Immunomodulatory extracts of bacteria lactobacillus and methods for preparing and using them

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to an extract of one or more bacterial strains Lactobacillus. The extract of one or more bacterial strains Lactobacillus representing a soluble extract, wherein the extract contains chemically modified bacterial molecules prepared by the action of an alkaline medium on one or more bacterial strains Lactobacillus; the extract is effective in treating diseases associated with the anti-inflammatory cytokine production imbalance. A method for preparing the extract of one or more bacterial strains Lactobacillus. A pharmaceutical composition effective for reducing at least one symptom associated with at least one condition specified in a respiratory disorder, an allergic condition, an urinary disorder and a gastric disorder, containing the extract. A nutritional composition. A pharmacological composition effective in treating the diseases associated with the anti-inflammatory cytokine production imbalance, containing the extract. A method of relieving the above symptoms. The extract prepared by the above method.

EFFECT: extract is effective in treating the diseases associated with the anti-inflammatory cytokine production imbalance.

21 cl, 7 dwg, 23 tbl, 5 ex

 

The scope to which the invention relates.

Embodiments of the present invention include extracts from bacteria Lactobacillus, which may exert immunomodulatory effects in individuals. Embodiments of the present invention can be applied, for example, as a nutraceutical or pharmaceutical products for the treatment of diseases, such as diseases associated with an imbalance of the production of anti-inflammatory or Pro-inflammatory cytokines, such as infections, allergic disorders, autoimmune disorders and inflammation, or as adjuvants, providing health benefits individuals. Along with other aspects, the invention also includes methods of preparation and use of such extracts. The invention also relates to specific bacteria strains Lactobacillus.

Background and brief description of the invention

Immunomodulation is a global term that refers to a wide range of immune intervention that alters normal or pathological immune responses. Microbes produce and secrete a wide range of molecules that can modulate eukaryotic immune responses (Lavelle et al., Curr Top Med Chem. 2004, 4(5), 499-508). They include factors that destroy the protective mechanisms for sod is istia colonization and persistence of the pathogenic agent. Were identified originating from viruses, bacteria and parasites molecules that can inhibit the inflammatory response. In addition to microbial factors that can suppress immune responses themselves potent immune activators may also be of microbial origin. They include bacterial enterotoxins, excretory-secretory products of parasitic origin and viral nucleic acids.

It is believed that a family of at least 11 of receptors, called toll-like receptors (TLR), and expressed by the host organism, plays a key role in the immunological detection and innate reactivity against microbes. In Fig. 1 presents a list of TLR ligands (Gay and Gangloff, Ann. Rev. Biochem., 2007, 76:141-65). TLR recognize a wide range of molecules, also known as associated with pathogen molecular types (PAMP), produced by viruses, bacteria and fungi (Tse and Horner, Ann Rheum Dis. 2007 Nov; 66 Suppl 3:iii77-80). Associated with TLR immunomodulation was used in the development of new treatments for a wide range of pathological conditions, including infectious, malignant, autoimmune and allergic diseases.

Agonists and antagonists of TLR were investigated as potential therapeutic agents for the prevention and treatment of diseases. In relatively small clinical COI is the tests, TLR agonists were used as adjuvants for vaccines intended to prevent infections, eliminate allergic hypersensitivity and removal of malignant cells. TLR agonists were also investigated as a means for monotherapy and adjunctive therapy for the treatment of patients and infectious, allergic and malignant diseases. The use of TLR antagonists have been investigated in preclinical studies and clinical trials as potential therapeutic agents about autoimmune diseases and sepsis.

Probiotics are live microorganisms that may have health effects on the individual with the introduction in adequate quantities (Mottet et al., Digestive and Liver Disease, 2005, 37: 3-6; Ezendam et al., Nutr Rev, Jan. 2006, 64(1): 1-14; Gill and Prasad, Adv Exp Med Biol, 2008, 606: 423-54). Biological mechanisms involved in stimulation of the immune response of probiotic microorganisms and certain cellular components of these microorganisms were involved in the study. For example, gram-positive bacteria have a characteristic macromolecules that make up the cell wall, such as lipoteichoic acid (LTA). LTA can be associated with immunostimulatory activity (for example, Bhakdi et al., Infect. Immun., 1991, 59: 4614-4620; Setoyama et al., J Gen Environ, 1985, 131 (9): 2501-2503; Cleveland et al., Infect Immun, 196, 64(6): from 1906-1912). Cm. (Deininger et al., Clin Vaccine Immunol, 2007, 14(2): 1629-1633). In addition, probiotic bacteria may contain a variety of TLR ligands with immunomodulatory characteristics. It was found that fragments of the cell wall of various bifidobacterial strains stimulate the production of interferon-gamma (IFN-γ) in vitro in murine splenocytes (T. Ambrouche, "Contribution a l etude du pouvoir immunomodulateur des bifidobacteries: Analyse in vitro et etude ex vivo des mecanismes moleculaires impliques," Ph.D. Thesis, Universite Laval, Quebec, 2005). Capsules made from fragments of the cell wall in the form of particles of certain lactic acid bacteria (Del-Immune V®, Pure Research Products, LLC, Colorado), are also designed to stimulate the immune system.

However, the ingestion of probiotic bacteria in live or killed form, or the ingestion of fragments of the cell wall in the form of particles such bacteria may not be the most effective way to ensure immunomodulatory effect in individuals. For example, the living cell extracts can contain large proteins and lipopetides, the size of which prevents effective absorption by the individual, thereby limiting the local concentration of useful molecules from probiotic bacteria in the body. Conditions inside the body can also destroy the active bacterial components or otherwise modify the chemical structure of these components, making the x inactive. The risks associated with oral introduction of live probiotic microorganisms (Lactobacillus), include bacteremia and septicaemia (Lactobacillus Sepsis Associated With Probiotic Therapy, Pediatrics, Jan. 2005, 115 (1): 178-181). Therefore, there is a need for other means to ensure the favorable effects of probiotic bacteria in in need of individuals.

The present invention relates to extracts of Lactobacillus, some embodiments of which can be high together with immunomodulating activity. For example, embodiments of the present invention are extracts from bacterial strains that may be useful as nutraceuticals or pharmaceuticals, in some cases for the treatment of infectious diseases, allergies, respiratory disorders and inflammatory pathological conditions, or to act as a Supplement in connection with the treatment Protocol. The present invention also relates to compositions containing extracts, and methods of producing extracts, for example, with environments that do not pose a risk Pranovich diseases. Methods in accordance with the invention include, for example, lysis of cells under alkaline conditions, or in alkaline conditions followed by acid conditions. In some embodiments, the implementation, the extracts according to the invention is predstavlyaet a soluble extracts meaning that they do not contain significant quantities of solid or particulate matter. In some embodiments, implementation, extracts contain chemically modified TLR ligands. In some embodiments, implementation, alkaline treatment may cause chemical modification of cellular materials, including TLR ligands, components of the cell wall, proteins, lipoteichoic acid, lipopetides and phospholipids.

Some embodiments of the invention may include extracts obtained from one or more of the following types:

Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus casei defensis, Lactobacillus casei ssp. casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus lactis and Lactobacillus delbrueckii.

In some embodiments, implementation, extracts contain at least one strain from each of the above types of bacteria, while in other embodiments implement one or more of certain strains of the above list can be deleted or substituted with one or more other strains. Some embodiments of the present invention include an extract obtained from one or more of the following bacterial strains: Lactobacillus fermentum I-3929, Lactobacillus rhamnosus 71.38, Lactobacillus plantarum 71.39, Lactobacillus johnsonii 103782 and Lactobacillus helveticus 03146. The above strains deposited according to the Budapest Treaty. Each of Lactobacillus fermentum I-3929, Lactobacillus rhamnosus 71.38, Lactobacillus plantarum 71.39, Lactobacillus johnsonii 103782 and Lactobacillus helveticus 103146 deposited in the National Collection of Cultures of Microorganisms of the Institute Pasteur Collection Nationale de Culture des Microorganismes at the lnstitut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France. Lactobacillus fermentum I-3929 was deposited on February 27, 2008, Other strains are among the collections of the Depositary and can be obtained by contact with the Depositary.

The present invention also, along with other aspects, refers to a strain of Lactobacillus fermentum I-3929, the extracts obtained from this strain, methods of obtaining such extracts and their application. This strain was obtained by providing opportunities for these strains of Lactobacillus plantarum and Lactobacillus fermentum be chromosomal exchange, thereby obtaining a new strain of Lactobacillus. It was found that the extracts obtained from Lactobacillus fermentum I-3929, active on several models in vivo and in vitro, correlating with infection and immunological disorders.

In some embodiments, the implementation, the extract obtained only from one particular bacterial strain. Alternatively, it may be more than one strain. In other embodiments, the implementation may add one or more extracts from another type of microorganism, for example, b is Karelenergo view other than Lactobacillus.

The extracts can be obtained by lysis of bacterial cells in certain conditions, after the cells are grown to a suitable concentration in the culture medium. In some embodiments, implementation, bacteria are grown in an environment that does not create risk associated with prion diseases or the risk of other diseases that can be transmitted through ingestion of products derived from environments on animal basis. For example, in some embodiments, the implementation for cell cultivation environment is using plant-based, such as soy-based.

Lysates (i.e., products of lysis of the cells) can also be filtered to remove nucleic acids and larger cell fragments, such as insoluble or having the form of a particulate material. In some embodiments, the implementation, the amount of nucleic acids present in the extracts, less than 100 µg/ml Therefore, in some embodiments, the implementation of the extract obtained contains soluble molecular components and does not contain significant quantities of insoluble or having the form of particles of material.

Molecules of membranes and cell walls, including lipoproteins, lipopetides, composition, lipopolysacharide, lipoteichoic acid and resemble teichoic acid, can bytestore or suspended in the extracts. During the process of lysis, the molecules in cells, such as membrane and cell wall, can be chemically modified, for example, split into smaller patterns, by treatment with alkali. In spite of such chemical modifications, embodiments of the invention can maintain its biological activity, compared with whole cells, or such embodiments of could even show increased biological activity compared to the whole cell.

For example, the alkali treatment can be used for lysis of the cells or may be applied to the cells, which were lysed in a different way. During alkaline processing in accordance with some variations of the invention, L-amino acids found in natural proteins and lipopetides, at least partially racemethionine in D-amino acids. D-amino acids may have a beneficial effect in increasing the duration of the effectiveness of the extracts, because they are not exposed to effective digestion in the gastrointestinal tract of mammals. D-amino acids can also protect smaller peptides and proteins from destruction during digestion. Examples of D-amino acids include associated protein D-amino acids and to a lesser extent lisinolril (deVrese et al., J Nutrition, 2000, 2026-2031). Thus, antigenic molecules in extracts, chemically modified at the time of lysis for the content of D-amino acids, can remain in the body for longer periods of time, potentially allowing more strong immunostimulating effect in some versions of the implementation.

In some embodiments, the implementation, the filtering process may also affect the properties of the obtained extracts, because the pore size of the filter, and in some cases chemical properties of the surface of the filter (i.e., polarity) can change the type of materials that are removed and kept. For example, in some embodiments, the implementation uses a filtering process that is designed to hold the interest of molecules, but the removal of other molecules, such as nucleic acid or insoluble or having the form of particles of materials.

Filtered extracts can also be further purified by organic extraction, organic-water extraction, chromatography, ultracentrifugation, ultrafiltration or a combination of both.

Brief description of drawings

Figure 1: a Ligand for a family of 11 toll-like receptors (TLR)expressed by the host organism.

Figure 2: a Drawing device for filtration tangential flow (TF) to obtain a bacterial extracts after lysis of the bacteria. The drawing shows two different configurations for filter: parallel type, where all filters simultaneously, and coil type, where the filters configured on the serial type.

Figure 3: Generalized correlation between functional parameters and flow indicating the area of the pressure control and adjustment of the mass transfer for a method of filtering in a tangential flow (TFF).

Figure 4: Stimulation of spleen cells, cultured for 48 h in the presence of various dilutions of the lysates AFer300, CFer300 and DFer300, and ARahr300, CRahr300 and DRahr300. After adding 30 μl/well of a solution of Alamar blue®, diluted in 1:1 ratio to the medium for cell culture, the cells further incubated (a) 8.5 hours (first experiment); (b) 24 h (second experiment). Shows the average emission at 590 nm ± standard deviation cultures in two repetitions.

Figure 5: Induction of nitric oxide production (NO) in mice treated with extracts of Lactobacillus fermentum I-3929 and Lactobacillus rhamnosus 71.38, (a) the first analysis, and (b) the second analysis. The results are expressed in μm of nitric oxide (NO) in the form of average value ± standard deviation.

6: effect of the extracts according to the invention on the Hyper-reactive Airways (AHR), defined by pletismography whole body (Emka) with increasing concentrations of respirable metacholine through one day after the latter of antigenic stimulation. The results (average value increased pause ± standard error of the mean) are shown for animals of the negative control group treated with saline phosphate buffer (PBS) (n=4), untreated animals with antigenic stimulation LACK (as positive control group, n=8), mice treated OM-1009A, with antigenic stimulation LACK (n=8)and mice treated OM-1009B, with antigenic stimulation LACK (n=7).

A detailed description of the invention

Definition

Extract: the Extract, as defined in the present description, means a material obtained after lysis of one or more bacterial strains. In some cases, the extract prepared from only one strain, whereas in the other extract is prepared from a mixture of several different strains.

In some cases, the extract is a soluble extract, meaning that it does not contain significant amounts presented in the form of particles and insoluble materials such as fragments in the form of particles or solid cell walls. Instead, the components of the cell walls, organelles and cell membranes may be contained in the extracts in the extent to which they are dissolved or suspended. For example, the extract may be treated for removal are presented in the form of particles and insoluble materials, for example, through the your filtering, centrifugation or other separation techniques.

Chemical lysis: This method of lysis of bacterial cells in basic, acidic and/or osmotic conditions.

The lysate Used in the present description, the term refers to the extract of the bacteria, the resulting procedure lysis of the cells.

Filtration: the filtration Process, as described in the present description, means the transmission of an extract or mixture of extracts through one or more filters, such as microfilters (i.e., microfiltration) and/or ultrafilter (i.e., ultrafiltration). Such filtering is not necessary to remove 100% of the components, to remove which it is intended, but may, in some embodiments, implementation, make extracts essentially free of these components. In some cases, filtering is repeated in several passages or cycles.

Initial pH: the term refers to the pH measured in the beginning of the procedure, such as lysis or filtering.

Sugars: Sugar, as defined in the present description includes monosaccharides, disaccharides and larger saccharides, such as linear and branched polysaccharides. Sugars also include substituted or chemically modified sugars, such as lipopolysaccharides (LPS) and their chemically modified variants.

Lipoproteins: This term refers to macromolecules, which include protein or peptide chain, and lipids, for example, a protein or peptide covalently linked to a lipid. Used in the present description, the term LP also includes lipopetides.

The composition: This term refers to polymers. Containing sugars and amino acids.

Lipoteichoic acid (LTA): This term refers to associate with the surface of the adhesive amphiphilic molecule present in gram-positive bacterial strains.

Tagaeva acid: This term refers to polymers of glycerophosphate or revitalizat connected together through fosfolipidnyh links.

D-amino acids: This term refers to amino acids that exist in programada isomeric forms, in contrast to the biosynthetic received L-amino acids that exist in levogyrate isomeric forms.

Racemization: This term indicates at least partial chemical modification of L-amino acids to D-amino acids.

Environment, which avoids the risk-based prion diseases, means the culture medium used at any stage of obtaining extracts that do not contain materials such as serum or meat extracts taken from animals such as cows or sheep or any other animal that can convey the basis of the data on prion diseases. Examples of such environments include synthetic environment plant-based, and also the environment with the use of horse serum or environment containing materials derived from animal species that do not transmit prion disease. The examples are based on the prion diseases include, for example, a spongiform encephalopathy of cattle, prurigo and disease of Creutzfeldt-Jakob disease.

Not the animal, the environment represents the environment on plant-based (i.e., plant) environment, such as soy medium and synthetic medium.

Used in the present description, the term nutraceutical refers to any composition that can have health effects at the individual after the introduction, where the composition is, for example, available to the individual without the recipe of the doctor.

The term treatment is used in a therapeutic context, in the present description means and the current treatment of diseases and disorders, as well as, for example, prevention or protection from the development of new diseases or disorders.

Used in the present description, the term adjuvant to describe embodiments of the invention relates to variants of the invention, carried out by the individual in conjunction with the medical plan of treatment.

Used in the present description the terms immunomodulation, immunomodulatory and the WMD like refer to the ability to modify immune responses in an individual so which may have health effects, for example, to provide anti-inflammatory or immune-stimulating effect.

Used in the present description, the terms anti-inflammatory and the like belong to the immunomodulatory effects, serving to reduce inflammation.

Used in the present description the terms immunostimulatory and the like belong to stimulate the immune system.

Used in the present description, the term protective immunity means that a variant embodiment of the invention applies an individual in order to provide protection against subsequent antigenic stimulation infectious agent or allergen. As a consequence, during antigenic stimulation level of an infectious agent or allergen in an individual has a sufficiently low concentration so as not to cause significant harm to the health of the individual. The length of time during which such effective protection against antigenic stimulation may be limited, for example, a period of several hours, days or weeks.

Used in the present description, the term individual means any individual animal, including mammals individuals, such as people and Pets. Pets can, for example, include mammals, such as dogs, cats, Loches is di, pigs, cows, sheep, goats or other livestock, and may not include mammals, such as birds, for example, chickens, ducks, geese, turkeys and other species of agricultural fowl.

It is clear that certain bacterial strains identified in the present description and used in the invention can include strain, obtained from the initial Deposit specified in the present description, or his genetic clone, including the strain, which was re-deposited at a later time under other Deposit code name, but which is genetically the same strain, as originally deposited version.

All numbers used in this description are approximate, subject to the errors inherent in their measurement, rounding and digit statistical significance.

Obtaining extracts

The present invention includes an extract of one or more bacterial strains Lactobacillus, where the extract is a soluble extract, and where the extract contains chemically modified bacterial molecules.

The extracts of the present invention can be obtained, for example, the cultivation of cells, followed by collecting the resulting biomass lysis and purification. For each strain to obtain enough material, fermentation culture on Intesa with the lot of the working sowing followed by the introduction of seed into a larger fermentation containers.

Your environment can be the same for each species. In some embodiments, implement, for growing all be used strains can be applied environment, which avoids the risk-based prion diseases.

After fermentation, the resulting biomass from one strain or set of strains can be inactivated by heat treatment, concentrated and frozen. Therefore, in some embodiments, the initial material used for production of extracts, may not be subjected to lysis of whole cells.

In other embodiments, the initial material used to obtain the extracts, can be a biomass obtained from cells already at least partially lysed mechanically, enzymatically or chemically. In some embodiments, the implementation, the source material may be a fraction of such pre-lysed cells, such as the fraction containing the cell wall.

In some embodiments, the initial material is processed in an alkaline environment, such as consisting of a strong base, such as hydroxide or other strong mineral or organic base. At this stage of lysis or processing base, not lysed cells in the source material lyse while in some embodiments, implementation, cellular components can be chemically modified. Therefore, in some embodiments, the implementation of the chemically modified bacterial molecules are obtained by treatment with a base, such as processing a strong basis of one or more bacterial strains Lactobacillus, which received the extract (i.e. processing base is not lysed cells or components or fractions of bacterial cells, as has just been explained).

In some embodiments, the implementation of the processing may be subjected to a concentration of dry matter of biomass from 2 to 90 g/l, for example, from about 2 to about 80 g/l, or from about 3 to about 40 g/l, for example, 3, 5, 10, 15, 20, 25, 30, 35, or 40 g/l, or even from 5 to 50 g/l or other ranges limited to the above concentrations. In some embodiments, implementation, processing the base are from about 40 to about 80 g/l, for example, 40, 50, 60, 70 or 80 g/l or other ranges limited to the above concentrations.

Dry weight of biomass is defined in the present description dry weight of material in grams per liter of sample. It can be measured by drying the sample in a small porcelain Cup at about 105°C until it reaches constant weight.

Temperatures can range from 30 to 60°C, for example, from 30 to 55°C, 30 to 50°C, from 3 to 45°C, from 30 to 40°C., or from 30 to 35°C. In some embodiments, the implementation, the processing temperatures may range from 35 to 60°C, for example, from 35 to 55°C, 35 to 50°C, 35 to 45°C or, for example, from 35 to 40°C. In some embodiments, the implementation, the processing temperatures may reach 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or even 40°C, or ranges limited to the above temperatures.

Processing time may vary from 2 hours to several days, for example, 1, 2, 3, 4, 5 or even 10 days, or from 3 to 120 hours, or from 3 to 48 hours, for example, 3, 5, 8, 15, 14, 16, 18, 20, 22, 24, 26, 28, 30, 36, 40, 44 or 48 h, or from 15 to 120 h, for example, from 60 to 120 hours, for example, 60, 72, 84, 96, 108 or 120 h, or ranges restricted to the above time intervals. It is clear that these time ranges include any fractional number of days, hours or minutes within them.

In some embodiments, implementation, use the concentration of a strong base from 0.001 N to 1.0 N, for example, from 0.001 N to 0.6 N, or from 0.10 N to 0.8 N, or from 0.6 N to 1.0 N, or range, starting or ending 0.001, of 0.002, 0.003 to 0.1 or N, or from 0.1 N to 0.6 N, or range, starting or ending from the 0.6 to 0.7, and 0.8, and 0.9, and 1.0 or 1.0 N or other ranges limited to the above concentrations. In some embodiments, implementation, used this concentration of the base. To access the e initial pH greater than 9,0, or a pH greater than 9.5, pH greater than 10.0 and less than 13,5, for example, more than 11.5, more than 12.0, more than 12,5, more than 13,0 or from pH 9.0 to pH of 13.5. In some embodiments, the implement may be used such concentration of the base so that, for example, to achieve an initial pH greater than 10.0 and less than 13,0 or from pH 9.0 to pH of 13.0.

In some embodiments, implementation, pH during processing may be reduced after extraction of soluble components. For example, the initial pH may be a basic pH, such as from pH 9.0 to pH of 13.0 or from pH 9.5 to pH of 12.5. Treatment may be given the opportunity to continue for a certain period of time, for example, from 3 to 120 h, for example, from 3 to 48 hours, or during the time periods listed above, at the above temperatures. Then, in some embodiments, implementation, possibly giving the pH by adding, for example, hydrochloric acid to obtain a pH of from 2.0 to 4.5 or pH from 2.5 to 4.5 or pH from 2.5 to 4.0, for example, 2,5, 3,0, 3,5, 4,0, or in the range bounded by any of the pH values listed above. The second treatment at low pH can be carried out at a temperature of from 30 to 60°C, 35 to 55 35 to 45°C, for example, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or even 45°C. the acid treatment Time can vary from 1 hour to several hours Ilido 72 h, for example, from 1 hour to 24 hours, or from 1 hour to 6 hours, or from 3 h to 48 h, or from 3 hours to 24 hours or from 4 to 72 h, or even from 24 h to 72 h, or in any range of time limited by the above time periods.

In some embodiments the invention, the alkaline treatment is performed on bacterial biomass, containing, for example, material from Lactobacillus fermentum, having a dry weight of biomass of 10 g/l to 40 g/L. In other embodiments the invention, the alkaline treatment is performed on bacterial biomass, containing a mixture of strains of Lactobacillus, and having a dry weight of biomass of 10 g/l to 40 g/L. In such scenarios, the implementation, the alkaline treatment may be performed at a concentration of ion hydrochloride from 0.025 to 0.25 N N or at a pH of from 9.5 to 12.5 at a temperature of from 35 to 45°C during the period of time from 3 h to 48 hours In some embodiments, implementation, alkaline treatment is performed on bacterial biomass, which contains material from one or more strains of Lactobacillus in ion concentration hydrochloride from 0.025 to 0.20 N N, from 0.025 to 0.15 N, from 0.025 to 0.10 N, 0.05 N to 0.25 N, from 0.05 to 0.20 N N, from 0.05 to 0.15 N, 0.05 N 0.10 N 0.10 N to 0.25 N, from 0.10 to 0.20 N N, from 0.10 to 0.15 N N, from 0.15 to 0.25 N N, from 0.15 to 0.20 N N or even from 0.20 to 0.25 N N. In such scenarios, implementation, pH may, for example, be from 9.5 to 12.0, from 9.5 to 11.5, 9.5 to 11.0, 9.5-10.5, 9.5 to 10.0, 10.0 to 12.5, 10.0 to 12.0, from 0.0 to 11.5, 10.0 to 11.0, 10.0 to 10.5, from 10.5 to 12.5, from 10.5 to 12.0, from 10.5 to 11.5, from 10.5 to 11.0, 11.0 to 12.5, 11.0 to 12.0, 11.0 to 11.5, 11.5 to 12.5, 11.5 to 12.0 or even pH ranging from 12.0 to 12.5. Time of the alkali treatment for such embodiments can be from 3 hours to 36 hours, from 3 h to 24 h, from 3 hours to 18 hours, from 3 h to 12 h, from 3 h to 6 h, 6 h to 48 h, from 6 hours to 36 hours, from 6 h to 24 h, 6 h to 18 h, 6 h, 12 h, 6 h to 8 h, 8 h to 48 h, 8 h to 36 h, from 8 h to 24 h, 8 h to 18 h, 8 h, 12 h, 12 h to 48 h, from 12 h to 36 h, 12 h to 18 h, 18 h to 48 h, 18 h and 36 h, 18 h d 24 h, 24 h to 48 h, 24 h and 36 h or 36 h to 48 hours Alkaline processing can be performed during any periods of time, close to the border with the above ranges, for example, 3, 6, 8, 12, 18, 24, 36 or even 48 hours such conditions can provide a reasonable alkaline processing.

In other embodiments, the implementation of 10 g/l and 40 g/l dry weight biomass of one or more strains of Lactobacillus can be subjected to ion concentration of hydroxide from 0.15 to 0.50 N N or pH 11.5 to 13.5 at a temperature of from 35 to 45°C over a period of time from 15 hours to 120 including, for Example, in some embodiments, the implementation of the concentration of hydroxide may range from 0.15 to 0.45 N N, from 0.15 to 0.40 N N 0.15 N to 0.35 N, from 0.15 to 0.30 N N 0.15 N to 0.25 N, from 0.15 to 0.20 N N, from 0.20 to 0.50 N N, from 0.20 to 0.40 N N, from 0.20 to 0.30 N N, from 0.25 to 0.50 N N, from 0.30 to 0.50 N N, from 0.30 to 0.40 N N or from 0.40 to 0.50 N. In this variant implementation of the ia pH may be, for example, from 11.5 to 13.0, 11.5 to 12.5, 11.5 to 12.0, ranging from 12.0 to 13.5, ranging from 12.0 to 13.0, ranging from 12.0 to 12.5, 12.5 to 13.5, from 12.5 to 13.0, of 13.0 to 13.5. The period of time for the alkali treatment can range from 15 h to 100 h, 15 h to 90 h, 15 h 75 h 15 h 60 h 15 h to 48 h, 15 h to 36 h from 24 h to 120 h from 24 h to 100 h, 24 h, 90 h, 24 h 75 h from 24 h to 60 h, 24 h to 48 h, 36 h to 120 h 36 h to 100 h, 36 h to 90 h, 36 h 75 h, 36 h and 60 h, 36 h to 48 h, 48 h, 120 h, 48 h to 100 h, 48 h to 90 h, 48 h 75 h, 48 h 60 h 60 h 120 h 60 h to 100 h 60 h 90 h 60 h 75 h 75 h to 120 h 75 h to 100 h 75 h 90 h 90 h to 120 h, or, for example, from 100 to 120 h Periods of time, also provided for the alkali treatment in the exercise of such options include 15, 24, 48, 60, 75, 90, 100 and 120 hours Such conditions can handle a strong alkali.

In other embodiments, implementation of 10 g/l to 40 g/l initial dry weight of the biomass can be processed by the concentration of hydroxide from 0.025 to 0.25 N N or pH from 9.5 to 12.5, at a temperature of from 35 to 45°C over a period of time from 3 h to 48 hours Then brought to a pH level of from 2.5 to 4.0 by adding acid, such as hydrochloric acid (HCl), which is an acid treatment. Acid treatment can be performed at a temperature of from 35 to 45°C over a period of time from 1 h to 24 h for Example, in such embodiments, implementation, alkaline obrabotkaponravilos biomass, containing one or more strains of Lactobacillus can be performed in hydroxide concentration from 0.025 to 0.20 N N, from 0.025 to 0.15 N, from 0.025 to 0.10 N, 0.05 N to 0.25 N, from 0.05 to 0.20 N N, from 0.05 to 0.15 N, 0.05 N 0.10 N 0.10 N to 0.25 N, from 0.10 to 0.20 N N 0.10 N to 0.15 N 0.15 N to 0.25 N, from 0.15 to 0.20 N N or even from 0.20 to 0.25 N In N. time of the alkali treatment in such scenarios, the implementation of the pH may, for example, be from 9.5 to 12.0, from 9.5 to 11.5, 9.5 to 11.0, 9.5-10.5, 9.5 to 10.0, 10.0 to 12.5, 10.0 to 12.0, 10.0 to 11.5, 10.0 to 11.0, 10.0 to 10.5, from 10.5 to 12.5, from 10.5 to 12.0, from 10.5 to 11.5, from 10.5 to 11.0, 11.0 to 12.5, 11.0 to 12.0, 11.0 to 11.5, 11.5 to 12.5, 11.5 to 12.0 or even pH ranging from 12.0 to 12.5. Time of the alkali treatment for such embodiments can be from 3 hours to 36 hours, from 3 h to 24 h, from 3 hours to 18 hours, from 3 h to 12 h, from 3 h to 6 h, 6 h to 48 h, from 6 hours to 36 hours, from 6 h to 24 h, 6 h to 18 h, 6 h, 12 h, 6 h to 8 h, 8 h to 48 h, 8 h to 36 h, from 8 h to 24 h, 8 h to 18 h, 8 h, 12 h, 12 h to 48 h, from 12 h to 36 h, 12 h to 18 h, 18 h to 48 h, 18 h and 36 h, from 18 h to 24 h, 24 h to 48 h, 24 h and 36 h or 36 h to 48 hours Alkaline processing can be performed during any period of time, close to the border with the above ranges, for example, 3, 6, 8, 12, 18, 24, 36 or even 48 hours and Then the pH can be brought up to a level of from 2.5 to 3.5, from 2.5 to 3.0, 3.0 to 4.0, from 3.0 to 3.5 or 3.5 to 4.0 by adding acid to the acid treatment after alkaline lysis. Acid clicks the processing can be performed during the period of time from 1 h to 18 h, from 1 h to 12 h from 1 h to 6 h from 1 h to 3 h, 3 h to 24 h, from 3 hours to 18 hours, from 3 h to 12 h, from 3 h to 6 h, 6 h to 24 h, 6 h to 18 h, 6 h, 12 h, 12 h to 24 h, 12 h to 18 h, 18 h to 24 h Periods of time, also provided for the acid treatment, include 1, 3, 6, 12, 18 and 24 hours

Lysates obtained after the above-described basic processing, can then be purified by centrifugation and/or filtration, for example, to delete presented in the form of particles and insoluble components. For example, lysates can centrifugeuse at 9000 x gravity followed by one or more cycles by filtration through a filter with pore size 0.2 μm. In some cases, it may be a consecutive cycles of filtration through filters with large pore size followed by filtration through a filter with pore size 0.2 μm. Can also be used in the methods of ultrafiltration to facilitate the extraction of soluble materials from the extract, for example, recirculation of the permeate of ultrafiltration for further filtration.

In some embodiments, the implementation of the filtering method can be used to filter the lysate and for the extraction of soluble molecules from larger cell fragments (figure 2). See, for example, the guide Separations Technology, Pharmaceutical and Biotechnology Applications, Wayne P. Olson, Editor, lnterpharm Press, Inc., Buffalo Grove, IL, U.S.A., p.126-135 - ISBN:0-935184-72-4. At the beginning of this process, assaulty bacterial lysate can be stored in the first tank. For example, if TFF extract may be subjected and filtration through a microfilter, and through an ultra-filter. For example, included a loop microfiltration (MF), and is pumping product. The resulting retentate MF is recycled, while the MF permeate is transferred to the second tank.

After achieving the appropriate degree of concentration included loop ultrafiltration (UF). The UF permeate may be recirculated back into the tank for continuous extraction solubilizing extracts from the lysate, while retentate UF is stored in the second tank. During continuous extraction, the volume in the tanks 1 and 2 can be selected by adjusting the flow rates of permeate microfiltration and ultrafiltration.

Can run multiple cycles of extraction, or TFF, or other means of filtration. In the variants of implementation, which is used TFF, at the end of the last cycle of the loop microfiltration can disconnect and loop microfiltration can be used separately, and the MF permeate can be transported in tank 2.

The terms of the transverse flux and transmembrane pressure are determined independent of pressure and adjustable mass transfer areas of film theory, described, for example, M. Cheryan (Ultrafiltration and Microfiltration Handbook, 2ndEd., Ch. 4, 1998). The flow of permea is a and the output extraction affect the filtering conditions (transmembrane pressure (TMP), transverse flow, temperature and so on). The filter type can also affect the filtration efficiency, as the system type plates (cartridge filter). Can be used in different configurations, including parallel type and coil type (see figure 2). Some were designed to be optimized for each used combination of regime type and the filter type.

Loop microfiltration can be equipped with filters with a pore size of 1.2 μm to 0.1 μm, such as filters with a pore size of from 0.65 to 0.2 μm or 0.45 μm. The cross-flow can be set from 100 to 3000 l/h m2(LHM), for example, from 300 to 2500 LHM or 2000 LHM when TMP is from 0.3 to 2 bar (from 31.5 to 210 N/m2). Loop ultrafiltration can be equipped with filters from 10 kDa to 1000 kDa, for example from 10 kDa to 100 kDa or 10 kDa to 30 kDa, or from about 30 kDa to 100 kDa. Transverse flow can range from 30 to 1000 LHM, for example, from 20 to 500 LHM in TMP from 0.2 to 1.5 bar (157,5 N/m2).

From 5 to 20 volumes of diafiltration can be used for the extraction of soluble components from the walls of the bacterial cells. In some embodiments, implementation, use 8 to 15 volumes. Therefore, for example, in some embodiments, the implementation can be used from 5 to 15 cycles of the filter, in some cases, from 8 to 15 cycles.

If desired, after filtration, the extract could critique the sustained fashion to concentrate or centrifugeuse. For example, there may be further microfiltration using a filter with smaller pore size, such as a filter with a pore size of 0.2 μm. After filtration, the extract may liofilizirovanny before his inclusion in the composition for application.

In some embodiments, implementation, after filtering, the extract can be purified to separate, remove or increase the concentration of one or more modified components in the extract. For example, removal of charged components can be used in stage a strong ion chromatography. Can use other cleaning methods, such as gel filtration, chromatography, ultracentrifugation, extraction and precipitation.

Chemical properties of bacterial extracts

Processing may lead to a variety of chemical modifications of cellular components. For example, in proteins: (1) the peptide bond can undergo partial decomposition, generating smaller polypeptides; (2) natural L-amino acids can be at least partially racemethionine in D-amino acids; and (3) aspartic and glutamic residues can be diaminononane, resulting in changes to the isoelectric point of proteins. Such molecules as lipoteichoic acid, lipopetides and phospholipids may be catalyzed by a base GI is rolito of ester groups and/or amide bonds, the resulting modified amphiphilic structures, which may have a new physico-chemical and immunological properties. Examples of other possible chemical modifications include partial solubilization of cell wall polysaccharides and complete hydrolysis of ribonucleic acid (RNA) in a separate ribonucleotides, including the restructuring of the phosphate groups.

Therefore, some or all of these chemical modifications can occur during the processing of the base cells of Lactobacillus as described in this application. Such molecular modification can affect the biological activity of extracts.

For example, the processing base of bacteria in accordance with the present invention can result in partial hydrolysis of proteins, as well as diaminononane, deletirovanie and/or partial racemization of amino acids from L to D. In one analytical study of the extract in accordance with the invention, were observed each of the peaks representing D-aspartic acid, D-glutamic acid, D-serine, D-methionine, D-gested, D-alanine, D-arginine, D-phenylalanine, D-tyrosine, D-leucine and D-lysine. The percentage of D-amino acids listed species in this study ranged from 3% to 40%. Therefore, some embodiments of the invention provide the possibility of racemization real the or more of the serine, threonine, histidine, alanine, arginine, tyrosine, phenylalanine, leucine and lysine, for example, all of the above amino acids, or any selection of more than one but less than all of the above amino acids, such as, for example, alanine, phenylalanine and lysine. In some embodiments, the implementation of at least one or more of the above amino acids can be racemethionine of D in L. In other embodiments, the implementation of at least 40% of one or more of the above amino acids can be racemethionine.

Thus, the extracts of the present invention may contain from 1 to 90% of D-amino acids, for example, from 1 to 80% or from 1%to 60%. In some embodiments, the implementation, the extract contains from 10 to 45% of D-amino acids, for example, from 25 to 35% of D-amino acids. The extracts according to the invention may contain at least one D-amino acid selected from the group consisting of D-aspartic acid, D-asparagine, D-glutamic acid, D-glutamine, D-serine, D-methionine, D-histidine, D-alanine, D-arginine, D-phenylalanine, D-tyrosine, D-leucine, D-lysine, D-valine and D-threonine. In some embodiments, the implementation of the concentration of any of the D-amino acids is from 1 to 50%, for example, from 10 to 40%, or even from 15 to 35%.

Some extracts of the present invention contain the bacterial cell wall and membrane components which you Lactobacillus, such as lipoteichoic acid, taikoubou acid, peptidoglycan or a combination of both. In some embodiments, implementation, these components are chemically modified. Some extracts also contain components of the cell wall and/or cell membrane, such as lipoproteins, which can be chemically modified. In some embodiments, implementation, components of the cell wall or components of the cell membrane, for example, lipoproteins, dissolve or suspendered in extracts and, thus, are not present in the form of particles or insoluble form.

In addition, the extract in accordance with the present invention may contain, for example, from 10 to 100 mg/ml soluble dry weight (SDW) of material, from 1 to 30 mg/ml of protein (Prot.), from 0.5 to 4.0 mg/ml of sugar and less than 100 mg/ml DNA. For example, some embodiments of contain from about 15 to 35 mg/ml of soluble dry matter, from 3 to 7 mg/ml protein, from 1.0 to 3.0 mg/ml of sugar and from 10 to 40 μg/ml DNA. The extract in accordance with the present invention may contain, for example, 30 mg/ml soluble dry weight, 9.6 mg/ml protein, 2.4 mg/ml of sugar and 33 μg/ml DNA, or in another example, contains 32.4 mg/ml of soluble dry matter, of 5.8 mg/ml protein, 2,3 mg/ml of sugar and less than 100 µg/ml DNA. Soluble Dry Weight (SDW) in g/l or mg/ml is determined by receiving 5 ml of races is Voronoi faction in the lysis or processing base and drying to constant weight in a porcelain Cup at 105°C.

In some embodiments, the implementation of the extracts contain at least 0.3 mg/ml saccharides, for example, from 0.3 to 4.5 mg/ml of sugars. In some embodiments, the implementation of at least one saccharide selected from monosaccharides, disaccharides and polysaccharides. Some of the extracts according to the invention contain at least one branched polysaccharide. In some embodiments, the implementation of at least one saccharide chemically modified.

Lysis or processing base of bacteria in accordance with the present invention can reduce the average molecular weight of the macromolecules of the component to the range, such as from 1 kDa to 300 kDa and 100 kDa, or up to a range from 1 kDa to 60 kDa and 10 kDa. In some embodiments, the implementation, the extract contains at least one protein with a molecular mass of less than 50 kDa, or less than 30 kDa, for example, less than 10 kDa.

The biological activity of bacterial extracts

The extracts according to the invention may possess immunomodulatory activity. For example, some extracts can stimulate the immune system. Some extracts may have anti-inflammatory activity. Certain effects of the extract may depend on the conditions of manufacture and type or strain of Lactobacillus or mixture of species and strains, which received extra is so Accordingly, some extracts in accordance with the invention can be powerful immunostimulirutuyu activity and, thus, ironically, can be used in the treatment of infections or as additions to such treatment, while other variants of the implementation can be more weak immunostimulirutuyu activity, but to show anti-inflammatory activity, thus being useful in the treatment of inflammatory disorders such as allergies, asthma, autoimmune disease, colitis, and inflammatory intestinal diseases, or as additions to such treatment.

Thus, some of the extracts according to the invention may be effective for treating patients suffering from disorders, including but not limited to microbial infection, allergic diseases, and disorders of the digestive tract. Some extracts in accordance with the invention can be provided to the patient in the form of nutraceuticals, for example, as adjuncts in the treatment of various conditions, including without limitation, microbial infection, allergic diseases and disorders of the digestive tract.

The range of types of biological activity of extracts can be determined by several in vitro and in vivo. For example, analysis of AlamarBlueTM-Assay includes fluorometric the/colorimetric growth indicator based on detection of metabolic activity indicator oxidation-reduction (REDOX) in response to chemical reduction in the growth of cells (example 4).

Cellular in vitro testing products of nitric oxide (NO) from murine macrophages and are used as a screening test to determine the ability of the extract to stimulate the immune system to destroy invasive bacteria (figure 5). In some embodiments, implementation, extracts can stimulate NO production in murine macrophages, leading to the measured NO concentrations from 3 μm to 60 μm, for example, from 5 μm to 40 μm. In some embodiments, implementation, NO concentration can be higher than 30 μm. The type of bacterial species may also affect these results. For example, extracts from Lactobacillus fermentum can cause large production of nitric oxide than Lactobacillus rahmnosus (see, for example, example 5 below). For screening of embodiments according to the invention to determine their immunostimulating or anti-inflammatory potential in vitro tests of the bacterial extracts according to the invention can be performed on mononuclear cells of peripheral blood (PBMC). Cm. publication Foligne et al. {World J Gastroenterol, 2007, 13(2):236-243). Can be measured release and IL12p70 (inflammatory cytokine), and IL10 (anti-inflammatory cytokine), and calculated the ratio of IL-10/IL-12 (example 6). Some embodiments of the invention provide a higher IL10/IL12 than control live Lactobacteria fermentum, thus suggesting that some who are extracts according to the invention can be equivalent to or even more powerful anti-inflammatory properties, than living maternal organism when introduced in vivo. Therefore, the invention includes extracts capable of reaching calculated relationship IL10/IL12 in mononuclear cells of peripheral blood, where the ratio is equal to or greater than the ratio IL10/IL12, achieved a live Lactobacillus strain from which the extract obtained.

The immune response of the extracts according to the invention can also be tested by the study of their effects on toll-like receptors (TLR), for Example, extracts can be tested in HEK293 cells in the presence or in the absence of TLR2 agonist Pam3Cys, or in the presence or in the absence of the TLR4 agonist LPS (example 7). Cell line HEK293 enables effective monitoring of TLR activity using ELISA assays, such as titration of IL-8 or based reporter systems that provide monitoring induced TLR activation of NF-κB. Some embodiments of the invention can act as antagonists of TLR 2/6 in HEK cells TLR 2/6. Thus, some embodiments of can be used to combat infections in the individual, while other variants of the implementation can be used against inflammation and/or autoimmune disorders.

The screening described in the present description extracts may also be conducted in relation to the activity of the receptor TLR and NOD2 (Example 8). Some embodiments of the invention activate the receptor (TLR) m/OR NOD2 in vitro, indicating that they may be able to activate the immune system, TLRs and/or NOD2.

Methods of plaque-forming cells (PFC) can be used to assess non-specific stimulation of B-lymphocytes (example 9). Certain lymphoid cells release hemolytic antibodies, which penetrate and cause lysis of adjacent erythrocytes education leisnig plaques in the presence of complement. Some embodiments of the invention can increase the secretion of immunoglobulins B cells, and, thus, can potentially be used prophylactically to premirovany immune system in individuals suffering from recurrent infections.

Anti-infective efficacy of the extracts of the present invention may be tested, for example, after infection with Salmonella individuals, such as infection of mice (example 10). Some embodiments of the invention can provide protective immunity against infections, such as bacterial infections, i.e. infections Salmonella. For example, some of the options for implementation may reduce mortality in mice induced by injection of Salmonella thyphimurium.

The combination of NO activity in vitro by determination of the activity in vivo, as measured, for example, in the mouse model you is private allergen asthma (example 11) may provide a more complete view of potential clinical activity described in this application extracts. On models LACK (protein from the parasite Leishmania major), the number of eosinophils found in liquids broncho-alveolar lavage, compared to control animals with asthma (not treated) can be reduced by a factor from 1 to 10, for example, reduced from 1.5 times to 5 times. Accordingly, in some embodiments, implementation, extract reduces the number of eosinophilic cells, polymorphonuclear cells, lymphocytic cells or any combination of them suffering from asthma murine individual by a factor of at least 1.5 to relatively non-treated control asthma. Some of the options for implementation may reduce eosinophilia in asthma individuals and at the same time to reduce the level of Th2 cytokines such as IL4, IL5, IL13), which are markers of asthma. Thus, such embodiments of, for example, may have anti-inflammatory activity in individuals suffering from immunological disorders such as allergic disorders, including asthma.

Compositions containing bacterial extracts

The extracts according to the invention can be prepared a number of different ways for the end of the introduction. For example, can be obtained oral tablets, capsules, pills, and liquid formulation or aerosols. Can be also obtained preparation is effective shape for infusion or injection.

Embodiments of the present invention can be prepared, for example, in the form of a solid dosage form or liquid dosage forms. Illustrative solid dosage forms may include, for example, tablet (e.g., coated tablet, chewable tablet, tablet, emit gas when dissolved, sublingual tablet, granular material, powder or capsule)containing the extract.

Solid dosage forms may also contain diluents, fillers and/or other excipients. Can add other excipients, as preservatives, coloring agents, flavorings and sweeteners.

Alternatively, capsules and tablets can be designed for oral route of administration.

The extracts of the present invention can be included in one or more nutraceutical compositions, such as nutrients and/or dietary supplements and food additives, or one or more pharmaceutical compositions.

Introduction bacterial extracts the individual

Dose containing at least one extract of the present invention, may be administered to an individual suffering from at least one disorder selected from disorders of the digestive tract, disorders of the respiratory tract, disorders of the urinary tract and allergies the state, or having the risk of their development. For example, in some embodiments, the implementation of the extracts can be administered to individuals suffering from infections of the upper and lower parts of the lung, obstructive pulmonary disease with acute infection of the lower respiratory tract, obstructive pulmonary disease in the acute stage, the common cold, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, laryngopharyngitis, influenza, pneumonia, bronchopneumonia, bronchitis, rhinitis, common cold, pharyngitis, sinusitis, tonsillitis, laryngitis, laryngotracheitis, bronchitis, allergic asthma, atopic dermatitis, urinary tract infections due to obstructive and reflux uropathy, urethritis, tubulo-interstitial nephritis, obstructive pyelonephritis, cystitis, including chronic cystitis syndrome pelvic pain in men, including prostatitis and chronic prostatitis, prostatitis, inflammatory diseases of the pelvic organs in women, or at risk of their development.

In some embodiments, implementation, extracts are introduced to the individual in the form of a nutraceutical composition, such as a nutritional Supplement and/or dietary Supplement. In other embodiments, implementation, extracts are introduced to the individual in the form of pharmaceutical compositions. The introduction may include the introduction of a single dose or m is divine doses.

In some embodiments, the implementation, the extract may be provided in a therapeutically effective dose for the treatment of an individual suffering from one or more of the above conditions. In some embodiments, the implementation, the extract may be provided in addition to other medical treatment.

WORKING EXAMPLES

EXAMPLE 1: Bacterial culture

EXAMPLE 1.1: Lactobacillus fermentum I-3929

The initial conditions of cultivation

Culture medium was obtained by dissolving in purified water the following components: sodium Chloride: 3 g/l; Monohydratefast sodium: 2 g/l; sodium Acetate: 1 g/l; Peptone soybean 50 g/l; Glucose: 12 g/l; calcium Chloride: 0.1 g/l; potassium Chloride: 0.1 g/l; sodium Bicarbonate: 0.5 g/l; pyruvate: 0.1 g/l; Glutamate: 0.2 g/l; the Solution of metals (copper sulfate: 3 mg/l; ferric chloride: 830 mg/l; zinc sulfate: 860 mg/l; sulfuric acid: 1.1 mg/l): 0.5 ml/l was Then added polypropylenglycol (0,02 ml/l, the density of 1,005 g/ml). After dissolution, the pH was not regulated. After sterilization, the medium, small Erlenmeyer flask separately seeded content of frozen vials containing 1.5 ml of frozen bacteria) and incubated at 37°C for 8 hours. Then aliquots of 20 ml of this culture was transferred into a larger Erlenmeyer flask containing 1000 ml of culture medium and again incubated in the same conditions. On the Le 16 h of growth, the contents of the Erlenmeyer flask with a capacity of 1 l is moved in OBRAZOVATEL.

The cultivation conditions in OBRAZOVATEL

20 liters of culture medium was obtained by dissolving in purified water the following components: sodium Chloride: 3 g/l; Monohydratefast sodium: 2 g/l; sodium Acetate: 1 g/l; Peptone soybean 50 g/l; Glucose: 12 g/l; calcium Chloride: 0.1 g/l; potassium Chloride: 0.1 g/l; sodium Bicarbonate: 0.5 g/l; pyruvate: 0.1 g/l; Glutamate: 0.2 g/l; the Solution of metals (copper sulfate: 3 mg/l; chloride iron: 830 mg/l; zinc sulfate: 860 mg/l; sulfuric acid: 1.1 mg/l): 0.5 ml/l was Then added polypropylenglycol (0,02 ml/l). After dissolution, the pH was not regulated. The incubation temperature was regulated at 37°C, under stirring at a speed of 100 rpm During the cultivation pH was not regulated. After 24 h 7 liters of vospriiatiia was transferred into a fermenter (optical density (OD) at 700 nm of the culture of OBRAZOVATEL after 24 h = 1,24). Culture of vospriiatiia in sterile conditions and transferred to fermenters.

Conditions of cultivation in fermenters

70 liters of culture medium was obtained in purified water the following components: sodium Chloride: 3 g/l; Monohydratefast sodium: 2 g/l; sodium Acetate: 1 g/l; Peptone soybean 50 g/l; Glucose: 12 g/l; calcium Chloride: 0.1 g/l; potassium Chloride: 0.1 g/l; sodium Bicarbonate: 0.5 g/l; pyruvate: 0.1 g/l; Glutamate: 0.2 g/l; the Solution of metals (copper sulfate: 3 the g/l; the ferric chloride: 830 mg/l; zinc sulfate: 860 mg/l; sulfuric acid: 1.1 mg/l): 0.5 ml/l was Then added polypropylenglycol (0,02 ml/l). After dissolution, the pH was not regulated.

After sterilization of the culture was added 8 g/l glucose. The incubation temperature was regulated at 37°C under stirring at a speed of 100 rpm and without aeration. During cultivation, the pH was regulated at the level of 5.7. After 16 h of culture (OD of the culture at 700 nm 2,30) iactiveaware heat treatment at 65°C for 35 minutes and was transferred to a tank for collecting the cell culture. After inactivation, the culture was transferred into a rack ultrafiltration to separate the biomass from the culture medium, concentrated and washed with NaCl (9 g/l) in treated water. The biomass was divided into aliquot amount (mass concentrated bacterial suspension 2000 when 31.8 mg dry weight of biomass per 1 gram of the concentrated bacterial suspension) and then frozen at -15°C.

EXAMPLE 1.2: Lactobacillus helveticus 103146

The initial conditions of cultivation

Culture medium was obtained by dissolving in purified water the following components: sodium Chloride: 3 g/l; Monohydratefast sodium: 2 g/l; sodium Acetate: 1 g/l; Peptone soybean 50 g/l; Glucose: 12 g/l; calcium Chloride: 0.1 g/l; potassium Chloride: 0.1 g/l; sodium Bicarbonate: 0.5 g/l; pyruvate: 0.1 g/l; Glutamate: 0.2 g/l; the Solution of the metal is (copper sulfate: 3 mg/l; the ferric chloride: 830 mg/l; zinc sulfate: 860 mg/l; sulfuric acid: 1.1 mg/l): 0.5 ml/l was Then added polypropylenglycol (0,02 ml/l). After dissolution, the pH was not regulated. After sterilization, the medium, small Erlenmeyer flask separately seeded content of frozen vials containing 1.5 ml of frozen bacteria) and incubated at 37°C for 9 hours. Then aliquots of 20 ml of this culture was transferred into a larger Erlenmeyer flask containing 1000 ml of culture medium and again incubated in the same conditions. After 15 h of growth, the contents of the Erlenmeyer flask with a capacity of 1 l is moved in OBRAZOVATEL.

The cultivation conditions in OBRAZOVATEL

20 liters of culture medium was obtained by dissolving in purified water the following components: sodium Chloride: 3 g/l; Monohydratefast sodium: 2 g/l; sodium Acetate: 1 g/l; Peptone soybean 50 g/l; Glucose: 12 g/l; calcium Chloride: 0.1 g/l; potassium Chloride: 0.1 g/l; sodium Bicarbonate: 0.5 g/l; pyruvate: 0.1 g/l; Glutamate: 0.2 g/l; the Solution of metals (copper sulfate: 3 mg/l; chloride iron: 830 mg/l; zinc sulfate: 860 mg/l; sulfuric acid: 1.1 mg/l): 0.5 ml/l was Then added polypropylenglycol (0,02 ml/l). After dissolution, the pH was not regulated. The incubation temperature was regulated at 37°C, under stirring at a speed of 100 rpm without aeration. During cultivation, the pH was not regulated. After 9 h 7 liters of vosberg the users was transferred into a fermenter (OD at 700 nm of the culture of OBRAZOVATEL through 9 h: 0,14). Culture of vospriiatiia in sterile conditions and transferred to fermenters.

Conditions of cultivation in fermenters

70 liters of culture medium was obtained in purified water the following components: sodium Chloride: 3 g/l; Monohydratefast sodium: 2 g/l; sodium Acetate: 1 g/l; Peptone soybean 50 g/l; Glucose: 12 g/l; calcium Chloride: 0.1 g/l; potassium Chloride: 0.1 g/l; sodium Bicarbonate: 0.5 g/l; pyruvate: 0.1 g/l; Glutamate: 0.2 g/l; the Solution of metals (copper sulfate: 3 mg/l; ferric chloride: 830 mg/l; zinc sulfate: 860 mg/l; sulfuric acid: 1.1 mg/l): 0.5 ml/l was Then added polypropylenglycol (0,02 ml/l). After dissolution, the pH was not regulated.

After sterilization of the culture was added 8 g/l glucose. The incubation temperature was regulated at 33°C, under stirring at a speed of 100 rpm and without aeration. At the beginning of the cultivation pH was regulated at the level of 6.7 CH3COOH. After 24 h of culture (OD of the culture at 700 nm 4,17) iactiveaware heat treatment at 65°C for 35 min and transferred into the reservoir for collection of cell cultures. After inactivation, the culture was transferred into a rack ultrafiltration to separate the biomass from the culture medium, concentrated and washed with NaCl (9 g/l) in purified water (9 g/l). The biomass was divided into aliquot amount (mass concentrated bacterial suspension 440 g of 19.1 mg dry weight of biomass per 1 gram of koncentrira is authorized bacterial suspension) and then frozen at -15°C.

EXAMPLE 1.3: Lactobacillus plantarum 71.39

The initial conditions of cultivation

Culture medium was obtained by dissolving in purified water the following components: sodium Chloride: 3 g/l; Monohydratefast sodium: 2 g/l; sodium Acetate: 1 g/l; Peptone soybean 50 g/l; Glucose: 12 g/l; calcium Chloride: 0.1 g/l; potassium Chloride: 0.1 g/l; sodium Bicarbonate: 0.5 g/l; pyruvate: 0.1 g/l; Glutamate: 0.2 g/l; the Solution of metals (copper sulfate: 3 mg/l; ferric chloride: 830 mg/l; zinc sulfate: 860 mg/l; sulfuric acid: 1.1 mg/l): 0.5 ml/l was Then added polypropylenglycol (0,02 ml/l). After dissolution, the pH was not regulated. After sterilization, the medium, small Erlenmeyer flask separately seeded content of frozen vials containing 1.5 ml of frozen bacteria) and incubated at 35°C for 9 hours. Then aliquots of 20 ml of this culture was transferred into a larger Erlenmeyer flask containing 1000 ml of culture medium and again incubated in the same conditions. After 15 h of growth, the contents of the Erlenmeyer flask with a capacity of 1 l is moved in OBRAZOVATEL.

The cultivation conditions in OBRAZOVATEL

20 liters of culture medium was obtained by dissolving in purified water the following components: sodium Chloride: 3 g/l; Monohydratefast sodium: 2 g/l; sodium Acetate: 1 g/l; Peptone soybean 50 g/l; Glucose: 12 g/l; calcium Chloride: 0.1 g/l; potassium Chloride: 0.1 g/l; sodium Bicarbonate: 0,5g/l; pyruvate: 0.1 g/l; Glutamate: 0.2 g/l; the Solution of metals (copper sulfate: 3 mg/l; ferric chloride: 830 mg/l; zinc sulfate: 860 mg/l; sulfuric acid: 1.1 mg/l): 0.5 ml/l was Then added polypropylenglycol (0,02 ml/l). After dissolution, the pH was not regulated. The incubation temperature was regulated at 37°C, under stirring at a speed of 100 rpm without aeration. During the cultivation the pH was not regulated. After 9 h 7 liters of vospriiatiia was transferred into a fermenter (OD at 700 nm of the culture of OBRAZOVATEL through 9 h = 1,62). Culture of vospriiatiia in sterile conditions and transferred to fermenters.

Conditions of cultivation in fermenters

70 liters of culture medium was obtained in purified water the following components: sodium Chloride: 3 g/l; Monohydratefast sodium: 2 g/l; sodium Acetate: 1 g/l; Peptone soybean 50 g/l; Glucose: 12 g/l; calcium Chloride: 0.1 g/l; potassium Chloride: 0.1 g/l; sodium Bicarbonate: 0.5 g/l; pyruvate: 0.1 g/l; Glutamate: 0.2 g/l; the Solution of metals (copper sulfate: 3 mg/l; ferric chloride: 830 mg/l; zinc sulfate: 860 mg/l; sulfuric acid: 1.1 mg/l): 0.5 ml/l was Then added polypropylenglycol (0,02 ml/l). After dissolution, the pH was not regulated.

After sterilization of the culture was added 8 g/l glucose. The incubation temperature was regulated at 35°C, under stirring at a speed of 100 rpm and without aeration. After 24 h of culture (OD of the culture at 700 nm = 6,36) iactiveaware heat of the processing at 65°C for 35 min and transferred into the reservoir for collection of cell cultures. After inactivation, the culture was transferred into a rack ultrafiltration to separate the biomass from the culture medium, concentrated and washed with NaCl (9 g/l) in treated water. The biomass was divided into aliquot amount (mass concentrated bacterial suspension 600 g of 60.7 mg dry weight of biomass per 1 gram of the concentrated bacterial suspension) and then frozen at -15°C.

EXAMPLE 1.4: Lactobacillus rhamnosus 71.38

The initial conditions of cultivation

Culture medium was obtained by dissolving in purified water the following components: sodium Chloride: 3 g/l; Monohydratefast sodium: 2 g/l; sodium Acetate: 1 g/l; Peptone soybean 50 g/l; Glucose: 12 g/l; calcium Chloride: 0.1 g/l; potassium Chloride: 0.1 g/l; sodium Bicarbonate: 0.5 g/l; pyruvate: 0.1 g/l; Glutamate: 0.2 g/l; the Solution of metals (copper sulfate: 3 mg/l; ferric chloride: 830 mg/l; zinc sulfate: 860 mg/l; sulfuric acid: 1.1 mg/l): 0.5 ml/L. After dissolution, the pH was not regulated. After sterilization, the medium, small Erlenmeyer flask separately seeded content of frozen vials containing 1.5 ml of frozen bacteria) and incubated at 35°C for 9 hours. Then aliquots of 20 ml of this culture was transferred into a larger Erlenmeyer flask containing 1000 ml of culture medium and again incubated in the same conditions. After 15 h of growth, the contents of the flask Erlenmeyer eat the awn 1 l is moved in OBRAZOVATEL.

The cultivation conditions in OBRAZOVATEL

20 liters of culture medium was obtained by dissolving in purified water the following components: sodium Chloride: 3 g/l; Monohydratefast sodium: 2 g/l; sodium Acetate: 1 g/l; Peptone soybean 50 g/l; Glucose: 12 g/l; calcium Chloride: 0.1 g/l; potassium Chloride: 0.1 g/l; sodium Bicarbonate: 0.5 g/l; pyruvate: 0.1 g/l; Glutamate: 0.2 g/l; the Solution of metals (copper sulfate: 3 mg/l; chloride iron: 830 mg/l; zinc sulfate: 860 mg/l; sulfuric acid: 1.1 mg/l): 0.5 ml/l was Then added polypropylenglycol (0,02 ml/l). After dissolution, the pH was not regulated. The incubation temperature was regulated at 35°C, under stirring at a speed of 100 rpm without aeration. During cultivation, the pH was not regulated. After 9 h 7 liters of vospriiatiia was transferred into a fermenter (OD at 700 nm of the culture of OBRAZOVATEL through 9 h: 3,75). Culture of vospriiatiia in sterile conditions and transferred to fermenters.

Conditions of cultivation in fermenters

70 liters of culture medium was obtained in purified water the following components: sodium Chloride: 3 g/l; Monohydratefast sodium: 2 g/l; sodium Acetate: 1 g/l; Peptone soybean 50 g/l; Glucose: 12 g/l; calcium Chloride: 0.1 g/l; potassium Chloride: 0.1 g/l; sodium Bicarbonate: 0.5 g/l; pyruvate: 0.1 g/l; Glutamate: 0.2 g/l; the Solution of metals (copper sulfate: 3 mg/l; ferric chloride: 830 mg/l; zinc sulfate: 860 mg/l; sulfuric acid: 1.1 mg/l): 0.5 ml/L. Then d is balali polypropylenglycol (0,02 ml/l). After dissolution, the pH was not regulated.

After sterilization of the culture was added 8 g/l glucose. The incubation temperature was regulated at 35°C, under stirring at a speed of 100 rpm and without aeration. After 14 h of culture (OD of the culture at 700 nm 5,43) iactiveaware heat treatment at 65°C for 35 min and transferred into the reservoir for collection of cell cultures. After inactivation, the culture was transferred into a rack ultrafiltration to separate the biomass from the culture medium, concentrated and washed with NaCl (9 g/l) in treated water. The biomass was divided into aliquot amount (mass concentrated bacterial suspension 2798 g of 51.7 mg dry weight of biomass per 1 gram of the concentrated bacterial suspension) and then frozen at -15°C.

EXAMPLE 1.5: Lactobacillus iohnsonii 103782

The initial conditions of cultivation

Culture medium was obtained by dissolving in purified water the following components: sodium Chloride: 3 g/l; Monohydratefast sodium: 2 g/l; sodium Acetate: 1 g/l; Peptone soybean 50 g/l; Glucose: 12 g/l; calcium Chloride: 0.1 g/l; potassium Chloride: 0.1 g/l; sodium Bicarbonate: 0.5 g/l; pyruvate: 0.1 g/l; Glutamate: 0.2 g/l; the Solution of metals (copper sulfate: 3 mg/l; ferric chloride: 830 mg/l; zinc sulfate: 860 mg/l; sulfuric acid: 1.1 mg/l): 0.5 ml/l was Then added polypropylenglycol (0,02 ml/l). After dissolution, the pH was not regulated is. After sterilization, the medium, small Erlenmeyer flask separately seeded content of frozen vials containing 1.5 ml of frozen bacteria), and incubated at 33°C for 10 hours. Then aliquots of 20 ml of this culture was transferred into a larger Erlenmeyer flask containing 1000 ml of culture medium and again incubated in the same conditions. After 14 h of growth, the contents of the Erlenmeyer flask with a capacity of 1 l is moved in OBRAZOVATEL.

The cultivation conditions in OBRAZOVATEL

20 liters of culture medium was obtained by dissolving in purified water the following components: sodium Chloride: 3 g/l; Monohydratefast sodium: 2 g/l; sodium Acetate: 1 g/l; Peptone soybean 50 g/l; Glucose: 12 g/l; calcium Chloride: 0.1 g/l; potassium Chloride: 0.1 g/l; sodium Bicarbonate: 0.5 g/l; pyruvate: 0.1 g/l; Glutamate: 0.2 g/l; the Solution of metals (copper sulfate: 3 mg/l; chloride iron: 830 mg/l; zinc sulfate: 860 mg/l; sulfuric acid: 1.1 mg/l): 0.5 ml/l was Then added polypropylenglycol (0,02 ml/l). After dissolution, the pH was not regulated. The incubation temperature was regulated at 35°C, under stirring at a speed of 100 rpm without aeration. the pH of the culture brought to 5.6 with acetic acid. After 24 h 7 liters of vospriiatiia was transferred into a fermenter (OD at 700 nm of the culture of OBRAZOVATEL after 24 h: 0,47). Culture of vospriiatiia in sterile conditions and transferred to fermenters.

Conditions of cultivation in fermenters

70 liters of culture medium was obtained in purified water the following components: sodium Chloride: 3 g/l; Monohydratefast sodium: 2 g/l; sodium Acetate: 1 g/l; Peptone soybean 50 g/l; Glucose: 12 g/l; calcium Chloride: 0.1 g/l; potassium Chloride: 0.1 g/l; sodium Bicarbonate: 0.5 g/l; pyruvate: 0.1 g/l; Glutamate: 0.2 g/l; the Solution of metals (copper sulfate: 3 mg/l; ferric chloride: 830 mg/l; zinc sulfate: 860 mg/l; sulfuric acid: 1.1 mg/l): 0.5 ml/l was Then added polypropylenglycol (0,02 ml/l). After dissolution, the pH was not regulated.

After sterilization of the culture was added 8 g/l glucose. The incubation temperature was regulated at 35°C, under stirring at a speed of 100 rpm and without aeration. After 24 h of culture (OD of the culture at 700 nm 0,174) iactiveaware heat treatment at 70°C for 39 min and transferred into the reservoir for collection of cell cultures. After inactivation, the culture was transferred into a rack ultrafiltration to separate the biomass from the culture medium, concentrated and washed with NaCl (9 g/l) in treated water. The biomass was divided into aliquot amount (mass concentrated bacterial suspension of 61.5 g of 20.2 mg dry weight of biomass per 1 gram of the concentrated bacterial suspension) and then frozen at -15°C.

EXAMPLE 2: Bacterial lysates

EXAMPLE 2.1

One aliquot of biomass Lactobacillus fermetum I-3929 from example 1.1, containing 6 g of bacterial dry mass, were thawed at room temperature, then was diluted with purified water to reach 12 g/l dry weight of bacterial biomass. Alkalization performed to 0.03 M sodium hydroxide. the pH measured at the beginning of the lysis, represented 10.3. Then the lysate was incubated for 6 h at 40°C with continuous stirring. After incubation, the pH was 9.9.

EXAMPLE 2.2

One aliquot of biomass Lactobacillus rhamnosus 71.38 from example 1.4, containing 20 g of bacterial dry mass, were thawed at room temperature, then was diluted with purified water to achieve a 40 g/l dry weight of bacterial biomass. Alkalization performed to 0.03 M sodium hydroxide. the pH measured at the beginning of the lysis, represented 10.3. Then the lysate was incubated for 6 h at 40°C with continuous stirring. After incubation, the pH was 9.7.

EXAMPLE 2.3

One aliquot of biomass Lactobacillus fermentum I-3929 from example 1.1, containing 6 g of bacterial dry mass, were thawed at room temperature, then was diluted with purified water to reach 12 g/l dry weight of bacterial biomass. Alkalization performed by 0.06 M sodium hydroxide. the pH measured at the beginning of the lysis, amounted to 12.3. Then the lysate was incubated for 6 h at 40°C with continuous stirring. After incubation, the pH was 11.8.

EXAMPLE 2.4

One aliquot of biomass Lactobacillus fermetum I-3929 from example 1.1, containing 6 g of bacterial dry mass, were thawed at room temperature, then was diluted with 0.2 N NaCl solution to achieve 12 g/l dry weight of bacterial biomass. The final concentration of NaCl was 0,15N. Alkalization performed by 0.06 M sodium hydroxide. the pH measured at the beginning of the lysis, was 6.4. Then the lysate was incubated for 6 h at 40°C with continuous stirring. After incubation, the pH was 6.3.

EXAMPLE 2.5

The aliquot of the lysate of example 2.1 was taken to continue the process of lysis. the pH of the total volume was brought up to 3.6 25% HCl and then incubated in a water bath at 40°C for 1 h under static conditions.

EXAMPLE 2.6

The aliquot of the lysate of example 2.4 was taken to continue the process of lysis. the pH of the total volume was brought up to 12.4 10N NaOH and then incubated in a water bath at 40°C for 1 h under static conditions.

EXAMPLE 2.7

One aliquot of biomass Lactobacillus plantarum 71.39 from example 1.3, containing 0.4 g of bacterial dry mass, were thawed at room temperature, then was diluted with purified water to achieve 7 g/l dry weight of bacterial biomass. Alkalization performed by 0.06 M sodium hydroxide. the pH measured at the beginning of the lysis, was 12.6. Then the lysate was incubated for 2 h at 40°C with continuous stirring. After incubation, the pH of 12.4.

EXAMPLE 2.8

One aliquot of biomass Lactobacillus johnsonii 103782 from approx the RA 1.5, containing 0.3 g of bacterial dry mass, were thawed at room temperature, then was diluted with purified water to achieve a 6 g/l dry weight of bacterial biomass. Alkalization performed by 0.06 M sodium hydroxide. the pH measured at the beginning of the lysis, was 12.5. Then the lysate was incubated for 2 h at 40°C with continuous stirring. After incubation, the pH was 12,3.

EXAMPLE 2.9

One aliquot of biomass Lactobacillus helveticus 103146 from example 1.2, containing 0.4 g of bacterial dry mass, were thawed at room temperature, then was diluted with purified water to achieve 8 g/l dry weight of bacterial biomass. Alkalization performed by 0.06 M sodium hydroxide. the pH measured at the beginning of the lysis, was 12.8. Then the lysate was incubated for 2 h at 40°C with continuous stirring. After incubation, the pH was 12,2.

EXAMPLE 2.10

One aliquot of biomass Lactobacillus fermentum I-3929 from example 1.1, containing 6.8 g of bacterial dry mass, were thawed at room temperature, then was diluted with purified water to achieve 7 g/l dry weight of bacterial biomass. Alkalization performed to 0.08 M sodium hydroxide. the pH measured at the beginning of the lysis, amounted to 12.2. Then the lysate was incubated for 7 h at 40°C with continuous stirring. After incubation, the pH was 12.0. the pH of the lysate was brought to 9.8 with HCl.

The lysate contained with globalisierung dry weight (SDW): 20,5 mg/g, proteins (Prot): 4,9 mg/g

EXAMPLE 2.11

One aliquot of biomass Lactobacillus fermentum I-3929 from example 1.1, containing 6.8 g of bacterial dry mass, were thawed at room temperature, then was diluted with purified water to achieve 7 g/l dry weight of bacterial biomass. Alkalization performed 0.15 M sodium hydroxide. the pH measured at the beginning of lysis was 13,0. Then the lysate was incubated for 63 h at 40°C with continuous stirring. After incubation, the pH was 12.6. the pH of the lysate was brought to 9.9 HCl.

The lysate contained SDW: of 23.7 mg/g Prot: 5.0 mg/g

EXAMPLE 2.12

One aliquot of biomass Lactobacillus fermentum I-3929 from example 1.1, containing 98 g of bacterial dry mass, were thawed at room temperature, then was diluted with purified water to 10 g/l dry weight of bacterial biomass. Alkalization performed to 0.08 M sodium hydroxide. the pH measured at the beginning of the lysis, was 12.0. Then the lysate were incubated for 24 h at 40°C with continuous stirring. After incubation, the pH was 11.1. the pH of the lysate was brought to 11.5 with NaOH.

The lysate contained SDW: of 23.7 mg/g

EXAMPLE 2.13

One aliquot of biomass Lactobacillus fermentum I-3929 from example 1.1, containing 39 g of bacterial dry mass, were thawed at room temperature, then was diluted 0,2N NaCl to achieve 7.6 g/l dry weight of bacterial biomass in order to obtain osmotic Lisa is and. the pH measured at the beginning of lysis was 4.4. Then bacterial osmotic lysate were incubated for 24 h at 40°C with continuous stirring. After incubation, the pH was 4.2. Then the bacterial lysate was made alkaline by adding NaOH to achieve 0,06 n pH measured at the beginning of the alkaline lysis was 10.6. The lysate re-incubated for 2.25 hours at 40°C with continuous stirring. After incubation, the pH was 10.2.

EXAMPLE 3: Purification of lysates

EXAMPLE 3.1

The lysate of example 2.5 centrifuged for 20 minutes at 3000×g. Then the pH of the supernatant was brought to 6.8 10N NaOH and filtered through successive filters with pore size of 0.45 μm and 0.2 μm, and finally sterilizing filter with a pore size of 0.22 μm. The concentrate contained Prot: 2.8 mg/ml; DNA: to 17.4 ág/ml Percentage of D-Amino acids: 14,9% D-AIa, 14.4% of D-Pro, 14.2% of D-Asp.

The NO production in mg dry weight/ml: 0.003 mg/ml (C1) - 0.03 mg/ml (C2) - 0.3 mg/ml (C3) of active dry weight/ml: C1: 3,3 µm, C2: 33,2 μm, C3: 52,5 mm. Active Dry Weight in g/l or mg/ml is a soluble dry weight in g/l or mg/ml minus the chloride content in g/l or mg/ml

EXAMPLE 3.2

The lysate of example 2.6 centrifuged for 20 minutes at 3000×g. Then the pH of the supernatant was brought to 7 with HCl and filtered through successive filters with pore size of 0.45 μm and 0.2 μm, and finally sterilizing filter with pore size 0.22 μm Concentrate contained Prot: 12,3 mg/ml; DNA: 27,7 µg/ml the Percentage of D-Amino acids: 15,9% D-Ala, 24.4% of D-Pro, 17.4% of D-Asp, 45,1% D-Ser, 10.1% of D-Met.

The NO production in mg dry weight/ml: 0.003 mg/ml (C1) - 0.03 mg/ml (C2) - 0.3 mg/ml (C3) of active dry weight/ml: C1: 8.0 µm, C2: 56,0 μm, C3: 94,3 microns.

EXAMPLE 3.3

300 ml of the lysate of example 2.1 first centrifuged at 3000×g for 20 minutes. the pH of the supernatant was brought to 7.1 HCl. The extract was concentrated through the membrane of polytonality (PES) (Sartorius Stedim Biotech GmbH) with a pore size of 0.45 μm at a constant flow 330-350 ml/min on a system of cross-flow on a Sartoflow® Slice 200 Benchtop Crossflow System. The volume of output was 75%. Finally, the concentrate was subjected to sterilizing filtration through PES membrane with a pore size of 0.2 μm (Nalgene).

The concentrate contained SDW: 30.0 mg/g; Prot: 9.6 mg/ml; DNA: 32,8 µg/ml, the Percentage of D-Amino acids: 14,3% D-Ala, 13.9% of D-Pro, 14.1% with D-Asp, 36.9% of D-Ser. The NO production in mg of active dry weight/ml: 0.003 mg/ml (C1) - 0.03 mg/ml (C2) - 0.3 mg/ml (C3) of active dry weight/ml: C1: of 5.1 μm, C2: 30,0 μm, C3: 64,0 mm.

EXAMPLE 3.4

pH 300 ml of lysate of example 2.2 was brought to 7.0 with HCl. The extract was filtered as described in example 3.3 with constant flow with a speed of 350 ml/mn. The volume of output accounted for more than 75%. Finally, the concentrate was subjected to sterilizing filtration through PES membrane with a pore size of 0.2 μm (Nalgene).

The concentrate contained SDW: 49.2 mg/g; Prot: 14,2 mg/ml; DNA: 34,1 µg/ml, the Percentage of D-Amino acids: 16,0% D-Ala, 13.3% of D-Pro, 14,2 D-Asp.

The NO production in mg of active dry weight/ml: 0.003 mg/ml (C1) - 0.03 mg/ml (C2) - 0.3 mg/ml (C3) of active dry weight/ml: C1: 0 μm, C2: 4,1 μm, C3: 26,3 mm.

EXAMPLE 3.5

300 ml of the lysate of example 2.3 first centrifuged at 3000×g for 20 minutes. the pH of the supernatant was brought to 7.1 HCl. The extract was filtered as described in example 3.3 with constant flow speed 330-350 ml/mn. The volume of output accounted for more than 75%. Finally, the concentrate was subjected to sterilizing filtration through PES membrane with a pore size of 0.2 μm (Nalgene).

The concentrate contained SDW: 34,5 mg/g; Prot: 8,9 mg/ml; DNA: 16,8 µg/ml, the Percentage of D-Amino acids: 16,4% D-Ala, 24,3% D-Pro, 17,3% D-Asp.

The NO production in mg of active dry weight/ml: 0.003 mg/ml (C1) - 0.03 mg/ml (C2) - 0.3 mg/ml (C3) of active dry weight/ml: C1: 3.0 mm, C2: 36,4 μm, C3: 69,5 mm.

EXAMPLE 3.6

1000 ml of the lysate of example 2.13 centrifuged for 20 minutes at 9384×g. The supernatant was filtered directly through a sterile filter with a pore size of 0.22 μm.

The concentrate contained SDW: to 32.7 mg/g; Prot: 6,5 mg/g; Sugar: 2.4 mg/g Concentration of sugar was analyzed according to the procedure described by Herbert et al. (Meth. Environ, 1971, 5B:266 et seq.).

Screening for biological activity performed on Mononuclear Cells of Peripheral Blood (PBMC) of a person in various concentrations as described in example 6. The results obtained when 0.5 mg of active dry weight/the l were as follows: TNF-α: 83 PCG/ml; IL-6: 898 PCG/ml; IL-12: 140 PCG/ml (in the presence of 10 ng/ml IFN-γ); and IL-10: 221 PCG/ml (in the presence of IFN-γ).

EXAMPLE 3.7

The bacterial lysates mixture of example 2.10 transferred to the tank for microfiltration (MF) after adjusting the pH to 10.2. In block microfiltration (MF) has been used filter for filtration tangential flow (TF) with a pore size of 0.45 μm (Sartocon Slice Sartorius). Cross-thread brought up to 290 l/h•mc2(LHM) and Transmembrane Pressure (TMP) - 0.4-0.5 bar (42-52,5 N/m2). The permeate is transferred to the tank for ultrafiltration (UF).

After the volume of lysate in the tank for microfiltration reached half of the original volume, run the UF unit. Permeate from UF filters was used as washing buffer reservoir MF. The volumes of tanks and MF, and UF maintained at the same level. After completion of 10 volumes of diafiltration, UF stopped, and the bacterial lysate was concentrated in tanks MF. Retrieved volumetric yield was 83%. the pH of the extracted extract in the tank UF brought to 7.3 with HCl and then filtered through a sterile filter with a pore size of 0.2 μm.

The concentrate contained SDW: 17.5 mg/g; Prot: 4.3 mg/g

EXAMPLE 3.8

The lysate of example 2.7 centrifuged for 15 minutes at 9384×g. Then the pH of the supernatant was brought to 7.3 with HCl and filtered through successive filters with the apostrophes pores of 0.45 μm and 0.2 μm, and finally sterilizing filter with a pore size of 0.22 μm.

Mononuclear Cells from Peripheral Blood (PBMC) with a 10 times diluted product: 777 PCG/ml IL-10, 26 PCG/ml IL-12, 13183 PCG/ml IL-6.

EXAMPLE 3.9

The lysate of example 2.8 centrifuged for 15 minutes at 9384×g. Then the pH of the supernatant was brought to 7.4 with HCl and filtered through successive filters with a pore size of 0.45 µm and 0.2 µm, and finally sterilizing filter with a pore size of 0.22 μm.

PBMC with a 10 times diluted product: 904 PCG/ml IL-10, 0 PCG/ml IL-12, 4995 PCG/ml IL-6.

EXAMPLE 3.10

The lysate of example 2.9 centrifuged for 15 minutes at 9384×g. Then the pH of the supernatant was brought to 7.6 with HCl and filtered through successive filters with a pore size of 0.45 µm and 0.2 µm, and finally sterilizing filter with a pore size of 0.22 μm.

PBMC with a 10 times diluted product: 476 PCG/ml IL-10, 0 PCG/ml IL-12, 4924 PCG/ml IL-6.

EXAMPLE 3.11

The lysate of example 2.11 transferred to the tank MF after adjusting the pH to 10.4. Installation TFF was analogous to example 3.7. Cross-thread brought up to 290 l/h m2and TMP - 0.4-0.5 bar (42-52,5 N/m2). UF was stopped after 10 volumes of diafiltration. The output of the recovered volume was 86%. the pH of the final product brought to 7.3 with HCl.

The resulting concentrate contained SDW: 24,2 mg/g; Prot: 4,8 mg/g

EXAMPLE 3.12

The lysate of example 2.12 transferred to the tank MF after adjusting the pH to 11.5. Install the CA TFF was analogous to example 3.7. Cross-thread brought up to 290 l/h m2and TMP - up to 0.4 bar (42 N/m2). UF was stopped after 10 volumes of diafiltration. The output of the recovered volume was 74%. the pH of the final product brought to 7.2 with HCl.

The resulting concentrate contained SDW: 32.4 mg/g; Prot: 5,8 mg/g; Sugar: 2,3 mg/g

Screening for biological activity performed on Mononuclear Cells of Peripheral Blood (PBMC) of a person in various concentrations as described in example 6. The results obtained when 0.5 mg of active dry weight/ml, were as follows: TNF-α: 37 PCG/ml; IL-6: 1092 PCG/ml; IL-12: 81 PCG/ml (in the presence of 10 ng/ml IFN-γ); and IL-10: 160 PCG/ml (in the presence of 10 ng/ml IFN-γ). The protein concentration was measured using a kit for analysis of protein DC Protein assay (BioRad DC Protein assay, kit no. 500-0116). Performed Protocol analysis on the microplate. Test samples (0.1 ml) were diluted with 1.9 ml of 25 mm phosphate buffer with a pH of 11. Protein standard curve constructed each time you run the analysis with a solution of bovine serum albumin with 2 mg BSA/ml (BSA, Pierce ref 23210). The BSA solution was diluted in 25 mm phosphate buffer with a pH of 11 to obtain the following concentrations: 0,18, 0,24, 0,3, of 0.36 and 0.42 mg BSA/ml Samples (20 ml) and BSA standards (20 ml) was added in four repetitions in a clean, dry title microplate. To each well was added A reagent (25 μl) of the test protein DC Protein assay kit. After 10 minutes, aidou well was added a reagent B. The content of the moon title of microplasma was mixed on a rotary tablet shaker for 5 seconds. After 20 minutes at room temperature were recorded spectral absorption capacity at 750 nm. The protein concentration in each sample was calculated using the slope of the linear regression on the protein standard curve:

OD at 750 nm = a*(protein concentration)+b mg protein in the sample = [20×(OD at 750 nm - b)]/a

Soluble dry weight (SDW) was determined by obtaining 5 ml of the soluble fraction in the lysis and drying it to constant weight in a porcelain Cup at 105°C.

EXAMPLE 4: Activation of murine splenic cells In Vitro

Immunostimulating effect of embodiments of the invention were analyzed in vitro by measuring the activation of Murine Splenic Cells (analysis using the dye Alamar blue).

Material and methods

Stimulation of splenic cells

Analysis of AlamarBlueTM-Assay designed for the quantitative measurement of the growth of human cells or animals. The analysis includes a fluorometric/colorimetric growth indicator based on detection of metabolic activity indicator oxidation-reduction (REDOX) in response to chemical reduction in the cell growth. Due to growth, an indicator of REDOX changes of oxidized (not fluorescent, blue) Faure what s in the reduced (fluorescent, red) form.

Balb/c mice (females, age 6-8 weeks) were obtained from Charles River Laboratories, Sulzfeld, Germany, and they were killed by dislocation of vertebra in the cervical spine. Spleens homogenized using ceramic homogenizer; cell suspension was washed by centrifugation (280×g, 4°C, 10 min) and resuspendable in 5 ml of medium RPMI 1640 containing 5% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin. 100 μl of the splenic cells (2×106/ml) were incubated with 50 μl of dilutions of the bacterial extract in 96-well plates (Falcon 3072) for 48 h at 37°C and 5% CO2in the medium for cell culture.

After adding 30 μl of a solution AlamarBlueTM, diluted 1:1 in culture medium, chemical recovery AlamarBlueTM measured by the reader Fluoroskan Ascent Reader (ThermoLabsystems, Frankfurt, Germany) at wavelengths of excitation 544 nm and wavelengths of emission of 590 nm.

Test products

Tested the following extracts:

Afer300: extract from Lactobacillus fermentum I-3929 (12 g/l), obtained as described in example 3.5 (31,6 mg of active dry weight/g);

Cfer300: extract from Lactobacillus fermentum I-3929 (12 g/l), obtained as described in example 3.3 (28.4 mg of active dry weight/g);

Dfer300: extract from Lactobacillus fermentum I-3929 (12 g/l), obtained as described in example 2.4, and purified using the same conditions as in example 3.3 (29,5 mg of active dry weight/g). This example is used in the AC is estwe not alkaline lysed control.

ARahr300: extract from Lactobacillus rhamnosus 71.38 (40 g/l)obtained by alkaline lysis NaOH 0,03 M at 40°C for 6 hours (to 49.3 mg of active dry weight/g);

CRahr300: extract from Lactobacillus rhamnosus 71.38 (40 g/l), as described in example 3.4 (46.4 mg of active dry weight/g);

DRahr300: extract from Lactobacillus rhamnosus 71.38 (40 g/l), lysed by osmotic stress 0.15 N NaCl solution at 40°C for 6 hours (46.2 mg of active dry weight/g). This example is used as a non-alkaline lysed control.

Negative controls

Aqua dest. (distilled water) or saline phosphate buffer (PBS).

Results

In vitro studies to determine the activation of splenocytes

The increase in metabolic activity of murine splenic cells after treatment with the extract was determined in 2 independent experiments analysis using Alamar Blue (see figa and 4b and table 1).

Splenic cells were cultured for 48 h in the presence of the extract. After adding a solution of Alamar blue®, was measured value of emission. As shown in figa-b, bacterial extracts were effective at dilutions of about 1:300. All groups were compared statistically with control groups using T-student test.

Table 1
P-value for bacterial extracts in 2 independent experiments analysis using the dye Alamar Blue (see figa and 4b)
PartyBreedingP-value test 1P-value test 2
Afer300(dilution 1:300)0,000016 (Figa)0,00016 (.4b)
Cfer300(dilution 1:300)0,00021 (Figa)0,048 (.4b)
Dfer300(dilution 1:300)0,00033 (Figa)0,00016 (.4b)
ARahr300(dilution 1:300)0,0029 (Figa)0,0012 (.4b)
CRahr300(dilution 1:300)0,0031 (Figa)0,12 (.4b)
DRahr300(dilution 1:300)0,0025 (Figa)0,08 (.4b)

Conclusion

Immunostimulating effect of bacterial extracts the analysis is listed in vitro by measuring the activation of murine splenic cells analysis of Alamar Blue. Thus, in two independent experiments, applicants compared the extract obtained by osmotic lysis (Dfer300), which includes intact, having the form of particles components of the cell walls. Two extracts in accordance with the present invention (AFer300 and CFer300). These three extracts were obtained from Lactobacillus fermentum I-3929. Each of the extracts AFer300, CFer300 and DFer300 were effective in stimulating the metabolism of the cells at a dilution of 1:300. The applicants did not observe differences between control osmotic lysis DFer300 and Afer300 and CFer300.

Applicants also compared the three extract derived from a second strain, Lactobacillus rhamnosus 71.38 (ARahr300, CRahr300, DRahr300). As in the previous set, ARahr300 and CRahr300 are within the scope of the invention, while DRahr is controlled osmotic lysis. In this case, the extract ARahr300 was effective in two independent analyses at a dilution of 1:300, whereas extracts CRahr300 and DRahr300 showed a weak but significant stimulation in one of the two tests at the same dilution. When comparing results for the six extracts, it appears that the extracts of Lactobacillus fermentum I-3929 were more effective in stimulating growth of the splenic cells, compared with extracts of Lactobacillus rhamnosus 71.38.

EXAMPLE 5: Production of nitric oxide by macrophages derived from bone marrow

Immunostimulating potential series the variants of implementation in accordance with the invention was tested by measuring the production of nitric oxide (NO) in mouse macrophages, derived from bone marrow.

Materials and methods

Six male mice C57/BL6 (six males, as SPF, Charles Rivier, FR) were killed by inhalation of CO2. Remove the hip joint, femur and tibia were removed from the back of the body. Bone marrow was extracted from the lumen of the injection of the modified Dulbecco eagle medium (DH) through the bone after you cut both end parts. After washing, the stem cells resuspendable (40000 cells/ml) in the medium DH with the addition of 20% horse serum and 30% supernatant of L929 cells. Cell suspension was incubated for 8 days in an incubator at 37°C in an atmosphere of 8% CO2and saturated. Then macrophages were separated ice-cold PBS, washed and resuspendable environment DH with the addition of 5% fetal calf serum (FCS), amino acids and antibiotics (Wednesday DHE). The density of cells brought up to 700,000 cells/ml Aqueous solutions of the extracts were serially diluted in medium DHE directly to the title microplate. The extracts according to the invention was tested in three repetitions, and every title microplate contained medium as a negative control. The final volume in each well was 100 μl. 100 μl of cell suspension was added to the diluted extracts and cells were incubated for 22 hours in an incubator at 37°C in a saturated state power are the th atmosphere of 8% CO 2. At the end of the incubation period, 100 μl of supernatant was transferred to another title microplate, and the concentration of the produced nitrite in each supernatant was determined by conducting the reaction Griss. 100 ál Griss (5 mg/ml sulfanilamide + 0.5 mg/ml of hydrochloride of N-(1-naphthyl)Ethylenediamine in 2.5% aqueous phosphoric acid was added to each well. Title microplate was read by a spectrophotometer (SpectraMax Plus, Molecular Devices) at 562 nm in comparison with a reference at 690 nm. The nitrite concentration was proportional to the amount of the formed oxide nitrite. The content of nitrite was determined using a standard curve of sodium nitrite (from 1 to 70 microns NaNO2). The results were presented in microns of nitric oxide (NO) in the form of average value ± standard deviation and were plotted on the graph as a curve dose-response.

Test products

Tested the following:

First analysis:

OP0701B4_CFer300: extract from Lactobacillus fermentum I-3929 (12 g/l), obtained as described in example 3.3.

OP0701B4_Dfer300: extract from Lactobacillus fermentum I-3929 (12 g/L), obtained as described in example 2.4, and purified using the same conditions as in example 3.3. This example is used as a non-lysed by alkali control.

OP0701B4_CRahr300: extract from Lactobacillus rhamnosus 71.38 (40 g/l), obtained as described in example 3.4.

OP0701B4_ DRhr300: extract from Lactobacillus rhamnosus 71,38 (40 g/l), obtained by the osmotic stress of 0.15 N NaCl solution at 40°C for 6 hours. This example is used as a non-lysed by alkali control.

Second analysis:

OP0701C_10G0.5P4H: extract from Lactobacillus fermentum I-3929 at a concentration of 10 g/l dry weight biomass 0,037M NaOH, incubated at 40°C for 4 hours.

OP0701C_10G1 P4H: extract from Lactobacillus fermentum I-3929 at a concentration of 10 g/l dry weight biomass 0,075M NaOH, incubated at 40°C for 4 hours.

OP0701C_10G2P4H: extract from Lactobacillus fermentum I-3929 at a concentration of 10 g/l dry weight biomass 0,150M NaOH, incubated at 40°C for 4 hours.

OP0701C_10G1P21H: extract from Lactobacillus fermentum I-3929 at a concentration of 10 g/l dry weight biomass 0,075M NaOH, incubated at 40°C for 21 hours.

OP0701C_10G0.5P21H: extract from Lactobacillus fermentum I-3929 at a concentration of 10 g/l dry weight biomass 0,037M NaOH, incubated at 40°C for 21 hours.

OP0701C_10G2P21 H: extract from Lactobacillus fermentum I-3929 at a concentration of 10 g/l dry weight biomass 0,150 M NaOH, incubated at 40°C for 21 hours.

OP0701C_10G2P45H: extract from Lactobacillus fermentum I-3929 at a concentration of 10 g/l dry weight biomass 0,150 M NaOH, incubated at 40°C for 45 hours.

The results of the first analysis

In the first analysis (tiga) observed that the extracts obtained from shchelochen lysis in accordance with the present invention, have the same activity as extracts, p is obtained as a result of osmotic lysis.

Applicants also observed that the activity of immune stimulation was associated with a selected strain. The extracts obtained from Lactobacillus fermentum I-3929, caused the production of a larger number of NO2and NO3than extracts obtained from Lactobacillus rhamnosus 78.31.

The results of the second analysis

In the second analysis (fig.5b), the applicants have observed that activity in vitro extract is correlated with the initial lysis conditions. The activity of the following bacterial strains are shown for the same strain of Lactobacillus fermentum I-3929, and for the same amount of 10 g dry weight of biomass, a liter of lysate: OP0701C_10G0.5P4H, OP0701C_10G1P4H, OP0701C_10G2P4H, OP0701C_10G1P21H, OP0701C_10G0.5P21H, OP0701C_10G2P21H and OP0701C_10G2P45H. Activity in vitro depended on the initial concentration of NaOH and duration of the lysis.

Conclusion

The results are presented of experiments showed that despite chemical modification generated by the process of the alkali treatment, the activity of embodiments is not decreased, compared with the control extract obtained by osmotic lysis, or compared to the live bacteria (with respect to this aspect, see Example 6 below).

EXAMPLE 6: in vitro Screening to identify Pro - and anti-inflammatory activity

For screening of the embodiments of the invention to identify their immunostimulating or protivovospalitel the potential in vitro, the tests were performed on a series of bacterial extracts on human PBMC. We measured the release of IL12p70 (inflammatory cytokine) and IL10 (anti-inflammatory cytokine)and IL-10/IL-12 was as described in the publication Foligne et al. World J Gastroenterol 2007 January 14; 13(2): 236-243).

The aim was to compare the production of IL-12p70 and IL-10 series of 6 extracts obtained by means of extraction/ purification. The ratio of IL-10/IL-12p70 obtained for each extract, can be correlated with anti-inflammatory potential of the extracts. Live bacteria were used as controls to compare with the activity of the extracts (2x107cfu/ml (colony forming units/ml) Lactobacillus fermentum I-3929)).

Materials and methods

Step number 6 bacterial extracts were diluted in medium for cell culture in the presence of 10 ng/ml IFN-γ, a cytokine that is known, increases the production of IL-12p70, starting with an initial dose of 1 mg/ml (the highest end tested dose). PBMC isolated from the blood of healthy donors were tested in serial dilutions of active dry weight of extracts from 100 ng/ml to 1 mg/ml

IFN-γ was added in Wednesday, at least 3 hours before lysates or live bacterial strain. Presents data of two independent experiments.

Obtaining PBMC person

PBMC were isolated from peripheral blood. Briefly,after centrifugation in a gradient of ficoll (Pharmacia, Uppsala, Sweden), mononuclear cells were collected, washed in RPMI medium 1640 (Live technologies, Paisley, Scotland) and their contents brought up to 2×106cells/ml in RPMI 1640 with the addition of gentamicin (150 µg/ml), L-glutamine (2 mmol/l) and 10% fetal calf serum (FCS) (Gibco-BRL).

Induction of cytokines

PBMC (2×106cells/ml) were sown in 24-well plates to tissue culture (Corning, NY). Cells stimulated. As explained above. After 24 h of stimulation at 37°C in an atmosphere of air with 5% CO2, cultural supernatant gathered, prosvetlili by centrifugation and stored at -20°C until analysis of cytokines. Cytokines were measured by ELISA with the use of pairs of antibodies firm Pharmingen (BD Biosciences, San Jose, Ca, USA) for IL-10 and IL-12p70, in accordance with the manufacturer's recommendations.

Test products

OP0701C_10G2P45H (A): extract from the lysate Lactobacillus fermentum I-3929 when 10 g dry weight of biomass per liter of lysate, 0.15 M NaOH at 40°C for 45 p.m.

OP0701C_1OG1P4H (B): extract of a lysate of Lactobacillus fermentum I-3929 when 10 g dry weight of biomass per liter of lysate, of 0.075 M NaOH at 40°C for 4 hours

OP0701C_5G4P21H (C): extract from the lysate Lactobacillus fermentum I-3929 when 5 g dry weight of biomass per liter of lysate, 0,300 M NaOH at 40°C for 21 hours

OP0701C_40G0.5P4H (D): extract of a lysate of Lactobacillus fermentum I-3929 at 40 g dry weight of biomass per liter of lysate, 0,037 M NaOH at 40°C for 6 hours

OP0701B4_CFer150 (E): extract from the lysate Lactobacillus fermentum I-3929 (12 g/l), obtained as described in the example is 3.1.

OP0701B4_BFer300 (F): extract of a lysate of Lactobacillus fermentum I-3929 when 6 g dry weight of biomass per liter of lysate, of 0.075 M NaOH at 40°C for 6 hours

Lactobacillus fermentum I-3929, live bacteria, frozen at -80°C with 2×107CFU/ml in 20% glycerol.

Results

The purpose of this test was to compare immunostimulating and anti-inflammatory potential in vitro embodiments of the invention with live Lactobacillus. For the analysis of various extracts and live bacteria, we compared the number of each of the cytokines IL-10 and IL-12 released from PBMC, and IL10/IL12.

If the concentration or IL-10 or IL-12, or both values were close to the levels without stimulation (i.e., below 10 pkg/l), the ratio was not considered calculated and were marked by the letters NC (not calculated value).

The results obtained in the PBMC expressed in PCG/ml, are shown in tables 2 and 3.

In the presence of IFNγ, the effect of 6 bacterial extracts according to the invention (with extract A extract F) compared with live form Lactobacillus fermentum (2×107bacteria/ml, tested twice as Living specimen 1 and the Living model 2) in human PBMC (reaction IL10 and IL12p70 in PCG/ml). The ratio of IL10/IL12 presented only for doses of extracts of 100 μg/ml and 1 mg/ml

Table 2
Pro-inflammatory (immune-stimulating) or anti-inflammatory (immunomodulatory) potential in vitro bacterial extracts on human PBMC
ABCDEF
IL10(+IFNγ)
1 mg/ml14610899953961549
100 µg/ml323211211066
IL12(+IFNγ)
1 mg/ml8781 947748
100 µg/ml111015916211
IL10/IL12(+IFNγ)
1 mg/mlN.C.14,03N.C.10,1712,4911,40
100 µg/mlN.C.2,11N.C.2,050,650,52
N.C. Not calculated value

In the presence of IFNγ, the effect of 6 bacterial extracts according to the invention (with extract A extract F) compared with live form Lactobacillus fermentum (2×107bacteria/ml, tested twice as Living specimen 1 and the Living model 2) in human PBMC (reaction IL10 and IL12p70 in PCG/ml). The ratio of IL10/IL12 presents only the for doses of extracts of 100 μg/ml and 1 mg/ml

Table 3
Pro-inflammatory (immune-stimulating) or anti-inflammatory (immunomodulatory) potential in vitro of a live strain I-3929 Lactobacillus fermentum on PBMC man
Lactobacillus fermentum I-3929
Live sample 1
Lactobacillus fermentum I-3929
Live sample 2
IL10(+IFNγ)
2×107CFU/ml386355
IL12(+IFNγ)
2×107CFU/ml187191
IL10/IL12(+IFNγ)
2×107CFU/mlto 2.06to 1.86

Based on the results presented in table 2 and table 3, IL10 production was decreased in the following order:

B≅E≅D>F>live Lactobacillus>A>C

Based on the results presented in table 2 and table 3, the production of IL12 decrease is alas in the following order:

live Lactobacillus>E>B>D>F>A>C

When considering correlations were observed following General type:

B≅D>E≅F>live Lactobacillus

Results correlations IL10/IL12 for extracts A and C are not shown, because it was found that these extracts are effective inducers of cytokines.

At concentrations lower than 100 µg/ml, the observed concentrations of cytokines were too low to draw any conclusions.

Conclusion

Correlations can indicate that in the experimental conditions used in example 6, some of the bacterial extracts according to the invention have a more pronounced immunomodulatory effects than the effects of live Lactobacillus fermentum. For example, extracts B, D, E and F showed a higher ratio of IL10/IL12p70 than the control in the form of live bacteria. Thus, these extracts can be more active. Than live probiotic bacteria against the States described in this application, such as an inflammatory condition.

EXAMPLE 7: Effect on toll-like receptors

Embodiments of the invention are obtained from gram-positive bacteria, and therefore it is expected that they act through TLR2 receptors. TLR receptors expressed primarily, but not exclusively, immune cells such as monocytes, macrophages, dendritic the yrs, T-cells, etc. and they are key sensors of microbial products that can be interpreted by the host as danger signals. Even though TLR receptors first run nonspecific innate immunity, their activation initiates a full immunological cascade that in the presence of antigens leads to the development of acquired immunity.

Cells that Express this functional gene TL, are valuable tools for many applications, such as the study of the mechanisms involved in the recognition or the TLR signaling, and development of new potential therapeutic drugs. In the following Experiments tested the activity of three bacterial extracts on these key adapters immune response.

Materials and methods

The reaction of the extracts according to the invention was tested (or as such to verify their agonistic effect, or in the presence of the TLR2 agonist Pam3Cys, or in the presence of the TLR4 agonist LPS to test the antagonistic activity) in the following two cellular systems:

(a) HEK-TLR2/6 (ELISA IL-8 after 24 h)

(b) HEK-MD2-TLR4-CD14 (ELISA IL-8 after 24 h)

(a) HEK-TLR2/6

The cell line HEK293 were chosen for their zero or low basal expression of TLR genes. These cells provide an effective monitoring activity TLR using analysis EISA, such as titration of IL-8 or based on receptor systems that monitor induced TLR activation of NF-κB.

Cells HEK-TLR2/6 (Invivogen, Toulouse, France) are established methods engineered HEK293 cells. Stably transfected multiple genes from the path TLR2/6, which include TLR2, and TLR6 genes involved in the recognition or involved in the cascade of signal transmission. These cells secrete IL-8 after stimulation of TLR2/6. The experiments were performed in accordance with the manufacturer's instructions.

Briefly, 2×104cells/well (200 μl RPMI medium) were incubated at 37°C for 3 days (5% CO2). The medium was removed and wells were added to 90 μl of RPMi+5% FCS. Then added agonists and control (10 µl/onions). Cells were returned to the incubator for 24 hours Supernatant collected and ELISA IL-8 was performed according to the manufacturer's instructions.

Test products

OP0701B4_Afer50: extract of a lysate of Lactobacillus fermentum I-3929 at a concentration of 12 g weight of dry biomass per liter of lysate, of 0.075 M NaOH at 40°C for 4 h and purified as described in example 3.6.

OP0701C-Bt1LAC: extract from Lactobacillus fermentum I-3929, obtained as described in example 3.7.

OP0701C-Bt2LAC: extract from Lactobacillus fermentum I-3929, obtained as described in example 3.11.

Results: secretion of IL-8

Results for controls (negative = K12 LPS ultrapure, a TLR4 agonist; and PAM3CSK4 = positive, the TLR2 agonist)are presented in tables 4-6. The results (expressed in PCG/ml IL-8) show the average secretion of IL-8, specific ELISA at 24 h after stimulation controls.

The cell line HEK TLR2/6 responded to the TLR2 agonist Pam3Cys. In contrast, TLR4 agonist of E coli (K12 LPS) was inactive even at a high dose of 0.01 mg/ml

Experiments with a single application of 3 extracts

3 bacterial extract tested in this case (OP0701B4Afer50, OP0701C-Bt1LAC and OP0701C-Bt2LAC), showed higher immune-stimulating properties than the TLR2 agonist Pam3Cys.

Experiments with three bacterial extracts + Pam3Cvs

As indicated above, three of the bacterial extract was tested to identify their alleged antagonistic or additive properties in comparison with Pam3Cys added directly after extracts.

Bacterial extract OP0701 B4 AFer50

The results presented in table 4, indicate that the extract OP0701B4_AFer50 caused the production of high levels of IL-8 (agonist TLR 2/6).

Bacterial extract OP0701C-BtILAC

The results presented in table 5, indicate that the extract OP0701C-Bt1LAC caused the production of high levels of IL-8 (agonist TLR 2/6).

Bacterial extract OP0701C-Bt2LAC

The results presented in table 6, indicate that the extract OP070C-Bt2LAC did not cause the production of IL-8 (agonist TLR 2/6), and in the presence of Pam3Cys had an antagonistic effect on TLR2/6.

Conclusion

Depending on the conditions of alkaline lysis (initial concentration dry weight biomass, initial concentration of the base and the processing base), bacterial extracts could have different types of actions.

OP0701B4_Afer50 and OP0701C-Bt1LAC were agonists of TLR2/6, but stronger alkaline lysis performed on the same bacterial strain, led to antagonistic activity against TLR2/6 (OP0701C-Bt2LAC).

(b) HEK-TLR4-MD2-CD14

Conducted extensive research TLR4, because he is the main receptor involved in the recognition of lipopolysaccharide (LPS), responsible for septic shock.

Cells HEK-TLR4-MD2-CD14 highly sensitive to LPS. They were obtained by stable transfection of HEK293 cells genes TLR4, MD2 and CD14 and reporter system induced NF-κB. These cells secrete IL-8.

Used the same experimental procedure as for the other cell line HEK TLR2/6, described above. Results for controls and 3 tested bacterial extracts according to the invention are shown in tables 7-9.

Results: Secretion of IL-8

Results for controls (positive = LPS K12C ultrapure, a TLR4 agonist; and PAM3CSK4 = negative, TLR2 agonist) are presented in tables 7-9. The results (expressed in PCG/ml IL-8) show the media what their values secretion of IL-8, specific ELISA at 24 h after stimulation controls.

Cell line clearly responded only to the TLR4 agonist LPS K12. On the contrary, as expected, the TLR2 agonist of Pam3Cys was inactive even at a high dose of 0.01 mg/ml

Experiments with a single application of 3 extracts

As expected from agonists gram-positive bacteria, these 3 tested bacterial extract (OP0701B4 Afer50: table 7, OP0701C-Bt1LAC: table 8 and OP0701C-Bt2LAC: table 9) did not show distinct immunostimulatory properties by way of TLR4.

Experiments with 3 bacterial extracts + K12 LPS

As indicated above, three of the extract according to the invention have only tested to determine their alleged antagonistic or additive properties in comparison with LPS added directly after the extracts. And again, at the level of receptor TLR4 effect was not observed (tables 7-9).

Bacterial extract OP0701B4 AFer50

The results indicate that the extract OP0701B4_AFer50 not activated receptor TLR4 (Pam3Cys: 138 PCG/ml).

Bacterial extract OP0701CBtI-LAC

Bacterial extract OP0701CBt2LAC

Conclusion

Taken together, results presented here, obtained in HEK cells, and the results obtained on cells from PBMC of man, testify, h is about Afer50 and BT1LAC capable of stimulating the immune system through the path of TLR2. In addition, BT2LAC can act as antagonist of TLR2/6. Therefore, the extracts according to the invention can stimulate the immune system through the path of TLR2 or act as antagonists TLR2/6, thus corraleros with anti-infective and anti-inflammatory activity in vivo.

EXAMPLE 8

Cells that Express this functional TLR gene, are valuable tools for many applications, such as the study of the mechanisms involved in the recognition or the TLR signaling, and development of new potential therapeutic drugs. Therefore, the aim of the experiments described below, is testing activity 4 bacterial extracts in relation to these key adapters immune response.

In this test, conducted screening 8 receptors TLR and NOD2 4 bacterial extracts, including extracts in accordance with the present invention.

Method

Bacterial extracts according to the invention was tested in a 96-well microplate. Extracts were diluted in culture medium DMEM, and 20 μl of each dilution was tested in two repetitions. Volume of 180 μl suspensions of HEK293 cells containing 25,000 or 50,000 cells in culture medium DMEM+10% FCS (one line HEK293 cells, specific for each TLR with a reporter gene, secretio the Ala alkaline phosphatase under the control of the NF-kB) were added to each well in two repetitions. After 16 hours of incubation with each cell line, from 20 to 50 µl of each supernatant was transferred into a 96-hole microplasma and completed a 200 ál Quantiblue (InVivoGen no REP-QB1). The enzymatic reaction with Sekretareva alkaline phosphatase was performed for 30-60 min for different series of cell lines expressing TLR. The reading was performed using a microplate reader at 630 nm.

Material

The list agonists positive control (and their respective concentrations)used in this screening analysis

TLR2 PAM2 100 ng/ml; TLR3 PoIy(I:C) 100 ng/ml; TLR4 E. coli K12 LPS1 µg/ml; flagellin TLR5 S. typhimurium flagellin 1 µg/ml; TLR7 R848 10 μg/ml; TLR8 R848 10 μg/ml; TLR9 CpG one 2006 10 μg/ml; NOD2 Muramyldipeptide 1 µg/ml.

Negative controls

Line recombinant cells HEK-293 only for reporter gene (NFkB) was used as negative control for cell lines TLR. The magnitude of the negative control for each clone was represented by the background signal is not induced clones. TNF-alpha was used as a positive control for this cell line not expressing TLR.

Tested products

Tested the following bacterial extracts:

OP0701B4_CFer300: extract from Lactobacillus fermentum I-3929 (12 g/l), obtained as described in example 3.3 (F);

OP0701B4_CRahr300: extract from Lactobacillus rhamnosus 71.38 (40 g/l)obtained, ka is described in example 3.4 (G);

OP0701D_10L1 PswitchA: extract from Lactobacillus fermentum I-3929 (10 g/l), obtained as described in example 3.12 (I); and

OP0701D_5LOSMOConc: extract result from osmotic lysis of Lactobacillus fermentum I-3929 (7.6 g/l)obtained by the osmotic stress 0.16 N NaCl solution at 40°C for 24 h (H) as a control.

Obtaining bacterial extracts

20 ál subject to testing of the sample used for the stimulation of all cell lines in a 200 μl reaction volume.

The screening was performed in the same concentration, usually 0.5 mg dry weight/ml Tests were performed in two repetitions.

Results

Bacterial extracts and controls were tested in two repetitions on line recombinant cells HEK-293, which functionally Express this protein TLR or NOD2, as well as reporter gene driven by the promoter of NFkB. Results activation of TLR and NOD2 are represented as values of optical density (OD). The results are shown in tables 10-13 and are summarized in table 15.

Bacterial extract F (OP0701B4Cfer300)

Table 10
Activation of receptors TLR and NOD2 extracts OP0701B4Cfer300
hTLR2hTLR3hTLR4hTLR5hTLR7 hTLR8hTLR9hNOD-2
The reference sample3,4932,0521,341to 3.6571,4802,7861,9550,576
Extract F3,9630,0510,7350,3040,0700,0800,0260,503

Bacterial extract F specifically activated hTLR2, hTLR4 and hNod2, and to a lesser extent, hTLR5.

Bacterial extract G (OP0701B4CRahr300)

Table 11
Activation of receptors TLR and NOD2 extracts OP0701 B4CRahr300
hTLR2hTLR3hTLR4hTLR5hTLR7hTLR8hTLR9hNOD-2
The reference sample3,493 2,0521,341to 3.6571,4802,7861,9550,576
Extract G0,8080,0030,1770,0170,0280,044-0,0320,369

Bacterial extract G activated hTLR2, hNOD2, and to a much lesser extent hTLR4.

Bacterial extract H (OP0701D 5LOSMOConc)

Table 12
Activation of receptors TLR and NOD2 extracts OP0701D_5LOSMOConc
hTLR2hTLR3hTLR4hTLR5hTLR7hTLR8hTLR9hNOD-2
The reference sample3,4932,0521,341to 3.6571,4802,7861,955 0,576
Extract H4,5370,0570,6380,050-0,0720,0540,0110,892

Bacterial extract H specifically activated hTLR2, hTLR4 and Nod2.

Bacterial extract I (OP0701D_10L1PswitchA)

Table 13
Activation of receptors TLR and NOD2 extracts OP0701D_10LPswitchA
hTLR2hTLR3hTLR4hTLR5hTLR7hTLR8hTLR9hNOD-2
The reference sample3,4932,0521,341to 3.6571,4802,7861,9550,576
The extract I4,1680,0960,3423,490 -0,0240,1320,4100,790

Bacterial extract I strongly activates hTLR2, hTLR5, hNOD2, and to a lesser extent, hTLR4 and hTLR9.

Table 15
Activation of receptors TLR and NOD2 different extracts obtained by other conditions of the method in accordance with the present invention
hTLR2hTLR3hTLR4hTLR5hTLR7hTLR8hTLR9hNOD-2
Extract F+-+±---+
Extract G+-±----+
Extract H +-+----+
The extract I+-±+--±+

Thus, the bacterial extracts according to the present invention can help to activate the immune system through various TLR and Nod2. Therefore, the extracts according to the invention can be good activators of the immune response.

When comparing extract H extract G & F, it becomes evident that the extracts have the same path TLR and Nod, as the extracts obtained by osmotic lysis. When comparing extract H and I extract, it was possible to observe the activation path TLR5 and to a lesser extent the way TLR9 adding acid after alkaline lysate lysate. Even extract F, resulting from the use of weak alkali, showed activation path TLR5, in contrast to the extract of H.

The extract I acted on TLR5, which indicates the potential use in radiation Ter the FDI. Indeed, radiation therapy is a well established and highly effective treatment for certain types of cancer. However, its side effects can be very severe, as it can destroy healthy cells in the body, particularly bone marrow cells and cells in the gastrointestinal tract. Recently, Burdelya et al. reported that the peptide CBLB502 binds to and activates TLR5 signaling of nuclear factor-κB, the way that cancer cells frequently activate in order to avoid cell death (Burdelya et al., "An agonist of toll-like receptor 5 has radioprotective activity in mouse and primate models," Science, 2008, 320:226-30). In mice and rhesus monkeys treated CBLB502 shortly before exposure to lethal doses of whole body radiation, showed less damage to healthy cells in the bone marrow and gastrointestinal tract, and they survived significantly longer than controls. It is important that the tumour mice, CBLB502 did not violate the antitumor efficacy of radiation therapy.

EXAMPLE 9: the Ability of the extract to produce plaque-forming cells against sheep red blood cells) in mice

Methods of plaque-forming cells (PFC) allows the evaluation of non-specific stimulation of B-lymphocytes. This technique was first described by Cunningham and Seenberg (Immunology, 1968, 14, 599). It was demonstrated the presence of hemolytic antibodies form around their antibodies cells, as is described below. Mouse lymphoma cells, and a dense population of foreign (RAM) cells, at the same time was placed on a microscope slide. Certain lymphoid cells release hemolytic antibodies, which penetrate and cause lysis of adjacent erythrocytes education lizinoj plaques in the presence of complement. At the end of the experiment, was calculated the number of PFC per 106cells or spleen.

Materials and methods

Tested products

Bacterial extract 1, obtained as described in example 3.5, and bacterial extract 2, obtained as described in example 3.4.

Animals

40 male Balb/C mice/experiment (IFFA-CREDO, St. Germain sur L Arbresle Cedex, France) aged 5 to 6 weeks with an average body weight of 20±2 g were divided into 5 groups of 8 animals each.

The structure of the experiment

Animals were divided into 5 groups as follows:

Group (a) 8 mice treated with 1.2 mg/mouse of extract 1, as described in example 3.5, volume = 0,2 ml

Group (b) 8 mice treated with 0.6 mg/mouse of extract 1, as described in example 3.5.

Group (c) 8 mice treated with 1.2 mg/mouse of extract 2, as described in example 3.4, volume = 0,2 ml

Group (d) 8 mice treated with 0.6 mg/mouse of extract 2, as described in example 3.4, volume = 0,2 ml

Group (e) 8 mice treated with 0.2 ml of isotonic NaCl solution.

We and received extracts, as described above, these doses daily per os (by mouth) for 5 consecutive days (from day 1 to day 5). Two intubations occurred in the 19th and 20th days. On the 29th day, the antigen (106sheep red blood cells) in 0.2 ml of isotonic saline (0.9% NaCl solution, Olivera) were injected with intravenously through the tail vein of the animal; after 4 days (33rd day) received suspensions of splenic cells.

Reagents and equipment

The solution Olivera free of endotoxin, (Sigma, 38299 St Quentin Fallavier, Cedex, France, ref. A 3351)

The primary environment Needle (BME, concentrated 10-fold) in not containing bicarbonate solution Needle (Bio-Merieux, 69280 Marcy I'etoile, France ref: 8 210 2)

Lyophilized serum of Guinea pigs (Bio-Merieux, Marcy I'etoile, France ref: 7 212 2)

Sheep erythrocytes (Bio-Merieux, Marcy I'etoile, France ref: 7214 1)

Tripney blue, sterile distilled water, NaCl 0,9%, tissue homogenizer, centrifuge, camera Neibauer to count cells, glass test tubes.

Treatment of test or control product

Control animals received only sheep erythrocytes (SRBC). Animals of other groups received oral bacterial extracts, as described previously. For this purpose, each extract was dissolved in water.

Suspension of splenic cells

4 days after the last injection of antigen (SRBC), a suspension of splenic cells were obtained for ka the DOI mouse in accordance with the following procedure:

The animal was narcoticyou ether and then squashed by the displacement of the cervical spine. The spleen was removed and crushed in a glass tissue homogenizer with 2 ml of BME at a pH of 6.75-6,80. Then added 3 ml of sterile distilled water, and the mixture was stirred for 20 seconds. The resulting slurry is then diluted with 10 BME and centrifuged at 1200 rpm for 10 min at 4°C. the Supernatant was removed, and the precipitate suspended in 2 ml of BME. The cells allowed to recover for 5-10 minutes on ice. The illumination of viable mononuclear cells was performed as follows:

Dilution 1/20 aliquot of cell suspension was received saline containing 0.1% solution Trypanosoma blue. Not stained cells, ie, the living cells were counted in the chamber of Neubauer.

The cell suspension was kept in melting ice during the counting and then immediately used for lysis plaques. This method prevents loss of viability, which can occur if too long waiting.

Getting slides

On a conventional glass slide, carefully sprinkled with powder and with any remote trace of fat, with intervals of about 1.5 cm was taped 3 parallel strips (thickness 0.1 mm) double-sided tape. Then the strips were covered with pre-purified patronymically (22 mm × 22 mm) for the formation of two "cameras" between the slide and cover glass.

Fraction suspensions of splenic cells brought up to 25000 mononuclear cells / mm3. In a small glass test tube mix the following items (added in that order) received by automatic pipettes:

1) 7.5 ml BME

2) 0.5 ml SRBC suspension, washed and centrifuged twice with saline, 50% residue

3), 0.4 ml of normal Guinea-pig serum, used as a source of complement

Finally, 50 μl of the suspension of the splenic cells at a density of 25,000 cells/mm3was added to 200 μl of the previous mixture. After gentle homogenization, the suspension was introduced in "Camera Cunningham" capillary action before sealing the free sides of the paraffin. Then slides were placed in a moist chamber in a thermostat at 37°C.

Annotation

After incubation for 1 hour in a thermostat, the slide was examined under a microscope with magnification of 100 times (eyepiece 10 X lens X 10).

Lytic plaques can be easily identified. 5 vertical optical strips were examined on each slide and counted the number of lytic plaques. The number of cells (N), forming a direct lytic plaques on 106cells were calculated by extrapolation. If x represents the number of observed plaques, and X represents the number of investigated cells, the number of direct l the political plaques on 10 6cells equal to:

N=(x/X)*106

where X=C*V=C*(n*e*l*L)

C = final concentration of the splenic cells

V = observed volume

n = the number of optical stripes

e = thickness of the adhesive tape

I = width of the optical field

L = length of the optical strips

The number of direct lytic plaques on 106cells were obtained from the following equation:

PFC (106cells)=(x*106)/(C*n*e*I*L)

Knowing the number of collected cells per spleen, it was possible to deduce the number of cells per spleen, forming lytic plaques.

In mice, was "absolute standard" spontaneous lytic plaques were observed.

Statistical analysis and results

The results were considered significant when p<0,05 (t student test). The results are shown in table 16.

Table 16
The ability of bacterial extracts 1 and 2 to produce plaque-forming cells against sheep red blood cells) in mice
GRMPFC/106cellFrequency PFC
Extract 1; see Example 3.5
(1.2 mg/mouse/introduction)
100%
100%
100%
124%
123%
114%
19%
143%
113%
Extract 1; see Example 3.5
(0.6 mg/mouse/introduction)
100%
100%
100%
117%
118%
112%
103%
139%
128%
Extract 2; see Example 3.4
(1.2 mg/mouse/introduction)
100%
100%
100%
119%
124%
117%
109%
131%
120%
Extract 2; see Example 3.4
(0.6 mg/mouse/introduction)
100%
100%
100%
117%
114%
113%
112%
119%
111%

It was found that the extracts according to the invention are activators of B-cells when tested at concentrations of 1.2 mg/mouse/day or 0.6 mg/mouse/day. There was a slight dose-dependent effect. Therefore, some of the extracts according to the invention could potentially be used for antigenic stimulation of the immune system in patients suffering from recurrent infections.

EXAMPLE 10: Effect CFer300 intraperitoneal infection with Salmonella typhimurium in mice balb/c

Protection of mice infected with Salmonella typhimurium, tested bacterial lysates obtained from Lactobacillus, after oral administration.

Materials and methods

Animals and their contents

Balb/c mice were kept in the laboratory is the second of the Institute of immunology in Moscow. For the experiment on the protection in vivo, not inbred white mice laboratory breed bought in a Pole in the Russian State Research Center of Biomedical Technologies (Russian Academy of Medical Sciences). Upon delivery, the body weight of mice was 12-14, for all experiments, mice were kept in the absence of pathogens on a standard diet for rodents and water.

The research group (main experiment)

Two groups of 22 mice used for testing anti-infective efficacy of bacterial extracts obtained using an experimental model of infection with Salmonella typhimurium in mice.

One group was treated with oral solution CFer300, and the second group received a false treatment (water) as a negative control.

0.5 ml of solution was injected into each mouse orally once daily for 10 consecutive days before all mice were subjected to antigenic stimulation Salmonella typhimurium:

Group 1: Mice treated CFer300, administered as a single oral dose of 2 mg (0.5 ml).

Group 2: Mice treated with a false treatment using oral administration of 0.5 ml of water daily for 10 days.

Test product

Tested bacterial extract was a OP0701B4CFer300 ("CFer300"); extract from Lactobacillus fermentum I-3929 (12 g/l)obtained the first, as described in example 3.3 (28.4 mg of active dry weight/g).

A preliminary experiment

The aim of the preliminary experiment was to determine the dose of the infectious agent that caused the mortality of close to 50%. 3 weeks after antigenic stimulation. As antigenic stimulation, a suspension of Salmonella enterica, serovar typhimurium strain 415 (Institute of Vaccines and Sera them. (ONU), Russian Academy of Medical Sciences) were intraperitoneally injected with each mouse. Dose antigenic stimulation was in the range of 103up to 105CFU Salmonellae on the mouse.

Observations and death registration

After antigenic stimulation, mice were kept in standard conditions for laboratory animals. Daily observations and death registration was carried out for a period of 21 days after infection. The anti-infective efficacy was evaluated in accordance with survival after infection (SR), average life expectancy after infection (ADL), protection factor (DF) and the performance index of the drug (El)calculated for each experimental group. For SR took the percentage of animals living in the experimental groups at day 21 after infection. ADL, DF and EI was calculated as follows:

ADL=(X1+X2+...+Xn)/N

where:

ADL is the average life expectancy

From X1 to Xn represent the period after infection have been the AI for experimental mice from 1 to n, and

N represents the total number of animals in the experimental group.

DF=CD/ED

where:

DF is the protection factor,

CD represents the percentage of dead animals in the control group, and

ED represents the percentage of dead animals in the experimental group.

EI=[(DF-1)/DF]×100%

where:

EI represents the index of effectiveness of the drug and

DF is the protection factor.

Results: tolerability of a medicinal product

The drug was administered orally once daily for 10 days. During the 10-day period prior treatment was not observed evidence of toxicity or side effects. And he was a good move.

Titration of Salmonellae

A preliminary experiment was performed to determine the dose of the antigenic stimulation of Salmonella typhimurium to cause mortality of approximately 50%. The results are shown in table 17.

Table 17
A preliminary experiment to determine the dose of antigenic stimulation Salmonella typhimurium, corresponding to a mortality of approximately 50%
Dose S.typhiMice/groupDays after infectionDead mice/total number of Lost (%)
12345678910111213
1056----12----1--4/667
1046-------1 -----1/617
1036-------------0/60

The underlined numbers represent the number of animals found dead on the specified day (after infection).

Conclusion

Based on these data, a dose of 105CFU of strain 415 Salmonella typhimurium strain (67% dead animals in bold) was selected for subsequent studies.

Main experiment: Antigenic stimulation of mice treated CFer300

Mice treated for 10 days prior drug treatment CFer300 (n=22), or in the control group, water (n=22), were subjected to antigenic stimulation in day after pre-treatment.

A suspension of Salmonella typhimurium was injected intraperitoneally in the form to the s antigenic stimulation 10 5CFU per mouse. Follow-up was performed within 21 days after infection. Mortality in groups of animals are shown in table 18.

Figures in italics represent the number of animals found dead on the specified day (after infection). These data were used to calculate SR, ADL, DF and EI (table 19).

Table 19
The survival rate during the observation period (21 days)
Pre-treatment substancesMortality (%)Survival (%)ADL (days)DFEI (%)
CFer 300277317,11,6740%
H2O455514,710

In the control group who received prior treatment with water, survival during the observation period (21 days) was 55%, and ADL was 14.7 days. At the dose of 2 mg/day, CFer300 showed protective efficiency is, what resulted SR=73% and ADL=17,1 day, ie, DF=1,67, and EI=40%.

In summary, a 10-day course of daily oral treatment CFer300 entered in a single dose of 2 mg, provided partial protection in mice infected with Salmonella typhimurium. As shown in this experiment, and shown previously in vitro and ex vivo disclosed in the present description extracts can be used in the further development of therapeutic drugs.

EXAMPLE 11: Effect of two extracts of Lactobacillus model of asthma caused by LACK

Two variants of the invention were tested in the mouse model induced by allergen asthma after oral administration (Julia et al., Immunity, 2002, 16:271-283).

Material and methods

Animals and maintenance

6-week-old female mice BALB/c ByJ purchased from Janvier, France. They were kept and fed under standard conditions.

The research group

The total number of 27 mice were divided into 4 groups as follows:

Group A: untreated sensitized LACK and stimulated saline mouse; LACK is a protein from the parasite Leishmania major (4 mice).

Group B: untreated sensitized LACK and subjected to antigenic stimulation mice (8 mice).

Group C: Treated OM-1009A (8 mg dry mass of the residue in the introduction), sensitised LACK and subjected to antigenic stimulation mice (8 mice).

Group D: Treated with treatment the OM-1009B (8 mg dry mass of the residue in the introduction), sensitized LACK and subjected to antigenic stimulation mice (7 mice).

The treatment and the circuit under test or control product

Mice were treated with -3 on the 22nd day. At day 0 and day 7, mice were senzibilizirani intraperitoneally (I.P. Pavlova.) 10 µg LACK in the presence of 2 mg of alum 3. From 17 to 21 days, mice were subjected to 20-minute exposure to antigenic stimulation aerosol solution LACK (0,15%) (Group B, C and D), or saline (Group A) as a control. On the 22nd day there was an analysis of the ability of mice to respond to inhalation of methacholine the development of AHR. On the 23rd day, the mice were killed and assessed the presence of pneumonia.

Methodology

Mice drug was treated three times as described above. Lavage cannula introduced into the trachea, served in individual mice after phlebotomy. The lungs were washed 3 times with 1 ml of warmed PBS. Cells were washed in PBS, resuspendable 300 ál and counted using a camera Barker-Turk. For differential counting of cells in BAL (broncho-alveolar lavage), preparations of cells after centrifugation were prepared and stained by Wright/Giemsa. Tested group consisted of mice wild-type (wt), sensitised LACK and subjected to false stimulation PBS (Control), sensitised LACK and subjected to antigenic stimulation of mouse wt (animals with asthma), wt mouse treated OM-1009A, and mouse wt, l the black OM-1009B. The total number of cells in BAL were determined by microscopic examination of preparations of cells after centrifugation and staining on the Wright/Giemsa.

Since we treated mice (see results below) observed a reduction in airway inflammation, then got lung extracts, and quantitative determination of IL-4, IL-5 and IL-13 were performed multiplex analysis (using software CBA).

Tested products

Tested the following two bacterial lysate: extract OM-1009A from Lactobacillus fermentum I-3929, obtained as described in example 3.7, and extract OM-1009B from Lactobacillus fermentum I-3929, obtained as described in example 3.11.

Results

a) Hyper-reactive Airways

On the 22nd day of the conducted analysis to determine the ability of mice to the development of AHR after inhalation of methacholine these doses. The results are presented in figure 5. The results show that OM-1009-B restored basal values Penh, similar to untreated animals suffering from asthma group treated with PBS. OM-1009-A was more effective, because the values obtained Penh were even lower than the values observed in not suffering asthma control group.

b) Total and differential cell number in liquids broncho-alveolar lavage

On the 23rd day, the mice were killed and evaluated pneumonia. The total number of cells in BAL pre is represented in figure 20 (right column), And differential cell number: the Number of eosinophils (Eo), the number of neutrophils (Neutro), the number of lymphocytes (Lympho) and the number of other cells (Other).

Table 20
Total and differential cell count in BAL after treatment two extracts according to the invention (Group C and D). Presents averages and standard errors of the average
AverageEoNeutroLymphoOtherThe total number of cells
(A) Ctrl PBS9013178117964912565000
(B) Ctrl LACK = mouse asthma3476915131785609301097785714
(C) OM-1009A+LACK1152272787954725158419356250
(D) OM-1009B+LACK 2038341421034523204575457143

SEMEoNeutroLymphoOtherThe total number of cells
(A) Ctrl PBS166906625555682887
(B) Ctrl LACK78464143921773678575135275
(C) OM-1009A+LACK3674110444270243429694905
(D) OM-1009B+LACK57955322067312531174992

Both extracts significantly reduced the total number of cells in BAL (p<0.01 for OM-1009A; and p<0.03 in for OM-1009B. Compared with group B). The number of eosinophils reduce the of n respectively 3 and 1.7 times. The number of neutrophils decreased, respectively, 1.8 and 3.6 times. The reduction in the number of eosinophils and neutrophils indicates a lower concentration of inflammatory cells in BAL animals receiving pre-treatment with the extracts.

c) Th2 cytokines detected in the lung extracts and quantified multiplex analysis (software CBA)

Table 21
Individual IL4 levels in the lungs of mice
PCG/mgmouse 1mouse 2mouse 3mouse 4mouse 5mouse 6mouse 7mouse 8Wednesday
(A) PBS00,00530,00710,01570,0070
(B) LACK
(mouse asthma)
0,03950,01410,0279 0,02030,03530,05740,09490,08060,0462
(C) PO OM 1009A0,03790,01200,00660,00560,01810,01460,03960,04630,0226
(D) PO OM 1009B0,04090,05150,04140,05110,04840,03000,02700,0415

Table 22
Individual levels of IL5 in the lungs of mice
PCG/mgmouse 1mouse 2mouse 3mouse 4mouse 5mouse 6mouse 7mouse 8Wednesday
(A) PBS 00,00040,004300,0012
(B) LACK
(mouse asthma)
0,11410,10020,15320,11150,30980,51730,53850,45470,2874
(C) PO OM 1009A0,18170,04450,05720,03640,16430,04880,19170,10570,1038
(D) PO OM 1009B0,15180,27980,17970,54390,36850,13200,17050,2609

Table 23
Individual levels of IL13 in the lungs of mice
PCG/mgmouse 1mouse 2mouse 3mouse 4mouse 5mouse 6mouse 7mouse 8Wednesday
(A) PBS0000,02080,0052
(B) LACK
(mouse asthma)
0,27270,08960,35420,23030,61781,13061,17681,02440,6120
(C) PO OM 1009A0,35980,12310,08880,09950,29750,19090,52130,5463 0,2784
(D) PO OM 1009B0,36280,89190,42910,76990,64410,49810,49720,5847

Levels of Th2 cytokines (IL4, IL5 and IL13) (see table 21, 22, 23), when compared to asthmatic control group (B)was reduced by the extract OM-1009A, but not extract OM-1009B. Therefore, bacterial extracts obtained by the treatment of a strong base (OM-1009-B), may be less active than extracts obtained in conditions of handling moderate base (OM-1009-A). Even though both of the extract were active in the analysis of factor Penh (6), OM-1009A may be a better candidate than the OM-1009B, for the treatment or prevention of conditions related to associated with Th2 diseases, such as allergic diseases and atopy, or their symptoms.

1. Extract one or more bacterial strains Lactobacillus, where the extract is a soluble extract, and where the extract contains chemically modified bacterial molecules, and the extract obtained by exposure to an alkaline environment on one or more bacterial strains Lactobacillus, and where the specified extract useful in the treatment of diseases connected to the imbalanced production of inflammatory cytokines.

2. The extract according to claim 1, where the extract possesses immunomodulatory activity from the individual.

3. The extract according to claim 2, where the extract possesses immunostimulatory activity from the individual.

4. The extract according to claim 2, where the extract has anti-inflammatory activity from the individual.

5. The extract according to claim 1, where one or more bacterial strains Lactobacillus contains one or more of Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus casei defensis, Lactobacillus casei ssp. casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus lactis and Lactobacillus delbrueckii.

6. The extract according to claim 1, where one or more bacterial strains Lactobacillus contains one or more of Lactobacillus fermentum 1-3929, Lactobacillus rhamnosus 71.38, Lactobacillus plantarum 71.39, Lactobacillus johnsonii 103782 and Lactobacillus helveticus 103146.

7. The extract according to claim 1, where the specified chemical modification leads to the fact that one or more of aspartic acid, glutamic acid, serine, histidine, alanine, arginine, tyrosine, methionine, phenylalanine, and lysine in the specified extract racemethionine at least 10%.

8. The extract according to claim 1, where the extract is able to achieve the calculated ratio IL10/IL12 in mononuclear cells of peripheral blood, and this ratio is equal to or greater than the ratio of IL10/IL12, achieved a live Lactobacillus, from which vtorogo the extract obtained.

9. The extract according to claim 1, where the extract reduces the number of eosinophilic cells, polymorphonuclear cells, lymphocytic cells, or any combination in asthmatic mice by a factor of at least 1,5 relative with asthma untreated control.

10. The method of obtaining the extract according to claim 1, including:
(a) culturing one or more bacterial strains Lactobacillus in the culture medium;
(b) exposure to an alkaline environment for each bacterial strain Lactobacillus; and
(c) treatment of the product of stage (b) to remove insoluble and having the form of particles of material.

11. The method according to claim 10, where stage (C) is performed by filtration tangential flow.

12. The method according to claim 10, where the phase (b) provides for the effect on each bacterial strain Lactobacillus pH greater than 9,0 sufficient for chemical modification of bacterial molecules.

13. The method according to claim 10, additionally comprising processing each strain at pH less than 4.5 after stage (b) and before stage (C).

14. The method according to claim 10, where the chemical modification includes racemization one or more of aspartic acid, glutamic acid, serine, histidine, alanine, arginine, tyrosine, methionine, phenylalanine, and lysine in the extract of at least 10%.

15. Pharmaceutical composition useful for reducing at least one symptom SV is connected, at least one state selected from respiratory disorders, allergic disorders, disorders of the urinary tract and digestive disorders containing extract according to claim 1.

16. Nutraceutical composition, useful in the treatment of diseases associated with an imbalance of the production of inflammatory cytokines, containing extract according to claim 1.

17. Pharmaceutical composition useful in treating diseases associated with an imbalance of the production of inflammatory cytokines, containing extract according to claim 1.

18. A method of reducing at least one symptom associated at least one condition selected from respiratory disorders, allergic disorders, disorders of the urinary tract and digestive disorders, including introduction to the individual a therapeutically effective amount of an extract according to claim 1.

19. The method according to p, where the individual is a human or a pet.

20. The extract obtained using the method according to any one of PP-14.

21. The extract according to claim 20, where the extract is a soluble extract.



 

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4 cl, 4 dwg, 3 ex

FIELD: biotechnologies.

SUBSTANCE: method provides for preparation of a nutrient medium on the basis of a dry nutrient medium MEDIA TW-50 with application of a salt additive into it, such as dibasic sodium phosphate at the specified ratio. The nutrient medium is sterilised, cooled down to the specified temperature and neutralised, with 20% solution of caustic soda, with establishment of the specified pH value of the medium to produce the nutrient medium. The acidophilic culture of the strain 126 and yeast autolysate are introduced at the specified ratio into the produced nutrient medium with subsequent cultivation. At the same time in process of cultivation the acidity of the nutrient medium is maintained with staged deoxidisation by the 20% solution of the caustic soda. The bacterial mass is cooled and separated. The bacterial mass is mixed with the protective medium containing saccharose, gelatine and water, where additionally lactulose syrup is introduced, as a symbiotic protective factor, ascorbic acid, as an anti-enzyme agent, talc powder as a drying and sliding agent at the specified ratio. It is homogenised, poured, frozen and dried with a sublimation method.

EFFECT: invention makes it possible to increase survival rate of acidophilous bacteria under sublimation and storage of a preparation and to reduce hygroscopicity of a finished preparation.

3 ex

FIELD: chemistry.

SUBSTANCE: three types of clay of different chemical composition is used with addition of molasses and Linex biopreparation in the following ratios, wt %: Dialbeculit clay -38-40; Irlit clay 1 - 28-32; Irlit clay 7 - 16-20; molasses - 8-12; Linex biopreparation -2-4. Adding such a composition to the soil reduces the amount of oil pollutants by 72%.

EFFECT: low toxicity of soil and expenses on amelioration.

1 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: method includes preparation of the inoculum of cells Streptomyces sp. MT246 (All-Russian Collection of Microorganisms Ac-2618D). Preparation of the nutrient medium containing one or more sources of carbon, nitrogen, metal ions in the form of soluble salts with fractional feeding of the carbon source during the productive stage. At that, as an additional carbon source the nutrient medium contains rhamnose, and a necessary component of the nutrient medium - MnS04 and sorbent from the group of amberlites XAD. Adding of the inoculate of cells Streptomyces sp. MT246 (All-Russian Collection of Microorganisms Ac-2618D) to the nutrient medium together with rhamnose and MnSO4 in a predetermined ratio and incubating of the medium at pH 7.0-7.5, temperature 25-35°C for 7-10 days. Separation of the residue is carried by centrifugation. The resulting residue is added to ethanol at a predetermined ratio, followed by extraction of tacrolimus at 30 for 2 hours and centrifuging to obtain the extract comprising tacrolimus.

EFFECT: invention enables to increase the yield of tacrolimus.

9 cl, 6 ex

Synbiotic mixture // 2495927

FIELD: chemistry.

SUBSTANCE: invention relates to a synbiotic preparation containing N-acetyl-lactosamine and/or an oligosaccharide containing N-acetyl-lactosamine and a probiotic strain Lactobacillus sp. The oligosaccharide containing N-acetyl-lactosamine is lacto-N-tetraose or lacto-N-neotetraose. The probiotic strain Lactobacillus sp. is Lactobacillus rhamnosus ATCC 53103, Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus reuteri ATCC 55730 or Lactobacillus reuteri DSM 17938.

EFFECT: synbiotic preparation is used in infant nutritional composition as well as in production of a nutritional composition for preventing or as a medicinal agent for preventing or treating pathogenic infections of the gastrointestinal tract and upper respiratory tracts.

6 cl, 1 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: method includes extraction of DNA from strains B.pertussis, performance of a polymerase chain reaction with simultaneous introduction of specific primers PF-PR for production of amplicons of a fragment of a promotor of pertussis toxin ptxP, restriction of produced amplicons with endonucleases Kpnl and Mlul and electrophoresis of restriction products in agarose gel. Differentiation of strains B.pertussis is carried out by comparison of size of amplicons of DNA fragments of promotor ptxP of investigated strains B.pertussis with reference samples of DNA strains B.pertussis.

EFFECT: invention may be used for accelerated detection of epidemic strains with modified genetic structure during microbiological and molecular-genetic monitoring of Bordetella pertussis strains and for further development of promising diagnostic and prevention preparations on their basis.

1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, namely immunology and may be used for immune system stimulation. That is ensured by administering a composition containing a complex of isolated carbonic anhydrase IX (CA9) protein and an antigen, wherein the isolated CA9 proteins and the antigen are noncovalently linked to each other, into an individual. The group of inventions also refers to a composition containing a purified population of dendrite cells coming into contact with the above composition.

EFFECT: group of inventions provides immune cell stimulation by enhancing the antigen immunogenicity ensured by adding the complex with the CA9 protein which can function as a chaperone protein.

18 cl, 6 dwg, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to pharmacology and medicine and concerns an immunostimulating composition, and methods for using it for enhancing the immune response, particularly for treating infections caused by bacterial and viral pathogens, to adjuvant compositions and methods for enhancing the antigen immunogenicity when used as an adjuvant of vaccines. The pharmaceutical composition of pattern-recognising receptor ligands contains muramyl peptide of structural formula N-acetylmuramyl-L-alanyl-D-isoglutamine and lipopolysaccharide or lipid A in the following proportions, wt %: muramyl peptide of structural formula N-acetylmuramyl-L-alanyl-D-isoglutamine 0.00001 to 0.01, lipopolysaccharide or lipid A 0.00001 to 0.01, a solvent - the rest.

EFFECT: group of inventions provides high immunostimulating activity and prolonged action of the composition.

7 cl, 5 ex, 1 tbl, 9 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to pulmonology and may be used in treating the patients with chronic bronchitis in combination with secondary immunodeficiency. For this purpose, the patient with the remitting disease undergoes 6 procedures of ozone therapy every second days by the intravenous introduction of ozonised physiological saline with the ozone concentration 600 mcg/l in the ozone-oxygen gas mixture. One week after the ozone therapy, patient's venous blood is sampled and exposed to UV light for 2 procedures every 7 days. At the first procedure, the blood volume to be exposed makes 0.8 ml/kg of patient's body weight, and at the second procedure - 1 ml/kg.

EFFECT: method provides the effective treatment in the given category of patients that is ensured by the activation of the cell, phagocytic and humoral immunity without the use of aq drug-induced therapy as a result of the combined selected regimen.

2 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry and represents using probiotic bifidus bacteria in preparing a nutritional composition for improving IgA secretion in the infants born by cesarean section for the first four months of life.

EFFECT: invention provides improving specific and non-specific IgA secretion thereby enhancing the mucosal immune protection in the infant.

11 cl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: given invention refers to medicine, biopharmaceutics, and may be used to prepare vaccines. For this purpose, the immunogenic composition contains: 1) an antigen Neisseria autotransporter protein representing NadA or Hsf; 2) an antigen Neisseria protein involved in iron absorption and representing Lipo28 or low-molecular or high-molecular TbpA; and 3) a vesicle preparation of an outer membrane containing immunotype L3 Neisseria lipopolysaccharide (LPS); and wherein the above antigens if present in the outer membrane vesicle are positively regulated in accordance with recombinant technology in the above outer membrane vesicle.

EFFECT: using the given vaccine that is a combination of the various class Neisseria antigens generates the immune response which occurs to be stronger if expressed in bactericidal units.

17 cl, 11 dwg, 21 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, and concerns a new multifunctional combined interferon drug preparation of broad spectrum of action. A preparation in the form of a suppository contains a synergistic combination of the immunomodulator interferon alpha-2b and the herbal biostimulant aloe extract; it contains vitamin E, glycine, buffer salts, tysol, lecithin and solid fat as a base in the specified proportions.

EFFECT: invention provides preparing the combined drug preparation in the form of long storage-stable suppository having the standard physical-chemical, structural and mechanical, multifunctional biological properties, higher bioavailability and synergistic therapeutic efficacy of interferon alpha-2b with the herbal biostimulant aloe extract.

1 cl, 5 tbl, 3 dwg

FIELD: chemistry.

SUBSTANCE: disclosed are peptides which are isolated from the FOXM1 protein and are capable of activating cytotoxic (killer) human T cells by forming an antigen-presenting complex with a HLA-A2 molecule. Disclosed are compositions which contain the disclosed peptides, use of a peptide to produce an agent for inducing cancer immunity, treating and preventing cancer, and for producing antibodies which selectivity bind the disclosed peptides. The invention describes an exosome and an isolated antigen-presenting cell, which present a complex of the disclosed peptide with a HLA-A2 molecule, for inducing cytotoxic T cells, methods of inducing a antigen-presenting cell and a cytotoxic T cell, as well as a method of damaging FOXM1 and HLA-A2 expressing cells.

EFFECT: invention can further be used in treating tumours that are characterised by high FOXM1 expression.

14 cl, 2 ex, 1 dwg, 1 tbl

FIELD: chemistry.

SUBSTANCE: disclosed are germanium complexes, having a general structural formula (I): Gex[AD][CA]y[AA]z (I), where AD denotes a purine nitrogenous base derivative, having antiviral activity; CA denotes a hydroxycarboxylic acid; AA denotes an amino acid selected from α-amino acids, where x=1÷2, y=2÷4, z=0÷2, wherein all CA in the complex are identical or different, all AA in the complex are identical or different. Also disclosed is a method of producing germanium complexes, a medicinal agent and an immunostimulant.

EFFECT: invention enables to obtain germanium complexes, having high antiviral and immunostimulating activity.

18 cl, 5 dwg, 3 tbl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, namely to paediatrics and may be used for preparing a drug or therapeutic nutritional composition for maturating an immune responses in a newborn infant. That is ensured by using an oligosaccharide specified in a group consisting of: lacto-N-tetrose, lacto-N-neotetrose, lacto-N-hexose, lacto-N-neohexose, para-lacto-N-hexose, para-lacto-N-neohexose, lacto-N-octose, lacto-N-neooctose, iso-lacto-N-octose, para-lacto-N-octose and lacto-N-decose. Also, the above oligosaccharide may be used for modulating the immune system of the newborn infant to ensure the developing beneficial intestinal microflora for the first weeks of life comparable to such found in breastfed infants.

EFFECT: group of inventions enables the developing beneficial intestinal microflora in the infant, and reduces the risk of a further allergy.

27 cl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology and veterinary medicine. Method of obtaining immunotropic medication for prevention and treatment of inflammatory processes of agricultural animals, includes mixing 90 wt.fr of 0.2-0.3% agar suspension, 2.5 wt.fr of concentrate of refined polysaccharide complex of yeast cells, 3.5 wt.fr. of (-)2,3,5,6-tetrahydro-6- phenylimidazo -[2,1-b]- thiazole hydrochloride and 0.2 wt.fr. of formalin, with additional introduction of 5.0 mln U of antibiotic kanamycin monosulfate and bringing volume of medication to 100 wt.fr.

EFFECT: increase of non-specific immunogenesis resistance, prevention and treatment of animal diseases.

2 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, namely to using at least one immunomodulatory compound of general formula (1) or a pharmaceutically acceptable, solvate or isomer thereof for preparing a pharmaceutical composition for treating a disease or disorder specified in asthma, atopic dermatitis, allergic rhinitis, inflammatory intestinal disease, diabetes or rheumatoid arthritis in homoiothermal animal, including a human. What is also presented is using (5S,11R)-1-amino-5-[(R)-3-dodecanoyloxytetradecanoylamino]-6-oxo-7-aza-11-[(R)-3-hydroxytetradecanoylamino]dodecan-12-ol-12-dihydrophosphate (OM-294-BA-MP (S,R)) or a pharmaceutically acceptable salt, solvate or isomer thereof for preparing the pharmaceutical composition.

EFFECT: group of inventions provides treating the above diseases by modulating the TH1/TH2 cytokine balance by reducing TH2-cytokine release and enhancing TH2-cytokine production.

11 cl, 16 dwg, 14 ex

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