中国农业科学 ›› 2021, Vol. 54 ›› Issue (4): 804-819.doi: 10.3864/j.issn.0578-1752.2021.04.012

• 园艺 • 上一篇    下一篇

不同茶叶中游离氨基酸的对映异构体

朱荫(),张悦,严寒,吕海鹏,林智()   

  1. 中国农业科学院茶叶研究所/农业部茶树生物学与资源利用重点实验室,杭州 310008
  • 收稿日期:2020-05-19 接受日期:2020-09-27 出版日期:2021-02-16 发布日期:2021-02-16
  • 通讯作者: 林智
  • 作者简介:朱荫,Tel:0571-87967281;E-mail: zhuy_scu@tricaas.com
  • 基金资助:
    国家自然科学基金(31701702);浙江省自然科学基金(LQ15C160007);现代农业产业技术体系建设专项资金(CARS-19);中国农业科学院创新工程(CAAS-ASTIP-2014-TRICAAS)

Enantiomeric Analysis of Free Amino Acids in Different Teas

ZHU Yin(),ZHANG Yue,YAN Han,LÜ HaiPeng,LIN Zhi()   

  1. Tea Research Institute, Chinese Academy of Agricultural Sciences/Key Laboratory of Tea Biology and Resource Utilization of Ministry of Agriculture, Hangzhou 310008
  • Received:2020-05-19 Accepted:2020-09-27 Online:2021-02-16 Published:2021-02-16
  • Contact: Zhi LIN

摘要:

【目的】游离氨基酸是茶叶的主要化学成分之一,与茶叶的滋味品质密切相关。大多数氨基酸具有手性中心,因此存在滋味特性及生理活性均可能截然不同的L型和D型两种对映异构体。由于检测技术的限制,过去的研究多集中在L型氨基酸上,而茶叶中D型氨基酸罕有系统的研究报道。因此,研究茶叶中游离氨基酸对映异构体,对茶叶化学理论体系的深化及滋味品质的提升与调控具有重要的推进作用。【方法】采用手性HPLC法、MTBSTFA硅烷衍生化法和酯化-PFP酰化法3种不同的分析方法对比茶叶中游离氨基酸对映异构体的分离性能,结果表明采用酯化-PFP酰化法结合手性气相色谱-质谱联用技术(gas chromatography-mass spectrometry,GC-MS),可实现茶叶中丙氨酸、缬氨酸、苏氨酸、脯氨酸、亮氨酸、异亮氨酸、丝氨酸、半胱氨酸、天冬氨酸、蛋氨酸、苯丙氨酸、谷氨酸、酪氨酸、赖氨酸及色氨酸等15种重要游离氨基酸对映异构体的有效分离。继而建立茶叶中游离氨基酸对映异构体的高效衍生化及定性定量分析方法,并查明市售11种代表性白茶、乌龙茶及普洱茶中游离氨基酸对映异构体的分布规律。【结果】衍生化条件为:将氨基酸反应液在100℃下酯化105 min,继而以四氢呋喃为反应溶剂,在100℃下五氟丙酰化10 min。游离氨基酸对映异构体的回收率介于75.26%—123.6%(低浓度)、81.23%—121.8%(高浓度),RSD值介于2.09%—13.12%(低浓度)、1.48%—10.59%(高浓度)。茶样分析结果表明,大部分氨基酸的1—2个对映异构体可以在茶样中检测出,且D-氨基酸普遍存在于茶样中,尤其是D-苏氨酸、D-天冬氨酸、D-半胱氨酸、D-苯丙氨酸、D-茶氨酸、D-谷氨酰胺及D-谷氨酸混合物在大部分茶样中均有分布,而D-蛋氨酸、D-赖氨酸及D-色氨酸在所有茶样中均未被检测出。在具体含量分布上,除了L-茶氨酸、L-谷氨酸及L-谷氨酰胺混合物之外,L-半胱氨酸(1.48—2.08 mg?g-1)、D-半胱氨酸(1.46—1.49 mg?g-1)、L-丝氨酸(0.15—1.80 mg?g-1)及D-天冬氨酸(1.02—1.14 mg?g-1)在白茶中的含量普遍较高;L-半胱氨酸(1.52—1.70 mg?g-1)、D-半胱氨酸(1.45—1.49 mg?g-1)、L-丝氨酸(1.03—1.50 mg?g-1)、L-蛋氨酸(1.03—1.52 mg?g-1)、L-酪氨酸(1.32—1.35 mg?g-1)及D-天冬氨酸(1.01—1.15 mg?g-1)在乌龙茶中具有较高含量。在普洱茶中,L-茶氨酸、L-谷氨酸及L-谷氨酰胺混合物的含量(1.04 mg?g-1)与其他氨基酸相比并无显著性差异,而L-苏氨酸、L-色氨酸及L-丝氨酸含量较高,介于0.61—0.84 mg?g-1。在氨基酸总量上,各茶类呈现出白茶(最高为40.61 mg?g-1)>乌龙茶(最高为25.43 mg?g-1)>普洱茶(8.01 mg?g-1)的分布规律。多元统计分析结果表明,L-茶氨酸、L-谷氨酸及L-谷氨酰胺混合物、L-色氨酸及L-天冬氨酸在部分白茶中的含量显著高于乌龙茶,而L-酪氨酸与L-蛋氨酸则在乌龙茶中含量较高。【结论】D-氨基酸存在于大部分茶叶中,对茶汤滋味品质存在潜在影响,但在本研究样例中,D-氨基酸的含量分布在不同茶类间未表现出显著性差异。

关键词: 茶叶, 游离氨基酸, 对映异构体, 气相色谱-质谱联用技术, 五氟丙酸酐

Abstract:

【Objective】 Free amino acids are the main chemical compositions in teas, and they are closely related to the taste quality of tea. In account of the existence of stereogenic centers, most amino acids contain two enantiomers (D and L configurations) with obviously different taste characteristics and biological activities. However, due to the limitation of detection technology, the studies of L-amino acids were focused in the previous work, and the D-amino acids have been rarely studied. Therefore, the study on the enantiomers of free amino acids in teas is very important for deepening the theoretical system of tea chemistry and is beneficial for improving and controlling of tea taste quality. 【Method】 In this study, the separation performance of free amino acid enantiomers (Chiral HPLC, Silanization of MTBSTFA and Esterification PFP acylation) was compared by using three different analytical methods. A total of 15 pairs of amino acids, including alanine, valine, threonine, isoleucine, leucine, proline, serine, cysteine, aspartic acid, methionine, phenylalanine, glutamic acid, tyrosine, lysine and tryptophan, were effectively separated by using esterification- pentafluoropropionic acylation combined with chiral gas chromatography-mass spectrometry (GC-MS). Furthermore, the efficient derivatization, qualitative and quantitative analysis approaches were established, and the distribution regularities of amino acids enantiomers in 11 representative commercial white, oolong and pu-erh teas were investigated. 【Result】 The derivatization conditions were showed as follows: the reaction mixture was esterified at 100oC for 105 min, and then further acylated by pentafluoropropionic anhydride using tetrahydrofuran as the solvent at 100oC for 10 min. Recoveries of the free amino acid enantiomers were ranged from 75.26% to 123.6% (low concentration) and from 81.23% to 121.8% (high concentration), and the corresponding RSDs were ranged from 2.09% to 13.12% (low concentration) and from 1.48% to 10.59% (high concentration). The analysis results indicated that 1-2 enantiomers of most amino acids could be detected, and D-amino acids were frequently distributed in most tea samples, especially D-threonine, D-aspartic acid, D-cysteine, D-phenylalanine, and the mixture of D-theanine, D-glutamic acid and D-glutamine. On the contrary, D-methionine, D-lysine and D-tryptophan were unable to be detected in all samples. As for the detailed content distributions, in addition to the mixture of L-theanine, L-glutamic acid and L-glutamine and L-aspartic acid, L-cysteine (1.48-2.08 mg?g-1), D-cysteine (1.46-1.49 mg?g-1) and D-aspartic acid (1.02-1.14 mg?g-1) presented higher contents in white teas, L-cysteine (1.52-1.70 mg?g-1), D-cysteine (1.45-1.49 mg?g-1), L-serine (1.03-1.50 mg?g-1), L-methionine (1.03-1.52 mg?g-1), L-tyrosine (1.32-1.35 mg?g-1) and D-aspartic acid (1.01-1.15 mg?g-1) were abundant in oolong teas. In pu-erh teas, no significant differences on the contents between the mixture of L-theanine, L-glutamic acid and L-glutamine (1.04 mg?g-1) and other amino acids, and L-threonine, L-tryptophan, and L-serine presented relatively high content levels ranging from 0.61 to 0.84 mg?g-1. The content distribution of the total amino acids were presented the following tendency: white tea (up to 40.61 mg?g-1) > oolong tea (up to 25.43 mg?g-1) > pu-erh tea (8.01 mg?g -1). Moreover, multivariate statistical analysis results indicated that the mixture of L-theanine, L-glutamic acid and L-glutamine, L-tryptophan, L-aspartic acid, L-tyrosine and L-methionine showed significant content differences, and the first three enantiomers were abundant in white teas, and other compounds were rich in oolong teas.【Conclusion】 D-amino acids could be detected in most teas, indicating their potential impact on the taste quality of tea infusion. However, no significant differences on the content distribution of D-amino acids were observed among different kinds of teas used in our study.

Key words: tea, free amino acids, enantiomers, gas chromatography-mass spectrometry, pentafluoropropionic anhydride