RU2261908C1 - BIFIDUS-BACTERIUM STRAIN Bifidobacterium bifidum USEFUL IN PREPARATION OF FERMENTED-MILK AND NONFERMENTED PRODUCTS, BIOACTIVE SUPPLEMENT, BIFIDOBACTERIA-CONTAINING PRODUCT, COSMETIC AND TOILETRY AGENTS - Google Patents

Abstract

FIELD: biotechnology, food processing industry, medicine.

SUBSTANCE: strain Bifidobacterium bifidum is obtained by sequential cycles of lyophilic drying and reducing on thioglycol cultural medium (50 cycles). Clones with excellent antioxidant activity have been selected according to maximum accumulation of bifidus-bacteria biomass in sterile defatted milk not less than lg 9 after cultivation for 16 h. Strain has superoxide dismutase activity not less than 19.7 unit/mg of microbial protein mass. Strain Bifidobacterium bifidum can effectively growth in cultivation media and accumulate necessary titer of viable bacteria, has high acid-forming activity, antagonistic action against pathogenic and conditional pathogenic microflora, and excellent antioxidantaction. Culture has marked probiotic action cased by its superoxide dismutase activity. Strain is useful in preparation of functional foodstuff, bioactive supplement, cosmetic and toiletry agents.

EFFECT: bifidus-bacterium strain for manufacturing of products with excellent probiotic and healthy effect.

2 tbl, 5 ex

Description

The invention relates to biotechnology, the food industry and can be used in the production of functional foods, unfermented foods, pharmaceuticals, in the preparation of cosmetic and hygiene products.

A wide range of biologically active and technologically advanced strains of bifidobacteria makes it possible to develop new mono-and complex products of functional nutrition, clinical nutrition and drugs that can provide high colonization resistance of the human intestine, restore its normal microflora, and reduce the incidence of various intestinal infections.

To date, a number of bacterial preparations and dietetic food products have been developed based on domestic strains of bifidobacteria Bifidobacterium bifidum 1, B. bifidum 791, B. bifidum VKPM S-1257, B. bifidum VKPM Ac - 1578, etc. (A.S. 343588, patent SU 1347221, patent SU 849779, RU 2152993).

The quality of functional food products and pharmaceuticals is determined by the properties of the producer strain, its specific activity, and production-valuable properties.

A known strain of Bifidobacterium bifidum 791 BAG is used to obtain a liquid concentrate of bifidobacteria, sour-milk therapeutic and dietary products and dietary supplements. A characteristic feature of the strain is its ability to utilize 9 free amino acids in sterile skim milk without growth additives: glycine, alanine, valine, methionine, isoleucine, leucine, phenylalanine, histidine and arginine. The strain exhibits high technological properties. The maximum titer of bifidobacteria in sterile skim milk without growth factors after 24 hours of cultivation is 1-2 × 10 ¹⁰ , after 30 days of storage in this medium - 1-2 × 10 ⁸ (RU 2165454 C1).

The strain Bifidobacterium bifidum 791 was selected as the closest analogue of the present invention. The strain was isolated from the intestines of a healthy infant, deposited at the All-Russian Research Institute of Genetics under the number B-3300 and is characterized by the following features.

Cultural and morphological characters.

Rod-shaped cells with V-shaped bifurcations at one or two ends, motionless, in a smear form clusters, gram-positive.

In 0.72% hepatic and hydrolyzate-milk agar forms colonies in the form of buckwheat grains or discs up to 3 mm in diameter. In liquid nutrient media it grows along the entire height of the volume, except for the aerobiosis zone. It does not form pigment.

Physiological and biochemical characteristics.

Obligate anaerobic, optimal temperature 37.5 ° C, pH 7.2-7.4.

It ferments lactose with the formation of acetic and L (+) lactic acids, acidifying the growth medium to pH 4.0. Arabinose, xylose, mannose, cellobiose, inulin, mannitol, sorbitol, salicin does not ferment.

It ferments milk with the formation of a stable clot by 16-18 hours. There is no gas-forming ability, catalose-forming ability, gelatin liquefaction.

Acid-forming activity: at 24 h - 70 ° T, at 72 h - 90-100 ° T (on the liver medium), on hydrolyzate-milk medium - up to 100 ° T and 200 ° T, respectively.

Antagonistic properties.

It shows antagonistic activity against Shigella Sonne, Flexner, enteropathogenic Escherichia coli, staphylococci.

The DNA of the strain belongs to the expressed GC type with a specificity coefficient of 1.58, the molecular percentage of the content of GC pairs in the DNA is 61.35.

Resistant to kanamycin, monomycin, sensitive to penicillin antibiotics, kefzol, gentamicin (patent 1347221).

The known strain is not resistant enough to the damaging factors of aggressive environments of the gastrointestinal tract (acids, proteolytic enzymes) during the passage of bifidobacteria of the strain in the gastrointestinal tract, which indicates its insufficient colonizing ability and viability.

The objective of the invention is the identification of a strain of Bifidobacterium bifidum 791-MB, which is resistant to a wide range of antibiotics, pronounced antioxidant activity, biological activity, high vitality and colonizing properties, which provides functional food products, products based on it and drugs with high therapeutic and prophylactic properties.

Strain B. bifidum 791-MB was obtained from breeding strain B bifidum 791 by sequential passaging on thioglycol medium and skim milk for 50 passages. Clones were selected based on the maximum accumulation of biomass of bifidobacteria in sterile skim milk for a period of not more than 18-20 hours of at least lg 9, as well as with pronounced antioxidant activity: superoxide dismutase activity of the strain of at least 23.3 u / mg protein.

As a result of the selection, the strain B. bifidum 791-MB was obtained, which is deposited and stored in the All-Russian collection of industrial microorganisms "VNIIGenetika" under the number VKPM As-1688 and is characterized by the following features:

Cultural and morphological characters.

Rod-shaped cells with V-shaped bifurcations at one or two ends, motionless, in the smear form clusters gram-positive.

Physiological and biochemical characteristics.

Obligate anaerobic, optimal temperature 37.5 ° C, pH 7.2-7.4.

It ferments lactose with the formation of acetic and L (+) - isomers of lactic acid.

Arabinose, xylose, mannose, mannitol, inulin, cellobiose, sorbitol and salicin do not ferment.

It ferments milk with the formation of a stable clot by 16-18 hours. Gas-forming ability, catalase-forming ability, liquefaction of gelatin are absent.

Acid-forming activity: at 24 h - 70 ° T, at 72 h - 90-100 ° T (on the liver medium), on hydrolyzate-milk medium - up to 100 ° T and 200 ° T, respectively.

The maximum titer of bifidobacteria in the hydrolyzate-milk agar medium after 24 hours of cultivation is 1 × 10 ¹⁰ ; the maximum titer of bifidobacteria in sterile skim milk without growth factors after 24 hours of cultivation is 1-3 × 10 ¹⁰ .

The strain grows on nutrient media generally accepted in production with biomass accumulation of at least lg of 8–9 microbial numbers for 18–20 h of cultivation.

The DNA of the strain is of a pronounced GC type with a specificity coefficient of 1.58, the molecular percentage of the content of GC pairs in the DNA is 61.35.

Antagonistic properties, antibiotic resistance:

The strain is resistant to antibiotics: kanamycin, lincomycin, amikocin, ampicillin, benzylpenicillin, streptomycin, tetracycline, as well as sulfadimezin (see table 1).

Determination of the resistance level of the strain to antibiotics was carried out by the method of serial dilutions in a liquid nutrient medium — sterile skim milk. Various concentrations of sterile antibiotic solutions and a 16-hour culture of bifidobacteria in the amount of 5% were introduced into the nutrient medium. Stability was determined by the change in titratable acidity in the samples after 48 h of incubation at 37-38 ° C.

Table 1 Antibiotic resistance of strain B. bifidum-791-MB Antibiotics, units of activity, Mkg / cm ³ dose Titratable acidity of milk after cultivation with strain, ° T Ampicillin 250 68-72 Benzylpenicillin, U / cm ³ 10,000 61-67 Kanamycin Sulfate twenty 95-97 Lincomycin 25 72-76 Streptomycin 10 107-113 Sulfademinisine fifty 52-56 Tetracycline 10 52-56 Oleandomycin 12.5 48-52

The data obtained indicate that the strain of B. bifidum 791-MB is resistant to the highest concentrations of the following antibiotics: ampicillin, kanamycin, lincomycin, streptomycin, benzylpenicillin.

The strain exhibits pronounced antagonistic activity against pathogenic and conditionally pathogenic microorganisms: E. coli, S. aureus, Candida aibirans, Proteus mirabilis, Klebsiella pneumonia, Shigella sonnei, Citrobacter freundii. Bacillus subtilis (see table 2).

table 2
Comparison of the antagonistic properties of strains of B. bifidum 791 * and B. bifidum 791-MB Bifidobacteria strain Zone of growth inhibition of test cultures (mm) Sh.sonne Salmonella typhimurium E.coli Candida albicans Proteus vulgarus S.aureus B. bifidum 791 10 10 10 10 10 10 B. bifidum 791-MB 11-12 9-10 11-12 10-11 10-11 11-12

(* data are specified in accordance with the requirements of FS 42-132BC-94 "Dry Bifidumbacterin").

The data obtained indicate that the strain has the greatest antagonistic effect on Sh.Sonne, E. coli, Candida typhimurium, Proteus vulgarus, S.aureus, which are most often the cause of food poisoning arising from the consumption of dairy products.

Storage conditions of the claimed strain.

Freshly prepared strain of B. bifidum 791-MB in a nutrient medium with a mass fraction of solids of 14-16% with the addition of a protective medium containing sucrose, gelatin, sodium citrate, is dried by freeze-drying in ampoules. The strain is well stored at 6-8 ° C without loss of activity titer during the year.

The main production and useful feature of strain B. bifidum 791-MB, distinguishing it from strain B. bifidum 791, is that the culture grown under stationary conditions on a thioglycol medium in a volume of 300 cm ³ has a pronounced superoxide dismutase activity. The SOD activity of strain B. bifidum 791-MB is 25.6 ± 2.3 U / mg of protein contained in the bacterial mass. The revealed SOD activity of the bifidobacteria culture indicates that the strain has a pronounced probiotic effect, which can manifest itself in both in vivo and in vitro conditions. The high activity of acid formation, the resistance of the strain to basic antibiotics, contributes to the effective restoration of normal intestinal microflora in the process of antibiotic therapy.

The inventive strain can be used as a biologically active principle of probiotic orientation in the production of various forms of bacterial preparations, biologically active food additives, dairy products, unfermented foods, in the preparation of cosmetic and hygiene products, for example: creams, napkins, lotions, tampons, nutritional masks, panty liners, soaps, cosmetic milk, etc., provides a probiotic and bacteriological effect, helps restore bifido Laura.

The invention also aims to expand the arsenal of similar tools. At the same time, the biological value of functional food products and preparations prepared on its basis increases. Eating foods using the claimed strain promotes colonization of the intestinal mucosa, displacement of pathogenic and conditionally pathogenic microflora, correction of intestinal dysbiosis, and helps restore bifidoflora.

It was found that the strain B. bifidum 791-MB is not bacteriogenic and does not have bacteriocin resistance to strains of bifidobacteria of the species B. longum, which makes it possible to use it when co-cultured in starter cultures, as part of a complex drug or a consortium of bifidobacteria, is also aimed at expanding the arsenal of similar agents .

Example 1. The use of strain B.bifidum 791-MB in the composition of the starter cultures for the preparation of fermented milk products.

1st passage A lyophilized strain of bifidobacteria in an amount of 0.1 g (1 dose) of dry biomass is titrated by tenfold dilutions to 10 ^-9 and grown at 37.5-38 ° C until separate colonies appear in the last dilutions. Or 1 dose of dry biomass is introduced into a container with a nutrient medium and grown until the appearance of individual colonies in the latest dilutions. Or 1 dose of dry biomass of the strain is introduced into a container with a nutrient medium and grown to obtain biomass with a cell content of at least 10 ⁸ / ml.

2nd passage A grown culture with a volume of not more than 10% is introduced into the growing medium and cultivated to obtain biomass with a microbial content of at least 10 ⁸ / ml.

3rd passage - (product). The resulting biomass in a volume of not more than 5% is introduced into sterile milk and cultured until a clot forms, which is poured into containers and cooled. The resulting product contains in 1 ml of at least 10 ⁸ live bifidobacteria.

Example 2. The use of strain B. bifidum 791-MB as part of a biologically active additive.

1st passage The lyophilized strain is activated by plating in a nutrient medium and grown at 37-38.5 ° C until biomass with a cell content of at least 10 ⁸ / ml is obtained.

2nd passage A grown culture in a volume of not more than 10% is introduced into the nutrient medium and cultured to obtain biomass with a cell number of at least 10 ⁸ / ml.

3rd passage The resulting uterine culture in an amount of not more than 5% is introduced into the nutrient medium and grown at 37.5-38 ° C.

Obtaining a biologically active additive - The increased biomass is a liquid concentrate with a high, at least 10 ⁸ / ml, titer of bifidobacteria. Various bifidogenic factors can be added to the concentrate, which contribute to the stabilization of the properties of the culture and better engraftment of the strain in the body. A concentrate spilled in a container can be directly used as a biologically active additive of therapeutic and prophylactic action. You can get a dry biological active additive, for this, protective drying media (sugar gelatin, skim milk) are added to the biomass of the 3rd passage, the suspension is frozen at a temperature of at least -45 ° C, lyophilized, sealed and a dry concentrate with a content of bifidobacteria of at least 10 ⁷ / ml, which can be used as a biological additive, introducing it into food raw materials immediately before use.

Example 3. The use of strain B. bifidum 791-MB to obtain a bifid-containing preparation.

1st passage The lyophilized strain is activated by plating in a nutrient medium and grown at 37-38.5 ° C until biomass with a cell content of at least 10 ⁸ / ml is obtained.

2nd passage A grown culture in a volume of not more than 10% is introduced into the nutrient medium and cultured to obtain biomass with a cell number of at least 10 ⁸ / ml.

3rd passage The resulting uterine culture in an amount of not more than 5% is introduced into the nutrient medium and grown at 37.5-38 ° C.

Obtaining a bifid-containing preparation - The grown biomass is a liquid concentrate with a high, at least 10 ⁸ / ml titer of bifidobacteria. Protective drying media (sugar-gelatin, skim milk) is added to the biomass of the third passage, the suspension is frozen at a temperature of at least -45 ° C, lyophilized, sealed and a dry concentrate with a content of bifidobacteria of at least 10 ⁸ / ml can be used, which can be used as bifid-containing drug.

Example 4. The use of strain B.bifidum 791-MB to obtain kvass or juice.

1st passage Lyophilized strain in the amount of 0.1 g of dry biomass is activated by plating in a nutrient medium. Grown at 37.5-38 ° C to obtain biomass with a content of microbial cells of at least 10 ⁸ / ml

2nd passage A grown culture in a volume of not more than 10% is introduced into the nutrient medium and cultured to obtain biomass with a cell content of at least 10 ⁸ / ml.

3rd passage (receipt of product). Increased biomass in the amount of not more than 5% is applied to bread kvass or juice after pasteurization, so that the content of bifidobacteria in 1 ml of kvass or juice is at least 10 ⁸ . Kvass or juice is cooled and consumed without subsequent fermentation.

Example 5. The use of strain B.bifidum 791-MB to obtain a cosmetic product.

1st passage Lyophilized strain in the amount of 0.1 g of dry biomass is activated by plating in a nutrient medium. Grown at 37.5-38 ° C to obtain biomass with a content of microbial cells of at least 10 ⁸ / ml

2nd passage A grown culture in a volume of not more than 10% is introduced into the nutrient medium and cultured to obtain biomass with a cell content of at least 10 ⁸ / ml.

3rd passage (receiving cosmetic products). The grown biomass in the amount of not more than 5% is introduced into the bioreactor with thioglycol medium and incubated at 37.5-38 ° C with gentle stirring until the bifidobacteria titer reaches 10 ⁹ cells / ml. Subsequently, the resulting cell suspension is destroyed by heating in an autoclave. The resulting suspension of biologically active components is cooled and mixed with a cream base in a ratio of 1: 2-1: 20, depending on the purpose of the manufactured cream.

Claims (1)

The strain of bifidobacteria Bifidobacterium bifidum 791-MB, VKPM As-1688, used for the preparation of dairy, non-fermented products, dietary supplements, bifidobacteria, cosmetics and hygiene products.