Abstract
The germination kinetics of Bacillus subtilis, Bacillus cereus, and Bacillus megaterium spores were comprehensively studied using nutrients, dodecylamine, exogenous dipicolinic acid (DPA), and the nonthermal technology of High Pressure Processing (HPP) as germinants at water activities aw=0.995–0.698 and DPA loss measured for spore populations and single spores. Primary findings were: i) germinant receptor (GR)-dependent germination was less sensitive to changes in aw than were other germination mechanisms; ii) HPP germination was less sensitive to changes in aw than was germination by other germinants, and iii) the irreversible commitment step was more sensitive to inhibition by lowering aw than were DPA release or cortex peptidoglycan hydrolysis. Presently, we use the three-step Quasi-chemical Germination Kinetics (QCGK) model, Transition State Theory, and Extrathermodynamics' Bunnett equation to determine the effects of humectants on spore germination rates. The QCGK model is an ordinary differential equation system that postulates spore germination occurs as a sequence of two productive steps with associated rate constants (k1 is irreversible and k2) and one nonproductive step (k3). The QCGK model fits the germination kinetics data well for changes in the observed dynamics with decreasing aw. Transition State Theory calculated negative activation volumes (ΔV∗) from the pressure-dependences of k1 and k2, and Bunnett equation calculations established linear relationships for the model's rate parameters, including rate constants and maximum germination rates, with variations in aw. Taken together, these results suggest that the irreversible step (k1) is rate-limiting and the inverse dependence of k1 and k2 on humectant concentration could occur through shielding of the activated complex by the solvated nonelectrolytes at low aw, which prevents the germination intermediate from linking to the aqueous environment.
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