Furthermore, to characterize these compounds, ethanol-added sake was separated into upper and flow-through fractions using a 3000-molecular weight cut-off membrane (Amicon Ultra-15; Merck KGaA, Darmstadt, Germany). Unfractionated sake was added to the upper fraction to adjust its volume to that of the flow-through fraction. As shown in Table 3, DMTS-pp values of each fraction before incubation were fairly similar. However, after incubation at 25°C for 7 days, the DMTS-pp values of ethanol-added sake and the upper fraction increased, whereas that of the flow-through fraction did not. Moreover, the upper fraction, which contained almost twice as much high molecular weight compounds as the control, had the highest DMTS-pp value. These results indicated that the upper fraction contained compounds that increased DMTS-pp after incubation. These compounds were more effective at 25°C than at 15°C and were inactivated by heat treatment.