Frank Schmid

Introducing a New Breed of Wine Yeast: Interspecific Hybridisation between a Commercial Saccharomyces cerevisiae Wine Yeast and Saccharomyces mikatae

Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment.

Viability of common wine spoilage organisms after exposure to high power ultrasonics.

Microbial spoilage of wine can lead to significant economic loss. At present sulfur dioxide is the main additive to juice/must/wine to prevent and control microbial spoilage. As an alternative, or complement to SO(2), high power ultrasonics (HPU) may be used to control microbes. Several wine spoilage yeasts and bacteria were treated with HPU in saline (0.9% w/v NaCl), juice and red wine to assess their susceptibility to HPU. Significant killing was seen across several yeasts and bacteria commonly associated with winemaking and wine spoilage. In general the viability of yeast was more affected than that of bacteria.

Genome-wide identification of the Fermentome; genes required for successful and timely completion of wine-like fermentation by Saccharomyces cerevisiae

BackgroundWine fermentation is a harsh ecological niche to which wine yeast are well adapted. The initial high osmotic pressure and acidity of grape juice is followed by nutrient depletion and increasing concentrations of ethanol as the fermentation progresses. Yeast’s adaptation to these and many other environmental stresses, enables successful completion of high-sugar fermentations. Earlier transcriptomic and growth studies have tentatively identified genes important for high-sugar fermentation. Whilst useful, such studies did not consider extended growth (>5 days) in a temporally dynamic multi-stressor environment such as that found in many industrial fermentation processes. Here, we identify genes whose deletion has minimal or no effect on growth, but results in failure to achieve timely completion of the fermentation of a chemically defined grape juice with 200 g L−1 total sugar.ResultsMicro- and laboratory-scale experimental fermentations were conducted to identify 72 clones from ~5,100 homozygous diploid single-gene yeast deletants, which exhibited protracted fermentation in a high-sugar medium. Another 21 clones (related by gene function, but initially eliminated from the screen because of possible growth defects) were also included. Clustering and numerical enrichment of genes annotated to specific Gene Ontology (GO) terms highlighted the vacuole’s role in ion homeostasis and pH regulation, through vacuole acidification.ConclusionWe have identified 93 genes whose deletion resulted in the duration of fermentation being at least 20% longer than the wild type. An extreme phenotype, ‘stuck’ fermentation, was also observed when DOA4, NPT1, PLC1, PTK2, SIN3, SSQ1, TPS1, TPS2 or ZAP1 were deleted. These 93 Fermentation Essential Genes (FEG) are required to complete an extended high-sugar (wine-like) fermentation. Their importance is highlighted in our Fermentation Relevant Yeast Genes (FRYG) database, generated from literature and the fermentation-relevant phenotypic characteristics of null mutants described in the Saccharomyces Genome Database. The 93-gene set is collectively referred to as the ‘Fermentome’. The fact that 10 genes highlighted in this study have not previously been linked to fermentation-related stresses, supports our experimental rationale. These findings, together with investigations of the genetic diversity of industrial strains, are crucial for understanding the mechanisms behind yeast’s response and adaptation to stresses imposed during high-sugar fermentations.

Relative Efficacy of High-Pressure Hot Water and High-Power Ultrasonics for Wine Oak Barrel Sanitization

A significant amount of capital and maintenance is invested in oak barrels, which contribute greatly to wine quality. If not cleaned correctly, barrels can accumulate tartrate deposits and wine spoilage organisms, which can be detrimental to wine quality. We examined the efficacy of using high-power ultrasonics (HPU) for sanitization of wine barrels using the spoilage yeast Dekkera (Brettanomyces) bruxellensis as the test organism and indirectly compared it to standard hot water washing practices. Both 1- and 3-year-old oak were investigated and no culturable cells were detected on the surface (0 to 2 mm) or subsurface (2 to 4 mm) of the oak after treatment with HPU in hot water (60°C). Additionally, wines stored over a 12-month period in barrels initially cleaned with hot water, cold water, or HPU did not differ in their extraction of oak compounds, nor could these wines be differentiated by a sensory panel. Thus, HPU did not adversely affect oak extraction into wine.

Use of fresh versus frozen or blast-frozen grapes for small-scale fermentation

Background: This paper firstly examines the validity of using laboratory-scale fermentations as a means of correlating winemaking outcomes with larger industrial scale fermentations. Secondly, conventional and blast-freezing of whole bunches were investigated for their relative suitability as methods of preservation as determined by the nature of the resulting wines. Methods: Red must fermentations were compared at the laboratory 80 kg scale, and the more industrially representative 500 kg pilot scale. Fermentation profiles and duration for both scales were found to be very similar. Whole bunches were either slow/conventionally frozen (−20°C), or quickly/blast-frozen (−25°C). Results: Wines made from frozen grapes compared well with the wine made from the fresh must. Color and chemical analyses of the wines revealed few differences. A duo-trio sensory evaluation showed that wine from blast-frozen grapes was more similar to the fresh wines than wines from conventional frozen grapes. Conclusion: The findings of this research suggest that whole-bunch blast-freezing of grapes is preferable to conventional freezing.

Optimisation of industrial strains of Saccharomyces cerevisiae using recombinant and non-recombinant methods

The production of fermented beverages such as beer and wine is typically achieved using strains of Saccharomyces cerevisiae, which have been selected for their reliable fermentative properties and desirable sensory contribution to the end product. Even so, these strains are not without shortcomings and new strains are constantly being sought. In aiming to provide such strains, working with industrial isolates has its own set of challenges. Wine strains of S. cerevisiae are typically of higher ploidy or aneuploid, frequently lack convenient selectable markers and are homothallic. Laboratory strains, along with their comprehensive set of genetic tools, might be a more convenient research vehicle, but the fact remains that such strains fare poorly under the highly stressful conditions of industrial fermentations: sugar contents of over 200 g/L; low nutrient availability and pH; and high final ethanol concentrations. More importantly, the wines produced by laboratory strains of yeast frequently display poor sensory properties. Put simply, laboratory yeast lack the desirable phenotypic characteristics needed to make wine of quality to enable industrially relevant studies.