Efficacy of heat and ethanol spore treatments for the isolation of psychrotrophic Clostridium spp. associated with the spoilage of chilled vacuum-packed meats

Abstract

Psychrotrophic Clostridium spp. associated with chilled meat spoilage are difficult to isolate and culture. In this study the kinetics of heat and ethanol spore inactivation was determined as a first step towards optimising the recovery of psychrotrophic clostridial spores from meat and environmental samples. To determine heat inactivation, spores of nine isolates associated with spoiled chilled or frozen meat and a psychrotrophic reference strain Clostridium algidicarnis NCFB 2931, suspended in phosphate buffer, were exposed to temperatures between 75°C and 95°C for 0 to 120 min using a submerged tube procedure. Survivors after various temperature-time combinations were enumerated on Peptone Yeast Extract Glucose Starch (PYGS) agar containing lysozyme. D-values and z-values for each spore suspension were determined from their respective survival curves. To determine ethanol inactivation, similar phosphate buffer spore suspensions were mixed with equal volumes of absolute ethanol, incubated at 20°C and survivors enumerated on lysozyme-containing PYGS agar after 0 to 300 min. Based on spore heat inactivation, the 10 isolates could be grouped as having either heat-sensitive or heat-resistant spores. For heat-sensitive spore types, 60 min ethanol treatment gave maximum spore recovery whereas for heat-resistant spore types, heat treatment at 80°C for 10 min gave the best recovery. When the spore heat-resistance type is unknown, as would be the case when attempting an isolation from spoiled product, both an ethanol treatment and a separate heat treatment should be used, to ensure maximum spore recovery.