A method of obtaining a bacterial preparation on the basis of bifidobacteria

 

(57) Abstract:

The invention relates to biotechnology, medical Microbiology, veterinary medicine and can be used for enrichment of biologically active selenium food and give them therapeutic and prophylactic value, contributing to the maintenance of the status syntrophic bifidobacteria adults, the normalization of the intestinal microflora of infants and patients with intestinal dysbiosis due to radiation sickness, the effects of chemotherapy and antibiotic therapy. A method of obtaining a bacterial preparation on the basis of bifidobacteria includes preparation of nutrient medium, cooling to a temperature of cultivation, the introduction of pure cultures of bacteria, cultivation, followed by separation of the biomass from the culture fluid. In a nutrient medium in the process of its preparation make Selenite sodium sulfate and magnesium in a ratio of 1 to 1.25 to 1; as pure cultures contribute at least one culture or the Association of bacteria, including Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium adolescentis, and/or Bifidobacterium breve, and/or Bifidobacterium infantis in the amount of from about 1.0 to 1.5. % each. The invention allows to expand the range of useful modulating the Invention relates to biotechnology, medical Microbiology, veterinary medicine and can be used for enrichment of biologically active selenium food and give them therapeutic and prophylactic value, contributing to the maintenance of the status commensal bifidobacteria adults, the normalization of the intestinal microflora of infants and patients with intestinal dysbiosis due to radiation sickness, the effects of chemotherapy and antibiotics.

Selenium is a vital element, whose participation in the metabolism intensively studied in recent years (1).

The applicant conducted a study of selenium consumption by bacteria from the environment of cultivation and the effect of selenium on the viability and acid-forming activity of the Association of bifidobacteria.

Known way of getting a bacterial drugs, the closest one is a method of obtaining a bacterial concentrate for the production of dairy products, including the preparation of the nutrient medium on the basis of the cheese with the whey and lactose, magnesium chloride, processing environment yeast-galactosidase, the cultivation of Bifidobacterium Bifidobacterium longum, followed by separation of the biomass, its mixing with the supports growth of bifidobacteria, who have a certain activity and viability, to reduce the duration of ripening milk.

However, therapeutic effect of known bacterial concentrate has a limited scope as the age of people who use these products and the spectrum of antibiotic resistance and antagonistic activity.

The problem to which the invention is directed, was to create the conditions to obtain the bacterial preparation, in which he contains cleocinonline selenium in biologically active form, and to expand the range of useful bifidobacteria inherent in the human body, depending on age.

According to the invention the task is solved as follows.

Method for obtaining bacterial preparation on the basis of bifidobacteria, including the preparation of the nutrient medium, cooling to a temperature of cultivation, the introduction of pure cultures of bacteria, cultivation, followed by separation of the biomass from the culture fluid, which according to the invention in a nutrient medium in the process of its preparation make sodium Selenite and cernosice bifidobacteria, comprising Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium adolescentis, and/or Bifidobacterium breve, and/or Bifidobacterium infantis in the amount of from 1.0 to 1.5 vol.% each.

The proposed method is as follows. Prepare a nutrient medium of the following composition (g/l):

peptone 10; lactose 10; sodium citrate 6; potassium dihydrophosphate 1; phosphate sodium 2; cysteine hydrochloride 0.15 or ascorbic acid, 0,5); agar 0,5; solution corn extract (1:6) 50 ml medium In the cultivation contribute magnesium sulfate in the amount of 0,012 g/l and sodium Selenite from 0,012 0,015 g/l Volume of the nutrient medium was adjusted to 1000 ml with distilled water. The initial pH of the medium to 7.0 to 7.2. The nutrient medium is sterilized at a temperature of 115-120oC, cooled to a temperature of cultivation 37-39oC and make 1,0-1,5% vol. pure cultures of Bifidobacterium longum, Bifidobacterium bifidum, and/or Bifidobacterium adolescentis, and/or Bifidobacterium breve, and/or Bifidobacterium infantis, taken, respectively, in the ratio 1: 1:1:1:1, and cultured until reaching titers of bacteria not less than 109. This ratio is introduced into the nutrient medium composition of selenium and sulfur allows to reach the maximum accumulation of intracellular biologically active selenium in organic form, mainly in the form of amino acids. With the authorized microflora, as well as high antibioticassociated that allows you to maintain the status of the intestinal microflora when conducting antibiotic therapy.

The data presented in table I, show the species composition of commensal bacteria, mainly inherent in the human body, depending on age. From table. I follows that infants are four species of bifidobacteria, children under 3 years old are five species of bifidobacteria, and people older than 3 years - three species of bifidobacteria.

The data presented in the table. II shows the sensitivity of the bacteria that make up the Association, to antibiotics. From table. II it follows that bifidobacteria included in the Association, provide mutual overlapping of the spectra of their antibiotic resistance.

The data presented in the table. III, show antagonistic activity of bifidobacteria. From table. III it follows that the presence in the cells of bifidobacteria selenium increases antagonistic activity as pure cultures and associations of bifidobacteria especially against S. Aureus and Pr. Vulgaris.

The data presented in the table. IV, show the life activity of acid activity of the Association of bifidobacteria containing botril is clear, increasing their viability by 20% acid-forming activity by 18%.

The data presented in the table. V show the effect of selenium on the duration of growth phases and the number of cells in the Association of bifidobacteria. From table. V, it follows that the introduction of selenium into the environment of growth of bifidobacteria significantly reduces the exponential phase of bacterial growth and prolong the stationary phase. This increases the accumulation of biomass of bifidobacteria (see K. O. E. - colonialera unit).

Example 1.

In the composition for the preparation of nutrient media contribute 0,012 g/l of magnesium sulphate and sodium Selenite. The solution volume was adjusted to 1000 ml with distilled water. Culture medium sterilized at 115-120oC for 20 minutes, cooled to a temperature of cultivation, make it a 1.0 to 1.5. percent pure culture of Bifidobacterium bifidum and cultured for 48-72 hours at a temperature of 37oC to achieve a titer of bacteria not less than 109. Antagonistic activity of bifidobacteria (POS. table 2. III) in the presence of selenium cells is increased by 5-23%, especially in relation to crop-antagonists Sh. sonnei and S. aureus.

Examples 2 to 5

Medium for cultivation of bifidobacteria goto the ium adolescentis, or Bifidobacterium breve or Bifidobacterium infantis. Antagonistic activity of bifidobacteria in the presence of selenium cells is increased by 15-20%, especially in respect of Sh. flexneri and S. aureus (POS. table 4. III); 11-20%, especially in respect of Sh. sonnei (POS. 6 table. III): 13-25%, especially in respect of Sh. sonnei (POS. 8 table. III); 7-36% especially against E. coli (Ref. 10 table. III).

Examples 6-8

In the nutrient medium composition contribute sodium Selenite at a concentration that provides optimal competitive relations with sulfur consumption of the environment of the cultivation, mainly of a 0.012 - 0.015 g/l and 0,012 g/l, respectively. In sterilized and cooled to the temperature of the cultivation environment contribute in a separate container pure culture.

As pure cultures using Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacteriurn infantis. In production, the additive is introduced with respect to their synanthropy depending on the age of the people and antibiotic resistance three, four or five strains in the ratio 1:1:1; 1:1:1:1; 1:1:1:1:1 respectively. Antagonistic activity of the Association of bifidobacteria (POS. 12, 14, 16 PL. III) increases two or three times in the presence of intracellular selenium in biologically active form.

Glodny food is brought by the Association of bacteria with respect to their synanthropy depending on the age of the people and antibioticassociated and number provide a title in the finished food product 107- 108.

Example 9.

Whole cow's milk normalize by the mass fraction of fat, warm and clean, homogenized at a pressure of 12,52,5 MPa and a temperature of from 45 to 79oC, pasteurized at a temperature of 922oC 3-5 min, cooled and sent to an intermediate tank. In chilled milk make the Association Bifidobacterium 5 (or Bifidobacterium 4, or Bifidobacterium 3), cells of which contain associated selenium, stirred for 3-5 min until a homogeneous consistency, is poured and cooled in the chamber to a temperature of not more than 42oC.

Example 10

In whole milk contribute to the growth of MMC-3 in an amount of 0.20 g/L. the Mixture was mixed thoroughly, cleaned, heated to a temperature of 422oC, purified by centrifugal malacocincla and homogenized at a pressure of 152.5 MPa and a temperature of from 45 to 85oC, the mixture is pasteurized at a temperature of 952oC aged from 30 to 40 minutes or sterilized in the fermenter at a pressure of 0.05 MPa, cooled to the fermentation temperature (392oC) and fermented Association of bifidobacteria containing intracellular biologically active selenium. The fermented mixture is stirred during knogo bunch acidity 604oSo At the end of ripening cool the product to a temperature of 4-6oC and poured.

Example 11.

In skim milk make growth - corn extract in the amount of 0,0027 l/L. the Mixture was mixed thoroughly, cleaned, heated to a temperature of 422oC, purified by centrifugal malacocincla and homogenized at a pressure of 152.5 MPa and a temperature of from 45 to 85oC. the Mixture is pasteurized at a temperature of 952oC aged from 30 to 40 minutes or sterilized in the fermenter at a pressure of 0.05 MPa, cooled to the fermentation temperature 392oC and fermented Association of bifidobacteria containing intracellular biologically active selenium. The fermented mixture is stirred for 102 minutes and left alone. The fermentation is carried out at a temperature of 371oC from 3 to 6 hours prior to the formation of milk clot acidity 604oSo At the end of ripening cool the product to a temperature of 4-6oC and poured.

Because the applicant conducts extensive research capabilities useful lives of the Association of bifidobacteria containing intracellular biologically active selenium, his claims are not limited to the above examples.

2. USSR author's certificate N 1686718 And 23 With 9/12, 1989.

1. A method of obtaining a bacterial preparation on the basis of bifidobacteria, including the preparation of the nutrient medium, cooling to a temperature of cultivation, the introduction of pure cultures of bacteria, cultivation, followed by separation of the biomass from the culture fluid, characterized in that in a nutrient medium in the process of its preparation make Selenite sodium sulfate and magnesium in a ratio of 1-1,25:1.

2. The method according to p. 1, characterized in that as pure cultures in the environment contribute, at least one culture or the Association of bacteria, including Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium adolescentis, and/or Bifidobacterium breve, and/or Bifidobacterium infantis in the amount of from 1.0 to 1.5 vol.% each culture.

 

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FIELD: milk industry, in particular, production of lactic-acid bifidus activating product for grownups and at least one-year-old children.

SUBSTANCE: method involves pasteurizing whole milk; cooling to fermentation temperature; introducing starter including bifidus bacteria and lactic streptococcuci; introducing vitamin additive; mixing; fermenting until live cell content in product is 109-1010 CFU/cm3 and acidity is 80-1000T; cooling; mixing and bottling. Vitamin additive is concentrated citrus juice, or Jerusalem artichoke juice, or Jerusalem artichoke powder juice, or Jerusalem artichoke syrup. Concentrated citrus juice is introduced in an amount of 30-50 ml per 1 l of milk, Jerusalem artichoke juice, powder juice or syrup is introduced in an amount of 0.3-0.5% by volume of milk. Method allows useful components to be extracted to maximal extent from basic product.

EFFECT: improved quality, increased assimilation of useful components by people of any age owing to development of natural microflora by product itself.

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