Ethanol fermentation procedure
The procedure for ethanol fermentation with cooking is shown in
Figure 1 (Koguchi et al., 2010). 30 g of unpolished black rice grains
and 50 ml of deionized water were dispensed into a 300 ml
Erlenmeyer flask and autoclaved at 121°C for 15 min. After cooling,
the cooked rice was mixed with 5 g of dried white koji as the
saccharifying agent, 40 ml of deionized water, and 10 ml of a yeast
suspension, which readily brought the population of yeast in the
initial mash to 3.0 x 107 cells/ml. Ethanol fermentation was
conducted at 25°C in the dark.
The procedure for ethanol fermentation without cooking is shown
in Figure 2. 30 g of unpolished raw black rice grains, 90 ml of
deionized water, 5 g of dried white koji, and 10 ml of a yeast
suspension were dispensed into a 300 ml Erlenmeyer flask. The
population of yeast in the initial mash was adjusted to 3.0 × 107
cells/ml, and fermentation was conducted in the same manner as for
the ethanol fermentation with cooking.
The decrease in weight of the Erlenmeyer flask and its contents
as a result of the evolution of CO2 gas was measured every 24 h.