Rapid method for measuring thearubigins and theaflavins in black tea using C18 sorbent cartridges

This communication describes the routine use of small inexpensive C₁₈ cartridges for rapid separation and accurate measurement of both thearubigins (TR) and theaflavins (TF) in a liquor brewed from black tea. The slow, traditional method overestimates TR content because of interference from flavonol glycosides. Comparison is also made favourably with other methods available for estimating total TF and sometimes TR in tea.     

The formation of thearubigin-like substances by in-vitro polyphenol oxidase-mediated fermentation of individual flavan-3-ols

An enzymic in-vitro model tea fermentation system has been used in combination with gradient elution reversed-phase high-performance liquid chromatography to study the formation from individual fiavan-3-ols of resolvable and unresolvable thearubigin-like substances. It has been found that in 30 min at pH 5.6 tea polyphenol oxidase (EC 1.14.18.1) produces a distinctive range of resolvable thearubigin-like products from each flavan-3-ot substrate examined ((+)-catechin, (?)-epicatechin, (?)-epigallocatechin, (?)-epicatechin gallate and (?)-epigallocatechin gallate). However, (?)-epicatechin, and to a much lesser extent (+ )-catechin, were the only flavan-3-ols converted to an unresolvable thearubigin-like hump under these conditions. In addition, it was demonstrated that varying the pH of incubation in the range 5.0–6.0 had little effect on the profile of products obtained from the simple catechins, but a pronounced effect in the case of the gallocatechins. It is concluded that the combination of (?)-epicatechin and polyphenol oxidase is the simplest model of tea fermentation that can be used to generate in quantity thearubigin-like materials for structure elucidation.     

Influence of region of production on clonal black tea chemical characteristics

Production of black tea from the same vegetatively propagated (VP) cultivars, in Kenya and Malawi, shows variations in both chemical composition and quality. Whereas it is possible to produce black teas with similar total theaflavins and individual theaflavins, brightness and total colour levels, black teas from Kenya generally have higher thearubigins, total volatile flavour compounds and flavour index. The black tea fermentation process is much faster in Malawi compared to that in Kenya, as evidenced by faster production of plain black tea chemical parameters, especially theaflavins. Consequently, in Malawi the maximum amount of theaflavins formation takes a shorter fermentation duration than in Kenya. Given ample fermentation duration, fermentation in Kenya produces a similar amount of theaflavins. This makes it necessary to optimise fermentation time, in different geographical regions even when the same cultivar is being processed. The other plain black tea quality parameters (thearubigins, brightness and total colour) were higher in black tea which was processed in Kenya than those processed in Malawi. However, the pattern in the changes in the individual theaflavins or theaflavins digallate equivalent followed that of total (Flavognost) theaflavins, suggesting that the flavan-3-ols patterns in tea leaf might not have been affected by the geographical area of production. The total volatile flavour compounds (VFC), Group I and II VFC and the flavour index were higher in black teas processed in Kenya, further demonstrating the fact that high grown Kenyan teas are more flavoury. In both Kenya and Malawi black teas, aroma quality declined with a long duration of fermentation. Short fermentation time is therefore a method of producing more aromatic black teas. The variations in black tea quality between Malawi and Kenya were possibly due to difference in environmental conditions, leading to different shoot growth rates and biochemical composition in the shoots.     

Evaluation of the taste contribution of theaflavins in black tea infusions using the taste activity concept

The contribution of theaflavins to the astringent taste of black tea infusions has been investigated. The major benzotropolons theaflavin, theaflavin-3-gallate, theaflavin-3-gallate, theaflavin-3,3-digallate, and theaflavic acid were synthetically prepared by enzymatic oxidation of binary mixtures of the corresponding flavan-3-ols, purified by high-speed countercurrent chromatography, and then sensorially evaluated by means of a comparative duo test. The sensory analysis revealed that the theaflavins imparted a mouth-coating, astringent, and long-lasting oral sensation at the very back of the throat, and demonstrated the theaflavins to have by far lower oral thresholds than the astringent catechins; for example, the threshold concentration of theaflavin (0.016 mmol/L) is by a factor of 33 and 58 lower than those of its precursors epigallocatechin and epicatechin, respectively. In order to study the importance of these theaflavins as contributors to tea astringency, these benzotropolons were quantified in tea infusions and taste activity values were calculated from the quotient of their concentration in tea and their taste detection thresholds. Relating the taste activity values of the individual theaflavins to the overall astringency of the teas revealed that the theaflavins accounted for less than 0.1% of the overall astringency of the teas investigated. In consequence, the theaflavin concentration seems not to be a suitable measure for taste quality of tea infusions.     

茶叶提取物体外抗氧化活性与其功能性成分含量的相关性研究

分别选取信阳毛尖、红碎茶、普洱熟茶为典型不发酵、全发酵及后发酵茶种,测定其醇提物及水提物中的主要成分,并以氧自由基的吸收能力(ORAC)和DPPH自由基的清除能力为指标,比较各样品体外抗氧化活性,采用功能化合物重组模型,探究茶叶提取物抗氧化活性的主要贡献物质。结果表明:茶叶醇提物中茶多酚、咖啡因及醇溶蛋白的含量较高,其ORAC值及DPPH自由基清除能力均较对应水提物强;茶叶水提物功能性组分以茶多酚、茶多糖及氨基酸为主,都具有一定的抗氧化活性。其中茶多酚含量,尤其是酯型儿茶素(ECG和EGCG)含量是影响茶叶提取物抗氧化活性的主要因素。重组试验结果表明,儿茶素和没食子酸是不发酵茶(信阳毛尖)提取物的主要抗氧化组分,但并不是红碎茶、普洱熟茶等发酵茶中的主要抗氧化成分。     

花果香与陈香型普洱茶生化成分与香气物质的比较

本研究选择同一企业生产的花果香型与陈香型普洱茶为材料,应用分光光度法、高效液相色谱法、气相色谱联用质谱(GC-MS)和感官审评等技术,研究比较该两种香型普洱茶的品质特征与物质基础。审评发现花果香型滋味浓厚酸醇,具有花果香或焦糖香,而陈香型滋味醇厚、陈香显著。分析结果表明花果香型的水浸出物、游离氨基酸、可溶性糖、没食子酸、茶黄素、茶红素、表没食子酸儿茶素的含量均高于陈香型(p<0.05),陈香型的表儿茶素含量高于花果香型(p<0.05)。GC-MS一共鉴定到532个化合物,显著差异化合物55个(vip>1.00,p<0.05,FC>1.50);花果香型具有花香、熟果香的(3R,6S)-2,2,6-三甲基-6-乙烯基四氢-2H-吡喃-3-醇、苯乙醇、氧化芳樟醇(呋喃类)、苄醇、芳樟醇、香叶醇等物质相对含量明显高于陈香型,而陈香型显陈香的1,2-3-三甲氧基苯、1,2,3-三甲氧基-5-甲基苯等甲氧基苯类及烷烃类物质相对含量高于花果香型。综上,花果香型普洱茶中没食子酸、水浸出物、游离氨基酸等生化成分含量显著高于陈香型,(3R,6S-2,2,6-三甲基-6-乙烯基四氢-2H-吡喃-3-醇等显花果香的物质相对含量高于陈香型;而甲氧基苯等显陈香的物质低于陈香型,构成了花果香型(BJ)普洱茶滋味浓厚酸醇、显花果香的品质特征。综合来看,花果香型普洱茶是一款内含成分更为丰富、香气独特的新型普洱茶产品。     

In-vitro studies on the effect of polyphenol oxidase and peroxidase on the formation of polyphenolic black tea constituents

Flavanols (catechins and gallocatechins) isolated from green tea leaves (Camellia sinensis) (L) O Kuntze) were treated in vitro with polyphenol oxidase, peroxidase and a combination of both. The flavanol levels and those of the products formed were monitored by reversed-phase high-performance liquid chromatography (HPLC using spectrophotometric detection at 280 and 380 nm. Flavanols and theaflavins were quantified, whilst integrated peak areas are given for the remaining compounds. After fermentation in the presence of polyphenol oxidase higher levels of theaflavins and resolved thearubigins were obtained, whilst peroxidase produced higher amounts of chromatographically unresolved thearubigins of higher molecular weight, observed as a rise in the HPLC baseline, in the presence of peroxidase a significant decrease in the levels of all flavonol glycosides was observed, whilst in the presence of polyphenol oxidase only myricetin glycoside levels decreased.     

The use of green tea (Camellia sinensis) leaf flavan-3-ol composition in predicting plain black tea quality potential

Reliable and quantifiable green leaf parameters for predicting black tea quality potential of tea bushes at the single bush stage are necessary to shorten tea breeding and clonal selection programmes. Green leaf flavan-3-ols (catechins) and plain black tea quality of 11 popular Kenyan clones were determined. The individual green leaf flavan-3-ols, and all the black tea quality parameters and sensory evaluations, except total flavan-3-ols were significantly different. The green leaf epigallocatechin gallate (EGCg) levels significantly correlated with black tea total theaflavin (r = 0.749, P ? 0.007), liquor brightness (r = 0.898, P ? 0.0001) levels and Taster B (r = 0.595, P ? 0.051) sensory evaluations, and negatively with thearubigins (r = −0.652, P ? 0.027), while green leaf epicatechin (EC) levels correlated positively with thearubigins (r = 0.694, P ? 0.016) and negatively with theaflavin digallate equivalent (r = −0.751, P ? 0.007), and sensory evaluation by Taster A (r = −0.785, P ? 0.003) and Taster B (r = −0.679, P ? 0.020). Thus, high levels of EGCg and low levels of EC in tea green leaf can be used as indicators of plain black tea quality potential of tea plants. The sum of gallated flavan-3-ols correlated significantly and positively with theaflavin digallate equivalent (r = 0.669, P ? 0.022) levels, but negatively correlated with the black tea thearubigins (r = −0.582, P ? 0.052). The sum of trihydroxyflavan-3-ols (gallocatechins) positively correlated with brightness (r = 0.750, P ? 0.007), and sensory evaluation by Taster A (r = 0.656, P ? 0.026), but negatively with thearubigins (r = −0.641, P ? 0.031). However, the sum of dihydroxyflavan-3-ols (simple catechins) correlated positively with thearubigins (r = 0.716, P ? 0.012) and negatively with total theaflavins (r = −0.6694, P ? 0.022), theaflavin digallate equivalent (r = −0.631, P ? 0.035), brightness (r = −0.843, P ? 0.001), Taster A (r = −0.775, P ? 0.004) and Taster B (r = −0.638, P ? 0.032) sensory evaluation. The ratios of trihydroxyflavan-3-ols to dihydroxyflavan-3-ols correlated positively with brightness (r = 0.678, P ? 0.020) and sensory evaluation of Taster A (r = 0.667, P ? 0.023), but negatively with thearubigins (r = −0.697, P ? 0.015). In addition to green leaf high levels of EGCg and low levels of EC, high levels of the sum of gallated catechins, trihydroxyflavan-3-ols, and the ratio of trihydroxyflavan-3-ols to dihydroxyflavan-3-ols are parameters in green tea leaf that may be used in predicting plain black tea quality potential of Kenyan tea clones.     

基于代谢组学的不同年份晒青红茶化学成分分析

为探究红茶在贮藏过程中化学成分的变化规律,以贮藏时间为0、1、3 a和4 a的晒青红茶为代表样品,采用基于超高效液相色谱-四极杆轨道阱质谱的代谢组学方法对其研究.结果表明,在不同年份晒青红茶中共鉴定出82种化合物,包括儿茶素类6种、二聚儿茶素类9种、氨基酸类5种、生物碱类8种、酚酸类8种、有机酸类3种、香气糖苷类2种、...     

Phenolic profile in Czech white wines from different terroirs

Grapevine cv. Riesling is a traditional variety of cool climate viticulture regions to which the Czech Republic belongs too. In total 35 wine samples from 9 different terroirs of wine-growing regions in the Czech Republic were evaluated for the content of 20 phenolic compounds comprising hydroxybenzoic acids, hydroxycinnamates, stilbenes, and flavan-3-ols. Phenolic compounds were evaluated by an HPLC method. Methods of multivariate statistical analysis were used to discriminate wine samples on the basis of their geographical origin. Canonical variate analysis proved that it is possible to differentiate wines according to their geographical origin by following authenticity markers: gallic acid, caffeic acid, caftartic acid, p-coutaric acid, ferulic acid ethylester, p-coumaric acid ethylester, (+)-catechin, and (?)-epicatechin. On the basis of statistical analyses, 100% wine samples were correctly classified. The results indicate that, for the case of white wines, mainly hydroxycinnamates and flavan-3-ols can be used for differentiation of their geographical origin.     

Biological and chemical factors affecting the valuation of North East Indian plains teas. III. Statistical Evaluation of the Biochemical Constituents and their Effects on Colour,Brightness and Strength of Black Teas

Regardless of tasters and methods of manufacture five biochemical factors, total oxygen uptake of unprocessed tea shoots and theaflavins (TF), thearubigins (TR), (?)-epigallocatechin gallate (EGCE) and theogallin (TG), of black tea are the main contributing factors of colour of North East India plains tea. Similarly, three constituents, TF, TR and (?)-epicatechin gallate (ECG) of black tea, are main the factors for brightness. Four factors, total oxygen uptake and ECG of unprocessed tea shoots and TF and moisture of black tea are the main contributing factors of strength of North East Indian plains tea.     

The phenolic pigment composition of black tea liquors—part II: Discriminating origin

The phenolic constituents of 95 black teas, from nine countries, were analysed by HPLC. The procedure resolved 38 coloured phenolic components which were classified as theaflavins (4), thearubigins (22) and flavonol glycosides (11). One of the thearubigin constituents was unresolved chromatographically, but nevertheless quantified as a hump. The relative levels of these substances for different regions and countries of origin were compared. Statistical analysis of the HPLC results with discriminant analysis or canonical variant analysis and ANOVA, highlighted the characteristic differences in phenolic substance levels which contributed most powerfully to the discrimination between origins. Discriminant analysis revealed a discrimination rule based on phenolic composition which achieved a country of origin classification success of 94%. This type of approach highlights which substances are most important in defining origin in terms of tasters' assessments.     

The chemical oxidation of catechins and other phenolics: A study of the formation of black tea pigments

The chemical oxidation of polyphenols has been used to produce black tea pigments. (+)-Catechin, (?)-epicatechin, (?)-epicatechin gallate and (?)-epigallocatechin gallate were isolated from convenient sources, their identity being confirmed by mass spectrometry and HPLC. The oxidation of a mixture of (?)-epicatechin gallate and gallic acid gave an oxidation product of (?)-epicatechin gallate in addition to epitheaflavic acid 3-gallate. The catechins were oxidised chemically and the products analysed by HPLC, the chromatograms being compared with a black tea chromatogram. The reactions gave both resolved and unresolved pigments, and many of the resolved pigments had HPLC retention times close to black tea pigments. Each catechin behaved differently, the chromatograms of the oxidation products from each starting material being clearly distinguishable. Resolved pigments were obtained by the chemical oxidation of other phenolic compounds. This work provides a convenient method for studying the formation of resolved and unresolved black tea pigments.     

Leaching of residues of certain pesticides from black tea to brew

Tea is the most commonly consumed beverage in the world. It is prepared after infusing processed black tea in hot water. During the process of brewing, along with flavour and aroma, the residues of plant protection chemicals may also be transferred into the tea brew or infusion. The leaching of certain pesticides, such as ethion, endosulfan, dicofol, chlorpyrifos, deltamethrin, hexaconazole, fenpropathrin, propargite, quinalphos and lambdacyhalothrin from powdered black tea into the brew was studied. The rate of transfer of the pesticide residue from black tea to the hot brew was largely influenced by physicochemical parameters, such as water solubility and octanol-water partition coefficient. Tea brews prepared from untreated black tea samples were fortified with standard solutions of the respective pesticides, extracted and analysed using GC and HPLC by following standardised methods. Results revealed that the rate of leaching of residues of these pesticides into the tea brew was low due to their low solubilities in aqueous medium and high octanol-water partitioning coefficients.     

黄金茶工夫红茶的甜味量化及其与主要滋味物质相关性分析

以黄金茶工夫红茶为研究对象,量化评价茶汤甜味强度,检测主要滋味物质含量,并分析两者相关关系。结果表明：不同厂家的黄金茶工夫红茶甜味强度差异明显;糖类甜味物质中麦芽糖含量最高,氨基酸类甜味物质中丝氨酸含量最高;甜味物质（包括单糖、双糖和甜味氨基酸）和茶氨酸含量均与甜味强度呈正相关,且糖总量与甜味强度呈显著（P＜0.05）正相关（r=0.781）;儿茶素、咖啡碱、没食子酸、茶黄素、茶红素含量与甜味强度呈负相关,且儿茶素总量、茶黄素含量与甜味强度呈显著（P＜0.05）负相关（r=－0.811和－0.781）,添加表没食子儿茶素没食子酸酯能降低糖类甜度。鉴于甜味是工夫红茶的分属滋味之一,甜味量化及相关性分析将为工夫红茶品质评判提供研究基础。     

ENZYMATIC SYNTHESIS OF THEAFLAVINS AND EPITHEAFLAVIC ACID FROM TEA CATECHINS AND THEIR ANTIOXIDANT ACTIVITY

A number of in vitro and in vivo studies have demonstrated that black tea polyphenols have significant biological activities, including antioxidant, antiinflammatory and anticancer activity. The theaflavins have received attention as one of responsible components for biological activities of black tea. However, little information is available for the biological activity of individual theaflavins and minor components in black tea. In the present study, theaflavins and epitheqflavic acid were synthesized from their parent flavan-3-ols catechins using an enzymatic oxidation method and employing crude polyphenol oxidase from banana fruit. The compounds were isolated using column chromatographic methods, and their structures confirmed by ¹H-NMR and APCI-MS. The peroxyl radical scavenging activities of theaflavins and epitheaflavic acids were measured using a modified ORAC method. The results indicated that theaflavins have higher peroxyl radical scavenging activity than EGCG. The hierarchy of radical scavenging activity of theaflavins, epitheaflavic acid and related compounds were in the order of: theaflavin-3, 3'-digallate ∼ theflavin-3-gallate ∼ theaflavin-3'-gallate ∼ theaflavin > epitheaflavic acid > EGCG > purpurogallin.     

不同冷冻和摇青工艺的红茶品质比较分析

该研究以传统加工的红茶为对照，分析了萎凋前冷冻处理及该处理与萎凋后一次、两次、三次摇青组合处理等新工艺对茶叶品质形成的影响，明确了最佳的工艺处理条件。结果表明，与对照相比，新工艺均可显著增加茶叶没食子酸、茶多酚、茶黄素、黄酮苷等含量（P<0.05），同时，冷冻处理会显著降低可溶性糖含量（P<0.05）（下降10.73%），冷冻和摇青组合处理可显著降低茶褐素含量（P<0.05）（平均下降6.77%）；与冷冻处理相比，冷冻和摇青组合工艺处理可显著降低茶多酚含量（P<0.05）（平均下降3.55%），并显著增加茶黄素含量（P<0.05）（平均增加18.06%）。各处理中以冷冻和两次摇青组合工艺处理的茶叶同时具有相对较高的氨基酸、茶黄素、顺-己酸-3-己烯酯，以及相对较低的没食子酸、儿茶素、黄酮苷、茶褐素、3-辛烯-2-酮，所制红茶呈现橙黄明亮、甜香、甜醇等色香味兼具的品质，感官总分为91.30，分别较对照和冷冻处理高出了2.40、2.60分。综上可知，冷冻和两次摇青的组合处理为最佳新工艺。     

Determination of free radicals and flavan-3-ols content in fermented and unfermented teas and properties of their infusions

The objective of this study was to analyse selected brands of fermented (red and black) and unfermented (white and green) teas for free radical content with the use of electron paramagnetic resonance (EPR) spectroscopy and for contents of flavan-3-ols by means of high-performance liquid chromatography. Analyses were also conducted for the polyphenolic profile of infusions of the analysed teas (with the Folin–Ciocalteu’s method) and their antioxidant activity (in reaction with a DPPH radical) at three brewing times (5, 10 and 15 min). The obtained results showed the possibility of using rapid spectroscopic method EPR to evaluate the oxidative changes in tea leaves caused by enzymatic fermentation. The number of free radicals in teas was negatively correlated with contents of flavan-3-ols, (?)-EGCG in particular. The main signals observed in EPR spectra of teas were attributed to semiquinone radicals; however, also signals attributed to carbohydrate radicals were detected. Regarding unfermented teas, it was ascertained that teas with the highest content of flavan-3-ols, (?)-EGCG in particular, were characterised by the lowest content of semiquinone radicals and a high content of carbohydrate radicals. The group of fermented teas demonstrated to contain mainly semiquinone radicals. The total phenolic content and antioxidant activity of the tea infusions were strongly diversified depending on the kind and brand of tea as well as on the extraction time. The predominating flavan-3-ol in the analysed teas was epigallocatechin gallate (?)-EGCG, the content of which was additionally highly correlated with the antioxidant activity of the tea infusions.     

Catechins depletion patterns in relation to theaflavin and thearubigins formation

The determination of the depletion pattern of catechins in black tea processing is important in achieving optimum tea quality. This study investigated catechins (unoxidised di- and trihydroxylated) depletion patterns in relation to theaflavin and thearubigin formation. It was during the process of green leaf fermentation at selected temperature and time combinations. The tea leaves were obtained from three clones (6/8, 303/577 and 311/287) within the Tea Research Foundation of Kenya. The results were showed that unequal depletion rates of di- and trihydroxylated catechins led to a decline in total theaflavin and an increase in thearubigins levels. An equitable decline in both groups of catechins corresponded to a subsequent rise in theaflavins content. The decline in the catechins levels was much faster at higher temperatures resulting in a shorter fermentation time to achieve a peak of the theaflavins content. Clone 311/287 had the highest mean theaflavins content (26.99 μmol/g) and the least mean percent thearubigins (15.02%) level. Theaflavins content correlated positively with liquor brightness determined by a spectrophotometer and tea tasters (r = 0.7221, p < .0001). The thearubigins content was however found to relate negatively with liquor brightness. It was concluded that the experimental conditions tested form a good basis for clonal specific processing conditions that can be utilised in manufacturing quality black tea.     

云南固态速溶普洱茶生产企业标准现状分析

目的了解速溶普洱茶行业现状,为规范固态速溶普洱茶产业,制定固态速溶普洱茶行业标准提供市场依据。方法通过对固态速溶茶国家标准与55个速溶普洱茶生产企业60个企业标准理化指标和安全性指标的综合分析,确定各理化指标的基本数值范围和企业标准涉及的安全性指标种类,并对国家标准与相关企业标准进行对比。结果水分含量在8%~15%的企业标准数量占调查企业标准总数的61.36%,总灰分含量在15%~25%的企业标准数量占调查企业标准总数的78.82%,茶多酚含量在10%~20%的企业标准数量占调查企业标准总数的69.35%;安全性指标可参考国家相关标准。结论固态速溶普洱茶行业标准可结合现有固态速溶茶国家标准和产业实际需求进行制定。