RU2491335C1 - Bifidobacterial and lactobacillary 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 and bacterial preparation for treating dysbiotic gastrointestinal conditions in children aged from three to fourteen - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: bifidobacterial and lactobacillary consortium contains Bifidobacterium bifidum 19, B. bifidum 791, B. longum B379M, B. longum OV-20, B. breve OV-12, L .helveticus "К3Ш24", L. helveticus NK-1, L. casei KHM-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 of the strains including the length of an exponential growth phase of each of them. Thereby 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 supplements, provides the more effective and fast correction of the child's gastrointestinal microflora.

4 cl, 10 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 the 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 and 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 9 strains of bifidobacteria B.bifidum 791, B.bifidum LVA-3, B.longum B379M, B.longum I-3, B.breve 79-119, B. breve 79-88, B.infantis 73-15, B.infantis 79-43, B.adolescentis G7513.

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.

In addition to bifidobacteria. in probiotic biological products, lactobacilli are widely used. These microorganisms have high antagonistic activity against a wide range of pathogenic and conditionally pathogenic microbes, stimulate nonspecific resistance of the human body, and have a pronounced effect on the enzymatic system. Possessing beta-galactosidase activity, level lactase deficiency, which is especially common in children of the first year of life. The use of the co-cultivation method requires the analysis of interstrain interactions of microorganisms in vitro [13]. Biocompatible strains, i.e. strains whose relationships are regarded as symbiosis.

Co-cultivation of two or more types of lactobacilli increases their manufacturability and expands the range of functional activity.

The experiments on the biocompatibility of various strains made it possible to distinguish the group of lactobacilli, the most promising in relation to co-cultivation. Of greatest interest from this group of lactobacilli are strains of L. helveticus K ₃ W ₂₄ , L. helveticus NK-1 and L.casei KHM-12. They have a pronounced antagonism towards opportunistic microorganisms and resistance to the spectrum of antibacterial agents. In addition, a high degree of biocompatibility of these strains was established, which determined the feasibility of their joint cultivation [14].

Known RF Patent No. 2180348 "Consortium of bifidobacteria and lactobacilli used for the preparation of bacterial preparations, starter cultures for fermented milk products, fermented and non-fermented food products, biologically active additives intended for the correction of microflora of children aged 3 to 12 years" [ fifteen].

This patent proposes a consortium of bifidobacteria and lactobacilli of the following composition: Bifidobacterium bifidum 1, B. bifidum 791, B. breve 79-88, B.infantis 79-43, B.longum B379M, Lactobacillus plantarum 8-PA-3, L.fermentum 90-TC-4, Lactobacillus acidophilus K3 w24.

Describing the RF Patent [15], it should be noted that the disadvantage of the method for producing the consortium claimed in it is that it is obtained by combining inoculum cultures of individual strains of bifidobacteria in equal proportions, followed by the addition of inoculum cultures of lactobacilli cultures in a ratio of 10: 1 and co-cultivation in a nutrient medium for 10-12 hours in a fermenter or 18-20 hours in stationary conditions at a temperature of 37-38 ° C.

Currently, various molecular genetic methods, including sequencing, multilocus genotyping, and others, have been developed and are widely used to study the taxonomy of representatives of microbiota, including bifidobacteria and lactobacilli, which significantly replenished our ideas about the species diversity of these bacteria, made it possible to reclassify the known and identify new species [16, 17]. 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. So, the strain L. fermentum 90-TC-4 showed itself as L. plantarum. Strain B.bifidum 1, 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 different authors vary from 17% to 70% [18, 19]. The species B. bifidum and B. longum are predominant in the microbiocenosis of the child [20]. There are observations of a high frequency of isolation of B.catenulatum and B. pseudocatenulatum species - from 72% [20] to 90% [18]. However, according to some authors, these species are associated with atopic eczema in children [21, 22]. Reiter [23] classifies them as a subspecies of B. adolescentis, which is not characteristic of 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 for strain B. breve OV-12 [24], strain B. bifidum OV-19 [25], strain B. longum OV-20 [26]. 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 with a diploma, which is included in 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. bifidum 1, which is a natural recombinant.

The objective of the present invention is the creation of a consortium of bifidobacteria and lactobacilli, 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 preparation, 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:

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

1. Bifidobacterium bifidum OV-19 - GKNM MNIIEM them. G.N. Gabričevskogo №216, isolated from the intestines of a healthy child. RU 2375444

2. Bifidobacterium B. bifidum 791-VNIIgenetics at TsMPM No. B-3300, GKNM MNIIEM im. G.N.Gabrichevsky №80, isolated from the intestines of a healthy child.

3. Bifidobacterium longum B379M-VNIIgenetics at TsMPM No. ..., GKNM MNIIEM named after G.N. Gabričevskogo №79., Isolated from the intestines of a healthy person.

4. Bifidobacterium longum OV-20 - GKNM MNIIEM them. G.N. Gabričevskogo No. 214, isolated from the contents of the intestines of a healthy person. RU 2375445

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

6. Lactobacillus helveticus K ₃ W ₂₄ - GKNM MNIIEM them. G.N. Gabričevskogo No. 42, isolated from the contents of the intestines of a healthy person, All-Russian Research Institute of Genetics No. VKPM B-3190.

7. Lactobacillus helveticus NK-1 - GKNM MNIIEM them. G.N. Gabričevskogo №54, isolated from the intestines of a healthy person.

8. Lactobacillus casei KHM-12 - GKNM MNIIEM them. G.N. Gabričevskogo No. 12, isolated from a dairy product.

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 and lactobacilli is the joint cultivation of certain strains, taking into account the duration of the exponential growth phase of each of them and the numerical ratio in the intestinal microbiocenosis of bifidobacteria and lactobacilli, on the basis of which the time point of inoculation of the nutrient medium during the phased introduction of monoculture inocula is determined.

The optical standard turbidity set the concentration of OE / ml, corresponding to 1 billion cells of bifidobacteria. Lactobacilli, being microaerophylls, absorbing residual oxygen from the medium, create favorable conditions for the development of bifidobacteria. The optical standard turbidity set the concentration of OE / ml, corresponding to 1 billion cells. When selecting the dose of the inoculum, it is also necessary to take into account the ratio of bifidobacteria and lactobacilli in human microbiocenosis (100: 1) and the higher kinetics of lactobacillus growth.

Sowing each of the cultures of bifidobacteria in the fermenter, 2% by 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 inoculation of the new strain to 6.8. When inoculating lactobacilli% inoculum is 0.1%; pH before sowing should be more acidic - 6.0.

The order of application of the inoculum to obtain this consortium is as follows:

1) Bifidobacterium bifidum OV-19 - 2%;

2) Bifidobacterium bifidum 791 - 2%:

3) Bifidobacterium longum OV-20 - 2%;

4) Bifidobacterium longum B379M - 2%;

5) Bifidobacterium breve OV-12 - 2%;

6) Lactobacillus helveticus K ₃ W ₂₄ - 2%;

7) Lactobacillus casei KHM-12 - 0.1%;

8) Lactobacillus helveticus NK-1 - 0.1%.

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.

Cultural and morphological signs of the consortium.

Cells represent Gram + polymorphic bacilli: bifidobacteria are located mainly in the form of clusters; in the stationary phase of growth, cells with bifurcations at the ends or with club-shaped thickenings are often observed. Lactobacilli - chains of large cells with rounded ends.

When grown in a fermenter in an exponential growth phase under microscopy, the culture has the appearance of even rods of different sizes, arranged in chains or clusters.

In semi-liquid nutrient media, the biomass of the consortium of bifidobacteria and lactobacilli 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.

Bifidobacteria - obligate anaerobes, lactobacilli - microaerophiles. Transparent colonies are formed on an agarized surface. The optimum temperature of cultivation is 37.0-38 ° C, the pH of the culture 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 pH 4.3-5.1.

The results of the cultivation of monocultures of bifidobacteria and lactobacilli and their joint cultivation are presented in 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 1.6 ± 0.1 × 10 ⁹ - 1.7 ± 0.2 × 10 ⁹ 90 ° B. bifidum 791 1.5 ± 0.3 × 10 ⁹ - 1.8 ± 0.1 × 10 ⁹ 100 ° B.longum B379 1.1 ± 0.2 × 10 ⁹ - 1.5 ± 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 1.5 ± 0.3 × 10 ⁹ - 1.8 ± 0.4 × 10 ⁹ 120 ° L. helveticus K ₃ W ₂₄ 1.0 ± 0.1 × 10 ⁹ - 1.1 ± 0.1 × 10 ⁹ 40 ° Lhelveticus NK-1 0.3 ± 0.2 × 10 ⁹ - 0.4 ± 0.1 × 10 ⁹ 180 ° L.casei KHM-12 0.1 ± 0.1 × 10 ⁹ - 0.1 ± 0.1 × 10 ⁹ 170 ° Consortium 8.6 ± 0.2 × 10 ⁹ 210 ° - -

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 7.6 ± 0.6 × 10 ⁹ 200 ° Hydrolyzed milk 6.9 ± 0.2 × 10 ⁹ 190 ° Medium for the cultivation of bifidobacteria and lactobacilli according to the invention 8.2 ± 0.3 × 10 ⁹ 210 °

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 Consortium of Bifidobacteria and Lactobacilli CFU / ml ° T Hydrolyzed milk 7.9 ± 0.2 × 10 ⁹ 180 ° The inventive environment 8.8 ± 0.3 × 10 ⁹ 210 °

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.

Table 6 Enzymatic activity of strains during separate and co-cultivation according to ANAEROtest-23 Active ingredient B.bifdum 791 B.bifdum OV-19 B.longum B 379M B.longum OV-20 B.breve OV-12 L.casei KNM-12 L.helveticus NK-1 L.helveticus K3SCH24 Consortium L-ramnose 0 ± 0 0 0 ± 0 0 + Dulcitol 0 0 0 0 0 0 ± 0 + Erythrol 0 0 0 0 0 0 ± 0 ± D-galactose + + + + + + + + + D-glucose + + + + + + + + + D-fructose + 0 + + + + + + + D-lactose + 0 + + + + + + + D-sucrose 0 0 + 0 + + + + + D-maltose 0 0 + 0 + + 0 + + D-raffinose ± 0 + + ± ± ± ± + L-arabinose ± 0 + ± ± + ± ± + D-xylose ± 0 0 ± 0 ± ± ± ± Salicin 0 0 0 0 0 0 0 + + D-cellobiosis 0 0 0 0 0 + 0 ± + D-mannose 0 0 0 0 0 + + ± + D-trehalose ± 0 0 0 0 + + + +

D-melecitosis 0 0 0 0 0 + + + + Inulin 0 0 0 0 0 0 0 0 0 D-beckons 0 0 0 0 0 0 0 + + D-sorbitol 0 0 + + 0 + 0 ± + Inositol 0 0 0 0 0 0 0 +

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 retardation of test cultures (in mm) in the presence of a consortium and individual strains of bifidobacteria and lactobacilli 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 L. helveticus K ₃ W ₂₄ 18 + 1 19 + 3 20 ± 1 27 ± 2 21 ± 2 L.acidophilus NK-1 27 ± 4 29 ± 1 29 ± 3 22 ± 1 30 ± 2 L.casei KHM-12 28 ± 2 25 ± 4 31 ± 3 30 ± 3 31 ± 1 Consortium of bifidobacteria and lactobacilli 34 ± 2 31 ± 2 33 ± 3 31 ± 2 35 ± 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 and lactobacilli, 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 and lactobacilli, 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 and lactobacilli in the consortium is presented in table 8.

Table 8 The ratio of individual species of bifidobactria and lactobacilli in the consortium No. 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 6. L. helveticus K ₃ W ₂₄ 10 ⁸ -10 ⁹ 3.0-3.5 7. L. helveticus NK-1 10 ⁸ -10 ⁹ 3.0-3.5 8. L. casei KNM-12 10 ⁸ -10 ⁹ 3.0-3.5

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, 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 - 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 cleansing of the body from toxins and undigested food due to stimulation of the contractility of the intestinal wall, there is a good laxative effect, 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.

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

1. A consortium of bifidobacteria and lactobacilli for the normalization of the microflora of the gastrointestinal tract of children from 3 to 14 years and the method for its preparation

1st passage - lyophilized material of strains B. bifidum OV-19, B. bifidum 791, B. longum B379M, B. longum OV-20, B. breve OV-12, L. helveticus K ₃ W ₂₄ , L.casei KHM-12, L.helveticus NK-1 are separately rehydrated with physiological saline in a volume of 1 ml and titrated by ten-fold dilution in nutrient medium to 10 ^7-8 . Incubated at a temperature of 37-38 ° C for 36 hours

2nd passage - each of the grown cultures from a dilution of 10 ^6-8 in a volume of 3% is introduced into the accumulation medium of the claimed composition (30 ml) and cultivated for 7 hours at 37-38 ° C.

3rd passage - each of the grown cultures is seeded in the claimed semi-liquid medium and grown for 17 hours at 37-38 ° C.

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 cultivation process is 9 hours at 37-38 ° C.

The composition of the growing medium (per 100):

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

2. Biologically active food supplement for children from 3 to 14 years and the method of its production

Supplements are made on the basis of a consortium of dry biomass of bifidobacteria and lactobacilli with the addition of functionally significant ingredients.

Consortium cultivation.

1st passage - lyophilized material of strains B. bifidum OV-19, B. bifidum 791, B. longum B379M, B. longum OV-20, B. breve OV-12, L.helveticus K ₃ W ₂₄ , L.casei KHM-12, L.helveticus NK-1 are separately rehydrated with physiological saline in a volume of 1 ml and titrated by ten-fold dilution in a nutrient medium to 10. Incubate at a temperature of 37-38 ° С for 36 hours.

2nd passage - each of the grown cultures from a dilution of 10 ^6-8 in a volume of 3% is introduced into the accumulation medium (30 ml) and cultivated for 7 hours at 37-38 ° C.

3rd passage - each of the grown cultures is seeded in a nutrient semi-liquid medium and grown for 17 hours at 37-38 ° C.

A consortium of bifidobacteria and lactobacilli is obtained by co-cultivation of the above strains. For this, a strictly quantitative ratio of seed of each strain is selected in accordance. The optical standard turbidity set the concentration of OE / ml, corresponding to 1 billion cells of bifidobacteria. Sowing crops in the fermenter is carried out in stages, with a frequency of 30 minutes according to the developed scheme, adjusting the pH of the culture fluid with 20% NaOH before sowing a new strain to 6.0. The cultivation process is 8 hours at 37-38 ° C.

A protective sucrose-gelatinous medium and skimmed 10% milk of 10% by volume of the biomass of bifidobacteria are added to the grown biomass and freeze-dried.

The technical result achieved by obtaining a biologically active additive using the inventive consortium is a more effective and quick correction of the microflora of the gastrointestinal tract of children.

After grinding, the biomass is mixed with vitamin-mineral premix, inulin, oligofructose and packaged in 0.25 g capsules, 20-60 pieces in sterile plastic jars with hermetically sealed lids. The can is put in a cardboard case along with instructions for use.

The formulation of biologically active additives are presented in table 9 (a, b).

a) Formulation of dietary supplements for food for children under 3 years of age (composition of 1 package 25

Table 9 No. Name of raw material (material) Weight, g (qty, pcs.) one. Dry biomass of a consortium of bifidobacteria and lactobacilli 4,5 2. In itamine-mineral premix 1,5 3. Inulin 3.0 four. Oligofructose 3.0

b) The ratio of parts of raw materials by weight in one spoon (0.5 g)

name of raw materials Weight in kg Dry biomass of a consortium of bifidobacteria and lactobacilli 0.1 Inulin 0.05 Oligofructose 0.05 Vitamin and Mineral Premix 0.05 Total: 0.5 g

Percentage of ingredients in 1 capsule:

Biomass of a consortium of bifidobacteria and lactobacilli - 40% Inulin - twenty% Oligofructose - twenty% Vitamin and Mineral Premix - twenty%

Table 10 The composition of the vitamin-mineral premix in terms of one dose (0.5 g) of dietary supplement: component The content of vitamins and minerals in the finished dietary supplement (mg in Daily intake (mg, in 2 spoonfuls) % of recommended daily intake of children

1 spoon-dose) 1-3 years Vitamin E 1.65 3.3 66 Vitamin B 1 275 mcg 550 mcg 69 Riboflavin 0.31 0.62 69 Vitamin B6 0.36 0.72 80 Vitamin B12 195 mcg 390 mcg 39 Folic acid 51.5 mcg 130 mcg 130 Pantothenic acid 1.71 3.42 171 Niacinamide 2.97 5.94 59 Biotin 25 mcg 50 mcg 625 Vitamin C 11.7 23,4 52 Zinc 2.25 4,5 45 Selenium 8.5 mcg 17 mcg 31 ** CustoMix IMMUNITY Vitamin and Mineral Premix is manufactured by DSM Nutritional Products Europe Ltd (CH - 4002 Basel), for use in the food industry to enrich food products. Certificate of state registration No. 77.99.11.9. U.2471.9.04 of September 7, 2004

3. A bacterial preparation for the treatment of gastrointestinal dysbiosis in children from 3 to 14 years of age 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, L...helveticus K ₃ W ₂₄ , L.casei KHM-12, L.helveticus NK-1 are separately rehydrated with physiological saline in a volume of 1 ml and titrated by ten-fold dilution in nutrient medium to 10 ^7-8 . Incubated at a temperature of 37-38 ° C for 24 hours

2nd passage - each of the grown cultures from a dilution of 10 ^6-8 in a volume of 3% is introduced into the inventive accumulation medium (30 ml) and cultivated for 7 hours at 37-38 ° C.

3rd passage - each of the grown cultures is seeded in the claimed semi-liquid medium and grown for 17 hours at 37-38 ° C.

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 cultivation process is carried out for 10 hours at 37-38 ° C. The composition of the growing medium (per 100):

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. Protective sucrose-gelatin medium and 10% skim milk at 10% by volume of biomass are added to the grown complex biomass of bifidobacteria and lactobacilli, 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 as an active substance is a more effective and quick correction of dysbiotic disorders of the gastrointestinal tract of children aged 3 to 14 years.

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

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Claims (4)

1. Consortium of bifidobacteria and lactobacilli Bifidobacterium bifidum OV-19, B. bifidum 791, B. longum B379M, B. longum OV-20, B. breve OV-12, L.helveticus K ₃ W ₂₄ , L.helveticus NK-1 , L.casei KHM-12, used for the preparation of bacterial preparations and dietary supplements designed to correct the microflora 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 of bifidobacteria is sown in a fermenter at 2% of the volume of culture medium in stages with a periodicity of 30 minutes, adjusting the pH the culture fluid of 20% NaOH before inoculation of each strain to 6.8, and when sowing strains of lactobacilli, the percentage of each strain in the fermenter spend 0.1% of the volume of the culture medium, adjusting the pH of the culture fluid d sowing of each strain of lactobacilli to 6.0, while the seeding priority of each strain is set depending 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 l 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 Bifldobacterium bifidum OV-19, B. bifidum 791, B. longum B379M, B. longum OV-20, B. breve OV-12, L.helveticus K ₃ W ₂₄ , L.helveticus NK-1, L.casei KHM-12, inulin, oligofructose, vitamin-mineral premix in the following percentage ratio:
Biomass of a consortium of bifidobacteria and lactobacilli 40% Inulin twenty% Oligofructose twenty% Vitamin and Mineral Premix 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 Bifldobacterium bifidum OV-19, B. bifidum 791, B. longum B379M, B. longum OV-20, B.breve OV-12, L.helveticus K ₃ W ₂₄ , L.helveticus NK-1, L. casei KHM-12 with a CFU / g content of at least 10 ⁸ .