Quantitative Evaluation of Clostridium botulinum Nonproteolytic Types B, E, and F Growth Risk in Fresh Salmon Tissue Homogenates Stored under Modified Atmospheres

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Abstract
The potential risk of C. botulinum growth in fresh fish stored under modified atmospheres (MA) remains unclear, as few qualitative studies have identified certain conditions leading to toxigenesis. This is the second paper of a series attempting to quantify the effect of selected parameters on the probability (P) of toxigenesis by one spore in fish. The factorially designed experiments included fresh salmon tissue homogenate with 3 levels of initial microbial flora (IMF) inoculated with a pool of spores of 13 nonproteolytic type B, E and F strains at 7 levels (10−2–104/3-g sample) and incubated at 1, 4, 8, 12, 16, and 30°C under 3 MA (vacuum, 100% CO2, 70% CO2 + 30% air) for up to 60 d. The earliest we observed toxicity at 30, 16, 12, 8 and 4°C irrespective of MA were 1, 2, 6, 9-12 and 15–>60 days and required 10°, 103–104, 102–103, 101–104 and 104 spores/sample, respectively. The probability of toxigenesis was affected significantly (P<0.05) by IMF, MA, storage temperature (T), storage time (ST) and the interactions T × ST, MA ×T, MA × ST, and IMF × T. Only type B toxin was detected in toxic samples. Using linear and logistic regression models, equations were derived which could estimate the length of the lag phase and the P of toxigenesis by one spore under a particular storage condition.