Short communicationInability of non-proteolytic Clostridium botulinum to grow in mussels inoculated via immersion and packaged in high oxygen atmospheres
Introduction
In 2010, aquaculture production of mussels worldwide approached 2 million metric tons (FAO, 2012), and hermetically sealed, enhanced-oxygen modified atmosphere packaging (MAP) is becoming the preferred method of packing mussels for better quality and extended shelf life (Bernardez and Pastoriza, 2011). While millions of live mussel MA-packages are sold in Europe each year (e.g., greater than 50 million kg/year in the Netherlands, J. Bol, pers. commun.), the package is prohibited in the U.S. due to the potential risk of botulism, which is a serious illness that can be fatal. Foodborne botulism has occurred in a few instances in cooked and home-prepared mussels (Lecour et al., 1988, Hauschild, 1993), and avian type E botulism has been associated with the consumption of fresh water mussels from anoxic sediments by birds (Getchell and Bowser, 2006). Botulism spores have been detected in clams and oysters from the Oregon coast (Craig et al., 1968), but no cases of human botulism have been observed from the consumption of packaged live mussels (not previously cooked and preserved). However, with the exception of studies by Newell et al. (2012), no previous inoculated-pack studies have been reported to determine if Clostridium botulinum growth and toxin production can occur in MA fresh live mussel packages under temperature abuse conditions. Such studies are needed to determine if raw (live) mussels packaged under MAP conditions are a potential risk for botulism (United States Food and Drug Administration, 2011). The main objective of this study was to determine if MA-packaged live mussels were prone to the development of botulism as a seafood hazard. In a previous study, inoculated pack studies were performed with live mussels that received uniform inocula of sufficient concentrations of non-proteolytic C. botulinum spores throughout the packages (Newell et al., 2012). However, in those studies, spores were introduced externally into the mussel package via a buffer solution. In these studies with the objective of simulating natural contamination of C. botulinum spores in mussels (Mytilus edulis) to ensure consistent uptake of C. botulinum spores in the digestive tracts, feeding studies were performed using cultured algae and a spore suspension in seawater.
Since no toxin was produced by any live mussels (even the controls) in the previous studies using live mussels (Newell et al., 2012), experiments were performed not only using an enhanced oxygen modified atmosphere, but also under a vacuum using cooked mussel meat and tuna as positive controls. Tuna was included in this study to serve as a control as it is prone to production of botulinum toxin by C. botulinum when the conditions are permissive (Lynt et al., 1975). Botulism challenge studies were performed at normal storage (4 °C) and abuse (12 °C) temperatures, with the objective of determining if botulism is a seafood risk in live mussels in an enhanced oxygen MAP package.
Section snippets
Uptake of C. botulinum spores by mussels using an immersion method
A mixture of 6 non-proteolytic C. botulinum strains was used: Beluga (type E), Minnesota (type E), 70 E (type E), 2 B (type B), 17 B (type B), and F202 (type F). A spore crop for each individual strain was prepared according to the method described by Peck et al., 1992. Approximately equal numbers of spores of each strain were mixed to prepare a master stock of 108 spores/ml and stored at −20 °C in sterile distilled water. To prepare the inocula, a master stock was diluted in sterile distilled
Uptake of spores
The spore uptake results revealed that a longer submersion time and higher concentration of C. botulinum spores (103 spores/ml) in the seawater solution with algae (104 cells/ml) resulted in a higher number of C. botulinum spores ingested by the mussels, either in the tissue (500/g) or in the GI tract (111/g) (Table 1). Blue mussels ingest bacteria (Birkbeck and McHenery, 1982), especially those species which have cell walls that are sensitive to bacteriolytic lysozymes. Martínez et al. (2009)
Conclusion
Based on the limited number of samples in this study, and previous studies (Newell et al., 2012), live mussels packed in MA-packages appear to be safe from the production of botulinum toxin, even under abusive storage temperature (12 °C) for at least 12 days which is beyond their shelf life. Additional studies are needed to isolate presumptive antimicrobial agents from live mussels or mussel extrapallial fluid.
Acknowledgments
We thank the Maine Technology Institute for a Seed Grant to Great Eastern Mussel Farms to investigate MAP, and Frank Simon, Chip Davison, Dave Preston and Peter Fischer from Great Eastern Mussel Farms. This material is based upon work supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under Agreement No. MEK 2004-02655. Manuscript preparation was supported by Canadian Cove Mussel Farms. We would also like to thank Mary Lositkoff and staff from the
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