Raboso Piave Research Articles

Decoding the Proanthocyanins Profile of Italian Red Wines

The Italian wine appellations system is organized in hundreds of origin wines, with unique characteristics that are protected with many denominations of origin. The aim of this work was to analyze and compare the proanthocyanin profile of 12 single-cultivar and single-vintage Italian red wine groups (Aglianico from Campania, Cannonau from Sardinia, Corvina from Veneto, Montepulciano from Abruzzo, Nebbiolo from Piedmont, Nerello Mascalese from Sicily, Primitivo from Apulia, Raboso Piave from Veneto, Sagrantino from Umbria, Sangiovese from Tuscany and Romagna, and Teroldego from Trentino), each one produced in their terroirs under ad hoc legal frameworks to guarantee their quality and origin. All wines were analyzed with a protocol that combined the phloroglucinolysis reaction with an LC-MS/MS instrument. The results underlined Sagrantino wines as the richest in proanthocyanins. Sangiovese, Montepulciano, Nerello, and Teroldego were the richest in B-ring trihydroxylated flavan-3-ols, and especially Nerello was the richest in prodelphinidins. Cannonau, Raboso Piave, Nerello, and Corvina were characterized by C-ring trans conformation flavan-3-ols. Nebbiolo and Corvina had high percentages of galloylated flavan-3-ols. Aglianico and Primitivo had the lowest percentages of B-ring trihydroxylated and C-ring trans conformation flavan-3-ols. This information should be useful in better understanding the Italian red wines and valorize them.

The macromolecular diversity of Italian monovarietal red wines

While red wine phenolics have been extensively studied, polysaccharides and proteins have not received the same level of attention, especially when considering Italian wines. In this study, for the first time, quantitative and qualitative data on the macromolecular (proteins and polysaccharides) and tannin composition of 110 monovarietal red wines from 11 of the most important Italian grape varieties are reported. The winemaking did not include any filtration, oak contact, fining treatments, or ageing on yeast lees. Results highlighted a great inter- and intra- varietal diversity. The protein content ranged between 0 and 159 mg/L, polysaccharides between 211 and 1081 mg/L and total tannins between 171 and 3746 mg/L, with averages of 41 mg/L, 497 mg/L and 1687 mg/L, respectively. Six varieties with protein content representative of the variability observed were selected and submitted to electrophoresis. Within each variety, the SDS-PAGE mobility of protein-tannin complexes was similar but showed two distinct patterns for wines of different varieties (higher mobility for Corvina, Teroldego and Raboso Piave, lower mobility for Nebbiolo, Aglianico, Cannonau), suggesting that the Italian monovarietal wines can be diverse also in their colloidal-forming structures. This can be explained by looking at the different percentages of protein-reactive tannins (TBSA) on the total tannin content (TMCP), which is a varietal characteristic.

Diversity of Italian red wines: A study by enological parameters, color, and phenolic indices

An extensive survey was conducted on 110 Italian monovarietal red wines from a single vintage to determine their standard compositional, color, and phenolic characteristics, analysing more than 35 parameters evaluated through methods commonly used in the wine industry. ‘Primitivo’ achieved the highest average alcohol strength (15.4% v/v) and dry extract values, while ‘Cannonau’ showed the lowest total acidity. ‘Corvina’ had the lowest phenolic content (1065 mg/L by Folin-Ciocalteu assay), remarkably different from the highest found in ‘Sagrantino’ (3578 mg/L), the latter being also the richest variety in both proanthocyanidins and vanillin-reactive flavanols. ‘Teroldego’ wines were the richest in both total and monomeric anthocyanins (702 and 315 mg/L, respectively), followed by ‘Aglianico’ and ‘Raboso Piave’, while ‘Corvina’, ‘Nebbiolo’, and ‘Nerello Mascalese’ were the poorest. ‘Montepulciano’ and ‘Sangiovese’ showed intermediate values for the majority of the parameters analyzed. A multivariate PCA-DA approach allowed achieving both a classification of the different wines as well as the discrimination of ‘Sangiovese’ wines produced in two regions (Emilia Romagna and Toscana) that returned a 42–66% success rate depending on the zone considered. Taking into account the number and diversity of the wines analyzed, a correlation study helped in better understanding the underlying relations between the most common and widespread analytical techniques for phenolic and color determinations.

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.

Insights on the stilbenes in Raboso Piave grape (Vitis vinifera L.) as a consequence of postharvest vs on-vine dehydration.

Grape withering is a process used to produce reinforced wines and raisins. Dehydration is usually carried out postharvest by keeping ripe grapes in special warehouses in controlled conditions of temperature, relative humidity (RH) and air flow. Alternatively, grape clusters can be left on the vines after the canes have been pruned. In general, dehydration increases stilbenes in grape, but there are few studies on the effects of on-vine withering. The stilbene profiles of Raboso Piave grape during postharvest and on-vine dehydration were studied here. High-resolution mass spectrometry (MS) was used to identify 19 stilbenes, including resveratrol monomers, dimers (viniferins), oligomers and glucoside derivatives. The two dehydration methods generally had different effects on the above nutraceuticals in grape. The samples kept in warehouses revealed significant increases in Z-ω-viniferin, E-ϵ-viniferin, δ-viniferin and another resveratrol dimer which were not observed in the plants. Trans-Resveratrol increased significantly only in samples dehydrated in the warehouse at 21 °C and 60-70% RH. The findings increase knowledge of stilbene composition in grapes subjected to withering on-vine. The choice of dehydration method affects the contents of these nutraceuticals in the grape and consequently in wines. Reasonably, it could also affect other secondary metabolites important for wine quality. © 2017 Society of Chemical Industry.

Anthocyanin and flavonol composition response to veraison leaf removal on Cabernet Sauvignon, Nero d’Avola, Raboso Piave and Sangiovese Vitis vinifera L. cultivars

The elimination of a certain number of leaves around bunches before veraison is a common practice in vineyards to increase berries sunlight exposure, which, if acting in synergy with temperature increase, may affect grape anthocyanin and flavonol composition and give rise to contradictory results. The aim of this study was to analyze the effect over two years of leaf removal on anthocyanin and flavonol composition at harvest in four red Vitis vinifera L. varieties: Cabernet Sauvignon, Nero d’Avola, Raboso Piave and Sangiovese, characterized by different anthocyanin and flavonol profiles. The concentration of total anthocyanins in berries did not vary among control and defoliated vines in all varieties in both vintages, while total flavonols strongly increased after the treatment. Our results showed a genotype-dependent response to leaf removal that may induce a strong enhancement of the di-substituted branch of the flavonoid pathway, with consequences on anthocyanins and flavonols profile.

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.

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

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. 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. 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. 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 grape bioactive stilbenes by suspect screening metabolomics

Stilbenes are one of the main classes of grape polyphenols associated with the beneficial effects of drinking wine. In the present study, a suspect screening metabolomics approach was used to study these grape compounds. Analysis was performed by Ultra High Performance Liquid Chromatography/Quadrupole Time-Of-Flight (UHPLC/QTOF) mass spectrometry and compounds were identified using the database GrapeMetabolomics expressly constructed. By this approach, a total of 18 stilbene derivatives was identified in two V. vinifera red grape varieties (cvs. Raboso Piave and Primitivo) on the basis of accurate mass measurements and isotopic patterns. Identifications were confirmed by multiple mass spectrometry (MS/MS). Bioactive stilbenes such as trans-resveratrol, Z- and E-piceid, piceatannol and Z- and E-astringin, and several resveratrol dimers, trimers and tetramers such as pallidol and pallidol-3-O-glucoside, E- and Z--viniferin, E- and Z-δ-viniferin, caraphenol B, parthenocissin A, E- and Z-miyabenol C, hopheapenol, ampelopsin H, vaticanol C-like or isohopeaphenol, were identified. In our knowledge, some of these resveratrol trimers and tetramers were reported in grape for the first time.

An innovative approach to grape metabolomics: stilbene profiling by suspect screening analysis

Suspect screening analysis is a targeted metabolomics approach in which identification of compounds relies on specific available information such as their molecular formula and isotopic pattern. This method was applied to the study of grape metabolomics with an UPLC/MS high-resolution Q-TOF mass spectrometer (nominal resolution 40,000) coupled with a Jet Stream ionization source. The present paper describes the detailed qualitative and quantitative study of grape stilbenes, the principal polyphenols associated with the beneficial effects of drinking wine. For identification of compounds, a new database was expressly constructed from the molecular information of potential metabolites of grape and wine from the literature and other electronic databases. Currently, GrapeMetabolomics contains about a thousand putative grape compounds. If untargeted analysis of a sample provides identification of a new compound with a sufficiently confident score, it is added to the database. Thus, by increasing the number of samples studied, GrapeMetabolomics can be expanded. This method is effective for identification of the molecular formulae of several hundred metabolites in two runs (positive and negative ionization) with minimal sample preparation, and can also be used to analyse some single classes of compounds involved in cell and tissue metabolism. With this approach, a total of 18 stilbene derivatives was identified in two grape samples (Raboso Piave and Primitivo) on the basis of accurate mass measurements and isotopic patterns, and identification was confirmed by MS/MS analysis. The approach can also potentially be applied to the metabolomics of other plant varieties.

Sensorial, biochemical and molecular changes in Raboso Piave grape berries applying “Double Maturation Raisonnée” and late harvest techniques

At ripening, Vitis vinifera cv Raboso Piave grapes have high acidity, which results in an astringent wine that is not easy to drink. To overcome this limitation, several researches have attempted to alter the polyphenols profile mainly by applying different harvest techniques.The aim of this work was to investigate sensorial, biochemical, and molecular changes in Raboso Piave grape berries subjected to delayed harvests as Late Harvest (LH) and “Double Maturation Raisonnée” (DMR) techniques. At the molecular level, a microarray study was conducted comparing Traditional Harvest berries (TH) to LH and DMR ones. Gene ontology enrichment analysis pointed out that LH and DMR techniques affected metabolism of acids, sugars and polyphenols. A Principal Component Analysis, performed on transcriptomic data, pointed out that malate catabolism as well as some branches of flavonoids biosynthesis are significantly affected by DMR. In DMR grape berries, the flavonol and catechin accumulations were induced and depressed, respectively. In parallel, the transcription of flavonol synthase and leucoanthocyanidin-reductase 2, the main genes responsible for flavonol and catechin biosynthesis, were similarly induced and down-regulated. These changes resulted in a brighter colored wine with lower astringency.

Phenol compound metabolism and gene expression in the skin of wine grape (Vitis vinifera L.) berries subjected to partial postharvest dehydration

Specific polyphenol compound concentrations and gene expression patterns were determined by both microarray and qRT-PCR analyses in the epicarp of red-skinned grape berries (Vitis vinifera L. cv ‘Raboso Piave’) dehydrated, after harvest, at slow (S) and rapid (R) rates of up to 10 and 30% weight loss (WL). Increases in flavonols (quercetin) and trans-resveratrol concentrations were observed in the skins of all dehydrated samples, whereas flavan-3-ols concentrations showed a decreasing trend, which was more pronounced in S samples. The decrease in flavan-3-ol concentrations was paralleled by a reduction in procyanidin B1 and, particularly B2. Computational analysis of microarray data revealed that several key genes of the flavonoid pathways were unaffected or down-regulated during berry dehydration, with the exception of flavonol synthase, which was induced as well as one MybB transcription factor. Chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), leucoanthocyanidin dioxygenase (LDOX) and MybA were markedly down-regulated, particularly in relation to 30% WL, whereas UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) was unaffected in all the samples considered. Specific genes involved in the lignin biosynthetic pathway, including laccase, were induced in the most dehydrated samples. Considering the accumulation pattern and gene expression profiles, stilbenes and flavonols may represent useful biomarkers to monitor postharvest dehydration.

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.

Parental relationships among three grape varieties studied by MALDI of grape seed protein profiles

Two Raboso cultivars, i.e. Raboso Veronese and Raboso Piave, are two black Vitis vinifera varieties. A genetic study suggested that Raboso Veronese is the progeny of a spontaneous cross between Raboso Piave and Marzemina Bianca cultivars. Parental relationships are usually investigated by genetic studies, which are effective to establish genetic links among different cultivars. Considering that proteome is the genome expression, in this article we evaluated the power of seed protein profiles obtained by matrix-assisted laser desorption/ionization (MALDI)/MS for parentage investigation. The three cultivars lead to very similar spectra with differences in the relative intensity of the most abundant species and the presence of very weak specific ions. In order to evaluate the analytical significance of these aspects, the variability due to instrumental factors and due to different harvesting areas and years of the same cultivars have been considered and measured by the calculation of discrepancy factor values. On one hand, the results obtained can be considered a valid confirmation of the genomic findings, whereas on the other hand, the results provide evidence for the ability of MALDI/MS to individuate minor differences in protein profiles of complex protein mixtures.

Gene pool variation and phylogenetic relationships of an indigenous northeast Italian grapevine collection revealed by nuclear and chloroplast SSRs

A germplasm safeguard programme was set up with 19 grapevine varieties considered as indigenous to northeastern Italy. To better estimate how genetic structure can be used to obtain a conservation perspective of local varieties, genetic variability was examined at 30 nuclear and 3 chloroplast polymorphic microsatellite loci in the native varieties plus 7 European cultivars taken as reference. The genetic profiles of all the cultivars were searched for possible parentage relationships and several suspected cases of the same variety having different names were investigated. The alleles shared at the loci suggest a parent-offspring relationship between Merlot and Cabernet Franc, 'Gruaja' and 'Negrara Veronese', and Marzemina Nera and Marzemina Bianca. Alleles at the 30 nuclear loci are consistent with Raboso Veronese being the progeny of Marzemina Bianca and Raboso Piave. Chloroplast-specific haplotypes were singled out for the first time in this indigenous germplasm and should be considered typical of the region. It is hypothesized that there are many specific haplotypes for the local varieties due to a past contribution of wild grapevine to the cultivated gene pool. The majority of investigated cultivars were demonstrated to constitute an independent source of genetic variation, and therefore a possible valuable resource of genetic traits for breeders.