Browsing by Author "Van der Westhuizen, Theunes Johannes"

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    Characterization and evaluation of indigenous Saccharomyces cerevisiae strains isolated from South African vineyards
    (Stellenbosch : Stellenbosch University, 1999-12) Van der Westhuizen, Theunes Johannes; Pretorius, I. S.; Augustyn, O. P. H.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH SUMMARY: The bioconversion of grape juice into wine by simply allowing the yeasts, associated with grape berries and winery equipment, to ferment the sugars to ethanol, carbon dioxide and other minor, but important metabolites, is an ancient process. The art and science of winemaking has been extensively studied since the time when Louis Pasteur demonstrated, for the first time, the relationship between yeast and alcoholic fermentation. It is now recognized that the fermentation of grape must and production of premium quality wines is a complex ecological and biochemical process involving the sequential development of microbial species, as affected by a particular environment. This complex heterogeneous microbiological process includes the interaction of many microbial species, represented by fungi, yeasts, lactic acid bacteria and acetic acid bacteria, as well as the mycoviruses and bacteriophages affecting these grape-associated microorganisms. However, of all these different microbes and viruses, yeast represent the heart of the harmonious biochemical interaction with the musts derived from the various varieties of Vitis species which, in turn, are largely products of their respective genetic make-ups and the terroir. These yeasts are significant in winemaking because they not only conduct the alcoholic fermentation, but can also spoil wine during conservation in the cellar and after packaging, and they affect wine quality through the production of fermentation metabolites and through autolysis. A sound understanding of yeast systematics, biogeography and ecology is therefore essential to endeavours to preserve and exploit the hidden oenological potential of the untapped wealth of yeast biodiversity in our wine-producing regions. One of the main thrusts of this kind of eco-taxonomic survey is to determine the actual contribution of the indigenous strains of the so-called wine yeast (Saccharomyces cerevisiae) and wild yeasts (non-Saccharomyces species) to the sensory properties of wines and to eventually develop new starter culture strains for guided fermentations, including mixed starter cultures tailored to reflect the characteristics of a given wine region. Against this background, a comprehensive, long-term biogeographical survey and strain development programme was launched. This dissertation represents the first phase of this long-overdue research programme aimed to systematically catalogue yeasts in different climatic zones of the 350-year-old wine-producing regions of the Western Cape and to develop new yeast starter cultures that would further increase the quality of South African wine. The specific aims of this dissertation included (i) the evaluation of yeast fingerprinting techniques for their suitability to accurately and rapidly differentiate amongst S. cerevisiae strains; (ii) the isolation and characterization of S. cerevisiae strains from the coastal regions of the Western Cape; (iii) to determine the natural population dynamics of S. cerevisiae strains in selected vineyards over a four-year period; (iv) to make a preliminary determination of the possible effect that these indigenous S. cerevisiae isolates may have on wine flavour, and (v) to breed new starter culture strains with improved characteristics. Eighteen strains of S. cerevisiae used for commercial production of wine in South Africa were characterized by means of long-chain fatty acid analysis, randomly amplified polymorphic DNA (RAPD-PCR) and electrophoretic karyotyping (CHEF-DNA analysis). Variations in DNA profiles of the strains were apparent in the number, position and intensity of bands. It was found that electrophoretic karyotyping, as a single technique, seemed to be the most useful method to be used for routine fingerprinting. However, it was proposed that the combined use of these three techniques would provide the most reliable means of differentiating amongst wine yeast strains. Two of these fingerprinting techniques, CHEF-DNA and RAPD-PCR analysis, were used to determine the geographic distribution of indigenous S. cerevisiae strains isolated from local vineyards. Grapes were aseptically harvested from 13 sites in five areas in the coastal regions of the Western Cape during 1995. These sites were Groot Constantia and Buitenverwachting in the Constantia area; Jordan, Lievland, Mont Fleur and Nietvoorbij in the Stellenbosch area; Vergelegen in Somerset West; De Rust, Oak Valley, White Hall and Wildekrans in the Elgin/Bot River area; and Bouchard Finlayson and Hamilton Russel in the Hermanus area. After fermentation, 30 yeast colonies per sample were isolated and examined for the presence of S. cerevisiae. Five sampling sites yielded no S. cerevisiae strains. Electrophoretic karyotyping revealed the presence of 46 unique karyotypes in eight of the remaining sites. No dominant strain was identified and each site had its own unique collection of strains. The number of strains per site varied from two to 15. Only in four cases did one strain appear at two sites, while only one instance of a strain occurring at three sites was recorded. All sites contained killer and sensitive strains, however, killer strains did not always dominate. Commercial strains were recovered from three sites. Although commercial yeasts dominated the microflora at two sites, it appears that fears of commercial yeasts ultimately dominating the natural microflora seem to be exaggerated. As an extension of the 1995 survey samples were taken from the same locations at Groot Constantia, Buitenverwachting, Jordan, Lievland, Mont Fleur, Vergelegen, Bouchard Finlayson and Hamilton Russel during 1996 to 1998. This was done in an effort to assess how the natural population dynamics of S. cerevisiae are affected over the long term by abiotic factors. Thirty colonies per site were isolated and the S. cerevisiae strains were characterized by electrophoretic karyotyping. The identity of strains appearing at more than one site in the same, or different years, was confirmed by RAPD-PCR analysis. Strain numbers per site varied over the four-year study period. Weather conditions resulting in severe fungal infestations and heavy applications of chemical sprays during 1996 and early 1997 dramatically reduced the numbers of S. cerevisiae strains recovered during 1997. A return to normal weather patterns during mid 1997 resulted in a gradual recovery of the indigenous population as noted during the 1998 harvest. Indications are that some of the strains isolated are widespread in the study area and may represent yeasts typical of the area. Again, commercial wine yeast strains were recovered in only a few instances and the likelihood that commercial yeasts will eventually replace the natural yeast microflora in vineyards therefore seems remote. As a preliminary study to determine the possible effect of these indigenous S. cerevisiae strains on wine flavour, 33 of the indigenous yeasts were allowed to ferment Chenin blanc wine in laboratory fermentations. The juice was analyzed. The ability to form esters, fatty acids and higher alcohols was compared to that of two local commercial yeasts. None of the indigenous strains were found to be suitable for fermenting white must at 15°C. Their ability to ferment red musts at much higher temperatures still needs to be assessed. Furthermore, differences noted indicate that some of these strains show potential to be included in our extensive yeast-breeding programme as this would broaden the genetic pool. In parallel with the search to isolate and identify indigenous S. cerevisiae strains with good oenological potential, an extensive selection and breeding programme with cultures from our strain collection was undertaken. The aim of this programme was to generate new strains that are better suited to New World winemaking styles and conditions prevailing in South Africa. As a result, 145 hybrids, differentiated by elecrophoretic karyotyping and long-chain fatty acid analysis, were produced. Fifty-eight of the hybrids were able to ferment juice to dryness at 15°C in less than 21 days during microvinification trials. Five of the strains were released for commercial use after extensive industrial-scale evaluation. Based on the success of these interstrain hybridizations, the breeding programme will now be expanded to include some of the indigenous S. cerevisiae strains. In conclusion, it is only when we have a much better understanding of yeast biodiversity, biogeography, ecology and the interaction within yeast communities that we will be able to optimally harness the genetic pool in our strain development programme, aimed to benefit both the wine producer and the consumer.
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    Genetic characterisation and breeding of wine yeasts
    (Stellenbosch : Stellenbosch University, 1990) Van der Westhuizen, Theunes Johannes; Pretorius, I. S.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: To remain competitive in the market place, the South African wine industry will have to direct well-planned yeast strain-development programmes. However, the winemaker can only benefit from the extensive biochemical and molecular information of the yeast cell and the impressive arsenal of genetic techniques available, if the wine industry defines its requirements in genetic terms. The successful application of these genetic and recombinant deoxyribonucleic acid (DNA) techniques in breeding programmes depends on the availability of rapid and reliable techniques to differentiate between parental and hybrid strains. Ten strains of Saccharomyces cerevisiae used for commercial production of wine in South Africa, were characterised by electrophoretic banding patterns of total soluble cell proteins, DNA restriction fragments and chromosomal DNA. Variations in the protein and DNA profiles of strains N6, N21, N66, N76, N95 and N97 were apparent in the number, position and intensity of the bands. Strains N93 and N181 originated from the same culture and, as expected, displayed the same characteristic protein, DNA restriction fragment and chromosomal banding patterns. Similar protein and DNA profiles were also obtained for killer strain N96 and strain N91. Strain N91 is a derivative of strain N96, cured of the K2 killer character. Results obtained by electrophoretic fingerprinting and karyotyping corresponded well, indicating that these techniques are valuable in the identification and quality control of industrial wine yeasts. The value of electrophoretic fingerprinting and karyotyping was also demonstrated in a breeding programme. The aim of this breeding programme was to obtain hybrids that combine the desired oenological characteristics of strains N76 and N96, and of strains N96 and N181. The protein banding patterns of hybrids USM21, USM22 and USM23 were identical and contained a combination of prominent unique bands present in the profiles of parental strains, N76 and N96H (N96H is a haploid derived from N96). The DNA restriction fragment profiles of hybrids USM21, USM22 and USM23 contained slight variations, whereas their profiles were quite different from those of their parental strains, N76 and N96H. The contour clamped homogeneous electric field (CHEF) karyotypes of hybrids USM21, USM22 and USM23 were identical but differed from those of their parental strains, N76 and N96H. The protein profiles of hybrid USM30 and its parental strains, N96H and N181, were similar, whereas their DNA restriction fragment banding patterns and CHEF karyotypes showed discrete differences. In conclusion, protein and DNA fingerprinting techniques were found to be valuable in selecting four hybrid killer strains after mass spore-cell mating. These four killer hybrids contain desirable oenological properties long sought after by the South African wine industry. Fermentation trials and evaluation of these hybrids were conducted independently by the Deparment of Oenology, University of Stellenbosch and by Stellenbosch Farmers' Winery and they have now been released for commercial wine production.