© 2015 Elsevier Ltd. The effect of pressure in the mixing vessel on the inactivation of enzymes and hiochi bacteria and on the change in free amino acid content in unpasteurized sake (UPS) using a two-stage system of low-pressure carbon dioxide microbubbles (MBCO2) was investigated. At a pressure of 0 MPa in the mixing vessel, hiochi bacteria in UPS were completely inactivated by heating at 65 °C for 1 s with two-stage MBCO2; α-amylase, glucoamylase and acid carboxypeptidase were inactivated after heating for 5 s, although α-glucosidase activity remained at approximately 13% even after 20 s. However, the inactivation efficiency increased with increasing pressure in the mixing vessel. In addition, the contents of aspartic acid, glutamic acid, alanine and arginine, key free amino acids involved in producing the taste of sake, in UPS tended to decrease by two-stage MBCO2 at all tested conditions, although these were minimally affected by pressure in the mixing vessel and exposure time in the heating coil.