| ISO analysis for Bacillus cereus is only suitable to a limited extent for vegetable and salad products with a high level of background flora, as it can produce false positive or false negative results. Furthermore, the method cannot differentiate between Bacillus thuringiensis (Bt) strains derived from biopesticides, such as Bt subsp. kurstaki or aizawai, and pathogenic B. cereus strains that cause emetic or enterotoxic food poisoning. As the EFSA requires this differentiation for biopesticides, a combined microbiological and molecular diagnostic workflow has been developed. This helps primary producers control biopesticide dosage and ensures compliance with legal limits in secondary production.
The workflow begins with microbiological quantification on modified Chromoselect Bacillus agar. Ces-PCR excludes emetic B. cereus strains, cry2-specific PCR identifies Bt biopesticide strains, and RAPD-PCR enables genetic strain matching. Advanced analyses include enterotoxin profiling, ONT sequencing and Illumina MiSeq sequencing. An LC-MS/MS method qualitatively and quantitatively determines Bt crystal proteins with detection limits in the ppb range.
The results demonstrate that only Bt strains are present in primary production, displacing other B. cereus strains. In secondary production, Bt strains rarely exceed the limits; however, the addition of raw materials increases the diversity of non-emetic B. cereus strains, with ST26 being particularly prevalent. Additionally, plant-pathogenic bacteria and hygiene indicators that cause crop losses, reduced shelf life and durability issues were detected.
This approach combines practical applicability with scientific precision, offering primary producers and food safety authorities powerful tools for risk assessment throughout the production process. |