【作者】 汪小钢；

【导师】 潘才元；

【作者基本信息】 中国科学技术大学 ， 高分子化学与物理， 2007， 博士

【摘要】 全球咖啡碱年销量约为12万吨，20％～25％用于工业，其余大部分用于医药和食品。近些年来，人工合成咖啡碱对人体和环境的毒副作用使得大部分发达国家明文禁止“合成咖啡碱不得加入食品饮料中”，从而为天然咖啡碱的开发带来无限商机。在国外，天然咖啡碱主要是来源于生产咖啡过程中的一种副产品。我国是产茶大国，各种制茶废弃物非常丰富，因而充分利用制茶废料提取咖啡碱，具有原料易得，工艺简便等特点。但由于天然咖啡碱的生产成本比人工合成咖啡碱要高很多，从而限制了天然咖啡碱的大规模生产和应用。因此，如何降天然咖啡碱的生产成本就显得十分重要。本论文首次采用微生物发酵技术来提高茶叶中咖啡碱含量，从而降低其生产成本。试验结果表明，微生物发酵确实能够较大幅度地增加茶叶中咖啡碱的含量，对发酵体系中的微生物进行跟踪检测，并与咖啡碱的含量变化进行综合分析，发现与咖啡碱含量变化相关的微生物主要是真菌和酵母，细菌几乎不起作用；进一步的单一微生物发酵试验表明，选用的霉菌能够提高茶叶中咖啡碱含量，而所选用的两种假丝酵母却使茶叶中咖啡碱含量降低。试验结果表明，黑曲霉发酵使茶叶中咖啡碱含量增加的幅度最大，在第16天，绿茶咖啡碱含量增加到9.63％，与最初的3.47％相比，增幅为177.5％；对试验结果进行综合分析，可以推断：微生物受茶叶中一些物质的诱导，产生了新的代谢途径，利用茶叶中的一些化合物合成咖啡碱，这些化合物大部分不溶于水，它们在绿茶中的含量远高于红茶；而且微生物代谢合成咖啡碱的最直接前提物可能是茶叶碱，而在茶树体中合成咖啡碱的最直接前提物是可可碱。然而，对于茶多酚制品而言，过多的咖啡碱残留会影响它在一些领域的应用，因此有必要降低茶多酚中的咖啡碱含量。现在常用的方法是用氯仿等有机溶剂脱除咖啡碱，也有用超临界CO₂来分离的，尽管很有成效，但都存在很多问题，比如有机溶剂的毒害与残留，超临界CO₂的一次性投资很大等。因此，有人用柱层析等色谱的方法来分离茶多酚和咖啡碱，取得了一些成果，比如用C₁₈色谱柱、凝胶色谱柱、高速逆流色谱等分离茶多酚和咖啡碱，但C₁₈色谱柱和凝胶色谱柱成本很高，高速逆流色谱制备规模太小，因此寻找合适的价格低廉的固定相十分重要。本论文根据已有文献的报道，采用分子印迹技术合成以咖啡碱为模板的分子印迹聚合物，通过它对分子大小和结构的特殊选择性来分离咖啡碱。结果表明，以咖啡碱为模板，甲基丙烯酸为功能单体，采用水溶液微悬浮聚合合成分子印迹聚合物是可行的，模板分子和功能单体的最佳摩尔比为1∶12，流动相宜采用甲醇的醋酸水溶液；咖啡碱分子印迹色谱柱分离没食子儿茶素没食子酸酯（EGCG）和咖啡碱以及分离茶多酚各组分的实验结果证明，用分子印迹聚合物脱除茶多酚中的咖啡碱是可以实现的，EGCG和咖啡碱之间的分离度高达2.39，但分离过程会造成一些组分的损失。表没食子儿茶素没食子酸酯（EGCG）是儿茶素的主体成分，具有很强的药理功能，因此它的分离提取一直受到各界的重视。目前常用的方法是用葡聚糖凝胶的方法分离制备EGCG，也有用高速逆流色谱来分离制备的，还有将两种方法结合起来应用，都取得了很好的效果。但制备的规模很小，且成本很高。在本论文中，分子印迹技术被应用于分离EGCG。试验结果表明，以EGCG为模板，4-乙烯基吡啶为功能单体，采用水溶液微悬浮聚合制备分子印迹聚合物是可行的，模板分子和功能单体的最佳摩尔比为1∶16：EGCG分子印迹色谱柱分离EC、ECG和GCG实验结果证明，用分子印迹聚合物分离旋光异构体具有较好的效果，EGCG和GCG的分离度为1.52，但对于空间结构相同、分子上官能团差异较小如EGCG和ECG时，分离度较小，仅为0.60；应用儿茶素分子印迹柱分离咖啡碱和EGCG具有很好的效果，它们之间的分离度高达2.30；对茶多酚的分离试验表明，可以应用EGCG分子印迹色谱柱从茶多酚中分离制备高纯度EGCG。茶色素是儿茶素的氧化产物，它对心血管和肿瘤等具有很好的防治效果，其主体成分是茶黄素和茶红素以及茶褐素。茶色素的制备方法有两种，一种是通过多酚氧化酶或过氧化物酶来氧化获得，另一种是通过铁氰化钾等化学氧化剂的氧化获得。多酚氧化酶和过氧化物酶有很好的选择性、专一性和较强的催化活性，但是很难获得，也不易回收再利用，因此成本很高。而人工模拟酶可以批量生产，既具有酶的催化能力，同时它的稳定性又大大优于天然酶蛋白，在有机相和水相中都不溶解，这样便于分离回收再利用。本论文以低分子量的壳聚糖为载体，通过与水杨醛反应合成壳聚糖希夫碱，然后与铜离子进行配位络合，最后用环氧氯丙烷进行交联，得到壳聚糖铜络合物。试验结果表明，形成的铜络合物具有与辣根过氧化物酶类似的催化能力，其活性较天然的过氧化物酶弱，如以焦性没食子酸为底物，壳聚糖希夫碱铜络合物的催化能力约为辣根过氧化物酶的28.28％。但它能够回收再利用，试验数据表明，重复使用4次后，模拟酶的活性几乎未发生变化，第四次催化形成的产物是第一次的86.18％，其损失可能是因为有一部分模拟酶在搅拌过程中发生粉碎，同时模拟酶对儿茶素还是有少量的吸附，这都会影响到模拟酶的活性。试验结果表明，用壳聚糖希夫碱铜络合物作为辣根过氧化物酶的模拟酶催化儿茶素的氧化制备茶黄素和茶红素是可行的。茶色素的另一种制备方法是用化学氧化剂氧化儿茶素，最常用的氧化剂是铁氰化钾，它的氧化产率较高，但氧化剂的分离回收困难，而且氧化反应后需通过酸化来终止反应，这一过程中易产生氢氰酸，对人体和环境具有潜在的危险；而且，铁氰化钾氧化还原后形成的亚铁氰化钾在有酸或酸性盐存在时，特别是加热时，也会分解而放出剧毒的氢氰酸。此外，铁氰化钾氧化后从体系中分离困难，既不利于反应的终止，也不利于产品安全。为了提高铁氰化钾的稳定性，减轻它对人体和环境的潜在危害，实现与产品的完全分离，我们尝试以离子交换树脂为载体，将铁氰根离子吸附固定在树脂上，用它作为高分子氧化剂，去氧化儿茶素制备茶色素。试验结果表明，强碱性阴离子交换树脂717固载的铁氰化钾能氧化儿茶素形成茶黄素，但它的氧化活性较游离的铁氰化钾弱很多，若以D，L-C和EGCG为反应底物，固载氧化剂主产物茶黄素单没食子酸酯的峰面积（510351.9）是游离铁氰化钾氧化的主产物峰面积（1026658.0）的49.71％。反应后的树脂固载氧化剂可以通过过氧化氢的氧化有效再生，这对于降低生产成本，解决环境污染等是十分有利的。更多还原

【Abstract】 Global consumption of caffeine has been estimated at 120000 tons per annum, 20%～25% of it is used in industry and the rest of it is used in food and medicine. In recent yeas, the synthetic caffeine is prohibited in the fields of food and drink in most of developed countries due to its harm on human health and environment. It brings limitless business opportunity to natural caffeine. In foreign countries, natural caffeine is mainly from the by-product which is produced during the processing of coffee. Our country is the biggest tea manufacturing country in the world, and is rich in all kinds of tea wastes which are produced during the manufacturing of tea. Therefore, extra-cting caffeine from the tea wastes features the advantages of ready availability of raw material and simplicity of procedure. But the cost of natural caffeine extracted from tea or coffee is much higher than that of the synthetic caffeine, which limits the pro-duction and application of natural caffeine in large scope. So it is necessary to lower the cost of natural caffeine. Microbial fermentation was applied in the paper to increase the caffeine content in tea, which would decrease the cost of natural caffeine. The results demonstrated that the microbial fermentation did increase the caffeine content of tea. The analysis of microorganisms and caffeine showed that the fungi and yeast had effects on the change of caffeine content, while the bacteria had no effect on the change of caffeine. The single microorganism was further used in the fermentation of black and green teas in order to find which microorganism has the biggest effect on the caffeine content increase. The results illustrated that the three molds applied in this study could enhance the caffeine content in green tea through fermentation, whereas the two yeasts had no this effect. Among the three moulds, Aspergillus niger van Tiegh em had the most positive effect on the increase of caffeine content. On the 16th day of fermentation with Aspergillus niger van Tieghem, the caffeine content reachs a maximum value of 9.63%. In comparison with an initial 3.47% in starting tea, the caffeine content increased by 177.5%. A conclusion can be drawn from the compre hensive analysis of the results: The microorganisms are stimulated to establish a new secondary metabolism route to biosynthesize caffeine in vivo after taking up the essential and especial components such as xanthosine from teas. These components are poorly soluble in water and the contents of them in black tea are much lower than that in green tea. The biosynthesis of caffeine in the microbial metabolism may be via the methylation of the theophylline, while the biosynthetic route of caffeine in the tea plant is via methylation of theobromine.However, more residue of caffeine in tea polyphenols has a negative influence on the application of tea polyphenols in some fields. So it is necessary to reduce the caffeine content in tea polyphenols. Now, the common method for the removal of caffeine from tea polphynols is the extraction by chloroform. The Supercritical carbon dioxide is also used for extraction of caffeine. Although they are effective in the removal of caffeine, there are many problems in these two methods such as the residue and harm of organic solvent and the big one-off investment of the equipments for supercritical carbon dioxide extraction of caffeine. Therefore, somebody separates caffeine from tea polyphenols with column chromatography and makes achievements. The C₁₈ column and Gel column and High-speed countercurrent chromatography are used for the separation of tea polyphenols and caffeine. But the costs of C₁₈ and gel column are high, while the High-speed countercurrent chromatography cannot reach a preparation scale. So it is important for us to find a kind of suitable and cheap solid phase for the separation. According to the relevant research, a molecularly imprinted polymer（MIP） is prepared with caffeine as the template molecule by the molecularly imprinting technique in the paper to separate caffeine from tea polyphenols with the selectivity of molecular weight and structure. The results indicate that the preparation of MIP using methacrylic acid（MAA） as the functional monomer, caffeine as the template is perfect by aqueous micro-suspension polymerization. The best ratio of template to functional monomer is 1:12, methanol to acetic acid to water as mobile phase. The results demonstrate that the MIP with caffeine as the template can separate caffeine from tea polphenols, the separation degree of caffeine and epigallocatechin gallate（EGCG） is 2.39. Of course the separation by MIP leads to the loss of some components of tea polyphenols.Epigallocatechin gallate（EGCG） which is the dominant component of catechins is gotten attention from all circles because of its stronger pharmacological function. The sephadex gels are used in the common method for preparation of EGCG. The high-speed countercurrent chromatography is also adopted to prepare EGCG. In addition, these two methods are combined into the procedure of preparing EGCG. All of them make good results. But the preparation scale is small and the cost is very high. In this paper, a molecularly imprinted polymer（MIP） was prepared with EGCG as the template molecule by the molecularly imprinting technique to separate EGCG from tea polyphenols. The results indicate that the preparation of MIP using 4-vinylpyridine as the functional monomer, EGCG as the template is perfect by aqueous microsuspension polymerization. The best ratio of template to functional monomer is 1:16. The results demonstrate that the MIP of EGCG can realize the chiral separation of enantiomers, the separation degree of EGCG and GCG is 1.52. The separation degree of EGCG and epicatechin gallate（ECG） on the MIP of EGCG is only 0.60 because they have more similar structure. The separation degree of caffeine and EGCG is 2.30. The result of separation of tea polyphenols with MIP of EGCG indicates that the preparation of EGCG through the MIP column is reliable.Tea pigments which include theaflavins, thearubigins and theabrownin are the oxidation products of catechins. They have good effects on cardiovascular diseases and tumor. The pigments could be obtained by two ways: oxidation of catechins by the catalysis of polyphenol oxidase and peroxidase or by the chemical oxidant such as potassium hexacyanoferrate（Ⅲ）. Although polyphenol oxidase and peroxidase have the specific oxidative ability for catechins, the cost of enzymatic reaction is very high because it is difficult to get and reuse the enzymes. Artificial enzyme mimics which not only have the ability of enzymatic catalysis but also have more stability than natureal enzymes could be prepared in batch. The enzyme mimics don’t dissolve in water and organic solvent, which is convenient for its recovery. In this paper, low-molecular weight chitosan was used to synthesize the chitosan schiff base via the reaction of chitosan with salicylaldehyde. And then chitosan schiff base copper complex was obtained by reaction of chitosan schiff base with copper（Ⅱ） salt. Crosslinked chitosan schiff base copper complex was prepared using epichlorohydrin（ECH） as cross linker. The results indicates that the copper complex has the same but low catalytic property as horseradish peroxidase, only 28.28% of the activity of horseradish peroxidase using pyrogallic acid as substrate. But the copper complex can be used again after filtration from the reaction system. The experimental data show that the activity of enzyme mimic（chitosan schiff base copper complex） decrease little and is 86.18% of the activity of the first time after four repeat. The loss of activity may be caused by the crush of enzyme mimic during the stirring and the adsorption of catechins by enzyme mimic. However, it is no problem for chitosan schiff base copper complex using as the mimetic enzyme of horseradish peroxidase to catalyze the oxidation of catechins to form theaflavins and thearubigins.The other method for preparation of tea pigments is through the oxidation of catechins by chemical oxidants. The common oxidant is potassium hexacyanoferrate（Ⅲ） which can yield more products. But it is difficult to recover it from the reaction system, which is not good for the timely stop of oxidation reaction and the safety of product. Meanwhile, the using of potassium hexacyanoferrate（Ⅲ） which can produce hydrocyanic acid after being treated with acids may harm the environment and the body. And then the potassium ferrocyanide which is the reduction product of potassium hexacyanoferrate（Ⅲ） also produce virulent hydrocyanic acid when meeting the acids or acidic salts especially at high temperature. In order to increase the stability of potassium hexacyanoferrate（Ⅲ） and reduce the harm of it and realize the complete separation from the product, the ion-exchange resin is used to adsorb ferricyanide by exchange and then to oxidize the catechins to form tea pigments. The results demonstrated that the theaflavins could be prepared by the oxidation of catechins using the ferricyanide supported by 717 strong base anion exchange resin as oxidant. The activity of polymer-supported oxidizer is much lower than the free potassium hexacyanoferrate（Ⅲ）. The peak area of main product by oxidation of DL-C and EGCG using polymer-supported oxidizer is 510351.9,49.71% relative to the peak area of the same product using free potassium hexacyanoferrate（Ⅲ） as oxidizer. The resin-supported oxidizer will be regenerated by oxidation of it with H₂O₂, which must be helpful for us to reduce the production cost and reduce the environmental pollution.更多还原