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•Released DPA increased with increased treatment time for each process temperature.•Positive correlation between DPA release and log reduction was observed.•Correlation (QC-B, 610-F, and Giorgio-A) after pressure-assisted thermal treatment.•No correlation was found for PA3679 after pressure-assisted thermal treatment.•DPA is found in unprocessed prepared spore crop suspensions in prolonged cold storage.

The microbial safety concern associated with thermally processed extended shelf life (ESL) refrigerated foods is based on adequate elimination of spore-forming pathogens such as nonproteolytic Clostridium botulinumtypes B, E, and F. These pathogens are traditionally regarded as targets for validation of thermally processed ESL foods. However, their use for research is restricted due to their designation as select agents. In this study, the thermal resistances of spores of 10 nonproteolytic C. botulinumtypes B and F and seven psychrotrophic Bacillus cereusstrains were evaluated in ACES (N-(2-acetamido)-2-aminoethanesulfonic acid) buffer (0.05 M, pH 7.00) and compared to determine whether any of the B. cereusstrains could serve as a nonselect agent for establishing thermal processes for ESL refrigerated foods. Thermal decimal reduction times (DT-values) of both nonproteolytic C. botulinumtypes B and F and psychrotrophic B. cereusstrains decreased as process temperature increased from 80 to 91°C, and the highest values were obtained at 80°C. All psychrotrophic B. cereusstrains tested were more thermally resistant than nonproteolytic C. botulinumtypes B and F. DT-values of nonproteolytic C. botulinumtypes B and F decreased to <1.0 min at 87°C, whereas all psychrotrophic B. cereusstrains had higher DT-values (i.e., 52.35 to 133.69 min) at the same temperature. Among all psychrotrophic B. cereusstrains tested, BC-6A16 had the highest DT-values at any given temperature. The DT-values indicated that the psychrotrophic B. cereusstrains were more thermally resistant than the nonproteolytic C. botulinumstrains and therefore may be potential target pathogens for thermal process validation of ESL refrigerated foods. However, further comparative challenge studies are needed with a model food system or an ESL refrigerated food to confirm these results.

The impact of high pressure processing on the inactivation of spores of nonproteolytic Clostridium botulinumis important in extended shelf life chilled low-acid foods. The three most resistant C. botulinumstrains (Ham-B, Kap 9-B, and 610-F) were selected for comparison of their thermal and pressure-assisted thermal resistance after screening 17 nonproteolytic C. botulinumstrains (8 type B, 7 type E, and 2 type F). Spores of strains Ham-B, Kap 9-B, and 610-F were prepared using a biphasic media method, diluted in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer (0.05 M, pH 7.00) to 105to 106CFU/mL, placed into a modified sterile transfer pipette, heat sealed, and subjected to a combination of high pressures (600 to 750 MPa) and high temperatures (80 to 91°C) using laboratory and pilot-scale pressure test systems. Diluted spores from the same crops were placed in nuclear magnetic resonance tubes, which were heat sealed, and subjected to 80 to 91°C in a Fluke 7321 high precision bath with Duratheram S oil as the heat transfer fluid. After incubation for 3 months, survivors in both studies were determined by the five-tube most-probable-number method using Trypticase–peptone–glucose–yeast extract broth. The highest (>5.0) log reductions in spore counts for Ham-B, Kap 9-B, and 610-F occurred at the highest temperature and pressure combination tested (91°C and 750 MPa). Thermal D-values of Ham-B, Kap 9-B, and 610-F decreased as the process temperature increased from 80 to 87°C, decreasing to <1.0 min at 87°C for these strains. Pressure-assisted thermal D-values of Ham-B, Kap 9-B, and 610-F decreased as the process temperature increased from 80 to 91°C with any pressure combination and decreased to <1.0 min as the pressure increased from 600 to 750 MPa at 91°C. Based on the pressure-assisted thermal D-values, pressure exerted a more protective effect on spores of Ham-B, Kap 9-B, and 610-F when processed at 83 to 91°C combined with pressures of 600 to 700 MPa when compared with thermal treatment only. No protective effect was observed when the spores of Ham-B, Kap9-B, and 610-F were treated at lower temperatures (80 to 83°C) in combination with 750 MPa. However, at higher temperatures (87 to 91°C) in combination with 750 MPa, a protective effect was seen for Ham-B, Kap9-B, and 610-F spores based on the calculated pressure-assisted thermal D-values.

The purpose of this study was to determine the inactivation kinetics of the spores of the most resistant proteolytic Clostridium botulinumstrains (Giorgio-A and 69-A, as determined from an earlier screening study) and of Clostridium sporogenesPA3679 and to compare the thermal and pressure-assisted thermal resistance of these spores. Spores of these strains were prepared using a biphasic medium method. C. sporogenesPA3679 spores were heat treated before spore preparation. Using laboratory-scale and pilot-scale pressure test systems, spores of Giorgio-A, 69-A, and PA3679 suspended in ACES [N-(2-acetamido)-2-aminoethanesulfonic acid] buffer (pH 7.0) were exposed to various combinations of temperature (93 to 121°C) and pressure (0.1 to 750 MPa) to determine their resistance. More than a 5-log reduction occurred after 3 min at 113°C for spores of Giorgio-A and 69-A and after 5 min at 117°C for spores of PA3679. A combination of high temperatures (93 to 121°C) and pressures yielded greater log reductions of spores of Giorgio-A, 69-A, and PA3679 compared with reduction obtained with high temperatures alone. No survivors from initial levels (>5.0 log CFU) of Giorgio-A and 69-A were detected when processed at a combination of high temperature (117 and 121°C) and high pressure (600 and 750 MPa) for <1 min in a pilot-scale pressure test system. Increasing pressure from 600 to 750 MPa at 117°C decreased the time from 2.7 to 1 min for a >4.5-log reduction of PA3679 spores. Thermal D-values of Giorgio-A, 69-A, and PA3679 spores decreased (i.e., 29.1 to 0.33 min for Giorgio-A, 40.5 to 0.27 min for 69-A, and 335.2 to 2.16 min for PA3679) as the temperature increased from 97 to 117°C. Pressure-assisted thermal D-values of Giorgio-A, 69-A, and PA3679 also decreased as temperature increased from 97 to 121°C at both pressures (600 and 750 MPa) (i.e., 17.19 to 0.15 min for Giorgio-A, 9.58 to 0.15 min for 69-A, and 12.93 to 0.33 min for PA3679 at 600 MPa). At higher temperatures (117 or 121°C), increasing pressure from 600 to 750 MPa had an effect on pressure-assisted thermal D-values of PA3679 (i.e., at 117°C, pressure-assisted thermal D-value decreased from 0.55 to 0.28 min as pressure increased from 600 to 750 MPa), but pressure had no effect on pressure-assisted thermal D-values of Giorgio-A and 69-A. When compared with Giorgio-A and 69-A, PA3679 had higher thermal and pressure-assisted thermal D-values. C. sporogenesPA3679 spores were generally more resistant to combinations of high pressure and high temperature than were the spores of the C. botulinumstrains tested in this study.

The potential threat of terrorist attacks against the United States food supply using neurotoxin produced by Clostridium botulinum(BoNT) has resulted in the need for studying the effect of various food process operations on the bioavailability of this toxin. The objective of this study was to evaluate C. botulinumtype A neurotoxin bioavailability after a simulated hot fill juice bottling operation. C. botulinumtype A acid mud toxin [106mouse lethal dose [MLD50]/ml] was deposited into juice bottles at an experimentally determined fastest cooling spot. Bottles [12 or 20 oz [355 and 592 ml]) were filled with either apple juice or an orange drink, at 80 or 85°C, in either upright or inverted orientations. Toxicity of the juice was evaluated as a function of holding time (1 to 2 min] by the mouse bioassay. The fastest cooling point in the upright orientation was determined to be at a bottle’s bottom rim. In the inverted orientation, the fastest cooling point was in the bottle cap region. With respect to these two points, the upright bottle cooled faster than the inverted bottle, which was reflected in a higher inactivation of BoNT in the latter. For the orange drink (pH 2.9) toxicity was reduced by 0.5 × 106MLD50/ml to a nondetectable level after 1 min in all bottle sizes, orientations, and temperatures as measured by the mouse bioassay. This indicates that there was at least a 0.5 × 106MLD50/ml reduction in activity. Inactivation in apple juice (pH 4.0), to the same degree as in the orange drink, was found only for the inverted orientation at 85°C. Complete inactivation in apple juice for all conditions was found at a lower added toxin level of 0.25 × 105MLD50/ml. In general, bottle inversion and filling at 85°C provided complete inactivation of BoNT to the 0.5 × 106MLD50/ml level. All experiments resulted in the inactivation of 2.5 × 104MLD50/ml of BoNT regardless of juice type, fill temperature, or bottle orientation and size.

The purpose of this study was to determine the effect of sporulation temperature on the resistance of Clostridium botulinumtype A spores of strains 62A and GiorgioA to thermal and high pressure processing (HPP). Spore crops produced in Trypticase-peptone-glucose-yeast extract broth at four incubation temperatures (20, 27, 37, and 41°C) were harvested, and heat resistance studies were conducted at 105°C (strain 62A) and 100°C (strain GiorgioA). Resistance to HPP was evaluated by subjecting the spores to a high pressure (700 MPa) and temperature combination (105°C, strain 62A; 100°C strain GiorgioA) in a laboratory-scale pressure test system. The decimal reduction time (D-value) was calculated using the log-linear model. Although the time to sporulation for GiorgioA was shorter and resulted in higher spore concentrations than for 62A at 20, 27, and 37°C, GiorgioA did not produce a sufficient spore crop at 41 °C to be evaluated. The heat resistance of 62A spores was greatest when produced at 27°C and decreased for spore crops produced above or below 27°C (D105°C-values: 20°C, 1.9 min; 27°C, 4.03 min; 37°C, 3.66 min; and 41°C, 3.5 min; P <0.05). Unlike 62A, the heat resistance behavior of GiorgioA spores increased with rising sporulation temperature, and spores formed at the organism's optimum growth temperature of 37°C were the most resistant (D100°C-values: 20°C, 3.4 min; 27°C, 5.08 min; and 37°C, 5.65 min; P <0.05). Overall, all spore crops were less resistant to pressure-assisted thermal processing than thermal treatment alone. Sporulation temperature has an effect on the resistance of C. botulinumspores to heat and HPP, and is characteristic to a particular strain. Knowledge of the effect of sporulation temperature on the resistance of C. botulinumspores is vital for the production of spores utilized in thermal and high pressure inactivation studies.

Several studies have been published on the inactivation of bacterial spores by using high pressure processing in combination with heat. None of the studies investigated the effect of the packaging system or the pressurizing fluid on spore inactivation. The objective of this study was to select and validate an appropriate packaging system and pressure transfer fluid for inactivation of Clostridium botulinumspores by using high pressure processing in combination with thermal processing. Inactivation of spores packaged in three packaging systems (plastic pouches, cryovials, and transfer pipettes) was measured in two pressure test systems (laboratory-scale and pilot-scale) at 700 MPa and >105°C. Total destruction (>6.6-log reduction) of the spores packaged in the graduated tube part of transfer pipettes was obtained after processing for up to 10 min at 118°C and 700 MPa in both pressure test systems, compared with the spores packaged either in plastic pouches or cryovials. Reduction of spores packaged in plastic pouches was lowest (<4.8 log) for both pressure test systems when processed at the same conditions (i.e., 700 MPa and 118°C). Within the pilot-scale pressure system, increasing the process temperature from 118 to 121°C at 700 MPa for 10 min resulted in only a small increase in spore reduction (<5.1 log) for spores packaged in plastic pouches, whereas there were no recoverable spores for either of the other two packaging systems. Use of plastic pouches for packaging spores in inactivation kinetic studies could lead to erroneous conclusions about the effect of high pressure in combination with heat. BioGlycol is the pressure–heat transfer fluid of choice, as compared with Duratherm oil, to maximize the temperature response rate during pressurization within the laboratory-scale pressure test system.

The ability of automated ribotyping to differentiate between major types and individual strains of Clostridium botulinumwas tested using the Qualicon Riboprinter Microbial Characterization System. Pure spores of C. botulinumtype A, proteolytic type B, nonproteolytic type B, and type E strains were inoculated onto modified anaerobic egg yolk agar and incubated 24 h at 35°C. Plates were rinsed with buffer (2mM Tris + 20mM EDTA) to remove vegetative cells that were heated for 10 min at 80°C, treated with a lysing agent, and ribotyped in the Qualicon Riboprinter utilizing the enzyme EcoRI. Riboprint patterns were obtained for 30 strains of the four major types of C. botulinummost commonly involved in human foodborne botulism. Proteolytic strains yielded the best and most consistent results. Fifteen ribogroups were identified among the 31 strains tested. Interestingly, in two cases, a single ribogroup contained patterns from isolates belonging to evolutionarily distinct Clostridiumlineages. This degree of differentiation between strains of C. botulinummay be useful in hazard analysis and identification, hazard analysis and critical control point monitoring and validation, environmental monitoring, and in inoculation studies.