Pulsed Power Applications for Protein Conformational Change and the Permeabilization of Agricultural Products.

Pulsed electric fields (PEFs), which are generated by pulsed power technologies, are being tested for their applicability in food processing through protein conformational change and the poration of cell membranes. In this article, enzyme activity change and the permeabilization of agricultural products using pulsed power technologies are reviewed as novel, nonthermal food processes. Compact pulsed power systems have been developed with repetitive operation and moderate output power for application in food processing. Firstly, the compact pulsed power systems for the enzyme activity change and permeabilization are outlined. Exposure to electric fields affects hydrogen bonds in the secondary and tertiary structures of proteins; as a result, the protein conformation is induced to be changed. The conformational change induces an activity change in enzymes such as α-amylase and peroxidase. Secondly, the conformational change in proteins and the induced protein functional change are reviewed. The permeabilization of agricultural products is caused through the poration of cell membranes by applying PEFs produced by pulsed discharges. The permeabilization of cell membranes can be used for the extraction of nutrients and health-promoting agents such as polyphenols and vitamins. The electrical poration can also be used as a pre-treatment for food drying and blanching processes. Finally, the permeabilization of cell membranes and its applications in food processing are reviewed.

Microbial current production from Streptococcus mutans correlates with biofilm metabolic activity.

Once pathogens form a biofilm, they become more tolerant to drugs and quicker to recover from physical removal than planktonic cells. Because such robustness of a biofilm is associated with the active metabolism of its constituent microbes, establishment of a direct assay quantifying biofilm's metabolic activity is important for developing antibiofilm substrates and techniques. Current production capability via extracellular electron transport (EET) was recently found in Gram-positive pathogens, which we hypothesized to correlate with the metabolic activity of their biofilm. Here, we identified current production from the biofilm of oral pathogen Streptococcus mutans that enables the electrochemical assessments of their metabolic activity in situ which conventionally require gene insertion for a fluorescent protein expression. Single-potential amperometry (SA) showed that S. mutans produced an anodic current and formed a biofilm within 8 h on a +0.4 V electrode vs a standard hydrogen electrode (SHE) in the presence of the electron donor glucose. Current production was significantly decreased by the addition of a metabolic inhibitor Triclosan. Furthermore, the anabolic activity of a single cell using high-resolution mass spectroscopy revealed that higher current production resulted in a higher metabolic fixation of an atomically labeled nitrogen 15N. These results demonstrate that current production in S. mutans reflects its metabolic activity. Given electrochemical impedance spectroscopy (EIS) helps quantifying the bacterial cell adhesion on the electrode, combination of EIS and SA could be a novel assay for EET capable pathogens for quantifying their time-dependent metabolic activity, cellular electrode coverage and physiological response to antibiofilm compounds.

Medium's conductivity and stage of growth as crucial parameters for efficient hydrocarbon extraction by electric field from colonial micro-algae.

The green algae Botryococcus braunii produces a high amount of extracellular hydrocarbon, making it a promising algae in the field of bio-fuels production. As it mainly produces squalene like hydrocarbons, cosmetic industries are also interested in its milking. Pulsed electric fields (PEF) are an innovative method allowing oil extraction from micro-algae. In common algae accumulating hydrocarbon inside cytoplasm (Chlorella vulgaris, Nannochloropsis sp., etc), electric fields can destroy cell membranes, allowing the release of hydrocarbon. However, for B.braunii, hydrocarbons adhere to the cell wall outside of cells as a matrix. In a previous article we reported that electric fields can unstick cells from a matrix, allowing hydrocarbon harvesting. In this work, we deeper investigated this phenomenon of cell hatching by following 2 parameters: the conductivity of the medium and the cultivation duration of the culture. Cell hatching is accurately evaluated by both microscopic and macroscopic observations. For high conductivity and a short time of cultivation, almost no effect is observed even after up to 1000 PEF pulses are submitted to the cells. While lower conductivity and a longer cultivation period allow strong cell hatching after 200 PEF pulses are applied to the cells. We identify 2 new crucial parameters, able to turn the method from inefficient to very efficient. It might help companies to save energy and money in case of mass production.

Spatio-temporal dynamics of calcium electrotransfer during cell membrane permeabilization.

Pulsed electric fields (PEFs) are applied as physical stimuli for DNA/drug delivery, cancer therapy, gene transformation, and microorganism eradication. Meanwhile, calcium electrotransfer offers an interesting approach to treat cancer, as it induces cell death easier in malignant cells than in normal cells. Here, we study the spatial and temporal cellular responses to 10 µs duration PEFs; by observing real-time, the uptake of extracellular calcium through the cell membrane. The experimental setup consisted of an inverted fluorescence microscope equipped with a color high-speed framing camera and a specifically designed miniaturized pulsed power system. The setup allowed us to accurately observe the permeabilization of HeLa S3 cells during application of various levels of PEFs ranging from 0.27 to 1.80 kV/cm. The low electric field experiments confirmed the threshold value of transmembrane potential (TMP). The high electric field observations enabled us to retrieve the entire spatial variation of the permeabilization angle (θ). The temporal observations proved that after a minimal permeabilization of the cell membrane, the ionic diffusion was the prevailing mechanism of the delivery to the cell cytoplasm. The observations suggest 0.45 kV/cm and 100 pulses at 1 kHz as an optimal condition to achieve full calcium concentration in the cell cytoplasm. The results offer precise levels of electric fields to control release of the extracellular calcium to the cell cytoplasm for inducing minimally invasive cancer calcium electroporation, an interesting affordable method to treat cancer patients with minimum side effects.

A new mechanism for efficient hydrocarbon electro-extraction from Botryococcus braunii.

BACKGROUND: Recent understanding that specific algae have high hydrocarbon production potential has attracted considerable attention. Botryococcus braunii is a microalga with an extracellular hydrocarbon matrix, which makes it an appropriate green energy source. RESULTS: This study focuses on extracting oil from the microalgae matrix rather than the cells, eliminating the need for an excessive electric field to create electro-permeabilization. In such a way, technical limitations due to high extraction energy and cost can be overcome. Here, nanosecond pulsed electric fields (nsPEF) with 80 ns duration and 20-65 kV/cm electric fields were applied. To understand the extraction mechanism, the structure of the algae was accurately studied under fluorescence microscope; extraction was quantified using image analysis; quality of extraction was examined by thin-layer chromatography (TLC); and the cell/matrix separation was observed real-time under a microscope during nsPEF application. Furthermore, optimization was carried out by screening values of electric fields, pulse repetition frequencies, and energy spent. CONCLUSIONS: The results offer a novel method applicable for fast and continues hydrocarbon extraction process at low energy cost.

E. coli electroeradication on a closed loop circuit by using milli-, micro- and nanosecond pulsed electric fields: comparison between energy costs.

One of the different ways to eradicate microorganisms, and particularly bacteria that might have an impact on health consists in the delivery of pulsed electric fields (PEFs). The technologies of millisecond (ms) or microsecond (µs) PEF are still well known and used for instance in the process of fruit juice sterilization. However, this concept is costly in terms of delivered energy which might be too expensive for some other industrial processes. Nanosecond pulsed electric fields (nsPEFs) might be an alternative at least for lower energetic cost. However, only few insights were available and stipulate a gain in cost and in efficiency as well. Using Escherichia coli, the impact of frequency and low rate on eradication and energy consumption by msPEF, µsPEF and nsPEF have been studied and compared. While a 1 log10 was reached with an energy cost of 100 and 158 kJ/L with micro- and millisecond PEFs respectively, nsPEF reached the reduction for similar energy consumption. The best condition was obtained for a 1 log10 deactivation in 0.5h, for energy consumption of 143 kJ/L corresponding to 0.04 W · h when the field was around 100 kV/cm. Improvement can also be expected by producing a generator capable to increase the electric field.