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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. more...

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-β-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. more...

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., (© 2023 John Wiley & Sons Ltd.) more...

The present study aims to both identify and quantify trans-sinapoylquinic acid (SiQA) regioisomers in green coffee by combined UHPLC-ESI-QqTOF-MS/MS and UHPLC-ESI-QqQ-MS/MS methods. Among the various mono-acyl chlorogenic acids found in green coffee, SiQA regioisomers are the least studied despite having been indicated as unique phytochemical markers of Coffea canephora (known as Robusta). The lack of commercially available authentic standards has been bypassed by resorting to the advantages offered by high-resolution LC-MS as far as the identification is concerned. SiQA regioisomers have been identified in several samples of Robusta and Coffea arabica (known as Arabica) commercial lots from different geographical origin and, for the first time, in different samples of coffee wild species (Coffea liberica and Coffea pseudozanguebariae). Quantification (total SiQA ranging from 3 to 5 mg/100 g) let to reconsider these chlorogenic acids as unique phytochemical markers of Robusta being present in the same quantity and distribution in C. liberica as well. Gardeniae Fructus samples (fruits of Gardenia jasminoides) have additionally been characterized as this matrix is recognized as one of the few naturally occurring SiQA sources. The SiQA regioisomer content (total SiQA about 80 mg/100 mg) fully supports the proposal to use this matrix as a surrogate standard for further studies., (© 2023 John Wiley & Sons Ltd.) more...

Introduction: Prosecco wine production has been strongly extended in the last decade and several new clones have been introduced. "Glera" (minimum 85%) and "Glera lunga" are grape varieties of great economic impact used to produce Prosecco wines. Study of grape berry secondary metabolites is effective in the classification of vine varieties and clones. High-resolution mass spectrometry provides complete panorama of these metabolites in single analysis and coupling to statistical multivariate analysis is successfully applied in vine chemotaxonomy., Objectives: update and deepen the knowledge on the "Glera" and "Glera lunga" berry grapes chemotaxonomy and investigate some of the most produced and marketed clones by using the modern analytical and statistical tools., Methods: five clones of "Glera" and two of "Glera lunga" grown in the same vineyard with same agronomical practices were studied for three vintages. Grape berry metabolomics was characterized by UHPLC/QTOF and multivariate statistical analysis was performed on the signals of main metabolites of oenological interest., Results: "Glera" and "Glera lunga" showed different monoterpene profiles ("Glera" is richer in glycosidic linalool and nerol) and differences in polyphenols (catechin, epicatechin and procyanidins, trans-feruloyltartaric acid, E-ε-viniferin, isorhamnetin-glucoside, quercetin galactoside). Vintage affected the accumulation of these metabolites in berry. No statistical differentiation among the clones of each variety, was found., Conclusions: Coupling HRMS metabolomics/statistical multivariate analysis enabled clear differentiation between the two varieties. The examined clones of same variety showed similar metabolomic profiles and enological characteristics, but vineyard planting using different clones can result in more consistent final wines reducing the vintage variability linked to genotype × environment interaction., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.) more...

Grapes represent a significant source of phenolic compounds known for their health-promoting properties, such as antioxidant capacity on normal cells and prooxidant activity on tumor cells. The genotype highly affects the polyphenolic composition in grapes and, consequently, the nutritional quality of berries. This work aimed to characterize the phenolic composition, the antioxidant, and anticancer activity of grape skin extracts (GSEs) of nine new table grape genotypes selected from a breeding program to obtain new cultivars of seedless table grapes, well adapted to the climatic change and with higher nutraceutical properties. The grape polyphenolic profile was characterized by Ultra-High-Performance Liquid Chromatography/Quadrupole-Time of Flight mass spectrometry analysis. GSE antioxidant activity was determined by the ABTS, DPPH, and ORAC assays; GSE cell growth inhibition test was carried out in the Caco2 human cancer cell line. The nine GSEs showed different flavonoid and non-flavonoid profiles, and all possessed antioxidant activity, with the 'Aika N.', 'Turese N.', and 'Egnatia N.' the most active. As anticancer activity against the tested cancer cell line, 'Daunia N.' and 'Apenestae N.' showed the EC50 after 24 h of 35.60 µg/mL and 150.91 µg/mL, respectively. The relationship between polyphenolic profile and the antioxidant and anticancer activity of GSE was also investigated. Interestingly, among the different classes of polyphenolics, flavan-3-ols e proanthocyanidins showed the highest positive correlation with the anticancer activity of extracts. These findings can be helpful for the preparation of new extracts for the pharmaceutical and nutraceutical industry and geneticists working in vine breeding programs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Milella, De Rosso, Gasparro, Gigante, Debiase, Forleo, Marsico, Perniola, Tutino, Notarnicola, Velasco and Flamini.) more...

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., (© 2022 Society of Chemical Industry.) more...

Introduction: The biochemical diversity of flavonoids is based on glycosylation, methylation, acylation, and many other modifications of the flavonoid backbone. Liquid chromatography coupled to high-resolution mass spectrometry demonstrated to be a powerful approach to gain new insights into the flavonoid composition of many plant species, including grapes., Objectives: Among different metabolomic approaches, suspect screening analysis relies on the construction of a specific database and on ultra-high performance liquid chromatography/quadrupole time-of-flight (UHPLC/QTOF) analysis to find new compounds of oenological interest., Methods: A homemade database containing mass data information retrieved from the literature specific for plant flavonoid derivatives (GrapeFlavMet) was constructed. Tandem mass spectrometry analysis of V. vinifera and hybrid grape extracts was performed, and MS/MS fragmentation allowed to assign the putative flavonoid chemical structure to various identification levels, as established by the Metabolomics Standard Initiative., Results: By this approach, putative flavonoid derivatives with different glycosylation and acylation patterns were identified. They include three pentoside derivatives of tetrahydroxy-flavone, tetrahydroxy-flavanone and myricetin isomers, a putative dihydrorhamnetin hexoside derivative, three cinchonain isomers (phenylpropanoid-substituted flavan-3-ols with antidiabetic properties), and two syringetin isomer derivatives (acetyl- and p-coumaroyl-hexoside). Two acetyl-hexoside derivatives of dihydrorhamnetin and pentahydroxy-methoxy-flavanone, and three derivatives of tetrahydroxy-dimethoxy-flavanone (acetyl, p-coumaroyl, and caffeoyl-hexoside) were tentatively annotated., Conclusions: Most of the compounds were identified in grape for the first time, while two putative syringetin derivatives previously proposed in the literature were confirmed. These findings deepen the current knowledge on grape flavonoids, suggesting more connections at the biochemical level., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.) more...

Grape aroma precursors have been extensively studied and many glycosidically-bound terpenols and C 13 -norisoprenoids were identified. Instead, these compounds were scarcely investigated in green Coffea arabica where just few glycosidic compounds were identified so far. By resorting to knowledge of glycoside aroma precursors in grape and the possibility to identify their structures using a high-resolution mass spectrometry database constructed for grape metabolomics, targeted investigation of glycoside precursors in green C. arabica from different geographical origins, was performed. High linalool hexose-pentose was found in all the investigated samples and hexosyl-pentoside derivatives of geraniol, linalooloxide and another linalool isomer, were identified. Moreover, two putative norisoprenoid glycosides were characterized. β-Damascenone was detected in the volatile fraction of the examined C. arabica coffees only after acid addition, however no signals of β-damascenone glycosides, were found. Findings suggests that this important aroma compound could form by hydrolysis and dehydration of a putative 3-hydroxy-β-damascone glycoside precursor identified for the first time in coffee. Aglycones released during the roasting process contribute to enrich the coffee aroma with their positive sensory notes and the identification of these glycosides can contribute to disclose the coffee biology including biochemical, physiological and genetic aspects., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.) more...

Along with the ongoing climate change, drought events are predicted to become more severe. In this context, the spray-induced gene silencing (SIGS) technique could represent a useful strategy to improve crop stress resilience. A previous study demonstrated that the Arabidopsis mutants for a glutathione S-transferase (GST) gene had increased abscisic acid (ABA) levels and a more activated antioxidant system, both features that improved drought resilience. Here, we used SIGS to target a putative grape GST gene (VvGST40). Then, ecophysiological, biochemical and molecular responses of 'Chardonnay' cuttings were analysed during a drought and recovery time-course. Gas exchange, ABA and t-resveratrol concentration as well as expression of stress-related genes were monitored in not treated controls, dsRNA-VvGST40- and dsRNA-GFP- (negative control of the technique) treated plants, either submitted or not to drought. VvGST40-treated plants revealed increased resilience to severe drought as attested by the ecophysiological data. Analysis of target metabolites and antioxidant- and ABA-related transcripts confirmed that VvGST40-treated plants were in a priming status compared with controls. SIGS targeting an endogenous gene was successfully applied in grapevine, confirming the ability of this technique to be exploited not only for plant protection issues but also for functional genomic studies., (© 2021 John Wiley & Sons Ltd.) more...

Phenolics are ubiquitous compounds that represent the most abundant and diverse class of plant metabolites. From an analytical point of view, phenolics can be divided into soluble phenolics such as phenolic acids, phenylpropanoids, flavonoids and quinones, and nonsoluble compounds such as proanthocyanidins, lignins, and cell wall-bound hydroxycinnamic acids. Extraction of phenolics from the sample material is the first step toward their analysis. Biochemical methods for determination of total phenolics content were widely used in the past but modern chromatographic and mass spectrometric methods for identification and quantification of individual compounds are available in recent years. In this chapter, we describe methods for phenolic compounds extraction used in our laboratories from berries of Vitis vinifera and analytical methods including HPLC coupled to DAD detector and Q-TOF LC/MS for their analysis., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.) more...

In the Valpolicella area (Verona, Italy) Vitis vinifera cv. Corvina is the main grape variety used to produce Amarone wine. Before starting the winemaking process, the Corvina grapes are stored in a withering (i.e., dehydrating) warehouse until about 30% of the berry weight is lost (WL). This practice is performed to concentrate the metabolites in the berry and enrich the Amarone wine in aroma and antioxidant compounds. In compliance with the guidelines and strict Amarone protocol set by the Consorzio of Amarone Valpolicella, withering must be carried out by setting the grapes in a suitable environment, either under controlled relative air humidity (RH) conditions and wind speed (WS)-no temperature modification is to be applied-or, following the traditional methods, in non-controlled environmental conditions. In general, the two processes have different dehydration kinetics due to the different conditions in terms of temperature, RH, and WS, which affect the accumulation of sugars and organic acids and the biosynthesis of secondary metabolites such as stilbenes and glycoside aroma precursors. For this study, the two grape-withering processes were carried out under controlled (C) and non-controlled (NC) conditions, and the final compositions of the Corvina dried grapes were compared also to evaluate the effects on the organoleptic characteristics of Amarone wine. The findings highlighted differences between the two processes mainly in terms of the secondary metabolites of the dried grapes, which affect the organoleptic characteristics of Amarone wine. Indeed, by the sensory evaluation, wines produced by adopting the NC process were found more harmonious, elegant, and balanced. Finally, we can state how using a traditional system, grapes were characterised by higher levels of VOCs (volatile compounds), whilst wines had a higher and appreciable complexity and finesse. more...

The Italian grape pomace distillate grappa is often refined by ageing in wooden barrels. Chemical changes of the volatile profile of two samples produced from a Cabernet Sauvignon/Merlot blend and Prosecco pomace both at two ethanol contents (55% and 68%) were studied during one-year ageing in cherry and oak barriques. Grappa variety, ethanol content and barrel type strongly affected the volatiles profile of the distillates. Oak-aged grappa contained 10-fold the wood volatiles of the cherry-aged one, but the latter had higher levels of syringaldehyde, coniferaldehyde, and 3,4,5-trimethoxyphenol; 55% ethanol extracted higher levels of wood compounds and ethoxy-compounds were higher in the 68%-ethanol distillates. Prosecco grappa extracted higher wood compounds and showed no significant changes in the levels of fruity/floral esters and terpenols. Findings of this study can be also useful in the development of new ageing processes of distillates for which cherry barrels are still not used (e.g., brandy and whisky)., (Copyright © 2020 Elsevier Ltd. All rights reserved.) more...

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., (© 2020 John Wiley & Sons, Ltd.) more...

Grapes contain many flavonoid and non-flavonoid compounds with anticancer effects. In this work we fully characterized the polyphenolic profile of two grape skin extracts (GSEs), Autumn Royal and Egnatia, and assessed their effects on Polyunsaturated Fatty Acid (PUFA) membrane levels of Caco2 and SW480 human colon cancer cell lines. Gene expression of 15-lipoxygenase-1 (15-LOX-1), and peroxisome proliferator-activated receptor gamma (PPAR-γ), as well as cell morphology, were evaluated. The polyphenolic composition was analyzed by Ultra-High-Performance Liquid Chromatography/Quadrupole-Time of Flight mass spectrometry (UHPLC/QTOF) analysis. PUFA levels were evaluated by gas chromatography, and gene expression levels of 15-LOX-1 and PPAR-γ were analyzed by real-time Polymerase Chain Reaction (PCR). Morphological cell changes caused by GSEs were identified by field emission scanning electron microscope (FE-SEM) and photomicrograph examination. We detected a different profile of flavonoid and non-flavonoid compounds in Autumn Royal and Egnatia GSEs. Cultured cells showed an increase of total PUFA levels mainly after treatment with Autumn Royal grape, and were richer in flavonoids when compared with the Egnatia variety. Both GSEs were able to affect 15-LOX-1 and PPAR-γ gene expression and cell morphology. Our results highlighted a new antitumor mechanism of GSEs that involves membrane PUFAs and their downstream pathways. more...