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.

The use of extracted-ion chromatograms to quantify the composition of condensed tannin subunits.

RATIONALE: The analysis of tannins is currently usually done by liquid chromatography after the chemical depolymerization of tannins. However, one of the main limitations of this method is the difficulty in specifically detecting each constitutive tannin monomer in a complex matrix, as numerous compounds co-elute with the monomers, thereby compromising the analysis. METHODS: The depolymerization of tannins under acidic conditions and in the presence of thioglycolic acid releases the various constitutive monomers, either as terminal units or as extension units. The tannin subunits were then quantified using extracted-ion chromatogram (EIC) mass spectrometry, which required the determination of the ionization efficiency of the monomers. Thus, we used AgBF4-assisted coupling for the hemisynthesis of the noncommercial epicatechin gallate dimer. RESULTS: The EIC showed that the derivatives of the extension units were twice as ionized as the terminal units. Unlike the UV chromatograms, this new EIC-based method is more specific and accurate because it is not impacted by the co-elution phenomenon. This result, when added to the linearity obtained on a large range of the calibration curves, allowed for quantification of tannin subunits from EIC in complex mixtures with less pretreatment of the samples. CONCLUSIONS: Due to its specificity and sensitivity, this EIC-based method represents a significant step toward improving the quantification of the tannin composition of samples. The results of this study should allow the oxidation markers to be quantified more accurately and taken into account, thereby providing a better indication of the actual tannin composition.

Analysis of targeted phenolic ageing markers in Syrah red wines during bottle ageing: Influence of cork oxygen transfer rate.

A Syrah red wine ageing experiment was set up during 24-months and the influence of four micro-agglomerated corks were investigated. Specific phenolic ageing markers were selected and hemi-synthesized: vitisin B, malvidin-ethyl-catechin, and epicatechin-sulfonate. A targeted quantification method of these markers was then developed and validated by using ultra-high performance liquid chromatography - triple quadrupole mass spectrometry (UHPLC-QqQ-MS) operating in MRM (Multiple Reaction Monitoring). Results showed a significant decline in native grape polyphenol levels (anthocyanins, flavanols) as ageing progresses while pyranoanthocyanins, ethyl-linked pigments, and flavanol-sulfonates content increased. The cork oxygen transfer rate emerged as a pivotal factor and had significant effects on polyphenolic concentration evolution but had no significant impact on flavanol-sulfonate formation. These results provide valuable insights into the chemical evolution ongoing during wine ageing, accentuating the pivotal role of cork stopper selection in preserving wine quality over time.

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.

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.

Study of the oxidative evolution of tannins during Syrah red wines ageing by tandem mass spectrometry.

Red wine is a very complex medium in which condensed tannins undergo many modifications during winemaking and bottle ageing. These reactions have an impact on the organoleptic properties. This work aimed to highlight tannins evolution related to wine evolution by studying three vintages of Syrah wines. An accelerated oxidation was also undertaken in order to evaluate the ability of this oxidation to imitate natural evolution. After chemical depolymerization of the tannins, the monitoring of 6 types of markers at two oxidation levels was investigated. An evolution of the tannin oxidation state during ageing evidenced by the increase of the markers of the second oxidation level was observed. In the 2018 oxidized wine sample, the first oxidation level markers were similar to the 2014 vintage but the second oxidation level markers were higher than other vintages, indicating a more advanced state of tannin oxidation.

Detection and Identification of Oxidation Markers of the Reaction of Grape Tannins with Volatile Thiols Commonly Found in Wine.

Condensed tannins undergo many modifications during winemaking and the aging of wine. These reactions take place between the various constituent units of the tannins as well with other types of molecules present in wines. This work aimed to highlight the potential reactions of tannins and aromas. After the monomeric standards of the reaction products were synthesized, the study of the reactivity of tannins (dimer B2, trimer C1, and grape seed tannins composed of 23% epicatechin-3-O-gallate) with aromas (3-sufanylhexan-1-ol, 4-methyl-4-sulfanylpentan-2-one, ethanethiol, and furfurylthiol) permitted identification of numerous reaction products. These tannin/aroma oxidation markers, detected after chemical depolymerization, allow for progress in the characterization of tannins in the oenological environment and the understanding of their reactivity toward the molecules present in the samples. A better characterization of tannins will make it possible to better measure the impact of these modifications on the organoleptic qualities of wines.

Ferulic acid content variation from wheat to bread.

The health-promoting effects of whole-grain consumption have been attributed in a large part to the phytochemical profile of the wheat grain, and particularly to the bioactive molecules present in bran. This study shed light on the impact of human practices, especially harvesting sites (terroirs) and wheat species and varieties, as well as bread-making conditions on the variation of the antioxidant and antimicrobial ferulic acid (FA) content. FA concentration in the bran of wheat species (durum and bread wheat) and varieties (Chevalier, Renan, Redon, Saint Priest le vernois rouge, Bladette de Provence, Pireneo, Rouge de Bordeaux, LA1823, Claudio et Bidi17) harvested in five sites in France on 2015 and 2017, has been evaluated. Statistical analysis showed significant differences in FA content for wheat varieties and terroirs. During bread making, baking and type of leaven impacted the FA content of dough and bread. The differences were not due to the type of fermentation (sourdough/commercial yeast) but rather to the diversity of fermenting microbial strains and flour used for backslopping.