茶氨酸提取纯化工艺研究
|
摘要
系统研究了从茶多酚工业废液(以下简称废液)中提取纯化茶氨酸的方法。茶多酚工业废液中含有大量的茶多酚、糖类及蛋白质等杂质，研究采用各种分离的手段，如絮凝、超滤、吸附、阳离子交换及重结晶的工艺来除去杂质，分离纯化茶氨酸。并对各分离工艺进行优化，得到最佳工艺参数。结果表明：
絮凝能有效去除工业废液中的多糖类与蛋白质等杂质。经絮凝后，茶多酚废液溶液由混浊变为澄清，O.D.650值由1.5降至0.2左右，蛋白质杂质减少了50％，可溶性糖的含量也降低20％，茶多酚含量变化不大，茶氨酸的回收率超过95％。最佳絮凝工艺条件为壳聚糖添加至浓度4mg／mL，溶液pH为3.0～4.0，温度为20-30℃。
超滤能进一步澄清溶液，降低溶液中蛋白质等大分子物质的含量。研究结果表明，超滤的最佳工艺为选择十万分子量的醋酸纤维素膜，处理液温度为35-45℃时，以0.1-0.15MPa的工作压力进行操作，可得到较好的处理效果。通过超滤，蛋白质含量减少48％，可溶性糖含量下降25％，茶氨酸有一定程度的被截留，茶氨酸回收率约为90％。
吸附能进一步去除色素、多酚类物质及大分子有机物。筛选出一种对杂质吸附较多，同时茶氨酸的回收率较高的树脂—AB-8。静态吸附实验表明在35℃、溶液pH在4.0左右时，AB-8树脂对多酚类等有机质的吸附量最大。通过动态吸附法考察了树脂在不同流速、不同上样浓度及不同树脂层高径比条件下茶多酚的穿透曲线。结果表明，采用1.0BV／h的流速，树脂层高径比在10：1左右时进行动态吸附，单位树脂的吸附量最大。70％的乙醇的解吸性能较好。
阳离子交换树脂能较专一吸附氨基酸，不同阳离子交换树脂在吸附氨基酸的选择性上相差不大。离子交换树脂以732树脂为宜。在上样液茶氨酸浓度为1.5mg／mL时，溶液pH在3.0左右时，控制流速1.0BV／h，单位体积树脂吸附容量较大。洗脱液选择1.5mol／L的氨水，控制流速为1.0 BV／h，收集1.5倍柱体积沈脱液。洗脱液60℃减压浓缩挥去氨后，溶液冷冻干燥得粗茶氨酸。
乙醇重结晶法可得到较高纯度的茶氨酸。10％(W／V)粗茶氨酸液加入4倍体积乙醇，在常温下振荡混匀静置1h后，离心分离去除产生的沉淀。离心清液60℃减压浓缩为80％(W／V)的溶液，冷却后加4倍体积冷乙醇，0℃下冷藏24h，可产生结晶状物质，4℃、8000rpm／min离心得类白色固体，经检测此固体物茶氨酸纯度为65％。将所得固体用少量热水溶解，同上述方法重结晶3次。所得晶体60℃下真空干燥，可得纯度＞90％的茶氨酸产品。
不吹士学位论文
通过以上絮凝*超滤、吸附*阳离子交换*重结晶的工艺，可得到纯度超过
%%的茶氨酸产品，茶氨酸回收率为35%，产品得率为0.8%。
Techniques of isolation and purification of theanine from tea polyphenol waste industrial liquid were studied in this paper. To get rid of tea polyphenol, protein, soluble sugar in waste industrial liquid, some of chemical technique for separation, such as flocculation, membrane ultra filtration, absorption, ion-exchange absorption and re-crystallization were employed. To optimize the techniques,conditions of techniques were studied . The result suggested that:
Focculation can efficiently remove protein. Effects of concentration of chitson, pH and temperature of solution were investigated. After flocculation, the solution become clear, the O.D.650 value decrease from 1.5 to 0.2, 50% protein and about 20% soluble sugar were removed, the content of tea polyphenol remain unchanged under the optimum conditions while the recovery rate of theanine was 95%. The optimum conditions were the mass concentration of chitson was 4mg/mL, pH was 3.0- 4.0, temperature was 20-30℃.
Memberane ultrafiltration can efficiently remove protein and polyphenol. Effect of different pore diameter, temperature of solution and work pressure were studied. The result showed that ultrafiltration can clarify the solution. 100k CA membrane may be suitable, 25-35℃ and 0.1-0.15Mpa work condition may be the fittest. Under the optimum conditions, the removal rate of protein was 48% and soluble sugar was 25%, the recovery rate of theanine was 90%.
4 kinds of adsorbents were used to study adsorption performance for TP. The result showed that AB-8 is a kind of good adsorption for TP. The pH, temperature of solution that affect adsorption performance were studied. The adsorptive capacities of AB-8 resin were the most, while elute speed was 1.0 BV/h and the ratio of height to diameter of column was 10:1. 70% ethanol was the efficient eluant.
The purification method of crude theanine was studied with ion exchange resin. Amino acid was primary purified from the extact of tea with cation resin. The optimal cation resin operation conditions were pH 3.0, 1.5mg/mL theanine solution, the elution rate 1.0BV/h. The saturated cation-exchange resin was eluted with different concentration solution of NH3.H2O. In view of the result, no matter what eluent concentrations of NH3H2O( from 1.5mol/L to 0.2mol/L) were adopted , the purity of theanine had no difference. Under the optimal condition, the rate of recovery of
theanine was 87% and purity of the product was 50%.
A crystallization method had been examined to obtain theanine with high purity. In this process, the crude theanine obtained by cation resin technique was dissolved in the 80% ethanol, and then theanine was crystallized from ethanol solution at 0℃ for 24 h. The rate of recovery of theanine was 70% and purity of theanine crystals was 65%. Through re-crystallization we can get theanine which purity was 90%.
Through this technique, the final yield and purity of the product were 0.8% and 90%.
絮凝能有效去除工业废液中的多糖类与蛋白质等杂质。经絮凝后，茶多酚废液溶液由混浊变为澄清，O.D.650值由1.5降至0.2左右，蛋白质杂质减少了50％，可溶性糖的含量也降低20％，茶多酚含量变化不大，茶氨酸的回收率超过95％。最佳絮凝工艺条件为壳聚糖添加至浓度4mg／mL，溶液pH为3.0～4.0，温度为20-30℃。
超滤能进一步澄清溶液，降低溶液中蛋白质等大分子物质的含量。研究结果表明，超滤的最佳工艺为选择十万分子量的醋酸纤维素膜，处理液温度为35-45℃时，以0.1-0.15MPa的工作压力进行操作，可得到较好的处理效果。通过超滤，蛋白质含量减少48％，可溶性糖含量下降25％，茶氨酸有一定程度的被截留，茶氨酸回收率约为90％。
吸附能进一步去除色素、多酚类物质及大分子有机物。筛选出一种对杂质吸附较多，同时茶氨酸的回收率较高的树脂—AB-8。静态吸附实验表明在35℃、溶液pH在4.0左右时，AB-8树脂对多酚类等有机质的吸附量最大。通过动态吸附法考察了树脂在不同流速、不同上样浓度及不同树脂层高径比条件下茶多酚的穿透曲线。结果表明，采用1.0BV／h的流速，树脂层高径比在10：1左右时进行动态吸附，单位树脂的吸附量最大。70％的乙醇的解吸性能较好。
阳离子交换树脂能较专一吸附氨基酸，不同阳离子交换树脂在吸附氨基酸的选择性上相差不大。离子交换树脂以732树脂为宜。在上样液茶氨酸浓度为1.5mg／mL时，溶液pH在3.0左右时，控制流速1.0BV／h，单位体积树脂吸附容量较大。洗脱液选择1.5mol／L的氨水，控制流速为1.0 BV／h，收集1.5倍柱体积沈脱液。洗脱液60℃减压浓缩挥去氨后，溶液冷冻干燥得粗茶氨酸。
乙醇重结晶法可得到较高纯度的茶氨酸。10％(W／V)粗茶氨酸液加入4倍体积乙醇，在常温下振荡混匀静置1h后，离心分离去除产生的沉淀。离心清液60℃减压浓缩为80％(W／V)的溶液，冷却后加4倍体积冷乙醇，0℃下冷藏24h，可产生结晶状物质，4℃、8000rpm／min离心得类白色固体，经检测此固体物茶氨酸纯度为65％。将所得固体用少量热水溶解，同上述方法重结晶3次。所得晶体60℃下真空干燥，可得纯度＞90％的茶氨酸产品。
不吹士学位论文
通过以上絮凝*超滤、吸附*阳离子交换*重结晶的工艺，可得到纯度超过
%%的茶氨酸产品，茶氨酸回收率为35%，产品得率为0.8%。
Techniques of isolation and purification of theanine from tea polyphenol waste industrial liquid were studied in this paper. To get rid of tea polyphenol, protein, soluble sugar in waste industrial liquid, some of chemical technique for separation, such as flocculation, membrane ultra filtration, absorption, ion-exchange absorption and re-crystallization were employed. To optimize the techniques,conditions of techniques were studied . The result suggested that:
Focculation can efficiently remove protein. Effects of concentration of chitson, pH and temperature of solution were investigated. After flocculation, the solution become clear, the O.D.650 value decrease from 1.5 to 0.2, 50% protein and about 20% soluble sugar were removed, the content of tea polyphenol remain unchanged under the optimum conditions while the recovery rate of theanine was 95%. The optimum conditions were the mass concentration of chitson was 4mg/mL, pH was 3.0- 4.0, temperature was 20-30℃.
Memberane ultrafiltration can efficiently remove protein and polyphenol. Effect of different pore diameter, temperature of solution and work pressure were studied. The result showed that ultrafiltration can clarify the solution. 100k CA membrane may be suitable, 25-35℃ and 0.1-0.15Mpa work condition may be the fittest. Under the optimum conditions, the removal rate of protein was 48% and soluble sugar was 25%, the recovery rate of theanine was 90%.
4 kinds of adsorbents were used to study adsorption performance for TP. The result showed that AB-8 is a kind of good adsorption for TP. The pH, temperature of solution that affect adsorption performance were studied. The adsorptive capacities of AB-8 resin were the most, while elute speed was 1.0 BV/h and the ratio of height to diameter of column was 10:1. 70% ethanol was the efficient eluant.
The purification method of crude theanine was studied with ion exchange resin. Amino acid was primary purified from the extact of tea with cation resin. The optimal cation resin operation conditions were pH 3.0, 1.5mg/mL theanine solution, the elution rate 1.0BV/h. The saturated cation-exchange resin was eluted with different concentration solution of NH3.H2O. In view of the result, no matter what eluent concentrations of NH3H2O( from 1.5mol/L to 0.2mol/L) were adopted , the purity of theanine had no difference. Under the optimal condition, the rate of recovery of
theanine was 87% and purity of the product was 50%.
A crystallization method had been examined to obtain theanine with high purity. In this process, the crude theanine obtained by cation resin technique was dissolved in the 80% ethanol, and then theanine was crystallized from ethanol solution at 0℃ for 24 h. The rate of recovery of theanine was 70% and purity of theanine crystals was 65%. Through re-crystallization we can get theanine which purity was 90%.
Through this technique, the final yield and purity of the product were 0.8% and 90%.
引文
1 K. Hetten. Ekborg-ett, Taylor. Armstrong. Agric Food Chem. 1997, 45: 353～362
2 乌居秀一．茶叶研究报告，1952，1：2
3 Juneja L R, Chu D-C, et al. L-theanine—a unique amino acid of green tea and its relaxation effect in humans. Trends in Food Science & Technology, 1999, 10:199～204
4 酒户弥二郎．农艺化学会志，1949，23：262
5 马家骧，高鹤娟等译．食品中添加剂的分析方法．1988
6 赵利涛．红绿茶加工过程中氨基酸变化与品质．氨基酸杂志，1990(3)
7 李布清，湖海明．茶叶中茶氨酸的快速测定．分析测试通报，1989，8(4)：51～53
8 郭升平．高效液相色谱法测定茶叶中茶氨酸的研究．色谱，1996，14(6)：464～465
9 周光明，齐东梅．高效液相色谱—钴离子催化化学发光抑制法测定茶叶中的茶氨酸．色谱．2002，20(6)：550～553
10 朱小兰，陈波．高效液相色谱法测定茶叶中的茶氨酸．色谱，2003，21(4)：400～402
11 Kiehne A, et al. Thermospray2LC2MS analysis of various groups of polyphenols in tea.. Catechins, flavonol O-glycosides and flavone C2glycosides. Z Lebensm Unter Forsch, 1996 (Jan), 202(1): 48～54
12 Aucamp J P, et al. Simultaneous analysis of tea catechins caffeine, gallic acid, theanine and ascorbic acid by micellarelectrokinetic capillary chromatography. J Chromatogr A, 2000, 876 (1-2): 235～242
13 杨贤强．茶树氮素代谢的研究．茶叶，1982，1：10～16
14 杨贤强．茶树中茶氨酸代谢的探讨．茶叶．1981，3：4～7
15 杨贤强，詹柏松．茶树的次级代谢．福建茶叶，1988，1：10～12
16 杨贤强．秋冬施肥对光合产物积累与分配的影响．浙江农业大学学报，1989,15(1)：83～86
17 杨贤强．人工光照对离体茶鲜叶氨基酸及香气成分的影响．福建茶叶，1991，1：11～21
18 Tsushida T. Agric. sci, 1987
19 向井俊博池．茶业研究报告，1992，76：45
20 王泽农主编．茶叶生物化学．农业出版社出版，1980
21 Matsuua T., Kakuda t. etc. Effect of precursor, temperature, and illumination on theanine accumulation in tea callus. Agric. Bio. Chem, 1990, 54(9), 2283～2286
22 Kiladze T. D., Tsanava V. P. etc. Biosynthesis and distribution of nitrogencontaining compounds in tea plants fed through roots on nitroyen-15 nitrate and carbon-14 sucrose. BA, 1990,115544
23 Shen Y., Yang x. etc. Effects of lateautumn fertilizer dressing on accumnlation of photo synthates in tea and its contribution to spring tea gualty. J Agrie.sci, 1990, 115(2), 233～238
24 Kitaoka S, Hayashi H, et al. Transmural potential changes associated with the in vitro absorption oftheanine in the guinea pig intestine. Biosci. Biotech. Biochem, 1996, 60(1): 1768～1771
25 Unno T, Suzuki Y, et al. Metabolism of theanine, γ-g101utamylethylamide in rats. J. Agric. Food Chem, 1999, 47(4): 1593～1596
26 Terashima T, Takido J, Yokogoshi H. Time-dependent changes of amino acids in the serum, liver, brain and urine of rats administered with theanine. Biosci. Biotech. Biochem., 1999,
63(4): 615～618
27 Yokogoshi H et al. Theanine-induced reduction of brain serotonin concentration in rats. Biosci Biotechnol Biochem, 1998, 62(4): 816-817
28 Yokogoshi H et al. Reduction effect of theanine on blood pressure and brain 5-hydroxyindoles in spontaneously hypertensive rats. Biosci Biotechnoi Biochem, 1995,59(4): 615～618.
29 Yokogoshi H et al. Hypotensive effect of gamma-glutamyl methylmide in spontaneously hypertensive rats. Life Sci,1998,62(12): 1065～1068.
30 Kakuda T, Yanase H, et al. Protective effect of (?)-glutamylethylamide (theanine) on ischemic delayed neuronal death in gerbils. Neurosci. Lett., 2000, 289(3): 189～192
31 木村良平，栗田正子，村田敏郞．药学杂志，1975，95：892
32 Kaduda T et al. lnhibiting effects oftheanine on caffeine stimulation evaluated by EEC in the rat. BiotechnolBiochem, 2000, 64(2): 287～293.
33 Yokogoshi H et al. Effect of theanine, r-glutamylethylamide,on brain monoamines and striatal dopamine release in conscious rats.. Neurochem Res, 1998, 23(5): 667～663.
34 小林加奈理．农化．1998，72：153
35 Sadzuka Yet al. Mokulation of cancer chem. otherapy by green tea. Clin Cancer Res, 1998,4(1): 153～156
36 Sugiyama T et al. Enhamcing effects of green tea components on the antitumor activity of adriamycin against M5076 ovarian sarcoma.. Cancer Lett, 1998,133(1): 19～26
37 Sugiyama T et al. Enhamcing effects of green tea components on the antitumor activity of adriamycin against M5076 ovarian sarcoma.. Cancer Lett, 1998,133 (1): 19～26
38 Sadzuka Y et al. Efficacies of tea components on doxorubicin induced antitumor activity and reversal of multidrug resistance. Toxicol Leff, 2000, 114(1-3): 155～162
39 Sadzuda Y et al. Improvement of idarubicin induced antitumor activity and bone marro suppression by theanine, a component of tea. Cancer Lett, 2000, 158(2): 119～124.
40 Sadzuka Yet al. The effects oftheanine as a novel biochemical moducator on the antiumor activity of adriamycin. Cancer LETT, 1996,105(2): 203～209.
41 矢崎一三．茶機能，村松敬一郎等编．东京：学会出版．2002：336～337
42 茶山和敏．茶機能，村松敬一郎等编．东京：学会出版．2002：205～209
43 杉山公男．茶機能，村松敬一郎等编．东京：学会出版．2002：230～233
44 Yokozawa T, Dong E. Influence of green tea and its three major components upon low-density lipoprotein oxidation. Exp. Toxicol. Pathol., 1997, 49(5): 329-335.
45 JunejaL R，人久保勉．绿茶驚効果．RYOKUCHA，2002，1：29～31
46 汤炎．茶氨酸的合成、蓟理及其在食品中的应用．茶业通报，2002，24(4)：19～21
47 赵丹，王朝旭．茶氨酸的国內外研究现状．食品科学，2002，23(5)：146～148
48 林智．从茶叶抗病毒的研究谈茶氨酸的生产与应用前景．中国茶叶，2003，25(3)：4～5
49 Jp 05,68,578, (1993)
50 Kwagishi, Het al. Biosci, Biotech. Biochem, 1992,56 (4): 689.
51 Abelian. V. H, et al. Biosci. Biotech. Biochem, 1993
52 Tachikis. T, et al, J. Gen. Appl. microbiol, 1986, 32: 545～548.
53 M. Arai. T, Matsuura. T, et al. Nippon Dojo Hiryogaku Zasshi (in Japanese). 1989, 60(8): 157～159
54 Matsuura. T, et al. Biosci. Biotech. Biochem, 1990
55 T. Matsuura T. Kakuda et al. Theanine formation by tea suspension cells. Biosci. Biotech.
Biochem, 1992, 56(8): 1179～1181
56 钟俊辉，陶文忻．茶愈伤组织培养及茶氨酸的累积．无锡轻厂业大学学报，1997，3(6)：33～35
57 陈瑛等．几种激素对茶愈伤组织合成茶氨酸的影响．无锡轻工业大学学报，1998，7(1)：74～77
58 T. Fumya et al. Production oftheanine and other gamma-glutamyl derivatives by camellia sinensis cuhural cells. J. BA, 1990, 10(2): 218～230
59 K. Sasaoka, M. Kito et al. Some propertites of the theanine synthesizing enzyme in tea seedlings. Bio. Chem, 1965, 29(11): 984～988
60 陈宗道，周才琼，重华荣编著．茶叶化学工程学．1999，5：36～37
61 《化学分离富集方法及应用》编委会编著。中南工业大学出版社，1996年12月第1版
62 沈金玉，熊训浩．絮凝与膜超滤耦合预处理谷氨酰胺发酵液．精细化工，2002，19(11)
63 骆广生，高春满．天然吸附剂—壳聚糖吸附性能的研究．天然产物研究与开发，1994，5(2)
64 马青山，贾瑟．絮凝化学与絮凝剂，1988，3
65 沈一丁 石珞．壳聚糖交联阳离子高分子絮凝剂的表征及应用．高分子材料科学与工程，2002，18(4)．-101-104
66 张宗恩．甲壳素、壳聚糖及其衍生物在水处理中的应用．环境保护，1996(8)．-13-14
67 张文清．金鑫荣．壳聚糖作为吸附絮凝剂的应用进展．食品工业，1994(4)．-51-52
68 李静，高玉洁．天然高分子絮凝剂壳聚糖．天津造纸，2003，25(1)．-33-34
69 夏涛．膜技术在茶叶深加工中的应用．茶叶科学技术，1996，2：10-13
70 孙福强，崔英德．膜分离技术及其应用前景展望．化工科技，2002，10(4)：58—63
71 尹军峰，权启爱．绿茶汁澄清工艺的膜分离技术研究．茶叶科学，2001，21(2)：116—119
72 尹军峰，权启爱．超滤膜澄清乌龙茶饮料技术的研究．茶叶科学2003，23(增)：58—62
73 薛怀德．超滤膜法简要介绍．膜科学与技术，1991，11(1，2)：171～177
74 姜玉玺．膜分离技术的应用与进展．黑龙江大学学报，2002，19(3)：98～103
75 吕晓龙．超滤膜孔径及其分布的测定方法．1995，21(3)：137～141
76 朱长乐，刘茉娥．膜科学技术．1992，4
77 叶振华．化工吸附分离过程．1992，12
78 徐世芳，毛丽珍．大孔吸附树脂及其在中药化学成分纯化中的应用．浙江省医药科学院学报，2001，6：45～47．
79 陈劲春，李一．四种吸附材料分离茶多酚的初步结果比较．北京化工大学学报(自然科学版)，2000，27(2)：95～96
80 董文宾，胡英．吸附树脂法制备茶多酚精品的工艺研究．食品科学，2002，23(11)：68～73
81 卢锦花．大孔吸附树脂对银杏叶黄酮类化合物吸附及解吸的研究．化学研究与应用，2002，14(2)：164～167
82 冉艳红，杨春哲．利用吸附树脂处理苹果酒．食品与发酵上业，2001，27(10)：49～52．
83 赵利，钱芳．大孔吸附树脂及其在蛋白质、氨基酸、多肽纯化中的应用．四川食品与发酵，2003，2：39-41
84 徐宇平，杨力．味精废液中氨基酸的色谱分离研究．离子交换与吸附，2002．18(5)：426～431
85 陈瑛等．离子交换法提取茶氨酸的研究．氨基酸和生物资源，1998，20(1)
86 武汉水利电力学院电厂化学教研室．热力发电厂水处理，水利电力出版社，1976
87 姜文涛．离子交换树脂的污染及其处理．质量天地，2003(10)：54～56
88 陈晓峰，黄箫．影响离子交换树脂使用寿命因素的探讨．炼油与化工，2003，14(3)：17～19
89 谭伟，李强．离子交换树脂的酸碱静态法再生．临床检验杂志，2003，21(3)：168～169
90 高永生，时丽秀．离子交换树脂再生的简便快速处理．内蒙古医学杂志，2003，35(2)：
142～145
91 王建国．固定床离子交换树脂复苏方法探讨．净水技术，2002，21(3)：46～47
92 冯容保．氨基酸的结晶工艺．发酵科技通讯，2002，31(4)
93 陈玉胜，张祥福．水析结晶精制对二氯苯研究．化学工程，2000，28(4)：46～48
94 崔现宝，杨志才．含麝香体系多元溶液固液平衡研究．化学丁程，2001，29(5)：61～65
2 乌居秀一．茶叶研究报告，1952，1：2
3 Juneja L R, Chu D-C, et al. L-theanine—a unique amino acid of green tea and its relaxation effect in humans. Trends in Food Science & Technology, 1999, 10:199～204
4 酒户弥二郎．农艺化学会志，1949，23：262
5 马家骧，高鹤娟等译．食品中添加剂的分析方法．1988
6 赵利涛．红绿茶加工过程中氨基酸变化与品质．氨基酸杂志，1990(3)
7 李布清，湖海明．茶叶中茶氨酸的快速测定．分析测试通报，1989，8(4)：51～53
8 郭升平．高效液相色谱法测定茶叶中茶氨酸的研究．色谱，1996，14(6)：464～465
9 周光明，齐东梅．高效液相色谱—钴离子催化化学发光抑制法测定茶叶中的茶氨酸．色谱．2002，20(6)：550～553
10 朱小兰，陈波．高效液相色谱法测定茶叶中的茶氨酸．色谱，2003，21(4)：400～402
11 Kiehne A, et al. Thermospray2LC2MS analysis of various groups of polyphenols in tea.. Catechins, flavonol O-glycosides and flavone C2glycosides. Z Lebensm Unter Forsch, 1996 (Jan), 202(1): 48～54
12 Aucamp J P, et al. Simultaneous analysis of tea catechins caffeine, gallic acid, theanine and ascorbic acid by micellarelectrokinetic capillary chromatography. J Chromatogr A, 2000, 876 (1-2): 235～242
13 杨贤强．茶树氮素代谢的研究．茶叶，1982，1：10～16
14 杨贤强．茶树中茶氨酸代谢的探讨．茶叶．1981，3：4～7
15 杨贤强，詹柏松．茶树的次级代谢．福建茶叶，1988，1：10～12
16 杨贤强．秋冬施肥对光合产物积累与分配的影响．浙江农业大学学报，1989,15(1)：83～86
17 杨贤强．人工光照对离体茶鲜叶氨基酸及香气成分的影响．福建茶叶，1991，1：11～21
18 Tsushida T. Agric. sci, 1987
19 向井俊博池．茶业研究报告，1992，76：45
20 王泽农主编．茶叶生物化学．农业出版社出版，1980
21 Matsuua T., Kakuda t. etc. Effect of precursor, temperature, and illumination on theanine accumulation in tea callus. Agric. Bio. Chem, 1990, 54(9), 2283～2286
22 Kiladze T. D., Tsanava V. P. etc. Biosynthesis and distribution of nitrogencontaining compounds in tea plants fed through roots on nitroyen-15 nitrate and carbon-14 sucrose. BA, 1990,115544
23 Shen Y., Yang x. etc. Effects of lateautumn fertilizer dressing on accumnlation of photo synthates in tea and its contribution to spring tea gualty. J Agrie.sci, 1990, 115(2), 233～238
24 Kitaoka S, Hayashi H, et al. Transmural potential changes associated with the in vitro absorption oftheanine in the guinea pig intestine. Biosci. Biotech. Biochem, 1996, 60(1): 1768～1771
25 Unno T, Suzuki Y, et al. Metabolism of theanine, γ-g101utamylethylamide in rats. J. Agric. Food Chem, 1999, 47(4): 1593～1596
26 Terashima T, Takido J, Yokogoshi H. Time-dependent changes of amino acids in the serum, liver, brain and urine of rats administered with theanine. Biosci. Biotech. Biochem., 1999,
63(4): 615～618
27 Yokogoshi H et al. Theanine-induced reduction of brain serotonin concentration in rats. Biosci Biotechnol Biochem, 1998, 62(4): 816-817
28 Yokogoshi H et al. Reduction effect of theanine on blood pressure and brain 5-hydroxyindoles in spontaneously hypertensive rats. Biosci Biotechnoi Biochem, 1995,59(4): 615～618.
29 Yokogoshi H et al. Hypotensive effect of gamma-glutamyl methylmide in spontaneously hypertensive rats. Life Sci,1998,62(12): 1065～1068.
30 Kakuda T, Yanase H, et al. Protective effect of (?)-glutamylethylamide (theanine) on ischemic delayed neuronal death in gerbils. Neurosci. Lett., 2000, 289(3): 189～192
31 木村良平，栗田正子，村田敏郞．药学杂志，1975，95：892
32 Kaduda T et al. lnhibiting effects oftheanine on caffeine stimulation evaluated by EEC in the rat. BiotechnolBiochem, 2000, 64(2): 287～293.
33 Yokogoshi H et al. Effect of theanine, r-glutamylethylamide,on brain monoamines and striatal dopamine release in conscious rats.. Neurochem Res, 1998, 23(5): 667～663.
34 小林加奈理．农化．1998，72：153
35 Sadzuka Yet al. Mokulation of cancer chem. otherapy by green tea. Clin Cancer Res, 1998,4(1): 153～156
36 Sugiyama T et al. Enhamcing effects of green tea components on the antitumor activity of adriamycin against M5076 ovarian sarcoma.. Cancer Lett, 1998,133(1): 19～26
37 Sugiyama T et al. Enhamcing effects of green tea components on the antitumor activity of adriamycin against M5076 ovarian sarcoma.. Cancer Lett, 1998,133 (1): 19～26
38 Sadzuka Y et al. Efficacies of tea components on doxorubicin induced antitumor activity and reversal of multidrug resistance. Toxicol Leff, 2000, 114(1-3): 155～162
39 Sadzuda Y et al. Improvement of idarubicin induced antitumor activity and bone marro suppression by theanine, a component of tea. Cancer Lett, 2000, 158(2): 119～124.
40 Sadzuka Yet al. The effects oftheanine as a novel biochemical moducator on the antiumor activity of adriamycin. Cancer LETT, 1996,105(2): 203～209.
41 矢崎一三．茶機能，村松敬一郎等编．东京：学会出版．2002：336～337
42 茶山和敏．茶機能，村松敬一郎等编．东京：学会出版．2002：205～209
43 杉山公男．茶機能，村松敬一郎等编．东京：学会出版．2002：230～233
44 Yokozawa T, Dong E. Influence of green tea and its three major components upon low-density lipoprotein oxidation. Exp. Toxicol. Pathol., 1997, 49(5): 329-335.
45 JunejaL R，人久保勉．绿茶驚効果．RYOKUCHA，2002，1：29～31
46 汤炎．茶氨酸的合成、蓟理及其在食品中的应用．茶业通报，2002，24(4)：19～21
47 赵丹，王朝旭．茶氨酸的国內外研究现状．食品科学，2002，23(5)：146～148
48 林智．从茶叶抗病毒的研究谈茶氨酸的生产与应用前景．中国茶叶，2003，25(3)：4～5
49 Jp 05,68,578, (1993)
50 Kwagishi, Het al. Biosci, Biotech. Biochem, 1992,56 (4): 689.
51 Abelian. V. H, et al. Biosci. Biotech. Biochem, 1993
52 Tachikis. T, et al, J. Gen. Appl. microbiol, 1986, 32: 545～548.
53 M. Arai. T, Matsuura. T, et al. Nippon Dojo Hiryogaku Zasshi (in Japanese). 1989, 60(8): 157～159
54 Matsuura. T, et al. Biosci. Biotech. Biochem, 1990
55 T. Matsuura T. Kakuda et al. Theanine formation by tea suspension cells. Biosci. Biotech.
Biochem, 1992, 56(8): 1179～1181
56 钟俊辉，陶文忻．茶愈伤组织培养及茶氨酸的累积．无锡轻厂业大学学报，1997，3(6)：33～35
57 陈瑛等．几种激素对茶愈伤组织合成茶氨酸的影响．无锡轻工业大学学报，1998，7(1)：74～77
58 T. Fumya et al. Production oftheanine and other gamma-glutamyl derivatives by camellia sinensis cuhural cells. J. BA, 1990, 10(2): 218～230
59 K. Sasaoka, M. Kito et al. Some propertites of the theanine synthesizing enzyme in tea seedlings. Bio. Chem, 1965, 29(11): 984～988
60 陈宗道，周才琼，重华荣编著．茶叶化学工程学．1999，5：36～37
61 《化学分离富集方法及应用》编委会编著。中南工业大学出版社，1996年12月第1版
62 沈金玉，熊训浩．絮凝与膜超滤耦合预处理谷氨酰胺发酵液．精细化工，2002，19(11)
63 骆广生，高春满．天然吸附剂—壳聚糖吸附性能的研究．天然产物研究与开发，1994，5(2)
64 马青山，贾瑟．絮凝化学与絮凝剂，1988，3
65 沈一丁 石珞．壳聚糖交联阳离子高分子絮凝剂的表征及应用．高分子材料科学与工程，2002，18(4)．-101-104
66 张宗恩．甲壳素、壳聚糖及其衍生物在水处理中的应用．环境保护，1996(8)．-13-14
67 张文清．金鑫荣．壳聚糖作为吸附絮凝剂的应用进展．食品工业，1994(4)．-51-52
68 李静，高玉洁．天然高分子絮凝剂壳聚糖．天津造纸，2003，25(1)．-33-34
69 夏涛．膜技术在茶叶深加工中的应用．茶叶科学技术，1996，2：10-13
70 孙福强，崔英德．膜分离技术及其应用前景展望．化工科技，2002，10(4)：58—63
71 尹军峰，权启爱．绿茶汁澄清工艺的膜分离技术研究．茶叶科学，2001，21(2)：116—119
72 尹军峰，权启爱．超滤膜澄清乌龙茶饮料技术的研究．茶叶科学2003，23(增)：58—62
73 薛怀德．超滤膜法简要介绍．膜科学与技术，1991，11(1，2)：171～177
74 姜玉玺．膜分离技术的应用与进展．黑龙江大学学报，2002，19(3)：98～103
75 吕晓龙．超滤膜孔径及其分布的测定方法．1995，21(3)：137～141
76 朱长乐，刘茉娥．膜科学技术．1992，4
77 叶振华．化工吸附分离过程．1992，12
78 徐世芳，毛丽珍．大孔吸附树脂及其在中药化学成分纯化中的应用．浙江省医药科学院学报，2001，6：45～47．
79 陈劲春，李一．四种吸附材料分离茶多酚的初步结果比较．北京化工大学学报(自然科学版)，2000，27(2)：95～96
80 董文宾，胡英．吸附树脂法制备茶多酚精品的工艺研究．食品科学，2002，23(11)：68～73
81 卢锦花．大孔吸附树脂对银杏叶黄酮类化合物吸附及解吸的研究．化学研究与应用，2002，14(2)：164～167
82 冉艳红，杨春哲．利用吸附树脂处理苹果酒．食品与发酵上业，2001，27(10)：49～52．
83 赵利，钱芳．大孔吸附树脂及其在蛋白质、氨基酸、多肽纯化中的应用．四川食品与发酵，2003，2：39-41
84 徐宇平，杨力．味精废液中氨基酸的色谱分离研究．离子交换与吸附，2002．18(5)：426～431
85 陈瑛等．离子交换法提取茶氨酸的研究．氨基酸和生物资源，1998，20(1)
86 武汉水利电力学院电厂化学教研室．热力发电厂水处理，水利电力出版社，1976
87 姜文涛．离子交换树脂的污染及其处理．质量天地，2003(10)：54～56
88 陈晓峰，黄箫．影响离子交换树脂使用寿命因素的探讨．炼油与化工，2003，14(3)：17～19
89 谭伟，李强．离子交换树脂的酸碱静态法再生．临床检验杂志，2003，21(3)：168～169
90 高永生，时丽秀．离子交换树脂再生的简便快速处理．内蒙古医学杂志，2003，35(2)：
142～145
91 王建国．固定床离子交换树脂复苏方法探讨．净水技术，2002，21(3)：46～47
92 冯容保．氨基酸的结晶工艺．发酵科技通讯，2002，31(4)
93 陈玉胜，张祥福．水析结晶精制对二氯苯研究．化学工程，2000，28(4)：46～48
94 崔现宝，杨志才．含麝香体系多元溶液固液平衡研究．化学丁程，2001，29(5)：61～65