RU2528862C1 - STRAIN Lactobacillus fermentum HAVING BROAD SPECTRUM OF ANTAGONISTIC ACTIVITY AND PROBIOTIC LACTOBACTERIUM CONSORTIUM FOR MANUFACTURING BACTERIAL PREPARATIONS - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain Lactobacillus fermentum RCM B-2793D is proposed, which has antagonistic activity against pathogenic and opportunistic microorganisms. Also the consortium of strains of Lactobacillus fermentum RCM B-2793D, Lactobacillus crispatus VKM B-2727D, Lactobacillus gasseri VKM B-2728D, Lactobacillus plantarum VKM B-273D is proposed, which has probiotic action and is used for production of bacterial preparations and lactic acid products of functional nutrition. The consortium has a higher antagonistic activity against pathogenic and opportunistic microorganisms compared to individual strains of lactobacilli in its composition.

EFFECT: group of inventions enables to expand the range of probiotic strains, the means for prevention and treatment of mastitis in breastfeeding mothers.

2 cl, 1 dwg, 5 tbl, 12 ex

Description

The invention relates to biotechnology, the medical and food industries and can be used in the production of bacterial preparations and functional food products intended for the prevention and treatment of mastitis in breast-feeding mothers.

The microflora of a person is a system that combines many microbiocenoses characterized by a certain composition and occupying the corresponding biotopes in the human body. The leading microbiocenoses are the gastrointestinal tract, urogenital tract, respiratory tract, and skin [1]. The microflora of the body is affected by many factors, such as antibiotics, stress, eating disorders, environmental disorders, various diseases, all of which can lead to the development of dysbiosis.

One of the consequences of violation of the microbiocenosis of the gastrointestinal tract, urogenital tract and skin in mothers who are breast-feeding, is the occurrence of mastitis. Acute mastitis occurs in women in the process of establishing breastfeeding. A complication of acute mastitis is recurrent mastitis, the etiological agent of which are Staphylococcus aureus and Staphylococcus epidermidis. Clinical isolates of these pathogens are characterized by resistance to a wide range of antibiotics [2] and the ability to use human milk oligosaccharides as growth factors [3].

In general, the use of probiotics is relevant to restore and maintain normal microbiota [4]. In particular, probiotic prophylaxis and therapy of mastitis, which, according to statistics, affect about 30% of women during lactation, are an important problem [2, 5].

Known strains of the species Lactobacillus fermentum, used for the correction of dysbiosis.

So, the known strain of Lactobacillus fermentum 90-Ts-4, characterized by high adhesive properties to the vaginal epithelium [6], however, the disadvantage of this strain is a narrow spectrum of application, limited only to the solution of gynecological problems. A known strain of Lactobacillus fermentum ME-3 DSM 14241 [7].

The strain is used for the preparation of a medicinal product for the prevention and treatment of intestinal and urogenital infections, and is also used for the preparation of functional nutrition products, however, data on the effectiveness of its use for the prevention and treatment of mastitis in breast-feeding mothers are not available.

A known strain of Lactobacillus fermentum TS 3-06 [8]. The strain is used for the manufacture of bacterial preparations and the production of liquid milk sourdough as a food product for therapeutic purposes. It has a high growth rate and resistance to extreme factors, such as low acidity and bile, but there is no evidence of its effectiveness in the prevention and treatment of mastitis in breast-feeding mothers.

Known strains of lactobacilli (Lactobacillus fermentum CECT 5716 and Lactobacillus salivaris CECT 5713), the oral administration of which reduces bacterial contamination of breast milk with staphylococci (Staphylococcus aureus and Staphylococcus epidermidis) [10]. At the same time, the therapeutic effect for infectious mastitis is manifested, however, there is no data on the duration of the disease remission.

A known strain of Lactobacillus fermentum Lc40, isolated from breast milk of a healthy woman, on the basis of which the Australian company Puremedic developed a probiotic drug QIARA, intended for the prevention and treatment of acute mastitis [11], but there is no data on the effectiveness of this drug in the treatment of recurrent mastitis.

The objective of the present invention is to expand the range of probiotic strains, in particular, with pronounced antagonistic properties against Staphylococcus aureus and Staphylococcus epidermidis, and probiotic consortia that can be used to obtain bacterial preparations and fermented milk functional food products intended, in particular, for treatment and prevention of infectious mastitis.

The problem is solved in that a probiotic strain of Lactobacillus fermentum 3872 was obtained, as well as a consortium of strains of Lactobacillus fermentum 3872, Lactobacillus crispatus 2029, Lactobacillus gasseri 2016, Lactobacillus plantarum 2025, which has probiotic activity, can be used and can direct starter cultures for the fermentation of milk and the production of fermented milk functional foods aimed at the prevention and treatment of mastitis in breast-feeding mothers.

Strain Lactobacillus fermentum 3872 was isolated from breast milk by studying the species spectrum of lactobacilli in a healthy mother-child pair. This strain was also found in the feces of the child and mother and the vaginal secretion of the mother.

The strain was identified by 16S rRNA gene sequencing method, 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) and has a registration number VKM B-2793D.

The strain 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, it forms medium, white, round, convex colonies with a smooth edge.

When grown in liquid medium MPC pH-6.5 (Himedia, India) or milk at 37 ° C for 24-48 hours, the cells of the strain represent short sticks ~ 3 μm in size, do not form chains.

Physiological and biological signs.

The strain is an optional anaerobic, temperature optimum 37 ± 2 ° C, grows at 42 ° C, weak growth at 30 ° C. The optimal pH of the medium is 5.5-6.0.

Biochemical properties.

The ability of the strain to ferment carbohydrates is evaluated in an API test in accordance with the manufacturer's instructions (BioMerieux, France).

The strain assimilates D-Ribose, D-Galactose, D-Glucose, D-Fructose, D-Maltose, D-Lactose, D-Melibiosis, D-Sucrose, D-Trehalose, D-Raffinose API test: Lactobacillus fermentum ID = 99.8 %; T = 0.89.

Genomic fingerprint

Strain-specific identification of Lactobacillus fermentum 3872 is carried out by the method of genomic fingerprint using primers based on diverged inverted repeats (DIR) in the coding regions of the genome [13]. DIR-PCR is carried out in 25 μl of a mixture of the following composition: 1x BioTaq polymerase buffer (17 mM (NH ₄ ) SO ₄ , 6 mM Tris-HCl, pH 8.8, 2.5 mM MgCl ₂ ), 7.5 nM dNTP, 50 ng of the DNA template, 10 pcM primer and 1.25 units BioTaq DNA Polymerase ("Dialat LTD", Russia). The temperature-time profile of the reaction was as follows: first cycle — 94 ° C × 30 sec; 30 ° C × 30 sec; 72 ° C × 1 min 30 sec; the next 43 cycles - 94 ° C × 5 sec, 30 ° C × 30 sec and 72 ° C × 1 min 30 sec; final elongation - 7 min at 72 ° C. Analysis of PCR products is carried out by electrophoresis in 1.5% agarose gel stained with ethidium bromide at a field strength of 6 V / cm.

The results shown in figure 1 show that the spectra obtained on DNA isolates of Lactobacillus fermentum isolated from breast milk, baby feces, vaginal secretions and mother's feces, are similar in number and size of fragments, indicating their complete identity. All four isolates are a strain of Lactobacillus fermentum 3872, which can live in various biotopes of the ecological system "mother-child".

Sensitivity to antibiotics.

Strain L.fermentum 3872 is resistant to metronidazole and ciprofloxacin.

The method, conditions and composition of the media for the propagation of the strain.

The strain Lactobacillus fermentum 3872 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.

For the cultivation of individual colonies of the strain using agarized medium MPC containing 1.4% agar (Difco, USA).

Method, conditions and composition of media for storage of strain

Strain Lactobacillus fermentum 3872 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;

- frozen at -70 ° C. Duration of storage is 6 months.

The strains of Lactobacillus crispatus 2029, Lactobacillus gasseri 2016, Lactobacillus plantarum 2025, included in the inventive consortium along with the strain Lactobacillus fermentum 3872, were isolated from the vagina of healthy women of reproductive age, identified by microorganism sequencing of the microorganism organism, and they were stored in a microorganism organism . G.K. Scriabin RAS (Moscow region, Pushchino) under the following registration numbers:

The strain Lactobacillus crispatus 2029 has a registration number VKM B-2727D;

The strain Lactobacillus gasseri 2016 has a registration number VKM B-2728D;

The strain Lactobacillus plantarum 2025 has a registration number VKM B-2731D.

The strains of Lactobacillus crispatus 2029, Lactobacillus gasseri 2016, Lactobacillus plantarum 2025 are characterized by the following features.

Cultural and morphological characters.

When growing on the surface of an agarized medium MPC pH-6.5 (Himedia, India) by the method of exhausting bar in a thermostat at 37 ° C for 24-48 hours:

- strain Lactobacillus crispatus 2029 - colonies are medium, pale white, round, flat with a smooth edge,

- Lactobacillus gasseri strain 2016 - small, pale white, round, flat colonies with a smooth edge.

- strain Lactobacillus plantarum 2025 - medium, white, round, convex colonies with a smooth edge.

When grown on liquid MPC pH-6.5 (Himedia, India) or milk at 37 ° C for 24-48 hours, the cells of the strain Lactobacillus crispatus 2029 are medium bacilli ~ 5-6 microns in size, form short chains; cells of the Lactobacillus gasseri 2016 strain are medium bacilli ~ 4-5 μm in size, form chains, cells of the Lactobacillus plantarum 2025 strain represent short bacilli ~ 3 μm in size, form short chains.

Physiological and biological signs.

All strains are facultative anaerobes, temperature optimum 37 ± 2 ° С, grow at 42 ° С, weak growth at 30 ° С. The optimal pH of the medium is 5.5-6.0.

Biochemical properties.

The ability of the consortium strains to ferment carbohydrates was evaluated in an API test in accordance with the manufacturer's instructions (BioMerieux, France).

Strain Lactobacillus crispatus 2029 assimilates D-Glucose, D-Fructose, D-Sazarose, Esculin iron citrate.

The strain of Lactobacillus gasseri 2016 assimilates D-Glucose, D-Fructose, N-Acetylglucosamine, Esculin iron citrate, D-Sazarose, D-Trehalose.

The strain Lactobacillus plantarum 2025 assimilates D-Ribose, D-Galactose, D-Glucose, D-Fructose, D-Mannitol, D-Sorbitol, Methyl α D-mannopyranoside, N-Acetylglucosamine, Amygdalin, Arbutin, Esculin iron citrate, Salicin, D , D-Maltose, D-Lactose, D-Melibiosis, D-Trehalose, D-Sazarose, D-Melicytosis, Gentiobiosis and has a taxonomic characteristic ID = 99.8%; T = 0.89.

Sensitivity to antibiotics.

Strain L. crispatus 2029 is resistant to lincomycin, metronidazole, gentamicin and ciprofloxacin.

Strain L.gasseri 2016 is resistant to metronidazole and ciprofloxacin.

Strain L.plantarum 2025 is resistant to metronidazole and ciprofloxacin.

The method, conditions and composition of the media for the propagation of strains of the consortium.

The strains of Lactobacillus crispatus 2029, Lactobacillus gasseri 2016 and Lactobacillus plantarum 2025 are grown at 37 ± 2 ° C in liquid MPC or 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.

For the cultivation of individual colonies of strains using agar medium MPC containing 1.4% agar (Difco, USA).

A consortium of strains of Lactobacillus fermentum 3872, Lactobacillus crispatus 2029, Lactobacillus gasseri 2016, Lactobacillus plantarum 2025 can be stored:

- 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;

- frozen at -70 ° C. Duration of storage is 6 months.

The claimed solution is illustrated by the following figure:

Figure 1. Genomic fingerprints for the Lactobacillus fermentum 3872 strain isolated from breast milk when studying the species spectrum of lactobacilli in a healthy pair of mother-child (1), feces of a baby (2), vaginal secretions and feces of a mother (3, 4), respectively; L-markers of molecular weights; k - control (water).

The invention is illustrated by the following examples.

Example 1. Cultivation of a strain of Lactobacillus fermentum 3872 in the laboratory.

To obtain seed in an ampoule containing 10 mg of a lyophilized culture of Lactobacillus fermentum 3872, 0.5 ml of a sterile solution of 0.9% sodium chloride is added and the culture is grown on the surface of MPC agarized medium by the method of exhaustion in a thermostat at 37 ° C for 18 hours . Then, the isolated colony is 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 added to bottles containing 500 ml of liquid MPC medium and grown at 37 ° C for 18 hours. The titer of the obtained culture 2.0 × 10 ⁹ CFU / ml By adjusting the cell concentration to 6.0 × 10 ⁸ CFU / ml, a standardized starting biomass of Lactobacillus fermentum 3872 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 a temperature of 4 ° C.

Example 2. Obtaining a bacterial preparation of a consortium of strains of Lactobacillus fermentum 3872, Lactobacillus crispatus 2029, Lactobacillus gasseri 2016, Lactobacillus plantarum 2025.

To obtain a bacterial preparation based on a consortium of lactobacillus strains, mechanical mixing of standardized liquid cultures of Lactobacillus fermentum 3872 strain and a consortium of Lactobacillus crispatus 2029, Lactobacillus gasseri 2016, Lactobacillus plantarum 2025 strains is carried out (hereinafter: consortium 1)

Consortium 1 is obtained as follows. The strains that make up consortium 1 are separately cultured in liquid MPC at 37 ° C for 18 hours, then adjusting the cell concentration to 6.0 × 10 ⁸ CFU / ml, a standardized starting biomass of each strain is obtained. The cultures are mixed in equal volumes and get the starting biomass of the consortium 1. The standard concentration of cells of each strain in the resulting biomass is 2 × 10 ⁸ CFU / ml

The starting biomass of the strain Lactobacillus fermentum 3872, obtained according to example 1, is mixed with the starting biomass of the consortium 1 in a ratio of 1: 3 by volume, which allows to obtain a consortium (hereinafter consortium 2), in which the strains of Lactobacillus fermentum 3872, Lactobacillus crispatus 2029 2016 Lactus , Lactobacillu splantarum 2025 are in equal concentrations, i.e. in a ratio of 1: 1: 1: 1.

The standard cell concentration of each strain in the resulting biomass (standardized starting biomass) is 1.5 × 10 ⁸ CFU / ml. The total titer is 6.0 × 10 ⁸ CFU / ml.

A consortium of strains of Lactobacillus fermentum 3872, Lactobacilltis crispatus 2029, Lactobacillus gasseri 2016, Lactobacillus plantarum 2025 can also be obtained by direct mixing of standardized biomass of all strains. At the same time, other ratios of the initial strains (from 1: 100 to 100: 1) can be used to obtain the consortium, but a ratio of 1: 1: 1: 1 is preferred.

The resulting consortium is lyophilized according to Example 1. In a freeze-dried state, the bacterial preparation can be stored for two years at a temperature of 4 ° C. The total titer of cells in one bottle is 6.0 × 10 ⁸ CFU.

This consortium can be used as a bacterial preparation or as a direct starter culture to produce fermented products.

Example 3. Assessment of the activity of acid formation.

The activity of acid formation is determined by the titrimetric method [14].

As the starter culture, use the standardized starting biomass of the strain Lactobacillus fermentum 3872 obtained in example 1, and the starting biomass of the consortium 1 and consortium 2 obtained in example 2.

Sourdough (strain and each consortium separately) in an amount of 1% is introduced into bottles containing 1.5% milk fat and cultured for 24-48 hours at 37 ° C. Measurements are carried out within 48 hours. The result is presented in degrees Turner (T ° is the value expressing the amount of 0.1 N alkali, which went to the titration of 100 ml of the test sample).

The results shown in table 1 show that the strain of Lactobacillus fermentum 3872 fermented milk in 48 hours, the acidity of the product is 70.2 ± 1.5 ° T.

Upon inoculation of consortium 2, clot formation occurs in 20 hours and by 48 hours the product acidity reaches 106.5 ± 1.4 ° T, which corresponds to the standards of fermented milk drinks and 70-140 ° T yoghurts. The resulting product has good taste properties and has a slight sour-milk smell. Studies have shown that the properties of the inventive consortium when used as a direct starter culture meet the technological requirements for the production of fermented milk products.

Example 4. The ability to produce H ₂ O ₂ .

The effective production of hydrogen peroxide (H ₂ O ₂ ) by probiotic lactobacilli is one of the main factors ensuring the colonization resistance of microbiocenoses of the body [15].

The quantitative determination of hydrogen peroxide (H ₂ O ₂ ) produced by lactobacilli cultures is carried out by the colorimetric method using the 3.3 ', 5,5'-tetramethylbenzidine chromophore (TMB) (Sigma) in the presence of horseradish peroxidase (Sigma) [16].

The results shown in table 2 show that the most effective production of H ₂ O ₂ was observed during the cultivation of consortium 2. The concentration of H ₂ O ₂ in the cultivation medium in this case was 148 mg / L.

Example 5. The study of the resistance of strains to gastric and intestinal stress (the ability to grow in an environment with a low pH or in an environment containing bile).

With oral administration of probiotic preparations, it is extremely important that the lactobacilli strains included in the consortium are resistant to gastric and intestinal stress.

In the study, the following methods are used.

In vitro imitation of gastric stress

Artificial Gastric Juice Composition:

NaCl (SigmaS9625) 2.2 g / l L-lactic acid (SigmaLl 750) 9.9 g / l (0.11 M) Pepsin (SigmaP7125) 3.5 g / l pH: 2.7 ± 0.02 (adjusted 35% NaOH) pH after 1/11 dilution: 3.10 ± 0.10 (control for each culture)

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

After incubation, the culture (each repetition and control) is diluted from 10 ² to 10 ⁹ or 10 ¹⁰ in MPC, dilutions are seeded 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 breeding.

In vitro imitation of intestinal stress

Artificial intestinal juice composition:

Bile salts (pig bile SigmaB8631) 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 cultures are incubated for 5 hours, at 37 ° C, 10% CO ₂ . As control use 1 ml of MPC. Measurements are performed in duplicate for each culture.

Cultures and controls are diluted from 10 ² to 10 ⁹ or 10 ¹⁰ in MRS, 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 dilution.

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

where: ∑C 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 shown in table 3 show that the strain of Lactobacillus fermentum 3872 is resistant to gastric and intestinal stress.

Thus, it was shown that all strains included in consortium 2 are resistant to extreme factors, such as low acidity and bile content, and therefore, consortium 2 is promising for use in the medical and food industries for the production of drugs and for the preparation of products functional nutrition.

Example 6. The study of the adhesion of the strain Lactobacillus fermentum 3872 to transplantable Hela cells.

The adhesion of probiotic bacteria is considered as a positive sign, which allows to protect the macroorganism from pathogenic and conditionally pathogenic microorganisms. In particular, probiotic lactobacilli with adhesiveness to the epithelium of the urogenital tract are promising for the prevention and treatment of urogenital infections [17].

Hela cell cultures were grown in DMEM with 10% fetal serum + 1% penicillin-streptomycin + 0.2 mM Hepes + 2 mM L-glutamine at 37 ° C in a CO ₂ incubator. Cells at a concentration of 3.0 × 10 ⁵ cells / ml were plated on 6-well plates in a volume of 2.0 ml per well and cultured for 24 hours. Upon reaching 70-80% of the monolayer, the cells are washed 3 times with serum-free DMEM and then DMEM with L-glutamine is added to them. Before bacteria were added to Hela cells (18 hour culture on MPC medium), they were washed twice with PBSpH 7.2, using centrifugation at 5000 rpm for 15 minutes, and resuspended in DMEM medium with L-glutamine. The concentration of bacteria to determine the ratio of bacterium / somatic cell is regulated by optical density (ODλ = 590 nm) and controlled by seeding on the corresponding solid medium. Multiplicity of infection (MOI) is 50 and 100 bacteria / cell. Hela cells with lactobacilli introduced to them are incubated for another 5 hours at 37 ° C in a CO ₂ incubator. After incubation, the cells are washed 3 times with PBS pH 7.2, then fixed in cold ethanol (3 min) and stained with Romanovsky-Giemsa stain for 30 minutes. Next, the tablets are washed with distilled water, dried at room temperature and examined microscopically. The number of Hela cells with attached bacteria (percent adhesion) and the number of bacteria attached to one cell (adhesion index) are counted.

Studies have shown that for the Lactobacillus fermentum 3872 strain, the adhesion percentage is 89 ± 4, the adhesion index was 29 ± 5, which indicates the expressed ability of the Lactobacillus fermentum 3872 strain to adhere to Hela cells.

Example 7. A study of the adhesion of a strain of Lactobacillus fermentum 3872 to buccal epithelial cells.

The study of adhesion to buccal epithelial cells is carried out according to the method described in [18]. The multiplicity of infection was 100 bacteria per cell. Buccal epithelial cells with lactobacilli introduced to them are incubated for 5 hours at 37 ° C in a CO ₂ incubator in an atmosphere containing 5% CO ₂ ).

The results showed that for the strain Lactobacillus fermentum 3872, the adhesion percentage is 94 ± 3, the adhesion index is 28 ± 7.

The data indicate the expressed ability of the strain Lactobacillus fermentum 3872 to adhesion on the surface of the buccal epithelium.

Example 8. The study of the antagonistic activity of the strain of Lactobacillus fermentum 3872, consortium 1 and consortium 2.

The study of the antagonistic activity of lactobacilli cultures is carried out by the method of two-layer agar [9]. The research results are presented in table 4.

Differences in antagonistic activity of strain L.fermentum 3872 and consortium 1 containing strains of L.crispatus 2029, L. gasseri 2012, L.plantarum 2025 in relation to S. aureus SA40 and S. epidermidis SE8 (clinical isolates from breast milk of women with a diagnosis recurrent mastitis) are statistically unreliable. The addition of strain L.fermentum 3872 to consortium 1 leads to a significant increase in the antagonistic activity of the new probiotic consortium 2 compared to consortium 1 and the separately used strain L.fermentum 3872 (p <0.05).

The antagonistic activity of consortium 1 in relation to G. vaginalis (the etiological agent of bacterial vaginosis) is significantly higher compared to the strain L.fermentum 3872 (p <0.05). With the combined use of the strain L.fermentum 3872 with strains of the consortium 1 L.crispatus 2029, L. gasseri 2012, L. plantarum 2025 (consortium 2), their action is synergistic. The antagonistic activity of consortium 2 is significantly higher compared to consortium 1 (p <0.05).

Thus, the results of studies indicate that consortium 2 exhibits a more pronounced antagonistic activity compared to consortium 1 and strain L.fermentum 3872.

Example 9. Preparation of fermented milk products using a strain of Lactobacillus fermentum 3872, consortium 1 and consortium 2.

Sour-milk products based on the Lactobacillus fermentum 3872 strain, consortium 1 or consortium 2 can be obtained using either a standardized liquid starting biomass or a lyophilized biological preparation. In this case, the functional nutrition product can be obtained both by adding the starter culture in a suitable medium (for example, sterilized milk with a fat content of 1.5%) and subsequent cultivation, and by introducing the starter culture into the finished fermented milk product immediately before use.

a) Standardized starting biomass of the strain Lactobacillus fermentum 3872 (according to example 1), consortium 1 and consortium 2 (according to example 2) (separately the strain and each consortium) in a volume of 5% is introduced into sterile milk, mixed and cultured at a temperature of 37 ° C in for 20-24 hours until a clot forms, after which the product is cooled to 4 ° C and packaged. As a result, fermented milk functional food products with a beneficial microflora content of at least 10 ⁸ CFU / ml are obtained.

b) Immediately before use, the product can be obtained as follows: in two bottles containing a lyophilized preparation, for example, the consortium 2 obtained according to example 2, add 1 ml of boiled warm water not higher than 37 ° C, the drug is dissolved, the contents are poured into 100 ml of kefir and mix. The resulting product is ready for use.

Example 10. The harmlessness of a consortium containing strains of Lactobacillus crispatus 2029, Lactobacillus gasseri 2016, Lactobacillus plantarum 2025, Lactobacillus fermentum 3872.

The fermented milk product based on consortium 2, obtained according to example 9a, was consumed by volunteers from the staff of the Department of Immune Biochemistry of the Institute of Engineering Immunology OJSC for two months. As a result of this study, no side effects caused by regular long-term consumption of a fermented milk product made using consortium 2 were detected.

Example 11. The use of fermented milk functional food product for the treatment and prevention of candidiasis (thrush) in women of reproductive age.

Twelve women volunteers aged 24-35 years with a diagnosis of candidiasis (thrush), confirmed by laboratory analysis, were divided into three groups, four in each group. The first group every day for 14 days drank during dinner 100 ml of fermented milk product obtained using consortium 2 according to example 9a. The second group daily drank 100 ml of fermented milk product obtained using the consortium 2 of Example 96 daily for 14 days. The third group (control) daily drank 100 ml of kefir that was not enriched in the consortium preparation during dinner for 14 days. After 14 days, the symptoms of candidiasis (thrush) disappeared in women of the first and second groups. The level of C. albicans in the vaginal discharge of the first and second groups decreased from 10 ⁵ CFU / ml to 10 ² CFU / ml, which is normal. Side effects from the intake of functional fermented milk products containing consortium 2 were absent. In the third group, the symptoms of candidiasis persisted.

The research results show the promise of using a consortium of probiotic strains Lactobacillus crispatus 2029, Lactobacillus gasseri 2016, Lactobacillus plantarum 2025, Lactobacillus fermentum 3872 for the production of fermented milk functional food products for the prevention and treatment of candidiasis. The method is simple, economical, affordable and can be effectively applied at home.

Example 12. A comparative evaluation of the effectiveness of the use of fermented milk products obtained when using Lactobacillus fermentum 3872, consortium 1 and consortium 2 as direct starter cultures in the complex treatment of recurrent mastitis.

The experiment involved twelve women volunteers aged 27-36 years with a diagnosis of recurrent mastitis, who had previously undergone a course of antibiotic therapy for 14 days under the supervision of a physician. Before antibiotic therapy, all women in breast milk were found to have Staphylococcus aureus (1.3 × 10 ⁴ -2.6 × 10 ⁴ KOE / ml) and Staphylococcus epidermidis (2.3 × 10 ⁴ -3.5 × 10 ⁴ CFU / ml). In the clinical microbiological laboratory, the sensitivity of the isolated isolates to various antibiotics was determined, and a course of antibiotic therapy for mastitis was carried out under the supervision of the attending physician. After three days of antibiotic therapy, the well-being of all patients improved and signs of recovery appeared, however, the traditional etiotropic course of treatment for mastitis continued for 14 days. After a course of antibiotic therapy, a decrease in the level of Staphylococcus aureus to 1.5 × 10 ² -2.4 × 10 ² CFU / ml and Staphylococcus epidermidis 2.6 × 10 ² -3.2 × 10 ² CFU was found in all patients in breast milk / ml This moment was the beginning of the experiment.

Women were divided into 4 groups of three participants in each group.

The first group daily drank 150 ml of fermented milk product during dinner for 10 days, in which the Lactobacillus fermentum 3872 strain was used as a direct starter culture.

The second group drank 150 ml of fermented milk product every evening for 10 days during dinner, in which consortium 1 containing Lactobacillus crispatus 2029, Lactobacillus gasseri 2012, Lactobacillus plantarum 2025 was used as a direct starter culture.

The third group drank 150 ml of fermented milk product every evening for 10 days, in which consortium 2 containing Lactobacillus crispatus 2029, Lactobacillus gasseri 2012, Lactobacillus plantarum 2025, Lactobacillus fermentum 3872 was used as a direct starter culture.

The fourth group (control) daily drank 150 ml of fermented milk product that did not contain Lactobacillus fermentum 3872, as well as lactobacilli, which are part of the consortia 1 and 2, during dinner for 10 days.

After the course of probiotic therapy, all participants in the experiment were monitored for 180 days. The effectiveness of the probiotic action of Lactobacillus fermentum 3872, consortium 1 and consortium 2 was evaluated by the duration of remission of recurrent mastitis. The research results are given in table.5.

In group 4 (control), the duration of remission was 36-54 days after antibiotic therapy. In groups 1 and 2, the duration of remission after probiotic therapy was 92-128 days and 85-116 days, respectively. In all women of group 3, recurrence of the disease was absent during 180 days of observation.

The research results indicate that the most promising for the complex treatment of recurrent mastitis is consortium 2, containing Lactobacillus crispatus 2029, Lactobacillus gasseri 2016, Lactobacillus plantarum 2025, Lactobacillus fermentum 3872.

Literature

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Claims (2)

1. The bacterial strain Lactobacillus fermentum BKM B-2793D, with antagonistic activity against pathogenic and conditionally pathogenic microorganisms. 2. A consortium of strains of Lactobacillus fermentum BKM B-2793D, Lactobacillus crispatus BKM B-2727D, Lactobacillus gasseri BKM B-2728D, Lactobacillus plantarum BKM B-2731D, which has a probiotic effect and is used for the manufacture of bacterial preparations and functional acid.