Factors influencing Clostridium botulinum spore germination, outgrowth, and toxin formation in acidified media

Clostridium botulinum type A spores were inoculated at a level of 10(7) spores per ml into sterile beef media with protein concentrations of 1, 2, 3, 4, or 6% and acidified to pH values of 2.01 to 4.75 with hydrochloric acid or 4.19 to 4.60 with citric acid. All experimental manipulations, including blending, acidification, inoculation, incubation (30 degrees C), and analyses, were conducted in an anaerobic chamber-incubator in which atmospheric oxygen levels were maintained below 2 ppm (2 microliters/liter). Under these strict anaerobic conditions (oxidation-reduction values in media ranging from -370 to -391 mV), C. botulinum spores were consistently found to germinate, grow, and produce toxin below pH 4.6. The boundary between toxic and atoxic samples in HC1-acidified beef media was mediated by titratable acidity, pH, and protein concentration. A limiting acidity was not established for the citrate-acidified samples; all blends tested (1, 2, 3, and 4% protein and titratable acidities of 0.091 to 0.453%) became toxic within 5 weeks. At the same pH and protein concentration, citric acid was less effective than HC1 in preventing the germination of C. botulinum spores. Higher levels of cell proliferation in the beef protein, as well as enhanced gas production and putrefactive degradation, indicated that beef was a better substrate than soy for C. botulinum spores under these conditions. Reducing the inoculum to 10(4) delayed but did not prevent spore outgrowth and toxin release at pH levels below 4.6.