RU2518282C1 - Nutrient medium for submerged cultivation of tularemia microbe - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: nutrient medium for submerged cultivation of tularemia microbe contains as a base, that provides the content of amino nitrogen in the medium of not less than 0.32%, dry enzymatic hydrolyzate of fibrin obtained from waste of whey-vaccine production, and the components - glucose and calcium pantothenate at a predetermined ratio.

EFFECT: invention enables to obtain a cost-effective and efficient in terms of productivity nutrient medium for accumulation of bacterial mass Francisella tularensis, which expands the range of nutrient media and for the first time enables the accumulation of its biomass in a liquid medium under conditions of submerged cultivation.

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Description

The invention relates to biotechnology, microbiology and can be used at the stage of accumulation of biomass of the producer strain in the production of live tularemia vaccine.

It is known that the causative agent of tularemia is demanding on the composition of nutrient media. For its cultivation, protein media based on natural substrates are usually used: egg yolk, blood, liver, brain, etc. [Olsufev I.G. Taxonomy, microbiology and laboratory diagnosis of the causative agent of tularemia. - M.: Medicine, 1975. - 190 p.]. Moreover, vitamin-mineral supplements are often used to stimulate growth, containing, in particular, calcium pantothenate, thiamine, cysteine or cystine, magnesium ions [Private medical microbiology with the technique of microbiological research: Textbook. / Ed. A.S. Labinskoy, L.P. Blinkova, A.S. Yeshchina.- M.: Publishing House "Medicine", 2005. - 600 p.].

Dense (agar) media containing fish or meat hydrolysates (autolysates), yeast extracts or autolysates with mandatory addition of cysteine (cystine) 0.1% and glucose 1% have found widespread use for the cultivation of tularemia bacteria. However, dense nutrient media are not suitable for deep cultivation and biomass accumulation of tularemia microbe in the production of medical immunobiological preparations.

In the production of live tularemia vaccine, until now, semi-liquid media have been used to accumulate biomass under deep cultivation conditions. The composition of the media includes hydrolysates of fresh fish, liver or meat - 20-30%, gelatin hydrolyzate - 10%, gelatin - 1.5%, sodium chloride - 0.5%, glucose - 1%, cystine - 0.1%; the pH of the medium is 7.2-7.3 [Kolyaditskaya L.S., Shmurygina A.A. Improvement of dry live tularemia vaccine. // ZhMEI. - 1957, No. 10. - S. 84-89]. The sowing dose is 0.5-1.0 billion sq.m. / ml of nutrient medium (according to the industry standard sample of turbidity OSO-42-28-85-P (10 ME) of the Federal State Budget Scientific Center for Health and Social Development of the Ministry of Health and Social Development of Russia, equivalent to a concentration of 5 billion m .k / ml). The effectiveness of the semi-liquid media used for the production of the vaccine is small, after 18-20 hours of cultivation, the density of the suspension increases 5-6 times [Olsufiev N.G. Tularemia. - 1960. - 167 p.].

Protein-based liquid media for the cultivation of tularemia microbe were tested in different ways, but, as a rule, growth was possible only with a massive inoculum dose [Olsufiev N.G. Taxonomy, microbiology and laboratory diagnosis of the causative agent of tularemia. - M.: Medicine, 1975. - 190 p.]. Chamberlain’s serum-glucose-cystine broth [Francis E. The amino-acid cistine in the cultivation of tularence // Public Health Reports. - 1923, Vol. 38 (3). - P.324-7.]. Reproduction of tularemia microbe in this medium is observed only when inoculating 1-2 billion microbial cells per 1 ml of medium (according to the turbidity standard).

Known multicomponent, synthetic liquid nutrient medium containing, in g / l: L-arginine - 0.2, L-cysteine - 1.5, L-histidine - 2.0, L-isoleucine - 0.3, DL-methionine - 1.0, DL-deicin - 0.4, L-lysine - 0.4, L-proline - 1.0, DL-threonine - 4.0, L-tyrosine - 0.2, L-valine - 0 , 8, glucose - 15.0, thiamine - 0.02, calcium pantothenate - 0.05, MgSO4 - 0.04, NaCl - 5.0, Na2HPO4 - 1.774, KH2PO4 - 2.837 [Maysky V.G., Shishov I. .N., Basilova G.I. Nutritional Needs Francisella tularensis II Microbiol. - 1984. - 6. - S.20-23]. The disadvantage of this environment is the high cost.

Known liquid semi-synthetic nutrient medium (pH 7.2), containing as a base yeast extract 5 g / l with the addition of salts (monosubstituted phosphate 12 g / l, sodium chloride 10 g / l, potassium hydroxide - 3.9 g / l, ferrous sulfate (II) heptahydrate 6 g / l), glucose 2 g / l and cysteine 0.1 g / l, which is proposed to be used for molecular genetic studies of tularemia microbe [Lapin A.A., Pavlov V.M., Domotenko L.V., Khramov M.V., Mokrievich A.N. Simple liquid culture medium for molecular genetic studies Francisella tularensis II Problems of especially dangerous infections. - 2009, No. 102. - S.66-67].

Known semi-synthetic nutrient medium T, containing as a nutrient basis a cardiac infusion of 10 g / l, bactotriptone 10 g / l, yeast extract 10 g / l, casamic acid 10 g / l; as a glucose-salt additive, a solution is used containing MgSO4, FeSO4, KCl, K2HPO4, glucose, cysteine, sodium citrate, which is added to the nutrient medium before use [Pavlovich N.V., Mishankin B.N. Transparent culture medium Francisella tularensis II Antibiotics and honey. biotechnol. - 1987, issue 32. - S.133-137].

The disadvantage of the above liquid nutrient media is their relatively high cost and lack of efficiency, in particular they do not provide the necessary growth of viable microorganisms in the logarithmic phase for 20-24 hours

In the patent RU 2451743 [BIPM No. 15 of May 27, 2012], to obtain biomass of tularemia microbe by the method of deep cultivation, a liquid nutrient medium is proposed containing, g / l: pancreatic hydrolyzate of fish meal 20.5, growth stimulator of hemophilic organisms 5, baker's yeast extract 2.3, magnesium sulfate 1.0, sodium sulfite 0.7, potassium dihydrogen phosphate 1, cysteine 0.5. When cultivating tularemia microbe, a solution of glucose-vitamin supplement (1.23 g / l) is added to the medium in an amount of 2 ml per 100 ml of medium. The accumulation of biomass in this medium after 18 ± 2 hours is 18 billion m.k. / ml. The disadvantage of this environment is its multicomponent nature and its insufficient effectiveness in its ability to accumulate the biomass of a microorganism in order to produce live tularemia vaccine.

The objective of the present invention is the construction of high-quality simple nutrient medium, which provides an increase in the biomass yield of tularemia microbe.

The technical result of the invention is to increase the efficiency of the nutrient medium for the accumulation of biomass suitable for the production of MIBP (including live tularemia vaccine), expanding the raw material base for the preparation of the protein base of the nutrient medium, reducing its cost through the use of waste from serum-vaccine production, and also ensuring the rational utilization of waste generated during the production of medicinal products.

The technical result is achieved in that the nutrient medium contains a dry enzymatic fibrin hydrolyzate prepared from a waste of whey vaccine production, glucose, calcium pantothenate, in the following ratio of components, g / l:

fibrin enzymatic hydrolyzate 50-55 (up to a content of at least 0.32% amine nitrogen) glucose 10 calcium pantothenate 0.05 distilled water rest pH 7.0-7.2

Dry enzymatic fibrin hydrolyzate prepared from waste whey vaccine production is used as a nutrient base of the claimed nutrient medium. Enzymatic fibrin hydrolyzate - pancreatic digestion of fibrillar blood protein is a valuable protein basis of nutrient media.

The preparation of a liquid fibrin enzymatic hydrolyzate is given in patent RU 2425866 [BIPM No. 22 08/10/2011]. Recently, liquid protein hydrolysates used as bases for culture media have been prepared in dry form. It is convenient to transport and store the dry hydrolyzate for a long time at room temperature while maintaining its quality, in contrast to the liquid form stored at (6 ± 2) ° C. The liquid form of the fibrin enzymatic hydrolyzate was concentrated 6-7 times by evaporation on a UVV-50 vacuum-evaporation unit and dried by convection on a KYaUL 101325.002 spray-type drying unit in a "pseudo-boiling layer".

According to the state of aggregation, the dried fibrin enzymatic hydrolyzate was a fine powder with a pale yellow tint. When analyzing the physical and chemical parameters, it was determined: total nitrogen content - (0.76 ± 0.03)%; amine nitrogen - (0.39 ± 0.03)%; the percentage of protein breakdown - (50.7 ± 1.7)%; peptone content (on the Difco scale) - (53.7 ± 1.5)%; traces of undigested protein were absent; dry residue - (4.7 ± 0.2)%; chlorides - (0.22 ± 0.01)%; humidity - (2.4 ± 0.2)%; pH - (6.9 ± 0.2), which meets the requirements for dry bases of nutrient media. The control of the dry form of the basis of the nutrient medium was carried out in accordance with regulatory documents [Methods for the control of bacteriological culture media: Guidelines MUK 4.2.2316-08. - M.: Rospotrebnadzor. - 2008. - 67 p .; Methods of control of medical immunobiological preparations administered to people: Guidelines MUK 4.1 / 4.2.588-96. - M.: Information and Publishing Center of the Ministry of Health of Russia. - 1998. - 128 p.].

A nutrient medium with a base of fibrin enzymatic hydrolyzate for growing tularemia microbe is prepared in the following way. The broth is cooked in the reactor: water, purified by distillation, is pumped in under vacuum, adding 10% to boil it, 50-55 g / l of dry enzymatic fibrin hydrolyzate is added in an amount ensuring a content of at least 0.32% of amine nitrogen in the medium. Then add glucose to a concentration of 1%. Everything is mixed and adjusted to pH (7.2 + 0.2) with a 20% sodium hydroxide solution and boiled for 30 minutes. The broth is filtered through a Salnikov filter at a temperature of (75 ± 5) ° C under a pressure of (2.5 ± 0.5) MPa. The liquid medium from the fibrin enzymatic hydrolyzate is sterilized with saturated steam at (110 + 2) ° C for 30 minutes, pressure (0.05 ± 0.02) MPa. Before the inoculum of the seed culture is added to the sterile medium, a sterile solution of calcium pantothenate is added at a concentration of 0.005% (0.05 g / l).

The possibility of carrying out the claimed invention is confirmed by the following example.

Example 1. Deep cultivation of tularemia microbe in a liquid nutrient medium.

To determine the effectiveness of the claimed medium, a producer strain of live tularemia vaccine Francisella tularensis 15 NIIEG was used. The sowing dose of the culture F. tularensis 15 NIIEG grown on a commercial dense nutrient medium FT-arape should be at least 1 billion sq.m. / ml (according to the industry standard sample turbidity OSO-42-28-85-P (10 ME) FSBI NTsESMP of the Ministry of Health and Social Development of Russia, equivalent concentration of 5 billion m.k. / ml).

The prepared culture of F. tularensis vaccine strain 15 NIIEG is grown by the method of deep cultivation in a bioreactor with automatic maintenance of the cultivation parameters: aeration of 0.5-0.7 l of air per 1 l of culture medium, stirring speed of 500 rpm for 20 ± 2 h at liquid nutrient medium of the following composition: fibrin hydrolyzate 5%, glucose 1%, calcium pantothenate 0.005% pH 7.2 at a temperature of 37 ° C. After completion of the cultivation process, the native culture of F. tularensis vaccine strain 15 NIIEG has the following characteristics: pH 7.0 ± 0.1, the concentration of microbial cells 35 ± 0.5 billion m.k. in ml of medium (according to the industry standard sample of turbidity OSO turbidity 42-28-85-P (10 ME) of FSBI NTsESMP of the Ministry of Health and Social Development of Russia, equivalent to a concentration of 5 billion cubic meters / ml).

The biomass of tularemia microbe obtained on the claimed nutrient medium, based on fibrin hydrolyzate, has morphological (cell shape and size) and cultural (colony shape) properties characteristic of the causative agent of tularemia. The obtained biomass of tularemia microbe has the following characteristics: viability coefficient 62 ± 0.5%, degree of dissociation 97 ± 1% SR (white) immunogenic colonies of the total number grown. Cultural and morphological signs typical of a tularemia microbe: small cocciform bacilli, motionless, gram-negative. The culture is agglutinated with tularemia serum to a titer of 1: 3200.

Thus, the claimed nutrient medium for the deep cultivation of tularemia microbe based on the enzymatic fibrin hydrolyzate allows you to accumulate and receive within 20 ± 2 hours a high concentration (up to 35 ± 0.5 billion m.k. in ml of medium) of viable F. tularensis biomass with a low degree of dissociation, in an amount exceeding the result on similar media, which can significantly reduce the cost of the nutrient medium and, accordingly, the final preparation of MIBP. The efficiency index of the media obtained on the basis of fibrin hydrolyzate is 2 times higher compared to the medium obtained on the basis of pancreatic hydrolyzate of fish bone meal. The increase in the biomass yield of microorganisms is due to the presence in the nutrient medium of a balanced and complete composition.

The invention expands the range of culture media for the accumulation of the bacterial mass of F. tularensis and for the first time allows the accumulation of its biomass in a liquid medium under conditions of deep cultivation. The use of fibrin hydrolyzate, which is a waste product of whey vaccine production, as a part of a liquid medium allows one to utilize waste and reduces environmental pollution, and also increases the profitability of vaccine production by using a cost-effective nutrient medium, and the scale of whey vaccine production allows to obtain a significant amount of such raw materials .

Claims (1)

A nutrient medium for the accumulation of biomass of tularemia microbe, containing a nutrient base, glucose, characterized in that the nutrient base contains a dry enzymatic fibrin hydrolyzate - waste whey vaccine production, and additionally contains calcium pantothenate, in the following ratio, g / l:
fibrin enzymatic hydrolyzate 50-55 (up to a content of at least 0.32% amine nitrogen) glucose 10 calcium pantothenate 0.05 distilled water rest pH 7.0-7.2