RU2562539C1 - Method for intestinal microbiocoenosis correction - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention represents a method for intestinal microbiocoenosis correction with the use of the microbial cultures E. coli M-17, B. subtilis "ТПИ" 13, B. licheniformis "ТПИ" 11, B. Bifidum 1, L. plantarum 8P-A3, B. cereus IP5832, B. subtilis 534 characterised by the fact that before use daily doses of the above microbial cultures are supposed to be mixed to perfluordecaline emulsion in normal saline, administered into small laboratory animals in an amount of 0.5 cm³, large domestic, farm and laboratory animals in an amount of 20.0 cm³, and humans in an amount of 10.0 cm³.

EFFECT: 1,2-1,5 times higher antagonist activity of the microbial preparations and 1,2-1,9 higher gastrointestinal survival of the probiotic cultures.

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Description

I. The technical field to which the invention relates.

The invention relates to microbiology, medicine, veterinary medicine and can be used for the correction of intestinal microbiocenosis in a complex of measures for the prevention and treatment of gastrointestinal disorders and immune status disorders caused by dysbiosis and their consequences.

II. State of the art

Known methods for the correction of intestinal microbiocenosis by using probiotics based on cultures of bifidobacteria and lactobacilli, bacilli, enterococci, etc. (Satellite symposium. Correction and prevention of dysbiosis. New approaches to the treatment of diseases of the gastrointestinal system. Edited by N. A. Tokareva. Effect, pharmacotherapy, Gastroenterology. 2011, No. 3. - P. 77-84; Panin AN, Malik NI, Ilayev OS Probiotics in animal husbandry - status and prospects / Veterinary medicine. 2012, No. 3. - S. 3-8; Intestinal microflora: an inside look. Collection of scientific articles. 2012, vol. . No. 1. - S. 3-99.)

The results of other studies (Bakulin V.M., Bakulin M.K., Ovsyannikov Yu.S., Tikhonov I.V. Use of perfluorocarbons in the technology of probiotic microbial cultures / Biosecurity and Biosafety. 2013, No. 4. - P. 49-52 .) showed in the study of perfluorocarbon compounds - perfluorodecalin (PFD) and carbogal (KG), that the introduction of 5 vol. % PFD into the liquid cultivation medium, while maintaining all other growth conditions, allowed to increase the yield of Escherichia coli M-17 cultures (probiotic strain of the drug Colibacterin), Bacillus subtilis TPI 13, Bacillus licheniformis TPI 11 (probiotic strains of the drug Biod-5) and Saccharomyces boulardii (probiotic strain of the drug Enterol) 1.55-1.67 times, simultaneously with an increase in the biomass of the studied cultures, their antibacterial activity also increased (1.6-1.75 times); According to the degree of activation of growth processes, CG was inferior to PFD and allowed to increase biomass yields by 1.26–1.46 times and increase the antibacterial activity of the resulting cultures by 1.2–1.4 times. In another work (Kuzin A.V. Perfluorocarbon compounds in the complex treatment of resistant forms of gastroduodenal ulcers: thesis for the degree of Candidate of Medical Sciences / M .: 2004. - 138 pp.) It is shown that the use of perfluorane irrigation (the active component of which is perfluorodecalin) gastroduodenal ulcers, led to their speedy healing.

The disadvantage of the used methods for the correction of intestinal microbiocenosis is that they have a delayed and weakened corrective effect due to the low engraftment of microorganisms in the biofilm and a reduced stimulating effect to restore normal resident microflora, which can be explained on the basis of one of the main environmental laws - the law of depletion of a heterogeneous substance in its island condensations G.F. Hilmi (Hilmi GF Fundamentals of biophysics of the biosphere. L .: Gidromethioizdat, 1966. - 272 p.). In accordance with this law, an individual system existing in an environment with a different level of organization gradually loses its structure, as it were dissolved in the environment. Law G.F. Hilmi is associated with another law - optimality (Reimers NF Ecology. Theories, laws, rules, principles and hypotheses. - M .: 1994. - 367 p.) And largely reflects the thermodynamics of a small system located in an alien environment. The most complete is the law of depletion of heterogeneous matter in its island condensations G.F. Hilmi appears when taken orally, i.e. a peculiar introduction of probiotics into the gastrointestinal tract of a patient with pronounced dysbiotic changes in the intestinal microflora and low resistance of the injected drugs to stressful chemical, physical and biological factors of the internal environment of the gastrointestinal tract (Pogorelsky I.P., Chicherin I.Yu., Lundovsky I. A. Ecological and functional marginality of probiotic microorganisms: Society, science, innovations: annual, open, scientific and technical conference, April 16-27, 2012: collection of materials from Vyatka State University; ed. S.G. Lit Vinets. Kirov. 1 electronic opt. Disk (CD-ROM). Section "Microbiology").

Common essential features with the claimed method of these works is the use of probiotics used to correct and regulate intestinal biocenosis, and perfluorocarbon compounds used for deep cultivation of probiotic cultures and therapy of gastroduodenal ulcers by irrigation.

III. The invention consists in the following.

The task was to develop a method for the correction of intestinal microbiocenosis by using probiotics in combination with perfluorocarbon compounds.

The problem is solved by oral administration of complexes created from commercial medical or veterinary probiotics with emulsified perfluorodecalin, using food lecithin as an emulsifier.

Probiotic preparations mixed with perfluorodecalin prepared in accordance with TU 95.1233-92 amended. 1, 2, emulsified in physiological sodium chloride solution using food lecithin as an emulsifier, when administered orally, they have a more effective corrective effect on intestinal microbiocenosis in the complex of measures for the prevention and treatment of gastrointestinal disorders and immune status disorders caused by dysbacteriosis and their consequences .

The essence of the technical solution, the method of correction of intestinal microbiocenosis is explained as follows. The ingress of microbial preparations into the gastrointestinal tract is a powerful stress factor for the autochthonous and allochthonous components of the corrected biocenosis, therefore, emulsified perfluorodecalin is introduced into the composition of the drug as a powerful anti-stress component, providing improved conditions for the correction of microbial biocenosis in this ecological niche, and increasing the effectiveness of probiotics (C .V. Darmova Organofluorine compounds, stresses and survival of microorgnisms / S.V. Darmova, M.K. Bakulin and others. Data List "Science - Production - Technology - Environment." - 2009. - P. 137-139).

With this method of correction of intestinal microbiocenosis by ingestion of complexes that use only certified preparations intended for medical, veterinary or food use, which have passed the appropriate certification, and whose safety and usefulness for humans and animals have been shown as a result of many years of practical use.

The perfluororganic compound intended for the implementation of this method is perfluorocarbon perfluorodecalin, the most studied of perfluorocarbons from the point of view of harmlessness and usefulness for humans in the development of the medicinal product "Perftoran" and probiotic microorganisms in the development of a method for their deep cultivation. Perfluorodecalin (PFD, chemical formula - C ₁₀ F ₁₈ ), TU 95.1233-92 colorless, transparent, odorless, non-combustible liquid, with a viscosity of 2.74 mm ² × cSt ^-1 at 25 ° C, a density of 1.945 g / cm ³ and a boiling point of 155 ° C. PFD does not serve as a nutrient, is not utilized by microorganisms, and is the best of organofluorine compounds for use in biotechnological processes as a powerful anti-stress factor and at the same time as an activator of the growth of microbial culture and their production of biologically active substances (Ivanitsky G.R. Biophysics at the threshold of the new millennium: perfluorocarbon media and gas transport blood substitutes / G.R. Ivanitsky // Biophysics. - 2001. - No. 1. - P. 5-33; Ivanitsky G.R.Nanocontainers based on perfluorocarbons with func her transfer of nitric oxide / G.R. Ivanitsky // Biophysics. - 2008. - No. 2. - P. 367-377; Bakulin MK. Theory and practice of using "blue blood" perfluorocarbons in deep cultivation of biodestructors / M.K. Bakulin and other Theoretical and Applied Ecology. - 2010. - No. 4. - S. 4-8).

For humans and animals with a large safety margin, the permissible dose of the commercial preparation of perfluorane is set at 30.0 cm ³ / kg or 3.0 cm ³ / kg of perfluorocarbons (2.1 cm ³ / kg of perfluorodecalin and 0.9 cm ³ / kg of perfluoromethylcyclohexylpiperidine) , which are an active component of perftoran. At this dose, as shown by numerous experiments on various animals and observations in the clinic, the drug does not cause hemolytic effects, anaphylactoid reactions, is not pyrogenic, does not inhibit hematopoiesis, is not teratogenic and is not embryotoxic, is not carcinogenic, and does not lead to pathological changes in organs and no more toxic than medical glucose (Medical and biological aspects of the use of perfluorocarbon emulsions / Under the editorship of Beloyartsev F.F. - Pushchino, 1983. - 186 p .; Ivanitsky G.R. Biophysical principles for the creation of perfluorocarbon media and ha of zootransport blood substitutes (review) / Ivanitsky GR // Organofluorine compounds in biology and medicine. - Pushchino, 2001. - P. 448).

With the proposed correction method, first, using mixing devices (mixers, homogenizers, etc.) or by simple vigorous shaking of the hand in a bottle for 2-3 minutes, an emulsion of perfluorodecalin containing 100 cm ³ is prepared:

10.0 grams of perfluorodecalin;

0.9 grams of NaCl;

0.1 grams of edible lecithin.

Then, one of the probiotic preparations used for correction is introduced into the resulting emulsion based on the content of 1 dose with the recommended instructions for the use of the drug in an amount intended for oral administration: 0.5 cm ³ for small laboratory animals (mice, hamsters, guinea pigs), by which the drug is administered using a needle with olive; 20.0 cm ³ for large domestic, agricultural and laboratory animals to which the drug is administered with food; 10.0 cm ³ for human intake in accordance with the recommendations for the main microbial preparation. Before use, the perfluorodecalin emulsion with the corrective microbial preparation introduced is shaken vigorously by hand in the vial for 10-20 seconds.

As a result of using the proposed method, the antagonistic activity of microbial preparations increases by 1.2-1.5 times, as shown in in vitro experiments with respect to enteropathogenic cultures of the test strains Staphylococcus aureus 209, Proteus vulgaris SX18 and Salmonella enteritidis var. Danysz increases the survival rate of probiotic cultures in the gastrointestinal tract of experimental animals (white mice and guinea pigs) by 1.2-1.9 times, as shown by the example of cultures of strains isolated from probiotics: Colibacterin (Escherichia coli M-17) Biod -5 (Bacillus subtilis TPI 13 and B. licheniformis TPI 11); Probifor (B. bifidum 1); Lactobacterin (Lactobacillus plantarum 8P-A3) Bactisubtil (B. cereus IP 5832) and Sporobacterin (B. subtilis 534).

The cultivation of probiotic microorganisms was carried out on solid nutrient media of the recommended composition, the survival of probiotic microorganisms in the gastrointestinal tract of experimental animals was evaluated in accordance with the recommended method (Ivanov V.P. Improving the methods for diagnosing large intestine dysbiosis: information letter / V.P. Ivanov, A.G. Boytsov, A.D. Kovalenko et al. - St. Petersburg: Center for State Sanitary and Epidemiological Supervision, 2002. - 31 p .; Likhacheva A.Yu., Bondarenko V.M., Sokolova K.Ya. Current status of the issue of taxonomy ba of the Lactobacillus genus / A.Yu. Likhacheva, V.M. Bondarenko, K.Ya. Sokolova // Journal of Microbiol. - 1992. - No. 9-10. - P. 74-78; Methodical recommendations "Safety assessment of nanomaterials" Approved by Order of the Rospotrebnadzor dated October 12, 2007 No. 280. - M .: Rospotrebnadzor, 2001; Darmov IV, Survival of microorganisms of probiotics in the gastrointestinal tract of experimental animals / IV Darmov, I.Yu. Chicherin, I.P. Pogorelsky et al. Journal of Infectology. - 2012. - T.4. - No. 1. - S. 68-74).

When growing probiotic microorganisms under microaerophilic conditions, the Anaerobic system Mark III - LE003 (Hi Media Laboratories Pvt. Ltd, Mumbai, India) with Hi Anaero Gas Facet gas generator bags was used.

To study the survival of probiotic microorganisms in the gastrointestinal tract of experimental animals, probiotic strains were labeled with resistance to 500 μg / cm ^{3 of} streptomycin, which made it possible to differentiate them in feces from bacterial cultures of their own indigenous intestinal microflora. The essence of the procedure for producing labeled derivatives of probiotic cultures of microorganisms is to use 2-4 reseeding them on solid nutrient media with increasing concentrations of streptomycin and the selection of spontaneous mutants that retain species characteristics.

The selected mutants of probiotic cultures resistant to streptomycin (Sm ^r mutants) stably retained the sign of antibiotic resistance. A study of the population composition of mutant bacteria on the basis of antibiotic resistance on solid nutrient media containing streptomycin at a concentration of 150 μg / cm ³ indicated that all populations of the studied bacteria retained the hereditarily fixed trait of streptomycin resistance (Sm ^r- sign).

Mutant bacteria retained all their original controlled cultural-morphological, tinctorial, antagonistic and biochemical characters. The marker trait helps to identify and identify bacteria of labeled probiotic cultures in feces when seeding samples on selective solid nutrient media on which microorganisms of indigenous microflora do not grow. These conditions are met by dense nutrient media containing 100 μg / cm ^{3 of} streptomycin, on which R ⁺ - (resistant to this drug) microorganisms are able to grow.

Antagonistic activity was determined on a solid medium by applying 50 μl of the grown probiotic culture with a strip along the diameter of the cup and sowing after 24 hours with 50 μl of a suspension of daily agar culture of the test strains at a concentration of 1 · 10 ⁷ CFU · cm ^{-3 by a} line perpendicular to the diametrical strip followed by growing culture for 2 days. The average size of the zones of growth inhibition in the strokes of the test cultures in comparison with the control was used as arbitrary units (UE) of the antibacterial activity of the culture.

Example 1. Determination of the survival rate of probiotic microorganisms (Sm ^r ) when passing through the gastrointestinal tract of white mice and guinea pigs when using probiotic cultures according to the accepted (previously known) and proposed correction methods (tables 1 and 2).

          Example 2. Determination of the antagonistic activity of microbial preparations in an in vitro test against cultures of enteropathogenic bacterial strains of Staphylococcus aureus 209, Proteus vulgaris SX18 and Salmonella enteritidis var. Danysz (table 3).

Claims (1)

A method for the correction of intestinal microbiocenosis using microbial cultures of E. coli M-17, B. subtilis TPI 13, B. licheniformis TPI 11, B. Bifidum 1, L. plantarum 8P-A3, B. cereus IP5832, B. subtilis 534, included the composition of the probiotic preparations Kolibacterin, Biod-5, Probifor, Lactobacterin, Bactisubtil, Sporobacterin, characterized in that the daily doses of these microbial cultures are mixed before use with perfluorodecalin emulsion in physiological saline containing 100 cm ³ :
10.0 grams of perfluorodecalin;
0.9 grams of NaCl;
0.1 grams of edible lecithin,
and then introduced to small laboratory animals in a volume of 0.5 cm ³ , large domestic, agricultural and laboratory animals - in a volume of 20.0 cm ³ , for human consumption - in a volume of 10.0 cm ³ .