Anthocyanin profile and antioxidant activity from 24 grape varieties cultivated in two Portuguese wine regions
Aims: The purpose of this work was to evaluate the general phenolic composition and the anthocyanin profile of 24 grape varieties from two Portuguese wine regions as well as their antioxidant activity in the different grape berry fractions (skins, pulps and seeds).
Methods and results: Individual anthocyanin composition of grape skin extracts was analyzed by HPLC, whereas total antioxidant activity was evaluated by two methods: DPPH and ABTS. In general, a high variability was found among the different autochthonous and non-autochthonous grape varieties in relation to the polyphenolic compounds analyzed, especially the individual anthocyanins. The individual anthocyanins in grape skin extracts were mainly malvidin (1.40-7.09 mg/g of skin), in particular malvidin-3-glucoside (0.62-6.09 mg/g of skin). The highest antioxidant activity was consistently detected in the seed extracts; however, it was not possible to establish a clear difference among the grape varieties analyzed.
Conclusion: High variability in polyphenolic content, individual anthocyanin composition and antioxidant activity was found among the diverse autochthonous and non-autochthonous grape varieties studied. Seeds showed the highest antioxidant activity, followed by skin and pulp, irrespective of the grape variety.
Significance and impact of the study: Most vineyards in Portugal grow Portuguese cultivars of Vitis Vinifera L. and other cultivars grown worldwide. The phenolic compounds and antioxidant activity of these grape cultivars have never been characterized under the environmental conditions of the Douro and Dão regions. The variability in phenolic content among the grape varieties studied confirms the hypothesis that genetic factors have an important role in the biosynthesis of these compounds and, consequently, in the antioxidant activity of grapes.
Grapes are the chief dietary sources of anthocyanins (Rechkemmer and Pool-Zobel, 1996). Anthocyanins are mainly localized in berry skin (Jordão et al., 1998a), where they are found at concentrations of 200 to 5000 mg/Kg of fresh grapes (Ribéreau-Gayon, 1982). Anthocyanin pigments are of noticeable importance in grapes and wines because of their dual role: first, they constitute an essential part of the sensory attributes, as their concentration, different forms and derivatives directly affect the color of the final wine and second, they are considered to have diverse biological properties and therefore are regarded as secondary metabolites with potential nutritional value. Grape anthocyanins are the 3-O-monoglucosides of delphinidin, cyanidin, petunidin, peonidin, and malvidin. Glucosylated derivatives of these anthocyanins, esterified at the C6 position of glucose with acetyl or coumaroyl groups, have also been detected, yet generally at low concentrations. Malvidin is the predominant monomeric anthocyanin in grapes and is the reddest of all anthocyanins, providing the characteristic color of young red wines.
Anthocyanin concentration and profile is influenced by several factors such as varietal diversity (Kallithraka et al., 2005), soil type (Yokotsuka et al., 1999), environmental conditions (Jackson and Lombard, 1993; Mateus et al., 2002), vineyard management (Jordão et al., 1998b), and grape maturity (Jordão et al., 1998a). Additionally, some grape varieties can have a high anthocyanin concentration but a low extractability index (Romero-Cascales et al., 2005).
According to several studies, flavan-3-ols, flavonols and anthocyanins are the most important compounds that contribute to red grape and wine antioxidant proprieties (Beecher, 2003; Simonetti et al., 1997). For Rivero-Pérez et al. (2008), the free anthocyanin fraction is mainly responsible for the total antioxidant capacity and scavenger activity in red wines. However, other authors have reported that there is no correlation between antioxidant activity and spectral anthocyanin content in grapes and finished wines (Arnous et al., 2002; Kallithraka et al., 2005). For example, considering some individual anthocyanins (cyanidin-3-glucoside, peonidin-3-glucoside and malvidin-3-glucoside) from Italian wines, Di Majo et al. (2008) reported low correlations between these individual compounds and their antioxidant capacity (ranging from 0.29 to 0.54). In addition, Jordão et al. (2012) reported differences in the correlation values between individual anthocyanins and antioxidant activity during the maceration process. Thus, there has been conflicting evidence regarding the contribution of anthocyanins to red grape and wine antioxidant properties.
Dão and Douro are two important wine-producing regions in northeastern Portugal. The Dão region is sheltered on nearly all sides by high granite mountains and is situated in the transition zone between maritime and continental climate, with abundant rainfall in the winter months and long warm dry summers leading up to harvest. The Douro region, which is situated around the Douro River basin, is sheltered from Atlantic winds by several mountains and has a continental climate, with hot and dry summers and cold winters. Although Portuguese Vitis vinifera L. autochthonous cultivars are the most frequently used cultivars for the production of high quality red wines in both regions, other varieties from other countries (especially from France) have been introduced in the past few years.
A few studies have been performed on the anthocyanin profile of Portuguese grape varieties. However, these studies were restricted to a small number of varieties from southern (Dallas and Laureano, 1994a; Jordão et al., 1998a) and northern Portugal (Dallas and Laureano, 1994a; Dopico-García et al., 2008; Mateus et al., 2002) and did not address the antioxidant activity of the different grape berry fractions.
Thus, the purpose of this work was to evaluate the anthocyanin profile in grape skin as well the antioxidant activity in different grape berry fractions (skins, pulps and seeds) of 24 grape varieties from both Portuguese wine regions. The results obtained in this work could be useful for selecting winemaking techniques in order to improve anthocyanin extraction and hence increase the antioxidant capacity of the wines produced in these two regions. Finally, we should note that this is the first report of the anthocyanin content and antioxidant activity of grape cultivars from the Dão and Douro wine regions.
Materials and Methods
In 2010, 24 grape varieties (Vitis vinifera L.) were harvested at technological maturity in good sanitary conditions from 6-year-old grapevines in two experimental vineyards of northeastern Portugal (one in the Douro region and the other in the Dão region). Grape berry samples (samples of 200 berries in duplicate) were picked randomly from 20 different vine plants in all possible conditions (e.g., different height and exposure to sunlight). All grape samples were kept frozen at -20 ºC until processing. The grape varieties studied are listed in Table 1.
Table 1. Grape variety and origin of the grape samples studied.
|Grape variety||Autochthonous/Non-autochthonous grape variety||Origin|
AG - autochthonous grape variety; NAG - non-autochthonous grape variety; * white grape variety with colored skin.
The evolutions of the climatic characteristics (rainfall, temperature and relative humidity) for the 2010 vintage in the two regions considered are summarized in Figure 1. In general, it was clear that during the three months (June, July and August) prior to sampling (at the end of September) the average temperature and average relative humidity was higher in the Dão region than in the Douro region. In addition, average rainfall for the months of July, August and September was lower in the Dão region.
Figure 1. Evolution of climatic characteristics (temperature, relative humidity and rainfall) for the 2010 vintage in the two regions considered.
2. General physico-chemical and phenolic parameters
Estimated alcohol degree, pH, titratable acidity and tartaric acid were measured directly from the must obtained after grape pressing, using the analytical methods recommended by the OIV (2006). Total phenolic compounds were determined by measuring absorbance at 280 nm (Ribéreau-Gayon et al., 1982). Non-flavonoids and flavonoids were determined using the method described by Kramling and Singleton (1969). Color intensity (A420 + A520 + A620) and hue (A420/A520) were estimated using the method described by the OIV (2006). All analyses were done in duplicate.
3. Sample preparation for antioxidant activity and individual anthocyanin composition analysis
Skins, pulps and seeds were removed manually from the berries, washed separately several times with distilled water, and dried on blotting paper. Before the extraction process, seeds were crushed. The extraction process followed the procedure described by Sun et al. (1996): 50 mL of 80 % methanol followed by 50 mL of 75 % acetone, each for 3 h with agitation. After clarification of the suspension by centrifugation (10 min at 3500 rpm), all the extracts were combined. Aliquots of each extract were filtered (Whatman 0.45 µm) and frozen at -20 ºC until processing for antioxidant activity (skin, pulp and seed extracts) and individual anthocyanin composition (skin extracts) analysis. Extractions were done in duplicate for each grape berry fraction.
4. Antioxidant activity determination
The total antioxidant activity of each grape berry fraction was determined from the extracts according to two previously described methods: ABTS and DPPH. The ABTS method is based on the discoloration that occurs when the radical cation ABTS.+ is reduced to ABTS (2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) (Re et al., 1999). The radical was generated by reaction of a 7 mM solution of ABTS in water with 2.45 mM potassium persulphate (1:1). The assay was made with 980 μL of ABTS.+ solution and 20 μL of sample (diluted 1:50 in water). The reaction was incubated for 15 min in darkness at room temperature, and then absorbance at 734 nm was measured.
The procedure using the DPPH method is described by Brand-Williams et al. (1995). Briefly, 0.1 mL of different sample concentrations was added to 3.9 mL of 2,2-diphenyl-1-picrylhydrazyl (DPPH) methanolic solution (25 mg/L). The DPPH solution was prepared daily and protected from the light. The reaction was carried out for 30 min at 20 ºC in closed Eppendorf tubes under shaking, and then absorbance at 515 nm was measured. Methanol was used as a blank reference. The antioxidant activity results were expressed as Trolox equivalents (TEAC mM), using the relevant calibration curve. All measurements were performed in duplicate.
5. Chromatographic analysis of individual anthocyanins
The analysis of individual anthocyanins from skin extracts was performed on a HPLC Dionex Ultimate 3000 Chromatographic System (Sunnyvale, California, USA) equipped with a quaternary pump Model LPG-3400 A, an auto sampler Model ACC-3000, a thermostated column compartment (adjusted to 25 ºC) and a multiple Wavelength Detector MWD-300. The column (250 x 4.6 mm, particle size 5 μm) was a C18 Acclaim® 120 (Dionex, Sunnyvale, California, USA) protected by a guard column of the same material. The solvents were (A) 40 % formic acid, (B) pure acetonitrile and (C) bidistilled water. The anthocyanin composition was analyzed by HPLC using the method described by Dallas and Laureano (1994b). Thus, initial conditions were 25 % A, 10 % B and 65 % C, followed by a linear gradient from 10 to 30 % B and 65 to 45 % C for 40 min, with a flow rate of 0.7 mL/min. The injection volume was 40 µL. Detection was made at 520 nm using Chromeleon 6.8 software (Sunnyvale, California, USA). Individual anthocyanins were quantified by means of calibration curve obtained with standard solutions of malvidin-3-glucoside chloride (>95 % purity) from Extrasynthese (Genay, France). The chromatographic peaks of anthocyanins were identified according to reference data previously described by Dallas and Laureano (1994b). All analyses were done in duplicate.
6. Statistical analysis
The data are presented as mean ± standard deviation. To determine whether there is a statistically significant difference between the data obtained for antioxidant activity and for the diverse phenolic compounds quantified in the different grape varieties, an analysis of variance (ANOVA, one-way) and comparison of treatment means were carried out using Statistica 7 software (StatSoft, Tulsa, USA). Scheffle’s test was applied to the data as comparison test to determine when samples are significantly different after ANOVA (p < 0.05). Principal component analysis (PCA) was used to analyze the data and study the relations between the grape varieties and their anthocyanin profile or antioxidant activity.
Results and discussion
1. General physico-chemical composition
The physico-chemical parameters of the grape varieties at technological maturity are summarized in Table 2. The estimated alcohol degree ranged from 9.53 % (v/v) (Grenache) to 14.84 % (v/v) (Malvasia Preta) with an average of 12.3 % (v/v). Titratable acidity, expressed as equivalent of tartaric acid, varied from 3.9 (Moreto Boal) to 13.5 g/L (Jean and Tinta Miúda) with an average of 7.3 g/L. The high titratable acidity and low pH values found in the majority of the grape varieties are probably a consequence of two main factors: their natural acidity and the ripening process (associated to low degradation rates of organic acids). In addition, these results are in agreement with the tartaric acid content of all grape varieties.
Table 2. General physico-chemical composition of grape varieties at technological maturity.
degree (%, v:v)
(g/L tartaric acid)
|Camarate||241 ± 10a||128 ± 5c||11.00 ± 0.02e||2.93 ± 0.04ef||5.1 ± 0.1abc||3.19 ± 0.23abc|
|Gewürztraminer||444 ± 1l||177 ± 1f||12.76 ± 0.01m||2.41± 0.00a||8.8 ± 0.2fgh||6.99 ± 0.02ijkl|
|Monvedro||273 ± 8bcd||130 ± 4cd||12.35 ± 0.01k||3.11± 0.00hij||4.7 ± 0.3ab||2.56 ± 0.02a|
|Moreto Boal||325 ± 2fg||152 ± 1e||13.59 ± 0.02p||3.17 ± 0.00ijk||3.9 ± 0.3a||3.35 ± 0.02bc|
|Negro Mole||318 ± 8efg||159 ± 4e||11.47 ± 0.01f||2.75 ± 0.00cd||6.2 ± 0.1cd||5.97 ± 0.08gh|
|Negro Mouro||319 ± 10efg||152 ± 5e||12.15 ± 0.02j||3.22 ± 0.00jk||4.1 ± 0.1a||3.38 ± 0.01bc|
|Alfrocheiro||260 ± 8ab||131± 6cd||11.96 ± 0.02h||2.91 ± 0.00ef||6.0 ± 0.4bcd||3.19 ± 0.23abc|
|Alvarilhão||297 ± 9def||144 ± 5de||13.45 ± 0.02o||2.83 ± 0.00de||8.0 ± 0.1ef||6.95 ± 0.06ijkl|
|Aramon||727 ± 6o||322 ± 3k||10.59 ± 0.02c||2.72 ± 0.01bc||9.5 ± 0.3gh||7.70 ± 0.12k|
|Bastardo||408 ± 2k||181 ± 1f||12.08 ± 0.02i||3.49 ± 0.00m||4.1 ± 0.2a||2.49 ± 0.68a|
|Cabernet Franc||268 ± 4abc||66 ± 0a||13.73 ± 0.01q||2.94 ± 0.01ef||6.0 ± 0.4bcd||5.56 ± 0.04fg|
|Carignan Noir||450 ± 3l||179 ± 1f||12.49 ± 0.02l||3.14 ± 0.01hij||8.5 ± 0.3fg||6.01 ± 0.11gh|
|Cornifesto||303 ± 6ef||108 ± 2b||14.01 ± 0.02r||3.26 ± 0.00k||6.0 ± 0.5bcd||3.35 ± 0.02bc|
|Gamay||487 ± 5m||228 ± 2h||11.67 ± 0.01g||3.09 ± 0.09hi||8.0 ± 0.1ef||6.38 ± 0.01hij|
|Grenache||378 ± 6ij||203 ± 4g||9.53 ± 0.01a||3.22 ± 0.07jk||10.0 ± 0.4h||6.22 ± 0.00ghi|
|Jean||387 ± 3jk||179 ± 1f||13.45 ± 0.02o||2.92 ± 0.01ef||13.5 ± 0.5i||7.04 ± 0.05jkl|
|Malvasia Preta||295 ± 8cde||129 ± 4cd||14.84 ±0.02s||3.38 ± 0.01l||8.5 ± 0.4fg||3.88 ± 0.17cd|
|Rufete||484 ± 8m||269 ± 5j||12.08 ± 0.01i||3.05 ± 0.01gh||5.5 ± 0.3bcd||2.87 ± 0.02ab|
|Sousão||336 ± 7gh||178 ± 4f||9.66 ± 0.02b||2.62 ± 0.01b||8.5 ± 0.1fg||7.24 ± 0.02kl|
|Tinta Amarela||530 ± 9n||270 ± 9j||10.93 ± 0.01d||2.91 ± 0.02ef||6.3 ± 0.1cd||3.50 ± 0.16bc|
|Tinta Barca||459 ± 9lm||230 ± 5hi||11.67 ± 0.02g||2.88 ± 0.01ef||9.0 ± 0.2fgh||6.53 ± 0.03ijk|
|Tinta Barroca||527 ± 7n||246 ± 3i||12.76 ± 0.01m||3.15 ± 0.01hij||5.5 ± 0.4bc||4.57 ± 0.37de|
|Tinta Miúda||358 ± 7hi||197 ± 4g||13.45 ± 0.02o||2.88 ± 0.00ef||13.5 ± 0.3i||7.25 ± 0.24kl|
|Tinto Cão||272 ± 6bcd||111 ± 2b||13.04 ± 0.01n||2.94 ± 0.00fg||6.8 ± 0.5de||4.96 ± 0.16ef|
a Grape berry weight and must volume of 200 berries; data are the average of two replicates ± standard deviation; different letters above means indicate statistically significant differences between grape varieties (p < 0.05).
2. General phenolic composition and anthocyanin profile
It is well known that the genetic potential for polyphenol biosynthesis and the degree of ripeness of individual grape varieties may affect the polyphenolic content in grape berries at harvest. In addition, the concentration of phenolic compounds is highly influenced by viticulture and environmental factors such as sunlight, temperature, altitude, soil type, and water and nutritional status (Jackson and Lombard, 1993; Yokotsuka et al., 1999). As expected, due to the reasons mentioned above, differences in phenolic composition were observed among grape varieties (Table 3).
Table 3. General phenolic composition of grape varieties at technological maturity.
(mg/L gallic acid)
(mg/L gallic acid)
(mg/L gallic acid)
|Camarate||989 ± 13a||946 ± 11abc||43 ± 2a||13.57 ± 0.83h||6.31 ± 0.35cde|
|Gewürztraminer||1124 ± 17ab||1031 ± 16bcd||93 ± 1abc||9.34 ± 0.24cde||7.96 ± 0.25ghi|
|Monvedro||1240 ± 14abc||1118 ± 13bcde||122 ± 1bcd||7.62 ± 0.16b||7.46 ± 0.15efgh|
|Moreto Boal||1032 ± 39a||961 ± 9abc||71 ± 3ab||5.21 ± 0.13a||11.33 ± 0.26j|
|Negro Mole||1375 ± 17abcd||1278 ± 16defg||97 ± 1abc||10.15 ± 0.38ef||5.54 ± 0.08abc|
|Negro Mouro||1593 ± 26def||1408 ± 23efgh||185 ± 3def||12.17 ± 0.11gh||6.46 ± 0.12cdef|
|Alfrocheiro||1492 ± 13bcd||1262 ± 11defg||229 ± 2efgh||9.94 ± 0.20def||7.79 ± 0.11fghi|
|Alvarilhão||1820 ± 39fgh||1582 ± 34ghi||238 ± 5efgh||9.17 ± 0.16cde||7.28 ± 0.22defgh|
|Aramon||1590 ± 13def||1341 ± 11fgh||248 ± 2fghi||8.56 ± 0.14bcd||8.74 ± 0.10i|
|Bastardo||2024 ± 37gh||1662 ± 30hi||362 ± 7k||9.93 ± 0.23def||8.59 ± 0.25hi|
|Cabernet Franc||1900 ± 17fgh||1535 ± 14fghi||365 ± 6k||13.26 ± 0.15h||8.26 ± 0.07hi|
|Carignan Noir||1476 ± 22bcd||1268 ± 19defg||208 ± 5efg||10.28 ± 0.87ef||8.35 ± 0.51hi|
|Cornifesto||1088 ± 18ab||874 ± 13a||214 ± 3efg||8.25 ± 0.04bc||6.82 ± 0.10cdefg|
|Gamay||1529 ± 18cde||1309 ± 12efgh||220 ± 2efgh||12.86 ± 0.43h||8.08 ± 0.48ghi|
|Grenache||1038 ± 52a||907 ± 9ab||131 ± 7bcd||5.41 ± 0.60a||7.43 ± 0.35efgh|
|Jean||1530 ± 24cde||1334 ± 10efgh||196 ± 5defg||10.99 ± 0.30fg||5.94 ± 0.29bcd|
|Malvasia Preta||1352 ± 28abcd||1083 ± 23bcde||269 ± 6ghij||10.05 ± 0.14ef||6.18 ± 0.27bcde|
|Rufete||1297 ± 33abc||1133 ± 28bcde||164 ± 4cde||11.16 ± 0.33fg||5.86 ± 0.00bc|
|Sousão||2130 ± 74h||1898 ± 12i||232 ± 8efgh||18.68 ± 0.21jk||5.78 ± 0.22bc|
|Tinta Amarela||1490 ± 28bcd||1197 ± 23cdef||294 ± 6hijk||16.22 ± 0.37i||4.86 ± 0.03ab|
|Tinta Barca||3033 ± 19i||2700 ± 16j||333 ± 13jk||19.15 ± 0.12k||5.99 ± 0.02bcd|
|Tinta Barroca||2783 ± 13i||2418 ± 18j||365 ± 15k||17.68 ± 0.10f||6.14 ± 0.16bcde|
|Tinta Miúda||2119 ± 37h||1857 ± 32i||263 ± 5ghij||21.68 ± 0.16l||4.19 ± 0.12a|
|Tinto Cão||1562 ± 33def||1254 ± 26defg||308 ± 11ijk||13.35 ± 0.42h||5.78 ± 0.18bc|
a abs.u. – absorbance units referred to a 1 mm path length cell used; data are the average of two replicates ± standard deviation; different letters above means indicate statistically significant differences between grape varieties (p < 0.05).
The total phenolic compounds, expressed as equivalent of gallic acid, ranged from 989 to 3033 mg/L with an average of 1608.5 mg/L. The highest concentration of total phenols (ranging from 2119 to 3033 mg/L) was detected in Tinta Barca, Tinta Barroca, Sousão and Tinta Miúda, while Camarate, Moreto Boal, Cornifesto and Grenache showed the lowest values (ranging from 989 to 1088 mg/L).
The interest of winemakers for grape polyphenolic content is increasing, as it offers tools to influence the color, bitterness, astringency, ‘mouth-feel’ and ‘age-ability’ of wines. Therefore, the color of red wines and its evolution depend not only on the winemaking and aging processes, but also on the potential concentration of anthocyanins in grape skin as well as the easiness of anthocyanin extraction from skin into must (Ortega-Regules et al., 2006).
The individual anthocyanin composition of the skin extracts of the grape varieties at technological maturity is presented in Table 4. Malvidin-3-glucoside was the major individual anthocyanin (concentrations ranging from 0.62 to 6.09 mg/g of skin) in all varieties except Alvarilhão and Rufete, where the major individual anthocyanins were peonidin-3-glucoside (1.04 mg/g of skin) and malvidin-3-p-coumaroyl glucoside (1.48 mg/g of skin), respectively. Malvidin-3-p-coumaroyl glucoside was the second main individual anthocyanin for the majority of the varieties and the values ranged from 0.12 to 3.44 mg/g of skin. These results are in agreement with previous findings in other grape varieties (Dallas and Laureano, 1994a; Jordão et al., 1998a; Kallithraka et al., 2005; Ortega-Regules et al., 2006). According to Ribéreau-Gayon et al. (2006), malvidin derivative forms are stable molecules and their presence give stability to the wine during the winemaking process because of their relative resistance to oxidation. Mateus et al. (2002) found that malvidin-3-glucoside and its acylated esters were the major anthocyanin monoglucosides in Touriga Nacional and Touriga Francesa at harvest date.
Table 4. Individual anthocyanins quantified in grape skin at technological maturity.
|Grape variety||Delp gluc||Cyan gluc||Petun gluc||Peon gluc||Malv gluc||
|Camarate||n.d.||0.05 ± 0.01e||0.22 ± 0.01defg||0.49 ± 0.02e||5.08 ± 0.02g||0.02 ± 0.0bc||0.02 ± 0.00a||0.12 ± 0.01g||1.29 ± 0.07i||0.12 ± 0.00g||0.11 ± 0.00def||0.90 ± 0.17def|
|Gewürztraminer||0.21 ± 0.05cd||0.01 ± 0.00a||0.44 ± 0.11j||0.26 ± 0.02cd||4.92 ± 0.04g||0.03 ± 0.00d||0.39 ± 0.00d||0.07 ± 0.00ef||1.54 ± 0.03jk||0.39 ± 0.00k||0.07 ± 0.00bcde||0.88 ± 0.13def|
|Monvedro||0.60 ± 0.00f||0.01 ± 0.01a||0.94 ± 0.02m||0.30 ± 0.05d||6.09 ± 0.04i||0.05 ± 0.00e||0.14 ± 0.00bc||0.01 ± 0.00a||1.65 ± 0.01k||0.11 ± 0.01g||0.09 ± 0.00cdef||1.48 ± 0.03efg|
|Moreto Boal||0.08 ± 0.02ab||0.01 ± 0.00a||0.06 ± 0.00ab||0.49 ± 0.03e||3.96 ± 0.08f||0.01 ± 0.00ab||0.06 ± 0.00a||0.11 ± 0.00g||1.01 ± 0.00h||0.10 ± 0.00fg||0.12 ± 0.01ef||1.27 ± 0.03efg|
|Negro Mole||0.04 ± 0.01ab||0.01 ± 0.00a||0.24 ± 0.05efgh||0.51 ± 0.10e||5.92 ± 0.00hi||0.02 ± 0.00bc||0.21 ± 0.00c||0.11 ± 0.00g||1.43 ± 0.07j||0.08 ± 0.00ef||0.11 ± 0.01def||2.21 ± 0.07gh|
|Negro Mouro||0.03 ± 0.01ab||n.d.||0.21 ± 0.00cdefg||0.54 ± 0.05ef||5.88 ± 0.04hi||0.02 ± 0.00bc||0.08 ± 0.01ab||0.10 ± 0.00fg||1.45 ± 0.04j||0.03 ± 0.01a||0.09 ± 0.03cdef||2.23 ± 0.05gh|
|Alfrocheiro||0.03 ± 0.01ab||n.d.||0.16 ± 0.01bcdef||0.14 ± 0.00b||2.90 ± 0.15e||n.d.||n.d.||0.04 ± 0.01abcd||0.18 ± 0.01bcde||0.06 ± 0.00cde||0.04 ± 0.01ab||1.46 ± 0.15efg|
|Alvarilhão||0.08 ± 0.01ab||0.24 ± 0.01h||0.12 ± 0.00bcd||1.04 ± 0.04h||0.99 ± 0.05ab||0.01 ± 0.00a||0.01 ± 0.00a||0.06 ± 0.00cde||0.05 ± 0.00ab||0.01 ± 0.00a||0.12 ± 0.01ef||0.12 ± 0.03a|
|Aramon||0.05 ± 0.00ab||0.03 ± 0.00bc||0.08 ± 0.00ab||0.17 ± 0.01bc||1.24 ± 0.01b||n.d.||0.01 ± 0.00a||0.01 ± 0.00a||0.15 ± 0.01bcde||0.03 ± 0.00a||0.01 ± 0.00a||0.32 ± 0.08bc|
|Bastardo||0.02 ± 0.00a||0.03 ± 0.00bc||0.01 ± 0.00a||0.13 ± 0.00ab||0.62 ± 0.00a||n.d.||n.d.||0.02 ± 0.00abc||0.05 ± 0.00ab||0.02 ± 0.00a||0.05 ± 0.00abc||0.16 ± 0.01a|
|Cabernet Franc||0.95 ± 0.00g||0.04 ± 0.00de||1.03 ± 0.01m||0.17 ± 0.02bc||4.10 ± 0.13f||0.01 ± 0.00a||0.02 ± 0.00a||0.20 ± 0.01i||0.27 ± 0.00ef||0.22 ± 0.01i||0.02 ± 0.00a||1.52 ± 0.10fgh|
|Carignan Noir||0.34 ± 0.05e||0.02 ± 0.01ab||0.57 ± 0.01k||0.56 ± 0.03ef||5.60 ± 0.37h||n.d.||n.d.||0.04 ± 0.01abcd||0.53 ± 0.01g||0.16 ± 0.01h||0.35 ± 0.03g||3.44 ± 0.43i|
|Cornifesto||0.03 ± 0.01a||n.d.||0.10 ± 0.00abc||0.56 ± 0.03ef||5.60 ± 0.37h||n.d.||n.d.||0.02 ± 0.00abc||0.17 ± 0.00bcde||0.06 ± 0.00cde||0.03 ± 0.00a||1.38 ± 0.13efg|
|Gamay||0.02 ± 0.01a||n.d.||0.08 ± 0.02ab||0.12 ± 0.00ab||1.77 ± 0.03c||n.d.||n.d.||0.04 ± 0.00abcd||0.23 ± 0.00cde||0.02 ± 0.00a||0.11 ± 0.02def||1.61 ± 0.01fgh|
|Grenache||0.25 ± 0.00cd||0.04 ± 0.00de||0.14 ± 0.01bcde||0.18 ± 0.00bcd||1.44 ± 0.01bc||n.d.||n.d.||n.d.||0.01 ± 0.00a||0.05 ± 0.00bcd||0.05 ± 0.00abc||0.17 ± 0.01a|
|Jean||0.09 ± 0.01b||0.02 ± 0.00ab||0.20 ± 0.01cdef||0.27 ± 0.01cd||2.75 ± 0.05de||0.02 ± 0.00bc||0.04 ± 0.00a||0.03 ± 0.03abc||0.89 ± 0.07h||0.04 ± 0.01abc||0.02 ± 0.00a||0.77 ± 0.02cde|
|Malvasia Preta||0.21 ± 0.00cd||0.06 ± 0.01ef||0.31 ± 0.00ghi||0.55 ± 0.02ef||2.32 ± 0.06d||n.d.||0.01 ± 0.00a||0.01 ± 0.00a||0.10 ± 0.00abc||0.02 ± 0.00a||0.04 ± 0.02abc||0.28 ± 0.05ab|
|Rufete||n.d.||n.d.||n.d.||0.01 ± 0.00a||1.39 ± 0.01bc||n.d.||n.d.||0.02 ± 0.00ab||0.14 ± 0.01bcde||0.03 ± 0.00a||0.05 ± 0.00abc||1.48 ± 0.03efg|
|Sousão||0.21 ± 0.01cd||0.09 ± 0.01g||0.33 ± 0.01hi||1.34 ± 0.01i||2.76 ± 0.05de||n.d.||n.d.||n.d.||0.06 ± 0.00ab||0.02 ± 0.00a||0.04 ± 0.00abc||0.18 ± 0.08a|
|Tinta Amarela||0.58 ± 0.02f||0.07 ± 0.01fg||0.74 ± 0.01l||0.69 ± 0.02g||2.91 ± 0.07e||0.02 ± 0.00bc||0.04 ± 0.01a||0.03 ± 0.01abcd||0.10 ± 0.01ab||0.05 ± 0.00bcd||0.06 ± 0.01abcd||0.65 ± 0.01cde|
|Tinta Barca||0.34 ± 0.00e||0.04 ± 0.00de||0.46 ± 0.01jk||0.23 ± 0.00bcd||2.41 ± 0.01d||n.d.||n.d.||0.05 ± 0.00bcde||0.13 ± 0.01bcd||0.04 ± 0.01abc||n.d.||0.98 ± 0.01def|
|Tinta Barroca||0.21± 0.01cd||0.06 ± 0.00ef||0.41 ± 0.01ij||0.57 ± 0.00efg||4.93 ± 0.05g||n.d.||n.d.||0.07 ± 0.00de||0.26 ± 0.00def||0.11 ± 0.01g||0.13 ± 0.01f||1.94 ± 0.03fgh|
|Tinta Miúda||0.20 ± 0.01c||0.01 ± 0.00a||0.25 ± 0.01fgh||0.64 ± 0.03fg||2.31 ± 0.06d||n.d.||0.02 ± 0.00a||0.04 ± 0.00bcde||0.26 ± 0.00def||0.02 ± 0.01a||0.11 ± 0.02def||0.41 ± 0.04bcd|
|Tinto Cão||0.27 ± 0.01d||0.01 ± 0.00a||0.39 ± 0.01ij||0.13 ± 0.02ab||2.65 ± 0.01de||n.d.||0.01 ± 0.00a||0.16 ± 0.01h||0.37 ± 0.00f||0.25 ± 0.01j||0.05 ± 0.03abc||2.57 ± 0.02hi|
Delp gluc, delphinidin-3-glucoside; Cyan gluc, cyanidin-3-glucoside; Petun gluc, petunidin-3-glucoside; Peon gluc, peonidin-3-glucoside; Malv gluc, malvidin-3-glucoside; Cyan acet-gluc, cyanidin-3- acetyl glucoside; Petun acet-gluc, petunidin-3-acetyl glucoside; Peon acet-gluc, peonidin-3-acetyl glucoside; Malv acet-gluc, malvidin-3-acetyl glucoside; Peon coum-gluc, peonidin-3-p-coumaroyl glucoside; Malv coum-gluc, malvidin-3-p-coumaroyl glucoside; individual anthocyanins are expressed as malvidin-3-glucoside equivalents (mg/g of skin); n.d., not detected; data are the average of two replicates ± standard deviation; different letters above means indicate statistically significant differences between grape varieties (p < 0.05).
In general, cyanidin derivatives (cyanidin-3-acetyl glucoside and petunidin-3-acetyl glucoside) were the less abundant individual anthocyanins (ranging from 0.01 to 0.05 and 0.01 to 0.39 mg/g of skin, respectively), which is in line with the results published by several authors (Dimitrovska et al., 2011; Ortega-Meder et al., 1994; Roggero et al., 1986). In addition, these two anthocyanins were not detected in a great number of the grape varieties studied. According to Di Stefano and Flamini (2008), cyanidin is the precursor of peonidin-3-glucoside by the action of the UFGT (which transform cyanidin into cyanidin-3-glucoside) and MT enzymes (which transform cyanidin-3-glucoside into peonidin-3-glucoside).
The glucoside group was the main anthocyanin chemical group in all grape varieties, ranging from 1.40 (Rufete) to 7.09 (Carignan Noir) mg/g of skin (sum of all five 3-glucoside anthocyanins). The coumaroyl glucoside group was the second most important group, ranging from 0.23 (Bastardo) to 2.87 (Tinto Cão) mg/g of skin, except in four varieties (Camarate, Gewürztraminer, Monvedro and Jean) where the acetyl glucoside group was the second main group. A high concentration of p-coumaroyl-anthocyanin derivatives, mainly from malvidin and petunidin, has been associated with warm climates (Downey et al., 2006). However, according to Férnandez-Lopez et al. (1998) it is commonly accepted that the anthocyanin composition of each cultivar is closely linked to its genetic inheritance and, from a qualitative point of view, is quite independent of seasonal conditions or production area.
The acetyl glucoside group, which participates in an intra-molecular copigmentation process leading to an increase in wine color intensity (Ortega-Regules et al., 2006), was the minor group, ranging from 0.01 (Grenache) to 2.03 (Gewürztraminer) mg/g of skin. Although the anthocyanin profile may be complex and quite different for each variety studied, in general Carignan Noir, Monvedro and Negro Mole showed the highest individual anthocyanin concentration while Bastardo, Aramon, Grenache and Alvarilhão showed the lowest concentration. It is important to note that grape anthocyanin concentration is largely due, among other factors, to the berry size of each grape variety. On the other hand, Gewürztraminer, Monvedro, Moreto Boal, Negro Mole, Alvarilhão, Cabernet Franc, Jean and Tinta Amarela presented the widest variety of individual anthocyanins while Rufete presented the narrowest variety of individual anthocyanins quantified.
To better understand the relationship between grape variety and anthocyanin concentration, a principal component analysis (PCA) was performed (Figure 2A). The first two principal components (PCs) explained 92.22 % of the total variance and showed that grape varieties can be distinguished according to their individual anthocyanin concentration. Figure 2A shows the corresponding loading plots that established the relative importance of each variable. The first principal component (PC1), which explained 81.89 % of the variance, was negatively correlated with the variables malvidin-3-glucoside and malvidin-3-p-coumaroyl glucoside. The second PC (PC2, 10.33 % of the variance) was positively correlated with the variable peonidin-3-glucoside and negatively correlated with malvidin-3-acetyl glucoside. Five groups could be distinguished. The varieties Camarate, Gewürztraminer, Monvedro, Negro Mole, Negro Mouro, Cabernet Franc, Carignan Noir, Cornifesto and Tinta Barroca were rather grouped on the negative side of PC1, due to their high content in malvidin-3-glucoside (ranging from 4.10 to 6.09 mg/g of skin) and malvidin-3-p-coumaroyl glucoside (ranging from 0.88 to 3.44 mg/g of skin). The varieties Moreto Boal, Monvedro, Gewürztraminer and Camarate appeared in the positive part of PC2, due to their high content in malvidin-3-acetyl glucoside (ranging from 1.01 to 1.65 mg/g of skin).
Figure 2. Principal component analysis score plot (PC1 and PC2) of grape varieties: individual anthocyanins (A) and antioxidant activity (B).
CA, Camarate; GW, Gewürztraminer; MN, Monvedro; MB, Moreto Boal; NM, Negro Mole; NMR, Negro Mouro; AF, Alfrocheiro; AV, Alvarilhão; AR, Aramon; BT, Bastardo; CF, Cabernet Franc; CN, Carignan Noir; CR, Cornifesto; GY, Gamay; GR, Grenache; J, Jean; MP, Malvasia Preta; R, Rufete; S, Sousão; TA, Tinta Amarela; TB, Tinta Barca; TBR, Tinta Barroca; TM, Tinta Miúda; TC, Tinto Cão.
Delp gluc, delphinidin-3-glucoside; Cyan gluc, cyanidin-3-glucoside; Petun gluc, petunidin-3-glucoside; Peon gluc, peonidin-3-glucoside; Malv gluc, malvidin-3-glucoside; Cyan acet-gluc, cyanidin-3 acetyl glucoside; Petun acet-gluc, petunidin-3-acetyl glucoside; Peon acet-gluc, peonidin-3-acetyl glucoside; Malv acet-gluc, malvidin-3-acetyl glucoside; Peon coum-gluc, peonidin-3-p-coumaroyl glucoside; Malv coum-gluc, malvidin-3-p-coumaroyl glucoside.
From the description presented above, anthocyanins could be considered useful markers to distinguish grape varieties; however, this characteristic should be used with caution since anthocyanin concentration is influenced not only by genetic background but also by agroecological factors, such as maturation (Conde et al., 2007; Jordão et al., 1998a), ripening stage (Segade et al., 2008), climate (Gil and Yuste, 2004), stress levels (Gatto et al., 2008) and cultural practices (Jordão et al., 1998b).
3. Antioxidant activity from different grape berry fractions
The data in Table 5 show the antioxidant activity quantified in the different grape berry fractions (pulps, skins and seeds) from the grape varieties studied. The highest antioxidant activity was found in seeds (ranging from 77.59 to 867.81 and from 75.52 to 363.47 µmol/g of seed, for ABTS and DPPH method, respectively), followed by skins (ranging from 1.13 to 292.05 and from 1.78 to 299.99 µmol/g of skin, for ABTS and DPPH method, respectively) and pulps (ranging from 0.04 to 4.80 and from 0.18 to 3.13 µmol/g of pulp, for ABTS and DPPH method, respectively). In addition, the average values were 314.17, 68.24 and 1.82 µmol/g, respectively for seeds, skins and pulps considering the ABTS method and 211.01, 89.78 and 1.72 µmol/g, respectively for seeds, skins and pulps considering the DPPH method. One possible explanation for this distribution in the different grape berry fractions could be the higher amount of polyphenols such as monomeric flavanols (catechin and epicatechin), dimeric, trimeric and polymeric proanthocyanidins, and phenolic acids in seeds compared to skins (Di Majo et al., 2008; Yilmaz and Toledo, 2004). These findings are in agreement with previous studies conducted with other red Vitis vinifera grape varieties (Poudel et al., 2008; Xu et al., 2010).
Table 5. Antioxidant activities of grape skin, pulp and seed extracts as measured by the ABTS and DPPH methods in grape varieties at technological maturity.
|Camarate||47.64 ± 0.62abc||58.14 ± 2.02efgh||2.57 ± 0.00kl||3.13 ± 0.27j||161.13 ± 7.23bcde||129.00 ± 18.91abc|
|Gewürztraminer||78.78 ± 7.45gh||45.30 ± 0.00defg||2.14 ± 0.07ijk||2.02 ± 0.05ef||276.21± 6.03fghi||330.89 ± 1.89ij|
|Monvedro||34.48 ± 0.62b||66.71 ± 0.00ghi||1.58 ± 0.02fghi||1.84 ± 0.07de||77.59 ± 0.00a||96.91 ± 11.35ab|
|Moreto Boal||93.28 ± 1.86h||9.63 ± 1.09ab||3.65 ± 0.02n||3.12 ± 0.03j||310.31 ± 1.21hi||210.56 ± 9.45def|
|Negro Mole||71.78 ± 4.96fg||88.11 ± 2.02hi||0.95 ± 0.04bcde||1.05 ± 0.09b||137.26 ± 4.82bcd||185.15 ± 15.13cde|
|Negro Mouro||57.74 ± 4.96cdef||48.60 ± 3.78defg||1.95 ± 0.07ijk||2.48 ± 0.01ghi||145.79 ± 14.47bcd||239.97 ± 39.71efg|
|Alfrocheiro||47.20 ± 3.72abc||15.34 ± 4.04bc||0.04 ± 0.03a||0.18 ± 0.06a||105.72 ± 1.21ab||229.27 ± 54.83def|
|Alvarilhão||9.03 ± 3.10a||38.79 ± 20.18defg||0.05 ± 0.03a||2.01 ± 0.06ef||174.77 ± 4.82bcde||300.14 ± 3.78ghij|
|Aramon||133.21 ± 4.96i||203.27 ± 16.40k||4.80 ± 0.22o||2.59 ± 0.07hi||505.52 ± 16.88l||314.84 ± 1.89hij|
|Bastardo||1.13 ± 0.62a||1.78 ± 0.01a||0.82 ± 0.07bcd||2.02 ± 0.05ef||410.05 ± 19.29jk||363.47 ± 11.82j|
|Cabernet Franc||52.91 ± 1.86bcdef||30.32 ± 1.01cde||1.71 ± 0.12fghi||1.48 ± 0.02cd||506.37 ± 10.85l||264.87 ± 4.73fghi|
|Carignan Noir||192.01 ± 9.93j||182.37 ± 1.26k||3.42 ± 0.13nm||2.25 ± 0.01fgh||867.81 ± 18.08n||130.34 ± 9.45abc|
|Cornifesto||52.91 ± 0.62bcdef||23.40 ± 3.47bcd||1.16 ± 0.07cdef||1.25 ± 0.08bc||306.89 ± 44.61hi||106.27 ± 5.67ab|
|Gamay||42.38 ± 3.10bc||68.22 ± 11.35ghi||1.92 ± 0.07hij||2.24 ± 0.02fgh||224.21 ± 19.29efgh||253.57 ± 19.24efgh|
|Grenache||33.16 ± 6.21b||299.99 ± 5.04l||2.95 ± 0.06lm||2.71 ± 0.05i||348.67 ± 14.47ij||75.52 ± 11.35a|
|Jean||63.88 ± 13.65defg||94.08 ± 12.61i||3.30 ± 0.56nm||1.12 ± 0.06bc||453.52 ± 20.49kl||122.31 ± 1.89abc|
|Malvasia Preta||1.57 ± 1.24a||3.21 ± 1.01a||0.55 ± 0.04ab||1.12 ± 0.06bc||237.85 ± 16.88efgh||95.57 ± 13.24ab|
|Rufete||37.55 ± 2.48bc||63.76 ± 7.57fghi||1.86 ± 0.03ghij||1.09 ± 0.05b||539.62 ± 69.92l||189.49 ± 13.24cde|
|Sousão||68.27 ± 0.00efg||43.88 ± 4.04defg||1.30 ± 0.16fgh||1.09 ± 0.02b||189.26 ± 1.21cdef||249.65 ± 5.67efg|
|Tinta Amarela||45.89 ± 1.86bcd||7.75 ± 0.96ab||0.63 ± 0.28abc||0.49 ± 0.00a||711.81 ± 14.47m||219.92 ± 0.00def|
|Tinta Barca||50.28 ± 0.62bcde||137.33 ± 7.06j||0.16 ± 0.06a||0.49 ± 0.01a||122.77 ± 3.62abc||161.09 ± 11.35bcd|
|Tinta Barroca||7.20 ± 1.96a||87.39 ± 1.01hi||1.24 ± 0.13defg||1.12 ± 0.00bc||206.31 ± 13.26defg||237.30 ± 1.89efg|
|Tinta Miúda||292.05 ± 3.72k||32.24 ± 6.73cdef||2.55 ± 0.06kl||2.14 ± 0.11efg||236.99 ± 22.91efgh||239.97 ± 1.89efg|
|Tinto Cão||123.55 ± 7.45i||25.33 ± 4.04bcd||2.40 ± 0.15jkl||2.48 ± 0.01ghi||283.88 ± 31.34ghi||318.35 ± 14.18hij|
Data are the average of two replicates ± standard deviation; values are expressed as µmol/g of skin, pulp or seed; different letters above means indicate statistically significant differences between grape varieties (p < 0.05).
Taking into account the antioxidant activity of the red grape varieties analyzed here, a large variation was found in the different grape berry fractions (Table 5). For seeds and considering the ABTS method, Carignan Noir, Tinta Amarela, Rufete, Cabernet Franc, Aramon, Jean and Bastardo showed the highest antioxidant activity values (ranging from 410.05 to 867.81 µmol/g of seed; p < 0.05) while Monvedro and Alfrocheiro showed the lowest antioxidant values (77.59 and 105.72 µmol/g of seed, respectively; p < 0.05). However, considering the DPPH method, Bastardo, Gewürztraminer, Tinto Cão, Aramon and Alvarilhão showed the highest antioxidant activity values (ranging from 300.14 to 363.47 µmol/g of seed; p < 0.05) while Monvedro, Grenache and Malvasia Preta showed the lowest antioxidant values (ranging from 75.52 to 96.91 µmol/g of seed; p < 0.05).
For skins and considering the ABTS method, Tinta Miúda, Carignan Noir, Aramon and Tinto Cão showed the highest antioxidant activity values (ranging from 123.55 to 292.05 µmol/g of skin; p < 0.05) while Bastardo, Malvasia Preta and Tinta Barroca showed the lowest antioxidant values (ranging from 1.13 to 7.20 µmol/g of skin, respectively; p < 0.05). The results obtained by the DPPH method showed similar distribution of high (Carignan Noir and Aramon) and low (Malvasia Preta and Bastardo) antioxidant activity values. Finally, for pulps, high antioxidant activity values were found for Aramon, Moreto Boal and Carignan Noir (4.80, 3.65 and 3.42 µmol/g of pulp for ABTS method, respectively) while low antioxidant values were found for Alfrocheiro, Alvarilhão, Tinta Barca and Malvasia Preta (0.04, 0.05, 0.16 and 0.55 µmol/g of pulp for ABTS method, respectively). Overall, there is no clear difference between the autochthonous and non-autochthonous grape varieties and the grape samples from the two wine regions considered. From these results, it is clear that the antioxidant activity values depend on the method used. According to Villaño et al. (2006), this divergence is due to the different reagents of the polyphenols with each method applied. For Wang et al. (2004), ABTS˙+ and DPPH radicals have a different stereochemical structure and a different method of genesis and thus they lend, after the reaction with the antioxidants, a qualitatively different response to the inactivation of their radical. Some authors reported that no single assay can provide all the information needed to evaluate antioxidant capacity, and multiple assays are therefore required to build up an antioxidant profile of particular foodstuffs (Rivero-Pérez et al., 2007).
To highlight the relation between grape varieties and their antioxidant activity in the different grape berry fractions (pulps, skins and seeds), a PCA was applied (Figure 2B). The PCA of the antioxidant activity values obtained by two methods showed that the first two PCs explained 84.47 % of the total variance. The first PC (70.21 % of the variance) was positively correlated with seeds ABTS and the second PC (14.26 % of the variance) was positively correlated with skins DPPH and negatively correlated with seeds DPPH. Grape varieties appeared grouped due to their different antioxidant activity of skins, seeds and pulps. The grape varieties Carignan Noir and Tinta Amarela were on the right side of PC1, disclosing a good correlation with seeds ABTS. However, the grape varieties Bastardo, Gewürztraminer, Alvarilhão and Tinto Cão were on the negative side of PC2, disclosing a good correlation with seeds DPPH.
A linear regression analysis was performed to determine the correlation between polyphenol composition and respective antioxidant activities in the different grape berry fractions. This analysis found no significant correlation (p > 0.05) between general phenolic parameters and antioxidant activity in the grape berry fractions. On the one hand, correlations between the antioxidant activity and the total polyphenolic content of a great number of grape seed and skin extracts from different grape varieties have been reported (Monagas et al., 2005; Xu et al., 2010). On the other hand, other authors (Bozan et al., 2008) reported no significant correlations between individual flavanols analyzed by HPLC or total polyphenols and antioxidant activity in seed extracts from several grape varieties. Thus, there is conflicting evidence in the literature about the correlation between polyphenol content and the antioxidant activity of grapes.
In this work, the influence of grape variety on the phenolic content, anthocyanin profile and antioxidant activity of the different grape berry fractions (skins, pulps and seeds) has been analyzed in twenty-four different grape varieties cultivated in two Portuguese wine regions (Douro and Dão). In general, a high variability was found among the diverse autochthonous and non-autochthonous grape varieties in relation to polyphenolic content, anthocyanin profile and antioxidant activity. Seeds were the grape berry fraction with the highest antioxidant activity, followed by skins and pulps, irrespective of the grape variety.
The results obtained for polyphenolic concentration, in particular individual anthocyanin concentration, and antioxidant activity must be considered in wineries, in order to apply the most appropriate winemaking techniques. Thus, from the data presented in this work, several specific winemaking techniques could be suggested depending on the grape variety: extended maceration could be used to optimise anthocyanin extraction and therefore improve color in Bastardo and Alvarilhão wines, whereas standard winemaking process could be a good option for Carignan Noir, Tinta Miúda and Tinto Cão. Increasing pumping over frequency during the maceration process can also increase the polyphenolic content and antioxidant capacity of wines, which is crucial for aging in oak barrels.
Finally, the variability found in phenolic content among grape varieties confirmed the hypothesis that genetic factors have an important role in the biosynthesis of these compounds and, consequently, in the antioxidant activity of grapes.
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