RU2746022C1 - Method for stabilizing bacterial cells of plague microbe before freeze-drying - Google Patents

Abstract

FIELD: biotech.SUBSTANCE: invention relates to biotechnology and medical microbiology. A method is proposed for stabilizing bacterial cells of a vaccine strain of the plague microbe before freeze-drying, including the preparation of a protective medium by lyophilization according to a certain mode of an aqueous solution containing 300 g of lactose, 30 g of thiourea, 30 g of ascorbic acid, 30 g of polyglucin in 610 cm3of distilled medium, and mixing 13 g of dry protective medium with 100 cm3of concentrated cell suspension.EFFECT: method provides protection of microorganisms from the effects of freezing and dehydration factors during freeze drying and can be used to obtain dry preparations based on living microorganisms.1 cl, 2 tbl, 2 ex

Description

The invention relates to biotechnology, in particular to methods of stabilizing bacterial cells with drying media that protect microorganisms from the effects of freezing and dehydration factors during freeze drying and can be used to obtain dry preparations based on living microorganisms.

Known methods for stabilizing microbial cells in a cell suspension before drying, including: preparing a protective medium in the form of an aqueous solution of protective components with the required concentration and adjusting the pH of the medium to values corresponding to the pH of the microbial suspension; mixing the cell suspension with a protective medium (usually in a 2: 1 ratio by volume) [E.E. Nikitin, I. V. Zvyagin Freezing and drying of biological preparations - M, 1971. - P. 169-181; B.I. Blankov, D.D. Klebanov Application of lyophilization in microbiology - M, 1961. - S. 161-172; K.E. Dolinov Fundamentals of technology of dry biological products, 1964. - S. 159-168].

At the same time, protective media that stabilize microbial cells before drying differ in the content and quantity of components, the choice of which, to a large extent, depends on the individual characteristics of microorganisms and the tasks to be solved.

The disadvantages of these methods include the introduction of an additional amount of water into the microbial suspension together with the protective components, as a result of which the water content in the suspension increases to 80%. This leads to the emergence of such effects on cells as dehydration and osmotic shock, and during freezing, introduced water serves as an additional source of the formation of ice crystals. These factors lead to the destruction of vital structures of microorganisms and a decrease in their survival rate during freeze drying [A.A. Belous, V.A. Bondarenko Structural changes in biological membranes during cooling - Kiev, 1982. - P. 221-224; N.S. Pushkar Theory and practice of cryogenic and sublimation preservation - Kiev, 1984. - pp. 109-114; A.M. Karpov, A.A. Ulumiev. Drying of products of microbiological synthesis, 1982. - P. 79; S.G. Ignatov Investigation of functional and structural changes in the membrane apparatus after low-temperature freezing // Biochemistry, 1981. - V. 46, no. - S. 996-2003].

As a prototype, a method for stabilizing microbial cells with a multicomponent aqueous solution containing, percent (by weight): lactose - 30; thiourea - 3; ascorbic acid - 3; polyglucin - 3; distilled water - 61 [V.V. Teterin, A.V. Yezhov [et al.] A method of obtaining a biological product based on a vaccine strain of the plague microbe. RF patent No. 2510825, publ. 04/10/2014].

The method consists in:

Preparation of the drying medium.

A weighed portion of polyglucin is pre-soaked in 100-200 cm3 of warm distilled water and kept at a temperature of 18-24 ° C. The resulting solution is filtered through a gauze filter. A portion of lactose is poured into 500 cm ^{3 of} distilled water and heated to a temperature of 80 ° C with constant stirring until complete dissolution. After that, the resulting solution is divided into two parts, in one part ascorbic acid is dissolved, and in the other thiourea, the dissolution of thiourea is carried out at a temperature of 70 ° C. A solution of ascorbic acid is neutralized with a 40% sodium hydroxide solution to a pH of 7.3-7.5 units. pH. The prepared solutions of thiourea and ascorbic acid are filtered through a gauze filter and mixed with a polyglucin solution. Correction of the pH value to values of 7.2-7.6 units. pH is produced with 10% sodium hydroxide solution.

Stabilization of bacterial cells.

A drying medium is added to a bottle with a concentrated suspension of the plague microbe in a 2: 1 ratio (2 parts of the concentrated suspension and 1 part of the drying medium). The contents of the bottle are thoroughly mixed by shaking.

The prototype and the claimed method have in common: the component composition of the protective medium (lactose, thiourea, ascorbic acid, polyglucin) and mixing of bacterial cells before drying with a protective medium to stabilize them.

The disadvantages of the prototype include:

as a result of the use of a liquid form of a protective medium, an increase in the volume of extracellular water occurs, which ultimately, as mentioned, with low-temperature freezing and drying, leads to a low survival rate of microbes of plague pathogens;

long sublimation process associated with the need to evaporate more water.

The object of the invention is to develop a method for stabilizing bacterial cells, which would provide the ability to carry out freeze drying in a shorter period of time and obtain a dry preparation with a high content of living cells.

The technical result is achieved due to the fact that in the claimed method, including the preparation of a protective environment and mixing a concentrated suspension with protective substances, to create the required concentration of them, providing stabilization and protection of cells at the stages of freezing and drying, the following differences are provided, consisting in using instead of a liquid protective environment of dry fast-dissolving medium, in the amount of 130 g per 1 dm ^{3 of the} suspension. In this case, the process of obtaining a dry medium includes the preparation of a liquid medium with a pH of 7.0 -7.2 units. pH and its sublimation dehydration.

The essence of the proposed method is as follows:

Preparation of a dry fast-dissolving protective medium.

An aqueous solution of protective components is prepared according to the recipe:

300 g lactose;

30 g thiourea;

30 g of ascorbic acid;

30 g polyglucin;

610 cm ^{3 of} distilled water.

The order of preparation of the solution is similar to the order of preparation of the drying medium of the prototype. Set the pH value of the solution from 7.0 to 7.2 units. pH.

The resulting solution is placed in flat-bottomed cuvettes with a layer of 7 mm, which are placed on the shelves of the drying chamber. Drying is carried out according to the following mode: the temperature of the freezing condenser minus 60 ° C, the material freezing temperature minus 40 ° C, the holding time of the material at the freezing temperature is 4 hours, the operating pressure in the sublimation chamber is no more than 20 Pa, the rate of temperature rise of the material is no more than 3 ° С / h, the maximum temperature of the material during drying is 25 ° С, the total drying time is 36 hours.

Get a dry form of the medium containing, percent (by weight): lactose - 76.9; thiourea - 7.7; ascorbic acid - 7.7; polyglucin - 7.7.

In a dry environment, determine the value of residual moisture according to the method [A.P. Kouzov, L. Ya. Scriabin. Methods for determining the physicochemical properties of powdery dust - L., 1983. - S. 83-84], which should be from 2.5 to 3.5%. Determine the degree of solubility of the dry protective medium in accordance with the requirements of [General Pharmacopoeia Monograph (OFS) 12.1.0005.11 "Solubility"], with a value of not more than 5 seconds - the dry medium is considered to be readily soluble in an aqueous medium.

The dry form of the protective environment is stored in a sealed container equipped with an inert gas injection system at a temperature of 20 to 25 ° C for one year.

Stabilization of bacterial cells.

A dry protective medium at the rate of 13 g per 100 cm ^{3 of} concentrated suspension is introduced into a container with a culture, shaken by hand and stirred until complete dissolution.

The presence of a causal relationship between the set of essential features of the claimed object and the achieved technical result is shown in Table 1.

The possibility of implementing the claimed invention is shown by the following examples.

Example 1.

Were prepared, according to previously described technologies, two protective environments:

dry protective environment containing, percent (by weight): lactose - 76.9; thio-urea - 7.7; ascorbic acid - 7.7; polyglucin - 7.7;

liquid protective medium containing, percent (by weight): lactose - 30; thio-urea - 3; ascorbic acid - 3; polyglucin - 3; distilled water - 61.

Then, the concentrated suspension of the vaccine strain of the plague microbe was stabilized with the prepared media. Used a concentrated suspension with a biological concentration of 94 billion live cells / cm ³ , a dry residue of 6.3%, obtained by the technology described in the prototype. Two series of stabilized suspensions were prepared:

series 1 - the concentrated suspension was stabilized with a dry form of a protective medium at the rate of 13 g of dry medium per 100 cm ^{3 of the} suspension;

series 2 - the concentrated suspension was stabilized with a liquid protective medium at the rate of 50 cm ^{3 of the} medium per 100 cm ^{3 of the} suspension.

Mixing of protective media with suspensions was carried out manually for 5 minutes. The results of evaluating the properties of stabilized suspensions are presented in Table 2.

To obtain dry preparations, freeze drying of stabilized suspensions was carried out in a MASS-5-02 installation. For this, 3 hours before loading, the shelves of the drying chamber were cooled to a temperature of minus 40 ° C, the condenser to minus 60 ° C. The suspensions were poured into metal cuvettes with a layer of no more than 7 mm, which were placed on the shelves of the drying chamber. The suspensions were frozen to a temperature of minus 40 ° C and kept at this temperature for 4 hours. Then, the working pressure in the sublimator was created from 20 to 10 Pa, 1 hour after the creation of a predetermined vacuum value, the shelves were heated and the temperature was gradually increased to 25 ° C. The material was dried at a temperature of 25 ° C. The total drying time was 36 hours.

As a result, 2 series of dry preparations were obtained: series 1 (using the claimed method) and series 2 (using the prototype method).

In dry preparations, the biological concentration was determined (by bacteriological method) and the survival rate of bacteria was calculated upon drying, the results are presented in Table 2.

For this kind of dry preparations, the main indicators are biological concentration (at least 100 billion live cells / g) and residual moisture (from 2 to 5%). According to table 2, the preparations obtained with 36-hour freeze drying were conditioned.

Example 2.

Were prepared, according to previously described technologies, two protective environment: dry protective environment containing (percentage by weight): lactose - 76.9; thio-urea - 7.7; ascorbic acid - 7.7; polyglucin - 7.7;

liquid protective medium containing (percentage by weight): lactose - 30; thio-urea - 3; ascorbic acid - 3; polyglucin - 3; distilled water - 61.

Then, the concentrated suspension of the vaccine strain of the plague microbe was stabilized with the indicated media. Used a concentrated suspension with a biological concentration of 105 billion live cells / cm ³ , a dry residue of 6.5%, obtained by the technology described in the prototype. Two series of stabilized suspensions were prepared:

series 3 - the concentrated suspension was stabilized by the dry form of the protective medium at the rate of 13 g of dry medium per 100 cm ^{3 of the} suspension;

series 4 - the concentrated suspension was stabilized with a liquid protective medium at the rate of 50 cm ^{3 of the} medium per 100 cm ^{3 of the} suspension.

Mixing of protective media with suspensions was carried out manually for 5 minutes. The results of evaluating the properties of stabilized suspensions are presented in Table 2.

The preparation of dry preparations was carried out according to the regimen described in example 1 with the difference that the total drying time was 26 hours.

As a result, 2 series of dry preparations were obtained: series 3 (using the claimed method) and series 4 (using the prototype method).

In dry preparations, the biological concentration was determined (by the plate method) and the survival rate of bacteria when dried was calculated, the results are presented in Table 2.

From the analysis of the data in Table 2, it follows that when using a dry protective medium, the survival of bacteria increases during drying, the biological concentration and content of cells in the preparation increase. At the same time, the conditioned values of residual moisture, from 2 to 5%, can be achieved after 26 hours of drying.

Claims (1)

A method for stabilizing bacterial cells of a vaccine strain of a plague microbe before freeze-drying, including preparing a protective medium and mixing it with a concentrated suspension of cells, characterized by obtaining a dry, rapidly dissolving form of a protective medium by lyophilizing an aqueous solution containing 300 g of lactose, 30 g of thiourea, 30 g of ascorbic acid, 30 g of polyglucin in 610 cm ^{3 of} distilled medium, according to the mode: condenser temperature minus 60 ° C, material freezing temperature minus 40 ° C, material holding time at freezing temperature 4 hours, operating pressure in the sublimation chamber no more than 20 Pa, temperature rise rate material is not more than 3 ° C / h, the maximum temperature of the material during drying is 25 ° C, and by mixing 13 g of dry protective medium with 100 cm ^{3 of} concentrated cell suspension.