EA041668B1 - THE CONSORTIUM OF BIFIDOBACTERIA USED FOR THE PREPARATION OF BIFID-CONTAINING PRODUCTS AND THE STRAINS INCLUDED IN THE CONSORTIUM - Google Patents

Description

SUBSTANCE: invention relates to biotechnology, food industry, pharmacology, cosmetology and can be used to obtain bifid-containing products.

Bifidobacteria are the most important symbionts of the human gastrointestinal tract. In children by the age of 3 months, their share in the microbiome is more than 90%, which indicates the leading role of this group of bacteria in the formation and development of human homeostasis systems from an early age. They have a morphokinetic effect, form enzymes involved in the metabolism of proteins, carbohydrates, lipids and nucleic acids, produce biologically active compounds, in particular vitamins PP, B1, B6, B9, B12. Bifidobacteria perform immunogenic and antimutagenic functions, and also participate in the detoxification of exogenous and endogenous toxic agents, which becomes especially relevant in modern conditions of massive anthropogenic impact on the biosphere, in which not only nature suffers, but also man himself.

A multifaceted positive effect on the human body allows us to consider bifidobacteria as an effective basis for designing bifid-containing products for various purposes based on promising strains of bifidobacteria.

In addition, representatives of the genus Bifidobacterium have been assigned the GRAS (generally recognized as safe) safety status, which confirms the possibility of using strains and metabolites (producers) of bifidobacteria strains in the production of safe food, medicines, dietary supplements, cosmetics and hygiene products.

In the human intestine, the biocenosis is dominated by the species Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium infantis (Bifidobacterium longum spp. infantis) and Bifidobacterium breve. It is these five species of bifidobacteria that are dominant and, in various combinations, create the conditions for the formation of the composition and normal state of the microbiota of a healthy person. (Kozlova E.P., Kurnosova N.A., Feklisova L.V. Theoretical and biomedical aspects of the use of various types of bifidobacteria as part of the means for the prevention and treatment of intestinal dysbacteriosis. Collection of works. Ministry of Health of the RSFSR. M .: 1986, page 67).

Known consortiums of probiotic strains of various combinations used for the preparation of probiotics, health food products, fermented milk products, biologically active additives and bifid-containing food products of foreign and domestic developers , RU 2261909 C1, RU 2491334 C1, RU 2491333 C, RU 2491332 C1, WO 2014021733, US 7807440 B2, EA 017595 B1).

Currently, in medicines, dietary supplements and probiotic products manufactured in Russia and abroad, a limited list of bifidobacteria strains is used.

It was found that the bifidoflora has a certain structure, according to which only a consortium of bifidobacteria has a dominant influence on the formation of the microbiota characteristic of a healthy intestine (proven in 88% of the cases studied), while the presence of one or two strains always leads to dysbiosis ( proved in 42% of cases of type 1-2 dysbiosis and in 86% of type 3 dysbiosis) (Ivanova E.V. The role of bifidoflora in the associative symbiosis of the human intestinal microbiota. Abstract of the thesis of a doctor of medical sciences. Chelyabinsk, 2009, p. 13) .

Of these, the most widely used are Bifidobacterium longum spp.longum strains ATCC 15707, RO23, BB536 (Morinaga Milk Industry), SBT-2928, I-3, B379M, DVA-13; Bifidobacterium longum spp. infantis strain RO3, 35624 (Procter &Gamble); Bifidobacterium longum spp. infantis strains 7315 and 79-43; Bifidobacterium bifidum strains 1 and 791, 791 BAG, LVA-3, RO71, 8-3; Bifidobacterium breve strain 79-88, 79-119, RO70; Bifidobacterium animalis spp. animalis strain DN-173010 (Danone/Dannon); Bifidobacterium animalis spp. lactis Bb12 (Chr. Hansen), HN019 (DR10) (Danisco); Bifidobacterium adolescentis strain G7513, GO-13, MC-42.

Bifidobacterium animalis subspecies lactis in various variations of the name, isolated from the rumen of an animal, has the smallest genome size. This is due to the fact that it lives in an environment with a constant and simple composition of the nutrient substrate, under conditions that do not require constant adaptation of the microbial cell. This is confirmed by the presence of only two complete copies of rRNA operons, while other species have up to four or five rRNA operons. Reduced adaptive properties of strains of B. animalis subsp. lactis do not lead to the formation of sustainable eubiosis (Bukharin O.V., Perumova N.B., Ivanova E.V. Bioflora in human associative symbiosis. Ekaterinburg: Ural Branch of the Russian Academy of Sciences, 2014, p. 11) .

Traditionally, in our country, when creating probiotics for humans, strains isolated from healthy donors are used.

Known polystrain preparations often represent a mechanical set of probiotic cultures without taking into account their symbiotic nature, the ability of strains introduced into preparations to develop together without suppressing each other. Therefore, manufacturers are forced to cultivate them separately and mix them immediately before freeze-drying. This leads to insufficient colonizing activity and viability of probiotic strains and weak resistance to damaging factors of aggressive environments of the gastrointestinal tract.

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The closest analogue (prototype) of the claimed consortium in terms of species composition is a consortium of five species of breeding strains of bifidobacteria Bifidobacterium bifidum 791-MB, Bifidobacterium longum B 379M-MB, Bifidobacterium adolescentis G 7513-MB, Bifidobacterium infantis 73-15-MB, Bifidobacterium 79-breve 119-MB used for the preparation of sour-milk, non-fermented products, dietary supplements, bifid-containing preparations, cosmetics and hygiene products (RU 2261909 C1). The strains that make up the consortium have similar cultural and biological properties; when co-cultivated, the consortium actively reproduces on nutrient media with the accumulation of production biomass in a short cultivation period, and has specific activity. The finished product contains 10 ⁹ living cells of bifidobacteria in 1 ml.

Individual strains of the consortium included in its composition were obtained from similar collection strains by successive 50 cycles of lyophilized drying and recovery.

The disadvantage of this consortium is the difficulty in obtaining selection strains, which involves 50 cycles of sequential lyophilized drying and recovery on thioglycol medium and skim milk, while the maximum titer of bifidobacteria is only 10 ⁹ living bacterial cells per 1 ml of the finished product.

The technical problem solved as a result of the creation of the present invention is the development of a consortium, including selection strains selected in composition not only taking into account biocompatibility, but also giving the consortium resistance to a wide range of antibiotics, damaging factors that ensure the process of digestion in the gastrointestinal tract, expressed antagonistic activity with the presence of a high titer of bifidobacteria of 10 ¹¹ CFU per 1 g of dry biomass and the production of bifid-containing products with high therapeutic properties on its basis, as well as simplification of obtaining a consortium.

The technical result lies in the fact that the proposed consortium of bifidobacteria used for the preparation of bifid-containing products, and the strains included in its composition, are able to actively accumulate microbial biomass on various nutrient media to a high concentration, have high antagonistic activity in relation to the main test cultures, which is the cause of food poisoning, and in addition, they have a high superoxide dismutase activity with a pronounced antioxidant activity, while the superoxide dismutase activity is not lower than 37 units / mg of microbial mass protein.

The strains included in the consortium can be easily identified based on the proposed nucleotide sequences of their DNA fragments.

The essence of the proposed technical solution is as follows.

A consortium of bifidobacteria is proposed containing the strain Bifidobacterium bifidum 791-BL deposited in the All-Russian Collection of Industrial Microorganisms of the Federal State Unitary Enterprise GosNIIgenetika of the Ministry of Education and Science (VKPM FSUE GosNIIgenetika) under registration No. No. Ac-2037, Bifidobacterium adolescentis strain G 7513BL, deposited with VKPM FSUE GosNIIgenetika under registration No. Ac-2038, Bifidobacterium infantis strain 73-15-BL, deposited with VKPM FSUE GosNIIgenetika under registration No. Ac-2039, strain Bifidobacterium breve 79-119 -BL deposited in VKPM Federal State Unitary Enterprise GosNIIgenetika under registration No. Ac-2036, used for the preparation of fermented, non-fermented food products, dietary supplements, bifid-containing preparations, cosmetic and hygiene products, medical preparations.

Proposed are the following original probiotic strains of bifidobacteria used to obtain the specified consortium.

The strain of bifidobacteria Bifidobacterium bifidum 791-BL, used to obtain a consortium, deposited in the VKPM Federal State Unitary Enterprise GosNIIgenetika under registration No. Ac-2035, and the nucleotide sequence of a fragment of the genome of the strain Bifidobacterium bifidum 791-BL SEQ ID NO: 1.

The strain of bifidobacteria Bifidobacterium longum B379M-BL, used to obtain a consortium, deposited in the VKPM Federal State Unitary Enterprise GosNIIgenetika under registration No. Ac-2037, and the nucleotide sequence of the genome fragment of this strain SEQ ID NO: 2.

The strain of bifidobacteria Bifidobacterium adolescentis G 7513-BL, used to obtain a consortium, deposited in the VKPM Federal State Unitary Enterprise GosNIIgenetika under registration No. Ac-2038, and the nucleotide sequence of the genome fragment of this strain SEQ ID NO: 3.

The strain of bifidobacteria Bifidobacterium infantis 73-15-BL, used to obtain a consortium, deposited in the VKPM Federal State Unitary Enterprise GosNIIgenetika under registration No. Ac-2039, and the nucleotide sequence of the genome fragment of this strain SEQ ID NO: 4.

The strain of bifidobacteria Bifidobacterium breve 79-119-BL, used to obtain a consortium, deposited in the VKPM Federal State Unitary Enterprise GosNIIgenetika under registration No. Ac-2036, and the nucleotide sequence of the genome fragment of this strain SEQ ID NO: 5.

To obtain the proposed consortium, the selection of known

- 2 041668 production strains of five species of bifidobacteria without the use of genetic modification methods as a result of 30 cycles of sequential lyophilized drying and recovery on the Bifilife M medium, and then through the co-cultivation of strains, a consortium is obtained.

The used method of selection of breeding strains took into account the study of their antibiotic resistance, cultural, antagonistic, morphological, physiological and biochemical properties, molecular genetic identification and biocompatibility in the co-cultivation of strains, taking into account the growth kinetics of each of them. This made it possible to create a consortium capable of correcting microecological disturbances more efficiently and safely.

Selection of strains and their characteristics.

1. To obtain the Bifidobacterium bifidum 791-BL strain, the Bifidobacterium bifidum 791-MB strain was selected; The organization that made the deposit is located at the address: 117545, Moscow, 1st Dorozhny proezd, 1. The original strain of Bifidobacterium bifidum 791-MB is protected by RF patent No. and restoration on a nutrient medium Bifilife M of the following composition, in g / l:

casein hydrolyzate of a shallow degree of cleavage, enzymatic - 15.0 g, yeast extract - 5.0 g, glucose - 5.0 g, sodium chloride - 2.5 g, cysteine hydrochloride - 0.75 g, sodium carbonate - 0.8 g, microbiological agar - 0.075 g,

MgSO4 - 0.011 g,

K ₂ HPO ₄ - 0.0001 g,

FeSO ₄ -H ₂ O - 0.00009 g,

MnSO ₄ -H ₂ O - 0.0001 g,

B1 (thiamine hydrochloride) - 0.0002 g,

B6 (pyridoxine hydrochloride) - 0.0002 g,

B3 (calcium pantothenate) - 0.001 g,

Bc (folic acid) - 0.00004 g,

PP (nicotinamide) - 0.002 g,

B2 (riboflavin) - 0.0002 g, glycine - 0.0056 g, glutamic acid - 0.0024 g, aspartic acid - 0.0003 g,

PABA - 0.1 g, citric acid - 0.0001 g.

The primary inoculum of the production strain Bifidobacterium bifidum 791-MB in a volume of 5% of the volume of the cultivation medium was introduced into the above medium and cultivated at a temperature of 37-38°C for 24 hours. Next, 30 cycles of drying-recovery were carried out on the Bifilife M nutrient medium.

As a result of the selection, a strain of Bifidobacterium bifidum 791-BL was obtained, which was deposited on November 16, 2016 in the VKPM Federal State Unitary Enterprise GosNIIgenetika under registration No. Ac-2035.

Cultural, morphological and biological characteristics of the strain Bifidobacterium bifidum 791-BL are rod-shaped variable cells with V-shaped bifurcations at one or two ends, immobile, form clusters in the smear, sometimes chains, gram-positive. Facultative anaerobe, optimum cultivation temperature is 37-38°C, pH 7.0±0.2.

Ferment milk with the formation of a stable clot by 16-18 hours. Gas-forming ability, catalase-forming ability, gelatin liquefaction are absent.

Acid-forming activity: by 24 h - 70°T, by 72 h - 90-100°T (on Blaurock's hepatic medium), on hydrolysate-milk medium up to 100°T and 180°T, respectively.

The maximum titer of bifidobacteria in hydrolysate-milk medium after 24 hours of cultivation is 1x10 ¹⁰ CFU/ml; the maximum titer of bifidobacteria in sterile skimmed milk without growth factors after 24 hours of cultivation is 1-3x10 ¹⁰ CFU/ml.

The strain grows on nutrient media used in production with biomass accumulation of at least lg 9.8-10.2 microbial number for 16-18 hours of cultivation.

The claimed strain has a pronounced antagonistic activity against pathogenic and conditionally pathogenic microorganisms: Shigella flexneri, Escherichia coli, Salmonella typhimurium, Shigella sonnei, Candida albicans, Proteus vulgaris, Staphylococcus aureus, Klebsiella pneumonia, Citrobacter freundii,

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bacillus subtilis. In table. 1 shows the antagonistic activity of the strain against some of the aforementioned microorganisms.

The antagonistic activity of a monostrain of bifidobacteria was determined by the zone of growth inhibition of test cultures of pathogenic and conditionally pathogenic microorganisms by the method of perpendicular strokes.

Table 1

Comparison of antagonistic properties of B. bifidum 791-MB and B. bifidum 791-BL strains

Bifidobacterium strain | Zone of growth inhibition of test cultures (mm) Shigella flexneri Escherichia coli Salmonella typhimurium Shigella sonnei candida albicans Proteus vulgaris Staphylococcus aureus B. bifidum 791-MB 10-12 11-12 9-10 10-12 10-11 10-11 11-12 B. bifidum 791-BL 15-25 15-18 13-15 18-22 15-17 18-22 16-17

Table data analysis. 1 shows that the claimed strain has a higher antagonistic activity against the main test cultures that cause food poisoning.

Determination of the level of resistance of the strain to antibiotics was carried out by the method of serial dilutions in a liquid nutrient medium - sterile skimmed milk. Various concentrations of sterile solutions of antibiotics and a 16-hour culture of bifidobacteria were introduced into the nutrient medium in an amount of 5% of the volume of the medium. Stability was determined by the change in titratable acidity in the samples after 48 h of incubation at a temperature of 37-38°C.

In addition, a production-useful feature of the B. bifidum 791-BL strain, which distinguishes it from the B. bifidum 791-MB strain, is that the culture grown under stationary conditions on Bifilife M medium in a volume of 300 cm ³ has an increased superoxide dismutase activity. SOD activity of B. bifidum 791-BL strain is 27.7 units/mg of protein contained in the bacterial mass.

The revealed SOD activity of the culture of bifidobacteria indicates that the strain has a pronounced probiotic effect, which can manifest itself both in vivo and in vitro. The high activity of acid formation, the resistance of the strain to the main antibiotics contributes to the effective restoration of the normal intestinal microflora against the background of antibiotic therapy.

Storage conditions of the proposed strain.

The freshly prepared B. bifidum 791-BL strain is stored on a nutrient medium with the addition of a protective medium containing sucrose, gelatin, sodium citrate, and freeze-dried in ampoules. The strain is well stored at a temperature of 6-8°C without loss of activity during the year.

Genetic identification of the strain was carried out using the analysis of 16S rRNA.

Conservative primers for 16S rDNA production:

8f - aga gtt tga tcc tgg etc ag

926r - ccg tea att cct ttr agt tt

1492r - ggt tac cct tgt tac gac tt

table 2

Homology of the sequenced DNA fragment of the deposited strain with the putative species and the closest species

Distance Matrix 1 2 3 4 5 6 7 8 9 10 eleven B. bifidum791-6H 1 — 0.912 0.970 0.919 0.970 0.919 0.901 0.970 0.912 0.898 0.966 D86194 2 0.088 — 0.939 0.911 0.939 0.911 0.924 0.939 1 0.906 0.934 B. bifid4 3 0.030 0.061 — 0.944 1 0.944 0.929 1 0.939 0.924 0.996 D89379 4 0.081 0.089 0.056 ... 0.944 1 0.929 0.944 0.911 0.940 0.939 B. bifid5 5 0.030 0.061 0.000 0.056 — 0.944 0.929 1 0.939 0.924 0.996 D89378 6 0.081 0.089 0.056 0.000 0.056 ... 0.929 0.944 0.911 0.940 0.939 D86190 7 0.099 0.076 0.071 0.071 0.071 0.071 — 0.929 0.924 0.911 0.924 B. bifidu 8 0.030 0.061 0.000 0.056 0.000 0.056 0.071 — 0.939 0.924 0.996 D86195 9 0.088 0.000 0.061 0.089 0.061 0.089 0.076 0.061 ... 0.906 0.934 D86184 10 0.102 0.094 0.076 0.060 0.076 0.060 0.089 0.076 0.094 — 0.924 B. bifid3 eleven 0.034 0.066 0.004 0.061 0.004 0.061 0.076 0.004 0.066 0.076 —

The studied strain of Bifidobacterium bifidum 791-BL belongs to the following systematic group: Bacteria; Actinobacteria; Actinobacteridae; Bifidobacteriales; Bifidobacteriaceae; Bifidobacterium, and the homology with the species Bifidobacterium bifidum is 97%. Analysis in the RDP II 16S rRNA database showed homology with the same bacterial species Bifidobacterium bifidum.

According to the analysis, a phylogenetic tree with homologous strains was constructed (Fig. 1).

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Table 3

B. bifid5 Bifidobacterium bifidum KSTS 3202 B. bifid4 Bifidobacterium bifidum KSTS 3202 B. bifid3 Bifidobacterium bifidum DSM 20456 (T) B. bifidu Bifidobacterium bifidum ATCC 29521 (T) D89379 Bifidobacterium subtile JCM 7109 D89378 Bifidobacterium subtile DSM 20096 D86190 Bifidobacterium bourn JCM 1211 D86195 Bifidobacterium pseudoIongum subsp. pseudoIongum JCM 1205 D86184 Bifidobacterium infantis ATCC 15697 (T) D86194 Bifidobacterium pseudoIongum subsp. globosum JCM 5820 B. bifidum 791-BL The studied strain of Bifidobacterium bifidum 791-BL

According to the results of the analysis of sequences of 16S rDNA variable regions, the tested strain Bifidobacterium bifidum 791-BL can be attributed to the species Bifidobacterium bifidum (97%).

Isolation of DNA for PCR.

PCR protocols. A guide to methods and applications.

Innis M., Gelfand D., Sninsky J. p. 14-15.

PCR conditions.

Catalog MBI Fermentas 1998/1999, 146-157.

Pavlicek A. et al. Fre-Tree-freeware program for construction of phylogenetic trees on the basis of distance data and bootstrap/jackknife analysis of tree robustness. Application in the RAPD analysis of genus Frenkelia Folia Biol (Praha) 1999 45(3) 97-9.

As a result, the nucleotide sequence of a fragment of the genome of the strain Bifidobacterium bifidum 791-BL SEQ ID NO: 1, identical to that presented on a machine-readable medium, was obtained.

Information about the safety of using the strain: the strain is not genetically modified and does not contain genes from other organisms; transferred resistance genes; genetic changes associated with the use of genetic engineering techniques; the strain is not zoopathogenic and phytopathogenic.

2. To obtain the Bifidobacterium longum B 379M-BL strain, the Bifidobacterium longum B 379M-MB strain was selected; The original strain is protected by the patent of the Russian Federation No. 2261907. The selection was carried out without the use of genetic modification methods by means of multiple cycles of lyophilized drying and recovery on the previously mentioned nutrient medium Bifilife M.

The primary inoculum of the production strain Bifidobacterium longum B 379M-MB in a volume of 5% of the volume of the cultivation medium was introduced into the above medium and cultivated at a temperature of 37-38°C for 24 hours. Next, 30 cycles of drying-recovery were carried out on the Bifilife M nutrient medium.

As a result of the selection, the strain Bifidobacterium longum B 379M-BL was obtained, which was deposited on November 16, 2016 in the VKPM Federal State Unitary Enterprise GosNIIgenetika of the Ministry of Education and Science under registration No. Ac2037.

Cultural, morphological and biological features - cells are Gram-positive polymorphic rods with a bifurcation or thickening at 1-2 ends, prone to the formation of clusters. In the hepatic environment, Blaurock forms colonies in the form of comets.

Facultative anaerobe, optimum temperature 37-38°C, pH 7.0±0.2.

Ferment milk with the formation of a stable clot by 16-20 hours. Gas-forming ability, catalase-forming ability, gelatin liquefaction are absent.

Acid-forming activity: when cultivating in sterile skimmed milk, the titratable acidity reaches 100°T by 18 hours, 120°T by 20 hours, and 145°T by 24 hours.

The maximum titer of bifidobacteria in the milk clot reaches 18 hours of cultivation and is ¹⁰⁹ CFU/ml.

The strain grows on nutrient media used in production with biomass accumulation of at least lg 9-10 microbial number for 16-18 hours of cultivation.

The strain has a pronounced antagonistic activity against pathogenic and conditionally pathogenic microorganisms: Shigella sonnei, Shigella flexneri, Salmonella typhimurium, Escherichia coli, Candida albicans, Proteus vulgaris., Staphylococcus aureus, Klebsiella pneumonia, Citrobacter freundii, Bacillus subtilis. In table. 4 shows antagonistic activity against some of the aforementioned micro-organisms.

The antagonistic activity of a monostrain of bifidobacteria was determined by the zone of growth inhibition of test cultures of pathogenic and conditionally pathogenic microorganisms by the method of perpendicular strokes.

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Table 4

Comparison of antagonistic properties of B. longum B379M-MB and B. longum B379M-BL strains

Bifidobacteria strain Zone of growth inhibition of test cultures (mm) | Shigella sonnei Shigella flexneri Salmonella typhimurium Escherichia coli candida albicans Proteus vulgaris Staphylococcus aureus B. longum B379MMB 15-17 18-20 15-17 17-19 15-17 18-20 17-19 V. longum B379MBL 26-30 32-34 20-22 26-28 20-22 30-31 25-26

Analysis of the data in the table shows that the claimed strain has a higher antagonistic activity against the main test cultures that cause food poisoning.

Determination of the level of resistance of the strain to antibiotics was carried out by the method of serial dilutions in a liquid nutrient medium - sterile skimmed milk. Various concentrations of sterile solutions of antibiotics and a 16-hour culture of bifidobacteria in the amount of 5% were introduced into the nutrient medium. Stability was determined by the change in titratable acidity in the samples after 48 h of incubation at a temperature of 37-38°C.

In addition, a production-useful feature of the B. longum B379M-BL strain, which distinguishes it from the B. longum B379M-MB strain, is that the culture grown under stationary conditions on a Bifilife M nutrient medium in a volume of 300 cm ³ has a pronounced superoxide dismutase activity. SOD activity of the B. longum B379M-BL strain is 32.0 U/mg of the protein contained in the bacterial mass. The revealed SOD activity of the culture of bifidobacteria indicates that the strain has a pronounced probiotic effect, which can manifest itself both in vivo and in vitro. The high activity of acid formation, the resistance of the strain to major antibiotics contributes to the effective restoration of normal intestinal microflora against the background of antibiotic therapy.

Storage conditions of the proposed strain.

Freshly prepared strain B. longum B379M-BL on a nutrient medium with the addition of a protective medium containing sucrose, gelatin, sodium citrate, freeze-dried in ampoules. The strain is well stored at a temperature of 6-8°C without loss of activity during the year.

Genetic identification of the strain was carried out using the analysis of 16S rRNA.

Conservative primers for 16S rDNA production:

8f - aga gtt tga tcc tgg etc ag

926r - ccg tea att cct ttr agt tt

1492r - ggt tac cct tgt tac gac tt

Table 5

Homology of the sequenced DNA fragment of the deposited strain with the putative species and the closest species

distance matrix

1 2 3 4 5 6 7 8 9 10 eleven B. longumB379M-EJI 1 ... 0.987 0.987 0.990 0.974 0.974 0.939 0.990 0.980 0.956 0.987 B. suis 2 0.013 — 1 0.998 0.982 0.982 0.953 0.998 0.987 0.969 1 B. indiem 3 0.013 0.000 ... 0.998 0.982 0.982 0.953 0.998 0.987 0.969 1 B. longum 4 0.010 0.003 0.003 ... 0.985 0.985 0.950 1 0.990 0.966 0.998 D89329 5 0.026 0.018 0.018 0.015 — 1 0.945 0.985 0.995 0.967 0.982 D89330 6 0.026 0.018 0.018 0.015 0.000 ... 0.945 0.985 0.995 0.967 0.982 D86186 7 0.061 0.047 0.047 0.050 0.055 0.055 — 0.950 0.945 0.966 0.953 B. plonpl 8 0.010 0.003 0.003 0.000 0.015 0.015 0.050 ... 0.990 0.966 0.998 D86196 9 0.021 0.013 0.013 0.010 0.005 0.005 0.055 0.010 — 0.972 0.987 D86187 10 0.044 0.031 0.031 0.034 0.034 0.034 0.034 0.034 0.028 — 0.969 D86184 eleven 0.013 0.000 0.000 0.003 0.018 0.018 0.047 0.003 0.013 0.031 —

The studied strain of Bifidobacterium longum B379M-BL belongs to the following systematic group: Bacteria; Actinobacteria; Actinobacteridae; Bifidobacteriales; Bifidobacteriaceae; Bifidobacterium. Analysis according to the RDP II 16S pRNA database showed the best homology with the bacterial species Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium suis, Bifidobacterium subtile, Bifidobacterium pseudolongum, Bifidobacterium pullorum. According to the analysis, a phylogenetic tree with homologous strains was constructed (Fig. 2).

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Table 6

D86184 Bifidobacterium infantis ATCC 15697 CD B. longum Bifidobacterium longum ATCC 15707 (T) B. suis Bifidobacterium suis ATCC 27533 (T) D86196 Bifidobacterium pullorum JCM1214 B. plonpl Bifidobacterium pseudoIongum subsp. pseudoIongum str. PNC-2-9G ATCC 25526 CD D86186 Bifidobacterium choerinum ATCC 27686 D86187 Bifidobacterium pseudocatenulatum str. type strain JCM 1200 B. indicm Bifidobacterium indicum ATCC 25912 (T) D89330 Bifidobacterium saeculare DSM 6533 D89329 Bifidobacterium subtile JCM 7109 V. longum V379M-BL The studied strain of Bifidobacterium longum V379M-BL

Based on the analysis of the sequences of the 16S rDNA variable regions, the Bifidobacterium longum B379M-BL strain can be assigned to the Bifidobacterium longum species (99%).

Isolation of DNA for PCR.

PCR protocols. A guide to methods and applications.

Innis M., Gelfand D., Sninsky J. p. 14-15.

PCR conditions.

Catalog MBI Fermentas 1998/1999, 146-157.

Pavlicek A. et al. Fre-Tree-freeware program for construction of phylogenetic trees on the basis of distance data and bootstrap/jackknife analysis of tree robustness. Application in the RAPD analysis of genus Frenkelia Folia Biol (Praha) 1999, 45(3) 97-9.

As a result, the nucleotide sequence of a fragment of the genome of the strain Bifidobacterium longum B379M-BL SEQ ID NO: 2, identical to that presented on a machine-readable medium, was obtained.

Information about the safety of using the strain: the strain is not genetically modified and does not contain genes from other organisms; transferred resistance genes; genetic changes associated with the use of genetic engineering techniques; the strain is not zoopathogenic and phytopathogenic.

3. To obtain the strain Bifidobacterium adolescentis G 7513-BL, the selection of the strain Bifidobacterium adolescentis G 7513-MB was carried out, which was deposited on July 15, 2004 in the VKPM FSUE State Research Institute of Genetics of the Ministry of Education and Science under No. Ac-1690. The original strain of Bifidobacterium adolescentis G 7513-MB is protected by RF patent No. 2261904. The selection was carried out without the use of genetic modification methods by means of multiple cycles of lyophilized drying and reconstitution on the previously mentioned nutrient medium Bifilife M.

The primary inoculum of the production strain Bifidobacterium adolescentis G 7513-MB in a volume of 5% of the volume of the cultivation medium was introduced into the above medium and cultivated at a temperature of 37-38°C for 24 hours. Then, 30 cycles of drying-recovery were carried out on the Bifilife M nutrient medium.

As a result of the selection, a strain of Bifidobacterium adolescentis G 7513-BL was obtained, which was deposited on November 16, 2016 in the VKPM Federal State Unitary Enterprise GosNIIgenetika of the Ministry of Education and Science under registration No. Ac2038.

Cultural, morphological and biological features - cells are Gram-positive polymorphic rods with a bifurcation or thickening at 1-2 ends, prone to cluster formation. In Blaurock's liver medium and corn-lactose medium, it forms clove-shaped colonies. Facultative anaerobe, optimum temperature 37-38°C, pH 7.0±0.2.

Ferment sterile milk with the formation of a stable dense clot by 22-24 hours. Gas-forming ability, catalase-forming ability, gelatin liquefaction are absent.

Acid-forming activity: when cultivated in sterile skimmed milk, titratable acidity reaches 70°T by 24 h and accumulates the microbial mass of bifidobacteria in 1 ml lg 9.09.4 microbial cells.

Acid-forming activity: by 24 h - 70°T, by 72 h - 90-100°T (on a liver medium), on a hydrolyzate-milk medium - up to 100°T and 180°T, respectively. The maximum titer of bifidobacteria in a hydrolysate-milk medium after 24 hours of cultivation is 1x10 ¹⁰ CFU/ml.

The strain grows on nutrient media used in production with biomass accumulation of at least lg 9.8-10.0 microbial number for 18-20 hours of cultivation.

The strain has a pronounced antagonistic activity against pathogenic and conditionally pathogenic microorganisms Shigella sonnei, Shigella flexneri, Escherichia coli, Proteus vulgaris, Staphylococcus aureus, Candida albicans, Klebsiella pneumonia, Citrobacter freundii, Bacillus subtilis. In table. 7 shows antagonistic activity against some of the aforementioned microorganisms.

The antagonistic activity of a monostrain of bifidobacteria was determined by the zone of growth inhibition of test cultures of pathogenic and conditionally pathogenic microorganisms by the method of perpendicular strokes.

- 7 041668

Table 7

Comparison of antagonistic properties of strains B. adolescentis G 7513-MB and B. adolescentis G 7513-BL

Bifidobacteria strain Zone of growth inhibition of test cultures (mm) Shigella sonnei Shigella flexneri Escherichia coli Proteus vulgaris Staphylococcus aureus B. adolescentis D 7513-MB 24.3 25.9 27.7 26.1 28.3 B. adolescentis G 7513-BL 26-30 26-30 26-30 26-30 28-30

Analysis of the data in the table shows that the claimed strain has a higher antagonistic activity against the main test cultures that cause food poisoning.

Determination of the level of resistance of the strain to antibiotics was carried out by the method of serial dilutions in a liquid nutrient medium - sterile skimmed milk. Various concentrations of sterile solutions of antibiotics and a 16-hour culture of bifidobacteria in the amount of 5% were introduced into the nutrient medium. Stability was determined by the change in titratable acidity in the samples after 48 h of incubation at a temperature of 37-38°C.

In addition, a production-useful feature of the B. adolescentis G 7513-BL strain, which distinguishes it from the B. adolescentis G 7513-MB strain, is that the culture grown under stationary conditions on a Bifilife M nutrient medium in a volume of 300 cm ³ has a pronounced superoxide dismutase activity. The SOD activity of the strain B. adolescentis G 7513-BL is 25.0 U/mg of the protein contained in the bacterial mass. The revealed SOD activity of the culture of bifidobacteria indicates that the strain has a pronounced probiotic effect, which can manifest itself both in vivo and in vitro. The high activity of acid formation, the resistance of the strain to the main antibiotics contributes to the effective restoration of the normal intestinal microflora against the background of antibiotic therapy.

Storage conditions of the proposed strain.

The freshly prepared B. adolescentis G 7513-BL strain is stored on a nutrient medium with the addition of a protective medium containing sucrose, gelatose, sodium citrate, and dried by sublimation in ampoules. The strain is well stored at a temperature of 6-8°C without loss of activity during the year.

Genetic identification of the strain was carried out using the analysis of 16S rRNA.

Conservative primers for 16S rDNA production

8f - aga gtt tga tcc tgg etc ag

926r - ccg tea att cct ttr agt tt

1492r - ggt tac cct tgt tac gac tt

Table 8

Homology of the sequenced DNA fragment of the deposited strain with the putative species and the closest species

distance matrix

1 2 3 4 5 6 7 8 9 10 eleven B.adolesr7513 En 1 — 0.936 0.958 0.958 0.967 0.944 0.947 0.944 0.950 0.977 0.960 D86194 2 0.064 — 0.936 0.938 0.927 0.936 0.947 0.931 0.936 0.930 0.938 B. longum 3 0.042 0.064 ... 0.955 0.947 0.947 0.941 0.942 0.957 0.955 0.999 D86183 4 0.042 0.062 0.045 — 0.965 0.951 0.951 0.963 0.958 0.972 0.957 D86197 5 0.033 0.073 0.053 0.035 — 0.941 0.941 0.966 0.969 0.983 0.949 D86188 6 0.056 0.064 0.053 0.049 0.059 ... 0.952 0.955 0.961 0.948 0.946 D86190 7 0.053 0.053 0.059 0.049 0.059 0.048 ... 0.958 0.951 0.944 0.943 D86187 8 0.056 0.069 0.058 0.037 0.034 0.045 0.042 ... 0.978 0.949 0.944 D86182 9 0.050 0.064 0.043 0.042 0.031 0.039 0.049 0.022 ... 0.952 0.958 B.adoles 10 0.023 0.070 0.045 0.028 0.018 0.052 0.056 0.051 0.048 — 0.955 D86184 eleven 0.040 0.062 0.001 0.043 0.051 0.054 0.057 0.056 0.042 0.045 —

The studied strain of Bifidobacterium adolescentis G7513-BL belongs to the following systematic group: Bacteria; Actinobacteria; Actinobacteridae; Bifidobacteriales; Bifidobacteriaceae; Bifidobacterium, and the homology with the species Bifidobacterium adolescentis is 97%.

According to the analysis, a phylogenetic tree with homologous strains was constructed (Fig. 3).

Table 9

sequence description

D86197 Bifidobacterium ruminantium JCM 8222 B.adoles Bifidobacterium adolescentis ATCC 15703 (T) D86183 Bifidobacterium dentium ATCC 27534 D86184 Bifidobacterium inf antis ATCC 15697 (T) D86190 Bifidobacterium boum JCM 1211 D86187 Bifidobacterium pseudocatenulatum str. type strain JCM 1200 D86182 Bifidobacterium angulatum str. type strain ATCC 27535 (T) B. longum Bifidobacterium longum ATCC 15707 (T) D86194 Bifidobacterium pseudoIongum subsp. globosum JCM 5820 D86188 Bifidobacterium indicum JCM 1302 B. adoles G7513-BL The studied strain of Bidobacterium adolescentis G7513-BL

- 8 041668

According to the results of the analysis of sequences of 16S rDNA variable regions, the tested strain of Bifidobacterium adolescentis G7513-BL can be attributed to the species Bifidobacterium adolescentis (97%).

Isolation of DNA for PCR.

PCR protocols. A guide to methods and applications.

Innis M., Gelfand D., Sninsky J. p. 14-15.

PCR conditions.

Catalog MBI Fermentas 1998/1999, 146-157.

Pavlicek A. et al. Fre-Tree-freeware program for construction of phylogenetic trees on the basis of distance data and bootstrap/jackknife analysis of tree robustness. Application in the RAPD analysis of genus Frenkelia Folia Biol (Praha) 1999, 45(3) 97-9.

As a result, the nucleotide sequence of a fragment of the genome of the strain Bifidobacterium adolescentis G 7513-BL SEQ ID NO: 3, identical to that presented on a machine-readable medium, was obtained.

Information about the safety of using the strain: the strain is not genetically modified and does not contain genes from other organisms; transferred resistance genes; genetic changes associated with the use of genetic engineering techniques; the strain is not zoopathogenic and phytopathogenic.

4. To obtain the strain Bifidobacterium infantis 73-15-BL, the selection of the strain Bifidobacterium infantis 73-15-MB was carried out, which was deposited on July 15, 2004 in the VKPM FSUE State Research Institute of Genetics of the Ministry of Education and Science under No. Ac-1692. The original strain of Bifidobacterium infantis 73-15-MB is protected by the patent of the Russian Federation No. 2261906. The selection was carried out without the use of genetic modification methods by means of multiple cycles of lyophilized drying and reconstitution on the previously mentioned nutrient medium Bifilife M.

The primary inoculum of the production strain Bifidobacterium infantis 73-15-MB in a volume of 5% of the volume of the cultivation medium was introduced into the above medium and cultivated at a temperature of 37-38°C for 24 hours. Then 30 cycles of drying-recovery were carried out on the Bifilife M nutrient medium.

As a result of the selection, a strain of Bifidobacterium infantis 73-15-BL was obtained, which was deposited on November 16, 2016 in the VKPM Federal State Unitary Enterprise GosNIIgenetika of the Ministry of Education and Science under registration No. Ac2039.

Cultural, morphological and biological features - cells are Gram-positive polymorphic rods with a bifurcation or thickening at 1-2 ends, prone to the formation of clusters. In Blaurock's hepatic medium, the strain forms colonies in the form of comets. In liquid nutrient media, it grows along the entire height of the volume, except for the zone of aerobiosis. Facultative anaerobe, optimum growing temperature 37-38°C, pH of nutrient media 7.0±0.2.

Ferment sterile milk by 24-28 hours with the formation of a dense clot. Gas-forming ability, catalase-forming ability, gelatin liquefaction are absent.

Acid-forming activity: when cultivated in sterile skimmed milk, titratable acidity reaches 80°T by 24 hours, and the strain accumulates the microbial mass of bifidobacteria in 1 ml lg 10.2-10.6 microbial cells. The strain grows on nutrient media used in production with biomass accumulation of at least lg 9-10 microbial number for 18-22 hours of cultivation.

The strain has a pronounced antagonistic activity against pathogenic and conditionally pathogenic microorganisms Shigella sonnei, Shigella flexneri, Escherichia coli, Proteus vulgaris, Staphylococcus aureus, Candida albicans, Proteus mirabilis, Klebsiella pneumoniae, Citrobacter freundii, Bacillus subtilis. In table. 10 shows antagonistic activity against some of the aforementioned microorganisms.

The antagonistic activity of a monostrain of bifidobacteria was determined by the zone of growth inhibition of test cultures of pathogenic and conditionally pathogenic microorganisms by the method of perpendicular strokes.

Table 10

Comparative data on the antagonistic activity of B. infantis 73-15-MB and B. infantis 73-15-BL strains

Bifidobacteria strain Zone of growth inhibition of test cultures (mm) Shigella sonnei Shigella flexneri Escherichia coli Proteus vulgaris Staphylococcus aureus B. infantis 73-15-MB 11.6 11.9 17 12.6 16.9 B. infantis 73-15-BL 18-20 23-25 17-21 19-25 17-19

Analysis of the data in the table shows that the claimed strain has a higher antagonistic activity against the main test cultures that cause food poisoning.

Determination of the level of resistance of the strain to antibiotics was carried out by the method of serial dilutions in a liquid nutrient medium - sterile skimmed milk. Various concentrations of sterile solutions of antibiotics and a 16-hour culture of bifidobacteria in the amount of 5% were introduced into the nutrient medium. Stability was determined by the change in titratable acidity in the samples after 48 h of incubation at a temperature of 37-38°C.

- 9 041668

In addition, a production-useful feature of the B. infantis 73-15-BL strain, which distinguishes it from the B. infantis 73-15-MB strain, is that the culture grown under stationary conditions on a nutrient medium Bifilife M in a volume of 300 cm ³ has pronounced superoxide dismutase activity. The SOD activity of the B. infantis 73-15-BL strain is 29 units/mg of the protein contained in the bacterial mass. The revealed SOD activity of the culture of bifidobacteria indicates that the strain has a pronounced probiotic effect, which can manifest itself both in vivo and in vitro. The high activity of acid formation, the resistance of the strain to the main antibiotics contributes to the effective restoration of the normal intestinal microflora against the background of antibiotic therapy.

Storage conditions of the proposed strain.

The freshly prepared strain of B. infantis 73-15-BL is stored on a nutrient medium with the addition of a protective medium containing sucrose, gelatin, sodium citrate, and freeze-dried in ampoules. The strain is well stored at a temperature of 6-8°C without loss of activity during the year.

Genetic identification of the strain was carried out using the analysis of 16S rRNA.

Conservative primers for 16S rDNA production:

8f - aga gtt tga tcc tgg etc ag

926r - ccg tea att cct ttr agt tt

1492r - ggt tac cct tgt tac gac tt

distance matrix

Table 11

Homology of the sequenced DNA fragment of the deposited strain with the putative species and the closest species

1 2 3 4 5 6 7 8 9 10 eleven B. infantis 73-15-BL 1 — 0.970 0.973 0.958 0.974 0.933 0.974 0.933 0.934 0.933 0.973 B. Iongu3 2 0.030 — 0.993 0.980 0.992 0.955 0.992 0.949 0.956 0.950 0.996 B. suis 3 0.027 0.007 — 0.981 0.999 0.954 0.999 0.951 0.958 0.955 0.997 B. indiem 4 0.043 0.020 0.019 — 0.980 0.955 0.980 0.970 0.940 0.949 0.984 B. longum 5 0.026 0.008 0.001 0.020 — 0.952 1 0.949 0.956 0.953 0.996 D86183 6 0.067 0.045 0.046 0.045 0.048 — 0.952 0.946 0.955 0.946 0.956 B. plonpl 7 0.026 0.008 0.001 0.020 0.000 0.048 ... 0.949 0.956 0.953 0.996 B.breve 8 0.067 0.051 0.049 0.030 0.051 0.054 0.051 — 0.940 0.939 0.953 D86187 9 0.066 0.044 0.043 0.060 0.044 0.045 0.044 0.060 ... 0.965 0.958 D86182 10 0.067 0.050 0.045 0.051 0.047 0.054 0.047 0.061 0.035 — 0.955 D86184 eleven 0.027 0.004 0.003 0.016 0.004 0.044 0.004 0.047 0.043 0.045 —

The studied strain of Bifidobacterium infantis 73-15-BL belongs to the following systematic group: Bacteria; Actinobacteria; Actinobacteridae; Bifidobacteriales; Bifidobacteriaceae; Bifidobacterium. Analysis according to the RDP II 16S rRNA database showed the best homology with the bacterial species Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium suis, Bifidobacterium pseudolongum.

According to the analysis, a phylogenetic tree with homologous strains was constructed (Fig. 4).

Table 12

D86184 Bifidobacterium infantis ATCC 15697 (T) B. longum Bifidobacterium longum ATCC 15707 (T) B. suis Bifidobacterium suis ATCC 27533 (T) B. plonpl Bifidobacterium pseudolongum subsp. pseudolongum str. PNC-2-9G ATCC 25526 (T) D86187 Bifidobacterium pseudocatenulatum str. type strain JCM1200 B. indiem Bifidobacterium indicum ATCC 25912 (T) B.breve Bifidobacterium infantis ATCC 15700 D86183 Bifidobacterium dentium ATCC 27534 D86182 Bifidobacterium angulatum str. type strain ATCC 27535 (T) B. Iongu3 Bifidobacterium longum ATCC 15708 B. infantis 73-15-BL The studied strain of Bifidobacterium infantis 73-15-BL

The studied strain of Bifidobacterium infantis 73-15-BL belongs to the species Bifidobacterium infantis. The species Bifidobacterium infantis is currently referred to as Bifidobacterium longum subsp. infantis. According to the results of the analysis of sequences of 16S rDNA variable regions, the tested Bifidobacterium infantis strain 73-15-BL can be attributed to the species Bifidobacterium longum subsp. infantis (97%).

Isolation of DNA for PCR.

PCR protocols. A guide to methods and applications.

Innis M., Gelfand D., Sninsky J. p.14-15.

PCR conditions.

Catalog MBI Fermentas 1998/1999, 146-157.

Pavlicek A. et al. Fre-Tree-freeware program for construction of phylogenetic trees on the basis of distance data and bootstrap/jackknife analysis of tree robustness. Application in the RAPD analysis of genus Frenkelia Folia Biol (Praha) 1999, 45(3) 97-9.

As a result, the nucleotide sequence of the genome fragment of the strain Bifidobacterium

- 10 041668 infantis 73-15-BL SEQ ID NO: 4 identical to that shown on the machine-readable medium.

Information about the safety of using the strain: the strain is not genetically modified and does not contain genes from other organisms; transferred resistance genes; genetic changes associated with the use of genetic engineering techniques; the strain is not zoopathogenic and phytopathogenic.

5. To obtain the strain Bifidobacterium breve 79-119-BL, the selection of the strain Bifidobacterium breve 79-119-MB was carried out, which was deposited on July 15, 2004 in the VKPM Federal State Unitary Enterprise GosNIIgenetika of the Ministry of Education and Science under registration No. Ac-1691. The original strain of Bifidobacterium breve 79-119-MB is protected by RF patent No. 2261905. The selection was carried out without the use of genetic modification methods through multiple cycles of lyophilized drying and reconstitution on the previously mentioned nutrient medium Bifilife M.

The primary inoculum of the production strain Bifidobacterium breve 79-119-MB in a volume of 5% of the volume of the cultivation medium was introduced into the above medium and cultivated at 3738°C for 24 hours. Next, 30 drying-recovery cycles were carried out on the Bifilife M nutrient medium.

As a result of the selection, a strain of Bifidobacterium breve 79-119-BL was obtained, which was deposited on November 16, 2016 in the VKPM Federal State Unitary Enterprise GosNIIgenetika of the Ministry of Education and Science under registration No. Ac2036.

Cultural, morphological and biological features - cells are gram-positive polymorphic rods with Y-shaped bifurcations at one or two ends; immobile, prone to the formation of clusters. In the hepatic environment, Blaurock forms colonies in the form of comets. In liquid nutrient media, it grows along the entire height of the volume, except for the zone of aerobiosis. Facultative anaerobe, optimum growing temperature 37-38°C, pH 7.0±0.2.

It ferments milk with the formation of a dense clot by 16-18 hours. Gas-forming ability, catalase-forming ability, gelatin liquefaction are absent.

Acid-forming activity: when cultivating in sterile skimmed milk, titratable acidity reaches 120°T by 18 hours, 140°T by 24 hours. The strain maximally accumulates the microbial mass of bifidobacteria by 18±2 hours of cultivation, reaching 109 CFU/ml in 1 ml.

The strain grows on nutrient media used in production with biomass accumulation of at least lg 10.2 microbial number for 16-18 hours of cultivation.

The strain has a pronounced antagonistic activity against pathogenic and conditionally pathogenic microorganisms Shigella sonnei, Shigella flexneri, Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Candida albicans, Klebsiella pneumoniae, Citrobacter freundii, Bacillus subtilis. In table. 13 shows antagonistic activity against some of the aforementioned microorganisms.

The antagonistic activity of a monostrain of bifidobacteria was determined by the zone of growth inhibition of test cultures of pathogenic and conditionally pathogenic microorganisms by the method of perpendicular strokes.

Table 13 Comparative data on the antagonistic activity of B. breve 79-119-MB and B. breve 79-119-BL strains

Bifidobacteria strain Zone of growth inhibition of test cultures (mm) Shigella sonnei Shigella flexneri Escherichia coli Staphylococcus aureus Proteus vulgaris B. breve 79-119-MB 16.0-16.7 18.2-18.9 18-18.5 16.9-17 19.2-20 B. breve 79-119-BL 26-30 33-35 26-28 21-25 28-30

Analysis of the data in the table shows that the claimed strain has a higher antagonistic activity against the main test cultures that cause food poisoning.

Determination of the level of resistance of the strain to antibiotics was carried out by the method of serial dilutions in a liquid nutrient medium - sterile skimmed milk. Various concentrations of sterile solutions of antibiotics and a 16-hour culture of bifidobacteria in the amount of 5% were introduced into the nutrient medium. Stability was determined by the change in titratable acidity in the samples after 48 h of incubation at a temperature of 37-38°C.

In addition, a production-useful feature of the strain B. breve 79-119-BL, which distinguishes it from the strain B. breve 79-119-MB, is that the culture grown under stationary conditions on a nutrient medium Bifilife M in a volume of 300 cm ³ has pronounced superoxide dismutase activity. The revealed SOD activity of the B. breve 79-119-BL strain indicates that the strain has a pronounced probiotic effect, which can manifest itself both in vivo and in vitro. The high activity of acid formation, the resistance of the strain to the main antibiotics contributes to the effective restoration of the normal intestinal microflora against the background of antibiotic therapy. The claimed strain has a pronounced antioxidant activity: the superoxide dismutase activity of the strain is 27.0 U/mg of microbial mass protein.

Storage conditions of the proposed strain.

The freshly prepared strain B. breve 79-119-BL is stored on a nutrient medium with the addition of a protective medium containing sucrose, gelatin, sodium citrate, and freeze-dried in ampoules. The strain is well stored at a temperature of 6-8°C without loss of activity during

- 11 041668.

Genetic identification of the strain was carried out using the analysis of 16S rRNA.

Conservative primers for 16S rDNA production:

8f - aga gtt tga tcc tgg etc ag

926r - ccg tea att cct ttr agt tt

1492 - ggt tac cct tgt tac gac tt

Table 14

Homology of the sequenced DNA fragment of the deposited strain with the putative species and the closest species

distance matrix

1 2 3 4 5 6 7 8 9 10 eleven B. breve79-119-B4 1 ... 0.943 0.974 0.936 0.930 0.930 0.977 0.931 0.943 0.944 0.970 B. longum 2 0.057 — 0.960 0.939 0.954 0.953 0.964 0.946 0.944 0.999 0.956 V. breve8 3 0.026 0.040 ... 0.954 0.948 0.948 0.996 0.949 0.960 0.962 0.996 D86186 4 0.064 0.061 0.046 — 0.940 0.930 0.957 0.944 0.960 0.941 0.949 D86183 5 0.070 0.046 0.052 0.060 ... 0.947 0.951 0.960 0.949 0.956 0.943 D89379 6 0.070 0.047 0.052 0.070 0.053 ... 0.951 0.945 0.941 0.954 0.943 B. breve 7 0.023 0.036 0.004 0.043 0.049 0.049 ... 0.952 0.964 0.965 0.991 D86187 8 0.069 0.054 0.051 0.056 0.040 0.055 0.048 ... 0.959 0.947 0.944 D86190 9 0.057 0.056 0.040 0.040 0.051 0.059 0.036 0.041 — 0.946 0.956 D86184 10 0.056 0.002 0.038 0.059 0.044 0.046 0.035 0.053 0.054 — 0.957 V. breve7 eleven 0.030 0.044 0.004 0.051 0.057 0.057 0.009 0.056 0.044 0.043 —

The studied strain Bifidobacterium breve 79-119-BL belongs to the following systematic group: Bacteria; Actinobacteria; Actinobacteridae; Bifidobacteriales; Bifidobacteriaceae; Bifidobacterium. Analysis in the RDP II 16S pRNA database showed homology with the same bacterial species Bifidobacterium breve.

According to the analysis, a phylogenetic tree with homologous strains was built (Fig. 5)

Table 15

B. breve Bifidobacterium infantis ATCC 15700 B. breveS Bifidobacterium infantis CIP 6469 (T) D86184 Bifidobacterium infantis ATCC 15697 (T) V. breve7 Bifidobacterium breve ATCC 15698 D86187 Bifidobacterium pseudocatenulatum str. type strain JCM1200 D86183 Bifidobacterium dentium ATCC 27534 B. longum Bifidobacterium longum ATCC 15707 (T) D86186 Bifidobacterium choerinum ATCC 27686 D86190 Bifidobacterium bourn JCM 1211 D89379 Bifidobacterium subtile JCM 7109 V. breve 79-119-BL The studied strain of Bifidobacterium breve 79-119-BL

According to the results of the analysis of sequences of 16S rDNA variable regions, the tested Bifidobacterium breve strain 79-119-BL can be attributed to the Bifidobacterium breve species (97%).

Isolation of DNA for PCR.

PCR protocols. A guide to methods and applications.

Innis M., Gelfand D., Sninsky J. p. 14-15.

PCR conditions.

Catalog MBI Fermentas 1998/1999, 146-157.

Pavlicek A. et al. Fre-Tree-freeware program for construction of phylogenetic trees on the basis of distance data and bootstrap/jackknife analysis of tree robustness. Application in the RAPD analysis of genus Frenkelia Folia Biol (Praha) 1999, 45(3) 97-9.

As a result, the nucleotide sequence of the genome fragment of the strain Bifidobacterium breve 79-119-BL SEQ ID NO: 5, identical to that presented on the machine-readable medium, was obtained.

Information about the safety of using the strain: the strain is not genetically modified and does not contain genes from other organisms; transferred resistance genes; genetic changes associated with the use of genetic engineering techniques; the strain is not zoopathogenic and phytopathogenic.

Obtaining a consortium and its characteristics.

1st passage - each strain of lyophilized monocultures of Bifidobacterium bifidum 791-BL, Bifidobacterium longum B379M-BL, Bifidobacterium adolescentis G 7513-BL, Bifidobacterium infantis 73-15-BL, Bifidobacterium breve 79-119-BL is resuspended on a nutrient medium Bifilife M to the eighth tube, followed by incubation at a temperature of 37-38°C for 48 hours.

2nd passage - the resulting cultures of each strain from tubes 10 ^-7 and 10 ^-8 (inoculation dose of 5% of the volume of the nutrient medium Bifilife M) are inoculated into separate containers. The cultivation time is determined in accordance with the duration of the exponential growth phase of each of them.

3rd passage - inoculation of inoculums of monocultures in the fermenter is carried out in stages in the amount of 3-5%

- 12 041668 of each strain from the volume of the nutrient medium. The process of co-cultivation lasts 10-12 hours, the pH of the finished biomass is 4.6-5.2. Protective media (sucrose-gelatin, skim milk) are added to the resulting biomass, the suspension is frozen at a temperature not exceeding minus 45°C, lyophilized, and a dry concentrate is obtained with a content of bifidobacteria of at least 10 ¹¹ CFU/g. The consortium is well stored at a temperature of 6-8°C without loss of activity within one year.

The created consortium of bifidobacteria showed a high compatibility of five strains of bifidobacteria with each other. Co-cultivation of a mixed population of all five strains proceeds much faster compared to separate cultivation of strains.

Cultural and morphological features of microorganisms included in the consortium - cells of strains of microorganisms of the consortium, are Gram-positive polymorphic rods with a bifurcation or thickening at 1-2 ends, prone to the formation of clusters. Dispute does not form. The morphology of the colonies is heterogeneous and depends on the strains included in the consortium, retaining the characteristics of each of them in the total culture. In liquid and semi-liquid nutrient media, they grow along the entire height of the volume, except for the aerobiosis zone.

Physiological and biochemical characteristics of microorganisms included in the consortium.

facultative anaerobes. Optimum growing temperature is 37-38°C, pH of nutrient media is 7.0±0.2. Catalase is not formed, there is no liquefaction of gelatin. The enzymatic activity of individual strains and the consortium according to ANAEROtest-23 is presented in Table. 16. As an illustration, the table shows how the spectrum of enzymatic activity of strains expands during co-cultivation: the number of assimilated substrates increases.

Table 16 Enzymatic activity of bifidobacteria strains in separate and joint cultivation according to ANAEROtest-23 data

Active Ingredient B. bifidum 791-BL V. longum V379M-BL V. breve 79-119-BL V. inf antis 73-15-BL B. adolescentis G7513-BL Consortium D-galactose + + + + + + D-glucose + + + + + + D-fructose + + + 0 + + D-lactose + + + + + + D-sucrose 0 + + 0 + + D-maltose 0 + + 0 + + D-raffinose ± + + ± + + L-arabinose ± + 0 ± ± ± D-xylose ± 0 ± ± ± ± Salicin 0 0 ± 0 + + D-cellobiose 0 ± + 0 + + D-mannose 0 0 + + 0 + D-manitol 0 0 + 0 0 + D-sorbitol 0 + + 0 0 +

Note: (+) -ferments; (-) - does not ferment; (±) - variable; 0 - no data.

Acid-forming activity: by 24 h 150°T, by 72 h - 180°T (on a nutrient medium Bifilife M), on a hydrolyzate-milk medium - up to 140-160°T and 170-190°T, respectively.

The consortium actively reproduces on media of different composition. In vitro on standard nutrient media, at a temperature of T 37°C, the consortium accumulates biomass to a high concentration of probiotic microorganisms 10 ¹¹ CFU per 1 g of biomass due to the absence of mutual inhibition of individual strains (Fig. 6).

The maximum titer of bifidobacteria of the consortium in sterile skimmed milk without growth factors by 22 hours is 2x10 ¹⁰ CFU / ml, on a corn-lactose medium by 20 hours it is 2x10 ¹⁰ CFU / ml, on a nutrient medium Bifilife M after lyophilization, a dry concentrate with a content of bifidobacteria not less than 10 ¹¹ CFU/g.

Bifidobacteria of the consortium are able to utilize free amino acids in sterile skimmed milk, such as valine, methionine, alanine, arginine, isoleucine, glycine.

The consortium exhibits antagonistic activity against pathogenic and conditionally pathogenic microorganisms Escherichia coli, Shigella sonnei, Staphylococcus aureus, Shigella fexneri, Candida albicans, Proteus vulgaris, Proteus mirabilis, Klebsiella pneumoniae, Citrobacter freundii, Bacillus subtilis. In table. 17 shows antagonistic activity against some of the aforementioned microorganisms.

The antagonistic activity of a consortium of bifidobacteria strains was determined by the method of developing mixed populations in co-cultivation with pathogenic microorganisms in the growth retardation zone of test cultures by the method of perpendicular strokes.

The synergistic effect of the consortium is also manifested in the greater antagonistic activity of the consortium compared to the prototype.

- 13 041668

Table 17

Bifidobacteria strain Zone of growth inhibition of test cultures (mm) Escherichia coli Shigella sonnei Staphylococcus aureus Shigella fexneri candida albicans Proteus vulgaris The proposed consortium Bifidobacterium bifidum 791BL, Bifidobacterium longum B 379M-BL, Bifidobacterium adolescentis G 7513-BL, Bifidobacterium infantis 73-15BL, Bifidobacterium breve 79119-BL, 26-30 26-30 28-30 33-35 20-22 30-31 Prototype included Bifidobacterium bifidum 791MB, Bifidobacterium longum B 379M-MB, Bifidobacterium adolescentis D 7513-MB, Bifidobacterium infantis 73-15MB, Bifidobacterium breve 79119-MB, 24-25 22-24 18-20 20-22 19-21 24-26

Analysis of the table data shows that the claimed consortium has the greatest antagonistic effect on the main test cultures that cause food poisoning.

Determination of levels of resistance of strains of bifidobacteria to antibiotics was carried out by the method of serial dilutions in a liquid nutrient medium for each strain separately. Sterile skimmed milk was used as a nutrient medium.

Various concentrations of sterile solutions of antibiotics and 16-hour cultures of bifidobacteria were introduced into the nutrient medium in an amount of 5% of the nutrient medium. Only the biomass of the studied strain of bifidobacteria was added to the control sample. Stability was determined by the change in titratable acidity in the samples after 48 h of incubation at a temperature of 37-38°C. The strain is resistant to antibiotics when the difference between the titratable acidity in the control and experimental samples is no more than 30°T.

In table. 18 shows antibiotic resistance of individual strains of the proposed consortium.

Table 18

Antibiotic resistance of strains of bifidobacteria that make up the proposed consortium

Antibiotics, dose activity units dose Titratable acidity of milk after cultivation with a strain in Turner degrees. Mg / cm ³ B. bifidum 791-BL V. longum V379M-BL B. adolescentis G7513-BL B. infantis 73-15-BL V. breve 79-119-BL Amikacin sulfate 10 76-81 78-82 53-58 77-82 82-89 Ampicillin 250 78-82 67-74 63-68 67-73 77-82 Benzylpenicillin, U / cm ³ 10000 71-76 78-82 59-63 83-88 101-105 Gentamicin 400 80-83 57-61 57-60.0 72-75 80-85 Doxycycline 10 45-50 47-51 42-46 53-59 57-59 Kanamycin sulfate 20 99-102 81-84 62-69 89-92 111-115 Chloramphenicol 50 37-41 45-52 38-41 30-34 37-40 Lincomycin 25 80-86 67-72 65-70 85-90 100-103 Oxacillin 10 38-42 40-46 40-46 30-35 40-43 Oleandomycin 12.5 57-62 67-71 50-56 55-60 65-71 Ofloxacin 20 47-51 56-62 60-66 46-51 39-45 Rifampicin 15 45-52 47-53 53-60 46-52 47-51 Streptomycin 10 89-92 57-61 59-64 71-76 105-111 Sulfadimidine 50 70-76 56-61 51-56 76-80 99-103 Tetracycline 10 71-76 61-66 55-60 83-88 86-91 Cefazolin 20 47-51 52-56 43-47 37-41 46-49 Benzathine benzylpenicillin (U / cm ³ ) 24000 46-48 56-60 37-39 31-33 42-46 Erythromycin 25 37-41 41-43 35-39 23-25 37-39 Control 101-105 85-93 70-74 90-97 110-115

Analysis of the data obtained indicates that the strains of bifidobacteria are resistant to the highest concentrations of the following antibiotics:

B. bifidum 791-BL - to amikacin, ampicillin, benzylpenicillin, gentamicin, kanamycin, lincomycin, tetracycline, sulfadimidine, streptomycin;

B. longum V379M-BL - to amikacin, ampicillin, benzylpenicillin, gentamicin, kanamycin, lincomycin, oleandomycin, ofloxacin, streptomycin, tetracycline, sulfadimidine, benzathine benzylpenicillin;

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B. adolescentis G7513-BL - to amikacin, ampicillin, benzylpenicillin, kanamycin, gentamicin, lincomycin, oleandomycin, tetracycline, ofloxacin, sulfadimidine, streptomycin, benzathine benzylpenicillin;

B. infantis 73-15-BL - to amikacin, ampicillin, benzylpenicillin, gentamicin, kanamycin, tetracycline, lincomycin, sulfadimidine, streptomycin;

B. breve 79-119-BL - to amikacin, ampicillin, benzylpenicillin, gentamicin, kanamycin, lincomycin, tetracycline, sulfadimidine, streptomycin.

A similar study on the claimed consortium of bifidobacteria showed that it was resistant to at least 12 antibiotics amikacin, tetracycline, benzylpenicillin, gentamicin, kanamycin, ofloxacin, sulfadimidine, streptomycin, ampicillin, lincomycin, oleandomycin and benzathine benzylpenicillin.

The ability of a consortium of five strains of bifidobacteria Bifidobacteruim bifidum 791-BL, Bifidobacterium longum B379M-BL, Bifidobacterium adolescentis G7513-BL, Bifidobacterium infantis 73-15-BL, Bifidobacterim breve 79-119-BL to actively accumulate microbial biomass on various nutrient media2 hours of cultivation, high antibiotic and superoxide dismutase activity with a pronounced antioxidant activity: superoxide dismutase activity of the consortium is not lower than 37 units/mg of microbial mass protein, the specific activity exhibited made it possible to use the consortium in the preparation of fermented, non-fermented food products, dietary supplements, bifid-containing preparations, cosmetic and hygiene products.

The conducted studies have shown that during the joint cultivation of a consortium of bifidobacteria on a nutrient medium Bifilife M, there is a more active development of the biomass of bifidobacteria in the consortium compared to separate cultivation and there is no mutual inhibition of cultures (Table 19).

Table 19

Comparative data of monocultures and a consortium of bifidobacteria

Name of culture B. bifidum 791-BL Cultivation of a consortium of bifidobacteria Separate cultivation of monocultures Number of CFU/g Number of CFU/g 10 ^p 2х1О ¹⁰ B. infantis 73-15-BL ZxYu ¹⁰ V. longum V379M-BL 1X10 ¹⁰ B. adolescentis G7513-BL 2x10 ⁹ V. breve 79-119-BL ZxYu ¹⁰

The consortium can be cultured on various nutrient media with consistently high CFU/ml of probiotic microorganisms and produced organic acids. The results of cultivation are presented in table. 20.

Table 20

The results of cultivation of the consortium on different nutrient media

Growing environment Bifidobacteria Consortium Number of CFU/g Blaurocca 5x10'° Hydrolyzate-milk ZxYu ⁹ Thioglycolic 7х1О ¹⁰ Wednesday "Bifilife M" 2x10 ^p

The resulting consortium and the strains that are part of the consortium can be used as a biologically active principle of a probiotic orientation in the production of food products, functional foods, dietary (therapeutic and preventive) foods, biologically active additives (BAA), cosmetics, hygiene products, medical and veterinary drugs.

At the same time, the biological value of functional food products and preparations made on its basis increases. Eating products made using the claimed consortium contributes to the colonization of the intestinal mucosa, the displacement of pathogenic and opportunistic microflora, the normalization of acidity, the correction of intestinal dysbiosis and the restoration of normal microflora.

The use of the declared consortium and the strains included in the consortium in the production of fermented, non-fermented food products, dietary supplements, bifid-containing preparations, cosmetic and hygiene products - creams, wipes, tampon lotions, nourishing masks, feminine pads, soap, cosmetic milk provides probiotic and bacteriological effect, contributes to the restoration of the normal microflora of the skin and mucous membranes, and is also aimed at expanding the arsenal of similar means.

The implementation of the appointment of the proposed consortium of bifidobacteria is presented in the following examples.

Example 1. The use of the proposed consortium of strains of bifidobacteria in the composition of the starter for the preparation of fermented milk products.

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Get the consortium as follows.

1st passage - each strain of lyophilized monocultures Bifidobacterium bifidum 791-BL, Bifidobacterium longum B 379M-BL, Bifidobacterium adolescentis G 7513-BL, Bifidobacterium infantis 73-15-BL,

Bifidobacterium breve 79-119-BL is resuspended up to the eighth test tube on the nutrient medium Bifilife

M followed by incubation at 37-38°C for 48 hours.

2nd passage - the obtained cultures of each strain from test tubes dilution 10 ^-7 and 10 ^-8 (inoculation dose of 5% of the volume of the nutrient medium Bifilife M) are inoculated into separate containers. The cultivation time is determined in accordance with the duration of the exponential growth phase of each of them.

3rd passage - inoculation of inoculums of monocultures in the fermenter is carried out in stages in the amount of 3-5% of each strain of the volume of the nutrient medium. The process of co-cultivation lasts 10-12 hours. The pH of the finished biomass is 4.6-5.2.

The biomass of the consortium of bifidobacteria, obtained as shown above, in a volume of not more than 5% (of the volume of milk) is introduced into sterile milk and cultivated until a clot is formed, which is poured into containers and cooled.

Example 2. The use of a consortium of strains of bifidobacteria in the composition of dietary supplements.

In the biomass of the consortium of bifidobacteria, obtained as shown in example 1, make various bifidogenic factors that help stabilize the properties of the culture and better survival of the strain in the body. The concentrate, poured into containers, can be directly used as a dietary supplement with a therapeutic and prophylactic effect.

You can get a dry biologically active additive, for this, protective media (sucrose-gelatin, skim milk) are added to the biomass obtained in example 1, the suspension is frozen at a temperature not exceeding -45 ° C, lyophilized, sealed and a dry concentrate is obtained with a bifidobacteria content of at least 10 ¹¹ CFU/g, which can be used as a biologically active additive by adding it to food raw materials immediately before use.

Example 3. Use of a consortium of strains of bifidobacteria to obtain a bifid-containing medical preparation.

In the biomass of the consortium of bifidobacteria obtained according to example 1, which is a liquid concentrate with a high titer of bifidobacteria, protective media (sucrose-gelatin, skim milk) are added. The resulting suspension is frozen at a temperature not exceeding -45°C, lyophilized, sealed and a dry concentrate is obtained with a content of bifidobacteria of at least 10 ¹¹ CFU/g, which can be used as a bifid-containing medical product.

Example 4. Use of a consortium of strains of bifidobacteria to produce kvass or juice.

The biomass of the consortium of bifidobacteria, obtained according to example 1, in a volume of not more than 5% of the volume of the normalized mixture (kvass or juice) is added to bread kvass or juice after pasteurization, so that the content of bifidobacteria in 1 ml of kvass or juice is at least 10 ⁷ cfu/ml Kvass or juice is cooled and consumed without subsequent fermentation.

Example 5. Use of a consortium of strains of bifidobacteria to obtain a cosmetic product.

The biomass of the consortium of bifidobacteria obtained according to example 1 is destroyed by heating in an autoclave or membrane filtration is carried out. The resulting biologically active components are cooled and mixed with a cream base in a ratio of 1:2-1:20, depending on the purpose of the cream being made.

Example 6. Use of a consortium of strains of bifidobacteria for the production of boiled smoked sausages.

The biomass of the consortium of bifidobacteria obtained according to example 1, in a volume of not more than 5% of the volume of minced meat, is added to the prepared sausage minced meat after adding salt, spices and sodium nitrite solution. After shaping, sausage sticks are sent to the sediment, which is carried out on frames at a temperature of 22°C, humidity 90%. The duration of precipitation is 6 hours. Cooking and smoking are carried out according to standard technology.

Example 7. Use of a consortium of strains of bifidobacteria for the production of smoked sausages.

The consortium of bifidobacteria in dry form, obtained according to example 2, is preliminarily restored in boiled chilled water at a temperature of 37°C, or activated in sterile cow's skimmed milk at a temperature of 37°C for 8-10 hours to an acidity of 60-65°T. The resulting biomass of the consortium of bifidobacteria is introduced into the prepared sausage mince, while the dosage of the introduced reduced consortium is 0.1-0.2% of the mince mass, and the dosage of the activated consortium is 2-5% of the mince mass. Then enriched sausage raw materials are processed according to the traditional scheme according to the technology for producing raw smoked sausages.

Example 8 Use of a consortium of bifidobacteria strains for the production of fortified dry infant formula.

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A small amount of dry milk mixture is thoroughly mixed with a dry concentrate of a consortium of bifidobacteria, obtained according to example 2, with a bifidobacteria content of at least 10 ¹¹ CFU/g at the rate of at least 100 g of concentrate per 1 ton of dry milk mixture. Then, the entire remaining batch of the milk mixture is thoroughly mixed with enriched dry milk mixture to achieve a content of bifidobacteria of at least 10 ⁷ CFU / g.

Example 9 Use of a consortium of bifidobacteria strains to produce fortified beer.

To 1 ton of low-alcohol beer add 100 g of the biomass of the consortium obtained in example 2, and mix thoroughly. The titer of bifidobacteria in the finished beer is at least 10 ⁷ CFU/ml. The taste, color, foaming and other characteristics of the beer do not change.

Example 10 Use of a consortium of bifidobacteria strains for the production of ice cream.

To obtain ice cream, cream is added to milk, the resulting mixture is heated to a temperature of 35 ° C, dry ingredients are added: granulated sugar, a stabilizer, heated to a temperature of 85 ° C, homogenized, cooled to a temperature of 6 ° C, a dry bacterial concentrate of a consortium of bifidobacteria is added, obtained according to example 2, with a content of bifidobacteria not less than 10 ¹¹ CFU/g in the amount of 1-5% by weight of the mixture.

Example 11. The use of a consortium of strains of bifidobacteria for the production of feed additives in animal husbandry and poultry farming.

To prepare 100 kg of a biologically active feed additive, a dry bacterial concentrate of a consortium of bifidobacteria, obtained according to example 2, with a content of bifidobacteria of at least 10 ¹¹ CFU/g, is added in an amount of 0.01-1% to the mass of the main feed and mixed thoroughly.

Example 12 Use of a consortium of bifidobacteria strains for the production of toothpaste.

Dry bacterial concentrate of a consortium of bifidobacteria, obtained according to example 2, with a bifidobacteria content of at least 10 ¹¹ CFU/g, is added to the toothpaste at the rate of 0.01 g of bacterial concentrate per 100 g of paste.

Example 13 Use of a consortium of bifidobacteria strains for the production of soft rennet cheese.

Normalized milk is subjected to heat treatment at a temperature of 74-76°C for 1-5 minutes and cooled to a fermentation temperature of 37-38°C, a 40% aqueous solution of calcium chloride is added at the rate of 50 ml per 100 kg of milk, 1% dry bacterial concentrate of a consortium of bifidobacteria obtained according to example 2, with a bifidobacteria content of at least 10 ¹¹ CFU/g. The mixture is stirred and a solution of a milk-clotting enzyme is added at the rate of 1.0-1.2 g per 100 kg of milk, then coagulation is carried out. The subsequent production of soft cheese is carried out according to standard technology.

Example 14 Use of a consortium of bifidobacteria strains for the production of brine mature cheese.

Coagulation of milk is carried out at a temperature of 32-34°C for 30-45 min, a combined starter is added in the amount of 1.0-2.0%, consisting of salt-tolerant pure cultures of mesophilic lactic acid streptococci; consortium of bifidobacteria obtained according to example 1; mesophilic lactic acid bacilli. Starter cultures of bifidobacteria, streptococci and mesophilic lactic acid bacilli are prepared separately. Streptococci, bifidobacteria and mesophilic lactic acid bacilli are added to the production starter. Then calcium chloride and rennet are added to the resulting mixture to obtain a clot. The subsequent production of brine mature cheese is carried out according to standard technology.

As a result of the claimed invention, a consortium based on five breeding strains of bifidobacteria was obtained, which is more resistant to damaging factors of the aggressive environment of the gastrointestinal tract during the passage of bifidobacteria in the gastrointestinal tract, which is expressed in increased colonizing activity. The consortium has a pronounced antioxidant activity and probiotic action in the absence of mutual inhibition of individual strains. The proposed consortium is high-tech, accumulates biomass on nutrient media in a short period of cultivation with a high concentration of probiotic microorganisms - 10 ¹¹ CFU/g.

The method used for selection of breeding strains, involving the study of their antibiotic resistance, cultural, antagonistic, morphological, physiological and biochemical properties and molecular genetic identification, made it possible to create a consortium capable of correcting microecological disorders more effectively and safely and expanding the arsenal of probiotic agents.

The list of nucleotide sequences of genome fragments of strains included in the consortium is presented in accordance with WIPO Standard ST 25. Certificates of deposit of strains and a list of nucleotide sequences on a machine-readable medium are attached.

Claims (6)

CLAIM 1. The strain of bifidobacteria Bifidobacterium bifidum 791-BL, used for the preparation of bifid-containing products, deposited in the VKPM Federal State Unitary Enterprise GosNIIgenetika under registration No. Ac-2035 and containing the nucleotide sequence of SEQ ID NO: 1. 2. The strain of bifidobacteria Bifidobacterium longum B 379M-BL, used for the preparation of bifid-containing products, deposited in the VKPM Federal State Unitary Enterprise GosNIIgenetika under registration No. Ac-2037 and containing the nucleotide sequence SEQ ID NO: 2. 3. The strain of bifidobacteria Bifidobacterium adolescentis G 7513-BL, used for the preparation of bifid-containing products, deposited in the VKPM Federal State Unitary Enterprise GosNIIgenetika under registration No. Ac-2038 and containing the nucleotide sequence SEQ ID NO: 3. 4. The strain of bifidobacteria Bifidobacterium infantis 73-15-BL, used for the preparation of bifid-containing products, deposited in the VKPM Federal State Unitary Enterprise GosNIIgenetika under registration No. Ac-2039 and containing the nucleotide sequence SEQ ID NO: 4. 5. The strain of bifidobacteria Bifidobacterium breve 79-119-BL, used for the preparation of bifid-containing products, deposited in the VKPM Federal State Unitary Enterprise GosNIIgenetika under registration No. Ac-2036 and containing the nucleotide sequence SEQ ID NO: 5. 6. Consortium of bifidobacteria containing strains according to claims 1-5 and used for the preparation of bifid-containing products.