Monoglucoside versus Diglucoside Anthocyanin Evolution of Red Wine Produced Using a Fungus-Resistant Grape Cultivar (Downy Mildew and Powdery Mildew) under Oxidative Conditions.

The need to reduce the use of pesticides in viticulture is increasing the interest in wines produced using fungal-resistant grapevine varieties, which are characterized by relevant contents of both monoglucoside and diglucoside anthocyanins. Aging in wooden barrels induces oxygen permeation into wine, but little is known about diglucoside anthocyanin evolution. Cabernet cortis wine was subjected to addition of oxygen and oak chips, and the anthocyanin changes were followed for 1 month. Decreases of 90% total monoglucosides, 80% acylated monoglucosides, 65% diglucosides, and 90% acylated diglucosides were observed. Monoglucosides formed pyranoanthocyanins, and the lower steric hindrance favored their polymerization with flavanols. Instead, the decrease in diglucosides was correlated to the number of hydroxyl groups of ring B, indicating the predominant oxidation of aglycones. However, three flavonol-anthocyanin-diglucoside derivatives named (epi)catechin-ethyl-Mv-dihexoside, (epi)catechin-ethyl-Pn-dihexoside, and (epi)catechin-Mv-dihexoside A-type were identified in wine for the first time. These research findings are useful for tuning suitable oenological practices to stabilize the color of these wines (type of barrel, aging times, oxygenation practices) and lower the malvin content, which currently is recommended by the OIV at a maximum of 15 mg/L and is a critical issue for their commercialization.

Study of Glycosidically Bound Volatile Precursors as Variety Markers to Reveal Not-Allowed Practices in White Wines Winemaking.

Liquid chromatography/high-resolution mass spectrometry (LC/HRMS) can provide identification of grape metabolites which are variety markers. White grapes are poorer in polyphenolics, and the main secondary metabolites which contribute the sensorial characteristics of wines are the glycosidically bound volatile precursors and their aglycones. The profiles of three white grape juices (Pinot grigio, Garganega, and Trebbiano) were characterized by LC/HRMS, and 70 signals of putative glycosidic terpenols, norisoprenoids, and benzenoids were identified. Four signals found only in Pinot grigio corresponded to a norisoprenoid hexose-hexose, 3-oxo-α-ionol (or 3-hydroxy-ß-damascone) rhamnosyl-hexoside, monoterpene-diol hexosyl-pentosyl-hexoside, and hexose-norisoprenoid; three signals were found only in Garganega (putative isopropyl alcohol pentosyl-hexoside, phenylethanol rhamnosyl-hexoside, and norisoprenoid hexose-hexose isomers), and a monoterpenol pentosyl-hexoside isomer only in Trebbiano. These variety markers were then investigated in juice blends of the three varieties. This approach can be used to develop control methods to reveal not-allowed grape varieties and practices in white wines winemaking.

The pivotal role of high-resolution mass spectrometry in the study of grape glycosidic volatile precursors for the selection of grapevines resistant to mildews.

A breeding program to produce new grape varieties tolerant to main vine fungal pathogens (Plasmopara viticola and Erysiphe necator) is carrying out by crossing Vitis vinifera cv. "Glera" with resistant genotypes such as "Solaris," "Bronner," and "Kunleany." Firstly, resistance gene-based markers analyses allowed the identification of five genotypes, which have inherited the resistance loci against mildews. To select those that also inherited the phenotype as close as possible to 'Glera' suitable to be introduced in the Prosecco wine production protocols, the grape glycosidic derivatives were studied by UHPLC/QTOF mass spectrometry. Targeted identification of the metabolites was performed using a database expressly constructed by including the glycosidic volatile precursors previously identified in grape and wine. A total of 77 glycosidic derivatives including many aroma precursors and some variety markers, were identified. Original resistant genotypes had distinct metabolomic profiles and different to 'Glera', while the crossings showed varying similarity degrees to V. vinifera parent. Findings demonstrated the Glera × Bronner and Glera × Solaris crossings are more suitable to produce high-sustainable Prosecco wines. Coupling of glycosidic volatile precursors profiling to multivariate statistical analysis was effective for phenotypic characterization of grapes and to evaluate their enological potential.

Mass spectrometry in the study of wood compounds released in the barrel-aged wine and spirits.

Aging of wines and spirits in wooden barrels is an industrial process used to stabilize the color, to improve the limpidity and to enrich the sensorial characteristics of the products. In red wines, the oxygen that permeates through the wood staves promotes the oxidization of polyphenols and the formation of new pigments with consequent stabilization of the wine color. Barrel aging of spirits, such as brandy, whisky, rum, and grappa is finalized to enrich their aroma and improve their sensorial characteristics by the contribute of the compounds released by the wood. Oak is the wood type mostly used in making barrels; however, an increasing interest in the use of chestnut, cherry, acacia, and in less extent, ash and mulberry, has been observed in the recent years. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry are the main techniques used to characterize respectively the volatile and polar metabolites released by the wood barrels in the products. In this article are reported the recent advancements in this field.

First investigation on polyphenols and glycosidic aroma precursors in a spontaneous colour mutant of 'Glera', the principal grape variety of Prosecco sparkling wine.

BACKGROUND: Somatic mutations in Vitis spp. are relatively frequent and can generate new agronomically interesting phenotypes. We report the discovery, genetic and chemical characterization of 'Glera rosa', a mutant for the berry skin colour of 'Glera', the main white cultivar used to produce Prosecco wine. RESULTS: We ascertained the relationship between the skin colour of 'Glera rosa' and the polymorphisms in the Myb-gene transcription factors involved in polyphenol biosynthesis. We established that VvMybA1 was homozygous (VvMybA1a/VvMybA1a) in 'Glera' but heterozygous (VvmybA1a/VvmybA1b) in the 'Glera rosa' mutant. We verified that the VvMybA1a non-functional allele contained Grapevine Retrotransposon 1 (Gret1), while in the VvmybA1b allele Gret1 was missing, and the gene function was partially restored. The effects of mutation on 'Glera rosa' grape metabolites were studied by high-resolution mass spectrometry and gas chromatography/mass spectrometry analysis. Fifteen anthocyanins and five unique flavonols were found in the 'Glera rosa' mutant. The mutation also increased the contents of trans-resveratrol and its derivatives (i.e., piceatannol, E-ε-viniferin, cis- and trans-piceid) and of some flavonols in grape. Finally, the mutation did not significantly affect the typical aroma precursors of Glera grape such as glycosidic monoterpenes, norisoprenoids and benzenoids. CONCLUSION: 'Glera rosa' could be an interesting genetic source for the wine industry to produce Prosecco DOC rosé typology (made by adding up to 15% of 'Pinot Noir'), which was introduced to the market in 2020 with a worldwide massive success. © 2022 Society of Chemical Industry.

Influence of Non-Saccharomyces on Wine Chemistry: A Focus on Aroma-Related Compounds.

Wine fermentation processes are driven by complex microbial systems, which comprise eukaryotic and prokaryotic microorganisms that participate in several biochemical interactions with the must and wine chemicals and modulate the organoleptic properties of wine. Among these, yeasts play a fundamental role, since they carry out the alcoholic fermentation (AF), converting sugars to ethanol and CO2 together with a wide range of volatile organic compounds. The contribution of Saccharomyces cerevisiae, the reference organism associated with AF, has been extensively studied. However, in the last decade, selected non-Saccharomyces strains received considerable commercial and oenological interest due to their specific pro-technological aptitudes and the positive influence on sensory quality. This review aims to highlight the inter-specific variability within the heterogeneous class of non-Saccharomyces in terms of synthesis and release of volatile organic compounds during controlled AF in wine. In particular, we reported findings on the presence of model non-Saccharomyces organisms, including Torulaspora delbrueckii, Hanseniaspora spp,Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia spp. and Candida zemplinina, in combination with S. cerevisiae. The evidence is discussed from both basic and applicative scientific perspective. In particular, the oenological significance in different kind of wines has been underlined.

Elucidations on the structures of some putative flavonoids identified in postharvest withered grapes (Vitis vinifera L.) by quadrupole time-of-flight mass spectrometry.

Grape dehydration is an oenological process used for production of high-quality reinforced and sweet wines. High-resolution mass spectrometry (ultrahigh-performance liquid chromatography/quadrupole time-of-flight [UHPLC/QTOF]) was used to deepen the characterization of some flavonoids previously proposed in Corvina and Raboso Piave withered grapes. By performing a targeted data analysis workflow and orthogonal identification approaches (tandem mass spectrometry [MS/MS]), in silico fragmentation, and calculation of putative retention time), elucidation on the structures of six compounds previously proposed was achieved (taxifolin-pentoside, two tetrahydroxyflavanone-hexoside derivatives, a tetrahydroxy-dimethoxyflavanone-hexoside derivative, a pentahydroxyflavone, and peonidin-O-pentoside); and the structures of four putative new grape flavonoids were characterized (dihydromyricetin-O-hexoside, taxifolin-di-O-hexoside, isorhamnetin, and a pinoquercetin isomer). Findings enlarge the panorama of flavonoids in grape and of their possible biosynthetic pathways.

Effect of pre-bloom leaf removal on grape aroma composition and wine sensory profile of Semillon cultivar.

BACKGROUND: Early leaf removal at pre-bloom is an innovative viticultural practice for regulating yield components and improving grape quality. The effects of this technique on vine performance, grape composition and wine sensory profile of Semillon variety were assessed. RESULTS: Pre-bloom leaf removal enhanced canopy porosity, total soluble solids in musts and reduced cluster compactness. This practice had a strong effect on glycoside aroma precursors, in particular by increasing glycoside terpenols and norisoprenoids. Metabolites of linalool were the most responsive to leaf removal. Wine produced from defoliated vines was preferred in tasting trials for its more intense fruity notes and mouthfeel attributes. CONCLUSION: Pre-bloom leaf removal is a powerful technique for modifying canopy microclimate, vine yield, grape composition and wine quality. The increase of glycoside aroma compounds in treated grapes has potential positive effect in improving the sensory profile of the resulting wines. © 2017 Society of Chemical Industry.

Changes in grape polyphenols (V. vinifera L.) as a consequence of post-harvest withering by high-resolution mass spectrometry: Raboso Piave versus Corvina.

Grape dehydration is an oenological process used for the production of high-quality reinforced and sweet wines. Corvina and Raboso Piave are two red grape varieties used for production of high-quality Italian wines, such as Recioto, Amarone di Valpolicella and Raboso Passito. Changes of polyphenolic composition of the grapes as a consequence of the withering were studied by ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC/QTOF); for identification of compounds a homemade HR-MS database of grape and wine metabolites, was used. Concomitant with trans-resveratrol and viniferins, relevant increases of other stilbenes (piceatannol, resveratrol trimers and tetramers) and antioxidant compounds (quercetin, syringetin and tamarixetin) were observed. These compounds are part of the induced metabolism occurring during the withering process and in general improve the nutraceutical properties of grapes and wines. On the other hand, longer processes showed to decrease flavan-3-ols and glycoside flavonols. Constant increase of E/Z ε-viniferin ratio was observed in all samples, and this parameter can be used to monitor the process. Copyright © 2016 John Wiley & Sons, Ltd.

Characterization of Non-Anthocyanic Flavonoids in Some Hybrid Red Grape Extracts Potentially Interesting for Industrial Uses.

Previous studies showed that hybrid grapes often have qualitatively and quantitatively higher polyphenolic contents than the common V. vinifera grape varieties. In general, these compounds are studied for grape chemotaxonomy and for nutraceutical purposes due to their relevant antioxidant activity. Non-anthocyanic flavonoid composition of five red hybrid grape varieties produced by crossing of V. vinifera, V. aestivalis, V. cinerea, V. berlandieri, V. labrusca, V. lincecumii, and V. rupestris were studied by liquid chromatography/high-resolution mass spectrometry. Thirty-one compounds were identified, including methylnaringenin, a tetrahydroxy-dimethoxyflavanone-hexoside, two flavonols (quercetin and a pentahydroxyflavone isomer), 20 glycoside flavonols (four quercetin, two myricetin, two kaempferol, three isorhamnetin, one laricitrin, two syringetin, one kaempferide and two dihydroflavonol derivatives; myricetin-glucoside-glucuronide; myricetin-diglucoside; syringetin-dihexoside), three flavan-3-ols (-)-epicatechin, (+)-catechin, (-)-epicatechin gallate) and four proantocyanidins (procyanidin B1, procyanidin B2, procyanidin B3 or B4/B5, procyanidin T2 or T3/T4/C1). Seibel 19881, Seyve Villard 12-347 and Seyve Villard 29-399 were particularly rich in polyphenols. These findings emphasize that these grapes are especially interesting for the production of antioxidant extracts for nutraceutical and pharmaceutical uses.

Putative identification of new p-coumaroyl glycoside flavonoids in grape by ultra-high performance liquid chromatography/high-resolution mass spectrometry.

RATIONALE: Grape polyphenols are antioxidant compounds, markers in vine chemotaxonomy, and involved in color stabilization of red wines. Sugar acylation usually confers higher stability on glycoside derivatives and this effect is enhanced by an aromatic substituent such as p-coumaric acid. Until now, only p-coumaroyl anthocyanins have been found in grape. METHODS: A method of 'suspect screening analysis' by ultra-high-performance liquid chromatography/high-resolution mass spectrometry (UHPLC/QTOFMS) has recently been developed to study grape metabolomics. In the present study, this approach was used to identify new polyphenols in grape by accurate mass measurement, MS/MS fragmentation, and study of correlations between fragments observed and putative structures. RESULTS: Three putative p-coumaroyl flavonoids were identified in Raboso Piave grape extract: a dihydrokaempferide-3-O-p-coumaroylhexoside-like flavanone, isorhamnetin-3-O-p-coumaroylglucoside, and a chrysoeriol-p-coumaroylhexoside-like flavone. Accurate MS provided structural characterization of functional groups, and literature data indicates their probable position in the molecule. A fragmentation scheme is proposed for each compound. CONCLUSIONS: Compounds were identified by overlapping various analytical methods according to recommendations in the MS-based metabolomics literature. Stereochemistry and the definitive position of substituents in the molecule can only be confirmed by isolation and characterization or synthesis of each compound. These findings suggest addressing research of acylated polyphenol glycosides to other grape varieties.

Study of combined effect of proteins and bentonite fining on the wine aroma loss.

The wine aroma loss as a consequence of treatments with bentonite is due to the occurrence of multiple interaction mechanisms. In addition to a direct effect of bentonite, the removal of aroma compounds bound to protein components adsorbed by the clay has been hypothesized but never demonstrated. We studied the effect of bentonite addition on total wine aroma compounds (extracted from Moscato wine) in a model solution in the absence and presence of total and purified (thaumatin-like proteins and chitinase) wine proteins. The results showed that in general bentonite alone has a low effect on the loss of terpenes but removed ethyl esters and fatty acids. The presence of wine proteins in the solution treated with bentonite tended to increase the loss of esters with the longest carbon chains (from ethyl octanoate to ethyl decanoate), and this was significant when the purified proteins were used. The results here reported suggest that hydrophobicity can be one of the driving forces involved in the interaction of aromas with both bentonite and proteins.

Applications of solid-phase microextraction and gas chromatography/mass spectrometry (SPME-GC/MS) in the study of grape and wine volatile compounds.

Volatile compounds are responsible for the wine "bouquet", which is perceived by sniffing the headspace of a glass, and of the aroma component (palate-aroma) of the overall flavor, which is perceived on drinking. Grape aroma compounds are transferred to the wine and undergo minimal alteration during fermentation (e.g., monoterpenes and methoxypyrazines); others are precursors of aroma compounds which form in winemaking and during wine aging (e.g., glycosidically-bound volatile compounds and C13-norisoprenoids). Headspace solid phase microextraction (HS-SPME) is a fast and simple technique which was developed for analysis of volatile compounds. This review describes some SPME methods coupled with gas chromatography/mass spectrometry (GC/MS) used to study the grape and wine volatiles.

Profiling of grape monoterpene glycosides (aroma precursors) by ultra-high performance-liquid chromatography-high resolution mass spectrometry (UHPLC/QTOF).

A 'suspect screening analysis' method for grape metabolomics by ultra-high performance-liquid chromatography (UHPLC) and high-resolution quadrupole-time of flight (QTOF) mass spectrometry was recently developed. This method was applied to study grape monoterpene glycosides, the main grape aroma precursors. Since standard compounds were not available, they were tentatively identified by overlapping various analytical approaches, in agreement with the indications recommended in mass spectrometry (MS)-based metabolomics. Accurate mass and isotopic pattern, MS/MS fragmentation, correlation between fragments observed and putative structures and between liquid chromatography coupled with mass spectrometry (LC/MS) and gas chromatography/mass spectrometry signals were studied. Seventeen monoterpene glycosides were identified without performing the hydrolytic artifacts commonly used to study these compounds which may affect sample profile. This is the first time that a detailed study of these aroma precursors has been carried out by direct LC/MS analysis.

Identification of saffron aroma compound ß-isophorone (3,5,5-trimethyl-3-cyclohexen-1-one) in some V. vinifera grape varieties.

Carotenoid-derived aroma compounds play an important role in the composition of aroma and grapes, and consequently of wine. The volatile composition of forty-five grape varieties harvested in 2011 and 2012 was investigated by solid phase extraction of samples and gas chromatography-mass spectrometry (GC-MS) analysis. In grape extracts of cultivars Barbera, Refosco dal Peducolo Rosso, Ribolla gialla and Rossese, the C9-norisoprenoid compound ß-isophorone (3,5,5-trimethyl-3-cyclohexen-1-one) was found and quantified. This carotenoid-derived compound is characterised by a saffron aroma and is here reported in grape for the first time.

Seed oil triglyceride profiling of thirty-two hybrid grape varieties.

Triglyceride profile of seed oil samples from 32 hybrid grape varieties not studied before was investigated. A new method for the analysis of triacylglycerols (TAGs) has been developed based on the direct infusion in the electrospray ionization (ESI) source and employing tetrahydrofuran/methanol/water (85:10:5 v|v|v) as solvent; the formation of [M + Na](+) ions in high yield has been observed. TAGs were identified by ESI-tandem mass spectrometry analysis, and the matrix-assisted-laser-desorption-ionization and time-of-flight profile of samples was determined. Six were the principal TAGs identified in seed oil: trilinolein (LLL) was the most abundant (43%), followed by dilinoleoyl-oleoylglycerol (LOL, 23%), and dilinoleoyl-palmitoylglycerol (LPL, 15%). Compounds present in lower concentration were LSL and LOO (11%), LOP (6%), and LSP (2%). Compared with seed oils produced from V. Vinifera grapes, some significant differences in the relative abundances of TAGs were found, in particular hybrid grape seed oils showed higher LOL and lower LPL content, respectively. Among the samples studied, a particularly high content of LLL (rich in unsaturated fatty acids) was found in seed oils from two red varieties.

A fast and selective method for anthocyanin profiling of red wines by direct-infusion pneumatic spray mass spectrometry.

RATIONALE: Anthocyanins confer the color to red wines. During wine aging, new anthocyanin derivatives with peculiar chromatic characteristics are formed. A fast and selective method for screening the anthocyanin composition of wine has been developed. METHODS: Direct-infusion electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of wine extracts has been performed by operating in positive ion mode with a low spray capillary voltage (0.5 kV) to detect only the species already present in ionic form. The method has been called direct-infusion pneumatic spray (DIPS) mass spectrometry. The anthocyanin profiles of thirteen red wines at different aging stages were studied. Using malvidin-3,5-diglucoside as internal standard, the indices of wine color and of wine color evolution were estimated. RESULTS: By decreasing the spray capillary voltage a dramatic increase in selectivity toward anthocyanins has been observed and the related peaks are the most abundant in the spectra. Several anthocyanins and anthocyanin derivatives (pyranoanthocyanins and derivatives formed by reaction with flavan-3-ols) have been identified in the wines. The younger wines showed higher total anthocyanin index. The aged wines showed color evolution index higher than 20% while it was lower in the younger samples. CONCLUSIONS: The anthocyanin fingerprint is effective for estimating the aging state of wine. The color evolution index can be correlated to barrel conditions during wine aging and used to distinguish between aged and younger wines. The study of pyranoanthocyanins and anthocyanin-flavanol derivatives can be useful to predict wine color stability.

Chemical characterization and enological potential of Raboso varieties by study of secondary grape metabolites.

DNA studies supported by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of seed proteins showed that the Vitis vinifera red grape variety Raboso Veronese is the progeny of a spontaneous cross between Raboso Piave (red) and Marzemina Bianca (white) varieties. In the present work, the main secondary grape metabolites of Raboso varieties were studied, and their enological potential was evaluated and compared with that of other red varieties reported in the literature. In general, Raboso grapes had high flavonoid contents and high percentages of polyphenols extractable in winemaking and substantial contents of norisoprenoid aroma precursors. Raboso Veronese stood out for its high content of cyanidin and had higher (+)-catechin and (-)-epicatechin contents than Raboso Piave and abundant quercetin glucoside, indicating substantial plant biosynthesis toward compounds dihydroxylated in the B-ring. Study of secondary grape metabolites is confirmed as an effective tool in differentiating similar varieties, in particular on the basis of polyphenol profiles.

A solid-phase microextraction gas chromatography/ion trap tandem mass spectrometry method for simultaneous determination of 'foxy smelling compounds' and 3-alkyl-2-methoxypyrazines in grape juice.

2'-Aminoacetophenone (o-AAP) was identified as the main cause of the aging note called 'hybrid note' or 'foxy smell' which is typical of non-Vitis vinifera grapes. Together with methyl anthranilate (MA), this compound contributes to the typical foxy taint of wines made with non-Vitis vinifera grapes. 3-Alkyl-2-methoxypyrazines in grapes, with their herbaceous note and very low sensory thresholds, contribute to the aroma of several wines. In this study, a solid-phase microextraction gas chromatography/ion trap tandem mass spectrometry (SPME-GC/IT-MS/MS) method for simultaneous detection of o-AAP, MA and ethyl-, isopropyl-, sec-butyl- and isobutylmethoxypyrazine in grape juice was developed. The method was applied to the study of several grape juices: the time required for analysis was less than 24 min, and the method was considered to be suitable for analysis of 'foxy compounds' and methoxypyrazines in grape juice. The high levels of MA and o-AAP found in Clinton and Siebel grapes confirmed the 'hybrid' character of these varieties.

Fast analysis of isobaric grape anthocyanins by Chip-liquid chromatography/mass spectrometry.

In a previous work, direct-infusion electrospray ionization ion trap tandem mass spectrometry (ESI-IT-MS/MS) was applied to the study of anthocyanins in extracts from the skins of Clinton grapes, a non-Vitis vinifera red grape variety qualitatively and quantitatively rich in anthocyanins. A good characterization of anthocyaninins was obtained, but it was impossible to differentiate some compounds with the same nominal mass but with different elemental composition. In this work, the capabilities of quadrupole time-of-flight mass spectrometry (QTOF-MS) coupled with Chip-liquid chromatography (LC-Chip) were applied to the study of Clinton anthocyanins and this method provided the complete sample anthocyanin fingerprint in less than 5 min. Multi-stage mass spectrometry (MS(n); n >2) was not necessary to identify isobaric compounds, nor were deuterium-exchange experiments necessary to distinguish between compounds containing the same aglycone. The fast separation bypasses the problem of petunidin-3-O-(6-O-acetyl)monoglucoside and delphinidin-3,5-O-diglucoside quantification, present in the direct-infusion ESI-ITMS approach, due to overlapping with matrix interferences.