Differentiation between Yeast Species, and Strains within a Species, by Cellular Fatty Acid Analysis. 2. Saccharomyces cerevisiae

Fatty acid extracts of 50 Saccharomyces cerevisiae strains, grown under rigidly standardised conditions, were subjected to capillary gas chromatographic analysis on a polar column. Strains contained saturated, mono-unsaturated and trace amounts of dienoic fatty acids. The mean relative percentages of 10 fatty acids were used to differentiate between the strains studied. Forty-six strains could be differentiated from all others in the group, based on the criterion that when comparing two strains the CFAP's were considered unique if the MRP's of at least one fatty acid differed at the 1 % level. Holman's Index of Relationship proved to be a useful tool for indicating degree of similarity between fatty acid proftles.Efforts to distinguish between the species Sacch. cerevisiae and Sacch. bayanus were not successful. More analyses on authentic strains (determined by DNA homology) are necessary to confirm whether such a separation is possible or not. Oenologically important changes that occurred in a commercial dried yeast during production were reflected in thefatty acid proftles of the dried products. Index of Relationship between the fatty acid profiles of five other dried yeast products, and the mother cultures from which they were produced, was very high indicating no change during the commercial production phase. Changes, or lack of change, were confirmed by fermentation studies

The Role and Use of Non-Saccharomyces Yeasts in Wine Production

The contribution by the numerous grape-must-associated non-Saccharomyces yeasts to wine fermentation has beendebated extensively. These yeasts, naturally present in all wine fermentations, are metabolically active and theirmetabolites can impact on wine quality. Although often seen as a source of microbial spoilage, there is substantialcontrary evidence pointing to a positive contribution by these yeasts. The role of non-Saccharomyces yeasts in winefermentation is therefore receiving increasing attention by wine microbiologists in Old and New World wineproducing countries. Species that have been investigated for wine production thus far include those from theCandida, Kloeckera, Hanseniaspora, Zygosaccharomyces, Schizosaccharomyces, Torulaspora, Brettanomyces,Saccharomycodes, Pichia and Williopsis genera. In this review the use and role of non-Saccharomyces yeast in wineproduction is presented and research trends are discussed

The Use of Candida pulcherrima in Combination with Saccharomyces cerevisiae for the Production of Chenin blanc Wine

Wine fermentations are conducted by naturally occurring or selected industrial wine yeast strains of Saccharomyces cerevisiae. However, non-Saccharomyces yeasts also occur naturally in fermenting grape musts, especially in the initial stages of the fermentation. It has been speculated that these yeasts can contribute to the overall characteristics of the wine. Generally, it is accepted that Kloeckera apiculata is the predominant non-Saccharomyces yeast species in grape must. However, it was shown previously that Candida pulcherrima was the predominant non-Saccharomyces yeast species in a grape must after sedimentation and prior to inoculation with commercial wine yeast. Subsequently, this yeast was investigated in laboratory and small-scale wine fermentations of Chenin blanc wine. As it could not ferment grape juice to dryness on its own, it was used in combination with a S. cerevisiae wine yeast strain. The effect of SO2, di-ammonium phosphate (DAP), pH and temperature on the growth of C. pulcherrima was also investigated. In combined fermentations, no change in overall fermentation rate or standard wine chemical analyses could be observed in comparison to a control S. cerevisiae fermentation.However, wine production in three consecutive years showed that the wine produced by the combined fermentation was of higher quality than that produced by the S. cerevisiae only

The Occurrence of Non-Saccharomyces cerevisiae Yeast Species Over Three Vintages in Four Vineyards and Grape Musts From Four Production Regions of the Western Cape, South Africa

The role of non-Saccharomyces yeasts in wine production has been extensively debated and there is growing evidence that non-Saccharomyces yeasts play an important role in wine quality. It has been suggested that metabolites formed by some non-Saccharomyces species may contribute to wine quality. Recently a comprehensive, longterm research programme was launched by role players in the South African wine industry, whose aims include the isolation, characterisation and preservation of the natural yeast biodiversity of the Western Cape. As part of the programme, this paper investigates the presence of non-Saccharomyces yeast species over three vintages in four vineyards and musts in four distinct areas of the Western Cape. Samples were taken and the non-Saccharomyces yeast isolates were characterised by biochemical profiling and pulse field gel electrophoresis. In total 720 yeasts representing 24 species were isolated. Predominant species found in the must samples, i.e. Candida stellata, Kloeckera apiculata, Candida pulcherrima and Candida colliculosa, should have the most impact on subsequent fermentation

Geographical Distribution of Indigenous Saccharomyces cerevisiae Strains Isolated from Vineyards in the Coastal Regions of the Western Cape in South Africa

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

Yeast Biodiversity in Vineyards and Wineries and Its Importance to the South African Wine Industry. A Review

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

Flavour Components of Whiskey. II Ageing Changes in the High-Volatility Fraction

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

The Value of Long-Chain Fatty Acid Analysis, Randomly Amplified Polymorphic DNA and Electrophoretic Karyotyping for the Characterization of Wine Yeast Strains

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

Flavour Components of Whiskey. I. Distribution and Recovery of Compounds by Fractional Vacuum Distillation

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

Seasonal Variation of Indigenous Saccharomyces cerevisiae Strains Isolated from Vineyards of the Western Cape in South Africa

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