真菌A-O1的鉴定、培养及发酵产物的初步研究
摘要
如今,由微生物发酵所得到的天然色素,因其自身具有来源丰富、安全且对环境无污染和可持续发展等诸多优点,被广泛使用。本论文以从土壤中分离得到的供试菌株为研究对象,对该菌株进行了鉴定并对其发酵条件和产物稳定性进行了研究,所得结果如下：
1、采用形态学和分子生物学鉴定的方法,对一株产酸碱指示剂的真菌进行了鉴定。结果表明,该菌株的无性态为串珠镰孢(Fusarium Moniliforme),接种后12小时左右,产孢细胞开始萌生,呈乳头状或角锥形,其形状上细下粗。小型分生饱子链状着生或假头状着生。通过该菌株rDNA ITS的PCR扩增和序列测定,与NCBI网站进行同源性比对分析,结果表明待测菌株与藤仓赤霉菌(Gibberella fujikuroi)同源性为100%,确定该菌株为藤仓赤霉菌。
2、为了进一步提高色素产量,对A-01菌株的培养条件进行了优化。以色素在紫外分光光度下OD485nm值为指标,筛选出的最佳培养条件为固体培养基是PDA,液体培养基是马铃薯葡萄糖培养基,最佳碳源为蔗糖,最佳氮源为鱼粉,最佳培养温度为28℃,最佳的pH值为6.5,最佳发酵时间为120h,最佳发酵转速为180r/min,最佳装液量为75mL/250mL,为了达到最佳配比,对碳源浓度、氮源浓度、温度、pH值四个因素进行了正交试验,其中温度对产色素的影响最大,其次依次是：碳源,pH值和氮源。正交试验的结果显示最佳配比是：发酵温度28℃,初始pH值6.5,2.5%蔗糖,2.5%鱼粉。
3、A-01菌株发酵产物的稳定性实验结果表明,该发酵产物的热稳定性较好,但随着温度的升高,色素的稳定性减弱。在偏中性条件下,色素稳定性较强,色素在强酸强碱条件下都不稳定。紫外线对色素的稳定性几乎没有影响,说明色素不会被紫外线分解。日光的照射不会对色素的稳定性造成影响。Na+、Fe3+对色素的稳定性有一定的破坏作用,其中K+、Mg2+对色素的稳定性具有较大的破坏作用,但不因时间的延长而产生进一步的破坏。而Ca2+对色素的稳定性没有影响,能起到一定的稳定色素的作用。柠檬酸对色素的稳定性无影响,苯甲酸钠对色素的稳定性有一定的破坏作用,但随着时间的延长并不会产生更大的破坏；而蔗糖、葡萄糖不但不会破坏色素的稳定性,相反,还有一定的增色作用,并且不随时间的延长而受影响。
Nowadays, the natural pigment obtained by microbial fermentation, for its rich resource, safety,no pollution to the environment and sustainable development are widely used. The tested strains were isolated from soil which have been identified, its fermentation conditions and the stability of product were studied,the results are as follows:
1.An identification of morphological and molecular biology methods, to produce an acid-base indicator fungus were identified. The results showed that the strain asexual state Fusarium is Fusarium Moniliforme, about12hours after inoculation, the spore cell begin to initiate, were papillary or pyramidal, its shape fine and coarse. Small conidia sub-chain with raw or slimy heads.Homology analysis by PCR amplification and sequencing of the rDNA ITS of the strains, with the NCBI website, the results show that the homology of the test strains and Fujikura Fusarium, is100%,determine the strain is Fujikura Fusarium.
2.In order to improve the production of pigment, the A-01strain culture conditions are optimized. OD485nm value indicators pigment under ultraviolet spectrophotometry, filter out the best culture conditions for the solid culture is PDA, the best liquid medium is potato dextrose medium, the best carbon source is sucrose, the best nitrogen source is fishmeal, the optimum temperature is28℃, the optimum pH value is6.5, the optimum fermentation time is120h, the optimum fermentation speed is180r/min, the optimum liquid volume is75mL/250mL.In order to obtain the best ratio,the four factors of carbon source concentration, nitrogen source concentration, temperature, pH value of the orthogonal experiment, in which the greatest impact on the production of pigment is temperature, followed in turn:carbon source, pH and nitrogen source.The orthogonal experiment results show that the best ratio is:fermentation temperature28°C, initial pH6.5,2.5%sucrose,2.5%fishmeal.
3.A-01fermentation product stability experiment results show that the thermal stability of fermentation product is stable, however, as the temperature rises, the stability of pigment is weaker. In neutral pH conditions, the stability of the pigment is more stable, the pigment is not stable in acid and alkali conditions. The stability of the pigment under UV has almost no effect, indicating the pigment does not decompose by UV. Sunlight radiation does not affect the stability of the pigment. Na+and Fe3+damage the stability of pigment,K+and Mg+greatly damage the stability of pigment, but not produce more damage with the extension of time. Ca2+has no effect on the stability of pigment, can play a role of the stability of pigment. Citric acid has no effect on the stability of pigment, sodium benzoate has effect on the stability of pigment, but not produce more damage with the extension of time; sucrose and glucose do not damage the stability of pigment, On the contrary, there is a certain hyperchromic effect and is not affected with the extension of time.
1、采用形态学和分子生物学鉴定的方法,对一株产酸碱指示剂的真菌进行了鉴定。结果表明,该菌株的无性态为串珠镰孢(Fusarium Moniliforme),接种后12小时左右,产孢细胞开始萌生,呈乳头状或角锥形,其形状上细下粗。小型分生饱子链状着生或假头状着生。通过该菌株rDNA ITS的PCR扩增和序列测定,与NCBI网站进行同源性比对分析,结果表明待测菌株与藤仓赤霉菌(Gibberella fujikuroi)同源性为100%,确定该菌株为藤仓赤霉菌。
2、为了进一步提高色素产量,对A-01菌株的培养条件进行了优化。以色素在紫外分光光度下OD485nm值为指标,筛选出的最佳培养条件为固体培养基是PDA,液体培养基是马铃薯葡萄糖培养基,最佳碳源为蔗糖,最佳氮源为鱼粉,最佳培养温度为28℃,最佳的pH值为6.5,最佳发酵时间为120h,最佳发酵转速为180r/min,最佳装液量为75mL/250mL,为了达到最佳配比,对碳源浓度、氮源浓度、温度、pH值四个因素进行了正交试验,其中温度对产色素的影响最大,其次依次是：碳源,pH值和氮源。正交试验的结果显示最佳配比是：发酵温度28℃,初始pH值6.5,2.5%蔗糖,2.5%鱼粉。
3、A-01菌株发酵产物的稳定性实验结果表明,该发酵产物的热稳定性较好,但随着温度的升高,色素的稳定性减弱。在偏中性条件下,色素稳定性较强,色素在强酸强碱条件下都不稳定。紫外线对色素的稳定性几乎没有影响,说明色素不会被紫外线分解。日光的照射不会对色素的稳定性造成影响。Na+、Fe3+对色素的稳定性有一定的破坏作用,其中K+、Mg2+对色素的稳定性具有较大的破坏作用,但不因时间的延长而产生进一步的破坏。而Ca2+对色素的稳定性没有影响,能起到一定的稳定色素的作用。柠檬酸对色素的稳定性无影响,苯甲酸钠对色素的稳定性有一定的破坏作用,但随着时间的延长并不会产生更大的破坏；而蔗糖、葡萄糖不但不会破坏色素的稳定性,相反,还有一定的增色作用,并且不随时间的延长而受影响。
Nowadays, the natural pigment obtained by microbial fermentation, for its rich resource, safety,no pollution to the environment and sustainable development are widely used. The tested strains were isolated from soil which have been identified, its fermentation conditions and the stability of product were studied,the results are as follows:
1.An identification of morphological and molecular biology methods, to produce an acid-base indicator fungus were identified. The results showed that the strain asexual state Fusarium is Fusarium Moniliforme, about12hours after inoculation, the spore cell begin to initiate, were papillary or pyramidal, its shape fine and coarse. Small conidia sub-chain with raw or slimy heads.Homology analysis by PCR amplification and sequencing of the rDNA ITS of the strains, with the NCBI website, the results show that the homology of the test strains and Fujikura Fusarium, is100%,determine the strain is Fujikura Fusarium.
2.In order to improve the production of pigment, the A-01strain culture conditions are optimized. OD485nm value indicators pigment under ultraviolet spectrophotometry, filter out the best culture conditions for the solid culture is PDA, the best liquid medium is potato dextrose medium, the best carbon source is sucrose, the best nitrogen source is fishmeal, the optimum temperature is28℃, the optimum pH value is6.5, the optimum fermentation time is120h, the optimum fermentation speed is180r/min, the optimum liquid volume is75mL/250mL.In order to obtain the best ratio,the four factors of carbon source concentration, nitrogen source concentration, temperature, pH value of the orthogonal experiment, in which the greatest impact on the production of pigment is temperature, followed in turn:carbon source, pH and nitrogen source.The orthogonal experiment results show that the best ratio is:fermentation temperature28°C, initial pH6.5,2.5%sucrose,2.5%fishmeal.
3.A-01fermentation product stability experiment results show that the thermal stability of fermentation product is stable, however, as the temperature rises, the stability of pigment is weaker. In neutral pH conditions, the stability of the pigment is more stable, the pigment is not stable in acid and alkali conditions. The stability of the pigment under UV has almost no effect, indicating the pigment does not decompose by UV. Sunlight radiation does not affect the stability of the pigment. Na+and Fe3+damage the stability of pigment,K+and Mg+greatly damage the stability of pigment, but not produce more damage with the extension of time. Ca2+has no effect on the stability of pigment, can play a role of the stability of pigment. Citric acid has no effect on the stability of pigment, sodium benzoate has effect on the stability of pigment, but not produce more damage with the extension of time; sucrose and glucose do not damage the stability of pigment, On the contrary, there is a certain hyperchromic effect and is not affected with the extension of time.
引文
[1]刘志皋,高彦徉.食品添加剂基础[M].北京：中国轻工业出版社,1994.
[2]阎炳宗.我国天然色素的现状及发展方针[J].精细与专用化学品,1999,9：8-9.
[3]刘志皋.食用色素发展概况[J].食品工业科技,1987,2：61-62.
[4]蔺定运.食用色素的识别与应用.北京：中国食品出版社,1987,2-12,119-129.
[5]车双辉.杜琪珍,夏明.天然蓝色素研究进展[J].食品研究与开发,2003,24(2)：18-20.
[6]石永峰.国外食品天然色素的研究进展[J].粮食与食品工业,1996,4：23-27.
[7]Armion D M.Handbook of U.S.Colorants for food drugs,and cosmetics,1984.
[8]黄小凤,李中林.天然食用色素的现状与展望[J].化工时刊,1991,12：12-16.
[9]张华,李景琳对食用天然色素研究与开发的思考[J].辽宁农业科学,1998,6：27-30.
[10]钟家珍.茶树食用色素的提取与研究[T].湖北林业科技,1994,3：7-9.
[11]魏春维,刘洪章,孙晓秋等.黑穗醋栗色索浸膏的色价测定及氨基酸分析[J].中国林副特产,1992.3：7-9.
[12]郑永军.一串红天然红色素的研究[J].食品业工科技,1997,6：37-38.
[13]杨志祥.枸杞子色素的研究[J].食品工业科技,1997,6：39-40.
[14]天津轻工业学院食品工业教学研究室编.食品添加剂[M].第2版.北京：轻工业出版社,1987.
[15]李爱华.浅谈我国食品添加剂的现状和发展趋势[J].食品科学,1995,(10)：5-9.
[16]聂晶.食用合成色素研究动态(综述)[J].中国食品卫生杂志,2002,(1)：58-60.
[17]潘淮.健康的美丽杀手——合成色素[J].广州食品工业科技,2003,(1)：5-55.
[18]Watford.J.Historican.Development of Food Coloration,In:Development in Food colors [M].London,UK:Applied Science publishers,1980,1:1-25.
[19]张华等.对食用天然色素研究与开发的思考[J].辽宁农业科学,1998,(6)：27-30.
[20]聂晶,齐兴娟.食用合成色素研究动态[J].中国卫生检验杂志.2002,14(1)：8-60.
[21]章钰.天然食品防腐剂[J].食品科技,1992,6：23.
[22]唐纯,丁建平.茶色素治疗冠心病心绞痛及高血脂血症的疗效[J].湖南医学,1999,16：38-39.
[23]Bartley G E,Scolnik P A.Plant carolenoids:pigments for photoprotection,visual attraction and human health[J].Plant Cell,1995,7:1027-1038.
[24]Niyogi K.K.Photoprotection revisited[J].Annu Rev Plant Physiol Plant Mol Biol,1999,50:391-417.
[25]Giovannucci E.Tomatoes,tomato-based products,lcopene and cancer:review of the Epiderniologi literature[J].J Natl Cancer lnst,1999,91:317-331.
[26]Arab L,Stech S.Lycopene and cardiovascular disease[J].Am J ClimNutr,2000,71:1691-1695.
[27]Franceschi S,Bidoli E.Tomatoes and risk of digestive-tract cancers[J].Int J Cancer,1994,59(2):181-184.
[28]Gerster H.Potential role of beta-carotene in the prevention of cardiovascular disease[J].Internat J Vit Nutr Res,1991,612:277-291.
[29]Heinonen O P,Alnanes D.The effect of vitamin E and beta-carotene on the incidence of lung cancer and other cancer in male smokers[J].New Eng Med,1994,330:1029-1035.
[30]Gilbert S.Effects of a combination of beta-carotene and vitamin A on lung cancer and cardiovascular disease[J].Eng J Med,1996,334:1150-1155.
[31]Anthony T D.Fat soluble vitamins,their biochemistry and application[J]. Trowbridge: Redwood Burn Ltd,1985,15:21-28.
[32]Blot W J,Li J Y.Nutrition intervention trials in Linxian,China:supplementation of specific vitamin/mineral combinations,cancer incidence and disease specific mortality in the general population[J].J Natl Cancer Inst,1993,85:14483-14492.
[33]Nagasawa H,Mitamura T,Sakamoto S.Effects of lycopene on spontaneous mammary tumor development in SHM vergin mice[J].Anticancer Res,1995,15:1173-1178.
[34]Sharoni Y, Giron E,Rise M.Effects of lycopene-enriched tomato[J].Cancer Detect Prev,1997,21:118-123.
[35]Clinton S k. Lycopene:chemistry,biology,and implications for human health and disease[J].Nutr Rev,,1998,56:35-51.
[36]Riso P.Pinder A.Does tomato consumption effectively increase the resistance of lymphocyte DNA to oxidative damage[J].Am J Clin Nutr,1999,69:712-718.
[37]Watzl B,Bub A.Modulation of human T-lymphocyte functions by the consumption of carotedoid—rich vegetables[J].Br J Nutr,1999,82:383-389.
[38]Porini M,Riso P. Lymphocyte lycopene concentration and DNA protection from oxidative damage is increased in women after a short period of tomato consumption[J].J Natr,2000,130:189-192.
[39]曾卫军.新疆天然食用植物色素资源的开发与利用.新疆师范大学学生科研论文,2000,3-9.
[40]Lapidot T,Hurel S,Ariri B,et al.pH-dependent forms of red wine anthocyanins as antioxidants[J].Agric Food Chem,1999,47:67-70.
[41]Saint C,Gaulejal N,Glories Y,et al.Free radical scavenging effect of anthocyanins in red wines[J].Food Research International,1999,32:327-333.
[42]Halliwell B.Free radicals and antioxidants:a personal view[J].Nutr Rev,1994, 52:253-265.
[43]Wang H,Gao G, Prior R 1.Oxygen radical absorbing capacity of anthocyanins[J].Agric Food Chem,1997,45:304-309.
[44]Yoshimoto M.Antimutagenicity of sweet potato roots[J].Biosci Biotechnol Biochem, 1999,63:537-541.
[45]Wang C J.Protective effect of hibiscus anthocyanins against tea-butyl hydroperoxide induced hepatic toxicity in rats[J].Food and Chemical Toxicology,2000,38:411-416.
[46]Igarashi k,Kimura Y. Takenaka anthocyanins On paraquat-induced A.Preventive effects of dietary cabbage acylated oxidative stress in rats[J].Biosci Biotechnol Biochem,2000,64:1600-1607.
[47]Wesche-Ebeling P Montgomery M W. Strawberry polyphenoloxidase:its role inanthocyanin degradation[J].Food Sci,1990,55:731-734.
[48]Cliford M N.Review:anthocyanins-nature,occurrence and dietary burden[J].Sci Food Agric,2000,80:1063-1070.
[49]Ghiselli A,Nardini M,Baldi A,et al. Antioxidant activity of different phenolic fractions separated from an Italian red wine[J].Agric Food Chem,1998,46:361- 367.
[50]Kayamori F, Igarashi K.Effects of dietary nasunin on the serum cholesterol level in rats[J].Biosci Biotechnol Biochem,1994,58:570-571.
[51]Nair M.A sweeter pill to swallow[J].Chem Brit 1999,35:16-18.
[52]Yoshimoto M.Antimutagenicity of sweetpotato roots[J].Biosci Biotechnol Biochem, 1999,63:537-541.
[53]Wang H,Gao G, Prior R L.Oxygen radical absorbing capacity of anthocyanins[J].J Agric Food them,1997,45:304-309.
[54]黄小凤,李中林.天然食用色索的现状与展望[J].化工时刊1991,12：12-16.
[55]Wang C J.Protective effect of hibiscus anthocyanins against tert-butyl hydroperoxide induced hepatic toxicity in rats[J].Food and Chemical Toxicology,2000,38:411-416.
[56]张立彦,丙汉明.食用色素的发展及在生产中的应用[J].食品与机械,1998,23(1)：32-34.
[57]胡隆基.食用天然色素发展动态[J].食品科技,1995,(2)：10-17.
[58]余焕玲,杨参.食用天然色素的应用[J].广州食品科技,2001,17(3)：63-65.
[59]刘毅,宁正祥.红曲色素在肉制品中的应用[J].食品与机械,1999,1：28-30.
[60]Lapidot T,Hurel S,Ariri B,et al.pH-dependent forms of red wine anthocyanins as antioxidants[J].Agric Food Chem,1999,47:67-70.
[61]Saint C,Gaulejal N,Glories Y,et al.Free radical scavenging effect of anthocyanins in red wines[J].Food Research International,1999,32:327-333.
[62]Yoshimoto M.Antimutagenicity of sweet potato roots[J].Biosci Biotech Biochem, 1999,63:537-541.
[63]Wang C J.Protective effect of hibiscus anthocyanins against tert-butyl hydroperoxide induced hepatic toxicity in rats[J].Food and Chemical Toxicology,2000,38:411-416.
[64]Igarashi K, Kumura Y, Takenaka A.Preventive effect of dietary cabbage acylate, anthochanins on paraquat-induced oxidative stress in rats[J].Biosci Biotechnol Biochem,2000,64:1600-1607.
[65]江苏南,杨计军.天然色素化学及其在纺织品上的应用(二)[J].印染,1997,23(7)：41-43.
[66]Leonid V Bystykh,Jan K Herrema,Wim Kruizinga,et al 5-Hydroxyaloesaponarin Ⅱ、a minor blue pigment in an actinorhodin-negative mutant of Streptomyces coelicolor A3(2)[J].Biotechnol Appl Biochem,1997,26:195-201.
[67]刘毅,宁正祥.红曲霉素发酵产物的保健作用及应用[J].广州食品工业科技,1999,15(2)：1-4.
[68]杨志孝.花生内衣色素提取[J].食品科学,1992,10：23-24.
[69]孙智达,何勇.汤饭子果实中天然红色素的研究[J].湖北农业科学,1996,5：53-56.
[70]周兆祥.乌饭树色素的研究[J].食品与发酵工业,1989,6：54-59.
[71]徐幸莲.鸟骨鸡黑素对延缓果蝇衰老的作用[J].南京农业大学学报：自然版.
[72]汪振诚,陈永刚,刘子贻等.虾壳红色素的抗氧化和抗肿瘤作用[J].东海海洋,1999,17(2)：26-29.
[73]龙盛京,马文力,农冠荣.黑豆色素及多糖对全血化学发光和活性氧的抑制作用[J].食品科学,1999,20(9)：9-12.
[74]钱萤.黑色食品与浅色食品抗氧自由基作用的比较[J].广西大学学报,1996, 21(3)：297.
[75]龙盛京.几种赤色与浅色食品抗活性氧效能的比较研究[J].营养学报,1997,19(4)：472.
[76]谢俊杰.食品防腐新领域一微生物天然防腐剂[J].食品科学,1999,(1)：13-14.
[77]王君,张宝善.微生物生产天然色素的进展[J].微生物学通报,2007,34(3)：580-583.
[78]Armstrong,G A,Alberti M,Leach F,et al.Nucleotide sequence,organization,and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter, Capsulatus[J].Mol Gen Genet,1989,216:254.
[79]Misawa N,Nakagawa M,Kobayashi K,et al.Elucidation Of the Erwiniauredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia[J]. J Bacteriol,1990,172.6704-6712.
[80]Salanoubat M,Genin S,Artiguenave F,et al.Genome sequence of the plant pathogen Ralstonia solanacearum[J].Nature,2002,415:497-502.
[81]Vasconeelos D F,Almeida m,Hungria C T,et al.The complete genome sequence of chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability[J].PNAS,2003,100:11660-11665.
[82]Ensley B D,Ratzkin B J,Osslund T D,et al.Expression of naphthalene Oxidation genes in Escherichia coli results in the biosynthesis of indigo [J].Science,1983,222: 167-169.
[83]Choi H S,Kim J K,Cho E H,et al.A novel flavin-containing monooxy genase from Methylophaga sp.strain SKI and its indigo synthesis in Escherichia coli[J].Biochem Bioph Res Com,2003,306:930-936.
[84]Del campo J A,Rodrfguez H,Moreno J,et al.Appl Microbiol Biotechnol.2004, 64:848-854.
[85]魏力,方加玮,周俊初等.一株海洋细菌的初步鉴定及其产黑色素相关新基因(簇)的分离[J].微生物学通报,2007(6)：1118-1122.
[86]李建波,宋欣,曲音波.微生物学通报,2004,31(1)：50-54.
[87]张亮,朱湘民.微生物学通报,1999,26(5)：332-335.
[88]袁保红,杜青平,蔡创华.海洋通报,2005,24(6)：92-96.
[89]仓一华,陆玲,王伟.一株产类胡萝卜素酵母Rhodotorula.sp色素的组分及摇瓶发酵研究[J],食品科学,2002,23(5)：34-38.
[90]Buzzini P.An optimization study of carotenoid production by Rhodotorula glutinis DBVPG 3853 from substrates containing concentrated rectified grape must as the sole carbohydrate source[J],J Ind Microbiol Biotechnol,2000,24(1):41-45.
[91]陈建军,杨济龙.产番茄红素红酵母培养条件研究[J],云南农业大学学报,2005,20(3)：381-383.
[92]曾强松,童海宝,徐静安.三孢布拉霉发酵生产p-胡萝卜素工艺研究[J],工业微生物,2002,32(4)：7-10.
[93]张雪霞,刘刚叁,李宁.中国新药杂志,2005,14(7)：880-881.
[94]Salguero A,Leon R,Mariotti A,et al.Appl Microbiol Biotechnol,2005,66:506-511.
[95]Fbregas J,Domnguez A,Maseda A,et al.Interactions between irradiance andnutrient availability during astaxanthin accumulation and degradation in Haematococcus pluvialis[J],Applied Microbiology and Biotechnology,2003,61(5-6):545-551.
[96]Simova E D,Frengova G I,Besh D M.Zeitschrift fur Naturforsch C.A Journal of Biosciences,2003,58:225-229.
[97]Squina F M,Mercadante A Z.Journal of Food Biochemistry,2005,29(6):638-652.
[98]Orosa M,Franqueira D,Cid A,et al.Bioresource Technology,2005,96:373-378.
[99]Bhosale P,Gadre R V.β-Carotene production in sugarcane molasses by a Rhodotorula glutinis mutant[J], Journal of Industrial Microbiology and Biotechnology, 2001,26(6):327-332.
[100]Silva C,Cabral J M S,Isolation of aβ-Carotene Over-Producing Soil Bacterium, Sphingomonas sp.[J],Keulen F V.Biotechnology Letters,2004,26(3):257-262.
[101]Kang C D,Lee J S,Park T H,et al.Comparison of heterotrophic and photo autotrophic induction on astaxanthin production by Haematococcus pluvialis[J], Applied Microbiology and Biotechnology,2005,68(2):237-241.
[102]王克明,庄树宏.PVA固定化红曲霉发酵生产红曲色素的研究[J],烟台大学学报(自然科学与工程版),1998,11(4)：306-309.
[103]张子儒,郑巧东,姚善泾.红曲霉菌微胶囊化培养[J],食品发酵与工业,2003,29(11)：1-4.
[104]Shin C S,Kim H J,Kim M J,et al.Biotechnology and Bioengineering,1998,59:576-581.
[105]Suh J H,Shin C S.FEMS Microbiology Letters,2000,190:241-245.
[106]Frengova G I,Emilina S D,Beshkova D M.Synthesis of carotenoids by Rhodotorula rubra GED8 co-cultured with yogurt starter cultures in which ultrafiltrate [J]. Zeitschrift fur Naturforschung.C.A Journal of science,2003,58:562-567.
[107]金敬宏,张卫明.桂油提取新工艺[J].现代科技,1998,10,(7)：36-39.
[108]余焕玲,杨参,阐建全.食品中食用天然色素的作用[J].广州食品工业科技,2001,17(3)：63-65.
[109]谭建军.食用色素的性质及其应用[J].化学教育,1998,(2)：1-4.
[110]杨志娟.我国天然色素的现状与发展方针[D].《食品研究与开发》,2003,4,(24)：2.
[111]刘志明,张瑞,张金利.落葵红色素作为酸碱指示剂的研究[J].农产品加工(学刊)2007(8)：21-23.
[112]魏景超.真菌鉴定手册[M].上海：上海科学技术出版社,1979.
[113]HL巴尼特,BB亨特.半知菌属图解[M].北京：科学出版社,1977.
[114]刘婵,王博,段青,等.滇山茶基因组DNA不同提取方法效果比较[J].江苏农业科学.2010(6)：49-50.
[115]Yu J N.Huang J.Wang Z N,et 11.An Na+/H+anfiporter gene from wheat plays an important role in stress tolerance[J].J Bioeei,2007,32(6):1153-1161.
[116]汪文俊,王广策,张宝玉,等.海带栽培品系和长海带ITS区的PCR扩增及序列分析.高技术通讯[J].2005(4)：93-101.
[117]李春喜,王志和,文林.生物统计学(第二版)[M].科学出版社.2000,149-156.
[2]阎炳宗.我国天然色素的现状及发展方针[J].精细与专用化学品,1999,9：8-9.
[3]刘志皋.食用色素发展概况[J].食品工业科技,1987,2：61-62.
[4]蔺定运.食用色素的识别与应用.北京：中国食品出版社,1987,2-12,119-129.
[5]车双辉.杜琪珍,夏明.天然蓝色素研究进展[J].食品研究与开发,2003,24(2)：18-20.
[6]石永峰.国外食品天然色素的研究进展[J].粮食与食品工业,1996,4：23-27.
[7]Armion D M.Handbook of U.S.Colorants for food drugs,and cosmetics,1984.
[8]黄小凤,李中林.天然食用色素的现状与展望[J].化工时刊,1991,12：12-16.
[9]张华,李景琳对食用天然色素研究与开发的思考[J].辽宁农业科学,1998,6：27-30.
[10]钟家珍.茶树食用色素的提取与研究[T].湖北林业科技,1994,3：7-9.
[11]魏春维,刘洪章,孙晓秋等.黑穗醋栗色索浸膏的色价测定及氨基酸分析[J].中国林副特产,1992.3：7-9.
[12]郑永军.一串红天然红色素的研究[J].食品业工科技,1997,6：37-38.
[13]杨志祥.枸杞子色素的研究[J].食品工业科技,1997,6：39-40.
[14]天津轻工业学院食品工业教学研究室编.食品添加剂[M].第2版.北京：轻工业出版社,1987.
[15]李爱华.浅谈我国食品添加剂的现状和发展趋势[J].食品科学,1995,(10)：5-9.
[16]聂晶.食用合成色素研究动态(综述)[J].中国食品卫生杂志,2002,(1)：58-60.
[17]潘淮.健康的美丽杀手——合成色素[J].广州食品工业科技,2003,(1)：5-55.
[18]Watford.J.Historican.Development of Food Coloration,In:Development in Food colors [M].London,UK:Applied Science publishers,1980,1:1-25.
[19]张华等.对食用天然色素研究与开发的思考[J].辽宁农业科学,1998,(6)：27-30.
[20]聂晶,齐兴娟.食用合成色素研究动态[J].中国卫生检验杂志.2002,14(1)：8-60.
[21]章钰.天然食品防腐剂[J].食品科技,1992,6：23.
[22]唐纯,丁建平.茶色素治疗冠心病心绞痛及高血脂血症的疗效[J].湖南医学,1999,16：38-39.
[23]Bartley G E,Scolnik P A.Plant carolenoids:pigments for photoprotection,visual attraction and human health[J].Plant Cell,1995,7:1027-1038.
[24]Niyogi K.K.Photoprotection revisited[J].Annu Rev Plant Physiol Plant Mol Biol,1999,50:391-417.
[25]Giovannucci E.Tomatoes,tomato-based products,lcopene and cancer:review of the Epiderniologi literature[J].J Natl Cancer lnst,1999,91:317-331.
[26]Arab L,Stech S.Lycopene and cardiovascular disease[J].Am J ClimNutr,2000,71:1691-1695.
[27]Franceschi S,Bidoli E.Tomatoes and risk of digestive-tract cancers[J].Int J Cancer,1994,59(2):181-184.
[28]Gerster H.Potential role of beta-carotene in the prevention of cardiovascular disease[J].Internat J Vit Nutr Res,1991,612:277-291.
[29]Heinonen O P,Alnanes D.The effect of vitamin E and beta-carotene on the incidence of lung cancer and other cancer in male smokers[J].New Eng Med,1994,330:1029-1035.
[30]Gilbert S.Effects of a combination of beta-carotene and vitamin A on lung cancer and cardiovascular disease[J].Eng J Med,1996,334:1150-1155.
[31]Anthony T D.Fat soluble vitamins,their biochemistry and application[J]. Trowbridge: Redwood Burn Ltd,1985,15:21-28.
[32]Blot W J,Li J Y.Nutrition intervention trials in Linxian,China:supplementation of specific vitamin/mineral combinations,cancer incidence and disease specific mortality in the general population[J].J Natl Cancer Inst,1993,85:14483-14492.
[33]Nagasawa H,Mitamura T,Sakamoto S.Effects of lycopene on spontaneous mammary tumor development in SHM vergin mice[J].Anticancer Res,1995,15:1173-1178.
[34]Sharoni Y, Giron E,Rise M.Effects of lycopene-enriched tomato[J].Cancer Detect Prev,1997,21:118-123.
[35]Clinton S k. Lycopene:chemistry,biology,and implications for human health and disease[J].Nutr Rev,,1998,56:35-51.
[36]Riso P.Pinder A.Does tomato consumption effectively increase the resistance of lymphocyte DNA to oxidative damage[J].Am J Clin Nutr,1999,69:712-718.
[37]Watzl B,Bub A.Modulation of human T-lymphocyte functions by the consumption of carotedoid—rich vegetables[J].Br J Nutr,1999,82:383-389.
[38]Porini M,Riso P. Lymphocyte lycopene concentration and DNA protection from oxidative damage is increased in women after a short period of tomato consumption[J].J Natr,2000,130:189-192.
[39]曾卫军.新疆天然食用植物色素资源的开发与利用.新疆师范大学学生科研论文,2000,3-9.
[40]Lapidot T,Hurel S,Ariri B,et al.pH-dependent forms of red wine anthocyanins as antioxidants[J].Agric Food Chem,1999,47:67-70.
[41]Saint C,Gaulejal N,Glories Y,et al.Free radical scavenging effect of anthocyanins in red wines[J].Food Research International,1999,32:327-333.
[42]Halliwell B.Free radicals and antioxidants:a personal view[J].Nutr Rev,1994, 52:253-265.
[43]Wang H,Gao G, Prior R 1.Oxygen radical absorbing capacity of anthocyanins[J].Agric Food Chem,1997,45:304-309.
[44]Yoshimoto M.Antimutagenicity of sweet potato roots[J].Biosci Biotechnol Biochem, 1999,63:537-541.
[45]Wang C J.Protective effect of hibiscus anthocyanins against tea-butyl hydroperoxide induced hepatic toxicity in rats[J].Food and Chemical Toxicology,2000,38:411-416.
[46]Igarashi k,Kimura Y. Takenaka anthocyanins On paraquat-induced A.Preventive effects of dietary cabbage acylated oxidative stress in rats[J].Biosci Biotechnol Biochem,2000,64:1600-1607.
[47]Wesche-Ebeling P Montgomery M W. Strawberry polyphenoloxidase:its role inanthocyanin degradation[J].Food Sci,1990,55:731-734.
[48]Cliford M N.Review:anthocyanins-nature,occurrence and dietary burden[J].Sci Food Agric,2000,80:1063-1070.
[49]Ghiselli A,Nardini M,Baldi A,et al. Antioxidant activity of different phenolic fractions separated from an Italian red wine[J].Agric Food Chem,1998,46:361- 367.
[50]Kayamori F, Igarashi K.Effects of dietary nasunin on the serum cholesterol level in rats[J].Biosci Biotechnol Biochem,1994,58:570-571.
[51]Nair M.A sweeter pill to swallow[J].Chem Brit 1999,35:16-18.
[52]Yoshimoto M.Antimutagenicity of sweetpotato roots[J].Biosci Biotechnol Biochem, 1999,63:537-541.
[53]Wang H,Gao G, Prior R L.Oxygen radical absorbing capacity of anthocyanins[J].J Agric Food them,1997,45:304-309.
[54]黄小凤,李中林.天然食用色索的现状与展望[J].化工时刊1991,12：12-16.
[55]Wang C J.Protective effect of hibiscus anthocyanins against tert-butyl hydroperoxide induced hepatic toxicity in rats[J].Food and Chemical Toxicology,2000,38:411-416.
[56]张立彦,丙汉明.食用色素的发展及在生产中的应用[J].食品与机械,1998,23(1)：32-34.
[57]胡隆基.食用天然色素发展动态[J].食品科技,1995,(2)：10-17.
[58]余焕玲,杨参.食用天然色素的应用[J].广州食品科技,2001,17(3)：63-65.
[59]刘毅,宁正祥.红曲色素在肉制品中的应用[J].食品与机械,1999,1：28-30.
[60]Lapidot T,Hurel S,Ariri B,et al.pH-dependent forms of red wine anthocyanins as antioxidants[J].Agric Food Chem,1999,47:67-70.
[61]Saint C,Gaulejal N,Glories Y,et al.Free radical scavenging effect of anthocyanins in red wines[J].Food Research International,1999,32:327-333.
[62]Yoshimoto M.Antimutagenicity of sweet potato roots[J].Biosci Biotech Biochem, 1999,63:537-541.
[63]Wang C J.Protective effect of hibiscus anthocyanins against tert-butyl hydroperoxide induced hepatic toxicity in rats[J].Food and Chemical Toxicology,2000,38:411-416.
[64]Igarashi K, Kumura Y, Takenaka A.Preventive effect of dietary cabbage acylate, anthochanins on paraquat-induced oxidative stress in rats[J].Biosci Biotechnol Biochem,2000,64:1600-1607.
[65]江苏南,杨计军.天然色素化学及其在纺织品上的应用(二)[J].印染,1997,23(7)：41-43.
[66]Leonid V Bystykh,Jan K Herrema,Wim Kruizinga,et al 5-Hydroxyaloesaponarin Ⅱ、a minor blue pigment in an actinorhodin-negative mutant of Streptomyces coelicolor A3(2)[J].Biotechnol Appl Biochem,1997,26:195-201.
[67]刘毅,宁正祥.红曲霉素发酵产物的保健作用及应用[J].广州食品工业科技,1999,15(2)：1-4.
[68]杨志孝.花生内衣色素提取[J].食品科学,1992,10：23-24.
[69]孙智达,何勇.汤饭子果实中天然红色素的研究[J].湖北农业科学,1996,5：53-56.
[70]周兆祥.乌饭树色素的研究[J].食品与发酵工业,1989,6：54-59.
[71]徐幸莲.鸟骨鸡黑素对延缓果蝇衰老的作用[J].南京农业大学学报：自然版.
[72]汪振诚,陈永刚,刘子贻等.虾壳红色素的抗氧化和抗肿瘤作用[J].东海海洋,1999,17(2)：26-29.
[73]龙盛京,马文力,农冠荣.黑豆色素及多糖对全血化学发光和活性氧的抑制作用[J].食品科学,1999,20(9)：9-12.
[74]钱萤.黑色食品与浅色食品抗氧自由基作用的比较[J].广西大学学报,1996, 21(3)：297.
[75]龙盛京.几种赤色与浅色食品抗活性氧效能的比较研究[J].营养学报,1997,19(4)：472.
[76]谢俊杰.食品防腐新领域一微生物天然防腐剂[J].食品科学,1999,(1)：13-14.
[77]王君,张宝善.微生物生产天然色素的进展[J].微生物学通报,2007,34(3)：580-583.
[78]Armstrong,G A,Alberti M,Leach F,et al.Nucleotide sequence,organization,and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter, Capsulatus[J].Mol Gen Genet,1989,216:254.
[79]Misawa N,Nakagawa M,Kobayashi K,et al.Elucidation Of the Erwiniauredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia[J]. J Bacteriol,1990,172.6704-6712.
[80]Salanoubat M,Genin S,Artiguenave F,et al.Genome sequence of the plant pathogen Ralstonia solanacearum[J].Nature,2002,415:497-502.
[81]Vasconeelos D F,Almeida m,Hungria C T,et al.The complete genome sequence of chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability[J].PNAS,2003,100:11660-11665.
[82]Ensley B D,Ratzkin B J,Osslund T D,et al.Expression of naphthalene Oxidation genes in Escherichia coli results in the biosynthesis of indigo [J].Science,1983,222: 167-169.
[83]Choi H S,Kim J K,Cho E H,et al.A novel flavin-containing monooxy genase from Methylophaga sp.strain SKI and its indigo synthesis in Escherichia coli[J].Biochem Bioph Res Com,2003,306:930-936.
[84]Del campo J A,Rodrfguez H,Moreno J,et al.Appl Microbiol Biotechnol.2004, 64:848-854.
[85]魏力,方加玮,周俊初等.一株海洋细菌的初步鉴定及其产黑色素相关新基因(簇)的分离[J].微生物学通报,2007(6)：1118-1122.
[86]李建波,宋欣,曲音波.微生物学通报,2004,31(1)：50-54.
[87]张亮,朱湘民.微生物学通报,1999,26(5)：332-335.
[88]袁保红,杜青平,蔡创华.海洋通报,2005,24(6)：92-96.
[89]仓一华,陆玲,王伟.一株产类胡萝卜素酵母Rhodotorula.sp色素的组分及摇瓶发酵研究[J],食品科学,2002,23(5)：34-38.
[90]Buzzini P.An optimization study of carotenoid production by Rhodotorula glutinis DBVPG 3853 from substrates containing concentrated rectified grape must as the sole carbohydrate source[J],J Ind Microbiol Biotechnol,2000,24(1):41-45.
[91]陈建军,杨济龙.产番茄红素红酵母培养条件研究[J],云南农业大学学报,2005,20(3)：381-383.
[92]曾强松,童海宝,徐静安.三孢布拉霉发酵生产p-胡萝卜素工艺研究[J],工业微生物,2002,32(4)：7-10.
[93]张雪霞,刘刚叁,李宁.中国新药杂志,2005,14(7)：880-881.
[94]Salguero A,Leon R,Mariotti A,et al.Appl Microbiol Biotechnol,2005,66:506-511.
[95]Fbregas J,Domnguez A,Maseda A,et al.Interactions between irradiance andnutrient availability during astaxanthin accumulation and degradation in Haematococcus pluvialis[J],Applied Microbiology and Biotechnology,2003,61(5-6):545-551.
[96]Simova E D,Frengova G I,Besh D M.Zeitschrift fur Naturforsch C.A Journal of Biosciences,2003,58:225-229.
[97]Squina F M,Mercadante A Z.Journal of Food Biochemistry,2005,29(6):638-652.
[98]Orosa M,Franqueira D,Cid A,et al.Bioresource Technology,2005,96:373-378.
[99]Bhosale P,Gadre R V.β-Carotene production in sugarcane molasses by a Rhodotorula glutinis mutant[J], Journal of Industrial Microbiology and Biotechnology, 2001,26(6):327-332.
[100]Silva C,Cabral J M S,Isolation of aβ-Carotene Over-Producing Soil Bacterium, Sphingomonas sp.[J],Keulen F V.Biotechnology Letters,2004,26(3):257-262.
[101]Kang C D,Lee J S,Park T H,et al.Comparison of heterotrophic and photo autotrophic induction on astaxanthin production by Haematococcus pluvialis[J], Applied Microbiology and Biotechnology,2005,68(2):237-241.
[102]王克明,庄树宏.PVA固定化红曲霉发酵生产红曲色素的研究[J],烟台大学学报(自然科学与工程版),1998,11(4)：306-309.
[103]张子儒,郑巧东,姚善泾.红曲霉菌微胶囊化培养[J],食品发酵与工业,2003,29(11)：1-4.
[104]Shin C S,Kim H J,Kim M J,et al.Biotechnology and Bioengineering,1998,59:576-581.
[105]Suh J H,Shin C S.FEMS Microbiology Letters,2000,190:241-245.
[106]Frengova G I,Emilina S D,Beshkova D M.Synthesis of carotenoids by Rhodotorula rubra GED8 co-cultured with yogurt starter cultures in which ultrafiltrate [J]. Zeitschrift fur Naturforschung.C.A Journal of science,2003,58:562-567.
[107]金敬宏,张卫明.桂油提取新工艺[J].现代科技,1998,10,(7)：36-39.
[108]余焕玲,杨参,阐建全.食品中食用天然色素的作用[J].广州食品工业科技,2001,17(3)：63-65.
[109]谭建军.食用色素的性质及其应用[J].化学教育,1998,(2)：1-4.
[110]杨志娟.我国天然色素的现状与发展方针[D].《食品研究与开发》,2003,4,(24)：2.
[111]刘志明,张瑞,张金利.落葵红色素作为酸碱指示剂的研究[J].农产品加工(学刊)2007(8)：21-23.
[112]魏景超.真菌鉴定手册[M].上海：上海科学技术出版社,1979.
[113]HL巴尼特,BB亨特.半知菌属图解[M].北京：科学出版社,1977.
[114]刘婵,王博,段青,等.滇山茶基因组DNA不同提取方法效果比较[J].江苏农业科学.2010(6)：49-50.
[115]Yu J N.Huang J.Wang Z N,et 11.An Na+/H+anfiporter gene from wheat plays an important role in stress tolerance[J].J Bioeei,2007,32(6):1153-1161.
[116]汪文俊,王广策,张宝玉,等.海带栽培品系和长海带ITS区的PCR扩增及序列分析.高技术通讯[J].2005(4)：93-101.
[117]李春喜,王志和,文林.生物统计学(第二版)[M].科学出版社.2000,149-156.