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Food and Bioprocess TechnologyAn International Journal ISSN 1935-5130 Food Bioprocess TechnolDOI 10.1007/s11947-013-1209-2

Winery Trial on Application of PulsedElectric Fields for Improving Vinification ofGarnacha Grapes

Elisa Luengo, Ernesto Franco, FernandoBallesteros, Ignacio Álvarez & JavierRaso

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ORIGINAL PAPER

Winery Trial on Application of Pulsed Electric Fieldsfor Improving Vinification of Garnacha Grapes

Elisa Luengo & Ernesto Franco & Fernando Ballesteros &Ignacio Álvarez & Javier Raso

Received: 7 March 2013 /Accepted: 30 September 2013# Springer Science+Business Media New York 2013

Abstract The potential of pulsed electric fields (PEF) toimprove polyphenol extraction during winemaking was in-vestigated in a winery trial. Four thousand five hundred kilo-grams of Garnacha grapes were treated with PEF (4.3 kV/cm,60 μs) at a flow of 1,900 kg/h using a collinear treatmentchamber. Wine obtained from PEF-treated grapes with a mac-eration time of 7 days was compared with wines obtainedfrom untreated and PEF-treated grapes with the current mac-eration time (14 days) used by the winery. After 7 days ofmaceration, the color intensity, anthocyanin content, and poly-phenol index in the tank containing grapes treated by PEFwere 12.5, 25, and 23.5% higher, respectively, than in the tankcontaining untreated grapes. However, after 14 days of mac-eration, no significant differences were observed between thecontrol wine and the wine obtained from grapes treated byPEF for these three indices. An HPLC analysis indicated thatthe concentrations of major individual phenolic compoundswere similar among the three wines at bottling. A sensoryanalysis revealed that the wine obtained from PEF-treatedgrapes macerated for 7 days was significantly preferable tothe other two wines.

Keywords PEF .Wine . Polyphenols . Extraction .

Maceration

Introduction

Phenolic compounds have a major influence on the quality ofred wines because of their contribution to organoleptic attri-butes, ageability, and health properties of wine. Anthocyanins,a type of phenolic compound, are pigments responsible for thered color in young wines. They contribute to the stabilization ofcolor when aging wine due to the development of polymericpigments (Guadalupe and Ayestarán 2008). Proanthocyanidins,which refer to a large class of polymerized flavanols that arealso known as condensed tannins, contribute to the astringencyand bitterness of a wine, in addition to its color (del Llaudy et al.2008). Several studies have suggested that some phenoliccompounds, specifically proanthocyanidins and catechins, areassociated with the health benefits deriving from moderatewine consumption due to their antioxidant and radical scaven-ger properties (Rice-Evans et al. 1996; Nichenametla et al.2006).

Although some phenolic compounds of red wine comefrom grape seeds, they mainly come from grape skin, whichcontains a large amount of different phenols, including antho-cyanins and proanthocyanidins located in the cell vacuoles(Pinelo et al. 2006). These compounds are transferred to themust during the fermentation step, which is conducted in thepresence of skins and seeds.

The phenolic composition of wine depends on both thegrapes and the winemaking technology used, which maysignificantly affect the extraction of phenols and their subse-quent stability in the wine (Sacchi et al. 2005). The extractionof anthocyanins and proanthocyanidins during fermentativemaceration is essentially a diffusion process, and the rate andextent of extraction are influenced by the integrity of the grapeskin cell envelopes.

Recently, the improved extraction of phenolic compoundsthrough electroporation of grape skin cells using moderatepulsed electric field treatments (<10 kV/cm) during the

E. Luengo : I. Álvarez : J. Raso (*)Food Technology, Faculty of Veterinary, University of Zaragoza,C/ Miguel Servet, 177, Zaragoza, Spaine-mail: jraso@unizar.es

E. FrancoCentro de Transferencia Agroalimentaria. Gobierno de Aragón,Zaragoza, Spain

F. BallesterosBodegas Aragonesas, Carretera Magallon s/n Fuendejalon,Zaragoza, Spain

Food Bioprocess TechnolDOI 10.1007/s11947-013-1209-2

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maceration–fermentation step of red winemaking has beeninvestigated (Puértolas et al. 2010). Studies conducted at alaboratory scale using a parallel-electrode treatment chamberwith different grape varieties (López et al. 2008; Donsi et al.2010a, b) and in continuous flow (118 kg/h) with CabernetSauvignon grapes (Puértolas et al. 2010) demonstrated thepotential of using pulsed electric fields (PEF) to obtain wineswith a higher content of phenolic compounds and reduce theduration of maceration during vinification.

In the last few years, many studies have demonstrat-ed that PEF is a suitable technology for improving therelease of the intracellular compounds of interest withlow energy input (Donsi et al. 2010a, b; Vorobiev andLebovka 2010). However, a lack of powerful PEF gen-erators prevented the application of the technology at anindustrial scale. Recently, pulse generators and treatmentchambers for industrial applications have become com-mercially available (Toepfl and Heinz 2011). Studies onthe implementation and performance of PEF systemsinto existing production lines and processing at capacityranges close to industrial requirements are necessary to eval-uate the industrial feasibility of PEF technology and identifypossible drawbacks (Sack et al. 2010; Jaeger et al. 2012; Turket al. 2012).

The market currently demands deeply-colored, full-bodiedwines. A procedure used in wineries to obtain this kind ofwine is to extend the maceration time longer than required bythe yeasts to convert the sugars of the must into ethanol.However, this practice requires wineries to increase theirnumbers of maceration–fermentation tanks; on the other hand,there is also a risk of obtaining bitter and astringent wine dueto overextraction of proanthocyanidins from grape seeds(Kontoudakis et al. 2010).

The objective of this study is to assess the feasibility of PEFfor improving extraction of phenolic compounds, in order toreduce maceration time when manufacturing Garnacha redwine. This study was performed at a winery that processesgrapes using PEF in a continuous system, at a flow rate of 1,900 kg/h.

Material and Methods

Grape Samples

Grapes from Vitis vinifera L. var. Garnacha, from the certifiedorigin Campo de Borja (Aragon, northeast Spain), wereharvested from the 2010 vintage. Seven thousand kilogramsof grapes were manually harvested during their optimal ripen-ing stage (25.3 °Brix, titratable acidity of 5.6 g tartaric acid/l)and in good sanitary conditions. The grapes were transported toa winery in 20-kg boxes.

PEF Equipment

The PEF equipment used in this investigation (Modulator PG,ScandiNova, Uppsala, Sweden) generates square waveformpulses 3 μs wide, with a frequency of up to 300 Hz. Themaximum output voltage and current were 30 kVand 200 A,respectively. The actual voltage and current intensity appliedwere measured using a high-voltage probe (Tektronix,P6015A, Wilsonville, OR, USA) and a current probe(Stangenes Industries Inc. Palo Alto, CA, USA), respectively,connected to an oscilloscope (Tektronix, TDS 220,Wilsonville,OR, USA).

This study utilized a collinear treatment chamber, whichconsisted of three stainless steel tubular electrodes and twomethacrylate tubular insulator bodies. Its design defines twotreatment zones of 3 cm between electrodes with an innerdiameter of 3 cm. Electric field strength was numericallysimulated through the finite elements method by using theComsol Metaphysics software (Comsol Inc., Stockholm,Sweden) to determine the distribution of electric fields in thetreatment zones. The electric field strength used to character-ize the applied PEF treatments corresponded to the valuefound in the midposition of the central axis in the treatmentzone (Toepfl et al. 2007).

PEF Processing

The grapes were crushed, destemmed, and then pumpedacross a 6-cm diameter tube with a peristaltic pump to themaceration–fermentation tanks. The PEF treatment chamberwas located between the peristaltic pump (Rotho, MS1Ragazzini, Faenza, Italy) and the tanks. The mass flow ratewas 1,900±50 kg/h, which corresponds to a medium resi-dence time in the treatment zone of 0.41 s. The PEF treatmentconsisted of 20 pulses of 3 μs with an electric field strength of4 kV/cm (1.5 kJ/kg). The treatment frequency was 250 Hz. Inthe control treatment, the grapes were also pumped throughthe PEF treatment chamber, but, in this case, the pulse gener-ator was turned off.

Winemaking

Vinifications were conducted using untreated grapes with amaceration time of 14 days (control) and with PEF-treatedgrapes with maceration times of 7 days (PEF-7 wine) and14 days (PEF-14 wine). Maceration time of control wine cor-responds with the maceration time typically employed at thewinery for the variety of grapes used in this study. All vinifi-cations were made in duplicate in 1,500-L stainless steel tanksusing 1,100 kg of grapes. Sodiummetabisulfite was added (8 gof SO2/100 kg of grapes) to all tanks before fermentation,which was conducted at 25±2 °C. Fermentations wereperformed with the yeast Saccharomyces bayanus (PB3089,

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Pascal, Biotech, Paris, France), which was added at a concen-tration of 10 g of dry yeast/100 kg of grapes. During thefermentation process, the temperature and must density weremonitored daily and the cap was punched twice a day. After 7and 14 days, the wine pomace was pressed. Free-run andpressed wine from each tank was mixed and stored at roomtemperature. The wines were decanted; the free SO2 levels wereadjusted to 30 mg/l, and finally, the wines were filtered andbottled.

Chemical Analysis

The chromatic characteristics of the wines were determined bydirectly measuring the absorbance of the wines at 420, 520,and 620 nm using a Unicam UV500 spectrophotometer(Unicam Limited, Cambridge, UK) with a 1-mm path-lengthquartz cuvette. Color intensity (CI) was calculated as the sumof absorbance at 420, 520, and 620 nm. Tint was determinedas the proportion of absorbance measured at 420 and 520 nm;the proportions of yellow color (%Ye), red color (%Rd), andblue color (%Bl) were determined as the relation between 420,520, and 620 nm, respectively, and color intensity (Glories1984; Sudrau 1958).

Total polyphenol index, tannin content, and anthocyaniccontent were determined using a Unicam UV500 spectropho-tometer. The total polyphenol index (TPI) was measured bydirectly reading the absorbance at 280 nm of diluted wine 1/100 (v /v ) (Ribéreau-Gayon et al. 2006). Tannin content wasmeasured according to Ribéreau-Gayon et al. (2006b) and wasexpressed as grams per liter of a standardized oligomericprocyanidin solution.

Total anthocyanin content (TAC), expressed in milligramsper liter of malvidin-3-glucoside, was analyzed by determin-ing the absorbance at 520 nm of diluted wine 1/100 (v /v ) with1 % (v /v ) HCl (Ruiz-Hernández 2004).

Analyses on total and volatile acidity, pH, ethanol concen-tration, and reduced sugars were performed according to thespecifications of the Organisation Internationale de la Vigne etdu Vin (2005).

HPLC Analysis of Phenolic Compounds

A Varian ProStar high performance liquid chromatograph(Varian Inc., Walnut Creek, CA, USA) consisting of aProStar 240 ternary pump, a ProStar 410 autosampler, and aProStar 335 photodiode array detector was used for this study.The software program Star Chromatography Workstationv.6.41 (Varian) was used to acquire and manage data. AMicrosorb-MV 100–5 C18 reversed-phase column (25×0.46 cm; 5-μm particle size) and a precolumn (5×0.46 cm;5-μm particle size) made from the same material were used.The temperatures of both the column and precolumn weremaintained at 40 °C.

An elution gradient consisting of formic acid/water(5 mL/100 ml) (solvent A) and acetonitrile (solvent B) wasapplied at a flow rate of 1 ml/min as follows: 2–6 % of solventB in 25 min, 6–15 % of solvent B in 15 min, 15–20 % ofsolvent B in 12 min, and 20–40 % of solvent B in 18 min.Before injecting the next sample, the columnwas washed withacetonitrile for 10 min and re-equilibrated with the zero-timesolvent mixture for 20 min.

The wine samples were filtered (0.2-μm sterile syringefilter of cellulose acetate, VWR, West Chester, PA, USA)before being injected (10 μl) into the chromatograph. Eachsample was analyzed at least twice. Chromatograms wererecorded at 280 nm (for flavan-3-ols and gallic acid),320 nm (hydroxycinnamic acids and their principal deriva-tives), 360 nm (flavonols), and 520 nm (monomeric anthocy-anins) in the photodiode array detector.

Phenolic compounds were tentatively identified accordingto the retention time and the UV–vis spectra of the purecompounds, when possible (quercetin-3-glucoside, myricetin,quercetin, kaempferol, and isorhamnetin from Fulka (Buchs,Switzerland); gallic acid, caffeic acid, p -coumaric acid, (+)-catechin, and (−)-epicatechin from Sigma-Aldrich (St. Louis,MO, USA); kaemferol-3-glucoside and isorhamnetin-3-glucoside from Extrasynthèse (Genay, France)), and also inagreement to their order of elution and their UV–vis spectralcharacteristics published in the literature (de Villiers et al. 2004;Gómez-Alonso et al. 2007; López et al. 2009; Monagas et al.2005).

The quantification of commercial compounds was carriedout with the calibration curves obtained from using the con-centrations normally present in wine. For the noncommercialcompounds, quantification was done by using the calibrationcurves of similar compounds: malvidin chloride (Sigma-Aldrich) for monomeric anthocyanins, quercetin-3-glucosidefor myricetin-3-glucoside, caffeic acid for t -caftaric acid, andp -coumaric acid for t -coutaric acid. The concentrations of thestudied compounds were expressed in milligrams per liter.

Sensory Analysis

After 2 months in bottle, triangle tests were conducted todetermine if panelists (nine trained judges) could detect dif-ferences in the elaborated wines. The panelists, who wereseated at partitioned booths, were presented three samples,two of which were identical and the other one different. Foreach comparison, panelists were presented with the six possi-ble combinations of the two samples using a completelyrandomized design, and samples were named with a randomthree-digit code. Two replicates of each set of samples wereanalyzed.

The panelists were asked to choose which sample wasdifferent and to indicate their preferred samples. Preferencewas taken into consideration only in those tests where

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difference was correctly assessed. Significant differences(α =0.01) were determined using corresponding tables (ISO4120 2004).

Statistical Data Treatment

The results represent the mean ± standard deviation of theanalysis performed on the two tanks containing samples re-ceiving the same treatment. A t test was conducted to assesssignificant differences between vinifications conducted withuntreated and PEF-treated gapes along maceration–fermenta-tion time. The differences were considered significant atp <0.05. One-way analysis of variance (ANOVA) using theTukey test was performed to evaluate whether the differencesbetween the mean values among the three different groupswere significant. These differences were considered signifi-cant at p <0.05. GraphPad PRISM (GraphPad Software, SanDiego, USA) was used to perform the statistical analysis.

Results and Discussion

In order to identify the advantages of applying a PEF treat-ment before the maceration–fermentation step of the grapepomace, the current vinification process performed at thewinery—with 14 days of maceration (control)—was com-pared with macerations of 7 and 14 days using grapes treatedby PEF.

Effect of PEF on the Evolution of Color Intensity, TotalAnthocyanin Content, and Total Polyphenol Index

Figure 1 compares the evolution of CI (1A), TAC (1B), andTPI (1C) in fermentation–maceration during the first 7 days ofvinifying untreated Garnacha grapes (control) and grapestreated by PEF. The value of these indices depends directlyon the extraction of polyphenols from the grape skins. In orderto monitor the must fermentation, Fig. 1a shows the rate ofdecrease in the density of fermenting must, as an indirectmeasurement of the evolution of the alcoholic fermentation.The density evolutions of the musts from untreated and PEF-treated grapes were similar, which indicates that the electro-poration of the grape skins by PEF did not affect the transfor-mation of sugars into ethanol by yeast. In order to evaluate thepotential of PEF for reducing maceration time, grape pomacewas removed from two tanks that contained grapes treated byPEF after 7 days. Fermentation of the must was completedwithout the grape pomace.

According to Fig. 1, the application of a PEF treatment tothe grapes before the fermentation–maceration step promotedhigher CI, TAC, and TPI from the beginning of the fermenta-tion–maceration step. These results are in agreement withprevious studies conducted with the same grape variety at a

laboratory scale, in which the PEF treatment was applied inbatches (López et al. 2008). The evolution of the three indicesfollowed the same pattern in the tanks containing untreatedand PEF-treated grapes. In both cases, the concentrations ofanthocyanins increased until reaching a maximum value after

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Fig. 1 Evolution of color intensity (CI) a total anthocyanin content(TAC), b total polyphenol index (TPI) (1C), along fermentation–macer-ation time during the first 7 days of vinification of untreated Garnachagrapes (empty bars) and grapes treated by PEF (full bars). Figure 1a alsoshows evolution of must density during fermentation. Error bars corre-spond to standard deviation of the mean

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5 days, which remained constant afterwards. After this time,the extension of the maceration time did not increase signifi-cantly (p >0.05) the content of anthocyanins on any of thestudied treatments. Just before removing the grape pomace,statistically significant differences (p <0.05) were found in thecontent of anthocyanins of the two studied musts. The TAClevels of the tanks that contained PEF-treated grapes were25 % higher than in the control tanks. In the case of TPI,similarly to TAC, a higher extraction of polyphenols occurredin the samples treated by PEF. However in this case, in boththe treated and untreated samples, the TPI increased duringmaceration–fermentation until reaching their highest val-ue at the seventh day. At this point, the TPI levels ofthe tanks that contained samples treated by PEF were23.5 % higher, being the differences statistically signif-icant (p <0.05). Generally, during the fermentation–mac-eration step, the TPI increases for a longer time periodthan TAC because anthocyanin extraction is not asethanol dependent as that of other polyphenolic compounds.The solubility of anthocyanins in water is greater than thesolubility of proantocyanidins; on the other hand, extraction ofproantocyanidins from seeds only occurs when the ethanolgenerated during fermentation disorganizes the lipidic layer ofthe seeds, which prevents the transfer of polyphenolic com-pounds (Busse-Valverde et al. 2010).

The effect of PEF on the extraction of polyphenolsfrom grape seeds has been investigated (Boussetta et al.2012). The permeabilization of the grape seed cellsrequired the application of treatment conditions much higher(8–20 kV/cm, 0–20 ms) than those necessary for electropora-tion of grape skin cells. According to these results, at theintensity used in this study (4 kV/cm for 60 μs), the PEFtreatment applied should not affect the extraction of polyphe-nols from the grape seeds.

Color is a key property of red wine quality; it mainlydepends on the extraction of anthocyanins during fermentativemaceration. When the evolutions of color intensity for thecontrol and PEF-treated samples were compared during mac-eration–fermentation, both showed an increase in color inten-sity, until reaching a maximum at the same time (5 days)—thiswas at the same point as when the maximumwas observed foranthocyanin extraction. However, while TAC remained con-stant, a slightly decrease in CI was observed in both cases. Adrop in the must's color intensity during fermentation haspreviously been reported by other authors (Bautista-Ortínet al. 2004; Romero-Cascales et al. 2012). In spite of this dropin CI, after 7 days of maceration–fermentation, the colorintensity of the fermenting must in the tank containing PEF-treated grapes was 12.5 % higher. Although previous work byLopez et al. (2009) found that applying a PEF treatment tograpes led to wines richer in color intensity after differentmaceration times, in this study no statistically significantdifference was found.

Characterization of Garnacha Wines Elaboratedwith Untreated and PEF-Treated Grapes

Table 1 shows the results obtained for different oenologicalparameters of the control wine and elaborated wines from PEF-treated grapes with 7 and 14 days of maceration just beforebottling. The application of a PEF treatment to the grapesbefore vinification did not significantly affect the ethanol con-tent, pH, volatility, or total acidity of the Garnacha wine. Thedifferences observed in these parameters between the controlwine and wines elaborated with PEF-treated grapes at differentmaceration times did not have practical implications. Theseresults agree with other studies performed on a laboratory scaleor in vinifications with 100 kg of grapes, which reported thatapplying a PEF treatment to grapes before vinification did notaffect these wine parameters (Puértolas et al. 2009). Concerningthe other parameters shown in Table 1, which are dependent onpolyphenol extraction (CI, TPI, TAC, and tannin concentra-tion), the TPI and TAC were slightly higher in the PEF-7 wine.However, these differences were not statistically significant(p >0.05), and they did not have practical implications.

According to these results, the application of a PEF treat-ment created wine with polyphenol extraction-dependentvalues after 7 days ofmaceration similar to those of the controlwine, in which the maceration was extended to 14 days. Theseresults, which were obtained under capacity ranges close toindustrial requirements, confirm the previous observations

Table 1 Chemical characteristic of wines before bottling

Control PEF-7 days PEF-14 days

Ethanol (%, v /v) 16.9±0.3 16.9±0.2 17.1±0.2

pH 3.5±0.1 3.5±0.1 3.5±0.1

Titratable acidity (g/L)a 5.9±0.2 5.8±0.3 5.7±0.4

Volatile acidity (g/L)b 0.4±0.1 0.4±0.1 0.5±0.1

TAC (mg/L)c 630.0±5 668.0±37 637.3±22

TPI (A.U.) 51.5±1.6 53.3±1.3 52.1±0.3

Tannin content (g/l)d 2.2±0.2 2.1±0.1 2.2±0.1

CI (A.U.) 15.4±2.6 15.0±1.6 14.8±0.42

Tint (A.U.) 0.5±0.1 0.4±0.2 0.5±0.1

%Ye (A.U.) 29.9±0.4 28.4±0.3 30.6±0.4

%Rd (A.U.) 59.9±0.6a 57.4±0.5b 60.7±0.3a

%Bl (A.U.) 10.1±0.2ab 9.6±0.1a 10.6±0.3b

Different letters within the same row represents significant differencesaccording to one-way ANOVA analysis (p <0.05)

TAC total anthocyanin content, TPI total popyphenol index, CI colorintensity, %Ye , %Rd , %Bl percentages of blue, red, and yellow colors,A.U. absorbance unitsa Expressed as tartaric acidb Expressed as acetic acidc Expressed as malvidin-3-glucosided Expressed as procyanidin

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conducted at laboratory and pilot plant scales with other grapevarieties (Puértolas et al. 2010). On the other hand, it wasobserved for the first time that, when the maceration is ex-tended beyond fermentation, no differences were observedbetween the control wine and wines elaborated from PEF-treated grapes. The degradation of grape skin cell envelopesthat occurs during maceration, particularly in the presence of

ethanol, may cause the initial effects of PEF electroporationon polyphenol extraction observed in the first stages of mac-eration–fermentation to disappear in vinifications with extend-ed macerations. Other authors (Bautista-Ortín et al. 2004) alsoreported that the positive effects of using maceration enzymeson polyphenol extraction at the beginning of the winemakingprocess diminished, as maceration progressed.

Table 2 Low molecular weightphenolic compounds (milligramsper liter) in wines before bottling

Each value represents mean ±standard deviation. Nonsignifi-cant different according to one-way ANOVA (p >0.05) werefound between values of the dif-ferent columns

nd not detected

Control PEF-7 days PEF-14 days

Anthocyanins Delphinidin-3-glucoside 25.9±4.8 28.0±4.0 26.9±3.6

Cyanidin-3-glucoside 2.0±0.4 2.1±0.3 2.2±0.3

Petunidin-3-glucoside 36.4±4.5 39.4±4.1 37.5±3.9

Peonidin-3-glucoside 22.1±3.5 27.7±4.8 25.5±4.8

Malvidin-3-glucoside 314.5±29 331.6±24 300.4±21.2

Delphinidin-3-acetylglucoside 2.7±0.4 2.7±0.4 2.5±0.2

Cyanidin-3-acetylglucoside 1.4±0.2 1.3±0.2 1.5±0.1

Petunidin-3-acetylglucoside 1.9±0.3 1.5±0.2 2.0±0.2

Malvidin-3-acetylglucoside +peonidin-3-acetylglucoside

7.6±0.8 7.6±0.6 7.1±0.4

Delphinidin-3-p-coumaroylglucoside 1.7±0.3 2.1±0.3 1.7±0.3

Cyanidin-3-p-coumaroylglucoside 1.7±0.3 1.8±0.3 1.6±0.2

Petunidin-3-p-coumaroylglucoside 1.0±0.2 0.9±0.2 0.8±0.1

Peonidin-3-p-coumaroylglucoside 3.0±0.5 3.8±0.5 2.9±0.5

Malvidin-3-p-coumaroylglucoside 19.7±2.4 21.8±2.0 17.6±2.0

Flavanols (+)-Catechin 102.8±10 100.1±7.8 98.9±4.9

(−)-Epicatechin 14.4±1.8 14.6±1.6 15.9±2.0

Flavonols Myricetin-3-glucoside 15.7±0.3 17.29±1.5 16.4±1.5

Quercetin-3-glucoside 5.7±0.5 6.0±0.5 5.8±0.43

Isorhamnetin-3-glucoside nd nd nd

Kaempferol-3-glucoside nd nd nd

Myricetin 0.93±0.1 0.75±0.1 1.1±0.13

Quercetin 2.3±0.1 2.4±0.2 2.4±0.15

Isorhamnetin nd nd nd

Kaempferol nd nd nd

Wine acids Gallic acid 53.8±7.4 44.4±6.2 59.6±8.15

t-Caftaric acid 61.1±5.6 71.9±6.6 60.8±6.20

t-Coutaric acid 8.4±0.9 9.7±1.0 7.9±1.10

Caffeic acid 1.6±0.2 1.3±0.2 1.4±0.23

p-Coumaric acid 0.31±0.1 0.22±0.1 0.40±0.1

Table 3 Percentage of correct responses identifying the odd wine sample in the triangle test and percentage of preference for each one of the comparisons

Triangle test (percentageof correct responses)

Preference test (percentageof preference)

Control PEF-7 days PEF-14 days

Control/PEF-7 days 77.7a 24.13b 75.87 –

Control/PEF-14 days 59.7 50.55 – 49.45

PEF-7 days/PEF-14 days 83.3a – 82.86b 17.14

a Significant differences between the samples (α=0.01)b Proportion of preference statistically different to 50 % according to chi-square test

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Phenolic Composition of Garnacha Wines Elaboratedwith PEF-Treated Grapes

The concentrations of major individual anthocyanins, mono-meric flavan-3-ols, flavonols, and phenolic acid in the threetypes of wines are presented in Table 2. Fourteen differentanthocyanic compounds were identified and quantified in thethree wines. The HPLC anthocyanic profiles were similaramong the analyzed wines; this confirmed the results obtainedby other authors, which indicate that PEF treatment did notproduce a selective effect on the extraction of any anthocyanin(López et al. 2009). The major individual anthocyanin foundin all three types of wines was malvidin 3-glucoside, whichrepresented more than 50 % of the total individual anthocya-nins for the three wines elaborated. No significant differencesin content of all individual anthocyanins were detected be-tween the three wines.

The concentrations of other individual phenols were verysimilar for the three wines. No significant differences (p >0.05)were detected between the control wine and the wines elabo-rated from PEF-treated grapes at both maceration times.Puertolas et al. (2009) found higher concentrations of flavan-3-ols flavonols and phenolic acids in Cabernet Sauvignonwines elaborated from PEF-treated grapes. However, in thatinvestigation, grape pomace was separated from the fermentingmust for two wines before finishing fermentation—one with amaceration time of 6 days as the control and the other obtainedfrom PEF-treated grapes and macerated for 4 days.

Sensory Evaluation

Table 3 shows the percentage of correct responses identifyingthe odd sample in the triangle test and the results of thepreference test. Significant sensory differences (1 % level(α =0.01)) between the PEF-7 wine and both the control andPEF-14 wine were detected. However, the panelists were notable to differentiate the two wines elaborated with a macera-tion time of 14 days. When the panelists were asked toindicate their preferred wines, the majority chose the wineelaborated from grapes treated by PEF with a maceration of7 days compared to the control wine and the wine elaboratedfrom grapes treated by PEF with a maceration of 14 days(Table 3). It is assumed that skin proanthocyanins confer asoftening effect on the wine's mouthfeel. However,proanthocyanins from seeds contribute to the bitterness andastringency of wine (Busse-Valverde et al. 2010). Longermacerations promote higher concentrations of seedproanthocyanidins in wine because their extraction requiresthe presence of ethanol, which helps to remove the protectivelayer of the seed (del Llaudy et al. 2008). Therefore, the higherpreference for the wine elaborated with grapes treated by PEFwith a maceration of 7 days could be related to the lowerconcentration of seed proanthocyanins in this wine.

In summary, the results of the sensory test indicated that theapplication of a PEF treatment to grapes before the macera-tion–fermentation step resulted in a wine, which, after 7 daysof maceration, had a color and pholyphenolic content similarto a control wine obtained after 14 days of maceration, butwith better sensory properties.

Conclusions

PEF processing at near-commercial scale on grapes beforevinification was effective in increasing the extraction of poly-phenols and color intensity of Garnacha wines during the first7 days of fermentative maceration, which confirmed previousobservations from experiments conducted at laboratory andpilot plant scales. This study demonstrates the potential ofPEF to obtain wine with a sufficient concentration of phenoliccompounds with moderate maceration times. Consequently,doing so avoids the overextraction of proanthocyanidins fromgrape seeds, which could have a negative impact on thesensory properties of a young wine.

The recent development of equipment with sufficient pow-er to process large quantities of product, the easy implemen-tation by wineries of treatment chambers into existing pro-cessing lines, and low energy consumption are keys for PEFtechnology to become a commercially-viable technology forwineries in the near future.

Acknowledgments E.L. gratefully acknowledges the financial supportfor her doctorate studies to the Department of Industry and Innovation ofthe Aragon Government. Authors also acknowledge to the Department ofIndustry and Innovation of the Aragon Government and “EuropeanSocial Fund (ESF)” for financial support to conduct this research.

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