RU127750U1 - BACTERIAL SYSTEM FOR NORMALIZING MICROBIOCENOSIS OF HUMAN AND ANIMAL ORGANISM - Google Patents

Abstract

1. A bacterial system for normalizing the microbiocenosis of the human and animal body, containing a carrier that is carbon mineral enterosorbent, eubiotics bacteria immobilized on it, components of a nutrient and protective medium, characterized in that the carbon mineral mineral enterosorbent is modified by oxidative treatment, and the titer of eubiotics bacteria is ready the preparation is 1-1 ∙ 10 CFU per gram of the bacterial system. 2. The bacterial system according to claim 1, characterized in that the carbon mineral enterosorbent is either enteramine or SUMS-1.3. The bacterial system according to claim 1, characterized in that the eubiotics cells are either bifidobacteria or lactobacilli, or a mixture thereof.

Description

The utility model relates to biotechnology, namely to medical and veterinary drugs, namely to bacterial systems designed to normalize the microbiocenosis of the human body and animals.

At present, the problem of dysbiosis and intestinal dysfunctions is still relevant, due to a variety of reasons of a different nature: environmental degradation, exposure to xenobiotics (industrial and household pollutants, biochemically foreign compounds, preservatives, pesticides, herbicides, nitrates, nitrites, etc. etc.), irrational and unbalanced nutrition (fiber deficiency, excess of canned and refined foods, vitamin deficiencies, etc.), increased chronicity and allergization of us elimination, growth of immunodeficiency states, stress, gross violations of microbiocenosis as a result of antibacterial, hormonal or radiation therapy, micro and macroelementoses, etc. The functions of normal microflora in the body are vital and very extensive: protective, synthesizing, regulatory, desensitizing, detoxifying, enzymatic, immunostimulating (Materials of the All-Russian Scientific and Practical Conference "Dysbacteriosis and Eubiotics" / ZhMEI, 1996, No. 5, p.124-125; "Intestinal microbiocenosis. Modern ideas about the norm and pathology. Principles of correction of disorders" / Methodical recommendations for doctors, under the editorship of S. A. Kurilovich; compiled by I. O. Svetlov, G. S. Soldatov, M. I. Losev, T.I. Pospelova, Novosibirsk, 1998 , 26 pp.).

Bacterial systems containing living bacterial cells of eubiotics are known: bifidobacteria, lactobacilli, colibacilli, lactic acid bacteria, enterococci, etc. (Application WO 85/03848, MKI A23C 9/12, publ. 09/12/85; EVP No. 0154614, MKI A23C 9/123, publ. 11/09/85)

The disadvantages of the known technical solutions are low detoxification activity and, as a consequence, low colonizing ability. The reasons for these shortcomings are as follows. Violations of normoflora, dysbacteriosis, intestinal dysfunctions, as a rule, are accompanied by toxicosis of one degree or another, which complicate the colonization of the intestinal mucosa by probiotic bacteria. And the detoxification activity of the above drugs develops only as the intestines are colonized with normal microflora. In addition, when passing through the stomach, a significant (overwhelming) part of the bacteria dies in an unfavorable gastric environment and under the influence of enzymes. Currently, such drugs are considered more as components of dietary and therapeutic nutrition, but are not used for medicinal purposes.

Bacterial systems are known in which probiotic bacteria are protected from the inactivating effect of the gastric environment ("Intestinal microbiocenosis. Modern ideas about the norm and pathology. Principles of correction of disorders" / Methodical recommendations for doctors, edited by S. A. Kurilovich; comp. I. O. Svetlova, G.S. Soldatova, M.I. Loseva, T.I. Pospelova / Novosibirsk, 1998, 26 pp.) ... Known bacterial systems are made in the form of enteric capsules. Their therapeutic efficacy is higher than that of dry and liquid concentrates. Currently, these are the most widely used drugs, examples: linex, bifinorm, bifikol, primadofilus bifidus, normospectrum, etc.

The principal disadvantages of known bacterial systems are low detoxification activity: it develops only with the colonization of the intestinal walls with beneficial microflora (as well as in unencapsulated forms of probiotics), as well as low colonization efficiency. The reason for the low colonizing efficacy is that the macroencapsulated forms of probiotics act mainly on the luminal microflora, and for the organism the microflora localized on the intestinal walls is decisive. Bacteria of the luminal microflora, together with the contents of the intestine, move through the intestines and are evacuated naturally, respectively, macrocapsulated forms of probiotics act locally and for a short time.

Violations of normoflora, dysbacteriosis, intestinal dysfunctions, as a rule, are accompanied by toxicosis of one degree or another, therefore treatment regimens for such disorders and dysfunctions involve the inclusion of detoxification therapy.

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, activated carbon, carbactin, carbolong, polyphepan, etc., natural materials and their analogues (microcrystalline cellulose, zeolites, chitosans, pectins, microsorb, etc.), synthetic mineral sorbents and others materials that are used to cleanse the body of toxins, heavy metals, radionuclides and other toxins. Enterosorbents reduce the effects of exo- and endotoxicoses.

The next step in the construction of effective probiotic preparations is the production of complex bacterial systems, which are enterosorbent with living eubiotics bacteria immobilized on it. The therapeutic effectiveness of such bacterial systems consists of the sorption (detoxification) activity and protective ability of the used enterosorbent, the latter is manifested in the functional and colonization activity of immobilized eubiotics.

A known bacterial system, including enterosorbent - activated carbon - and microbial eubiotics cells immobilized on it (RF Patent No. 2017486, IPC ⁵ A61K 31/00, published on 08/15/94).

The disadvantage of the known bacterial system is the low colonization activity, which is caused by the disadvantages of activated carbon as an enterosorbent: activated carbons quickly clog up, actively absorb low molecular weight hormones, enzymes and intestinal gases, which impairs intestinal function. In addition, the protective properties of activated carbon are insufficient. This is due to the fact that the activated carbon used as a carrier with a particle size of less than 30 μm is a finely porous sorbent with a predominance of micropores and a high specific surface. As a result, microbial cells are located on the external surface of the sorbent particles and are not sufficiently protected from adverse environmental influences, including the inactivating effect of the gastric environment.

A known bacterial system, including carbon-mineral enterosorbent and components immobilized on it (eubiotic cells immobilized on it together with a nutrient medium) is a prototype (RF Patent No. 21118535, IPC ⁶ A61K 35/74, C12N 11/14, publ. 10.09.98 g .).

The disadvantages of the known bacterial system are insufficient colonization and functional activity, in addition, the adsorption (detoxification) ability of the enterosorbent in it is significantly reduced due to sorption of the components of the nutrient medium with eubiotics. All this does not contribute to high therapeutic activity.

The authors know one of the areas of improvement of bacterial systems. This direction consists in increasing the content of eubiotics in the bacterial systems desorbed from the surface of the sorbent, which would increase the total colonization and functional activity. The authors of the following patents that created the Ecoflor dietary supplement went this way: RF Patent No. 2164801, IPC А61К 35/74, А23С 9/12, С12N 1/08, publ. 04/10/2001, RF patent No. 2317089, IPC ⁶ A61K 35/74, A61P 43/00 publ. 02/20/2008, 10 ⁸ -10 ¹⁰ CFU / g of bifidobacteria are washed off the obtained preparations. As a result, the adsorption (detoxification) ability of the used enterosorbent SUMS-1 (enterumin) is reduced even in comparison with the prototype. The approach to the construction of bacterial systems proposed in the above patents is good for bacterial systems used as biologically active food supplements (BAA) for the prevention of dysbiosis in relatively healthy people. In diseases, an important factor stabilizing dysbiosis is intoxication. In these cases, it is highly desirable to enhance the detoxification (adsorption) activity of the bacterial system.

The objective of the utility model is to enhance the detoxification (adsorption) activity of the bacterial system and increase the functional and colonizing activity of immobilized eubiotics.

The problem is solved in that in the inventive bacterial system containing a carrier that is a carbonmineral enterosorbent, immobilized eubiotics bacteria, components of a nutrient and protective medium, the carbonmineral enterosorbent is modified by oxidative treatment, and the titer of the eubiotics bacteria in the finished product is 1 - 1 × 10 ⁷ CFU per gram of drug. At the same time, the carbon-mineral enterosorbent is either enterumin or SUMS-1, eubiotics cells are either bifidobacteria, or lactobacilli, or a mixture thereof.

The technical effect of the claimed utility model is to increase the detoxification activity (sorption ability), to increase the functional and colonizing activity of immobilized eubiotics.

The essence of the utility model: to obtain the inventive bacterial system to normalize the microbiocenosis of the human and animal organisms, a carbon mineral sorbent of either Enterumin or SUMS-1 type is used, which is subjected to oxidative modification, and immobilized live lyophilized dried eubiotics in a concentration known to be lower than that of the prototype - 1-1 × 10 ⁷ CFU / g. Such mild surface oxygenation increases the surface tropism of the sorbent to immobilized bacteria and increases the viability of these bacteria. Increased tropism and vitality can reduce the concentration of bacteria in the preparation to 1-1 × 10 ⁷ CFU / g, and thus increase the sorption (detoxification) activity of the bacterial system compared to the prototype to normalize the microbiocenosis of the human and animal organisms. As a result, the functional and colonization activity of immobilized bacteria in the bacterial system increases to normalize the microbiocenosis of the human and animal organisms.

The essence of the utility model is further illustrated by examples.

A method of obtaining a bacterial system.

Surface modification and cell immobilization is carried out according to moisture capacity. By definition, moisture capacity is the total amount of liquid (water, solution) required to completely wet the surface and fill the pores of the sorbent. It is measured empirically, for a sorbent like Enterumin or SUMS-1 it is in the range of 650-750 ml / kg of sorbent.

1 kg of Entermin type carbon-mineral enterosorbent is impregnated with a moisture capacity of 700 ml of a 3-5% hydrogen peroxide solution. It is stirred until smooth and kept at room temperature for 30-40 minutes or at an elevated temperature of 50-60 ° C for 10-15 minutes, then washed 1-2 times with 1 liter of distilled water, after which the water is drained, the sorbent is dried at a temperature of 70-100 ° C to a free-flowing state and residual humidity of not more than 7%. Ready modified sorbent is cooled to a temperature below 10 ° C, and impregnated with a moisture capacity of 700 ml of a solution of a liquid concentrate containing eubiotics (bifidobacteria) bacteria and components of a nutrient and protective environment. Stirred until smooth and completely moistened, then subjected to freezing and freeze drying. After drying, a ready-made bacterial system is obtained to normalize the microbiocenosis of the human body and animals. All work is carried out in compliance with aseptic measures. The titer of bacteria in the finished bacterial system to normalize the microbiocenosis of the human body and animals in CFU / g (colony forming units per gram of preparation) is determined by the standard method of biotitration on nutrient media. Depending on the titer of the initial concentrate used, the titer is 1-1 × 10 ⁷ CFU / g.

A comparative study of the adsorption activity of bacterial systems to normalize the microbiocenosis of humans and animals.

The adsorption activity of bacterial systems was compared on a model of sorption of the substrate - vitamin B ₁₂ , under standardized conditions. Experiment design: after 3 g of a dry preparation, 30 ml of vitamin B ₁₂ solution is pumped in a column with a concentration of 200 mg of vitamin per liter of solution at a circulation rate of 60 ml / min. After 10 minutes and 30 minutes, samples of the solution are taken, the residual concentration of vitamin in the solution is measured spectrophotometrically, and the adsorption capacity in% is calculated. The measurement of optical density is carried out under conditions corresponding to turbid solutions. The results are presented in table 1 for bacterial systems to normalize the microbiocenosis of the human body and animals, including bifidobacteria and in table 2 for bacterial systems to normalize the microbiocenosis of the human body and animals, including a mixture of bifidobacteria and lactobacilli.

Comparison of the functional activity of immobilized bifidobacteria (eubiotic bacteria) in bacterial systems.

The functional activity of immobilized bifidobacteria was determined by the standard method according to the activity of acid formation. Experimental design: identical amounts of the bacterial system (3 g each) were added to 25 ml of Blaurock medium, incubated at 37 ° C for 48 hours with periodic stirring. At the end of the incubation, 2 samples of 10 ml were taken and titrated with a solution of caustic soda in a concentration of 0.1 mol / l to pH = 8.5, the values of the obtained titers were averaged. The activity index was expressed in degrees Turner, calculated by the formula: T ° = A × K × 10, where A is the number of ml of caustic soda solution used for titration; K - correction to the titer of sodium hydroxide solution; 10 - correction factor for the mass of the analyzed sample. The results are presented in table 3.

Comparison of the relative colonization activity of bacterial systems.

The relative colonization activity of bifidobacteria in bacterial systems was evaluated by their ability to colonize a certain volume of the nutrient medium under the same standardized conditions. Experimental design: the same dose of bacterial systems to normalize the microbiocenosis of humans and animals (3 g) was added to 25 ml of Blaurock medium, incubated at 37 ° C for 72 hours with periodic stirring. At the end of the incubation, samples were taken and ground using the standard method of serial dilution on thioglycol medium to determine the concentration of bacteria in the culture medium in CFU / ml. Based on these titers, a volume of nutrient medium of 25 ml and taking into account dilution (3 grams per 25 ml of medium), the total growth of bifidobacteria was calculated (times, conditional colonization activity). The results are presented in table 4 for bacterial systems to normalize the microbiocenosis of the human body and animals, including bifidobacteria and table 5 for bacterial systems to normalize the microbiocenosis of the human body and animals, including a mixture of bifidobacteria and lactobacilli.

The advantages of the claimed bacterial system for the normalization of the microbiocenosis of the human body and animals are as follows:

- increase the sorption (detoxification) ability of the bacterial system to normalize the microbiocenosis of the human body and animals,

- an increase in the functional and colonizing activity of immobilized bacteria of the eubiotics of the bacterial system to normalize the microbiocenosis of the human body and animals.

All this helps to increase therapeutic efficacy.

Table 1 Bacterial systems for the normalization of the microbiocenosis of the human body and animals containing bifidobacteria No. Samples The content of bifidobacteria in the bacterial system, CFU / g Adsorption capacity for vitamin B ₁₂ ,% in 10 minutes in 30 minutes one Prototype 1.2 × 10 " 40 65 2 The inventive bacterial system to normalize the microbiocenosis of the human body and animals 1 2.5 × 10 ⁶ 55 78 3 The inventive bacterial system to normalize the microbiocenosis of the human body and animals 2 7.5 × 10 ² 67 91 four The inventive bacterial system for the normalization of the microbiocenosis of the human body and animals 3 "one"* 71 93 5 Bacterial system on an unmodified sorbent 2.0 × 106 52 74 “1” * - conditional concentration, the sample was obtained by washing the previous sample 2 with physiological solution until there were no living bacteria in the washing solution. That is, according to formal features, the sample does not contain bacteria. However, after the addition of the nutrient medium to the sample, an intensive growth of bacteria is observed, which indicates the presence in its composition of firmly bound non-absorbable bacteria, which, nevertheless, remain in a viable state and actively multiply. Existing methods of analysis do not allow to establish the exact number (concentration) of such bacteria. A conditional concentration of “1 CFU / g” means that the sample contains at least one viable bacterium.

table 2 Bacterial system for normalizing the microbiocenosis of the human and animal body, containing a mixture of bifidobacteria and lactobacilli No. Samples The total content of lactic acid bacteria in the bacterial system, CFU / g Adsorption capacity for vitamin B ₁₂ ,% in 10 minutes in 30 minutes one Prototype 1.6 × 108 39 63 2 The inventive bacterial system for the normalization of the microbiocenosis of the human body and animals 4 7.0 × 10 ⁷ 53 74 3 The inventive bacterial system for the normalization of the microbiocenosis of the human body and animals 5 2.5 × 10 ³ 65 87 four The inventive bacterial system for the normalization of the microbiocenosis of the human body and animals 6 "one"* 71 93 5 Bacterial system on an unmodified sorbent 3.0 × 10 ⁶ 51 72 “1” * - conditional concentration, the sample was obtained by washing the previous sample 5 with physiological saline until there were no living bacteria in the washing solution. That is, according to formal features, the sample does not contain bacteria. However, after the addition of the nutrient medium to the sample, an intensive growth of bacteria is observed, which indicates the presence in its composition of firmly bound non-absorbable bacteria, which, nevertheless, remain in a viable state and actively multiply. Existing methods of analysis do not allow to establish the exact number (concentration) of such bacteria. A conditional concentration of “1 CFU / g” means that the sample contains at least one viable bacterium.

Table 3 Bacterial systems for the normalization of the microbiocenosis of the human body and animals containing bifidobacteria No. Samples The content of bifidobacteria in the bacterial system, CFU / g Acid production, degrees Turner, T ° one Prototype 1.2 × 10 ⁸ 65,2 2 The inventive bacterial system 1 2.5 × 10 ² 99,4 3 The inventive bacterial system 2 7.5 × 10 ² 71.5 four The inventive bacterial system 3 "one"* 67.0 5 The drug is on an unmodified sorbent 2.0 × 10 ⁶ 63.5 “1” * - see note to table 1.

Table 4 Bacterial systems for the normalization of the microbiocenosis of the human body and animals containing bifidobacteria No. Samples The content of bifidobacteria in the bacterial system, CFU / g Biotiter of culture solutions, CFU / ml The total growth of bacteria, times one Prototype 1.2 × 10 ⁸ 2.4 × 10 ⁸ twenty 2 The inventive bacterial system to normalize the microbiocenosis of the human body and animals 1 2.5 × 10 ⁶ 7.8 × 10 ⁸ 2.5 × 10 ³ 3 The inventive bacterial system to normalize the microbiocenosis of the human body and animals 2 7.5 × 10 ² 8.5 × 10 ⁷ 9.4 × 10 ⁵ four The inventive bacterial system for the normalization of the microbiocenosis of the human body and animals 3 "one"* 6.2 × 10 ⁶ 5.1 × 10 ⁷ 5 Bacterial system on an unmodified sorbent 2.0 × 10 ⁶ 3.5 × 10 ⁷ 1.5 × 10 ² “1” * - see note to table 1.

Table 5 Bacterial system for normalizing the microbiocenosis of the human and animal body, containing a mixture of bifidobacteria and lactobacilli No. Samples The total content of lactic acid bacteria in the bacterial system, CFU / g Biotiter of culture solutions, CFU / ml The total growth of bacteria, times one Prototype 1.6 × 10 ⁸ 8.2 × 10 ⁸ 42 2 The inventive bacterial system 4 7.0 × 10 ⁷ 7.2 × 10 ⁸ 8.6 × 10 ¹ 3 The inventive bacterial system 5 2.5 × 10 ³ 1.5 × 10 ⁸ 5.0 × 10 ⁵ four The inventive bacterial system 6 "one"* 2.1 × 10 ⁶ 1.7 × 10 ⁷ 5 Bacterial system on an unmodified sorbent 9.1 × 10 ⁶ 5.7 × 10 ⁷ 5.1 × 10 ¹ “1” * - see the note to table 2.

Claims (3)

1. A bacterial system for normalizing the microbiocenosis of the human and animal body, containing a carrier that is carbon mineral enterosorbent, eubiotics bacteria immobilized on it, components of a nutrient and protective medium, characterized in that the carbon mineral mineral enterosorbent is modified by oxidative treatment, and the titer of eubiotics bacteria is ready the preparation is 1-1 ∙ 10 ⁷ CFU per gram of the bacterial system. 2. The bacterial system according to claim 1, characterized in that the carbon-mineral enterosorbent is either enteramine or SUMS-1. 3. The bacterial system according to claim 1, characterized in that the eubiotics cells are either bifidobacteria or lactobacilli, or a mixture thereof.