RU2491334C1 - Bifidobacterial consortium for preparing bacterial preparations and dietary supplements for correcting gastrointestinal microflora in children aged from three to fourteen, and method for preparing it, dietary supplement for correcting gastrointestinal microflora in children aged from three to fourteen and bacterial preparation for treating dysbiotic gastrointestinal conditions in children aged from three to fourteen - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: bifidobacterial consortium contains Bifidobacterium bifidum OV- 19, B. bifidum 791, B. longum B379M, B. longum OV-20, B. breve OV-12; it is used for preparing bacterial preparations and dietary supplements for correcting the microflora in the children aged from three to fourteen. The consortium is high-technology; it stores a biomass on nutrient mediums over a short period of time with a high concentration of probiotic microorganisms, possesses the acid-forming and antagonist properties in relation to the pathogenic and opportunistic microflora. The consortium is prepared by the co-culture including the length of an exponential growth phase of each of them whereby a priority of the nutrient medium inoculation of the specific composition is determined.

EFFECT: using the consortium as a part of the bacterial preparation for treating the dysbiotic gastrointestinal conditions in the children aged from three to fourteen, and the dietary supplement provides the more effective and fast correction of the child's gastrointestinal microflora in the children of the given age group.

4 cl, 9 tbl, 3 ex

Description

The invention relates to medicine and biotechnology and can be used for the preparation of prophylactic therapeutic agents intended for the correction of dysbiotic disorders of the microflora of the gastrointestinal tract and the maintenance of its colonization resistance in children aged 3 to 14 years. Means for prevention and treatment are designed primarily on the basis of two groups of microorganisms - bifidobacteria and lactobacilli. Bifidobacteria are the most significant representatives of the microflora of the human body - both in specific gravity in the composition of microbiocenosis and in their multifunctional role in maintaining homeostasis of the body. In breast-fed children, they make up almost 100% of the colon microflora. Bifidobacteria have a positive effect on the formation of the digestive system, immunity, and hematopoiesis of a child [1, 2, 3]. Bifidobacteria provide colonization resistance against pathogenic and conditionally pathogenic microorganisms; have an immunomodulatory effect; synthesize amino acids, vitamins, volatile fatty acids, enzymes involved in digestion and metabolism; contribute to the absorption of iron, calcium, maintenance of water-salt balance [4, 5]. Partial or complete elimination of bifidobacteria leads to inhibition of the body's immune forces, the development of dysbiosis.

The multifactorial positive effect on the child’s body allows us to consider bifidobacteria as an effective biocorrector and the basis for creating drugs that have a regulatory and stimulating effect on the body.

In Russia, the traditional approach to bacteriotherapy is the administration to patients of drugs based on cultures of bifidobacteria strains with proven probiotic action mainly in the form of monocultures of strains of Bifidobacterium bifidum 1, B. bifidum 791, B. bifidum LVA-3, B.longum B379M, B.adolescentis MC- 42. This is, first of all, the widely used therapeutic drug "Bifidumbacterin" [6].

It should be noted that in the microbiocenosis of the human intestine, bifidobacteria are represented simultaneously by many species - both individual and in associations [7]. This suggests that the introduction of several types of bifidobacteria into the composition of probiotic preparations will help to increase their therapeutic and prophylactic effectiveness.

A similar approach is proposed in the Russian patent [8] concerning a consortium based on bifidobacteria strains for the correction of gastrointestinal microflora in children aged 3 to 12 years old, a biological product based on it and dietary supplement for food.

At the same time, this consortium does not take into account that a rational approach to the design and application of new microflora correction tools should be based on such a phenomenon as autogenous succession - a sequence of changes in biocenoses in a certain ecological niche [9, 10]. During this process, in the species community, successive displacement of some species by others due to their bioecological benefits under certain conditions. In this case, the so-called succession series is formed with the formation of the final - sustainable biocenosis. All biocenoses in wildlife develop according to the laws of succession. As for the microecology of the human body, this pattern can be seen in the change in the species composition of bifidoflora during ontogenesis, which coincides with the critical periods of development in childhood and adolescence: the formation of the immune and enzymatic systems, a change in the hormonal background, and a change in diet. At the same time, the species composition of bifidoflora in adults is relatively stable [11, 12].

Thus, the preparations should be associations of dominant microorganisms, which are as close as possible in composition to the natural microbiocenosis of the human gastrointestinal tract in a certain age period. This is a reasonable approach that ensures the individualization of bacteriotherapy and increases its effectiveness, contributing to the stable localization or new population of the biotope with those types of bifidobacteria that prevail in the microbiocenosis of a particular person.

Currently, various molecular genetic methods have been developed and are widely used to study the taxonomy of representatives of microbiota, including bifidobacteria and lactobacilli, including sequencing, multilocus genotyping, and others, which significantly replenished our ideas about the species diversity of these bacteria and made it possible to reclassify the known and identify new species [13, 14]. This is closely related to the development of new probiotic drugs with immunomodulatory effects, a wide range of properties that determine metabolic and antagonistic activity.

So, the study of the above strains by molecular genetic methods showed a mismatch in the species affiliation of some strains previously identified using biochemical tests. Thus, the B. bifidum 1 strain, widely used in the production of probiotics, turned out to be a natural recombinant consisting of two clones: B. bifidum and B. breve. This information indicates the need for new approaches to the construction of multiprobiotic preparations.

The study of the species composition of bifidoflora in children over 3 years of age based on the amplification and sequencing of fragments of two genes: 16S ribosomal RNA (16S) and transaldolase (tal) showed that the B.infantis species is absent in the microbiocenosis of the gastrointestinal tract of children. The species B. breve is present in the microbiocenosis of this age group, but the data of various authors vary from 17% to 70% [15, 16]. The species B. bifidum and B. longum are predominant in the microbiocenosis of the child [17]. There are observations of a high frequency of isolation of B.catenulatum and B. pseudocatenulatum species - from 72% [18] to 90%. However, according to some authors, these species are associated with atopic eczema in children [19, 20]. Reiter [20] classifies them as a subspecies of B. adolescentis, which is uncharacteristic of the microbiocenosis of children. Such species are not physiological for children and cannot be used as part of microflora correction agents of this age group.

The data presented convincingly show that for the effective correction of dysbiotic disorders of the microbiocenosis of children of this age group, the preparation should include strains with a proven probiotic effect and, in a species ratio, complementary to bifidoflora of children. Of great importance is the inclusion in the composition of the microflora correction means of strains isolated from healthy children in the current period of time, because most production strains have an origin of 50 years or more.

This applies to comprehensively studied and patented strains of bifidobacteria isolated from healthy children: strain B. breve OV-12 [21], strain B. bifidum OV-19 [22], strain B. longum OV-20 [23]. Strain B. breve OV-12 is characterized by higher antagonistic activity to pathogenic and opportunistic microorganisms than strains B. breve 79-88 and B. breve 79-119. RF patent No. 2373274 for strain B. breve OV-12 is marked by a diploma, as one of the 100 best inventions of 2011 under No. 39-Strain B. longum OV-20 is characterized by a wide range of antibiotic resistance. Strain B. bifidum OV-19 has a stable genotype, in contrast to strain B. bifidumi, which is a natural recombinant.

The objective of the present invention is to create a consortium of bifidobacteria in composition as close as possible to the microbiocenosis of children aged 3 to 14 years, as well as the development of a method for its production, taking into account the biocompatibility of the strains during cultivation and further storage. The technical result of the present invention is the fact that the inventive consortium is high-tech, accumulates biomass on nutrient media in a short time with a high concentration of probiotic microorganisms, has acid-forming and antagonistic properties against pathogenic and opportunistic microflora. The synergistic effect of the joint use of strains in the consortium is manifested in an increase in all of these properties compared with individual strains.

The proposed consortium has the following species composition.

Consortium of bifidobacteria for drugs and dietary supplements intended for children from 3 to 14 years old:

1. Bifidobacterium bifidum OV-19 - GKNM MNIIEM them. G.N. Gabrichevsky

No. 216, isolated from the contents of the intestines of a healthy child RU 2375444.

Bifidobacterium B. bifidum 791 - VNIIgenetika in TsMPM No. B-3300, GKNM MNIIEM them. G.N.Gabrichevsky №80, isolated from the intestines of a healthy child.

2. Bifidobacterium longum B379M - All-Russian Research Institute of Genetics at TsMPM No. ..., GKNM MNIIEM named after G.N. Gabričevskogo No. 79, isolated from the contents of the intestines of a healthy person.

3. B.longum OV-20 - GKNM MNIIEM them. G.N. Gabričevskogo No. 214, isolated from the intestines of a healthy child. KP 2375445.

4. Bifidobacterium breve OV-12 - GKNM MNIIEM them. G.N.Gabrichevsky №217, isolated from the intestines of a healthy child. RU 2373274.

The present invention is also the development of a specific scheme for the joint cultivation of individual cultures to obtain a consortium.

The technical result of the proposed cultivation scheme of individual strains to obtain a consortium is the absence of mutual inhibition of strains and the expansion of the spectrum of enzymatic and antagonistic activity.

The method of obtaining the consortium is based on a preliminary study and selection for joint cultivation of strains on the principle of biocompatibility and taking into account the growth kinetics of each of them.

An algorithm for creating a consortium of bifidobacteria is the joint cultivation of the strains included in it, taking into account the duration of the exponential growth phase of each of them, based on which the time point of inoculation of the nutrient medium during the phased introduction of monoculture inocula is determined.

For this, a strictly quantitative ratio of the seed of each strain is selected. The optical standard turbidity set the concentration of OE / ml, corresponding to 1 billion cells of bifidobacteria.

Sowing each of the cultures in the fermenter, 2% of the volume of the culture medium, is carried out in stages, with a frequency of 30 minutes, adjusting the pH of the culture fluid with 20% NaOH before sowing a new strain to 6.8. The seeding sequence depends on the duration of the exponential growth phase of each strain and in this consortium is as follows:

1) B. bifidum OV-19 - 2%,

2) B. bifidum 791 - 2%,

3) B. longum OV-20 - 2%,

4) B. longum B379M - 2%,

5) B. breve OV-12-2%.

The process lasts 10-12 hours; The pH of the final product is 5.1-5.5.

Cultural and morphological signs of the consortium.

Cells represent Gram + polymorphic rods, located primarily in clusters; in the stationary phase of growth, cells with bifurcations at the ends or with club-shaped thickenings are often observed. When grown in a fermenter in an exponential growth phase under microscopy, the culture has the appearance of even sticks arranged in chains or clusters.

In semi-liquid nutrient media, the biomass of the consortium of bifidobacteria grows throughout the entire volume of the nutrient medium, except for the aerobiosis zone. The morphology of the colonies is heterogeneous and depends on the strains included in the consortium, preserving the characteristics of each of them in the total culture.

Physiological and biochemical signs of microorganisms included in the consortium.

Obligatory anaerobes. The optimum temperature of cultivation is 37.5-38 ° C, the pH of the nutrient medium is 7.2-7.4. Catalase is not formed, gelatin is not liquefied, gas formation is absent. Glucose is fermented to form acid without gas formation, acidifying the growth medium to a pH of 4.3-5.1.

Comparative data on the cultivation of individual cultures and their consortium show that during joint cultivation with the proposed scheme for the introduction of seed material, mutual inhibition of cultures does not occur (table 1).

Table 1 Comparative data on the cultivation of monocultures and a consortium of bifibacteria and lactobacilli Name of culture co-cultivation separate cultivation CFU / ml ° T CFU / ml ° T B. bifidum OV-19 1.8 ± 0.2 × 10 ⁹ - 1.9 ± 0.2 × 10 ⁹ 100 ° B. bifidum 791 1.5 ± 0.3 × 10 ⁹ - 1.8 ± 0.1 × 10 ⁹ 100 ° B.longum B379 1.4 ± 0.2 × 10 ⁹ - 1.6 ± 0.3 × 10 ⁹ 120 ° B.longum OV-20 1.5 ± 0.3 × 10 ⁹ - 1.8 ± 0.4 × 10 ⁹ 120 ° B. breve OV-12 1.6 ± 0.4 × 10 ⁹ - 2.0 ± 0.16 × 10 ⁹ 110 ° Consortium 7.8 ± 0.6 × 10 ⁹ 160 ° - -

The consortium is cultivated at 37 ° C with periodic stirring for 8-10 hours; The pH of the culture fluid after the end of the process is 3.9-4.3.

The consortium can be cultivated on various nutrient media with consistently high rates of CFU / ml of probiotic microorganisms and produced organic acids (table 2).

table 2 The results of the cultivation of consortia on different nutrient media

Growing medium Consortium of Bifidobacteria and Lactobacilli CFU / ml ° T Blaurocka 7.4 ± 0.2 × 10 ⁹ 180 ° Hydrolyzed milk 6.5 ± 0.6 × 10 ⁹ 160 ° Culture medium according to the invention 7.1 ± 0.3 × 10 ⁹ 180 °

As can be seen from the results presented in tables 1 and 2, there is no mutual inhibition of cultures. At the same time, there is a tendency to a synergistic effect.

The most promising in the production of drugs and dietary supplements for children is the hydrolyzate-milk environment. Currently, intolerance to cow's milk is widespread among children. This medium is hypoallergenic, since its basis is a milk hydrolyzate, consisting of low molecular weight peptides and amino acids that are easily absorbed by the child's body. The environment, made on the basis of milk hydrolyzate, has the following composition (calculated per 100 l):

Table 3 The composition of the hydrolyzate-milk environment Milk hydrolyzate 50 l Hydrochloric cysteine 20 g Lactose 1000 g Peptone enzymatic 200 g Microbiological agar agar 100 g Sodium chloride 400 g Purified tap water up to 100 l

The environment is easy to prepare, cost-effective; at the same time, as can be seen from the results presented in table 2, inferior to other media in growth properties.

To eliminate this drawback, it additionally included additives that stimulate the growth of probiotic microorganisms - prebiotics inulin and oligofructose and yeast autolysate containing B vitamins necessary for their development and ascorbic acid as an antioxidant. The composition of the environment thus improved is presented in table 4.

Table 4 The composition of the claimed medium (per 100 l): Milk hydrolyzate 50 l Yeast autolysate 500 g Hydrochloric cysteine 20 g Lactose 1000 g Peptone enzymatic 200 g Microbiological agar agar 100 g Sodium chloride 400 g Vitamin C 250 g Inulin 500 g Oligofructose 500 g Purified tap water up to 100 l

When studying the growth properties of the claimed medium, it was shown that they exceed the growth properties of the hydrolyzate-dairy medium, i.e. The desired result has been achieved. The results are presented in table 5.

Table 5 Comparative results of the cultivation of consortia on hydrolyzate-milk and the claimed medium

Growing medium Bifidobacteria Consortium CFU / ml ° T Hydrolyzed milk 7.2 ± 0.6 × 10 ⁹ 140 ° The inventive environment 8.2 ± 0.3 × 10 ⁹ 180 °

Thus, the claimed environment is promising in the production of drugs and dietary supplements for children of this age group.

The synergistic effect, which was noted during cultivation of the consortium, is also manifested in determining the spectrum of biochemical activity.

As an illustration, Table 6 shows how the spectrum of enzymatic activity of the strains expands during co-cultivation — the number of assimilated substrates increases.

The synergistic effect of the consortium is also manifested in the greater antagonistic activity of the consortium compared to monocultures, which increases the probiotic significance of the drug. This effect can be observed in the zones of growth inhibition of test cultures (Table 7).

Table 7 Zones of growth inhibition of test cultures (in mm) in the presence of a consortium and individual strains of bifidobacteria Object of study Sh. sonnel 5063 Sh. flexneri l70 E..coli 157 Staph. aureus 209 Proteus vulgaris 177 B. bifidum OV-19 19 ± 2 16 ± 1 17 ± 3 14 ± 2 18 ± 2 B. bifidum 791 17 ± 3 15 ± 2 15 ± 3 15 ± 1 17 ± 2 B. longum B379M 24 ± 2 20 ± 2 23 ± 2 18 ± 3 26 ± 3 B. longum OV-20 25 ± 2 23 ± 1 25 ± 2 24 ± 3 25 ± 2 B.breve OV-12 29 ± 2 25 ± 2 27 ± 3 25 ± 3 31 ± 1 Bifidobact Consortium 31 ± 1 27 ± 3 29 ± 1 28 ± 2 32 ± 1

As the data presented in table 7 show, the consortium is superior to individual strains in its composition in terms of antagonistic activity against test strains of pathogenic microorganisms.

Thus, the obtained results indicate not only the possibility of co-cultivation of various types of bifidobacteria, but also the synergistic effect of a consortium of strains, which allows us to solve the complex biotechnological problem of obtaining multicomponent probiotic preparations with a specific composition and purpose.

In the proposed consortium, in addition to a specific species composition of bifidobacteria, a certain quantitative ratio of microbial cultures after cultivation is noted. Quantitative accounting of the share of individual strains is made by counting the number of colonies grown in a semi-liquid medium. The culture of each strain has colonies of a certain morphology and size, which allows them to be distinguished. The ratio of individual species of bifidobacteria in the consortium is presented in table 8.

Table 8 The ratio of individual species of bifidobacteria in the consortium No. p / p Microorganism strain CFU / g Mass fraction of microorganisms,% one. B. bifidum OV-19 10 ⁹ -10 ¹⁰ 18.0-20.0 2. B. bifidum 791 10 ⁹ -10 ¹⁰ 20.0-18.0 3. B. longum B379M 10 ⁹ -10 ¹⁰ 14.5-12.0 four. B. longum OV-20 10 ⁹ -10 ¹⁰ 23.5-23.0 5. B. breve OV-12 10 ⁹ -10 ¹⁰ 24.0-27.0

When compiling the formulation of preparations for the correction of microflora, it was necessary to take into account the fact that the most appropriate is the combined use of a consortium of probiotic microorganisms together with prebiotics inulin and oligofructose and apple pectin, which stimulate the growth of both indigenous and microflora, which is part of the drug. In addition, prebiotics, encapsulating microbial cells, protect them during the passage of the gastrointestinal tract from the aggressive effects of the environment. The technical result of this invention is the use of a consortium as an active substance in combination with prebiotics - apple pectin, inulin and oligofructose. The presence of nutrient substrates contributes to the effective colonization of the biotope. Saccharolytic microorganisms, while increasing the production of organic acids, contribute to better absorption of calcium, iron, magnesium. In addition, inulin accelerates the body's cleansing of toxins and undigested food due to stimulation of the contractility of the intestinal wall, a good laxative effect is noted, it has an antitoxic and protective effect. Inulin has an “enveloping effect”, protecting the mucous membranes of the stomach and partially the intestines from mechanical irritation by food. The use of a diet with inulin supplementation inhibits the development of tumors and reduces the amount of ammonia in the intestine, an increased concentration of which promotes the growth of tumors. Inulin improves carbohydrate and lipid metabolism and normalizes blood sugar in patients with diabetes mellitus. Oligofructose has a pronounced choleretic effect, which is enhanced in connection with the facilitation of the outflow of bile from the liver and gall bladder to the duodenum, due to improved bowel movement.

In addition to the effects described, these ingredients have a high sorption capacity. Pectin is capable of forming complexes to remove heavy metals (lead, mercury, zinc, cobalt, molybdenum, etc.) and long-lived (with a half-life of several decades) isotopes of cesium, strontium, yttrium, etc. from the human body. In addition, pectin can absorb and remove biogenic toxins, anabolics, xenobiotics, metabolic products and biologically harmful substances that can accumulate in the body: cholesterol, bile acids, urea, and mast cell products. Of particular note is the multifaceted effect of pectins in dysbiosis. They completely naturally cleanse the intestines, gently envelop the mucous membrane, protect it and contribute to its rapid recovery. In addition, pectins shift the pH of the large intestine to the more acidic side. Pathogenic bacteria in such an environment die, and the beneficial microflora begins, on the contrary, to actively multiply. Experiments comparing the sorption efficiency of enterosorbents of different classes, carried out with lead and copper salts, showed that low-esterified pectins were recognized as the most effective. It is these unique natural substances that are recognized as the most effective for cleansing the body of harmful substances, parasites and their waste products.

Another option for a rational approach to the correction of dysbiotic conditions is the combined use of micronutrients and probiotic microorganisms. When such a complex is introduced into the human body, the disturbed indigenous microflora is replenished. Probiotic microflora increases the bioavailability of micronutrients and reduces the deficiency of vitamins and minerals. This, in turn, should contribute to a more effective restoration of indigenous microflora and metabolic balance in the body.

Examples

4. A consortium of bifidobacteria for bacterial preparations and dietary supplements intended for children from 3 to 14 years old, and a method for producing it

1st passage - lyophilized material of strains B. bifidum OV-19, B. bifidum 791, B. longum B379M, B. longum OV-20, B. breve OV-12 individually rehydrated with physiological saline in a volume of 1 ml and titrated by the method ten-fold dilution in culture medium to 10 ^7-8 . Incubated at a temperature of 37.5-38 ° C for 36 hours

2nd passage - each of the grown cultures from a dilution of 10 ^6-8 in a volume of 5% is seeded in a nutrient semi-liquid medium of the claimed composition (30 ml) and cultivated for 7 hours at 3 7.0-3 8 ° C.

3rd passage - each of the grown cultures is sown in the claimed medium and grown for 16 hours at 37.0-38 ° C. Consortium cultivation.

A consortium of bifidobacteria is obtained by co-culturing the above strains on a nutrient medium containing the following ingredients (per 100 liters):

Milk hydrolyzate - 50 l Yeast Autolysate - 500 g Hydrochloric cysteine - 20 g Lactose - 1000 g Peptone enzymatic - 200 g Agar agar 100 g microbiological - Sodium Chloride - 400 g Vitamin C - 250 g Inulin - 500 g Oligofructose - 500 g Purified tap water -  up to 100 l

For this, a strictly quantitative ratio of the seed of each strain is selected. The optical standard turbidity set the concentration of OE / ml, corresponding to 1 billion cells of bifidobacteria.

Sowing cultures in the fermenter at 2% each to the volume of the sowing medium is carried out sequentially, with a frequency of 30 minutes, adjusting the pH of the culture fluid with 20% NaOH before sowing a new strain to 6.8: 1) B. bifidum OV-19 - 2%, 2 ) B. bifidum 7P7-2%, 3) B. longum OV-20 -2%, 4) B. longum B379M - 2%, 5) B. breve OV-12 - 2%.

The cultivation process is carried out at 37-38 ° C until the pH of the final product is 5.1.

2. Biologically active food supplement to normalize the microflora of children aged 3 to 14 years and the method of its production

1st passage - lyophilized material of strains B. bifidum OV-19, B. bifidum 791, B. longum B379M, B. longum OV-20, B. breve OV-12 individually rehydrated with physiological saline in a volume of 1 ml and titrated by the method ten-fold dilution in culture medium to 10 ^7-8 . Incubated at a temperature of 37.5-38 ° C for 24 hours

2nd passage - each of the grown cultures from a dilution of 10 ^6-8 in a volume of 5% is seeded in a nutrient semi-liquid medium (30 ml) and cultivated for 6-8 hours at 3 7.0-38 ° C. (uterine cultures).

3rd passage - each of the grown cultures is seeded in a nutrient semi-liquid medium and grown for 16 hours at 37.0-38 ° C.

Consortium cultivation.

A consortium of bifidobacteria is obtained by co-culturing the above strains. For this, a strictly quantitative ratio of the seed of each strain is selected according to the developed scheme. The optical standard turbidity set the concentration of OE / ml, corresponding to 1 billion cells of bifidobacteria.

The composition of the growing medium (per 100 l):

Milk hydrolyzate 50 l Yeast autolysate 500 g Hydrochloric cysteine 20 g Lactose 1000 g Peptone enzymatic 200 g Microbiological agar agar 100 g Sodium chloride 400 g Vitamin C 250 g Inulin 500 g Oligofructose 500 g Purified tap water up to 100 l

The cultivation process is 10 hours at 37-38 ° C. To the grown biomass of bifidobacteria, a protective sucrose-gelatin medium and 10% skim milk at 10% by volume of the biomass of bifidobacteria are added and freeze-dried.

The technical result achieved by obtaining a biologically active food supplement using the inventive consortium is a more effective and quick correction of microflora and the functional state of the gastrointestinal tract of children aged 3 to 14 years. After grinding, the biomass of bifidobacteria is mixed with apple pectin, inulin and oligofructose. The mixture is packed in capsules of 0.25 g each and placed in 20-60 pieces in sterile plastic jars with hermetically sealed lids, packed in a pencil case in which the instructions for use are inserted.

The formulation of the drug is presented in table 9 (a, b).

Table 9 (a, b) Raw material consumption per pack (30 capsules) No. p / p Name of raw material (material) Weight, g (qty, pcs.) one. 2,4 2. 1.7 3. 1,6 four. 1.8

Single dose - 0.25 g capsule

name of raw materials Mass in g Bifidobacteria biomass 0.05 Apple Pectin 0.1 Inulin and 0.05 Oligofructose 0.05 Total: 0.25 * CFU / g bifidobacteria content of at least 10 ⁸

Percentage of components:

Bifidobacteria biomass - twenty% Apple Pectin - 40% Inulin - twenty% Oligofructose - twenty%

3. A bacterial drug for the treatment of dysbiotic disorders of the gastrointestinal tract in children from 3 to 14 years and the method of its production

1st passage - lyophilized material of strains B. bifidum OV-19, B. bifidum 791, B. longum B379M, B. longum OV-20, B. breve OV-12 individually rehydrated with physiological saline in a volume of 1 ml and titrated by the method ten-fold dilution in culture medium to 10 ^7-8 . Incubated at a temperature of 37.5-38 ° C for 24 hours

2nd passage - each of the grown cultures from a dilution of 10 ^6-8 in a volume of 5% is seeded in a nutrient semi-liquid medium (30 ml) and cultivated for 6 hours at 37.0-38 ° C (uterine cultures).

3rd passage - each of the grown cultures is seeded in a nutrient semi-liquid medium and grown for 16 hours at 3 7.0-38 ° С.

Consortium cultivation.

A consortium of bifidobacteria is obtained by co-cultivation of the above strains. For this, a strictly quantitative ratio of the seed of each strain is selected according to the developed scheme. The optical standard turbidity set the concentration of OE / ml, corresponding to 1 billion cells of bifidobacteria. The composition of the growing medium (per 100 l):

Milk hydrolyzate 50 l Yeast autolysate 500 g Hydrochloric cysteine 20 g Lactose 1000 g Peptone enzymatic 200 g Microbiological agar agar 100 g Sodium chloride 400 g Vitamin C 250 g Inulin 500 g Oligofructose 500 g Purified tap water up to 100 l

The cultivation process is carried out for 10 hours at 37-38 ° C. A protective sucrose-gelatin medium is added to the grown biomass of bifidobacteria and 10% fat-free milk is 10% by volume of the biomass of bifidobacteria, 5 ml are poured under aseptic conditions into glass penicillin vials and freeze-dried.

The technical result achieved by obtaining a bacterial preparation using the inventive consortium of bifidobacteria is a more effective and quick correction of gastrointestinal disorders in children aged 3 to 14 years.

* The content of CFU / g of bifidobacteria in the finished product is not less than 10 ⁸ .

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21. RF patent No. 2373274 dated 11/20/2009 The strain Bifidobacterium breve OV-12, used to obtain bacterial preparations, biologically active food additives, fermented and non-fermented foods, hygiene and cosmetics. Application No. 2008128539 of July 15, 2008.

22. RF patent No. 2375444 of 12/10/2009 The strain Bifidobacterium bifidum OV-19, used to obtain bacterial preparations, biologically active food additives, fermented and non-fermented foods, hygiene and cosmetics. Application No. 2008128540 of July 15, 2008.

23. RF patent No. 2375445 dated 12/10/2009 The strain Bifidobacterium longum OV-20, used to obtain bacterial preparations, dietary supplements, fermented and non-fermented foods, hygiene and cosmetics. Application No. 2008128542 of July 15, 2008.

Claims (4)

1. A consortium of bifidobacteria Bifidobacterium bifidum OV-19, B. bifidum 791, B. longum B379M, B. longum OV-20, B. breve OV-12 for the preparation of bacterial preparations and biologically active food additives intended for the correction of gastrointestinal microflora the intestinal tract of children aged 3 to 14 years. 2. The method of obtaining the consortium according to claim 1, providing for the joint cultivation of the strains included in it in a nutrient medium, characterized in that each strain is sown in a fermenter at 2% by volume of the culture medium in stages, with a periodicity of 30 minutes, adjusting the pH of the culture liquid 20% NaOH before inoculation of each strain to 6.8, while the sequence of inoculation of each strain depends on the duration of the exponential phase of its growth in a nutrient medium containing the following ingredients per 100 l:
Milk hydrolyzate 50 l Yeast autolysate 500 g Hydrochloric cysteine 20 g Lactose 1000 g Peptone enzymatic 200 g Microbiological agar agar 100 g Sodium chloride 400 g Vitamin C 250 g Inulin 500 g Oligofructose 500 g Purified tap water Up to 100 3. Biologically active food supplement for the correction of the microflora of the gastrointestinal tract of children from 3 to 14 years old, characterized in that it contains dry biomass of the consortium Bifidobacterium bifidum OV-19, B. bifidum 791, B. longum B379M, B. longum OV-20, B. breve OV-12, apple pectin, inulin, oligofructose in the following percentage ratio:
Bifidobacteria biomass twenty% Apple Pectin 40% Inulin twenty% Oligofructose twenty% 4. A bacterial preparation for the treatment of dysbiotic conditions of the gastrointestinal tract of children aged 3 to 14 years, characterized in that it contains dry biomass of the consortium of bifidobacteria Bifidobacterium bifidum OV-19, B. bifidum 791, B. longum B379M, B longum OV-20, B. breve OV-12 with a CFU / g content in the finished product of at least 10 ⁸ .