UA72065C2 - Strain aerococcus viridans 167k imb b-7069 with antibacterial activity and "viabac" preparation comprising this strain - Google Patents

Abstract

The invention relates to the novel strains of the probiotic bacteria and the medical preparations employing these strains. The strain Aerococcus viridans 167K IMB B-7069 possesses antibacterial activity and is suitable for preparing the medicinal compositions for treating the infectious diseases of various etiologies.

Description

The cell wall of aerococci contains glucose, galactose, and galactosamine and does not contain rhamnose, glycerol, or ribitol. The same content of HC pairs in the DNA of aerococci and pediococci was determined, equal to 41-4395. The content of HC pairs in the DNA of aerococci ranged from 39.5 to 42.095. In aerococci isolated from breast milk, the content of HC pairs is 39.9-42.0905, from cow's milk - 39.5-41.695.

Biological properties

Антагоністичну активність штаму Аегососсив мігічдап5 167К (р.н. ІМВ В-7069) перевіряють по відношенню 9 тест-штамів - зіарпуіососси5 ацгеиз5 209р, Ргоїеи5 миїЇдагі5 401, Ргоївеи5 тігаріїї5 Н2г091, КіерзіеПа рпештопіає 320, Рзендотопах аєгадіпоза 1312, ЗпідеПа зоппеї, ЗпідеПа Пехпегі 1046 , ZaItopeiIa iurpitigpt 5710, Mirpo

Maya r.6078 - by the stroke method on a dense nutrient medium in Petri dishes.

The ampoule with the culture is diluted with a 0.995 sodium chloride solution, based on the content of 2-10 aerococci in 1 ml, and inoculated with a stroke along the diameter of a cup with MPA containing 195 g of glucose and 0.4 mg/ml of potassium iodide. After incubating the cultures in a thermostat at a temperature of (36:2-1)"C for 48 hours, a culture of test strains is sown with a loop of Henle. After 24 hours of incubation at a temperature of (36541)"C, the growth retardation zone of the test culture measured from the edge of the stroke to the beginning of growth of the test culture.

The zones of inhibition of the growth of the test strains should be at least 7mm for Rzeidotopavh aegadiposa 1312 and at least 15mm for all other cultures, compared to 5mm and 10mm of the prototype.

Oxidase activity. The indicator of oxidase activity, which is determined by the ability of aerococci to change the color of starch in a nutrient medium with potassium iodide, correlates with the antagonistic activity of the strain. The diameter of the bluing of the starch around the stroke of Aegosossa5 migichap5 167K (born IMV B-7069) is at least ZOmm.

The adhesion ability of migo was studied on the surviving culture of intestinal epitheliocytes of rabbits and white outbred mice according to the generally accepted methodology. After an hour's incubation at 37"C, cultures of epitheliocytes together with aerococci of strains Aegosossi5 migidap5 167K (born IMV B-7069) and Aegosossi5 migidap5 167 (born IMV B-7041) were prepared for electron microscopy.

The degree of adhesion was assessed by smears stained with 195 toluidine blue solution. At the same time, the number of cells in contact with aerococci and intact cells was determined. The results are shown in the table.

Table 1

Adsorption capacity of strains Aegosossiv migidap5 167K (born IMV B-7069) and Aegosossiv migidap5 167 (born IMV B-7041) to enterocytes of rabbits and white mice. Indicator keejinwe Inn | « v-7069

A. Migidan5 167 (b. IMV, Vol

A. migidap5 167K (born IMV lov 0 (olomysh | vav vzoneelnne nn | t 8v-7041

According to the given data, both strains of aerococci were equally actively adsorbed on the enterocytes of both rabbits and mice, but the declared strain demonstrated higher efficiency compared to the prototype.

Effect on phagocytic activity of polymorphonuclear neutrophils of peripheral blood. Metabolism products of A. miidap5 167K (born IMV B-7069) are substances necessary in the process of phagocytic function - hydrogen peroxide, superoxide dismutase, lysozyme-like substances, toxic forms of oxygen, as well as hydroxyl radicals.

The activity of neutrophil granulocytes was assessed by phagocytic activity and the index of completion of phagocytosis - killing, which most fully characterizes the functional capacity of neutrophil phagocytes due to the formation of toxic forms of oxygen and hydroxyl radical by granulocytes.

Superoxide dismutase binds this oxygen to hydrogen, forming hydrogen peroxide. The processes of peroxidation lead to damage to the membranes of microorganisms, causing their death.

Two series of ip migo experiments were conducted with the aim of clarifying the effect of the enzyme complex A. migidap5 167K (b.

IMV B-7069) on the phagocytic function of polymorphonuclear neutrophils. The results are presented in tables.

Table 2

The effect of the enzyme complex A. migidap5 167K (born IMV B-7069) in a dose of 0.01 ml on the phagocytic activity of polymorphonuclear neutrophils in the experiment ip myo (Mat) (pe) FA | FU | Phi | Phage | FI | Fo | zF idosld. | 66 | 615 | 406 | 60 | 378 | 227 | 163

2.control./// | 45 | 411 | 185 | 60 | 558 | z3yZz | 074 experiment. | 70 | 387 | 270 | 80 | 455 | 364 | 085

Note: "p"e0.05;

FA - phagocytic activity;

FU - phagocytic number;

FI - phagocytic index;

ISF - the ratio of FU through ZOhv and HF after 2 hours.

As the given data show, in 4 out of 5 conducted experiments, a statistically significant increase in the index of completion of phagocytosis was noted when adding 0.0 ml of the enzyme complex A. migidap5 IMV B-7069.

Table C

The effect of the enzyme complex A. migidape5 167K (born IMV B-7069) in a dose of O0.02 ml on the phagocytic activity of polymorphonuclear neutrophils in the experiment of ip myo (Mat) (pe8) FAF | FI | Phi | FAZ" | FI | F | zF

M.okontrol/// | 60 | 475 | 285 | 66 | 748 | 494 | 063

Note: "p"e0.05;

FA - phagocytic activity;

FU - phagocytic number;

FI - phagocytic index;

ISF - the ratio of FU through ZOhv and HF after 2 hours.

Comparing the data presented in Tables 2 and 3, we can conclude about the dose-dependent stimulating effect of the enzyme complex A. migidap5 167K (born IMV B-7069) on the most important, final stage of phagocytosis - killing. A dose of 0.01 ml stimulates keeling without inhibiting adhesion and absorption functions.

Another object of this invention is the drug Vyabak (Miaras), which contains a therapeutically effective amount of lyophilized vegetative cells of the strain A. migidap5 167K (born IMV B-7069) as part of a bioconcentrate. According to the usual pharmaceutical practice, the bioconcentrate is preferably introduced into the composition together with one or more carriers (various medicinal, flavoring, food additives and fillers). Lists of components suitable for this purpose are well known to those skilled in the art, as are methods of mixing them. In particular, they can be milk sugar, fruit pectin, polyvinylpyrrolidone, cellulose derivatives, magnesium stearate, vitamins, fruit powders, etc. For oral use, the bioconcentrate according to the present invention can be incorporated into capsules, tablets or other oral dosage forms.

For external use, it can be used in the form of ointments, medical and preventive toothpastes, etc.

The following examples illustrate the invention, but do not limit it in any way.

Example 1. Preparation of lyophilized bioconcentrate of A. migidap5 167K (born IMV B8-7069) (hereinafter bioconcentrate).

Optimal growth of aerococci is observed after 24 hours at (36:1)"C on meat-peptone agar.

The composition of the medium for strain cultivation is as follows: microbiological agar GOST 17206-84 22g; glucose GOST 603 8-79 4g; peptone dry fermentative for bacteriological purpose GOST 13805-76 10g; distilled water GOST 6709-72 1 l.

Distilled water is mixed with peptone and agar to prepare the medium. The mixture is kept for 20 minutes at room temperature to swell the agar and boiled for 15-20 minutes until it is completely melted. Next, pH-3.8 is set with 3095 acetic acid solution. After settling for an hour, the medium is decanted through a cotton-gauze filter and glucose is added, after which it is sterilized at a temperature of (110-271)"C for 30 minutes.

Colonies with a diameter of 0.5-2.0 mm in the form of flat colorless discs grow on the nutrient medium.

After incubation, the microbial mass is washed off in aseptic conditions by adding an aqueous solution of the stabilizer.

They carry out bacterioscopic and bacteriological control of the collected microbial suspension and lyophilize it.

The dried suspension is crushed and packaged in a sterile container and sealed.

1 g of bioconcentrate contains up to 5x10!9 viable cells.

The obtained bioconcentrate is a mixture of freeze-dried live vegetative cells of the bacterium; stabilizer, for example gelatin and sucrose; dry components of the nutrient medium and biologically active substances produced by the strain.

In the future, the obtained bioconcentrate can be used by itself or as part of a medicinal product for the prevention and treatment of various painful conditions.

For the cultivation of A. migidap5 167K (b. IMV 8-7069) it is also possible to use a liquid nutrient medium, in particular MPB with a nitrogen-ammonia additive (not less than 12wt.9o). A good crop is obtained if this medium additionally contains Tmeep-80 and nicotinic acid. Correction of the pH of the solution is carried out with the help of 1095 ammonia and brought to a value of 6.2-6.4.

For industrial production, the most attractive for growing A. migidap5 167K (b. IMV B-7069) is the use of mushroom decoction, which is explained by its moderate price and industrially significant yield of the finished product. Which will ultimately affect the price reduction of the medicinal product based on A. miidaps 167K (born IMV B-7069).

Example 2. Use of A. migidap5 167K bioconcentrate (born IMV B-7069) to obtain a medicinal product in the form of a tablet.

Mix 20.0g of glucose, 20.0g of apple pectin and 32.0g of lactose. The resulting mixture is moistened with a solution of barley-malt extract (4.5 g of extract and 1.2 g of water), granulated, dried, granulated again, 1 g of the bioconcentrate obtained in Example 1, 1.3 g of ascorbic acid and 0.9 g of calcium stearate are added.

The mixture is thoroughly mixed and tableted, obtaining pleasant-tasting tablets containing 4.2x108 cells of A. miidaps 167K (born IMV B-7069).

In the future, the resulting tablets can be coated with a pharmaceutically acceptable film coating or used to obtain a powder or microgranules that can be filled into gelatin capsules.

Example 3. Clinical application of A. migidap5 167K (born IMV B-7069). 3.1. To evaluate the therapeutic properties of A. migidap5 167K (born IMV B-7069), an experiment was conducted with simulation of dysbacteriosis in rats. The study of the quantitative and qualitative composition of the microflora of the stomach, small and large intestines of rats was carried out by generally accepted methods.

As a result of the use of bioconcentrate A. migidap5 167K (born IMV B-7069) as part of the feed, the restoration of the normal coenosis of the intestine of experimental rats, a decrease in the number of opportunistic bacteria in the large intestine, Escherichia coli with unchanged enzymatic properties was sown in 105-105, enterococci were noted in 10, hemolytic forms of bacteria were practically absent, spore-forming forms of bacteria were sown both in control animals and in those that received ordinary feed (table 4).

Thus, the bioconcentrate of A. migidap5 167K (born IMV B8-7069) had a positive effect on the parameters of the microflora of the stomach, small and large intestines of experimental animals.

Indicators of microflora in animals that received the studied bioconcentrate as a biologically active additive to feed differed significantly from those in the control, which allows us to draw a conclusion about its beneficial effect on the quantitative and qualitative composition of the microflora of the gastrointestinal tract.

Table 4

Indicators of the composition of the microflora in rats based on 1 g of feces after 6 months of the experiment " bioconcentrate with enzymatic activity of microorganisms

Note; xx the difference is statistically significant at p»0.05 7 between animals receiving special feed and intact animals "x between animals receiving special feed and special feed with BAD. 3.2. Application of bioconcentrate A. migidap5 167K (born IMV B-7069 ) for the treatment of dental infections. In the treatment of teeth with chronic periodontitis in remission after medical and mechanical treatment of root canals and opening of apical holes in root canals, turundas with a bioconcentrate solution were left (2 doses of bioconcentrate (4.2x108 cells), 2ml of distilled water, 0 .5 ml of dimexide) under an airtight bandage.After two days, in the absence of clinical symptoms, the root canals were sealed with paste-like materials.

Treatment of acute and chronic periodontitis in the exacerbation stage was as follows. In the first visit, carious cavities were opened, mechanical and medicinal treatment of root canals. At the last stage, the root canals are washed with a bioconcentrate solution. The tooth was left open in order to drain the exudate. After three days, in the absence of symptoms of inflammation, the tubes with the bioconcentrate solution were left in the canals under a hermetic bandage. After 2-3 days, in the absence of inflammation, sealing was performed.

The conducted studies showed the high efficiency of the bioconcentrate in the treatment of periodontitis of the teeth: 138 teeth with chronic periodontitis in the remission stage were cured in two visits, 150 teeth with acute and chronic periodontitis in the exacerbation stage were cured in three visits and only 12 teeth whose X-ray showed the presence of granuloma size 0.5 by 0.5 cm, were cured after four visits. 3.3. Use of A. migidap5 167K bioconcentrate (born IMV B-7069) for the treatment of skin infections. Bioconcentrate A. migiChap5 167K (born IMV 8-7069) is used in the department of newborn pathology in the treatment of babies with third-degree diaper rash with necrotic changes. After irrigation of the affected areas with a bioconcentrate solution, rapid healing was noted with subsequent epithelization without the use of stereotypical treatment methods.

The duration of treatment was seven days, that is, twice as fast as when using traditional methods. 3.4. Treatment of dysbiosis.

Patients with dysbacteriosis caused by pathogens such as 5iari. atsgei5, Spigobrasieg, Kierzieva and Rgoieu5 utsIdagi5, Egehorasieg prescribed preparations of A. migidap5 167K (born IMV B-7069) for internal use in quantities that depended on the severity of the patient's condition. In all cases, the positive effect of such treatment came already on the second day. 3.5. Treatment of acute dysentery, escherichia and salmonellosis.

A. migidap5 167K (born IMV B-7069) was prescribed to patients on the background of traditional therapy to stop damage to the mucous membrane of the large intestine and intestinal dysbiosis. The drugs were administered internally and rectally in doses that depended on the severity of the course of the disease. The results are positive. 3.6. The use of A. migidap5 167K (born IMV B-7069) in gynecology also gave good results. Patients aged 4 to 17 years with intravaginal yeast, chlamydial, and trichomonad infections were prescribed douching with a bioconcentrate solution twice a day. After the course of treatment, in the vast majority of cases, the results were satisfactory - smears of the second and third degree of purity.

The strain A. migidap5 167K (born IMV B-7069), as well as the drug Vyabak (miarbas) based on lyophilized vegetative cells of the strain A. migidap5 167K (born IMV B-7069), exhibits antimicrobial activity and is effective for the treatment of infectious lesions of various localization.