Inactivation of Peach Polyphenoloxidase by Exposure to Pulsed Electric Fields

The effect of exponential decay pulsed electric fields on peach (var. Miraflores) polyphenoloxidase (PPO) was evaluated. Electric field intensities ranged from 2.18 to 24.30 kV/cm. Pulses of 0.02 and 0.08 ms width were applied in mono‐ and bipolar mode. A 70% maximum reduction in enzymatic activity was achieved after 5 ms using 0.02 ms width pulses in bipolar mode at 24.30 kV/cm. Peach PPO activity decreased following an exponential decay kinetic model. Rate constants ranged from 9.0 to 138 μS^‐1 in monopolar mode, and from 8.7 to 235 μS^‐1 in bipolar mode. An exponential decay model may describe the relationship between residual PPO and input electric energy.     

Models in a Bayesian framework for inactivation of pectinesterase in a commercial enzyme formulation by pulsed electric fields

The inactivation of pectinesterase (PE) in a commercial enzyme formulation (CEF) under pulsed electric fields (PEF) was studied. Samples of an aqueous solution of the CEF were exposed to exponential waveform pulses for up to 16 ms at electric field intensities ranging from 5 to 24 kV cm^–1. Pulses were delivered in monopolar mode. The observed inactivation of the enzyme was described by several kinetics and regarding the input of electrical energy density (Q) models using Bayesian non-linear regression. Posterior distributions of the characteristic parameters for each kinetic model (based on the Hülsheger or the Weibull equation) and each Q model (based on exponential decay or the Weibull equation) were obtained. Kinetics models based on the Weibull equation showed better goodness and accuracy than the other models.     

High-intensity pulsed electric field variables affecting Staphylococcus aureus inoculated in milk

Staphylococcus aureus is an important milk-related pathogen that is inactivated by high-intensity pulsed electric fields (HIPEF). In this study, inactivation of Staph. aureus suspended in milk by HIPEF was studied using a response surface methodology, in which electric field intensity, pulse number, pulse width, pulse polarity, and the fat content of milk were the controlled variables. It was found that the fat content of milk did not significantly affect the microbial inactivation of Staph. aureus. A maximum value of 4.5 log reductions was obtained by applying 150 bipolar pulses of 8 μs each at 35 kV/cm. Bipolar pulses were more effective than those applied in the monopolar mode. An increase in electric field intensity, pulse number, or pulse width resulted in a drop in the survival fraction of Staph. aureus. Pulse widths close to 6.7 μs lead to greater microbial death with a minimum number of applied pulses. At a constant treatment time, a greater number of shorter pulses achieved better inactivation than those treatments performed at a lower number of longer pulses. The combined action of pulse number and electric field intensity followed a similar pattern, indicating that the same fraction of microbial death can be reached with different combinations of the variables. The behavior and relationship among the electrical variables suggest that the energy input of HIPEF processing might be optimized without decreasing the microbial death.     

Inactivation of Yersinia enterocolitica by pulsed electric fields

The influence of growth conditions, treatment medium characteristics and PEF process parameters on the lethal effect on Yersinia enterocolitica of pulsed electric fields (PEF) treatments in batch has been investigated. Growth phase, temperature of growth, pH, conductivity of the treatment medium, pulse width and frequency of pulses did not influence the sensitivity of Y. enterocolitica to PEF. However, an A_w decrease from >0.99 to 0.93 of the treatment medium increased the PEF resistance of Y. enterocolitica with 3.5 log₁₀ cycles after a treatment of 22 kV/cm, 800 μs and 880 kJ/kg. Inactivation of Y. enterocolitica increased with the field strength, treatment time and total specific energy up to a maximum of 6 log₁₀ cycles after 28 kV/cm, 2000 μs and 3559 kJ/kg. A nonlinear relationship was found among the survival fraction and the treatment time or the specific energy that was accurately described by a mathematical model based on the Weibull distribution. The inactivation of Y. enterocolitica by PEF was characterized by maximum field strength thresholds. Above these thresholds, specific energy necessary to obtain a given level of inactivation scarcely decreased by increasing the electric field strength, and inactivation of Y. enterocolitica only depended on the specific energy applied.     

Inactivation effect of an 18-T pulsed magnetic field combined with other technologies on Escherichia coli

The inactivation effect of 18 T pulsed magnetic fields in combination with selected non-thermal technologies was studied on Escherichia coli ATCC 11775. The bacteria were subjected to a treatment of either ultrasound (20 kHz, 70 W, 242 μm), high hydrostatic pressure (207 MPa, 5 min), pulsed electric field (6.25 kV/cm, 5.6 ms), or anti-microbials (Nisin 77.5 mg/l, lysozyme 1 mg/ml) and 50 magnetic field pulses (18 T, 30 μs). No additional inactivation or cell damage due to exposure to the pulsed magnetic field at 42°C was observed.     

Influence of pulsed electric field energy on the damage degree in alfalfa tissue

The objectives of this study were to investigate the influence of pulsed electric field (PEF) parameters on the damage degree of alfalfa mash, and to determine the relationship between the maximum damage degree and the energy used. Alfalfa mash was treated with PEF at various electric field strengths of 1.25, 1.90, and 2.50 kV/cm. The capacitance of the discharge capacitor was varied from 0.5 to 1.5 μF in steps of 0.5 μF. The pulse number was increased gradually to the point where the impedance became constant. There was no significant increase in the rate of damage beyond 0.5 kJ applied energy. The rate of change of the damage degree at 0.5 kJ was highest when the capacitance was 1.5 μF for all the voltages. Increase in the electric field strength led to decrease in energy needed to obtain the maximum damage degree. To achieve an efficient result for alfalfa juice extraction, the capacitance of the discharge capacitor should preferably be 1 μF or more. In order to minimize energy consumption for a given damage degree in alfalfa, it is desirable to have the highest energy per pulse and fewer number of pulses.     

Impact of high-intensity pulsed electric field variables affecting peroxidase and lipoxygenase activities of watermelon juice

The effects of pulse frequency (50-250 Hz), pulse width (1.0-7.0 μs) and polarity (monopolar or bipolar) of high-intensity pulsed electric field (HIPEF) treatments (35 kV/cm and 1000 μs total treatment time) on peroxidase (POD) and lipoxygenase (LOX) activities were evaluated using a response surface methodology. Second-order expressions were accurate enough to fit experimental results. HIPEF bipolar treatments resulted to be more effective than monopolar treatments in reducing POD and LOX activities. Watermelon juice LOX was more resistant to HIPEF than POD within the range of assayed conditions. HIPEF treatments set at 50 Hz for 1.0 μs pulse width could attain minimum residual POD activity values up to 10%, whereas the highest POD activity reduction was reached by combining high frequencies and pulse widths. Thus, POD could be totally inactivated by applying 7.0-μs bipolar pulses at frequencies higher than 114 Hz. In addition, the effect of frequency on the LOX activity was highly affected by the pulse width of the treatment. Thus, treatments conducted at 220 Hz with bipolar pulses of 1.0 μs led to the lowest residual LOX activity (50%).     

Changes of health-related compounds throughout cold storage of tomato juice stabilized by thermal or high intensity pulsed electric field treatments

The effect of high intensity pulsed electric fields (HIPEF) processing (35 kV/cm for 1500 μs in bipolar 4-μs pulses at 100 Hz, with an energy density of 8269 kJ/L) on the main bioactive compounds and antioxidant capacity of tomato juice was investigated and compared to heat pasteurization (90 °C for 1 min or 30 s) having the fresh juice as a reference. HIPEF and heat treated tomato juices showed higher lycopene and lower vitamin C levels than the untreated juice. However, no significant changes in the total phenolic content and antioxidant capacity were observed between treated and fresh juices just after processing. Lycopene, vitamin C and antioxidant capacity of both treated and untreated juices decreased exponentially during storage following a first order kinetics (R² = 0.763–0.987), whereas tomato juices maintained their initial phenolic content. HIPEF-treated tomato juice maintained higher lycopene and vitamin C content than the thermally treated juices during the storage time. Hence, the application of HIPEF may be appropriate to achieve nutritious and fresh like tomato juice.

Industrial relevance

HIPEF processing can lead to tomato juice with higher nutritional value than that thermally processed. HIPEF-treated (35 kV/cm for 1500 μs with 4-μs bipolar pulses at 100 Hz, energy input of 8269 kJ/L) tomato juice shows greater lycopene, vitamin C and antioxidant capacity just after the treatment and during the storage time than heat treated (90 °C­30 s and 90 °C­60 s) tomato juice. Therefore, HIPEF technology is a feasible alternative to thermal treatment to obtain tomato juice with a high presence of health-related compounds.     

Influence of high-intensity pulsed electric field processing on lipoxygenase and β-glucosidase activities in strawberry juice

The influence of high-intensity pulsed electric field (HIPEF) parameters, pulse frequency, pulse width and pulse polarity in strawberry juice lipoxygenase (LOX) and β-glucosidase (β-GLUC) was studied using a response surface methodology. The studied parameters affected on both residual enzymatic activities at unchanging electric field strength of 35 kV/cm and treatment time for 1000 μs. The contour plots showed a minimum defined space where residual activity of LOX remained at 65% and 70% in monopolar and bipolar mode, respectively. Low pulse frequencies (up to 61.6 Hz) in monopolar treatments as well as pulse frequencies and widths higher than 218 Hz and 5.4 μs in bipolar treatments did not have any effect on LOX inactivation. On the other hand, the higher the pulse frequency and pulse width, the higher the β-GLUC inactivation obtained. Moreover, when the HIPEF treatment was applied in monopolar mode, an enhancement in β-GLUC activity was observed in most of the experimental range. HIPEF treatments have demonstrated adequately that can reduce activity of enzymes that are involved in the formation of desirable flavor compounds, helping processors to obtain juices that keep their fresh flavor.

Industrial relevance

High-intensity pulsed electric fields (HIPEF) have proved to be effective in the interaction of microorganisms and enzymes in juices, maintaining their quality and freshness.HIPEF juice processing has demonstrated to have some advantages with regard to conventional thermal treatment. HIPEF treatments can reduce adequately enzymes that are involved in the formation of desirable flavor or color compounds. Thus, HIPEF technology can help processors to obtain juices that keep their fresh flavor by achieving optimal inactivation of related enzymes. This would prevent the product from undesirable off-flavor formation, which in turn would result in greater acceptability by consumers.     

Influence of Pulsed Electric Field Protocols on the Reversible Permeabilization of Rucola Leaves

Reversible electropermeabilization of plant tissues with heterogeneous structure represents a technological challenge as the response of the different structures within the same specimen to the application of electric field may differ due to different cell sizes, extracellular space configurations, and electrical properties. The influence of five different pulsed electric field protocols with different pulse polarity, number of pulses (25, 50, 75, 100, 250, and 500), and intervals between pulses (no intervals and 1- and 2-ms intervals) on the reversible permeabilization of rucola (Eruca sativa) leaves was investigated. The electric field intensity was 600 V/cm. Electrical resistance of the bulk tissue was measured before and after electroporation, and propidium iodide was used to analyze the electroporation at the surface of the leaf. Leaf viability was assessed from survival in storage, and cell viability was investigated with fluorescein diacetate. Results indicate that the viability of the leaves could not be predicted by measurements of electrical resistance or permeabilization levels of the leaf surface. Higher survival rate was demonstrated when applying bipolar pulses compared with monopolar pulses, but the latter proved to be more effective than bipolar pulses for permeabilizing the surface of the leaves. Longer intervals between bipolar pulses resulted in increased viability preservation, while the number of electroporated cells on the leaf surface was comparable for all tested protocols.     

Biochemical and thermal characterization of crude exo-polygalacturonase produced by Aspergillus sojae

Crude exo-polygalacturonase enzyme (produced by Aspergillus sojae), significant for industrial processes, was characterized with respect to its biochemical and thermal properties. The optimum pH and temperature for maximum crude exo-polygalacturonase activity were pH 5 and 55 °C, respectively. It retained 60–70% of its activity over a broad pH range and 80% of its initial activity at 65 °C for 1 h. The thermal stability study indicated an inactivation energy of E_d = 152 kJ mol⁻¹. The half lives at 75 and 85 °C were estimated as 3.6 and 1.02 h, respectively. Thermodynamic parameters, ΔH^*, ΔS^* and ΔG^*, were determined as a function of temperature. The kinetic constants K_m and V_max, using polygalacturonic acid as substrate, were determined as 0.424 g l⁻¹ and 80 μmol min⁻¹, respectively. SDS-PAGE profiling revealed three major bands with molecular weights of 36, 53 and 68 kDa. This enzyme can be considered as a potential candidate in various applications of waste treatment, in food, paper and textile industries.     

In vitro evaluation of red and green lettuce (Lactuca sativa) for functional food properties

Lettuce (Lactuca sativa) is an important leafy vegetable consumed fresh or in salad mixes. We have compared the functional food properties of selected commercial red and green lettuce varieties grown under field conditions. Both lettuce cultivars were extracted with water at biological (38 °C) and room temperatures (22 °C) at pH 2. The residues from each extraction were further extracted, sequentially with methanol and ethyl acetate. The extracts were evaluated for their in vitro lipid peroxidation (LPO) and cyclooxygenase enzyme (COX) inhibitory activities. Amongst the extracts tested, all three extracts of red lettuce showed higher LPO and COX-1 and -2 enzyme inhibitory activities than did the green lettuce extracts. Red lettuce contained a single anthocyanin, cyanidin-3-O-(6″-malonyl-β-glucopyranoside) (1), which immediately converted to cyanidin-3-O-(6″-malonyl-β-glucopyranoside methyl ester) (2) and cyanidin-3-O-β-glucopyranoside (3) under laboratory conditions. Anthocyanins 1 and 2 inhibited LPO by 88% and 91.5%, respectively, at 0.25 μM concentration. Also, they inhibited COX-2 enzyme by 78.9% and 84.3% and COX-1 by 64% and 65.8%, respectively, at 5 μM. The chicoric acid (4), amongst other phenolics, such as quercetin glucoside, ferulic and caffeic acids, isolated from both green and red lettuce, showed 85.6%, 45.6% and 94% of LPO, COX-1 and -2 enzyme inhibitions at 50 μM, respectively. This is the first report of the LPO, COX-1 and -2 enzyme inhibitory activities of compounds 1, 2 and 4. The variation of phenolics in the red and green lettuces, and specifically the lack of anthocyanins in green lettuce, might account for the higher biological activity obtained with the red variety in our study.     

Inactivation of tomato juice peroxidase by high-intensity pulsed electric fields as affected by process conditions

A response surface was used to establish the high-intensity pulsed electric fields (HIPEF) conditions in processing tomato juice to obtain the greatest peroxidase (POD) inactivation. Keeping constant the electric field strength at 35 kV/cm and the temperature below 35 °C, the treatments were set at pulse frequency from 50 to 250 Hz, pulse width from 1 to 7 μs and treatment time from 1000 to 2000 μs, using monopolar or bipolar mode. The effect of these parameters on POD inactivation was evaluated through a second order model that adequately fitted the experimental data (p = 0.0001), with a determination coefficient (R²) of 0.85. HIPEF treatment resulted to be more effective in bipolar than monopolar mode to reduce POD activity and the longer the treatment time, the greater the reduction on the enzyme activity. A pulse frequency of 200 Hz was enough to reach a minimum value of residual POD activity. The significant interaction term pulse frequency and treatment time was included in the model, showing that different combinations of both variables can lead to the same level of residual POD activity. The effect of pulse width was enhanced by using a bipolar mode, being feasible to maximize POD inactivation selecting pulse width higher than 5.5 μs in bipolar mode.     

Inactivation and kinetic model for the <Emphasis Type="Italic">Escherichia coli</Emphasis> treated by a co-axial pulsed electric field

Inactivation of Escherichia coli CGMCC1.90 inoculated into carrot juice by a co-axial pulsed electric field was investigated and the fitting of its inactivation kinetics was performed by the Hülsheger and Peleg models. The average electric field strength ranged from 5 to 25 kV/cm and the number of pulses was from 207 to 1449 pulses. The level of E. coli inactivation increased with the increment of the electric field strength and the number of pulses. At the same specific energy input level, higher electric field strength caused higher microbial reduction. As the number of pulses increased, the kinetic constants b _E and calculated based on Hülsheger model varied from 0.3116 to 0.3790 and from 4.0565 to 1.6121 kV/cm, obtained by Peleg model from 8.0412 to 2.5676 kV/cm, but the k value changed little from 2.4213 to 2.6624, respectively. The model performance evaluation was assessed by using a series of indices including accuracy factor, bias factor, the sum of the squares of the differences of the natural logarithm of observed and predicted values, root mean square error and correlation coefficient R ² between observed and predicted values. According to these parameters, Hülsheger model fitted better to the inactivation by PEF than Peleg model in the study.     

Optimizing critical high-intensity pulsed electric fields treatments for reducing pectolytic activity and viscosity changes in watermelon juice

A response surface methodology was used to determine the combined effect of high-intensity pulsed electric fields (HIPEF) variables such as frequency, pulse width and polarity on the inactivation of pectolytic enzymes involved in viscosity changes of juices. Pectin methylesterase (PME) and polygalacturonase (PG) activities as well as viscosity were determined in watermelon juices processed at pulse frequencies from 50 to 250 Hz and pulse widths ranging from 1.0 to 7.0 μs in monopolar or bipolar mode. Electric field strength and total treatment time were maintained constant in all treatments at 35 kV/cm and 1,000 μs. Second-order expressions were accurate enough to fit the experimental results. The great PME reduction contrasted with the low impact of HIPEF on the PG activity of watermelon juice within the range of assayed conditions. Minimal residual PME activity values (15%) were obtained by selecting pulse widths higher than 5.5 μs at 250 Hz in bipolar mode, whereas the lowest PG residual activities (60%) were achieved after applying 7.0-μs bipolar pulses at 250 Hz. Moreover, watermelon juice viscosity increased throughout the range of studied conditions. The highest viscosity observed in the juice after applying 7.0-μs bipolar pulses at 250 Hz was related to the lowest PME activities obtained in the product treated under those conditions. Hence, the HIPEF processing optimization through frequency, pulse width and polarity could contribute to assure enzymatic inactivation while keeping valuable attributes of juices.     

Water-extractable phytochemicals from Phyllanthus niruri exhibit distinct in vitro antioxidant and in vivo hepatoprotective activity against paracetamol-induced liver damage in mice

The antioxidative and hepatoprotective potential of Phyllanthus niruri, a widely used medicinal plant in Brazil, were investigated. The aqueous extracts of leaves showed inhibition against thiobarbituric acid-reactive species (TBARS), induced by different pro-oxidants (10 μM FeSO₄ and 5 μM sodium nitroprusside) in rat liver, brain and kidney homogenates. The extracts also lowered the formation of TBARS in phospholipids extracted from egg yolk. The plant exhibited strong antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl free radical (IC₅₀, 43.4 ± 1.45 μg/ml) and iron chelation assay. The hepatoprotective activity of the extracts were also demonstrated in vivo against paracetamol-induced liver damage, as evidenced by the decrease in serum glutamate oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT), and increased catalase activity in the liver in treatment groups, compared to the control. The results of the present study suggest the potential use of P. niruri in the treatment of various diseases, among which liver disease is the most important, due to its ability to act as an antioxidant. Furthermore, since the treatment of human intoxications with paracetamol is always limited to the administration of N-acetyl-cysteine, additional studies would be important to determine whether aqueous extracts of P. niruri could increase the efficacy of N-acetyl-cysteine against paracetamol acute hepatotoxicity.     

Non-nutritive functional agents in rattan-shoots, a food consumed by native people in the Philippines

The tender shoots of Calamus ornatus, one of the food items consumed by the native people, Kanawan Aytas, in the Bataan region of the Philippines, have not been studied before. A bioassay-guided investigation of its methanolic extract afforded non-nutritive functional agents (NFAs), steroidal saponins 1–3, along with its aglycone (4). The NFAs 1–4 inhibited cyclooxygenase enzymes, COX-1 and -2, by 47%, 43%, 33%, and 53% and 71%, 75%, 78%, and 73%, respectively, at 28.2, 24.2, 21.2 and 60.4 μM. Treatment of breast (MCF-7), CNS (SF-268), lung (NCI-H460), colon (HCT-116) and gastric (AGS) cancer cell lines with the extract at 100 μg/ml reduced cell proliferation. Similarly, the pure NFAs 2 and 3 reduced the cell viability of breast, CNS, lung, colon and gastric cancer cell lines by 37.5%, 22.4%, 53.3%, 58.2%, 40.3% and 29.8%, 21.3%, 45.6%, 37.1%, 25.0%, respectively, at 24.2 and 21.2 μM. The 50% reduction in cell viability (IC₅₀) concentrations of 2 and 3 against these cancer cell lines were 8.8, 6.1, 7.5, 23.8, 12.1 and 3.8, 7.1, 3.3, 14.3, 12.1 μM, respectively. This is the first report on the isolation of steroidal saponins from C. ornatus shoots and their antiinflammatory and tumor cell proliferation inhibitory activities. Therefore, our results suggest that the Kanawan Aytas may yield health benefits from rattan-shoots in their diet.     

Production and stability of chlorine dioxide in organic acid solutions as affected by pH, type of acid, and concentration of sodium chlorite, and its effectiveness in inactivating Bacillus cereus spores

We studied the production and stability of chlorine dioxide (ClO₂) in organic acid solutions and its effectiveness in killing Bacillus cereus spores. Sodium chlorite (5000, 10,000, or 50,000 μg/ml) was added to 5% acetic, citric, or lactic acid solution, adjusted to pH 3.0, 4.0, 5.0, or 6.0, and held at 21 °C for up to 14 days. The amount of ClO₂ produced was higher as the concentration of sodium chlorite was increased and as the pH of the acid solutions was decreased. However, the stability in production of ClO₂ was enhanced by increasing the pH of the organic acid solutions. To evaluate the lethal activity of ClO₂ produced in various acid solutions as affected by acidulant and pH, suspensions of B. cereus spores were treated at 21 °C for 1, 3, 5, or 10 min in hydrochloric acid or organic acid solutions (pH 3.0, 4.0, 5.0, or 6.0) containing ClO₂ at concentrations of 100, 50, or 25 μg/ml. Populations of viable spores treated with ClO₂ at concentrations of 100 or 50 μg/ml in organic acid solutions decreased more rapidly than populations treated with the same concentrations of ClO₂ in HCl. Rates of inactivation tended to increase with higher pH of ClO₂ solutions. Results show that ClO₂ formed in organic acid solutions has higher stability and is more lethal to B. cereus spores than ClO₂ formed at the same concentration in HCl solution. This finding emphasizes the benefits of using organic acid solutions to prepare ClO₂ intended for use as an antimicrobial.     

Microbial inactivation by pulsed electric fields in a batch treatment chamber: effects of some electrical parameters and food constituents

The inactivation of Escherichia coli by high voltage pulsed electric fields in a batch treatment chamber was studied in liquid, solid and semisolid foods or model systems. Treatment heterogeneity was demonstrated and found to be due to the presence of an air bubble trapped inside the chamber. Agitation of the inoculated liquid samples (16 mM sodium phosphate buffer, pH 7, ρ=460 Ωcm) during pulse processing resulted in efficient microbial inactivation (five log cycles at 33 kV/cm and 25°C after 261 μs of cumulated pulses). A slower inactivation rate was observed in inoculated solid agar gels of the same pH and resistivity, under the same pulse processing conditions. The inactivation of E. coli in inoculated dairy cream (33% fat, pH 6.8, ρ=370 Ωcm), ovalbumin solution (10% protein w/v, pH 6.7, ρ=370 Ωcm) or fish egg suspension (pH 6.8, ρ=400 Ωcm) was almost identical to that in 16 mM phosphate buffer, pH 7. Thus emulsified lipids, soluble proteins or conductive food particulates do not appear to protect against microbial inactivation by electric pulses.