The combined effect of sub-optimal temperature and sub-optimal pH on growth and toxin formation from spores of Clostridium botulinum

Low-acid foods (pH greater than or equal to 4.5) are not sufficiently acidic to prevent growth of Clostridium botulinum in otherwise optimal conditions. The combination of sub-optimal pH and sub-optimal temperature may, however, result in a very significant reduction in the risk of growth of this bacterium compared with the risk in optimal conditions. The combined effect of incubation temperatures of 12 degrees and 16 degrees C and pH values between 5.2 and 5.5 on growth and toxin production from spores of Cl. botulinum during incubation for 28 d has been investigated. Growth and formation of toxin (type B) were detected only in medium at pH 5.5 and incubated at 16 degrees C, corresponding to a probability of growth from a single spore within 14 d of 1.6 x 10(-5). The probability of growth in 28 d in the remaining conditions was less than 9 x 10(-6). After transfer of inoculated media from 12 degrees to 30 degrees C growth occurred at pH 5.2-5.5 within 19 d. After transfer of inoculated media from 12 degrees to 20 degrees C growth occurred at pH 5.5 and 5.4 but not at pH 5.3 or 5.2 in 40 d. Growth at pH 5.2-5.5 was accompanied by formation of toxin, in most cases of types A or B. In addition to the effect of sub-optimal temperature and pH, chelation of divalent metal ions by citrate may have contributed to inhibition.