BG67244B1 - Immunomodulating synbiotic composition - Google Patents

Abstract

The invention relates to an immunomodulating synbiotic composition comprising an active formula based on a probiotic component of 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-NBIMCC 8760, Lactobacillus acidophilus LLA-10-NBIMCC 8242, Lactobacillus rhamnosus LLR-L1-NBIMCC 8769 and Lactobacillus fermentum LLF-01-NBIMCC 8781 or combinations thereof,or the prebiotic- Siberian larch bark (Larix gmelinii) and colostrum rich in proline-containing peptides and IgG at the amounts described in mg. The present invention also relates to a probiotic bacterial composition with immunomodulating 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-NBIMCC 8918, Lactobacillus helveticus LLH-108-NBIMCC 8760, Lactobacillus acidophilus LLA-10-N NBIMCC 8242, Lactobacillus rhamnosus LLR-L1-NBIMCC 8769, Lactobacillus fermentum LLF-01-NBIMCC 8781 or combinations thereof at the amounts described in mg according to the present invention, as well as to the bacterial strain Lactobacillus fermentum LLF-01 NBIMCC № 8781 and the strain Lactobacillus delbrueckii subsp. bulgaricus LLB-06 NBIMCC 8918 that produce bacterial composition with immunomodulating action. The compositions, according to the present invention, are intended for the preparation of a food supplement and / or a pharmaceutical product 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 normalization and balancing. the function of the gastrointestinal tract, including after prolonged use of antibiotics, with symptoms of intestinal dysbiosis and inflammatory bowel syndrome.

Description

The invention relates to an immunomodulatory synbiotic composition, in particular a probiotic bacterial composition, further enriched with a prebiotic and biologically active substances beneficial to human health in the prevention and / or treatment of inflammatory and immune diseases of the gastrointestinal tract, incl. in symptoms of intestinal 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 speed 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 weakened immunity. 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. Lactic acid bacteria are a tool for enriching the gastrointestinal tract with beneficial bacteria.

Lactic acid bacteria, in recent years, have been used for the prevention or concomitant treatment of immune diseases. It is known from WO 2015/046407 a prophylactic agent in the form of a food 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 immune growth cells IL-10 / 1L-12 (associated with enteritis, rheumatoid arthritis, immune diseases such as hepatitis).

Also known from EP 2482826 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 powder and / or 20-50% dry lactose; sweetening agents of 10-30%; dried fruit fillers of 5-20%; 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), incl. arabinogalactan and vitamin complex of 0-5%; 0-20% oligosaccharides and 0-5% structurants.

From BG 65614 is known a synbiotic, which is a mixture of monocultures Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Bifidobacterium

B1 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 of the effect of the probiotic.

Technical essence of the invention

These problems are solved according to the invention with an immunomodulatory synbiotic composition - an 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-НБПМКК 8918,

Lactobacillus helveticus LLH-108-NBPMKK 8760,

Lactobacillus acidophilus LLA-10-NBPMKK 8242,

Lactobacillus rhamnosus LLR-Ll-NBPMCC 8769 and

Lactobacillus fermentum LLF-01-NBPMKK 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 lactation in female milk, , 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-NBPMCC 8918 from 30 to 120 mg;

Lactobacillus helveticus LLH-108-NBPMCC 8760 - from 55 to 220,

Lactobacillus acidophilus LLA-10-NBPMKK 8242 - from 55 to 220,

Lactobacillus rhamnosus LLR-Ll-NBPMCC 8769 - from 55 to 220,

Lactobacillus fermentum LLF-01-NBPMCC 8781 - 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 of 10 ⁸ to 10 ¹⁰ CFU bacteria / g.

The newly isolated Lactobacillus delbrueckii subsp. bulgaricus LLB-06-NBPMCC 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? from glucose, forms metachromatin 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 agar. 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 intensive growth at 37 ° C, and weaker at 15 ° C, produces CO? from glucose. with intensive growth of lactic medium with formation of 40% D (-) and 60% L (+) lactic acid during 24-hour fermentation. The culture maintains good growth of MRS agar.

Carbohydrate metabolism of strains:

Name of monoculture 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-glucosidine esculin, salicin, cellobiose, maltose, melibiose, sucrose, trehalose, inulin, melisitose, raffinose, amidone, glycogen, xylitol, β gentibiose, turanose, lixose, tagatose, fucose, arabitol, gluconate, k 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-0-mannoside and methyl-oglucoside, за acetylglycine cellobiose, maltose, trehalose, inulin, melisitose, amidone, glycogen, xylitol, β gentobiose, turanose, lyxose, tagatose, fucose, arabitol, keto gluconate

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The remaining monocultures of Lactobacillus helveticus strain LLH-108-NBPMKK 8760, Lactobacillus acidophilus LLA-10-NBPMKK 8242 and Lactobacillus rhamnosus LLR-Ll-NBPMKK 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 storage, which in turn ensures the proper functioning of the digestive processes and maintaining the balance of intestinal peristalsis.

In order to achieve a product with the target properties, the strains are selected by the criterion:

- antimicrobial activity,

- survival in simulated intestinal juice,

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- in vitro adhesion to a human cell line; and - immunomodulatory activity.

The strains are not 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 nutrient medium based on milk or broth, followed by lyophilization and digestion. Preferred ingredients for such an environment are skim milk, yeast extract, casein peptone, meat extract, D-glucose. Salts such as sulphate (magnesium and manganese) and buffers such as sodium acetate, sodium phosphate, potassium phosphate are added to the medium. The optimal content of skim milk varies between 12-18% (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, CHsCOONa, MgSO4 and MnSCL. To the basic medium is added a buffer-activating mixture, representing an aqueous solution of CH 2 COONa, CH 2 PO 4, PagNPO 4, MgSO 4, MnSO 4 and the yeast extract is in an amount of 0.2% (v / v) to 2.0%, in particular 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 6.66.8 and sterilized at 107 ° C for 3 minutes. Prior to inoculation, the culture medium was 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, respectively, to encapsulation, dosing in sachet forms or tabletting. To prepare tablets, the resulting bacterial mixture is sieved, sieved and the remaining ingredients and excipients are added sequentially. 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-NBPMCC 8918.

Lactobacillus helveticus LLH-108-PBPMCC 8760.

Lactobacillus acidophilus LLA-10-N NBPMCC 8242,

Lactobacillus rhamnosus LLR-L1-NBPMCC 8769,

Lactobacillus fermentum LLF-01-NBPMCC 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 with the NBMPMC under № 8781 dated 21.05.2013 and strain Lactobacillus delbrueckii subsp.

BG 67244 Bl bulgaricus LLB-06, registered with the NBMPMC under № 8918 of 01.02.2016, producers of a 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 NBPMCC № 8781 and a strain of Lactobacillus delbrueckii subsp. bulgaricus LLB-06 / NBMPMK № 8918, 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 (immunomodulator at the level of mucosal immune system and innate system), as well as to normalize and balance the function of the gastrointestinal tract, incl. after prolonged use of antibiotics, with 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 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 various 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 release in the intestinal tract. The enriched multifunctional formula of the synbiotic composition according to the invention, comprising proline-rich polypeptides (4.5%) and 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 ^s to 10 ¹⁰ CFU bacteria / g of the composition and a shelf life of the finished product up to 24 months.

The administered oral synbiotic composition in healthy people demonstrates 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 fecal samples, and in patients with Irritable bowel syndrome (IBS) reduces the inflammatory activity expressed in the reduction of fecal calprotectin and causes a reduction or even disappearance of clinical symptoms.

Explanation of the attached figures

Figure 1. Survival of lactobacilli strains in simulated intestinal juice.

Figure 2. Adhesion ability of lactobacilli strains to human colorectal adenocarcinoma cell line HT-29 (AASS HTV 38).

Figure 3. Immunomodulatory activity of monocultures on TNF-α obtained from Balb / c mice.

Figure 4. Immunomodulatory activity of monocultures on IL-6 obtained from Balb / c mice.

Figure 5. Immunomodulatory activity of monocultures on IL-10 obtained from Balb / c mice.

Figure 6. Immunomodulatory activity with respect to TNF-α, IL-6 and IL-10 secretion obtained from 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 formula based on a probiotic, containing the active monoculture of the lactobacillus strain Lactobacillus delbrueckii subsp. bulgaricus in mg.

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

Active bacterial mixture ___

Lactobacillus delbrueckii subsp. bulgaricus LLB-Ob 30 60 120

B / Composition of the active probiotic-based formula 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

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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 1.С.1. 1.C.2 1.C.3 1.C.4 Lactobacillus delbrueckii subsp. 30 60 75 120 bulgaricus LLB -06- NBPMCC 8918 Lactobacillus helveticus LLH-108- NBPMCC 8760 55 110 137.5 220 Lactobacillus acidophilus LLA-10- NBPMCC 8242 55 110 137.5 220 Lactobacillus rhamnosus LLR-L1- NBPMCC 8769 55 110 137.5 220 Lactobacillus fermentum LLF-01- NBPMCC 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 strain and species are prepared. then 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-NBPMKK 8918 and Lactobacillus helveticus LLH-108-PBPMKK 8760.

100 l of reconstituted skimmed milk powder (14% dry matter) with 0.1% yeast extract and 2.0 l of buffer activating mixture, representing an aqueous solution of CH ₂ COONa, CH 2 PO 4, Na 2 HPC> 4, MgSO 4, MnSO 4 and the yeast extract were sterilized at 107 ° C for 3 min. 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 h 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-Ll-NBPMKK 8769, Lactobacillus acidophilus LLA-10-NBPMKK 8242 and Lactobacillus fermentum LLF-01-NBPMKK 8781.

100 1 reconstituted skimmed milk powder / 14% dry matter / with the addition of 0.1% yeast extract, 0.1% (w / v) MRS broth and 2.0 1 buffer activating mixture, representing an aqueous solution of CH ₃ COONa, CN ₂ PO4, Na ₂ HPO4, MgSO4, MnSO4 and yeast extract were sterilized at 107 ° C for 3 min. 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 h 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:

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Component / example Synbiotic composition 2.1. 2.2 2.3 2.4 Lactobacillus delbrueckii subsp. 30 60 75 120 bulgaricus LLB -06- NBPMCC 8918 Lactobacillus helveticus LLH-108- NBPMCC 8760 55 110 137.5 220 Lactobacillus acidophilus LLA-10- NBPMCC 8242 bb 110 137.5 220 Lactobacillus rhamnosus LLR-L1- NBPMCC 8769 55 110 137.5 220 Lactobacillus fermentum LLF-01- NBPMCC 8781 ex 110 137.5 220 Arabinogaiactans (from Larix gmelinii | 100 200 400 Colostrum ASP6O-2O1- 50 100 125 200

(from a cow containing min, 20% IgG and min. 4.5% PRPs)

Total / mg

400 800 1000 1600

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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 .......... ZŠ __________

Lactobacillus delbrueckii subsp.75 bulgaricus LLB -О6-НБПМКК 8918

Lactobacillus helveticus LLH-108- NBPMCC 8760 137.5

Lactobacillus acidophilus LLA-10- NBPMCC 8242137.5

Lactobacillus rhamnosus LLR-L1- NBPMCC 8769137.5

Lactobacillus fermentum LLF-01- NBPMCC 8781137.5

Arabinogalactans (Ametis JSC, from Lanx gmeliniO 250

Colostrum ASP6O-2O: 1 ·· 125 (from a cow containing min. 20% IgG and min. 4.5% PRPs

Example 4.

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

Lactobacilli strains have been tested for:

- antimicrobial activity,

- survival in simulated intestinal juice,

- In vitro adhesion to human cell line and

- immunostimulatory activity, as the record holders of all tested strains are presented in Fig. 1-5 and are selected for the synbiotic composition of 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 systems broth and milk with acidic (A) and neutralized (N) cell-free supernatants from exponential cultures of lactobacilli, as indicated by results with (++) - show 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 adherent cells cfu / ml of the recorders Lactobacillus helveticus LLH-108 and Lactobacillus fermentum LLFF identifies them as potential colonizers of the intestinal tract. The results are presented in Figs. 2.

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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 derived from Balb / c mice according to FIG. 3-5. Reducing the secretion of anti-inflammatory cytokines TNF-α and IL-6, and stimulating the secretion of anti-inflammatory cytokine IL-10 affects the direction of 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 (1 x 10 ^s cfu / ml) were incubated with splenocytes isolated from the spleens of Balb / c 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 obtained from Balb / c 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.

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. Test results of the synbiotic composition according to the invention on healthy people, proving an immunomodulatory effect.

The obtained 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 IL6 and IFN-γ in feka 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 / 400 mg in the morning and 2 capsules / 400 mg 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 cytokines 1L-6 and IFN-γ by enzyme immunoassay (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 cytokines 1L-6 and IFN-γ, as 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 anti-inflammatory cytokine IL-6 and IFN-γ levels in faecal samples.

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

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

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 - 300 pg / 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.

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Table № 1 · Antimicrobial activity of active monocultures

ВвлШам StapkylmMteM oureta T «h mikrairgyamshm Cuntli.Lt aUfuuns NBIMCC 3 ~ 0 * КШМССЗ ~ О2 NBIMCC ~ 4 Lb.buigarKu * LLB-06 A '' - ' A. Ν Lb. Ai & lilm LLU-1 & 8 /(-A.- A Lb. aridapkilu * LLA-10 A ..... A A Lb. rbamn & uis LLR L1 Lb. enzyme LLF-01 Λ '. V A *

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

MM—! L Oosh npouvifi on NK klevkn Before taking c-knbnotmk 16.1 * 1.2 (S.1-25 Μ <lsl reception of snnoiappch 14.6 * 1.2 (5.1-25.7) Statistical calculation, p 0 ^ 073 1 Act mpmra mi 4.U. 6 NK carrier cells (1.3-9.9) (HLA DR «) 7.1 * 0.7 : (2.6-13.5) 0.03 1

Table №3- Immunomodulatory effect of the synbiotic composition according to the invention on healthy people with respect to 11-6 and IFN-γ

Before taking X. t »~. «Λ After taking Statistical synbnotic synbnotic difference, p IL-6, pguil 5.6 ± 0.2 3.1 ± 0.1 0.000 (3.9-6.75) (2.3-5.5) IF5g. pg ml ~ i.2 = 2.6 43.6 ± 3.9 0.000 i (55.5-92.5) (28.3-99)

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BG 67244 Bl

Claims (8)

1. An immunomodulatory synbiotic composition comprising a probiotic. prebiotic and colostrum, characterized in that the probiotic component consists of live cells of a biologically pure culture of a bacterial strain with activity in the range of 10 ⁸ to 10 ¹⁰ CFU bacteria / g and an amount in mg for each of them selected from the group of: Lactobacillus delbrueckii subsp. bulgaricus LLB-06-NBPMCC 8918 - from 30 to 120, Lactobacillus helveticus LLH-108-NBPMCC 8760 - from 55 to 220, Lactobacillus acidophilus LLA-10-NBPMKK 8242 - from 55 to 220, Lactobacillus rhamnosus LLR-Ll-NBPMCC 8769 - from 55 to 220 and Lactobacillus fermentum LLF-01-NBPMKK 8781 - from 55 to 220, or combinations thereof, the prebiotic being arabinogalactan from Siberian larch bark (Larix gmelinii) - from 100 mg to 400 mg, and colostrum from 50 mg to 200 mg, containing at least 4.5% proline-containing peptides and at least 20% IgG. Immunomodulatory probiotic composition, characterized in that it contains live cells of a biologically pure culture of a bacterial strain or a combination thereof, with activity in the range of 10 ⁸ to 10 ¹⁰ CFU bacteria / g and an amount in mg for each strain selected from the group of: Lactobacillus delbrueckii subsp. bulgaricus LLB-06-NBPMCC 8918 - from 30 to 120, Lactobacillus helveticus LLH-108-NBPMCC 8760 - from 55 to 220, Lactobacillus acidophilus LLA-10-NBPMKK 8242 - from 55 to 220, Lactobacillus rhamnosus LLR-Ll-NBPMCC 8769 - from 55 to 220 and Lactobacillus fermentum LLF-01-NBPMCC 8781 - from 55 to 220. 3. Lactobacillus fermentum strain LLF-01, registered with the NBMPMC under № 8781 dated 21.05.2013. 4. Lactobacillus delbrueckii subsp. bulgaricus LLB-06, registered with the NBPMCC under № 8918 dated 01.02.2016. Use of an immunomodulatory synbiotic composition according to claim 1, 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, and for normalizing and balancing function of the gastrointestinal tract, incl. after prolonged use of antibiotics, with symptoms of intestinal dysbiosis and inflammatory bowel syndrome. Use of an immunomodulatory probiotic bacterial composition according to claim 2, 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, and for normalization and balancing the function of the gastrointestinal tract, incl. after prolonged use of antibiotics, with symptoms of intestinal dysbiosis and inflammatory bowel syndrome. Use of a strain of Lactobacillus fermentum LLF-01 NBMPMK № 8781 according to claim 3, 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 of the function of the gastrointestinal tract, incl. after prolonged use of antibiotics, with symptoms of intestinal dysbiosis and inflammatory bowel syndrome. 8. Use of a strain of Lactobacillus delbrueckii subsp. bulgaricus LLB-06, registered with the NBMPMC under № 8918. according to claim 4, 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, incl. after prolonged use of antibiotics, with symptoms of intestinal dysbiosis and inflammatory bowel syndrome.