EA006509B1 - Consortium of bifidobacteria bifidobacterium bifidum 791-mb, bifidobacterium longum b 379m-mb, bifidobacterium adolescentis g 7513-mb, bifidobacterium infantis 73-15-mb, bifidobacterium breve 79-119-mb used for preparing sour milk, nonfermented products, biologically-active additives, bifido-containing preparations, cosmetic and hygiene agents - Google Patents

Abstract

The invention relates to biotechnology, in particular, to consortium of bifidobacteria strains, which can be used for preparing of different forms of bacterial preparations, preparations-probiotics based thereon, functional food products, biologically-active additives, nonfermented products, sour milk products, cosmetic and hygiene agents. The following bifidibacteria strains are used to prepare the consortium: Bifidobacterium bifidum 791-MB, Bifidobacterium longum b 379M-MB, Bifidobacterium adolescentis G 75130-MB, Bifidobacterium infantis 73-15-MB, Bifidobacterium breve 79-119-MB, the typical feature of which is high antibiotic and superoxide dismutase activity with expressed anti-oxidant activity. The superoxide dismutase activity of the consortium is at least 24.9 unit/mg protein of microbial mass. Exhibited specific activity, ability to utilizing a wide range of amino acids, high antibiotic-stability permitted to increase the consortium stability to damaging factors of aggressive medium of gastrointestinal tract during passing bifidobacteria in the gastrointestinal tract and thus increasing its colonizing activity.

Description

The invention relates to biotechnology, food industry and can be used to obtain a consortium of bifidobacteria, in the production of functional foods and probiotic preparations, non-fermented products, in the preparation of cosmetic and hygiene products.

Preparations and bifidobacteria-based products are widely used for the treatment and prevention of dysbacteriosis of various etiologies, the normalization of the gastrointestinal tract of humans and animals, stress effects, long-term antibiotic chemotherapy.

As is known, in the intestine of an adult with the highest frequency in the biocenosis, species dominate |

Consortiums of various combinations of bifidobacteria strains are known, used for the preparation of probiotics, clinical nutritional products, sour-dairy products, dietary supplements and bifidus fermented and non-fermented products of foreign and domestic development companies (US Patent 6335040, 01.01.2002, US application 2002015990, 07.02 .2002, VI 2109054 C1, 04/20/1998, AO 98/44090, 08.10.1998, VI 2196173 C1, 10.01.2003, VI 2196174 C1, 10.01.2003).

The closest analogue of the claimed invention is a consortium of 5 strains of bifidobacteria: Efs 78nits, 791, B | -dairy, non-fermented foods, biologically active food additives and bacterial preparations. The strains have similar cultural, physiological and biochemical properties, the consortium actively propagates in nutrient media with the accumulation of production biomass in a short period of cultivation, and has a specific activity. The finished product contains in 1 ml of 10 ⁹ living bacteria cells. The content in 1 dose of the drug is not less than 10 ⁷ living cells of bifidobacteria (VI 2109054 C1, 04.20.1998).

A disadvantage of the known consortia can be considered their weak resistance to the damaging factors of aggressive media of the gastrointestinal tract (acids, proteolytic enzymes) during the passage of bifidobacteria in the gastrointestinal tract, which indicates insufficient colonizing activity and viability during the passage of the digestive tract.

The technical result of the claimed invention is the creation of a consortium of strains of bifidobacteria, more resistant to damaging factors aggressive environments of the gastrointestinal tract during the passage of bifidobacteria in the gastrointestinal tract and increase, thus, its colonizing activity during the passage of the digestive tract, expressed antioxidant activity and probiotic organism. conditions like ίπ νί \ Ό. and ίπ νίΙΐΌ.

The consortium was also obtained by combining the five strains of bifidobacteria B | Pébas1epit Hybite 791-MB, VKPM Ac-1688; B | BOBI CEPT 1OPD B 379M-MB, VKPM Ac-1689; C | POSBETAIT ABO1SE8SEPY8 G 7513-MB, VKPM AC-1690; , В | Побас1епит шГапШ 73-15-МБ, VKPM Ас-1692; В | Бобас1епит Ьгеуе 79-119-МБ, VKPM АС-1691, the characteristic feature of which is high antibiotic resistance with pronounced antioxidant activity: the superoxide dismutase activity of the consortium is not lower than 24.9 u / mg of protein and the probiotic effect of the cultures used in production is manifested during the consumption of food products and functional food products.

Strain B | Pébélépété Pétité 791-MB was obtained from collection B | Pébacélépét CU 791 by sequential repassigning on thioglycolic medium and skimmed milk (50 passages), deposited with VKPM VNIIgetika under the number VKPM Ac - 1688.

Strain B | Pébélénépit 1opdít 379M-MB was obtained from the collection strain B | Pábésélépit 1pdit by successive cycles of lyophilization drying and reconstitution on a thioglycolic medium (50 drying-rejuvenating cycles) deposited with VKPM VNIIgetik under the number VKPM Ac1689.

Strain B | PIBA 1AlE8SepJ8G 7513-MB was obtained from the collection strain B | PIBO1AbO1E8SepI8 G 7513 by successive lyophilization drying and reconstitution cycles on a thioglycol medium (50 drying-rejuvenation-reduction cycles) deposited with VKPM VIHI VIHI 13

Strain B | Pocaclepit shGAPSCH 73-15-MB was obtained from collection B | Pabactepit 1pGapΙίδ 73-15 by successive cycles of lyophilization drying and reconstitution on a thioglycolic medium (50 drying-reinstatement cycles) deposited with VKPM VNIIgetika under the number VKPM Ac1692.

Strain B | Boclertéré Béboué 79-119-MB was obtained from the collection strain; B | Bocabiépit Béboué 79-119 by successive cycles of lyophilization drying and reconstitution on a thioglycolic medium (50 cycles of bake-restoration), deposited with VKPM VNIIgetika, by the number of VKPM and by the number of VIKIMK,

The created consortium of bifidobacteria showed a high compatibility of the five strains of bifidobacteria with each other, the joint cultivation of a mixed population of all 5 strains of specimens

- 1 006509 proceeds positively in a manner similar to the separate cultivation of strains. On average, the number of cells compared to the initial number increased hundreds of times in 14-16 hours of cultivation at the optimum temperature. The consortium retained its specific properties for 2 years.

Cultural and morphological features of the consortium

Cells of microorganism strains are gram-positive polymorphic rods with bifurcation or thickening at 1-2 ends, prone to the formation of clusters. In liquid nutrient media grow over the entire height of the volume, except for the zone of aerobiosis. The logarithmic phase ends by 24 h.

Physiological and biochemical characteristics of the consortium

Obligate anaerobic. The optimum growing temperature is 37.5 ° С, the pH of the nutrient media is 7.2-7.4. Catalase does not form, there is no liquefaction of gelatin.

Ferments lactose to form acetic and b + lactic acid without the formation of gas, acidifying the growth medium to a pH of 3.9-41. Mannose, cellobiose, xylose, mannitol, sorbitol, salicin, inulin, arabinose do not ferment.

Acid-forming activity: by 24 h - 105 ° T, to 72 h -160-195 ° T (on thioglycolic medium), on hydrolyzate-dairy medium - up to 140-160 ° T and 200-220 ° T, respectively.

The consortium actively multiplies in environments of different composition. The maximum titer of bifidobacteria consortium in sterile skimmed milk without growth factors to 20 h is 2x10 ⁸ CFU / ml. The maximum titer of bifidobacteria on corn-lactose medium is observed by 16 h and is 2x10 ⁹ CFU / ml, on thioglycolic medium by 16 h it accumulates up to 1-3x10 ⁹ CFU / ml.

Consortium bifidobacteria are capable of utilizing free amino acids in sterile skimmed milk, such as valine, methionine, alanine, arginine, isoleucine, glycine.

DNA refers to a pronounced HZ-type with a specificity coefficient of 1.57, the molar percentage of HZ content in DNA is 61.3.

The consortium exhibits antagonistic activity in relation to the test cultures obtained from the gisk named after him. Tarasovich: E. Soi B-125, 81PY1OssossiG aigeis 209-P, Shigella Sonne, Flexner. (tab. 1). The consortium is resistant to amycocin, benzylpenicillin, gentamicin, streptomyce, kanamycin, tetracycline, ofloxalin (Table 2).

Capacity of the Bib for 16-20 h of cultivation, high antibiotic and superoxide dismutase activity with pronounced antioxidant activity: the superoxide dismutase activity of the consortium is not lower than 24.9 u / mg microbial protein protein, the specific activity shown allowed to use e in the preparation of fermented milk, non-fermentable products, biologically active additives, bifido preparations, cosmetic and hygiene products.

Studies have shown that when co-cultivated on a thioglycolic medium at a pH of 7.0-7.3 and a corn-lactose medium at a pH of 6.8-7.0 under stationary conditions, the studied strains of bifidobacterium antagonism in relation to each other do not show . It was noted that in the course of joint development, the best development of the B. ao1e§septCG G 5315-MB, V. Uyyit 791-MB, B. 1ppd B 379M-MB and the smallest - V. tGapyz 73-15-MB and B. Igee 79-119 MB.

Antagonistic activity of a consortium of strains of bifidobacteria was determined by the method of developing mixed populations when co-grown with pathogenic test microorganisms in the zone of growth inhibition of test microbes by the method of bar codes.

Table 1

Consortium The zone of growth inhibition of test cultures (mm) E. co1 8th 8oppe 8. aigeis SapsNOa a1Bisapz claimed consortium 24-24,8 21.7-23.2 17.6- 19.2 18.8-20.3 analogue 22.7-23.1 20.4-21.5 18.4-19.0 18.3-19.9

The consortium has the greatest antagonistic effect on 81ARY1osossiig aegeis and we will burst, which are most often the cause of food poisoning arising from the consumption of sour-milk products.

Determination of the levels of resistance of antibiotic strains of bifidobacteria was performed according to the method of serial dilutions in a liquid nutrient medium for each strain separately. Stability was determined on sterile skimmed milk. Various concentrations of sterile solutions of antibiotics and 16-hour cultures of bifidobacteria in the amount of 5% were introduced into the nutrient medium. In the control sample was made only the biomass of the studied strain of bifidobacteria. Stability was determined by the change in titratable acidity in the samples after 48 h incubation at a temperature of

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37-38 ° C. The strain is resistant to antibiotics when the difference between the titrated acidity in the control and experimental samples is not more than 30 ° T.

Table 2. Antibiotic resistance of strains of bifidobacteria that make up the consortium claimed

Antibiotics, Dose activity units Titrated acidity of milk after cultivation with strain, ⁰ T Mcg / cm ³ i.v. V. c. at.

vyMit 1op§itDeckup tGapise Lege

791-МB В 379М-МБ Г7513-МБ 73-15-МБ 79-119-МБ

Amikocin sulfate 10 38-42 69-71 113-119 68-72 73-79 Ampicillin 250 68-72 57-63 54-58 57-63 68-72 Benzylpeny- Zillin, U / cm ³ 10,000 61-67 68-72 85-91 84-88 93-95 Gentamicin 400 31-33 38-42 38-40.0 33-35 42-46 Doxycillin ten 36-4.0 39-41 32-36 43-49 47-49 Kanamycin Sulfate 20 95-97 129-131 128-132 120-124 115-117 Levomycetin 50 29-31 38-42 29-31 21-23 26-30 Lincomycin 25 72-76 58-62 77-83 77-79 91-93 Oxacillin ten 29-31 32-36 32-36 23-25 31-33 Oleandomycin 12.5 48-52 59-61 42-46 46-50 55-61 Ofloxalin 20 39-41 48-52 62-66 39-41 32-36 Rifampicin 15 38-42 38-42 45-51 38-42 39-41 Streptomycin ten 107-116 49-51 50-53 62-66 105-111 Sulfadimezin 50 52-56 47-49 42-46 27-29 40-44 Tetracycline ten 52-56 43-45 38-40 44-48 39-41 Furadonin five 37-39 32-36 34-38 29-31 34-38 Furazolidone five 37-38 28-32 27-29 26-30 32-36 Cefazolin 20 38-42 44-48 35-37 29-31 39-41 Extensillin U / cm ³ 24,000 39-41 48-52 29-31 23-25 34-38 Erythromycin 25 28-32 33-35 28-32 15-17 29-31 Control 93-99 77-83 52-56 81-87 103-109

Analysis of the data suggests that strains of bifidobacteria are resistant to the highest concentrations of the following antibiotics:

B. Yybit MB-791 - ampicillin, benzylpenicillin, kanamycin, lincomycin, streptomitsi ⁿ y;

В.1пдит В379М-МБ - to amicocin, ampicillin, benzylpenicillin, kanamycin, lincomycin, oleandomycin, oflaksacin, streptomycin, extensillin;

Vaiole8sep118 G7513-MB - to amicocin, ampicillin, benzylpenicillin, kanamycin, lincomycin, oleandomycin, oflaksacin, streptomycin, extensillin;

VlpGapys 73-15 -MB - to amicocin, ampicillin, benzylpenicillin, kanamycin, lincomycin, streptomycin;

V. lgeue 79-119-MB - for amycocin, ampicillin, benzylpenicillin, kanamycin, lincomycin, streptomycin.

A similar study in relation to the stated consortium of bifidobacteria showed that it is resistant to at least 6 antibiotics: amycocin, tetracycline, benzylpenicillin, gentamicin, kanamycin, oflaksacin, streptomycin.

Storage conditions of the proposed consortium

The consortium is stored on a nutrient medium with a mass fraction of dry substances of 14-16% with the addition of a protective medium containing sucrose, gelatose, sodium citrate, dried by the sublimation method in ampoules. The consortium is well kept at 6-8 ° C without loss of activity titer throughout the year.

The main production-useful feature of the proposed consortium of bifibobacteria that distinguishes it from the known ones should be considered to be a pronounced superoxide dismutase activity, which is 24.9 u / mg of protein contained in the microbial mass.

The revealed SOD-activity of the consortium of bifidobacteria indicates its pronounced probiotic action, which can manifest itself in both ν νο and w uigo conditions. High activity of acid formation, antibiotic resistance to a wide range of basic antibiotics, contributes to the effective restoration of normal intestinal microflora in the process of antibiotic therapy.

- 3 006509

The resulting consortium can be used as a biologically active agent of probiotic orientation in the production of various forms of bacterial preparations, dietary supplements, sour-dairy products, non-fermented foods, in the preparation of cosmetics and hygiene products. This increases the biological value of functional food products and drugs prepared on its basis. Eating foods using the stated consortium contributes to colonization of the intestinal mucosa, displacing pathogenic and conditionally pathogenic microflora, correcting intestinal dysbiosis, and restoring bifidoflora.

The use of the consortium claimed in the production of sour-milk, non-fermented products, dietary supplements, bifidus-containing preparations, cosmetic and hygiene products, such as creams, napkins, tampon lotions, nourishing masks, women's pads, soap, cosmetic milk, etc., provides a probiotic and bacteriological effect, helps to restore normal microflora, skin and mucous membranes, and also aims to expand the Arsenal of similar means.

Example 1. The use of a consortium of strains of bifidobacteria in the composition of the starter for the preparation of sour-milk products.

1st passage: Lyophilized B1BoBaS strains Eggish 791-MB, B1Chp 73BoBs, 1pcs B 379M-MB, 1BoBs, 7513-MB, B1BoB, B1bOrseps1111 G 7513-MB, B1Boys! -MB in an amount of 0.1 g (1 dose) of dry biomass is applied separately to the growing medium and titrated using the tenfold dilution method to 10 ^-9. It is grown until individual colonies appear in the last dilution at 37.5-38 ° С or 1 dose of dry biomass each strain is introduced into two tubes with 30 ml of nutrient medium in each. Grown to obtain biomass with a cell content of at least 10 ⁸ / ml.

2nd passage: Grown culture of each of the strains with a volume of not more than 10% is introduced into the growing medium and cultured until biomass with a microbial content of at least 10 ⁸ / ml is obtained.

3rd passage (product): The obtained biomass of each of the strains is mixed, receiving a consortium, and in a volume of not more than 5% is introduced into sterile milk and cultured until a clot is formed, which is poured into containers and cooled. The resulting product contains in 1 ml of at least 10 ⁸ live bifidobacteria.

Example 2. The use of a consortium of strains of bifidobacteria in the composition of biologically active additives.

1st passage: Lyophilized strains B1BoB! they are activated by planting in a nutrient medium and grown at 37-38.5 ° C to obtain biomass with a cell content of at least 1 0 ⁸ / ml.

2nd passage: The grown biomass of each of the strains in a volume of not more than 10% is introduced into a nutrient medium and cultured to obtain biomass with a cell count of at least 10 ⁸ / ml.

Getting biologically active additives

The obtained biomass of each of the strains is mixed, getting a consortium, contribute to the nutrient medium not more than 5% and grow to obtain a biomass with a high, not less than 10 ⁸ / ml, titer of bifidobactria. Various bifidogenic factors can be introduced into the concentrate, contributing to the stabilization of the properties of the culture and the better strain survival in the body. The concentrate poured in the tank can be directly used as a dietary supplement with a therapeutic preventive effect. A dry biologically active additive can be obtained, for this purpose, protective drying media (sucrozogelatin, skimmed milk) are added to the obtained biomass, the suspension is frozen at a temperature of at least -45 ° C, lyophilized, sealed and a dry concentrate is obtained with a content of bifidobacteria of at least 1 ⁷ / ml, which can be used as a biological additive, making it in food raw materials immediately before use.

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

1 st passage:!!! Lyophilized strains V1yboas egsht Yybit 791-MB, V1yboas etsht 1oidit The 379M-MB V1yboas etsht abokzsepTsz D 7513-MB, V1yboas etsht ίηΓηηΙίκ 73-15-MB V1yboas egit geue 79-119! -MB is activated by planting in a nutrient medium and grown at 37-38.5 ° C until biomass is obtained with a cell content of at least 10 ⁸ / ml.

2nd passage: The grown biomass of each of the strains in a volume of not more than 10% is introduced into a nutrient medium and cultured to obtain biomass with a cell count of at least 10 ⁸ / ml.

Getting bifidus drug

To the grown biomass of the consortium, which is a liquid concentrate with a high, not less than 10 ⁸ / ml, titer of bifidobacteria, add protective drying medium (sucrozelatine, skim milk), the suspension is frozen at a temperature of at least -45 ° C, lyophilized, sealed and get dry concentrate with a content of bifidobacteria of at least 10 ⁸ / ml, which can

- 4 006509 but used as a bifidobacteria preparation. According to the NTD for dry bifidumbacterin (FS-1.32 BC 94), a dose of at least 10 ⁷ living cells of bifidobacteria is allowed in 1 dose of the preparation

Example 4. The use of a consortium of strains of bifidobacteria to obtain kvass or juice.

1st passage: Lyophilized strains of B1BIs1gshsh Ybish 791 -MB, PszhaevaBasoTeLiPeWSB 379M-MB, B | nutrient medium and grown at 37-38,5 ° C to obtain biomass with a cell content of at least 10 ⁸ / ml.

2nd passage: The grown biomass of each of the strains in a volume of not more than 10% is introduced into a nutrient medium and cultured to obtain biomass with a cell count of at least 10 ⁸ / ml.

3rd passage (product receipt): The grown biomass of each of the strains is mixed to form a consortium, and in a volume of not more than 5% is applied to bread kvass or juice after pasteurization, so that the content of bifidobacteria in 1 ml of kvass or juice is at least 10 ⁸ / ml. Kvass or juice is cooled and consumed without subsequent fermentation.

Example 5. The use of a consortium of strains of bifidobacteria to obtain cosmetics.

1st passage: Lyophilized strains of the Ombish 791-MB, Unified Citizens' Units of the Units and grown at 37-38,5 ° C to obtain biomass with a cell content of at least 10 ⁸ / ml.

2nd passage: The grown biomass of each of the strains in a volume of not more than 10% is introduced into a nutrient medium and cultured to obtain biomass with a cell count of at least 10 ⁸ / ml.

3rd passage (obtaining a cosmetic): The grown biomass is mixed, receiving a consortium, and in a volume of not more than 5% is introduced into the bioreactor with a thioglycolic medium and incubated at 37.538 ° C with weak agitation until the bifidobacteria titer of 10 ⁹ cells / ml is reached. Subsequently, the resulting cell suspension is destroyed by heating in an autoclave. The resulting suspension of biologically active components is cooled and mixed with a cream base in the ratio of 1: 2-1: 20, depending on the purpose of the cream being manufactured.

Claims (1)

CLAIM Consortium of Bifidos , bifidobacteria drugs, cosmetics and hygiene products.