INTRODUCTION
The development and subsequent quality assessment
of functional foods is one of the priorities of healthy
nutrition [1]. Functional foods with a programmed
chemical composition can be fortified with important
nutrients and are suitable for various categories of
population, e.g. athletes, lactating and pregnant women,
senior citizens, children, etc. [2].
However, priority goes to gastrointestinal and
allergic patients and professional athletes. Their nutrition
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Gavrilova K.V. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
requires scientific approaches, since their diet should
contain a complex of peptides and free amino acids, as
well as simple and complex carbohydrates [3].
Foods fortified with proteins, especially those
containing essential amino acids, contribute to the rapid
and effective recovery of muscle tissue after intense
physical exertion. Peptides and polypeptides are known
to accelerate metabolic processes, hormone production,
and muscle tissue growth [4].
Food intolerance and allergic reactions are another
problem of modern society. Some people are allergic to
products that contain proteins of animal or plant origin.
Hence, a new generation of food products with easyto-
digest nutrients remains an important objective of food
industry. Modern studies confirm that plant raw materials
– and legumes in particular – are suitable for isolation
and modification of proteins, short peptides, and amino
acids [5, 6]. These groups of compounds are widely used
as dietary supplements and ingredients for functional
products [7–9]. Pea protein has a better nutritional value,
amino acid composition, and anti-nutrients than soybeans,
beans, and other legumes [10, 11].
There are many methods to isolate protein from
plant materials for subsequent hydrolysis [12–14].
However, most of them remain inefficient for enzymatic
transformations of heterogeneous substrate. Preliminary
mechanical activation means that raw material has to
be processed in specially designed energy-stressed
activator mills. The procedure makes it possible to
control the reactivity of solid substrates. In addition, it
increases the speed and yield of water-soluble products
for commercial purposes [15, 16].
However, the process of enzymatic reactions
after preliminary mechanical activation remains
understudied. A series of studies on the hydrolysis of
cellulose showed that the increase in specific surface
area and the degree of crystallinity of the substrate
affected the rate and yield of enzymatic hydrolysis [17].
In addition, it is important to study the elusive transfer
of mechanochemical processes from lignocellulose to
protein and starch.
The research objective was to study the mechanically
activated hydrolysis of pea biomass, as well as to
obtain a hydrolysate fortified with free amino acids and
peptides to be used in functional foods.
STUDY OBJECTS AND METHODS
The experiment featured dry biomass of split pea
seeds harvested in 2017. The peas corresponded with
State Standard 6201-68*, Class I, and was produced by
OOO ECO-PAK (Novosibirsk region, Russia). Before
the experiment, the pea biomass was subjected to rough
grinding in a knife mill to the size of ≤ 2 mm. The
ground biomass was vacuum-packed, stored at room
temperature, and used for further experiments.
* State Standard 6201-68. Polished pea. Specifications. Moscow:
Standartinform; 2010. 3 p.
Protosubtilin G3h was used as enzyme preparation
(OOO Sibbiopharm, Berdsk, Russia). The complex
was chosen for its catalytic activity and availability
for further technological application. This industrially
available enzyme preparation contains a complex of
enzymes that consists of neutral and alkaline proteases
and glycosidases, i.e. ≈ 11000 U/g of protease, ≤ 150 U/g
of xylanase, ≤ 200 U/g of β-glucanase, and ≤ 300 U/g
of α-amylase [18]. Protosubtilin belongs to the group
of enzyme feed additives that are able to break down
high-molecular proteins. This enzyme preparation is
produced by Bacillus subtilis.
Gravimetric methods were used to assess moisture
and ash content in the plant materials and processed
products, respectively [19, 20].
X-ray diffraction and thermal desorption of gases
were employed to measure the degree of crystallinity
and specific surface area according to the methods
described in [17] and [21], respectively.
The method described by Fadeeva et al. was used
to perform the elemental analysis that made it possible
to determine the quantitative protein content in the
peas. After that, the protein content was determined
using the nitrogen content with conversion factor of
6.25 according to the Kjeldahl method [22–24].
The mass fraction of soluble substances was
determined by the method of exhaustive extraction in a
Soxhlet extractor for 24 h. Distilled water was used as
extractant. The yield of water-soluble substances was
measured according to the reduction of the mass after
the extraction.
The content of free amino acids was defined at the
Centre of Mass Spectrometry Analysis (Institute of
Chemical Biology and Fundamental Medicine). An
optimised standard procedure was used as in [25]. A
set of isotope-labelled amino acids and acyl carnitines
No. 55000 (Chromsystems Instruments & Chemicals,
Germany) served as internal standards and solutions. An
Agilent-1200 chromatographic system with an Agilent
6410 QQQ mass spectrometer (Agilent Technologies,
USA) was employed as an HPLC-MS/MS system. A
quantitative analysis was performed in the mode of
multiple reactions monitoring; the total analysis time
was 2.5 min. The obtained data were processed using
MassHunter v.1.3 software.
The molecular weight of protein molecules was
measured using the Laemmli SDS PAGE procedure
[26]. For pre-denaturation, proteins were treated with
1.4-dithiothreitol at a 1:1 ratio. After that, they were
placed in a thermoshaker at 95°C (Biosan, Latvia) for
7 min. An Elf-4 power source was used to create electric
field (DNA-Technology, Russia). The concentrations of
polyacrylamide in the concentrating and separating gel
were 4% and 18%, respectively. The pre-phoresis stage
lasted 15 min. The current force was 15 mA, while
during the phoresis stage it was 35 mA.
To identify the zones of proteins after the
electrophoresis, they were stained with Coomassie
R-250 pigment according to the procedure described by
39
Gavrilova K.V. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
56%
21%
4%
12%
7%
Grains and legumes Sugar beet
Sunflower Potato
Vegetables
5%
10%
4% 4%
Dyballa et al. [27]. Protein markers were represented
by Unstained protein MW marker (Thermo Fisher
Scientific, USA) with protein molecular weight of
14.5–116 kDa and Unstained protein ladder (Thermo
Fisher Scientific, USA) with protein molecular weight of
5–250 kDa. MultiChrom-Planar programme processed
the mathematical data [28].
The fractionation of the plant material was
conducted according to the method described in [29, 30].
The initial crushed pea biomass was extracted in
alkaline water. A 1M solution of sodium hydroxide
was added to the pre-ground pea biomass. The solution
consisted of 2.5 ml of solution per 1 gram of biomass
(pH 9.0). The suspensions were placed in a WSB-30
water bath at 45°C and 180 rpm for 30 min (DAIHAN
Scientific, Korea). After the extraction, the soluble
portion was separated by centrifugation at 6000 rpm for
20 min. The precipitate was used in the next extraction
cycle under the same conditions. The extracted
components were precipitated with a threefold volume of
cooled ethanol and dried in a laboratory frost dryer Iney
4 (Institute for Biological Instrumentation of the Russian
Academy of Sciences, Russia).
After three extraction cycles (fraction No. 4), the
insoluble residue – a carbohydrate fraction – was washed
twice with chilled ethanol and dried under similar
conditions.
The mechanical activation of the plant material
with enzymes was carried out in an RM-20 roller millactivator
(5.5 kW), which was equipped with a water
cooling device (Fig. 1). The pea biomass was mixed
with a dry enzyme preparation and processed in an
activator at a rotor speed of 1450 rpm. The mixture of
raw materials and enzymes was supplied automatically
at a rate of 3 kg/h.
The spray-drying was performed in a Mini Spray
Dryer B-290 (Büchi, Switzerland) in the following
conditions: nozzle temperature = 110°C, cyclone
temperature = 70°C, gas flow rate = 700 L/h, feed rate =
5 mL/min.
The enzymatic hydrolysis l asted 7 h a t 5 0°C.
50 ml of distilled water was added to 15 g of initial or
mechanically activated pea biomass with a certain
amount of the enzyme preparation. Enzyme loading
equalled 0.5%–3%. Suspensions were thoroughly mixed
until uniform. For enzymatic hydrolysis, the suspensions
were placed in a WSB-30 water bath (DAIHAN
Scientific, Korea) at 50°C and 120 rpm. After enzymatic
hydrolysis, the supernatant was centrifuged at 6000 rpm
for 20 min. No enzyme preparation was added to the
control samples.
RESULTS AND DISCUSSION
Suitable protein plant materials were selected
for the mechanoenzymatic processing to be used in
functional, special, and therapeutic food products. The
physical and chemical characteristics are given below.
In addition, the selection was based on an analysis of
the existing market for high-protein plant materials,
state statistics, distribution of croppage, and percentage
of various cultures in Russian regions. This approach
made it possible to identify raw material with suitable
physicochemical parameters, as well as to determine its
prospects in subsequent processing and implementation.
Figure 2 shows a distribution diagram of croppage
in Russia in 2017. The diagram was based on the data
obtained from the Federal State Statistics Service [31].
Cereals and legumes clearly prevail over other cultures.
Legumes are richer in protein than grains. An analysis
of the distribution of croppage within the group of
leguminous crops showed that a large proportion (77%)
belongs to peas (Fig. 3).
Figure 1 Scheme of the roller type activator mill RM-20
Plant raw material
Rolls
Rotator
Mechanically processed product
Starter
Figure 2 Croppage distribution in Russia
56%
21%
4%
12%
7%
Grains and legumes Sugar beet
Sunflower Potato
Vegetables
5%
10%
4% 4%
40
Gavrilova K.V. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
Thus, legumes proved to be the most advantageous
source of vegetable protein in Russia, especially peas,
which contain about 25% of protein. In spite of the
fact that soy contains up to 35% of protein, it was not
considered in this study since it is rich in anti-nutritional
substances, Moreover, it has a low consumer loyalty,
which cannot be ignored in product development [5–7,
10, 32]. The protein content in peas varies greatly
according to genotypic characteristics and the
cultivation conditions. Leguminous proteins are poor
in methionine and cysteine. This is typical of plant
proteins. For instance, grain crops are poor in lysine
and threonine. However, the biological value of products
obtained from them can be fortified by a limiting amino
acid or other types of plant materials.
The present research involved a comparative
analysis of the protein content and amino acid
composition together with its coefficient of imbalance
and functionality in high-protein plant raw materials.
Peas demonstrated the highest functionality ratio of
amino acid composition (FRAAC) – 0.6, while soybeans
had 0.4 and beans and lentils had 0.3. This indicated
that peas possessed the optimal ratio of amino acids if
compared with reference chicken egg protein.
Thus, pea biomass appeared to have a high
nutritional value and a balanced amino acid
composition, which made it an optimal research subject.
Its physical and chemical patterns can subsequently
be transferred to other types of biomass. The samples
obtained after fractionation (Fig. 4) and freeze drying
were analysed for the protein content in the dry product.
The results are presented in Table 1. A polyacrylamide
gel electrophoresis defined the molecular weight of the
proteins in the fractions.
The obtained data are consistent with those already
published Mession et al.: the pea biomass contained
23–24.4% of protein and 48–60.3% of starch [33].
Fractions No. 1 and 2 isolated from the biomass were
fortified with proteins, while fraction No. 3 was fortified
with proteins and carbohydrates, and fraction No. 4 – with
carbohydrates.
The electrophoregram (Fig. 5) shows that fractions
1–3 contained proteins with molecular weight =
5–135 kDa, which corresponded to molecules that
consisted of 50–1350 amino acid residues. The
predominating molecules were those with molecular
weight = 24–135 kDa (240–1350 amino acid
residues). They were most likely to be sub-units of
11S-globulins [34]. Both the elemental analysis and the
gel electrophoresis showed that the content of protein
molecules in fraction 4 was at the level of trace amounts.
As proved by cellulose processing, enzyme
preparation increases the efficiency of subsequent
enzymatic hydrolysis, if added at the stage of mechanicchemical
processing [34]. The enzyme complex
used in the present research had a suitable catalytic
activity profile and was cheaper than its analogues,
such as proteases AP1, Alcalase, Savinase, Esperase,
and Neutrase (Shandong Longda Bio-Products and
Novozymes).
A set of experiments made it possible to determine
the effect of the conditions of mechanical activation
on the subsequent enzymatic hydrolysis. The pea
biomass was subjected to mechanical activation 1)
without enzymes and 2) with an insufficient amount
Figure 3 Percentage ratio of the croppage of legumes in
Russia
21%
Grains and legumes Sugar beet
Sunflower Potato
Vegetables
77%
5%
10%
4% 4%
Peas
Lentils
Chick peas
Grain vetch (Vicia L.) and vetch-prevailing mixes
Grain forage lupine (Lupinus L.)
0
20
40
60
80
100
0
3
6
9
12
0.60 0.90 1.40 2.10 3.15 4.75 7.20 10.90 16.50 24.90 37.70
Differential distribution, %
Fraction size, μm
Integral distribution, %
21%
Grains and legumes Sugar beet
Sunflower Potato
Vegetables
77%
5%
10%
4% 4%
Peas
Lentils
Chick peas
Grain vetch (Vicia L.) and vetch-prevailing mixes
Grain forage lupine (Lupinus L.)
0
20
40
60
80
100
0
3
6
9
12
0.60 0.90 1.40 2.10 3.15 4.75 7.20 10.90 16.50 24.90 37.70
Differential distribution, %
Fraction size, μm
Integral distribution, %
Figure 4 Frozen fractionation products before freeze drying:
1–4 are numbers of corresponding fractions
Table 1 Protein content in the initial raw material and in the
fractions
Sample Protein
content, %
Fraction content in
the raw material, %
Raw material 24.3 –
Fraction No. 1 97.1 19.0
Fraction No. 2 86.7 6.5
Fraction No. 3 45.7 0.4
Fraction No. 4 Trace 74.1
21%
Grains and legumes Sugar beet
Sunflower Potato
Vegetables
77%
5%
10%
4% 4%
Peas
Lentils
Chick peas
Grain vetch (Vicia L.) and vetch-prevailing mixes
Grain forage lupine (Lupinus L.)
0
20
40
60
80
100
0
3
6
9
12
0.60 0.90 1.40 2.10 3.15 4.75 7.20 10.90 16.50 24.90 37.70
Differential distribution, %
Fraction size, μm
Integral distribution, %
41
Gavrilova K.V. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
of enzymes (1%) in relation to the substrate. The
subsequent hydrolysis and complete extraction (Table 2)
showed that the mechanical activation without enzymes
barely increased the yield of the subsequent hydrolysis.
However, the mechanical activation with enzymes
increased the yield during subsequent hydrolysis from
18% to 60%, i.e. by ≥ 3 times
The results can be explained by the fact that a
simultaneous activation of substrate and enzymes
produced mechanocomposite. The mechanocomposite
was an intermediate solid-phase product with a high
reactivity. In such mechanocomposites, enzyme particles
are distributed non-diffusively, or mechanically, over the
surface of the substrate, which was disordered during
the activation process. Similar effects were observed in
other cases of activation of food and non-food plant raw
materials [35, 36]. When mechanocomposite is formed,
it usually increases the rate and yield of the subsequent
proteolytic and glycolytic processes. In this case, a
preliminary chemical interaction preceded the mixing of
the enzymes and the substrate. This interaction resulted
from a significant increase in surface area, which
enlarged from 0.6 to1.9 m2/g, and an extra disordering
of the substrate structure, whose crystallinity decreased
from 25% to 14%.
The conversion of enzymatic hydrolysis was studied
under the same conditions, according to the substrate –
enzyme ratio. The enzyme preparation was added in 0.5,
1, 2, 2.5, and 3% (Table 3).
Table 3 shows that the amount of water-soluble
substances increased, as the amount of enzymes
increased from 0.5% to 2%. The water-soluble
substances included reducing carbohydrates, which are
low molecular weight products of starch hydrolysis.
When the load of the enzyme complex increased to
2.5%–3%, the number of reaction products did not
increase. This might have been caused by the fact that
the sorption sites of the substrate were completely
filled with enzymes. The situation was fully consistent
with the idea that the heterogeneous stage of enzymatic
hydrolysis has a limiting effect.
The polyacrylamide gel electrophoresis was
used to study the changes in the molecular weight
during the enzymatic hydrolysis. Figure 6 shows the
electrophoregram of the proteins contained in the
hydrolysate 1–7 h after the hydrolysis. The data prove
that the amount of the original protein molecules
significantly decreased within 7 h. As a rule, proteins
degrade within 2 h. The molecular weight of the
degradation products of the original polypeptide
proteins revealed no significant changes after 4 h. After
Figure 5 Electrophoregram (A) and MWD profilograms (B) of proteins in the fractions; 1, 2, and 3 – fraction numbers. Fraction
No. 4 is not represented as it appeared to have no proteins in its composition
116 kDa
69 kDa
45 kDa
35 kDa
25 kDa
18 kDa
14 kDa
Table 2 Yield of water-soluble substances according to the processing conditions
Extraction from
the initial raw
material
Extraction from the product
of mechanical activation
without enzymes
Product after mechanical
activation (without
enzymes) and hydrolysis
Product after mechanical
activation with 1% of enzyme
preparation after hydrolysis
Yield of water-soluble
substances, %
18.0 18.5 25.1 60.6
Table 3 Yield of water-soluble substances and reducing
carbohydrates according to the amount of enzyme preparation
Enzyme
preparation, %
Yield of water-soluble
substances, %
Yield of reducing
carbohydrates, %
0.0 25.8 1.5
0.5 38.7 4.0
1.0 55.4 7.6
2.0 78.5 16.3
2.5 78.5 16.3
3.0 78.5 16.3
42
Gavrilova K.V. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
7 h, there remained an insignificant amount of stable
polypeptides with a molecular weight of ≈ 20 kDa.
These polypeptides were associated with the polypeptide
chain of legumin, which is resistant to neutral and
alkaline proteases [33].
Thus, an enzymatic hydrolysis that exceeded 4 h is
ineffective, since the number of low molecular weight
polypeptides did not increase much after that time.
Figure 6 Electrophoregram (A) and profilogram (B) of the molecular weight distribution of proteins in the hydrolysate after 0, 1, 2,
3, 4, and 7 h
69 kDa
45 kDa
35 kDa
25 kDa
18 kDa
Table 4 Content of essential amino acids in the hydrolysate
and in the control sample
Amino acid Amino acid content, μg/g
Pea biomass extract Hydrolysed pea biomass
Ile+Leu 515 10479
Met 110 1320
Phe 514 7681
Val 441 3921
Figure 7 Scanning electron microscopy of the hydrolysate
after spray-drying
μm
Figure 8 Granulometric composition of the hydrolysate after spray-drying
56%
21%
4%
12%
7%
Grains and legumes Sugar beet
Sunflower Potato
Vegetables
77%
5%
10%
4% 4%
Peas
Lentils
Chick peas
Grain vetch (Vicia L.) and vetch-prevailing mixes
Grain forage lupine (Lupinus L.)
0
20
40
60
80
100
0
3
6
9
12
0.60 0.90 1.40 2.10 3.15 4.75 7.20 10.90 16.50 24.90 37.70
Differential distribution, %
Fraction size, μm
Integral distribution, %
43
Gavrilova K.V. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
A mass-spectrometric analysis of amino acids was
performed to study the low molecular weight products
of the enzymatic hydrolysis. Table 4 shows that the
hydrolysis resulted in a significant increase in the
number of essential amino acids in comparison with the
control sample obtained without enzymes.
For the hydrolysates to be widely implemented,
there have to be new ready-made food products with
prolonged shelf life. Thus, a set of experiments on
spray-drying had to be performed [37]. The spraydrying
process can be easily scaled and is widely used in
food industry to produce dry enzymes, foodstuffs, and
unstable compounds [38–40].
The product obtained by spray drying (Figs. 7
and 8) had a monomodal particle size distribution.
The main share belonged to spherical particles with a
diameter of 5–20μм. The size was associated with the
characteristics of the equipment: the nozzle opening was
25μм in diameter.
Most of the particles were concave, which made it
possible to describe the mechanism of drying. Initially,
a powerful inward-directed deformation removed the
solvent from the surface of the drop. As a result, there
formed a layer of the product. The solvent diffused
the layer of the dry product, after which the particle
deformed and collapsed.
In the control experiment, vacuum drying without
splashing the hydrolysate resulted in the formation of
a layer that was not dispersed into individual particles.
An electron scanning microscopy of the ground product
(Fig. 9) showed that it had a dense structure without
pores. This confirms the spray-drying mechanism:
the drying occurs on the surface, while the dry layer
captures the solvent, and a high mechanical tension
deforms the particle, giving it a concave shape.
CONCLUSION
Thus, the paper featured the process of mechanical
activation and subsequent enzymatic hydrolysis of pea
proteins. The original pea biomass was described using
modern chemical methods. The protein content was
24.3%, and MWD was 5–135 kDa.
The fractionation produced four fractions of
biopolymers with various contents of protein and
carbohydrate molecules. The experiment made it
possible to define the optimal conditions for the
mechanical activation performed together with
proteolytic enzymes. The enzymes were obtained
from the complex enzyme preparation Protosubtilin
G3x. When both the substrate and the enzymes were
mechanically activated, it produced mechanocomposite.
As a result, the specific surface area increased by
3.2 times, while the crystallinity decreased by 2 times,
which raised the yield of the subsequent enzymatic
hydrolysis from 18% to 61%.
During hydrolysis, protein broke down within
2 h, and there was almost no change after 4 h. The
experiment detected non-hydrolysed protein molecules
with a molecular weight of ≈ 20 kDa. They presumably
corresponded with legumin, which is resistant to neutral
and alkaline proteases.
The research involved an experiment on spraydrying
of the obtained hydrolysates for their potential
use as food components. The resulting product had a
monomodal particle size distribution. The particles had a
spherical shape with a diameter of 5–20 μ.
CONFLICT OF INTEREST
The authors declare that there is no conflict of
interest related to this article.
ACKNOWLEDGEMENTS
The authors would like to express their deepest
gratitude to V.I. Berezin, G.N. Nesterova, I.V. Ivanov,
S.Yu. Abramov, and I.B. Orekhov (Institute of Solid
State Chemistry and Mechanochemistry), A.G. Ogienko
and N.F. Beizel (Institute of Inorganic Chemistry), and
L.N. Rozhdestvenskaya (Novosibirsk State Technical
University).
FUNDING
The research was funded by the Russian Science
Foundation, Project No. 17-73-10223: ‘Processes of
mechanical activation and enzymatic hydrolysis of plant
raw material polymers for obtaining low-molecular
components of functional foods’.

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