine is a particular non-protein amion acid in tea,in addition to this, the main free amino acids in tea include Glu, Gln, Ala, Ser, Asp and so on. And amin acids have close relation to flavor of tea, the content of amino acids influence the quality of tea directly. As the methods are improved rapidly in recent years, and currently we primarily use instrument for the determination and identification of amino acids. But there is no report that use Gas Chromatography-Mass Spectrometry(GC-MS) determination of main free amino acids in tea. The dissertation consists of two parts : Part one is research and established the mothed about Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography-Flame Ionization Detector (GC-FID) in determination of main free amino acids in tea. The derivative reagent used in this study was N-tertbutyldimethylsily N-methytrifluroacetemide (MTBSTFA,≥95%) with tert-butyldimethylsilyl chloride (TBDMSCl, 1%). Conditions of reagent, temperature, time, and acidity for derivatization were optimized to give high efficiency. The optimized procedure was as follows: dried amino acids standards or tea extract (water) under N2 gas were redissolved in acetonitrile (ACN) and derivatized with MTBSTFA (1:1 to ACN) at 110℃for 30 min. Analysis of pure amino acid standards showed good linearity (r2≥0.99) from 10 to 1000 nmol. Three peaks were detected for theanine with GC and MS data indicated that only the third peak was the completely derivatized product while the second peak was a product of one N-end derivatization. Free amino acids in tea sample were quantified by the developed methods and the results were comparable to those analyzed by Amino Acids Autoanalyzer. Recoveries of theanine, alanine, serine, aspartate, glutamate, and glutamine were 85%-108% range for spiked tea extract.Part two is about use Gas Chromatography-Mass Spectrometry (GC-MS) analysis the main free amino acids in tea (Thea, Ala, Glu, Gln )which absorbed different nitrogen source labelled stable isotope 15N. The application of stable isotopic tracer technology is universally, but the study of isotopomer distributions in tea has not been reported. GC-MS is a rapid method that provides rich information on isotopomer on isotopomer distributions for metabolic flux analysis.After derivatized with MTBSTFA , analysed the characteristic ion peak M-57, mass spectroscopy gave rise to characteristic spectra for all analysed compounds and established the detection method preliminary. Used Amino acid standards enriched with different proportions (0, 20, 40, 60, 80 and 100%) of universally (U) 15N-labelled Ala, Glu, Gln.(amion acids containing > 98% 15N at all nitrogen positions), were used to calibrate the method for isotopic enrichment of the individual amino acids. The tea plant were placed in a greenhouse ,and were supplied with 15NH4+ for 48h. A comparative analysis between control and 48h show that there are difference in labelled amino acids (theanine not very obviously). The developed GC-MS method provided a potential methodology to investigate nitrogen metabolism in tea plants by quantifying the abundance of 15N labeled amino acids.更多还原