Cécile Thibon

Identification and characteristics of new volatile thiols derived from the hop (Humulus luplus L.) cultivar Nelson Sauvin.

Nelson Sauvin (NS) is a unique hop cultivar that was bred and grown in New Zealand. This hop gives a specific flavor (exotic fruit-like, white wine-like) to finished beers. However, the key compounds of this flavor have not yet been identified. We have attempted to identify the specific flavor compounds derived from NS. We focused on certain volatile thiols that are well-known to contribute to wine flavors, especially Sauvignon Blanc. The product made from NS (NS product) lost its specific flavor by contact with copper. Copper is well-known as an absorber of thiols in the field of wine flavor investigations. Therefore, it might point to the existence of thiols. We analyzed the NS product by GC-FPD, GC-olfactometry and GC-MS, and identified two new volatile thiols, 3-sulfanyl-4-methylpentan-1-ol (3S4MP), and 3-sulfanyl-4-methylpentyl acetate (3S4MPA). These compounds have a grapefruit-like and/or rhubarb-like odor, similar to that of Sauvignon Blanc. We quantified these compounds in the NS products and determined their thresholds. As a result, 3S4MP contained about 2-fold of its threshold in beers, and 3S4MPA was included below its threshold. However, it was confirmed that 3S4MP enhanced the flavors of 3S4MPA by synergy. Therefore, we concluded that both of the new volatile thiols would contribute to the specific odor of beers produced with NS.

Analysis of the diastereoisomers of the cysteinylated aroma precursor of 3-sulfanylhexanol in Vitis vinifera grape must by gas chromatography coupled with ion trap tandem mass spectrometry

The diastereoisomeric distribution of S-3-(hexan-1-ol)cysteine (P-3SH), the cysteinylated precursor of 3-sulfanylhexan-1-ol (3SH) in Vitis vinifera grape juice, was determined by a new method. This procedure is based on the purification of P-3SH in a small volume of must (500 microL) by affinity chromatography, followed by the separation of chiral molecules in derivative forms by gas chromatography coupled with ion trap tandem mass spectrometry (GC-MS/MS). The diastereoisomers were easily separated using heptafluorobutyric anhydride and heptafluorobutanol (HFBA and HFOH) as derivatization reagents. Method validation was conducted using samples of grape juice, synthetic must, fermenting must, and wine that were fortified with P-3SH at concentrations of 0.6 and 2.5 microM. The relative standard deviation (RSD) and limit of detection (LOD) of the GC-MS/MS method were 4.6% and 1.5 nM, respectively. P-3SH assays in Bordeaux white grape juice affected by Botrytis cinerea showed an unusually increased proportion of the RS form of the precursor (approximately RR:RS=30:70) as compared to a diastereoisomer ratio (in the vicinity of 50:50) in healthy grape juice.

3-Sulfanylhexanol precursor biogenesis in grapevine cells: the stimulating effect of Botrytis cinerea.

Volatile thiols, compounds that contribute strongly to the varietal aroma, are present in much higher concentrations in sweet wines than in dry wines. This positive effect, due to the presence of Botrytis cinerea on the berries, in fact results from a strong enrichment of cysteine S-conjugate precursors in botrytized berries. In the present study, a convenient model was investigated to reproduce and therefore study this phenomenon. A Vitis vinifera cell culture was used as a simple model, and we focused on S-3-(hexan-1-ol)-l-cysteine (P-3SH), the cysteinylated precursor of 3-sulfanylhexanol. We demonstrated that grapevine cells were able to produce P-3SH and that the presence of B. cinerea considerably increased the precursor level (up to 1000-fold). This positive result was determined to be due to metabolites secreted by the fungus. These molecules were temperature sensitive, unstable over time, and their production was activated in the presence of grapevine cells. Moreover, part of the pathway leading to P-3SH was deciphered: it was directly derived from the cleavage of S-3-(hexan-1-ol)-l-glutathione, which itself was generated after a conjugation of glutathione on (E)-2-hexenal.

Surprising Structural Lability of a Cysteine‐S‐Conjugate Precursor of 4‐Methyl‐4‐sulfanylpentan‐2‐one, a Varietal Aroma in Wine of Vitis vinifera L. cv. Sauvignon Blanc

4‐Methyl‐4‐sulfanylpentan‐2‐one (1; 4MSP) provides a characteristic aroma compound of wines made from Vitis vinifera L. cv. Sauvignon blanc. 4MSP has a strong box‐tree odor with a very low perception threshold and is derived from the cysteinylated precursor S‐(1,1‐dimethyl‐3‐oxobutyl)cysteine (4; P‐4MSP). P‐4MSP is transformed into 4MSP during alcoholic fermentation and is an excellent marker of varietal aroma potential. An improved synthesis of P‐4MSP as well as of its deuterium‐labeled analogue [D6]‐P‐4MSP is described. Several analytical methods (NMR, IR, LSI‐MS, GC/MS, ESI‐MSn) were combined to elucidate spontaneous reversible structural changes of P‐4MSP at different pH values. At low pH, P‐4MSP has a linear keto form. The keto–enol tautomerism was observed at neutral pH. At pH 8, the formation of N‐substituted intramolecular hemiaminal was characterized by ESI‐MS and ESI‐MSn experiments. The hemiaminal loses H2O at high pH to produce a cycloimine, which is easily opened by acid hydrolysis. The keto–enol tautomerism explained the incorporation of only six D‐atoms during the preparation of the P‐4MSP deuterated standard even if [D10]mesityl oxide was used. Derivatization conditions for GC/MS analysis strongly affected the ratio of the monosilylated intramolecular cyclic form and the disilylated linear form of P‐4MSP. The structural changes of P‐4MSP may have a considerable impact on the development of methods of measuring varietal aroma potential.

Identification of S-3-(hexanal)-glutathione and its bisulfite adduct in grape juice from Vitis vinifera L. cv. Sauvignon blanc as new potential precursors of 3SH.

Two main precursors (S-3-(hexan-1-ol)-l-cysteine and S-3-(hexan-1-ol)-l-glutathione) of 3-sulfanylhexanol (3SH, formerly named 3-mercaptohexanol) have been identified so far in grape juice but a correlation between precursor concentrations in grape juices and 3SH concentrations in wines is not always observed. This suggests that there may be other compounds associated with the aromatic potential. In this work, S-3-(hexanal)-glutathione (Glut-3SH-Al) and its bisulfite (Glut-3SH-SO3) adduct were identified in Sauvignon blanc grape juice by liquid chromatography coupled to Fourier transform mass spectrometry experiments. A partial purification of the compounds was carried out by Medium Pressure Liquid Chromatography (MPLC) on the reverse phase using 5L of grape juice. The addition of synthetized Glut-3SH-Al and Glut-3SH-SO3 in the synthetic medium induced a significant release of 3SH after fermentation. Moreover, we demonstrate that Glut-3SH-Al and its bisulfite adduct are present in grape juice and could be considered as new direct 3SH precursors with molar conversion yields close to 0.4%.

Involvement of Dimethyl Sulfide and Several Polyfunctional Thiols in the Aromatic Expression of the Aging Bouquet of Red Bordeaux Wines

The development of an aromatic bouquet during fine wine aging depends on complex transformations occurring in a reductive atmosphere, favorable to the formation and preservation of sulfur odorants, such as dimethyl sulfide (DMS) and polyfunctional thiols. The aim of this study was to address their role in the occurrence, evolution, and perceived sensory nuances of the aging bouquet of red Bordeaux wines. These compounds were quantified in 24 wines and scored by a professional wine panel for the degree to which they reflected the aging bouquet olfactory concept. Partial least square (PLS) analysis, combining sensory and quantitative chemical data, predicted that DMS, 2-furanmethanethiol, and 3-sulfanylhexanol concentrations correlated with the typicality score, discriminating highly-typical wines from less-typical ones. Several vintages from three vineyards were then subjected to sensory and chemical analysis to determine how aging bouquet typicality and the intensity of five key aromatic notes (undergrowth, truffle, fresh fruit, toasted, and empyreumatic) evolved during bottle storage in relation to these three sulfur odorants. PCA analysis emphasized their combined impact on aging bouquet typicality and their contribution to undergrowth, truffle, and empyreumatic attributes.

Vine nitrogen status and volatile thiols and their precursors from plot to transcriptome level

BackgroundVolatile thiols largely contribute to the organoleptic characteristics and typicity of Sauvignon blanc wines. Among this family of odorous compounds, 3-sulfanylhexan-1-ol (3SH) and 4-methyl-4-sulfanylpentan-2-one (4MSP) have a major impact on wine flavor. These thiols are formed during alcoholic fermentation by the yeast from odorless, non-volatile precursors found in the berries and the must. The present study investigates the effects of vine nitrogen (N) status on 3SH and 4MSP content in Sauvignon blanc wine and on the glutathionylated and cysteinylated precursors of 3SH (Glut-3SH and Cys-3SH) in the berries and the must. This is paralleled by a RNA-seq analysis of gene expression in the berries. The impact of N supply on the expression of the glutathione-S-transferase 3 and 4 (VviGST3 and VviGST4) and the γ-glutamyltranspeptidase (VviGGT), considered as key genes in their biosynthesis, was also evaluated.ResultsN supply (N100 treatment) increased the 3SH content in wine while no effect was noticed on 4MSP level. Furthermore, N supply increased Glut-3SH levels in grape berries at late berry ripening stages, and this effect was highly significant in must at harvest. No significant effect of N addition was noticed on Cys-3SH concentration. The transcript abundance of the glutathione-S-transferases VviGST3 and VviGST4 and the γ-glutamyltranspeptidase (VviGGT), were similar between the control and the N100 treatment. New candidate genes which might be implicated in the biosynthetic pathway of 3SH precursors were identified by whole transcriptome shotgun sequencing (RNA-seq).ConclusionsHigh vine N status has a positive effect on 3SH content in wine through an increase of Glut-3SH levels in grape berries and must. Candidate GSTs and glutathione-S-conjugates type transporters involved in this stimulation were identified by RNA-seq analysis.

Comparison of electron and chemical ionization modes for the quantification of thiols and oxidative compounds in white wines by gas chromatography-tandem mass spectrometry.

A rapid, sensitive method for assaying volatile impact compounds in white wine was developed using gas chromatography-tandem mass spectrometry (GC-MS/MS) technology, with a triple quadrupole analyzer operating in chemical ionization and electron impact mode. This GC-MS/MS method made it possible to assay volatile thiols (3SH: 3-sulfanylhexanol, formerly 3MH; 3SHA: 3-sulfanylhexyl acetate, formerly 3MHA; 4MSP: 4-methyl-4-sulfanylpentan-2-one, formerly 4MMP; BM: benzenemethanethiol; E2SA: ethyl 2-sulfanylacetate; and 2FM: 2-furanmethanethiol) and odoriferous oxidation markers (Sotolon: 4,5-dimethyl-3-hydroxy-2(5)H-furanone, methional, and phenylacetaldehyde) simultaneously in dry white wines, comparing electron impact (EI) and chemical ionization (CI) modes. More molecular ions were produced by CI than protonated molecules, despite the greater fragmentation caused by EI. So, even using the best reactant gas giving the highest signal for thiols, EI was the best ionization mode, with the lowest detection limits. For all compounds of interest, the limits of quantification (LOQ) obtained were well below their detection thresholds (ranging from 0.5 to 8.5ng/L for volatile thiols and 65-260ng/L for oxidation markers). Recovery rates ranged from 86% to 111%, reproducibility (in terms of relative standard deviation; RSD) was below 18% in all cases, with correlation coefficients above 0.991 for all analytes. The method was successfully applied to the analysis of compounds of interest in Sauvignon Blanc wines from a single estate and ten different vintages.

Vine Nitrogen Status Does Not Have a Direct Impact on 2-Methoxy-3-isobutylpyrazine in Grape Berries and Wines.

Methoxypyrazines (MP) constitute a large family of compounds that contribute to the vegetative varietal aroma of many grapevine varieties and wines. The berry content in 2-methoxy-3-isobutylpyrazine (IBMP), a major MP reminiscent of green-pepper aroma, can be influenced by environmental factors or cultural practices such as water status or mineral nutrition. To date, no study has investigated a possible direct effect of nitrogen (N) on IBMP synthesis without possible interference from water status and vigor variations. In this study, only vine nitrogen status was significantly different among treatments. Water status was controlled during the season, and vine vigor was similar among treatments. IBMP level was maximal at bunch closure and decreased during the season. There was no significant effect of nitrogen nutrition on this metabolite. Moreover, the expression profiles of VvOMT3 and VvOMT4, key genes in the IBMP biosynthetic pathway, were similar between treatments. This result indicates that when an effect of N on IBMP was found in previous studies, it was likely mediated through the modification of bunch-zone microclimate, induced by the higher vigor of high N-status vines.