Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/25/2010
Publication Date: 11/5/2010
Citation: Rasooly, R., Do, P.M. 2010. Clostridium botulinum neurotoxin type B is heat-stable in milk and not inactivated by pasteurization. Journal of Agricultural and Food Chemistry. 58:12557-12561. Interpretive Summary: Botulinum neurotoxins (BoNT), produced by some bacteria, are a cause of food poisoning. Because these toxins are extremely poisonous, they might be used for intentional contamination of food in an incident of terrorism. This paper describes simultaneously the detection and distinguishing of BoNT serotypes A and B in milk, using an improved method. This method is based on two unique internally quenched fluorogenic peptides that are converted by the toxins into a highly fluorescent dye. Since each peptide is labeled with different fluorophores we were able to detect and distinguish BoNT serotypes A and B simultaneously in one reaction and sample. The improved method is sensitive enough to detect very small amounts of BoNT that are dangerous to humans. Because it is relatively fast and inexpensive, this method could be used for large scale screening of BoNT in food. It could also replace the widely-used live mouse test for BoNT.
Technical Abstract: Foodborne botulism is caused by the ingestion of foods containing botulinum neurotoxins (BoNTs). Currently, the only accepted assay to detect active C. botulinum neurotoxin is an in vivo mouse bioassay, which raises ethical concerns with regard to the use of experimental animals. Therefore, there is a need to develop fast and effective methods to detect active BoNTs. This study reports the successful use of an enzymatic activity assay employing internally quenched fluorogenic peptides corresponding to SNAP-25, for BoNT-A, and VAMP2, for BoNT-B, as an alternative method to the mouse bioassay. Since each peptide is labeled with different flurophores we were able to detect and distinguish BoNT serotypes A and B simultaneously in one reaction and sample. This study also reports the commonly used food processes such as acidity and pasteurization, which are known to inhibit C. botulinum growth and toxin production, are more effective in inactivating BoNT serotype A. We also report that milk protects BoNT-B activity from pasteurization.