Tris(2-hydroxyethyl)ammonium arylchalcogenylacetates, growth stimulants of alcohol yeast Saccharomyces cerevisiae

Article abstract of DOI:10.1007/s11172-017-1893-6

A number of tris(2-hydroxyethyl)ammonium arylchalcogenylacetates ArYCH₂CO₂ ^–? ?HN⁺(CH₂CH₂OH)₃ (1—4) (Ar = aryl; Y = O, S, SO₂) were synthesized. Their structure was es

Full text of DOI:10.1007/s11172-017-1893-6

Russian Chemical Bulletin, International Edition, Vol. 66, No. 7, pp. 1320—1324, July, 2017
1320
Tris(2ꢀhydroxyethyl)ammonium arylchalcogenylacetates,
growth stimulants of alcohol yeast Saccharomyces cerevisiae
E. A. Privalova,a N. P. Tiguntseva,a S. N. Adamovich,b,c R. G. Mirskov,b,c and A. N. Mirskovab,c
aIrkutsk National Research Technical University,
83 ul. Lermontova, 664074 Irkutsk, Russian Federation.
Eꢀmail: epriv@istu.edu
bA. E. Favorsky Irkutsk Institute of Chemistry, Siberian Branch of the Russian Academy of Sciences,
1 ul. Favorskogo, 664033 Irkutsk, Russian Federation.
Eꢀmail: mir@irioch.irk.ru
cIrkutsk Scientific Center of the Siberian Branch of the Russian Academy of Sciences,
134 ul. Lermontova, 664033 Irkutsk, Russian Federation.
–
A number of tris(2ꢀhydroxyethyl)ammonium arylchalcogenylacetates ArYCH2CO2
•
•HN+(CH2CH2OH)3 (1—4) (Ar = aryl; Y = O, S, SO2) were synthesized. Their structure
was established by IR and 1H, 13C, 15N NMR spectroscopy. The influence of compounds 1—4
on the growth and development of yeast fungi Saccharomyces cerevisiae was studied. Comꢀ
pounds 1—4 were found to be efficient, safe, and available synthetic stimulants of these
processes. The use of compounds 1—4 in low concentrations (from 10–8 to 10–4 wt.%,
depending on the time of cultivation, concentration, and the anion type) reduced the lagꢀ
phases of development and increased the generation rate of yeast cells by 2—5 times. The
amount of yeast cells increased by 2—3 times, while the content of protein in the biomass
increased by 13—15%. The fermentation of sugarꢀ and starchꢀcontaining raw material with
yeast Saccharomyces cerevisiae increased the yield of ethyl alcohol (biofuel) by 5—9%.
Key words: arylchalcogenylacetic acids, tris(2ꢀhydroxyethyl)ammonium salts, stimulants,
cultivation, Saccharomyces cerevisiae yeast, growth rate, biomass, fermentation, ethanol
production.
Microbiological synthesis is the basis for obtaining
We used biologically active arylchalcogenylacetic acꢀ
ids and biogenic ethanolamines (triethanolamine) to synꢀ
thesize5—10 a number of tris(2ꢀhydroxyethyl)ammonium
arylchalcogenylacetates (1—4) by the following Scheme:
food acids, vitamins, alcohols (biofuels). The most largeꢀ
tonnage product obtained by this method is ethyl alcoꢀ
hol, the biosynthesis of which is most often carried out by
cultures of yeast fungi of the Saccharomyces cerevisiae
species. The fermentation of different carbohydrates by
yeast requires from 60 to 70 hours and gives about 60%
yield of ethyl alcohol.1—3 Along with ethanol, a significant
amount of sedimentary yeast is formed, which is a valuable
protein supplement to the fodder for livestock and raw
materials for the production of proteinꢀcontaining agents.1,2
The activation of the metabolic processes of the yeast
cells directed on the increase of the formation of the tarꢀ
get fermentation products will make more efficient the
use of carbohydrates of the nutrient medium and increase
the productivity of yeast, while reducing the consumpꢀ
tion of fermented sugars and the total time of cultivation.
The stimulation of growth and ethanolꢀdirected oriꢀ
entation of yeast metabolism can be achieved by appꢀ
lication of biostimulants. Nowadays, expensive and poorly
soluble natural stimulants such as biotin, ferulic and gibꢀ
berellic acids are used in the industrial production.4
ArYCH2CO2H + N(CH2CH2OH)3
ArYCH2CO2–HN+(CH2CH2OH)3
1—4
where Ar — aryl; Y = O, S, SO2.
The composition and a tricyclic "atrane" structure of
obtained compounds were established by IR and NMR
spectroscopy, elemental analysis, and Xꢀray diffracꢀ
tion.11—16
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1320—1324, July, 2017.
1066ꢀ5285/17/6607ꢀ1320 © 2017 Springer Science+Business Media, Inc.

Growth stimulants of alcohol yeast
Russ. Chem. Bull., Int. Ed., Vol. 66, No. 7, July, 2017
K
1321
It is possible that due to the unique structure, comꢀ
pounds 1—4 possess a synergistic effect and exhibit bioꢀ
logical activity exceeding activity of the parent acids and
amines. Thus, among compounds 1—4 there were found
nontoxic (LD50 = 1300—6000 mg kg–1) compounds,
which are promising for medicine, clinical microbiology
and biotechnology with antioxidant, immunotropic, anꢀ
tiallergenic, antitumor, antimetastatic, protective, growthꢀ
and enzymeꢀstimulating action.5—10
1
2
3
4
3.5
3.0
2.5
2.0
1.5
1.0
The purpose of the present work is the synthesis and
–
1•10–4
1•10–5 1•10–6
1•10–7 1•10–8 C/wt.%
study
of
compounds
4ꢀClꢀC6H4SCH2CO2–
HN+(CH2CH2OH)3 (1), 4ꢀClꢀC6H4SO2CH2CO2–
HN+(CH2CH2OH)3 (2), 2ꢀClꢀC6H4OCH2CO2–
HN+(CH2CH2OH)3 (3) and 4ꢀClꢀC6H4OCH2CO2
HN+(CH2CH2OH)3 (4) as stimulants of growth of race
XII alcohol yeast Saccharomyces cerevisiae.
Fig. 2. The number of cells growth factor (К) in the media with
addition of compounds 1—4 as compared to the control.
nounced effect only in the concentrations of 1•10–8
—
1•10–7 wt.% (Fig. 2). The stimulating effect started to
operate after 10—12 h from the beginning of cultivation,
and after 36 h the amount of yeast cells increased as
compared to the control by 1.8—2.9 times for compounds
1 and 2 and by 2.4—2.8 times for compound 4.
Microscoping of cultural media did not show significant
difference in the viability of yeast cells when compounds 1—4
were present in the medium as compared to the control.
Since the methods used gave only estimated results,
we obtained curves of microorganisms growth for sepaꢀ
rate concentrations of compounds 1—4 (Fig. 3). As it is
seen from Fig. 3, starting from 10—14 hours of cultivaꢀ
tion the growth rate of microorganisms in the media conꢀ
taining compounds 1 and 4 increases by 2—5 times as
Results and Discussion
The results of the influence of compounds 1 and 2,
which are the most efficient in stimulation of the yeast
cells growth within the entire range of concentrations
studied, are shown in Fig. 1.
Compound 3 did not exert significant influence on
the biomass growth, while compound 4 possessed a proꢀ
n/mln.pcs. mL–1
20
a
16
12
8
1•10–4 1•10–5 1•10–6 1•10–7 1•10–8 Control
n/mln.pcs. mL–1
a
1•10–4
1•10–5
1•10–6
Control
20
15
10
5
4
2
6
12
18
36
t/h
n/mln.pcs. mL–1
20
b
12
22
32
42
52
62
t/h
n/mln.pcs. mL–1
16
12
8
b
1•10–4 1•10–5 1•10–6 1•10–7 1•10–8 Control
1•10–7
1•10–8
Control
20
15
10
5
4
2
6
12
18
36 t/h
12
22
32
42
52
62
t/h
Fig. 1. The yeast biomass growth at different concentrations
(wt.%) of compounds 1 (a) and 2 (b) (n is the number of cells,
t is the cultivation time).
Fig. 3. The accumulation of yeast biomass in the presence of
compounds 1 (a) and 4 (b) at different concentrations (wt.%).

1322
Russ. Chem. Bull., Int. Ed., Vol. 66, No. 7, July, 2017
Privalova et al.
[C2H5OH] (vol.%)
4
compared to the control. A reduction of the lagꢀphases is
observed for compounds 1—4, i.e., the yeast cells reach
the exponential phase of growth faster. In the initial (lagꢀ
phase) and in the final (stationary phase) periods, the
rates of cell generation in the media containing comꢀ
pounds 1—4 and in the control group are similar. It is
reasonable to use the indicated specific features of the
culture growth in continuous cultivation. The introducꢀ
tion of compounds 1—4 in nutrient medium can result in
a considerable growth of the biomass of yeast for a shorter
period of time. Note that compounds 1 and 2, which bear
a sulfurꢀcontaining anion (Y = S, SO2), stimulate the
yeast biomass growth within the whole range of studied
concentrations, while compounds 3 and 4 (Y = O) exhibꢀ
ited activity in the narrower range.
The presence of compounds 1—4 in the nutrient meꢀ
dium composition stimulates the accumulation of proꢀ
tein compounds in the cell (Fig. 4). The greatest effect
was shown by compounds 2 (Y = SO2) and 3 (Y = O) in
the concentration of 1•10–6 and 1•10–5 wt.%, respecꢀ
tively. In their presence, the content of protein in the
biomass increased by 13—15% to the dry mass as comꢀ
pared to the control.
It should be noted that compound 3 efficiently stimuꢀ
lates accumulation of protein in the cell, exerting no proꢀ
nounced influence on the growth of the amount of cells.
The fermentation of synthetic Reeder medium with
the content of sucrose increased to 10 wt.% in the presꢀ
ence of compounds 1—4 leads to the increase in the rate
and the fermentation degree of sugars. Thus, the fermenꢀ
tation degree increases by 8—30%, while the formation of
ethanol calculated on assimilated sugar increases in the
case of compound 1 by 1.1—1.4 times (Figs 5 and 6). The
efficience of fermentation of Reeder medium without adꢀ
dition of compounds 1—4 was low. The yield of ethanol
was only 37%. The introduction of compounds 1—4 in
the medium led to the increase in the ethanol production
to 58% from theoretical yield (Table 1).
1
2
3
3
2
1
4
12
24
36
48
60
72
84
t/h
Fig. 5. The dynamics of the formation of ethanol in the presꢀ
ence of compound 1 in different concentrations (wt.%): 1•10–4 (1),
1•10–5 (2), 1•10–6 (3), as well as in the absence of the stimuꢀ
lant (4 is the control group).
Formation of ethanol/mg/100 mg
40
30
20
10
1•10–4
1•10–5
1•10–6
Control
Fig. 6. The formation of ethanol (mg per 100 mg of assimilated
sugar) in the medium containing compound 1 in the concenꢀ
trations 1•10–4, 1•10–5, 1•10–6 wt.% and in the control sample
after 96 h of fermentation.
The flour medium, as compared to Reeder medium, is
more favorable for cultivation of race XII yeast. The ferꢀ
Table 1. The results of fermentation of Reeder nutrient mediꢀ
um in the presence of compounds 1—4
Content (%)
50
Comꢀ
pound
Concentration
in nutrient
medium (wt.%) fermentation (%) theoretical)
Degree
of sugar
Yield of ethanol
(% from
40
30
20
10
1
1•10–4
1•10–5
1•10–6
1•10–6
1•10–4
1•10–5
1•10–7
1•10–7
1•10–8
—
66.2
66.8
66.5
49.6
73.9
73.7
73.3
50.2
58.6
41.6
54
47
42
41
58
58
56
42
46
37
2
3
1
2
3
4
Control
4
Fig. 4. The content of protein in yeast biomass grown using
compounds 1 (1•10–4 wt.%), 2 (1•10–6 wt.%), 3 (1•10–5 wt.%),
and 4 (1•10–7 wt.%).
Control

Growth stimulants of alcohol yeast
Russ. Chem. Bull., Int. Ed., Vol. 66, No. 7, July, 2017
1323
Table 2. The results of fermentation of flour nutrient medium
in the presence of compounds 1—4
were confirmed by elemental analysis, IR spectroscopy, 1H,
13C, 15N (relative to MeNO2) NMR spectroscopy, and Xꢀray
diffraction.
Tris(2ꢀhydroxyethyl)ammonium (4ꢀchlorophenylsulfanyl)ꢀ
Comꢀ
pound
Concentration
in nutrient
medium (wt.%) fermentation (%) theoretical)
Degree
of sugar
Yield of ethanol
(% from
acetate (1). The yield was 95%. M.p. 78—79 °C. IR, ν/cm–1
:
1362 (νs COO–); 1588 (νas COO–); 2920 (br, N+—H); 3150
(br, OH). 1H NMR, δ: 7.89—7.22 (m, 4 H, C6H4); 3.94 (t, 6 H,
OCH2, J = 5.0 Hz); 3.64 (s, 2 H, SCH2); 3.46 (t, 6 H, NCH2,
J = 5.0 Hz). 13C NMR, δ: 176.71 (C=O); 134.68—128.97 (C6H4);
55.19 (OCH2); 54.93 (NCH2); 38.15 (SCH2). 15N NMR, δ:
–338.90.
1
1•10–4
1•10–5
1•10–6
1•10–4
1•10–8
—
96.6
97.1
96.1
96.2
96.5
96.5
66
70
66
70
65
61
2
3
4
Tris(2ꢀhydroxyethyl)ammonium (4ꢀchlorophenylsulfonyl)ꢀ
acetate (2). The yield was 96%. M.p. 94 °C. IR, ν/cm–1: 1125
(ν SO2); 1362 (ν SO2); 1367 (ν COO–); 1611 (ν COO–);
Control
s
as
s
as
2925 (br, N+—H); 3210 (br, OH). H NMR, δ: 7.42—6.70 (m,
4 H, C6H4); 4.55 (s, 2 H, SO2CH2); 3.80 (t, 6 H, OCH2,
J = 5.0 Hz); 3.42 (t, 6 H, NCH2, J = 5.0 Hz). 13C NMR, δ:
178.05 (C=O), 150.09—127.25 (C6H4), 64.00 (SO2CH2), 59.06
(OCH2), 57.60 (NCH2). 15N NMR, δ: –335.15.
1
mentation of such medium based on wheat flour results
in the higher fermentation degree of sugars for a shorter
period of time in both the control group and in the media
containing compounds 1—4. The experiment resulted also
in the higher yield of ethanol. Thus, the yield in the conꢀ
trol was 61% and increased by 5—9% when compounds
1—4 were introduced in the flour medium (Table 2).
In conclusion, we found that tris(2ꢀhydroxyethyl)ꢀ
ammonium arylchalcogenylacetates 1—4 are efficient
synthetic stimulants of growth and development of race
XII yeast Saccharomyces cerevisiae and make it possible
to intensify the process of their cultivation. This is reꢀ
flected in the biomass growth and increase in the rate of
the culture growth in the logarithmic phase by 2—5 times.
The content of protein in the yeast biomass increases by
2—15%. The highest growth of the content of protein
compounds is observed when compound 2 is introduced
in the nutrient medium in a concentration of 1•10–6 wt.%.
A positive influence of compounds 1—4 on the yeast
cells allows increasing the depth of sugar fermentation
and enhancing ethanolꢀdirected metabolism of yeast cells.
The yield of ethanol in the presence of compounds 1—4
increases by 5—9%. The use of biostimulants in producꢀ
tion of ethanol (biofuel), as well as of fodder and baker´s
yeast, will increase the efficiency of the processes and the
yields of the target products per mass unit of consumed
sugar. The advantage of synthetic biostimulants 1—4 is
their availability, low cost, good solublility in water,
stability on storage, nontoxicity, and efficiency in low
concentrations (1•10–8—1•10–4 wt.%).
Tris(2ꢀhydroxyethyl)ammonium (2ꢀchlorophenyloxy)acetate
(3). The yield was 95%. M.p. 80—81 °C. IR, ν/cm–1: 1404
(ν COO–); 1590 (νas COO–); 2925 (br, N+—H); 3182 (br, OH).
s
1H NMR, δ: 7.25—6.80 (m, 4 H, C6H4); 4.48 (s, CH2CO); 3.92
(t, 6 H, OCH2, J = 5.0 Hz); 3.42 (t, 6 H, NCH2, J = 5.0 Hz).
13C NMR, δ: 177.00 (C=O), 156.02—111.39 (C6H4), 66.94
(CH2CO), 55.14 (OCH2), 54.89 (NCH2). 15N NMR, δ: –339.00.
Tris(2ꢀhydroxyethyl)ammonium (4ꢀchlorophenyloxy)acetate
(4). The yield was 94%. M.p. 83 °C. IR, ν/cm–1: 1397 (ν COO–):
s
1606 (νas COO–); 2920 (br, N+—H); 3156 (br, OH). 1H NMR,
δ: 7.18—6.70 (m, 4 H, C6H4); 4.45 (s, CH2CO); 3.93 (t, 6 H,
OCH2, J = 5.0 Hz); 3.44 (t, 6 H, NCH2, J = 5.0 Hz). 13C NMR,
δ: 176.51 (C=O), 154.61—112.31 (C6H4), 66.98 (CH2CO),
55.03 (OCH2), 54.77 (NCH2). 15N NMR, δ: –338.60.
Biological tests. The yeast Saccharomyces cerevisiae of race
XII were cultivated on a synthetic Reeder medium of the folꢀ
lowing composition (g L–1): sucrose — 20; (NH4)2SO4 — 3;
MgSO4 — 0.7; NaCl — 0.5; KH2PO4 — 1; K2HPO4 — 0.1. The
dosage of compounds 1—4 into the nutrient medium was carꢀ
ried out as follows. A matrix solution with a concentration of
2 mg mL–1 (compounds 1—4 (200 mg) were dissolved in disꢀ
tilled water (100 mL)) was prepared. Then, a serial dilution of
the matrix solution was carried out to a concentration of
1•10–7—1•10–2 wt.%. The diluted matrix solution was dosed
into the nutrient medium in an amount necessary to obtain the
desired concentration. The inoculum of the yeast Saccharoꢀ
myces cerevisiae of race XII was prepared by seeding on sloping
wortꢀagar with further rinsing of the grown culture with the
Reeder medium. The content of yeast cells in the inoculum was
(7—8)•106 cell mL–1; the dosage of inoculum is 5% to the
volume of the nutrient medium. The cultivation was carried
out for 20—36 (72) h at 30 °C. The total amount of yeast cells in
the culture medium was determined by optical method on
a KFKꢀ3 photoelectrocolorimeter (ZOMS) at 490 nm. The
viability of the yeast was assessed by the presence of living and
budding cells. The content of living cells was determined by
staining with methylene blue, that of the budding cells by microꢀ
scopy. Control experiments were carried out with the addition
of distilled water to the nutrient medium instead of the solution
of compounds 1—4. The total biomass of yeast was determined
by gravimetric method, the protein content in the biomass was
determined by colorimetry with amidoꢀblack.17 The alcohol
Experimental
Synthesis of compounds 1—4 (general procedure). A soluꢀ
tion of tris(2ꢀhydroxyethyl)amine (triethanolamine) and the
corresponding acid in ethyl alcohol (molar ratio 1 : 1) was
heated for 15—30 min at 65 °C and maintained for 1 h at 20—22 °C.
The mixture was poured into diethyl ether (anhydrous) and
maintained for 12 h at 5—10 °C. A precipitate was collected by
filtration, washed with ether, and dried in vacuo. The products
were isolated as colorless powders, well soluble in water and
ethanol. The composition and the structure of compounds 1—4

1324
Russ. Chem. Bull., Int. Ed., Vol. 66, No. 7, July, 2017
Privalova et al.
fermentation of the Reader medium with a sucrose content of
10% in the presence of compounds 1—4 was carried out for
96 h at 30 °C, with the norm of application of the yeast seed
material being 5% to the volume of the nutrient medium. The
fermentation progress was monitored by determining the conꢀ
tent of residual sugar by the Dubois method.18 The accumulaꢀ
tion of ethanol was determined by GCꢀMS on an Agilent Techꢀ
nologies 7820 A gas chromatograph with an HР 5975 selective
mass spectrometer detector. The experiment conditions: the
ionization energy 70 eV, the separator temperature 280 °C, the
source of ions temperature 230 °C; a 30000×0.25ꢀmm quartz
column with a stationary phase (5% diphenyl polysiloxane);
the column temperature 50 °C. The components were identiꢀ
fied using the NIST11 library of mass spectra. The quantity of
the components was calculated from the peak areas using corꢀ
rection sensitivity coefficients. The simulation of the industrial
process of alcohol fermentation was carried out on a nutrient
medium containing 10% of the 1st grade wheat flour. To obtain
fermentable sugars, the kneading flour was saccharified with
enzymatic agents Amlosubtilin G3x and Glukavamorin manuꢀ
factured by Sibbiofarm Ltd. (Berdsk). The dosage of the agents
and the conditions of the enzymatic hydrolysis were selected in
accordance with the manufacturer’s recommendations. The ferꢀ
mentation time was 72 h. The remaining conditions were simiꢀ
lar to the fermentation of the Reader medium.
Materials and Ethanolꢀcontaining Products], Pishch. promꢀst´,
Moscow, 2005, 72—97 (in Russian).
3. V. A. Polyakov, L. V. Rimareva, Proizvodstvo spirta i likeroꢀ
vodochnykh izdeliy [Production of Ethanol and Alcoholic Bevꢀ
erages], 2001, 1, 6 (in Russian).
4. S. I. Khorunzhina, Biokhimicheskiye i fizikoꢀkhimicheskiye
osnovy tekhnologii soloda i piva [Biochemical and Physicoꢀ
chemical Foundations of Malt and Beer Technology], Kolos,
Moscow, 1999, 311 pp. (in Russian).
5. A. N. Mirskova, G. G. Levkovskaya, R. G. Mirskov, M. G.
Voronkov, Russ. J. Org. Chem., 2008, 44, 1478.
6. A. N. Mirskova, G. G. Levkovskaya, O. P. Kolesnikova,
O. M. Perminova, E. V. Rudyakova, S. N. Adamovich,
Russ. Chem. Bull., 2010, 59, 2236.
7. A. N. Mirskova, R. G. Mirskov, S. N. Adamovich, M. G.
Voronkov, Chem. Sustainable Dev., 2011, 19, 429.
8. S. N. Adamovich, A. N. Mirskova, R. G. Mirskov, U. Schilde,
Chem. Cent. J., 2011, 5, 23.
9. A. N. Mirskova, S. N. Adamovich, R. G. Mirskov, U. Schilde,
Chem. Cent. J., 2013, 7, 34.
10. A. N. Mirskov, S. N. Adamovich, R. G. Mirskov, M. G.
Voronkov, Russ. Chem. Bull., 2014, 63, 1869.
11. V. E. Shklover, G. V. Gridunova, Yu. T. Struchkov, M. G.
Voronkov, Yu. I. Kryukova, A. N. Mirskova, Dokl. Akad.
Nauk SSSR, 1983, 269, 387 [Dokl. Chem. (Engl. Transl.),
1983, 269].
The work was performed within the framework of the
Integration Program of the Irkutsk Scientific Center of
the Siberian Branch of the Russian Academy of Sciences
"Fundamental Studies and Breakthrough Technologies
as a Basis for Advanced Development of Baikal Region
and its Interregional Connections".
12. N. N. Chipanina, T. N. Aksamentova, S. N. Adamovich,
A. L. Albanov, A. N. Mirskova, R. G. Mirskov, M. G.
Voronkov, Comput. Theor. Chem., 2012, 985, 36.
13. M. G. Voronkov, V. S. Fundamenskiy, S. N. Adamovich,
E. A. Zel´bst, A. A. Kashayev, V. A. Bruskov, R. G. Mirskov,
A. N. Mirskova, J. Struct. Chem., 2013, 54, 182.
14. I. A. Ushakov, V. K. Voronov, D. S. Grishmanovskii, S. N.
Adamovich, R. G. Mirskov, A. N. Mirskova, Russ. Chem.
Bull., 2015, 64, 58.
References
15. A. N. Mirskova, S. N. Adamovich, R. G. Mirskov, O. P.
Kolesnikova, Uwe Schilde, Open Chem., 2015, 13, 149.
16. I. A. Ushakov, V. K. Voronov, S. N. Adamovich, R. G.
Mirskov, A. N. Mirskova, J. Mol. Struct., 2016, 1103, 125.
17. G. A. Buzun, K. M. Dzhemukhadze, L. F. Mileshko, Fizꢀ
iologiya rasteniy [Plant Physiology], 1982, 29, No. 1, 196
(in Russian).
1. V. L. Yarovenko, V. A. Marinchenko, Tekhnologiya spirta
[Technology of Ethanol], Kolos, Moscow, 2001, 464 pp.
(in Russian).
2. D. V. Arsen´yev, A. A. Yezhkov, Tekhnologiya etilovogo spirꢀ
ta na osnove sopryazheniya protsessov brozheniya i distillyatsii.
O sostoyanii na napravleniyakh razvitiya proizvodstva spirta
etilovogo iz pishchevogo syr´ya i likerovodochnoy produktsii
[Technology of Ethyl Alcohol on the Basis of a Combination of
Fermentation and Distillation Processes. On the State of the
Development of Ethyl Alcohol Production from Food Raw
18. M. Dubois, Analyt. Chem., 1956, 28, 350.
Received December 1, 2016;
in revised form February 18, 2017