2. Materials and methods
2.1. Raw material
Waste wheat–rye bread, the most common bread type in Poland, was obtained from a large local producer. The material constituted returns of unsold bread from shops, it did not shown signs of surface mold spoilage. The whole loafs were manually cut into ca. 2–4 cm dices and dried in a forced air oven (WTC Binder, Germany) at 40 C for 12 h. Afterward the material was ground in a knife mill (Rotary Mill, Brabender, Germany) with 1.5 mm internal mesh sieve and stored at room temperature in airtight jar until used. The moisture content in raw material was measured using WPS 50P weighing dryer (Radwag, Poland) and it ranged 41.43 ± 1.38 g kg1 (mean value ± standard deviation at n = 3). Starch content in the substrate was measured using Ewers polarimetric method [25] and it ranged 689.13 ± 4.52 g kg1 of dry matter. The content of total sugars in waste bread sample was measured using the DNS method [26] after mild acid hydrolysis (70 C, 10 min) with 80 g L1 HCl solution (1:7.5 m/v raw material to acid solution ratio). Total sugars content in the raw material ranged 866.87 ± 2.85 g kg1 dry matter.
2.2. Enzymes and yeast
Thermostable a-amylase (EC 3.2.1.1) preparation Termamyl SC DS was used in the enzymatic liquefaction step in the amount of
1.25 mL kg1 of raw material dry matter. Glucoamylase ( EC 3.2.1.3) preparation SAN Extra L and protease (EC 3.4.23.28) Neutrase 0.8 L were used in the simultaneous saccharification and fermentation. The enzyme doses were 1.25 and 0.9 mL kg1 of dry matter for SAN Extra L and Neutrase 0.8 L respectively. All enzymes were kindly provided by Novozymes (Denmark). Active dry yeast Saccharomyces cerevisiae Ethanol Red were purchased from Fermentis (France). The yeast were rehydrated in sterile distilled water (1:10 m/v yeast to water ratio) for 30 min at ca. 30 C prior to inoculation.
2.3. Determination of thermal properties of gelatinization of starch present in the raw material
The temperatures of phase transitions of starch present in waste bread were determined using differential scanning calorimetry (DSC) as described by Zie˛ba et al. [27]. Briefly 10 mg (dry matter basis) amount of waste bread was weighed into semi-pressure aluminum crucible (ME-2990, Mettler-Toledo) and bidistilled water was added to obtain raw material to water ratio of 1:3 m/v. The crucibles were sealed and conditioned for 1 h at room temperature. The analysis were performed using DSC 822e differential calorimeter (Mettler-Toledo, Switzerland) in a temperature range of 25–100 C at 4 C min1 heating rate. Empty crucible was used as the reference sample. The analysis was performed in triplicate and obtained thermograms were integrated using Stare software ( Mettler-Toledo, Switzerland). The initial temperature of gelatinization (T0), endotherm peak temperature (Tp) and gelatinization final temperature (Tf) were obtained which ranged: T0 – 45.63 ± 0.07 C; Tp – 52.35 ± 1.16 C; Tf – 58.59 ± 1.49 C.