Masters Degrees (Institute for Wine Biotechnology)

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Browsing Masters Degrees (Institute for Wine Biotechnology) by browse.metadata.advisor "Du Plessis, H. W."

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    Molecular typing of wine yeasts : evaluation of typing techniques and establishment of a database
    (Stellenbosch : Stellenbosch University, 2012-03) Hoff, Justin Wallace; Du Plessis, H. W.; Bauer, Florian; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: The yeast species, Saccharomyces cerevisiae and S. bayanus are well known for the key role they play during alcoholic fermentation in both wine and beer industries. These yeasts are available in pure active dried form and can be used to produce different wine styles and to manage quality. There are more than 200 commercial wine yeast strains on the market and include naturally isolated strains and hybrids. With all these commercial yeasts available, strain authenticity is very important to the manufacturer of active dried wine yeasts (ADWY) because it can prevent commercial losses and maintain market credibility. It is as important to the winemaker as it may impact wine quality. Various traditional and molecular techniques have been successfully applied to perform quality control of wine yeast strains. The aims of this study were to evaluate electrophoretic karyotyping (CHEF) and PCRbased methods to distinguish between Saccharomyces wine yeast strains and to establish a database containing molecular profiles of commercial strains. CHEF karyotyping was chosen because it is generally used in the wine industry to distinguish between wine yeast strains, but can be time-consuming. Alternatively, PCR-based methods are considered to be reliable and fast. These PCR methods included the evaluation of interdelta regions, multiplex-PCR of miniand microsatellites, MET2 gene RFLP analysis and the use of several species-specific primers. In this study, 62 commercial wine yeast strains, were randomly selected from various manufacturers of ADWY, and two reference strains, S. bayanus CBS 380 and S. cerevisiae CBS 1171, were evaluated. CHEF karyotyping could successfully differentiate between all 64 yeast strains. The two primer sets used for interdelta amplifications, delta1-2 and delta12-21, yielded 59 and 62 profiles, respectively. Yeast strains considered to be similar or identical according to interdelta amplification results, were resolved with CHEF karyotyping. CHEF karyotyping was proven to be more accurate than interdelta amplifications in distinguishing between commercial wine yeast strains. However, the results of interdelta amplifications were very useful and less time-consuming. The multiplex-PCR of mini- and microsatellite primers only succeeded in identifying a specific band within 55 of the 64 yeast strains including the S. cerevisiae reference strain, a possible indication of species specificity. However, oenological designation using MET2 gene RFLP analysis and species-specific primers indicated that all the commercial strains in this study had a S. cerevisiae ancestry. Restriction analysis of the MET2 gene with EcoRI also successfully identified AWRI Fusion and Zymaflore X5 as hybrid yeast strains. A wine yeast database was created and contains three libraries, i.e. CHEF karyotypes, delta1-2 and delta12-21 electrophoretic profiles. The database was proven to be functional and showed great accuracy in grouping and identifying test strains. The database has many possible applications, but there is still some optimisation and refinement needed.