Characteristics of Quinoa Protein Isolate Treated by Pulsed Electric Field.

The aim of this study was to investigate the impact of a pulsed electric field (PEF) on the structural and functional properties of quinoa protein isolate (QPI). The findings revealed a significant alteration in the secondary structure of QPI following PEF treatment, converting the random coil into the ß-sheet, resulting in an improvement in structure orderliness and an enhancement of thermal stability. The PEF treatment led to a reduction in particle size, induced structural unfolding, and increased the surface hydrophobicity, resulting in a statistically significant enhancement in the solubility, foaming, and emulsifying properties of QPI (p < 0.05). Specifically, PEF treatment at 7.5 kV/cm for 30 pulses was identified as the optimal condition for modifying QPI. This study provides a basis for the precision and range of application of pulsed electric field treatment and offers the possibility of improving the physical and chemical properties of quinoa protein.

Effect of magnetic field-assisted fermentation on the in vitro protein digestibility and molecular structure of rapeseed meal.

BACKGROUND: There has been a significant growth in demand for plant-derived protein, and this has been accompanied by an increasing need for sustainable animal-feed options. The aim of this study was to investigate the effect of magnetic field-assisted solid fermentation (MSSF) on the in vitro protein digestibility (IVPD) and functional and structural characteristics of rapeseed meal (RSM) with a mutant strain of Bacillus subtilis. RESULTS: Our investigation demonstrated that the MSSF nitrogen release rate reached 86.3% after 96 h of fermentation. The soluble protein and peptide content in magnetic field feremented rapeseed meal reached 29.34 and 34.49 mg mL-1 after simulated gastric digestion, and the content of soluble protein and peptide in MF-FRSM reached 61.81 and 69.85 mg mL-1 after simulated gastrointestinal digestion, which significantly increased (p > 0.05) compared with the fermented rapeseed meal (FRSM). Studies of different microstructures - using scanning electron microscopy (SEM) and atomic force microscopy (AFM) - and protein secondary structures have shown that the decline in intermolecular or intramolecular cross-linking leads to the relative dispersion of proteins and improves the rate of nitrogen release. The smaller number of disulfide bonds and conformational alterations suggests that the IVPD of RSM was improved. CONCLUSIONS: Magnetic field-assisted solid fermentation can be applied to enhance the nutritional and protein digestibility of FRSM. © 2024 Society of Chemical Industry.

Selective extraction of recombinant membrane proteins from Hansenula polymorpha by pulsed electric field and lytic enzyme pretreatment.

BACKGROUND: In yeast, recombinant membrane proteins including viral scaffold proteins used for the formation of enveloped Virus-like particles (eVLPs) typically accumulate intracellularly. Their recovery is carried out by mechanical disruption of the cells, often in combination with detergent treatment. Cell permeabilization is an attractive alternative to mechanical lysis because it allows for milder and more selective recovery of different intracellular products. RESULTS: Here, we present a novel approach for extraction of integral membrane proteins from yeast based on cell envelope permeabilization through a combination of pulsed electric field and lytic enzyme pretreatment of the cells. Our primary experiments focused on Hansenula polymorpha strain #25-5 co-expressing the integral membrane small surface protein (dS) of the duck hepatitis B virus and a fusion protein of dS with a trimer of a Human papillomavirus (HPV) L2-peptide (3xL2-dS). Irreversible plasma membrane permeabilization was induced by treating the cell suspension with monopolar rectangular pulses using a continuous flow system. The permeabilized cells were incubated with lyticase and dithiothreitol. This treatment increased the cell wall permeability, resulting in the release of over 50% of the soluble host proteins without causing significant cell lysis. The subsequent incubation with Triton X-100 resulted in the solubilization and release of a significant portion of 3xL2-dS and dS from the cells. By applying two steps: (i) brief heating of the cells before detergent treatment, and (ii) incubation of the extracts with KSCN, an 80% purity on the protein level has been achieved. Experiments performed with H. polymorpha strain T#3-3, co-expressing dS and the fusion protein EDIIIWNV-dS consisting of dS and the antigen from the West Nile virus (WSV), confirmed the applicability of this approach for recovering dS. The treatment, optimal for solubilization of 3xL2-dS and a significant part of dS, was not effective in isolating the fused protein EDIIIWNV-dS from the membranes, resulting in its retention within the cells. CONCLUSIONS: This study presents an alternative approach for the recovery and partial purification of viral membrane proteins expressed in H. polymorpha. The factors influencing the effectiveness of this procedure and its potential use for the recovery of other integral membrane proteins are discussed.

Application of electric field treatment (EFT) for microbial control in water and liquid food.

Water disinfection and food pasteurization are critical to reducing waterborne and foodborne diseases, which have been a pressing public health issue globally. Electrified treatment processes are emerging and have become promising alternatives due to the low cost of electricity, independence of chemicals, and low potential to form by-products. Electric field treatment (EFT) is a physical pathogen inactivation approach, which damages cell membrane by irreversible electroporation. EFT has been studied for both water disinfection and food pasteurization. However, no study has systematically connected the two fields with an up-to-date review. In this article, we first provide a comprehensive background of microbial control in water and food, followed by the introduction of EFT. Subsequently, we summarize the recent EFT studies for pathogen inactivation from three aspects, the processing parameters, its efficacy against different pathogens, and the impact of liquid properties on the inactivation performance. We also review the development of novel configurations and materials for EFT devices to address the current challenges of EFT. This review introduces EFT from an engineering perspective and may serve as a bridge to connect the field of environmental engineering and food science.

Insights of keratin geometry from agro-industrial wastes: A comparative computational and experimental assessment.

Understanding the structural properties of keratin is of great importance to managing their potential application in keratin-inspired biomaterials and its management of wastes. In this work, the molecular structure of chicken feather keratin 1 was characterized by AlphaFold2 and quantum chemistry calculation. The predicted IR spectrum of the N-terminal region of feather keratin 1, consisting of 28 amino acid residues, was used to assign the Raman frequencies of the extracted keratin. The MW of experimental samples were 6 & 1 kDa while the predicted MW (∼10 kDa) of ß-keratin. Experimental analysis shows the magnetic field treatment could affect the functional and surface structural properties of keratin. The particle size distribution curve illustrates the dispersion of particle size concentration, while TEM analysis demonstrates the reduction of particle diameter to 23.71 ± 1.1 nm following treatment. High-resolution XPS analysis confirmed the displacement of molecular elements from their orbital.

Combination with litchi procyanidins under PEF treatment alters the physicochemical and processing properties of inulin.

A novel alternative to prepare the inulin-procyanidin complex assisted by pulsed electric field (PEF) treatment was explored in this study. Results showed that the optimal condition of PEF treatment enhanced the adsorption rate of procyanidins to inulin from 78.56 to 103.46 µg/mg. Based on well fitted by Redlich-Peterson model and spectral analysis including UV and FT-IR, the interaction between inulin and procyanidin was evidenced to be dominated by hydrogen bonds. The DSC curve and the SEM spectrum displayed better stability of the PEF-treated inulin-procyanidin complex than the untreated complex. The PEF-treated complex had lower solubility but higher water-holding capacity than inulin, which exhibited stronger shear-thinning property and more stable flow behavior referring to rheological analysis. Furthermore, the gel formed from the PEF-treated complex possessed greater hardness, chewiness and viscosity, with no significant effects noted in terms of springiness, cohesiveness and resilience.

Biological signalling supports biotechnology - Pulsed electric fields extract a cell-death inducing factor from Chlorella vulgaris.

Compared to mechanical extraction methods, pulsed electric field (PEF) treatment provides an energy-efficient and gentle alternative. However, the biological processes involved are poorly understood. The unicellular green microalga Chlorella vulgaris was used as model organism to investigate the effect of PEF treatment on biological cells. A viability assay using fluorescein diacetate measured by flow cytometry was established. The influence of developmental stage on viability could be shown in synchronised cultures when applying PEF treatment with very low specific energies where one part of cells undergoes cell death, and the other part stays viable after treatment. Reactive oxygen species generation after similar low-energy PEF treatment could be shown, indicating that PEFs could act as abiotic stress signal. Most importantly, a cell-death inducing factor could be extracted. A water-soluble extract derived from microalgae suspensions incubated for 24 h after PEF treatment caused the recipient microalgae to die, even though the recipient cells had not been subjected to PEF treatment directly. The working model assumes that low-energy PEF treatment induces programmed cell death in C.vulgaris while specifically releasing a cell-death inducing factor. Low-energy PEF treatment with subsequent incubation period could be a novel biotechnological strategy to extract soluble proteins and lipids in cascade process.

Operando Investigation of Locally Enhanced Electric Field Treatment (LEEFT) Harnessing Lightning-Rod Effect for Rapid Bacteria Inactivation.

The growth of undesired bacteria causes numerous problems. Here, we show that locally enhanced electric field treatment (LEEFT) can cause rapid bacteria inactivation by electroporation. The bacteria inactivation is studied in situ at the single-cell level on a lab-on-a-chip that has nanowedge-decorated electrodes. Rapid bacteria inactivation occurs at the nanowedge tips where the electric field is enhanced due to the lightning-rod effect. Electroporation induced by the locally enhanced electric field is the predominant mechanism. The antimicrobial performance depends on the strength of the enhanced electric field instead of the applied voltage, and no generation of reactive oxygen species (ROS) is detected when >90% bacteria inactivation is achieved. Quick membrane pore closure under lower voltages confirms that electroporation is induced in LEEFT. This work is the first-time visualization and mechanism elucidation of LEEFT for bacteria inactivation at the single-cell level, and the findings will provide strong support for its future applications.

Demonstrative Experiment on the Favorable Effects of Static Electric Field Treatment on Vitamin D3-Induced Hypercalcemia.

The purpose of this study was to elucidate the effects of static electric field (SEF) treatment on vitamin D3 (Vit D3)-induced hypercalcemia and renal calcification in mice. The mice were assigned to three groups: Vit D3-treated mice, mice treated with Vit D3 and SEF (Vit D3 + SEF), and untreated mice. After the administration of Vit D3, the Vit D3 + SEF-treated mice were exposed to SEF treatment by a high-voltage alternating current over five days. Serum biochemical examinations revealed that both the creatinine and blood urea nitrogen concentrations were significantly higher in the Vit D3-treated group. Significantly, decreased Cl concentrations, and increased Ca and inorganic phosphorus concentrations, were found in the Vit D3-treated group. In the Vit D3 + SEF-treated group, these parameters returned to the levels of the untreated group. In the Vit D3-treated group, histopathological examinations showed marked multifocal calcification in the lumens of the renal tubules and the renal parenchyma. The myocardium was replaced by abundant granular mineralization (calcification), with degeneration and necrosis of the calcified fibers. The stomach showed calcification of the cardiac mucosa. SEF treatment remarkably attenuated the Vit D3-induced hypervitaminotic injuries. In conclusion, this study provides important evidence that SEF treatment can reduce hypercalcemia and remove calcium deposits from the renal, cardiac, and gastric tissues. SEF treatment is useful in the regulation of disorders caused by an imbalance of serum electrolytes.

A German Prospective Study of the Safety and Efficacy of a Non-Invasive, High-intensity, Electromagnetic Abdomen and Buttock Contouring Device.

OBJECTIVE: This study evaluated the efficacy of a novel treatment method utilizing high-intensity focused electromagnetic field (HIFEM) in reducing fat and strengthening the muscles in the abdominal and gluteal areas. METHODS: A total of 14 patients, aged 23 to 49 years (mean: 33.2 years) received four treatments on the abdomen and/or buttocks, each lasting for 30 minutes. Ultrasound imaging was performed at baseline, immediately post-treatment, and eight weeks after the last treatment to examine the changes in thickness of the rectus abdominis and subcutaneous fat. Additionally, waist circumference, digital photographs, and patient satisfaction were collected at the same time points. The improvement in digital images was evaluated by practitioners and the patients themselves. RESULTS: Two months after the treatments, ultrasound measurements showed, on average, a 15.7-percent reduction of subcutaneous adipose tissue and an average increase in the thickness of the rectus abdominis muscle by 26.1 percent. The abdominal circumference of the treated patients showed an average decrease of 2.84 centimeters. Immediately following the final session, the patient's satisfaction was 87.5 percent; eight weeks later, it was 100 percent. During the patient evaluation of photographs, 25 percent of the patients rated their appearance as improved, 66.7 percent as significantly improved, and 8.3 percent as greatly improved. Using the Global Aesthetic Improvement Scale, the medical practitioners rated the patients as 100-percent improved. No relevant side effects of the treatment were documented. CONCLUSION: The data collected on the HIFEM procedure evaluated here are in line with the results from clinical studies and applications of this treatment in the United States. These results support the efficacy and tolerability of the HIFEM procedure. The treatment achieved high doctor and patient satisfaction.

Evaluation of stabilizing material and stabilization efficiency through comparative study of toxic heavy metal transfer between corn and peanut grown in stabilized field soil.

Soil contaminated with toxic heavy metals (THMs) was stabilized by adding a combination of waste resources in 7.0 wt%, including coal-mine drainage sludge, waste cow bone, and steelmaking slag, in the ratio of 5:35:60. Subsequently, corn and peanut were cultivated in treated soil to investigate the effects of the waste resources on THM mobility in soil and translocation to plants. Sequential extraction procedures (SEP) was used to analyze mobile phase THMs which could be accumulated in the plants. SEP shows that mobile Pb, Cd, Cu, Zn, Ni, Cr, and As were reduced by 8.48%, 29.22%, 18.85%, 21.66%, 4.58%, 62.78%, and 20.01%, respectively. The bioaccumulation of THMs was clearly hindered by stabilization; however, the increment in the amount of immobile-phase THMs and change in the amount of translocated THMs was not proportional. The corn grains grown above the soil surface were compared with the peanut grains grown beneath the soil surface, and the results indicating that the efficiency of stabilization on THM translocation may not depend on the contact of grain to soil but the nature of plant. Interestingly, the results of bioaccumulation with and without stabilization showed that the movement of some THMs inside the plants was affected by stabilization.

Electric-field enhanced microalgae inactivation using a flow-through copper ionization cell.

Using copper (Cu) to treat algal blooms is a commonly accepted method worldwide. However, the release of Cu may cause environmental and health risk. It is required to exploit an efficient way to reduce the Cu concentration but improve the algicidal effectiveness. Here, a Cu ionization cell (CIC) was designed and utilized in a flow-through system for inactivation of two bloom-forming microalgae species, Chlorella vulgaris and Microcystis aeruginosa. The results showed that the in-situ Cu release in the CIC treatment cause efficient microalgae inactivation. The 96 h-growth inhibition for C. vulgaris and M. aeruginosa reached 98.5 ± 3.1 % and 75.9 ± 2.0 % at a flow rate of 5 mL/min with the effluent Cu concentration of 554 ± 9 µg/L and 613 ± 17 µg/L, respectively. The maximum quantum yield (Fv/Fm) inhibitions of C. vulgaris and M. aeruginosa were 37.0 ± 1.6 % and 70.9 ± 2.1 %. The electric field enhanced CIC treatment has a locally higher Cu level because of the in-situ release. The CIC improved the microalgae inactivation performance by increasing the microalgae cell membrane permeability with excessive Cu uptake. The energy consumption was only 16.8 J/L. The in-situ Cu treatment in this work provides a microalgae inactivation method with the more environment-friendly and cost-effective prospect.

The Effect of Traditional and Non-Thermal Treatments on the Bioactive Compounds and Sugars Content of Red Bell Pepper.

The aim of the study was an investigation of the effect of traditional and non-thermal treatment on the bioactive compounds of red bell pepper. As a thermal process, blanching in water and in steam was studied, while for non-thermal the sonication, pulsed electric field treatment and their combination were used in this experiment. The red bell peppers were evaluated based on quality attributes such as: total carotenoids content; polyphenols; vitamin C; antioxidant activity and sugars content. Vitamin C and sugar content were analyzed using liquid chromatography and other measurements were determined based on the spectrophotometric method. Results showed that the blanching in water or in steam reduced bioactive compounds concentration; whereas non-thermal treatments as pulsed electric field (PEF) applied separately or in combination with ultrasound (US + PEF) let to obtain similar or slightly lower content of bioactive compounds in comparison to untreated peppers. When sonication (US) and combined treatment as PEF + US were applied; in most cases reduction of bioactive compounds concentration occurred. This effect was probably related to the effect of relatively long (30 min) ultrasound treatment. The application of appropriate parameters of non-thermal processing is crucial for the high quality of processed material.

Evaluation of a tumor electric field treatment system in a rat model of glioma.

OBJECTIVE: Glioma is a devastating disease lacking effective treatment. Tumor electric field therapy is emerging as a novel non-invasive therapy. The current study evaluates the efficacy and safety of a self-designed tumor electric field therapy system (TEFTS ASCLU-300) in a rat orthotopic transplantation model of glioma. METHODS: A model of intracranial orthotopic transplantation was established in rats using glioma C6 cells. For electric field therapy, glioma-bearing rats were exposed to alternating electric fields generated by a self-developed TEFTS starting on either 1st (Group 2) or 3rd (Group 3) day after transplantation, while other conditions were maintained the same as non-treated rats (Group 1). Glioma size, body weight, and overall survival (OS) were compared between groups. Immunohistochemical staining was applied to access tumor cell death and microvessel density within the tumor. In addition, the systemic effects of TEFTS on blood cells, vital organs, and hepatorenal functions were evaluated. RESULTS: TEFTS treatment significantly elongated the OS of tumor-bearing rats compared with non-treated rats (non-treated vs treated: 24.77 ± 7.08 days vs 40.31 ± 19.11 days, P = .0031). Continuous TEFTS treatment starting on 1st or 3rd day significantly reduced glioma size at 2 and 3 weeks after tumor cell inoculation (Week 2: Group 1:289.95 ± 101.69 mm3 ; Group 2:70.45 ± 17.79 mm3 ; Group 3:73.88 ± 33.21 mm3 , P < .0001. Week 3: Group 1:544.096 ± 78.53 mm3 ; Group 2:187.58 ± 78.44 mm3 ; Group 3:167.14 ± 109.96 mm3 , P = .0005). Continuous treatment for more than 4 weeks inhibited tumor growth. The TEFTS treatment promoted tumor cell death, as demonstrated by increased number of Caspase 3+ cells within the tumor (non-treated vs treated: 38.06 ± 10.04 vs 68.57 ± 8.09 cells/field, P = .0007), but had minimal effect on microvessel density, as shown by CD31 expression (non-treated vs treated: 1.63 ± 0.09 vs 1.57 ± 0.13% of positively stained areas, P > .05). No remarkable differences were observed in hepatorenal function, blood cell counts, or other vital organs between non-treated and treated groups. CONCLUSION: The TEFTS developed by our research team was proved to be effective and safe to inhibit tumor growth and improve general outcomes in a rat model of brain glioma.

Analysis of damage-associated molecular pattern molecules due to electroporation of cells in vitro.

Background Tumor cells can die via immunogenic cell death pathway, in which damage-associated molecular pattern molecules (DAMPs) are released from the cells. These molecules activate cells involved in the immune response. Both innate and adaptive immune response can be activated, causing a destruction of the remaining infected cells. Activation of immune response is also an important component of tumor treatment with electrochemotherapy (ECT) and irreversible electroporation (IRE). We thus explored, if and when specific DAMPs are released as a consequence of electroporation in vitro. Materials and methods In this in vitro study, 100 µs long electric pulses were applied to a suspension of Chinese hamster ovary cells. The release of DAMPs - specifically: adenosine triphosphate (ATP), calreticulin, nucleic acids and uric acid was investigated at different time points after exposing the cells to electric pulses of different amplitudes. The release of DAMPs was statistically correlated with cell permeabilization and cell survival, e.g. reversible and irreversible electroporation. Results In general, the release of DAMPs increases with increasing pulse amplitude. Concentration of DAMPs depend on the time interval between exposure of the cells to pulses and the analysis. Concentrations of most DAMPs correlate strongly with cell death. However, we detected no uric acid in the investigated samples. Conclusions Release of DAMPs can serve as a marker for prediction of cell death. Since the stability of certain DAMPs is time dependent, this should be considered when designing protocols for detecting DAMPs after electric pulse treatment.

Impact of incubation conditions on protein and C-Phycocyanin recovery from Arthrospira platensis post- pulsed electric field treatment.

Pulsed electric field (PEF) was conducted for the extraction of proteins/C-Phycocyanins from Arthrospira platensis. The cyanobacterial suspension was treated with 1 µs long pulses at an electric field strength of 40 kV·cm-1 and a treatment energy of 114 kJ·kgsus-1 and 56 kJ·kgsus-1. For benchmarking, additional biomass was processed by high pressure homogenization. Homogeneity of the suspension prior to PEF-treatment influenced the protein/C-phycocyanin extraction efficiency. Stability of C-Phycocyanin during post-PEF incubation time was affected by incubation temperature and pH of the external medium. Biomass concentration severely affect proteins/C-Phycocyanins extraction yield via PEF-treatment. The optimum conditions for extraction of proteins/C-Phycocyanin was obtained at 23 °C while incubating in pH 8-buffer. The energy demand for PEF-treatment amounts to 0.56 MJ·kgdw-1 when processing biomass at 100 gdw·kgsus-1. PEF treatment enhances the protein/C-Phycocyanin extraction yield, thus, it can be suggested as preferential downstream processing method for the production of C-Phycocyanin from A. platensis biomass.

Extraction of Proteins and Other Intracellular Bioactive Compounds From Baker's Yeasts by Pulsed Electric Field Treatment.

Yeasts are rich source of proteins, antioxidants, vitamins, and other bioactive compounds. The main drawback in their utilization as valuable ingredients in functional foods and dietary supplements production is the thick, indigestible cell wall, as well as the high nucleic acid content. In this study, we evaluated the feasibility of pulsed electric field (PEF) treatment as an alternative method for extraction of proteins and other bioactive intracellular compounds from yeasts. Baker's yeast water suspensions with different concentration (12.5-85 g dry cell weight per liter) were treated with monopolar rectangular pulses using a continuous flow system. The PEF energy required to achieve irreversible electropermeabilization was significantly reduced with the increase of the biomass concentration. Upon incubation of the permeabilized cells in water, only relatively small intracellular compounds were released. Release of 90% of the free amino acids and low molecular UV absorbing compounds, 80% of the glutathione, and ∼40% of the total phenol content was achieved about 2 h after pulsation and incubation of the suspensions at room temperature. At these conditions, the macromolecules (proteins and nucleic acids) were retained largely inside. Efficient protein release (∼90% from the total soluble protein) occurred only after dilution and incubation of the permeabilized cells in buffer with pH 8-9. Protein concentrates obtained by ultrafiltration (10 kDa cut off) had lower nucleic acid content (protein/nucleic acid ratio ∼100/4.5) in comparison with cell lysates obtained by mechanical disintegration. The obtained results allowed to conclude that PEF treatment can be used as an efficient alternative approach for production of yeast extracts with different composition, suitable for application in food, cosmetics and pharmaceutical industries.

Efficacy and safety of ingenol mebutate gel in field treatment of actinic keratosis on full face, balding scalp, or approximately 250 cm2 on the chest: A phase 3 randomized controlled trial.

BACKGROUND: Ingenol mebutate (IngMeb) 0.015% or 0.05% is approved for actinic keratosis (AK) areas of 25 cm2 or less; some patients require treatment of larger fields. OBJECTIVE: To determine efficacy and safety of IngMeb 0.027% in areas of AK of up to 250 cm2 during an 8-week initial assessment period and extended 12-month follow-up. METHODS: This phase 3, randomized, double-blind, vehicle-controlled trial (NCT02361216) enrolled adult patients with 5 to 20 AK lesions on the face/scalp (25-250 cm2) or chest (approximately 250 cm2). Patients received once-daily IngMeb or vehicle for 3 consecutive days on the full face, full balding scalp, or approximately 250 cm2 on the chest. The primary endpoint was complete AK clearance (AKCLEAR 100; week 8). Additional endpoints included partial AK clearance (AKCLEAR 75), recurrence, patient satisfaction, cosmetic outcome, and safety. RESULTS: IngMeb was superior to vehicle for complete AK clearance (21.4% vs 3.4%, P < .001) and AK clearance of 75% or greater (59.4% vs 8.9%, P < .001) at week 8. Probability of sustained clearance during the 12-month follow-up was 22.9% for patients treated with IngMeb. Increased treatment satisfaction and cosmetic outcomes were observed with IngMeb versus vehicle. No unexpected safety signals were identified. LIMITATIONS: Localized skin responses hindered maintenance of double-blinding. CONCLUSIONS: IngMeb 0.027% was superior to vehicle for treatment of AK areas of up to 250 cm2. The safety profile of IngMeb was as expected.