EA040504B1 - STRAIN LACTOBACILLUS ACIDOPHILUS N. V. EP 317/402 "NARINE" AAAA, INTENDED TO RESTORE NORMAL HORMONAL REGULATION AND INTESTINAL MICROFLORA AND IMPROVE THE BODY IMMUNITY - Google Patents

Description

The invention relates to biotechnology and relates to a strain of lactobacilli that has the ability to produce interferon in human unicellular cells, to influence the physiological and immunological processes in the body. The strain can be used to prepare products, including fermented milk products, designed to reduce the effects of dysbacteriosis and restore normal intestinal microflora.

The causes of dysbacteriosis are known: these are various infectious diseases, antibiotics, chemotherapy, hormone and radiation therapy, psycho-emotional stress and unfavorable environmental conditions, social factors, alcoholism and others.

They directly or indirectly contribute to the imbalance of the microbiocenosis of the body and, first of all, the intestinal microflora of the human body with an increase in the activity of a relatively wide range of opportunistic microorganisms of 3-4 pathogenicity groups.

In this regard, an urgent task is to identify new strains of lactobacilli and develop drugs and food products based on them.

A known strain of Lactobacillus acidophilus n.v. 317/402 Narine INMI-9602. The strain produces a significant amount of antibiotic substances harmless to children and adults that inhibit the growth and development of both gram-positive and gram-negative bacteria that cause acute forms of gastrointestinal diseases (SU 163357, class d2N 1/20, 1964). However, the strain has a slow acid-forming ability and the absence of phage lysis in the strain L. acidophilus n.v. 317/402 Narine. Whole milk product is prepared by fermentation of whole milk at 28-40°C, 1 wt.% strain. Clotting occurs after 5-8 hours, the acidity of the product is 60-90°T. This product is suitable as a starter, as well as a therapeutic and prophylactic agent for diseases of the gastrointestinal tract. The product can be used in medical and veterinary practice. However, the product obtained using the strain L. acidophilus n.v. 317/402 Narine, acidity not higher than 90°T, does not provide a person with the entire complex of amino acids, vitamins necessary for the correction of dysbacteriosis and various gastrointestinal diseases.

The closest is the bacterial strain Lactobacillus acidophilus n.v. EP 317/402 Narine AAA VKPM B-7747, used in the preparation of drugs, dietary and therapeutic products for the treatment of dysbacteriosis and its consequences (SU 2146454, class d2N 1/20, 2000). The strain exhibits high acid-forming activity and pronounced antagonistic properties against opportunistic and pathogenic microorganisms, which have a high vitamin-forming ability, increased productivity of α- and γ-interferon in single-celled human cells as factors that play a decisive role in antiviral and anticancer protection, affecting physiological processes in the body.

However, this strain is not sufficiently active against a number of opportunistic microorganisms of 3-4 pathogenicity groups.

In this regard, the claimed invention is directed to obtaining a new strain of lactobacilli, in which the positive properties of the closest analogue would be supplemented by its activity against strains that cause dysbacteriosis, such as Proteus mirabilis 160205, collection number B1267, Bacillus cereus ATCC 10702, collection number B- 1367, Streptococcus faecalis 555, B-330, Salmonella abony 103/39, collection number B-1364, Staphylococcus epidermidis ATCC 14990 collection number B-1363, Staphylococcus haemolyticus MRS collection number B-1348, Pseudomonas aeruginosa collection number B-1295, Shigella sonnei 32 collection B-582, Staphylococcus aureus ATCC 6538, collection B-1266, Enterococcus faecium ATCC 19434, collection B1268, Shigella sonnei 5063, collection B-1335 Staphylococcus aureus 1850 MRSA, collection B-1352 Salmonella interica 441 No. 14, collection number B-1337, Candida albicans 620, collection number Y-583.

Also, the invention is directed to increasing activity against strains that cause dysbacteriosis, such as Staphylococcus aureus 1721 (ATCC 43300) MRSA, collection number B-1349, Staphylococcus epidermidis 1827 MRSE, collection number B-1350, Staphylococcus epidermidis 1833 MRSE, collection number B- 1351, Bacillus subtilis ATCC 6633, collection number B-654, Escherichia coli 6645 ATCC 25922, collection number B-655, Pseudomonas aeruginosa ATCC 9027, collection number B-656, Mycobacterium smegmatis GK, collection number B-836, Klebsiella pneumonia 378/ collection number B-378, Salmonella typhimurium 2606, collection number B-581, Serratia marcescens d, collection number B-1, Bacillus cereus, collection number B-277.

The strain Lactobacillus acidophilus n.v. EP 317/402 Narine AAAA, deposited in the FGUN GNTs VB Vektor under the number B-1321 and in the All-Russian Collection of Microorganisms IBPM named after. G.K.Skryabin RAS under the number B-3468D, obtained by selection of the original culture of Lactobacillus acidophilus n.v. EP 317/402 Narine AAA VKPM V-7747 (patent for the invention of the Russian Federation 2146454, class d2N 1/20, 2000) and directed selection according to indicators: increased antibiotic activity against pathogens of gastrointestinal diseases, antibiotic resistance, dynamics of reproduction of the bacterial population in the nutrient environment, vitamin-forming capacity and the number of viable bacteria.

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Comparative data on the antagonistic activity of the proposed and known strains are given in table. 1. Brief description of the strains presented in table. 1.

Escherichia coli strain (Group 4) (B-655), type strain, is used as a test culture in determining the antimicrobial activity of drugs and to determine the suitability of culture media. Escherichia coli can cause escherichiosis - various infectious diseases that occur with intoxication, fever, usually with damage to the gastrointestinal tract, less often urinary, biliary tract, other organs, or with the development of sepsis.

The strain of the genus Klebsiella 378 (Group 4) (B-378), a facultative anaerobe, grows well at 37°C on simple nutrient media. In humans, Klebsiella can cause various inflammatory processes (septicemia, meningitis, pyemia, etc.). The pathogenicity of Klebsiella is associated with the presence of a capsule.

Mycobacterium smegmatis strain (Group 4) (B-836). The strains of this group are gram-positive, acid-resistant, polymorphic cells: weak branching, formation of filaments, coccal forms are possible. Colonies may have a yellow pigment. Saprophytes capable of forming intermediate forms that cause diseases. Growth is slow, aerobic, growth temperature for different strains is from 30 to 40°C.

Strains of the genus Proteus (Group 4) (P.mirabilis B-1267), Gram-negative polymorphic rods, facultative anaerobes, grow well at 37°C on simple nutrient media. Proteus lives in the human intestine, is excreted during food intoxication, dyspepsia, otitis and other diseases.

Pseudomonas aeruginosa strains No. B-656, B-1295 (group 4) (blue-purulent bacillus). The pathogenic gram-negative strain is an obligate aerobe, the causative agent of Pseudomonas aeruginosa; the optimum growth temperature is 37°C, it produces the pigment pyocyanin, it grows on simple nutrient media. It is one of the most common causative agents of nosocomial infections, and infections caused by this microorganism are characterized by a severe course and are associated with high mortality.

Strains of the genus Salmonella (group 3) (B-581, B-1364, B-1337) include about 2000 serovars. Many of them parasitize in humans, domestic and wild animals, causing diseases of the intestinal tract. Salmonella is a large group of enterobacteria, among which various serotypes are the causative agents of typhoid fever, paratyphoid A, B and C and the most common foodborne diseases - salmonellosis.

Strains of the genus Serratia (Group 4) (B-1) are represented by enzyme producers and test strains; cultivation conditions: LB, RPA, 37°C; facultative anaerobes, gram-negative. Bacteria of the genus Serratia are often the cause of purulent-inflammatory, urological, gynecological and intestinal diseases. Also, infectious processes caused by these bacteria often develop in young children, cancer patients and are the cause of nosocomial infections.

Strains of the genus Staphylococcus (group 4) are represented in the work by 7 strains (Table 1); are facultative anaerobes, gram-positive. Staphylococci are pyogenic bacteria capable of causing various inflammatory processes. They produce enterotoxin, which can cause severe food poisoning and leukocidin, a toxin that destroys leukocytes and leads to the formation of abscesses.

Strains of Streptococcus faecalis, (Group 4) (B-330) are inhabitants of the intestines of humans and warm-blooded animals, they are rounded gram-positive cells arranged in pairs or in short chains, they are also isolated in diseases of the gallbladder, urinary tract, they are facultative anaerobes that grow well in temperature of 37°C on simple nutrient media. Fecal enterococcus can be the causative agent of various infections: urinary tract, intra-abdominal, pelvic organs, wounds, endocarditis. Fecal enterococci are the most pathogenic species among enterococci, they account for 80-90% of all enterococci isolated in human clinical material. Fecal enterococci are often the cause of nosocomial infections.

Shigella sonnei strain (group 3) (B-1335, B-582) is a facultative anaerobe, available strains are used as test cultures in determining the antimicrobial activity of drugs and for testing nutrient media. Represented by gram-negative immobile rods without capsules, growing well at a temperature of 37 ° C on simple nutrient media. Microorganisms of this species are the causative agents of intestinal infections in primates and humans. All types of shigella can be the causative agents of an infectious disease - bacterial dysentery (also called shigellosis), which occurs with symptoms of intoxication.

Bacillus cereus strain (group 4) (B-277, B-1367) - gram-positive rods that form endospores, grow at temperatures of 35-45 ° C on simple nutrient media, hydrolyze starch and casein, break down tyrosine, are capable of growing in anaerobic conditions, restore nitrates. Microorganisms of this species can cause food poisoning.

Strains of the genus Candida (Group 4) (C.albicans No. Y-583, Candida sp. Ft-5), yeast-like fungi,

- 2 040504 multiplying by budding, forming pseudomycelium, under adverse conditions cause candidiasis of the skin, mucous membranes and internal organs. The strain available in the collection is used as a test culture in determining the antimicrobial activity of drugs and to determine the suitability of nutrient media.

Strains of the genus Aspergillus (Group 4) (F-1366, F-1319) are widespread saprophytic molds that can cause aspergillosis disease, which has various clinical manifestations: invasive aspergillosis, pulmonary aspergilloma and allergic bronchopulmonary aspergillosis. The progressive course of the disease and its resistance to treatment is partly due to the ability of the pathogen to rapidly grow and invade blood vessels. There are reports of the occurrence of aspergillosis of the larynx, trachea and bronchi in patients with impaired immunity, AIDS, solid organ transplantation, long-term therapy with corticosteroids. May be present in airborne aerosols.

Strains of saprophytic fungi of the genus Trichoderma (Group 4) (F-899) are ubiquitous, can cause allergic diseases of the upper respiratory tract in humans, are associated with peritonitis and infections in immunosusceptible patients with hematological malignant tumors or during organ transplantation. Mold fungi, which include fungi of the genus Trichoderma, are most dangerous in urban areas; indoor dampness is a significant risk factor for respiratory and other diseases.

Strains of Penicillium sp. (Group 4) (F-1320) belong to fungi that are ubiquitous in the external environment, participating in biocorrosion and biodegradation of various materials, primarily of plant origin. Representatives of the genus Penicillium are one of the most common laboratory contaminants that can cause endocarditis, post-traumatic endophthalmitis. Fungi of the genus Penicillium are often associated with allergic diseases and may cause clinical symptoms in sensitized individuals.

Determination of the antimicrobial activity of the selected strain L. acidophilus n.v. EP 317/402 Narine AAAA, deposited in the FGUN GNTs VB Vektor under the number B-1321 and in the All-Russian Collection of Microorganisms IBPM named after. G.K. Scriabin RAS under the number B-3468D, and the control strain Lacidophilus n.v. EP 317/402 Narine AAA, deposited in VKPM under the number B-7747, was carried out by the diffusion method of delayed antagonism. The studied strains are grown on a milk medium, bifidus medium or other composition for 2 days, then applied as a stroke on the surface of the nutrient medium in a Petri dish, kept in a thermostat for 48 hours at a temperature of 37°C. Further, suspensions of pathogen strains are sown to the stroke of the studied strains with perpendicular strokes. The cups inoculated in this way are placed in a thermostat and incubated at a temperature of 37°C for 24-48 hours. To control growth, test strains are sown on cups without a stroke of the studied strains and cultivated in a similar way. A positive result is taken into account by the presence of a zone of inhibition of the growth of test strains of pathogenic microorganisms in the zone of diffusion of metabolites of the studied strains.

The data obtained indicate a high antimicrobial activity of the selected strain of Lactobacillus acidophilus n.v. EP 317/402 Narine AAAA, deposited in the FGUN GNTs VB Vektor under the number B-1321 and in the All-Russian Collection of Microorganisms IBPM named after. G.K. Scriabin RAS under the number B-3468D, in relation to gram-positive and gram-negative pathogenic bacteria.

Under the influence of metabolites of the claimed strain under the conditions of the experiments, growth inhibition of all tested 25 pathogenic gram-positive and gram-negative bacterial test strains was observed, expressed to varying degrees.

Of the studied 7 test strains, they were the most susceptible to the antibiotic action of the selected strain of Lactobacillus acidophilus n.v. EP 317/402 Narine AAAA, deposited in the FGUN GNTs VB Vektor under the number B-1321 and in the All-Russian Collection of Microorganisms IBPM named after. G.K. Scriabin RAS under number B-3468D (Staphylococcus aureus 1721 (ATCC 43300) MRSA, Escherichia coli 6645 ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Bacillus cereus, Klebsiella pneumonia 378, Salmonella typhimurium 2606, Serratia marcescens), growth inhibition zones for them were 17-34 mm.

Для 12 тест-штаммов (Staphylococcus epidermidis 1827 MRSE, Staphylococcus epidermidis 1833 MRSE, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 10702, Mycobacterium smegmatis GK, Staphylococcus haemolyticus MRS, Shigella sonnei 32, Staphylococcus aureus ATCC 6538, Enterococcus faecium ATCC 19434, Shigella sonnei 5063, Staphylococcus aureus 1850 MRSA, Salmonella interica 441 No. 14), the zones of growth inhibition were somewhat smaller, amounting to 15-29 mm, which is also a fairly effective action.

The remaining five strains of bacteria were more resistant to the effects of metabolites of the claimed selected strain, the zones of growth inhibition were 10-14 mm.

In the study of the antifungal activity of the claimed selected strain under the conditions of the experiment, a slight inhibition of the growth of yeast strains by its metabolites was found.

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C. albicans 620, which became invisible on the third day of cultivation, then similar typical colonies of fungi formed in the experiment and control.

At the same time, the study showed the possibility of inhibiting the declared twelve pathogenic gram-positive and gram-negative bacterial test strains, such as Proteus mirabilis 160205, Bacillus cereus ATCC 10702, Streptococcus faecalis 555, Salmonella abony 103/39, Staphylococcus epidermidis ATCC 14990, Staphylococcus Memo Pseudomonas aeruginosa, Shigella sormei 32, Staphylococcus aureus ATCC 6538, Enterococcus faecium ATCC 19434, Shigella sonnei 5063, Staphylococcus aureus 1850 MRSA, Salmonella interica 441 no. n.v. EP 317/402 Narine AAA VKPM V-7747.

Thus, we can conclude that the Lactobacillus acidophilus n.v. strain is highly active. EP 317/402 Narine AAAA, deposited in the FGUN GNTs VB Vektor under the number B-1321 and in the All-Russian Collection of Microorganisms IBPM named after. G.K. Scriabin RAS under the number B-3468D, in relation to the above representatives of the enteropathogenic intestinal microflora.

When fermenting carbohydrates with a strain of Lactobacillus acidophilus n.v. EP 317/402 Narine AAAA, deposited in the FGUN GNTs VB Vektor under the number B-1321 and in the All-Russian Collection of Microorganisms IBPM named after. G.K. Scriabin RAS under the number B-3468D, mainly lactic acid and a small amount of volatile acids are formed, not exceeding 5-6% of the total amount of titratable substances. The strain ferments milk for 5-12 hours at a temperature of 38-40°C. The strain is homofermative, capable of forming flaprotein proteases. Synthesizes vitamins of groups B and C, folic acid, thiamine, riboflavin, has cellulose activity.

The method, conditions and composition of the media for long-term storage of the strain is carried out: in test tubes in agar medium MRS with a single subculture within 1 month; in liquid nitrogen at -193°C; in a freeze-dried state, the culture can be stored for up to 2 years.

Method, conditions and composition of propagation media: MRS agar medium, 37-40°C, for 2472 hours.

Conditions and composition of the medium for fermentation: fermentation is carried out in whole milk.

Genetic features of the strain (auxotrophy, resistance to antibiotics, phages, etc.): the strain is resistant to a wide range of antibiotics, phages are not known for this type of strain.

Cultural and morphological features of the selected strain L. acidophilus n.v. EP 317/402 NARINE AAAA, deposited with the FGUN GNTs VB Vektor under the number B-1321 and in the All-Russian Collection of Microorganisms IBPM named after. G.K. Scriabin RAS under the number B-3468D: homofermentative immobile Gram-positive microaerophilic rods, elongated cells ranging in size from 2 to 20x0.8 μm, have high phenol resistance up to 0.4-0.5%, phthalazoles up to 1%, synthomycin resistance up to 0.003%, capable of producing significant the amount of harmless antibiotic substances that inhibit the growth and development of many both gram-positive and gram-negative pathogenic bacteria, including dysentery pathogens.

The strain does not produce catalase, does not reduce nitrates, does not form indole and skatole, does not liquefy gelatin, does not possess hemolytic ability; ferments lactose, glucose, sucrose, fructose, mannose, levulose, galactose, maltose, raffinose, starch, dextrin, sorbitol, mannitol, dulcite without gas formation.

The strain has a slow acid-forming ability; minimum activity 90°T, optimal activity 120-160°T, maximum activity up to 360°T; the number of viable lactobacilli in 1 ml of the strain is 150-250 million living cells of lactobacilli. Determination of the activity of the strain is carried out according to the McCready method.

The strain is not zoopathogenic, phytopathogenic and does not pose a danger for other reasons.

Recommended storage conditions: lyophilization, low-temperature freezing in a cryoprotectant.

In table. 1 presents comparative data on the antimicrobial activity of a selected strain of Lactobacillus acidophilus n.v. EP 317/402 Narine AAAA, deposited in the FGUN GNTs VB Vektor under the number B-1321 and in the All-Russian Collection of Microorganisms IBPM named after. G.K. Scriabin RAS under number B-3468D, and strain Lactobacillus acidophilus n.v. EP 317/402 Narine AAA, B-7747 (patent No. 2146454), investigated by the diffusion method of delayed antagonism.

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Table 1

Comparative table of antimicrobial activity of the selected strain Lactobacillus acidophilus n.v. EP 317/402 Narine AAAA, deposited in the FGUN SSC VB Vector under the number B-1321 and in

All-Russian Collection of Microorganisms IBFM named after A.I. G.K. Scriabin RAS under number B-3468D, and strain Lactobacillus acidophilus n.v. EP 317/402 Narine AAA, B-7747 (patent No. 2146454), ______ studied by the diffusion method of delayed antagonism ______

Strain Lactobacillus acidophilus N.V. EP 317/402 Narine Strain Lactobacillus aci- AAAA, deposited in the FGUN SSC VB "Vector" under the number B-1321 and in the All-Russian Collection of Microorganisms IBFM them. G.K.Scriabin RAS under the number B-3468D dophilus N.V. EP 317/402 Narine AAA, B-7747 (RF patent No. 2146454) No. p/n Col. No. test- strain Name of the test strain Pathogen streak growth inhibition zone (mm) Operating environment Pathogen streak growth inhibition zone (mm) 1 B-1349 Staphylococcus aureus 1721 (ATCC 43300) MRSA 34 Wednesday on milk 28-32 2 B-1350 Staphylococcus epidermidis 1827 MRSE 18 Wednesday on milk 15 3 B-1351 Staphylococcus epidermidis 1833 MRSE 20 Wednesday on milk 18 4 B-654 Bacillus subtilis ATCC 6633 17 Wednesday on milk 13 5 B-655 Escherichia coli 6645 ATCC 25922 32 Wednesday on milk 26-30 6 B-656 Pseudomonas aeruginosa ATCC 9027 34 Wednesday on milk 27-32 7 B-1267 Proteus mirabilis 12 Wednesday on absent

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160205 milk 8 B-277 Bacillus cereus 31 Wednesday on milk 27-29 9 B-1367 Bacillus cereus ATCC 10702 16 Wednesday on milk absent 10 B-836 Mycobacterium smegmatis GK 20 Wednesday on milk 2 eleven B-330 Streptococcus faecalis 555 10 Wednesday on milk absent 12 B-1364 Salmonella abony 103/39 eleven Wednesday on milk absent 13 B-1363 Staphylococcus epidermidis ATCC 14990 10 Wednesday on milk absent 14 B-1348 Staphylococcus haemolyticus MRS 20 Wednesday on milk absent 15 B-1295 Pseudomonas aeruginosa 12 Wednesday on milk absent 16 B-582 Shigella sonnei 32 23 Wednesday on milk absent 17 B-378 Klebsiella pneumonia 378 31 Wednesday on milk 26-28 18 B-1266 Staphylococcus aureus ATCC 6538 22 Wednesday on milk absent 19 B-1268 Enterococcus faecium ATCC 19434 25 Wednesday on milk absent 20 B-581 Salmonella typhimurium 2606 thirty Wednesday on milk 24-27 21 B-1335 Shigella sonnei 5063 23 Wednesday on milk absent 22 IN 1 Serratia marcescens d 33 Wednesday on milk 27-30 23 B-1352 Staphylococcus aureus 1850 MRSA 20 Wednesday on milk absent 24 B-1337 Salmonella interica 441 №14 18 Wednesday on milk absent 25 Y-583 Candida albicans 620 5 Wednesday on milk absent

The strain Lactobacillus acidophilus n.v. EP 317/402 Narine AAAA was used for the production of an experimental batch of Narine Raduzhny. It is vacuum-dried biomass of acidophilic lactobacilli Lactobacillus acidophilus n.v. EP 317/402 Narine AAAA.

Experiments were carried out on the influence of Narine Raduzhny backbone produced on the basis of a strain of Lactobacillus acidophilus n.v. EP 317/402 Narine AAAA, deposited in the FGUN GNTs VB Vektor under the number B-1321 and in the All-Russian Collection of Microorganisms IBPM named after. G.K. Scriabi-6 040504 at the Russian Academy of Sciences under the number B-3468D, on the course of alloxan diabetes in experimental animals.

The work was carried out on the basis of the laboratory of endocrinology of the GU NCCEM SB RAMS and the laboratory of ultrastructural studies of the GU NII CEL SB RAMS (January-March 2019).

The experiments were carried out on mature male Wistar rats obtained from the nursery of the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences (Novosibirsk). The maintenance, care of animals and their removal from the experiment was carried out in accordance with the Rules for carrying out work using experimental animals (Appendix to the order of the Ministry of Health of the USSR dated 12.08.1977 No. 755). The animals were kept in individual cages on a standard vivarium diet with free access to water. The rats received food at 14-16 hours daily.

4 groups of experimental animals were formed:

intact animals, untreated (n=3);

rats treated daily for 3 weeks in the morning per os with 0.5 ml of bacterium (n=6), group Narine Raduzhny;

rats with alloxan diabetes (n=4), group Al;

rats with alloxan diabetes treated from the next day after the administration of alloxan for 3 weeks with the bacterium according to the above scheme (n=9), group Al+Narine Raduzhny.

Experimental diabetes mellitus was caused by a single intraperitoneal injection of a diabetogenic dose of alloxan - 17 mg/100 g of body weight.

In the dynamics of the development of diabetes mellitus, body weight, the volume of water drunk, diuresis, and glucosuria were measured. At the end of the experiment, the weight of the body and internal organs was measured in all animals; levels of glucose, cholesterol, triglycerides, immunoreactive insulin (IRI); activity of enzymes - aspartate aminotransaminase (ACT) and alanine aminotransaminase (ALT) in blood serum; a morphometric study of the pancreas and the mucous membrane of the small and large intestines was performed.

The concentration of glucose, cholesterol, triglycerides in the blood serum was determined using the FLUTEST GLU, FLUTEST CHOL, Biosub TG kits (Biocon, Germany), the activity of enzymes in the liver - using the Biozyme kits (Biocon, Germany). The content of immunoreactive insulin (IRI) in blood serum was measured by radioimmunoassay using the rio-INS-PG-1251 HOPIBOH kit (Belarus), the glucose-insulin index was calculated as the ratio of glucose concentration in mmol/l to the concentration of IRI in pmol/l.

The pancreas was fixed in Bouin's fluid for 24 hours at +4°C, dehydrated in a series of alcohols of increasing concentration, and embedded in a histoplast. Sections 5 µm thick were stained with hematoxylin and eosin. A morphometric study of the endocrine part of the pancreas was carried out: the number of islets of Langerhans on a standard area of 10 mm ² was counted, their individual sizes (area, mm ² ), and the total relative volume (Vv%) were determined by point counting.

Ileum and colon samples were fixed in 1% OsO ₄ solution in phosphate buffer (pH 7.4), dehydrated in increasing concentration of ethanol, and embedded in epon. Semithin sections 1 μm thick were prepared from the obtained blocks, stained with toluidine blue, and examined under a light microscope. The number of goblet cells, interepithelial lymphocytes and globular leukocytes per 100 enterocytes in the epithelial lining of the mucous membrane of the intestinal villi of the ileum was determined. In the colon epithelium, the number of goblet cells and interepithelial lymphocytes per 100 colonocytes was studied. The percentage of fibroblasts, macrophages, eosinophils, mast cells, plasma cells and lymphocytes in the lamina propria of the ileum and colon was determined.

Statistical processing of the material was carried out using the Statistica 6 software package (StatSoft USA). One-way analysis of variance was performed, followed by post hoc comparison using the Newman-Keuls test or the non-parametric Mann-Whitney test. The probability of validity of the null hypothesis was taken at a 5% significance level.

Data in tables and text are presented as arithmetic mean and standard error of the mean (M±m).

Research results.

Lifetime parameters, values of hormonal and metabolic parameters of blood and mass indices of internal organs of experimental animals.

In table. 2 shows the results of measuring intravital parameters in experimental animals.

The values of intravital parameters in rats treated with the bacterium did not differ from those in intact animals. In rats of the Al + Narine Raduzhny group, statistically significant differences in lifetime parameters from animals of the Al group were also not revealed, although a tendency to greater body weight gain, less volume of water consumed and diuresis, lower values of glucosuria can be noted, which in general allows us to speak of more mild course of the disease in animals treated with the bacterium.

In table. 3 presents data on measuring the levels of metabolites, hormones and activity of fer

- 7 040504 cops in the blood serum of rats at the end of the experiment. In rats treated with the bacterium, cholesterol content (by 14.5%) and serum ALT activity (by 2 times) decreased in blood serum compared to intact animals. In the same group of animals, a tendency to a decrease in the concentration of triglycerides was noted (see Table 3). In rats with diabetes, as well as in healthy animals, a decrease in the content of triglycerides, cholesterol in the blood serum, and a decrease in ALT activity were noted against the background of taking the bacterium (Table 3).

table 2

The values of intravital parameters in experimental animals

Intact Narine Rainbow Al Al+Narine Rainbow Body weight gain in 3 weeks, g 65±16 77±13 22±16* ⁺ 25±16 * ⁺ Body weight gain for 3 weeks, % 30.8±10.3 36.3±6.6 10.8±9.2 ♦+ 13.3±7.4 * ⁺ Daily water intake 1 and 2 weeks after the start of the experiment, ml/day 6±1 (average over two terms) 8±1 (average over two terms) 130±25* 120±35* 90±20* ⁺ 90±20* ⁺ Daily diuresis after 1 and 2 weeks after the start of the experiment, ml/day 6±2 (average over two terms) 6±2 (average over two terms) 92±28* 92±23* 66±14* ⁺ 66±12* ⁺ Glucosuria 1 and 2 weeks after beginning of the experiment, g/day 0.002±0.001 (average over two terms) 0.005±0.00 2 (average over two terms) 5.2±2.4* 7.8±2.7* 4.1±0.6* ⁺ 5.6±1.3* ⁺ The concentration of glucose in the urine through 1 and 2 weeks after the start of the experiment, mmol/l 3.0±1.4 (average over two terms) 5.0±0.8 (average over two terms) 458±34* 324±40* 390±70* ⁺ 249±63* ⁺

Note.

The differences are statistically significant (p<0.05):

* - from intact animals;

- from the Narine Raduzhny group.

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Table 3

Content of metabolites, IRI and activity of enzymes in blood serum

experimental animals Index Intact Narine Rainbow Al Al+Narine Rainbow Glucose, mmol/l 6.8+0.9 7.2+0.2 25.9+2.4 * 20.4+2.0 * ⁺ Triglycerides, mmol/l 3.68+0.68 2.65+0.33 4.52+0.58 2.78+0.23 ^# Cholesterol, mmol/l 2.82+0.12 2.41+0.11* 3.52+0.35 3.22+0.20 ⁺ ACT, U/l 94.8+6.13 83.7+10.0 116.4+30.1 126.5+25.2 ALT, U/l 59.4+11.8 28.3+1.2 * 57.6+2.8 41.5+1.8 * ^+# IRI, pmol/l 165.7+13.0 161.5+25.7 79.1+3.6 * 101.3+8.1 * ⁺ Glucose/IRI 0.042+0.008 0.048+0.006 0.326+0.015 * 0.219+0.031 * ^+#

Note.

The differences are statistically significant (p<0.05):

* - from intact animals;

⁺ - from the Narine Raduzhny group;

# - from the Alloxan group.

3 weeks after the administration of alloxan, rats showed a clear form of experimental diabetes mellitus, which was characterized by a threefold increase in blood glucose levels compared to healthy animals, and in rats from the Al + Narine Raduzhny group, a tendency to lower values of this indicator was noted compared to animals from the group Al. The content of IRI in the blood of rats from the groups Al and Al + Narine Raduzhny was significantly lower than in healthy animals, however, in rats treated with the bacterium, the level of the hormone tended to increase, and the average value of the glucose-insulin index was significantly lower compared to animals from the group Al (see Table 3). The obtained data suggest that rats from the Al + Narine Raduzhny group, compared with animals from their Al group, have more pronounced signs of the beginning of the process of restoring disorders in the hormonal regulation of carbohydrate metabolism.

From Table. 4, which presents data on the mass indices of internal organs, it follows that the relative mass of fat depots in rats treated with the bacterium is 27% less than in intact animals. An increase in diuresis and a decrease in body weight in rats with alloxan diabetes (see Table 2) correspond to data on an increase in the mass index of the kidneys and a decrease in the relative mass of fat depots (see Table 4). There was no difference in the average values of these indicators in the Al and Al + Narine Raduzhny groups.

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Table 4

Mass indices of internal organs of experimental animals

Index Intact Narine Rainbow Al Al+Narine Rainbow Heart, g/100g of body weight 0.29+0.01 0.31+0.01 0.34+0.02 0.33+0.02 Kidney, g/100g body weight (1) 0.61+0.05 0.64+0.01 1.20+0.25 * 0.97+0.06 * ⁺ Spleen, g/100g body weight 0.52+0.04 0.62+0.07 0.62+0.06 0.57+0.05 Fat depots: epididymal and retroperitoneal white fat, g/100g body weight 3.33+0.33 2.42+0.15 * 1.05+0.42 * 1.27+0.28 * ⁺ Thymus, g/100g body weight 0.15+0.03 0.13+0.01 0.06+0.01♦ 0.09+0.01 * ⁺ Adrenal glands, mg/100g body weight 12.29+0.5 2 13.32+1.26 26.24+2.38* 20.05+1.21 * ^+# Thyroid gland, mg/100g body weight 5.56+1.12 4.44+0.44 5.94+0.36 4.78+0.22

Note.

The differences are statistically significant (p<0.05):

* - from intact animals;

⁺ - from the Narine Raduzhny group;

# - from the Alloxan group.

In diabetic rats, the relative weight of the thymus almost halved, and the relative weight of NP increased by 63-113% compared with intact animals. At the same time, the mass index of NP in rats from the Al + Narine Raduzhny group was significantly lower than in rats from the Al group (see Table 4).

Thus, restoration of the disturbed hormonal regulation of carbohydrate metabolism in diabetic rats on the background of taking the bacterium was faster. This is evidenced by data on the content of IRI in the blood serum and the values of the glucose-insulin index. The results obtained also allow us to assume that the intake of the bacterium contributes to the enhancement of lipid metabolism in the body of experimental animals.

Morphometric study of the pancreas.

In table. 5 shows the results of a morphometric study of the islet apparatus of the pancreas of rats, in table. 6 - data on the number of islets of different sizes on a standard cut area.

Table 5

Results of a morphometric study of the pancreas of experimental animals

Indicators Intact Narine Rainbow Al Al+Narine Rainbow Number of islets 12.34+0.68 10.64+0.83 4.70+0.76 5.72±0.85* ⁺ (N/IOmm ² ) ♦ Relative volume of islands (Vv%) 1.25+0.10 0.94+0.10 * 0.30+0.05 * 0.41±0.07 * ⁺

Note.

The differences are statistically significant (P<0.05):

* - from intact animals;

⁺ - from the Narine Raduzhny group.

In rats treated with the bacterium, there was a tendency to a decrease in the number density of the islets of Langerhans in the pancreas and a significant decrease in their total relative volume (see Table 5). An analysis of the individual sizes of islets on a standard area showed that in these animals the number of large functionally more active islets on a standard section area decreased (Table 6).

In animals with alloxan diabetes, both the numerical density and the total relative volume of pancreatic islets were significantly reduced, while many of the islets showed signs of destruction. The largest decrease in the number was noted for large islands.

In animals from the Al + Narine Raduzhny group, the number and relative volume of islets of Langerhans were less than in animals from the Narine Raduzhny group, however, compared with the Al group, twice as many large islets were noted (Table 5).

Table 6

The number of islets of different sizes on a standard section of the pancreas ____________________ experimental animals ____________________

Island size, mm ² Intact Narine Rainbow Al Al+Narine Rainbow 2* ^10'3 (small) 2.62+0.65 2.81+0.52 1.29+0.39 1.59+0.37 4* ^10'3 (medium) 2.50+0.60 2.42+0.19 1.03+0.24* 0.85+0.14 * ⁺ 5* ^10'3 (medium) 2.95+0.69 2.71+0.26 1.75+0.37 2.14+0.33 > 10 * 10' ³ (large) 4.28+0.84 2.70+0.46* 0.64+0.17 * 1.27+0.31 * ⁺

Note.

The differences are statistically significant (P<0.05):

* - from intact animals;

- from the Narine Raduzhny group.

Thus, the intake of bacterium by healthy animals was accompanied by a decrease in the volume of the endocrine part of the pancreas. In rats with alloxan diabetes, the backproduct did not have a pronounced effect on the restoration of the number and volume of pancreatic islets. The observed trend towards an increase in the proportion of actively functioning large islets in the pancreas is consistent with the data on a higher level of IRI in the blood of rats from the Al + Narine Raduzhny group compared to animals from the Al group.

Morphometric study of the mucous membrane of the small and large intestines of experimental animals.

In table. Figures 7 and 8 show the results of a morphometric study of the epithelium of the villi and the lamina propria of the mucous membrane of the ileum of rats.

In rats treated with the bacterium, an increase in the number of goblet cells by 27% was noted, the content of interepithelial lymphocytes decreased by 35%. In the epithelium of the intestinal villi of the ileum, in addition to interepithelial lymphocytes, globular leukocytes were observed - small cells located near the basement membrane, having a small number of large granules, intensely stained with toluidine blue. Their number was the same in rats from all experimental groups.

Table 7

The results of a morphometric study of the epithelium of the ileum villi of experimental animals ____________________

Index Intact Narine Rainbow Alloxan Alloxan + Narine Rainbow goblet cells 14.82+1.04 18.86+1.31* 17.58+0.90* 15.30+0.96 Interepithelial lymphocytes 25.14+1.77 16.24+1.89* 26.73+1.97 18.13+1.63** Globular leukocytes 5.29+0.75 5.96+0.72 6.53+0.73 5.17+0.91

Notes. The number of goblet cells, interepithelial lymphocytes and globular leukocytes is calculated per 100 enterocytes.

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Table 8

The results of a morphometric study of the lamina propria of the mucosa of ___________ ileum of experimental animals ___________

The differences are statistically significant (P<0.05):

* - from intact animals;

# - from the Alloxan group.

Index Intact Narine Rainbow Al Al+Narine Rainbow fibroblasts (%) 20.76+0.93 29.84+1.42* 21.43+0.99 20.91+1.64 Fibrocytes (%) 2.98+0.53 2.44+0.50 4.37+0.68 5.26+0.78* Macrophages (%) 18.91+1.16 19.94+1.12 19.93+0.85 16.30+1.44# Eosinophils (%) 8.10+0.83 9.74+0.88 6.98+0.69 7.52+1.28 Mast cells (%) 1.82+0.30 2.26+0.39 2.42+0.34 2.03+0.44 Plasma cells (%) 7.96+0.98 7.33+0.92 12.31+0.84* 9.56+1.98 Lymphocytes (%) 39.12+1.81 27.42+1.71* 32.08+1.33* 38.27+2.08#

Notes.

The percentage of each cell type was calculated.

The differences are statistically significant (P<0.05):

* - from intact animals;

# - from the Al-loxan group.

In the lamina propria of the mucous membrane of the intestinal villi, fibroblasts were observed that differ in shape depending on the degree of maturity. Fibroblasts and fibrocytes (definitive forms of fibroblasts) were isolated. Macrophages, plasma cells, lymphocytes, eosinophils, and mast cells were noted. Neutrophils and globular leukocytes were rare. Blood capillaries had small gaps. Lymphatic capillaries were located in the center of the intestinal villi and had wide openings.

In rats treated with the bacterium, the content of fibroblasts in the lamina propria of the small intestine mucosa increased by 44%. The lumens of the blood and lymphatic capillaries were dilated. As well as in the epithelial lining, the number of lymphocytes decreased in the lamina propria (by 30%).

An increase in the content of goblet cells that produce mucus may indicate an increase in the efficiency of the digestion process and an increase in the barrier properties of the epithelium of the villi in rats treated with Narine Raduzhny.

In rats with alloxan diabetes, the number of goblet cells in the epithelium of the intestinal villi increased by 18.6% and there was a trend towards an increase in the content of globular leukocytes (see Table 7). In the lamina propria, the number of plasmocytes increased by 54.6% and the number of lymphocytes decreased by 18%, which may indicate the activation of humoral immunity. The ratio of other cells of the lamina propria of the mucous membrane of the intestinal villi of the small intestine in rats with alloxan diabetes did not differ significantly from the corresponding values in the control (see Table 8).

In rats with alloxan diabetes treated with the bacterium, the content of goblet cells in the epithelium of the villi was slightly lower than in the Al group and did not differ significantly from the corresponding value in the control. The content of interepithelial lymphocytes was lower than the control value by 28% and less than in rats with alloxan diabetes by 33% (see Table 7).

In the lamina propria of the mucous membrane of the ileum of rats from the Al + Narine Raduzhny group, an increase in the number of fibrocytes was noted. The same trend was noted in rats from the Al group. The content of lymphocytes in the lamina propria corresponded to the value in the control (see Table 8). Comparison of these data with a decrease in the number of interepithelial lymphocytes indicates a lower stress on immune homeostasis in rats from the Al + Narine Raduzhny group than in rats from the Al group. Perhaps for the same reason, the number of macrophages decreased by 18%.

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Thus, the use of the buccal product contributed to a decrease in the content of lymphocytes in the epithelium and the lamina propria of the small intestine mucosa. At the same time, there was an increase in the number of goblet cells in the epithelium and fibroblasts in the lamina propria of the mucous membrane of the small intestine, as a result of an increase in the barrier properties of the organ. The use of the bacterium against the background of alloxan diabetes had some corrective effect on the structure of the mucous membrane of the small intestine.

In table. Figure 9 shows the results of a morphometric study of the rat colon mucosa.

Table 9 Results of morphometric analysis of the condition of the colon mucosa of experimental animals

Indicators Intact Narine Rainbow Al Al + Narine Rainbow Goblet cells (N) 163.2±32.12 319.9 ± 51.88* 163.3± 24.19 163.6 ± 14.75 Interepithelial lymphocytes (N) 6.0±2.29 5.0±2.12 9.1 ± 1.88 6.1 ± 1.41 Macrophages (%) 28.0 ± 2.59 23.4±2.04 14.2±2.09* 10.0 ± 1.63* Plasma cells (%) 9.3±2.41 12.9 ± 1.28 14.6±1.81 7.8±2.19 ^# Lymphocytes (%) 15.3±2.66 8.1 ± 1.10* 9.9 ± 1.27 16.9±4.08 Mast cells (%) 1.36±0.64 3.2 ± 0.65* 3.02±0.65 2.3±0.78 Fibroblasts (%) 37.5 ± 3.42 42.4±2.72 50.0 ±3.45* 56.8 ±4.41* Eosinophils (%) 8.5 ± 2.42 9.9±0.90 7.4 ± 1.37 6.2±1.03

Notes.

The number of goblet cells and interepithelial lymphocytes is calculated per 100 enterocytes; the number of cells of each type in the lamina propria was calculated as a proportion (%) of the total number of cells in the test area.

The differences are statistically significant (P<0.05):

* - from intact animals;

# - from the Alloxan group.

Animals treated with the bacterium showed a 2-fold increase in the number of goblet cells in the epithelium of the colon mucosa.

At the same time, the content of interepithelial lymphocytes remained at the level of control indicators.

In the lamina propria of the colon mucosa, a 2-fold increase in the number of mast cells and a decrease in the number of lymphocytes by 47% were observed.

In the study of the mucous membrane of the large intestine of rats with alloxan diabetes, no significant differences were observed in the ratio of cells in the epithelial lining. However, interstitial edema, a 49% decrease in the content of macrophages, and an increase in the number of fibroblasts by 33% were noted in the mucosal lamina propria compared to the corresponding values in intact rats. The structure of the blood vessels differed little from those of the large intestine in intact rats, but the appearance of bacteria in the lumen of the capillaries of both the mucous and submucosal layers was noted. Accumulations of lymphocytes were observed in the lymphatic vessels.

In the study of the mucous membrane of the colon in rats with alloxan diabetes treated with Narine Raduzhny, no significant differences were observed in the ratio of cells in the epithelial lining. In the lamina propria of the large intestine of these animals, a decrease in the number of macrophages by 64% and an increase in fibroblasts by 51% were observed compared with intact animals. In its own plate, a decrease in the number of plasma cells by 47% was noted compared with animals from the Al group. In the lumen of the vessels of the submucosal layer, bacteria were observed, as well as in animals with alloxan diabetes.

Thus, with the prophylactic use of Narine Raduzhny, the content of goblet cells in the epithelium of the colon mucosa increased, and the number of interepithelial lymphocytes did not change. In the lamina propria of the colon mucosa, an increase in the number of mast cells and a decrease in the number of lymphocytes were observed. In rats with alloxano-

Claims (1)

With diabetes treated with Narine Raduzhny, in the lamina propria of the mucous membrane there was a decrease in the number of macrophages and plasma cells, an increase in the number of fibroblasts. The data obtained indicate a pronounced effect of the intake of the bacterium on the structure of the intestinal mucosa of healthy and sick rats. An example of the preparation of a working starter for obtaining a fermented milk product based on the Narine AAAA strain. To prepare a working starter, sterilized or pasteurized goat milk is fermented at a temperature of 38-40°C with a culture of Lactobacillus acidophilus n.v. EP 317/402 Narine AAAA at the rate of 2-3% of the total amount of milk. The mixture is incubated for 5-8 hours. The fermented starter is cooled to 6°C, kept at the same temperature for 5 hours. The acidity of the clot is 160°T. The number of living cells in 1 ml of liquid sourdough is up to 260 million cells. The obtained working sourdough can be used as a bacterial preparation with high selection properties for obtaining dietary, health-improving food products. The product in the form of dry sourdough is used as a dietary, functional food at any age, especially for infants in the absence of mother's breast milk, as well as complementary foods for children. The product is effective for animal health. An example of the preparation of a fermented milk product based on the Narine AAAA strain. For the preparation of a fermented milk product, whole goat milk chilled to 38-40 ° C is used, where 2-3% of the working starter of the Lactobacillus acidophilus n.v. strain is added. EP 317/402 Narine AAAA. The mixture is stirred and maintained at a temperature of 38-40°C for 5-8 hours until a clot with an acidity of 80-90°T is formed, then placed in a refrigerator for 2-3 hours for maturation. The quality of the prepared drink is preserved for 7-10 days at a temperature of +4-6°C. A product based on a strain of Lactobacillus acidophilus n.v. EP 317/402 Narine AAAA, has a nutritional value of up to 900 cal. The acidity of the finished product is 130°T, nitrogen% 8.7, amine nitrogen mg% 23. CLAIM Bacterial strain Lactobacillus acidophilus n.v. EP 317/402 Narine AAAA, deposited in the FGUN GNTs VB Vektor under the number B-1321 and in the All-Russian Collection of Microorganisms IBPM named after. G.K. Scriabin RAS under the number VKM B-3468D, designed to restore hormonal regulation disorders of carbohydrate metabolism and intestinal microflora, as well as increase the body's immunity. ^8j) Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2