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SUPPLEMENTARY MATERIAL
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Antioxidant capacity, radical scavenger activity, lipid oxidation protection
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analysis and antimicrobial activity of red grape extracts from different varieties
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cultivated in Portugal
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Ana C. Correia and António M. Jordão*
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Polytechnic Institute of Viseu (Centre for the Study of Education, Technologies and Health),
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Agrarian Higher School, Estrada de Nelas, Quinta da Alagoa, Ranhados, 3500-606 Viseu, Portugal.
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*Corresponding author
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Email: antoniojordao@esav.ipv.pt
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antoniojordao@portugalmail.com
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Abstract
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The aim of this study was to investigate the antioxidant capacity, radical scavenger activity, lipid oxidation
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protection and antimicrobial activity of grape extracts from 12 different red grape varieties cultivated in
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Portugal. The mean values of total phenolic content quantified in grape extracts varied from 833.7 to 2005.6
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mg/L gallic acid. Antioxidant capacity results showed different values for each grape variety ranging from
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3.96 to 32.96 mm/L Fe (II). The scavenger activity values ranged from 15.99% to 54.82% for the superoxide
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radical and from 11.79% to 29.67% for the hydroxyl radical. The grape extracts with the highest antioxidant
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capacity had a positive effect on the lipid oxidation protection and induced low peroxide values in butter
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samples. Finally, concerning antimicrobial activity, grape extracts from Touriga Nacional and Tinta Roriz
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grape varieties had significant antimicrobial activity, especially notable for total mesophilic aerobics.
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Keywords: antioxidant capacity; antimicrobial activity; grapes; lipid oxidation; scavenger activity.
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Experimental
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Sample preparation
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Twelve Portuguese red grape varieties belonging to the Vitis specie (subsp. vinifera) were harvested at
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technological maturity in 2011 vintage, from an experimental vineyard in the northeast of Portugal. The red
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grape varieties studied are from vines with the following standard name and voucher specimen number (in
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brackets): Mourisco Tinto (FRA 139), Touriga Franca (24 JBP), Touriga Nacional (19 ISA), Syrah (INRA-
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ENTAV 470), Pinot Noir (INRA-ENTAV 872), Tinta Roriz (110 JBP), Castelão (29 EAN), Merlot
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(ERSAFVG 353), Cabernet Sauvignon (INRA-ENTAV 169) and Alicant Boushet (INRA-ENTAV 803).
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Baga and Touriga Fêmea red grape varieties, until now don’t have a voucher specimen number attributed.
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Grapes (samples of 200 berries in duplicate) were kept frozen at -20 ºC until processing. For the grape
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varieties studied, sampling was done at technological maturity and in good sanitary conditions.
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Each grape berry sample was prepared as described by Carbonneau & Champagnol (1993). Thus, all
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analyses were done on the extract obtained from a maceration of crushed grapes at 25 ºC for 24 h in a pH 3.7
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buffer. All measurements were performed in duplicate.
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Total phenolic content
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Total phenolic compounds were determined by measuring absorbance at 280 nm using the methodology
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described by Singleton & Rossi (1965).
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Antioxidant capacity
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The FRAP method was used to measure the reductive power of a sample, according to the method described
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by Rivero-Pérez et al. (2008). It is based on increased absorbance at 593 nm due to the formation of
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tripyridyl-s-triazine complexes with iron (II) (TPTZ-Fe(II)) in the presence of a reductive agent. The reactive
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mixture was prepared by mixing 25 mL of sodium acetate buffer solution (0.3 mol/L, pH 3.6), 2.5 mL TPTZ
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(10 mmol/L), 2.5 mL FeCl3 (20 mmol/L) and 3 mL water. An aliquot of 30 μL of the diluted grape extract
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sample (diluted in water at 1:50) was added to 970 μL of the latter reactive mixture and incubated at 37 ºC
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for 30 min. The results were expressed as mmol/L of Fe(II), using the linear calibration obtained with
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different concentrations of FeSO4 (0.0 to 1.2 mmol/L).
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Radical scavenger activity
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Hydroxyl radical-scavenging activity (HRSA): Fenton reaction was used for measuring HRSA (Halliwell et
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al. 1987). The hydroxyl radicals (HO•) were generated through the following system: 10 L of FeCl3 (0.1
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mmol/L), 10 L of ascorbic acid (0.1 mmol/L), 10 L of H2O2 (1 mmol/L), and 10 L of EDTA (0.1
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mmol/L). Samples (15 L at a dilution of 1:50 in distilled water) were incubated at 37 ºC for 1h, with 20 L
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of desoxyribose (1 mmol/L final concentration) and 980 µL of potassium phosphate buffer solution (pH 7.4)
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in the presence of FeCl3, ascorbic acid, H2O2, and EDTA. 1.5 mL of trichloroacetic acid (28 %, w/v) and 1
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mL of thiobarbituric acid (1 %, w/v, 0.05 mol/L NaOH) were added to 1 mL of the sample under incubation
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and held for 15 min at 100 ºC, after which it was left to cool to room temperature. The malondialdehyde
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formed from the decay of desoxyribose was evaluated in a reaction with thiobarbituric acid and measured at
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532 nm. The final results were expressed as inhibition percent in relation to a control test (without the
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sample).
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Superoxide radical-scavenging activity (SRSA): The superoxide radical reacts with 4-nitroblue tetrazolium
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chloride to generate a colored compound with absorbance to 560 nm (Liu et al. 1997). The antioxidant
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scavenging superoxide radical values are associated with the coloration formed during the reaction process.
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The reactive solution was made with 50 L of nicotinamide (77 mol/L), 50 L of 4-nitroblue tetrazolium
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chloride (50 mol/L) and 5 L of phenazin methosulfate (3.3 mol/L final concentration) in a medium
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containing 16 mmol/L Tris–HCL at pH 8 and 10 L of the sample. The results were expressed as a
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percentage of inhibition in relation to a control test (without the sample).
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Lipid oxidation protection
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In order to analyze the lipid oxidation protection of the different grape extracts studied, peroxide values of
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butter samples containing red grape extracts were measured for 14 storage days at room temperature (20 ºC)
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using the method described by AOCS (1990).
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Antimicrobial activity
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Antimicrobial activity was screened against Staphylococcus aureus and total mesophilic aerobic
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microorganisms. The inoculated plates containing each grape extract were incubated in duplicate for 48 h at
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30 ºC (for total mesophilic aerobics microorganisms) and until 96 h at 37 ºC (for S. aureus). At the end of the
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incubated period, the inhibition zones formed on the mediums were evaluated.
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Statistical analysis
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The data are presented as mean ± standard deviation. To determine whether there is a statistically significant
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difference between the data obtained from the different grape varieties, an analysis of variance and
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comparison of treatment means were carried out using the Statistica 7 Software (Stat Soft, Tulsa, U.S.A.).
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Fisher's Least Significant Difference test was applied to the data as a comparison test to determine when
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samples are significantly different (p<0.05).
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References
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AOCS. 1990. Official methods and recommended practices of the American Oil Chemists’ Society. 4th ed.
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105
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Champaign, IL: American Oil Chemists’ Society.
Carbonneau A, Champagnol F. 1993. New systems of integral culture of the vineyard. Report of Programme
AIR-3-CT93.
Halliwell B, Gutteridge JMC, Aruoma OI. 1987. The deoxyribose method: a simple ‘test-tube’ assay for
determination of rate constants for reactions of hydroxyl radicals. Anal Biochem. 165:215–219.
Liu F, Ooi VEC, Chang ST. 1997. Free radical scavenging activity of mushroom polysaccharide extracts.
Life Sci. 60:763–771.
Rivero-Pérez MD, González-Sanjosé ML, Ortega-Herás M, Muñiz P. 2008. Antioxidant potential of singlevariety red wines aged in the barrel and in the bottle. Food Chem. 111:957–964.
Singleton VL, Rossi JA. 1965. Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid
reagents. Am J Enol Vitic. 16:144–158.