老白干酒曲中酵母菌多样性分析
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摘要
采用微生物分离培养与分子鉴定相结合的方法,在对酒曲中的酵母菌进行分离、纯化和形态学鉴定基础上,进行菌株核糖体26S rDNA聚合酶链式反应-限制性片段长度多态性分析技术结合的分子鉴定。并采用非培养方法,直接提取酒曲中的总DNA进行聚合酶链式反应扩增、电泳和切胶与回收,通过聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)法鉴定酵母菌。结果表明,采用培养方式分离到57株6种不同形态的酵母菌,经鉴定为5种分子类型,分别为奥默毕赤酵母(Pichia kudriavzevii)、葡萄牙棒孢酵母(Clavisporalusitaniae)、异常威克汉姆酵母(Wickerhamomycesanomalus)、胶红酵母(Rhodotorula mucilaginosa)、阿氏丝孢酵母(Trichosporon asahii)。经PCR-DGGE法鉴定得到10株酵母菌,分别为W. anomalus、Wickerhamomyces sp.、T. asahii、热带假丝酵母(Candida tropicalis)和Candida sp.各1株,Hyphopichiaburtonii 2株,另有unculturedSaccharomycopsis3株。培养与非培养两种方式共鉴定得到10株酵母菌,分别属于8个属,包括P. kudriavzevii、C. lusitaniae、W. anomalus、R. mucilaginosa、T. asahii、uncultured Saccharomycopsis、H. burtonii、Wickerhamomyces sp.、C. tropicalis、Candida sp.,该研究结果可为进一步研究酒曲的发酵能力提供参考。
In this study, yeasts were isolated and purified from the traditional fermentation starter(Daqu) for Laobaigan liquor. The isolates were identified by morphology as well as polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) analysis of ribosomal 26 S rDNA. Meanwhile, yeasts were identified by culture-independent method, namely polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE). Total DNA was extracted from Daqu and amplified by PCR. The PCR products were fingerprinted by denaturing gradient gel electrophoresis(DGGE) and were excised and recovered from the gel. The results showed that a total of 57 strains of yeast assigned to 6 kinds of morphology were isolated by traditional culture method and were identified as Pichia kudriavzevii, Clavispora lusitaniae, Wickerhamomyces anomalus, Rhodotorula mucilaginosa, and Trichosporon asahii. A total of 10 strains of yeast were obtained by PCR-DGGE, including one strain of W. anomalus, one strain of Wickerhamomyces sp., one strain of T. asahii,one strain of Candida tropicalis, one strain of Candida sp. two strains of Hyphopichia burtonii and three strains of uncultured Saccharomycopsis. Ten yeast strains belonging to eight genera were identified by culture-dependent and culture-independent methods, including P. kudriavzevii, C. lusitaniae, W. anomalus, R. mucilaginosa, T. asahii, uncultured Saccharomycopsis, H. burtonii, Wickerhamomyces sp., C. tropicalis, and Candida sp.. The results from this study provide a basis for further study on the fermentation performance of Daqu.
In this study, yeasts were isolated and purified from the traditional fermentation starter(Daqu) for Laobaigan liquor. The isolates were identified by morphology as well as polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) analysis of ribosomal 26 S rDNA. Meanwhile, yeasts were identified by culture-independent method, namely polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE). Total DNA was extracted from Daqu and amplified by PCR. The PCR products were fingerprinted by denaturing gradient gel electrophoresis(DGGE) and were excised and recovered from the gel. The results showed that a total of 57 strains of yeast assigned to 6 kinds of morphology were isolated by traditional culture method and were identified as Pichia kudriavzevii, Clavispora lusitaniae, Wickerhamomyces anomalus, Rhodotorula mucilaginosa, and Trichosporon asahii. A total of 10 strains of yeast were obtained by PCR-DGGE, including one strain of W. anomalus, one strain of Wickerhamomyces sp., one strain of T. asahii,one strain of Candida tropicalis, one strain of Candida sp. two strains of Hyphopichia burtonii and three strains of uncultured Saccharomycopsis. Ten yeast strains belonging to eight genera were identified by culture-dependent and culture-independent methods, including P. kudriavzevii, C. lusitaniae, W. anomalus, R. mucilaginosa, T. asahii, uncultured Saccharomycopsis, H. burtonii, Wickerhamomyces sp., C. tropicalis, and Candida sp.. The results from this study provide a basis for further study on the fermentation performance of Daqu.
引文
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[19]王海燕,唐洁,徐岩,等.清香型小曲白酒中微生物组成及功能微生物的分析[J].酿酒科技,2012,35(12):48-52.DOI:10.13746/j.njkj.2012.12.043.
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[22]程才璎,刘晓风,袁月祥,等.酱香型白酒酒曲和连续七轮次堆积酒醅的细菌群落结构[J].应用与环境生物学报,2014,20(5):825-831.DOI:10.3724/SP.J.1145.2014.03035.
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[27]徐艳文,杨莹,薛军侠,等.26S rDNA-RFLP分析在非酿酒酵母菌分类研究中的应用[J].微生物学杂志,2007,27(4):23-26.
[28]张红霞.PLFA对清香大曲微生物群落结构分析及产酯酵母的筛选应用研究[D].临汾:山西师范大学,2016:26-30.
[29]JEYARAM K,SINGH W M,CAPECE A,et al.Molecular identification of yeast species associated with‘Hamei’-a traditional starter used for rice wine production in Manipur,India[J].International Journal of Food Microbiology,2008,124(2):115-125.DOI:10.1016/j.ijfoodmicro.2008.02.029.
[30]惠丰立,柯涛,褚学英,等.大曲中酵母菌种群结构及多样性分析[J].食品与生物技术学报,2009,28(1):102-106.
[31]蒋文鸿.昌黎与济源产区高产β-葡萄糖苷酶野生非酿酒酵母的筛选及鉴定[D].咸阳:西北农林科技大学,2014:23-25.
[32]袁晓华.β-葡萄糖苷酶产生菌的筛选、培养条件优化及β-葡萄糖苷酶应用研究[D].济南:山东大学,2009:7-10.
[33]王玉霞.阿氏丝孢酵母(Trichosporon asahii)β-葡萄糖苷酶及葡萄糖苷类风味物质水解机制的研究[D].无锡:江南大学,2012:17-22.
[34]明红梅,周健,陈蒙恩,等.大曲中异常威克汉姆酵母发酵产苯乙醇的条件优化[J].湖北农业科学,2015,54(14):3492-3496.DOI:10.14088/j.cnki.issn0439-8114.2015.14.041.
[35]高亦豹.聚合酶链式反应-变性梯度电泳技术(PCR-DGGE)研究中国白酒大曲中微生物群落结构[D].无锡:江南大学,2010:32-33.
[36]彭杨,张奶英,何利,等.基于PCR-DGGE技术的四川麸醋固态发酵过程中微生物群落分析[J].现代食品科技,2016(8):171-177.DOI:10.13982/j.mfst.1673-9078.2016.8.026.
[37]周剑忠,董明盛,江汉湖.PCR-DGGE指纹技术与分离技术结合筛选藏灵菇奶发酵过程的优势菌[J].中国农业科学,2006,39(8):31-31.
[2]李艳,董振玲,牟德华.羊羔美酒大曲中酵母菌多样性及分子鉴定[J].食品科学,2014,35(5):144-149.DOI:10.7506/spkx1002-6630-201405029.
[3]陈金丽,郭阳,薛洁,等.优良野生葡萄酒酵母的筛选及性能评价[J].食品与发酵工业,2012,38(5):106-111.DOI:10.13995/j.cnki.11-1802/ts.2012.05.044.
[4]杨雪峰,苏龙,刘树文.利用WL营养培养基鉴定葡萄酒中的相关酵母菌[J].中外葡萄与葡萄酒,2006(4):4-7.DOI:10.13414/j.cnki.zwpp.2006.04.001.
[5]范佳,武伟伟,李艳.26S rDNA序列分析法鉴定开菲尔发酵液中的酵母菌[J].酿酒科技,2014,35(11):14-17.DOI:10.13746/j.njkj.2014.0248.
[6]PALLMANN C L,BROWN J A,OLINEKA T L,et al.Use of WLmedium to profile native flora fermentations[J].American Journal of Enology&Viticulture,2016,52(3):198-203.
[7]LI Z H,FENG X M,LU S Q,et al.Molecular phylogeny of Pneumocystis based on 5.8S rRNA gene and the internal transcribed spacers of rRNA gene sequences[J].Science in China Series C:Life Sciences,2008,51(5):445-452.DOI:10.1007/s11427-008-0057-1.
[8]ESTEVE-ZARZOSO B,BELLOCH C,URUBURU F,et al.Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers[J].International Journal of Systematic and Evolutionary Microbiology,1999,49(1):329-337.DOI:10.1099/00207713-49-1-329.
[9]马凯,刘光全,李金霞,等.基于26S rDNA D1/D2区序列分析的15株白地霉分子分类学研究[J].微生物学报,2007,47(2):359-362.
[10]徐艳文,杨莹,薛军侠,等.26S rDNA-RFLP分析在非酿酒酵母菌分类研究中的应用[J].微生物学杂志,2007,27(4):23-26.
[11]王洁,胡然,姜虹伶,等.腐败葡萄中酵母菌的分离及鉴定[J].酿酒科技,2015(5):29-33.DOI:10.13746/j.njkj.2014446.
[12]曾骏,陈安均,蒲彪,等.传统四川泡菜中酵母菌的动态变化规律[J].食品科学,2014,35(7):81-85.DOI:10.7506/spkx1002-6630-201407017.
[13]孙剑秋,刘雯雯,臧威,等.基于26S rDNA D1/D2序列分析酱香型白酒酒醅中酵母菌的群落结构[J].微生物学报,2012,52(10):1290-1296.DOI:10.13343/j.cnki.wsxb.2012.10.008.
[14]赵丽丽,陈存社,郭凤莲.26SrDNA序列分析法鉴定酵母菌[J].中国酿造,2008,27(15):49-51.
[15]FLóREZ A B,MAYO B.Microbial diversity and succession during the manufacture and ripening of traditional,Spanish,blue-veined Cabrales cheese,as determined by PCR-DGGE[J].International Journal of Food Microbiology,2006,110(2):165-171.DOI:10.1016/j.ijfoodmicro.2006.04.016.
[16]MU Z,YANG X J,YUAN H.Detection and identification of wild yeast in Koumiss[J].Food Microbiology,2012,31(2):301-308.DOi:10.1016/j.fm.2012.04.004.
[17]董红红,杨盼,吕杰,等.基于PCR-DGGE的咖啡湿法发酵酵母菌的分离鉴定[J].云南农业大学学报(自然科学),2014,29(2):251-257.DOI:10.3969/j.issn.1004-390X(n).2014.02.018.
[18]乌日娜,于美玲,孟令帅,等.PCR-DGGE分析东北自然发酵酸菜中的微生物多样性[J].现代食品科技,2014,30(10):8-12.DOI:10.13982/j.mfst.1673-9078.2014.10.002.
[19]王海燕,唐洁,徐岩,等.清香型小曲白酒中微生物组成及功能微生物的分析[J].酿酒科技,2012,35(12):48-52.DOI:10.13746/j.njkj.2012.12.043.
[20]DURAND N,SHEIKHA A F E,SUAREZ-QUIROS M L,et al.Application of PCR-DGGE to the study of dynamics and biodiversity of yeasts and potentially OTA producing fungi during coffee processing[J].Food Control,2013,34(2):466-471.DOI:10.1016/j.foodcont.2013.05.017.
[21]SHA S P,ANUPAMA A,PRADHAN P,et al.Identification of yeasts by polymerase-chain-reaction-mediated denaturing gradient gel electrophoresis in marcha,an ethnic amylolytic starter of India[J].Journal of Ethnic Foods,2016,3(4):292-296.DOI:10.1016/j.jef.2016.11.009.
[22]程才璎,刘晓风,袁月祥,等.酱香型白酒酒曲和连续七轮次堆积酒醅的细菌群落结构[J].应用与环境生物学报,2014,20(5):825-831.DOI:10.3724/SP.J.1145.2014.03035.
[23]谭映月,胡萍,谢和.应用PCR-DGGE技术分析酱香型白酒酒曲细菌多样性[J].酿酒科技,2012(10):107-111.DOI:10.13746/j.njkj.2012.10.024.
[24]姚淑敏,陈璐,闫华文.应用PCR-DGGE方法研究甜酒曲中真菌多样性[J].中国酿造,2016,35(11):44-48.DOI:10.11882/j.issn.0254-5071.2016.11.009.
[25]闫华文.甜酒曲中真菌多样性的研究及甜酒酿营养成分分析[D].济宁:曲阜师范大学,2015:16.
[26]赵东.西藏青稞酒酒曲微生物多样性的研究[D].泰安:山东农业大学,2011:32-35.
[27]徐艳文,杨莹,薛军侠,等.26S rDNA-RFLP分析在非酿酒酵母菌分类研究中的应用[J].微生物学杂志,2007,27(4):23-26.
[28]张红霞.PLFA对清香大曲微生物群落结构分析及产酯酵母的筛选应用研究[D].临汾:山西师范大学,2016:26-30.
[29]JEYARAM K,SINGH W M,CAPECE A,et al.Molecular identification of yeast species associated with‘Hamei’-a traditional starter used for rice wine production in Manipur,India[J].International Journal of Food Microbiology,2008,124(2):115-125.DOI:10.1016/j.ijfoodmicro.2008.02.029.
[30]惠丰立,柯涛,褚学英,等.大曲中酵母菌种群结构及多样性分析[J].食品与生物技术学报,2009,28(1):102-106.
[31]蒋文鸿.昌黎与济源产区高产β-葡萄糖苷酶野生非酿酒酵母的筛选及鉴定[D].咸阳:西北农林科技大学,2014:23-25.
[32]袁晓华.β-葡萄糖苷酶产生菌的筛选、培养条件优化及β-葡萄糖苷酶应用研究[D].济南:山东大学,2009:7-10.
[33]王玉霞.阿氏丝孢酵母(Trichosporon asahii)β-葡萄糖苷酶及葡萄糖苷类风味物质水解机制的研究[D].无锡:江南大学,2012:17-22.
[34]明红梅,周健,陈蒙恩,等.大曲中异常威克汉姆酵母发酵产苯乙醇的条件优化[J].湖北农业科学,2015,54(14):3492-3496.DOI:10.14088/j.cnki.issn0439-8114.2015.14.041.
[35]高亦豹.聚合酶链式反应-变性梯度电泳技术(PCR-DGGE)研究中国白酒大曲中微生物群落结构[D].无锡:江南大学,2010:32-33.
[36]彭杨,张奶英,何利,等.基于PCR-DGGE技术的四川麸醋固态发酵过程中微生物群落分析[J].现代食品科技,2016(8):171-177.DOI:10.13982/j.mfst.1673-9078.2016.8.026.
[37]周剑忠,董明盛,江汉湖.PCR-DGGE指纹技术与分离技术结合筛选藏灵菇奶发酵过程的优势菌[J].中国农业科学,2006,39(8):31-31.