Research Articles (Viticulture and Oenology)

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Flavour components of whiskey. I. distribution and recovery of compounds by fractional vacuum distillation
(South African Society for Enology and Viticulture, 2001) MacNamara, K.; Van Wyk, C. J.; Augustyn, O. P. H.; Rapp, A.
A vacuum fractional distillation procedure is described for separating both the matrix components and flavour compounds of a whiskey into well-defined groups based on differences in azeotropic boiling points. The distillation was carried out at near ambient temperatures to accommodate both unaged and aged whiskies. Analytical and sensory data indicated good recovery of congeners. Individual fractions were reconstituted with ethanol and water to the original volume and strength dimensions of the whiskey. Undesirable thermal changes in the aged products were minimised by the low temperature fractionation and allowed changes in the flavour composition of whiskey due to maturation to be investigated for such unaged and aged reconstituted pairs.
Flavour components of whiskey. II. ageing changes in the high-volatility fraction
(South African Society for Enology and Viticulture, 2001) MacNamara, K.; Van Wyk, C. J.; Augustyn, O. P. H.; Rapp, A.
The volatile compounds isolated from whiskey by fractional vacuum distillation were identified by two-dimensional capillary gas chromatography/mass spectrometry. Changing levels with ageing were quantified for the most abundant compounds by direct split injection of whiskeys on a gas chromatograph equipped with a flame ionisation detector. The ageing decreases in volatile sulfides were similarly determined using a sulfur chemiluminescence detector. Large volume headspace injection sufficiently reproduced the distillation enrichment to allow direct twodimensional determination of similar ageing changes for other trace compounds. Seven compounds at μg/L and low mg/L levels were monitored and quantified.
Flavour components of whiskey. III. ageing changes in the low-volatility fraction
(South African Society for Enology and Viticulture, 2001) MacNamara, K.; Van Wyk, C. J.; Brunerie, P.; Augustyn, O. P. H.; Rapp, A.
The low-volatility wood-originating compounds isolated from whiskey by vacuum fractional distillation were analysed by high-resolution gas chromatography and mass spectrometry (GC-MS). Three phenolic esters previously unreported in whiskey were identified and confirmed by synthesis. Formation profiles for sixteen compounds were established in whiskeys aged for periods from 1.5 to 10 years in second-fill heavy-charred American Bourbon barrels. These profiles indicated significant increases for several compounds, especially in the older whiskeys. Ratios of aromatic phenolic aldehydes, and similar ratio changes during ageing, were different from reported data relating to other wood types and treatments. Further preparative separation by high-pressure liquid chromatography (HPLC) of the wood fraction followed by GC-MS allowed retention and mass spectral characterisation of additional compounds originating from wood. Sensory investigation indicated different and unique contributions from the HPLC cuts. Spiking of the three phenolic esters into a young whiskey gave a detectable increase in maturation intensity.
Geographic distribution and evaluation of saccharomyces cerevisiae strains isolated from vineyards in the warmer, Inland Regions of the Western Cape in South Africa
(South African Society for Enology and Viticulture, 2000) Khan, W.; Augustyn, O. P. H.; Van der Westhuizen, T. J.; Lambrechts, M. G.; Pretorius, I. S.
The aim of this study was to examine the geographic distribution of Saccharomyces cerevisiae strains indigenous to 19 sites in the warmer, inland regions of the Western Cape in South Africa. These strains were compared to those isolated previously from the cooler, coastal regions of the same province by subjecting both sets of organisms to the same characterisation procedures. Thirty isolates per sampling site were isolated and the S. cerevisiae strains subjected to the following characterisation procedures; karyotyping using pulse field gel electrophoresis (CHEF), randomly amplified polymorphic DNA, the polymerase chain reaction technique (RAPD-PCR), sugar fermentation ability, flocculation ability, stress resistance/response and extracellular enzyme activity. When considering biodiversity per sampling site, CHEF karyotypes indicated the recovery of 30 S. cerevisiae strains. This number was reduced to 21 when comparing banding patterns over sites. Addition of RAPD-PCR data expanded the number of unique strains to 29. Subsequent consideration of sugar fermentation data indicated that one of the strians with exactly equivalent CHEF and RAPD-PCR patterns was in fact galactose positive while the other was galactose negative. These data clearly indicate that characterisation of yeast strains by application of a single technique is not a sound practice. None of the S. cerevisiae strains isolated in this study occurred in the coastal regions. In addition, each site sampled in this study had its own unique collection of wine yeast strains and no strain common to all sites in the study region was found. Survival mechanisms of S. cerevisiae are obscure. Although we found that many of the isolated strains could grow invasively/form pseudohyphae and that these abilities could therefore contribute to the organism's overwintering ability, other mechanisms must also be involved.
Geographical distribution of indigenous saccharomyces cerevisiae strains isolated from vineyards in the coastal regions of the Western Cape in South Africa
(South African Society for Enology and Viticulture, 2000) Van der Westhuizen, T. J.; Augustyn, O. P. H.; Pretorius, I. S.
Notwithstanding numerous studies on the yeast biota of grapes and grape must, the origin of the primary wine yeast Saccharomyces cerevisiae has been rather controversial. One school of thought claims that the primary source of S. cerevisiae is the vineyard, whereas another believes that ecological evidence points to a strict association with artificial, man-made environments such as wineries and fermentation plants. One of the main thrusts of these kinds of investigations is to understand the succession of yeasts during fermentation of wine and to determine the actual contribution of indigenous strains of S. cerevisiae and wild yeast species to the overall sensorial quality of the end product, even in guided fermentations using selected S. cerevisiae starter cultures. There is increasing interest within the wine community in the use of indigenous strains of S. cerevisiae and mixed starter cultures, tailored to reflect the characteristics of a given region. Against this background we have launched a comprehensive and long overdue biogeographical survey systematically cataloging yeasts in different climatic zones of the 350-year-old wine-producing regions of the Western Cape. The present paper represents the first phase of this programme aimed at preserving and exploiting the hidden oenological potential of the untapped yeast biodiversity in South Africa's primary grape-growing areas. Grapes were aseptically harvested from 13 sites in five areas in the coastal regions of the Western Cape. After fermentation, 30 yeast colonies per sample were isolated and examined for the presence of S. cerevisiae. Five sampling sites yielded no S. cerevisiae. CHEF-DNA analysis 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.
Seasonal variation of indigenous saccharomyces cerevisiae strains isolated from vineyards of the Western Cape in South Africa
(South African Society for Enology and Viticulture, 2000) Van der Westhuizen, T. J.; Augustyn, O. P. H.; Khan, W.; Pretorius, I. S.
There is strong support for the use of naturally-occurring Saccharomyces cerevisiae strains that improve the sensory quality of wines and reflect the characteristics of a given region. Contrary to popular belief, S. cerevisiae is found at very low numbers on healthy, undamaged grapes and is rarely isolated from intact berries. The majority of studies on the population kinetics and geographic distribution of indigenous S. cerevisiae strains have not adequately focused on the variation in their numbers over a longer period of time. This paper discusses the results obtained in the first phase of a comprehensive research programme aimed at assessing how the natural population dynamics of S. cerevisiae are affected over the long term by abiotic factors. Indigenous strains of S. cerevisiae were aseptically isolated from eight sites in four areas in the coastal regions of the Western Cape, South Africa, during 1995 through 1998. Thirty colonies per site were isolated and the S. cerevisiae strains were characterised by electrophoretic karyotyping. Strain numbers per site varied over the four-year study period. Weather conditions resulting in severe fungal infestations and heavy applications of chemical sprays dramatically reduced the numbers of S. cerevisiae strains recovered during 1997. A return to normal weather patterns in 1998 resulted in a gradual recovery of the indigenous population. Indications are that some of the strains isolated are widespread in the study area and may represent yeasts typical of the area. 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 seems remote.
The value of long-chain fatty acid analysis, randomly amplified polymorphic DNA and electrophoretic karyotyping for the characterization of wine yeast strains
(South African Society for Enology and Viticulture, 1998) Van der Westhuizen, T. J.; Augustyn, O. P. H.; Pretorius, I. S.
Wine yeast strains of Saccharomyces had previously been classified into several different species or varieties. This classification system was based mainly on sugar fermentation and assimilation patterns. Subsequently, most of these species were reclassified as Saccharomyces cerevisiae. The assignment of the majority of wine yeast strains to a single species does, however, not imply that all stains of S. cerevisiae are equally suitable for wine fermentation. These physiological strains of S. cerevisiae differ significantly in their fermentation performance and their ability to contribute to the final bouquet and quality of the various types of wine and distillates. Therefore, to ensure strain authenticity, security and proper strain management, it is of cardinal importance to have reliable taxonomic techniques available to identify and characterize individual strains of commercial cultures. In this study, 18 commercial wine yeast strains were characterized in order to evaluate and compare three taxonomic techniques, namely long-chain fatty acid analysis, randomly amplified polymorphic DNA (RAPD) and electrophoretic karyotyping. As a single identification technique, electrophoretic karyotyping seems to be the most useful method for routine fingerprinting of wine yeast strains. However, we propose that the combined use of these three techniques provides the most reliable means of differentiating amongst commercial wine yeast strains.
Yeast biodiversity in vineyards and wineries and its importance to the South African wine industry : a review
(South African Society for Enology and Viticulture, 1999) Pretorius, I. S.; Van der Westhuizen, T. J.; Augustyn, O. P. H.
The art of winemaking is as old as human civilization and the use of yeast in this complex ecological and biochemical process dates back to ancient times. Traditionally, yeasts associated with grape berries were simply allowed to ferment the sugars to ethanol, carbon dioxide and other minor, but important, metabolites. Spontaneous fermentations are still being used in boutique wineries that depend more on vintage variability. Various microbes found on the surface of grape skins and the indigenous microbiota associated with winery surfaces participate in these natural wine fermentations. Yeasts of the genera Kloeckera, Hanseniaspora and Candida predominate in the early stages, followed by several species of Metschnikowia and Pichia (including those species that were previously assigned to the genus Hansenula) in the middle stages when the ethanol rises to 3-4%. The latter stages of natural wine fermentations are invariably dominated by the alcohol-tolerant strains of Saccharomyces cerevisiae. However, other yeasts, such as species of Brettanomyces, Kluyveromyces, Schizosaccharomyces, Torulaspora and Zygosaccharomyces also may be present during the fermentation and can occur in the resultant wine. By contrast, the rule, rather than the exception, for modern wineries depending on reliable fermentation and the production of wines with predictable quality, is the use of specially selected starter cultures of Saccharomyces. However, the use of such cultures may not necessarily prevent the growth and metabolic activity of indigenous, winery associated strains of S. cerevisiae or other wild yeasts such as Kloeckera apiculata, Hanseniaspora uvarum, Candida stellata and Torulaspora delbrueckii. It is therefore clear that both spontaneous and inoculated wine fermentations are affected by the diversity of yeasts associated with the vineyard (natural habitat) and winery (man-made niche). In light of this, focused taxonomic surveys within an ecological framework are essential to preserve and exploit the hidden oenological potential of the untapped wealth of yeast biodiversity in our wine-producing regions. To achieve this, yeast taxonomists need to continue to isolate and characterize new yeast species and strains, while wine microbiologists develop improved identification techniques that differentiate more efficiently among individual strains. At the same time such biological surveys will complement strain development and the current international effort of molecular biologists to assign a biological function to the products of each of the 6000 genes identified by computer analysis of the nucleotide sequence of the 16 chromosomes of a laboratory strain of S. cerevisiae. Furthermore, only when we have a much better understanding of yeast biodeversity, biogeography, ecology and the interaction within yeast communities will we be able to optimally harness gene technology that will benefit both the wine producer and the consumer.

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