RU2375444C1 - Bifidobacterium bifidum OV-19 STRAIN USED TO PREPARE BACTERIAL PREPARATIONS, BIOLOGICALLY ACTIVE FOOD ADDITIVES, FERMENTED AND UNFERMENTED FOODSTUFF, HYGIENIC AND COSMETIC PRODUCTS - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: Bifidobacterium bifidum OV-19 strain is recovered from gut contents of a breast-fed infant, deposited and stored in the State Collection of Microorganisms of Normal Microflora G.N.Gabrichevsky Moscow Scientific Research Institute of Epidemiology and Microbiology of Federal Supervision Agency for Customer Protection and Human Welfare under No. 216. The strain is advanced, accumulates biomass on nutrient mediums in a relatively short time with high concentration of bifidus bacteria, manifests acid-forming and antagonistic properties with respect to pathogenic and opportunistic microflora, resists wide range of high-acid antibiotics. It is used to sour milk herewith forming an even clot. It is nontoxic.

EFFECT: invention allows using B bifidum OV-19 strain in manufacturing of bacterial preparations, biologically active food additives, fermented and unfermented foodstuff, hygienic and cosmetic products ensuring probiotic effect and microbiocoenosis normalisation in a human body, including gastroenteric and urogenital tracts, skin and mucous covers and extends the range of products 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, fermented and non-fermented 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.

At the same time, the rationalization of the approach to the design and application of new microflora correction tools should be based on the laws associated with the stabilization of normobiocenosis, in particular, take into account the phenomenon of autogenous succession - a successive change in microbiocenoses during ontogenesis, as a result of which some species are replaced microorganisms by others due to their bioecological benefits under the given conditions. In this case, a succession series is formed - a series with the final formation of a relatively stable biocenosis. This pattern can be seen in the change in the species composition of bifidoflora during ontogenesis, which coincides with critical periods of development in childhood and adolescence; at the same time, in adults, the composition of bifidoflora is relatively stable [8].

Studies of the species composition of bifidoflora, previously carried out using biochemical methods of identification, led to the conclusion that bifidobacteria in the human body are represented by five main 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; B. adolescentis species 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, 9].

Species of B. catenulatum, B. pseudocatenulatum, B. dentium, B. animalis, B. angulatum are characteristic of microbiocenosis of young children. At the same time, evidence has already been confirmed at a new methodological level that the species B. bifidum and B. longum remain the dominant representatives of human bifidoflora in all age periods of life. This means that the optimal formula of a probiotic agent should include both the “mobile” part that changes during ontogenesis, which is complementary to the microbiocenosis of a certain age group of people, and the “conservative” one, which consists of species that are stably preserved during ontogenesis. In this regard, the selection of promising strains of bifidobacteria, belonging to the species B. bifidum and B. longum, as universal and most common, is of particular interest in the creation of microflora correction tools.

The objective of the present invention is to obtain a strain of B. bifidum OV-19 with antagonistic activity, resistance to external influences and antibacterial therapy, high kinetics of biomass accumulation on nutrient media in industrial cultivation.

Strain B. bifidum OV-19 was isolated from the intestinal contents of a healthy infant who was breastfed and deposited in the State collection of microorganisms of normal microflora. G.N.Gabrichevsky Rospotrebnadzor under No. 216.

The inventive strain can be used in the production of bacterial preparations in various forms (capsules, tablets, suspensions, suppositories, aerosols), biologically active food additives, fermented and non-fermented foods, including baby food, juices, drinks, etc. Also, the strain B. bifidum OV-19 can be used in the production of hygiene and cosmetics, for example creams, gels, and cosmetic masks.

The technical result of the claimed invention is to obtain a strain of B. bifidum OV-19, which can be used as a biologically active principle of probiotic orientation in the composition of bacterial preparations, biologically active additives, fermented and non-fermented foods, hygiene and cosmetics. The strain B. bifidum OV-19 in the course of the technological process on production media in a short time accumulates biomass with a high content of bifidobacteria, has antagonistic activity against pathogenic and opportunistic microorganisms, a wide spectrum of resistance to antibacterial agents, acidic environment, is non-toxic, which provides 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 correcting 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 modern man.

The inventive strain has the following properties.

Morphological properties

The cells of strain B. bifidum OV-19 are Gram + polymorphic, undisputed motionless thin, slightly curved sticks with thickenings at both ends, located singly, in pairs at an angle of 60 ° to each other or in the form of a tetrahedron.

In semi-liquid nutrient media, cells grow throughout the entire volume except the aerobiosis zone; separately grown colonies after 16-24 hours of growth have the form of carnations.

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 3.5-4.0. Ferments galactose, esculin, raffinose, arabinose, xylose. Does not ferment maltose, fructose, lactose, melecitose, sucrose, salicin, trehalose, mannitol, rhamnose, mannose, cellobiose, sorbitol. For urease, nitrates and indole - the test is negative.

Genotyping of B. bifidum OV-19 strain was carried out on the basis of amplification, cloning, and subsequent sequencing of gene fragments encoding 16S ribosomal RNA and transaldolase. The read DNA sequences are registered in GenBank under the numbers EU 272013 and EU 272019.

Cultural properties

The strain actively accumulates biomass in various production environments 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 B. bifidum OV-19 strain on different growth media Culture medium CFU / ml ⁰ T The medium for the cultivation of bifidobacteria (BS) 5.0 × 10 ⁹ 174 IFA 5.6 × 10 ⁹ 205 Hydrolyzate-milk environment 6.0 × 10 ⁹ 210 Hepatic environment 5.8 × 10 ⁹ 210 Skimmed reconstituted milk 5.0 × 10 ⁸ 90

The data presented in table 1 show that the strain B. bifidum OV-19 accumulates biomass of bifidobacteria on production media with a CFU / ml content of at least lg 9. Moreover, high titratable acidity values are also noted, which indicates an active accumulation of organic acids. Milk fermented by 18 hours of cultivation with the formation of an even serum-free clot with a sour-milk smell and taste. When cultivated in milk, the acidity is not more than 90 ° T.

Table 2 shows the cultivation of strain B. bifidum OV-19 for 96 hours in skim milk and bifidum medium.

Table 2. The cultivation of strain B. bifidum OV-19 for 96 hours Growth time after sowing, (h) Cultivation on milk Growing on BS environment ⁰ T CFU / ml ⁰ T CFU / ml 0 eighteen 5.0 × 10 ⁶ 34 2.1 × 10 ⁶ 24 85 2.0 × 10 ⁸ 168 5.0 × 10 ⁹ 36 90 2.0 × 10 ⁸ 178 4.6 × 10 ⁹ 48 90 2.1 × 10 ⁸ 188 4.9 × 10 ⁹ 72 96 1.8 × 10 ⁸ 195 4.8 × 10 ⁹ 96 1.8 × 10 ⁸ 201 4.8 × 10 ⁹

The data presented in table 2 demonstrate that during prolonged cultivation of strain B. bifidum OV-19 with an increase in the content of organic acids in the culture fluid and skim milk, there is no decrease in the content of viable bifidobacteria cells, which indicates a high stability of strain B bifidum OV-19 to acidic environment.

Thus, it can be seen from the above data that the strain B. bifidum OV-19 is highly adaptable; when cultured on production media, it accumulates biomass of bifidobacteria and organic acids in a short time and with high rates. Resistant to acidic environment. It ferments milk, which makes it possible to manufacture fermented dairy products on its basis with valuable therapeutic and organoleptic properties.

The antagonistic activity of strain B. bifidum OV-19 was studied by the method of delayed antagonism with test cultures of opportunistic and pathogenic microorganisms (table 3).

Table 3. Antagonistic activity of strain B. bifidum OV-19 No. The strain of the test microorganism The zone of growth retardation in the presence of strain B. bifidum OV-19, mm one Staphylococcus aureus ATCC 653 8-P 15 ± 4 2 Shigella flexneri 337 19 ± 3 3 Shigella sonnei 177 18 ± 5 four Proteus vulgaris 1274 18 ± 3 5 Escherichia coli 157 12 ± 3

The results presented in table 3 showed that when applying test cultures to the daily culture of strain B. bifidum OV-19 on an agar medium (perpendicular stroke method), their growth is inhibited. The size of the growth inhibition zones indicates that B. bifidum OV-19 strain exhibits high antagonistic properties with respect to the tested cultures.

Sensitivity to antibiotics of the strain B. bifidum OV-19 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. bifidum OV-19 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 30 ± 2 2 Oxacillin ≤10 11-13 ≥14 10 15 ± 0 3 Ampicillin ≤10 11-14 ≥16 10 25 ± 2 four Carbenicillin ≤14 15-18 ≥19 25 40 ± 0 5 Azlocillin ≤13 14-16 ≥16 75 28 ± 2 6 Cephalotin ≤14 15-18 ≥19 thirty 36 ± 2 7 Cephalexin ≤14 15-18 ≥19 thirty 35 ± 0 8 Cefazolin ≤14 15-18 ≥19 thirty 25 ± 2 9 Ceftazidime ≤14 15-17 ≥18 thirty 25 ± 2 10 Cefotaxime ≤14 15-20 ≥21 thirty 35 ± 2 eleven Imipenem ≤13 14-15 ≥16 10 35 ± 0 12 Streptomycin ≤16 17-19 ≥20 thirty 15 ± 0 13 Neomycin ≤12 13-16 ≥17 thirty 10 ± 0 fourteen Kanamycin ≤14 15-18 ≥19 thirty 0 ± 0 fifteen Gentamicin ≤12 13-14 ≥15 10 8 ± 0 16 Amikacin ≤14 15-16 ≥17 thirty 0 ± 0 17 Tetracycline ≤16 17-21 ≥22 thirty 28 ± 0 eighteen Doxycycline ≤15 16-19 ≥20 10 35 ± 0 19 Erythromycin ≤17 18-21 ≥22 fifteen 30 ± 0 twenty Azithromycin ≤13 14-17 ≥18 fifteen 25 ± 0 21 Clarithromycin ≤13 14-17 ≥18 fifteen 35 ± 0 22 Roxithromycin ≤13 15-18 ≥19 thirty 35 ± 0 23 Lincomycin ≤19 20-23 ≥24 fifteen 35 ± 0 24 Clindamycin ≤14 15-20 ≥21 2 35 ± 2 25 Vancomycin ≤14 15-16 ≥17 thirty 25 ± 2 26 Chloramphenicol (Levomycetin) ≤15 16-18 ≥19 thirty 35 ± 2 27 Rifampicin ≤12 13-15 ≥16 5 35 ± 0 28 Polymyxin ≤11 12-14 ≥15 300ED 0 ± 0 29th Nalidixic acid ≤12 13-17 ≥18 thirty 8 ± 0 thirty Ciprofloxacin ≤15 16-20 ≥21 5 15 ± 3 31 Pefloxacin ≤14 15-21 ≥22 10 8 ± 0 32 Lomefloxacin ≤18 19-21 ≥22 10 20 ± 2

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

Harmlessness

In the experience of studying the acute toxicity of strain B. bifidum OV-19, its complete harmlessness was shown: when white mice were injected with per os microbial suspension of bifidobacteria in an 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 B. bifidum OV-19 strain is highly technological, actively increases the biomass of bifidobacteria on production media with a bifidobacterium content of at least 10 ⁹ CFU / ml, forms organic acids during cultivation, and has high antagonistic activity in relation to opportunistic and pathogenic microorganisms, resistance to a wide range of antibiotics, resistant to high acidity. Fermenting milk with the formation of a clot. Harmless when administered to laboratory animals.

The presented data on the study of strain B. bifidum OV-19 allow us to conclude that it is promising when used as a basis for bacterial preparations, dietary supplements, fermented and non-fermented foods, as well as hygiene and cosmetic products.

Example 1. The use of strain B. bifidum OV-19 to obtain a biologically active food supplement in liquid form.

1st passage: lyophilized starter culture of strain B. bifidum OV-19 is rehydrated with cultivation medium or physiological saline in 1 ml, and titrated by ten-fold dilutions 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 bifidobacteria content of at least 10 ⁹ CFU / ml, which can be directly used as a biologically active therapeutic and prophylactic additive. The finished product is poured into 100 ml vials and hermetically sealed.

Example 2. The use of strain B. bifidum OV-19 as an ingredient in an unfermented food product.

1st passage: lyophilized starter culture of strain B. bifidum OV-19 is rehydrated with culture medium or 1 ml physiological saline and titrated by 10-fold dilutions 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 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. bifidum OV-19 to obtain a bacterial preparation.

1st passage: lyophilized starter culture of strain B. bifidum OV-19 is rehydrated with culture medium or 1 ml saline and titrated by ten-fold dilutions 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 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 ⁹ CFU / ml bifidobacteria.

Sugar-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 in 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. bifidum OV-19 to obtain an 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 ⁸ CFU of viable bifidobacteria.

Example 5. The use of strain B. bifidum OV-19 to obtain hygiene and cosmetics.

Dry biomass of bifidobacteria strain B. bifidum OV-19 with a bifidobacteria content 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.

Example 6. The use of strain B. bifidum OV-19 to obtain a dairy product.

1st passage: lyophilized starter culture of strain B. bifidum OV-19 is rehydrated with culture medium or physiological saline in a volume of 1 ml and titrated by ten-fold dilution in nutrient medium 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 not more than 5% and incubated at 37-38 ° C (industrial leaven).

4th passage: production ferment is introduced into sterile skim milk in an amount of 5% of its volume and cultured at 37-38 ° C until ripening (within 16-18 hours). The finished product is cooled and poured into 0.2 l in plastic cups and corked with metal foil lids. The product contains at least 10 ⁸ CFU / ml bifidobacteria.

Bibliography

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 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, pp. 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 for the 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.

8. Sokolova K.Ya., Solovieva I.V. / Dysbacteriosis. Theory and practice. N. Novgorod, 1999, 198 p.

9. Shkoporov A.N., Efimov B.A., Volodin N.N., Kafarskaya L.I. / Bifidobacteria: a traditional view and modern genetic research. The journal "Issues of Practical Pediatrics", 2007, v.2, No. 5, pp. 76-79.

Claims (1)

The strain Bifidobacterium bifidum OV-19 collection number No. 216, is stored in the State collection of microorganisms of normal microflora FSUE MNIIEM them. G.N.Gabrichevsky Rospotrebnadzor used to obtain bacterial preparations, dietary supplements, fermented and non-fermented foods, hygiene and cosmetics.