Yeasts are predominant in the ancient and complex process of winemaking. In spontaneous fermentations, there is a progressive growth pattern of indigenous yeasts, with the final stages invariably being dominated by the alcohol-tolerant strains of Saccharomyces cerevisiae. This species is universally known as the ‘wine yeast’ and is widely preferred for initiating wine fermentations. The primary role of wine yeast is to catalyze the rapid, complete and efficient conversion of grape sugars to ethanol, carbon dioxide and other minor, but important, metabolites without the development of off-flavours. However, due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing quest for specialized wine yeast strains possessing a wide range of optimized, improved or novel oenological properties. This review highlights the wealth of untapped indigenous yeasts with oenological potential, the complexity of wine yeasts’ genetic features and the genetic techniques often used in strain development. The current status of genetically improved wine yeasts and potential targets for further strain development are outlined. In light of the limited knowledge of industrial wine yeasts’ complex genomes and the daunting challenges to comply with strict statutory regulations and consumer demands regarding the future use of genetically modified strains, this review cautions against unrealistic expectations over the short term. However, the staggering potential advantages of improved wine yeasts to both the winemaker and consumer in the third millennium are pointed out. Copyright # 2000 John Wiley & Sons, Ltd.

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Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking

The most mysterious aspect of wine is the endless variety of flavours that stem from a complex, completely non-linear system of interactions among many hundreds of compounds. In its widest sense, wine flavour refers to the overall impression of both aroma and taste components. Aroma is usually associated with odorous, volatile compounds; the bouquet of wine refers to the more complex flavour compounds which evolve as a result of fermentation, elevage and ageing. With the exception of terpenes in the aromatic grape varieties and alkoxypyrazines in the herbaceous cultivars, perceived flavour is the result of absolute amounts and specific ratios of many of these interactive compounds, rather than being attributable to a single "impact" compound. Without underestimating the complexity of these interactive effects or negating the definitive role played by the accumulated secondary grape metabolites in the varietal character of wine, this review will focus mainly on the contribution of yeast fermentation to the sensorial quality of the final product. Yeast and fermentation conditions are claimed to be the most important factors influencing the flavours in wine. Both spontaneous and inoculated wine fermentations are affected by the diversity of yeasts associated with the vineyard and winery. During the primary alcoholic fermentation of sugar, the wine yeast, Saccharomyces cerevisiae, together with other indigenous non-Saccharomyces species, produce ethanol, carbon dioxide and a number of by-products. Of these yeast-derived metabolites, the alcohols, acetates and C4-C8 1tfatty acid ethyl esters are found in the highest concentration in wine. While the volatile metabolites contribute to the fermentation bouquet ubiquitous to all young wines, the production levels of these by-products are variable and yeast strain specific. Therefore, this article also highlights the importance of untapping the hidden wealth of indigenous yeast species present on grapes, and the selection and genetic development of yeast starter culture strains with improved flavour profiles. In the future, some winemakers may prefer to use mixtures of indigenous yeast species and tailored S. cerevisiae strains as starter cultures to reflect the biodiversity and stylistic distinctiveness of a given region. This will help winemakers to fullfil the consumer's demand for individual wines with intact local character and to ensure the survival of wine's most enthralling aspect - its endless variety.

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Yeast and its Importance to Wine Aroma - A Review

Production of bioethanol from forest and agricultural products requires a fermenting organism that converts all types of sugars in the raw material to ethanol in high yield and with a high rate. This review summarizes recent research aiming at developing industrial strains of Saccharomyces cerevisiae with the ability to ferment all lignocellulose-derived sugars. The properties required from the industrial yeast strains are discussed in relation to four benchmarks: (1) process water economy, (2) inhibitor tolerance, (3) ethanol yield, and (4) specific ethanol productivity. Of particular importance is the tolerance of the fermenting organism to fermentation inhibitors formed during fractionation/pretreatment and hydrolysis of the raw material, which necessitates the use of robust industrial strain background. While numerous metabolic engineering strategies have been developed in laboratory yeast strains, only a few approaches have been realized in industrial strains. The fermentation performance of the existing industrial pentose-fermenting S. cerevisiae strains in lignocellulose hydrolysate is reviewed. Ethanol yields of more than 0.4 g ethanol/g sugar have been achieved with several xylose-fermenting industrial strains such as TMB 3400, TMB 3006, and 424A(LNF-ST), carrying the heterologous xylose utilization pathway consisting of xylose reductase and xylitol dehydrogenase, which demonstrates the potential of pentose fermentation in improving lignocellulosic ethanol production.

Towards industrial pentose-fermenting yeast strains

Saccharomyces cerevisiae and grape juice are ‘natural companions’ and make a happy wine marriage. However, this relationship can be enriched by allowing ‘wild’ non- Saccharomyces yeast to participate in a sequential manner in the early phases of grape must fermentation. However, such a triangular relationship is complex and can only be taken to ‘the next level’ if there are no spoilage yeast present and if the ‘wine yeast’ – S. cerevisiae – is able to exert its dominance in time to successfully complete the alcoholic fermentation. Winemakers apply various ‘matchmaking’ strategies (e.g. cellar hygiene, pH, SO2, temperature and nutrient management) to keep ‘spoilers’ (e.g. Dekkera bruxellensis ) at bay, and allow ‘compatible’ wild yeast (e.g. Torulaspora delbrueckii, Pichia kluyveri, Lachancea thermotolerans and Candida/Metschnikowia pulcherrima ) to harmonize with potent S. cerevisiae wine yeast and bring the best out in wine. Mismatching can lead to a ‘two is company, three is a crowd’ scenario. More than 40 of the 1500 known yeast species have been isolated from grape must. In this article, we review the specific flavour-active characteristics of those non- Saccharomyces species that might play a positive role in both spontaneous and inoculated wine ferments. We seek to present ‘single-species’ and ‘multi-species’ ferments in a new light and a new context, and we raise important questions about the direction of mixed-fermentation research to address market trends regarding so-called ‘natural’ wines. This review also highlights that, despite the fact that most frontier research and technological developments are often focussed primarily on S. cerevisiae , non- Saccharomyces research can benefit from the techniques and knowledge developed by research on the former.

Not your ordinary yeast: non-Saccharomyces yeasts in wine production uncovered

The microbial ecology of wine grape berries.

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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Yeast biodiversity in vineyards and wineries and its importance to the South African Wine Industry. A review

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.

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Geographical distribution of indigenous Saccharomyces cerevisiae strains isolated from vineyards in the coastal regions of the Western Cape in South Africa.

Despite our limited knowledge of the genetic make-up of commercial wine yeasts and the fact thatthe advantages of genetic manipulation of wine yeasts have not yet been demonstrated in practice, the wine industry has to realise that "the name of the game" is recombinant DNA and that the pace of progress is fast. The technology is so powerful that it now enables manipulation of the genome in ways hard to imagine only a decade ago. In this review we highlight the importance of the wine yeast to the wine industry and the necessity for well-planned breeding programmes. First, we summarise reliable taxonomic methods that are useful as diagnostic techniques in such breeding strategies. Second, we emphasise the complexity of the genetic features of commercial wine-yeast strains. Third, we review the genetic techniques available and point out the potential of these techniques (individually and in combination) in strain-development programmes. Finally, we attempt to stimulate interest in the genetic engineering of wine yeasts by discussing a few potential targets of strain development. The impact of yeast genetics and recombinant DNA technology on the wine industry promises to be impressive.

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The impact of yeast genetics and recombinant DNA technology on the wine industry - a review

Electrophoretic banding pattens of total soluble cell proteins, DNA restriction fragments and chromosomal DNA were used to characterise ten strains ofSaccharomyces cerevisiae used for commercial production of wine. These fingerprinting procedures provided unique profiles for all the different yeast strains and can therefore be used to identify and control industrial strains. Furthermore, the protein profiles, restriction fragments banding patterns and electrophoretic karyotyping by contour clamped homogeneous electric field electrophoresis (CHEF), were valuable to differentiate hybrid and parental strains in yeast breeding programmes. Hybrid strains, with desirable oenological properties, were obtained by mass spore-cell mating between a heterothallic killer yeast and two homothallic sensitive strains and all were shown to have unique DNA fingerprints and electrophoretic karyotypes.

The value of electrophoretic fingerprinting and karyotyping in wine yeast breeding programmes.

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.

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T.J. van der Westhuizen

Papers

Yeast biodiversity in vineyards and wineries and its importance to the South African Wine Industry. A review

Geographical distribution of indigenous Saccharomyces cerevisiae strains isolated from vineyards in the coastal regions of the Western Cape in South Africa.

The impact of yeast genetics and recombinant DNA technology on the wine industry - a review

The value of electrophoretic fingerprinting and karyotyping in wine yeast breeding programmes.

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