The chemical composition of raw materials
The varietal characteristics, the course of the growing season, agrotechnics, and the conditions of malting process are the key factors determining the quality of the cereals and the malts produced from them [32–36].
The chemical composition of the tested raw materials (Table 1) shows that the moisture content in the tested raw materials amounted (88.0 ± 3.56) g kg−1 unmalted rye grain, whereas in cereal malts was between (35.0 ± 1.85) g kg−1 barley malt and (50.0 ± 2.85) g kg−1 wheat malt. Water content provides an indicator of the storability of cereals. Its content in malts should usually be below 5 % .
The main criterion for the selection of raw materials for efficient ethanol production is sugar content. As a distillers’ raw material, cereal grain is valued for its high efficiency because it is rich in starch . The starch content in the used in our study rye grains Dańkowskie Złote cv. was at the level of (621.0 ± 15.4) g kg−1, which is consistent with data published in the literature . In turn, the starch content in cereal malts was lower and ranged from (505.0 ± 13.6) g kg−1 wheat malt to (542.7 ± 15.5) g kg−1 rye malt. The reason for the lower concentrations of starch after malting is due to the development of enzymes hydrolyzing the starch to soluble sugars [39, 40]. As a consequence, the obtained results show a significant difference between the amount of reducing sugars contained in unmalted rye and cereal malts. In unmalted rye grain (Dańkowskie Diament cv.), reducing sugars content amounted to (14.7 ± 0.6) g kg−1, whereas cereal malts contained significantly higher concentrations of these sugars, ranging between (165.7 ± 13.0) g kg−1 barley malt and (188.9 ± 18.9) g kg−1 wheat malt.
As regards protein content, it was higher in unmalted rye than in rye malt or others malts. This is due, among others, to the fact that some proteins are used during the controlled germination of cereals in the malting process . Proteins are essential for the quality of malt. First, high protein content decreases available carbohydrates, adversely influencing the fermentation process, and second, proteolysis (protease hydrolysis producing amino acids and peptides) during malting and mashing is necessary for yeast metabolism [42, 43]. Yeast growth requires the uptake of nitrogen for the synthesis of protein and other nitrogenous components of the cell, but yeasts can only utilize low molecular weight nitrogenous materials such as inorganic ammonium ion, urea, amino acids and small peptides. The yeast cannot take up proteins or break down peptides larger than tripeptides . As an essential nutrient, free amino nitrogen (FAN) is especially important for yeast growth in the beginning of the fermentation process . The extracellular proteolytic activity of yeast is negligible. Proteolysis does not occur in the fermentation, unless the yeast has autolyzed. Nitrogen uptake slows or ceases later in the fermentation as yeast multiplication stops. When the sugar content is high and vital nutrients are lacking, a stuck fermentation can occur [46, 47].
Considering the above-mentioned aspects, to ensure appropriate conditions for the yeast fermentation activity, and to limit the development of bacterial infections, mashes were supplemented with ammonium ion, supplied as phosphate salt (NH4)2HPO4.
As regards the amylolytic enzymes activities in used raw materials, it was found that unmalted rye (Dańkowskie Diament cv.) was characterized by a low activity of α-amylase (0.83 ± 0.007) CU g−1 and a significantly higher β-amylase activity at the level of (4.96 ± 0.38) BU g−1. Among the malts used in this study, the highest amylolytic activity showed the wheat malt, while the lowest was observed in the barley malt. This is consistent with the manufacturer’s declaration, concerning the diastatic power of the tested malts expressed in Windisch–Kolbach units .
The chemical composition of mashes before and after fermentation
The reference mash was prepared from unmalted rye grain with application of amylolytic preparations. Digestion with the enzymes contained the preparations was conducted sequentially. After the liquefaction of starch by bacterial α-amylase (preparation Termamyl SC) in the time of 30 min, subsequent stages of the process were conducted according to the principles of SSF process, i.e., the mash was cooled to approximately 60 °C, supplemented with the saccharifying glucoamylase (preparation SAN Extra) and immediately cooled to the temperature at which fermentation was started (30 °C). Due to the procedure used, prepared rye mash was characterized by a higher content of dextrins (64.9 ± 2.5) g L−1 than reducing sugars (56.2 ± 1.8) g L−1. The principal benefits of performing the saccharification together with the fermentation are the reduced end-product inhibition of the enzymatic hydrolysis, and the reduced investment costs  should also be remembered that the saccharification stage is ideally suited for growth of very heat tolerant Lactobacillus strains known to create the lactic acid in fuel alcohol plants that interferes with yeast metabolism. Simultaneous saccharification and fermentation (SSF) technologies were developed for batch fermentation to eliminate bacterial contaminations and the resultant loss of ethanol yields .
Hydrolysis of starch to the fermentable carbohydrates glucose, maltose, and maltotriose is an important aspect of brewing as well as of distilling technology. Starch hydrolysis is carried out by the malt enzymes α-amylase, β-amylase, limit dextrinase, and α-glucosidase . Because the activity of the different enzymes is highly dependent on temperature, the manipulation of such variable is the main control mechanism for the mashing process. The mashing in brewing technology often consists of several temperature steps, each favoring different malt enzyme activities. The lowest temperature (45–50 °C) is the optimal temperature for cell wall degrading enzymes, β-glucanases, while the proteases works best at 52 °C, the β-amylase at 60–65 °C, and the α-amylase at 72 °C. The last step in the mashing is inactivation of the enzymes at 78 °C. Two major starch-digesting enzymes are released from malt, α-amylase and β-amylase. Temperatures of 60–65 °C maximize the activity of β-amylase, while temperature 72 °C is optimal to α-amylase activity [51, 52].
An energy-saving and brewing cycle time reducing solution is a simple heated step mashing at 60 °C . Analogously, the distillery mashes with the application of cereal malts were prepared.
The differences in the chemical composition of the prepared distillery mashes, depending on the type and amount of malts used, were observed (Table 2). The presence of wheat and barley malts (30 or 50 %) in sweet mashes did not significantly change their total extract content relative to the reference mash originating from unmalted rye alone (p > 0.05). Higher extract values between (200.0 ± 5.6) g kg−1 and (266.6 ± 6.6) g kg−1 were observed in the mashes containing 30 or 50 % rye malt. The obtained results stay in accordance with the ones described by Hübner et al.  who stated that the extract contents in rye malt-based worts are significantly higher than in barley malt-based worts, because rye lucks the husk which represent about 10 % of barley dry weight.
The content of total sugars (after acid hydrolysis) in the mash obtained from unmalted rye was (128.3 ± 4.5) g glucose L−1 mash, while in mashes containing rye malt it ranged from (155.0 ± 5.5) g glucose L−1 mash (30 % rye malt) to (172.0 ± 5.8) g glucose L−1 mash (50 % rye malt). The concentrations of reducing (fermentable) sugars were twice as high in mashes containing 30 % cereal malts as in the reference sample. An increase in the content of cereal malt in mashes from 30 to 50 % did not improve significantly the initial degree of starch saccharification (p > 0.05). That led to a higher concentration of dextrins, especially in mashes containing rye malt despite the relatively high activity of amylolytic enzymes in this malt. It was probably that the amylolytic enzyme activities could be inhibited by high sugar content.
The chemical analysis of mashes after fermentation consisted of the determination of apparent and real extract, as well as the concentration of ethanol, reducing sugars, total sugars (after acid hydrolysis), and dextrins (Table 2).
During Scotch whisky production, the fermentation process is usually allowed to proceed to a point at which the specific gravity of fermented mash drops to below 1.0 . Apparent extract (measured in the presence of ethanol which decreases medium density) for well-fermented distillery mashes with an initial extract of approximately 180 g kg−1 should not exceed 10–15 g kg−1 .
After 72-h fermentation, the tested mashes were characterized by higher values of apparent extract. In the mash originating from unmalted rye, it amounted (18.5 ± 0.5) g kg−1, whereas in samples of mashes with 30 % cereal malt it ranged from (19.2 ± 1.0) to (28.0 ± 1.2) g kg−1. Samples containing 50 % malt were characterized by an apparent extract in the range of (22.0 ± 0.9) to (26.0 ± 1.3) g kg−1.
In brewing technology, the suitability of the extracted compounds for ethanol production is reflected by the parameter called fermentability. This factor is defined as the percentage of extracted compounds, which can be fermented into ethanol by yeast. The fermentability values measured in rye malts ranged from approximately 73 to 77 %  and are considerably lower than in barley malt (typically >80 %) or wheat malt (>78 %) . This is explained by the higher contents of FAN in barley malt .
In our research, the highest concentration of ethanol (8.40 ± 0.25) % vol. was found in the mash containing 50 % rye malt, while the lowest (6.60 ± 0.12) % vol. in the mash containing 50 % barley malt (Table 2).
As regards the residual sugars, the mash prepared exclusively from unmalted rye was marked after fermentation by the lowest content of reducing sugars and dextrins. However, it should be noted that the above mash was also characterized by the lowest content of sugar before fermentation. The decrease in dextrins content can be attributed to the continuous combined action of α-amylase, β-amylase, and limit dextrinase. The activity of α-amylase results in an increase in (shorter) dextrins; while β-amylase rapidly removes a maltose moiety from the non-reducing end of all dextrins. If the concerted activity of malt α- and β-amylases was sufficiently random, then it could be expected that this would result in a general decrease in the concentration of dextrins .
The highest amounts of non-hydrolyzed dextrins, between (12.6 ± 0.5) and (14.7 ± 0.6) g L−1, remained in the mashes containing 30 % barley malt and 30 % rye malt, respectively. The relatively low barley malt β-amylase activity (Table 1) could be the probable cause of incomplete starch hydrolysis and low ethanol production in the mash prepared with this malt. In turn, the high concentration of unhydrolyzed dextrins in the mash containing 30 % rye malt could be a consequence of too high content of starch relative to the amylolytic activities present in the applied dose of the malt.
At higher malt dosages, malt provides a rich source of α-amylase, and it has a greater effect than β-amylase does, thus resulting in lower levels of maltose and higher levels of glucose and maltotriose. It is clear that the addition of malted cereals leads to higher levels of soluble sugars and also that the addition of unmalted cereals brings in a certain level of carbohydrate. It is also important to consider the hydrolytic effects, which the endogenous malt starch-degrading enzymes bring into the system .
In order to evaluate fermentation results, the degree of sugar utilization and the efficiency of ethanol biosynthesis (expressed as percent of the theoretical amount) were calculated (Fig. 1). The highest sugar consumption rate (94.19 ± 2.35) % was found for the mash prepared only from unmalted rye and treated with amylolytic preparations. In the mashes prepared from unmalted rye with the addition of cereal malts, sugar consumption ranged from (83.74 ± 2.09) % for the mash containing 30 % barley malt to (93.08 ± 2.33) % for the mash containing 50 % rye malt. In turn, fermentation efficiency (real yield relative to the theoretical amount) ranged from (75.39 ± 1.88) % for the mash consisting of 50 % unmalted rye and 50 % rye malt to (87.92 ± 2.19) % for the mash consisting of 50 % unmalted rye and 50 % wheat malt. Higher ethanol yields were found for mashes prepared with wheat malt (p < 0.05), whereas the lowest yield was observed for mashes containing rye malt. A likely reason for this may be the fact that despite its high α-amylase activity, rye malt often adversely affects the course of fermentation as it contains compounds that inhibit the yeast activity. Cereal grains contain various amounts of non-starch polysaccharides (NSPs), which are composed predominantly of arabinoxylans (pentosans), β-glucans, and cellulose . The detrimental effect of soluble NSPs is mainly associated with their viscosity and physiological effects on the digestive medium. The content and type of NSPs differ among cereals. NSP content relative to dry matter is lower in wheat kernels (11.4 %) than in rye (13.2 %). Arabinoxylans (AX) are the predominant NSP in wheat (6–8 %) and rye (8.9 %), while β-glucans are the predominant NSP in barley (7.6 %) . Most of the arabinoxylans found in cereal grains are insoluble in water, but those not bounded to the cell walls, which can form highly viscous solutions and absorb an amount of water equivalent to about ten times their weight, are named as water-extractable AX (WEAX). These compounds are important for brewing purpose and agricultural distillates production. Soluble NSPs cause an increase in the viscosity of the medium, generally hampering the digestion process, whereas insoluble NSPs impede the access of endogenous enzymes to their substrates by physical entrapment [45, 60].
Summing up, the obtained results indicate that under the conditions set in our experiments ethanol yield from 100 kg of starch was between (54.26 ± 1.36) L A100 (for the mash containing 50 % rye malt) and (63.30 ± 1.58) L A100 (for the mash containing 50 % wheat malt) (Fig. 1).
Chemical composition of the obtained distillates
During the fermentation process, yeasts produce ethanol and carbon dioxide, which promote the synthesis of alcohols, esters, organic acids, and these compounds determine the flavor and aroma of the alcoholic beverages [61–63]. The pathways of synthesis, type and concentration of these compounds also depend on the microorganisms present during the fermentation process , the chemical composition of the raw materials, ratio of C/N, and the environmental conditions [65, 66].
A comparison of the chemical composition of the obtained distillates demonstrates a significant effect of the cereal malts used in the preparation of sweet mashes (Tables 3, 4).
The main volatile compounds occurring in alcoholic beverages are aldehydes—intermediates in two-step decarboxylation of alpha-keto acids to alcohols as well as indirect products during the synthesis and oxidation of alcohols. Aldehydes, which represent a rather widely described class of compounds, as aroma compounds are often noted to have a negative influence on the quality characteristics of spirits. The concentrations of carbonyl compounds in agricultural distillates depend on the quality of raw materials, their chemical composition, the conditions of technological processes and microbial contamination .
Acetaldehyde is the predominant aliphatic carbonyl compound contained in raw spirits. Among the produced distillates, the highest concentration of acetaldehyde at (135.82 ± 2.56) mg L−1, 100 % vol. alcohol was found in the distillate from unmalted rye (p < 0.05). A comparison of distillates from mashes containing 30 and 50 % malts shows considerable variation in acetaldehyde concentrations. This could be due to the quality and type of malts used. In the case of raw spirits obtained from mashes containing wheat malt, the concentration of acetaldehyde tended to decrease when the content of that malt in sweet mashes was increased (p < 0.05). In contrast, distillates produced from mashes containing rye malt exhibited an inverse tendency. Of note is the distillate derived from the mash containing 50 % unmalted rye and 50 % wheat malt, which revealed the lowest acetaldehyde content at (36.06 ± 0.35) mg L−1 100 % vol. alcohol (Table 3).
Also, the presence of aldehydes such as furfural, hexanal, and benzaldehyde was determined in the studied distillates. Furfural is a compound formed during the dehydration of pentoses in technological processes carried out at elevated temperatures . The lowest concentration of this aldehyde (18.29 ± 1.39) mg L−1 100 % vol. alcohol was found in the distillate derived from the mash containing unmalted rye only. A higher malt content in the mashes was reflected in an increase in the concentration of furfural in the obtained distillates. The highest concentration of this compound (117.23 ± 0.80) mg L−1 100 % vol. alcohol was found in the distillate from the mash containing 50 % rye malt. The studied cereal distillates also contained small amounts of hexanal and benzaldehyde (Table 3).
Esters (mostly ethyl esters of monocarboxylic acids) are an important group of flavor compounds found in spirits such as whisky, cognac, and rum. Total ester content varies widely in strong spirits. Ethyl acetate is quantitatively the most important component of the ester fraction, usually accounting for over 50 % of the total. Many short-chain esters, such as isobutyl acetate, ethyl 3-methylbutyrate, ethyl n-butyrate, 2-methylbutyl acetate, and 3-methylbutyl acetate, have fairly strong odors. Therefore, their occurrence in whisky, cognac, and rum has been investigated extensively . In whisky, the concentration of long-chain carboxylic acid esters increases from ethyl hexanoate up to ethyl decanoate and then declines with C18 ethyl esters typically being the longest esters to be detected [70, 71].
Determination of the content of esters in the analyzed distillates shows that their concentrations were higher (p < 0.05) in distillates from mashes containing cereal malts than in raw spirits obtained from unmalted rye only. In this group of compounds, ethyl acetate is predominant. The highest concentrations of ethyl acetate were found in distillates from fermented mashes containing 50 % unmalted rye and 50 % rye malt (Table 3).
The tested distillates exhibited small amounts of esters of higher carboxylic acids and ethanol, i.e., ethyl caproate (ethyl hexanoate) and ethyl caprylate (ethyl octanoate) as compared to the predominant ethyl acetate. In the distillate obtained from unmalted rye, the concentration of those esters was similar (p > 0.05). In the majority of spirit distillates obtained from mashes containing cereal malts, the concentrations of ethyl caproate were lower than in control (the distillate obtained from unmalted rye only), (p < 0.05). On the other hand, a higher content of wheat and barley malts in mashes led to an increase in the concentration of ethyl caprylate in the distillates. Trace amounts of hexyl acetate were also found in all raw spirit samples (Table 3).
One of the undesirable compounds in spirit distillates is methanol, which is generated through hydrolysis of methylated pectins present in the plants and fruits. Methanol will be an unavoidable component of fermented mashes from fruits with high pectin contents such as Williams pears, plums, and mirabelles. This compound is produced in the process of hydrolysis of pectins by specific pectolytic enzymes, and in particular pectin methylesterase . While methanol does not directly affect the flavor of the distillate, it is subjected to restrictive controls owing to its high toxicity . Nevertheless, due to the fact that methanol arises as a result of de-esterification of pectins, which are naturally present in fruits, its presence at limited concentrations in fruit-derived distillates proves the identity of those products . Small pectic deposits have been reported in barley coleoptiles  and in wheat grain . It is also believed that some yeast strains (among others assigned to the genus Saccharomyces) have pectin methyl esterases [77, 78].
Based on the literature data , methanol concentrations for different samples of Scotch whiskies (single malt, single grain, blended) ranged between 4.7 and 16.4 g per 100 L absolute alcohol (i.e., between 47 mg and 164 mg L−1).
EU Regulation no. 110/2008  defines acceptable concentrations of methanol in ethyl alcohols of agricultural origin, wine spirits, and fruit spirits, but does not set any limits on the content of this compound in distillates of agricultural origin and grain spirits. Methanol concentrations in the obtained distillates (raw spirits) varied widely (p < 0.05) between (52.67 ± 2.63) mg L−1 100 % vol. alcohol (70 % unmalted rye and 30 % rye malt) and (118.7 ± 4.75) mg L−1 100 % vol. alcohol (50 % unmalted rye and 50 % wheat malt). The greatest differences (p < 0.05) in the concentration of methanol, as compared to the control (the distillate obtained from unmalted rye only), were noted in distillates from fermented mashes containing wheat malt (especially at a concentration of 50 %), (Table 4). It should, however, be noted that all obtained distillates meet the requirements of the mentioned EU Regulation , which stipulates that the maximum methanol content in ethyl alcohol of agricultural origin shall amount to 30 g hL−1 100 % vol. alcohol (i.e., 300 mg L−1).
From a quantitative point of view, the most important group of fermentation by-products is higher alcohols, represented mainly by: n-propanol, amyl alcohol, and its isomers, i.e., 2-methyl-1-butanol, 3-methyl-1-butanol. The regulation of the biosynthesis of higher alcohols is complex, since they may be produced as by-products of amino acid catabolism or via pyruvate derived from carbohydrate metabolism . These compounds play an important role in the formation of flavor qualities in spirits, such as whisky and Starka. Scotch malt whiskies are rich in higher alcohols, with contents often well over 2 g L−1 . According to the recommendations of the Polish Standard , the maximum concentration of these compounds in the agricultural distillates used for Starka production is 5 g L−1 100 % vol. alcohol.
Aylott and MacKenzie  carried out research aimed at developing of analytical strategies to confirm the authenticity of Scotch whisky. Blended Scotch whiskies, being combinations of many different Scotch malts and grains, are diverse and show representative analytical profiles of their constituent parts. The concentrations of 2- and 3-methyl butanol in Grain Scotch whisky were relatively low compared to n-propanol and isobutanol. The Malt Scotch whiskies were rich in these higher alcohols, with the average 2- and 3-methyl butanol concentration being 190 g/100 L absolute alcohol, compared to only 30 g/100 L absolute alcohol in the Grain Scotch whiskies.
In the obtained distillates, the most abundant among higher alcohols was 3-methyl-1-butanol. In the trials obtained from unmalted rye, it was followed by 2-methyl-1-butanol and 2-methyl-1-propanol, while the distillates from mashes containing cereal malts exhibited higher concentrations of 2-methyl-1-propanol than 2-methyl-1-butanol (Table 4).
The highest concentrations of amyl alcohol isomers (i.e., 2-methyl-1-butanol and 3-methyl 1-butanol) were found in the distillate obtained from unmalted rye mash. In turn, distillates originating from fermented mashes containing cereal malts exhibited higher concentrations of 1-propanol, 1-butanol, 1-hexanol, and benzyl alcohol (p < 0.05). The differences found in higher alcohol concentrations in the tested distillates are probably the consequence of the type of raw material. This is confirmed by report by Kłosowski et al. , who studied influence of various yeast strains and selected starchy raw materials on production of higher alcohols during the alcoholic fermentation process. The authors observed that among the distillates obtained from different raw materials, the highest final total content of higher alcohols (ca. 5.46 mg L−1 100 % vol. alcohol, on average) was found in the distillates produced from the maize mashes, while in the rye and amaranth distillates the concentrations of these compounds were similar and reached the level of ca. 3.44 mg L−1 100 % vol. alcohol.