Effects of spore purity on the wet heat resistance of Clostridium perfringens, Bacillus cereus and Bacillus subtilis spores

Heat resistance of spores of Clostridium perfringens 8238 (Hobbs Serotype 2), Bacillus cereus NCTC 11143 (4810/72), and Bacillus subtilis PS533, an isogenic derivative of strain PS832 (a 168 strain) was determined in ground beef at 95 °C. Spore purification was by centrifugation and washing with sterile distilled water (dH2O), followed by sonication and then Histodenz centrifugation for B. subtilis and C. perfringens, and centrifugation and washing with sterile dH2O followed by Histodenz centrifugation for B. cereus. Bags containing inoculated beef samples were submerged in a temperature-controlled water bath and held at 95 °C for predetermined lengths of time. Surviving spore populations were enumerated by plating on mannitol egg yolk polymyxin agar (MYP) agar plates for B. cereus and B. subtilis, and on tryptose-sulfite-cycloserine agar (TSC) agar plates for C. perfringens. Survivor curves were fitted to linear, linear with tail, and Weibull models using the USDA Integrated Pathogen Modeling Program (IPMP) 2013 software. The Weibull model provided a relatively better fit to the data since the root mean square error (RMSE), mean square error (MSE), sum of squared errors (SSE), and Akaike information criterion (AIC) values were lower than the values obtained using the linear or the linear with tail models. Additionally, the Weibull model accurately predicted the observed D-values at 95 °C for the three spore-formers since the accuracy factor (A ) values ranged from 1.03 to 1.08 and the bias factor (B ) values were either 1.00 or 1.01. Times at 95 °C to achieve a 3-log reduction decreased from 206 min for C. perfringens spores purified with water washes alone to 191 min with water washes followed by sonication and Histodenz centrifugation, from 7.9 min for B. cereus spores purified with water washes alone to 1.4 min with water washes followed by Histodenz centrifugation, and from 20.6 min for B. subtilis spores purified with water washes alone to 6.7 min for water washes followed by sonication and Histodenz centrifugation. Thermal-death-time values reported in this study will assist food processors to design thermal processes to guard against bacterial spores in cooked foods. In addition, clearly spore purity is an additional factor in spore wet heat resistance, although the cause of this effect is not clear.