Effect of Equilibrated pH and Indigenous Spoilage Microorganisms on the Inhibition of Proteolytic Clostridium botulinum Toxin Production in Experimental Meals under Temperature Abuse

Clostridium botulinum is a foreseeable biological hazard in prepared refrigerated meals that needs to be addressed in food safety plans. The objective of this study was to evaluate the effect of product composition and storage temperature on the inhibition of botulinum toxin formation in nine experimental meals (meat, vegetable, or carbohydrate based). Treatments were inoculated with proteolytic C. botulinum, vacuum packaged, cooked at 90°C for 10 min, and assayed for botulinum toxin in samples stored at 25°C for up to 96 h for phase 1, or at 25°C for 12 h and then transferred to 12.5°C for up to 12 and 6 weeks in phases 1 and 2, respectively. For phase 1, none of the treatments (equilibrated pH 5.8) supported toxin production when stored at 25°C for 48 h, but toxin production was observed in all treatments at 72 h. For the remaining experiments with storage at 12.5°C, toxin production was dependent on equilibrated pH, storage time, and growth of indigenous spoilage microorganisms. In phase 1, no gross spoilage and no botulinum toxin was detected for any treatment (pH ≤5.8) stored at 12.5°C for 12 weeks. In phase 2, gross spoilage varied by commodity, with the brussels sprouts meal with pH 6.5 showing the most rapid spoilage within 2 weeks and botulinum toxin detected at 5 and 6 weeks for the control and cultured celery juice treatments, respectively. In contrast, spoilage microbes decreased the pH of a pH 5.9 beef treatment by 1.0 unit, potentially inhibiting C. botulinum through 6 weeks at 12.5°C. None of the other treatments with pH 5.8 or below supported toxin production or spoilage. This study provides validation for preventive controls in refrigerated meals. These include equilibrated product pH and storage temperature and time to inhibit toxin formation by proteolytic C. botulinum, but the impact of indigenous microflora on safety and interpretation of challenge studies is also highlighted.