pe

Lu

, Avdlbace

e gof

ncr

of the bioactive compounds gallic acid, (+)-catechin, ()-epicatechin, and (E)-resveratrol. Anthocyaninscontent also increased at an average of 50 mg/l. The volatile prole of wines analysed by SBSEGCMS

nd marive in

play a fundamental role in dening the polyphenol and volatileproles of wines. Moreover, biochemical and chemical reactionstaking place during winemaking and ageing, such as oxidation,polymerization and complexation reactions, have a signicant ef-fect on the wine prole (Bayonove, Baumes, Crouzet, & Gnata,2003; Cheynier, Moutounet, & Sarni-Manchado, 2003).

Young red wines have a characteristic bright red colour associ-ated to a higher content of anthocyanins in an equilibrium state

and norisoprenoids play a fundamental role (Ebeler & Thorngate,2009; Francis & Newton, 2005; Zalacain, Marn, Alonso, & Salinas,2007). These compounds have been associated with the fruity, o-ral and citrus aroma of wine and some authors have related themwith the expression of terroir or typicity from particular viticulturalregions (Bayonove et al., 2003). Given its importance and consider-ing that most regular quality wines have a limited concentration ofsuch compounds, strategies for increasing their concentration inregular quality wines are important.

The use of additives is becoming a common practice in the wineindustry for improving the sensory prole of wines in order to

Corresponding author. Tel.: +34 967599310; fax: +34 96 599338.

Food Chemistry 136 (2013) 224236

Contents lists available at

he

lseE-mail address: Amaya.Zalacain@uclm.es (A. Zalacain).quality parameters. However, many wine cellars deal with limitedbudgets, where little is left for approaching constantly changingconsumer demands. Sometimes this translates into accumulationof out-of-trend wines before bottling, generating a low revenueperspective. Therefore, considering strategies to overcome thisproblem should be taken into account by regulating authorities.

Many sensory and quality parameters of wines are related tothe composition and concentration of avonoids, phenolic acids,and volatile compounds extracted from grapes during winemaking(Ribreau-Gayon, Dubourdieu, Donche, & Lonvaud, 2004). Har-vesting and oenological techniques used for producing wine also

shorter time in standard wines than correct, high quality, and pre-mium wines (Zamora, 2003). The decline of their overall attributesis partly related to a low concentration of polyphenols, as well as tooxidative degradation and polymerization-condensation phenom-ena (Cheynier et al., 2003; Li, Guo, & Wang, 2008). Moreover, sincemost standard and low quality wines are made from grapes withmoderate or decient contents of polyphenols, they are doomedto the use of widely recognized additives like SO2 and ascorbic acidto reduce oxidative reactions (Oliveira et al., 2011).

The volatile prole of wines is also considered one of the mainquality attributes, where varietal compounds like monoterpenesKeywords:Waste grape skinsWineColourPhenolic compoundsVolatilesPre-bottling

1. Introduction

Dynamic consumer preferences aproducers to be constantly innovat0308-8146/$ - see front matter 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.07.110was only moderately inuenced by the treatments. Mixtures of dehydrated waste grape skins were usefulto improve the colour and polyphenol prole of red wines, considering them a useful tool for correctingcolour loss before bottling.

2012 Elsevier Ltd. All rights reserved.

ket trends require wineterms of sensory and

called copigmentation (Boulton, 2001), as well as to a lower expo-sure to oxygen and derived colour degradation reactions than agedwines (Oliveira, Ferreira, De Freitas, & Silva, 2011). The optimal sen-sorial attributes, where colour has a major role, is known to last forAccepted 25 July 2012Available online 4 August 2012

component. Total polyphenols mean increase was 10% with a maximum value of 20%. Analysis of lowmolecular weight phenolic compounds by HPLCDAD showed a signicant (p < 0.05) content increasePre-bottling use of dehydrated waste graand aroma composition of red wines

Miguel Angel Pedroza a, Manuel Carmona b, GonzaloAmaya Zalacain a,a Escuela Tcnica Superior de Ingenieros Agrnomos, Universidad de Castilla-La ManchabAlbacete Science & Technology Park Foundation, Universidad de Castilla La Mancha, A

a r t i c l e i n f o

Article history:Received 16 April 2012Received in revised form 21 June 2012

a b s t r a c t

Different dehydrated wastwines as an innovative waycolour intensity of wines i

Food C

journal homepage: www.ell rights reserved.skins to improve colour, phenolic

is Alonso a, Maria Rosario Salinas a,

a. de Espaa, s/n, Albacete E-02071, Spainte E-02071, Spain

rape skins from the juice industry were added into aged and young redcompensating for colour loss before bottling. After addition of grape skins,eased a mean 11% and a maximum of 31% with predominance of the red

SciVerse ScienceDirect

mistry

vier .com/locate / foodchem

hemmake them more competitive and to reduce defects. Wood chips,enzymes, enological tannin, are some examples of internationallyapproved enological practices (OIV, 2012) which modify the com-position and sensory characteristics of wines. On the other hand,other products obtained from grape skins like additive E-163, alsoknown as Anthocyanins or Enocyanin, are currently commercializedglobally for use in the food industry. Recently, consumer demandfor labeling wine ingredients and additives has brought to debatewhether wine cellars should indicate their use. In this direction,the use of natural and vegetal based additives, such as enologicaltannin from grape seeds and grape skins, is commonly better per-ceived by consumers (Cheng, Bekhit, Sedcole, & Hamid, 2010).

Process sustainability is progressively becoming a mandatorystandard in developed countries making relevant the proposalsfor reusing or exploiting current industrial wastes. In the case ofthe wine industry, one of the major wastes is grape marc (mixtureof grape skins, seeds and stalks). This waste and its componentshave been recently studied for their potential use as raw materials,ingredients and antioxidants (Arvanitoyannis, Ladas, & Mavroma-tis, 2006; Bekhit et al., 2011; Casazza, Aliakbarian, De Faveri, Fiori,& Perego, 2011; Fiori, 2010; Pinelo, Arnous, & Meyer, 2006; Ping,Brosse, Chrusciel, Navarrete, & Pizzi, 2011; Spigno & De Faveri,2007). Waste grape skins from the juice industry are commonlyobtained after shorter processing time (4 days of maceration) thanthose from red winemaking. This fact implies that juice industrygrape marc is not as exhausted as that from winemaking and thusa richer source of several compounds.

Recently Pedroza, Carmona, Salinas, and Zalacain (2011), evalu-ated the production of ros wines by macerating dehydrated wastegrape skins into white wines, resulting in a stable product with tai-lor made characteristics according to those obtained in model winesolutions. Furthermore, this approach made it possible to reuse anagroindustrial byproduct offering an alternative for commerciali-zation of both; the white wine (converted into ros) and the wastegrape skins. The aim of this work was to use dehydrated wastegrape skins as a new oenological tool for compensating colour,phenolic and aroma degradation in red wines before bottling. Mix-tures of waste grape skins were assayed for producing differentcompositional proles, evaluating their immediate impact andstorage behaviour.

2. Materials and methods

2.1. Dehydrated waste grape skins (DWGS)

Waste grapemarcs of Vitis vinifera Bobal (red variety) and amix-ture of 70% Airn (white variety) with an unknown red variety,namely AMIX, were obtained from a juice concentrate factory inCastilla-La Mancha (Julian Soler, Cuenca, Spain). Samples obtainedimmediately after pressing the macerated must were collected inplastic bags (60 kg) and frozen at 20 C. In the laboratory, wastegrapemarcswere thawed at 25 C and ovendried at 60 C, accordingto Pedroza, Carmona, Pardo, Salinas, and Zalacain (2012) until a con-stant weight was achieved (35% moisture content). Dried sampleswere sieved through 3 mm mesh to remove seed and stalks. Dehy-dratedwaste grape skins (DWGS)were then ground in a cuttingmillMS 100 (Retsch, GmbH & Co. KG, Denmark) and sieved to 1.0 mmparticles. Four types of DWGS were then prepared: 100% Bobal (Bo-bal), 100% AMIX (AMIX), 75% AMIX + 25% Bobal (GM75), and 50%AMIX + 50% Bobal (GM50). The later mixtures were selected to ob-tain intermediate values between plain AMIX and Bobal DWGS.

2.2. Wines

M.A. Pedroza et al. / Food CTwo aged (A05 and A07) and two young (Y08 and Y09) redwines were provided by a local winery. Young wines correspondedto table wines commercialized as bulk product with a shelf life nolonger than three years after bottling. These wines were resultfrom traditional winemaking (510 days of maceration; fermenta-tion temperature 25 C in stainless steel tanks) of several red grapevarieties cultivated regionally (Tempranillo, Cabernet Sauvignon,Merlot, etc.). Aged wines were produced similarly as young wines,but with longer maceration times (1020 days) and subjected to atleast 6 months of storage in American oak barrels (250 L). All wineshad pH range = 3.53.9 and alcohol content = 13.514% vol. In or-der to obtain representative samples of wines before bottling, sam-pling was performed at the bottling line in 750 ml amber glassbottles (four bottles of each wine with synthetic cork closures).Wines were then taken to the laboratory and prepared for itstreatment.

2.3. Maceration conditions

DWGS were macerated according to conditions established byPedroza et al. (2011). Briey, a dosage of 5 g DWGS/L was macer-ated into each wine during 3 days at 18 C. Amber crystal asksof 125 ml with screwcap plastic closures and 5 0.5 ml headspacewere used during the experiment. After maceration, DWGS wereremoved with a strainer and the wines stored in the same typeof crystal bottles at 18 C. Wine evolution analysis started immedi-ately after maceration, and then after 3 and 6 months (T0, T3, andT6 respectively).

2.4. Chemicals and standards

FolinCiocalteu reagent from Merck (Darmstadt, Germany). So-dium Carbonate from Panreac (Barcelona, Spain). Caffeic acid, (+)-catechin, p-coumaric acid, ()-epicatechin, gallic acid, and (E)-res-veratrol, from SigmaAldrich (Steinheim, Germany) were used asstandards for low molecular weight phenolic compound analysis.Malvidin-3-glucoside (Mv-3-G) standard from Extrasynthse (Gen-eay, France) was used for anthocyanins quantication. HPLC-gradeacetonitrile was from Panreac (Barcelona, Spain). Eugenol, farnesol,1-hexanol, b-ionone, isoamyl acetate, D-limonene, nerolidol, (E)-whiskylactone, (Z)-whiskylactone, supplied by SigmaAldrich(Steinheim, Germany), and b-damascenone supplied by Firmenich(Geneva, Switzerland) were used as calibration standards in winemodel solution (12% v/v ethanol, pH = 3.6, 5 g/l tartaric acid) forvolatile analysis.

2.5. Sample characterization

2.5.1. UVvis spectrophotometryStandard colour parameters and total phenolic compoundswere

measured in a Lambda 25 UVVis spectrophotometer (Perkin El-mer, Norwalk, CT) with quartz cells. All samples were rst lteredthrough a PVDF Durapore lter of 0.45 lm (Millipore, Bedford,MA). Colour was determined following Glories method (Glories,1984), measuring absorbance at 420, 520, and 620 nm. Total poly-phenols (TP) were determined at 750 nm according to Singletonand Rossi (Singleton & Rossi, 1965) and expressed as mg/l of gallicacid equivalents (GAE) according to a calibration curve with theequation TP = (0.2735 Absorbancy 0.036) 1000), R2 = 0.994and with a mean relative standard deviation below 5%.

2.5.2. Phenolic compounds determination by HPLCDADPhenolic compound analysis was carried out according to Coz-

zolino et al. (2004). The samples were ltered through a PVDFDurapore lter of 0.45 lm (Millipore, Bedford, MA) and injected

istry 136 (2013) 224236 225into an Agilent 1100 HPLC chromatograph (Palo Alto, CA) equippedwith a Phenomenex (Torrance, CA) Synergi 4 l Hydro-RP column(4 lm particle size, 80 pore size, 150 2.0 mm) at 25 C.

In red wines, Shade represents a measure of colour degradation

A07, where evolution was mostly stable. All second order interac-

hemSolvents were: (A) 1% v/v acetonitrile, 1.5% v/v phosphoric acid inwater and (B) 20% v/v solvent A, 80% v/v acetonitrile. Gradient elu-tion at a constant ow rate of 0.4 ml/min was: 0 min (14.5% solventB), 18 min (27.5% solvent B), 20 min (27.5% solvent B), 21 min(50.5% solvent B), 22 min (50.5% solvent B), 26 min (100% solventB), and 28 min (100% solvent B). The injection volume used was20 ll. Compound detection was carried out with a diode arraydetector by comparison with the corresponding UVvis spectraand retention time of pure standards in the chromatogram. Gallicacid, (+)-catechin, vanillic acid, syringic acid and ()-epicatechinwere identied at 280 nm; ferulic acid and caffeic acid were iden-tied at 324 nm, (trans)-resveratrol and p-coumaric acid wereidentied at 308 nm; malvidin-3-G, was identied at 520 nmwhiledelphinidin-3-G, cyanidin-3-G, petunidin-3-G, peonidin-3-G wereidentied according to the literature (Alcalde-Eon, Escribano-Bai-lon, Santos-Buelga, & Rivas-Gonzalo, 2006) and quantied as mal-vidin-3-G equivalents. Quantication was based on 5-pointcalibration curves of respective standards (R2 > 0.95, tted to ori-gin) in wine model solution previously described. Relative stan-dard deviation of all calibration points was below 1%. Datareported represent the mean of two replicates.

2.5.3. Volatile compound determination by SBSEGCMSTen millilitres of wines were used in duplicate to determine the

free volatile fraction (Pedroza, Zalacain, Lara, & Salinas, 2010) byimmersion of a polydimethylsiloxane coated stir bar [Twister,0.5 mm lm thickness, 10 mm length from Gerstel, (Mlheim ander Ruhr, Germany)] and stirring at 500 rpm during 1 h at 25 C.After this time, the stir bar was removed from samples, rinsed withdistilled water, dried with cellulose tissue and nally transferredinto thermal desorption tubes for the GC/MS analysis.

Volatile compounds were desorbed from the stir bar in an ATD400 (Perkin Elmer, Norwalk, CT) under the following conditions:oven temperature at 290 C; desorption time, 4 min; cold trap tem-perature, 30 C; helium inlet ow, 45 ml min1. After this, thecompounds were transferred into the HewlettPackard 6890 (PaloAlto, CA) gas chromatograph coupled to an HewlettPackard 3Dmass detector (Palo Alto, CA) with a fused silica capillary columnSGE BP21 (stationary phase 30 m length, 0.25 mm i.d., and0.25 lm lm thickness) (Ringwood, Australia). The chromato-graphic program was set at 40 C (held for 2 min), raised to230 C at 10 C min1 and held for 15 min. Electron impact modeat 70 eV was used for mass spectrometry analysis. The mass rangevaried from 35 to 500 u (Scan Monitoring) and the detector tem-perature was 150 C. Identication was carried out using the NISTlibrary and standard spectra. Quantication was based on 5-pointcalibration curves of respective standards (R2 > 0.95, tted to ori-gin) in synthetic wine solution previously described. Mean relativestandard deviation of calibration curves was in all cases below 6%except for 1-hexanol (45%) and farnesol (15%). Data reported rep-resent the mean of two replicates. To avoid matrix interferencesbetween the volatile compounds, the MS quantication was car-ried out in the single ion monitoring (SIM) mode using their char-acteristic m/z values reported in the NIST library and those fromZalacain et al. (2007).

2.6. Statistical analysis

An experimental design considering wine (A05, A07, Y08, Y09),DWGS type (control, AMIX, Bobal, GM75, GM50), and storage time(0, 3, 6 months) as categorical factors. Main effect of each factorand second order interactions between factors on the dependentvariables (composition of samples) were evaluated by means of

226 M.A. Pedroza et al. / Food Cmultifactor analysis of Variance (ANOVA) with Statgraphics Centu-rion 16.1. SPSS Statistics 19.0 Software (Chicago, IL) was used foridentifying homogeneous subsets according to ANOVA post hoctions for CI and shade were statistically signicant, however, inter-action plots comparing wine and grape skins revealed that thesince it is the balance between the yellow and red colour (Zamora,2003) where the higher the value, the higher the inuence of yel-low component and thus lower quality. All DWGS produced a dim-inution of Shade, where Bobal had smaller values than the rest oftreatments, although slight differences were found with the othergrape skins (Table 1). Shade changes were slightly more positive inA07, Y08 and Y09 than in A05, where GM50 and GM75 had theclosest values compared to those of control. It is important to notethat A05 control wine had the highest shade value, with a predom-inant yellow component and thus with higher colour degradation.

Evaluation of CI and Shade at T3 and T6 revealed that DWGShad different evolution trends in each type of wine. CI predomi-nantly increased with time in all samples, where Bobal had thehighest average increase (16% at T3 and 20% at T6). The most inu-enced wine was Y09 at T6, where GM75 reached a maximum in-crease of 45% with respect to control wine (Table 1). DWGS alsocaused a signicant increase in A05 wines after 6 months (2125%). In contrast, the use of DWGS had a low increase of CI inTukeys test. ANOVA was performed using two sided p 6 0.05 andLevenes test for assessing equality of variance.

3. Results and discussions

Colour loss is a natural process experienced by all types ofwines due to chemical and biochemical transformations relatedwith the polyphenols content and the presence of oxygen. Suchchanges are particularly important for red wines because colouris an important quality parameter that may inuence consumerpreferences (Parpinello, Versari, Chinnici, & Galassi, 2009). Previ-ous work on dehydration and characterization of waste grape skinsfrom the juice industry (Pedroza et al., 2012) and extraction condi-tions in wine model solution and white wines (Pedroza et al., 2011)pointed out favourable parameters for releasing colour, polyphe-nols and aroma compounds. These works also suggested that theuse of DWGS mixtures could be studied in order to t the needsof certain types of wines like those having colour loss before bot-tling. The following ndings evaluated the effect of DWGS in differ-ent types of red wine that experienced colour loss before bottling.

3.1. Colour

The addition of DWGS into wines caused a signicant impact inthe Colour Intensity (CI) and Shade of all wines (Table 1). BobalDWGS produced the highest CI increase with respect to control,having an overall average increase of 15% followed by GM50(13%), GM75 (10%), and nally AMIX (5%). The maximum CI in-crease was observed in Bobal-Y09 (31%). The improvement of CIwas mainly ascribed to the increase of absorbance at 520 nm (datanot shown), which is commonly related with the amount of antho-cyanins of wines and in this case with those released by each typeof DWGS. Grape skins had the least inuence on CI of A07 wine, asno signicant increase with respect to control was observed. Onthe other hand, all types of DWGS produced a signicant increaseof CI in Y09 and A05 wines. According to these results, it seems thatthe type of wine regulates the impact of DWGS, where the initial CIis not a restrictive variable for colour release. Moreover, this re-marks that the treatment may improve the colour of both agedand young wines.

istry 136 (2013) 224236highest impact of grape skins was achieved in Y09 wine, where Bo-bal and GM75 were the DWGS producing the highest values duringthe whole experiment.

Table 1UVvis determinations of Colour and Total polyphenols from young (Y08, Y09) and aged (A05, A07) red wines exhibiting colour loss and added with Dehydrated Waste Grape Skins (GM75: mixture of 75% AMIX + 25% Bobal; GM50:mixture of 50% AMIX and 50% Bobal).

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Table 2Monomeric Antocyanin glycosides composition (mg/l) from young red wines (Y08, Y09) added with Dehydrated Waste Grape Skins (GM75: mixture of 75% AMIX + 25% Bobal; GM50: mixture of 50% AMIX and 50% Bobal).

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Table 3Monomeric Antocyanin glycosides composition (mg/l) from aged red wines (A05, A07) added with Dehydrated Waste Grape Skins (GM75: mixture of 75% AMIX + 25% Bobal; GM50: mixture of 50% AMIX and 50% Bobal).

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Table 4Low molecular weight phenolic compounds composition (mg/l) of young red wines (Y08, Y09) added with Dehydrated Waste Grape Skins (GM75: mixture of 75% AMIX + 25% Bobal; GM50: mixture of 50% AMIX and 50% Bobal).

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Table 5Low molecular weight phenolic compounds composition (mg/l) of aged red wines (A05, A07) added with Dehydrated Waste Grapeskins (GM75: mixture of 75% AMIX + 25% Bobal; GM50: mixture of 50% AMIX and 50% Bobal) (Seeabove-mentioned references for further information).

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Table 6Volatile compounds (lg/l) of young red wines (Y08, Y09) added with Dehydrated Waste Grape Skins (GM75: mixture of 75% AMIX + 25% Bobal; GM50: mixture of 50% AMIX and 50% Bobal).

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Table 7Volatile compounds (lg/l) of aged red wines (A05, A07) added with Dehydrated Waste Grape Skins (GM75: mixture of 75% AMIX + 25% Bobal; GM50: mixture of 50% AMIX and 50% Bobal). (See above-mentioned references for furtherinformation.).

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and nally Bobal (Table 4). It was noted that AMIX, GM50, andGM75 produced always higher yields than Bobal, regardless of

hemOn the other hand, Shade values of wines with DWGS had a ten-dency to increase with time while control wines were more stable.This result was mainly attributed to the increase of the yellowcomponent which may be ascribed to oxidative browning of poly-phenols (Li et al., 2008). Such browning may be directly relatedwith the decrease of total polyphenols subject of the followingdiscussion.

3.2. Total polyphenols

After maceration (T0), Total polyphenols (TP) were signicantlyincreased (520%) by all treatments, where Bobal had the higherimpact, closely followed by the mixtures GM75 and GM50 (Ta-ble 1). It was appreciated that DWGS released the highest amountof TP in A05 wines (98 6 mg GAE/L) in contrast with Y09 wines(31 3 mg GAE/L). A previous work macerating Bobal and AMIXin wine model solution and using the same extraction parameters(Pedroza et al., 2011), showed that both DWGS were able to releaseup to 180 mg GAE/L. This fact suggests that the maximum releaseof TP may be primarily controlled by the matrix, leaving a residualrole to the grape skin type. Moreover, when evaluating whitewines, it was observed that DWGS released up to 391 mg GAE/L,indicating that white wine matrix (with lower content of polyphe-nols) have more favourable equilibrium conditions thus acceptinga higher amount of polyphenols in solution. It appears that afterDWGS addition, TP reached an equilibrium concentration similarwithin all types of wines regardless of treatment, where an averagevalue of 618 11 mg GAE/L could be characteristic of our samples.However, these results remark the potential of DWGS for improv-ing the phenolic content of wines to the extent of matching theconcentration of aged wines to that of treated young wines.

Regarding evolution of TP, DWGS-wines had a similar behaviouras control ones in all treatments (Table 1). This was in agreementwith previous observations on the evolution of TP in ros wineselaborated with DWGS (Pedroza et al., 2011). A higher decreaseof TP was observed in Y09 after 6 months where AMIX treatmenthad the higher loss. Such a decrease could be related to the in-crease of the shade values previously discussed. It can also benoted from the evolution data (Table 1) that TP may increase atT3 with a following decrease after T6, remarking the continuouslychanging dynamic equilibrium occurring during wine storage. Re-sults suggest that using DWGS do not cause particular alteration inthe evolution of total polyphenols, and that their positive effect isstable for up to 6 months. All second order interactions were statis-tically signicant, showing that all wines treated with grape skinshad superior mean TP values than control wines during the wholeexperiment. In addition, the concentration of TP was similar be-tween all wines-grape skin combinations, where bobal had not sig-nicant superior values.

3.3. Anthocyanins

After maceration, DWGS released into all wines an average of50 mg/l of total monoglucoside anthocyanins. Bobal was the DWGSproducing the highest release (63 mg/l) in A05 wine, while AMIXhad the lowest (37%) in A07 wine (Table 2). However, when eval-uating the average release of each DWGS in all wines, it was foundthat Bobal and GM50 had similar yields (52 mg/l) followed byAMIX (47 mg/l) and GM75 (46 mg/l). It was remarkable that thesevalues were similar in all wines since the release of anthocyaninsdepends on the equilibrium concentration of each wine, and itwould be expected that young wines, with higher endogenous con-centration of anthocyanins before treatment, had a less efcient

234 M.A. Pedroza et al. / Food Cextraction than aged wines (with signicantly lower anthocyaninsand therefore a less saturated solution). This average anthocyaninsyield was similar to that obtained in model wine solutions withthe type of wine. This was in agreement with previous ndingson model wine solutions where AMIX released a higher amountof LMWPC, (Pedroza et al., 2011; Pedroza et al., 2012) thereforemixtures of DWGS are a good strategy for balancing the decien-cies of composition. Since LMWPC relate to sensory attributes likethe acid taste of wines and bitterness, the use of DWGS may allowwinemakers to design particular taste proles by adjusting theproportion of different grape skins. Regarding the copigmentationphenomena, Boulton (2001) suggests that an increase in the con-centration of co-factor molecules, such as gallic acid, catechin, caf-feic acid, epicatechin, etc., during maceration of wines may play acentral role for increasing the solubility of anthocyanins in thewine solution and therefore achieving higher extraction yield ofanthocyanins and improved colour. Moreover, the increase of co-factor concentration has been considered to prevent colour degra-dation in grape juice (Brenes, Del Pozo-Insfran, & Talcott, 2005).

Main LMWPC released by DWGS in wines were gallic acid, (+)-catechin, ()-epicatechin, and (E)-resveratrol (Tables 4 and 5). Gal-lic acid had the highest increase in Y09 wine (7074%). Catechinand ()-epicatechin were better released in A05 (74353% and168263% respectively). The former three compounds have beenreported to have antioxidant activity with suitable use as dietarysupplements (Yilmaz & Toledo, 2004). Caffeic and coumaric acidshad signicantly higher yields in both young wines. (E)-resveratrolhad a signicant increase in Y08 (115136%). The later molecule isof signicant relevance since it is being attributed with health pro-Bobal (50 mg/l) and lower to that obtained when macerating AMIXwith white wines (68 mg/l) (Pedroza et al., 2011) Such facts sug-gest that the white wine matrix has other variables participatingin the solubility of anthocyanins.

Anthocyanins experienced a signicant decrease during storage,accounting for a 5070% loss after 3 months and a complementary640% loss after 6 months (Tables 2 and 3). Evolution patterns ofthe different anthocyanins were similar within all wines. AMIXand GM75 had the lowest decrease of anthocyanins (77 2%) inA05, A07, and Y08. Apparently, DWGS with higher amount of whitegrape skins were able to keep a higher concentration of free antho-cyanins in solution for a longer time. However, this behaviour notobserved in Y09 wine, as it had the highest decrease (8589%) ofanthocyanins regardless of DWGS type. Observed changes maybe ascribed either to polymerization-stabilization reactions (Boul-ton, 2001; Cheynier et al., 2003) and/or degradation due to chem-ical oxidation phenomena (Li et al., 2008). Since the colour ofsamples does not change as abruptly as the anthocyanins concen-tration, we considered that the rst hypothesis may predominantlyoccur, supporting the concept of the partial role of monoglucosideanthocyanins in colour (Zamora, 2003). In spite of the degradation,samples with DWGS had signicantly higher concentration of totalanthocyanins at the end of the experiment. All factor interactionswere statistically signicant, however, the most important differ-ences were noted in the concentration of anthocyanins betweentreated and control wines, regardless of the grape skin type.

3.4. Low molecular weight phenolic compounds

After maceration treatment (T0), DWGS were able to signi-cantly increase the concentration of total low molecular weightphenolic compounds (LMWPC) in young and aged wines (Tables4 and 5). This was particularly important for A05 wine, whereGM50 produced the highest increase, followed by AMIX, GM75

istry 136 (2013) 224236moting properties such as prevention of cardiovascular diseasesand antioxidant activity (Fernndez-Mar, Mateos, Garca-Parrilla,Puertas, & Cantos-Villar, 2012; Frombaum, Le Clanche,

ero, Pardo, Alonso, & Salinas, 2002; Garde-Cerdn & Ancn-Azpilic-

the best results were observed during the rst 3 months when vol-

because of its novelty, probably, the most important challenges forits aproval as wine additive will come from political and cultural

hemBonnefont-Rousselot, & Borderie, 2012). Coumaric acid concentra-tion decreased after DWGS addition in A07 (Table 4).

In general, after 6 months of storage all wines with DWGS hadsuperior content of total LMWPC than control wines. DWGS withhigher proportion of white grape skins continued having the higheramount of LMWPC. This was also conrmed by interaction plotscomparing average values of total LMWPC during the whole exper-iment. It was observed that the composition of aged wines wasmore unstable than that of young wines; the evolution of Y08wines showed that gallic acid, (+)-catechin and caffeic acid re-mained stable during the trial, while ()-epicatechin, and (E)-res-veratrol reached a maximum concentration at T6. On the otherhand, Y09 wines with DWGS had higher concentration of caffeicacid (T6), ()-epicatechin (T3), and (E)-resveratrol (T6). Increasingwith time of the later compound is of particular interest since theymight be released by hydrolysis from their glycosilated precursors(Gmez-Gallego, Garca-Carpintero, Snchez-Palomo, Hermosn-Gutirrez, & Gonzlez Vias, 2012). Interaction plots revealed thatA05 was the most affected wine during storage, although it wasalso the wine most favoured by the DWGS treatment.

3.5. Volatile composition

The volatile composition of wines was modied after additionof DWGS by the increase of b-ionone (oral descriptor). This com-pound is an important impact odourant because of its low olfactorythreshold and appreciated descriptor. The increase of b-ionone wasof the most importance in the case of aged wines (Table 7), wherethis compound was absent or below its olfactory threshold (OT)(0.09 lg/l (Francis & Newton, 2005)) in control wine and afteraddition of DWGS, it increased to a concentration 26 times overthe OT. Bobal was the DWGS releasing the highest amount of b-io-none (0.54 lg/l in A05 wine). Wine-time was the only statisticallysignicant interaction of this compound, describing a decreasewith time, particularly important in the case of young wines.

On the other hand, nerolidol and b-damascenone concentrationdecreased after treatment regardless of the DWGS type. This phe-nomena was also observed when adding DWGS to white wines(Pedroza et al., 2011) However, in the case of b-damascenone,the concentration (0.772.33 lg/l) remained over the OT(0.05 lg/l (Francis & Newton, 2005)), thus its characteristic oral-fruity note (Bayonove et al., 2003) may persist in the wine.Although statistically signicant interactions (wine-DWGS andwine-time) were observed for this compound, the most importantdifferences were always associated to control wines having highermean concentration than those with DWGS.

The herbaceous compounds represented by 1-hexanol re-mained mostly without signicant differences or in some casesin a lower concentration than control wines. Also this compoundwas not over its OT. Nerolidol and Farnesol were not detected inY09 after addition of DWGS, indicating that in this particular winethese compounds could be transformed during treatments as a re-sult of hydrolysis and cyclation reactions taking place at wine con-ditions (Marco, 2006). This was not observed in the rest of wines,thus this behaviour was not clear. When evaluating the evolutionof wines in terms of chemical families (Tables 6 and 7), an increasewith time of isoamyl acetate (banana note) was observed in alltreated wines, but no particular DWGS seems to induce suchbehaviour. Since this compound is produced by yeast (Styger, Prior,& Bauer, 2011), this could be related with refermentation of wines,although no visual sign of this was observed. Terpenes in A05, A07and Y08 increased after 6 months of storage. Increasing of thesecompounds with storage may be due to their existence in glycosi-

M.A. Pedroza et al. / Food Clated forms and subsequent chemical hydrolysis. It may as well berelated to their susceptibility to different reactions and transfor-mations (isomerization, cyclation, oxidation, etc.) (Gnata, 2003).concerns related with the usage conditions.

Acknowledgements

M.A.P. has received a CONACYT grant from the Mexican Govern-ment. This Study has been funded by Junta de Comunidades deCastilla-La Mancha (Project PAI08-0148-9842). Thanks to Ana Solerfrom Julian Soler S.A. Juice Concentrate Factory for supplying wastegrape skins. Thanks to Kathy Walsh for proofreading themanuscript.

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4. Conclusions

Reaching a conclusion, the use of dehydrated waste grape skinsfrom the juice industry proved being a useful tool for amelioratingquality parameters of colour and phenolic compounds before bot-tling. Firstly, the red colour of wines increased by all DWGS regard-less of the wine type, together with Total polyphenols content. Thecomplementary mixing of red and white grape skins was useful torelease signicant amounts of anthocyanins and low molecularweight phenolic compounds, characteristic each grape skin variety.Important bioactive compounds such as (E)-resveratrol and cate-chins were signicantly released by all types of DWGS. The volatileprole of wines was moderately inuenced by DWGS althoughquality impact odourants like b-ionone were released. Moreovercompounds related to wood ageing such as whiskylactones andeugenol were not particularly affected by treatment. The type ofwine inuenced the release of compounds quantitatively, indicat-ing that chemical equilibrium of each wine may help or hinderthe extraction. Addition of DWGS allowed preventing signicantloss of phenolic compounds occurring during storage. Althoughthe use of DWGS is currently not authorized as enological practiceueta, 2006).The dehydration pretreatment of DWGS was a cause of the low

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Pre-bottling use of dehydrated waste grape skins to improve colour, phenolic and aroma composition of red wines1 Introduction2 Materials and methods2.1 Dehydrated waste grape skins (DWGS)2.2 Wines2.3 Maceration conditions2.4 Chemicals and standards2.5 Sample characterization2.5.1 UVvis spectrophotometry2.5.2 Phenolic compounds determination by HPLCDAD2.5.3 Volatile compound determination by SBSEGCMS

2.6 Statistical analysis

3 Results and discussions3.1 Colour3.2 Total polyphenols3.3 Anthocyanins3.4 Low molecular weight phenolic compounds3.5 Volatile composition

4 ConclusionsAcknowledgementsReferences