RU2165454C1 - Strain of bacterium bifidobacterium bifidum n 791/bag used for preparing bifidobacteria liquid concentrate, fermented-milk curative-dietetic foodstuffs and biologically active additions - Google Patents

Abstract

FIELD: biotechnology, medicinal microbiology. SUBSTANCE: strain Bifidobacterium bifidum N 791/BAG is obtained by selection of the strain B. bifidum N 791. Selection is carried out without use of any chemical and physical mutagens by a long time by turn culturing in two different nutrient media: Blaurock's medium and sterile defatted milk without growth factors for 50 passages by morphocultural criterions. Strain is more resistant to impairment factors of cultured media and aggressive media of human gastroenteric tract, it provides utilization of the more broad spectrum of amino acids, i. e. it shows the more high viability and colonizing properties that provides preparing preparations and foodstuffs on its base with the more long storage time and the more high curative-prophylactic properties. Invention can be used for preparing liquid concentrate of bifidobacteria and fermented- -milk curative-dietetic foodstuffs and bifido-containing bifidobacteria biologically active additions based on liquid concentrate of bifidobacteria. EFFECT: strain of bacterium indicated above, valuable properties. 10 tbl, 7 ex

Description

 The invention relates to biotechnology and medical microbiology and is a strain of bifidobacteria of the species Bifidobacterium bifidum, which can be used to obtain a liquid concentrate of bifidobacteria, and also for the preparation of sour-milk therapeutic and dietary products, bifidoboron biologically active additives (BAA) and probiotic preparations based on the specified liquid concentrate of bifidobacteria.

 The functions of normal microflora in the body are vital and very extensive: synthesizing, regulatory, desensitizing, detoxifying, enzymatic, protective and immunostimulating. Preparations and products containing bifidobacteria are widely used for the prevention and treatment of dysbiosis and the normalization of the intestinal microflora of people who have undergone stressful effects, antibiotic and chemotherapy.

 Probiotics and medical nutrition products based on bifidobacteria strains of the species B. bifidum, B. longum, B. adolescentis, B. breve, B. infantis are widely used abroad (French patent N 2336886; French patent N / 2411008; Swiss patent N 637297; German patent 2421066; Japanese patent N 05227946; US patent N 4645667).

 However, the strains of bifidobacteria of foreign developers are focused on the creation of mainly therapeutic and prophylactic drugs and dietary supplements in dry form (tablets, capsules). These strains have significant drawbacks if they are used for the preparation of preparations and fermented milk products in liquid form: the short shelf life of these preparations and products, low organoleptic qualities that are not essential for preparations in dry form.

 A known strain of the species B. bifidum N1 isolated from the intestines of an infant, which is widely used in Russia for the manufacture of preparations based on it: dry and liquid bifidumbacterin, fermented milk bifidumbacterin.

 The disadvantage of B. bifidum N 1 strain is that, along with slow enzymatic activity against milk fermentation without growth factors, the strain also has weak antagonistic activity against pathogenic microorganisms: Staphylococcus aureus, Shigella, Proteus and pathogenic Escherichia coli.

 The closest analogue (prototype) of the present invention is a strain of B. bifidum N 791 (RF Patent N 1347221, IPC A 61 K 35/74, publ. 01.07.1991). The strain Bifidobacterium bifidum N 791 was isolated from the intestines of a healthy infant, was deposited in the Central Museum of Industrial Microorganisms of the Institute "VNIIgenetika" under the numbers CMPM B-3300 and is characterized by the following features.

 Cultural and morphological characters.

 Rod-shaped cells with V-shaped bifurcations at one or two ends, motionless, in the smear form clusters gram-positive.

 In 0.72% liver and hydrolyzate-milk agar forms colonies and in the form of buckwheat grains or discs up to 3 mm in diameter. In liquid nutrient media it grows along the entire height of the volume, except for the aerobiosis zone. It does not form pigment.

Physiological and biochemical characteristics
Obligatory anaerobic, optimal temperature 37.5 ^o C, pH 7.2-7.4. It ferments lactose with the formation of acetic and + lactic acids, acidifying the growth medium to pH 4.0. Arabinose, xylose, mannose, cellobiose, inulin, mannitol, sorbitol, salicin does not ferment.

 It ferments milk with the formation of a stable clot by 16-18 hours. There is no gas-forming ability, catalase-forming ability, gelatin liquefaction.

Acid-forming activity: by 24 hours - 70 ^° Turner, by 72 hours - 90-100 ^° Turner (on the liver medium), on hydrolyzate-milk medium - up to 100 ^° Turner and 200 ^° Turner, respectively.

Antagonistic properties
It is active against Shigella Sonne, Flexner, enteropathogenic Escherichia coli, staphylococci.

 The strain DNA is of a pronounced GC type with a specificity coefficient of 1.58, the molar percentage of the GC content in the DNA is 61.35. Resistant to kanamycin, monomycin, sensitive to penicillin antibiotics, kefzol, gentamicin.

 However, the strain B. bifidum N 791 is not sufficiently resistant to the damaging factors of cultivation media and aggressive environments of the gastrointestinal tract (acids, proteolytic enzymes) during the promotion of bifidobacteria of the strain in the gastrointestinal tract, and therefore its viability and colonizing properties are insufficient. In addition, the disadvantages of B. bifidum strain N 791 include the fact that the cells of these bifidobacteria utilize mainly 4 amino acids (out of 14): Proline, Tyrosine, Lysine, Tryptophan and synthesize other amino acids from them in transamination reactions, which is energetically unjustified, how the same amino acids are in free form in a nutrient medium.

 The objective of the invention is the creation of a strain of the species B. bifidum N 791 / BAG, which would be more resistant to the damaging factors of culture media and hostile environments of the gastrointestinal tract of a person, would ensure the utilization of a wider range of amino acids, i.e. would have higher viability and colonizing properties, which would ensure the receipt of drugs and products based on it with longer shelf life and higher therapeutic and prophylactic properties.

To obtain B. bifidum strain N 791 / BAG, B. bifidum strain N 791 was selected. The selection was carried out without the use of any chemical and physical mutagenic agents by continuous alternating cultivation in two different nutrient media: Blaurock medium and sterile skim milk without growth factors for 50 passages according to the following morphological and cultural criteria:
- characteristic morphology for cultures of B. bifidum species: in the Blaurock environment, gr + sticks with bifurcation, and sticks in milk without growth factors;
- the coagulation rate of sterile milk is not more than 20-22 hours;
- the achievement of the titer of bifidobacteria should be at least lg 10 for a cultivation period of not more than 24 hours;
- the shelf life of bifidobacteria in liquid form should be at least 2 months in sterile skim milk without growth additives.

The inventive strain of Bifidobacterium bifidum N 791 BAG is stored in a lyophilized form and deposited in the collection of research institutes of culture collections of microorganisms SSC VB "Vector" under N В-686 (certificate of depositor N 0599 dated 10/14/1999 g attached to the application materials) and is characterized by the following features:
Cultural and morphological features
Rod-shaped cells of homogeneous morphology, V-shaped bifurcations at one or two ends are extremely rare, motionless, in the smear form clusters gram-positive.

 In a hydrolyzate-milk agarized medium, colonies are formed in the form of clear cloves with a length of 5-10 mm, a thickness of 1 - 2 mm.

In liquid nutrient media it grows along the entire height of the volume, except for the aerobiosis zone. It does not form pigment. Physiological and biochemical characteristics: obligate anaerobic, optimal temperature 37.5 ^o C, pH 7.2-7.4. It ferments lactose with the formation of acetic and + lactic acids (acidifying the growth medium to pH 4.0). Arabinose, xylose, mannose, cellobiose, inulin, mannitol, sorbitol, salicin does not ferment.

It ferments milk without growth factors with the formation of a stable clot by 16-18 hours. No gas-forming ability, catalase-forming ability, gelatin liquefaction:
Acid-forming activity: to 24 hours - 100 ^° Turner, to 72 hours - 150-200 ^° Turner (on the liver medium), on hydrolyzate-milk medium - up to 160 ^° Turner and 250 ^° Turner, respectively.

The maximum titer of bifidobacteria after 24 hours of growth in a hydrolyzate-milk agar medium is 1-5 · 10 ¹⁰ .

The maximum titer of bifidobacteria in a dairy medium with growth factors: milk hydrolyzate and yeast autolysate after 24 hours of growth is - (5-10) · 10 ¹⁰ , after 30 days of storage in this medium - 2-5 · 10 ¹⁰ .

The maximum titer of bifidobacteria in a dairy medium (sterile skim milk) without growth factors after 24 hours of growth is 1-2 · 10 ¹⁰ , after 30 days of storage in this medium - 1-2 · 10 ⁸ .

 Bifidobacteria of the strain completely utilize 9 free amino acids in sterile skim milk without growth additives: glycine, alanine, valine, methionine, isoleucine, leucine, phenylalanine, histidine, arginine.

 The strain DNA is of a pronounced GC type with a specificity coefficient of 1.58, the molar percentage of the GC content in the DNA is 61.35.

Antagonistic properties, antibiotic resistance:
It is active against Shigella Sonne, Flexner, enteropathogenic Escherichia coli, staphylococci.

 Resistant to cananicin, monomycin, sensitive to antibiotics of the penicillin series, kefzol, gentamicin (see table 1).

 The analysis of table 1 shows that the claimed strain and the strain of the prototype are equally resistant to antibiotics.

 Storage conditions (preservation and de-preservation) of the claimed strain.

Strain B. bifidum N 791 BAG 1-2-3 passages were cultured in milk growth medium at 37.5 - 38 ^o C for 16-18 hours until the formation of a clot, acidity 100 - 120 ^o C Turner. A protective drying medium with pH 7.4 (sucrose 10%, gelatin 7-10%, peptone 1-3%, ascorbic acid) in an amount of 25% of the biomass volume and skim milk in an amount of 25% biomass are added to the grown biomass. The resulting suspension is poured into 5 ml in penicillin vials, frozen at a temperature of minus 40 - 45 ^o C and lyophilized from a frozen state in a vacuum dryer for 24 to 36 hours. The resulting preparation contains 0.5-1 · 10 ⁹ living cells of bifidobacteria. According to the scientific and technical documentation for dry bifidumbacterin (FS 42-132 BC-94), a content of at least 10 ⁷ living bifidobacteria cells in one dose of the drug is allowed.

The lyophilized B. bifidum strain N 791 BAG in penicillin vials is well preserved at 6-8 ^° C (for 3-4 years without loss of bifidobacteria titer).

 Stability of the strain is maintained from the lyophilized preparation after 6-12 months by 5 transfers (passages) in test tubes on the Blaurock liver environment. At the 6th passage, reseeding is done in a milky growth medium with growth additives of the above composition in order to obtain biomass for subsequent freeze drying and storage in the dried state.

Marker signs of the strain B. bifidum N 791 BAG, distinguishing it from the prototype strain B. bifidum N 791 (CMPM B-3300), are as follows:
- rod-shaped cells of homogeneous morphology;
- in a hydrolyzate-milk agarized medium, colonies are formed in the form of clear cloves 5-10 mm long, 1 - 2 mm thick;
- acid-forming activity: by 24 hours - 100 ^° Turner, by 72 hours - 150-200 ^° Turner (on the liver medium), on hydrolyzate-milk medium - up to 160 ^° Turner and 250 ^° Turner, respectively;
- the maximum titer of bifidobacteria after 24 hours of growth in a hydrolyzate-milk agar medium is 1-5 · 10 ¹⁰ ;
- the maximum titer of bifidobacteria in a dairy medium with growth factors: milk hydrolyzate and yeast autolysate after 24 hours of growth is - (5-10) · 10 ¹⁰ , after 30 days of storage in this medium - 2-5 · 10 ¹⁰ ;
- the maximum titer of bifidobacteria in a dairy medium (sterile skim milk) without growth factors after 24 hours of growth is 1-2 · 10 ¹⁰ , after 30 days of storage in this medium - 1-2 · 10 ⁸ ;
- bifidobacteria of the claimed strain completely utilize 9 free amino acids in sterile skim milk without growth additives: glycine, alanine, valine, methionine, isoleucine, leucine, phenylalanine, histidine, arginine.

 Example 1. To identify the technological features and properties of the claimed strain, studies were conducted of B.bifidum N 791 and B.bifiduin N 791 BAG strains in a dairy medium without growth factors.

 Below (table 2) the growth and technological characteristics of the strains of Bifidumbifidum 791 and Bifidumbifidum 791 BAG in a dairy medium without growth factors are given.

 Analysis of table 2 shows that the claimed strain in comparison with the prototype most fully uses the growth resources of a relatively poor growth medium (skim milk without growth factors), has higher growth and technological characteristics compared to the prototype strain, namely: reduced ripening time by 4 hours, the titer in the finished product increased by an order of magnitude, and after 30 days of storage in a refrigerator, the biological activity of the claimed strain was two orders of magnitude higher. In addition, the claimed strain in comparison with the prototype has a high activity of acid formation, which contributes to the accumulation of volatile acids in a concentrate of bifidobacteria and, therefore, increased antagonistic activity of products obtained using this strain. Moreover, as can be seen from table 2, the claimed strain is more resistant to the action of volatile fatty acids, because for a long time it retains a high number of viable cells during storage (100 times higher than the prototype strain). This is very important in the production of therapeutic and dietary fermented milk products that contain acidophilus bacteria or Bulgarian bacillus, which are very active acidifiers.

 In addition, the growth properties of B. bifidum 791 and B. bifidum 791 BAG strains and hydrolyzate-milk agar medium were studied. The data are shown in table 3.

Thus, it can be seen from table 3 that the growth activity of strain 791 BAG in a hydrolyzate-milk agar medium is significantly higher at lower final acidity (135 T) instead of (159 T). With the same acidity, the titer of strain 791 BAG is 1 order higher
Example 2. The use of strain B.bifidum N 791 BAG for the preparation of a liquid concentrate of bifidobacteria (cholelithiasis)
Strain B. bifidum 791 / BAS is grown in skim milk enriched with hydrolyzate-milk and autolysate growth supplement, at 37.5 - 38 ^o C for 16-18 hours to an acidity of 100 - 130 ^o Turner. By 16-18 hours of cultivation, the titer of active bifidobacteria cells in 1 ml of medium is lg 10.5-11 (using B.bifidum 791 strain, biomass accumulation occurs by 18-24 h and is lg. 9.0-9.3).

 The biologically active additive (BAA) "liquid concentrate of bifidobacteria" is used as an independent therapeutic and prophylactic agent, as a biologically active additive, and also as a starter for the manufacture of bifid-containing fermented milk products.

New useful biosynthetic and anabolic properties of the selected strain made it possible to obtain a liquid concentrate of bifidobacteria with enhanced biological characteristics:
- the titer of bifidobacteria in the product increased from 1 · 10 ⁹ to 3 - 5 · 10 ¹⁰ CFU / ml;
- balanced intake and amino acid content in the final product.

Example. 3. The study of the amino acid composition of products prepared on the basis of strain B.bifidum 791 BAG
Studies of the amino acid composition of products prepared on the basis of the B. bifidum 791 BAG strain were carried out on a US Biotromik amino acid analyzer.

Experiment Conditions
1. Sample preparation: precipitation of the total protein with sulfosalicylic acid; protein removal by centrifugation.

2. The determination of the content of free amino acids in the sample was carried out on the Biotronik LC 5001 amino acid analyzer with the following parameters:
- Column 3.2x385 mm "Biotronik resin 2710",
- Mobile phase: a sequence of five citrate buffer solutions of varying acidity and ionic strength;
- Flow rate: buffer - 0.3 ml / min, reagent - 0.55 ml / min;
- Sample volume: 50 μl.

 - Quantification using a Hamilton amino acid calibration solution (USA).

- Detection: post-column staining with a solution of ninhydrin in DMSO, L _vis - 570 nm.

 The results of the analysis of the amino acid composition of a liquid concentrate of bifidobacteria are shown in table 4.

 In a liquid concentrate of bifidobacteria based on the B. bifidum 791 BAG strain, the content of free amino acids is increased and reaches about 14.7%, of which the proportion of essential amino acids reaches 50.01%.

 The increased amount of essential amino acids from the total amount of free amino acids, especially such as: valine - 8.3%, leucine - 13.4%, lysine - 7.7%, as well as methionine - 3.1%, branched chain amino acids, has important physiological significance, since these amino acids are most actively involved in protein synthesis processes. Thus, a lack of valine leads to a disorder of coordination of movements, a lack of leucine causes changes in the kidneys and thyroid gland, a lack of lysine leads to disruption of blood formation processes. In a liquid concentrate of bifidobacteria, the arginine content is increased to 7.7%. An increased arginine content is biologically important, since free arginine helps stimulate the body’s immune system, in addition, it has antioxidant properties, which has a beneficial effect on increasing the shelf life of bifidobacteria liquid concentrate.

 Despite the increased utilization of B. bifidum 791 / BAG strains of free amino acids during the growth of bifidobacteria in a milk growth medium enriched with growth factors, the final product, a liquid concentrate of bifidobacteria, remains a fairly large amount of free amino acids - 418.39 mg / l, which are especially irreplaceable . In addition, the set of free amino acids is physiological, as evidenced by the ratio of amino acid concentrations of methionine / isoleucine, equal to a coefficient of 0.83, quite close to a coefficient of 1 (optimal physiological ratio).

 Table 5 shows the amino acid analysis data in a milk cultivation medium (skim milk 11% COM without growth factors) after 24 hours of cultivation of B. bifidum N 791 and B. bifidum N 791 BAG strains.

 The content of free amino acids in the hydrolyzate-milk culture medium after 24 hours of cultivation of strains of B. bifidum 791 and B. bifidum 791 BAG are shown in table 6.

 Analysis of table 5 shows that the claimed strain in comparison with the prototype most fully uses the growth resources of a relatively poor dairy environment (skim milk without growth factors), making the most of a set of amino acids, some amino acids are utilized 15-20 times better than that of the prototype strain (except for proline). Thus, the claimed strain has good technological characteristics when growing on simple dairy environments without the addition of specialized growth additives, which makes it possible to reduce the cost of production of a liquid concentrate of bifidobacteria.

 From table 6 it follows that the noted high utilizing ability of amino acids from plain milk medium (in skim milk without growth additives) practically does not affect the amino acid composition of products obtained using milk hydrolysates and yeast autolysates. This allows you to get enriched with vitamins and amino acids, biologically active additives and food products based on this strain in the same way as using the strain of the prototype.

Example 4. The study of the antagonistic activity of strain B. bifidiim 791 / BAG
The antagonistic activity of B. bifidum 791 / BAG strain was studied in vitro when co-grown with pathogenic test strains in the zone of growth inhibition of test microbes by the barcode method.

 The antagonistic activity of B. bifidum791 BAG strain and its colonization activity were most fully studied in vivo in an organism-population model - laboratory mice in a chronic experiment.

 Studies were conducted on tribal mice of both sexes of the ICR colony for 3 years on 18 generations of random shredding in the amount of 900 females and 450 males.

 Bifidobacteria liquid concentrate was produced on the basis of B. bifidum 791 BAG strain on a dairy medium with the addition of growth factors.

 Bifidobacteria concentrate was used at a dose of 0.1 ml per head per day for 1 month daily.

 Bacteriological studies of feces of mice were carried out in accordance with the guidelines "Intestinal dysbiosis and methods for its laboratory diagnosis."

 Surveillance of survival and weight of mice was performed.

 Based on the data on the number of corpus luteum and implantation sites of live and dead embryos, post- and preimplantation mortality of the embryos was determined.

The results of a bacteriological study of young generation 1 received from parents who received a liquid concentrate of bifidobacteria showed that there was not only a significant decrease in the level of pathogenic and conditionally pathogenic microflora, but also an increase in bifidobacteria and lactobacilli almost equally from 10 ⁵ to 10 ⁸ - 10 ⁹ microbial bodies in 1 g of feces (Table 8).

 Bacteriological studies of the feces of pedigree young generations of the next generations showed the presence of the formed barrier effect of gastro-intestinal microflora with respect to pathogenic microorganisms such as Salmonella, Staphylococcus aureus, Clostridium digficile, Candida, Proteus, etc. fungi. This is the result of the intestinal colonizing ability of mice with B. bif strain 791 BAG. The earlier formation of the microflora of newborns through the correction of the microflora of the parents provided good body protection and the ability to resist infection from the outside.

 The effect of a liquid concentrate of bifidobacteria on the fecundity of animals was investigated (Table 9). The litter of mice is determined by the number of ovulated eggs, the death of embryos before and after implantation. The death of embryos at an early stage before implantation may increase with an increase in the degree of inbreeding. Late embryonic mortality is an indicator of the state of the body and manifests itself in late pregnancy. As can be seen from table 9, the intake of bifidobacteria concentrate based on the new strain led to the complete exclusion of late embryonic mortality after 3 years, whereas at the beginning of the experiments (1 year) it was 14.9%.

 Analysis of tables 8 and 9 shows that the use of a liquid concentrate of bifidobacteria for 3 years, made on the basis of the inventive strain of bifidobacteria B. Bifidum N 791 BAG, led to the elimination of mice from the intestines of pathogenic microflora and stable colonization by bifidobacteria, which ultimately allowed significantly reduce mortality and late embryonic mortality in mice.

Example 5. The study of the duration of storage of a strain of bifidobacteria
B.bifldum 791 / BAG in bifidobacteria liquid concentrate
The increase in the shelf life of the liquid concentrate of bifidobacteria is associated with the solution to the problem of neutralizing the active acidity of the drug observed during storage of the drug, which leads to the death of bifidobacteria cells and, consequently, to a decrease in their titer in the drug. The solution to this problem is to fulfill at least two conditions:
- reduction of initial acidity in the finished concentrate of bifidobacteria to values of the order of 140 ^o T;
- obtaining the initial titer of bifidobacteria (not lower than 3-5 · 10 ¹⁰ CFU / ml).

 However, the simultaneous fulfillment of these two conditions encounters a contradiction, since an increase in the titer of bifidobacteria leads to a natural increase in the products of their metabolism - lactic and acetic acids, therefore, to an increase in the acidity of the concentrate of bifidobacteria (if you do not resort to artificial correction of the pH of the product, neutralizing the acids with alkaline solutions).

 The above problems are solved by selection when obtaining the inventive strain of B. bifidum 791 / BAG, which is confirmed by the data shown in table 10.

As shown by experiments on a concentrate of bifidobacteria obtained using B. bifidum strain N 791 / BAG, neutralization of active acidity from pH 5.6 units to pH 7.0 units (using 10% NaHCO ₃ solution) does not significantly affect the safety of bifidobacteria strain B. bifidum N 791 / BAG (see tab. 10). However, the addition of soda sharply reduces organoleptic characteristics, creating a bitter aftertaste. The difference in the titers of bifidobacteria in the experimental samples of bifidobacteria concentrates prepared using the inventive strain does not differ significantly during storage, which indicates a significant acid resistance of the strain. This property allows the use of the inventive strain for the production of products with a long shelf life without quenching the acidity of alkaline agents.

Example 6. The use of a liquid concentrate of bifidobacteria as a dietary supplement for immunocorrection of the human body
In the species of bifidobacteria B. bifidum, the presence of certain immunostimulating and antiviral properties was found in case of viral (influenza) infection in preschool children. A decrease in the incidence of acute viral diarrhea (rotaviruses) has also been observed in infants in the United States after food fortification with bifidobacteria of the species B. bifidum (Biotechnol. News, 1994, N 14, p. 3).

 In this regard, the use of the preparation of cholelithiasis based on the B. bifidum strain N 791 BAG in the clinic on volunteers was carried out in early childhood departments and the endocrine department of the Novosibirsk Regional Hospital. Bifidobacteria concentrate was prescribed not only to normalize the intestinal microflora, but also as an immunomodulator.

 The rationale for therapeutic doses of bifidobacteria concentrate for humans, as well as the duration of their use, was carried out experimentally in animals and based on an analysis of the literature data regarding the use of these products.

 The course of treatment was 4 weeks. Bifidoconcentrate was administered orally into the body of children at a dose of lg 10 CFU / kg of body weight. In 80% of patients of the department of early childhood, the elimination of dystrophic syndrome was observed (10 people out of 15). In 60% of patients, a tendency to an increase in serum albumin in the blood by 10 - 13%. This indicates the normalization of the liver and spleen, strengthening their protective functions.

 Moreover, the liquid concentrate of bifidobacteria based on the strain B. bifidum N 791 BAG had the properties of immunocorrection. One patient out of 12 had a complete normalization of immunological parameters (for IgG) of blood. A decrease in glycemia from 5.0 to 9.6 mmol / L was observed in all patients. In 4 patients out of 12, normalization of the total number of Escherichia coli was noted. In 4 patients out of 12, the number of lactose-negative enterobacteria decreased. In 8 patients out of 12, the number of bifidobacteria returned to normal.

 Experimental animal studies, prevention and treatment of sick people in a hospital and on an outpatient basis show that the bifidobacteria concentrate, made on the basis of the inventive strain, has high antagonistic activity and an increased shelf life with a high titer, which allows the use of cholelithiasis as a serial preparation for immunocorrection of the body, treat bacterial and viral dysbiosis, dyspepsia and dysentery without antibiotics.

Example 7. Obtaining a therapeutic and dietary fermented milk product based on a concentrate of bifidobacteria B. bifidum N 791 / BAG
Sourdough - the main part of the primary microflora of fermented milk products, enriches milk with microorganisms more than 100 times. The production process and the quality of fermented milk products depend on its properties. A liquid bifidobacteria concentrate, prepared on the basis of a new B. bifidum strain N 791 / BAG, was used to prepare bifid-containing fermented milk products of dietetic food.

As a basis, milk is used, which is normalized by the mass fraction of fat. Then the milk is subjected to "deep" pasteurization at a temperature of (92 ± 2) ^o C with holding for at least 15 min and cooled to a fermentation temperature of 35 ^o C. Milk is skimmed in fermented milk beverage tanks with a cooled jacket equipped with special mixers for uniform mixing of milk clot. In milk make industrial yeast based on a liquid concentrate of bifidobacteria (B. bifidum strain N 791 / BAG) in an amount of 2-5% by weight of the normalized mixture. The resulting mixture is fermented at a temperature of 35 ± 1 ^o C for 4-5 hours. The end of the fermentation is determined by the formation of a sufficiently strong clot and acidity, which should be no more than 80 ^o T. Upon completion of the fermentation, the product is cooled to 25-30 ^o C, feeding in the interwall space of the reservoir an ice ox. Then the clot is stirred for 10-15 minutes. The mixed clot is immediately fed to the bottling and then cooled to (6 ± 2) ^o C in a refrigerated chamber in a packaged form. The shelf life of a fermented milk product packaged in containers with hermetic closure is 5 days. The product was certified at the Food Certification Center of the Department of State Sanitary and Epidemiological Supervision of the Ministry of Health of the Russian Federation, it received a Hygienic Certificate agreed by TU and TI. Thus, it follows from the foregoing that a new, selected strain of bifidobacteria Bifidobacterium bifidum N 791 BAG is proposed for patenting, which possesses a stable manifestation of the above properties during subsequent passage of the museum culture of the strain on the Blaurock liver medium. In total, the claimed strain passed 70 passages without changing the selected properties. The proposed strain of bifidobacteria Bifidobacterium bifidum N 791 BAG in comparison with the prototype strain has a higher biosynthetic and growth activity, increased utilization of free amino acids of milk culture media, which increased by 9-13 times in relation to most free amino acids. The safety of live bifidobacteria in the milk culture medium increased by 1 - 2 orders of magnitude. The inventive strain has higher therapeutic and prophylactic properties and longer shelf life in a liquid concentrate of bifidobacteria.

Claims (1)

 The strain Bifidobacterium bifidum 791 / BAG (collection V-686 Scientific Research Institute of the collection of cultures of microorganisms of the State Scientific and Scientific Center for Seventh Century "Vector") used to obtain a liquid concentrate of bifidobacteria, fermented milk therapeutic and prophylactic products and biologically active additives.

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