The inactivation effect of carbonation under heating (CH) followed by heating (HT2) in the presence of monoglycerol monocaprate (MC10) or monoglycerol monolaurate (MC12) on Bacillus subtilis spores was investigated. CH (80 degrees C, 5MPa, 10 min) followed by HT2 (90 degrees C, 10 min) induced an approximately 5 log order of inactivation in the presence of MC10 or MC12 at 0.05% (w/v). MC12 appeared to have a greater effect on increasing the inactivation effect of CH followed by HT2 than MC10. Heating, pressurization, and pH downshift involved in CH may play important roles in the high inactivation effect of CH followed by HT2. As indicated by increased DAPI stainability, CH in the presence of MC10 forced germination. The results show that heating after CH in the presence of MC10 and MC12 effectively inactivated B. subtilis spores under milder heating conditions than conventional autoclaving.