For 79% of strains, crude supernatant fluids, not concentrated by ammonium sulphate precipitation, were cytotoxic on Caco cells. Once diluted twofold, 30% of the supernatant fluids were cytotoxic. Supernatant fluids of two strains were still active after an eightfold dilution. Supernatant fluids heated for 40 min at 58 °C lost cytotoxicity. Most strains able to grow at 5 °C and 10 °C within 3 days, and unable to grow at 42 °C, were not cytotoxic to Caco cells ( Table 5). Strains isolated from pasteurized purées were more cytotoxic than strains isolated from cooked chilled vegetables; 44% of the strains from purées were cytotoxic even when their supernatant fluid was diluted (10% of the strains from cooked chilled vegetables), and all the strains active with supernatant fluid diluted fourfold and eightfold were from the pasteurized purées stored at room temperature (data not shown). Out of the 15 non-cytotoxic strains, 10 were positive by both TECRA-BDE and BCET-RPLA tests, and three were positive by one test. Therefore, the majority of these negative strains were nevertheless producing enterotoxins. When supernatant fluids were concentrated 10-fold by ammonium sulphate precipitation, all the negative strains became cytotoxic, confirming that enterotoxins were produced, but at a low level.
Table 5. Cytotoxicity of supernatant fluids of Bacillus cereus isolates in relation to their growth temperatures