RU2687373C2 - Method of obtaining brucellar biomass of vaccine strains at deep cultivation using a minimally invasive liquid nutrient medium - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a method of producing brucell biomass of vaccine strains. Method involves deep cultivation of the brucella culture on a liquid nutrient medium minimized by content of the protein component under conditions of regulation of partial pressure dissolved in the medium of oxygen and dosed supply of glucose according to the scheme: introduction of 10.0 g/l of glucose into the nutrient medium before seeding, then continuous dosing thereof at 1.5 g/l per hour from 16 to 19 hours of growth and 2.0 g/l per hour from 20 to 24 hours of growth.

EFFECT: method provides higher yield of Brucella biomass.

1 cl, 3 tbl

Description

TECHNICAL FIELD

The invention relates to the field of biotechnology and can be used to obtain Brucella biomass in the technology of preparation of biological products based on them.

PRIOR ART

As the basis for the preparation of various drugs for the diagnosis of brucellosis, the vaccine strain 19BA Brucella abortus is now widely used [RU 2163141 C1, 20.02.2001]. In addition, live vaccines from strains 19 B. abortus and Rev-1 B. melitensis are used for the specific prevention of brucellosis in farm animals [Manual on the use of vaccines live dry against brucellosis on farm animals from Brucella abortus 19 (1996), Manual on the use of vaccines live native strain Rev-1 brucella melitenzis for immunizing rams against infectious epididymitis (1995)]. The main stage of the manufacturing technology of these drugs is the process of obtaining microbial biomass of the causative agent of brucellosis with the required concentration and in sufficient volume. In biotechnology, to obtain standardized cultures of microbes, it has been developed quite deeply and has found wide practical application of the method of periodic deep-seated cultivation.

For deep cultivation of brucella in the technology of preparation of biological products most often use liquid nutrient media based on Hottinger's parvar with a high content of the protein component with the addition of glycerin, glucose and yeast extract [D. Devrishov, Kuznetsov S.M. et al. Comparative assessment of the growth properties of the nutrient medium for the preparation of live Brucella vaccine from the Rev-1 strain // Veterinary Medicine. - 2007. - №1. - p. 6-7, Luzan N.S. Improvement of industrial technologies for the production of Brucella vaccine and diagnostics from Brucella abortus 19 strain. - Diss ... Cand. biol. sciences. - Shchelkovo, 2001. - 124 p.]. However, it should be noted that the high content of the hydrolyzate of a valuable food product in the composition of the medium significantly increases the cost of its cost and determines the presence of a large amount of ballast impurities in the resulting cultures.

The available experimental data on the dynamics of utilization of free amino acids in the process of growing brucella suggests that most nutrient media used in obtaining biologics contain protein hydrolysates in concentrations that significantly exceed the nutritional needs of this microorganism [Yudin YI, Zhavoronkova L.V., Shabalin B.A. et al. Assessing the possibility of using “minimized” nutrient media based on various hydrolyzates for deep-growing brucella // Materials of the All-Russian Scientific and Practical Conference dedicated to the 60th anniversary of the branch of FGU “48 Central Research Institute of the Ministry of Defense of Russia - TsVTP BZ” population. Diagnosis, treatment and prevention of dangerous infectious diseases. Epidemiology and epizootology. Microbiology. Biotechnology. Ecology ”, July 16, 2009, Ekaterinburg. - Ekaterinburg, 2009. - P. 111-113]. This circumstance indicates the fundamental possibility of reducing the amount of the protein component in the nutrient medium used.

A method of obtaining Brucella antigen from the strain Brucella abortus 19 for the manufacture of a single Brucella antigen for the agglutination reaction (RA), the complement fixation reaction (RAC) and the reaction of the long complement fixation (RDSK), a part of which is to obtain microbial culture by deep growing in a bioreactor [ RU 2085212 C1, 08.15.1996]. The method requires the use of expensive nutrient medium, while the level of accumulation of microbial cells in deep culture (about 30 billion microbes / ml - the value is determined in the analysis of the technological chain of the preparation of the drug) is average and can be increased.

A method of obtaining a Brucella antigen strain Brucella abortus 19 for the manufacture of a single Brucella antigen for RA, RSK and RDSK, Brucella antigen for Rose-Bengal serum test (RBP) and Brucella antigen for ring reaction (CR) with milk, a method of manufacturing a brucellosis diagnostic serum sets [RU 2361610 C1, 03.20.2008]. In the above group of inventions described is the closest to the claimed existing deep method of obtaining microbial culture of strain B. abortus 19, which has several disadvantages: the relatively high consumption of protein hydrolyzate (Hottinger digest) based on expensive food raw material for the preparation of the medium, the content of which is 25% volume; high content of ballast impurities due to the high concentration of the protein component in the nutrient medium used (the content of amino nitrogen is from 1.40 to 1.60 g / l); the use of high concentrations of inoculum (from 3.5 to 5.0 billion microbes / ml) at the stage of industrial cultivation. In addition, in this method, the well-known method of feeding the microbial culture with the substrate during the deep growing process, which ensures a stable increase in the cell concentration in the resulting culture, is not implemented.

SUMMARY OF INVENTION

The objective of the proposed method is to increase the yield of microbial cells in deep culture, reducing the cost of the nutrient medium used, reducing the content of ballast impurities in the resulting preparations.

The technical result is to ensure sufficient accumulation of biomass of Brucella vaccine strains in deep culture, reducing the cost of the nutrient medium used and reducing the content of ballast impurities.

To perform the stated technical result is proposed:

the use of minimized content of the protein component of the liquid nutrient medium;

cultivating brucella vaccine strains in a bioreactor under dosing conditions of glucose according to a given pattern; maintaining the pH of the medium at a certain level by automatically administering a dosed solution of ammonia and regulating the partial pressure of oxygen dissolved in the culture fluid throughout the cultivation process.

The minimized liquid nutrient medium has the following composition: enzymatic hydrolyzate of meat until the content of amino nitrogen in the medium is from 0.45 to 0.55 g / l; dry yeast extract - 2.50 g / l; Fe ₂ (SO ₄ ) ₃ · 3H ₂ O - 7.00 mg / l; MnSO ₄ · 5H ₂ O - 2.00 mg / l; KH ₂ PO ₄ - 1.13 g / l; MgSO ₄ · 7H ₂ O - 0.23 g / l; nicotinic acid - 2.00 mg / l; thiamine chloride - 0.20 mg / l; calcium pantothenate - 1.00 mg / l; glucose (total amount per growing cycle) - 26.00 g / l, distilled water - to the calculated volume; pH of the medium is from 6.8 to 7.2 units. pH

The scheme of the metered introduction of glucose provides for the simultaneous introduction of it into the nutrient medium before seeding at a concentration of 10.0 g / l, from 16 to 19 h of growth - continuous introduction at 1.5 g / l per hour, from 20 to 24 h of growth - continuous introduction at 2.0 g / l per hour. The optimum pH of the culture fluid in the range from 6.8 to 7.2 units. The pH is maintained by automatic dosed administration of 12.5% aqueous ammonia.

In relation to the existing method, the proposed method has a number of differences (table 1).

The application of the proposed method provides for the accumulation of microbial cells in deep culture at a concentration of up to 85-90 billion microbes / ml, reducing the cost of the used liquid nutrient medium and reducing the content of protein ballast impurities in microbial culture. The duration of the submerged cultivation process is from 26 to 28 hours.

IMPLEMENTATION OF THE INVENTION

The claimed invention is confirmed by the following examples of practical application.

Example 1

Enzyme hydrolyzate of meat and distilled water are poured into the digester through a nylon sieve in the calculation of the final concentration of amine nitrogen in the medium from 0.45 to 0.55 g / l. The mixture is thoroughly mixed, boiled for 5 minutes, alkalinized with 20% NaOH solution to a pH of 6.8 to 7.2 units. pH, add weighed salt, boil for 20-25 minutes, after which the medium is filtered through brightening plates and poured into bottles with a capacity of 20 liters. The liquid nutrient medium is transferred to a bioreactor and sterilized at a temperature of from 121 to 125 ° C for 45 minutes, then cooled to a temperature of from 36 to 38 ° C. A glucose solution with the addition of vitamins and a solution of yeast extract are prepared separately, sterilized at a temperature of 108 to 112 ° C for 20 minutes and injected into a sterile environment immediately before it is sown. The estimated concentration of glucose in the nutrient medium before seeding is 10.0 g / l. The sowing of the nutrient medium in the bioreactor is carried out by microbial culture of the 19BA strain of B. abortus grown in a liquid nutrient medium based on an enzymatic meat hydrolyzate of similar composition. Sowing dose (concentration of seed) is from 0.5 to 1.0 billion microbes / ml according to the turbidity optical standard for determining the concentration of microbes CCA 42-28-85-2013.

Deep cultivation in the bioreactor is carried out according to the following mode:

cultivation temperature - from 36.5 to 37.5 ° C;

the pH value of the culture fluid is from 6.8 to 7.2 units. pH;

the partial pressure of dissolved oxygen (as a percentage of saturation) is maintained according to the scheme: from 1 to 11 h of growth - from 20 to 25 pO ₂ ; from 12 to 23 h of growth - from 40 to 45 pO ₂ ; from 24 to 26-28 hours of growth - from 30 to 35 pO ₂ ;

the introduction of glucose in the process of growing is carried out according to the scheme: from 16 to 19 h of growth - continuously at 1.5 g / l per hour; from 20 to 24 hours of growth continuously at 2.0 g / l per hour.

The technological parameters of the submerged cultivation process are supported by the following control actions:

temperature maintenance - by supplying temperature-controlled water to the bioreactor jacket;

maintaining the pH value of the culture fluid - automatic dosed introduction of 12.5% aqueous ammonia solution;

maintaining the partial pressure of dissolved oxygen - by regulating the flow of air for aeration and by changing the speed of rotation of the mixer.

The introduction of a 12.5% aqueous solution of ammonia into the submerged culture is carried out by the method of automatic dosing with a decrease in the pH value of the culture fluid of less than 6.8 units. pH At the end of the cultivation process with the stabilization of the pH value of the culture fluid at a level of from 7.0 to 7.2 units. The pH of the stirring is stopped and it is aerated for 0.5-3 hours. The criterion for the completion of the deep-growing process is a spontaneous increase in the pH of the culture fluid to a value of 7.4 units. pH

In the obtained depth culture of the 19BA strain of B. abortus, the typicality of microbial morphology and the presence of extraneous microflora are assessed by Gram smear microscopy, the total microbial concentration is determined using the optical turbidity standard for determining the microbial concentration of the CCA 42-28-85-2013 and the content of live microbes by sowing the appropriate dilutions on a dense nutrient medium in Petri dishes.

Using the proposed method were obtained 3 series of deep culture of the strain 19VA B. abortus. The duration of cultivation ranged from 26 to 28 hours

Table 2 presents the results of the evaluation of the biological properties, antigenic and immunogenic characteristics of a series of deep-seated cultures of B. abortus strain 19BA, taking into account their intended purpose for use in the preparation of vaccine and diagnostic products.

When assessing the immunogenicity, guinea pigs are immunized subcutaneously at a dose of 2.0 billion microbes according to the optical turbidity standard to determine the microbial concentration of the CCA 42-28-85-2013, after 28 days. after immunization, animals are infected with a culture of a virulent strain 16M B. melitensis at a dose of 100 live microbes. The immunogenicity of the culture is assessed by the proportion of non-diseased animals on the basis of bacteriological and serological examination data after 30 days. after infection.

When assessing the resistance to cryopreservation, the culture is stabilized with a protective medium, frozen at a temperature of minus 18 to minus 22 ° C, stored at the specified temperature for 3 days, then reactivated for 40 minutes at a temperature of 36 to 38 ° C and the survival rate is determined microbes.

Example 2

Deep cultivation of brucella culture is carried out analogously to example 1, but instead of the 19BA strain of B. abortus, the Rev-1 strain of B. melitensis is used.

In the deep culture of the Rev-1 strain B. melitensis, the typicality of microbial morphology and the presence of extraneous microflora are assessed by Gram smear microscopy, the total microbial concentration is also determined using the turbidity optical standard for determining the microbial concentration of the CCA 42-28-85-2013 and the content live microbes by sowing appropriate dilutions on a dense nutrient medium in Petri dishes.

Using the proposed method were obtained 3 series of deep culture of the strain of the strain Rev-1 B. melitensis. The duration of cultivation ranged from 26 to 28 hours

Table 3 presents the results of the evaluation of the biological properties, antigenic and immunogenic characteristics of a series of deep-seated cultures of the Rev-1 strain of B. melitensis with regard to their intended use for use in the preparation of vaccine and diagnostic preparations. Assessment of immunogenicity and resistance to cryopreservation carried out analogously to example 1.

Claims (4)

The method of obtaining biomass of Brucella vaccine strains, which consists in the fact that submerged cultivation is carried out at a temperature of from 36.5 to 37.5 ° C in minimized on the content of the protein component of a liquid nutrient medium of the following composition: enzymatic hydrolyzate of meat and distilled water in the amount of the final concentration of amine nitrogen in the medium from 45.0 to 55.0 mg%; dry yeast extract - 2.5 g / l; Fe ₂ (SO ₄ ) ₃ ⋅3H ₂ O - 7.0 mg / l; MnSO ₄ .5H ₂ O - 2.0 mg / l; KH ₂ PO ₄ - 1.13 g / l; MgSO ₄ · 7H ₂ O - 0.23 mg / l; nicotinic acid - 2.0 mg / l; thiamine chloride - 0.2 mg / l; calcium pantothenate - 1.0 mg / l; with the introduction of 10.0 g / l of glucose in a nutrient medium before seeding and continuous dosing of 1.5 g / l per hour from 16 to 19 hours of growth and 2.0 g / l per hour from 20 to 24 hours of growth; maintaining the pH of the culture fluid in the range of 6.8 to 7, 2 units. pH by dosing 12.5% aqueous ammonia; with regulation of the partial pressure of oxygen dissolved in the medium from 20 to 25 pO ₂ from 1 to 11 h of growth, from 40 to 45 pO ₂ from 12 to 23 h of growth, from 30 to 35 pO ₂ from 24 to 26-28 h of growth. 2. A method of producing biomass of Brucella vaccine strains under item 1, characterized in that the concentration of inoculum is from 0.5 to 1.0 billion microbes / ml of medium.