Enzymes and Lactobacillus fructivorans in unpasteurized sake were inactivated by a two-stage method with low-pressure carbon dioxide microbubbles (two-stage MB-CO2), and the quality of the treated sake was evaluated by the measurement of volatile compounds and by sensory tests. No surviving L. fructivorans cells were detected, and α-amylase, glucoamylase and acid carboxypeptidase were completely inactivated after two-stage MB-CO2 treatment. However, the relative residual activities of α-glucosidase were 28%-36% and 1%-11% after the two-stage MB-CO2 treatment at 45 C and 50 C, respectively. The effectiveness increased with temperature and exposure time of the heating coil. Ethyl butyrate, isoamyl acetate, ethyl hexanoate and ethyl octanoate in unpasteurized sake were decreased to 28%-49% by two-stage MB-CO2, but these losses could be prevented by passing the two-stage MB-CO2-treated sake through a cooling coil. Furthermore, the sensory scores relating to the total aroma and total taste of the MB-CO 2-treated sake were higher than those of unpasteurized and heat-treated sakes. Industrial relevance: Sake is a traditional alcoholic beverage in Japan. Unpasteurized sake is generally subjected to heat treatment to prevent the growth of microorganisms and enzymatic activity, although heat treatment often causes undesirable changes in sake quality. Non-thermal processes using supercritical carbon dioxide (SC-CO2) have been widely studied as alternative processes. However, high pressure is necessary for effective pasteurization with SC-CO2 treatment and the equipment is prohibitively expensive. In this study, we proposed a pasteurization technique that uses low-pressure CO2 microbubbles as a novel technique, which could inactivate microorganisms and enzymes in unpasteurized sake at moderate temperature and lower pressure. © 2013 Elsevier Ltd. All rights reserved.