Inability of non-proteolytic Clostridium botulinum to grow in mussels inoculated via immersion and packaged in high oxygen atmospheres

Highlights

• Clostridium botulinum was introduced into mussels by immersion in seawater with algae.

• No toxin was detected in inoculated and spoiled mussel packages devoided of oxygen.

• Botulinum toxin was produced in cooked mussel and tuna controls.

• The studies indicate that live MAP mussels should not be a botulism hazard.

Abstract

A series of botulism challenge studies were conducted to determine if botulinum toxin would be produced in mussels (Mytilus edulis) inoculated with non-proteolytic Clostridium botulinum spores and held under modified atmosphere (MA) packaging conditions at normal (4 °C) and abusive (12 °C) temperatures. Spore mixtures of six strains of non-proteolytic C. botulinum were introduced into live mussels through immersion in a seawater solution with cultured algae. Mussels were packed in a commercial high-oxygen (60–65% O₂) MA-package with a buffer, and also packed under a vacuum. Feeding live mussels cultured algae (10⁴ cells/ml) with a C. botulinum spore suspension (10³ spores/ml) in seawater at 4 °C for 6 h resulted in the uptake of spores into mussel tissue (500/g) and the mussel GI tract (100/g). Under all of the experimental conditions evaluated, none of the fresh mussels became toxic, even after spoilage and in the absence of oxygen. However, control samples using tuna or cooked mussel meats became toxic in the absence of oxygen. Botulinum toxin was not produced in fresh mussels packaged under the MA-packaging conditions evaluated, even at an abusive storage temperature (12 °C) for at least 12 days or at normal storage temperate (4 °C) for at least 21 days, which is beyond their shelf life.

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.