RU2736064C1 - Protective medium for stabilization of tularemia pathogen during preparation and storage of dry preparations - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention refers to biotechnology, namely to a protective medium for stabilization of tularemia pathogen during preparation and storage of dry preparations. Protective medium for stabilizing cells of tularemia pathogen during preparation and storage of dry preparations contains, by weight, thiocarbamide – 3.3 %, ascorbic acid – 1.8 %, distilled water – 84.1 %, 10 % lactose and 0.8 % polyglucin.

EFFECT: said protective medium promotes stabilization of tularemia pathogen cells, providing reduced drying time and high bacterial survival during dry preparations lyophilisation and storage in a wide temperature range, specifically, the disclosed composition of the protective medium enables to obtain a high-quality product with residual moisture of 2_9 to 4_2 % during cell survival from 86 to 92 % in a shorter period.

1 cl, 2 tbl, 3 ex

Description

DESCRIPTION OF THE INVENTION

The invention relates to biotechnology, in particular to compositions and formulations of protective stabilizing media designed to preserve the viability of bacterial cells at the stages of drying and storage and can be used to obtain dry cultures and biological products containing live bacteria of the tularemia pathogen.

It is known that in the process of lyophilization, a certain part of microorganisms dies off, most during freezing, and a smaller part during sublimation and drying. The storage process of dry preparations is also accompanied by gradual cell death. The purpose of preserving microorganisms in a viable state is special protective media, of various compositions, widely used and guaranteeing a certain level of bacterial survival at the stages of processing.

The causative agent of tularemia belongs to a group of bacteria with an average resistance to freezing and dehydration factors. The survival rate when these microorganisms are dried without the use of protective media ranges from 10 to 30%. The use of cell stabilization with drying media increases the survival rate up to 40-60%.

Known protective drying medium used to obtain by the method of sublimation dry vaccine preparations against plague, tularemia, brucellosis and tuberculosis, containing: 10% sucrose or lactose, from 0.1 to 0.2% agar-agar, from 1 to 1.5% gelatin and from 88.3 to 88.9% of water (disaccharide-agar-gelatin medium) [Faibich M.M. Stabilization of vaccine preparations during drying and storage // Journal of Microbiology, Epidemiology and Immunology - 1968, No. 2. - S. 59-65]. When using the above protective medium, with freeze drying, up to 50% of the bacteria of the tularemia vaccine culture survived.

The disadvantages of this environment include: the absence in the composition of antioxidants - substances that can neutralize or slow down the oxidation processes of vital cell structures (lipids and enzymes) when exposed to free radicals of air oxygen [Emanuel N.M. Free radicals in biology / Per. from English. - M.-. Institute of Chemical Physics of the USSR Academy of Sciences, 1981. - S. 68-70].

Also known protective drying environment, designed to obtain dry tularemia vaccines, containing: 0.3% sucrose and 5 or 10% gelatin [Olsufiev NG. Taxonomy, microbiology and laboratory diagnostics of tularemia causative agent. - M .: Medicine, 1975 - S. 67]. When using the specified composition of the protective medium in the process of freeze drying, 50 to 60% of the bacterial cells of the vaccine survived.

The disadvantages of this protective environment include: the use of gelatin in high concentrations gives the medium a gel-like consistency, which creates difficulties when washing the culture from the surface of the agar, and also prevents the uniform distribution of bacteria in the volume of the medium [Blanks BI, Klebanov D.L. The use of lyophilization in microbiology - M .: Medgiz, 1961 - S. 170]; lack of antioxidants in this environment.

The closest to the declared is a protective environment, which is an aqueous solution containing 10% sucrose, 0.5% thiourea, 0.5% ascorbic acid, 1% gelatin, 0.05% peptone and 87.95% distilled water [Nikitin E .E., Zvyagin I.V. freezing and drying biological products. - M .: Kolos, 1971 - S. 264].

Common to the claimed invention is the multicomponent composition of an aqueous solution and the use of thiourea and ascorbic acid in the medium.

The disadvantages of this environment, drying can be attributed: the use of sucrose, capable of hydrolysis with the formation of glucose and fructose, which reduces its stabilizing efficiency [Ackerman Y. Biophysics / Per. from English. V.A. Otroshchenko, V.N. Soyfer. - M .: Mir - S. 338]; the presence of gelatin leading to gelling in a protective environment; not the most optimal content in the environment of antioxidants - thiourea and ascorbic acid [Zvyagin IV, Khorkov IA guidelines for the development of freezing-drying modes of bacterial preparations. M .: Glavmikrobioprom, 1981. - S. 35]; the use of hydroscopic substances sucrose, gelatin and peptin leads to the need to lower the freezing temperature of the stabilized suspension to minus 40 ° C, while the increased content of hydrophilic substances delays the drying process and can create increased residual moisture in dry culture [Blankov B.I., Klebanov D. L. The use of lyophilization in microbiology - M .: Medgiz, 1961 - P. 164].

The objective of the invention is to develop a composition of a protective environment for stabilizing the cells of the causative agent of tularemia, providing a reduction in drying time and high survival of bacteria during lyophilization and storage of dry preparations in a wide temperature range.

The problem is solved due to the fact that the component composition of the protective medium is balanced and optimized for the cells of the causative agent of tularemia, the composition of the drying medium includes the following differences: sucrose is replaced by lactose, gelatin is replaced by polyglucin, peptone is excluded, and the content of thiourea and ascorbic acid is increased.

The use of lactose and polyglucin, which are structure-forming and cryoprotectants, makes it possible to form the structure of the dried material, regulate the rate of dehydration of microbial cells and significantly reduce their destruction during freezing and dehydration. The exclusion of sucrose, gelatin and peptone from the medium will allow freezing of the stabilized cell suspension at a temperature of minus 28 ° C (versus minus 40 ° C using the prototype medium), while the drying process will be reduced from 45 to 36 hours. This, in combination with an increase in the content of thiourea and ascorbic acid, which increase the protection of lipids and cell enzymes from air oxygen and free radicals, will ensure the survival of microbes when dried at 80-90%, with low-temperature storage - 80-85%, when stored at room temperature - not less than 50%.

The composition of the protective environment, percentage (by weight): lactose - 10; thiourea -3.3; ascorbic acid - 1.8; polyglucin - 0.8; distilled water 84.1. The pH of the medium is from 7.0 to 7.2 units. pH.

The protective environment is prepared in the following order. Calculated samples of the components are weighed with an accuracy of 0.1 g, distilled water is taken by volume.

A weighed portion of polyglucin is soaked in water (1/6 of the calculated volume) and kept at room temperature for at least four hours (until complete dissolution). A weighed portion of ascorbic acid is dissolved in water (1/6 of the calculated volume) by heating in a water bath to a temperature of no more than 30 ° C and stirring. The resulting solution of ascorbic acid is cooled in air to a temperature of 20-25 ° C and with the help of a 40% sodium hydroxide solution, its pH value is brought to values of 6.7-6.9 units. pH.

A portion of lactose is dissolved in the remaining distilled water by heating in a water bath to a temperature not exceeding 80 ° C and stirring. Then the lactose solution is cooled in air to a temperature of 40 ° C and a sample of thiourea is dissolved in it.

In a cooled solution of lactose and thiourea, solutions of polyglucin and ascorbic acid are added, mixed and the pH value is determined. If necessary, the pH value of the medium is adjusted with 5% sodium hydroxide solution to values of 7.0-7.2 units. pH. The prepared protective medium is a light, transparent liquid. The medium is not stored, prepared and used on the day of washing of the tularemia causative agent colonies grown on the surface of a dense nutrient medium.

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.

Two drying media were prepared with the following compositions:

1) protective environment of the prototype, percentage (by weight):

sucrose - 10;

thiourea - 0.5;

ascorbic acid - 0.5;

gelatin - 1;

peptone - 0.05;

distilled water - 87.95;

2) the declared protective environment, percentage (by weight):

lactose - 10;

thiourea - 3.3;

ascorbic acid - 1.8;

polyglucin - 0.8;

distilled water - 84.1.

Mattresses with colonies of tularemia causative agent that grew on the surface of a dense nutrient medium were divided into two equal groups before washing. Condensation was removed from the mattresses under aseptic conditions. Then, sterile porcelain balls with a diameter of 7 mm (10 g each) were introduced into each mattress, and 10 cm ^{3 of a} protective medium was also added using a sterile pipette. In this case, the protective environment of the prototype was introduced into the first group of mattresses. The claimed protective environment was introduced into the second group. The mattresses were closed with cotton-gauze plugs, and by rocking them manually, the colonies were washed off the surface of the dense nutrient medium. The resulting suspensions of microbial cells were collected in two sterile flasks.

Stabilized cell suspensions were poured no more than 8 mm in height into metal cuvettes, which were installed on the shelves of the sublimation chamber of the MASS-5-02 installation (OOO Biomashpribor, Yoshkar-Ola).

Receiving dry preparations, series 1 (the inventive environment) and series 2 (prototype environment), based on the causative agent of tularemia, was carried out at a suspension freezing temperature of minus 28 ° C according to the following regime:

freezing temperature of the stabilized suspension - minus 28 ° С,

the duration of the holding of the material at the freezing temperature before the start of creating a vacuum in the sublimation chamber - 4 hours;

condenser temperature - freezer - from minus 52 to minus 60 ° С;

the value of the working vacuum in the sublimation chamber - from 30 to 20 Pa;

the rate of temperature rise of the material - no more than 3 ° C / h

material temperature during drying - 24 ° С;

total drying time - 36 hours.

In dry cultures, the concentration of cells was determined (by bacteriological method) and the survival rate of bacteria upon drying was calculated. The results are shown in Table 2.

The preparation of series 2 did not meet the requirements for dry bacterial preparations in terms of residual moisture, the values of which are from 1 to 6%, are the safest for the viability of bacteria [Blankov BI, Klebanov D.L. The use of lyophilization in microbiology. -M .: Medgiz, 1961. - S. 164].

Example 2.

The composition of protective media and the procedure for obtaining stabilized suspensions using the prototype medium and the inventive medium are presented in example 1.

Receiving dry preparations, series 3 (the inventive environment) and series 4 (prototype environment), based on the causative agent of tularemia, was carried out at a suspension freezing temperature of minus 40 ° C according to the following regime:

freezing temperature of the stabilized suspension - minus 40 ° С,

the duration of the holding of the material at the freezing temperature before the start of creating a vacuum in the sublimation chamber - 4 hours;

condenser temperature - freezer - from minus 52 to minus 60 ° С;

the value of the working vacuum in the sublimation chamber - from 30 to 20 Pa;

the rate of temperature rise of the material - no more than 3 ° C / h

material temperature during final drying - 24 ° С;

total drying time is 45 hours.

The results of the analysis of the properties of dry preparations are presented in table 2.

From the results presented in table 2, it can be seen that the claimed composition of the protective environment makes it possible to obtain a high-quality product upon drying, with a residual moisture content of 2.9 to 4.2% with a cell survival rate of 86 to 92% in a shorter time.

Example 3.

The obtained dry preparations, series 1-4, were stored in metal sealed glasses under stationary conditions in three temperature-time modes:

temperature from minus 15 to minus 9 ° С for 3 months;

temperature from 0 to 4 ° C for 1 month;

temperature from 18 to 22 ° C for 3 days.

After storage, the concentration of living cells in the preparations was determined (by bacteriological method) and the survival rate of bacteria after storage was calculated. The results are shown in Table 2.

The data presented in Table 2 indicate that the claimed composition of the protective environment allows maintaining up to 85% of viable cells at temperatures from minus 15 to minus 9 ° C for 3 months, up to 69% of cells at temperatures from 0 to 4 ° C for 1 month and up to 53% of cells at a temperature of 18 to 22 ° C for 3 days.

Claims (1)

A protective environment for stabilizing tularemia pathogen cells during the preparation and storage of dry preparations, including thiourea, ascorbic acid and distilled water, which differs in the content, by weight, of thiourea - 3.3%, ascorbic acid - 1.8%, distilled water - 84, 1%, as well as the introduction of 10% lactose and 0.8% polyglucin into the composition.