RU2451736C2 - Bifidobacterium bifidum STRAIN USED TO OBTAIN BIFIDO-CONTAINING PRODUCT - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to food, cosmetic, biotechnology and medical industry. The Bifidobacterium bifidum 74-22 strain has acid-forming activity and antagonistic activity on pathogenic and opportunistic microorganisms, and is deposited in the National Collection of Normal Microflora of the G.N.Gabrichevsky Moscow Research Institute of Epidemiology and Microbiology under registration number 244.

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, 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 mostly 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, RF 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 74-22 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 74-22 was isolated from the intestines of a healthy infant and was deposited in the State collection of normal microflora (GKNM) FSUE MNIIEM them. G.N.Gabrichevsky Rospotrebnadzor under No. 244.

The inventive strain can be used in the production of dairy products, fermented foods, such as kvass, beer, etc., non-fermented foods, such as juices, drinks, etc., biologically active food additives. The strain Bifidobacterium bifidum 74-22 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 74-22 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 74-22, which can be used as a biologically active substance in dairy, fermented and non-fermented foods, hygiene and cosmetics, dietary supplements and bacterial preparations. The strain Bifidobacterium bifidum 74-22 has biologically and technologically significant properties, such as high acid-forming activity and the ability to build up microbial biomass in a short period of cultivation. The strain Bifidobacterium bifidum 74-22 is antagonistically active against pathogenic and opportunistic microorganisms. This will allow the use of the strain Bifidobacterium bifidum 74-22 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 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 an elongated form of 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 4.3-3.9.

Ferments lactose, fructose, galactose, sucrose. Does not ferment sorbitol, arabinose, salicin, inulin, cellobiose, xylose, mannose, beckons. The strain Bifidobacterium bifidum 74-22 has antagonistic activity against pathogenic and conditionally pathogenic microflora (table 1).

Table 1 Antagonistic activity of strain B. bifidum 74-22 Strain Zone of growth inhibition of test cultures mm / M ± t Sh. sonnet Sh. flexneri E. coli S. aureus Pr. vulgaris B. bifidum 74-22 28 ± 2.6 20 ± 2.8 12 ± 2.4 18 ± 1.6 16 ± 2.2

The strain actively increases the 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) of strain B. bifidum 74-22 in the dynamics of development NUTRITION TIME OF CULTIVATION OF BIOLOGICAL ACTIVITY 12 h 24 h 48 h CFU T ° CFU T ° CFU T ° GM 7.6 92 8.0 106 7.0 118 Bf 8.2 86 9,4 118 7.6 134

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 74-22 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, hygiene, cosmetics, dietary supplements and bacterial preparations containing strain B. bifidum 74-22 can be widely used in the food, medical, biotechnological and cosmetic industries.

The ability of strain B. bifidum 74-22 to increase biomass in a nutrient medium in a short time of cultivation, 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 the species B. bifidum, 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 74-22 in the composition of biologically active additives.

1 passage: lyophilized strain of B. bifidum 74-22 in the amount of 0.1 g of dry biomass is introduced into test tubes with nutrient medium and titrated by ten-fold dilutions to 10 ^-9 . Incubated at a temperature of 38.0 ± 0.5 ° C until the formation of visible growth in the last dilutions.

2 passage: a 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. When listening to the concentrate with protective media (sucrose-gelatin medium, skim milk or others), a dry biologically active additive with a content of bifidobacteria of at least 10 ⁷ / ml is obtained.

Example 2. The use of strain B. bifidum 74-22 for the preparation of fermented and non-fermented foods.

1 passage: lyophilized strain of B. bifidum 74-22 in the amount of 0.1 g of dry biomass is introduced into test tubes with nutrient 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.

2 passage: a 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 74-22 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 74-22 from the last dilutions or from a test tube with grown biomass is added to 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 obtained biomass of strain B. bifidum 74-22 (passage No. 2) is added to the fermented milk product in an amount of 5-10% by volume, obtaining at least 10 ^6-7 / ml in the finished product of bifidobacteria. The number of passages No. 2 in this case can be increased depending on the volume of production.

Example 4. The use of strain B. bifidum 74-22 to obtain bacterial preparations.

The culture of the strain of the 1st or 2nd passage (example No. 1) is introduced into the nutrient medium, cultivated at a temperature of 38.0 ± 0.5 ° C until 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 scientific and technical documentation for a similar bifidobacterium preparation "Bifidumbacterin dry" and is sufficient to use the resulting agent as a drug - eubiotics.

Example 5. The use of strain Bifidobacterium bifidum 74-22 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 74-22 is introduced into the composition of the nutrient cream in an amount of not more than 1% of the 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 No. 244, deposited in GKNM FSUE MNIIEM them. G.N.Gabrichevsky Rospotrebnadzor and used to obtain bifidobacteria.