Dimitra L. Capone

Evolution of 3-mercaptohexanol, hydrogen sulfide, and methyl mercaptan during bottle storage of Sauvignon blanc wines. Effect of glutathione, copper, oxygen exposure, and closure-derived oxygen.

The effects of wine composition and postbottling oxygen exposure on 3-mercaptohexanol (3-MH), hydrogen sulfide (H2S), and methyl mercaptan (MeSH) were investigated. A Sauvignon blanc wine with initial copper concentration of 0.1 mg/L was treated with copper sulfate and/or glutathione (GSH) prior to bottling to give final concentrations of 0.3 and 20 mg/L, respectively. The wines were bottled with a synthetic closure previously stored in either ambient air or nitrogen to study the effect of the oxygen normally present in the closure. Bottled wines were stored for 6 months in either air or nitrogen to study the effect of oxygen ingress through the closure. Copper addition resulted in a rapid initial decrease in 3-MH. During storage, a further decrease of 3-MH was observed, which was lower with GSH addition and lowered oxygen exposure. H2S accumulated largely during the second 3 months of bottle storage, with the highest concentrations attained in the wines treated with GSH and copper. Lower oxygen from and through the closure promoted H2S accumulation. The concentration of MeSH was virtually not affected by the experimental variables at 6 months, although differences were observed after 3 months of storage. The implications for wine quality are discussed.

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

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.

Effects of Transporting and Processing Sauvignon blanc Grapes on 3-Mercaptohexan-1-ol Precursor Concentrations

The effects of different processing treatments on thiol precursor concentrations have been investigated through studies involving transportation of machine-harvested Sauvignon blanc fruit and assessment of different applications of antioxidants, along with juice preparation and enzyme inhibition experiments. The influence these trials had on 3-S-cysteinylhexan-1-ol (Cys-3-MH) and 3-S-glutathionylhexan-1-ol (Glut-3-MH) concentrations in juices is discussed. Very interesting findings included the large increase in precursor concentrations after transportation, particularly for Cys-3-MH, and the limited formation of Glut-3-MH when grape proteins were precipitated during processing. The various results provided information about the ability to modulate precursor concentrations depending on the processing technique employed. Additionally, a conjugated aldehyde, which is the obvious missing link between the reaction of (E)-2-hexenal and glutathione in the formation of Glut-3-MH, has been tentatively identified for the first time. Deuterium-labeled 3-S-glutathionylhexanal (Glut-3-MHAl) was produced through the addition of labeled (E)-2-hexenal to grapes, followed by grape crushing, and detected in the juice by HPLC-MS/MS, along with the corresponding labeled Glut-3-MH.

Introducing a New Breed of Wine Yeast: Interspecific Hybridisation between a Commercial Saccharomyces cerevisiae Wine Yeast and Saccharomyces mikatae

Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment.

Volatile and Color Composition of Young and Model-Aged Shiraz Wines As Affected by Diammonium Phosphate Supplementation Before Alcoholic Fermentation

A Shiraz must with low yeast assimilable nitrogen (YAN) was supplemented with two concentrations of diammonium phosphate (DAP) and then fermented with maceration on grape skins. The nonvolatile, volatile, and color composition of the final wines were investigated. Ethanol and residual sugars were not affected by DAP supplementation, while glycerol, SO 2, and residual YAN increased and acetic acid decreased. DAP-supplemented treatments gave rise to higher concentrations of acetates, fatty acids, and fatty acid ethyl esters but lower concentrations of branched-chain fatty acids and their ethyl esters. No major difference between treatments was observed for higher alcohols, monoterpenes, norisoprenoids, and low-molecular-weight sulfur compounds. DAP-supplemented fermentations resulted in wines with higher concentrations of malvidin-3-glucoside, higher color intensity, and altered color tonality. Model aging studies indicated that higher concentrations of esters are still present in wines from the DAP-treated fermentations after aging. DAP supplementation also resulted in increased concentrations of dimethyl sulfide after model aging. It can be concluded that DAP treatment of a low YAN must fermented by maceration on skins can significantly affect wine color, aroma, and flavor.

Engineering Saccharomyces cerevisiae To Release 3-Mercaptohexan-1-ol during Fermentation through Overexpression of an S. cerevisiae Gene, STR3, for Improvement of Wine Aroma

ABSTRACT Sulfur-containing aroma compounds are key contributors to the flavor of a diverse range of foods and beverages. The tropical fruit characters of Vitis vinifera L. cv. Sauvignon blanc wines are attributed to the presence of the aromatic thiols 3-mercaptohexan-1-ol (3MH), 3-mercaptohexan-1-ol-acetate, and 4-mercapto-4-methylpentan-2-one (4MMP). These volatile thiols are found in small amounts in grape juice and are formed from nonvolatile cysteinylated precursors during fermentation. In this study, we overexpressed a Saccharomyces cerevisiae gene, STR3, which led to an increase in 3MH release during fermentation of a V. vinifera L. cv. Sauvignon blanc juice. Characterization of the enzymatic properties of Str3p confirmed it to be a pyridoxal-5′-phosphate-dependent cystathionine β-lyase, and we demonstrated that this enzyme was able to cleave the cysteinylated precursors of 3MH and 4MMP to release the free thiols. These data provide direct evidence for a yeast enzyme able to release aromatic thiols in vitro that can be applied in the development of self-cloned yeast to enhance wine flavor.

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.