WO2009051511A1

WO2009051511A1 - Method for producing a liquid or dried bifidobacteria concentrate or a starter

Info

Publication number: WO2009051511A1
Application number: PCT/RU2007/000565
Authority: WO
Inventors: Aleksei Vladimirovich Molokeev; Aleksandr Georgievich Kusliy; Vladimir Ivanovich Baybakov; Tatyana Leonidovna Karikh; Leonid Georgievich Nikulin; Vyacheslav Vladimirovich Mironenko; Galina Vladimirovna Yasudis; Irina Aleksandrovna Guseva; Raisa Mikhailovna Yatsentyuk
Original assignee: Obschestvo S Ogranichennoy Otvetstvennostyu 'sibbiosan'
Priority date: 2007-10-15
Filing date: 2007-10-15
Publication date: 2009-04-23

Abstract

The invention relates to biotechnology, medical microbiology and the food industry. The inventive method for producing a liquid or dried bifidobacteria concentrate or a starter, involves introducing a seed dose of strain consortium of bifidobacteria Bifidobacterium bifidum 8-3 8-3 (deposited in VNIIgenetics in VKPM 'All-Russian Collection of Industrial Organisms' under N°Ac-1248 ), Bifidobacterium longum Ya3 (deposited in VNIIgenetics in VKPM 'All-Russian Collection of Industrial Organisms' under N°Ac-1252 ) and Bifidobacterium adolescentis B1 (deposited in VNIIgenetics in VKPM 'All-Russian Collection of Industrial Organisms' under N°Ac-1243), in the form of a base, into a nutrition medium comprising skimmed milk, the content of MSNF in which is equal to or less than 8.0%. The nutrition medium comprises: 5.0-7.0 mass% lactopeptone, 0.001-0.005 mass% muriatic cysteine and 2.0-4.0 mass% autolysate or baker's yeast extract, the rest being the skimmed milk, the content of NSNF in which is equal to or less than 8.0%. The bifidobacteria biomass is cultivated at a temperature of (37.0-38.0)±0.5°C until the bifidobacteria concentrate of at least of 1011 CFU/ml is obtained. The product is cooled and packed, or a protectivemedium is introduced therein, andis dried in sterile conditions.

Description

A method of obtaining a liquid or dry concentrate or yeast of bifidobacteria

Technical field

The invention relates to methods for producing, on the basis of a consortium of strains of bifidobacteria, liquid or dry concentrates or starter cultures of bifidobacteria, dry preparations of bifidobacteria, fermented milk, medical and dietary products, bifid-containing biologically active additives (BAA) and probiotic preparations based on the specified liquid concentrate and bifid can be used in biotechnology, medical microbiology and food industry.

The functions of normal microflora in the body are vital and very extensive: synthesizing, regulatory, desensitizing, detoxifying, enzymatic, protective and immunostimulating.

Preparations and products containing bifidobacteria are widely used for the prevention and treatment of dysbiosis and the normalization of the intestinal microflora of people who have undergone stressful effects, antibiotic and chemotherapy. State of the art

Probiotics and medical nutrition products are widely used abroad based on consortia of bifidobacteria strains of the species B. bifidum, B. lopgum, B. adolesceptis, B. breve, B. ipfaptis in different combinations (US Patent Yas5843922, IPC С 12 N 1 / 20, A 61 K 31/715, publ. 12/01/1998; Application US JCH22002015990, IPC C12 N 1/20, publ. 02/07/2002; US patent JY26335040, IPC A 23 C 19/32, publ. 01.01.2002) , as well as on the basis of consortia of strains of bifidobacteria and lactobacilli (Application US JY220020090416, IPC A 23L 1 / 0O ₅ publ. 07/11/2002; US patent _N≥ 5866385, IPC C 12 N 1/21, A 23 C 9/123, publ. 02.02.1999). However, consortia of strains of bifidobacteria and consortia of strains bifidobacteria and lactobacilli of foreign developers are focused on the creation of mainly therapeutic and prophylactic drugs and dietary supplements in dry form (tablets, capsules). These consortia of strains have significant drawbacks if they are used for the preparation of preparations and fermented milk products in liquid form: short shelf life of these preparations and products, low organoleptic qualities that are not essential for preparations in dry form.

Known consortia of strains of bifidobacteria and lactobacilli used for the production of sour-milk and non-fermented products in liquid form (RF Patent JY ° 2180348, IPC C 12 N 1/20, publ. 2002; RF patent JV22180914, IPC C 12 N 1/20 , publ. 2002; RF patent JN22180915, IPC C 12 N 1/20, publ. 2002).

The main disadvantage of fermented milk products made on the basis of consortia of strains of bifidobacteria and lactobacilli is the insufficient shelf life (not more than 1 month) due to the fact that lactobacilli during storage of the product contribute to its rapid acidification. The titer of bifidobacteria in the acidified product quickly drops, which reduces the therapeutic properties of this product.

A known consortium of five types of strains of bifidobacteria (WO 98/44090, IPC C 12 N 1/20, publ. 08.10.1998), which is intended for the preparation of fermented milk, non-fermented food products and bacterial preparations. The consortium was obtained by a combination of B. bifidum 791, B. lopgum, B379M, B. breve 79-119, B. iplesptis 73-15, B. adolesceptis G7513 strains of bifidobacteria, which have similar cultural and physiological and biochemical properties, as well as those with fermenting activity. A consortium of strains of five species of bifibacteria actively propagates in nutrient media with the accumulation of industrial biomass in a short cultivation period. It ferments milk with the formation of a clot with good taste and organoleptic properties, has a specific activity. Consortium of strains recommended as a part of starter cultures for the preparation of sour-milk therapeutic and dietary products, unfermented food products and bacterial preparations. The finished product contains 1 ml of 10 ⁹ live bacteria. In 1 dose of the drug, at least 10 ⁷ living bifidobacteria cells. However, such a consortium of bifidobacteria strains is not sufficiently resistant to the damaging factors of cultivation media and aggressive environments of the gastrointestinal tract (acids, proteolytic enzymes) during storage and promotion of the bifidobacteria of the consortium in the gastrointestinal tract, and therefore its viability and colonizing properties are insufficient when using products based on consortium.

A known method of producing a concentrate of bifidobacteria, including the deep cultivation of a culture of bifidobacteria on a nutrient medium containing (mass.%): The basis of up to 100%; casein pancreatic hydrolyzate 1.5-2.0; baker's yeast autolysate 0.3-0.5; glucose 0.25-0.95; monosubstituted potassium phosphate 0.1-0.2; ammonium citric trisubstituted 0.2-0.3; sodium chloride 0.3-0.4; magnesium sulphate heptahydrate 0.1-0.2; and cystine 0.025-0.03. The culture of bifidobacteria is grown in 2 stages lasting 19-24 hours to obtain a biomass concentrate, followed by dehydration in the presence of a protective medium and drying of the product (RF patent N ° 2005119, IPC C 12 N 1/20, publ. 30.12.93). Based on the specified concentrate of bifidobacteria, bifid-containing fermented milk products can be prepared.

However, this method is difficult to implement, because can be carried out only under conditions of deep cultivation in a fermenter in 2 stages lasting up to 24 hours with continuous mixing of the medium, instrument tracking and periodic adjustment of a number of parameters of the culture medium. In addition, the bifidobacteria concentrate obtained by this method has an insufficient titer and can be stored for a long time only in dry form. The liquid form of such a product quickly loses its activity during storage.

The closest analogue (prototype) of the proposed consortium is a consortium of three strains of bifidobacteria (RF Application JY22003107387, IPC C 12 N 1/20, publ. 20.12.2004). Consortium of bifidobacterial strains: Bifidobacterium bifidum N2791BAG (collection N2B-686 Research Institute of Microorganism Culture Collections SSC VB Vector, Koltsovo), Bifidobacterium bifidum 8-3 (deposited at the All-Russian Research Institute of Genetics and Medicine All-Russian Research Institute of Genetics in VKPM under NeB 5894) used to obtain a liquid concentrate of bifidobacteria, sour-milk, non-fermented therapeutic and prophylactic products, dietary supplements and bacterial preparations. The consortium is resistant to the damaging factors of cultivation media and aggressive media of the human gastrointestinal tract, and ensures utilization of a wide range of amino acids, i.e. It has high viability and colonizing properties, which ensures the production of drugs and products based on it with longer shelf life while maintaining high therapeutic and prophylactic properties.

The closest way to obtain a concentrate or yeast of bifidobacteria, including a consortium of these bacteria, is a method that includes the introduction of a seed dose of a culture of bacteria Bifidobacterium bifidum in a nutrient medium containing the basis of a nutrient medium with additives of cystine hydrochloride and baker's yeast autolysate and cultivation of Bifidifactum (37-38) ± 0.5 ° C until a bifidobacteria concentrate is obtained, cooling and packaging of the finished product (RF Patent N22104706, IPC A61 K 35/74, publ. 02.20.1998). D (-) - lactose, ascorbic acid, pancreatic hydrolyzate of milk are additionally introduced into the nutrient medium, sterile milk with a dry milk content (COMO) of 11-14% is used as the basis of the nutrient medium in the following ratio nutrient components, May:

Pancreatic milk hydrolyzate 7-9

D (-) - Lactose 0.05-0.15

Hydrochloric cystine 0.001-0.007 Ascorbic acid 0.003-0.009

Baker's yeast autolysate 0.5-2.0

Sterile milk (base), rest up to 100%

Moreover, the inoculated dose of Bifidobacterium bifidum bacteria culture is applied in a volume of 5 - 7% of the volume of the nutrient medium with a titer (5 10 ⁷ - 5 10 ⁸ ) CFU / ml, and cultivation is carried out for a time sufficient to obtain a bifidobacteria concentrate with a biological titer of at least (0.8-1.0) 10 ¹⁰ CFU / ml and acidity 100 - 12O ⁰ T. Such a consortium is obtained using a growth nutrient medium enriched with protein substances (12% dry milk residue with an excess of protein). However, along with the positive qualities of such a consortium

(ensuring long shelf life in liquid form, good organoleptic) there are negative properties. Low antibiotic resistance. In the production of a dry concentrate of bifidobacteria and dry bifidobacteria on the basis of this consortium, instability and heterogeneity of the biomass of bacteria are observed during its drying (due to the high protein content) and, as a result, the concentration of bifidobacteria in the dry preparation is not high enough. In addition, the manufacture of a cultivation medium for this consortium requires the use of high-quality skimmed milk powder. Disclosure of invention

The technical result of the invention is the higher resistance of the proposed consortium of bifidobacteria strains to antibiotics with a specific composition of the strains, expanding its probiotic therapeutic and prophylactic properties (colonization and antagonistic) and providing the possibility of stable homogeneous biomass of bifidobacteria in a relatively “poor” cultivation medium to obtain dry preparations with a high titer of bifidobacteria.

The specified technical result is achieved by the fact that a consortium of strains of bifidobacteria has been created: Bifidobacterm bifϊdum 8-3

(deposited at the All-Russian Research Institute of Genetics at VKPM under N ° Ac 1248), Bifidobacterium lopgum ID (deposited at the All-Russian Research Institute of Genetics at VKPM under JVГeAc-1252),

Bifidobacterium adolescentis Bl (deposited at the All-Russian Research Institute of Genetics at VKPM under NaAc-1243), used to obtain a liquid or dry concentrate or starter culture of bifidobacteria.

The indicated technical result is also achieved by the fact that in the method for producing a liquid or dry concentrate or sourdough of bifidobacteria, comprising administering a seed dose of a bifidobacteria culture into a nutrient medium containing a nutrient medium with additives of hydrochloric acid cysteine, baker's yeast autolysate and growing bifidobacteria biomass at (37-38 ) ± 0,5 ⁰ C to obtain a concentrate of bifidobacteria, cooling and packing the finished liquid product, or introduction to the liquid product and the protective atmosphere in the freeze-drying ste Under the conditions of the invention, the hydrolyzate of milk lactopeptone is additionally introduced into the nutrient medium, milk return with a low milk solids content (COMO) of not more than 8.0% is used as the basis for the nutrient medium in the following ratio of the components of the nutrient medium (May. %): milk back hydrolyzate (lactopeptone) 5.0 - 7.0 Cysteine hydrochloride 0.001-0.005

Baker's Yeast Autolysate 2.0 - 4.0

Milk return (base) with COMO no more than 8%, rest up to 100% A consortium is used as a seed dose of bifidobacteria Bifidobacterium strains according to claim 1 of the present claims: Bifidobacterium bifidum 8-3, Bifidobacterium lumphase ID, Bifidobacterium adolescentis B 1.

In the inoculum dose, the strains of Bifidobacterium bifidum 8-3, Bifidobacterium lupus ID, Bifidobacterium adolescentis B 1 are in a ratio of 3: 1: 1.

A seed dose of bifidobacteria is applied in a volume of 5.0-7.0% of the volume of the nutrient medium with a titer of bifidobacteria of at least 10 ⁹ CFU / ml, cultivation is carried out for a time sufficient to obtain a concentrate of bifidobacteria with a biological titer of at least (0.8- 1.0) 10 ¹¹ CFU / ml and acidity 100 - 12O ⁰ T, and the protective medium is introduced into the finished liquid product in an amount of at least 40% by weight of the latter.

The protective medium for drying biomass of a concentrate or starter culture based on a consortium of bifidobacteria contains sodium chloride, peptone, sucrose, gelatin, ascorbic acid in the following components (wt.%): Sodium chloride 0.4-0.5 peptone 0.7-1, 0 sucrose 6.0-7.0 gelatin 0.4-0.5 ascorbic acid 0.07-0.1 milk with COMO 12.0% rest up to 100%

Before drying, after applying the protective medium, the acidity of the biomass of bifidobacteria is corrected with NaOH solutions to pH = 6.5-7.0.

After drying the product, it has a titer of bifidobacteria of at least 10 ¹⁰ CFU / ml.

Information about the deposit of strains of the consortium of bifidobacteria:

Strain Bifidobastrium bifidum 8-3. Received from the VKPM collection.

Isolated from human feces and selected in

UKRNIIMASOMOLPROM. Deposited at the All-Russian Research Institute of Genetics in the VKPM collection under N ^o Ac-1248. Designed for the production of fermented dairy products. Copy of certificate attached

The strain Bifidobacterium lapgum ID (deposited at the All-Russian Research Institute of Genetics in the VKPM collection under JVaAc-1252). Designed for the production of fermented dairy products. A copy of the certificate is attached. The strain Bifidobacterium adolescentis Bl (deposited at the All-Russian Research Institute of Genetics in the VKPM collection under NsAc-1243. It is intended for the production of fermented dairy products. A copy of the certificate is attached. The control of cultures of bifidobacteria is carried out in accordance with MUK 4.2.577-96

“Methods of microbiological control of baby food, therapeutic nutrition and their components)).

Cultural and morphological features of the consortium: Rod-shaped cells, V-shaped bifurcations at one or two ends are extremely rare, motionless, in the smear form clusters gram-positive. In a hydrolyzate-milky, agarized medium, colonies are formed in the form of clear cloves 5 to 10 mm long and 1 to 2 mm thick. 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 ⁰ C, pH 1.2-1, A. Fermentes lactose with the formation of acetic and lactic acids, acidifying the growth medium to pH 4.0. Arabinose, xylose, mannose. cellobiose, inulin, mannitol, sorbitol, salicin does not ferment. The consortium strains ferment milk without growth factors with the formation of. To 16-18 hours a stable clot with an acidity of 140-160 ⁰ T, which practically does not increase even during the 5-month shelf life. There is no gas-forming ability, catalase-forming ability, gelatin liquefaction.

Acid-forming activity: at 24 h - 100 ° Turner, at 72 h - 150-

200 ° Turner (on the hepatic environment), on hydrolyzate-milk medium - up to 160 ° Turner and 250 ° Turner, respectively. The maximum titer of bifidobacteria of the consortium in skimmed natural milk (with 8% COM) with growth factors: milk hydrolyzate and yeast extract after 24 hours of growth is - (2-5) ^'10 , after 90-120 days of storage in this medium - (2- 5) x 10 ¹⁰ CFU / ml. Thus, the inventive consortium has a longer shelf life compared to the consortium of the prototype in 1.5 times.

The antagonistic properties of the consortium

The consortium is active in relation to Shigella Sonne, Flexner, enteropathogenic Escherichia coli, staphylococci.

The antagonistic activity of the consortium (table 1) of bifidobacteria strains was studied using the ip vitro method when co-grown with pathogenic test strains in the zone of growth inhibition of test microbes by the barcode method

Table 1

Data on growth inhibition of pathogenic and opportunistic microorganisms

Consortium study on antibiotic resistance

The inventive consortium is resistant to kanamycin, monomycin. This makes it easy to isolate bifidobacteria from a mixture of them with lactobacilli. Sensitive to penicillin antibiotics, kefzol, gentamicin. The ability of the inventive consortium of strains of bifidobacteria to grow a dense microbial mass in a relatively poor nutrient medium, to ferment milk in a short time, to exhibit specific activity makes it possible to use it in the preparation of fermented milk, non-fermented food products and dry bacterial preparations. Table 2 Comparative sensitivity of the consortium to antibiotics

Notes:

MIC - the minimum inhibitory concentration of MAC - the maximum permissible concentration in the blood

Thus, the consortium of strains is less sensitive to antibiotics kanamycin and monomycin (than, for example, the well-known consortium prototype of bifidobacteria strains) and products based on it can be used simultaneously with these antibiotics in their treatment, thus preventing the development of dysbiosis from antibiotic therapy.

Example 1. The use of a consortium of strains of bifidobacteria for the preparation of a liquid concentrate of bifidobacteria (JCB). 1st passage

Liquid cultures of strains: B. bifidum 8-3, B. lopgum ID, and B. adolesceptis Bl in an amount of 1 ml are introduced into 2 test tubes with 9 ml in a hepatic growth medium (for each strain separately). Strain the strains in test tubes for 15-18 hours at 37.5-38 ⁰ C. 2nd passage

The grown culture of each of the strains, separately in a volume of 5-10%, is added to 0.5 L of a growth milk production culture medium and cultivated for 16-18 hours at 37.5-38 ⁰ C to obtain laboratory starter cultures of each strain.

Then the biomass of starter cultures of each of the strains is mixed in the ratio (3: 1: 1, respectively): - 6 parts of B. bifidum 8-3 bifidobacteria biomass, - 2 parts of B. lumum YaZ biomass, and - 2 parts of B. adolesceptis B 1 biomass.

3rd passage The resulting mixed biomass in a volume of 5-10% is introduced into a production dairy medium, in a volume of 10 liters, and cultivated in a thermostatic way in glass bottles at 37.5-38 ° C, without adjusting the process for acidity for 15-18 hours until a clot forms. Get the production yeast of the consortium in liquid form. The content of probiotic microorganisms in the obtained biomass of the symbiotic association is Ig 9-10 in 1 ml.

A method of obtaining a liquid concentrate or yeast of bifidobacteria

The method includes the introduction of a sowing dose of industrial yeast of the inventive consortium of cultures of bifidobacteria with a titer of at least 10 ⁹

CFU / ml in a nutrient medium in an amount of 5.0-7.0.%, Of its volume. The nutrient medium contains (mass.%): Milk hydrolyzate (lactopeptone) 5.0 - 7.0 Cysteine hydrochloride 0.001 - 0.005

Baker's Yeast Autolysate 2.0 - 4.0

Milk return (base) with COMO no more than 8%, rest up to 100%

The content of total amine nitrogen in the medium is 500 mg%. The hydrolyzate of milk proteins provides an enrichment of the nutrient medium with a mixture of high molecular weight and low molecular weight peptides, which are effective stimulators of bifidobacteria growth. Yeast extract contains B vitamins and stimulating growth factors. bifidobacteria, as well as a high content of amino nitrogen, which provides the most favorable growth conditions for bifidobacteria. Amine nitrogen is a source of ready-to-assimilate nitrogenous nucleotide bases necessary for the synthesis of nucleic acids of bifidobacteria during their intensive reproduction. The components providing the reducing properties of the claimed medium are mainly cysteine, as well as the amino acids glutamine and asparagine, which have antioxidant properties, which are also rich in yeast extract. The biomass of bifidobacteria is grown in a volume of 10 liters and cultivated in a thermostatic way in glass bottles at 37.5-38 ⁰ C, without regulating the process of acidity for 15-18 hours until a clot forms. The liquid concentrate or yeast of bifidobacteria has a biological titer of at least (0.8-1.0) 10 ¹¹ CFU / ml and acidity of 100 - 12O ⁰ T. The resulting product is cooled and packaged in a container.

The liquid product can be used as a biologically active additive for independent use or for enrichment of sour-milk products for the purpose of correcting dysbacterioses, as well as for obtaining dry biomass by freeze-drying and spray drying. Example 2. ^' The method of drying a liquid concentrate or sourdough bifidobacteria

In a liquid product obtained in accordance with example 1, a protective medium is introduced in an amount of at least 40% by weight of the latter. A protective medium for drying biomass of a concentrate or starter culture based on a consortium of bifidobacteria contains (mass%): sodium chloride 0.4-0.5 peptone 0.7-1.0 sucrose 6.0-7.0 gelatin 0.4-0 5, ascorbic acid 0.07-0.1 milk with COMO 12.0% rest up to 100%

After introducing the protective medium, the acidity of the biomass of bifidobacteria is corrected with a NaOH solution to pH = 6.5-7.0. The resulting suspension is poured into 2 ml ampoules or 5 ml into vials, frozen at t = minus 40-45 ⁰ C and lyophilized from a frozen state in vacuum dryers. After drying the product, it has a titer of living bifidobacteria cells of at least 10 ¹⁰ - 10 ¹¹ CFU / ml, and contains a moisture content of not more than 0.1-0.2%.

In addition, the liquid product can be dried in a standard spray dryer under sterile conditions. For this, the liquid product is subjected to spray drying in an upward flow of air heated to 50-55 ⁰ C.

The resulting dry product contains not less than 10 ¹⁰ - 10 ^minutes of living bifidobacteria cells in 1 g Residual moisture - 1-2%.

Example 3. The use of liquid starter culture for the preparation of symbiotic fermented milk products of therapeutic and functional nutrition

The liquid starter culture of the consortium prepared according to Example 1 (after the 3rd passage) in the amount of 0.2-0.3% is introduced into pasteurized milk together with kefir or acidophilic starter culture in the amounts of starter culture intended for the preparation of bifidokefir or bifacil, mixed and cultured 4 - 18 hours until a clot forms, after which the product is cooled and packaged in consumer packaging. The result is a therapeutic and prophylactic dairy products with a useful bifidoflora content of at least Ig 7-8 in 1 ml of symbiotic product. The resulting products have a pronounced immunocorrective effect.

Example 4. The use of dry sourdough direct application for the preparation of symbiotic sour-milk products of therapeutic and functional nutrition Dry sourdough of the consortium, in the amount of 0.005-0.05 mass% they are directly introduced into pasteurized milk along with kefir or acidophilic starter culture in the amounts of starter cultures intended for the preparation of bifidokefir or bifacil, mixed and cultured for 4-18 hours until a clot forms, after which the product is cooled and packaged in consumer packaging. The result is a therapeutic and prophylactic sour-milk products containing live bifidobacteria not less than Ig 6-7 in 1 ml of symbiotic product.

The resulting products have a pronounced immunocorrective effect.

The study of the amino acid composition of a liquid concentrate of bifidobacteria, prepared on the basis of the inventive consortium

Table 3 shows the amino acid analysis data.

Table 3

Thus, a liquid concentrate of bifidobacteria, prepared on the basis of the inventive consortium, has high biological value, and is a supplier of essential amino acids that are in easily digestible form. A rich set of the most important essential amino acids, such as: valine - 8.3%, leucine - 13.4%, lysine - 7.7%, and methionine - 3.1%, has important physiological significance, since lack of valine leads to impaired coordination of movements, lack of leucine causes changes in the kidneys and thyroid gland, lack of lysine leads to disruption of blood formation.

In a liquid concentrate of bifidobacteria, the arginine content was increased to 7.7% against 2.0% in a liquid concentrate of bifidobacteria, obtained on the basis of a prototype consortium and the technology for its preparation.

An increase in arginine content is very important, since this amino acid stimulates the body's immune system, in addition, it has antioxidant properties, which allows to increase the shelf life of the product.

Study of the duration of storage of a consortium of strains of bifidobacteria in a liquid concentrate of bifidobacteria

The duration of storage of a consortium of strains of bifidobacteria in a liquid concentrate of bifidobacteria was studied (table 4). A significant period (up to 3 months) of storing bifidobacteria liquid concentrate with a titer of at least 10 ¹⁰ CFU / ml is associated with ensuring the initial acidity of the preparation of the bifidobacteria consortium no more than (140-145) ° T, obtaining the initial titer of bifidobacteria (at least 10 ¹¹ CFU / ml). Table 4

Data on the safety of the proposed consortium of bifidobacteria in cholelithiasis

The study of the activity of a dry concentrate of bifidobacteria, obtained on the basis of the inventive consortium

The study subjected to a dry concentrate of bifidobacteria obtained in accordance with example 2.

Table 5

Research data on the activity of a consortium of bifidobacteria in a dry concentrate obtained by spraying or lyophilic methods

After freeze-drying ZhKB (table 5), the activity of dry ZhKB on the basis of the inventive consortium is five times higher, and after spray drying, two orders of magnitude compared with the prototype.

Industrial applicability. The above examples show that the inventive method for producing a concentrate or yeast provides active propagation of a consortium of strains of 3 types of bifidobacteria in relatively poor nutrient media with the accumulation of industrial biomass in a short cultivation period. The product has specific activity. Used media and reagents are publicly available.

Claims

Claim

1. A method of producing a liquid or dry concentrate or starter culture of bifidobacteria, including the introduction of a seed dose of a culture of bifidobacteria in a culture medium containing the basis of the nutrient medium with the addition of hydrochloric acid cysteine, autolysate or baker’s yeast extract and growing biomass of bifidobacteria at (37-38)

± 0.50 ^° C until a bifidobacterium concentrate is obtained, cooling and packing the finished liquid product or introducing a protective medium into the liquid product and freeze-drying it under sterile conditions, characterized in that lactopeptone is additionally introduced into the nutrient medium, and fat-free as the basis of the nutrient medium milk with a dry milk content of not more than

8.0% in the following ratio of components of the nutrient medium, (May.%): Lactopeptone 5.0 - 7.0

Hydrochloric cysteine 0.001-0.005

Autolysate or baker's yeast extract 2.0 - 4.0 Skim milk with a skimmed milk powder content of not more than 8.0%, the rest is up to 100%, and a consortium of Bifidobacterium bifidobacterium bifidum 8-3, Bifidobacterium lumumum Bifidobacterium adolescentis B 1

2. The method according to p. 1, characterized in that the seed dose of the strains

Bifidobacterium bifidum 8-3, Bifidobasterium lapgum ID, Bifidobasterium adolescentis B 1 are in a ratio of 3: 1: 1.

3. The method according to p. 1, characterized in that the inoculated dose of bifidobacteria is applied in a volume of 5.0-7.0% of the volume of the nutrient medium with a titer of bifidobacteria of at least 10 ⁹ CFU / ml, the cultivation is carried out for a time sufficient to obtain concentrate Bifidobacteria biological titer of not less than (0.8-1.0) 10 ^minutes cfu / ml and the acidity of 100 - 12O ⁰ T, and the protective medium is introduced into the finished liquid product in an amount not less than 40% by weight of the latter.

4. The method according to p. 1, characterized in that the protective environment for drying the biomass of the concentrate or starter culture based on a consortium of bifidobacteria contains sodium chloride, peptone, sucrose, gelatin, ascorbic acid with the following components (wt.%): Sodium chloride 0, 4-0.5 peptone 0.7-1.0 sucrose 6.0-7.0 gelatin 0.4-0.5 ascorbic acid 0.07-0.1 milk with COMO 12.0% rest up to 100%

5. The method according to p. 1, characterized in that before drying after applying the protective medium, the acidity of the biomass of bifidobacteria is adjusted with a NaOH solution to pH = 6.5-7.0.