INTRODUCTION
Recent studies have proven the effectiveness of
mechanical and enzymatic treatment of grains (at
temperatures under 100°C) with complex enzyme
preparations in ethanol production [1–4]. This “soft”
technology for grain wort preparation can significantly
reduce heat and power consumption and increase
profitability. It is based on controlled biocatalytic
conversion of grain polymers (starch, xylans, β-glucans,
and proteins) with the formation of easily digestible
carbohydrates and nitrogenous substances that yeast
cells need for normal metabolism [2, 4–7].
However, the studies hardly took into account
the presence of phytic acid and its salts (phytates) in
grains that contain up to 80% of phosphorus in a bound
state [8–10]. The bioavailability of phosphorus can be
increased by using phytolytic enzymes. Grains contain
enzymes that catalyze the destruction of intracellular
polymers. Normally in a latent state, these enzymes
are activated during germination. Studies show that the
amount of phytolytic enzymes in grain is insufficient for
the complete release of phosphorus [11, 12]. Therefore,
researchers have recently focused on obtaining
enzyme preparations – sources of phytases – based on
microorganisms. Using genetic engineering, they have
developed highly productive recombinant strains of
fungi, yeasts, and bacteria that synthesize phytolytic
enzymes [13–15].
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Foods and Raw Materials, 2022, vol. 10, no. 1
E-ISSN 2310-9599
ISSN 2308-4057
128
Rimareva L.V. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 117–126
Phytolytic enzymes of microbial origin have been
widely studied, namely their use in fodder production
for the release of phytate phosphorus. The biocatalytic
conversion of plant phytates has been shown to improve
the digestibility of nutrients in fodder and stimulate
the growth of farm animals and poultry [16–18]. Much
attention has been paid to the use of phytases in the food
industry to increase the digestibility and bioavailability
of food components [19–21]. However, there is a lack
of data concerning the catalytic action of microbial
phytases on grain polymers in fermentation.
Previous studies have shown a positive effect of
phytases on the growth and development of yeast cells
cultivated on grain media, including beer production
from sorghum [22–24]. Some authors who studied the
effect of phytolytic enzymes reported better rheological
properties of rye and triticale wort, as well as improved
fermentation [6, 25, 26].
However, there is a lack of research into their effect
on the biochemical parameters of grain wort and mash,
especially those from wheat and corn [2, 23]. What
needs studying is changes in the ionic composition of
grain wort depending on the substrate specificity of
enzyme systems used for its preparation, as well as
the assimilation of the released phytate phosphorus
by the Saccharomyces cerevisiae yeast during ethanol
fermentation [1–3, 22–25]. Hardly studied is the effect
of phytases on the hydrolytic capacity of enzymes with
substrate specificity in relation to starch and proteins,
on the conversion of these polymers in grain wort
preparation, and on the efficiency of yeast generation
and ethanol fermentation.
Thus, literature analysis revealed a lack of studies
into the potential of phytic substances with bound
phosphorus for the conversion of grain to ethanol.
A number of papers reported that phytates – strong
chelating agents – bind not only phosphorus, but also
metal cations (calcium, magnesium, manganese, zinc,
iron, etc.) [19, 21, 27–29]. Low bioavailability of macroand
microelements in grains can have a negative effect
on the supply of yeast with minerals and on the catalytic
activity of some metal-dependent enzymes.
The S. cerevisiae yeast is a key factor in the
efficiency of ethanol fermentation. The yield and
quality of ethanol, as well as the process duration,
largely depend on the fermentation activity and yeast
productivity. The metabolism of S. cerevisiae is
significantly affected not only by the strains’ genetic
characteristics, but also by the conditions of their
cultivation (nutrient medium with easily assimilated
nutrition) [2, 30–32].
Metabolism involves all enzymatic reactions
that occur in the cell to regulate the composition
and synthesis of target and secondary metabolites.
Therefore, there is a need for research to select effective
enzyme systems that contribute to deep destruction
of grain polymers. It is especially true of phytolytic
enzymes. Recent studies have revealed that phytic
acid forms stable complexes with carbohydrates and
proteins [33–35]. Apparently, this can reduce the
hydrolytic action of carbohydrases and proteases on
carbohydrate and peptide polymers. Phosphate groups
of phytic acid bind to basic amino acids (arginine,
histidine, and lysine) and form strong protein-phytate
complexes.
However, the studies of phytate-carbohydrate
complexes have ambiguous results. They show that the
interaction occurs either through direct binding to starch
or indirectly, through starch-associated proteins [34–37].
Therefore, to ensure a steadily high yield of ethanol, it
is important to select optimal parameters for preparing
grain media to produce high-quality wort [1–3].
In connection with the above, we can assume that
the biocatalytic destruction of phytic substances will
contribute to the release of phosphorus and other
valuable microelements. It will also stimulate the
conversion of carbohydrate and protein polymers of
grain through the use of hydrolases with substrate
specificity in relation to the main polymers of grain.
We aimed to study the effect of hydrolytic enzymes
with proteases and phytases on the efficiency of yeast
generation and metabolism during the fermentation of
wheat and corn wort.
STUDY OBJECTS AND METHODS
Our study objects included wheat and corn wort
prepared with enzyme preparations that served as
sources of hydrolases with different substrate specificity
and action. They were used for:
‒ dextrinization and saccharification of starch α-amylase
(EC 3.2.1.1.) and glucoamylase (EC 3.2.1.3.);
‒ destruction of xylanase non-starch polysaccharides
(EC 3.2.1.8, 3.2.1.32, 3.2.1.37, 3.2.1.72);
‒ hydrolysis of protein substances of the protease
complex (EC 3.4.11-3.4.15, EC 3.4.21-3.4.24); and
‒ hydrolysis of phytase substances (EC 3.1.3.8).
Yeast, organic acids, and dietary supplements
were obtained from the Biotechnology Department.
Ultraconcentrates of culture liquids were used
as enzyme preparations to produce thermostable
α-amylase (Bacillus licheniformis sp., Amilolikheterm),
glucoamylase and xylanase (VKM F-4278D,
a recombinant strain of Aspergillus awamori,
Glucavamorin-Xyl), a protease complex (VKPM F-931,
a mutant strain of Aspergillus oryzae, Protoorizin), and
phytase (Phytaflow, Novozymes, Denmark) [38–40].
Enzyme activity was determined by the existing
methods. A unit of amylolytic activity (AA) was defined
as an amount of enzyme that catalyzes the hydrolysis
of 1 g of soluble starch to dextrins of various molecular
weights under standard conditions (30°C, pH 6.0,
10 min). A unit of glucoamylase activity (GA) was an
amount of enzyme that is capable of catalyzing starch
hydrolysis at 30°C (pH 4.7) and releasing 1 μmol of
glucose per minute. A unit of xylanase activity (XA)
was an amount of enzyme that acts on xylan from birch
wood and releases 1 μmol of reducing sugars (in glucose
equivalent) per minute under standard conditions
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Rimareva L.V. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 117–126
(50°C, pH 5.0). A unit of total proteolytic activity (PA)
was an amount of enzyme that brings hemoglobin into
a TCA non-precipitated state equivalent to 1 μmol of
tyrosine per minute (30°C, pH 5.3). A unit of phytase
activity (PhA) was an amount of enzyme that catalyzes
the hydrolysis of sodium phytate to produce 1 μmol
of inorganic phosphate per minute under standard
conditions (37°С, pH 5.5, 15 min).
Grain wort was prepared by the enzymatichydrolytic
processing of grain (40–90°C, hydromodule
1:3). For this, 50 g of grain flour and 150 cm3 of water
were mixed in 750-cm3 Erlenmeyer flasks and regularly
stirred in a PE-4300 water bath (Ekros, Russia).
The water-grain mixture was prepared for 2 h
(40–90°C) using a thermostable α-amylase (0.6 AA
units/g starch) to liquefy starch. When the mixture was
cooled to 60°C (56–60°C, 1 h), EPs with glucoamylase
(9.0 GA units/g starch) and xylanase (0.4 XA units/g
grain) activity were used to saccharificate starch and
hydrolyze non-starch polysaccharides. They served as a
control. The experimental samples contained EPs with
phytase (1.0–3.0 PhA units/g grain) and proteolytic
activity (0.1 PA units/ g grain), in addition to those with
amylolytic, glucoamylase, and xylanase activity.
Wort was fermented using a selected race of the
Saccharomyces cerevisiae 985-T yeast with thermotolerant
and osmophilic properties (34–35°C, 68 h) [41].
Methods. The Instructions for the Technochemical
Control of Alcohol Production were followed to
determine the contents of starch, protein, and nonstarch
polymers of grain, the concentrations of yeast
cells and reducing carbohydrates in the grain mash,
as well as ethanol concentration and yield [42]. The
concentration of amine nitrogen in the grain wort was
measured by iodometric titration (Pharmaceutical
Regulations 1.2.3.0022.15). The ionic composition of
wheat and corn wort, as well as mash, was determined
using a PrinCE-560 capillary electrophoresis system
(Netherlands) equipped with a conductometric detector.
The composition and content of volatile metabolites
synthesized by yeast were measured using an HP
Agilent 6850 gas chromatograph (USA).
Data obtained in triplicate were statistically
processed in Microsoft Excel using the Student’s
coefficient with a 0.95 confidence interval.
RESULTS AND DISCUSSION
Ground wheat and corn were used as a substrate
to prepare wort. Table 1 compares the compositions of
the main polymers contained in the grains. According
to their caryopsis composition, wheat and corn, like all
grains, are classified as multicomponent starchy plant
raw materials, in which starch is the main polymer that
determines ethanol yield [10, 25, 42].
Corn had a higher content of starch (65.8%), while
wheat was richer in protein (13.9%). In addition, the
grains under study contained non-starch polysaccharides
(hemicelluloses, cellulose), protein substances, and
phytates (Table 1). This multicomponent composition
of grain polymers determined the selection of enzyme
preparations with a given substrate specificity to prepare
grain wort (Table 2). Amilolicheterm (thermostable
α-amylase, 330 AA units/g) was used to liquefy and
dextrinize starch. Glucavamorin-Xyl (glucoamylase,
7700 GA units/g; xylanase, 350 XA units/g) was
used to saccharify starch and hydrolyze non-starch
polysaccharides. Protoorizin (protease, 580 PA
units/g) was used for protein proteolysis and Phytaflow
(30 000 PhA units/g) was selected to convert phytic
substances.
Protoorizin contained at least five proteolytic
enzymes that differ in action [39]. In previous studies,
the authors used neutral protease or Glucavamorin-Xyl,
which contained proteinases to catalyze the hydrolysis
of proteins to peptides with different molecular
weights [24, 25]. In contrast to them, Protoorizin
contains a complex of proteinases and peptidases that
hydrolyze proteins to low-molecular-weight peptides and
free amino acids [40, 43].
Table 1 Biochemical composition of wheat and corn
Components Content, %
Wheat Corn
Proteins 13.9 ± 0.5 10.2 ± 0.4
Mono- and disaccharides 3.0 ± 0.1 4.1 ± 0.2
Starch 57.4 ± 2.6 65.8 ± 3.2
Hemicellulose 4.4 ± 0.2 3.0 ± 0.1
Cellulose 2.9 ± 0.1 3.3 ± 0.1
Phytates 1.30 ± 0.05 2.30 ± 0.09
Values are expressed as means ± SD
Table 2 Enzyme preparations by activity of major and minor enzymes
Enzyme preparation Enzyme activity, units/g
АA GA XA PA PhA
Amilolicheterm 330 ± 8 0 25 ± 5 7.0 ± 0.1 0
Glucavamorin-Xyl 110 ± 4 7700 ± 240 350 ± 13 6.0 ± 0.1 0
Protoorizin 0 0 0 580 ± 26 0
Phytaflow 0 0 0 0 30000 ± 1200
AA – Amylolytic activity; GA – Glucoamylase activity; XA – Xylanase activity; PA – Proteolytic activity; PhA – Phytase activity
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Enzyme preparations were used at the stage of
grain wort preparation in the amounts specified above
(see “Study objects and methods”). The control wort
contained Amilolicheterm and Glucavamorin-Xyl
preparations, the sources of carbohydrases that catalyze
the hydrolysis of polysaccharides. Their proteolytic
enzymes had practically no effect on the degree of
protein conversion due to the low level of their activity.
The concentration of proteases in the control was under
0.02 PA units/g grain, which was 5 times lower than
in the experimental samples. Therefore, Protoorizin
(0.1 PA unit/g grain) and Phytaflow (1.0–3.0 PhA units/g
grain) were additionally added to the experimental
samples to ensure an efficient conversion of grain
polymers and activate yeast generation and ethanol
fermentation.
We found that the ionic composition of grain
wort changed depending on the type of grain and the
substrate specificity of the enzymes used to hydrolyze
grain polymers. In the wheat wort treated with a
complex of amylolytic and xylanase enzymes (AA +
GA + XA), the phosphate content was 2.7 times higher
than in the corn wort (Fig. 1). The concentrations of
other cations, such as potassium, calcium, sodium, and
magnesium, were also different. The wheat wort was
richer in potassium, while the corn wort had a higher
content of calcium and magnesium (Table 3).
Phytolytic enzymes had a more significant effect
on the release of phytate phosphorus. Increasing their
amount to 1.5–2.0 PhA units/g grain led to higher
concentrations of released phosphates due to the
catalytic destruction of phytic substances. We found that
the content of phosphorus ions increased 1.6 times in
the wheat wort and 3.8 times in the corn wort. A further
increase in the phytase concentration to 3.0 PhA units/g
grain had practically no effect on the content of soluble
phosphates in the wort (Fig. 1).
The catalytic synergism of phytases and proteases
stimulated the release of phosphorus ions. Their content
increased 1.8 times in the wheat wort and 4.9 times in
the corn wort (Table 3).
In addition to the release of phosphorus ions,
phytases increased the concentration of cations in the
wheat and corn wort samples: potassium by 12 and 13%,
AA – amylolytic activity; GA – glucoamylase activity; XA – xylanase activity; PhA – phytase activity
Figure 1 Effects of phytolytic enzymes on phosphate release in wheat (1) and corn (2) wort
Table 3 Effects of hydrolytic enzymes on amine nitrogen and basic ions in wheat and corn wort
Enzyme composition Type of
wort
Amine nitrogen,
mg%
Ion content, mg/dm3
Phosphorus Potassium Calcium Sodium Magnesium
Control (АA+GA+XA) Wheat 53.0 ± 2.5 787.1 ± 28.2 811.4 ± 33.4 12.1 ± 0.2 16.5 ± 0.4 131.1 ± 3.7
Corn 40.3 ± 1.2 290.0 ± 13.2 780.3 ± 24.7 16.1 ± 0.4 16.1 ± 0.3 179.6 ± 5.4
Control+0.1 PA units Wheat 85.7 ± 3.2 960.5 ± 29.4 825.1 ± 34.5 12.9 ± 0.3 16.9 ± 0.5 141.9 ± 4.2
Corn 70.4 ± 2.6 330.4 ± 13.3 834.6 ± 28.8 18.0 ± 0.4 19.0 ± 0.7 199.3 ± 8.2
Control+1.5 PhA units Wheat 53.5 ± 1.8 1242.1 ± 44.2 907.0 ± 34.9 13.9 ± 0.5 23.0 ± 0.8 151.8 ± 5.8
Corn 41.3 ± 1.6 1109.0 ± 38.4 878.0 ± 26.3 21.8 ± 0.6 20.9 ± 0.7 218.5 ± 8.9
Control+2.0 PhA units Wheat 53.9 ± 2.1 1232.3 ± 51.2 909.3 ± 31.4 14.1 ± 0.3 23.9 ± 0.8 152.1 ± 6.7
Corn 41.1 ± 1.6 1112.3 ± 36.7 881.0 ± 28.4 22.9 ± 0.7 21.7 ± 0.7 222.3 ± 6.4
Control +1.5 PhA
units+0.1 PA units
Wheat 93.9 ± 3.8 1379.6 ± 51.4 918.7 ± 34.6 14.0 ± 0.4 24.2 ± 0.8 152.9 ± 4.8
Corn 76.5 ± 2.6 1423.6 ± 56.7 888.9 ± 42.5 23.4 ± 0.8 23.9 ± 0.7 225.6 ± 7.4
AA – amylolytic activity; GA – glucoamylase activity; XA – xylanase activity; PA – proteolytic activity; PhA – phytase activity
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AA+GA+XA
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calcium by 15 and 35%, sodium by 39 and 30%, and
magnesium by 16 and 22%, respectively (Table 3).
We found that the content of cations depended on
the substrate specificity of the enzymes involved in the
bioconversion of grain polymers. The combined action
of proteolytic and phytolytic enzymes contributed not
only to the accumulation of phosphates and minerals,
but also to a significant increase in amine nitrogen. Its
concentration was 1.8 times as high in the wheat wort
and 1.9 times as high in the corn wort, compared to the
control.
Grain wort enriched with phosphates and minerals
was used as a nutrient medium to cultivate the ethanol
yeast Saccharomyces cerevisiae (race 985-T). We
studied the process of yeast generation on grain wort
prepared with hydrolytic enzymes differing in substrate
specificity and found that the presence of phytase in the
enzyme complex had a positive effect on the growth and
reproduction of yeast cells (Fig. 2).
As we know, mineral and nitrogenous substances
are essential for the biochemical reactions of yeast
cells [24, 25, 43]. Magnesium and calcium ions
activate the catalytic ability of almost all intracellular
metalloenzymes, including phosphofructokinase,
which is involved in glucose metabolism. Potassium,
sodium, and calcium ions have a regulatory effect on
the metabolism of yeast cells. Potassium also plays an
essential role in oxidative phosphorylation and glycolysis
processes [43]. It activates yeast aldolase and, together
with magnesium ions, catalyzes pyruvate carboxylase.
Like nitrogen, potassium can also affect yeast lipid
metabolism.
We analyzed the processes of yeast generation and
carbohydrate consumption and found that the presence
of phosphorus and other minerals in the medium
intensified the growth of S. cerevisiae yeast. In the lag
phase (first 18–24 h of growth), the concentration of
yeast cells increased 1.2–1.3 times, alongside a rising
rate of carbohydrate consumption. A 1.3-fold increase
in phytolytic enzymes (up to 2.0 PhA units/g grain)
during grain wort preparation had no significant effect
on the yeast growth and the assimilation of reducing
carbohydrates (Fig. 2).
The catalytic synergism of phytase and protease
significantly enriched the wheat wort with mineral and
nitrogenous nutrition and resulted in the most active
growth of yeast cells (2-fold) and a more intensive
consumption of carbohydrates (Fig. 2, curves 4 and 8).
A similar pattern was observed with the corn wort.
Thus, our results confirmed that the nutrient
medium has a significant effect on yeast generation
and physiological activity, particularly the presence of
soluble macro- and microelements in addition to easily
digestible carbohydrates and nitrogenous substances.
At the next stage, we analyzed changes in the
concentration of phosphates in the mash against the
amount of phytolytic enzymes used in the preparation
of wheat and corn wort (Figs. 3a and 3b). We found that
the content of phosphorus ions significantly decreased
during the fermentation of the control wort, which was
not treated with phytases. In the logarithmic phase
of yeast cell growth (on day 1), it declined 1.9 times
in the wheat wort and 2.3 times in the corn wort. The
wort samples treated with protease in addition to
carbohydrases showed the same trend – a sharp decrease
in phosphates after 24 h of fermentation, followed by a
slight rise.
In the experimental samples treated with phytolytic
enzymes, the concentration of phosphates hardly
changed during the fermentation of grain wort. This
might be due to the ongoing biocatalytic hydrolysis of
phytic substances and an extra release of phosphorus.
By the end of fermentation, the content of phosphates
slightly increased, which might be associated with
autolytic processes in the cell (Fig. 3).
We found an increase of 2.4–2.6 times and 4.3–
5.1 times in the residual content of phosphates in the
wheat mash and the corn mash, respectively. This
indicated that phytolytic enzymes not only enriched
the grain wort with assimilable phosphorus and other
valuable minerals, but also improved the value of grain
stillage, a waste product of ethanol production that is
used in the diet of farm animals.
Apart from the main fermentation products
(ethanol and carbon dioxide), yeast cells synthesize
accompanying metabolites: secondary (organic
acids, aldehydes, and esters) and by-products (higher
alcohols) [43].
We studied the metabolism of S. cerevisiae 985-T
yeast during its cultivation on wheat and corn wort
treated with phytolytic enzymes and found a decrease
of 18–21 and 20–23%, respectively, in total metabolite
formation that accompanies ethanol synthesis (Fig. 4).
1 and 5 – treated with amylases and xylanase (Control);
2 and 6 – treated with phytases (Control + 1.5 PhA units/g grain);
3 and 7 – treated with phytases (Control + 2.0 PhA units/g grain);
4 and 8 – treated with phytases and proteases
(Control + 1.5 PhA units + 0.1 PA units)
PA – proteolytic activity; PhA – phytase activity
Figure 2 Changes in carbohydrate consumption (1–8)
and Saccharomyces cerevisiae 985-T yeast growth (5–8)
during wheat wort fermentation for 68 h
2.5
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1
2 3 4
0
300
600
900
1200
1500
АС+ГлС+КС
(К)
К+0,1 ед.ПС К+1,5 ед.ФС К+1,5
ед.ФС+0,1
ед.ПС
mg/dm3
1 2
600
800
1000
mg/dm3
1
2
3
4
5
6
7
8
0
20
40
60
80
100
120
140
8 16 24 32 40 72
cm3 mln./cm3
h
3 4
ГлС+КС
К)
К+0,1 ед.ПС К+1,5 ед.ФС К+1,5
ед.ФС+0,1
ед.ПС
(К)+1.0
ед.ФС
(К)+1.5
ед.ФС
(К)+2.0
ед.ФС
(К)+2.5
ед.ФС
(К)+3.0
ед.ФС
1
2
3
4
5
6
7
8
0
20
40
60
80
100
120
140
0
2
4
6
8
10
12
14
16
18
0 8 16 24 32 40 72
g/100 cm3 mln./cm3
h
К+0,1 ед.ПС К+1,5 ед.ФС К+1,5 ед.ФС+0,1
ед.ПС
1
2 3 4
0
300
600
900
1200
1500
АС+ГлС+КС
(К)
К+0,1 ед.ПС К+1,5 ед.ФС К+1,5
ед.ФС+0,1
ед.ПС
mg/dm3
К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5
ед.ФС+0,1ед.ПС
0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5
ед.ФС+0,1ед.ПС
К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.Ф(СК )+1,5 ед.ФС+0,1ед.ПС
68
132
Rimareva L.V. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 117–126
These results indirectly confirmed the improvement of
phosphorus metabolism in the cell.
The use of proteases reduced the synthesis of side
and secondary metabolites by more than 30%. The
greatest effect was caused by the combined catalytic
action of phytases and proteases during yeast generation
on the media enriched with assimilable phosphates
and amine nitrogen. In particular, the total content
of accompanying volatiles in the wheat and corn mash
decreased by 44 and 42%, respectively, compared to the
control.
Higher alcohols (mostly isoamylol, isobutanol,
and 1-propanol) dominated among side and secondary
metabolites synthesized by S. cerevisiae 985-T during
the wort fermentation. In addition, the mash contained
aromatic alcohols (β-phenylethyl, p-hydroxyphenylethyl)
and a small amount of secondary by-products, primarily
aldehydes and esters (Fig. 5).
Our study showed that treating the nutrient media
with phytolytic and proteolytic enzymes decreased
the synthesis of higher and aromatic alcohols (1.9 and
1.4 times, respectively) (Fig. 5). Exposure to phytases
reduced the amount of higher alcohols and proteases
(1.3–1.4 and 1.5–1.6 times, respectively). In addition, we
observed a slight decrease in aldehydes and esters.
Thus, our study showed that providing a yeast cell
with a balanced nitrogenous and mineral nutrition
created conditions for synthesizing ethanol with a
reduced amount of fermentation by-products. By
regulating yeast metabolism we can improve the quality
and sensory properties of the target product.
According to our results, the biochemical changes
in the grain wort affected the yield of ethanol, the
main fermentation product. The destruction of phytic
AA – amylolytic activity; GA – glucoamylase activity; XA – xylanase activity; PA – proteolytic activity; PhA – phytase activity
Figure 4 Total side metabolites during the cultivation of Saccharomyces cerevisiae 985-T yeast on wheat (1) and corn (2) wort
1
2
0
200
400
600
800
1000
1200
1400
АС+ГлС+КС
(К)
(К)+1.0
ед.ФС
(К)+1.5
ед.ФС
(К)+2.0
ед.ФС
(К)+2.5
ед.ФС
(К)+3.0
ед.ФС
mg/dm3
1
2
3
4
0
2
4
6
8
10
12
14
16
18
0 8 16 24 g/100 cm3 1
2 3 4
0
300
600
900
1200
1500
1800
АС+ГлС+КС (К) К+0,1 ед.ПС К+1,5 ед.ФС К+1,5 ед.ФС+0,1
ед.ПС
mg/dm3
1
2 3 4
0
300
600
900
1200
1500
АС+ГлС+КС
(К)
К+0,1 ед.ПС К+mg/dm3
1 2
0
200
400
600
800
1000
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5
ед.ФС+0,1ед.ПС
mg/dm3
1
2 3 4
100
200
300
400
500
600
700
mg/dm3
1
2 3 4
0
300
600
900
1200
1500
1800
АС+ГлС+КС (К) К+0,1 ед.ПС К+1,5 ед.ФС К+1,5 ед.ФС+0,1
ед.ПС
mg/dm3
1
2 3 4
0
300
600
900
1200
1500
АС+ГлС+КС
(К)
К+0,1 ед.ПС К+1,5 ед.ФС К+1,5
ед.ФС+0,1
ед.ПС
1 2
0
200
400
600
800
1000
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5
ед.ФС+0,1ед.ПС
mg/dm3
1
2 3 4
0
100
200
300
400
500
600
700
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5
ед.ФС+0,1ед.ПС
mg/dm3
1
2
3 4
0
100
200
300
400
500
600
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.Ф(СК )+1,5 ед.ФС+0,1ед.ПС
mg/dm3
AA+GA+XA
Control
Control+0.1 PA
units
Control+1.5 PhA
units
Control+1.5
PhA units+0.1
PA units
Control+2.0 PhA
units
AA – amylolytic activity; GA – glucoamylase activity; XA – xylanase activity; PA – proteolytic activity; PhA – phytase activity
Figure 3 Changes in the concentration of phosphates (mg/dm3) after 0 (1), 24 (2), 48 (3), and 68 h (4) during the fermentation
of wheat (a) and corn (b) wort depending on the amount of enzymes
0
200
АС+ГлС+КС
(К)
(К)+1.0
ед.ФС
(К)+1.5
ед.ФС
(К)+2.0
ед.ФС
(К)+2.5
ед.ФС
(К)+3.0
ед.ФС
1
3
4
0
2
4
0 8 16 24 1
2 3 4
0
300
600
900
1200
1500
1800
АС+ГлС+КС (К) К+0,1 ед.ПС К+1,5 ед.ФС К+1,5 ед.ФС+0,1
ед.ПС
mg/dm3
1
2 3 4
0
300
600
900
1200
1500
АС+ГлС+КС
(К)
К+0,1 ед.ПС mg/dm3
1 2
0
200
400
600
800
1000
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5
ед.ФС+0,1ед.ПС
mg/dm3
1
2 3 4
0
100
200
300
400
500
600
700
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5
ед.ФС+0,1ед.ПС
mg/dm3
1
2
3 4
0
100
200
300
400
500
600
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.Ф(СК )+1,5 ед.ФС+0,1ед.ПС
mg/dm3
(К)+1.5
ед.ФС
(К)+2.0
ед.ФС
(К)+2.5
ед.ФС
(К)+3.0
ед.ФС
1
2
3
4
5
6
7
8
0
20
40
60
80
100
120
140
0
2
4
6
8
10
12
14
16
18
0 8 16 24 32 40 72
g/100 cm3 mln./cm3
h
ПС К+1,5 ед.ФС К+1,5 ед.ФС+0,1
ед.ПС
1
2 3 4
0
300
600
900
1200
1500
АС+ГлС+КС
(К)
К+0,1 ед.ПС К+1,5 ед.ФС К+1,5
ед.ФС+0,1
ед.ПС
mg/dm3
(К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5
ед.ФС+0,1ед.ПС
(К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5
ед.ФС+0,1ед.ПС
(К)+1,5 ед.ФС (К)+2,0 ед.Ф(СК )+1,5 ед.ФС+0,1ед.ПС
1
2
0
200
400
600
800
1000
1200
АС+ГлС+КС
(К)
(К)+1.0
ед.ФС
mg/dm3
1
2 3 4
0
300
600
900
1200
1500
1800
АС+ГлС+КС (К) К+0,1 mg/dm3
1 2
0
200
400
600
800
1000
АС+ГлС+КС (К) (К)+0,1 ед.ПС mg/dm3
1
2 3 4
0
100
200
300
400
500
600
700
АС+ГлС+КС (К) (К)+0,1 ед.ПС mg/dm3
1
2
200
300
400
500
600
mg/dm3
1
2
0
200
400
600
800
1000
1200
1400
АС+ГлС+КС
(К)
(К)+1.0
ед.ФС
(К)+1.5
ед.ФС
(К)+2.0
ед.ФС
(К)+2.5
ед.ФС
(К)+3.0
ед.ФС
mg/dm3
1
2
3
4
5
6
7
8
0
20
40
60
80
100
120
140
0
2
4
6
8
10
12
14
16
18
0 8 16 24 32 40 72
g/100 cm3 mln./cm3
h
1
2 3 4
0
300
600
900
1200
1500
1800
АС+ГлС+КС (К) К+0,1 ед.ПС К+1,5 ед.ФС К+1,5 ед.ФС+0,1
ед.ПС
mg/dm3
1
2 3 4
0
300
600
900
1200
1500
АС+ГлС+КС
(К)
К+0,1 ед.ПС К+1,5 ед.ФС К+1,5
ед.ФС+0,1
ед.ПС
mg/dm3
1 2
0
200
400
600
800
1000
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5
ед.ФС+0,1ед.ПС
mg/dm3
1
2 3 4
0
100
200
300
400
500
600
700
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5
ед.ФС+0,1ед.ПС
mg/dm3
1
400
500
600
mg/dm3
(а)
(b)
AA+GA+XA
Control
Control+0.1 PA
units
Control+1.5 PhA
units
Control+1.5 PhA
units+0.1 PA units
AA+GA+XA
Control
Control+0.1 PA
units
Control+1.5 PhA
units
Control+1.5
PhA units+0.1
PA units
133
Rimareva L.V. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 117–126
substances enriched the wort with mineral nutrition
and activated the physiological activity of yeast cells.
It also led to a slight increase in ethanol yield during
the fermentation of the wheat and corn wort (by 2.3–
2.4 and 1.5–1.7%, respectively) (Table 4). The ethanol
concentration in the mash varied due to a higher starch
content in corn (Tables 1 and 4).
The metabolic processes in yeast cells improved
on the media treated with amylolytic, xylanase, and
proteolytic enzymes, as well as a complex of enzymes
with phytases. This improvement contributed to a
complete fermentation of carbohydrates and an increase
in ethanol yield, with a simultaneous decrease in
associated metabolites (Table 4, Figs. 4 and 5).
The highest concentration of ethanol generated in
the wort treated with a full complex of enzymes was
11.15% in the wheat mash and 12.74% in the corn mash.
The ethanol yield from the fermentation of wheat and
corn wort increased by 3.2 and 2.6%, respectively, when
treated with proteases and by 4.3 and 3.2%, respectively,
when treated with proteases and phytase (Table 4).
The rise in ethanol synthesis in the experimental
samples was probably associated with an improved
conversion of polymers of grain wort and its enrichment
with assimilable amine nitrogen. Another reason was
a release of macro- and microelements that was vital
for yeast cells. These findings were consistent with a
number of previous studies [2, 7, 25–27].
AA – amylolytic activity; GA – glucoamylase activity; XA – xylanase activity; PA – proteolytic activity; PhA – phytase activity
Figure 5 Concentrations of higher alcohols (1), aromatic alcohols (2), esters (3), and aldehydes (4) during yeast cultivation
on wheat (a) and corn (b) wort treated with various enzymatic complexes
Table 4 Ethanol yield during fermentation of wheat and corn wort treated with various enzymatic complexes
Enzyme composition Ethanol concentration in mash, % vol. Ethanol yield, cm3/100 g starch
Wheat Corn Wheat Corn
АA+GA+XA (Control) 9.80 ± 0.04 11.81 ± 0.05 65.6 ± 0.3 64.8 ± 0.4
Control + 0.1 PA units 10.87 ± 0.06 12.57 ± 0.07 67.7 ± 0.2 66.5 ± 0.3
Control + 1.5 PhA units 10.65 ± 0.05 12.20 ± 0.06 67.1 ± 0.1 65.8 ± 0.3
Control + 2.0 PhA units 10.72 ± 0.05 12.21 ± 0.07 67.2 ± 0.3 65.9 ± 0.3
Control + 1.5 PhA units + 0.1 PA 11.15 ± 0.06 12.74 ± 0.07 68.4 ± 0.2 66.9 ± 0.2
AA – amylolytic activity; GA – glucoamylase activity; XA – xylanase activity; PA – proteolytic activity; PhA – phytase activity
0
200
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5
ед.ФС+0,1ед.ПС
1
2 3 4
0
100
200
300
400
500
600
700
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5
ед.ФС+0,1ед.ПС
mg/dm3
1
2
3 4
0
100
200
300
400
500
600
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.Ф(СК )+1,5 ед.ФС+0,1ед.ПС
mg/dm3 (а)
(b)
AA+GA+XA
Control
Control+0.1 PA
units
Control+1.5 PhA
units
Control+1.5
PhA units+0.1
PA units
Control+2.0 PhA
units
0
200
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5
ед.ФС+0,1ед.ПС
1
2 3 4
0
100
200
300
400
500
600
700
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5
ед.ФС+0,1ед.ПС
mg/dm3
1
2
3 4
0
100
200
300
400
500
600
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.Ф(СК )+1,5 ед.ФС+0,1ед.ПС
mg/dm3
1
2 3 4
0
300
600
900
1200
1500
1800
АС+ГлС+КС (К) К+0,1 ед.mg/dm3
1 2
0
200
400
600
800
1000
АС+ГлС+КС (К) (К)+0,1 ед.ПС mg/dm3
1
2 3 4
0
100
200
300
400
500
600
700
АС+ГлС+КС (К) (К)+0,1 ед.ПС mg/dm3
1
2
3 4
0
100
200
300
400
500
600
АС+ГлС+КС (К) (К)+0,1 ед.ПС mg/dm3
1
2 3 4
0
300
600
900
1200
1500
1800
АС+ГлС+КС (К) К+0,1 ед.ПС К+1,5 ед.ФС К+1,5 ед.ФС+ед.ПС
mg/dm3
1 2
0
200
400
600
800
1000
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС ед.ФС+mg/dm3
1
2 3 4
0
100
200
300
400
500
600
700
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (ед.ФС+mg/dm3
1
2
3 4
0
100
200
300
400
500
600
АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.Ф(СК )+1,5 mg/dm3
AA+GA+XA
Control
Control+0.1 PA
units
Control+1.5 PhA
units
Control+1.5
PhA units+0.1
PA units
Control+2.0 PhA
units
CONCLUSION
We found almost no changes in the concentration of
phosphates during the fermentation of phytases-treated
wort, with a slight increase by the end of the process.
It was probably caused by the continuing biocatalytic
hydrolysis of phytic substances and the release of
phosphorus, as well as autolytic processes in the cell.
The control samples (without phytolytic enzymes) had
a significantly lower residual content of phosphates in
the wheat and corn mash (2.4–2.6 and 4.3–5.1 times,
respectively).
Our results confirmed that nitrogen and phosphorus
nutrition played a regulatory role in the generation and
metabolism of ethanol yeast. The catalytic action of
phytases and proteases ensured the accumulation of
easily assimilable phosphates, minerals, and amino
acids in the wort. Also, it intensified the growth of
yeast cells and increased the rate of carbohydrate
consumption. Finally, it decreased the formation of side
metabolites 1.7–1.9 times, mainly due to higher and
aromatic alcohols. At the same time, the Saccharomyces
cerevisiae 985-T yeast synthesized ethanol, whose yield
increased by 1.5–4.3%, depending on the type of grain
and enzyme complex. The greatest effect was achieved
by a full complex of enzymes (carbohydrase, protease,
and phytase).
CONTRIBUTION
The authors were equally involved in writing the
manuscript and are equally responsible for plagiarism.
CONFLICT OF INTEREST
The authors declare that there is no conflict of
interest.

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