The starch liquefaction was conducted for
60 min. Then the samples were cooled to 55 C, its pH was adjusted
to 5.8 with 7.11 mol L1 H2SO4, glucoamylase (SAN Extra L,
1.25 mL kg1 of raw material dry matter) and protease (Neutrase
0.8 L, 0.875 mL kg1 of raw material dry matter) were added and
the hydrolysis was conducted for 90 min. After this time the samples
were cooled to 20 C, the pH of the samples was adjusted to
4.5 using 1 mol L1 H2SO4 solution, and total weight of the mashes
was adjusted to 250 g using distilled water resulting in a final raw
material loading at 150 g kg1 of mash. 200 g aliquots of the
mashes were transferred to 300 ml conical flasks, inoculated with
2 ml of yeast slurry (yeast concentration of 1 g kg1 of mash) and
sealed with rubber stopper with fermentation tube and sampling
port. Further procedure was the same as described in Section 2.3.1.