Sake was brewed on a small scale by the addition of raw materials in three steps (first addition Soe, second addition Naka, and third addition Tome) using three 70% polished rice samples (Yamadanishiki (Hiroshima), Gohyakumangoku (Fukui), and Gohyakumangoku (Niigata)). The moisture of the polished rice sample was adjusted to 13.6–13.8% (w/w) in a humidity chamber. Tap water was used for mashing. The composition of raw materials is shown in Table 1. Three kilograms of rice were fermented in two stainless vessels (1.5 kg per vessel), and the two sake mashes were mixed after fermentation. The pretreatment of the milled rice grains was as follows. The rice was washed with water for 1 min, drained for 30 s, washed again with water for 1 min, and drained again for 30 s. Next, the rice was steeped in water for 20–30 min, drained for 15–20 h at 15°C, and then steamed for 50 min. Sake-koji was made from steamed rice according to a standard procedure. Aspergillus oryzae spores (106 conidiospores/g polished rice grains) were inoculated onto the steamed rice grains. The temperature of the rice grains during koji making was adjusted to approximately 31°C at the time of inoculation and gradually increased to 42°C as the A. oryzae mycelia grew. The duration of the koji making was 46 h, and the ratio of the weight of the produced koji to that of the original milled rice was 118–120%. A yeast inoculum, which consisted of 2 × 109 cells of Saccharomyces cerevisiae K-701, was used as the seed mash (starter mash) and was allowed to ferment at 25°C for 4 days. The main mash temperatures were 15°C at the Soe addition, 10–11°C at the Naka addition, and 8–9°C at Tome addition. Then, the fermentation temperature was increased by 1.0°C per day to a maximum temperature of 15°C, which was then maintained for 7 days and gradually decreased to 10–11°C. To account for total Cs, the sake mash was not sampled during fermentation. Instead, the fermentation process was monitored by measuring the weight of the evolved carbon dioxide. Both the milled rice samples, after washing and steeping in water, and the steamed rice samples were prepared in the same way for mashing. The sake was separated from the sake cake in two steps. In the first step, the sake was separated from the solids by centrifugation (2630 ×g for 20 min) on day 20 after the third addition. In the second step, the precipitate was pressed for 15 h using a compact sake mash press machine (Toyoshoukai, Siga, Japan) at a maximum pressure of 20 MPa, after which the additional sake was separated from the sake cake. The sake obtained from the two steps was mixed and analyzed.