摘要
酵母菌种资源是重要的生物资源,是维持生物多样性和工业酵母改良的物质基础,是发酵工业的基本生产资料。菌种资源的丰富程度是认识酵母、科学地实施酵母菌鉴定、分类和系统发育研究的先决条件。新疆地处欧亚大陆腹地,距海遥远,四周高山环绕,水汽稀少。这里地域辽阔,地形复杂,人口稀少,生态系统保持相对完整。这种独特的生态环境孕育了许多具有特色的动植物和微生物资源。新疆的生物多样性在中国具有独特性、复杂性,对该地区微生物资源特别是作为模式生物的酵母资源研究具有一定的理论和实际意义。
从新疆28个市(或县)收集到的52份土壤(主要是葡萄园)中分离出198株酵母菌,根据形态学特征挑取部分菌株,运用形态学结合26S rDNA Dl/D2序列分析方法进行鉴定,共鉴定出12属29个种(或变种),其中优势属为假丝酵母属Candida、毕赤酵母属Pichia和隐球酵母属Cryptococcus,广布种为Galactomyces geotrichum和Issatchenkia orientalis。经与国内已发表的酵母种进行比较,发现中国新纪录种6个,分别是分离于喀什泽普的Cryptococcus uzbekistanensis,分离于哈密的Issatchenkia occidentalis,分离于吐鲁番鄯善、石河子、博乐精河的Issatchenkia terricola,分离于博乐精河的Pichia mexicana,分离于库尔勒、博乐精河的Phichia galeiformis,分离于奇台的Rhodosporidium kratochvilovae。实验结果表明,新疆喀什地区酵母菌种多样性程度略高于吐鲁番地区。
抽取25株酵母菌进行糖发酵、耐糖、耐NaCl及温度生长实验发现,供试菌株对极限生长条件具有较高的耐性。实验中发现三株菌能在46℃下生长,经鉴定为Issatchenkia orientalis;一株菌耐NaCl达到14%,经过鉴定为德尔布有孢酵母Torulaspora delbrueckii。这四个菌株具有进一步研究的意义。
从新疆分离得到两株产香酵母菌株,分别为石河子的菌株XJB45和喀什的菌株XJA58,根据形态特征,生理生化特征及26S rDNA Dl/D2区序列分析将其均鉴定为Galactomyces geotrichum,GC-MS分析表明,这两株菌具有较强的产酯能力,其中菌株XJB45产酯15种,菌株XJA58产酯16种,两株菌所产主体酯亦不尽相同。同时两株菌也均合成大量β-苯乙醇。两株菌生长迅速,产酯能力强,发酵周期短,具有潜在的应用价值。
The resources of yeast species is a significant bio-resources and lays a solid substance foundation for maintaining bio-diversity and modifying industrial yeast .The abundance extent of species resources is the premise conditions for yeast identification , classification as well as phylogenetic study scientifically.Xinjiang,which is far from the sea and all surrounded by high mountains that lead to scarce water vapor, is located at the hinterland of Eurasian-Continent.It owns vast area, complex landform, sparse population and maintains relatively complete ecosystem. A lot of special animals, plants as well as microbiology species (or varieties) were created under the unique ecological environment. It is of certain theoretical and practical significance to explore the yeast resources of Xinjiang because of its unique and complexity of bio-diversity in China.
198 yeast strains were isolated from 52 soil samples (mainly from grape orchards of 28 cities or counties) collected from Xinjiang, then parts of the strains were selected according to morphologic characteristics. Among them 29 species (or variety) belonging to 12 genera were identified based on the combination of morphology and 26S rDNA Dl/D2 domain sequence analysis. The dominant genera identified were Candida, Pichia and Cryptococcus. Galactomyces geotrichum and Issatchenkia orientalis were the most widely distributed species.Compared with yeast species published domestically,6 China new record species were discoverd there which were Cryptococcus uzbekistanensis from Kashi, Issatchenkia occidentalis from Hami, Issatchenkia terricola from Turpan,Shihezi and Bole, Pichia mexicana from Bole, Phichia galeiformis from Kuerle and Bole, Rhodosporidium kratochvilovae from Qitai,respectively.Based on the experimental results, the yeast species diversity in Kashi district was a little bit higher than that of Turpan district.
25 yeast strains were chosen randomly to measure their capability of sugar fermentation, tolerance of glucose and NaCl, as well as growth temperature. The data indicated that the tested strains have a relatively good extreme growth ability on the whole.3 strains which were identified as Issatchenkia orientalis can grow at 46℃;One strain identified as Torulaspora delbrueckii can tolerate 14% NaCl. Therefore it makes sense to further study the four strains for their useful characteristics.
Two strains producing fruity aroma, which are respectively XJB45 from Shihezi and XJA58 from Kashi, were identified as Galactomyces geotrichum based on morphology, physiology and 26S rDNA Dl/D2 domain sequence analysis. GC-MS analysis results show that both strains have strong esters-producing capability but a little difference, strain XJB45 can generate 15 kinds of esters compared with strain XJA58 with 16 kinds of ones. A great amounts ofβ-Phenethyl alcohol can also be produced by both strains. The two strains have a potential value of application because of their rapid growth ability, high ester-producing capability and short fermentation period.
引文
[1] 张纪忠.微 生物分类学[ M].上 海: 复旦大学出版社,1990
[2] Lodder, J. (Ed.). The Yeasts, A Taxonomic Study, 2nd edn[M]. North-Holland Publishing Company, Amsterdam. 1970
[3] J.A.巴尼特,胡瑞卿译.酵母菌的特征与鉴定手册[M].青岛海洋大学出版社,1990
[4] Kreger-van Rij.. The Yeast, A Taxonomy Study, 3rd edn[M] Elsevier Science Publishers, Amsterdam, 1984
[5] 白逢彦,梁慧燕,贾建华.表型相似的热带假丝酵母和麦芽糖假丝酵母在脉冲电泳核型上的差异[J].微生物学通报,2001,(20)1:31-43
[6] 李明霞,唐荣观.不同类群酵母胞壁甘露聚糖核磁共振氢谱的比较研究[J].真菌学报,1989 ,8(4):296-303
[7] 严健,周与良.假丝酵母属可溶性蛋白及酯酶的聚丙烯酰胺凝胶电泳[J].南京大学学报(自然科学)1995,28 (2) :54-59
[8] Valente,P,Ramos,J.P,Leoncini. Sequencing as a tool in yeast molecular taxonomy [J]. Can .J.Microbio,1999,145:949-958
[9] Mara R.D., Jack W.,et al. Molecular analyses of the IGS & ITS regions of rDNA of the psychrophilic yeasts in the genus Mrakia[J].Antonie van Leeuwenhoek,2000, 77: 7–12
[10] Takashima M.,Nakase T. Four new species of the genus Sporobolomyces isolated from leaves in Thailand[J]. Mycoscience,2001,41,357-369
[11] Sugita T.,Cafete-Gibas,Takashima ,et al. Three new species of Bullera isolated from leaves in the Ogasawara Islands[J]. Mycoscience,1999,40: 491-501
[12] Nisiotou,G.R. Gibson. Isolation of culturable yeasts from market winesand evaluation of the 5.8S-ITS rDNA sequence analysis for identification purposes[J]. Letters in Applied Microbiology,2005, 41:454–463
[13] Gutell R. R., Fox G E..Compilation of large subunit RNA sequences presented in a structural format[J]. Nucleic Acids Res,1988,16:175-269
[14] Kurtzman, C. P., Robnett. Identification and ascomycetous yeasts from analysis of nuclear large subunit (26S) phylogeny of ribosomal DNA partial sequences [J]. Antonie van Leeuwenhoek, 1998, 73: 331-371
[15] Sugita,T.,Nishikawa,A..Fugal identification method based on DNA sequence analysis: reassessment of the methods of the pharmaceutical society of Japan and the Japanses pharmacopoeia[J].Journal of Health Science,2003,49(6):531-533
[16] Lachance M.A., Daniel H.M.. The D1/D2 domain of the large-subunit rDNA of the yeast species Clavispora lusitaniae is unusually polymorphic [J].FEMS Yeast Research,2003,4 : 253-258
[17] Swann,E.C. and Taylor,J. Phylogenetic perspectives on basidiomycete systematics: evidence from the 18S rRNA gene[J]. Can. J. Bot. ,1995 ,73 (Suppl. 1):862-S868
[18] Takashima,M.,Suh, S.-O., Nakase, T..Phylogenetic relationships among species of the genus Bensingtonia and related taxa based on the small subunit ribosomal DNA sequences[J]. J. Gen. Appl.Microbiol.,1995,41:131-141
[19] Takashima,M.,Nakase, T..A phylogenetic study of the genus Tilletiopsis, Tilletiaria anomala and related taxa based on the small subunit ribosomal DNA sequences[J]. J. Gen. Appl. Microbiol,1996,2:421-42 9
[20] Kreger-van Rij, N. J. W. (Ed.) The Yeast, A Taxonomy Study, 3rd edn [M]. Elsevier Science Publishers, Amsterdam, 1984
[21] Takahito W., Yoshinori M., Syuichi O,et al. A new approach to species determination for yeast strains: DNA microarray-based comparative genomic hybridization using a yeast DNA microarray with 6000 genes[J]. Yeast,2004,1: 351–365
[22] Iwaguchi, S.-L, Homma, M,et al.Variation in the electrophoretic karyotype analyzed by the assignment of DNA probes in Candida albicans[J]. Gen. Microbiol, 1990,6: 2433-2442
[23] Doi M., Homma, M., Chindamporn, A .,et al. Estimation of chromosome number and size by pulsed-field gel electrophoresis (PFGE) in medically important Candida species[J].Gen. Microbiol,1992,38: 2243-2251
[24]白逢彦,贾建华.有不同碳源利用方式的酿酒酵母菌株的脉冲电泳核型分析[J].菌物系统, 2000 ,9(1):65 - 71
[25] Patricia Romero A., Belen P., Manuel Q. Diferential detection of Debaryomyces hansenii isolated from intermediate-moisture foods by PCR-RFLP of the IGS region of rDNA[J].FEMS Yeast Research,2005,5 : 455–461
[26]刘杨,崔晓龙,李文均等.RNA 二级结构在微生物发育分析上的应用[J].微生物学通报,2006, 33(2):147-150
[27] 许超德.云南野生隐球酵母菌的分类研究[D].昆明:云南大学.2004,41-45
[28] 华炳生.酵母菌在医药工业业中的应用[J].工业微生物,1987,17: 39-40
[29] 莫湘筠,黄玲.产麦角固醇酵母的选育及其发酵条件的研究[J].食品与发酵工业,1990,16: 20-23
[30] Mercier J, Wilson C.L. Effect of wound moisture on the biocontrol by Candida oleophila of grey mold (Botrytis cinerea)of apple[J]. Postharvest Biol Technol. 1995,6:9-15
[31] Zhou T., Northover J., Schneider K.E..Biological control of post-harvest diseases of peach with phyllosphere isolates of Pseudomonns syringae[J]. Can J Plant Pathol,1999, 21: 375-381
[32] 杨清香,贾振杰,潘峰等.酵母菌在废水处理中的应用[J].环境污染治理技术与设备, 2005,(6)2:1-5
[33] 尹平,白逢彦,周培瑾.降解三硝基甲苯的酵母和类酵母菌的研究[J].微生物学报,1998,38 (4) :295-298
[34] 吴丽娟,蒋建新,驻佩芳等.酵母表达系统及其应用研究[J].生命的化学,2003,23(1):46-49
[35] 李明霞,唐荣观.我国各类基物上的酵母菌分布及其尿素酶活性[J].真菌学报,1983,2(4):228-23
[36] 周与良,史国利,周菊岩等. 渤海海水中酵母菌的种类[J].真菌学报. 1991 ,10(1):36-42
[37] 周与良,李明林,王红梅等.渤海沿岸不同基物上的酵母菌种类[J].南开大学学报(自然科学),1990,4:57-61
[38] 周与良,史国利,周菊岩等.黄海海水中酵母菌的种类[J].真菌学报,1993, 12(4):327-329
[39] 魏艳敏,但汉斌,周与良.长江水中红酵母菌种类调查初探[J].河北农业大学学报,1998,(21)2:51-54
[40] 李绍兰,陈有为,杨丽源等.云南酵母菌的研究I:西双版纳热带雨林中的酵母菌[J].云南大学学报(自然科学版),2002,24:378-380
[41] 李绍兰,陈有为,杨丽源等.云南酵母菌的研究Ⅱ:白马雪山自然保护区的酵母菌[J].云南大学学报(自然科学版),2002,24:469-470
[42] 许超德,李绍兰,杨丽源等.云南杜鹃花上的酵母菌物种多样性[J].菌物研究,2003,1(1):37-39
[43] 陆惠中,王启明,白逢彦等.秦岭地区子囊菌酵母物种多样性研究[J].菌物学 报,2004,23(2):183-187
[44] Bai F-Y, Takashima M, Hamamoto M & Nakase T. Sporobolomyces yunnanensis sp. nov., a Q-10(H2)-containing yeast species with a close phylogenetic relationship to Erythrobasidium hasegawianum[J]. Int. J. Syst. Evol. Microbiol. 2001,51: 231-235
[45] Bai F-Y, Takashima M & Nakase T. Description of Bullera kunmingensis sp. nov.,and clarification of the taxonomic status of Bullera sinensis and its synonyms based on molecular phylogenetic analysis[J]. FEMS Yeast Res. 2001,1: 103-109
[46] Bai F-Y, Takashima M & Nakase T. Phylogenetic analysis of strains originally assigned to Bullera variabilis: descriptions of Bullera pseudohuiaensis sp. nov., Bullera komagatae sp. nov. and Bullera pseudoschimicola sp. nov[J]. Int. J. Syst. Evol. Microbiol. 2001,51: 2177-2187
[47] Bai F-Y, Takashima M, Jia J-H & Nakase T. Dioszegia zsoltii sp. nov., a new ballistoconidium-forming yeast species with two varieties[J]. J. Gen. Appl. Microbiol. 2002,48: 17-23
[48] Bai F-Y, Zhao J-H, Takashima M, Jia J-H, Boekhout T & Nakase T. Reclassification of the Sporobolomyces roseus and Sporidiobolus pararoseus complexes, with the description of Sporobolomyces phaffii sp. nov[J]. Int. J. Syst. Evol. Microbiol. 2002,52: 2309-2314
[49] Zhao J-H, Bai F-Y, Guo L-D & Jia J-H. Rhodotorula pinicola sp. nov., a basidiomycetous yeast species isolated from xylem of pine twigs[J]. FEMS Yeast Res. 2002,2: 159-163
[50] Bai F-Y, Takashima M, Zhao J-H, Jia J-H & Nakase T. Bullera anomala sp. nov. and Bullera pseudovariabilis sp. nov., two new ballistoconidium-forming yeast species from Yunnan, China[J]. Antonie van Leeuwenhoek2003,83: 257-263
[51] Wang Q-M, Bai F-Y, Zhao J-H & Jia J-H. Bensingtonia changbaiensis sp. nov. and Bensingtonia sorbi sp. nov., novel ballistoconidium-forming yeast species isolated from plant leaves[J]. Int. J. Syst. Evol. Microbiol. 2003, 53: 2085-2089
[52] Wang Q-M, Bai F-Y, Zhao J-H & Jia J-H. Dioszegia changbaiensis sp. nov., a basidiomycetous yeast species isolated from northeast China[J]. J. Gen. Appl. Microbiol. 2003,49(5): 295-299
[53] Zhao J-H, Bai F-Y, Wang Q-M & Jia J-H. Sporobolomyces bannaensis, a new ballistoconidium-forming yeast species in the Sporidiobolus lineage[J]. Int. J. Syst. Evol. Microbiol. 2003,53: 2091-2093
[54] Fungsin B, Takashima M, Bai F-Y, Artjariyasripong S & Nakase T. Bullera panici sp. nov. and Bullera siamensis sp. nov., two new yeasts in the Bullera variabilis cluster isolated in Thailand[J]. Mircobiol. Cult. Coll. 2003, 19: 23-32
[55] Wang Q-M & Bai F-Y. Four new yeast species of the genus Sporobolomyces from plant leaves[J]. FEMS Yeast Res. 2004,4: 579-586
[56] Lu H-Zh, Jia J-H, Wang Q-M & Bai F-Y. Candida asparagi sp. nov., Candida diospyri sp. nov. and Candida qinlingensis sp. nov., novel anamorphic ascomycetous yeast species[J]. Int. J. Syst. Evol. Microbiol. 2004,54: 1409-1414
[57] Wang Q-M, Bai F-Y, Lu H-Zh, Jia J-H & Takashima M. Bullera cylindrica, Bullera hubeiensis and Bullera nakasei, ballistoconidium-forming yeast species isolated from plant leaves[J]. Int. J. Syst. Evol. Microbiol. 2004,54: 1877-1882
[58] Wu Z-W & Bai F-Y. Kazachstania aquatica sp. nov. and Kazachstania solicola sp. nov., new ascomycetous yeast species[J]. Int. J. Syst. Evol. Microbiol. 2005. 55 :2219-2224
[59] Yarrow D..Method for the isolation , maintenance and identification of yeasts.In the yeasts : a taxonomic study , 4th ed.[M] Amsterdam :Elsevier Sciences,1998
[60] (美)F.奥斯伯,R.E.金斯顿,J.G.塞德曼等著,颜子颖,王海林译.《精编分子生物学实验指南》[M].科学出版社,1998:522-523
[61] Fell JW,Boekhout T,Fonseca A et al, 2000.Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/ D2 domain sequence analysis[J].Int J Syst Evol Microbiol,50:l351-1371
[62] 冯克宽,王明谊,曾家豫.酵母菌的分离及鉴定[J].西北师范大学学报(自然科学版),1997(33)2:56-59
[63] Takashi, Yoshinobu. Induction and Characterization of Artificial Diploids from the Haploid Yeast Torulaspora delbrueckii[J]. Applied and environmental microbiology , 1987,(53)7: 1504-1511
[64] Yujioda, Mihe Yabuki, Kenzo Tonomura etal. Reexamination of Yeast Strains Classified as Torulaspora delbrueckii (Lindner)[J]. International Journal of Systematic Bacteriology, 1997,(47)4:1102-1106
[65] Maria Jose, Joaquin Panadero, Jose Antonio,etal. Regulation of Salt Tolerance by Torulaspora delbrueckii Calcineurin Target Crz1p [J]. EUKARYOTIC CELL,. 2006 (5)3 :469-479
[66] R.Boutrou,M.Gueguen.Interests in Geotrichum candidum for cheese technology [J].International Journal of Food Microbiology, 2005,102:1- 20
[67] S.C.B. Gopinath, A. Hilda,etal. Purification of lipase from Geotrichum candidum: conditions optimized for enzyme production using Box-Behnken design [J]. World Journal of Microbiology & Biotechnology,2003,19: 681-689
[68] Maria Cklia Bertolini, etal.Expression and characterization of Geotrichum candidum lipase I gene comparison of specificity profile with lipase II[J]. Eur. J. Biochem,1995, 228:863-869
[69] Abadulia E,Tzanov T,Costa S.et a1.Decolorization and detoxification of textile dye with a laccase from Trametes[J].Appl.Environ.Microbio1.,2000,66(8):3357-6
[70] 曲静然,刘玉,宋俊梅.豆腐废水发酵生产白地霉的研究[J].食品研究与开发 ,2005,(26)3:99-102
[71] 贺淹才,张敬标.白地霉和产朊假丝酵母[J].中国饲料,1997,7:38-40