RU1792330C - Method of obtaining chicken colibacillosis vaccine - Google Patents

Abstract

Usage: microbiological industry, veterinarians, immunoprophylaxis of colibacteriosis of chickens. Summary of the invention: E. coli is grown in a deep way with constant aeration, stirring and automatic pH adjustment after 5 h of growth in the range of 7.0-7.2. Inactivation and deposition of cells is carried out in two volumes of acetone, followed by centrifugation, drying and grinding of the pellet. 2 tab.

Description

The invention relates to the microbiological industry, in particular to the development of a technology for the production of colivectin, which can be used in veterinary medicine for the immunoprophylaxis of colibacillosis of chickens.

A known method for the preparation of coli vaccine by growing Escherlchla coli in culture medium of the following composition, May. %: agar-agar 1.0-2.0, peptone 0.1-0.5, glucose 0.1-0.5, aspartic acid 0-0.4, sodium desoxycholate 0-0.2, yeast extract 0-0.1, mineral salts (MgS04, MnCl2, FeCl, CaCl2) 0.001-0.00001.

Colibacilli are grown by surface method at a temperature of 37 ° C, after the bacteria are washed off with a buffer solution from the surface of the agar to a cell concentration of 109 to 1010 / ml. The next step in the preparation of the vaccine is the inactivation of bacterial cells by chemical and physical means. Formaldehyde is used as a chemical agent, and gamma irradiation with an additive of oxide is used as a physical factor.

aluminum or saponin. After inactivation of the cells, separation is carried out.

The disadvantage of this method is the use of the method of surface cultivation of colibacilli, a small yield of the number of cells and the preparation of the vaccine in liquid form. °

The closest in technical essence to the invention is to obtain a vaccine against colibacteriosis of birds when separately growing strains E, coli No. 211 and No. 216 by a deep method under aeration conditions to obtain a bacterial mass of 20-30 billion microbial bodies / ml in Hottinger broth with the addition of glucose, formalin inactivation in an amount of 0.2-0.4% for 3-4 days at a temperature of 37-38 ° C.

Bouillon cultures were centrifuged for 35-40 minutes, the supernatant was drained, the precipitate was diluted with buffered saline pH 7.2-7.9. The resulting suspension of strain cultures is combined in equal volumes (1: 1) and canned

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a solution of thiomersan in an amount of 0.005-0.01%.

The content of the components in the resulting vaccine is as follows.

Vaccine components Content in 1 L Escherich antigens Minimum, maximum

(cells): E. coll No. 211

E. coll No. 216 Formalin (see Thiomersan (cm3) Buffered saline

4.10135.1013

2.10132.5.1013

2.10132.5.1013

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up to 1 l

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The disadvantage of this method is the use of an expensive nutrient medium - Hotginger broth, obtaining a small yield of the number of cells, obtaining a vaccine in liquid form, the complexity and duration of the process.

The purpose of the invention is to increase the yield of the vaccine, simplify and speed up the method.

This is achieved by growing Ecsherichia coli in a deep manner by controlling the aeration and pH of the medium in a culture medium of the following composition, May. %: Sugar-dried starch1.5 Peptone -1.5 Corn extract 0.75 Tap water The rest The concentration of the culture fluid, the inactivation and sedimentation of colibacillus cells with two volumes of acetone cooled to -10 ° C, centrifugation of the precipitate and drying in a vacuum dryer at 35- 40 ° C.

A significant difference between the claimed method for producing colivaccine is the regulation of aeration and pH, which ensures the acceleration of bacterial growth.

For 8 hours, air is supplied at a rate of 10 m3 per hour per 1 m3 of culture fluid, then the air supply is increased to 15 m3 / hour,

The essence of adjusting the pH of the medium in the range of 7.0–7.2 is that when colibacteria are grown without pH adjustment, the culture medium is acidified to 4.5–4.0, which causes a slowdown in growth. The number of cells is 5-10 billion / ml. The supply of ammonia to the fermenter after 5 hours of growth and maintaining the pH at the same level dramatically increases the number of cells to 30-40 billion, / ml.

At the 12th hour of growth, the pH in the fermenter spontaneously alkalizes to 7.6-7.8, after which the cultivation is stopped.

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Another difference is the level of the technological process, which includes the use of two volumes of acetone as an inactivator and precipitator of colibacillus cells, which provides (after centrifugation and drying) a dry colivaccine preparation and simplifies its widespread use in veterinary medicine.

Example. For the deep cultivation of colibacilli, the following medium was used, May. %: Candy starch1.5 Peptone 1.5 Corn extract 0.75 Tap water 96.25 1. Preparation of a nutrient medium.

a) saccharification of starch

75 kg of insoluble corn starch is poured with 1 m of tap water, heated to 40 ° C, 375 g of amylosubtiline GXx are added, the pH is adjusted to 6.6, heated to 85 ° C and kept under stirring for 30 minutes. Then it is boiled for one hour, cooled to 75 ° C, 175 g of amylosubtiline GXx are added, the quality of saccharification with iodine is checked (should not give a blue color), the pH is adjusted to 5.0 with phosphoric acid, cooled to 58 ° C, 12.5 L of glucavamorin Gx with an activity of 50 U / ml was added and kept at 58 ° C for 6 hours. Thus prepared saccharified starch is sterilized at a temperature of 120 ° C for 30 minutes.

b) preparation of a peptone solution 75 kg of peptone are dissolved with stirring in 1 m of tap water.

c) preparation of a complete nutrient medium

37.5 kg of corn extract and 1 m of peptone solution are added to 1 m of tap water with stirring. The total volume of water was adjusted to 4 m3 (taking into account 20% condensate). Solutions of corn extract and peptone are sterilized at a temperature of 130 ° C for 30 minutes.

After sterilization, the components of the nutrient medium, that is, the corn extract, peptone and saccharified starch are combined, cooled to 38 ° C, adjusted to pH 7.6 and seeded with 0.1% volume culture of Escherichia coli.

2. Obtaining biomass of colibacilli.

The formation of E. cofi is carried out in a fermenter with a capacity of 10 m3 with a working volume of the nutrient medium 5 m in a deep way at 38 ° C for 12 hours with constant stirring and aeration, and in the first 8 hours

supplied at the rate of 10 m3 per hour and 1 m3 of culture fluid, then the air supply is increased to 15 m3 / hour. After 5 hours of growth, the pH drops sharply to 7.0. To maintain a pH between 7.0 and 7.2, an ammonia feed is automatically turned on. At the 12th hour of growth, the pH in the fermenter spontaneously alkalizes to 7.6, after which the cultivation is stopped.

From 5 hours of age, the population and number of cells in one ml of culture fluid are determined every hour using an industry standard turbidity sample (TOC 42-28-85-86).

3. Getting the target product.

After stopping the growth, the culture fluid is concentrated, cooled to 4 ° C and precipitated with 2 volumes of acetone cooled to -10 ° C. Acetone is added in small portions with continuous stirring. The resulting precipitate is centrifuged, dried in a vacuum dryer at and crushed,

The dry vaccine obtained by this method determines the number of cells, which is 3-10 10 per gram (average data from 5 experiments).

The physico-chemical characteristics of colivacin should correspond to the requirements presented in table. 1.

The appropriateness of using two volumes of acetone to produce colivaccine is shown in Table. 2

As can be seen from the data presented in table. 2, the optimal yield of the target product was obtained by precipitation with two volumes of acetone.

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The application of the proposed technological process provides an opportunity to increase the yield by 100 times. The number of colibacillus cells and reduce the technological process.

The preparation of a coli vaccine as a dry preparation simplifies its widespread use in veterinary medicine for the immunoprophylaxis of colibacillosis of chickens.

Claims (1)

The claims A method for producing a vaccine against colibacteriosis of chickens, comprising deep growing a culture of Escherichia coli in a nutrient medium under aeration conditions, inactivating the obtained culture, concentrating it by subsequent separation of the target product, characterized in that, in order to increase the vaccine yield, simplify and speed up the method , use the nutrient medium of the following composition, May. %: Candy starch1.5 Peptone 1.5 Corn extract 0.75 Water Else, when growing a crop in the first eight hours, air is supplied at the rate of 10 m3 / h per 1 m3 of culture liquid, and then the air supply is increased to 15 m / h, while during the growing process, the pH of the medium is maintained within the range of 7.0-7.2, the inactivation and concentration of the culture are carried out simultaneously with two volumes of acetone, the precipitate is separated by centrifugation and the obtained target product is dried and crushed, Table 1 Physico-chemical indicators of coli vaccine Indicators Characteristic Appearance Color Mass fraction of moisture, % no more Grinding size: sieve passage No. 38,% no less than sieve residue No. 27,% no more number of cells per gram the drug Fine powder Light brown 10 65 twenty 3-10 1.2 Test method According to GOST 20264.1-74 According to GOST 20264.1-74 According to GOST 20264 1-74 According to GOST 4403-77 According to GOST 4403-77 OSO-42-59-86 The number of cells during the deposition of bacteria of different volumes acetone Table 2