Heat-resistance of spores of non-proteolytic Clostridium botulinum estimated on medium containing lysozyme

Abstract

Heat treatment of spores of non‐proteolytic strains of Clostridium botulinum at 75–90°C, and enumeration of survivors on a nutrient medium containing lysozyme gave biphasic survival curves. A majority of spores were inactivated rapidly by heating, and the apparent heat‐resistance of these spores was similar to that observed by enumeration on medium without lysozyme. A minority of spores showed much greater heat‐resistance, due to the fact that the spore coat was permeable to lysozyme, which diffused into the spore from the medium and replaced the heat‐inactivated germination system. The proportion of heated spores permeable to lysozyme was between 0.2 and 1.4% for spores of strains 17B (type B) and Beluga (type E), but was about 20% for spores of strain Foster B96 (type E). After treatment of heated spores with alkaline thioglycolate, all were permeable to lysozyme. D‐values for heated spores that were permeable to lysozyme (naturally and after treatment with thioglycolate) were: for strain 17B, D85°C, 100 min; D90°C, 18.7 min; D95°C, 4.4 min; for strain Beluga, D85°C, 46 min; D90°C, 11.8 min; D95°C, 2.8 min. The z‐values for these spores of strains 17B and Beluga were 7.6°C and 8.3°C.

Thermal Inactivation of Nonproteolytic Clostridium botulinum Type E Spores in Model Fish Media and in Vacuum-Packaged Hot-Smoked Fish Products

“…}, growth observed in rainbow trout medium within 90 days at 30°C, {, no growth observed in rainbow trout medium; F, growth observed in whitefish medium within 90 days at 30°C, E, no growth observed in whitefish medium; ----, 6D heat treatments (i.e., heat treatments proposed to eliminate 10 6 nonproteolytic spores) recommended by the Advisory Committee on the Microbiological Safety of Foods (1); ------, 6D heat treatments recommended by the European Chilled Food Federation (17 (3), and 2.8 min at 95°C (37), as opposed to 0.07 to 6.6 min at 82°C without lysozyme (7,11,27,32,44,47). The heat-resistant spore fraction was estimated to be on the order of 0.1%, while in phosphate buffer percentages of 0.1 to 1.0% and sometimes up to 20% have been reported (37). The presence of a heat-resistant spore fraction may therefore greatly complicate the safe processing of REPFEDs, potentially substantially extending the required process times.…”

“…Lower z values of around 6 to 7°C have been reported for other seafoods, in fish (7,11,32) and in phosphate buffers (34,37). Such z values have been recommended for use in calculating pasteurization values related to heat processing in the production of REPFEDs at temperatures below 90°C (1,17).…”

“…Biphasic survivor curves indicate that from 0.1 to 20% of a spore population is permeable to lysozyme and is more heat resistant than the nonpermeable fraction (37,45). Without the addition of lysozyme to the recovery medium, decimal reduction times (D values) for spores of type E strains of 0.07 to 6.6 min at 82°C, depending on the heating medium, have been reported (7,11,27,32,41,44,47); with lysozyme, higher D values of 48.3, 12.6, and 3.17 min at 85, 90, and 95°C, respectively, have been determined (37).…”

Laboratory Diagnostics of Botulism

“…Heating at 60°C for 10 to 20 min is thus a safer choice for group II spores. The addition of a heat-resistant lytic enzyme, such as lysozyme (5 g/ml), to the culture medium may enhance germination of heat-stressed spores (169,170). Routine liquid media include chopped-meat-glucose-starch medium (39); cooked-meat medium (175,176); broths containing various combinations of tryptone, peptone, glucose, yeast extract, and trypsin (e.g., tryptone-peptone-glucose-yeast extract medium) (129); reinforced clostridial medium (79); and fastidious anaerobe broth (179).…”

“…Group I organisms seem to be more of terrestrial origin and are present in temperate climates, whereas group II strains, particularly type E, are frequently found in aquatic environments in the Northern hemisphere (Table 1). Differences in spore heat resistance and growth temperatures are responsible for the safety risks posed by C. botulinum groups I and II in the food industry; group I spores, which have a high heat resistance (112,138,180,184,192,202), cause problems in canning and home preservation of vegetables and meat, whereas group II spores, with somewhat lower spore heat resistance (135,140,(168)(169)(170), are of great concern in minimally processed packaged foods that have extended shelf lives at refrigerated temperatures (134,167).…”