Ijciet 10 01_158-2

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International Journal of Civil Engineering and Technology (IJCIET)
Volume 10, Issue 01, January 2019, pp. 1718-1730, Article ID: IJCIET_10_01_158
Available online at http://www.iaeme.com/ijciet/issues.asp?JType=IJCIET&VType=10&IType=01
ISSN Print: 0976-6308 and ISSN Online: 0976-6316
© IAEME Publication Scopus Indexed
IMPACT OF MECHANICAL ACTIVATION ON
THE PREBIOTIC PROPERTIES OF PLANT
BIOLOGICAL RESOURCES
Natalia S. Rodionova, Evgeny S. Popov, Vyacheslav Yu. Kustov, Aleksandr A.
Rodionov, Ekaterina A. Pozhidaeva, Natalia A. Rodionova, Andrey A. Dyakov
Voronezh State University of Engineering Technologies, Voronezh, Russia
ABSTRACT
The manuscript investigates the effect of mechanical activation by the wave-
disintegration method on the pre-biotic properties of the dietary supplement
composition of wheat germ (Vitazar dietary supplement), arabinogalactan of
Dahurian larch (Flarabin dietary supplement), and selenium xanthan (Seleksen
dietary supplement). The authors revealed the changes in the phase state of the
crystal lattices of the nanopowders of the studied biological objects. The manuscript
determines the parameters of the accumulation of lactobacilli and bifidobacteria
biomass during the fermentation of substrates containing milk, and the composition of
dietary supplements in the concentration range of 1-5%. The manuscript shows the
possibility of reducing the accumulation time of active cells to concentrations of not
less than 109 CFU/g of lactobacilli L. Сasei subsp. Rhamnosus, as well as consortia
consisting of lactobacilli: L. acidophilus, L. plantarum, L. fermentum, of
bifidobacteria: Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium
adolescentis; of lactobacilli and bifidobacteria: L. Сasei subsp. Rhamnosus, L.
acidophilus, L. plantarum, L. fermentum, Bifidobacterium bifidum, Bifidobacterium
longum, Bifidobacterium adolescentis L. The manuscript studies the process of
biomass accumulation of probiotic microflora and its metabolites (lactic acid) on
substrates containing the composition of dietary supplements in mechanically
activated form. The authors established that being a source of dietary fibers, poly-
and oligosaccharides, vitamins, mineral substances, antioxidants, the composition
under study can support the active development of lactobacilli and bifidobacteria,
forming a favorable environment for them. The authors recorded accumulation of
microflora metabolites by an increase in the titration rate, as well as by a change in
CFU in 1 g of the fermented substrate. In the study of the fermentation process'
kinetics, the main stages of the fermentation process were identified, the lag-phase
time was reduced to 1-1.2 hours, activation of the exponential phase of the process,
and a rather long period of growth retardation and a stable state of the biosystem
with preservation of probiotic microorganism concentration not less than 109 CFU/g.

Impact of Mechanical Activation on the Prebiotic Properties of Plant Biological
Resources
Keywords: Biological Objects, Lactobacilli and Bifidobacteria, Prebiotic Properties,
Mechanical Activation, Microflora
Cite this Article: Natalia S. Rodionova, Evgeny S. Popov, Vyacheslav Yu. Kustov,
Aleksandr A. Rodionov, Ekaterina A. Pozhidaeva, Natalia A. Rodionova, Andrey A.
Dyakov. Impact of Mechanical Activation on the Prebiotic Properties of Plant
Biological Resources, International Journal of Civil Engineering and Technology,
10(1), 2019, pp. 1718-1730.
http://www.iaeme.com/IJCIET/issues.asp?JType=IJCIET&VType=10&IType=01
1. INTRODUCTION
In modern conditions of widespread use of preservatives in food technology, the gradual
introduction of antibiotics in agricultural technologies, an increase in the number of diseases
of the population is observed due to a weakening of the immune system, an increase in
dysbacteriosis and allergic reactions. There is an acute problem of finding innovative
technical solutions aimed at restoring the microecology and homeostasis of the organism
through the introduction of new natural biocorrectors into the diet. Considering the needs of
modern consumers, it is necessary to develop new product lines that differ from traditional
ones with a higher content of probiotic microorganisms in the active form, with the inclusion
of immunomodulators, antioxidants, and organic selenium compounds [1, 2, 3].
Probiotic microflora is currently regarded as an extracorporal human organ that performs
essential functions for the body. The state of the indigenous microbiota is adversely affected
by antibiotics, radiation and chemotherapy, nutritional imbalances, the presence of heavy
metals preservatives in food, high physical exertion, and stress [4, 5, 6, 7, 8]. Another
unfavorable factor is the introduction of intensive agricultural technologies, leading to a
significant reduction in the composition of plant and animal raw materials content of essential
nutrients - vitamins, macro - and microelements, which are necessary not only for the
macroorganism, but also provide the required level of the nutrient substrate for the intestinal
microflora. In this regard, the development of prescription-technological solutions for
synbiotic products containing prebiotics, active forms of symbiotic probiotics, as well as
metabiotics, further enriched with biologically active ingredients of natural origin, is relevant.
For example, in articles [9, 10, 11, 12], the process of fermentation by probiotic
microorganisms was carried out on milk-vegetable substrates with the inclusion of inulin,
soy. Probiotic products are also known to be produced on milk-free substrates using legumes,
cereals (oatmeal, wheat bran), rice, fruits (guava, orange, passion fruit), and vegetables [13,
14, 15, 16]. In these experimental studies, plant materials used for the synthesis of probiotic
biomass as a substrate were introduced in a non-activated form, which does not allow the
biopotential to be fully revealed and the prebiotic properties of these components to be used.
In this regard, many researchers have substantiated the prospects for the use of mechanical
activation, as a containing no reagents and environmentally friendly method of exposure
concerning various biological objects in order to enhance or modify their functional and
biologically active properties. Mechanical activation of natural biopolymers is a complex
physicochemical phenomenon consisting in the accumulation of potential energy by a
substance and an increase in its chemical activity as a result of an increase in the internal
structure energy and surface energy under the action of mechanical forces in the process of
intensive nanodispersion. Thanks to the mechanical activation effect, it is possible to
significantly increase the level of functional, including prebiotic, effects of biologically active
ingredients introduced into the formulations of synbiotic products [17].

Rodionov, Ekaterina A. Pozhidaeva, Natalia A. Rodionova, Andrey A. Dyakov.
A composition based on Vitazar, Flarabin, and Seleksen dietary supplements was chosen
as an enriching prebiotic component. It is known that, among all, Vitazar [18], contains
protein - 25-37%, carbohydrates - 19-25% (including sucrose, maltose, pentosans,
oligosaccharides), dietary fiber - 1.8-4.2%, fats 6.0 -8.1% (including polyunsaturated fatty
acids), and mineral substances 4.0-7.7%. Vitazar is a unique source of mineral bioelements
necessary for the vital activity of the body: phosphorus, calcium, magnesium, potassium,
iron, manganese, and zinc. The composition of the fat fraction of Vitazar includes vitamins
A, E, D, policosanol, which has a pronounced cholesterol-lowering effect on the body.
Seleksen dietary supplement is an organic compound (22.8-23.9%), a compound of
selenium, and xanten. Seleksen does not cause adverse effects, even at concentrations that
significantly exceed the maximum permissible level, in contrast to inorganic forms of
selenium, which have specific toxicity. Favorable pharmacological properties of Seleksen are
clinically confirmed: high antioxidant activity, increased immunity, strengthening the
cardiovascular system, reducing the risk of developing cancer, protecting stem cells during
cytostatic, chemo- and radiotherapy.
Flarabin dietary supplement is produced of Dahurian larch, contains 90 ± 10%
arabinogalactan, and 0-10% dihydroquercetin. Its immunomodulatory, antiviral, antioxidant
effect, membranotropic, and prebiotic properties have been clinically proven. As a
technological component, Flarabin dietary supplement has emulsifying properties, is well
soluble in milk, and is heat resistant. It is essential to study the effect of wave-disintegration
on the activation of the prebiotic properties of the listed plant sources of biologically active
substances in order to obtain enriched synbiotic products of a functional orientation.
The enrichment of probiotic products with the composition of plant components is
essential for increasing their functionality, which is formed mainly by the presence of the
functional properties of these prebiotics. However, data on the effect of the composition
based on Vitazar, Flarabin, and Seleksen dietary supplements on the development of
consortia of bifidobacteria and lactobacilli with a microbial number of at least 109
CFU/ml
are not available. Cultivation of bifidobacteria and lactobacilli, when introduced into the milk
base of the composition under consideration, will provide a new product with complex
functional action, due to the presence of probiotic microflora and prebiotics.
2. CONCEPT HEADINGS
The purpose of the research was to study the effect of mechanical activation by disintegrative
wave dispersion on the fermentation process of dairy-plant systems containing the
composition of wheat germ flour, arabinogalactan from Dahurian larch, selenium xanten,
probiotic microorganisms, and their consortia.
The research relates to consortia of lacto - and bifidobacteria, produced by Bioproduct
LLC, intended for the production of the following products: Immunolact (Str. thermophilus,
L. сasei subsp., L. rhamnosus), Laktinal (Str. thermophilus, L. acidophilus, L. plantarum, L.
fermentum), Bifilyuks (Str. thermophilus, B. bifidum, B. longum, B. adolescentis), and
BioMatrix-LB1 (Str. thermophilus, L. сasei subsp., L. rhamnosus, L. acidophilus, L.
plantarum, L. fermentum, B. bifidum, B. longum, B. adolescentis). These microorganisms
and consortia have a pronounced lactase activity and, when cultured in a dairy medium, the
concentration of active cells can reach 109
CFU/g. Direct application starters with the
following indicators were used for the fermentation: mass fraction of moisture - not more
than 5.0%; the number of lactic acid bacteria (CFU), in 1 g, not less than 5.0 billion; the
number of thermophilic streptococci in 1 g, not less than 1.0 million; citrate-fermenting
lactococci in 1 g, not less than 3.0 billion; bifidobacteria, in 1 g, not less than 3.0 billion;

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yeast and mold in 1 g, not more than 5.0. Pathogenic microorganisms (including Salmonella),
E. coli bacteria were absent. The starter, activated for 2 hours, was introduced into
pasteurized (92 ± 2ºС, exposure for 2 min) and cooled (37-40 º
С) nutrient medium - skimmed
milk containing 1-5% of the studied composition BAA or nanopowder of the composition
and the resulting one-, two-, three, or fourfold wave-disintegrating effects using a fine
grinding disintegrator “Kedr 3601” (linear speed 300 m/s), with a weakly modulating effect
of SHF-EHF radiation. Skimmed milk served as the control element. The phase composition
and structure of the obtained nanopowders were investigated by the method of x-ray phase
analysis using electron microscopy with the INGA Energy X-ray microanalysis system. The
average particle size of the samples was determined on a JSM-6380LV electron microscope.
Sample preparation and microscopy were carried out following the instructions for the
device.
Fermentation of the control and experimental samples was carried out at 37-40ºС for 18
hours. In the course of thermostating, titrated acidity of the medium and pH were controlled,
the process was carried out until the growth of titrated acidity was slowed down. The
organoleptic evaluation was performed, and microbiological indicators (CFU/g) were
determined in the final products: the amount of bifidobacteria and lactobacilli.
3. RESULT
The results of the study of changes in the microstructure of the composition as a result of the
wave-disintegration effect are presented in Figure 1.
a b

Figure 1 The microstructure of the composition depending on the number of wave-disintegrating
effects: a - control; b - single, c - double; d - threefold; e - fourfold
When grinding the bioactive composition in the disintegrator, significant changes in the
microstructure of the object under study were observed, depending on the frequency of the
wave disintegration effect. After a triple exposure, a qualitative change in the structure of the
nanopowder is observed (Figure 1, d), which is maintained during subsequent exposure
(Figure 1, e).
The method of X-ray phase analysis provided the data on the phase changes of the
resulting biocomposite nanopowder. The shape of the diffraction peaks on the X-ray pattern
of the composition that has not been subjected to mechanical activation indicates a weak
crystallization of the lattices of the compounds making up the composition (Figure 2, a). As a
result of mechanical activation, changes in the shape of the peaks (Figure 2, b, c, d, e) are
noted. The peaks become higher and narrower, which indicates changes in the phase state of
the following bio-elements in the nanopowder: calcium, potassium, zinc, phosphorus, and
copper. Also, double peaks of the same element are present on the X-ray patterns, which
indicates the presence of isoform states, for example, calcium, potassium, and copper. It
should be noted that as a result of three- and fourfold nanodispersion, an amorphous
biopolymer structure is also formed, as evidenced by some smoothing of the peaks occurring
c d
e

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on the radiographs of samples after 3 and 4-fold dispersion, which is also confirmed by
changes in the microstructure of the samples.

Figure 2 The X-ray patterns of the study of changes in the structural-phase state of the bio-elements
of the composition depending on the number of wave-disintegrating effects: a - control; b - single, c -
double; d - threefold; e - fourfold
The obtained data indicate significant changes in the phase state of the bio-composition,
which may affect its functional and biological properties. The results of the development of
probiotic microorganisms consortia studied in dairy systems containing a nanopowder bio-
composition with different X-ray characteristics are shown in Figure 3.

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Figure 3 The change in titration in the milk-plant systems containing non-activated composition (%):
1 – 1.0, 2 – 2.5, 3 – 5.0; nanopowder of the composition (%): 4 – 1.0, 5 – 2.5, 6 – 5.0; 7 – 0 (control)
in the process of fermentation of probiotic microorganisms by consortia
The obtained data shows that the accumulation of acidity begins to change intensively
after 3-3.5 hours from the start of the process in control, after 2-2.5 hours in systems with a
non-activated composition and after 1.5-2 hours in samples with a nanopowder bio-
composition, which indicates a more rapid transition of the system from the lag-phase to the
exponential with the introduction of non-activated composition into milk and the
intensification of this process with the introduction of the composition in the form of
nanopowder. More active acid formation should be noted in samples containing consortia of
lactobacilli, and less active - in samples containing consortia of bifidobacteria, and
intermediate for consortia containing lactobacilli and bifidobacteria.
Gel formation, due to the achievement of the isoelectric point of milk caseins (pH 4.6), is
noted at various time intervals from the beginning of the process (Table 1).

Table 1 Duration of reaching the isoelectric point of casein milk by the milk-plant systems, hour
Consortium name Control
The content of the
non-activated
composition, %
The content of the
composition's
nanopowder, %
1.0 2.5 5.0 1.0 2.5 5.0
Str. thermophilus, L. сasei
subsp., L. rhamnosus
6.5 6.3 5.8 6.0 6.2 5.5 5.6
Str. thermophilus, L.
acidophilus, L. plantarum, L.
fermentum
6.0 5.9 5.4 5.6 5.7 5.0 5.2
Str. thermophilus, B.
bifidum, B. longum, B.
adolescentis
7.3 7.1 6.8 6.9 7.0 6.5 6.6
Str. thermophilus, L. сasei
subsp., L. rhamnosus, L.
acidophilus, L. plantarum, L.
fermentum, B. bifidum, B.
longum, B. adolescentis
6.7 6.6 6.3 6.4 6.5 6.0 6.2
The resulting clot had a uniform, stretching thick consistency. The introduction of
biocomposite in milk in concentrations up to 3% intensified acid formation in the system.
When the concentration increased to 4-5%, a slight "cereal" taste and smell appeared. Also,
the intensity of acidity increased, which may be due to an increase in osmotic pressure in the
system, which adversely affects the development of microorganism consortia under study.
With further thermostatic control, the growth of acidity was less intense; after 16 hours of
fermentation of the system with non-activated composition, clot compaction and syneresis
were observed, which reduced the visual appeal of the fermented milk product. Syneresis in
the control sample was observed after 14 hours of temperature control, which indicates a
positive effect of the biocomposite on the moisture-binding capacity of the plant-casein gel.
This allows for increasing the duration of the cultivation of probiotic microflora with the
accumulation of its metabolites, and the mechanical activation of the studied composition had
a clear intensifying effect, as evidenced by the reduction in time to reach the gelation point.
The concentration of probiotic microorganisms after 6-8 hours of the process reached 108
-109
CFU/ml and then it remained at this level for 10-12 hours of cultivation.

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4. DISCUSSION
The obtained data confirm the intensifying effect of carbohydrates of plant compositions on
the growth of biomass of lactic acid microorganisms and the activation of their properties,
including exopolysaccharide activity. Many researchers have evaluated the primary carbon
sources for lactic acid bacteria - lactose, glucose, galactose, sucrose, fructose, and mannose,
as well as their combinations. For example, the yield of glycans was higher in the medium
containing glucose and fructose, rather than in the medium containing lactose or sucrose
when cultured with S. salivarius ssp. thermophilus, even though the reproduction of
microorganisms themselves slowed down.
The nitrogen source in the nutrient substrate has a significant influence on the
exopolysaccharide activity of lactic acid bacteria. It is known that an increase in the
concentration of whey proteins has a beneficial effect on the synthesis of polysaccharides by
bacteria of the strain L. rhamnosus [19] and L. delbrueckii ssp. Bulgaricus [20]. As a nitrogen
source for biomass synthesis and activation of exopolysaccharide activity, the Vitazar dietary
supplement protein can be considered in connection with its significant amounts of 33-39%,
various fractional and amino acid composition.
Vitamins and minerals have an equally strong effect on the growth of the biomass of
lactic acid bacteria and the synthesis of exopolysaccharides. The introduction of CaCl2 and
H2O2 when L. casei is cultivated in a mixture for fermentation increases the yield of
exopolysaccharides more than nine times [21]. The Vitazar dietary supplement shows
magnesium content at about 38 mg/100 g [18].
The enrichment of the mixture with B group vitamins increases the biosynthesis of
exopolysaccharides by the L. delbrueckii strain, subsp. bulgaricus NCFB 2772 from 130 to
250 mg/l. Yeast extract containing vitamins of this group (2775 mg/l) [22] had a beneficial
effect on the synthesis of L. rhamnosus L. rw-9595 M exopolysaccharides. The Vitazar
dietary supplement contains B group vitamins (mg%): В1 – 5, В2 – 0.6, В3 – 94, В5 – 6.0, В9
– 1.0 [18].
5. CONCLUSION
The experimental studies resulted in establishing the possibility of activating the prebiotic properties
of the plant bio-composition, consisting of the dietary supplements Vitazar, Flarabin, and
Seleksen. The conditions and mode parameters of the cultivating biomass of probiotic
microorganisms consortia consisting of Str. thermophilus, L. сasei subsp., L. rhamnosus; Str.
thermophilus, L. acidophilus, L. plantarum, L. fermentum; Str. thermophilus, B. bifidum, B.
longum, B. Adolescentis; Str. thermophilus, L. сasei subsp., L. rhamnosus, L. acidophilus, L.
plantarum, L. fermentum, B. bifidum, B. longum, B. adolescentis.
The possibility of obtaining biomass data consortia with the content of active cells of at
least 109
CFU/ml. The optimal concentration of nanopowder in synbiotic drinks is 3-3.5%,
the fermentation process duration (hour) before gelation (pH 4.6) comprised the following
number for the consortium of Str. thermophilus, L. сasei subsp. – 15 h, for the consortium
of Str. thermophilus, L. acidophilus, L. plantarum, L. fermentum – 13 h, for the consortium
of Str. thermophilus, B. bifidum, B. longum, B. adolescentis – 14 h, for the consortium of Str.
thermophilus, L. сasei subsp., L. rhamnosus, L. acidophilus, L. plantarum, L. fermentum, B.
bifidum, B. longum, B. adolescentis – 13 h. The clot is dense, slightly stretching, the taste
and smell of the product are traditional, milky with a light cereal aroma, the color is light
cream. The products had high levels of active form of probiotic microorganisms, as well as
prebiotic components, which makes them promising for use in special diets of athletes,
military personnel, persons subjected to antibiotic chemotherapy and radiation therapy, as

well as experiencing regular high physical activity and stress. The developed products are
intended both for direct consumption and in the form of ingredients of a wide range of recipes
and products for healthy nutrition - beverages, sauces, desserts, bakery, confectionery,
chopped meat products, and pastes.
ACKNOWLEDGMENT
The research was performed within the framework of applied scientific research and
experimental development of the Presidential grant (No. 075-02-2018-333).
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