功能性食品添加剂—谷胱甘肽分离纯化工艺的研究
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摘要
谷胱甘肽(GSH)是一种生物活性三肽。GSH在生物体内具有多种重要的生理功能,在临床医药、食品工业、体育运动及有关生物研究领域都有着广泛的用途。本文对产朊假丝酵母(Candida utilisWSH 02-08) GSH积累株酵母细胞中GSH的提取分离和纯化进行了研究。研究的主要内容如下:
1.研究确定了实际体系中影响谷胱甘肽稳定性的主要因素是pH值和温度:pH值为1～2谷胱甘肽较稳定;谷胱甘肽在常温下保藏两天相对较稳定,在20min内100℃下提取液GSH时,其损失率较小。因此,在对GSH进行分离纯化过程中应尽量避免较高pH和高温。
2.研究了应用超滤技术去除富含谷胱甘肽的产朊假丝酵母提取液中的蛋白质的最佳工艺操作参数;同时为提高谷胱甘肽回收率,比较了间歇稀释过滤(DD)和连续稀释(CD)过滤两种稀释方法的优缺点,最终确定采用间歇稀释过滤和超滤相结合的方法分离纯化谷胱甘肽。另外,实验还确定了超滤膜最佳的清洗方法。
3.本实验研究了超滤技术对谷胱甘肽在阳离子交换树脂(001×7)上的交换行为的影响。同时对影响固定床离子交换吸附的条件的研究,确定了最佳上柱和洗脱的工艺参数,最终的GSH回收率达到80.2%,蛋白质去除率60%,GSH浓缩倍数达2.5倍(高峰时达到8.3倍),纯化倍数为3.9倍,获得了良好的分离效果。
4.在上述研究的基础上,通过改进提取工艺,对谷胱甘肽的工业化生产路线进行了简化,使得富含GSH的产朊假丝酵母经提取、离交、浓缩和干燥简单四步主要单元操作,得到含量为11.3%谷胱甘肽复合产品。
Glutathione, bio-synthesized from L-glutamic acid, L-cysteine and glycine in two consecutive steps byγ-glutamyl-cysteine synthase (GSH I, EC 6.3.2.2) and glutathione synthase (GSH II, EC 6.3.2.3), is one of the major non-protein thiol compounds. GSH (the reduced form of glutathione) is widely distributed in nature and plays many important physiological roles in living cells, such as enhancing the immunity of the body promptly and maintaining the normal redox environment of cells as an antioxidant. Since GSH is widely used in many fields, i.e. clinical medicine, food industry, athletic sports and biological research, the demand of GSH has been expanding. In this paper, the separation and purification process of glutathione from Candida utilis WSH 02-08 were investigated in detail. The main results were described as follows:
1. It was found that the stability of GSH was determined by pH and temperature in the extracting solution. In addition, a high GSH stability was observed when pH value at the range of 1 to 2 and at room-temperature for two days. Furthermore, only a little of GSH was degraded when GSH extract was treated by 100℃for 20 minutes. Those results indicted that, an optimization pH and temperature is required for GSH separation and purification.
2. An optimal operation parameters for removing protein from Candida utilis WSH 02-08 extraction by ultrafiltraion were determined. At the same time, in order to further increase the recovery rate of GSH, the advantages and disadvantages of dormancy dilution (DD) and continuous dilution (CD) were compared. Base on those results, an integrated processing of DD and ultrafiltraion was determined and as the most effective method to separate and purify. Furthermore, the cleaning process of ultrafiltration membrane was also determined in this paper.
3. The affects of ultrafiltraion on the exchange action of GSH on cation exchange resin(001×7)were studied. The optimal parameters of adsorption and elution were determined through the study of conditions affecting ion exchanging and adsorption of fixed bed .The results was fellow: the recovery rate of GSH 80.2%, the removal rate of protein 60%, the concentration ratio of GSH 2.5:1 (at most 8.3:1), the purification rate 3.9. With those optimal conditions, an efficient GSH separation was achieved.
4. Based on those results, an industrialized process for GSH extraction was developed. A end-product which contain 11.3% GSH was obtained, after Candida utilis WSH 02-08 was treated by extracted, ion exchanged, concentrated and dried.
1.研究确定了实际体系中影响谷胱甘肽稳定性的主要因素是pH值和温度:pH值为1～2谷胱甘肽较稳定;谷胱甘肽在常温下保藏两天相对较稳定,在20min内100℃下提取液GSH时,其损失率较小。因此,在对GSH进行分离纯化过程中应尽量避免较高pH和高温。
2.研究了应用超滤技术去除富含谷胱甘肽的产朊假丝酵母提取液中的蛋白质的最佳工艺操作参数;同时为提高谷胱甘肽回收率,比较了间歇稀释过滤(DD)和连续稀释(CD)过滤两种稀释方法的优缺点,最终确定采用间歇稀释过滤和超滤相结合的方法分离纯化谷胱甘肽。另外,实验还确定了超滤膜最佳的清洗方法。
3.本实验研究了超滤技术对谷胱甘肽在阳离子交换树脂(001×7)上的交换行为的影响。同时对影响固定床离子交换吸附的条件的研究,确定了最佳上柱和洗脱的工艺参数,最终的GSH回收率达到80.2%,蛋白质去除率60%,GSH浓缩倍数达2.5倍(高峰时达到8.3倍),纯化倍数为3.9倍,获得了良好的分离效果。
4.在上述研究的基础上,通过改进提取工艺,对谷胱甘肽的工业化生产路线进行了简化,使得富含GSH的产朊假丝酵母经提取、离交、浓缩和干燥简单四步主要单元操作,得到含量为11.3%谷胱甘肽复合产品。
Glutathione, bio-synthesized from L-glutamic acid, L-cysteine and glycine in two consecutive steps byγ-glutamyl-cysteine synthase (GSH I, EC 6.3.2.2) and glutathione synthase (GSH II, EC 6.3.2.3), is one of the major non-protein thiol compounds. GSH (the reduced form of glutathione) is widely distributed in nature and plays many important physiological roles in living cells, such as enhancing the immunity of the body promptly and maintaining the normal redox environment of cells as an antioxidant. Since GSH is widely used in many fields, i.e. clinical medicine, food industry, athletic sports and biological research, the demand of GSH has been expanding. In this paper, the separation and purification process of glutathione from Candida utilis WSH 02-08 were investigated in detail. The main results were described as follows:
1. It was found that the stability of GSH was determined by pH and temperature in the extracting solution. In addition, a high GSH stability was observed when pH value at the range of 1 to 2 and at room-temperature for two days. Furthermore, only a little of GSH was degraded when GSH extract was treated by 100℃for 20 minutes. Those results indicted that, an optimization pH and temperature is required for GSH separation and purification.
2. An optimal operation parameters for removing protein from Candida utilis WSH 02-08 extraction by ultrafiltraion were determined. At the same time, in order to further increase the recovery rate of GSH, the advantages and disadvantages of dormancy dilution (DD) and continuous dilution (CD) were compared. Base on those results, an integrated processing of DD and ultrafiltraion was determined and as the most effective method to separate and purify. Furthermore, the cleaning process of ultrafiltration membrane was also determined in this paper.
3. The affects of ultrafiltraion on the exchange action of GSH on cation exchange resin(001×7)were studied. The optimal parameters of adsorption and elution were determined through the study of conditions affecting ion exchanging and adsorption of fixed bed .The results was fellow: the recovery rate of GSH 80.2%, the removal rate of protein 60%, the concentration ratio of GSH 2.5:1 (at most 8.3:1), the purification rate 3.9. With those optimal conditions, an efficient GSH separation was achieved.
4. Based on those results, an industrialized process for GSH extraction was developed. A end-product which contain 11.3% GSH was obtained, after Candida utilis WSH 02-08 was treated by extracted, ion exchanged, concentrated and dried.
引文
[1] Penninckx M J, Elskens M T. Metabolism and functions of glutathione in micro-organisms. Adv Microbial Physiol, 1993, 34: 239-301
[2] Meister A. Glutathione, ascorbate, and cellular protection. Cancer Res, 1994, 54(7): 1969-1975
[3] Penninckx M J. An overview on glutathione in Saccharomyces versus non-conventional yeasts. FEMS Yeast Research, 2002, 2: 295-305
[4] Sies H. Glutathione and its role in cellular functions. Free Radical Bio Med, 1999, 27(9-10): 916-921
[5] 欧阳平凯. 化工产品手册?生物化工产品手册. 北京: 化学工业出版社, 1999, 152-153
[6] Meister A, Anderson M E. Glutathione. Ann Rev Biochem, 1983, 52: 711-760
[7] 韩秀文, 屈铭, 缪希茄, 倪坚毅, 胡皆汉. 谷胱甘肽在水溶液中的构象研究. 化学通报, 1994, 3: 36-40
[8] 刘振玉. 谷胱甘肽的研究与应用. 生命的化学, 1995, 15(1): 19-21
[9] Cooper A J L, Kristal B S. Multiple roles of glutathione in the central nervous system. Biol Chem, 1997, 378(8): 793-802
[10] Smith J E. Relationship of in vivo erythrocyte glutathione flux to the oxidized glutathione transport system. J Lab Clin Med, 1974, 83: 444-450
[11] 沈亚领, 李爽, 迟莉丽, 雷肇祖. 谷胱甘肽的应用与生产. 工业微生物, 2000, 30(2): 41-45
[12] Izawa S, Inoue Y, Kimura A. Oxidative stress response in yeast: effect of glutathione on adaptation to hydrogen peroxide stress in Saccharomyces cerevisiae. FEBS Lett, 1995, 368: 73-76
[13] Jahoor F, Jackson A, Gazzard B, Philips G, Sharpstone D, Frazer M E, Heird W. Erythrocyte glutathione deficiency in symptom-free HIV infection is associated with decreased synthesis rate. Am J Physiol, 1999, 276(1): E205-E211
[14] 卓肇文. 还原型谷胱甘肽的功能与应用. 氨基酸杂志, 1989, 1: 41-42
[15] 袁尔东, 郑建仙. 功能性食品基料?谷胱甘肽的研究进展. 食品与发酵工业, 1999, 25(5): 52-56
[16] Sen C K. Nutritional biochemistry of cellular glutathione. Nutr Biochem, 1997, 8: 660-672
[17] Harington C R, Mead T H, Synthesis of Glutathione, Biochem J, 1935, 29(3): 1602～1611
[18] Maki Haruhiko, Fukuda Hideki, Purification Of Glutathione, Patent Of Japan, Pn62-246594, 1987, 10, 21
[19] Maki Haruhiko, Fukuda Hideki, Purifying Method For Glutathione, Patent Of Japan, Pn:63-088196,1988 ,4, 19
[20] Maki Haruhiko, Imai Satoshi, Method For Purifying Glutathione, Patent Of Japan, Pn: 61-027999, 1986, 2, 7
[21] Matsui Katsuaki, Yoshiya Toyofumi, Purification Of Glutathione, Patent Of Japan, Pn: 62-292797, 1987, 12, 19
[22] Haruhiko Maki, Hideki Fukuda, the Separation of Glutathione and Glutami cacidusing A Simulatednoving-Bed Absorber System.J Fermend, 1987, 65(1): 61～70
[23] Kataoka Katsuyuki, Senoo Yoshimi, Kawabata Takashi, Etal, Purification Of Glutathione, Patent Of Japan, Pn:06～056884, 1994,3,1
[24] 陶祖贻,赵爱民,离子交换平衡及其动力学,原子能出版社,1989,27～84
[25] 王辉,冯万祥,含汞树脂分离提纯谷胱甘肽,华东理工大学学报,1996,22(6):717～721
[26] 王辉,冯万祥,几种国产树脂对谷胱甘肽的分离性能测定 ,离子交换与吸附,1997,13(3),318～321
[27] Penninckx M, A short review on the role of glutathione in the response of yeast to nutritional, environmental, and oxidative stresses, Enzyme Microb Technol, 2000, 26(9-10): 737～742
[28] 沈亚领, 李爽, 迟莉丽, 雷肇祖. 谷胱甘肽的应用与生产. 工业微生物, 2000, 30(2): 41-45
[29] 范崇东.酵母细胞中谷胱甘肽的分离纯化.2004.3
[30] Ellman G L. A Calorimetric Method for Determining Low Concenrrations of Mercaptans[J].Arch Of Biochem &Biophys, 1959, 82:70～77.
[31] 林加涵,魏文玲,彭宣宪,Experiment Of Modern Biology,北京:高等教育出版社,Springer,2001.10:36～38
[32] Munir Cheryan. Ultrafiltration Handbook.Technomic publishing CO. INC, 1986
[33] Marcel Mulder. Basic Priciples of Membrane Technology.清华大学出版社,1999.
[34] Butterworth T A, Wang D I C, In: Terui G, ed, Fermentation Technology today[J].Soxc. Ferment. Technol. Japan, 1972, 19.
[35] Tetsuya Kawakita, Tadako Ogura, Masaru Saeki And Hideo Hayasaki, Selectivity Coefficients Of Amion Acids For The Ammonium Ion On A Strong Cation Exchange Resin, Agic.Biol.Chem,1990, 54(1): 1～8
[36] 钱庭宝,刘维琳,离子交换树脂应用手册,天津:南开大学出版社,1989
[37] 孙彦,生物分离工程,北京:化学工业出版社,1998.10
[38] 赵利,王璋,许时婴,大孔吸附树脂对酪蛋白非磷酸肽吸附性的研究,食品工业技术,2002,12:28～31
[39] Jung.G, Breitmaier, E, and Voelter, W, Dissoziationgs gleichgewichte won Glutathione, Eine Fourier- Transform -13C-NMR sprktroskopische untersuchung, Eur.J.Biochem, 1972, 24:438～444
[40] 姜志新,湛竞清,宋正孝编著,离子交换分离工程,天津大学出版社,1992
[41] Uotani Osamu, Tani Hideo, Mizushima Shinji Et Al. Purification Of Glutathione, Patent Of Japan, Pn: 52～073821, 1977.06.21
[42] Kimura Hikari, Inoue Yoshiharu, Kobayashi Susumu, Production Of Glutathione, Patent Of Japan, Pn:08-070884, 1996,03,19
[43] 氨基酸发酵工艺,中国轻工业出版社,1989
[2] Meister A. Glutathione, ascorbate, and cellular protection. Cancer Res, 1994, 54(7): 1969-1975
[3] Penninckx M J. An overview on glutathione in Saccharomyces versus non-conventional yeasts. FEMS Yeast Research, 2002, 2: 295-305
[4] Sies H. Glutathione and its role in cellular functions. Free Radical Bio Med, 1999, 27(9-10): 916-921
[5] 欧阳平凯. 化工产品手册?生物化工产品手册. 北京: 化学工业出版社, 1999, 152-153
[6] Meister A, Anderson M E. Glutathione. Ann Rev Biochem, 1983, 52: 711-760
[7] 韩秀文, 屈铭, 缪希茄, 倪坚毅, 胡皆汉. 谷胱甘肽在水溶液中的构象研究. 化学通报, 1994, 3: 36-40
[8] 刘振玉. 谷胱甘肽的研究与应用. 生命的化学, 1995, 15(1): 19-21
[9] Cooper A J L, Kristal B S. Multiple roles of glutathione in the central nervous system. Biol Chem, 1997, 378(8): 793-802
[10] Smith J E. Relationship of in vivo erythrocyte glutathione flux to the oxidized glutathione transport system. J Lab Clin Med, 1974, 83: 444-450
[11] 沈亚领, 李爽, 迟莉丽, 雷肇祖. 谷胱甘肽的应用与生产. 工业微生物, 2000, 30(2): 41-45
[12] Izawa S, Inoue Y, Kimura A. Oxidative stress response in yeast: effect of glutathione on adaptation to hydrogen peroxide stress in Saccharomyces cerevisiae. FEBS Lett, 1995, 368: 73-76
[13] Jahoor F, Jackson A, Gazzard B, Philips G, Sharpstone D, Frazer M E, Heird W. Erythrocyte glutathione deficiency in symptom-free HIV infection is associated with decreased synthesis rate. Am J Physiol, 1999, 276(1): E205-E211
[14] 卓肇文. 还原型谷胱甘肽的功能与应用. 氨基酸杂志, 1989, 1: 41-42
[15] 袁尔东, 郑建仙. 功能性食品基料?谷胱甘肽的研究进展. 食品与发酵工业, 1999, 25(5): 52-56
[16] Sen C K. Nutritional biochemistry of cellular glutathione. Nutr Biochem, 1997, 8: 660-672
[17] Harington C R, Mead T H, Synthesis of Glutathione, Biochem J, 1935, 29(3): 1602～1611
[18] Maki Haruhiko, Fukuda Hideki, Purification Of Glutathione, Patent Of Japan, Pn62-246594, 1987, 10, 21
[19] Maki Haruhiko, Fukuda Hideki, Purifying Method For Glutathione, Patent Of Japan, Pn:63-088196,1988 ,4, 19
[20] Maki Haruhiko, Imai Satoshi, Method For Purifying Glutathione, Patent Of Japan, Pn: 61-027999, 1986, 2, 7
[21] Matsui Katsuaki, Yoshiya Toyofumi, Purification Of Glutathione, Patent Of Japan, Pn: 62-292797, 1987, 12, 19
[22] Haruhiko Maki, Hideki Fukuda, the Separation of Glutathione and Glutami cacidusing A Simulatednoving-Bed Absorber System.J Fermend, 1987, 65(1): 61～70
[23] Kataoka Katsuyuki, Senoo Yoshimi, Kawabata Takashi, Etal, Purification Of Glutathione, Patent Of Japan, Pn:06～056884, 1994,3,1
[24] 陶祖贻,赵爱民,离子交换平衡及其动力学,原子能出版社,1989,27～84
[25] 王辉,冯万祥,含汞树脂分离提纯谷胱甘肽,华东理工大学学报,1996,22(6):717～721
[26] 王辉,冯万祥,几种国产树脂对谷胱甘肽的分离性能测定 ,离子交换与吸附,1997,13(3),318～321
[27] Penninckx M, A short review on the role of glutathione in the response of yeast to nutritional, environmental, and oxidative stresses, Enzyme Microb Technol, 2000, 26(9-10): 737～742
[28] 沈亚领, 李爽, 迟莉丽, 雷肇祖. 谷胱甘肽的应用与生产. 工业微生物, 2000, 30(2): 41-45
[29] 范崇东.酵母细胞中谷胱甘肽的分离纯化.2004.3
[30] Ellman G L. A Calorimetric Method for Determining Low Concenrrations of Mercaptans[J].Arch Of Biochem &Biophys, 1959, 82:70～77.
[31] 林加涵,魏文玲,彭宣宪,Experiment Of Modern Biology,北京:高等教育出版社,Springer,2001.10:36～38
[32] Munir Cheryan. Ultrafiltration Handbook.Technomic publishing CO. INC, 1986
[33] Marcel Mulder. Basic Priciples of Membrane Technology.清华大学出版社,1999.
[34] Butterworth T A, Wang D I C, In: Terui G, ed, Fermentation Technology today[J].Soxc. Ferment. Technol. Japan, 1972, 19.
[35] Tetsuya Kawakita, Tadako Ogura, Masaru Saeki And Hideo Hayasaki, Selectivity Coefficients Of Amion Acids For The Ammonium Ion On A Strong Cation Exchange Resin, Agic.Biol.Chem,1990, 54(1): 1～8
[36] 钱庭宝,刘维琳,离子交换树脂应用手册,天津:南开大学出版社,1989
[37] 孙彦,生物分离工程,北京:化学工业出版社,1998.10
[38] 赵利,王璋,许时婴,大孔吸附树脂对酪蛋白非磷酸肽吸附性的研究,食品工业技术,2002,12:28～31
[39] Jung.G, Breitmaier, E, and Voelter, W, Dissoziationgs gleichgewichte won Glutathione, Eine Fourier- Transform -13C-NMR sprktroskopische untersuchung, Eur.J.Biochem, 1972, 24:438～444
[40] 姜志新,湛竞清,宋正孝编著,离子交换分离工程,天津大学出版社,1992
[41] Uotani Osamu, Tani Hideo, Mizushima Shinji Et Al. Purification Of Glutathione, Patent Of Japan, Pn: 52～073821, 1977.06.21
[42] Kimura Hikari, Inoue Yoshiharu, Kobayashi Susumu, Production Of Glutathione, Patent Of Japan, Pn:08-070884, 1996,03,19
[43] 氨基酸发酵工艺,中国轻工业出版社,1989
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