Comparison of Thermal and Pressure-assisted Thermal D-Values of Non-proteolytic Clostridium botulinum Types B and F

Introduction: The impact of high pressure processing (HPP) on the inactivation of nonproteolytic spores of Clostridium botulinum is important for extended shelf life refrigerated low-acid foods.

Purpose: Study and compare the thermal and pressure-assisted thermal resistances of the most resistant nonproteolytic C. botulinum strains as selected from our previous screening study of 17 strains.

Methods: Spores of nonproteolytic C. botulinum strains, Ham-B, Kap 9-B and 610-F were prepared using biphasic media and diluted in ACES buffer (0.05M, pH 7) to 10⁵-10⁶ CFU/ml and placed into a modified sterile transfer pipette, heat-sealed and subjected to a combination of temperatures (80-91°C) and high pressures (600-750 MPa) in a laboratory and pilot-scale high pressure test systems. Another set of diluted spores were placed in NMR tubes which were heat-sealed at both ends, and subjected to various temperatures (80-91°C) in a Fluke 7321 High Precision Bath with Duratherm S as heat transfer fluid.  Survivors were determined by 5-tube MPN method using TPGY broth after incubation for 3 months at ambient temperature.

Results: Both thermal and pressure-assisted thermal D-values (min) of Ham-B, Kap 9-B, and 610-F decreased as the process temperature increased. The highest log reduction of spores (> 5.0) occurred at 91°C and with the high temperature and pressure combination (91°C and 750 MPa). Pressure-assisted thermal D-values were consistently higher for Ham-B, Kap 9-B, and 610-F when processed at any temperature/pressure combination when compared to those processed at any temperature without application of pressure. Thermal D-values at 87°C for Ham-B, Kap 9-B and 610-F were 0.48, 0.53, and 0.37, respectively, compared to those at the 87°C combined with 600 MPa which were higher (i.e., 3.52 for Ham-B, 3.33 for Kap 9-B, and 5.34 for 610-F).

Significance: Overall, pressure-assisted thermal D-values of Ham-B, Kap 9-B, and 610-F were higher compared to their thermal D-values at the same temperature.  It appears that within the range of pressures evaluated, combination of high pressure with temperature has a protective effect on spores of C. botulinum nonproteolytic types B and F strains based on the thermal and pressure-assisted D-values.