## 3 Results and Discussion
### Chemical Characteristic of Mashes and Fermentation Results  
The chemical composition of the prepared distillery mashes varied according to the type of bread used and the method of mash preparation. The obtained results are presented in Table 2.  
The pH of mashes prepared from wheat bread was found to be 5.1, while those prepared from wheat-rye bread had a pH of 4.5. This difference can be attributed to the higher concentration of lactic and acetic acids present in the used raw material (see Table 1). Due to the fact that the optimal pH range for yeast growth can vary from pH 4.00 to 6.00 [35], the pH of the prepared bread-based mashes was not adjusted in order to limit the number of additional treatments and reagents used. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Parameters** & \multicolumn{2}{c}{**Wheat Bread**} & \multicolumn{2}{c}{**Wheat–Rye Bread**} \\  & \multicolumn{2}{c}{**Content [g/100 g Dry Mass]**} \\ \hline  & **Mean** & **SD** & **Mean** & **SD** \\ \hline Moisture * & 24.35 b  & 0.15 & 19.80 a  & 0.37 \\ Total protein (Nx6.25) & 7.08 b  & 0.01 & 8.18 a  & 0.05 \\ Total reducing sugars expressed as glucose, incl.: & 3.03 a  & 0.20 & 2.02 b  & 0.31 \\ Maltotriose & 0.20 a  & 0.01 & 0.21 a  & 0.01 \\ Maltose & 0.22 a  & 0.01 & 0.12 b  & 0.01 \\ Glucose & 2.58 a  & 0.24 & 1.66 b  & 0.20 \\ Starch & 71.70 b  & 1.71 & 77.16 a  & 4.98 \\ Xylose & 0.16 b  & 0.03 & 0.49 a  & 0.02 \\ Arabinose & 0.11 b  & 0.04 & 0.27 a  & 0.00 \\ Succinic acid & 0.04 a  & 0.01 & 0.04 a  & 0.01 \\ Lactic acid & 0.05 b  & 0.01 & 0.10 a  & 0.01 \\ Acetic acid & 0.04 b  & 0.01 & 0.07 a  & 0.01 \\ Glycerol & 0.04 b  & 0.01 & 0.14 a  & 0.01 \\ \hline \hline \end{tabular}  
* g/100 g the results expressed as mean values \(\pm\) SD (n = 3); mean values with different letters (a, b) within the same row are significantly different (ANOVA, \(p<0.05\)).  
\end{table} 
Table 1: Chemical composition of tested raw materials. Despite the same proportions of the raw material and water employed in the preparation of mashes by all of the aforementioned methods, statistically significant differences (\(p\leq 0.05\)) in the extract content of the obtained mashes were observed. The PLS-SHF method resulted in the preparation of wheat and wheat-rye bread-based sweet mashes with a higher extract content (\(p\leq 0.05\)) than those that were prepared using the PLS-SSF method. This may be attributed to the release of greater quantities of sugars into the medium during the separate saccharification stage, as evidenced by the total reducing sugars content in the obtained mashes (Table 2). It is notable that the highest extract content was observed in samples prepared using the NSH method, which involved native starch hydrolysis. It is possible that the heterogeneity of the bread portions and the progressive drying process may have contributed to the observed differences in the extract content. The fermentable sugars present in sweet mashes prepared by all used methods, i.e., PLS-SSF, PLS-SHF, NSH-A and NSH-N/A consisted mainly of glucose at concentrations ranging from 77.28 \(\pm\) 1.62 g/L (wheat-rye bread, PLS-SSF) to 147.81 \(\pm\) 0.51 g/L (wheat-rye bread, NSH-A). However, significantly higher (\(p\leq 0.05\)) glucose concentrations were determined in the mashes in which separate hydrolysis/saccharification of dextrins was carried out than in the samples saccharified during fermentation (SSF). Furthermore, relatively high concentrations of maltose (ranging from 33.79 \(\pm\) 1.70 to 35.10 \(\pm\) 3.74 g/L) were also determined in the mashes prepared from both types of bread using the PLS-SSF method. This is due to the fact that the conditions of separate hydrolysis at a temperature of 65 \({}^{\circ}\)C favour the hydrolysis of dextrins into monosaccharides. Additionally, all mashes contained small amounts of maltotriose, with the majority of these concentrations remaining below 1 g/L.