Yoji Hayasaka

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.

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.

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.

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.

Identification of a β-D-glucopyranoside precursor to guaiacol in grape juice following grapevine exposure to smoke

The presence of the beta-D-glucopyranoside of guaiacol (glucoside) in juice of grapes following grapevine exposure to smoke was investigated. The glucoside was synthesized as a reference compound and an HPLC-MS/MS method was developed for its detection in juice. The glucoside was found in the juice extracts of grapes exposed to bushfire smoke, as well as grapes experimentally exposed to smoke. Compared to the control (unsmoked) juice sample, the experimentally smoked juice contained a significant amount of the glucoside, indicating glucosylation of guaiacol occurred following grapevine smoke exposure. The reference compound, and the glucoside found in the smoked juice samples were less susceptible to acid treatment but virtually disappeared after enzyme treatment with beta-glucosidase. The susceptibility of the glucoside to enzyme hydrolysis could be one reason for the release of guaiacol from smoke affected grapes during fermentation.

Rapid Isolation of Red Wine Polymeric Polyphenols by Solid-Phase Extraction

A rapid technique for the isolation of polymeric polyphenols from red wine has been developed and validated. A copolymer reversed-phase SPE cartridge was utilized in conjunction with predominantly organic eluents to provide three phenolic fractions from red wine without the need for sample pretreatment. The first fraction contained the bulk of the monomeric and oligomeric phenolic material, while the second and third fractions contained the polymeric polyphenolic compounds, as determined by HPLC analysis. The two polymeric polyphenolic fractions differed in their solubility and extent of pigmentation, and the differences appeared to be related to wine age. This method contrasted with other available fractionation techniques because the interfering, nonpolymeric material can be removed in a single wash fraction, while the polymeric material is separated into two distinct fractions based on their diverse physicochemical properties. It is anticipated that the rapid access to discrete polymeric fractions afforded by this method will be of benefit in furthering the understanding of red wine polymeric polyphenols.

Use of electrospray mass spectrometry for mass determination of grape (Vitis vinifera) juice pathogenesis-related proteins: a potential tool for varietal differentiation.

Methods based on liquid chromatography-mass spectrometry (LC-MS) and protein trap mass spectrometry (trap-MS) were developed to determine the complement of pathogenesis-related (PR) proteins in grape juice. Trap-MS was superior to LC-MS in terms of simplicity, rapidity, and sensitivity. Proteins with a wide range of masses (13--33 kDa) were found in the juices of 19 different varieties of grape (Vitis vinifera) and were identified as mostly PR-5 type (thaumatin-like) and PR-3 type (chitinases) proteins. Although the PR proteins in juices of grapes are highly conserved, small consistent differences in molecular masses were noted when otherwise identical proteins were compared from different varieties. These differences persisted through different harvest years and in fruits grown in different Australian locations. With the definition of four different masses for PR-5 proteins (range = 21,239--21,272 Da) and nine different masses of PR-3 proteins (range = 25,330--25,631 Da) and using statistical analysis, the methods developed could be used for varietal differentiation of grapes grown in several South Australian locations on the basis of the PR protein composition of the juice. It remains to be seen whether this technology can be extended to grapes grown worldwide and to wine and other fruit-derived products to assist with label integrity to the benefit of consumers.

Two-step purification of pathogenesis-related proteins from grape juice and crystallization of thaumatin-like proteins

Grape thaumatin-like (TL) proteins and chitinases play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. A two-step method is described that highly purified hundreds of milligrams of TL proteins and chitinases from two juices by strong cation exchange (SCX) and hydrophobic interaction chromatography (HIC). The method was fast and separated isoforms of TL proteins and chitinases from within the same juice, in most cases to >97% purity. The isolated proteins were identified by peptide nanoLC-MS/MS and crystallized using a high-throughput screening method. Crystals from three protein fractions produced high-resolution X-ray crystallography data.