A Preliminary Study On The Variation Of The Asinensins And The A Flavins Transformed From Catechins

With the development of material and spiritual life, a growing number of people choose drinkingtea as one of their life-style. Teas are generally classified into six categories: green tea, black tea, oolongtea, dark tea, white tea and yellow tea. Black tea is one of the most popular beverages in the world. Thetheasinensins and theaflavins are producted by enzymatic oxidation of catechins, which related to thequality of black tea. The catechins, theaflavins and theasinensins were taken as indexes in this paper, thechanges in black tea processing and vitro enzymatic oxidation experiments were system researched onthe foundation of establishing method of high performance liquid chromatography (HPLC) andoptimization of extraction conditions. The results were as follows:1. A High Performance Liquid Chromatography (HPLC) method was established for thesimultaneous determination of theasinensins (theasinensin A, theasinensin B, theasinensin C) andtheaflavins (theaflavin, theaflavin-3-gallate, theaflavin-3’-gallate, theaflavin-3,3’-digallate) in tea. Thedetermination was performed on a cosmosil5C18-AR-Ⅱcolumn (4.6mm×250mm),50mM H3PO4and100%acetonitrile were used as the mobile phases with a gradient elution. The flow rate was0.8mL/min. The column temperature was35℃. The UV detection wavelength was at280nm. Theexternal standard method was used for the quantitative analysis. The method is reproducible, accurate,stable and suitable for the simultaneous determination of theasinensins and theaflavins in tea.2. In order to study the of extraction conditions on theasinensins extraction in tea, severalimportant factors such as extraction time, extraction temperature, liquid-solid ratio and extractionsolvent concentration were studied by one factor at-a-time. By keeping extraction time10min,liquid-solid ratio (10:1-40:1mL/g), extraction solvent concentration (40%-70%) and extractiontemperature (30℃-70℃) was chosen as the factors of three-factor second order general rotationcombination experiment. The experiment results indicated: the extraction solvent concentration,extraction temperature had extremely significant effect on theasinensins extraction in tea, respectively;the liquid-solid ratio had significant effect on theasinensins extraction in tea; the influencing sequencefrom significant to common is extraction solvent concentration, extraction temperature, liquid-solidratio. The optimum extraction parameters were as follows: extraction temperature70℃, liquid-solidratio29.41:1mL/g, extraction solvent concentration48%methanol, extraction time10min. Bysubstituting levels of the factors into the quadratic regression equation, the maximum predictableresponse for theasinensins extraction was calculated and was experimentally verified. The maximumtheasinensins extraction obtained experimentally using the optimized extraction conditions was11.63mg/g, which was in correlation with the predicted value of11.70mg/g by the response surfacemethodology regression study.3. We confirmed that the best way to store and dry samples were cryopreservation (liquid nitrogen)and vacuum freeze-drying by comparing the effect of difference fixing methods and storing samplemethods on the contents of theasinensins. We compared the effects of difference fermentation temperatures and drying temperatures on the synthesis of theaflavins and theasinensins from catechins.23℃was favorable for the synthesis of theaflavins. In contrast,31℃was favorable for the synthesisof theasinensins. Compared with the other drying temperature, the maximum value of theaflavins wasgenerated when drying temperature is110℃. In contrast, the maximum value of theasinensins wasgenerated when drying temperature is110℃or140℃. Compared with the content of theasinensinsat the end stage of tea fermentation, the content of theasinensins was increased when drying temperatureis110℃,140℃,170℃. In contrast, the content of theasinensins is changeless when dryingtemperature is50℃,80℃.4. We compared the activity and catechin oxidation abilities of PPO from difference sources. Theresults indicated: compared with the other enzymatic source, the enzyme activity of housi was not high,but had strong catechin oxidation abilit ies and yields the maximum value of theaflavins andtheasinensins. So we chose housi as the best enzyme source. Apple, banana, laccase were favorable forthe formation of theaflavin and theasinensin B. In contrast, housi were favorable for the formation oftheasinensin C, theaflavin-3-gallate and theaflavin-3,3’-gallate. By comparing the effects of differencefermentation temperatures and pH values on the synthesis of theaflavins and theasinensins fromcatechins, we think the lower fermentation temperature (27℃and32℃) is favorable for theformation of theaflavins. In contrast, the higher fermentation temperature (37℃and42℃) isfavorable for the formation of theasinensins. Theaflavins and theasinensins are stable in acid but areextensively degraded at conditations neutral pH. In neutral pH, theasinensins are more stable thantheaflavins.5. We study the effect of pH on the oxidation of homogenous catechin reactants (EGC, EGCG) andheterogenous mixtures (ECG+EGC, ECG+EGCG, EGC+EC, EGCG+EC, EGCG+EGC) in vitroenzymatic oxidation. The results indicated: Acid condition were advantageous to the synthesis andaccumulation of TFDG and neutral conditions were conducive to the formation and degradation of TF,TF-3-G, TF-3’-G, TSA, TSB and TSC; When EGC and EGCG as the enzymatic oxidation ofsubstrates at the same concentration, the rate of synthesis of theasinensins from high to low was TSC,TSB, TSA.