RU2694597C1 - Method for preparation of seed culture in technology of anthrax vaccines - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention relates to the biotechnology. Disclosed is a method for preparing crop cultures of anthrax microbe. Method comprises culturing a reference culture of Bacillus anthracis STI-1 strain using a deep-well method at temperature of 32–34 °C for 36–40 hours during aeration and stirring using culture media based on fish course hydrolysates. When 70–75 % freely spores and forespores are found in culture, content of fermenter is heated to 36–37 °C and held without aeration for 3–5 hours to achieve 90 % spores stained by Ziehl-Nielsen, then concentrated and washed native culture.

EFFECT: method provides increased quantity of seed grass.

1 cl, 1 dwg, 2 tbl, 2 ex

Description

DESCRIPTION OF THE INVENTION

The invention relates to the technology of medical immunobiological preparations and is intended for the production of anthrax vaccines containing live spores of vaccine strains of Bacillus anthracis.

In the production technology of vaccines against anthrax (USSR author's certificate No. 125875, 30h, 6, 1960), sowing cultures prepared by the “surface” method are traditionally used. This method consists of two main stages:

- obtaining the vegetative form of the anthrax microbe in a liquid nutrient medium;

- obtaining a spore form on a solid nutrient medium (Voronin ES Biotechnology. - M .: GIORD, 2005. - 762 p., the regulations of PR 08461522-12-14).

The disadvantages of the traditional method of obtaining crops in the production of anthrax vaccines are the laboriousness and duration of the cultivation process, low productivity and the need to use a large number of inoculants, which significantly increases the risk of contamination of crops with extraneous microflora, as well as using a sufficiently large amount of nutrient medium.

The main disadvantages are due to the fact that a large number of glass containers (mattresses) are used to grow crops of the anthrax microbe, a multistep process with a large number of manipulations during sowing and washing of the spore culture from mattresses, which often leads to the appearance of extraneous microflora, and also using enough large amount of nutrient medium.

Most of the stated disadvantages of the traditional method are devoid of the deep method of preparing sowing crops in fermenters, which is successfully used in the production of various biotechnological products.

Known methods of deep cultivation of seed mycelium edible mushrooms of the genus Azospirillum and bacteria of the genus Brucella (see, for example, RF Patent RU 2361610 C1, March 20, 2008, RF Patent RU 2249614 C2, March 21, 2003).

These methods cannot be applied to the cultivation of B. anthracis due to the difference in nutritional needs and the generic affiliation of these microorganisms.

The objectives of the invention are to increase the concentration of spores of the B. anthracis strain STI-1 in sowing cultures, reduce the risk of contamination of sowing cultures with extraneous microflora, reduce the duration of its preparation process using a smaller amount of nutrient medium, increase the number of sowing doses, and reduce the marriage due to a smaller number inoculators and manipulations in the proposed implementation method.

Problem solving is achieved by using the in-depth method of preparing sowing crops in a laboratory fermenter, followed by concentration on the microfiltration unit and washing the spores with distilled water.

The technical result, which can be achieved using the proposed method, allows you to:

- reduce the duration of the preparation of sowing crops;

- to increase the number of spores in 1 cm ^{3 by} concentrating the native culture in a microfiltration unit;

- reduce the marriage by reducing the risk of contamination of crops with extraneous microflora at the stages of its preparation;

increase the number of seed doses and, accordingly, the cultivation cycles in an industrial fermenter;

- reduce the consumption of the nutrient medium.

The possibility of implementing the claimed invention is shown in the following examples:

Example 1. Preparation of seed cultures for anthrax vaccines (existing method).

To prepare one batch of an anthrax microbe seed culture in a “superficial” way, the reference culture is seeded in two bottles of Hottinger broth for at least one day, then they are reseeded on an agar medium based on Hottinger's digestion. And for this they use at least 100 mattresses containing at least 30 dm ^{3 of} dense nutrient medium. At 4-5 days of cultivation, the microbial culture is checked for completeness of sporulation, and in the presence of 90% of the normally painted Ziehl-Nielsen spores, the culture is washed out from the surface of the dense nutrient medium. As a result, approximately 800 cm ^{3 of} spore culture are obtained with a total concentration of about 3.5 billion spores per cm ³ , which corresponds to 135-220 seed doses for a cultivator apparatus with a volume of 100 dm ³ .

Example 2. Preparation of seed crops for anthrax vaccines (the proposed method).

To obtain a series of sowing crops using laboratory fermenter AK-0,012. 6 dm ^{3 of} sterile liquid nutrient medium based on 1% hydrochloric acid hydrolyzate of fish meal (containing total nitrogen (1.36 ± 0.20) g / l and amine nitrogen (0.45 ± 0.15) g / The apparatus is seeded with the reference culture of the B. anthracis strain STI-1, obtained from the LA Tarasevich gisk named after L. Tarasevich, for which the reference culture is resuspended in distilled water and heated at (70 ± 1) ° C for 30 minutes to activate spore germination. The cultivation process is carried out at a temperature of (33 ± 1) ° C for 36-40 hours, with aeration of 0.2-0.4 Mov of atmospheric sterile air per unit volume of the nutrient medium per minute and stirring the culture fluid with a frequency of 300-320 rpm.

The completeness of sporulation is evaluated microscopically in smears stained with Ziehl-Nielsen. If 70-75% of the spores and prospor are freely located in the culture, the contents of the fermenter are heated to a temperature of 36-37 ° C and kept at this temperature without aeration for (4 ± 1) hours.

In the presence of at least 90% of spores, normally stained by Ziehl-Nielsen, the native culture is concentrated and washed with distilled water on a Sartokon-mini microfiltration unit using polypropylene modules with a pore diameter of 0.2 μm.

Comparative characteristics of spore suspensions of B. anthracis strain STI-1 for the preparation of seed cultures obtained by the existing and declared methods are presented in table 1.

Spore suspension is mixed with distilled water and glycerol and poured into ampoules. Sowing cultures obtained in this way have characteristic cultural-morphological immunobiological and biochemical properties with high immunogenicity (at least 25,000 conventional units for guinea pigs) characteristic of the capsule-free vaccine strain of B. anthracis STI-1 and low reactogenicity in the absence of proteolytic activity.

As a result of additional electron-microscopic comparative studies of ultrathin sections of spores of seed crops produced by B.anthracis production STI-1, prepared using various methods of cultivation, it was shown that exosporium, spore shells, cortex, sporoplasm and spore nucleoid were grown by deep-growing in apparatus. the cultivator and on the surface of the agar nutrient medium, do not differ in ultrastructural organization (Fig. 1).

When using crops grown according to the claimed method, experimental series of anthrax vaccine were obtained, not differing in quality indicators from those produced by the existing technology (Table 2). Live anthrax vaccine is a porous mass of yellowish-white color with a brown tinge, dissolving within five minutes. After dissolution - an opaque homogeneous suspension of grayish-white color without any inclusions.

The inventive deep method of preparing sowing crops allows you to get anthrax vaccine that meets the requirements of the pharmacopoeial article of the enterprise P 00 001273 / 01-230911 "Vaccine anthrax live" and production regulations PR 08461522-12-14 "Live anthrax vaccine, lyophilisate for the preparation of suspensions for the suspension, for the preparation of suspensions for pending percutaneous scarification application.

Claims (1)

The method of preparation of sowing cultures of anthrax microbe using nutrient media based on hydrolyzed fish meal, wherein the reference culture of Bacillus anthracis strain STI-1 is resuspended in distilled water and heated at a temperature of 69-71 ° C for 30 minutes and cultured in a deep method laboratory fermenter with a capacity of 12 dm ³ at a temperature of 32-34 ° C for 36-40 hours with aeration of 0.2-0.4 volumes of sterile atmospheric air per unit volume of the nutrient medium per minute and stirring 300-320 rpm, then m, when reaching in culture 70-75% of spore and spore freely located, the contents of the fermenter are heated to 36-37 ° C and kept without aeration for 3-5 hours until reaching 90% of spores stained by Tsil-Nielsen, then they are concentrated and washed distilled water on the Sartokon-mini microfiltration unit using polypropylene modules with a pore diameter of 0.2 μm.

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