Influence of pH and Temperature on the Growth of and Toxin Production by Neurotoxigenic Strains of Clostridium butyricum Type E

Abstract

Strains of Clostridium butyricum that produce botulinal toxin type E have been implicated in outbreaks of foodborne botulism in China, India, and Italy, yet the conditions that are favorable for the growth and toxinogenesis of these strains remain to be established. We attempted to determine the temperatures and pH levels that are most conducive to the growth of and toxin production by the six strains of neurotoxigenic C. butyricum that have been implicated in outbreaks of infective and foodborne botulism in Italy. The strains were cultured for 180 days on Trypticase–peptone–glucose–yeast extract broth at various pHs (4.6, 4.8, 5.0, 5.2, 5.4, 5.6, and 5.8) at 30°C and at various temperatures (10, 12, and 15°C) at pH 7.0. Growth was determined by checking for turbidity; toxin production was determined by the mouse bioassay. We also inoculated two foods: mascarpone cheese incubated at 25 and 15°C and pesto sauce incubated at 25°C. The lowest pH at which growth and toxin production occurred was 4.8 at 43 and 44 days of incubation, respectively. The lowest temperature at which growth and toxin production occurred was 12°C, with growth and toxin production first being observed after 15 days. For both foods, toxin production was observed after 5 days at 25°C. Since the strains did not show particularly psychrotrophic behavior, 4°C can be considered a sufficiently low temperature for the inhibition of growth. However, the observation of toxin production in foods at room temperature and at abused refrigeration temperatures demands that these strains be considered a new risk for the food industry.