Phenolic and colour characteristics of must and wine obtained from red grapes treated by pulsed electric fields. Efficacy of PEF to reduce maceration time in elaboration of red wines

Pulsed electric fields effect was studied on the physico-chemical and general phenolic composition as colour characteristics and stilbene content in must and wine. For this purpose, a continuous pulsed electric fields equipment was used to treat three red grape varieties of DOCa Rioja. Graciano, Tempranillo and Grenache wines from these grapes were elaborated with different maceration times, 2 days in the untreated sample (control) and the PEF-treated sample (PEF), and normal maceration time in another untreated sample (control-NM). Parameters as colour intensity, anthocyanin content, total polyphenol index and tannin content showed no differences between the PEF sample with 2 days of maceration and the control-NM sample, except in the case of Tempranillo wines. Total stilbenes, trans-resveratrol and trans-piceid of Graciano wines elaborated from PEF samples showed a higher concentration than the control wines. Alternatively, PEF wines and control-NM wines showed no differences between them. Tempranillo variety wines presented no differences between the three types of samples. In the Grenache variety, only trans-piceid levels showed differences between control and PEF wines. Moreover, relationship between must and wine characteristics was evaluated and compared between different samples. The trend lines obtained for the CI, TPI and AC parameters for samples of Graciano, Tempranillo and Garnacha indicate that the initial content of compounds extracted significantly affected the days of maceration necessary to obtain the appropriate wine. The results obtained increase the knowledge of pulsed electric fields as a technology available for use in the winery to elaborate red wines with reduced maceration time.


Introduction
The extraction of phenolic compounds and shorten the time of maceration to obtain these compounds during fermentation process to obtain red wines is one of the most important issues for the wine industry. Currently, the contact time of the skins with the must during fermentation is long. The goal is to achieve high quality red wines adapted to the consumer. However, this is costly, both in energy and in use of the tanks in winery.
Key components linked to the protective effects of moderate wine consumption include phenolic compounds from grapes [1]. Resveratrol, a prominent member of the stilbene family, has been attributed beneficial properties as anticancer capacity, antioxidant, cardioprotective, anti-ageing or neuroprotective [2,3]. Stilbene compounds are in nature as two isomeric forms, cis and trans. Both can be linked to a glucose molecule. In grapes, the most common form is the glycosylated, known as piceid [4,5].
Pulsed electric field technology is considered a non-thermal method that implies the application of pulses a high strength electric field of microsecond to a product placed between two electrodes. External electric field cause an 1 3 electrical breakdown of the membrane. that leads to a permeabilization of the membranes. Permeabilization could be transitory or permanent [6]. Several studies indicate that the extraction of phenolic compounds can increase with application of pulsed electric fields [7][8][9][10][11][12] and aromatic compounds [12,13] from grapes.
The existing literature has shown the importance of grape variety to enhance the process of diffusion of these compounds from grape skins to must by using pulsed electric fields technology [7][8][9][14][15][16]. In other techniques, such as cold maceration, the same occurs [17][18][19]. The different results yielded by the different varieties may be due to differences in the skin cell layers, size of cells and content of these compounds in cells. Furthermore, to date, few of the studies carried out have evaluated compounds such as stilbenes with reduced maceration time on a pilot scale in different grape varieties.
Previous research from our group investigated which could be the best of four pulsed electric fields treatments to apply in musts of same grape varieties to enhance the extraction of phenolic compounds, including stilbenes [11], also aromatic compounds [13]. Those studies served to select the best pulsed electric fields treatment to be used in this study.
The aim of this study was to determine the effect of pulsed electric fields to improve the extraction of compounds of skins of grapes to shorten the maceration during fermentation step in the red winemaking of Graciano, Tempranillo and Grenache varieties to obtain a product with the same quality as current wines, and with special emphasis on the extraction of stilbenes. Also, the study defines the trend line between must and wine with and without PEF treatment and different conditions of maceration.

Characteristics of the samples
Graciano, Tempranillo and Grenache varieties of grapes from the experimental vineyard of ICVV (Instituto de Ciencias de la Vid y del Vino, La Rioja, Spain), D.O.Ca. Rioja were used. For each variety, 400 kg of grapes were harvested at their optimum maturity. Subsequently, grapes were destemmed, crushed and sulphited with 70 mg/kg SO 2 . Must with skins were located into 9 stainless steel vats of 25 l. 3 of them were used for samples treated by pulsed electric fields and 6 vats were used for untreated samples (control).

Characteristics of pulsed electric fields equipment and treatments
Pulsed electric fields equipment and treatments were exposed in López-Giral et al. [20]. The equipment used in this study was an ELCRACK-HVP5 unit (DIL, Germany). The pulsed electric fields chamber was a co-linear chamber ELCRACK DN25 (2.50 cm of diameter; 2.38 cm distance between electrodes; 4.45 cm 2 of electrode area). Pulses of 7.4 kV/cm, 20 µs of pulse width and frequencies of 400 Hz were applied. The total mean residence time was determined by the flow rate and unit volume. It was 0.09-0.10 s. A membrane pump was used to pump the crushed grapes from each variety (PV8 Saniflo, Wilden, USA) into the treatment chamber of pulsed electric fields.

Vinification
For each variety, 9 stainless steel vats of crushed grapes were filled (6 untreated and 3 with PEF treatments) and these were inoculated with yeast Uvaferm VRB (Lallemand, St. Simon, France) to produce alcoholic fermentation. Untreated and PEF treated grapes were macerated during fermentation for 2 days, control and PEF-treated (denominated PEF) and for normal maceration was 5 days (denominated control-NM). Completion of the fermentation was considered when the content of sugar was less than 2.5 g/l. Alcohol monitoring was performed by daily density control.

Analysis of oenological parameters
To determine the oenological parameters in the must, untreated (control) and PEF samples were analyzed. And, also in wines, oenological parameters were analyzed at the end of the alcoholic fermentation as control, PEF and control-NM samples. To calculate the stilbene content, aliquots of each sample were frozen, including must (control and PEF) and wine (control, PEF and control-NM).
The methods established by the ECC [21] was used to determine pH, probable alcohol, total acidity, malic acid and tartaric acid. Colour intensity (CI) and tonality were analyzed by measurements of absorbance at 420, 520 and 620 nm using the Glories method [22]. The method to determine total polyphenol index (TPI) was used by measuring the absorbance at 280 nm after conventional dilution of samples [23]. Anthocyanin content (AC) was analyzed by decolouration with sulphur dioxide [24]. The method used to determine the tannin content (TC) was described by Ribéreau-Gayon et al. [25]. Ionization index (II) was determined according to the Glories method [26] and the polymerization index (PI) was calculated by 1 3 measurements of absorbance at 520 nm after one hour of decolouration with SO 2 [27]. Tannin content, ionization index and polymerization index only were determined in wine. The treatments were performed in triplicate. Therefore, the mean of the results of the three different analysis was used (n = 3).

Analysis of stilbenes by SPE-HPLC
Stilbenes were analysed using the method described by Garde-Cerdán et al. [5]. Briefly, 10 ml of sample were passed through the solid phase extraction (SPE) cartridge. Then, three fractions of water were used to wash. The cartridge was dried by letting air pass through it for 30 min. The stilbenes were eluted with 0.7 ml of methanol. The filtered eluate obtained was diluted with water (60 methanol:40 water (v/v)). For the wines samples, it was only necessary to carry out their filtration and was injected into the HPLC system.
Stilbenes were determined by reverse-phase HPLC (Agilent 1100 Series liquid chromatograph, Palo Alto, USA). The injected amount was 30 μl and the column temperature were 25 ºC. All separations were performed on a ZORBAX Eclipse Plus C18 (150 × 3.0 mm, I.D. 3.5 µm) column (Agilent) with an Eclipse XDB-C18 precolumn (12.5 × 4.6 mm, I.D. 5 µm). Three eluents were used as mobile phases: A: water, and acetic acid (98:2, v/v); B: water, acetic acid, and acetonitrile (78:2:20, v/v/v); and C: methanol. Detection was performed by an FLD detector (λ excitation = 310 nm, λ emission = 403 nm), and a DAD detector (λ = 280 nm, 310 nm, and 325 nm). Standards (Sigma-Aldrich) were used to identify the target compounds taking to account the retention times and UV-Vis spectral characteristics. Calibration graphs of the respective standards were used to quantify (R 2 > 0.99), which underwent the same process of SPE extraction as the samples. In the case of wines, quantification was based on the calibration curves of the respective standards (R 2 > 0.99) in a 12% v/v ethanol in water at pH 3.6 (model wine).
The treatments were performed in triplicate. Therefore, the mean of the results of the three different analysis was used (n = 3).

Statistical analysis
Data management and analysis were performed using SPSS Version 21.0 (Chicago, USA). ANOVA were used to compare the oenological parameters and stilbene compounds. Results were expressed as means ± standard deviation. A p value ≤ 0.05 was considered significant (Duncan test). The statistical analysis was performed for must considering paste without and with pulsed electric fields treatments. In wine, the statistical analysis was performed comparing wine untreated (control), PEF and control-NM samples. Table 1 presents the oenological parameters of musts in contact with skins of the control and PEF samples. Results were previously published [20]. That results are important to compare and relate the results obtained in different wines. The oenological parameters showed no differences between Table 1 Oenological parameters of the control and the PEF treated Graciano, Tempranillo, and Grenache must samples All parameters are listed with their standard deviation (n = 3). For each grape variety, different letters indicate significant differences between control and PEF samples (p ≤ 0.05) a As g/l tartaric acid the control and PEF musts of Graciano. However, a significant increase after the pulsed electric fields treatment in all parameters was observed in the Tempranillo must. The oenological parameters in Grenache must showed differences in pH, total acidity, and tartaric acid. Despite pH showing significant differences, the pH increase in Tempranillo and Grenache varieties was less than 5%, and in all cases below 3.9 for Tempranillo, 3.4 for Graciano and 3.5 for Grenache. Increases in total acidity were below 8% and fell within the standard values for the variety. In the case of Graciano, total acidity parameter were no significant differences between samples. In both cases, the parameter was the highest with respect to the other varieties. The rest of parameters, potassium, CI, TPI and AC varied after pulsed electric fields application, all increasing with pulsed electric fields treatment, except the tonality value that decreased in the PEF must. Musts of the three varieties showed improvements in TPI and AC, respectively of 52 and 84% in Graciano, 94 and 184% in Tempranillo and 99 and 459% in Grenache. Similarly, CI improved in PEF musts, with increases of 103%, 184% y 235%, respectively for Graciano, Tempranillo and Grenache. Further, the red tonality parameter was higher. The extractive improvement was not the same percentage in all cases, although it, pulsed electric fields effect was considerable for all the indicated compounds. As Maza et al. [12] indicated, studies made with several grape varieties harvested in different countries have concluded that pulsed electric fields increase the permeability of the cytoplasmic membranes of the hypodermal cells of grapes. Thus, the liberation of the polyphenols placed in the cytoplasm was facilitated. Table 2 presents the general oenological parameters of the wines elaborated with control and PEF must, both with two days of maceration with skins, and wines from must without pulsed electric fields and traditional maceration time with skins (control-NM). Regarding alcohol content and pH, no significant difference between the three groups was evident (control, PEF and control-NM samples) for each variety. Despite alcohol content showing significant differences in the PEF samples, the increase was less than 4%. In all cases, the alcohol content was between 12 and 15% v/v. The Grenache variety showed the highest values for alcohol content with 14-15% v/v. For the three varieties, pH of wines was between 3.2 and 3.4. Also, for total acidity, volatile acidity and malic acid differences observed for each variety and treatment (control, PEF and control-NM samples) were low, even though in some cases, for the Tempranillo and Grenache varieties, the results showed significant differences. The highest values of total acidity were for Graciano, between 7.2 and 7.9 g/l. Also, the highest values of volatile acidity were for Tempranillo, between 0.24 and 0.32 g/l. Similarly, the highest content of malic acid was for Tempranillo, between 3.2 and 3.8 g/l. In all cases, values obtained for the parameters studied were included within the normal values for each variety in the La Rioja region.

Effect of pulsed electric fields on the oenological parameters in wines
As for colour parameters, CI, TPI, AC and TC were significantly different between the samples studied (control regarding the control-NM and PEF) in the Graciano and Grenache varieties, with the control samples showing the lowest values.
In the Graciano wines, CI, TPI, AC and TC increased in control-NM and PEF about the control by between 65-68, 47-60, 56-62 and 56-89%, respectively. The effect of pulsed electric fields treatment in this variety was studied by López et al. [15] during the maceration-fermentation process keeping the skins in contact throughout the fermentation. In that study, differences were also noted for all the parameters sampled between the control and PEF. Similarly, the CI, TPI and AC of the PEF sample at 3 days was of the same order as in the control with skin until the end of fermentation (8 days).
In the Grenache wines, the improvements were 96, 61-62, 70-72 and 56-72% for CI, TPI, AC and TC, respectively. Interestingly, Grenache is the most studied grape variety into PEF treatment, even at the industrial level. In this regard, Maza et al. [12] observed in Grenache wines, at high volume, CI, TPI and AC values of 12.1, 51.3 and 778 mg/l for PEF wines with 3 days of maceration. In this study, values for PEF samples with 2 days of maceration for CI (10.8), TPI (45.9) and AC (613 mg/l) were slightly lower. However, TC values were higher (2.460 mg/l) than those of the study cited [12] (1.408 mg/l). On the other hand, a comparison of the two research, current and Maza et al. [12], reveals higher improvements in present research. These results further support the idea of influence of different maceration times, raw material, area of harvest and grape maturity.
No significant differences were showed between control and control-NM for CI in Tempranillo wines. However, a significant difference was showed between them (control-NM and control) and PEF; in these, the CI parameter increased by 25%. As was the case for the AC parameter, the lowest content was for control-NM, which showed differences with regards to the control (11%) and pulsed electric fields treatments (16%). The decrease in AC during long macerations throughout the fermentation period has already been previously cited by some authors [14]. Nevertheless, the TPI and TC parameters were of the same order in the control-NM and PEF samples and higher than in the control. Improvements were between 22 and 31% for TPI and 49-55% for TC among control and the other samples. The TPI of PEF wine was 76.4, while in the control it was 58.4.

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Other previous pulsed electric fields studies in this variety have yielded contradictory results. These results are consistent with López et al. [15], which showed the advantages of applying PEF treatments in grapes to obtain red wine. By contrast, Saldaña et al. [28] observed no such benefits. In that case, the pulsed electric fields treatments applied to the grapes did not significantly increase TPI during macerationfermentation process. López et al. [15] observed improvements in TPI of up to 29% at 4 days of the maceration fermentation process between control and PEF samples, results that are of the same order as in this study, with improvements of around 22-31% for TPI.
Furthermore, the TC of PEF wine was 4.230 mg/l and 2.730 mg/l in the control. As TC content was higher for PEF wine and control-NM (4.073 mg/l). These results would be very suitable to develop wines with long ageing [29]. In the parameter of tonality, the variation between samples were no significant in the Graciano and Tempranillo wines, with values between 0.41 and 0.54. The tonality of the Grenache control was higher than the PEF and control-NM samples. Table 2 presents the related index of red wine with polyphenols (anthocyanins and tannins) and colour stabilization as the ionization index (II). The index indicates that part of the anthocyanins contributes to the colour of wine [30]. Somers [31] reported medium ionization index values for Merlot wine of around 49, and for Syrah wine of about 52. In this study, the ionization index of the wines was highly dependent on the variety, with the highest values being for Graciano wines, with values between 55 (control-NM) and 59 (control). Wines of Tempranillo and Grenache varieties showed Table 2 Oenological parameters of the control, PEF and control-NM wines of Graciano, Tempranillo, and Grenache varieties All parameters are listed with their standard deviation (n = 3). For each grape variety, different letters indicate significant differences between control, PEF treated and control-NM samples (p ≤ 0.05) a As g/l tartaric acid b As g/l acetic acid Another important index to evaluate the ability of wine to age is the polymerization index (PI). This is indicated by the PI-TPI relation. A high product of PI x TPI indicates a better ability of the wine to age [29]. Comparison of the PI of different varietal wines elaborated, showed that the Graciano wines obtained the highest PI values. As was the case with II (ionization index), no significant differences were found among the three types of wines, values of samples were 1.63 (control), 2.40 (PEF) and 1.95 (control-NM). PI of Tempranillo wines was significantly higher in the PEF samples (1.62), as compared to control (1.12) and control-NM (1.09). In contrast, in the PI values of Grenache wines, no significant differences were found between control-NM (1.15) and PEF (0.98) samples. Neither control (0.69) and PEF wines showed significant differences. Ruiz-Hernández [29] indicated that to define whether a wine is valid to obtain a good young wine, the PI and TPI values f should be between 0.4 and 0.9 for PI and 38-45 for TPI For aging values should be up to 0.9 for PI and 45 for TPI and for reserve wines, values of up to 2.0 for PI and 55 for TPI. By comparing the PI and TPI results for the control, PEF and control-NM wines for each grape variety, it can be observed that PI and TPI were within the ranges indicated [29] for the usual types of wines (young, aging and reserve) Among the three grape varieties treated, Grenache wines presented the lowest PI-TPI values, with only control-NM and PEF wines being valid for aging. For Tempranillo wines, all could be assigned for aging and in Graciano wines, PEF wines obtained PI and TPI values of 2.4-57.3. Hence, it can be said that Graciano PEF wines could be assigned to elaborate reserve wines.

Comparative effect of pulsed electric fields treatment between must and wine
In this investigation, the percentage improvement after pulsed electric fields treatment application in CI, TPI and AC parameters was reduced in wine ( Table 2) as regards the percentage improvement obtained in must in all varieties (Table 1). These results are consistent with Delsart et al. [9]. They also observed that differences were reduced in the winemaking process on Cabernet Sauvignon variety. They observed a TPI increase in must of 55% for a 4 kV/ cm treatment, while the improvement in wine obtained with 12 days of contact the skins with must was 11%. These results are in line with the percentage of improvement in our study for must of Graciano (52%), though the percentage of improvement in wine obtained with contact of the skins with must for 2 days was 47%. Similar results were observed for the AC and CI parameters, with an increase of 84 and 104%, respectively in PEF must about control and 61 and 65% for AC and CI parameters, respectively, in PEF wine compared with control wine. The advantage of pulsed electric fields treatment in must of Tempranillo variety in AC, TPI and CI parameters were of 84, 94 y 184%, respectively, while the benefits of treatment in wines were 5, 31 y 25% in AC, TPI and CI parameters, respectively. Likewise, in the Grenache variety, the benefits of treatment in must in AC, TPI and CI parameters were 99, 459 and 235%, respectively; while differences between control wine and PEF wine were 62, 70 and 65% respectively for AC, TPI and CI parameters. The higher TPI benefit percentages in our study with respect to those obtained by Delsart et al. [9] are probably due to the reduced contact time between skins and must in this study. Also, the percentage of improvement is largely dependent on variety. In general, a comparison of the two wines, PEF and control-NM reveals no significant differences in the AC, TPI and CI parameters. However, the difference in maceration time between both wines was 3 days. Likewise, the consequence of pulsed electric fields treatment on a small scale by removing the skins from the must during fermentation at 48, 72, 96 and 268 h in the Cabernet Sauvignon variety was evaluated by López et al. [7]. Differences of 20% with 48 h of maceration were observed in the CI parameter, which was higher in the PEF samples. Alternatively, López et al. [15] studied the consequence of pulsed electric fields treatment on a small scale in Graciano and Grenache, and they observed that after 72 h of maceration during vinification process, the CI, AC and TPI parameters were practically the same as in the control wines with skins left until the end of fermentation. Later, the effect of treatments on a pilot plant scale in the Cabernet Sauvignon, Merlot and Syrah grape varieties was evaluated by Puértolas et al. [32]. They observed after 96 h of maceration the maximum value for the AC in the control sample (1216 mg/L) in the Cabernet Sauvignon variety. While a similar AC value was achieved in the samples treated by pulsed electric fields and with contact with skins during vinification of less than 50 h. Finally, Ruiz-Hernández [29] consider as a "good wine for aging" that whose content in tannins is higher than 3.000 mg/l, while to obtain a young wine, 2.000 mg/L of tannins are sufficient. In this study, all control-NM and PEF wines obtained values of tannins above 2.000 mg/l, although this was not the case with the control wines of Graciano and Grenache. Further, the Tempranillo control-NM and PEF wines exceeded values of 4.000 mg/l of tannins (TC). In all cases, all parameters were in normal ranges for red wines, even though the control wine of Grenache obtained low IC, TPI and AC values.
Correlations (R 2 ) between must and wine in CI, TPI and AC for control, PEF and control-NM samples for the three grape varieties studied, taking into account the maceration time in days are shown in Fig. 1. Equations and R 2 1 3 of the trend line are shown in the different figures. In the Graciano and Grenache varieties, quite similar trend lines were detected for the control, PEF and control-NM samples. The equation of the trend line showed a slope of the same order, although the trend line slopes in the PEF samples were less pronounced. This is because that parameter in must and wine is quite similar, and similar to the control-NM, that is, electroporation practically managed to already extract in the must the amount of colour of the final wine. The R 2 of CI were quite good for control and control-NM samples for the Graciano, Tempranillo and Grenache. The worst correlations were for the PEF samples of Graciano and Tempranillo, because practically no differences in CI were observed between must and wine. For TPI, the lowest slopes were observed for control and PEF samples from Graciano. Samples presented an R 2 above 0.78, except the control and PEF from Graciano with an R 2 under 0.55. In Tempranillo, the behaviour for TPI was somewhat strange, with the initial extraction being very fast, which was clearly observed for the control and PEF samples, and slower for the control-NM. In the Grenache variety, the slopes were very similar across the different samples. For AC, values of the same order were observed in the Grenache and Graciano samples. The control-NM slope from Grenache was slightly lower than for the other 2 samples, since probably after 4 days it had reached the maximum extraction. For the Graciano variety, the slope of AC samples was slightly higher for the PEF sample than for the control and control-NM samples, which showed the same slope, which indicated a sustained extraction over time. In all cases, R 2 were considered quite good, with values above 0.77. In general, the trend lines obtained for the CI, TPI and AC parameters for samples of Graciano, Tempranillo and Garnacha indicate that the initial amount of compounds extracted significantly affected the days of maceration necessary to obtain the appropriate wine.

Effect of pulsed electric fields treatments on stilbene content in musts and wines
The concentrations of total stilbenes, trans-resveratrol, trans-and cis-piceid from each of the three grape varieties in must (control and PEF) and wines (control, PEF and control-NM) are shown in Fig. 2. Among the three grape varieties treated, Graciano presented the highest total stilbenes, trans-resveratrol, trans-and cis-piceid in both must and wines. This variety stands out for its high content of phenolic compounds [33], and among these, it has a significant concentration of these compounds which are important due to their healthy properties [33]. PEF application in must enhanced the extraction of total stilbenes in the three varieties (Fig. 2a): 33% for Graciano, 68% for Tempranillo, and 218% for Grenache. In Fig. 2b of trans-resveratrol and Fig. 2c of trans-piceid, PEF treatments increased their extraction in Tempranillo (169 and 66%, respectively) and Grenache (263 and 164%, respectively) grape varieties. Also, the extraction of cis-piceid was improved in the Graciano (37%) and Grenache (305%) grape varieties (Fig. 2d).
In all varieties, concentration of total stilbenes, trans-resveratrol, and trans-piceid was increased after alcoholic fermentation; that is, these compounds were extracted from the skins during alcoholic fermentation (Figs. 2a-c). However, concentration of cis-piceid was not modified during alcoholic fermentation in the Graciano and Tempranillo grape varieties (Fig. 2d). Comparing wines, PEF and control-NM samples provided a higher concentration of total stilbenes (44-61%), trans-resveratrol (60-89%), and trans-piceid (53-72%), but not cis-piceid, than control wine for Graciano (Fig. 2). In the Tempranillo and Grenache varieties, the difference between control and PEF/control NM wines was no significant (Fig. 2). Therefore, shortening maceration time using the pulsed electric fields treatment yielded the same results as normal maceration in control wines for Graciano. However, in Tempranillo and Grenache, it was not possible to reduce the maceration time by using pulsed electric fields to improve stilbene content in wines. (c) ( d) Fig. 2 Concentration of total stilbenes, trans-resveratrol, trans-and cis-piceid (mg/l) from must and wines of Graciano, Tempranillo and Grenache grape varieties. In must, samples were control and PEF. In wines, samples were control, PEF and control-NM. Parameters (n = 3) with their standard deviation are listed. Three grape varieties and moment (must or wine) are indicated with different lowercase letters for significant differences between control and PEF samples; different capital letters represent significant differences between control must and control wine, and between PEF must and wine; and different Greek letters represent significant differences between wines (control, PEF and control-NM) (p ≤ 0.05) 1 3

Conclusions
The results of this paper provide evidence that pulsed electric fields technology could be useful to shorten the usual time of maceration of skins with must during red winemaking process. It could be adequate on the three grape varieties studied, Graciano, Tempranillo and Grenache. The results corroborate the effectiveness of pulsed electric fields treatment. However, the final values of the different parameters studied such as CI, AC, TPI, TC, II and PI depends on the initial grape composition and the variety. With regard to stilbene content, shortening maceration time using the pulsed electric fields treatment yielded the same results as control NM wines for Graciano; however, in Tempranillo and Grenache, the increase in maceration time or use of pulsed electric fields treatments were not effective ways of enhancing stilbene content. On the other hand, taking into account the must and wine parameters of CI, TPI and AC and their trend lines, could be possible predict the time needed to achieve adequate maceration. Nevertheless, more research is needed to obtain a good prediction model. Industrially, the competitiveness of wineries could be clearly improved because of increasing production capacity and reducing the workforce and energy consumption.

Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval This article does not contain any studies with human or animal subjects.
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