New Fungus-Resistant Grapevine Vitis and V. vinifera L. × M. rotundifolia Derivative Hybrids Display a Drought-Independent Response in Thiol Precursor Levels.

The use of new disease-resistant grapevine varieties is a long-term but promising solution to reduce chemical inputs in viticulture. However, little is known about water deficit effects on these varieties, notably regarding berry composition. The aim of this study was to characterize the primary metabolites and thiol precursors levels of 6 fungi-resistant varieties and Syrah. Vines were grown under field conditions and under different water supply levels, and harvested at the phloem unloading arrest. A great variability among varieties regarding the levels of thiol precursors was observed, with the highest concentration, of 539 µg/kg, being observed in 3176-N, a hybrid displaying red fruits. Water deficit negatively and equally impacted the accumulation of sugars, organic acids, and thiol precursors per berry and per plant, with minor effects on their concentration. The observed losses of metabolites per cultivation area suggest that water deficits can lead to significant economic losses for the producer.

Evolution of Markers Involved in the Fresh Mushroom Off-Flavor in Wine During Alcoholic Fermentation.

The fresh mushroom off-flavor (FMOff) is due to several C8 compounds such as 1-octen-3-one, 1-octen-3-ol and 1-hydroxyoctan-3-one, among others. Recently, glycosidic precursors of some FMOff compounds have been identified in grape musts, but the evolution of such compounds during alcoholic fermentation (AF) remains poorly studied. Therefore, the aim of this work was to monitor both FMOff glycosidic precursors and volatile compounds during AF by comparing healthy and Crustomyces subabruptus-contaminated musts. For the first time, glycosidic analysis revealed the presence of 1-hydroxyoctan-3-one glycosides in the laboratory-contaminated musts, together with other FMOff glycosidic fractions already described in the literature. During AF, the FMOff glycosidic fraction decreased, even more in the case of 1-hydroxyoctan-3-one precursors. For the volatile FMOff compounds, their evolutions were both compound- and matrix-dependent except for 1-hydroxyoctan-3-one, which seemed to reach an identical threshold concentration in wine regardless of its initial level in contaminated musts.

Evidence of Enzymatic and Chemical Interconversions of Barley Malt 3-Sulfanylhexanol Conjugates during Mashing.

Recent studies have highlighted in malt the occurrence of the glutathionylated precursor of 3-sulfanylhexanol (G-3SHol) at concentrations reaching hundreds of µg/kg. Here, SIDA-LC-MS/MS was used to investigate the potential conversion of G-3SHol to its dipeptide and cysteinyl analogues during mashing. At 45 and 55 °C, malt γGT and carboxypeptidase activities quickly degrade G-3SHol (up to 90% loss), first to the cysteinylglycine conjugate and then to the cysteine conjugate (up to 205% increase). No γ-glutamylcysteine S-conjugate formation is observed. At 80 °C, despite enzyme inactivation, the G-3SHol level decreases steadily because of suspected imine formation with wort aldehydes at pH 5.5. More surprisingly, CysGly-3SHol is still generated at 80 °C. This indicates the presence in the wort of as yet unidentified precursors.

New Insights on the Scalping Phenomenon of Volatile Sulphur Compounds on Micro-Agglomerated Wine Closures.

Flavour scalping in wine is a well-known phenomenon that is defined as the sorption of flavour compounds on wine closures. While the impact of closure type was the object of several studies, no research has addressed the impact of wine closure permeability on flavour scalping. For that purpose, the adsorption of volatile sulphur compounds (VSCs) on four micro-agglomerated wine cork closures was investigated by soaking them in model and Shiraz wines for 7 days. From a kinetic point of view, most of the VSCs were quickly scalped after 1 h of soaking, and this effect increased after 6 h until reaching a plateau. Most importantly, no significant impact of the closure on the kinetics and adsorption rates of the VSCs was found. As to the quantitative aspects, VSC sorption on closures accounted for 1% to 5% of the initial VSCs present in the wines only, meaning that the impact was negligible under oenological conditions.

Unravelling copper effect on the production of varietal thiols during Colombard and Gros Manseng grape juices fermentation by Saccharomyces cerevisiae.

Nowadays the rapidly increasing organic vineyard management with the utilization of copper as sole fungal control pesticide against downy mildew raises once again the question of copper impact on varietal thiols in wine. For this purpose, Colombard and Gros Manseng grape juices were fermented under different copper levels (from 0.2 to 3.88 mg/l) to mimic the consequences in must of organic practices. The consumption of thiol precursors and the release of varietal thiols (both free and oxidized forms of 3-sulfanylhexanol and 3-sulfanylhexyl acetate) were monitored by LC-MS/MS. It was found that the highest copper level (3.6 and 3.88 mg/l for Colombard and Gros Manseng respectively) significantly increased yeast consumption of precursors (by 9.0 and 7.6% for Colombard and Gros Manseng respectively). For both grape varieties, free thiol content in wine significantly decreased (by 84 and 47% for Colombard and Gros Manseng respectively) with the increase of copper in the starting must as already described in the literature. However, the total thiol content produced throughout fermentation was constant regardless of copper conditions for the Colombard must, meaning that the effect of copper was only oxidative for this variety. Meanwhile, in Gros Manseng fermentation, the total thiol content increased along with copper content, resulting in an increase up to 90%; this suggests that copper may modify the regulation of the production pathways of varietal thiols, also underlining the key role of oxidation. These results complement our knowledge on copper effect during thiol-oriented fermentation and the importance of considering the total thiol production (reduced+oxidized) to better understand the effect of studied parameters and differenciate chemical from biological effects.

New Light on the Varietal Thiols Pathway during Alcoholic Fermentation: Role of 3-S-(N-Acetyl-cysteinyl)-hexan-1-ol (NAC3SH).

For many years, knowledge on thiol precursors has been limited to S-conjugates of glutathione (G3SH), cysteine (Cys3SH), and later on the dipeptides γ-GluCys and CysGly. In this work, we took the parallel between precursor degradation and the glutathione-mediated detoxification pathway a step further by considering a new type of derivative, 3-S-(N-acetyl-l-cysteinyl)hexanol (NAC3SH). This compound was synthesized and then added to the existing liquid chromatography with tandem mass spectrometry (LC-MS/MS) method of thiol precursors. This intermediate was only identified during alcoholic fermentation in synthetic must spiked with G3SH (1 mg/L or 2.45 µmol/L) in the presence of copper with concentration above 1.25 mg/L, which demonstrates for the first time the existence of this new derivative (until 126 µg/L or 0.48 µmol/L) and the capacity of the yeast to produce such a compound. Its status as a precursor was also studied during fermentation, in which a release of 3-sulfanylhexanol was noted corresponding to a conversion yield close to 0.6%. This work completed the thiol precursor's degradation pathway in Saccharomyces cerevisiae in synthetic conditions with a new intermediate, confirming its connection with the xenobiotic detoxification pathway and giving new insights on the precursor's fate.

First identification of a new molecule involved in the fresh mushroom off-flavor in wines: 1-hydroxyoctan-3-one.

The fresh mushrooms off-flavor (FMOff) has been appearing in wines since the 2000 s; the C8 compounds, 1-octen-3-one, 1-octen-3-ol and 3-octanol are involved in this specific taint, yet they alone do not fully explain its occurrence. The objective of this work was to identify by GC-MS new FMOff markers in contaminated matrices, to correlate compound levels with wine sensory characterization and to determine the sensory attributes of 1-hydroxyoctan-3-one, a new candidate involved in FMOff. In practice, grape musts were artificially contaminated with Crustomyces subabruptus, and fermented to obtain tainted wines. GC-MS analysis of contaminated musts and wines revealed the presence of 1-hydroxyoctan-3-one only in contaminated musts, and not in the healthy control. In a selection of 16 wines affected by FMOff, the level of 1-hydroxyoctan-3-one correlated significantly (r2 = 0.86) with sensory analysis scores. Finally, 1-hydroxyoctan-3-one was synthesized and found to generate a fresh mushroom aroma in a wine matrix.

Impact of accentuated cut edges, yeast strain, and malolactic fermentation on chemical and sensory profiles of Sauvignon blanc wine.

This pioneering investigation involved the application of accentuated cut edges (ACE) technique to Sauvignon blanc winemaking. The concentration of varietal thiol precursors in juice was significantly higher for ACE treatment compared to conventional crushing, with two-way or three-way interactions of the experimental factors, which included yeast strain and malolactic fermentation, being determined from the wine data. ACE yielded higher concentrations of 4-methyl-4-sulfanylpentan-2-one (4-MSP) and enantiomers of 3-sulfanylhexanol (3-SH) and 3-sulfanylhexyl acetate (3-SHA) in wines that were more abundant in phenolic compounds. Compared to Sauvy yeast strain, VIN13 produced greater amounts of 3-SH and 3-SHA but less 4-MSP with wines exhibiting lower intensity 'floral' and 'fruity' notes. MLF increased 3-SH and 4-MSP concentrations and led to wines that exhibited more non-fruity sensory attributes. The study revealed the potential of ACE for increasing varietal thiol concentrations in Sauvignon blanc wine and altering overall sensory profiles, with interactions involving yeast strain and MLF.

Identification of a Glycosylated Fraction Involved in Mushroom Off-Flavors in Grapes: Influence of Gray Rot, Powdery Mildew and Crustomyces subabruptus.

An organoleptic defect, termed fresh mushroom off-flavor and mainly caused by the C8 compounds 1-octen-3-one, 3-octanol and 1-octen-3-ol, has been identified in wines and spirits since the 2000s. The aim of this work was to identify the presence of glycosidic precursors of these C8 compounds and to evaluate the influence of different molds on the glycosylated fractions of three grape varieties. Must samples contaminated by molds (gray rot, powdery mildew and Crustomyces subabruptus) and three levels of attack severity (from healthy to 10-15%) were studied. After a ß-glycosidase treatment on Meunier and Pinot noir musts contaminated by Crustomyces subabruptus, 1-octen-3-one, 1-octen-3-ol and 3-octanol were identified by GC-MS, proving the existence of glycosidic fractions in the musts. A Pinot noir must contaminated by Crustomyces subabruptus displayed a 230% increase in the glycosylated fraction responsible for 1-octen-3-one in comparison with an uncontaminated sample. Powdery mildew did not appear to affect the levels of the studied glycosidic fractions in Chardonnay musts. Gray rot on Meunier and Pinot noir musts had opposite effects depending on glycoside type, decreasing the 1-octen-3-one fraction and increasing the 1-octen-3-ol fraction.

Impact of accentuated cut edges (ACE) technique on volatile and sensory profiles of Shiraz wines.

Varietal thiols are important wine aroma compounds that are generally less abundant in red wines. Accentuated cut edges (ACE), known for accelerating phenolic extraction, was applied to Shiraz winemaking and compared with conventional crushing (NOACE) to examine the effects on varietal thiol precursor extraction and thiol formation. Water addition to grape must and skin contact time (SCT) during fermentation were also assessed. Although there was no difference for precursors in the must, ACE significantly decreased 3-S-glutathionylhexan-1-ol concentration during fermentation. 3-Sulfanylhexan-1-ol and ethyl esters were significantly influenced by crushing method and/or SCT, with NOACE or shorter SCT yielding higher concentrations. Acetates, higher alcohols, fatty acids, and isoprenoids differed according to the interaction of crushing method and SCT, with ACE and shorter SCT significantly enhancing all groups except acetates. Volatiles in Sauvignon blanc and Pinot noir wines produced at commercial scale with ACE were briefly evaluated, suggesting an impact of grape variety.

Chiral analysis of cis-2-methyl-4-propyl-1,3-oxathiane and identification of cis-2,4,4,6-tetramethyl-1,3-oxathiane in wine.

cis-2-Methyl-4-propyl-1,3-oxathiane (cis-2-MPO), arising from 3-sulfanylhexan-1-ol (3-SH) and acetaldehyde, was recently identified in wine, but the enantiomeric distribution was unknown. Such information could reveal influences on wine aroma, given the impact of chirality on odorant molecules. Herein, a stable isotope dilution assay employing headspace solid-phase microextraction with chiral gas chromatography-mass spectrometry was developed, validated, and applied to a selection of wines. Studies with (3R)-3-SH revealed the elution order of the cis-2-MPO enantiomers and the concentrations of (2R,4S)-2-MPO and (2S,4R)-2-MPO in the studied wines ranged from undetected to 250 ng/L and 303 ng/L, respectively. Strong positive correlations were found between (3R)-3-SH and (2S,4R)-2-MPO (r = 0.654), and (3S)-3-SH and (2R,4S)-2-MPO (r = 0.860). Additionally, cis-2,4,4,6-tetramethyl-1,3-oxathiane, constituted from acetaldehyde and 4-methyl-4-sulfanylpentan-2-ol (4-MSPOH), was identified in wine for the first time. This new 1,3-oxathiane, which presents a novel fate for 4-MSPOH, was detected in wines as a single enantiomer at up to 28 ng/L.

Spotlight on release mechanisms of volatile thiols in beverages.

Volatile thiols are very strong-smelling molecules that can impact the aroma of numerous beverages. Several thiols and thiol precursors have been reported previously in different plants used as raw material for beverages, some of which are fermented. We focused on thiols in beverages and their release mechanisms from precursors during processing. Volatile thiols in beverages can be classified aslow molecular weight volatile thiols (e.g. H2S, methanethiol) which impact the smell negatively, and volatile thiols with higher boiling points that contribute positively to the aroma profile. The first part of this review is devoted to volatile thiols, without considering small malodorous molecules. The second part deals with thiol precursors and the different release mechanisms induced by processing (e.g. extraction, roasting or fermentation) and by the growing methods (e.g. viticulture), which can impact on amounts of thiols and their precursors.

Evolution and Correlation of cis-2-Methyl-4-propyl-1,3-oxathiane, Varietal Thiols, and Acetaldehyde during Fermentation of Sauvignon blanc Juice.

cis-2-Methyl-4-propyl-1,3-oxathiane (cis-2-MPO) was recently identified in wine and proposed to arise from the reaction of 3-sulfanylhexan-1-ol (3-SH) and acetaldehyde. However, the evolution profile of cis-2-MPO during alcoholic fermentation (AF) and storage and its relationship with varietal thiols and acetaldehyde production were unknown. These aspects were investigated by fermenting Sauvignon blanc juice with J7 and/or VIN13 yeast strains and assessing the stability of cis-2-MPO during wine storage. Moderate to strong Pearson correlations verified similar evolution trends between acetaldehyde, 3-sulfanylhexyl acetate, and cis-2-MPO, with initial increases and a peak during the early to middle stages of AF before consecutive decreases until the end. Contrarily, 3-SH correlated moderately only at the end of AF. A consistent decrease observed for cis-2-MPO when spiked into Sauvignon blanc wine and assessed during 1-year storage revealed its general instability, but acetaldehyde addition (100 mg/L), pH 3.0, and storage at 4 °C all appeared to retain cis-2-MPO. These results have implications for wine aroma and the potential for cis-2-MPO to act as a sink (or source) for 3-SH in wine over time.

Impact of harvest maturity on the aroma characteristics and chemistry of Cascade hops used for dry-hopping.

The impact of ripening on the dry-hop aroma potential and chemical development of Cascade hops is not well understood. Therefore, 5-6 weekly hop samples were collected over the 2014, 2015 and 2016 harvests. Concentrations of humulones did not change as a function of harvest date, while total hop essential oil content displayed significant positive trends. Concentrations of thiol precursors decreased over harvest while concentrations of free thiols increased. These weekly samples were used to dry-hop an unhopped base beer. Overall hop aroma intensity and citrus quality attributed to beer during dry-hopping increased as a function of harvest date. These results suggest that for brewers to maximize the efficiency of hop usage, early harvested Cascades might be better for bittering, while, later harvested Cascades might be better for dry-hopping or aroma additions because they attributed more intense citrusy aromas to beer and had higher concentrations of free thiols and terpene alcohols.

Revisiting the evaluation strategy of varietal thiol biogenesis.

The varietal thiols 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), and 4-mercapto-4-methylpentan-2-one (4MMP) are key aroma compounds in wine due to the tropical notes they impart. They are released by yeast during alcoholic fermentation from different precursors. However, a large part of 3MH origin remains unknown. In this study, we focused on dipeptide forms arising from glutathione S-conjugates to 3MH and 4MMP. Using labelled tracers, we showed in spiked must the release of varietal thiols from 4 different compounds. We highlighted the interconversion between different forms of precursors under defined enological conditions. Cysteinyl-glycine S-conjugates are partially degraded into cysteine S-conjugates, contrary to γ-glutamyl-cysteine S-conjugates. Glutathione S-conjugate to 3MH can be partially degraded to γ-glutamyl-cysteine S-conjugate to 3MH. For the first time, all these labeled forms of precursors were found to release 3MH or 4MMP between 0.17 and 1% molar conversion yield. Two different yeasts were compared without any significant difference.

First identification and quantification of S-3-(hexan-1-ol)-γ-glutamyl-cysteine in grape must as a potential thiol precursor, using UPLC-MS/MS analysis and stable isotope dilution assay.

Varietal thiols are key aroma compounds in wine issued from multiple and complex origins. Several precursor families have been identified in grapes and must and have been widely studied. But a large part of thiol origin still remains unknown. Thus, we only have an incomplete picture of thiol precursors and there is a lack of knowledge on pre-fermentative mechanisms that can impact their levels. Our study focused on the formal identification and the quantification of new varietal thiol precursors in must. First of all, we synthesized natural and labeled standards using an original multi-step strategy, then we developed and validated a UPLC-MS/MS method that allowed us to identify and quantify for the first time a dipeptide S-conjugate to 3MH, the γGluCys-3MH, in Sauvignon B. We observed the S-4-mercapto-4-methylpentan-2-one-l-cysteinyl-glycine (CysGly-4MMP) and S-4-mercapto-4-methylpentan-2-one-N-(l-γ-glutamyl)-l-cysteine (γGluCys-4MMP) but at too low concentration to be quantified.

Innovative analysis of 3-mercaptohexan-1-ol, 3-mercaptohexylacetate and their corresponding disulfides in wine by stable isotope dilution assay and nano-liquid chromatography tandem mass spectrometry.

Both 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA) were largely studied for the last 20 years due to their pleasant olfactory notes conferred to wine. Until now, many analytical methods focused only on the free forms of both 3MH and 3MHA in wine that provided partial information in the wine aroma evolution. Our study proposes new analytical measurements which allow quantification of both free and disulfide forms of 3MH and 3MHA to better understand the redox phenomenon occurring in wines and further, to orientate wine aroma evolution. Free thiols were analyzed by an original method based on maleimide derivatization allowing in-situ disulfide reduction followed by SIDA-LC-MS/MS analyses exhibiting excellent performances. Indeed, the accuracy ranged from 95 to 110% in three different wine matrices and the repeatability and intermediate reproducibility were inferior to 15% (RSD measurements). Our method exhibited very low limits of detection, which are below to 0.5ng/L and inferior to the perception thresholds of both compounds. Then, this method was applied to three different wines exposed to several oxidative conditions. On the one hand, it was demonstrated that copper sulfate treatment firstly destroyed the total amount of free 3MH to the benefit of thioether and disulfides compounds, with proportions that could be slightly modified by glutathione addition. On the other hand, oxygenation of wines resulted in partial free 3MH destruction to the benefit of thioether compounds. We proposed for the first time an innovative analysis that gives a complete picture of wine aroma, which can be really useful to winemakers to manage wine aroma evolution and to take advantage of the disulfide reservoir.

Development and validation of a high-throughput analysis of glutathione in grapes, musts and wines by Stable Isotope Dilution Assay and LC-MS/MS.

For the first time, we proposed a high-throughput method to quantify glutathione in grapes, musts and wines for all grape varieties using Stable Isotope Dilution Assay (SIDA). Indeed, the use of SIDA as a quantification method is essential to overcome the chemical instability of glutathione. In practice, glutathione was derivatized in-situ with N-ethylmaleimide to block the cysteine residue and to enhance its lipophilic properties. After quenching with acetic acid, samples were directly analyzed by LC-MS/MS (run of 13 min) in Multiple Reaction Monitoring mode using labeled glutathione as internal standard. The validation according to the International Organization of Vine and Wine recommendations demonstrated the high sensitivity (LOD=45 µg L(-1)), accuracy (recovery=112%) and intermediate reproducibility (RSD=12%) of the method. This high-throughput method that requires only 1 mL of matrix, allowed us to analyze 70 samples per day for a moderate cost.

Development of a routine analysis of 4-mercapto-4-methylpentan-2-one in wine by stable isotope dilution assay and mass tandem spectrometry.

The 4-mercapto-4-methylpentan-2-one (4MMP) is a key aroma compound in wines, especially in Sauvignon Blanc ones. Its accurate quantification is quite difficult due to its traces levels and its reactivity in wine conferred by the thiol function. In this paper, we proposed a new method for its quantification in wine without any sample preparation, based on automated derivatization procedure by methoximation and SIDA-SPME-GC-MS/MS analysis. The derivatization procedure was adapted from a previously published method in order to decrease the amount of reagents and the volume of wine (only 3mL are required). The use of SPME and the detection conditions have also been optimized to reach the best sensitivity as possible. The method was then validated according to the International Organization of Vine and Wine recommendations and exhibited excellent performances. Indeed, this method allowed us to quantify the 4MMP in wine at traces levels (LOD=0.19 ng L(-1)) with reproducible results (RSD<15%) and a very good accuracy (recovery=102%).

Analysis of ochratoxin A in grapes, musts and wines by LC-MS/MS: first comparison of stable isotope dilution assay and diastereomeric dilution assay methods.

Ochratoxin A (OTA) exhibits potent nephrotoxic, carcinogenic and teratogenic effects and its maximum level in wines has been set to 2 µg L(-1) by regulation. Consequently, the analytical procedures for OTA determination in wines have to be both very sensitive and reliable. In this paper, we compared two quantification methods: the stable isotope dilution assay (SIDA) and the diastereomeric dilution assay (DIDA). For this purpose, non-natural analogues of OTA were synthesized: the labeled OTA (OTA-d4) as a diastereomeric mixture for the SIDA and one non-natural OTA's diastereomer (OTA-dia) for the DIDA. To quantify OTA in red grapes, musts or wines, the sample preparation was optimized using immunoaffinity column extraction and the analysis was performed by LC-MS/MS in Multiple Reaction Monitoring mode. A validation procedure in agreement with the International Organization of Vine and Wine recommendations was conducted. It appeared that SIDA quantification exhibited excellent sensitivity (LOD<1 ng L(-1)), accuracy (recovery=98%), repeatability (RSD<3%) and intermediate reproducibility (RSD<4%) compared to quantification by DIDA. Indeed, DIDA method did not provide satisfactory results demonstrating that immunoaffinity extraction is exclusively selective for the natural OTA and not for its diastereomer, which therefore cannot be considered as a good internal standard for this particular method.