Personalised diet improve intestine microbiotaand metabolism of obese rats

Recent research on human microbiome provide opportunities to develop functional foods of new gen-

eration that can regulate intestinal microbiota and the biochemical status of the individual. The aim of the

study was to determine the effect of individually designed nutrition on the intestinal microbiota and metabolic

parameters of rats. Outbred laboratory rats with obesity were randomly divided into 9 groups (n = 12) de-

pending on the type of food ingredients taken orally for three months. The ratio of the intestinal commensal

microorganisms main groups, as well as the lipid profile and the content of glucose, urea, calcium in the

serum of animals were determined. It was shown that cholesterol level in the serum was reduced in experi-

mental groups after consumption of lactobacilli suspension, blueberry juice, fermented milk drink based on

lactobacilli, fermented milk drink with blueberry juice, sauerkraut. In most cases, the gut microbiome of ex-

perimental animals was characterized by a consistently high level of lacto and other beneficial bacteria and

decreased amount of opportunistic microorganisms at the end of the experiment compared with animals in

the control group. Based on the obtained data, we first proposed the principles of creating functional products

by synergistically combining components of edible plants that act as prebiotics and microorganisms that act

as probiotics for personalized use, targeted correction of intestinal microbiome and prevention of noncom-

municable diseases.

K e y w o r d s: functional foods, lipid profile, cholesterol, obesity, intestine microbiota, prebiotic and probio-tic components.

The fact that co-evolutionary relationships

between nutrition, gut microbiota, stress,

lifestyle, and environmental factors lead to

epigenetic influence on human health outcomes is

already widely accepted [1, 2]. Nowadays, it is ob-

vious that changes in gut microbiota often act as a

trigger of noncommunicable diseases connected to

low-grade inflammation and metabolic disorders,

such as obesity, type 2 diabetes (T2D), cardiovascu-

lar diseases, and others. Recently, clear evidence that

patients with such metabolic diseases face changes

in specific groups of gut microbiome has been re-

ported. In addition, most of the mechanisms of

commensal microorganisms’ modulation of human

health are being investigated [3-5]. For example, en-

teric carriage of a community of Clostridium species

induces IL-10 secreting Foxp3 Tregs in the colon,

probably via the induction of transforming growth

factor beta [6].

Recent scientific advances point to the fact that

low-grade inflammation is related to gut microbiota

dysfunction caused by microorganism’ imbalance

and contributing to obesity. This is currently one of

the most serious public health challenges worldwide

because of its increasing prevalence and its contribu-

tion to a complex of symptoms collectively called the

“metabolic syndrome” and other comorbidities, such

as type 2 diabetes [7, 8]. Diabetic individuals have lower counts of Bifidobacterium and Faecalibacte-

rium microbial representatives beneficial for gut [9].

The beneficial effect of the “healthy gut microbial

composition” and microorganisms’ ratio are recon-

sidered because of their different functional features

[10].

Nowadays, one of the commonly known and

most widely used approaches to correct human mi-

crobiota is to prescribe various biopharmaceuticals

like pre-, pro-, and synbiotics, or pharmabiotics,

if their efficacy is clinically proven. Among the

numerous gut microbial species, certain commen-

sal bacteria are known to provide health benefits to

the host when administered in adequate amounts,

and, as such, they are labeled “probiotics” [11]. In

addition to the reported limited success of biophar-

maceuticals’ use [12] in medical practice, which is

potentially increased through prescription personali-

zation [13], in our opinion, the most promising is still

a more integrated, “natural”, and targeted prognos-

tic correction of microbiota via the newly developed

functional nutrition of new generation [14].

One of the recognized beneficial mechanisms

of commensal gut microbiota effect on the host is

the production of specific short-chain fatty acids

(butyrate non acetate and propionate) during the fer-

mentation of dietary fibers. These acids have multi-

ple beneficial effects on the host’s energy metabo-

lism regulation on the whole: they not only improve

gut condition, but also directly affect all the other pe-

ripheral tissues of the host, including muscles, liver,

and nerves [15-17].

It has been proven that dietary microbiome ad-

justment prevents overweight and obesity. One of the

good examples is the “Mediterranean diet” based on

the use of bread, pasta, rice, corn porridge, cereals

and potatoes, fruits, vegetables, legumes, olive oil,

yogurt, and fish [18, 19]. However, data concern-

ing its efficacy still requires further confirmation by

properly arranged clinical trials [20].

Interestingly, individual members of the gut

microbiome can also have profound effects on

host mucosal homeostasis, and specific microbes

have been found to promote inflammatory [21, 22]

or anti-inflammatory [23, 24] responses in the gut

[25]. Hence, the interaction between the microbi-

ome and the gut immune system is crucial for the

maintenance of mucosal homeostasis. For example,

Bacteroides fragilis produces a polysaccharide A

which induces Tregs that secrete IL-10 and inhibit

gut inflammation [5, 26]. Additionally, B. fragilis

can produce a-galactosylceramide (a-Gal-CerBf), a

glycosphingolipid which is capable of binding CD1d

and activating invariant natural killer T cells [27].

Microbiota of every individual is unique and

has the function of modulating the immune system.

Therefore, individually designed diets that take into

account a person’s characteristics and are able to pre-

vent the development of infectious and diet-related

somatic pathologies can be considered promising.

Modern food is considered not only as com-

position of nutrients. It must also comply with in-

dividual requirements and have a positive effect at

the biochemical, cell, tissue, organ, and organism

levels. For example, Chardonnay grape seed flour

can modulate the gut microbiota while lowering ex-

cessive plasma cholesterol and improving the state

of the vascular wall [28]. Moreover, it is better to

choose food from a local source [29] so that it was

ethnic, tasty, and cheap. If we know the key micro-

organisms or their associations that are markers of a

certain disease, we may choose the food developed

to specifically modulate gut microbiota.

The aim of this study was to investigate the im-

pact of individually designed nutrition on gut micro-

biota and metabolism of rats.

Materials and Methods

Bacterial strains and animal groups. In the

study, we used our original strains sequenced and

documented in the Depositary of Microorganisms

of the D.K. Zabolotny Institute of Microbiology

and Virology of the NASU, namely: Lactobacillus

casei IMB B-7412 (isolated from sauerkraut), Lac-

tobacillus plantarum IMB B-7414 (isolated from

sauerkraut), Lactobacillus paracasei IMB B-7483

(isolated from Sautéed pickled green beans), and

L. plantarum KR-1 (isolated from Kvass southern).

Bacterial strains were obtained from fermented

products and were chosen based on their anti-inflam-

matory features, ability to specifically modulate lo-

cal immune response, and regulate gut microbiota

representatives, which was demonstrated in vitro

[30]. Pro- and anti-microbial activity of the pre- and

probiotic components of the complex novel foods

was investigated in vitro [31, 32].

All experiments in rats were performed in

accordance with the international principles out-

lined by the European Convention for the Protec-

tion of Vertebrate Animals Used for Experimental

and Other Scientific Purposes (Strasbourg, 1986)

signed by the Verkhovna Rada of Ukraine in 2002, Law of Ukraine No. 3447 - IV “On the Protection

of Animals from Cruelty”, meeting minutes of the

Bioethics Commission of the Medical Faculty of the

State University “Uzhhorod National University”

(Minutes No. 1, dated May 24, 2019).

In this study, nine groups of 12 white laborato-

ry rats aged 22-24 months, with male and female rats

being equally presented in each group, were formed.

For the experiments we have used rats additionally

fed with a “fat-rich” diet (in analogues to high-fat

diet rodent models [33]) for the induction of obesity.

We have specifically chosen the old rat generation

for the personalized diet experiments in order to ad-

dress “age” relevant issues and to be able to provoke

the “type 2 diabetes” like human condition connect-

ed with microbiome and metabolomic profile” that

are relevant to chronic inflammation changes and

which are often initiated in elderly people. In the

experimental groups, each animal received tested

ingredient(s) in the amount of 0.5 ml daily during 12

weeks in addition to the standard food received by

the control group. During the experiment, all ani-

mals were orally consuming different ingredient(s)

depending on the experimental group: Group 1 –

suspension of lactobacilli (L. casei IMB B-7412,

L. plantarum IMB B-7414, L. paracasei IMB

B-7483, and L. plantarum KR‐1); Group 2 – blueber-

ry juice (Vaccinium murtillus); Group 3 – fermented

milk drink with strains of lactobacilli (L. paracasei

IMB B-7483, L. casei IMB B-7412, L. plantarum

IMB B-7414, and L. plantarum KR-1) and blueberry

juice (the ratio of the fermented drink and blueberry

juice was 4:1); Group 4 – fermented milk drink with

strains of lactobacilli (L. paracasei IMB B-7483,

L. casei IMB B-7412, L. plantarum IMB B-7414,

and L. plantarum KR‐1) without plant components;

Group 5 – sauerkraut juice with L. casei IMB B-7412

and L. plantarum IMB B-7414; Group 6 – persimmon

juice (Diospyros kaki); Group 7 – lignin; Group 8 –

pectin (15%); Group 9 – control group, standard vi-

varium diet food. A suspension of microorganisms

was prepared daily before the administration using

48 h pure cultures of tested microorganisms taken at

the concentration of no less than 1.5×108

CFU/ml ac-

cording to McFarland. Body weight of each animal’s

group was determined twice, at the start and at the

end of experiment (Table 1, Fig. 1).

Study of the biochemical and microbiological

parameters. Before and after the gavage, the weight

of the all the experimental animals was monitored

and blood sampling was taken from the tail vein

using the Microvette® capillary blood collection

system for biochemical study. The lipid profile,

namely total lipids, triglycerides, low-density lipo-

proteins (LDL), cholesterol, urea, calcium, and glu-

cose, was measured by colorimetric analysis using

ready-made reagents manufactured by “Philisit-

Diagnostics,” LLC.

In order to detect changes in major gut micro-

biota representatives and their composition under the

influence of tested ingredients of the newly develo-

ped functional nutrition in obese rats in real time,

the samples were taken on the third, seventh, 14th,

21st, 28th, 35th, 42nd, 49th, 56th, 63rd, 70th, 77th, 84th,

and 91st days from the beginning of the experiment.

Therefore, on every seventh day of the experiment

1 g of feces was collected from the experimental

animals and mixed with 1 ml of PBS. Ten-fold serial

dilution of samples was performed and plated cor-

respondingly on the chromogenic, typical, and se-

lective growth media: MacConkey agar, Blood agar,

Nutrient agar, Mannitol salt agar, Wilson-Blair agar,

Sabouraud dextrose agar, Lactobacillus MRS agar,

Bifidobacterium agar, Anaerobic blood agar, Bile

esculin agar, Streptococcus selective agar, Bacte-

roides bile esculin agar (all above mentioned growth

media produced by HiMedia Laboratories, India),

UriSelectTM 4 Medium (Bio-Rad Laboratories, Inc,

USA), or Blaurock semi-liquid modified hepatic me-

dium (Liofilchem, Italy).

For Bacteroides’ isolation, of 10-5–10-7 dilutions

of each sample, 10 μl were plated on the surface of

Bacteroides bile esculin agar and incubated for 4-5

days under anaerobic conditions. After microscopy,

the morphology of the microorganisms was evalua-

ted. Gram-negative polymorphic anaerobes can

be preassigned to Bacteroides. Lactobacilli were

isolated from 10-2–10-8 dilutions. Seeding was car-

ried out in the amount of 10 μl on MRS broth or

MRS agar and incubated for 2–3 days under anaero-

bic conditions.

Enterococci, staphylococci, and streptococci

were isolated by plating 10 μl of a 10-2–10-8 dilution

on bile esculin agar, mannitol salt agar, or strepto-

coccus selection agar respectively and incubated

at 37°C in a thermostat for 18-24 h. Blood agar,

nutrient agar, and UriSelect were also used for strep-

tococci isolation. Preliminary differential diagnosis

of Staphylococcus aureus was performed using tests

for catalase, hemolytic, and coagulase activity.

Yeast-like fungi were isolated by plating the

10‐2–10-8 dilution on the surface of sabouraud dex-

trose agar. After 2–3 days, white-matte colonies

were selected for identification. The total amount of

aerobic bacteria and their hemolytic properties was

determined by plating 10-5 and 10-7 suspension dilu-

tions on blood agar. The total amount of enterobac-

teria was determined by plating 10 μl of the suspen-

sion of 10-5–10-8 dilutions on MacConkey Agar.

Spore-forming bacteria, including clostridia,

were isolated by seeding 10 μl of 10-3–10-5 and 10-7

dilutions on Wilson-Blair agar. After 48 h of incuba-

tion, the number of black colonies in the agar depth

was calculated and formation of the gas separating

the nutrient medium was recorded.

Counting of all types of microorganisms was

carried out according to the formula: CFU/g = a×b×c,

where: a stands for the number of colonies grown on

the nutrient medium; b stands for dilution coefficient

dose (when plating 100 μl, a = 10; when seeding

50 μl, a = 5; when plating 10 μl, a = 100); and c

stands for dilution factor.

Identification of isolated microorganisms was

performed using biochemical test systems ANAE-

RO-23, ENTERO-24, NEFERM-test, Candida-23,

STAPHYtest 16, and STREPTOtest 24 (Erba La-

chema s.r.o., Czech Republic).

Statistical analysis. The experiments were per-

formed in triplicate. For the mathematical analysis of

the data, licensed software SPSS 17.0 was used. Par-

ametric results were reported as means with SD. The

normality of distribution was determined using the

Lilliefors criterion. In the group differences before

and after the intervention were evaluated using the

Wilcoxon rank-sum test. The differences between

the groups were evaluated using the Mann–Whitney

U test. P < 0.05 was considered significant. Correla-

tion relationships were determined using the Pearson

coefficient.

Results and Discussion

Under the influence of different ingredients –

potential components of the new generation func-

tional foods – body weight of the experimental ani-

mals significantly decreased (P < 0.05) in all groups

except the control one, where it grew by (20 ± 4) g,

and the fourth group of rats that followed the fer-

mented milk drink diet without plant components,

where we noted an increased body weight (P < 0.05)

by (15 ± 4) g (Fig. 1). LDL significantly decreased

(P < 0.05) under the influence of lactobacilli, 15%

apple pectin concentrate, lignins, and sauerkraut

juice, while in other experimental groups there were

no significant changes observed (P > 0.05) com-

pared to the control group where this parameter

significantly increased (P < 0.05), namely 2.5 times

(Fig. 2, B).

As a result of the introduction of the diets we

developed, total lipid content significantly decreased

(P < 0.001) in all animal groups except three groups:

one of them being the control group, where a signifi-

cant increase (P < 0.01) in the total lipid content was

noted, and in two experimental groups of animals

consuming apple pectin and lignin (Fig. 2, C). Cho-

lesterol reduction (P < 0.05) occurred in all experi-

mental groups of animals (Fig. 2, D). Interestingly,

only in case of apple pectin consumption triglyceride

concentration decreased (Fig. 2, A). A decrease in

the concentration of blood urea from (3.57 ± 0.03)

mmol/l to (0.9 ± 0.01) mmol/l (P < 0.01) was ob-

served only in the group of animals following a diet

enriched with probiotic bacterial strains (Fig. 3, A).

Glucose content decreased significantly (P < 0.01)

under the effect of blueberry juice and fermented

milk drink without plant components (Fig. 3, C),

but not when fermented milk drink with blueberry

extract was used separately. An increase in calcium

from (0.68 ± 0.03) mmol/l to (0.79 ± 0.05) mmol/l

(P < 0.01) was observed under the composition of

fermented milk drink without plant components.

When using all other diets, a decrease in calcium

levels was observed (Fig. 3, B).

Despite the increasing weight of animals in the

control group and animals consuming fermented

milk drink with lactobacilli without plant compo-

nents, these two groups significantly differed in other

lipid profile indices. In contrast to the control group,

animals consuming fermented milk drink without

plant components had an improvement in almost all

registered biochemical parameters (including a slight

decrease in total blood plasma lipids and a similar

increase in calcium levels).

In the study, we used elderly white rats with

obesity caused by age-related changes and feeding

them high-fat diet. Existing at the beginning of the

experiment and progressive metabolic disorders led

to an increase in the weight of animals in the con-

trol group in the absence of treatment and resulted

in a difference between the biochemical parameters

of the rats’ blood before and after the experiment.

The increase in weight of experimental animals of

group 4 is explained by the fact that long-term con-sumption of lactic acid products (on the example of our studied fermented beverage – fermented milk

product with selected LAB strains) leads to weight

gain due to their high fat and caloric content. There-

fore, when prescribing or selecting products, even a

healthy diet should be guided by their defined norms,

limited amount or fat content, and to ensure a balan-

ced diet, it is necessary to predict the effects of ex-

posure, which can be provided by our correlations of

prognostic corrections of the microbiome.

Our study demonstrated that animals can be

overweight not only because of increasing levels

of opportunistic microorganisms such as Staphylo-

coccus nepalensis and Enterococcus faecalis, but also because of the high level of the probiotic strain Bifidobacterium breve. Confirmed results of the

ability of blueberry juice (from 3.3 to 1.38 mmol/l)

and developed fermented milk drink (from 2.8 to

1.38 mmol/l) to regulate blood glucose levels in vivo

may have a prognostic value for recommending this

diet to patients with type 2 diabetes.

During the whole study we observed the fol-

lowing changes in rats’ gut microbiota under the in-

fluence of different diets. A consistently high level

of lactic acid and other beneficial bacteria and a de-

creasing level of opportunistic microorganisms were

detected in almost all experimental groups compared

to the control group.

Consumption of almost all of the developed

food components (diets) caused a decrease in the

concentration of E. faecalis and different Staphylo-

coccus species compared to that in the control group.

The level of Klebsiella pneumoniae and Morganella

morganii decreased significantly under the effect of

blueberry juice. The level of B. subtilis remained al-

most unchanged during the whole experiment under

the influence of the test samples based on plant ex-

tracts, plant pectins, and lignins.

Oral administration of lactobacillus strains sus-

pension (L. casei ІМВ В-7412, L. plantarum IMB

B-7414, L. paracasei IMB B-7483, and L. plan-

tarum KR-1) demonstrated an antagonistic activity

against Staphylococcus aureus, Peptostreptococcus

anaerobius, and E. faecalis and led to a decrease in

Escherichia coli and Enterobacter cloacae levels in

obese rats during the whole experiment (Fig. 4, A).

The concentration of commensal K. pneumo-

niae, M. morganii, E. coli, Actinomyces naeslundii,

and Bacteroides significantly decreased under the

influence of a blueberry juice-based diet. In this

study group, complete elimination of Streptococcus

parvulus and increase in E. faecalis and staphylo-

cocci were observed (Fig. 4, B).

Reduction in opportunistic microorganisms and

increase in beneficial microbiota indicated a positive

effect of consumption of the developed fermented

milk product with blueberry juice. Throughout the

study, we observed an increase in commensal lac-

tobacilli, elimination of Staphylococcus spp. and

E. faecalis, and a decreased concentration of E. coli

in this experimental group compared to the control

group (Fig. 5, A).

It was demonstrated that the 12-weeks oral ad-

ministration of fermented milk drink with strains of

lactobacilli (L. paracasei IMB B-7483, L. casei IMB

B-7412, L. plantarum IMB B-7414, and L. plantarum

KR-1) eliminated M. morganii and E. faecalis and

reduced E. coli and Actinomyces israelii concentra-

tions in obese rats (Fig. 5, B).

In this study, increased commensal lactoba-

cilli concentration, partial elimination of E. coli and

Bacillus subtilis, as well as complete elimination of

cocci were observed under the influence of sauer-

kraut juice (Fig. 6, A).

It should be noted that persimmon juice, in case

of long-term oral administration to animals, dem-

onstrated a significant antagonistic effect against

Staphylococcus cohnii and led to a decrease in the

levels of E. coli, Proteus mirabilis, E. cloacae, and

E. faecalis. In addition, consistently high levels of

microorganisms, such as Lactobacillus acidophilus,

Bifidobacterium longum, and B. subtilis ((5 ± 0.3)

×108CFU/g), were observed in case of persimmon

juice consumption compared to the control group

(Fig. 6, B).

As most vegetables and fruits contain dietary

fiber (pectin, lignin, cellulose, and hemicellulose),

which is a natural enterosorbent and can affect quan-

titative and qualitative composition of gut microbio-

ta, we investigated apple pectin and lignin effect in a

chronic experiment on obese rats.

The reduction in enterococci, B. subtilis,

E. coli, and P. mirabilis with a slight increase in

commensal lactobacilli concentration were observed

under the influence of lignin (Fig. 7, A).

Oral administration of pectin-based diet (apple-

pectin concentrate) demonstrated complete elimina-

tion of E. faecalis and a decrease in the concentra-

tion of commensal Bifidobacterium, staphylococci,

E. coli, and B. subtilis (Fig. 7, B).

Changes in gut microbiota of the control group

of animals demonstrated a decrease in the concen-

tration of E. faecalis and B. subtilis, as well as an

increase in the concentration of P. mirabilis (Fig. 8).

The reduction in Lactobacillus spp., B. subtilis,

and P. mirabilis in the gut microbiome may serve

as a biomarker of triglyceride increase and LDL

and cholesterol reduction (and vice versa). The in-

crease in B. breve and B. subtilis is a biomarker of

decreased plasma calcium (and vice versa).

In order to confirm the possibility of using

these microbial and biochemical parameters as bio-

markers, we searched for correlation between them

and classical disease signs, both at the beginning of

the study and after it.

In our study, such an indicator of lipid metabo-

lism as triglycerides closely correlates with LDL

(Pearson correlation coefficient is r = 0.7506) and

Lactobacillus spp. (r = 0.7130) in the gut microbiome

of rats. In turn, LDL inversely correlates with cho-

lesterol (r = -0.6772), while the inverse correlation of

cholesterol with the triglycerides (r = -0.7402) was

also observed. Overweight, as a baseline indicator

of abdominal fat distribution, directly correlates with

B. breve (r = 1,000) and S. nepalensis (r = 0.9244).

A correlation was found between B. breve and

E. faecalis (r = 0.9917) as well as between E. faecalis

and S. nepalensis (r = 1,000). In its turn, S. nepalen-

sis inversely correlates with B. breve (r = -0.5379).

Correlation was also found between P. mirabilis and

Lactobacillus spp. (r = 0.6776). In this study, the

change in the ratio of E. coli isolates with typical

and atypical enzymatic activity was investigated,

with the number of isolates with atypical enzymatic

activity predominating. It is interesting that the con-

centration of lactose-negative E. coli strains cor-

relates with the number of lactobacilli (r = 0.7608)

and Staphylococcus spp. (r = -0.7608) while Lacto-

bacillus reuteri correlates with Staphylococcus spp.

(r = 0.7456).

We also observed correlation between L. aci-

dophilus and S. aureus (r = 0.9979) in turn, S. au-

reus correlated with Staphylococcus epidermidis

(r = -0.8520), and S. epidermidis inversely correlated

with L. acidophilus (r = -1,000). A correlation be-

tween P. anaerobius and E. cloacae was established

(r = 0.6727).

In this study we detected correlation between

Bacteroides distasonis and M. morganii (r = 0.6811)

in turn, M. morganii correlated with K. pneumoniae

(r = 0.6811) and K. pneumoniae was negatively corre-

lated with B. distasonis (r = 1,000) while A. naeslun-

dii (r = 1,000) and A. naeslundii negatively correlated

with S. parvulus (r = -1,000). Negative correlation

of B. distasonis with S. parvulus (r = -1,000) and

S. parvulus with K. pneumoniae (r = 1,000) was also

established.

We found a direct correlation between the

amount of L. acidophilus and S. aureus in the in-

testinal contents of rats using the tested products.

Other scientists have studied that in obesity and ir-

ritable bowel syndrome observed the growth of op-

portunistic pathogens, including staphylococci and a

decrease in lactobacilli [34].

Also we can say that blood glucose is an am-

biguous indicator. Today, there is evidence that dif-

ferent strains of different LABs regulate blood glu-

cose levels differently - from no effect to a decrease

or increase in serum levels. In particular, according

to the European Journal of Clinical Nutrition [35],

men were given lactobacilli-based yogurt (L. acido-

philus) for three weeks and as a result the level of

HDL cholesterol, triglycerides and blood glucose in

the serum remained unchanged.

Another study shows that the strain of Lactoba-

cillus rhamnosus causes a decrease in glucose levels

[36]. There is a study according to which the level of

glucose depends on the content of calcium ions and

its reduction should not be interpreted unambiguous-

ly [37]. Therefore, to assess the effectiveness against

obesity, we first considered the expected (desired)

changes in lipid metabolism.

In fact, there are virtually no publications

linking biochemical and microbial indicators. These

parameters should be correlated with biochemical

and immune parameters. Our goal was a compre-

hensive study of all indicators to determine dynamic

changes. This direction was initiated by us and simi-

lar approaches were developed by us in the corre-

sponding articles [38].

The results of our study demonstrated positive

effects of separate components as well as the novel

functional food itself, namely fermented milk drink

with strains of lactobacilli and blueberry juice. Ap-

parently, this is due to the fact that probiotic strains

of lactobacilli and bifidobacteria create unfavorable

conditions for pathogens’ colonization by decreas-

ing their adhesive features. Additionally, probiotic

strains and plant extracts have the ability to adjust

and stimulate the beneficial gut microbiota of the ex-

perimental animals.

While analyzing the results of this study, it can

be argued that the suspension of lactobacilli L. para-

casei ІМВ В-7483, L. casei ІМВ В-7412, L. plan-

tarum ІМВ В-7414, and L. plantarum KR-1 demon-

strated the best hypocholesterolemic activity since

the long-term oral administration of suspension of

these strains led to a decrease in cholesterol levels

from 2.95 to 0.84 mmol/l.

As the glucose level in the serum of obese ani-

mals decreased due to oral administration of blue-

berry juice, as well as fermented milk drink with

strains of L. casei IMB B-7412, L. plantarum IMB

B-7414, L. paracasei IMB B-7483, and L. plantarum

KR-1, these particular components can be offered to

patients with type 2 diabetes.

It is interesting that the use of suspensions of

lactobacilli L. paracasei ІМВ В-7483, L. casei ІМВ

В-7412, L. plantarum ІМВ В-7414, and L. plantarum

KR-1, sauerkraut juice (enriched with strains of

L. plantarum IMB B-7414 and L. casei IMB B-7412),

15% apple pectin concentrate, and lignin cause a de-

crease in LDL levels. However, it should be noted

that a 15% apple pectin concentrate has the highest

hypolipidemic activity since the long-term oral ad-

ministration of this component leads to a decrease in

not only LDL, but also triglycerides and cholesterol.

That is why these components can be used to prevent

atherosclerosis and cardiovascular disease.

Conclusion. The results obtained during the

study, as well as the correlation between the changes

in key representatives of gut microbiota and values

of biochemical indicators of the organism’s state give

the possibility to develop the new generation func-

tional foods able to regulate gut microbiota balance

and prevent the development of diet-associated pa-

thologies.

Conflict of interest. Authors have completed

the Unified Conflicts of Interest form at http://ukr-

biochemjournal.org/wp-content/uploads/2018/12/

coi_disclosure.pdf and declare no conflict of interest.

Funding. This work is funded under the State

governmental budget of Ministry of Education and

Science, Topic: “Composite biological products from

microorganisms, plants and nanocompounds” Regis-

tration number 0113U002369.

Персоніфікована дієта

покращує мікробіоту

кишечника та метаболізм

щурів з ожирінням

В. В. Баті1, Т. В. Мелешко1

, О. В. Паллаг1,

І. П. Заячук2, Н. В. Бойко11

НДНЦ молекулярної мікробіології та

імунології слизових оболонок, Ужгородський

національний університет, Україна; 2

Кафедра фізіології та патофізіології, Ужгородський

національний університет, Україна;

e-mail: victoria.bati@uzhnu.edu.ua

Сучасні дослідження мікробіому людини

уможливлюють конструювання функціональ-

них продуктів харчування нового покоління,

здатних регулювати кишкову мікробіоту та

біохімічний статус індивідууму. Метою дослі-дження було з’ясувати вплив індивідуально роз-

робленого харчування на мікробіоту кишечника

та показники метаболізму в щурів. Безпородних

щурів із ожирінням було випадковим чином роз-

ділено на дев’ять груп (n = 12) залежно від типу

перорально вживаних протягом трьох місяців

інгредієнтів харчування. Визначали співвідно-

шення основних груп кишкових коменсальних

мікроорганізмів, а також ліпідний профіль та

вміст глюкози, сечовини, кальцію у сироватці

крові тварин. Встановлено зниження рівня хо-

лестеролу в сироватці крові дослідних тварин

після випоювання їм суспензії лактобактерій,

соку чорниці, кисломолочного напою на основі

лактобактерій, кисломолочного напою з соком

чорниці, квашеної капусти. У більшості випад-

ків кишковий мікробіом експериментальних

тварин характеризувався стабільно високим

рівнем лакто- та інших корисних бактерій та

зменшенням кількості умовно-патогенних мі-

кроорганізмів наприкінці експерименту порів-

няно з тваринами контрольної групи. На основі

одержаних даних нами вперше запропоновано

принципи створення функціональних продук-

тів нового покоління шляхом синергідного по-

єднання компонентів їстівних рослин (що діють,

як пребіотики) та мікроорганізмів (що діють, як

пробіотики) з метою їх прогностичного та пер-

соніфікованого застосування для попередження

виникнення некомунікативних захворювань,

шляхом спрямованої корекції мікробіому ки-

шечника макроорганізму, а відтак і його біохі-

мічного статусу.

К л ю ч о в і с л о в а: функціональні про-

дукти харчування, ліпідний профіль, холесте-

рол, ожиріння, мікробіота кишечника, пребіо-

тичні та пробіотичні компоненти.

References

1. D'Argenio V, Salvatore F. The role of the gut

microbiome in the healthy adult status. Clin

Chim Acta. 2015; 451(Pt A): 97-102.

2. Dickerson F, Severance E, Yolken R. The

microbiome, immunity, and schizophrenia and

bipolar disorder. Brain Behav Immun. 2017; 62:

46-52.

3. Ascher S, Reinhardt C. The gut microbiota:

An emerging risk factor for cardiovascular and

cerebrovascular disease. Eur J Immunol. 2018;

48(4): 564-575.

4. Virgin HW, Todd JA. Metagenomics and

personalized medicine. Cell. 2011; 147(1): 44-56.

5. Round JL, Mazmanian SK. Inducible Foxp3+

regulatory T-cell development by a commensal

bacterium of the intestinal microbiota. Proc Natl

Acad Sci USA. 2010; 107(27): 12204-12209.

6. Atarashi K, Tanoue T, Shima T, Imaoka A,

Kuwahara T, Momose Y, Cheng G, Yamasaki S,

Saito T, Ohba Y, Taniguchi T, Takeda K,

Hori S, Ivanov II, Umesaki Y, Itoh K, Honda K.

Induction of colonic regulatory T cells by

indigenous Clostridium species. Science. 2011;

331(6015): 337-341.

7. Isolauri E, Rautava S, Collado MC, Salminen S.

Role of probiotics in reducing the risk of

gestational diabetes. Diabetes Obes Metab.

2015; 17(8): 713-719.

8. Sanz Y, Santacruz A, Gauffin P. Gut microbiota

in obesity and metabolic disorders. Proc Nutr

Soc. 2010; 69(3): 434-441.

9. Furet JP, Kong LC, Tap J, Poitou C, Basdevant A,

Bouillot JL, Mariat D, Corthier G, Doré J,

Henegar C, Rizkalla S, Clément K. Differential

adaptation of human gut microbiota to bariatric

surgery-induced weight loss: links with

metabolic and low-grade inflammation markers.

Diabetes. 2010; 59(12): 3049-3057.

10. Lloyd-Price J, Abu-Ali G, Huttenhower C. The

healthy human microbiome. Genome Med. 2016;

8(1): 51.

11. Sassone-Corsi M, Raffatellu M. No vacancy: how

beneficial microbes cooperate with immunity to

provide colonization resistance to pathogens. J

Immunol. 2015; 194(9): 4081-4087.

12. Pat. No 90789 A Technique for the prevention

of purulent complications and wound infection

after tooth extraction with bacterial suspension

based on Bacillus subtilis 090 / Petrov VO,

Rusyn VI, Boiko NV. Publ. 10.06.2014 Bul.

No 11. (In Ukrainian).

13. Pat. No 93301. Composite biological product

for the treatment of inflammation of

periodontal tissues and correction of associated

gastroduodenal disorders of the intestine in

children / Melnyk VS, Diachuk EY, Bati VV,

Levchuk OB, Boiko NV. Publ. 25.09.2014, Bul.

No 18. (In Ukrainian).

14. Ercolini D, Fogliano V. Food Design To Feed the

Human Gut Microbiota. J Agric Food Chem.

2018; 66(15): 3754-3758.

91

15. Kasubuchi M, Hasegawa S, Hiramatsu T,

Ichimura A, Kimura I. Dietary gut microbial

metabolites, short-chain fatty acids, and host

metabolic regulation. Nutrients. 2015; 7(4):

2839-2849.

16. Donohoe DR, Garge N, Zhang X, Sun W,

O'Connell TM, Bunger MK, Bultman SJ. The

microbiome and butyrate regulate energy

metabolism and autophagy in the mammalian

colon. Cell Metab. 2011; 13(5): 517-526.

17. Desbonnet L, Clarke G, Traplin A, O'Sullivan O,

Crispie F, Moloney RD, Cotter PD, Dinan TG,

Cryan JF. Gut microbiota depletion from early

adolescence in mice: Implications for brain and

behaviour. Brain Behav Immun. 2015; 48: 165-

173.

18. Enck P, Zimmermann K, Rusch K, Schwiertz A,

Klosterhalfen S, Frick JS. The effects of

maturation on the colonic microflora in infancy

and childhood. Gastroenterol Res Pract. 2009;

2009: 752401.

19. Trichopoulou A, Naska A, Orfanos P,

Trichopoulos D. Mediterranean diet in relation

to body mass index and waist-to-hip ratio: the

Greek European Prospective Investigation into

Cancer and Nutrition Study. Am J Clin Nutr.

2005; 82(5): 935-940.

20. Garcia-Mantrana I, Selma-Royo M, Alcantara C,

Collado MC. Shifts on Gut Microbiota

Associated to Mediterranean Diet Adherence

and Specific Dietary Intakes on General Adult

Population. Front Microbiol. 2018; 9: 890.

21. Ivanov II, Atarashi K, Manel N, Brodie EL,

Shima T, Karaoz U, Wei D, Goldfarb KC,

Santee CA, Lynch SV, Tanoue T, Imaoka A,

Itoh K, Takeda K, Umesaki Y, Honda K,

Littman DR. Induction of intestinal Th17 cells

by segmented filamentous bacteria. Cell. 2009;

139(3): 485-498.

22. Ivanov II, Frutos RdeL, Manel N, Yoshinaga K,

Rifkin DB, Sartor RB, Finlay BB, Littman DR.

Specific microbiota direct the differentiation of

IL-17-producing T-helper cells in the mucosa

of the small intestine. Cell Host Microbe. 2008;

4(4): 337-349.

23. Mazmanian SK, Round JL, Kasper DL. A

microbial symbiosis factor prevents intestinal

inflammatory disease. Nature. 2008; 453(7195):

620-625.

24. Round JL, Mazmanian SK. Inducible Foxp3+

regulatory T-cell development by a commensal

bacterium of the intestinal microbiota. Proc Natl

Acad Sci USA. 2010; 107(27): 12204-12209.

25. McDermott AJ, Huffnagle GB. The microbiome

and regulation of mucosal immunity.

Immunology. 2014; 142(1): 24-31.

26. Mazmanian SK, Round JL, Kasper DL. A

microbial symbiosis factor prevents intestinal

inflammatory disease. Nature. 2008; 453(7195):

620-625.

27. Wieland Brown LC, Penaranda C,

Kashyap PC, Williams BB, Clardy J,

Kronenberg M, Sonnenburg JL, Comstock LE,

Bluestone JA, Fischbach MA. Production of

α-galactosylceramide by a prominent member

of the human gut microbiota. PLoS Biol. 2013;

11(7): e1001610.

28. Kim H, Kim DH, Seo KH, Chon JW, Nah SY,

Bartley GE, Arvik T, Lipson R, Yokoyama W.

Modulation of the intestinal microbiota is

associated with lower plasma cholesterol and

weight gain in hamsters fed chardonnay grape

seed flour. J Agric Food Chem. 2015; 63(5):

1460-1467.

29. Deller S, Canto A, Brown L. Food access, local

foods, and community health. Community Dev.

2017; 48(5): 657-680.

30. Bati VV, Boyko NV. The biological properties

of lactоbacilli strains isolаted from food of plant

origin and edible plants. ScienceRise. 2016;

8(1(25)): 6-14. (In Ukrainian).

31. Bati VV, Boyko NV. Microbiological analysis

of sauerkraut in the process of its fermentation

according to the traditional and modernized

technologies. Microbiol Biotechnol. 2017;

(2(38)): 90-100.

32. Bati VV, Boyko NV. Novel functional food for

prevention of non-communicable diseases. Biol

Stud. 2019; 13(1): 71-84.

33. Buettner R, Parhofer KG, Woenckhaus M,

Wrede CE, Kunz-Schughart LA, Schölmerich J,

Bollheimer LC. Defining high-fat-diet rat

models: metabolic and molecular effects of

different fat types. J Mol Endocrinol. 2006;

36(3): 485-501.

34. Grygoruk GV, Myshchuk VG, Tserpiak NV.

Changes in gut microbiota and blood lipid profile

in patients with irritable bowel syndrome in

association with obesity. Bukovin Med Herald.

2019; 23(1(89)): 32-38. (In Ukrainian).

35. Schaafsma G, Meuling WJ, van Dokkum W,

Bouley C. Effects of a milk product, fermented

by Lactobacillus acidophilus and with fructo-

V. V. Bati, T. V. Meleshko, O.V. Pallah et al.

92

ISSN 2409-4943. Ukr. Biochem. J., 2021, Vol. 93, N 4

oligosaccharides added, on blood lipids in male

volunteers. Eur J Clin Nutr. 1998; 52(6): 436-

440.

36. Farida E, Nuraida L, Giriwono PE, Jenie BSL.

Lactobacillus rhamnosus Reduces Blood

Glucose Level through Downregulation

of Gluconeogenesis Gene Expression in

Streptozotocin-Induced Diabetic Rats. Int J

Food Sci. 2020; 2020: 6108575.

37. Bartlett PJ, Gaspers LD, Pierobon N, Thomas AP.

Calcium-dependent regulation of glucose

homeostasis in the liver. Cell Calcium. 2014;

55(6): 306-316.

38. Golubnitschaja O, Topolcan O, Kucera R,

Costigliola V, EPMA. 10th Anniversary of the

European Association for Predictive, Preventive

and Personalised (3P) Medicine - EPMA World

Congress Supplement 2020. EPMA J. 2020;

11(Suppl 1): 1-133.