**6. A case study - red wine vinification**

A promising application of PEF pretreatment of vegetable tissue is in the vinification process of red wine. Grapes contain large amounts of different phenolic compounds, especially located in the skin, that are only partially extracted during traditional winemaking process, due to the resistances to mass transfer of cell walls and cytoplasmatic membranes. In red wine, the main phenolic compounds are anthocyanins, responsible of the color of red wine, tannins and their polymers, that instead give the bitterness and astringency to the wines (Monagas et al., 2005). In addition, polyphenolic compounds also contribute to the health beneficial properties of the wine, related to their antioxidant and free radical-scavenging properties (Nichenametla et al., 2006).

The phenolic content and composition of wines depends on the initial content in grapes, which is a function of variety and cultivation factors (Jones and Davis, 2000), but also on the winemaking techniques (Monagas et al., 2005). For instance, increasing fermentation temperature, thermovinification and use of maceration enzymes can enhance the extraction of phenolic compounds through the degradation or permeabilization of the grape skin cells (Lopez et al., 2008b). Nevertheless, permeabilization techniques suffer from some drawbacks, such as higher energetic costs and lower stability of valuable compounds at higher temperature (thermovinification), or the introduction of extraneous compounds and

Mass Transfer Enhancement by Means of Electroporation 169

This is particularly evident in Fig. 12, where the kinetic constant *kd* (Fig. 12a) and the equilibrium concentration *y∞* (Fig. 12b) are reported as a function of the total specific energy delivered by the PEF treatment. While both *kd* and *y∞* increased for Aglianico grapes at increasing the specific energy, for Piedirosso the estimated values of both *kd* and *y<sup>∞</sup>* remained constant and independent on the PEF treatments. This is even more remarkable if considering that PEF treatments, under the same operative conditions, caused a significant increase of the permeabilization index *Zp* on both grape varieties, as shown in Fig. 12c. In particular, for a total specific energy *WT*> 10 kJ/kg a complete permeabilization (*Zp* ≈ 1) was obtained for Piedirosso and an almost complete permeabilization for Aglianico (*Zp* ≈ 0.8).

0 5 10 15 20 25

a

b

c

0 5 10 15 20 25

Wt (kJ/kg) 0 5 10 15 20 25

Fig. 12. Kinetic constant *kd* (a), equilibrium polyphenolic concentration *y∞* (b) estimated through Eq. 15 from maceration data and permeabilization index *Zp* (c) of different untreated and PEF-treated grape varieties, Aglianico and Piedirosso (Donsì et al., 2010a).

*kd* (d-1)

0.4 0.6 0.8 1.0 1.2 1.4 1.6

*y*

*∞*

*Zp*

0.0 0.2 0.4 0.6 0.8 1.0 Aglianico Piedirosso

general worsening of the wine quality (Spranger et al., 2004). Therefore, PEF treatment may represent a viable option for enhancing the extraction of phenolic compounds from skin cells during maceration steps, without altering wine quality and with moderate energy consumption.

From a technological prospective, great interest was recently focused on the application of PEF for the permeabilization of the grape skins prior to maceration. The enhancement of the rate of release of phenolic compounds during maceration offers several advantages. In case of red wines obtained from grapes poor in polyphenols, it can avoid blending with other grape varieties richer in phenolic compounds, or use of enzymes. Moreover, it can reduce significantly the maceration times (Donsì et al., 2010a; Donsì et al., 2010b).

The main effect of PEF treatment of grape skins or grape mash is the increase of color intensity, anthocyanin content and of total polyphenolic index with respect to the control during all the vinification process on different grape varieties (Lopez et al., 2008a; Lopez et al., 2008b; Donsì et al., 2010a). Furthermore, it was reported that PEF did not affect the ratio between the components of the red wine color (tint and yellow, red and blue components) and other wine characteristics such as alcohol content, total acidity, pH, reducing sugar concentration and volatile acidity (Lopez et al., 2008b). In particular, Fig. 11 shows the evolution of total polyphenols concentration in the grape must during the fermentation/maceration stages of two different grape varieties, Aglianico and Piedirosso. Prior to the fermentation/maceration step, the grape skins were treated at different PEF intensities (*E* = 0.5 – 3 kV/cm and total specific energy from 1 to 25 kJ/kg), with their permeabilization being characterized by electrical impedance measurements. Furthermore, the release kinetics of the total polyphenols were characterized during the fermentation/maceration stage by Folin-Ciocalteau colorimetric methods. It is evident that on Aglianico grape variety the PEF treatment caused a significant permeabilization that enhanced the mass transfer rates of polyphenols through the cellular barriers. Moreover, higher intensity of PEF treatment resulted in both faster mass transfer rates and higher final concentration of polyphenols (Fig. 11a). In contrast, the PEF treatment of Piedirosso variety did not result in any effect on the release kinetics of polyphenols, with very slightly differences being observable between untreated and treated grapes (Fig. 11b).

Fig. 11. Evolution over time of total polyphenols concentration in the grape must during fermentation/maceration of two Italian grape varieties: Aglianico (a) and Piedirosso (b) (Donsì et al., 2010a).

general worsening of the wine quality (Spranger et al., 2004). Therefore, PEF treatment may represent a viable option for enhancing the extraction of phenolic compounds from skin cells during maceration steps, without altering wine quality and with moderate energy

From a technological prospective, great interest was recently focused on the application of PEF for the permeabilization of the grape skins prior to maceration. The enhancement of the rate of release of phenolic compounds during maceration offers several advantages. In case of red wines obtained from grapes poor in polyphenols, it can avoid blending with other grape varieties richer in phenolic compounds, or use of enzymes. Moreover, it can reduce

The main effect of PEF treatment of grape skins or grape mash is the increase of color intensity, anthocyanin content and of total polyphenolic index with respect to the control during all the vinification process on different grape varieties (Lopez et al., 2008a; Lopez et al., 2008b; Donsì et al., 2010a). Furthermore, it was reported that PEF did not affect the ratio between the components of the red wine color (tint and yellow, red and blue components) and other wine characteristics such as alcohol content, total acidity, pH, reducing sugar concentration and volatile acidity (Lopez et al., 2008b). In particular, Fig. 11 shows the evolution of total polyphenols concentration in the grape must during the fermentation/maceration stages of two different grape varieties, Aglianico and Piedirosso. Prior to the fermentation/maceration step, the grape skins were treated at different PEF intensities (*E* = 0.5 – 3 kV/cm and total specific energy from 1 to 25 kJ/kg), with their permeabilization being characterized by electrical impedance measurements. Furthermore, the release kinetics of the total polyphenols were characterized during the fermentation/maceration stage by Folin-Ciocalteau colorimetric methods. It is evident that on Aglianico grape variety the PEF treatment caused a significant permeabilization that enhanced the mass transfer rates of polyphenols through the cellular barriers. Moreover, higher intensity of PEF treatment resulted in both faster mass transfer rates and higher final concentration of polyphenols (Fig. 11a). In contrast, the PEF treatment of Piedirosso variety did not result in any effect on the release kinetics of polyphenols, with very slightly

significantly the maceration times (Donsì et al., 2010a; Donsì et al., 2010b).

differences being observable between untreated and treated grapes (Fig. 11b).

t (d) 0 2 4 6 8 10 12

Untreated E=1.5 kV/cm Wt

a b

E=3 kV/cm Wt

E=3 kV/cm Wt

Total polyphenols (g/L)

=10 kJ/kg

Fig. 11. Evolution over time of total polyphenols concentration in the grape must during fermentation/maceration of two Italian grape varieties: Aglianico (a) and Piedirosso (b)

=10 kJ/kg

=20 kJ/kg

0.0

0.5

1.0

1.5

2.0

2.5

t (d) 0123456789

Untreated E=0.5 kV/cm Wt

E=1 kV/cm Wt

E=1 kV/cm Wt

E=1.5 kV/cm Wt

=1 kJ/kg

=10 kJ/kg

=5 kJ/kg

=25 kJ/kg

consumption.

Polyphenols (g/L)

0

(Donsì et al., 2010a).

2

4

6

8

This is particularly evident in Fig. 12, where the kinetic constant *kd* (Fig. 12a) and the equilibrium concentration *y∞* (Fig. 12b) are reported as a function of the total specific energy delivered by the PEF treatment. While both *kd* and *y∞* increased for Aglianico grapes at increasing the specific energy, for Piedirosso the estimated values of both *kd* and *y<sup>∞</sup>* remained constant and independent on the PEF treatments. This is even more remarkable if considering that PEF treatments, under the same operative conditions, caused a significant increase of the permeabilization index *Zp* on both grape varieties, as shown in Fig. 12c. In particular, for a total specific energy *WT*> 10 kJ/kg a complete permeabilization (*Zp* ≈ 1) was obtained for Piedirosso and an almost complete permeabilization for Aglianico (*Zp* ≈ 0.8).

Fig. 12. Kinetic constant *kd* (a), equilibrium polyphenolic concentration *y∞* (b) estimated through Eq. 15 from maceration data and permeabilization index *Zp* (c) of different untreated and PEF-treated grape varieties, Aglianico and Piedirosso (Donsì et al., 2010a).

Mass Transfer Enhancement by Means of Electroporation 171

Assuming that the resistance to mass transfer through the vacuole membrane is the rate determining step, the fact that the mass transfer rates are enhanced only for Aglianico and not for Piedirosso can be explained only inferring that, due to biological differences, the applied PEF treatments were able to permeabilize the vacuole membrane only of Aglianico

In summary, PEF treatments of the grape skins resulted able to affect the content of polyphenols in the wine after maceration, depending on the grape variety. For Piedirosso grapes, the PEF treatment did not increase the release rate of polyphenols. On the other hand, PEF treatment had significant effects on Aglianico grapes, with the most effective PEF treatment inducing, in comparison with the control wine, a 20% increase of the content of polyphenols and a 75% increase of anthocyanins, with a consequent improvement of the color intensity (+20%) and the antioxidant activity of the wine (+20%). Moreover, in comparison with the use of a pectolytic enzyme for membrane permeabilization, the most effective PEF treatment resulted not only in the increase of 15% of the total polyphenols, of 20% of the anthocyanins, of 10% of the color intensity and of 10% of the antioxidant activity, but also in lower operational costs. In fact, the cost for the enzymatic treatment is of about 4 € per ton of grapes (the average cost of the enzyme is about 200 €/kg, and the amount used is 2 g per 100 kg of grapes), while the energy cost for the PEF treatments, calculated as (specific energy)·(treatment time)·(energy cost), was estimated in about 0.8 € per ton of grapes (with the energy costs assumed to be 0.12 €/kWh) in the case of the most effective

PEF technology is likely to support many different mass transfer-based processes in the food industry, directed to enhancing process intensification. In particular, the induction of membrane permeabilization of the cells through PEF offers the potential to effectively enhance mass transfer from vegetable cells, opening the doors to significant energy savings in drying, to increased yields in juice expression, to the recovery of valuable cell metabolites, with functional properties, or even to the functionalization of foods. For instance, PEF treatment of the grape pomaces during vinification can significantly increase the polyphenolic content of the wine, thus improving not only the quality parameters (i.e. color, odor, taste…) but also the health beneficial properties (i.e. antioxidant activity). Furthermore, PEF treatments can also be applied to enhance mass transfer into the food matrices, by permeabilization of the cell membranes and enhanced infusion of functional compounds or antimicrobial into foods, minimally altering their organoleptic attributes. In consideration of the fact that energy requirements for PEF-assisted permeabilization are in the order of about 10 kJ/kg of raw material, it can be concluded that PEF pretreatments can represent an economically viable option to other thermal or chemical permeabilization techniques. However, further research and development activities are still required for the optimization of PEF technology in process intensification, especially in the development of

Ade-Omowaye B.I.O, Angersbach A., Eshtiaghi N.M., Knorr D. (2001). Impact of high

intensity electric field pulses on cell permeabilisation and as pre-processing step in coconut processing. *Innovative Food Science & Emerging Technologies*, 1, 203-209.

industrial-scale generators, capable to provide the required electric field.

grape skin cells and not of Piedirosso grape skin cells.

treatment (Donsì et al., 2010a).

**8. References** 

**7. Conclusions and perspectives** 

Fig. 13, which reports a scheme of a grape skin cell, may help in clarifying the discrepancies observed between measured permeabilization and mass transfer rates in the case of Piedirosso and to explain the mechanisms of PEF-assisted enhancement of polyphenols extraction. Polyphenols and anthocyanins are mainly contained within the vacuoles of the cells, and therefore their extraction encounters two main resistances to mass transfer, which are formed respectively by the vacuole membrane and the cell membrane. PEF treatment causes permanent membrane permeabilization provided that a critical trans-membrane potential is induced across the membrane by the externally applied electric field (Zimmermann, 1986). Since for a given external electric field the trans-membrane potential increases with cell size (Weaver and Chizmadzhev, 1996), the critical value of the external electric field *Ecr* required for membrane permeabilization will be lower for larger systems. Therefore, it can be assumed that the critical electric field for cell membrane permeabilization, *Ecr1*, will be lower than the one for vacuole membrane permeabilization, *Ecr2*. Therefore, in agreement with the reported data, it can be assumed that the applied electric field *E > Ecr1* already at *E* = 1 kV/cm and that the extent of cell membrane permeabilization depends only on the energy input. Whereas, in the case of the vacuole membrane permeabilization, the critical value *Ecr2* is probably in the range of the applied electric field, and the increase of the intensity of *E* (from 0.5 to 3 kV/cm) can also increase the permeabilization of the membrane of smaller vacuoles. For the above reasons, it can be concluded that the permeabilization index *Zp* takes into account the permeabilization of the cell membrane and therefore suggests that cell permeabilization occurred both for Aglianico and Piedirosso grapes.

Fig. 13. Simplified scheme of the effect of PEF treatments with electric field intensity *E* on the structure of a grape skin cell. *Ecr1*: critical electric field for cell membrane permeabilization; *Ecr2*: critical electric field for vacuole membrane permeabilization.

Fig. 13, which reports a scheme of a grape skin cell, may help in clarifying the discrepancies observed between measured permeabilization and mass transfer rates in the case of Piedirosso and to explain the mechanisms of PEF-assisted enhancement of polyphenols extraction. Polyphenols and anthocyanins are mainly contained within the vacuoles of the cells, and therefore their extraction encounters two main resistances to mass transfer, which are formed respectively by the vacuole membrane and the cell membrane. PEF treatment causes permanent membrane permeabilization provided that a critical trans-membrane potential is induced across the membrane by the externally applied electric field (Zimmermann, 1986). Since for a given external electric field the trans-membrane potential increases with cell size (Weaver and Chizmadzhev, 1996), the critical value of the external electric field *Ecr* required for membrane permeabilization will be lower for larger systems. Therefore, it can be assumed that the critical electric field for cell membrane permeabilization, *Ecr1*, will be lower than the one for vacuole membrane permeabilization, *Ecr2*. Therefore, in agreement with the reported data, it can be assumed that the applied electric field *E > Ecr1* already at *E* = 1 kV/cm and that the extent of cell membrane permeabilization depends only on the energy input. Whereas, in the case of the vacuole membrane permeabilization, the critical value *Ecr2* is probably in the range of the applied electric field, and the increase of the intensity of *E* (from 0.5 to 3 kV/cm) can also increase the permeabilization of the membrane of smaller vacuoles. For the above reasons, it can be concluded that the permeabilization index *Zp* takes into account the permeabilization of the cell membrane and therefore suggests that cell permeabilization occurred both for Aglianico

Nucleus Vacuole

Fig. 13. Simplified scheme of the effect of PEF treatments with electric field intensity *E* on

the structure of a grape skin cell. *Ecr1*: critical electric field for cell membrane permeabilization; *Ecr2*: critical electric field for vacuole membrane permeabilization.

*E < Ecr*<sup>1</sup>

*Ecr1 < E < Ecr2*

*E > Ecr2*

Membrane

and Piedirosso grapes.

Assuming that the resistance to mass transfer through the vacuole membrane is the rate determining step, the fact that the mass transfer rates are enhanced only for Aglianico and not for Piedirosso can be explained only inferring that, due to biological differences, the applied PEF treatments were able to permeabilize the vacuole membrane only of Aglianico grape skin cells and not of Piedirosso grape skin cells.

In summary, PEF treatments of the grape skins resulted able to affect the content of polyphenols in the wine after maceration, depending on the grape variety. For Piedirosso grapes, the PEF treatment did not increase the release rate of polyphenols. On the other hand, PEF treatment had significant effects on Aglianico grapes, with the most effective PEF treatment inducing, in comparison with the control wine, a 20% increase of the content of polyphenols and a 75% increase of anthocyanins, with a consequent improvement of the color intensity (+20%) and the antioxidant activity of the wine (+20%). Moreover, in comparison with the use of a pectolytic enzyme for membrane permeabilization, the most effective PEF treatment resulted not only in the increase of 15% of the total polyphenols, of 20% of the anthocyanins, of 10% of the color intensity and of 10% of the antioxidant activity, but also in lower operational costs. In fact, the cost for the enzymatic treatment is of about 4 € per ton of grapes (the average cost of the enzyme is about 200 €/kg, and the amount used is 2 g per 100 kg of grapes), while the energy cost for the PEF treatments, calculated as (specific energy)·(treatment time)·(energy cost), was estimated in about 0.8 € per ton of grapes (with the energy costs assumed to be 0.12 €/kWh) in the case of the most effective treatment (Donsì et al., 2010a).
