Next Article in Journal
A Control Alternative for the Hidden Enemy in the Wine Cellar
Previous Article in Journal
Pesticide Residues and Stuck Fermentation in Wine: New Evidences Indicate the Urgent Need of Tailored Regulations
Previous Article in Special Issue
Free Amino Nitrogen in Brewing

Fermentation 2019, 5(1), 24;

Effects of Ultradisperse Humic Sapropel Suspension on Microbial Growth and Fermentation Parameters of Barley Distillate
Department of Food Biotechnology for Plant Origin Products, Faculty of Food Biotechnologies and Engineering, St. Petersburg National Research University of Information Technologies, Mechanic, and Optics (ITMO University), 9 Lomonosova street, St. Petersburg 191002, Russia
International Laboratory “Solution Chemistry of Advanced Materials and Technologies” (SCAMT), St. Petersburg National Research University of Information Technologies, Mechanic, and Optics (ITMO University), 9 Lomonosova street, St. Petersburg 191002, Russia
Correspondence: [email protected] or [email protected]; Tel.: +7-9819182731
These authors contributed equally to this work.
Present address: Department of Food Science, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand.
Received: 4 February 2019 / Accepted: 20 February 2019 / Published: 26 February 2019


Barley and other cereal grains can be used in the production of ethanol. The quality and safety of the grains utilized have enormous effects on the overall yield and quality of the final product (ethanol). Therefore, the present paper seeks to elucidate the antimicrobial activities of ultradisperse humic sapropel suspensions (UDHSS) on barley, wort, fermentation, and the quality of the final product. A standard microbiological method was used to assess the biocidal activities. Physicochemical parameters and volatile compounds were determined. Treated samples exhibited least microbial growth (for grain: 1.145 ± 0.120 × 104 cfu/g) when compared to the control (3.425 ± 0.33 × 105 cfu/g). Mash from the treated sample had less Free Amino Nitrogen (35.14 ± 0.02 mg/L) than the control experiment (41.42 ± 0.01). However, the levels of °Brix and Free Amino Nitrogen (FAN) were unaffected by the UDHSS treatments. After the chromatographic analysis, it was revealed that the barley distillate obtained from treated grains had high volatiles concentration when compared to the control experiment. The volume of the methanol quantified in the distillate was low, and hence safe, and might find applications in the food industries or in domestic consumption after rectification.
antimicrobial effects; mashing; distillation; volatile compounds; gas chromatography; reactive oxygen species

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 Na2HPO4, 0.24 g KH2PO4, 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 (CO2) was determined. Each handmade fermenter was equipped with a rubber hose (Figure 2), which was dipped in water to allow CO2 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 CO2 eluting from the fermenters was then quantified using Equation (1).
X = ( m m 1 m 2 ) × 100 ,
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 CO2 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/dm3.

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.

3. Results and Discussion

3.1. Microbial Assessment

The incidence of microbial load for the treated sample ranged from 1.145 ± 0.120 × 104 cfu/g, 1.55 ± 0.212 × 103 cfu/mL, and 2.07 ± 0.127 × 102 cfu/mL for grains, mashed wort, and wash (sampled during fermentation), respectively. Whereas the control differs from 3.425 ± 0.33 × 105 cfu/g, 2.904 ± 0.141 × 104 cfu/mL, and 3.335 ± 0.205 × 103 cfu/mL for grains, mashed wort and wash, respectively (Table 1). With respect to the control (experiment) grains had the highest microbial load followed by the mashed wort and the least been the wash. On the other hand, microbial growth was recorded with the treated samples where grains showed the highest growth followed by the mashed wort and the wash. From these results, it can be concluded that the UDHSS exhibited some antimicrobial properties, which had inhibited/reduced the proliferation of the microbes on the treated samples, as compared with the control.
UDHSS, and its isomers, were reported previously to exhibit antibacterial and antifungal properties [8,9,17], the biocidal actions were attributed to the fulvic acids (FA), humic acids (HA), mumie, and humin which are the principal constituents of HS in UDHSS [18,19,20,21]. Furthermore, HS has been documented to have inflicted damage on DNA with further growth arrest and apoptosis. This damage was ascribed to the reactive oxygen species (ROS) generated as the result of the HS [22]. Microbial contamination of grains is inevitable since the entire production process (during crop growth, harvesting, postharvest drying, and storage) is a possible source of contamination [23]. Contaminated grains could ruin (i.e., spoilage or off-flavour generation in beverage) an entire production line, thus causing financial loss to the brewer/distillers and dissatisfaction to the consumers since it is unpleasant to drink poorly flavored beverages. However, the presence of alcohol, the bitter compounds in hops, have low pH and exert antimicrobial effects on microbes in the products [24,25]. UDHSS has great potential in the fermentation since it was able to reduce the microbial load on the treated sample, and furthermore, its application will not create resistant strains and has no effects on the environment and to the consumer when compared with conventional antibiotics. The potency of the UDHSS could have been improved by evaporation to increase the concentration to 20%. For instance, 10 mL of UDHSS would have been more effective than the current 20 mL of the 10% applied.

3.2. Moisture and Starch Content of Barley Grains

The starch content of the treated and the control were 58.8 ± 0.2 and 58.3 ± 0.3, respectively (Table 2), with a 0.5% increase after treatment, which was not significantly different (P > 0.05). Starch is the major source of carbohydrates in cereals and therefore plays an important role in the production of alcohol. The hydrolysis of starch to glucose requires enzymes such as alpha and beta-amylase, glucoamylase, etc. The starch content of barley accounts for 55–70% of the total carbohydrates [26]. However, the starch content varies due to (1) genetics, (2) geographical location, (3) length of exposure to light (photoperiodism), and other such factors [27]. According to Patron et al. [28], the mutation of the lys5 gene resulted in a drastic decrease in starch content. The alpha-amylase utilized is of a bacterial source and is thermostable hydrolyzing α-1, 4 bonds at random points on the starch molecule to rapidly reduce the viscosity of gelatinized starch solutions. It is a metal ion-containing enzyme requiring a calcium ion as its cofactor for maximum activity and stability.
The second enzyme used, glucoamylase, hydrolyzes the maltose and dextrins from the non-reducing end of the molecule. Sammartino [29] has reported that glucoamylase hydrolyzes both α-1, 4 and α-1, 6 bonds to completely degrade the dextrins to glucose.
The moisture content of the treated grains (10.57 ± 0.03) was found to be lower when compared to the control (P < 0.05). The treated samples were dried after treatment and this could have caused a decrease in the moisture, as seen in Table 2. The low moisture content of malt or barley is good for brewers as it impedes the growth of microorganisms, thereby minimizing the risk of contamination. In contrast, high moisture content in grains supports the growth of fungi and other spoilage organisms, resulting in contamination of the grains. Contaminated grains are not utilized in ethanol production since it could affect the quality, contaminate the production lines, and decrease the overall yield and profit of a company. The moisture content of cereals indicates their safety, the quality, and shelf life [30]. As reported by Belitz and colleague [31], the moisture content of barley grains is in the range of 11–14%.

3.3. °Brix

The general overview of the fermentable sugars accumulation in the mash was measured, and the results are recorded in Figure 3. After 30 min of the mashing, the sugars were low in the control (5.967 ± 0.058) and the treated sample (6.133 ± 0.058) and this could be attributed to the fact that mashing began at low temperature, which had little influence on the enzymatic activities. However, an increase in temperature resulted in the drastic increase in enzymatic activities, consequently increasing the concentration of the fermentable sugars in the mash. This trend was consistent in the control and the treated sample. There is direct correlation between temperature and the rate of enzymatic activity coupled with the pH of the mash.
At the end of the mashing, the treated mash had slightly higher (21.267 ± 0.058) °Brix when compared to the control (20.533 ± 0.115). This could be attributed to the fact that the HS might slow the rate of the exogenous enzyme on the treated grains. According to Brigg et al. [32], 90–92% of the solids in the brewing wort are carbohydrates, which consist of sucrose, fructose, glucose, maltose, maltotriose, as well as dextrin. Moreover, 95% of those carbohydrates are products of the starch hydrolysis, which takes place in the mash tun.
The composition of carbohydrates in wort and its utilization by yeast has significant effects on fermentation efficiency and yeast metabolism, as well as the organoleptic profile of the final product [33]. β-amylase is not a thermostable enzyme and therefore prone to rapid denaturation at temperatures above 55 °C. Therefore, a thermostable α-amylase is employed to carry the reaction forward at a higher temperature to increase the yield of the ethanol [34].

3.4. Free Alpha Amino Nitrogen (FAN) of Mash

In the present study, the concentration of FAN in the treated sample was less (35.14 ± 0.02) than that of the control sample (41.42 ± 0.01) (Table 3). The amino acids constitute an important fraction of the wort and the determination of FAN is of interest in experimental work and in routine control of products in order to establish its bioavailability. Yeasts consume amino acids as a source of nitrogen during fermentation. The determination of FAN is required to assess yeast performance [35].
The yeast cells require nitrogenous compounds (e.g., individual amino acids, ammonium ions, and small peptides) for basic metabolism. Assimilable nitrogen or FAN, which can be defined as the sum of the individual wort amino acids, ammonium ions, and low molecular weight peptides [36]. The formation of volatiles is related to the concentration of these nitrogenous compounds, and their presence is not only vital for yeast performance, but also to obtain quality products. The low concentration of FAN in mash could be attributed to the humic substances (HA, FA, and humin) in the applied UDHSS. Proteases are vital mashing enzymes. During the mashing, proteases break down proteins into amino acids (via proteolysis). Proteolysis of the treated sample was inhibited due to the presence of HA. According to Ladd and Butler [37], HA inhibit proteolytic enzymes by binding to proteases via a cation-exchange mechanism, which allows the amino groups to link with the humic carboxyl groups. The inhibition of proteases affected proteolysis, thereby decreasing the amino acid concentration of the mash.

3.5. Variation of Carbon Dioxide during Fermentation

The mass (g) of CO2released during fermentation was quantified and recorded in Table 4. The CO2 released from the control fermenter (6.950 ± 0.031 g) was slightly higher when compared to the treated vessel (6.870 ± 0.020 g) after 24 h of fermentation. However, the dynamic changed after 48 h of fermentation where the treated vessel recorded the higher release (8.508 ± 0.022) than the control (8.474 ± 0.012 g). After 72 h, 8.846 ± 0.04 and 8.824 ± 0.013 g of CO2 was released from the control and treated vessel, respectively.
During fermentation, fermentable sugars are transformed to CO2 and alcohol. The metabolic activity of yeast could be related to the amount of CO2, alcohol, and energy released during fermentation. The increase in the evolving CO2 in the present work showed that the yeast performance (i.e., metabolic activity) was high and could be molded to increase yield. However, HS (HA and humin), or other elements present in UDHSS [38,39], do not seem to have significant effects on the released CO2.

3.6. Titratable Acidity

During fermentation, the TA of the samples were determined and the results were recorded in Table 5. The TA of both samples was low 0.443 ± 0.040 and 0.396 ± 0.015 on the first day, however, the control showed an increase, while the treated sample remained unchanged until the third day when it increased to 0.510 ± 0.010. There was a drastic decrease of TA (control) from 0.443 ± 0.040 to 0.406 ± 0.015 on the third day.
TA correlates to the acid taste of a product. The lower the pH value, the higher the TA [40,41,42,43]. The increment of TA is very important during fermentation because it affects the pH of the mash. Yeast metabolites, i.e., lactic, malic, citrus, and acidic acids could be attributed to the changes in the TA [41]. Low pH promotes the growth of yeasts because they flourish best in pH as low as 2.0. The yeast, as a living organism, can regulate its own intracellular pH [44]. On the other hand, bacteria cannot tolerate acidic conditions. The increase in TA during fermentation inhibits the growth of bacterial curbing the menace of cross contamination in a production line. Lower beverage (i.e., beer) pH is one of the essential properties, which gives it microbial and physical stability [45].

3.7. Ethyl Alcohol Analysis: Volatile Compounds Determination

A total of 13 compounds were identified including two aldehydes, one ester, and 10 alcohols. Twelve volatiles were detected in the product produced from the treated samples, whereas 13 were detected in the control. Among the compounds, only 1-butanol was not identified in the treated samples, and on the other hand, 1-pentanol and hexanol were detected in the product (control). The concentration of volatile compounds in treated grains is higher than the control (Table 6). Acetaldehyde, methyl acetate, ethyl acetate, methanol, 2-propanol, 1-propanol, isobutyl alcohol, n-butanol, and isoamyl alcohol are the main volatiles detected in the in spirit drinks. The high concentration of volatiles in the treated sample could be attributed to the fact that UDHSS had elevated the amount of sugars in the grain hence increase the amount of the compounds detected. Higher alcohols (HA) are compounds that have more carbon atoms than ethanol and contribute to beverage flavor due to their solvent-like aroma resulting in a warm mouthfeel [46,47]. The efficient uptake and utilization of amino acid and sugar determined the concentration of HA [42]. The precursors for the formation of HA are formed or synthesized during proteolysis of proteins to amino acids in the mash. The type of mashing protocol adopted could significantly affect the quantity of HA [48].
HA was as follows: Isoamylol (0 and 38.495 mg/dm3), isobutanol (31.0407 and 14.8179 mg/dm3), 1-propanol (30.0025 and 16.1658 mg/dm3), ethanol (7.6 and 5.0 mg/dm3), 2-propanol (1.1315 and 0.6711 mg/dm3), 1-pentanol (0 and 0.1992 mg/dm3), methanol (0.0004 and 0.0002 mg/dm3), and hexanol, (0.4580 and 0 mg/dm3) in the treated and the control, respectively. HA, also called fusel oil, has a negative impact on the quality products [48,49]. It can be observed that the ethanol content in the treated was more than 20% higher when compared to the control and the possible hypothesis is attributed to the number of nutrients in the UDHSS applied. The minerals, vitamins, etc., might have provided well-balanced nutritional requirements for the yeast resulting in rapid fermentation thus forming more ethanol than in the control. The nutritional composition of UDHSS was previously reported [8]. Similarly, Reference [50] identified 40 volatiles in spirit drinks.
The concentration of methanol (wood alcohol) detected was low in treated (0.0004% v/v) and the control (0.0002% v/v). Methanol is a toxic compound and is lethal to the consumer when a high volume (30–50 g) is ingested [51]. However, the amount detected in this study is low to cause any complication when consumed. Therefore, the distillate is safe for drinking after rectification and further analysis.
Esters are volatiles formed during a vigorous phase of fermentation by the enzymatic chemical condensation of acids and alcohols [32]. The quantity of ester (ethyl acetate) identified was 0.3 mg/L and 1 mg/L in the treated and control sample, respectively. Ethyl acetate is the most abundant ester in alcoholic beverages. The concentration of esters in spirits depends on the type of raw material, yeast strain employed, cleanliness of the environment, and the mash pH. The low concentrations of ethyl acetate mask bad flavor in beverages. However, at high concentrations, it gives a ‘vinegar flavor’ to spirits [52,53,54].
According to Ferreira et al. [55], aldehydes can be synthesized by the direct reaction of sugar (precursor) with amino acids or via transition metal ion-catalyzed oxidation of the amadori compound. 0.2 mg/L of acetaldehyde was detected in the treated sample, whereas 0.3 mg/L was found in the control experiment. Notably, benzaldehyde a predominant aldehyde in beer was 16 and 14 mg/L for the treated and the control experiment, respectively. Aldehydes (acetaldehyde and others) could negatively affect the quality of raw spirits. As reported by Reference [56], aldehydes cause an unpleasant taste and odor in spirits even when present at low concentrations. In previous studies [57,58], phenylacetaldehyde was detected in lager beer. Analysis of agricultural distillates (spirits) is of the utmost importance not only for the legal requirements that need be fulfilled to use them in the production of spirit-based beverages, but also because it is affecting the quality [59] and consequently consumer preference for a particular product.

4. Conclusions

In this study, the antimicrobial potency of UDHSS on barley grain, mash, and wash was assessed as well as parameters of wort and fermented wash during fermentation. The results revealed that UDHSS could replace conventional antibiotic currently employed in curbing the menace of microbial contamination of grains in the food and fermentation industries. However, a comparative study with traditional antibiotics is recommended in the future. The application of UDHSS is regarded as safe and poses no threat to the consumer and the environment, coupled with it effectively unleashing potent ROS, thus killing the microbes. The antimicrobial properties of UDHSS make it a promising agent in food industries. However, an overdose during application could also cause other spoilage organisms to grow because of the nutritional composition in the UDHSS [8] itself. Hence, an extensive study is required to establish the right volume to apply since the efficacy of the antimicrobial activities is also dependent on it. The study also showed that UDHSS affected some parameters of barley, fermentation, and the final product (ethanol). The results of gas chromatography prove that UDHSS had played a role in the increment of volatile compounds. Comparing the control and the treated sample revealed that the amount of methanol was too low to cause any complication when ingested. Moreover, the concentration in the former was high when compared to the latter. The lower concentration of volatile compounds in the distillate from the treated sample proves that it can find application in the food industry. After rectification, it could be used as drinking alcohol. However, rigorous and extensive study is required to decipher the effects of UDHSS on fermentation and the formation volatiles in the final product based on different types of barley.

Author Contributions

Conceptualization, D.N., P.A., and N.V.B.; Methodology, D.N., P.A., and N.V.B.; Software, P.A.; Validation, P.A. and M.V.U.; Formal Analysis, D.N. and P.A.; Investigation, D.N., P.A. and M.V.U.; Resources, N.V.B.; Writing–Original Draft Preparation, D.N., and P.A.; Writing–Review & Editing, P.A.; Visualization, M.V.U.; Supervision, N.V.B.; Funding Acquisition, N.V.B.


The government of the Russian Federation, Grant 08-08, financially supported this work.


The authors are grateful to the Institute of Limnology, Russian Academy of Sciences, Saint Petersburg, Russian Federation for providing the Ultradisperse Humic Sapropel Suspension (UDHSS).

Conflicts of Interest

The authors declare no conflicts of interest.


  1. Raneses, A.; Hanson, K.; Shapouri, H. Economic impacts from shifting cropland use from food to fuel biomass bioenergy. Biomass Bioenergy 1998, 15, 417–422. [Google Scholar] [CrossRef]
  2. Sheetal, B.G.; Patil, I.D. Utilization of cereal grains for bioethanol production. IJSSBT 2015, 3, 5–9. [Google Scholar]
  3. Murghagh, J.E. Production of neutral spirits and preparation of gin and vodka. In The Alcohol Textbook: A Reference for the Beverage, Fuel and Industrial Alcohol Industries, 3rd ed.; Jacques, K.A., Lyons, T.P., Kelsall, D.R., Eds.; Nottingham University Press: Nottingham, UK, 1999; pp. 195–210. [Google Scholar]
  4. Bischoff, K.M.; Liu, S.; Leathers, T.D.; Worthington, R.E.; Rich, J.O. Modelling bacterial contamination of fuel ethanol fermentation. Biotechnol. Bioeng. 2009, 103, 117–122. [Google Scholar] [CrossRef] [PubMed]
  5. Narendranath, N.V.; Hynes, S.H.; Thomas, K.C.; Ingledew, M.W. Effects of lactobacilli on yeast-catalyzed ethanol fermentations. J. Appl. Environ. Microbiol. 1997, 63, 4158–4163. [Google Scholar]
  6. Lushia, W.; Heist, P. Antibiotic-resistant bacteria in fuel ethanol fermentations. Ethanol. Prod. Mag. 2005, 80–82. [Google Scholar]
  7. Kireicheva, L.V.; Khokhlova, O.B. Sapropels: Composition, Properties, Applications; Roma: Amsterdam, The Netherlands, 1998; p. 120. Available online: (accessed on 26 February 2019). (In Russian)
  8. Barakova, N.V.; Sharova, N.Y.; Juškauskaite, A.R.; Mityukov, A.S.; Romanov, V.A.; Nsengumuremyi, D. Fungicidal activity of ultradisperse humic sapropel suspensions. Agron. Res. 2017, 15, 639–648. [Google Scholar]
  9. Nsengumuremyi, D.; Barakova, N.V.; Romanov, V.A.; Mityukov, A.S.; Guzeva, A.V. The effect of sapropel extracts on microflora and physicochemical parameters of dried distillers grain. Agron. Res. 2018, 16, 1457–1465. [Google Scholar]
  10. Adadi, P.; Obeng, A.K. Assessment of bacterial quality of honey produced in Tamale metropolis (Ghana). J. Food Drug Anal. 2017, 25, 369–373. [Google Scholar] [CrossRef] [PubMed][Green Version]
  11. International Organization for Standardization Technical Committee 34 (ISO/TC 34). ISO 712: 2009 Cereals and Cereal Products Determination of Moisture Content. 2009. Available online:!iso:std:44807:en (accessed on 8 October 2018).
  12. International Organization for Standardization Technical Committee 93 (ISO/TC 93). ISO10520:1997 Native Starch Determination of Starch Content, Ewers Polarimetric. 1997. Available online: (accessed on 26 February 2019).
  13. Lie, S. The EBC-Ninhydrin method for determination of free alpha amino nitrogen. J. Inst. Brew. 1973, 79, 37–41. [Google Scholar] [CrossRef]
  14. GOST 12788-87. Methods of Determining Acidity in Beer. Determination of Acidity by Direct Titration of the Sample with Phenolphthalein. 1987. Available online:ГОСТ_12788-87 (accessed on 8 October 2018). (In Russian).
  15. GOST 30536-2013. Vodka and Ethyl Alcohol: Vodka and Ethanol from Food Raw Material. Gas-Chromatographic Express-Method for Determination of Toxic Microadmixtures Content. 2013. Available online: (accessed on 8 October 2018). (In Russian).
  16. GOST 32039-2013. Vodka and Ethanol from Food Raw Material. Gas-Chromatographic Method for Determination of Authenticity. 2013. Available online: (accessed on 8 October 2018). (In Russian).
  17. Wu, M.; Song, M.; Liu, M.; Jiang, C.; Li, Z. Fungicidal activities of soil humic/fulvic acids as related to their chemical structures in greenhouse vegetable fields with cultivation chronosequence. Sci. Rep. 2016, 6, 32858. [Google Scholar] [CrossRef] [PubMed][Green Version]
  18. Shtin, S.M. Lake Sapropels Complex Utilization; MSU: Moscow, Russia, 2005; p. 213. (In Russian) [Google Scholar]
  19. Kosov, V.I. Sapropel. Resources, Engineering, Geoecology, Saint Petersburg, Nauka, Russia. 2007, p. 244. Available online: https://xn--90ax2c.xn--p1ai/catalog/000200_000018_RU_NLR_bibl_1156546/ (accessed on 26 February 2019). (In Russian).
  20. Schepetkin, I.; Khlebnikov, A.; Kwon, B.S. Medical drugs from humus matter: Focus on mumie. Drug Dev. Res. 2002, 57, 140–159. [Google Scholar] [CrossRef]
  21. Buzlama, A.V.; Chernov, Y.N. Pharmacological properties, action pathways and prospectives of humic substances in medicine: An analysis. Eksperimental’naia i Klinicheskaia Farmakologiia 2010, 73, 43–48. (In Russian) [Google Scholar] [PubMed]
  22. Cheng, M.L.; Ho, H.Y.; Huang, Y.W.; Lu, F.J.; Chiu, D.T.Y. Humic acid induces oxidative DNA damage, growth retardation, and apoptosis in human primary fibroblasts. Exp. Biol. Med. 2003, 228, 413–423. [Google Scholar] [CrossRef]
  23. Magan, N.; Aldred, D. Managing microbial spoilage in cereals and baking products. In Food Spoilage Microorganisms; de Blackburn, C., Ed.; Woodhead Publishing Ltd.: Cambridge, UK, 2006; pp. 194–212. [Google Scholar]
  24. Hill, A.E. Microbiological stability of beer. In Beer a Quality Perspective; Bamforth, C., Ed.; Academic Press: Cambridge, UK, 2009; pp. 163–184. [Google Scholar]
  25. Suzuki, K. 125th Anniversary review: Microbiological instability of beer caused by spoilage bacteria. J. Inst. Brew. 2011, 117, 131–155. [Google Scholar] [CrossRef]
  26. Goesaert, H.; Brijs, C.; Veraverbeke, W.S.; Courtin, C.M.; Gebruers, K.; Delcour, J.A. Wheat constituents: How they impact bread quality, and how to impact their functionality. Trends Food Sci. Technol. 2005, 16, 12–30. [Google Scholar] [CrossRef]
  27. Zhu, X.; Cui, W.; Zhang, E.; Sheng, J.; Yu, X.; Xiong, F. Morphological and physicochemical properties of starches isolated from three taro bulbs. Starch/Stärke 2018, 70, 1700168. [Google Scholar] [CrossRef]
  28. Patron, N.J.; Greber, B.; Fahy, B.; Laurie, D.A.; Parker, M.L.; Denyer, K. The lys5 mutations of barley reveal the nature and importance of plastidial ADP-Glc transporters for starch synthesis in cereal endosperm. Plant Physiol. 2004, 135, 2088–2097. [Google Scholar] [CrossRef] [PubMed]
  29. Sammartino, M. Enzymes in Brewing. MBAA TQ 2015, 52, 156–164. [Google Scholar]
  30. Nelson, O.A.; Hulett, G.A. The moisture content of cereals. J. Ind. Eng. Chem. 1920, 12, 40–45. [Google Scholar] [CrossRef]
  31. Belitz, H.D.; Grosch, W.; Schieberle, P. Cereals and cereal products. In Food Chemistry, 4th ed.; Belitz, H.-D., Grosch, W., Schieberle, P., Eds.; Springer: Berlin, Germany, 2009; pp. 670–675. [Google Scholar]
  32. Briggs, D.E.; Boulton, C.A.; Brookes, P.A.; Stevens, R. Brewing: Science and Practice; Woodhead: Cambridge, UK, 2004; Volume 1, p. 881. [Google Scholar]
  33. Boulton, C.; Quain, D. Brewing Yeast and Fermentation, 1st ed.; Blackwell Science: Oxford, UK, 2001; p. 659. [Google Scholar]
  34. Muller, R.E. The effects of mashing temperature and mash thickness on wort carbohydrate composition. J. Inst. Brew. 1991, 97, 85–92. [Google Scholar] [CrossRef]
  35. Heggart, H.M.; Margaritis, A.; Pilkington, H.; Stewart, R.J.; Dowhanick, T.M.; Russell, I. Factors affecting yeast viability and vitality characteristics: A review. Master Brew. Assoc. Am. Tech. Q. 1999, 36, 383–406. [Google Scholar]
  36. He, Y.; Dong, J.; Yin, H.; Zhao, Y.; Chen, R.; Wan, X.; Chen, P.; Hou, X.; Liu, J.; Chen, L. Wort composition and its impact on the flavour-active higher alcohol and ester formation of beer—A review. J. Inst. Brew. 2014, 120, 157–163. [Google Scholar] [CrossRef]
  37. Ladd, J.N.; Butler, J.H.A. Inhibition by soil humic acids of native and acetylated proteolytic enzymes. Soil Biol. Biochem. 1971, 3, 157–160. [Google Scholar] [CrossRef]
  38. Bobrov, V.A.; Fedorin, M.A.; Leonova, G.A.; Markova, Y.N.; Orlova, L.A.; Krivonogov, S.K. Investigation into the elemental composition of sapropel from Lake Kirek (West Siberia) by SR XFA technique. J. Surf. Investig. X-ray Synchrotron Neutron Tech. 2012, 6, 458–463. [Google Scholar] [CrossRef]
  39. Platonov, V.V.; Chunosov, S.N.; Fridzon, K.Y. Biological action of sapropel. Fund Res. 2014, 9–11, 2474–2480. (In Russian) [Google Scholar]
  40. Tyl, C.; Sadler, G.D. pH and Titratable Acidity. In Food Analysis; Nielsen, S., Ed.; Springer: Basel, Switzerland, 2017; pp. 389–405. [Google Scholar]
  41. Adadi, P.; Kovaleva, E.G.; Glukhareva, T.V.; Shatunova, S.A.; Petrov, A.S. Production and analysis of non-traditional beer supplemented with sea buckthorn. Agron. Res. 2017, 15, 1831–1845. [Google Scholar]
  42. Adadi, P.; Kovaleva, E.G.; Glukhareva, T.V.; Shatunova, S.A. Biotechnological production of non-traditional beer. AIP Conf. Proc. 2017, 1886, 020098. [Google Scholar] [CrossRef]
  43. Adadi, P.; Kovaleva, E.G.; Glukhareva, T.V.; Barakova, N.V. Production and investigations of antioxidant rich beverage: Utilizing Monascus purpureus IHEM LY2014-0696 and various malts. Agron. Res 2018, 16, 1312–1321. [Google Scholar]
  44. Gray, W.D. Studies on the alcohol tolerance of yeasts. J. Bacteriol. 1941, 42, 561–574. [Google Scholar] [PubMed]
  45. O’Rourke, T. The role of pH in brewing. Brewer Int. 2002, 2, 21–23. [Google Scholar]
  46. Lewis, M.J.; Young, T.W. Brewing, 2nd ed.; Springer: Berlin, Germany, 2002; p. 398. [Google Scholar]
  47. André, M.O.M. Continuous Fermentation of Alcohol-Free Beer—Bioreactor Hydrodynamics and Yeast Physiology. Ph.D. Thesis, University of Minho, Braga, Portugal, 2012. [Google Scholar]
  48. Charapitsa, S.V.; Kavalenka, A.N.; Kulevich, N.V.; Makoed, N.M.; Mazanik, A.L.; Sytova, S.N.; Zayats, N.I.; Kotov, Y.N. Direct determination of volatile compounds in spirit drinks by gas chromatography. J. Agric. Food Chem. 2013, 61, 2950–2956. [Google Scholar] [CrossRef] [PubMed]
  49. Patil, A.G.; Koolwal, S.M.; Butala, H.D. Fusel oil: Composition, removal and potential utilization. Int. Sugar J. 2002, 104, 51–58. [Google Scholar]
  50. Stanisz, M.; Sapińska, E.; Pielech-Przybylska, K. Characteristics of contaminants present in raw spirits. Zeszyty Naukowe Chemia Spożywczai Biotechnologia 2009, 73, 105–121. [Google Scholar]
  51. Tarko, T. Components of the aroma of alcoholic beverages. Laboratorium 2006, 11, 39–42. (In Polish) [Google Scholar]
  52. Briggs, D.E.; Boulton, C.A.; Brookes, P.A.; Stevens, R. Metabolism of Wort by Yeast: Brewing Science and Practice; CRC Press: New York, NY, USA, 2004. [Google Scholar]
  53. Vallejo-Cordoba, B.; González-Córdova, A.F.; del Carmen Estrada-Montoya, M. Tequila volatile characterization and ethyl ester determination by solid phase microextraction gas chromatography/mass spectrometry analysis. J. Agric. Food Chem. 2004, 52, 5567–5571. [Google Scholar] [CrossRef] [PubMed]
  54. Branyik, T.; Vicente, A.A.; Dostalek, P.; Teixeira, J.A. A review of flavour formation in continuous beer fermentations. J. Inst. Brew. 2008, 114, 3–13. [Google Scholar] [CrossRef]
  55. Ferreira, I.M.; Luís, F.; Guido, L.F. Impact of wort amino acids on beer flavour: A review. Fermentation 2018, 4, 23. [Google Scholar] [CrossRef]
  56. Berry, D.R.; Slaughter, J.C. Alcoholic beverage fermentations. In Fermented Beverage Production, 2nd ed.; Lea, A.G.H., Piggott, J.R., Eds.; Springer Science & Business Media: New York, NY, USA, 2003; pp. 25–39. [Google Scholar]
  57. Vesely, P.; Lusk, L.; Basarova, G.; Seabrooks, J.; Ryder, D. Analysis of aldehydes in beer using solid-phase microextraction with on-fiber derivatization and gas chromatography/mass spectrometry. J. Agric. Food Chem. 2003, 51, 6941–6944. [Google Scholar] [CrossRef] [PubMed]
  58. Saison, D.; De Schutter, D.P.; Uyttenhove, B.; Delvaux, F.; Delvaux, F.R. Contribution of staling compounds to the aged flavour of lager beer by studying their flavour thresholds. Food Chem. 2009, 114, 1206–1215. [Google Scholar] [CrossRef]
  59. Wiśniewska, P.; Śliwińska, M.; Dymerski, T.; Wardencki, W.; Namieśnik, J. The analysis of raw spirits—A review of methodology. J. Inst. Brew. 2016, 122, 5–10. [Google Scholar] [CrossRef]
Figure 1. Hand-made mash tun in a water bath during the mashing process.
Figure 1. Hand-made mash tun in a water bath during the mashing process.
Fermentation 05 00024 g001
Figure 2. The hand-made fermenters with pipes in the incubator during fermentation.
Figure 2. The hand-made fermenters with pipes in the incubator during fermentation.
Fermentation 05 00024 g002
Figure 3. °Brix of the mash during the mashing of processes.
Figure 3. °Brix of the mash during the mashing of processes.
Fermentation 05 00024 g003
Table 1. Microbial of load samples after treatment (Mean ± S.D × CFU/mL (g)).
Table 1. Microbial of load samples after treatment (Mean ± S.D × CFU/mL (g)).
Sampling PointsTreatedControl
Grains1.145 ± 0.120 × 104 cfu/g3.425 ± 0.33 × 105 cfu/g
Mashed wort1.55 ± 0.212 × 103 cfu/mL2.904 ± 0.141 × 104 cfu/mL
Wash2.07 ± 0.127 × 102 cfu/mL3.335 ± 0.205 × 103 cfu/mL
S.D = Standard deviation. Cfu/mL= colony forming unit per milliliter. Cfu/g = colony-forming units per gram.
Table 2. Moisture and starch content of barley grains before and after treatment.
Table 2. Moisture and starch content of barley grains before and after treatment.
SampleStarch Content (%)Moisture Content (%)
Treated58.8 ± 0.210.57 ± 0.03
Control58.3 ± 0.311. 62 ± 0.02
Table 3. Free Amino Nitrogen of the mash.
Table 3. Free Amino Nitrogen of the mash.
ParameterControl, mg/LTreated, mg/L
Free Amino Nitrogen (FAN)41.42 ± 0.0135.14 ± 0.02
Table 4. Carbon dioxide released during fermentation (g of CO2 per 100 g of mash).
Table 4. Carbon dioxide released during fermentation (g of CO2 per 100 g of mash).
Time (hours)Control (g)Treated (g)
246.950 ± 0.0316.870 ± 0.020
488.474 ± 0.0128.508 ± 0.022
728.846 ± 0.048.824 ± 0.013
Table 5. Titratable acidity of treated and untreated fermented wash during fermentation.
Table 5. Titratable acidity of treated and untreated fermented wash during fermentation.
DayControl (°)Treated (°)
10.396 ± 0.0150.443 ± 0.040
20.443 ± 0.0400.443 ± 0.040
30.406 ± 0.0150.510 ± 0.010
Table 6. Volatile compounds found in distillate.
Table 6. Volatile compounds found in distillate.
NumberCompoundsControl Sample, mg/dm3Treated Sample, mg/dm3
2Ethyl acetate0.27840.9619
3Methanol *0.00020.0004
5Ethanol *5.08847.6540
* Methanol and ethanol were determined in % v/v (volume per volume). (×) indicate absent of volatile compound.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (
Back to TopTop