RU2261906C1 - BIFIDUS-BACTERIUM STRAIN Bifidobacterium infantis 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 infantis 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 25.9 unit/mg of microbial protein mass. Strain B. onfantis 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.

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Description

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

A known strain of Bifidobacterium infantis 79-43, used to obtain bifidobacterium preparations of eubiotics and medical diet food. The strain exhibits high technological capabilities, actively increases the microbial mass on nutrient media adopted in the production of bacterial preparations for 16-24 hours of cultivation with the content of microbial cells lg 7.88-8.03, has acid-forming and inhibitory properties against pathogenic and conditionally pathogenic microorganisms resistant to several antibiotics (RU 2111248 C1, 05.20.1998).

As the closest analogue of the invention, the selected strain of Bifidobacterium infantis 73-15. The strain is isolated from the intestinal contents of a healthy infant, stored in a lyophilized state and deposited under No. 155 in the State collection of microorganisms - representatives of normal human microflora (GNKM) of the Moscow Research Institute of Epidemiology and Microbiology named after 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 - 143 ° T, at 24 h - 167 ° T on hydrolyzed-milk medium, on sterile milk - up to 82 ° T and 95 ° T, respectively (RU 2112034 C1, 05/10/1997).

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 activity during the passage of the digestive tract.

The objective of the invention is the identification of a strain of Bifidobacterium infantis 73-15-MB, with a high acid-forming ability, resistance to a wide range of antibiotics, pronounced antagonistic and antioxidant activity, biological activity, high viability and colonizing properties, which provides functional food products, products on it basis and bacterial preparations with high probiotic and therapeutic properties.

Strain B.infantis 73-15-MB, obtained from the collection strain B.infantis 73-15 by successive cycles of lyophilization drying and recovery on nutrient thioglycol medium (50 drying-recovery cycles). Clones were selected according to the maximum accumulation of biomass of bifidobacteria in sterile skim milk by 14-18 hours of cultivation of at least lg 9, as well as pronounced antibiotic and antioxidant activity: superoxide dismutase activity of the strain of at least 25.9 u / mg microbial protein.

As a result of the selection, the strain B.infantis 73-15-MB was obtained, which is deposited and stored in the All-Russian collection of industrial microorganisms "VNIIGenetika" under the number VKPM As-1692 and is characterized by the following features.

Cultural and morphological characters.

Cells are gram-positive polymorphic bacilli with bifurcation or thickening at 1-2 ends, prone to the formation of clusters. In the hepatic environment of Blaurock, the strain forms colonies in the form of comets. The logarithmic phase of growth ends by 24 hours. In liquid nutrient media it grows along the entire height of the volume, except for the aerobiosis zone.

Physiological and biochemical characteristics.

Fixed, undisputed obligate anaerobic. The optimum temperature of cultivation is 37.5-38.0 ° C, the pH of the nutrient medium is 7.2-7.4. Catalase does not form, gelatin does not thin. It ferments glucose to form acetic and L (+) - isomers of lactic acid without gas formation, acidifying the growth medium to pH 4.1. Ferments lactose, xylose, mannose, variable - mannitol, inulin, does not ferment arabinose, cellobiose, salicin, sorbitol.

Fermented sterile milk by 14-20 hours with the formation of a dense clot.

Acid-forming activity: when cultured in sterile skim milk, titratable acidity reaches up to 100 ° T by 16 hours, 160 ° T by 20 hours, and 180 ° T by 24 hours. The strain maximally accumulates the microbial mass of bifidobacteria by 16 hours of cultivation, reaching 8.3 lg CFU / cm ^{3 of} microbial cells in 1 ml, maximally accumulates the microbial mass of bifidobacteria on thioglycol medium and corn-lactose medium by 20 hours of cultivation, respectively 2 × 10 ⁸ CFU / cm ³ , but by 24 hours cell death was already observed.

The strain grows on culture media generally accepted in production with biomass accumulation of at least lg of 8–9 microbial numbers for 18–22 hours of cultivation.

Antagonistic properties, resistance to antibiotics.

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

Determination of the level of resistance of the strain to antibiotics was carried out by the method of serial dilution 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. infantis 73-15-MB

Antibiotics, units of activity, mcg / cm ³ Dose Titratable acidity of milk after cultivation with strain, ° T Ampicillin 250 57-63 Benzylpenicillin, U / cm ³ 10,000 84-88 Kanamycin Sulfate twenty 120-124 Lincomycin 25 77-79 Streptomycin 10 62-66 Amicocin sulfate 10 68-73 Oleandomycin 12.5 46-50 Tetracycline 10 44-48

The data obtained indicate that the strain B.infantis 73-15-MB is resistant to the highest concentrations of the following antibiotics: amikocin, ampicillin, benzylpenicillin, kanamycin, lincomycin, streptomycin, tetracycline, oleandomycin.

The strain has a pronounced antagonistic activity against pathogenic and conditionally pathogenic microorganisms: E. coli, S. aureus, Candida albikans, Proteus mirabilis, Proteus vulgaris, Klebsiella pneumoniae, Shigella sonnei, Citrobacter freundii, Bacillus subtilis, which are most often the cause of food poisoning when consuming fermented milk products (table 2).

table 2

Comparative data on the antagonistic activity of strain B.infantis 73-15-MB and B.infantis 73-15

Strain Zones of growth inhibition of test cultures (mm) bifidobacteria Sh.Sonnei Sh. Flexneri E.coli S.aureus Pr.vulgaris B.infantis 73-15 eleven 12 16 12.57 16.5 B.infantis 73-15-MB 11.6 11.9 17 12.6 16.9

Analysis of the data in the table shows that the inventive strain has a higher antagonistic activity in relation to the main test cultures.

The claimed strain showed a pronounced antioxidant activity: superoxide dismutase activity of the strain - 25.9 + 2.0 u / mg protein of microbial mass. Storage conditions of the claimed strain.

Freshly prepared strain of B.infantis 73-15-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 sign of strain B.infantis 73-15-MB, distinguishing it from strain B.infantis 73-15, is that the culture grown under stationary conditions in sterile skim milk has a pronounced superoxide dismutase activity. The SOD activity of strain B. infantis 73-15-MB is 25.9 units / mg of protein contained in the microbial mass.

The revealed SOD activity of the bifidobacteria culture indicates that the strain has a pronounced probiotic effect, which can occur under 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, fermented milk products, unfermented food products, and in the preparation of cosmetic and hygiene products. 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.infantis 73-15-MB is not bacteriogenic and does not have bacteriocin resistance to strains of bifidobacteria of the species B. bifidum and B. longum, which allows its use in joint cultivation in starter cultures, as part of a complex drug or a consortium of bifidobacteria.

The use of the claimed strain in the production of sour-milk, non-fermentable products, biologically active additives, bifid-containing preparations, cosmetic and hygiene products, for example: creams, napkins, lotions, tampons, nourishing masks, women’s pads, soaps, cosmetic milk, etc., provides a probiotic and bacteriological effect, helps restore normal microflora, skin and mucous membranes, and is also aimed at expanding the arsenal of similar drugs.

Example 1. The use of strain B.infantis 73-15-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 dilution 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.infantis 73-15-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.

3-passage - Received uterine culture in an amount of not more than 5% contribute to 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. 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.infantis 73-15-MB 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.

3-passage - Received uterine culture in an amount of not more than 5% contribute to 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.infantis 73-15-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.infantis 73-15-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 (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 Bifldobacterium infantis 73-15-MB, VKPM Ac-1692 used for the preparation of dairy, non-fermented products, dietary supplements, bifidobacteria, cosmetics and hygiene products.