Pulsed Electric Fields Resistance Research Articles

Genotypic and phenotypic characterization of a Salmonella Typhimurium strain resistant to pulsed electric fields

Pulsed Electric Fields (PEF) technology is regarded as one of the most interesting alternatives to current food preservation methods, due to its capability to inactivate vegetative microorganisms while leaving the product's organoleptic and nutritional properties mostly unchanged. However, many aspects regarding the mechanisms of bacterial inactivation by PEF are still not fully understood. The aim of this study was to obtain further insight into the mechanisms responsible for the increased resistance to PEF of a Salmonella Typhimurium SL1344 variant (SL1344-RS, Sagarzazu et al., 2013), and to quantify the impact that the acquisition of PEF resistance has on other aspects of S. enterica physiology, such as growth fitness, biofilm formation ability, virulence and antibiotic resistance. WGS, RNAseq and qRT-PCR assays indicated that the increased PEF resistance of the SL1344-RS variant is due to a higher RpoS activity caused by a mutation in the hnr gene. This increased RpoS activity also results in higher resistance to multiple stresses (acidic, osmotic, oxidative, ethanol and UV-C, but not to heat and HHP), decreased growth rate in M9-Gluconate (but not in TSB-YE or LB-DPY), increased ability to adhere to Caco-2 cells (but no significant change in invasiveness) and enhanced antibiotic resistance (to six out of eight agents). This study significantly contributes to the understanding of the mechanisms of the development of stress resistance in Salmonellae and underscores the crucial role played by RpoS in this process. Further studies are needed to determine whether this PEF-resistant variant would represent a higher, equal or lower associated hazard than the parental strain.

Inactivation of vegetative cells, germinated spores, and dormant spores of Bacillus atrophaeus by pulsed electric field with fixed energy input

AbstractIncreasing the electric energy input during pulsed electric field (PEF) treatment is a straightforward technique to boost the efficiency of microbial inactivation. However, drawbacks such as electrode corrosion, arc formation, and high cost are also involved. This study aims to investigate the impact of medium conductivity (), electric field strength (), and the number of pulses () on the inactivation of vegetative cells, germinated spores, and dormant spores of Bacillus atrophaeus with fixed energy input. Our results indicated that (1) vegetative cells were susceptible to PEF treatment, and the relative importance of , , and was ; (2) germinated spores were more resistant to PEF treatment than vegetative cells, and the relative importance of , , and was ; and (3) similar to the thermal resistance property, dormant spores were also highly resistant to PEF treatment and were not significantly inactivated in our experiment setting. It is concluded that the degree of PEF resistance of cell at different physiological states may be related to its structure and size. This study added to the body of knowledge on selecting PEF treatment settings and gave insight into the scaling up of PEF applications in the food processing industry.Practical ApplicationsIn the food processing industry, eliminating contaminants such as bacterial spores is essential, and PEF processing is a common nonthermal technique. While the traditional method of increasing the electric energy input to increase microbial inactivation efficiency is beneficial, it is not cost‐effective. The results of this investigation demonstrated that decreasing spore resistance through germination followed by parameter‐optimized PEF treatments is a practical method for improving pasteurization efficiency. This novel technique might be used in the food sector.

Membrane fatty acids composition and fluidity modification in Salmonella Typhimurium by culture temperature and resistance under pulsed electric fields

SummaryEffects of various growth temperatures on cell membrane fatty acid composition of Salmonella Typhimurium (S. Typhimurium) and the resistance to Pulsed Electric Fields (PEF) treatments, as well as PEF combining with mild‐thermal (35, 45, 55 °C) treatments were investigated. Results indicated that the PEF resistance of S. Typhimurium at stationary phase was varied markedly at different growth temperatures. S. Typhimurium grown at 45 °C exhibited greater PEF resistance than cells grown at a lower temperature. Alteration of membrane fatty acid composition decreased in the unsaturated to saturated fatty acids ratio (UFA/SFA) and increased in cyclopropane fatty acids (CFA) proportion as growth temperature increased. It was found that the PEF resistance of S. Typhimurium at stationary phase was in a membrane fluidity dependent manner. Thermal combined PEF treatment improved the PEF lethality which indicated that the PEF resistance of S. Typhimurium was greatly affected by the fluidity of cytomembrane.

Effect of ethanol adaption on the inactivation of Acetobacter sp. by pulsed electric fields

The effects of low concentrations of ethanol (3% to 9%, v/v) on cell membrane fatty acid composition, membrane lipid conformation, membrane fluidity and pulsed electric fields (PEF) resistance of Acetobacter sp. in the stationary phase were investigated. Results showed that the growth of Acetobacter sp. was inhibited, and analysis of gas chromatography–mass spectrometry, Raman spectroscopy and fluorescence polarization indicated that membrane fluidity of Acetobacter sp. cells was significantly increased by modifying membrane fatty acid composition and membrane lipid conformation after exposure to ethanol. Moreover, results showed that cells cultivated in the medium with relatively high ethanol concentrations were more vulnerable to PEF. For example, the inactivation of Acetobacter sp. (109 CFU/mL) cultivated with 9% ethanol by PEF (20.0 kV/cm, 6.0 ms) could reach 5.17 log, significantly higher than those cultivated without ethanol (3.22 log). This was confirmed by morphological analysis, showing various changes on the cell surface after PEF treatment. Acetobacter sp. is one of spoilage microorganisms in winemaking, and it is able to convert the ethanol produced by yeasts into acetic acid and thereby increase volatile acidity in wine. In this study, the effect of ethanol as a growth substrate on PEF resistance in Acetobacter sp. cells was investigated. Results here reported will contribute to better understand the influencing mechanisms of ethanol on the inactivation of Acetobacter sp. cells by PEF treatment and further develop the application of PEF for inactivating acetic acid bacteria (AAB) in the wine industry.

Variations in cellular membrane fatty acid composition ofEscherichia coliin resistance to pulsed electric fields induced by eugenol

Eugenol (EUG; 4-allyl-2-methoxyphenol) is a natural antibacterial component from essential oil. The effects of EUG on cell membrane fatty acid composition, membrane fluidity and resistance to pulsed electric field (PEF) of Escherichia coli ATCC 8,739 (E. coli) were investigated. Results indicated that the PEF resistance of E. coli decreased as EUG concentration increased, which may be related to the alterations in membrane fatty acid composition and fluidity. Gas chromatography−mass spectrometry and micro-Raman spectroscopy showed that EUG increased membrane fluidity by notably increasing the proportion of unsaturation fatty acids and simultaneously decreasing the content of cyclic fatty acids. These results revealed that PEF resistance of E. coli was influenced by EUG due to alterations of fatty acid composition and membrane fluidity. Practical applications Pulsed electric field (PEF), as a widely used non-thermal sterilization technology, has gained much attention and increasingly focused on improving the efficiency to inactivate micro-organisms. Eugenol (EUG; 4-allyl-2-methoxyphenol) derived from cloves is a natural phenol essential oil and exhibits several pharmacological properties such as being an antimicrobial. Our results revealed the underlying mechanism that EUG reduced PEF resistance of E. coli cells and contributed to achieve better understanding of microbial inactivation by PEF. Therefore, a new route for the application of eugenol to improve the efficiency of PEF in the food industry was created.

Influence of acid and low-temperature adaptation on pulsed electric fields resistance of Enterococcus faecium in media of different pH

Abstract This investigation was undertaken to study the inactivation of Enterococcus faecium ATCC 49624 by pulsed electric fields (PEF) treatments after exposure to sublethal acid and cold stresses. Cells were grown in Brain Heart Infusion (BHI) buffered to pH 7.4 (non-acid-adapted cells) and BHI acidified to pH 5.4 with acetic, ascorbic, citric, lactic, malic and hydrochloric acids (acid-adapted cells). Fresh and refrigerated cultures (kept at 4 °C for up to 1 month) were treated by PEF at 37 kV/cm in citrate-phosphate buffer of different pH (4.0, 5.5 and 7.0), at specific energies ranging from 79 to 1580 kJ/kg. Results demonstrated that acid adaptation exerted a different effect on the PEF resistance of E. faecium depending on the pH of the treatment medium. Thus, when treated in a medium of pH 7.0, previous acid adaptation, regardless of the type of acidulant used, reduced the calculated δ values (specific energies required to inactivate the first log10 cycle of the population) by approximately two-fold. By contrast, in a medium of pH 4.0, acid-adapted cells showed a higher PEF resistance than non-adapted cells. On the other hand, exposure to 4 °C reduced the PEF resistance of acid-adapted cells, but not that of non-acid-adapted cells. Results obtained in this study suggested that, after the same PEF treatments, the number of electroporated cells was lower for non-acid-adapted cells, but that acid-adapted cells displayed a superior ability to repair sublethal damages caused by PEF. Nevertheless, exposure to refrigeration conditions would significantly reduce this damage repair ability. Finally, FT-IR spectroscopy measurements indicated that growth of E. faecium cells under acid conditions caused an increase in membrane fluidity. It was also observed that refrigerated storage resulted in a significant (p Industrial relevance Given the inability of PEF to inactivate spores, pasteurization of acid products such as fruit juices appears as one of the more feasible applications for this technology. Microbial cells present in these products would have, very likely, undergone a process of adaptation to this acid pH; however, the impact of this phenomenon on microbial PEF resistance has only been scarcely investigated. Thus, the objective of this work was to study the inactivation of Enterococcus faecium by PEF treatments after sequential exposure to sublethal acid and cold stresses. This work will provide very valuable data for process design to further develop PEF application and improve understanding of microbial inactivation.

Effect of cell membrane fatty acid composition of Escherichia coli on the resistance to pulsed electric field (PEF) treatment

The underlying relationships between growth temperature, membrane fatty acid composition (mFAC) and PEF resistance of Escherichia coli (E. coli) were explored. Results indicated the PEF resistance of E. coli at stationary stage was directly related to mFAC. For the cells at stationary stage, as growth temperature decreased from 37 °C to 15 °C, the PEF (23.3 kV/cm, 1.2 ms) lethality of E. coli was increased 1.5 log10, reached 3.2 log10. Meanwhile, the proportion of unsaturated fatty acid (UFA) was increased from 20.07 to 47.65%. And the fluidity index (FI) of the cell membrane was also increased from 0.42 to 1.42. It revealed that the PEF resistance of E. coli at stationary stage was determined by the mFAC and fluidity of the cell membrane. Those conclusions were also confirmed by cytomembrane fluidity determined by fluorescence probe (DPH) and the different PEF-induced electro-permeabilization of dioleoyl-sn-glycero-3-phosphocholine (DOPC) and Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles. On the other hand, for the cell at exponential stage, the PEF resistance of E. coli was rarely affected by mFAC. The PEF (23.3 kV/cm, 1.2 ms) lethality of cells was almost the same (3.2 log10) at different growth temperature.

Influence of growth and treatment temperature on Staphylococcus aureus resistance to pulsed electric fields: Relationship with membrane fluidity

The influence of growth and treatment temperature (10–42 °C) on Staphylococcus aureus resistance to Pulsed Electric Fields (PEF) was investigated. No statistical differences in resistance to PEF (26 kV/cm) (p > 0.05) were detected between S. aureus cells grown at 10, 20, 37, or 42 °C. In contrast, suspensions grown at 30 °C displayed significantly (p < 0.05) higher resistance than suspensions grown at the other 4 temperatures tested. On the other hand, an increase of treatment temperature from 10 to 42 °C resulted in a progressive decrease in PEF resistance for all suspensions. It is noteworthy that this decrease was of similar magnitude regardless of the growth temperature of S. aureus cells. The increase in treatment temperature also led to a fluidization of S. aureus cell membranes, measured by fluorescence anisotropy. However, this increase in fluidity was greater for cells grown at lower temperatures (10–20 °C) than for cells grown at higher ones (30–42 °C). Cells grown at 30 °C, which had shown a higher PEF tolerance, did not have a more rigid membrane than cells grown at 37 or 42 °C. The addition of benzyl alcohol (20 mM), which led to a fluidization of bacterial membranes, did not render S. aureus cells more PEF sensitive. Altogether, the results obtained cast doubt on the existence of a direct causal relationship between average membrane fluidity and the PEF resistance of bacterial cells. Understanding the mechanisms of bacterial resistance to PEF is essential for in Pulsed Electric Fields process optimization and PEF-based combined processes design. Form a practical point of view, results here reported will contribute to understand the mechanisms of treatment temperature dependent bacterial sensitization to PEF. This is of particular interest regarding combined processes involving heat and PEF.

Salmonella typhimurium resistance on pulsed electric fields associated with membrane fluidity and gene regulation

Abstract Effects of different growth temperatures on cytoplasmic membrane fluidity and phospholipids phase transition temperature (T m ) of Salmonella typhimurium and resistance to pulsed electric field (PEF) inactivation, as well as the expression of stress-related genes and fatty acid biosynthesis-associated genes were investigated. Results indicated that the PEF resistance of S . typhimurium increased as growth temperature increased. S . typhimurium cultivated at 10 °C exhibited the lowest PEF resistance with the reduction of 4.23 log 10 CFU/mL, while the reduction of 2.10 log 10 CFU/mL was found in S . typhimurium cultivated at 45 °C under the same PEF treatment, due to the up-regulation of the expression of fabA gene, which was characterized by the lowest T m of membrane phospholipids and the greatest membrane fluidity. Although the expression of alternative sigma factors were altered by growth temperature, these genes were not essential for S . typhimurium to develop PEF resistance, suggesting that the PEF resistance modified by growth temperature could be caused by alterations in membrane fluidity. Industrial relevance Pulsed electric fields (PEF) treatment has been widely applied in nonthermal pasteurization and increasingly focused on synergistic combinations with other techniques such as thermal treatment, sonication and antibacterial agents to improve the efficacy of PEF to inactivate micro-organisms. Our results indicated that S . typhimurium cultivated at relatively lower temperature was easily inactivated by PEF, due to the up-regulation of the expression of fabA gene, which was characterized by the lowest phase transition temperature of cytoplasmic membrane phospholipids and the greatest membrane fluidity. Therefore, the underlying mechanism of alterations in PEF resistance of S . typhimurium induced by growth temperature was explored to achieve better understanding of microbial inactivation by PEF.

A Comparison of Pulsed Electric Field Resistance for Three Microorganisms with Different Biological Factors in Grape Juice via Numerical Simulation

Pulsed electric field (PEF) is a promising nonthermal food preservation technology that is based on the use of electric field to eradicate spoilage and pathogenic microorganisms in food products. The effect of various biological factors on the transmembrane potential of different microorganisms (Staphyloccocus aureus, Escherichia coli DH5α, and Saccharomyces cerevisiae) was investigated by means of both numerical simulation and experimental method. The PEF resistance of different microorganisms in grape juice was compared by applying field strength of 12–24 kV/cm, treatment time of 30–180 μs, and an initial temperature of 30 oC. The results showed that S. cerevisiae exhibited the least resistance to PEF treatment, E. coli DH5α the second, and S. aureus the third. The simulation results indicated that larger cells like S. cerevisiae presented the higher values of transmembrane potential and induced field strength around the cells compared to E. coli DH5α and S. aureus, which led to a less resistance to PEF treatment. The effect of cell orientation on the induced transmembrane potential was very slight (1.67 % for E. coli DH5α and 3.43 % for S. cerevisiae). The thicker cell membrane caused concentrated electric field in the cell membrane, which enhanced the sensitivity of microorganism to PEF treatment. However, both transmembrane potential and electric field strength decreased with the thickness of cell wall increasing. According to both experimental and simulation results, it was evident that there was significant difference in the inactivation rate between different microorganisms, which could be largely attributed to the biological factors of different microorganisms.

Effects of pulsed electric fields on cytomembrane lipids and intracellular nucleic acids of Saccharomyces cerevisiae

In this study, pulsed electric fields (PEF) treatments at 20 kV/cm for 0–500 μs were applied to Saccharomyces cerevisiae to investigate the effects of PEF on the cytomembrane and intracellular nucleic acids. The change in composition of membrane lipids characterized by decrease in the ratio of contents of unsaturated fatty acids versus saturated fatty acids induced by PEF was observed, which was related with the increase in rigidness of cytomembrane (lower cytomembrane fluidity and higher cytomembrane viscosity) after PEF. PEF treatment caused no significant change in DNA but disruption of RNA of S. cerevisiae, which was also confirmed by the increase of PEF resistance and no occurrence of injured microbial cells by supplementation of RNA stabilizer, magnesium. The results showed the damage of cytomembrane and RNA were associated with the sublethally injured S. cerevisiae, suggesting cytomembrane and RNA were primary targets for PEF induced microbial damage.

Comparing the pulsed electric field resistance of the microorganisms in grape juice: Application of the Weibull model

Pulsed electric field (PEF) technology is a nonthermal food preservation technology that is based on the use of electric field to eradicate microorganisms in foods. In present study, the PEF resistance of three strains of microorganisms (Staphyloccocus aureus, Escherichia coli DH5α and Saccharomyces cerevisiae) in grape juice was investigated by applying PEF treatments ranging from 9 kV/cm to 27 kV/cm and from 34 μs to 275 μs at an initial treatment temperature of 40 °C. The temperature measurement at particular locations in the processing line showed a general trend for all the composites: greater temperature increasing as the electric field strength and treatment time values increased. In all experimental conditions used in this investigation temperature increments were always lower than 8.8 °C. The obtained survival curves indicated that the inactivation of three strains increased with electric field strength and treatment time, or specific energy. The S. cerevisiae exhibited the least resistance to PEF treatments, S. aureus the second, E. coli DH5α the third. The experimental kinetics of inactivation by PEF of these microorganisms has been described by mathematical equations based on the modified Weibull model, using treatment time and specific energy as the control parameters. Both R2 and RMSE indicated a slightly higher degree of correlation between experimental and fitted values was achieved for models in terms of specific energy. Finally, the predicted inactivation curves from the established model were validated to evaluate the performance of the model using a total of 36 datasets for three microorganisms. Models built in terms of specific energy showed better accuracy than those in terms of treatment time. Whether treatment time or specific energy was used as control parameter of the model, all the bias factors ranged within the proposed acceptable limits. Overall, 80.6% and 85% of the mean prediction errors for above two models were in the acceptable prediction zone, more than 70% of which was considered as a threshold for a simulation model to provide acceptable predictions. The validation results indicated that the Weibull models were safe for predicting inactivation curves of S. aureus, E. coli DH5α and S. cerevisiae by PEF in grape juice. Therefore, the developed tertiary models were useful for assessing the lethal effects of different parameters on PEF treatment results and evaluating the relation between cost and benefit of a potential implementation of PEF technology in industrial production processes.

Emergence of pulsed electric fields resistance in Salmonella enterica serovar Typhimurium SL1344

In this investigation we selected and isolated a culture derived from Salmonella enterica serovar Typhimurium SL1344 with stable increased resistance to pulsed electric fields (PEF) after repeated rounds of PEF treatment and outgrowth of survivors. The resulting culture showed a higher resistance to PEF treatments under different treatment conditions. The acquisition of PEF resistance was only observed in stationary phase cells. The cytoplasmic membrane of the resistant variant showed a higher resilience against PEF treatments, since a lower permeabilization degree was observed after PEF treatments, in comparison to the parental strain. Resistance to PEF was also accompanied by a higher tolerance to acidic pH, hydrogen peroxide and ethanol, but not to heat. The occurrence of a PEF resistant variant in S. enterica serovar Typhimurium SL1344 emphasizes the need to further study the mechanisms of inactivation and resistance by PEF for an adequate design of safe treatments.

Acquisition of pulsed electric fields resistance in Staphylococcus aureus after exposure to heat and alkaline shocks

Staphylococcus aureus cells were exposed to acid (4.5) and alkaline (9.5) pH, hydrogen peroxide (50μM), sodium chloride (10%), heat (45°C) and to a cold shock (4°C) for 2h and then treated by pulsed electric fields (PEF). Exposing S. aureus cells to acidic, oxidative, hyperosmotic or cold shock conditions resulted in no protection to PEF. By contrast, heat and alkaline shocks increased the resistance of S. aureus cells to PEF, regardless the electric field strength applied, the pH and aw of the treatment medium and the strain and growth phase investigated. A ΔsigB deletion mutant was also able to develop these cross-resistance responses demonstrating that the σB factor is not necessary for the acquisition of resistance to PEF after exposure to heat and alkaline shocks. When cells were recovered in selective medium (with NaCl added) alkaline-shocked, but not heat-shocked cells, also showed higher PEF resistance than control cells. This investigation contributes to increase the knowledge on the factors determining bacterial PEF resistance and may help in the design of new preservation methods involving PEF.

SigB absence decreased Listeria monocytogenes EGD-e heat resistance but not its Pulsed Electric Fields resistance

This study shows the behaviour of Listeria monocytogenes under heat and Pulsed Electric Fields (PEF) treatments, as well as the influence of sigB in bacterial resistance and recovery. Absence of the sigB gene resulted in a decreased heat tolerance, showing that σ B influences L. monocytogenes heat survival. Heat treatments at 60 °C (40 s) caused sublethal membrane injuries in 99.99% of survivors. The repair of heat damage required energy production and lipid, protein, and RNA synthesis, and it lasted 6 h. Furthermore, deletion of sigB did not affect the heat injuries repair. PEF resistance at pH 4.0 and 7.0 was not influenced by sigB. Sublethal damage after PEF treatments was only detected when PEF-treated cells had previously been heat-shocked (45 °C/1 h). The membrane repair only required energy production, and it was independent of sigB. Although both heat and PEF treatments have an effect on cellular membrane, the repair of the sublethal damages suggests different membrane targets, and thus we propose a different mechanism of inactivation by these food preservation technologies.

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 Log 10 reductions were obtained after 30 pulses (90 μs) at 15, 27, 38 and 50 °C, respectively. At pH 7.0, around two Log 10 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.

Selection of surrogate bacteria in place of E. coli O157:H7 and Salmonella Typhimurium for pulsed electric field treatment of orange juice

Pulsed electric field (PEF) technology has been used for the inactivation of microorganisms and to prevent flavor loss in liquid foods and beverages in place of thermal pasteurization. When used to pasteurize orange juice, PEF may prevent loss of volatile sensory attributes. Enterohemorrhagic E. coli O157:H7 (EHEC), two strains of Salmonella Typhimurium, and twenty strains of non-pathogenic bacteria were screened for inactivation in orange juice by PEF at 22 and 20 kV/cm at 45 and 55 °C, respectively. Higher populations of both salmonellae were inactivated (2.81 and 3.54 log CFU/ml) at 55 °C, in comparison with the reduction of EHEC (2.22 log). When tested under the same conditions, inactivation of EHEC was slightly greater than that of a non-pathogenic E. coli (NPEC) ATCC 35218 (2.02 log). NPEC was further tested as a surrogate for EHEC by comparing inactivation kinetics at 45, 50 and 55 °C at field strengths of between 7.86 and 32.55 kV/cm. Statistical comparison of revealed that EHEC and NPEC inactivation curves were homogeneous at outlet temperatures of 45 and 50 °C; however, EHEC was slightly more sensitive to PEF than the surrogate NPEC at 55 °C. The higher PEF resistance of non-pathogenic E. coli 35218 at 55 °C may provide a desirable margin of safety when used in pilot plant challenge studies in place of E. coli O157:H7.

Inactivation kinetics of pulsed electric field-resistant strains of Listeria monocytogenes and Staphylococcus aureus in media of different pH

A study of the effect of pulsed electric fields (PEF) on the inactivation of Listeria monocytogenes STCC 5672 and Staphylococcus aureus STCC 4459 in McIlvaine buffer covering a range from pH 3.5 to 7.0 was conducted. Mathematical models based on the Weibull distribution were developed to describe the influence of the electric field strength, treatment time and pH of the treatment medium on the lethality of both Gram positive pathogenic bacteria after PEF treatments. Both microorganisms were more sensitive to PEF in media of low pH, although the influence of the pH on the PEF resistance was more significant in S. aureus. In the best cases scenario, the highest inactivation levels achieved were 3.3 and 6.1 log 10 cycles for L. monocytogenes and S. aureus respectively in pH 3.5 after 500 μs of 35 kV/cm. Based on these results and those observed in literature, L. monocytogenes STCC 5672 at any pH investigated has been shown as one of the most PEF resistant microorganism. Therefore, this microorganism should be considered as a possible target microorganism to define process criterion for PEF pasteurization.

Modeling inactivation kinetics and occurrence of sublethal injury of a pulsed electric field-resistant strain of Escherichia coli and Salmonella Typhimurium in media of different pH

A study of the effect of pulsed electric fields (PEF) on the kinetics of inactivation and the occurrence of cell damage in Escherichia coli O157:H7 and Salmonella Typhimurium 878 treated in McIlvaine buffer covering a range from pH 3.5 to 7.0 was conducted. Mathematical equations based on the Weibull distribution were developed to describe the influence of the electric field strength, treatment time and pH of the treatment medium on the lethality and generation of cell damage of both Gram negative pathogenic bacteria after the application of PEF treatments. E. coli O157:H7 was more PEF resistant than Salmonella Typhimurium at all pH investigated. PEF resistance of E. coli was influenced by the pH but the pH hardly affected the PEF resistance of Salmonella Typhimurium 878. After 150 μs at 35 kV/cm, 1 and 5 log10 cycles of inactivation of E. coli O157:H7 were observed in the range of pH 3.5–4.5 and 5.5–6.5, respectively. Cell damage increased with the field strength and treatment time. A maximum cell damage level of 4.2 and 2.7 log10 cycles for E. coli O157:H7 and Salmonella Typhimurium was observed respectively after a treatment of 30 kV/cm at pH 3.5. PEF induced cell damage was not detected at pH higher than 5.0 for both microorganisms. The developed equations can be applied to design combining processes which can increase the lethality of PEF or to reduce the intensity of PEF treatments to achieve a determine level of microbial inactivation. This study demonstrates that when the influence of several factors on the microbial behavior is investigated, the development of mathematical models is a very useful tool to evaluate the influence of each parameter and their interactions. In this study, it has been mathematically described for first time the influence of the pH of the treatment medium and the occurrence of sublethal injury in a wide range of electric field strengths and treatment times in two Gram negative pathogenic bacteria, Escherichia coli O157:H7 and Salmonella Typhimurium 878. These models would also be of interest for engineering design, evaluation and optimization of PEF process as a new technique for food preservation.

Organic acids make Escherichia coli more resistant to pulsed electric fields at acid pH

Stationary growth phase cells of Escherichia coli were more pulsed electric fields (PEF) resistant in citrate–phosphate McIlvaine buffer at pH 4.0 than at pH 7.0. The greater PEF resistance was also confirmed in fruit juices of similar acid pH. In this work we studied whether the higher PEF resistance of E. coli at acid pH was due to the low pH itself or to the interaction of the components of the treatment medium with the cells. The protective effect on E. coli cells was due to the presence of organic acids such as citric, acetic, lactic or malic at pH 4.0. The protective effect of citric acid at pH 4.0 depended on its concentration. A linear relationship was observed between the Log 10 of the citric acid concentration and the degree of inactivation. Organic acids contained in laboratory treatment media (citrate–phosphate buffer) or in fruit juices did not sensitize E. coli cells to PEF but, on the contrary, they induced a protective effect that made E. coli cells more resistant at pH 4.0 than at neutral pH. This work could be useful for improving food preservation by PEF technology and it contributes to the knowledge of the mechanism of microbial inactivation by PEF.