Research On Polyphenol Oxidase In Liupao Tea And The Application Of PPO Producing Microorganism To Tea Quality

Used the large unfermented tea leaves from Wuzhou,Liupao tea is a kind of dark tea which processed according to specific initial and refined processes.It has the fragrance named Liupao and its aqueous solution tastes smooth.Fermentation is the key to the quality formation of Liupao tea.Under high temperature and humidity conditions,microorganisms grow and propagate.A series of reactions,such as catalytic oxidation of tea polyphenols,take place with tea as substrate.At present,there are few studies on the polyphenol oxidase and microorganisms in Liupao tea.This paper links the changes of polyphenol oxidase,physical and chemical components of tea and the number of microorganisms in order to understand the formation process of Liupao tea quality.Polyphenol oxidase isozymes were isolated and purified from Liupao tea fermentation samples.The enzymatic properties of polyphenol oxidase isozymes were studied,which provided theoretical guidance for the establishment of optimal reaction system in the enzymatic synthesis of tea pigment and enzyme immobilization.In addition,polyphenol oxidase-producing thermophilic bacteria isolated from Liupao tea were applied to simulated solid-state fermentation of tea.The effects of sterility,traditional fermentation and single strain fermentation on the transformation of tea components were compared,and the formation mechanism of color,aroma and taste of Liupao tea was further explained.The experimental results obtained in this study are as follows:1.Water extract is the main flavoring and coloring substance of tea soup,and it has a high correlation with polyphenol oxidase(r=0.8776).The content of tea polyphenols decreased from 16.6%to 8.2%in the fermentation period.With the increase of polyphenol oxidase activity in the later period,tea polyphenols decreased faster.At the same time,free amino acids decreased from 40.1mg/g to 9.9mg/g.During the pile up of Liupao tea,theaflavin and thearubin decreased from 0.12%to 0.05%and from 5.24%to 0.74%,respectively.Thearubigins increased from 2.53%to 5.77%,which was due to the increased activity of polyphenol oxidase,catalyzing the further oxidation of theaflavins and thearubin.In addition,the activity of polyphenol oxidase was positively correlated with the quantity of molds(r = 0.9192).The activity of polyphenol oxidase showed an upward trend in general,but the polyphenol oxidase before fermentaion had been inactivated by the process of hgh temperature.It is speculated that the new polyphenol oxidase may be produced by microorganism.In summary,polyphenol oxidase plays an important role in the formation of the quality of Liupao tea.2.The extraction of polyphenol oxidase from Liu Bao tea was optimized,and then the crude enzyme solution of two kinds of tea samples before and after pile fermentation was fractionated by ammonium sulfate precipitation.After Salting out,the enzyme were concentrated.Then it were purified by gel filtration chromatography.Finally,the samples were analyzed by SDS-PAGE.The molecular weight of PPO was about 59 kDa in front of the fermentation,while the bands of 59 kDa and 70 kDa in the end of the fermentation appeared respectively.The reason for this was that microbial action produced new PPO during the piling up process,and this PPO may be laccase produced by fungi.The optimum pH value of PPO 1 with molecular weight of 70 kDa is 6.5,the optimum temperature is 40 ?,and the optimum substrate(catechol)concentration is 0.5 mol/L.The optimum pH,temperature and substrate(catechol)concentration of PP02 with molecular weight of 59 kDa were 6.0,35 ? and 0.5 mol/L respectively.3.Thermophilic bacteria were isolated and identified for the first time from the Liupao tea fermentation samples by traditional culture method,including 5 genera and 14 species of bacteria,5 genera and 9 species of mold.Three Polyphenol oxidase producing thermophilic were screened by selective medium color change and liquid culture enzyme activity test.They are Lichtheimia corymbifera,Aspergillus tubingensis and Aspergillus fumigatus.In addition,the enzyme production curves of the five most active Polyphenol Oxidase Producing Strains Isolated from Liupao tea by our research group were plotted by liquid fermentation and tea juice culture.4.Penicillium griseofulvum,Aspergillus Niger and Aspergillus fumigatus were selected for solid-state fermentation of tea.The application of polyphenol oxidase producing bacteria in bioconversion of tea was studied.The experimental samples were divided into sterile,traditional and three single-strain fermentation groups.The changes of tea pigment,tea polyphenols and polyphenol oxidase activities were determined within 40 days.In terms of growth,Aspergillus Niger and Aspergillus fiumigatus can grow well in high temperature and humidity solid tea environment than Penicillium gray and non-sterilized Liupao tea microorganisms.The enzyme activities of the three strains fermented group were higher than those of the traditional fermentation and aseptic fermentation groups.With the increase of simulated fermentation time,the content of tea polyphenols in each group decreased in varying degrees,especially in Aspergillus fumigatus fermentation group,followed by the traditional fermentation group.Theaflavins content of each fermentation group decreased with time.The content of thearubin and theafuscin in traditional fermentation group,aseptic group and Penicillium gray fermentation group showed an obvious growth trend.However,Aspergillus fumigatus and Aspergillus niger decreased the content of theaflavins and increased the content of tea brown.