Gordon M. Elsey

Engineering volatile thiol release in Saccharomyces cerevisiae for improved wine aroma

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. Copyright

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.

First Identification of 4-S-Glutathionyl-4-methylpentan-2-one, a Potential Precursor of 4-Mercapto-4-methylpentan-2-one, in Sauvignon Blanc Juice

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.

Determination of rotundone, the pepper aroma impact compound, in grapes and wine.

Shiraz, also known as Syrah or Hermitage, is one of Australias 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.

Synthesis of Wine Thiol Conjugates and Labeled Analogues: Fermentation of the Glutathione Conjugate of 3-Mercaptohexan-1-ol Yields the Corresponding Cysteine Conjugate and Free Thiol

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.

Occurrence, sensory impact, formation, and fate of damascenone in grapes, wines, and other foods and beverages

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.

Modulation of volatile thiol and ester aromas by modified wine yeast

The volatile thiols, in particular 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA) are potent aroma shown to contribute strongly to the varietal aroma of Sauvignon Blanc wines. The thiols 4MMP and 3MH exist as non-volatile, aroma-inactive cysteine bound conjugates in the grape must and during fermentation the thiol is cleaved from the precursor. However, no cysteine conjugate for 3MHA has been identified. In this work we showed that 3MHA is formed from 3MH by the wine yeast Saccharomyces cerevisiae during fermentation. Furthermore, the alcohol acetyltransferase, Atf1p, the enzyme involved in the formation of the ester ethyl acetate, was shown to be the main enzyme responsible for the formation of 3MHA. Both a laboratory yeast and a commercial wine yeast overexpressing the ATF1 gene produced significantly more 3MHA than the wild-type. Although an atf1Δ laboratory yeast strain showed reduced 3MHA formation, it was not abolished, indicating that other enzymes are also responsible for its formation. Therefore, overexpression of the ATF1 gene in a wine yeast presents the possiblity of modulating both the thiol and ester aromas in wine.

Synthesis of the Individual Diastereomers of the Cysteine Conjugate of 3-Mercaptohexanol (3-MH)

The individual diastereoisomers of the cysteine conjugate of 3-mercaptohexanol (4) were synthesized with high isomeric purity (>98%). On treatment with Apotryptophanase enzyme, the 3R diastereoisomer of 4 gave an 82% yield of the R enantiomer of 1, with no trace of the 3S enantiomer present. Conversely, the 3S diastereoisomer of 4 gave the 3S enantiomer of 1 (43%) accompanied by a trace of the 3R form (S/R = 98.5:1.5), reflecting the diastereomeric purity of the cysteine conjugate. The same stereochemical outcome was observed when the individual diastereoisomers of 4 were added to fermentations with the Saccharomyces cerevisiae AWRI 1655 yeast strain, which gave 1 in 1% yield. A d(10)-analogue of 1 was synthesized and used as an internal standard to determine, by gas chromatography-mass spectrometry (GC-MS), the amounts of 1 formed in these transformations.

Hydroxycinnamic Acid Ethyl Esters as Precursors to Ethylphenols in Wine

A method for determining ethyl coumarate and ethyl ferulate in wine using GC-MS with deuterium-labeled analogues has been developed and used to measure the evolution of these two esters during the production of two commercial monovarietal red wines, cv. Grenache and Shiraz. During fermentation, the concentration of ethyl coumarate rose from low levels to 0.4 mg/L in Grenache and 1.6 mg/L in Shiraz wines. These concentrations then increased further during barrel aging to 1.4 and 3.6 mg/L, respectively. The concentration of ethyl ferulate was much lower, reaching a maximum of only 0.09 mg/L. Conversion of ethyl coumarate and ethyl ferulate to their corresponding ethylphenols was observed during fermentations of a synthetic medium with two strains of Dekkera bruxellensis (AWRI 1499 and AWRI 1608), while a third (strain AWRI 1613) produced no ethylphenols at all from these precursors. Strains AWRI 1499 and 1608 produced 4-ethylphenol from ethyl coumarate in 68% and 57% yields, respectively. The corresponding yields of 4-ethylguaiacol from ethyl ferulate were much lower, 7% and 3%. Monitoring of ethyl coumarate and ethyl ferulate concentration during the Dekkera fermentations showed that the selectivity for ethylphenol production according to yeast strain and the precursor was principally a result of variation in esterase activity. Consequently, ethyl coumarate can be considered to be a significant precursor to 4-ethylphenol in wines affected by these two strains of Brettanomyces/Dekkera yeast, while ethyl ferulate is not an important precursor to 4-ethylguaiacol.

Evolution and Occurrence of 1,8-Cineole (Eucalyptol) in Australian Wine

A new method has been developed for the quantitation of 1,8-cineole in red and white wines using headspace solid-phase microextraction (SPME) combined with stable isotope dilution analysis (SIDA) and gas chromatography-mass spectrometry (GC-MS). An extensive survey of Australian wines (44 white and 146 red) highlighted that only red wines contained significant amounts of 1,8-cineole (up to 20 μg/L). Hydrolytic studies with limonene and α-terpineol, putative precursors to 1,8-cineole, showed a very low conversion into 1,8-cineole (< 0.6%) over a 2 year period, which does not account for the difference between white and red wines. 1,8-Cineole was chemically stable in model wine solution over 2 years, and absorption from a Shiraz wine by bottle closures was most evident for a synthetic closure only (14% absorption after 1 year). Two commercial ferments at two different locations were monitored daily to investigate the evolution of 1,8-cineole throughout fermentation. Both ferments showed daily increases in 1,8-cineole concentration while in contact with grape solids, but this accumulation ceased immediately after pressing. This observation is consistent with the extraction of 1,8-cineole into the ferment from the solid portions of the grape berries.