Bifidobacterium bifidum strain used to obtain bifido-containing product

FIELD: chemistry.

SUBSTANCE: invention relates to food, cosmetic, biotechnology and medical industry. The Bifidobacterium bifidum 79-93 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 246 and can be used in producing fermented milk products, food products, sanitary and cosmetic agents, biologically active additives and bacterial preparations.

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.

2 tbl, 5 ex

 

The invention relates to the food, cosmetic, biotech and medical industries, in particular used for the preparation of fermented milk, fermented and non-fermented food, hygiene and cosmetics, dietary supplements and bacterial preparations

Due to unfavorable environmental conditions, stress, prolonged antibiotic, radiotherapy and chemotherapy, dysbiosis resulting from illness, an increasing number of people suffering from disorders of the normal microflora. For the treatment and prevention of these disorders is gaining attention as drugs, with probiotic action, and food products containing bifidobacteria.

In the human intestine, basically, there are 5 species of bifidobacteria - B.bifidum, B.longum, B.adolescentis, B.breve and B.infantis. Of them with the greatest frequency in the biocenosis detected species B.bifidum and B.longum. View B.adolescentis more common in adults, and species B.breve and B.infantis - predominantly in infants.

Well-known foreign mono - and combination preparations-eubiotics and dairy products, including bifidobacteria species B.bifidum, B.lonqum, B.adolescentis, B.breve (patent 2336886, patent 2411008, France; patent 637297, Switzerland; patent 2134179, patent 242106, Germany; patent 05227946, patent 0430642, Japan; patent 4645667, U.S. patent 6063935, Japan, the patent 5922375, U.S. patent 5902743, U.S. patent 6746672, U.S. patent 6548057, USA). In our country for the preparation of bacterial preparations, dietary supplements, medicinal products-dietary use of domestic strains of bifidobacteria species B.bifidum 1,791,792; B.lonqum B379M, I-2, I-4; B.adolescentis MC-42, G 7513; .breve 79-119; B.infantis 79-43 (copyright certificate №343588, RF patent 1347221, 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 drugs is largely due to the properties of the producer strain - specific activity and adaptability. A large range of biologically active and technologically advanced strains of bifidobacteria makes possible the development of new mono - and integrated products for medical nutrition and bakpreparatov, capable of providing high colonization resistance of the human intestine, to restore the normal microflora and reduce the incidence of various intestinal infections.

The present invention is to obtain a strain of microorganism of the species B.bifidum 79-93 with high biological activity, resistance to environmental conditions, with a stabilizing kislotoobrazovanie in the whole temperature range times the development of cells, antagonistically active against pathogenic and conditionally pathogenic microorganisms proliferating on artificial nutrient media.

Strain Bifidobacterium bifidum 79-93 isolated from the contents of the intestines of healthy infant and deposited in the Public collections of the normal microflora (HCNM) fsri mniam them. Hingamisega of the CPS No. 246.

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 additives to food. Strain Bifidobacterium bifidum 79-93 can be widely used for the production of hygienic and cosmetic products such as creams, lotions, wipes, Soaps, powders, tampons, nourishing masks, gels, feminine sanitary pads, skin milk, various powders, etc.

In addition, the strain of Bifidobacterium bifidum 79-93 can be used in the production of bacterial preparations in the form of candles, oral, and other tools.

The technical result of the claimed invention to provide a strain of Bifidobacterium bifidum 79-93, which can be used as biologically active substances in the composition of fermented milk, fermented and non-fermented food is aktov, hygienic and cosmetic products, biologically active additives and bacterial preparations. Strain Bifidobacterium bifidum 79-93 has biologically and technologically important properties, such as high acid activity and its ability to increase microbial biomass in a short period of cultivation. Strain Bifidobacterium bifidum 79-93 antagonistically active against pathogenic and conditionally pathogenic microorganisms. This will allow the use of Bifidobacterium bifidum 79-93 as a biologically active component in the composition of different probiotic means of ensuring the normalization of microbiocenosis of the human body, including the gastrointestinal and urogenital tracts, skin and mucous membranes of the skin, and broaden the Arsenal of similar means.

Cultural and morphological properties of the strain. Cells are gr+polymorphic sticks with bifurcation or thickening at 1-2 ends, in a liquid nutrient medium to grow over the entire height of the volume, except for the zone of aerobiosis. Separately grown colonies on hepatic nutrient medium have an elongated form colonies. Logarithmic growth phase ends at 12-24 hours of cultivation.

Physical and biochemical properties of the strain

Non-sporulating, still gone anaerobic. The optimum temperature for growing 38,0±0,5°C. Catalase does not form, gelatino not p is Sziget. Sprayway glucose with the formation of acetic and L + lactic acid, without the formation of gas, sakissa environment growing to a pH of 4.3-3,9.

Ferments lactose, fructose, galactose, sucrose. Not ferments sorbitol, arabinose, salicin, inulin, cellobiose, xylose, mannose, beckons. Strain Bifidobacterium bifidum 79-93 has an antagonistic activity against pathogenic and conditionally pathogenic microflora (table 1).

Table 1
Antagonistic activity of strain .bifidum 79-93
StrainThe zone of growth inhibition of the test cultures, mm/ M±M
Sh.sonneiSh.flexneriE.coliS.aureusPr.vulgaris
.bifidum 79-9318±2,820±2,616±2,622±1,820±2.0

The strain is expanding microbial mass and increase hydrolysate-dairy medium (GM), Bifidum environment (BF), thus producing acid (table 2).

Table is CA 2
The accumulation of microbial biomass (CFU / ml 10 lg) and the activity of acid generating (in degrees Turner) strain .bifidum 79-93 in the dynamics of development
NUTRIENT MEDIUMTIME of CULTIVATION
BIOLOGICAL ACTIVITY
12 hours24 hours48 h
SOMESOMESOME
GM7,8988,21087,2106
BF8,4949,61227,8122

The maximum strain accumulates microbial mass to 12-24 hours, reaching in 1 ml lg 8-9 microbial cells that are actively producing acid.

Thus, the proposed strain .bifidum 79-93 tech, because it breeds in a variety of is nutritional the x environments with the accumulation of production of biomass with a high concentration of bifidobacteria; has acid-forming activity and antagonistic activity against pathogenic and conditionally pathogenic microorganisms.

Fermented milk, fermented, unfermented food, hygiene, cosmetics, dietary supplements and bacterial preparations containing strain .bifidum 79-93, can be widely used in food, medical, biotechnology and cosmetic industries.

The ability of strain .bifidum 79-93 to increase the biomass in the nutrient medium in a short period of cultivation, acid-forming activity, antagonistic activity against pathogenic and conditionally pathogenic microflora, as well as other useful for the human body properties, typical for the species .bifidum, make possible the use of the strain in the various corrective means of therapeutic and prophylactic purposes.

Example 1. The use of strain .bifidum 79-93 in the composition of dietary supplements.

1 passage: lyophilized strain .bifidum 79-93 in the amount of 0.1 g dry biomass contribute in tubes with nutrient medium and titrated by the method of ten-fold dilutions up to 10-9. Incubated at a temperature 38,0±0.5°C prior to the formation of visible growth in the last dilutions.

2 passage: a grown culture of 10-7-9dilutions in the volume of d is 10% contribute to the nutrient medium and cultured at a temperature 38,0±0,5°C for 12-24 h before formation of the dense growth of the visible area of aerobiosis.

3 passage: the biomass in an amount up to 10% of the volume contribute to the accumulation and cultivate 12-24 h at 38,0±0.5°C, to obtain a biomass content of live bifidobacteria not less than 108/ml.

The result is biologically active additive, which is a liquid concentrate with a high, not less than 108/ml, content of bifidobacteria. When listening to concentrate with protective environments (saharso-gelatin medium, skim milk or other) get dry biologically active Supplement containing bifidobacteria not less than 107/ml.

Example 2. The use of strain .bifidum 79-93 for the preparation of fermented and non-fermented foods.

1 passage: lyophilized strain Century bifidum 79-93 in the amount of 0.1 g dry biomass contribute in tubes with nutrient medium and titrated by the method of ten-fold dilutions up to 10-9. Incubated at 38,0±0.5°C prior to the formation of visible growth in the last dilutions.

2 passage: a grown culture of 10-7-9dilutions up to 10% contribute to the nutrient medium and cultured at a temperature 38,0±0,5°C for 12-24 h before formation of dense growth with a highly visible area of aerobiosis.

3 passage: the biomass in an amount up to 10% of the volume contribute to the accumulation and cultivate 12-24 h at 38,0±0.5°C, to obtain a biomass containing the receiving of live bifidobacteria not less than 10 8/ml.

Obtaining a fermented or non-fermented food: grown biomass bring in food raw materials (milk, juices, beer, kvass, etc. up to 10% of the volume, mix and pour into containers. The result is a product with a concentration of live bifidobacteria not less than 106/ml.

Example 3. The use of strain .bifidum 79-93 for the preparation of a fermented milk product.

1 passage: lyophilized strain introduced into the environment of cultivation and titrated by the method of ten-fold dilutions up to 10-9. Incubated at 38,0±0.5°C prior to the formation of visible growth in the last dilutions. Or dry lyophilized biomass of bifidobacteria contribute in 1-2 doses in the tube with 30 ml of culture medium. Cultivated at a temperature 38,0±0.5°C, to obtain a biomass content of live bifidobacteria not less than 107/ml.

2 passage: a grown culture of strain .bifidum 79-93 of the last dilution or test-tube with the increased biomass contribute to the accumulation of up to 10% and cultured at a temperature 38,0±0.5°C, to obtain a biomass containing bifidobacteria not less than 108/ml.

3 passage: the biomass of the strain .bifidum 79-93 (passage No. 2) enter in the composition of the fermented milk product in the amount of 5-10% by volume, getting in the finished product bifidobacteria not less than 106-7/ml. Quantity p is Saga No. 2 may be increased depending on the volume of production.

Example 4. The use of strain .bifidum 79-93 to obtain bacterial preparations.

The culture of the strain of the 1st or 2nd passage (example No. 1) contribute to the nutrient medium, cultured at a temperature 38,0±0,5°C to obtain the biomass of bifidobacteria with the contents of living cells is not less than 108in 1 ml. To the obtained biomass add a protective environment drying (saharso-gelatin medium, skim milk). The resulting suspension is poured into ampoules, vials and other containers, frozen at minus 35-45°C and lyophilizers from the frozen state in a vacuum drying apparatus. The resulting preparation contains not less than 107living cells of bifidobacteria. The content of bifidobacteria is consistent with the scientific and technical documentation on similar bifidobakterii drug "Bifidumbacterin dry" and is sufficient to use the funds received as a drug - eubiotics.

Example 5. The use of Bifidobacterium bifidum 79-93 to get hygienic and cosmetic products. The culture of the strain of the 1st or 2nd passage (example 1) contribute to the nutrient medium, cultured at a temperature of 37-38 degrees Celsius to obtain the biomass of bifidobacteria with the contents of living cells is not less than 1081 ml, dried by spray or freeze drying.

The obtained dry biomass b is phytobacteria Bifidobacterium bifidum 79-93 enter into the composition of the nutrient cream in an amount of not more than 1% by volume.

Safety: with the introduction of three outbred white mice dose per os microbial culture of strain in an amount of 5×107SOME of the animal's death, deviations in health status of the mice during the observation period is not marked.

Strain Bifidobacterium bifidum No. 246, deposited in the Public collections of microorganisms of the normal microflora (HCNM) fsri mniam them. Hingamisega Rospotrebnadzor and used to obtain bifidobacteria products.



 

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