Production Of Theaflavins Research Articles

Comparison of the Activity for Theaflavin Production by a Recombinant Polyphenol Oxidase and a Commercial Enzyme

AbstractTheaflavins (TFs) contribute greatly to the color and flavor of black tea, and have various bioactivities beneficial to human health. This research compared the activity for TF production from tea polyphenols of recombinant polyphenol oxidase (Malus domestica, GenBank login number LT718523.1, MdPPO2) with that of commercial polyphenol oxidase (Agaricus bisporus, AbPPO) in both free and immobilized forms. Enzyme assays by LC-MS revealed that the production of TFs by the commercial enzyme AbPPO was almost five times as high as that of free recombinant MdPPO2. When immobilized on mesoporous silica, however, the activity of recombinant MdPPO2 increased significantly, whereas AbPPO almost lost its activity. In terms of the relative enzyme activity, the immobilized recombinant MdPPO2 had the highest relative enzyme activity, which was more than six times higher than that of free AbPPO. Among the TFs that were produced, TF3 was the most abundant, followed by TF2a, TF1, and TF2b.

Research on Rapid Detection Methods of Tea Pigments Content During Rolling of Black Tea Based on Machine Vision Technology.

As a crucial stage in the processing of black tea, rolling plays a significant role in both the color transformation and the quality development of the tea. In this process, the production of theaflavins, thearubigins, and theabrownins is a primary factor contributing to the alteration in color of rolled leaves. Herein, tea pigments are selected as the key quality indicators during rolling of black tea, aiming to establish rapid detection methods for them. A machine vision system is employed to extract nine color feature variables from the images of samples subjected to varying rolling times. Then, the tea pigment content in the corresponding samples is determined using a UV-visible spectrophotometer. In the meantime, the correlation between color variables and tea pigments is discussed. Additionally, Z-score and PCA are used to eliminate the magnitude difference and redundant information in original data. Finally, the quantitative prediction models of tea pigments based on the images' color features are established by using PLSR, SVR, and ELM. The data show that the Z-score-PCA-ELM model has the best prediction effect for tea pigments. The Rp values for the model prediction sets are all over 0.96, and the RPD values are all greater than 3.50. In this study, rapid determination methods for tea pigments during rolling of black tea are established. These methods offer significant technical support for the digital production of black tea.

Non-Targeted Metabolomics Reveals the Effects of Different Rolling Methods on Black Tea Quality.

A non-targeted metabolomics approach and sensory evaluation, coupled with multivariate statistical analysis, systematically uncover the impact of the rolling time on the quality parameters of black tea. GC-MS analysis reveals that a moderate extension of rolling time favorably contributes to the accumulation of characteristic aroma components in black tea. The volatile components reach their highest concentration in black tea samples processed during an 80-min rolling period. UHPLC-Q-TOF/MS analysis demonstrates a substantial decrease in the contents of catechins and flavonoids with an increase in rolling time. Simultaneously, the production of theaflavins, coupled with the degradation of green bitterness volatiles (GBVs), significantly contributes to the formation of endogenous aroma components in black tea. These findings underscore the close relationship between rolling time control and black tea quality, emphasizing that a moderate extension of the rolling time fosters the development of improved black tea flavor quality. The comprehensive quality evaluation indicates that the optimal duration is 80 min. However, the initial 0 to 20 min of rolling is a crucial phase for the genesis and transformation of black tea quality. This study offers valuable insights into the influence of rolling time on black tea quality, potentially enhancing future studies of rolling technology. It provides theoretical guidelines for optimizing the processing of Gongfu black tea.

Proteolytic activation and characterization of recombinant polyphenol oxidase from Rosa chinensis for efficient synthesis of theaflavins

Although the oxidation of tea polyphenols catalyzed by polyphenol oxidases (PPOs) is an attractive method for the synthesis of theaflavins (TFs), PPOs for synthesizing TFs were impure, and either not activated or activated by classical chemical methods. Herein, high pure PPO from Rosa chinensis (RcPPO) was cleanly and efficiently activated, and the MBP-RcPPO zymogen was successfully expressed in E. coli BL21 (DE3) with a high pure yield of 37.20 mg/L. Enzymatic activity of the RcPPO after proteolytic activation with trypsin in vitro increased to 5.52 times compared with the MBP-RcPPO zymogen, obtaining a 1625.00 U/L yield of activated enzyme with a specific activity of 480.77 U/mg for 25 mmol/L catechol, and could be enhanced by CuSO4, MgCl2, DMSO and ethanol. The MBP-RcPPO zymogen was specifically cleaved at the peptide bond of R (−5)-I (−4) for N-terminal, but not specifically cleaved at multiple cleavage sites for C-terminal during the activation with trypsin. RcPPO has been classified as tyrosinase with the obvious preference for diphenol compound substrates. Moreover, the four major TFs were synthesized from tea polyphenols extract by RcPPO with a higher efficiency than native PPO from Camellia sinensis, and the yield of TFs monomers could be furtherly improved using the corresponding purified tea polyphenols as substrate. The results indicated that activated RcPPO may serve as a promising biocatalyst for commercial-scale production of TFs.

Influence of enzyme source and catechins on theaflavins formation during in vitro liquid-state fermentation

Theaflavins (TFs) are the main components responsible for the bright, orange-yellow color of liquor and fresh, brisk, and astringent taste of black tea. TFs are formed from the enzymatic oxidative condensation of catechins. In this study, we aimed to optimize the production of TFs from catechins in an in vitro liquid-state fermentation system. We examined (1) three different enzyme sources (2) two different times for the addition of catechins and (3) three different ratios (1:2, 1.5:1.5, and 2:1) of key catechins [(−)-epigallocatechin (EGC); (−)-epigallocatechin gallate (EGCG)]. The results indicated that the use of polyphenol oxidase (PPO) promoted a rapid and uniform consumption of catechins, resulting in a sustained high-level production of TFs. The batchwise addition of catechins was the best method for sustained and high-efficiency TFs formation. An EGC/EGCG ratio of 1:2 led to the formation of relatively higher amounts of theaflavin-3ʹ-gallate (TF-3ʹ-G) and TF-3,3ʹ-digallate (TF-D-G), which resulted in a significantly higher total TFs content. These results demonstrate that the batchwise addition of catechin substrates with a high EGCG content using PPO as an enzyme source can improve the formation of TFs. Our results provide a theoretical basis for understanding the mechanisms of TFs synthesis.

Genome-wide analysis and metabolic profiling unveil the role of peroxidase CsGPX3 in theaflavin production in black tea processing

Plucked tea leaves can be processed into black tea (Camellia sinensis), which is rich in health-promoting molecules, including flavonoid antioxidants. During black tea processing, theaflavins (TFs) and thearubigins (TRs) are generated via the successive oxidation of catechins by endogenous polyphenol oxidase (PPO)- or peroxidase (POD)-mediated reactions. This process must be well controlled to achieve the proper TF/TR ratio, which is an important quality parameter of the tea beverage. However, little is known about the POD/PPO catalyzed TF formation process at the molecular genetic level. Here, we identified and characterized the POD genes responsible for TF production in tea. Genome-wide analysis of POD/PPO family genes, metabolite profiling, and expression analysis of PPO/POD genes in tea leaves enabled us to select several PPO/POD genes potentially involved in TF production. Differential gene expression in plant tissues and enzyme activity in several tea varieties traditionally used for processing of various beverage types indicate that black tea processing primarily depends on PPO/POD activity. Among these POD/PPO genes, the POD CsGPX3 is involved in the generation of TFs during black tea processing. The capacity of PPO/POD-catalysed TF production is potentially used for controlling catechin oxidation during black tea processing and could be used to create molecular markers for breeding of tea plant varieties suitable for the production of high-quality black tea beverages.

Transcriptome and Metabolic Profiling Unveiled Roles of Peroxidases in Theaflavin Production in Black Tea Processing and Determination of Tea Processing Suitability

Theaflavins (TFs) are generated by endogenous polyphenol oxidase (PPO)- and peroxidase (POD)-catalyzed catechins oxidation during black tea processing, which needs to be well-controlled to obtain a proper TFs/thearubigins (TRs) ratio for better quality. Not all leaves from any tea plant cultivars or varieties are suitable for making high-quality black teas, regardless of the processing techniques. The mechanisms underlying TFs formation and the main factors determining the tea leaf processing suitability are not fully understood. We here integrated transcriptome and metabolite profiling of tea leaves to unveil how enzymes or metabolites in leaves are changed during black tea processing. The information enabled us to identify several PPO and POD genes potentially involved in tea processing for TF production. We characterized a POD gene, whose recombinant enzyme showed TF creation activity. The capacity for POD-catalyzed TF production could be used as a molecular marker for breeding tea plant varieties suitable for high-quality black tea production.

Recombinant polyphenol oxidases for production of theaflavins from tea polyphenols

Theaflavins (TFs) have attracted much attention due to their various bioactivities in black tea. This paper describes the first trial for enzymatic production of TFs by recombinant polyphenol oxidases (PPOs). PPO genes were cloned from nine species and expressed in E. coli. Crude enzyme assays by LC-MS revealed that eight recombinant PPOs were active for TFs production from tea polyphenols as substrates. Much higher activities were observed for crude enzymes of Md2 from Malus domestica (apple), Pp4 from Pyrus pashia (pear), and Ej2 from Eriobotrya japonica (loquat). When immobilized on mesoporous silica, crude Md2 was most active. The purified Md2 was immobilized and showed almost twice activity as high as its free enzyme. While the maximum activity of free enzyme was found at pH 5 and 10–30 °C, the immobilized enzyme had broader range of pH 4–6 and 10–40 °C. The activity of immobilized enzyme was relatively constant during the pH and thermal stability test. When used at 0.2 mg/ml in the beginning, the immobilized enzyme retained approximately 40% of its initial activity after 8 cycles of operation.

Effects of Variety, Season, and Region on Theaflavins Content of Fermented Chinese Congou Black Tea

The effects of variety, region, and season on the theaflavins content of fermented Congou black tea were determined. Fresh tea leaves from 5 varieties, 3 tea-growing regions, and 3 harvesting seasons were treated by withering, rolling, fermenting, and drying. Contents of theaflavins, polyphenol oxidase (PPO), peroxidase (POD), and individual catechins in each treatment process were determined. The results showed that the varieties of Yinghong 9 and Hongyan 12 produced high concentrations of theaflavin, as much as 0.5% and 0.6%, respectively. The production capacity of theaflavin in different regions had the trend of Yingde > Changsha > Hangzhou. Fresh leaves plucked in spring season produced the strongest theaflavins. In tea leaves, high ratio of PPO to POD activity and high contents of certain individual catechins lead to high yield of theaflavin production during the whole process.

Synthesis of Theaflavins and Their Functions.

Numerous epidemiological and interventional clinical studies have consistently reported that black tea is good for human health. The polyphenolic compound, theaflavin, and its galloyl esters (theaflavins) are the primary red pigments in black tea that possess several health benefits, including fat-reducing and glucose-lowering capabilities and lifestyle-related disease prevention related to anti-obesity, anticancer, anti-atherosclerotic, anti-inflammatory, antiviral, antibacterial, anti-osteoporotic, and anti-dental caries properties. These compounds are produced by key enzymes, such as polyphenol oxidase and peroxidase, from parent green tea catechins present in fresh green tea leaves during the production of black tea leaves or the fermentation of green tea. However, theaflavins are only present in low concentrations in black tea; thus, their extraction from black tea leaves at sufficient levels for use in medical studies has been difficult. To circumvent this issue, different procedures for the synthesis of theaflavins using chemical oxidizing reagents or enzymes have been studied; however, low yields have limited their utility. Recently, however, several biosynthetic methods have been developed for the mass production of theaflavins. Using these methods, the physiological functions of theaflavins in lifestyle-related diseases in mice and humans have also been studied. In this review, we present the synthesis of theaflavins and their health benefits.

Clonal Variations in the Response of Hard Physically Withered Leaf to Rehydration Following Long Chemical Wither Durations

Long chemical wither durations and hard physical wither reduce the quality of plain black tea, through reduction in total theaflavins production while encouraging thearubigins formation. Such quality reduction may vary with genetic makeup of the tea plant. However, rehydration reactivates activities of oxidative enzymes responsible for producing plain black tea quality parameters. Influence of rehydrating hard physically withered leaf that had undergone long chemical wither durations on two clonal plain black tea quality parameters were assessed. Rehydration restored the formation of plain tea quality parameters from hard physically withered leaf. However, quality deterioration due to long chemical wither duration could not be reversed by rehydration. The patterns of responses of the clones used were similar. Thus in the processing of plain black tea, efforts should be made to maintain chemical wither durations to below 30 hours. However, rehydration reverses reduced plain black tea quality parameters by hard physical withers to equivalent of chemical wither.

Optimization of theaflavin biosynthesis from tea polyphenols using an immobilized enzyme system and response surface methodology

Theaflavins were synthesized from tea polyphenols extracted from green tea using an immobilized polyphenol oxidase system. To optimize the production of theaflavins, response surface methodology was applied to determine the effects of five critical variables and their mutual interactions on theaflavin biosynthesis at five levels. A total of 52 individual experiments were performed and a statistical model predicted that the highest theaflavin concentration was 0.766 mg ml(-1) at optimized conditions. Using these optimal parameters under experimental conditions in three independent replicates, the average value of the biosynthesized theaflavin concentration reached 0.75 +/- 0.017 mg ml(-1) and matched the value predicted by the model.

In vitro oxidation of flavanols from tea leaf

The six major flavanols present in fresh shoots of tea ( Camellia sinensis L.) were extracted, purified, and oxidized in vitro with a soluble and partially purified polyphenol oxidase preparation, also extracted from fresh tea shoots. Besides different forms of theaflavin gallate, two species of theaflavin were observed. When the six flavanols were oxidized together, epigallocatechin limited total theaflavin production.