Showing papers by "Isak S. Pretorius published in 2005"

Yeast and bacterial modulation of wine aroma and flavour

Abstract: Wine is a highly complex mixture of compounds which largely define its appearance, aroma, flavour and mouth-feel properties. The compounds responsible for those attributes have been derived in turn from three major sources, viz. grapes, microbes and, when used, wood (most commonly, oak). The grape-derived compounds provide varietal distinction in addition to giving wine its basic structure. Thus, the floral monoterpenes largely define Muscat-related wines and the fruity volatile thiols define Sauvignon-related wines; the grape acids and tannins, together with alcohol, contribute the palate and mouth-feel properties. Yeast fermentation of sugars not only produces ethanol and carbon dioxide but a range of minor but sensorially important volatile metabolites which gives wine its vinous character. These volatile metabolites, which comprise esters, higher alcohols, carbonyls, volatile fatty acids and sulfur compounds, are derived from sugar and amino acid metabolism. The malolactic fermentation, when needed, not only provides deacidification, but can enhance the flavour profile. The aroma and flavour profile of wine is the result of an almost infinite number of variations in production, whether in the vineyard or the winery. In addition to the obvious, such as the grapes selected, the winemaker employs a variety of techniques and tools to produce wines with specific flavour profiles. One of these tools is the choice of microorganism to conduct fermentation. During alcoholic fermentation, the wine yeast Saccharomyces cerevisiae brings forth the major changes between grape must and wine: modifying aroma, flavour, mouth-feel, colour and chemical complexity. The wine bacterium Oenococcus oeni adds its contribution to wines that undergo malolactic fermentation. Thus flavour-active yeasts and bacterial strains can produce desirable sensory results by helping to extract compounds from the solids in grape must, by modifying grape-derived molecules and by producing flavour-active metabolites. This article reviews some of the most important flavour compounds found in wine, and their microbiological origin.

Yeast modulation of wine flavor.

Abstract: Publisher Summary This chapter reviews the scientific knowledge of the role of microorganisms, especially yeast, in the development of wine flavor. Specific attention is given to the contribution of esters, higher alcohols, volatile thiols, volatile phenols, and monoterpenoids to the flavor profile. It is well known that grapes of different cultivars display characteristic aromas that are distinctive of the wines. However, it can be shown that although some volatile aroma substances arise from components of the grapes, many of these compounds are changed and a substantial portion of wine flavor substances are formed during yeast fermentation. Therefore, wine has more flavor than the grape juice it is fermented from, and the importance of yeast and other wine-related microorganisms is central to the development of wine flavor. Furthermore, different biosynthetic pathways are interactive during the formation of the aroma of alcoholic beverages, and different factors play their part in the formation of the total aroma.

The effect of sulphur dioxide and oxygen on the viability and culturability of a strain of Acetobacter pasteurianus and a strain of Brettanomyces bruxellensis isolated from wine

Abstract: Aims: The objective of this study was to investigate the effects of free molecular and bound forms of sulphur dioxide and oxygen on the viability and culturability of a selected strain of Acetobacter pasteurianus and a selected strain of Brettanomyces bruxellensis in wine. Methods and Results: Acetic acid bacteria and Brettanomyces/Dekkera yeasts associated with wine spoilage were isolated from bottled commercial red wines. One bacterium, A. pasteurianus strain A8, and one yeast, B. bruxellensis strain B3a, were selected for further study. The resistance to sulphur dioxide and the effect of oxygen addition on these two selected strains were determined by using plating and epifluorescence techniques for monitoring cell viability in wine. Acetobacter pasteurianus A8 was more resistant to sulphur dioxide than B. bruxellensis B3a, with the latter being rapidly affected by a short exposure time to free molecular form of sulphur dioxide. As expected, neither of these microbial strains was affected by the bound form of sulphur dioxide. The addition of oxygen negated the difference observed between plate and epifluorescence counts for A. pasteurianus A8 during storage, while it stimulated growth of B. bruxellensis B3a. Conclusions: Acetobacter pasteurianus A8 can survive under anaerobic conditions in wine in the presence of sulphur dioxide. Brettanomyces bruxellensis B3a is more sensitive to sulphur dioxide than A. pasteurianus A8, but can grow in the presence of oxygen. Care should be taken to exclude oxygen from contact with wine when it is being transferred or moved. Significance and Impact of the Study: Wine spoilage can be avoided by preventing growth of undesirable acetic acid bacteria and Brettanomyces/Dekkera yeasts through the effective use of sulphur dioxide and the management of oxygen throughout the winemaking process.

Genetic determinants of volatile-thiol release by Saccharomyces cerevisiae during wine fermentation

Abstract: 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.

Grape and wine biotechnology: Challenges, opportunities and potential benefits

Abstract: 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.

Characteristics of Flo11-dependent flocculation in Saccharomyces cerevisiae.

Abstract: The FLO11-encoded flocculin is required for a variety of important phenotypes in Saccharomyces cerevisiae, including flocculation, adhesion to agar and plastic, invasive growth, pseudohyphae formation and biofilm development. We present evidence that Flo11p belongs to the Flo1-type class of flocculins rather than to the NewFlo class. Both Flo1-type and NewFlo yeast flocculation are inhibited by mannose. NewFlo flocculation, however, is also inhibited by several other carbohydrates including glucose, maltose and sucrose. These differences have in at least one case been shown to reflect differences in the structure of the carbohydrate-binding site of the flocculins. We report that Flo11p-dependent flocculation is inhibited by mannose, but not by glucose, maltose or sucrose. Furthermore, Flo11p contains a peptide sequence highly similar to one that has been shown to characterise Flo1-type flocculins. Further characterisation of the properties of Flo11p-dependent flocculation revealed that it is dependent on calcium, occurs only at cell densities greater than 1 · 10 8 ml 1 , and only occurs at acidic pH.

Mss11p is a central element of the regulatory network that controls FLO11 expression and invasive growth in Saccharomyces cerevisiae.

Abstract: The invasive and filamentous growth forms of Saccharomyces cerevisiae are adaptations to specific environmental conditions, under particular conditions of limited nutrient availability. Both growth forms are dependent on the expression of the FLO11 gene, which encodes a cell-wall-associated glycoprotein involved in cellular adhesion. A complex regulatory network consisting of signaling pathways and transcription factors has been associated with the regulation of FLO11. Mss11p has been identified as a transcriptional activator of this gene, and here we present an extensive genetic analysis to identify functional relationships between Mss11p and other FLO11 regulators. The data show that Mss11p is absolutely required for the activation of FLO11 by most proteins that have previously been shown to affect FLO11 expression, including the signaling proteins Ras2p, Kss1p, and Tpk2p, the activators Tec1p, Flo8p, and Phd1p, and the repressors Nrg1p, Nrg2p, Sok2p, and Sfl1p. The genetic evidence furthermore suggests that Mss11p activity is not dependent on the presence of any of the above-mentioned factors and that the protein also regulates other genes involved in cellular adhesion phenotypes. Taken together, the data strongly suggest a central role for Mss11p in the regulatory network controlling FLO11 expression, invasive growth, and pseudohyphal differentiation.

Development and assessment of a recombinant Saccharomyces cerevisiae wine yeast producing two aroma-enhancing β-glucosidases encoded by the Saccharomycopsis fibuligera BGL1 and BGL2 genes

Abstract: The distinctive varietal flavour of grapes and wine is affected by the absolute and relative concentrations of many com- pounds, including monoterpene alcohols such as citronellol, geraniol, hotrienol, linalool, nerol and α-terpineol. Monoterpenols in grapes and wine can occur either as free volatile and odorous molecules, or as glycosidically bound, odourless, non-volatile complexes. β-Glu- cosidases constitute one group of glycosidases that can help to unleash this latent pool of grape-derived volatile aglycons and provide an additional source of wine aroma and flavour compounds. However, yeast (Saccharomyces cerevisiae) and bacteria (Oenococcus oeni) most commonly used to initiate alcoholic and malolactic fermentation during winemaking have only limited ability to liberate the aro- matic terpenols as well as other aglycones bound to saccharides. Therefore, the purpose of this study was to clone, integrate and express two β-glucosidase genes (BGL1 and BGL2) from the dimorphic yeast, Saccharomycopsis fibuligera, in a commercially available wine yeast strain, VIN13. Using p-nitrophenyl-β-D-glucopyranoside as a synthetic substrate, enzyme assays and kinetic studies indicated that both these two extracellularly produced β-glucosidases were able to cleave glycosidic bonds efficiently. Subsequently, wine from Chenin blanc, Gewurztraminer and Pinotage grapes was made with the transformed and untransformed S. cerevisiae VIN13 strains, and compared. A series of analyses indicated that wines produced by the β-glucosidase-producing VIN13 strain contained slightly high- er levels of terpenols in comparison with the wines produced by the untransformed control VIN13 strain. Surprisingly, the wines pro- duced by the transformed strain also contained increased ester concentrations. Many of these fragrant compounds, when produced in appropriate concentrations, would contribute to the fermentation bouquet of wine. The extent to which this acquired capacity of the transformed wine yeast is of practical significance in large-scale wine production is at present unclear but presents a worthwhile propo- sition for further investigation.

Olfaction and taste: Human perception, physiology and genetics

Abstract: Drinking wine is a manifestly sensuous experience. Wine stimulates most of our senses; particularly smell (olfaction) and taste, and, to a lesser extent, sight and touch. These sensory inputs interact with the limbic system in our brain which is associated with emotions and memory. Agreeable smells can thus evoke feelings of enjoyment and nostalgia. But how are tastants and aromas detected? Over the past few years a great deal has been learnt about how small tastant and odorant molecules are detected by specific protein receptors located in our mouth and in nasal cavities, respectively. Indeed, we have a vast array of olfactory receptors encoded by a group of genes that represent a significant part of the human genome. Interestingly, many more aroma compounds can be detected and discriminated than can be accounted for by the number of olfactory receptors that are encoded by our genes. Such disparity implies a level of complexity in this system which is not yet fully understood. Sophistication in olfaction is something that winemakers (and drinkers!) have long appreciated, and now scientists are beginning to unravel some of the underlying mysteries. Discovering why some individuals are more receptive to different tastes and smells than others will help wine producers understand variation in consumer preferences between different parts of the world, and possibly capture new opportunities in a changing global marketplace. Given such prospects, this present review offers a timely summary of some key developments in our understanding of odorant and tastant detection. We also consider how genetic components in human olfaction might be utilised to develop a ‘biosensor’ for aroma detection and discrimination.

Domain engineering of Saccharomyces cerevisiae exoglucanases

Abstract: To illustrate the effect of a cellulose-binding domain (CBD) on the enzymatic characteristics of non-cellulolytic exoglucanases, 10 different recombinant enzymes were constructed combining the Saccharomyces cerevisiae exoglucanases, EXG1 and SSG1, with the CBD2 from the Trichoderma reesei cellobiohydrolase, CBH2, and a linker peptide. The enzymatic activity of the recombinant enzymes increased with the CBD copy number. The recombinant enzymes, CBD2-CBD2-L-EXG1 and CBD2-CBD2-SSG1, exhibited the highest cellobiohydrolase activity (17.5 and 16.3 U mg −1 respectively) on Avicel cellulose, which is approximately 1.5- to 2-fold higher than the native enzymes. The molecular organisation of CBD in these recombinant enzymes enhanced substrate affinity, molecular flexibility and synergistic activity, contributing to their elevated action on the recalcitrant substrates as characterised by adsorption, kinetics, thermostability and scanning electron microscopic analysis.

Amylolytic enzymes from the yeast Lipomyces kononenkoae

Abstract: The Lipomyces kononenkoae α-amylases LKA1 and LKA2 belong to the glycoside hydrolase family 13 and exhibit specificity towards α-1,4 and α-1,6 linkages in starch and related substrates. LKA1 exhibits specificity towards α- 1,4 and α-1,6 linkages and large amounts of reducing sugars are liberated from highly branched amylopectin and glycogen and linear amylose. LKA2, on the other hand, shows high reactivity towards lintner starch, dextrin and amylase, although only small amounts of reducing sugars are liberated from branched substrates, such as amylopectin and glycogen. These enzymes share the four conserved segments of the catalytic domain found in other members of the family, but have some major variant amino acids within these segments. In addition, LKA1 consists of an N-terminal starch-binding domain (SBD). This is the only α-amylase known to possess this N-terminal domain and it exhibits homology to the N-terminal SBD of Rhizopus oryzae glucoamylase. It shares no homology with the C-terminal starch-binding domains present in the cyclodextrin glucanotransferases, glucoamylases or α-amylases. The evolutionary tree based on the sequence alignment of SBDs reveals that the N-terminal SBDs are separated from the C-terminal SBDs.