Thesis (PhD (Genetics. Plant Biotechnology))--Stellenbosch University, 2008. / Wine quality is greatly influenced by the number of microorganisms, which occur
throughout the winemaking process. These microorganisms are naturally present on
the grapes and in the cellar from where they can be introduced to the winemaking
process at any given time and consequently impart specific contributions to the wine
quality. However, these microorganisms can be seen either as beneficial or as wine
spoilage microorganisms, depending on the conditions under which they can
proliferate during the winemaking process. Wine yeasts (Saccharomyces spp.) are
typically responsible for the alcoholic fermentation; lactic acid bacteria (LAB) are
responsible for malolactic fermentation (MLF), while acetic acid bacteria (AAB) and
other wild yeasts (non-Saccharomyces spp.) are typically associated with the
formation of off-flavours under poorly controlled winemaking conditions.
In recent years, evidence from the wine industry has highlighted a specific group
of non-Saccharomyces yeast species as a serious cause for wine spoilage that
required more research investigations. Yeast of the genus Brettanomyces or its
teleomorph Dekkera has been identified as one of the most controversial spoilage
microorganisms during winemaking as they can produce several compounds that are
detrimental to the organoleptic quality of wine. This has triggered the research
initiative behind this doctoral study on the significance of Brettanomyces and Dekkera
yeasts during winemaking.
In this dissertation, various aspects of the detection, isolation and identification
methods of Brettanomyces yeast from the winemaking environment were
investigated. As a first objective, a culture collection of Brettanomyces bruxellensis
wine isolates had to be established. This followed after the isolation of
Brettanomyces yeasts from various red wine cultivars from South African wineries
from different stages of the winemaking process. Different conventional
microbiological methods such as plating on selective agar media and microscopy
were investigated along with molecular identification techniques such as the
polymerase chain reaction (PCR) in this regard.
Other focus areas of this study aimed at performing genetic characterisation and
differentiation studies of B. bruxellensis wine isolates. For this purpose, different
intraspecific identification methods were investigated on several strains, including
strains of European origin. The application of molecular techniques allowing strain
identification aided in the selection of specific strains that were evaluated for volatile
phenol production in synthetic media and wine. The results obtained from this work
indicated that a large degree of genetic diversity exists among B. bruxellensis strains
and that the volatile phenol production differed between the strains after evaluation in
synthetic media and wine.
In addition to the molecular intraspecific strain identification techniques that were
investigated, a feasibility study was also performed that focused on evaluating Fourier transform infrared (FTIR) spectroscopy combined with chemometrics as an
alternative approach for differentiating between B. bruxellensis strains.
The two approaches of FTIR spectroscopy that were investigated involved the
use of firstly, Fourier transform mid-infrared (FTMIR) spectroscopy to obtain spectral
fingerprints of spoiled wines by different B. bruxellensis strains; and secondly,
Attenuated total reflectance (FTIR-ATR) to obtain spectral fingerprints from whole
cells of B. bruxellensis on microbiological agar media. The results of this study
illustrated the potential of FTIR spectroscopy to become a reliable alternative to
molecular based methods for differentiating between B. bruxellensis strains and for
characterisation studies.
The formation of volatile phenols in wine by species of the genera Brettanomyces
and Dekkera is one of the primary reasons for their classification as wine spoilage
yeasts. The enzymatic activities of this reaction have been identified and involve a
phenyl acrylic (phenolic) acid decarboxylase (PAD) and a vinyl phenol reductase
(VPR). However, only a limited amount of information is available about these
enzymes from Brettanomyces/Dekkera yeasts and no genetic data have been
described. It was therefore imperative that this dissertation should include a genetic
investigation into the phenylacrylic (hydroxycinnamic) acid decarboxylase from the
species B. bruxellensis involved in the formation of volatile phenols. Strategies that
were investigated included various molecular DNA techniques and protein purification
procedures to obtain either genetic or protein sequence data. The decarboxylase
activity of this yeast species towards p-coumaric acid was demonstrated and
substantial genetic sequence data was obtained.
The results from this dissertation made a substantial contribution to the current
available knowledge about Brettanomyces/Dekkera spp. and led to a better
understanding of this wine spoilage yeast. This research developed a platform from
which further investigations could follow and the knowledge gained will be invaluable
for future Brettanomyces research projects at the Institute for Wine Biotechnology at
Stellenbosch University.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/1239 |
| Date | 12 1900 |
| Creators | Oelofse, Adriaan |
| Contributors | Du Toit, M., Pretorius, I. S., Lonvaud-Funel, A., Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology . |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | Stellenbosch University |
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