Grape proteins aggregate in white wine to form haze. A novel method to prevent haze in wine is the use of haze protective factors (Hpfs), specific mannoproteins from Saccharomyces cerevisiae, which reduce the particle size of the aggregated proteins. Hpf1p was isolated from white wine and Hpf2p from a synthetic grape juice fermentation. Putative structural genes, YOL155c and YDR055w, for these proteins were identified from partial amino acid sequences of Hpf1p and Hpf2p, respectively. YOL155c also has a homologue, YIL169c, in S. cerevisiae. Comparison of the partial amino acid sequence of deglycosylated-Hpf2p with the deduced protein sequence of YDR055w, confirmed five of the 15 potential N-linked glycosylation sites in this sequence were occupied. Methylation analysis of the carbohydrate moieties of Hpf2p indicated that this protein contained both N- and O-linked mannose chains. Material from fermentation supernatant of deletion strains had significantly less activity than the wild type. Moreover, YOL155c and YIL169c overexpressing strains and a strain overexpressing 6xHis-tagged Hpf2p produced greater haze protective activity than the wild type strains. A storage trial demonstrated the short to midterm stability of 6xHis-tagged Hpf2p in wine.

Slow denaturation of wine proteins is thought to lead to protein aggregation, flocculation into a hazy suspension and formation of precipitates. The majority of wine proteins responsible for haze are grape-derived, have low isoelectric points and molecular weight. They arc grape pathogenesis-related (PR) proteins that arc expressed throughout the ripening period post veraison, and are highly resistant to low pH and enzymatic or non-enzymatic proteolysis. Protein levels in un-fined white wine differ by variety and range up to 300 mg/L. Infection with some common grapevine pathogens or skin contact, Such as occurs during transport of mechanically harvested fruit, results in enhanced concentrations of some PR proteins in juice and wine. Oenological control of protein instability is achieved through adsorption of wine proteins onto bentonite. The adsorption of proteins onto bentonite occurs within several minutes, suggesting, that a continuous contacting process could be developed. The addition of protcolytic enzyme during short term heat exposure, to induce PR protein denaturation, showed promise as an alternative to bentonite fining. The addition of haze-protective factors, yeast mannoproteins, to wines results in decreased particle size of haze, probably by competition with wine proteins for other non-proteinaceous wine components required for the formation of large insoluble aggregations of protein. Other wine components likely to influence haze formation are ethanol concentration, pH, metal ions and phenolic compounds.

The image of wine as a harmonious blend of nature, art and science invites tension between tradition and innovation, and no tension in the business of making wine is greater than that brought into play by the potential afforded by 21st Century grape and wine biotechnology. The challenge is to realise the potential of technological innovation without stripping the ancient art of grapegrowing and winemaking of its charm, mysticism and romanticism. Equally challenging is the multitude of complex and interconnected agronomic, business, regulatory and social obstacles currently blocking commercial availability of transgenic grapes, wine yeast and malolactic bacterial starter strains. While the need to assess rigorously the potential negative impacts of new technologies is self-evident, over the long term, failure to overcome these hurdles will disadvantage the international wine sector and consumers alike. This contention is illustrated with reference to recent examples of genetically improved grapevine, yeast and bacterial prototypes showing potential for enhanced, cost-effective production of wine with minimised resource inputs, improved quality and low environmental impact.

In recognition of the importance of assimilable nitrogen in the successful completion of several fermentation processes we have sought to develop yeast strains that utilize this typically limited nutrient more efficiently.With the aid of transposon mutagenesis and a high throughput method for analysis of multiple fermentations, we have identified 'nitrogen efficient' mutants that catabolise more sugar for a given amount of nitrogen utilized.In this way we identified two genes, NGR1 and GID7, whose disruption leads to enhanced catabolism of glucose by an industrial strain and/or laboratory strain, during anaerobic growth in a medium with limiting nitrogen.Fermentation profiles were also evaluated with moderate or excess nitrogen supplied as either ammonia, or a mix of amino acids and ammonia.Deletion of NGR1 or GID7 altered the amounts in which major metabolites were produced, and in ngr1 strains, decreased biomass yield.Transcript levels of a selection of key central nitrogen metabolism genes were determined by real time PCR for fermentation samples.Deletion of either GID7 or NGR1 caused an increase in transcript abundance of both GLT1 and GLN1 and a decrease of GDH1, thereby suggesting an affect on central nitrogen metabolism.In addition, the deletion of GID7 caused an increase in the transcript abundance of GDH2.

Volatile thiols, particularly 4-mercapto-4-methylpentan-2-one (4MMP), make an important contribution to the aroma of wine. During wine fermentation, Saccharomyces cerevisiae mediates the cleavage of a nonvolatile cysteinylated precursor in grape juice (Cys-4MMP) to release the volatile thiol 4MMP. Carbon-sulfur lyases are anticipated to be involved in this reaction. To establish the mechanism of 4MMP release and to develop strains that modulate its release, the effect of deleting genes encoding putative yeast carbon-sulfur lyases on the cleavage of Cys-4MMP was tested. The results led to the identification of four genes that influence the release of the volatile thiol 4MMP in a laboratory strain, indicating that the mechanism of release involves multiple genes. Deletion of the same genes from a homozygous derivative of the commercial wine yeast VL3 confirmed the importance of these genes in affecting 4MMP release. A strain deleted in a putative carbon-sulfur lyase gene, YAL012W, produced a second sulfur compound at significantly higher concentrations than those produced by the wild-type strain. Using mass spectrometry, this compound was identified as 2-methyltetrathiophen-3-one (MTHT), which was previously shown to contribute to wine aroma but was of unknown biosynthetic origin. The formation of MTHT in YAL012W deletion strains indicates a yeast biosynthetic origin of MTHT. The results demonstrate that the mechanism of synthesis of yeast-derived wine aroma components, even those present in small concentrations, can be investigated using genetic screens.

In the first decade of the twenty-first century, the potential therapeutic effects of regular moderate wine consumption are being increasingly acknowledged. They include a reduction in the risk of, and death from, cardiovascular disease, which accounted for 40% of all Australian deaths in 2000. The reduction in risk for wine consumers is similar to that of consumers of fruits, grains and vegetables, which, together with wine, are the core components of a 'Mediterranean-style diet. The chemical components of wine considered primarily responsible for this therapeutic effect are ethanol, and the phenolic compounds and their polyphenolic forms. indeed, moderate wine consumption has been observed to Supplement the cardioprotective effects of an already high phenolic diet, and more importantly, to counter the harmful effects of a high fat diet on blood clotting, endothelial function and lipid oxidation, which contribute to the development of cardiovascular disease.This paper explores both the viticultural and vinification factors that influence phenolic concentration in grapes and wine. The synthesis and accumulation of phenolic compounds in grapes is primarily dependent upon varietal factors, the expression of which is influenced by a combination of climatic and viticultural factors Such as sunlight and temperature during ripening, as well as ripeness at harvest. While the maximum possible concentration of phenolic compounds in a wine will be determined by the content in the constituent grapes, factors which influence the extraction of the phenolic compounds from the skins and seeds primarily influence their concentration in the juice, must and wine. Once harvested, the concentration of phenolic compounds in grapes is invariate, but extraction efficiency can vary during vinification. Accordingly, this paper also explores innovative techniques and technologies that can increase the phenolic content of the resultant wine. At best, winemaking can only extract at 50% of the total phenolic compounds accumulated in the grapes. Therefore, the phenolic content of the resultant wine can only be increased by supplementation Of the must during fermentation with additional Sources of phenolic compounds. Alternatively, a grape seed extract could be added to wine post fermentation to supplement its phenolic content, although this same grape seed extract may also be added to other foods such as yoghurt, from which the phenolic compounds are readily absorbed. Regular and moderate consumption of wine by Consumers Should, however, be placed in context with the other constituents and characteristics of a healthy diet and lifestyle. Indeed, wine consumers generally have fewer risk factors for cardiovascular disease compared with beer and spirits Consumers, which is reflected in an approximately 25% to 35% lower risk of cardiovascular disease for wine consumers compared to consumers of beer and spirits, respectively.

ABSTRACTVolatile thiols, particularly 4-mercapto-4-methylpentan-2-one (4MMP), make an important contribution to the aroma of wine. During wine fermentation,Saccharomyces cerevisiaemediates the cleavage of a nonvolatile cysteinylated precursor in grape juice (Cys-4MMP) to release the volatile thiol 4MMP. Carbon-sulfur lyases are anticipated to be involved in this reaction. To establish the mechanism of 4MMP release and to develop strains that modulate its release, the effect of deleting genes encoding putative yeast carbon-sulfur lyases on the cleavage of Cys-4MMP was tested. The results led to the identification of four genes that influence the release of the volatile thiol 4MMP in a laboratory strain, indicating that the mechanism of release involves multiple genes. Deletion of the same genes from a homozygous derivative of the commercial wine yeast VL3 confirmed the importance of these genes in affecting 4MMP release. A strain deleted in a putative carbon-sulfur lyase gene, YAL012W, produced a second sulfur compound at significantly higher concentrations than those produced by the wild-type strain. Using mass spectrometry, this compound was identified as 2-methyltetrathiophen-3-one (MTHT), which was previously shown to contribute to wine aroma but was of unknown biosynthetic origin. The formation of MTHT in YAL012W deletion strains indicates a yeast biosynthetic origin of MTHT. The results demonstrate that the mechanism of synthesis of yeast-derived wine aroma components, even those present in small concentrations, can be investigated using genetic screens.

LL IN ACCORD with Louis Pasteur’s famous dictum that there is not pure science and applied science but only the application of science, useful practical outcomes often start with a twinkle in a basic researcher’s blurred, but alert, eye. Pasteur’s own research reflects an impressive synthesis of goals of understanding to reach the applied goals of preventing spoilage in milk, vinegar, beer and wine, and of conquering flacherie in silkworms, anthrax in sheep and cattle, cholera in chickens, and rabies in animals and humans. The history of science is full of examples where an open mind has made important insights in areas not originally intended at the outset of the research. However, as Albert Einstein once said, in science, chance favours the prepared mind. In addition to the benefits of defined outcomes formulated by appropriate practitioner/researcher interactions (see Hoj et al. 2003), the occasional serendipitous uncovering of phenomena is one of the important but often overlooked benefits of both public and industry investment in R&D. Alexander Flemming’s discovery of penicillin represents a famous and monumental example of such serendipity at play. Another example of famous spin-offs from ongoing fundamental research was the development of the worldwide web which owes much of its existence to the need for physicists from a multitude of nations to communicate effectively in relation to the conduct of experiments at the CERN particle physics facilities in Europe. During The Australian Wine Research Institute’s ongoing research into the causes and potential prevention of protein hazes in wine, unintended discoveries, although on a much less monumental scale than that of penicillin and the Web, have been made in relation to haze-forming proteins. It has been discovered that haze-forming proteins have a potential use in varietal identification of grapes/juices and hence, by implication, also label integrity programs. Varietal authentication of grapes, juices, musts and wines is important, not only to grapegrowers and winemakers, but also to the marketing of wines, since wine labelling laws and trade regulations now demand that varietals be correctly identified (Pinder and Meredith 2003). This article describes a prime example of an effort that brings together informed judgments of scientific promise of basic research and of industry need for varietal identification.