PaPer Ital. J. Food Sci., vol. 27 - 2015 505 - Keywords: antioxidant activity, ABTS, DPPH, FRAP, wine, sulfites - EFFECT OF SULFITES ON THE IN VITRO ANTIOXIDANT ACTIVITY OF WINES C. D. DI MATTIA, A. PIVA, M. MArTUSCELLI, D. MASTrOCOLA and G. SACCHETTI* Faculty of Bioscience and Technology for Agriculture, Food and Environment, University of Teramo, Via C.R. Lerici 1, Mosciano S. Angelo, 64023 Teramo, Italy *Corresponding author: Tel. +39 0861 266913, Fax +39 0861 266915, email: gsacchetti@unite.it AbstrAct the objective of this study was to assess the contribution of sO 2 to the overall antioxidant activ- ity of wines. In this study, white, red, and model wines, with increasing sulfite content, were used. the radical scavenging activity of the wines was evaluated by Abts and DPPH assays, while the reducing capacity of the wines was assessed by the FrAP assay. sO 2 positively affected the antiox- idant properties of the wines and, in some cases, its contribution to the overall antioxidant activi- ty of wines was higher than that of naturally occurring antioxidants. Depending on the assay, sO 2 showed both synergistic and antagonistic effects with the antioxidants naturally present in wines. 506 Ital. J. Food Sci., vol. 27 - 2015 INtrODUctION Wine is one of the most important dietary sources of antioxidants with both in vitro and in vivo antioxidant activity (MANZOccO et al., 1998; FrANKEL et al., 1995; VINsON and HONtZ, 1995; sErAFINI et al., 1998; tsANG et al., 2005). the in vitro antioxidant activity of wine, which has been studied in depth for decades, is highly cor- related to its phenolic content (sIMONEttI et al., 1997; bUrNs et al., 2000; ALONsO et al., 2002; FErNÁNDEZ-PAcHÓN et al., 2004; YILDIrIM et al., 2005). In vivo studies have shown that the con- sumption of wine modulates the serum non-en- zymatic antioxidant capacity in humans. Howev- er, the direct antioxidant effect of polyphenols in vivo is still under debate (sErAFINI et al., 2011; HOLLMAN et al., 2011). Even though the antioxidant activity of wines has been mostly attributed to the presence of phenolic compounds (MANZOccO et al., 1998; YILDIrIM et al., 2005; bUrNs et al., 2001; VILLA- ÑO et al., 2006), exogenous antioxidants, such as sulfites, are added during the wine-making process. sulfur dioxide is one of the most com- monly used additives in the food and beverage industries (WHO, 1998) due to its antioxidant, antiseptic, and preservative properties (brANEN et al., 2002). In wines, sulfur dioxide has pos- itive effects by inhibiting oxidation and micro- bial growth, increasing pigment extraction, and reducing color loss and phenolic polymeriza- tion (rIbÉrEAU-GAYON et al., 2000). However, the use of sulfites in certain food products has either been banned (FDA, 1986) or strictly lim- ited (EEc, 1995) and is currently under regula- tion due to its allergenic effects in hypersensi- tive individuals (EFsA, 2004). Even though sulfites have reducing and an- tioxidant properties, there are contradictory findings on the contribution of sufur dioxide to the overall antioxidant capacity of wines. some studies have reported that sulfur dioxide reacts with DPPH radicals and improves the radical scavenging activity of wines (AbrAMOVIc et al. 2015). Other studies have found that sulfur di- oxide plays a minor role in the antioxidant ca- pacity of wines (MANZOccO et al., 1998; cIM- INO et al., 2007). Additionally, as reported by KILMArtIN et al. (2001), the contribution of sul- fur-containing antioxidants is lost when their reducing properties are determined by cyclic voltammetry methods equipped with glassy car- bon electrodes. On the other hand, authors have reported that sulfur dioxide might play a significant role in the antioxidant activity of beverages and sauces (LONG et al., 2000; LAcHMAN et al., 2009; MIt- sUHAsHI et al., 2001), especially of white wines, which contain high sulfite levels and low natural antioxidant levels. Additionally, the in vivo effect of sulfites on the antioxidant activity of foods is still unknown (cAMPANELLA et al., 2004; LAG- GNEr et al., 2005). the objective of this study was to evaluate the contribution of sulfur dioxide to the over- all in vitro antioxidant capacity of wines using the Abts, DPPH, and Ferric reducing Antioxi- dant Power (FrAP) assays. these assays differ in several properties including mechanism of ac- tion (radical or redox reaction) and environmen- tal conditions (solvent polarity and pH). In this study, three wine types (white wine, red wine, and a model wine) were used. this experimen- tal approach was used to assess possible matrix effects which, to the best of the authors’ knowl- edge, have not been evaluated. MAtErIALs AND MEtHODs Materials three types of wines were analyzed: white wine (trebbiano d’Abruzzo Pietrosa, 2003 vin- tage, winery Dora sarchese), red wine (Mon- tepulciano d’Abruzzo, 2004 vintage, Miglian- ico social winery), and a model wine made from distilled water, ethanol (12% v/v), and tartaric acid (0.033 M, pH 3.6). the content of alcohol, total polyphenols, and sufur diox- ide, and the pH value of the three wines are shown in table 1. Different levels of sodium metabisulphite (K 2 s 2 O 5 ) were added to the wines. All reagents used in this study were of analytical grade. Chemical and chemico-physical analyses Alcohol content, total and free sulfur dioxide content, and pH values were determined by of- ficial EU methods (EEc, 1990). total polyphe- table 1 - total and free sulphur dioxide content, pH, alcohol amount, total polyphenol index and total dry extracts of the white and red wines under investigations. Samples SO 2 TOT SO 2 FREE pH Alcohol TPI Total dry extract (mg l-1) (mg l-1) (%v/v) (mg GAE l-1) (g l-1) white wine 77 65 3.24 12.90 1052 22.80 red wine 70 54 3.27 13.05 1837 23.55 Data coefficient of variation <2%. Ital. J. Food Sci., vol. 27 - 2015 507 nol content was determined by the method re- ported by sINGLEtON and rOssI (1965). ABTS assay the radical-scavenging activity of the samples was determined by the Abts (2,2’-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid) radi- cal decolorization assay (rE et al., 1999). the bleaching rate of the Abts radical in the pres- ence of sample was monitored at 734 nm. Abts radical solution (2.97 mL; Abs = 0.70±0.02) was mixed with 30 µL of diluted wine samples (1:2, 1:5, 1:10, and 1:20) using the model wine as diluent. Abts radical bleaching was monitored at 25°c for 60 min; the decoloration degree after 5 min was used as an indicator of antioxidant activity. In the dilution range considered, the Abts radical bleaching was proportional to the concentration of sample added to the medium; a dose-response curve was fitted to a linear mod- el. Antioxidant activity, which was calculated as the ratio between the regression coefficient of the dose-response curve of the sample and the regression coefficient of the dose-response curve of trolox (hydrophilic homologue of to- copherol), was expressed as µmoles of trolox equivalents per mL of sample (tEAc Abts : trolox Equivalent Antioxidant capacity). FRAP assay the reducing activity of the samples was de- termined according to the method described by bENZIE and strAIN (1996), with slight modifi- cations. sample (0.1 mL) was mixed with FrAP reagent (2.9 mL) obtained by mixing 300 mM ac- etate buffer (pH 3.6), 10 mM tPtZ (2,4,6-tripy- ridyl-s-triazine) solubilized in 40 mM Hcl, and 20 mM Fecl 3 in a 10:1:1 ratio. Absorbance was measured at 593 nm for 6 min. A calibration plot was generated based on FesO 4 7H 2 O; the results were expressed as mM Fe2+. DPPH• assay the antiradical activity of the samples was measured by the DPPH (2,2-diphenyl-1-pic- rylhydrazyl) decolorization assay as reported by brAND-WILLIAMs et al. (1995), with slight modifications in data computation. A dose-re- sponse curve was generated by adding 0.1 mL of sample at different dilutions (1:2, 1:5, 1:10, and 1:20) to 2.9 mL of a 6.1⋅10-5 M DPPH-meth- anol solution. radical bleaching was moni- tored at 25°c for 60 min. Dilutions were per- formed with the model wine as diluent. the tEAc DPPH value was calculated as the ratio be- tween the regression coefficient of the dose-re- sponse curve of the sample and the regression coefficient of the dose-response curve of trolox and expressed as µmoles of trolox equivalents per ml of sample. Statistical analyses three aliquots were sampled from each wine; each aliquot had different levels of sodium meta- bisulfite. All analytical determinations were car- ried out in triplicate. Data were reported as mean ± standard deviations. Linear regression was ap- plied to assess the relationship between sulfite content and antioxidant activity; the goodness of fit was evaluated by the coefficient of deter- mination (r2). the antioxidant activity of wines in the absence of sulfites was obtained by ex- trapolation of the intercept value; the accuracy of the predicted values was assessed from the standard deviation. All statistical analyses were performed with statistica for Windows (stat- soft, tulsa, OK). rEsULts AND DIscUssION the proximate composition and sulfite content of the wines are shown in table 1. the polyphe- nol content of the white wine was quite high be- cause the wine was processed by cryo-macera- tion, while that of red wine was relatively low be- cause it was a ‘cerasuolo-type’ red wine. these two types of wine were selected for this study because they had similar alcohol and total dry extract contents. the sulfite content of the three wines in- creased with increasing sodium metabisulfite addition. the total sulfur dioxide content, which was assessed by titration, was 50, 100, 150, and 200 mg L-1 in the model wine; 77, 113, 125, 153, 185, and 209 mg L-1 in the white wine; and 70, 100, 125, 150, 175, and 200 mg L-1 in the red wine. the amount of sodium metabisulfite added to the wines was calculated using data in table 1. the free sulfur dioxide content was measured immediately and 2 h after sodium metabisulfite addition; no significant changes in bound sulfilte levels were obtained between these two time points. this time lapse is usu- ally required for antioxidant activity determi- nations. Antioxidant activity was determined by Abts (tEAc method), DPPH, and FrAP assays. the Abts and DPPH assays have similar mech- anism of action towards Ar-OH, because they can be neutralized either by direct reduction via electron transfer or by radical quenching via H atom transfer (PrIOr et al, 2005), even though in the case of DPPH radical, the hydro- gen atom removal from Ar-OH could be con- sidered as a marginal reaction because it oc- curs very slowly in strong hydrogen bond-ac- cepting solvents such as methanol (HUANG et al., 2005). the environmental conditions of the two radical scavenging assays are quite differ- ent because the Abts assay is performed in aqueous media versus pure methanol in the DPPH assay. 508 Ital. J. Food Sci., vol. 27 - 2015 Radical scavenging activity as determined by the ABTS radical decolorization assay the antiradical activity of the wines, as de- termined by the Abts decolorization assay, is shown in Fig. 1. Antiradical activity improved with increasing sulfur dioxide concentration. the model wine containing 50 ppm sO 2 , an amount that is likely to occur in real wines, was charac- terized by a tEAc Abts value of 0.85 µmoles trolox equivalents per ml of sample, while the 200 ppm model wine had a tEAc Abts value of 3.48 µmoles trolox equivalents per ml of sample. taking into account the fact that the anti- oxidant activity of the white wine measured by the Abts assay may vary between 0.8 and 4.24 µmoles trolox equivalents per mL (ALONsO et al., 2002; DE bEEr et al., 2003; VILLAÑO et al., 2004), these results suggest that sulfites may play a more significant role in the antirad- ical properties of wines than polyphenols. How- ever, all wine samples had sO 2 added in its free form; commercial wines are likely to have sO 2 bound to different compounds. to evaluate the effect of sulfur dioxide on the antioxidant capacity in a real wine, the antirad- ical activity determinations were carried out in white wine with different contents of total sulfur dioxide. the results revealed that the white wine, with a total sO 2 content of 77 mg L-1, was char- acterized by a tEAc Abts value of 1.16 µmoles of trolox equivalents per ml. taking into account that 77 mg L-1 of sulfur dioxide in the model wine exerted a tEAc value of 1.30, it can be hy- pothesized that most of the antioxidant capaci- ty of white wine is attributed to its sulfur diox- ide content. by extrapolating the antioxidant ac- tivity of white wine without sulfites from the re- gression curve (Fig. 1), the wine had a tEAc Abts value of 0.40 µmoles of trolox equivalents per ml of sample. therefore, sulfur dioxide contrib- uted to the antioxidant activity of white wine to such an extent that an amount of 50 mg L-1 sul- fur dioxide can double the tEAc Abts value. these results were in agreement with those obtained by LONG et al. (2000), who reported that small quantities of sulfites can affect the total antiox- idant activity of the product. In white wine, an increase in sulfur dioxide concentration from 77 to 200 mg L-1 doubled its antioxidant activity. this result is quite signifi- cant because most wine research studies have not evaluated sulfite interference or sulfite con- tribution to the overall wine antioxidant activity (VILLAÑO et al., 2006; LAcHMAN et al., 2009; 24, VILLAÑO et al., 2004; ArNAO, 2000), even when the fractionation of polyphenolic compounds could not explain the overall antioxidant activ- ity of the samples (FErNÁNDEZ-PAcHÓN et al., 2004). the regression coefficient of the dose-re- sponse curve of the white wine was lower than that of the model wine (Fig. 1), which could be attributed to matrix effects. to further investigate the matrix effect on the antioxidant capacity of sulfur dioxide, the anti- radical activity was also determined in red wine. Fig. 1 shows that the red wine, with a sulfur di- oxide content of 70 mg L-1, had a tEAc Abts val- ue of 1.70 µmoles of trolox equivalents per ml. considering that the model wine with similar sulfur dioxide content had a tEAc Abts value of 1.18, it could be hypothesized that a consider- able percentage of the antiradical activity of red wine is attributed to its sulfur dioxide content. However, when the antioxidant activity of the red wine with no sulfites was extrapolated in the re- gression curve (Fig. 1), the tEAc Abts value was 1.52. taking into account the regression equa- Fig. 1 - Antiradical activity as evaluated by the Abts radical decolorization assay of the model wine solutions and of the white and red wines as a function of free sO 2 concentration. Ital. J. Food Sci., vol. 27 - 2015 509 tion, the sulfur dioxide contribution to the over- all antiradical capacity was 10–38% in the test- ed concentration range, which was lower than that of white wine. In decreasing order of regression coefficient magnitude, the wines were model wine > white wine > red wine. this result confirmed the pres- ence of a matrix effect on the determination of antioxidant activity; this matrix effect was high- er in the red wine than in the white wine. It has been extensively reported that sulfites in wine can bind to several compounds such as acetal- dehyde and polyphenols. Polyphenol content is usually much higher in red wines than in white wines (ALONsO et al., 2002; DE bEEr et al., 2003). Additionally, red wines contain a high amount of anthocyanins, which bind to sulfur dioxide (ANtONELLI and ArFELLI, 1993; tIMbEr- LAKE and brIDLE, 1967). However, in this study, the free sulfite content was taken into consid- eration (Fig. 1); therefore, in our experimental conditions it could be assumed that natural an- tioxidants and sulfites interfered with the anti- oxidant activity assays. In fact, both synergis- tic and antagonistic effects among antioxidants were observed in different in vitro antioxidant activity assays. Reducing activity as determined by the FRAP method the antioxidant properties of the wines were evaluated with the FrAP assay (bENZIE and strAIN, 1996). In contrast with the previously described methods, this method is based on the reducing capacity of a compound rather than its antiradical activity. the FrAP values of the model, white, and red wines are shown in Fig. 2. the model wine had low reducing activity; however, the addition of sulfites (70–200 ppm) resulted in a threefold in- crease relative to the initial value. the addition of sodium metabisulfite to the white and red wines increased their reducing power. the higher the sulfite content, the higher the reducing proper- ties, likely due to the protective role of sulfur di- oxide against polyphenol oxidation. An increase in sulfur dioxide from 71 to 200 ppm contribut- ed to a 73% and 158% increase in the reducing capacity of the red and white wines, respectively. In the red and model wines, it was possible to extrapolate the FrAP value without sulfite ad- dition. based on the results, sulfur dioxide is responsible for most of the reducing power of the wine samples. However, with respect to the white wine without sulfite addition, the exper- imental data did not allow an accurate estima- tion of the FrAP value because of a non-linear response (Fig. 2). this result could be attribut- ed to synergistic effects between natural antioxi- dants and sulfur dioxide. the synergistic effects between natural antioxidants and sulfur diox- ide could account for a non-linear response be- tween the FrAP assay and the sO 2 dose, which was evident in the red and white wines (Fig. 2). If the individual effect of an antioxidant on FrAP is linear within a certain concentration range, the synergistic effect of two antioxidants could show an increase or decrease in the response due to variations in their molar ratios (HIDAL- GO et al., 2010). In order of decreasing regression coefficient magnitude, the wines were red wine >white wine > model wine. there were no negative matrix ef- fects in the red and white wines. contrary to the results obtained from the Abts assay, sulfites Fig. 2 - reducing capacity as evaluated by the FrAP method of the model wine solutions and of the white and red wines as a function of free sO 2 concentration. 510 Ital. J. Food Sci., vol. 27 - 2015 and naturally occurring antioxidants (i.e., poly- phenols) had a synergistic effect on the reducing power of wines (Fig. 3). this result may be at- tributed to several factors: (i) in the experimen- tal conditions of the FrAP assay (pH= 3), there is a lower amount of bound sO 2 (rIbÉrEAU-GAY- ON et al., 2000) than in the Abts assay (pH= 7); (ii) free sO 2 could scavenge hydrogen peroxides produced via the Fenton reaction from catecho- ls; and (iii) polyphenols could prevent the proox- idant action of peroxomonosulfate radicals re- sulting from Fe(III)-initiated bisulfite oxidation (DANILEWIcZ, 2007; DANILEWIcZ et al., 2008). the synergistic effect between sulfites and poly- phenols support the facts that sO 2 and catecho- ls are not individual antioxidants, and that the antioxidant activity of wine is a result of multi- ple antioxidants (DANILEWIcZ et al., 2008). Fig. 4 - Antiradical activity as evaluated by the DPPH radical decolorization assay of the model wine solutions (secondary y axe) and of the white and red wines (primary y axe) as a function of free sO 2 concentration. Fig. 3 - reducing capacity of the samples with no sulfites (*obtained by extrapolation of data linear regression) and in the presence of 150 and 200 mg l-1 total sulphur dioxide content. Radical scavenging activity as determined by the DPPH• radical cation decolorization assay the antiradical activity of sulfur dioxide was evaluated by the DPPH decolorization assay (brAND-WILLIAMs et al., 1995), which relies on a methanol-soluble stable radical in an amphi- philic environment. Dose response curves were generated with different dilutions of hydroalco- holic solutions containing increasing amounts of total sulfur dioxide. Fig. 4 shows the antiox- idant activities of the model wine, expressed as tEAc DPPH , plotted against the total sulfur diox- ide content (25–200 mg L-1). the model wine containing sulfites had lim- ited antiradical activity in the amphiphilic envi- ronment (MANZOccO et al., 1998). Water-metha- Ital. J. Food Sci., vol. 27 - 2015 511 nol mixtures are not ideal solutions; in such mix- tures, small volumes of water in methanol result in the stabilization of methanol clusters as a re- sult of hydrophobic and hydrogen bond interac- tions (tAKAMUKU et al., 2000; WAKIsAKA et al., 1998; OKAsAKI et al., 1984), resulting in a phase separation that could limit the contact between the water soluble antioxidant (sulfites) and the methanol-soluble radicals with negative effects on the estimation of the antioxidant capacity. Fig. 4 shows the dose response curves of the white and red wines. In the white wine, increas- ing sulfur dioxide content from 77 to 200 mg L-1 caused a slight increase in the antiradical activ- ity (+13%). In the red wine, the increase in sul- fur dioxide contributed to increased but fluctu- ating antiradical activity values. A reduction in antioxidant activity due to sulfite addition (>150 mg L-1) was observed in the model wine (Fig. 4). based on the results obtained from the DPPH• assay, there were no negative matrix effects on the antiradical activity of sulfur dioxide. White and red wines, as opposed to the model wine of this study, contain phenolic compounds, which exhibit a surface activity that affects their rad- ical scavenging efficiency in multiphasic sys- tems (DI MAttIA et al., 2009; 2010). A possible explanation for this result is that amphiphilic compounds like polyphenols could have act- ed as surfactants allowing sulfur dioxide to ex- ert its antiradical activity with positive effects on the tEAc DPPH value. In this case, the inter- facial effect of polyphenols may justify the pos- itive combined effects between the two antioxi- dants. Another possible explanation is the syn- ergistic effects between polyphenols and sO 2 on reducing activity. the synergistic effects between natural anti- oxidants and sulfur dioxide could account for a non-linear response between the DPPH assay re- sults and the sO 2 dose, which was evident in the red wine (Fig. 4). In the case of the FrAP assay, a non-linear response was observed in the white wine, which contains less polyphenols (Fig. 2), while in the case of the DPPH• assay, a non-lin- ear response was evident in the red wine, which contains more polyphenols (Fig. 4). this result could be due to the different solvents used in the two assays: hydrophilic solvents in the FrAP as- say and amphiphilic solvents in the DPPH• as- say. 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