Establishment Of A Standard For Determination Of Theaflavins And Enzymatic Synthesis Of Theaflagallins

Theaflavins, are benzotropolone-containing pigments with an important contribution to the appearance and mouthfeel of black tea, the applied basic research of which have attracted extensive studies since they have powerful biological activity, particularly antioxidative activity and anticancer. The main contents and results of this paper are summarized as follows:A determination method for four major theaflavins in tea and tea products using high pressure liquid chromatography was established. The method used gradient elution with C18 column, detection wavelength was set at 280 nm. The linearity was obtained over the range of 0.1 ?g/mL?40 ?g/mL(R2>0.9999). The average recovery of sample was in the range of 90.03%?105.05%, with relative standard deviation of less than 5%. The reproducibilities among three laboratories were good eno?gh. This method can be simple, rapid and accurate detection for four major theaflavins in tea and tea products.On the basis of the above-described method, the research was conducted by using microporous menbranes made of six kinds of materials and in two different pore sizes made by five different manufacturers to investigate the effects of microporos filtrating membrane in HPLC quantitative analysis of the main components (theaflavins, catechins, caffeine) in black tea and black tea products. The results showed that 10 ?g/mL theaflavin (TF) standard product was almost completely absorbed by Nylon membrane, of which the recovery rate was less than 1%. Meanwhile, the recovery rate of TF was more than 97% in the Hydrophilic polytetrafluoroethylene (PTFE) or poly vinyl idene fluoride (PVDF) membrane. In addition, the properties of membrane filters made by different manufacturers differ dramatically. For example, the recovery rate of TF were 11.86% and 99.95% by the the same kind of microporos filtrating membrane (Acetate polyether sulfone) from factory 1 and factory 3, respectively. Therefore, the use of the microporos filtrating membranes is a key pre-processing step for accurately measuring theaflavins and catechins, etc.The enzymatic synthesis of theaflagallin-3-gallate with horseradish peroxidase catalyzed in the ethanol/water medium was studied. The influence fators including pH, H2O2 concentration, temperature and molar ratio of substrates on the enzymatic synthesis of theaflagallin-3-gallate were systematically examined. The product is characterizatized by LC-MS, the result show that the product is theaflagallin-3-gallate. The theaflagallin-3-gallate yield in ethanol/water system was much higher than that in fully aqueous medium.The method of one pot enzymatic synthesis of theaflagallin was developed. Epigallocatechin (EGC) was obtained from the hydrolysis of epigallocatechin gallate (EGCG) catalyzed by tannase, and subsequently oxidatied by Horseradish peroxidase with gallic acid (GA). In this paper, firstly, we tried to use EGCG and pyrogallol synthesize theaflagallin-3-gallate by peroxidase, and then through tannase form theaflagallin. The optimization of the hydrolytic reaction conditions of EGCG catalyzed by tannase was investigated. As a result, the yield of 98% was achieved by adding 50mg tannase/250mg EGCG at the concentration of 0.5% under pH 5.0, at 35? for 10 min.