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Cincia e ecnologia de Alimentos ISSN 0101-2061

Recebido para publicao em 2/12/2008Aceito para publicao em 29/6/2009 (003971)1 Programa de Ps-graduao de Cincia dos Alimentos, Departamento de Cincia e Tecnologia de Alimentos, Rod. Admar Gonzaga, n. 1346, CEP 88034-001,

Itacorubi, Florianpolis - SC, Brasil, E-mail: [email protected] Empresa de Pesquisa e Extenso Agropecuria de Santa Catarina, Estao Experimental, Videira - SC, Brasil*A quem a correspondncia deve ser enviada

Colour, phenolic content and antioxidant activity o grape juice

Cor, contedo fenlico e atividade antioxidante de sucos de uva

Vvian Maria BURIN1, Leila Denise FALCO1, Luciano Valdemiro GONZAGA1,

Roseane FE1

, Jean Pierre ROSIER2

, Marilde erezinha BORDIGNON-LUIZ1

*

1 Introduction

Phenolic compounds are widely distributed in theplant kingdom. Tey are ound in many oods in dierentconcentrations (SCALBER; WILLIAMSON, 2000. In grapes,they are present in the pulp, skin, and seeds. Tey are consideredone o the most important quality parameters o grapes and theirproducts since they contribute to their colour and organolepticcharacteristics such as avour, bitterness, and astringency.Phenolic compounds are also eective antioxidants (DANGLES;WIGAND; BROUILLARD, 1992; GMEZ-CORDOVS;GONZLEZ-SANJOS, 1995.

Grapes, and in particular red grape products, are rich inphenolic compounds (FULEKI; RICARDO-DA-SILVA, 2003.

Phenolic compounds have drawn attention not only becauseo their important role in the development o grape products,but also or their benecial health eects (OBYRNE et al.,2002; BUB et al., 2003. hey have antioxidant propertiesand can prevent the oxidative damage o cells (CAVALLINI;

BINDOLI; SILIPRANDI, 1978; LOPEZ-VELEZ; MARNEZ-MARNEZ; DEL VALLE-RIBES, 2003; PARK et al., 2003.Several epidemiological and clinical studies have reported thebenets o consuming ruits and vegetables high in polyphenols,resulting in reduced risks o developing cardiovascular diseasesand several types o cancers (GARCA-ALONZO et al., 2004.Polyphenols are multiunctional, and their antioxidant activitymay be due to their ability to act as reducing agents by donatinghydrogen, capturing singlet oxygen (HANASAKI; OGAWA.;FUKUI, 1994; LUGASI; HVARI, 2003, or acting as chelators(BUB et al., 2003.

he eect o consuming ood and beverages rich inpolyphenols in terms o preventing diseases such as cancer andcoronary diseases (LAPIDO et al., 1999; LAZZ et al., 2003 isquite signicant. Polyphenols can also reduce damage to DNAand production o ree radicals in the body (BUB et al., 2003.Many o the avonoids ound in grape juice, such as catechin,

Resumo

As prticas vitcolas mostram crescimento econmico no Estado de Santa Catarina, com produo de uvas que so utilizadas para produzirvinhos e sucos. As uvas so ricas em compostos enlicos e tm chamado a ateno no somente devido ao importante papel no desenvolvimentode produtos derivados de uvas, mas tambm por seu potencial eeito benco para a sade. O objetivo deste trabalho oi avaliar sucos de uvacomercial, orgnico e artesanal produzidos no Estado de Santa Catarina. Os sucos de uvas oram analisados quanto ao contedo de polienoltotal, colorao e atividade antioxidante. Os sucos comerciais tiveram os maiores valores mdios de antocianinas monomricas totais e depolienis totais. Foi obtida tima correlao (R = 0,9566 entre a atividade antioxidante e o contedo de polienis totais nos sucos comerciais.A Anlise de Componentes Principais (ACP mostrou haver orte correlao entre a cor vermelha e as antocianinas monomricas totais. Noentanto, entre as antocianinas monomricas e as polimricas ocorreu correlao negativa.Palavras-chave: antocianinas; compostos fenlicos; Vitis labrusca.

Abstract

Viticultural practices in the State o Santa Catarina, Brazil, have shown economic growth, with the production o grapes used to producewines and grape juice. Grapes are rich in phenolic compounds which have drawn attention not only because o their important role in thedevelopment o products derived rom grapes, but also or their potential benecial health eects. Te objective o this study was to evaluatecommercial, organic and homemade grape juices produced in Santa Catarina. Grape juices were analyzed or total phenolic content, colour,and antioxidant activity. Te commercial juices had the highest average values or total monomeric anthocyanins and total phenolics. Terewas a strong positive correlation (R = 0.9566 between the antioxidant activity and total phenolic content or the commercial juice. In addition,the Principle Components Analysis showed a strong positive correlation between the red colour and total monomeric anthocyanins. However,the total monomeric anthocyanis and polymeric anthocyanins showed a negative correlation.Keywords: anthocyanins; phenolics compounds; Vitis labrusca.

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Characterization o grape juice

method (SINGLEON; ROSSI, 1965, and the results wereexpressed as gallic acid equivalent (mgGAE.L1.

Determination of total monomeric anthocyanins

he total anthocyanins were determined by the pH-dierential method described by Giusti and Wrolstad (2001).

For this purpose, aliquots o the juices were adjusted to pH1.0 and 4.5 with buers. Te absorbance o each solution wasmeasured at wavelength o maximum absorption and 700 nm.Te dierence in the absorbance values pH 1.0 and 4.5 wasdirectly proportional to total anthocyanin concentration, whichwas expressed in malvidin 3.5-diglucoside.L1.

Antioxidant capacity

Te antiradical activities o the grape juices were determinedusing the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPHaccording to Kim, Guo and Packer (2002 with modications.A sample o100M o DPPH was dissolved in 80% aqueous

methanol. Te samples (0.1 mL were added to 2.9 mL omethanol DPPH solution. Te decrease in the absorbance othe resulting solution was monitored at 517 nm or 30 minutes.Te DPPH radical scavenging activity o the grape juice wasexpressed as rolox Equivalent Antioxidant Capacity (EAC(mM.

Colour measurements

a Monomeric and polymeric anthocyanins: the contributiono monomeric anthocyanins to the grape juice colour atpH 3.6 (% monomeric, and the degree o anthocyaninpolymerization (% polymerization were determined using

the colorimetric eects that SO2 and acetaldehyde have in theorms o anthocyanins, ollowing the method proposed byLevengood and Boulton (2004. Te analysis was conducted byspectrophotometry, and the absorbance (520 nm was measuredin a 1 mm cuvette. Te reading was corrected by multiplying by10. Colour ractions o the monomeric (MA% and polymerizedanthocyanins (PA% were determined through the ollowingcalculations (Equations 1 and 2:

MA% = (A20 - ASO2) / Aacet 100 (1)

PA% = ASO2/ Aacet 100 (2)

b Colour Intensity (CI, int (: CI and were

determined using the spectrophotometric absorbance o thegrape juices at 420, 520, 620 nm (GLORIES, 1984, and theollowing calculations (Equations 3 and 4:

CI = Abs 420 + Abs 520 + Abs 620 (3)

T = Abs 420/ Abs 520 (4)

Statistical analysis

Analysis o variance (ANOVA, Principal ComponentsAnalysis (PCA, and correlation analysis were conducted usingStatistica 6.0 (2001 (StatSo Inc., ulsa, OK, USA.

epicatechin, quercetin, and anthocyanins are known to haveantioxidant, anti-inammatory, and platelet inhibitory eects,as well as or being able to reduce LDL oxidation and oxidativedamage to DNA, both in vitro and in animal studies (FRANKELet al., 1998; SINGLEARY et al., 2003.

Colour is the most important attribute used, along withother variables, as an indicator o the quality o grape juice. Tis

characteristic is directly dependent on the phenolic compositiono the juice and the anthocyanins present in the grape skin(MAZZA; MINIAI, 1993. Te anthocyanins participate inmany reactions that promote changes in the colour o grapeproducts, mainly through copigmentation and ormation opolymeric pigments (WROLSAD; DURS; LEE, 2005.

Te quantity and composition o phenolic compounds andanthocyanins dier according to the species, variety, maturityo the grapes, weather, viticultural practices, and the regionwhere the grapes are grown (MAZZA, 1995; BAUISA-ORN et al., 2007. Dierent methods and treatments usedduring the production o grape juice also aect signicantly

the nal phenolic composition, compared to that in naturaruit. Tese include the type o extraction and contact time, aswell as heat and enzymatic treatments (FRANKEL et al., 1998.Te high temperatures used during the extraction, storage,and pasteurization led to the degradation o anthocyaninsand, consequently, a decrease in the colour and total phenolicscontent (MORRIS; SISRUNK; SIMS, 1986.

Te objective o this study was to evaluate the total phenoliccontent, colour, and antioxidant activity o commercial, organic,and homemade grape juices produced in the State o SantaCatarina.

2 Materials and methods

2.1 Grape juice samples

Te analysis was carried out on 12 grape juice samplesproduced in the State o Santa Catarina, Brazil, comprising7 commercial juices (C, 2 organic grape juices (CO, and3 homemade grape juices (A. All juices were manuactured withBrdograpes (Vitis labrusca in the months o February andMarch 2007. All reagents were o analytical grade. All analyseswere perormed in triplicate, and the results were expressed asmeans S.D.

2.2 Analytical determinations

Physical-chemical analysis

Te samples o grape juice were analyzed or soluble solidscontent (Brix, pH, titratable acidity measurements (g tartaricacid.L1, and total solids (% according toAmerine and Ough(1976.

Determination of total phenols

Te amounts o total phenols in the grape juices weredetermined according to the Folin-Ciocalteu colorimetric


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anthocyanin levels reported or grape juice samples, the valueswere ound to be close to, although higher than, those oundin the wine samples.

Total Phenolics (TP)

Te otal Phenolic content (P ranged rom 3433.04 to1117.10 mg GAE.L1 in the commercial juice samples C2 andC1, respectively, as shown in able 2. All commercial juicesamples showed signicant dierences (p < 0.05, which wasnot observed or the organic juices. Tis statistical dierencebetween the grape juices could be due to dierences in thejuice processing techniques, such as extraction type, time andtemperature, and the addition, or not, o enzymes, as well asother actors related to the grapes, such as the grape maturityand cultivation practices.

3 Results and discussion

Physico-chemical analysis

Te results or the physico-chemical analysis o the grapejuices are shown in able 1. Te commercial grape juice sampleshad higher mean values or total acidity than those o homemadegrape juice. For the Brix and S values, the homemade grapejuice had the highest values indicating that these juices containa greater amount o solids.

Brazilian Legislation (BRASIL, 2000 requires that grape juices have a minimum o 14 Brix, however, some juicespresented lower values (able 1. Regarding total acidity, thesame legislation establishes a minimum value o 0.41. In thiscase, all samples were in accordance with the legislation.

Te Brix/total acidity represents the balance between theacid and sweet taste o grape juice, and thus, it indicates thequality o the grape juice. Brazilian Legislation establishes limitvalues between 15 and 45. Te high Brix/total acidity valuesobtained may be due to the low acidity o the grape juices.

Total monomeric anthocyanins

Te determination o total anthocyanins was perormed bythe pH-dierential method, and the results are shown in able 2.Tere was a signicant dierence (p < 0.05 between the sampleso the same type o juice. It was observed that anthocyanin valuesranged rom 25.56 to 460 mg.L1 or the commercial sample (C4and homemade grape juice sample (A3, respectively.

Te determinant actors in the variation o anthocyaninconcentration in grape juices are the techniques used inthe juice processing, such as high temperatures during the

extraction and pasteurization, as well as the dierent orms ostorage (FRANKEL et al., 1998. Grape juices may be packed intransparent bottles and exposed to the light, which aects thephenolic content, especially in the anthocyanins, which couldlead to a reduction in its colouring level (MORRIS; SISRUNK;SIMS, 1986.

Malacrida and Motta (2005 determined total anthocyaninsin commercial grape juice. he anthocyanin mean valueswere 28.7 mg.L1 (juice and 17.3 mg.L1 (reconstitute juice,that is, around ten times lower than those ound in our study.his dierence may be due to the type o grape used, thejuice processing, and storage methods. Te temperature usedduring the processing and storage o juices can cause changesin the colour o the product. Tis is due to the ormation ochalcones (colourless causing loss o colour due to anthocyanindegradation (JACKMAN; SMIH, 1996.

Research studies carried out with Italian wines producedrom dierent varieties o the grape Vitis vinifera (n = 7have shown that total anthocyanin levels ranged rom 102to 405 mg.L1 (CIMINO et al., 2007. Munz-Espada et al.(2004 quantied the level o total anthocyanin monomers bythe pH-dierential method in wines manuactured with threenon-Vitis vinifera grapes (Marechal Foch, Norton, and Concordvarieties. Concord wine showed a mean value o 170 mg.L1and Marechal Foch wine o 140 mg.L1. Comparing the total

Table 1. Physicochemical analysis o grape juice samples.

Juices** pH* SS(Brix*

itratableacidity*

otal solids(g%*

SS/titratableacidity (g%*

A1 3.30a 10.0a 8.20 0.09a 12.03 0.23 12.1

A2 3.51a 19.0b 6.87 0.09b 24.55 0.09b 27.1

A3 3.64a 21.5c 6.44 0.08c 33.87 1.6c 33.4

C1 3.30a 15.5ad 9.84 0.15a 14.51 0.09 15.8

C2 3.56b 12.0b 7.35 0.09b 11.86 0.03b 16.3

C3 3.34c 9.5c 8.62 0.08c 12.59 0.09c 11.0

C4 3.51b 14.5 7.72 0.09d 14.40 0.04 18.7

C5 3.43c 16.0d 5.60 0.07e 17.15 0.27d 28.6

C6 3.42c 15.5ad 9.25 0.09 15.5 0.13e 16.7

C7 3.25d 12.5b 9.41 0.08 11.64 0.29b 13.3

CO1 3.40a 15.0 a 9.63 0.10a 14.66 0.06 15.6

CO2 3.32b 12.0b 8.03 0.09b 11.18 0.21b 14.9**homemade grape juice (A; commercial grape juice (C, and organic grape juice (CO;*Mean values or triplicates S.D. Dierent letters in the same column are signicantlydierent at the 5% level (ukey HSD est. SS, total Soluble Solids (Brix; itratableacidity (g tartaric acid.L1; and total solids (g%.

Table 2. Determination o otal Anthocyanins (A, otal Polyphenols(P and Antioxidant Activity (DPPH o homemade (A, Commercial(C and organic (CO grape juices.

Juices A (mg.L1* P (mg.L1* DPPH (mM*

A1 241.22 0.15a 2509.06 18.26 8.23 0.17

A2 207.99 2.94b 21374.56 23.21b 7.32 0.48

A3 460.45 10.01c 235.09 10.13b 7.94 0.45

C1 42.45 0.33 1117.10 4.38 2.51 0.03C2 336.31 1.38b 3433.04 4.988b 11.05 0.13b

C3 431.11 1.55c 3228.36 18.26c 9.57 0.17c

C4 25.56 0.86d 1257.60 41.45d 5.53 0.11d

C5 124.95 1.12e 2286.82 57.52e 7.26 0.16e

C6 79.99 1.77 1737.13 10.13 6.22 0.59

C7 307.73 2.50g 2918.42 17.54g 9.22 0.21c

CO1 221.51 3.09 2634.79 26.79 8.24 0.19

CO2 168.50 1.80b 2643.57 41.45 9.08 0.16b

*Mean values or triplicates S.D. Dierent letters in the same column are signicantlydierent at the 5% level (ukey HSD est; A (mg malvidin 3.5 diglucoside.L1; P(mg GAE.L1; DPPH (Expressed as rolox Equivalent Antioxidant Capacity (EAC(mM.

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It is known that the antioxidant activity o grape products isinuenced by its phenolic composition (DVALOS; BAROLOM;GMEZ-CORDOVS, 2005. In order to investigate thecontribution o phenolic constituents to the antioxidant activity ingrape juices, a linear regression was obtained between the DPPHvalues, otal Phenolic content (P, and otal Anthocyanin (Ain commercial juices. Higher correlations were ound between

P content and DPPH (R = 0.9566 when compared with Aand DPPH (R = 0.8307. Tis is also shown in Figure 1, in whichthe mathematical model proved well adjusted or polyphenoltotal and antioxidant capacity. In a study with sixteen samples ored wine correlating antioxidant activity (DPPH and phenoliccontent (P, the correlation coefcient o 0.8237 was reported(FERNNDEZ-PACHN et al., 2004, which is less than thevalues obtained with grape juice.

Dani et al. (2007, researching dierent red grape juices,also observed a positive correlation between the total phenoliccontent (Folin-Ciocalteu and the antioxidant activity (DPPH.Te same authors have shown that or grape juices made romVitis labrusca grapes, considered an antioxidant source, the

biological activity is inuenced not only by the phenolic levels,but also by the agricultural method utilized.

Grape juice colour

Te results or the colour characterization o the homemade(A, commercial (C, and organic (CO grape juices arerepresented in able 3.

Most commercial juices and all o the homemade juicesamples showed signicant dierences (p < 0.05 or monomericand polymeric anthocyanins, with higher percentages omonomeric anthocyanins. Teir contribution to the colorationo commercial juices ranged rom 35.71 to 69.48%; however,the highest percentages were observed in homemade grape(76.39%. Te polymeric anthocyanins, present in all juices,showed mean values ranging rom 31.73 to 65.71%. Malacridaand Motta (2005 have reported that an average grape juice valueo 81.6% or polymeric anthocyanins is related to juice colour.

Te total polyphenol contents have also been comparedin studies carried out with dierent juice types produced romwhite and red grapes obtained through conventional or organicagricultural methods. It was observed that the red grape juiceshad higher phenolic contents than those o white grape juices,and organic juices had higher phenolic contents than those ojuices made rom conventionally armed grapes (DANI et al.,

2007. In this study, the mean P values in the organic juice(2639.18 mg GAE.L1 were also higher than those ound in thecommercial juice (2282.64 mg GAE. L1. In similar studies onP content in grape juices using the Folin-Ciocalteu methodcarried out by Frankel et al. (1998, the results or Concordgrape juice ranged between 1654 - 1971 mg GAE.L1; and orjuices produced with a greater variety o grapes, the P rangewas 1407-1541 mg GAE.L1. Te same authors mention thatthe phenolic contents o grape juice may be inuenced by theprocedures employed in the juice production and reactionsoccurring during storage.

Cabernet Sauvignon wines (Vitis vinfera o the 2004 and2005 vintages rom our dierent places in Santa Catarina Statewere analyzed or the total phenolic contents, showing P valuesranging rom 1.008 to 1.597 mg GAE.L1 (FALCO et al., 2007.Tese values are lower than the grape juice samples analyzedin this study, also originating rom Santa Catarina State. Tisshows that grape juice can be considered as a source o phenoliccompounds without being alcoholic.

In the technological context, the prole o the phenoliccomposition o grapes represents a determinant actor inwine and grape juice quality. Tis prole can also be used tomonitor the production process and in the quality control othose beverages.

Scavenging capacity

he antioxidant capacity evaluated using the DPPHmethod ranged rom 2.51 to 11.05 mM (able 2 or all juices.No signicant dierences between the samples o homemadejuice (p < 0.05 were observed; however, in the commercial juicesamples dierences occurred.

In studies carried out with wine made rom non-Vitisvinifera grapes, Munz-Espada et al. (2004 showed that theaverage antioxidant activity values were 0.85 mM, which arelower than those ound in the grape juice study here reported.In another study, the antioxidant activity was determinedby the DPPH method in sixteen samples o red wine, and itwas observed that the values ranged rom 6.10 to 17.41 mM(FERNNDEZ-PANCHN et al., 2004. Tese results are closerto those obtained or the samples o grape juice here reported.

Vinson, euel and Wu (2001 carried out a study comparingthe action o red wine, dealcoholized wine, and red grape juicein relation to atherosclerosis inhibition in hamsters. heydemonstrated that all tested beverages produced signicantinhibition o atherosclerosis. However, when comparingthe inhibition eect o the three samples, all with the samepolyphenol concentration, the grape juice yielded the best result.Tese data suggest that grape juice is an excellent alternative tored wine or people who need to avoid alcoholic beverages.

Figure 1.Interaction between otal Phenolics (P and antioxidant activity(DPPH in Commercial (C and organic (CO grape juice samples.


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During the juice processing stages and, particularly, duringtheir storage, the anthocyanin content decreased progressiveand irreversibly orming more stable polymeric pigments. Tesepigments are responsible or changing the grape juice aroma,colour, and avour (FRANCIA-ARICHA et al., 1997.

he commercial juice samples showed a signiicantdierence (p < 0.05 regarding Colour Intensity (CI. Te CI

represents the amount o colour present in the juices. Te CIvalues ranged rom 5.37 to 21.12.

Figure 2 shows the percentage o yellow, red, and blue colouror commercial, homemade, and organic grape juices. All juicesshowed a higher percentage o red colour, ollowed by yellow andblue. Tese data are correlated with the results or the tint ( othe juice samples, being inversely proportional (able 3.

Principal Components Analysis (PCA (Figure 3 wasconducted on the correlation matrix using all commercialgrape juice (C and CO colour attributes. Figure 3 shows thatthe juices were clearly separated by two actors, with the axeso Factor 1 Factor 2 explaining 96.47% o the total varianceamong the data. Te rst axis represents 76.27% and the secondaxis 20.76% o the total dispersion.

Figure 3 shows that the monomeric anthocyanins, totalanthocyanins (mg.L1, and red color (% are negativelycorrelated with Factor 1, in which the monomeric anthocyaninsand red color (% were both strongly correlated. It was alsoobserved that the values or Polymeric Anthocyanins (PA,int (, and yellow colour percentage were strongly positivelycorrelated with Factor 1. With regard to Factor 2, the percentageo blue colour and Colour Intensity (CI were negativelycorrelated with this actor. It can be observed that the red colourwas negatively correlated with , and that monomeric was

negatively correlated with polymeric anthocyanin.For the commercial juices, samples C5 and CO2 showed

higher correlations with blue colour and . Samples C7 and C2were correlated with total anthocyanins and colour intensity,and sample C3 indicated a strong correlation with monomericanthocyanins.

4 Conclusions

In the grape juice samples high concentrations o totalanthocyanins and polyphenols content were observed, valuessimilar to those reported or red wine.

Te content o total phenols, total monomeric anthocyanins,and antioxidant activity diered signiicantly among thecommercial juice samples.

A strong correlation was observed between the antioxidantactivity and phenolic content in the commercial grape juicesamples, which indicates that grape juice is a source o thesecompounds providing an alternative option or people whoneed to avoid alcohol.

Te results suggest that the total polyphenol o grape juicecan be used to determine the characteristics o this product,and that it can also be used as a parameter or monitoring theproductive process, or even or studies on quality control.

Figure 3. Principal Components Analysis (PCA o grape juice colourparameters.

Table 3. Characterization o grape juice colour.

Juice** CI* * PA(%* MA (%*A1 16.32 0.01a 0.75 0.01a 44.15 0.01a 57.33 0.01a

A2 8.16 0.01b 0.65 0.01b 31.73 0.01b 76.39 0.01b

A3 16.62 0.02c

0.56 0.02c

25.9 0.04c

66.7 0.04c

C1 5.37 0.02a 0.78 0.02ab 51.06 0.04a 50.2 0.04ab

C2 20.58 0.01b 0.78 0.01b 42.7 0.02b 58.4 0.03c

C3 21.12 0.02c 0.57 0.03c 31.54 0.01c 69.48 0.01d

C4 6.72 0.01d 0.91 0.02d 65.71 0.02d 35.71 0.02a

C5 12.68 0.01e 1.04 0.01e 60.42 0.01e 42.55 0.51bd

C6 9.31 0.01 0.76 0.02a 54.6 0.01 46.13 0.01d

C7 18.34 0.02g 0.70 0.02a 44.27 0.02g 55.12 0.02e

CO1 13.11 0.02a 0.77 0.01a 46.85 0.02a 55.02 0.01a

CO2 16.81 0.01b 0.98 0.01b 59.82 0.01b 40.33 0.01b

**homemade grape juice (A; commercial grape juice (C, and organic grape juice (CO;*Mean values between o S.D. Dierent letters in the same column are signicantlydierent at the 5% level (ukey HSD est. CI, colour intensity; , tint; PA, polimericanthocyanins; MA, and monomeric anthocyanins.

Figure 2. Representation o colour (yellow, red, and blue in homemade(A, commercial (C, and organic (CO juices.

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Reerences

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