Improvement of antioxidant activity of peptides with molecular weights ranging from 1 to 10 kDa by PEF technology

Egg white protein powder was hydrolyzed with Alcalase to produce antioxidant peptides. Then, the peptides were fractionated with ultrafiltration membranes. The peptides (1-10 kDa) were further treated by pulsed electric field (PEF) to investigate its effect on the antioxidant activity of the peptides. Antioxidant activity was evaluated using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical inhibition assay. The results indicated that optimal electric field intensity and standing times of PEF can enhance the antioxidant activity of the peptides. Therefore, a Box-Behnken design (BBD) with three independent variables including concentration, electric field intensity and pulse frequency was used to establish the regression equation of second-order response surface. The optimal conditions were as follows: concentration 8 mg/ml, electric field intensity 10 kV/cm and pulse frequency 2400 Hz. Under these conditions, the peptides antioxidant activity was 62.64% ± 0.98%. The present study demonstrated that the antioxidant activity of peptides (1-10 kDa) could be improved using PEF.     

Optimized extraction of calcium malate from eggshell treated by PEF and an absorption assessment in vitro

Under optimized pulsed electric field (PEF) treatment for production of eggshell calcium malate (ESCM) by one-factor-at-a-time test and ternary quadratic regression orthogonal combination design (TQROCD), an absorption assessment of ESCM treated by the best conditions of PEF were performed in male mice with apparent calcium absorption rate (ACAR), serum alkalinity phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), serum calcium and serum phosphorus, length of femurs and skeletal calcium content were studied. The highest dissoluble calcium malate content (7.075 mg/mL) was obtained with the 6.0% malic acid, the electric field intensity of 20 kV/cm, and pulse duration of 24 μs. In vitro, ESCM prepared by the best conditions of PEF at doses of 133.0 mg kg(-1) d(-1) for 70 d not only significantly improve the ALP activity, the femur length and calcium content of bone of the mice (P<0.05) but also decreased the levels of TRAP (P<0.05). The ratio of calcium and phosphorus was in the normal range. PEF could be taken as a highly effective, environmentally friendly and energy-saving method for preparation of ESCM, which treated by PEF could promote the absorption of calcium in vitro, extraordinary can promote bone development and a healthy bone.     

Optimized PEF treatment for antioxidant polypeptides with MW 10-30kDa and preliminary analysis of structure change

Antioxidant polypeptides of molecular weight (MW) ranging from 10 to 30kDa were produced from egg-white protein powder by enzyme hydrolysis and ultrafiltration (UF). Ferric reducing antioxidant potential (FRAP) value (mmol Fe(2+)/g) was used to evaluate the antioxidant activity. One-factor-at-a-time (OFAT) tests and Box-Behnken design (BBD) of response surface methodology (RSM) were used to investigate the effect of pulsed electric field (PEF) treatment parameters on antioxidant activity of polypeptides. The optimal conditions were as follows: concentration 8mg/mL, electric field intensity 10kV/cm, and pulse frequency 2000Hz, under which, the FRAP value increased 44.23%, compared to the antioxidant activity of the polypeptides without PEF treatment. Both near-infrared spectroscopy (NIR) and mid-infrared spectroscopy (MIR) were used to analyze the change of functional groups.     

高压脉冲电场法提取干松针总黄酮及其体外抗氧化性检测

主要研究了高压脉冲电场法提取干松针总黄酮的工艺条件,并对松针总黄酮体外抗氧化性能进行初步分析.结果表明,最优的高压脉冲电场提取条件为:电场强度20 kV/cm,脉冲数8个,料液比1:50.松针总黄酮提取率的影响因素:料液比＞电场强度＞脉冲数.松针落叶中总黄酮物质对自由基清除能力随浓度的增加而升高,松针总黄酮对Fe~(3+)的还原能力比抗坏血酸强.     

Effects of pulsed electric fields on mouse spleen lymphocytes in vitro

The effects of pulsed electric fields on cell membranes were investigated. In vitro exposure of mouse splenocytes to a single high-voltage pulse resulted in an increase in membrane permeability that was dependent on both the electric field strength and the pulse duration. Exposure to a 2 μs, 3.0 kV/cm pulse resulted in the induction of a 1.26 V transmembrane potential, and elicited a 50% loss of intracellular K⁺. These results are in agreement with previous studies of the effects of pulsed electric fields on erythrocytes and microorganisms. The effect of pulsed electric fields on the functional integrity of lymphocytes was i vestigated by measuring [³H]thymidine incorporation by cells cultured in the presence and absence of various mitogens following exposure to an electrical pulse. No statistically significant effects on the response of mouse spleen lymphocytes to concanavalin A, phytohemagglutinin or lipopolysaccharide were observed following exposure to 2 μs electric pulses at amplitudes of up to 3.5 kV/cm. Exposure to a single 10 μs pulse of 2.4–3.5 kV/cm produced a statistically significant reduction in the response of lymphocytes to lipopolysaccharide stimulation that was attributed to cell death.     

Study of mechanisms of electric field-induced DNA transfection. III. Electric parameters and other conditions for effective transfection.

Electric parameters, osmolality, temperature, and pH of the suspending medium and the growth phase of cells, etc., are known to influence the efficiency of the pulsed electric field (PEF)-induced DNA transfection of cells. PEF-induced transfection of Escherichia coli JM105 by plasmid DNA PUC18, PUC19, PBR322, and PMSG has been used as a model system to establish quantitative relationships between these parameters and transfection efficiency. The main findings are summarized for experiments using unipolar square wave PEF. (a) For a given field strength (up to 6 kV/cm), the transfection efficiency (TE) was linearly dependent on the pulse width (up to 1 ms). (b) When field strength is fixed, Log [TE] correlated with the number of pulses applied. Similarly, when field duration was fixed, Log [TE] correlated with the number of pulses. (c) In the absence of MgCl2, TE showed a maximal value at 50 mM sucrose and was reduced by several fold at lower and higher sucrose concentrations. Cell survival was nearly constant in the range 1-300 mM sucrose. (d) E. coli in the early and mid-exponential growth phases was more susceptible to PEF for DNA transfection than it was in the stationary phase. (e) For a given set of electric parameters, TE was the highest at neutral pH and was greatly reduced at acidic and alkaline pH. (f) Increasing the temperature from 0 to 37 degrees C resulted in the reduction of TE by three orders of magnitude. This could reflect a rapid shrinking of pores at higher temperatures. (g) TE was inversely proportional to the square of the size of the plasmid DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of pulsed electric fields on inactivation and metabolic activity of Lactobacillus plantarum in model beer

AIMS: Inactivation and sublethal injury of Lactobacillus plantarum at different pulsed electric field (PEF) strengths and total energy inputs were investigated to differentiate reversible and irreversible impacts on cell functionality. METHODS AND RESULTS: Lactobacillus plantarum was treated with PEF in model beer (MB) to determine critical values of field strength and energy input for cell inactivation. Below critical values, metabolic activity and membrane integrity were initially reduced without loss of viability. Above critical values, however, irreversible cell damage occurred. Presence of nisin or hop extract, during PEF treatment, resulted in an additional reduction of cell viability by 1;5 log cycles. Also, addition of the hop extract resulted in an additional two log cycles of sublethal injury. Partial reversibility of membrane damage was observed using propidium iodide (PI) uptake and staining. Inoculated MB containing hops was stored after PEF to evaluate the efficacy of such treatment for beer preservation. CONCLUSION: Cells were inactivated only above critical values of 13 kV x cm(-1) and 64 kJ x kg(-1); below these values cell damage was reversible. Storage experiments revealed that surviving cells were killed after 15 h storage in MB containing hops. SIGNIFICANCE AND IMPACT OF THE STUDY: Both reversible and irreversible cell damage due to PEF treatment was detected, depending on specific treatment conditions. The combination of PEF and hop addition is a promising nonthermal method of preservation for beer.     

Electric pulses to prepare feeder cells for sustaining and culturing of undifferentiated embryonic stem cells

Current challenges in embryonic-stem cell (ESC) research include the inability of sustaining and culturing of undifferentiated ESCs over time. Growth-arrested feeder cells are essential to the culture and sustaining of undifferentiated ESCs, and they are currently prepared using gamma-radiation and chemical inactivation. Both techniques have severe limitations. In this study, we developed a new, simple and effective technique (pulsed electric fields, PEFs) to produce viable growth-arrested cells (RTS34st) and used them as high-quality feeder cells to culture and sustain undifferentiated zebrafish ESCs over time. The cells were exposed to 25 sequential 10-ns electric pulses (10nsEPs) of 25, 40 and 150 kV/cm with 1-s pulse interval, or 2 sequential 50-μs electric pulses (50μsEPs) of 2.83, 1.78 and 0.78 kV/cm with 5-s pulse interval, respectively. We found that the cellular effects of PEFs depended directly upon the duration, number and electric field strength of the pulses, showing the feasibility of tuning them to produce various types of growth-arrested cells for culturing undifferentiated ESCs. Both 10nsEPs of 40 kV/cm produced by a 10nsEP generator and 50μsEPs of 1.78 kV/cm provided by inexpensive and widely available conventional electroporators, generated high-quality growth-arrested feeder cells for proliferation of undifferentiated ESCs over time. PEFs can therefore be used to replace radiation and chemical inactivation methods for preparation of growth-arrested feeder cells for advancing ESC research.     

Permeabilization of tumor cells induced by pulsed electric fields in vitro

The permeabilization of tumor cells in vitro under the action of pulsed electric fields with a duration of 6 mks in the range of amplitudes 1-7 kV/cm was studied. In the mode of excitation in the ambience of localized plasma discharge in a chamber of special design, an enhanced damage to cells in suspension was observed. It is assumed that the enhancement is due to the synchronous action of the electric field and acoustic shock wave pulses. In the mode without the plasma breakdown of ambience, when the pulse duration of electric field of intensity of 1-2 kV/cm was increased to 60 mks, the efficiency of permeabilization increases nearly by one order. The experimental results are compared with the known theoretical models of cell membrane electroporation.     

Inactivation of Mycobacterium paratuberculosis by pulsed electric fields

The influence of treatment temperature and pulsed electric fields (PEF) on the viability of Mycobacterium paratuberculosis cells suspended in 0.1% (wt/vol) peptone water and in sterilized cow's milk was assessed by direct viable counts and by transmission electron microscopy (TEM). PEF treatment at 50 degrees C (2,500 pulses at 30 kV/cm) reduced the level of viable M. paratuberculosis cells by approximately 5.3 and 5.9 log(10) CFU/ml in 0.1% peptone water and in cow's milk, respectively, while PEF treatment of M. paratuberculosis at lower temperatures resulted in less lethality. Heating alone at 50 degrees C for 25 min or at 72 degrees C for 25 s (extended high-temperature, short-time pasteurization) resulted in reductions of M. paratuberculosis of approximately 0.01 and 2.4 log(10) CFU/ml, respectively. TEM studies revealed that exposure to PEF treatment resulted in substantial damage at the cellular level to M. paratuberculosis.     

Releasing profiles of gene products from recombinant Escherichia coli in a high-voltage pulsed electric field

Release of recombinant proteins from gene-engineered Escherichia coli by applying a pulsed electric field (PEF) to a cell suspension was studied. When E. coli/pNC1, which produces beta-glucosidase and accumulates it in cytoplasm, was exposed to PEF, the most effective release of this enzyme was achieved in the cell suspension of 5% glycine and 15% PEG solution under 10kV/cm and 280J/ml of a PEF in a needle-plate electrode chamber. However, the amount of released beta-glucosidase by PEF treatment was only 26% of that by ultrasonic treatment. On the other hand, alpha-amylase produced by E. coli/pHI301A and accumulated in the periplasmic space could be easily released by PEF treatment. When this recombinant E. coli was suspended in 0.9% NaCl and 10% PEG solution and exposed to 10kV/cm and 200J/ml of a PEF in a plate-plate electrode chamber, 89% of intracellular alpha-amylase with nine-times higher specific activity compared with that by ultrasonic treatment was released. The release tendency of cellobiohydrolase, produced by E. coli/pNB6 and accumulated in both the cytoplasm and periplasmic space, was intermediate between those of beta-glucosidase and alpha-amylase. In this case, 70% of cellobiohydrolase with 1.9-times higher specific activity compared with that by ultrasonic treatment could be released when E. coli/pNB6 was suspended in 15% PEG and 10kV/cm and 200J/ml of a PEF was applied in a needle-plate electrode chamber. These results indicated that PEF treatment could easily disrupt the outer membrane, but it was difficult to disrupt the cytoplasmic membrane simultaneously. Therefore, PEF treatment is useful for easy release of periplasmic protein with selectivity.     

Quantitative studies on the viability of yeast protoplasts following dielectrophoresis

Abstract Protoplasts from Saccharomyces cerevisiae and Saccharomyces diastaticus were collected in a non-homogeneous alternating electric field. The dependence of the viability of the protoplasts on different conditions of collection was tested by determining the regeneration rates in each case. The parameters varied in collection were the field strength (0.33 kV/cm–6.67 kV/cm), the frequency of the alternating field (1–2 MHz) and the collection time (2–10 min). The introduction of a new type of fusion chamber (meander chamber) permitted, for the first time, quantitative exposure of protoplasts to the electric field as well as their complete transference into the regeneration medium. The regeneration rates of yeast protoplasts collected under those conditions employed for electrofusion did not differ from those of protoplasts which had been maintained under the same experimental conditions but were not subject to the influence of an alternating electric field. The two yeast strains were fused together (collection 1 kV/cm; pulse 15 kV/cm; duration of pulse 40 μs) and the fusion products were introduced into a selection medium for regeneration. The fusion rate was about 4.8 × 10⁻⁴; on average 272 colonies grew on the selection medium for each chamber filling.     

Selection and identification of a Listeria monocytogenes target strain for pulsed electric field process optimization

Nine Listeria monocytogenes strains were treated individually with a continuous pulsed electric field (PEF) apparatus, and their sensitivities to the treatment were compared at 25 kV/cm. When cell suspensions of these strains in 0.1% NaCl (pH 7.0) were treated at 23 degrees C for 144 micro s, inactivation ranged from 0.7 to 3.7 log(10) CFU/ml. Inactivation by 72- micro s PEF treatments at 37 degrees C ranged from 0.3 to 2.5 log(10) CFU/ml. L. monocytogenes OSY-8578 was substantially more resistant than other strains when cells were PEF treated in 0.1% NaCl, whereas Scott A was one of the most sensitive strains. The superiority of OSY-8578's resistance to that of Scott A was confirmed in 50% diluted acid whey (pH 4.2). Changes in sensitivity to PEF during phases of growth were minimal in OSY-8578 and substantial in Scott A. Use of L. monocytogenes OSY-8578, therefore, is recommended in studies to optimize PEF processes that target L. monocytogenes. The nine L. monocytogenes strains were genotyped with pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) techniques. These strains were better differentiated with PFGE than with AP-PCR. The target strain (OSY-8578) was characterized by both molecular typing techniques, but resistance to PEF, in general, was not associated with a particular genotype group.     

Relationship between Sublethal Injury and Inactivation of Yeast Cells by the Combination of Sorbic Acid and Pulsed Electric Fields

The objective of this study was to investigate the occurrence of sublethal injury after the pulsed-electric-field (PEF) treatment of two yeasts, Dekkera bruxellensis and Saccharomyces cerevisiae, as well as the relation of sublethal injury to the inactivating effect of the combination of PEF and sorbic acid. PEF caused sublethal injury in both yeasts: more than 90% of surviving D. bruxellensis cells and 99% of surviving S. cerevisiae cells were sublethally injured after 50 pulses at 12 kV/cm in buffer at pHs of both 7.0 and 4.0. The proportion of sublethally injured cells reached a maximum after 50 pulses at 12.0 kV/cm (S. cerevisiae) or 16.5 kV/cm (D. bruxellensis), and it kept constant or progressively decreased at greater electric field strengths and with longer PEF treatments. Sublethally PEF-injured cells showed sensitivity to the presence of sorbic acid at a concentration of 2,000 ppm. A synergistic inactivating effect of the combination of PEF and sorbic acid was observed. Survivors of the PEF treatment were progressively inactivated in the presence of 2,000 ppm of sorbic acid at pH 3.8, with the combined treatments achieving more than log₁₀ 5 cycles of dead cells under the conditions investigated. This study has demonstrated the occurrence of sublethal injury after exposure to PEF, so yeast inactivation by PEF is not an all-or-nothing event. The combination of PEF and sorbic acid has proven to be an effective method to achieve a higher level of yeast inactivation. This work contributes to the knowledge of the mechanism of microbial inactivation by PEF, and it may be useful for improving food preservation by PEF technology.     

Enhanced freeze tolerance of baker’s yeast by overexpressed trehalose-6-phosphate synthase gene (TPS1) and deleted trehalase genes in frozen dough

Several recombinant strains with overexpressed trehalose-6-phosphate synthase gene (TPS1) and/or deleted trehalase genes were obtained to elucidate the relationships between TPS1, trehalase genes, content of intracellular trehalose and freeze tolerance of baker’s yeast, as well as improve the fermentation properties of lean dough after freezing. In this study, strain TL301_TPS1 overexpressing TPS1 showed 62.92 % higher trehalose-6-phosphate synthase (Tps1) activity and enhanced the content of intracellular trehalose than the parental strain. Deleting ATH1 exerted a significant effect on trehalase activities and the degradation amount of intracellular trehalose during the first 30 min of prefermentation. This finding indicates that acid trehalase (Ath1) plays a role in intracellular trehalose degradation. NTH2 encodes a functional neutral trehalase (Nth2) that was significantly involved in intracellular trehalose degradation in the absence of the NTH1 and/or ATH1 gene. The survival ratio, freeze-tolerance ratio and relative fermentation ability of strain TL301_TPS1 were approximately twice as high as those of the parental strain (BY6-9α). The increase in freeze tolerance of strain TL301_TPS1 was accompanied by relatively low trehalase activity, high Tps1 activity and high residual content of intracellular trehalose. Our results suggest that overexpressing TPS1 and deleting trehalase genes are sufficient to improve the freeze tolerance of baker’s yeast in frozen dough. The present study provides guidance for the commercial baking industry as well as the research on the intracellular trehalose mobilization and freeze tolerance of baker’s yeast.     

Inactivation and injury of Escherichia coli O157:H7 and Staphylococcus aureus by pulsed electric fields

Two pathogenic microorganisms Escherichia coli O157:H7 and Staphylococcus aureus, suspended in peptone solution (0.1% w/v) were treated with 12, 14, 16 and 20 kV/cm electric field strengths with different pulse numbers up to 60 pulses. Pulsed electric field (PEF) treatment at 20 kV/cm with 60 pulses provided nearly 2 log reduction in viable cell counts of E. coli O157:H7 and S. aureus. S. aureus cells were slightly more resistant than E.coli O157:H7 cells. The results related to the effect of initial cell concentration of E. coli O157:H7 on the PEF inactivation showed that more inactivation was obtained by decreasing initial cell concentration. Any possible injury by PEF was also investigated after applying 20 kV/cm electric field to the microorganisms. As a result, it was determined that there was 35.92 to 43.36% injury in E. coli O157:H7 cells, and 17.26 to 30.86% injury in S. aureus cells depending on pulse number. The inactivation results were also described by a kinetic model.     

Evidence for contribution of neutral trehalase in barotolerance of Saccharomyces cerevisiae

In yeast, trehalose accumulation and its hydrolysis, which is catalyzed by neutral trehalase, are believed to be important for thermotolerance. We have shown that trehalose is one of the important factors for barotolerance (resistance to hydrostatic pressure); however, nothing is known about the role of neutral trehalase in barotolerance. To estimate the contribution of neutral trehalase in resisting high hydrostatic pressure, we measured the barotolerance of neutral trehalase I and/or neutral trehalase II deletion strains. Under 180 MPa of pressure for 2 h, the neutral trehalase I deletion strain showed higher barotolerance in logarithmic-phase cells and lower barotolerance in stationary-phase cells than the wild-type strain. Introduction of the neutral trehalase I gene (NTH1) into the deletion mutant restored barotolerance defects in stationary-phase cells. Furthermore, we assessed the contribution of neutral trehalase during pressure and recovery conditions by varying the expression of NTH1 or neutral trehalase activity with a galactose-inducible GAL1 promoter with either glucose or galactose. The low barotolerance observed with glucose repression of neutral trehalase from the GAL1 promoter was restored during recovery with galactose induction. Our results suggest that neutral trehalase contributes to barotolerance, especially during recovery.     

Relationship between sublethal injury and inactivation of yeast cells by the combination of sorbic acid and pulsed electric fields

The objective of this study was to investigate the occurrence of sublethal injury after the pulsed-electric-field (PEF) treatment of two yeasts, Dekkera bruxellensis and Saccharomyces cerevisiae, as well as the relation of sublethal injury to the inactivating effect of the combination of PEF and sorbic acid. PEF caused sublethal injury in both yeasts: more than 90% of surviving D. bruxellensis cells and 99% of surviving S. cerevisiae cells were sublethally injured after 50 pulses at 12 kV/cm in buffer at pHs of both 7.0 and 4.0. The proportion of sublethally injured cells reached a maximum after 50 pulses at 12.0 kV/cm (S. cerevisiae) or 16.5 kV/cm (D. bruxellensis), and it kept constant or progressively decreased at greater electric field strengths and with longer PEF treatments. Sublethally PEF-injured cells showed sensitivity to the presence of sorbic acid at a concentration of 2,000 ppm. A synergistic inactivating effect of the combination of PEF and sorbic acid was observed. Survivors of the PEF treatment were progressively inactivated in the presence of 2,000 ppm of sorbic acid at pH 3.8, with the combined treatments achieving more than log10 5 cycles of dead cells under the conditions investigated. This study has demonstrated the occurrence of sublethal injury after exposure to PEF, so yeast inactivation by PEF is not an all-or-nothing event. The combination of PEF and sorbic acid has proven to be an effective method to achieve a higher level of yeast inactivation. This work contributes to the knowledge of the mechanism of microbial inactivation by PEF, and it may be useful for improving food preservation by PEF technology.     

Evidence for Contribution of Neutral Trehalase in Barotolerance of Saccharomyces cerevisiae

In yeast, trehalose accumulation and its hydrolysis, which is catalyzed by neutral trehalase, are believed to be important for thermotolerance. We have shown that trehalose is one of the important factors for barotolerance (resistance to hydrostatic pressure); however, nothing is known about the role of neutral trehalase in barotolerance. To estimate the contribution of neutral trehalase in resisting high hydrostatic pressure, we measured the barotolerance of neutral trehalase I and/or neutral trehalase II deletion strains. Under 180 MPa of pressure for 2 h, the neutral trehalase I deletion strain showed higher barotolerance in logarithmic-phase cells and lower barotolerance in stationary-phase cells than the wild-type strain. Introduction of the neutral trehalase I gene (NTH1) into the deletion mutant restored barotolerance defects in stationary-phase cells. Furthermore, we assessed the contribution of neutral trehalase during pressure and recovery conditions by varying the expression of NTH1 or neutral trehalase activity with a galactose-inducible GAL1 promoter with either glucose or galactose. The low barotolerance observed with glucose repression of neutral trehalase from the GAL1 promoter was restored during recovery with galactose induction. Our results suggest that neutral trehalase contributes to barotolerance, especially during recovery.     

Nanosecond pulsed electric field generators for the study of subcellular effects

Modeling and experimental studies have shown that pulsed electric fields of nanosecond duration and megavolt per meter amplitude affect subcellular structures but do not lead to the formation of large pores in the outer membrane. This "intracellular electromanipulation" requires the use of pulse generators which provide extremely high power but low energy pulses. In this study, we describe the concept of the required pulsed power sources, their design, operation, and the necessary diagnostics. Two types of pulse generators based on the Blumlein line principle have been developed and are described here. One system is designed to treat a large number of cells in cuvettes holding volumes from 0.1 to 0.8 ml. Pulses of up to 40 kV amplitude, with a duration of 10 ns and a rise time close to 1 ns can be applied to the cuvette. For an electrode gap of 1 mm this voltage corresponds to an average electric field of 40 MV/m. The second system allows for real time observation of individual cells under a microscope. It generates pulses of 10-300 ns duration with a rise time of 3.5 ns and voltage amplitudes up to 1 kV. Connected to a microreactor with an electrode gap of 100 microm, electric fields up to 10 MV/m are applied.