RU2452770C2 - Bifidobacterium bifidum STRAIN USED FOR PRODUCTION OF BIFIDO-CONTAINING PRODUCTS - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: Bifidobacterium bifidum 79-94 strain possessing acid-forming activity, antagonistic activity with regard to pathogenic and opportunistic microorganisms is deposited in the State Collection of Normal Microflora Microorganisms of Federal State Academic Institution "G. N. Gabrichevsky Moscow Research Institute of Epidemiology and Microbiology" under the Federal Service for Consumer Rights Protection and Human Welfare (Rospotrebnadzor) under Registration Number 231 and may be used for production of cultured milk products, food products, hygienic and cosmetic aids, dietary supplements and bacterial preparations.

EFFECT: invention allows to produce cultured milk, fermented and non-fermented food products, hygienic and cosmetic aids, dietary supplements and bacterial preparations ensuring normalisation of microflora in the human organism.

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Description

The invention relates to the food, cosmetic, biotechnological and medical industries, in particular it is used for the preparation of sour-milk, fermented and non-fermented foods, hygienic and cosmetic products, biologically active additives and bacterial preparations.

Due to adverse environmental conditions, stress, prolonged antibacterial, radiation and chemotherapy, dysbiosis resulting from past diseases, the number of people suffering from disorders of the normal microflora of the body increases. For the treatment and prevention of these disorders, increasing attention is being paid to both drugs with a probiotic effect and food products containing bifidobacteria.

In the human intestine, mainly 5 species of bifidobacteria are found - B. bifidum, B. longum, B. adolescentis, B. breve and B. infantis. Of these, the species B. bifidum and B. longum are found with the greatest frequency in the biocenosis. Species B. adolescentis is more common in adults, while species B. breve and B. infantis are found mainly in infants.

Foreign mono- and complex eubiotics and fermented milk products are known, including bifidobacteria of the species B. bifidum, B. lonqum, B. adolescentis, B. breve (patent 2336886, patent 2411008, France; patent 637297, Switzerland; patent 2134179 patent 2421066, Germany; patent 05227946, patent 0430642, Japan; patent 4645667, USA, patent 6063935, Japan, patent 5922375, USA, patent 5902743, USA, patent 6746672, USA, patent 6548057, USA). In our country, domestic strains of bifidobacteria species B. bifidum 1,791,792 are used for the preparation of bacterial preparations, biologically active additives, and dietetic food products. B. lonqum B379M, I-2, I-4; B. adolescentis MC-42, G 7513; B. breve 79-119; B.infantis 79-43 (copyright certificate No. 343588, RF patent 1343421, RF patent 849779, RF patent 2108383, RF patent 2152993, patent 863639, RF patent 2120991, RF patent 2078814, RF patent 2111248).

The quality of medicinal products and preparations is largely due to the properties of the producer strain - its specific activity and adaptability. A large assortment of biologically active and technologically advanced strains of bifidobacteria makes it possible to develop new mono-and complex medical nutrition products and drugs that can provide high colonization resistance of the human intestine, restore its normal microflora and reduce the incidence of various intestinal infections.

The objective of the present invention is to obtain a strain of microorganisms of the species B. bifidum 79-94 with high biological activity, resistance to environmental conditions, with stabilizing acid formation in the entire temperature range of cell development, antagonistically active against pathogenic and conditionally pathogenic microorganisms, multiplying rapidly by artificial nutrient media.

The strain Bifidobacterium bifidum 79-94 isolated from the intestines of a healthy infant and deposited in the State collection of normal microflora (GKNM) FSUE MNIIEM them. G.N.Gabrichevsky Rospotrebnadzor under No. 231.

The inventive strain can be used in the production of fermented milk products, fermented foods, for example: kvass, beer, etc., unfermented foods, for example: juices, drinks, etc., starter cultures for the preparation of fermented milk products and biologically active additives to food. The strain Bifidobacterium bifidum 79-94 can also be widely used for the production of hygiene and cosmetics, for example: creams, lotions, napkins, soaps, powders, tampons, nourishing masks, gels, women's pads, cosmetic milk, various powders, etc.

In addition, the strain Bifidobacterium bifidum 79-94 can be used in the production of bacterial preparations, in the form of suppositories, oral and other drugs.

The technical result of the claimed invention is to obtain a strain of Bifidobacterium bifidum 79-94, which can be used as a biologically active substance in dairy, fermented and non-fermented foods, starter cultures, hygiene and cosmetics, dietary supplements and bacterial preparations. The strain Bifidobacterium bifidum 79-94 has biologically and technologically significant properties, such as high acid-forming activity and the ability to increase microbial biomass in a short cultivation period. The strain Bifidobacterium bifidum 79-94 is antagonistically active against pathogenic and conditionally pathogenic microorganisms. This will allow the use of the strain Bifidobacterium bifidum 79-94 as a biologically active component in various probiotic agents that ensure the normalization of microbiocenoses of the human body, including gastrointestinal and genitourinary tracts, skin and mucous membranes, and will expand the arsenal of similar tools.

Cultural and morphological properties of the strain.

Cells are gr + polymorphic bacilli with bifurcation or thickening at the 1-2 ends, in liquid nutrient media they grow along the entire height of the volume, except for the aerobiosis zone. Separately grown colonies on the hepatic nutrient medium have comet-shaped colonies. The logarithmic growth phase ends at 12-24 hours of cultivation.

Physico-biochemical properties of the strain.

Indisputable, motionless anaerobic. The optimum growing temperature is 38.0 ± 0.5 ° C. Catalase does not form, gelatin does not thin. It ferments glucose with the formation of acetic and L + lactic acids, without the formation of gas, acidifying the growth medium to a pH of 4.2-3.8. Ferments lactose, fructose, galactose, sucrose. Does not ferment sorbitol, arabinose, salicin, inulin, cellobiose, xylose, mannose, beckons. The strain skim milk for 24-36 hours.

The strain Bifidobacterium bifidum 79-94 has antagonistic activity against pathogenic and conditionally pathogenic microflora (Table 1).

Table 1 Antagonistic activity of strain B. bifidum 79-94 Strain Zone of growth inhibition of test cultures mm / M ± m Sh.sonnei Sh.flexneri E.coli S.aureus Pr.vulgaris B. bifidum 79-94 20 ± 2.6 16 ± 2.2 12 ± 1.8 22 ± 1.0 24 ± 2.8

The strain actively increases microbial mass during growth on hydrolyzate-milk medium (GM), Bifidum medium (BP), producing acids (Table 2).

The strain maximally accumulates microbial mass by 12-24 hours, reaching 1-9 lg of 8-9 microbial cells, actively producing acids.

Thus, the proposed strain of B. bifidum 79-94 is technologically advanced, because it multiplies in various nutrient media with the accumulation of industrial biomass with a high concentration of bifidobacteria; possesses acid-forming activity, as well as antagonistic activity in relation to pathogenic and conditionally pathogenic microorganisms.

Sour-milk, fermented, non-fermented foods, hygienic, cosmetics, dietary supplements and bacterial preparations containing B. bifidum 79-94 strain can be widely used in the food, medical, biotechnological and cosmetic industries.

The ability of B. bifidum 79-94 strain to increase biomass in a nutrient medium in a short cultivation period, acid-forming activity, antagonistic activity against pathogenic and conditionally pathogenic microflora, as well as other properties useful for the human body that are characteristic of B. bifidum species, make it is possible to use the strain as part of various corrective agents for therapeutic purposes.

Example 1. The use of strain B. bifidum 79-94 in the composition of biologically active additives.

1 passage: lyophilized strain of B. bifidum 79-94 in the amount of 0.1 g of dry biomass is introduced into test tubes with nutrient medium and titrated by ten-fold dilution to 10 ^-9 . Incubated at a temperature of 38.0 ± 0.5 ° until the formation of visible growth in the last dilutions.

2 passage: the grown culture from 10 ^-7-9 dilutions in a volume of up to 10% is introduced into the nutrient medium and cultivated at a temperature of 38.0 ± 0.5 ° C for 12-24 hours until a dense growth with a visible aerobiosis zone is formed.

3 passage: the resulting biomass in an amount of up to 10% of the volume is introduced into the accumulation medium and cultured for 12-24 hours at 38.0 ± 0.5 ° C until biomass with a content of live bifidobacteria of at least 10 ⁸ / ml is obtained.

The result is a biologically active additive, which is a liquid concentrate with a high, at least 10 ⁸ / ml, content of bifidobacteria.

Example 2. The use of strain B. bifidum 79-94 for the preparation of fermented and unfermented foods.

1 passage: lyophilized strain of B. bifidum 79-94 in the amount of 0.1 g of dry biomass is introduced into test tubes with nutrient medium and titrated by ten-fold dilution to 10 ^-9 . Incubated at 38.0 ± 0.5 ° C until the formation of visible growth in the last dilutions.

2 passage: the grown culture from 10 ^-7-9 dilutions in a volume of up to 10% is introduced into the nutrient medium and cultivated at a temperature of 38.0 ± 0.5 ° C for 12-24 hours until a dense growth with a well-visible aerobiosis zone is formed.

3 passage: the resulting biomass in an amount of up to 10% of the volume is introduced into the accumulation medium and cultured for 12-24 hours at 38.0 ± 0.5 ° C until biomass with a content of live bifidobacteria of at least 10 ⁸ / ml is obtained.

Getting a fermented or non-fermented food product: the grown biomass is introduced into food raw materials (milk, juices, beer, kvass, etc.) in an amount up to 10% of the volume, mixed and poured into containers. The result is a product with a concentration of live bifidobacteria of at least 10 ⁶ / ml.

Example 3. The use of strain B. bifidum 79-94 for the preparation of fermented milk product.

1 passage: lyophilized strain is introduced into the culture medium and titrated by ten-fold dilutions to 10 ^-9 . Incubated at 38.0 ± 0.5 ° C until the formation of visible growth in the last dilutions. Or dry lyophilized biomass of bifidobacteria is added in an amount of 1-2 doses in a test tube with 30 ml of culture medium. Cultivate at a temperature of 38.0 ± 0.5 ° C to obtain biomass containing live bifidobacteria not less than 10 ⁷ / ml.

2nd passage: a grown culture of strain B. bifidum 79-94 from the last dilutions or from a test tube with grown biomass is introduced into the accumulation medium with a volume of up to 10% and cultivated at a temperature of 38.0 ± 0.5 ° C until biomass with a content of bifidobacteria of at least 10 ⁸ / ml.

3 passage: the resulting biomass of strain B. bifidum 79-94 in a volume of up to 10% is introduced into sterile milk, cultivated until a clot forms (fermented milk product). The number of passages No. 2 can be increased depending on the volume of production.

Example 4. The use of strain B. bifidum 79-94 to obtain bacterial preparations.

The culture of the strain of the 1st or 2nd passage (Example 1) is introduced into the nutrient medium, cultivated at a temperature of 38.0 ± 0.5 ° C until a biomass of bifidobacteria is obtained with a living cell content of at least 10 ⁸ in 1 ml. Protective drying media (sucrose-gelatin medium, skim milk) are added to the resulting biomass. The resulting suspension is poured into ampoules, vials and other containers, frozen at a temperature of minus 35-45 ° C and lyophilized from a frozen state in vacuum drying machines. The resulting preparation contains at least 10 ⁷ living bifidobacteria cells. This content of bifidobacteria is consistent with the scientific and technical documentation for a similar bifidobacterium-containing drug - "Bifidumbacterin dry" and is sufficient to use the resulting product as a drug - eubiotics.

Example 5. The use of strain Bifidobacterium bifidum 79-94 to obtain hygiene and cosmetics. The culture of the strain of the 1st or 2nd passage (Example 1) is introduced into the nutrient medium, cultivated at a temperature of 37-38 degrees Celsius to obtain biomass of bifidobacteria with a content of living cells of at least 10 ⁸ in 1 ml, dried by spraying or lyophilization.

The resulting dry biomass of bifidobacteria of the strain Bifidobacterium bifidum 79-94 is introduced into the composition of the nutrient cream in an amount of not more than 1% by volume.

Harmlessness: when three outbred white mice were injected with a dose of per os microbial culture of the strain in an amount of 5 × ^{10 7} CFU per animal death, deviations in the health status of mice were not observed during the observation period.

Claims (1)

The strain Bifidobacterium bifidum 79-94, used to obtain bifidobacteria products, is deposited in the State collection of normal microflora (GKNM) FSUE MNIIEM them. G.N. Gabrichevsky Rospotrebnadzor, registration number 231.