Extraction And Characterization Of Polyphenol Oxidase In Tea Leaves

Tea is the main production and sales economic crops in China.A large difference between the quality of tea produced by the traditional process and less high-quality tea is produced.The quality of tea comes from the various chemical changes that occur during the processing of tea.The reaction of tea polyphenol oxidase catalyzing oxidation tea polyphenols into tea pigment forms the basis quality of tea.During the tea processing,activate of enzyme activity in order to improve the quality of ferment tea,such as black tea.Inhibition of enzyme activity to ensure the green tea does not become red.Control enzyme activity within a certain range in order to reach the production requirements of semi-fermented tea,such as oolong.Regulating the biochemical reactions of polyphenol oxidase catalyzing tea polyphenols is the key to the formation of a good quality of tea.In order to know better about the enzyme changing trends in different conditions(especially temperature),to improve the quality of the flavor,aroma,appearance and other quality requirements in tea,to meet the requirement of standardized and large-scaled tea production,we choose new hybrid varieties of oolong tea(Camellia sinensis cv.Jinguanyin)as raw materials,the main results are as follows:The optimize process of extraction and purification of polyphenol oxidase from tea:compare the enzyme activity by different extract methods of tea polyphenol oxidase to determine the best extraction method.It turns out to be the combination method of acetone powder extraction and homogenized extraction is the best extraction method: the fresh tea is freeze dried,mashed in a chilled acetone and washed until colorless by a certain concentration of cold acetone.The acetone powder is volatilized of acetone then preparing to be used.Add buffer,polyvinylpyrrolidone(PVP),quartz sand to the acetone powder in a mortar and ground under the ice bath,filtered after centrifugation to obtain a crude enzyme solution.The degree of saturation range of ammonium sulfate precipitation is 50~85%.Ammonium sulfate powder was added slowly to the crude enzyme solution to a saturation of 50%,standing for 8 h under 4 °C.It is centrifuged for at 9000×g 25 min at 4 °C,discard the pellet and the supernatant was collected and set the volume.The ammonium sulfate is added to the supernatant to 85% saturation in the same way.The fraction precipitated at 85% saturation was separated by centrifugation at12000×g for 25 min at 4 °C.The precipitate was dissolved in a small amount of phosphate buffer,pH 7.2,and dialyzed overnight at 4 °C in the water.Study on enzymatic characteristics of polyphenol oxidase of tea(Camellia sinensis cv.Mingke1).The enzyme reaction rate increases with the increasing of substrate concentration when the substrate concentration under 0.03 mol/L.The enzymatic reaction rate reaches a maximum when the substrate concentration is 0.03 mol/L.Increase the substrate concentration and enzyme reaction rates are no longer increased.The optimum temperature of polyphenol oxidase is 40 °C.Its thermal stability is poor.The enzyme activity declines for 20% after 50 °C thermal treatment for 1 h.The enzyme activity is less than 20% after 70 °C thermal treatment for1 h.High temperature heat treatment can effectively inhibit polyphenol oxidase activity.The optimum pH is 6.9,was weakly acidic,too acidic or alkaline will inhibit the enzyme activity.Effect of heat treatment on polyphenol oxidase in fresh tea leaves: After 1 h heat treatment at 25 °C to 50 °C,polyphenol oxidase activity is higher than 0.5 h.After heat treatment between25~50 °C for 1 h,the residual activity of polyphenol oxidase is the highest,followed by 0.5 h,the residual activity of polyphenol oxidase after 2 h heat treatment is the minimum.When the time of heat treatment is 0.5 h and 2 h,enzyme activities increased rapidly in the range of25~30 °C,gently in 30~40 °C.T On the contrary,when the time of heat treatment is 1 h,the activity of polyphenol oxidase rises more slowly under in the range of 25~30 °C,more quickly in the range of 30~40 °C.Higher than 40 °C heat treatment,enzyme activity is significantly decreased.