BG112471A - Immunomodulating synbiotic composition - Google Patents

Abstract

The invention relates to an immunomodulating synbiotic composition, in particular a probiotic bacterial composition, further enriched with a prebiotic and biologically active substances that have a beneficial effect on human health in the prevention and/or therapy of inflammatory and immune diseases in gastrointestinal tract, including symptoms of intestinal dysbiosis and inflammatory bowel syndrome. In particular, the composition comprises the probiotic component of living cells of a biologically pure culture of a bacterial strain selected from the group of Lactobacillus delbrueckii subsp. bulgaricus LBB-06-НБПМК 8918, Lactobacillus helveticus LLH-108-НБПМК 8760, Lactobacillus acidophilus LLA-10-НБПМК 8242, Lactobacillus rhamnosus LLR-L1-НБПМК 8769 и Lactobacillus fermentum LLF-01-НБПМК 8781 or combinations thereof, prebiotic-arabinogalactan and colostrum. The used lactic acid bacteria strains are selected so that in combination with each other and in combination with the other components - the prebiotic and the colostrum, they give high survival, achieve the specific probiotic effect and the guaranteed antimicrobial activity against human pathogens, as adhesive and immunomodulating properties of the composition. The indigestible dietary fiber of arabinogalactan, thanks to its immunomodulating (antivirus and antitumor) activity, combined in the synbiotic formula, form a protective layer around probiotics and control their release into the intestinal tract. The enriched multifunctional formula of the synbiotic composition according to the invention, including proline polypeptides rich (4.5%) and IgG (minimum 20%) colostrum content, supplies the necessary immune, growth factors and a balanced combination of vitamins and minerals. The orally administered synbiotic composition demonstrates an immunomodulating effect, shown in an increase in % of activated NK (natural killer) cells in the peripheral blood and a decrease in the levels of anti-inflammatory cytokines in fecal samples, and in patients with irritable bowel syndrome (IBS) in reduction of faecal calprotectin to a reduction or even disappearance of clinical symptomatology.

Description

IMMUNOMODULATING SYNBIOTIC COMPOSITION

FIELD OF THE INVENTION

The invention relates to an immunomodulatory synbiotic composition, in particular a probiotic bacterial composition, further enriched with prebiotic and biologically active substances, beneficial to human health in the prevention and / or treatment of inflammatory and immune diseases of the gastrointestinal tract, including symptoms of gastrointestinal tract. dysbiosis and inflammatory bowel syndrome.

BACKGROUND OF THE INVENTION

The immune system is a complex multicomponent system consisting of several subsystems that protect the host's body from infectious agents and tumor cells. The immune system controls and is responsible for attacking pathogens, recognizing them and removing them from the body. By maintaining the acquired immunity, the immune system constantly increases its immune response in terms of the possible rate of destruction of the foreign antigen in the body.

On the other hand, the gastrointestinal tract contains a rich flora of over 500 different types of bacteria important for its health. When the normal state of health changes, some bacteria can overgrow and cause infections and weaken the immune system. Weakening and imbalance of the natural defense system can allow potentially pathogenic microorganisms to cause clinical infections. Maintaining the balance of the gastrointestinal tract is of particular importance to man and he is responsible for the proper functioning and nutrition of every cell in the body. Soft acid bacteria are a tool for enriching the gastrointestinal tract with beneficial bacteria.

Lactic acid bacteria, in recent years, have been used to prevent or accompany the treatment of immune diseases. It is known from WO 2015/046407 a prophylactic agent in the form of a nutritional composition, and as a medicament containing lactic acid bacteria, in particular strains of Lactobacillus helveticus, intended for the prevention of immune diseases by inhibiting the proliferation of IL-10 / IL-12 immune growth cells. (associated with enteritis, rheumatoid arthritis, immune diseases such as hepatitis).

Also known from EP2482826 is a composition comprising arabinogalactan intended to support the adaptive immune response against foreign antigens following vaccination and administered in the form of functional compositions or dietary supplements.

It is also known from RU 2 599 544 and a dry milk-based tablet composition for oral and / or oral administration, containing skimmed milk powder and / or whey and / or dry lactose of 20-50%; sweetening agents of 10-30%; dried fruit fillers of 520%; lyphilized probiotic cultures selected from Propionibacterium freudenreichii subsp. shermanii and / or Bifidobacterium adolesescentis and / or Lactobacillus acidophilus and / or Lactobacillus casei, and / or Lactobacillus rhamnosus a total of 0-20%; biologically active substances (BAB) including arabinogalactan and vitamin complex of 0-5%; 020% oligosaccharides and 0-5% structurants.

From BG 65 614 a synbiotic is known, which is a mixture of monocultures of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Bifidobacterium bifidum, Bifidobacterium longum and inulin and optionally bee pollen or royal jelly in the amounts described.

It is also known from CZ 28707 and a dairy probiotic fermented product with a positive effect on the rheology of the product, containing 1-10% colostrum, 1-2% probiotic culture of Bifidobacterium animalis subsp. lactis, Enterococcus faecium and L. acidophilus, 0, 01-4% yogurt or cream-kefir culture, 1-7% prebiotic selected from fructooligosaccharide or inulin and up to 100% goat / cow milk.

A disadvantage of the described dairy probiotic product is its insufficient biological value of the selected probiotic cultures, as well as their insufficient survival when entering the gastrointestinal tract.

The problem to be solved is to expand the range of functional dairy products, but also to create a fundamentally new form of product with immunomodulatory action, which will enrich and balance the gastrointestinal tract with lactic acid bacteria with specific probiotic action and their products from their vital activity, as well as to provide conditions for development, multiplication and increase the effect of the probiotic.

• :: ’· ·; · / ·· · ··· ♦ ·

SUMMARY OF THE INVENTION

These problems are solved according to the invention with an immunomodulatory synbiotic composition-active formula based on a probiotic containing live cells of a biologically pure culture of a bacterial strain selected from the group of: Lactobacillus delbrueckii subsp. bulgaricus LLB -06-NBIMCC 8918, Lactobacillus helveticus LLH-108- NBPMK 8760, Lactobacillus acidophilus LLA-10- NBPMK 8242, Lactobacillus rhamnosus LLR-L1- NBPMK 8769 and

Lactobacillus fermentum LLF-01- NBPMK 8781 or their combinations, as well as a prebiotic in the form of indigestible dietary fiber isolated from the bark of Siberian larch polysaccharide arabinogalactan (Arabinogalactans from Larix gmelinii) and primary milk from lactating women. , rich in proline-containing peptides with immunomodulatory effect (min. 4.5% PRPs) and IgG (min. 20%), where the amount in mg of the components is respectively:

Lactobacillus delbrueckii subsp. bulgaricus LLB -06-NBIMCC 8918 from 30 to 120 mg;

Lactobacillus helveticus LLH-108- NBPMK 8760- from 55 to 220, Lactobacillus acidophilus LLA-10- NBPMK 8242- from 55 to 220, Lactobacillus rhamnosus LLR-L1- NBPMK 8769- from 55 to 220, Lactobacillus fermentum LF1LF1LF - from 55 to 220, Arabinogalactan - from 100 to 400 and

Colostrum - from 50 to 200.

Each bacterial strain of the probiotic of the composition has activity in the range from 10 ⁸ to 10 ¹⁰ CFU bacteria / g.

The newly isolated Lactobacillus delbrueckii subsp. bulgaricus LLB-06- NBIMCC 8918 is a milk isolate, in the form of Gram / + /, catalase-negative medium to long rods, aerotolerant, with intensive growth at 45 ° C but not at 15 ° C, does not produce CO 2 from glucose, forms metachronatin granules depending on the age of the culture, with intensive growth of lactic medium with the formation of exclusively D (-) lactic acid during 24-hour fermentation. The culture does not support good growth of MRS arap. The other newly isolated strain of Lactobacillus fermentum LLF-01, isolated from the saliva of a healthy person, is Gram / + /, catalase-negative rods with pronounced polymorphism, aerotolerant, with intense growth at 37 ° C, and weaker at 15 ° C, produces CO ₂ of glucose, with intensive growth of lactic medium with formation of 40% D (-) and 60% L (+) lactic acid during 24-hour fermentation. The culture supports good growth of MRS arap.

carbohydrate metabolism of strains:

Name monoculture on Ferments Does not ferment Lactobacillus delbrueckii subsp. bulgaricus LLB-06 glucose, fructose, mannose, lactose glycerol, erythrol, arabinose, ribose, xylose, adonitol, β methyl-xyloside, galactose, sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, and methyl-D-mannoside, and methyl-O-glucosine , esculin, salicin, cellobiose, maltose, melibiose, sucrose, trehalose, inulin, melisitose, raffinose, amidone, glycogen, xylitol, β gentibiose, turanose, lyxose, tagatose, fucose, arabitol, gluconate Lactobacillus fermentum LLF-01 ribose, galactose, glucose, fructose, mannose, lactose, melibiose, sucrose, raffinose, gluconate glycerol, erythrol, arabinose, xylose, adonitol, β methyl-xyloside, sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, and methyl-O-mannoside, and methyl-D-glucoside, за arcinylbutyline, acetylglycin , cellobiose, maltose, trehalose, inulin, melisitose, amidone, glycogen, xylitol, β gentobiose, turanose, lyxose, tagatose, fucose, arabitol, keto gluconate _________

The remaining monocultures of Lactobacillus helveticus strain LLH-108-NBIMCC 8760, Lactobacillus acidophilus strain LLA-10-NBIMCC 8242 and Lactobacillus rhamnosus LLR-L1-NBIMCC 8769, respectively, are known and participate in other formulations developed.

The strains have technological stability with proven good survival during lyophilization, as well as high viability during 0 storage, which in turn ensures the proper course of the digestive processes and maintaining the balance of intestinal peristalsis.

In order to achieve a product with the target properties, the strains were selected by the criterion: - antimicrobial activity, - survival in simulated intestinal juice, - in-vitro adhesion to a human cell line and - immunomodulatory activity. The strains are not 5 genetically modified and have no proven transmissible antibiotic resistance.

The process for preparing the synbiotic composition of the present invention consists of the following. Each of the five types of bacteria is cultured separately in the appropriate: ^::: 5: · *. · · · * ·· * ···· * ♦ · for the type of nutrient medium based on milk or broth medium, followed by lyophilization and digestion. Preferred ingredients for such an environment are skim milk, yeast extract, casein peptone, meat extract, D-glucose. To the medium are added salts such as sulfate (magnesium and manganese) and buffers such as sodium acetate, sodium phosphate, potassium phosphate. The optimal skimmed milk content varies between 1218% (w / v). The amount of yeast extract varies from 0.06% (w / v) to 0.5%. Optionally, the addition of dry (MRS) medium in amounts of 0.1-1.0% (w / v) also has a beneficial effect on the biological activity of lactobacilli.

Preferred medium ingredients with added dry MRS include D-glucose, casein peptone, meat extract and yeast extract, as well as Tween-80, diammonium hydrogen citrate, CH3COOA, MgSO ₄ and MnSO ₄ . To the basic medium is added a buffer-activating mixture, representing an aqueous solution of CH3C001Ma, KN2PO ₄ , Na2HPO ₄ , MgSO ₄ , MnSO ₄ and the yeast extract is in an amount of 0.2% (v / v) to 2.0%, more especially 0.5% (v / v). The components of the medium thus described and selected are mixed and dissolved in water.

After complete dissolution and homogenization of the ingredients used, the medium is adjusted to pH 6.6-6.8 and sterilized at 107 ° C for 3 minutes. Before inoculation, the culture medium is cooled to 38 ° C. The amount of inoculating culture varies from 1 to 5% (v / v) of the total amount of culture medium. The fermentation is carried out at a temperature of 37 ° C for 16-24 hours. The resulting active culture at the end of the fermentation process is mixed with a protecting medium containing 10% skimmed milk powder and 10% sucrose and lyophilized. The resulting dry culture was ground and stored at -20 ° C until mixing. The resulting mixture is homogenized and subjected to encapsulation, dosing or tabletting, respectively. To prepare the tablets, the resulting bacterial mixture is sieved, sieved and the other ingredients and excipients are successively added. Take 2 capsules / tablets or 1 sachet per dose once or twice a day. The use can be unlimited. Store in a cool, dry place.

According to another aspect, the present invention relates to a probiotic bacterial composition with immunomodulatory action, comprising an effective amount of live cells of a biologically pure culture of a bacterial strain selected from the group of; Lactobacillus delbrueckii subsp. bulgaricus LLB -06- NBPMK 8918, Lactobacillus helveticus LLH-108- NBPMK 8760, Lactobacillus acidophilus LLA-10- N NBPMK 8242,:: * ··; 6 ·::,: ·

Lactobacillus rhamnosus LLR-L1- NBPMK 8769,

Lactobacillus fermentum LLF-01-NBPMK 8781, or combinations thereof, at the amounts described in mg according to the present invention.

According to another further aspect, the present invention relates to a bacterial strain of Lactobacillus fermentum LLF-01 registered in the NBPMK under № 8781 of 21.05.2013 and a strain of Lactobacillus delbrueckii subsp. bulgaricus LLB -06, registered in NBPMK under № 8918 from 01.02.2016 producers of bacterial composition with immunomodulatory action.

According to another further aspect, the present invention relates to the use of an immunomodulatory synbiotic bacterial composition, a probiotic bacterial composition, a strain of Lactobacillus fermentum LLF-01 NBPMK № 8781 and a strain of Lactobacillus delbrueckii subsp.bulgaricus LLB, yielding -06 / 89B in the form of a food supplement and / or a pharmaceutical product intended for the prevention and / or concomitant therapy of inflammatory and immune diseases of the gastrointestinal tract (immunomodulator at the level of mucosal immune system and innate immune system), as well as for normalizing and balancing gastrointestinal function including after prolonged use of antibiotics, in symptoms of intestinal dysbiosis and inflammatory bowel syndrome.

The advantages of the invention are based on the properties of the used strains of lactobacilli (including independently of the newly isolated), selected so that in combination with each other and in combination with the other components - prebiotics and colostrum, to give high survival, to achieve specific probiotic effect and guaranteed antimicrobial activity against human pathogens, adhesive and immunomodulatory properties. The multibacterial formula combines strains in a unique way with: - a wide range of antimicrobial activity, making them natural antagonists of potential pathogenic bacteria; - good transit tolerance, allowing the strains to survive the passage through the different sections of the gastrointestinal tract and to reach vital and active to their target site of action in the lower parts of the small intestine; and - high adhesion ability, ensuring permanent colonization of the intestinal tract and interaction of microorganisms with the mucous surface, as well as providing their immunomodulatory activity.

The indigestible dietary plant fibers from arabinogalactan, due to their immunomodulatory (antiviral and antitumor) activity combined in the synbiotic formula form a protective layer around the probiotics and control their:: ' ⁷ ·: ·:: * release in the intestinal tract. The enriched multifunctional formula of the synbiotic composition according to the invention, comprising proline-rich polypeptides (4.5%) and an content of IgG (minimum 20%) colostrum, provides the necessary immune, growth factors and a balanced combination of vitamins and minerals.

The resulting composition according to the present invention has a high activity in the range of 10 ⁸ to 10 ¹⁰ CFU bacteria / g of the composition and a shelf life of the finished product up to 24 months.

The orally synbiotic composition administered to healthy people demonstrated an immunomodulatory effect by increasing the% of activated (natural killer) NK cells in the peripheral blood and lowering the levels of anti-inflammatory cytokines (IL-6 and IFN-γ) in faecal samples, and in 10 patients with the syndrome. of irritable bowel syndrome (IBS), reduces the inflammatory activity, expressed in a decrease in fecal calprotectin and causes a reduction or even disappearance of clinical symptoms.

BRIEF DESCRIPTION OF THE FIGURES

15 FIG. 1: Survival of lactobacilli strains in simulated intestinal juice; FIG. 2: Adhesion ability of lactobacilli strains to human colorectal adenocarcinoma cell line HT-29 (AASS NTV 38); FIG. 3: Immunomodulatory activity of monocultures on TNF-α derived from Balb / c mice; 20 FIG. 4: Immunomodulatory activity of monocultures on 1L-6 derived from Balb / c mice; c FIGURE 5: Immunomodulatory activity of monocultures on IL-10 derived from Balb / c mice; FIG. 6: Immunomodulatory activity with respect to the secretion of TNF-α, IL-6 and IL-10, 25 Balb / c mice of probiotic composition and synbiotic composition; FIGURE 7: Faecal calpotrotectin (PCP) of the synbiotic composition in patients with irritable bowel syndrome (IBS).

EXAMPLES OF THE INVENTION

EXAMPLE 1.

A / Composition of the active probiotic-based formula containing the active monoculture of the lactobacillus strain Lactobacillus delbrueckii subsp. bulgaricus in mg.

·· · ···· · ·

Component / Example 1. A.l. 1.A.2 1.A.Z

Active bacterial mixture Lactobacillus delbrueckii subsp. bulgaricus LLB-06 30 60 120

B / Composition of the active formula based on a probiotic containing the active monoculture of the lactobacillus strain Lactobacillus fermentum in mg.

Component / example 1. B.1. 1.В.2 1.В.З

Active bacterial mixture Lactobacillus fermentum LLF-01 55 110 220

C / Composition of the probiotic-based active formula containing the five active monocultures.

The active bacterial mixture in one dose contains the following components: dry lyophilisate active monocultures of five strains of lactobacilli in mg as follows:

Component / example Active bacterial mixture l.C.l. 1.C.2 1.C.3 1.C.4 Lactobacillus delbrueckii subsp. 30 60 75 120 bulgaricus LLB -06- NBPMK 8918 Lactobacillus helveticus LLH-108- NBPMK 8760 55 110 137.5 220 Lactobacillus acidophilus LLA-10- NBPMK 8242 55 110 137.5 220 Lactobacillus rhamnosus LLR-L1- NBPMK 8769 55 110 137.5 220 Lactobacillus fermentum LLF-01- NBPMK 8781 55 110 137.5 220 Total / mg 250 500 625 1000

Each of the five monocultures of lactobacilli is cultured separately, for which purpose the necessary nutrient media specific to the given strain and species are prepared, after which they are lyophilized and digested.

The cultivation of each monoculture takes place in a pre-prepared liquid nutrient medium (culture medium), to which the main components stimulating the fermentation process of the respective monoculture are dissolved and resuspended.

Obtaining a pure culture of Lactobacillus delbrueckii subsp. bulgaricus LLB -06- NBPMK 8918 and Lactobacillus helveticus LLH-108- NBPMK 8760.

100 l of reconstituted skimmed milk powder (14% dry matter) with the addition of 0.1% yeast extract and 2.0 l of buffer activating mixture representing an aqueous solution of CH3COOA, KN2PO4, Na ₂ HPO ₄ , MgSO ₄ , MnSO ₄ and the yeast extract were sterilized at 107 ° C for 3 minutes. The medium is cooled to 37 ° C and inoculated with 4 l of refreshed laboratory culture. Incubate in a fermenter at 37 ° C for 16 hours at pH 5.6 - 5.7 in automatic mode. The resulting culture was cooled to 4 ° C, mixed with 10 L of protecting medium containing 10% skimmed milk powder, 10% sucrose and lyophilized. The resulting dry culture was ground and stored at -20 ° C until mixing.

Preparation of pure culture from Lactobacillus rhamnosus LLR -L1-NBPMK 8769, Lactobacillus acidophilus LLA-10-NBPMK 8242 and Lactobacillus fermentum LLF-01-NBIMCC 8781.

100 l of reconstituted skimmed milk powder (14% dry matter) with the addition of 0.1% yeast extract, 0.1% (w / v) MRS broth and 2.0 l of buffer activating mixture representing an aqueous solution of CH ₃ COONa, CN ₂ PO ₄ , Na ₂ HPO ₄ , MgSO ₄ , MnSO ₄ and yeast extract were sterilized at 107 ° C for 3 minutes. The medium was cooled to 37 ° C and inoculated with 4 L of refreshed laboratory culture. Incubate in a fermenter at 37 ° C for 16 hours at pH 5.6 - 5.7 in automatic mode. The resulting culture was cooled to 4 ° C, mixed with 10 L of protecting medium containing 10% skimmed milk powder, 10% sucrose and lyophilized. The resulting dry culture was ground and stored at -20 ° C until mixing.

EXAMPLE 2

Exemplary synbiotic composition in a single capsule dose containing the active formula based on pro, -prebiotic and colostrum in mg as follows:

Component / example Synbiotic composition 2.1. 2.2 2.3 2.4 Lactobacillus delbrueckii subsp. bulgaricus LLB -06- NBPMK8918 30 60 75 120 Lactobacillus helveticus LLH-108- NBPMK8760 55 110 137.5 220 Lactobacillus acidophilus LLA-10- NBPMK8242 55 110 137.5 220 Lactobacillus rhamnosus LLR-L1- NBPMK 8769 55 110 137.5 220 Lactobacillus fermentum LLF-01- NBPMK 8781 55 110 137.5 220 Arabinogalactans (from Larix gmelinii) 100 200 250 400 Colostrum ASP60-201- (from a cow containing at least 20% IgG and min. 4.5% PRPs) ___________________ 50 100 125 200

Total / mg

400 800 1000 1600 • W · '--- _e . _φ :: ίλ:. :: * · · · · / ·· · ···· · J

The resulting multicomponent mixture of lyophilized monocultures is mixed with the other components of the prebiotic arabinogalactan and colostrum in the proportions according to the present invention, homogenized and is suitable for dosing the required oral form. The resulting composition is distributed in gastro-resistant vegan capsules selected from DR caps TM, Capsugel, intended for oral administration.

Each capsule contains 250 million live bacteria at the time of production, the resulting composition is stable with a shelf life of 24 months.

EXAMPLE 3

Synbiotic composition of finished product, lyophilized culture per 1000 g of finished product in g, as follows:

Component / example

Synbiotic composition 3.1.

Lactobacillus delbrueckii subsp.75 bulgaricus LLB -06-NBIMCC 8918

Lactobacillus helveticus LLH-108- NBPMK 8760137.5

Lactobacillus acidophilus LLA-10- NBPMK 8242137.5

Lactobacillus rhamnosus LLR-L1- NBPMK 8769137.5

Lactobacillus fermentum LLF-01- NBPMK 8781137.5

Arabinogalactans (Ametis JSC, from Larix gmelinii) 250

Colostrum ASP60-201-125 (from a cow containing min. 20% IgG and min. 4.5% PRPs

EXAMPLE 4

4.a. Results of tests performed on active lactobacilli monocultures.

Lactobacilli strains were tested for: - antimicrobial activity, - survival in simulated intestinal juice, - In vitro adhesion to human cell line, and - immunostimulatory activity, with the record holders of all tested strains presented in Figures 1-5 and respectively. the synbiotic composition according to the present invention.

The survival of the individual strains was demonstrated in simulated intestinal juice, as activity with the number of cells at the beginning of the test and after 2 hours in 0.3% bile salts pH 7, the results are presented in Fig.1.

The antimicrobial activity is presented in table. 1 and tested against human pathogens Staphylococcus aureus, Escherichia coli and Candida albicans by the method of wells with two model • ♦ · * · * ··· · · a ·:: 11 .:. :: ·. · _:: ·· * ···· · · * broth and milk systems with acidic (A) and neutralized (N) cell-free supernatants from exponential cultures of lactobacilli, the indicated results with (++) - showing strong inhibition, and (+ ) - significant and (-) - no inhibition.

The adhesion ability of the monocultures was tested against human colorectal adenocarcinoma cell line HT-29 (AACC HTB 38) by quantifying the number of adhered cells cfu / ml of the recorders Lactobacillus helveticus LLH108 and Lactobacillus fermentum and LLF as potential colonizers of the intestinal tract. The results are presented in Figs. 2.

The immunomodulatory properties of the monocultures were tested for comparative immunomodulatory activity with respect to the secretion of cytotoxic signal peptides by splenocytes with respect to TNF-α, IL-6 and IL-10 obtained from Balb / c mice according to FIG. 3-5. Reducing the secretion of the anti-inflammatory cytokines TNF-α and IL-6, and stimulating the secretion of the anti-inflammatory cytokine IL-10 affects the local immune tolerance and favors the intestinal microenvironment, which is constantly under attack by various food antigens and microorganisms. Thermally inactivated cells obtained from an exponential culture of the respective lactobacilli were used for the determination. Killed cells at a given concentration (1x10 ⁸ cfu / ml) were incubated with splenocytes isolated from the spleens of Balb / mice (6-8 weeks old) for 20 h / 37 ° C. Supernatants obtained from splenocyte treatment were used to determine the concentration of cytokines TNF-α, IL-6 and IL-10 produced by enzyme-linked immunosorbent assay (ELISA kits).

4.6. Test results of the active probiotic component and the active probiotic component with added arabinogalactan and colostrum.

The obtained probiotic component containing a mixture of the five strains of lactobacilli, as well as the probiotic component with added arabinogalactan and colostrum obtained according to the invention (Example 1.C and Examples 2) were tested for comparative immunomodulatory activity with respect to the secretion of cytotoxic signal peptides from splenocytes with respect to TNF-α, IL-6 and IL-10 derived from Balb / s mice according to FIG. 6. The addition of arabinogalactan and colostrum significantly reduces the synthesis of the anti-inflammatory cytokine IL-6 compared to the pure probiotic.

: Hz.:.: ·, *: ·:

The determination of the immunomodulatory properties of the probiotic component and the synbiotic mixture was performed as above in the evaluation of the individual monocultures. The lactobacilli in the probiotic component are represented in equal proportions.

4.c Results of tests of the synbiotic composition according to the invention on healthy people, demonstrating an immunomodulatory effect.

The resulting synbiotic product, dosed in capsules according to the invention, containing the 5 strains of lactobacilli, arabinogalactan and colostrum was tested for the following components of the immune system - natural killer cells (NK) in the peripheral circulation, test for anti-inflammatory cytokines IL-6 and IFN- y in faecal samples in 20 healthy volunteers (7 men and 13 women) with a mean age of 35 ± 2 (19-66) years after 21 days of synbiotic composition with 2 capsules / 400mg in the morning and 2 capsules / 400mg in the evening . Blood samples were tested for total and activated NK cells by cytometry (FACS Calibur, BD, USA) with monoclonal antibodies (anti CD3 FITC, anti-CD16 / 56 PE, anti-HLA-DR PE Cy5, BD, USA). One gram of the faecal samples is homogenized in 1000 ml of phosphate buffer. The supernatants obtained from the faecal extracts after centrifugation were tested for the presence of the cytokines IL-6 and IFN-γ by enzyme-linked immunosorbent assay (Human ELISA kits, Gen Probe Diaclone SAS, FR). The results before and after administration of the synbiotic composition with respect to NK cells are presented in table. 2, and the cytokines IL-6 and IFN-γ, as a concentration in pg / ml are presented in Table 2, respectively. 3. Administration of the synbiotic product reported an immunomodulatory effect after oral administration of the synbiotic according to the present description, expressed in a statistically significant increase in% of NK cells in peripheral blood and a decrease in levels of anti-inflammatory cytokines IL6 and IFN-γ in faecal samples.

4.s. Test results of the synbiotic composition according to the invention on patients with irritable bowel syndrome (IBS).

The obtained synbiotic product, dosed in capsules according to the invention, containing the 5 strains of lactobacilli, arabinogalactans and colostrum was tested for the components of the immune system - the marker for the assessment of intestinal inflammation - fecal calpotrotectin (PCP) from faecal samples in 20 patients with IB 6 men and 14 women) with a mean age of 37 ± 2 (24-52) years after 21 days of synbiotic composition with 2 capsules / 400mg in the morning and 2 capsules / 400mg in the evening.

: из.:.:: · .. · f ·· · ···· · ·

PCP was assayed by immunochromatographic assay for the quantification of calprotectin in human fecal samples (Quantum Blue, Buehlmann Laboratories AG, Switzerland). The final result is presented in micrograms per gram of faeces (pg / g). The sensitivity of the test is in the range of 30 pg / g - 300pg / g. Normal PCP levels in this study were below 50 pg / g, indicating a “gray area up to 60 pg / g ·

The results before and after administration of the synbiotic composition as described for PCP are presented in FIG. 7, with a statistically significant decrease in the inflammatory activity after the intake of the synbiotic, expressed in a decrease in PCP and reduction, even disappearance of the clinical symptoms.

Table № 1 - Antimicrobial activity of active monocultures

Test microorganism

Staphylococcus aureus; I _ NBIMCC 3703_______i Escherichia coE ___NBIMCC 3702 Candida albicans NBIMCC 74 Lb.bulgaricus LLB-06 \ A ~: N ⁺⁺ i Α ~ Λ N ⁺ ------------------------------ b- . _ ................... - Lb. hebedcus LLH-108 | A ^ N ** '‘’ l · A ++ '. i Lb. acidophilus LLA-10 !! A; N ++ A ** _______________ Lb. rhamnosus ULR-L1 I A **; N **; A ** A ⁺ --------- —.......-.....---- and Lb.fermentum LLF-01 1 A-; A ” -

Table № 2- Immunomodulatory effect of the synbiotic composition according to the invention on healthy people against NK cells

Healthy individual »

Before taking a synbiotic After taking a synbiotic __________ Statistical 1 _ difference, p ___________ 1 Total percentage of Ϊ6.1 ± Ϊ.2 14.6 ± 1.2 0.073 1 Vests (8.1-25.5) i (5.1-25.7) _ ______________________________ Activated 4.9 ± 0.6; 7.1 ± 0.7 NK carrier cells (1.3-9.9) | (2.6-13.5) 1 (HLA-DR +) ........................................— ____________-.......................... — ......—----------

Table №3- Immunomodulatory effect of the synbiotic composition according to the invention on healthy people with respect to IL-6 and IFN-γ

Healthy individuals

Before taking a synbiotic After taking a synbiotic Statistical difference, p IL-6, pg / ml 5.6 ± 0.2 (3.9-6.75) 3.1 ± 0.1 (2.3-5.5) 0.000 IFNg, pg / ml 73.2 ± 2.6 (55.5-92.5) _________ 43.6 ± 3.9 ¢ 28.3-99) __________ 0.000

Survival in simulated intestinal juice

Lb. bulgaricus LLB-Ob

Lb. helveticusLLH-108

Lb. acidophilusLLA-10

Lb. rhamnosus LLR-L1

Lb fermentum LLF-01

2 4 6 8 10 12

Log cfu. gr initial cell number and Log cfu / gr after 2h in 0.3% bile salts, pH7

Figure 1

Adhesion to HT-29 cell line (%)

Lb. bulgaricus LLB-06

Lb. helveticus LLH-108

Lb. acidophilus LLA-10

Lb. rhamnosus LLR-L1

Lb.fermentunt LLF-01

I — I) -2%

3.4%

0 .:

16.8%

123456789

Log cGi / pinachal ^u Log cfu ml adhesion cells

Figure 2

TNF-apg / ml

22.3

0.6 (-) control

control

Lb. Lb. Lb. Lb. Lb.

bulgaricus helveticus acidophilus rhamnosus fermentui h

LLB-06 LLH-108 LLA-10 LLR-L1 LLF-01

Fig.Z

56.9

and I1.-6 pg / ml

123.5

rhatftnasus LL.R-L1 LLF-O1

Figure 4

Figure 5

Fig.6

OKJI (mg / kg)

Claims (14)

PATENT CLAIMS An immunomodulatory synbiotic composition comprising προ-, prebiotic and colostrum, characterized in that it comprises a probiotic component consisting of live cells of a biologically pure culture of a bacterial strain selected from the group of: Lactobacillus delbrueckii subsp. bulgaricus LLB -06- NBPMK 8918, Lactobacillus helveticus LLH-108- NBPMK 8760, Lactobacillus acidophilus LLA-10- -NBPMK 8242, Lactobacillus rhamnosus LLR-L1- -NBPMK 8769 and Lactobacillus fermentum LLF-01-NBPMK 8781 or combinations thereof and prebioticcarbinogalactan. Immunomodulatory synbiotic composition according to claim 1, characterized in that the amount in mg of the components is respectively: Lactobacillus delbrueckii subsp. bulgaricus LLB -06- NBPMK 8918 - from 30 to 120, Lactobacillus helveticus LLH-108- NBPMK 8760 - from 55 to 220, Lactobacillus acidophilus LLA-10- NBPMK 8242 - from 55 to 220, Lactobacillus rhamnosus LLR-L1- L1- H1- N1 from 55 to 220, Lactobacillus fermentum LLF-01- NBPMK 8781 - from 55 to 220, Arabinogalactan - from 100 to 400 and colostrum - from 50 to 200. Immunomodulatory synbiotic composition according to claim 1, characterized in that the arabinogalactan is derived from the bark of Siberian larch (Larix gmelinii). Immunomodulatory synbiotic bacterial composition according to claim 1, characterized in that the colostrum contains at least 4.5% proline-containing peptides and at least 20% IgG. 5. Immunomodulatory probiotic composition, characterized in that it contains live cells of a biologically pure culture of a bacterial strain selected from the group of: Lactobacillus delbrueckii subsp. bulgaricus LLB -06- NBPMK 8918, Lactobacillus helveticus LLH-108- NBPMK 8760, Lactobacillus acidophilus LLA-10- NBPMK 8242, Lactobacillus rhamnosus LLR-L1- NBPMK 8769, Lactobacillus fermentum LLF-01- NBPMK 8781 or combinations thereof. Immunomodulatory probiotic composition according to claim 5, characterized in that it contains an effective amount of a bacterial mixture of: Lactobacillus delbrueckii subsp. bulgaricus LLB -06- NBPMK 8918, Lactobacillus helveticus LLH-108- NBPMK 8760, Lactobacillus acidophilus LLA-10- NBPMK 8242, Lactobacillus rhamnosus LLR-L1- NBPMK 8769 and Lactobacillus fermentum LLF-01- NBPMK 8781. Immunomodulatory probiotic bacterial composition according to claims 5 and 6, characterized in that the amount in mg of the components of the bacterial mixture is respectively: Lactobacillus delbrueckii subsp. bulgaricus LLB -06- NBPMK 8918 - from 30 to 120, Lactobacillus helveticus LLH-108- NBPMK - from 55 to 220, Lactobacillus acidophilus LLA-10- NBPMK 8242 - from 55 to 220, Lactobacillus rhamnosus LLR-L1- NBPMK 8769 - from 55 to 220, Lactobacillus fermentum LLF-01- NBPMK 8781 - from 55 to 220. Immunomodulatory probiotic bacterial composition according to claims 1 to 7, characterized in that it has an activity in the range from 10 ⁸ to ¹⁰ CFU bacteria / g of the composition. 9. Lactobacillus fermentum strain LLF-01, registered with the NBPMK under № 8781 dated 21.05.2013. 10. Strain of Lactobacillus delbrueckii subsp. bulgaricus LLB -06, registered with the NBPMK under № 8918 dated 01.02.2016. Use of an immunomodulatory synbiotic composition according to claims 1-4 for the preparation of a product in the form of a food supplement and / or a pharmaceutical product intended for the prevention and / or concomitant therapy of inflammatory and immune diseases of the gastrointestinal tract, as well as for normalization and balancing the function of the gastrointestinal tract, including after prolonged use of antibiotics, in symptoms of intestinal dysbiosis and inflammatory bowel syndrome. Use of an immunomodulatory probiotic bacterial composition according to claims 5-8 for the preparation of a product in the form of a food supplement and / or a pharmaceutical product intended for the prophylaxis and / or concomitant therapy of inflammatory and immune diseases of the gastrointestinal tract, as well as for normalization and balancing of the function of the gastrointestinal tract, including after long-term use of antibiotics, symptoms of intestinal dysbiosis and inflammatory bowel syndrome. Use of a strain of Lactobacillus fermentum LLF-01 NBPMK № 8781 according to claim 9, for the preparation of a product in the form of a food supplement and / or a pharmaceutical product intended for the prophylaxis and / or concomitant therapy of inflammatory and immune diseases of the gastrointestinal tract, as well as to normalize and balance the function of the gastrointestinal tract, including after prolonged use of antibiotics, in symptoms of intestinal dysbiosis and inflammatory bowel syndrome. 14. Use of a strain of Lactobacillus delbrueckii subsp. bulgaricus LLB -06, registered in the NBMPM under № 8918, according to claim 10, for the preparation of a product in the form of a food supplement and / or a pharmaceutical product intended for the prophylaxis and / or concomitant therapy of inflammatory and immune diseases of the gastrointestinal tract, and to normalize and balance the function of the gastrointestinal tract, including after prolonged use of antibiotics, in symptoms of intestinal dysbiosis and inflammatory bowel syndrome.