Mark A. Sefton

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.

Determination of oak lactones in barrel-aged wines and in oak extracts by stable isotope dilution analysis

The cis- and trans-isomers of 5-butyl-4-methyl-4,5-dihydro-2(3H)-furanone, the so-called oak lactones, are derived from oakwood, and the cis-isomer is an important contributor to wine flavour. Their deuterium-labelled forms, [2H4]cis-oak lactone and [2H4]trans-oak lactone, were synthesised from the unlabelled analogues, and were utilised in a new method employing gas chromatography-mass spectrometry to determine the concentration of these compounds in wine or extracts of oak shavings in a single analysis. The method can employ either liquid-liquid extraction or solid-phase microextraction, and is both rapid and accurate. There was some artefactual generation of cis-oak lactone during the analysis of model wine extracts of unheated oak shavings when diethyl ether extraction and injector block temperatures at or above 225 degrees C were employed.

Analysis of precursors to wine odorant 3-mercaptohexan-1-ol using HPLC-MS/MS: Resolution and quantitation of diastereomers of 3-S-cysteinylhexan-1-ol and 3-S-glutathionylhexan-1-ol

A method has been developed and validated for the analysis of the individual diastereomers of 3-S-cysteinylhexan-1-ol (Cys-3-MH) and 3-S-glutathionylhexan-1-ol (Glut-3-MH) extracted from grape juice and wine. The method uses stable isotope dilution analysis (SIDA) combined with solid-phase extraction (SPE) and reversed-phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for quantitation. These compounds have been considered as potential precursors to the important wine odorant 3-mercaptohexan-1-ol (3-MH). This constitutes the first analytical method where (1) Glut-3-MH has been accurately quantified in grape juice and wine and (2) the individual Cys- and Glut-3-MH diastereomers were separated and quantified by a single HPLC-MS/MS method. The use of deuterium-labeled internal standards has resulted in an accurate and precise method that can achieve quantitation limits of <0.5 microg/L for the individual Cys- and Glut-3-MH diastereomers in grape juice and white wine. The method has been applied to the determination of 3-MH precursor diastereomers in various white juice and wine samples. Overall, Glut-3-MH was always more abundant than Cys-3-MH in the juices and wines examined, regardless of grape variety. Stereochemically, (S)-Glut-3-MH generally dominated over the (R)-diastereomer in the juices and wines, but there was not such a marked difference between the distribution of Cys-3-MH diastereomers. These results have important implications for understanding the formation of wine flavor, and the application of this method will allow further exploration of precursors to the varietal thiol 3-MH.

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.

Application of a modified method for 3-mercaptohexan-1-ol determination to investigate the relationship between free thiol and related conjugates in grape juice and wine.

A method has been developed for determining 3-mercaptohexan-1-ol (3-MH) in wine and grape juice using gas chromatography with conventional electron ionization (EI) mass spectrometry. The limit of quantitation of 40 ng/L was achieved with excellent precision using stable isotope dilution analysis (SIDA) combined with headspace solid-phase microextraction (SPME) of derivatized 3-MH. This method was used in combination with HPLC-MS/MS analysis of the individual diastereomers of 3-S-cysteinylhexan-1-ol (Cys-3-MH) and 3-S-glutathionylhexan-1-ol (Glut-3-MH), which are known precursors of the volatile thiol 3-MH. Commercial and small-lot winemaking trials were evaluated to determine the concentrations of precursors and free 3-MH at various stages of grape processing and winemaking. Five Sauvignon blanc clones were also assessed for precursors and free thiol during ripening, revealing the presence of 3-MH in the unfermented juices and a stark increase in precursor concentrations in the latter stage of ripening. Additionally, differences due to sample freezing and mode of juice preparation were revealed for the precursors, and a set of commercially available wines was analyzed to investigate the amounts of precursors and free 3-MH in Sauvignon blanc and other white wine varieties. There was seemingly no relationship between precursor concentrations in juice and 3-MH concentrations in wine. This was somewhat understandable, because the formation of precursors appears to be a dynamic process affected by a multitude of factors, beginning with grape ripening and continuing during vinification.

Precursors of damascenone in fruit juices

Abstract The acid catalysed reactions of 6,7-megastigmadiene-3,5,9-triol and the β-D-glucosides of 5-megastigmen-7-yne-3,9-diol and 3-hydroxyl-β-damascone have been studied in relation to the formation of damascenone. The results show that hydrolysis of the allene triol could account for damascenone formation in the juices of grapes and other fruits.

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.