和-技术分析葡萄酒相关酵母菌多样性

ARISA和T-RFLP技术分析葡萄酒相关酵母菌多样性

ANALYSIS OF WINE-RELATED YEAST DIVERSITY BY ARISA AND T-RFLP TECHNIC

作者：孙　悦　　导师：刘延琳

西北农林科技大学　　发酵工程2011届硕士

摘　要

酵母菌是葡萄酒生产中最重要的微生物有机体，它们通过自身的一系列代谢活动和其多样性及群体组成影响着葡萄酒的质量。葡萄酒相关酵母菌的多样性调查对于葡萄酒生产具有重要的意义，既能够防治微生物病害，监控发酵过程，还能为选育优良性状酵母菌株提供基础。

ARISA（automated ribosomal intergenic spacer analysis）和T-RFLP（terminal restriction fragment length polymorphism）技术在研究微生物群体多样性方面有着独特的优势，是国外广泛用于微生物调查研究的非培养方法，但在国内外还未见应用于葡萄酒酵母菌研究的相关报道。本研究对葡萄酒相关酵母菌作了ARISA分析、T-RFLP分析，进行了酵母菌群落结构多样性的研究，以期为国内葡萄酒相关酵母菌的调查引入快速、精确、简单的新方法，补充并完善我国对葡萄酒酵母菌群体多样性的研究工作。主要研究结果如下：

1.　纯种酵母菌的ARISA和T-RFLP分析。以5.8 S rDNA-ITS序列为目标片段，使用荧光标记引物ITS1-HEX和ITS4进行PCR扩增反应，得到目的条带后对PCR产物及其经过HaeⅢ和HinfⅠ的酶切消化产物（即荧光标记的PCR产物和T-RFs）分别进行毛细管电泳分析，得到ARISA分析（PCR产物）和T-RFLP分析（T-RFs）结果，以此进行相应数据库的构建。结果表明，ARISA和T-RFLP技术重现性好。对39种91株酵母菌的分析表明，根据PCR产物的长度，可将供试菌株中的10个种进行区分；在T-RFLP分析中，根据HaeⅢ酶切产生的T-RFs长度，可将供试菌株中的14个种进行区分，根据HinfⅠ酶切产生的T-RFs长度，可将供试菌株中的11个种进行区分。综合分析酵母菌的ARISA和T-RFs数据，对供试菌株中的15个种进行区分，分别为：Brettanomyces bruxellensis;Candida apicola; Candida boidinii ; Candida norvegica; Candida stellata;Candida zemplinina; Debaryomyces hansenli var. hansenli;Metschnikowia pulcherrima;Pichia anomala;Pichia fermentans;Pichia membranifaciens; Pichia kluyveri;Saccharomyces ludwigii; Torulaspora delbrueckii; Zygosaccharomyces bailii。单独使用T-RFLP技术对供试菌株中的14个种进行区分，不能对C. stellata进行区分；对于S. ludwigii;T. delbrueckii和Z. bailii来说只有HaeⅢ的酶切结果可以对其进行区分；其余的11个种都可以用HinfⅠ酶切分析进行区分。

2.　自然发酵过程中酵母菌的T-RFLP分析。对雷司令和霞多丽葡萄品种自然发酵过程的5个时期进行取样，样品不经稀释直接涂在YEPD平板以获得总酵母菌群，用于T-RFLP分析。结合酵母菌WL营养培养基（Wallerstein Laboratory Nutrient Agar）分类鉴定方法和5.8 S rDNA-ITS序列的HaeⅢ和HinfⅠ酶切分析，研究酒精发酵各时期酵母菌种类和数量。对于雷司令葡萄自然发酵过程，T-RFLP分析比常规分析方法鉴定出的酵母菌种类要多，M. pulcherrima,C. zemplinina和300±1bp代表的酵母菌种类，在酵母菌的常规分析方法中都没有鉴定出来；对于霞多丽葡萄自然发酵过程， T-RFLP分析比常规分析方法鉴定出的酵母菌种类要少。WL分类表明霞多丽自然发酵过程中还存在M. pulcherrima和P. minuta var.minuta， 而在T-RFLP图谱中没有检测出来，可能是因为取样不均匀造成的。T-RFs为300±1bp的酵母菌在常规分析方法中没有被检测到。此外，S. cerevisiae是酒精发酵中期和后期的优势菌，随着发酵的进行，S. cerevisiae在发酵液中数量增加，到发酵后期分离到的S. cerevisiae比例达到100%，成为主要发酵动力群体并最终主导发酵完成。

总之，本文首次将ARISA和T-RFLP两种不依赖于培养的技术应用于葡萄酒相关酵母菌多样性研究中，初步建立了纯培养酵母菌ARISA和T-RFLP分析的数据库，探索了二者在酒精发酵过程的酵母菌多样性研究中的应用价值；加强了人们对酒精发酵过程的认识和理解，为建立葡萄酒相关酵母菌群生态与葡萄酒品质之间的对应关联奠定基础。

关键词　ARISA　T-RFLP　酵母菌多样性　免培养方法

Abstract

Yeasts are the most important microbial organisms during winemaking. The quality of wine is affected by the metabolic activities，the diversity and the composition of the yeast micropopulation. Studying the diversity of wine-related yeasts is of great significance for wine production,both to control microbial diseases,monitoring the fermentation process,but also for the basis to select yeast strains with good characters.

Automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphism (T-RFLP) technologies possess unique advantage in the study of microbial diversity,which are widely used in microbiological research abroad as culture-independent methods. However,they have not been used in the study of wine-related yeasts. In this study,we performed ARISA and T-RFLP analysis on wine-related yeasts to study the diversity of yeast community structure,and to introduce fast,simple new methods to improve the research work of wine-related yeast diversity in China. The main findings are as follows:

1.　ARISA and T-RFLP were adapted to pure cultures of yeasts. PCR amplification targeted for 5.8 S rDNA-ITS was performed with the primer ITS1 having its 5′ end labeled with HEX and ITS4. The mixture of amplicons was subjected to a restriction reaction with restriction endonuclease HaeⅢ and HinfⅠ. Following the restriction reaction,the mixture of fluorescent fragments was separated using capillary electrophoresis to generate the database of ARISA analysis and T-RFs. The result showed good repeatability for both ARISA and T-RFLP analysis. 91 yeast stains belonged to 39 species were analyzed by both ARISA and T-RFLP. 10 yeast species could be distinguished according to the lengths of PCR products. For T-RFLP analysis,14 and 11 yeast species could be distinguished according to the lengths of T-RFs using HaeⅢ and HinfⅠ,respectively. In summary,15 yeast species could be identified by the ARISA analysis along with T-RFLP analysis: Brettanomyces bruxellensis; Candida apicola; Candida boidinii; Candida norvegica; Candida stellata;Candida zemplinina; Debaryomyces hansenli var. hansenli;Metschnikowia pulcherrima;Pichia anomala;Pichia fermentans;Pichia membranifaciens; Pichia kluyveri;Saccharomyces ludwigii; Torulaspora delbrueckii; Zygosaccharomyces bailii. 14 yeast species except for C. stellata could be identified using T-RFLP technique alone. For S. ludwigii;T. delbrueckii and Z. bailii,only the result of HaeⅢ digestion could distinguish them. T-RFLPwith HinfⅠdigestion could distinguish the other 11 yeast species.

2.　Samples of five stages were collected during spontaneous fermentation of Chardonney and Riesling. DNA from total biomass grown on Yeast Extract Peptone Dextrose Medium (YEPD) without dilution was extracted for T-RFLP analysis. Combined with Wallerstein Laboratory Nutrient Agar analysis and restriction reaction 5.8 S rDNA-ITS using HaeⅢ and HinfⅠ,we revealed the yeast population and amount during the stages of alcoholic fermentation more quickly and accurately. During the spontaneous fermentation of Riesling,T-RFLP analysis had detected the existence of M. pulcherrima;C. zemplinina as well as the yeast with the T-RF lengths of 300±1bp. During the spontaneous fermentation of Chardonney,the yeast species detected by T-RFLP analysis were less yeast than the traditional methods. M. pulcherrima and P. minuta var.minuta were detected by WL,while T-RFLP did not due to the nonuniform samples. T-RFLP analysis had detected the existence of other yeasts with the T-RF lengths of 300±1bp. S. cerevisiae dominated the middle and the end fermentation. As the fermentation process went on,S. cerevisiae increased. At the end stage of fermentation,the proportion of S. cerevisiae reached to 100% and dominated then finished the fermentation.

In a word,this is the first time to use the two culture-independent methods,ARISA and T-RFLP,to study the diversity of wine-related yeasts. We also established the database of ARISA analysis and T-RFs using pure cultures and study their potential to determine yeast composition during wine fermentation. And their application enhanced our understanding of fermentation process,forming the basis for studying relations between yeast ecology and quality of wine.

Key words　ARISA　T-RFLP　Yeast diversity　Culture-independent methods