Rafael Pagán

Relationship between Membrane Damage and Cell Death in Pressure-Treated Escherichia coli Cells: Differences between Exponential- and Stationary-Phase Cells and Variation among Strains

ABSTRACT The relationship between membrane damage and loss of viability following pressure treatment was examined in Escherichia coli strains C9490, H1071, and NCTC 8003. These strains showed high, medium, and low resistance to pressure, respectively, in stationary phase but similar resistance to pressure in exponential phase. Loss of membrane integrity was measured as loss of osmotic responsiveness or as increased uptake of the fluorescent dye propidium iodide. In exponential-phase cells, loss of viability was correlated with a permanent loss of membrane integrity in all strains, whereas in stationary-phase cells, a more complicated picture emerged in which cell membranes became leaky during pressure treatment but resealed to a greater or lesser extent following decompression. Strain H1071 displayed a very unusual pressure response in stationary phase in which survival decreased to a minimum at 300 MPa but then increased at 400 to 500 MPa before decreasing again. Membranes were unable to reseal after treatment at 300 MPa but could do so after treatment at higher pressures. Membrane damage in this strain was thus typical of exponential-phase cells under low-pressure conditions but of stationary-phase cells under higher-pressure conditions. Heat shock treatment of strain H1071 cells increased pressure resistance under low-pressure conditions and also allowed membrane damage to reseal. Growth in the presence of IPTG (isopropyl-β-d-thiogalactopyranoside) increased resistance under high-pressure conditions. The mechanisms of inactivation may thus differ at high and low pressures. These studies support the view that membrane damage is an important event in the inactivation of bacteria by high pressure, but the nature of membrane damage and its relation to cell death may differ between species and phases of growth.

Influence of different factors on the output power transferred into medium by ultrasound

The influence of several factors (amplitude of ultrasonic waves, external static pressure, temperature and viscosity of medium) acting, either individually or in combination, on the amount of power transferred to a liquid medium during ultrasonication (power output) was measured by calorimetry. At constant amplitude (150 microns) and pressure (200 kPa), the power output decreased as the temperature was raised. The effect of temperature could be compensated by increasing pressure. The magnitude of the increase in power output due to raising the pressure depended on the pressure range and the treatment temperature. At all temperatures and pressures studied, the power output increased exponentially when the amplitude was increased linearly. The magnitude of this power output did not depend on the temperature or pressure of treatment. At 40 degrees C the magnitude of the increase in power output due to increasing the pressure was not influenced by the amplitude of sonic waves. The power output increased as the viscosity of the medium was increased. The magnitude of this effect did not depend on the amplitude but on the static pressure.

Inactivation of Bacillus subtilis spores by combining ultrasonic waves under pressure and mild heat treatment

The inactivation of Bacillus subtilis spores by ultrasonic treatments under static pressure (Mano‐Sonication, MS) and a combined MS/heat treatment (Mano‐Thermo‐Sonication) was investigated. The sporicidal effect of MS treatments depended on static pressure, amplitude of ultrasonic waves and treatment temperature. At 70 °C, pressure increments up to 500 kPa caused progressively more inactivation. An MS treatment at 500 kPa and 117 μm of amplitude for 12 min inactivated approximately 99% of the B. subtilis spore population. Over 500 kPa, further increments in pressure did not increase the percentage of inactivation. In the range 90–150 μm, an exponential relationship was observed between the amplitude of ultrasonic waves under pressure and the number of survivors. While an MS treatment (20 kHz, 300 kPa, 70 °C, 12 min) at 90 μm inactivated 75% of the B. subtilis spore population, the same treatment at 150 μm inactivated 99·9% of this population. The MS treatments at temperatures higher than 70 °C (MTS) led to more spore inactivation. In the range 70–90 °C, the combination of heat with an MS treatment (20 kHz, 300 kPa, 117 μm, 6 min) had a synergistic effect on spore inactivation. The inactivating effect of ultrasound was due neither to titanium particles eroded from the sonication tip, nor to free radicals released during ultrasonic treatment. The MS treatments sensitized spores of B. subtilis to lysozyme.

Occurrence of sublethal injury after pulsed electric fields depending on the micro-organism, the treatment medium ph and the intensity of the treatment investigated

Aims:  The objective was to investigate the occurrence of sublethal injury after pulsed electric field (PEF) depending on the treatment time, the electric field strength and the pH of the treatment media in two Gram‐positive (Bacillus subtilis ssp. niger, Listeria monocytogenes) and six Gram‐negative (Escherichia coli, Escherichia coli O157:H7, Pseudomonas aeruginosa, Salmonella serotype Senftenberg 775W, Salmonella serotype Typhimurium, Yersinia enterocolitica) bacterial strains.

Inactivation of Escherichia coli by citral

Aims:  The aim was to evaluate (i) the resistance of Escherichia coli BJ4 to citral in a buffer system as a function of citral concentration, treatment medium pH, storage time and initial inoculum size, (ii) the role of the sigma factor RpoS on citral resistance of E. coli, (iii) the role of the cell envelope damage in the mechanism of microbial inactivation by citral and (iiii) possible synergistic effects of mild heat treatment and pulsed electric fields (PEF) treatment combined with citral.

The influence of process parameters for the inactivation of Listeria monocytogenes by pulsed electric fields

The influence of the electric field strength, the treatment time, the total specific energy and the conductivity of the treatment medium on the Listeria monocytogenes inactivation by pulsed electric fields (PEF) has been investigated. L. monocytogenes inactivation increased with the field strength, treatment time and specific energy. A maximum inactivation of 4.77 log(10) cycles was observed after a treatment of 28 kV/cm, 2000 micros and 3490 kJ/kg. The lethal effect of PEF treatments on L. monocytogenes was not influenced by the conductivity of the treatment medium in a range of 2, 3 and 4 mS/cm when the total specific energy was used as a PEF control parameter. A mathematical model based on the Weibull distribution was fitted to the experimental data when the field strength (15-28 kV/cm), treatment time (0-2000 micros) and specific energy (0-3490 kJ/kg) were used as PEF control parameters. A linear relationship was obtained between the log(10) of the scale factor (b) and the electric field strength when the treatment time and the total specific energy were used to control the process. The total specific energy, in addition to the electric field strength and the treatment time, should be reported in order to evaluate the microbial inactivation by PEF.

Pulsed electric fields cause sublethal injury in Escherichia coli

Aims: The objective was to investigate the occurrence of sublethal injury in Escherichia coli by pulsed electric fields (PEF) at different pH values.

Inactivation of Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Senftenberg by ultrasonic waves under pressure.

The resistance of Salmonella Enteritidis (ATCC 13076), Salmonella Typhimurium (ATCC 13311), and Salmonella Senftenberg 775W (ATCC 43845) to ultrasonic waves under pressure treatments, at sublethal (manosonication) and lethal temperatures (manothermosonication) in citrate-phosphate buffer and in liquid whole egg was investigated. The influence of treatment parameters on the inactivation rate of manosonication was also studied. Decimal reduction times (Dt) of Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Senftenberg 775W corresponding to a heat treatment at 60 degrees C in pH 7 buffer and in liquid whole egg were 0.068, 0.12, and 1.0 min for buffer, and 0.12, 0.20, and 5.5 min for liquid whole egg. Those corresponding to a manosonication treatment (117 microns, 200 kPa, 40 degrees C) in both media were 0.73, 0.78, and 0.84 min, and 0.76, 0.84, and 1.4 min, respectively. When the amplitude of ultrasonic waves was increased linearly, the inactivation rate of manosonication increased exponentially. The inactivation rate also increased when pressure was raised. However, the magnitude of this increase was progressively smaller at higher pressures. The magnitude of the influence of the amplitude of ultrasonic waves and static pressure on the inactivation rate of manosonication was the same in the three serotypes investigated. Whereas a heat treatment at 60 degrees C only attained a 1/2-log cycle reduction in the number of Salmonella Senftenberg 775W survivors, a manothermosonication treatment (117 microns and 200 kPa) at this temperature attained a 3-log cycle reduction.

Effect of environmental factors and cell physiological state on Pulsed Electric Fields resistance and repair capacity of various strains of Escherichia coli.

The aim was to determine the resistance variation of four strains of Escherichia coli to Pulsed Electric Fields (PEF), the role of the sigma factor RpoS in PEF resistance, as well as the influence of several environmental factors and the cell physiological state on the PEF resistance and repair capacity. The rpoS null mutant, E. coli BJ4L1, exhibited decreased PEF resistance as compared with its wild-type parent, BJ4. W3110 and O157:H7 were the most PEF-resistant strains: whereas 2 and more than 3 Log10 cycles of BJ4 and BJ4L1 cells, respectively, were inactivated after 50 pulses at 35 kV/cm, only 0.5 Log10 cycle of inactivation of W3110 and O157:H7 was attained. A different pattern was observed and the resistance variation among strains was largely reduced, when selective recovery media were used. At exponential growth phase, the resistance of the four strains was lower, and more than 4 Log10 cycles of inactivation of all strains tested were attained at 30 kV/cm. Previous heat and cold shock treatments scarcely influenced cell PEF resistance. PEF survival increased with the reduction in water activity of the treatment medium to 0.94: the occurrence of sublethally injured cells was negligible, and less than 1 Log10 cycle of inactivation was attained at 35 kV/cm. PEF-treated cells were sensitive to a subsequent storage at pH 4.0 or in the presence of sorbic acid, attaining a final inactivation of 4-5 Log10 cycles after 24 hour-incubation. In conclusion, the work confirms the role of rpoS in PEF resistance. E. coli strains exhibit large differences in PEF resistance. These differences were less important when cells were recovered under selective conditions. Both resistance variation among strains and occurrence of sublethal damage were noticeably influenced by the environmental factors tested.

Inactivación de formas esporuladas de Bacillus subtilis mediante campos eléctricos pulsantes de alta intensidad en combinacion con otras tecnicas de conservacion de alimentos/Inactivation of Bacillus subtilis spores using high intensity pulsed electric fields in combination with other food conservation technologies

The inactivation of Bacillus subtilis spores using high intensity pulsed electric fields (HIPEF) was investigated. Spores were not inactivated when HIPEF treatment (60 kV/cm, 75 pulses) was used alone. The combination of-HIPEF and moderate temperatures around 60°C, and/or the activation of spore suspension prior to HIPEF treatment, and/or the use of up to 5000 IU/ml lysozyme, did not inactivate spores. High hydrostatic pressure (1500 atm, 30 min, 40°C) resulted in the initiation of germination of more than five log cycles in the number of spores, making them sensitive to subsequent pasteurization heat treatment, whereas they were not sensitive to subsequent HIPEF treatment at temperatures less than 40 °C. An intermediate step is needed which allows the outgrowth of spores to vegetative cells. Thus, the combination of high hydrostatic pressure and HIPEF treatment offers an attractive alternative to the stabilization of food products by heat to inactivate spores.