RU2296156C2 - Strain bifidobacterium lactic 672 used to prepare lactic therapeutic and prophylactic products, fermented and non-fermented food products, biologically active additives, probiotic, and cosmetics - Google Patents

Abstract

FIELD: biotechnological methods.

SUBSTANCE: invention is intended for use in, food processing, cosmetics, biotechnological, medicinal industries, and veterinary in order to strengthen health, in particular when preparing lactic fermented and non-fermented food products, cosmetics, biologically active additives, and probiotic. Strain Bifidobacterium lactis 672 BKPM As-1677 is isolated from contents of healthy baby bowels. Invention enhances biological activity of the strain and its resistance to environmental conditions, accelerates growth on artificial nutrient media and acid-forming and antagonistic activity with regard to pathogenic and conditionally pathogenic microflora. Preparation of strains possessing above properties promotes extension of assortment of probiotics used to maintain health, in particular to normalize microflora gastroenteric and urogenital tracts, human and animal cutaneous coverings and mucous membranes.

EFFECT: increased assortment of bacterial preparations.

2 tbl, 6 ex

Description

The field of technology.

The invention relates to new strains of bifidobacteria and their use in food, cosmetic, biotechnological, medical industry and veterinary medicine with the aim of improving health.

The inventive strain, in particular, can be used in the preparation of fermented, fermented and non-fermented foods, starter cultures, cosmetics, dietary supplements and bacterial preparations.

The level of technology.

Due to adverse environmental conditions, prolonged and sometimes unsystematic antibiotic therapy, the number of people and animals suffering from disturbances in the normal microflora of the body is increasing. 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.

Products containing bifidobacteria are an effective means of prevention, as well as the treatment of various gastrointestinal diseases, as well as intestinal dysbiosis caused by intestinal and other diseases, prolonged antibiotic therapy, radiation and chemotherapy, stress exposure, adverse environmental conditions and other factors.

The resistance of bacteria to the conditions of the gastrointestinal tract is an important criterion for the selection of strains that promote health.

The quality of therapeutic and prophylactic products and preparations is largely due to the properties of the producer strain - its specific activity and manufacturability. A large assortment of biologically active and technologically advanced strains of bifidobacteria makes it possible to develop new mono-and complex products of therapeutic and prophylactic nutrition and drugs that can provide high colonization resistance of the intestines of humans and animals, restore their normal microflora and reduce the incidence of various intestinal infections.

Foreign mono-and complex preparations are known - eubiotics and fermented milk products, which include bifidobacteria of the species: Bifidobacterium bifidum (patent FR 2336886, 07.29.1977, A 23 C 9/12), Bifidobacterium bifidum, Bifidobacterium longum, Bifidobentererum 2411008, 07/06/1979, A 61 K 35/74), Bifidobacterium longum, Bifidobacterium breve (patent CH 637297, 07/29/1983, A 61 K 39/02), Bifidobacterium lactis (patent US 6379663, 03/14/2002, A 01 N 63/00.

In our country, for the preparation of bacterial preparations and dietetic food products, strains of bifidobacteria of the species: Bifidobacterium bifidum VKPM Ac-1578 (patent RU 2152993, 20.07.2000, C 12 N 01/20), Bifidobacterium bifidum VKPM S-1257 (RU patent 2108383, 04/10/1998, C 12 N 01/20), Bifidobacterium longum B379M, Bifidobacterium bifidum 791 (patent RU 2092064 10.10.1997, A 23 C 09/12), Bifidobacterium adolescentis MC-42 (copyright certificate SU 863639, 15.09. 1981, C 12 N 15/00). Foreign strains are also used in fermented milk products enriched with bifidobacteria, for example, Bifidobacterium lactis HN-019 (patent RU 2208632. 07/20/2003, C 12 N 01/20), Bifidobacterium lactis 420 (specification for strain, Bifidobacterium lactis 420 of DANISCO company , dated May 2, 2003).

To date, nothing is known about the use of domestic strains of Bifidobacterium lactis in the above areas.

Closest to the claimed strain is a strain of Bifidobacterium lactis 420 (prototype) (specification for the strain, Bifidobacterium lactis 420 of the company "DANISCO", 05/02/2003). The strain has antagonistic activity against certain pathogenic and conditionally pathogenic microorganisms.

For a number of tested properties, the known strain of Bifidobacterium lactis 420 is inferior to the claimed strain of Bifidobacterium lactis 672, in particular, it is more sensitive to phenol, bile and less active against certain pathogenic microorganisms (see table 1 and table 2).

Disclosure of the invention.

The objective of the present invention is to obtain a strain of microorganisms of the species Bifidobacterium lactis for use in the manufacture of therapeutic and prophylactic dairy products, fermented and non-fermented foods, starter cultures, dietary supplements, bacterial preparations, cosmetics, etc. agents containing bifidobacteria.

When solving this problem, the following technical results are achieved: the strain has high biological activity, in particular, high antioxidant activity (superoxide dismutase activity) in comparison with the information available in literature, resistance to environmental conditions, multiplies rapidly on artificial nutrient media, has acid-forming and antagonistic activity against pathogenic and conditionally pathogenic microflora. Obtaining strains with similar properties helps to expand the assortment of probiotics used to maintain health, in particular, to normalize the microflora of the gastrointestinal and genitourinary tracts, skin and mucous membranes of humans and animals.

The problem is achieved by using the strain of Bifidobacterium lactis 672 (Ac-1677 VKPM) as a part of starter cultures, food products, bacterial, cosmetic products, food additives, etc.

The strain Bifidobacterium lactis 672 can be used in a mono- or compositional probiotic concept in the preparation or manufacture of various starter cultures, sour-milk, fermented and non-fermented foods, dietary supplements, bacterial preparations, probiotics, cosmetics.

The strain Bifidobacterium lactis 672 was isolated from the intestines of a healthy child and deposited in VKPM (Russia, Moscow, 113545, 1st Dorozhny proezd, 1, GNIIG-VKPM) under No. Ac-1677.

The inventive strain survives in vitro that mimic the conditions of the gastrointestinal tract (GIT), in particular in the presence of 40% bile, 0.4% phenol, gastrointestinal enzymes and at low pH values.

The inventive strain can be used in the production of fermented milk products, fermented foods, such as: kvass, etc., unfermented foods, such as juices, drinks, etc., starter cultures and biologically active food additives. Also, the strain Bifidobacterium lactis 672 can be widely used for the manufacture of cosmetics, for example: creams, lotions, nourishing masks, cosmetic milk, etc.

In addition, the strain of Bifidobacterium lactis 672 can be used in the production of bacterial preparations, in the form of suppositories, oral and other drugs.

Cultural, morphological and physiological-biochemical properties of the strain Bifidobacterium lactis 672.

Cells are gram-positive, motionless, non-spore-forming, catalase-negative, anaerobic, 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; when growing on an agarized medium, GMCs under anaerobic conditions form small (up to 1 mm in diameter) round colorless colonies.

The optimum temperature of cultivation is 37-39 ° C.

The strain ferments glucose, lactose, sucrose, maltose, salicin, xylose, arabinose, raffinose with the formation of acetic and lactic acids, without the formation of gas;

skim milk at 22 h with the formation of a clot;

actively increases microbial mass when growing on corn-lactose medium (GMK-1) (composition GMK-1: corn-milk mixture - 15 g; peptone - 15 g; lactose - 9 g; ascorbic acid - 0.5 g; sodium citrate trisubstituted - 0.6 g; magnesium sulfate - 0.12 g; potassium phosphate monosubstituted - 2 g; sodium phosphate disubstituted - 1 g; agar - 3 g; distilled water - 1 l), bifidum medium (BP) (composition of bifidum medium : glucose - 7.5 g; lactose - 2.5 g; pancreatic casein hydrolyzate - 30 g; sodium chloride - 2.5 g; magnesium sulfate - 0.5 g; L-cysteine - 0.5 g; ascorb new acid - 0.5 g; yeast extract - 5 g; sodium acetate - 0.3; agar - 0.75 g; distilled water - 1 liter);

maximally accumulates microbial mass by 12-24 h, reaching 1-9 lg CFU (colony forming units) of 8-9;

exhibits antagonistic properties against pathogenic and conditionally pathogenic microflora (tables 1 and 2 are given at the end of the description), namely: the number of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae decreases more than 10 times, the number of Proteus mirabilis decreases more than 100 times , Proteus vulgaris, Candida albicans, and the number of Shigella sonnei, Citrobacter freundii, Bacillus subtilis is reduced by more than 1000 times when co-cultured in vitro compared to the control.

Thus, the proposed strain of Bifidobacterium lactis 672 is technologically advanced, since it actively propagates in a nutrient medium with the accumulation of production biomass in short (12-24 h) cultivation periods with a high concentration of microbial cells (log CFU ~ 9), has acid-forming and inhibitory properties against pathogenic and opportunistic microflora, high antioxidant activity (superoxide dismutase activity, SOD activity is 95.6 ± 0.2 in units / mg protein).

In addition, a study of the sensitivity spectrum to antibiotics and antibacterial drugs revealed that the strain of Bifidobacterium lactis 672 is naturally resistant to: aztreonam, kanamycin, lincomycin, neomycin, netilmicin, sulfanilamide, cefazolin, tobramycin, and carboxymene, vancocin, levicin, carb meropenem, oxacillin, oleandomycin, tetracycline, piperacillin, rifampicin, furagin, furadonin, furazolidone, cephalotin, ceftazidime, cefepime, cefotaxime, ceftriaxone, cefuroxime inu.

Thus, according to a number of properties and their combination, the strain of Bifidobacterium lactis 672 has greater biological activity compared with the known strains of Bifidobacterium lactis.

The strain Bifidobacterium lactis 672 is resistant to phenol and bile; resistance to low pH and gastrointestinal enzymes; marked antagonistic activity against Proteus vulgaris, Proteus mirabilis, Klebsiella pneumoniae, Shigella sonnei, Citrobacter freundii, Bacillus subtilis, natural resistance to: aztreonam, kanamycin, lincomycin, neomycin, netilmicin, sulfanilamine, cef; good adaptability (the ability of the strain to grow in a large titer equal to log KOE ~ 9 on selected media and is well preserved in fermented milk products for the required long shelf life); pronounced antioxidant activity.

The ability of the strain Bifidobacterium lactis 672 to grow in a large titer in a nutrient medium during short cultivation periods, its antagonistic, acid-forming, antioxidant activity, the ability to persist for a long time in dairy products, the ability to synthesize certain vitamins and other useful properties make it possible to use the strain in various food and medical supplies.

The implementation of the invention.

Example 1. The use of strain Bifidobacterium lactis 672 in the composition of biologically active additives.

1 passage: the lyophilized strain of Bifidobacterium lactis 672 in the amount of ~ 0.1 g of dry biomass is introduced into test tubes with nutrient medium and titrated by ten-fold dilution to 10 ^-9 . Incubated at a temperature of 37-38 ° C until the formation of visible growth in the last dilutions.

2nd passage: a grown culture of 10 ^-7-9 dilutions in a volume of up to 10% is introduced into the nutrient medium and cultivated at a temperature of 37-38 ° C for 12-24 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 12-24 hours at 37-38 ° C until biomass with a live content of Bifidobacterium lactis 672 of at least 10 ⁸ CFU / ml is obtained.

The result is a biologically active additive, which is a suspension of Bifidobacterium lactis 672 bifidobacteria cells with a titer of at least 10 ⁸ CFU / ml, if necessary (using concentration), a higher concentration of bifidobacteria cells can be obtained.

Example 2. The use of strain Bifidobacterium lactis 672 for the preparation of unfermented foods.

1 passage: the lyophilized strain of Bifidobacterium lactis 672 in the amount of ~ 0.1 g of dry biomass is introduced into test tubes with nutrient medium and titrated by ten-fold dilution to 10 ^-9 . Incubated at 37-38 ° C until the formation of visible growth in the last dilutions.

2nd 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 37-38 ° C for 12-24 hours until a dense growth with a well-visible zone of aerobiosis 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 12-24 hours at 37-38 ° C until biomass with a content of live bifidobacteria of at least 10 ⁸ CFU / ml is obtained.

Obtaining an unfermented food product: the grown and, if necessary, concentrated biomass of Bifidobacterium lactis 672 is introduced into the food product (milk, juices, etc.), mixed and poured into containers. The result is a product with a concentration of live bifidobacteria of at least 10 ⁶ CFU / ml.

Example 3. Preparation of a fermented milk product for therapeutic purposes using strain Bifidobacterium lactis 672 as a component of starter cultures.

1 passage: lyophilized strain is introduced into the culture medium and titrated by ten-fold dilutions to 10 ^-9 . Incubated at 37-38 ° C until the formation of visible growth in the last dilutions.

2nd passage: the grown culture of the strain Bifidobacterium lactis 672 from the last dilutions is added to the accumulation medium with a volume of up to 10% and cultivated at a temperature of 37-38 ° C until biomass with a content of bifidobacteria of at least 10 ⁸ CFU / ml is obtained.

3 passage: the resulting biomass in an amount of up to 10% of the volume is introduced into the accumulation medium and cultured for 12-24 hours at 37-38 ° C until biomass with a content of live bifidobacteria of at least 10 ⁸ CFU / ml is obtained.

Preparation of a fermented milk product: the obtained biomass of the strain Bifidobacterium lactis 672 in a volume of up to 10% is introduced into a pasteurized milk mixture at a temperature of 37-38 ° C along with other starter cultures used at normal rates, cultivated until a clot forms (pH 4.5-4.6 ) A product with a concentration of live bifidobacteria Bifidobacterium lactis 672 of at least 10 ⁶ CFU / ml is cooled to 22 ° C, poured into a container and further cooled to 6 ° C. The number of passages can be increased depending on the volume of production.

Example 4. The use of strain Bifidobacterium lactis 672 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 37-38 ° C until a biomass of bifidobacteria is obtained with a living cell content of at least 10 ⁸ CFU / ml. Protective drying media (e.g., 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 40-45 ° C and lyophilized from a frozen state in vacuum drying machines. The resulting preparation contains at least 10 ⁸ living cells of bifidobacteria in 1 g. By forming doses of an adequate level of consumption of probiotic microorganisms per day equal to at least 5 × 10 ⁸ -5 × 10 ¹⁰ CFU (for bifidobacteria), you can use the resulting preparation as a probiotic preparation .

Example 5. The use of strain Bifidobacterium lactis 672 to obtain 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 37-38 ° C until a biomass of bifidobacteria is obtained with a living cell content of at least 10 ⁸ KOE / ml.

Preparation of cosmetics: the grown biomass (if necessary a concentrate) of the strain Bifidobacterium lactis 672 is introduced into the composition of the cosmetic.

Example 6. The use of a strain of bifidobacteria Bifidobacterium lactis 672 to obtain a fermented product.

The culture of the strain of the 1st or 2nd passage (Example 1) is introduced into the nutrient medium, cultivated at a temperature of 37-38 ° C until a biomass of bifidobacteria is obtained with a living cell content of at least 10 ⁸ KOE / ml.

To obtain a fermented product such as kvass, the grown biomass (if necessary, a concentrate) of the bifidobacterium strain Bifidobacterium lactis 672 is introduced into the prepared base (water, sugar, honey, must, ascorbic acid) together with Lactobacillus plantarum and Lactobacillus fermentum, mixed and cultured at 37 ° C to obtaining a drink with titratable acidity of 65-85 ° T. Then the drink is cooled and bottled. The result is a finished therapeutic product with a content of at least 10 ⁷ live probiotic microorganisms in 1 ml.

Claims (1)

The strain of bifidobacteria Bifidobacterium lactis 672 VKPM Ac-1677, used for the preparation of sour-milk therapeutic and prophylactic products, fermented and non-fermented foods, biologically active additives, probiotics and cosmetics.