RU2451726C2 - Bifidobacterium adolescentis STRAIN USED TO OBTAIN BIFIDO-CONTAINING PRODUCT - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to food, cosmetic, biotechnology and medical industry. The Bifidobacterium adolescentis 79-66 strain, having acid-forming activity, antagonistic activity on pathogenic and opportunistic microorganisms, is deposited in the National Collection of Normal Microflora of the G.N.Gabrichevsky Moscow Research Institute of Epidemiology and Microbiology under registration number 232.

EFFECT: invention enables to obtain fermented milk products, fermented and unfermented food products, sanitary and cosmetic agents, biologically active additives and bacterial preparations, which enable to normalise microbiocenosis of the human body, including gastrointestinal and urogenital tracts, skin and mucous covers.

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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 dairy, fermented and non-fermented foods, hygiene and cosmetics, dietary supplements 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 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 mostly in infants.

Foreign mono-and complex preparations-eubiotics and dairy 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, for the preparation of bacterial preparations, biologically active additives, medical dietetic foods, domestic B. bifidum species of B. bifidum species 1,791,792 are used; B.lonqum B379M, I-2, I-4; B. adolescentis MS-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. adolescentis 79-66 with high biological activity, resistance to environmental conditions, with stabilizing acid formation in the entire temperature range of cell development, fermenting milk and propagating on artificial nutrient media of various compositions.

The strain Bifidobacterium adolescentis 79-66 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. 232.

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 adolescentis 79-66 can also be 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 adolescentis 79-66 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 adolescentis 79-66, 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 adolescentis 79-66 has biologically and technologically significant properties, such as acid-forming activity, the ability to increase microbial biomass in various nutrient media. The strain Bifidobacterium adolescentis 79-66 is antagonistically active against pathogenic and conditionally pathogenic microorganisms. This will make it possible to use it as a biologically active component in various probiotic agents that ensure the normalization of microbiocenoses of the human body, 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 temperature of cultivation is 37-39 degrees Celsius. 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 4.6-4.2. Ferments lactose, arabinose, xylose, cellobiose. inulin, mannitol, salicin. Does not ferment sorbitol, inositol. The strain fermented milk for 24-36 hours of cultivation.

The strain Bifidobacterium adolescentis 79-66 has antagonistic activity against pathogenic and conditionally pathogenic microflora (Table 1).

Table 1 Antagonistic activity of strain B. adolescentis 79-66 Strain Zone of growth inhibition of test cultures mm / M ± m Sh.sonnei Sh.flexneri E.coli S.aureus Pr.vulgaris B. adolescentis 79-66 10 ± 2.0 8 ± 1.4 8 ± 1.2 16 ± 2.6 8 ± 2.8

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

table 2 The accumulation of microbial mass (CFU / ml lg 10) and the activity of acid formation (in degrees Turner) strain B. adolescentis 79-66 in the dynamics of development NUTRITION CULTIVATION TIME
BIOLOGICAL ACTIVITY 12 h 24 h 48 h CFU T ° CFU T ° CFU T ° GM 7.6 68 8.4 86 8.0 108 Bf 7.8 74 8.8 88 8.6 118

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

Thus, the proposed strain B. adolescentis 79-66 is technologically advanced, because it multiplies in nutrient media with the accumulation of industrial biomass with a high concentration of bifidobacteria; possesses acid-forming activity, is capable of fermenting milk.

Sour-milk, fermented, non-fermented foods, hygienic, cosmetics, dietary supplements and bacterial preparations containing strain B. adolescentis 79-66, can be used in the food, medical, biotechnological and cosmetic industries.

The ability of the strain B. adolescentis 79-66 to increase biomass in various nutrient media, acid-forming activity, antagonistic activity against pathogenic and conditionally pathogenic microflora, as well as other properties useful to the human body, characteristic of the species B. adolescentis, make it possible to use the strain as part of various corrective, therapeutic agents.

Example 1. The use of strain B. adolescentis 79-66 in the composition of biologically active additives.

1 passage: lyophilized strain of B. adolescentis 79-66 in the amount of 0.1 g of dry biomass is introduced into test tubes with nutrient medium and titrated by the method of ten-fold dilutions to 10 ^-9 . Incubated at a temperature of 37-38 degrees Celsius 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 37-38 degrees Celsius 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 cultivated for 12-24 hours at 37-38 degrees Celsius 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. When drying the concentrate with protective media (sucrose-gelatin medium, skim milk or others), a dry dietary supplement with bifidobacteria containing at least 10 ⁷ / ml is obtained.

Example 2. The use of strain B. adolescentis 79-66 for the preparation of fermented and non-fermented foods.

1 passage: lyophilized strain of B. adolescentis 79-66 in the amount of 0.1 g of dry biomass is introduced into test tubes with nutrient medium and titrated by the method of ten-fold dilutions to 10 ^-9 . Incubated at 37-38 degrees Celsius 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 37-38 degrees Celsius for 12-24 hours until a dense growth with a well-visible zone of aerobiosis is formed.

3 passage: the resulting biomass in an amount of up to 10% of the volume is introduced into the accumulation medium and cultivated for 12-24 hours at 37-38 degrees Celsius 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. adolescentis 79-66 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 37-38 degrees Celsius 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 37-38 degrees Celsius to obtain biomass containing live bifidobacteria not less than 10 ⁸ / ml.

2 passage: the grown culture of strain B. adolescentis 79-66 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 37-38 degrees Celsius until a biomass with a content of bifidobacteria of at least 10 ⁸ / ml

3rd passage: the resulting biomass of strain B. adolescentis 79-66 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. adolescentis 79-66 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 37-38 degrees Celsius 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 degrees Celsius and lyophilized from a frozen state in vacuum-drying bifidobacteria. 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 adolescentis 79-66 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 in 1 ml, dried by spraying or lyophilization.

The resulting dry biomass of bifidobacteria of the strain Bifidobacterium adolescentis 79-66 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 adolescentis 79-66, used to obtain bifidobacteria products, was deposited in the State collection of microorganisms of normal microflora (GKNM) FGUN MNIIEM them. G.N.Gabrichevsky Rospotrebnadzor, registration number 232.