Study On Coupled Technology Of Biosynthesis Of Theaflavins By Resin Immobilized Polyphenol Oxidase And Separation

Enzyme engineering technology is one of the modern five biological engineering technology and its applications range widely focusing on separation and extraction of enzymes, exploitation production of various enzymes, immobilization technology research and development,preparation of enzyme reactor and so on. Theaflavins are of a class of pigment material that polymerization of catechis during the processig of tea by polyphenol oxidase and peroxidase as catalyst. Recently years, research found that the antioxidant activity better than polyphenols and other medical health care value. Currently, available Theaflavins material are obtained from extraction of black tea and its low content and yield that restricted its apllications and exploitation in medical care.With the development of Immobilized enzyme technology rapidly in recent years,Immobilization of polyphenol enzymes and Biosynthesis of TFs with IPPO become a feasible and effective option.In this paper,we obtained immobilized laccase with method of pre-researcher.According to characteristics of IPPO and conditions of enzymatic biosynthesis of theaflavins, designed column packed bed reactor to achieve continuous dynamic preparation theaflavins by IPPO.Combining with column chromatography technology,we gained the coupling of dynamic preparation and separation of theaflavins and obtained a large-scale theaflavins preparation craftwork parameters.The apparatus of continuous preparation system was designed and maded by ourself. Continuous preparation of theaflavins by immobilized polyphenol enzyme in packed bed bioreactor was performed.Four mainly facts were considerated in this experiment as following: temperature, substrate flow ratio,oxygen flow rate,pH and subsrate concentration.As a results,we selected the temperature at 28℃,substrate flow ratio 2.0mL/min, oxygen flow rate 50mL/min,pH 5.7 and substrate concentration 10mg/mL as a optimized condition to biosynthesize theaflavins at a highly efficient.Combining with column chromatography technology,We achieved the coupling of dynamic preparation and separation of theaflavins.We gained a certain percentage of content and ester-type theaflavins products. We discussed how different materials impact the preparation of TFs and varied feeding cycles ways impact on the yield of TFs.Under the conditions of substrate flow ratio 11.0mL/min, temperature 28℃, oxygen flow rate 50mL/min, reaction system pH 5.0, 1L substrate concentration 10mg/mL of tea polyphenols continuous injectioned packed reactor and 30% ethanol eluent circulation process was carried out,The ratio of content and ester-type TFs up to 80% and its yield was 11.64%. The overall yield of theaflavins up to 20.10% and The yields of TFs increased 57%.On the base of these experiments above.The coupling of continuous preparation and separation of TFs experiment was magnified 4 fold shown that the proper magnified scale of the reaction did better to prepare TFs and The yields of TFs increased 87%. The dryness of different collections of fraction of separation,we gained different percentage of content and ester-type theaflavins products.All in all,The coupled of preparation and separation of TFs by IPPO was achieved via experiments from small scale to large scale and the utilizing efficient of catechins was increasing.