RU2169574C1 - Method of biopreparation preparing and dry biopreparation - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: method involves preparing the tableted biopreparation by trituration method. Active substance is prepared by culturing microorganisms in nutrient medium and auxiliary substances, polyglucin and sodium alginate are added. Obtained mixture is placed in matrix and subjected for sublimating drying at temperature from -10 to -50 C and pressure 4-12 Pa for at least 48 h. The obtained preparation has living microorganisms (bacteria or viruses). The end product has the following composition, wt. -%: polyglucin, 2-3; sodium alginate, 5-6; auxiliary substances, 2-5; microorganisms-containing nutrient medium, the balance. Invention can be used in food industry, medicine, veterinary science and adjacent branches. EFFECT: high activity and stability of preparation in storage. 8 cl, 1 tbl, 5 ex

Description

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

 Dry biological products based on living microorganisms, in particular bacteria and viruses, are known (Heb. Pat. N 0097484, 1984, class A 23 C 1/05; auth. St. USSR N 1459239, 1986, class C 12 n 7/00 ; US patent N 4289888, 1979, class A 23 C 9/123; N. G. Rybalsky and others. Vaccines as an object of the invention, M., VNIIGPE, 1988, pp. 192-258). The preparation contains cells of microorganisms, the remains of a nutrient medium (PS) -cultural (QL) or allantoic (AL) fluid, fats, proteins and other auxiliary additives. The technology includes, as a rule, the preparation of a solution or suspension of the biomass of microorganisms, the introduction of special additives into it, drying and obtaining the finished product. The process conditions and composition are determined by the characteristics of the microorganisms used and the nature of the task.

 The disadvantages of preparations containing live microorganisms are, as a rule, instability during storage and often an inconvenient form of use for humans (powder or solution).

 The prototype of the present invention with respect to the “substance” is a preparation of Lactobacillus acidophilus cells with PS residues and additives, in particular a protective medium containing molasses, sugar, pectin and a number of other compounds (ed. St. USSR N 1227145, 1983, class. A 23 C 9/123).

 The disadvantage of the prototype is the inability to obtain a highly active drug in tablet form due to the deactivation of microorganism cells at the stages of drying on a spray dryer and pressing.

 Currently, tabletting processes, as a rule, include the preparation of raw materials, the introduction of auxiliary substances into it and its compression (Technology of dosage forms. Edited by L.A. Ivanova, M., Medicine, 1991, p. 132). The disadvantage of this method is the large loss of active principle during compression in the case of labile microorganisms.

 The prototype of the proposed method is the technology of trituration tablets. Preparation includes the preparation of finely divided powders of the active principle and excipients, their mixing with a solvent (alcohol solutions), the introduction of lactose or sucrose, rubbing the resulting mixture into a matrix (tablet form), air drying and preparation of a finished form (Technology of dosage forms. Ed. L.A. Ivanova, M., Medicine, 1991, p. 201). The method is effective for nitroglycerin and the like. labile chemical compounds, but when switching to biological preparations it becomes unpromising due to the difficulties in obtaining fine powders while maintaining the activity of microorganisms, as well as relatively low productivity.

 The task facing the authors was to modify the technology for producing trituration tablets for biological products and to create new, more active and storage-stable biological preparations containing living microorganisms.

It was found that the problem can be solved by using a water-containing mixture of microorganisms as a feedstock with residues of a nutrient medium and auxiliary substances, introducing a mixture of polyglucin and sodium alginate into the resulting mixture as structure-forming agents and drying the material placed in the biomaterial matrix by freeze drying at a temperature of -10 to -55 ^o C and a pressure of 5-12 PA for at least 36 hours.

 The creation of a new method made it possible, on the basis of the property of a mixture of polyglucin and sodium alginate, established by the authors, to create a mesh spatial multifunctional structure in a viscous medium, in the nodes of which microgranules, which are microorganisms protected from the effects of negative environmental factors, are protected by a protective shell of residues nutrient medium and excipients. This structure allows relatively easy dehydration of the resulting mixture, which allowed the use of freeze drying technology.

The resulting preparation has the following composition of the final product (wt.%):
polyglucin - 2-3
sodium alginate - 5-6
excipients - 2-5
culture medium containing microorganisms - rest
As microorganisms, it contains bacteria, for example strains of Lactobacillus acidophilus, or viruses, in particular duck hepatitis virus.

 Sweeteners, for example megasvit, colorants, in particular sublimated beet juice and other substances, are used as auxiliary substances.

 The introduction of larger or smaller amounts of excipients is possible, but practically not necessary.

 When the concentration of polyglucin and sodium alginate exceeds the declared parameters, the internal structure of the drug is violated and either the tablet does not form, or when drying, the death of microorganisms increases significantly.

 The invention is illustrated by the following examples.

Example 1. In ampoules with lyophilized standards of production strains of Lactobacillus acidophilus A-91 and H-91 make 1 ml of distilled water, then mix thoroughly and separately into test tubes with skim milk and incubated at 37 ± 2 ^o C for 16- 20 hours

Inoculum is introduced into flasks of 100 ml of milk-hydrolyzate medium containing 0.2 g of peptone, 0.06 g of sodium chloride, 0.002 g of cysteine and 0.1 g of lactose in a pancreatic milk hydrolyzate and kept at 37 ± 2 ^o C for 16-20 hours.

 Ready seed is obtained by mixing separately grown cultures so that the cell ratio of individual strains is close to 1: 1 according to optical density at a wavelength of 600 nm.

The native culture is grown on a medium containing 1 g of pancreatic hydrolyzate of milk 2 g of peptone, 0.6 g of sodium chloride, 0.02 g of cysteine, 4 g of calcium carbonate and 1 g of lactose at 38 ± 2 ^o C for 16-20 hours.

 A 3-component medium consisting of sucrose, gelatin and OSM was added to the culture liquid in a ratio of 10%: 2%: 4% calculated on the dry matter of the final product as a suspension in water (living cell content was 3.0 billion CFU / g) , poured into 50 ml containers and 0.4 g sodium alginate, 0.2 g polyglucin, 0.2 g freeze-dried beet juice and 10 mg megasvit were introduced into each container.

The resulting mixture was poured into matrix sterile aluminum molds with a diameter of 20 mm and a height of 7 mm using a dispenser of 2 cc per mold. Then the molds were cooled to -10 ^o C and placed in a sublimation chamber for 2.0-2.5 hours at -25-27 ^o C, after which the temperature decreased to -47-50 ^o C while reducing the pressure to 10-12 Pa. The molds were kept for 33-43 hours with a gradual increase in temperature to -25-27 ^o C and a decrease in pressure to 5-6 Pa. When the specified conditions are reached in the chamber, the mold is exposed for another 3 hours, after which the vacuum is released and the tablets are unloaded from the molds. The resulting tablets have a residual moisture content of not more than 2.1%. The content of polyglucin is 2.4%, sodium alginate - 5.6%, beet juice - 2.6%, megasvit - 0.15%. The dry product titer is 1.8 billion CFU / g. (When using the procedure for producing tablets by pressing, the titer was 0.5 billion CFU / g).

 Example 2. In the conditions of example 1, tabletting was carried out when additives were added to the QOL to achieve a final concentration of sodium alginate - 6%, polyglucin - 2%, freeze-dried beet juice - 1.9% and megasvit - 0.1% CFU / g.

 The initial activity of the culture is 3.5 billion CFU / g. The activity of the final drug is 1.4 billion.

 Example 3. In the conditions of example 1, tabletting was carried out with the introduction of additives in the QOL to achieve a final concentration of sodium alginate - 5%, polyglucin - 3%, freeze-dried beet juice - 2.9%, megasvit - 0.2%, gelatin - 1.9%. The initial activity of the culture is 3.0 billion CFU / g. The activity of the final drug is 1.5 billion CFU / g.

Example 4. Obtained as a result of freeze-drying in examples 1-3, the tablets were laid for "accelerated" storage for 3 months at 40 ^o C according to the methodology (IV Zvyagin and other Methodological recommendations on the development of freeze-drying modes of biological preparations. M., Glavmikrobioprom, 1981, 35 pp.) The test results are shown in table 1.

Example 5. Production strain "K-UNIIP" duck hepatitis virus was bred in physiological saline 1: 100 in a volume of 0.2 cubic meters. cm and passivated on 8.5-9 day old chicken embryos. Embryos were incubated at 37.5 ^° C. for 96 hours. The resulting virus-containing liquid was mixed in a 1: 1 ratio with a solution of a drying medium consisting of equal amounts of peptone and sorbitol, after which 0.8% sodium alginate, 0.4% polyglucin, and 0.6% glucose were added.

The resulting material was poured and subjected to further processing according to the method of example 1 at a condenser temperature of -50 ^o C, an operating pressure of 8-10 Pa, a final pressure of 3-5 Pa for 48 hours.

The residual moisture content of the samples was 2.2%. The virus ELD ₅₀ before drying was 10 to the degree of -4.47 / cc, after drying, 10 to the degree of -4.35 / cc. see. Inactivation degree 0.45 lg. In the control, the degree of inactivation is 0.75 log.

 The results obtained indicate the high stability and effectiveness of the technology for producing tablets and the activity of drugs obtained on their basis.

Claims (8)

1. A method of obtaining a biological product in a trituration method, including preparing the active principle, mixing it with excipients, introducing sugars into the mixture, placing it in a matrix, drying and preparing the finished form, characterized in that the active principle is obtained by culturing microorganisms on a nutrient medium, auxiliary substances and sugars are introduced directly into the mixture obtained by cultivation, 2–3 wt.% based on the final product of polyglucin and 5–6 wt.% based on the final are added as sugars Recreatives Products sodium alginate and adjuvants are administered in an amount of 2 - 5 wt% based on the final product, and the resulting mixture was placed in a die and subjected to sublimation drying at -10 to -50 ^o C and a pressure of 4 -. 12 Pa at least 48 hours  2. Dry biological product containing live microorganisms, a nutrient medium and excipients, characterized in that it additionally contains polyglucin and sodium alginate in the following ratio of ingredients in the final product, wt.%: Polyglukin - 2 - 3, sodium alginate - 5 - 6 , excipients 2 - 5, a nutrient medium containing microorganisms - the rest.  3. The dry biological product according to claim 2, characterized in that it contains bacteria as microorganisms.  4. The dry biological product according to claim 2 or 3, characterized in that it contains Lactobacillus acidophilus cells as bacteria.  5. The dry biological product according to claim 2, characterized in that it contains viruses as microorganisms.  6. The dry biological product according to claim 2 or 5, characterized in that it contains duck hepatitis virus as viruses.  7. The dry biological product according to claim 2, characterized in that it contains sublimated beet juice as auxiliary substances.  8. The dry biological product according to claim 2, characterized in that it contains megasvit as auxiliary substances.

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