Atmospheric oxygen and other conditions affecting the production of cereulide by Bacillus cereus in food

Factors influencing the production of cereulide, the emetic toxin of Bacillus cereus in food and laboratory media were investigated, using liquid chromatography–ion trap mass spectrometry and sperm motility inhibition bioassay for detection and quantitation. Oxygen was essential for production of the emetic toxin by B. cereus. When beans, rice or tryptic soy broth were inoculated with cereulide producing strains B203, B116 (recent food isolates) or the strain F-4810/72, high amounts (2 to 7 μg ml−1 or g−1 wet wt) of cereulide accumulated during 4-day storage at room temperature. In parallel cultures and foods, stored under nitrogen atmosphere (>99.5% N2), less than 0.05 μg of cereulide ml−1 or g−1 wet wt accumulated. The outcome of the bioassay matched that of the chemical assay, with no indication of interference by substances in the rice or beans. Boiling for 20 to 30 min did not inactivate cereulide or cereulide producing strains in rice or the beans. Adding l-leucine and l-valine (0.3 g l−1) stimulated cereulide production 10- to 20-fold in R2A and in rice water agar. When the B. cereus strains were grown on agar media under permissive conditions (air, room temperature), cereulide was produced overnight with little or no increase when the incubation was extended to 4 days. In broth culture, the production of cereulide started later than 16–24 h. Anoxic storage prevented cereulide production also when the amino acids had been supplied. Packaging with modified atmosphere low in oxygen may thus be used to reduce the risk of cereulide formation during storage of food.