Pulsed Electric Fields to Obtain Healthier and Sustainable Food for Tomorrow

Pulsed Electric Fields to Obtain Healthier and Sustainable Food for Tomorrow illustrates innovative applications derived from the use of pulsed electric fields beyond microbial inactivation. The book begins with an introduction on how pulsed electric fields work and then addresses the impact of pulsed electric fields on bioaccessability/bioavailability and the development of nutraceuticals and food additives. Other sections explore the reduction of contaminants and assess the improvement of industrial process efficiency. A final section explores patents and commercial applications.

This book will be a welcomed resource for anyone interested in the technological, physiochemical and nutritional perspectives of product development and the reduction of food toxins and contaminants. The concepts explored in this book could have a profound impact on addressing the concept of "food on demand," a concept that is a top priority in industry.

• Explores how pulsed electric field treatment affects nutrients and the retention of bioactive compounds
• Identifies PEF approaches and optimized, targeted processing conditions to improve food quality, bioavailability and bioaccessibility of nutrients and bioactive compounds
• Highlights the mechanisms influencing the reduction of toxins and contaminants during pulsed electric fields processing
• Explains how pulsed electric fields design can enhance sustainability throughout the food chain

• Language: English
• Publisher: Academic Press
• Release date: Apr 17, 2020
• ISBN: 9780128172643

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Part I

Introduction

Outline

1 How does pulsed electric field work?

2 An overview of the potential applications to produce healthy food products based on pulsed electric field treatment

1

How does pulsed electric field work?

Urszula Tylewicz,    Department of Agricultural and Food Sciences, Alma Mater Studiorum, University of Bologna, Campus of Food Science, Cesena, Italy

Abstract

The electroporation phenomena are related to the cell membrane permeability, which is altered when the biological cells are exposed to external electric fields. This alteration of the cell membrane integrity could lead to the formation of transient or permanent pores, translated into the reversible or irreversible electroporation. Therefore it is of fundamental importance to understand the basic mechanism involved in this process.

This chapter makes a short overview of basics insights of pulsed electric field (PEF) treatment, as well as the immediate consequences of the PEF application on membrane and cellular organization. It gives the main theories of the PEF impact on biological cells induced by local and structural changes of the cell membrane and describes the electroporation phenomenon. Moreover, this chapter describes the key parameters that influence the efficiency of the PEF treatment.

Keywords

Pulsed electric fields; electroporation; membrane permeability; biological tissue; PEF mechanism

1.1 Introduction

The electroporation phenomenon of cell membranes has been known for several decades and the intensive research on pulsed electric field (PEF) technology has been performed as a promising nonthermal technology for both microbial inactivation and mass transfer enhancement (Barba et al., 2015; Donsì, Ferrari, & Pataro, 2010; Tylewicz et al., 2017). Moreover, recently PEF has received increasing attention, because of the possibility for the manipulation of biological cells and tissues (Faurie, Golzio, Phez, Teissié, & Rols, 2005; Gómez Galindo, 2017; Poojary et al., 2017).

The electroporation occurs when the biological cells are exposed to the external electric field in the form of short and intense electric pulses, with the intensity higher than threshold value for the electroporation. Electroporation of the cell membranes could promote transient or permanent pore formation, depending on different PEF parameters applied (electric field strength, pulse duration, pulse number, total time of treatment, etc.) and on the characteristics of the raw materials such as cell size and shape. With low electric field strength a reversible electroporation could be achieved, which means that the created pores reseal after removing the electrical field. This kind of electroporation could be used to incorporate a different functional substance or drugs into the biological tissue, assuring the survival of the electrically stimulated cells. When high electric field strength is used, the irreversible tissue permeabilization (permanent membrane damage) and consequently the cell death occur (Donsì et al., 2010; Weaver & Chizmadzhev, 1996).

There are several theories explaining the mechanism of the reversible electroporation and/or the electrical membrane breakdown; however, the exact mechanism of electroporation is not yet fully understood.

In the first part of the chapter a basic mechanism of electroporation is explained along with the introduction of membrane permeability, transmembrane potential, and the relation between these parameters. In the second part, key parameters that can have an influence on the efficiency of the PEF treatment are described.

1.2 Cell membrane permeabilization

The real mechanism of action of PEF is still not well understood. According to the empirical descriptions of Gossling (1960) and Doevenspeck (1961), there is a disruptive effect of electric fields on biological cells. Subsequently, the theory of dielectric breakdown of cell membranes has been raised by Zimmermann, Pilwat, and Riemann (1974) and Neumann and Rosenheck (1972).

Biological cells, when exposed to external electric fields, revealed specific changes related to membrane permeability. These changes could be associated with the formation of transient pores in the membrane and consequently compromising its semipermeability (Balasa, 2017).

According to Teissie, Golzio, and Rols (2005), the permeabilization of a cell membrane is achieved by five different steps: induction (trigger) (μm), expansion (ms), stabilization (ms), resealing (s), and memory (h). First of all, the formation of pores occurs when the externally applied electric field is above the electroporation threshold value, which is related to the transmembrane voltage. When transmembrane voltage, the sum of induced potential difference across the cell membrane and resting membrane potential, exceeds certain critical value, the electroporation takes place (Kranjc & Miklavčič, 2017).

During the application of PEF on the cell, an induced transmembrane voltage (ΔVi) is created, which is locally associated with the dielectric properties of the plasma membrane. Using a physical model based on a thin, weakly conductive shell (the membrane with the conductivity λm), filled with an internal conductive medium (the cytoplasm with the conductivity λi), and immersed in an external conductive medium (conductivity λe), the induced transmembrane voltage could be explained by the following Laplace differential equation:

(1.1)

where M is the point on the cell that is considered, t is the time after application of electric field, f is a factor depending on the cell geometry, rcell is the radius of the cell, Ee is the external electric field strength, and θ(M) is the angle between the direction of the field and the normal of the cell surface in M.

g(λ) is related to the different conductivities as (Zimmermann et al., 1974)

(1.2)

where d is the thickness of the membrane (nm).

The characteristic time constant of the membrane charging (τm) can be calculated by (Kinosita & Tsong, 1977)

(1.3)

where Cm (0.5–1.0 μF/cm²) is the specific membrane capacitance. For mammalian cells, τm is calculated in the submicrosecond time range and it strongly depends on the buffer composition as the internal composition is fixed by the cell metabolism.

The electroporation occurs as long as the field is maintained at an overcritical value (expansion step). Several authors observed that even though the leaky state of cell membrane was induced during the onset of the pulse, the structural reorganization of the membrane was observed on a much longer time scale (Hibino, Itoh, & Kinosita, 1993; Hibino, Shigemori, Itoh, Nagayama, & Kinosita, 1991; Teissie et al., 2005). The stabilization step is an important issue of cell membrane electroporation; in fact, the pores need to be stable enough to allow interaction of the intra- and extracellular media (Toepfl, 2006). Gabriel and Teissie (1999) reported that as soon as a field strength was subcritical, a strong decrease in the flow of the polar molecules was observed, even though the cell membrane remained permeable to polar compounds. The stabilization step is followed by slow resealing of the cell membrane (for seconds to minutes) and recovery of its semipermeability. It has been shown that the decrease in the number of permeabilized cells with postpulse incubation time was a first-order process and depended strongly on temperature (Rols & Teissie, 1990). Moreover, during the resealing process the production of reactive oxygen species in the permeabilized part of the cell surface was observed (Teissie et al., 2005). Finally, the memory effect was observed, which means that some changes in the membrane properties remained present on a time scale of hours, but finally the cell behavior was back to normal.

The resealing of the membrane is able to preserve the biological cell from lysis in most pulsing conditions. However, different cellular alterations may be induced making impossible the cell resealing, leading to cell death on the long term (Teissie et al., 2005).

In general, the detection of the cellular tissue electroporation represents a difficult issue, mainly because the time range of pore formation is really short (submicrosecond) and also the pore area is extremely low, covering just 0.1% of the total membrane surface (Toepfl, 2006).

In biological cells the cell membrane plays an important role in the transport of different components. The biological membrane is a complex assembly between proteins and a mixture of lipids, which are nonhomogenously distributed as it happened in fluid matrix but are accumulated locally. Moreover, the balance between active pumping and spontaneous leaks creates the ionic gradient across the membrane (Teissie, 2014). The cell membrane can act as a capacitor filled with dielectric material of low electrical conductance and a dielectric constant of about 2 (Zimmermann et al., 1974). The opposite polarity charges accumulate on both sides of the membrane, which induce perpendicular transmembrane potential of about 10 mV. When the external electrical field is applied to the biological material, an additional potential is created by movement of charges along the electric field lines (Toepfl, 2006). In Fig. 1.1 a scheme of impact of the cell membrane exposure to the external electric field is illustrated.

Figure 1.1 Scheme of mechanism of the cell membrane permeabilization induced by an external electrical field (Ee). Ec, Critical electric field strength. Source: Adapted from Donsì, F., Ferrari, G., & Pataro, G. (2010). Applications of pulsed electric field treatments for the enhancement of mass transfer from vegetable tissue. Food Engineering Reviews, 2(2), 109–130. https://doi.org/10.1007/s12393-010-9015-3.

As described in Fig. 1.1, the exposure of cells to external electric field (Ee) can lead to three different outcomes of electroporation process defined by three different threshold values.

• Ee<Ec: The electric field applied is below the critical value (Ec) and no electroporation process occurs.

• Ee>Ec: The electric field strength exceeds Ec values and temporary membrane permeabilization takes place. However, the electric field is still below irreversible electroporation threshold and cells can recover their integrity and remain viable after the end of electric field exposure.

: The electric field strength exceeds greatly Ec values and permanent membrane permeabilization takes place. This phenomenon leads to extensive leakage of intracellular content and cell death.

In some cases, when the external electric field exceeds the threshold values of Ethermal, the electric field establishes high electric currents causing temperature increase and thermal damage to the cell (Kranjc & Miklavčič, 2017).

Low-intensity PEF treatment with relatively low values of Ee (≈20–100 V/cm) can cause electroporation to some extent. In this case the process of resealing can be very quick in order to repair the membranes immediately after the turn of the electric field strength. This kind of electroporation is called reversible electroporation (Barba et al., 2015).

The application of moderate PEF treatment can cause a loss of the permeability in some of the cells, while other are able to be resealed, and the insulating properties of the cell membrane can be recovered within several seconds after PEF treatment.

In reversible electroporation, transient pores of small size are formed that reseal when the electric field is not supplied any more. Reversible permeabilization of cell membrane has been widely studied and used in biotechnology for the transfer of genetic materials (DNA) inside bacterial cells as well to improve fusion of cells (Chang, Chassy, Saunders, & Sowers, 1992). Electroporation is also used in biomedicine to allow the permeation of cytotoxin through the membranes of cancerous cells and to increase the concentration of the anticancer agent in solid tumors. Electrochemotherapy is now widely used, starting from the first clinical trials on head and neck tumors by Belehhradek et al. (1993).

The reversible electroporation can also be applied in food processing. Pereira, Galindo, Vicente, and Dejmek (2009) studied the reversibility of the electroporation of potato cells, and they observed transient changes in the viscoelastic properties after PEF application with single 10−5–10−3 s rectangular pulses at electric field of 30–500 V/cm. Tylewicz et al. (2017) observed the reversibility of the electroporation of strawberry tissue by preservation of the integrity of cellular structure by time-domain nuclear magnetic resonance and by maintenance of cell viability by fluorescence staining observed by fluorescence microscope. This reversibility was observed when 100 V/cm was applied and was compromised when higher electric field strength of 200 V/cm was used.

On the other hand, high-intensity PEF treatment causes an irreversible damage of the cell membrane. Long-term changes in tissue electrical conductivity after PEF treatment application can also be related to osmotic flow and moisture redistribution inside the sample (Lebovka, Bazhal, & Vorobiev, 2001). This kind of electroporation has been widely investigated in food science, especially in extraction process (Barba et al., 2015; Parniakov, Barba, Grimi, Lebovka, et al., 2015; Parniakov, Barba, Grimi, Marchal, et al., 2015; Vorobiev & Lebovka, 2010) and microbial inactivation (Arroyo & Lyng, 2017; Saldaña, Álvarez, Condón, & Raso, 2014).

1.3 Critical value/dielectric breakdown

In order to create a local dielectric rupture of the membrane and consequently inducing the formation of a pore, acting as a conductive channel, the overall potential should exceed a critical value of about 1 V. Also, the membrane properties should be considered as the compressibility, the permittivity, and the initial thickness (Schoenbach, Peterkin, Alden, & Beebe, 1997). This increase in permeability reestablishes the equilibrium of the electrochemical and electric potential differences of the cell plasma and the extracellular medium (Glaser, Leikin, Chernomordik, Pastushenko, & Sokirko, 1998). This equilibrium is known as a Donnan equilibrium, indicating dielectric breakdown (Zimmermann, Pilwat, Beckers, & Riemann, 1976).

1.3.1 Models applied for dielectric breakdown

An electromechanical model was developed, suggesting that the membrane, considered as a capacitor containing a perfectly elastic dielectric, subjected to an external electrical field is subjected to the mechanical compression. The mechanical instability occurs when there is an increase of the transmembrane potential, which causes an increase of the compression forces.

In a model system of phosphatidylcholine bimolecular lipid layers, a good agreement between predicted breakdown voltage and assumed elastic parameters was observed by Crowley (1973).

The electromechanical model is still one of the most accepted theories to explain the effect of external electrical fields on biological cells. The electric breakdown could be considered reversible if the pores induced are small in comparison to the membrane area, which is also related to the application of low electric field strength. Increasing the treatment intensity promotes formation of large pores and the reversible damage will turn into irreversible breakdown (Toepfl, 2006).

Experimental studies have been performed in order to support this electromechanical compression model. A critical electric field strength, depending on the size and geometry of a cell, was found to be in the range of 1–2 kV/cm for plant cells and 10–14 kV/cm for microbial cells (e.g., Escherichia coli).

The gap of the electromechanical model is its too high simplification; in fact, the subsequent behavior such as resealing of pores, membrane conductance course, and transport phenomena are not considered. Therefore several other models have been proposed to predict the mechanisms at a molecular level, for example, the fluid mosaic model of a lipid bilayer with protein units embedded. These theories include the occurrence of membrane deteriorations and reorientations on the lipid bilayer and the protein channels as cause of increase in permeability (Toepfl, 2006).

An extension of the electromechanical model was described by Dimitrov (1984). This model was used to describe the time course of field-induced breakdown of membranes, considering different parameters of cell membrane such as the viscoelastic properties, membrane surface tension, and molecular rearrangements, as well as pore expansion. Other models are based on molecular reorientation and localized defects within the cell membrane which are expanded and destabilized by exposure to an electric field. The presence of small pores of hydrophobic nature fluctuating in the lipid matrix was suggested to be the initial structural basis of electroporation (Chernomordik, 1992). The application of external electrical field could transform them into hydrophilic pores by reorientation. This could happen with increasing of pore radius above the value where the pore energies of both orientations coincide. If the pore radius is small, the formation of hydrophobic pores is more favorable, but at a range of 0.5 nm the pore energies of hydrophobic and hydrophilic pores become equal and pore inversion may occur (Glaser et al., 1998). These pores might also cause a loss of ability to regulate the intracellular pH (Simpson, Whittington, Earnshaw, & Russel, 1999) and short circuit of protein-pumps (Chernomordik, 1992).

Both lipid domain and protein channels could be a site of the electroporation, since their functionality is influenced by the transmembrane potential. The gating potential for protein channels is in the range of 50 mV, which is smaller than the dielectric strength of a phospholipid bilayer. However, even though protein channels are opened by the application of electric field, it may not be sufficient to prevent the development of a transmembrane potential above the breakdown potential of the lipid bilayer (Toepfl, 2006).

1.4 Electroporation on different systems

The study on the electroporation has been conducted on different systems, such as (1) individual cells, (2) cell suspensions, and (3) tissue. The common important feature of each system is the fact that the cell membrane plays a great role in amplifying the applied electric field (Weaver & Chizmadzhev, 1996).

1.4.1 Spherical cells

In the case of spherical cell with a nonconducting membrane that is exposed to external electric field (Ee), the transmembrane potential distribution in the region surrounding the cell could be explained by the following Laplace equation with appropriate boundary conditions.

(1.4)

where ΔVi is the transmembrane voltage, rcell is the radius, and θ is the angle between the site on the cell membrane where ΔVi is measured and the direction of Ee.

At the poles (θ=0,π) the potential drop of about 75% occurs across the membrane in the region near the cell, and the transmembrane electric field (Em) is higher than Ee. The amplification associated with this field concentration is Em/Ee=1.5rcell/h=2×10³ for rcell=10 μm, considering the membrane thickness (h) as 5×10−7. Therefore as an example, when 10 μm radius cell is exposed to Ee, in order to achieve the transmembrane voltage of 0.5 V, the Ee of about 300 V/cm needs to be applied (Weaver & Chizmadzhev, 1996).

According to Eq. (1.4), the critical transmembrane potential is attained with the external electric field decreasing with the cell radius. In order to promote the electroporation of cells in plant tissue, which are quite large (about 100 μm), the electric field required is of 0.5–5 kV/cm (Donsì et al., 2010); however, even lower electric field strength (0.1–0.4 kV/cm) has been proved to provoke the electroporation of the plant tissue, in particular in apple (Dellarosa et al., 2016), strawberry (Tylewicz et al., 2017), and kiwifruits (Traffano-Schiffo, Laghi, Castro-Giraldez, Tylewicz, Ragni, et al., 2017; Traffano-Schiffo, Laghi, Castro-Giraldez, Tylewicz, Romani, et al., 2017; Traffano-Schiffo et al., 2016).

For the small microbial or algal cells, with the dimensions of about 1–10 μm, a higher electric field is required (10–80 kV/cm) in order to promote the electroporation of their membrane (Barba et al., 2015).

1.4.2 Nonspherical geometrically regular cells

In more generalized models the sphere can be replaced by spheroid (e.g., an oblate spheroid as a model of an erythrocyte) or by ellipsoid (a geometrical body in which each of its three orthogonal projections is a different ellipse).

A description of a cell is geometrically realistic if the thickness of its membrane is uniform, as it is in the case of spheres but not with spheroids or ellipsoids. In fact, the thickness of the membrane modeled in spheroidal or ellipsoidal coordinates is necessarily nonuniform, and by solving Laplace’s equation in these coordinates, the spatial distribution of the electric potential in a nonrealistic setting is obtained. However, in the case of cells surrounded by a physiological medium and with intact membranes that are nonporated the electric conductivity of the membrane can be neglected (i.e., the membrane is treated as an insulator). The ΔVi obtained in this way is still realistic, since the electric potential in each part of the cytoplasm is constant, and the geometry of the inner surface of the membrane does not affect the potential distribution outside the cell (Kotnik & Miklavčič, 2000).

1.4.3 Irregularly shaped cells

For an irregularly shaped cell the ΔVi cannot be solved as an elementary mathematical function but can be determined numerically by using modern computers and the finite-elements method implemented in software packages such as COMSOL Multiphysics (Pucihar, Kotnik, Valič, & Miklavčič, 2006; Pucihar, Miklavčič, & Kotnik, 2009). With these methods it is possible to obtain ΔVi in quite accurate way, considering a sufficiently accurate determination of three-dimensional shape of the cell and using sufficiently fine spatial and temporal resolution (Kotnik, 2017).

1.4.4 Cells in dense suspensions and tissues

In real conditions the cells are rarely isolated. When they are sufficiently close to each other, there is a mutual distortion of the field caused by their proximity, which needs to be considered. Often, the cells are also in direct contact, forming two-dimensional (monolayers attached to the bottom of a dish) or three-dimensional (tissues) structures, and they can even be electrically interconnected.

In dilute cell suspensions the distance between the cells is much larger than the cells themselves, causing that the local field outside each cell is almost unaffected by the presence of other cells. Thus for cells representing less than about 1% of the suspension volume (e.g., for spherical cells with radius of about 10 μm, this corresponds to up to 2 million cells/mL), the deviation of the actual ΔVi from the one predicted is negligible. However, when there is an increase of the volume fraction occupied by the cells, the distortion of the local field around each cell by the presence of other cells in the neighborhood becomes more pronounced. In this case the ΔVi starts to differ noticeably from the predicted values, and an accurate estimation of the ΔVi must be assessed either numerically or by analytical approximations (Pavlin, Pavšelj, & Miklavčič, 2002; Susil, Šemrov, & Miklavčič, 1998). The most appropriate model of dense cell suspensions is the one that resembles a face-centered cubic lattice, with uniform cell arrangement (Pavlin et al., 2002; Pucihar, Kotnik, Teissie, & Miklavčič, 2007).

For larger volume fractions of the cells, the electrical properties of the suspension start to approach that of a tissue but only to a certain extent. The arrangement of cells in tissues does not necessarily resemble a face-centered lattice, and the uniform electroporation is difficult to obtain in tissues, because they generally consist of diversely shaped cells, various cell types (including vascularization), and cells connection through gap junctions, resulting in spatially varying and often anisotropic electrical properties. Therefore the exposure of the tissue to homogeneous external electric field does not mean that inside the tissue the field is distributed homogeneously; in fact, some cells are almost unavoidably electroporated more intensely than others (Dymek et al., 2015). In order to reduce field inhomogeneity, the electric field delivery to a tissue must be carefully designed by building a numerical model of the tissue, taking into account its particular structure; the number, size, shape, and positioning of the electrodes. These parameters need to iteratively optimize until sufficient field homogeneity is achieved inside the tissue or in a subtissue of interest. Once the tissue cells are electroporated, the electric conductivity and dielectric permittivity of the tissue change affecting the electric field distribution. In particular, when a train of pulses is delivered, these dynamic changes must also be considered for optimization of the results. In such applications the real-time measurements of tissue conductivity need to be performed to complement the numerical modeling, allowing subsequent pulses to be adapted to the detected increase of conductivity reflecting the extent of electroporation (Kotnik et al.,