Effects of Pulsed Electric Field on the Physicochemical and Structural Properties of Micellar Casein.

Pulsed electric field (PEF) as a green processing technology is drawing greater attention due to its eco-friendliness and potential to promote sustainable development goals. In this study, the effects of different electric field strengths (EFS, 0-30 kV/cm) on the structure and physicochemical features of casein micelles (CSMs) were investigated. It was found that the particle sizes of CSMs increased at low EFS (10 kV/cm) but decreased at high EFS (30 kV/cm). The absolute ζ-potential at 30 kV/cm increased from -26.6 (native CSMs) to -29.5 mV. Moreover, it was noticed that PEF treatment leads to changes in the surface hydrophobicity; it slightly increased at low EFS (10 kV/cm) but decreased at EFS > 10 kV/cm. PEF enhanced the protein solubility from 84.9 (native CSMs) to 87.1% (at 10 kV/cm). PEF at low EFS (10 kV/cm) intensified the emission fluorescence spectrum of CSMs, while higher EFS reduced the fluorescence intensity compared to native CSMs. Moreover, the analysis of the Amide Ι region showed that PEF-treated CSMs reduced the α-helix and increased the ß-sheet content. Raman spectra confirmed that PEF treatment > 10 kV/cm buried tyrosine (Tyr) residues in a hydrophobic environment. It was also found that PEF treatment mainly induced changes in the disulfide linkages. In conclusion, PEF technology can be employed as an eco-friendly technology to change the structure and physiochemical properties of CSMs; this could improve their techno-functional properties.

Investigation of osmotic shock effect on pulsed electric field treated S. cerevisiae yeast cells.

Pulsed electric field (PEF) treatment is known to cause plasma membrane permeabilization of microorganisms, an effect known as electroporation. PEF treatment is very attractive since it can achieve permeabilization with or without lethal damage in accordance with desired results. This study aimed to expand the accomplishment of electroporation outcomes by applying sudden post-PEF osmotic composition change of the media. Changes in yeast cells' viability, size and plasma membrane regeneration rate were evaluated. However, we still have questions about the intracellular biochemical processes responsible for plasma membrane recovery after electroporation. Our suggested candidate is the high osmolarity glycerol (HOG) kinase pathway. The HOG pathway in Saccharomyces cerevisiae yeasts is responsible for volume recovery after dangerous shape modifications and intracellular water disbalance caused by environmental osmotic pressure changes. Thus, we evaluated the HOG pathway inactivation effect on S. cerevisiae's reaction to PEF treatment. Results showed that Hog1 deficient S. cerevisiae cells were considerably more sensitive to electric field treatment, confirming a link between the HOG pathway and S. cerevisiae recovery process after electroporation. By suddenly changing the osmolarity of the media after PEF we influenced the cells' plasma membrane recovery rate, severity of permeabilization and survivability of yeast cells. Studies of electroporation in combination with various treatments might improve electric field application range, efficiency, and optimization of the process.

Development of a Disposable Polyacrylamide Hydrogel-Based Semipermeable Membrane for Micro Ag/AgCl Reference Electrode.

Currently, Ag/AgCl-based reference electrodes are used in most electrochemical biosensors and other bioelectrochemical devices. However, standard reference electrodes are rather large and do not always fit within electrochemical cells designed for the determination of analytes in low-volume aliquots. Therefore, various designs and improvements in reference electrodes are critical for the future development of electrochemical biosensors and other bioelectrochemical devices. In this study, we explain a procedure to apply common laboratory polyacrylamide hydrogel in a semipermeable junction membrane between the Ag/AgCl reference electrode and the electrochemical cell. During this research, we have created disposable, easily scalable, and reproducible membranes suitable for the design of reference electrodes. Thus, we came up with castable semipermeable membranes for reference electrodes. Performed experiments highlighted the most suitable gel formation conditions to achieve optimal porosity. Here, Cl- ion diffusion through the designed polymeric junctions was evaluated. The designed reference electrode was also tested in a three-electrode flow system. The results show that home-built electrodes can compete with commercial products due to low reference electrode potential deviation (~3 mV), long shelf-life (up to six months), good stability, low cost, and disposability. The results show a high response rate, which makes in-house formed polyacrylamide gel junctions good membrane alternatives in the design of reference electrodes, especially for these applications where high-intensity dyes or toxic compounds are used and therefore disposable electrodes are required.

Pulsed Electric Field: Fundamentals and Effects on the Structural and Techno-Functional Properties of Dairy and Plant Proteins.

Dairy and plant-based proteins are widely utilized in various food applications. Several techniques have been employed to improve the techno-functional properties of these proteins. Among them, pulsed electric field (PEF) technology has recently attracted considerable attention as a green technology to enhance the functional properties of food proteins. In this review, we briefly explain the fundamentals of PEF devices, their components, and pulse generation and discuss the impacts of PEF treatment on the structure of dairy and plant proteins. In addition, we cover the PEF-induced changes in the techno-functional properties of proteins (including solubility, gelling, emulsifying, and foaming properties). In this work, we also discuss the main challenges and the possible future trends of PEF applications in the food proteins industry. PEF treatments at high strengths could change the structure of proteins. The PEF treatment conditions markedly affect the treatment results with respect to proteins' structure and techno-functional properties. Moreover, increasing the electric field strength could enhance the emulsifying properties of proteins and protein-polysaccharide complexes. However, more research and academia-industry collaboration are recommended to build highly effective PEF devices with controlled processing conditions.

Antimicrobial photodynamic therapy (aPDT) for biofilm treatments. Possible synergy between aPDT and pulsed electric fields.

Currently, microbial biofilms have been the cause of a wide variety of infections in the human body, reaching 80% of all bacterial and fungal infections. The biofilms present specific properties that increase the resistance to antimicrobial treatments. Thus, the development of new approaches is urgent, and antimicrobial photodynamic therapy (aPDT) has been shown as a promising candidate. aPDT involves a synergic association of a photosensitizer (PS), molecular oxygen and visible light, producing highly reactive oxygen species (ROS) that cause the oxidation of several cellular components. This therapy attacks many components of the biofilm, including proteins, lipids, and nucleic acids present within the biofilm matrix; causing inhibition even in the cells that are inside the extracellular polymeric substance (EPS). Recent advances in designing new PSs to increase the production of ROS and the combination of aPDT with other therapies, especially pulsed electric fields (PEF), have contributed to enhanced biofilm inhibition. The PEF has proven to have antimicrobial effect once it is known that extensive chemical reactions occur when electric fields are applied. This type of treatment kills microorganisms not only due to membrane rupture but also due to the formation of reactive compounds including free oxygen, hydrogen, hydroxyl and hydroperoxyl radicals. So, this review aims to show the progress of aPDT and PEF against the biofilms, suggesting that the association of both methods can potentiate their effects and overcome biofilm infections.

Electroporation Assisted Improvement of Freezing Tolerance in Yeast Cells.

Prolonged storage of frozen dough worsens the structure of thawed dough. The main reason is the inhibition of yeast activity. In this study we investigated applicability of pulsed electric field treatment for introduction of cryoprotectant into yeast cells. We showed that pre-treatment of cells suspended in a trehalose solution improves freezing tolerance and results in higher viability after thawing. Viability increased with rise in electric field strength (from 3 to 4.5 kV/cm) and incubation time (from 0 to 60 min) after exposure. Pretreatment resulted in lower decrease in the viability of thawed cells, viability of untreated cells dropped to 10%, while pre-treatment with PEF and trehalose tripled the viability.

Mediated amperometry as a prospective method for the investigation of electroporation.

Pulsed electric field effects induced in a membrane, as well as intracellular structures, depend on cell type, field and media parameters. To achieve desired outcomes, membranes should be permeabilized in a controlled manner, and thus efficiency of electroporation should be investigated in advance. Here, we present a framework for using mediated amperometry as a prospective method for the investigation of electroporation and its effects on cellular machinery. Whole-cell sensors with single mediator systems comprised of hydrophilic or lipophilic mediators were successfully employed to investigate membrane permeability as well as cellular responses. Exposure of yeast cells to single electric field pulse (τ = 300 µs, E = 16 kV/cm) resulted in up to tenfold increase of current strength mediated with hydrophilic mediators. Exposure to PEF resulted in decrease of menadione mediated current strength (from 138 ± 15 to 32 ± 15 nA), which could be completely compensated by supplementing electrolyte with NADH.

The link between yeast cell wall porosity and plasma membrane permeability after PEF treatment.

An investigation of the yeast cell resealing process was performed by studying the absorption of the tetraphenylphosphonium (TPP+) ion by the yeast Saccharomyces cerevisiae. It was shown that the main barrier for the uptake of such TPP+ ions is the cell wall. An increased rate of TPP+ absorption after treatment of such cells with a pulsed electric field (PEF) was observed only in intact cells, but not in spheroplasts. The investigation of the uptake of TPP+ in PEF treated cells exposed to TPP+ for different time intervals also showed the dependence of the absorption rate on the PEF strength. The modelling of the TPP+ uptake recovery has also shown that the characteristic decay time of the non-equilibrium (PEF induced) pores was approximately a few tens of seconds and this did not depend on the PEF strength. A further investigation of such cell membrane recovery process using a florescent SYTOX Green nucleic acid stain dye also showed that such membrane resealing takes place over a time that is like that occurring in the cell wall. It was thus concluded that the similar characteristic lifetimes of the non-equilibrium pores in the cell wall and membrane after exposure  to  PEF indicate a strong coupling between these parts of the cell.

Pulsed electric field effects on inactivation of microorganisms in acid whey.

Prospects of pulsed electric field technology application on acid whey concentrate pretreatment were analyzed. Stationary and flow pre-treatment systems were combined with different treatment parameters: electric field strength (E = 39 kV/cm, 95 kV/cm, 92 kV/cm), pulse duration (τ = 60 ns, 90 ns, 1000 ns) and pulse number (pn = up to 100 pulses). Isolates of Saccharomyces sp. and Lactobacillus sp. were predominant in concentrate. Significant non-thermal inactivation effect was achieved after PEF treatment. Exposure to short pulses selectively inactivated yeast cells, as a result PEF technology can be applied for low-energy acid whey processing.

Caspase dependent apoptosis induced in yeast cells by nanosecond pulsed electric fields.

Saccharomyces cerevisiae yeast cells were used as a model organism to investigate the effects of various pulsed electric fields on the programed death of such cells. These were exposed to electric field pulses with field strengths (E) of up to 220kV/cm. The effects of square shaped pulses having different durations (τ=10-90ns) and different pulse numbers (pn=1-5) were then analysed. The obtained results show that nanosecond pulses can induce the death of such cells, which in turn is dependent on the electric field pulse parameters and increase with the rise in E, τ and pn. The decrease of the cells' viability was accompanied by an increase in the active form of intracellular yeast metacaspases. It was thus shown that nanosecond electric field pulses induced the caspase-dependent yeast cell death.