Research On The Biosynthesis Of Theaflavins By Immobilized Polyphenol Oxidase From Microorganism And Fruits

Theaflavins(TFs) are important tea functional components, however, the content in black tea is small(0.2～2.0%) and the yield is low. So, extraction of TFs from black tea is not a practical choice. Chemical oxidation method is a second choice, however, it would cost more on liquid waste treatment; for a lack of substrate specificity, chemical method brings more byproducts. Oxidation enzyme in vitro is an appropriate method for TFs production, however, enzyme application from fresh tea leaves is limited in some seasons like winter and affected greatly by tea cultivars. The technology of producing TFs from microorganism and fruits was studied in this paper, and the enzyme was immobilized in order to provide a basis for industral production.1. The coupled reaction in the production of TFs and TRs from tea polyphenol (TP) with polyphenol oxidase (PPO) and peroxidase (POD) catalysis was analyzed. It showed that Km value of PPO, POD with TP and POD with TFs were 1.00×10⁶, 2.26×10⁶, 22.54×10⁶. It indicated that affinity of PPO with substrates TP was higher than that of POD, and affinity of PPO with TP and TFs had little differences. It was concluded that POD only played a minor role in producing TFs and PPO was a primary enzyme in producing TFs.2. The capacity of TFs catalysed by PPO from microorganism Trametes trogii and Trametes versicolor was studied. After constant temperature culture for several days, enzyme solution was extracted and purified. The analysis indicated that enzyme from Trametes trogii had a higher enzyme activity than that from Trametes versicolor, and reached the peak of capacity 2～4 days earlier than Trametes versicolor. As most of the enzyme from microorganism were extracellular enzyme, it was not necessary to disrupt cell when extracted. Optimum pH value of solution was 4.5. However, the percentage of the TFs transfomation was only 0.2%, far away from prediction. Therefore, the technical route for TFs production from microorganism could not meet the industrial needs.3. A series of experiments for identifying the effect of TFs production catalysed by enzyme solution from various fruits were carried out. It was concluded that the effect of TFs production was determined by fruit varietis, homogenate method, pH as well as reaction time. The best conditon was as followings: variety N1, stone removed, pH4.6 and 2 hours. 4. The relationship of enzyme immobilization to enzyme activity and TFs production was studied. It showed that the activity of PPO from fruit N1 was twice as much as that from fresh tea leaves, however, both of them decreased by 50% after immobolized. Amount of TFs produced in fruit N₁ was lower than that in fresh tea leaves. Stability of immobilized PPO (IPPO) was also discussed. Enzyme activity stayed in 95.6% and 92.6% after using 30 times and kept in 0.1M citric acid buffer solution(pH4.6,4℃) for 30 days respectively. It is confirmed that the IPPO could be used repeatedly and stably.So, it is feasible to use PPO from fruits to produce TFs instead of that from tea leaves in off-seasons. After immobilized, IPPO was more suitable for TFs industrialized production.