Molecular typing of wine yeasts : evaluation of typing techniques and establishment of a database

[摘要] ENGLISH ABSTRACT:The yeast species, Saccharomyces cerevisiae and S. bayanus are well known for the key rolethey play during alcoholic fermentation in both wine and beer industries. These yeasts areavailable in pure active dried form and can be used to produce different wine styles and tomanage quality. There are more than 200 commercial wine yeast strains on the market andinclude naturally isolated strains and hybrids. With all these commercial yeasts available, strainauthenticity is very important to the manufacturer of active dried wine yeasts (ADWY) because itcan prevent commercial losses and maintain market credibility. It is as important to thewinemaker as it may impact wine quality. Various traditional and molecular techniques havebeen successfully applied to perform quality control of wine yeast strains.The aims of this study were to evaluate electrophoretic karyotyping (CHEF) and PCRbasedmethods to distinguish between Saccharomyces wine yeast strains and to establish adatabase containing molecular profiles of commercial strains. CHEF karyotyping was chosenbecause it is generally used in the wine industry to distinguish between wine yeast strains, butcan be time-consuming. Alternatively, PCR-based methods are considered to be reliable andfast. These PCR methods included the evaluation of interdelta regions, multiplex-PCR of miniandmicrosatellites, MET2 gene RFLP analysis and the use of several species-specific primers.In this study, 62 commercial wine yeast strains, were randomly selected from variousmanufacturers of ADWY, and two reference strains, S. bayanus CBS 380 and S. cerevisiaeCBS 1171, were evaluated. CHEF karyotyping could successfully differentiate between all 64yeast 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 identicalaccording to interdelta amplification results, were resolved with CHEF karyotyping. CHEFkaryotyping was proven to be more accurate than interdelta amplifications in distinguishingbetween commercial wine yeast strains. However, the results of interdelta amplifications werevery useful and less time-consuming. The multiplex-PCR of mini- and microsatellite primers onlysucceeded 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, oenologicaldesignation using MET2 gene RFLP analysis and species-specific primers indicated that all thecommercial strains in this study had a S. cerevisiae ancestry. Restriction analysis of the MET2gene with EcoRI also successfully identified AWRI Fusion and Zymaflore X5 as hybrid yeaststrains. 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 andshowed great accuracy in grouping and identifying test strains. The database has manypossible applications, but there is still some optimisation and refinement needed.

[发布机构] Stellenbosch University