Bifidobacterium longum strain, suitable for application in immunomodulation, induction of cytokins production, treatment of autoimmune disease, control of il - 10:il - 12 ratio, and application thereof

FIELD: medicine.

SUBSTANCE: group of inventions relates to the strain of Bifidobacterium longum NCIMB 41675, a composition, containing thereof, and a food product, containing the said strain or composition. The claimed strain possesses an ability to induce the production of cytokins and control IL-10:IL-12 ratio is suitable for application in immunomodulation, treatment of an autoimmune disease.

EFFECT: claimed strain, composition and food product possess probiotic properties.

25 cl, 16 dwg, 4 tbl, 8 ex

 

The technical field to which the invention relates

The present invention relates to a strain of bifidobacteria {Bifidobacterium) and its use as probiotic bacteria, in particular, as immunomodulatory biotherapeutic agents.

The level of technology

The defense mechanisms of the gastrointestinal tract from colonization by intestinal bacteria are extremely complex and include immune and non-immune interaction (1). Innate protective mechanisms include the low pH of the stomach, bile salts, peristalsis, satinowye layers and antimicrobial substances and compounds, such as lysozyme (2). Immune mechanisms include specialized lymphoid formation, underlying the M-cell (also called complexes of Payer) distributed throughout a thin and rectum (3). Luminal antigens that are present in these areas, encourage the development of subpopulations of T - and b-cells with the formation of cytokine networks and secretion of antibodies into the gastrointestinal tract (4). Furthermore, antigens can be accessed through the epithelial cells to intraepithelial lymphocytes and immunocytes under their own plate mucous membranes (5). Thus, a person spends a considerable force on the immune protection of the gastrointestinal tract. Because the mucosa of the intestine is the most �big surface, through which man comes into contact with the environment, it required specific mechanisms used for regulating the immune response to 100 tons of food that passes through the gastro-intestinal tract in the course of the average lifetime of a person. In addition, the intestine is inhabited by over 500 species of bacteria, the concentration of which in the rectum is 1011-1012/G. In this regard, the regulatory mechanisms are not able to distinguish between pathogenic bacteria that normally live in the intestine, from the invasions of pathogens that can cause serious problems for the owner. The intestinal flora is actively involved in the protection of the owner, struggling with falling through the digestive system of potentially pathogenic microorganisms.

It is believed that some species of probiotic bacteria are more effective when they are derived from types, treatment which is intended, or from closely related species. There is therefore a need for probiotic strains derived from companion animals, and distinct from strains obtained from humans, which could be used for the treatment of companion animals.

In the publication WO 01/90311 described probiotic microorganisms isolated from samples of faeces of cats and having probiotic activity. However, these bacteria were obtained from fecal about�ascov, so they may not be a part of the natural flora present in the upper gastrointestinal tract.

Accordingly, there is a need for strains of bacteria obtained by separation from the natural flora present in the upper gastrointestinal tract, especially suitable for cats and possessing probiotic properties and ability to survive in the course of their treatment. There is also a need for compositions containing such strains and convenient to use.

Summary of the invention

The present invention provides a selected strain of bifidobacteria No. 41675 on NCIMB.

Strain of bifidobacteria may be in the form of living cells. Strain of bifidobacteria may be in the form of living cells.

Bifidobacteria can be isolated from tissue samples of the colon healthy cats, taken by biopsy.

Strain of bifidobacteria may have significant immunomodulation action in its oral consumption.

In the present invention it is also proposed that the composition containing the strain of bifidobacteria.

The composition may further comprise a probiotic material. The composition may further contain a prebiotic material. The composition may further contain a carrier for oral administration. Carrier for oral administration m�can be one of pharmaceutically acceptable carriers, such as, for example, a capsule, a tablet, a powder. A carrier for oral administration may also be a food product, such as, for example, oily suspension, suspension on the basis of milk, cheese, consisting of cocoa butter, sauce and/or composition on the basis of yogurt.

In the present invention it is also proposed that a food product containing a strain of Bifidobacterium or composition according to the present invention.

The food product can be can be dry food. The food product may be a moist food product. The food product may be food for companion animals.

The present invention proposes the strain Bifidobacterium, or composition, or a food product in accordance with the present invention that can be used as a drug.

The present invention proposes the strain Bifidobacterium, or composition, or a food product in accordance with the present invention that can be used for the prevention and/or treatment of undesirable inflammatory processes.

The present invention proposes the strain Bifidobacterium, or composition, or a food product in accordance with the present invention that can be used for the prevention and/or treatment of undesirable inflammatory processes in gastrointestinal� tract.

The present invention proposes the strain Bifidobacterium, or composition, or a food product in accordance with the present invention that can be used for the prevention and/or treatment of autoimmune disorders that cause unwanted inflammatory processes.

The present invention proposes the strain Bifidobacterium, or composition, or a food product in accordance with the present invention that can be used for the prevention and/or treatment of diarrhoeal States caused undesirable inflammatory processes.

The present invention proposes the strain Bifidobacterium, or composition, or a food product in accordance with the present invention, for correcting or improving the immune system of companion animals.

The present invention proposes the strain Bifidobacterium, or composition, or a food product in accordance with the present invention that can be used for the prevention and/or treatment of autoimmune diseases in companion animals.

The present invention proposes the strain Bifidobacterium, or composition, or a food product in accordance with the present invention that can be used for the prevention and/or treatment of inflammation in companion animals.

In the present invention �predlagaetsja strain of bifidobacteria AH121A (number NCIMB 41675), as well as its mutants and variants.

The mutant may be a genetically modified mutant. The variant may be a naturally occurring variant of bifidobacteria.

The strain may be obtained by isolating it from the cut and washed feline gastrointestinal tract.

Strain can be a probiotic. The strain may be in the form of a biologically pure culture.

In the present invention it is also proposed that the selected strain of bifidus bacteria, NCIMB 41675.

Strains of bifidobacteria can be in the form of living cells. Alternatively, strains of bifidobacteria can be in the form of living cells.

Typically, the probiotic bacteria are used in the form of living cells. However, it is possible to use them in the form of living cells, for example, in the form of dead cultures or compositions containing beneficial factors expressed by the probiotic bacteria. Slain culture can include microorganisms that are killed when exposed to high temperature, extreme pH, or pressure. When not using live cells, the manufacture of the final product is simplified, they can be included in a wide variety of pharmaceutical forms, and conditions of storage of medicines from them are significantly less stringent than for preparations containing living cells. For example, in U.S. patent 4347240 about�isano successful use of dead high temperature cells of the strain Lactobacillus casei YIT 9018 for the treatment and/or prevention of tumor growth.

The present invention also proposed the use of bacteria, obtained by separation of cut and washed feline gastrointestinal tract, to maintain and improve the health of the animal companion, and the use of formulations containing lactic acid bacteria.

In the present invention it is also proposed that the composition containing the strain of bifidobacteria. The composition may include another probiotic material. The composition may include a prebiotic material.

Bifidobacteria are symbiotic microorganisms. They were isolated from the microflora of the gastro-intestinal tract of humans. The immune system of the gastrointestinal tract does not give a severe reaction to the representatives of the microflora, otherwise arisen inflammatory reaction would destroy also the host cell and disrupted the functioning of tissues. Therefore, there are certain mechanisms by which the immune system can recognize pathogenic members of the family of gastro-intestinal microflora and to distinguish them from pathogenic organisms. This ensures the integrity of host cells on the one hand, and maintaining the required protective barrier from pathogens, on the other hand.

In the context of the present description, the terms "mutant", "variant" and "genetically modified m�Thant include a strain of bifidobacteria, genetic and phenotypic properties of which differ from the corresponding properties of the parent strain. The term "naturally occurring variant of the strain Bifidobacterium longum" means organisms that are selected through selection of the organisms in which there are spontaneous changes in target properties. Intentional changes in the properties of the parent strain are conventional genetic engineering techniques (in vitro), such as the gap genes, conjugational transfer and other. The term "genetic modification" includes the introduction of exogenous and/or endogenous DNA sequences in the genome of the strain of Bifidobacterium, for example by insertion into the genome of the bacterial strain by means of vectors based on plasmid DNA or bacteriophage.

The terms "natural mutation" and "induced mutations" include changes at least one base through deletions, insertions, treatment or other changes to DNA that may lead to changes in the amino acid sequence encoded by the DNA sequence.

The terms "mutant", "variant" and "genetically modified mutant" also include the strain of bifidobacteria, which was subjected to genetic changes that accumulate in the genome to the extent that in essence and in nature the same for all microorganisms, and/or genetic�climate change, that occur as a result of spontaneous mutation of gains and/or losses that cannot be obtained through intentional (in vitro) transformations of the genome, but which can be obtained by means of natural selection of variants and/or mutants, and which provide the desired benefits that increase the viability of the bacteria when exposed to aggressive environmental factors, such as, for example, antibiotics. The mutant can be created by intentional (in vitro) the insertion of certain genes into the genome, which radically alter the biochemical functionality of the body, but the expression products of which can be used for identification or breeding of bacteria, for example, which give resistance to antibiotics.

Well-versed in the art would understand that a mutant or variant strains of bifidobacteria can be identified through analysis of their DNA sequence on homology with the parent strain. Strains of bifidobacteria that have a high degree of identity of the DNA sequence with the parent strain, can be considered a mutant or variant strains. The strain of Bifidobacterium having a degree of identity (homology) DNA sequence corresponding to the sequence of the parent strain, which is 96% or more, 97% or more, 98% or more, �whether 99% or more, could be considered a mutant or variant strain. The sequence homology can be determined using the BLAST program, available on the website http://www.ncbi.nlm.nih,gov/BLAST/.

Mutants of the parent strain include derivative strains of bifidobacteria that have homology to the intergenic spacer polynucleotide sequence 16s-23s constituting at least 85%, at least 90% or at least 95% relative to the corresponding sequence of the parent strain. Such mutants may optionally contain DNA mutations in other DNA sequences in the bacterial genome.

Brief description of the drawings

The present invention will be more apparent from the following detailed description of its embodiments, given only as examples, accompanied by the attached drawings.

Fig.1. Photo B. longum AH121A grown on agar with the dye Congo red.

Fig.2. Bar chart showing the ratio of expression of IL-10:IL-R in mononuclear cells of peripheral blood, stimulated and stimulated by the strain Bifidobacterium longum AH121A;

Fig.3. Bar graph showing the survival rate of the bacteria strain AH121A at low pH values (the bacteria were in an environment with a pH of 2.5 to 6 hours; survival was determined by the method of bell counting).

Fig.4-6. Columnar� chart showing the production of cytokines in cultures in vitro of peripheral blood mononuclear cells.

Fig.7A-7E. Graphs induction of IL-10 (PG/ml) in peripheral blood mononuclear cells after stimulation in vitro, depending on the number of bacteria in the hole, for strains AH121A and Bifidobacterium 35624;

Fig.8A-8D. Graphs induction of IL-1β (PG/ml) in peripheral blood mononuclear cells after stimulation in vitro, depending on the number of bacteria in the hole, for strains AH121A and Bifidobacterium 35624;

Fig.9A-9D. Graphs induction of IL-6 (PG/ml) in peripheral blood mononuclear cells after stimulation in vitro, depending on the number of bacteria in the hole, for strains AH121A and Bifidobacterium 35624;

Fig.10A-10D. Graphs induction of IL-8 (PG/ml) in peripheral blood mononuclear cells after stimulation in vitro, depending on the number of bacteria in the hole, for strains AH121A and Bifidobacterium 35624;

Fig.11A-11D. Graphs induction of IL-12p70 (PG/ml) in peripheral blood mononuclear cells after stimulation in vitro, depending on the number of bacteria in the hole, for strains AH121A and Bifidobacterium 35624;

Fig.12A-12E. Graphs induction of TNF-α (PG/ml) in peripheral blood mononuclear cells after stimulation in vitro, depending on the number of bacteria in the hole, for strains AH121A and Bifidobacterium 35624;

Fig.13A-13C. Graphics the induction of IFN-γ (PG/ml) in peripheral blood mononuclear cells after stimulation in vitro, depending on the number of bacteria in the hole, �La strains AH121A and Bifidobacterium 35624;

Fig.14A-14D. Graphics the induction of G-CSF (PG/ml) in peripheral blood mononuclear cells after stimulation in vitro, depending on the number of bacteria in the hole, for strains AH121A and Bifidobacterium 35624;

Fig.15. Bar chart showing the influence of AH121A on the production of IL-10 and IL-12p70 in human myeloid dendritic cells type.

Fig.16. Bar chart showing the influence of AH121A on human T cells, not exposed to CD4.

Detailed description of the invention

Strain Bifidobacterium longum AH121A was deposited in the National collection of industrial and marine bacteria (NCIMB - Aberdeen, UK) 5 November 2009, giving it the number NCIMB 41675.

Strain Bifidobacterium longum UCC35624 was deposited in the National collection of industrial and marine bacteria (NCIMB - Aberdeen, UK) 13 January 1999, giving it the number NCIMB 41003.

Strain Bifidobacterium longum may be a genetically modified mutant or naturally occurring variant.

Strain Bifidobacterium longum preferably has the shape of living cells.

Alternatively, the strain Bifidobacterium longum may be in the form of non-living cells.

In the context of the present description, the term "animal companion" means a domestic animal. Preferably, the term "animal companion" means a domestic animal of the family Felidae (cat) canine (dog), rabbit�, ferret, horse, cow and similar animals. Most preferable the "animal companion" means a domestic animal of the cat family.

Strains of bifidobacteria lactic acid

In the first type of embodiments of the present invention, allowing for the many variations without departing from the idea and scope of the present invention, it is proposed that strain of lactic acid bacteria of the genus Bifidobacteria, which can be obtained by isolating it from the cut and washed feline gastrointestinal tract, and which has a probiotic effect in relation to animals. Probiotics are microorganisms, live or dead, their constituents such as proteins, carbohydrates, or purified fractions of bacterial ferments that positively impact the animal host. In addition, this term may also be extended to non-living cells, for example, destroyed the culture, or formulations or compositions containing beneficial factors produced by probiotic bacteria. They can include microorganisms, killed under the influence of high temperature, extreme pH, or pressure. For the purposes of the present description assumes that the term "probiotics" additionally includes the metabolites produced by microorganisms in accordance with the present invention during broze�Oia, if not otherwise specified. Such metabolites may be released by them in the fermentation medium, or stored in the microorganisms. In the context of the present description, the term "probiotic" includes bacteria, homogenized bacteria, extracts of bacteria, the bacterial supernatants of enzymes and their mixtures, which have a beneficial effect on the animal host when feeding the animal at therapeutic doses.

It was determined that the lactic acid bacteria of the genus Bifidobacteria, selected directly from dissected and washed gastrointestinal tract of mammals, have affinity for the gastrointestinal tract when fed to their respective mammal in the form of live bacterial cells, and they also have a significant immunomodulatory effect in the mammal when fed in the form of living cells, living cells, or in fraktsionirovanii. Although theoretically this is not necessary, it is believed that probiotic bifidobacteria in accordance with the present invention cause a favorable reaction in the host organism, what is their probiotic action. It was determined that probiotic bifidobacteria isolated from dissected and washed gastrointestinal tract, can modulate the host immune system due to the immediate�public interaction with the mucous membrane of the epithelium and cells of the immune system of the host. This immunomodulatory effect, in combination with other kinds of influences, traditionally associated with probiotic bacteria, in particular, prevention of affinity to intestinal pathogens through direct impact on them or due to competition for nutrients, provides exceptionally high efficiency bifidobacteria in accordance with the present invention as a probiotic organism.

Bifidobacteria according to the present invention, obtained by extraction from dissected and washed feline gastrointestinal tract, possess antimicrobial activity in vitro against several pathogenic strains/species of bacteria. Although theoretically this is not necessary, such an antimicrobial effect in vitro indicates their potential probiotic activity in animals in vivo, preferably in relation to animal companions, such as animals of the cat family. Lactic acid bacteria according to the present invention preferably has an antimicrobial effect against Salmonella typhimurium, Listeria monocytogenes, Listeria innocua or Eschericia coli, preferably against a mixture of these strains, and even more preferably against all of these strains.

Although theoretically this is not necessary, it is believed that the antimicrobial action of lactic acid bacteria in accordance with the USA�AI with the present invention may be the result of different interactions involving lactic acid bacteria according to the present invention. Well-versed in the art has previously suggested that some strains of bifidobacteria isolated from faeces samples, have a probiotic effect in the gastro-intestinal tract after oral consumption due to the fact that they form a barrier layer on the intestinal mucosa and thus prevent the attachment to the mucous membrane of pathogenic organisms. This requires the consumption of "live", rather, viable cells of bacteria, so that in the intestine caught colony of such bacteria. We, however, believe that bifidobacteria according to the present invention, obtained by extraction from dissected and washed feline gastrointestinal tract, may have potent probiotic action as their consumption in a viable form, and not in a viable form by developing, during fermentation in vitro of a substance or substances that inhibit the growth of pathogenic microorganism, or kill pathogenic microorganisms, and/or enhance the immune abilities of the animal host. This form of probiotic action is very desirable, because the bacteria in accordance with the present invention may be given to the animal in the form of live cultures, a lifeless sack�ur or purified products of fermentation, nevertheless, to provide for the animal-owner of a beneficial therapeutic effect.

Preferably, the lactic acid bacteria according to the present invention can remain viable after passing through the stomach. This is desirable in order to live cultures of bacteria can be taken orally, and after passing through the esophagus and the stomach, they could colonize the gastro-intestinal tract. Colonization of the gastrointestinal tract lactic acid bacteria according to the present invention is desirable in order that they could have on the animal host positive long-term probiotic effect. Oral supplementation is not viable cells or selected components of them also gives a temporary advantage, but because the bacteria are not viable, they can't grow and constantly to exert a probiotic effect in situ. As a result, may require constant doses of bifidobacteria owner to maintain that they provide health benefits for the owner. In contrast, viable cells that are able to pass through the gastrointestinal tract and remain viable, colonizes mucous membranes of the intestine due to the adhesion to and subsequent expansion, thereby providing�ivut permanent probiotic effect in situ. Therefore, it is preferable that the lactic acid bacteria according to the present invention maintained their viability after the environment-suspension having pH 2.5, for 1 hour. In the context of the present description, the term "viable" means that at least 25% of bacteria, originally placed in suspended culture medium remain viable on the results of the method of flow calculation, known versed in the art. The term to remain viable" preferably means that at least 50% of bacteria, originally placed in suspended culture medium remain viable. In this case it is also preferable that the lactic acid bacteria according to the present invention maintained their viability when exposed to environments with low pH values, as they reflect the influence of gastric juice in the stomach and upper intestine in vivo during oral their consumption of animals.

The strain of lactic acid bacterium types Bifidobacteria obtainable by isolation from dissected and washed feline gastrointestinal tract, can be used to ensure probiotic effects when oral consumption of animals, preferably animals-companions, or person. Not limiting approx�ry beneficial effects of oral consumption of the proposed bifidobacteria on the components of the health and physiology of the animal include therapeutic withdrawal symptoms, or prevention of disease States such as inflammatory disorders, immunodeficiency, a disease of inflamed bowel, irritable bowel syndrome, cancer (particularly neoplasms of the gastrointestinal and immune systems), diarrhoeal condition, diarrhea associated with taking antibiotics, appendicitis, autoimmune disorders, multiple sclerosis, Alzheimer's disease, amyloidosis, rheumatoid arthritis, arthritis, abnormal joint mobility, diabetes mellitus, insulin resistance, bacterial infections, viral infections, fungal infections, periodontal disease, urogenital diseases, injuries caused by surgical interventions, metastatic disease due to surgery, sepsis, weight loss, obesity, excessive accumulation of adipose tissue, anorexia, seizures, fever, cachexia, wound healing, ulcers, infections of the gastrointestinal tract, allergies, asthma, respiratory disease, disorders of the circulatory system, ischaemic heart disease, anaemia, disorders of the device blood clotting, kidney disease, disease of the Central nervous system, liver disease, nutritional disorders, osteoporosis, endocrine diseases, diseases of the skin. Most preferred is the application of strain to treat diseases of the gastrointestinal tract, including Le�ood and prevention of diarrhoea correction of the immune system, preferably the treatment or prevention of autoimmune diseases and inflammations; the preservation or improvement of the health of the skin and wool, preferably the treatment or prevention of atopic diseases of the skin, eliminate or reduce the effects of aging, including level of consciousness and mental activity; the prevention of disorders associated with the axis hypothalamus-pituitary-adrenal gland, improve the condition of joints, including their mobility.

The results of treatment of the above diseases can be assessed using methods known to one versed in the art. So, for example, inflammatory disorders, including autoimmune diseases, can be detected and observed with the use of such tests on the immune system in vivo as blastogenic lymphocytes, the activity of natural killer cells, production of antibodies in response to vaccines, delayed hypersensitivity, and combinations thereof. Below is a brief description of these methods, although they are well known to one versed in the art.

1. Blastogenic lymphocytes. During this test measured the proliferative response in vitro of lymphocytes isolated from fresh whole blood of test and control animals at various mitogens, and is an overall assessment of the options�functioning of T and b cells. To conduct this test from whole blood was isolated peripheral blood mononuclear cells using the methods of centrifugation and separation by density Ficoll-Hypaque. Dedicated peripheral blood mononuclear cells were washed twice in medium RPMI 1640 with the addition of HEPES, L-glutamine and penicillin/streptomycin. The washed cells were resuspended in medium RPMI 1640, determined their number and brought to the desired concentration of cells. 2×105cells were exposed to the effect of a range of concentrations (from 0.1 mcg/ml to 100 μg/ml) of various mitogens, some examples of which include the mitogen Phytolacca (manufactured by Gibco), phytohemagglutinin (Gibco) and concanavalin And (manufactured by Sigma), three times for 72 hours at 37°C in atmosphere containing 5% CO2with the addition of 10% fetal bovine serum (manufactured by Sigma). After 54 hours the cells were irradiated H-thymidine activity 10 μci, after which cells were collected and carried to the counting of scintillations with the help of the device TopCount NXT at 72 hour intervals.

2. The activity of natural killer cells. As described in U.S. patent 6310 090, this test allows you to measure activity in vitro natural killer cells isolated from fresh blood of experimental and control animals. Natural killer (NK) cells are components of the innate immune function of a mammal. For the assessment�Ki cytotoxic activity of NK-cells was used for cell adenocarcinoma of the thyroid gland cats. Previously it was shown that the cell line was sensitive, that is, killed NK cell cats. Target cells were grown in a flask Kzt75 containing 20 ml of minimal medium (MEM production Sigma Chem. Co., St. Louis, Missouri, USA) with 10% fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin. After reaching confluently target cells were treated with trypsin, washed three times and were resuspended in complete medium (RPMI-1640+10% fetal bovine serum +100 units/ml penicillin +100 µg/ml streptomycin) to a concentration of 5×105cells/ml On three aliquots of target cells with a volume of 100 μl was applied with a pipette on the tablet with 96 holes with a U-shaped bottom (manufactured by Costar, Cambridge, mA) and incubated for 8 hours in preparation of cells for better adhesion to each other. After that, the target cells were added to the lymphocytes (cells-effectors, 100 μl) isolated by centrifugation on Ficoll-Hypaque (as described above), so that the ratio of the number of cells-effectors to the number of target cells (the ratio (E:T) was 10:1. After 10 hours of incubation at 37°C was added 20 μl of substrate, containing 5 mg of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT). The mixture was incubated for 4 hours at 37°C, after which not metabolized MTT OTS�see a significant. The formazan crystals were dissolved by adding 200 μl of 95% ethanol. The optical density was measured at a wavelength of 570 nm using a reader for microplates. The percentage of specific lysis of NK-cells was calculated as follows:

Specific cytotoxicity (%)=

100×{1-[(ODtarget cells and cells-effectors-ODcells-effectors)/(ODtarget cells)]}

3. The development of antibodies in response to vaccines. Experimental subjects were given a set of (up to 5 vaccines) after at least 12 weeks after feeding with probiotic or control feeding. The vaccine may be a mixture of vaccine for primary and booster vaccination. Non-limiting examples of sets of vaccines that can be used for this test may include a mixture of vaccines manufactured by Fort Dodge Animal Health. He limiting examples of vaccines that can be used for this test may include vaccines against feline plague, adenovirus infection, corona virus infection, parainfluenza, and parvovirus. A specific type of vaccines used also determines the history of vaccination of the experimental subject. The level of antibodies specific to the vaccine in the blood was measured for 3 weeks, and thus determined the duration and strength of the immune response in the control group and the group treated with probiotic food.

4. G�perusteellisesti delayed type. This test is a non-invasive method of assessing the state of the immune system in vivo. The test is performed subcutaneous injection of polyclonal mitogen "phytohemaglutinin" (PHA) in combination with sheep red blood cells, as a multivalent vaccine, histamine (100 μl histamine-phosphate 0,0275 g/l production Greer, Lenoir, NC, USA) or saline phosphate buffer (PBS 8.5 g/l production Sigma). Immune response was measured and recorded in the form of the thickness of skin folds with calipers after 0, 24, 48 and 72 hours after injection. The increased thickness of the skin fold greater hypersensitivity immune response, which can be reduced by treatment of the bacteria in accordance with the present invention.

Additional methods to assess the effect of the use of bifidobacteria in accordance with the present invention are described in U.S. patents 6133323 and 6310090.

In addition, the efficiency of elimination of the effects of aging can be determined by dual x-ray absorptiometry or computed tomography scanning, which allows to determine the mass of body fat and bone mineral content. Similarly, this method can also be used to determine anatomical changes, such as losing weight or changing tightly�T. bone tissues in the test subjects after infection.

Bifidobacteria according to the present invention can also be used to reduce the level of stress in companion animals. To determine the level of stress and also during the subsequent adjustments and the decrease can be measured levels of stress hormones in the blood, such as epinephrine, norepinephrine, dopamine, cortisol, C-reactive protein and other acute phase proteins. These hormones are recognized biomarkers of stress, and their content can be easily measured by methods well known to one versed in the art. In addition, with the help of computer tomography can be settled directly measured the size of the adrenal gland as an indicator of the activity of the axis hypothalamus-pituitary-adrenal in vivo.

Furthermore, by assessing the state of the skin of the coat of the animal companion, conducted by an experienced professional can be identified problems with the skin and wool cover, and observed progress in improving the condition of skin and a wool cover. Examples of assessment criteria skin and a wool cover may include: a) the rate of shedding is determined according to the standard procedure by which to comb the hair, harvested and weighed brushed hair, and their weight compare experience with animal control; (b) subjective evaluation of skin and fur is is an experienced specialist in this fact�frames, as shedding, dandruff, Shine, uniformity, softness and density of the wool; (C) the functional assessment of the barrier function of the skin is performed by wiping the skin area with gauze soaked in acetone. In this method, the skin is removed unicellular layer, resulting in that section of the skin is broken its barrier function is provided by the lipid fractions of the stratum corneum. Breach the barrier function is characterized quantitatively by measuring the increase in transepidermal water loss (TEWL) and the degree of redness on the damaged skin of ways well known to one versed in the art. Redness is measured in points using a special camera and lighting system. Indicators of water loss (TEWL) and erythema in scores measured before skin treatment, immediately after treatment, at the end of the 5th and the 24th hour after treatment, and based on these data, assessed the status of the protective and healing functions of the skin.

In addition, treatment of infections of the gastrointestinal tract of companion animals may include improvement of their bacterial flora. Improvement of the microflora in companion animals preferably includes reduction of the number of pathogenic bacteria in the feces of companion animals. The content of pathogenic bacteria in the feces of companion animals �can be quantified using a standard method of flow calculation, known versed in the art. To pathogenic bacteria, preferably bacteria are selected from the group consisting of bacteria species of Clostridia, Escherichia, Salmonella, and similar mixtures thereof. Non-limiting examples of suitable strains of pathogenic bacteria include C. perfringens, C. difficile, Eschericia coli, Salmonella typhimurium, and mixtures thereof.

A method of using bacteria in accordance with the present invention may also include the prevention or treatment of diseases of the urinary tract of domestic animals, preferably companion animals. Non-limiting examples of treatment or prevention of diseases of the urinary tract may include treatment or prevention of infections of the urinary tract, the treatment or prevention of kidney diseases, including stone disease, treatment or prevention of bladder infections and similar diseases. Although theoretically this is not necessary, the authors believe that bifidobacteria according to the present invention may be useful to prevent the above disease States due to its ability to decompose oxalic acid that has been shown in experiments in vitro. Oxalic acid is a byproduct of metabolic processes occurring in the urinary tract, and its salts can give insoluble residues, which can lead to infections of the kidneys, urinary PU�Ira and other departments of the urinary tract. Due to decomposition of the salts of oxalic acid and the prevention of their accumulation and precipitation in the urinary tract, bacteria in accordance with the present invention can be used for the treatment and prevention of infections and other painful conditions of the urinary tract. The decomposition of oxalic acid can be measured in vitro using the # 755699 offered by Boehringer Mannheim/R-Biopharm.

Bifidobacteria according to the present invention can be used to preserve and improve the health of an animal companion by improving the digestion of fibers. Improve the digestion of fibers is very desirable, as it contributes to the growth mentioned bifidobacteria and the favorable development of the endogenous microflora, which in turn contributes to the suppression of some potentially pathogenic bacteria. In addition, it was reported about the decrease in the number of toxic metabolites and detrimental enzymes produced in the fermentation processes, caused by pathogenic microorganisms in humans (6). The degree of digestion of fibers can be determined by the method described by Vickers and co-authors (7), with the difference that instead inoculation of diluted faeces samples should be used pure cultures of the studied bacterial strains.

Strains of probiotic bacteria isolated from animals seme�STV cat, can be used to reduce fetid odors of feces and urine from the trays for toilet companion animals, by reducing the formation of compounds in the feces and urine that cause unpleasant odors. Non-limiting examples of compounds that cause bad odors include ammonia, indoles, phenols, amines, fatty acids with branched chains and volatile compounds containing sulphur. Although theoretically this is not necessary, it can be expected that the decrease in the content of these compounds in the feces or urine of an animal companion will lead to the weakening of unpleasant smells from urine and feces.

A method of using lactic acid bacteria in accordance with the present invention generally includes oral consumption of animals. Oral intake can be a part of a normal diet, or may be conducted in addition to it. Oral consumption is held at least once a month, preferably at least once a week, and even more preferably at least once a day. Lactic acid bacteria according to the present invention may be given to a pet, preferably an animal companion, in a therapeutically effective amount sufficient to maintain or improve the condition of his health. In Kontek�those of the present description, the term "therapeutically effective amount" in relation to lactic acid bacteria means, the number of bacteria is sufficient to provide a desired effect on the animal requiring such treatment, and at the same time small enough to avoid side effects such as toxicity, irritation or allergic reaction, that is, provide a reasonable ratio of benefit/risk when receiving bacteria in accordance with the present invention. The specific "therapeutically effective amount" in each case will depend on factors such as the specific diagnosis of the animal, the physical condition of the animal, duration of treatment, prescribed dose, the possible presence of both the treatment used by the media, the solubility of the form of a specific drug and dosage regimen.

Lactic acid bacteria is preferably assigned to an animal companion at a dose of 1.0×104CFU per day to 1.0×1014SOME day, more preferably from 1.0×106CFU per day to 1.0×1012CFU per day. The composition according to the present invention preferably contains at least 0.001% of lactic acid bacteria, i.e. from 1.0×104CFU/g to 1.0×1011CFU/g of lactic acid bacteria of the genus Bifidobacteria isolated from dissected and washed feline gastrointestinal tract. Lactic acid bacteria can be fed to the animal in the form of living cells, mersul�nnyh cells distillates, isolates or other fractions of the fermentation products of lactic acid bacteria according to the present invention, or any mixtures thereof.

Preferably, the lactic acid bacteria, or isolated or purified fractions thereof were used to prepare compositions that are designed to maintain or improve the health status of the animal. As mentioned above, the composition may be a part of a normal diet, or given to the animal advanced. In cases where the composition is part of a usual diet, it can be in the form of dry fodder for animals, for example, biscuits or pellets or feed treated grain, moist pet food, yogurts, sauces, products for chewing, treats and other things.

Such compositions may contain additional components. Such additional components may be useful for inclusion in the proposed formulations, but in the context of the present invention, they are only optionally available. So, for example, food compositions preferably should be balanced in nutritional value. In one of the embodiments of the nutritional compositions may contain (in terms of dry substance) from about 20% to about 50% crude protein, preferably from about 22% to about 40% crude protein, total weight with food�of the support. The crude protein material may include any material, the protein content of which is at least 15% by weight, and non-limiting examples of such materials include materials of plant origin containing protein, such as soy beans, cotton seeds, and peanuts, animal material containing protein, such as casein, albumin and products on the basis of muscle tissue. Non-limiting examples of products on the basis of muscle tissue, which can be used in accordance with the present invention, include fresh meat and dried meat and melted products, such as fishmeal, feed the birds, meat food, animal feed, bones and the like. Other types of suitable sources of protein include wheat gluten or corn, as well as proteins extracted from sources on the basis of microorganisms, such as yeast.

In addition, the food compositions may contain, based on dry substance, from about 5% to about 35% fat, preferably from about 10% to about 30% fat by weight of the total weight of the food composition. In addition, the nutritional compositions containing lactic acid bacteria according to the present invention, can also contain from about 4% to about 25% total dietary fiber. The compositions may also contain a source of starch to�described in WO 99/51 108.

The compositions according to the present invention may further contain a source of hydrocarbons. Examples of such sources are cereals or cereal, such as rice, corn, barley, alfalfa, wheat and similar products. In addition, the composition may also contain other materials, such as whey powder or other dairy products.

Compositions containing bacteria according to the present invention, can also contain a prebiotic. The term "prebiotic" includes substances or compounds that are fermented by the intestinal flora of the animal companion and thereby contribute to the growth or development of lactic acid bacteria in the gastrointestinal tract of an animal companion at the expense of pathogenic bacteria. As a result of this fermentation produces fatty acids, in particular produced short-chain fatty acids in the colon. The result is a reduction of the pH in the colon. Non-limiting examples of suitable prebiotics include oligosaccharides, such as inulin and its hydrolysis products, known as fructooligosaccharides, galacto-oligosaccharides, Xylo-oligosaccharide and oligo derivatives of starch. The prebiotic may be in the composition in any suitable form. For example, the prebiotic may be in the composition in the form of plant material, with�holding the fiber. Suitable plant materials include asparagus, artichokes, onions, wheat, or chicory, or products remaining in the processing data of plant materials. Alternatively, the prebiotic fiber can be in the composition in the form hinolinovogo extract, for example, suitable extracts of chicory. Suitable inulinase extracts offers Orafti SA (Belgium) under the trade name "Raftiline". So, for example, as a source of inulin suitable drug Raftiline (g) ST, which represents a fine white powder containing from about 90% to 94% by weight of inulin, up to about 4% by weight of glucose and fructose and from about 4% to about 9% sucrose.

Alternatively, the fiber may be present in the form of FOS, such as "Raftilose" offered by Orafti SA (Belgium). Is suitable, for example, the product Raftilose (g) P95. Alternatively, fructo-oligosaccharides can be obtained by hydrolysis of inulin, enzymatic methods or by use of microorganisms.

A suitable process of producing dry pet food for companion animals is boiling with extrusion, although can be used and sintering, and other suitable processes. When using extrusion cooking process usually turns out dry food for companion animals in the form of granules. When using prebiotic he could be mixed with other components of the animal feed prior to further processing. A suitable process is described, for example, in European patent application No. 0850569. When using a probiotic microorganism preferably, the body was deposited in the form of a coating layer on dry food for companion animals, or from the inside was filled with dry food for companion animals. A suitable process is described, for example, in European patent publication EP 0862863.

If the composition has the appearance of wet food, for the manufacture of meat products containing meat and stimulated bifidobacteria can be used in the processes described in U.S. patents 4781939 and 5132137. Can also be used and other processes for the manufacture of products type of whole meat, such as cooking in a steam oven. Alternatively, can be produced by products such as sausage meat by emulsification of a suitable meat material, adding a suitable gelling agent, and heating the meat emulsion with the gelling agent and the subsequent filling of cans or other suitable containers. Generally wet formulations for animal feed can contain from about 7% to about 15% protein, from about 1% to about 10% fat, and from about 1% to about 7% fiber. Non-limiting examples of ingredients that can be used in wet food compositions that include chicken, Turkey poults�well, beef, white fish, chicken broth, Turkey broth, beef broth, liver, chicken, brewer's rice, corn bran, fishmeal, egg, beet pulp, chlorides, linseed mass, mutton, waste production of beef or chicken, and mixtures thereof. In another embodiment of the compounds used as food additives, such as biscuits, chewing foods or other "treats" may contain, based on dry substance, from about 20% to about 60% protein, or from about 22% to about 40% protein by weight by weight of the composition. In another example, the compounds used as food additives may contain, based on dry substance, from about 5% to about 35% fat, or from about 10% to about 30% fat by weight, of the total weight of the composition used as an additive. In General, the nutritional compositions and formulations that are used as supplements designed for felines, well known to one versed in the art.

Food for companion animals may contain other active substances such as fatty acids with long chains and zinc. Suitable fatty acids are long chains include the α-linoleic acid, γ-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid. A suitable source of eicosapentaenoic acid and docosahexaenoic acid is �yby fat.

Suitable sources of γ-linolenic acid are oil borage, oil black currant seed and evening primrose oil. Safflower oil, sunflower oil, corn oil and soybean oil are suitable sources of linoleic acid. These oils can also be used for coating substrates as described above. The zinc may be in the composition in any suitable form, e.g., in the form of zinc sulfate or zinc oxide. In addition, most of the ingredients traditionally used in animal feed, are sources of fatty acids and zinc. It was observed that the combination of chicory, a source of prebiotic, oil, rich in linoleic acid, such as, for example, soybean oil, provides unexpected advantages, presumably explain the synergistic effect.

If the composition has the form of a sauce, it preferably contains at least 10% of the broth, non-limiting examples of which include the broth, vegetables, beef, chicken or ham. A typical composition in the form of a sauce may contain from about 0.5% to about 5% crude protein, from about 2% to about 5% crude fat and from about 1% to about 5% fiber.

Other non-limiting examples of additives that may be used in accordance with the present invention include powders, oil suspensions, suspense� milk-based, the cheeses, the compositions based on cocoa butter, pills and capsules. If the composition has the form of a tablet, to keep it solid and compressed form, we need the appropriate binder. Non-limiting examples of suitable binders include natural resins, such as xanthan gum, pectin, lecithin, alginates and other compounds known to one versed in the art. If the composition has the form of a capsule, it must be enclosed in a shell, as is known versed in the art. Non-limiting examples of suitable materials for membranes include polyvinyl alcohol (PVA), polyvinylpyrrolidone, alginates and gelatin. The compositions of yogurt can contain from about 1% to about 5% protein, from about 10% to about 20% of hydrocarbon, from about 1% to about 5% fiber, from about 1% to about 5% fat and from about 50% to about 90% of liquid carrier, such as milk.

Examples

The following examples are given only for illustration of the present invention, and not meant in any way to limit the scope of the present invention.

Example 1. The allocation of Bifidobacterium longum AN

Strain Bifidobacterium longum AH121A was isolated from tissue samples of the cat's intestines.

Tissue samples of the cat's intestines were obtained from healthy cats, converted to local veterinarians for mtan�Ziya at the initiative and with the approval of the owners. All animals were healthy and did not have any disease. Each cat cut thick, blind and ileum and dissected them to expose the mucosa.

The tissue samples mechanically homogenized vigorously shaken for 1 minute, and collected the supernatants. Each supernatant were applied to the agar de Mann Rogosa Sharpe (MRS), and then incubated for 48 hours at 37°C in the instrument Anerocult GasPak. Obtained after incubation the colony of cups-passaged touch tighter and environment MRS again grown under the same anaerobic conditions. Received after this colony-passaged touch 4 more times, until, so far, has not received a pure colony of the same strain. Under a microscope, studied the morphological properties of the colony. Suitable isolates were tested for gram reaction and catalase activity. Testing on active pharmaceutical ingredients (API set 5 OCHL production BioMerieux) showed that bacteria are gram-positive and catalase-negative. The collected cells were washed twice with 0.05 M phosphate buffer (pH 6.5) and cysteine-HCl (500 mg/l), and then destroyed by ultrasound. Using centrifugation removed the remains of the cells. The supernatants were incubated with NaF (6 mg/ml) and iodoacetate sodium (10 mg/ml) for 30 min at 37°C. the Reaction was terminated by incubation with hydroxylamine-HCl (pH 6.5) in those�Linux for 10 minutes at room temperature. Was added HCl (4M), FeCl3·6H2O (5% weight/volume in 0.1 M HCl) and fructose-6-phosphate sodium, and watched the color change. Redness testified about the formation of acetyl phosphate from iron chelate through hydroxylation.

Identification of species of bacteria

For additional identification of the selected bifidobacteria was carried out the sequencing of the intergenic spacer region 16s (hereinafter referred to as IGS) by the following method. From bacteria strain AH121A DNA was extracted using 100 μl of extraction solution and 25 µl preparaciones solution for fabric (set of reagents XNAT2 production Sigma). The samples were incubated for 5 min at room temperature, then 2 hours at a temperature of 95°C, after which was added 100 μl of neutralizing solution from the same set Sigma XNAT2. Determined the amount of genomic DNA in solution using the Nanodrop spectrophotometer. The obtained samples were stored at 4°C. was Performed polymerase chain reaction with IGS-specific primers: IGS L: 5'-GCTGGATCACCTCCTTTCT-3' (identity No. 3, resulting in a received sequence identification number 1) and IGS R: 5'-CTGGTGCCAAGGCATCCA-3' (identification No. 4, resulting in a received sequence ID 2). The reaction was carried out under the following cycle parameters: 94°C for 3 minutes (1 cycle), 94°C for 30 seconds, 53°C in the Techa�s 30 seconds 72°C for 30 sec (28 cycles). The reaction mixture for PCR contained 4 ál (50 ng) DNA, PCR XNAT2 production Sigma, 0.4 µm of primers IGS IGS L and R (MWG Biotech, Germany). Reactions were carried out on the amplifier Eppendorf. For analysis of gene expression IGS PCR products (10 µl) were separated in 2% agarose gel with TAE buffer, stained with ethidium bromide, with a molecular weight marker (in increments of 100 pairs, production Roche). Separate the amplification products corresponding to the individual bands after separation in the gel, purified using the kit reagents Wizard PCR production Promeg. Purified PCR products are sequenced using IGS-specific primers, the sequence of which is given above. Decoded sequence was tested on a database of nucleotide sequences NCBI for homology with known sequences using the standard program BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/). After finding the closest sequences produced their alignment with the target by using the DNASTAR program MegAlign. The obtained sequence (direct sequence IGS, the identification number 1 and return a sequence of IGS, ID 2, is given in the list of sequences). Search our database NCIMB showed that the strain AH121A characterized by a unique last�dovalidate IGS (direct and inverse), having a high degree of homology with the corresponding sequences of the bacteria Bifidobacterium longum.

Example 2. Screening on agar with the dye Congo red

For phenotypic screening of bacterial strains expressing EPS (extracellular polysaccharides) test was used for the agar with the dye Congo red. The procedure consisted in the following. In 10 ml of modified (addition of 0.05% cysteine) broth medium Rogosa were aseptically inoculable freshly grown colony of bacterial strain, after which incubation was carried out anaerobically at 37°C until the turbidity (approximately 16 to 24 hours). Broth culture was aseptically stehovani on cups with agar containing the dye Congo red and incubated at 37°C for 48 hours. It is believed that EPS produced as a by-product of growth and/or metabolism of certain strains of bacteria, prevents the uptake of the dye Congo red, resulting in a colony of a milky white colour. Strains that produce less EPS, stronger stained with the dye Congo red, resulting in a colony has a color between pink and red. Strains that do not produce EPS, become red and look almost transparent against the red agar.

As shown in Fig.1: colonies of B. longum AH121A have no�club mucoid morphology and a bright white color.

Example 3. The stability of B. longum AH121A against bile salts and low pH

This experiment was conducted to determine the stability of the feline isolates of bacteria B. longum AH121A against different concentrations of bile, as well as to assess the survival of bacteria at a pH of 2.5 to 6 hours.

Experimental design

Resistance of bacteria of the strain B. longum AH121A against bile was determined by sowing lyophilized bacteria on cups with agar MRS/RCA, to which was added feline bile (at a concentration of 0,5; 1,0; 2,0%). Was also evaluated the survival rate of strain at a pH of 2.5 to 6 hours, meth flow counting for 5 minutes before incubation and after 5, 30, 60, 120, 180 and 360 min after the start of incubation.

Detailed description of the method

The procedure for determining the resistance of bacteria against feline bile was as follows.

Cups with different concentrations of bile was prepared from concentrated 45% solution of synthetic bile, which was subjected to heat treatment at 80°C for 10 min to kill any foreign organisms.

Medium with different concentrations of bile was prepared as follows:

Medium with 2% bile: 6,67 ml concentrate bile +143,33 ml agar

Medium with 1% bile: 3,33 ml concentrate bile +146.67 ml agar

Wednesday with 0.5% bile: 1,67 ml concentrate bile +148.33 ml agar

For each dilution was removed Koli�the ETS agar after autoclaving, and added instead the proper amount of concentrate bile.

Fresh cups of bile was prepared daily (although they can be stored for weeks). Pre-determined the number of CFU/g of each sample was lyophilized bacteria method of smudging on the glass.

Bacteria were applied to the cups containing bile agar by resuspension of the lyophilized bacteria in 10 ml of sterile PBS buffer (at a concentration of 109CFU/ml), separation of the Cup into 4 equal sectors, and drawing on the Cup 4 strain samples (10 μl of cell suspension).

The cups were dried for 30 minutes (or until dry spots suspension on the agar), and then incubation was performed bacteria under appropriate conditions.

Procedure for assessing the survival of bacteria of the strain AH121A in conditions of low pH (pH 2.5) was as follows.

The acidification of the medium was performed by adding 6 M HCl. First acid was added to 100 ml of the broth was determined by how much acid to bring the pH of such amount of broth to 2.5. The corresponding amount of acid was added to the remainder of the MRS broth (4×100 ml). Pre-determined the number of CFU/g of each sample was lyophilized bacteria method of smudging on the glass.

Dry lyophilized bacteria were resuspended in acidified medium to obtain concentrations 10sup> 9CFU/ml and incubated under anaerobic conditions. Through 5, 30, 60, 120, 180, 240 and 360 after the start of incubation took aliquots, were applied to glass and determined the number of CFU in a milliliter of sample.

Results

Table 1
Growth of isolates AH121A in the presence of a feline bile
% bile (weight/volume)0,00,51,02,0
The survival of strain AH121A+++++++++++
+++= very good growth (100%)
++= good growth (66%)

Conclusions

The effects feline bile on the growth of bacteria of the strain AH121A presented in Table 1. As the table shows, the bacteria of this strain is able to withstand concentrations feline bile, less 2%.

Fig.3 shows the resistance of the strain to an environment with a pH of 2.5.

Example 4. The influence of V. lonsum AH121A on the ratio of expression of IL-10:IL-12

From the peripheral blood of healthy people mononuclear cells were isolated using preparazioni Vacutainer tubes production�TWA BD (catalog number 362761), in accordance with the manufacturer's instructions. Peripheral blood mononuclear cells were washed and were resuspended in modified medium Glutamax™ production Dulbecco containing glutamax (substitute glutamine) + pyruvate +4.5 g/l glucose (Gibco, catalog number 10569-010)+10% fetal bovine serum (Sigma, catalog number F4135), with the addition of 1% penicillin/streptomycin (Sigma, catalog number R). Peripheral blood mononuclear cells were incubated in the flat-bottomed plates with 96 wells (2×105cells in the hole) with the addition of 20 µl of bacterial suspension (concentration of bacteria 1×107CFU/ml) for 48 hours at 37°C in atmosphere containing 5% CO2. After incubation for 2 days cups centrifuged at 300 g, the supernatant was removed, and the obtained samples were stored at -80°C until further experiments. The levels of interleukin 10 (IL-10) and interleukin-l (IL-12p70) in the supernatant of the culture was determined using a kit with 96-hole production Meso Scale Discovery (Gaithersburg, Maryland, USA; catalog number KV-1).

Bacteria for joint cultivation experiments were prepared in two ways: (a) freshly grown bacteria were grown in medium Difco MRS and collected immediately after entering stationary phase. All cells were grown under anaerobic conditions at 37°C. (b) the Lyophilized bacteria expressed�ivali under anaerobic conditions at 37°C in medium Difco MRS and collected immediately after entering stationary phase. From each preparation of bacteria was prepared as a lyophilized powder and stored at -80°C in a separate container in the form of aliquots of 100 mg. Immediately before use, one aliquot of each strain was removed from the refrigerator and allowed to warm up to room temperature. For each experiment used a new aliquot. Each strain was washed 3 times in a test tube with a capacity of 10 ml and then centrifuged. For each of the growth conditions built calibration curves (the dependence of optical density on the number of living cells), and the drug with the washed cells normalized to obtain the desired number of cells before adding it to the peripheral blood mononuclear cells. In all the experiments, a control, which does not contain bacteria. All assays were performed in triplicate.

The results of experiments showing the influence of strain AH121A on the ratio of the expression levels of IL-10:IL-12, shown in Fig.2. The results are almost the same for freshly grown and lyophilized cultures.

Example 5. The influence of strain AH121A on the production of IL-10

The availability of adequate immune response to microbes is an important indicator of the overall health of the host body. Over-reaction can cause inflammatory conditions (e.g., colitis), while insufficient immune response leads to survival and races�ostranenie pathogens in the body. Described below are methods of analysis are well represented in the literature and are useful techniques for the determination of immunological activity of the host body in response to different microbes.

For experiments was collected peripheral blood mononuclear cells (monocytes, dendritic cells, b cells and T cells) in healthy volunteers and stimulated them in vitro bacterial strains. The supernatants of cultures were collected and identified them in the levels of cytokines.

IL-10 is an important cytokine that prevents aberrant proinflammatory immune response. In animals off the expression of IL-10 was developed colitis, and tumors of the gastrointestinal tract. To prevent such potentially dangerous immune responses regulatory cells of the immune system creating and using an IL-10. Increased expression of this cytokine is protected from excessive inflammation and excessive immune response to pathogens.

The production of IL-10 in peripheral blood mononuclear cells in vitro was determined 48 hours after co-incubation with bacterial strains. Strains AH121A 35624 and induced the production of IL-10 to the same extent (Fig.4-6).

Example 6. Bifidobacterium longum EN 1714 has immunomodulatory effects in a joint incubation with the cells of the human immune system that is different from the action of Bifidobacterim longum AH35624.

Materials and methods

Strain Bifidobacterium longum infantis UCC35624 (V) and a strain of Bifidobacterium longum AH121A were analyzed for induction of cytokines in mononuclear cells of peripheral blood. Bacteria for co-cultivation was prepared as follows. Bacteria were grown under anaerobic conditions at 37°C in medium Difco MRS and collected immediately after entering stationary phase. From each type of bacteria was prepared as a lyophilized powder and stored at -80°C in a separate vessel in the form of aliquots of 100 mg. Immediately before use, one aliquot of each strain was removed from the refrigerator and allowed to warm up to room temperature. For each experiment used a new aliquot. Each strain was washed 3 times in a test tube with a capacity of 10 ml and then centrifuged.

Conducted direct counting of cells under the microscope in a counting chamber Petrov-Hausser in accordance with the manufacturer's instructions. Before using the cells for analysis of cytokines in peripheral blood mononuclear cells, the solution is washed cells normalized to obtain the same number of cells per unit volume. In each cell for analysis was added 20 μl of a solution of bacteria in a saline solution with phosphate buffer (PBS), to obtain the total number of bacteria indicated in each experiment.

Analysis of the induction of cytokines in Mononoke�Arah peripheral blood

Peripheral blood mononuclear cells were isolated from peripheral blood of healthy people using preparazioni Vacutainer tubes BD production (catalog number 362761), in accordance with the manufacturer's instructions. Peripheral blood mononuclear cells were washed and were resuspended in a modified atmosphere Modified Eagle Medium Glutamax™ production Dulbecco containing glutamax (substitute glutamine) + pyruvate +4.5 g/l glucose (Gibco, catalog number 10569-010)+10% fetal bovine serum (Sigma, catalog number F4135), with the addition of 1% penicillin/streptomycin (Sigma, catalog number R). Peripheral blood mononuclear cells were incubated in plates with 96 flat-bottomed wells (2×105cells in the hole) with the addition of 20 µl of bacterial suspension with different concentrations of bacteria: a 2.5×108, And 1.0×108That is 5.0×107And 2.5×107, And 1.0×107and 1.0×106(up to 6 concentrations) for 48 hours at 37°C in atmosphere containing 5% CO2. The levels of interleukin 10 (IL-10) and interleukin-l (IL-12p70) in the supernatant of the culture was determined using a kit with 96 units of production Meso Scale Discovery (Gaithersburg, Maryland, USA; catalog number KV-1). The levels of human interleukin 1β (II-1b), human interleukin-6 (11-6), human interleukin 8 (11-8) human interleukin 10 (11-10), human interleukin� R (IR), human interferon-gamma (IFN-γ), human tumor necrosis factor alpha (TNF-α) and human G-CSF (granulocyte colony-stimulating factor) was quantified and expressed in pilgrimag per milliliter (PG/ml). For each sample was carried out for 3-5 experiments (corresponding to graphs A-E in the drawings).

Results

Two independent cultures (1 and 2) strain of Bifidobacterium longum infantis UCC35624 (V) and a strain of Bifidobacterium longum AH1714 investigated for immunomodulating properties analysis, induction of cytokines in peripheral blood mononuclear cells when adding bacteria in different concentration of 2.5×108, And 1.0×108That is 5.0×107And 2.5×107, And 1.0×107and 1.0×106(up to 6 concentrations). The supernatants were examined for the levels of various cytokines, including IL-1β, -6, -8, -10 and -12, IFN-α, IFN-γ and G-CSF. The measurement results are presented as mean values (PG/ml) ± standard deviation for different samples (A to E, that is up to 5 pieces).

Compared to the strain 35624 strain AH121A also gave a strong induction of the production of most cytokines, including IL-10, but some great, namely, increased levels of cytokines IL-6 and IL-8.

IL-10: incubation with AH121A induces increasing with the dose of bacteria the production of anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells after stimulation in vitro (Fig.7) the nature of the induction of IL-10 are qualitatively and quantitatively similar to the induction by incubation with strain 35624. Maximum induction of IL-10 is not observed until the number of bacteria, which were about 1.0×109in the hole.

IL-1β: incubation with AH121A induces increasing with the dose of bacteria production of the proinflammatory cytokine IL-1β in peripheral blood mononuclear cells after stimulation in vitro (Fig.8). The nature of induction of IL-1β qualitatively and quantitatively similar to the induction by incubation with strain 35624. Maximum induction of IL-1β was not observed until the number of bacteria, which were about 1.0×109in the hole.

IL-6: incubation with AH121A induces increasing with the dose of bacteria the production of cytokine IL-6 in peripheral blood mononuclear cells after stimulation in vitro (Fig.9). The nature of induction of IL-6 quantitatively different from induction by incubation with strain 35624: incubation with strain AH121A gives higher levels of IL-6, especially when small quantities of bacteria in the hole.

IL-8: incubation with AH121A induces increasing with the dose of bacteria the production of cytokine IL-8 in peripheral blood mononuclear cells after stimulation in vitro (Fig.10). The nature of induction of IL-8 quantitatively different from induction by incubation with strain 35624: incubation with strain AH121A gives higher levels of IL-8 in all doses of bacteria in the hole.

IL-12: incubation with AH121A induces increasing with the dose of bacteria production of the proinflammatory cytokine IL-12 in peripheral blood mononuclear cells after stimulation in itro (Fig.11). The nature of the modulation of the production of IL-12 has a bell-shaped form during incubation with both strains: AH121A and 35624: gradually reaches a peak and then begins to fall at higher concentrations of bacteria. Quantitatively, the expression of IL-12 is highly variable between different samples, but the average expression with bacteria AH121A similar expression with 35624 bacteria.

TNF-α: incubation with AH121A induces increasing with the dose of bacteria the production of proinflammatory cytokine TNF-α in peripheral blood mononuclear cells after stimulation in vitro (Fig.12). The nature of induction of TNF-α are qualitatively and quantitatively similar to the induction by incubation with strain 35624 in three of the 5 samples, and in 2 of 5 samples somewhat stronger induction (Fig.12C and 12E). The maximum induction occurs when concentrations of bacteria and 1.0×108in the hole.

INF-γ: incubation with AH121A induces increasing with the dose of bacteria the production of Pro-inflammatory cytokine INF-γ in peripheral blood mononuclear cells after stimulation in vitro (Fig.13). Quantitatively the expression of INF-γ is highly variable between different samples, but the average expression with bacteria AH121A similar expression with 35624 bacteria.

G-CSF: incubation with AH121A induces increasing with the dose of bacteria production of the cytokine G-CSF in peripheral blood mononuclear cells after stimulation in vitro (Fig.14). The nature of the induction of G-CSF qualitatively and quantitatively CX�well with induction by incubation with strain 35624.

Example 7. The influence AH121A on the production of cytokines in dendritic cells

Summary

The immune response is a carefully regulated process, which in normal operation provides protection against infection and tolerance to harmless antigens in the environment. However, when inflammation is excessively activated immune response leads to chronic Pro-inflammatory state characterized by activation of innate immunity and growth of a subpopulation of polarized T cells. Currently the treatment of inflammatory diseases is focused on the inhibition of key inflammatory mediators, i.e. populations of inflammatory cells. However, the use of this approach provides only a temporary suppression of symptoms of inflammation. Successful treatment or prevention, providing long-term relief from the symptoms of inflammation, can only be achieved by the activation of regulatory processes in cells that help protect the body from destroying its proinflammatory reactions. Bifidobacterium AH121A are probiotic microorganisms, selectively stimulating the production of IL-10 in the system of innate immunity (dendritic cells) and inducing the polarization of Foxp-3+regulatory T-cells in vitro. In vivo generation of IL-10 and regulatory T-cells are powerful supersolubility inflammatory response.

Regulatory T cells (Treg-cell)

The main role of regulatory T-cells (Treg-cells), which consists in the maintenance of immune tolerance has been demonstrated in several animal models in which adoptive the introduction of Treg-cells, or the intentional increase in the population of Treg-cells contributed to the prevention or cure a number of diseases provoked by T-cells, including allergies, asthma, inflammatory lung disease, autoimmune disease and allograft rejection, due to restoration of tolerance to allergens, auto-antigens and ALLO-antigens [8]. Described multiple mechanisms of immune suppression, the mediator of which are Tregcells, and in which a special role is played by the generation of IL-10 [9]. The absence or improper functioning of Treg-cells also correlates with the syndrome increase the level of IgE, gipereozinofiliya and autoimmune States, while the presence of Treg-cells correlates with immune tolerance [10]. Studies of the mechanisms by which the immune response to not pathological antigens in some cases leads to allergies, while others harmless to the immune response showed that Treg-cells that produce allergen-specific IL-10 (TR1-cells) are the dominant subpopulation of T-cell�OK in healthy people [11, 12]. Repeated exposure of bee venom antigens in healthy, non-allergic beekeepers during the season of harvest is a valuable in vivo model to confirm the mechanisms of immune tolerance to bee venom antigens [13]. After multiple bee stings are specific to antigens of bee venom TH1 and TH2-cells are transformed into TR1-cells producing IL-10. This occurs in parallel with the suppression of the skin response to allergens and late phase inhibition of allergen-specific TH1 and TH2-cells. These processes occur, while keeping the impact on beekeeper bee venom, and 2-3 months after the season of harvest, the expression levels of the respective cells are returned to the initial level.

Currently explores various strategies of stimulating Treg-cells in vivo. Such strategies include the adoptive transfer-induced or constitutive Tregcells and their stimulation of specific adjuvants or immunomodulators. These approaches look more attractive in comparison with traditional methods of treatment, as the suppression of the activity of antigen-specific Treg-cells leads to a General suppression of the immune system and at the same time provides long-term antigen-specific reg�the stimulation of immunity in vivo. Moreover, it is possible to individual (specific) treatment of patients with minimal side effects. Many immunomodulators, recently developed or currently under development, such as rapamycin, abatacept (blocker co-stimulation of CD80/CD86:CD28 offered Orencia), a non-mitogenic anti-CD3 monoclonal antibody, monoclonal antibodies capable of cleaving the T cells and directed against tumor necrosis factor (TNF-α has a direct or indirect effect on Tregcells in the form of stimulation or suppression of their function [14-18]. Studies in mice showed the feasibility of selective increase of population of Treg-cells that target the body (or lymph nodes, which removes the lymph from the body) due to the recognition of the allergen or auto-antigen, expressed in the fevered body [19]. Thus, the transfer of an organ-specific Treg-cells may be effective in suppressing the current of the disease, and carried Treg-cells do not necessarily have to recognize exactly the same antigen, the auto-aggressive effector T cells [20]. This has direct implications for treatment strategies aimed at the mechanism of immune tolerance to allergens, auto-antigens and antigens in transplantation, which involved Treg-cells. The possibility of adoptive transfer of Treg -cells or low molecular weight compounds that stimulate Tregcells in the tissues currently being investigated [19], but double-blind trials with placebo control have not been reported. At the moment the used antigen-specific approach, providing activation of development Treg-cells in humans, is a method for suppressing the initiation of transcription (allergen-SIT). Allergen-SIT activates Treg-cells and TR1-cells, and it seems that the treatment of adrenergic β2 antagonists increases the number and activity of this type of cells [21-23]. Recently it was reported on significant elements of transcription, regulating the expression of the Foxp3 promoter, and chances are they will become new targets for the development of new methods of treatment [24, 25]

Germs as new treatment methods

Interest in the intentional purpose of the microorganisms or products of their metabolism for the treatment of aberrant inflammatory activity is gaining momentum. Typical microorganisms studied currently include bifidobacteria, lactobacilli, pathogenic strains of E. coli and bacteroids [26-31]. The protective effect associated with these microorganisms probably provides a variety of mechanisms involving epithelial cells, dendritic cells and T cells. However, more importantly, what is increasingly reported�. recently, is their common ability to activate Treg cells. For example, studies in mice have shown that symbiotic microorganisms (probiotic cocktail VSL # 3), which enters the gastrointestinal tract, contribute to the development of Treg-cells in the mucosa, which reduces inflammation in colitis [32]. Moreover, the consumption of the strain Bifidobacterium infantis leads to transformations Tregcells and protects against induced by lipopolysaccharides activate NF-κ in vivo, and Lactobacillus reuteri induce Treg-cells that protect against allergic reactions in the respiratory tract in mice [33, 34]. Treg-cells are produced in the thymus, but their production can also be induced in peripheral organs, including the intestinal mucosa [35, 36]. CD103-positive dendritic cells in General are responsible for the transformation Treg-cells through processes that are dependent on transforming growth factor TGF-β and retinoic acid [37, 38]. It seems that the driving force behind such transformations are specific environmental factors of the gastrointestinal tract, associated with the presence of a large number of symbiotic microorganisms. It is unlikely, however, that all symbiotic microorganisms are equally effective in the induction of Treg-cells in vivo. A recent model study on mice, in which we compared various si�biotic microorganisms (Bifidobacterium longum EN 1206, Bifidobacterium breve AN and Lactobacillus salivarius AN), showed that the bacteria Bifidobacterium longum EN 1206 induced Tregcells and protected from the influx of eosinophils into the lungs and blocked the induction of serum IgE [39]. Other types of bacteria in such models has not been effective in the induction of Treg-cells and could not protect against allergic reactions. Therefore, the induction of Treg-cells can be considered as a critical feature of healthy microbiota that helps protect the body from possible development of aberrant immunological response to potential allergens. In addition to the use of living organisms for the treatment of allergies another interesting approach is the determination of microbial factors responsible for the beneficial effect provided by these microorganisms, and the use of only these selected factors. For example, the polysaccharide And isolated from Bacteroides fragilis, contributes to the achievement of a proper balance with TH1/TH2 in dendritic cells of intestinal mucosa in mice that do not contain microbes, and, as shown in animal studies, contributes to the protection from colitis by producing IL-10 in CD4-positive T-cells [29, 40]. Continuing the search for new microorganisms, inducing tolerogenic dendritic cells and activating Tregcells, the UN�but will also help to find new molecules, with healing properties.

Differentiation and maturation of dendritic cells

Human monocytes were isolated from blood using the method of representing a combination of density separation by centrifugation (Ficoll) and division of cells using a magnetic microbasin covered with CD14-specific antibodies (MiltentyiBiotec). The purity of the selected fraction of CD14+cells was above 90% in all experiments. To obtain immature dendritic cells (iDC), purified CD14+cells were grown for 5 days in the presence of IL-4 (R&D systems) and GM-CFS (R&D systems), so that they differentiated into myeloid dendritic cells (MDDC). On the sixth day, the cells are not subjected to stimulation, some stimulated by lipopolysaccharide (1 mg/ml), and 5×105cells MDDC stimulated with bacterial cells (in various quantities) within 24 hours. In particular, stimulation were performed with cells of strain In longum AH121A in the ratio of 10:1 (bacteria/cells, i.e. 5×106bacteria), and in the proportion of 1:1 (bacteria/cells, i.e. 5×105bacteria) within 24 hours. As expected, the addition of antibiotic bacterial growth during this period was not observed. Supernatants were isolated and analyzed them for levels of IL-10 and IL-12p70 using multiplex Luminex platform.

The allocation of human CD4+T-cells and and� culturing with dendritic cells

Peripheral blood mononuclear cells were isolated by centrifugation of leukocyte films in the Lymphoprep gradients. CD4-positive T cells were separated by negative selection on a column for affinity chromatography of production R&D Systems according to manufacturer's instructions. After the division of the T cells were washed and were resuspended in medium RPMI 1640 for cultures with addition of 10% fetal bovine serum inactivated by heat, 100 IU/ml penicillin, 100 µg/ml streptomycin and 3 mmol/l L-glutamine. Purified CD4+T cells (1×106/ml) stimulated by a combination of monoclonal antibodies: immobilized anti-CDS (1 μg/ml) and soluble aHTH-CD28 (5 μg/ml) (manufactured by Pharmingen). After that purified CD4+T cells were incubated with dendritic cells obtained as described above. After 48 hours of CD4+T-cells were permeabilityof and stained for CD25 and Foxp-3. Then analyzed the cells by flow cytometry.

The results show that dendritic cells, being stimulated by bacteria AH121A, trigger polarization and/or expansion of a population of Treg cells, which expresses CD25 and Foxp-3.

Probiotics have different ability to induce the production of cytokines by dendritic cells

Type of produced cytokines can influence the polarization of T cells. To explore Taco�about the impact we have analyzed the production of IL-12p70 and IL-10 into myeloid dendritic cells 24 hours after treatment by bacteria as described above. Lipopolysaccharides were strong inducers of all types of the examined cytokines, while AH121A induced the production of various cytokines in different degrees (Fig.15). So, for example, bacteria AH121A induced lower levels of IL-10 compared with lipopolysaccharides, and has not induced the production of IL-12p70 at a detectable level. That is, bacteria AH121A have a potential ability to decrease inflammation.

The difference in the production of cytokines reflects the different ability to polarization of T cells

The development of certain types of cytokines by dendritic cells is important for the launch of the polarization of T cells towards Th1, Th2, Thl7, or Treg cells. Given the difference in the levels of produced cytokines, we further investigated whether myeloid dendritic cells stimulated AH121A, the ability to induce Foxp3+Treg-cells. Dendritic cells were incubated with AH121A for 5 days, and then co-cultivated with non-alogena CD4+T-cells. After that analyzed the population of CD4+Foxp3+Treg-cells by sorting the cells with fluorescence excitation. The results show that dendritic cells stimulated by bacteria AH121A, can run the polarization of a subpopulation of Treg-cells expressing CD25 and Foxp-3 (Fig.16).

We pislik conclusion, what bacteria AH121A developed tolerogenic dendritic cells, which, in turn, induced the generation of CD4+Foxp3+Treg-cells. Indeed, we have shown that myeloid dendritic cells grown with AH121A, transformed CD4+CD25-T cells in CD4+CD25'Foxp3+T cells. That is, bacteria AH121A possess strong immunomodulatory effects, since they regulate in the direction of increasing the production of Treg-cells of myeloid dendritic cells in vivo. The results indicate the development of CD4+CD25"Foxp3+Treg-cells in response to AH121A in vitro, and this effect may be therapeutically useful, as it provides modulation of inflammatory immune disorders in vivo (Fig.16).

It has been previously shown that some probiotics (such as L. casei and L. reuteri) can induce the development of Tregcells (32, 41, 42). It was shown that Bifidobacterium A was a powerful mediator of the activation of Tregcells in three model animal studies. Moreover, the consumption of Bifidobacterium IN protected from the influx of eosinophils into the lungs and blocked the induction of serum IgE. It can be argued that Treg cells play an important role in the regulation of allergen-specific immune responses (39). Numerous studies on animals show that CD4 CD25 Rohr-cell naberhaus� to the lungs and waste the lymph nodes, and can suppress caused by allergens in the airway eosinophilia, hypersecretions mucous membranes and hypersensitivity of the respiratory tract (43-48).

Numerous studies on humans and animals show that the disease of inflamed bowels occurs from loss of tolerance to symbiotic bacteria. Research Round and Mazmanian (49) shows that the PSA directs the development of Treg-cells in the treatment and prevention of colitis. These findings are consistent with those of studies in which it was shown that generation of IL-10 Foxp3+T-cells are important for tolerance on the surfaces of mucous membranes and to prevent inflammation of the intestine (49, 50).

Example 8. Examples of formulations

The following are examples of compositions in the form of dry granules containing probiotic bifidobacteria in accordance with the present invention.

IngredientsThe percentage by weight
Example 1Example 2Example 3Example 4
Grain cerealto 100to 100to 100 to 100
By-products of avian43,5404535
Fat bird1,281,021,16Of 1.35
Egg products2,42,12,52,2
Chicken liver1,01,01,01,0
The dry yeast1,01.01,01,0
Monolatrist1,01,01,01,0
Calcium carbonate0,80,80,80,8
Potassium chloride0,60,60,6 0,6
Vitamins0,40,40,40,4
Choline chloride0,30,30,30,3
Minerals0,30,30,30,3
DL-methionine0,10,10,10,1
Sodium chloride0,030,030,030,03
Probiotic (1×1010CFU/g NCIMB 41675 in sunflower oil)10,5-0,6
Probiotic (1×1010CFU/g NCIMB 41675 in sunflower oil)0,510,4

The following are examples of compositions for the preparation of wet animal feed containing p�abioticheskie bacteria Bifidobacteria longum in accordance with the present invention.

IngredientsThe percentage by weight
Example 5Example 6Example 7
Water3847up to 50
Chicken liver25to 2015
Products chicken252020
Rice malting5710
Egg products32,51,5
Fat chicken2,93,03,2
Chicken broth0,60,70,9
Taurine0,1 0,10,1
Vitamins0,050,10,1
Minerals0,050,10,1
Probiotic (1×1010CFU/g456
NCIMB41675)

The following are examples of compositions for the preparation of food additives in the form of yogurt containing probiotic bacteria are Bifidobacteria longum in accordance with the present invention.

IngredientsThe percentage by weight
Example 8Example 9Example 10
Milk384248
Sugar121210
Modified starch 1,00,80,8
Prebiotic0,250,30,5
Probiotic (1×1010CFU/g NCIMB41675)456

Although in this document are illustrated and described specific embodiments of the present invention, one versed in the art it will be obvious that it's possible to make other changes and modifications that do not violate the idea and the purpose of the invention. To this end he had in mind in the appended claims to represent all possible such changes and modifications within the scope of the present invention.

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1. The selected strain Bifidobacterium longum AH121A deposited at NCIMB under the number 41675, suitable for use in immunomodulation, induction of cytokine production, the treatment of autoimmune diseases and control the relationship of IL-10:IL-12.

2. Strain Bifidobacterium longum according to claim 1, characterized in that it is in the form of viable cells.

3. Strain Bifidobacterium longum according to claim 1, characterized in that it is in the form of non-viable cells.

4. Strain Bifidobacterium longum according to claim 1, characterized in that it is isolated from the obtained by biopsy sample of tissue of the large intestine of an animal of the cat family.

5. Strain Bifidobacterium longum according to claim 1, characterized in that it has a significant immunomodulation action in its oral consumption.

6. Strain Bifidobacterium longum according to any one of claims.1-5, characterized in that it is intended for use as a medicament.

7. Strain Bifidobacterium longum according to any one of claims.1-5, characterized in that it is intended for use in the prevention and/or treatment of undesirable inflammatory processes.

8. Strain Bifidobacterium longum according to any one of claims.1-5, characterized in that it is intended for use in the prevention and/or treatment of undesirable inflammatory processes in infection�chno gastrointestinal tract.

9. Strain Bifidobacterium longum according to any one of claims.1-5, characterized in that it is intended for use in the prevention and/or treatment of autoimmune disorders that cause unwanted inflammatory processes.

10. Strain Bifidobacterium longum according to any one of claims.1-5, characterized in that it is intended for use in the prevention and/or treatment of diarrhoeal States caused undesirable inflammatory processes.

11. Strain Bifidobacterium longum according to any one of claims.1-5, characterized in that it is intended for use in the correction or improvement of the immune system of companion animals.

12. Strain Bifidobacterium longum according to any one of claims.1-5, characterized in that it is intended for use in the prevention and/or treatment of autoimmune diseases in companion animals.

13. Strain Bifidobacterium longum according to any one of claims.1-5, characterized in that it is intended for use in the prevention and/or treatment of inflammation in companion animals.

14. Composition, suitable for use in immunomodulation, induction of cytokine production and control relationship of IL-10:IL-12 containing a strain of Bifidobacterium longum according to any one of claims.1-5, and one or more ingredients selected from a probiotic material, prebiotic material, a carrier for oral administration.

15. The composition according to claim 14, characterized in that it contains politicalcultural.

16. Composition according to any one of claims.14 or 15, characterized in that it contains a prebiotic material.

17. Composition according to any one of claims.14 or 15, characterized in that it comprises a carrier for oral administration.

18. The composition according to claim 17, characterized in that the carrier for oral administration is a pharmaceutically acceptable carrier such as a capsule, tablet or powder.

19. The composition according to claim 17, characterized in that the carrier is for oral administration selected from dry granules, wet pet food, yogurt, oil slurries, suspensions milk, cheese, composition based on cocoa butter, sauce and/or composition based on yoghurt and any combination thereof.

20. A food product containing a strain of Bifidobacterium longum according to any one of claims.1-5 or a composition according to any one of claims.14-19.

21. The food product according to claim 20, characterized in that it is a dry food product.

22. The food product according to claim 20, characterized in that it is a wet food product.

23. The food product according to any one of claims.20-22, characterized in that it further comprises a probiotic material.

24. The food product according to any one of claims.20-22, characterized in that it further comprises a prebiotic material.

25. The food product according to any one of claims.12-14, characterized in that it is food for companion animals.



 

Same patents:

FIELD: biotechnology.

SUBSTANCE: method of production of probiotic preparation of immobilised bifidobacteria for feeding beef breed cattle is proposed. The bifidobacteria of strain Bifidobacterium longum are grown on a nutrient medium to obtain biotitre of 108-109 CFU/ml. The medium is prepared using the electro-activated cathode water with pH 8-9 and redox potential -400…-500 mV, and with the addition of a stabiliser - serine in an amount of not less than 0.01 wt %. Then bifidobacteria together with the components of the nutrient medium are immobilised on enterosorbent polyphepan.

EFFECT: improved appetite of cattle, digestibility of nutrients of the ration, increase in average daily gain, stimulation of rumen digestion, normalisation of all types of metabolism, acceleration of the settlement of the gastrointestinal tract with normal microflora, reduction of excretion with faeces of pathogenic and opportunistic pathogenic bacteria.

4 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology. Disclosed are versions of a method of producing arginine through fermentation of agroindustrial wastes, including starch-containing wastes, to obtain fermented liquid containing arginine and separating the arginine from the fermented liquid. Enzymatic hydrolysis of agroindustrial wastes can be carried out before fermentation to convert the wastes into reducing sugars. Fermentation is carried out in aerobic conditions at pH 5.5-7.5, at 27-36°C for 1-10 days in the presence of Corynebacterium glutamicum ATCC 21831 or Corynebacterium glutamicum ATCC 21493. The agroindustrial wastes used are pulp, seed powder, cassava pulp and/or jackfruit seed powder.

EFFECT: group of inventions provides higher arginine output.

20 cl, 6 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions relate to the field of biotechnology and deal with a method of preventing or treatment of a disease in a subject, caused by a pathogenic organism, by the introduction of a vaccine composition, the vaccine composition and its application. The characterised vaccine composition contains bacteria, attenuated by mutation in a gene, coding the ABC-peptide transporter protein OppD, which can persist in the subject. The claimed inventions can be applied in immunology to obtain and apply vaccines.

EFFECT: said mutation makes the coded ABC-peptide transporter protein non-functional.

19 cl, 10 dwg, 21 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates for composition for treatment or prevention of disorders, associated with reduced level of defensins. Composition contains from 0.005 to 1000 mg of Lactobacillus johnsonii Lal (NCC533, No CNCM 1-1225) per a daily dose, with at least 90% of L. johnsonii Lal (NCC533, No CNCM 1-1225) being transferred into state in which they become non-replicating at temperature110-140°C for 5-30 s.

EFFECT: invention provides enhancement of expression of defensin hBD1 mRNA.

5 cl, 2 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and specifically to a fermentation medium and a method of producing recombinant proteins using said medium. A fermentation medium for producing recombinant proteins selected from a group including GM-CSF, streptokinase and lipase, using microorganisms selected from a group including: E. Coli, Streptomyces sp. and Rhizomucor sp., is characterised by keeping concentration of urea or derivatives thereof in the range of 0.5 g/l to 2 g/l. The medium contains, per litre of water, basic salts in the following amounts: orthophosphoric acid (85%) 2.67-133.5 ml, calcium sulphate 0.093-4.65 g, potassium sulphate 1.82-91 g, magnesium sulphate-7H2O 1.49-74.5 g, potassium hydroxide 0.413-20.65 g, glycerine 4-200 g. The medium contains, per litre of water, trace elements in the following amounts: copper sulphate-5H2O 0.6-30 g, sodium iodide 0.008-0.4 g, manganese sulphate-H2O 0.3-15 g, sodium molybdate-H2O 0.02-1 g, boric acid 0.002-0.1 g, cobalt chloride 0.05-2.5 g, zinc chloride 2-100 g, iron (II) sulphate-7H2O 6.5-325 g, biotin 0.02-1 g, sulphuric acid 0.5-25 ml.

EFFECT: invention increases the output of end products.

6 cl, 27 dwg, 3 tbl, 16 ex

FIELD: biotechnology.

SUBSTANCE: strain of bacterium Bacillus subtilis RNCIM B-11964 is proposed, which is a highly active producer of pectolytic enzymes macerating plant tissue.

EFFECT: strain exhibits high pectate-lyase and pectin-lyase activity with respect to pectins from different sources.

2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to biotechnology and microbiology. There are presented strain Bacillus sp. KCCM11143P possessing antifungal activity on Saprolegnia sp., culture fluid produced by culturing the strain, probiotic composition, feed additive, antifungal agent and agent for improving quality of water containing the strain Bacillus sp. KCCM11143P or its culture fluid. There are also presented method for culturing fish or shell fish, method for preventing saprolegniosis in animals and method for improving quality of water with using the strain Bacillus sp. KCCM11143P or its culture fluid.

EFFECT: strain Bacillus sp KCCM11143P has a high production level of siderophores inhibiting a high ability of iron uptake and thereby inhibits the growth of other pathogens and Saprolegnia sp, promotes a higher rate of food consumption and weight gain by fish, as well as reducing an excretion level that promotes higher quality of water.

10 cl, 4 dwg, 11 tbl, 15 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Disclosed is a Microbacterium species VKM Ac-2614D strain for cleaning contaminated and chronically contaminated fresh water bodies in the temperature range of +2°C to +25°C.

EFFECT: invention enables to remove petroleum hydrocarbons from water and bottom deposits with low oxygen concentration in the water and in high latitude conditions.

3 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the hydrolysis industry, particularly methods of removing acetone-butyl-alcohol fermentation inhibitors from hydrolysates of lignocellulose material, and can be used in preparing culture media for producing biethanol, biobutanol and acetone. The method includes treating hydrolysates with a group of microorganisms, wherein the consortium of microorganisms used is active sludge from sewage treatment facilities which is pre-adapted to a growth substrate based on substances which inhibit acetone-butyl-alcohol fermentation. The method employs active sludge from urban sewage treatment facilities, the bacterial component of which is represented by Pseudomonas (64%), Bacillus (18%), Zooglea (7%), Micrococcus (5%), Chromobacterium (3%), Acinetobacter (2%) and Citrobacter (1%) bacteria. The method also employs active sludge from sewage treatment facilities of pig-breeding farms, the bacterial component of which is represented by Nocardia (35%), Rhodococcus (28%), Micrococcus (18%), Pseudomonas (13%) and Bacillus (6%) bacteria.

EFFECT: invention eliminates inhibiting factors in the process of fermentation of hydrolysates of lignocellulose material, enables to avoid corrosion of the hydrolysis equipment and enables to optimise the microbial culture.

3 cl, 4 ex

FIELD: biotechnology.

SUBSTANCE: strain of bacteria Pseudomonas aeruginosa No.6-DEP is deposited in the collection of FSBI "VGNKI". When using the vaccine based on the proposed strain, the titre of antibodies to somatic antigen O6 Pseudomonas aeruginosa in blood serum of rabbits and pigs, 14 days after the last immunisation is 1:819.2 and 1:1024, respectively.

EFFECT: strain has high immunogenic activity and is intended for production of a vaccine against pseudomonosis of pigs.

3 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, namely to internalisation of therapeutic molecules into cell, and can be applied in medicine. Obtained is composition for delivering molecules of nucleic acids into cells, containing at least one peptide with at least 92% identity to GAAEAAARVYDLGLRRLRQRRRLRRERVRA (SEQ ID NO: 2); IREIMEKFGKQPVSLPARRLKLRGRKRRQR (SEQ ID NO: 3); or YLKVVRKHHRVIAGQFFGHHHTDSFRMLYD (SEQ ID NO: 4), bound to one or several molecules of nucleic acids.

EFFECT: invention makes it possible to increase efficiency of delivery of molecules of nucleic acids into mammalian cell due to peptide, capable of internalisation into mammalian cell with efficiency, constituting at least 200% of efficiency of internalisation of peptide TAT, which has amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO: 1).

8 cl, 16 dwg, 1 tbl, 8 ex

Crystals // 2556206

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention describes crystals of 2-{4-[N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino]butyloxy}-N-(methylsulphonyl)acetamide ("compound A"), as a form I of the compound A crystal, which shows diffraction peaks at 9.4 degrees, 9.8 degrees, 17.2 degrees and 19.4 degrees in its power X-ray diffraction spectrum, as a form II of the compound A crystal, which shows diffraction peaks at 9.0 degrees, 12.9 degrees, 20.7 degrees and 22.6 degrees in its power X-ray diffraction spectrum, as a form III of the compound A crystal, which shows diffraction peaks at 9.3 degrees, 9.7 degrees, 16.8 degrees, 20.6 degrees and 23.5 degrees in its power X-ray diffraction spectrum. There are also described methods for producing the forms I, II and III of the compound A crystal, based pharmaceutical composition and PGI2 receptor agonist agent, an accelerating agent for angiogenic therapy, gene engineering or autoimmune bone marrow transplantation, and an accelerating agent for angiogenesis for peripheral artery recovery or angiogenic therapy on the basis thereof; there are also described a preventive or therapeutic agent for a wide range of diseases and conditions.

EFFECT: preparing the new therapeutic agent for the wide range of diseases and conditions.

11 cl, 6 dwg, 6 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented invention refers to immunology. What is presented is a monoclonal anti-IFNAR1 antibodies with L234F, L235E and P331S Fc mutations of human IgG1 possessing a lower affinity to Fcgamma RI, Fcgamma RIIIA and c1q receptors as compared to a non-modified antibody. There are described the recovered nucleic acid providing expression of the above antibody containing a nucleotide sequence coding the antibody, and a pharmaceutical composition based on the above antibody.

EFFECT: using the invention provides the antibody possessing the lower affinity to Fcgamma RI, Fcgamma RIIIA and c1q receptors that provides reducing the undesired effector functions in treating chronic inflammation and autoimmune conditions.

9 cl, 34 dwg, 7 tbl, 36 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates for composition for treatment or prevention of disorders, associated with reduced level of defensins. Composition contains from 0.005 to 1000 mg of Lactobacillus johnsonii Lal (NCC533, No CNCM 1-1225) per a daily dose, with at least 90% of L. johnsonii Lal (NCC533, No CNCM 1-1225) being transferred into state in which they become non-replicating at temperature110-140°C for 5-30 s.

EFFECT: invention provides enhancement of expression of defensin hBD1 mRNA.

5 cl, 2 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel N-containing heteroaryl derivatives of formula I or II or their pharmaceutically acceptable salts, which possess properties of JAK kinase, in particular JAK3, and can be applied for treating such diseases as asthma and chronic obstructive pulmonary disease (COPD). In formulae A represents carbon and B represents nitrogen or A represents nitrogen and B represents carbon; W represents CH or N; R1 and R2, independently represent hydrogen, C1-4alkyl, halogenC1-4alkyl, -CN; R3 represents C1-4alkyl, R9-C1-4alkyl, Cy1, where Cy1 is optionally substituted with one or several substituents R10; R4 represents hydrogen, C1-4alkyl, R12R7N-C0alkyl, where one of R7 and R12 represents hydrogen, and the other represents C1-4alkyl or group R13, which is selected from C1-5alkyl, Cy2-C0alkyl; R5 represents hydrogen; R6 represents hydrogen, C1-4alkyl, C1-4alkoxyC1-4alkyl, hydroxyC1-4alkyl, R12R7N-C1-4alkyl, R16CO-C0alkyl, Cy1; R7 represents hydrogen or C1-4alkyl; R9 represents halogen, -CN, -CONR7R12, -COR13, CO2R12, -OR12, -SO2R13, -SO2NR7R12, -NR7R12, -NR7COR12; R10 represents C1-4alkyl or R9-C0-4alkyl; R11 represents C1-4alkyl, halogen, -CN, -NR7R14; R12 represents hydrogen or R13; R13 represents C1-5alkyl, hydroxyC1-4alkyl, cyanoC1-4alkyl, Cy2-C0alkyl or R14R7N-C1-4alkyl; where Cy2 is optionally substituted with one or several constituents R11; R14 represents hydrogen or C1-4alkyl; R16 represents C1-4alkyl, halogenC1-4alkyl, C1-4alkoxyC1-4alkyl, hydroxyC1-4alkyl or cyanoC1-4alkyl; Cy1 represents monocyclic carbocyclic unsaturated or saturated ring, selected from C3-C6cycloalkyl, phenyl, or saturated monocyclic 4-6-membered heterocyclic ring, containing from 1 to 2 heteroatoms, selected from N and S, or partially unsaturated 10-membered bicyclic heterocyclic ring, containing oxygen atom as heteroatom, which can be substituted with group R11, where said ring is bound with the remaining part of molecule via any available C atom, and where one or several ring C or S atoms are optionally oxidised with formation of CO or SO2; and Cy2 represents monocyclic carbocyclic unsaturated ring, selected from C3-C6cycloalkyl, or aromatic monocyclic 4-6-membered heterocyclic ring, containing from 1 to 2 heteroatoms, selected from N and S, or unsaturated 10-membered bicyclic heterocyclic ring, containing oxygen atom as heteroatom, which can be substituted with group R11, where said ring is bound with the remaining part of molecule via any available atom C or N.

EFFECT: obtaining novel heteroaryl derivatives.

27 cl, 41 ex

FIELD: medicine.

SUBSTANCE: invention refers to biochemistry, particularly to affinity-matured CRIg versions. Declared is the CRIg versions, which is an alternative complement pathway inhibitor at least twice stronger than human CRIg with a native sequence, and optionally possesses C3b-bindingaffinity at least twice as much. There are also declared a chimeric molecule and a pharmaceutical composition, both containing the above CRIg version. The CRIg version can be used for preparing a therapeutic agent for treating a complement-associated disease or condition.

EFFECT: invention enables improving the therapeutic effectiveness of CRIg polypeptides.

17 cl, 17 dwg, 4 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to quinolines substituted by phosphorus-containing group of formula and applicable in medicine, wherein Z represents V1 and V2 are independently specified in hydrogen or halogen; one of R and R` represent phosphorus-containing substitute Q; the other one is specified in hydrogen or methoxyl; wherein the phosphorus-containing substitute Q represents A represents O; L represents C1-6alkyl; J represents NH or C3-6heterocycloalkyl and J is optionally substituted by G3; X is absent or represents -C(=O)-; X is absent or represents C1-6alkyl; each of R1 and R2 are independently specified in C1-6alkyl or C1-6alkoxy; G3 represents C1-6alkyl, R3S(=O)m-, R5C(=O)- or R3R4NC(=O)-; R3, R4 and R5 are independently specified in 3 or C1-6alkyl; m is equal to 0-2.

EFFECT: there are presented new protein kinase inhibitors effective for treating the diseases associated with abnormal protein kinase activity.

20 cl, 42 ex, 8 tbl, 3 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to biotechnology, namely to novel IL-17-inhibiting polypeptides, corresponding to fused proteins, to compositions and their application for medicinal purposes. Polypeptide contains amino acid sequence, which is selected from group, consisting of GVTLFVALYD YKAFWPGDLS FHKGEKFQIL RTSDGDWWEA RSLTTGETGY IPSNYVAPVD SIQ (SEQ ID NO: 39), GVTLFVALYD YKAFWPGDIS FHKGEKFQIL RTSDGEWWVA RSLTTGEEGY IPSNYVAPVD SIQ (SEQ ID NO: 57) or GVTLFVALYD YKAFWPGDIS FHKGEKFQIL RTSDGEWWIA RSLTTGEEGY IPSNYVAPVD SIQ (SEQ ID NO: 107); amino acid sequence, which has, at least, 80%, preferably, at least, 90%, more preferably, at least, 95% identity of amino acid sequence with SEQ ID NO: 39, SEQ ID NO:57 or SEQ ID NO: 107; fragment or functional derivative of SEQ ID NO: 39, SEQ ID NO: 57 or SEQ ID NO: 107, obtained due to substitution, addition and/or removal of not more than 5 amino acids.

EFFECT: invention makes it possible to bind IL-17 with high specificity and affinity.

33 cl, 17 dwg, 3 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: invention refers to immunology. What is disclosed is using a peptide with an amino acid sequence GLAGGSAQSQRAPDRVL for selecting CεmX-specific antibodies and antigen-binding fragments of these antibodies. What is presented is the CεmX-specific antibody, as well as a method providing selecting the antibodies specifically binding the peptide according to the invention, and a method of treating IgE-mediated diseases involving administering the antibody into an individual according to the invention.

EFFECT: it has been disclosed that the monoclonal antibodies specifically binding the CεmX segment GLAGGSAQSQRAPDRVL can effectively bind to mIgE in human B-cells and are applicable for targeting to these B cells for treating IgE-mediated diseases.

14 cl, 5 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of immunology. Claimed is isolated antibody to ICOS protein of people with increased effector function. Also described are cell and method of obtaining antibody in accordance with claimed invention, pharmaceutical composition, method of treating autoimmune disease or disorder, transplant rejection and malignancy of human T-cells, as well as method of depletion of ICOS-expressing T-cells, method of destroying germ centre structure in secondary lymphoid organ of primates, methods of depleting B-cells of germ centre of secondary lymphoid organ and circulating B-cells, which have undergone class switching, in primates.

EFFECT: invention can be further applied in therapy of diseases, mediated by T-cells.

33 cl, 21 dwg, 3 tbl

FIELD: biotechnology.

SUBSTANCE: method of production of probiotic preparation of immobilised bifidobacteria for feeding beef breed cattle is proposed. The bifidobacteria of strain Bifidobacterium longum are grown on a nutrient medium to obtain biotitre of 108-109 CFU/ml. The medium is prepared using the electro-activated cathode water with pH 8-9 and redox potential -400…-500 mV, and with the addition of a stabiliser - serine in an amount of not less than 0.01 wt %. Then bifidobacteria together with the components of the nutrient medium are immobilised on enterosorbent polyphepan.

EFFECT: improved appetite of cattle, digestibility of nutrients of the ration, increase in average daily gain, stimulation of rumen digestion, normalisation of all types of metabolism, acceleration of the settlement of the gastrointestinal tract with normal microflora, reduction of excretion with faeces of pathogenic and opportunistic pathogenic bacteria.

4 tbl, 2 ex

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