1. Introduction
Thermal-pressure processing is a common method in agricultural distilleries which use starchy raw materials. Its technological benefits include sterilization, which eliminates process-dangerous lactic acid bacteria on cereal grains [
1], and the liberation of starch from plant cells. Moreover, high temperatures change the physical properties of starch, enabling more effective enzymatic hydrolysis. However, heat treatment of starchy materials can lead to a number of chemical changes, involving reducing sugars, amino acids, and peptides, which are abundant in cereal raw materials [
2]. During heating, reactions may occur between the carbonyl or hemiacetal groups in reducing sugars and the amino groups in amino acids and peptides. These can then initiate a series of reactions, referred to collectively as Maillard reactions, which produce compounds with strong sensory properties. Maillard reaction products (MRPs) affect the taste, smell, and color of sweet mash obtained using the thermal-pressure method [
3,
4,
5,
6,
7]. Moreover, Maillard reactions involve reducing sugars, which might otherwise have provided a potential substrate for ethanol production, as well as amino acids with peptides, which could have been a valuable nitrogen source for yeast in the fermentation process. Some MRPs, including furfural and 5-hydroxymethylfurfural (HMF), have been shown to affect the fermentation process [
8,
9,
10,
11]. Furfural and HMF are sugar degradation products, formed by dehydration of pentoses and hexoses, respectively, at high temperature and pressure [
12,
13]. Both are known to have a negative effect on
Saccharomyces cerevisiae yeast. However, furfural is considered a much stronger inhibitor than HMF in terms of yeast growth and the productivity of ethanol. This is related to its effect on glycolytic activity and the tricarboxylic acid cycle, as well as to its causing oxidative stress and reducing the activity of various dehydrogenases in yeast cells [
12,
14]. Agricultural distilleries using starchy raw materials therefore need to control the steaming time carefully, to minimize the concentration of MRPs.
Steaming of cereal raw materials—such as rye, wheat, or triticale—is carried out at pressures of 0.3–0.4 MPa and temperatures of 144–152 °C for about 30–40 min. The steaming of barley grain requires harsher parameters (0.5 MPa, 159 °C), due to the presence of an additional fibrous layer around the kernel, which develops as the flower buds grow together with the kernel during the grain’s puberty [
15]. A well-steamed mass should have a light-yellow color, and the content of the kernel hulls should flow out freely.
Apart from furfural and 5-hydroxymethylfurfural (HMF), MRPs notably include aldehydes, for example phenylacetaldehyde and benzaldehyde, as well as pyrazines [
16,
17], other furans [
18], pyridine, melanoidins [
19], and ketones [
6]. In research into the formation of carbonyl compounds in Maillard reactions, Rooney et al. [
20] confirmed that, in the presence of amino acids—such as alanine, valine, leucine, isoleucine, phenylalanine, and methionine—the corresponding aldehydes are formed—i.e., acetaldehyde, isobutyraldehyde, isovaleraldehyde, 2-methylbutanal, phenylacetaldehyde, and methional. Moreover, small amounts of other carbonyl compounds may be detected, including acetone. Ketones were also identified among MRPs studied by Cui K. et al. [
18]. Mansour et al. [
21], in an investigation into the effects of glucose, fructose, and sucrose on the flavor of extruded wheat semolina, likewise detected ketones.
In the next stage of the process, ethanol fermentation, the MRPs present in distillery mashes are transformed into other compounds by yeast. Aldehydes will be reduced to corresponding alcohols, which then participate in the synthesis of esters. Acetone is also reduced to the corresponding alcohol, in this case 2-propanol (syn. isopropyl alcohol). 2-propanol belongs to the group of higher alcohols. Higher alcohols are the dominant volatile compounds present in fermented mashes. Some of them, such as 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-1-propanol, 1-propanol, and 2-phenylethanol, are synthesized by the yeast from amino acids [
22,
23] and/or simple sugars [
24,
25]. However, the first pathway is dominant. Synthesis of other higher alcohols, such as 2-butanol, 2-propanol, 1-pentanol, and 1-hexanol, which occur in lower concentrations, only takes place via the latter pathway [
26]. Bacteria are also able to synthesize secondary and primary higher alcohols [
27,
28]. Bacteria, as well as yeast, are present in distillery mashes [
1,
22]. The most common are lactic acid bacteria [
29,
30], for which close to optimal growth conditions occur during the fermentation process. Therefore, particular attention should be given to the temperature during fermentation and the initial pH of the sweet mash. High values for each of these parameters affect the growth of lactic acid bacteria [
31], which poses a real threat to the proper course of the process, since acetic acid and lactic acid are well known yeast inhibitors [
32,
33]. Moreover, lactic acid bacteria are well known for their ability to synthesize volatile compounds such as alcohols, aldehydes, ketones, esters, alkanes, alkenes, terpenes, furans, and sulfur compounds [
34,
35,
36,
37]. Some of these negatively affect the quality of the agricultural distillates obtained [
38,
39].
In our previous research [
40], we found 2-propanol in distillates obtained from rye- and potato-based mashes prepared using both thermal-pressure (TP) and pressureless (PLS) methods for starch liberation. It was noticed that the concentration of 2-propanol was substantially higher in distillates obtained from mashes prepared using the TP method. The low boiling point of 2-propanol compared to other higher alcohols (C3-C8) and its ability to form azeotropes makes the removal of this compound from the spirit during rectification very difficult. Therefore, it is necessary to limit its synthesis during fermentation. According to EU Regulations [
41], the higher alcohols level (expressed in 2-methyl-1-propanol) in ethyl alcohol of agricultural origin is limited to a maximum of 0.5 g per hectoliter of 100% vol. alcohol, while to meet non-EU standards [
42] the concentration of other alcohols, especially isopropyl alcohol, should be also taken into account, due to the difficulty of removing it in the distillation processes.
According to the literature [
27,
28,
36,
37], the synthesis of 2-propanol takes place via acetone reduction catalyzed by primary and/or secondary alcohol dehydrogenase. For acetone synthesis, in turn, the main substrate is acetyl-CoA. Two molecules of acetyl-CoA are condensed to one molecule of acetoacetyl-CoA (by acetoacetyl-CoA synthase), from which acetoacetate is formed (by acetoacetyl-CoA transferase) and finally acetoacetate is decarboxylated to acetone (by acetoacetate decarboxylase) [
27]. Acetic acid, which is used by yeast and bacteria to synthesize acetyl-CoA, is already present in sweet mash, and is also formed during the fermentation process [
1,
43]. According to Martins et al. [
5] and Davídek et al. [
43], acetic acid is formed during Maillard reaction as the result of the degradation of Amadori products. Based on studies by Rooney et al. [
20], Davidek et al. [
43], and Cui et al. [
18], it can further be assumed that acetone may be present in sweet mash (i.e., before fermentation) prepared by using the TP method, as a result of Maillard reactions. According to Davídek et al. [
43], acetone formation occurs as a result of hydrolytic α-dicarbonyl cleavage of 2,4-pentanedione. However, the literature does not provide adequate reasons for the synthesis of 2-propanol during the ethanol fermentation of distillery sweet mashes. Differences between the technologies used for sweet mash production, as well as the complexity of the biochemical processes which occur during fermentations involving both yeast and other microorganisms (mainly lactic acid bacteria), combined with the action of multiple factors (including pH, temperature, sugars content, time), make it difficult to clearly explain the presence of 2-propanol.
The purpose of the present study was therefore to evaluate whether and in what quantities acetone is present in distillery sweet mashes prepared using the TP method. We then investigated the effects of S. cerevisiae yeast and lactic acid bacteria on acetone metabolism in distillery mashes, and on 2-propanol content in the fermented mashes, depending on the fermentation temperature.
3. Materials and Methods
3.1. Industrial Scale Pressure-Thermal Treatment and Mashing Process
Rye grains were treated on an industrial scale, in a tapered cylindrical steamer (total volume 3.75 m3). The steamer was filled with water (1.75 m3), which was heated to boiling point by injecting superheated steam. Then, 0.7 t of rye grains was poured in. After venting, the steamer was closed and the pressure inside was increased gradually to 0.42–0.45 MPa. The raw material was steamed at this pressure for 50 min, with periodical circulation of the content. The whole process was carried out with manual control of the steaming parameters (pressure and time) and circulation. Upon completion, the content of the steamer was transferred to a cylindrical steel-mashing vessel.
The steamed mass was stirred continuously in a steel mashing vessel and cooled to 90 °C, at which point liquefying Termamyl S.C. preparation was added (0.13 L/t of starch, Novozymes, Bagsværd, Denmark). The mixture was kept at this temperature for 15 min, then cooled to 65 °C before the addition of saccharifying SAN Extra preparation (0.6 L/t of starch, Novozymes, Denmark). After the addition of SAN Extra, the mash was cooled further, to 32–34 °C. This procedure was repeated 37 times. All the mash samples (n = 37) were frozen immediately for transportation from the agricultural distillery to the laboratory. The mash samples were assessed in terms of their color and chemical composition.
Mash samples from the yeast tank used for yeast propagation were also frozen and transported to the laboratory (after the 1st and 10th cycles). Prior to inoculation with the yeast, the sweet mash was acidified to pH 3.5.
3.2. Laboratory Scale Pressure-Thermal Treatment, Mashing, and Fermentation Processes
Pressure-thermal processing and mashing (with enzyme preparations) were performed according procedures described previously [
1]. Rye grains were used as the raw material. The samples of mash produced on a laboratory scale were used immediately to prepare fermentation trials.
The pH of the sweet mash before fermentation was set at 5.0 using sulfuric acid (25% w/w solution). Two variants of the sweet mash were then prepared, with different microorganisms used for inoculation:
Ethanol Red yeast (Saccharomyces cerevisiae, Fermentis Division S.I. Lesaffre, Marcq-en-Barœul, France) (1.3×107 CFU/mL of mash), with the addition of 80 mg/L IsoStab® hop α-acid preparation (BetaTec GmbH, Nürnberg, Germany), to protect the process from bacterial contamination and prevent microbial infections;
a mixture of lactic acid bacteria strains Lactococcus lactis ssp. lactis ŁOCK0877 and Lactobacillus casei ŁOCK0901, obtained from the ŁOCK Pure Culture Collection (final quantity 4.0×106 CFU/mL of mash), with the addition of nystatin, to prevent yeast growth.
Before fermentation, all of the mash samples (1000 mL) were supplemented with diammonium phosphate (0.2 g/L). The glass flasks (2000 mL) were closed with a fermentation tube filled with glycerin. Fermentations were performed in two thermostatic rooms, with the temperatures set to 27 and 35 °C. Fermentation was continued for 72 h. Before and after fermentation, the mash samples were taken for gas chromatographic analysis (HS-GC-MS). All experiments were prepared in triplicate.
3.3. Color Measurement
Tests were performed using a CHROMA METER CR-5 device (Konica Minolta, Osaka, Japan), using the CIE Lab scale. Three color components were measured: L* (darkness or lightness of color ranges from black (0) to white (100)), a* (+red to −green chromatic components), and b* (+yellow to −blue chromatic components). Based on the L* and b* parameters, the yellowness index (YI) was calculated as Equation (1) [
56]
Prior to analysis, samples of the mashes were centrifuged at 23,300× g for 10 min at 15 °C. The final result was the arithmetic mean of three measurements for each sample.
3.4. Determination of Total Sugars and Extract Content in the Mashes
The concentration of total sugars in the sweet and fermented mashes prepared on the laboratory scale was determined according to the method described previously [
1]. The content of extract in the sweet mashes prepared at both the industrial and laboratory scales was measured using a hydrometer, according to the method described previously [
57].
3.5. HPLC Analysis of Fermented Mashes
The concentrations of ethanol and lactic acid in the mashes were evaluated using HPLC, according procedures described previously [
1].
Fermentation efficiency was calculated according to method described previously [
57].
3.6. Gas Chromatographic Analysis (HS-GC-MS) of Sweet and Fermented Mashes
Qualitative analysis was performed using a GC apparatus (Agilent 7890A, Agilent Technologies, Santa Clara, CA, USA) coupled to a mass spectrometer (Agilent MSD 5975C, Agilent Technologies, Santa Clara, CA, USA). A capillary column was used to separate the compounds (TGWAX-MS, Thermo, Scientific Fisher, Pittsburgh, PA, USA; 60 m × 0.32 mm × 0.50 μm). The GC oven temperature was programmed to increase from 40 °C (5 min) to 80 °C at a rate of 5 °C/min, then to 220 °C at a rate of 10 °C/min, where it was maintained for 5 min. The flow rate of the carrier gas (helium) through the column was 1.1 mL/min. The temperature of the injector (split/splitless) was 250 °C. Injections of the tested samples were made in the split mode (10:1) using a headspace analyzer (Agilent 7697A, Agilent Technologies, Santa Clara, CA, USA). The temperatures of the MS ion source, transfer line, and quadrupole were 230, 250, and 150 °C, respectively. The ionization energy was 70 eV. Prior to analysis, a 20 mL headspace vial was filled with 7 mL of mash and closed tightly using an aluminum cap and septa.
Headspace conditions:
Temperature settings: oven temperature 50 °C, loop temperature 60 °C, transfer line temperature 70 °C.
Timing settings: vial equilibration time 20 min, injection duration 0.7 min, GC cycle time 47 min.
Vial and loop settings: vial shaking 136 shakes/min, fill pressure 15 psi, vial pressurization gas helium.
Acetone, MRPs (furfural and hydroxymethylfurfural), and 2-propanol were identified based on a comparison of their mass spectra with those of standard compounds and with the mass spectra in the NIST/EPA/NIH Mass Spectra Library (2012; Version 2.0g).
Quantitative analysis of the sweet and fermented mashes was performed to determine the concentrations of the identified compounds. Headspace and GC conditions were the same those used for qualitative analyses. The acetone, furfural, and 2-propanol were quantified using calibration curves in the selected ion monitoring mode (SIM). Quantitative analysis was performed using Agilent MassHunter software (Agilent Technologies, Santa Clara, CA, USA). The results were expressed in mg/L of mash. All analyses were performed in triplicate.
3.7. Statistical Analysis
Statistical calculations were performed using STATISTICA 6.0 software (Tibco Software, Palo Alto, CA, USA). To evaluate the differences between the tested sweet mash samples, analysis of variance (ANOVA) was conducted with a 0.05 significance level. When statistical differences were detected (p < 0.05), means were compared using the post hoc Duncan test with a 0.05 significance level. Correlation and regression analyses were used to determine the relationship between the test color components and the concentrations of acetone and furfural. Significance tests were performed with a 0.05 significance level.