RU2373274C1 - Bifidobacterium breve ov-12 strain, used for making bacterial preparations, bioligically active additives to food, fermented and unfermented food products, hygienic and cosmetic agents - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: Bifidobacterium breve OV-12 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.217. 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 and is not toxic.

EFFECT: possibility of using B brever OV-12 strain in making bacterial preparations, biologically active additives to food, fermented and unfermented food products, hygienic and cosmetic agents, which provide for probiotic effect and normalisation of microbiocenoses of the human body, including gastrointestinal and urogenital tracts, skin and mucous coatings, and also widens the range of similar agents.

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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 with 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 methods of identification, allowed us to conclude 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].

The use of modern approaches to the genotyping of bifidobacteria confirmed the presence of B. breve in the microflora of infants [2, 5, 6, 8].

The inclusion of new production strains of bifidobacteria will expand the range of developed bifidobacteria for the correction of microecological disturbances in various biotopes and will allow these measures to be carried out more rationally.

The aim of the present invention is a strain of Bifidobacterium breve OV-12, which is isolated from the intestines of a healthy infant, breastfed, and deposited in the State collection of microorganisms of normal microflora FGUN MNIIEM them. G. N. Gabrichevsky Rospotrebnadzor under the number 217.

The inventive strain can be used in the production of bacterial preparations in various forms, including dietary supplements, capsules, tablets, suspensions, suppositories; fermented and unfermented foods, including baby food, juices, drinks, etc. Also, strain B. breve OV-12 can be used in the production of hygiene products, for example creams, gels, cosmetic masks.

The technical result of the claimed invention is the production of bacterial preparations, biologically active food additives, fermented and non-fermented foods, hygienic and cosmetic products, which contain the strain B. breve OV-12 as an active principle of probiotic orientation, during the technological process on production media in short terms accumulating biomass with a high content of bifidobacteria, which has antagonistic activity against pathogenic and clearly pathogenic microorganisms, broad spectrum resistance to antibacterial agents, organic acids, as well as external influences that provides active colonization biotope and restoring the 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 strains of bifidobacteria, 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 strain B. breve OV-12 are Gram + polymorphic, non-spore motionless large sticks with bifurcation at one end, arranged in pairs crosswise in the form of a chromosome, the letters X, T, “snail’s horn”.

In semi-liquid nutrient media, cells grow throughout the entire volume except the aerobiosis zone; separately grown colonies look like “comets” in the form of fir-trees up to 1 cm long. The logarithmic growth phase ends by 16-24 hours.

Physiological and biochemical properties

Obligate anaerobic. The optimum cultivation temperature is 37.5-38 ° С. 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 3.9-4.1. It ferments maltose, fructose, galactose, lactose, melecitose, sucrose, mannose, variably ferments raffinose, xylose, arabinose. Does not ferment salicin, trehalose, rhamnose, mannose, esculin, cellobiose, sorbitol. On urease, nitrates and indole, the reaction is negative.

Genotyping of strain B. breve OV-12 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 272015 and EU 272021.

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 accumulation of biomass and organic acids during the cultivation of strain B. breve OV-12 on various nutrient media. Culture medium CFU / ml ° T The medium for the cultivation of bifidobacteria (BS) 5.0 × 10 ⁹ 178 IFA 5.6 × 10 ⁹ 205 Hydrolyzate-milk environment 6.0 × 10 ⁹ 210 Hepatic environment 5.8 × 10 ⁹ 210 Skimmed reconstituted milk 2.0 × 10 ⁸ 135

The data presented in table 1 show that the strain B. breve OV-12 accumulates biomass of bifidobacteria on production media with a content of at least 10 ⁹ CFU / ml and organic acids are valuable metabolic products, with which, mainly, the level of antagonistic activity of the strain is associated . Milk ferments by 24 hours to 90 ° T with the formation of a dense clot. The maximum acidity in milk is 150 ° T.

Data from the study of the dynamics of the accumulation of microbial mass and organic acids (metabolic products) during cultivation are presented in table 2.

table 2 The dynamics of the cultivation of strain B. breve OV-12 in milk and medium for isolation and cultivation of bifidobacteria in stationary conditions Growth time after sowing, (h) Cultivation on milk Growing on BS environment ° T CFU / ml ° T CFU / ml 0 eighteen 1.2 × 10 ⁷ 34 2, lxl0 ⁶ 24 90 2.0 × 10 ⁸ 140 3,5x10 ⁹ 48 135 2.2 × 10 ⁸ 260 4.0x10 ⁹

As can be seen from table 2, in the process of cultivation there is a dynamic accumulation of microbial mass and organic acids. The maximum values of these indicators were observed at 24 hours of cultivation.

Thus, it can be seen from the above data that the strain B. breve OV-12 is highly technological, actively increasing the biomass of bifidobacteria on production and laboratory media with high values of CFU / ml.

The antagonistic activity of the strain was studied on solid nutrient media by the method of delayed antagonism with test cultures of opportunistic microorganisms.

The results presented in table 3 showed that B. breve OV-12 shows high antagonistic properties in relation to the tested strains.

Table 3 Antagonistic activity of strain B. breve OV-12 No. The strain of the test microorganism The zone of growth retardation in the presence of strain B. breve OV-12, mm one Staphylococcus aureus ATCC 6538-P 20 ± 3 2 Shigella flexneri 337 28 ± 3 3 Shigella sonnei 177 26 ± 5 four Proteus vulgaris 1274 28 ± 2 5 Escherichia coli 157 26 ± 3

Sensitivity to antibiotics of strain B. breve OV-12 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. breve OV-12 No. Antibiotic name Zone Interpretation The content of the drug in the disk, mcg The diameter of the zone of growth inhibition in mm Steady Intermediate resistant Sensitive one Benzylpenicillin ≤19 20-27 ≥28 10 37 ± 1 2 Oxacillin ≤10 11-13 ≥14 10 35 ± 0 3 Ampicillin ≤10 11-14 ≥16 10 40 ± 2 four Carbenicillin ≤14 15-18 ≥19 25 42 ± 2 5 Azlocillin ≤13 14-16 ≥16 75 40 ± 2 6 Cephalotin ≤14 15-18 ≥19 thirty 40 ± 2 7 Cephalexin ≤14 15-18 ≥19 thirty 32 ± 2 8 Cefazolin ≤14 15-18 ≥19 thirty 30 ± 2 9 Ceftazidime ≤14 15-17 ≥18 thirty 33 ± 2 10 Cefotaxime ≤14 15-20 ≥21 thirty 35 ± 2 eleven Imipenem ≤13 14-15 ≥16 10 40 ± 0 12 Streptomycin ≤16 17-19 ≥20 thirty 15 ± 2 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 12 ± 2 16 Amikacin ≤14 15-16 ≥17 thirty 0 ± 0 17 Tetracycline ≤16 17-21 ≥22 thirty 27 ± 2 eighteen Doxycycline ≤15 16-19 ≥20 10 34 ± 0 19 Erythromycin ≤17 18-21 ≥22 fifteen 30 ± 0 twenty Azithromycin ≤13 14-17 ≥18 fifteen 20 ± 2 21 Clarithromycin ≤13 14-17 ≥18 fifteen 25 ± 3 22 Roxithromycin ≤13 15-18 ≥19 thirty 37 ± 2 23 Lincomycin ≤19 20-23 ≥24 fifteen 35 ± 1 24 Clindamycin ≤14 15-20 ≥21 2 32 ± 2 25 Vancomycin ≤14 15-16 ≥17 thirty 38 ± 3 26 Chloramphenicol (Levomycetin) ≤15 16-18 ≥19 thirty 21 ± 2 27 Rifampicin ≤12 13-15 ≥16 5 35 ± 2 28 Polymyxin ≤11 12-14 ≥15 300 units 0 ± 0 29th Nalidixic acid ≤12 13-17 ≥18 thirty 0 ± 0 thirty Ciprofloxacin ≤15 16-20 ≥21 5 12 ± 2 31 Pefloxacin ≤14 15-21 ≥22 10 0 ± 0 32 Lomefloxacin ≤18 19-21 ≥22 10 25 ± 2

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

Harmlessness

The experience of studying the acute toxicity of strain B. breve OV-12 showed its complete harmlessness: when white mice were injected with a dose of 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 above data that the new strain B. breve OV-12 is highly technological; when cultivated on production media, it actively accumulates biomass of bifidobacteria at least 10 ⁹ CFU / ml and organic acids, and has high antagonistic activity with respect to pathogenic and pathogenic microorganisms, resistant to a wide range of antibiotics. Harmless when administered to laboratory animals.

The presented data on the study of strain B. breve OV-12 allow us to conclude that it is promising for use as a basis for bacterial preparations, dietary supplements, fermented and non-fermented foods, as well as hygienic and cosmetic products.

Example 1. The use of strain B. breve OV-12 to obtain a biologically active food supplement in liquid form

1st passage: lyophilized starter culture of strain B. breve OV-12 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 (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. breve OV-12 as an ingredient in an unfermented food product

1st passage: lyophilized starter culture of strain B. breve OV-12 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 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. breve OV-12 to obtain a bacterial preparation

1st passage: lyophilized starter culture of strain B. breve OV-12 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 CFU / ml of bifidobacteria.

To the finished biomass add sucrose-gelatin protective drying environment in the amount of 10-15% and 15-20% skim milk. 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 CFU / g of living bifidobacteria cells.

Example 4. The use of strain B. breve OV-12 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. 0.5% aerosil and 1% ascorbic acid are added to the mixture based on the resulting mass. After thorough mixing, they are encapsulated in capsules No. 0 of 0.4 g each. B1 g of dietary supplement contains at least 10 ⁸ CFU of viable bifidobacteria.

Example 5. The use of strain B. breve OV-12 to obtain hygiene and cosmetics

Dry biomass of bifidobacteria of strain B. breve OV-12 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.

Example 6. The use of strain B. breve OV-12 to obtain a dairy product

1st passage: lyophilized starter culture of strain B. breve OV-12 is rehydrated with culture medium or with physiological saline in a volume of 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 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.

Literature

1. Bondarenko V.M., Mapulevich 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 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., 52 p.

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

Claims (1)

The strain Bifidobacterium breve OV-12, collection number No. 217, is stored in the State collection of microorganisms of normal microflora Federal State Medical Research Institute named after G.N.Gabrichevsky Rospotrebnadzor used to obtain bacterial preparations, dietary supplements, fermented and non-fermented foods, hygiene and cosmetics.