RU2317089C2 - Complex preparation-probiotic in immobilized and lyophilized form and method for its obtaining - Google Patents

Abstract

FIELD: medicine, veterinary science, agriculture, biotechnology.

SUBSTANCE: the present innovation refers to preparations of medicinal and veterinary indication and methods for their obtaining. The suggested complex preparation-probiotic includes a carrier being a porous aluminum oxide-based enterosorbent SUMS-1 and cells of bacteria-eubiotics with nutritive and protective media immobilized upon the mentioned carrier-enterosorbent. The cells of bacteria-eubiotics with nutritive medium are being a lyophilized concentrate of the consortium of either bifidobacteria or lactobacteria, or their mixture at titer being 10⁸-10¹⁰ CFU/g, and as a porous carbon-modified enterosorbent SUMS-1 it contains enterosorbent SUMS-1 additionally treated with the solution of metal salt at the following ratio of the preparation components in lyophilized form, weight%: cells with nutritive medium of the consortium of bifido-, or lactobacteria-eubiotics, or their mixture with titer of 10⁸-10¹⁰ CFU/g 1.0-10.0; protective medium 0.1-10.0; enterosorbent SUMS-1 additionally treated with the solution of metal salt - the rest up to 100%. Method for obtaining the suggested preparation deals with treating a porous enterosorbent SUMS-1 with the solution of metal salt at volumetric ratio being 1:1, not more and its washing against a reagent with 2-3-fold volumes of distilled water followed by mixing enetrosorbent and bacteria-eubiotics with nutritive and protective media at the ratio of 1:1, not more to keep the mixture till complete immobilization of bacterial cells onto enterosorbent with its subsequent freezing and freeze drying before sterilization. As bacteria one should apply the concentrate of the consortium of bifidobacteria, or lactobacteria, or their mixture at titer being 10⁸-10¹⁰ CFU/g. The suggested complex preparation-probiotic provides higher efficiency of sorption and desorption of bacterial cells with enterosorbent SUMS-1 that shows its clinical efficiency and higher colonization activity of bacteria-eubiotics.

EFFECT: higher efficiency.

9 cl, 5 ex, 2 tbl

Description

The invention relates to drugs for medical and veterinary use and methods for their preparation and can be used in biotechnology, medicine and agriculture.

Enterosorbents are widely used for the prevention and treatment of many pathological conditions caused by a violation of endoecology. These are various types of activated carbons, for example carbactin, carbolong, microsorb, polyphepan, etc. (Senov P.L. Pharmaceutical chemistry. - M .: Medicine, 1978, p.132-133; RF patent N2029546, IPC ⁶ A61K 9/16, publ. 02.27.95), natural materials and their analogues (crystalline microcellulose, zeolites, chitosans, pectins, etc.), synthetic mineral sorbents and other materials that are used to cleanse the body of toxins, radionuclides, heavy metals and toxins. Enterosorbents reduce the effects of exo- and endotoxicoses.

However, enterosorbents do not eliminate the causes of the effects of exo- and endotoxicosis, as a rule, they are not selective, work mainly on excess substrate, can have a negative effect due to sorption of metabolites (vitamins, hormones) necessary for the body and do not actively normalize microbiocenosis .

The functions of normal microflora in the body are vital and very extensive: synthesizing, regulatory, desensitizing, detoxifying, enzymatic, protective and immunostimulating. To normalize microbiocenosis, widespread preparations of live bacterial cells: bifidobacteria (international application (WO) No. 85/03848), MKI A23C 9/12, publ. 09/12/85), lactobacilli (application EPO No. 0154614, MKI A23C 9/123, publ. 09/11/85), colibacteria, etc.

The disadvantages of these drugs are their rather short shelf life if they are in liquid form, or their high cost if they are made in dry form, as well as poor resistance to inactivating factors of the external environment and the gastrointestinal tract of the body when used.

A complex preparation is known, including a porous inorganic carrier and microorganism cells immobilized on it (application of Germany No. 3410650, MKI C12N 11/14, publ. 03.10.85). As carriers, stalactite substrates with a double pore structure are used. The substrates have through macropores, which make possible the free exchange of liquid and gas from the inside of the carrier into the environment. The size of the macropores lies within the cell size of the microorganisms.

The disadvantage of this drug is that it is not intended for use as a medical or veterinary drug due to the lack of data on the effect of a stalactite carrier with microorganisms deposited on it on the microbiocenosis of the gastrointestinal tract of a human or animal organism.

A complex pharmaceutical preparation is known, including a pharmaceutically acceptable carrier and microorganisms immobilized on it (international application (WO) No. 91/09608, MKI AK61K 35/74, publ. 11.07.91). The carrier does not violate the viability of microorganisms. As microorganisms, the strain L1a Streptococcus lactis is used. The drug is intended to combat pathogenic microflora of the intestines of humans and animals, causing diarrhea and other gastrointestinal disorders.

The disadvantage of the drug is that the strain L1a Streptococcus lactis used in it mainly affects the pathogenic microflora of the intestines of humans and animals, i.e. exhibits antagonistic properties, but is poorly involved in the restoration of the microflora of the gastrointestinal tract. In addition, there is no data on the antacid properties of the carrier, which indicates a weak carrier protection of immobilized Streptococcus lactis cells under the action of the internal environment of the body with low pH values.

A complex bacterial preparation is known, including a carrier and microbial cells immobilized on it in a concentration of 1 mm ² 2.1 × 10 ³ -1.0 × 10 ⁵ (RF patent No. 2017486, IPC ⁶ A61K 31/00, publ. 15.08.94 g.). Activated carbon in the form of a powder with a particle size of less than 30 microns is used as a carrier, and lactobacilli and Escherichia coli are used as microorganisms.

The disadvantage of this drug is that the activated carbon used as a carrier is a finely porous sorbent with a predominance of micropores and a high specific surface area. As a result, microbial cells are located on the surface of the sorbent particles and are not adequately protected under adverse environmental influences. Activated carbons actively absorb low molecular weight biologically active substances, vitamins and intestinal gases, which impairs peristalsis. They quickly become clogged and work mainly in the upper parts of the gastrointestinal tract. In addition, coals do not have antacid properties, which does not contribute to the survival of cells deposited on them under the influence of media with low pH values. The drug does not provide for the simultaneous use of several strains of microorganisms of different types, for example a mixture of bifidobacteria and lactobacilli, which significantly reduces the effectiveness of the drug, especially when restoring the microflora of the gastrointestinal tract.

A known probiotic preparation in immobilized form, comprising a carrier, which is a sorbent, and eubiotics cells immobilized on the carrier, the cells of eubiotics used in conjunction with a nutrient medium, and as a sorbent use a material with antacid properties, developed meso- and macroporous structure and macropore volume of at least 0.01 cm ³ / g in the form of powder with particle size of not more than 0.1 mm, or in the form of granules of (0.1-5.0) mm or as tablets with the following quantitative ratio of components etc. Paratov wt. %: cells of eubiotics with a nutrient medium and a titer of 10 ⁸ -10 ⁸ CFU / ml - 1.0-50.0; carrier sorbent - the rest is up to 100%.

As the sorbent, a porous alumina-based material with hydrophilic-hydrophobic surface topochemistry is used, for example, carbon-modified SUMS-1 enterosorbent or SIAL type modified with an organosilicon polymer type enterosorbent. As eubiotics, bifidobacteria or lactobacilli or a mixture thereof are used (RF patent No. 21118535, IPC ⁶ A61K 35/74, publ. 09/10/98).

However, the sorbents used in this preparation have insufficient efficiency of sorption and desorption of eubiotics bacteria, and also have low colonization activity of the eubiotics bacteria that are part of the preparation.

A known method of obtaining a comprehensive preparation of bacteria-eubiotics (RF patent No. 2067114, IPC АКК 35/74, publ. 09/27/96), including growing in deep cultivation cultures of strains of bifidobacteria and streptococcus, mixing biomass with a protective medium of the following composition (wt. .%): sucrose 25-30; gelatin 2-3; milk powder 3-5; monosodium glutamate 0.5-1 and contact-sorption dehydration of the target product, which is carried out chilled to minus 8-10 ° C with a sorbent, for example aluminum oxide, with a residual moisture content of less than 1% at a mass ratio of 1: 10-12, respectively, and drying carried out for 18-20 hours at a temperature of 0-5 ° C in a closed volume.

However, the sorbent used in this method has insufficient efficiency of sorption and desorption of eubiotics, and also has low colonization activity of eubiotics.

The closest technical solution (prototype) is a probiotic preparation in an immobilized and lyophilized form (RF patent No. 2164801, IPC АКК 35/74, publ. 10.04.01). The preparation includes a carrier, which is a sorbent, and probiotic cells immobilized on the indicated carrier, the probiotic cells being used in conjunction with a nutrient medium, and a material with antacid properties, a developed meso- and macroporous structure and a macropore volume of at least 0.01, is used as a sorbent cm ³ / g in the form of a powder with a particle size of not more than 0.1 mm, or in the form of granules with a size of (0.1-5.0) mm, or in the form of tablets, according to the invention, the preparation additionally contains a protective medium, and the cells of probiotics with feeder second medium are dry concentrate of bifidobacteria based on milk with a titer of 10 ⁸ to 10 ¹⁰ CFU / g and the amino acid content (mg / 100 g dry biomass): glutamic acid - 15,0-68,0; glutamine - 8.0-20.0; leucine - 10.0-50.0; arginine - 20.0-30.0; cysteine - 50.0-80.0 in the following ratio of the components of the drug in dry form wt.%: cells of eubiotics with a nutrient medium and a titer of 10 ⁸ -10 ¹⁰ CFU / g - 0.1-10.0; protective environment - 0.1-10.0; carrier sorbent - the rest is up to 100%. Gelatin, sucrose, ascorbic acid and sodium chloride are used as a protective medium in the following components, wt.%: Gelatin - 8.5-11.5; ascorbic acid - 0.8-1.2; sodium chloride - 1.7-3.0; sucrose - the rest is up to 100%. As the sorbent, a porous alumina-based material with hydrophilic-hydrophobic topochemistry is used, for example, carbon-modified SUMS-1 enterosorbent. The drug as probiotic bacteria additionally contains dry sourdough of viscous strains of acidophilic bacteria Lactobacillus acidophilus type AB, the ratio of which with a dry concentrate of bifidobacteria is 1: (8-9), respectively. The bacteria Bifidum bifidum No. 1, or No. 791, or LVA-3, or Bifidobacterium longum 379, or Bifidobacterium adolescentis MC-42, or a mixture of bacterial cells of these strains taken in equal weight fractions are used as bifidobacteria in a dry concentrate.

The closest method (prototype) is the method of obtaining a complex preparation of probiotic in immobilized and lyophilized form, described in the description of the patent of the Russian Federation No. 2164801, IPC A61K 35/74, publ. 04/10/2011, including washing and sterilizing the porous carbon-modified SUMS-1 enterosorbent and cooling it to room temperature, mixing the enterosorbent and eubiotics with nutrient and protective media in a ratio of not more than 1: 1 and keeping the mixture for at least 1 hours with stirring and a temperature of + 4 ° C until the bacterial cells are completely immobilized onto enterosorbent, washing the resulting complex preparation in isotonic saline solution, followed by freezing and freeze drying.

However, the sorbents used in the indicated preparation and the method for its preparation also have insufficient efficiency of sorption and desorption of eubiotics bacteria. In addition, there is a low colonization activity of the eubiotics bacteria that make up the preparation.

The technical result of the claimed technical solutions is the creation of such a comprehensive probiotic preparation with a method for its preparation, which would provide higher efficiency of sorption and desorption of bacterial cells by the SUMS-1 enterosorbent, which determines its clinical effectiveness, and higher colonization activity of eubiotics.

The specified technical result is achieved in that in a complex probiotic preparation in an immobilized and lyophilized form, including a carrier, which is a porous enterosorbent SUMS-1 based on aluminum oxide, modified with carbon with hydrophilic-hydrophobic topochemistry of the surface, and cells of eubiotics bacteria with nutrient and protective medium immobilized on the specified carrier-enterosorbent, according to the invention, the cells of bacteria-eubiotics with a nutrient medium are lyophilized concentrate of a consortium of bifidobacteria, or lactobacilli, or a mixture thereof with a titer of 10 ⁸ -10 ¹⁰ CFU / g, and as a porous carbon-modified enterosorbent SUMS-1, it contains enterosorbent SUMS-1, additionally treated with a metal salt solution in accordance with the method according to .p.5 and 6 of the claims in the following ratio of the components of the drug in lyophilized form, wt.%:

cells with a nutrient medium of the consortium bifido-, or lactobacilli-eubiotics, or a mixture thereof with a titer of 10 ⁸ -10 ¹⁰ CFU / g 1.0-10.0 protective environment 0.1-10.0 SUMS-1 enterosorbent, additionally processed metal salt solution the rest is up to 100%.

A preparation with a microbial biomass content of less than 0.1 wt.% Has a low biotiter with a predominance of cells firmly bound to the sorbent, i.e. acts primarily as an enterosorbent. The number of microbial cells introduced into the preparation does not exceed 15 wt.%, As a result of which the entire biomass of the bacteria of the preparation is in a protected state. At the same time, the SUMS-1 enterosorbent in the preparation retains its basic properties, which ensure the binding and elimination of toxins, products of incomplete metabolism and pathogenic microorganisms from the intestine.

As a consortium of bifidobacteria, it contains strains of bifidobacteria: Bifidobacterium bifidum 8-3, Bifidobacterium longum TWA-13 and Bifidobacterium adolescentis GO-13 in a ratio of 8: 1: 1.

As a consortium of lactobacilli, it contains strains of lactobacilli Lactobacillus acidophilus 57S, Lactobacillus plantarum P-75, Lactobacillus casei C6 in a ratio of 4: 1.0: 1.0.

A mixture of consortia of bifidobacteria and lactobacilli has a ratio of 1: 1 to 3: 1.

The specified technical result is also achieved by the fact that in the method for producing a complex probiotic preparation in an immobilized and lyophilized form, including washing and sterilizing the porous carbon-modified SUMS-1 enterosorbent and cooling it to room temperature, mixing the enterosorbent and eubiotics bacteria with nutrient and protective media in a ratio of not more than 1: 1 and maintaining the mixture for at least 1 hour with stirring and at a temperature not exceeding + 4 ° C until the bacterial cells are completely immobilized to enterosorbent, washing the resulting complex preparation in isotonic saline followed by freezing and freeze-drying, according to the invention, before sterilization, the SUMS-1 enterosorbent is treated with a metal salt solution in a volume ratio of no more than 1: 1 and washed with a 2-3-fold volume from the reagent distilled water, and as bacteria-eubiotics use a concentrate of a consortium of bifidobacteria, or lactobacilli, or a mixture of them with a titer of 10 ⁸ -10 ¹⁰ CFU / g

As a solution of a metal salt using a 3% solution of magnesium sulfate.

Strains of bifidobacteria are used as a consortium of bifidobacteria: Bifidobacterium bifidum 8-3, Bifidobacterium longum TWA-13, Bifidobacterium adolescentis GO-13 in a ratio of 8: 1: 1, and Lactobacus bacterium Lactobacillus bacteria Lactobacillus 57 and Lactobacillus casei C6 in a ratio of 4: 1.0: 1.0. A mixture of consortia of bifidobacteria and lactobacilli has a ratio of 1: 1 to 3: 1.

The strain of bifidobacteria: Bifidobacterium bifidum 8-3 was deposited at the All-Russian Research Institute of Genetics at VKPM under No. B 5799. The strain Bifidobacterium longum TWA-13 was deposited at the All-Russian Research Institute of Genetics at No. B 5894. The strain Bifidobacterium adolescentis no.

The strain of lactobacilli Lactobacillus acidophilus 57S was deposited at the All-Russian Research Institute of Genetics at VKPM under No. 5863. The strain Lactobacillus plantarum P-75 was deposited at the All-Russian Research Institute of Genetics at No. 3962. The strain Lactobacillus casei C6 was deposited at All-Russian Scientific Research Institute of Genetics at no.

Introduction to the preparation of dry microbial biomass with a biotiter of 10 ⁸ -10 ¹⁰ CFU / g provides him with optimal biological activity. If the initial microbial biomass has a titer of less than 10 ⁸ CFU / g, the preparation has insufficient biological activity and, when introduced into the gastrointestinal tract, exhibits a weak therapeutic effect. A titer of dry microbial biomass of more than 10 ¹⁰ CFU / ml significantly increases the cost of the drug without a significant increase in the therapeutic effect.

The basis of the liquid bacterial suspension for drying is eubiotics cells in a milk nutrient medium with an increased titer of cells (10 ⁹ -10 ¹⁰ CFU / ml) - a liquid concentrate of bifidobacteria. Milk culture medium with eubiotics cells contains an increased amount of protein (at least 12% COM - dry milk residue). This allows you to increase the antibiotic properties of probiotic bacteria in the drug. In addition, an increased amount of protein increases the antacid properties of the drug. A high concentration of cells in a liquid bacterial suspension allows their use in the preparation in a dry form at a concentration of 1.0-10.0% with a titer of 10 ⁸ -10 ¹⁰ CFU / ml.

The sorbent carrier is mechanically strong powder particles, or granules, or tablets made of alumina, the surface of which is modified with carbon upon receipt of a black SUMS-1 sorbent. Sorbents have a specific surface area of up to 300 m ² / g and a developed structure of meso and macropores. The sizes of the macropores of the sorbent are comparable with the sizes of microbial cells, which allows filling the macropores with the indicated cells. By controlling the percentage of carbon in the sorbent, followed by its redox treatments, a carrier is obtained with desired hydrophilic-hydrophobic and other topochemical characteristics. Sorbents of the SUMS-1 type, to a lesser extent than other similar materials, extract vitamins and hormones from the body and do not violate peristalsis, which makes them possible to use for long courses. The presence of alumina in the sorbent gives its surface certain buffered antacid properties, which contributes to better preservation of activity and survival of microbial cells immobilized on it, for example, when the drug passes through the stomach (eliminates the negative effect of gastric juice on microbial cells) or under other adverse conditions (low pH).

The macroporous structure of the sorbent carrier, the cells of which are filled with microbial cells, also helps to protect these immobilized cells from inactivating environmental factors. Moreover, microbial cells located in the pores and cells located close to the outer surface of the sorbent carrier differ in their desorption abilities, and such heterogeneity of the cell properties ensures a prolonged action of the drug and promotes the colonization of not only the upper, but also the lower intestines. The porous carrier, as an enterosorbent, removes the effects of local (and through it general) toxicosis, which contributes to the survival of the drug bacteria and intestinal microflora, and also reduces the burden on the detoxification organs of a person or animal. In addition, when using a consortium of bifidobacteria or a consortium of lactobacilli, or a mixture of consortia of bifidobacteria and lactobacilli as microbial cells in the preparation, not only displacement of pathogenic microorganisms from the gastrointestinal tract is ensured (due to the antagonistic properties of these bacteria), but also the restoration of greatly reduced intestinal microflora with dysbiosis and other diseases.

The following are examples of the compositions of the proposed complex preparation of eubiotics bacteria in immobilized and lyophilized form.

Example 1. The probiotic preparation "Ecoflor" in the form of a powder with a particle size (40-100) microns (wt.%):

cells with lactobacilli consortium nutrient medium with a titer of 10 ¹⁰ CFU / g 1,0 protective environment, including: gelatin 0,085 vitamin C 0.008 sodium chloride 0.017 sucrose 0.89 SUMS-1 enterosorbent, additionally processed 3% solution of magnesium sulfate the rest is up to 100%.

Example 2. The probiotic preparation "Ecoflor" in the form of a powder with a particle size (40-100) microns (wt.%):

cells with a nutrient medium of a consortium of bifidobacteria with a titer of 10 ⁹ CFU / g 7.0 protective environment, including: gelatin 0.85 vitamin C 0.08 sodium chloride 0.17 sucrose 8.9 SUMS-1 enterosorbent, additionally processed 3% solution of magnesium sulfate the rest is up to 100%.

Example 3. The probiotic preparation "Ecoflor" in the form of granules with a size of (0.5-5.0) mm (wt.%):

cells with culture medium and titer of 10 ⁸ CFU / g of a mixture of consortia bifidobacteria and lactobacilli in 1: 1 ratio 10.0 protective environment, including: gelatin 0.575 vitamin C 0.06 sodium chloride 0.15 sucrose 4,215 SUMS-1 enterosorbent, additionally processed 3% solution of magnesium sulfate the rest is up to 100%.

Example 4. The probiotic preparation "Ecoflor" in the form of granules with a size of (0.5-5.0) mm (wt.%):

cells with culture medium and titer 10 ⁹ CFU / g mixtures of consortia of bifidobacteria and lactobacilli in 3: 1 ratio 5,0 protective environment, including: gelatin 0.85 vitamin C 0.08 sodium chloride 0.17 sucrose 8.9 SUMS-1 enterosorbent, additionally processed 3% solution of magnesium sulfate the rest is up to 100%.

Example 5. The technology of obtaining the finished form of the drug "Ecoflor".

Using a special technology, initial SUMS-1 sorbents are prepared (RF patent No. 2026733), in which alumina is modified with carbon, with the required porous structure, chemical surface nature and particle or granule size. Next, a measured amount of SUMS-1 enterosorbent is poured into a cylindrical container, filled with distilled water in a volume ratio of 1: 1 and mixed thoroughly. Wash at least 3 times with distilled water until the carbon film and suspension disappear completely. Next, the SUMS-1 enterosorbent is treated with a solution of a metal salt, namely a 3% solution of magnesium sulfate in a volume ratio of 1: 1. Wash off sulfate ions with a 2-3-fold volume of distilled water. After treating the SUMS-1 enterosorbent with a 3% solution of magnesium sulfate, its biosorption efficiency increases by 1.4 times, and the biodesorption efficiency, which determines the clinical effect of the drug, is 1.

Sterilization of SUMS-1 is carried out by dry heat in an oven in bulk with a layer of not more than 1.5-2.0 cm at a temperature of (160 ± 2) ° C for 2 hours. Sterility control SUMS-1 is determined microbiologically. The control of the sterilization process SUMS-1 is carried out using chemical tests.

The process of immobilization of SUMS-1 is carried out under aseptic conditions. For immobilization, take equal in volume (1: 1) amounts of SUMS-1 enterosorbent and culture of microorganism consortia. For immobilization use a consortium of bifidobacteria (with a titer of at least 10 ¹⁰ CFU / ml), or a consortium of lactobacilli (with a titer of at least 10 ⁸ CFU / ml), or their mixture in a volume ratio of 9: 1, respectively, in accordance with examples 1-3. Bacterial biomass is prepared on a milk nutrient medium containing an increased amount of protein (at least 12% COM - the dry residue of milk) according to standard technology.

A mixture of SUMS-1 enterosorbent with cells of eubiotics bacteria is thoroughly mixed for 5 minutes and placed on a shaker for 1 hour at an intensity of 100 vibrations per minute at a temperature of 6 ± 2 ° С. The process of immobilization can be carried out in glassware on a roller installation. To determine the quality of immobilization from the initial titer of bifidobacteria and lactobacilli in the concentrate of bifidobacteria and lactobacilli, the obtained biotitration result is subtracted. The process is considered completed if no more than 50% of the initial amount of bifidobacteria and lactobacilli remains in the solution.

Next, the product is washed once in an isotonic NaCl solution. The turbid solution is sterilely drained, samples are taken and the completeness of the immobilization process is monitored in accordance with MUK 4.2.577-96 (Methods of microbiological control of baby food, therapeutic nutrition and their components ", clause 7.1 and clause 7.10). The process is considered complete if in solution no more than 50% of the initial amount of bifidobacteria and lactobacilli remained.

The washed product is frozen in a Kelvinator type low-temperature refrigerator at a temperature of minus (40-45) ° С for (24-48) hours. Cassettes with pre-frozen preparation are loaded into the drying chamber when the temperature of the plates reaches minus (20-30) ° C and the chamber is evacuated. The finished preparation "Ecoflor" in dry immobilized form is Packed in bottles and hermetically closed with corks. Mass fraction of moisture is not more than 3.0 wt.%.

Table 1 shows the adsorption capacities of SUMS-1 after surface modification (measured by adsorption of methylene blue according to FS 42-3283-96).

Table 1
Data on the adsorption capacity of the SUMS-1 enterosorbent after treatment with metal salts No. p / p Name of salts Adsorption capacity sorbent pH one SUMS-1 clean 0.017 ± 0.002 g 8.2 2 SUMS-1 + AlCl ₃ (0.36%) 0.018 ± 0.002 g 6.1 3 SUMS-1 + AlCl ₃ (0.75%) 0.018 ± 0.002 g 5.8 four SUMS-1 + AlCl ₃ (3%) 0.012 ± 0.001 g 3.26 5 SUMS-1 + MgSO ₄ (1%) 0.017 ± 0.001 g 6.7 6 SUMS-1 + MgSO ₄ · 6H ₂ O (3%) 0.024 ± 0.002 g 6.5 7 SUMS-1 + MgSO ₄ · 6H ₂ O (5%) 0.027 ± 0.003 g 6.3 8 SUMS-1 + MgCl ₂ (1%) 0.015 ± 0.002 g 6.5 9 SUMS-1 + MgCl ₂ (3%) 0.018 ± 0.002 g 6.6 10 SUMS-1 + NiCl ₂ (3%) 0.016 ± 0.002 g 5.9 eleven SUMS-1 + ZnCl ₂ (3%) 0.019 ± 0.001 g 5.5

The treated surface of SUMS-1 was evaluated in biological tests by:

1) the effectiveness of biosorption (the ratio of the number of adsorbed cells to the total number of bacteria in the initial suspension),

2) the effectiveness of biodesorption (the ratio of the number of bacteria desorbed from the surface of SUMS-1 to the number of sorbed bacteria).

The effectiveness of biodesorption is an important indicator that largely determines the clinical effectiveness of IKE. The results of biological tests are given in table. 2.

table 2
Biological testing data of SUMS enterosorbent after treatment with metal salts Surface modification Effective. desorption LgT4 / LgT3 Effective. sorption of LgT1 / LgT3 The initial titer of T1 (CFU / ml) Sorption T3 = (T1-T2) T4 desorption T3 T2 Immobilization of a consortium of bifidobacteria AlCl ₃ - 0.01% 0.8714 1.001 1 · 10 ¹⁰ 4 · 10 ⁸ 9.610 ⁹ 5 · 10 ⁸ AlCl ₃ - 0.36% 0.8781 1,0004 1 · 10 ¹⁰ 7 · 10 ⁷ 9.9310 ⁹ 6 · 10 ⁸ AlCl ₃ - 0.75% 0.9009 1,000 1 · 10 ¹⁰ 1 · 10 ⁸ 9.9910 ⁹ 1 · 10 ⁹ MgSO ₄ · 6H ₂ O - 3% 1,0000 1.015 1 · 10 ¹⁰ 3 · 10 ⁹ 7 · 10 ⁹ 7 · 10 ⁹ MgSO ₄ · 6H ₂ O - 3% 0.9344 1.004 1 · 10 ¹⁰ 1 · 10 ⁹ 9 · 10 ⁹ 2 · 10 ⁹ Immobilization of a mixture by a consortium of bifidobacteria and lactobacilli MgSO ₄ · 6H ₂ O - 3% 0.9068 1.007 1 · 10 ¹⁰ 1.610 ⁹ 8.410 ⁹ 1 · 10 ⁹ Immobilization of a consortium of lactobacilli MgSO ₄ · 6H ₂ O - 3% 0.7839 1.005 6 · 10 ⁸ 6 · 10 ⁷ 5,410 ⁸ 7 · 10 ⁶ Note:
T1 (CFU / ml) is the initial titer of bacterial biomass;
T2 (CFU / ml) - titer of the biomass of bacteria remaining after immobilization on enterosorbent;

T3 = T1-T2 - titer (CFU / ml) of bacterial cells immobilized on a sorbent;
T4 - titer (CFU / ml) of bacterial cells after desorption, which determines the clinical effect of the drug

Thus, according to the results of studies of the treated surface of SUMS-1 with metal salts, option No. 6 (Table 1) of the treatment with MgSO ₄ · 6H ₂ O (3%) is the most optimal. After treatment of the SUMS-1 enterosorbent with a 3% solution of magnesium sulfate, its biosorption efficiency increases by 1.4 times (the ratio of sorbed cells to the total number of bacteria in the initial suspension), and the biodesorption efficiency, which determines the clinical effect of the action of the complex preparation (ratio of bacteria desorbed from the surface of SUMS-1 to the number of bacteria adsorbed on it) is 1, which indicates that almost all bacteria can be desorbed in the gastrointestinal tract.

Preclinical Studies

Studies have confirmed that the complex preparation "Ecoflor" is non-toxic for laboratory animals, restores intestinal normobiocenosis, eliminates opportunistic and pathogenic microflora, does not cause adverse and unforeseen adverse reactions, stimulates the body's adaptation to adverse environmental factors. The main results of the study:

1) the morphometric indicators of animals in the experimental groups increased in accordance with the population standard;

2) laboratory blood tests remained within the physiological norm;

3) histological examination of organs showed the absence of toxic effects;

4) confirmed the absence of a damaging effect on the structure of intestinal epithelium from the side of the drug;

5) pathological anatomical autopsy confirmed the condition of the organs is normal. The results obtained indicate that the complex preparation "Ecoflor" successfully passed preclinical trials.

The results of clinical trials of the complex drug "ECOFLOR"

All patients (45 people - the experimental group and 25 people - the comparison group) had a chronic pathology: atopic dermatitis - in 44.4% of cases; acute allergosis - in 11.1% of cases; secondary chronic pyelonephritis - in 22.2% of cases; diabetes mellitus - in 11.1% of cases; bronchial asthma - in 11.1% of cases.

After the experimental course of treatment with the complex drug Ecoflor, the following results were obtained:

1) the drug "Ecoflor" helps to reduce abdominal pain, manifestations of dyspeptic syndrome, normalization of intestinal motor function, regression of skin rashes;

2) the use of "Ecoflora" helps to reduce subjective clinical (malaise, weakness, impaired appetite) and objective laboratory criteria (normalization of the leukocyte intoxication index and hematological intoxication index, relief of cytolysis syndrome and normalization of total serum protein, as well as a decrease in the severity of urinary syndrome), reflecting endogenous intoxication syndrome;

3) when using the Ecoflor drug in 62.2%, a complete restoration of the functions of digestion and absorption in the intestine is observed, in other patients there is a tendency to normalization of indicators;

4) the drug "Ecloflor" helps to restore intestinal microflora: E. coli, bifidoflora; decrease in coccal forms, E. coli with weak enzyme properties, lactose-negative enterobacteria;

5) when taking the drug, no side effects and complications were detected.

Thus, the drug has successfully passed clinical trials, is a harmless and highly effective tool for restoring intestinal normobiocenosis and stopping endogenous intoxication syndrome.

Claims (9)

1. A complex probiotic preparation in an immobilized and lyophilized form, comprising a carrier, which is a porous enterosorbent SUMS-1 based on aluminum oxide, modified with carbon with hydrophilic-hydrophobic topochemistry, and eubiotic bacteria cells with nutrient and protective media immobilized on the indicated enterosorbent, characterized in that the cells of the bacteria-eubiotics with a nutrient medium are a lyophilized concentrate of a consortium of bifidobacteria, or lactobacilli, or mixture with a titer of 10 ⁸ to 10 ¹⁰ cfu / g, and as modified porous carbon enterosorbent SUMS-1 it comprises enterosorbent SUMS-1 further treated with a solution of metal salts in the following ratio of the drug in dry form, wt.%: cells with a nutrient medium of the consortium bifido-, or lactobacilli-eubiotics, or a mixture thereof with a titer of 10 ⁸ -10 ¹⁰ CFU / g 1.0-10.0 protective environment 0.1-10.0 SUMS-1 enterosorbent, additionally processed metal salt solution rest 2. The complex probiotic preparation according to claim 1, characterized in that as a consortium of bifidobacteria, it contains strains of bifidobacteria: Bifidobacterium bifidum 8-3, Bifidobacterium longum TWA-13 and Bifidobacterium adolescentis GO-13 in a ratio of 8: 1: 1. 3. The complex probiotic preparation according to claim 1, characterized in that as a consortium of lactobacilli it contains strains of lactobacilli Lactobacillus acidophilus 57S, Lactobacillus plantarum P-75, Lactobacillus casei Sat in a ratio of 4: 1.0: 1.0. 4. The complex probiotic preparation according to claim 1, characterized in that the mixture of consortia of bifidobacteria and lactobacilli has a ratio of 1: 1 to 3: 1. 5. A method of obtaining a complex probiotic preparation in an immobilized and lyophilized form, including washing and sterilizing the porous carbon-modified SUMS-1 enterosorbent and cooling it to room temperature, mixing enterosorbent and eubiotics with a nutrient and protective medium in a ratio of not more than 1 : 1 and keeping the mixture for at least 1 hour with stirring and at a temperature not exceeding + 4 ° C until the bacterial cells are completely immobilized on an enterosorbent, washing the resulting complex preparation ATA in isotonic saline, followed by freezing and freeze drying, characterized in that before sterilization, the SUMS-1 enterosorbent is treated with a metal salt solution in a volume ratio of not more than 1: 1 and washed with a 2-3 times volume of distilled water from the reagent, and as eubiotic bacteria use a concentrate of a consortium of bifidobacteria, or lactobacilli, or a mixture thereof with a titer of 10 ⁸ -10 ¹⁰ CFU / g. 6. The method according to claim 5, characterized in that as a solution of a metal salt using a 3% solution of magnesium sulfate. 7. The method according to claim 5, characterized in that as a consortium of bifidobacteria, strains of bifidobacteria are used: Bifidobacterium bifidum 8-3, Bifidobacterium longum TWA-13, Bifidobacterium adolescentis GO-13 in a ratio of 8: 1: 1. 8. The method according to claim 5, characterized in that as a consortium of lactobacilli, lactobacillus strains Lactobacillus acidophilus 57S, Lactobacillus plantarum P-75 and Lactobacillus casei Sat are used in a ratio of 4: 1.0: 1.0. 9. The method according to claim 5, characterized in that the mixture of consortia of bifidobacteria and lactobacilli has a ratio of from 1: 1 to 3: 1.