Effect of processing variables on the outgrowth of Clostridium sporogenes PA 3679 spores in comminuted meat cured with sorbic acid and sodium nitrite.

The effects of the initial pH and a "short pump" on the outgrowth of Clostridium sporogenes PA 3679 spores in comminuted cured pork were studied. Fresh ground pork was cured with salt, sugar, phosphate, ascorbate, and varying amounts of sodium nitrite and sorbic acid. The product was comminuted and inoculated with 1,000 spores of C. sporogenes per g. The meat was stuffed into 1-ounce (ca. 28.4-g) aluminum tubes, cooked to 58.5 degrees C, cooled, and incubated at 27 degrees C to observe for swells. Product cured with 0.2% sorbic acid in combination with 40 ppm sodium nitrite (40 microgram/g) had better clostridium inhibition than did product cured with 120 ppm nitrite within a pH range of 5.0 to 6.7. The sorbic acid-40 ppm nitrite combination also gave better clostridial protection than did the 120 ppm nitrite alone when reduced amounts of curing ingredients were present.     

Effect of processing variables on the outgrowth of Clostridium sporogenes PA 3679 spores in comminuted meat cured with sorbic acid and sodium nitrite.

The effects of the initial pH and a "short pump" on the outgrowth of Clostridium sporogenes PA 3679 spores in comminuted cured pork were studied. Fresh ground pork was cured with salt, sugar, phosphate, ascorbate, and varying amounts of sodium nitrite and sorbic acid. The product was comminuted and inoculated with 1,000 spores of C. sporogenes per g. The meat was stuffed into 1-ounce (ca. 28.4-g) aluminum tubes, cooked to 58.5 degrees C, cooled, and incubated at 27 degrees C to observe for swells. Product cured with 0.2% sorbic acid in combination with 40 ppm sodium nitrite (40 microgram/g) had better clostridium inhibition than did product cured with 120 ppm nitrite within a pH range of 5.0 to 6.7. The sorbic acid-40 ppm nitrite combination also gave better clostridial protection than did the 120 ppm nitrite alone when reduced amounts of curing ingredients were present.     

Monoclonal antibodies for use in detection of Bacillus and Clostridium spores.

Five monoclonal antibodies against bacterial spores of Bacillus cereus T and Clostridium sporogenes PA3679 were developed. Two antibodies (B48 and B183) were selected for their reactivity with B. cereus T spores, two (C33 and C225) were selected for their reactivity with C. sporogenes spores, and one (D89) was selected for its reactivity with both B. cereus and C sporogenes spores. The isotypes of the antibodies were determined to be immunoglobulin G2a (IgG2a) (B48), IgG1 (B183), and IgM (C33, C225, and D89). The antibodies reacted with spores of B. cereus T, Bacillus subtilis subsp. globigii, Bacillus megaterium, Bacillus stearothermophilus, C. sporogenes, Clostridium perfringens, and Desulfotomaculum nigrificans. Antibody D89 also reacted with vegetative cells of B. cereus and C. sporogenes. Analysis of B. cereus spore extracts showed that two of the antigens with which the anti-Bacillus antibodies reacted had molecular masses of 76 kDa and approximately 250 kDa. Immunocytochemical localization indicated that antigens with which B48, B183, and D89 react are on the exosporium of the B. cereus T spore. Antibody D89 reacted with the exosporium and outer cortex of C. sporogenes spores in immunocytochemical localization studies but did not react with extracts of C. sporogenes or B. cereus spores in Western blotting. Some C. sporogenes antigens were not stable during long-term storage at -20 degrees C. Antibodies B48, B183, and D89 should prove to be useful tools for developing immunological methods for the detection of bacterial spores.     

The effect of transition metal ions on the resistance of bacterial spores to hydrogen peroxide and to heat.

The presence of 10 microM-Cu2+ increased the lethal effect of hydrogen peroxide on spores of Clostridium bifermentans but not on those of Clostridium sporogenes PA 3679, Clostridium perfringens, Bacillus cereus or Bacillus subtilis var. niger. Cu2+ at 100 muM also increased the lethal effect of heat on spores of C. bifermentans but not on those of B. sutilis var. niger. The rate and extent of Cu2+ uptake by spores of C. bifermentans and B. subtilis var. niger were similar, but examination of unstained sections of spores by electron microscopy suggested that Cu2+ is bound by the protoplasts of spores of C. bifermentans but not of B. subtilis var. niger.     

The Effect of Sodium Chloride on Radiation Resistance and Recovery of Irradiated Anaerobic Spores

Sodium chloride (3% and 6% w/v) were without effect on the radiation resistance of spores of Clostridium sporogenes and Cl. oedematiens type C. Increasing doses of gamma radiation rendered spores increasingly sensitive to post radiation inhibition by sodium chloride in the plating medium.     

Nisin: a possible alternative or adjunct to nitrite in the preservation of meats.

Nisin at 75 ppm (75 microgram/g) was superior to 150 ppm of nitrite in inhibiting outgrowth of Clostridium sporogenes PA3679 spores in meat slurries, which had been heated to simulate the process used for cooked ham. The inhibitory activity of nisin decreased as the spore load or pH of the slurries increased. Unlike nitrite, inhibition by nisin was unaffected by high levels of iron either as a constituent of meats or when added as an iron salt. In slurries treated with 75 ppm of nisin, refrigerated storage for 56 days resulted in depletion of nisin to a level low enough to allow outgrowth within 3 to 10 days if the slurries were subsequently abused at 35 degrees C. In contrast, a combination of 40 ppm of nitrite and either 75 or 100 ppm of nisin almost completely inhibited outgrowth in these slurries. The nisin-nitrite combination appeared to have a synergistic effect, and the low concentration of nitrite was sufficient to preserve the color in meats similar to that of products cured with 150 ppm of nitrite.     

The Influence of High Concentrations of Carbon Dioxide on the Germination of Bacterial Spores

The influence of carbon dioxide at 1–55 atm on the germination of Clostridium sporogenes, Clostridium perfringens and Bacillus cereus spores in a complex medium was studied. The germination studies at atmospheric pressure were done in the pH range 5.2–6.7. Controls at the same pH were done in 100% nitrogen. Carbon dioxide at atmospheric pressure (1 atm) inhibited the spore germination of B. cereus spores but strongly enhanced the germination rate of those of the clostridia. Spore germination of Cl. sporogenes and Cl. perfringens was inhibited completely at 10 atm and at 25 atm, respectively. The germination rate in carbon dioxide or nitrogen was generally higher at pH 6.7 than at 5.2–6.0.     

Effect of pH on the Heat Resistance of Clostridium sporogenes Spores in Minced Meat

S ummary : The effect of the pH value of minced meat on the heat resistance of Clostridium sporogenes spores inoculated in it was examined. A drop in pH from 6·0 to 4·8 decreased the decimal reduction time by 40%, irrespective of the heating temperature. In other words, the z value is independent of pH.     

Synergism between Ultrasonic Waves and Hydrogen Peroxide in the Killing of Micro-organisms

The lethal effect on different micro-organisms of ultrasonic waves and hydrogen peroxide separately and in combination was examined. Ultrasonic waves were able to disintegrate Fusobacterium nucleatum within 3 min and to kill Veillonella parvula after 15 min and Streptoccus sanguis after 20 min; 20 vols H₂O₂ (6% w/v) killed V. parvula, Strep. sanguis and Staphylococcus aureus after 5 min treatment, and Clostridium sporogenes spores after 25 min. Sonication of Cl. sporogenes spores, Bacillus cereus spores and Candida albicans in 20 vols H₂O₂, using an ultrasonic probe, was lethal to the organisms after 15, 10 and 10 min, respectively. The latter 2 organisms were not killed by 30 min exposure to either agent separately. Similar results were obtained when an ultrasonic tank was used for sonication.     

Fluorescent Studies in the Genus Clostridium. I. The location of antigens on the surface of Clostridium sporogenes during sporulation and germination

S ummary : Fluorescent labelled specific antisera against spores and vegetative cells have been used as stains to follow the antigenic changes that occur on the surface of Clostridium sporogenes during sporulation and germination.     

Association of vegetative antigens with the spores of Clostridium sporogenes

Clean spores of Clostridium sporogenes stored for two years continued to elicit the production of specific antibodies in immunized rabbits. Disintegrated spores stimulated not only antibodies specific for the spore component, but also those directed against 'H' and 'O' antigens of the sporangium cells.     

System for evaluating clostridial inhibition in cured meat products.

A method for evaluating inhibition of Clostridium botulinum, C. sporogenes, and C. perfringens in cured meat products was developed. This system can easily be used in the microbiology laboratory using aluminum ointment tubes as the product container. Swells caused by gas production by the organism are easily observed by using the aluminum tubes. Results obtained confirmed earlier work on the inhibitory effect of sodium nitrite and sorbic acid against the clostridia in cured meat products.     

System for evaluating clostridial inhibition in cured meat products.

A method for evaluating inhibition of Clostridium botulinum, C. sporogenes, and C. perfringens in cured meat products was developed. This system can easily be used in the microbiology laboratory using aluminum ointment tubes as the product container. Swells caused by gas production by the organism are easily observed by using the aluminum tubes. Results obtained confirmed earlier work on the inhibitory effect of sodium nitrite and sorbic acid against the clostridia in cured meat products.     

Effects of inoculum level and pressure pulse on the inactivation of Clostridium sporogenes spores by pressure-assisted thermal processing

The effects of initial concentration and pulsed pressurization on the inactivation of Clostridium sporogenes spores suspended in deionized water were determined during thermal processing (TP; 105 degrees C, 0.1 MPa) and pressure-assisted thermal processing (PATP; 105 degrees C and 700 MPa) treatments for 40 min and 5 min holding times, respectively. Different inoculum levels (10(4), 10(6), and 10(8) CFU/ml) of C. sporogenes spores suspended in deionized water were treated at 105 degrees C under 700 MPa with single, double, and triple pulses. Thermally treated samples served as control. No statistical significances (p > 0.05) were observed among all different inoculum levels during the thermal treatment, whereas the inactivation rates (k1 and k2) were decreased with increasing the initial concentrations of C. sporogenes spores during the PATP treatments. Double- and triple-pulsed pressurization reduced more effectively the number of C. sporogenes spores than single-pulse pressurization. The study shows that the spore clumps formed during the PATP may lead to an increase in pressure-thermal resistance, and multiple-pulsed pressurization can be more effective in inactivating bacterial spores. The results provide an interesting insight on the spore inactivation mechanisms with regard to inoculum level and pulsed pressurization.     

The Effect of Sodium Chloride on Heat Resistance and Recovery of Heated Spores of Clostridium sporogenes (PA 3679/S2)

S ummary . In 3% and 6% (w/v) sodium chloride solutions the heat resistances of spores of Clostridium sporogenes were the same as in water. Heating to give a 0.1% survival rendered the surviving spores more sensitive to inhibition by sodium chloride in the recovery medium.     

An evaluation of the sporicidal activity of ozone.

This study was undertaken to determine the feasibility of sterilizing surfaces with ozone-saturated water by the methods of the Association of Official Analytical Chemists (AOAC). Initially, it was determined that there was no apparent difference in ozone resistance between spores of Bacillus subtilis and Clostridium sporogenes when they are suspended in water. Both species were inactivated by a 10-min exposure at ambient temperature. Resistance was increased when the spores were dried on AOAC carriers. Viable organisms were recovered after an exposure of 40 min at ambient temperature. An increase in the reactor water temperature to 60 degrees C did not improve the effectiveness of the ozone in sterilizing AOAC carriers. Dried spores of C. sporogenes were more resistant than B. subtilis spores because of a greater accumulation of organic matter on the carriers. No significant sporicidal activity was demonstrated after 40 min for spores of either species when they were inoculated on silk suture loops. The data suggest that organic loading and poor ozone penetrability are key factors in effecting the ability of ozone to sterilize surfaces rapidly.     

An evaluation of the sporicidal activity of ozone

This study was undertaken to determine the feasibility of sterilizing surfaces with ozone-saturated water by the methods of the Association of Official Analytical Chemists (AOAC). Initially, it was determined that there was no apparent difference in ozone resistance between spores of Bacillus subtilis and Clostridium sporogenes when they are suspended in water. Both species were inactivated by a 10-min exposure at ambient temperature. Resistance was increased when the spores were dried on AOAC carriers. Viable organisms were recovered after an exposure of 40 min at ambient temperature. An increase in the reactor water temperature to 60 degrees C did not improve the effectiveness of the ozone in sterilizing AOAC carriers. Dried spores of C. sporogenes were more resistant than B. subtilis spores because of a greater accumulation of organic matter on the carriers. No significant sporicidal activity was demonstrated after 40 min for spores of either species when they were inoculated on silk suture loops. The data suggest that organic loading and poor ozone penetrability are key factors in effecting the ability of ozone to sterilize surfaces rapidly.     

Effect of Temperature on Germination of Spores from Some Bacillus and Clostridium Species

The effect of temperature on germination of spores of Bacillus subyilis, B. megaterium. B. cereus, Clostridium sporogenes, Cl. butyricum and Cl. bifermentans was studied. At lower temperatures (+5°C to +10°C) the three Glostridium species germinated to a less extent than the three Bacillus. species. The optimum temperature for germination of the six species varied between +35°C and +45°C. The Clostridium species were more tolerant to heat than the Bacillus species.     

Identification of Possible Artifacts in the Association of Official Analytical Chemists Sporicidal Test

Two laboratories tested four different brands of alkaline 2% glutaraldehyde sterilants by the Association of Official Analytical Chemists sporicidal test. Each laboratory found survival of Clostridium sporogenes spores on spore-labeled unglazed porcelain penicylinders (cylinders) to vary from test to test, and survival did not always correlate with increasing sterilant exposure time. These results were consistent with a theory that there may be random conditions within the test that prevent the sterilant from contacting all spores. Further studies indicated that the prior history of the unglazed porcelain cylinders and whether the C. sporogenes culture grown in egg-meat media had been processed (homogenized) to eliminate visible pieces of egg-meat media were important factors affecting the results and repeatability of this test.     

The bacteriocidal effects of transition metal complexes containing the NO+ group on the food-spoilage bacterium Clostridium sporogenes

The chemical and molecular mechanism of toxicity of nitrite towards food-spoilage bacteria such as Clostridium botulinum or Clostridium sporogenes is not well understood. In order to discover the active species and explore its chemistry, a number of compounds related to nitrite were synthesized. Their bacteriocidal effects on C. sporogenes were investigated in Oxoid nutrient broth No. 2 growth medium at pH 7.0. Inhibition of cell growth, expressed as the concentration which causes 50% cell inhibition, was observed with nitrite at 10 mM, whereas [Fe4S3(NO)7]-(the anion of Roussin's black salt) and (Fe2(SCH2CH2OH)2(NO)4] (a water-soluble Roussin's red salt ester) were found to be effective at 0.001 mM and 0.005 mM, respectively, confirming previous reports that iron-sulphur-nitrosyl complexes are much more toxic to these organisms than nitrite itself. The nitroprusside anion, [Fe(CN)5NO]2- was found to be toxic at 0.030 mM and the corresponding chromium species, [Cr(CN)5NO]3-, at 0.1 mM. Therefore, on the basis of the number of NO groups present, the nitrosylcyano complexes are comparable in activity with the iron-sulphur-nitrosyl compounds. These results show that neither iron nor sulphur are essential for the bacteriostatic effect of the Roussin's type compounds. The property that all these compounds have in common is that they contain NO+. It is proposed that this is the active species responsible for the preservative effect of nitrite, and that a relationship may exist between the N-O stretching frequency, a measure of the NO+ character, and the toxicity of these NO(+)-containing complexes.