BG113505A - Bifidobacterium longum ssp. longum strain and bifidobacterial preparation with hypolypidemic effect, anti-inflammatory immunomodulatory effect and insulin resistance abatement effect - Google Patents

Abstract

The present invention relates to a new strain of Bifidobacterium longum ssp. longum 3/15, deposited at the National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC) of the Republic of Bulgaria under reference number NBIMCC 9085, which possesses a complex of beneficial health properties: hypolipidemic activity, anti-inflammatory immunomodulatory activity, capability to abate insulin resistance, strong adhesion to epithelial cells and high survival rate in the gastrointestinal tract. The subject of the present invention is also a bifidobacterial preparation obtained by cultivating a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085, resulting in a clinically proven set of functional effects, including improvement of lipid indicators, abatement of insulin resistance and favorable effect on the markers of inflammatory status. The present invention also relates to the use of said novel strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 and said bifidobacterial preparation for the prevention and treatment of atherosclerosis, cardiovascular and inflammatory diseases as well as for the reduction of diabetic risk.

Description

FIELD OF ENGINEERING

The present invention relates to the field of biotechnology and, in particular, to a probiotic strain of bifidobacterium, which possesses a complex of beneficial properties for human health and its use in probiotic preparations for the prevention of metabolic, cardiovascular and inflammatory diseases.

PRIOR ART

Bifidobacteria are considered probiotics because they are living microorganisms that have a beneficial effect on human health when taken in sufficient quantities. A minimum level of approximately 10 ⁶ -10 ⁷ viable bifidobacteria cells per gram of intestinal contents has been suggested. [1]

Bifidobacteria are one of several dominant culturable bacterial groups present in the colonic microflora, playing an important role in maintaining a balanced healthy intestinal microflora. There is compelling evidence for the interrelationship between gut microflora, obesity, the endocannabinoid system, systemic low-grade inflammation, lipopolysaccharides, intestinal permeability, arterial stiffness.

Probiotics, in addition to improving the composition of the intestinal microflora, also act on other goals such as improving immunity, favorably affecting inflammatory and metabolic diseases, reducing the risk of developing atherosclerosis. Of extreme importance for the manifestation of their beneficial effects are the adhesive properties of the bacterial cells, as well as their survival and ability to colonize the gastrointestinal tract.

The beneficial health properties demonstrated by probiotics are strain-specific, and even strains of the same genus and species exhibit different properties and to varying degrees.

The beneficial effect of bifidobacteria against intestinal inflammatory diseases such as ulcerative colitis and Crohn's disease has been proven, and the treatment is probiotic preparations accompanying drug therapy. The mechanisms of this beneficial effect on intestinal inflammations are prevention of the adhesion of pathogens on the intestinal epithelium; immunomodulatory effect by inducing the synthesis of anti-inflammatory cytokines IL-10 (interleukin 10) and/or TGF-beta (transforming growth factor beta), reduction of the synthesis by epithelial cells of the proinflammatory cytokine IL-8 (interleukin 8), absence or reduced induction of TNFα (tumor necrosis factor alpha), IL-1 (interleukin 1) and/or IL-6 (interleukin 6); proteolytic degradation of antigens with proinflammatory properties; elimination of toxins, etc. [2]

Increased induction of proinflammatory cytokines, especially TNFα, stimulates epithelial cells to produce interleukin IL-8, which stimulates neutrophil cell activity. [3] With persistent increased activity of neutrophils, ulcers may form, intestinal bleeding may occur [4], conditions found in inflammatory bowel disease.

The immunomodulatory effect is strictly strain-specific. Lactobacilli and bifidobacteria strains were studied, some of which lead to an increase in the level of TNFa and IL-6, others to a decrease in TNFa and an increase in IL-10. [5]

It has been established that some strains of bifidobacteria have the ability to lower serum cholesterol, whose high levels are the cause of a number of diseases such as hyperlipidemia, atherosclerosis, lipid metabolic syndrome. The mechanisms for this are through the direct adsorption of cholesterol to lipoprotein fractions of the bacterial cell walls or through the significant hydrolase activity towards bile salts (BSH activity) possessed by some probiotic strains. Through their BSH activity, probiotic strains lower the concentration of bile salts, which is compensated by synthesizing new amounts of bile acids from cholesterol and conjugating these bile acids with taurine or glycine to new stores of bile salts, replacing the deconjugated bile salts. The main property in the selection of bacterial strains for the development of probiotic preparations with a beneficial effect on cholesterol metabolism is the presence of significant hydrolase activity towards bile salts.

It is also known that good levels of the enzyme "bile salt hydrolase" in bifidobacteria are associated not only with its ability to reduce cholesterol, but also to reduce inflammation.

Korean patent KR102224072B1 [6] describes a strain of Bifidobacterium longum ssp. longum possessing a combination of two main functional characteristics: direct reduction of total serum cholesterol and immune regulation by decreasing the secretion of pro-inflammatory cytokines TNFα and IL-6 and increasing the secretion of the anti-inflammatory cytokine IL-10. These effects were confirmed by in vitro functional assessment of the strain and in vivo mouse experiment.

It is known that cardiovascular diseases are a leading cause of morbidity and mortality worldwide, and the main reason for their occurrence, development and subsequent complications is atherosclerosis. The main risk factors, the combination of which leads to the appearance and accelerated development of the atherosclerotic process, are hypertension, dyslipidemia, obesity and pro-inflammatory status, diabetes mellitus or impaired glucose metabolism, immobility. Often these factors combine in the same patient, which sharply increases the risk of cardiovascular disease. The phenomenon is well known in patients with metabolic syndrome. The more of the risk factors that exist in a patient, the greater the likelihood of cardiovascular disease, because their harmful effects are multiplied when they are combined. Therefore, it is important to reduce the combined influence of the available risk factors by influencing their totality simultaneously, rather than individually.

An actual problem is the selection of a probiotic strain of Bifidobacterium longum ssp. longum, which, in addition to immune regulation and direct reduction of cholesterol, also exhibits hydrolase activity against bile salts.

Another current problem is proving the hypolipidemic and anti-inflammatory and immunomodulatory activities of a strain of Bifidobacterium longum ssp. longum through a clinical trial of the application of the strain in patients at risk of developing atherosclerosis.

TECHNICAL ESSENCE OF THE INVENTION

The subject of the present invention is a new strain of Bifidobacterium longum ssp. longum 3/15, deposited under number NBIMCC 9085 on January 26, 2022 in the National Bank for Industrial Microorganisms and Cell Cultures (NBPMKK) of the Republic of Bulgaria, possessing a complex of beneficial properties for health - hypolipidemic activity, anti-inflammatory immunomodulatory activity, ability to reduce insulin resistance, strong adhesion to epithelial cells and high survival in the gastrointestinal tract.

The subject of the present invention is also a bifidobacterial preparation obtained from a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 and possessing a clinically proven set of health benefits: improvement of lipid parameters, reduction of insulin resistance, beneficial effect on markers of inflammatory status.

The subject of the invention is also the use of the new strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 and of the bifidobacterial preparation obtained from a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 for the simultaneous beneficial effect of multiple health risk factors such as the prevention of both a specific inflammatory or metabolic disease, but also of diseases caused by these risk factors in their totality, in the specific case for the prevention of atherosclerosis and cardiovascular diseases.

A strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 was isolated from fecal samples of a 28-year-old healthy volunteer who had not used antibiotics in the past 1 year and had not used probiotics in the past six months. The sample taken is serially diluted in anaerobized peptone water in an anaerobic box to a dilution of 10' ⁶ and plated on a non-selective medium for anaerobes Blood Liver agar supplemented with 5.0% defibrinated horse blood. Cultivation is anaerobic at 37°C for 72 hours. Colonies, with typical Bifidobacterium genus morphology, were inoculated into MRS broth with L-cysteine and lithium chloride and cultivated in an anaerobic environment at a temperature of 37°C for 24 hours. For belonging to the genus Bifidobacterium, the Gram-positive cultures grown with a typical rod shape were tested by genus-specific PCR. Species affiliation and strain identity of the strain were established using precise DNA-based methods (Figure 1) such as 16S rDNA sequencing, Pulse Electrophoresis. A strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is gram-positive, catalase-negative, rod-shaped. A photomicrograph of the strain is presented in Figure 2.

Isolation of the strain from the intestinal tract of a healthy volunteer proves its GRAS (generally recognized as safe) status.

A strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is capable of significantly lowering serum cholesterol concentration. The strain exhibits two main activities related to the reduction of the cholesterol level - direct lowering of the cholesterol level, as well as its indirect reduction as a consequence of the hydrolase activity of the strain towards bile salts (BSH activity). The extent of both strain activities was determined in vitro.

The direct elimination of cholesterol is mainly due to its adsorption on components of the cell wall of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085. In the determination of this activity, residual cholesterol is measured in medium with added water-soluble cholesterol. Measurements were performed by gas chromatography and showed an average of 62.7% ± 1.6% direct cholesterol lowering. (Example 1.1)

A strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 demonstrated high hydrolase activity towards bile salts, through which it indirectly affects the serum cholesterol level. As a result of the deconjugation of bile salts, their concentration decreases, which is compensated by the synthesis of new bile acids and the conjugation of these bile acids to new bile salts. The participation of serum cholesterol in the synthesis of new bile acids reduces its concentration.

The determination of the BSH activity of a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is reverse phase liquid chromatography (RP-HPLC) with UV detection at 210 nm. The strain cultured in medium supplemented with the bile salts sodium taurocholate (TCA) and sodium glycocholate (GCA) achieved 72.0% ± 1.9% deconjugation of the bile salts present in the medium. (Example 1.2)

The ability of a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 to reduce cholesterol was also proven by the decrease in the values of lipid indicators in the clinical observation after administration of the bifidobacterial preparation obtained from a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085. (Example 3.1)

At the same time, a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 possesses anti-inflammatory immunomodulatory activity, which is manifested by the strain's ability to favorably alter the induction of pro-inflammatory and anti-inflammatory cytokines.

The anti-inflammatory immunomodulatory ability of the strain was determined in vitro by quantifying the expression of the signal peptides TNF-α, IL-8, IL-6, TGF-β and IL-10 from a combined system of two cell lines: the human monocyte cell line THP- 1 and the epithelial cell line HT-29 separated by a semipermeable membrane. (Example 1.3)

The results of the in vitro evaluation of the induction of signal peptides from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 showed a decrease in the expression of the pro-inflammatory cytokines IL-8, IL-6, TNF-α, as well as an increase in the synthesis of the anti-inflammatory cytokines TGF-β and IL-10. The ability to significantly decrease the level of interleukin IL-8 was demonstrated, from 2734 ± 149 pg/ml in the control to 112 ± 13 pg/ml in the strain evaluation, which is an essential factor in the prevention and therapy of inflammatory diseases of the intestinal tract.

A beneficial effect on indicators of inflammatory status was also found in the clinical observation with intake of the bifidobacterial preparation obtained from a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085: the levels of the pro-inflammatory cytokines TNFα, IL-Ιβ and IL-6 are decreased, and the level of the anti-inflammatory cytokine TGF-β is increased. (Example 3.3)

Simultaneously, a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 exhibits strong adhesion to epithelial cells, as well as a high level of survival in the gastrointestinal tract, which enhances the impact of the beneficial effects of the strain on the human organism.

The ability of a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 to adhere to epithelial cells was determined in vitro using the epithelial cell lines CaCo-2 and HT-29. The results show that a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 has very strong adhesion with an average number of more than 15 adhered bacterial cells per eukaryotic cell. (Example 1.4)

The survival of a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 was determined in the clinical monitoring of the administration of the bifidobacterial preparation of the strain. Faecal samples from nine volunteers, patients in the clinical observation, after 30 days of taking the bifidobacterial preparation were examined, and an average colony count of 2.2x10 ⁶ CFU/g was found. (Example 3.4)

The bifidobacterial preparation, subject of the present invention, is obtained by cultivating a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085. (Example 2)

It can be administered in liquid or dry (lyophilized) form, and the lyophilized bifidobacterial preparation can be administered both in powder and capsule form.

The liquid bifidobacterial preparation contains live cells of a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 at least 1x10 ⁹ CFU/g.

The lyophilized bifidobacterial preparation contains live cells of a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 at least 5xl0 ⁹ CFU/g. Residual moisture content is 4% ± 1%.

The bifidobacterial preparation, lyophilized or liquid, can be used as an additive to products in dry and/or liquid form. When adding it to lactic acid products, it is added in an amount of 0.5% - 2%.

The lyophilized bifidobacterial preparation can be encapsulated. Before encapsulation, an anti-caking agent, in particular microcrystalline cellulose, is added to it. Each capsule contains no less than 2x10 ⁹ live cells of a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085

On the application of the bifidobacterial preparation from the specially selected strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 was conducted in a double-blind placebo-controlled clinical trial. During clinical observation, the hypolipidemic and anti-inflammatory effect of the bifidobacterial preparation was evaluated in patients with a high atherosclerotic vascular risk or atherosclerotic disease - all of them had mild and moderate lipid disorders, overweight or obesity, more than half of them had arterial hypertension under therapeutic control, type 2 diabetes under therapy, as well as proven hepatic steatosis. (Example 3)

During the clinical observation, a very good tolerability and absence of unwanted side effects were found when taking the bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085.

The performed blood tests on a number of biochemical, metabolic and hormonal indicators prove statistically significant favorable metabolic effects on lipid metabolism after 30 days of taking the probiotic bifidobacterial preparation: a decrease in total serum cholesterol; a decrease in LDL cholesterol (cholesterol in low-density lipoproteins) and Non-HDL cholesterol, which confirmed the hypolipidemic effect of the bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085. (Example 3.1)

An important part of the conducted clinical studies are the analyzes of the levels of vascular risk markers, respectively cytokines and inflammatory factors, whose pathophysiological role is of great importance in the light of considering the metabolic syndrome and obesity as a state of low-grade inflammation leading to endothelial dysfunction.

The results of the clinical observation show a favorable change of the studied cytokines and confirm the anti-inflammatory immunomodulating effect of the bifidobacterial preparation. (Example 3.3)

At the same time, an additional effect was reported during the clinical observation, namely a statistically significant improvement in the state of insulin resistance in the patients receiving the bifidobacterial preparation. A decrease in basal glycemia and insulinemia, as well as an index of insulin resistance, was reported, establishing a new effect reducing diabetes risk and improving diabetes control. (Example 3.2)

The results of the medical research within the framework of the conducted clinical observation show the simultaneous favorable influence of three risk factors for the development of atherosclerosis, namely the lipoproteinemia, the state of insulin resistance and the inflammatory status. Thus, the clinical observation of the effects upon application of the bifidobacterial preparation from a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 confirmed the established in vitro functional characteristics of the strain, namely hypolipidemic and anti-inflammatory immunostimulatory properties, as well as establishing a new functional effect reducing diabetes risk.

Based on this, the bifidobacterial preparation of strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is recommended for successful use in medical practice in the primary and secondary prevention and therapy of atherosclerosis, cardiovascular and inflammatory diseases and diabetes risk.

EXPLANATIONS OF THE ATTACHED FIGURES

Figure 1. Pulse-electrophoretic profile of strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 c the enzyme AsnI. M and m-molecular markers. Unfolding conditions: initial pulse 2s, final pulse 15s, voltage 5V/cm, duration 24h. The molecular weights of the DNA fragments of strain Bifidobacterium longum ssp. longum LBB 3/15 NBIMCC 9085 in kbp ca: 199, 136, 112.99, 93,73,61,57,51,47,38.7, 26, 22.2, 20.5, 17, 9, 14.2, 13.5, 11.6, 9.0 ,8.5,8.0, 7.5,6.8.

Figure 2. Photomicrograph of a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085

Figure 3. Change in the level of cytokine TNFa upon intake of a bifidobacterial preparation from a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 compared to placebo group.

Figure 4. Change in the level of cytokine IL-Ιβ when taking a bifidobacterial preparation from a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 compared to placebo group.

Figure 5. Change in the level of cytokine IL-6 upon intake of a bifidobacterial preparation from a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 compared to placebo group.

Figure 6. Change in the level of cytokine TGF-βΙ upon intake of a bifidobacterial preparation from a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 compared to placebo group.

EXAMPLES OF IMPLEMENTATION OF THE INVENTION

Example 1. In vitro functional evaluation of a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085.

1.1. Assessment of direct cholesterol reduction capacity

A strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 was cultivated anaerobically at 37°C for 18 hours in MRS broth with Lcysteine, the medium containing 0.3% bile salts and 100 pg/ml water-soluble cholesterol (cholesterol-PEG600). The inoculum is 0.5% and contains 1x10 ⁹ CFU/ml. After incubation, the cells were pelleted by centrifugation and 0.5 ml of the supernatant was treated with 4 ml of 2M KOH in methanol at 80°C for 20 minutes. Extraction followed with 5 ml of hexane and 4 ml of the hexane layer was transferred to test tubes and evaporated with a stream of nitrogen at a temperature of 60°C. The dry residue was dissolved in 0.5 ml of isopropanol and the amount of cholesterol contained was determined by gas chromatography. The degree of cholesterol reduction was determined based on an uninoculated control medium.

Measurements are performed three times and the results are averaged. Table 1 shows the results of the direct adsorption of cholesterol, expressed as a percentage of cholesterol reduction.

Table 1

Strain % lowering of cholesterol Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 62.7 ± 1.6

1.2. Evaluation of bile salt hydrolase activity (BSH activity)

A strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 was cultured anaerobically at 37°C for 18 hours in MRS broth with L-cysteine, with the addition of the two most important bile salts: sodium taurocholate (TCA) and sodium glycocholate (GCA) at a concentration of 1 tM for each of them. The inoculum was 0.5% and contained 1x10 ⁹ CFU/ml, and uninoculated medium was used as a control.

Determination is reverse phase liquid chromatography (RP-HPLC) with UV detection at 210 ppm. Free and conjugated bile acids are detected in reverse-phase gradient mode. Solvent A was 65% methanol in 0.03 M sodium acetate at pH 4.3 adjusted with phosphoric acid. Solvent B is HPLC grade methanol. The eluent program is an isocratic step for 8 min at 15% eluent B and then a linear gradient to 85% in 17 min. This is followed by an isocratic step of 5 minutes at 85%. The injected amount is 5 μΐ.

Table 2 presents the results of determining the BSH activity of a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 as the percentage of hydrolysis of the bile salts present in the medium, after triplicate testing and averaging the results.

Table 2

Strain % deconjugation of bile salts Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 72.0 ± 1.9

1.3. Evaluation of anti-inflammatory immunomodulatory activity

The anti-inflammatory immunomodulatory activity was evaluated by quantifying the expression of pro-inflammatory and anti-inflammatory signal peptides from a combined system of two cell lines: the human monocyte cell line THP-1 and the epithelial cell line HT-29, separated by a semipermeable membrane. The neat system of the two cell lines proved suitable in assessing the induction of both the pro-inflammatory cytokines TNF-α, IL-8 and IL-6, and in determining the induction of the anti-inflammatory cytokines IL-10 and TGF-β. Both cell lines were maintained at 37°C and 5% CO on RPMI 1640 medium for THP-1 cell line and DMEM medium for HT-29 cell line. Both media were supplemented with 10% fetal bovine serum, 4.5 g/μl glucose, 10 μM HEPES, 1 mM sodium pyruvate, 2 μM L-glutamine, 1.5 g/μl sodium bicarbonate, 100 U/ml penicillin, 100 μg/ml streptomycin. In a 48-well plate for cell lines, in the lower wells, 1 x 10 ⁶ per milliliter THP-1 cells (5 x 10 ³ per well) were differentiated into macrophages using 5 pg/ml PMA (phorbol 12-myristate13-acetate) added to the growth medium, for 24 hours in a COg incubator. Epithelial HT-29 cells were grown as a monolayer in semipermeable membrane inserts and matured for 14 days with daily DMEM medium changes. Differentiated adherent macrophages were washed twice with PBS buffer (Dulbecco's phosphate-buffered saline) and incubated with fresh medium without PMA for 24 hours. Salmonella lipopolysaccharide was added at a concentration of 1 pg/ml in RPMI 1640 medium and incubation continued for an additional 4 hours. After aspirating the medium, 0.5 ml per well of bacterial cells of strain Bifidobacterium longum ssp were added to the differentiated macrophages. longum 3/15 NBIMCC 9085 c concentration lx10 ⁶ CFU/ml suspended in RPMI 1640 medium. Incubate for 24 hours in a CO2 incubator.

The evaluation of the induction of the signal peptides TNF-α, IL-8, IL-6, TGF-β and IL-10 by the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 was performed by sandwich ELISA. After the end of the incubation, the supernatant was collected and centrifuged to eliminate the bacterial and human cells. For determining the signal peptides, 100 µl of the obtained supernatants are used. Assessment of the induction of each cytokine was performed in triplicate. Table 3 shows the average results of the triplicate induction test for the five cytokines.

Table 3

Control / Strain IL-10 pg/ml TNF-α pg/ml TGF-β pg/ml IL-6 pg/ml IL-8 pg/ml control (the neat system of 121 477 1242 2238 2734 TNR-1 and HT-29) ±14 ±31 ±79 ±135 ±149 Bifidobacterium longum ssp. 237 281 1655 1846 112 longum 3/15 NBIMCC 9085 ±25 ±23 ±84 ±117 ±13

1.4. Assessment of adhesion to epithelial cells

To determine the adhesion of a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 for epithelial cells, the epithelial cell lines CaCo-2 and HT-29 were used, which were cultured as a monolayer of mature epithelial cells in DMEM (Dilbecco's modified Eagle's medium) medium supplemented with 10% fetal bovine serum. Incubation is at 37°C in a water-saturated atmosphere containing 5% CO2. At about 90% established monolayer, cells were passaged by incubation with 0.25% trypsin and 10 mM EDTA solution at 37°C for 10 minutes. Eukaryotic cells were seeded in 6-well clusters at a concentration of 2x10 ⁵ cells/ml. The medium was changed every 2 days for a total of 14 days. In this period, the monolayer is built (for about 3-4 days) and cell receptors mature. The monolayers were then washed twice with PBS buffer and the bacterial culture of strain Bifidobacterium longum ssp was added to them. longum 3/15 NBIMCC 9085 c concentration IxlO ⁸ CFU/ml in 2 ml DMEM without antibiotics. Bacterial and eukaryotic cells were co-incubated for 60 minutes at 37°C in a water-saturated atmosphere containing 5% CO2. This was followed by washing the wells five times with PBS, fixing with methanol for 3 minutes, Gram staining and microscopic evaluation of adhesion. The number of adherent bacterial cells per 20 epithelial cells was determined by counting 10 microscopic fields. The adhesion assay was performed in triplicate on both Saso-2 and HT-29 cell lines, and the results were averaged.

The results of determining the adhesion of a strain of Bifidobacterium longum ssp. longum 3/15, NBIMCC 9085, expressed as the average number of adherent bacterial cells per eukaryotic cell, are presented in Table 4.

Table 4

Strain average number of adhered bacteria on a Saso-2 eukaryotic cell average number of adhered bacteria on an HT-29 eukaryotic cell Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 16 19

Example 2. Preparation of bifidobacterial preparation

The bifidobacterial preparation is obtained by cultivating a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085.

To obtain mother yeast, the strain is cultivated in an MRS broth medium containing lithium chloride and L-cysteine in anaerobic conditions at a temperature of 37°C for 18-24 h. With the resulting mother culture, containing a biomass of about 1x10 ⁹ CFU/ml, a specially prepared medium was inoculated in an industrial fermenter to carry out the fermentation process of a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085. The medium included 8% dry milk, 2% dry whey, 2% lactose, 0.5% yeast extract, 0.5% peptone casein and 2% fructooligosaccharide. Adjust the pH of the medium to pH 6.8 and sterilize in a fermenter at 120 °C for 15 minutes. Cool to 37°C and inoculate with 5% mother culture. Fermentation is carried out at a temperature of 37°C for 14-16 hours with continuous blowing with carbon dioxide. After reaching pH 4.9-5.0, the fermentation process is stopped by cooling with ice water and subsequently adjusting the pH to pH 5.8-6.2.

The resulting biomass is a liquid bifidobacterial preparation containing no less than 1x10 ⁹ CFU/g live cells of a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085.

To obtain a lyophilized bifidobacterial preparation, the cryoprotectant medium with 10% sucrose and 0.5% pectin is added to the fermented medium in a ratio of 1:10 and dosed in an industrial lyophilizer.

Sublimation drying takes place at a temperature of minus 28°C to minus 30°C with a layer thickness of no more than 1.2 cm. Secondary drying is at a temperature of 30°C. After the process is completed, the vacuum in the sublimation chamber is compensated with nitrogen. The lyophilized bifidobacterial preparation from strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is packaged under an inert (nitrogen) atmosphere. The finished product contains at least 5x10 ⁹ CFU/g live cells of a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 and its water content is 4% ± 1%.

The lyophilized bifidobacterial preparation can be encapsulated on an encapsulation machine by first adding an anti-caking agent such as microcrystalline cellulose. Each capsule contains not less than 2x10 ⁹ CFU/g live cells of a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085.

Example 3. Clinical observation of the effects upon application of a bifidobacterial preparation from a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 as a probiotic

The aim of the clinical observation is to evaluate the hypolipidemic and anti-inflammatory immunomodulatory effect of the bifidobacterial preparation in patients with dyslipoproteinemia and high atherosclerotic vascular risk or atherosclerotic disease, and to develop recommendations for its use in primary and secondary prevention of cardiovascular diseases.

The clinical studies were performed in the conditions of a double-blind, placebo-controlled clinical observation in a group of 60 patients, 42 women and 18 men, with mild to moderate dyslipoproteinemia, respectively with elevated cholesterol, low-density lipoproteins and/or triglycerides. During the clinical observation, the patients did not take hypolipidemic drugs or other nutritional supplements having metabolic effects. The age of the patients is between 18 and 75 years. They were divided into two groups, a group of 30 patients who took the probiotic bifidobacterial preparation, tentatively called the "probiotic group," and a group of 30 patients who took a placebo product, tentatively called the "placebo group."

The clinically observed product is a bifidobacterial preparation from the probiotic strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085, c amount of live cells not less than 5x10 ⁹ CFU/g. The bifidobacterial preparation is taken in the form of capsules, each of which contains no less than 2x10 ⁹ live cells of the probiotic strain.

The placebo product is a capsule that contains dry milk without bacterial cells of the probiotic strain Bifidobacterium, longum ssp. longum 3/15 NBIMCC 9085.

All patients took 1 capsule 3 times a day, respectively, with a bifidobacterial preparation or a placebo product, for 30 days as an adjunct to a recommended hypolipidemic dietary regimen under the conditions of a double-blind placebo-controlled observation. All patients underwent a clinical examination before and after completion of observation. Twice, before and after the clinical observation, fasting venous blood was taken to study hematological, biochemical, metabolic, including lipid and hormonal parameters, as well as specific markers for vascular risk assessment, incl. cytokines. The observed and evaluated parameters were complete lipid profile, serum levels of markers of vascular inflammation (Reactive protein, cytokines TNFα, IL-Ιβ, IL-6, TGF-β), as well as fasting serum insulin level and insulin resistance by HOMA- IR index.

The obtained results were processed statistically with the methods of descriptive statistics, Wilcoxon Signed Ranks Test and parametric T-test.

Analysis of the patient groups showed a similar gender and age distribution. Patients in the "probiotic group" were 23 women and 7 men, aged 21 to 70 years, with a mean age of 54.37 ± 11.91 years. Patients in the "placebo group" were 19 women and 11 men, aged 22 up to 75 years, with a mean age of 49.60 ± 13.79 years. All patients had mild and moderate lipid disorders, increased levels of total and LDL cholesterol, decreased HDL cholesterol, increased triglycerides. All had metabolic syndrome, overweight or obesity, two-thirds had arterial hypertension under therapeutic control, 35 patients had type 2 diabetes under therapy, and 29 patients had proven hepatic steatosis.

All patients tolerated the administered capsules very well and none of them reported adverse effects. During clinical monitoring, no adverse side effects were detected.

3.1. Assessment of lipid indicators

Table 5 shows the dynamics of the changes in the lipid parameters of the patients in the "probiotic group".

Table 5

Indicator Before clinical observation After clinical observation p * Total cholesterol, mM/L 5.803 ±0.941 5.587 ± 0.973 <0.05 LDL cholesterol, mM/L 3.429 ±0.810 3,203 ± 1,000 <0.05 HDL cholesterol, mM/L 1.426 ±0.306 1.380 ±0.318 n.s. VLDL cholesterol, mM/L 0.950 ± 0.286 0.968 ± 0.284 n.s. Non HDL cholesterol, mM/L 4.375 ± 0.882 4.206 ± 0.923 <0.05 Triglycerides, mM/L 1.560 ±0.653 1.600 ±0.672 n.s.

* statistical reliability

Table 6 shows the dynamics of the changes in the lipid parameters of the patients in the "placebo group".

Table 6

Indicator Before clinical observation After clinical observation R Total cholesterol, mM/L 5.442 ± 0.900 5.543 ±0.834 <0.05 LDL cholesterol, mM/L 3.080 ±0.710 3.238 ±0.651 <0.05 HDL cholesterol, mM/L 1.316 ±0.362 1.297 ±0.347 n.s. VLDL cholesterol, mM/L 1.010 ±0.495 1.177 ±0.465 <0.05 Non HDL cholesterol, mM/L 4.076 ± 1.175 4.066 ± 1.012 n.s. Triglycerides, mM/L 2.102 ±0.653 1.900 ± 1.001 n.s.

The summarized results of the study of lipid indicators show that in the "probiotic group", compared to the "placebo group", a significant favorable reduction of total cholesterol by 5.57%, of LDL cholesterol by 11.71%, and of Non HDL cholesterol by 3.86%. VLDL cholesterol in the "placebo group" increased by 0.167 mM/L, while in the "probiotic group" the increase was only 0.018 mM/L. The average levels of HDL cholesterol and triglycerides before the clinical observation were in target levels and did not suffer a significant change after one month of taking the bifidobacterial preparation.

The analysis of individual changes in the lipid parameters of the patients in the "probiotic group" showed that more than half of them had favorable changes - total cholesterol decreased in 53.33% of them, LDL cholesterol decreased in 50% of patients, VLDL cholesterol decreased in 56.66% of patients and in 26.66% of patients HDL cholesterol increased.

3.2. Assessment of other biochemical and metabolic indicators

Table 7 presents the results of other investigated biochemical and metabolic indicators in the patients in the "probiotic group".

Table 7

Indicator Before clinical observation After clinical observation p * Uric acid, μΜ/L 349.759 ± 114.510 337.690 ± 99.549 <0.05 Glucose, mM/L 6.24 ±3.15 5.84 ± 1.78 <0.05 Insulin, mE/L 14.04 ±8.38 12.66 ±6.81 <0.05 HOMA-IR 3.67 ± 2.47 3.43 ± 2.03 <0.05 AC AT, mE/L 18.69 ±4.52 19.06 ±6.38 n.s. TOOL, mE/L 26.34 ± 14.24 28.58 ± 15.18 n.s. GGTP, mE/L 33.25 ± 16.48 33.41 ± 15.94 n.s. CRP, mg/dL 0.33 ± 0.39 0.35 ± 0.41 n.s.

* statistical reliability

Table 8 presents the results of other investigated biochemical and metabolic indicators in patients in the "placebo group".

Table 8

Indicator Before clinical observation After clinical observation p * Uric acid, μΜ/L 342.929 ± 90.25 338.864 ± 119.75 n.s. Glucose, mM/L 5.540 ± 1.45 5.52 ±0.98 n.s. Insulin, mE/L 16.180 ± 17.59 13.06 ±8.36 n.s. HOMA-IR 4.07 ± 4.42 4.44 ± 5.43 n.s. ASAT, mE/L 19.89 ± 6.42 18.89 ± 5.18 n.s. TOOL, mE/L 27.25 ± 16.75 28.78 ± 14.60 n.s. GGTP, mE/L 35.18 ± 15.39 33.66 ± 14.47 n.s. CRP, mg/dL 0.35 ±0.38 0.43 ±0.431 n.s.

* statistical reliability

The analysis shows that in the "probiotic group" a statistically significant reduction in the state of insulin resistance accompanying the metabolic syndrome was achieved, with blood glucose decreasing by 6.41%, basal insulinemia by 9.81% and HOMA-IR index by 6.53 %, while the changes of these indicators in the "placebo group" are not statistically significant. These results add a very significant beneficial effect of the application of the bifidobacterial preparation regarding the improvement of the state of insulin resistance. The changes in serum uric acid were similar, with its values falling significantly by 3.45% in the "probiotic group" patients, while the "placebo group" patients had insignificant changes in the uric acid level. The monitored levels of serum transaminases, GGTP and CRP were within reference values in both groups and remained without significant changes.

The levels of C-reactive protein (CRP) at baseline in both groups were within reference limits and remained within such limits, not undergoing significant change after 30 days of administration of the bifidobacterial preparation and the placebo product, (Table 7 and Table 8)

3.3. Evaluation of the levels of cytokines and inflammatory factors

The results of the studied cytokines are presented in figure 3, figure 4, figure 5 and figure 6. The achieved changes in their levels were statistically significant for the "probiotic group", and after 30 days of taking the bifidobacterial preparation, a decrease in the level of pro-inflammatory cytokines TNFa was found by 6.56% (Figure 3), IL-Ιβ by 3.34% (Figure 4) and IL-6 by 7.60% (Figure 5), as well as an increase in the anti-inflammatory cytokine TGF-βi by 14.87% (Figure 6). These data point to a beneficial effect of the bifidobacterial preparation on the proinflammatory status in metabolic syndrome by modulating the cytokine profile.

The results of the conducted clinical trial categorically confirm the anti-inflammatory immunomodulatory effect of the bifidobacterial preparation - a decrease in TNFa, IL-Ιβ, IL-6 and a moderate increase in ΤϋΡβΙ.

3.4. Evaluation of the survival of a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 in the gastrointestinal tract

After the clinical observation, fecal samples were taken from 9 volunteers, patients of the "probiotic group", and were processed according to the described procedure for the isolation of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085. Colonies of the genus Bifidobacterium in faecal samples that show the strain-specific DNA profile of a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 obtained by the Pulse Electrophoresis method (Figure 1) averaged 2.2x10 ⁶ CFU/g.

The results presented in Table 9 show a high level of survival of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 in the gastrointestinal tract.

Table 9

Strain mean number of colonies in faecal samples of volunteers, CFU/g, n=9 minimum number of colonies in faecal samples of volunteers, CFU/g, n=9 maximum number of colonies in faecal samples of volunteers, CFU/g, n=9 Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 2.2x10 ⁶ 6.8x10 ⁵ 6.4x10 ⁶

APPLICATION (USE) OF THE INVENTION

A strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 and the bifidobacterial preparation from strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 have a unique combination of probiotic properties, suitable for the prevention of atherosclerosis and cardiovascular diseases, inflammatory diseases, as well as for reducing the risk of diabetes.

REFERENCES

1. Paineau D, Carcano D, Leyer G, Darquy S, Alyanakian M, Simoneau G,

Bergmann J, Brassart D, Bomet F & Ouwehand A (2008) Effects of seven potential probiotic strains on specific immune responses in healthy adults: a double-blind, randomized, controlled trial. FEMS Immunol Med Microbiol 53: 107-113

2. Hedin, M. Mullard, E. Sharratt et al., (2010). Probiotic and prebiotic use in patients with inflammatory bowel disease: a case-control study. Inflammatory Bowel Diseases, vol. 16, no. 12, pp. 2099-2108

3. Papadopoulos, J., Karpouzis, A., Tentes, J., Kouskoukis, C. (2014). Assessment of Interleukins IL-4, IL-6, IL-8, IL-10 in Acute Urticaria. J Clin Med Res. 6(2): 133-137

4. Belkaid Y, and Hand T. (2014) Role of the microbiota in immunity and inflammation. Cell, vol. 157, no. 1, pp. 121-141

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6. Patent KR102224072B1. Bifidobacterium longum subsp. longum having both abilities of reducing total cholesterol in serum and immune regulation and its application.

Claims (10)

1. Strain Bifidobacterium longum ssp. longum 3/15 deposited in NBPMCC under number NBIMCC 9085 with a strain-specific DNA profile with molecular masses of its DNA fragments in kbp: 199, 136, 112, 99, 93, 73, 61, 57, 51, 47, 38.7, 26, 22.2, 20.5, 17, 9, 14.2, 13.5, 11.6, 9.0, 8.5, 8.0, 7.5, 6.8. 2. Strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085, according to claim 1, characterized in that it has simultaneously hypolipidemic activity by direct reduction of cholesterol and by hydrolysis of bile salts, anti-inflammatory immunomodulatory activity, ability to reduce insulin resistance, high adhesion to epithelial cells and high survival rate in the gastrointestinal tract. 3. Bifidobacterial preparation, characterized in that it contains a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 c live cell count at least 1x10 ⁹ CFU/g. 4. Bifidobacterial preparation, according to claim 3, characterized in that it simultaneously exhibits a hypolipidemic effect, an anti-inflammatory immunomodulatory effect and an insulin resistance-reducing effect. 5. Bifidobacterial preparation, according to claim 3, characterized in that it is in the form of a lyophilizate or a liquid culture. 6. Use of the bifidobacterial preparation, according to Claim 3, as a probiotic. 7. Use of a bifidobacterial preparation, according to Claim 6, as a food additive and/or in any form in food for humans and animals. 8. Use of the bifidobacterial preparation, according to Claim 6, as an additive to a starter culture for food production. 9. Use of the bifidobacterial preparation, according to Claim 6, in the composition of functional products and/or pharmaceutical preparations, intended to influence health indicators of humans or animals. 10. Bifidobacterial preparation, according to Claim 3, for use for the prevention and/or therapy of inflammatory diseases and/or atherosclerosis and/or cardiovascular diseases and/or for reducing the diabetes risk.