【作者】 梁燕；

【导师】 王岳飞；

【作者基本信息】 浙江大学 ， 茶学， 2008， 硕士

【摘要】 茶黄素是茶叶中一种重要的功能成分,在红茶中的含量仅为0.2~-2.0%,因而从红茶中直接提取制备茶黄素分离纯化的难度大,且得率低;采用化学氧化法,需要投入更多的废水处理费用,并且由于化学法缺乏底物专一性,副产品复杂,产品纯度受到一定限制;体外酶促氧化法则是比较合适的方法,但茶鲜叶来源的酶受季节、茶树品种等因素限制,且提取方法较难。针对上述问题,本研究探索了以微生物、水果作为酶源,催化生产茶黄素的技术条件,并对得到的酶液进行固定化,以期为工业化生产高品质茶黄素提供前期工作基础。结果表明:1.研究了茶多酚形成茶黄素、茶红素的藕联反应,分析了多酚氧化酶、过氧化物酶催化生成茶黄素的能力。结果显示,多酚氧化酶、过氧化物酶与茶多酚,以及过氧化物酶与茶黄素反应的酶动力学米氏常数Km值分别为1.00×10~6,2.26×10~6和22.54×10~6,表明多酚氧化酶对底物茶多酚的亲和力要明显高于过氧化物酶,而过氧化物酶对茶多酚和茶黄素的亲和力差异较小,对茶黄素的生成起不到积极作用。所以在进一步生产茶黄素试验中选择多酚氧化酶作为酶促反应的主要酶。2.研究了微生物来源的多酚氧化酶催化茶多酚生产茶黄素的效果。选择产PPO的硬毛栓菌、彩绒革盖菌,恒温培养数日后提取并纯化酶液,并以此酶液催化茶多酚生产茶黄素。结果表明,硬毛栓菌的酶活性普遍高于彩绒革盖菌,且达到产酶高峰的时间要早2～4天;该菌产的酶主要是胞外酶,在提取酶时不需要进行细胞破碎;反应生成茶黄素的最适pH为4.5,但催化生成茶黄素的转化率仅为0.2%,与预期目标相差甚远。因而,采用微生物源的多酚氧化酶生产茶黄素的技术路线不能满足工业化生产的要求。3.研究了水果N来源的酶液催化生产茶黄素的效果。不同水果品种,匀浆方法,pH值及反应时间对茶黄素形成有显著影响,生产茶黄素的最佳条件为:将品种N1果实,去核不去皮后匀浆,在pH=4.6的反应体系中反应2小时。4.研究了酶固定化技术对酶活、茶黄素产量的影响。结果表明,与茶鲜叶相比,水果N1来源的多酚氧化酶活性是茶鲜叶来源的酶的2倍,固定化后两者的活性均降为固定化前的1/2。从茶黄素的生成量来看,茶鲜叶来源的酶液催化形成茶黄素的能力强于水果N1来源的酶。从固定化酶的稳定性来看,该固定化酶经过30次的重复使用,酶活依然保持原有水平的95.6%。在0.1M柠檬酸磷酸盐缓冲液(pH=5.6,4℃)中贮存30天后,酶活为原有水平的92.6%,可以认为该酶的重复利用性以及稳定性都较好。所以用水果N来源的多酚氧化酶在茶鲜叶缺乏的季节代替茶鲜叶来源的酶生产茶黄素是可行的。酶液经过固定化后,能稳定地重复使用,适于工业化生产茶黄素。更多还原

【Abstract】 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.更多还原