RU2291194C2 - Consortium of bifidobacterium strains used in preparing liquid concentrate of bifidobacterium, fermented-milk, nonfermented, curative-prophylactic foodstuffs, biologically active supplements and bacterial preparations - Google Patents

Abstract

FIELD: biotechnology, medicinal microbiology.

SUBSTANCE: invention represents a consortium of strains of bifidobacteria. The consortium comprises the following strains of bifidobacteria: Bifidobacterium bifidum № 791 BAG, Bifidobacterium bifidum 8-3, Bifidobacterium longum DVA-13 used in preparing a liquid concentrate of bifidobacteria, fermented-milk, nonfermented curative-prophylactic foodstuffs, biologically active supplements and bacterial preparations. The ratio of strains in the consortium is 8:1:1, respectively. The consortium of strains of bifidobacteria shows enhanced resistance against damaging factors of cultured media and aggressive media of human gastrointestinal digestive tract that provides utilization of broader spectrum of amino acids, i. e. it possesses higher viability and colonizing properties that, in turn, provides preparing preparations and foodstuffs based on thereof showing prolonged storage times in retaining high curative-prophylactic properties. The proposed consortium strains of bifidobacteria can be used in preparing liquid concentrate of bifidobacteria and in preparing fermented-milk, nonfermented curative-dietetic foodstuffs, bifido-containing biologically active supplements and preparation-probiotics based on the indicated liquid concentrate of bifidobacteria.

EFFECT: improved preparing method, valuable properties of consortium.

2 cl, 5 tbl, 5 ex

Description

The invention relates to biotechnology and medical microbiology and is a consortium of strains of bifidobacteria, which can be used to obtain a liquid concentrate of bifidobacteria, as well as for the preparation of fermented milk, non-fermented therapeutic and dietary products, bifidobacteria, biologically active additives (BAA) and probiotic preparations based on the specified liquid concentrate of bifidobacteria.

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 therapeutic food products are widely used abroad based on consortia of bifidobacteria strains of the species B. bifidum, B. longum, B. adolescentis, B. breve, B. infantis in various combinations (US Patent No. 5843922, IPC C 12 N 1 / 20, A 61 K 31/715, publ. 12/01/1998; Application US No. 20052010, IPC C 12 N 1/20, publ. 02/07/2002; US patent No. 63,350,040, 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 No. 2002090416, IPC A 23 L 1/00, publ. 07/11/2002; US patent No. 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 of 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 No. 2180348, IPC C 12 N 1/20, publ. 2002; RF patent No. 2180914, IPC C 12 N 1/20 , publ. 2002; RF patent No. 2180915, 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.

The closest analogue (prototype) of the present invention is a consortium of strains of bifidobacteria (WO 98/44090, IPC C 12 N 1/20, publ. 08.10.1998). A consortium of strains of bifidobacteria of five types is intended for the preparation of dairy, non-fermented food products and bacterial preparations. The consortium was obtained by a combination of B. bifidum 791, B. longum, B379M, B. breve 79-119, B.infantis 73-15, B. adolescentis 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 period of cultivation. It ferments milk with the formation of a clot with good taste and organoleptic properties, has a specific activity. A consortium of strains is 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 (prototype) 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 bifidobacteria of the consortium in the gastrointestinal tract, and therefore its viability and colonizing properties are insufficient Consortium based products.

Disclosure of invention

The technical result of the invention is the creation of such a consortium of strains of bifidobacteria, which would be more resistant to the damaging factors of culture media and hostile environment of the human gastrointestinal tract, would ensure the utilization of a wider range of amino acids, i.e. would have a higher viability and colonizing properties, which would ensure the receipt of drugs and products based on it with longer shelf life while maintaining high therapeutic and prophylactic properties.

The specified technical result is achieved by a consortium containing the following strains of bifidobacteria: Bifidobacterium bifidum No. 791 BAG, Bifidobacterium bifidum 8-3, Bifidobacterium longum TWA-13, used to obtain a liquid concentrate of bifidobacteria, fermented milk, non-fermented, therapeutic, therapeutic and therapeutic agents. The ratio of strains in the consortium is 8: 1: 1, respectively.

Information about the deposit of strains of the consortium

1. Bifidobacterium bifidum No. 791 BAG (collection No. B-686 Scientific research institute of collections of cultures of microorganisms SSC VB "Vector", Koltsovo).

2. Bifidobacterium bifidum 8-3 (deposited at the All-Russian Research Institute of Genetics at VKPM under No. B 5799).

3. Bifidobacterium longum DVA-13 (deposited at the All-Russian Research Institute of Genetics at VKPM under No. B 5894).

The properties of the consortium are determined by the strain of bifidobacteria Bifidobacterium bifidum No. 791 BAG, the content of which in the consortium is 80%.

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-milk agarized medium, colonies are formed in the form of clear cloves 5-10 mm long, 1-2 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 ° C, pH 7.2-7.4. Ferments 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.

It ferments milk without growth factors with the formation by 16-18 hours of a stable clot with a relatively low acidity - 120-150 ° T, which practically does not increase even during the 5-month shelf life. The gas-forming ability, catalase-forming ability, liquefaction of gelatin are absent.

Acid-forming activity: by 24 hours - 100 ° Turner, by 72 hours - 150-200 ° Turner (on the liver medium), on hydrolyzate-milk medium - up to 160 ° Turner and 250 ° Turner, respectively.

The maximum titer of bifidobacteria after 24 hours of growth in a hydrolyzate-milk agar medium is 1-5 × 10 ¹⁰ .

The maximum titer of bifidobacteria of the consortium in a dairy medium with growth factors: milk hydrolyzate and yeast autolysate after 24 hours of growth is - (5-10) · 10 ¹⁰ , after 30 days of storage in this medium - 2-5 × 10 ¹⁰ .

The maximum titer of bifidobacteria of the consortium in a dairy medium (sterile skim milk) without growth factors after 24 hours of growth is 1-2 × 10 ¹⁰ , after 30 days of storage in this medium, 1-2 × 10 ⁸ .

Bifidobacteria of the consortium completely utilize 9 free amino acids in sterile skim milk without growth additives: glycine, alanine, valine, methionine, isoleucine, leucine, phenylalanine, histidine, arginine. The strain DNA is of a pronounced GC type with a specificity coefficient of 1.58, the molar percentage of the GC content in the DNA is 61.35.

Antagonistic properties, resistance to antibiotics of the consortium:

It is active against Shigella Sonne, Flexner, enteropathogenic Escherichia coli, staphylococci.

It is resistant to cananicin, monomycin, sensitive to penicillin antibiotics, kefzol, gentamicin.

The ability of a consortium of strains of four types of bifidobacteria to increase microbial mass in nutrient media, 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 bacterial preparations.

Research results confirming the beneficial properties of the inventive consortium of bifidobacteria strains

The choice of the composition of the strains of the consortium. To identify the technological features and properties of the inventive consortium of strains of bifidobacteria, comparative studies were conducted during the cultivation of several variants of consortia, as well as their storage. The cultivation of the consortia was carried out in a dairy environment with growth factors, made according to the patent of the Russian Federation No. 2169763 (wt.%):

Pancreatic Hydrolyzate milk back  7.0 Hydrochloric cysteine (L-cystine)  0.003 D (-) - lactose  0.09 Sodium Chloride  0.05 Enzymatic yeast autolysate  2.0 Milk return (base), normalized by dry residue up to 12%  the rest is up to 100%

The content of amine nitrogen in the medium is 300 mg%.

The introduction into the environment of pancreatic hydrolyzate of milk protein proteins enriches it with a mixture of high molecular weight and low molecular weight peptides, which are effective stimulators of bifidobacteria growth. The enzymatic yeast autolysate contains vitamins and stimulating growth factors of bifidobacteria, as well as the optimal content of amine nitrogen, which provides the most favorable growth conditions for bifidobacteria.

D (-) - lactose is a moderate stress factor for bifidobacteria cells and provides the initiation of polymorphism of bifidobacteria cells (rod-shaped, coccoid and transitional forms between them). The polymorphism of cells in a population gives it additional resistance to adverse environmental factors, i.e. provides longer shelf life of the drug bifidobacteria in liquid form without loss of activity.

In this medium, an enzymatic autolysate of yeast was used as a bifid-stimulating additive, which increases the content of B vitamins in the medium, and is also the only source of nitrogen nucleotide bases ready for assimilation, which are necessary for the rapid synthesis of bifidobacteria nucleic acids 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 enzymatic autolysate.

According to the data presented below in table 1, we can conclude that the best consortium corresponds to option No. 8, containing 3 strains: B.bif. 791 BAG (as the basis of the consortium), B. bif 8-3 and B. long. DVA-13 in a ratio of 8: 1: 1.

In this regard, option 8 of the consortium was adopted for patenting.

The study of the antagonistic activity of the proposed consortium of strains of bifidobacteria

The antagonistic activity of the consortium (table 2) of strains of bifidobacteria was studied in vitro by co-cultivation with pathogenic test strains in the zone of growth inhibition of test microbes by the barcode method.

table 2
Inhibition of the growth of pathogenic and opportunistic microorganisms Name of the test product Zones of growth inhibition of test cultures (mm) Sh. sonne Salmonella typhim Esherichia colli Candida albicans Liquid concentrate of bifidobacteria based on the inventive consortium 21.0-23.0 19.0-20.0 22.0-24.0 19.0-20.0

The antagonistic activity of the bifidobacteria of the consortium and its colonization activity was most fully studied in vivo in the organism-population model - laboratory mice in a chronic experiment.

- Studies were conducted on tribal mice of both sexes of the ICR colony for 3 years on 6 generations of mice in the amount of 900 females and 450 males.

- A liquid concentrate of bifidobacteria was produced on the basis of a consortium of strains of bifidobacteria on a dairy medium with the addition of growth factors.

- Bifidobacterium concentrate was used at a dose of 0.1 ml per head per day for 1 month daily.

- Bacteriological studies of feces of mice were carried out in accordance with the guidelines "Intestinal dysbiosis and methods for its laboratory diagnosis."

- Surveillance of survival, weight of mice was carried out.

- Based on the data on the number of corpus luteum and implantation sites of living and dead embryos, the post- and preimplantation mortality of the embryos was determined.

The results of a bacteriological study of young generation 1, received from parents who received a liquid concentrate of bifidobacteria, showed that there was not only a significant decrease in the level of pathogenic and conditionally pathogenic microflora, but also an increase in bifidobacteria and lactobacilli almost equally from 10 ⁵ to 10 ⁸ - 10 ⁹ microbial bodies in 1 g of feces (Table 2).

Bacteriological studies of the feces of breeding young stocks of the next generations showed the presence of the formed barrier effect of gastro-intestinal microflora in relation to pathogenic microorganisms such as Salmonella, Staphylococcus aureus, Clostridium digficile, Candida, Proteus, etc. The earlier formation of the microflora of newborns through the correction of the microflora of the parents provided good body protection and the ability to resist infection from the outside.

The effect of a liquid concentrate of bifidobacteria on the fecundity of animals was investigated (Table 3). The litter of mice is determined by the number of ovulated eggs, the death of embryos before and after implantation. The death of embryos at an early stage before implantation can increase with an increase in the degree of inbreeding. Late embryonic mortality is an indicator of the state of the body and manifests itself in late pregnancy. As can be seen from Table 3, the intake of bifidobacteria concentrate based on a new consortium of strains led to the complete exclusion of late embryonic mortality after 3 years, whereas at the beginning of the experiments (1 year) it was 14.9%.

Analysis of tables 3 and 4 shows that the use of a liquid concentrate of bifidobacteria for 3 years, made on the basis of the inventive consortium of strains of bifidobacteria, led to the elimination of intestinal mice of pathogenic microflora and stable colonization of bifidobacteria, which ultimately significantly reduced mortality and late embryonic mortality in mice.

Examples of preferred implementation

Example 1. The use of a consortium of strains of bifidobacteria in the composition of the starter culture for the preparation of a liquid concentrate of bifidobacteria (JCB)

1st passage

Museum liquid cultures of B. bifidum 791 BAG, B. longum DVA-13, B. bifidum 8-3 strains in an amount of 1 ml are introduced into 2 tubes with 9 ml of hydrolyzate-milk or liver growth medium (for each strain). Strain the strains in test tubes separately for 15-18 hours at 37.5-38 ° C.

2nd passage

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

Then the biomass of starter cultures of each strain is mixed in the following ratio: 8 parts of B. bifidum 791 BAG biomass biomass, 1 part of B. longum TWA-13 biomass, 1 part of B. bifidum 8-3 biomass.

The resulting total biomass in the amount of 5-10% is introduced into the production special milk medium manufactured according to the patent of the Russian Federation No. 2169763 (the composition and characteristics of the medium are given above) in the volume of 10 liters and cultivated by thermostatic method 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 resulting biomass of the symbiotic association is lg 8-9 in 1 ml.

3rd passage

The resulting biomass of the production leaven of the consortium in the amount of not more than 5% is introduced into the production special milk medium in the amount of 10 liters and cultivated by thermostatic method in glass bottles at 37.5-38 ° C, without adjusting the process for acidity for 15-18 hours until clot. Get a liquid concentrate-starter culture of the bifidobacteria of the consortium (ZhKB), which can be used as a biologically active additive for independent use or for the enrichment of unfermented foods or feed additives in order to correct dysbacteriosis.

The content of probiotic microorganisms in the resulting biomass of the symbiotic association is lg 9-10 in 1 ml.

Example 2. The use of the consortium as a starter for the preparation of symbiotic fermented milk products of therapeutic and functional nutrition - Bifidokefira or Bifacil

The consortium sourdough 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 sourdough in the volumes of sourdoughs provided 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 lg 7-8 in 1 ml of a symbiotic product.

The resulting products have a pronounced immunocorrective effect.

Example 3. The use of a consortium of strains of bifidobacteria to obtain a bacterial preparation

To the grown biomass of the consortium of Example 1 (passage 3), protective drying media (sucrose 10% and gelatin 5%) are added. The resulting suspension is poured into 2 ml ampoules or 5 ml into vials, frozen at t = minus 40-45 ° C and lyophilized from a frozen state in vacuum dryers. The resulting preparation contains at least 10 ⁹ living bifidobacteria cells.

The production technology of the consortium as part of the products does not require expensive equipment (fermenters) and allows you to get large quantities of biomass concentrate of bifidoconsortium in a simple thermostatic way without pH correction and the use of gas exchange systems. At the same time, the concentration of bifidobacteria in the biomass of the concentrate of the bifidobacteria consortium reaches its maximum value of log 10. The acidity of the biomass of the concentrate of bifidobacteria even remains almost unchanged for 5 months when stored in a liquid state.

As a result, the proposed consortium is more technologically advanced and economical in comparison with other forms of consortia, and is very convenient in use, storage, and transportation.

Example 4. The 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 5). A significant period (up to 5 months) of storage of a liquid concentrate of bifidobacteria with a titer of at least 10 ⁸ CFU / ml is associated with ensuring the initial acidity of the preparation of a consortium of bifidobacteria not more than (140-145) ° T, obtaining the initial titer of bifidobacteria (not less than 10 ¹⁰ CFU / ml ) and the use of a nutrient medium according to the patent of the Russian Federation No. 2109054.

Table 5
Data on the safety of bifidobacteria in the inventive consortium in a liquid concentrate of bifidobacteria Sample No. ZhKB on the basis of the proposed consortium T ° initial T ° final
5 months Title, initial CFU / ml Caption after 4 months. CFU / ml Caption after 5 months. CFU / ml one 136 160 2 × 10 ¹⁰ 1 × 10 ¹⁰ 3 × 10 ⁸ 2 110 - 2 × 10 ¹⁰ 2 × 10 ⁹ 1 × 10 ⁸ 3 125 165 5 × 10 ¹⁰ 2 × 10 ⁹ 2 × 10 ⁸ four 130 157 4 × 10 ¹⁰ 4 × 10 ¹⁰ 7 × 10 ⁸ 5 140 167 2 × 10 ¹⁰ 3 × 10 ⁹ 3 × 10 ⁹ 6 145 170 4 × 10 ⁹ 2 × 10 ⁹ - 7 145 - 5 × 10 ⁹ 4 × 10 ⁹ - 8 143 152 5 × 10 ⁹ 6 × 10 ⁸ - 9 152 160 3 × 10 ¹⁰ 3 × 10 ⁹ - 10 135 - 6 × 10 ⁹ 1 × 10 ¹⁰ -

Example 5. The use of a consortium in the composition of a liquid concentrate of bifidobacteria for the correction of disorders of the intestinal biocenosis in viral hepatitis

Clinical testing was carried out in the clinic of the Department of Infectious Diseases of the Vladivostok State Medical University on the basis of the infectious diseases department of MSC fishermen.

Currently, a violation of intestinal biocenosis is considered as one of the factors determining the occurrence of chronic inflammatory processes in both the small and large intestines. At the same time, intestinal dysbiosis is a complication, manifestation or outcome of other diseases, among which are viral hepatitis. Intestinal dysbacteriosis adversely affects the metabolic processes in the body (on the synthesis of amino acids, the exchange of bile and fatty acids, vitamin-forming function, etc.), contributes to the allergization of the body. Therefore, the prevention and treatment of disorders of intestinal biocenosis is an object of special attention.

The liquid concentrate of the consortium of bifidobacteria is a microbial mass of live bifidobacteria, made according to example 1 and restores the normal composition of the intestinal microflora. Under observation were 26 patients aged 20 to 65 years, suffering from chronic hepatitis in combination with dysbacteriosis of 2-3 degrees. The comparison group consisted of 30 patients receiving conventional treatment for viral hepatitis.

The clinical symptoms of the disease before treatment were typical: discomfort in the middle and lower third of the abdomen, bloating and rumbling in the abdomen, stool disorders with a predominance of constipation. In the microbial composition of feces in the examined patients, the average number of bifidobacteria and lactobacilli was reduced and the number of conditionally pathogenic strains of Escherichia coli, Proteus and fungi was increased. Changes in the intestinal microflora in patients with viral hepatitis were detected regardless of the etiological factor and treatment methods and were recorded both in the period of jaundice and in the period of early convalescence. In addition, changes in the colon biocenosis persisted 2-3 months after discharge from the hospital. In some patients, these changes did not have clinical manifestations, in some cases flatulence, unstable stools, poor tolerance of coarse fiber and protein were noted.

The drug was prescribed 4 ml orally 20 minutes before meals in combination with enzymatic preparations, sorbents for 15 days.

The use of a liquid concentrate of a consortium of bifidobacteria in complex treatment had a positive effect on the course of the disease: intestinal function improved, discomfort, bloating and rumbling in the abdomen disappeared in 22 patients. In 5 patients, diarrhea stopped on the first day, in 18 patients constipation was stopped. In all patients observed, the composition of the colon microflora improved. By the end of treatment, in 24 patients, the number of anaerobic microorganisms of bifidobacteria and lactobacilli significantly increased, the number of opportunistic microorganisms decreased.

Thus, for the correction of dysbiotic disorders in the clinic of viral hepatitis, it is advisable and pathogenetically justified to prescribe a probiotic liquid concentrate of a consortium of bifidobacteria.

Industrial applicability

The above examples show that the proposed consortium of strains of four types of bifidobacteria actively propagates in nutrient media with the accumulation of industrial biomass in a short period of cultivation. The product ferments milk with the formation of a clot with good taste and organoleptic properties; has specific activity. A consortium of strains can be recommended as part of starter cultures for the preparation of a liquid concentrate of bifidobacteria, sour-milk therapeutic and dietary products, unfermented food products and bacterial preparations.

The description of the invention sets forth the entire process for obtaining a consortium. Used media and reagents are publicly available. Tools and techniques for obtaining and growing the biomass of the consortium, including in the form of laboratory and industrial starter cultures, as well as tools and techniques for freezing and lyophilization of a dry bacterial preparation, are traditionally used in the medical industry and biotechnology.

Claims (2)

1. A consortium of strains of bifidobacteria: Bifidobacterium bifidum No. 791BAG, a collection of microorganism cultures of the Federal State Institution Scientific Center for Science and Technology “Vector”, collection number B-686, Bifidobacterium bifidum 8-3, VKPM B-5799, Bifidobacterium longum DVA-13, VKPM used to obtain a liquid concentrate of bifidobacteria, dairy, non-fermented therapeutic and prophylactic products, dietary supplements and bacterial preparations. 2. The consortium according to claim 1, characterized in that it contains the above strains in a ratio of 8: 1: 1, respectively.