Isolation of Taste-Active Triterpenoids from Quercus robur: Sensory Assessment and Identification in Wines and Spirit.

Six new triterpenoids (1-6), two known genins (7 and 8), and five known functionalized triterpenoids (9-13) were isolated from a Quercus robur heartwood extract. The purification protocol was guided by LC-HRMS by searching for structural analogues of bartogenic acid on the basis of their putative empirical formula. The structures of the new compounds were unequivocally elucidated using HRESIMS and 1D/2D NMR experiments. Sensory analyses were performed in water and in a non-oaked white wine on the pure compounds 1-13 at 5 mg/L. All molecules were perceived as bitter in water and wine, but they were mostly reported as modifying the wine taste balance. Using LC-HRMS, compounds 1-13 were observed in oaked red wine and cognac and were semiquantified in oak wood extracts. The influence of two cooperage parameters, oak species and toasting process, on compounds 1-13 content was studied. All compounds were found in quantities significantly higher in pedunculate than in sessile oak wood. Toasting is a key step in barrel manufacture and modulates the concentration of the discussed compounds. Significantly higher quantities were observed in untoasted wood compared to medium or highly toasted wood. These findings provide new insights into the molecular origin of taste changes due to oak aging.

Natural allelic variations of Saccharomyces cerevisiae impact stuck fermentation due to the combined effect of ethanol and temperature; a QTL-mapping study.

BACKGROUND: Fermentation completion is a major prerequisite in many industrial processes involving the bakery yeast Saccharomyces cerevisiae. Stuck fermentations can be due to the combination of many environmental stresses. Among them, high temperature and ethanol content are particularly deleterious especially in bioethanol and red wine production. Although the genetic causes of temperature and/or ethanol tolerance were widely investigated in laboratory conditions, few studies investigated natural genetic variations related to stuck fermentations in high gravity matrixes. RESULTS: In this study, three QTLs linked to stuck fermentation in winemaking conditions were identified by using a selective genotyping strategy carried out on a backcrossed population. The precision of mapping allows the identification of two causative genes VHS1 and OYE2 characterized by stop-codon insertion. The phenotypic effect of these allelic variations was validated by Reciprocal Hemyzygous Assay in high gravity fermentations (> 240 g/L of sugar) carried out at high temperatures (> 28 °C). Phenotypes impacted were mostly related to the late stage of alcoholic fermentation during the stationary growth phase of yeast. CONCLUSIONS: Our findings illustrate the complex genetic determinism of stuck fermentation and open new avenues for better understanding yeast resistance mechanisms involved in high gravity fermentations.

Triterpenoids from Quercus petraea: Identification in Wines and Spirits and Sensory Assessment.

Eight new triterpenoids (1-8), the known genin (9), and two known functionalized triterpenoids (10 and 11) were isolated from a Quercus petraea heartwood extract. The structures of the new compounds were unequivocally elucidated using HRESIMS and 1D/2D NMR experiments. Sensory analyses were performed in a non-oaked wine on the pure compounds 1-11. Except compounds 1 and 11, all molecules exhibited a sweet taste at 5 mg/L that was particularly intense for compounds 3 and 9. Using LC-HRMS, compounds 1-11 were observed in an oak wood extract and in oaked red wine and cognac. They were also semiquantified in several samples of sessile ( Q. petraea) and pedunculate ( Q. robur) oak wood extract. All compounds were found in quantities significantly higher in sessile than in pedunculate oak wood. These results support the hypothesis of their contribution to the increase in sweetness during oak aging and show that they can be used as chemical markers to identify the species of oak used for cooperage.

Development of a quantitation method to assay both lyoniresinol enantiomers in wines, spirits, and oak wood by liquid chromatography-high resolution mass spectrometry.

Wine taste balance evolves during oak aging by the release of volatile and non-volatile compounds from wood. Among them, an enantiomer of lyoniresinol, (+)-lyoniresinol, has been shown to exhibit bitterness. To evaluate the impact of (+)-lyoniresinol on wine taste, a two-step quantitation method was developed and validated. First, (±)-lyoniresinol was assayed in wines, spirits, and oak wood macerates by C-18 liquid chromatography-high resolution mass spectrometry (LC-HRMS). Then, the lyoniresinol enantiomeric ratio was determined by chiral LC-HRMS in order to calculate the (+)-lyoniresinol content. In red and white wines, the average concentrations of (+)-lyoniresinol were 1.9 and 0.8 mg/L, respectively. The enantiomer proportions were not affected by bottle aging, and lyoniresinol appeared to remain stable over time. The sensory study of (+)-lyoniresinol established its perception threshold at 0.46 mg/L in wine. All the commercial wines quantitated were above this perception threshold, demonstrating its impact on wine taste by an increase in bitterness. In spirits, (+)-lyoniresinol ranged from 2.0 to 10.0 mg/L and was found to be released continuously during oak aging. Finally, neither botanical origin nor toasting was found to significantly affect the (+)-lyoniresinol content of oak wood. Graphical abstract From oak wood to wine: evaluation of the influence of (+)-lyoniresinol on the bitterness of wines and spirits.

Taste-Guided Isolation of Bitter Lignans from Quercus petraea and Their Identification in Wine.

Quercoresinosides A and B (1 and 2), two new lignans, were isolated from a toasted Quercus petraea heartwood extract along with a known compound, 3-methoxy-4-hydroxyphenol 1-O-ß-d-(6'-O-galloyl)glucopyranoside (3). The purification protocol was based on a taste-guided approach that sought to reveal new bitter compounds released from oak wood into wines and spirits. HRMS and NMR data were used to establish that compounds 1 and 2 are lignan derivatives bearing a glucosyl unit and a galloyl unit at the same positions. Hydrolysis of these compounds showed that they could be distinguished by the absolute configuration of their respective lyoniresinol genin as determined by chiral LC-HRMS in comparison with (+)- and (-)-lyoniresinol standards. Sensory analyses were performed in a non-oaked wine on the pure compounds 1-3. The three molecules exhibited a bitter taste at 2 mg/L that was particularly intense for compounds 2 and 3. Finally, LC-HRMS demonstrated the occurrence of compounds 1-3 in oaked wine and brandy, which supports the hypothesis of their contributions to the increase in bitterness during oak aging.

Identification of new natural sweet compounds in wine using centrifugal partition chromatography-gustatometry and Fourier transform mass spectrometry.

Sweetness contributes notably to the taste-balance of dry wines and increases during oak-barrel aging owing to the release of natural sweeteners from wood. The search for such taste-active molecules, which are sometimes present at very low concentrations in wine or other complex matrixes, requires both reliable purification tools and powerful identification techniques. Here, we report the development of an original inductive method using centrifugal partition chromatography (CPC) and sensorial analysis. This method, called CPC-gustatometry, was implemented to isolate a sweet fraction with only four compounds from a complex oak wood extract. The recently developed Fourier transform mass spectrometry (FT-MS, Orbitrap analyzer) was used jointly with two-dimensional nuclear magnetic resonance (2D (1)H and (13)C NMR) to obtain the structural elucidation of the purified compounds. The tandem mass spectrometry (MS/MS) spectra obtained with resonant and nonresonant fragmentation modes were compared, thus providing complementary information about the molecular structure. Two oleanane-type triterpenoids substituted with galloyl and glucosyl moieties were identified, one of which exhibits sweet properties. We term these compounds which have never been reported, Quercotriterpenoside I and II.

Impact of Closure OTR on the Volatile Compound Composition and Oxidation Aroma Intensity of Sauvignon Blanc Wines during and after 10 Years of Bottle Storage.

The oxygen transfer rate (OTR) of closures is a well-known parameter impacting the quality of Sauvignon blanc wines (SBw) within the first years of storage, but little research has been published on its long-term effects. The chemical changes in oxidation odor intensity in three SBw sealed with natural cork and other closures that had different known OTRs, ranging from <0.1 to 4.6 mg/year, were monitored over a 10 year period. During aging, free SO2 and 3-sulfanylhexanol loss, concomitant with increases in dissolved O2, OD420, and sotolon, were correlated with closure OTR levels. After 10 years of aging, sensory analysis was conducted, supported by additional chemical analysis of aroma impact markers, including methional, phenylacetaldehyde, 2-furanmethanthiol, 4-methyl-4-sulfanylpentan-2-one, ethyl-2-sulfanylacetate, and hydrogen sulfide, as well as total SO2 and dissolved CO2. These analyses revealed that selected SBw were protected from oxidation over a 10 year aging period, provided that the closure OTR did not exceed 0.3 mg/year.

Genetic improvement of thermo-tolerance in wine Saccharomyces cerevisiae strains by a backcross approach.

During red wine fermentation, high temperatures may cause stuck fermentation by affecting the physiology of fermenting yeast. This deleterious effect is the result of the complex interaction of temperature with other physicochemical parameters of grape juice, such as sugar and lipid content. The genetic background of fermenting yeast also interacts with this complex matrix and some strains are more resistant to high temperatures than others. Here, the temperature tolerance of nine commercial starters was evaluated, demonstrating that, at high sugar concentrations, half of them are sensitive to temperature. Using a classical backcross approach, one thermo-sensitive commercial starter was genetically improved by introducing quantitative trait loci conferring resistance to temperature. With this breeding program it is possible to obtain a thermo-resistant strain sharing most of its genome with the initial commercial starter. The parental and improved strains were compared for population growth and fermentation ability in various conditions. Despite their common genetic background, these two strains showed slight physiological differences in response to environmental changes that enable identification of the key physiological parameters influencing stuck fermentation.

Taste-guided isolation of sweet-tasting compounds from grape seeds, structural elucidation and identification in wines.

This work aimed at improving knowledge about sweetness in dry wines. Following on from the empirical observations of winegrowers, we assessed the contribution of grape seeds to wine sensory properties. An inductive fractionation method guided by gustatometry was used to isolate and characterize sweet-tasting compounds from grapes. Fractionation of grape seed macerates was achieved by liquid-liquid extraction, centrifugal partition chromatography (CPC) and preparative HPLC. Then, the structures of the purified compounds were elucidated by use of FTMS and NMR. Five compounds were identified: two new compounds, 2-hydroxy-3-methylpentanoic-2-O-ß-glucopyranoside (H3MP-G) and 2-hydroxy-4-methylpentanoic-2-O-ß-glucopyranoside acids (H4MP-G), along with gallic-4-O-ß-glucopyranoside acid (AG-G), 3-indolyl-(2R)-O-ß-d-glycoside lactic acid (ILA-G) and epi-DPA-3'-O-ß-glucopyranoside acid (epi-DPA-G). These compounds exhibited various levels of sweetness in a hydro-ethanolic solution and in white and red wines. Additionally, H3MP-G, H4MP-G and epi-DPA-G were identified for the first time in grapes and wines, whereas AG-G has already been reported in white grapes but never in wine.

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.

Impact of mixed Torulaspora delbrueckii-Saccharomyces cerevisiae culture on high-sugar fermentation.

Conventional wine yeasts produce high concentrations of volatile acidity, mainly acetic acid, during high-sugar fermentation. This alcoholic fermentation by-product is highly detrimental to wine quality and, in some cases, levels may even exceed legal limits. In this study, a non-conventional species, Torulaspora delbrueckii, was used, in pure cultures and mixed with Saccharomyces cerevisiae yeast, to ferment botrytized musts. Fermentation rate, biomass growth, and the formation of volatile acidity, acetaldehyde, and glycerol were considered. This study demonstrated that T. delbrueckii, often described as a low acetic producer under standard conditions, retained this quality even in a high-sugar medium. Unlike S. cerevisiae, this species did not respond to the hyper-osmotic medium by increasing acetic production as soon as it is inoculated into the must. Nevertheless, this yeast produced low ethanol and biomass yields, and the fermentation was sluggish. As a result, T. delbrueckii fermentations do not reach the required ethanol content (14%vol.), although this species can survive at this concentration. A mixed culture of T. delbrueckii and S. cerevisiae was the best combination for improving the analytical profile of sweet wine, particularly volatile acidity and acetaldehyde production. A mixed T. delbrueckii/S. cerevisiae culture at a 20:1 ratio produced 53% less in volatile acidity and 60% less acetaldehyde than a pure culture of S. cerevisiae. Inoculating S. cerevisiae after 5 days' fermentation by T. delbrueckii had less effect on volatile acidity and acetaldehyde production and resulted in stuck fermentation. These results contribute to a better understanding of the behaviour of non-Saccharomyces and their potential application in wine industry.

Changes in the sotolon content of dry white wines during barrel and bottle aging.

GC-MS in electron ionization mode (EI) was used as a simple, sensitive method for assaying sotolon [4,5-dimethyl-3-hydroxy-2(5) H-furanone] in various dry white wines. The impact of barrel-aging conditions, that is, whether yeast lees were present or not, on the formation of sotolon in dry white wines was then studied. The sotolon content was highest in dry white wines aged in new barrels without lees, often exceeding the perception threshold (8 microg/L). These results demonstrated that yeast lees were capable of minimizing the formation of sotolon in dry white wines during aging. The sotolon and oxygen contents of several bottle of the same white wine were also compared 7 years after bottling. At the range of dissolved oxygen concentrations generally measured, between 5 and 100 microg/L, the sotolon content remained below its perception threshold in wine. The perception threshold was exceeded only in wines with oxygen concentrations above 500 microg/L. The presence of dissolved oxygen in the wine samples analyzed also resulted in a decrease in their free sulfur dioxide content.

Distribution and organoleptic impact of sotolon enantiomers in dry white wines.

The enantiomers of sotolon, a flavor compound typical of oxidized white wines, were separated by preparative HPLC to determine their perception thresholds and distribution in wines. The enantiomeric ratios of chiral sotolon were evaluated in several dry white wines using gas chromatography and a chiral column (beta-cyclodextrin) connected to a 2 m precolumn (BP20). The perception threshold of (S)-sotolon (0.8 microg/L) in model wine solution was 100 times lower than that of the (R) form (89 microg/L), indicating that (S)-sotolon contributes to the characteristic aroma of prematurely aged dry white wines. Both enantiomers are detected in white wines. Analysis of commercial dry white wines from various vintages and origins revealed three types of distribution patterns: the racemic form, an excess of R, and an excess of S. The proportions found in these wines may be partially explained by the slow racemization kinetics (20 months) of optically active sotolon.

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 [D(6)]-P-4MSP is described. Several analytical methods (NMR, IR, LSI-MS, GC/MS, ESI-MS(n)) 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-MS(n) experiments. The hemiaminal loses H(2)O 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 [D(10)]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.

Assay of glutathione in must and wines using capillary electrophoresis and laser-induced fluorescence detection. Changes in concentration in dry white wines during alcoholic fermentation and aging.

Glutathione (GSH) was assayed in must and wine using capillary electrophoresis coupled with laser-induced fluorescence (LIF) detection. Sample preparation involved conjugating thiols with monobromobimane (MBB) in a 2-(N-cyclohexylamino)ethanesulfonic acid [CHES] buffer (179mM). The electrophoretic conditions were 30kV with a capillary length of 105cm from the inlet to the detector (120cm total length) and a 50microm inner diameter. Under these conditions, the complete separation from the other main non-volatile thiols took less than 20min. We also described the optimum conditions for derivatizing wine samples with MBB to increase eletrophoretic sensitivity. The detection limit for glutathione assay is 65nmol/L. This simple, sensitive method provides a specific assay of glutathione in reduced form, as the sample preparation technique does not modify the balance of oxidized and reduced forms. We used this method to monitor changes in the reduced glutathione content of a white wine during alcoholic fermentation and barrel aging.

New methodology for removing carbonyl compounds from sweet wines.

Sweet white wines from botrytized grapes present high SO2 levels because of their high sulfur dioxide binding power. The objective of this work was to develop a new method for reducing this binding power by partially eliminating the carbonyl compounds naturally present in these wines that are responsible for this phenomenon. A selective liquid-solid removal technique was developed. Phenylsulfonylhydrazine was selected as the best candidate for removing carbonyl compounds. Its reactivity in the presence or absence of sulfur dioxide was verified in model media containing acetaldehyde, pyruvic acid, and 2-oxoglutaric acid, some of the main carbonyl compounds responsible for the SO2 binding power of sweet wines. The scavenging function was grafted on porous polymer supports, and its efficiency was evaluated in model wines. Dependent upon the supports used, different quantities of carbonyl compounds (over 90% in some cases) were removed in a few days. The presence of sulfur dioxide delayed removal without changing its quality. The results obtained showed that the method removed carbonyl compounds efficiently and was applicable to wines at any stage in winemaking.

Which impact for beta-damascenone on red wines aroma?

beta-Damascenone, a C-13 norisoprenoid compound, is usually presented as an impact odorant in red wines. Its direct contribution to their aroma was investigated. Both free beta-damascenone and beta-damascenone precursors were isolated from various French red wines and then analyzed by gas chromatography-mass spectrometry, revealing concentrations in the vicinity of 1 and 2 microg/L for free compounds and both forms, respectively. Gas chromatography-olfactometry analyses were also performed on dilutions of both red wine extracts and pure beta-damascenone. The very low detection threshold in olfactometry for this compound explains why it is found at the highest dilution factor in aroma extract dilution analysis methods. Moreover, determination of beta-damascenone's odor thresholds confirmed the huge importance of the matrix: beta-Damascenone is characterized by a very low perception threshold in hydroalcoholic solution as compared to red wine, where it is over 1000-fold higher. In hydroalcoholic solution, beta-damascenone enhanced fruity notes of ethyl cinnamate and caproate and masked the herbaceous aroma of IBMP. Globally, these results suggested that beta-damascenone has more an indirect than a direct impact on red wine aroma.

Odorous impact of volatile thiols on the aroma of young botrytized sweet wines: identification and quantification of new sulfanyl alcohols.

Specific extraction of volatile thiols using sodium p-hydroxymercuribenzoate revealed the presence of three new sulfanylalcohols in wines made from Botrytis-infected grapes: 3-sulfanylpentan-1-ol (II), 3-sulfanylheptan-1-ol (III), and 2-methyl-3-sulfanylbutan-1-ol (IV). The first two have citrus aromas, whereas the third is reminiscent of raw onion. In addition, 2-methyl-3-sulfanylpentan-1-ol, which has a raw onion odor, was tentatively identified. Like 3-sulfanylhexan-1-ol (I), already reported in Sauternes wines, compounds II, III, and IV were absent from must. They were found in wine after alcoholic fermentation, and their concentrations were drastically higher when Botrytis cinerea had developed on the grapes. In the commercial botrytized wines analyzed, the mean levels of II, III, and IV were 209, 51, and 103 ng/L, respectively. Despite their low odor activity values, sensory tests showed additive effects among I, II, and III, thus confirming their olfactory impact on the overall aroma of botrytized wines.

Study of the contribution of massoia lactone to the aroma of Merlot and Cabernet Sauvignon musts and wines.

Organic extracts of musts and red wines marked by dried fruit and cooked fruit aromas were analyzed by gas chromatography coupled to olfactometry and mass spectrometry. Thanks to this analytical approach we identified a fragrant lactone corresponding to an odorant zone reminiscent of coconut and dried figs as 5,6-dihydro-6-pentyl-2H-pyran-2-one (C10 massoia lactone). Using chiral GC-GC-MS, we show that only the (R)-C10 massoia lactone is found in musts and wines. Its detection thresholds were 10µg/L and 11µg/L in must and wine model solution, respectively. In Merlot and Cabernet Sauvignon musts marked by dried fruit flavors from overripe grapes, its concentration reached 68µg/L. In contrast, in wines marked by these flavors, it never exceeded 20µg/L. We show that (R)-C10 massoia lactone is reduced to (R)-δ-decalactone during alcoholic fermentation. In addition, we underline the contribution of temperature during the growing season on its level in old red wines.

Toward a Molecular Understanding of the Typicality of Chardonnay Wines: Identification of Powerful Aromatic Compounds Reminiscent of Hazelnut.

Chardonnay wines impart a unique complex aroma characterized by its buttery, yellow stone fruit, melon, bready, and woody notes. Among the terms used in the sensory analysis of these wines, this study investigated hazelnut-like attributes. Multidimensional gas chromatography coupled to olfactometry identified five pyrroles reminiscent of hazelnut: 1-ethylpyrrole-2-carboxaldehyde, 1H-pyrrole, 2-acetyl-1H-pyrrole (first identification in wine), 1-methylpyrrole-2-carboxaldehyde, and 1H-pyrrole-2-carboxaldehyde. Quantitative analyses demonstrated their significantly higher abundance in Chardonnay wines. However, they proved irrelevant in sensory terms, given the low amounts measured in wine compared to their olfactory detection threshold. Nevertheless, the presence of methanethiol derivatives from these pyrroles was investigated in wine. 1-Methylpyrrole-2-methanethiol and 1-ethylpyrrole-2-methanethiol were identified and exhibited hazelnut-like aroma. These compounds, which have not been observed in natural products to date, are potent volatile compounds with detection thresholds of 0.7 and 1.4 ng/L, respectively, in model wine. These findings open up promising perspectives concerning the interpretation of the typical aromatic nuances of some Chardonnay wines.