Open access peer-reviewed chapter

Pulsed Electric Fields as a Green Pretreatment to Enhance Mass Transfer from Grapes of Bioactive Molecules: Aromatic, Phenolic, and Nitrogen Compounds

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Teresa Garde-Cerdán, Eva P. Pérez-Álvarez, Pilar Rubio-Bretón and Noelia López-Giral

Submitted: 23 February 2022 Reviewed: 22 March 2022 Published: 27 May 2022

DOI: 10.5772/intechopen.104609

From the Edited Volume

Trends and Innovations in Food Science

Edited by Yehia El-Samragy

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The objective of this chapter was to summarize the effects of four pulsed electric field (PEF) treatments on the chemical composition of three grape varieties. To this end, Graciano, Tempranillo, and Grenache grapes were destemmed and crushed and then were treated using a PEF continuous system. Phenolic and nitrogen compounds were analyzed by HPLC and volatile compounds by GC-MS. The results showed that the influence of PEF treatments on these bioactive molecules was different depending on the grape variety and PEF treatment applied. This non-thermal technology improved Grenache aromatic composition, but Tempranillo and Graciano volatile composition was not affected by PEF. The treatment with the highest time and energy was the most effective on the total stilbene extraction, greatly increasing the content of these compounds in all grape varieties. Moreover, all PEF treatments enhanced yeast assimilable nitrogen (YAN) and total amino acids of Grenache grape variety, while for Graciano and Tempranillo, the PEF treatments hardly affected its nitrogen compounds content. In conclusion, from the point of view of the chemical composition and taking into account the treatments used in this study, it can be concluded that PEF is an appropriate technology to improve the quality of Grenache variety.


  • volatile compounds
  • stilbenes
  • free amino acids
  • must
  • PEF
  • varietal aroma
  • flavor
  • resveratrol
  • piceid
  • YAN

1. Introduction

Modern winemaking requires elaborating quality wines, but also beneficial to health. The winemaking industry, to be competitive, has to develop and to take new processes that allow to get these requirements maintaining the sensory quality at the highest possible level. Therefore, it is particularly important to have new technologies that allow to improve the processes and to optimize the quality. Pulsed electric field technology (PEF) is considered one of the non-thermal methods for inactivating microorganisms in foods and also enhances mass transfer by electroporation of the cytoplasmatic membranes [1, 2]. In this way, in the extraction of grape bioactive components, some studies have been carried out on the effect of this technology on the extraction of several compounds, most of which are focused on the study of phenolic compounds [3, 4]. In addition, studies using continuous semi-industrial systems are scarce [3, 4, 5]. The three families of compounds that most influence grape quality are: volatile compounds, phenolic compounds, and nitrogenous compounds. Grape volatile composition is one of the key parameters determining must and wine quality [6].

These volatile compounds are located in the grape both in the pulp and in the skin and depend fundamentally on the variety, cultural practices, soil type, and geographical place [7, 8]. They are responsible for the varietal or primary wine aroma and are composed of several hundreds of compounds of different chemical groups integrated as monoterpenoids, C13 norisoprenoids, and benzenoid compounds from the aroma of the grape [9, 10, 11]. Two groups of most odoriferous which give floral aroma are monoterpenoids and C13 norisoprenoids [12].

On the other hand, C6 compounds are the major group of volatile compounds formed in the pre-fermentative stage, and they can have a negative effect on wine quality due to their herbaceous odors [13]. Resveratrol is a stilbene that has been the most widely studied phenolic compound due to its beneficial properties attributed to it, such as cardioprotective capacity, antioxidant, anticancer, antidiabetic, neuroprotective, and anti-aging activities [14, 15].

In nature, resveratrol can be found in two isomeric forms, cis and trans. Also, the glycosylated form, known as piceid, is the most abundant [16, 17].

Nitrogen compounds are quantitatively the second most abundant compounds in grapes, after sugars. This fraction is present in different forms, ammonium, amino acids, peptides, and proteins [18]. The quantity and quality of these compounds, mainly ammonium and amino acids, determine the growth of yeast and the fermentation rate [19, 20, 21, 22, 23]. Moreover, esters, higher alcohols, volatile fatty acids, and carbonyls are important contributors to the wine fermentation bouquet [24]. These compounds principally arise as metabolites of yeast sugar and amino acids [25], and their formation is affected by the nitrogen compounds present in the initial must [26, 27]. Therefore, the study of the amino acids content of the grape juice is relevant to estimating the aromatic profile of wine [28].

For these reasons, this work aimed to study the composition of must and wine from grapes treated by different PEF conditions using a continuous system of pilot scale.


2. Materials and methods

2.1 Graciano, Tempranillo, and Grenache grape samples

The study was carried out with three red grape varieties from the D.O.Ca. Rioja: Graciano, Tempranillo, and Grenache. A total of 400 kg of grapes of each variety were harvested at their optimum technology maturity. Then grapes were processed as in industry, were destemmed, crushed, and sulphited with 70 mg/kg S02. A total 10 stainless steel vats were filled with 25 L of the must with their skins; 2 vats were used for each PEF treatment applied (4 treatments); and 2 vats were used for untreated samples.

2.2 Pulsed electric fields (PEF) extraction treatments

PEF extraction treatments were exposed in López-Giral et al [3]. The PEF equipment used was an ELCRACK-HVP5 unit (DIL, Germany) with a co-linear PEF treatment chamber ELCRACK DN25 of 2.50 cm of diameter and 2.38 cm distance between electrodes (4.45 cm2 of electrode area). Pulses of 7.4 kV/cm were applied with frequencies of 300 and 400 Hz and a pulse width of 10 and 20 μs. Denomination of PEF treatments was:

Treat1 (10 μs–300 Hz), Treat2 (10 μs–400 Hz), Treat3 (20 μs–300 Hz), and Treat4 (20 μs–400 Hz).

The crushed grapes from each variety were pumped with a membrane pump (PV8 Saniflo, Wilden, USA) to the PEF treatment chamber.

After treatments, the samples were collected in a stainless steel vat. After 6 h skins and seeds were separated of the must in all samples. Then, the pomace was pressed with a little water press. Aliquots of each sample (control and Treat1–4) were frozen in order to subsequently analyze their aromatic, phenolic, and nitrogen composition.

2.3 Determination of grape volatile compounds by HS-SPME-GC-MS

The grape volatile composition was analyzed according to the methodology exposed by Garde-Cerdán et al. [2]. The SPME fiber used was divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS, 50/30 μm) (Supelco, Bellenfonte, USA). Fibers were thermally conditioned (270°C, 60 min). A total 2 g of NaCl were added to 12 ml of sample into a 20 ml vial for the extraction of volatiles from the different samples. Samples were conditioned for 15 min/60°C with stirring. Subsequently, the extraction was performed for 105 min at this temperature. The SPME fiber containing the volatile compounds was placed in the SPME holder (Supelco) and was manually introduced into the GC injection port at 250 °C (equipped with a glass liner, 0.75 mm I.D. (Supelco), and ThermogreenTM LB-2 septum (Supelco)) and kept during 15 min for desorption. A blank test was carried out to check possible carry-over. The desorbed compounds were separated in an Agilent (Palo Alto, USA) gas chromatograph system (GC) coupled to a mass spectrometric detector (MS) equipped with a SPBTM-20 fused silica capillary column (30 m × 0.25 mm I.D. × 0.25 μm film thickness) (Supelco). Carrier gas used was helium (purity = 99.999%; 1.2 ml/min). The injections were performed in splitless mode (1 min). The program to separate volatile compounds consisted of an initial oven temperature of 40°C for 5 min, a temperature gradient of 2°C/min to a final temperature of 220°C, and a final time of 20 min (total run time = 115 min). The acquisitions were performed in Full Scan (35–300 m/z). NIST library was used for identification by comparison with the mass spectrum and retention index of chromatographic standards. The GC peak area of each compound was obtained from the ion extraction chromatogram by selecting target ions for each one. The analyses were done in duplicate. Hence, the results of volatile compounds correspond to the average of four analyses (n = 4).

2.4 Analysis of grape stilbenes by SPE-HPLC

The determination of these phenolic compounds was performed by the method described by Garde-Cerdán et al. [29]. Briefly, to carry out the extraction of stilbenes from must samples, a Discovery® DCS-18 (100 mg/1 ml) cartridge (Sigma-Aldrich, Madrid, Spain) was employed. The cartridge was conditioned by rinsing with 4 ml of methanol, followed by 4 ml of water. An amount of 10 ml of sample (centrifuged at 10,000 rpm for 10 min) was passed through the solid phase extraction (SPE) cartridge. Then, a washing step was carried out with three fractions of water. Then, 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 to a proportion of 60:40 (v/v) of methanol/water. The final sample was injected into the HPLC system. Stilbenes were analyzed by reverse-phase HPLC using a liquid chromatograph Agilent 1100 Series. The injected amount was 30 μl and the column temperature was 25 °C. All separations were performed on a ZORBAX Eclipse Plus C18 (150 × 3.0 mm, I.D. 3.5 μm) column (Agilent) with pre-column Eclipse XDB-C18 (12.5 × 4.6 mm, I.D. 5 μm). Three eluents were used as mobile phases: eluent A: water, and acetic acid (98:2, v/v); eluent B: water, acetic acid, and acetonitrile (78:2:20, v/v/v); and eluent C: methanol. The flow rate was 0.9 ml/min. Detection was performed by a fluorescence detector (FLD) and a diode array detector (DAD). The target compounds were identified according to the retention times and UV-Vis spectral characteristics of corresponding standards (Sigma-Aldrich). Quantification was done using the calibration graphs of the respective standards. The SPE-HPLC determinations were carried out in duplicate and, as treatments were performed in duplicate, the results for stilbenes correspond to the mean of four analyses (n = 4).

2.5 Grape amino acids determination by HPLC

The samples amino acids analysis was performed by the method described by Garde-Cerdán et al. [30]. Free amino acids were analyzed using the same HPLC equipment as for stilbenes determination, using both detectors (FLD and DAD). The pure reference compounds and internal standards were obtained from Sigma-Aldrich.Samples were centrifugated at 4.000 rpm/10 min/20 °C. Then, 5 ml of sample was mixed with 100 μl of norvaline and 100 μl of sarcosine (internal standards) and filtered through a 0.45 μm OlimPeak filter (Teknokroma, Barcelona, Spain). Afterward, samples were submitted to automatic precolumn derivatization with o-phthaldialdehyde (OPA Reagent, Agilent) and with 9-fluorenylmethylchloroformate (FMOC Reagent, Agilent). 10 μl at 40°C were injected from the derivatized samples. All separations were made on a Hypersil ODS (250 × 4.0 mm, I.D. 5 μm) column (Agilent).

Eluents used as a mobile phases were: eluent A: 75 mM sodium acetate, 0.018% triethylamine (pH 6.9) + 0.3% tetrahydrofuran; eluent B: water, methanol, and acetonitrile (10:45:45, v/v/v).

Identification of compounds was performed by comparison of their retention times with their pure reference standards. Also the quantification of different amino acids was made by preparing solutions of reference compounds and internal standards in HCl at 0.1 N in the range of the amino acid concentrations usually found in musts Yeast assimilable nitrogen (YAN), was determined according to the method described by Aerny [31]. The results for amino acids and YAN correspond to the mean of four analyses (n = 4) because treatments were performed in duplicate and also analyses of them were carried out in duplicate.

2.6 Statistical analysis

Data management and analysis were performed using SPSS 21.0 (Chicago, USA). ANOVA was used to compare the volatile, phenolic, and nitrogen compounds data. Results were expressed as means ± standard deviation. A p-value ≤ 0.05 was considered significant (Tukey test). In figures all parameters are listed with their standard deviation. In figures for each grape variety, different letters indicate significant differences. Discriminant analyses were done with the volatile compounds areas and stilbenes and amino acids concentration in the different samples.


3. Results and discussion

3.1 Effect of PEF treatments on volatile composition of Graciano, Tempranillo, and Grenache samples

3.1.1 Monoterpenoids

Monoterpenoids play a significant role in the wine varietal aroma, contributing to its floral and citrus character [12]. The results of monoterpenoids in the control and the samples after each of the four treatments by PEF for the three grape varieties are shown in Figure 1. For Graciano, the content of most of monoterpenoids and the total monoterpenoids was higher in control and treatment with the highest energy (Treat4) than in PEF treatments at low energies (Treat1-3). There was an exception in the case of α-terpineol, citronellol, and p-cymene, which were not affected by any of the treatments applied to the Graciano variety, except for citronellol in Treat1. In the case of Tempranillo, total monoterpenoids were higher in the treated samples at low energies (Treat1-3). However, when considered individually, these compounds showed no clear trend. In the case of citronellol, nerol, and geraniol, Treat 2 and 3 favored their presence (Figure 1d, e, and g). In the case of linalool, neral, geranyl acetone, and γ-geraniol, no significant differences were observed when comparing control with treated samples. Regarding α-terpineol, its presence in the control was higher than in samples treated with Treat1, 3, and 4 (Figure 1c). Moreover, p-cymene decreased its amount in the samples with Treat2, 3, and 4 (Figure 1k). On the other hand, the presence of total monoterpenoids for Grenache was higher in the samples treated by PEF than in the control one (Figure 1a). The same behavior was observed for linalool, citronellol, nerol, geraniol, isogeraniol, and γ -geraniol, with the exception of Treat3 for linalool and Treat2 for isogeraniol, which showed no differences with respect to the control. These results do not match with those obtained by Maza et al. [32], which did not find an increment in the concentration of monoterpenoids on wines of the Grenache variety by application of a PEF treatment. On the other hand, Comuzzo et al. [33] also observed that the concentration of geraniol, significantly, increased after PEF pre-treatment of white grapes of the Garganega variety. Comparing the control must samples with the samples from PEF treatments, Treat1-3 decreased the total monoterpenoids in Graciano by approximately 18% and increased it by about 16% in Tempranillo. PEF increased total monoterpenoids by approximately 50% in Grenache, regardless of the treatment intensity. The overall proportion of monoterpenoids with regard to the total amount of volatile compounds in untreated must samples was of 1.4% in Graciano, 0.9% in Tempranillo, and 4.2% in Grenache. The relative proportion of these compounds in the treated must samples was similar to the control for each of the three grape varieties. Hence it can be said that the effect of PEF technology on monoterpenoids was not selective, as observed by Puértolas et al. [34] and López et al. [35] for phenolic compounds. Monoterpenoids are mainly found in skin, although their distribution between pulp and skin depends on the grape variety and compound [36]. The results obtained suggest that monoterpenoids present in the pomace of Grenache were extracted more easily by applying PEF than those present in Tempranillo and Graciano skin because, in this work, PEF application was made in the presence of skin. Other authors have reported that phenolic compounds extraction from grape varieties depends on the morphology and skin composition [37]. Probably, for this reason, a significant increase in monoterpenoids in presence of skin was found in Grenache after PEF treatments. In this regard, Delsart et al. [38] studied two PEF treatments (4 kV/cm; 1 ms and 0.7 kV/cm; 200 ms) on Cabernet Sauvignon grapes. The first PEF treatment, had an impact mainly on vacuolar tannins, whereas the second PEF treatment had a greater impact on the parietal tannins and the skin cell walls. This enhanced polyphenol extraction kinetics. That means the degree of compound extraction from the skin is not only influenced by the grape variety but also by the type of pulse applied.

Figure 1.

Monoterpenoids average area in control and PEF treatments 1–4 in Graciano (Gr), Tempranillo (T), and Grenache (G) grape varieties (From Garde-Cerdán et al. [5]).

3.1.2 C13 norisoprenoids

Figure 2 shows the results of C13 norisoprenoids in the control and the samples after each of the four treatments by PEF for the three grape varieties [5]. In the case of Graciano, the total C13 norisoprenoids and (E)-β-damascenone decreased with the lowest energy treatment (Treat1). However, their presence was maintained after treating the samples at higher energy (Treat2–4). The presence of methyl jasmonate decreased by PEF application, except with Treat3 (Figure 2f). The (Z)-β-damascenone, β-ionone, and β-cyclocitral contents were not affected by any of the treatments applied to Graciano. For Tempranillo, PEF technology was detrimental to the total content of C13 norisoprenoids, and the two isomers of β-damascenone (Figures 2ac). The PEF treatments also decreased the presence of β-ionone upon Treat1 and 4 (Figure 2d) but they did not influence the β-cyclocitral and methyl jasmonate amount. For Grenache, only β-ionone and β-cyclocitral were affected by PEF treatments. In general, PEF favored their presence in the musts with the exception of Treat2 and 4 for the β-ionone and Treat2 for the β-cyclocitral (Figure 2de). These results are in agreement with those obtained by Maza et al. [32], which observed that the concentration of β-ionone, associated with the floral aroma of “violets”, which had gone undetected in the control wines, was indeed observed at concentrations greatly exceeding the odor threshold in the wines obtained from Grenache grapes treated by PEF.

Figure 2.

C13 norisoprenoids average area in control and PEF treatments 1–4 in Graciano (Gr), Tempranillo (T), and Grenache (G) grape varieties (From Garde-Cerdán et al. [5]).

Treat1 decreased the presence of total C13 norisoprenoids in Graciano by 28%, whereas, in the case of Tempranillo, all the treatments reduced the presence of these compounds by around 37%. However, in Grenache, there was no effect. C13 norisoprenoids are distributed in both pulp and skin, unlike monoterpenoids, which are predominantly present in the skin and it could be the cause that no increase was observed with PEF treatments.

In contrast to these results, Comuzzo et al. [11] observed that PEF processing of white grapes (cv. Garganega) after crushing, significantly increased the concentration of norisoprenoid glycosides in the juice of this white grape variety. In control samples, the total concentration of C13 norisoprenoids in Graciano, Tempranillo, and Grenache respectively was 7.3%, 2.0%, and 3.2%. The two compounds most abundant in all varieties were β-damascenone (both isomers) and β-ionone. β-Damascenone sum was 97% in Graciano, 92% in Tempranillo, and 98% in Grenache, while the proportion of β-ionone was 1.9% in Graciano, 5.2% in Tempranillo, and 1.3% in Grenache. These proportions varied little upon PEF treatments.

PEF effect was not selective for norisoprenoids. β-Damascenone and β-ionone.

3.1.3 Benzenoid compounds

Figure 3 shows the results for benzenoid compounds in the control and in the samples after each of the four treatments by PEF for the three grape varieties [5]. Benzenoid compounds, particularly, 2-phenylethanol and eugenol, confer a desirable aroma to the wine, with rose and clove aroma descriptors [17, 39, 40].

Figure 3.

Benzenoid compounds average area in control and PEF treatments 1–4 in Graciano (Gr), Tempranillo (T), and Grenache (G) grape varieties (From Garde-Cerdán et al. [5]).

In the case of Graciano, treatments had neither effect on total benzenoids nor benzyl alcohol. The content of 2-phenylethanol increased with Treat1, while eugenol was only detected in this grape variety, showing that Treat4 favored its presence. For the Tempranillo variety, Treat1 and 4 resulted in a decrease in the presence of total benzenoids and 2-phenylethanol. On the other hand, benzyl alcohol was found in higher amounts in grapes treated with Treat2. For Grenache, PEF favored the presence of total benzenoids and 2-phenylethanol regardless of the treatment, and Treat1 increased the presence of benzyl alcohol with respect to the control samples. By contrast, Comuzzo et al. [33] observed a slightly decreased of 2-phenylethanol in white wines obtained by PEF processing, but this appears to have a notably low potential impact on sensory perception. By comparing the content of total benzenoids, no loss nor gain was observed upon Graciano samples treated by PEF treatments.

However, in Tempranillo, Treat1 and 4 decreased the presence of these compounds by 24%. For Grenache, all treatments, except Treat2, increased their presence by 45%. The extraction of benzenoid compounds was increased in Grenache by PEF treatments.

3.1.4 Esters

The results of esters in the control and the samples after each of the four treatments by PEF for the three varieties are shown in Figure 4. In the case of Graciano, only Treat1 favored the presence of total esters and hexyl acetate. The four PEF treatments resulted in a decrease in 2-hexen-1-ol acetate; and the presence of methyl salicylate was enhanced by Treat2 and 4. For Tempranillo, the treatments applied had no effect on the presence of esters, except for total esters and hexyl acetate in Treat3, which favored its presence; and methyl hexanoate in Treat1-3. For the latter compound, Treat1 decreased its amount, while Treat2 and 3 increased its content in the musts. On the other hand, the presence of total esters for Grenache grape variety was favored by Treat2-4. Moreover, all treatments favored the presence of methyl hexanoate, and an increase for methyl salicylate in the samples was obtained with Treat3 and 4. Maza et al. [32] obtained different results since they did not observe an increment in the concentration of total esters in wines of the Grenache grape variety by application of a PEF treatment. Esters are mainly formed during alcoholic fermentation and play an important role in wine aroma [41]. In control samples, the proportion of total esters with regard to the total amount of volatile compounds was 0.9% in Graciano, 1.1% in Tempranillo, and 0.4% in Grenache. By comparing the contents of these compounds, it can be observed that the application in the Graciano grape variety of Treat1 increased the content of esters by approximately 62%, while Treat3 resulted in a decrease of 23%. In Tempranillo, the application of Treat3 resulted in an increase of these compounds by 29%. Finally, Treat2-4 improved its presence in Grenache by about 32%. Differences depending on grape variety were also found in the study of Fauster et al. [42], where the effects of a PEF treatment on white wine mash were significantly higher for the wines obtained from Traminer variety than those from Grüner Veltliner.

Figure 4.

Esters average area in control and PEF treatments 1–4 in Graciano (Gr), Tempranillo (T), and Grenache (G) grape varieties (From Garde-Cerdán et al. [5]).

3.1.5 C6 compounds

Figure 5 shows the results for C6 compounds in the control and the samples after each of the four treatments by PEF for the three grape varieties [5]. When these compounds are at low levels contribute positively to wine aroma; while, at high levels, they are responsible for herbaceous flavors [33].

Figure 5.

C6 compounds average area in control and PEF treatments 1–4 in Graciano (Gr), Tempranillo (T), and Grenache (G) grape varieties (From Garde-Cerdán et al. [5]).

The presence of (E)-2-hexen-1-ol in must samples of Treat1 of Graciano was diminished. However, Treat2 in Tempranillo enhanced the content of total C6 compounds, n-hexanol, (Z)-3-hexen-1-ol, and (E)-2-hexenal; while Treat1 decreased the content of hexanal. Treat1 in Grenache favored the presence of total C6 compounds and (Z)-3-hexen-1-ol. Also Treat1 and 3 enhanced the content of hexanal, and Treat2 favored the presence of (E)-2-hexenal. In general, and matching with the Comuzzo et al. [33] results, the PEF treatments hardly affected the amounts of C6 compounds.The PEF samples increased of total C6 compounds in 72% after Treat1 in Grenache and 31% upon Treat2 in Tempranillo. On the other hand, the most abundant C6 compounds were in control samples accounting for 87%, 95%, and 91% in Graciano, Tempranillo, and Grenache respectively. The PEF treatments did not affect the relative abundance of C6 compounds in must samples.

3.2 Influence of PEF treatments on resveratrol and piceid content in Graciano, Tempranillo, and Grenache samples

The concentration of total stilbenes, trans-resveratrol, trans- and cis-piceid from each of the three grape varieties in the control and the four PEF treatments tested were shown in Figure 6. Among the three grape varieties treated, Graciano presented the highest total stilbenes concentration (Figure 6a), is the only one in which trans-resveratrol was detected (Figure 6b). For its part, cis-resveratrol was not detected in any of the samples because it is usually absent in grapes [43]. The total stilbene content in the PEF treated Tempranillo samples increased more (up to 200%, ranging from 90% to 200%) with respect to the untreated samples that both, the Graciano (40% for Treat2 and Treat3, and 60% for Treat4) and Grenache (50% with the Treat4) samples. Thus, the Treat4, the one with the highest time and energy, was the most effective on the total stilbene extraction for the three grape varieties (Figure 6a). Therefore, as the phenolic compounds are found mainly in the grape skins, the application of PEF treatments increased their extraction although the efficiency and the effect of this PEF on the stilbenes depend on the grape variety applied, which plays an important role in the grapes stilbene concentration [44]. The piceids, which are extracted from the grape skins before extracting the aglycones, resveratrols since the presence of ethanol is required to increase their solubility and mobility [45], were in higher concentration (Figure 6c and d) than resveratrol (Figure 6b). This matched with those results reported by Romero-Pérez et al. [46]. Besides, trans-piceid was the most abundant stilbene in the three cultivars, improving its extraction in Tempranillo with all the PEF applied treatments, while in the case of the Graciano and Grenache grape varieties, increases its content only after applying the highest energy and timing treatment (Treat4) (Figure 6c). trans-Piceid is the precursor of trans-resveratrol, in which content did not increase in Graciano grapes with the application of PEF treatments (Figure 6b). On the other hand, Treat3 and Treat4 increased the cis-piceid extraction in Graciano (Figure 6d), meanwhile, as observed with the trans-isomer for the Tempranillo grape variety, the cis-piceid extraction increased with all the PEF treatments for this grape variety. Regarding the effects of the PEF on Grenache cis-piceid content, no one change was observed (Figure 6d). Similar as what occurs with the trans isomer, cis-piceid is the precursor of cis-resveratrol by hydrolysis during fermentation. Besides, the final concentration of these compounds is conditioned by the reaction of trans-cis isomerization, especially in relation to the aglycons, due to the instability of the cis isomer, so the trans is the highest and most stable isomer [17].

Figure 6.

Stilbene concentration (mg/l) in control and PEF treatments 1–4 in Graciano (Gr), Tempranillo (T), and Grenache (G) grape varieties (From López-Alfaro et al. [4]).

3.3 Effect of PEF treatments on vnitrogen compounds of Graciano, Tempranillo, and Grenache samples

The content of total amino acids, total amino acids without proline, and YAN from each of the three grape varieties in the control and the four PEF treatments tested were shown in Figure 7. The effect of PEF treatments in the extraction of nitrogen compounds was different between the three grape varieties. Control samples of Graciano only presented significant differences of total amino acids with samples of Treat1 (lower than control) (Figure 7a). However, in Tempranillo, samples of control, Treat1 and 3 showed no significant differences between them, with higher amino acids content only in samples treated with Treat2. In the Grenache grape variety, the total amino acids content in all PEF samples was significantly different and higher than the control ones. Samples with the highest total amino acids content were those treated with Treat3 and 4 for this grape variety. In the case of the total amino acids without proline (Figure 7b), no differences were observed between control and PEF treated samples in Graciano. In Tempranillo, only samples of Treat2 showed positive differences with regard to control ones. Meanwhile, in Grenache, all the PEF treated samples obtained significantly higher values of total amino acids without proline than the control, being Treat3 the best treatment, matching with that observed for total amino acids content. The ratio of total amino acids without proline with regard to total amino acids (in %) was dependent on the grape variety. Grenache was the variety most benefited by the application of PEF treatments, being all the PEF treatments applied significantly advantageous with respect to not applying them. The percentage of improvement of the Grenache samples treated by PEF with respect to the control samples ranged between 15% and 22% for the total amino acids and from 16 to 33% for the total amino acids without proline. Regarding the effect in of PEF treatments on YAN content, in Graciano the YAN content was around 200 mg N/l, being the samples of Treat1 the ones with significantly lower YAN content (Figure 7c). In Tempranilllo, all samples presented YAN values between 140 and 162 mg N/l, being only in the samples from Treat2 and 4 superiors to 150 mg N/l, which is the minimum value to achieve the correct development of the fermentation, according to Blouin and Peynaud [47] and Bell and Henschke [48]. Tempranillo samples treated with Treat1 presented the lowest YAN content and it was significantly different from the rest of the treatments. In Grenache, samples treated by PEF did not show any differences in the YAN content between them with values around 292 mg N/l. However, Grenache control samples had less YAN content (266 mg N/l) than the PEF treated samples. YAN is composed of ammonium ions and free amino nitrogen. These compounds are the main sources of nitrogen for Saccharomyces cerevisiae and their content affects the kinetics of fermentation [49, 50]. Figure 8 shows the free amino acids found in Graciano, Tempranillo, and Grenache musts from control and PEF treatments (Treats 1-4). Proline (Figure 8t) and arginine (Figure 8i) were the two majority amino acids in grape musts. Graciano was the grape variety with more proline, with contents of about 570–840 mg/l, and Grenache was the variety with less proline, with contents of about 280–420 mg/l. With respect to the arginine content, Grenache had more content than Tempranillo and Graciano, with values about 190–315 mg/l, 120–155 mg/l, and 140–230 mg/l, respectively. The characteristic index based on the ratio of the proline and arginine is used to indicate the differential accumulation of these amino acids by different grape cultivars. The proportion of non-assimilable (proline) to assimilable nitrogen (arginine) is indicated by this index, which provides a useful indication of the likely nutritional value for yeast metabolism of the grape must [27]. Regarding the effects of the PEF treatments on the proline and arginine content, in Graciano, there were significant differences of proline content between samples of Treat4, which had the highest values, and the rest of the treatments. For arginine, the highest content was observed for the samples treated by PEF with the Treat2, and the lowest content was for Treat4 samples. In Tempranillo, samples of Treat1 had significantly less content of proline than the rest of the samples, except Treat3, among which there were no differences. On the other hand, all Tempranillo PEF treated samples were richer in arginine content than the control one, being those of Treat1 and 4, the ones with more content of this amino acid. In Grenache, proline content of Treat1 and 4 was higher than control samples and for arginine concentration, content in Treat2 and 3 were higher with respect to control ones. The highest arginine/proline ratio was observed in the Grenache variety with values between 0.57 and 0.98. The highest value of this ratio was for Treat3 samples. Values of this ratio for Graciano were between 0.19 and 0.35 and the best ratio was for Treat2 samples. Values of this ratio for Tempranillo were between 0.27 and 0.34 and the best ratio was for Treat1 samples. Thus, as the proline to arginine ratio was lower than 1 for the three grape varieties, that suggested that all red varieties are proline accumulators, as reported by authors as Garde-Cerdán et al. [50], and Pérez-Álvarez et al. [51] for cv. Tempranillo, and that the proline to arginine ratio is influenced by nitrogen nutrition [52] and depends on the variety [48]. Amino acids concentration at harvest depends on climatic conditions and agronomic practices, while amino acid profile mainly depends on variety and zone [53, 54] and also the maturity. For example, Grenache at an early stage of maturity (19.7 °Brix) was classified as a high arginine accumulator, but by 24 °Brix it accumulated predominantly more proline than arginine [55]. In this study, Grenache with 24 °Brix, also presented more content of proline than arginine. Leaving aside the proline and arginine which have already been said were the majority amino acids in samples, in Graciano, the four amino acids with more content, from highest to lowest content, were alanine, glutamic acid, serine, and threonine, all of them with contents from 60 mg/l to 156 mg/l. In Tempranillo, glutamic acid, alanine, serine and histidine, with contents between 35 and 85 mg/l, and in Grenache, glutamic acid, histidine, alanine, and threonine, with contents from 48 to 172 mg/l were the amino acids majority. Among the sources that produce rapid yeast growth are amino acids such as glutamine, asparagine, glutamic acid, and alanine. These produce carbon derivatives, which are rapidly integrated into the fermentative metabolism of yeasts. In this study, except for asparagine, whose concentrations were low (except in Grenache, with a content ranging between 40 and 50 mg/l), both, alanine and glutamic acid were between the first and third place of the amino acids in the three grape varieties (without taking into account proline and arginine). On the other hand, among the sources that produce lower growth are the aromatic amino acids (tryptophan, tyrosine, and phenylalanine) and branched (leucine, isoleucine, and valine) that produce ketoacid complexes, which must be converted into aldehyde complexes and higher alcohols for their elimination [56]. In the case of these latter groups of amino acids, the fact of producing carbon skeletons that are not easily assimilated also makes these amino acids the most interesting in the production of aromas [57]. In this study, the concentration of these amino acids was low for the three grape varieties: so, their content was lower than 20 mg/l, 31 mg/l, and 40 mg/l in Graciano, Tempranillo, and Grenache must samples, respectively. Threonine, methionine, and serine are also considered aroma precursors. The concentrations of some of the volatiles correlated well with the aromatic composition of the equivalent wines. Development of models by chemometric analysis showed that threonine and serine affected corresponding fatty acid esters and alcohols, and methionine strongly affected methionol concentration [48]. In this study, threonine content in Graciano ranged between 60 and 85 mg/l, in Grenache around 50 mg/l, and Tempranillo between 25 and 35 mg/l. For methionine, the variety with the highest content was Grenache with values around 15 mg/l. Serine content in Graciano, Tempranillo, and Grenache was higher than 60, 35, and 40 mg/l, respectively. These amino acids are very important because higher alcohols come from them directly and some esters indirectly, as they come from these higher alcohols. For example, n-propanol comes from threonine, 2-methyl-1-butanol from isoleucine, 3-methyl-1-butanol from leucine, isobutanol from valine, 2-phenylethanol from phenylalanine, and methionol from methionine [58]. With respect to the effect of the four PEF treatments on the content of amino acids in the musts of the three grape varieties, the results were very dependent on the grape variety. In PEF treated samples of Graciano, only threonine content (Figure 8g) in Treat3 samples was superior to control ones. In Tempranillo, the amino acids contents of the Treat2 samples were higher than the control one (for aspartic acid, asparagine histidine, threonine, tyrosine, valine, leucine, phenylalanine, and lysine). No differences or even reduction of some amino acids contents were produced by Treat1, 3, and 4. In some cases, positive differences were obtained with Treat4 respect to the control. In Grenache, in general, all the samples treated by PEF showed higher or equal amino acids contents than the control ones (Figure 8).

Figure 7.

Total amino acids (mg/l), total amino acids without proline (mg/l) and yeast assimilable nitrogen (YAN) (mg N/l) in Graciano (Gr), Tempranillo (T), and Grenache (G) grapes for control and PEF treatments (Treats 1–4).

Figure 8.

Free amino acids concentration (mg/l) in Graciano (Gr), Tempranillo (T), and Grenache (G) grapes for control and PEF treatments (Treats 1–4).


4. Conclusions

The technology of pulsed electric fields (PEF) affected the volatile composition of Graciano, Tempranillo, and Grenache depending on the grape variety. However, the flavor profile of the samples was not affected in any case. The volatile composition of grape juice was enhanced by PEF application in Grenache, without significant improvement in Graciano and Tempranillo. However, the highest energy PEF treatment improved the stilbene total content in musts from the three grape varieties in an important way. Moreover, the four PEF treatments enhanced YAN and the total amino acids content in Grenache, while for Graciano and Tempranillo grape varieties, the PEF treatments hardly influenced the grape nitrogen composition. In conclusion, PEF is a good tool in order to improve the quality of the Grenache grape variety.



This research was funded by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) to project RTA201100070-00-00 and the Navarra Government to project IIQ14037.RI1.


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Written By

Teresa Garde-Cerdán, Eva P. Pérez-Álvarez, Pilar Rubio-Bretón and Noelia López-Giral

Submitted: 23 February 2022 Reviewed: 22 March 2022 Published: 27 May 2022