Effect of sporulation medium and its divalent cation content on the heat and high pressure resistance of Clostridium botulinum type E spores

The variability of times to detect growth from individualClostridium botulinumtype E endospores is differentially affected by high pressure treatments

“…Additionally, 600 MPa at 80°C and 800 MPa at 60°C were used resulting in approximately 5 and 4 log reduction of viable spore counts, respectively. [11] We detected no growth in minimum 27, maximum 34, and a mean number of 32 wells per plate (94 inoculated wells). Assuming that the number of spores per well follows a Poisson distribution as a result of the dilution process, this would correspond to 1.25, 1.02, and 1.08 viable spores per well on average.…”

“…Growth conditions, harvesting, and purification of spore suspensions were performed essentially identical to previous experiments. [11] Briefly, sporulation in Sediment Fish Extract medium for 5-6 d, harvesting, washing, and ethanol treatment, and final washing steps resulted in the production of spore suspensions with > 99% phase bright spores. Finally, spores were resuspended in 50 mM imidazole phosphate buffer, pH 7, adjusting the initial viable spore count to approximately 5 × 10 7 spores/mL.…”

“…Determination of the detection time was essentially done as described by Stinger et al [16] Treated and untreated samples were serially diluted using S + (saline plus surfactant) [11] and TPYC medium (tryptone, peptone, yeast extract medium [17] + sugar mix) in the final dilution step to give approximately 20 viable spores/mL. Fifty microliter of a diluted spore suspension was placed in one well of a 96-well plate containing 200 µL TPYC.…”

“…Independently from food matrix-related effects on spore inactivation, we previously showed that HPT could be generally used to effectively reduce C. botulinum type E viable spore counts by several log cycles. [11] However, available data on the effect of HPT processing on the distribution of times to detect growth from individual spores are limited to Bacillus spores, [12] and single spore detection times of Clostridium species after HPT treatments have up to now not been examined. Here, we aimed at contributing in closing this gap of knowledge, which is important since (i) the success of a preservation process is reflected by its effectiveness in decreasing the probability that a single spore grows out, [10] (ii) common C. botulinum contamination levels in food are presumably low, i.e., growth is likely to initiate from only a few spores, [13] (iii) even a small inoculum of C. botulinum may develop into a hazard, [14] and (iv) spore populations are intrinsically heterogeneous, i.e., individual lag or detection times cannot be predicted from population measurements.…”

Construction of Nontoxigenic Mutants of Nonproteolytic Clostridium botulinum NCTC 11219 by Insertional Mutagenesis and Gene Replacement

“…However, the increasing consumer demand for fresh-tasting, healthy ready-to-eat foods that have been minimally processed and contain less salt and no artificial preservatives yet have a long shelf-life represents a challenge for the food industry in view of these botulinum safety guidelines (10,11,12). Furthermore, novel food processing and preservation technologies (e.g., high-pressure or pulsed electric field treatment and natural preservatives) have found their way to commercial food production, but data regarding their efficiency in controlling gIICb is scarce or lacking (13,14,15,16,17). This is in sharp contrast to the attention given in this context to other pathogens, like Listeria monocytogenes, Salmonella, and enterohemorrhagic Escherichia coli.…”