- Email: [email protected]
Antioxidant activity and phenolic composition of organic and conventional grapes and wines
Journal of Food Composition and Analysis 23 (2010) 569–574
Contents lists available at ScienceDirect
Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca
Original Article
Antioxidant activity and phenolic composition of organic and conventional grapes and wines Juana Mulero a,*, Francisco Pardo b, Pilar Zafrilla a a b
Department of Food Technology and Nutrition, Catholic University of San Antonio, Avda Los Jero´nimos s/n, Murcia 30107, Spain Cooperativa de Bodegas San Isidro, Jumilla, Murcia, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 February 2009 Received in revised form 20 April 2010 Accepted 17 May 2010
The phenolic compounds and antioxidant activity from Monastrell variety grapes obtained by organic and conventional agriculture during the last month of ripening and the wines obtained from them were studied. Samples of grapes were collected from the last month of ripening to full maturity in each plot, and winemaking was carried out on the day of the final collection of grape samples, coinciding with the maturity of the grapes. The antioxidant activity a month before harvesting was higher in the organic grapes (5.7 0.03 mM Trolox/g) than in the conventional ones (4.40 0.05 mM Trolox/g), although these differences disappear in the moment of harvesting. Similarly the total amount of phenolic compounds a month before harvesting was higher in the organic grapes (974.2 54.4 mg/kg) than in the conventional grapes (447.7 27.8 mg/kg), although these differences disappear at the moment of harvesting. In wine, phenolic compounds and the antioxidant activity were slightly higher in organic wine than in conventional wine, although the differences were not significant. ß 2010 Elsevier Inc. All rights reserved.
Keywords: Antioxidant activity Phenolic compounds Grape Wine Organic agriculture Anthocyanins Horticulture and biodiversity Food composition Food analysis
1. Introduction Organic agriculture is a horticultural production management system that promotes and enhances biodiversity, biological cycles and soil biological activity (Research Institute of Organic Farming, 2000; Alternative Farming Systems Information Center, 2005). Organic is a labelling term that denotes products produced under the authority of the Organic Foods Production Act (CEE, 1991; Le Guillou and Scharpe´, 2001). The primary goal of organic agriculture is to optimize the health and productivity of interdependent communities of soil life, plants, animals and humans. Grapes contain a large amount of different phenolic compounds in skin, pulp and seeds and these are the main compounds responsible of colour, taste, mouth feel, oxidation and other chemical reactions in wine. Vineyard location and specific site are very important. In dry climates irrigation generally increases the grapevine vigour, berry size and yield (Roggero et al., 1986). Within the same fruit type, the growing season, variety, environmental and climatic conditions, plant disease, soil type,
* Corresponding author. Tel.: +34 968278705; fax: +34 968278666. E-mail addresses: [email protected], [email protected] (J. Mulero). 0889-1575/$ – see front matter ß 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2010.05.001
geographic locations, and maturity seem to influence the concentration of phenolic compounds (Subramani et al., 2002). These compounds are an integral part of the human diet considered as biologically active non-nutrient compounds (Subramani et al., 2002). Among these phenolic substances, flavonoids, and in particular anthocyanins, are of interest because of their high occurrence in foods. The presence of hydroxycinnamic acids and flavonols in grapes is well documented (Ribe´reau-Gayon, 1972; Wulf and Nagel, 1980). The flavonols are localized in the solid parts of the cluster. In red grapes, flavonols are present in much smaller quantities than anthocyanins and in most cases they have been considered to be negligible. As polyphenolic compounds, flavonoids share the ability to act as antioxidants by a free radical scavenging mechanism and metal ion chelation (Lodovici et al., 2001). Growing evidence of the role of radicals and antioxidants in the health and ageing has promoted a great interest on these compounds. A wide range of studies have shown that the antioxidative properties of these compounds may protect against arteriosclerosis and coronary heart disease (Struch, 2000; Sun et al., 2002). The aim of this work was to study the behaviour of phenolic compounds and antioxidant activity during ripening and processing of conventional and organically cultured grapes.
570
J. Mulero et al. / Journal of Food Composition and Analysis 23 (2010) 569–574
2. Materials and methods
degree) were determined following the procedures of OIV (1990).
2.1. Reagents 2.5. Extraction of phenolic compounds Formic acid and methanol (MeOH) of analytical grade were supplied by Merck (Darmstad, Germany). Milli-Q system (Millipore Corp., Bedford, MA) ultrapure was used throughout this research. Cyanidin 3-rutinoside was purchased from Polyphenols A.S. (Sandnes, Norway). Rutin was purchased from Merck (Darmstadt, Germany). Chlorogenic acid, Trolox (6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid) and 2,2-diphenylpicrylhydrazyl (DPPH) were purchased from Sigma (Madrid, Spain). 2.2. Grape samples Grape samples were collected during the last month of ripening. 30 days before harvest, 14 days before harvest, and the last date of collection coincided with the technological maturity of the grapes. At this time samples were collected for analysis of grapes and for the winemaking. One hundred berries were randomly taken from each sample from different parts of various clusters in every vineyard during the early morning hours, transported to the laboratory and processed on the same day. They came from a smallholding representative of the area. The selection of neighbouring parcels allowed us to compare organic and conventional vineyards in the same climate and soil conditions. Parcels P1 and P2 (conventionally cultured) and P3 and P4 (organic cultured) are located in the same spot. Organic grapes were treated with natural pesticides such as dry flowable sulphur, copper salts and oligoelements. Conventional production used fertilizers and pesticides to control weeds, pests, and diseases; these chemicals are subject to rigorous testing and authorization procedures before they can be used, and winegrowers have respected the guidelines and restrictions to use them.
Grapes. Skin represented approximately 13% of the total fresh weight of the grape berry. The grapes were peeled to complete a sample of 5 g of grape skins from which it was extracted the phenolic compounds. Phenolic compounds in grapes were analysed only in skin. Samples were homogenized in an Ultraturrax T-25 equipment (Janke and Kunkel, Ika-Labortechnick) at 24,000 rpm for 1 min after addition of 4 mL of a solution of MeOH/formic acid (97:3) per gram of skin. The extracts were centrifuged at 5000 g for 5 min in a Centromix centrifuge (Selecta, Barcelona), filtered through a 0.45 nm membrane filter Millex HV13 (Millipore), and analysed by HPLC. Three extracts were made for each sample collected and sampling was done in triplicate. Wine phenolic compounds. Before HPLC analysis, ecological and traditional red wines were filtered through a 0.45 mm filter (type Millex HV13, Millipore Corp, Bedford, MA). 2.6. HPLC-DAD analysis of phenolics 20 mL of every sample were injected for HPLC analysis using a Merck-Hitachi pump L-7100 (Merck-Hitachi, Darmstadt, Germany) and a diode array detector Merck-Hitachi 7455, employing a reversed-phase column Lichrochart 100 RP-18 column (Merck, Darmstadt, Germany) (25 cm 0.4 cm, 5 mm particle size), and water plus 5% formic acid (solvent A) or HPLC grade methanol (solvent B) as solvents at a flow rate of 1 mL min 1. Elution was performed with a gradient starting with 2% B to reach 32% B at 30 min, 40% B at 40 min, and 95% B at 50 min, becoming then isocratic for 5 min (Cantos et al., 2000). HPLC experiments were repeated three times. Phenolic content was expressed as mg/kg of fresh weight taking into account the percentage represented by the skin out of the total berry weight. Chromatograms were recorded at 510, 320 and 360 nm.
2.3. Vinification 2.7. Phenolic compounds identification and quantification The vinification process was carried out in a pilot plant the same day of harvesting. The grape clusters were squeezed in a squeezing roller. After eliminating the stalks the squeezed harvest was transferred to fermentation tanks, (tree tanks for each grape samples) where 80 and 70 mg/kg of SO2 were added in conventional and ecological vinification respectively to prevent the development of microorganisms as well as oxidation. The wine was kept in vats at 25 8C for 10 days to allow the processes of fermentation and maceration. Throughout this period daily mass homogenizations were performed to dissolve the cap of the wine, like in industrially performed processes. Density and temperature records were also daily accomplished to control possible fermentation arrests. Once maceration was completed, the must-wine was taken out of the vats and pressed, discarding the marcs and recovering the wine. The wine recovered was decanted 10 days later, discarding dregs. Finally, the wine was bottled and labelled according to its origin.
The phenolic compounds in grapes and wine were identified by their UV–vis spectra, recorded with a diode array detector and, when possible, by chromatographic comparison with authentic markers. Individual anthocyanins were quantified by comparisons with an external standard of cyanidin 3-rutinoside at 510 nm. Flavonols were quantified as rutin at 360 nm and the hydroxycinnamic acid derivatives at 320 nm as chlorogenic acid. All analyses were repeated three times and the results were expressed as mean values in mg/kg of grape SD. The reproducibility of the HPLC analyses was within 5% error. The total phenolic compounds were calculated by adding the amounts of anthocyanins, flavonols and hydroxycinnamic acids detected in each chromatogram, as previously reported (Cantos et al., 2000). All analyses were done in triplicate and the results expressed as mean values SD. The reproducibility of the HPLC analysis was within 6%. 2.8. Statistical data treatment
2.4. Physicochemical analysis Grapes. Baume´ degree, pH, total acidity, were determined following the procedures of OIV (Office International de la Vigne et du vin) (1990). Wines. The characteristic ecological parameters of elaborated wines (pH, density, volatile acidity, total acidity and alcoholic
In order to assess the relationships between the compounds quantified and the antioxidant properties a statistical analysis was performed using SPSS v:12.0. Data are presented as mean SE (standard error). Significance of differences was determined by analysis of variance (ANOVA). A p-value of p < 0.05 was considered statistically significant.
[(Fig._1)TD$IG]
J. Mulero et al. / Journal of Food Composition and Analysis 23 (2010) 569–574
571
2.9. Antioxidant activity The samples were analysed according to the technique reported by Brand-Williams et al. (1995). Briefly, a volume of 5 mL methanol extract (1:1, v/v with methanol) was added to a volume of 2,2diphenyl-1-picrylhydrazyl (DPPH) (Sigma, Steinheim, Germany) 0.094 mM in methanol up to completing 1 mL. The free radical scavenging activity using the free radical DPPH reaction was evaluated by measuring the absorbance at 515 nm after 60 min of reaction at 20 8C in a spectrophotometer (Varian Cary 50-Bio, Victoria, Australia). The reaction was carried out in closed Eppendorf tubes shaken at 20 8C. The results were expressed as mmol/L Trolox equivalents, a vitamin E analogue (Yamaguchi et al., 1998). Fig. 1. Evolution of the antioxidant activity in conventional and organic grapes during ripening.
3. Results and discussion 3.1. Physicochemical analysis Grapes. Berry weight and soluble solids increased over the ripening period. These trends were accompanied by a concomitant increase in pH and a decreased total acidity (Table 1). No significant differences in the physicochemical parameters were observed in both types of grapes. Their evolution during ripening was similar to that found by Pririe and Mullins (1977), and also to that reported for the period of ripening in grapes of the Shiraz and Cabernet sauvignon varieties and in Monastrell variety by Aleixandre et al. (2002). Wine. All parameters are in the normal range for good quality wine (Pardo, 1996). These values were similar to those obtained by Aleixandre et al. (2002) in wines made from Monastrell grapes. No differences were found between the physicochemical parameters of organic wine and traditional wine (Table 2). 3.2. Antioxidant activity Grapes. The antioxidant activity in stage 1 of organic grapes, (5.70 0.03 mM Trolox/g) was higher than that observed in conventional grapes (4.40 0.05 mM Trolox/g) showing significant differences (p = 0.046). However, such difference disappeared when Table 1 Enological parameters of conventional and organic grapes. Sample
Weight
8Be
TAc
pH
Stage 1 CG OG
121.4 126.6
11.05 10.95
7.65 8.16
3.41 3.60
Stage 2 CG OG
134.4 129.6
11.20 11.70
6.91 7.03
3.46 3.55
Stage 3 CG OG
147.5 139.4
12.00 12.20
5.41 6.58
3.70 3.64
TAc, total acidity (g/L tartaric acid); weight, 100 berry (g); 8Be, Baume degree; CG, conventional grapes; OG, organic grapes; Stage 1, first date of berry samples; Stage 2, second date of berry samples; Stage 3, third date of berry samples.
Table 2 Physicochemical parameter of conventional and organic wines. Sample
Density
pH
Alcoholic degree
VA
TA
CW OW
0.95 0.93
3.08 3.26
10.7 11.9
0.220 0.384
6.94 6.00
CW, conventional wine, average values obtained from the results in wines made from the two conventional vineyards; OW, organic wine, average values obtained from the results in wines made from the two organic vineyards; VA, volatile acidity expressed in g/L acetic acid; TA, total acidity expressed in g/L tartaric acid.
the final antioxidant activity was considered (stage 3) (4.70 0.11 mM Trolox/g in organic grapes and 4.80 0.25 mM Trolox/g in conventional grapes) (Fig. 1). No significant differences were established between the antioxidant activity in organic and conventional grapes in the moment of harvest. These values were higher to the reported by Yilmaz and Toledo ˜ oz-Espada et al. (2004) in Chardonnay and Merlot varieties and Mun (2004) in Norton, Concord and Marechal varieties by, and lower than the values described in the Muscadina variety by Pastrana-Bonilla et al. (2003). These variations in the evolution of the antioxidant activity of the grapes have been previously reported by Lee and Talcott (2004) in Muscadina red grapes. Wine. The antioxidant activity is similar in both types, organic and traditional, 6.78 0.45 mM Trolox/ml and 6.02 0.44 mM Trolox/ml respectively. These values are lower than those obtained by other authors (Rice-Evans et al., 1996; Simonetti et al., 1997; Pellegrini et al., 2000; Gambuti et al., 2004). 3.3. Anthocyanins Ten anthocyanin-glucosides (five of them acylated) were identified in organic and conventional red grapes skins and their wines: delphinidin 3-glucoside, cyanidin 3-glucoside, petunidin 3glucoside, peonidin 3-glucoside, malvidin 3-glucoside, cyanidin 3-pcoumaroylglucoside, petunidin 3-p-coumaroylglucoside, peonidin 3-p-coumaroylglucoside, petunidin 3-p-coumaroylglucoside, and malvidin 3-p-coumaroylglucoside, as described by Ferna´ndez-Lo´pez et al. (1992) and Cantos et al. (2003). The acylated derivatives were globally quantified due to the small individual concentrations observed. Grapes. Malvidin 3-glucoside was the major anthocyanin in both types of grapes, with mean contributions of about 42.0% and 29.0% of the total anthocyanins in conventional and organic grapes, in stage 3 respectively (Table 3). These results agree with those presented previously by Ferna´ndez-Lo´pez et al. (1992) and Cantos et al. (2003). The concentration of anthocyanins was higher in organic grapes (700.0 31.6 mg/kg) than in conventional ones (329.6 17.07 mg/ kg) in stage 1, with significant differences (p = 0.011) which remained in stage 2 (p = 0.012); these results disagree with Vian et al. (2006). However, these differences disappeared in stage 3 with the grapes ripening, showing mean concentrations of anthocyanins up to 706.5 35.82 mg/kg in conventional grapes and 741.9 38.82 mg/ kg in organic grapes (Table 4). The evolution of the average concentration of total anthocyanins in conventional grapes during ripening showed an increase in the concentration of anthocyanins during this period. These results agree with those obtained by other authors (Lanaridis and BenaTzourou, 1997; Esteban et al., 2001; Kennedy et al., 2002; Pe´rez-
572
J. Mulero et al. / Journal of Food Composition and Analysis 23 (2010) 569–574
Table 3 Anthocyanins. Composition of conventional and organic grapes during ripening. Sample
Dp-3-glc
Cy-3-glc
Pt-3-glc
Pn-3-glc
Mv-3-glc
Acyl derv.
Stage 1 CG OG
40.83 3.42 101.5 4.87
29.53 1.33 106.9 4.59
39.45 0.88 87.98 5.12
32.55 1.44 113.6 5.37
132.2 5.35 201.1 7.79
54.63 4.80 88.96 5.28
Stage 2 CG OG
43.38 2.83 91.83 4.77
40.25 2.16 113.8 6.1
42.89 1.59 84.10 6.33
40.05 2.22 292.5 6.66
134.8 6.45 199.4 8.17
75.63 6.00 95.28 3.20
Stage 3 CG OG
72.68 2.96 96.85 5.33
78.00 3.51 107.3 7.18
82.5 3.55 76.88 4.05
108.9 6.17 152.5 8.55
252.5 8.18 213.5 10.28
113.0 11.49 95.43 3.45
Values are expressed as mg/kg fresh weight of total grape berry. Mean values are shown (n = 3) standard error. CG, conventional grapes; OG, organic grapes; Stage 1, first date of berry samples; Stage 2, second date of berry samples; Stage 3, third date of berry samples; Dp-3-glc, delphinidin 3-glucoside; Cy-3-glc, cyanidin 3-glucoside; Pt-3-glc, petunidin 3glucoside; Pn-3-glc, peonidin 3-glucoside; Mv-3-glc, malvidin 3-glucoside; Acyl derv., acylated derivatives. Table 4 Phenolics compounds of maturity grapes and wines obtained from them. Sample
Anthocyanins
Hydroxycinnamic acids derivatives
Flavonols
Total phenolic compounds
CG OG CW OW
706.5 35.82 741.9 38.82 296.6 17.22 344.7 14.78
16.98 0.57 22.81 0.24 49.6 2.68 43.02 2.66
249.6 20.61 217.3 19.91 150.0 13.27 170.5 7.75
973.1 57.00 982.0 58.97 496.3 33.17 558.3 25.19
Values of grapes are expressed as mg/kg fresh weight of total grape berry standard error. Values of wine are expressed as mg/L of wine standard error. CG, conventional grapes; OG, organic grape; CW, conventional wine; OW, organic wine.
˜ o and Gonza´lez-San Jose´, 2004) who noted that the Magarin content of anthocyanins in the grapes skins was clearly influenced by the ripening of the grapes, and the beginning in the veraison, displaying the highest content at the moment of harvesting. Wine. The average content of anthocyanins was lower in conventional wine than organic wine, and the anthocyanin concentrations in both types of wine was higher than mentioned by other authors (Teissedre and Landrault, 2000; Arnous et al., 2001; Sa´nchez-Moreno et al., 2003). The malvidin 3-glucoside was the major anthocyanin in both types of wine.
conventional ones (16.98 0.57 mg/kg) although these differences are not significant (p > 0.05). This difference stayed without significant variation during ripening of the grapes and there were lower than those obtained by Kammerer et al. (2004) in different varieties of red grapes. Wine. Concentration of hydroxycinnamic derivatives was similar in traditional and organic wine (Table 4). Trans-Caffeoyltartaric acid was the majority compound in both types of wine with a concentration of 21.40 1.31 mg/L in conventional wine and 24.40 0.41 mg/L in organic wines.
3.4. Hydroxycinnamic acids
3.5. Flavonols
Three hydroxycinnamic derivatives were quantified in grapes and wine: trans-caffeoyltartaric acid, trans-p-coumaroyltartaric acid and an unidentified third derivative, which we have been named compound 2. Grapes. In both types of grapes the major hydroxycinnamic acids derivatives were trans-p-coumaroyltartaric and trans-caffeoyltartaric acid (Table 5). The concentration of hydroxycinnamic acids derivatives in stage 1 was higher in organic grapes (22.81 0.24 mg/kg) than in
Six flavonols were identified in organic and conventional red grapes skins and wine: myricetin 3-glucoside, quercetin 3glucoside, quercetin 3-rutinoside, kaempferol 3-glucoside, myricetin and quercetin. Grapes. In both types of grapes, conventional and organic, myricetin 3-glucoside, quercetin 3-glucoside, and quercetin 3rutinoside were the most abundant flavonols, followed by kaempferol 3-glucoside, myricetin and quercetin (Table 6). The concentration of flavonols in stage 1 was higher in organic grapes (251.2 20.7 mg/kg) than in conventional ones (101.6 10.90 mg/kg) with significant differences (p = 0.021). Such differences disappeared with the ripening of the grapes (Table 4). The concentration of flavonols observed was slightly higher to that found in Monastrell grapes by Cantos et al. (2003). Nevertheless, they are within the concentrations described by Cho et al. (2003) in different varieties of red grapes and by Lee and Talcott (2004) in Muscadina grapes. Wine. Content of flavonols was lower in conventional wine than organic wine (Table 4), although there were no significant differences. Myricetin 3-glucoside and quercetin 3-glucoside was majority flavonols (37.0 3.5 and 40.2 3.1 mg/L respectively for conventional wine and 41.9 1.2 and 41.1 1.6 mg/L respectively for organic wine).
Table 5 Evolution of the hydroxycinnamic acids derivatives composition in conventional and organic grapes during the ripening. Sample
Trans-Caffeoyltartaric
Compound 2
Trans-p-Coumaroyltartaric
Stage 1 CG OG
4.79 0.32 6.24 0.38
3.57 0.18 5.17 0.23
8.63 0.07 11.41 0.09
Stage 2 CG OG
4.20 0.33 4.92 0.7
3.33 0.27 5.04 0.20
8.15 0.10 11.44 0.23
Stage 3 CG OG
3.58 0.25 4.69 0.50
2.78 0.35 4.60 0.06
10.28 0.20 11.58 0.15
Values are expressed as mg/kg fresh weight of total grape berry. Mean values are shown (n = 3) standard error. CG, conventional grapes; OG, organic grape; Stage 1, first date of berry samples; Stage 2, second date of berry samples; Stage 3, third date of berry samples.
3.6. Total phenolic compounds Grapes. The concentration of the total phenolic compounds in the first sampling was of 974.2 54.46 mg/kg in organic samples
J. Mulero et al. / Journal of Food Composition and Analysis 23 (2010) 569–574
573
Table 6 Evolution of the flavonols composition in conventional and organic grapes during ripening. Sample
Myr-3-glc
Q-3-glc
Q-3-Rut
Kp-3-glc
Myricetin
Quercetin
Stage 1 CG OG
23.53 1.88 54.18 4.23
27.70 3.40 64.38 4.43
41.90 4.90 113.0 9.46
2.45 0.11 4.73 0.33
4.28 0.33 13.18 1.85
1.76 0.29 1.90 0.48
Stage 2 CG OG
22.35 4.28 36.36 3.25
23.90 4.00 46.18 6.15
43.18 4.00 107.8 4.25
3.48 0.25 4.90 0.38
4.73 0.30 13.95 2.10
0.93 0.22 1.38 0.06
Stage 3 CG OG
67.60 4.62 44.65 4.60
34.98 2.20 39.04 6.87
118.0 7.50 110.7 5.99
9.38 1.98 4.78 0.83
16.13 3.98 14.85 1.16
3.50 0.35 3.25 0.48
Values are expressed as mg/kg fresh weight of total grape berry. Mean values are shown (n = 3) standard error. CG, conventional grapes; OG, organic grapes; Stage 1, first date of berry samples; Stage 2, second date of berry samples; Stage 3, third date of berry samples; Myr-3-glc, myricetin 3-glucoside; Q-3-glc, quercetin 3-glucoside; Q-3-Rut, quercetin 3rutinoside; Kp-3-glc, kaempferol 3-glucoside.
[(Fig._2)TD$IG] Grayer, 1992). The differences found between both types of grapes may be due to different treatment of crops, since organic grapes were not treated with fertilizers and pesticides which defend the plant against attacks. The differences found between both types of grape in antioxidant activity in first sampling coincides with the highest concentration of phenolic compounds in organic grapes, since these are the makers of the antioxidant activity of grape. Acknowledgments We are grateful to Bodegas San Isidro of Jumilla (Murcia) for supplying wine and grapes. The authors acknowledge the financial support of Consejeria de Ciencia, Tecnologı´a, Industria y Comercio de la Regio´n de Murcia and Catholic University San Antonio de Murcia. Fig. 2. Evolution of the total phenols in conventional and organic grapes during ripening.
and 447.7 27.85 mg/kg in conventional grapes, showing significant differences (p = 0.016). This higher concentration of phenolic compounds in the organic grapes disappeared during ripening (Fig. 2). These values are lower than those obtained by Valls et al. (2000) in Tempranillo and Cabernet sauvignon varieties. These changes in the concentration of phenolic compounds in different harvests have been previously reported by several authors (Riu-Aumatell et al., 2002). On the other hand, Hidalgo (2003) pointed out that the concentration of polyphenolic compounds of the skin changes greatly depending on the variety and also on the grapes ripening stage. Other authors, such as Bautista-Ortin et al. (2003) stated that the stage of ripening not only influences the phenolic content of the variety, but also its degree of extraction, which will lately condition the presence in the wine of phenolic compounds after the winemaking process. Wine. Content of total phenolic compounds is slightly higher in organic wines than in conventional wine. However, when applying the analysis of variance (ANOVA) to the different phenolic compounds quantified, there was no significant differences between the concentrations found in conventional and ecological red wine at confidence level of 95%. 4. Conclusion In both types of grapes, organic and conventional, only significant differences were found in the phenolic compounds in the first sampling, and these differences have disappeared with maturation. Phenolics have a number of important roles to play in viticulture and oenology including UV protection, disease resistance, and defence against predation in plants (Harborne and
References Aleixandre, J.L., Lizama, V., Alvarez, I., Garcı´a, M.J., 2002. Varietal differentiation of red wines in the Valencian Regio´n (Spain). J. Agric. Food Chem. 50, 751–755. Alternative Farming Systems Information Center, 2005. Organic Agricultural Products: Marketing and Trade Resources. NAL, USDA. Arnous, A., Makris, D.P., Kefalas, P., 2001. Effect of principal polyphenolic components in relation to antioxidant characteristics of aged red wines. J. Agric. Food Chem. 49, 5736–5742. Bautista-Ortin, A.B., Pe´rez-Prieto, L.J., Romero-Cascales, I., Ferna´ndez-Ferna´ndez, J.I., Lo´pez-Roca, J.M., Go´mez-Plaza, E., 2003. Influencia del estado de maduracio´n de la uva de la variedad Monastrell en el color de los vino. In: II Congreso Nacional ˜ a. de Ciencia y Tecnologı´a de los Alimentos, Orihuela, Espan Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. Lebensm. Wiss. Technol. 28, 25–30. Cantos, E., Espı´n, J.C., Ferna´ndez, M.J., Oliva, J., Toma´s-Barbera´n, F.A., 2003. Postharvest UV-C-irradiated grapes as a potential source of producing stilbeneenriched red wines. J. Agric. Food Chem. 51, 1208–1214. Cantos, E., Garcı´a-Viguera, C., De Pascual-Teresa, S., 2000. Effect of postharvest ultraviolet irradiation on resveratrol and other phenolics of Cv. Napoleo´n table grapes. J. Agric. Food Chem. 48, 4606–4612. CEE, Reglamento n8 2092/91 del CONSEJO de 24 de junio de 1991. Cho, M.J., Howard, L.R., Prior, R.L., Clarck, J.R., 2003. Flavonoid glycosides and antioxidant capacity of various blackberry, blueberry and red grape genotypes determined by higt-performance liquid chromatography/mass spectrometry. J. Sci. Food Agric. 84 (13), 1771–1782. Esteban, M.A., Villanueva, M.J., Lissarrague, J.R., 2001. Effect of irrigation on changes in the antocyanin composition of the skin of cv Tempranillo (Vitis vinifera L.) grape berries during ripening. J. Sci. Food Agric. 81, 409–420. Ferna´ndez-Lo´pez, J.A., Hidalgo, V., Almela, L., Lo´pez-Roca, J.M., 1992. Quantitative changes in anthocyanin pigments of Vitis vinifera cv Monastrell during maturation. J. Sci. Food Agric. 58, 153–155. Gambuti, A., Strollo, D., Ugliano, M., Lecce, L., Moio, L., 2004. Trans-Resveratrol, quercetin, (+)-catechin, and ( )-epicatechin content in South Italian monovatietal wines: relationship with macertion time and marc pressing during winemaking. J. Agric. Food Chem. 52, 5747–5751. Harborne, J.B., Grayer, R.J., 1992. Flavonoids and insects. In: Harborne, J.B. (Ed.), The Flavonoids Advances in Research Since 1986. Chapman and Hall, London, UK. Hidalgo, J., 2003. Tratado de Enologı´a. Mundi-prensa, Madrid. Kammerer, D., Claus, A., Carle, R., Schieber, A., 2004. Polyphenol screening of pomace from red and white grape varieties (Vitis vinifera L.) by HPLC-DAD– MS/MS. J. Agric. Food Chem. 52, 4360–4367. Kennedy, J.A., Matthews, M.A., Waterhouse, A., 2002. Effect of maturity and vine water status on grape skin and wine flavonoids. Am. J. Enol. Vitic. 53 (4), 268–274.
574
J. Mulero et al. / Journal of Food Composition and Analysis 23 (2010) 569–574
Lanaridis, P., Bena-Tzourou, I., 1997. Etude des variations des anthocyanes pendant la maturation des raisins de cinq ce´pages rouges, cultive´s en Gre`ce. J. Int. Sci. Vigne Vin 31, 205–212. Le Guillou, G., Scharpe´, A., 2001. La agricultura ecolo´gica. Guı´a sobre la normativa comunitaria. Comisio´n Europea, Direccio´n General de Agricultura, Oficina de Publicaciones Oficiales de las Comunidades Europeas, Luxemburgo. Lee, J.H., Talcott, T., 2004. Fruit maturity and juice extraction influences ellagic acid derivatives and other antioxidant polyphenolics in Muscadine grapes. J. Agric. Food Chem. 52, 361–366. Lodovici, M., Guglielmi, F., Casalini, C., Meoni, M., Cheynier, V., Dolara, P., 2001. Antioxidant and radical scavenging properties in vitro of polyphenols extracts from red wine. Eur. J. Nutr. 402, 74–77. ˜ oz-Espada, A.C., Wood, K.V., Bordelon, B., Watkins, B.A., 2004. Anthocyanin Mun quantification and radical scavenging capacity of Concord, Norton, and Marechal Foch grapes and wines. J. Agric. Food Chem. 52, 6779–6786. Office International de la Vigne et du Vin, 1990. Recueil des me´thodes Internationales d’analyse des vins et des mouts. OIV, Parı´s. ˜ as, bodegas y vinos. Consejerı´a de medio Ambiente, Pardo, F., 1996. Jumilla. Vin Agricultura y Agua, Com. Aut., Murcia. Pastrana-Bonilla, E., Ago, C.C., Sellappan, S., Krewer, G., 2003. Phenolic content and antioxidant capacity of Muscadine grapes. J. Agric. Food Chem. 51, 5497–5503. Pellegrini, N., Simonetti, P., Gardana, C., Brenna, O., Brighenti, F., Pietta, P., 2000. Polyphenol content and total antioxidant activity of Vini novelli (young red wines). J. Agric. Food Chem. 48, 732–735. ˜ o, S., Gonza´lez-San Jose´, M.L., 2004. Evolution of flavanols, anthocaPe´rez-Magarin nins, and their derivatives during the aging of red wines elaborated from grapes harvested at different stages of ripening. J. Agric. Food Chem. 52, 1181–1189. Pririe, A., Mullins, M.G., 1977. Interrelationships of sugars, anthocyanins, total phenols and dry weight in the skin of grape berries during ripening. Am. J. Enol. Vitic. 28 (4), 204–209. Research Institute of Organic Farming, 2000. Organic Farming Enhances Soil Fertility and Biodiversity: Results from a 21 Year Old Field Trial. FiBL, Frick, Switzerland. Ribe´reau-Gayon, P., 1972. E¨volution des composes phe´noliques au cours de la maturation du raisin. II. Discusio´n des re´sultants obte´nus en 1969, 1970 et 1971. Int. Sci. Vigne Vin 2, 161–175.
Rice-Evans, C.A., Miller, N.J., Paganga, G., 1996. Structure antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20, 933–956. Riu-Aumatell, M., Lo´pez-Barajas, E., Lo´pez-Tamames, E., Buxaderas, S., 2002. Influence of yield and maturation index on polysaccharides and other compounds of grape juice. J. Agric. Food Chem. 50, 4604–4607. Roggero, J.P., Coen, S., Ragonnet, B., 1986. High performance liquid chromatography survey on the changes in pigment content in ripening grapes of Syrah. An approach to anthocyanin metabolism. Am. J. Enol. Vitic. 37, 77–83. Sa´nchez-Moreno, C., Cao, G., Ou, B., Prior, R.L., 2003. Anthocyanin and proanthocyanidin content in selected white and red wines. Oxygen radical absorbance capacity comparison with nontraditional wines obtained from higbush blueberry. J. Agric. Food Chem. 51, 4889–4896. Simonetti, P., Pietta, P., Testolin, G., 1997. Polyphenolic content and total antioxidant potential of selected Italian wines. J. Agric. Food Chem. 45, 1152–1155. Struch, R., 2000. Wine and cardiovascular disease. Food Res. Int. 333 (4), 219–223. Subramani, S., Casimir, C.A., Krewer, G., 2002. Phenolic compounds and antioxidant capacity of Georgia-grown blueberries and blackberries. J. Agric. Food Chem. 50, 2432–2438. Sun, A.Y., Simonyi, A., Sun, G.Y., 2002. The ‘‘French paradox’’ and beyond: neuroprotective effects of polyphenols. Free Radic. Biol. Med. 324, 314–318. Teissedre, P.L., Landrault, N., 2000. Wine phenolics: contribution to dietary intake and bioavailability. Food Res. Int. 33, 461–467. Valls, J., Lampreave, M., Nadal, M., Arola, L., 2000. Importancia de los compuestos feno´licos en la calidad de los vinos tintos de crianza. Alimentacio´n, equipos y tecnologı´a, Marzo, pp. 119–124. Vian, M.A., Tomao, V., Coulomb, P.O., Lacombe, J.M., Dangles, O., 2006. Comparison of the anthocyanin composition during ripening of Syrah grapes grown using organic or conventional agricultural practices. J. Agric. Food Chem. 54, 5230–5235. Wulf, L.W., Nagel, C.W., 1980. Identification and changes of flavonoids in Merlot and Cabernet Sauvignon wines. J. Food Sci. 45, 479–484. Yamaguchi, T., Takamura, H., Matob, T., Terao, J., 1998. HPLC method for evaluation of the radical-scavenging activity of foods by using 1,1-2-diphenyl-2-picrylhydrazyl. Biosci. Biochem. 62, 1201–1204. Yilmaz, Y., Toledo, R., 2004. Major flavonoids in grape seeds and skins: antioxidant capacity of catechin, epicatechin, and gallic acid. J. Agric. Food Chem. 52, 255–260.
Contents lists available at ScienceDirect
Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca
Original Article
Antioxidant activity and phenolic composition of organic and conventional grapes and wines Juana Mulero a,*, Francisco Pardo b, Pilar Zafrilla a a b
Department of Food Technology and Nutrition, Catholic University of San Antonio, Avda Los Jero´nimos s/n, Murcia 30107, Spain Cooperativa de Bodegas San Isidro, Jumilla, Murcia, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 February 2009 Received in revised form 20 April 2010 Accepted 17 May 2010
The phenolic compounds and antioxidant activity from Monastrell variety grapes obtained by organic and conventional agriculture during the last month of ripening and the wines obtained from them were studied. Samples of grapes were collected from the last month of ripening to full maturity in each plot, and winemaking was carried out on the day of the final collection of grape samples, coinciding with the maturity of the grapes. The antioxidant activity a month before harvesting was higher in the organic grapes (5.7 0.03 mM Trolox/g) than in the conventional ones (4.40 0.05 mM Trolox/g), although these differences disappear in the moment of harvesting. Similarly the total amount of phenolic compounds a month before harvesting was higher in the organic grapes (974.2 54.4 mg/kg) than in the conventional grapes (447.7 27.8 mg/kg), although these differences disappear at the moment of harvesting. In wine, phenolic compounds and the antioxidant activity were slightly higher in organic wine than in conventional wine, although the differences were not significant. ß 2010 Elsevier Inc. All rights reserved.
Keywords: Antioxidant activity Phenolic compounds Grape Wine Organic agriculture Anthocyanins Horticulture and biodiversity Food composition Food analysis
1. Introduction Organic agriculture is a horticultural production management system that promotes and enhances biodiversity, biological cycles and soil biological activity (Research Institute of Organic Farming, 2000; Alternative Farming Systems Information Center, 2005). Organic is a labelling term that denotes products produced under the authority of the Organic Foods Production Act (CEE, 1991; Le Guillou and Scharpe´, 2001). The primary goal of organic agriculture is to optimize the health and productivity of interdependent communities of soil life, plants, animals and humans. Grapes contain a large amount of different phenolic compounds in skin, pulp and seeds and these are the main compounds responsible of colour, taste, mouth feel, oxidation and other chemical reactions in wine. Vineyard location and specific site are very important. In dry climates irrigation generally increases the grapevine vigour, berry size and yield (Roggero et al., 1986). Within the same fruit type, the growing season, variety, environmental and climatic conditions, plant disease, soil type,
* Corresponding author. Tel.: +34 968278705; fax: +34 968278666. E-mail addresses: [email protected], [email protected] (J. Mulero). 0889-1575/$ – see front matter ß 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2010.05.001
geographic locations, and maturity seem to influence the concentration of phenolic compounds (Subramani et al., 2002). These compounds are an integral part of the human diet considered as biologically active non-nutrient compounds (Subramani et al., 2002). Among these phenolic substances, flavonoids, and in particular anthocyanins, are of interest because of their high occurrence in foods. The presence of hydroxycinnamic acids and flavonols in grapes is well documented (Ribe´reau-Gayon, 1972; Wulf and Nagel, 1980). The flavonols are localized in the solid parts of the cluster. In red grapes, flavonols are present in much smaller quantities than anthocyanins and in most cases they have been considered to be negligible. As polyphenolic compounds, flavonoids share the ability to act as antioxidants by a free radical scavenging mechanism and metal ion chelation (Lodovici et al., 2001). Growing evidence of the role of radicals and antioxidants in the health and ageing has promoted a great interest on these compounds. A wide range of studies have shown that the antioxidative properties of these compounds may protect against arteriosclerosis and coronary heart disease (Struch, 2000; Sun et al., 2002). The aim of this work was to study the behaviour of phenolic compounds and antioxidant activity during ripening and processing of conventional and organically cultured grapes.
570
J. Mulero et al. / Journal of Food Composition and Analysis 23 (2010) 569–574
2. Materials and methods
degree) were determined following the procedures of OIV (1990).
2.1. Reagents 2.5. Extraction of phenolic compounds Formic acid and methanol (MeOH) of analytical grade were supplied by Merck (Darmstad, Germany). Milli-Q system (Millipore Corp., Bedford, MA) ultrapure was used throughout this research. Cyanidin 3-rutinoside was purchased from Polyphenols A.S. (Sandnes, Norway). Rutin was purchased from Merck (Darmstadt, Germany). Chlorogenic acid, Trolox (6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid) and 2,2-diphenylpicrylhydrazyl (DPPH) were purchased from Sigma (Madrid, Spain). 2.2. Grape samples Grape samples were collected during the last month of ripening. 30 days before harvest, 14 days before harvest, and the last date of collection coincided with the technological maturity of the grapes. At this time samples were collected for analysis of grapes and for the winemaking. One hundred berries were randomly taken from each sample from different parts of various clusters in every vineyard during the early morning hours, transported to the laboratory and processed on the same day. They came from a smallholding representative of the area. The selection of neighbouring parcels allowed us to compare organic and conventional vineyards in the same climate and soil conditions. Parcels P1 and P2 (conventionally cultured) and P3 and P4 (organic cultured) are located in the same spot. Organic grapes were treated with natural pesticides such as dry flowable sulphur, copper salts and oligoelements. Conventional production used fertilizers and pesticides to control weeds, pests, and diseases; these chemicals are subject to rigorous testing and authorization procedures before they can be used, and winegrowers have respected the guidelines and restrictions to use them.
Grapes. Skin represented approximately 13% of the total fresh weight of the grape berry. The grapes were peeled to complete a sample of 5 g of grape skins from which it was extracted the phenolic compounds. Phenolic compounds in grapes were analysed only in skin. Samples were homogenized in an Ultraturrax T-25 equipment (Janke and Kunkel, Ika-Labortechnick) at 24,000 rpm for 1 min after addition of 4 mL of a solution of MeOH/formic acid (97:3) per gram of skin. The extracts were centrifuged at 5000 g for 5 min in a Centromix centrifuge (Selecta, Barcelona), filtered through a 0.45 nm membrane filter Millex HV13 (Millipore), and analysed by HPLC. Three extracts were made for each sample collected and sampling was done in triplicate. Wine phenolic compounds. Before HPLC analysis, ecological and traditional red wines were filtered through a 0.45 mm filter (type Millex HV13, Millipore Corp, Bedford, MA). 2.6. HPLC-DAD analysis of phenolics 20 mL of every sample were injected for HPLC analysis using a Merck-Hitachi pump L-7100 (Merck-Hitachi, Darmstadt, Germany) and a diode array detector Merck-Hitachi 7455, employing a reversed-phase column Lichrochart 100 RP-18 column (Merck, Darmstadt, Germany) (25 cm 0.4 cm, 5 mm particle size), and water plus 5% formic acid (solvent A) or HPLC grade methanol (solvent B) as solvents at a flow rate of 1 mL min 1. Elution was performed with a gradient starting with 2% B to reach 32% B at 30 min, 40% B at 40 min, and 95% B at 50 min, becoming then isocratic for 5 min (Cantos et al., 2000). HPLC experiments were repeated three times. Phenolic content was expressed as mg/kg of fresh weight taking into account the percentage represented by the skin out of the total berry weight. Chromatograms were recorded at 510, 320 and 360 nm.
2.3. Vinification 2.7. Phenolic compounds identification and quantification The vinification process was carried out in a pilot plant the same day of harvesting. The grape clusters were squeezed in a squeezing roller. After eliminating the stalks the squeezed harvest was transferred to fermentation tanks, (tree tanks for each grape samples) where 80 and 70 mg/kg of SO2 were added in conventional and ecological vinification respectively to prevent the development of microorganisms as well as oxidation. The wine was kept in vats at 25 8C for 10 days to allow the processes of fermentation and maceration. Throughout this period daily mass homogenizations were performed to dissolve the cap of the wine, like in industrially performed processes. Density and temperature records were also daily accomplished to control possible fermentation arrests. Once maceration was completed, the must-wine was taken out of the vats and pressed, discarding the marcs and recovering the wine. The wine recovered was decanted 10 days later, discarding dregs. Finally, the wine was bottled and labelled according to its origin.
The phenolic compounds in grapes and wine were identified by their UV–vis spectra, recorded with a diode array detector and, when possible, by chromatographic comparison with authentic markers. Individual anthocyanins were quantified by comparisons with an external standard of cyanidin 3-rutinoside at 510 nm. Flavonols were quantified as rutin at 360 nm and the hydroxycinnamic acid derivatives at 320 nm as chlorogenic acid. All analyses were repeated three times and the results were expressed as mean values in mg/kg of grape SD. The reproducibility of the HPLC analyses was within 5% error. The total phenolic compounds were calculated by adding the amounts of anthocyanins, flavonols and hydroxycinnamic acids detected in each chromatogram, as previously reported (Cantos et al., 2000). All analyses were done in triplicate and the results expressed as mean values SD. The reproducibility of the HPLC analysis was within 6%. 2.8. Statistical data treatment
2.4. Physicochemical analysis Grapes. Baume´ degree, pH, total acidity, were determined following the procedures of OIV (Office International de la Vigne et du vin) (1990). Wines. The characteristic ecological parameters of elaborated wines (pH, density, volatile acidity, total acidity and alcoholic
In order to assess the relationships between the compounds quantified and the antioxidant properties a statistical analysis was performed using SPSS v:12.0. Data are presented as mean SE (standard error). Significance of differences was determined by analysis of variance (ANOVA). A p-value of p < 0.05 was considered statistically significant.
[(Fig._1)TD$IG]
J. Mulero et al. / Journal of Food Composition and Analysis 23 (2010) 569–574
571
2.9. Antioxidant activity The samples were analysed according to the technique reported by Brand-Williams et al. (1995). Briefly, a volume of 5 mL methanol extract (1:1, v/v with methanol) was added to a volume of 2,2diphenyl-1-picrylhydrazyl (DPPH) (Sigma, Steinheim, Germany) 0.094 mM in methanol up to completing 1 mL. The free radical scavenging activity using the free radical DPPH reaction was evaluated by measuring the absorbance at 515 nm after 60 min of reaction at 20 8C in a spectrophotometer (Varian Cary 50-Bio, Victoria, Australia). The reaction was carried out in closed Eppendorf tubes shaken at 20 8C. The results were expressed as mmol/L Trolox equivalents, a vitamin E analogue (Yamaguchi et al., 1998). Fig. 1. Evolution of the antioxidant activity in conventional and organic grapes during ripening.
3. Results and discussion 3.1. Physicochemical analysis Grapes. Berry weight and soluble solids increased over the ripening period. These trends were accompanied by a concomitant increase in pH and a decreased total acidity (Table 1). No significant differences in the physicochemical parameters were observed in both types of grapes. Their evolution during ripening was similar to that found by Pririe and Mullins (1977), and also to that reported for the period of ripening in grapes of the Shiraz and Cabernet sauvignon varieties and in Monastrell variety by Aleixandre et al. (2002). Wine. All parameters are in the normal range for good quality wine (Pardo, 1996). These values were similar to those obtained by Aleixandre et al. (2002) in wines made from Monastrell grapes. No differences were found between the physicochemical parameters of organic wine and traditional wine (Table 2). 3.2. Antioxidant activity Grapes. The antioxidant activity in stage 1 of organic grapes, (5.70 0.03 mM Trolox/g) was higher than that observed in conventional grapes (4.40 0.05 mM Trolox/g) showing significant differences (p = 0.046). However, such difference disappeared when Table 1 Enological parameters of conventional and organic grapes. Sample
Weight
8Be
TAc
pH
Stage 1 CG OG
121.4 126.6
11.05 10.95
7.65 8.16
3.41 3.60
Stage 2 CG OG
134.4 129.6
11.20 11.70
6.91 7.03
3.46 3.55
Stage 3 CG OG
147.5 139.4
12.00 12.20
5.41 6.58
3.70 3.64
TAc, total acidity (g/L tartaric acid); weight, 100 berry (g); 8Be, Baume degree; CG, conventional grapes; OG, organic grapes; Stage 1, first date of berry samples; Stage 2, second date of berry samples; Stage 3, third date of berry samples.
Table 2 Physicochemical parameter of conventional and organic wines. Sample
Density
pH
Alcoholic degree
VA
TA
CW OW
0.95 0.93
3.08 3.26
10.7 11.9
0.220 0.384
6.94 6.00
CW, conventional wine, average values obtained from the results in wines made from the two conventional vineyards; OW, organic wine, average values obtained from the results in wines made from the two organic vineyards; VA, volatile acidity expressed in g/L acetic acid; TA, total acidity expressed in g/L tartaric acid.
the final antioxidant activity was considered (stage 3) (4.70 0.11 mM Trolox/g in organic grapes and 4.80 0.25 mM Trolox/g in conventional grapes) (Fig. 1). No significant differences were established between the antioxidant activity in organic and conventional grapes in the moment of harvest. These values were higher to the reported by Yilmaz and Toledo ˜ oz-Espada et al. (2004) in Chardonnay and Merlot varieties and Mun (2004) in Norton, Concord and Marechal varieties by, and lower than the values described in the Muscadina variety by Pastrana-Bonilla et al. (2003). These variations in the evolution of the antioxidant activity of the grapes have been previously reported by Lee and Talcott (2004) in Muscadina red grapes. Wine. The antioxidant activity is similar in both types, organic and traditional, 6.78 0.45 mM Trolox/ml and 6.02 0.44 mM Trolox/ml respectively. These values are lower than those obtained by other authors (Rice-Evans et al., 1996; Simonetti et al., 1997; Pellegrini et al., 2000; Gambuti et al., 2004). 3.3. Anthocyanins Ten anthocyanin-glucosides (five of them acylated) were identified in organic and conventional red grapes skins and their wines: delphinidin 3-glucoside, cyanidin 3-glucoside, petunidin 3glucoside, peonidin 3-glucoside, malvidin 3-glucoside, cyanidin 3-pcoumaroylglucoside, petunidin 3-p-coumaroylglucoside, peonidin 3-p-coumaroylglucoside, petunidin 3-p-coumaroylglucoside, and malvidin 3-p-coumaroylglucoside, as described by Ferna´ndez-Lo´pez et al. (1992) and Cantos et al. (2003). The acylated derivatives were globally quantified due to the small individual concentrations observed. Grapes. Malvidin 3-glucoside was the major anthocyanin in both types of grapes, with mean contributions of about 42.0% and 29.0% of the total anthocyanins in conventional and organic grapes, in stage 3 respectively (Table 3). These results agree with those presented previously by Ferna´ndez-Lo´pez et al. (1992) and Cantos et al. (2003). The concentration of anthocyanins was higher in organic grapes (700.0 31.6 mg/kg) than in conventional ones (329.6 17.07 mg/ kg) in stage 1, with significant differences (p = 0.011) which remained in stage 2 (p = 0.012); these results disagree with Vian et al. (2006). However, these differences disappeared in stage 3 with the grapes ripening, showing mean concentrations of anthocyanins up to 706.5 35.82 mg/kg in conventional grapes and 741.9 38.82 mg/ kg in organic grapes (Table 4). The evolution of the average concentration of total anthocyanins in conventional grapes during ripening showed an increase in the concentration of anthocyanins during this period. These results agree with those obtained by other authors (Lanaridis and BenaTzourou, 1997; Esteban et al., 2001; Kennedy et al., 2002; Pe´rez-
572
J. Mulero et al. / Journal of Food Composition and Analysis 23 (2010) 569–574
Table 3 Anthocyanins. Composition of conventional and organic grapes during ripening. Sample
Dp-3-glc
Cy-3-glc
Pt-3-glc
Pn-3-glc
Mv-3-glc
Acyl derv.
Stage 1 CG OG
40.83 3.42 101.5 4.87
29.53 1.33 106.9 4.59
39.45 0.88 87.98 5.12
32.55 1.44 113.6 5.37
132.2 5.35 201.1 7.79
54.63 4.80 88.96 5.28
Stage 2 CG OG
43.38 2.83 91.83 4.77
40.25 2.16 113.8 6.1
42.89 1.59 84.10 6.33
40.05 2.22 292.5 6.66
134.8 6.45 199.4 8.17
75.63 6.00 95.28 3.20
Stage 3 CG OG
72.68 2.96 96.85 5.33
78.00 3.51 107.3 7.18
82.5 3.55 76.88 4.05
108.9 6.17 152.5 8.55
252.5 8.18 213.5 10.28
113.0 11.49 95.43 3.45
Values are expressed as mg/kg fresh weight of total grape berry. Mean values are shown (n = 3) standard error. CG, conventional grapes; OG, organic grapes; Stage 1, first date of berry samples; Stage 2, second date of berry samples; Stage 3, third date of berry samples; Dp-3-glc, delphinidin 3-glucoside; Cy-3-glc, cyanidin 3-glucoside; Pt-3-glc, petunidin 3glucoside; Pn-3-glc, peonidin 3-glucoside; Mv-3-glc, malvidin 3-glucoside; Acyl derv., acylated derivatives. Table 4 Phenolics compounds of maturity grapes and wines obtained from them. Sample
Anthocyanins
Hydroxycinnamic acids derivatives
Flavonols
Total phenolic compounds
CG OG CW OW
706.5 35.82 741.9 38.82 296.6 17.22 344.7 14.78
16.98 0.57 22.81 0.24 49.6 2.68 43.02 2.66
249.6 20.61 217.3 19.91 150.0 13.27 170.5 7.75
973.1 57.00 982.0 58.97 496.3 33.17 558.3 25.19
Values of grapes are expressed as mg/kg fresh weight of total grape berry standard error. Values of wine are expressed as mg/L of wine standard error. CG, conventional grapes; OG, organic grape; CW, conventional wine; OW, organic wine.
˜ o and Gonza´lez-San Jose´, 2004) who noted that the Magarin content of anthocyanins in the grapes skins was clearly influenced by the ripening of the grapes, and the beginning in the veraison, displaying the highest content at the moment of harvesting. Wine. The average content of anthocyanins was lower in conventional wine than organic wine, and the anthocyanin concentrations in both types of wine was higher than mentioned by other authors (Teissedre and Landrault, 2000; Arnous et al., 2001; Sa´nchez-Moreno et al., 2003). The malvidin 3-glucoside was the major anthocyanin in both types of wine.
conventional ones (16.98 0.57 mg/kg) although these differences are not significant (p > 0.05). This difference stayed without significant variation during ripening of the grapes and there were lower than those obtained by Kammerer et al. (2004) in different varieties of red grapes. Wine. Concentration of hydroxycinnamic derivatives was similar in traditional and organic wine (Table 4). Trans-Caffeoyltartaric acid was the majority compound in both types of wine with a concentration of 21.40 1.31 mg/L in conventional wine and 24.40 0.41 mg/L in organic wines.
3.4. Hydroxycinnamic acids
3.5. Flavonols
Three hydroxycinnamic derivatives were quantified in grapes and wine: trans-caffeoyltartaric acid, trans-p-coumaroyltartaric acid and an unidentified third derivative, which we have been named compound 2. Grapes. In both types of grapes the major hydroxycinnamic acids derivatives were trans-p-coumaroyltartaric and trans-caffeoyltartaric acid (Table 5). The concentration of hydroxycinnamic acids derivatives in stage 1 was higher in organic grapes (22.81 0.24 mg/kg) than in
Six flavonols were identified in organic and conventional red grapes skins and wine: myricetin 3-glucoside, quercetin 3glucoside, quercetin 3-rutinoside, kaempferol 3-glucoside, myricetin and quercetin. Grapes. In both types of grapes, conventional and organic, myricetin 3-glucoside, quercetin 3-glucoside, and quercetin 3rutinoside were the most abundant flavonols, followed by kaempferol 3-glucoside, myricetin and quercetin (Table 6). The concentration of flavonols in stage 1 was higher in organic grapes (251.2 20.7 mg/kg) than in conventional ones (101.6 10.90 mg/kg) with significant differences (p = 0.021). Such differences disappeared with the ripening of the grapes (Table 4). The concentration of flavonols observed was slightly higher to that found in Monastrell grapes by Cantos et al. (2003). Nevertheless, they are within the concentrations described by Cho et al. (2003) in different varieties of red grapes and by Lee and Talcott (2004) in Muscadina grapes. Wine. Content of flavonols was lower in conventional wine than organic wine (Table 4), although there were no significant differences. Myricetin 3-glucoside and quercetin 3-glucoside was majority flavonols (37.0 3.5 and 40.2 3.1 mg/L respectively for conventional wine and 41.9 1.2 and 41.1 1.6 mg/L respectively for organic wine).
Table 5 Evolution of the hydroxycinnamic acids derivatives composition in conventional and organic grapes during the ripening. Sample
Trans-Caffeoyltartaric
Compound 2
Trans-p-Coumaroyltartaric
Stage 1 CG OG
4.79 0.32 6.24 0.38
3.57 0.18 5.17 0.23
8.63 0.07 11.41 0.09
Stage 2 CG OG
4.20 0.33 4.92 0.7
3.33 0.27 5.04 0.20
8.15 0.10 11.44 0.23
Stage 3 CG OG
3.58 0.25 4.69 0.50
2.78 0.35 4.60 0.06
10.28 0.20 11.58 0.15
Values are expressed as mg/kg fresh weight of total grape berry. Mean values are shown (n = 3) standard error. CG, conventional grapes; OG, organic grape; Stage 1, first date of berry samples; Stage 2, second date of berry samples; Stage 3, third date of berry samples.
3.6. Total phenolic compounds Grapes. The concentration of the total phenolic compounds in the first sampling was of 974.2 54.46 mg/kg in organic samples
J. Mulero et al. / Journal of Food Composition and Analysis 23 (2010) 569–574
573
Table 6 Evolution of the flavonols composition in conventional and organic grapes during ripening. Sample
Myr-3-glc
Q-3-glc
Q-3-Rut
Kp-3-glc
Myricetin
Quercetin
Stage 1 CG OG
23.53 1.88 54.18 4.23
27.70 3.40 64.38 4.43
41.90 4.90 113.0 9.46
2.45 0.11 4.73 0.33
4.28 0.33 13.18 1.85
1.76 0.29 1.90 0.48
Stage 2 CG OG
22.35 4.28 36.36 3.25
23.90 4.00 46.18 6.15
43.18 4.00 107.8 4.25
3.48 0.25 4.90 0.38
4.73 0.30 13.95 2.10
0.93 0.22 1.38 0.06
Stage 3 CG OG
67.60 4.62 44.65 4.60
34.98 2.20 39.04 6.87
118.0 7.50 110.7 5.99
9.38 1.98 4.78 0.83
16.13 3.98 14.85 1.16
3.50 0.35 3.25 0.48
Values are expressed as mg/kg fresh weight of total grape berry. Mean values are shown (n = 3) standard error. CG, conventional grapes; OG, organic grapes; Stage 1, first date of berry samples; Stage 2, second date of berry samples; Stage 3, third date of berry samples; Myr-3-glc, myricetin 3-glucoside; Q-3-glc, quercetin 3-glucoside; Q-3-Rut, quercetin 3rutinoside; Kp-3-glc, kaempferol 3-glucoside.
[(Fig._2)TD$IG] Grayer, 1992). The differences found between both types of grapes may be due to different treatment of crops, since organic grapes were not treated with fertilizers and pesticides which defend the plant against attacks. The differences found between both types of grape in antioxidant activity in first sampling coincides with the highest concentration of phenolic compounds in organic grapes, since these are the makers of the antioxidant activity of grape. Acknowledgments We are grateful to Bodegas San Isidro of Jumilla (Murcia) for supplying wine and grapes. The authors acknowledge the financial support of Consejeria de Ciencia, Tecnologı´a, Industria y Comercio de la Regio´n de Murcia and Catholic University San Antonio de Murcia. Fig. 2. Evolution of the total phenols in conventional and organic grapes during ripening.
and 447.7 27.85 mg/kg in conventional grapes, showing significant differences (p = 0.016). This higher concentration of phenolic compounds in the organic grapes disappeared during ripening (Fig. 2). These values are lower than those obtained by Valls et al. (2000) in Tempranillo and Cabernet sauvignon varieties. These changes in the concentration of phenolic compounds in different harvests have been previously reported by several authors (Riu-Aumatell et al., 2002). On the other hand, Hidalgo (2003) pointed out that the concentration of polyphenolic compounds of the skin changes greatly depending on the variety and also on the grapes ripening stage. Other authors, such as Bautista-Ortin et al. (2003) stated that the stage of ripening not only influences the phenolic content of the variety, but also its degree of extraction, which will lately condition the presence in the wine of phenolic compounds after the winemaking process. Wine. Content of total phenolic compounds is slightly higher in organic wines than in conventional wine. However, when applying the analysis of variance (ANOVA) to the different phenolic compounds quantified, there was no significant differences between the concentrations found in conventional and ecological red wine at confidence level of 95%. 4. Conclusion In both types of grapes, organic and conventional, only significant differences were found in the phenolic compounds in the first sampling, and these differences have disappeared with maturation. Phenolics have a number of important roles to play in viticulture and oenology including UV protection, disease resistance, and defence against predation in plants (Harborne and
References Aleixandre, J.L., Lizama, V., Alvarez, I., Garcı´a, M.J., 2002. Varietal differentiation of red wines in the Valencian Regio´n (Spain). J. Agric. Food Chem. 50, 751–755. Alternative Farming Systems Information Center, 2005. Organic Agricultural Products: Marketing and Trade Resources. NAL, USDA. Arnous, A., Makris, D.P., Kefalas, P., 2001. Effect of principal polyphenolic components in relation to antioxidant characteristics of aged red wines. J. Agric. Food Chem. 49, 5736–5742. Bautista-Ortin, A.B., Pe´rez-Prieto, L.J., Romero-Cascales, I., Ferna´ndez-Ferna´ndez, J.I., Lo´pez-Roca, J.M., Go´mez-Plaza, E., 2003. Influencia del estado de maduracio´n de la uva de la variedad Monastrell en el color de los vino. In: II Congreso Nacional ˜ a. de Ciencia y Tecnologı´a de los Alimentos, Orihuela, Espan Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. Lebensm. Wiss. Technol. 28, 25–30. Cantos, E., Espı´n, J.C., Ferna´ndez, M.J., Oliva, J., Toma´s-Barbera´n, F.A., 2003. Postharvest UV-C-irradiated grapes as a potential source of producing stilbeneenriched red wines. J. Agric. Food Chem. 51, 1208–1214. Cantos, E., Garcı´a-Viguera, C., De Pascual-Teresa, S., 2000. Effect of postharvest ultraviolet irradiation on resveratrol and other phenolics of Cv. Napoleo´n table grapes. J. Agric. Food Chem. 48, 4606–4612. CEE, Reglamento n8 2092/91 del CONSEJO de 24 de junio de 1991. Cho, M.J., Howard, L.R., Prior, R.L., Clarck, J.R., 2003. Flavonoid glycosides and antioxidant capacity of various blackberry, blueberry and red grape genotypes determined by higt-performance liquid chromatography/mass spectrometry. J. Sci. Food Agric. 84 (13), 1771–1782. Esteban, M.A., Villanueva, M.J., Lissarrague, J.R., 2001. Effect of irrigation on changes in the antocyanin composition of the skin of cv Tempranillo (Vitis vinifera L.) grape berries during ripening. J. Sci. Food Agric. 81, 409–420. Ferna´ndez-Lo´pez, J.A., Hidalgo, V., Almela, L., Lo´pez-Roca, J.M., 1992. Quantitative changes in anthocyanin pigments of Vitis vinifera cv Monastrell during maturation. J. Sci. Food Agric. 58, 153–155. Gambuti, A., Strollo, D., Ugliano, M., Lecce, L., Moio, L., 2004. Trans-Resveratrol, quercetin, (+)-catechin, and ( )-epicatechin content in South Italian monovatietal wines: relationship with macertion time and marc pressing during winemaking. J. Agric. Food Chem. 52, 5747–5751. Harborne, J.B., Grayer, R.J., 1992. Flavonoids and insects. In: Harborne, J.B. (Ed.), The Flavonoids Advances in Research Since 1986. Chapman and Hall, London, UK. Hidalgo, J., 2003. Tratado de Enologı´a. Mundi-prensa, Madrid. Kammerer, D., Claus, A., Carle, R., Schieber, A., 2004. Polyphenol screening of pomace from red and white grape varieties (Vitis vinifera L.) by HPLC-DAD– MS/MS. J. Agric. Food Chem. 52, 4360–4367. Kennedy, J.A., Matthews, M.A., Waterhouse, A., 2002. Effect of maturity and vine water status on grape skin and wine flavonoids. Am. J. Enol. Vitic. 53 (4), 268–274.
574
J. Mulero et al. / Journal of Food Composition and Analysis 23 (2010) 569–574
Lanaridis, P., Bena-Tzourou, I., 1997. Etude des variations des anthocyanes pendant la maturation des raisins de cinq ce´pages rouges, cultive´s en Gre`ce. J. Int. Sci. Vigne Vin 31, 205–212. Le Guillou, G., Scharpe´, A., 2001. La agricultura ecolo´gica. Guı´a sobre la normativa comunitaria. Comisio´n Europea, Direccio´n General de Agricultura, Oficina de Publicaciones Oficiales de las Comunidades Europeas, Luxemburgo. Lee, J.H., Talcott, T., 2004. Fruit maturity and juice extraction influences ellagic acid derivatives and other antioxidant polyphenolics in Muscadine grapes. J. Agric. Food Chem. 52, 361–366. Lodovici, M., Guglielmi, F., Casalini, C., Meoni, M., Cheynier, V., Dolara, P., 2001. Antioxidant and radical scavenging properties in vitro of polyphenols extracts from red wine. Eur. J. Nutr. 402, 74–77. ˜ oz-Espada, A.C., Wood, K.V., Bordelon, B., Watkins, B.A., 2004. Anthocyanin Mun quantification and radical scavenging capacity of Concord, Norton, and Marechal Foch grapes and wines. J. Agric. Food Chem. 52, 6779–6786. Office International de la Vigne et du Vin, 1990. Recueil des me´thodes Internationales d’analyse des vins et des mouts. OIV, Parı´s. ˜ as, bodegas y vinos. Consejerı´a de medio Ambiente, Pardo, F., 1996. Jumilla. Vin Agricultura y Agua, Com. Aut., Murcia. Pastrana-Bonilla, E., Ago, C.C., Sellappan, S., Krewer, G., 2003. Phenolic content and antioxidant capacity of Muscadine grapes. J. Agric. Food Chem. 51, 5497–5503. Pellegrini, N., Simonetti, P., Gardana, C., Brenna, O., Brighenti, F., Pietta, P., 2000. Polyphenol content and total antioxidant activity of Vini novelli (young red wines). J. Agric. Food Chem. 48, 732–735. ˜ o, S., Gonza´lez-San Jose´, M.L., 2004. Evolution of flavanols, anthocaPe´rez-Magarin nins, and their derivatives during the aging of red wines elaborated from grapes harvested at different stages of ripening. J. Agric. Food Chem. 52, 1181–1189. Pririe, A., Mullins, M.G., 1977. Interrelationships of sugars, anthocyanins, total phenols and dry weight in the skin of grape berries during ripening. Am. J. Enol. Vitic. 28 (4), 204–209. Research Institute of Organic Farming, 2000. Organic Farming Enhances Soil Fertility and Biodiversity: Results from a 21 Year Old Field Trial. FiBL, Frick, Switzerland. Ribe´reau-Gayon, P., 1972. E¨volution des composes phe´noliques au cours de la maturation du raisin. II. Discusio´n des re´sultants obte´nus en 1969, 1970 et 1971. Int. Sci. Vigne Vin 2, 161–175.
Rice-Evans, C.A., Miller, N.J., Paganga, G., 1996. Structure antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20, 933–956. Riu-Aumatell, M., Lo´pez-Barajas, E., Lo´pez-Tamames, E., Buxaderas, S., 2002. Influence of yield and maturation index on polysaccharides and other compounds of grape juice. J. Agric. Food Chem. 50, 4604–4607. Roggero, J.P., Coen, S., Ragonnet, B., 1986. High performance liquid chromatography survey on the changes in pigment content in ripening grapes of Syrah. An approach to anthocyanin metabolism. Am. J. Enol. Vitic. 37, 77–83. Sa´nchez-Moreno, C., Cao, G., Ou, B., Prior, R.L., 2003. Anthocyanin and proanthocyanidin content in selected white and red wines. Oxygen radical absorbance capacity comparison with nontraditional wines obtained from higbush blueberry. J. Agric. Food Chem. 51, 4889–4896. Simonetti, P., Pietta, P., Testolin, G., 1997. Polyphenolic content and total antioxidant potential of selected Italian wines. J. Agric. Food Chem. 45, 1152–1155. Struch, R., 2000. Wine and cardiovascular disease. Food Res. Int. 333 (4), 219–223. Subramani, S., Casimir, C.A., Krewer, G., 2002. Phenolic compounds and antioxidant capacity of Georgia-grown blueberries and blackberries. J. Agric. Food Chem. 50, 2432–2438. Sun, A.Y., Simonyi, A., Sun, G.Y., 2002. The ‘‘French paradox’’ and beyond: neuroprotective effects of polyphenols. Free Radic. Biol. Med. 324, 314–318. Teissedre, P.L., Landrault, N., 2000. Wine phenolics: contribution to dietary intake and bioavailability. Food Res. Int. 33, 461–467. Valls, J., Lampreave, M., Nadal, M., Arola, L., 2000. Importancia de los compuestos feno´licos en la calidad de los vinos tintos de crianza. Alimentacio´n, equipos y tecnologı´a, Marzo, pp. 119–124. Vian, M.A., Tomao, V., Coulomb, P.O., Lacombe, J.M., Dangles, O., 2006. Comparison of the anthocyanin composition during ripening of Syrah grapes grown using organic or conventional agricultural practices. J. Agric. Food Chem. 54, 5230–5235. Wulf, L.W., Nagel, C.W., 1980. Identification and changes of flavonoids in Merlot and Cabernet Sauvignon wines. J. Food Sci. 45, 479–484. Yamaguchi, T., Takamura, H., Matob, T., Terao, J., 1998. HPLC method for evaluation of the radical-scavenging activity of foods by using 1,1-2-diphenyl-2-picrylhydrazyl. Biosci. Biochem. 62, 1201–1204. Yilmaz, Y., Toledo, R., 2004. Major flavonoids in grape seeds and skins: antioxidant capacity of catechin, epicatechin, and gallic acid. J. Agric. Food Chem. 52, 255–260.