【作者】 王丽璞；

【导师】 成浩； 黎星辉；

【作者基本信息】 南京农业大学 ， 茶学， 2011， 硕士

【摘要】 本文研究一条以成本较低且多酚含量较高的绿茶粉为原料,利用生物酶法制备TFs并采用大孔吸附脂树脂法纯化TFs的工艺。工艺流程主要分为多酚氧化酶(PPO)酶源物质的筛选：提取梨、苹果等七种果蔬中PPO并测定其活性,选择活性高的果蔬作为酶源物质；不同金属离子对PPO活性的影响：测定不同浓度的金属离子对PPO活性的影响,选择PPO的激活剂；制备TFs的反应条件的优化选择：对TFs的合成条件做单因素试验和正交试验,选择最佳条件；纯化TFs的条件的优化选择。(1)选择梨、苹果、马铃薯、茶鲜叶、茄子、甘薯和藕等七种不同的果蔬为酶源物质,测定其PPO活性。通过对其活性的比较,筛选活性最高者作为制备茶黄素的最佳酶源。各种酶源物质活性依次为：茄子中PPO的酶活为174U；马铃薯中PPO的酶活为101.43U；甘薯中PPO的酶活为58U；梨中PPO的酶活为37U；藕中PPO的酶活为32U；苹果中PPO的酶活为24.67U；茶鲜叶中PPO的酶活为10U。选择茄子中PPO作为最佳酶源。(2)选择铜离子、锰离子、镁离子、钙离子、钠离子、铝离子和铁离子七种离子作为研究对象,通过在粗酶液中加入不同浓度的七种金属离子,研究七种金属离子对茄子PPO活性的影响。研究表明,浓度为3mmol/L的铜离子对PPO有显著激活作用,而钙离子、钠离子、铝离子和铁离子对PPO则有抑制作用。其中,浓度为5mmol/L的铁离子是PPO的最佳抑制剂。在此条件下PPO的活性可被抑制96.6%。(3)通过对合成TFs的不同条件(pH值、温度、底物浓度、酶添加量和反应时间)的研究,在单因素试验和正交试验的基础上,确定合成TFs的最佳条件。结果表明,反应体系的最佳条件为：pH值为4.5,温度为25℃,底物浓度为2.5g/L,酶添加量为35m1,反应时间为40分钟。其中,pH值和底物浓度是反应体系中重要的两个因素。(4)采用大孔吸附树脂法对在最佳条件下制备到得的TFs进行纯化,通过对不同吸附条件(树脂型号、pH值、上样速度、除杂用洗脱液浓度、洗脱液体积和洗脱液浓度)的选择,确定纯化TFs的最佳工艺条件。结果表明,纯化的最佳条件为：pH值为3,上样速度为2BV/h,除杂用洗脱液浓度为15%洗脱液体积为3BV,洗脱液浓度为90%。pH值和上样速度是影响纯化效果的重要因素。更多还原

【Abstract】 This article reported a new process applied bio-enzyme method that green tea p owder with low cost and high polyphenol as raw material to synthesize theaflavins, then macroporous adsorptive resin was done to purify theaflavins in earlier studies. The technological process was divided into polyphenol oxidase(PPO) selection, extra cted PPO from seven plants such as pear、apple and so on and measured their acti vity, choose the material with highest activity as the best enzyme source; the effects of different metal ions on PPO, different metal ion with different concentration wer e measured to choose activator of PPO; the selection of reaction conditions of synth esizing theaflavins, optimum synthesis condition were determined on the basis of sin gle factor and orthogonal experiments; the selection of optimum condition to purify theaflavins.(1) Pear, apple, potatoes, tea leaves, eggplant, sweet potato and lotus root seve n plants were chosen as enzyme source materials, the buffer solution method were used to extract and measure the activity of PPO. By comparing the activity of diffe rent enzyme source materials, the material with the highest activity was chosen as t he best enzyme source material for theaflavin synthesis. The PPO activity in differe nt source material was:174U in eggplant,101.43U in potato,58U in sweet potatoes, 37U in pear,32U in lotus roots,24.67U in apple,10U in tea leaves. Eggplant wa s choosen as the best enzyme source material.(2) Copper ion, manganese ion, magnesium ion, calcium ion, sodium ion,alumi num ion and ferric ion at different concentrations were added into crude eggplant P PO enzyme.The results showed that copper ion at a concentration of 3mmol/L can s ignificantly activate PPO, while calcium ion, sodium ion, aluminum ion and ferric i on inhibited the activity of PPO. Among them, ferric ion at concentration of 5mmol /L can significantly inhibit the activity of PPO, which can be inhibited 96.6% on th is condition.(3) Different synthesis conditions (pH value, temperature, concentration of subs trate, enzyme concentration and reaction time) were determined on the basis of singl e factor and orthogonal experiments. The results showed that the best conditions of reaction System were a:pH value of 4.5, temperature of 25℃, substrate concentrati on of 2.5g/L, enzyme additive amount of 35ml and a reaction time of 40min. Am ong these conditions, pH value and concentration of substrate were two the most i mportant factors in this reaction system.(4) Macroporous adsorptive resin was selected to putify theaflavins that synthesi zed on the best conditions included the type of resin, pH value, concentration of sa mple, the flow rate, the effluent volume and the concentration of effluent.The results showed that the best condition to purify the theaflavins were:pH valve of 3, samp le filling rate of 2BV/h,15% effluent of eliminating impurity volume, effluent volu me of 3 BV and an effluent concentration of 90%. pH value and sample filling rat e were two important factors in the purification system.更多还原