1. Introduction
Ethanol or ethyl alcohol is a type of alcohol and its production is nothing new. In ancient times, Egyptians produced ethanol from vegetables while the Chinese discovered the technique of distillation, which increases the concentration of alcohol in fermented wash [
1]. Ethanol can be produced from different grains such as corn, wheat, barley, sorghum, oat, and rice [
2]. Distillation is used to produce rectified spirits. The latter is highly concentrated ethanol (drinking alcohol), which has been purified by means of rectification (repeated distillation). Rectified spirits, produced from grain, sugar beets, or potatoes, are used for multiple purposes, namely in the production of whiskey, brandy, gin, vodka, liqueurs, for medicinal purposes, and so on [
3].
The safety of raw materials utilized in the production of ethanol technology has a significant effect on the quality and yield of ethanol as well as the by-products (distillers’ grain). Maximizing the yield of ethanol is the main priority of every ethanol producer. However, yeasts are not the only living organisms that use the sugar or other nutrients in the wort. When contaminated grains are used to produce mash for ethanol production, bacteria and fungi compete with yeasts for the nutrients, thereby decreasing the yield and quality of the ethanol produced. The goal of every producer is to maximize profit. Encountering lower yields due to contamination by unwanted organisms will not guarantee this outcome, but rather lead to loss of profit, which could collapse an enterprise. According to Bischoff et al. [
4], the class of lactic acid bacteria (LAB) that includes
Lactobacillus,
Pediococcus,
Leuconostoc, and
Weissella causes the most problems during fermentation. During fermentation, yeast converts fermentable sugars (from starch degradation) to ethanol. Conversely, bacteria transform the same sugars to lactic or acetic acid. When bacteria are not controlled, yields can drop significantly, which is regarded as a huge economic loss for producers [
5].
This has led to the wide application of antibiotics. Antibiotics such as penicillin, virginiamycin, erythromycin, tylosin, and tetracycline are effective against these LABs, killing them and leaving yeast unharmed. The most commonly used antibiotics in ethanol production are penicillin and virginiamycin [
6]. Continuous use of these antibiotics can lead to the development of resistant strains, which could be difficult to manage. Therefore, using sapropel extracts as an alternative measure when dealing with the menace of contamination during ethanol production was proposed.
Sapropel is defined as the benthos, found in fresh water, formed under anaerobic conditions from a dead organic matter of hydrobiotic microflora and microfauna. It is principally composed of nutrients (i.e., sugars, minerals, lipids, etc.) and organic compounds known as humic substances (HS).
Sapropels and sapropel extracts have been previously reported to exhibit some antibacterial and antifungal properties, hence could be used as an alternative and novel antibiotic. The antimicrobial properties of sapropels can be attributed due to the presence of HS [
7,
8,
9].
Therefore, the purpose of this paper is to study the antimicrobial potency of ultradisperse humic sapropel suspensions (UDHSS) and its effects on the chemical composition of barley grains, parameters of wort during and after mashing, fermentation, and on volatile compounds of ethyl alcohol.
2. Materials and Methods
The objects of the study were ultradisperse humic sapropel suspensions (UDHSS) obtained from the Russian Academy of Sciences (RAS) Limnology Institute, St Petersburg, Russia. The source of the sapropel is Seryodka Lake, Pskov, Russia.
The sapropel used was extracted via the hot method at 40 °C (104 °F) at pH 11.8 and 3.7 of the concentration of dry matter. Barley grains were purchased from the Narovny market, St Petersburg, Russia.
An amount of 20 mL of UDHSS 10% dry matter and pH solution of 6.7 was sprinkled on 100 g of barley grains followed by uniform mixing. The treated grains and the control samples were allowed to rest period (undisturbed) for 24 h. The treated sample was then air-dried in cabinet dryer ES-4610 (Reaktivsnab, Shymkent, Kazakhstan) at a temperature of 50 °C to 10–12% moisture content. Both treated and the control samples (10 g) were suspended in 100 mL sterile phosphate buffer solution (PBS (8 g NaCl, 0.2 g KCl, 1.44 g Na
2HPO
4, 0.24 g KH
2PO
4, 1 L distilled water, pH = 7.4) and mixed for 30 min on a shaker. Again, 1 mL of mash and the wash (treated and control) were aseptically pipetted into 100 mL sterile PBS. The mixtures were homogeneously mixed with the aid of a sterile glass rod. Serial dilution, inoculation, and quantification were carried out according to the method previously described in Reference [
10]. Beef extract agar medium (Research Center for Pharmacotherapy, Saint Petersburg, Russia) was utilized in this study.
Moisture analyzer MOC-120H (Shimadzu, Tokyo, Japan) was used in assessing the moisture of the barley grains and flour based on the method previously described by ISO/TC 34 [
11].
The starch content of the grains was determined using an optical activity AA-55 automatic Polarimeter (Optical Activity Limited, Cambridgeshire, UK) from the recommendations of the ISO/TC 93-Ewers polarimetric method [
12].
The treated and untreated grains were milled separately using a coffee grinder (Sinbo SCM-2929, Istanbul, Turkey). Milled grain (500 g) was measured and transferred into hand-made mash tuns filled with 1.25 L of warm water (45 °C). The mixture was then placed in a water bath equipped with temperature regulators and a heating system (
Figure 1). Enzyme preparation was then done by adding α-amylase (2.5 unit/1 g of starch) and Xylanase (1 unit/1 g of grains), in warm water (45 °C), to the mixture (Erbslöh, Geisenheim, Germany). The ratio of grains to water was 1:2.5. The temperature of the mash was then increased to 50 °C for 30 min followed by 70 °C with a rest time of 4 h.
During mashing, a portion of the mash was collected every 30 min and centrifuged with a centrifuge (ULAB, Beijing, China) at 5000 rpm for 10 min. The °Brix was measured using a refractometer PTR-46 (Shimadzu, Tokyo, Japan). The Free Amino Nitrogen (FAN) was determined by the Ninhydrin method [
13].
Glucoamylase enzyme (7 unit/g of starch (Erbslöh, Geisenheim, Germany) was added to the mash after it was left to cool down. The yeast was reactivated 15 min before pitching (1 g per 10 mL of warm water (35 °C). The cool mash was pitched with Saccharomyces cerevisiae (1 g of yeasts per 1 L of mash (Lallemand, WI, USA), and kept in an incubator (Guangzhou Kenton Apparatus Company Limited, Guangzhou, China) and allowed to ferment at 30 °C for 72 h.
The degree of carbon dioxide (CO
2) was determined. Each handmade fermenter was equipped with a rubber hose (
Figure 2), which was dipped in water to allow CO
2 to escape while preventing oxygen from entering the fermenter. The mass of each fermenter was measured before and during fermentation at 24 h intervals. The mass of the CO
2 eluting from the fermenters was then quantified using Equation (1).
where
X: Mass of carbon dioxide;
m: Mass of fermenter and mash before fermentation;
m1: Mass fermenter and mash during fermentation;
m2: Mass of mash; and 100: Conversion of the mass of CO
2 in 100 g.
The titratable acidity (TA) of the wash was determined according to the method previously described in Reference [
14] with some modifications. TA was determined by direct titration of the samples with phenolphthalein as the indicator until a slight pink coloring remains for 30 s.
After fermentation, the distillation was performed by measuring 100 mL of fermented wash into a EV311 rotary vacuum evaporator (Lab Tech, Milan, Italy). 60 mL of distilled water was used to rinse the measuring cylinder. The mixture (fermented wash and distilled water) was then transferred to a round-bottom flask, which was then connected to the distillation setup and evaporated at 75 °C at 70 rpm. The distillation continued until 95 mL of distillate (alcohol and water) was obtained. Distilled water (5 mL) was then added to get 100 mL of distillate.
Volatile compounds were determined according to Reference [
15] and Reference [
16]. The method is based on the chromatographic separation of micro impurities in a sample of alcohol-containing liquid and their subsequent detection by a flame ionization detector (FID). Gas chromatography “Crystal 5000.2”, equipped with capillary column HP-FFAP (Santa Clara, CA, USA) 50 m × 0.32 mm × 0.52 μm, was used during the analysis. The temperature of the column prior to and at the end of the experiment was 76 °C and 200 °C, respectively. The temperature of the column thermostat was set to 5 °C/min up to 90 °C, and finally, it was set to 20 °C/min to 200 °C. The evaporation temperature was 180 °С. An injector with flow division: coefficient of the flow division was 1:26.7. The flame ionization detector (FID): Detector temperature was 210 °C. The air consumption was 200 mL/min, hydrogen consumption was 20 mL/min and blowing was 25 mL/min. The initial pressure of the carrier gas—compressed nitrogen (of particular purity) was 60 kPa. After 8.5 min, the pressure increases with a gradient of 30 kPa per minute up to 145 kPa. Without pre-treatment 0.3 μL of the ethanol was injected in the splitless mode (vent time, 60 s) and the compounds were identified by comparing the mass spectra obtained with Mass Spectral Library of the National Institute of Standards and Technology (NIST). The range of measured volume fractions of methanol was from 0.0001% to 0.0500% and the mass concentrations of other toxic micro-impurities from 0.5 to 10.0 mg/dm
3.
Data Analysis
Data generated were subjected to analysis of variance (ANOVA) using Origin statistical software (version 8.1 (Northampton, MA, USA) at 5% significance. All measurements were made in at least triplicate. Results were reported as means ± standard deviations.