The method of obtaining the bank autochthonous strains of microorganisms for the recovery of intestinal microbiocenosis person

 

(57) Abstract:

The invention is intended for the prevention and treatment of a dysbacteriosis of intestines of humans. The method includes sampling of faeces from the same organism during its clinically healthy, since 7-15 days after birth and throughout life periodically not more than 1 time per year. Trial distinguish and identify autotomy of the normal intestinal microflora. The biomass of each species of bacteria separately accumulating on selective nutrient media to titer not less than 103-109cells/ml of the Obtained biomass combined and add the stabilizing composition. The mixture was divided into samples. Each sample shall be preserved and stored over the life of a person with a periodic control biotite. In infants up to 1 year as of the normal intestinal microflora produce mainly bifidobacteria species Bifidobacterium bifidum, Bifidobacterium brevis, Bifidobacterium infantis and Lactobacillus species Lactobacillus acidophilus, Lactobacillus fermenti. At the age of 1 year as of the normal intestinal microflora produce mainly bifidobacteria species Bifidobacterium longum, Bifidobacterium adolescentis, Lactobacillus species Lactobacillus acidophilus, Lactobacillus fermenti, Lactobacillus Plantarum, strains of the bacteria Escherichia coli and the breast is the process of storage after controlling for biotite additionally combined in equal proportions of the specimens of autostartup bacteria, isolated from the normal intestinal flora of man at different periods of his life. Introduction in the human gut samples Bank of autosomal microorganisms simultaneously affect different parts of the disturbed bacteriocins intestine through the use of a full spectrum of normal intestinal microflora. 6 C.p. f-crystals.

The invention relates to the field of Microbiology and medicine and can be used for the prevention and treatment of a dysbacteriosis of intestines of man or animal.

Full range of normal intestinal microflora is an essential component in many metabolic reactions of the microorganism and, above all, prevents the development of pathogenic and conditionally pathogenic microorganisms in the gastrointestinal tract. Microflora involved in the enterohepatic circulation of the major components of bile, enzymes flora in the distal part of the intestine inactivate biologically active compounds (biogenic amines) released by the body with digestive juices; intestinal flora disposes of undigested food substances with the formation of amines, organic acids and other compounds that affect the metabolism in the body. OOI reactivity of organism. The result of antigenic stimulation (AutoPlay), primarily by intestinal bacteria in the body creates a common pool of immunoglobulins.

There is a method of prophylaxis of a dysbacteriosis of intestines of newborn children, including the introduction of the child through 30-120 min after birth orally, once the strains of bifidobacteria in the dose of 108- 109microbial cells obtained from the mother (ed. St. 1743607, class a 61 K 35/74, publ. 1992). This method is effective for prophylaxis of a dysbacteriosis of intestines of a child born by caesarean section [1].

However, for prophylaxis of a dysbacteriosis is not the whole range of healthy intestinal flora, and only a monoculture of bifidobacteria. In addition, the bacteria culture is not adapted to the intestines of the child, may there not to settle down, so as not taken from the intestines of the child (i.e., is not autostartup), so this method of prevention is not effective enough. In oral introduction bacteria of them dies during the passage of various sections of the gastrointestinal tract.

A method of obtaining and application of complex bacterial preparation for correction of intestinal dysbiosis, including the allocation from the body is organizma based. As the basis of using activated carbon, the surface of the particles which are combined with microbial cells at a concentration of 1 mm22,1103- 105. When activated carbon is used in the form of powder with particle size less than 30 microns. The resulting preparation is administered orally in the human body three times over several days at a dose of 0.3-1.0)102a MIC. CL /g (RF patent 2017486, class a 61 K 31/00, publ. 1994) [2].

However, for prophylaxis of a dysbacteriosis is not the entire range of healthy human intestinal microflora, and only the monoculture of lactic acid bacteria or E. coli, resulting in reduced efficacy of prophylaxis of a dysbacteriosis.

There is a method of treatment of a dysbacteriosis of intestines of humans, including the sampling of faeces from the body, the titration of the investigated material sterile saline solution, seeding it from different dilutions on nutrient medium, the identification, selection autostartup normal intestinal microflora containing bifido - and lactobacteria, separate the time between the biomass of each species of bacteria and their subsequent mixing, short-term storage at a temperature of +4-7oAnd periodic oral introduction of the mixture of the decree(ed.St. 1286212, class a 61 K 35/74, publ. 1987) [3].

However, for the treatment of dysbacteriosis is not the entire range of healthy human intestinal microflora, and only autotomy bifidobacteria and lactobacilli, resulting in reduced efficacy of treatment of a dysbacteriosis. In addition, the selection of autostartup is performed before antibiotic therapy, when a normal part of healthy microflora person is in a depressed condition. In oral introduction bacteria of them perish with the passage of the different sections of the gastro-intestinal tract, which reduces the effectiveness of treatment of a dysbacteriosis and increases the recovery time of the body.

Sources of scientific, technical and patent information is not known statement of the problem associated with the creation and use of Bank autochthonous strains of microorganisms for the recovery of the intestinal microflora of humans.

Thus, development of a method of obtaining a Bank autochthonous strains of microorganisms for the recovery of intestinal microbiocenosis person is a new problem, and its solution proposed in the present application materials, is not known from modern technology (except for methods of sampling and Krotkova [2,3] for the treatment of dysbacteriosis of various etiologies), has no prototype and meets all the eligibility criteria of the invention.

The objective of the proposed invention is a first method of obtaining the Bank autochthonous strains of microorganisms, which would allow to get more persistent and pronounced therapeutic effect when it is applied by simultaneous exposure to different parts of the disturbed bacteriocins the human intestine through the use of a full spectrum of normal (healthy) autochthonous intestinal flora.

This task is solved in that in the method of obtaining the Bank autochthonous strains of microorganisms for the recovery of the intestinal microflora of humans, according to the invention, produce samples of faeces from the same organism during its clinically healthy, since 7-15 days after birth and throughout life periodically not more than 1 time per year, from samples distinguish and identify autotomy normal intestinal microflora, the biomass of each species of bacteria separately accumulating on selective nutrient media to titer not less than 103- 109cells/ml, obtained biomass combined and add the stabilizing composition, the floor is a mini control biotite.

After 7-15 days after birth ends with the formation of his intestinal microflora. The use of the Bank autochthonous strains of microorganisms, including the full range of normal intestinal autosecretary man, provides a comprehensive biological action (with the introduction of a mixture of strains in the intestine), designed to accelerate the normalization of microflora.

In infants up to 1 year as of the normal intestinal microflora produce mainly bifidobacteria species Bifidobacterium bifidum, Bifidobacterium brevis, Bifidobacterium infantis and Lactobacillus species Lactobacillus acidophilus, Lactobacillus fermenti.

At the age of 1 year as of the normal intestinal microflora produce mainly bifidobacteria species Bifidobacterium longum, Bifidobacterium adolescentis, Lactobacillus species Lactobacillus acidophilus, Lactobacillus fermenti, Lactobacillus plantarum, strains of the bacteria Escherichia coli and lactic acid streptococci mainly species Streptococcus faecium, Streptococcus faecalis, Streptococcus avium, Streptococcus salivarius and Streptococcus bovis.

To create a Bank of autochthonous strains of microorganisms it is necessary to have samples of strains from different periods of human life to obtain drugs over a wide range and complex actions.

The Association of biomass autostartlist individual strains in the mixture during preservation and storage.

As a stabilizing composition use saharso gelatin or Saharsa-starch protective environment in the amount of 5-10 wt.% from biomass mixture autostartup. Lowering the concentration of the stabilizer in biomass significantly increase the loss of biological activity during drying and subsequent storage.

Preservation of the samples of the mixture of autostartup produced by lyophilization.

During storage after controlling for biotite additionally combined in equal proportions of the specimens of autostartup bacteria isolated from the normal intestinal flora of man at different periods of his life, thereby increasing the biological activity of the drug and the settling down of autobatterie in the human gut.

Using the full range of normal (healthy) autochthonous intestinal flora, you can affect different parts of the disturbed bacteriocins intestine in the process of treatment of a dysbacteriosis or prevent violations of bacteriocins bowel in the prevention of the specified diseases.

Technology of preparation of the Bank of samples of autochthonous strains of microorganisms are given in examples 1-4.

her than 7-15 day after his birth. The investigated material is titrated with a sterile saline solution, making serially tenfold diluted to 10-8. Various dilutions specified material inoculated on nutrient medium, Blaurock, aloes, Endo, Viburnum, Mrs-4, blood agar with polymyxin to determine the baseline levels of coliform bacteria, lactobacilli, bifidobacteria, enterococci, staphylococci and other bacteria with subsequent identification and selection of autostartup normal intestinal microflora, the corresponding breast to the child's age and contains bifidus and Lactobacillus. Two days material from the isolated colonies of lactobacilli (Lactobacillus acidophilus, Lactobacillus fermenti) grown on medium Mrs-4, as well as from isolated colonies of bifidobacteria (Bifidobacterium bifidum, Bifidobacterium brevis, Bifidobacterium infantis), grown on the environment Blaurock again subcultured onto appropriate selective medium. Grew up two days after the second passage of the isolated colonies of bifidobacteria and lactobacilli subcultured (separately) by the above nutrient medium, Blaurock, Mrs-4 and cultured by standard methods to obtain the biomass of microorganisms. For example, the biomass of bifidobacteria (Bifidobacterium bifidum, Bifidobacte - 109- 1010cells/ml Then the biomass of each species autochthonous strains of microorganisms administered in equal ratio to the reactor and stirred to obtain a mixture. In the mixture add saharso gelatin protective environment (stabilizing composition) in an amount of 5-10 % by volume of the mixture of biomass. The biomass autostartup poured into vials and dried the standard method of freeze-drying and subsequent seal and store at a temperature not higher than minus 18oTo preserve the activity of the drug over a long period of time, for example during the period of a person's life. Periodically (at least 1 time per year) control (check) biotar one or more samples of the Bank of autostartup microorganisms through the passages on selective nutrient media mixture of autostatus and titration standard method for each strain. In case of reduction of biotite stored drug the entire volume of dry biomass is activated by passages on differentiated nutrient medium to maximize biotite. Later in the biomass type stabilizer, poured into vials and dried by lyophilization method and continue to keep at temperature not higher than minus 18oC.

The investigated material is titrated with a sterile saline solution, making serially tenfold diluted to 10-8. Various dilutions specified material inoculated on nutrient medium, Blaurock, aloes, Endo, Viburnum, Mrs-4, blood agar with polymyxin to determine the baseline levels of coliform bacteria, lactobacilli, bifidobacteria, enterococci, staphylococci and other bacteria with subsequent identification and selection of autostartup normal intestinal microflora containing bifidobacteria and lactobacilli, coliforms, lactic acid streptococci, the lactic acid paloc the tobacillus plantarum), grown on the environment Mrs-4, isolated from colonies of bifidobacteria (Bifidobacterium longum, Bifidobacterium adolescentis), grown on the environment Blaurock from isolated colonies enterococci (Streptococcus faecium, Streptococcus faecalis, Streptococcus avium, Streptococcus salivarius and Streptococcus bovis), grown on medium Kalina, from isolated colonies of Escherichia coli (E. coli) grown on blood agar with polymyxin again subcultured onto appropriate selective medium. Grew up two days after the second passage of the isolated colonies of bifidobacteria and lactobacilli, enterococci, Escherichia coli subcultured (separately) by the above nutrient medium, Blaurock, Mrs-4, Kalina, blood agar with polymyxin and cultured by standard methods to obtain the biomass of microorganisms. For example, the biomass of bifidobacteria has a titer not less than 109cells/ml, biomass lactobacilli 109- 1010cells/ml, Escherichia coli biomass of not less than 107- 108cells/ml, and biomass Streptococcus faecium is not less than 109cells/ml Then the biomass of each species autochthonous strains of microorganisms administered in equal ratio to the reactor and stirred to obtain a mixture. As the stabilizing composition can be used Saharsa-brahmanbaria, storage and control of biotite mixture of autostartup similar to example 1. Samples of the mixture of autostartup also stored throughout the life of this man.

Example 3. Additionally, this same person from time to time (not more than 1 time per year) make sampling of faeces at an older age (aged 30-45 years and older) in the period of its clinically healthy and normal functioning of the intestines. The technology of obtaining samples of the mixture of biomass data autostartup microorganisms and methods for their preservation and storage are similar to examples 1 and 2. Samples of the mixture of autostartup microorganisms obtained in example 3 is also stored in the person's life.

Example 4. During storage after controlling for biotite additionally combined in equal proportions of the specimens of autostartup bacteria isolated from the normal intestinal flora of man at different periods of his life and prepared in accordance with examples 1, 2 or 2 3 or 1-3, thereby increasing the biological activity of the mixture of the newly obtained sample autostartup microorganisms and the settling down of autobatterie in the human gut.

The set of stored samples of the preparations obtained in accordance with the laws the surveillance of the intestinal microflora of humans.

Ways to use Bank autochthonous samples of the drug are given in examples 5 and 6.

Example 5. For the prevention or treatment of dysbacteriosis one of the vials of sample mixture autostartup obtained, for example, in examples 1-3, reveal, a mixture of autostartup activated by passages on differentiated nutrient medium. The passage of autostatus stop in the logarithmic growth phase is not earlier than 7 h after inoculation on differentiated nutrient medium. Next, the activated drug in liquid form is injected into the rectal body in the region of the rectum by means of a probe in a dose of not less than 105- 107each species of microbe cells per 1 kg of body weight in 2-3 days. Thus, the preparation containing autochthonous strains of the normal intestinal microflora, bypassing the stomach directly into the intestine of man, resulting in preventing the decrease in its biotite.

Example 6. For treatment of a dysbacteriosis of intestines of humans, for example, after antibioticotherapy, radiation therapy, etc., when there is complete or partial destruction of normal intestinal microflora, take a sample autostartup obtained in example 4 from the intestines of the body in different periods is acii on differentiated nutrient medium. Next, the activated drug in liquid form is injected into the rectal body in the region of the rectum by means of a probe in a dose of not less than 107- 109each species of microbe cells per 1 kg of body weight for 4-5 days. Thus, the preparation containing autochthonous strains of the normal intestinal microflora, bypassing the stomach directly into the intestine of man, resulting in preventing the decrease in its biotite.

Improving the efficiency of the proposed method for the prevention and treatment of dysbacteriosis in comparison with the known analogues is its simultaneous impact on the various links broken bacteriocins intestine through the use of a full spectrum of normal (healthy) autochthonous microflora of the intestine, rectal insertion in the human intestine.

The proposed method provides a comprehensive biological action, designed to accelerate the normalization of microflora, accelerate the recovery of the morphological structure of the mucous membrane and cellular factors of local immunity, correction immunobiological reactivity and the number of indicators of homeostasis, increase nonspecific resistance to adverse medication, eeenie can be used in medicine and Microbiology.

1. The method of obtaining the Bank autochthonous strains of microorganisms for the recovery of intestinal microbiocenosis of a person, characterized in that make sampling of faeces from the same organism during its clinically healthy state from 7 to 15 days after birth and throughout life periodically not more than 1 time per year, from samples distinguish and identify autotomy normal intestinal microflora, the biomass of each species of bacteria separately accumulating on selective nutrient media to titer not less than 103- 109cells/ml, obtained biomass combined and add the stabilizing composition, the resulting mixture was divided into samples, each of which is canned, and stored over the life of a person with a periodic control biotite.

2. The method according to p. 1, characterized in that in infants up to 1 year as of the normal intestinal microflora produce mainly bifidobacteria species Bifidobacterium bifidum, Bifidobacterium brevis, Bifidobacterium infantis and Lactobacillus species Lactobacillus acidophilus, Lactobacillus fermenti.

3. The method according to p. 1, characterized in that the age of 1 year as of the normal intestinal microflora produce mainly bifidobacteria species Bifidobacterium longum, Bifidobacterium a is the mainly Streptococcus species Streptococcus faecium, Streptococcus faecalis, Streptococcus avium, Streptococcus salivarius and Streptococcus bovis.

4. The method according to p. 1, characterized in that the combination of biomass autostartup each species is carried out in equal proportions.

5. The method according to p. 1, characterized in that as a stabilizing composition use saharso gelatin or Saharsa-starch protective environment in quantities of 5 to 10 wt.% from biomass mixture autostartup.

6. The method according to p. 1, characterized in that the preservation of the samples of the mixture of autostartup produced by lyophilization.

7. The method according to p. 1, characterized in that during storage after controlling for biotite additionally combined in equal proportions of the specimens of autostartup bacteria isolated from the normal intestinal flora of man at different periods of his life.

 

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SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.

EFFECT: valuable medicinal properties of strain.

5 cl, 8 dwg, 10 ex

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