RU2213140C2 - Strain of bacterium bifidobacterium infantis 37 b for preparing biologically active supplements regulating intestine microflora in infants - Google Patents

Abstract

FIELD: medicine, biotechnology, medicinal microbiology, pediatrics. SUBSTANCE: strain Bifidobacterium infantis 37 B (VKPM Ac-1636) is isolated from digestive tract of one year old health infant. The strain is able to inhibit the growth of opportunistic microorganism effectively and shows good acid- forming capacity and natural resistance to some antibiotics. The strain is non-pathogenic, apyrogenic for humans and animals and able to normalize digestive tract microflora in children. The strain is ready to usage in pediatric practice as biologically active supplements. Invention can be used for correction of disorders in microbiocenosis of digestive tract in children as biologically active supplements. EFFECT: valuable medicinal properties of strain. 3 tbl

Description

 The invention relates to medical microbiology, namely to pediatric practice, in particular, can be used in the manufacture of biologically active additives, as well as bacterial preparations for the normalization of microflora in cases of intestinal microbiocenosis disorders in young children.

 It is known that for the correction of dysbiotic changes in the gastrointestinal tract, the currently existing biological product Bifidumbacterin (Goncharova G.I., Dyakova E.I., Semenova L.P. and others) is used. The new domestic preparation Dry Bifidumbacterin and its effectiveness in intestinal diseases of children and adults. // All-Russian Congress of Epidemiologists, Microbiologists and Infectious Diseases: Abstracts. Kazan, 1972. - P.37-38), which includes representatives of the normal intestinal microflora. Biologically active additives are widely used abroad, including bifidobacteria, lactobacilli, and combinations thereof (Elmer GW, Mc Farland LW, Surawicz CM Biotherapeutic Agents and Infection Diseases. Human Press. 1999. - 316 P .; Fuller R. A review: Probiotics in man and animals. J. Appl. Bacteriol. l989.-66. P.365-378). But these drugs are developed without taking into account the characteristics of the species composition of the intestinal microflora in children. There are currently no drugs for the prevention and correction of dysbacteriosis in young children, which include a complex of microorganism strains that are most typical for microflora in children.

 The aim of the invention is to obtain a strain of bifidobacteria with antagonistic activity in vitro and in vivo, for use in the production of biologically active additives effective for the prevention and correction of dysbiotic disorders of intestinal microflora in young children.

 This goal was achieved in that a strain of Bifidobacterium infantis 37 B was isolated from the intestines of a clinically healthy child of the first year of life, belonging to the species characteristic of children of this age group, which has significant antagonistic activity against opportunistic microorganisms, good acid-forming function and natural resistance to a number of antibiotics.

 The strain is deposited in the All-Russian collection of industrial microorganisms "GosNIIGenetika" and is characterized by the following properties.

 Cultural and morphological properties. When cultivated in a liquid selective medium, Blaurock under anaerobic conditions forms a loose precipitate and uniform turbidity after 24 hours. When cultured in a column of 0.75% agar, Blaurock forms colonies in the form of buckwheat grains or discs up to 0.5-1 mm in diameter for 2 days. Blaurocca forms S-type colonies on a dense medium, round, with smooth edges, homogeneous, opaque, they do not form pigment, the profile of the colonies is convex, without gloss, and can easily be removed by a loop from the surface of a dense nutrient medium.

 In smears, the cells are arranged in the form of "Chinese characters", they look like straight or branched sticks with Y-shaped bifurcations at one or both ends, sometimes they form thickenings at the ends. Fixed, smeared randomly in the smear. They are stained gram-positive, sometimes granularity is revealed inside the cells. They do not form a dispute.

Physiological properties. Obligate anaerobic, grows in a high column of agar in vitro, in tubes with liquid medium and on Petri dishes with dense medium under anaerobic conditions. When cultured in a liquid medium under anaerobic conditions, 18 • 10 ⁸ -5.0 • 10 ⁸ live microbial cells grow in 1 ml of medium 18–20 hours after plating. The optimum growth at a temperature of 37.5 ^o .

 Biochemical signs. Synthesizes the enzyme fructose-6-phosphate phosphoketalase. Catalase does not form. It ferments fructose, galactose, glucose, lactose, ribose, raffinose, xylose, sucrose, maltose, and melibiosis. It does not ferment arabinose, cellobiose, melesitosis, mannose, trehalose, sorbitol, mannitol, inulin, salicin. Gelatin does not thin. Indole does not produce, does not reduce nitrates.

Acid formation when grown in Blaurock medium - 159.4 ^o (according to Turner) (table 2).

 Sensitivity to antibiotics is presented in table 1.

 Laboratory testing showed the non-pathogenicity of the Bifidobacterium infantis 37 B strain for experimental animals (mice), as well as for humans (in experiments on volunteers).

 The use of the strain is illustrated by the following examples.

 Example 1. The study of the antagonistic activity of the strain of Bifidobacterium infantis 37 In vitro.

 The determination of antagonistic activity was carried out in relation to opportunistic microorganisms: Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus faecalis, Clostridium perfringens. Bactofock agar (Hydrobiosis, Moscow) was used as a nutrient medium for the growth of bifidobacteria, and semi-liquid (0.75%) Brain Heart Infusion agar (BBL, USA) was used for indicator microorganisms.

 When studying the antagonistic activity of the Bifidobacterium infantis 37 B strain, 3.5 ml of 0.75% BHI agar, which was previously added with 0.1 ml of the daily culture of the indicator strain, was applied to plates with grown colonies of bifidobacteria.

After an 18-hour incubation at 37 ^° C under anaerobic conditions for clostridia and aerobic conditions for the remaining indicator microorganisms, the radii of the zones of growth inhibition of the indicator strains around the bifidobacteria colonies were measured.

 The experimental results showed that Bifidobacterium infantis 37B inhibits the growth of Escherichia coli (radius of the growth inhibition zone is 12.4 mm), Klebsiella pneumoniae (16.0 mm), Staphylococcus aureus (13.3 mm), Streptococcus faecalis (6.7 mm ), Clostridium perfringens (10.9 mm) (see table 3).

 The strain Bifidobacterium infantis 37 B actively inhibits the growth of opportunistic microorganisms in vitro.

 Example 2. The study of pathogenicity, pyrogen-free and antagonistic activity of strain Bifidobacterium infantis 37 In the intestines of volunteers.

The Bifidobacterium infantis 37 B strain was received by a volunteer for 20 years for 10 days at a dose of 1.0 • 10 ⁹ cells per day. Assessed the general condition, temperature in the morning and evening during the entire period of receiving a suspension of microorganisms. In addition, we conducted a bacteriological study of the contents of the colon before and on the second day after the end of the suspension of bifidobacteria.

 Another volunteer, 27 years old, received Bifidumbacterin as a control. He also had a bacteriological study of the colon microflora before and on the second day after taking the drug.

 During the entire observation period, the Bifidobacterium infantis 37 B strain did not cause any side effects, the subject did not complain, the body temperature remained normal, sleep and appetite were not disturbed, there were no dyspeptic disorders.

Analysis of bacteriological data revealed positive changes in the quantitative composition of microorganisms present in the intestines of the volunteer. There was an increase in the level of bifidobacteria from 2.0 • 10 ⁶ colony forming units per gram of intestinal contents (CFU / g) to 1.0 • 10 ⁸ CFU / g and lactobacilli from 3.0 • 10 ⁶ CFU / g to 1.5 • 10 ⁷ CFU / g, as well as a decrease in the level of enterococci from 2.9 • 10 ⁸ CFU / g to 4.9 • 10 ⁷ CFU / g, staphylococci from 1.0 • 10 ⁴ CFU / g to 2.3 • 10 ³ CFU / g, yeast fungi of the genus Candida from 2.3 • 10 ⁴ CFU / g to 1.1 • 10 ³ CFU / g.

The volunteer who took the drug "Bifidumbacterin" had an increase in the level of bifidobacteria from 7.7 • 10 ⁹ CFU / g to 1.1 • 10 ¹⁰ CFU / g. But at the same time, the level of opportunistic microorganisms did not change significantly.

 The strain Bifidobacterium infantis 37 B is non-pathogenic and pyrogen-free for humans and has antagonistic activity against opportunistic microorganisms not only in vitro, but also in vivo.

 Example 3. The study of the effect of the strain of Bifidobacterium infantis 37 B on the microflora of the intestines of the child.

The strain of Bifidobacterium infantis 37 B was received by a child of 3 years for 10 days at a dose of 1.0 • 10 ⁹ cells per day. A bacteriological study of the contents of the colon was performed before and on the second day after the end of the suspension of bifidobacteria.

 Another child received Bifidumbacterin for control. He also had a bacteriological study of the colon microflora before and on the second day after taking the drug.

Data analysis bacteriological examination showed positive changes in the quantitative composition of microorganisms present in the infant gut, who took the strain Bifidobacterium infantis B. 37 was observed with increasing levels of bifidobacteria 5,1 • September ¹⁰ colony forming units per gram of the material (cfu / g) to 9.9 • 10 ¹⁰ CFU / g and lactobacilli from 7.9 • 10 ⁷ to 3.6 • 10 ⁸ CFU / g.

The volunteer who took the drug "Bifidumbacterin" had an increase in the level of bifidobacteria from 2.6 • 10 ⁹ CFU / g to 1.1 • 10 ¹⁰ CFU / g. At the same time, the level of lactobacilli did not significantly change.

 Despite its apparent simplicity, the restoration of intestinal microflora in young children is a complex microbiological and pediatric problem. Isolation from the intestines of a clinically healthy child of the first year of life, which is promising for the production of a strain of bifidobacteria, which is most characteristic in contrast to the already known strains for the intestinal microflora of children of this age group, and the study of its properties is the fruit of a long research work. As a result of this, the strain Bifidobacterium infantis 37 B was obtained for the first time, possessing high antagonistic activity against opportunistic microorganisms, significant acid-forming function, and resistance to a number of antibiotics. The strain is tested for pathogenicity and pyrogen-free for animals and humans.

 All these properties make it possible to use the Bifidobacterium infantis 37 B strain for the production of biologically active additives for regulating the microflora of the gastrointestinal tract in young children.

 Strain Bifidobacterium infantis 37 B is completely ready for use.

Claims (1)

 The bacterial strain Bifidobacterium infantis VKPM Ac-1636 for the preparation of biologically active additives that regulate the intestinal microflora in young children.

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