Effect of Toxin Concentration on the Heat Inactivation of Staphylococcal Enterotoxin A in Beef Bouillon and in Phosphate Buffer

The slope of the thermal inactivation curve of enterotoxin A in beef bouillon (initial pH 6.2) was found to be approximately 27.8 C (50 F) with three different concentrations of toxin. Inactivation by heat was dependent on the concentration of enterotoxin A heated. Enterotoxin A was inactivated by less heat in a pH 7.2 phosphate buffer than in beef bouillon as detected by serology. Results indicate that natural (usually low) levels of toxin rather than concentrated enterotoxin A should be used in heat inactivation studies.     

Heat Inactivation of Enterotoxin A from Staphylococcus aureus in Veronal Buffer

Serological tests were used to determine the slope of the thermal inactivation curve of crude enterotoxin A in Veronal buffer (pH 7.2), and the resulting z value was 27.8 C. (50 F). Serological assays also showed that the heat inactivation at each time-temperature depended on the original concentration of enterotoxin A. The usefulness of the Oudin tube serological test for determining end points of inactivation of naturally produced enterotoxin A (not concentrated) is discussed. We concluded that this test cannot be used to determine end points of heat inactivation for enterotoxin A in the minute quantities naturally produced in foods.     

Thermal inactivation and injury of Moraxella-Acinetobacter cells in ground beef.

The thermal inactivation and injury (sensitivity to 0.8% NaCl) of a radiation-resistant culture of Moraxella-Acinetobacter mixed in minced beef were determined. Survival curves for Moraxella-Acinetobacter cells in beef had an initial shoulder preceding a logarithmic decline when the cells were heated at 65, 70, and 75 degrees C, but not at 80 degrees C. In all cases, the experimental points not included in the shoulder were linearized by means of a least-squares straight line, and the latter was used to determine D values. Shoulder values of 12.2, 4.1, and 0.6 min at temperatures of 65, 70, and 75 degrees C were added to the respective D values of 35.4, 6.6, and 1.4 min to determine the time required to destroy one log cycle. The Z value was 7.3 degrees C. Moraxella-Acinetobacter cells in meat were more rapidly injured than inactivated, on initial exposure to heat. The number of cells injured by this initial exposure increased as the temperature was increased. At 65 degrees C the percentage of injured cells increased more rapidly with exposure time than did the inactivated cells. As the temperature was increased, the rates of inactivation and injury became more and more similar.     

Heat Resistance of Spores of Marine and Terrestrial Strains of Clostridium botulinum Type C

Resistance to heat of spores of marine and terrestrial strains of Clostridium botulinum type C in 0.067 m phosphate buffer (pH 7.0) was determined. The marine strains were 6812, 6813, 6814, and 6816; the terrestrial strains were 468 and 571. The inoculum level equaled 10(6) spores/tube with 10 replicate tubes for each time-temperature variable. Heating times were run at three or more temperatures to permit survival of some fraction of the inoculum. Survivors were recovered at 85 F (30 C) in beef infusion broth containing 1% glucose, 0.10% l-cysteine hydrochloride, and 0.14% sodium bicarbonate. D values were calculated for each fractional survivor end point after 6 months of incubation. Thermal resistance curves were constructed from the D value data. D(220) (104 C) values for spores of 468 and 571 equaled 0.90 and 0.40 min, respectively. The corresponding values for spores of 6812, 6813, 6814, and 6816 were 0.12, 0.04, 0.02, and 0.08 min. The z values for the thermal resistance curves ranged from 9.0 to 11.5 F (5.0 to 6.2 C).     

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 D _t 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 D _t values and longer shoulders. D _t 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.     

The influence of pH and temperature on the properties of myosin

1. The rate of denaturation of myosin solutions at temperatures between 32 degrees and 45 degrees and at pH values between 5.3 and 6.2 has been studied, by using adenosine-triphosphatase activity and solubility in m-potassium chloride at pH6.1 as criteria. 2. Myosin, when heated, loses its adenosine-triphosphatase activity before it becomes insoluble. 3. The loss of adenosine-triphosphatase activity and solubility are both first-order and pH-dependent reactions. Myosin, however, becomes insoluble only when heated within a narrow range of pH values. 4. The thermodynamic functions found for the two processes of denaturation are compared and discussed. 5. The possibility is discussed that, in muscle undergoing rigor, conditions may obtain that would denature myosin.     

Thermal inactivation of susceptible and multiantimicrobial-resistant salmonella strains grown in the absence or presence of glucose

The heat resistance of susceptible and multiantimicrobial-resistant Salmonella strains grown to stationary phase in glucose-free tryptic soy broth supplemented with 0.6% yeast extract (TSBYE-G; nonadapted), in regular (0.25% glucose) TSBYE, or in TSBYE-G with 1.00% added glucose (TSBYE+G; acid adapted) was determined at 55, 57, 59, and 61 degrees C. Cultures were heated in sterile 0.1% buffered peptone water (50 microl) in heat-sealed capillary tubes immersed in a thermostatically controlled circulating-water bath. Decimal reduction times (D values) were calculated from survival curves having r(2) values of >0.90 as a means of comparing thermal tolerance among variables. D(59 degrees C) values increased (P < 0.05) from 0.50 to 0.58 to 0.66 min for TSBYE-G, TSBYE, and TSBYE+G cultures, respectively. D(61 degrees C) values of antimicrobial-susceptible Salmonella strains increased (P < 0.05) from 0.14 to 0.19 as the glucose concentration increased from 0.00 to 1.00%, respectively, while D(61 degrees C) values of multiantimicrobial-resistant Salmonella strains did not differ (P > 0.05) between TSBYE-G and TSBYE+G cultures. When averaged across glucose levels and temperatures, there were no differences (P > 0.05) between the D values of susceptible and multiantimicrobial-resistant inocula. Collectively, D values ranged from 4.23 to 5.39, 1.47 to 1.81, 0.50 to 0.66, and 0.16 to 0.20 min for Salmonella strains inactivated at 55, 57, 59, and 61 degrees C, respectively. z(D) values were 1.20, 1.48, and 1.49 degrees C for Salmonella strains grown in TSBYE+G, TSBYE, and TSBYE-G, respectively, while the corresponding activation energies of inactivation were 497, 493, and 494 kJ/mol. Study results suggested a cross-protective effect of acid adaptation on thermal inactivation but no association between antimicrobial susceptibility and the ability of salmonellae to survive heat stress.     

Effect of culture age, pre-incubation at low temperature and pH on the thermal resistance of Aeromonas hydrophila

S. CONDON, M.L. GARCIA, A. OTERO AND F.J. SALA. 1992. The thermal resistance of Aeromonas hydrophila strain NCTC 8049 was determined within the range 48°-65°C with a thermoresistometer TR-SC and McIlvaine buffer. The effects of culture age, pre-incubation at 7°C and the pH of the heating menstruum were evaluated. The pattern of thermal death was dependent on culture age. Cells heated in the late logarithmic growth phase (15 h at 30°C) were twice as resistant as those in the early stage (5 h at 30°C), and the maximum D -value was obtained after 72 h incubation (5.5 total increase). The age of the cells did not affect z -values significantly. The heat resistance of cells incubated for 48 h at 30°C increased (twice) after holding at 7°C for 72 h. Pre-incubation at low temperature of older cultures (72 h, 30°C) did not influence their D -values. Maximum heat resistance was found at pH 6.0 and minimal at pH 4.0. Decreasing the pH from 6.0 4.0 reduced D -values by a factor of 5. Although the strain studied was heat-sensitive ( D _55°C= 0.17 min; z = 5.11°C), survivor curves of cultures older than 50 h showed a significant tailing. Organisms surviving in the tails were only slightly more resistant than were the original population.     

Effect of pH, Protein Concentration, and Ionic Strength on Heat Inactivation of Staphylococcal Enterotoxin B

Heat treatment of highly purified staphylococcal enterotoxin B causes a more rapid loss of immunological activity at 70 to 80 C than at 90 to 100 C. Toxicological results based on intravenous injection of dogs paralleled the results obtained by immunological means (single gel diffusion). The loss of immunological activity did not follow first-order kinetics. Results are given on the effects on heat inactivation of changing pH, ionic strength, and initial concentration of enterotoxin. Disc-gel electrophoresis of purified enterotoxin B showed a major and minor band. The minor band was a size isomer of the major band.     

Effect of beef broth protein on the thermal inactivation of staphylococcal enterotoxin B1.

Enterotoxin B produced by Staphylococus aureus 243 in brain heart infusion broth was concentrated by dialysis against 40% polyethylene glycol (20 M), partially purified on a Sephadex G-100 column and heated at 110 degrees C in thermal death time cans. Various heating menstrua included 0.04 M Veronal buffer (pH 7.4), beef broth, and fractions of beef broth obtained by ultrafiltration or precipitation with ammonium sulfate. The toxin was assayed serologically using the microslide gel double-diffusion method. The time requiring for 90% inactivation at 110 degrees C (D110 value) obtained in buffer and in beef broth was 18 and 60 min, respectively. When the concentration of beef broth was increased fivefold, the D110 increased to 78 min. The apparent protective effect or protein was further investigated using beef broth protein obtained by precipitation with (NH4)2SO4. The D110 values were 51 and 70 min when the protein concentration in the heating menstruum was 3.8 and 7.7 mg/ml, respectively. However, when the beef broth protein was dialyzed against buffer before use as a heating menstrum, the D110 was only 39 or 41 min at comparable protein concentrations. Results indicated a dialyzable factor, whose protective effect was partially destroyed by trypsin and chymotrypsin but did not by disodium ethylenediaminetetraacetate, was involved in the protection of enterotoxin B during heating.     

Effect of beef broth protein on the thermal inactivation of staphylococcal enterotoxin B1.

Enterotoxin B produced by Staphylococus aureus 243 in brain heart infusion broth was concentrated by dialysis against 40% polyethylene glycol (20 M), partially purified on a Sephadex G-100 column and heated at 110 degrees C in thermal death time cans. Various heating menstrua included 0.04 M Veronal buffer (pH 7.4), beef broth, and fractions of beef broth obtained by ultrafiltration or precipitation with ammonium sulfate. The toxin was assayed serologically using the microslide gel double-diffusion method. The time requiring for 90% inactivation at 110 degrees C (D110 value) obtained in buffer and in beef broth was 18 and 60 min, respectively. When the concentration of beef broth was increased fivefold, the D110 increased to 78 min. The apparent protective effect or protein was further investigated using beef broth protein obtained by precipitation with (NH4)2SO4. The D110 values were 51 and 70 min when the protein concentration in the heating menstruum was 3.8 and 7.7 mg/ml, respectively. However, when the beef broth protein was dialyzed against buffer before use as a heating menstrum, the D110 was only 39 or 41 min at comparable protein concentrations. Results indicated a dialyzable factor, whose protective effect was partially destroyed by trypsin and chymotrypsin but did not by disodium ethylenediaminetetraacetate, was involved in the protection of enterotoxin B during heating.     

Thermal Inactivation Characteristics of Bacterial Spores at Ultrahigh Temperatures

The thermal inactivation characteristics of Bacillus stearothermophilus (1518) spores and putrefactive anaerobe (PA) 3679 (NCA) spores suspended in skim milk were determined after treatment in pilot-plant ultrahigh-temperature (UHT) processing equipment. Temperature-survivor curves were constructed from survival data to emphasize the critical nature of temperature control in process evaluation. Time-survivor curves for PA 3679 spores were concave upward, and decimal reduction time (DRT) curves for these spores supported the observation of a protective response occurring at the longest exposure times. However, exposure time did not markedly affect the extremely high z(D) value obtained for PA 3679 spores. The substitution of Gelysate for Trypticase and Thiotone as the peptone in the sporulation medium increased the relative heat resistance of B. stearothermophilus spores, but lowered the z(D) value from 16 F to 12 F. The DRT curves in all cases were linear, but the z(D) values observed in this study differed considerably from those reported by other workers.     

Studies on the Xylanase from Streptomyces

Xylanase from Streptomyces xylophagus nov. sp. has been purified by ammonium sulfate fractionation and chromatography on DEAE-cellulose column. The purification of the enzyme was 276-fold with a yield of 18.6% on the basis of the activity per weight of total nitrogen. The purified enzyme was homogeneous on moving-boundary electrophoresis. Optimum pH and temperature for the enzyme activity were 6.2 and 55~60°C, respectively. The enzyme was stable up to 40°C and in the range of pH from 5.3 to 7.3, but inactivated at higher than 50°C and at extreme pH values of 2.4 and 9.4. Hydrolyzed products of xylan by the enzyme were xylose and xylobiose.     

Thermal Inactivation Characteristics of Bacillus subtilis Spores at Ultrahigh Temperatures

The thermal inactivation characteristics of Bacillus subtilis A spores suspended in skim milk with the use of large-scale ultrahigh temperature (UHT) processing equipment were investigated in terms of survival as measured with two plating media. Data on survival immediately after UHT treatments were recorded in temperature-survivor curves, time-survivor curves, and decimal reduction time (DRT) curves. The temperature-survivor curves emphasized that inactivation is accelerated more by increases in the treatment temperature than by increases in the exposure time. Time-survivor curves and DRT curves were not linear. Generally, exceedingly concave time-survivor curves were observed with the standard plating medium; however, only slightly concave curves were observed when CaCl(2) and sodium dipicolinate were added to the medium. For a given UHT sample, larger D values were obtained by use of the medium with the added CaCl(2) and sodium dipicolinate. The DRT curves of all data were concave and appeared to have two discrete slopes (z(D) values). The z(D) values observed in the upper UHT range (above 260 F; 127 C) were twice those observed at lower test temperatures.     

Studies on D-tetrose metabolism. VI. Crystallization and some properties of D-erythrulose reducatase from beef liver.

D-Erythrulose reductase of beef liver was crystallized from ammonium sulfate solution at pH 8.17. The crystals are needle-shaped. The enzyme protein contains 851 amino acid residues per mole of the enzyme: Lys28, His11, Arg52, Asp79, Thr58, Ser56, Glu68, Pro20, Gly80, Ala107, Val112, Met24, Ile31, Leu88, Tyr7, Phe22, Trp4, and Cys4. The enzyme is inactivated by exposure to temperatures below 12degrees. The inactivation is accelerated by increasing the salt concentration and decreasing the enzyme concentration. The pH of the medium also has a pronounced effect, the maximum stability of the enzyme is obtained at pH 8.5. NADP+ protected the enzyme from cold inactivation at all stages of the process and also afforded protection against inactivation by heat and pH. The cold inactivation of the enzyme is accompanied by dissociation of the enzyme protein to subunits.     

Thermal Inactivation of Staphylococcal Enterotoxins B and C

Thermal inactivation profiles of staphylococcal enterotoxins B (SEB) and C (SEC) at 80, 100, and 121 C showed that SEC is more resistant than SEB to heat. After 24 h of incubation at 25 C, some reactivation (recovery of serological reactivity) occurred in toxins that had been inactivated by heat. If the toxin was stirred during heating, reactivation did not occur. An examination of the reactivation kinetics of heat-treated SEC showed that reactivation was temperature dependent. At 25 C, the incubation temperature of heat-treated crude SEC (80 C for 10 min), 100% reactivation occurred after 24 h, whereas at 4 C only slight reactivation was observed. We and others observed that heat-treated toxins initially lost more serological activity when heated at a low temperature (80 C) than at a higher temperature (100 C); in the present study we demonstrate that this is a reversible phenomenon.     

Detection of staphylococcal enterotoxin in foods.

A method has been developed for the detection of staphylococcal enterotoxin A in the boiled rice extract. The procedure utilized was the batch adsorption of enterotoxin from the cell-free culture supernatant by CG-50 ion exchange resin at pH 5.6. The enterotoxin was eluted by various concentrations of elution solution with different pH values. The lyophilized eluate was dissolved in Phosphate Buffer Saline (PBS) solution and analyzed with a quantitative double diffusion method. The desorption of enterotoxin from ion exchange resin appeared to be less effective by increasing the concentration of elution solution than by elevating the pH value of elution solution. The pH below 6.2 seemed to lose the ability to elute the enterotoxin from ion exchanger but enough to elimate non-specific extra proteins. The quantitative double diffusion method was able to detect enterotoxin in food with approximation in quantitation.     

Effect of thermal treatments in oils on bacterial spore survival

The heat resistance of Bacillus cereus F4165/75, Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores suspended in buffer (pH 7.2), olive oil and a commercial oil (a mixture of rapeseed oil and soy oil) was investigated. Linear survivor curves were obtained with B. cereus spores in the three menstrua and with 62A and PA 3679 spores suspended in buffer. However, the inactivation kinetics of the clostridial spores suspended in oils were concave upward with a characteristic tailing-off for 62A spores suspended in olive oil. These deviations from the semi-log model could not be ascribed to a heterogeneity in heat resistance of the spore population or to the variation of aw during heating. Spore resistance to heat increased in the order: buffer much less than commercial oil less than olive oil. The greater heat resistance of oil-suspended spores was ascribed to the low aw (0.479 and 0.492 for commercial oil and olive oil, respectively) and to the composition of the oils. The difference in z values (ca 28 degrees C in oils and 10 degrees-12 degrees C in buffer) suggested that the mechanism of inactivation differs for spores suspended in lipids and in aqueous systems. The thermodynamic data were consistent with this hypothesis.     

Identification of histidine residues at the active site of Megalobatrachus japonicus alkaline phosphatase by chemical modification

Alkaline phosphatase from Megalobatrachus japonicus was inactivated by diethyl pyrocarbonate (DEP). The inactivation followed pseudo-first-order kinetics with a second-order rate constant of 176 M(-1) x min(-1) at pH 6.2 and 25 degrees C. The loss of enzyme activity was accompanied with an increase in absorbance at 242 nm and the inactivated enzyme was re-activated by hydroxylamine, indicating the modification of histidine residues. This conclusion was also confirmed by the pH profiles of inactivation, which showed the involvement of a residue with pK(a) of 6.6. The presence of glycerol 3-phosphate, AMP and phosphate protected the enzyme against inactivation. The results revealed that the histidine residues modified by DEP were located at the active site. Spectrophotometric quantification of modified residues showed that modification of two histidine residues per active site led to complete inactivation, but kinetic stoichiometry indicated that one molecule of modifier reacted with one active site during inactivation, probably suggesting that two essential histidine residues per active site are necessary for complete activity whereas modification of a single histidine residue per active site is enough to result in inactivation.     

Modelling the effect of the redox potential and pH of heating media on Listeria monocytogenes heat resistance

Aims:  Study the effect of redox potential and pH of the heating media on Listeria monocytogenes heat resistance and model its action at fixed temperature.
Methods and Results:  The heat resistance of Listeria monocytogenes at 58°C was studied in Brain Heart Infusion broth as a function of pH (from 5·0 to 7·0) and redox potential ( E _h7). The media redox was adjusted with nitrogen gas, potassium ferricyanide and dithiothreitol. A Weibull model was used to fit survival curves. The heat resistance parameter (δ_58°C) was estimated from each inactivation curve. A major effect of pH was observed. Bigelow model was used to describe the effect of redox potential on the apparent L. monocytogenes heat resistance. The highest δ_58°C values have been obtained at pH 7·0 and oxidizing conditions.
Conclusions:  The developed model indicates that the E _h7 has a significant effect and varied depending on the pH of the heating media. The z _redox values, calculated from δ_58°C allowed quantifying the influence of heating media redox potential on L. monocytogenes thermal inactivation.
Significance and Impact of the Study:  The obtained model shows the action of redox potential on L. monocytogenes thermal destruction and might be useful to take into account in food thermal processes.