Effects of Pulsed Electric Field on Meat Tenderization and Microbial Decontamination: A Review

The tenderizing and bactericidal effects of PEF are generally attributed to cell membrane breakdown induced by electroporation (Zimmermann, 1996). When a cell is exposed to an electric field, micropores are simultaneously created in the phospholipid bilayer of the cell membrane, resulting in increased cell membrane permeability. The cell membrane, which consists of a phospholipid bilayer naturally has a specific transmembrane potential of approximately 10 mV between its inner and outer layers. When the membrane is exposed to an external electric field, the potential induced by the electric field increases until it reaches a critical value. As the potential increases, the membrane reorganizes the charges on both sides, leading to a viscoelastic deformation of the membrane due to the attraction of charges with opposite polarity. This process continues until the critical value is reached. If the external electric field surpasses the critical value of the transmembrane potential, an electro-compression force is generated on both sides of the membrane, initiating the formation of pores.

Detailed information on the PEF system and its components is provided in Fig. 1A. The PEF system typically consists of two main components: the pulse generator and the treatment chamber. The inclusion of other subcomponents depends on whether the system operates in continuous mode (for both liquid and solid samples) or batch mode (single batch). In the continuous mode, a pump or conveyor is necessary to transport the sample to the treatment chamber. Treatment chambers come in three common types: parallel plates, co-axial, and co-linear chambers (Arshad et al., 2020). For the treatment of liquid food in continuous mode, heat exchangers are additionally used to preheat the sample before treatment and cool it down afterward (Toepfl et al., 2014). In contrast, the single batch mode does not require these additional components. The batch chamber is similar to the parallel plates chamber but lacks the flow of the sample. This type of chamber is typically used for solid foods and is suitable for bench-scale applications. PEF treatment for solid foods is conducted in the batch chamber, which is filled with a low-conductive medium such as tap water. Unlike continuous chambers, batch chambers have been modified in various forms (Jeong et al., 2023; Li et al., 2020; Mok et al., 2017). The adjustable chamber illustrated in Fig. 1A was designed to allow for the adjustment of electrode gap (Jeong et al., 2023). This setup can be used to treat samples without the need for a medium and enables direct contact with the chamber without space between the electrode and the sample. While the use of batch chambers is advantageous for tailoring conditions at the bench scale, it is important to note that, in contrast to the continuous mode, treatment in batch mode is more susceptible to raising the sample temperature due to the high electric field strength (Toepfl et al., 2014). Therefore, batch treatment should be carried out at an acceptable level of PEF intensity to minimize heating.

Fig. 1. Description of PEF system and components (A) and parameters of PEF treatment and oscilloscope image (B). PEF, pulsed electric field.

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The effect of PEF on tissue is determined by both the properties of the sample and the PEF parameters. From the perspective of suitability for PEF processing, the electrical conductivity of the samples is a crucial factor because it influences temperature increases (Athmaselvi et al., 2014). The parameters in the PEF system are detailed in Fig. 1B. The electric field strength (E, kV/cm) is a primary factor that determines the extent of the PEF effect. The electric field strength is calculated using the following Eq. (1):

A sufficient field strength is necessary to reach the threshold for entering an electroporated state (irreversible state). The required level varies for each sample and depends on the cell size. Solid foods with large cells typically require a relatively lower field strength for electroporation compared to microbes. The pulse width, measured in microseconds, represents the duration during which the high voltage is maintained for each pulse in the form of a square wave. Short pulse widths are beneficial in preventing temperature increases during PEF treatment. The pulse frequency is a factor that determines the treatment time in terms of the number of pulses. These parameters can be expressed as a specific energy (Q, kJ/kg) using the following Eq. (2) (Zhang et al., 1995):

where V is the output voltage (kV), t is the treatment time (number of pulses/frequency), R is the resistance (ohm), and m is the mass of sample (kg). In the case of PEF treatments for solid foods, field strength is commonly used as a variable rather than specific energy, as the pulse width and treatment time are often held at fixed values.

The mechanism of electroporation can be theoretically explained; however, the evidence of cell membrane permeability is not entirely clear at the cellular level. This uncertainty arises because, while we can observe the irreversible state of the cell disrupted by PEF, we cannot easily observe the reversible state. At the tissue level, such as in vegetables or meat with a heterogeneous distribution of cell sizes, observing the reversible state can be quite challenging. The effects of PEF on tissues are observed as macroscopic phenomena, unlike at the cellular level, and these phenomena are dependent on both PEF intensity and tissue properties. When tissue is exposed to PEF, some cells within the tissue are damaged by the electric field. The distribution of damaged cells varies depending on the intensity and cell size. In other words, damaged and intact cells coexist heterogeneously. The ratio of damaged cells can be assessed by measuring an electric signal that results from ion release from the cells. This signal change is expressed in terms of electric conductivity (S/m). The PEF treatment selectively affects the cell membrane inside the tissue, leading to changes in ion release. Morphologically, it can be challenging to distinguish between intact and treated tissue. For example, in the microstructure of PEF-treated tissue, the cell wall may appear intact, whereas the cell membrane and organelles may exhibit some degree of damage (Kim et al., 2019). Similarly, in muscle tissue where there is no cell wall, morphological differences can be difficult to discern. This selective effect can result in a more flexible texture, caused by the release of turgor pressure within the cell. Therefore, PEF can soften the texture and improve processing suitability. Furthermore, the damaged cell membrane can create porosity in the tissue, increasing mass transport channels and accelerating mass transfer. Damaged organelles, such as vacuoles, can also facilitate the extraction of bio compounds.

Lebovka et al. (2002) introduced an effective tool for assessing the degree of tissue rupture using conductivity. The authors proposed evaluating the degree of rupture caused by PEF treatment through the cell disintegration index Z (CDI), which compares intact cells with completely ruptured cells. This tool has gained widespread acceptance in evaluating tissue permeability in numerous studies. Similar to the CDI concept, other evaluation tools have been developed to assess relative disintegration of mass transfer, texture, and acoustics. These methods provide a representation of macroscopic change of the tissue rather than focusing on the cellular level.

The underlying mechanisms through which PEF influences microscopic phenomena, as well as at the cellular level, remain unclear. However, recent efforts have been made to substantiate existing hypotheses. Genovese et al. (2021) utilized non-destructive MRI analysis to observe changes in moisture distribution in plant tissues after PEF treatment. Plant tissues exhibit heterogeneous moisture distribution due to variations in cell size. This heterogeneity leads to varying effects of PEF on each cell. Irreversible deformation induced by PEF results in a homogeneous distribution of moisture, which is confirmed through T₂ mapping and MRI tomography (Genovese et al., 2023). Some ruptured cells release cytoplasm into intracellular voids, leading to a homogeneous moisture distribution as the cytoplasm fills the voids. This phenomenon alters the osmotic pressure in adjacent cells. This non-destructive analysis significantly enhances our understanding of cell permeabilization and complements conductivity analysis.

Tenderness is a critical factor when evaluating sensory quality, and the primary effect of PEF on meat is its tenderization. The degree of tenderization at a macroscopic scale is typically assessed through mechanical measurements such as shear force or meat hardness. However, various studies utilizing different meat materials, PEF intensities, and sample sizes have produced conflicting results regarding the tenderization effects of PEF. Tables 1 and 2 categorize these studies based on the presence or absence of a tenderization effect following PEF treatment. The studies listed in Table 1 demonstrate that PEF treatment did not lead to a significant tenderization effect. These studies share common characteristics, including the use of commercial PEF systems and direct contact with the electrodes. Interestingly, low PEF intensity did not result in a tenderization effect (Bhat et al., 2019a; Khan et al., 2017). Similarly, studies employing high-intensity PEF treatment, which should be sufficient for cell disruption, did not impact meat tenderness (Arroyo et al., 2015a; Arroyo et al., 2015b; Faridnia et al., 2014; Faridnia et al., 2015; McDonnell et al., 2014; O’Dowd et al., 2013). In contrast, in the case of plant tissue, treatment with an electric field strength of approximately 1.0 kV/cm resulted in increased flexibility and softness in texture (Fauster et al., 2018; Lee et al., 2022). These results may be because the PEF effect is masked when using excessively large sample sizes for treatment and measuring texture at excessively high temperatures.

Large sample sizes require a considerable amount of energy to reach the necessary PEF intensity for tenderization. Additionally, the outcome is influenced by sample properties such as fat composition or connective tissue. Bhat et al. (2019a) and Khan et al. (2017) did not observe a significant reduction in shear force due to insufficient PEF intensity. However, Suwandy et al. (2015c) observed a tenderization effect with a sufficient PEF intensity, even when using samples of the same size as the former studies. A sufficient PEF intensity can ensure that the cell permeability induced by PEF is homogeneous. Generally, the presence or absence of a PEF effect is indirectly validated through changes in electric signals. Sufficient PEF intensity increases the electric conductivity of muscle tissue because damaged cells release intracellular fluid, which is an electrolyte. However, it can be challenging to determine whether a homogeneous PEF effect has been achieved for each sample immediately after PEF treatment, as some previous studies did not provide this information (Arroyo et al., 2015a; Arroyo et al., 2015b; McDonnell et al., 2014). Conversely, in a recent study, Jeong et al. (2023) used smaller sample sizes and clearly observed a positive linear relationship between PEF intensity and electric conductivity. While this relationship can be observed through changes in texture, an increase in conductivity can also occur due to changes in microstructure, regardless of the tenderization effect. According to O’Dowd et al. (2013), PEF treatment reduced the diameter of muscle fiber bundles and increased conductivity. Similar results were reported by Khan et al. (2017).

Most of the studies listed in Tables 1 and 2 heated the meat samples for measuring shear force or texture profiles. While Jeong et al. (2023) assessed the texture of raw beef Semitendinosus muscle, it is important to note that eating raw meat is not a common practice worldwide, except in some East Asian countries. Additionally, the texture of raw meat can vary depending on its temperature. Due to these factors, meat texture is generally evaluated using cooked meat, typically prepared in a water bath at 80°C until the internal temperature reaches approximately 70°C. However, this cooking process can potentially negate the effects of PEF treatment. Connective tissues in meat tend to contract at temperatures over 65°C (Tornberg, 2005), resulting in a tougher texture. Tenderization by PEF may not be observed when the PEF effect is not uniformly distributed throughout the meat. This phenomenon can be confirmed by comparing the studies listed in Tables 1 and 2, as they provide evidence of this effect. Furthermore, studies that have explored the combination of PEF and sous-vide cooking have observed a tenderization effect (Jeong et al., 2020; Karki et al., 2023). According to Jeong et al. (2020), the PEF effect observed in raw beef Semitendinosus muscle was maintained even after sous-vide cooking (60°C, 1 h to 24 h). Karki et al. (2023) conducted the PEF treatment and sous-vide cooking (60°C, 24 h and 36 h) on short ribs. The high-energy PEF-treated (0.7 kV/cm, 5,200 pulses) and longer-cooked (36 h) samples exhibited a significant reduction in tenderness compared to the control (p<0.001).

Several studies have demonstrated that proteolysis is facilitated by PEF treatment, regardless of the mechanical texture measurements. Table 3 summarizes the findings of studies on proteolysis induced by PEF, which were approached from three different perspectives: the level of myofibril structure (Chian et al., 2019; Mungure et al., 2020; O’Dowd et al., 2013), structural proteins (Bhat et al., 2019b; Bhat et al., 2019c; Jeong et al., 2023; Suwandy et al., 2015a; Suwandy et al., 2015b), and myofibrillar protein (MP) extracts (Dong et al., 2020; Wang et al., 2022).

In a study by O’Dowd et al. (2013), myofibrils were extracted through a four-step filtration process, and their particle size was measured. The final filtration fraction, from which connective tissues and debris had been removed, exhibited a smaller particle size in the PEF-treated sample compared to the control and heated samples. PEF treatment also resulted in the rupture and fragmentation of myofibril structure (Chian et al., 2019; Mungure et al., 2020). While these phenomena could be observed under a microscope, further measurements are needed to examine finer changes at the level of structural proteins. The degradation of troponin-T and desmin has been associated with tenderization (Marino et al., 2013; Yates et al., 1983). Calpain activity also plays a crucial role in meat protein degradation (Coria et al., 2018). Jeong et al. (2023) investigated the proteolysis effect of PEF treatment (0.5 to 2.0 kV/cm) and found that PEF treatment did not significantly affect μ-calpain but resulted in a significant decrease in troponin-T. Calpain activity was slightly increased by PEF treatment in deer longissimus lumborum muscle (Bhat et al., 2019c). Likewise, troponin-T and desmin were degraded by PEF on different muscles (Bhat et al., 2019b; Bhat et al., 2019c; Suwandy et al., 2015a; Suwandy et al., 2015b). Dong et al. (2020) and Wang et al. (2022) used MP extracts to observe structural changes in the protein, rather than focusing on a specific protein. Unlike other studies, they treated the samples with high-intensity PEF exceeding 10 kV/cm. Their results indicated that the microstructure of MP was deformed, and the hydrophobicity of the MP surface was enhanced by PEF. These structural changes induced modifications in the rheological properties of MP (Wang et al., 2022).

In addition to meat protein degradation, changes in structure and increased permeability induced by PEF treatment can facilitate the aging process. As evidence of the tenderization effect, PEF treatment may not produce immediate results in the absence of an aging process (O’Dowd et al., 2013), but its effect becomes evident with extended aging periods (Suwandy et al., 2015a; Suwandy et al., 2015b). Therefore, in addition to mechanical texture properties, PEF treatment can facilitate tenderization through proteolysis. Further research should explore approaches in which PEF improves end-product properties as a pre-treatment and investigates whether the PEF effect persists after subsequent processes or enhances the efficiency of the process.