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RESULTS: The white commercial sparkling wines tested had much less total phenolic, proanthocyanidin content, antioxidant capacity and scavenger activity ...
Research Article Received: 8 December 2009

Revised: 31 May 2010

Accepted: 1 June 2010

Published online in Wiley Interscience: 9 July 2010

(www.interscience.wiley.com) DOI 10.1002/jsfa.4064

Proanthocyanidin content, antioxidant capacity and scavenger activity of Portuguese sparkling wines (Bairrada Appellation of Origin) ´ ˜ a∗ Fernando J Gonc¸alves,a Ana C Correia,a Joao ˜ Cantao, ˜ a Antonio M Jordao, ´ b and Maria L Gonzalez ´ M Dolores Rivero-Perez SanJose´ b Abstract BACKGROUND: The main object of the present study was to investigate the different proanthocyanidin fraction (monomeric, oligomeric and polymeric fraction) contents, antioxidant capacity and scavenger activity of the most important and representative commercial sparkling wines available in Bairrada Portuguese Appellation of Origin. RESULTS: The white commercial sparkling wines tested had much less total phenolic, proanthocyanidin content, antioxidant capacity and scavenger activity than the sparkling red wines. For all white and red sparkling wines the polymeric fraction of proanthocyanidins was the most abundant fraction quantified. The antioxidant capacity was positively correlated with the different proanthocyanidin fractions studied. However, in general, higher correlations between total polyphenols, different proanthocyanidin fractions and antioxidant capacity were found only for red sparkling wines. CONCLUSION: The results confirm that Portuguese sparkling wines from Bairrada Appellation of Origin are good sources of antioxidants when compared with other wines elaborated from other grapes varieties and from other regions. At same time, good linear correlations between the levels of each different proanthocyanidin fractions and total polyphenols with antioxidant capacity were found for the commercial sparkling wines analysed. c 2010 Society of Chemical Industry  Keywords: antioxidant capacity; proanthocyanidins; scavenger activity; sparkling wines

INTRODUCTION

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Sparkling wine produced in Portugal, and especially in the Bairrada region (north central area of Portugal), is produced according to the m´ethode traditionelle champenoise. Among the different processes of wine-making, sparkling wine production is a very distinctive one. Grapes are harvested at lower sugar content, and the must obtained by pressing is first fermented in temperaturecontrolled tanks, followed by a second fermentation in the bottle. The pressure applied to the grapes to obtain the must (first and second pressings), the grape varieties used, and the ageing period during the second fermentation in contact with lees are factors that affect the quality of the final product. Sparkling wine production is very important for the economy of the Bairrada area in Portugal. This region is traditionally the main Portuguese wine region for sparkling wine production (especially for white wines) and has begun to export to other countries, especially the European Union. Traditionally, four different varieties of autochthonous grapes (Bical, Cercial, Maria Gomes and Baga) are used for making sparkling wines. Over the last decade, health effects of wine consumption have been studied in depth. Wine is an important component in the Mediterranean dietary tradition because it is very rich in antioxidant compounds. The antioxidant potential of red wines is

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due to the presence of phenolic compounds, which may inhibit platelet aggregation,1 prevent oxidation of the human low-density lipoproteins (LDL),2 and decrease inflammatory and carcinogenic processes.3 Phenols are considered to be free radical scavengers, and their antioxidant properties depend on their chemical structure. Specifically, these properties depend on their ability to donate hydrogen or electron and their ability to delocalize the unpaired electron within the aromatic structure.4 Flavan-3-ols, including proanthocyanidins, flavonols, and anthocyanins, are the most important compounds that contribute to red wine antioxidant properties.5,6 The phenolic composition of wine varies not only



Correspondence to: Ant´onio M Jord˜ao, Polytechnic Institute of Viseu (Centre for the Study of Education, Technologies and Health), Agrarian Higher School, Department of Food Industries, Estrada de Nelas, Quinta da Alagoa, Ranhados 3500-606 Viseu, Portugal. E-mail: [email protected]

a Polytechnic Institute of Viseu, Agrarian Higher School, Department of Food Industries, Estrada de Nelas, Quinta da Alagoa, Ranhados 3500-606 Viseu, Portugal b Department of Biotechnology and Food Science, University of Burgos, Plaza Misael Ba˜nuelos s/n 09001 Burgos, Spain

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Proanthocyanidin content of Portuguese sparkling wines

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France39 and Brazil40 . Portuguese wines8,11,41 as well as red and white grape cultivars42,43 were also studied for their proanthocyanidin contents. However, for antioxidant capacity and scavenger activity of Portuguese wines, there is a considerable lack of information. The latter is particularly poor in respect of sparkling wines. Thus the main object of the present study was to investigate the different proanthocyanidin fraction (monomeric, oligomeric and polymeric fraction) content, antioxidant capacity and scavenger activity of the most important and representative commercial sparkling wines available in Bairrada Portuguese Appellation of Origin. This paper will help towards a better understanding of the quality of current sparkling wines from these Portuguese wine appellation origins.

MATERIAL AND METHODS Wine samples A total of 24 most representative commercial Portuguese sparkling wines from Bairrada Appellation of Origin with different residual sugar contents (classified as brut, half-dry and sweet) were tested. All wines were elaborated from the most characteristic red and white grape varieties of Vitis vinifera cultivated in the region. The sparkling wines were produced according to the m´ethode traditionelle champenoise. The different commercial sparkling wines tested in this study are listed in Table 1. Chemicals Ethyl acetate, diethyl ether and methanol were purchased from Flucka-Biochemika (Buchs, Switzerland). ABTS, 6-hidroxyl2,5,7,8-tetramethyl-2-carboxylic acid (Trolox), 2,4,6-tris(2-pyridyls-triazine) (TPTZ), 2-desoxy-D-ribose and phenazine methosulfate (PMS) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Potassium persulfate (K2 O8 S2 ), iron(III) chloride 6-hydrate (FeCl3 .6H2 O), iron(II) sulfate 7-hydrate (FeSO4 .7H2 O), hydrogen peroxide (H2 O2 ), L-ascorbic acid and trichloroacetic acid (TCA) were obtained from Panreac (Barcelona, Spain). Thiobarbituric acid (TBA) was purchased from Merck (Darmstadt, Germany), nicotinamide adenine dinucleotide disodium salt (NADH) and 4nitroblue tetrazolium chloride (NBT) from Roche (Indianapolis, USA). Finally, EDTA tetrasodium salt was purchased from Amresco (Cleveland, OH, USA). The following parameters in the sparkling wines were determined in quadruplicate, except for general chemical analysis, which were determined in duplicate. Additionally, all wine samples were degasified before analysis. General chemical analysis The sparkling wine samples tested in our study were analysed for pH, titratable acidity, alcohol level, free and total SO2 levels, total anthocyanins, colour density and hue, using the analytical methods recommended by the OIV.44 Total phenolic content The total phenolic content of the sparkling wine samples was determined with Folin–Ciocalteu reagent, using gallic acid as standard. The Singleton and Rossi45 improved method was applied. The results were expressed as gallic acid equivalents (mg L−1 ).

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with the variety of grape used but also with cultivation technique, maturity, oenological technique and the ageing process.7 – 11 One of the most important phenolic compounds present in wines are proanthocyanidins. These compounds are present in the solid parts of grape clusters (skins, seeds and stems) and in traces in the pulp.12 Proanthocyanidins are of undoubted importance in oenology because they contribute to the sensory properties of wine, and they have an important role in the process of maturation and ageing of red wines. During wine ageing, these molecules, which are highly reactive species, undergo several reactions, such as polymerization,13 oxidation,14 and interactions with polysaccharides, proteins and other substances.15 Additionally, it was been shown that wine astringency depends on the structural characteristics of proanthocyanidins, such as mean degree of polymerization and percentage of galloylation.16 Grape proanthocyanidins have been reported to possess a broad spectrum of biological, pharmacological and therapeutic activities against free radicals and oxidative stress, as shown by in vitro and in vivo studies.17 Several authors have demonstrated that the peroxy radicalscavenging capacities of red wines are related to the amount of total proanthocyanidins.18,19 According to some authors,20 catechin is one of the main antioxidants in red wine, while caffeic acid may be considered one of the main antioxidants in white wine. However, there is no consensus whether the antioxidant properties of wines are linked to the total phenol concentration rather than individual phenols.21 Recently, Sun et al.22 evaluated the in vitro antioxidant activity of several red wine polyphenolic fractions, including monomers, oligomers, polymers, anthocyanins and complexes. According to these authors, the phenolic complex fractions and newly formed condensation products between epicatechin and malvidin-3-glucoside maintain antioxidant activities as strong as those of their compositional phenolics. There are various methods to evaluate the antioxidant capacity of different foods, such as wine, which involve different mechanisms.6,19 Chemical methods are based on the scavenging of reactive nitrogen and oxygen species such as peroxynitrite,23 hydroxyl radical and superoxide.24 Other methods measure the disappearance of free radicals such as ABTS (2,2 -azinobis-(3ethyl-benzothiazoline-6-sulfonate) cation radical)25 or DPPH (2,2diphenyl-1-picrylhydrazyl)26 through spectrophotometric measurement. Other assays to determine the total antioxidant power include techniques such as the ferric reducing/antioxidant power method (FRAP method)27 or use in situ electrochemically generated bromine.28 According to Benzie and Strain,27 the FRAP assay offers a putative index of antioxidant, or reducing, potential of samples. At low pH, when a ferric tripyridyltriazine (Fe(III) – TPTZ) complex is reduced to the ferrous (Fe(II)) form, an intense blue colour with an absorption maximum at 593 nm develops. The antioxidant capacity values changes according to the method used. The lack of strong correlation between different methods, e.g. DPPH and ABTS,29 is probably attributable to the fact that every individual phenol compound contained in wine causes a different response to the specific radical used in the assay. Thus the use of a single method cannot provide a comprehensive prediction of antioxidant efficacy of the different compounds.30 In recent years several authors have reported the phenolic composition and antioxidant capacity of wines from different countries, namely from Austria,31 Croatia,32 Czech Republic,29 Italy,19,33 USA,34 China,35 South Africa,36 Greece,37 Spain,20,21,38

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Table 1. Origin, classification and varietal composition of commercial sparkling wines tested from Bairrada Appellation of Origin Wine sample coding

Commercial wine designation

Residual sugar content

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 HD1 HD2 HD3 HD4 HD5 HD6 HD7 S1 S2 S3 S4 S5 S6

S˜ao Jo˜ao Trono Real EVB Valdarcos Alto Viso Diamante Azul M&M Bairrada Bruto Quinta Mata Fidalga Conselheiro Alianc¸a Reserva Danubio ´ Coroa de Rei Torre Ouro Reserva 46 Car´ıcia Caves Barroc˜ao Neto Costa Eldorado Neto Costa Danubio ´ Flor Azul Montanha Caves Primavera

Brut (50 g L−1 )

Vintage year 1999 2005 2002 2002 2006 2004 2005 2005 2004 2003 2005 2001 2004 2005 2003 2005 2004 2001 2001 2003 2006 2006 2005 2006

Grape cultivar(s) Bical and Maria Gomesa Bical and Maria Gomesa Bical and Maria Gomesa Bical and Maria Gomesa Bical, Cercial and Maria Gomesa Bical and Maria Gomesa Bical, Cercial and Maria Gomesa Bagab Bagab Bagab Bagab Bical, Cercial and Maria Gomesa Bical and Maria Gomesa Bical and Maria Gomesa Bical and Maria Gomesa Cercial and Maria Gomesa Cercial and Maria Gomesa Cercial and Maria Gomesa Bical, Cercial and Maria Gomesa Bical, Cercial and Maria Gomesa Bical and Cerciala Bical and Cerciala Bical and Cerciala Bical, Cercial and Maria Gomesa

White grape variety. Red grape variety.

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Fractionation of proanthocyanidins according to their polymerization degree For fractionation of wine proanthocyanidins according to their polymerization degree, catechins (monomers), oligomeric (degree of polymerization ranging from 2 to 12–15) and polymeric (degree of polymerization >12–15) fractions, a C18 Sep-Pack column was used, following the method described by Sun et al.43 Thus each sample was passed through the two preconditioned neutral Sep-Pack cartridges connected in series. To eliminate phenolic acids, 4 mL de-alcoholized medium was adjusted to pH 7.0 and then passed through the two connected Sep-Pack cartridges preconditioned with 10 mL water adjusted to pH 7.0. After drying the column with N2 , elutions were carried out first with 25 mL ethyl acetate to elute catechins and oligomeric proanthocyanidins, and then the polymeric fraction was eluted with 10 mL methanol. To separate the monomeric from oligomeric fraction, the ethyl acetate fraction was evaporated to dryness under vacuum at 25 ◦ C, dissolved in distilled water and then redeposited onto the same connected cartridges preconditioned with distilled water. After drying the cartridges with N2 , catechins and oligomeric proanthocyanidins were eluted sequentially with 25 mL diethyl ether (catechin fraction) and finally with 10 mL methanol (oligomeric fraction). For each fraction obtained previously, flavanols were quantified using the modified vanillin assay described by Sun et al.46 Thus the vanillin reaction with catechin fraction was carried out in a 30 ◦ C water bath for 15 min, and measurement of A500 was also performed at 30 ◦ C. For oligomeric and polymeric fractions, both the vanillin reaction and measurement of A500 were performed

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at room temperature and the maximum A500 was taken as the measured value.

FRAP method This method was used to measure the reductive power of a sample.27 It is based on increased absorbance at 593 nm due to formation of tripyridyl-s-triazine complexes with iron(II) (TPTZFe(II)) in the presence of a reductive agent. The reactive mixture was prepared by mixing 25 mL sodium acetate buffer solution (0.3 mol L−1 , pH 3.6), 2.5 mL TPTZ (10 mmol L−1 ), 2.5 mL FeCl3 (20 mmol L−1 ) and 3 mL water. 30 µL of diluted wine sample (diluted in water at 1 : 50 and 1 : 5, for red and white wines, respectively) was added to 970 µL of the latter reactive mixture and incubated at 37 ◦ C for 30 min. The results were expressed as mmol L−1 of Fe(II), using linear calibration obtained with different concentration of FeSO4 (0.0 to 1.2 mmol L−1 ).

ABTS •+ method This assay is based on decoloration that occurs when the radical cation ABTS •+ is reduced to ABTS.25 The radical was generated by reaction of a 7 mmol L−1 solution of ABTS in water with 2.45 mmol L−1 potassium persulfate (1 : 1). The assay was made up with 980 µL ABTS •+ solution and 20 µL of the sample (at a dilution of 1 : 50 in water). The reaction takes place in darkness at room temperature. Absorbance measurements at 734 nm were made after 15 min reaction time. The results were expressed in mmol L−1 of Trolox, using the relevant calibration curve.

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Table 2. General chemical and phenolic composition of the commercial sparkling wines tested from Bairrada Appellation of Origin Wine sample coding B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 HD1 HD2 HD3 HD4 HD5 HD6 HD7 S1 S2 S3 S4 S5 S6

Alcohol content (%, v/v)

Total acidity (g L−1 tartaric acid)

pH

Total SO2 (mg L−1 )

11.8 ± 0.2 12.5 ± 0.1 11.2 ± 0.1 11.7 ± 0.3 11.6 ± 0.1 11.6 ± 0.2 12.1 ± 0.04 11.6 ± 0.1 11.7 ± 0.2 12.0 ± 0.0 11.4 ± 0.2 11.3 ± 0.2 11.5 ± 0.2 11.2 ± 0.1 10.2 ± 0.05 11.7 ± 0.3 11.5 ± 0.4 12.0 ± 0.1 11.2 ± 0.2 11.5 ± 0.2 11.5 ± 0.1 11.5 ± 0.2 11.0 ± 0.2 11.4 ± 0.2

5.25 ± 0.20 6.37 ± 0.52 6.22 ± 0.81 5.77 ± 0.30 5.62 ± 0.09 5.40 ± 0.14 5.47 ± 0.44 5.33 ± 0.34 4.72 ± 0.13 4.95 ± 0.08 5.10 ± 0.45 5.25 ± 0.56 5.02 ± 0.87 6.90 ± 0.45 4.50 ± 0.43 5.77 ± 0.15 5.63 ± 0.56 4.20 ± 0.43 4.50 ± 0.21 4.95 ± 0.78 5.17 ± 0.10 5.02 ± 0.29 5.25 ± 0.34 5.85 ± 0.76

3.18 ± 0.03 3.22 ± 0.05 3.06 ± 0.01 3.20 ± 0.07 3.17 ± 0.02 2.93 ± 0.01 3.05 ± 0.02 3.26 ± 0.03 3.28 ± 0.04 3.31 ± 0.05 3.20 ± 0.02 2.95 ± 0.03 2.97 ± 0.01 2.54 ± 0.02 3.14 ± 0.02 2.96 ± 0.05 2.74 ± 0.03 3.21 ± 0.01 2.90 ± 0.02 2.89 ± 0.04 2.76 ± 0.02 2.78 ± 0.03 2.89 ± 0.03 2.71 ± 0.01

121.6 ± 8.53 99.2 ± 4.98 73.6 ± 4.59 67.2 ± 3.87 147.2 ± 3.24 128.0 ± 3.57 118.4 ± 6.98 67.2 ± 4.67 70.4 ± 6.21 44.8 ± 3.93 64.0 ± 2.24 140.8 ± 5.82 76.8 ± 7.81 128.0 ± 9.10 38.4 ± 2.32 67.2 ± 3.22 73.6 ± 5.76 48.0 ± 3.91 86.4 ± 4.56 118.4 ± 7.12 80.0 ± 6.78 121.6 ± 7.89 112.0 ± 2.45 44.8 ± 4.52

Free SO2 (mg L−1 )

Total polyphenols (mg L−1 )a

Total anthocyanins (mg L−1 )b

Colour density (abs. units)c

Colour hue (abs. units)

9.6 ± 0.15 12.8 ± 1.63 12.8 ± 1.18 9.8 ± 0.74 19.2 ± 1.11 16.0 ± 4.20 12.8 ± 2.43 16.0 ± 2.78 16.0 ± 3.98 9.6 ± 0.54 16.0 ± 3.21 19.2 ± 3.98 6.4 ± 1.09 28.8 ± 4.98 9.0 ± 0.98 10.3 ± 1.54 9.6 ± 1.39 4.0 ± 0.43 9.6 ± 1.21 28.8 ± 4.21 9.6 ± 2.19 16.0 ± 1.98 25.6 ± 3.56 7.8 ± 2.81

208.8 ± 5.3 182.1 ± 0.4 137.6 ± 8.6 157.9 ± 7.0 275.5 ± 1.7 202.6 ± 1.2 369.9 ± 9.7 1926.6 ± 2.9 3069.7 ± 23.1 1788.1 ± 4.6 2523.8 ± 54.3 230.4 ± 1.7 254.9 ± 2.8 171.2 ± 0.8 212.6 ± 1.62 168.9 ± 4.0 217.0 ± 4.8 54.8 ± 1.8 126.8 ± 2.2 54.8 ± 1.8 147.8 ± 3.9 91.3 ± 8.49 119.8 ± 8.7 108.9 ± 2.7

ND ND ND ND ND ND ND 129.3 ± 6.8 70.3 ± 5.1 55.7 ± 3.4 92.1 ± 3.7 ND ND ND ND ND ND ND ND ND ND ND ND ND

0.26 ± 0.02 0.10 ± 0.10 0.12 ± 0.05 0.18 ± 0.07 0.23 ± 0.04 0.26 ± 0.07 0.18 ± 0.03 1.71 ± 0.05 1.19 ± 0.04 1.41 ± 0.06 0.81 ± 0.01 0.12 ± 0.02 0.27 ± 0.02 0.15 ± 0.01 0.35 ± 0.08 0.12 ± 0.06 0.28 ± 0.04 0.16 ± 0.02 0.12 ± 0.05 0.08 ± 0.02 0.12 ± 0.07 0.07 ± 0.01 0.09 ± 0.02 0.18 ± 0.06

ND ND ND ND ND ND ND 0.86 ± 0.05 1.87 ± 0.09 0.87 ± 0.03 0.77 ± 0.01 ND ND ND ND ND ND ND ND ND ND ND ND ND

a

Values expressed in gallic acid equivalents. Values expressed in malvidin-3-glucoside equivalents. c 1 cm path length cell for white sparkling wines; 1 mm path length cell for red sparkling wines. ND, values not determined; values are given as the mean ± SD of the two experiments. b

Hydroxyl radical-scavenging activity (HRSA) Desoxyribose (2-desoxy-D-ribose) decays when exposed to hydroxyl radicals generated by the Fenton reaction.47 The hydroxyl radicals (HO •) were generated though the following system: 10 µL FeCl3 (0.1 mmol L−1 ), 10 µL ascorbic acid (0.1 mmol L−1 ), 10 µL H2 O2 (1 mmol L−1 ) and 10 µL EDTA (0.1 mmol L−1 ). Samples (15 µL at a dilution of 1 : 50 in water) were incubated at 37 ◦ C for 1 h, with 20 µL desoxyribose (1 mmol L−1 final concentration) in the presence of FeCl3 , ascorbic acid, H2 O2 , and EDTA. A 1.5 mL amount of TCA (28%, w/v) and 1 mL TBA (1%, w/v, 0.05 mol L−1 NaOH) were added to 1 mL of the sample under incubation and held for 15 min at 100 ◦ C, after which it was left to cool to room temperature. The malondialdehyde (MDA) formed from the decay of desoxyribose was evaluated in reaction with TBA and measured at 532 nm. The result was expressed as inhibition percent in relation to a control test (without the sample).

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Statistical analysis The same samples were analysed in four replications and the results were expressed as mean value ± standard error. The coefficient variation and correlation coefficient between antioxidant capacity values and the content of phenolic composition (total polyphenols and the different proanthocyanidin fractions) were performed using Microsoft Excel 2003 software. In order to determine whether there was a statistically significant difference between the results obtained for different proanthocyanidin fraction contents and for antioxidant capacity, hydroxyl and superoxide radical scavenger activities, an analysis of variance and comparison of treatment means (ANOVA, one-way) were carried out using SPSS version 11.0 (SPSS Inc., Chicago, IL, USA). Differences between means were tested using Duncan’s test (α = 0.05).

RESULTS AND DISCUSSION General chemical and phenolic composition The general chemical composition (alcohol content, total acidity, pH and SO2 levels) of all samples is summarized in Table 2. The alcohol content of all commercial sparkling wines analysed ranged from 10.2% (HD4) to 12.5% (B2) and the average value was 11.5%.

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Superoxide radical-scavenging activity (SRSA) The superoxide radical reacts with NBT to generate a coloured compound with absorbance to 560 nm.48 The antioxidant scavenging superoxide radical values are associated with the coloration formed during the reaction process. The reactive solution was made with 50 µL NADH (77 µmol L−1 ), 50 µL NBT (50 µmol L−1 ) and 5 µL PMS (3.3 µmol L−1 final concentration) in a medium of 16 mmol L−1 Tris-HCL, pH 8, and 10 µL of the sample. The result

was expressed as inhibition percent in relation to a control test (without the sample).

www.soci.org For total acidity the values quantified varied from 4.20 (HD7) to 6.90 g L−1 (HD3) in equivalent tartaric acid and the average value was 5.34 g L−1 in equivalent tartaric acid. The high total acidity and, in particular, the low pH values are typical of sparkling wines because the low values of these parameters have a positive impact on the sensorial evaluation of this wine category, especially in the freshness characteristics of white sparkling wines. In general, these pH low values are a consequence of two main factors: the natural acidity of the grape varieties used and the higher ascorbic and/or citric acid content added during the wine-making process. Table 2 also shows the general phenolic composition (total polyphenols, total anthocyanins, colour density and colour hue) obtained for the sparkling wine samples. There was a wide range of total polyphenolic concentration in the wines tested. As expected, the sparkling red wines had higher amounts of total polyphenols (average value 2327.0 mg L−1 ) and colour density (1.28 abs. units) than the sparkling white wines (average value 174.6 mg L−1 for total polyphenols and 0.17 abs. units for colour density). This is explained, namely, by the grape skin (grape pigments or anthocyanins are present in red grapes only) and seed contact time (e.g., maceration process), and by the high temperature used during the first fermentation in the red sparkling wine process. The results obtained in the Portuguese sparkling wines studied presented, in general, higher amounts of total polyphenols than several Spanish sparkling wines (Cavas) reported by Satu´e-Garcia et al.20 In this study total polyphenols ranged from 148.9 to 186.1 mg L−1 in equivalent gallic acid. Many factors affect the quantitative phenolic composition, such as the grape variety, pressure applied to the grapes to obtain the must, the clarification process, and the ageing period in the second fermentation during contact with lees.20,49,50

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Proanthocyanidin content according to their polymerization degree The wines, especially the red wines, represent a rich source of polyphenols, e.g. catechins, proanthocyanidins and other phenolics – all potent antioxidants possessing biological properties that may protect against cardiovascular disease.17 Monomeric, oligomeric and polymeric fractions of proanthocyanidins quantified in the different commercial red and white sparkling wines tested are shown in Table 3. There was a wide range of concentration of the different proanthocyanidin fractions in selected sparkling wines. As expected, the red wines had higher significantly amounts for all three proanthocyanidins fractions studied when compared with white sparkling wines. At same time, the relative concentration of each proanthocyanidin fraction decreased in the order polymeric > oligomeric > monomeric fraction. These results are in agreement with those available in the literature for white and red table wines.8,41,43 Thus the content of monomeric fraction varied from 0.7 (B4) to 5.5 mg L−1 (B5), averaging 2.8 mg L−1 , for the white sparkling wines, and from 9.6 (B10) to 13.5 mg L−1 (B9), averaging 11.2 mg L−1 , for the red sparkling wines. For the oligomeric fraction, we obtained a variation from 13.7 (B4) to 25.2 mg L−1 (B7), averaging 20.4 mg L−1 , for white sparkling wines, and a variation from 49.7 (B10) to 60.9 mg L−1 (B9), averaging 53.7 mg L−1 , for red sparkling wines. Finally, the content of polymeric fraction varied from 25.3 (B4) to 55.9 mg L−1 (HD2), averaging 41.3 mg L−1 , for the white sparkling wines, and from 330.7 (B10) to 420.3 mg L−1

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Table 3. Monomeric, oligomeric and polymeric fraction of proanthocyanidin values of commercial sparkling wines tested from Bairrada Appellation of Origin Wine sample coding

Monomeric fraction (mg L−1 )

Oligomeric fraction (mg L−1 )

Polymeric fraction (mg L−1 )

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 HD1 HD2 HD3 HD4 HD5 HD6 HD7 S1 S2 S3 S4 S5 S6 AV (w) CV % (w) R (w) AV (r) CV % (r) R (r)

1.5 ± 0.3a 2.0 ± 0.5a 2.5 ± 0.4a 0.7 ± 0.1a 5.5 ± 1.1b 3.6 ± 1.5a 3.6 ± 1.0a 10.2 ± 2.1c 13.5 ± 1.9c 9.6 ± 1.5c 11.7 ± 3.1c 3.0 ± 0.5a 4.5 ± 1.1b 1.1 ± 0.2a 3.2 ± 0.5a 1.5 ± 0.1a 2.6 ± 0.3a 2.4 ± 0.5a 3.5 ± 0.2a 2.0 ± 0.4a 4.6 ± 0.5b 3.1 ± 0.8a 3.4 ± 0.6a 3.1 ± 0.2a 2.8 ± 0.56 42.4 0.7–5.5 11.2 ± 2.15 15.5 9.6–13.5

18.5 ± 1.2a 18.9 ± 1.8a 17.4 ± 2.1a 13.7 ± 0.5a 25.0 ± 1.9a 20.2 ± 2.1a 25.2 ± 0.9a 50.9 ± 3.4b 60.9 ± 2.5b 49.7 ± 3.0b 53.3 ± 3.6b 24.2 ± 0.6a 24.7 ± 0.8 a 17.1 ± 1.4a 21.4 ± 0.7a 15.3 ± 0.4a 24.8 ± 2.5a 19.8 ± 1.0a 20.2 ± 0.7a 18.6 ± 0.3a 21.5 ± 0.8a 20.2 ± 0.9a 22.4 ± 2.3a 20.3 ± 1.4a 20.4 ± 1.12 16.0 13.7–25.2 53.7 ± 3.12 9.4 49.7–60.9

35.3 ± 4.2a 37.2 ± 2.5a 30.4 ± 0.9a 25.3 ± 1.2a 52.7 ± 3.4b 36.2 ± 3.5a 45.1 ± 0.5b 370.9 ± 2.0c 420.3 ± 6.4c 330.7 ± 5.4c 405.1 ± 4.3c 47.2 ± 2.1b 55.9 ± 1.5b 30.3 ± 0.7a 43.0 ± 3.4b 25.7 ± 1.8a 55.1 ± 4.5b 42.7 ± 2.1b 44.2 ± 1.4b 30.1 ± 2.4a 53.1 ± 0.9b 47.1 ± 1.6b 46.7 ± 2.1b 44.0 ± 0.5b 41.3 ± 1.97 23.2 25.3–55.9 381.7 ± 4.52 10.4 330.7–420.3

Values are given as the mean ± SD of the four experiments; different letters in a column indicate statistically significant differences according to the Duncan test (α = 0.05); AV (w), average values for white sparkling wines; CV % (w), coefficient of variation for white sparkling wines; R (w), range for white sparkling wines; AV (r), average values for red sparkling wines; CV % (r), coefficient of variation for red sparkling wines; R (r) range for red sparkling wines.

(B9), averaging 381.7 mg L−1 , for the red sparkling wines. In general, the sparkling wines studied contained lower concentrations of the different proanthocyanidin fractions than in red and white table Portuguese wines, as quantified by other authors.42,43 This discrepancy reflects the behaviour of wines from different sources produced by using different technologies of wine-making and maturation. For example, it should be emphasized that during the making of sparkling wines (especially white sparkling wines) the pressure applied to the grapes to obtain the must is lower than the usual pressure used during the production of table wines. Furthermore, sparkling wines are usually made from very clean musts, which are intensively clarified. Thus these differences are some of main factors that could explain the lower proanthocyanidin concentration quantified in the sparkling wines studied.

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Table 4. Antioxidant capacity (FRAP and ABTS methods), hydroxyl (HRSA) and superoxide radical (SRSA) scavenger activities values of commercial sparkling wines tested from Bairrada Appellation of Origin Wine sample coding

FRAP (mmol L−1 Fe(II))a

ABTS (TEAC mmol L−1 )b

HRSA (%)

SRSA (%)

3.56 ± 0.09a 3.72 ± 0.08a 2.56 ± 0.09a 3.13 ± 0.08a 6.09 ± 0.14a 3.85 ± 0.07a 4.71 ± 0.13a 34.07 ± 0.48b 56.48 ± 0.38b 36.08 ± 0.84b 40.57 ± 1.00b 5.23 ± 0.06a 5.95 ± 0.03a 4.46 ± 0.15a 5.01 ± 0.05a 3.09 ± 0.04a 4.13 ± 0.02a 4.49 ± 0.07a 5.44 ± 0.14a 4.49 ± 0.07a 6.20 ± 0.07a 5.09 ± 0.02a 5.52 ± 0.08a 5.01 ± 0.07a 4.51 ± 1.02 26.80 2.56–6.20 41.80 ± 9.11 24.30 34.07–56.48

1.67 ± 0.08a 1.93 ± 0.09a 1.43 ± 0.02a 1.80 ± 0.10a 2.80 ± 0.10a 2.12 ± 0.04a 2.35 ± 0.05a 30.89 ± 1.56b 37.42 ± 1.49b 25.60 ± 0.41b 28.66 ± 0.46b 1.78 ± 0.14a 2.27 ± 0.05a 1.24 ± 0.19a 1.79 ± 0.07a 0.87 ± 0.16a 2.82 ± 0.12a 1.02 ± 0.10a 2.10 ± 0.04a 1.02 ± 0.10a 2.27 ± 0.08a 1.97 ± 0.09a 2.16 ± 0.05a 1.95 ± 0.11a 1.86 ± 0.48 29.15 1.02–2.82 30.64 ± 4.59 16.36 25.21–37.42

63.17 ± 1.71a 67.91 ± 0.94a 66.72 ± 0.28a 63.06 ± 0.98a 62.04 ± 1.27a 70.82 ± 1.78a 72.68 ± 3.17a 90.70 ± 2.01b 73.37 ± 2.26a 89.61 ± 0.13b 80.96 ± 2.82b 65.61 ± 2.26a 67.10 ± 2.54a 63.67 ± 4.55a 65.01 ± 1.86a 65.52 ± 3.71a 69.70 ± 0.82a 50.14 ± 3.37a 65.45 ± 3.88a 50.14 ± 3.37a 75.50 ± 1.54a 64.50 ± 3.61a 73.30 ± 0.87a 62.38 ± 1.26a 65.22 ± 6.42 9.79 50.14–75.50 83.66 ± 7.50 18.60 73.37–90.70

43.36 ± 1.23a 49.80 ± 5.45a 31.72 ± 2.01b 36.81 ± 3.02b 72.33 ± 0.34c 48.78 ± 3.29a 30.77 ± 3.06b 70.68 ± 4.55c 70.62 ± 1.90c 77.13 ± 0.64c 77.96 ± 1.15c 42.40 ± 1.39a 25.34 ± 1.82b 24.48 ± 2.96b 29.68 ± 3.78b 23.74 ± 3.26b 49.46 ± 1.31a 31.67 ± 1.72b 49.40 ± 1.72a 31.67 ± 1.72b 49.13 ± 2.05 a 50.12 ± 1.91a 50.90 ± 1.25a 41.76 ± 1.14a 40.66 ± 19.12 30.07 23.74–72.33 74.09 ± 4.24 5.40 70.62–70.68

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 HD1 HD2 HD3 HD4 HD5 HD6 HD7 S1 S2 S3 S4 S5 S6 AV (w) CV % (w) R (w) AV (r) CV % (r) R (r)

mmol L−1 of Fe(II); b Trolox equivalent; values are given as the mean ± SD of the four experiments; different letters in a column indicate statistically significant differences according to the Duncan test (α = 0.05); AV (w), average values for sparkling white wines; CV % (w), coefficient of variation for sparkling white wines; R (w), range for white sparkling wines; AV (r), average values for red sparkling wines; CV % (r), coefficient of variation for red sparkling wines; R (r) range for red sparkling wines.

a

J Sci Food Agric 2010; 90: 2144–2152

anthocyanins and complexes) and newly formed condensation products between epicatechin and malvidin-3-glucoside formed during wine storage and ageing maintain antioxidant activities as strong as those of their compositional phenolics. Thus the antioxidant capacity, considering the FRAP method, varied from 2.56 (B3) to 6.20 mmol L−1 Fe(II) (S3), averaging 4.51 mmol L−1 Fe(II), for the white sparkling wines and from 34.07 (B8) to 56.48 mmol L−1 Fe(II) (B9), averaging 41.80 mmol L−1 Fe(II), for the red sparkling wines. At same time, considering the results obtained using the ABTS antioxidant capacity method, similar relative results were obtained. The hydroxyl and superoxide radicals are also extremely reactive free radicals formed in biological systems and have been implicated as two highly damaging species in free radical pathology, capable of damaging almost every molecule found in living cells. The hydroxyl radical (HRSA) and superoxide radicalscavenging activities (SRSA) of all red and white sparkling wines tested are shown in Table 4. The results obtained varied from 50.14% (HD7) to 75.50% (S3), averaging 65.22% HRSA for the white sparkling wines and from 73.37% (B9) to 90.70% (B8), averaging 83.66% HRSA for the red sparkling wines. For superoxide radical, we

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Antioxidant capacity, scavenger activity and correlation with phenolic composition The antioxidant capacity evaluated by two different methods (FRAP and ABTS) in the commercial white and red sparkling wines tested is presented in Table 4. The significantly higher values obtained in sparkling red wines for antioxidant capacity is easily explained by the higher levels of polyphenols (including proanthocyanidins and anthocyanins) that were quantified in red sparkling wines. Previous studies reported that flavan-3-ols are the main phenolic fraction with higher potent antioxidant activity in wine.49,51 Additionally, although some authors6,52 reported strong antioxidant activities of anthocyanins and good correlation between anthocyanin concentration and antioxidant,53 few studies have verified the antioxidant activity of individual anthocyanins or the purified anthocyanin fractions. These purified anthocyanin fractions are obtained by the usual extraction methods and contain important amounts of proanthocyanidins (monomeric and polymeric fractions), and thus the latter compounds would contribute significantly to the total antioxidant activity of the extract.52 Recently, Sun et al.22 showed that different red wine polyphenolic fractions (including monomers, oligomers, polymers,

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Table 5. Linear correlations between the levels of each different proanthocyanidin fraction and total polyphenols with antioxidant capacity of commercial sparkling wines tested from Bairrada Appellation of Origin Correlation coefficient (adjusted R2 ) Antioxidant method Variables Sparkling white wines MF OF PF TP

FRAP

ABTS

0.62 0.56 0.64