sulfites addition in comparison to conventional red wines

Food Chemistry 179 (2015) 336–342

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Polyphenols content, phenolics profile and antioxidant activity of organic red wines produced without sulfur dioxide/sulfites addition in comparison to conventional red wines Ivana Garaguso, Mirella Nardini ⇑ Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria (CRA) – Centro di Ricerca per gli Alimenti e la Nutrizione (CRA-NUT), via Ardeatina 546, Roma RM 00178, Italy

a r t i c l e

i n f o

Article history: Received 28 November 2014 Received in revised form 30 January 2015 Accepted 31 January 2015 Available online 9 February 2015 Chemical compounds studied in this article: Caffeic acid (PubChem CID: 689043) Ferulic acid (PubChem CID: 445858) 40 -Hydroxycinnamic acid (PubChem CID: 637542) Syringic acid (PubChem CID: 10742) Catechin (PubChem CID: 9064) Resveratrol (PubChem CID: 445154) Rutin (PubChem CID: 5280805) Myricetin (PubChem CID: 5281672) Quercetin (PubChem CID: 5280343)

a b s t r a c t Wine exerts beneficial effects on human health when it is drunk with moderation. Nevertheless, wine may also contain components negatively affecting human health. Among these, sulfites may induce adverse effects after ingestion. We examined total polyphenols and flavonoids content, phenolics profile and antioxidant activity of eight organic red wines produced without sulfur dioxide/sulfites addition in comparison to those of eight conventional red wines. Polyphenols and flavonoids content were slightly higher in organic wines in respect to conventional wines, however differences did not reach statistical significance. The phenolic acids profile was quite similar in both groups of wines. Antioxidant activity was higher in organic wines compared to conventional wines, although differences were not statistically significant. Our results indicate that organic red wines produced without sulfur dioxide/sulfites addition are comparable to conventional red wines with regard to the total polyphenols and flavonoids content, the phenolics profile and the antioxidant activity. Ó 2015 Elsevier Ltd. All rights reserved.

Keywords: Organic wine Sulfites Polyphenols Antioxidant activity

1. Introduction Oxidative stress is involved in the pathology of many diseases, such as atherosclerosis, diabetes, neurodegenerative diseases, aging and cancer (Aruoma, 1998). Dietary antioxidants may afford protection against oxidative stress-related diseases. Among dietary antioxidants, phenolics compounds are by far the most abundant in most diets (Aruoma, 1998; Block, Patterson, & Subar, 1992). Epidemiological studies strongly suggest that long-term consumption of polyphenols-rich foods offers some protection against the development of cancer, cardiovascular diseases, diabetes, osteoporosis and degenerative diseases (Aruoma, 1998). For individuals regularly consuming wine, coffee, beer and tea, these beverages will likely be the major sources of phenolics. It has been recognized that wine can have beneficial effects on human health when drunk ⇑ Corresponding author. Tel.: +39 06 51494454; fax: +39 06 51494550. E-mail address: [email protected] (M. Nardini). http://dx.doi.org/10.1016/j.foodchem.2015.01.144 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

in moderation. Epidemiological studies from numerous different populations show that individuals with the habit of moderate daily wine consumption present significant reduction in all-cause and particularly cardiovascular mortality when compared to abstainers or those who drink excess alcohol (Guilford & Pezzuto, 2011; Hertog, Feskens, Hollman, Katan, & Kornhout, 1993; Renaud & de Lorgeril, 1992). Human intervention studies investigating moderate levels of consumption also support the health benefits of wine. Hypolipidemic, hypotensive and anti-atherosclerotic effects, antioxidant status improvement and reduction in oxidation biomarkers have been described (Cooper, Chopra, & Thurnham, 2004; Cordova, Jackson, Berke-Schlessel, & Sumpio, 2005; PerezJimenez & Saura-Calixto, 2008). The total amount of polyphenols in red wines has been estimated to range from 2000 to 6000 mg/L (Quideau, Deffieux, Douat-Casassus, & Pouysegu, 2011). Wine polyphenols have been reported to be bioavailable in several studies (Bitsch, Netzel, Frank, Strass, & Bitsch, 2004; Gonthier et al., 2003; Nardini et al.,

I. Garaguso, M. Nardini / Food Chemistry 179 (2015) 336–342

2009; Vitaglione et al., 2005). These compounds are directly related to the quality of wines, so phenolic analysis can be used as an effective tool in characterizing different wines. Many factors can influence the phenolic composition of wines, including grape variety and the technology applied (Mulero, Zafrilla, Cayuela, Martinez-Cacha, & Pardo, 2011). Nevertheless, wine may also contain some components negatively affecting the health of moderate wine drinkers, such as pesticides, preservatives (sulfur dioxide, sulfites), trace metals and various compounds produced by different microorganisms during winemaking, including neurotoxins (ochratoxin A), potential carcinogens (ethyl carbamate) and allergens (biogenic amines) (Pozo-Bayon, Monagas, Bartolomé, & MorenoArribas, 2012). Among these, sulfur dioxide and sulfites are widely used during the different steps of winemaking and storage, for their sterilizing and antibacterial properties. Moreover, for their antioxidant properties, they can act against non-enzymatic and enzymatic oxidation of wines. Although sulfur dioxide and sulfites are widely used as preservatives in food, beverage and pharmaceutical industries, the use of these additives is strictly controlled due to the risks for human health derived from their consumption. In addition, high doses of sulfur dioxide/sulfites can cause organoleptic alterations in the final product (indesirable aromas of the sulfurous gas and the reduction products hydrosulfate and mercaptans). Sulfites may induce relevant adverse effects after their ingestion, such as anaphylactic shock, asthmatic attacks, urticaria, angioedema, nausea, abdominal pain, diarrhea and even death (DaltonBunnow, 1985; Vally & Thompson, 2003; Yang & Purchase, 1985). A considerable percentage of consumers show intolerance or high sensitivity to sulfites, with increasing risk in asthmatic and children. Recently, Laggner, Hermann, Sturm, Gmeiner, and Kapiotis (2005) reported that sulfite at concentrations found in vivo strongly promote low-density lipoprotein (LDL) oxidation by Cu2+ and stimulate the LDL-oxidase activity of ceruloplasmin, acting as a pro-atherogenic agent in the presence of transition metal. The consumption levels of sulfites show that the risk of exceeding the Acceptable Daily Intake concerns only regular consumers of alcoholic beverages, such as cider, beer and, particularly, wine, the main vector (Bemrah et al., 2012; Mareschi, Francois-Collange, & Suschetet, 1992). According to European Commission regulations (Ruling n° 606/ 2009) (EC, 2009), the total sulfur dioxide content cannot exceed 150 mg/L in conventional red wines, and 200 mg/L in conventional white wines. In organic wines, the total sulfur dioxide content cannot exceed 100 mg/L in red wines and 150 mg/L in white wines. These legislative rules, attracted the interest of scientific research to reduce and replace the amount of sulfites in wines. Methods for sulfites removal or reduction during wine-making, as absorption through the use of anion and cation exchangers or membranes, the use of electrochemical treatments, lysozyme, bacteriocins or other chemical additives such as dimethyldicarbonate have been described (Garcia-Ruiz et al., 2008; Pozo-Bayon et al., 2012). Recently, special attention has been paid to the potential use of some natural constituents of grapes and wines, such as phenolic compounds, as an alternative to sulfites (Garcia-Ruiz, Moreno-Arribas, Martin-Alvarez, & Bartolomé, 2011). Nevertheless, more studies are necessary to evaluate the potential use of these technologies in winemaking. In the recent years, wines produced using an environmentally sustainable approach, such as organic wines, have enjoyed increasing popularity, due to the growing demands for healthy products. In particular, the production of organic wines with no added sulfur dioxide/sulfites has gained considerable interest. This study aims to investigate the total polyphenols and flavonoids (a class of polyphenols family) content, the phenolics profile and the antioxidant activity of organic red wines obtained without

337

sulfur dioxide/sulfites addition in comparison to those of conventional red wines. 2. Materials and methods 2.1. Materials Caffeic acid, catechin, syringic acid, p-coumaric acid, ferulic acid, resveratrol, myricetin, quercetin, trolox, gallic acid, potassium peroxodisulfate, 2,4,6-tris(2-pyridyl)-S-triazine, 2,20 -azino-bis(3ethyl benzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and EDTA were from Sigma (St. Louis, MO, USA). Rutin, hydroxytyrosol and isoferulic acid were from Extrasynthese (Genay Cedex, France). Ascorbic acid and all organic solvents were obtained from Carlo Erba (Milano, Italy). Stock solutions of standard phenolics were prepared in methanol (1 mg/ml), stored at 80 °C and used within 1 week. Working standard solutions were prepared daily by dilution in sample buffer (1.25% glacial acetic acid, 7% methanol in twice-distilled water). 2.2. Wines Both organic and conventional red wines used in this study were produced in Italy and purchased at local markets and wine shops. All organic wines were produced according to the official organic farming practices (Italian Association for Organic Farming, AIAB, Italy), which typically excludes the use of artificial chemical fertilizers, pesticides, fungicides and herbicides, and have certificate of organic production. Although vinifications were performed before the final approval of the Reg. EC 203/2012, the winemaking protocol used suited the requisites of the EU regulation for organic wine production (EC, 2012). All the organic red wines analyzed in this study were obtained without sulfur dioxide/sulfites addition during winemaking processes. Wine bottles were stored in the dark and analyzed immediately after opening, upon appropriate dilution (in the range 125–2000 fold dilution in final samples). Aliquots were frozen at 80 °C for phenolics determination. 2.3. Wines analyses Total acidity and total sulfur dioxide were measured according to European official methods (EC, 1990). Total polyphenols were evaluated by the Folin–Ciocalteu method (Singleton & Rossi, 1965), using gallic acid as reference compound. Results are expressed by reference to the calibration curve as milligrams of Gallic Acid Equivalents per liter of wine. The total flavonoids content was determined by a colorimetric method previously described (Dewanto, Wu, Adom, & Liu, 2002), using catechin as reference compound. Results are expressed by reference to the calibration curve as milligrams of catechin equivalents per liter of wine. 2.4. Wine treatment for phenolics determination by high performance liquid chromatography (HPLC) Wine (1 ml aliquots) was added with isoferulic acid (40 lg) as internal standard and NaCl (300 mg) and phenolic compounds extracted with diethylether and diethylacetate as described by Pozo-Bayon, Hernandez, Martin-Alvarez, and Polo (2003). Then, the extract was evaporated under nitrogen stream. For total phenolic acids determination (caffeic acid, ferulic acid, p-coumaric acid and syringic acid), alkaline hydrolysis treatment in the presence of ascorbate and EDTA was performed on wine sample prior to the extraction procedure (Nardini, Cirillo, Natella, & Scaccini, 2002).

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After hydrolysis, the sample was acidified at pH 3.0 with concentrated HCl, added with NaCl (300 mg) and extracted as above reported. Extract was evaporated under nitrogen stream and the dried residue dissolved in 0.1 ml methanol, vortexed for 4 min, then EDTA (0.5 M, 40 ll), ascorbic acid (5% w/v, 0.2 ml) and twice-distilled water, up to 1 ml final volume, were added. Samples were vortexed for 4 min, filtered and analyzed by HPLC after appropriate dilution. Recovery experiments performed adding known amounts of pure phenolic compounds to wine samples followed by extraction procedure showed an almost complete recovery of all phenolic compounds under study (range 90.8–105.8%). For alkaline hydrolysis treatment, the recovery for the four phenolic acids under study (caffeic acid, ferulic acid, p-coumaric acid, syringic acid) and for isoferulic acid (used as internal standard) was in the range 96.2– 105.8%. 2.5. Antioxidant activity The total antioxidant activity of wines was measured by the Ferric Reducing Antioxidant Power (FRAP) assay (Benzie & Strain, 1996). The reducing capacity of wine tested was calculated with reference to the iron sulfate calibration curve (range 10–100 lM). The antioxidant activity of wine is expressed as mM Fe2+ equivalent/L wine. The free radical scavenging capacity of wines was also studied using the ABTS radical cation decolorization assay, according to Re et al. (1999). The reducing capacity of wine was calculated with reference to the trolox calibration curve. The antioxidant activity of wine is expressed as mM Trolox equivalent/L wine. All solutions were prepared daily.

kin–Elmer Series 4 Liquid Chromatograph (Perkin–Elmer Norwalk, CT, USA) with gradient pump, column thermoregulator, autosampling injector (Gilson, Beltline, Middleton, WI, USA) equipped with diode array detector (DAD) (Perkin–Elmer Norwalk, CT, USA). Operating conditions were as follows: column temperature, 30 °C; flow rate: 1 ml/min; injection volume, 100 ll; detector at 280 nm. Chromatographic separation was performed on a Supelcosil LC18 column (5.0 lm particle size, 250  4.6 mm ID) including a guard column (C18, 5.0 lm particle size, 20  4.0 mm ID; both Supelco, Bellefonte, PA, USA). For gradient elution, mobile phase A and B were employed. Solution A contained 1.25% glacial acetic acid in twice-distilled water, solution B was absolute methanol. The following gradient was used: 0–30 min, from 98% A, 2% B to 80% A, 20% B, linear gradient; 31–60 min, from 80% A, 20% B to 76% A, 24% B, linear gradient; 61–70 min, from 76% A, 24% B to 50% A. 50% B, linear gradient; 71–90 min, 40% A, 60% B; 91– 120 min, 98% A, 2% B.

2.7. Statistical analysis All measurements were made in triplicate. Data presented are means ± standard error. Statistical analysis was performed by student t-test, using a statistical package running on a PC (KaleidaGraph 4.0, Synergy Software, Reading, PA, USA). Student’s t test was used for regression analyses. The probability of P < 0.05 was considered statistically significant.

3. Results and discussion 3.1. Wines characterization

2.6. HPLC instrumentation Phenolics in food, beverage and human plasma extracts are routinely detected in our laboratory by HPLC (Nardini et al., 2002, 2009; Piazzon, Forte, & Nardini, 2010). The HPLC consists of a Per-

The characteristics of both organic and conventional red wines are reported in Table 1. All wines were produced in Italy, particularly in the northern regions of Veneto, Piedmont, Trentino-Alto Adige and Friuli-Venezia Giulia, from the 2011 and 2012 vintages.

Table 1 Characteristics of organic and conventional red wines. Growing regions (Italy)

Grape variety

Vintage

Alcoholic strength (%)

Total aciditya (tartaric acid) (g/L)

Organic wines 1R Cabernet Sauvignon 2R Monferrato

Veneto Piedmont

2012 2011

12.0 13.0

5.51 5.70

3R 4R 5R 6R 7R

Rosso Veronese Merlot Refosco Cabernet Franc Teroldego

Veneto Friuli-Venezia Giulia Veneto Friuli-Venezia Giulia Trentino-Alto Adige

2011 2012 2012 2012 2011

13.5 12.5 12.5 12.5 12.5

4.87 6.19 5.47 5.10 5.74

8R

Refosco

Friuli-Venezia Giulia

100% Cabernet Sauvignon 80% Barbera 20% Cabernet Sauvignon 100% Corvina 100% Merlot 100% Refosco (red stalk-peduncle) 100% Cabernet Franc 80% Teroldego 20% Lagrein 80% Refosco (red stalk-peduncle) 20% other red wines

2012

12.5

6.04

100% Barbera 100% Teroldego 100% Refosco (red stalk-peduncle) 70% Corvina 15% Corvinone 15% Rondinella 100% Cabernet-Sauvignon 100% Cabernet-Sauvignon 100% Merlot 100% Refosco (red stalk-peduncle)

2011 2011 2011 2012

13.0 13.0 12.5 12.0

5.40 4.95 5.55 6.00

2012 2012 2012 2012

12.0 12.5 12.0 12.0

5.62 5.74 5.74 5.02

Code

Wine

Conventional wines AR Barbera BR Teroldego CR Refosco DR Valpolicella

Piedmont Trentino-Alto Adige Friuli-Venezia Giulia Veneto

ER FR GR HR

Veneto Veneto Veneto Friuli-Venezia Giulia

Cabernet-Sauvignon Cabernet-Sauvignon Merlot Refosco

Organic wines were produced without sulfur dioxide/sulfites addition. a Total acidity values are mean of three determinations (SE 6 0.15).

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Alcoholic strength was in the range 12–13% for both organic and conventional red wines. For total acidity, the lowest values measured were 4.87 and 4.95 g/L for organic and conventional wines, respectively, while the highest values measured were 6.19 and 6.00 g/L for organic and conventional wines, respectively. Total acidity values were not significantly different in organic red wines in respect to conventional red wines (Table 3). Total sulfur dioxide content was 62.0 mg/L for organic red wines and 46.9 ± 5.3 mg/L for conventional red wines.

3.2. Total polyphenols and flavonoids content of wines Table 2 lists the results obtained from total polyphenols and flavonoids measurements for each red wine. The total polyphenols and flavonoids concentrations found are in agreement with data from the literature (Quideau et al., 2011; Stockham et al., 2013; Yoo, Prenzler, Saliba, & Ryan, 2011). Among the organic wines, Cabernet-Sauvignon (1R) showed the lowest values of all parameters tested, while Rosso Veronese (3R) showed the highest values. Among the conventional group, Barbera (AR) exhibited the lowest polyphenols content and Merlot (GR) exhibited the lowest flavonoids content, while Cabernet-Sauvignon (FR) and Refosco (CR) showed the highest polyphenols and flavonoids content, respectively. The comparison between organic and conventional red wines showed that the total polyphenols content was not significantly different in organic wines in respect to conventional wines, although the mean value was somewhat higher (+4.5%) in organic wines (4417 ± 318 mg/L) in respect to conventional wines (4225 ± 317 mg/L) (Table 3). Data concerning the flavonoids con-

tent of wines gave a similar trend: the total flavonoids content of organic wines (1447 ± 119) was found not statistically different, although a little bit higher (+7.9%), from the level measured in conventional wines (1341 ± 82) (Table 3).

3.3. Wines antioxidant activity Table 2 lists the results obtained from antioxidant activity measurements for each red wine, evaluated by both the FRAP method, based upon the ferric ion reduction, and the ABTS assay, based on free radical scavenging. The antioxidant activity values obtained are in agreement with data from the literature (Kostadinovic et al., 2012; Pulido, Hernandez-Garcia, & Saura-Calixto, 2003; Zhu, Zhang, Deng, Li, & Lu, 2012). In the organic group, Rosso Veronese (3R) showed the highest antioxidant activity measured by both FRAP and ABTS assay, while Cabernet-Sauvignon (1R) showed the lowest antioxidant activity. In the conventional group, Merlot (GR) exhibited the lowest values of antioxidant activity, while Teroldego (BR) and Cabernet-Sauvignon (FR) the highest values. As shown in Table 3, the overall results obtained from the antioxidant activity measurements showed higher values for organic red wines (FRAP 23.1 ± 1.8 mM, ABTS 23.7 ± 2.1 mM) in comparison to conventional wines (FRAP 20.3 ± 1.1, ABTS 18.8 ± 1.4). From our data, measured FRAP and ABTS values were about 14% and 26% higher, respectively, in organic wines produced without sulfur dioxide/sulfites addition in respect to conventional wines. However, these differences did not reach statistical significance, although the P value of 0.0744 obtained for ABTS assay was quite close to the threshold value (0.05) of significativity (Table 3).

Table 2 Antioxidant potential, total polyphenols and flavonoids content of organic and conventional red wines. Code

Wine

Polyphenols gallic acid Eq. (mg/L)

Flavonoids catechin Eq. (mg/L)

FRAP Fe2+ Eq. (mM)

ABTS trolox Eq. (mM)

Organic wines 1R 2R 3R 4R 5R 6R 7R 8R

Cabernet-Sauvignon Monferrato Rosso Veronese Merlot Refosco Cabernet Franc Teroldego Refosco tricanus

3007 ± 70 4756 ± 90 5653 ± 50 4230 ± 87 3799 ± 97 4463 ± 92 5560 ± 68 3868 ± 6

910 ± 8 1576 ± 25 2013 ± 5 1510 ± 11 1329 ± 11 1141 ± 22 1417 ± 45 1683 ± 36

15.4 ± 0.6 27.6 ± 0.1 31.4 ± 0.8 24.9 ± 0.3 18.7 ± 0.6 21.2 ± 1.0 25.4 ± 0.6 19.9 ± 0.1

15.2 ± 0.4 23.8 ± 1.1 32.2 ± 0.3 25.4 ± 1.0 21.6 ± 0.4 24.2 ± 0.4 30.5 ± 1.5 16.4 ± 0.3

Conventional wines AR BR CR DR ER FR GR HR

Barbera Teroldego Refosco Valpolicella Cabernet-Sauvignon Cabernet-Sauvignon Merlot Refosco

3043 ± 51 5183 ± 52 4144 ± 145 3434 ± 141 4222 ± 93 5775 ± 10 3692 ± 63 4309 ± 101

1180 ± 14 1446 ± 11 1662 ± 28 1411 ± 16 1169 ± 33 1632 ± 24 1024 ± 7 1205 ± 18

16.4 ± 0.1 21.2 ± 0.4 22.5 ± 1.5 18.8 ± 0.3 20.6 ± 0.1 26.1 ± 0.9 16.7 ± 0.7 20.4 ± 1.6

18.8 ± 0.1 26.4 ± 0.3 16.9 ± 0.03 16.7 ± 0.2 15.5 ± 0.9 23.2 ± 0.1 14.6 ± 0.1 18.4 ± 0.8

Organic wines were produced without sulfur dioxide/sulfites addition. FRAP, Ferric Reducing Antioxidant Power; ABTS, 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt. Values are mean ± SE, n = 3.

Table 3 Comparison between organic and conventional red wines.

Total Total FRAP ABTS Total

polyphenols (gallic acid Eq. mg/L) flavonoids (catechin Eq. mg/L) (Fe2+ Eq. mM) (trolox Eq. mM) acidity (tartaric acid Eq. g/L)

Organic wines

Conventional wines

P

4417 ± 318 1447 ± 119 23.1 ± 1.8 23.7 ± 2.1 5.6 ± 0.1

4225 ± 317 1341 ± 82 20.3 ± 1.1 18.8 ± 1.4 5.6 ± 0.1

0.6762 0.4748 0.2247 0.0744 0.9198

Organic wines were produced without sulfur dioxide/sulfite addition. FRAP, Ferric Reducing Antioxidant Power; ABTS, 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt. Data are mean ± SE, n = 8. Statistical analysis has been performed using the Student t test.

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In our study, a linear correlation between total polyphenols content and antioxidant activity values was found both in organic and conventional red wines, with correlation coefficient of 0.8917 and 0.8791, respectively (P = 0.0029 and 0.0040 respectively) for FRAP assay, and correlation coefficient of 0.936 and 0.72, respectively (P = 0.0006 and 0.0044 respectively) for ABTS assay, indicating that both in organic and conventional groups total polyphenols are mainly responsible for the antioxidant activity of wines (Fig. 1). Positive and significant correlations between polyphenols and antioxidant activity of red wines have been already reported in the literature (Yoo et al., 2011). Overall, our results suggest that total polyphenols and flavonoids levels and antioxidant activity in organic red wines produced without sulfur dioxide/sulfites addition are comparable to those of conventional red wines. 3.4. Phenolics profile analyses Due to the role of phenolic compounds as natural antioxidant affecting the quality of wines, further analyses to determine the profile of simple phenols, phenolic acids and flavonoids were carried out by HPLC. Table 4 lists the concentration of single phenolic compounds, representative of the different classes of polyphenols, measured by HPLC in both organic and conventional red wines. The

hydroxycinnamic acid derivatives caffeic, ferulic and vanillic acids; the hydroxybenzoic acid derivative syringic acid; the flavonoids catechin (flavanol), rutin, myricetin and quercetin (flavonols); the alcohol hydroxytyrosol and the stilbene derivative resveratrol were analyzed in both organic and conventional red wines. For phenolic acids, due to the fact that they are present in wine mainly as esterified forms (particularly with tartaric acid), we measured both the level of the free forms and the total amounts (free plus conjugated forms), obtained after alkaline hydrolysis. The levels of wine phenolics measured in this study are in agreement with those reported in the literature (Garcia-Ruiz et al., 2011; PozoBayon et al., 2003; Yoo et al., 2011). In the most of organic wines, the concentrations of total caffeic acid (5 out of 8) and of the flavonoids rutin (5 out of 8), myricetin (5 out of 8) resveratrol (5 out of 8) and quercetin (6 out of 8) were higher in respect to those measured in conventional wines. As shown in Table 5, however, these differences did not reach statistical significance. Also, the concentration of total measured phenolics in organic wines did not significantly differs from that found in conventional wines. Overall, our results suggest that the phenolics profile of organic wines, produced without sulfur dioxide/sulfites addition, is not qualitatively and quantitatively different from that of conventional wines. Castellari et al. reported that sulfur dioxide addition during the prefermentative phase of vinification reduced resveratrol oxidation

Fig. 1. Relationship between total polyphenols content (Folin–Ciocalteu) and antioxidant activity measured by both FRAP and ABTS assays of organic (panels A and C) and conventional (panels B and D) red wines. GAE, Gallic Acid Equivalents; FRAP, Ferric Reducing Antioxidant Power; ABTS, 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt. Data were analyzed for correlation by Student t test.

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I. Garaguso, M. Nardini / Food Chemistry 179 (2015) 336–342 Table 4 Phenolics composition of organic and conventional red wines (mg/L). Compound

Hydroxy-tyrosol Catechin Caffeic acid Free Total Syringic acid Free Total p-Coumaric acid Free Total Ferulic acid Free Total Resveratrol Myricetin Rutin Quercetin

Organic wines 1-R

2-R

4-R

5-R

6-R

7-R

8-R

5.9 ± 0.6 58.1 ± 2.5

11.0 ± 1.1 76.5 ± 3.2

12.1 ± 0.1 71.2 ± 2.4

13.1 ± 0.5 25.7 ± 0.6

6.2 ± 0.5 39.8 ± 1.8

5.1 ± 0.2 15.1 ± 0.02

6.2 ± 0.5 16.2 ± 0.9

15.7 ± 0.2 69.1 ± 1.4

12.9 ± 0.9 32.2 ± 0.1

41.6 ± 1.7 52.7 ± 1.0

19.1 ± 0.9 51.6 ± 2.0

7.6 ± 0.1 9.7 ± 0.3

12.5 ± 1.3 18.7 ± 1.9

2.1 ± 0.03 37.2 ± 1.9

4.7 ± 0.03 28.9 ± 0.8

9.6 ± 0.4 30.6 ± 0.9

7.1 ± 0.1 18.4 ± 0.6

4.5 ± 0.2 14.3 ± 0.2

6.5 ± 0.01 13.8 ± 0.05

2.7 ± 0.1 6.0 ± 0.2

6.1 ± 0.1 13.8 ± 0.9

4.3 ± 0.1 14.8 ± 1.1

2.2 ± 0.2 7.7 ± 0.2

4.0 ± 1.1 7.5 ± 0.1

6.9 ± 0.5 39.7 ± 1.1

5.1 ± 0.01 40.1 ± 0.6

3.1 ± 0.1 44.8 ± 1.3

8.6 ± 0.04 28.4 ± 3.6

3.4 ± 0.01 66.1 ± 2.8

3.1 ± 0.1 89.4 ± 1.8

3.3 ± 0.01 52.5 ± 2.1

0.26 ± 0.03 1.2 ± 0.03 1.1 ± 0.9 4.9 ± 0.2 12.2 ± 0.3 16.6 ± 0.5

0.5 ± 0.02 0.8 ± 0.01 0.14 ± 0.01 2.2 ± 0.1 0.9 ± 0.1 4.8 ± 0.4

0.4 ± 0.01 2.8 ± 0.1 1.6 ± 0.1 7.6 ± 0.1 6.1 ± 0.2 8.6 ± 0.1

0.4 ± 0.05 1.9 ± 0.2 5.6 ± 0.1 12.0 ± 0.8 27.0 ± 0.6 7.9 ± 0.3

0.57 ± 0.03 1.6 ± 0.1 2.8 ± 0.1 7.2 ± 0.2 19.9 ± 0.7 14.7 ± 0.7

D-R

E-R

F-R

G-R

H-R

0.7 ± 0.1 1.6 ± 0.1 1.1 ± 0.01 1.5 ± 0.05 3.4 ± 0.2 1.7 ± 0.1

0.8 ± 0.01 2.6 ± 0.1 0.39 ± 0.03 4.8 ± 0.05 7.0 ± 0.1 2.8 ± 0.2

3-R

24.1 ± 1.0 215.7 ± 3.3 0.8 ± 0.01 3.3 ± 0.1 2.0 ± 0.04 5.9 ± 0.07 6.8 ± 0.1 5.8 ± 0.1

Conventional wines

Hydroxy-tyrosol Catechin Caffeic acid Free Total Syringic acid Free Total p-Coumaric acid Free Total Ferulic acid Free Total Resveratrol Myricetin Rutin Quercetin

A-R

B-R

C-R

9.3 ± 1.0 57.1 ± 5.7

6.6 ± 0.2 87.5 ± 1.6

12.1 ± 0.4 51.2 ± 2.3

8.3 ± 0.2 40.6 ± 0.3

7.2 ± 0.2 49.5 ± 2.2

3.0 ± 0.1 57.3 ± 1.2

6.2 ± 0.3 58.3 ± 2.3

16.3 ± 0.2 68.6 ± 0.8

7.1 ± 0.5 61.6 ± 4.6

11.5 ± 0.4 33.6 ± 0.8

8.7 ± 0.03 36.1 ± 1.5

3.1 ± 0.1 14.8 ± 0.3

5.2 ± 0.2 21.6 ± 0.5

12.0 ± 0.6 32.7 ± 0.9

5.3 ± 0.1 18.5 ± 0.4

15.1 ± 0.4 21.3 ± 1.2

7.9 ± 0.4 16.1 ± 1.4

7.9 ± 0.1 23.9 ± 0.6

5.1 ± 0.2 16.1 ± 0.4

5.0 ± 0.04 13.7 ± 0.4

5.9 ± 0.3 19.5 ± 0.5

3.2 ± 0.2 8.3 ± 0.3

3.3 ± 0.2 10.5 ± 0.9

4.0 ± 0.2 51.9 ± 5.1

8.1 ± 0.1 96.0 ± 2.3

24.9 ± 0.3 255.9 ± 8.1

4.6 ± 0.2 47.0 ± 1.0

2.7 ± 0.1 36.8 ± 0.4

3.4 ± 0.1 58.9 ± 0.3

3.3 ± 0.2 42.4 ± 0.8

10.5 ± 0.1 47.0 ± 2.6

1.4 ± 0.1 2.6 ± 0.2 0.21 ± 0.05 2.45 ± 0.3 6.35 ± 0.7 1.8 ± 0.2

0.48 ± 0.02 1.8 ± 0.1 1.1 ± 0.03 9.2 ± 0.2 2.6 ± 0.05 3.6 ± 0.2

0.51 ± 0.04 2.7 ± 0.04 1.5 ± 0.05 1.8 ± 0.1 5 .7 ± 0.1 2.5 ± 0.2

0.4 ± 0.01 1.7 ± 0.05 0.92 ± 0.01 2.1 ± 0.1 2.1 ± 0.2 1.5 ± 0.2

0.51 ± 0.01 1.7 ± 0.01 1.6 ± 0.1 9.4 ± 0.5 1.6 ± 0.1 5.5 ± 0.4

2.1 ± 0.05 6.2 ± 0.2

0.6 ± 0.04 1.9 ± 0.1 1.0 ± 0.04 5.5 ± 0.2 6.7 ± 0.2 6.4 ± 0.2

0.55 ± 0.05 1.8 ± 0.1 2.9 ± 0.1 4.6 ± 0.3 10.3 ± 0.1 5.3 ± 0.1

1.1 ± 0.1 2.2 ± 0.1 1.8 ± 0.04 4.5 ± 0.2 3.6 ± 0.2 5.3 ± 0.1

Organic wines were produced without sulfur dioxide/sulfites addition. Data are means ± SE, n = 3. Table 5 Comparison between phenolics composition of organic and conventional red wines. Compound Hydroxy-tyrosol Catechin Caffeic acid Free Total Syringic acid Free Total p-Coumaric acid Free Total Ferulic acid Free Total Resveratrol Rutin Myricetin Quercetin Total phenolics (analyzed)

Organic wines (mg/L)

Conventional wines (mg/L)

P

9.4 ± 1.4 46.5 ± 9.0

8.6 ± 1.4 58.7 ± 5.0

0.7065 0.2528

13.8 ± 4.4 32.7 ± 5.2

8.5 ± 1.4 30.0 ± 5.3

0.2735 0.7240

4.7 ± 0.6 12.0 ± 1.6

5.1 ± 0.8 14.3 ± 2.1

0.6960 0.3996

7.2 ± 2.5 72.1 ± 21.6

7.7 ± 2.6 79.5 ± 26.0

0.8978 0.8296

0.6 ± 0.1 2.0 ± 0.3 1.8 ± 0.6 10.4 ± 3.1 5.8 ± 1.2 7.9 ± 1.9 273.7 ± 31.1

0.7 ± 0.1 2.1 ± 0.1 1.4 ± 0.3 4.9 ± 1.0 4.9 ± 1.1 4.0 ± 0.7 285.5 ± 32.0

0.4017 0.8227 0.5063 0.1166 0.6121 0.0763 0.7952

Organic wines were produced without sulfur dioxide/sulfites addition. Values are mean ± SE, n = 8. Statistical analysis has been performed using the Student t test.

(Castellari, Spinabelli, Riponi, & Amati, 1998). Due to the antioxidant properties of sulfur dioxide/sulfites, organic wines produced without sulfur dioxide/sulfites addition might be expected to result in lower resveratrol, and more generally, phenolics levels in respect to conventional wines. However, from our data, the

content of characteristic phenolic compounds, representative of the different polyphenols classes, in organic wines produced without sulfur dioxide/sulfites addition during winemaking is similar to that of conventional red wines and no significant differences are observed.

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