© 2014 American Chemical Society. To determine optimal temperature of a two-stage system of low pressure carbon dioxide microbubbles (MB-CO2) for inactivating enzymes in unpasteurized sake (UPS), the effect of two-stage MB-CO2 containing a heating coil at various temperatures on the inactivation of the α-glucosidase in UPS was investigated, and the quality of the sake treated by two-stage MB-CO2 was estimated by sensory evaluation and component analysis. α-Glucosidase activity in the UPS was completely inactivated by two-stage MB-CO2 with a heating coil at 45 °C for 50 min, 55 °C for 5 min, 65 °C for 10 s (MB65), and 75 °C for 1 s, respectively. The quality of the MB65's sake was determined to be significantly excellent by the sensory evaluation. The reason was suggested to be due to relatively low contents of free amino acids, change in organic acid balance, and less damage to volatile compounds.