Alkaline phosphatase and microbial inactivation by pulsed electric field in bovine milk

The effects of pulsed electric field (PEF) treatments at field intensities of 25–37 kV cm^− 1 and final PEF treatment temperatures of 15 °C and 60 °C on the inactivation of alkaline phosphatase (ALP), Total Plate Count (TPC), Pseudomonas and Enterobacteriaceae counts were determined in raw skim milk. At 15 °C, PEF treatments of 28 to 37 kV cm^− 1 resulted in 24–42% inactivation in ALP activity and < 1 log reduction in TPC and Pseudomonas count, while the Enterobacteriaceae count was reduced by at least 2.1 log units to below the detection limit of 1 CFU mL^− 1. PEF treatments of 25 to 35 kV cm^− 1 at 60 °C resulted in 29–67% inactivation in ALP activity and up to 2.4 log reduction in TPC, while the Pseudomonas and Enterobacteriaceae counts were reduced by at least 5.9 and 2.1 logs, respectively, to below the detection limit of 1 CFU mL^− 1. Kinetic studies suggested that the effect of field intensity on ALP inactivation at the final PEF treatment temperature of 60 °C was more than twice that at 15 °C. A combined effect was observed between the field intensity and temperature in the inactivation of both ALP enzyme and the natural microbial flora in raw skim milk.Industrial relevanceMilk has been pasteurised to ensure its safety and extend its shelf life. However, the need for retaining heat-sensitive nutrient and sensory properties of milk has resulted in interest in the application of alternative technologies. The results of the current study suggest that PEF as a non-thermal process can be employed for the treatment of raw milk in mild temperature to achieve adequate safety and shelf life while preserving the heat-sensitive enzymes, nutrients and bioactive compounds.     

Effect of high intensity pulsed electric fields and heat treatments on vitamins of milk

The effects of high intensity pulsed electric field (HIPEF) treatments at room or moderate temperature on water-soluble (thiamine, riboflavin, ascorbic acid) and fat-soluble vitamins (cholecalciferol and tocopherol) were evaluated and compared with conventional thermal treatments. Vitamin retention was determined in two different substrates, milk and simulated skim milk ultrafiltrate (SMUF). Samples were subjected to HIPEF treatments of up to 400 micros at field strengths from 18.3 to 27.1 kV/cm and to heat treatments of up to 60 min at temperatures from 50 to 90 degrees C. No changes in vitamin content were observed after HIPEF or thermal treatments except for ascorbic acid. Milk retained more ascorbic acid after a 400 microstreatment at 22.6 kV/cm (93.4%) than after low (63 degrees C-30 min; 49.7% retained) or high (75 degrees C-15s; 86.7% retained) heat pasteurisation treatments. Retention of ascorbic acid fitted a first-order kinetic model for both HIPEF and thermal processes. First-order constant values varied from 1.8 x 10.4 to 1.27 x 10(-3) micros(-1) for the HIPEF treatments (18.3-27.1 kV/cm) and, for thermal processing ranged from 5 x 10(-3) to 8 x 10(-2) min(-1) (50-90 degrees C). No significant differences were found between the results obtained after applying HIPEF treatments at room or moderate temperature. However, results depended on the treatment media. A beneficial effect of natural skim milk components, mainly proteins, was observed on the preservation of ascorbic acid, since skim milk retained more ascorbic acid than SMUF after HIPEF treatments.     

Reduction of protease activity in milk by continuous flow high-intensity pulsed electric field treatments

High-intensity pulsed electric field (HIPEF) is a non-thermal food processing technology that is currently being investigated to inactivate microorganisms and certain enzymes, involving a limited increase of food temperature. Promising results have been obtained on the inactivation of microbial enzymes in milk when suspended in simulated milk ultrafiltrate. The aim of this study was to evaluate the effectiveness of continuous HIPEF equipment on inactivating a protease from Bacillus subtilis inoculated in milk. Samples were subjected to HIPEF treatments of up to 866 micros of squared wave pulses at field strengths from 19.7 to 35.5 kV/cm, using a treatment chamber that consisted of eight colinear chambers connected in series. Moreover, the effects of different parameters such as pulse width (4 and 7 micros), pulse repetition rates (67, 89, and 111 Hz), and milk composition (skim and whole milk) were tested. Protease activity decreased with increased treatment time or field strength and pulse repetition rate. Regarding pulse width, no differences were observed between 4 and 7 micros pulses when total treatment time was considered. On the other hand, it was observed that milk composition affected the results since higher inactivation levels were reached in skim than in whole milk. The maximum inactivation (81%) was attained in skim milk after an 866-micros treatment at 35.5 kV/cm and 111 Hz.     

Kinetic models for pulsed electric field and thermal inactivation of Escherichia coli and Pseudomonas fluorescens in whole milk

Inactivation of Escherichia coli ATCC 11775 and Pseudomonas fluorescens ATCC 948 in UHT whole (4% fat) milk during thermal processing at 56–62 °C and pulsed electric field (PEF) processing at 30 or 35 kV cm⁻¹ at approximately 30, 40 or 50 °C was investigated. E. coli ATCC 11775 was more heat-resistant than P. fluorescens ATCC 948, but more susceptible to PEF processing. All inactivation kinetics showed strong deviations from log-linearity. Thus, a simplified logistic (log-decay) regression model was used to accurately predict thermal and PEF inactivation of E. coli ATCC 11775 and P. fluorescens ATCC 948 under various treatment conditions. This is a useful tool for identifying processing conditions to inactivate pathogenic and spoilage microorganisms in whole milk at sub-pasteurisation temperatures.     

Effect of thermosonication,pulsed electric field and their combination on inactivation of Listeria innocua in milk

The impact of thermosonication (TS) and pulsed electric field (PEF), individually and combined, on the survival of Listeria innocua 11288 (NCTC) in milk was investigated. TS (400 W, 160 s) without pre-heating reduced L. innocua by 1.2 log₁₀ cfu mL^?1, while shorter treatment times produced negligible inactivation, suggesting TS to be a hurdle rather than an effective standalone treatment. PEF (30 and 40 kV cm^?1, 50 μs) at 10 °C caused a reduction of L. innocua of 1.1 and 3.3 log cycles, respectively. The highest field strength (40 kV cm^?1) combined with TS (80 s) led to 6.8 log₁₀ cfu mL^?1 inactivation. Milk pre-heated to 55 °C (over 60 s) prior to TS followed by PEF (30 and 40 kV cm^?1) showed inactivation between 4.5 and 6.9 log₁₀ cfu mL^?1, the latter being comparable (P > 0.05) with thermal pasteurisation. The data indicate that TS followed by PEF represents a valid alternative for L. innocua inactivation in milk.     

pH-changes during pulsed electric field treatments — Numerical simulation and in situ impact on polyphenoloxidase inactivation

Pulsed electric field (PEF) treatment of liquid media was investigated with focus on the pH shifts occurring in a batch parallel plate electrode treatment chamber.A numerical simulation of pH-shifts during PEF application was performed, which was experimentally verified by the application of an optical method based on digital image processing.Numerical, as well experimental results showed pH-shifts of up to 4.04 units already after a treatment time of 34 μs at electric field strength of 10 kV/cm. As a result, pH values of 10.9 and 3.3 were observed at the cathode and anode respectively after PEF treatment of a salt solution with an initial pH of 7.1. Furthermore, it was shown that the PEF treatment may cause partial PPO inactivation not directly related to the electric field impact on the enzyme. This finding contributes to the understanding of PEF side effects, such as electrochemical reactions and pH changes during PEF treatments.

Industrial Relevance

Microbial and enzyme inactivation by PEF processing depends not only on treatment parameters, such as electric field strength, pulse number, temperature and frequency, but also on food properties, such as electrical conductivity, ion strength and pH. It has been widely reported that pH alters the microbial inactivation by PEF and also that PEF can produce pH-shifts. The presented investigation contributes to the understanding of pH-shifts by the application of PEF processing and the application of a simple digital imaging method for measuring and analyzing the pH during the PEF treatment, as well as, the understanding on PPO inactivation due to pH-shifts during PEF treatments.     

Inactivation of wine-associated microbiota by continuous pulsed electric field treatments

Four different treatments of pulsed electric field (PEF) technology were tested in a continuous-flow system for inactivating 25 species of yeast, lactic acid bacteria and acetic acid bacteria associated with wines. Overall, the reached inactivation varied from 0.64 to 4.94 log units. Curiously, it was determined that the application of higher specific energy did not provide significant declination of yeast population, although significantly different inactivations were determined for bacteria. The inactivation of bacteria highly depended on each species and results corroborated different sensitivity of two Oenococcus oeni strains what could be related with their genetic characteristics.     

Influence of pulsed electric field treatments on the volatile compounds of milk in comparison with pasteurized processing

Effects of pulsed electric field (PEF) treatments on the volatile profiles of milk were studied and compared with pasteurized treatment of high temperature short time (HTST) (75 °C, 15 s). Volatile compounds were extracted by solid-phase micro-extraction (SPME) and identified by gas chromatography/mass spectrometry (GC-MS) and gas chromatography-olfactometry (GC-O). A total of 37 volatile compounds were determined by GC-MS, and 19 volatile compounds were considered to be major contributors to the characteristic flavor of milk samples. PEF treatment resulted in an increase in aldehydes. Milk treated with PEF at 30 kV/cm showed the highest content of pentanal, hexanal, and nonanal, while heptanal and decanal contents were lower than in pasteurized milk, but higher than in raw milk. All the methyl ketones detected in PEF milk were lower than in pasteurized milk. No significant differences in acids (acetic acid, butanoic acid, hexanoic acid, octanoic acid, and decanoic acid), lactones, and alcohols were observed between pasteurized and PEF-treated samples; however, 2(5H)-furanone was only detected in PEF-treated milk. Although GC-MS results showed that there were some volatile differences between pasteurized and PEF-treated milk, GC-O data showed no significant difference between the 2 samples.     

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.     

Advances in innovative processing technologies for microbial inactivation and enhancement of food safety – pulsed electric field and low-temperature plasma

The need for enhancing microbial food safety and quality, without compromising the nutritional, functional and sensory characteristics of foods, has created an increasing world-wide interest in low-temperature innovative processes for food preservation. In contrast, to the traditional thermal processes, these emerging technologies are predominantly reliant on physical processes, including high hydrostatic pressures, pulsed electric fields and low-temperature plasmas that inactivate microorganisms at ambient or moderately elevated temperatures and short treatment times. The current review presents the latest developments in the two most recent of these technologies, pulsed electric field and low-temperature plasma treatments for food preservation and disinfection of food contact surfaces.     

Effects of pH,temperature, and pre-pulsed electric field treatment on pulsed electric field and heat inactivation of Escherichia coli O157:H7

This investigation was undertaken to study the inactivation of Escherichia coli O157:H7 by pulsed electric field (PEF) treatment and heat treatment after exposure to different stresses. E. coli O157:H7 cells exposed to different pHs (3.6, 5.2, and 7.0 for 6 h). different temperatures (4, 35, and 40 degrees C for 6 h), and different pre-PEF treatments (10, 15, and 20 kV/cm) were treated with PEFs (20, 25, and 30 kV/cm) or heat (60 degrees C for 3 min). The results of these experiments demonstrated that a pH of 3.6 and temperatures of 4 and 40 degrees C caused significant decreases in the inactivation of E. coli O157:H7 by PEF treatment and heat treatment (P < 0.05). Pre-PEF treatments, pHs of 5.2 and 7.0, and a temperature of 35 degrees C, on the other hand, did not result in any resistance of E. coli O157:H7 cells to inactivation by PEF treatment and heat treatment (P > 0.05).     

Critical factors determining inactivation kinetics by pulsed electric field food processing

The potential to commercialize the non-thermal pulsed electric field (PEF) technology as a new method to preserve food products has caught the attention of the food industry that wishes to fulfil consumers demands for fresh products. In recent years, numerous research groups have demonstrated the possibility to inactivate a range of microorganisms both in buffer systems and in food products using different PEF systems. In this review, we survey the critical process factors and main characteristics of food products that determine the microbial inactivation kinetics.     

低电压脉冲电场对花楸果汁大肠杆菌灭活工艺优化

为探寻低电压脉冲电场对花楸果汁中大肠杆菌的灭活效果,实验考察了在指数衰减波的作用下,电压、电容、电阻、介质电导率及处理次数对大肠杆菌致死率的影响。经单因素及正交优化实验得到最佳处理工艺为:电压400 V,电容300μF,介质电导率0.5 m S/cm,处理次数3次,该条件下大肠杆菌致死率达到2.242。实验结果表明,低电压脉冲电场可以达到有效杀灭果汁中微生物的目的,这为该技术的进一步研究提供了理论基础。      

Comparative study on shelf life of whole milk processed by high-intensity pulsed electric field or heat treatment

The effect of high-intensity pulsed electric fields (HI-PEF) processing (35.5 kV/cm for 1,000 or 300 μ with bipolar 7-μs pulses at 111 Hz; the temperature outside the chamber was always < 40° C) on microbial shelf life and quality-related parameters of whole milk were investigated and compared with traditional heat pasteurization (75° C for 15 s), and to raw milk during storage at 4° C. A HIPEF treatment of 1,000 μ ensured the microbiological stability of whole milk stored for 5 d under refrigeration. Initial acidity values, pH, and free fatty acid content were not affected by the treatments; and no proteolysis and lipolysis were observed during 1 wk of storage in milk treated by HIPEF for 1,000 μ. The whey proteins (serum albumin, β-lactoglobulin, and α-lactalbumin) in HIPEF-treated milk were retained at 75.5, 79.9, and 60%, respectively, similar to values for milk treated by traditional heat pasteurization.     

Modeling high-intensity pulsed electric field inactivation of a lipase from Pseudomonas fluorescens

The inactivation kinetics of a lipase from Pseudomonas fluorescens (EC 3.1.1.3.) were studied in a simulated skim milk ultrafiltrate treated with high-intensity pulsed electric fields. Samples were subjected to electric field intensities ranging from 16.4 to 27.4 kV/cm for up to 314.5 μS, thus achieving a maximum inactivation of 62.1%. The suitability of describing experimental data using mechanistic first-order kinetics and an empirical model based on the Weibull distribution function is discussed. In addition, different mathematical expressions relating the residual activity values to field strength and treatment time are supplied. A first-order fractional conversion model predicted residual activity with good accuracy (A_f = 1.018). A mechanistic insight of the model kinetics was that experimental values were the consequence of different structural organizations of the enzyme, with uneven resistance to the pulsed electric field treatments. The Weibull model was also useful in predicting the energy density necessary to achieve lipase inactivation.     

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.     

Enhancing inactivation of Staphylococcus aureus in skim milk by combining high-intensity pulsed electric fields and nisin

High-intensity pulsed electric fields (HIPEF) can be used as a nonthermal preservation method that is believed to enhance the effect of nisin on microorganisms such as Staphylococcus aureus. The survival of S. aureus inoculated into skim milk and treated with nisin, with HIPEF, or with a combination of nisin-HIPEF was evaluated. Nisin dose, milk pH, and HIPEF treatment time were the controlled variables that were set up at 20 to 150 ppm, pH 5.0 to 6.8, and 240 to 2,400 micros, respectively. HIPEF strength and pulse width were kept constant at 35 kV/cm and 4 micros, respectively. No reduction in S. aureus concentration was observed in skim milk at its natural pH after treatment with nisin, but 1.1 log units were recovered after 90 min of treatment at pH 5.0 with 150 ppm nisin. A reduction in viable S. aureus counts of 0.3 and 1.0 log unit in skim milk treated with HIPEF at its natural pH was observed at 240 and 2,400 micros, respectively. The nisin-HIPEF treatment design was based on a response surface methodology. The combined effect of nisin and HIPEF was clearly synergistic. However, synergism depended on pH. A maximum microbial inactivation of 6.0 log units was observed at pH 6.8, 20 ppm nisin, and 2,400 micros of HIPEF treatment time, whereas a reduction of over 4.5 log units was achieved when pH, nisin concentration, and HIPEF treatment times were set at 5.0, 150 ppm, and 240 micros, respectively.     

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.     

Growth of pulsed electric field exposed Escherichia coli in relation to inactivation and environmental factors

Pulsed electric fields (PEF) have been proven to inactivate microorganisms during nonthermal conditions and have the potential to replace thermal processing as a method for food preservation. However, there is a need to understand the recovery and growth of survivors and potentially injured microorganisms following PEF processing. The purpose of this investigation was to study the growth of Escherichia coli at 10 degrees C following exposure to electrical field strengths (15, 22.5 and 30 kV/cm) in relation to inactivation and the amount of potentially sublethally injured cells. One medium was used as both a treatment medium and an incubation medium, to study the influence of environmental factors on the inactivation and the growth of the surviving population. The pH (5.0, 6.0 and 7.0) and water activity (1.00, 0.985 and 0.97) of the medium was varied by adding HCl and glycerol, respectively. Growth was followed continuously by measuring the optical density. The time-to-detection (td) and the maximum specific growth rate (micromax) were calculated from these data. Results showed that the PEF process did not cause any obvious sublethal injury to the E. coli cells. The number of survivors was a consequence of the combination of electrical field strength and environmental factors, with pH being the most prominent. Interestingly, the micromax of subsequent growth was influenced by the applied electrical field strength during the process, with an increased micromax at more intense electrical field strengths. In addition, the micromax was also influenced by the pH and water activity. The td, which could theoretically be considered as an increase in shelf life, was found to depend on a complex correlation between electrical field strength, pH and water activity. That could be explained by the fact that the td is a combination of the number of survivors, the recovery of sublethal injured cells and the growth rate of the survivors.