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.

Unambiguous NMR Structural Determination of (+)-Catechin-Laccase Dimeric Reaction Products as Potential Markers of Grape and Wine Oxidation.

(+)-Catechin-laccase oxidation dimeric standards were hemi-synthesized using laccase from Trametes versicolor in a water-ethanol solution at pH 3.6. Eight fractions corresponding to eight potential oxidation dimeric products were detected. The fractions profiles were compared with profiles obtained with two other oxidoreductases: polyphenoloxidase extracted from grapes and laccase from Botrytis cinerea. The profiles were very similar, although some minor differences suggested possible dissimilarities in the reactivity of these enzymes. Five fractions were then isolated and analyzed by 1D and 2D NMR spectroscopy. The addition of traces of cadmium nitrate in the samples solubilized in acetone-d6 led to fully resolved NMR signals of phenolic protons, allowing the unambiguous structural determination of six reaction products, one of the fractions containing two enantiomers. These products can further be used as oxidation markers to investigate their presence and evolution in wine during winemaking and wine ageing.

Multimethod Approach for Extensive Characterization of Gallnut Tannin Extracts.

Gallotannins extracted from gallnuts are commonly added to wine to improve its properties. They consist of mixtures of galloylester derivatives of glucose. However, their composition and properties are not well-established. In this study, methods based on liquid chromatography coupled to ultraviolet-visible detection and mass spectrometry, size-exclusion chromatography, and one-dimensional (31P) and two-dimensional (1H diffusion ordered spectroscopy, 31P total correlated spectroscopy, and 1H/13C heteronuclear single-quantum correlation and heteronuclear multiple-bond correlation) nuclear magnetic resonance spectroscopies have been implemented for extensive chemical characterization of three commercial gallnut tannin extracts. Differences in the proportions of the different constituents (gallic, digallic, and trigallic acids and galloylglucose derivatives) and in the structure and molecular weight distributions of gallotannins were demonstrated between the three extracts, with chains containing 8.5, 12.2, and 12.4 galloyl groups on average for TAN A, TAN B1, and TAN B2, respectively. The antioxidant capacities of the extracts, evaluated using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) method, were similar and related mostly to their total tannin content, with only a limited impact of the tannin composition.

New flavanol O-glycosides in grape and wine.

The presence of monomeric and dimeric flavan-3-ol monohexosides was investigated in grapes and wines. Polyphenol extracts were prepared from grape seeds and skins (Syrah, Merlot, and Cabernet-Sauvignon) sampled at three different developmental stages. Different wines (Tannat, Alicante, Syrah, Merlot, and Grenache) were also studied. The different samples obtained were analyzed by UPLC-ESI-IT-MS. Specific molecular ions corresponding to flavan-3-ol hexosides were detected by using targeted molecular ions with specific m/z values: 451 for (epi)catechin hexosides, and 739 for procyanidin dimer hexosides. 4'-O-ß-glucosyl-(+)-catechin and 7-O-ß-glucosyl-(+)-catechin were obtained by using a glucosyl transferase from apple, UGT71A15, and their structures determined by NMR. These glucosylated monomers and the dimers were identified in all analyzed grape seed and several skin extracts at the different stages and in wines made from different varieties.

Quantification of hydroxycinnamic derivatives in wines by UHPLC-MRM-MS.

A UHPLC-MS/MS method was developed for the quantification of the main compounds involved in oxidation reactions occurring in white musts and wines such as hydroxycinnamic acids, their glutathione and cysteinylglycine adducts (GRP, GRP2, 5-(S-glutathionyl)-trans-caftaric acid, 2-(S-cysteinylglycyl)-trans-caftaric acid, and 2-(S-glutathionyl)-trans-caffeic acid), and reduced and oxidized glutathione (GSH, GSSG) in wine. Since oxidation is the main concern in white wine-making, directly affecting its quality, the developed method was then applied in a series of white wines made with different pre-fermentation treatments to limit oxidation at must stage. The glucose esters and/or glucosides of hydroxycinnamic acids were quantified as glucogallin equivalent. The developed method led to an overall improvement in the limits of detection (LODs) and quantification (LOQs) for all the compounds studied in comparison to other methods such as high-performance liquid chromatography with fluorescence detection (HPLC-FLD) or diode array UV detection (HPLC-DAD). LOD values ranged from 0.0002 to 0.0140 mg/L and LOQs from 0.0005 to 0.0470 mg/L. The recoveries ranged between 80 and 110% in wines, and the relative standard deviation (RSD) for precision intra- and inter-day was below 15%. The accuracy and intra- and inter-day precision met the acceptance criteria of the AOAC international norms. As far as we know, this study is the first report of quantification of GRP, 2-(S-cysteinylglycyl)-trans-caftaric acid, and 2-(S-glutathionyl)-trans-caffeic acid using these non-commercially available compounds as external standards. Those compounds represent a significant proportion of hydroxycinnamic acid derivatives in wines. The methodology described is suitable for the analysis of hydroxycinnamic derivatives in wines.

The host plant Pinus pinaster exerts specific effects on phosphate efflux and polyphosphate metabolism of the ectomycorrhizal fungus Hebeloma cylindrosporum: a radiotracer, cytological staining and 31 P NMR spectroscopy study.

Ectomycorrhizal (ECM) association can improve plant phosphorus (P) nutrition. Polyphosphates (polyP) synthesized in distant fungal cells after P uptake may contribute to P supply from the fungus to the host plant if they are hydrolyzed to phosphate in ECM roots then transferred to the host plant when required. In this study, we addressed this hypothesis for the ECM fungus Hebeloma cylindrosporum grown in vitro and incubated without plant or with host (Pinus pinaster) and non-host (Zea mays) plants, using an experimental system simulating the symbiotic interface. We used 32 P labelling to quantify P accumulation and P efflux and in vivo and in vitro nuclear magnetic resonance (NMR) spectroscopy and cytological staining to follow the fate of fungal polyP. Phosphate supply triggered a massive P accumulation as newly synthesized long-chain polyP in H. cylindrosporum if previously grown under P-deficient conditions. P efflux from H. cylindrosporum towards the roots was stimulated by both host and non-host plants. However, the host plant enhanced 32 P release compared with the non-host plant and specifically increased the proportion of short-chain polyP in the interacting mycelia. These results support the existence of specific host plant effects on fungal P metabolism able to provide P in the apoplast of ectomycorrhizal roots.

Synthesis, Identification, and Structure Elucidation of Adducts Formed by Reactions of Hydroxycinnamic Acids with Glutathione or Cysteinylglycine.

Grape polyphenols, especially hydroxycinnamic acids such as caftaric and caffeic acid, are prone to enzymatic oxidation reactions during the winemaking process, forming o-quinones and leading to color darkening. Glutathione is capable of trapping these o-quinones and thus limiting juice browning. In this study, the addition of glutathione or cysteinylglycine onto caftaric or caffeic acid o-quinones formed by polyphenoloxidase-catalyzed reactions was investigated by UPLC-DAD-ESIMS and NMR data analyses. Complete identification of adducts has been achieved via NMR data. The results confirmed that the favored reaction is the substitution of the sulfanyl group of cysteine at C-2 of the aromatic ring. Several minor isomers, namely, the cis-isomer of the 2-S adduct and trans-isomers of the 5-S and 6-S adducts, and the 2,5-di-S-glutathionyl adducts were also identified and quantified by qNMR. With the exception of 2-(S-glutathionyl)- and 2,5-di(S-glutathionyl)-trans-caftaric acid, these products had never been formally identified. In particular, the 5-S and 6-S derivatives are reported here for the first time. The first formal identification of 2-S cis-derivatives is also provided. Moreover, NMR and UPLC-DAD-ESIMS analysis showed that signature UV and MS spectra can serve as markers of the conformation and substitution position in the aromatic ring for each of the isomers.

A comprehensive investigation of guaiacyl-pyranoanthocyanin synthesis by one-/two-dimensional NMR and UPLC-DAD-ESI-MS(n).

In red and rosé wines, the grape anthocyanins are progressively converted to more stable pigments, including phenylpyranoanthocyanins. One-/two-dimensional NMR and UPLC-DAD-ESI-MS(n) measurements were used to monitor the synthesis of guaiacylpyranomalvidin 3-O-glucoside from malvidin 3-O-glucoside and vinylguaiacol in model solutions and identify the products formed during the reaction. The highest conversion rates (30%, determined by (1)H qNMR) were obtained with a small excess of vinylguaiacol in methanol/water (70/30) at pH 3 and 35°C. Two reaction pathways competed with the formation of guaiacylpyranomalvidin 3-O-glucoside. The first one only concerns malvidin 3-O-glucoside and consists in C-ring cleavage with formation of malvone and smaller molecular weight breakdown products. This pathway is favored at higher pH and incubation temperature. At lower pH values or in the presence of large vinylguaiacol excess, faster consumption of malvidin 3-O-glucoside resulted from the formation of more complex pyranoanthocyanins substituted by vinylguaiacol oligomers.

Straightforward synthesis of deuterated precursors to demonstrate the biogenesis of aromatic thiols in wine.

Straightforward synthesis of labeled S-3-(hexan-1-ol)-glutathione and S-4-(4-methylpentan-2-one)-glutathione has been developed through a conjugate addition optimization study. Sauvignon blanc fermentation experiments with the [(2)H(10)] S-4-(4-methylpentan-2-one)-glutathione used as a tracer released the corresponding deuterated thiol, thus proving the direct relationship with the 4-mercapto-4-methylpentan-2-one under enological conditions. The conversion yield of such transformation was estimated to be close to 0.3%, opening an avenue for additional study on varietal thiol biogenesis.

Characterization of a colorless anthocyanin-flavan-3-ol dimer containing both carbon-carbon and ether interflavanoid linkages by NMR and mass spectrometry.

Direct addition of anthocyanins and flavan-3-ols was investigated in a model system by incubating malvidin 3-glucoside and (-)-epicatechin in ethanol. Analysis of reaction products by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC/ESI-MS) before and after thiolysis showed the formation of colorless dimers detected at m/z 781 in the negative ion mode, with retention times and spectroscopic characteristics identical to those of compounds detected in wine, which contain one malvidin 3-glucoside unit and one flavanol unit. On the basis of their resistance to thiolysis, these compounds were postulated to be bicyclic dimers linked with both carbon-carbon and ether bonds as observed in the case of A type proanthocyanidins. The major dimer analyzed by NMR experiments was identified as malvidin 3-glucoside(C2-O-C7,C4-C8)epicatechin, confirming this hypothesis. A similar assay was performed with (+)-catechin instead of (-)-epicatechin, and the formation of bicyclic dimers was also observed.