多酚氧化酶和过氧化物酶催化不同儿茶素制备茶褐素及其理化性质和抑菌机理

doi:10.1016/j.jia.2023.07.004

摘要/Abstract

摘要：

茶褐素（TBs）是普洱茶、青砖茶等黑茶的特征性功能与品质成分。茶褐素（TBs）是在黑茶的渥堆发酵加工过程中，茶多酚发生酶促和非酶促氧化聚合反应产生的一类多分子量分布、水溶性的褐色聚合物。一直以来，茶褐素的制备是以黑茶为原料进行提取纯化，但制得的茶褐素含有结合态的多糖、咖啡碱等异质成分，难以除去。分离纯化工艺繁琐，有机溶剂使用多，这使其一直难于产业化。本研究分别以表没食子儿茶素（EGC）、表没食子儿茶素没食子酸酯（EGCG）、表儿茶素没食子酸酯（ECG）、EGC/EGCG（质量比1:1）、EGCG/ECG（质量比1:1）、EGC/ECG（质量比1:1）、EGC/ECG（质量比1:1）和EGC/EGCG/ECG（质量比1:1:1）为底物，依次经多酚氧化酶（PPO）和过氧化物酶（POD）催化制得茶褐素，命名为TBs-dE-1、TBs-dE-2、TBs-dE-3、TBs-dE-4、TBs-dE-5、TBs-dE-6和TBs-dE-7。研究了TBs-dE（1-7）的理化特性和抑菌活性与机理。感官及色差测定表明7个茶褐素样品均显示不同程度的棕褐色色度。pH3.0-9.0的水溶液中的Zeta电位表明，TBs-dE（1-7）带负电荷，电势随pH增大而增大。紫外可见扫描光谱（UV-vis）显示，TBs-dE（1-7）在208 nm和274 nm处有特征吸收峰；傅里叶红外光谱（FT-IR）表明其为多酚类化合物。TBs-dE（1-7）对大肠杆菌DH 5α（E.coli DH 5α）均具有显著的抑制作用。其中TBs-dE-3的抑菌作用最强（其最小抑菌浓度MIC为1.25 mg/mL，最小杀菌浓度MBC为10 mg/mL），其次是TBs-dE-5、TBs-dE-6。选取这三种TBs-dE进一步探究其抑菌机理。结果表明，TBs-dE通过破坏大肠杆菌的胞外膜，使内容物泄漏，同时增加细胞内活性氧含量，致使因氧化应激引起细胞代谢异常起到抑菌作用。研究结果为茶褐素的产业化制备及产品开发提供理论依据。

Abstract:

Theabrownins (TBs) are the characteristic functional and quality components of dark teas such as Pu’er tea and Chin-brick tea. TBs are a class of water-soluble brown polymers with multi-molecular weight distribution produced by the oxidative polymerisation of tea polyphenols during the fermentation process of dark tea, both enzymatically and non-enzymatically. TBs have been extracted and purified from dark tea all the time, but the obtained TBs contain heterogeneous components such as polysaccharides and caffeine in the bound state, which are difficult to remove. The isolation and purification process was tedious and required the use of organic solvents, which made it difficult to industrialise TBs. In this study, epigallocatechin (EGC), epigallocatechin gallate (EGCG), epigallocatechin gallate (ECG), EGC/EGCG (mass ratio 1:1), EGCG/ECG (mass ratio 1:1), EGC/ECG (mass ratio 1:1) and EGC/EGCG/ECG (mass ratio 1:1:1) as substrates and catalyzed by polyphenol oxidase (PPO) and peroxidase (POD) in turn to produce TBs, named TBs-dE-1, TBs-dE-2, TBs-dE-3, TBs-dE-4, TBs-dE-5, TBs-dE-6 and TBs-dE-7. The physicochemical properties and the antibacterial activity and mechanism of TBs-dE-1–7 were investigated. Sensory and colour difference measurements showed that all seven tea browning samples showed varying degrees of brownish hue. Zeta potential in aqueous solutions at pH 3.0–9.0 indicated that TBs-dE-1–7 was negatively charged and the potential increased with increasing pH. The characteristic absorption peaks of TBs-dE-1–7 were observed at 208 and 274 nm by UV-visible (UV-vis) scanning spectroscopy. Fourier transform infrared (FT-IR) spectra indicated that they were phenolic compounds. TBs-dE-1–7 showed significant inhibition of Escherichia coli DH5α (E. coli DH5α). TBs-dE-3 showed the strongest inhibitory effect with minimum inhibitory concentration (MIC) of 1.25 mg mL^–1 and MBC of 10 mg mL^–1, followed by TBs-dE-5 and TBs-dE-6. These three TBs-dEs were selected to further investigate their inhibition mechanism. The TBs-dE was found to damage the extracellular membrane of E. coli DH5α, causing leakage of contents, and increase intracellular reactive oxygen content, resulting in abnormal cell metabolism due to oxidative stress. The results of the study provide a theoretical basis for the industrial preparation and product development of TBs.

Key words: theabrownins , catechin , enzymatic oxidation , physicochemical properties , antibacterial mechanism

CHEN Xiao-qiang, LIU Jia-yan, HUANG Xue-jun, WEI Yan-an, SHAO Rui-xiang, CHEN Ting-ting, XIE Jian-chun. 多酚氧化酶和过氧化物酶催化不同儿茶素制备茶褐素及其理化性质和抑菌机理[J]. Journal of Integrative Agriculture, 2023, 22(9): 2905-2916.

CHEN Xiao-qiang, LIU Jia-yan, HUANG Xue-jun, WEI Yan-an, SHAO Rui-xiang, CHEN Ting-ting, XIE Jian-chun . Physicochemical properties and antibacterial mechanism of theabrownins prepared from different catechins catalyzed by polyphenol oxidase and peroxidase[J]. Journal of Integrative Agriculture, 2023, 22(9): 2905-2916.

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