高产海藻糖菌株的诱变育种、培养基优化及提取纯化的研究
|
摘要
海藻糖是一种非还原性双糖，其独特的保护功能能使生物大分子(如蛋白质、核酸等)在外界高温、脱水等恶劣环境下免受伤害，近几年成为中外科学家研究的热点。
本文以安琪活性干酵母复壮后的鲜酵母为出发菌株，在确定测定方法和摇瓶培养观察的前提下，分别对其进行紫外、化学和微波诱变。在紫外诱变过程中，对出发菌株共进行了三轮紫外诱变，照射时间是40秒～60秒，每轮照射后，从15株菌中筛选正突变高的菌株进行下一轮的诱变。在进行的第三轮诱变时筛选到一株高产菌株，其海藻糖的产量为19.52g／100g干酵母。化学诱变联合使用了两种低毒性诱变剂，两种诱变剂的最佳配比是：5—溴尿嘧啶300μ mol／L和盐酸平阳霉素5μ mol／L，诱变菌株中最高海藻糖产量为15.98g／100g干酵母。经微波诱变后获得的高产菌株海藻糖的产量为17.01g／100g干酵母。
本文在诱变的同时对酵母菌的操作条件和培养基也进行了优化，在找到海藻糖发酵体系操作最佳条件即最适摇床转速为240r／min、最适温度为35℃的前提下，对培养基中的碳源浓度、氮源浓度、氮碳比、无机盐和微量元素的组成和含量也进行了探讨，从而找出最优培养基配方。
对从活性干酵母中提取海藻糖的工艺进行了研究。作者用蒸馏水从活性干酵母中提取海藻糖的最佳条件是：温度80℃、料液比1：20(干酵母的质量和提取剂的体积比，g／ml)，提取1个小时，海藻糖的产量是16.32g海藻糖／100g干酵母，pH值对提取率没有影响。并比较了不同提取剂对海藻糖提取的影响，其中用1％的氧化钙溶液提取，产量可达21.54g海藻糖／100g干酵母。
在研究从啤酒废酵母中提取海藻糖的工艺时，对啤酒废酵母进行了特殊的处理，即把废酵母在烘箱中烘干一定时间后，用粉碎机制成废酵母粉。并比较了传统提取方法(用冷凝回流装置提取)和索式提取法的产量。实验表明，用索式提取海藻糖的产量约是传统提取产量的3倍，提取工艺为：料液比3：50(废酵母粉的质量和提取剂的体积比，g／ml)，恒温水浴温度为80℃，真空度0.09Mpa下，提取一小时，产量为6.505g海藻糖／kg干废酵母，而所测啤酒废酵母中海藻糖的总含量是7.98g海藻糖／kg干废酵母。
研究了提取液的纯化、精制及结晶工艺。用碱金属除蛋白、活性炭脱色、离子交换树脂除杂离子和进一步脱色。离子交换树脂采用001×7型阳离子交换树脂和201×7型阴离子交换树脂组成的混合床，阴阳离子交换树脂的体积比为2：1。精制后的提取液浓缩到质量百分数为15％左右，用4倍体积的无水乙醇结晶一周，可得柱状晶体，结晶得率为80％。
Trehalose is a nonreducing disaccharide of glucose.It has an excellent ability to protect biological cells(such as protein,nucleic acid,etc)from damage caused by drying and dehydration,so it has been the studing focus for scientists in the recent years.
In this paper taking AnQi active fresh yeast after rejuvenation as starting strains,mutation was obtained by UV, chemical reagents and microwave heating on the base of making sure the measuring method and observation of fermentation culture.During UV mutation process,there were three turns of UV-mutation on the starting strains in all and the mutation time was 40-60 seconds.After one turn of mutation , strains with high mutation ratio were selected from fifteen strains to receive the next turn for mutation.In the three turns of mutation a high yielding trehalose strain was screened and its trehalose yeilds was 19.52g /100g dry yeast.During chemical mutation two chemical reagents with low toxicity were used together and the best proportion of the two reagents were described as follow: 5-Bu was 300 μ mol/L and Pingyangmycin hydrochloride was 5 μ mol/L. Of the chemical mutation the highest yielding of strain was 15.98g / 100g dry yeast.Through microwave mutation the highest yielding of strain was 17.01g / 100g dry yeast.
At the same time the operation conditions and cultrue medium of the yeast strain were also optimized.The best operation conditions for trehalose fermentation system was described as follows: the best ration speed was 240r/min and temperature was 35℃.On this base,the carbon source concentration nitrogen source concentration , proportion of carbon and nitrogen source concentration, constitute and content of inorganic salt and microelement also were investigated to find the best culture medium in which the yeast strain synthesize trehalose furthest.
Technology of extraction trehalose from active dry yeast was studied.Taking distilled water as extraction solvent,the maximum extraction ratio of trehalose from active yeast that was 16.32gtrehalose / 100g dry yesat was obtained when the extraction temperature was 80℃, ratio of dry yeast weight and distilled water volum was 1:20 and the extraction time was one hour and pH had no effect on extraction ratio of trehalose.By comparing the effects of different extraction solvent on the extraction ratio of trehalose, high yield of trehalose ,21.54g trehaolse /100g dry yeast, could be obtained when 1 % GaO solution as extraction solvent.
To study the technology of extraction trehalose from waste beer yeast,the material was dealed with in a peculiar way in which waste yeast was heated in oven for a period of time then made into powder and compared traditional extraction method with Suoshi extraction
method.The experimental results in this paper showed that the extraction ratio of trehalose by Suoshi method was almost three times as much as by traditional method.The extraction technology was described as follow: ratio of waste yeast weight and distilled water volum was 3:50,temperature was 80℃, degree of vacuum was 0.09Mpa, the time of extraction was one hour and the yield of trehalose was 6.505gtrehalose / kg dry waste yeast,however the all content of trehalose in waste beer yeast was 7.98g trehalose/kg dry waste yeast.
Purification , refining and crystallization of prosess were also studied.Alkali metals were used to remove protein, active carbon was used to remove pigments and ion exchange resin to remove motley ions and pigments further. 001×7 cation exchange resin and 201×7 anion exchange resin composed the mixed ion exchange column and the volume proportion of 201×7 anion exchange resin and 001×7 cation exchange resin was 2:1.When extraction solvent containing trehalose after refining was condensed to almost mass proporation 15 % and crystalled in ethanol of four times as much as extraction solvent volum for one week ,crystal of trehalose with prismatic shape could be obtained and the crystal ratio was 80%.
本文以安琪活性干酵母复壮后的鲜酵母为出发菌株，在确定测定方法和摇瓶培养观察的前提下，分别对其进行紫外、化学和微波诱变。在紫外诱变过程中，对出发菌株共进行了三轮紫外诱变，照射时间是40秒～60秒，每轮照射后，从15株菌中筛选正突变高的菌株进行下一轮的诱变。在进行的第三轮诱变时筛选到一株高产菌株，其海藻糖的产量为19.52g／100g干酵母。化学诱变联合使用了两种低毒性诱变剂，两种诱变剂的最佳配比是：5—溴尿嘧啶300μ mol／L和盐酸平阳霉素5μ mol／L，诱变菌株中最高海藻糖产量为15.98g／100g干酵母。经微波诱变后获得的高产菌株海藻糖的产量为17.01g／100g干酵母。
本文在诱变的同时对酵母菌的操作条件和培养基也进行了优化，在找到海藻糖发酵体系操作最佳条件即最适摇床转速为240r／min、最适温度为35℃的前提下，对培养基中的碳源浓度、氮源浓度、氮碳比、无机盐和微量元素的组成和含量也进行了探讨，从而找出最优培养基配方。
对从活性干酵母中提取海藻糖的工艺进行了研究。作者用蒸馏水从活性干酵母中提取海藻糖的最佳条件是：温度80℃、料液比1：20(干酵母的质量和提取剂的体积比，g／ml)，提取1个小时，海藻糖的产量是16.32g海藻糖／100g干酵母，pH值对提取率没有影响。并比较了不同提取剂对海藻糖提取的影响，其中用1％的氧化钙溶液提取，产量可达21.54g海藻糖／100g干酵母。
在研究从啤酒废酵母中提取海藻糖的工艺时，对啤酒废酵母进行了特殊的处理，即把废酵母在烘箱中烘干一定时间后，用粉碎机制成废酵母粉。并比较了传统提取方法(用冷凝回流装置提取)和索式提取法的产量。实验表明，用索式提取海藻糖的产量约是传统提取产量的3倍，提取工艺为：料液比3：50(废酵母粉的质量和提取剂的体积比，g／ml)，恒温水浴温度为80℃，真空度0.09Mpa下，提取一小时，产量为6.505g海藻糖／kg干废酵母，而所测啤酒废酵母中海藻糖的总含量是7.98g海藻糖／kg干废酵母。
研究了提取液的纯化、精制及结晶工艺。用碱金属除蛋白、活性炭脱色、离子交换树脂除杂离子和进一步脱色。离子交换树脂采用001×7型阳离子交换树脂和201×7型阴离子交换树脂组成的混合床，阴阳离子交换树脂的体积比为2：1。精制后的提取液浓缩到质量百分数为15％左右，用4倍体积的无水乙醇结晶一周，可得柱状晶体，结晶得率为80％。
Trehalose is a nonreducing disaccharide of glucose.It has an excellent ability to protect biological cells(such as protein,nucleic acid,etc)from damage caused by drying and dehydration,so it has been the studing focus for scientists in the recent years.
In this paper taking AnQi active fresh yeast after rejuvenation as starting strains,mutation was obtained by UV, chemical reagents and microwave heating on the base of making sure the measuring method and observation of fermentation culture.During UV mutation process,there were three turns of UV-mutation on the starting strains in all and the mutation time was 40-60 seconds.After one turn of mutation , strains with high mutation ratio were selected from fifteen strains to receive the next turn for mutation.In the three turns of mutation a high yielding trehalose strain was screened and its trehalose yeilds was 19.52g /100g dry yeast.During chemical mutation two chemical reagents with low toxicity were used together and the best proportion of the two reagents were described as follow: 5-Bu was 300 μ mol/L and Pingyangmycin hydrochloride was 5 μ mol/L. Of the chemical mutation the highest yielding of strain was 15.98g / 100g dry yeast.Through microwave mutation the highest yielding of strain was 17.01g / 100g dry yeast.
At the same time the operation conditions and cultrue medium of the yeast strain were also optimized.The best operation conditions for trehalose fermentation system was described as follows: the best ration speed was 240r/min and temperature was 35℃.On this base,the carbon source concentration nitrogen source concentration , proportion of carbon and nitrogen source concentration, constitute and content of inorganic salt and microelement also were investigated to find the best culture medium in which the yeast strain synthesize trehalose furthest.
Technology of extraction trehalose from active dry yeast was studied.Taking distilled water as extraction solvent,the maximum extraction ratio of trehalose from active yeast that was 16.32gtrehalose / 100g dry yesat was obtained when the extraction temperature was 80℃, ratio of dry yeast weight and distilled water volum was 1:20 and the extraction time was one hour and pH had no effect on extraction ratio of trehalose.By comparing the effects of different extraction solvent on the extraction ratio of trehalose, high yield of trehalose ,21.54g trehaolse /100g dry yeast, could be obtained when 1 % GaO solution as extraction solvent.
To study the technology of extraction trehalose from waste beer yeast,the material was dealed with in a peculiar way in which waste yeast was heated in oven for a period of time then made into powder and compared traditional extraction method with Suoshi extraction
method.The experimental results in this paper showed that the extraction ratio of trehalose by Suoshi method was almost three times as much as by traditional method.The extraction technology was described as follow: ratio of waste yeast weight and distilled water volum was 3:50,temperature was 80℃, degree of vacuum was 0.09Mpa, the time of extraction was one hour and the yield of trehalose was 6.505gtrehalose / kg dry waste yeast,however the all content of trehalose in waste beer yeast was 7.98g trehalose/kg dry waste yeast.
Purification , refining and crystallization of prosess were also studied.Alkali metals were used to remove protein, active carbon was used to remove pigments and ion exchange resin to remove motley ions and pigments further. 001×7 cation exchange resin and 201×7 anion exchange resin composed the mixed ion exchange column and the volume proportion of 201×7 anion exchange resin and 001×7 cation exchange resin was 2:1.When extraction solvent containing trehalose after refining was condensed to almost mass proporation 15 % and crystalled in ethanol of four times as much as extraction solvent volum for one week ,crystal of trehalose with prismatic shape could be obtained and the crystal ratio was 80%.
引文
[1] 聂凌鸿．海藻糖的生物保护作用[J]．生命的化学，2001，21(3)：206—208
[2] G. Dupont et R. Locquin. Traite de chimie organique(TomeⅧ). Masson et Cie Editors, 1994
[3] Thevelein. Regulation of trehalose mobilization in fungi. Microbiol Rev, 1984,48: 42-59
[4] Elbein A D. The metabolism of α, α-trehalose. Adv Carbohydrate Chem Biocbem, 1974,30: 227-256
[5] 尤新．淀粉糖品生产与应用手册．北京：中国轻工业出版社，1997：295—311
[6] 任小青．海藻糖的研究进展及其开发前景．郑州工程学院学报，2001，22(1)
[7] 张树珍．海藻糖的研究进展及其应用前景．华南热带农业大学学报，2003，6(3)：22—29
[8] 葛宇，袁勤生．海藻糖对生物活性物质的保护作用机理研究进展．药物生物技术．2002，9(5)：297—300
[9] Crowe J H et al. Science, 1984, 223: 701-703
[10] Colaca C et al. Biotechnology, 1992, 10(9): 701-703
[11] Green J L et al. J Phys Chem. 1989, 93: 2880-2882
[12] Sussich F et al. J Am Chem Soc. 1998, 120: 7893-7899
[13] Timesheff S N, Annu Rev Biophys Biomol Struct. 1993, 22: 67
[14] Singer M A, Lindquist S. The Ying and yang of thermotolerance affecting trehalose[J].Tibtech, 1998, 16: 460
[15] Miller D P. Rational design of protective agents and processes for the stabilization of biologicals[D]. University of Wisconsin-Madison
[16] 简文杰，庞杰．海藻糖的生产及应用．杭州食品科技．2003，1：1—4
[17] 周青峰．海藻糖在食品加工上的应用．中国食品工业．1996(2)：22—23
[18] 王蕾，郑璞，史朝辉．新型食品添加剂海藻糖的生产方法及应用．食品科技，2002(2)：16—17
[19] Koichi Y,I Iiroe Y,I Iiromuk, et al. Protection by trehalose of DNA from radiation damage. Biosci Biotech Biochem. 1997, 61(10:160-161
[20] 戴玉秀，沈义国，周坚．海藻糖对抗逆乳酸菌的保护研究．微生物学通报，2001，8(2)：46-50
[21] 颜栋美，周青峰，姚汝华．海藻糖对载药脂质体保护作用的初步研究．广西大学学报，1998，23(4)：393-396
[22] 李群，袁勤生，李永丰．海藻糖对酶热稳定性保护作用的研究．药物生物技术，1997，4(1)：28-31
[23] 葛宇，赖承光，张文艳，等．海藻糖对保加利亚乳杆菌保护应用的研究．食品科学，2002，23(7)：55-58
[24] 王辉，李克寒，吴兵，等．海藻糖对冻干胸腺肽生物活性保护作用的研究．兰州大学学报，2001，37(6)：72-75
[25] 杨基础，杨小民，等．海藻糖对固定化酶的保护作用．化工学报，2000，51(2)：193-197
[26] 唐传核，葛文光．海藻糖对果胶酶在干燥过程中的保护作用．四川食品与发酵，1998(1—2)：42-44
[27] 陈黎忠，李羲光，边光珠，等．海藻糖对酶冻干品的保护作用．上海医学检测杂志，2001，16(3)：166-167
[28] 陈黎忠，边光珠，李羲光，等．海藻糖对人血清酶抗冻结性保护研究．医学研究通讯，2001，30(1)：
44-46
[29] 权国波．海藻糖在哺乳动物细胞保存中的作用机制及其应用现状．临床输血与检验，2003，5(1)：78—80
[30] 黄成垠，安国瑞，王庆敏，等．海藻糖对医涌诊断工具酶活性保护研究．微生物学通报．1997，24(6)：341-343
[31] Roser B. Chemistry of protein stabilization by trehalose[J].1991,5: 44-53
[32] 罗明典．微生物生产海藻糖及其应用前景．微生物学通报，1996，23(4)：252-254
[33] Roser B. Extraordinary stability of enzymes dried in trehalose: simplified molecular biology[J]. Bio Technology, 1992,10(9): 1007-1011
[34] 戴玉秀，程萍，周坚，等．海藻糖的生理功能／分子生物学研究及应用前景．微生物学通报，1995，22(2)：102-104
[35] 李滢冰．海藻糖在灰树花深层发酵中的积累及多糖的提取[J]．食品与发酵工业，2000，26(2)：11-15
[36] 杉木利行．海藻糖的开发和利用[J]．食品工业，1997，10(3)：1—4
[37] 刘传斌，云战友，冯朴荪等．海藻糖的制备及其对双歧杆菌活性的保护作用．中国生物制品学杂志．1997，10(2)：118
[38] 程池．天然生物保存物质—海藻糖的特性及应用[J]．食品与发酵工业，1996(1)：59—63
[39] 杨荣华，林家莲，周凌霄．海藻糖在食品工业中的应用．山西食品工业，1999(4)：27—28
[40] Bhandal I S. Trehalose as cryoprotectant for freeze preservation of carrot and tobacco cell, Plant Physiol, 1985, 28: 430-432
[41] George Kidd. Trehalose is a sweet target for agbiotech. Biotechnology, 1994,12: 1328-1329
[42] 邓如福，裴炎，王瑜宁，等．海藻糖对水稻幼苗抗寒性研究．西南农业大学学报，1991，13(3)：347-349
[43] 王三根，邓如福，裴炎，等．海藻糖提高绵阳15号小麦幼苗耐盐能力的研究．西南农业大学学报，1992，14(2)：182-185
[44] 裴炎，邓如福，王瑜宁，等．海藻糖对冷冻和冷冻干燥后绿豆苗Mg~(2+)／K~+ -ATPase活性和TTC还原的影响．植物学报，1994，36(增刊)：211-214
[45] Crowe J H, Crowe L M, Chapman D. Membrane integrity in anhydrobiotic organisms: towards a mechanism of stabilizing dry cells. Water and life, 1992: 87-103
[46] Crowe J H, Crowe L M, Chapman D. Interaction of carbohydrates with dry dipalmitoryl phosphatidylcholine. Arch Biochem Biophys, 1985, 236: 289-296
[47] Crowe J H, Crowe L M, Chapman D. Preservation of membranes in anhydrobiotic organism: the role of trehalose. Science, 1984, 223: 701-703
[48] Crowe J H, Crowe L M, Chapman D. Preservation of structural and functional activity in lyophilized sarcoplasmic reticulum, Arch. BioChem Biophys, 1983, 220: 477-484
[49] Kassen I, MeDougall J, Strom A R. Molecular cloning and physical mapping of the OtsBA genes. Which encode the osmoregulatory trehalose pathway of Escherichia coli. Journal of Bacteriol, 1992,174: 889-898
[50] Kjell-ove Holmstrom, Einar Mantyla, Biorn Welin. et al. Drought tolerance in tobacco.Nature, 1996, 379(22): 683-684
[51] Saito K, Kase T, Takahashi E. et al. Purification and characterization of a trehalose synthase from the basidiomycete grifola frondosa. Appl. Environ Bicrobiol, 1998, 64(1): 4340-4345
[52] Holmstrom-K O. Engineering plant adaptation to water-stress. Acta-Universitatis Agriculture-Sueciae-Agraia, 1998, 84: 49
[53] J. Am. Chem. Soc. 1950:2159-2261
[54] 陈秀萍．酵母综合利用产品——提取高效天然生物保存剂海藻糖．甘蔗糖业，2002(3)：40—43
[55] 郑玉娟，张红缨等．胶样菌CB39产海藻糖的研究．微生物学通报，2000，40(2)：204—208
[56] 李永红，段作营，毛忠贵．海藻糖的研究进展．淀粉与淀粉糖，2002(2)：14—19
[57] Hideki Kizwa et al., Biosci. Biotech. Biochem., 1995, 59(10): 1908-1912
[58] Junko Doi et al., Biosci. Biotech. Bi ochem., 1998, 62(4): 735-739
[59] Sawao Murao et al., Agric. Biol. Chem., 1985, 49(7): 2113-2118
[60] 荣绍丰，张海平，等．合成海藻糖的新型磷酸化酶．生物工程进展，2001，2l(2)：54—57
[61] Maruta, T Nakada, M Kubota et al. Purification and characterization of a novel enzyme, maltooligosyl trehalose trehalohydrolase, from Arthrobacter sp Q36. Biosci.Biotech. Biochem., 1995, 59(12): 2210-2214
[62] 李小东．以淀粉为原料利用微生物酶生成海藻糖的新方法．食品与科技，2000(75)：35—37
[63] 萧风岐．海藻糖生产技术的重大突破[J]．淀粉与淀粉酶，1996(2)：42
[64] 刘传斌，云战友，鲁济青，等．海藻糖的分析方法[J]．食品与发酵工业，1998，24(5)：40
[65] 于兰，肖冬光．纸层析分离洗脱法定量测定海藻糖．生物技术，2002，12(3)：27—29
[66] Thomas H, Paul S, Anders W. Heat-induced accumulation and futile cycling of trebalose in Saccbaromyces cerevisiae[J]. Journal of Bacteriology, 1987, 169(12): 5518
[67] 毛贵忠，朱利丹，邓绍荣．用薄层层析法分析海藻糖[J]，食品与发酵工业，1997，23(6)：20
[68] 周坚，吴大鹏，程萍，等．薄层层析法测定海藻糖[J]．微生物学通报，1997，24(2)：125
[69] 葛宇，袁勤生．海藻糖的定量分析方法比较．药物生物技术，2001，8(6)：348—351
[70] John E H, Naresh M. Culture age, temperature and pH affect the polyol and trehalose contents of fungal propagules[J]. Applied and Environmental Microbiology, 1996, 62(7): 2435
[71] Hanne M, Olaf B, Styrvold I K, et al. Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli[J]. Journal of Bacteriology, 1988, 170(1): 2841
[72] 汪云．糖品组分及糖替代品的气相色谱分析[J]．冷饮与速冻食品工业，1998，3：26
[73] 张惟杰．复合多糖生化研究技术．上海科学技术出版社，1987：6—7
[74] 王兰，肖冬光，张正．酶-DNS比色法测定酵母海藻糖的研究．工业微生物，2002，32(2)：18-21
[75] 颜栋美．生物材料中海藻糖的酶法分析．食品科学，1999(9)：62-64
[76] 张厚程，刘娟，池振明．扣囊复膜酵母菌高产突变株转化可溶性淀粉生产海藻糖的研究．食品与发酵工业，28(1)：1—3
[77] 章名春．工业微生物诱变育种．科学出版社，1984．
[78] 周建琴，高荣梅．乐霉素C产生菌的微波诱变．物学杂志，2002，19(4)：12-13
[79] 尤新．海藻糖．精细与专用化学品，2002(9)：21
[80] 姚汝华，周青峰．海藻糖及其应用前景．广州食品工业科技，1995，11(4)：29—31
[81] Yoshikawa Y, Matsumoto K, Nagato K, et al. Extraction of trehalose from baker's yeast{J}. Biosci. Biotech. Biochem, 1994, 58(7): 1226-1230
[82] 刘传斌，李宁，白凤武，等，酵母胞内海藻糖微波破细胞提取海藻糖与传统提取比较．大连理工大学学报，2001，41(2)：169-172
[83] 刘传斌，白凤武，等．酵母胞内海藻糖的微波破细胞提取．化工学报，2000，51(6)：810-813
[84] 章银良，刘庭淼，张鑫，等，微波破碎酵母细胞提取海藻糖的研究．郑州轻工业学院学报，2001，16(4)：51-53
[85] 刘洋，张红缨，高俊芳，等．酵母菌中海藻糖的几种提取方法的比较．中国生化药物杂志，1999。20(1)：15—17
[86] 刘占莲，潘鼎．中孔活性炭纤维的研究．材料导报，2003，17(2)：40—43
[87] 吴雪辉，李琳，郭祀远，等．糖品生产过程中的离子交换问题与分析．福建糖业，1997，11(3)：11—14
[88] 周德庆．微生物学教程，高等教育出版社，1996
[89] Trevelyan W E. Harrison J S. Studies on Yeast Metabolism. Biochem J, 1956,62: 177～182
[90] Lillie S H, Ringle J R. Reserve Carbohydrat Metabolism in Saccharomyces Cerevisiae: Responses to Nutrient Limiyation. J Bacteriol, 1980, 143(3): 1384～1394
[91] 沈萍，等．微生物实验．第3版．北京：高等教育出版社，1999。
[92] 梁海曼，周菊华．广西植物，1992，12(1)：64～756
[93] 周建琴，高荣梅．康乐霉素C产生菌的微波诱变．生物学杂志，2002，4
[94] Plant Propagators Society, 1982, 32: 45～55
[95] 方开泰．均匀设计与均匀设计表．科学出版社，1994
[96] 李永泉，翁醒华，贺筱蓉．微波诱变结合化学诱变选育酸性蛋白酶高产菌．微生物学报，1999，39(2)：181-184
[97] 俞俊堂，唐孝宣．生物工艺学，上海：华东理工大学出版社，1991，12
[98] 戚以政，汪叔雄．生化反应动力学与生物反应器，北京：化学工业出版社，1996
[2] G. Dupont et R. Locquin. Traite de chimie organique(TomeⅧ). Masson et Cie Editors, 1994
[3] Thevelein. Regulation of trehalose mobilization in fungi. Microbiol Rev, 1984,48: 42-59
[4] Elbein A D. The metabolism of α, α-trehalose. Adv Carbohydrate Chem Biocbem, 1974,30: 227-256
[5] 尤新．淀粉糖品生产与应用手册．北京：中国轻工业出版社，1997：295—311
[6] 任小青．海藻糖的研究进展及其开发前景．郑州工程学院学报，2001，22(1)
[7] 张树珍．海藻糖的研究进展及其应用前景．华南热带农业大学学报，2003，6(3)：22—29
[8] 葛宇，袁勤生．海藻糖对生物活性物质的保护作用机理研究进展．药物生物技术．2002，9(5)：297—300
[9] Crowe J H et al. Science, 1984, 223: 701-703
[10] Colaca C et al. Biotechnology, 1992, 10(9): 701-703
[11] Green J L et al. J Phys Chem. 1989, 93: 2880-2882
[12] Sussich F et al. J Am Chem Soc. 1998, 120: 7893-7899
[13] Timesheff S N, Annu Rev Biophys Biomol Struct. 1993, 22: 67
[14] Singer M A, Lindquist S. The Ying and yang of thermotolerance affecting trehalose[J].Tibtech, 1998, 16: 460
[15] Miller D P. Rational design of protective agents and processes for the stabilization of biologicals[D]. University of Wisconsin-Madison
[16] 简文杰，庞杰．海藻糖的生产及应用．杭州食品科技．2003，1：1—4
[17] 周青峰．海藻糖在食品加工上的应用．中国食品工业．1996(2)：22—23
[18] 王蕾，郑璞，史朝辉．新型食品添加剂海藻糖的生产方法及应用．食品科技，2002(2)：16—17
[19] Koichi Y,I Iiroe Y,I Iiromuk, et al. Protection by trehalose of DNA from radiation damage. Biosci Biotech Biochem. 1997, 61(10:160-161
[20] 戴玉秀，沈义国，周坚．海藻糖对抗逆乳酸菌的保护研究．微生物学通报，2001，8(2)：46-50
[21] 颜栋美，周青峰，姚汝华．海藻糖对载药脂质体保护作用的初步研究．广西大学学报，1998，23(4)：393-396
[22] 李群，袁勤生，李永丰．海藻糖对酶热稳定性保护作用的研究．药物生物技术，1997，4(1)：28-31
[23] 葛宇，赖承光，张文艳，等．海藻糖对保加利亚乳杆菌保护应用的研究．食品科学，2002，23(7)：55-58
[24] 王辉，李克寒，吴兵，等．海藻糖对冻干胸腺肽生物活性保护作用的研究．兰州大学学报，2001，37(6)：72-75
[25] 杨基础，杨小民，等．海藻糖对固定化酶的保护作用．化工学报，2000，51(2)：193-197
[26] 唐传核，葛文光．海藻糖对果胶酶在干燥过程中的保护作用．四川食品与发酵，1998(1—2)：42-44
[27] 陈黎忠，李羲光，边光珠，等．海藻糖对酶冻干品的保护作用．上海医学检测杂志，2001，16(3)：166-167
[28] 陈黎忠，边光珠，李羲光，等．海藻糖对人血清酶抗冻结性保护研究．医学研究通讯，2001，30(1)：
44-46
[29] 权国波．海藻糖在哺乳动物细胞保存中的作用机制及其应用现状．临床输血与检验，2003，5(1)：78—80
[30] 黄成垠，安国瑞，王庆敏，等．海藻糖对医涌诊断工具酶活性保护研究．微生物学通报．1997，24(6)：341-343
[31] Roser B. Chemistry of protein stabilization by trehalose[J].1991,5: 44-53
[32] 罗明典．微生物生产海藻糖及其应用前景．微生物学通报，1996，23(4)：252-254
[33] Roser B. Extraordinary stability of enzymes dried in trehalose: simplified molecular biology[J]. Bio Technology, 1992,10(9): 1007-1011
[34] 戴玉秀，程萍，周坚，等．海藻糖的生理功能／分子生物学研究及应用前景．微生物学通报，1995，22(2)：102-104
[35] 李滢冰．海藻糖在灰树花深层发酵中的积累及多糖的提取[J]．食品与发酵工业，2000，26(2)：11-15
[36] 杉木利行．海藻糖的开发和利用[J]．食品工业，1997，10(3)：1—4
[37] 刘传斌，云战友，冯朴荪等．海藻糖的制备及其对双歧杆菌活性的保护作用．中国生物制品学杂志．1997，10(2)：118
[38] 程池．天然生物保存物质—海藻糖的特性及应用[J]．食品与发酵工业，1996(1)：59—63
[39] 杨荣华，林家莲，周凌霄．海藻糖在食品工业中的应用．山西食品工业，1999(4)：27—28
[40] Bhandal I S. Trehalose as cryoprotectant for freeze preservation of carrot and tobacco cell, Plant Physiol, 1985, 28: 430-432
[41] George Kidd. Trehalose is a sweet target for agbiotech. Biotechnology, 1994,12: 1328-1329
[42] 邓如福，裴炎，王瑜宁，等．海藻糖对水稻幼苗抗寒性研究．西南农业大学学报，1991，13(3)：347-349
[43] 王三根，邓如福，裴炎，等．海藻糖提高绵阳15号小麦幼苗耐盐能力的研究．西南农业大学学报，1992，14(2)：182-185
[44] 裴炎，邓如福，王瑜宁，等．海藻糖对冷冻和冷冻干燥后绿豆苗Mg~(2+)／K~+ -ATPase活性和TTC还原的影响．植物学报，1994，36(增刊)：211-214
[45] Crowe J H, Crowe L M, Chapman D. Membrane integrity in anhydrobiotic organisms: towards a mechanism of stabilizing dry cells. Water and life, 1992: 87-103
[46] Crowe J H, Crowe L M, Chapman D. Interaction of carbohydrates with dry dipalmitoryl phosphatidylcholine. Arch Biochem Biophys, 1985, 236: 289-296
[47] Crowe J H, Crowe L M, Chapman D. Preservation of membranes in anhydrobiotic organism: the role of trehalose. Science, 1984, 223: 701-703
[48] Crowe J H, Crowe L M, Chapman D. Preservation of structural and functional activity in lyophilized sarcoplasmic reticulum, Arch. BioChem Biophys, 1983, 220: 477-484
[49] Kassen I, MeDougall J, Strom A R. Molecular cloning and physical mapping of the OtsBA genes. Which encode the osmoregulatory trehalose pathway of Escherichia coli. Journal of Bacteriol, 1992,174: 889-898
[50] Kjell-ove Holmstrom, Einar Mantyla, Biorn Welin. et al. Drought tolerance in tobacco.Nature, 1996, 379(22): 683-684
[51] Saito K, Kase T, Takahashi E. et al. Purification and characterization of a trehalose synthase from the basidiomycete grifola frondosa. Appl. Environ Bicrobiol, 1998, 64(1): 4340-4345
[52] Holmstrom-K O. Engineering plant adaptation to water-stress. Acta-Universitatis Agriculture-Sueciae-Agraia, 1998, 84: 49
[53] J. Am. Chem. Soc. 1950:2159-2261
[54] 陈秀萍．酵母综合利用产品——提取高效天然生物保存剂海藻糖．甘蔗糖业，2002(3)：40—43
[55] 郑玉娟，张红缨等．胶样菌CB39产海藻糖的研究．微生物学通报，2000，40(2)：204—208
[56] 李永红，段作营，毛忠贵．海藻糖的研究进展．淀粉与淀粉糖，2002(2)：14—19
[57] Hideki Kizwa et al., Biosci. Biotech. Biochem., 1995, 59(10): 1908-1912
[58] Junko Doi et al., Biosci. Biotech. Bi ochem., 1998, 62(4): 735-739
[59] Sawao Murao et al., Agric. Biol. Chem., 1985, 49(7): 2113-2118
[60] 荣绍丰，张海平，等．合成海藻糖的新型磷酸化酶．生物工程进展，2001，2l(2)：54—57
[61] Maruta, T Nakada, M Kubota et al. Purification and characterization of a novel enzyme, maltooligosyl trehalose trehalohydrolase, from Arthrobacter sp Q36. Biosci.Biotech. Biochem., 1995, 59(12): 2210-2214
[62] 李小东．以淀粉为原料利用微生物酶生成海藻糖的新方法．食品与科技，2000(75)：35—37
[63] 萧风岐．海藻糖生产技术的重大突破[J]．淀粉与淀粉酶，1996(2)：42
[64] 刘传斌，云战友，鲁济青，等．海藻糖的分析方法[J]．食品与发酵工业，1998，24(5)：40
[65] 于兰，肖冬光．纸层析分离洗脱法定量测定海藻糖．生物技术，2002，12(3)：27—29
[66] Thomas H, Paul S, Anders W. Heat-induced accumulation and futile cycling of trebalose in Saccbaromyces cerevisiae[J]. Journal of Bacteriology, 1987, 169(12): 5518
[67] 毛贵忠，朱利丹，邓绍荣．用薄层层析法分析海藻糖[J]，食品与发酵工业，1997，23(6)：20
[68] 周坚，吴大鹏，程萍，等．薄层层析法测定海藻糖[J]．微生物学通报，1997，24(2)：125
[69] 葛宇，袁勤生．海藻糖的定量分析方法比较．药物生物技术，2001，8(6)：348—351
[70] John E H, Naresh M. Culture age, temperature and pH affect the polyol and trehalose contents of fungal propagules[J]. Applied and Environmental Microbiology, 1996, 62(7): 2435
[71] Hanne M, Olaf B, Styrvold I K, et al. Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli[J]. Journal of Bacteriology, 1988, 170(1): 2841
[72] 汪云．糖品组分及糖替代品的气相色谱分析[J]．冷饮与速冻食品工业，1998，3：26
[73] 张惟杰．复合多糖生化研究技术．上海科学技术出版社，1987：6—7
[74] 王兰，肖冬光，张正．酶-DNS比色法测定酵母海藻糖的研究．工业微生物，2002，32(2)：18-21
[75] 颜栋美．生物材料中海藻糖的酶法分析．食品科学，1999(9)：62-64
[76] 张厚程，刘娟，池振明．扣囊复膜酵母菌高产突变株转化可溶性淀粉生产海藻糖的研究．食品与发酵工业，28(1)：1—3
[77] 章名春．工业微生物诱变育种．科学出版社，1984．
[78] 周建琴，高荣梅．乐霉素C产生菌的微波诱变．物学杂志，2002，19(4)：12-13
[79] 尤新．海藻糖．精细与专用化学品，2002(9)：21
[80] 姚汝华，周青峰．海藻糖及其应用前景．广州食品工业科技，1995，11(4)：29—31
[81] Yoshikawa Y, Matsumoto K, Nagato K, et al. Extraction of trehalose from baker's yeast{J}. Biosci. Biotech. Biochem, 1994, 58(7): 1226-1230
[82] 刘传斌，李宁，白凤武，等，酵母胞内海藻糖微波破细胞提取海藻糖与传统提取比较．大连理工大学学报，2001，41(2)：169-172
[83] 刘传斌，白凤武，等．酵母胞内海藻糖的微波破细胞提取．化工学报，2000，51(6)：810-813
[84] 章银良，刘庭淼，张鑫，等，微波破碎酵母细胞提取海藻糖的研究．郑州轻工业学院学报，2001，16(4)：51-53
[85] 刘洋，张红缨，高俊芳，等．酵母菌中海藻糖的几种提取方法的比较．中国生化药物杂志，1999。20(1)：15—17
[86] 刘占莲，潘鼎．中孔活性炭纤维的研究．材料导报，2003，17(2)：40—43
[87] 吴雪辉，李琳，郭祀远，等．糖品生产过程中的离子交换问题与分析．福建糖业，1997，11(3)：11—14
[88] 周德庆．微生物学教程，高等教育出版社，1996
[89] Trevelyan W E. Harrison J S. Studies on Yeast Metabolism. Biochem J, 1956,62: 177～182
[90] Lillie S H, Ringle J R. Reserve Carbohydrat Metabolism in Saccharomyces Cerevisiae: Responses to Nutrient Limiyation. J Bacteriol, 1980, 143(3): 1384～1394
[91] 沈萍，等．微生物实验．第3版．北京：高等教育出版社，1999。
[92] 梁海曼，周菊华．广西植物，1992，12(1)：64～756
[93] 周建琴，高荣梅．康乐霉素C产生菌的微波诱变．生物学杂志，2002，4
[94] Plant Propagators Society, 1982, 32: 45～55
[95] 方开泰．均匀设计与均匀设计表．科学出版社，1994
[96] 李永泉，翁醒华，贺筱蓉．微波诱变结合化学诱变选育酸性蛋白酶高产菌．微生物学报，1999，39(2)：181-184
[97] 俞俊堂，唐孝宣．生物工艺学，上海：华东理工大学出版社，1991，12
[98] 戚以政，汪叔雄．生化反应动力学与生物反应器，北京：化学工业出版社，1996