RU2453592C1 - Strain bifidobacterium bifidum used for preparing bifidus products - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: strain Bifidobacterium bifidum B-UH is recovered from a healthy infant, actively propagates in a nutrient medium with accumulation of an production biomass in a short 12-18-hour period of cultivation time of the high lactic acid bacillus concentration, is able to sour milk. The strain Bifidobacterium bifidum B-UH exhibits acid-forming activity, pathogen and opportunistic pathogen antagonist activity. The strain Bifidobacterium bifidum B-UH is deposited in Russian National Collection of Industrial Microorganisms, No. Ac-1875. That allows the strain Bifidobacterium bifidum B-UH being used in making bifidus products: bacterial preparations, biologically active food additives, fermented and non-fermented foodstuff, hygienic and cosmetic agents.

EFFECT: normalised microbiocenosis of a human body.

2 tbl, 5 ex

Description

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). 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 I-2, I-4; B. breve 79-119; B. infantis 79-43 and others (copyright certificate No. 343588, RF patent No. 1347221, Russian patent No. 849779, Russian patent No. 2108383, Russian patent No. 2152993, patent No. 863639, Russian patent No. 2120991, Russian patent No. 2078814, Russian patent No. 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 Bifidobacterium bifidum B-UH 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 microflora, well fermenting milk and breeding rapidly on artificial nutrient media.

The strain Bifidobacterium bifidum B-UH was isolated from a healthy infant, deposited at VKPM, Federal State Unitary Enterprise GosNIIgenetika, collection number Ac-1875.

The inventive strain can be used in the production of fermented milk products, fermented foods, such as kvass, beer, etc., unfermented foods, such as juices, drinks, etc., starter cultures for the preparation of fermented milk products and biologically active food additives. Also, the strain Bifidobacterium bifidum B-UH can be used for the production of hygienic and cosmetic products, 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 bifidum B-UH 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 B-UH, which can be used as a biologically active component in various probiotic means of correction of microbiocenoses of the human body, which has biologically and technologically significant properties, such as high acid-forming activity, the ability to increase microbial biomass in artificial nutrient media for short periods of cultivation, ferment milk, antagonistically active on relation to pathogenic and conditionally pathogenic microorganisms.

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 Bifidum medium have a comet-shaped colony shape. The logarithmic phase of growth ends by 12-18 hours of cultivation.

Physico-biochemical properties of the strain.

Indisputable, motionless anaerobic. The optimal 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 4.1-3.9. It ferments lactose, fructose, galactose, does not ferment sorbitol, arabinose, salicin, inositol, inulin, cellobiose, xylose, mannose, mannitol. The strain fermented milk at 16-24 hours.

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

Table 1 Antagonistic activity of strain B. bifidum B-UH Strain Zone of growth inhibition of test cultures mm / M ± m Sh. sonnci Sh. flexneri E. coli S. aureus Pr. vulgaris B. bifidum B-UH 22 ± 2.0 25 ± 1.8 18 ± 1.6 20 ± 1.2 25 ± 1.4

The strain actively increases the microbial mass during growth on hydrolyzate-milk medium (GM), bifidum medium (BP), milk (M), 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 the strain Bifidobacterium bifidum B-UH in the dynamics of development NUTRITION CULTIVATION TIME
BIOLOGICAL ACTIVITY 12 h 24 h 48 h CFU ° T CFU ° T CFU ° T GM 8.2 94 9.0 114 7.8 162 Bf 8.4 86 9.2 108 7.6 142 M 8.2 82 8.6 112 8.0 138

The strain maximally accumulates microbial mass by 12-18 hours, reaching 8-9 microbial cells in 1 ml of lg, actively producing acids.

Thus, the proposed strain of Bifidobacterium bifidum B-UH 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 foods, hygiene, cosmetics, dietary supplements and bacterial preparations containing the Bifidobacterium bifidum B-UH strain can be widely used in the food, medical, biotechnological and cosmetic industries.

The ability of the Bifidobacterium bifidum B-UH strain to increase biomass in a nutrient medium during short cultivation periods, acid-forming, antagonistic activity with respect to pathogenic and conditionally pathogenic microflora, as well as other properties useful to the human body characteristic of the species Bifidobacterium bifidum, make it possible to use the strain as part of various corrective agents for therapeutic purposes.

Example 1. The use of a strain of Bifidobacterium bifidum B-UH in the composition of biologically active additives.

1 passage: a lyophilized strain of Bifidobacterium bifidum B-UH 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 ± 1 ° 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 ± 1 ° C for 12-18 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 12-18 hours at 38 ± 1 ° 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 a strain of Bifidobacterium bifidum B-UH for the preparation of fermented and non-fermented foods.

1 passage: a lyophilized strain of Bifidobacterium bifidum B-UH 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 ± 1 ° 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 ± 1 ° C for 12-18 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 12-18 hours at 38 ± 1 ° 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 a strain of Bifidobacterium bifidum B-UH for the preparation of a dairy product.

1 passage: lyophilized strain is introduced into the culture medium and titrated by ten-fold dilutions to 10 ^-9 . Incubated at 38 ± 1 ° 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 ± 1 ° C to obtain biomass containing live bifidobacteria not less than 10 ⁷ / ml.

2nd passage: the grown culture of the Bifidobacterium bifidum B-UH strain 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 ± 1 ° С until biomass with a content of bifidobacteria of at least 10 ⁸ / ml is obtained.

3 passage; the resulting biomass of the strain Bifidobacterium bifidum B-UH 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 a strain of Bifidobacterium bifidum B-UH 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 ± 1 ° С until the biomass of bifidobacteria with the content of living cells of at least 10 ⁸ in 1 ml is obtained. 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 a strain of Bifidobacterium bifidum B-UH 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 38 ± 1 ° С until the biomass of bifidobacteria with the content of living cells of at least 10 ⁸ in 1 ml is obtained.

The resulting biomass of bifidobacteria of the strain Bifidobacterium bifidum B-UH is introduced in an amount of not more than 1% in the composition of soap or cream.

The clinical efficacy of Bifidobacterium bifidum was studied on the basis of the Sechenov Moscow Medical Academy as part of a liquid dietary supplement, Normoflorin. Under observation were 20 adults with inflammatory bowel disease (IBD) - ulcerative colitis, Crohn's disease, chronic colitis. Patients, along with basic therapy, received "Normoflorin" 1 tablespoon before meals for 30 days. As a result, in patients with the use of Normoflorin supplementation, a decrease in the negative manifestations of dyspeptic syndrome (nausea, belching, decreased appetite), abdominal syndrome (bloating, flatulence, frequency and nature of the stool) was noted. When using Normoflorin supplementation, no adverse events were observed.

Claims (1)

The strain Bifidobacterium bifidum VKPM Ac-1875 used to obtain bifidobacteria products.