Quantifying permeabilization and activity recovery of Bacillus spores in adverse conditions for growth

Heat treatment is the main hurdle used to eliminate spores in foods but the pH conditions which spores encounter after the treatment have a tremendous impact on the spore ability to germinate, outgrow and grow. The aim of this work was to discriminate the inactive permeable spores and the active spores in unfavorable acidic conditions, after a heat treatment. In this study, Bacillus weihenstephanensis KBAB4 was used as model micro-organism for psychrotrophic Bacillus. The spores were heat treated to inactivate 90% of the population, 12 min at 85 °C, or 2 min at 95 °C. After each treatment the spores were incubated at pH 5.50 or pH 7.40. The evolution of dormant spores, permeable spores, germinated and vegetative cells was monitored by flow cytometry using a double staining. LDS 751, stains in red all the permeable cells, and CFDA stains in green cells presenting an esterase activity. Dormant spores did not show neither red fluorescence nor green fluorescence. Permeabilized spores which did not recover metabolic activity were red fluorescent but not green fluorescent. Germinated spores (permeabilized and having an esterase activity) appeared red fluorescent and green fluorescent due to their permeability and their metabolic activity. This method allowed the differentiation of the impact of heat treatment and post-treatment incubation pH on the two first steps of germination: spore permeabilization and activity recovery. Having a better understanding of spore germination at unfavorable post-treatment pH allows a better control of spore forming bacteria in foods.