RU2261904C1 - BIFIDUS-BACTERIUM STRAIN Bifidobacterium adolescentis 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 adolescentis 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 B. adolescentis 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 make it possible to develop new mono-and complex products of functional nutrition, medical nutrition and bacterial preparations that can provide high colonization resistance of the human intestine, restore its normal microflora, and reduce the incidence of various intestinal infections.

A number of bacterial preparations and dietetic food products based on domestic strains of bifidobacteria Bifidobacterium adolescentis МС-42 and Bifidobacterium adolescentis G 7513 (SU 863639 A1, 09.15.1981, RU 2120991 C1, 10.27.1998, RU 2109054 C1, 20.04. .1998).

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

The closest analogue of the invention is a strain of Bifidobacterium adolescentis G 7513, used to obtain bifidobacterium-eubiotics and products of therapeutic and prophylactic nutrition. The strain was isolated from the contents of the intestines of an adult in 1975, stored in a lyophilized state and deposited under No. 158 in the State collection of microorganisms that are representatives of normal human microflora GKNM MNIIEM them. G.N.Gabrichevsky. A characteristic feature of the strain is its ability to actively reproduce on nutrient media adopted in the production of bacterial preparations with a high content of microbial cells for 16-24 hours of cultivation with the formation of a clot of tender consistency. The strain is an energetic acidifier. Acid-forming activity: at 16 h - 160 ° T, at 24 h - 164 ° T on corn-lactose medium, on hydrolyzate-milk medium - up to 166 ° T and 202 ° T, respectively (RU 2120991 C1, 10.27.1998).

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 adolescentis G 7513-MB, which is resistant to a wide range of antibiotics, pronounced antioxidant activity, biological activity, high viability and colonizing properties, which provides functional food products, products based on it and drugs with high therapeutic preventative properties.

The strain B. adolescentis G 7513-MB was obtained from the selection strain B. adolescentis G 7513 by successive cycles of lyophilization drying and recovery on nutrient thioglycol medium (50 drying-restoration cycles). Clones were selected based on the maximum accumulation of biomass of bifidobacteria in sterile skim milk by 16 hours of cultivation of at least lg 9, as well as with pronounced antibiotic and antioxidant activity: superoxide dismutase activity of the strain of at least 19.7 u / mg microbial protein.

As a result of the selection, the strain B. adolescentis G 7513-MB was obtained, which is deposited and stored in the All-Russian collection of industrial microorganisms "VNIIGenetika" under the number VKPM As-1690 and is characterized by the following features:

Cultural and morphological characters.

Cells are gram-positive polymorphic bacilli with bifurcation or thickening at the 1-2 ends, prone to the formation of clusters. In the hepatic environment Blaurock and corn-lactose medium forms a colony in the form of cloves.

Physiological and biochemical characteristics.

Obligate anaerobic, optimal temperature 37-38 ° C, pH 7.2-7.4.

It ferments glucose to form acetic and L (+) - isomers of lactic acid without gas formation, acidifying the medium to pH 4.0-4.1. It ferments lactose, arabinose, xylose, cellobiose, salicin, does not utilize mannose, mannitol, inulin, sorbitol. It ferments sterile milk with the formation of a stable dense clot by 16-20 hours. Gas-forming ability, catalase-forming ability, liquefaction of gelatin are absent.

Acid-forming activity: when cultivated in sterile skim milk, titratable acidity reaches 96 ° T by 16 hours, and 100 ° T by 24 hours. The strain maximally accumulates the microbial mass of bifidobacteria by 16-20 hours of cultivation, reaching 9.0-9.4 microbial cells in 1 ml of log; maximally accumulates the microbial mass of bifidobacteria in sterile skim milk without growth factors after 24 hours of cultivation, reaching 1 ml 1-3 lg of 9 microbial cells.

Acid-forming activity: by 24 hours - 70 ° T, by 72 hours - 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 CFU / cm ³ ;

- the maximum titer of bifidobacteria in sterile skim milk without growth factors after 24 hours of cultivation is 1-3 × 10 ¹⁰ CFU / cm ³ .

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 maximum number of cells accumulated on thioglycol medium and corn-lactose medium is very close to 20 hours and amounts to 2 × 10 ⁹ CFU / cm ³ .

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

Antagonistic properties, resistance to antibiotics.

The strain is resistant to antibiotics: kanamycin, lincomycin, amikocin, ampicillin, benzylpenicillin, streptomycin, tetracycline, gentamicin, 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 an 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. adolescentis G 7513-MB Antibiotics, units of activity, Mkg / cm ³ Dose Titratable acidity of milk after cultivation with strain, ° T Ampicillin 250 54-58 Benzylpenicillin, U / cm ³ 10,000 85-91 Kanamycin Sulfate twenty 128-132 Lincomycin 25 77-83 Streptomycin 10 51-53 Sulfadimezin fifty 42-44 Tetracycline 10 38-40 Oleandomycin 12.5 42-46 Ofloxacin twenty 62-66 Rifampicin fifteen 45-51

The data obtained indicate that the strain B. adolescentis G 7513-MB is resistant to the highest concentrations of the following antibiotics: kanamycin, benzylpenicillin, lincomycin, ofloxaline, ampicillin, streptomycin.

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

table 2
Comparison of the antagonistic properties of strains of B. adolescentis G 7513 and B. adolescentis G 7513-MB Bifidobacteria strain Zone of growth inhibition of test cultures, (mm) Sh.Sonnei Sh. Flexneri E.coli Proteus vulgarus S.aureus B. adolescentis D 7513 23,2 25.5 27,2 25.7 27.5 B. adolescentis D 7513-MB 24.3 25.9 27.7 26.1 28.3

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

Storage conditions of the claimed strain.

Freshly prepared strain B. adolescentis G 7513-MB is stored in a nutrient medium with a mass fraction of solids of 14-16% with the addition of a protective medium containing sucrose, gelatose, sodium citrate, freeze-dried 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 the strain B. adolescentis G 7513-MB, distinguishing it from the strain B. adolescentis G 7513, 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. SOD - the activity of strain B. adolescentis G 7513 MB is 19.7 ± 1.6 units / 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 flora.

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 established that the strain B. adolescentis G 7513-MB is not bacteriogenic and does not have bacteriocin resistance to strains of bifidobacteria of the species B. longum and B. bifidum, which allows its use in joint cultivation in starter cultures, as part of a complex drug or a consortium of bifidobacteria.

Example 1. The use of strain B. adolescentis G 7513-MB in the composition of the starter cultures for the preparation of fermented milk products.

1st passage - 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.adolescentis G 7513-MB as part of a biologically active additive.

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

2nd passage - A grown culture in a volume of not more than 10% is applied to 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 biologically active additives - The grown 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. It is possible to obtain a dry biological active additive for this purpose, 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 is} obtained / ml, which can be used as a biological additive, introducing it into food raw materials immediately before use.

Example 3 Use of B.adolescentis G 7513-MB strain to obtain a bifid-containing preparation.

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

2nd passage - A grown culture in a volume of not more than 10% is applied to 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 is obtained with a content of bifidobacteria of at least 10 ⁸ / ml, which can be used as bifid-containing drug.

Example 4. The use of strain B.adolescentis G 7513-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 the 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.adolescentis G 7513-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 containing microbial cells, not less than 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 (obtaining a cosmetic product) - Increased 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 adolescentis G 7513-MB, VKPM Ac-1690, used for the preparation of dairy, non-fermented products, dietary supplements, bifidobacteria, cosmetics and hygiene products.