EDIBLE FRUITS EXTRACTS AFFECT INTESTINALMICROBIOTA ISOLATED FROM PATIENTS WITHNONCOMMUNICABLE DISEASES ASSOCIATEDWITH CHRONIC INFLAMMATION

The aim of our study was to investigate the gut microbiota in patients with noncommunicable diseases associated with chronic inflammation, namely obesity, type 2 diabetes, atherosclerosis, and cardiovascular disease as well as to find out potential ability of edible plants fruits extracts to inhibit the

growth of selected conditionally pathogenic microorganisms.

Limited clinical trial was performed and gut microbiota analysis was done using routine methods and qPCR. The antibacterial properties of edible plants fruits in relation to the selected potentially patho genic microorganisms were studied.

The composition of the intestinal microbiota of obese patients was characterized by an increase in thenumber of Enterococcus spp. and Lactobacillus spp. along with a decrease in the amount of Escherichiacoli. Decreases in E. coli and lactobacilli were observed in patients with type 2 diabetes. In atherosclerosis,an increase in streptococci, enterococci, and enterobacteria was observed, whereas in patients with car-diovascular disease there was an additional increase in staphylococci and candida along with a decrease in E. coli. Decreases in Bifidobacterium spp., Bacteroides spp., Roseburia intestinalis and Akkermansia muciniphila were observed in patients of all groups. The growth of Klebsiella spp. was inhibited by red currant and plum extracts; Enterobacter spp. — sweet cherry extract; Proteus spp. — extracts of blue-berry and cornelian cherry; Staphylococcus spp. — the extracts of black currant, sweet cherry, plum, jostaberry, alycha and cornelian cherry.

The obtained data can be used for early diagnosis of noncommunicable diseases and for their prevention with the help of personalized nutrition.

According to the World Health

Organization (WHO), non-communicable

diseases (NCDs) are chronic diseases that

are not transmitted from person to person,

have a long course, and progress slowly. In

the late twentieth century, NCDs turned

into a global epidemic and one of the greatest

threats to human life and health. According

to the WHO, 40 million people die annually

from NCDs, which accounts for 70% of all

deaths in the world [1]. NCDs result from a

combined influence of genetic, physiological,

environmental, and behavioral factors [2].

Studies of changes in the intestinal

microbiome and its role in the occurrence

of NCDs have become extremely relevant in

recent years [3]. Microbiome is part of human

physiology and is significantly involved in a

wide range of vital physiological processes,

including energy homeostasis and metabolism,

synthesis of vitamins and other important

nutrients, endocrine signaling, prevention

of colonization by pathogens, regulation

of immune function, and metabolism of

xenobiotics, carcinogens, and other harmful

compounds [4].

Persistent low-grade inflammatory

response underscores metabolic syndrome and

is a risk factor for cardiovascular diseases

(CVDs) [5, 6]. Inflammatory markers are

associated with obesity and the risk of obesity-

related CVDs [7]. Perturbation of intestinal

microbiota and changes in gut permeability

are triggers for the chronic inflammatory

state [8]. “Metabolic endotoxaemia” is a term

used to describe a link between gut bacteria,

endotoxins, and their circulating levels, with

inflammatory-induced obesity and metabolic

diseases linking it to CVDs [9].

Some research studies [10] demonstrated

that intestinal microbiota changes related to

obesity lead to threshold inflammation. In

obese people, intestinal microbiota changes

stimulate the absorption of monosaccharides

due to the increased number of capillaries

in the small intestine epithelium [11] and

significantly increase the ability to obtain

more energy from food by increasing the

number of microorganisms capable of

fermenting indigestible carbohydrates in the

colon [12, 13]. Obesity is a major risk factor

for the development of type 2 diabetes (T2D),

which leads to the destruction of insulin

receptors and causes resistance to insulin. In

turn, patients with diabetes also tend to suffer

from comorbidities, such as hypertension and

dyslipidemia, which further accelerates the

atherosclerotic process, and, therefore, such

patients have an extremely high cardiovascular

risk [14]. Atherosclerosis is a major risk

factor for CVDs assuming accumulation of

cholesterol and macrophages on arteries

walls, thus contributing to the formation

of atherosclerotic plaques [15]. Recent

studies suggest that intestinal microbiota

disruption may also enhance development of

atherosclerosis and CVDs [16, 17].

In our opinion, among numerous NCDs, it

is necessary to distinguish a group of diseases

directly relating to changes in the microbiome

and the main trigger of which is chronic

inflammation, that is obesity, T2D mellitus,

atherosclerosis, and CVDs.

Nutrition is the most important factor

that regulates gut microbiota composition.

Personalized nutrition is one of the most

effective approaches for prevention and

treatment of NCDs [18]. The edible plants’

fruits which are characterized by high

biologically active compounds (BAC)

contents and ability to stimulate the growth

of beneficial microorganisms and inhibit

the growth of conditionally pathogenic

microorganisms could be perspective

components for personalized nutrition.

Therefore, the aim of our research was to

study intestinal microbiota in patients with

NCDs related to chronic inflammation, namely

obesity, T2D mellitus, atherosclerosis, and

CVDs as well as to find out potential ability

of edible plants fruits extracts to inhibit the

growth of selected conditionally pathogenic

microorganisms.

Materials and Methods

Participants and study design

In order to study gut microbiota in patients

with NCDs related to chronic inflammation,

we performed a limited clinical case study, in

which four groups were formed: 1 — patients

with obesity; 2 — patients with type 2 diabetes;

3 — patients with atherosclerosis; 4 — patients

with cardiovascular diseases. In order to

achieve this goal, we examined 10 people from

each group.

The inclusive criteria for obesity were the

value of the body mass index (BMI), which

exceeds () 30 kg/m2 [19]; signed informed

consent to participate in the study. Exclusion

criteria: smoking, alcohol or drug use, diabetes

mellitus, CVDs, clinically significant kidney

or liver disease (or other organs and organ

systems), acute inflammatory diseases at

the time of examination, cancer; significant

lifestyle changes, mainly of dietary habits and

physical activity in the period shorter than 6

months.

Patients with T2D were selected according

to the criteria typical of this nosology [20]:

fasting plasma glucose  6.1 mmol/l; impaired

glucose tolerance — two hours after the oral

dose a plasma glucose 7.8–11.1 mmol/l;

glycated hemoglobin (HbA1c)  6.5%; signed

informed consent to participate in the study.

Exclusionary criteria were smoking, alcohol,

or drug abuse; pregnancy; an unstable medical

status; significant lifestyle changes, mainly

of dietary habits and physical activity in the

period shorter than 6 months. No participants

had clinically significant cardiovascular, renal

or liver disease or a history of cancer.

Inclusion criteria for atherosclerosis were

[21]: patients with a BMI in the range of normal

weight; low cardiovascular risk (SCORE  1%);

total cholesterol level below 8 mmol/l; total triglycerides levels below 2.3 mmol/l; signed

informed consent to participate in the study.

Exclusion criteria: patients receiving lipid-

lowering therapy (statins, ezetimibe, etc.) or

patients who do not meet the minimum period

of 3 months of discontinuation of therapy;

the lipid profile outside the inclusion criteria;

diabetes mellitus; SCORE > 1%; proven

secondary causes of dyslipidemia; presence of

manifest cardiovascular system disease in the

form of coronary artery disease, past stroke,

TIA, MI, etc.; presence of acute diseases,

chronic deterioration, or presence of infection,

which may distort the laboratory parameters;

significant lifestyle changes, mainly of dietary

habits and physical activity in the period

shorter than 6 months.

The following inclusion criteria were

used to select patients with CVDs: diagnosed

coronary heart disease, stroke, carotid artery

stenosis [22]; SCORE 5%; hyperlipidemia;

signed informed consent to participate in

the study. Exclusion criteria were smoking,

alcohol, or drug abuse; pregnancy; an unstable

medical status; clinically significant renal or

liver disease, acute inflammatory diseases at

the time of examination or a history of cancer;

significant lifestyle changes, mainly of dietary

habits and physical activity in the period

shorter than 6 months.

The Transcarpathian Regional Clinical

Cardiology Dispensary was the place of

inpatient examination of patients diagnosed

with atherosclerosis and CVDs, and for

patients with obesity and T2D — the

therapeutic department of the Mukachevo

Central District Hospital.

According to the conclusions of the

Commission on Biomedical Ethics (Protocol

No6/1 of 26.05.2020), all studies were

performed in compliance with the basic

provisions of the Good Clinical Practice

(GMP) (1996), Convention on Human Rights

and Biomedicine of the Council of Europe

(04.04.1997), the World Medical Association

Declaration of Helsinki — Ethical Principles

for Medical Research Involving Human

Subjects (1964–2013), and the orders of

the Ministry of Health of Ukraine No690 of

23.09.2009 and No616 of 03.08.2012, in which

a person is an object of research. All patients

gave informed consent to participate in the

study.

Analysis of gut microbiota

In order to study gut microbiota the

faecal samples were diluted with pre-reduced

phosphate-buffered saline (PBS), then the ten-

fold serial dilution of samples was performed

in PBS and plated correspondingly on the

following nutrient media: Mitis Salivarius

Agar, Bile Esculin Agar, Mannitol Salt

Agar, Endo Agar, Bismuth Sulphite Agar,

HiCrome Clostridial Agar, Sabouraud

Dextrose Agar, Lactobacillus MRS Agar,

Bifidobacterium Agar, Bacteroides bile

esculin agar, Propionibacter Isolation Agar,

L.D. Esculin HiVegTM Agar (manufactured

by HiMedia Laboratories, India), UriSelectTM

4 Medium (Bio-Rad Laboratories, Inc, USA),

and Blaurock semi-liquid modified hepatic

medium (manufactured by Liofilchem, Italy).

Identification of isolated microorganisms was

performed using biochemical test systems

ANAERO-23, ENTERO-24, NEFERM-test,

Candida-23, STAPHY-16, and STREPTO test

24 (Erba Lachema s.r.o., Czech Republic).

Real-time polymerase chain reaction

(qPCR) was performed on an AriaMx

instrument (manufactured by Agilent

Technologies, USA) using specific primers

(Table 1). Isolation of bacterial DNA was

performed using the ZymoBIOMICS DNA

Mini Kit (Zymo Research, USA) according to

the instructions for use. The concentration of

isolated DNA in the samples was checked on

a DeNovix DS-11 FX + spectrophotometer/

fluorometer (DeNovix Inc., USA).

Extracts preparation

Using GrindomixTM electric mixer,

we obtained native homogenates of the

following edible plants’ fruits (grown in the

mountainous regions of Zakarpattia): Ribes

rubrum (red currant), Prunus avium (sweet

cherry), Prunus x domestica (plum), Ribes x

nidigrolaria (jostaberry), Vaccinium myrtillus

(blueberry), Ribes nigrum (black currant),

Prunus cerasifera (alycha) and C rnus mas L.

(cornelian cherry). The obtained homogenates

were filtered through nylon nanofilters with a

pore width of 44 μm (BD Falcon, USA).

We studied the antibacterial properties of

the above-mentioned edible plants fruits in

relation to the selected microorganisms such

as Escherichia coli, Enterobacter cloacae,

Klebsiella pneumoniae, Klebsiella oxytoca,

Proteus mirabilis, Pseudomonas aeruginosa,

Streptococcus pyogenes, Staphylococcus

aureus, Enterococcus faecalis, Candida

albicans by culturing them in extracts obtained

from these edible plants’ fruits [23]. The initial

concentration of the selected bacterial strains

was 1.5108 CFU/ml. After 24, 48 and 72

hour of their co-incubation the ten-fold serial

dilution of samples was performed and plated

correspondingly on an appropriate nutrient

medium. The test cultures of microorganisms

without edible plants fruits extracts were the

control in the study.

Data analysis

Statistical analyses were performed using

the statistical program Origin 2019 (OriginLab

Corporation, USA). All data are presented as

median and interquartile range or the mean

± SD. Nonparametric comparisons were done

using multiple comparisons Kruskal-Wallis

ANOVA with Dunn’s Test as post-hoc analysis.

P values < 0.05 were considered statistically

significant. Normally disturbed date were

compared using student’s t-test.

Results and Discussion

Among the coccal microorganism forms

of the intestinal microbiota of obese patients,

there was a significant increase in the

amount of enterococci, while the amounts of

streptococci and staphylococci were within

the norm. The level of bacteria of the genera

Enterococcus spp., Streptococcus spp., and

Staphylococcus spp. in the gut microbiota

of patients with T2D was within the norm.

In patients with atherosclerosis, intestinal

microbiota demonstrated an increase in the

amounts of enterococci and streptococci

along with the normal value of staphylococci

amount. Under CVDs, patients showed an

increase in the amounts of bacteria of the

genera Enterococcus spp., Streptococcus spp.,

and Staphylococcus spp. in gut microbiota

(Fig. 1).

Analysis of the obtained data reveals

that an significant increase in the amount of

staphylococci within intestinal microbiota was

characteristic only of the group of patients

with CVDs, while an increase in the amount

of streptococci was observed in patients with

atherosclerosis and CVDs. An increase in the

amount of enterococci was observed in patients

with obesity, atherosclerosis, and CVDs.

Therefore, an increase in the amount of coccal

microorganism forms, namely Enterococcus

spp., Streptococcus spp., and Staphylococcus

spp., within gut microbiota may indicate the

development of atherosclerosis and CVDs.

In the intestinal microbiota of patients

with obesity and T2D, Enterobacteriaceae

demonstrated a significant decrease in the

amount of normally fermenting Escherichia

coli at the normal concentration of Proteus

vulgaris, Klebsiella spp., and Enterobacter

spp. In patients with atherosclerosis, gut

microbiota demonstrated a significant

increase in the amount of Klebsiella spp. and

Enterobacter spp., while the amounts of E. coli

and P. vulgaris slightly exceeded the norm.

In the intestinal microbiota of patients with

CVDs, there was an increase in the amounts of

Enterobacter spp. and P. vulgaris, a decrease

in the value of E. coli, and a normal amount of

Klebsiella spp. (Fig. 2).

According to the data obtained in the

study, an increase in the amount of Klebsiella

spp. was characteristic only of patients with

atherosclerosis, while an increase in the

amounts of P. vulgaris and Enterobacter spp.

was observed in patients with atherosclerosis

and CVDs. The concentration of E. coli was

below the norm in patients with obesity, T2D,

and CVDs, but in patients with atherosclerosis

there was a slight excess of this bacterium.

Given the above, an increase in the amount of

enterobacteria, especially Klebsiella spp. and

Enterobacter spp., indicates the development

of atherosclerosis.

Anaerobic and facultative-anaerobic gut

microbiota of obese patients was characterized

by an increase in the value of Lactobacillus

spp., normal values of Clostridium spp.,

Faecalibacterium prausnitzii and yeast-

like fungi of the genus Candida, as well as a

decrease in the levels of Bifidobacterium spp.,

Bacteroides spp., Roseburia intestinalis, and

Аkkermansia muciniphila.

In the intestinal microbiota of patients

with T2D, there was a decrease in the amounts

of Bifidobacterium spp., Lactobacillus spp.,

Bacteroides spp., F. prausnitzii, R. intestinalis,

and A. muciniphila, while the amounts of

yeasts of the genus Candida and Clostridium

spp. were within the norm. In the intestinal

microbiota of patients with atherosclerosis

and CVDs, there was a decrease in the

values of Bifidobacterium spp., Bacteroides

spp., F. prausnitzii, R. intestinalis, and

A. muciniphila, as well as normal values

of Lactobacillus spp., Clostridium spp.

An significant increase in the amount of

Candida spp. within intestinal microbiota was

characteristic only of the group of patients

with CVDs (Fig. 3 and Fig. 4).

While analyzing the data obtained, we could

see that an increase in the concentration of

lactobacilli within gut microbiota is observed in

obese patients, a decrease in the concentration

of Lactobacillus spp. is characteristic of

the microbiota of patients with T2D, while

in patients with atherosclerosis and CVDs

the amount of lactobacilli is within the

norm. Intestinal microbiota of patients

of all groups was characterized by normal

amounts of Clostridium spp., while the normal

concentration of F. prausnitzii was observed

only in patients with obesity. Decrease in the

amounts of Bifidobacterium spp., Bacteroides

spp., R. intestinalis, and A. muciniphila within

gut microbiota was observed in patients of all

nosological groups.

In previous studies, we obtained data

demonstrating the content of biologically

active compounds of selected edible plants

fruits and their potential ability to stimulate

the growth of lactic acid bacteria [29]. Here

we present the results of the studied effect of

edible plants fruits extracts on commensal,

beneficial, potentially pathogenic bacterial

strains (Table 2).

According to the data obtained (Table 2), the

red currant extract totally inhibited the growth

of K. pneumoniae, K. oxytoca, and P. aeruginosa

on 48 h of co-cultivation as well as S. aureus

on 72 h of co-cultivation. The extract of black

currant totally inhibited growth of S. aureus

after 48 h of co-cultivation and K. pneumoniae,

K. oxytoca, and P. aeruginosa on 72 h of co-

cultivation. The sweet cherry extract totally

inhibited growth of E. cloacae and S. aureus on

48 h of co-cultivation as well as K. pneumoniae,

K. oxytoca, P. aeruginosa, E. faecalis, and P.

mirabilis on 72 h of co-cultivation. The growth

of such bacterial strains as K. pneumoniae,

K. oxytoca, S. aureus, and C. albicans was

totally inhibited by plum extract on 48 h of

co-cultivation, while the strains of E. coli, E.

cloacae, P. mirabilis, P. aeruginosa, and S.

pyogenes strains were totally inhibited after

72 h of co-cultivation. The jostaberry extract

totally inhibited growth of P. aeruginosa

and S. aureus after 48 h of co-cultivation and

E. cloacae after 72 h of co-cultivation. The

extract of blueberry on 48 h of co-cultivation

totally inhibited growth of P. mirabilis as well

as s K. pneumoniae, K. oxytoca, and S. aureus

after 72 h of co-cultivation. The growth of P.

aeruginosa and S. aureus was totally inhibited

by alycha extract on 48h of co-cultivation,

while such bacterial strains as E. cloacae,

K. pneumoniae, K. oxytoca, P. mirabilis,

E. faecalis, and C. albicans totally inhibited

after 72 h of co-cultivation. The cornelian

cherry extract totally inhibited growth of P.

mirabilis, S. aureus, and C. albicans after 48 h

of co-cultivation.

Analyzing the data obtained in these

study, it can be concluded that extracts

of red currant and plum can be used for

inhibition of the growth of Klebsiella spp.;

the extract of sweet cherry can be used for

inhibition of Enterobacter spp. growth; the

extracts of blueberry and cornelian cherry

are effective growth inhibitors of Proteus

spp.; the extracts of plum and cornelian

cherry can be used for growth inhibition of

Candida spp.; the extracts of black currant,

sweet cherry, plum, jostaberry, alycha

and cornelian cherry are effective growth

inhibitors of Staphylococcus spp.

Current research studies consider

a potential role of gut microbiota in

the development of obesity and related

comorbidities. Gut microbiota can influence

energy extraction from food, lipid metabolism,

immune response, and endocrine functions

and its profile has shown differences between

obese and non-obese subjects [30]. Our study

revealed that intestinal microbiota of obese

patients was characterized by a sharp increase

in the amount of enterococci and a decrease in

the amounts of normally fermenting E. coli

and bifidobacteria, which are early diagnostic

markers of metabolic disorders [31].

A close relationship between the

gut microbe-dependent production of

trimethylamine-N-oxide (TMAO), derived

from specific dietary nutrients, such

as choline and carnitine, and future

cardiovascular events has been widely

recognized [32]. Trimethylamine (TMA),

which is produced by gut microbial enzymes

TMA lyases, is a precursor of TMAO. As

different gut microbial compositions generate

different levels of TMAO [33], higher blood

TMAO levels and an increased development

of atherosclerosis and CVDs risk can be

attributed to a TMA-producing microbiome harboring TMA lyases. Our research results

demonstrate that intestinal microbiota of

patients with atherosclerosis and CVDs is

characterized by an increase in the amounts

of Streptococcus spp., E. coli, and Klebsiella

spp. This is confirmed by the fact that these

bacteria are able to produce TMA [34].

One of the most important metabolic

activity of gut microbiota is the production

of non-gaseous SCFAs (acetate, propionate,

and butyrate), through fermentation of

microbiotaaccessible, complex carbohydrates

(e.g., oligosaccharides, resistant starch,

and plant cell wall materials) [35]. Butyrate

plays a significant role in the maintenance

of intestinal epithelial cell integrity with

important functions in the prevention

of ‘leaky gut’ associated with diabetes.

Therefore, the role of SCFA, particularly

butyrate and butyrate-producing bacteria

such as Bifidobacterium spp., Bacteroides

spp., F. prausnitzii and R. intestinalis are

crucial for health in obesity and diabetes [36].

Taking into account this fact we can conclude

that the decreased level of butyrate-producing

bacteria indicates inflammation processes

which are associated with NCDs.

Thus, from the work presented here, it

can be concluded that the gut microbiota

alteration contributes to the development

of NCDs such as obesity, T2D mellitus,

atherosclerosis, and CVDs. Thus, such

knowledge can be applied in early

diagnosis of those diseases. Analyzing the

experimental data obtained, and taking

into account results of our previous

studies, we can suggest that selected

edible plants fruits extracts can be used as

components of personalized nutrition for

prevention and treatment of NCDs related

to chronic inflammation. However, in vivo

investigations are necessary to confirm the

interactions between microbiota modulating

and intestinal beneficial effects.

This work was supported by the Ministry

of Education and Science of Ukraine, grant

no. 0117U000379 the introduction of new

approaches to the creation and use of modern

pharmabiotics.

ЕКСТРАКТИ ЇСТІВНИХ ПЛОДІВ

ВПЛИВАЮТЬ НА КИШКОВУ

МІКРОБІОТУ, ІЗОЛЬОВАНУ

У ПАЦІЄНТІВ З НЕКОМУНІКАТИВНИМИ

ЗАХВОРЮВАННЯМИ, ПОВ’ЯЗАНИМИ

З ХРОНІЧНИМ ЗАПАЛЕННЯМ

Т. В. Мелешко , О. В. Паллаг

Р. О. Рукавчук , Л. С. Юсько , Н. В. Бойко

Ужгородський національний університет,

кафедра клініко-лабораторної діагностики

та фармакології, стоматологічний факультет,

Україна

Ужгородський національний університет,

науково-дослідний і навчальний центр

молекулярної мікробіології та імунології

слизових оболонок, Україна

E-mail: meleshkotv@ukr.net

Метою роботи було дослідити кишкову

мікробіоту у пацієнтів з некомунікативними

захворюваннями, пов’язаними з хронічним

запаленням, зокрема ожирінням, цукровим

діабетом 2-го типу, атеросклерозом та сер-

цево-судинними захворюваннями, а також

з’ясувати потенційну здатність екстрактів

плодів їстівних рослин пригнічувати ріст ок-

ремих умовно-патогенних мікроорганізмів.

В обмеженому клінічному дослідженні

аналіз мікробіоти кишечника проводили ру-

тинним методом, а також за допомогою qPCR.

Вивчено антибактеріальні властивості екстрак-

тів плодів їстівних рослин стосовно відібраних

умовно-патогенних мікроорганізмів.

Склад кишкової мікробіоти пацієнтів з

ожирінням характеризувався збільшенням

кількості Enterococcus spp. та Lactobacillus

spp. поряд зі зменшенням кількості

Escherichia coli. Зниження рівня E. coli та лактобактерій спостерігали у пацієнтів з цук-

ровим діабетом 2-го типу. За атеросклерозу

відзначали збільшення стрептококів, ентеро-

коків та ентеробактерій, тоді як у пацієнтів

із серцево-судинними захворюваннями на-

явним було додаткове підвищення кількості

стафілококів та кандид поряд зі зниженням

E. coli. Зменшення кількості Bifidobacterium

spp., Bacteroides spp., Roseburia intestinalis

та Аkkermansia muciniphila спостерігали у

пацієнтів усіх груп. Ріст Klebsiella spp. при-

гнічували екстракти червоної смородини і

сливи; Enterobacter spp. — екстракт череш-

ні; Proteus spp. — екстракти чорниці та ки-

зилу; Staphylococcus spp. — екстракти чорної

смородини, черешні, сливи, йошти, аличі та

кизилу.

Отримані дані можуть бути використані

для ранньої діагностики некомунікативних

захворювань та для їхньої профілактики за

допомогою персоніфікованого харчування.

T. V. Meleshko1, 2 1 Uzhhorod National University, Department

O. V. Pallah1, 2 of Clinical Laboratory Diagnostics and Pharmacology,

R. O. Rukavchuk2 Faculty of Dentistry, Ukraine

L. S. Yusko1, 2 2 Uzhhorod National University,

N. V. Boyko1, 2 Research Development and Educational Centre

of Molecular Microbiology and Mucosal Immunology, Ukraine

E-mail: meleshkotv@ukr.net

Received 27.07.2020

Revised 08.10.2020

Accepted 31.10.2020

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