Food Chemistry 177 (2015) 382–389

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Maceration with stems contact fermentation: Effect on proanthocyanidins compounds and color in Primitivo red wines Serafino Suriano a, Vittorio Alba b,⇑, Luigi Tarricone a, Domenico Di Gennaro a a Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Unità di Ricerca per l’Uva da Tavola e la Vitivinicoltura in Ambiente Mediterraneo, Cantina Sperimentale di Barletta, Via Vittorio Veneto 26, 76121 Barletta, Italy b Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Unità di Ricerca per l’Uva da Tavola e la Vitivinicoltura in Ambiente Mediterraneo, Via Casamassima 148, 70010 Turi, BA, Italy

a r t i c l e

i n f o

Article history: Received 1 December 2014 Received in revised form 8 January 2015 Accepted 9 January 2015 Available online 17 January 2015 Chemical compounds studied in this article: (+)-Catechin (PubChem CID: 9064) ( )-Epicatechin (PubChem CID: 72276) Epigallocatechin (PubChem CID: 72277) Epigallocatechin gallate (PubChem CID: 65064) Gallocatechin (PubChem CID: 65084) Gallocatechin gallate (PubChem CID: 199472) Malvidin-3-Glucoside (PubChem CID: 443652) Procyanidin B1 (PubChem CID: 11250133) Procyanidin B2 (PubChem CID: 122738) Tartaric acid (PubChem CID: 875)

a b s t r a c t Three Primitivo (Vitis vinifera, cv.) red wines were microvinified by means of different winemaking technologies: no stem-contact fermentation destemming 100% of grapes (D100); stem-contact fermentation destemming 75%, 50% of grapes (D75–D50) respectively. The objectives of this work were to improve proanthocyanidins content in wine, to monitor the relationships between anthocyanins/tannins and to detect the effects on the polymerization state of polyphenols after 12 months storage of wines. D100 showed higher anthocyanins content but lower color intensity and phenolic compounds content with respect to the theses D75 and D50, the last two showing lower anthocyanins content due their partial adsorption by grape stems during the fermentation. D75 gave the best results in terms of anthocyanins/color intensity balance and showed a better wine tannin component with respect to D50. Moreover after 12 months storage D50 reached a more advanced and stable polymerization state of colored pigments than the other wines. Ó 2015 Elsevier Ltd. All rights reserved.

Keywords: Primitivo Stem-contact Anthocyanins Color intensity Proanthocyanidins

1. Introduction Primitivo cv. (Vitis vinifera L.) is an early and vigorous wine grape variety wide spread in Apulia region, South-Italy. Primitivo adapts well to dry lands, with low farming needs. In presence of calcareous soils it shows a more dense and vivid berry color, while on deeper soils it tends to develop a high potential alcohol in wine.

⇑ Corresponding author. Tel.: +39 080 8915711; fax: +39 080 4512925. E-mail addresses: serafi[email protected] (S. Suriano), vittorio.alba@entecra. it (V. Alba), [email protected] (L. Tarricone), [email protected] (D. Di Gennaro). http://dx.doi.org/10.1016/j.foodchem.2015.01.063 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

Primitivo can be vinified in purity or mixed in various blends according to the production disciplinaries for protected designation of origin (PDO). Although a great development of viticulture/oenology in Apulia Region over the past 20 years, knowledge on phenolic, color and flavans assets on Primitivo wine as referred to winemaking processes and conservation need to be deepened. It is common among winemakers and consumers to consider Primitivo wine rich in color intensity but scarce in tannins content (Lovino, Baiano, Pati, Faccia, & Gambacorta, 2006), leading to not very structured, short-lived wines with reduced chromatic stability over time. Therefore, Primitivo winemaking has to take in consideration

S. Suriano et al. / Food Chemistry 177 (2015) 382–389

anthocyanins, and flavans from grape skins, seeds and stems. An adequate content of anthocyanins and tannins allows the formation of stable pigments that ensure greater color stability in wines (Scollary, 2010). The anthocyanin profile linked to the five main mono-glucosylated anthocyanins, acetylated and coumarylated forms has an important role in the determining wine chemical properties. On average, more than 50% anthocyanin profile in Primitivo grapes is characterized by malvidin 3-glucoside, about 5% by delphinidin 3g, around 6% by peonidin and petunidin 3-g, traces of cyanidin 3-g, 30% by anthocyanins coumarylated form and 7% by acetylated form (Lovino, La Notte, Suriano, Savino, & Dimitri, 2001; Tamborra & Esti, 2010). In particular, given the high content of malvidin 3-g, very resistant to oxidation, and a low amount of cyanidin 3-g easily attacked by oxygen, Primitivo wine show enviable color component if compared to other Italian and Apulian wines (Tamborra & Esti, 2010). Tannins play an important role in both grapes and wines. In wine, they are responsible for astringency since they bind with salivary proteins that precipitate, resulting in lack of lubrification in mouth. Tannins in grapes are mainly composed by proanthocyanidins and are present in the skins, seeds and stems. Seed proanthocyanidins are made up of (+)-catechin, ( )-epicatechin and ( )-epicatechin-3-gallate (Kennedy, Matthewa, & Waterhouse, 2002), whereas skin proanthocyanidins also contain ( )-epigallocatechin and a much lower proportion of ( )-epicatechin-3-gallate (Gonzalez-Manzano, Rivas-Gonzalo, & SantosBuelga, 2004; Souquet, Cheynier, Brossaud, & Moutounet, 1996). Stem proanthocyanidins are made up of four monomers: (+)-catechin, ( )-epicatechin, ( )-epicatechin-3-gallate and ( )-epigallocatechin (Souquet, Cheynier, & Moutonet, 2000; Vivas et al., 2004). It has been reported that seed proanthocyanidins present a lower mean degree of polymerization (mDP) than skin proanthocyanidins (Moutonet et al., 2002; Souquet et al., 2000). According to Souquet et al. (2000) stem and seed proanthocyanidins show similar mDP, while other authors (Vivas et al., 2004) report higher seed proanthocyanidins mDP than skin proanthocyanidins (Sun & Spranger, 2005). It has been reported that not-well-ripen grapes show lower extractability for anthocyanins and proanthocyanidins from skins despite a higher extractability from seeds (Canals, Llaudy, Valls, Canals, & Zamorav, 2005; Romeyer, Macheix, & Sapis, 1986) with respect to well-ripen grapes. Consequently, it is generally considered that not-well-ripen grapes may produce more astringent wines because their seeds can release a higher amount of proanthocyanidins, which are highly galloylated (Romeyer et al., 1986). It has also been reported that stems can release highly astringent, herbaceous and bitter proanthocyanidins (Boulton, Singleton, Bisson, & Kunkee, 1995). All this considered, grapes are usually destemmed in red winemaking. Moreover, grapes with high degree ripeness facilitate the extraction of tannins from skins, while this is more difficult in seeds due to their highly lignified seed coat. Furthermore, the formation of the complex tannins–anthocyanins is closely linked to the type of tannins present in grapes as evidenced by other authors (Poinsaut & Gerland, 1999). Only condensed tannins are able to form stable complexes with anthocyanins, which are responsible, among other compounds, of wine color stability over time. The enrichment of tannins content in wine during winemaking can be achieved by different commercial additives derived from grapes, oak, and/or exotic woods or better employing wood chips of various nature (oak extract, chestnut extract, quebracho extract), or aging wine in oak barrels. Beside this, the use of cluster stems left to macerate with juice and skins during all the period of fermentation is a rediscovered biological and traditional method to increase tannins in wine.

383

In this paper the best winemaking conditions in Primitivo wine were explored by comparing 3 different winemaking protocols defined to assure adequate amounts of anthocyanins during wine fermentation and subsequently by aging wine in order to increase the natural concentration of tannins thus encouraging polymerization processes and improving stabilization of chromatic components over time. The 3 investigated theses were: (1) winemaking destemmed 100% of grapes cluster (D100); (2) winemaking destemmed 75% of grapes cluster and stem-contact for all time fermentation (D75); (3) winemaking destemmed 50% of grapes cluster and stem-contact for all time fermentation (D50). 2. Materials and methods 2.1. Experimental design and winemaking The research was conducted in 2012 on Primitivo black grapes. Grapes came from a vineyards located in the territory of Sava (Apulia region-Italy). Vines were grown under organic farming conditions with Guyot pruning system. Grapes had the following chemical/physical characteristics: 34.40 °Brix, pH 3.95 and total acidity 3.84 g/L. Three treatments were performed: No stem-contact fermentation by destemming 100% grapes (D100). By totally destemmed/crushed grapes were collected into 100 kg capacity stainless steel tanks, sulfidated with 60 mg/L of SO2. Total acidity was corrected to 5.80 g/L and then inoculated with selected yeasts Lalvin V116 (Lallemmand – 20 g/100 kg). The skin maceration and fermentation lasted 10 days with twice daily pumping over at an average temperature of 25 °C and final racking and pressing in a vertical idropress 2.5 Bar. With stem-contact fermentation destemmed 75% of grapes (D75). Predicted the destemming/crushing of 75% of total mass grapes and only crushing of remaining part of the 25% with stems of cluster grapes. The pressed with stems 25% was transferred into stainless steel tank addition SO2, total acidity corrected a 5.8 g/L and followed the same steps of the previous thesis. With stem-contact destemmed 50% of grapes (D50). Predicted the destemming/crushing of 50% of total mass grapes and only crushing of remaining part of the 50% with stems of cluster grapes. The pressed with stems 50% was transferred into stainless steel tank, addition SO2, total acidity corrected a 5.8 g/L and followed the same steps of the first thesis. All vinifications and each treatments were performed in three replicates, in 100 kg capacity steel tank. During the fermentative pomace contact period (10 days in all vinifications) the temperature and must density were recorded. After racking, wines were stored at room temperature. One month later, the wines were racked and sulfur dioxide was added, no malolactic fermentation occurred. After the wines were cold stabilized ( 5 °C) for 1 month and then bottled. All analyses were made in triplicate at racking and after 6 months in the bottle (after 12 months storage period). 2.2. Chemicals and reference compounds (+)- Catechin, ( )- epicatechin, procianidin B1, procianidin B2, +epigallocatechin, epigallocatechin gallate were supplied by Extrasynthese. Standards purities were all over 95%. All the solvents (methanol, acetonitril, ethyl acetate, diethyl ether, phosphoric acid) supplied by Carlo Erba (Milan, Italy) were HPLC grade. All the solutions were obtained with distilled deionized water using Carlo Erba reagents.

384

S. Suriano et al. / Food Chemistry 177 (2015) 382–389

2.3. Wine composition Total acidity, volatile acidity, reducing sugar, pH value, SO2 total, alcohol and total dry extract were all determined on wine in according to EEC, regulation 1676/90.

2007; López, Puértolas, Hernández-Orte, Álvarez, & Raso, 2009). Detected compounds were quantified by integration as peak area. In order to calibrate the chromatographic response and to express the results in terms of concentration (mg/L), malvidin chloride was used (Sigma–Aldrich).

2.4. Spectrophotometric analysis

2.6. Statistical analysis

Phenolic composition and analysis procedures for flavonoids (F), anthocyanins (A), totalpolyphenols (TP), proanthocyanidins (P) and flavans reactive with vanillin (FRV) were determined in according to Di Stefano, Ummarino, and Gentilini (1997) and Di Stefano and Cravero (1989). The color intensity and hue were estimated by measuring absorbance at 420, 520 and 620 nm according to EU Regulation 1990.

Chemical analyses were repeated three times for each sample. The one way analysis of variance (ANOVA), and Duncan multiple comparison test to measure variation between treatments at a probability level of p < 0.05 was applied. Moreover, different principal component analysis (PCA) were carried out on phenolic compounds, on chromatic characteristic and on catechins and oligomeric procyanidins both at racking and after 12 months storage. Since the use of different measure units resulted in the entirely different types of scales for all data, which had the unequal weight, data were first standardized in order to transform all characters to a comparable scale and then PCA was performed with Statgraphics Centurion XV ver. 15.1.02.

2.5. HPLC analysis For flavans determination wines were subjected to fractionation by separation of two fractions containing individual catechins and oligomeric proanthocyanidins respectively using C18 1 g Sep-Pak cartridge as described by Sun, Pinto, Leandro, Ricardo-Da-Silva, and Spranger (1999). Each fraction was evaporated and dissolved in methanol, followed by HPLC analysis. An HPLC 1100 series Agilent technologies with binary pump and diode array detector (DAD) was used with a Thermo ODS RP-C18 Hypersil 200  2.1 (5 lm) column with a guard ODS Hypersil 20  2.1 mm (5 lm) was used for flavans analysis. 2 mL of each of the fractions extracted was filtered and immediately injected according to Squadrito’s method (Squadrito, Corona, Ansaldi, & Di Stefano, 2007). Separation was carried out at 30 °C, the flow rate was 0.25 mL/min and the injection volume 10 ll. The detection was at 280 nm, using phosphoric acid 10 3 M (solvent A) and acetonitrile (solvent B). The gradient elution program was: from 91% to 86% A in ten minutes; from 86% to 82% A in ten minutes; from 82% to 60% A in ten minutes; from 60% to 40% A in five minutes; from 40% to 91% A in five minutes; equilibration time of five minutes. Peaks identification was performed comparing the retention times and absorption spectra of pure compounds (supplied by the firm Extrasynthese) and analogs to those reported in literature (Garcia-Beneytez, Cabello, & Revilla, 2003; Ricardo Da Silva, Bourzeix, Cheynier, & Moutounet, 1991; Sun, Leandro, Ricardo da Silva, & Spranger, 1998). A Phenomenex Synergi 4u Hydro-RP 80A (250  4.60 mm, 4 micron) with guard column was used for organic acids analysis. The analysis was performed with an isocratic elution with H3PO4 10 3 M at 25 °C, 0.7 mL/min flow rate and detector was set at 210 nm in according to the method proposed by Cane (1990). Anthocyanins were isolated directly from the wine by passing 3 mL of wine through C 18 Sep-pak cartridge previously conditioned and eluted with 3 mL methanol, was evaporated to dryness and redissolved in a mixture of 1 mL methanol: H3PO4 10 3 M (40:60) before injection into chromatographic system in according to Squadrito’s method (Squadrito et al., 2007). The samples were injected into a Thermo ODS RP-C18 Hypersil 100  2.1 (5 lm) column with a guard ODS Hypersil 20  2.1 mm (5 lm). Separation was carried out at 30 °C, the flow rate was 0.25 mL/min and the injection volume 10 ll. The detection was at 520 nm, using solvent: a formic acid 10%; B formic 10% and methanol 50%. Linear gradients from 72% to 55% A in fifteen minutes; from 55% to 30% A in twenty minutes; from 30% to 10% A in ten minutes; from 10% to 1% A in five minutes; from 1% to 72% A in five minutes; equilibration time five minutes. The different analyzed anthocyanins compounds were tentatively identified according to the retention time and the UV–vis spectral characteristics described in the literature (Gómez-Alonso, García-Romero, & Hermosín-Gutiérrez,

3. Results and discussion 3.1. Wine chemical composition Grapes were harvested on August 20th 2012, earlier if compared to previous years. A very high sugar content in berries was reached due to the particular Apulian climatic conditions, with a dry summer and high temperatures during the ripening phase. The fundamental components of the different types of wines are shown in Table 1. Among the key components, ethyl alcohol was present in greater quantities. Its content is related directly to grape/must sugar concentration. Indeed, all wines reported very high alcoholic content. Ethyl alcohol is an important component in wine because it gives a distinctive flavor, slightly sweet, it is also an excellent solvent of aromatic substances and helps to enhance wine aroma and bouquet. The increase of tannic component in wine attributed to the presence of stems contact during the maceration/fermentation served to counteract the notes due to alcohol excess. The highest alcohol concentration was recorded in D50. No difference between D50, D75 and D100 was evidenced for pH and total acidity. Volatile acidity is an indicator of the healthy status of a wine, since high values can reveal anomalies in fermentation and during storage. All wines had medium/high volatile acidity Table 1 Mean ± standard deviation of chemico-physical composition of three different Primitivo wines: destemmed 100% (D100), destemmed 75% (D75), destemmed 50% (D50) at racking.

Alcohol (%) Reducing sugars (g/L) Total dry extract (g/L) Reduced extract (g/L) pH Total acidity (g/L) Volatile acidity (g/L) Total SO2 (mg/L) Free SO2 (mg/L) Tartaric acid (g/L) Lactic acid (g/L) Malic acid (g/L) Shikimic acid (mg/L) Citric acid (g/L) Succinic acid (g/L)

D100

D75

D50

19.67 ± 0.05b 3.2 ± 0.70 44.2 ± 1.90 41.2 ± 1.80 3.91 ± 0.10 6.15 ± 0.28 0.75 ± 0.03a 96 ± 3.3a 24 ± 0.18a 1.60 ± 0.15 0.04 ± 0.01 1.73 ± 0.12 18.4 ± 1.62b 1.14 ± 0.10 1.28 ± 0.13

19.38 ± 0.06c 2.8 ± 0.90 43.4 ± 2.80 41.4 ± 2.60 3.84 ± 0.08 6.38 ± 0.25 0.75 ± 0.03a 77 ± 3.1b 9 ± 0.10c 1.53 ± 0.14 0.05 ± 0.01 2.4 ± 0.14 23.7 ± 1.68a 0.96 ± 0.10 1.23 ± 0.05

20.05 ± 0.06a 3.4 ± 0.70 45.5 ± 1.90 43.1 ± 1.80 3.90 ± 0.09 6.3 ± 0.28 0.66 ± 0.30b 64 ± 3.1c 10 ± 0.12b 1.69 ± 0.15 0.06 ± 0.01 1.7 ± 0.13 22.4 ± 1.55a 0.97 ± 0.10 1.33 ± 0.06

Different letters in the same line indicate significant difference (P < 0.05) based on Duncan test.

S. Suriano et al. / Food Chemistry 177 (2015) 382–389 Table 2 Mean ± standard deviation (n = 3) of polyphenolic composition (mg/L) of three different Primitivo wines: destemmed 100% (D100), destemmed 75% (D75), destemmed 50% (D50) at racking and after 12 months storage. D100

D75

D50

Total polyphenols Total flavonoids Flavonoids–anthocyanins Flavans, vanillin Proanthocyanidins Total anthocyanins Vanillin/proant.

At racking 2685 ± 23b 2472 ± 31b 1891 ± 27b 1078 ± 16c 1744 ± 36c 401 ± 15a 0.62

3127 ± 29a 2838 ± 33a 2294 ± 29a 1333 ± 18b 2180 ± 43b 374 ± 18ab 0.61

3164 ± 30a 2875 ± 27a 2298 ± 33a 1587 ± 16a 2275 ± 44a 368 ± 20b 0.69

Total polyphenols Total flavonoids Flavonoids–anthocyanins Flavans, vanillin Proanthocyanidins Total anthocyanins Vanillin/proant.

After 12 months 1857 ± 21c 1949 ± 26c 1596 ± 22c 812 ± 21c 1297 ± 34c 250 ± 12a 0.62

2311 ± 19b 2453 ± 28a 2013 ± 18a 932 ± 15b 1754 ± 28b 206 ± 11b 0.53

2410 ± 21a 2035 ± 20b 1760 ± 21b 1013 ± 19a 1989 ± 35a 194 ± 13b 0.51

Different letters in the same line indicate significant difference (P < 0.05) based on Duncan test.

values positively correlated with must sugar concentration used as substrate in alcoholic fermentation by yeasts. 3.2. Polyphenolic composition and wine color Table 2 shows the polyphenolic composition of wines at racking and after 12 months storage. In general, the higher the percentage of stems used during maceration/fermentation, the higher the content of total polyphenols, total flavonoids, flavans reactive with vanillin and proantocyadins. The main objective of this work was to enrich wines in phenolic compounds, mainly in tannins. The extraction processes on stems during fermentation determine an enrichment of polyphenols in wine. At racking, D50 and D75 contained higher concentration of flavans reactive with vanillin (low molecular tannins) and proanthocyanidins (high molecular tannins) respect to D100, in agreement with previously published findings (Castellari, Arfelli, Riponi, & Amati, 1998; Spranger et al., 2004; Sun, Spranger, Roque-do-Vale, Leandro, & Belchior, 2001). Flavans, essentially consisting of monomers and oligomers, have low molecular weight. They are responsible for astringency and result very reactive and very unstable, leading to wine browning. Conversely, proanthocyanidins have high molecular weight which in acid environment and under heating evolve into the more polymerized and stable compounds cyanidin and delphinidin which confer a softer taste to wine. The V/L ratio, representing the condensation tannins degree, was higher at racking and declined after 12 months of storage, while resulted stable only in D100. Initially the tannins are in the less evolved and reactive forms, while after 12 months they acquire more advanced forms thanks to polymerization processes needed to ensure wine stability over the time. The higher the V/L ratio, the lower is the average degree of polymerization of tannins, which means that a wine has high probabilities to undergo alteration of color and structure. Therefore, a low V/L index resulted in a reduction of the reactivity of these molecules and in a predisposition to chromatic and tannic stabilization of wines. During aging, wine parameters relating to polyphenolic compounds normally undergo a natural evolution, evidenced by a progressive decay of total flavonoids, total polyphenols, flavans reactive to vanillin and of pro-anthocyanins (Salinas, Garijo, Pardo, Zalacain, & Gonzalo, 2003). The reductions were smaller for flavans reactive with vanillin and greater for proanthocyanidins in D100 compared to D50 and D75. In fact, losses in proanthocyani-

385

dins were of 25.7% in D100, of 19.6% in D75 and of 12.6% in D50. Therefore, D50 and D75 determined a greater reduction of low molecular weight tannins (more reactive and more astringent) and a contained losses of high molecular weight tannins (softer and less astringent tannins). Beside this, the presence of high molecular weight tannins promoted polymerization reactions between acetaldehyde–tannin–anthocyanin, determining a better stabilization in regards to wine color. Table 3 shows the results of the chromatic characteristics in the different types of Primitivo wines. D100 revealed highest statistically different levels in monomer and total anthocyanins. Small not significant differences were present between the D50 and D75. At racking D100 wine had total anthocyanin content of 401 mg/L, higher than D50 (368 mg/L) and D75 (374 mg/L), this trend also confirmed for what concerns monomer anthocyanins. This is probably due to a partial adsorption by stems of anthocyanins derived from skins extraction during the process of fermentation/maceration. Spranger et al. (2004) suggest that anthocyanins can irreversibly be adsorbed by stems and with them removed at racking after fermentation, as in the case of D50 and D75. In general, in red wines, color intensity is positively correlated to anthocyanins concentration (Gómez-Plaza, Gil-Muñoz, LópezRoca, Martínez-Cutillas, & Fernández, 2001; Gòmez-Mìguez & Heredia, 2004; Suriano, Ceci, & Tamborra, 2012), in contrast with our findings, for which D50 and D75 had lower anthocyanin content but higher color intensity with respect to D100 as reported in Table 3. Color intensity was 11.97 in D100, lower with respect to 15.90 in D75 and 15.20 in D50, probably due to a must oxygenation favored by better aeration related to the presence of stems. The oxygenation increases the strength of color and promotes processes of condensation between anthocyanins–tannins– acetaldehyde, very important for chromatic stability. Table 3 reports the influence of the winemaking techniques on the concentration of monomeric anthocyanins determined by HPLC. As shown, anthocyanin contents especially in red young wines depends significantly on the type of winemaking technology (Castillo-Sànchez et al., 2008), while it decreases notably after 12 months of aging. For all the theses, the most abundant between glycosylated anthocyanins was the malvidin-3-glucoside, followed by petunidin-3-g, peonidin-3-g, delphinidin-3-g and cyanidin-3-g, according to the anthocyanic profile for Primitivo red grape previously defined (Tamborra & Esti, 2010), confirming that although a technique of vinification may modify a wine anthocyanin content, the grape variety effect prevails (González-Neves, Ferrer, & Gil, 2012; González-Neves, Gil, Favre, & Ferrer, 2012; Favre et al., 2014). The levels of malvidin-3-glucoside and of coumarylated forms were lower in all stem-contact wines with respect to D100, suggesting on one side a decreasing effect of yeast, pomace and stems in adsorbing anthocyanins and their degradation and condensation with tannins (Ribéreau-Gayon & Markakis, 1982). At racking, coumarylated forms prevailed on acylated ones. After 12 months storage, a drastic reduction of coumarilated forms led the concentration of acetylated forms in D75 to prevail respect to 12 months before. The di-substituted Anthocyanins (cyanidin3-g. and peonidin-3-g.), highly susceptible to oxidation processes, were present in smaller quantities than the tri-substituted anthocyanins. Therefore, given D75 and D50 with good content in malvidin-3-g. resistant at oxidation processes, considered the tannins increase, we have obtained a more stable product in regards of oxidative profile. To assess the contribution to the color of wine of the different forms of combination between anthocyanic and flavanic pigments sensitive to pH and SO2 (Table 3), the indices dAl% (anthocyanin monomers), dAT% (polymeric pigments sensitive to SO2), dTAT% (stable polymeric pigments not sensitive to SO2) were adopted. They reflect the contribution of monomers anthocyanins and

386

S. Suriano et al. / Food Chemistry 177 (2015) 382–389

Table 3 Mean ± standard deviation (n = 3) of chromatic characteristics of three different Primitivo wines: destemmed 100% (D100), destemmed 75% (D75), destemmed 50% (D50) at racking and after 12 months storage. D100

D75

D50

Total anthocyanins (mg/L) Monomer anthocyanins (mg/L) k max wine TQ (nm) Col. Int. 420 + 520 + 620 (P.O. 1 cm) Tint 420/520 (P.O. 1 cm) Delphinidin-3-G (mg/L) Cyanidin-3-G (mg/L) Petunidin-3-G (mg/L) Peonidin-3-G (mg/L) Malvidin-3-G (mg/L) Total acetylated forms (mg/L) Total coumarylated forms (mg/L) dAl 1 mm wine pH (%) dAT 1 mm wine pH (%) dTAT 1 mm wine pH (%)

At racking 401 ± 15a 258 ± 11a 530 ± 3 11.97 ± 0.31c 0.72 ± 0.04 5.67 ± 0.15a 0.77 ± 0.01a 15.31 ± 0.56a 8.72 ± 0.45a 181.37 ± 5.22a 19.6 ± 0.74b 26.44 ± 1.32a 24.26 ± 1.2a 64.02 ± 2.10a 11.71 ± 0.94b

374 ± 18ab 221 ± 12b 531 ± 2 15.9 ± 0.40a 0.67 ± 0.3 4.42 ± 0.12b 0.66 ± 0.01b 12.6 ± 0.68b 7.51 ± 0.44b 150.50 ± 4.75b 21.65 ± 0.82a 22.98 ± 1.15b 21.30 ± 1.3b 60.60 ± 2.20b 18.20 ± 1.30a

368 ± 20b 215 ± 13b 531 ± 4 15.2 ± 0.27b 0.70 ± 0.04 4.3 ± 0.15b 0.54 ± 0.01c 12.25 ± 0.52b 7.31 ± 0.39b 149.21 ± 6.80b 18.27 ± 0.95b 22.36 ± 1.34b 20.80 ± 1.60b 60.20 ± 2.30b 18.90 ± 1.10a

Total anthocyanins (mg/L) Monomer anthocyanins (mg/L) k max wine TQ (nm) Col. Int. 420 + 520 + 620 (P.O. 1 cm) Tint 420/520 (P.O. 1 cm) Delphinidin-3-G (mg/L) Cyanidin-3-G (mg/L) Petunidin-3-G (mg/L) Peonidin-3-G (mg/L) Malvidin-3-G (mg/L) Total acetylated forms (mg/L) Total coumarylated forms (mg/L) dAl 1 mm wine pH (%) dAT 1 mm wine pH (%) dTAT 1 mm wine pH (%)

After 12 months 250 ± 14a 85 ± 10 520 ± 3 10.88 ± 0.28c 0.71 ± 0.03 2.12 ± 0.11a 0.30 ± 0.01c 5.44 ± 0.21a 3.20 ± 0.15b 61.72 ± 2.20a 5.78 ± 0.31 6.50 ± 0.34a 16.50 ± 1.10a 45.30 ± 1.80a 38.20 ± 1.70b

206 ± 12b 68 ± 8 520 ± 2 13.83 ± 0.32a 0.75 ± 0.03 2.28 ± 0.10a 0.51 ± 0.01a 4.35 ± 0.25b 4.05 ± 0.18a 47.4 ± 1.86b 6.10 ± 0.38 3.27 ± 0.28b 12.80 ± 1.10b 43.60 ± 2.00b 43.60 ± 2.40b

194 ± 14b 71 ± 4 520 ± 2 12.99 ± 0.31b 0.69 ± 0.04 1.56 ± 0.07b 0.23 ± 0.01b 4.01 ± 0.24b 2.46 ± 0.10c 50.67 ± 2.46b 5.76 ± 0.25 6.28 ± 0.36a 12.60 ± 1.00b 42.70 ± 1.70b 44.70 ± 2.70a

Different letters in the same line indicate significant difference (P < 0.05) based on Duncan test.

Table 4 Mean ± standard deviation (n = 3) of concentration (mg/L) of monomer catechins and oligomeric procyanidins of three different Primitivo wines: destemmed 100% (D100), destemmed 75% (D75), destemmed 50% (D50) at racking and after 12 months storage. D100

D75

D50

(+)-Catechin ( )-Epicatechin Gallocatechin Epicatechin gallate Epigallocatechin Epigallocatechin gallate Procyanidin B1 Procyanidin B2 Procyanidin B3 Procyanidin B4 Procyanidin B2 gallate Procyanidin T2 (trimer) Procyanidin C1 (trimer)

At racking 17.78 ± 0.74c 12.40 ± 0.21c 6.20 ± 0.25a 19.30 ± 0.75b 23.80 ± 0.38c 6.73 ± 0.75b 25.21 ± 0.44c 45.87 ± 1.24c 2.28 ± 0.07c 18.89 ± 0.37c 48.30 ± 1.24c 8.53 ± 0.72b 5.32 ± 2.34b

21.20 ± 0.83b 17.61 ± 0.31b 3.30 ± 0.10c 19.60 ± 0.67b 25.81 ± 0.41b 7.55 ± 0.83a 33.40 ± 0.53b 62.30 ± 1.88b 3.10 ± 0.05b 28.40 ± 0.46a 61.30 ± 1.08b 8.95 ± 0.83a 6.49 ± 0.74a

28.30 ± 0.88a 19.62 ± 0.29a 4.02 ± 0.03b 21.40 ± 0.98a 30.30 ± 1.04a 7.6 ± 0.78a 39.40 ± 0.67a 68.31 ± 1.93a 5.20 ± 0.04a 26.33 ± 0.63b 76.24 ± 2.34a 9.64 ± 0.78a 6.71 ± 0.64a

(+)-Catechin ( )-Epicatechin Gallocatechin Epicatechin gallate Epigallocatechin Epigallocatechin gallate Procyanidin B1 Procyanidin B2 Procyanidin B3 Procyanidin B4 Procyanidin B2 gallate Procyanidin T2 (trimer) Procyanidin C1 (trimer)

After 12 months 12.12 ± 0.64c 11.00 ± 0.24c 2.13 ± 0.14b 14.14 ± 0.45c 22.48 ± 0.44c 6.64 ± 0.61a 23.74 ± 0.37c 40.00 ± 1.33c 2.10 ± 0.06b 17.50 ± 0.38c 45.74 ± 1.32c 7.66 ± 0.60a 2.42 ± 0.27b

15.11 ± 0.33b 12.40 ± 0.41b 1.60 ± 0.02c 14.90 ± 0.44b 25.10 ± 0.58b 5.98 ± 0.67b 25.03 ± 0.55b 53.80 ± 1.76b 2.44 ± 0.04b 22.80 ± 0.41a 57.16 ± 1.16b 6.93 ± 0.65b 5.17 ± 0.68a

19.90 ± 0.74a 16.15 ± 0.24a 3.25 ± 0.03a 15.62 ± 0.66a 28.54 ± 1.06a 4.98 ± 0.62c 30.42 ± 0.63a 55.36 ± 0.92a 2.51 ± 0.04a 19.92 ± 0.49b 73.12 ± 2.12a 6.92 ± 0.61b 5.20 ± 0.64a

Different letters in the same line indicate significant difference (P < 0.05) based on Duncan test.

anthocyanin–tannin complexes to the absorbance at 520 nm. In young wines dAl and dAT have high values and in general dAT is greater than dTAT, since anthocyanin monomers give a fundamental contribution to the red color of the wines. In older wines, the best conditions are when dTAT is maximum and higher than the other two indexes dAl and dAT. This means that the pigments reached a molecular complexity no more affected by changes in pH and SO2). At racking, dAl% and dAT% were higher respect to dTAT% in all wines. After 12 months aging, all wines had similar evolution with a steady diminution of absorbancy fraction at 520 nm attributable to momoner anthocyanins (dAl%) and a further diminution of the dAT fraction, while the pigments not bleachable by SO2 (dTAT%) increased. Similar results were obtained by other authors (Bosso, Guaita, Panero, Borsa, & Follis, 2009; Suriano et al., 2012) who detected higher dTAT% levels for wine aged with other of V. vinifera varieties. D100 during fermentation was clearly different and higher respect to D75, D50 in chromatic indices dAl% and dAT%. D75 and D50 had higher concentration of red pigments (absorbing at 520 nm) not bleachable by SO2 (dTAT%) than D100. Therefore, D75 and D50 had a negative effect on the concentration of free anthocyanins, but increased the percentage of pigments not bleachable by SO2 (dTAT%) thus improving color stability during aging. Supplementary Material 1 (SM 1) shows the absorption spectra at visible wavelengths at racking and after 12 months storage. It can be noted that D75 presented higher absorbance than D50, this latter higher than D100. After 12 months storage, the differences between D75 and D50 tended to increase, while the differences between the D50 and D100 remained unaltered. D75 favored a greater absorbance at different wavelengths, determining an increase of color.

387

S. Suriano et al. / Food Chemistry 177 (2015) 382–389

Table 5 Principal component analysis (PCA): eigenvalues, eigenvectors and percent of variation based on polyphenolic composition, color composition and concentration (mg/L) of monomer catechins and oligomeric procyanidins of three different Primitivo wines: destemmed 100% (D100), destemmed 75% (D75), destemmed 50% (D50), analyzed at racking and after 12 months storage. PC1

PC2

At racking

After 12 months storage

Eigenvalue Variance (%) Cumulative variance (%)

Polyphenolic composition 5.84 97.29 –

0.16 2.71 100.00

Flavans–vanillin Flavonoids–anthocyanins Proanthocyanidins Total anthocyanins Total flavonoids Total polyphenols

Eigenvectors 0.388 0.409 0.414 0.414 0.413 0.412

Eigenvalue Variance (%) Cumulative variance (%)

Color composition 10.74 89.46 –

Col. Int. 420 + 520 + 620 Cyanidin 3-G dAl 1 mm wine pH dAT 1 mm wine pH Delphinidin 3-G dTAT 1 mm wine pH Malvidin 3-G Monomer anthocyanins Peonidin 3-G Petunidin 3-G Total acetylated forms Total coumarylated forms

Eigenvectors 0.293 0.277 0.305 0.305 0.305 0.305 0.304 0.305 0.305 0.305 0.002 0.305

Eigenvalue Variance (%) Cumulative variance (%)

Concentration (mg/L) of monomer catechins and oligomeric procyanidins 11.42 1.58 9.72 87.85 12.15 80.98 – 100.00 –

(+)-Catechin ( )-Epicatechin Epicatechin gallate Epigallocatechin Epigallocatechin gallate Gallocatechin Procyanidin B1 Procyanidin B2 Procyanidin B2 gallate Procyanidin B3 Procyanidin B4 Procyanidin C1 (trimer) Procyanidin C2 (trimer)

Eigenvectors 0.281 0.293 0.258 0.279 0.278 0.236 0.296 0.293 0.292 0.276 0.242 0.286 0.286

0.865 0.390 0.006 0.013 0.206 0.240 1.26 10.54 100.00 0.249 0.373 0.016 0.018 0.033 0.025 0.072 0.012 0.014 0.010 0.889 0.023

0.248 0.105 0.388 0.261 0.276 0.479 0.023 0.114 0.125 0.285 0.458 0.203 0.203

PC1

PC2

4.70 78.28 –

1.30 21.72 100.00

0.427 0.362 0.438 0.454 0.283 0.457 6.18 61.77 – 0.395 0.146 0.399 0.364 0.088 0.390 0.399 – 0.052 0.382 – 0.251

0.302 0.320 0.316 0.314 0.192 0.309 0.170 0.315 0.292 0.302 0.292 0.292 0.302

0.333 0.543 0.274 0.159 0.691 0.128 3.82 38.24 100.00 0.097 0.477 0.069 0.217 0.499 0.126 0.066 – 0.507 0.161 – 0.399 2.28 19.02 100.00 0.223 0.047 0.109 0.134 0.531 0.181 0.561 0.120 0.274 0.224 0.274 0.273 0.223

Values with higher weights in the determination of PCA axes are reported in bold.

3.3. Monomer catechins and oligomer procyanidins The levels of monomer catechins and oligomer procyanidins in Primitivo wines made by different winemaking techniques are presented in Table 4 and determined by HPLC. All wines were characterized by the absolute dominance of epigallocatechin among monomer favans. The (+)-catechin was quantitatively the second constituent in descending order, while gallocatechin was the monomer present in lesser amounts. Among dimer procyanidins B2 gallate was present in greater quantities, significantly higher in D50. The trimer procyanidins were detected lower than dimers in all wines. At racking, stems-contact wines D50 and D75 were statistically different for a higher concentration in monomers, dimers and galloilate forms with respect to D100, thanks to the contribution of flavans present in grape stems during winemaking processes. These results also agree with those previously published

(Spranger et al., 2004; Sun et al., 2001). D50 wine recorded the richest content of almost all flavans constituents, with the exception of procyanidin B4, higher in D75 both at racking and after 12 months storage. It can be argued that the extraction from stems regards both monomer catechins and oligomeric procyanidins and is not selective on individual compounds like monomer and dimer procyanidins. Therefore, stems can be considered a source of natural exploitable tannins. These results obtained by HPLC are in line with those obtained spectrophotometrically by flavans reactive to vanillin and proanthocyanidins assay. Moreover, Primitivo wines considered tannin lacking, can be brilliantly enriched by the use of cluster stems during fermentation processes. Anyway, a higher amount of tannins found in D50 and D75, although present in optimal concentrations, did not store the character of astringency typically due to the presence of tannin

388

S. Suriano et al. / Food Chemistry 177 (2015) 382–389

Fig. 1. Principal component analyses of three different Primitivo wines: destemmed 100% (D100), destemmed 75% (D75), destemmed 50% (D50), analyzed at racking (left side) and after 12 months storage (right side) based on polyphenolic composition (A), color components (B) and concentration (mg/L) of monomer catechins and oligomeric procyanidins (C).

compounds. Volatile compounds and sensory analysis are thereby in course and will furnish further information on taste and olfactory notes. 3.4. Comparing wines at racking and after 12 months storage by PCA analysis Table 5 and Fig. 1 report the data concerning the Principal Component Analysis conducted on the three Primitivo winemaking theses at racking and after 12 months storage for polyphenolic compounds, color composition and mono e oligomeric procyanidins. In particular, in all cases and for all classes of compounds analyzed, two axes were sufficient to discriminate the three theses. For what concerns phenolic compounds (Table 5 and Fig. 1A), the first two axes described 100% of total variation at racking and after 12 months storage. At racking, all the traits loaded on the first axis with the exception of Flavan–vanilline which prevailed on axis two. After 12 months storage a reversal of the weights of Flavonoids–anthocyanins and Total flavonoids on axes can be seen. Indeed, Fig. 1A compares PCA at racking and after 12 months storage, revealing a similar distribution of the theses with the exception of the inversion of positions for D50–D75. Total anthocyanins loading on axis 1 remained unaltered at racking and after 12 months storage, suggesting that its difference in content in the theses investigated was imputable to the winemaking technique and was not affected by storage. For what concerns color components (Table 5 and Fig. 1B), the first two axes explained 89.46% and 61.77% of total variation at

racking and after 12 months storage, respectively. At racking, all traits loaded on axis 1 but Total acetylated forms, which revealed to be one of the most discriminant trait at racking for the theses investigated, together with color intensity, D100 showing the lowest values. At racking D50 and D75 resulted very close one each other, while they differed mainly for Total acetylated forms. After 12 months storage Total acetylated forms resulted statistically identical in the three theses and then removed from the analysis. Table 5 and Fig. 1C report PCA for monomer catechins and oligomeric procyanidins. At racking, axis 1, principally determined by different Procyanidins, described 87.85% of total variance while a further 12.15% variance was explained by axis 2, on which Epicatechin gallate and Gallocatechin resulted the most discriminant traits. In this case, as in the rest of the previous PCAs, very few changes occurred from racking to 12 months storage, since the position of the three theses in the graphs remained substantially unaltered. After 12 months storage PCA for monomer catechins and oligomeric procyanidins reported a reduction of total variation to 80.98% for axis 1 with respect to racking, while the second axis grew to 19.02% of total variation, principally indicating an increase of the load of eigenvector for Epigallocatechin gallate. 4. Conclusion The maceration in presence of stems in red winemaking is an important technique to naturally enrich tannins in wine and increase the color stability over time in wines obtained by Primitivo cv. which normally shows high anthocyanins concentrations.

S. Suriano et al. / Food Chemistry 177 (2015) 382–389

The completely de-stemmed wine (D100) wine showed highest concentrations of total anthocyanins and monomers both at racking and after 12 months storage. D50 and D75 acquired color intensity with the flowing of time, since condensation processes between anthocyanin–tannin–acetaldehyde. In particular, D50 and D75 were richer in all the phenolic components, especially tannins. Flavans amounts in wine seemed to be in parallel with an increasing stems percentage during fermentation, in particular individual catechins and dimer procyanidins grew significantly. Due to the presence of a good level of anthocyanins and optimal tannins concentrations, stem contact theses D50 and D75 favored condensation processes between these compounds and acetaldehyde during the 12 months storage, enhancing color intensity and improving the state of molecular complexity of pigments responsible for stability of wines to be aged. Acknowledgments The authors thank Apulia Region for the financial support in the Regional Development Program 2007/2013, Axis I Improvement of competitiveness in agricultural and forestry sectors, Integrated Projects of the Production Chain – Measure 124. Authors also thank GianFranco Fino for grapes and for support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2015. 01.063. References Bosso, A., Guaita, M., Panero, L., Borsa, D., & Follis, R. (2009). Influence of two winemaking techniques on polyphenolic composition and color of wines. American Journal of Enology and Viticulture, 60(3), 379–385. Boulton, R. B., Singleton, V. L., Bisson, L. F., & Kunkee, R. E. (1995). Principles practices of winemaking. New York: Chapman and Hall, p. 88. Canals, R., Llaudy, M. C., Valls, J., Canals, J. M., & Zamorav, F. (2005). Influence of ethanol concentration on the extraction of color and phenolic compounds from the skin and seed of Tempranillo grapes at different stages of ripening. Journal of Agricultural and Food Chemistry, 53, 4019–4025. Cane, P. (1990). Il controllo della qualità dei vini mediante HPLC: Determinazione degli acidi organici. L’Enotecnico, 26(1–3), 69–72. Castellari, M., Arfelli, G., Riponi, C., & Amati, A. (1998). Evolution of phenolic compounds in red winemaking affected by must oxidation. American Journal of Enology and Viticulture, 49(1), 91–94. Castillo-Sànchez, J. X., Garcìa-Falcon, M. S., Garrido, J., Martìnez-Garballo, E., Martins-Dias, L. R., & Mejuto, X. C. (2008). Phenolic compounds and colour stability of Vinhao wines: Influence of wine-making protocol and fining agents. Food Chemistry, 106, 18–26. Di Stefano, R., & Cravero, M. C. (1989). I composti fenolici e la natura del colore dei vini rossi. L’Enotecnico, 15(10), 81–87. Di Stefano, R., Ummarino, I., & Gentilini, N. (1997). Alcuni aspetti del controllo di qualità nel campo enologico. Lo stato di combinazione degli antociani. Annali Istituto Sperimentale. Enologia di Asti, 105–121. EEC (1990). European Communities. Commission regulation No 2676/90 on ‘‘community analysis methods to use in wine sector’’. Official Journal of European Communities. No. L272/3.10.90. Favre, G., Peña-Neira, A., Baldi, B., Hernández, N., Traverso, S., Gil, G., et al. (2014). Low molecular-weight phenols in Tannat wines made by alternative winemaking procedures. Food Chemistry, 158, 504–512. Garcia-Beneytez, E., Cabello, F., & Revilla, E. (2003). Analysis of grape and wine anthocyanins by HPLC–MS. Journal of Agricultural and Food Chemistry, 51, 5622–5629. Gómez-Alonso, S., García-Romero, E., & Hermosín-Gutiérrez, V. (2007). HPLC analysis of diverse grape and wine phenolics using direct injection and multidetection by DAD and fluorescence. Journal of Food Composition and Analysis, 20, 618–626. Gòmez-Mìguez, M., & Heredia, F. J. (2004). Effect of the maceration technique on the relationships between anthocyanins composition and objective color of Syrah wines. Journal of Agricultural and Food Chemistry, 52, 5117–5123.

389

Gómez-Plaza, E., Gil-Muñoz, R., López-Roca, J. M., Martínez-Cutillas, A., & Fernández, J. I. (2001). Phenolic compounds and colour stability of red wines: Effect of skin maceration time. American Journal of Enology and Viticulture, 53(3), 266–270. Gonzalez-Manzano, S., Rivas-Gonzalo, J. C., & Santos-Buelga, C. (2004). Extraction of flavan-3-ols from grape seed and skin into wine using simulated maceration. Analytica Chimica Acta, 513, 283–289. González-Neves, G., Ferrer, M., & Gil, G. (2012). Differentiation of Tannat, Cabernet Sauvignon and Merlot grapes from Uruguay according to their general composition and polyphenolic potential. Comunicata Scientiae, 3(1), 41–49. González-Neves, G., Gil, G., Favre, G., & Ferrer, M. (2012). Influence of grape composition and winemaking on anthocyanin composition of red wines of Tannat. International Journal of Food Science and Technology, 47, 900–909. Kennedy, J. A., Matthewa, M. A., & Waterhouse, A. L. (2002). Effect of maturity and vine water status on grape skin and wine flavonoids. American Journal of Enology and Viticulture, 53(4), 268–274. López, N., Puértolas, E., Hernández-Orte, P., Álvarez, I., & Raso, J. (2009). Effect of a pulsed electric field treatment on the anthocyanins composition and other quality parameters of Cabernet Sauvignon freshly fermented model wines obtained after different maceration times. LWT – Food Science and Technology, 42, 1225–1231. Lovino, R., Baiano, A., Pati, S., Faccia, M., & Gambacorta, G. (2006). Phenolic composition of red grapes grown in southern Italy. Italian Journal of Food Science, 2(18), 177–186. Lovino, R., La Notte, E., Suriano, S., Savino, M., & Dimitri, P. (2001). Caratterizzazione polifenolica di uve nere da vino di vitigni autoctoni dell’Italia Meridionale. Proceedings of 2nd Workshop POM Misura 2, Progetto B 35, Foggia, 1° Giugno. . Moutonet, M., Fulcrand, H., Sarni-Manchado, P. J., Souquet, M., Atanalsova, V., Labarbe, B., Maury, C., Vidal, S., & Cheynier, V. (2002). Wine tannins and their role in astringency perception. 13ème Symposium International d’œnologie, Montpellier (pp. 737–747). Poinsaut, P., & Gerland, C. (1999). Les tanins: Synergies entre tanins des raisins et tanins oenologiques. Revue des Oenologues, 93, 11–12. Ribéreau-Gayon, P., & Markakis, P. (1982). Anthocyanins of Food Colors. New York: Academic Press. Chapter 8. Ricardo Da Silva, J. M., Bourzeix, V., Cheynier, V., & Moutounet, M. (1991). Procyanidin composition of chardonnay, Mauzac and Grenache blanc grapes. Vitis, 30, 245–252. Romeyer, F. M., Macheix, J. J., & Sapis, F. J. (1986). Changes and importance of oligomeric procyanidins during maturation of grape seed. Phytochemistry, 25(1), 219–221. Salinas, M. R., Garijo, J., Pardo, F., Zalacain, A., & Gonzalo, L. A. (2003). Color, polyphenol, and aroma compounds in rosè wines after prefermentative maceration and enzymatic treatments. American Journal of Enology and Viticulture, 54(3), 195–202. Scollary, G. R. (2010). GWRDC Tannin Review. Grape and wine research & development corporation. Australian Government. 132 pp. URL: Accessed on 07.01.15. Souquet, J. M., Cheynier, V., Brossaud, F., & Moutounet, M. (1996). Polimeric proanthocyanidins from grape skins. Phytochemistry, 43, 509–512. Souquet, J. M., Cheynier, V., & Moutonet, M. (2000). Les proanthocyanidins du raisin. Bulletin de l’OIV, 73, 601–609. Spranger, M. I., Climaco, M. C., Sun, B., Nilza, E., Fortunato, C., Nunes, A., et al. (2004). Differentiation of red winemaking technologies by phenolic and volatile composition. Analytica Chimica Acta, 513, 151–161. Squadrito, M., Corona, O., Ansaldi, G., & Di Stefano, R. (2007). Relazione fra i percorsi biosintetici degli HCTA, dei flavonoli e degli antociani nell’acino d’uva. Rivista di Viticoltura e Enologia, 60(3), 59–70. Sun, B., Leandro, C., Ricardo da Silva, J. M., & Spranger, M. I. (1998). Separation of grape and wine proanthocyanidins according to their degree of polymerization. Journal of Agricultural and Food Chemistry, 46, 1390–1396. Sun, B., Pinto, T., Leandro, M. C., Ricardo-Da-Silva, J. M., & Spranger, M. I. (1999). Transfer of catechins and proanthocyanidins from solid parts of the grape cluster into wine. American Journal of Enology and Viticulture, 50(2), 179–183. Sun, B., & Spranger, M. I. (2005). Changes in phenolic composition of Tinta Miúda red wines after 2nd years of ageing in bottle: Effect of winemaking technologies. European Food Research Technology, 221, 305–312. Sun, B., Spranger, M. I., Roque-do-Vale, F. M., Leandro, C., & Belchior, P. (2001). Effect of different winemaking technologies on phenolic composition in Tinta Miúda red wines. Journal of Agricultural and Food Chemistry, 49(12), 5809–5816. Suriano, S., Ceci, G., & Tamborra, P. (2012). Impact of different winemaking techniques on polyphenols compounds of nero di Troia wines. Italian Food Beverage Technology, 70, 5–15. Tamborra, P., & Esti, M. (2010). Authenticity markers in Aglianico, Uva di Troia, Negroamaro and Primitivo grapes. Analytica Chimica Acta, 660, 221–226. Vivas, N., Nonier, M. F., Vivas de Gaulejac, N., Absalon, C., Bertrand, A., & Mirabel, M. (2004). Differentiation of proanthocyanidin tannins from seeds, skins and stems of grape (Vitis vinifera) and heatwood of Quebracho (Shinopsis balansae) by matrix assisted laser desorption/ionisation time of flight mass spectrometry and thioacidolysis/liquid chromatography/electrospray ionisation mass spectrometry. Analytica Chimica Acta, 513, 147–156.