高产谷胱甘肽新菌种的选育及其工艺条件的研究
|
摘要
以乙硫氨酸和氯化锌为筛选标记，对出发菌株SL21通过紫外线诱变处理，运用推理育种技术，选育到一株抗乙硫氨酸和氯化锌的突变株HY78，该突变株经摇瓶发酵谷胱甘肽的产量为76.0mg／L，干细胞谷胱甘肽的含量为10.0mg／g，分别比出发菌株SL21提高268.9％和263.0％。该菌株经10次传代培养，谷胱甘肽产量仅下降了13.0％，是一株性状较稳定的优良菌株。
运用单因素实验和正交实验设计对突变株HY78产谷胱甘肽的发酵条件进行了研究，研究表明该突变株产谷胱甘肽的最佳发酵条件为培养温度30℃、初始pH7.0、摇床转速180r／min、接种龄18h、接种量10％，摇瓶装液量30ml／250ml；最佳培养基组成是：糖蜜40g，牛肉膏20g，Cys·HCl 1.5g，MgSO_4·7H_2O 9g，KH_2PO_4 1g，ZnSO_45mg，水1000ml，结果表明突变株HY78在该优化条件下摇瓶发酵，发酵液中谷胱甘肽的含量可达182.6mg／L，较优化前提高了140.3％。
在摇瓶培养的基础上，通过分批培养和补料分批培养，在3.7L自动生物发酵罐中对突变株HY78生物合成谷胱甘肽进行了扩大培养，结果表明分批培养较摇瓶培养谷胱甘肽的产量下降了15.9％，仅有157.6mg／L，而在补料分批培养中，由于流加了培养液，谷胱甘
浙江工必大学硕士格义
肤的产量较分批培养提高了59.7%，为251.7mg/L。
A Ethionine- and Zinc Chloride-resistant mutant HY78 was obtained from its parent strain SL21 by UV treatment and rational screening. The glutathione productivity of the mutant reached 76.0mg/L by flask culture, which was 268.9% higher than the parent strain. And the glutathione content in the dried cells reached 10.0mg/L, which was 263.0% higher than the parent strain. A descend of only 13.0% in the glutathione yield of the mutant was observed after ten times of subculture. Therefore, the obtained mutant is a stable strain.
Studies were carried out on the fermentation condition through Single Factor design and Orthogonal Test design, which showed that the optimum fermentation condition was culture temperature 30 ℃, initial pH7.0, rotate speed 180r/min, inoculum age 18h, inoculum size 10%, medium volumes 30ml/250ml. And the optimum medium composition was molasses 40g, beef extract 20g, Cys HCl 1.5g, MgSO4 7H2O 9g, KH2PO4 1g , ZnSO4 5mg, water 1000ml. In this condition the glutathione content came to 182.6mg/L, which was 140.3% higher than that of non-optimized medium.
In a 3.7L scale batch bioreactor, the rules of glutathione fermentation were studied by batch and fed-batch operation. The result showed that the glutathione content in the batch culture was only 157.mg/L, which was 15.9% lower than shake flask culture. While the glutathione content in the fed-batch culture reached
251.7mg/L, which was 59.7% higher than batch culture.
运用单因素实验和正交实验设计对突变株HY78产谷胱甘肽的发酵条件进行了研究，研究表明该突变株产谷胱甘肽的最佳发酵条件为培养温度30℃、初始pH7.0、摇床转速180r／min、接种龄18h、接种量10％，摇瓶装液量30ml／250ml；最佳培养基组成是：糖蜜40g，牛肉膏20g，Cys·HCl 1.5g，MgSO_4·7H_2O 9g，KH_2PO_4 1g，ZnSO_45mg，水1000ml，结果表明突变株HY78在该优化条件下摇瓶发酵，发酵液中谷胱甘肽的含量可达182.6mg／L，较优化前提高了140.3％。
在摇瓶培养的基础上，通过分批培养和补料分批培养，在3.7L自动生物发酵罐中对突变株HY78生物合成谷胱甘肽进行了扩大培养，结果表明分批培养较摇瓶培养谷胱甘肽的产量下降了15.9％，仅有157.6mg／L，而在补料分批培养中，由于流加了培养液，谷胱甘
浙江工必大学硕士格义
肤的产量较分批培养提高了59.7%，为251.7mg/L。
A Ethionine- and Zinc Chloride-resistant mutant HY78 was obtained from its parent strain SL21 by UV treatment and rational screening. The glutathione productivity of the mutant reached 76.0mg/L by flask culture, which was 268.9% higher than the parent strain. And the glutathione content in the dried cells reached 10.0mg/L, which was 263.0% higher than the parent strain. A descend of only 13.0% in the glutathione yield of the mutant was observed after ten times of subculture. Therefore, the obtained mutant is a stable strain.
Studies were carried out on the fermentation condition through Single Factor design and Orthogonal Test design, which showed that the optimum fermentation condition was culture temperature 30 ℃, initial pH7.0, rotate speed 180r/min, inoculum age 18h, inoculum size 10%, medium volumes 30ml/250ml. And the optimum medium composition was molasses 40g, beef extract 20g, Cys HCl 1.5g, MgSO4 7H2O 9g, KH2PO4 1g , ZnSO4 5mg, water 1000ml. In this condition the glutathione content came to 182.6mg/L, which was 140.3% higher than that of non-optimized medium.
In a 3.7L scale batch bioreactor, the rules of glutathione fermentation were studied by batch and fed-batch operation. The result showed that the glutathione content in the batch culture was only 157.mg/L, which was 15.9% lower than shake flask culture. While the glutathione content in the fed-batch culture reached
251.7mg/L, which was 59.7% higher than batch culture.
引文
[1]Meister, A., Anderson, M. Glutathione. ANNU, REV, Biochem. 1983,52:711-760.
[2]Hopkins F G. On an Autoxidisable Constituent of the Cell. Biochem J, 1921,15:286-305.
[3]Du Vigneaud, V., Miller, G., J. Bio. Chem., 1936,116:469-416.
[4]Harington C R, Mead T H. The Synthesis of Glutathione. Biochem. J. 1935,29:1602-1611.
[5]张洪渊．生物化学教程．成都：四川大学出版社，1988．
[6]郑云朗．谷胱甘肽的生物学功能．生物学通报，1995，30(5)：22-24．
[7]程元凯．谷胱甘肽的解毒作用与毒性代谢物．生物化学与生物物理进展，1994，21(5)：395-399．
[8]张平，孟宪钧，宋旭华，李新建．多器官衰竭端时谷胱甘肽代谢变化与细胞损伤的实验研究．中华医学杂志，1987，69(12)，713—714．
[9]Ziegler, D., Role of Reversible Oxidation-Reduction of Enzyme. Thiols-Disulfides in Metabolic Regulation. ANN. Rev. Biochem. 1985,54:305-309
[10]Kosower, N., S., Kosower, E., M., The glutathione status of cells. Int. Rev. Cytol., 1978,54:109-160.
[11]王正刚．啤酒酵母深加工及在食品医药工业中的应用．水解工业，1999，3：37-39．
[12]刘振玉．谷胱甘肽的研究与应用．生命的化学，1995，15(1)：19-21．
[13]王海鸥．面包酵母中谷胱甘肽对面团流变性质的影响．无锡轻工业大学学报，1999，18(3)：29-32．
[14]胡学智．日本的保健食品．工业微生物，2000，30(3)：44-49．
[15]赵克健．中国化学药品大全．北京：新时代出版社，1990．
[16]胡文铎，崔乃杰，高仲阳．国家基本药物及新特药临床指南．天津：天津科技翻译出版公司，1996．
[17]J. Rudinger, Chem. Commun. 1951,16:214.
[18]郑建仙．功能性食品．北京：中国轻工业出版社，1997．
[19]沈蓓英，江志炜．具有生物活性新型功能性食品添加剂—谷胱甘肽．粮食与油脂，1993(2)：27-32．
[20]曹新志．四川食品工业科技，1996(2)：17-20．
[21]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)：52-57．
[22]赵旭东，魏东芝，俞俊棠．固定化酵母细胞生物合成谷胱甘肽的研究．华东理工大学学报，2000，26(1)：29-32．
[23]Fujio, T., Hayashi, M., Compound from its precursor using the Enzymic Activity of a Bacterium, Eur, Pat. 1985, EP146265.
[24]吴坚平，林建平，岑沛霖．谷胱甘肽的生物合成．化工进展，1999(1)：38-41．
[25]储炬，李友荣．现代工业发酵调控学．北京：化学工业出版社，2002．
[26]Langer R S, Hamilton B K, Gardner C R. Enzymatic Regeneration of ATP, I. Alternative Routes. Atche J. 1976(22): 1079-1082.
[27]Whitesides, G., M.,Siegel, M., Garrelt,P. J. Org. Chem. 1975(40):2516-2519.
[28]Gardner, C., Colton, C. Langer, R. Enzymatic Regeneration of ATP from AMP and ADP: Part Ⅰ, Thermodynamics, Kinetics and process development, Enzyme Engineering, Plenum press,NewYork, 1974(2):209-217.
[29]Tochikura, T., Kariya, Y., Amino Acid and Nucleic Acid. 1974(29):59-74
[30]Fahey R C, Neuton G L. "Occurrence of low molecular thiols in biological systems"in Functions of Glutathione. Biochemical, Pysiological, Toxicological and Chinocal Aspect. New York:Larrson S, 1983.
[31]詹谷宇，田萍，刘卫东．酵母菌生物合成谷胱甘肽．药学学报，1990，25(7)：494-499．
[32]Kono G, Harada M, Sugisaki K. High Glutathione-containing Yeast. JP 1976-4-13.
[33]Ikeno Y, Tanno K, Omori Ⅰ. Glutathione. JP87296, 1976-1-16.
[34]施碧红，黄建忠，施巧琴．啤酒酵母(Saccharamyces cerevisiae)M-05变株合成谷胱甘肽的研究Ⅰ．菌种的选育．福建师范大学学报(自然科学版)，1996，12(4)：91-95．
[35]Lang-Hinrichs, Christine, Jansen-Weismann. Zink-resistent Hefen zur Herstellung von Glutathion. 1993, DE4219381.
[36]Murata K., Kimura A. Some Properties of Glutathione Biosyntesis-deficient Mutant of E. coli.B.J. Gen. Microbiol. 1982,128(5):1047-1052.
[37]Gushima H, Miya T, Mutara K. Construction of Glutathione-producing Strains of E. coliB by Reombinant DNA Techiques. J. Appl. Biochem. 1983,5(1):43-52.
[38]Watanabe K, Yamata Y, Murata K. Glutathione Producing by E. coli Cells with Hybrid
Plasmid Containing Tandemly Polymerized Genes for Glutalhione Synthase. Appl. Microbiol. Biotechnol. 1986,24(3):375-378.
[39]陈坚，李寅．发酵过程优化原理与实践．北京：化学工业出版社，2002．
[40]Chi-Hsien Liu, Chin-Fa Hwang, Chii-Cherng Liao. Medium Optimization for Glutathione Production by Saccharamyces cerevisiae. Process Biochemistry, 1999,34:17-23.
[41]施碧红，黄建忠，施巧琴．啤酒酵母(Saccharamyces cerevisiae)M-05变株合成谷胱甘肽的研究Ⅱ发酵条件的研究．福建师范大学学报(自然科学版)，2000，16(4)：74-78．
[42]李寅，陈坚，周楠迪．环境条件及摇瓶补糖策略对谷胱甘肽发酵的影响．生物工程学报．1998，14(2)：147-152．
[43]陶锐，吴梧桐，许激扬．酶法制备谷胱甘肽工艺的研究．药物生物技术，1999，6(4)：203-207．
[44]Alfafara, C. G.., Kanda A., Shioi, T. Effect of Amino Acids on Glutathione Production by Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 1992, 36(4):538-540.
[45]Miyamoto，S．，Mitsuba，N．，Takemoto，H．Glutathione by cultivating yeast on Methanol．公开特许公报，1977，77．156994．
[46]Alfafara C G, Miura K, Shimizu H. Cysteine Addition Strategy for Glutathione Production in Fed-batch Culture of Saccharomyces cerevisiae. Appl. Microbio. Biotech. 1992, 37:141-146.
[47]李寅，陈坚，阮文权，伦世仪．氨基酸对面包酵母发酵生产谷胱甘肽影响的初步研究．第八届化学工程论文报告会论文集，1996，pp1332—1335．
[48]李寅，陈坚，周楠迪，伦世仪．氨基酸和酵母膏对谷胱甘肽发酵的影响．中国医药工业杂志，1998，29(12)：537-542．
[49]Watanabe, K., Yamano, Y., Murata, K., Glutathione production by Escherichia coli. Cells with Hybrid plasmid containing Tandemly polymerized Genes for glutathione synthetase. Appl.Microbiol. Biotechnol. 1986,24:375-378.
[50]Takesue，H．，Fujii，K．，Miura，Y．Glutathione．公开特许公报，1979，79-86691．
[51]Hirakawa，K．Lactic acid for high yield glutathione production by the yeasts．公开特许公报1985，60-244284．
[52]Yamada, Y., Tani Y., Kamihara, T. Production of extarcellular glutathione by andida.tropicalis pk233. J. Gen. Microbiol, 1984,130(12):3275-3278.
[53]Alfafara, C., Miura, K., Shimizu, H., Fuzzy control of Ethanol concentration and its application to maximum glutathione production in yeast fed-batch culture. Biotechnol,
Bioeng. 1993,41(4):493-501.
[54]Sakato K, Tanaka H. Advanced Control of Glutathione Fermentation. Process Biotechnology and Bioengineering. 1992(40):904-912.
[55]李寅，陈坚，伦世仪．高密度培养工程菌生产谷胱甘肽．中国医药杂志，1999，30(1)：1-4．
[56]周楠迪，李寅，陈坚．从酵母中提取谷胱甘肽的初步研究．生物技术，1997，7(4)：30-33．
[57]曹新志．谷胱甘肽的分离和干燥方法．广州食品工业科技，1996，12(2)：44-45．
[58]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．
[59]朱自强，关怡新，李勉．双水相分配技术提取生物小分子的进展．化工进展，1996(4)：29-34．
[60]梅乐和，林东强，朱自强．双水相分配结合温度诱导相分离从酵母中提取谷胱甘肽．化工学报，1998，49(4)：470-474．
[61]周楠迪，李寅，陈坚．从酵母中提取谷胱甘肽的初步研究．生物技术，1997，7(4)：30-33．
[62]Zouhair Aherrahrou, XuJiyang, Wu Wutong. Determination of Glutathione in Biosynthesis Reaction by Paper Chromatography. Journal of China Pharmaceutical University, 1997,28(3):176-179.
[63]王荣民，曹新志，萧刚．谷胱甘肽测定方法初探．食品与发酵工业，1992(6)：34-37．
[64]程敬君，匡培根，张凤英．高效液相色谱—电化学检测法测定鼠脑中透析液中谷胱甘肽和半胱氨酸．色谱，1998，16(2)：167-169．
[65]Brent A., Tetri, Joseph F. Glutathione Measurement by High-performance Liquid Chromatorgraphy Separation and Fluorometry Detection of Glutathione-orthopthalaldehyde Adduct. Anal. Bioehem. 1989, 179:236-241
[66]夏弈明．总谷胱甘肽的测定方法．营养学报，1990，12(1)：18-22．
[67]张少辉．谷胱甘肽片的紫外分光光度测定，辽宁大学学报，1992，19(4)：66-69．
[68]Gowda. Micro. Chem., 1982,27:87-96.
[69]曹新志，陈彦．荧光法测定黄瓜中的谷胱甘肽．资源开发与市场，2000，16(5)：272-273．
[70]曹新志，陈彦．荧光法测定小麦胚中的谷胱甘肽．粮食与饲料工业．2001(11)：46-47．
[71]徐汉生，奚农葆．“抗氧化物之王”谷胱甘肽的研究与开发，湖北化工，2000，5：1-3
[72]沈萍，范广容，李广武．微生物实验．北京：高等教育出版社，1999．
[73]R．E．布坎南，N．E．吉本斯．伯杰细菌鉴定手册．北京：科学出版社，1984．
[74]陈思妘，萧熙佩．酵母生物化学．济南：山东科学技术出版社，1990．
[75]周德庆．微生物学教程．北京：高等教育出版社，1993．
[76]周润兰，喻胜华．应用概率统计．北京：科学出版社，1999．
[77]范代娣．通气与放大．北京：化学工业出版社，2003．
[78]林瑾琳，邱志成．味精生产技术．南昌：江西人民出版社，1985．
[2]Hopkins F G. On an Autoxidisable Constituent of the Cell. Biochem J, 1921,15:286-305.
[3]Du Vigneaud, V., Miller, G., J. Bio. Chem., 1936,116:469-416.
[4]Harington C R, Mead T H. The Synthesis of Glutathione. Biochem. J. 1935,29:1602-1611.
[5]张洪渊．生物化学教程．成都：四川大学出版社，1988．
[6]郑云朗．谷胱甘肽的生物学功能．生物学通报，1995，30(5)：22-24．
[7]程元凯．谷胱甘肽的解毒作用与毒性代谢物．生物化学与生物物理进展，1994，21(5)：395-399．
[8]张平，孟宪钧，宋旭华，李新建．多器官衰竭端时谷胱甘肽代谢变化与细胞损伤的实验研究．中华医学杂志，1987，69(12)，713—714．
[9]Ziegler, D., Role of Reversible Oxidation-Reduction of Enzyme. Thiols-Disulfides in Metabolic Regulation. ANN. Rev. Biochem. 1985,54:305-309
[10]Kosower, N., S., Kosower, E., M., The glutathione status of cells. Int. Rev. Cytol., 1978,54:109-160.
[11]王正刚．啤酒酵母深加工及在食品医药工业中的应用．水解工业，1999，3：37-39．
[12]刘振玉．谷胱甘肽的研究与应用．生命的化学，1995，15(1)：19-21．
[13]王海鸥．面包酵母中谷胱甘肽对面团流变性质的影响．无锡轻工业大学学报，1999，18(3)：29-32．
[14]胡学智．日本的保健食品．工业微生物，2000，30(3)：44-49．
[15]赵克健．中国化学药品大全．北京：新时代出版社，1990．
[16]胡文铎，崔乃杰，高仲阳．国家基本药物及新特药临床指南．天津：天津科技翻译出版公司，1996．
[17]J. Rudinger, Chem. Commun. 1951,16:214.
[18]郑建仙．功能性食品．北京：中国轻工业出版社，1997．
[19]沈蓓英，江志炜．具有生物活性新型功能性食品添加剂—谷胱甘肽．粮食与油脂，1993(2)：27-32．
[20]曹新志．四川食品工业科技，1996(2)：17-20．
[21]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)：52-57．
[22]赵旭东，魏东芝，俞俊棠．固定化酵母细胞生物合成谷胱甘肽的研究．华东理工大学学报，2000，26(1)：29-32．
[23]Fujio, T., Hayashi, M., Compound from its precursor using the Enzymic Activity of a Bacterium, Eur, Pat. 1985, EP146265.
[24]吴坚平，林建平，岑沛霖．谷胱甘肽的生物合成．化工进展，1999(1)：38-41．
[25]储炬，李友荣．现代工业发酵调控学．北京：化学工业出版社，2002．
[26]Langer R S, Hamilton B K, Gardner C R. Enzymatic Regeneration of ATP, I. Alternative Routes. Atche J. 1976(22): 1079-1082.
[27]Whitesides, G., M.,Siegel, M., Garrelt,P. J. Org. Chem. 1975(40):2516-2519.
[28]Gardner, C., Colton, C. Langer, R. Enzymatic Regeneration of ATP from AMP and ADP: Part Ⅰ, Thermodynamics, Kinetics and process development, Enzyme Engineering, Plenum press,NewYork, 1974(2):209-217.
[29]Tochikura, T., Kariya, Y., Amino Acid and Nucleic Acid. 1974(29):59-74
[30]Fahey R C, Neuton G L. "Occurrence of low molecular thiols in biological systems"in Functions of Glutathione. Biochemical, Pysiological, Toxicological and Chinocal Aspect. New York:Larrson S, 1983.
[31]詹谷宇，田萍，刘卫东．酵母菌生物合成谷胱甘肽．药学学报，1990，25(7)：494-499．
[32]Kono G, Harada M, Sugisaki K. High Glutathione-containing Yeast. JP 1976-4-13.
[33]Ikeno Y, Tanno K, Omori Ⅰ. Glutathione. JP87296, 1976-1-16.
[34]施碧红，黄建忠，施巧琴．啤酒酵母(Saccharamyces cerevisiae)M-05变株合成谷胱甘肽的研究Ⅰ．菌种的选育．福建师范大学学报(自然科学版)，1996，12(4)：91-95．
[35]Lang-Hinrichs, Christine, Jansen-Weismann. Zink-resistent Hefen zur Herstellung von Glutathion. 1993, DE4219381.
[36]Murata K., Kimura A. Some Properties of Glutathione Biosyntesis-deficient Mutant of E. coli.B.J. Gen. Microbiol. 1982,128(5):1047-1052.
[37]Gushima H, Miya T, Mutara K. Construction of Glutathione-producing Strains of E. coliB by Reombinant DNA Techiques. J. Appl. Biochem. 1983,5(1):43-52.
[38]Watanabe K, Yamata Y, Murata K. Glutathione Producing by E. coli Cells with Hybrid
Plasmid Containing Tandemly Polymerized Genes for Glutalhione Synthase. Appl. Microbiol. Biotechnol. 1986,24(3):375-378.
[39]陈坚，李寅．发酵过程优化原理与实践．北京：化学工业出版社，2002．
[40]Chi-Hsien Liu, Chin-Fa Hwang, Chii-Cherng Liao. Medium Optimization for Glutathione Production by Saccharamyces cerevisiae. Process Biochemistry, 1999,34:17-23.
[41]施碧红，黄建忠，施巧琴．啤酒酵母(Saccharamyces cerevisiae)M-05变株合成谷胱甘肽的研究Ⅱ发酵条件的研究．福建师范大学学报(自然科学版)，2000，16(4)：74-78．
[42]李寅，陈坚，周楠迪．环境条件及摇瓶补糖策略对谷胱甘肽发酵的影响．生物工程学报．1998，14(2)：147-152．
[43]陶锐，吴梧桐，许激扬．酶法制备谷胱甘肽工艺的研究．药物生物技术，1999，6(4)：203-207．
[44]Alfafara, C. G.., Kanda A., Shioi, T. Effect of Amino Acids on Glutathione Production by Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 1992, 36(4):538-540.
[45]Miyamoto，S．，Mitsuba，N．，Takemoto，H．Glutathione by cultivating yeast on Methanol．公开特许公报，1977，77．156994．
[46]Alfafara C G, Miura K, Shimizu H. Cysteine Addition Strategy for Glutathione Production in Fed-batch Culture of Saccharomyces cerevisiae. Appl. Microbio. Biotech. 1992, 37:141-146.
[47]李寅，陈坚，阮文权，伦世仪．氨基酸对面包酵母发酵生产谷胱甘肽影响的初步研究．第八届化学工程论文报告会论文集，1996，pp1332—1335．
[48]李寅，陈坚，周楠迪，伦世仪．氨基酸和酵母膏对谷胱甘肽发酵的影响．中国医药工业杂志，1998，29(12)：537-542．
[49]Watanabe, K., Yamano, Y., Murata, K., Glutathione production by Escherichia coli. Cells with Hybrid plasmid containing Tandemly polymerized Genes for glutathione synthetase. Appl.Microbiol. Biotechnol. 1986,24:375-378.
[50]Takesue，H．，Fujii，K．，Miura，Y．Glutathione．公开特许公报，1979，79-86691．
[51]Hirakawa，K．Lactic acid for high yield glutathione production by the yeasts．公开特许公报1985，60-244284．
[52]Yamada, Y., Tani Y., Kamihara, T. Production of extarcellular glutathione by andida.tropicalis pk233. J. Gen. Microbiol, 1984,130(12):3275-3278.
[53]Alfafara, C., Miura, K., Shimizu, H., Fuzzy control of Ethanol concentration and its application to maximum glutathione production in yeast fed-batch culture. Biotechnol,
Bioeng. 1993,41(4):493-501.
[54]Sakato K, Tanaka H. Advanced Control of Glutathione Fermentation. Process Biotechnology and Bioengineering. 1992(40):904-912.
[55]李寅，陈坚，伦世仪．高密度培养工程菌生产谷胱甘肽．中国医药杂志，1999，30(1)：1-4．
[56]周楠迪，李寅，陈坚．从酵母中提取谷胱甘肽的初步研究．生物技术，1997，7(4)：30-33．
[57]曹新志．谷胱甘肽的分离和干燥方法．广州食品工业科技，1996，12(2)：44-45．
[58]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．
[59]朱自强，关怡新，李勉．双水相分配技术提取生物小分子的进展．化工进展，1996(4)：29-34．
[60]梅乐和，林东强，朱自强．双水相分配结合温度诱导相分离从酵母中提取谷胱甘肽．化工学报，1998，49(4)：470-474．
[61]周楠迪，李寅，陈坚．从酵母中提取谷胱甘肽的初步研究．生物技术，1997，7(4)：30-33．
[62]Zouhair Aherrahrou, XuJiyang, Wu Wutong. Determination of Glutathione in Biosynthesis Reaction by Paper Chromatography. Journal of China Pharmaceutical University, 1997,28(3):176-179.
[63]王荣民，曹新志，萧刚．谷胱甘肽测定方法初探．食品与发酵工业，1992(6)：34-37．
[64]程敬君，匡培根，张凤英．高效液相色谱—电化学检测法测定鼠脑中透析液中谷胱甘肽和半胱氨酸．色谱，1998，16(2)：167-169．
[65]Brent A., Tetri, Joseph F. Glutathione Measurement by High-performance Liquid Chromatorgraphy Separation and Fluorometry Detection of Glutathione-orthopthalaldehyde Adduct. Anal. Bioehem. 1989, 179:236-241
[66]夏弈明．总谷胱甘肽的测定方法．营养学报，1990，12(1)：18-22．
[67]张少辉．谷胱甘肽片的紫外分光光度测定，辽宁大学学报，1992，19(4)：66-69．
[68]Gowda. Micro. Chem., 1982,27:87-96.
[69]曹新志，陈彦．荧光法测定黄瓜中的谷胱甘肽．资源开发与市场，2000，16(5)：272-273．
[70]曹新志，陈彦．荧光法测定小麦胚中的谷胱甘肽．粮食与饲料工业．2001(11)：46-47．
[71]徐汉生，奚农葆．“抗氧化物之王”谷胱甘肽的研究与开发，湖北化工，2000，5：1-3
[72]沈萍，范广容，李广武．微生物实验．北京：高等教育出版社，1999．
[73]R．E．布坎南，N．E．吉本斯．伯杰细菌鉴定手册．北京：科学出版社，1984．
[74]陈思妘，萧熙佩．酵母生物化学．济南：山东科学技术出版社，1990．
[75]周德庆．微生物学教程．北京：高等教育出版社，1993．
[76]周润兰，喻胜华．应用概率统计．北京：科学出版社，1999．
[77]范代娣．通气与放大．北京：化学工业出版社，2003．
[78]林瑾琳，邱志成．味精生产技术．南昌：江西人民出版社，1985．