RU2373277C1 - Bifidobacterium angulatum ov-15 strain, used for making bacterial preparations, bioligically active additives to food, unfermented food products, hygienic and cosmetic agents - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: Bifidobacterium angulatum OV-15 strain is extracted from intestine contents of a healthy nursing infant, deposited and stored at the State collection of microorganisms normal microflora of the Federal State Research Institution Moscow G. N. Gabrichevskii Research Institute of Epidemiology and Microbiology Rospotrebnadzor under No. 212. The strain suits modern requirements, accumulates biomass on culture media in short periods with high concentration of bifidobacteria, has acid-forming and antagonist properties towards pathogenic and opportunistic microflora, is resistant to a wide range of antibiotics, acidic medium and is not toxic. This allows for using B. angulatum OV-15 strain for making bacterial preparations, biologically active additive to food, unfermented food products, hygienic and cosmetic agents.

EFFECT: provision for probiotic effect and normalisation of microbiocenoses of the human body, including gastrointestinal and urogenital tracts, skin and mucous coatings, as well as wider range of agents for correcting microflora of the human body.

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Description

The invention relates to biotechnology, food, cosmetic and medical industries and can be used in the production of bacterial preparations, dietary supplements, unfermented foods, hygiene and cosmetics.

Means containing bifidobacteria with probiotic properties are intended to normalize the microflora of various biotopes of the human body damaged by the adverse effects of environmental factors, diseases, long-term use of antibiotics and other drugs, radiation and chemotherapy and many other reasons [1, 4 ].

The effectiveness of medicinal products and biological products depends primarily on the characteristics of the producer strain, its technological properties and specific activity. A large assortment of production strains possessing valuable biomedical properties makes it possible to design new microflora correction tools, both mono-and multicomponent, capable of providing high colonization resistance of the microbiocenosis of the gastrointestinal tract, skin, urogenital area and eliminate dysbiotic disorders.

Studies of the species composition of bifidoflora, previously carried out using biochemical identification methods, have led to the conclusion that bifidobacteria in the human body are represented by five species - Bifidobacterium bifidum, B. longum, B. adolescentis, B. breve and B. infantis (RF patents 1347221 , 849779, 2108383, 2152993, 863639, 2120991, 2078814, 2111248). Moreover, the analysis of the data showed that the species B. bifidum and B. longum are detected in healthy people of all age groups, but with age, their share in the composition of microbiocenosis gradually decreases. The species B. adolescentis is peculiar only to adults and older children; in older people it becomes predominant. Species of B. infantis and B. breve are found only in infants [3, 6].

DNA analysis, begun in the 70s, made it possible to identify new species of bifidobacteria (B. catenulatum, B. pseudocatenulatum, B. dentium, B. animalis, B. angulatum.) And expand ideas about the species composition of human bifidoblora and the laws of its formation [ 2, 5, 8].

This gives reason to believe that when designing and assigning microflora correction means, it is necessary to take into account the biodiversity of the species composition of bifidoflora. The inclusion of new production strains of bifidobacteria will expand the nomenclature and increase the effectiveness of the developed bifidobacteria for the correction of microecological disturbances in various biotopes and will allow these measures to be carried out more rationally.

The objective of the present invention is to obtain a strain of Bifidobacterium angulatum OV-15 with high biological activity, resistance to external influences and antibacterial therapy, high kinetics of biomass accumulation on nutrient media in industrial cultivation.

The strain B. angulatum OV-15 was isolated from the intestinal contents of a healthy infant who was breastfed, and deposited in the State collection of microorganisms of normal microflora FSUE MNIIEM them. G.N.Gabrichevsky Rospotrebnadzor under No. 212.

The inventive strain can be used in the production of bacterial preparations in various forms (capsules, tablets, suspensions, suppositories, aerosols), biologically active food additives, unfermented food products, including baby food, juices, drinks, etc. Also, the strain B. angulatum OV-15 can be used in the production of hygiene and cosmetics, for example creams, gels, cosmetic masks.

The technical result of the claimed invention is to obtain a strain of B. angulatum OV-15, which can be used as a biologically active principle of probiotic orientation in the composition of bacterial preparations, biologically active additives, unfermented foods, hygiene and cosmetics.

The strain B. angulatum OV-15 during the process in a short time on production media accumulates biomass with a high content of bifidobacteria, has antagonistic activity against pathogenic and opportunistic microorganisms, a wide range of resistance to antibacterial agents, to an acidic environment, is non-toxic, which ensures active colonization of the biotope and restoration of resident microflora.

The use of this strain will expand the nomenclature of the developed bifid-containing means for the correction of human microflora and will create a formula for the symbiotic association of bifidobacteria species, which is closest to the composition of the intestinal microbiocenosis of a modern healthy person.

The inventive strain has the following properties.

Morphological properties

The cells of the strain Bifidobacterium angulatum OV-15 are Gram + polymorphic, non-spore motionless rods with thickenings at both ends. Cells form clusters in the form of Chinese characters, are rarely found alone.

In semi-liquid nutrient media it grows throughout the entire volume except the aerobiosis zone; separately grown culture colonies after 16-24 hours of growth are in the form of a drop 3-5 mm long.

Physiological and biochemical properties

Obligate anaerobic. The optimum temperature of cultivation is 37-38 ° C. Catalase does not form, gelatin does not thin. It ferments glucose to form acetic and L + lactic acid without gas formation, acidifying the medium to pH 3.8-4.1. It ferments lactose, maltose, sucrose, salicin, esculin, galactose, melecitose, raffinose, celobiose, salicin, selectively trehalose, rhamnose, xylose, rhamnose, does not ferment fructose, mannitol, mannose, sorbitol. On urease, the indole reaction is negative.

Genotyping of the strain Bifidobacterium angulatum OV-15 was carried out on the basis of amplification, cloning and subsequent sequencing of fragments of genes encoding 16S ribosomal RNA and transaldolase. The read DNA sequences are registered in GenBank under the numbers EU 272012 and EU 272020

Cultural properties

The strain actively accumulates biomass on production media of various compositions to high levels of CFU / ml of bifidobacteria and organic acids (table 1). The logarithmic growth phase ends at 16-24 hours.

Table 1 The content of bifidobacteria and organic acids during the cultivation of strain B. angulatum OV-15 on different nutrient media Culture medium CFU / ml ° T Cultivation medium 5.0 × 10 ⁹ 134 bifidobacteria (EU) IFA 3.0 × l0 ⁹ 185 Hydrolyzate-milk environment 1.2 × 10 ⁹ 180 Hepatic environment 1.8 × 10 ⁹ 186 Skimmed reconstituted milk 5.0 × 10 ⁸ fifty

The data presented in table 1 show that the strain B. angulatum OV-15 accumulates biomass of bifidobacteria on the production media with a content of not less than 10 ⁹ CFU / ml and organic acids are valuable metabolic products, which are mainly associated with the level of antagonistic activity of the strain . Milk does not ferment; when cultured in milk, the acidity is not more than 50 ° T.

Table 2 presents the cultivation of strain B. angulatum OV-15 for 96 hours in skim milk and bifidum medium.

table 2 Indicators of cultivation of strain B. angulatum OV-15 for 96 hours Growth time after sowing, h Cultivation on milk Growing on BS environment ° T CFU / ml ° T CFU / ml 0 eighteen 5.0 × 10 ⁶ 44 2.1 × 10 ⁶ 24 46 4,5 × 10 ⁸ 120 5.0 × 10 ⁹ 36 48 4.0 × 10 ⁸ 124 5.0 × 10 ⁹ 48 49 5.0 × 10 ⁸ 128 4,5 × 10 ⁹ 72 fifty 5.0 × 10 ⁸ 134 4.0 × 10 ⁹ 96 fifty 5.0 × 10 ⁸ 136 4.0 × 10 ⁹

The data presented in table 2 demonstrate that during prolonged cultivation of strain B. angulatum OV-15 with an increase in the content of organic acids in the culture fluid, the content of viable bifidobacteria cells does not decrease, which indicates its high resistance to an acidic environment.

Thus, it can be seen from the above data that the strain B.angulatum OV-15 is highly adaptable; when cultivated on production media, it accumulates biomass of bifidobacteria and organic acids in a short time and with high rates. Resistant to acidic environment.

The antagonistic activity of the strain was studied by the method of delayed antagonism with test cultures of pathogenic and conditionally pathogenic microorganisms (table 3).

Table 3 Antagonistic activity of strain B. angulatum OV-15 No. The strain of the test microorganism The zone of growth retardation of pathogenic strains in the presence of B. angulatum OV-15, mm one Staphylococcus aureus ATCC 653 8-P 26 ± 3 2 Shigella flexneri 337 30 ± 2 3 Shigella sonnei 177 31 ± 3 four Proteus vulgaris 1274 32 ± 3 5 Escherichia coli 157 26 ± 4

The results presented in table 3 showed that when applying test cultures to the daily culture of strain B. angulatum OV-15 grown on an agar medium (perpendicular strokes method), their growth is inhibited. The size of the zones of growth retardation indicates that the strain exhibits high antagonistic properties in relation to the tested cultures.

Sensitivity to antibiotics of the strain B. angulatum OV-15 was studied on a model of 32 antimicrobial drugs used in medical practice. The study was carried out by the disk-diffusion method in the interpretation developed in the laboratory of biology of bifidobacteria of the Federal State Medical Research Institute named after G. N. Gabrichevsky, taking into account the recommendations of the MUK 4.2.1890-04 [7]. The data obtained are presented in table 4.

Table 4 Antibiotic sensitivity of strain B. angulatum OV-15 No. Name of antibiotic Interpretation of zones, mm The content of active substance in the disk, mcg The diameter of the zone of growth inhibition, mm Steady Intermediate resistant Sensitive one Benzylpenicillin ≤19 20-27 ≥28 10 28 ± 2 2 Oxacillin ≤10 11-13 ≥14 10 12 ± 0 3 Ampicillin ≤10 11-14 ≥16 10 22 ± 2 four Carbenicillin ≤14 15-18 ≥19 25 26 ± 1 5 Azlocillin ≤13 14-16 ≥16 75 26 ± 2 6 Cephalotin ≤14 15-18 ≥19 thirty 28 ± 2 7 Cephalexin ≤14 15-18 ≥19 thirty 21 ± 2 8 Cefazolin ≤14 15-18 ≥19 thirty 16 ± 2 9 Ceftazidime ≤14 15-17 ≥18 thirty 20 ± 2 10 Cefotaxime ≤14 15-20 ≥21 thirty 25 ± 0 eleven Imipenem ≤13 14-15 ≥16 10 23 ± 1 12 Streptomycin ≤16 17-19 ≥20 thirty 8 ± 0 13 Neomycin ≤12 13-16 ≥17 thirty 0 ± 0 fourteen Kanamycin ≤14 15-18 ≥19 thirty 0 ± 0 fifteen Gentamicin ≤12 13-14 ≥15 10 0 ± 0 16 Amikacin ≤14 15-16 ≥17 thirty 0 ± 0 17 Tetracycline ≤16 17-21 ≥22 thirty 28 ± 2 eighteen Doxycycline ≤15 16-19 ≥20 10 25 ± 2 19 Erythromycin ≤17 18-21 ≥22 fifteen 25 ± 0 twenty Azithromycin ≤13 14-17 ≥18 fifteen 17 ± 1 21 Clarithromycin ≤13 14-17 ≥18 fifteen 27 ± 2 22 Roxithromycin ≤13 15-18 ≥19 thirty 25 ± 0 23 Lincomycin ≤19 20-23 ≥24 fifteen 25 ± 0 24 Clindamycin ≤14 15-20 ≥21 2 28 ± 0 25 Vancomycin ≤14 15-16 ≥17 thirty 20 ± 0 26 Chloramphenicol (Levomycetin) ≤15 16-18 ≥19 thirty 34 ± 1 27 Rifampicin ≤12 13-15 ≥16 5 34 ± 1 28 Polymyxin ≤11 12-14 ≥15 300ED 0 ± 0 29th Nalidixic acid ≤12 13-17 ≥18 thirty 0 ± 0 thirty Ciprofloxacin ≤15 16-20 ≥21 5 10 ± 0 31 Pefloxacin ≤14 15-21 ≥22 10 0 ± 0 32 Lomefloxacin ≤18 19-21 ≥22 10 10 ± 0

As can be seen from table 4, the strain is resistant to neomycin, kanamycin, gentamicin, polymyxin, amikacin, pefloxacin, nalidixic acid, ciprofloxacin, lomefloxacin, streptomycin, is intermediate resistant to oxacillin, cefazolin, azithromycin. It is sensitive to other antibiotics used in the study.

Harmlessness

In the study of acute toxicity of the strain Bifidobacterium angulatum OV-15, its complete harmlessness was shown: when white mice were introduced per os microbial suspension of bifidobacteria in the amount of 5 × 10 ⁷ CFU per animal, the death of mice was not observed.

Thus, it can be seen from the data that the new strain of B. angulatum OV-15 is highly adaptable, actively increases the biomass of bifidobacteria at least 10 ⁹ CFU / ml, forms organic acids during cultivation, and has a high antagonistic activity against opportunistic and pathogenic microorganisms, resistant to a wide range of antibiotics, and to the increased acidity of culture media. Harmless when administered to laboratory animals.

The presented data on the study of the strain B. angulatum OV-15 allow us to conclude that it is promising when used as a basis for bacterial preparations, biologically active food additives, unfermented food products, hygiene and cosmetic products.

Example 1. The use of strain B. angulatum OV-15 to obtain a biologically active food supplement in liquid form.

1st passage: lyophilized starter culture of strain B. angulatum OV is rehydrated with culture medium or 1 ml saline, and titrated by 10-fold dilution in nutrient medium up to 10 ^7-8 times. Incubated at a temperature of 37-38 ° C for 48 hours.

2nd passage: the grown culture from a dilution of 10 ^6-8 times in a volume of 5% is introduced into the accumulation medium (1 L) and cultured for 16-24 hours at 37-38 ° C (laboratory sourdough).

3rd passage: laboratory leaven is introduced into the accumulation medium (20 liters) in a volume of 5% and incubated at 37-38 ° C (industrial leaven).

4th passage: production ferment is introduced in the amount of 3-5% into the apparatus for cultivation with a sterile hydrolyzate-milk medium of the following composition (per 1 liter of medium): milk hydrolyzate - 0.5 l, peptone - 2 g, lactose - 10 g, sodium chloride - 5 g, cysteine - 0.2 g, agar-agar - 1 g, distilled water - 0.5 liters; pH of the medium is 7.0-7.2. Cultivation is carried out for 16-24 hours at a temperature of 37-38 ° C. The grown biomass is a liquid concentrate of bifidobacteria with a content of bifidobacteria of at least 10 ⁹ CFU / ml, which can be directly used as a biologically active therapeutic and preventive supplement. The finished product is poured into 100 ml vials and hermetically sealed.

Example 2. The use of strain B. angulatum OV-15 as an ingredient in an unfermented food product.

1st passage: lyophilized starter culture of strain B. angulatum OV-15 is rehydrated with cultivation medium or physiological saline in 1 ml, and titrated by ten-fold dilution in nutrient medium up to 10 ^7-8 times. Incubated at a temperature of 37-38 ° C for 48 hours.

2nd passage: the grown culture from a dilution of 10 ^6-8 times in a volume of 5% is introduced into the accumulation medium (0.2 L) and cultured for 16-24 hours at 37-38 ° C (laboratory leaven).

3rd passage: laboratory leaven is introduced into the accumulation medium (4 L) in a volume of 5% and incubated at 37-38 ° C (industrial leaven).

4th passage: production ferment is introduced into the hydrolyzate-milk environment in an amount of 5% by volume of the medium and cultured at 37-38 ° C for 24 hours. The finished biomass contains at least 10 ⁹ CFU / ml of bifidobacteria.

Ready biomass is used to enrich food products. It is introduced into milk, juices, baby food, ice cream and other products, mixed and poured or Packed in containers, receiving a product with a concentration of bifidobacteria 10 ^6-7 CFU / ml or CFU / g

Example 3. The use of strain B. angulatum OV-15 to obtain a bacterial preparation.

1st passage: lyophilized starter culture of strain B. angulatum OV-15 is rehydrated with culture medium or 1 ml saline and titrated by ten-fold dilution in nutrient medium up to 10 ^7-8 times. Incubated at a temperature of 37-38 ° C for 48 hours.

2nd passage: the grown culture from a dilution of 10 ^6-8 times in a volume of 5% is introduced into the accumulation medium (0.2 L) and cultured for 16-24 hours at 37-38 C (laboratory souring).

3rd passage: laboratory leaven is introduced into the accumulation medium (4 L) in a volume of not more than 5% and incubated at 37-38 ° C (industrial leaven).

4th passage: production ferment is introduced into casein-yeast medium in an amount of 5% by volume of the medium and cultured at 37-38 ° C for 24 hours. The finished biomass contains at least 10 CFU / ml of bifidobacteria.

A sucrose-gelatin protective drying environment in the amount of 10-15% and 15-20% skim milk is added to the finished biomass. The resulting suspension is poured into 5 ml into penicillin vials, frozen at -40-45 ° C and lyophilized from a frozen state in vacuum dryers. The resulting preparation has at least 10 ⁸ CFU / g of living bifidobacteria cells.

Example 4. The use of strain B. angulatum OV-15 to obtain the encapsulated form of a biologically active food supplement.

For encapsulation, a mixture is prepared that includes the following ingredients: 1 part dry biomass containing at least 10 ⁹ CFU / g bifidobacteria, 6 parts inulin and 3 parts apple pectin. To the mixture, based on the resulting mass, add 0.5% aerosil and 1% ascorbic acid. After thorough mixing, they are encapsulated in capsules No. 0 of 0.4 g each. 1 g of dietary supplement contains at least 10 CFU of viable bifidobacteria.

Example 5. The use of strain B. angulatum OV-15 to obtain hygiene and cosmetics.

Dry biomass of bifidobacteria strain B. angulatum OV-15 with a content of bifidobacteria of at least 10 ⁹ CFU / g is introduced into the composition of the nutrient cream at the rate of 1 g per 100 g of cream.

Literature

1. Bondarenko V.M., Matsulevich T.V. Intestinal dysbiosis as a clinical and laboratory syndrome: current state of the problem. - M.: GEOTAR-Media, 2007, p. 300.

2. Voronina O. L., Subbotina M. E., Lunin V. G., Zhilenkova O. G., Amerkhanova A. M. / Differentiation of species of bifidobacteria based on sequencing of fragments of 16S rRNA and transaldolase genes. Scientific and practical journal "Clinical Nutrition", 2007, No. 1-2, p.31.

3. Goncharova G.I., Vilypanskaya F.L. - J. Microbiol., 1968, No. 7, p. 55.

4. Doronin A.F., Shenderov B.A. / Functional nutrition. -M .: Grant, 2002, p.295.

5. Karzanova M.E., Voronina O. L., Lunin V. G., Zhilenkova O. G., Amerkhanova A. M. / Determination of the species affiliation of bifidobacteria strains based on sequencing of fragments of 16S rRNA and transaldolase genes. - Scientific and theoretical journal "Reports of the Russian Academy of Agricultural Sciences", 2006, No. 5, p.9-12.

6. Kozlova EP, Kurnosova N.A., Feklisova L.V. / Theoretical and biomedical aspects of the use of various types of bifidobacteria as part of the means of prevention and treatment of intestinal dysbiosis. - Sat scientific works MNIIEM them. G.N. Gabrichevsky, 1986, p. 64-69.

7. Determination of the sensitivity of microorganisms to

antibacterial drugs. - MUK 4.2.1890-04, 2004., 52 p.

8. Shkoporov A.N., Efimov B.A., Volodin N.N., Kafarskaya L.I. / Bifidobacteria: a traditional view and modern genetic research. - The journal "Questions of practical pediatrics", 2007, v.2, No. 5, p.76-79.

Claims (1)

The strain Bifidobacterium angulatum OV-15, collection number No. 212, is stored in the State collection of microorganisms of normal microflora Federal State Medical Research Institute named after G.N.