Nonisothermal heat resistance determinations with the thermoresistometer Mastia.

AIMS: To design and build a thermoresistometer, named Mastia, which could perform isothermal and nonisothermal experiments. METHODS AND RESULTS: In order to evaluate the thermoresistometer, the heat resistance of Escherichia coli vegetative cells and Alicyclobacillus acidoterrestris spores was explored. Isothermal heat resistance of E. coli was characterized by D(60 degrees C) = 0.38 min and z = 4.7 degrees C in pH 7 buffer. When the vegetative cells were exposed to nonisothermal conditions, their heat resistance was largely increased at slow heating and fast cooling rates. Isothermal heat resistance of A. acidoterrestris was characterized by D(95 degrees C) = 7.4 min and z = 9.5 degrees C in orange juice. Under nonisothermal conditions, inactivation was reasonably well predicted from isothermal data. CONCLUSIONS: The thermoresistometer Mastia is a very suitable instrument to get heat resistance data of micro-organisms under isothermal and nonisothermal treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: The thermoresistometer Mastia can be a helpful tool for food processors in order to estimate the level of safety of the treatments they apply.

Application of artificial neural networks to describe the combined effect of pH and NaCl on the heat resistance of Bacillus stearothermophilus.

A model for prediction of bacterial spore inactivation was developed. The influence of temperature, pH and NaCl on the heat resistance of Bacillus stearothermophilus spores was described using low-complexity, black box models based on artificial neural networks. Literature data were used to build and train the neural network, and new experimental data were used to evaluate it. The neural network models gave better predictions than the classical quadratic response surface model in all the experiments tried. When the neural networks were evaluated using new experimental data, also good predictions were obtained, providing fail-safe predictions of D values in all cases. The weights and biases values of neurons of the neural network that gave the best results are presented, so the reader can use the model for their own purposes. The use of this non-linear modelling technique makes it possible to describe more accurately interacting effects of environmental factors when compared with classical predictive microbial models.

Impact of Moderate Heat, Carvacrol, and Thymol Treatments on the Viability, Injury, and Stress Response of Listeria monocytogenes.

The microbial safety and stability of minimally processed foods are based on the application of combined preservative factors. Since microorganisms are able to develop adaptive networks to survive under conditions of stress, food safety may be affected, and therefore understanding of stress adaptive mechanisms plays a key role in designing safe food processing conditions. In the present study, the viability and the sublethal injury of Listeria monocytogenes exposed to moderate heat (55 °C) and/or essential oil compounds (carvacrol and thymol, 0.3 mM) treatments were studied. Synergistic effects were obtained when combining mild heat (55 °C) with one or both essential oil compounds, leading to inactivation kinetics values three to four times lower than when using heat alone. All the treatments applied caused some injury in the population. The injury levels ranged from around 20% of the surviving population under the mildest conditions to more than 99.99% under the most stringent conditions. Protein extracts of cells exposed to these treatments were analysed by two-dimensional gel electrophoresis. The results obtained revealed that stressed cells exhibited differential protein expression to control cells. The proteins upregulated under these stressing conditions were implicated, among other functions, in stress response, metabolism, and protein refolding.

Influence of pH on heat resistance of Bacillus licheniformis in buffer and homogenised foods.

The influence of pH of heating menstruum (McIlvaine buffer) on the heat resistance of Bacillus licheniformis was investigated and compared with the heat resistance in homogenised tomato and asparagus at pH 7 and 4 in a wide range of temperatures. Heat resistance was in all mestrua smaller at acid pH. At 99 degrees C and pH 4, heat resistance was 1/20 lower than at pH 7. However, the magnitude of this effect decreased as heat treatment temperatures were increased almost disappearing at 120 degrees C. z values increased from 6.85 at pH 7, to 10.75 at pH 4. At 99 degrees C the effect of pH on heat resistance was constant along the range of pH's tested. The increase of one pH unit increased D99 by 180%. At pH 7 and 4, heat resistance was the same in buffer as in tomato and asparagus homogenates at all temperatures tested. The diminishing influence of the acidification of some foods on the heat resistance of B. licheniformis sterilisation temperatures should be taken into account when a raise in temperature is considered to shorten the duration of heat processes.

Influence of different factors on the inactivation of Salmonella senftenberg by pulsed electric fields.

The influence of growth phase, cell concentration, pH and conductivity of treatment medium on the inactivation of Salmonella senftenberg by high electric field pulses (HELP) was studied. Cells were more resistant to HELP treatments at the beginning of the logarithmic phase and at the stationary phase. Microbial inactivation was not a function of the initial cell concentration. At constant input voltage, electric field strength obtained in the treatment chamber depended on medium conductivity. At the same electric field strength, conductivity did not influence S. senftenberg inactivation. At the same conductivity, inactivation of S. senftenberg was bigger at neutral than acidic pH.

Survival of heated Bacillus coagulans spores in a medium acidified with lactic or citric acid.

The influence of the intensity of heat treatments on the capacity of citric or lactic acid to prevent growth of survivors of Bacillus coagulans spores after 10 days storage at 35 degrees C was studied. In most cases, the number of survivors during storage decreased. The extent of this spore inactivation depended on the intensity of previous heat treatment and the pH of the medium and the acidulant used. The inactivating effect of storage was pronounced even at pH values less acidic than those used by the canning industry. Citric acid was more effective than lactic acid on spores given only low heat treatments, but lactic was more effective against those given more severe heat treatments. The severity of heat treatment required for lactic to be more effective than citric acid increased with pH of the medium. Heat treatment also required increased pH for heated spores to grow. pH 4.6, regardless of acidulant used, was unable to prevent growth of unheated spores but a less acidic pH (pH 5.2) did prevent growth even when spores had been given only mild heat treatments (10 s at 100 degrees C).

Influence of pH on heat resistance of spores of Bacillus coagulans in buffer and homogenized foods.

The influence of pH of heating menstruum (McIlvaine buffer) on the heat resistance of Bacillus coagulans spores has been investigated and compared with the heat resistance in homogenized tomato and asparagus at pH 7 and 4 at a wide range of temperatures. Spores were less heat resistant in all menstrua at acid pH. The magnitude of this effect was greatest at the lowest heating temperatures tested. z values in buffer increased from 8.9 degrees C at pH 7 to 10.5 degrees C at pH 4. pH of menstrua was the main influencing factor, but media composition also influenced heat resistance: at pH 7 heat resistance was similar in all menstrua (D111 degrees C = 1.6 min) but at pH 4 the heat resistance in homogenized foods (D111 degrees C = 0.26 min in tomato and D111 degrees C = 0.28 min in asparagus) was lower than in buffer (D111 degrees C = 0.49 min). The reduced influence of the acidification of media on the heat resistance of B. coagulans at higher temperatures should be taken into account when a rise in the temperature of treatment for canned vegetables is considered to shorten duration of heat processes.

Heat resistance of Alicyclobacillus acidocaldarius in water, various buffers, and orange juice.

The effect of the pH or the composition of the heating medium and of the sporulation temperature on the heat resistance of spores of a thermoacidophilic spore-forming microorganism isolated from a dairy beverage containing orange fruit concentrate was investigated. The species was identified as Alicyclobacillus acidocaldarius. The spores showed the same heat resistance in citrate-phosphate buffers of pH 4 and 7, in distilled water, and in orange juice at any of the temperatures tested (D120 degrees C = 0.1 min and z = 7 degrees C). A raise in 20 degrees C in the sporulation temperature (from 45 to 65 degrees C) increased the heat resistance eightfold (from D110 degrees C = 0.48 min when sporulated at 45 degrees C to 3.9 min when sporulated at 65 degrees C). The z-values remained constant for all sporulation temperatures. The spores of this strain of A. acidocaldarius were very heat resistant and could easily survive any heat treatment currently applied to pasteurize fruit juices.

Resistance of heat-shocked cells of Listeria monocytogenes to mano-sonication and mano-thermo-sonication.

Heat shocks did not increase the resistance of Listeria monocytogenes to an ultrasonication treatment under pressure (Mano-Sonication; MS). While heat-shocked cells (180 min, 45 degrees C) became sixfold more heat resistant than native cells (D62 = 1.8 min vs D62 = 0.24 min), the resistance of native and heat-shocked cells to MS (200 kPa, 117 microns) was the same (DMS = 1.6 min). The inactivation rate of non-heat-shocked cells of L monocytogenes by a combined heat/ultrasonication treatment under pressure (Mano-Thermo-Sonication; MTS) was an additive effect. On the contrary, on heat-shocked cells, the inactivation rate of MTS was greater than that of heat added to the inactivation rate of MS (synergistic effect) in the range 62-68 degrees C.

Heat resistance of native and demineralized spores of Bacillus subtilis sporulated at different temperatures.

Demineralization reduced heat resistance of B. subtilis spores, but the pattern and magnitude of the reduction depended on sporulation temperature and on heating menstruum pH. The differences in heat resistance of native spores caused by sporulation temperature almost disappeared after demineralization. Demineralized spores were still susceptible to the heat-sensitizing effect of acidic pH.

Inactivation of enzymes within spores of Bacillus megaterium ATCC 19213 by hydroperoxides.

The organic hydroperoxides t-butyl hydroperoxide, cumene hydroperoxide, and peracetic acid were found to act similarly to hydrogen peroxide in causing inactivation of enzymes within intact spores of bacillus megaterium ATCC 19213 concomitant with mortality. Spores treated with lethal levels of the agents were germinated and permeabilized for enzyme assays. The hierarchy of sensitivities among enolase, glucose-6-phosphate dehydrogenase (G6Pdh), and pyruvate kinase to inactivation varied somewhat with the specific hydroperoxide used, possibly because of the differences in the types of radicals generated. However, each agent inactivated each of the enzymes, albeit at different rates. Comparative assessments of enzyme inactivation by lethal levels of H2O2 or by moist heat showed that some enzymes, such as G6Pdh, are highly sensitive to inactivation, while others, such as ATPases, are much more resistant. The enzymes G6Pdh and aldolase were highly sensitive to hydroperoxide inactivation and also to moist heat, while pyruvate kinase was much more sensitive to hydroperoxides than to moist heat. Our overall interpretation of the findings is that hydroperoxides and moist heat can produce cumulative damage to sensitive enzymes within spores, which progressively diminishes the capacities of the cells to undergo the outgrowth required for return to vegetative life.

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 degrees C, pressure increments up to 500 kPa caused progressively more inactivation. An MS treatment at 500 kPa and 117 microns 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 microns, 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 degrees C, 12 min) at 90 microns inactivated 75% of the B. subtilis spore population, the same treatment at 150 microns inactivated 99.9% of this population. The MS treatments at temperatures higher than 70 degrees C (MTS) led to more spore inactivation. In the range 70-90 degrees C, the combination of heat with an MS treatment (20 kHz, 300 kPa, 117 microns, 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 a Highly Heat-Sensitive Spore Subpopulation of Bacillus coagulans STCC 4522 and Its Conversion to a More Heat-Stable Form.

The profile of the survival curves, at different heating temperatures, of B. coagulans STCC 4522 sporulated at 52(deg)C has been studied, focusing on the early moments of treatment. A highly heat-sensitive spore subpopulation that includes more than 90% of the total spore population has been found. This heat-sensitive spore fraction was inactivated after 2 s of treatment at 111(deg)C. Its heat resistance was as much as 200-fold lower than that of the heat-resistant spore fraction (D(inf111(deg)C) of 0.01 min for the heat-sensitive spore fraction compared with D(inf111(deg)C) of 2 min for the heat-resistant fraction). The shape of the survival curve at 108.5(deg)C was modified after a sublethal heat shock at 80(deg)C for 3.5 h, resulting in a straight-line survival curve. The temperature of treatment also influenced the shape of the survival curves. The conversion of the highly heat-sensitive spore subpopulation to a more heat-stable form is discussed.

[Rhinitis with intolerance to non-steroidal anti-inflammatory agents. Report of 3 cases]. / Rinitis con intolerancia a antiinflamatorios no esteroideos. Communicación de tres casos.

Lumry described 6 patients who presented hypertrophic rhinosinusitis, positive nasal eosinophilia and intolerance to nonsteroidal antiinflammatory drugs, manifested exclusively with naso-ocular symptomatology. We present three patients with clinical manifestations of chronic rhinitis who had noticed before their first visit that several nonsteroidal antiinflammatory drugs precipitated their nasal symptomatology. None of them had ever presented with asthma symptoms. All of them had nasal polyps. The nasal smear showed eosinophilia of 20 to 45%. All three had sinusitis radiologically. The spirometric values were within normal limits (V.C., FEV1, MMEF25-75%). Skin tests with different inhalants antigens using the prick test technique as well as skin tests with pyrazolones (Phenyldimetrylpyrazolone: 25 and 250 mg./ml.; dipyrone: 4 and 44 mg./ml.; amidopyrine: 2.2 and 22 mg./ml.) using the intradermal technique were negative. Serum IgE (Phadezym IgE-Pharmacia) showed values of 23.9, 17.1 and 25.8 IU/ml. respectively. The bronchial inhalation challenge test with methacholine was positive with PD20FVE1 of 14 and 4.8 mg./ml. in two of our patients. Different nonsteroidal antiinflammatory drugs were administered to each patient in different days orally, with intervals of 7 and 25 days (aspirin 500 mg., dipyrone 575 mg., indomethacin 25 mg., naproxen 500 mg.) as well as tartrazine (50 mg.), paracetamol (500 mg.) and lactose as placebo. With 30 minutes intervals and up to three hours after drug administration, the symptoms were observed and spirometry was carried out. Steroids and antihistamines were suspended at least 48 hours before the test. Acetyl-salicylic acid, dipyrone, indomethacin and naproxen produced naso-ocular symptomatology without any objective reduction of FEV1; but paracetamol and tartrazine were well tolerated.(ABSTRACT TRUNCATED AT 250 WORDS)

[Francisella tularensis: update on microbiological diagnosis after an epidemic outbreak]. / Francisella tularensis: puesta a punto en el diagnóstico microbiológico tras un brote epidémico.

AIMS: Francisella tularensis is a facultative intracellular gramnegative aerobic coccobacilli which is difficult to isolate. The appearance of an epidemic outbreak of tularemia in Castilla y Leon (Spain) during the first months of 1998 led bur microbiology laboratory to develop the diagnostic methods of this disease which, up to then, were unknown by the authors. METHODS: During the months of January and February, 1998, 25 samples were processed for culture (17, adenopathy pus and 8 necrotic skin ulcers) using enriched culture mediums. In 20, direct immunofluorescence was performed on the sample spread. Serologic study was carried out in 352 patients by the agglutination technique in tubes, with titers > or = 1/160 being considered as positive. Susceptibility tests were performed by the disk plate technique in chocolate agar. RESULTS: Three isolates of F. tularensis were achieved (12%). Seven samples (38.8%) were positive by direct immunofluorescence. By serology 149 negative (42%) and 203 positive (58%) results were obtained. Seroconversion was observed in 53 patients (increase in titer value 4-fold above the basal determination). The strains isolated were sensitive to third generation cephalosporins, aminoglycosides, macrolides and quinolones. CONCLUSIONS: F. tularensis is a microorganism which is highly demanding of its culture. Serology and direct immunofluorescence are more effective techniques for the diagnosis of tularemia than cultures. The strains presented high sensitivity to the antibiotics tested. Since the appearance of this outbreak, tularemia should be included in the differential diagnosis of the infectious processes observed in Spain.