Characterization of Wet-Heat Inactivation of Single Spores of Bacillus Species by Dual-Trap Raman Spectroscopy and Elastic Light Scattering

Dual-trap laser tweezers Raman spectroscopy (LTRS) and elastic light scattering (ELS) were used to investigate dynamic processes during high-temperature treatment of individual spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis in water. Major conclusions from these studies included the following. (i) After spores of all three species were added to water at 80 to 90 degrees C, the level of the 1:1 complex of Ca2+ and dipicolinic acid (CaDPA; similar to 25% of the dry weight of the spore core) in individual spores remained relatively constant during a highly variable lag time (T-lag), and then CaDPA was released within 1 to 2 min. (ii) The T-lag values prior to rapid CaDPA release and thus the times for wet-heat killing of individual spores of all three species were very heterogeneous. (iii) The heterogeneity in kinetics of wet-heat killing of individual spores was not due to differences in the microscopic physical environments during heat treatment. (iv) During the wet-heat treatment of spores of all three species, spore protein denaturation largely but not completely accompanied rapid CaDPA release, as some changes in protein structure preceded rapid CaDPA release. (v) Changes in the ELS from individual spores of all three species were strongly correlated with the release of CaDPA. The ELS intensities of B. cereus and B. megaterium spores decreased gradually and reached minima at T-1 when similar to 80% of spore CaDPA was released, then increased rapidly until T-2 when full CaDPA release was complete, and then remained nearly constant. The ELS intensity of B. subtilis spores showed similar features, although the intensity changed minimally, if at all, prior to T-1. (vi) Carotenoids in B. megaterium spores' inner membranes exhibited two changes during heat treatment. First, the carotenoid's two Raman bands at 1,155 and 1,516 cm(-1) decreased rapidly to a low value and to zero, respectively, well before T-lag, and then the residual 1,155-cm(-1) band disappeared, in parallel with the rapid CaDPA release beginning at T-lag.