RU2641258C1 - Probiotic strain lactobacillus gasseri and its composition with lactoferrin for prevention of diarrhea, nerrocessing enterherolite and sepisis caused by escherichia coli strains in prematurely born children - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: Lactobacillus gasseri strain of BK-2918D VKM having antagonistic activity against Escherichia coli strains and a composition containing the strain of Lactobacillus gasseri ECM B-2918D and lactoferrin have been claimed. Probiotic strain lactobacillus gasseri and its composition with lactoferrin for prevention of diarrhea, nerrocessing enterherolite and sepsis caused by escherichia coli strains in prematurely born children.

EFFECT: expansion of the assortment of probiotic drugs and nutrient mixtures that provide the formation of normal microflora of the gastrointestinal tract in prematurely born children.

2 cl, 3 dwg, 3 tbl, 7 ex

Description

The invention relates to biotechnology and the medical industry and can be used in the production of bacterial preparations intended for the prevention of diarrhea, necrotizing enterocolitis and sepsis caused by Escherichia coli strains in premature babies.

In women of reproductive age, one of the main causes of miscarriage and premature birth is urogenital infectious disease. These diseases are associated with impaired microbiocenosis of the reproductive system of women. They are characterized by a decrease in the level of vaginal lactobacilli that control the species composition of this biotope and an increase in the level of opportunistic microorganisms [Ankirskaya A., 1995; Reis A., 1997; Reid G., 2008; Kuzmin V., 2011; Prilepskaya V., Bayramova G., 2011; Petrova M., 2015]. Therefore, a prematurely born baby when passing through the birth canal does not receive the microflora necessary for it, which is usually found in the reproductive system of a healthy mother. This leads to a violation of the first stage of the vertical physiological transfer of microorganisms from mother to child in the microecological system of mother-child. Normally, during urgent delivery, the baby is immediately applied to the mother’s chest. It has been established that during urgent delivery, lactobacilli accumulate in the mother’s breast nipples, which, when ingested in the newborn’s mouth, subsequently participate in the formation of a healthy microbiocenosis of his digestive system (second stage of vertical transmission of microorganisms from mother to child in the microecological mother-child system), in particular , stimulate the growth of bifidobacteria and accelerate the process of colonization of the intestine by these microorganisms, and also inhibit the process of colonization of the intestine by Escherichia coli [Martin R., et al., 2003]. In premature birth, there is no accumulation of lactobacilli in the nipples of the mother intended for transmission to the child, which indicates a violation of the second stage of the vertical transmission of microorganisms from mother to child in the microecological system of mother-child. In case of urgent delivery, immediate application of the baby to the mother’s breast stimulates the mother’s lactation process and filling the breast with initial colostrum milk. Colostrum contains a multifunctional protein of the transferrin family of lactoferrin (LF) at a maximum concentration of 7 mg / ml [Masson P., Heremans J., 1971]. By the time of birth, the baby does not yet have its own protection against intestinal infections. Lactobacilli transmitted from the mother stimulate the maturation of the intestinal mucosa and the formation of innate immunity of the newborn. LF also stimulates the maturation of the gastrointestinal mucosa, provides immunological protection and is involved in providing homeostasis of iron ions in the small intestine. The main mechanism of iron transport in the small intestine is carried out by the DMT-1 system (transporter 1 of divalent metals), which operates without the participation of LF [AndrewsN, 2000; Fleming M., et al., 1997; Gunshin H., et al., 1997]. An excess of iron ions in the intestines of a newborn is very harmful, as it leads to the activation of the growth of opportunistic and pathogenic microorganisms, as well as to the destruction of intestinal epithelial cells under the influence of oxidative stress induced by free radicals. LF controls the level of iron ions in the intestine. The increased concentration of LF in colostrum, as well as its significant content in breast milk (1 mg / ml) [Masson P., Heremans J., 1971] indicate that this protein belongs to the molecular factors of innate immunity and serves as the first line protection of the mucous surface of various organs, especially the gastrointestinal tract of the newborn, from various bacterial, fungal, viral pathogens. LF also protects intestinal epithelial cells from damage caused by free radicals [Hanson L., et al., 2002; Bellamy W., et al., 1993; Valenti P., Antonini G., 2005; Sanchez L., et al., 1992; Bullen J., 1972; Buldwin D., et al., 1984]. Primary expression of the LF gene is observed at the stage of 2-4 cells of the early period of mammalian embryonic development and continues until the end of the blastocyst formation stage. Further expression of the LF gene is terminated until the late stage of fetal development, when this protein begins to be synthesized in the epithelial cells of the digestive and respiratory systems, as well as in blood neutrophils [Ward P., et al., 1999]. In the adult animal organism, the LF gene is expressed by the epithelial cells of the internal glands, followed by the secretion of the protein into the mucosa and various biological fluids. LF expression in the mammary gland is controlled by the hormone prolactin, and in the reproductive organs by estrogen [Teng S., 2002]. Protection of the intestinal mucosa is provided by the bacteriostatic properties of LF, which are due to its ability to limit the intake of iron ions by pathogenic and conditionally pathogenic microorganisms [Sanchez L., et al., 1992]. LF also exhibits direct bactericidal activity. It binds directly to the outer membrane of gram-negative bacteria, which leads to the rapid release of microbial lipopolysaccharide, subsequent destruction of the membrane and the death of a bacterial cell [Ellison R., et al., 1988]. LF can exhibit antimicrobial properties through its proteolytic activity. LF hydrolyzes microbial adhesin proteins, which play a key role in bacterial colonization of the intestine by enteropathogenic E. coli strains; Shigella flexneri; Haemophilus influenzae [Plant A., et al., 2003; Ochoa T., et al., 2003; Gomez H., et al., 2003; Qiu J., et al., 1998].

Thus, a baby born prematurely does not receive from the mother a functional composition consisting of lactobacilli and LF, which controls the formation of a normal microbiota in its gastrointestinal tract and has a prophylactic effect on diarrhea, necrotizing enterocolitis and sepsis caused by E. coli strains . The patent WO 2009118771 A2 is known, in which human LF is patented, as well as a composition containing human LF and the probiotic Lactobacillus rhamnosus ATCC 53103 (LGG) for the manufacture of a medicament for the prevention of diarrhea, necrotizing enterocolitis and sepsis in the complex treatment of premature babies. The disadvantage of the above drug is the lack of a stimulatory effect of LF on the growth of L. rhamnosus ATCC 53103 (LGG) cells in the small intestine, which has been established in animal experiments [ShermanM., Et al., 2004].

The patent WO 2011051482 A1 is known in which compositions containing human or bovine LF, probiotics, prebiotics, a complex of vitamins and microelements for the production of drugs as means for the prevention of diarrhea, necrotizing enterocolitis and sepsis in the complex treatment of premature babies and children born in are patented. term. In the patented compositions, Lactobacillus rhamnosus CGMCC 1.3724, Bifidobacterium lactis CNCM 1-3446 (sold inter alia by the Christian Hansen company of Denmark), Bifidobacterium longum ATCC BAA-999 sold by Morinaga Milk Industry Co. are declared as probiotic components Ltd. of Japan, Lactobacillus paracasei CNCM 1-2116, Lactobacillus johnsonii CNCM 1-1225, Lactobacillus fermentum VRI 003 sold by Probiomics (Australia), Bifidobacterium longum CNCM 1-2170, Bifidobacterium longum CNCM 1-2618, Bifidobacter danve breve sold , Bifidobacterium breve sold by Morinaga (Japan) and the strain of Bifidobacterium breve sold by Institut Rosell (Lallemand), Lactobacillus paracasei CNCM 1-1292, Lactobacillus rhamnosus ATCC 53103 obtainable inter alia from Valio Oy of Finland, Enterococcus faecium. strains of Lactobacillus rhamnosus CGMCC 1.3724, as well as strains of Lactobacillus reuteri ATCC 55730, ATCC PTA 6475, ATCC PTA 4659 and ATCC PTA 5289, L. reuteri DSM 17938 company Biogaia AB (Kungsbroplan 3A) are claimed for inclusion in the pharmaceutical compositions.

In the above patents, strains of lactobacilli belonging to the species Lactobacillus gasseri are not included in the composition of the patented preparations. Strains of this species are found much more often than strains of lactobacilli of other species in breast milk, in the vaginal microbiota of healthy women of reproductive age and in the microecological system of mother-child [Ravel. J., et al., 2011; Martin R., et al., 2003; Jeurin J.V., et al., 2013], which indicates the evolutionary adaptation of this species of lactobacilli to the natural formation of the composition of L. gasseri + LF in breast milk of the mother. In this regard, to improve probiotic drugs and nutritional mixtures intended for the prevention of diarrhea, necrotizing enterocolitis and sepsis in premature babies, it is also advisable to use L. gasseri strains with probiotic properties together with LF.

The objective of the present invention is to expand the range of probiotic drugs for the prevention of diarrhea, necrotizing enterocolitis and sepsis caused by E. coli strains in premature babies.

The problem is solved in that a probiotic strain L. gasseri VKM B-2918D was obtained, as well as its composition L. gasseri VKM B-2918D + LF for the manufacture of bacterial preparations used to prevent diarrhea, necrotizing enterocolitis and sepsis induced by strains E. coli in premature babies. The strain L. gasseri VKM B-2918D was isolated from the stool of a healthy child at the age of 10 days when studying the species spectrum of lactobacilli circulating in the microecological system of mother-child. This strain was also sown from breast milk and vaginal discharge of the mother. The strain L. gasseri VKM B-2918D was identified by 16S rRNA gene sequencing and deposited in the All-Russian collection of microorganisms of the Institute of Biochemistry and Physiology of Microorganisms named after G.K. Scriabin RAS, (Moscow region, Pushchino).

The strain of Lactobacillus gasseri VKM B-2918D is characterized by the following features.

Cultural and morphological characters.

When grown on the surface of an agar medium MPC pH-6.5 (Himedia, India) by the method of exhausting bar in a thermostat at 37 ° C for 24-48 hours, the strain L. gasseri VKM B-2918D forms small, pale white, round colonies, flat with a smooth edge.

When grown on liquid MPC pH-6.5 (Himedia, India) or milk at 37 ° C for 24-48 hours, cells of the strain L. gasseri VKM B-2918D are gram-positive bacilli ~ 5-6 μm in size, form chains .

Physiological and biological signs.

Strain L. gasseri VKM B-2918D is an optional anaerobic, temperature optimum 37 ± 2 ° C, growing at 42 ° C, weak growth at 30 ° C. The optimal pH value is 5.5-6.2.

Biochemical properties.

The ability of the L. gasseri VKM B-2918D strain to ferment carbohydrates is evaluated in an API test in accordance with the manufacturer's instructions (BioMerieux, France).

The strain L. gasseri VKM B-2918D assimilates D-glucose, D-fructose, N-acetylglucosamine, D-sucrose, D-trehalose, hydrolyzes esculin.

Sensitivity to antibiotics.

The strain L. gasseri VKM B-2918D is sensitive to gentamicin, chloramphenicol, ofloxacin, tetracycline and is resistant to vancomycin.

The method, conditions and composition of culture media for propagating the strain of Lactobacillus gasseri VKM B-2918D.

The strain L. gasseri VKM B-2918D is grown at 37 ± 2 ° C in liquid medium MPC or in skim milk (0.01-0.03% fat).

Commercial skim milk (0.01-0.03% fat) is sterilized for 20 minutes by autoclaving at a pressure of 0.5 atm and a temperature of 110 ° C.

To grow isolated colonies of the L. gasseri VKM B-2918D strain, agarized MPC medium containing 1.4% agar (Difco, United States) is used.

The method, conditions and composition of media for storage of a strain of Lactobacillus gasseri VKM B-2918D.

Strain L. gasseri VKM B-2918D can be stored:

- in sterile skim milk at 4 ° C with periodic reseeding once every 15-20 days;

- in a lyophilized state in sealed ampoules (protective medium during drying - sucrose 10%, pH 7.0 or sterile skim milk) for 2 years at a temperature of 4 ° C.

The claimed solution is illustrated by the following figures:

FIG. 1. An analysis in the Scatchard coordinates of the specific binding of ¹²⁵ I-LF to Caso-2 epithelial cells.

- активированные клетки Сасо-2 (выращенные в среде ДМЕМ с добавкой культуральной жидкости штамма Lactobacillus gasseri ВКМ B-2918D).X-axis: the amount of labeled LF bound to the cells (in moles). Y-axis: the ratio of the concentration of bound and free labeled LF. • - intact Caco-2 cells (grown in DMEM, control),

- activated Caco-2 cells (grown in DMEM medium supplemented with culture fluid of Lactobacillus gasseri strain VKM B-2918D).

Note: human LF was used in experiments (Sigma, USA).

FIG. 2. The effect of Lactobacillus gasseri VKM B-2918D and a composition containing Lactobacillus gasseri VKM B-2918D + LF on the growth of lactic acid bacteria in various sections of the small intestine of newborn rat pups.

- p<0,05 достоверность различий между группами крысят, получавшими штамм L. gasseri ВКМ В-2918D и получавшими композицию, содержащую L. gasseri ВКМ B-2918D + ЛФ.1 - proximal (jejunum), 2 - distal (ileum). On the ordinate axis is the growth of lactic acid bacteria from homogenates of various departments of the small intestine, expressed in CFU in terms of gram of intestine / cm length of intestine. Control - intact rat pups. * - p <0.05, the significance of differences between the intact group and the group of rat pups treated with the strain L. gasseri VKM B-2918D; ** - p <0.01, the significance of differences between the control group and rats receiving a composition containing L. gasseri VKM B-2918D + LF;

- p <0.05, the significance of differences between groups of rat pups receiving L. gasseri VKM B-2918D strain and receiving a composition containing L. gasseri VKM B-2918D + LF.

Note: human LF was used in experiments (Sigma, USA).

FIG. 3. The effectiveness of inhibiting the growth of Escherichia coli in various parts of the small intestine of rat pups, which received intragastrally Lactobacillus gasseri VKM B-2918D, a composition containing Lactobacillus gasseri VKM B-2918D + LF, LF preparation.

On the ordinate axis is the growth of E. coli from homogenates of various departments of the small intestine, expressed in CFU in terms of gram of intestine / cm of intestinal length.

- p<0,05 достоверность различий между уровнем Е. coli в тощей кишке в контроле и у крысят, получивших профилактическую дозу штамма L. gasseri ВКМ B-2918D (LG) или профилактическую дозу препарата ЛФ;

- p<0,01 достоверность различий между уровнем Е. coli в тощей кишке в контроле и у крысят, получивших профилактическую дозу композиции, содержащей Lactobacillus gasseri ВКМ B-2918D + ЛФ; * - p<0,05 достоверность различий между уровнем Е. coli в подвздошной кишке в контроле и у крысят, получивших профилактическую дозу штамма L. gasseri ВКМ B-2918D (LG) или профилактическую дозу препарата ЛФ; ** - p<0,01 достоверность различий между уровнем Е. coli в подвздошной кишке в контроле и у крысят, получивших профилактическую дозу композиции, содержащей Lactobacillus gasseri ВКМ B-2918D + ЛФ.

- p <0.05, the significance of differences between the level of E. coli in the jejunum in the control and in rat pups who received a prophylactic dose of L. gasseri VKM B-2918D (LG) strain or a prophylactic dose of LF;

- p <0.01, the significance of differences between the level of E. coli in the jejunum in the control and in rat pups who received a prophylactic dose of a composition containing Lactobacillus gasseri VKM B-2918D + LF; * - p <0.05, the significance of differences between the level of E. coli in the ileum in the control and in rat pups who received a prophylactic dose of L. gasseri VKM B-2918D (LG) strain or a prophylactic dose of LF; ** - p <0.01, the significance of differences between the level of E. coli in the ileum in the control and in rat pups who received a prophylactic dose of a composition containing Lactobacillus gasseri VKM B-2918D + LF.

Note: human LF was used in experiments (Sigma, USA).

The invention is illustrated by the following examples.

Example 1. Cultivation of a strain of Lactobacillus gasseri VKM B-2918D in the laboratory.

To obtain inoculum in an ampoule containing 10 mg of a lyophilized culture of L. gasseri VKM B-2918D strain, 0.5 ml of a 0.9% sterile sodium chloride solution is added and the culture is grown on the surface of MPA agar medium by a depleting bar in an incubator at 37 ° C for 18 hours. Then, the isolated colony was placed in a bacteriological tube containing 7 ml of liquid MPC medium and grown in an incubator at 37 ° C for 15 hours.

The resulting seed in an amount of 1% is introduced into vials containing 500 ml of liquid MPC medium and grown at 37 ° C for 18 hours. A culture is obtained with a titer of 0.5 × 10 ⁹ CFU / ml. By adjusting the cell concentration to 1.0 × 10 ⁹ CFU / ml, a standardized starting biomass of L. gasseri VKM B-2918D strain is obtained.

For long-term storage, the cells are freed from the components of the growth medium by centrifugation, transferred to a drying medium (protective medium during drying is sterile skim milk), 1 ml is packaged in penicillin vials and lyophilized. In the freeze-dried state, the starting biomass can be stored for two years at 4 ° C.

Example 2. The study of the resistance of the strain of Lactobacillus gasseri VKM B-2918D to gastric and intestinal stress (the ability to grow in an environment with a low pH or in an environment containing bile).

In the study, the following methods are used.

In vitro imitation of gastric stress

Artificial Gastric Juice Composition:

NaCl (Sigma S9625) 2.2 g / l L-lactic acid (Sigma L1750) 9.9 g / l (0.11 M) Pepsin (Sigma P7125) 3.5 g / l pH: 2.7 ± 0.02 (adjusted 35% NaOH) pH after 1/11 dilution: 3.10 ± 0.10

1 ml of artificial gastric juice is added to 100 μl of the culture in the stationary phase (1/11 dilution). The culture is incubated for 10 minutes, 30 minutes and 60 minutes at 37 ° C, 10% CO ₂ . As a control, instead of artificial gastric juice, 1 ml of MPC is used. Measurements are performed in duplicate.

After incubation, the culture (each repetition and control) is diluted from 10 ² to 10 ¹⁰ in MPC, dilutions are plated on plates with MPC-agar and incubated for 24-48 hours at 37 ° C, 10% CO ₂ ). The titer of the culture is determined under stress and without stress by counting the number of colonies for dilutions.

In vitro imitation of intestinal stress

Artificial intestinal juice composition:

bile salts (pig bile Sigma B8631) 3.3 g / l (final concentration: 0.3%),

NaHCO ₃ carbonate buffer (Sigma S8875) 16.5 g / l (final concentration: 1.5%) pH: 6.3.

1 ml of artificial intestinal juice is added to 100 μl of the culture in the stationary phase (1/11 dilution). The culture is incubated for 5 hours at 37 ° C, 10% CO ₂ . As control use 1 ml of MPC. Measurements are performed in duplicate.

Culture and control are diluted from 10 ² to 10 ¹⁰ in MPC, dilutions are plated on plates with MPC agar and incubated for 24-48 hours at 37 ° C, 10% CO ₂ . The titer is determined under stress and without stress by counting the number of colonies for dilutions.

The calculation of microorganisms in a milliliter of CFU culture is carried out according to the formula:

ΣC / (n ₁ +0.1 n ₂ ) d

where: ΣС is the sum of all characteristic colonies counted on all plates containing from 15 to 300 colonies;

n ₁ is the number of cups in the lowest dilution (2 cups per dilution);

n ₂ is the number of cups in the highest dilution (2 cups per dilution);

d is the value of the first dilution (low dilution) taken for counting.

To determine the resistance of lactobacilli to gastric and intestinal stresses, cultures are used in the stationary phase of growth grown on liquid medium MPC, pH 6.5 for 18 hours at 37 ° C, 10% CO ₂ .

The results are shown in table. 1 show that the strain L. gasseri VKM B-2918D is resistant to gastric and intestinal stress.

Example 3. The study of the antagonistic activity of the strain of Lactobacillus gasseri VKM B-2918D against enteropathogenic strains of Escherichia coli.

The study of antagonistic activity is carried out by the method of two-layer agar [Ermolenko E. et al., 2004] using indicator cultures of E. coli.

The antagonistic activity of lactobacilli is determined by the zone of growth inhibition of indicator strains around the colonies of individual strains of lactobacilli.

The research results are shown in table 2.

The results indicate that the claimed strain of L. gasseri VKM B-2918D exhibits pronounced antagonistic activity against strains of E. coli.

Example 4. A study of the adhesion of Lactobacillus gasseri VKM B-2918D on epithelial cells of Saso-2 small intestine of a person.

The adhesion of probiotic lactobacilli is considered as a positive sign, which allows to protect the gastrointestinal tract from pathogenic and conditionally pathogenic microorganisms. Probiotic lactobacilli intended for the prevention and complex treatment of intestinal infectious diseases should have adhesion to human enterocytes.

The ability of lactobacilli to adhere to the transplanted cell line of Caco-2 human small intestine is assessed by the method described in [Coconnier et al., 1993].

Caco-2 is grown in DMEM medium containing additives (10% fetal calf serum, 1% penicillin-streptomycin, 0.2 mM Hepes, 2 mM L-glutamine) at 37 ° C in a CO ₂ incubator. Saso-2 cells at a concentration of 3.0 × 10 ⁵ cells / ml were plated on 24-well plates and cultured at 37 ° C in a CO ₂ incubator. Upon reaching 70-80% of the monolayer, the cells are washed 3 times with DMEM without additives, then DMEM with L-glutamine is added to them and used to evaluate the adhesive properties of lactobacilli. Lactobacilli L. gasseri VKM B-2918D (test) and L. rhamnosus ATCC 53103 (LGG), used as a reference preparation, were grown (each strain in two tubes) in MPC medium at 37 ° C for 16 hours. Then, LF is added to one of the tubes of each strain to a final concentration of 0.25 mg / ml and cultivation is continued for another 2 hours. Then, the lactobacilli are washed twice with PBS pH 7.2 by centrifugation at 5000 rpm for 15 minutes and resuspended in DMEM with L-glutamine. The concentration of lactobacilli is determined by optical density (ODλ = 590 nm) and is controlled by seeding on an agarized medium MPC. The multiplicity of infection (MOI) is 100 bacteria / cell. Saso-2 cells with lactobacilli introduced to them are incubated for another 5 hours at 37 ° C in a CO ₂ incubator. After incubation, Caco-2 cells were washed 3 times with PBS pH 7.2, then fixed in cold ethanol (3 min) and stained with Romanovsky-Giemsa stain for 30 min. Next, the tablets are washed with distilled water, dried at room temperature and examined microscopically. Adhesion of lactobacilli on Caso-2 epithelial cells is evaluated by two indicators.

1. Adhesion activity - the number of epithelial cells with adhered lactobacilli out of 100 counted epithelial cells,%.

2. Adhesive number - the average number of lactobacilli adhered to one epithelial cell, conv. units

The results of studies evaluating the ability of L. gasseri VKM B-2918D and L. rhamnosus ATCC 53103 (LGG) to adhere to the surface of Caco-2 epithelial cells are summarized in Table 3. The introduction of LF into the culture medium of MPC during the cultivation of L. gasseri VKM B- 2918D significantly increases the ability of lactobacilli of this strain to adhere to the surface of Caco-2 epithelial cells and does not affect the adhesion indicators of the L. rhamnosus ATCC 53103 strain (LGG). The in vitro results suggest that in the in vivo system, LF will stimulate the adhesion of cells of the strain L. gasseri VKM B-2918D on the surface of small intestine epithelial cells.

Example 5. Evaluation of the specific interaction of ¹²⁵ I-LF with receptors on the surface of transplantable epithelial cells of Saso-2 of the small intestine of a person. To determine the specific binding parameters of ¹²⁵ I-LF with Caco-2 epithelial cells, a graph is plotted of the relationship between the molar concentration of bound labeled ¹²⁵ I-LF and free labeled ¹²⁵ I-LF (B / F) versus the molar concentration of bound labeled ¹²⁵ I-LF (B ) [Tkachuk V.A., 1983]. The specific binding of ¹²⁵ I-LF to Caco-2 cells is evaluated by the difference between its “total” binding (labeled ¹²⁵ I-LF + Caco-2 cells) and “non-specific” binding (labeled ¹²⁵ I-LF + Caco-2 + cells unlabeled LF). To obtain ¹²⁵ I-LF, a ³⁰ Na ¹²⁵ I radioactive label (Amersham) and a label reagent for Iodo-Gen protein (Picrce, Rockford, IL) are used. Labeled protein is separated from the free label by chromatography on a Sephadex G-25.

Caco-2 cells (intact cells, control) are grown in DMEM containing additives (10% fetal calf serum, 1% penicillin-streptomycin, 0.2 mM Hepes, 2 mM L-glutamine) at 37 ° C in CO ₂ - incubator for 24 hours. After 18 hours of cultivation, 50 μl of the culture fluid of the L. gasseri VKM B-2918D strain are added to the tablets containing the Caco-2 cells of the experimental group and they continue to be grown in the CO ₂ incubator for another 4 hours. The total time for growing Saso-2 cells of the experimental and control groups is 24 hours. The cells of the experimental and control groups were washed 3 times with sterile 50 mM Tris-buffer containing 0.15 M NaCl, pH 7.5 and used to study the reception of ¹²⁵ I-LF. The binding reaction of labeled ¹²⁵ I-LF with Caco-2 cells was carried out at 4 ° C for 30 minutes in 50 mM Tris buffer containing 0.15 M NaCl, pH 7.5. The results of the assessment of specific binding are presented in FIG. 1. The specific interaction of ¹²⁵ I-LF with high affinity receptors on the surface of Caco-2 cells is characterized by Kd = (1.7 ± 0.5) × 10 ^-9 M. Addition of culture fluid of L. gasseri VKM B-2918D strain to Caco-cells 2 experimental group 2 in the process of growing increases the number of sites of specific binding of ¹²⁵ I-LF. These data indicate that the strain L. gasseri VKM B-2918D is able to increase the colonization resistance of epithelial cells of the small intestine of a person by stimulating screening of their surface using the molecular factor of innate immunity - LF.

Example 6. Evaluation of the ability of the strain of Lactobacillus gasseri VKM B-2918D and the composition containing the strain of Lactobacillus gasseri VKM B-2918D + LF to colonize various sections of the small intestine of newborn rat pups.

In the experiments, rats of Wistar females were used and rat pups at two days of age.

The rat model is used due to the fact that LF is not contained in rat milk [Masson PL, Heremans JF, 1971]. Cubs are divided into three groups of 6 animals each. The first group of rat pups is injected intragastrally with a culture of L. gasseri VKM B-2918D strain at a dose of 10 ⁷ CFU / kg on the third and fourth day from birth. The second group of rat pups is administered intragastrically on the third and fourth day from birth a composition containing 10 ⁷ CFU / kg L. gasseri VKM B-2918D + LF (500 mg / kg / day) (Sigma, USA). The third group of pups serves as an intact control. On the fifth day, rat pups are euthanized, the small intestine is isolated and it is divided into the proximal (jejunum) and distal (ileum). Both parts of the small intestine are washed with sterile saline and homogenized in 1 ml of saline. Homogenates of the proximal and distal parts of the small intestine are seeded on MRS agar, Himedia, India. The cultivation is carried out in a CO ₂ incubator in an atmosphere of 5% CO ₂ at 37 ° C for 72 hours. The content of lactic acid bacteria in the homogenates of various sections of the small intestine is expressed in CFU in terms of gram of intestine / cm of intestinal length. The research results are shown in FIG. 2. In the experimental groups of rat pups after enteral therapy with L. gasseri VKM B-2918D or a composition containing the L. gasseri VKM B-2918D + LF strain, the content of lactic acid bacteria significantly increased on the fifth day of life compared with the control group of animals. LF stimulated the growth of lactic acid bacteria in various parts of the small intestine. In the group of control animals, on the fifth day of life, lactic acid bacteria identified by sequencing of the 16 SrRNA gene 16 as Lactococcus were detected in the homogenates of the small intestine. In experimental groups of animals, L. gasseri strain was found in homogenates of the proximal and distal small intestines. In the homogenates of the small intestine of rat pups of the control group, the strain L. gasseri was not detected.

Example 7. Evaluation of the ability of a strain of Lactobacillus gasseri VKM B-2918D and a composition containing a strain of Lactobacillus gasseri VKM B-2918D + LF to inhibit the growth of Escherichia coli ATCC 25922 in the small intestine of newborn rats.

In the experiments, rats of Wistar females were used and rat pups at two days of age. Cubs are divided into four groups of 6 animals each. On the third and fourth day, the rat pups of the first experimental group were injected intragastrally with a culture of L. gasseri VKM B-2918D strain at a dose of 10 ⁷ CFU / kg. On the third and fourth day, the rat pups of the second experimental group were administered an intragastric composition containing 10 ⁷ CFU / kg L. gasseri VKM B-2918D + LF (500 mg / kg / day) (Sigma, USA). On the third and fourth day, pups of the third experimental group were administered intragastrally LF (500 mg / kg / day) (Sigma, USA). The fourth group of pups serve as an intact control. On the fifth day, all rat pups are infected by intragastric administration of E. coli ATCC 25922 at a dose of 10 ¹² CFU / kg. 16 hours after infection, rats are euthanized, the proximal and distal small intestines are isolated, washed with sterile saline and homogenates are prepared in 1 ml of saline. Homogenates are seeded on an agarized selective medium and cultivated in an incubator for 48 hours at 37 ° C. The content of E. coli is expressed in CFU in terms of gram of intestine / cm of intestinal length. The research results are shown in FIG. 3. In homogenates of various departments of the small intestine of experimental groups of rat pups who received an intragastric prophylactic dose of L. gasseri VKM B-2918D strain, a prophylactic dose of a composition containing L. gasseri VKM B-2918D + LF strain or a prophylactic dose of LF, a significant decrease in the number E was found coli compared with the control group. The most effective inhibition of E. coli growth in the small intestine was observed in the group of rat pups who received an intragastric prophylactic dose of a composition containing the L. gasseri BKM B-2918D + LF strain. The research results indicate the promise of using the L. gasseri VKM B-2918D strain and a composition containing the L. gasseri VKM B-2918D + LF strain as the basis for the creation of probiotic preparations for the prevention of diarrhea, necrotizing colitis and sepsis caused by E. coli strains in prematurely born children.

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Claims (2)

1. The bacterial strain Lactobacillus gasseri VKM B-2918D, having antagonistic activity to strains of Escherichia coli. 2. A composition consisting of a bacterial strain Lactobacillus gasseri VKM B-2918D and lactoferrin for the manufacture of a bacterial preparation for the prevention of diarrhea, necrotizing colitis and sepsis caused by Escherichia coli strains in premature babies.

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