RU2317326C1 - Strain bifidobacterium longum po-6 used for preparing fermented-milk, fermented and nonfermented foodstuffs, ferments, hygienic and cosmetic agents, biologically active supplements and bacterial preparations - Google Patents

Abstract

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to the strain Bifidobacterium longum PO-6 that is isolated from the health adult human intestine content and can be multiplied in different nutrient media with accumulation of industrial biomass with the high concentration of bifidobacteria. The strain Bifidobacterium longum PO-6 possesses acid-forming activity, antagonistic activity with respect to pathogenic and opportunistic microorganisms and ferments milk. Invention provides normalization of microbiocenosis in human body and among them digestive and urogenital tracts, cutaneous and mucosa tissues, and expand assortment of similar agents also.

EFFECT: valuable properties of strain.

5 ex

Description

The invention relates to the food, cosmetic, biotechnological and medical industries, in particular, it is used for the preparation of fermented, fermented and non-fermented foods, hygienic and cosmetic products, 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 eubiotics and fermented milk products are known, including bifidobacteria of the species B. bifidum, B. longum, 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.longum 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 wide range 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. longum PO-6 with high biological activity, resistance to environmental conditions, having 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 longum PO-6 was isolated from the contents of the intestines of a healthy adult and deposited in the Museum of Microorganisms FSUE MNIIEM them. G.N.Gabrichevsky Rospotrebnadzor under No. 208.

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 longum PO-6 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 of Bifidobacterium longum PO-6 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 longum PO-6, 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 longum PO-6 has biologically and technologically significant properties, such as acid-forming activity, the ability to increase microbial biomass in various nutrient media. The strain Bifidobacterium longum PO-6 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, 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. The logarithmic growth phase ends at 16-24 hours of cultivation.

Physico-biochemical properties of the strain.

Indisputable, motionless anaerobic. The optimum growing temperature is 37-39 ° 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 4.2-3.9. It ferments lactose, raffinose, arabinose, xylose, inulin, does not ferment mannitol, sorbitol, inositol, cellobiose, mannose.

The strain fermented milk by 18-24 hours of cultivation.

The strain Bifidobacterium longum PO-6 has antagonistic activity against pathogenic and conditionally pathogenic microflora (Table 1).

Table 1.
Antagonistic activity of strain B. longum PO-6 Strain Zone of growth inhibition of test cultures mm / M ± m Sh.sonnei Sh.flexneri E.coli S.aureus Pr.vulgaris B.longum PO-6 12 ± 1.2 18 ± 1.8 14 ± 1.6 12 ± 1.4 8 ± 2.8

The strain actively increases microbial mass during growth on hydrolyzate-milk nutrient 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. longum PO-6 in the dynamics of development NUTRITION CULTIVATION TIME BIOLOGICAL ACTIVITY 12 h 24 h 48 h CFU T ° CFU T ° CFU T ° GM 8.8 102 9.2 118 7.4 162 Bf 8.4 96 8.8 104 7.6 152

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

Thus, the proposed strain .longum PO-6 is technologically advanced, because it multiplies in various nutrient media with the accumulation of production 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 food products, hygienic, cosmetics, biologically active additives and bacterial preparations containing B. longum PO-6 strain can be used in the food, medical, biotechnological and cosmetic industries.

The ability of the strain B. longum PO-6 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 that are characteristic of the species B. longum, make it possible to use the strain as part of various corrective agents for therapeutic purposes.

Example 1. The use of strain B. longum PO-6 in the composition of biologically active additives.

1 passage: lyophilized strain of B. longum PO-6 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 ° C until the formation of visible growth in the last dilutions.

2nd passage: a grown culture of 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 ° C for 12-24 hours until a dense growth with a visible aerobiosis zone is formed.

3 passage: the resulting biomass in an amount up to 10% of the volume is introduced into the accumulation medium and cultured for 12-24 hours at 37-38 ° C until biomass is obtained with the content of live bifidobacteria not less than 10 ⁸ / ml.

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. longum PO-6 for the preparation of fermented and non-fermented foods.

1 passage: lyophilized strain of B. longum PO-6 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 ° 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 37-38 ° C 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 up to 10% of the volume is introduced into the accumulation medium and cultured for 12-24 hours at 37-38 ° C until biomass is obtained with the content of live bifidobacteria not less than 10 ⁸ / ml.

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. longum PO-6 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 ° 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 37-38 ° C to obtain biomass with a content of live bifidobacteria not less than 10 ⁷ / ml.

2nd passage: a grown culture of B. longum PO-6 strain 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 37-38 ° C until biomass with a content of bifidobacteria of at least 10 ⁸ / ml is obtained .

3rd passage: the resulting biomass of strain B.longum PO-6 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. longum PO-6 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 ° 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 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 B. longum PO-6 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 ° C until a biomass of bifidobacteria is obtained with a living cell content of at least 10 ⁸ in 1 ml.

The obtained biomass of bifidobacteria strain B. longum PO-6 is introduced into the composition of a hygienic or cosmetic product.

Claims (1)

The strain Bifidobacterium longum PO-6 (State collection of microorganisms of normal human microflora GKNM FGUN MNIIEM them.G. Gabrichevsky Rospotrebnadzor, No. 208), used to obtain fermented milk, fermented and non-fermented foods, starter cultures, hygienic and cosmetics, biologically active additives and bacterial preparations.