RU2261905C1 - BIFIDUS-BACTERIUM STRAIN Bifidobacterium breve 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 breve 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 22.2 unit/mg of microbial protein mass. Strain B. breve 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 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 medical dietetic foods, dietary supplements, unfermented foods based on domestic strains of bifidobacteria Bifidobacterium breve 79-88, B breve 79-119 (RU 2176270 C1, 27.11.2001, RU 2078815 C1, 05/10/1997).

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 known strain of Bifidobacterium breve 79-119, used for the preparation of therapeutic and prophylactic bifidobacteria, dairy, unfermented foods and dietary supplements, is the closest analogue to the claimed strain. The strain was isolated from the intestines of a healthy infant in 1979, stored in a lyophilized state and deposited under No. 153 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 - 156 ° T, at 24 h - 164 ° T on hydrolyzate-milk medium, on sterile milk - up to 114 ° T and 120 ° T, respectively (RU 2078814 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 to identify a strain of Bifidobacterium breve 79-119-MB, which has 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 ensures the receipt of functional nutrition products, products on it basis and bacterial preparations with high probiotic and therapeutic properties.

The strain B.breve 79-119-MB was obtained from the collection strain B.breve 79-119M by successive cycles of lyophilization drying and recovery on nutrient thioglycol medium (50 drying-restoration cycles). Clones were selected according to 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 22.2 u / mg protein.

As a result of the selection, strain B breve 79-119-MB was obtained, which is deposited and stored in the All-Russian Collection of Industrial Microorganisms "VNIIGenetika" under the number VKPM As-1691 and is characterized by the following features:

Cultural and morphological characters.

Cells are gram-positive polymorphic rods, elegant, with U-shaped bifurcations at one or two ends; motionless, prone to the formation of clusters. Blaurocca forms colonies in the form of comets in the hepatic environment. 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.

Obligatory anaerobic, optimal cultivation temperature 37.5-38.0 ° С, pH 7.2-7.4. Catalase does not form, gelatin does not thin. It ferments glucose with the formation of acetic and L (+) - isomers of lactic acid without the formation of gas, acidifying the growth medium to 4.1. It ferments lactose, mannose, cellobiose, salicin, does not ferment arabinose, xylose, inulin, mannitol, sorbitol. Ferments milk with the formation of a tight clot by 16-24 hours.

Acid-forming activity: when cultured in sterile skim milk, titratable acidity reaches 134 ° T by 16 hours, 148 ° T by 20 hours, and 164 ° T by 24 hours. The strain maximally accumulates the microbial mass of bifidobacteria by 16 hours of cultivation, reaching in 1 ml of 8.3 lg 9 microbial cells; maximally accumulates the microbial mass of bifidobacteria in sterile skim milk without growth factors after 24 hours of cultivation - 1-3 lg 9.

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, antibiotic resistance:

The strain is resistant to antibiotics: kanamycin, lincomycin, amikocin, ampicillin, benzylpenicillin, streptomycin, as well as sulfadimezin (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.breve 79-119-MB Antibiotics, units of activity, mcg / cm ³ dose Titratable acidity of milk after cultivation with strain, ° T Ampicillin 250 72-68 Benzylpenicillin, U / cm ³ 10,000 95-93 Kanamycin Sulfate twenty 117-115 Lincomycin 25 97-91 Streptomycin 10 111-105 Amicocin sulfate 10 79-73

The data obtained indicate that the strain B. breve 79-119-MB is resistant to the highest concentrations of the following antibiotics: amikocin, ampicillin, benzylpenicillin, kanamycin, lincomycin, streptomycin.

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.breve 79-119-MB and B.breve 79-119 Zones of growth inhibition of test cultures (mm) Bifidobacteria strain Sh.Sonnei Sh. Flexneri E.coli S.aureus Pr.vulgaris B.breve 79-119 16,2 18.7 18.2 16.7 19.2 B.breve 79-119-MB 16.0-16.7 18.2-18.9 18-18,5 16.9-17 19,2-20

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 - 22.2 u / mg protein of microbial mass.

Storage conditions of the claimed strain.

Freshly prepared strain of B. breve 79-119-MB is stored on 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 strain B. breve 79-119-MB, distinguishing it from strain B. breve 79-119, is that the culture grown under stationary conditions in sterile skim milk has a pronounced superoxide dismutase activity: SOD activity of strain B. breve 79-119-MB is 22.2 ± 1.8 u / 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 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, 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. breve 79-119-MB is not bacteriogenic and does not have bacteriocin resistance to strains of bifidobacteria of the species B. bifidum, which allows its use in joint cultivation in starter cultures, as part of a complex bacterial preparation or a consortium of bifidobacteria.

The use of the claimed strain in the production of fermented milk, non-fermented products, biologically active additives, bifid-containing preparations, cosmetic and hygiene products, for example: creams, napkins, lotions of tampons, nourishing masks, women's pads, soap, cosmetic milk, etc., provides probiotic and bacteriological effect, helps to 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.breve 79-119-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.breve 79-119-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 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 ⁸ / ml, which can be used as a biological additive, adding it to food raw materials immediately before use.

Example 3. The use of strain B.breve 79-119-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.breve 79-119-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.breve 79-119-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 breve 79-119-MB, VKPM Ac-1691 used for the preparation of dairy, non-fermented products, dietary supplements, bifidobacteria, cosmetics and hygiene products.