S. Condón

Membrane damage and microbial inactivation by chlorine in the absence and presence of a chlorine-demanding substrate.

ABSTRACT The relationship between cell inactivation and membrane damage was studied in two gram-positive organisms, Listeria monocytogenes and Bacillus subtilis, and two gram-negative organisms, Yersinia enterocolitica and Escherichia coli, exposed to chlorine in the absence and presence of 150 ppm of organic matter (Trypticase soy broth). L. monocytogenes and B. subtilis were more resistant to chlorine in distilled water. The addition of small amounts of organic matter to the chlorination medium drastically increased the resistance of both types of microorganisms, but this effect was more marked in Y. enterocolitica and E. coli. In addition, the survival curves for these microorganisms in the presence of organic matter had a prolonged shoulder. Sublethal injury was not detected under most experimental conditions, and only gram-positive cells treated in distilled water showed a relevant degree of injury. The exposure of bacterial cells to chlorine in distilled water caused extensive permeabilization of the cytoplasmic membrane, but the concentrations required were much higher than those needed to inactivate cells. Therefore, there was no relationship between the occurrence of membrane permeabilization and cell death. The addition of organic matter to the treatment medium stabilized the cytoplasmic membrane against permeabilization in both the gram-positive and gram-negative bacteria investigated. Exposure of E. coli cells to the outer membrane-permeabilizing agent EDTA increased their sensitivity to chlorine and caused the shoulders in the survival curves to disappear. Based on these observations, we propose that bacterial envelopes could play a role in cell inactivation by modulating the access of chlorine to the key targets within the cell.

Influence of the incubation temperature after heat treatment upon the estimated heat resistance values of spores of Bacillus subtilis

S. CONDÓN, A. PALOP, J. RASO AND F.J. SALA. 1996. The influence of the incubation temperature on the estimated heat resistance for survivors after heat treatment was investigated. The survival curves and the Dt values of spores of Bacillus subtilis heated at different temperatures in pH 7 buffer, obtained after incubating survivors at different temperatures (30, 37, 44 or 51°C), were compared. The incubation temperature influenced the profile of survival curves. Lower incubation temperatures led to bigger Dt values and longer shoulders. Dt values obtained after incubating at 30°C were higher (x3 approx.) than those obtained by incubating at 51°C. The incubation temperature did not modify z values (z= 9.1). These results show that shoulders are not only due to the activation of dormant spores but also to heat damage repair mechanisms. From the profile of survival curves at different incubation temperatures it would seem that heat damage is accumulative. Cells can repair the initial heat injury, but the accumulation of injuries would eventually make the damage irreversible.

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.

Pulsed electric fields inactivation of wine spoilage yeast and bacteria.

The use of the pulsed electric fields technology (PEF) as an alternative system of microbiological control in wineries was evaluated. The PEF resistance of different wine spoilage microorganisms such as Dekkera anomala, Dekkera bruxellensis, Lactobacillus hilgardii and Lactobacillus plantarum in both must and wine was investigated by applying treatments ranging from 16 to 31 kV/cm and from 10 to 350 kJ/kg at 24 degrees C. The non-exponential kinetics of inactivation by PEF of these microorganisms in both products has been described by mathematical equations based on the Weibull distribution. An optimum treatment of 186 kJ/kg at 29 kV/cm has been established which permitted to reduce the 99.9% of the spoilage flora of must and wine, limiting the risk of alteration of these products by microorganisms of genera Brettanomyces and Lactobacillus.

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.

Comparing predicting models for the Escherichia coli inactivation by pulsed electric fields

Abstract Inactivation of Escherichia coli by pulsed electric field treatments (PEF) between 15 and 28 kV/cm in citrate–phosphate McIlvaine buffer (pH 7, 2 mS/cm) was studied. At all electric field strengths investigated the shape of the survival curves was concave upwards. A two-term exponential model for mixed cell populations, a model based on a Weibull distribution of resistances within the bacterial population, a sigmoidal equation also justified by the existence of a resistance distribution, and a purely empirical equation were used to fit the observed survival curves. The three last models were simpler than the first one and allowed to develop secondary models to estimate the influence of the electric field strength on the inactivation of E. coli. A validation study showed that the performance of models derived from the Weibull distribution and the empirical equation were better than the derived from the sigmoidal equation.

Microbial heat resistance determinations by the multipoint system with the thermoresistometer TR-SC Improvement of this methodology

The thermoresistometer TR-SC for microbial heat resistance determinations by the multipoint system was improved. In manual mode (for the determination of high Dt values) the need of pipetting was eliminated: Selected volumes of heated menstruum were collected directly into plating medium by means of an automatic sampling valve operated by a solenoid activated by a timer. In the automatic mode, used for the determinations of very low Dt values, the inoculation of menstruum, equiliberated at the required temperature and the collection of samples during the experiment, were made fully automatic. The injection of the inoculum was made with a solenoid operated syringe, and samples were collected, with a specially adapted sample collector, directly into tubes containing melted plating medium. The maximum number of samples per unit of time was increased to 52 samples/s. The minimum detectable Dt value was reduced from 0.009 to 0.0009 min without decreasing precision (SD = 0.00017). This improvement eliminated the chance of handling errors, and reduced time and effort required for heat resistance determinations. This modification makes this method especially suitable for the determination, by the multipoint system, of very low Dt values as needed in experiments at ultrahigh temperatures.

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.