RU1792969C - Method of bacterial spore concentration - Google Patents

Abstract

Field of application: microbiologists, concentration of sp6p, spore vaccines, anthrax vaccine. Summary of the invention: polyethyleneimine is added to a spore suspension to a final concentration of 0.05.-6.1%. Settling is carried out at a temperature of 1-15 ° C and a pH of 6.0-8.0. The spore deposition time is 4-5 days. The method provides optimal conditions for maintaining the viability of microorganisms. 1 tab.

Description

The invention relates to the biological industry and can be used in the manufacture of live spore lyophilized vaccines and concentrates of live spore liquid vaccines against bacterial diseases.

At present, in the manufacture of live spore lyophilized vaccines, they are used to co-concentrate spores by precipitation by settling.

Precipitation of bacterial spores obtained on solid nutrient media and re-suspended in physiological saline takes 10 or more days.

The precipitation of spores obtained by the suspension method in the reactor takes much longer (more than 35 days), which is explained by the increased viscosity of the liquid phase due to the presence of a protein substrate in the liquid, a product of lysis of bacterial cells and not utilized components of the nutrient medium.

Since the biological industry is switching to a more progressive suspension method of manufacturing vaccines, it is not technologically feasible to use such a long (more than a month) to center the spore material.

It is also not practical to use the known method for concentrating spore material by centrifugation. When centrifuging, especially with large volumes of spore-containing liquid, it is very difficult to create conditions in which the contamination of the separated spores (prefabricated vaccine) with air microflora and the release of spore material into the environment would be completely excluded.

The closest is a method for concentrating small microorganisms by settling using an alkaline protein extract as an adjuvant.

The disadvantages of this method include the fact that the deposition is carried out under conditions

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(temperature 90 ° C and pH 3.0), which are detrimental to live microorganisms and therefore this method cannot be used in the manufacture of live bacterial vaccines.

The aim of the present invention is: to increase the viability of microorganisms.

The goal is achieved in that when separating microorganisms from the liquid phase, including precipitation using an auxiliary substance, polyethyleneimine (-CHz CH2NH-) n at an optimum concentration of 0.05 - 0.1% is used as an auxiliary substance. Another difference is that the deposition is carried out at a temperature of 1-15 ° С ...

In addition, precipitation was carried out at a pH of 6.0-8.0. ..-::. . , /.

Example 1. A suspension containing in 1 ml of 215 million viable spores of the vaccine OHworo anthrax sibira strain 55-VNI-IVViM, obtained in a reactor having a pH of 7.0 ± 0.2, is poured, observing sterility, in 2 two-liter vials of 2 liter in each. To one (test) vial was added 10 ml of a 10% aqueous solution of polyethyleneimine (PE) sterilized at a temperature of 120 ± 2 ° C for 30 minutes, while the concentration of PEI-in spore-containing liquid was 0.05%. The second bottle is used as a control. An initial sample of 1 ml was taken from a control vial and diluted 10 times in physiological saline followed by inoculation into Petri dishes with 2% m-peptone agar, incubating them at 37 ± 1 ° C for 20-24 hours and counting the grown colonies, the number of viable spores in 1 ml of the sample is determined. Both flasks are placed on the input; dylnik at a temperature of 4 ± 2 ° C. Within 5 days, every 24 hours, 1 ml of spore suspension samples are taken from the upper, middle and lower parts of the test vial, and they are combined into an average sample, in which the number of viable spores is determined by the method described above. In the same way, the number of spores in the samples from the control vial is determined.

The results are shown in the table. . -;. . :,;

From the data table. 1 shows that in the test vial after 4 days of standing in. only 10 million spores / ml remained from the supernatant of 215 million spores / ml; therefore, the remaining spores were sedimented and their amount was 95% of the total

spore in suspension. Over the next day, the percentage of spores in the supernatant did not increase. In the control vial, spore deposition was practically not recorded during the 5-day period. It was kept for another 30 days in the refrigerator, but even in this case there was no more than 50% of the spores in the sediment, moreover, the sediment was loose and easily broken., - ... .... .. .-; -.

When comparing the appearance of the suspension after 4 days of settling in the experimental and control vials, it was recorded that good bacterial deposition was carried out in the experimental vial

the spore supernatant was almost transparent, and the sediment was dense. In a control vial, signs of precipitation were not expressed.

At the end of the deposition (4 days) from the experimental vial, observe sterility,

decant the supernatant. A precipitate in an amount of 80-100 ml, containing 4-5 billion viable spores / ml, is used to make a vaccine concentrate or to prepare a lyophilized vaccine against anthrax in animals,

In the practice of biological enterprises during the deposition of bacterial spores, it is advisable to use 18-liter sterile cylinders with siphons for pumping liquids.

When PEI is added to the spore suspension in an amount of 0.1%, the precipitation of bacterial spores proceeds as well as when adding 0.05%. After 4 days

precipitation at 4 ± 2 ° C. 95% of the spores are in the sediment.

When PEI is added to a spore suspension in an amount of 0.0125% or less or 0.4% or more, Spore precipitation occurs

slow. After 5 days of settling at a temperature of 4 ± 2 ° C, less than 30% of bacterial spores are in the sediment.

When using a temperature of 16 ° C or higher, the deposition of bacterial spores does not slow down. However, under these conditions, part of the microorganisms can go from spore to vegetative form, which is unacceptable in the manufacture of a vaccine. When using temperatures below

At 0 ° C, the spore suspension crystallizes, which can lead to the death of some microorganisms. . :

At pH 5.0 or 9.0, the precipitation of bacterial spores does not slow down. However sour

or an alkaline environment is an unfavorable factor and can lead to the death of some microorganisms. :

Using the proposed method for concentrated bacterial spores, according

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Compared with existing methods, it provides the following advantages:

 a) as an adjuvant used to separate spores from the liquid phase, polyethyleneimine is used in an optimum concentration of 0.05-0.1% ;, .-. ;

I b) allows the concentration of bacterial spores at a temperature of 1% ° C;

1792969

c) allows the separation of spores from the liquid phase at pH 6.0-8.0;

d) allows to obtain, under sterile conditions, from a spore-containing liquid a concentrated spore material - a semi-finished product for the manufacture of lyophilized vaccines or a liquid vaccine concentrate;

e) the settling time of bacterial spores is 4-5 days.

Claims (1)

The claims I The method concentrates bacterial spores, including introducing an auxiliary substance into the spore suspension, followed by settling of the obtained cv and separating the precipitate; In order to increase the viability of microorganisms, polyethyleneimine is used as an auxiliary substance, they are introduced into the suspension to a final concentration of 0.05 - 0.1%, and sedimentation is carried out at a temperature of 1-15 ° СрН6-8.