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.

Aims: The objective of this study was to investigate what types of enzymes are being produced by non-Saccharomyces yeasts isolated from grapes in South Africa vineyards and clari®ed grape juice. These enzyme pro®les could pave the way for attributing speci®c effects in wine to some of these enzymes produced by so-called wild yeasts associated with grape must. Methods and Results: In this study 245 yeast isolates, belonging to the genera Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora and Kluyveromyces were screened for the production of extracellular pectinases, proteases b-glucanases, lichenases, b-glucosidases, cellulases, xylanases, amylases and sulphite reductase activity. These yeasts, representing 21 species, were previously isolated from grapes and clari®ed grape juice. The production of all extracellular hydrolytic enzymes screened for was observed except b-glucosidase activity. The amount and range of enzymes produced varied with different isolates of the same species. Conclusion: This study clearly revealed the potential of non-Saccharomyces wine yeasts to produce a wide range of useful extracellular enzymes during the initial phase of wine fermentation. Signi®cance and Impact of the Study: Enzymes produced by indigenous yeasts associated with grapes and juice might be harnessed to catalyse desired biotransformations during wine fermentation.

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

Research has shown that non-Saccharomyces yeast strains can be detected throughout wine fermentation. Non-Saccharomyces yeasts can therefore influence the course of fermentation and also the character of the resultant wine. Previously it was shown that four non-Saccharomyces species, i.e. Kloeckera apiculata, Candida stellata, Candida pulcherrima and Candida colliculosa, predominated in grape must at the start of fermentation. In this study these four yeasts were used singularly and in combination with an industrial wine yeast (Saccharomyces cerevisiae strain VIN 13) to ferment must on a laboratory scale. The resultant wine was analysed for ethanol, volatile acidity, total S02 and glycerol. Results show that, in comparison with the industrial wine yeast, the non-Saccharomyces yeast strains could not ferment all the sugar. Furthermore, while the individual non-Saccharomyces-fermented wines had different chemical analyses, the wines fermented by the combinations were similar to the wine produced by the industrial yeast only. In subsequent, small-scale winemaking trials some of the wines produced by combined fermentations were judged to be of better quality than those produced by the S. cerevisiae only. However, this quality increase could not be linked to increased ester levels.

The fission yeast Schizosaccharomyces pombe has been widely used to study eukaryotic cell biology, but almost all of this work has used derivatives of a single strain. We have studied 81 independent natural isolates and 3 designated laboratory strains of Schizosaccharomyces pombe. Schizosaccharomyces pombe varies significantly in size but shows only limited variation in proliferation in different environments compared with Saccharomyces cerevisiae. Nucleotide diversity, π, at a near neutral site, the central core of the centromere of chromosome II is approximately 0.7%. Approximately 20% of the isolates showed karyotypic rearrangements as detected by pulsed field gel electrophoresis and filter hybridization analysis. One translocation, found in 6 different isolates, including the type strain, has a geographically widespread distribution and a unique haplotype and may be a marker of an incipient speciation event. All of the other translocations are unique. Exploitation of this karyotypic diversity may cast new light on both the biology of telomeres and centromeres and on isolating mechanisms in single-celled eukaryotes.

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.

The present paper describes the behaviour of Lachancea thermotolerans and Saccharomyces cerevisiae in pure, co-cultured and sequential fermentations in cv. Emir grape must. Faster fermentation rates were observed in wine made with a pure culture of S. cerevisiae and wine produced with simultaneously inoculated cultures of L. thermotolerans and S. cerevisiae. Both L. thermotolerans and S. cerevisiae gave high population numbers. The use of L. thermotolerans in mixed and sequential cultures led to an increase in final total acidity content in the wines, varying in the range 5.40-6.28 g/l (as tartaric acid), compared to pure culture S. cerevisiae, which gave the lowest level of total acidity (5 g/l). The increase was in the order of 1.18-2.06 g/l total acidity. Increase in final acidity by the use of L. thermotolerans might be useful to improve wines with low acidity due to global climate change. Volatile acidity levels (as acetic acid) were in the range 0.53-0.73 g/l, while the concentration of ethyl alcohol varied in the range 10.76-11.62% v/v. Sequential fermentations of wines and pure culture fermentation of L. thermotolerans resulted in reduction in the concentrations of acetaldehyde and higher alcohols, with exception of N-propanol and esters. According to the sensory analysis, wine obtained with sequential inoculation of L. thermotolerans followed by inoculation of S. cerevisiae after 24 h, and simultaneous inoculation of these yeasts, was the most preferred.

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