Kevin H. Pardon

Quantitative analysis of 4-ethylphenol and 4-ethylguaiacol in red wine

2,3,5,6-[2H4]-4-Ethylphenol (d4-4-ethylphenol) was synthesised for use as an internal standard in a new, rapid and accurate analytical method, employing gas chromatography-mass spectrometry to determine the concentration of the important aroma compounds 4-ethylphenol and 4-ethylguaiacol in red wine. The concentrations of both compounds in wine stored in 44 American and 47 French new and used oak barrels from several suppliers were measured. Wine stored in shaved and refired oak barrels contained up to 85% less 4-ethylphenol and 4-ethylguaiacol than wine stored in normal barrels of the same age that were not shaved. The concentration of 4-ethylphenol found in 61 bottled commercial Australian red wines of various ages ranged from 2 microg/l in a Merlot up to 2660 microg/l in a Shiraz, with a mean concentration of 795 microg/l. 4-Ethylguaiacol was also detected in every red wine analysed, ranging in concentration from 1 microg/l (in a Pinot Noir) up to 437 microg/l (in a Merlot) with a mean concentration of 99 microg/l.

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

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.

Structures and colour properties of new red wine pigments

Abstract Two new red pigments were synthesized by nucleophilic addition of vinylphenols to malvidin 3-glucoside. The structures of the resulting pyranoanthocyanins were confirmed by electrospray-mass spectrometry and NMR spectroscopy (gHMQC, gHMBC and CIGAR experiments). By means of UV–vis spectroscopy the colour properties of the pigments were characterized; it could be demonstrated that the pyranoanthocyanins retained their red colour at pH 3.6 in model wine and were resistant to bisulfite-mediated bleaching. Finally, HPLC–MS analysis confirmed the presence of both anthocyanin-derived pigments in red wine.

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.

Glycosylation of smoke-derived volatile phenols in grapes as a consequence of grapevine exposure to bushfire smoke.

The presence of glycosides of smoke-derived volatile phenols in smoke-affected grapes and the resulting wines of Chardonnay and Cabernet Sauvignon was investigated with the aid of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). All volatile phenols studied (phenol, p-, m-, and o-cresols, methylguaiacol, syringol, and methylsyringol) could be detected as glycosylated metabolites in smoke-affected grapes in a similar fashion to that previously reported for guaiacol. These phenolic glycosides were found in smoke-affected grapes and wines at significantly elevated levels compared to those in non-smoked control grapes and wines. The extraction of these glycosides from grapes into wine was estimated to be 78% for Chardonnay and 67% for Cabernet Sauvignon. After acid hydrolysis, a large proportion of these phenolic glycosides in grapes (50%) and wine (92%) disappeared but the concentrations of volatile phenols determined by gas chromatography-mass spectrometry (GC-MS) were lower than expected. In the case of wine, the majority of the glycosides of phenol, cresols, guaiacol, and methylguaiacol were decomposed upon acid hydrolysis without releasing their respective aglycones, while syringol and methylsyringol were more effectively released.

Investigation into the Formation of Guaiacol Conjugates in Berries and Leaves of Grapevine Vitis vinifera L. Cv. Cabernet Sauvignon Using Stable Isotope Tracers Combined with HPLC-MS and MS/MS Analysis

Fermentation of grapes that had been exposed to bushfire smoke can potentially yield unpalatable, smoke-affected wine. Guaiacol and its glucoconjugate were previously found in smoke-affected grapes at an elevated concentration. To find and identify further guaiacol conjugates in smoke-affected grapes, a stable isotope feeding experiment combined with extensive HPLC-MS and MS/MS investigations was carried out. Leaves and berries of a potted grapevine were placed in contact with an aqueous mixture of d(0)- and d(3)-guaiacol for 1-2 days and collected 5 weeks later. Screening for potential guaiacol conjugates in the leaves and berries was facilitated by monitoring the unique mass spectrometric signature of an isotopic doublet separated by 3 Da. Seven different conjugates were detected in leaves and berries and were tentatively identified as mono- and diglycosides of guaiacol. Quantitative analysis demonstrated that the guaiacol conjugates were translocated between leaves and berries to a very limited extent and were also present as low-level natural compounds of untreated leaves and berries. The same guaiacol conjugates were also found at a considerably elevated concentration in leaves and berries obtained from grapevines exposed to bushfire smoke.

Contribution of several volatile phenols and their glycoconjugates to smoke-related sensory properties of red wine.

Guaiacol and 4-methylguaiacol are well-known as contributors to the flavor of wines made from smoke-affected grapes, but there are other volatile phenols commonly found in smoke from forest fires that are also potentially important. The relationships between the concentration of a range of volatile phenols and their glycoconjugates with the sensory characteristics of wines and model wines were investigated. Modeling of the attribute ratings from a sensory descriptive analysis of smoke-affected wines with their chemical composition indicated the concentrations of guaiacol, o-cresol, m-cresol, and p-cresol were related to smoky attributes. The best-estimate odor thresholds of these compounds were determined in red wine, together with the flavor threshold of guaiacol. Guaiacol β-D-glucoside and m-cresol β-D-glucoside in model wine were found to give rise to a smoky/ashy flavor in-mouth, and the respective free volatiles were released. The study indicated that a combination of volatile phenols and their glycosides produces an undesirable smoke flavor in affected wines. The observation of flavor generation from nonvolatile glycoconjugates in-mouth has potentially important implications.

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.

Determination of the importance of in-mouth release of volatile phenol glycoconjugates to the flavor of smoke-tainted wines.

The volatile phenols guaiacol, 4-methylguaiacol, syringol, 4-methylsyringol, o-, m-, and p-cresol, as well as their glycoconjugates, have previously been shown to be present in elevated concentrations in smoke-tainted wine. Sensory descriptive analysis experiments, with addition of free volatile phenols in combination with their glycosidically bound forms, were used to mimic smoke taint in red wines. The addition of volatile phenols together with glycoconjugates gave the strongest off-flavor. The hydrolysis of glycosidically bound flavor compounds in-mouth was further investigated by in vitro and in vivo experiments. The results indicate that enzymes present in human saliva are able to release the volatile aglycones from their glycoconjugates even under low pH and elevated ethanol conditions, confirming that in-mouth breakdown of monosaccharide and disaccharide glycosides is an important mechanism for smoke flavor from smoke affected wines, and that this mechanism may play an important general role in the flavor and aftertaste of wine.