Theaflavin Research Articles

Comparison of the activity of theaflavins produced by recombinant polyphenol oxidase and commercial enzyme

Theaflavins (TFs) contribute greatly to the colour and flavor of black tea, and have various bioactivities beneficial to human health. This research compared the activity for enzymatic production of TFs from tea polyphenols by recombinant polyphenol oxidase (Malus domestica, GenBank login number LT718523.1, MdPPO2) versus commercial enzyme (Agaricus bisporus, AbPPO) in both free and immobilized form. It describes the difference in activity for TFs production between recombinant and natural polyphenol oxidase. Enzyme assays by LC-MS revealed that the TFs production by AbPPO was almost 5 times as high as recombinant MdPPO2 in free enzyme. When immobilized on mesoporous silica, the activity of recombinant MdPPO2 increased significantly, while AbPPO almost lost its activity. By comparing relative enzyme activity, the immobilization of recombinant MdPPO2 has the highest relative enzyme activity, which was above 6 times higher than free AbPPO. Among these TFs, TF3 was the most abundant, followed by TF2a, TF1 and TF2b.

Fabrication of pH-responsive whey protein/sodium alginate composite hydrogel beads for theaflavins

In this study, pH-responsive whey protein (WP)/sodium alginate (SA) composite hydrogel beads using the ionic gelation method were described and evaluated for their potential as delivery carriers for theaflavin (TF). Fourier transform infrared spectroscopy (FTIR) confirmed the interaction between WP and SA in the hydrogel beads is driven primarily by the strong intermolecular interactions, including hydrogen bonding and electrostatic attractions. The swelling ratio of the beads possesses good pH dependence and pH reversibility, effectively preventing the release of TF in the gastric environment. The encapsulation efficiency (EE) of TF in hydrogel beads ranged from 94.995 ± 0.03 to 95.709 ± 0.14, with a loading capacity (LC) of 27–34 mg/g. In in vitro digestion simulations, owing to the pH responsiveness, hydrogel beads released minimal TF throughout gastric digestion and were fully released in the intestine phase. Additionally, the release kinetics of TF from the beads were further examined in a simulated intestinal environment. These findings suggest that the hydrogel bead system is a promising carrier for encapsulating TF, offering a theoretical and experimental basis for its future application in the food industry.

Changes in the structural, aggregation behavior and gel properties of pork myofibrillar protein induced by theaflavins

This study explores the effect of different theaflavins (TFs) concentrations (0, 100, 300, 600 and 900 mg/L) on the structure, aggregation behavior and gelation properties of pork myofibrillar protein (MP). The protein structure and aggregation behavior were characterized by free sulfhydryl groups, surface hydrophobicity, fluorescence emission spectra, particle size and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gel properties of samples were characterized by gel strength, cooking loss, microstructure and gel supernatant SDS-PAGE. The results showed a significant decrease in free thiol content with increasing TFs concentration, suggesting thiol-quinone covalent interaction between TFs and thiol group of MP. Intrinsic fluorescence spectroscopy confirmed a static quenching between TFs and MP. And TFs reduced the particle size of MP suspension and caused no protein aggregation bond in SDS-PAGE. For gel properties, TFs caused a decrease of gel strength from 96.77 g to 21.91 g and an increase in cooking loss from 40.34 % to 71.15 %. The bond of protein aggregates in gel supernatants SDS-PAGE revealed that some protein aggregates formed by disulfide bonding were not involve in gel formation with TFs addition. In conclusion, TFs cause thiol loss of MP and impaired MP gelling ability by interfering with disulfide bond formation during gelation.

Stability and gastrointestinal digestion behaviour of theaflavins encapsulated in W/O Pickering emulsions

Theaflavins (TFs) are naturally bioactive compounds with low stability and poor bioavailability. In this study, the stability of TFs during storage at 25 °C for 21 d and thermal treatments at 65 °C, 85 °C and 105 °C for 0.5–4 h of water in oil (W/O) Pickering emulsions composed of TFs (0–9 mg/g), ursolic acid, oil and water were investigated. The TFs incorporation at 9 mg/g had the smallest droplet size and the highest G′ and G″ values. After storage for 21 days and thermal treatment at 105 °C for 4 h, the TFs retention in emulsions (9 mg/g) were 1.2-fold and 1.4-fold higher than aqueous solution, respectively, which might be attributed to the encapsulation of TFs in water droplets. During digestion, TFs emulsions provided a greater surface area for lipid digestion and led to a higher release of free fatty acids than emulsions without TFs. Moreover, there was sustained release of TFs in the emulsion, resulting in significantly enhanced TFs bioaccessibility (57.8 ± 9.2%) compared with aqueous solution (17.7 ± 1.1%) and Oil-Water mixture (32.6 ± 2.3%) after intestinal digestion. This study provides insights for food industry in designing W/O emulsion systems for the delivery of hydrophilic bioactive components.

Non-covalent interaction between lactoferrin and theaflavin: Focused on the structural changes, binding mechanism, and functional properties

In this study, non-covalent binding mechanism of lactoferrin (LaF)-theaflavin (TF) complex and its functional properties were investigated. Multi-spectroscopic analyses showed that the secondary structure of LaF was altered with increasing TF concentration. The non-covalent binding of TF to LaF resulted in a reduction in the content of the α-helix and β-sheet, as well as a decrease in the fluorescence intensity of LaF. DSC result showed that non-covalent binding of TF improved thermal stability of LaF. Molecular dynamics simulations confirmed that the stable binding of LaF-TF was driven by hydrogen bonding and hydrophobic interactions. Additionally, non-covalent binding of TF increased the antioxidant capacity and emulsifying properties of LaF. Dynamic interfacial tension indicated that the strong interaction between LaF and TF reduced the interfacial tension, but improved the rheological properties of LaF. The functional characteristics of the non-covalent complex was effectively enhanced, paving the way for its potential use in the food industry.

Oxidation characteristics of catechins in suspended fermentation of different varieties’ tea leaves

This study focused on the efficiency of enzymatic synthesis for theaflavins (TFs) and SII types of thearubigins (SII-TRs) in suspended fermentation of different varieties of tea leaves. The results showed that the content of catechins in tea variety-Xiaoxianghong 21–3 (XXH21–3) was the highest among the five kinds of fresh tea leaves, and it had the strongest ability of transforming catechins to TFs and SII-TRs. The total amount of catechins for XXH 21–3 decreased rapidly and the conversion ratio of TFs reached the highest level of 13.96 % after 20 min, the residual catechins was the lowest (3.27 %) and the conversion rate of SII-TRs was the highest (13.02 %) after 120 min during the suspended fermentation of five varieties tea leaves. Among six types of catechins, epicatechin gallate (ECG), epigallocatechin gallate (EGCG) and epigallocatechin (EGC) were consumed in large quantities, and the residues were all less than 5 % in the final stage of fermentation, of which ECG consumption was the fastest, followed by EGCG. These results demonstrated that the concentration of catechins and PPO activities were positively correlated with the formation of TFs and SII-TRs, POD activity was positive correlation with synthesis of SII-TRs and negative correlation with TFs.

Theaflavins mitigate diabetic symptoms in GK rats by modulating the INSR/PI3K-Akt/GSK-3 pathway and intestinal microbiota

Dietary management and interventions are crucial in the clinical management of diabetes. Numerous active dietary components in black tea have demonstrated positive effects on blood glucose levels and metabolic functions. However, limited research has explored the potential of theaflavins (TF), polyphenols in black tea, for diabetes management. In this study, high-purity TF was administered to Goto–Kakizaki (GK) diabetic model rats for four weeks to investigate its impact on diabetic pathology and analyze the underlying mechanisms through liver transcriptomics, hepatocyte metabolomics, and gut microbiome analysis. The findings indicated that continuous administration of TF (100 mg/kg) significantly suppressed blood glucose levels, reduced insulin resistance, and decreased the expression of oxidative stress indicators and inflammatory factors in GK rats. Further analysis revealed that TF might alleviate insulin resistance by improving hepatic glycogen conversion and reducing hepatic lipid deposition through modulation of key pathways, such as peroxisome proliferator-activated receptors and PI3K/AKT/GSK-3 pathways within the liver, thereby ameliorating diabetic symptoms. Additionally, TF intake facilitated the restoration of the intestinal microbial community structure by reducing the abundance of harmful bacteria and increasing the abundance of beneficial bacteria. It also reduced endotoxin lipopolysaccharide production, thereby lowering the chances of insulin resistance development and enhancing its efficacy in regulating blood glucose levels. These findings offer a novel perspective on the potential of black tea and its active constituents to prevent and treat diabetes and other metabolic disorders, providing valuable references for identifying and applying active dietary components from tea.

Development of theaflavins-loaded albumin nanoparticles and in silico analysis of theaflavins: BSA interaction

Theaflavins (TFs), bioactive compounds present in black tea manufactured from Camellia sinensis leaves have potential health benefits. Due to their instability and low bioavailability, we have encapsulated TFs into albumin nanoparticles (Alb-TFs-NP) and characterized them using different techniques. The mean particle size of Alb-TFs-NP was 125.9 nm with a low (0.23) polydispersity index and Zeta potential of -76.8 mV. Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) analysis revealed a spherical shape of Alb-TFs-NP with a smooth surface. The fluorescence of bovine serum albumin (BSA) and TFs was at 280 nm and 660 nm excitation and 690-700 nm emission respectively. The BSA-TFs complex interacts at distinct amino acid binding sites with good docking energy of TF1 (-10.5 kcal/mol-1), TF2 (-11.1 kcal/mol-1), TF2a (-11.2 kcal/mol-1) and TF3 (-10.4 kcal/mol-1) at the BSA active site. Primarily hydrogen bonding was involved in the BSA-TFs interaction. The study could further research the transport distribution and bioactivities of TFs in the human body.

Effect of sodium alginate‐locust bean gum ice glazing containing different antioxidants on quality of mackerel (Pneumatophorus japonicus) during frozen storage

SummaryThe quality of fish is easily damaged during the frozen process, and the ice glazing technology can better maintain the quality of frozen fish. However, the traditional ice glazing technology is not yet stable and some of the properties need to be improved. This research optimised ice glazing technology by adding different antioxidants to the traditional ice glazing, which improved the stability of the ice glazing and lessened the dry consumption of mackerel during frozen storage. The lipid oxidation and protein denaturation of mackerel were inhibited with the addition of antioxidants to the ice glazing, the freshness of mackerel was maintained better, and the production of histamine was also decreased during frozen storage. It was found that the ice glazing with the addition of antioxidant bamboo leaves (AOB) had better water retention, lower TVB‐N value, and histamine content compared to the addition of theaflavins (TF) and sodium isovitamin (VC). Moreover, the sample treated by the ice glazing with AOB showed less lipid oxidation and protein denaturation. Therefore, ice glazing with the addition of AOB could be an optional method for relieving the quality deterioration of mackerel during frozen storage. This research also provides insights into the coupling of antioxidants and ice‐coating techniques.

Effects of conjugation of egg lysozyme to theaflavins on their structure and function

Proteins modified by polyphenols have more advantages. In this study, we investigated the effect of covalent conjugation of egg white lysozyme (LYZ) with theaflavin (TF) on its structure and related functions. The LYZ-TF covalent conjugation was prepared by an alkaline method (pH 9.0) with a branching rate of about 80%. The results such as SDS-PAGE and UV spectroscopy proved the formation of the covalent conjugation. The results of fluorescence and infrared spectroscopy showed that the conformation of the LYZ-TF coupling was altered, with an increase in α-helix content by more than 10% and an increase in surface hydrophobicity compared to LYZ. The results of relevant functional characterization experiments showed the thermal stability and antioxidant properties of LYZ modified by TF, but its enzymatic activity and in vitro antimicrobial properties were inhibited. Notably, the amount of TF addition also affected the structure and function of the conjugates. These results provide some theoretical guidance for the development of novel food functional materials with enhanced properties.

Improvement of Theaflavins on Glucose and Lipid Metabolism in Diabetes Mellitus.

In diabetes mellitus, disordered glucose and lipid metabolisms precipitate diverse complications, including nonalcoholic fatty liver disease, contributing to a rising global mortality rate. Theaflavins (TFs) can improve disorders of glycolipid metabolism in diabetic patients and reduce various types of damage, including glucotoxicity, lipotoxicity, and other associated secondary adverse effects. TFs exert effects to lower blood glucose and lipids levels, partly by regulating digestive enzyme activities, activation of OATP-MCT pathway and increasing secretion of incretins such as GIP. By the Ca2+-CaMKK ꞵ-AMPK and PI3K-AKT pathway, TFs promote glucose utilization and inhibit endogenous glucose production. Along with the regulation of energy metabolism by AMPK-SIRT1 pathway, TFs enhance fatty acids oxidation and reduce de novo lipogenesis. As such, the administration of TFs holds significant promise for both the prevention and amelioration of diabetes mellitus.

Co-immobilization of laccase-mediator system to catalyze the synthesis of theaflavins from tea polyphenols

The co-immobilized laccase-mediator system (LMS) has broad development prospects in the fields of biocatalysis, biochemistry, and environmental remediation. In this study, laccase and mediator molecules were co-immobilized in functionally modified calcium alginate microspheres to improve their catalytic activity and stability and to systematically characterize the physicochemical properties and evaluate the catalytic performance of the co-immobilized laccase-mediator system. The co-immobilized LMS was used to catalyze the synthesis of theaflavins (TFs) from tea polyphenols, improve the catalytic efficiency, realize the recycling of the system, and explore the safety of the catalytic products. The results showed that the catalytic efficiency of the co-immobilized LMS was significantly improved and the relative activity remained above 50% after 10 cycles. The in vitro safety of the catalytic product was verified by Human hepatocellular carcinoma (HepG2) cell-related indexes. The results show that the co-immobilized laccase mediator system has great development potential in the industrial application of theaflavins.

Chemical, sensory and biological variations of black tea under different drying temperatures

As the final processing step, drying temperature between 90 and 140 ℃ is usually applied to terminate enzymatic activities and improve sensory characteristics of black tea. Liquid chromatography tandem mass spectrometry (LC-MS) based non-targeted and targeted metabolomics analyses combined in vitro biological assays were adopted to investigate the chemical and biological variations after drying. Fifty-nine differentially expressed metabolites including several hydroxycinnamic acid derivatives and pyroglutamic acid–glucose Amadori rearrangement products (ARPs) were identified, the latter of which was correspondingly accumulated with increasing temperature. The levels of theaflavins (TFs), thearubigins (TRs), monosaccharides and free amino acids gradually decreased with increasing temperature. Furthermore, the bioassays of black tea showed that drying under 110 ℃ provided the highest antioxidant capacities, but the inhibitory effects on α-glucosidase and α-amylase were decreasing along with increasing drying temperature. These results are valuable for optimizing drying process to obtain superior sensory properties and preserve bioactivities of black tea.

Efficient enzymatic synthesis of theaflavin and its production mechanism.

In this study, a novel preparation method of theaflavin (TF) has been established. Our findings indicated that the formation of TF was significantly enhanced by using an ice bath (2-3°C). Additionally, increasing the ratio of (-)-epigallocatechin (EGC) under the ice bath could further improve its yield. This approach prevented the appearance of a dark solution within 3h, effectively protecting TF from oxidation. Our study on the generation mechanism of TF suggested that EGC-quinone I (EGC-Q-I) with two carbanions could potentially serve as one of synthons based on the retrosynthetic analysis of the bicyclo[3.2.1]octane-type intermediate. Subsequently, quantum mechanical calculations further supported this hypothesis. Practical Application: In this study, we have developed a novel method for the synthesis of theaflavin (TF), demonstrating that the use of ice bath significantly enhanced its yield. Increasing the ratio of (-)-epigallocatechin (EGC) under the ice bath further improved TF yields and prevented darkening of the solution for at least 3h, thereby protecting TF from oxidation. Our study suggested that EGC-quinone I is a potential synthon based on the retrosynthetic analysis of the bicyclo[3.2.1]octane-type intermediate (BOI). This hypothesis is supported by QM calculations.

The substitution sites of hydroxyl and galloyl groups determine the inhibitory activity of human pancreatic α-amylase in twelve tea polyphenol monomers

Tea polyphenols have been reported as potential α-amylase inhibitors. However, the quantitative structure–activity relationship (QSAR) between tea polyphenols and human pancreas α-amylase (HPA) is not well understood. Herein, the inhibitory effect of twelve tea polyphenol monomers on HPA was investigated in terms of inhibitory activity, as well as QSAR analysis and interaction mechanism. The results revealed that the HPA inhibitory activity of theaflavins (TFs), especially theaflavin-3′-gallate (TF-3′-G, IC50: 0.313 mg/mL), was much stronger than that of catechins (IC50: 18.387–458.932 mg/mL). The QSAR analysis demonstrated that the determinant for the inhibitory activity of HPA was not the number of hydroxyl and galloyl groups in tea polyphenol monomers, while the substitution sites of these groups potentially might play a more important role in modulating the inhibitory activity. The inhibition kinetics and molecular docking revealed that TF-3′-G as a mixed-type inhibitor had the lowest inhibition constant and bound to the active sites of HPA with the lowest binding energy (−7.74 kcal/mol). These findings could provide valuable insights into the structures-activity relationships between tea polyphenols and the HPA inhibitors.

Effects of Pulsed Electric Field Pretreatment on Black Tea Processing and Its Impact on Cold-Brewed Tea.

This study applied pulsed electric fields (PEFs) to accelerate the withering and drying processes during cold-brewed black tea production. PEF pretreatment was administered at 1.0, 1.5, and 2.0 kV/cm electric field strengths, combined with varying withering times from 8 to 12 hr. During the 12-hour withering process, the redness value (a*) and total color change (∆E) of PEF-treated leaves significantly increased (p < 0.05). Furthermore, the homogenous redness of tea leaves during fermentation depended on the PEF strength applied. In addition, PEF pretreatment remarkably reduced the drying time, up to a 50% reduction at a 2.0 kV/cm field strength. Additionally, the 2.0 kV/cm PEF-pretreated black tea exhibited a notable 42% increase in theaflavin (TF) content and a 54% increase in thearubigin (TR) content. Sensory evaluation scores were highest for black tea that received PEF pretreatment at 2.0 kV/cm. These findings highlight the significant potential of PEFs in enhancing the efficiency of withering and drying processes while positively impacting the physicochemical and sensory properties of cold-brewed black tea.

Theaflavin pretreatment ameliorates renal ischemia/reperfusion injury by attenuating apoptosis and oxidative stress in vivo and in vitro

Oxidative stress-induced apoptosis is an important pathological process in renal ischemia/reperfusion injury (RIRI). Theaflavin (TF) is the main active pigment and polyphenol in black tea. It has been widely reported because of its biological activity that can reduce oxidative stress and protect against many diseases. Here, we explored the role of theaflavin in the pathological process of RIRI. In the present study, the RIRI model of 45 min ischemia and 24 h reperfusion was established in C57BL/6 J male mice, and theaflavin was used as an intervention. Compared with the RIRI group, the renal filtration function, renal tissue damage and antioxidant capacity of the theaflavin intervention group were significantly improved, while the level of apoptosis was reduced. TCMK-1 cells were incubated under hypoxia for 48 h and then reoxygenated for 6 h to simulate RIRI in vitro. The application of theaflavin significantly promoted the translocation of p53 from cytoplasm to nucleus, upregulated the expression of glutathione peroxidase 1 (GPx-1) in cells, and inhibited oxidative stress damage and apoptosis. Transfection with p53 siRNA can partially inhibit the effect of theaflavin. Thus, theaflavin exerted a protective effect against RIRI by inhibiting apoptosis and oxidative stress via regulating the p53/GPx-1 pathway. We conclude that theaflavin has the potential to become a candidate drug for the prevention and treatment of RIRI.

Tea Harvesting and Processing Techniques and Its Effect on Phytochemical Profile and Final Quality of Black Tea: A Review.

Tea (Camellia sinensis) has grown for over 300 years and is recognized worldwide as among other well-renowned crops. The quality of black tea depends on plucking (method, standard, season, and intervals), withering and rolling (time and temperature), fermentation (time, temperature, and RH), drying (temperature and method), and storage conditions, which have a high influence on the final quality of black tea. At the rolling stage, the oxidation process is initiated and ends at the early drying stage until the enzymes that transform tea polyphenols into thearubigins (TRs) and theaflavins (TFs) are denatured by heat. By increasing fermentation time, TRs increased, and TF decreased. Each is liable for black tea's brightness, taste, and color. The amino acids and essential oils also grant a distinctive taste and aroma to black tea. Throughout withering, rolling, and fermentation, increases were found in essential oil content, but during drying, a decrease was observed. However, the Maillard reaction, which occurs when amino acids react with sugar during drying, reimburses for this decrease and enhances the flavor and color of black tea. As compared to normal conditions, accelerated storage showed a slight decrease in the total color, TF, and TRs. It is concluded that including plucking, each processing step (adopted technique) and storage system has a remarkable impact on black tea's final quality. To maintain the quality, an advanced mechanism is needed to optimize such factors to produce high-quality black tea, and an objective setting technique should be devised to attain the desirable quality characteristics.

Quality analysis of steamed beef with black tea and the mechanism of action of main active ingredients of black tea on myofibrillar protein

In this study, we analyzed the tea polyphenol composition, volatile flavor composition and storage stability of steamed beef with black tea. The molecular docking and dynamics were used to elucidate the interaction mechanism between the active components of black tea and myofibrillar proteins. The highest content of caffeine (CAF) was found in black tea steamed beef products, followed by catechin (C), epicatechin gallate (ECG), epicatechin gallate (EGCG) and theaflavins (TF). Steamed beef with black tea showed low ΔE* value, low TBARS value, low carbonyl content as well as high sulfhydryl content during storage. The addition of C, CAF, ECG, EGCG and TF enhanced the oxidative stability of myofibrillar protein. In this study, the effects of active components of black tea on the oxidative stability of myofibrillar protein and their interactions were determined, which could provide a reference for the application of black tea and its active components in meat products. At the same time, it can provide new ideas for the development of new meat products.

The influence of rolling pressure on the changes in non-volatile compounds and sensory quality of congou black tea: The combination of metabolomics, E-tongue, and chromatic differences analyses

Rolling represents an essential stage in congou black tea processing. However, the influence of rolling pressure on tea flavor and non-volatile compounds remains unclear. Herein, a combination of untargeted metabolomics, tea pigments quantification, E-tongue, colorimeter and sensory evaluation was used to evaluate the effect of rolling pressure on black tea quality. As the rolling pressure increased, theaflavins (TFs), thearubigins (TRs), and theabrownins (TBs) significantly elevated. The tea metabolic profiles fluctuated and 47 metabolites were identified as key differential metabolites including flavan-3-ols, flavonol/flavone glycosides, phenolic acids, amino acids. These substances altered possibly due to the variations in enzymatic oxidation of tea phenolics and amino acids. Overall, black tea with moderate rolling pressure presented higher sweetness, lower bitterness, and higher quality index (10 TFs + TRs)/TBs. The results were verified by a validation batch. This study provided new insights into the regulation of rolling pressure and a guidance for black tea processing.