紫丁香蘑液体发酵海藻糖的提取纯化及应用研究
|
摘要
紫丁香蘑(Lepista nuda)为伞菌目,白蘑科,香蘑属的一种药食皆用的野生真菌。目前,对紫丁香蘑的林地栽培获得子实体的试验进行了研究,而对紫丁香蘑的液体发酵及有效成分的提取研究的比较少。食药用菌中占70%以上的游离糖和游离糖醇是由一二种糖和糖醇组成的,紫丁香蘑中主要以海藻糖为主,含量较高。海藻糖可应用于食品、医药、化妆品等多方面的研究。我国食用菌资源丰富,借助食用菌开发海藻糖具有独特的优越性。本文对液体发酵紫丁香蘑海藻糖的提取、纯化、鉴定以及应用进行研究。本研究主要试验结果如下:
1.以菌丝体干重和海藻糖的含量为测定指标,确定紫丁香蘑液体发酵培养基的组成。试验得出,最适氮源为酵母膏,碳源为葡萄糖,最初pH值为7.0。
2.紫丁香蘑菌丝体最佳提取工艺参数研究。通过对紫丁香蘑菌丝体进行单因素和3因素3水平的正交试验,研究提取时间、温度、乙醇浓度对紫丁香蘑菌丝体海藻糖提取的影响。影响提取的各因素顺序:温度>乙醇浓度>提取时间。最佳提取参数最优组合为A_2B_1C_2,即提取时间为1h,温度为70℃,乙醇浓度30%。得到的海藻糖的含量为12.92%,蛋白含量0.62%。
3.海藻糖的纯化。采用Sevag法除蛋白。Sevag法中海藻糖溶液与氯仿-正丁醇溶液的比例不同时,不但对蛋白去除率有影响,而且对海藻糖的损失程度也不同,综合考虑,最后确定除蛋白的最佳海藻糖溶液与氯仿及正丁醇的体积比为5:1:0.25。通过测定蛋白含量,从而选择了除蛋白的次数为二次,经二次除蛋白后的海藻糖溶液中的蛋白含量为0.3%。通过单因素和4因素3水平试验,对除蛋白后的海藻糖溶液进行活性炭脱色,活性炭脱色的最佳工艺为:调节pH值为4,加入3%的活性炭,60℃下搅拌10min,脱色率达到72.04%,海藻糖含量为8.06%。脱盐的最佳工艺条件为:1mL/min,pH值6,此时脱色率在90%以上,脱盐率在97%以上。
4.薄层层析鉴定海藻糖。通过在不同的展开剂中Rf值的测定,确定展开剂为正丁醇:吡啶:水=15:3:2,点样量1.0ul,硫酸和甲醇配制成1:4的溶液,105℃的干燥箱中加热使之显色。结晶海藻糖得率为2.24%,海藻糖含量92.56%。
5.海藻糖应用的研究表明,海藻糖具有一定的抗冷冻和抗疲劳作用。在抗冷冻方面,通过观察不同时间鱼糜色泽、气味、水质三个因素的变化。在抗疲劳方面,海藻糖对小鼠游泳试验、爬绳试验、肝糖原、血清尿素氮都有显著影响。
Lepista nuda was belong to Agaricales of Tricholomtaceae.Lepista nuda not only has edible value,but also has officinal value.The research of experiment on collecting fruit body by woodland culture of lepista nuda has been test.For the liquid fermnentation of Lepista nuda and it's extraction of active component, there are few reaserch about this topic.In domestic fungus,more than 70%of lib-sug and lib-sugar alcohol are made up of one or two kinds of sugar.The main component in Lepista nuda is Trehalose,and it's content is rather great.The Trehalose can be used to study food,cosmetic and medicine.In our country,the domestic fungus are very plenteous,so developing the Trehalose have great advantage by domestic fungus. In this study,considering the Lepista nuda-hyphal as my studying object,the extraction,purification, assessment and application for the Trehalose of the Lepista nuda liquid fermentation hyphostroma were studied in this exprement.The result that was investigated are following:
1.According to the dry weight of hyphostroma and cont of trehalose confirm the medium's composition in the lepista nuda liquid fermentation.The experiment shows that the most compatible nitrogen source is yeast extract,and the carbon source is glucose,and the inital pH is 7.
2.The study of best extract craft parameter for the Lepista nuda hyphostroma.Through single-factor and the orthogonal design,which is to three factors and three levels,I study the extracting time and temperature,and study the effect,which alcohol saturation impact the extraction of Trehalose from Lepista nuda.The best extracting parameter is A_2B_1C_2 The data I have studied as follows:time is 1h;the temperature is70℃;concentration of ethanol is 30%.The cont of Trehalose I gained is 12.92%,and protein level is 0.62%.
3.Purification of Trehalose.And I adopted the Sevag to deproteinize.In this experiment,I found as follows:in Sevag,when the ratio of sample volume between Trehalose and chloroform n-butanol is different,it does't only effect rate of deproteinization,but also effect the lossing degree of Trehalose.I sumed up the conclusion from the materials above as follows:the best volume ratio between the Trehalose, chloroform and n-butanol is 5:1:0.25.According determining protein level,I choose the time of deproteinization is twice.After twice deproteinization,the protein level in the Trehalose solution is 0.3%. Using four factors and three levels for activated carbon decolorization,after the addition of the Trehalose solution,the optimal craft of decaloration by activated carbon was:adjusting pH to 4,put in 3%activated carbon,agitating 10min in 60℃.The bleaching rate is 72.04%;cont of Trehalose is 8.06%.The best craft condition is 1mL/min;pH is 6.In this time,the bleaching rate is more than 90%,and desalting rate is more than 97%.
4.Using TLC to accredit Trehalose.spreading agent:n-butanol:Pyr:water=15:3:2;Sample is 1.0ul.The liquor rate:vitriol:Meoh =1:4.heat it under 105℃in baking oven in order to colorate.The ratio of crystallization is 2.24%,and the content of Trehalose is 92.56%.
5.The study shows that the Trehalose has the function of anti- freezing and anti-fatigue.In the part of anti-freezing,I observe the color,and smell and water quality of Fish wasteful in different time.In the part of anti-fatigue,the Trehalose can have the influence on the Comparison of time to grasp the rope of mice, Comparison of time to swim of mice,serum urea nitrogen and hepatic glycogen.
1.以菌丝体干重和海藻糖的含量为测定指标,确定紫丁香蘑液体发酵培养基的组成。试验得出,最适氮源为酵母膏,碳源为葡萄糖,最初pH值为7.0。
2.紫丁香蘑菌丝体最佳提取工艺参数研究。通过对紫丁香蘑菌丝体进行单因素和3因素3水平的正交试验,研究提取时间、温度、乙醇浓度对紫丁香蘑菌丝体海藻糖提取的影响。影响提取的各因素顺序:温度>乙醇浓度>提取时间。最佳提取参数最优组合为A_2B_1C_2,即提取时间为1h,温度为70℃,乙醇浓度30%。得到的海藻糖的含量为12.92%,蛋白含量0.62%。
3.海藻糖的纯化。采用Sevag法除蛋白。Sevag法中海藻糖溶液与氯仿-正丁醇溶液的比例不同时,不但对蛋白去除率有影响,而且对海藻糖的损失程度也不同,综合考虑,最后确定除蛋白的最佳海藻糖溶液与氯仿及正丁醇的体积比为5:1:0.25。通过测定蛋白含量,从而选择了除蛋白的次数为二次,经二次除蛋白后的海藻糖溶液中的蛋白含量为0.3%。通过单因素和4因素3水平试验,对除蛋白后的海藻糖溶液进行活性炭脱色,活性炭脱色的最佳工艺为:调节pH值为4,加入3%的活性炭,60℃下搅拌10min,脱色率达到72.04%,海藻糖含量为8.06%。脱盐的最佳工艺条件为:1mL/min,pH值6,此时脱色率在90%以上,脱盐率在97%以上。
4.薄层层析鉴定海藻糖。通过在不同的展开剂中Rf值的测定,确定展开剂为正丁醇:吡啶:水=15:3:2,点样量1.0ul,硫酸和甲醇配制成1:4的溶液,105℃的干燥箱中加热使之显色。结晶海藻糖得率为2.24%,海藻糖含量92.56%。
5.海藻糖应用的研究表明,海藻糖具有一定的抗冷冻和抗疲劳作用。在抗冷冻方面,通过观察不同时间鱼糜色泽、气味、水质三个因素的变化。在抗疲劳方面,海藻糖对小鼠游泳试验、爬绳试验、肝糖原、血清尿素氮都有显著影响。
Lepista nuda was belong to Agaricales of Tricholomtaceae.Lepista nuda not only has edible value,but also has officinal value.The research of experiment on collecting fruit body by woodland culture of lepista nuda has been test.For the liquid fermnentation of Lepista nuda and it's extraction of active component, there are few reaserch about this topic.In domestic fungus,more than 70%of lib-sug and lib-sugar alcohol are made up of one or two kinds of sugar.The main component in Lepista nuda is Trehalose,and it's content is rather great.The Trehalose can be used to study food,cosmetic and medicine.In our country,the domestic fungus are very plenteous,so developing the Trehalose have great advantage by domestic fungus. In this study,considering the Lepista nuda-hyphal as my studying object,the extraction,purification, assessment and application for the Trehalose of the Lepista nuda liquid fermentation hyphostroma were studied in this exprement.The result that was investigated are following:
1.According to the dry weight of hyphostroma and cont of trehalose confirm the medium's composition in the lepista nuda liquid fermentation.The experiment shows that the most compatible nitrogen source is yeast extract,and the carbon source is glucose,and the inital pH is 7.
2.The study of best extract craft parameter for the Lepista nuda hyphostroma.Through single-factor and the orthogonal design,which is to three factors and three levels,I study the extracting time and temperature,and study the effect,which alcohol saturation impact the extraction of Trehalose from Lepista nuda.The best extracting parameter is A_2B_1C_2 The data I have studied as follows:time is 1h;the temperature is70℃;concentration of ethanol is 30%.The cont of Trehalose I gained is 12.92%,and protein level is 0.62%.
3.Purification of Trehalose.And I adopted the Sevag to deproteinize.In this experiment,I found as follows:in Sevag,when the ratio of sample volume between Trehalose and chloroform n-butanol is different,it does't only effect rate of deproteinization,but also effect the lossing degree of Trehalose.I sumed up the conclusion from the materials above as follows:the best volume ratio between the Trehalose, chloroform and n-butanol is 5:1:0.25.According determining protein level,I choose the time of deproteinization is twice.After twice deproteinization,the protein level in the Trehalose solution is 0.3%. Using four factors and three levels for activated carbon decolorization,after the addition of the Trehalose solution,the optimal craft of decaloration by activated carbon was:adjusting pH to 4,put in 3%activated carbon,agitating 10min in 60℃.The bleaching rate is 72.04%;cont of Trehalose is 8.06%.The best craft condition is 1mL/min;pH is 6.In this time,the bleaching rate is more than 90%,and desalting rate is more than 97%.
4.Using TLC to accredit Trehalose.spreading agent:n-butanol:Pyr:water=15:3:2;Sample is 1.0ul.The liquor rate:vitriol:Meoh =1:4.heat it under 105℃in baking oven in order to colorate.The ratio of crystallization is 2.24%,and the content of Trehalose is 92.56%.
5.The study shows that the Trehalose has the function of anti- freezing and anti-fatigue.In the part of anti-freezing,I observe the color,and smell and water quality of Fish wasteful in different time.In the part of anti-fatigue,the Trehalose can have the influence on the Comparison of time to grasp the rope of mice, Comparison of time to swim of mice,serum urea nitrogen and hepatic glycogen.
引文
[1]陈士瑜,陈惠.菇菌栽培手册.科学技术文献出版社:2003,562-566.
[2]卯晓岚.中国经济真菌[M].科学出版社:1998:129,127-130.
[3]庄司当.紫丁香蘑林地栽培试验[J].特产情报,1988(3):63-65.
[4]戴彩云,崔晓明.茶园自生食用菌紫丁香蘑的驯化栽培[J].贵州茶叶,1994,1(77):24-26.
[5]高锦明,董泽军,杨雪,等.紫丁香蘑的化学成分[J].中草药,2002,33(5):398-401.
[6]Olof Hjelm,Emma Johansson,Gunilla Oberg.Production of organically bound halogens by the litter-degrading fungus Lepista nuda[J].Soil Biology and Biochemistry,1999,31:1509-1515.
[7]Crowe J H,Hoekstra FA,Crowe LM.Rev Physiol,1992,54:579-599.
[8]TSCHIRCH A.Handbuch der Pharmakognosie Bdll[M].Juliuse Hofbuchandlung:1992,147.
[9]涂国云.海藻糖的性质、生产及应用[J].江西食品科学,2003(3):33-35.
[10]张玉华,凌沛学,籍保平.海藻糖的研究现状及其应用前景[J].食品与药品,2005,7(3):8-9.
[11]李爱民,张银志,徐晖.海藻糖的研究与应用[J].汉中师范学院学报,2003,21(2):89-94.
[12]尤新.淀粉糖品生产与应用手册[M].中国轻工出版社:1997.
[13]Barrett,J.Anhydrobiotic nematodes.Agriculturalzoology Reviews,1991,4:161-176.
[14]刘传斌,谢健,苗蔚荣,等.海藻糖的性质及其广阔应用前景[J].中国微生物杂志,1998,10(6):377-379.
[15]程池.天然保存剂—海藻糖的特性和应用[J].食品与发酵工业,1996(1):59-64.
[16]王俊,尹华,李明玉.富产海藻糖合成酶菌株的筛选[J].暨南大学学报,2004,25(3):361-369.
[17]Elbein A D.The metabolism of trehalose[J].Adv in Carbohyd Chem Biochem,1974,30:227-229.
[18]Kane SM,Roth R.Carbohydrate metabolism during ascospore development in yeast.J.Bacterial,1974,118:8-14.
[19]ZimmermannFK Yeast Sugar Metabolism[M].Lancaster U.S.A:Technic Publishing Company Inc,1997,28(6):285-303.
[20]Lillie S H,et al Reserved carbohydrate metabolism in Saccharomyces Cerevisiae;Response to nutrient limitation[J].J.Bacterial,1980,26(4):1384-1394
[21]戴秀玉,程苹,周坚,等.海藻糖的生理功能、分子生物学研究与应用前景[J].微生物学通报,1995,22(2):102-104.
[22]WELSHE T,RONDNEYA H Osmotically induced intracellular trehalose,but not glycine betaine accumulation promotes desiccation tolerance in Escherichia coli[J].FEMS Microbiology Lesstters.1999,174(1):57-63.
[23]Wyatt GR,Kalf GF,The chemistry of insect hemolymph.Ⅱ Trehalose and other crbohydrates.Journal of General Physiology,1957,40:833-847
[24]聂凌鸿,宁正祥.海藻糖的生物保护作用[J].生命的化学,2001(3):206-208.
[25]Hounsa CG,Brandt EV,Thevelein J,et al.Role of trehalose in surival of Sacchromyces cerevisiae under osmotic stress.Microbiology,1998,144:671-680.
[26]Eroglu A,mehmet T,thomas LT.Beneficial effect of microinjected trehalose on the ctyosurival of human oocytes.Fertihty and Sterility,2002,77:152-158.
[27]李桂芹.海藻糖的生物学功能及其应用[J].安徽农业科学,2007,35(24):7724-7725
[28]PAUL V A.Trehalose accumulates in Saccharomyces ceretislae during expose to agents that nduce heat shock response[J].FEBS Letters,1987,225(1);259-263.
[29]LAERE A V.FRANCOIS A,OVERLOOP K,et al.Relation between germinination trehalose and the status of water in Phycomyces blakeleeanus as measureal by proton NMR[J].J Gener Microbiol,1987,133:239-245.
[30]Colaco CALS,Roser B.Trehalose-a multifunctional additive for food preservation.Food packaging and preservation,1995,123-140
[31]HOTTIGER T,SCHMUTZP,WIEMKEN A.Heat induced accmulation and futile cycling of trehalose in Saccharomyces cervisiae[J].J Bacterial,1987,169(12):5518-5520.
[32]Colaco C,Sen S,ThangavelnM,et al.Estraordinary stabilityof enzymes dried in trehalose:simplified molecular biology[J].Biotechnology,1992,10:1007-1011.
[33]唐传核.海藻糖及其应用[J].食品研究与开发,1998,20(4):16.
[34]李滢冰,冯梦醒,徐继祖.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.
[35]Allen-ind.Isolation of trehalose by alcohol crystallization-trehalose purification from fermentation broth.Derwent Biotechnol Abst,1995,14(2):01165.
[36]Murao S,Nagano S,et al.En2ymatic synthesis of trehalose form maltose.Agric Biol Chem.1985,49(7):2113-2118.
[37]缪静.海藻糖的制备方法[J].烟台师范学院学报(自然科学版),2000,15(1):60-63.
[38]陈杰,何聪芬,叶兴国,等.海藻糖及其在生物工程方面的应用[J].生物技术通报,2005,(6):29-33.
[39]Elbein AD,Pan YT,Pastuszak l,et al.New insights on trehalose:a multifunctionalmolecule.Glycobiology,2003,13:17-27.
[40]Kato M,Miura Y,Kettoku M,et al.Purification and characterization of new trehalose producing enzymes isolated from the hyperthermophilic archae,Sulfolobus solfataricus KMI.Biosciences,Biotechnology and Biochemistry,1996,60:546-550.
[41]简文杰,庞杰.海藻糖的生产及其应用[J].杭州食品科技,2003,(1):1-4.
[42]赵晓峰,吴荣书.海藻糖的功能特性及其应用[J].广州食品工业科技,2004,20(2):151-155.
[43]张树珍.海藻糖的研究进展及其应用前景[J].华南热带农业大学学报,2000(3):22-29.
[44]Garg AK,Kim JK,Owens TG,et al.Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses[J].Proc Natl Acad Sci USA,2002,99(25):15898 -15903.
[45]Guo N,Puhlev I,Brown DR,et al.Trehalose expression confers desiccation tolerance on human cells[J].Nat Biotechnol,2000,18(2):168-171.
[46]Matsuo T.Trehalose protects corneal epithelial cells from death by drying[J].Br.J.Ophthalmol,2001,85(5):610-612.
[47]袁勤生.隐生生物中的典型化合物海藻糖[J].中国生化药物杂志,1999,20(1):48-50.
[48]刘欣,周爱梅,赵力超,等.海藻糖、乳酸钠对冷藏鳙鱼鱼糜蛋白抗冷冻效果的影响[J].食品与发酵工业,2007,30(8):63-64
[49]高茹菲,陈舜胜.海藻糖抗冻作用研究[J].渔业现代化,2005,(4):48-49.
[50]戴秀玉,沈义国,周坚.海藻糖对乳酸菌的抗逆保护研究[J].微生物学通报,2001,4(2):465-470.
[51]李浩明,高蓝.海藻糖在生物制品干燥与保存中的研究[J].食品与发酵工业,1994,4(49):49-51.
[52]Colaco C.A.L.S,Smith C.J.S,Sen S.Chemistry of pritein stabilization by trehalose[J].American Chemical Society,1994,(567):222-240.
[53]周青峰.海藻糖在食品加工上的应用[J].中国食品工业,1996(2):22-23.
[54]陈黎忠,李羲光,边光珠,等.海藻糖对酶冻干品的保护作用[J].化工学报,2000,51(2):1931-97.
[55]权国波.海藻糖在哺乳动物细胞保存中的作用机制及应用现状[J].临床输血与检验,2003,5(1):78-80.
[56]唐传核,葛文光.从活性干酵母中提取海藻糖的工艺[J].无锡轻工业大学学报,1998,17(3):37-40.
[57]傅琳琳,傅开科.海藻糖的研究现状及其应用前景[J].江西科学,2000,18(3):182-85.
[58]魏杰.天然保存剂—海藻糖[J].安徽科技,1999,(3):24.
[59]宋祺,陈坚,伦世仪.海藻糖对面包酵母耐贮力的影响[J].无锡轻工业大学学报,1997,16(3):32-36.
[60]杨荣华,林家莲,周凌霄.海藻糖在食品工业中的应用[J].山西食品工业,1999,(4):27-28.
[61]James S.Cellular responses to extreme water loss[J].Cryobiology,1982,19:306-308.
[62]王岁楼.食品生物技术[M].海洋出版社:1998
[63]Richards AB,Krakowka S,Dexter LB,et al.Trehalose:a review of multiple safety studies.Food and Chemical Toxicology,2002,40:871-898.
[64]OKU T,MITSUKO O.Transitory laxative threshold of trehalose and lactulose in healthy women[J].J Nutr Sci&Vitaminol,1998,44(6) Dec:787-798.
[65]Uritani M,Takal M,Yoshinage K.Protective effect of disaccharides on restricition endonucleases during drying under vacuum.Jounal of Biochemistry,1995,117:774-779.
[66]李桂芹.海藻糖的生物学功能及其应用[J].安徽农业科学,2007,35(24):7724-7725.
[67]Hata Y,Nishimori Y,Okochi Y,et al.JP:4-290808.
[68]阚红玲,孙洪涛,董健军.海藻糖在化妆品中的应用[J].食品与药品,2005,7(9):48-50.
[69]衫本利行.食品工业,1997,10(3):1-4.
[70]Crowe J H,Crowe L M.Preservation of membranes in an hydrobiotic organism.the role of trehalose J.Science,1984,223:701-703.
[71]马文锦,刘树兴,潘巨忠,等.海藻糖的生产制备及应用前景[J].现代农业科技,2007,(24):199-201
[72]胡宗利,夏玉先,陈国平,等.海藻糖的生产制备及其应用前景[J].中国生物工程杂志,2004,(4):44-48.
[73]James S.Cellular responses to extreme water lossJ.Cryobiology,1982,19:306-308.
[74]Colaco C,Sen S,Thangaveln M,et al.Estraordinary stability of enzymes dried intrehalose.simplified molecular biologyJ.Biotechnology,1992,10:1007~1011.
[76]徐浩,王丽,江慧修,等.葡萄酒活性干酵母的生物学和工艺的研究[J].真菌学报,1990,9(1):73-78.
[77]Yokomise H,inui K,wada H,et al.Longterm eryopreservation can prevent rejection of canine tracheal allografts with preservation of graft viability.Journal of Throacic and Cardiovascular Surgergy,1996,111:930-934
[78]ADRIDGE S.Trehalose boots prospects for improved biopharmaceuticals and VaccinesJ.Cenetic Engineering News,1995,15:10-11.
[79]Garg AK,Kim JK,Owens TG,et al.Trehalose accumulation in rice Plantsconfers high tolerancelevels to different abiotic stresses.PNAS,2002,99(25):15898-15903.
[80]SERRANO R,FRANCISCO A,CULIANZ-MACIA,et al.Genetic engineering of salt and drought tolerance withyeast regulatory genes[J].Scientia Horticulturae,1999,78(1):261-269.
[81]俞建瑛,蒋宇,王善利.生物化学试验技术[M].化学工业出版社:2005.
[82]刘洋,张红缨,高俊芳,等.酵母中海藻糖的几种提取方法的比较[J].中国生化药物杂志,1999,20(1):15-16.
[83]ZHAN Li-ming(张立名).Analysis Principle of the Modem Biology Chemistry(现代生物化学分析原理)[M].China University of Science and Technology Press,1991.(in Chinese)
[84]杨云,刘福勤,冯卫生,等.碱法提取大枣渣多糖及活性炭脱色的工艺研究[J].食品与发酵工业,2004,30(7):30-32.
[85]王元凤,金征宇.茶多糖脱色的研究[J].食品与发酵工业,2004,8:61-65.
[86]彭卫红,罗婷,甘炳成,等.碳源和氮源对紫丁香蘑菌丝生长的影响研究[J].西南农业学报,2004,17:419-422.
[87]Lillie SH,Ringle JR.Reserverd carbohydrat metabolism in Saccharomyces cerevisiae:Responses to nutrient limitation.J Bacteriol,1980,143(3):1384-1394.
[88]周伟.从活性干酵母中提取海藻糖的工艺研究:[硕士学位论文].浙江:浙江大学,2001.
[89]何芸,姚开,贾冬英,等.抗鱼肉蛋白冷冻变性机理的研究进展[J].氨基酸和生物资源,2007,29(2):36-39
[90]何来英,严卫星,楼密密,等.保健食品抗疲劳作用试验方法研究[J].中国食品卫生杂志,1997,9(4):1-6.
[2]卯晓岚.中国经济真菌[M].科学出版社:1998:129,127-130.
[3]庄司当.紫丁香蘑林地栽培试验[J].特产情报,1988(3):63-65.
[4]戴彩云,崔晓明.茶园自生食用菌紫丁香蘑的驯化栽培[J].贵州茶叶,1994,1(77):24-26.
[5]高锦明,董泽军,杨雪,等.紫丁香蘑的化学成分[J].中草药,2002,33(5):398-401.
[6]Olof Hjelm,Emma Johansson,Gunilla Oberg.Production of organically bound halogens by the litter-degrading fungus Lepista nuda[J].Soil Biology and Biochemistry,1999,31:1509-1515.
[7]Crowe J H,Hoekstra FA,Crowe LM.Rev Physiol,1992,54:579-599.
[8]TSCHIRCH A.Handbuch der Pharmakognosie Bdll[M].Juliuse Hofbuchandlung:1992,147.
[9]涂国云.海藻糖的性质、生产及应用[J].江西食品科学,2003(3):33-35.
[10]张玉华,凌沛学,籍保平.海藻糖的研究现状及其应用前景[J].食品与药品,2005,7(3):8-9.
[11]李爱民,张银志,徐晖.海藻糖的研究与应用[J].汉中师范学院学报,2003,21(2):89-94.
[12]尤新.淀粉糖品生产与应用手册[M].中国轻工出版社:1997.
[13]Barrett,J.Anhydrobiotic nematodes.Agriculturalzoology Reviews,1991,4:161-176.
[14]刘传斌,谢健,苗蔚荣,等.海藻糖的性质及其广阔应用前景[J].中国微生物杂志,1998,10(6):377-379.
[15]程池.天然保存剂—海藻糖的特性和应用[J].食品与发酵工业,1996(1):59-64.
[16]王俊,尹华,李明玉.富产海藻糖合成酶菌株的筛选[J].暨南大学学报,2004,25(3):361-369.
[17]Elbein A D.The metabolism of trehalose[J].Adv in Carbohyd Chem Biochem,1974,30:227-229.
[18]Kane SM,Roth R.Carbohydrate metabolism during ascospore development in yeast.J.Bacterial,1974,118:8-14.
[19]ZimmermannFK Yeast Sugar Metabolism[M].Lancaster U.S.A:Technic Publishing Company Inc,1997,28(6):285-303.
[20]Lillie S H,et al Reserved carbohydrate metabolism in Saccharomyces Cerevisiae;Response to nutrient limitation[J].J.Bacterial,1980,26(4):1384-1394
[21]戴秀玉,程苹,周坚,等.海藻糖的生理功能、分子生物学研究与应用前景[J].微生物学通报,1995,22(2):102-104.
[22]WELSHE T,RONDNEYA H Osmotically induced intracellular trehalose,but not glycine betaine accumulation promotes desiccation tolerance in Escherichia coli[J].FEMS Microbiology Lesstters.1999,174(1):57-63.
[23]Wyatt GR,Kalf GF,The chemistry of insect hemolymph.Ⅱ Trehalose and other crbohydrates.Journal of General Physiology,1957,40:833-847
[24]聂凌鸿,宁正祥.海藻糖的生物保护作用[J].生命的化学,2001(3):206-208.
[25]Hounsa CG,Brandt EV,Thevelein J,et al.Role of trehalose in surival of Sacchromyces cerevisiae under osmotic stress.Microbiology,1998,144:671-680.
[26]Eroglu A,mehmet T,thomas LT.Beneficial effect of microinjected trehalose on the ctyosurival of human oocytes.Fertihty and Sterility,2002,77:152-158.
[27]李桂芹.海藻糖的生物学功能及其应用[J].安徽农业科学,2007,35(24):7724-7725
[28]PAUL V A.Trehalose accumulates in Saccharomyces ceretislae during expose to agents that nduce heat shock response[J].FEBS Letters,1987,225(1);259-263.
[29]LAERE A V.FRANCOIS A,OVERLOOP K,et al.Relation between germinination trehalose and the status of water in Phycomyces blakeleeanus as measureal by proton NMR[J].J Gener Microbiol,1987,133:239-245.
[30]Colaco CALS,Roser B.Trehalose-a multifunctional additive for food preservation.Food packaging and preservation,1995,123-140
[31]HOTTIGER T,SCHMUTZP,WIEMKEN A.Heat induced accmulation and futile cycling of trehalose in Saccharomyces cervisiae[J].J Bacterial,1987,169(12):5518-5520.
[32]Colaco C,Sen S,ThangavelnM,et al.Estraordinary stabilityof enzymes dried in trehalose:simplified molecular biology[J].Biotechnology,1992,10:1007-1011.
[33]唐传核.海藻糖及其应用[J].食品研究与开发,1998,20(4):16.
[34]李滢冰,冯梦醒,徐继祖.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.
[35]Allen-ind.Isolation of trehalose by alcohol crystallization-trehalose purification from fermentation broth.Derwent Biotechnol Abst,1995,14(2):01165.
[36]Murao S,Nagano S,et al.En2ymatic synthesis of trehalose form maltose.Agric Biol Chem.1985,49(7):2113-2118.
[37]缪静.海藻糖的制备方法[J].烟台师范学院学报(自然科学版),2000,15(1):60-63.
[38]陈杰,何聪芬,叶兴国,等.海藻糖及其在生物工程方面的应用[J].生物技术通报,2005,(6):29-33.
[39]Elbein AD,Pan YT,Pastuszak l,et al.New insights on trehalose:a multifunctionalmolecule.Glycobiology,2003,13:17-27.
[40]Kato M,Miura Y,Kettoku M,et al.Purification and characterization of new trehalose producing enzymes isolated from the hyperthermophilic archae,Sulfolobus solfataricus KMI.Biosciences,Biotechnology and Biochemistry,1996,60:546-550.
[41]简文杰,庞杰.海藻糖的生产及其应用[J].杭州食品科技,2003,(1):1-4.
[42]赵晓峰,吴荣书.海藻糖的功能特性及其应用[J].广州食品工业科技,2004,20(2):151-155.
[43]张树珍.海藻糖的研究进展及其应用前景[J].华南热带农业大学学报,2000(3):22-29.
[44]Garg AK,Kim JK,Owens TG,et al.Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses[J].Proc Natl Acad Sci USA,2002,99(25):15898 -15903.
[45]Guo N,Puhlev I,Brown DR,et al.Trehalose expression confers desiccation tolerance on human cells[J].Nat Biotechnol,2000,18(2):168-171.
[46]Matsuo T.Trehalose protects corneal epithelial cells from death by drying[J].Br.J.Ophthalmol,2001,85(5):610-612.
[47]袁勤生.隐生生物中的典型化合物海藻糖[J].中国生化药物杂志,1999,20(1):48-50.
[48]刘欣,周爱梅,赵力超,等.海藻糖、乳酸钠对冷藏鳙鱼鱼糜蛋白抗冷冻效果的影响[J].食品与发酵工业,2007,30(8):63-64
[49]高茹菲,陈舜胜.海藻糖抗冻作用研究[J].渔业现代化,2005,(4):48-49.
[50]戴秀玉,沈义国,周坚.海藻糖对乳酸菌的抗逆保护研究[J].微生物学通报,2001,4(2):465-470.
[51]李浩明,高蓝.海藻糖在生物制品干燥与保存中的研究[J].食品与发酵工业,1994,4(49):49-51.
[52]Colaco C.A.L.S,Smith C.J.S,Sen S.Chemistry of pritein stabilization by trehalose[J].American Chemical Society,1994,(567):222-240.
[53]周青峰.海藻糖在食品加工上的应用[J].中国食品工业,1996(2):22-23.
[54]陈黎忠,李羲光,边光珠,等.海藻糖对酶冻干品的保护作用[J].化工学报,2000,51(2):1931-97.
[55]权国波.海藻糖在哺乳动物细胞保存中的作用机制及应用现状[J].临床输血与检验,2003,5(1):78-80.
[56]唐传核,葛文光.从活性干酵母中提取海藻糖的工艺[J].无锡轻工业大学学报,1998,17(3):37-40.
[57]傅琳琳,傅开科.海藻糖的研究现状及其应用前景[J].江西科学,2000,18(3):182-85.
[58]魏杰.天然保存剂—海藻糖[J].安徽科技,1999,(3):24.
[59]宋祺,陈坚,伦世仪.海藻糖对面包酵母耐贮力的影响[J].无锡轻工业大学学报,1997,16(3):32-36.
[60]杨荣华,林家莲,周凌霄.海藻糖在食品工业中的应用[J].山西食品工业,1999,(4):27-28.
[61]James S.Cellular responses to extreme water loss[J].Cryobiology,1982,19:306-308.
[62]王岁楼.食品生物技术[M].海洋出版社:1998
[63]Richards AB,Krakowka S,Dexter LB,et al.Trehalose:a review of multiple safety studies.Food and Chemical Toxicology,2002,40:871-898.
[64]OKU T,MITSUKO O.Transitory laxative threshold of trehalose and lactulose in healthy women[J].J Nutr Sci&Vitaminol,1998,44(6) Dec:787-798.
[65]Uritani M,Takal M,Yoshinage K.Protective effect of disaccharides on restricition endonucleases during drying under vacuum.Jounal of Biochemistry,1995,117:774-779.
[66]李桂芹.海藻糖的生物学功能及其应用[J].安徽农业科学,2007,35(24):7724-7725.
[67]Hata Y,Nishimori Y,Okochi Y,et al.JP:4-290808.
[68]阚红玲,孙洪涛,董健军.海藻糖在化妆品中的应用[J].食品与药品,2005,7(9):48-50.
[69]衫本利行.食品工业,1997,10(3):1-4.
[70]Crowe J H,Crowe L M.Preservation of membranes in an hydrobiotic organism.the role of trehalose J.Science,1984,223:701-703.
[71]马文锦,刘树兴,潘巨忠,等.海藻糖的生产制备及应用前景[J].现代农业科技,2007,(24):199-201
[72]胡宗利,夏玉先,陈国平,等.海藻糖的生产制备及其应用前景[J].中国生物工程杂志,2004,(4):44-48.
[73]James S.Cellular responses to extreme water lossJ.Cryobiology,1982,19:306-308.
[74]Colaco C,Sen S,Thangaveln M,et al.Estraordinary stability of enzymes dried intrehalose.simplified molecular biologyJ.Biotechnology,1992,10:1007~1011.
[76]徐浩,王丽,江慧修,等.葡萄酒活性干酵母的生物学和工艺的研究[J].真菌学报,1990,9(1):73-78.
[77]Yokomise H,inui K,wada H,et al.Longterm eryopreservation can prevent rejection of canine tracheal allografts with preservation of graft viability.Journal of Throacic and Cardiovascular Surgergy,1996,111:930-934
[78]ADRIDGE S.Trehalose boots prospects for improved biopharmaceuticals and VaccinesJ.Cenetic Engineering News,1995,15:10-11.
[79]Garg AK,Kim JK,Owens TG,et al.Trehalose accumulation in rice Plantsconfers high tolerancelevels to different abiotic stresses.PNAS,2002,99(25):15898-15903.
[80]SERRANO R,FRANCISCO A,CULIANZ-MACIA,et al.Genetic engineering of salt and drought tolerance withyeast regulatory genes[J].Scientia Horticulturae,1999,78(1):261-269.
[81]俞建瑛,蒋宇,王善利.生物化学试验技术[M].化学工业出版社:2005.
[82]刘洋,张红缨,高俊芳,等.酵母中海藻糖的几种提取方法的比较[J].中国生化药物杂志,1999,20(1):15-16.
[83]ZHAN Li-ming(张立名).Analysis Principle of the Modem Biology Chemistry(现代生物化学分析原理)[M].China University of Science and Technology Press,1991.(in Chinese)
[84]杨云,刘福勤,冯卫生,等.碱法提取大枣渣多糖及活性炭脱色的工艺研究[J].食品与发酵工业,2004,30(7):30-32.
[85]王元凤,金征宇.茶多糖脱色的研究[J].食品与发酵工业,2004,8:61-65.
[86]彭卫红,罗婷,甘炳成,等.碳源和氮源对紫丁香蘑菌丝生长的影响研究[J].西南农业学报,2004,17:419-422.
[87]Lillie SH,Ringle JR.Reserverd carbohydrat metabolism in Saccharomyces cerevisiae:Responses to nutrient limitation.J Bacteriol,1980,143(3):1384-1394.
[88]周伟.从活性干酵母中提取海藻糖的工艺研究:[硕士学位论文].浙江:浙江大学,2001.
[89]何芸,姚开,贾冬英,等.抗鱼肉蛋白冷冻变性机理的研究进展[J].氨基酸和生物资源,2007,29(2):36-39
[90]何来英,严卫星,楼密密,等.保健食品抗疲劳作用试验方法研究[J].中国食品卫生杂志,1997,9(4):1-6.