Spectrofluorimetric assessment of bacterial cell membrane damage by pulsed electric field

A rapid fluorescence staining technique was used to assess cell membrane damage and ensuing injury and death caused by pulsed electric field (PEF) treatment. Cell suspensions of Lactobacillus leichmannii ATCC 4797, Listeria monocytogenes Scott A and Escherichia coli O157:H7 ATCC 35150 were subjected to PEF for145.6 μs at field strengths of 5–20 kV/cm. Immediately after PEF treatment, cells were stained with propidium iodide (PI), and changes in fluorescence intensity were measured with a spectrofluorimeter. Increase in field strength decreased the count of survivors and proportionally increased the fluorescence intensity, confirming that cell inactivation by PEF is caused by membrane damage. Cells of E. coli O157:H7 were incubated with or without EDTA before exposure to PEF, but similar inactivation was observed, regardless of the EDTA pre-treatment. Increase in the fluorescence intensity, however, was appreciable in the EDTA-PEF-treated cells. The fluorescence staining technique, therefore, revealed membrane-related injury when EDTA pre-treated cells were PEF-treated. In conclusion, the fluorescence staining technique can be used to assess membrane damage associated with PEF treatments and is potentially useful in determining the relative sensitivity of microorganisms to PEF or monitoring the efficacy of such treatments.     

Bacterial resistance after pulsed electric fields depending on the treatment medium pH

The objective was to evaluate and compare the pulsed electric field (PEF) resistance of four Gram-positive (Bacillus subtilis, Listeria monocytogenes, Lactobacillus plantarum, Staphylococcus aureus) and four Gram-negative (Escherichia coli, E. coli O157:H7, Salmonella serotype Senftenberg 775W, Yersinia enterocolitica) bacterial strains under the same treatment conditions. Microbial characteristics such as cell size, shape or type of the cell envelopes did not exert the expected influence on microbial PEF resistance. The most PEF resistant bacteria depended on the treatment medium pH. For instance, L. monocytogenes, which showed the highest PEF resistance at pH 7.0, was one of the most sensitive at pH 4.0. The most PEF resistant strains at pH 4.0 were the Gram-negatives E. coli O157:H7 and S. Senftenberg. A subsequent holding of PEF-treated cells in pH 4.0 for 2 h increased the degree of inactivation up to 4 extra Log₁₀ cycles depending on the bacterial strain investigated. Under these treatment conditions, the most PEF resistant bacterial strains were still the pathogens S. Senftenberg and E. coli O157:H7.

Industrial relevance

The design of appropriate food preservation processes by PEF requires the selection of an adequate target bacterial strain, which should correspond to the most PEF resistant microorganism contaminating food. This study indicates that the pH of the treatment medium plays an important role in determining this target bacterial strain. On the other hand, the combination of PEF and subsequent holding under acidic conditions has been proven to be an effective method in order to achieve a higher level of microbial inactivation.     

Membrane permeabilization and cellular death of Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae as induced by high pressure carbon dioxide treatment

In this study, the relationship between (irreversible) membrane permeabilization and loss of viability in Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae cells subjected to high pressure carbon dioxide (HPCD) treatment at different process conditions including temperature (35–45 °C), pressure (10.5–21.0 MPa) and treatment time (0–60 min) was examined. Loss of membrane integrity was measured as increased uptake of the fluorescent dye propidium iodide (PI) with spectrofluorometry, while cell inactivation was determined by viable cell count. Uptake of PI by all three strains indicated that membrane damage is involved in the mechanism of HPCD inactivation of vegetative cells. The extent of membrane permeabilization and cellular death increased with the severity of the HPCD treatment. The resistance of the three tested organisms to HPCD treatment changed as a function of treatment time, leading to significant tailing in the survival curves, and was dependent on pressure and temperature. The results in this study also indicated a HPCD-induced damage on nucleic acids during cell inactivation. Transmission electron microscopy showed that HPCD treatment had a profound effect on the intracellular organization of the micro-organisms and influenced the permeability of the bacterial cells by introducing pores in the cell wall.     

Component release after exposure of Staphylococcus aureus cells to pulsed electric fields

The objective of this work was to get further insights on the mechanism of inactivation of bacterial cells by pulsed electric fields (PEF) through the study of the release of intracellular components after exposing Staphylococcus aureus cells in McIvlaine buffer (pH 7.0, 2 mS/cm) to PEF treatments of different intensity (18 and 25 kV/cm) and treatment times (from 20 to 400 μs). Release of most compounds, except proteins, was almost immediate after the treatment, but the relative amount released depended on the molecule studied. A good correlation between the release of the smallest components studied (particularly ions) and membrane permeabilization (as measured by NaCl sensitization and PI entry) was observed. On the other hand, results obtained suggested that S. aureus inactivation by PEF would be related to the exit of cytoplasmic proteins of a molecular weight higher than 6 kDa. Results obtained in this work indicated that increasing PEF treatment time would reduce the capability of S. aureus cells to repair the electropores formed and suggested that this might be due to the formation of pores of a larger size, which S. aureus cells would be unable to reseal in a situation of homeostasis loss.Industrial relevanceResults reported here can help to design more effective treatments for microbial inactivation using PEF on food, and therefore facilitate its industrial implementation.     

Inactivation of papain by pulsed electric fields in a continuous system

Papain (E.C. 3.4.22.2), a cysteine protease in papaya, was activated with reducing agents and treated with pulsed electric fields (PEF) in a continuous system at 10°C. Irreversible reduction of activity was observed in PEF-treated papain after 24 h storage at 4°C. Oxidation of papain active site, a cyteine residue, was not the major cause of papain inactivation by PEF. Temperature did not increase over 35°C during PEF treatment. The pH of papain solution was not changed after PEF treatment. Structural change was observed in PEF-treated papain by CD analysis. Inactivation of PEF-treated papain was related to the loss of α-helix structure. Heating at 60-80°C for 2 min did not significantly reduce the activity of papain.     

Screening for Listeria monocytogenes surrogate strains applicable to food processing by ultrahigh pressure and pulsed electric field

Ultrahigh pressure (UHP) and pulsed electric field (PEF) are emerging processing technologies developed to enhance the safety while maintaining the fresh-like quality of food. For each food and process combination, a pathogen of concern (i.e., target pathogen) must be determined, and a low-risk microorganism that serves as the pathogen surrogate for process validation must be identified. The objective of this study was to identify a surrogate for Listeria monocytogenes for UHP and PEF process validation. Potential surrogates tested include four Lactobacillus spp., a Pediococcus sp., and a Listeria innocua strain. These were compared with nine L. monocytogenes strains, with regard to sensitivity to UHP and PEF processing. For UHP treatment, the strains were suspended in citrate-phosphate buffer (pH 7.0 or 4.5), sweet whey, or acidified whey and pressure processed at 500 MPa for 1 min. For PEF treatment, the strains were suspended in NaCl solution, acid whey, or sweet whey and processed at 25 kV/cm. The lethality of UHP or PEF treatment varied considerably, depending on medium types and pH and the treated strain. Treating the tested microorganisms with UHP inactivated 0.3 to 6.9 log CFU/ml for L. monocytogenes strains and 0.0 to 4.7 log CFU/ml for the potential surrogates. When PEF was employed, populations of tested microorganisms decreased < 1.0 to 5.3 log CFU/ml. L. monocytogenes V7 and OSY-8578 were among the most resistant strains to UHP and PEF treatments, and thus are candidate target strains. Lactobacillus plantarum ATCC 8014 demonstrated similar or greater resistance compared with the target organisms; therefore, the bacterium is proposed as a surrogate of L. monocytogenes for both processes under the conditions specified in the food matrices tested in this study.     

Permeabilization of plant tissues by monopolar pulsed electric fields: effect of frequency

Pulsed electric fields (PEF) nonthermally induce cell membrane permeabilization and thereby improve dehydration and extraction efficiencies in food plant materials. Effects of electrical field strength and number of pulses on plant tissue integrity have been studied extensively. Two previous studies on the effect of pulse frequency, however, did not provide a clear view: one study suggested no effect of frequency, while the other found a greater impact on tissue integrity at lower frequency. This study establishes the effect of pulse frequency on integrity of onion tissues. Changes in electrical characteristics, ion leakage, texture parameters, and percent weight loss were quantified for a wide range of pulse frequencies under conditions of fixed field strength and pulse number. Optical microscopy and viable-cell staining provided direct visualization of effects on individual cells. The key finding is that lower frequencies (f < 1 Hz) cause more damage to tissue integrity than higher frequencies (f = 1 to 5000 Hz). Intriguingly, the optical microscopy observations demonstrate that the speed of intracellular convective motion (that is, cytoplasmic streaming) following PEF application is strongly correlated with PEF frequency. We provide the first in situ visualization of the intracellular consequence of PEF at different frequencies in a plant tissue. We hypothesize that cytoplasmic streaming plays a significant role in moving conductive ionic species from permeabilized cells to the intercellular space between plant cells, making subsequent pulses more efficacious at sufficiently low frequencies. The results suggest that decreasing the pulse frequency in PEF may minimize the number of pulses needed to achieve a desired amount of permeabilization, thus lowering the total energy consumption. Practical Application: PEF cause pores to be formed in plant cell membranes, thereby improve moisture removal and potential extraction of desirable components. This study used in situ microscopic evaluation of onion cells, as they were pulsed with electric fields at different frequencies, to determine whether frequency was an important parameter. We illustrate that membranes were more effectively broken at lower frequencies as compared to higher frequencies. Application of this information will allow for improved design of PEF systems for more energy efficient dehydration or extraction of plant tissues.     

Mechanism of action of Spanish oregano, Chinese cinnamon, and savory essential oils against cell membranes and walls of Escherichia coli O157:H7 and Listeria monocytogenes

The mechanism of the antimicrobial action of Spanish oregano (Corydothymus capitatus), Chinese cinnamon (Cinnamomum cassia), and savory (Satureja montana) essential oils against cell membranes and walls of bacteria was studied by the measurement of the intracellular pH and ATP concentration, the release of cell constituents, and the electronic microscopy observations of the cells when these essential oils at their MICs were in contact with Escherichia coli O157:H7 and Listeria monocytogenes. E. coli O157:H7 and L. monocytogenes, two pathogenic foodborne bacteria, were used as gram-negative and gram-positive bacterial models, respectively. Treatment with these essential oils at their MICs affected the membrane integrity of bacteria and induced depletion of the intracellular ATP concentration. Spanish oregano and savory essential oils, however, induced more depletion than Chinese cinnamon oil. An increase of the extracellular ATP concentration was observed only when Spanish oregano and savory oils were in contact with E. coli O157:H7 and L. monocytogenes. Also, a significantly higher (P < or = 0.05) cell constituent release was observed in the supernatant when E. coli O157:H7 and L. monocytogenes cells were treated with Chinese cinnamon and Spanish oregano oils. Chinese cinnamon oil was more effective to reduce significantly the intracellular pH of E. coli O157:H7, whereas Chinese cinnamon and Spanish oregano decreased more significantly the intracellular pH of L. monocytogenes. Electronic microscopy observations revealed that the cell membrane of both treated bacteria was significantly damaged. These results suggest that the cytoplasmic membrane is involved in the toxic action of essential oils.     

Inactivation of Escherichia coli, Listeria innocua and Saccharomyces cerevisiae in relation to membrane permeabilization and subsequent leakage of intracellular compounds due to pulsed electric field processing

Membrane permeabilization, caused by pulsed electric field (PEF) processing of microbial cells, was investigated by measurement of propidium iodide (PI) uptake with flow cytometry. Inactivation of Escherichia coli, Listeria innocua and Saccharomyces cerevisiae was determined by viable counts, and leakage of intracellular compounds, such as ATP and UV-absorbing substances, was measured in the extracellular environment. Electrical field strength and pulse duration influenced membrane permeabilization of all three tested organisms of which S. cerevisiae was the most PEF sensitive, followed by E. coli and L. innocua. It was shown by viable counts, PI uptake and leakage of intracellular compounds that L. innocua was the most resistant. Increased inactivation corresponded to greater numbers of permeabilized cells, which were reflected by increased PI uptake and larger amounts of intracellular compounds leaking from cells. For E. coli and L. innocua, a linear relationship was observed between the number of inactivated cells (determined as CFU) and cells with permeated membranes (determined by PI uptake), with higher number of inactivated cells than permeated cells. Increased leakage of intracellular compounds with increasing treatment severity provided further evidence that cells were permeabilized. For S. cerevisiae, there was higher PI uptake after PEF treatments, although very little or no inactivation was observed. Results suggest that E. coli and L. innocua cells, which took up PI, lost their ability to multiply, whereas cells of S. cerevisiae, which also took up PI, were not necessarily lethally permeabilized.     

Isolation and preliminary characterization of a bacteriocin produced by Lactobacillus plantarum N014 isolated from nham, a traditional Thai fermented pork

Lactobacillus plantarum N014 was isolated from nham, a traditional Thai fermented pork, and exhibited antimicrobial activity against Listeria monocytogenes. Its bacteriocin had a broad inhibitory spectrum toward both gram-positive and gram-negative bacteria. The bacteriocin activity was sensitive to all proteolytic enzymes used in this study, including papain, pepsin, pronase E, proteinase K, and trypsin, but was resistant to the other enzymes, such as alpha-amylase, lipase A, and lysozyme. Furthermore, activity was stable over various heat treatments and pH values. The bacteriocin exerted a bacteriolytic mode of action. It was produced during the exponential growth phase and reached its highest level as producer cells entered the stationary phase. Adsorption of the bacteriocin onto producer cells was pH-dependent. No bacteriocin adsorption was detected at pH 1 to 3, whereas 100% bacteriocin adsorption was found at pH 7. Plasmid isolation revealed that L. plantarum N014 contained no plasmids. From Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and growth inhibition testing against L. monocytogenes, the estimated molecular mass of L. plantarum N014 bacteriocin was 8 kDa.     

Observations on the water distribution and extractable sugar content in carrot slices after pulsed electric field treatment

The impact of pulsed electric field (PEF) processing conditions on the distribution of water in carrot tissue and extractability of soluble sugars from carrot slices was studied. Time domain NMR relaxometry was used to investigate the water proton mobility in PEF-treated carrot samples. Three distinct transverse relaxation peaks were observed in untreated carrots. After PEF treatment only two slightly-overlapping peaks were found; these were attributed to water present in the cytoplasm and vacuole of carrot xylem and phloem tissues. This post-treatment observation indicated an increase in water permeability of tissues and/or a loss of integrity in the tonoplast. In general, the stronger the electric field applied, the lower the area representing transverse relaxation (T₂) values irrespective of treatment duration. Moreover an increase in sucrose, β- and α-glucose and fructose concentrations of carrot slice extracts after PEF treatment suggested increases in both cell wall and vacuole permeability as a result of exposure to pulsed electric fields.     

Modelling inactivation of Listeria monocytogenes by pulsed electric fields in media of different pH

A study of the effect of square-wave pulsed electric fields (PEF) on the inactivation of Listeria monocytogenes in McIlvaine buffer of different pH (3.5-7.0) was conducted. L. monocytoges was more PEF sensitive at higher electric field strengths (E) and in media of low pH. A treatment at 28 kV/cm for 400 mus that inactivated 1.5, 2.3 and 3.0 Log10 cycles at pH 7.0, 6.5 and 5.0 respectively destroyed almost 6.0 Log10 cycles at pH 3.5. The general shape of survival curves of L. monocytogenes PEF treated at different pH was convex/concave upwards. A mathematical model based on the Weibull distribution accurately described these survival curves. At each pH, the shape parameter (n value) did not depend on E. The relationship between n value of the Weibull model and the pH of the treatment medium was described by the Gompertz equation. A multiple linear regression model using three predictor variables (E, E2, pH2) related the Log10 of the scale paramenter (b value) of the Weibull model with E and pH of the treatment medium. A tertiary model developed using McIlvaine buffer as treatment medium predicted satisfactorily the inactivation of L. monocytogenes in apple juice.     

Disruption of Escherichia coli, Listeria monocytogenes and Lactobacillus sakei cellular membranes by plant oil aromatics

The role of membrane disruption in the bactericidal activity of the plant oil aromatic compounds eugenol, carvacrol and cinnamaldehyde was investigated using confocal laser scanning microscopy, changes in ATP levels and cell viability. In 25 mM HEPES buffer pH 7 at 20 degrees C, 10 mM eugenol or carvacrol increased uptake of propidium iodide by Escherichia coli, Listeria monocytogenes and Lactobacillus sakei over a 10-min period. The same treatments resulted in lowered viability, rapid depletion of cellular ATP and release of ATP, with the exception of Lb. sakei treated with carvacrol. Eugenol or carvacrol at 5 mM to 10 mM inhibited E. coli and L. monocytogenes motility. Lb. sakei was resistant to cinnamaldehyde. Thus, its effects were only studied on E. coli and L. monocytogenes. At 10 mM, cinnamaldehyde caused a slight but statistically significant increase in propidium iodide staining of E. coli, but had no effect on L. monocytogenes. Cinnamaldehyde treatment of E. coli at 10 mM and L. monocytogenes at 40 mM resulted in decreased cellular ATP, but there was no concomitant release of ATP. Cinnamaldehyde at 5 and 10 mM inhibited E. coli and L. monocytogenes motility. Results for eugenol and carvacrol are consistent with non-specific permeabilization of the cytoplasmic membrane. Evidence for increased membrane permeability by cinnamaldehyde is less conclusive. The release of ATP from eugenol and carvacrol-treated cells and absence of release from cinnamaldehyde-treated cells could indicate that eugenol and carvacrol possess ATPase inhibiting activity. Secondary effects would also be consistent with membrane disruption.     

Mechanism of action of pulsed high electric field (PHEF) on the membranes of food-poisoning bacteria is an 'all-or-nothing' effect

Salmonella typhimurium (CRA 1005) was more sensitive than Listeria monocytogenes (NCTC 11994) to pulsed high electric field (PHEF) treatment in distilled water (10, 15 and 20 kV/cm), 10 mM tris-maleate buffer pH 7.4 (15 kV/cm) and model beef broth (0.75% w/v: 15 kV/cm). Sublethal injury could not be detected using a selective medium plating technique, indicating that bacterial inactivation by PHEF may be an 'all-or-nothing' event. PHEF-induced membrane permeabilization resulted in increased UV-leakage and a decreased ability of L. monocytogenes to maintain a pH gradient.     

Enhanced inactivation of foodborne pathogenic and spoilage bacteria by FD&C Red no. 3 and other xanthene derivatives during ultrahigh pressure processing

Variability among microorganisms in barotolerance has been demonstrated at genus, species, and strain levels. Identification of conditions and additives that enhance the efficacy of ultrahigh pressure (UHP) against important foodborne microorganisms is crucial for maximizing product safety and stability. Preliminary work indicated that FD&C Red No. 3 (Red 3), a xanthene derivative, was bactericidal and acted synergistically with UHP against Lactobacillus spp. The objective of this study was to determine the antimicrobial efficacy of Red 3 and other xanthene derivatives, alone and combined with UHP, against spoilage and pathogenic bacteria in citrate-phosphate buffer (pH 7.0). Xanthene derivatives tested were fluorescein, Eosin Y, Erythrosin B, Phloxine B, Red 3, and Rose Bengal. Halogenated xanthene derivatives (10 ppm) were effective at reducing Listeria monocytogenes survivors but ineffective against Escherichia coli O157:H7. When combined with UHP (400 MPa, 3 min), the presence of derivatives enhanced inactivation. Because Red 3 was the only xanthene derivative to produce synergistic inactivation of both pathogens, further studies using this colorant were warranted. Efficacy of Red 3 against gram-positive bacteria (Lactobacillus plantarum and L. monocytogenes) was concentration dependent (1 to 10 ppm). E. coi O157: H7 strains were resistant to Red 3 concentrations up to 300 ppm. When Red 3 was combined with UHP, the lethality against gram-positive and gram-negative bacteria was dose dependent, with synergy being significant for most strains at > or = 3 ppm. Additional gram-positive and gram-negative bacteria showed lethalities similar to those observed for L. plantarum or L. monocytogenes, and E. coli O157:H7, respectively. Red 3 is a potentially useful additive to enhance the safety and stability of UHP-treated food products.     

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.     

Lactobacillus plantarum inhibits growth of Listeria monocytogenes in an in vitro continuous flow gut model, but promotes invasion of L. monocytogenes in the gut of gnotobiotic rats

The ability of the pediocin AcH producing Lactobacillus plantarum DDEN 11007 and its non-producing plasmid-cured isogenic variant, DDEN 12305 to prevent the persistence and growth of Listeria monocytogenes EP2 in two gastrointestinal (GI) tract models was examined. In vitro studies conducted in a two-stage continuous flow system showed that L. plantarum DDEN 11007 inhibited L. monocytogenes EP2 under these conditions, while less effect was seen of the non-bacteriocin producing variant. The inhibitory effect was more pronounced at pH 5 than at pH 7. No effect on persistence of L. monocytogenes in the GI tract was seen in gnotobiotic rats colonized with either the pediocin AcH producing or the non-bacteriocin producing variant of L. plantarum when compared to rats inoculated with L. monocytogenes EP2 alone. Surprisingly, inoculation of the gnotobiotic animals with either of the L. plantarum strains prior to inoculation with L. monocytogenes EP2 resulted in increased occurrence of L. monocytogenes in liver and spleen when compared to the animals inoculated with L. monocytogenes EP2 alone. Our results indicate that the presence of L. plantarum in the gut of gnotobiotes facilitates L. monocytogenes invasion by an unknown mechanism. This observation is however not necessarily specifically related to L. plantarum, and should not be interpreted as the expected effect in animals carrying a conventional intestinal microflora.     

Disintegration efficiency of pulsed electric field induced effects on onion (Allium cepa L.) tissues as a function of pulse protocol and determination of cell integrity by ¹H-NMR relaxometry

The influence of electrical pulse protocol parameters on cell rupture of onion tissues was investigated in order to improve fundamental understanding and to enhance the processing of plant tissues with pulsed electric fields (PEFs). The impact of PEF parameters on cell integrity of 20 mm dia, 4-mm thick disks of Don Victor onions (Allium cepa L.) was determined by ion leakage measurements. Electric field strength, pulse width, total pulse duration, and frequency effects were determined in relation to their effects on cell damage as a function of pulse protocol. Electric field strengths up to 500 V/cm increased the damage efficiency but there was no significant difference in efficiency beyond this field strength. Larger pulse widths increased the degree of tissue disintegration at a constant pulse number. Higher PEF efficiency was achieved with shorter pulse widths and a larger number of pulses at a constant total treatment time. Lower frequencies caused a greater degree of disintegration at constant number of pulses. 1H-NMR experiments were performed to determine the proton relaxation components of the PEF-treated onion samples and to obtain cell damage information nondestructively. Paramagnetic ion uptake by the onion sample was used to identify different proton relaxation components. Five different proton relaxation components were observed and changes in the 2 components representing different proton environments showed high correlations with ion leakage results (R2= 0.99), indicating that T(2) distributions can be used to obtain information about cell membrane integrity in PEF-treated samples. 1H-NMR proved to be an effective method for nondestructive quantification of cell membrane rupture in onions.     

Effect of a pulsed electric field treatment on the anthocyanins composition and other quality parameters of Cabernet Sauvignon freshly fermented model wines obtained after different maceration times

The influence of the application of a pulsed electric field (PEF) treatment (5 kV/cm, 2.1 kJ/kg) to the grape pomace on different quality parameters and anthocyanins content of Cabernet Sauvignon wines obtained after different maceration times (48, 72, 96 and 248 h) has been investigated.Regardless of the maceration time, the application of a PEF treatment led to freshly fermented model wines that were richer in colour intensity, total polyphenols index (TPI), tannins and showed better visual characteristics. It was observed that the concentration of anthocyanins compounds in the freshly fermented model wines obtained from PEF-treated pomace was higher, as compared to the control wine. The HPLC anthocyanic profiles of freshly fermented model wines obtained from PEF-treated pomace were similar to those of control wine indicating that the permeabilization of the cell membranes of pomace did not produce a selective effect on any anthocyanin. Malvidin-3-glucoside and Malvidin-3-glucoside acetate were the predominant anthocyanins in both control and PEF wine.According to the results obtained in this investigation the application of a PEF treatment permitted to reduce the maceration time during vinification of Cabernet Sauvignon grapes from 268 to 72 h.     

Comparing the PEF resistance and occurrence of sublethal injury on different strains of Escherichia coli,Salmonella Typhimurium,Listeria monocytogenes and Staphylococcus aureus in media of pH 4 and 7

The effect of pulsed electric fields (PEF) on the reduction of the population and on the occurrence of sublethal injury in five strains of two Gram-positive (Listeria monocytogenes and Staphylococcus aureus) and two Gram-negative (Escherichia coli and Salmonella Typhimurium) microorganisms was investigated in media of pH 4.0 and 7.0. Samples were subjected to 50 exponential waveform pulses of 15, 20, 25 and 30 kV/cm at a repetition rate of 1 Hz.PEF resistance and occurrence of sublethal injury depended on the electric field strength and pH of the treatment medium and varied widely among the 20 strains investigated. In general, the increment of the electric field strength from 20 to 30 kV/cm had a significant effect on the viability loss of all the strains. However this increment hardly affected the viability of E. coli W3110 and O157:H7 in the medium of pH 4.0 and of L. monocytogenes 4031, 5672 and 4032 in the medium of pH 7.0. At 30 kV/cm a population reduction higher than 4-Log₁₀ cycles was observed in two strains of S. Typhimurium (878, 4594) and in three strains of S. aureus (976, 4465, 4466) in the medium of pH 4.0. In the medium of pH 7.0, this level of reduction was observed in two strains of E. coli (471, BJ4L1), in three strains of S. Typhimurium (443, 880, 722) and in three strains of S. aureus (976, 4465, 4466).In general, important sublethal injury was not observed for the strains of the two-Gram positive microorganisms in the media of pH 4.0 and 7.0 and for the strains of S. Typhimurium treated in the media of pH 7.0. For E. coli sublethal injury was detected for all the strains investigated, except the strain BJ4L1 treated at 30 kV/cm in the medium of pH 7.0.Industrial relevance. The identification of the most PEF resistant microorganisms of public health concern is necessary to establish the treatment conditions for PEF pasteurization. Data obtained in this investigation demonstrates that the PEF resistance and the occurrence of sublethal injury may vary greatly among different strains of a bacteria and both depend on the pH of the treatment medium. Therefore the most resistant microorganisms of public health significance can be expected to vary for different foods depending of their pH.