Study On Theaflavins Synthesis By Screening And Using Microorganism Polyphenol Oxidase

Theaflavins (TFs) is the key influence factor on quality of black tea. It has some powerful pharmacological effects. There are extensive application prospects in the fields of natural medicine, functional foods, daily chemical industry, functional beverage, etc. However, the contents of theaflavins in black tea is lower (0.2-2.0%). It is difficult to separate and purify theaflavins from black tea and the yield is so low, that seriously affects the industrialized production and wide application of TFs. Biosynthesis of TFs in vitro is an effective method to produce TFs in large quantities. But lack of polyphenol oxidase (PPO) restricts biosynthesis of TFs in vitro. In order to solve this key problem, this research based on the enzyme resources of microorganism, because microorganism can be cultured easily, and can easy separate PPO in high quantity from the culture medium. This study firstly was involved with selecting high quality stain, which can produce high quantity PPO. And further used PPO produced by selected stain to synthesize theaflavins in vitro.(1) This paper made investigation of Asepergillus cristatus, Aniger, Trametes trogii from the catechol enduring ability, characters of colonies growing on catechin medium and fermentation in liquid. The research showed Asepergillus cristatus had some enduring ability in catechol environment. It grew well on 0.2% catechin medium, but could not produce color circle on this medium. Also the quantity of PPO was low when it fermented in deep liquid. Aniger had a strong enduring ability in catechin environment, and it grew very well on 0.1-0.4% catechin medium and could make transparent hydrolysis circle on the medium, but it could not produce PPO in deep liquid fermentation medium. Trametes trogii grew well on 0.1-0.2% catechin medium and produced yellow-red oxidized circle, and it could produce PPO in liquid fermentation medium (enzyme quantity was 328 U/mL). So Trametes trogii was chosen as a good enzyme resource.(2) After deep liquid fermentation medium optimized, the composition of best culture medium for Trametes trogii included: 2.0% potato, 0.3% KH₂PO₄, 0.3% MgSO₄, 0.001% V_B, 0.005% CaCl₂, 1.0% wheat bran, 0.6% yeast-extract, 0.001mol/L L-Tyrosine. The best rotation speed of shaker was 110r/min, and the best quantity of inoculation was 4.215 ×10⁶/50mL. High quantity of PPO could be produced at this circumstance. Under this improved culture condition, the enzyme activity reached 711 U/mL, and it was much higher than 328 U/mL, which produced in unimproved...