Thesis (MSc)--Stellenbosch University, 2012. / 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. / AFRIKAANSE OPSOMMING: Die Saccharomyces sensu stricto kompleks, is bekend vir die belangrike rol wat hierdie giste
speel tydens alkoholiese fermentasie in biede wyn en bier industrieë. Dit is om hierdie rede dat
kelders rein aktief gedroogte wyngis gebruik vir die produksie van spesifieke wynstyle, asook
kwaliteit. Daar is meer as 200 kommersiële wyngiste op die mark beskikbaar en dit sluit
natuurlike isolate en hibriede in. Daarom is gisras verifikasie baie belangrik vir die vervaardiger
van aktief gedroogde wyngiste asook die wynmaker om finansiële verliese te voorkom en mark
vertrouenswaardigheid te handhaaf. Verskeie tradisionele en molekulêre metodes word
suksesvol toegepas vir gehalte beheer van die gisrasse.
Die doel van hierdie studie was om elektroforetiese kariotipering (CHEF) en PKR
gebaseerde tegnieke se vermoë om tussen Saccharomyces wyngiste te onderskei, te
ondersoek. Ook deel van die doelwitte was om ‘n databasis te skep wat die verskillende
elektroforetiese profiele van die kommersiële gisrasse bevat. Tydens hierdie studie is 62
kommersiële gisrasse van verskeie vervaardigers ewekansig geselekteer. Saccharomyces
bayanus CBS 380 en S. cerevisiae CBS 1171 is as verwysingsrasse gebruik. Elektroforetiese
kariotipering (CHEF) is gekies omdat dit een van die mees algemeenste tegnieke is wat gebruik
word om tussen wyngiste te onderskei, maar dit word as tydrowend en arbeidsintensief beskou.
As ‘n alternatief is daar na PKR gebaseerde tegnieke gekyk. Hierdie tegnieke word as
betroubaar en vinnig beskou. Verskeie PKR gebaseerde tegnieke is ondersoek, naamlik PKR
van interdelta areas, multipleks-PKR van mini- en mikrosatelliete, MET2 geen RFLP analise en
die gebruik van spesie-spesifieke inleiers. Interdelta amplifikasies en mini- en makrosatelliet
inleiers is geselekteer as gevolg van hul vermoë om Saccharomyces wyngiste tot op spesie en
ras vlak te onderskei. Die MET2 geen en spesie-spesifieke inleiers is geselekteer om die
kommersiele wyngis as S. cerevisiae, S. bayanus of as hibriede te klassifiseer.
CHEF kariotipering kon tussen al 64 giste onderskeid tref. Die twee stelle inleiers wat vir
interdelta amplifikasie gebruik was, delta1-2 en delta12-21, het onderskeidelik 59 en 62 profiele
gelewer. Gis rasse wat identiese profiele met die delta inleiers gelewer het, kon egter met CHEF
kariotipering onderskei word. Die resultate het getoon dat CHEF kariotipering beter tussen die
kommersiële wyngiste kon onderskei as die interdelta amplifikasies, maar dat die interdelta
amplifikasies nogsteeds goeie onderskeiding toon en dat dit minder tydrowend is. Die
multipleks-PKR van mini- en mikrosatelliete kon slegs ‘n enkele band in 55 van die 64 giste uit
lig. ‘n Moontlike aanduiding van spesie spesifiekheid. Die oenologiese groepering volgens
MET2 geen analise en spesies-spesifieke inleiers dui aan dat al die kommersiele wyngiste wat
in hierdie studie gebruik is, moontlik van S. cerevisiae afkomstig is. Restriksie analise van die
MET2 geen met EcoRI het ook AWRI Fusion en Zymaflore X5 as hibriede geïdentifiseer. Die
CHEF kariotipes en interdelta elektroforetiese profiele is gebruik om ‘n databasis van die
kommersiële Saccharomyces wyngiste op te stel. Die databasis is funksioneel en het die toets rasse akkuraat geïdentifiseer en korrek gegroepeer. Die databasis moet egter nog verdere
optimisering en verfyning ondergaan.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/19942 |
| Date | 03 1900 |
| Creators | Hoff, Justin Wallace |
| Contributors | Du Plessis, H. W., Bauer, F. F., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 75 p. : ill. |
| Rights | Stellenbosch University |
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