Pulsed Electric Fields Effects on Proteins: Extraction, Structural Modification, and Enhancing Enzymatic Activity

J Marín-Sánchez; A Berzosa; I Álvarez; C Sánchez-Gimeno; J Raso

doi:10.1089/bioe.2024.0023

. 2024 Sep 16;6(3):154–166. doi: 10.1089/bioe.2024.0023

Pulsed Electric Fields Effects on Proteins: Extraction, Structural Modification, and Enhancing Enzymatic Activity

J Marín-Sánchez ¹, A Berzosa ¹, I Álvarez ¹, C Sánchez-Gimeno ¹, J Raso ^1,^✉

PMCID: PMC11447477 PMID: 39372091

Abstract

Pulsed electric field (PEF) is an innovative physical method for food processing characterized by low energy consumption and short processing time. This technology represents a sustainable procedure to extend food shelf-life, enhance mass transfer, or modify food structure. The main mechanism of action of PEF for food processing is the increment of the permeability of the cell membranes by electroporation. However, it has also been shown that PEF may modify the technological and functional properties of proteins. Generating a high-intensity electric field necessitates the flow of an electric current that may have side effects such as electrochemical reactions and temperature increments due to the Joule effect that may affect food components such as proteins. This article presents a critical review of the knowledge on the extraction of proteins assisted by PEF and the impact of these treatments on protein composition, structure, and functionality. The required research for understanding what happens to a protein when it is under the action of a high-intensity electric field and to know if the mechanism of action of PEF on proteins is different from thermal or electrochemical effects is underlying.

Keywords: pulsed electric fields, protein extraction, protein modification, enzymatic activity

Introduction

Proteins are remarkably complex long-chain molecules consisting of amino acid residues linked by peptide bonds, likely constituting the most versatile compound found in food. From a nutritional standpoint, proteins are macronutrients essential in relatively substantial quantities to support bodily growth and repair, furnish energy, and supply crucial amino acids that must be obtained from the diet since the human body cannot produce them.¹ Proteins are present in a diverse array of traditional foods, encompassing animal protein sources like meat, dairy, fish, and eggs, as well as plant protein sources such as beans, lentils, and nuts. Beyond their nutritional contributions, proteins serve as fundamental ingredients in the food industry, leveraging specific functional properties that facilitate processing and form the foundation for product performance.² The primary functional properties of proteins include gelation, emulsification, foaming, water binding, coagulation, and flavor-binding capabilities. These attributes, influencing protein behavior during food processing, arise from complex interactions among protein composition, structural conformation, physiochemical properties, and other components in the food matrix.³

Enzymes, which catalyze chemical reactions, are proteins synthesized by all living organisms. Frequently, endogenous enzymes within foods accelerate chemical transformations leading to alterations in flavor, color, and texture, thereby diminishing the overall edibility of the food.⁴ Nevertheless, these enzymes also serve a technological role by expediting various reactions in the processing of food commodities or ingredients.⁵

The configuration of protein structures hinges on the sequence of amino acids and the chemical bonds within both the polypeptide backbone and the side chains of amino acids. From a structural perspective, proteins exhibit the following four levels of organization: primary, secondary, tertiary, and quaternary. The structural arrangement of a protein plays a pivotal role in its functionality, as it determines the protein’s ability to interact with other molecules. Any alteration in the shape of the protein at any structural level is termed denaturation. Denatured enzymes may lose their functionality, whereas, conversely, the functional properties of proteins may necessitate the maintenance of their structure or a previous denaturation.⁶

Food processing significantly influences the physical, chemical, and functional characteristics of proteins. Throughout this process, the intrinsic interactions among amino acids may be altered, resulting in protein denaturation. In addition, amino acids may engage in reactions with other components present in the food matrix. These reactions can lead to diminished bioavailability of amino acids, potentially reducing digestibility.⁷ However, they also contribute to color and flavor development in various foods, exemplified by processes such as the Maillard reaction.⁸ Furthermore, endogenous enzymes in raw materials or commercial enzymes used during processing may induce proteolytic events, breaking down proteins into peptides and amino acids.^9,10

In recent years, innovative food processing technologies such as pulsed electric fields (PEF) have emerged with the aim of efficiently manufacturing and preserving food, thereby reducing energy costs and enhancing the sustainability of the food sector.¹¹ The main goal of using PEF in food processing is to induce electroporation in microbial cells, as well as cells from vegetable and animal tissues. This aims to achieve microbial inactivation, enhance mass transfer, or improve process efficiency by inducing alterations in the textural and rheological properties of the products.¹²

PEF treatment is typically applied using two metallic electrodes that are in direct contact with the liquid food product or with the liquid in which solid products are submerged. To generate the necessary electric field for inducing cell electroporation, several kV/cm must be applied.¹³ This process involves the passage of electrical current through the product being treated. The current that flows in the electrodes consists of free electrons and in the material placed between the electrodes of charged particles such as ions.¹⁴

The impact of different stresses such as heating acidification, pressure, and so on imposed during food processing on protein composition, structure, and functionality has been extensively explored and is currently well-understood.^15–17 However, a more comprehensive understanding of the effect of PEF on the functional and biological properties of proteins is necessary.

This article seeks to provide a critical review of the recent research conducted on the effect of PEF in protein processing, including protein extraction, its impact on physicochemical and functional properties, and its effect on enzyme activity. These aspects need to be investigated more deeply to understand what happens to the effects of high-intensity electric fields on proteins.

Fundamentals of Pulsed Electric Fields Processing

PEF technology constitutes a physical treatment method involving the intermittent application of high-voltage (kV) pulses of a duration ranging from microseconds to milliseconds to a product positioned between two electrodes. The applied voltage generates an electric field, the intensity of which is contingent upon the gap between the electrodes.¹¹

The key process parameters defining the efficiency of a PEF treatment include the electric field strength, the processing time (which relies on the number of pulses delivered and the pulse width), and the pulse frequency, denoting the number of pulses administered per second (Fig. 1). Another key parameter is the total specific energy (kJ/kg) dispensed in the treatment chamber to generate the electric field, which is determined by the applied voltage, processing time, and the electrical resistance of the treatment chamber that depends on its dimension and conductivity of the product. While the mechanism by which PEF exerts its effect on cells does not rely on heating, the conduction of electric current through the food material results in some Joule heating.¹⁸

Fig. 1. — Main process parameters of PEF technology. PEF, pulsed electric field.

The main effect of the application of an external electric field to a biological material is the electroporation phenomenon. Electroporation consists of the formation of local defects or pores in the lipid bilayer of the cytoplasmic membrane enveloping the cytoplasm of eukaryotic or prokaryotic cells. Consequently, the cytoplasmic membrane becomes permeable to molecules that would otherwise be unable to traverse it in its intact state.¹⁹ Depending on the intensity of the treatment applied (electric field strength, processing time, specific energy) and cell characteristics (size, shape, orientation in the electric field), the electroporation of the lipid bilayer can be either reversible or irreversible.¹³ It is reversible if the bilayer returns spontaneously to its pre-breakdown state by recovering membrane integrity. If structural changes in the lipid bilayer due to PEF treatment are permanent, electroporation is irreversible.

For electroporation to occur in a cell subjected to an external electric field, the transmembrane potential must surpass a specific threshold, resulting from the buildup of oppositely charged ions on either side of the nonconductive membrane. The transmembrane potential induced by an external electric field varies based on factors such as cell size and the intensity of the applied electric field. As a result, the critical electric field strength necessary to induce electroporation in larger cells, such as those found in plant or animal tissues (0.5–1 kV/cm), is lower than that required for microbial cells (10–15 kV/cm).^20,21

The electroporation of microbial membranes results in the loss of their selective permeability, leading to microbial death at temperatures lower than those utilized in traditional thermal processing.^22–24 In addition, electroporation of the cytoplasmic membrane proves to be an effective method for enhancing mass transfer phenomena across cell membranes. This process finds applications in various food industry operations, aiming to obtain specific intracellular compounds of interest such as proteins or to remove water from foods through drying.^25,26 Alterations in the textural and rheological properties of products, where the structure is largely dependent on cell integrity, may be achieved by irreversible electroporation of cells of food material. The modification of textural properties in plant and animal tissues serves as the basis for diverse PEF applications, including reducing the energy required for cutting, facilitating the peeling of fruits and vegetables, and tenderizing meat.^27–29

The primary objective of processing through PEF is to generate a sufficiently large electric field to cause cell electroporation to achieve microbial inactivation, enhance mass transfer, or modify the food structure. However, generating a high-intensity electric field necessitates the flow of electric current from one electrode to another through the treated product. This current flow may lead to undesirable side effects such as electrochemical reactions, electrophoresis-based phenomena, and an increase in the sample’s temperature.³⁰ These side effects may result in electrode fouling due to the formation of a particle film on the electrode surface, migration of electrode material, and modification of components that may impact the nutritional, organoleptic, or functional properties of the processed product.³¹,³² In contrast, the lack of homogeneity in the distribution of the electric field in the treatment zone may cause over- or under-processing or dielectric breakdowns.³³ Consequently, any study aiming to understand the effect of PEF on protein processing requires a correct definition of the processing conditions (electric field strength, pulse width and frequency, total specific energy, etc.) to reduce the unwanted effects caused by the current flow and an appropriate design of the treatment chamber.³⁴ These issues are essential to prevent methodological artifacts and to minimize the unwanted effects that may occur during the application of PEF.

Extraction of Proteins Assisted by PEF

The escalating demand for total dietary protein, driven by the ongoing global population growth, necessitates the exploration of sustainable and nutritious protein sources. This exploration extends to alternative sources such as microorganisms, insects, seaweed, microalgae, and by-products generated during food processing.³⁵ Typically, these nontraditional protein sources require prior protein extraction to enhance digestibility and bioavailability.

The efficiency of protein extraction is augmented through cell disruption methods. Mechanical grinding, osmotic shock, high-pressure homogenization, ultrasound, and enzymatic hydrolysis represent various physical and chemical techniques used for this purpose.³⁶ While these methods effectively release proteins from cellular compartments by inducing complete cell disruption, they often result in nonselective extraction of compounds and micronization of cellular debris. This undesired outcome impedes the attainment of a pure protein-rich extract. In contrast, PEF increases the permeability of the cytoplasmic membrane through electroporation, facilitating protein extraction without disintegrating the cell structure at a minimum processing time and energy consumption compared with other techniques.³⁷

Variables such as the strength of the electric field and the duration of treatments significantly influence the extraction process.^38–40 Furthermore, extraction conditions following PEF treatment, such as pH, temperature, or extraction time, have been found to impact protein extraction efficiency.^41–43 Due to the huge number of factors influencing extraction assisted by PEF, optimizing protein extraction conditions should involve the use of response surface methodology, allowing for the simultaneous optimization of individual factors along with their possible interactions, rather than the more time-consuming one-factor-at-a-time approach typically used.⁴⁴

The interest in the application of PEF to improve the extraction of proteins from microbial cells, especially yeast and microalgae, is growing (Table 1). The cytoplasm of microorganisms contains proteins of varying molecular weights and structural conformations. Consequently, the size of pores generated by PEF treatment significantly influences the extraction process. Using various, fluorescent-labeled dextran molecules with different molecular weights, it has been observed that the radius of membrane pores may range from 0.77 to 5.11 nm, depending on the electric field and the duration of the pulses.⁴⁵ This finding, coupled with the fact that PEF-induced pores allow access to the cytoplasm without disintegrating the cell wall, offers the possibility of sequentially extracting peptides and proteins based on their molecular weight. Approximately one hour after the treatment, amino acids and small peptides are released from the cytoplasm, whereas macromolecules like proteins and nucleic acids are retained inside the cytoplasm.⁴⁶ Extending the incubation time is required for efficient protein release. Consequently, an electroporated cytoplasmic membrane could be used as an ultrafiltration unit for separating compounds of different molecular weights. Combining treatments that cause membrane pores of different sizes with extended incubation times could permit obtaining protein fractions of varying molecular weights.⁴⁷

Table 1.

Recent Studies on the Application of Pulsed Electric Fields (PEF) for Improving Protein Extraction from Microorganisms

Microorganism	Treatment conditions	Protein extraction after PEF treatment	Reference
Nannochloropsis sp.	20 kV/cm, 400 pulses of 10 µs, 4 ms treatment time (53.1 kJ/kg)	6% of total proteins (no incubation period)	⁸⁴
Haematococcus pluvialis	3 kV/cm, 9 pulses of 2 ms, total treatment time 18 ms, square wave bipolar pulses (8 kJ/kg)	8-fold increase in protein extraction after 24 h of incubation compared to untreated cells	⁸⁵
Chlorella vulgaris	17.1 kV/cm, pulses of 5 µs, 200 Hz, square wave monopolar pulses (111 kJ/kg)	5% of total proteins (no incubation period)	⁸⁶
Chlamydomonas reinhardtii	7.5 kV/cm, 5 pulses of 100 µs, total treatment time 500 µs, square wave monopolar pulses (180 kJ/kg)	23% of total proteins (1 hour of incubation)	⁸⁷
Chlorella vulgaris	20 kV/cm, pulses of 5 µs, 1000 Hz, square wave monopolar pulses (100 kJ/kg)	5.2% of total proteins (1 hour of incubation)	⁸⁸
Chlorella vulgaris	20 kV/cm, 2 pulses of 50 µs, total treatment time 100 µs, exponential decay pulses (7.76 kJ/kg)	30% of total proteins (24 h of incubation)	⁵¹
Arthrospira platensis	40 kV/cm, pulses of 1 µs, 6 Hz, square wave monopolar pulses (122 kJ/kg)	100% of total proteins (6 h of incubation)	⁸⁹
Chlorella vulgaris	40 kV/cm, 47 pulses of 1 µs, total treatment time 47 µs, 4.5 Hz, square wave monopolar pulses (150 kJ/kg)	50% of total proteins (24 h of incubation)	⁴⁷
Arthrospira platensis	20 kV/cm, 105 pulses of 5 µs, total treatment time 525 µs, square wave pulses (100 kJ/kg)	17% of total proteins (3 h of incubation)	⁹⁰
Saccharomyces cerevisiae	3.3 kV/cm, 15 pulses of 0.8 ms, total treatment time 12 ms, square wave monopolar pulses (120 kJ/kg)	90% of total proteins (16 h of incubation)	⁴⁶
Arthrospira maxima	25 kV/cm, 300 Hz, square wave bipolar pulses (100 kJ/kg)	72% of total proteins (2 h of incubation)	⁹¹
Haematococcus pluvialis	1 kV/cm, 9 pulses of 0.22 ms, total treatment time 20 ms, 9.62 Hz, square wave monopolar pulses (200 kJ/kg)	46% of total proteins (45 min of incubation)	⁴⁰
Saccharomyces cerevisiae	20 kV/cm, 400 pulses of 0.6 µs, total treatment time 240 µs, 1 Hz, square wave monopolar pulses	70% of total proteins (24 h of incubation)	⁴⁸
Saccharomyces cerevisiae	15 kV/cm, 50 pulses of 3 µs, total treatment time 150 µs, 119.5 Hz, square wave monopolar pulses (87.7 kJ/kg)	66% of total proteins (24 h of incubation)	⁷⁵
Hansenula polymorpha	5.85 kV/cm, 30 pulses of 0.7 ms, total treatment time 21 ms, square wave monopolar pulses (158.8 kJ/kg)	30% of total proteins (2 h of incubation)	²⁵

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Yeast has become one of the most utilized hosts to produce recombinant proteins for industrial and pharmaceutical use. Recently, the applicability of PEF for improving the extraction of recombinant proteins accumulated in the cell cytoplasm from Saccharomyces cerevisiae, Pichia pastoris, or Hansenula polymorpha has been evaluated.^25,48,49 In contrast, the efficiency of PEF has also been demonstrated in cases where recombinant proteins are secreted by the yeast into the growth media. Electroporation of the yeast biomass once the production process has finished allows the extraction of proteins that remain in the cytoplasm, improving the yield of the process.⁵⁰ Large intracellular proteins may require subsequent incubation with lytic enzymes to increase cell wall porosity for efficient recovery.²⁵

The possibility of inducing reversible electroporation by applying PEF treatments of moderate intensity opens the doors to designing more eco-friendly biotechnological processes. This approach requires adjusting the intensity of the PEF treatment to reversibly increase the permeability of the cytoplasmic membrane, facilitating the release of proteins from the cytoplasm without killing the cells. This approach has been demonstrated for the biocompatible extraction of proteins from the microalgae Haematococcus pluvialis and Chlorella vulgaris.^40,51

PEF technology also emerges as an innovative tool capable of embodying the principles of a circular economy by facilitating the extraction of proteins from by-products generated in the food industry that represents a significantly underutilized source of protein. Larger animal and plant cells require lower electric field strength for electroporation compared with microbial cells. PEF, coupled with aqueous extraction, enabled obtaining high-purity protein extracts from complex animal and vegetable by-product matrices.^27,37 The energy efficiency and scalability of PEF in reclaiming proteins from chicken waste have been highlighted.⁵² Compared with traditional methods, PEF consumed significantly less energy, positioning it as a more sustainable and environmentally friendly alternative. The scalability of PEF enhances its potential for widespread adoption in industrial protein extraction processes. Mirroring its success in meat processing, PEF’s versatility is further demonstrated in the valorization of fish processing by-products such as fishbone or viscera isolating collagen and proteins with improved emulsifying properties compared with conventional enzymatic methods.⁵³ In emerging protein sources such as insects, PEF enables enhanced cell permeabilization and biomass disintegration, facilitating the production of high-protein, dry, defatted insect-based food while maintaining protein integrity.⁵⁴

PEF represents a useful tool as a pretreatment to improve the extraction of proteins from alternative sources and by-products of the food industry. The scalability of PEF enhances its potential for widespread adoption in industrial protein extraction processes. However, further studies on the optimization of extraction conditions to design efficient and reproducible extraction processes for industrial applications accompanied by the evaluation of the impact of extraction on subsequent purification operations are required.

Effect of PEF on Structure and Functional Properties of Proteins

The exploration of PEF for altering protein structure and, consequently, functional properties has attracted considerable attention. Investigations encompass proteins from both animal and plant sources, with studies aimed at comprehending the mechanisms underlying the effects of electric fields on proteins isolated from diverse food sources.

Whey proteins, caseins, ovalbumin, and soybean protein isolates have frequently served as models for assessing the impact of PEF on protein structure and functionality (Table 2). As shown in the table, different studies have found that PEF may modify the physicochemical and functional properties of proteins by improving solubility, water-holding capacity, emulsifying, foaming, and gelation properties. Fundamental studies conducted with sophisticated techniques such as Raman, FTIR, or fluorescent spectroscopy conclude that PEF may alter the secondary and tertiary structure of proteins by reducing the α-helix content and loss in β-sheets.^55–57 The mechanisms underlying the effects of electric fields on proteins remain unclear. Nonetheless, it has been suggested that during PEF treatment, polar groups within proteins absorb energy, leading to the generation of free radicals. These free radicals are believed to disrupt intramolecular interactions among protein molecules.^29,58

Table 2.

Recent Studies on the Effect of Pulsed Electric Fields (PEF) on Protein Structure and Functional Properties Modification

Protein	Treatment conditions	Changes on structure	Influence on protein	Reference
Canola protein	35 kV/cm, pulses of 8 µs, 600 Hz, square wave monopolar pulses	Secondary and tertiary structures altered by changing α-helices and β-sheets, increasing amount of free sulfhydryl groups and surface hydrophobicity	Significant increment in solubility, water-holding capacity, oil-holding capacity, emulsifying capacity, emulsion stability, foaming capacity, and foam stability	⁵⁸
Pine nut protein	5–20 kV/cm, 1800–2400 Hz, square wave bipolar pulses	Altered α-helix, β-sheet, β-turn, and random coils of peptides	Increased antioxidant activity	⁹²
Ovalbumin	16 kV/cm, pulses of 40 µs, 1 Hz, square wave monopolar pulses	The combined effect of metal ions and PEF on the surface hydrophobicity and surface tension increased with pulsed time ﬁrst and then decreased	Enhanced surface properties	⁹³
β-Lactoglobulin	20 kV/cm, 30 pulses of 10 µs, total treatment time 300 µs, 1 Hz	Increased α-helix, decreased β-sheet and random coil elements	Increased tryptic and chymotryptic hydrolysis of β-Lactoglobulin	⁹⁴
Pea, rice, and gluten concentrates	1.65 kV/cm, 60000 pulses of 5 µs, total treatment time 300 ms, 400 Hz, square wave monopolar pulses	PEF induced unfolding, intramolecular rearrangement, and aggregate formation, altering protein structure	Increased water and oil holding capacity and solubility in gluten concentrate	⁹⁵
Ovomucin	10–40 kV/cm, 2000 Hz	Significant alterations in the primary, secondary, and tertiary structures of the protein	Changes in the spatial conformation could reduce sensitization	⁹⁶
Wheat gluten	2.5–12.5 kV/cm, total treatment time 2–9.0 s, square wave monopolar pulses (1.650x10³-2.475x10⁴ kJ/kg)	As field strength increased, α-helix reduced, β-sheet increased, while random coils remained stable	Significant effect on solubility, water-holding capacity, oil-holding capacity, foamability, foam stability, and emulsion stability	⁹⁷
Whey protein isolate	10 kV/cm, 1000 Hz, square wave bipolar pulses	Surface hydrophobicity, exposed sulfhydryl, and total sulfhydryl decreased	Improved succinylation	⁹⁸
Micellar casein isolate	16 kV/cm, pulses of 2 µs, total treatment time 6–31 µs, 100 Hz, square wave monopolar pulses (24–100 kJ/kg)	Induced reorganization in the configuration of the micell	Enhanced protein digestibility and peptide formation, potentially enhancing its nutritional value	⁹⁹

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The aforementioned PEF effects on proteins that may cause modifications in surface hydrophobicity, protein denaturation, and protein aggregation affecting functional properties are similar to those caused by thermal or chemical effects.^29,59 Generally, the modification of the physicochemical and functional properties of proteins requires the application of very intense PEF with an electric field strength and duration much longer than those required for electroporation of cell membranes (Table 2). Under these intense treatments, the spatial and temporal distribution of the electric field strength and temperature inside the treatment chamber, especially in continuous flow treatment, may make it difficult to discern the effect of the electric field from other unwanted side effects such as temperature increments and electrochemical reactions.

The potential of PEF to alter the secondary and tertiary structure of proteins has led to investigations into the modification of allergenic properties in food proteins. Results on this matter suggest that PEF treatments do not significantly reduce the allergenicity of plant or animal proteins.^60,61 Studies have shown that PEF treatment did not induce structural modifications in allergenic proteins found in peanuts and apples.⁶² While the primary allergenic protein in peaches was denatured by a combination of PEF treatment (25 kV/cm) and moderate temperatures (50°C), in vitro allergenicity remained unaffected.⁶⁰ Conversely, it has been observed that the immunoreactivity of egg proteins was only minimally impacted by the application of very intense electric fields (35 kV/cm).⁶³

Currently, results presented in the literature on protein modification by PEF are challenging to compare and sometimes contradictory. Different experimental approaches used by various authors and the lack of a comprehensive description of treatment conditions make it difficult to replicate experiments in other laboratories.

Since PEF processing at an industrial scale requires the application of treatment in a continuous flow, many studies on the effect of PEF on proteins have been conducted using continuous treatment chambers. However, using this approach, the distribution of treatment time is not homogeneous due to the flow velocity and the residence time distribution.³³ Basic studies aiming to understand the mechanism of action of PEF on food proteins should be conducted with protein isolates using a batch parallel treatment chamber that permits a uniform distribution of the electric field, treatment time, and strictly controlled treatment conditions.⁶⁴ The extent of electrochemical reactions and temperature increments occurring during PEF treatment depends on the total amount of electrical charge passing through the electrodes during the pulse treatment. Different strategies can effectively reduce electrochemical reactions and temperature increments. These include keeping the applied voltage, pulse width, and frequency at the minimum necessary for generating a sufficient electric field. In addition, using electrode materials that minimize electrochemical reactions can further mitigate these issues.³⁴ The integration of molecular dynamics simulation of the effect of the electric field on protein structure, with numerical simulation techniques that permit the evaluation of the distribution of temperature, treatment time, and electric field strength in the treatment chamber and the occurrence of electrochemical reactions, could also result in a very useful approach to understanding what happens to a protein when it is under the action of a high-intensity electric field.⁶⁵

Therefore, before exploiting the potential of PEF as a tool for improving protein functionality, more basic research is needed to understand the exact mechanisms by which the application of an external electric field affects food proteins.

Inactivating, Boosting, and Triggering Enzymatic Activity by PEF

It is widely acknowledged that endogenous enzymes can expedite undesirable reactions in foods, leading to spoilage.⁶⁶ However, the remarkable catalytic efficiency of these ubiquitous biomolecules has been harnessed to enhance processing in the food and biotechnology industries for centuries.⁶⁷ While the potential of PEF technology to decrease or impair enzymatic activity has been extensively explored, its ability to positively modify enzymatic activity has received comparatively less attention.

Several studies have demonstrated the efficacy of PEF in inactivating various enzymes that may cause undesirable changes in food attributes such as loss of texture, color, or flavor. Table 3 presents examples of recent PEF applications for enzyme inactivation in both model systems and foods. It is shown that different sensitivities of enzymes to PEF, along with processing conditions such as electric field strength, frequency, pulse width, treatment time, specific energy, temperature, and characteristics of the media in which enzymes are suspended, significantly impact enzyme inactivation. However, as it happens with the effect of PEF on protein functionality, the mechanism of enzyme inactivation by PEF is not yet clearly understood. Destruction of enzymatic activity by PEF has been linked to structural modification and protein unfolding due to changes in the secondary and tertiary structure rather than the primary structure.²⁹ Similar to the effect of PEF on protein structure in general, the application of very intense PEF treatments is required to significantly impair enzymatic activity by PEF. Although thermal effects can strongly influence the impact of PEF treatment on enzyme inactivation, most studies in the literature do not distinguish between electric field and thermal effects. Some authors have reported that the inevitable increment of temperature that occurs during the application of the intense PEF treatments or the combined effect of the temperature and the electric field is the major reason for enzymatic inactivation.³³

Table 3.

Recent Studies on Enzyme Inactivation in Model Systems and Food by Pulsed Electric Fields (PEF)

Matrix	Enzyme	Treatment conditions	Observations	Reference
Sodium-caseinate hydrolysates	Commercial protease Protamex^™	14–18.2 kV/cm, 100–900 pulses of 20 µs, total treatment time 2–18 ms, 10 Hz	The maximum reductions in enzyme activity were 66% and 72% at 14 and 18.2 kV/cm for 900 and 500 pulses, respectively	¹⁰⁰
Unpasteurized sake	α-amylase Acid carboxypeptidase	40 kV/cm, 300–900 pulses of 10 µs, total treatment time 3–9 ms, 1 Hz	PEF caused slight inactivation of α-amylase Acid carboxypeptidase was inactivated by PEF at 4°C, but activated at 25°C	¹⁰¹
Bovine milk	Alkaline phosphatase Xanthine oxidase Plasmin	25.7 kV/cm, pulses of 20 µs, total treatment time 34 µs, 20 Hz, square wave bipolar pulses	Alkaline phosphatase activity decreased by 96–97%, xanthine oxidase activity by 30%, and plasmin activity by 7% after PEF treatment	¹⁰²
Carrot and apple mashes	Peroxidase (POD) Polyphenol oxidase (PPO)	0.8 kV/cm, 50 pulses of 10 µs, total treatment time 0.5 ms, exponential decay pulses (0.5 kJ/kg)	PEF treatment at 20°C and 40°C had no significant impact on POD activity PEF treatment at 80°C resulted in a 90% reduction in both POD and PPO activities compared with the control	¹⁰³
Phosphate buffer	Papain	10–13 kV/cm, 96–432 pulses, square wave monopolar pulses	Maximum inactivation (64%) was achieved using 13 kV/cm, 288 pulses, and a flow rate of 0.2L/min	⁷²
Apple juice	Polyphenol oxidase (PPO) Peroxidase (POD) Pectin methylesterase (PME)	12.5–40 kV/cm, 50–200 pulses of 2 µs, total treatment time 100–400 µs, 62–94 Hz, square wave bipolar pulses (76.4–132.5 kJ/kg)	12.5 kV/cm (76.4 kJ/L) resulted in a 36% reduction in PPO activity and a 49% reduction in POD activity 12.5 kV/cm (132.5 kJ/L) led to the inactivation of PPO, POD, and PME by over 90%	¹⁰⁴

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PEF technology has been found to both decrease and enhance enzymatic activity depending on the conditions, with enhancement achieved through mechanisms such as triggering or boosting enzymatic activity. The boosting of enzymatic activity through PEF is associated with structural modifications of the enzyme. However, triggering occurs due to cell electroporation, causing the enzyme to uncouple from cell organelles, facilitating contact with the substrate.

The impact of PEF on enzymatic activity can yield opposing outcomes, either stimulating or inhibiting enzymatic activity, depending largely on the electric field intensity. A gradual increase occurred in enzyme activity of α-amylase, β-glucosidase, alcalase, or pectinase with electric field intensity up to 12–15 kV/cm, beyond which the activity declined.^68–71 In contrast, papain activity was inhibited at a relatively lower electric field intensity of 10 kV/cm.⁷² This illustrates that the enzyme type and specific conditions of the PEF treatment can substantially influence the enzyme’s response. While the precise mechanisms behind the PEF-induced increase in enzymatic activity remain elusive, the mentioned studies suggest that these changes likely result from modifications in the secondary and tertiary structures of the enzymes. These modifications can lead to the creation of additional active sites, alterations in the enzyme’s overall globular structure, or modifications of existing active sites. Such changes can also lower the free energy of activation in enzymatic reactions by fostering proper substrate–enzyme orientation.⁷³

The potential of PEF to trigger endogenous enzymatic activity in various cell types has been demonstrated in several studies. Electroporation of the cytoplasmic membrane of S. cerevisiae by PEF not only facilitated the release of intracellular compounds but also triggered yeast autolysis, representing the self-degradation of cellular constituents by its own enzymes.^74,75 This effect is attributed to the release of hydrolytic enzymes such as proteases and β-glucanase from plasmolyzed vacuoles after electroporation. The influx of water, a consequence of uncontrolled molecular transport through the electroporated cytoplasmic membrane of the yeast, decreases the osmotic pressure of the cytoplasm, causing vacuole plasmolysis and enzyme release (Fig. 2).

Fig. 2. — Schematic diagram illustrating the improvement of the extraction of cell compounds by triggering endogenous enzymatic activity through PEF treatment: **(A)** extraction of intracellular and cell wall compounds and **(B)** extraction of lipophilic compounds from fresh biomass using ethanol as a green solvent.

The rapid liberation of mannoproteins from the cell wall of yeast treated by PEF was correlated with the increment of β-glucanase and protease activity in the supernatant containing the electroporated cells (Fig. 2A).⁷⁶ The potential of PEF to reduce the duration of the “aging on the lees” step by triggering S. cerevisiae autolysis has been demonstrated in both white and red wine.^76,77 In contrast, the fact that the amount of total amino acids released from yeast biomass treated by PEF along the incubation time was higher than the total content of amino acids determined before the application of the treatment has been associated with the hydrolysis of proteins by endogenous proteases released from plasmolyzed vacuoles (Fig. 2A).^74,75 Promoting proteolysis by endogenous proteases during extraction by incubating cells under optimal protease conditions could permit obtaining protein-hydrolyzed compounds like essential amino acids or peptides that possess biofunctional properties such as antihypertensive, antioxidant, and antimicrobial effects.^56,78

The extraction of carotenoids from PEF-treated fresh biomass of yeasts and the microalgae using ethanol as a green solvent was also correlated with triggering esterase activity after electroporation.^79–81 Ethanol was ineffective for extracting carotenoids when the electroporated biomass was suspended in this solvent just after the treatment, as the interaction between ethanol and carotenoids was too weak to disrupt the linkage of carotenoids with the cell lipids. However, ethanol was effective after incubation of the PEF-treated biomass in an aqueous medium for several hours. This effect was correlated with the triggering of esterase activity in the electroporated cells, which hydrolyzed the association of carotenoids with lipids (Fig. 2B). In this way, the ethanol-carotenoids complex could diffuse across the electroporated cell membrane driven by a concentration gradient. Esterase activity triggered by PEF also resulted in a positive improvement in the extraction of carotenoids from the dry biomass of electroporated cells yeast using as a solvent ethanol or eutectic mixtures.⁸² The successful enhancement of the lipid bioaccessibility of C. vulgaris biomass by PEF also required incubating the biomass suspension after PEF treatment. Proteome analysis identified four endogenous cell wall-degrading enzymes that may be involved in cell wall lytic activity during incubation after PEF.⁸³

The triggering of the activity of endogenous enzymes enables the extraction of cellular compounds without the need for commercial enzymes and with a minimal environmental footprint for extracting lipophilic compounds, such as carotenoids, from both fresh and dried biomass using eco-friendly solvents like ethanol or eutectic solvents. Consequently, in addition to improving mass transfer through the cytoplasmic membrane, electroporation presents a promising tool to modulate the activity of endogenous enzymes, with wide-ranging applications in the food and biotechnological industries.

Conclusions

PEF represents a valuable tool for the food and biotechnological industry for enhancing the recovery of valuable proteins, improving protein functionality, and inactivating, boosting, or triggering enzymes. These effects may contribute to enhancing food shelf life and quality, as well as improving the sustainability of the food sector by reducing energy costs and contributing to the reuse of by-products generated during food processing.

The mechanisms of action of PEF in improving protein extraction and triggering the enzymatic activity of endogenous enzymes located in the vacuoles are associated with the well-understood electroporation phenomenon. However, broadening its scope as a procedure for modification of the structures and technological functionalities of proteins requires more in-depth studies on the mechanisms involved under appropriately controlled treatment conditions that can discriminate the effects of electric fields from other unwanted side effects that may occur during PEF processing.

Data Availability

No data were used for the research described in the article.

Authors’ Contributions

J.M.-S.: Conceptualization, writing—original draft, and visualization. A.B.: Supervision. I.Á.: Supervision. C.S.-G.: Conceptualization, writing—review and editing, visualization, and supervision. J.R.: Conceptualization, writing—review and editing, visualization, and supervision.

Author Disclosure Statement

The authors affirm that the research was carried out without any affiliations or financial ties that might be perceived as potential conflicts of interest.

Funding Information

The authors express their appreciation for the financial backing received from the PEFREV project (Grant ID: PID2020-113620RB-I00) of the Spanish Research Agency. J.M.-S. and A.B. are thankful for the financial assistance granted by the Ministerio de Ciencia e Innovación and Ministerio de Universidades, (Spain), respectively, which supported their academic pursuits.

Declaration of Generative AI and AI-Assisted Technologies in the Writing Process

During the preparation of this work, the authors used ChatGPT 3.5 in order to improve readability. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

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Data Availability Statement

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PERMALINK

Pulsed Electric Fields Effects on Proteins: Extraction, Structural Modification, and Enhancing Enzymatic Activity

J Marín-Sánchez, MS

A Berzosa, MS

I Álvarez, PhD

C Sánchez-Gimeno, PhD

J Raso, PhD

Abstract

Introduction

Fundamentals of Pulsed Electric Fields Processing

Fig. 1.

Extraction of Proteins Assisted by PEF

Table 1.

Effect of PEF on Structure and Functional Properties of Proteins

Table 2.

Inactivating, Boosting, and Triggering Enzymatic Activity by PEF

Table 3.

Fig. 2.

Conclusions

Data Availability

Authors’ Contributions

Author Disclosure Statement

Funding Information

Declaration of Generative AI and AI-Assisted Technologies in the Writing Process

References

Associated Data

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Cite

Add to Collections

PERMALINK

Pulsed Electric Fields Effects on Proteins: Extraction, Structural Modification, and Enhancing Enzymatic Activity

J Marín-Sánchez, MS

A Berzosa, MS

I Álvarez, PhD

C Sánchez-Gimeno, PhD

J Raso, PhD

Abstract

Introduction

Fundamentals of Pulsed Electric Fields Processing

Fig. 1.

Extraction of Proteins Assisted by PEF

Table 1.

Effect of PEF on Structure and Functional Properties of Proteins

Table 2.

Inactivating, Boosting, and Triggering Enzymatic Activity by PEF

Table 3.

Fig. 2.

Conclusions

Data Availability

Authors’ Contributions

Author Disclosure Statement

Funding Information

Declaration of Generative AI and AI-Assisted Technologies in the Writing Process

References

Associated Data

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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