Pulsed electric fields (PEF): technology, role in food science and emerging applications

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Recommendations guidelines on the key information to be reported in studies of application of PEF technology in food and biotechnological processes. The application of pulsed electric field PEF technology as a non-thermal cell membrane permeabilization treatment, was widely demonstrated widely to be effective in microbial inactivation studies, as well as to increase the rates of heat and mass transfer phenomena in food and biotechnological processes drying, osmotic treatment, freezing, extraction, and diffusion.

As a result, the biological material is exposed to an electric field whose intensity depends on the voltage across the electrodes, as well as on the geometry of both the electrodes and the interelectrode space. E-mail address: justin. Depending on the intensity of the treatment applied external electric field, single pulse duration, treatment time and the cell characteristics size, shape, orientation in the electric field , the viability of the electroporated cell can be preserved by recovering the membrane integrity, or the electroporation can permanently lead to cell death.

Cell size differences between plant and microbial cells, give a wide range of treatment intensity: 0. Reversible "electroporation" is a procedure usually used in molecular biology and clinical biotechnological applications in vivo to gain access to the cytoplasm in order to introduce or deliver in vivo drugs, oligonucleotides, antibodies, plasmids, etc. Miklavcic, Mali,. Technical limitations impeded the exploitation of PEF at an industrial level during many years.

The lack of reliable and viable industrial equipment was indeed a critical factor to support up-scaling of the volumes to be treated from mL to m3 Sack et al. Large treatment volumes required a shift from the well-established batch methodologies used in basic science towards the flow processes, which is nowadays possible due to the recent developments in pulsed power generators. Other critical aspects that have contributed to limit the spread of PEF technology, are the poor description of the operating protocols, the control and monitoring of the pulse parameters, and the lack of a standardized way of reporting treatment conditions.

As the first commercial applications of PEF technology are now available Golberg et al. Lack of such information is a barrier for the development and wide use of the technology. Variability in results obtained in different laboratories on PEF research may be a consequence of a number of reasons including differences in PEF equipment and PEF treatment conditions. Additionally, in microbial inactivation works and in algae processing, the growth or cultivation conditions of the microorganisms, the treatment medium and the recovery conditions, can play an important role to the outcome of the process.

Furthermore, pre and post-treatment conditions can considerably influence the efficiency of the PEF-assisted mass transfer processing. Different aspects of experimental procedures biological and engineering must be described in sufficient details to allow the work to be reproduced in other laboratories. All data must be obtained by paying attention to statistical detail in the planning stage. If a sufficiently large number of replicates are not organized before the experiment is undertaken, biological variation is not eliminated satisfactorily.

Replicate design has been recognized to be important to biological experiments for a considerable time Dhand, ; McNutt, Specifically, it has been adopted by the committee within the Working group of Food and Biotechnology of electro-poration, in COST action TD EP4Bio2Med Miklavcic, , and is in the series of publications that addresses the same topic in Preclinical research in electroporation as well as in the field of medical use of elec-troporation Campana et al.

The objective of this paper is to provide recommendation guidelines on the key information that should be reported in studies regarding the application of PEF for microbial inactivation or PEF-assisted processing in food and biotechnological field. These guidelines are intended to facilitate the comparison of data, to create a reliable basis for a better. It can also be expected that this report may help new researchers in the field to obtain data which are repeatable, reproducible and free from methodological errors.

The most typical process parameters that characterize PEF technology are amplitude of electric pulses U , electric field strength E , treatment time t , pulse shape, pulse width t , number of pulses n , pulse specific energy W and pulse repetition frequency f. The electric field strength and the treatment time are the main process parameters that define the PEF treatment intensity. Electric field strength refers to the field strength locally present in the treatment chamber during the sample treatment, and depends on the voltage applied between the electrodes, geometry of the treatment chamber, and the spatial distribution of dielectric properties of the material between the electrodes.

In contrast, other chamber configurations, such as co-linear electrode configuration Fig. Treatment time refers to the number of pulses applied multiplied by the pulse width or pulse duration :. As it is shown in Fig. Voltage and current waveforms of the electric pulses delivered in the treatment chamber, should be monitored continuously using high voltage and fast high current probes located as close as possible to the treatment chamber, in order to precisely define the treatment intensity. Generally, in fact, the voltage output from the pulse power is lower than the voltage measured in the treatment chamber, especially for chamber configuration characterized by a low intrinsic electrical resistance.

Thus, in order to accurately describe processing conditions, pulse characteristics, including peak voltage, pulse shape, pulse width, and pulse polarity, should be reported. To this purpose, a snapshot of the monitored pulses voltage, current delivered to the treatment chamber, should be provided, which implies that a digitized recording is included in the set-up of the PEF system.

In the case of exponential decay. Schematics of parallel plate and co-linear treatment chamber configuration with qualitative distribution of the electric field in the treatment zone. Frequency and protocol of application of the series of pulses should be also documented. The specification of the pulse frequency is important, since it determines the. This is a very important issue, since the metal released may affect microbial inactivation or may further react with the biomaterials present in the bulk, also after the application of the pulse treatment, as well as negatively affect the efficiency of the PEF treatment with time, and reduce the electrode lifetime corrosion.

In addition to pulse frequency, pulse protocol should be also described in detail. For batch treatment, number of pulses applied per each train, number of trains of pulses and time interval between two consecutive trains, should be reported. For continuous flow treatment, the number of recirculation of the treated product through the PEF chamber, should also be specified. Moreover, it is worth remembering that, in batch treatment the number of electric pulses to be applied is set directly by the user.

In continuous flow process, instead, it is a function of the pulse frequency and residence time tr of the product in the treatment chamber, which depends on the flow rate F and volume v of the treatment zone, according to the following equation:. It depends on the electrical properties of the treated product and on the pulse shape including peak voltage and pulse width. The electrical properties of treated product are changing —. Due to the losses through the connections and the components of the discharge circuit, the value of W is generally different from the energy output from the pulse generator.

Moreover, waveforms of voltage and current can considerably deviate from the ideal square or exponential shapes. Therefore, according to Eq. R in fl is the electrical resistance of the treatment chamber, which can be calculated according to the following equation:. In any case, in batch treatments the initial temperature of the product as well as the final temperature after the application of the PEF treatment should be documented.

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An adequate description of the methods used for pre-heating the product before entering the PEF treatment chamber, as well as for cooling the treated product at the exit of the PEF chamber, should also be provided. Moreover, it is also necessary to specify the location of the temperature sensors in relation to the treatment chamber. When the experimental setup consists in several continuous flow treatment chambers connected in series, the temperature sensors should be located immediately before and after each treatment chamber, especially when the treated product is cooled in heat exchangers placed in between two consecutive treatment chambers.

Temperature sensors whose measurement is not influenced by the electric field should be used. If a direct temperature measurement is not possible, the resulting temperature increase of the treated product can be estimated based on a calculation of the total specific energy and assuming adiabatic heating, i. Particularly, total specific energy input should be preferred instead of treatment time especially when exponentially decay pulses are applied. Furthermore, the specification of the total energy input will also give an estimation of the energy consumption due to the PEF process.

Temperature is also a critical parameter that influences the efficacy of PEF treatment. On the other hand, the dissipation of the electrical energy delivered to the product during PEF treatment increases the temperature of the product, which in turn increases the electrical conductivity and may modify product viscosity.

As a consequence, the increment of the product temperature may lower the resistance of the treatment chamber, leading to a decrease of the applied field strength, unless the external voltage is not increased accordingly. In addition, in continuous processes, flow rate and residence time of the processed product in the treatment chamber may also change as a result of the product temperature increase. It has been demonstrated that using static parallel electrode treatment chamber with temperature-controlled electrodes allows to obtain data on microbial inactivation at different temperatures at quasi-isothermal conditions preventing the artefacts caused by temperature increase when no temperature control of electrodes is used Saldana et al.

Temperature increase, as a consequence of Joule heating, is enhanced at higher electric fields, total specific energy, frequencies and pulse widths. Therefore, optimal processing conditions for studying. An appropriate description of the PEF generator and treatment chamber used to conduct the experiments should be provided. For commercial equipment, the name of the supplier company and the model should be specified. If the PEF generator is a laboratory prototype or specially fabricated unit, an adequate description of the components power supply, capacitors, switches, transformers, etc , electrical configuration, measurement and data acquisition systems, and any other pertinent information that characterizes the equipment to reproduce exposure of sample to pulsed electric field should be provided.

Laboratory studies on either microbial inactivation or improving mass transfer phenomena by PEF may be conducted in batch or in continuous flow treatment chambers that should be described in details.

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The two most used treatment chamber designs considered for application of PEFs in continuous flow are parallel plate electrodes and colinear configurations Fig. Parallel plate electrode configuration is the simplest in design and consists of a rectangular parallelepiped shape channel of insulating material with two electrodes on opposite sides.

As previously reported, this configuration typically provides uniform electric field in the treatment zone, with the applied electric field being perpendicular to the product flow. However, because it is characterized by a large electrode surface and low intrinsic electrical resistance, it generally operates at high current, which also may facilitate the triggering of undesired electrochemical phenomena at the electrode-liquid food interface of the PEF chamber electrode corrosion. In the co-linear configuration, couples of tubular electrodes are spaced with insulator spacer tubes.

The product is treated as it flows from one electrode to the other, parallel to the electric field. Such configuration has advantageous fluid dynamics, highly desiderate for food processing and convenient for cleaning in place, as well as a high intrinsic resistance due to the low effective area of the cross section of the tubular electrodes.

Thus, this device typically operates at lower current than the parallel plate configuration, which makes it suitable for limiting the occurrence of electrode reactions, as well as for the connection of multiple co-linear units in parallel from the electrical viewpoint. The main problem of this configuration is in-homogeneity in the electric field strength and temperature distribution in the treatment zone during PEF processing. Therefore, an adequate chamber design is required in order to ensure more uniform distribution of the electric field. A further cause of treatment inhomogeneity in both parallel plate electrode and co-linear treatment chamber, which is most important in microbial inactivation studies, is the existence of laminar flow into the treatment zone.

This is because the higher flow rate required to promote turbulent flow conditions needs a higher pulse modulator power, as well as a higher commutation rate of the switching devices, in order to deliver the required amount of energy per volume element. As already recommended for power supplies, the name of the supplier company and the model number should be specified for commercial treatment chambers. If the treatment chamber is a prototype or specially fabricated, an adequate description is required.

A schematic drawing of the treatment chamber details, which describes the geometrical shape of insulator and electrode along the boundary to the material to be treated, should be provided. Additionally, the material of the electrodes and insulators, and the most relevant sizes such as electrode gap, surface area or dimensions of the electrodes e.

PEF processing for industrial applications requires continuous flow processing, thus the results obtained in batch treatments need to be validated in a continuous flow installation before they can be successfully implemented on a large scale. In this frame, however, further studies based on the development and application of characteristic dimensionless numbers are necessary. A detailed knowledge or a good estimation of the values assumed to be the critical process parameters inside the treatment chamber during processing of biological matrices is required.

However, the small dimensions of the treatment chambers may make impossible to perform adequate measurements of the process parameters inside the treatment chamber with the corresponding probes without perturbation of the flow, temperature, and electric field distribution Jaeger et al.

Therefore, it is recommended to use numerical simulation techniques to provide information on the spatial and temporal distribution of the electric field strength, temperature, and flow velocity inside the treatment chambers. Moreover, it is worth noting that a numerical approach would also allow the use of local and time resolved information, which could help in obtaining insight in the mechanisms of action of the PEF technology, with respect to an analyses based only on integral values.

Many studies on microbial inactivation by PEF have been conducted and reported in the literature. The technology of PEF follows general principles, however the numerous factors affecting microbial inactiva-tion by PEF, the broad experimental conditions used by different research groups and the diversity of equipment available limits the comparison of results, the standardization of experimental procedures used in different laboratories and obtaining solid conclusions in this topic.

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Due to the difficulty to standardize experimental procedures used in different laboratories, information that should be provide for researchers in any study of microbial inactivation by PEF are shown in Table 1. Therefore the strain of the. Genus, species and strain of the microorganisms Culture conditions Initial inoculum o Description of the procedure for microbial cultivation. Recommended information to be reported in studies of PEF-assisted processing for improving heat and mass transfer phenomena in food and biotechnological processes.

Raw material Origins, variety, maturation and storage conditions of plant matrices and cell microbial, algae suspensions. It should be desirable that that the strain or strains used in the study should be available for other researchers. The preparation of the microbial culture and the storage conditions can significantly affect the microbial sensitivity to PEF. Therefore, the cultivation of the microorganism should be standardized to minimize its influence on variability between repeated experiments either from day to day, or from test period to test period.

Initial inoculum, growth medium composition, growth temperature, time of incubation and growth phase of the cells used for inactivation experiments should be reported. Lipid composition variations in the cytoplasmic membrane induced by modifications of the growth temperature have been suggested as the origin of the distinct PEF sensitivity. At lower growth temperatures the degree of fatty acid insaturations of the phospholipids of the cell membrane raises which could increase the fluidity of the bacterial cell membrane and increased its sensitivity to electroporation.

The treatment medium used for the inactivation studies should be well defined to allow reproduction in other laboratories. Composition of the treatment medium should be reported and factors that may affect microbial inactivation such as pH, conductivity, activity of water osmo-larity , as well as presence of preservatives should be measured and reported. For inactivation studies it has been recommended a minimum of three replicate sets per trial repeated on separated days in order to be able to measure the experimental error as well as differences.

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For testing microbial resistance to a lethal treatment such as PEF, acquisition of multiple data points along the time for a given electric field strength is preferred because they give more information than end-point measurements based on the inactivation produced by a given treatment. The acquisition of multiple data points permits the elaboration of the survival curves in which the logarithmic of survivors is plotted against inactiva-tion time for a given treatment intensity. Modelling kinetics data obtained under different experimental conditions and developing of predictive models are very useful tools for quantifying the influence of different factors on microbial inactivation by PEF, as well as to define equivalent treatment conditions to achieve a given level of inactivation.

Quantification of the survivors after the treatment is one of the most important factors in estimating the efficacy of an inactivation technique such as PEF.

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It is important to use procedures that recover the greatest number of microorganisms. Recovery medium, incubation time and temperature during incubation should be reported because they have a significant effect on the number of microorganisms recovered after the PEF treatment. The time and storage conditions between treatment and microbiological analysis should also be reported.

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Comparison of cell counts of PEF treated samples on selective and nonselective media is the most conventional technique to detect suble-thal injury. Sublethally injured population fails to survive and multiply in harsh environments tolerant by untreated cells Mackey, If the existence of sub-lethal injured microorganisms is detected by adding selective agents in the recovery medium it is necessary to establish previously the maximum concentration of the selective agent that has not inhibitory effect on untreated cells. The selective agent and the concentration used for detecting sub-lethal injured microorganisms need to be given.

Generally longer incubation times are required when microorganism are plated on selective media because inactivation may be overestimated when the incubation time is the same in nonselective and selective media. PEF-assisted processing for improving mass transfer phenomena in food and biotechnological processes. The application of PEF as a mild cell disintegration technique for improving food quality, accelerate heat transfer process, as well as mass transfer efficiency of target compounds from matrices of biological.

However, similarly to the application of PEF for microbial inactiva-tion, it is difficult to compare between studies of different groups, due to the large number of parameters, which are interrelated, as well as the large variety of experimental conditions and equipment used by several researchers. For these reasons, in Table 2 we summarize the main information regarding raw material characteristics, the upstream processes e. This information is essential to allow standardization of experimental procedures and reproducibility of the experiments in view of the utilization of bench-scale data on PEF assisted processing to define processing conditions in commercial size equipment.

Information on raw materials is very important since it can contribute to define the optimal processing conditions as well as the properties of the final product. Therefore, for the case of raw material of plant origin, information such as geographical origin, variety, degree of ripeness, moisture content, as well as storage time and conditions e. Similarly, in the case of cell microbial, algae in suspension, detailed description of the genus, species, strain number and source of supply, as well as growth or cultivation conditions should be provided, as reported in detail in the Section 3.

Raw materials are typically pre-treated before PEF-assisted processing with the aim of softening or hydrating biomaterial, reducing the particle size, increasing the surface-volume-ratio, or to induce mixture densification. For example, raw materials of plant origin are typically subjected to peeling, slicing, mechanical grinding or pre-heating. In the case of cell suspensions, a concentration step of the biomass could be required. Therefore, detailed information on the features of the equipment used name of the manufacturer and model and processing conditions should be specified.

In the case of cell suspensions, the type, pH and electrical conductivity of suspending medium, as well as the cell concentration for studies on microbial cells , should be reported. For microalgae processing the component yield per kg of biomass dry weight in suspension is in focus. Thus, the content of biomass dry weight in the treated suspension is a mandatory value to be reported in experimental studies. Also component yields have to be related to the processed biomass dry weight, as usual in the microalgae processing community. Finally, it is worth noting that raw material pre-treatment may also cause partial or total cell disintegration Jaeger et al.

Therefore, the impact of conventional pre-treatment on cell membrane disruption should be reported in order to be discriminated from that of the PEF treatment. As previously reported, an appropriate description of the PEF generator, treatment chamber and auxiliary devices e. Moreover, also initial voltage applied, pulse shape, pulse width, number of pulses or treatment time, frequency, pulse protocol, initial and final temperature for batch processes, and mass flow, residence time, inlet and outlet temperature for continuous flow chamber, should be also specified.

The design and the operating mode of the equipment to be used for processing of the electroporated matrices, may play an important role for the exploitation of the potential benefits that may result from PEF pre-treatment. In addition, the results achieved from the PEF-assisted processing investigations that are typically used to compare data obtained from different studies, are generally collected after the characterization of the final product. It is, therefore, crucial to provide detailed information on the equipment manufacturer, model , the experimental conditions, the protocol analysis and methods used in the downstream process Table 2.

For example, in the case of the extraction processes by mechanical pressing, a detailed description of the type of press as well as the pressing conditions e. When the extraction process following the PEF pre-treatment is carried out by using solvents, a detailed description of the type of extractor, as well as information on the type of solvent e.

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If the extract solution requires a further purification stage before analyses, detailed information on the type of the devices and protocols of purification adopted, should be also reported. In PEF assisted drying processes thermal drying, osmotic dehydration, freeze-drying information on the type of dryer, initial temperature and moisture content of the biomaterial, thermodynamic properties of hot air e. In this paper, basic principles of PEF technology and its application in food and biotechnological processes have been reviewed, and the main problems that a researcher may encounter when conducting experiments with the PEF technology, have been described.

This paper provides recommendations for standardization of research methodology, as well as key information that should be reported in studies regarding. It is expected that this paper will contribute to improve the current state of knowledge on electroporation mechanisms, and to identify the critical factors affecting electropora-tion, with final objective of extending the commercial exploitation of PEF processing in the food and biotechnological industries.

Environmental factors influencing the inactivation of Listeria monocytogenes by pulsed electric fields. Letters in Applied Microbiology, 35, Balasubramaniam, V. Recommended laboratory practices for conducting high-pressure microbial inactivation experiments. Barba, F. Current applications and new opportunities for the use of pulsed electric fields in food science and industry. Food Research International, 77, Application of pulsed electric field in the production of juice and extraction of bioactive compounds from blueberry fruits and their by-products.

Journal of Food Science and Technology.

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Campana, L. Sersa, G. Recommendations for improving the quality of reporting clinical electrochemotherapy studies based on qualitative systematic review. Longer pulses permit reducing the number of pulses to be applied for a given treatment time. Therefore in a continuous process, where the flow processed is determined by the frequency applied by the PEF generator, it is possible to increase the processing capacity of the PEF installation. National Center for Biotechnology Information , U. Sponsored Document from. Innov Food Sci Emerg Technol.

Author information Article notes Copyright and License information Disclaimer. Javier Raso: se. This article has been cited by other articles in PMC. Industrial relevance Benefits from PEF treatment of the grapes before the maceration step in the vinification process have been demonstrated. Introduction A driving force to maintain and enhance the competitiveness of food industry is technological innovation.

Material and methods 2. Grape samples Grapes from Vitis vinifera L. Each vinification was carried out by triplicate. Results and discussion 3. Grape characterization Experiments to define PEF processing parameters that cause the required electroporation in the grape skin cell to obtain a significant improvement polyphenol release during vinification were conducted with three different grape varieties and, in the case of Grenache, with grapes harvested in two different moments.

Open in a separate window. Influence of PEF treatments of different electric field strength and pulse width on the extraction of polyphenols from different grapes varieties in ethanol solution Fig. References Abram F. Modelling and optimization of inactivation of Lactobacillus plantarum by pulsed electric field treatment. Journal of Applied Microbiology. Bazhal M. Optimisation of pulsed electric field strength for electroplasmolysis of vegetable tissues.

Biosystems Engineering. Cholet C. Structural and biochemical changes induced by pulsed electric field treatments on cabernet sauvignon grape berry skins: Impact on cell wall total tannins and polysaccharides. Journal of Structure and Food Chemistry.

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