Effect of lysozyme concentration, heating at 90 degrees C, and then incubation at chilled temperatures on growth from spores of non-proteolytic Clostridium botulinum.

The heat treatment necessary to inactivate spores of non-proteolytic Clostridium botulinum in refrigerated, processed foods may be influenced by the occurrence of lysozyme in these foods. Spores of six strains of non-proteolytic Cl. botulinum were inoculated into tubes of an anaerobic meat medium, to give 10(6) spores per tube. Hen egg white lysozyme (0-50 micrograms ml-1 was added, and the tubes were given a heat treatment equivalent to 19.8 min at 90 degrees C, cooled, and incubated at 8 degrees, 12 degrees, 16 degrees and 25 degrees C for up to 93 d. In the absence of added lysozyme, neither growth nor toxin formation were observed. A 6-D inactivation was therefore achieved. In tubes to which lysozyme (5-50 micrograms ml-1 had been added prior to heating, growth and toxin formation were observed. With lysozyme added at 50 micrograms ml-1, growth was first observed after 68 d at 8 degrees C, 31 d at 12 degrees C, 24 d at 16 degrees C, and 9 d at 25 degrees C. Thus, in these circumstances, a heat treatment equivalent to 19.8 min at 90 degrees C was not sufficient, on its own, to give a 6-D inactivation. A combination of the heat treatment, maintenance at less than 12 degrees C, and a shelf-life not more than 4 weeks reduced the risk of growth of non-proteolytic Cl. botulinum by a factor of 10(6).