A method of producing a biological product and a dry inoculant

 

(57) Abstract:

The invention relates to biotechnology. Can be used in food industry, medicine, veterinary science and related industries. Get tablet biologic trituration way. The active principle is obtained by cultivation of microorganisms in a nutrient medium, introducing auxiliary substances, poliglyukin and sodium alginate. The resulting mixture is placed in a die and subjected to freeze-drying at a temperature of from -10 to -50C and a pressure of 4-12 PA for at least 48 hours Received biological product contains live microorganisms (bacteria or viruses). The final product has the following composition, wt.%: poliglyukin 2-3, sodium alginate 5-6, excipients 2-5, a nutrient medium containing microorganisms else. The product has a high activity and stability during storage. 2 S. and 6 C.p. f-crystals, 1 table.

The invention relates to the field of biotechnology, namely to dry preparations and methods for their preparation, and can be used in food industry, medicine, veterinary science and related industries.

Known dry biological products on the basis of living organisms, in particular bacteria and VirusWall - as the object of the invention, M, unuigbe, 1988, S. 192-258). The product contains cells of microorganisms, the remains of the nutrient medium (PS)-cultural (QOL) or lantoine (RH) liquids, fats, proteins, and other adjuvants. The technology includes, as a rule, the preparation of a solution or suspension of the biomass of microorganisms, the introduction of its special additives, drying and getting the finished product. Process conditions and compositions are determined by the characteristics of microorganisms and the nature of the task.

Disadvantages preparations containing live microorganisms is typically instability during storage and often inconvenient form of application for a person (powder or solution).

The prototype of the present invention with respect to the "substance" is the product of cells of Lactobacillus acidophilus with the remnants of TS additives and auxiliary substances, in particular a protective environment containing molasses, sugar, pectin and a few other compounds (ed.St. USSR N 1227145, 1983, CL A 23 C 9/123).

The disadvantage of the prototype is the inability to obtain a highly active drug in tablet form because of the deactivation of the cells of the microorganisms in the stages of drying in spray is gotowka feedstock, the introduction of auxiliary substances and pressing Technology of medicinal forms. Ed. by L. A. Ivanova, M., Medicine, 1991, S. 132). The disadvantage of this method is the large loss of the active agent during the extrusion in the case of labile microorganisms.

The prototype of the proposed method is a technology of obtaining trituration tablets. Preparation includes the preparation of finely dispersed powders of the active agent and excipients, the mixture with the solvent (alcohol solution), the introduction of lactose or sucrose, rubbing the resulting mixture in the matrix (tablet form), air drying and getting ready forms (Technology of medicinal forms. Ed. by L. A. Ivanova, M., Medicine, 1991, S. 201). The method is effective for nitroglycerin, etc., labile chemical compounds, but in the transition to biological drugs becomes unviable due to difficulty obtaining fine powders while maintaining the activity of microorganisms, and relatively low productivity.

The challenge faced by the authors, was the modification of technology for trituration tablets for biologics and the creation of new, more active but that problem can be solved by using as the feedstock slurry of a mixture of microorganisms with the remnants of the nutrient medium and auxiliary substances, the introduction of the mixture as the builders of the mixture poliglyukina and sodium alginate, and conducting drying, placed in a matrix of a biomaterial method of freeze drying at a temperature of from -10 to -55oC and a pressure of 5-12 PA for at least 36 hours.

Creating a new way was possible on the basis established by the authors of the properties of the mixture poliglyukina and sodium alginate to create a viscous environment spatial mesh polyfunctional structure, the nodes of which, apparently, are microspheres representing the microorganisms are protected from exposure to adverse environmental factors protective sheath from the remnants of the nutrient medium and auxiliary substances. This structure allows relatively easy dehydration of the mixture, which allowed us to use the technology of freeze drying.

The resulting product has the following composition of the final product (wt.%):

poliglyukin - 2-3

sodium alginate - 5-6

scragginess it contains bacteria, for example, strains of Lactobacillus acidophilus, or viruses, in particular the duck virus hepatitis.

As auxiliary substances used sweeteners, such as megasweet, dyes, in particular freeze-dried beet juice and other substances.

The introduction of larger or smaller amounts of auxiliary substances may, for almost no need.

When the concentrations poliglyukina and sodium alginate for the claimed parameters violated the internal structure of the drug and either not formed tablet, or during drying increases significantly the death of microorganisms.

The invention is illustrated by the following examples.

Example 1. In ampoules with liofilizovannye standards of industrial strains of Lactobacillus acidophilus A-91 and H-91 make 1 ml of distilled water, and then mix thoroughly and make separately in tubes with skim milk and incubated at 372oC for 16-20 hours.

The seed material is injected into the flask 100 ml hydrolysate-dairy medium containing pancreatic hydrolysate of milk 0.2 g of peptone, 0.06 g of sodium chloride, 0.002 g of cysteine and 0.1 g of lactose and incubated at 372oC for 16-20 hours.

The finished item is part of the strains was close to 1:1 according to optical density at wavelength 600 nm.

Growing native culture is produced in the medium containing 1 l of pancreatic hydrolysate of milk, 2 g peptone, 0.6 g of sodium chloride, 0.02 g of cysteine, 4 g of calcium carbonate and 1 g of lactose at 382oC for 16-20 hours.

To the culture fluid type 3-component medium consisting of sucrose, gelatin and OSM in the ratio of 10%:2%:4% calculated on dry substance of the final product in the form of a suspension in water (contents of living cells was 3.0 billion CFU/g) was poured in the container of 50 ml and was injected in every capacity of 0.4 g of sodium alginate, 0.2 g poliglyukina, 0.2 g of freeze-dried beet juice and 10 mg mahasweta.

The resulting mixture was poured into sterile matrix aluminum forms with a diameter of 20 mm and a height of 7 mm by means of a metering device 2 CC in the form. The forms are then cooled to -10oC and placed in the freeze-drying chamber at 2.0-2.5 hours at-25-27oC, after which the temperature was lowered to-47-50oC while reducing the pressure to 10-12 PA. Form was maintained during 33-43 hours while gradually raising the temperature up to-25-27oC and pressure up to 5-6 PA. When a camera these conditions form exhibited a further 3 hours, after which resets the vacuum and osushestvimiy - 2.4 per cent, of sodium alginate and 5.6%, beet juice - 2.6%, mahasweta - 0.15%. The title of the dry drug - 1.8 billion CFU/g (using the procedure of getting the tablet by pressing the titer was $ 0.5 billion CFU/g).

Example 2. In the conditions of example 1 were carried out tableting with the introduction of QOL additives to achieve the final concentration of sodium alginate - 6%, poliglyukina - 2%, freeze-dried beet juice - 1.9% and mahasweta - 0.1% CFU/g

The initial activity of the culture - 3.5 billion CFU/g Activity of the final product - 1.4 billion

Example 3. In the conditions of example 1 were carried out tableting with the introduction of QOL additives to achieve the final concentration of sodium alginate and 5%, poliglyukina - 3%, freeze-dried beet juice - 2.9%, mahasweta - 0.2%, gelatin - 1.9%. The initial activity of the culture - 3.0 billion CFU/g Activity of the final preparation - 1.5 billion CFU/g

Example 4. The resulting freeze-drying in examples 1-3 tablets were laid on "accelerated" storage for 3 months at 40oC according to the method (I. Zvyagin Century and other Methodological recommendations on the development of modes freeze-drying of biological products. M, Glavmikrobioprom, 1981, 35 C.) the Results are given in tablestar 1:100 in a volume of 0.2 cubic cm and passivatable at 8.5-9 day old chick embryos. Embryos were incubated at 37.5oC for 96 hours. Received vaccinated fluid was mixed in a 1:1 ratio with a solution environment drying, consisting of equal amounts of peptone and sorbitol, and then added 0.8% of sodium alginate, 0.4% poliglyukina and 0.6% glucose.

The resulting material was drawn off and subjected to further processing by the method of example 1 at a condenser temperature of -50oC, working pressure 8-10 PA final pressure 3-5 PA within 48 hours.

Residual moisture of the samples was 2.2%. EDS50the virus before drying was 10 degree -4.47/CC, after drying is 10 to the -4.35/cubic see the Degree of inactivation 0.45 lg. In control - the degree of inactivation 0.75 lg.

The results obtained indicate a high stability and efficiency of technology for tablets and activity obtained on the basis of drugs.

1. A method of producing a biological product trituration way, including the preparation of the active agent, its mixed with auxiliary substances, the introduction of a mixture of sugars, the location in the matrix, drying and getting ready form, characterized in that the active Nacht directly into the mixture, the resulting cultivation, as sugars are 2 to 3 wt.% in calculating the final product poliglyukina and 5 - 6 wt.% in calculating the final product of sodium alginate, and auxiliary substances injected in amounts of 2 to 5 wt.% calculated on the final product, after which the resulting mixture is placed in a die and subjected to freeze-drying at a temperature of from -10 to -50oC and a pressure of 4 to 12 PA for at least 48 hours

2. Dry inoculant containing live microorganisms, nutrient medium and excipients, characterized in that it further comprises poliglyukin and sodium alginate in the following ratio of ingredients in the final product, wt.%: poliglyukin - 2 - 3, sodium alginate - 5 - 6, auxiliary substances 2 to 5, a nutrient medium containing the microorganisms, and the rest.

3. Dry biological product under item 2, characterized in that the microorganisms it contains bacteria.

4. Dry biological product under item 2 or 3, characterized in that as the bacteria it contains cells of Lactobacillus acidophilus.

5. Dry biological product under item 2, characterized in that the microorganisms it contains viruses.

6. Dry biological product under item 2 or 5, o p. 2, characterized in that, as auxiliary substances it contains freeze-dried beet juice.

8. Dry biological product under item 2, characterized in that as auxiliary substances it contains megasweet.

 

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