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70
INNOVATIVE TRENDS IN THE DEVELOPMENT OF ADVANCED
TRITICALE GRAIN PROCESSING TECHNOLOGY
E. P. Meleshkina, G. N. Pankratov, I. S. Vitol*,
R. H. Kandrokov, and D. G. Tulyakov
All-Russian Research Institute of Grain and its Processing Products,
Dmitrovskoye Highway 11, Moscow, 127434, Russian Federation
* e-mail: vitolis@yandex.ru
Received May 17, 2017; Accepted in revised form August 10, 2017; Published December 26, 2017
Abstract: The study has been carried out at the All-Russian Research Institute of Grain and Its Processing Products. This
paper describes the formation of new grades of triticale flour based on the cumulative ash curves the analysis of
technological and biochemical indicators of which showed that flour of the grades T-60, T-70 and T-80 obtained from
endosperm can be used directly in bakery, flour of the grades T-120 and T-220 obtained from peripheral parts and triticale
bran can be limitedly used in bakery, and are mainly raw materials for further processing. On the basis of the study of the
kinetics and efficiency of the effect of proteolytic and cellulolytic enzyme preparations (EP) and their compositions,
optimal conditions for enzymatic modification (the EP dosage is 0.5–0.75 units of PA/g of flour, 0.3...0.4 units of CA/g of
bran, the optimum temperature is 40–50C, pH is 5.0 and 3.5, the duration of reactions is 1.5 and 2 hours) have been
determined. It has been shown using the gel-chromatography method that the use of multienzyme compositions (MEC) of
proteases allowed to hydrolyze triticale flour proteins completely and to use the obtained hydrolyzate as a component of
hypoallergenic and gluten-free flour products. The use of cellulolytic EP allowed to increase the amount of reducing
substances and soluble protein by 1.5–2.5 times in comparison with the control sample. The biomodified bran obtained
using the MEC "Shearzyme 500 L" + "Neutrase 1.5 MG" and "Viscoferm L" + "Distizym Protacid Extra" has a high
degree of hydrolysis of non-starch polysaccharides and proteins, is characterized by a certain ratio of high-, medium-,
low-molecular peptides and amino acids, has different functional and technological properties. They can be used in the
production of a wide range of general-purpose, functional and treatment-and-prophylactic food products.
Keywords: Triticale grain, flour, bran, grain processing technology, enzyme preparations, modified grain processing
products, functional and technological properties
DOI 10.21603/2308-4057-2017-2-70-82 Foods and Raw Materials, 2017, vol. 5, no. 2, pp. 70–82.
INTRODUCTION
The relevant trends in the development of flour
technology include both the improvement of traditional
methods and the development of technologies of products
with a high biological and nutritional value, the use of
biotechnological methods in the technology of advanced
processing products, the creation of technologies of new,
non-traditional products, etc. The final objective of the
technologies being developed is to obtain products with
the specified composition and properties.
All-Russian Research Institute of Grain and Its
Processing Products conducts fundamental and applied
studies to develop the basic methods for managing
technological processes of the preparation and grinding
of grain of various crops in order to obtain products with
the specified chemical composition and properties. Thus,
using the example of processing of triticale grain into
flour and cereals, principles of the formation of stable
streams of flour from various anatomical parts of grains
have been developed, which allows to form various
types of flour with the specified properties. The
application of the developed technologies allows to
obtain such products from triticale grain as: graded
baker's flour, cereals for children's and dietary nutrition
and grits for pasta [1, 2, 3, 4].
Triticale is a new crop, this is the first grain crop
obtained by crossing wheat (Triticum) with rye (Secale).
The first report on the receipt of a wheat-rye hybrid was
published in 1875 [5]. The main manufacturers of
triticale in the world are Poland, Germany, France and
Belarus, moreover, the cultivation area of this most
promising culture expands both in the world and in
Russia. The croppage in Russia was 624 thousand tons
in 2017, according to Roskomstat. The average yield of
triticale in Russia in 2016 is 27.8 c/ha, which is the
largest value for the period of 2009-2016, and is also
4.7 c/ha more than in 2015 [6]. 75 grades of winter
triticale and 14 grades of spring triticale have been added
to the State Register of Selection Achievements
approved for use in Russia (2017). All new grades are
recommended for food purposes [7].
The biotopotential of triticale grain depends primarily
on: varietal features and growing conditions. The
nutritional value is related to a high protein content,
essential amino acids and a balanced amino acid
composition. The biological value of triticale grain
depends on the predominance of water and salt-soluble
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71
protein fractions and, as a consequence, a higher degree
of assimilation of triticale proteins, as well as the
presence of vitamins, macro- and micronutrients [4, 8, 9].
However, at present, in Russia, triticale is used
mainly in the production of mixed fodder and alcohol.
Perspective is the application of flour from triticale
grain as a component of raw materials in the
production of confectionery products: biscuits, cakes
and crackers. It is possible to use triticale flour in the
production of fast breakfasts or in the production of
dietary bread, including multi-grain bread and that
from whole grains [9, 10, 11]. There is no production
of bread from graded triticale flour currently in Russia.
The use of methods for a biotechnological effect on
various crops and their processing products with
obtaining general-purpose, functional and treatment
and prophylactic food products is a promising and
relevant trend of scientific research for the
technological development of the milling branch. At
present, the use of enzymatic hydrolysis of
biopolymers of food raw materials of both animal and
vegetable origin is being actively and comprehensively
studied and introduced into the practice of food and
processing industries [12, 13, 14, 15, 16].
The use of modern biotechnological methods
allows to develop methods for enzymatic modification
of grain processing products (flour of various types,
including that with a high content of peripheral parts,
bran) using multienzyme compositions (MEC) based
on proteolytic and cellulolytic enzyme preparations; to
obtain modified products (protein hydrolyzate,
structurally modified flour, biomodified bran) with
various values of degree and depth of hydrolysis of
proteins and non-starch polysaccharides with various
functional and technological properties.
The study aims at developing a flexible technology
based on the division of triticale grain into anatomical
parts to obtain new general-purpose and special
products with a high nutrition and biological value and
to obtain components with specific functional and
technological properties. The implementation of the
taken aim will allow to design food products from
grain with the specified composition and properties.
STUDY OBJECTS AND METHODS
The experimental studies have been carried out at
the Federal State Budgetary Scientific Institution "All-
Russian Research Institute of Grain and its Processing
Products". In this paper, flour was used from of triticale
grain of new grades formed on the basis of cumulative
ash curves. Since the studied samples of triticale grain
did not contain any foreign and grain impurities, the
technological process of preparing triticale grain for
milling included only hydrothermal treatment: the
grain was moistened up to 14–15% and softened for
12 hours [3]. The technological process of grain
grinding included 4 break, 6 reduction and 2 scratch
systems. The parameters and grinding regimes
corresponded to the recommended "Rules for the
organization and conduct of a technological process at
flour mills" for graded wheat milling according to a
short process scheme. 6 samples of triticale grain of
different grades were isolated for laboratory milling:
Topaz (2011, 2012); Skolot (2012); Vocaliz (2012);
Tribun (2012) and Donslav (2012). Thus, the range of
values of the quality indicators of the studied samples
was: glassiness is 55–72%, the natural weight is
715–737 g/l, the weight of 1000 grains is 40–44 g, the
ash content is 1.85–1.89%, the crude gluten content is
17–24%, the gluten quality is 46–64 units of GDI, the
falling number is 74–175 s and the protein content is
12–13% [1].
Figure 1 presents the process of grinding and
forming the quality of flour in the form of cumulative
ash curves. The presence of 3 stages of flour formation
has been established, which is clearly seen from the
graphs of cumulative curves (Fig. 1). In addition, the
statistical analysis has shown the reliability of
representation of cumulative curves in the form of three
linear sections.
Fig. 1. Ash content cumulative curves.
0.45
0.55
0.65
0.75
0.85
0.95
0 102030405060708090
Donslav Skolot Topaz 2012 Topaz 2011 Tribun Vocaliz
Flour yield, %
Ash content of flour, %
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72
The first stage of flour formation consisted in
extracting the central part of endosperm with a flour
yield of 40–45% and an ash content of 0.60% and
included the 1st, 2nd, and 3rd reduction systems. The
letter designation A has conditionally been assigned
to the given flour stream. The second stage consisted
of 5–7 technological systems and was characterized
by a yield of triticale flour in the amount of 25–26%
and ash content of 0.91%. The letter designation
B has conditionally been assigned to the given flour
stream. The third stage consisted of scraping with a
flour yield of 5–7% and ash content of 2.20% and
included the 6th reduction system and scratch
systems. The conventional designation of flour stream
is C. Further on, the flour of each of the stages was
mixed to obtain individual flour grades, which
resulted in obtaining 5 flour grades. The conventional
designation of the grades includes the index T which
stands for triticale, and a number which stands for the
value of ash content × 100. Thus, flour T-60 was the
stream A with an ash content of 0.60%, flour T-70
was a mixture of the streams A+B, flour T-80 was a
mixture of the streams A+B+C, flour T-120 was a
mixture of the streams B+C and flour T-220 was the
stream C.
The soluble protein content was determined using the
Lowry method [17] and the protease activity - using the
modified Anson method [18], bovine serum albumin
was used as the standard substrate, amine nitrogen -
using the formol titration method, and reducing
substances (RS) - using the Bertrand method [19].
Determination of the fractional composition of proteins
according to Osborne: albumins were isolated using
distilled water, globulins - using a 10% NaCl solution,
prolamines - using 70% ethanol, and glutelins - using a
0.2% NaOH solution. The proteins and the products of
proteolysis of triticale flour and bran were fractionated
by molecular weight using the gel chromatography
method with a column with Sephadex G-75 and
Toyopearl gel HW-55F [19].
The following were used as proteolytic and
cellulolytic enzymatic preparations: "Neutrase
1.5 MG" - a bacterial metalloproteinase (Zn)
produced by Bacillus amyloliquefaciens, "Alcalase
FG" - a bacterial proteinase produced by Bacillus
licheniformis (Novozymes, Denmark); "Distizym
Protacid Extra" - a fungal protease produced by
Aspergillus niger (Döhler, Germany), "Protease
GC-106" - a fungal protease produced by Aspergillus
oryzae (Genencor, USA), "Shearzyme 500L" - a
purified xylanase produced by Aspergillus oryzae and
Aspergillus aculeatus, "Viscoferm L" - a balanced
mixture of xylanase, β-glucanase, cellulase and
α-amylase produced by Aspergillus aculeatus
(Novozymes, Denmark). All the preparations are
recommended for the hydrolysis of biopolymers of
grain raw materials [20, 21].
The functional and technological properties were
determined using the methods described in [22] and
in [23, 24]. The water absorption capacity (WAC) was
determined as the amount of water adsorbed by the
modified triticale bran after centrifugation. To determine
the fat emulsifying capacity (FEC), 50 ml of distilled
water was added to the weighed amount of 1 g of
modified triticale bran and suspended at 4000 rpm for
1 minute. Then 10 ml of refined sunflower oil was added
to the mixture and emulsified for 5 minutes at a rate of
8000 rpm. The obtained emulsion was centrifuged for
5 minutes at 2000 rpm. FEC was calculated as a ratio of
the emulsion volume and the overall system volume
expressed as a percentage. The emulsion stability (ES)
was determined by heating the emulsion for 30 min at
80°С, then cooled and centrifuged at 2000 rpm. ES was
calculated as a ratio of the emulsion volume and the
overall system volume expressed as a percentage. To
determine the fat binding capacity (FBC), the weighed
amount was put into a pre-weighed centrifuge tube, 5 ml
of refined sunflower oil was added and mixed for
1 minute at 1000 rpm, then centrifuged for 15 minutes at
4000 rpm. The unadsorbed oil was drained, the tubes
were weighed and the FBC was calculated as a ratio of
the weight of the bound oil to the weighed amount. The
foaming capacity (FC) was determined by mixing a
weighed amount in 25 ml of distilled water in a
graduated cylinder and thoroughly mixed, the volume
was made up to 300 ml and shaken for 1 min. FC was
expressed as a ratio of a foam height (mm) to a liquid
height (%).
The analyses were performed in triplicate, presenting
the results as average arithmetic ones. The discrepancy
between parallel assays did not exceed 3% of the
average arithmetic value with the confidence probability
P = 0.95.
RESULTS AND DISCUSSION
Starting to develop methods for enzymatic
modification of biopolymers of vegetable raw
materials, it is necessary to consider the following main
factors: first of all, these are the features of
biopolymers of the given vegetable raw materials, the
heterogeneity of a substrate, the presence of various
kinds of effectors capable of activating or inhibiting
both endogenous enzymes and enzymes in the
composition of enzyme preparations, the presence
concomitant enzymes in addition to the basic activity
of enzymes, etc.; secondly, the conditions for
enzymatic modification, the main kinetic parameters of
enzymatic reactions involving the studied enzyme
preparations, which may differ from the kinetic
characteristics obtained in the studies of purified
enzymes using standard substrates.
At the first stage of the study, the main
technological and biochemical characteristics of the
study objects were studied, namely, the flour samples
formed on the basis of cumulative ash curves and
triticale bran (Table 1, 2 and 3).
The flour sample T-60, which is a fraction of the
central part of endosperm, and is significantly different
in whiteness, ash content, quantity and quality of
gluten, had the best technological properties, as shown
in Table 1. The obtained data allow to estimate the
technological properties of new grades from triticale
grain flour as high, with the prevalence of a wheat
phenotype. It has been established that triticale grain is
characterized by the absence of a significant
dependence between the content of gluten and protein,
both in grain and in single flour streams. The expected
tendency of increasing the protein content in the
systems of final grain grinding has been revealed.
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73
Table 1. Quality of the formed triticale flour grades
Flour sample,
grade
Moisture
content, %
Whiteness, units
of RZ-BPL device
Ash
content, %
Amount of gluten,% Quality of gluten,
units of GDI
Crude Dry
Т-60 12.0 53.75 0.63 22.7 8.24 70 I – sufficient
Т-70 12.1 49.75 0.72 21.0 7.96 66 I – sufficient
Т-80 12.1 42.2 0.85 21.7 8.20 66 I – sufficient
Т-120 11.7 29.95 1.14 15.8 6.10 57 I – sufficient
Т-220 11.3 -8.675 1.99 0.4 0.08 89 II – satisfactory weak
Table 2. Chemical composition of new grades of
triticale flour
Flour sample,
grade
Protein
(N×6.25), %
Starch,
%
Fat,
%
Т-60 10.14 82.28 1.00
Т-70 12.23 81.11 1.14
Т-80 16.84 77.68 1.25
Т-120 17.65 75.60 1.60
Т-220 24.88 47.34 2.90
Table 2 presents the analysis of the total content of
the main grain biopolymers in the formed grades of
triticale flour.
The data presented in Table 2 show that the studied
samples, especially the sample T-220, despite a high
protein content, are characterized by low baking
qualities, as evidenced by trial laboratory baking [1],
but can be used as valuable food ingredients.
The study of the quantitative ratio and properties of
various fractions of soluble grain albumins is, along
with theoretical interest, of great practical interest for
the technologies that use grain as the main raw
material. Despite the fact that the separation of protein
substances by solubility is rather relative, nevertheless,
it is used quite widely at the present time. However,
there are a lot of questions that remain unclear to this
day. This is due, most often, to a difference in the
methodological approach of different researchers.
The study of the fractional composition of the
soluble proteins of the formed grades of triticale flour
showed that the samples of T-60 and T-70 differ in the
lowest content of albumins and globulins, but the
highest content of prolamins and glutelins that are
concentrated in endosperm and form gluten. The main
part of albumins and globulins is found in the samples
T-120 and T-220, this is apparently due to the presence
of the refined germ and the aleuron layer in the flour
samples. In the sample T-80 flour, the percentage of all
fractions is approximately the same and is 20–25%, the
given sample has been formed by mixing 3 main flour
streams, which are characterized by a different
composition of anatomical parts of the grains (Table 3).
Table 4. Proteolytic activity of the formed grades of
triticale flour
Flour
sample,
grade
Protein,
mg/ml
Proteolytic power (PP)
Acid
proteinases,
units of PP/mg
of protein
Neutral
proteinases,
units of
PP/mg of
protein
Т-60 0.080 0.60 0.85
Т-70 0.080 0.80 1.20
Т-80 0.100 1.40 1.80
Т-120 0.160 1.40 2.10
Т-220 0.400 0.80 1.00
It is known that proteolytic enzymes play an
important part in the processes that proceed in grain
when stored and processed. The flour obtained by
effecting the grain, violating its integrity and, to a
certain extent, by destroying the cellular structure, is a
completely different object of study from a
biochemical point of view. The object in which the
oxidative and hydrolytic processes are primarily
activated, including the processes related to the
proteolysis of endogenous proteins.
The proteolytic enzymes of triticale grain and triticale
flour have been studied poorly [25], much less than the
parent proteases - that of wheat [26, 27] and rye [28]. The
studies carried out at the Federal State Budgetary
Scientific Institution "All-Russian Research Institute of
Grain and Its Processing Products" on the p0roteolytic
enzymes of triticale grain, revealed the presence of three
types of proteinases that actively hydrolyze bovine serum
albumin (a standard substrate) and self-proteins: acid
proteinases with an optimum pH of 3.5; neutral
proteinases with an optimum pH of 6.5 and alkaline
proteinases with an optimum pH of 9.5 [29].
Table 4 presents data on the activity of acidic and
neutral proteinases of the formed grades of triticale
flour. The proteases were extracted as described in the
paper [29]. Determination of protease activity using the
modified Anson method.
Table 3. Fractional composition of soluble proteins of the formed grades of triticale flour
Flour sample,
grade
Fractional composition of proteins,% of the total protein content
Albumins Globulins Prolamins Glutelins Insoluble residue
Т-60 11.05 17.82 39.25 28.08 3.80
Т-70 12.00 18.14 36.78 26.64 6.44
Т-80 20.58 22.24 25.68 23.47 8.03
Т-120 72.02 12.04 4.08 3.50 8.30
Т-220 43.79 28.95 12.53 6.78 7.95
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74
Table 5. Biochemical composition of triticale grain and triticale bran of different grades
Grade name Protein (N×6.25), % Starch, % Reducing sugars, % Fiber, %
grain
b
ran grain
b
ran grain
b
ran grain
b
ran
Topaz 7.6 18.11 68.1 24.84 0.20 12.35 2.08 12.35
Skolot 14.5 18.81 62.6 25.25 0.20 14.85 2.40 14.85
Donslav 14.2 15.90 64.8 28.26 0.24 14.42 2.46 14.42
Vocaliz 12.5 17.06 66.4 32.72 0.28 14.68 2.40 14.68
Table 6. Fractional composition of triticale bran proteins, % of the total protein content
Grade name Albumins Globulins Prolamins Glutelins Insoluble residue
Topaz 36.8 24.0 9.6 14.0 20.4
Skolot 38.6 22.2 10.2 14.6 19.8
Donslav 34.0 22.6 9.8 14.8 20.4
Vocaliz 38.0 22.4 10.0 14.6 20.0
The analysis of activity of acidic and neutral
proteinases in the formed flour grades indirectly indicates
that part of the proteolytic activity in triticale grain is
related to gluten proteins, but still the highest activity was
noted for the samples T-80 and T-120, that is, these are
more likely the proteins of the germ and the subaleurone
layer. At the same time, the activity of neutral proteases is
1.5–2.0 times higher than that of acid proteases. The value
of proteolytic activity in the formed grades of triticale
flour is, in addition to other biochemical indicators, of key
importance, since proteinases are able to actively
hydrolyze their own proteins, including glutens, which
ultimately effects the technological process and the
finished product. In addition, proteolytic enzymes
participate in the regulation of the activity of other
enzyme systems, for example, amylases.
The activity of amylolytic enzymes of grain and
flour is another important technological and
biochemical characteristic that determines along with
other indicators the baking advantages of flour. It was
estimated using the method for determining a falling
number (FN). FN was 294 s for T-60; 266 s for T-70;
272 s for T-80; 245 s for T-120 and 174 s for T-220.
The falling number value for wheat flour at a level
of 230–330 s characterizes the normal amylolytic
activity of wheat flour, this value for rye flour is about
100 seconds less. The falling number values obtained
in the study of triticale flour samples show that the
activity of amylases (excluding the flour sample T-220)
is similar to the activity of these enzymes in wheat
flour, and along with other indicators confirms the
predominance of the wheat phenotype in the triticale
grain being studied.
Table 5 presents the biochemical composition of
triticale bran. The comparative analysis of the main
components of triticale grain and bran indicates a regular
increase in the content of crude protein in bran - up to
15.90 ... 20.56%, of fiber - up to 10.68 ... 14.85% and a
decrease in the starch content up to 32.72 ... 22.62%.
The significant increase in sugars in bran fractions
compared with whole grains is due, most likely, to the
presence of a refined germ [1]. It should also be taken
into account that the carbohydrate complex of triticale
grain contains a significant amount of insoluble dietary
fiber - hemicelluloses (up to 30%) [5].
The analysis of the fractional composition of
soluble proteins (Table 6) showed that the proteins of
bran from triticale grain differ in a relatively high total
content of albumins and globulins, which is generally
characteristic of triticale grain proteins, while the
number of globulins is 3 to 3.5 times higher than in
whole grain (7–8% of the total protein content). When
in a dissolved state, they are actively hydrolyzed by
endogenous proteolytic enzymes, giving a large
number of hydrolysis products with different molecular
weights. The content of prolamines is 2–2.5 times
lower than in whole grain (23.6–25.0%).
The study of streams of flour from triticale grain
allowed to reveal the most promising streams for
obtaining advanced processing products [1, 4].
The scheme of advanced triticale grain processing
(Fig. 2), which includes the stages of preparing grain
for processing, namely: the selection of grain according
to certain quality criteria, the formation of mill
mixtures, cleaning and hydrothermal treatment and the
division into anatomical parts.
Fig. 2. Scheme of advanced triticale grain processing.
Obtaining fractions that consist of: a - the
central part of endosperm; b - the intermediate
part of endosperm; c - the peripheral part of
endosperm; (d) the aleuron layer and the seed
coat; e - the seed and fruit coats.
Preparation
for processin
g
Re
q
uirements for
g
rain
q
ualit
y
Formation of mill mixtures
Cleanin
g
and h
y
drothermal
p
retreatmen
t
Separation into anatomical parts
a, b and c are
bread flour
a and b are
confectionery flour
a and b are
macaroni flour
c and d are biomodified
flour and bran
d and e are
dietary fiber
Triticale grain
ISSN 2308-4057. Foods and Raw Materials, 2017, vol. 5, no. 2
75
Table 7. Characteristics of the enzyme preparations of proteases during the hydrolysis of triticale flour proteins
Indicator “Neutrase
1.5МG”
“Alcalase
FG”
“Protease
GC-106”
“Distizym
Protacid Extra”
Initial velocity, V0 (min) 30 30 30 30
Optimum temperature, °С 50 45 50 40
Optimum рН 5.5 6.5 5.5-6.0 3.5
Optimum amount of enzyme preparation,
units of PA/g of flour 0.50 0.5 0.75 0.75
Saturated substrate concentration, mg/cm3 100 100 100 100
The flour of the samples A, AB and ABC (T-60,
T-70 and T-80) was obtained from endosperm and
can be used directly in bakery, which was confirmed
by trial baking [1], and also after enzymatic
modification, as the components of special products
that have specific functional and technological
properties. The flour of the samples BC and C (T-120
and T-220) from the peripheral parts of endosperm,
including the aleurone layer and the seed coat, may be
limitedly used in baking, and is mainly a raw material
for further processing.
At the second stage of the study, a study was
carried out of the effectiveness of proteolytic and
cellulolytic enzymatic preparations and the main
kinetic parameters of enzymatic reactions in which
different types of flour and triticale bran were used as a
substrate. The enzymatic modification of proteins of
vegetable raw materials, including proteins of grain
crops, is an important stage in advanced technologies
of advanced processing of grain raw materials. As a
result of modification of the protein components of
grain and flour with the use of proteolytic enzymatic
preparations, hydrolysis products with a certain profile
of peptides and a number of amino acids with specific
properties can be obtained.
In case of the traditional characteristics of enzyme
preparations, the optimum temperature and pH, as well
as other kinetic parameters, is detected using a standard
substrate [30]. At the same time, in production, in the
conditions of a specific food production technology, a
complex heterogeneous system acts as the latter, which
leads to a change in the basic kinetic parameters of the
enzymatic reaction. The composition of the grain
substrate can effect the course of the proteolysis
process and change the optimum values of temperature
and pH [20].
Table 7 presents the main kinetic characteristics of
the enzymatic reaction of hydrolysis of triticale flour
proteins using bacterial and fungal proteolytic enzyme
preparations. The hydrolysis was carried out at the
optimum pH and temperature for 30 minutes. It has been
previously established that the reaction is zero order for
30 min. The enzyme preparations were added in the
amounts from 0.25 to 1.5 units of PA/g of flour, the
substrate concentration varied from 20 to 120 mg/ml.
Taking into account the complex structure of the
cell wall (the main component of bran), enzyme
preparations with a whole complex of activities are
required to degrade it and increase the degree of
protein extraction: cellulase, hemicellulase and
pectolytic activity [31].
Table 8. Characteristics of the enzymatic preparations
"Shearzyme 500 L" and "Viscoferm L" when effecting
the non-starch polysaccharides of triticale bran
Indicator "Shearzyme 500 L" "Viscoferm L"
Initial velocity,
V0 (min) 30 30
Optimum
temperature, °С 50 50
Optimum рН 5.5 3.5
Optimal amount
of enzyme
preparation,
units/g of bran
0.3 units
of xylanase
ability/g of bran
0.4 units
of cellulolytic
ability/g of bran
Table 8 presents the characteristics of the
enzymatic reaction of hydrolysis of non-starch
polysaccharides of triticale bran when effected by the
enzymatic preparations "Shearzyme 500 L" and
"Viscoferm L". The composition of the incubation
mixture is the following: milled triticale bran and
water (the hydromodule is 1 : 10), a phosphate-citrate
buffer 0.1 M (20% of volume) and an enzyme
preparation with the activity from 0.1 to 0.5 activity
units/g of bran. It has been established that the
reaction is zero order for 30 min. The optimum
temperature and pH were revealed when studying the
activity of the enzyme preparations under study in the
range of 20–70°C and pH of 3.0–6.0. The hydrolysis
efficiency was estimated by RS accumulation using
the Bertrand method.
Similar results were obtained using the flour
samples T-120 and T-220 as a substrate. Thus, optimal
conditions for the hydrolysis of non-starch
polysaccharides of triticale bran and flour with a high
content of peripheral parts of grains using the
enzymatic preparations "Shearzyme 500 L" and
"Viscoferm L" were selected.
The enzymatic hydrolysis of triticale bran proteins
using enzyme protease preparations was carried out
under the following conditions: the enzyme
preparations "Neutrase 1.5 MG" and "Distizym
Protacid Extra" were applied in the amounts
from 0.25 to 1.5 units of PA/g of bran; the
substrate concentration varied from 20 to 120 mg/ml
(Table 9).
ISSN 2308-4057. Foods and Raw Materials, 2017, vol. 5, no. 2
76
Table 9. Characteristics of the enzyme preparations
"Neutrase" and "Distizym Protacid Extra" when
effecting triticale bran proteins
Indicator "Neutrase
1.5 MG"
"Distizym
Protacid
Extra"
Initial velocity, V0 (min) 30 30
Optimum temperature, °С 50 40
Optimum рН 5.5 3.5
Optimal amount of
enzyme preparation,units
of PA/g of bran
0.50
0.75
Saturated substrate
concentration, mg/cm3
100 100
To estimate the efficiency of the studied enzyme
preparations, the enzymatic hydrolysis was carried out
under the optimal conditions, which were selected
experimentally. The incubation mixture consisted of
triticale bran, water (the hydromodule is 1 : 10), the
appropriate buffer (20% of volume) and an enzyme
preparation based on the final concentration of the
corresponding optimum. Sampling was carried out
every 30 minutes for 2 hours, the samples were
transferred to centrifugal glasses and centrifuged at
6000 rpm for 10 minutes. The supernatant was used to
determine the reducing sugars (reducing substances)
using the Bertrand method and the amount of soluble
protein using the Lowry method.
The hydrolysis efficiency was estimated by the
accumulation of RS and soluble protein. The results are
shown in Fig. 3 and 4. It has been shown that the
enzymatic preparation "Shearzyme 500 L" increases the
amount of RS and soluble protein by 2 times; and the
preparation "Viscoferm L" increases the amount of RS
by 1.5 times and the amount of soluble protein by
2.5 times. The obtained data indirectly indicate the
possibility of a significant increase in the nutritional
value of secondary products of grain triticale processing.
The flour, obtained from different parts of
endosperm, was modified using the multienzyme
compositions (MEC) based on bacterial and fungal
microbial enzyme protease preparations.
Fig. 3. Accumulation of RS during the hydrolysis of
nonstarch polysaccharides of triticale bran using the
preparations Shearzyme 500L and Viscoferm L.
Fig. 4. Accumulation of soluble protein during the
hydrolysis of non-starch polysaccharides of triticale bran
using the preparations Shearzyme 500L and Viscoferm L.
The enzymatic hydrolysis of triticale flour when
effected by the preparations "Neutrase 1.5 MG" and
"Protease GC-106" was carried out for 2 hours. The
suspension was then centrifuged at 6000 rpm for
15 minutes. 5 ml of supernatant was applied to a column
filled with the gel Sephadex G-75. The elution was carried
out using distilled water. The volume of aggregated
fractions is 4 ml. The optical density of the eluate in the
fractions was registered with a wavelength of 280 nm.
A water extract of triticale flour was used as a control
sample. The elution profiles are shown in Figure 5.
Fig. 5. Fractionation of the products of triticale flour proteolysis using preparations of microbial proteases on a column
with Sephadex G-75.
0
50
100
150
200
250
30 60 90 120
RS, % of theinitial value
Hydrolysis time, min
Shearzyme 500L Viscoferm L
0
50
100
150
200
250
300
30 60 90 120
Protein, % of the initial
value
Hydrolysis time, min
Shearzyme 500L Viscoferm L
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20 25 30 35 40 45 50
А280
Fraction number
Control Neutrase 1.5 MG Protease GC-106
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77
Table 10. Fractionation of the products of proteolysis of triticale flour proteins
Fraction Molecular weight, Da % of the total
Control “Neutrase 1.5 MG” “Protease GC-106”
11–20 ≥ 70000 42.71 24.76 19.10
21–25 40000 ÷ 30000 6.49 5.30 4.02
26–32 30000 ÷ 20000 3.36 20.50 7.68
33–36 20000 ÷ 10000 14.18 10.45 6.98
37–45 ≤ 3000 34.12 38.91 62.14
Table 10 presents data on the molecular weight, the
products of proteolysis of triticale flour proteins formed
when applying the preparations of bacterial and fungal
proteases, and the percentage of different fractions.
The comparative analysis of the elution profiles
presented in Figure 5 and the data of Table 10 shows
that the application of preparations of bacterial and
fungal proteases does not only change the ratio of high,
medium and low molecular weight proteolysis
products, but also largely changes the pattern of
elution: the nature of distribution of the proteolysis
products formed as a result of the use of
different preparations is completely different in
fractions.
Thus, in case of the enzymatic hydrolysis of
triticale flour proteins using the preparation "Neutrase
1.5 MG", there is a decrease in the high-molecular
fraction (with a molecular weight of more than
70000 Da) by 42.03%, then, when effected by the
preparation "Protease GC-106", - by 55.28%. The
increase in the low molecular weight fraction (the
molecular weight is less than 3000 Da) is 16.51% and
35.21%, respectively.
When using "Neutrase 1.5 MG", the amount of the
formed medium molecular weight peptides with a
molecular weight from 30000 to 20000 Da is
approximately 2.5–3 times higher as compared to
"Protease GC-106"; in turn, when effected by "Protease
GC-106", the amount of low-molecular peptides (the
molecular weight is 20000 ÷ 10000 Da) is 5.8 times
higher than when effected by "Neutrase 1.5 MG".
Table 11. Fractionation of products of proteolysis of
triticale flour proteins obtained using MEC
Fraction Molecular
weight, Da
% of the total
Control MEC
11 – 20 ≥ 70000 33.56 5.36
21 – 25 40000 ÷ 30000 8.54 4.82
26 – 32 30000 ÷ 20000 14.01 18.94
33 – 36 20000 ÷ 10000 4.57 30.92
37 – 45 ≤ 3000 39.29 40.01
On the basis of the studies carried out, multitalzyme
compositions have been compiled to obtain products of
proteolysis of triticale flour proteins with a different
degree of hydrolysis, and, consequently, with various
functional and technological functions [32].
The use of MEC, which includes proteolytic
enzymes with a different specific effect (the bacterial
protease preparations "Neutrase 1.5 MG" and
"Alcalase FG" and the fungal protease preparation
"Protease GC-106"), allowed to hydrolyze proteins
almost completely, as evidenced by this fractionation
of products of triticale flour proteolysis using the gel
chromatography method on a column with Sephadex
G-75 (Fig. 6).
Thus, there are practically no high molecular
weight fraction with a molecular weight of more than
70,000 and fraction with a molecular weight of
40,000–30,000 Da, while the amount of low molecular
weight peptides and amino acids in the hydrolyzate has
increased approximately by 2.5–3.0 times in
comparison with the control sample.
Fig. 6. Fractionation of products of proteolysis of triticale flour proteins obtained using MEC on a column with
Sephadex G-75.
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78
The obtained data allowed to position the
hydrolyzate obtained with the use of MEC on the basis
of enzyme protease preparations as a possible
component of hypoallergenic and gluten-free products
used for the therapeutic and prophylactic purpose.
Bran and flour with a high content of peripheral
parts containing a large number of non-starch
polysaccharides, in turn, were modified using MEC
based on cellulolytic and proteolytic enzymatic
preparations. As a result, products of enzymatic
modification of flour and bran from triticale grain with
a different degree of hydrolysis of proteins and non-
starch polysaccharides and various functional and
technological properties have been obtained [21, 31].
The composition of 2 multi-enzyme compositions
used for the enzymatic modification of triticale bran
and flour with a high content of peripheral parts
included: "Shearzyme 500 L" + "Neutrase 1.5 MG"
(MEC-1) and "Viscoferm L" + "Dystizym Protacid
Extra" (MEC-2). The choice of enzyme preparations
is caused by various specific effects and
approximately the same effect optima: the optimum
temperature is 50°C; pH is 5.5–6.0 for MEC-1 and
40°C; pH is 3.5 for MEC-2. The hydrolysis was
carried out in 2 stages. At the first stage, a cellulolytic
enzyme preparation was applied. At the second stage,
a proteolytic enzyme preparation was applied. The
dosage of enzyme preparations, the substrate
concentration and the duration of each stage were
selected experimentally [4]. Figures 7, 8 and Table 12
present the results of fractionation of the products of
proteolysis using the gel chromatography method on a
column with Toyopearl gel HW-55F.
The obtained experimental data on the kinetics of
enzymatic reactions of hydrolysis of biopolymers of a
grain substrate (different types of flour and triticale
bran); the degrees of hydrolysis and the ratio of
fractions with different molecular weights using the gel
chromatography method on a column with Toyopearl
gel HW-55F have formed the basis for the
development of biotechnological methods for
modifying the products of triticale grain processing.
The developed methods for modifying the products
of triticale grain processing include the following stages:
– the preparation of a suspension - triticale flour, bran:
water (the hydromodule is 1 : 4);
– the preparation of solutions of enzyme preparations;
the creation of MEC;
– the enzymatic hydrolysis using MEC under the
developed conditions (the substrate concentration, the
dosage of enzyme preparations, the optimum
temperature and pH);
– the inactivation of enzyme preparations; the product
being obtained is hydrolyzed flour or bran (an
unclarified hydrolyzate);
– centrifugation;
– the product being obtained is a hydrolyzate
(a supernatant) and paste (a precipitate);
– drying;
– the product being obtained is a dry hydrolyzate and
hydrolyzed flour and bran;
To estimate the possibility of using the products
obtained in food branches, their functional and
technological properties have been studied.
Fig. 7. Fractionation of the products of proteolysis of triticale bran proteins of MEC-1 using the gel chromatography
method on a column with Toyopearl gel HW-55F.
Fig. 8. Fractionation of the products of proteolysis of triticale bran proteins of MEC-2 using the gel chromatography
method on a column with Toyopearl gel HW-55F.
0
0.1
0.2
0.3
0.4
0 5 10 15 20 25 30 35 40 45
А280
Fraction number
Control MEC 1
0
0.1
0.2
0.3
0.4
0 5 10 15 20 25 30 35 40 45
А280
Fraction number
Control MEC 2
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79
Table 12. Fractionation of the products of proteolysis of triticale bran proteins using MEC
Fraction Molecular weight, Da % of the total
Control MEC-1 MEC-2
Peak I 6 – 13 ≥ 700000 (blue dextran yield) 35.81 2367 19.55
Peak II 14 – 15 450000 ÷ 350000 13.26 14.79 12.62
Peak III 16 – 19 300000 ÷ 100000 9.95 26.04 3.20
Peak IV 20 – 22 100000 ÷ 50000 13.26 0 0
Peak V 23 – 26 50000 ÷ 25000 10.08 5.02 1.77
Peak VI 27 – 30 25000 ÷ 1500 5.31 2.54 0
Peak VII 31 – 36 ≤ 1000 (tyrosine yield) 12.33 51.06 62.63
Table 13. Functional properties of the modified triticale bran
Note. * Control C 1 - bran; Experiment 1 - bran + MEC1; Experiment 2 - bran + MEC2
A wide range of physico-chemical characteristics
that determine the behavior in heterogeneous food
systems during processing, storage and consumption,
and also provide the desired structure, technological
and consumer properties of food products are to be
meant by the functional and technological properties of
proteins and protein preparations. Vegetable proteins,
as well as proteolysis products with various values of
molecular weight, can act as the ingredients of general-
purpose, treatment-and-prophylactic and special food
products. This is due to the inherent unique functional
properties [33]. Depending on the amino acid and
fractional composition, molecular weights, the
presence of charged and uncharged groups, hydrophilic
and hydrophobic groups and other structural features,
proteins can serve as gelling agents, foaming agents
and form and stabilize suspensions and emulsions,
etc. [34, 35].
The requirements for the functional properties of
proteins are specific for a certain scope and type of
product. For example, when making meat products, the
most important are the water- and fat-retaining
abilities, gelling, the emulsifying and adhesive
properties; in bakery - the water-binding, emulsifying
and foaming abilities; the main criterion for choosing a
protein preparation in the production of beverages is
solubility. To solve the problem of the applicability of
specific proteins for obtaining various food products, it
is necessary to know how their functional and
technological properties change depending on a
number of physico-chemical factors: the nature and
concentration of proteins in the system, the
temperature, pH, the presence and concentration of
concomitant biopolymers and low molecular weight
substances [33, 36].
In some cases, to improve and regulate the
functional properties in order to expand the scope of
these or other protein preparations, they are modified
using physical, chemical, enzymatic and other
methods.
The enzymatic method for the modification of
vegetable proteins is preferable to physico-chemical
modification, since its advantage are soft reaction
modes, the ability to regulate the degree of hydrolysis,
its specific directivity and the retention of the
biological value [32, 33, 37–40].
Tables 13 and 14 present the water binding capacity
(WBC); the fat binding capacity (FBC); the fat
emulsifying capacity (FEC); the emulsion stability
(ES); the foam forming capacity (FFC) and the foam
stability (FS) of the modified triticale bran.
The functional properties of bran from triticale
grain and the hydrolyzed samples obtained using
MEC1 and MEC2 differ from each other. Thus, the
water-binding capacity of the hydrolysed bran in the
first option increases by 16%, in option 2 - on the
contrary, it decreases by 12.6% with respect to the
unhydrolyzed triticale bran. The similar pattern can be
seen with respect to the foam forming capacity
(Experiment 1: an increase of 18.0%; Experiment 2: a
decrease of 16.1%). The fat binding and fat
emulsifying capacity increases in both experimental
options by 13.6% and 6.1% and by 19.2% and 7.7%
respectively.
The stability of the emulsion and foam of the
modified triticale bran is reduced: ES - by 8.7%; FS -
by 12.5% (Experiment 1) and ES - by 20.7%; FS - by
25.0% (Experiment 2).
Similar studies were carried out using flour samples
with a high content of peripheral parts (Table 2).
There is a tendency for samples of the flour modified
using MEC1 of an increase in WBC by 21.3 ... 26.0%; in
FBC by 13.8 ... 16.0%; in FEC by 74 ... 9.0%. There is,
on the contrary, a tendency for samples of the flour
modified using MEC2 of a decrease in these functional
characteristics: in WBC by 11.8 ... 18.3%; in FBC by
6.7 ... 22.3%; in FEC by 3.8 ... 4.0%.
The stability of the emulsion and foam of the
modified flour from triticale grain is also reduced, as in
the case of the modified triticale bran: ES - by 8.7%;
FS - by 13.4% (Experiment 3) and ES - by 20.7%; FS -
by 26.7% (Experiment 4); ES - by 9.1%; FS - by
27.3% (Experiment 5) and ES - by 8.0%; FS - by
30.2% (Experiment 6).
Sample* WBC, g/g FBC, g/g FAC , % ES , % FFC, % FS, %
Control - C 1 1.56 1.32 52 58 50 32
Experiment 1 - E1 1.80 1.50 62 53 59 28
Experiment 2 - E2 1.20 1.40 56 46 42 24
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80
Table 14. Functional properties of the modified flour from triticale grain with a high content of peripheral parts
Sample WBC, g/g FBC, g/g FAC, % ES, % FFC, % FS, %
Control - C2 0.56 0.52 50 52 80 65
Experiment 3 - E3 0.67 0.59 54 50 83 55
Experiment 4 - E4 0.54 0.48 48 42 55 43
Control - C3 0.64 0.54 52 55 86 63
Experiment 5 - E5 0.80 0.62 57 50 98 58
Experiment 6 - E6 0.52 0.41 50 46 64 44
Note. * Control C2 - Flour T-120; Experiment 3 - T-120 + MEC 1; Experiment 4 - T-120 + MEC2; Control C3 - Flour T-220; Experiment 5 - T-220 +
MEC 1; Experiment 6 - T-220 + MEC2
It is known that the functional properties of the
products of enzymatic hydrolysis of protein raw
materials depend on the physico-chemical properties of
the initial protein, the specificity of the proteases used,
the composition of MEC used, the conditions for
hydrolysis, the degree of hydrolysis and the ratio of the
fractions of proteolysis products with different
molecular weights [36, 37].
The revealed differences in the functional
properties in the initial and modified products of
triticale grain processing are related, first of all, to the
conditions for enzymatic modification (of the pH
medium), the composition and specific effect of the
enzymes that are part of the composition of MEC;
obtaining products of various degrees of hydrolysis,
and the number of high-, medium- and low-molecular
compounds; an increase or decrease in free polar
(charged) aggregations, hydrophilic and/or
hydrophobic groups, providing interactions with
different types of substances.
The obtained results indicate that the use of MEC
on the basis of cellulolytic and proteolytic enzyme
preparations allows for an advanced destruction of
proteins of the products of triticale grain processing; to
obtain products with various degrees of hydrolysis and
the ratio of components by molecular weight, which
leads to a change in the functional and technological
properties of the initial flour and will allow to find its
new scopes in food products. Thus, the samples with
the pH values close to the neutral ones (modified using
MEC1), taking into account the values of the foam
forming and fat emulsifying capacities, can be used in
foam-emulsion systems, bakery products, cakes and
biscuits. The samples with low pH values (modified
using MEC2), taking into account their functional
properties, can be used to enrich fruit beverages,
fermented milk products, salad dressings, sauces, etc.
At the same time, it should be taken into account that
with low pH values the rate of the Maillard reaction
significantly decreases, which can have both negative
and positive effects depending on the specific food
technology, namely: the retention or reduction of the
amount of amino acids and reducing sugars; the
formation of melanoidins and a complex of aromatic
compounds.
CONCLUSION
In general, the proposed technology allows to form
various grades of triticale flour (bread, confectionery,
macaroni flour, etc.) and cereals such as "semolina"; to
carry out advanced processing of triticale bran and
flour, including that with a high content of peripheral
parts, using biotechnological methods (enzymatic
modification); to receive valuable components for the
enrichment and creation of new products with the
given properties and composition, contributing thereby
to the expansion of not only the raw material base, but
also the range of the output products.
The studies carried out have shown that the
functional and technological properties of the modified
products of triticale grain processing finally depend on
the specificity of enzyme preparations and the
composition of MEC. The use of MEC on the basis of
preparations of microbial proteases allows to hydrolyze
triticale flour proteins almost completely, and to position
the obtained hydrolyzate as a possible component of
hypoallergenic and gluten-free flour products.
The use of cellulolytic and proteolytic enzyme
preparations in the hydrolysis of biopolymers of triticale
bran allowed to increase the amount of reducing
substances (reducing sugars) by 1.5–2.0 times, soluble
protein - by 2.0–2.5 times, and the use of MEC on their
basis showed that the obtained hydrolysates have a high
degree of hydrolysis of non-starch polysaccharides and
proteins, a specific ratio of high-, medium- and low-
molecular weight peptides and amino acids.
To solve the issue of the applicability of specific
products whose proteins are modified, it is necessary to
know in various food technologies not only a chemical
composition, but also functional and technological
properties. The obtained experimental data on the study
of the water binding, fat binding, fat emulsifying and
foam forming capacities, as well as the emulsion
stability and foam stability of the modified triticale bran
and flour with a high content of peripheral parts with the
use of 2 multi-enzyme compositions showed that
enzymatic modification leads to certain changes in the
functional and technological properties of the initial
flour and bran from triticale grain; and allow to find new
and more rational scopes of the modified products as
enrichers and as functional and technological
components. Thus, the samples with the pH values close
to the neutral ones (modified using MEC1), taking into
account the values of foam forming and fat emulsifying
capacities, can be used in the production of bakery
products, cakes and biscuits. The samples with low pH
values (modified using MEC2), taking into account their
functional properties, can be used to enrich fruit
beverages, fermented milk products, salad dressings,
sauces, etc. The results of the studies have formed the
basis for the development of methods for the enzymatic
modification of triticale flour and bran. Hydrolysates,
structurally modified flour and biomodified bran, which
can be used in the production of a wide range of general-
purpose, functional and treatment and prophylactic food
products have been obtained.
ISSN 2308-4057. Foods and Raw Materials, 2017, vol. 5, no. 2
81
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Please cite this article in press as: Meleshkina E.P., Pankratov G.N., Vitol I.S., Kandrokov R.H., and Tulyakov D.G.
Innovative Trends in the Development of Advanced Triticale Grain Processing Technology. Foods and Raw Materials, 2017,
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