RU2748307C1 - Method for producing nutrient substrate for growing anthrax microbe - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method is proposed for producing a nutrient substrate for growing the STI-1 strain of the anthrax microbe, including dehydration on a spray-type drying unit of a liquid nutrient substrate containing enzymatic casein hydrolysate and a solution of processed corn extract in a ratio of 70% and 30%, at a feed rate of 20 to 25 dm³·h^-1, an air temperature at the inlet from 118 to 122ºC and at the outlet from 85 to 90ºC.

EFFECT: method provides a technologically advanced nutrient substrate with an extended service life in the production of anthrax vaccine.

1 cl, 5 tbl

Description

The invention relates to biotechnology, in particular to methods for producing the bases of nutrient media and can be most effectively used in the production of anthrax vaccines.

Currently, there is a known method of obtaining a nutrient substrate for the preparation of a nutrient medium (PS) in the production of an anthrax vaccine (prototype [1]). The method consists in the sequential preparation of an enzymatic hydrolyzate of casein (FPS), a solution of processed corn extract (CE) and mixing the liquid components in a calculated ratio based on the value of total nitrogen.

The disadvantage of this method is the liquid form of the base, which excludes the possibility of its long-term use and storage. The main properties of the nutrient substrate are preserved in a narrow range of storage temperatures - from 5 to 10 ° C, no more than one month.

According to studies [2, 3], dry nutrient bases and media, in comparison with liquid ones, are much more effective due to their longer shelf life, standardization and ease of use. We propose a method for preparing a dry nutrient substrate, consisting of a mixture of FHA and a solution of treated EC in a ratio of 70% and 30%, dehydrated in a spray drying unit A1-FMU with technological parameters: the rate of drug supply for drying is from 20 to 25 dm ³ h ^-1 , inlet air temperature from 118 to 122 ° С, outlet air temperature from 85 to 90 ° С.

The proposed method allows you to obtain a dry nutrient substrate with desired properties, with a shelf life of three years and an extended storage temperature range - from 0 to 30 ° C.

The presence of a primary-consequence relationship between the set of essential features of the proposed invention and the achieved technical result is shown in table 1.

The problem solved by the invention is the dehydration of the nutrient substrate containing 70% FPS and 30% solution of the treated CE, in a spray drying unit.

This problem is solved due to the fact that the claimed method of obtaining a nutrient substrate provides for enzymatic hydrolysis of casein, processing of corn extract (condensed), mixing liquid components (enzymatic hydrolyzate of casein and a solution of processed corn extract) in a ratio of 70% and 30% and dehydration of the resulting liquid a nutrient substrate on a spray-type drying unit at a drug feed rate for drying from 20 to 25 dm ³ ⋅h ^-1 , an inlet air temperature from 118 to 122 ° C, an outlet air temperature from 85 to 90 ° C.

The technical result of the invention is a dry nutrient substrate with desired properties, with a shelf life of up to three years and extended temperature storage conditions - from 0 to 30 ° C. The prepared dry nutrient substrate is easy to use, which makes it possible to shorten the preparation time for PS from it.

The method for preparing a nutrient substrate is as follows.

Preparation of FGK. The calculated amount of water is poured into the reactor and the prepared sample of casein is loaded in a ratio of 10: 1. The mixture to be hydrolyzed is heated to a temperature of (80 ± 1) ° С and incubated for 2 h with constant stirring. Then the mixture is cooled to a temperature of (50 ± 1) ° C and, with the mixer operating, add minced pancreas (PZh) in a casein: PZh ratio of 2: 1 and ferment for 2 hours at the indicated temperature. The concentration of hydrogen ions is corrected to a value of (8.1 ± 0.1) units. pH 40% sodium hydroxide solution during the entire hydrolysis process. After the end of fermentation, the hydrolysis is clarified first at pH 4.1-4.5 with concentrated hydrochloric acid, then at pH 7.8-8.0 with 40% sodium hydroxide solution, boiled at each of the indicated values for 5 minutes, filtered.

The characteristics of the enzymatic hydrolyzate of casein in terms of physicochemical parameters are presented in table 2.

Preparation of the treated CE solution. The calculated amount (4.25 liters) of corn extract is placed in a digester, then 17 liters of distilled water are added. The resulting solution is stirred until the original EC is completely dissolved and the mixture is left to stand for 20 hours. After settling, the resulting supernatant liquid (untreated EC) is decanted and the total nitrogen content is determined in it, which should be from 3000 to 3500 mg%. A solution of untreated CE and distilled water are poured into the digester, according to the calculation of the total nitrogen content. The resulting solution is heated to a temperature of (62 ± 5) ° C and the concentration of hydrogen ions is adjusted with a 20% sodium hydroxide solution to (7.1 ± 0.2) units. pH. Then it is heated to a temperature of (85 ± 5) ° С and filtered with a suction filter through cardboard. The resulting solution of treated and filtered EC is poured into a glass container and sterilized at a temperature of (123 ± 3) ° С for 30 min. In appearance, the sterile solution of the treated EC is a clear, dark brown liquid with a characteristic odor.

The characteristics of the treated CE solution in terms of physicochemical parameters are presented in Table 3.

Preparation of a liquid nutrient substrate. According to the production technology of the anthrax vaccine, the finished PS used for growing the STI-1 strain of the anthrax microbe must contain FGK (70%) and a solution of treated EC (30%).

Calculated quantities (based on the total nitrogen content) of FGK and a solution of the treated EC are poured into the digester. The resulting mixture is thoroughly mixed and adjusted with a 20% sodium hydroxide solution to a concentration of hydrogen ions (7.3 ± 0.4) units. pH. The contents of the kettle are heated with continuous stirring to a boil. The hot nutrient substrate is filtered through cardboard on a Nutsche filter.

The characteristics of the liquid nutrient substrate in terms of physicochemical parameters are presented in table 4.

Preparation of a dry form of a nutrient substrate.

Drying of the nutrient substrate is carried out on a spray drying plant of the A1-FMU type with a vibro-boiling layer of granules of inert material with the following technical parameters:

inlet air temperature - from 118 to 122 ° С;

outlet air temperature - from 85 to 90 ° С;

the rate of feeding the preparation for drying is from 20 to 25 dm ³ ⋅h ^-1 ;

The technological parameters of drying ensure the yield of the finished dry product in the amount of 80 g from 1 dm ^{3 of the} liquid substrate. The nutrient substrate is a hygroscopic, finely dispersed, homogeneous light brown powder; the drug in an amount of 2 g dissolves 98 cm ^{3 of} distilled water when boiled for 1 min; a two percent aqueous solution of the drug is transparent, brown in color, there is a slight precipitate.

Characteristics of the prepared dry nutrient substrate in terms of physical and chemical parameters:

loss in mass on drying - 2.91%;

total nitrogen concentration - 10.13%;

amine nitrogen concentration - 4.44%;

hydrogen ion concentration

two percent aqueous solution - 7.0 units. pH.

According to regulatory requirements in dry hydrolysates used in the technology of immunobiological preparations (IBP) [1], the loss in weight during drying should not exceed 5%. Consequently, spray drying at the selected technological parameters makes it possible to obtain a product with the required residual moisture.

Table 5 shows the comparative characteristics of liquid and dry nutrient substrates in terms of physical and chemical parameters.

Thus, the claimed spray drying of a liquid nutrient substrate containing 70% FPS and 30% treated EC on a spray drying plant A1-FMU with technological parameters: the rate of drug supply for drying from 20 to 25 dm ³ ⋅h ^-1 , compressed air pressure on nebulizers 0.02 MPa, air temperature at the inlet from 118 to 122 ° C, air temperature at the outlet from 85 to 90 ° C provides a dry nutrient base for bacteriological media with desired properties.

The studies carried out to determine the shelf life of the nutrient substrate using the accelerated aging method [4] showed that storage conditions under which the quality of the dry nutrient substrate does not change throughout the warranty period can be created in a room with a temperature from 0 to 30 ° C and a relative air humidity not more than 70%. The shelf life of the drug under these storage conditions is three years.

In addition, the dry nutrient substrate is technologically advanced in use, which makes it possible to shorten the preparation time for PS from it.

Thus, the proposed method for obtaining a nutrient substrate for the preparation of PS on a spray-type drying unit will allow obtaining a dry form of the preparation, thereby increasing the shelf life of the product from one month to three years, significantly expanding the storage temperature range of the product from 0 to 30 ° C, while ensuring its standardization and manufacturability in application.

Literature

1. Regulation: PR No. 08461522-26-19 / Industrial Regulations PR No. 08461522-011-09 for the production of live lyophilisate anthrax vaccine for the preparation of a suspension for subcutaneous administration, skin scarification application. - Approved. early Research Center "48 Central Research Institute" of the Ministry of Defense of Russia, Kirov, 2019.

2. Zaitsev, V.F. Some directions of standardization of nutritious bases from casein / Zaitsev V.F. [and others] // Sat. scientific. Proceedings for the 75th anniversary of the Research Institute of Microbiology of the Ministry of Defense of the Russian Federation, April 14-16, 2003. - P. 145.

3 Telishevskaya A.Ya. Protein hydrolysates: preparation, composition, application. - M .: "Rosselkhozakademiya", 2000. - 296 p.

4. Determination of the shelf life of dry microbiological media and nutrient bases by the method of "accelerated storage" at elevated temperatures: Methodical recommendations. - Makhachkala, 1987 .-- 6 p.

Claims (1)

A method of obtaining a nutrient substrate for growing a strain STI-1 anthrax microbe, providing for enzymatic hydrolysis of casein, processing of condensed corn extract, mixing liquid components of enzymatic hydrolyzate of casein and a solution of processed corn extract in a ratio of 70% and 30%, characterized in that the liquid nutrient substrate is exposed to dehydration in a spray-type drying unit at a drug feed rate for drying from 20 to 25 dm ³ ⋅h ^-1 , an inlet air temperature from 118 to 122 ° C, an outlet air temperature from 85 to 90 ° C.