Microbiological and enzymatic activity of bovine whole milk treated by pulsed electric fields

Pulsed electric field (PEF)‐treated milk (25.7 kV/cm for 34 μs after preheating to 55 °C and holding for 24 s) was microbiologically stable for 21 days at 4 °C, and similar to thermally treated milk (63 °C for 30 min or 73 °C for 15 s). Alkaline phosphatase inactivation was comparable after PEF (preheating followed by PEF) and both thermal treatments. PEF treatment initially reduced xanthine oxidase (30%) and plasmin (7%) activities, but after 21 days of refrigerated storage these activities were similar to the initial untreated milk. During refrigerated storage of PEF (preheating followed by PEF) and thermally treated milk, lipolytic activity increased and pH levels decreased.     

Combined effect of temperature and pulsed electric fields on soya milk lipoxygenase inactivation

Pulsed electric fields (PEF) were applied to freshly prepared soya milk using a laboratory scale continuous PEF system to study the feasibility of inactivating lipoxygenase (LOX). Square wave PEF using different combinations of pre-treatment temperature, electric field strength and treatment time were evaluated in this study. Inactivation curves for the enzyme were plotted for each parameter and inactivation kinetics were calculated and modelled. Results showed the highest level of inactivation (84.5%) was obtained using a combination of preheating to 50 °C, and a PEF treatment time of 100 μs at 40 kV/cm. Inactivation of LOX activity as a function of treatment time could be described using a first order kinetic model. Calculated D values following pre-heating to 50 °C were 172.9, 141.6 and 126.1 μs at 20, 30 and 40 kV/cm, respectively.     

The effect of pulsed electric fields on the inactivation and structure of lysozyme

The effect of pulsed electric fields (PEF) on the activity and structure of lysozyme selected as a model enzyme was investigated. The inactivation of lysozyme in phosphate buffer was a function of electric field strength, treatment time, electrical conductivity, and enzyme concentration. No significant (p > 0.05) change in the activity of PEF-treated lysozyme was found after storage for 12, 24 and 48 h at 4 °C. The effect of PEF on tertiary structure of lysozyme was demonstrated by second-derivative UV spectra and intrinsic fluorescence. The results indicated that the unfolding of tertiary structure was induced by PEF treatment at 35 kV/cm for 1200 μs, and more tyrosine residues were buried inside the protein after PEF treatment, accompanied by the exposure of more tryptophan residues. CD spectra suggested that the inactivation of lysozyme by PEF was closely related to the loss of α-helix of secondary structure.     

Comparative study of inactivation and conformational change of lysozyme induced by pulsed electric fields and heat

A comparative study of inactivation and conformational change of lysozyme induced by pulsed electric fields (PEF) at 35 kV/cm and heat at 100 °C was carried out. The results showed that both PEF- and heat-induced inactivation of lysozyme followed a first-order model. While the relative residual activity (RRA) values of lysozyme induced by PEF and heat were similar, the conformational changes in tertiary and secondary structures were different in the two treatments demonstrated by 8-aniline-1-naphthalene sulfonate-binding and intrinsic fluorescence and circular dichroism analysis, indicating the different enzyme inactivation mechanisms. The tertiary and secondary structures of lysozyme unfolded coincidentally with the decrease of lysozyme RRA values from 92 to 62% induced by PEF, suggesting that lysozyme treated with PEF did not form a molten globule. However, in heat treatment, lysozyme with RRA values from 90 to 78% had the same backbone secondary structure as the native protein, but with significant changes in tertiary structure.     

Freezing of potato tissue pre-treated by pulsed electric fields

The effects of pulsed electric fields (PEF) pre-treatment on the freezing, freeze-drying and rehydration behavior of potato were studied. Potato samples (26 mm diameter, 10 mm high) were treated by PEF (400 V/cm) for various durations between 10⁻⁴ and 0.3 s. The degree of tissue damage was quantified by the change in electrical conductivity. PEF treated and untreated samples were either frozen in an air-blast freezer with air at −35 °C and 2 m/s velocity or freeze-dried at 0 °C and 0.04 mbar pressure and then rehydrated in water at 25 °C. The freezing times for PEF pre-treated samples reduced as the PEF-induced tissue damage increased. Scanning electron microscope images of the air-blast frozen and then freeze-dried samples showed increased deformation of cells and larger intercellular spaces (frozen samples only) for the PEF pre-treated samples. However, PEF pre-treatment improved the rate of freeze-drying and improved the quality and rehydration of the samples.     

高强脉冲电场对食品中大肠埃希氏菌基因水平传递的影响

目的:探讨高强脉冲电场对大肠埃希氏菌的基因获得及水平传递中的作用。方法:采用接合转移试验,研究了PEF强电场作用对大肠埃希氏菌基因水平传递的影响,并以未经处理的标准菌株为对照,通过PCR方法进行基因检测分析。结果:所有经PEF处理的菌株均由正常的接合转移率42.4%～68.5%上升到100%。PCR检测结果显示,所得接合子中int基因的扩增产物大小仍为923bp,与正常未处理int基因大小一致。结论:PEF处理使得细菌接合转移率明显提高,PEF处理对传递到接合子中的基因未能产生影响。     

Acceleration of soluble matter extraction from chicory with pulsed electric fields

This work discusses effects of thermal and PEF treatments on efficiency of soluble solids extraction from chicory tissues. The effects of temperature, PEF electric field strength and time of treatment on damage degree and acceleration of the soluble matter extraction kinetics are discussed. The PEF treatment with field strength 100–600 V/cm, treatment time 10⁻³–50 s, and temperature 20–80 °C, was applied. The activation energy of usual thermal damage was rather high (W_τ ≈ 263 kJ/mol), however, it can be noticeably reduced to W_τ = 30–40 kJ/mol by application of PEF treatment. The moderate electric energy consumption (U < 10 kJ/kg) at room temperatures demands application of relatively high electric field strength (400–600 V/cm), however, the value of U noticeably decreases with temperature increase. The benefits of PEF application for enhancement of the soluble matter extraction from chicory were demonstrated. The PEF-pretreatment noticeably accelerated diffusion even at low temperatures within 20–40 °С. Proposed technique appears to be promising for future industrial applications of “cold” soluble matter extraction from chicory roots.     

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.     

Milk thermization by pulsed electric fields (PEF) and electrically induced heat

Whole milk was processed using selected combinations of pulsed electric fields (PEF) and thermal treatments to inactivate Listeria innocua. Electric field intensities of 30 and 40 kV/cm were applied at selected number of pulses (1–30) and temperatures (20–72 °C) for less than 10 s. A maximum microbial reduction of 4.3 log cycles was achieved using 10, 17.5, 20 and 25 pulses, when processing milk at 30 kV/cm and initial temperatures of 43, 33, 23 and 13 °C, respectively. Around 4.3 log cycles of L. innocua was observed when treating milk at 40 kV/cm using 3, 10, 12.5, 15, and 20 pulses and 53, 33, 23, 15, and 3 °C, respectively. Milk treated with 40 kV/cm of electric field intensity, few pulses, and initial temperature close to 55 °C showed the best balance between L. innocua inactivation and energy-consumption. An energy expenditure of around 244 J/mL was achieved, which can be further reduced to 44 J/mL using a thermal regeneration system.     

Inactivation of soybean lipoxygenase in soymilk by pulsed electric fields

The inactivation of soybean lipoxygenase by pulsed electric fields (PEF) was studied. Effects of PEF parameters (treatment time, pulse strength, pulse frequency and pulse width) were evaluated. Soymilk was exposed to pulsed strengths from 20 to 42 kV/cm for up to 1036 μs treatment time in square wave pulse of bipolar mode. Moreover, pulse frequency (100–600 Hz) and pulse width (1–5 μs) was also tested at constant pulsed treatment time of 345 μs and strength of 30 kV/cm. Residual activity of soybean lipoxygenase decreased with the increase of treatment time, pulse strength, pulse frequency and pulse width. The maximum inactivation of soybean lipoxygenase by PEF achieved 88% at 42 kV/cm for 1036 μs with 400 Hz of pulse frequency and 2 μs of pulse width at 25 °C. Inactivation of soybean lipoxygenase by pulsed electric fields was modeled using several kinetic models. Weibull distribution function was most suitable model describing the inactivation of soybean LOX as a function of pulsed electric fields process parameters. Moreover, reduction of soybean LOX activity related to the electric field strength could be well described by the Fermi model.     

Effects of combined exposure of micrococcus luteus to nisin and pulsed electric fields

Death and injury following exposure of Micrococcus luteus to nisin and pulsed electric field (PEF) treatment were investigated in phosphate buffer (pH 6.8, σ=4.8 ms/cm at 20°C). Four types of experiment were carried out, a single treatment with nisin (100 IU/ml at 20°C for 2 h), a single PEF treatment, a PEF treatment followed by incubation with nisin (as before) and addition of nisin to the bacterial suspension prior to the PEF treatment. The application of nisin clearly enhanced the lethal effect of PEF treatment. The bactericidal effect of nisin reduced viable counts by 1.4 log₁₀ units. Treatment with PEF (50 pulses at 33 kV/cm) resulted in a reduction of 2.4 log₁₀ units. PEF treatment followed by nisin caused a reduction of 5.2 log₁₀ units in comparison with a 4.9 log₁₀ units reduction obtained with nisin followed by PEF. Injury of surviving cells was investigated using media with different concentrations of salt. Sublethally damaged cells of M. luteus could not be detected by this means, following PEF treatment.     

Combined effect of temperature and pulsed electric fields on apple juice peroxidase and polyphenoloxidase inactivation

Pulsed electric fields (PEF) were applied to freshly prepared apple juice using a laboratory scale continuous PEF system to study the feasibility of inactivating peroxidase (POD) and polyphenoloxidase (PPO). Square wave PEF using different combinations of electric field strength, pre-treatment temperature and treatment time were evaluated in this study and compared to conventional pasteurisation (72 °C; 26 s). Inactivation curves for the enzyme were plotted for each parameter and inactivation kinetics were calculated. Results showed the highest level of decrease in the enzymatic activity of 71% and 68%, for PPO and POD, respectively, were obtained by using a combination of preheating to 50 °C, and a PEF treatment time of 100 μs at 40 kV/cm. This level of inactivation was significantly higher (P < 0.05) than that recorded in juice processed by conventional mild pasteurisation where the activity of PPO and POD decreased by 46% and 48%, respectively. The kinetic data for the inactivation of both enzymes could be described using a 1st-order model (P < 0.001).     

高强脉冲电场对过氧化物酶的影响

本文采用高强脉冲电场对过氧化物酶进行处理。研究了电场强度，脉冲数目和频率等因素对酶活性的影响，并与热处理过氧化物酶进行对比。结果表明：高强脉冲电场可引起过氧化物酶的活性变化。且失活率与电场强度和脉冲数目成正比。频率对活性的影响不明显。     

高强脉冲电场对脂肪酶的影响研究

采用高强脉冲电场对脂肪酶进行处理,研究了电场强度、脉冲数目、温度、介质环境等因素对酶活的影响.结果表明,高强脉冲电场可引起脂肪的失活,且失活率与电场强度和脉冲数目呈正比,但酶对电场的抑制比微生物要强,热效应和溶液离子浓度对酶活的影响很小;不同介质环境的影响表明酶在环境中的三维结构状态是影响电场处理效果的最可能的原因.     

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.     

Influence of Pulsed Electric Field on Escherichia coli and Saccharomyces cerevisiae

Heat sterilization destroys the color, smell, taste, and nutrients of food. Pulsed electric field can effectively kill microorganisms for many foods at room temperature without compromising sensory and nutritional quality. The effect of pulsed electric field on the sterilization rates of Escherichia coli and Saccharomyces cerevisiae was investigated. The fundamental mechanism of pulsed electric field sterilization on S. cerevisiae was briefly discussed. The inactivation of E. coli and S. cerevisiae increased as the electric field strength and treatment time increased. Pulsed electric field treatments at 35 kV/cm and 90 μs reduced S. cerevisiae and E. coli by 5.30 and 5.15 log numbers, respectively. The inactivation of E. coli and S. cerevisiae was not remarkable at increased conductivity and the same electric field strength. Concaves and holes in S. cerevisiae cell surface, protoplast deletion, overflow of substances from cells, and denatured DNA were observed. These results supported the hypothesis of “membrane perforation” to explain the mechanism underlying pulsed electric field sterilization.     

Evaluation of selected mathematical models to predict the inactivation of Listeria innocua by pulsed electric fields

The inactivation of Listeria innocua ATCC 51742 by pulsed electric fields was investigated at 35, 40 and 45 kV/cm. Results indicate that at treatment times shorter than 37 μs at 40 and 45 kV/cm, and 49 μs at 35 kV/cm, there is a linear relationship between the logarithm of the survivor fraction and the treatment time. However, longer times result in an abrupt increase in the slope of the inactivation curve and in inactivation values greater than six logarithmic cycles. A model based on Weibull's survival function was used to describe microbial inactivation and then compared to a first-order kinetic model. Distribution parameters of Weibull's survival function and kinetic constant for the first-order kinetic model were calculated by fitting experimental data. Calculated mean times for microbial inactivation from Weibull's distribution were 11.55, 8.65 and 5.39 μs at 35, 40 and 45 kV/cm, respectively. The goodness-of-fit between experimental and predicted values was determined using an accuracy factor. The model based on the Weibull survival distribution provided better accuracy factors than first-order kinetics. The model based on Weibull's survival function seems promising for describing survival curves that exhibit concavity.     

Evaluation of a static treatment chamber to investigate kinetics of microbial inactivation by pulsed electric fields at different temperatures at quasi-isothermal conditions

A static parallel electrode treatment chamber with tempered electrodes has been designed to obtain kinetics data on microbial inactivation by pulsed electric fields (PEF) at different temperatures at quasi-isothermal conditions. Distribution of the electric field strength and temperature within the treatment zone was estimated by a finite element method. A good agreement was observed between the temperatures estimated by numerical simulation and temperatures measured by a thermocouple in the treatment zone before and after the PEF treatments (values of RMSE below 3%). Influence of the treatment temperature on PEF inactivation (30 kV/cm) of Salmonella typhimurium was investigated at temperatures between 4 and 50 °C in media of pH 3.5 and 7.0. Treatment temperature had an important effect on microbial inactivation for both values of pH. At pH 3.5 the inactivation of S. typhimurium was irrelevant at 4 °C but about 1.5, 2.9, 4.0 and 5.0 Log₁₀ reductions were obtained after 30 pulses (90 μs) at 15, 27, 38 and 50 °C, respectively. At pH 7.0, around two Log₁₀ cycles of inactivation were observed after 50 pulses (150 μs) at 4 °C. At temperatures in the range between 15 and 50 °C the treatment temperature practically did not influence PEF resistance of S. typhimurium. A model based on the Weibull distribution adequately described kinetics of inactivation of S. typhimurium at different temperatures. The treatment chamber designed in the investigation could be useful to obtain kinetics data on PEF destruction of microorganisms or other components of interest at a uniform distribution of electric field strength and homogeneous and quasi-isothermal conditions in a wide range of temperatures.     

Evaluation of phenolic extraction during fermentation of red grapes treated by a continuous pulsed electric fields process at pilot-plant scale

The feasibility of processing red grapes by pulsed electric fields (PEF) at pilot-plant scale to improve the extraction of anthocyanins and phenols during the maceration-fermentation step of the winemaking process has been investigated. With this general purpose a colinear continuous treatment chamber was developed. The influence of field strength (2, 5 and 7 kV/cm) and grape variety (Cabernet Sauvignon, Syrah and Merlot) in the extraction kinetics was studied. Extraction curves were described by an exponential equation that permits to estimate the extraction rate (k) and the maximum extraction yield (Y_max). An increment of the electric field from 2 to 7 kV/cm increased the extraction rate of anthocyanins and total phenols for the three varieties investigated. The increment of Y_max due to the application of PEF was more remarkable in Cabernet Sauvignon than in Merlot and Syrah. The continuous PEF system presented in this work constitutes an important step for the application of PEF technology at commercial scale.     

Modeling within the Bayesian framework, the inactivation of pectinesterase in gazpacho by pulsed electric fields

The inactivation of pectinesterase (PE) activity in gazpacho (a Spanish ready-to-use cold vegetable soup) under pulsed electric fields (PEFs) was studied. Samples were exposed to 4 μs monopolar or bipolar square-wave pulses at 5–35 kV cm⁻¹ electric field intensity for up to 1500 μs and 200 Hz (temperature below 40 °C). Inactivation of PE was greater when treatment time and electric field intensity increased, and bipolar pulses were more effective inactivating PE than monopolar ones. Estimation of parameters and quantities involved in the tested models were performed within the Bayesian framework. The kinetic evolution of the enzyme was explained using a 3-parameter biexponential model based on a mechanism involving two irreversible consecutive steps. Rate constant k₁ was not dependent on neither electric field intensity nor pulse polarity. Rate constant k₂ and ratio between the activities of intermediate forms and the native ones of the enzyme Λ were affected by those conditions.