The bactericidal activity of a methyl and propyl parabens combination: isothermal and non-isothermal studies

D. GILLILAND, A. LI WAN PO AND E. SCOTT. 1992. The effect of temperature on the kill rate of Escherichia coli by methyl and propyl parabens was studied. The kill kinetics was first order. It was shown that the Arrhenius equation provided a good model for describing the relationship between the first order rate constant and the temperature. The activation energy was found to be 274 kJ/mol for exponential phase cells and 168 kJ/mol for stationary phase cells. Exponential phase cells were much more susceptible to the lethal effects of the parabens than were the stationary phase cells. For example, at 34°C stationary phase cells, in chemically defined media, had a kill rate constant of 0.072/h while the corresponding value for exponential phase cells was 0.238/h. In water the rate of kill for exponential phase cells was even faster giving a rate constant of 5.25/h at 34°C. Non-isothermal kinetic testing was not found to be useful for modelling bacterial kill kinetics because we could not achieve the precision required in bacterial enumeration.     

Temperature dependence of mammalian muscle contractions and ATPase activities

Isolated rat and mouse extensor digitorum longus (EDL) and soleus muscles were studied under isometric and isotonic conditions at temperatures from approximately 8 degrees -38 degrees C. The rate constant for the exponential rise of tension during an isometric tetanus had a Q10 of approximately 2.5 for all muscles (corresponding to an enthalpy of activation, delta H = 66 kJ/mol, if the rate was determined by a single chemical reaction). The half-contraction time, contraction time, and maximum rate of rise for tension in an isometric twitch and the maximum shortening velocity in an isotonic contraction all had a similar temperature dependence (i.e., delta H approximately 66 kJ/mol). The Mg++ ATPase rates of myofibrils prepared from rat EDL and soleus muscles had a steeper temperature dependence (delta H = 130 kJ/mol), but absolute rates at 20 degrees C were lower than the rate of rise of tension. This suggests that the Mg++ ATPase cycle rate is not limiting for force generation. A substantial fraction of cross-bridges may exist in a resting state that converts to the force-producing state at a rate faster than required to complete the cycle and repopulate the resting state. The temperature dependence for the rate constant of the exponential decay of tension during an isometric twitch or short tetanus (and the half-fall time of a twitch) had a break point at approximately 20 degrees C, with apparent enthalpy values of delta H = 117 kJ/mol below 20 degrees C and delta H = 70 kJ/mol above 20 degrees C. The break point and the values of delta H at high and low temperatures agree closely with published values for the delta H of the sarcoplasmic reticulum (SR) Ca++ ATPase. Thus, the temperature dependence for the relaxation rate of a twitch or a short tetanus is consistent with that for the reabsorption rate of Ca++ into the SR.     

Kinetic evaluation of claimed synergistic paraben combinations using a factorial design.

The antimicrobial effects of methyl and propyl parabens are investigated, with Escherichia coli as test organism, with a view to determining whether the parabens act synergistically. At appropriate concentrations, the parabens killed E. coli cells according to first order kinetics and the bactericidal effects were quantified by the first order kill rate constants. Combinations of methyl or propyl parabens, at concentrations which slow down or inhibit bacterial growth when used singly, produced definite kill. In this sense, the parabens are therefore synergistic since in combination they produce an effect which is not observed when they are used singly. This effect is not true synergism as shown by the results of our experiments with a factorial design. Analysis of variance indicated no significant interaction between the two parabens.     

Kinetic evaluation of claimed synergistic paraben combinations using a factorial design

D. GILLILAND, A. LI WAN PO AND E. SCOTT. 1992. The antimicrobial effects of methyl and propyl parabens are investigated, with Escherichia coli as test organism, with a view to determining whether the parabens act synergistically. At appropriate concentrations, the parabens killed E. coli cells according to first order kinetics and the bactericidal effects were quantified by the first order kill rate constants. Combinations of methyl or propyl parabens, at concentrations which slow down or inhibit bacterial growth when used singly, produced definite kill. In this sense, the parabens are therefore synergistic since in combination they produce an effect which is not observed when they are used singly. This effect is not true synergism as shown by the results of our experiments with a factorial design. Analysis of variance indicated no significant interaction between the two parabens.     

Kinetics of internalization and degradation of asialo-glycoproteins in isolated rat hepatocytes

The rate constants for internalization of surface-bound asialo-orosomucoid by hepatocytes were 0.040 min-1 at 20 degrees C, 0.18 min-1 at 30 degrees C and 0.28 min-1 at 40 degrees C. At 40 degrees C, internalization accounted for most of the increase in cell-associated radioactivity. The activation energy over the temperature range 20 to 40 degrees C was 68 +/- 7 (S.D.) kJ/mol. At 10 degrees C, most of the cell-associated asialo-orosomucoid was bound to the cell surface in a reaction which followed ordinary chemical kinetics. Pre-incubation of hepatocytes with a large concentration of unlabelled asialo-orosomucoid did not influence the uptake of subsequently added 125I-asialofetuin; neither was degradation of 125I-asialo-fetuin affected in this experiment. The fractional rate of degradation (the fraction of cell-associated asialo-fetuin which was degraded per unit time) was constant over a twelve-fold range of intracellular asialo-fetuin concentrations. Increasing the temperature from 20 to 30 degrees C produced approximately a ten-fold increase in the rate of degradation of either asialo-fetuin or asialo-orosomucoid. The average activation energies of degradation over the range 20 to 40 degrees C were 125 kJ/mol for asialo-fetuin and 149 kJ/mol for asialo-orosomucoid; however, the Arrhenius plots were not straight lines over this temperature range.     

NH(4)OH-KOH pulping mechanisms and kinetics of rice straw

The mechanisms and kinetics of NH(4)OH-KOH mixture pulping rice straw were studied. When aqueous ammonia was mixed with a small amount of caustic potash (ratio of 1:5), three distinct delignification phases were observed in the pulping process: a bulk delignification phase from the beginning of the cooking period to 100 degrees C, a supplementary delignification phase from 100 degrees C to 155 degrees C lasting a further 45 min, and a residual delignification phase until the end of the cooking period. There were two silica removal phases; the first phase was from the beginning of the cooking period to 100 degrees C and the second phase was from 100 degrees C to the end of the cooking period. The rate of delignification reaction was first order with respect to residual lignin and 0.3 order with respect to [OH(-)]. The silica removal was pseudo-first-order with respect to residual silica and 0.6 order with respect to [OH(-)]. The activation energies of the delignification and removal of silica reactions were 35.6 and 30.9 kJ/mol, respectively.     

Kinetically robust monomeric protein from a hyperthermophile

Equilibrium and kinetic studies were carried out under denaturation conditions to clarify the energetic features of the high stability of a monomeric protein, ribonuclease HII, from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII). Guanidine hydrochloride (GdnHCl)-induced unfolding and refolding were measured with circular dichroism at 220 nm, and heat-induced denaturation was studied with differential scanning calorimetry. Both GdnHCl- and heat-induced denaturation are very reversible. It was difficult to obtain the equilibrated unfolding curve of Tk-RNase HII below 40 degrees C, because of the remarkably slow unfolding. The two-state unfolding and refolding reactions attained equilibrium at 50 degrees C after 2 weeks. The Gibbs energy change of GdnHCl-induced unfolding (DeltaG(H(2)O)) at 50 degrees C was 43.6 kJ mol(-1). The denaturation temperature in the DSC measurement shifted as a function of the scan rate; the denaturation temperature at a scan rate of 90 degrees C h(-1) was higher than at a scan rate of 5 degrees C h(-1). The unfolding and refolding kinetics of Tk-RNase HII were approximated as a first-order reaction. The ln k(u) and ln k(r) values depended linearly on the denaturant concentration between 10 and 50 degrees C. The DeltaG(H(2)O) value obtained from the rate constant in water using the two-state model at 50 degrees C, 44.5 kJ mol(-1), was coincident with that from the equilibrium study, 43.6 kJ mol(-1), suggesting the two-state folding of Tk-RNase HII. The values for the rate constant in water of the unfolding for Tk-RNase HII were much smaller than those of E. coli RNase HI and Thermus thermophilus RNase HI, which has a denaturation temperature similar to that of Tk-RNase HII. In contrast, little difference was observed in the refolding rates among these proteins. These results indicate that the stabilization mechanism of monomeric protein from a hyperthermophile, Tk-RNase HII, with reversible two-state folding is characterized by remarkably slow unfolding.     

The temperature-dependence of adenylate cyclase from baker''s yeast.

The Michaelis constant of membrane-bound adenylate cyclase increased from 1.1 to 1.8 mM between 7 and 38 degrees C (delta H = 13 kJ/mol). Over this temperature range, the maximum velocity increased 10-fold, and the Arrhenius plot was nearly linear, with an average delta H* of 51 kJ/mol. The temperature-dependence of the reaction rate at 2 mM-ATP was examined in more detail: for Lubrol-dispersed enzyme, Arrhenius plots were nearly linear with average delta H* values of 45 and 68 kJ/mol, respectively, for untreated and gel-filtered enzymes; for membrane-bound enzyme, delta H changed from 40 kJ/mol above about 21 degrees C to 62 kJ/mol below 21 degrees C, but this behaviour does not necessarily indicate an abrupt, lipid-induced, transition in the reaction mechanism.     

Effect of temperature and pH on carbamoylation and phosphorylation of serum cholinesterases. Theoretical interpretation of activation energies in complex reactions

1. The effect of temperature and pH was studied on the kinetics of inhibition of horse serum and human serum cholinesterase by four organophosphorus compounds and five carbamates. 2. For all compounds, and at each pH and temperature, the inhibition followed the kinetics of a bimolecular reaction with the inhibitor in excess, and with a negligible concentration of the Michaelis complex. 3. The second-order rate constants (k(a)) for inhibition of human serum cholinesterase by one organophosphate and one carbamate increased from 5 degrees to 40 degrees C with an apparent activation energy of 46kJ/mol (11kcal/mol). 4. The k(a) constant for inhibition of horse serum cholinesterase increased with temperature from 5 degrees to 30 degrees C, and then decreased from 30 degrees to 40 degrees C. The theoretical interpretation of such an unusual effect of temperature is derived. 5. The increase of k(a) with pH (human serum cholinesterase) followed the dissociation curve for a single group on the enzyme (pK7.5). 6. Rate constants for decarbamoylation (k(+3)) were determined, and the time-course of inhibition was calculated from the k(a) and k(+3) constants.     

Spontaneous lipid peroxidation in rabbit and mouse epididymal spermatozoa: dependence of rate on temperature and oxygen concentration

The rate of spontaneous lipid peroxidation, as measured by formation of malonaldehyde (MA), was determined as a function of O2 concentration and temperature in mouse and rabbit spermatozoa released from the cauda epididymidis. The peroxidation rate was linear in O2 concentration in the suspending medium up to 210 microM (the concentration at PO2 of ambient air at 34 degrees C) for sperm from both species over the temperature range 34-40 degrees C. This is the range over which the reaction is measurable for both species: below 34 degrees C, the rates become too slow to be measured accurately for rabbit sperm by our methods, while above 40 degrees C the rates for mouse sperm become too rapid. This narrow range is characteristic of a high activation energy (EA) for the peroxidation process. Values of EA were calculated from plots of kox versus (T)-1, where kox is a second order rate constant with the units (10(8) cells/ml)-1 min-1. It is defined by the equation: vma = kox (Sp) (O2), where vma is the rate of malonaldehyde production, (Sp) is concentration of sperm cells and (O2) is the O2 concentration in the suspending medium. For mouse sperm, EA was calculated to 78.7 kcal/mol (329 KJ/mol); for rabbit sperm, the value was 77.6 kcal/ml (324 KJ/mol). These high EAs and consequent steep dependence of the spontaneous lipid peroxidation rates on temperature favor long sperm life in the epididymis at around 32 degrees C and low PO2 in these scrotal animals, while allowing for a relatively short life at 37 degrees C at higher PO2 in the oviduct.     

Thermal adaptation in CHO cells at 40 degrees C: the influence of growth conditions and the role of heat shock proteins

The kinetics of thermal adaptation at the nonlethal temperature of 40 degrees C was studied in CHO (Chinese hamster ovary) cells in vitro. Thermal resistance, demonstrated as an increase in mean 45 degrees C killing time or as an increase in the shoulder of the 45 degrees C survival curve, was fully developed by 2 h. Control cells in early logarithmic phase were more heat sensitive than those in stationary phase. Corresponding 45 degrees C killing time frequency distributions were unimodal with an increase in mean killing time from early logarithmic to stationary phase. Cells which were thermally adapted at 40 degrees C for 6 h had biphasic 45 degrees C killing time frequency distributions, and as cells progressed from early logarithmic to stationary phase the heat-sensitive subpopulation progressively declined. Exposure to 40 degrees C produced a 30% increase in total protein synthesis. Proteins with molecular weights 72, 89, and 109 kDa which correspond to those induced by lethal heat shock were synthesized at 40 degrees C, but there was no close temporal correlation between the development of heat resistance at 40 degrees C and synthesis of the heat shock proteins. Cycloheximide (100 micrograms/ml) reduced the mean 45 degrees C killing time but did not totally prevent the development of heat resistance at 40 degrees C.     

The recovery of Arcobacter butzleri NCTC 12481 from various temperature treatments

AIMS: The aim of this study was to determine the growth and survival characteristics for Arcobacter butzleri NCTC 12481. METHODS AND RESULTS: The temperature and pH growth ranges were 15-39 degrees C and pH 6.0-8.0, as determined using impedance microbiology. The maximum specific growth rate was 00.57 h(-1) at 30 degrees C, pH 7.0. Arcobacter butzleri harvested from the exponential phase was more resistant to heat treatment than stationary phase cells (D55 1.1 and 0.4 min, respectively). Fluorescent dye uptake, and the release of UV-absorbing material, increased in heat-treated cells. After 21 d storage at 4 and -20 degrees C, A. butzleri was recovered on blood agar, but not on the isolation media CAT or CCDA. CONCLUSION: Arcobacter butzleri cells from the exponential phase were less heat sensitive than those from the stationary phase. The organism was able to survive cold storage for at least 3 weeks. SIGNIFICANCE AND IMPACT OF THE STUDY: The growth and survival characteristics have been quantified thus providing a greater understanding of this newly emerging pathogen.     

Pressure perturbation and differential scanning calorimetric studies of bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius

Differential scanning calorimetry (DSC) and pressure perturbation calorimetry (PPC) were used to characterize thermal phase transitions, membrane packing, and volumetric properties in multilamellar vesicles (MLVs) composed of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius grown at different temperatures. For PLFE MLVs derived from cells grown at 78 degrees C, the first DSC heating scan exhibits an endothermic transition at 46.7 degrees C, a small hump near 60 degrees C, and a broad exothermic transition at 78.5 degrees C, whereas the PPC scan reveals two transitions at approximately 45 degrees C and 60 degrees C. The endothermic peak at 46.7 degrees C is attributed to a lamellar-to-lamellar phase transition and has an unusually low DeltaH (3.5 kJ/mol) and DeltaV/V (0.1%) value, as compared to those for the main phase transitions of saturated diacyl monopolar diester lipids. This result may arise from the restricted trans-gauche conformational changes in the dibiphytanyl chain due to the presence of cyclopentane rings and branched methyl groups and due to the spanning of the lipid molecules over the whole membrane. The exothermic peak at 78.5 degrees C probably corresponds to a lamellar-to-cubic phase transition and exhibits a large and negative DeltaH value (-23.2 kJ/mol), which is uncommon for normal lamellar-to-cubic phospholipid phase transformations. This exothermic transition disappears in the subsequent heating scans and thus may involve a metastable phase, which is irreversible at the scan rate used. Further, there is no distinct peak in the plot of the thermal expansion coefficient alpha versus temperature near 78.5 degrees C, indicating that this lamellar-to-cubic phase transition is not accompanied by any significant volume change. For PLFE MLVs derived from cells grown at 65 degrees C, similar DSC and PPC profiles and thermal history responses were obtained. However, the lower growth temperature yields a higher DeltaV/V ( approximately 0.25%) and DeltaH (14 kJ/mol) value for the lamellar-to-lamellar phase transition measured at the same pH (2.1). A lower growth temperature also generates a less negative temperature dependence of alpha. The changes in DeltaV/V, DeltaH, and the temperature dependence of alpha can be attributed to the decrease in the number of cyclopentane rings in PLFE at the lower growth temperature. The relatively low DeltaV/V and small DeltaH involved in the phase transitions help to explain why PLFE liposomes are remarkably thermally stable and also echo the proposal that PLFE liposomes are generally rigid and tightly packed. These results help us to understand why, despite the occurrence of thermal-induced phase transitions, PLFE liposomes exhibit a remarkably low temperature sensitivity of proton permeation and dye leakage.     

Bypass of the ts block of tsJT60, a G0-specific ts mutant from rat fibroblasts, by fetal bovine serum and epidermal growth factor

tsJT60, a temperature-sensitive (ts) G0-mutant cell line from a Fischer rat, grows normally in the exponential growth phase at 34 degrees C and 39.5 degrees C, but when stimulated with fetal bovine serum (FBS), from the G0 phase they reenter the S phase at 34 degrees C but not at 39.5 degrees C. The ts-block was bypassed when G0-arrested tsJT60 cells were stimulated at 39.5 degrees C with FBS plus epidermal growth factor (EGF). The presence of EGF for the first 6 h after serum stimulation caused tsJT60 cells to enter the S phase in the presence of FBS at 39.5 degrees C. When EGF was added 6 h after serum stimulation, entrance into the S phase was delayed by about 6 h. The sequential presence of two growth factors, EGF without FBS for 6 h then FBS without EGF, or the reversed sequence, failed to initiate DNA synthesis at 39.5 degrees C. The binding of EGF was not temperature sensitive. The amounts of RNA and protein present doubled after stimulation with both FBS and EGF at 39.5 degrees C. These and other findings suggest that EGF bypasses only some specific event in the entire prereplicative process that operates operating in serum-stimulated cells at 39.5 degrees C.     

Novel direct access to enantiomerization barriers from peak profiles in enantioselective dynamic chromatography: enantiomerization of dialkyl-1,3-allenedicarboxylates

The axially chiral allenes dimethyl-1,3-allenedicarboxylate 1 and diethyl-1,3-allenedicarboxylate 2 show characteristic plateau formation during enantioselective GC separation on the chiral stationary liquid phase Chirasil-beta-Dex. The elution profiles, obtained from temperature-dependent dynamic GC (DGC) experiments (1: 100-140 degrees C; 2: 110-150 degrees C) were evaluated with the recently derived approximation function (AF) k1(approx) = f(t(R)(A),t(R)(B),w(h)(A),h(plateau), N) to yield the enantiomerization rate constant directly k(1). These values were compared with those obtained by computer-aided simulation with ChromWin. The Eyring activation parameters of the experimental interconversion profiles were determined to be: DeltaG(#)(298.15 K) = 103.6 +/- 0.9 kJ mol(-1), DeltaH(#) = 44.7 +/- 0.4 kJ mol(-1), DeltaS(#) = -198 +/- 7 J K(1) mol(-1) for dimethyl-1,3-allenedicarboxylate 1, and DeltaG(#)(298.15 K) = 103.5 +/- 1.1 kJ mol(-1), DeltaH(#) = 44.7 +/- 0.5 kJ mol(-1), DeltaS(#) = -197 +/- 9 J K(-1) mol(-1) for diethyl-1,3-allenedicarboxylate 2. The approximation function (AF) presented here allows the fast determination of rate constants k(1) and activation barriers of enantiomerization DeltaG(#) from chromatographic parameters without extensive computer simulation.     

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.     

Multi-method global analysis of thermodynamics and kinetics in reconstitution of monellin

Accurate and precise determinations of thermodynamic parameters of binding are important steps toward understanding many biological mechanisms. Here, a multi-method approach to binding analysis is applied and a detailed error analysis is introduced. Using this approach, the binding thermodynamics and kinetics of the reconstitution of the protein monellin have been quantitatively determined in detail by simultaneous analysis of data collected with fluorescence spectroscopy, surface plasmon resonance and isothermal titration calorimetry at 25 degrees C, pH 7.0 and 150 mM NaCl. Monellin is an intensely sweet protein composed of two peptide chains that form a single globular domain. The kinetics of the reconstitution reaction are slow, with an association rate constant, k(on) of 8.8 x 10(3) M(-1) s(-1) and a dissociation rate constant, k(off) of 3.1 x 10(-4) s(-1). The equilibrium constant K(A) is 2.8 x 10(7) M(-1) corresponding to a standard free energy of association, DeltaG degrees , of -42.5 kJ/mol. The enthalpic component, DeltaH degrees , is -18.7 kJ/mol and the entropic contribution, DeltaS degrees , is 79.8 J mol(-1) K(-1) (-TDeltaS degrees = -23.8 kJ/mol). The association of monellin is therefore a bimolecular intra-protein association whose energetics are slightly dominated by entropic factors.     

Fluorescence decay of 1-methylpyrene in small unilamellar l-alpha-dimyristoylphosphatidylcholine vesicles. A temperature and concentration dependence study

The fluorescence decay kinetics of 1-methylpyrene in small unilamellar l-alpha-dimyristoylphosphatidylcholine vesicles above the phase transition temperature has been studied as a function of concentration and temperature. When the 1-methylpyrene/phospholipid ratio equals 1:2000 no excimer is observed and the fluorescence decay is monoexponential. When this ratio is equal to or higher than 1 200, excimer is observed and the monomer and excimer decays can be adequately described by two exponential terms. The deviation of the monomer decays from monoexponentiality cannot be described by a model where the diffusion-controlled excimer formation is time dependent. The observed decays are compatible with the excimer formation scheme which is valid in an isotropic medium. The activation energy of excimer formation is found to be 29-9 +/-1.4 kJ mol . The (apparent) excimer formation constant and the excimer lifetime at different temperatures have been determined. The diffusion coefficient associated with the excimer formation process varies between 2 x 10(-10) m(2)/s at 70 degrees C to 4 x 10(-11) m(2)/s at 25 degrees C.     

Temperature compensation of postsynaptic currents from the extraocular muscle of temperate teleost fishes

Miniature end plate currents were recorded from white inferior oblique extraocular muscle fibres of one temperate marine teleost (Aldrichetta forsteri, Family Mugilidae) and two temperate freshwater teleosts (Galaxias fasciatus, Family Galaxiidae and Oncorhynchus mykiss, Family Salmonidae). Miniature end plate currents were digitised and averaged over a temperature range of 5-25 degrees C. For each species, decay of miniature end plate currents was exponential and exhibited a strong temperature dependence. Lower temperatures resulted in prolonged decay phases, which decreased exponentially as a function of absolute temperature. Although values of the exponential time constant tau (tau) obtained for each species at 5 degrees C, 15 degrees C and 25 degrees C were significantly different, at any given temperature, there were no significant differences between tau values for the three species, despite differences in phylogeny (different families) and habitat (marine versus freshwater). At their normal temperature of 15 degrees C, mean values of tau for the three species ranged from 840 micros to 940 micros, and apparent activation energies ranged from -41 kJ mol(-1) deg(-1) to 50 kJ mol(-1) deg(-1). These observations confirm earlier reports that teleost miniature end plate currents are consistently shorter than those of other vertebrates.     

Effects of pulsed electric fields on inactivation kinetics of Listeria innocua

The effects of pulsed electric field (PEF) treatment and processing factors on the inactivation kinetics of Listeria innocua NCTC 11289 were investigated by using a pilot plant PEF unit with a flow rate of 200 liters/h. The electric field strength, pulse length, number of pulses, and inlet temperature were the most significant process factors influencing the inactivation kinetics. Product factors (pH and conductivity) also influenced the inactivation kinetics. In phosphate buffer at pH 4.0 and 0.5 S/m at 40 degrees C, a 3. 0-V/microm PEF treatment at an inlet temperature of 40 degrees C resulted in > or = 6.3 log inactivation of strain NCTC 11289 at 49.5 degrees C. A synergistic effect between temperature and PEF inactivation was also observed. The inactivation obtained with PEF was compared to the inactivation obtained with heat. We found that heat inactivation was less effective than PEF inactivation under similar time and temperature conditions. L. innocua cells which were incubated for a prolonged time in the stationary phase were more resistant to the PEF treatment, indicating that the physiological state of the microorganism plays a role in inactivation by PEF. Sublethal injury of cells was observed after PEF treatment, and the injury was more severe when the level of treatment was increased. Overall, our results indicate that it may be possible to use PEF in future applications in order to produce safe products.