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Polyakov V.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
INTRODUCTION
Modern alcohol technologies are based on complex
and deep processing of agricultural raw materials
aimed at improving production profitability. The
effectiveness of biotechnological processing is achieved
by developing new biocatalysts of various action and
substrate specificity. This ensures deep hydrolysis of
high molecular weight polymers of grain, especially rye
with its high content of non-starch polysaccharides, gum
substances, and mucus.
As shown by many studies, the use of complex
enzyme preparations with broad substrate specificity
can increase the depth of hydrolysis of grain polymers
into ethanol, especially when making concentrated grain
wort [1–4]. The complex should contain amylolytic,
proteolytic, and hemicellulase enzymes. Amylolytic
enzymes play a part in starch conversion: α-amylase in
starch dextrinization and liquefaction and glucoamylase
in its saccharification). Proteases are beneficial for the
generation and metabolism of alcoholic yeast, since
their catalytic effect on protein enriches the wort with
easily digestible amino acids assimilated by yeast [5].
Hemicellulases (β-glucanases and xylanases), catalysing
the hydrolysis of non-starch polysaccharides, decrease
the wort viscosity and lead to the formation of additional
fermented carbohydrates due to the destruction of grain
xylans and glucans. The synergic action of amylolytic,
proteolytic and hemicellulase enzymes improve the
quality of grain wort and its rheological properties,
especially when processing difficult-to-ferment raw
materials, such as rye and barley. Improved biocatalytic
conversion of grain polymers intensifies alcohol
Research Article DOI: http://doi.org/10.21603/2308-4057-2019-2-Х-Х
Open Access Available online at http:jfrm.ru
Effects of a complex phytase-containing enzyme preparation
on the rye wort fermentation process
Viktor A. Polyakov , Elena M. Serba* , Marina B. Overchenko, Nadezhda I. Ignatova,
and Liubov V. Rimareva
Russian Scientific Research Institute of Food Biotechnology –
a Branch of Federal Research Centre of Nutrition and Biotechnology, Moscow, Russia
* e-mail: serbae@mail.ru
Received October 11, 2017; Accepted in revised form March 20, 2018; Published Х Х, 2019
Abstract: A complex of amylases, proteases, and hemicellulases is known to enhance deep conversion of polysaccharides and
proteins, especially in the processing of difficult-to-ferment raw materials, such as rye, providing grain wort with soluble
carbohydrates, amino acids, and peptides. Grain is also a source of phosphorus, whose bioavailability can be increased by
hydrolysing the grain with phytase-containing enzyme preparations. However, their catalytic action during the preparation of
grain wort for alcohol production has hardly been studied. This study aimed to investigate the effect of a new complex phytasecontaining
enzyme preparation on yeast metabolism and the efficiency of rye wort fermentation. The work was carried out in the
Russian Research Institute of Food Biotechnology. The Glucavamorin complex enzyme preparations derived from recombinant
strains were the object of our research. The preparations differed in the activity level of the main enzyme, glucoamylase, and
minor hemicellulase enzymes, as well as in the presence of phytase. The results confirmed their biocatalytic ability to efficiently
hydrolyse polymers of rye grain. An increased content of hemicellulases in Glucavamorin-Xyl improved the rheological properties
of rye wort. The greatest effect was achieved with the phytase-containing Glucavamorin-Ply. This preparation improved the
phosphorus nutrition of yeast, which increased its biomass by 30% and decreased the level of fermentation by-products by 18–20%.
Alcohol yield tended to increase and its strength reached 10.5–10.9% vol. When using a phytase-containing enzyme complex, it
was possible to reduce the amount of the main enzyme, glucoamylase, without causing the key fermentation indicators to degrade.
Keywords: Rye wort, phytase, enzyme preparations, yeast, ethanol, fermentation, metabolites
Please cite this article in press as: Polyakov V.A., Serba E.M., Overchenko M.B., Ignatova N.I., and Rimareva L.V. Effects of
a complex phytase-containing enzyme preparation on the rye wort fermentation process. Foods and Raw Materials, 2019, vol. 7,
no. 2, pp. Х–Х. DOI: http://doi.org/10.21603/2308-4057-2019-2-Х-Х.
Copyright © 2019, Polyakov et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International
License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix,
transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.
Foods and Raw Materials, 2019, vol. 7, no. 2
E-ISSN 2310-9599
ISSN 2308-4057
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Polyakov V.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
fermentation, increasing the target product (ethanol)
yield and decreasing side metabolites formation.
A number of studies into the phytase effect on
the processing of sorghum and corn for lager beer
production showed a potential possibility of improving
nutritional conditions for yeast during the fermentation
of raw whole grains [6]. Some researchers noted a
positive effect of phytase treatment on the embryo
and fibre yield during the dry grinding of yellow dent
corn [7]. A study of the phytolytic effect on the quality
of wheat bread enriched with bran revealed an increase
in the bioavailability of iron contained in it [8]. To make
the conversion of phytin-containing raw materials more
efficient, microorganism strains with phytase activity
were selected and identified [9, 10]. Considerable
research was conducted into the use of phytase to
improve the digestibility of feed nutrients, including
phytate phosphorus [11, 12]. However, there is a lack
of studies into the effectiveness of phytolytic enzymes
during the preparation of concentrated grain wort for
alcohol production, especially from rye.
Currently, extensive studies are underway to obtain
enzyme preparations based on recombinant strains
of microscopic fungi using genetic engineering and
mutagenesis [13–15]. The preparations contain a
complex of enzymes with an increased biocatalytic
capacity for xylanase, β-glucanase, and cellulase, and
they can be used in the alcohol industry [16]. New highly
active multienzyme preparations can contribute towards
the implementation of innovative technologies for deep
conversion of grain into ethanol.
Of particular interest are phytolytic enzymes.
Phytase is an enzyme that breaks down phytic acid.
Phytic acid in the form of myo-inositol hexaphosphoric
acid or phytate (acid salt) is the main form of mineral
phosphorus in plant tissues [17]. Cereal grains have a
particularly high content of phytic acid [18]. Phosphorus
is essential for yeast cells to grow and develop. Under
anaerobic conditions, yeast assimilates phosphorus
mainly in the initial period of fermentation when its
consumption is 80–90% of the maximum content in
yeast. Young, actively breeding yeast cells are richer in
phosphorus than non-budding old cells. For example,
after 6 hours of fermentation, yeast cells accumulate
2.15% of phosphorus per dry matter, while this value
is only 1% at the end of fermentation. Therefore,
when making grain wort, it is important to enrich it
with phosphorus to ensure a stable process of yeast
generation and alcoholic fermentation.
Cereals are the main source of phosphorus, whose
bioavailability can be enhanced by hydrolysis of grain
with phytase-containing enzyme preparations. Phytate
hydrolysis helps reduce the consumption of enzyme
preparations, as it inhibits many enzymes and enables
the release of valuable trace elements, such as calcium,
magnesium, zinc, etc. [19, 20]. This way, it provides
alcoholic yeast with additional nutrition.
Apart from generating the main products of
fermentation, namely alcohol and carbon dioxide, yeast
cells synthesise metabolites called secondary products
or by-products of fermentation. The biosynthesis of
by-products is associated with the cell’s regulatory
functions. By-products formation depends on the
medium composition, the level of nitrogen, carbon,
and phosphorus in the medium, the conditions of
yeast cultivation and the genetic characteristics of
the strain used [3, 4]. One of the ways to improve the
efficiency of alcohol production is to create conditions
to reduce carbohydrate expenditure for the formation
of fermentation by-products through the use of media
with a balanced amino acid composition. The amount
of ethanol impurities can also be reduced by regulating
technological processes in such a way that conditions are
provided to promote ethanol synthesis with decreased
formation of fermentation by-products [4]. Therefore,
complex enzyme preparations contribute to a more
rational use of high-molecular components of grain raw
materials.
Our aim was to study the effect of a new complex
phytase-containing preparation on yeast metabolism and
the efficiency of rye wort fermentation.
STUDY OBJECTS AND METHODS
This research was conducted at the Department for
Biotechnology of Enzyme Preparations, Yeast, Organic
Acids and Biologically Active Substances of the Russian
Research Institute of Food Biotechnology, a branch of
the Federal Research Centre of Nutrition, Biotechnology
and Food Safety.
The study objects included rye grain, the
Saccharomyces cerevisiae 985T alcohol yeast, and
complex enzyme preparations (EP), Glucavamorin
Xyl and Glucavamorin Ply. The preparations were
obtained in the Laboratory of New Enzyme Producers
of the Russian Research Institute of Food Biotechnology
from the transformants of the commercial Aspergillus
awamori strain [21]. They differed in the activity level
of the major enzyme (glucoamylase) and minor enzymes
(hemicellulases).
We used the following methods to determine the
catalytic activity of the enzyme preparations.
Amylolytic and glucoamylase activity was determined
according to State Standard 54330-2011*. The method
for determining amylolytic activity is based on the
quantification of starch hydrolysed by amylolytic enzymes
to dextrins of various molecular weight under standard
conditions (temperature 30°С; pH 6.0 for bacterial and
4.7 for fungal α-amylase; hydrolysis duration 10 min). The
method for determining glucoamylase activity is based
on the quantification of glucose formed during starch
hydrolysis by glucoamylase under standard conditions
(temperature 30°С; pH 4.7; hydrolysis duration 10 min).
β-glucanase activity (β-GcS) was determined
according to State Standard 53973-2010**. The method
* State Standard 54330-2011. Enzyme preparations for food
industry. Methods for determination of amylase activity. Moscow:
Standartinform, 2012.
** State Standard 53973-2010. Enzyme preparations for food industry.
Methods for determination of β-glucanase activity. Moscow:
Standartinform, 2011.
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Polyakov V.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
is based on the quantitative determination of reducing
sugars resulting from β-glucanase action on β-1,4 bonds
of β-glucan under standard conditions (temperature
50°С; pH 4.7; hydrolysis duration 10 min).
Cellulase activity was determined according to
State Standard 55293-2012 (Enzyme preparations for
food industry. Method for determination of cellulase
activity. Moscow: Standartinform, 2014). The method
is based on the quantitative determination of reducing
sugars resulting from cellulase action on the substrate of
sodium carboxymethylcellulose (CMC) at 50°C.
Xylanase activity was determined according to State
Standard 55302-2012***. The method is based on the
quantitative determination of reducing sugars resulting
from xylanase (exoxylanase) action on β-1,4 bonds of xylan.
Phytase activity was determined according to State
Standard 31487-2012****. One unit of phytase activity
is the amount of enzyme that catalyses the hydrolysis of
sodium phytate to form 1 μmol of inorganic phosphate
per minute under standard conditions (temperature
37°C; pH 5.5; hydrolysis duration 15 min).
Rye, used as a raw material to make grain wort, was
prepared by “soft” enzymatic cooking at a water ratio
of 1 : 3. At the mash stage, thermostable α-amylase was
used for starch dextrinization at the rate of 0.5 unit/g
starch. At the saccharification stage, the test samples
included complex enzyme preparations, namely
Glucavamorin Xyl and Glucavamorin Ply in an amount
of 6–10 units/g starch each. The grain wort used as a
control sample was made with commercial enzyme
preparations (EP) without phytase: Glucomil L-706 as a
source of glucoamylase and BrewZyme BGX as a source
of xylanase, β-glucanase, and cellulase. The amount
of EPs for the biocatalysis of rye wort polymers in the
control sample was 10; 0.4; 0.05; and 0.2 unit/g starch
for glucoamylase, xylanase, β-glucanase, and cellulase,
respectively.
The wort was fermented with the Saccharomyces
cerevisiae 985T alcohol yeast, which has thermotolerant
and osmophilic properties, by the fermentation samples
*** State Standard 55302-2012. Enzyme preparations for food
industry. Method for determination of xylanase activity. Moscow:
Standartinform, 2013.
**** State Standard 31487-2012. Enzyme preparations. Methods of
phytase enzyme activity determination. Moscow: Standartinform, 2012.
method. The fermentation was carried out at 35°C
for 72 hours. The Guidelines for the Technical and
Chemical Control of Alcohol Production were followed
to determine the biochemical indicators of rye grain, wort
concentration, the number of yeast cells, the percentage of
budding cells, the content of total and residual reducing
carbohydrates (RS, reducing substances), and ethanol
concentration and yield [3, 5]. The composition and
level of side metabolites formed during fermentation
was analysed on an Agilent 6850 gas chromatograph
according to State Standard 55792-2013*****.
RESULTS AND DISCUSSION
Ground rye grain, whose biochemical composition
is given in Table 1, was used as a substrate for
fermentation. Rye is known to be a multicomponent
substrate characterised by a high content of
hemicelluloses and gum substances. The studied rye
grain contained 56.4% of starch, 8.4% of hemicellulose,
and 2.2% of cellulose. The presence of non-starch
polysaccharides complicates the process of preparing
concentrated wort that has good rheological properties
and contains soluble carbohydrates in a form that is
accessible to yeast cells.
Therefore, new enzyme preparations of
glucoamylase and hemicellulase action were tried to
prepare media ensuring stable yeast generation and
alcohol fermentation.
To prepare the grain for fermentation, we used the
Glucavamorin complex enzyme preparations based
on A. Аwamori recombinant strains and containing
xylanase, β-glucanase, and cellulase, along with
amylolytic enzymes.
The Glucavamorin enzyme preparations had
glucoamylase (GlS), xylanase (XS), β-glucanase
(β-GcS), and cellulase (ClS) activities. Of particular
interest was the Glucavamorin-Ply preparation, which
additionally exhibited a high level of phytase (PhS)
activity (4,300 units/g). Glucavamorin-Xyl had a
higher level of xylanase (810 units/g) and glucoamylase
(9,640 units/g) activity. The results are in Table 2.
The complex enzyme preparations were used as
sources of glucoamylase and concomitant enzymes.
***** State Standard 55792-2013. Brew from food raw material.
Gas-chromatographic method for determination of volatile organic
admixtures. Moscow: Standartinform, 2014.
Table 1. Biochemical indicators of rye grain
Indicator Content, %
Proteins 15.7
Mono- and disaccharides 1.5
Starch 56.4
Cellulose 2.2
Hemicellulose 8.4
Gum substances 2.5
β-glucan 0.18
Arabinoxylan 0.65
Table 2. Comparative activity of enzyme preparations derived
from Aspergillus awamori recombinant strains
and commercial preparations
Enzyme preparation Enzyme activity, units/g
GlS XS ClS β-GcS PhS
Glucavamorin-Xyl 9,640 810 120 285 0
Glucavamorin-Ply 7,580 260 130 310 4,300
BrewZyme BGX 0 3,600 2,000 500 0
Glucomil L-706 8,000 0 0 0 0
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Polyakov V.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
Therefore, their amount was based on the glucoamylase
quantity of 6–10 units/g starch. The level of concomitant
enzymes contained in the complex EPs was also
monitored (Table 3).
In the control sample (No. 1) we used Glucomil, for
starch saccharification, and BrewZyme, for the catalytic
hydrolysis of hemicelluloses. Glucavamorin-Xyl was
used in test samples No. 2, 3, and 4 in an amount of 6.0,
8.0, and 10.0 units/g starch, respectively, at the stage of
saccharification at 58–60°C. Glucavamorin-Ply was
used in test samples No. 5, 6, and 7 in an amount of
6.0–10.0 units/g starch.
The studies showed that the concentration of soluble
solids (SS) in the rye wort prepared with various amounts
of the Glucavamorin complex enzymes was 25.8–26.7%,
and the content of reducing carbohydrates was 14.9–15.9%.
The highest rates of reducing substances were achieved
with the use of Glucavamorin-Ply. Apparently, this was
due to a higher β-glucanase activity of the preparation.
The catalytic action of β-glucanase contributed to
the hydrolysis of grain glucans and the formation of
additional reducing carbohydrates (Table 3).
Further studies showed that the quality of the grain
wort made with the complex enzyme preparations
affected the processes of yeast generation and alcohol
fermentation.
The comparative studies into the fermentation of
rye wort treated with phytase-free enzyme preparations
revealed a higher efficiency of Glucavamorin-Xyl,
especially in sample No. 4, where it was used at the
maximum amount (10 units/g starch). As seen in
Table 4, the yield of ethanol reached 65.7 cm3/100 g
starch, exceeding the rate in the control sample
(65.3 cm3/100 g starch).
Thus, the above confirmed that the synergic action
of the enzymes was determined by their catalytic effect
on the grain structural polymers interrelated with
each other. We found that to improve the technological
parameters of concentrated grain wort, it was necessary
to use a complex of hemicellulase enzymes (xylanase,
β-glucanase, and cellulase), along with the traditionally
used amylases. The effective destruction of non-starch
polymers improved the rheological properties of the rye
wort, which had a positive effect on the fermentation
Table 3. Effects of enzyme complexes derived from recombinant strains on the concentration of rye wort and content of reducing
carbohydrates
Rye wort
samples
Enzyme complex and amount, unit/g Rye wort indicators
Glucomil +BrewZyme
(control)
Glucavamorin-
Xyl
Glucavamorin-
Ply
Soluble
solids (SS), %
Reducing
carbohydrates (RC), %
1 GlS – 10.00
XS – 0.40
ClS – 0.22
β-GcS – 0.05
– – 26.2 15.5
2 – GlS – 6.00
XS – 0.50
ClS – 0.08
β-GcS – 0.18
– 25.8 14.9
3 – GlS – 8.00
XS – 0.70
ClS – 0.10
β-GcS – 0.23
– 26.1 15.3
4 – GlS – 10.00
XS – 0.81
ClS – 0.12
β-GcS – 0.31
– 26.4 15.7
5 – – GlS – 6.00
XS – 0.21
ClS – 0.12
β-GcS – 0.24
PhS – 3.41
26.2 15.0
6 – – GlS – 8.00
XS – 0.31
ClS – 0.14
β-GcS – 0.32
PhS – 4.52
26.4 15.5
7 – – GlS – 10.00
XS – 0.37
ClS – 0.18
β-GcS – 0.40
PhS – 5.63
26.7 15.9
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Polyakov V.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
process. The use of Glucavamorin-Xyl with an increased
concentration of hemicellulases contributed to a slight
rise in ethanol yield compared to the control sample with
the same amount of glucoamylase (10 units/g starch;
Table 4).
The greatest effect was achieved with Glucavamorin-
Ply, which included phytase, apart from a hemicellulase
complex. For example, the use of Glucavamorin-Ply
in samples No. 5, 6, and 7 in amounts of 6.0; 8.0; and
10.0 units/g starch, respectively, intensified the processes
of yeast generation and alcohol fermentation, which
increased the ethanol yield to 66.0 cm3/100 g and
decreased the concentration level of residual carbohydrates
in the mash to 0.64 g/100 cm3 (Fig. 1, Table 4).
Such enzyme complexes contribute to a more
rational use of grain components, reduce the wort
viscosity, enrich the wort with nutrients, increase the
physiological and fermentation activity of yeast and, as
a result, accelerate the processes of yeast generation and
alcohol fermentation.
The phytolytic action of the preparation had a
positive effect on yeast generation and led to a higher
concentration of yeast cells compared to the control (I)
and those samples where a phytase-free glucoamylase
EP was used (Table 2, samples No. 2–4). There was
a tendency towards an increase in ethanolyield to
65.7–66.0 cm3/100 g starch, with alcohol concentration
of 10.5–10.9% vol., even though the amount of
glucoamylase was reduced by 20–40% (from 10.0 to
6.0–8.0 units/g starch) in samples No. 2 and 4.
By catalysing the hydrolysis of phytic acid in the
raw material, phytase contained in Glucavamorin-Ply
appeared to release additional mineral phosphorus,
assimilated by alcohol yeast. This improved the growth,
activity and productivity of yeast cells.
We studied Saccharomyces cerevisiae 985T
yeast cultured under anaerobic conditions on rye
media with enzyme preparations that differed in the
content of phytolytic enzymes. The study showed that
Glucavamorin-Ply contributed to increased physiological
activity of yeast cells (Fig. 1).
As seen in Fig. 1, the presence of phosphorus in the
medium led to intensified yeast development, especially
in the lag phase (first 18–24 hours of growth), accelerated
Table 4. Effects of enzyme complexes derived from recombinant strains on the rye wort fermentation
Rye wort
samples
Enzyme complex and amount, unit/g Fermentation indicators per 72 h
Glucomil +
BrewZyme
(control)
Glucavamorin-
Xyl
Glucavamorin-
Ply
Yeast, mln/cm3 Soluble solids,
g/100 cm3
Alcohol concentration,
% vol.
Ethanol yield,
18 h 44 h cm3/100 g starch
1 GlS – 10.00
XS – 0.40
ClS – 0.22
β-GcS – 0.05
– – 85 94 0.80 10.2 65.3
2 – GlS – 6.00
XS – 0.50
ClS – 0.08
β-GcS – 0.18
– 68 83 0.74 10.2 65.3
3 – GlS – 8.00
XS – 0.70
ClS – 0.10
β-GcS – 0.23
– 82 100 0.72 10.3 65.5
4 – GlS – 10.00
XS – 0.81
ClS – 0.12
β-GcS – 0.31
– 100 107 0.68 10.5 65.7
5 – – GlS – 6.00
XS – 0.21
ClS – 0.12
β-GcS – 0.24
PhS – 3.41
108 135 0.68 10.5 65.7
6 – – GlS – 8.00
XS – 0.31
ClS – 0.14
β-GcS – 0.32
PhS – 4.52
115 138 0.66 10.8 65.9
7 – – GlS – 10.00
XS – 0.37
ClS – 0.18
β-GcS – 0.40
PhS – 5.63
120 140 0.64 10.9 66.0
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Polyakov V.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
Higher alcohols Aldehydes Esters Organic acids
Sample 1 Sample 3 Sample 6
carbohydrate consumption, and increased concentration
of yeast cells (1.4–1.5 times). The fermentation
process was more complete, with the minimal
amount of residual carbohydrates (0.64 g/100 cm3)
and the maximum ethanol yield (Table 4).
The analysis of the phytolytic effect of the
enzyme preparations on the metabolism of yeast cells
showed that Glucavamorin-Ply contributed to an
18–20% decrease in the formation of side metabolites
accompanying ethanol synthesis, thereby improving the
quality of the target product (Fig. 2).
We compared the metabolites synthesised during the
fermentation of rye wort made with the glucoamylase
enzyme preparations. We found that the phytasecontaining
Glucavamorin-Ply (sample No. 4) lowered the
content of volatile substances by the end of fermentation,
compared to the control (sample No. 1) and the sample
with the phytase-free Glucavamorin-Xyl. It did so by
reducing the synthesis of major impurities: higher
alcohols, aldehydes, and esters (Fig. 3). This improved
the organoleptic and analytical indicators of the final
product, i.e. ethanol.
CONCLUSION
The study showed that the use of the Glucavamorin
complex enzyme preparations, derived from Aspergillus
awamori recombinant strains, at the stage of preparing
rye wort for fermentation enhanced the efficiency
of yeast generation and alcohol fermentation. The
increased content of minor hemicellulase enzymes in
Glucavamorin-Xyl improved the rheological properties
of the rye wort and had a positive effect on the
fermentation process.
The catalytic effect of the phytase-containing
Glucavamorin-Ply enzyme preparation improved
the phosphorus nutrition of yeast. This intensified
yeast generation, increased the concentration of yeast
cells in the rye wort by 30%, reduced the level of side
metabolites by 18–20%, and enhanced ethanol yield.
The study revealed that using a phytase-containing
enzyme complex made it possible to reduce the amount
of the main enzyme, glucoamylase, from 10.0 to
6.0–8.0 units/g starch without causing the key
fermentation indicators to degrade.
Thus, the study confirmed that the synergic effect
of enzymes with different substrate specificity on the
polymers of grain raw materials enhanced the efficiency
of their conversion when fermenting rye wort.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
The study was financed with a federal subsidy within
the framework of the Programme for Basic Scientific
Research of the State

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