An obscure sesquiterpene, rotundone, has been identified as a hitherto unrecognized important aroma impact compound with a strong spicy, peppercorn aroma. Excellent correlations were observed between the concentration of rotundone and the mean 'black pepper' aroma intensity rated by sensory panels for both grape and wine samples, indicating that rotundone is a major contributor to peppery characters in Shiraz grapes and wine (and to a lesser extent in wine of other varieties). Approximately 80% of a sensory panel were very sensitive to the aroma of rotundone (aroma detection threshold levels of 16 ng/L in red wine and 8 ng/L in water). Above these concentrations, these panelists described the spiked samples as more 'peppery' and 'spicy'. However, approximately 20% of panelists could not detect this compound at the highest concentration tested (4000 ng/L), even in water. Thus, the sensory experiences of two consumers enjoying the same glass of Shiraz wine might be very different. Rotundone was found in much higher amounts in other common herbs and spices, especially black and white peppercorns, where it was present at approximately 10000 times the level found in very 'peppery' wine. Rotundone is the first compound found in black or white peppercorns that has a distinctive peppery aroma. Rotundone has an odor activity value in pepper on the order of 50000-250000 and is, on this criterion, by far the most powerful aroma compound yet found in that most important spice.

Shiraz, also known as Syrah or Hermitage, is one of Australia's most popular red wine varieties both domestically and internationally. Black pepper aroma and flavor are important to some Australian Shiraz red wine styles. Recently, rotundone (a bicyclic sesquiterpene) was identified as the potent aroma compound responsible for pepper aromas in grapes, wine, herbs, and spices, including peppercorns. Here the development, optimization, and validation of the analytical method for the quantitative determination of rotundone in grapes and wine are described and discussed. The method is precise, accurate, robust, and sensitive with a subpart per trillion limit of quantitation. The method uses stable isotope dilution analysis with d(5)-rotundone as internal standard, solid-phase extraction and microextraction, and gas chromatography-mass spectrometry.

Volatile thiols, such as 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA), are among the most potent aroma compounds found in wine and can have a significant effect on wine quality and consumer preferences. At optimal concentrations in wine, these compounds impart flavours of passionfruit, grapefruit, gooseberry, blackcurrant, lychee, guava and box hedge. The enzymatic release of aromatic thiols from grape-derived, non-volatile cysteinylated precursors (Cys-4MMP and Cys-3MH) and the further modification thereof (conversion of 3MH into 3MHA) during fermentation, enhance the varietal characters of wines such as Sauvignon Blanc. Wine yeast strains have limited and varying capacities to produce aroma-enhancing thiols from their non-volatile counterparts in grape juice. Even under optimal fermentation conditions, the most efficient thiol-releasing Saccharomyces cerevisiae wine strain known realizes less than 5% of the thiol-related flavour potential of grape juice. The objective of this study was to develop a wine yeast able to unleash the untapped thiol aromas in grape juice during winemaking. To achieve this goal, the Escherichia coli tnaA gene, encoding a tryptophanase with strong cysteine-β-lyase activity, was cloned and overexpressed in a commercial wine yeast strain under the control of the regulatory sequences of the yeast phosphoglycerate kinase I gene (PGK1 ). This modified strain expressing carbon-sulphur lyase activity released up to 25 times more 4MMP and 3MH in model ferments than the control host strain. Wines produced with the engineered strain displayed an intense passionfruit aroma. This yeast offers the potential to enhance the varietal aromas of wines to predetermined market specifications.

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 carbonsulfur 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.

Synthesis of the putative wine thiol precursor 3-S-glutathionylhexan-1-ol (Glut-3-MH) has been undertaken to provide pure reference materials for the development of HPLC-MS/MS methods for precursor quantitation in grape juice and wine, and for use in fermentation experiments. Labeled thiol conjugates were also prepared for use as internal standards. Purification and fermentation of a single diastereomer of Glut-3-MH with VIN13 (CSL1) yielded not only the (R)-enantiomer of the wine impact odorant 3-mercaptohexan-1-ol (3-MH) but also the cysteine conjugate intermediate as a single (R)-diastereomer, as determined by HPLC-MS/MS. Chiral GC-MS was used to quantify the total amount of (R)-3-MH released from the ferments, resulting in a molar conversion yield of the glutathione conjugate of about 3%. Enzymatic degradation of the single (R)-Glut-3-MH diastereomer with a gamma-glutamyltranspeptidase confirmed the stereochemical relationship to the related cysteine conjugate. This is the first demonstration that Glut-3-MH can liberate 3-MH under model fermentation conditions, where the cysteine conjugate is also formed in the process. This furthers our understanding of the nature of wine thiol precursors and opens avenues for additional studies into formation and interchange of wine thiols and their precursors.

The volatile thiol 4-mercapto-4-methylpentan-2-one (4MMP) is a potent contributor to wine aroma. In grape juice, 4MMP is bound to cysteine as a non-volatile compound and requires the action of yeast during fermentation to release the aroma active thiol. A method was developed to measure 4MMP release from the precursor by headspace solid-phase microextraction and separation by gas chromatography with atomic emission detection to screen the ability of wine yeast to release 4MMP. Yeast commonly used in white wine making were grown with the precursor at two different temperatures, and the amount of 4MMP released was measured. The results demonstrate that yeast strain selection and fermentation temperature can provide an important tool to enhance or modulate the grape-derived aromas formed during wine fermentation.

Among plant-derived odorants, damascenone is one of the most ubiquitous, sometimes occurring as an apparent natural product but more commonly occurring in processed foodstuffs and beverages. It has been widely reported as a component of alcoholic beverages, particularly of wines made from the grape Vitis vinifera . Although damascenone has one of the lowest ortho- and retronasal detection thresholds of any odorant, its contribution to the sensory properties of most products remains poorly understood. Damascenone can be formed by acid-catalyzed hydrolyses of plant-derived apocarotenoids, in both aglycon and glycoconjugated forms. These reactions can account for the formation of damascenone in some, but not all, products. In wine, damascenone can also be subject to degradation processes, particularly by reaction with sulfur dioxide.

The identification of 4-S-glutathionyl-4-methylpentan-2-one (glut-4-MMP) by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) experiments in a Sauvignon Blanc juice extract is described. Synthesis of an authentic reference compound enabled confirmation of the presence of glut-4-MMP in a Sauvignon Blanc juice for the first time. The juice extract was obtained by batch-wise percolation of 6 L of juice through a sintered glass funnel packed with C18 sorbent, followed by further purification using low-pressure chromatography on C18. Analysis of the juice extract revealed a chromatographic peak with the same retention time and mass spectrum as the synthetic reference compound, and spiking experiments verified the findings. The presence of glut-4-MMP in grape juice may be related to the biosynthesis of the relevant S-cysteinyl conjugate and, subsequently, to the formation of aroma-active 4-mercapto-4-methylpentan-2-one (4-MMP). This compound has a very low reported sensory threshold (3 ng/L) in wine and is partially responsible for the aromas that are important to the quality and style of some wine varieties.