Analysis of theaflavins and thearubigins from black tea extract by MALDI-TOF mass spectrometry

Black tea contains two major groups of pigments, theaflavins (TFs) and thearubigins (TRs). TFs contain a bis-flavan substituted 1,2-dihydroxy-3,4-benzotropolone moiety. Unlike the TFs, TRs have not yet been characterized. The chemical structure of the TRs remains a mystery. The present paper reports our effort to study the structure of TFs and TRs using delayed pulsed ion extraction of ions generated via the matrix-assisted laser desorption ionization (MALDI) technique, on line with a Linear time-of-flight (TOF) mass spectrometer. Spectra of standard TFs show not only pseudomolecular ions but also ions resulting from fragmentation. The analysis of MALDI-TOF spectra of black tea fractions shows the structure of some TRs, which are similar to those of TFs because the same loss of mass is observed.     

Determination of theaflavins including methylated theaflavins in black tea leaves by solid-phase extraction and HPLC analysis

A quantitative method for four theaflavins and two methylated theaflavin derivatives in black tea leaves was developed by solid-phase extraction and a high-performance liquid chromatographic method with photodiode array detection. The theaflavins in black tea leaves were extracted three times with 40 vol 50% aqueous ethanol (mg dry tea powder/mL) containing 2% ascorbic acid. The ethanol extracts were diluted 4-fold with distilled water. All diluted extracts were directly applied to the solid-phase C18 cartridge column without concentration. The fraction of theaflavins was obtained by 40% ethanol extraction after rinsing with water followed with 15% ethanol extraction. An aliquot of theaflavins after concentration was injected onto an ODS C18 reversed-phase column, and four theaflavins and two methylated theaflavins were sufficiently separated by a linear gradient system using distilled water and acetonitrile with 0.5% acetic acid. This analytical method is sensitive for the determination of a small amount of methylated theaflavins, since various interfering substances produced during the fermentation process were eliminated in advance by solid-phase extraction. Using this analytical method, we also demonstrated that methylated theaflavins were easily produced during the manufacture of black tea.     

Molecular binding of black tea theaflavins to biological membranes: relationship to bioactivities

Molecular dynamics simulations were used to study the interactions of three theaflavin compounds with lipid bilayers. Experimental studies have linked theaflavins to beneficial health effects, some of which are related to interactions with the cell membrane. The molecular interaction of theaflavins with membranes was explored by simulating the interactions of three theaflavin molecules (theaflavin, theaflavin-3-gallate, and theaflavin-3,3'-digallate) with a mixed bilayer composed of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE). The simulations show that the theaflavins evaluated have an affinity for the lipid bilayer surface via hydrogen bonding. The molecular structure of theaflavins influenced their configuration when binding to the bilayer surface, as well as their ability to form hydrogen bonds with the lipid headgroups. The theaflavin-bilayer interactions studied here help to define structure-function relationships of the theaflavins and provide a better understanding of the role of theaflavins in biological processes. The significance of the results are discussed in the context of black tea composition and bioactivity.     

Role of polyphenol oxidase and peroxidase in the generation of black tea theaflavins.

It has been reported earlier that when macerated tea leaf is fermented at lower pH, the resultant black tea contains increased levels of theaflavin, an important quality marker in black tea. In an attempt to investigate the biochemistry and chemistry underlying this observation, in vitro oxidation experiments using polyphenol oxidase (PPO) from fresh tea leaves, horseradish peroxidase (POD), and tea catechins, precursors for theaflavins, were carried out. In vitro oxidation experiments using crude tea PPO resulted in higher content of theaflavins at pH 4.5 in comparison with pH 5.5, the normal pH of the macerated tea leaf. When purified PPO was used in the in vitro system, surprisingly a reversal of this trend was observed, with more theaflavins being formed at the higher pH. A combination of pure tea PPO and POD led to an observation similar to that with the crude enzyme preparation, suggesting a possible role for POD in the formation or degradation of theaflavin. POD was observed to oxidize theaflavins in the presence of H(2)O(2), leading to the formation of thearubigin, another black tea pigment. This paper demonstrates that tea PPO, while oxidizing catechins, generates H(2)O(2). The amount of H(2)O(2) produced is greater at pH 5.5, the optimum pH for PPO activity, than at pH 4.5. Hence, an observed increase of theaflavins in black teas fermented at pH 4.5 appears to be due to lower turnover of formed theaflavins into thearubigins.     

alpha-Glucosidase inhibitory profile of catechins and theaflavins

To clarify the postprandial glucose suppression effect of flavonoids, the inhibitory effects of catechins and theaflavins against alpha-glucosidase (AGH) were examined in this study. It was initially demonstrated that theaflavins and catechins preferentially inhibited maltase rather than sucrase in an immobilized AGH inhibitory assay system. For the maltase inhibitory effects of theaflavins, the effects were observed in descending order of potency of theaflavin (TF)-3-O-gallate (Gal) > TF-3,3'-di-O-Gal > TF-3'-O-Gal > TF. This suggests that the AGH inhibition induced by theaflavins is closely associated with the presence of a free hydroxyl group at the 3'-position of TF as well as the esterification of TF with a mono-Gal group. In addition, the R-configuration at the 3'-position of TF-3-O-Gal showed a higher inhibitory activity than the S-configuration. As a result of a single oral administration of maltose (2 g/kg) in rats, a significant reduction in blood glucose level was observed at a dose of 10 mg/kg of TF-3-O-Gal, demonstrating for the first time that TF-3-O-Gal can suppress glucose production from maltose through inhibition of AGH in the gut.     

Antioxidative activities of volatile extracts from green tea, oolong tea, and black tea

Antioxidative activities of volatile extracts from six teas (one green tea, one oolong tea, one roasted green tea, and three black teas) were investigated using an aldehyde/carboxylic acid assay and a conjugated diene assay. The samples were tested at levels of 20, 50, 100, and 200 micrograms/mL of dichloromethane. The results obtained from the two assays were consistent. All extracts except roasted green tea exhibited dose-dependent inhibitory activity in the aldehyde/carboxylic acid assay. A volatile extract from green tea exhibited the most potent activity in both assays among the six extracts. It inhibited hexanal oxidation by almost 100% over 40 days at the level of 200 micrograms/mL. The extract from oolong tea inhibited hexanal oxidation by 50% in 15 days. In the case of the extract from roasted green tea, the lowest antioxidative activity was obtained at the level of 200 micrograms/mL, suggesting that the extract from roasted green tea contained some pro-oxidants. The extracts from the three black teas showed slight anti- or proactivities in both assays. The major volatile constituents of green tea and roasted green tea extracts, which exhibited significant antioxidative activities, were analyzed using gas chromatography-mass spectrometry. The major volatile chemicals with possible antioxidative activity identified were alkyl compounds with double bond(s), such as 3,7-dimethyl-1,6-octadien-3-ol (8.04 mg/kg), in the extract from green tea and heterocyclic compounds, such as furfural (7.67 mg/kg), in the extract from roasted green tea. Benzyl alcohol, which was proved to be an antioxidant, was identified both in a green tea extract (4.67 mg/kg) and in a roasted tea extract (1.35 mg/kg).     

Isolation of black tea pigments using high-speed countercurrent chromatography and studies on properties of black tea polymers

Isolation of theaflavins and epitheaflavic acids from black tea using high-speed countercurrent chromatography (HSCCC) on a preparative scale is demonstrated. HSCCC also enabled the isolation of a polymeric fraction from black tea. According to Roberts' classification, the polymeric fraction mainly consisted of SII thearubigins (TR). HPLC analysis showed that the isolated material is free of any known chromatographically resolved tea constituents and eluted from reversed-phase packings as a convex "hump" (a broad signal). The antioxidant activity of the TR fraction was 3.6 mmol of Trolox equivalents per gram. The total phenolic content of this fraction was determined to be 34.7 g/100 g (as gallic acid equivalents).     

电子舌在红茶饮料区分辨识中的应用

为检验电子舌在茶饮料方面的辨识能力,该文应用法国Alpha M.O.S公司生产的传感器型电子舌对中国市场上已有的7种红茶饮料进行检测,所得数据用主成分分析法（PCA）和聚类分析法（CA）进行分析。结果表明,该电子舌可以很好的区分这7种红茶饮料。且在PCA第1主成分和第2主成分得分图上,区别指数达95.38。由此可见,电子舌在茶饮料的品牌区分、质检以及真伪辨识中有巨大的应用潜力。同时,该研究也初步探讨了聚类分析法在电子舌数据处理方面的应用。     

去除冷却猪肉辐照异味方法初探

为了探索去除冷却猪肉辐射后异味的方法,研究了气调处理,吸附剂处理及气调与吸附剂组合处理对异味去除的效果,对吸附剂种类,添加量,吸附进程也作了分析。结果表明,预冷猪肉经60% O2 30% CO2 10% N2气调包装,或内置0.01 g/(50 g)肉小包装吸附剂（氯化锌/氧化锌）的薄膜包装后,辐照2.8 kGy,辐照后及冷藏期感觉不出辐照异味,猪肉品质正常。二者组合处理经4.2 kGy辐照后异味达不可察觉程度。     

植物物料红茶的浸提动力学研究

分析了植物物料溶质浸出过程中在高阻力区及低阻力区中的质量传递性质,据此建立了具有快速项和慢速项的两项浸提动力学模型。建立了浸提平衡浓度、快速溶质比例及半平衡时间的三项指标评价体系,全面评价溶质浸提动力学过程。试验表明干燥方法影响着Freedom红茶产品的容积密度,导致组织收缩特性不同,溶质的浸提动力学性能亦不同。流化床干燥、薄层干燥的红茶具有优良的浸提动力学性能,真空干燥红茶的浸提动力学性能较好,盘式干燥红茶的浸提动力学性能较差,微波干燥红茶的浸提动力学性能很差。流化床干燥温度变化不影响红茶的浸提动力学性能。较小颗粒红茶具有更好的浸提动力学性能。     

Effect of milk and brewing method on black tea catechin bioaccessibility

The aim of this study was to investigate whether milk reduces the bioaccessibility of tea catechins, which would compromise tea beneficial effects ascribed to polyphenols. Adding milk to black tea has been shown to lead to polyphenol-protein complexes. So far, data on the intestinal stability of polyphenol-protein complexes are scarce. English black tea (0.93 ± 0.06 mol/L total catechins) and Indian black tea (1.83 ± 0.08 mol/L catechins) were prepared with skimmed or full-fat milk and subjected to simulated gastric, small intestinal, and brush border digestion. Adding milk (5.6-40%) to tea results in a decrease of total catechin (TCAT) recovery. However, the bioaccessibilities of TCAT of tea with milk versus tea controls were comparable (p > 0.05). The type of milk did not influence TCAT recovery during all digestive stages (p > 0.05). Polyphenol-protein complexes are degraded during digestion. It is very unlikely that consumption of tea with or without milk will result in differences in catechin plasma concentration.     

基于数据融合策略的红茶发酵程度判别

发酵是红茶加工过程中关键的一道工序,对红茶的品质形成有着重要影响。该研究以大叶种英德红茶中的英红九号为研究对象,试验收集了204份不同发酵时间的红茶样品并使用便携式近红外光谱仪和工业相机获取红茶发酵中的信息,基于近红外光谱数据、图像数据和数据融合策略分别建立了红茶发酵程度判别模型。通过分析茶多酚和儿茶素类含量的变化,将红茶的发酵划分为3个阶段,即发酵不足、发酵适度和发酵过度。采用Savitzky-Golay光滑对原始光谱进行预处理,利用竞争自适应重加权采样（Competitive Adaptive Reweighted Sampling, CARS）、连续投影算法（Successive Projections Algorithm, SPA）和主成分分析（Principal Components Analysis, PCA）对近红外光谱变量进行降维处理;相应地,图像进行去阴影后提取了9个颜色特征变量,采用皮尔森（Pearson）相关分析和主成分分析进行特征变量提取。最后采用线性判别分析（Linear Discriminant Analysis, LDA）和支持向量机（Support Vector Machine, SVM）分别建立了基于近红外、图像和两者数据融合的分类模型。结果表明,在建模数据相同的条件下,非线性的支持向量机模型性能优于线性判别分析模型。单一传感器数据建模效果不佳,近红外光谱和图像判别模型的预测集最大准确率仅为83.82%和73.53%。低层次数据融合建模效果较单一传感器数据建模无明显提升,而中层次的数据融合建模效果比单一数据建模均有显著提高,其中SPA提取光谱变量结合Pearson提取图像变量建立的判别模型效果较佳,校正集和预测集准确率分别达到了97.06%和95.59%。研究表明,近红外光谱和视觉结合的中层次融合策略可以作为一种快速判别红茶发酵程度的方法,研究结果为红茶发酵程度构建等级模型与判别奠定了一定的理论基础,为红茶发酵的自动化检测提供了重要依据。     

Effects of infusion time and addition of milk on content and absorption of polyphenols from black tea

Epidemiological studies assessing the health benefits of drinking black tea are equivocal. Such disparity may reflect an inability of semiquantitative assessment to consider how infusion time and addition of milk affect the bioavailability of potentially beneficial antioxidant polyphenols. Six brands of tea demonstrated similar increases in antioxidant capacity and total phenolic and catechin contents with increasing infusion time. These results were unaffected by the addition of milk. Consumption of black tea (400 mL) was associated with significant increases in plasma antioxidant capacity (10%) and concentrations of total phenols (20%), catechins (32%), and the flavonols quercetin (39%) and kaempferol (45%) (all p < 0.01) within 80 min. This was unaffected by adding milk. Infusion time may therefore be a more important determinant in the absorption of polyphenols from black tea. Observational studies assessing the health benefits of tea consumption require recording of brewing methods as well as frequency of consumption.     

红茶在超微-酶解反应器中的萃取特性研究

本文通过研究红茶在超微－酶解反应器(超微粉碎与酶解反应耦合)中作用所得茶汤的品质变化情况,探讨超微－酶解反应器在红茶萃取方面的优越性,并寻求一条生产茶汤的新途径。结果表明：经过超微－酶解反应器后的茶汤的可溶性固形物含量和茶多酚含量,随着剪切温度、剪切频率、剪切时间、加酶量的增大呈增大的趋势;较过胶体磨后的茶汤,二者含量分别提高了6.7%～12.6%和1.5%～8.0%;较直接热水萃取,二者含量分别提高了16.0%～20.1%和6.2%～24.4%。红茶加酶后在反应器中反应较不加酶所得茶汤茶多酚含量显著提高23.3%～35.6%。     

Breast cancer prevention by green tea catechins and black tea theaflavins in the C3(1) SV40 T,t antigen transgenic mouse model is accompanied by increased apoptosis and a decrease in oxidative DNA adducts

Tea consumption is associated with a reduced risk of mammary cancer as reflected by epidemiological studies and experiments in carcinogen-induced rodent models of mammary carcinogenesis. We tested the hypothesis that green tea catechins (GTC) or theaflavins from black tea (BTT) interfere with mammary carcinogenesis in C3(1) SV40 T,t antigen transgenic multiple mammary adenocarcinoma (TAg) mice and that GTC/BTT affect tumor survival or oxidation status. TAg mice received GTC/BTT (0.05%) in drinking water for their lifetime. As compared to control mice, they survived longer and had smaller tumors. On microscopic inspection, the size of the largest tumor per mouse was decreased by 40-42% (p<0.01). GTC (0.01%) and BTT (0.05%) increased levels of cleaved caspase 3 in tumor tissue by 67 and 38%, respectively (p<0.05), intimating increased apoptosis. Tumor levels of the malondialdehyde-DNA adduct M1dG in mice receiving GTC or BTT (0.05%) were reduced by 78 (p<0.001) or 63% (p<0.05), respectively, as compared to controls. The results render the exploration of the breast cancer chemopreventive properties of tea preparations in humans worthwhile.     

基于机器视觉形状特征参数的祁门红茶等级识别

外形是评价茶叶质量的重要指标之一,目前主要依赖人工审评的方法,客观、准确的评价外形指标对茶叶加工、销售有重要的意义。该研究提出了一种基于形状特征直方图结合支持向量机的茶叶等级识别方法。以7个等级的祁门工夫红茶为研究对象,构建图像采集系统,标定相机参数,采集各等级的茶叶图像。对图像进行灰度化、二值化处理,提取叶片的6个绝对形状特征:长度、宽度、面积、周长、最小外接矩长、宽,在此基础上计算狭长度,矩形度2个相对形状特征,生成形状特征的直方图。以直方图分布为特征向量,构建了基于BP神经网络,极限学习机(extreme learning machine,ELM),支持向量机(support vector machine,SVM),最小二乘支持向量机(least squares support vector machine,LS-SVM)的等级识别模型,并对比了不同模型的识别效果。结果表明,该文构建的图像采集系统测量精度0.3 mm,能够准确提取形状特征参数;基于形状特征直方图的LS-SVM模型识别效果最好,识别精度为95.71%,测试集决定系数为96.2%,具有算法复杂度低,易于求解的优点。研究结果为实现茶叶的客观、数字化等级鉴定,提供了试验数据和参考方法。     

Characterization of corrinoid compounds from a Japanese black tea (Batabata-cha) fermented by bacteria

A Japanese fermented black tea (Batabata-cha) contained a considerable amount of vitamin B(12) (456 +/- 39 ng per 100 g dry tea leaves and 2.0 +/- 0.3 ng per 100 mL of tea drink). A corrinoid compound was partially purified and characterized from the tea leaves. The patterns of the purified compound by the silica gel 60 thin-layer chromatography and C18 reversed phased high-performance liquid chromatography were identical to those of authentic vitamin B(12). When 20 week old vitamin B(12) deficient rats, which excreted substantial amounts (about 250 mg/day) of methylmalonic acid in urine as an index of vitamin B(12) deficiency, were fed the tea drink (50 mL/day, 1 ng of vitamin B(12)) for 6 weeks, urinary methylmalonic acid excretion (169 +/- 29 mg/day) of the tea drink-supplemented 26 week old rats decreased significantly relative to that (250 +/- 32 mg/day) of the deficient rats. The results indicate that the vitamin B(12) found in the fermented black tea is bioavailable in mammals.     

Characterization of the key aroma compounds in the beverage prepared from Darjeeling black tea: quantitative differences between tea leaves and infusion

By application of the aroma extract dilution analysis on the volatile fraction isolated from a black tea infusion (Darjeeling Gold Selection), vanillin (vanilla-like), 4-hydroxy-2,5-dimethyl-3(2H)-furanone (caramel), 2-phenylethanol (flowery), and (E,E,Z)-2,4,6-nonatrienal (oat-flake-like) were identified with the highest flavor dilution (FD) factors among the 24 odor-active compounds detected in the FD factor range of 4-128. Quantitative measurements performed by means of stable isotope dilution assays and a calculation of odor activity values (OAVs; ratio of concentration to odor threshold in water) revealed, in particular, the previously unknown tea constituent (E,E,Z)-2,4,6-nonatrienal as a key odorant in the infusion and confirmed the important role of linalool and geraniol for the tea aroma. An aroma recombinate performed by the 18 odorants for which OAVs > 1 were determined in their "natural" concentrations matched the overall aroma of the tea beverage. In the black tea leaves, a total of 42 odorants were identified, most of which were identical with those in the beverage prepared thereof. However, quantitative measurements indicated that, in particular, geraniol, but also eight further odorants were significantly increased in the infusion as compared to their concentration in the leaves.     

机械超微粉碎与不同粒度常规粉碎对红茶理化特性的影响

为探究机械超微粉碎对茶叶物性及功能成分浸出特性的影响,该文通过超微粉碎和常规粉碎2种方法制备超微红茶粉和4种粒度的粗粉红茶粉,研究了5种茶粉的粒度分布、微观形貌、比表面积、结晶度、表面氧碳元素比、中红外图谱和功能成分的浸出特性。结果表明:常规粉碎不同粒度茶粉间差异较小,而超微粉碎较大程度地改变了茶叶的物料性质。超微粉碎后,茶粉粒度减小至微米级,比表面积增大,茶粉的结晶度降低,表面暴露的纤维素和半纤维素相对含量增多,而中红外谱图显示超微粉碎茶粉的官能团结构并没有改变。沸水浸提过程中茶多酚、咖啡因、可溶性糖和水浸出物的溶出结果表明:5种茶粉功能成分的浸出量都随时间的增加而增加,经过一段时间后达到平衡。在达到浸出平衡时,不同粒度茶粉的茶多酚和咖啡因含量差异不显著,超微茶粉的可溶性糖含量为4种粗粉的2倍左右,水浸出物比粗粉茶粉提高10%以上。上述研究结果表明:超微粉碎能够有效减小茶粉的粒度,破坏植物的细胞壁结构,使内部成分暴露于表面,此外,组织结构和细胞壁的破坏使不溶的纤维成分转化成可溶性成分,可溶性糖和水浸出物显著增多,超微粉碎能够在一定程度上提高茶粉的有效利用率。