## 3 Results and Discussion
### Chemical Characteristic of Mashes and Fermentation Results
In the majority of fermentation trials, low concentrations of reducing sugars were identified, namely maltotriose (from 0.02 \(\pm\) 0.00 to 0.21 \(\pm\) 0.00 g/L), maltose (from 0.14 \(\pm\) 0.02 to 0.90 \(\pm\) 0.01 g/L) and glucose (from 0.01 \(\pm\) 0.00 to 0.08 \(\pm\) 0.02 g/L), which indicates their high utilisation during fermentation. It is noteworthy that only in samples of wheat bread mashes prepared by the PLS-SHF and NSH-N/A methods higher concentrations of glucose were determined, amounting to 3.79 \(\pm\) 0.42 g/L and 2.07 \(\pm\) 0.15 g/L, respectively (see Table 3). In the aforementioned PLS-SHF sample, a relatively high concentration of lactic acid (2.61 \(\pm\) 0.14 g/L) in the mash after fermentation was recorded, which may indicate the development of undesirable microorganisms, such as lactic acid bacteria, during a separate saccharification step [44]. With regard to the non-hydrolysed dextrins present in mashes upon completion of fermentation, an interesting phenomenon was observed. The concentration of these compounds was found to be relatively high (ranging from 1.87 \(\pm\) 0.56 to 2.52 \(\pm\) 0.58 g/L) in mashes prepared from both wheat and wheat-rye bread using the PLS-SSF and PLS-SHF methods, in comparison to the samples prepared using the native starch hydrolysis method. Moreover, pentose sugars, namely xylose and arabinose, were also identified in trace amounts in all of the fermented mashes. The yeast of the _S. cerevisiae_ genus used in this study metabolizes pentoses, but without converting them into ethanol [45]. All samples after fermentation contained organic acid, i.e., succinic acid, lactic acid, formic acid, and acetic acid. These acids are regarded as fermentation by-products [46]. Succinic acid is an intermediate product of the tricarboxylic acid (TCA) cycle and constitutes one of the end products of the anaerobic metabolism of yeast [47]. Moreover, all tested mashes exhibited relatively elevated concentrations of glycerol, which is one of the products of yeast metabolism. The primary function of this compound is to provide protection for yeast against environmental stressors [48]. In order to evaluate the results of fermentation process, the degree of sugar utilisation and the efficiency of ethanol biosynthesis (expressed as a percentage of the theoretical amount) were determined. The utilisation of sugars was found to be high in all fermentation trials, regardless of the type of bread used and the method of mash preparation. The degree of sugar utilisation ranged from 96.03 \(\pm\) 0.48% to 99.17 \(\pm\) 0.50% (see Figure 3). The lowestvalue of this indicator was determined for the wheat bread-based mash prepared by the PLS-SHF method, which contained relatively high concentration of unutilised glucose, as mentioned above (see Table 3). A comparison of the fermentation efficiency revealed that the PLS-SSF method was the optimal method for processing both types of bread. The fermentation efficiency for these mashes was found to be at the same level of 96.1 \(\pm\) 0.48% of the theoretical. The application of the PLS-SHF method for the preparation of the mashes did not result in an increase in fermentation efficiency. In fact, the process efficiencies were lower than those observed in the PLS-SSF method (from 85.75 \(\pm\) 0.43 to 88.60 \(\pm\) 0.44%). With regard to the NSH-N/A method, the fermentation of wheat bread-based mashes exhibited a higher efficiency, with a value of 87.85 \(\pm\) 0.43% of the theoretical, than that observed for the wheat-rye bread-based mash sample (83.74 \(\pm\) 0.43% of theoretical). It may be hypothesised that the reason for the lower ethanol yields obtained from wheat-rye bread is the more intensive phenomenon of starch retrogradation. The resulting starch complexes hinder the hydrolysis process, creating a barrier to the action of amyloid enzymes [39].