2.3.5. Separate hydrolysis and fermentation (SHF) (Variant V)
Waste bread samples (37.5 g dry matter basis) were weighted
into the mashing cups and slurred with ca. 180 mL of distilled
water. pH of the slurry was adjusted to 6.0 using 1 mol L1
H2SO4 or NaOH solutions. The cups were placed in a LB 12 laboratory
mashing device (Lochner Labor + Technik, Germany) preheated
to 45 C. The mashing was conducted with 150 rpm
(2.5 Hz) stirring speed and 2 C min1 heating and cooling rate.
When the temperature of the slurry reached 60 C thermostable
a-amylase (Termamyl SC DS, 2 mL kg1 of raw material dry matter)
was added and the bath of the mashing device was further
heated to 85 C. 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.