RU2218394C2 - Strain of bacterium lactobacillus acidophilus 27l for preparing biologically active supplements regulating intestine microflora in children of early age - Google Patents

Abstract

FIELD: biotechnology, medicinal microbiology. SUBSTANCE: strain Lactobacillus acidophilus 27L is isolated from intestine of health child of the 1-st year life. The strain shows good acid- -forming properties, able to inhibit opportunistic microorganisms and shows resistance to some antibiotics. The strain has no pathogenic and pyrogenic properties for humans and animals, normalizes digestive tract microflora in children. The strain is deposited in VKPM at = --8237. The strain is ready for application in pediatric practice as biologically active supplements. Invention can be used as biologically active supplements for correction of disorders in biocenosis of digestive tract in children. EFFECT: valuable medicinal properties of strain. 2 tbl, 3 ex

Description

 The present invention relates to medical microbiology, in particular to pediatric practice, in particular, can be used in the manufacture of biologically active additives to normalize microflora in cases of intestinal microbiocenosis disorders in young children.

 The use of biologically active additives, including bifidobacteria, lactobacilli, and combinations thereof can serve as one of the methods for correcting intestinal microflora (Elmer G.W., Mc Farland L.W., Surawicz C. M. Biotherapeutic Agents and Infection Diseases. Human Press. - 1999. - 316 p.). However, complex preparations created taking into account the peculiarities of the species composition of bifidoflora and lactoflora of children are currently not available.

 In our country, traditionally, for the correction of dysbiotic changes in the gastrointestinal tract, the existing biological product Lactobacterin is used (Tarasova NB, Sokolova K.Ya. New drug lactobacterin for the prevention and treatment of intestinal dysbiosis .// All-Union Congress of Epidemiologists, Microbiologists and Infectious Diseases, 11th: Abstracts. - Tbilisi. - 1970. - 40 p.), Which includes lyophilized strains of bacteria that are representatives of the normal intestinal microflora (Efimov B.A., Korshunov V.M., Kafarskaya L.I., Tarabrina N.P., Gladko I.A. Dia Gnostics prophylaxis and treatment of intestinal dysbiosis. Methodical recommendations. - M.: MH RF. - 1991. - P.9). But these strains of microorganisms are isolated from adults and therefore not effective enough to regulate microflora in young children. Complex preparations, including strains of microorganisms that are most characteristic of children of this age group, are currently not available in Russia.

 The aim of the invention is to obtain a strain of lactobacilli, most typical of the intestinal microflora of young children, used in the production of biologically active additives for the regulation of dysbiotic disorders.

 This goal was achieved in that a strain of Lactobacillus acidophilus 27L was isolated from the intestines of a clinically healthy child of the first year of life, which belongs to the species most characteristic of children of this age group, which has a significant acid-forming function, good antagonistic activity against opportunistic microorganisms 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 cultured in a liquid selective medium, MRS (Mans, Rogosa and Sharpe broth) forms a loose precipitate and uniform turbidity after 24 hours. On a dense medium, MRS forms round colonies with uneven edges, homogeneous, opaque, they do not form pigment, the colony profile is flat, without gloss, and can easily be removed by a loop from the surface of a dense nutrient medium.

 In smears, cells look like short and long straight rods arranged in parallel, forming chains. They are painted gram-positive. Stationary, do not form a dispute.

Physiological properties. Microaerophil, growth on solid nutrient media is usually enhanced in anaerobiosis or under reduced oxygen pressure. The optimum growth at a temperature of 37.5 ^o . It grows well at a temperature of 45 ^o C. The optimum pH of 6.2-6.4.

 Biochemical signs. Catalase does not form. It ferments cellobiose, fructose, galactose, glucose, lactose, mannose, sucrose, trehalose, ribose, raffinose, maltose, salicin. Does not ferment arabinose, melesitosis, melibiosis, rhamnose, xylose, sorbitol, mannitol, adonitol, dulcitol, inositol, inulin. Gelatin does not thin. Indole does not produce.

Acid formation when grown in MRS medium is 256.4 ^° (according to Turner).

 Laboratory testing showed the non-pathogenicity of the Lactobacillus acidophilus 27L strain for experimental animals (mice), as well as for humans (in experiments on volunteers).

 Sensitivity to antibiotics is presented in table 1.

 Antagonistic activity against opportunistic microorganisms is presented in table 2.

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

 Example 1. The study of the acid-forming activity of a strain of Lactobacillus acidophilus 27L.

The activity of acid formation was determined by the titrometric method. To this end, 1 ml of a 1 billionth suspension of the daily culture of lactobacilli was introduced into test tubes with 9 ml of liquid MPC medium and cultured at 37.5 ^° C in anaerostats purged with a gas mixture. After 48-72-168 hours from the start of cultivation, two tubes were removed, from which 5 ml of culture liquid were taken and titrated with 0.1 N alkali solution. The amount of alkali used for titration corresponds to the amount formed of 0.1 n. acid in 5 ml of culture fluid. The final result was expressed in degrees Turner: T ^o = A • K • 20, where A is the amount of 0.1 N. alkali, which went on titration of 5 ml of the test liquid, K is the correction to the titer, determined by titration of 0.1 N. alkali solution 0.1 n succinic acid, T ^o - the value expressing the amount of 0.1 N. alkali, which went on titration of 100 ml of the test sample.

As a result of the studies, it was found that by 48 hours there was a significant acidification of the culture fluid (acidity was 174.7 ^o T). In the following hours, there was a slower increase to a maximum of 168 hours (acidity was 256.4 ^o T).

 The strain of lactobacilli Lactobacillus acidophilus 27L has a high acid-forming activity.

 Example 2. The study of pathogenicity, pyrogen-free and antagonistic activity of the strain Lactobacillus acidophilus 27L in the intestines of volunteers.

The Lactobacillus acidophilus 27L strain was received by a volunteer for 27 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 lactobacilli.

 Another volunteer, 24 years old, received Lactobacterin 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 Lactobacillus acidophilus 27L 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 2.4 • 10 ⁸ colony forming units per gram of test material (CFU / g) to 9.3 • 10 ⁹ CFU / g and lactobacilli from <10 ⁴ to 6.0 • 10 ⁶ CFU / g, and also a decrease in the level of enterococci from 1.1 • 10 ⁶ CFU / g to 5.0 • 10 ⁴ CFU / g and staphylococci from 1.0 • 10 ⁴ CFU / g to 2.2 • 10 ³ CFU / g.

A volunteer taking Lactobacterin showed an increase in the level of lactobacilli from 3.0 • 10 ⁶ CFU / g to 1.5 • 10 ⁷ CFU / g. But at the same time, the level of opportunistic microorganisms did not change significantly.

 The strain Lactobacillus acidophilus 27L 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 Lactobacillus acidophilus 27L on the microflora of the intestines of the child.

The strain of Lactobacillus acidophilus 27L 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 lactobacilli.

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

An analysis of bacteriological data revealed positive changes in the quantitative composition of microorganisms present in the intestines of a child taking the Lactobacillus acidophilus 27L strain. There was an increase in the level of bifidobacteria from 9.5 • 10 ⁹ colony forming units per gram of test material (CFU / g) to 8.1 • 10 ¹⁰ CFU / g and lactobacilli from 5.2 • 10 ⁵ CFU / g to 1.3 • 10 ⁷ CFU / g.

The volunteer taking the Lactobacterin preparation showed an increase in the level of lactobacilli from <10 ⁴ CFU / g to 2.4 • 10 ⁵ CFU / g. At the same time, the level of bifidobacteria did not change significantly.

 Despite its apparent simplicity, the restoration of intestinal microflora in young children through the use of biologically active additives 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 lactobacilli, 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 Lactobacillus acidophilus 27L was first obtained, which has a significant acid-forming function, high antagonistic activity against opportunistic microorganisms and natural resistance to a number of antibiotics.

 All these properties make it possible to use the Lactobacillus acidophilus 27L strain as biologically active additives for regulating the microflora of the gastrointestinal tract in young children.

 Strain Lactobacillus acidophilus 27L is completely ready for use.

Claims (1)

The bacterial strain Lactobacillus acidophilus VKPM B-8237 for the preparation of biologically active additives that regulate the intestinal microflora in young children.

Images