Analysis Of Wine-Related Yeast Diversity By ARISA And T-RFLP Technic

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 HaeⅢand 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 repeatalility for both ARISA and T-RFLP analysis. 91 yeast stains belonged to 39 species were analysed 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 HaeⅢand 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 HaeⅢdigestion could distinguish them. T-RFLP with 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 HaeⅢand HinfⅠ, we revealed the yeast polulation and amount during the stages of alcoholic fermentation more quickly and accurately. During the spontaneous fermentation of Riesling, T-RFLP analysis had detectd 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 detectd by T-RFLP analysis were less yeast than the traditional methods. M. pulcherrima and P. minuta var.minuta were detectd by WL, while T-RFLP did not due to the nonuniform samples. T-RFLP analysis had detectd 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.