Nutrient medium for submerged cultivation of tularemia microbe. RU patent 2518282.
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| Preparation for purification of water and soil from oil pollution and method of obtaining thereof / 2516412 Group of inventions relates to field of biochemistry, ecology, protection of the environment. Claimed is preparation for purification of water and soil from oil pollution, which contains microorganisms, oil destructors, sorbent, cryoprotector - glycerol, microfertilisers - sodium nitrate 0.5% and potassium phosphate 0.5%. As oil destructor, it contains association of oil-oxidising microorganisms: Bacillus subtilis VKM B-81, Pseudomonas spp. VKM B-892, Pseudomonas putida VKM B-1301, Rhodococcus sp. VKM Ac-950, Mycobacterium flavescens VKM Ac-1415 in amount 75-85% of the total number of cells, as well as soil bacteria Agrobacteium radiobacter VKM B-1219 in amount 15-25% of the total number of cells. Sorbent in preparation represents finely-dispersed dehydrated zeolite with size of granules 0.1-0.5 mm, powdered with Aerosil A-300 nanoparticles. Ratio of components in preparation (wt %) is the following: zeolite - 94±1, Aerosil A-300 - 3±0.5, glycerol - 1±0.2, sodium nitrate - 0.5±0.2, potassium phosphate- 0.5±0.2, association of oil-oxidising microorganisms with Agrobacteium radiobacter in efficient quantity 2-3*108 kl/g - 1±0.5. Also claimed is method of preparation obtaining. Assocoation of oil-oxidising bacteria and fraction of soil bacteria are grown separately. After that, two obtained culture liquids are mixed in ratio 75-85% of association of oil-oxidising bacteria of the total quantity of cells and 15-25% of soil bacteria of the total quantity of cells. Suspension is concentrated to concentration 2*1011 kl/ml. Zeolite is preliminarily crushed to granules with size 0.1-0.5 mm, kept in furnace at temperature 250°C to the stage of swelling, cooled to temperature 20°C at humidity 10-12% and mixed with Aerosil A-300. Glycerol, sodium nitrate, potassium phosphate are introduced into concentrated suspension, and after that mixed with zeolite. Obtained zeolite and concentrated suspension are mixed with ratio 9:1. After that process of contact-chemical dehydration is carried out. |
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| Strain of fungus stagonospora cirsii davis having herbicidal activity against canada thistle / 2515899 Strain of the fungus Stagonospora cirsii Davis of All-Union Research Institute of Plant Protection 1.42 has herbicidal activity against canada thistle and species closely related to it - S. Cirsii. It was deposited in the State Collection of Microorganisms of All-Union Research Institute of Plant Protection under the collection number of Stagonospora cirsii Davis of All-Union Research Institute of Plant Protection 1.42 and can be used for biological control of canada thistle. |
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| Strain of filamentous fungus aspergillus oryzae - producer of maltogenic alpha-amylase / 2514224 Strain of fungus Aspergillus oryzae Amy T-52-3-21 produces maltogenic α-amylase, and it is deposited in the All-Russian Collection of Microorganisms Institute of Biochemistry and Physiology of Microorganisms n.a. GK Scriabin RAS under the number F-4476D. The strain is made on the basis of strain Aspergillus oryzae of All-Russian Collection of Microorganisms F-3927D using the genetic engineering methods. Activity of α-amylase at 120 h of growth of the strain is 600-640 units/ml. |
| Strain of bacteria bacillus vallismortis - destructor of oil and oil products / 2513702 Strain of bacteria Bacillus vallismortis VKPM V-11017 is proposed - destructor of oil and oil products. Strain may within short period of time in the wide range of temperatures from +8 to +37°C degrade oil by 78.3%. |
| Nutrient medium for extraction of bacteria of shigella kind from water objects / 2517750 Nutrient medium is proposed for extraction of Shigella kind bacteria from water objects. The nutrient medium contains the following components: 0.1% alcohol solution of bromcresol green - 9.0 ml; 0.2% alcohol solution of methyl red - 3.0 ml; fermentative peptone - 0.5 g; extract of fodder yeast - 4.5 g; potassium dihydrophosphate - 8.7 g; caustic soda - 1.4 g; sodium chloride - 5.0 g; distilled water - up to 1000 ml; medium pH is 6.70-6.85. |
| Method to prepare medicated product from live strains and microorganisms of lactobacilli and bifidobacteria "lb-complex plus" / 2517734 Method provides for cultivation at 37±1°C of strains of lactobacilli and bifidobacteria in the medium and packing of liquid product with account of daily dose necessary for patients. The medium contains components in the following quantities: caseine hydrolysate dissolved by distilled water, 0.33-0.4 g/l, sodium chloride 5 g/l, fructose 10 g/l, peptone 2 g/l, agar-agar 0.75 g/l for lactobacilli, for bifidobacteria - 1.0 g/l, ascorbic acid 0.25 g/l, distilled water 0.67-0.6 g/l. Strains-producers are Lactobacillus plantarum 8 RA-3, Lactobacillus fermentum 39, Lactobacillus fermentum 90 TC-4, Bifidobacterium bifidum 791, Bifidobacterium longum 379, Bifidobacterium bifidum 1. For lactobacilli and bifidobacteria they prepare accordingly media with different content of agar-agar, amine nitrogen in caseine hydrolysate 160-170 and 180-200 mg% and medium pH 7.8-8.0 and 8.5-8.6. Starting from the first generation the bifidobacteria and lactobacilli are cultivated for 24±1 hours, two strains together, one separately. The produced biomass is mixed with the fresh nutrient medium at the ratio of 1:1000, and strains of lactobacilli are cultivated 24±1 hours, strains of bifidobacteria - 48±1 hours. Upon completion of cultivation of biomass of strains they are mixed at the ratio of 2:1:2:1. |
| Nourishing composition, including probiotics and improving sleep pattern / 2517616 Invention relates to application of probiotic bacterial strain for production of a probiotic composition for reducing sleep disorders and/or improvement of sleep quality in people and animals. As a bacterial strain used is Lactobacillus reuteri DSM 17938 or Bifidobacterium longum NCC 3001 (ATCC BAA-999). |
| Strain of fungus stagonospora cirsii davis having herbicidal activity against canada thistle / 2515899 Strain of the fungus Stagonospora cirsii Davis of All-Union Research Institute of Plant Protection 1.42 has herbicidal activity against canada thistle and species closely related to it - S. Cirsii. It was deposited in the State Collection of Microorganisms of All-Union Research Institute of Plant Protection under the collection number of Stagonospora cirsii Davis of All-Union Research Institute of Plant Protection 1.42 and can be used for biological control of canada thistle. |
| Medication (versions), composition (versions) and application of medication (versions) for reduction of halitosis / 2515113 Invention relates to versions of medication for reduction of halitosis, versions of compositions based on said medications and versions of application of said medications. Device for reduction of halitosis represents strains of microorganisms Lactobacillus acidophilus, selected from group Lactobacillus acidophilus DSM 19825, Lactobacillus acidophilus DSM 19826 and Lactobacillus acidophilus DSM 19827. Version of said medication represents culture supernatant of said strains. Also claimed are compositions, which contain said microorganisms, and their application for reduction of halitosis. |
| Method of culturing bifidobacteria in milk / 2515048 Invention relates to biotechnology and can be used in the dairy industry. Pasteurised milk is cooled to a given temperature and MnSO4, ZnSO4, KJ, CuSO4, FeSO4 and selexen are then added in a given ratio, followed by addition of bifidobacteria. The mixture is stirred and soured until a clot of given titratable acidity is obtained. |
| Method of obtaining toxin actinobacillus pleuropneumoniae apxi, applying culture medium, containing calcium-borogluconate complex / 2514667 Claimed invention relates to field of microbiology and deals with method of obtaining RTX-toxin ApxI. Claimed method is realised by cultivation of bacteria Actinobacillus pleuropneumoniae in culture medium, which provides growth of bacteria, and said culture medium contains borogluconate in concentration lower than 60 mmol/l in order to form in medium calcium-bologluconate complex. |
| Hafnia alvei bacteria strain, capable of producing thermolabile lt-enterotoxin / 2514656 Invention relates to biotechnology and can be used to produce thermolabile enterotoxin (LT-enterotoxin) and Hafnia alvei anatoxin when producing a vaccine. The strain is deposited in the State Collection of Pathogenic Microorganisms of FBSI Scientific Centre for Evaluation of Medical Products of the Ministry of Public Health and Social Development of Russia under number 294. |
| Biological preparation for leguminous crop of liquid form based on nodule bacteria / 2514217 Biological preparation comprises the culture fluid containing the strain of nitrogen-fixing nodule bacteria with a titre of not less than 107 CFU/ml, and an aqueous solution containing (wt %): sodium carboxymethyl cellulose (2.0-5.0), potassium sorbate (0.5-3.0) and glycerol (1.0-5.0), mixed in a ratio of from 1:1 to 1:2. |
| Bacterial strain bacillus thuringiensis varthuringiensis n800/15 as agent for preparing entomocidal biopreparation / 2514211 Invention refers to microbiology, particularly to the protection of agricultural, medicinal and forest crops. The strain Bacillus thuringiensis var. thuringiensis No.800/15 (BtH1 800/15) possesses the entomocidal activity on depredators - phytophage insects. The strain is deposited in State Scientific All-Russia Research Institute for Agricultural Microbiology (GNU VNIISHM), No. 611. |
 Strain of lactobacilli lactobacillus paracasei cncm i-2116 (ncc 2461) eliciting ability to prevent intestine colonization with pathogenic microorganisms causing diarrhea, supernatant of its culture and oral agent for prophylaxis and/or treatment of diarrhea-associated disorders / 2243779Invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea. |
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.
1 ex
The invention relates to biotechnology, Microbiology and may be used at the stage of accumulation of biomass producer strain in the production of tularemia live vaccine.
It is known that the tularemia demanding to the nutrient media. For its cultivation usually use the protein environment based on natural substrates: egg yolk, blood, liver, brain, and other [Olsufiev I.G. Taxonomy, Microbiology and laboratory diagnostics of tularemia. - M: Medicine, 1975. - 190 S.]. It is often to promote growth are used vitamin and mineral Supplement containing, in particular, calcium Pantothenate, thiamine, cysteine or cystine, magnesium ions [Private medical Microbiology techniques microbiological studies: the manual. Ed. Aserinsky, Lpeinado, Asesinos.- M: OJSC Publishing house "Medicine", 2005. - 600 C.].
For the cultivation of tularemia bacteria are widely used dense (agarwood) containing media as the basis hydrolysates (autolysates) fish or meat extracts or autolysates yeast with the mandatory addition of cysteine (cysteine) 0.1% and glucose 1%. However, nutritionally dense environment is not suitable for deep cultivation and accumulation of biomass tularemia pathogen in production of medical immunobiological preparations.
In the production of tularemia live vaccine to date for the accumulation of biomass in conditions of deep cultivation used semi environment. In the medium composition includes hydrolysates fresh fish, liver or meat - 20-30%, hydrolyzed gelatin - 10%, gelatin - 1,5%, sodium chloride 0.5 percent, glucose - 1%, cystine - 0,1%; pH environment of 7.2-7.3 for [Kosticka PS, Shmurygina A.A. Improvement of preparation of dry tularemia live vaccine. // IMAI. - 1957, №10. - P.84-89]. Sowing dose is 0.5-1.0 mlgc/ml of culture medium (industry standard sample turbidity special 42-28-85-P (10 ME) fgbu ncesmp Ministry of Russia, the equivalent concentration of 5 mlrd/ml). The effectiveness of semi-liquids used for the production of vaccines, small, after 18-20 h of cultivation density of suspended solids increases 5-6 times [Olsufiev N.G. Tularemia. - 1960. - 167 C.].
Liquid albumen, for the cultivation of tularemia pathogen, experienced in various forms, but as a rule, growth was possible only with a massive planting dose [Olsufiev N.G. Taxonomy, Microbiology and laboratory diagnostics of tularemia. - M: Medicine, 1975. - 190 S.]. As a liquid environment Chamberlain used 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 pathogen in this environment is observed only at inoculation 1-2 billion microbial cells per 1 ml environment (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-datin - 0,4, L-lysine is 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 [may V.G., Shishov I.N., Bashilova GI Nutritional requirements Francisella tularensis II Microbiol. - 1984. - 6. - 20-23]. The disadvantage of this environment is the high cost.
Famous semi-synthetic liquid nutrient medium (pH 7,2), containing as a basis yeast extract 5 g/l, with the addition of salts (phosphate one-deputizing 12 g/l, sodium chloride 10 g/l, potassium hydroxide of 3.9 g/l, sulfate iron (II) as of 6 g/l)glucose 2 g/l and cysteine 0.1 g/l, which is proposed to use for molecular genetic studies of tularemia pathogen [Lapin A.A. Pavlov, V.M., Domotenko L.V., Temples M.V., Makarevich A.N. Simple liquid nutrient medium for molecular genetic studies Francisella tularensis II Problems of especially dangerous infections. - 2009, №102. - P.66-67].
Famous semi-synthetic nutrient medium T, containing as nutrition cardio-cerebral infusion of 10 g/l, bakterien 10 g/l, yeast extract 10 g/l, Kazarinova acid 10 g/l; as glucose and electrolyte additives use a solution containing MgSO4, FeSO4, KCl, K2HPO4, glucose, cysteine, sodium citrate, which is added into the medium before using [Pavlovich N.V., Michaniki B.N. Transparent nutrient media for cultivation of Francisella tularensis II Antibiotics and honey. biotechnol. - 1987, vyp. - S-137].
The problem with the above liquid media is relatively high cost and lack of effectiveness, in particular, they do not provide the necessary growth of viable organisms in the logarithmic stage for 20-24 hours
In the patent RU 2451743 [BIPM №15 27.05.2012,] to obtain biomass tularemia pathogen by the method of deep cultivation proposed liquid nutrient medium, containing (g/l: pancreatic digest of rabotoy flour 20,5, growth factors Haemophilus organisms 5, Baker's yeast extract 2,3, magnesium sulfate 1,0, sodium sulfite 0,7, potassium dihydrophosphate 1, cysteine of 0.5. At cultivation of tularemia pathogen on Wednesday add a solution of glucose-vitamin additives (1,23 g/l) in the amount of 2 ml per 100 ml of medium. The accumulation of biomass in this environment through 18 yo 2 hours is 18 mlrd/ml. the Disadvantage of this environment is the complexity and lack of effectiveness in their ability to accumulate biomass of microorganisms for the production of tularemia live vaccine.
The present invention is to construct a qualitative simple medium, which provides the increase of biomass yield tularemia pathogen.
The technical result of the invention consists in increasing the efficiency of nutrient medium for accumulation of biomass suitable for production mibp (including tularemia live vaccine), the expansion of raw materials for making a protein basic nutritional environment, the cheaper it through the use of waste serum-vaccine production, and ensuring the rational utilization of waste generated in the production of medical preparations.
The technical result is achieved by the fact that the culture medium includes dry enzymatic hydrolysate of fibrin made from waste serum-vaccine production, glucose, calcium Pantothenate, in the following ratio, g/l:
enzymatic hydrolysate of fibrin
50-55(content no less than 0,32% amino nitrogen)
glucose 10calcium Pantothenate
0,05distilled water
else pH 7,0-7,2Dry enzymatic hydrolysate of fibrin made from waste serum-vaccine production, used as a nutritional basis claimed nutrient medium. Enzymatic hydrolysate fibrin - pancreatic prewar fibrillar the blood protein is good protein nutrient media.
Cooking liquid enzymatic hydrolysate of fibrin given in the patent RU 2425866 [BIPM №22 10.08.2011 year]. Recently liquid protein hydrolysates, used as the basis for nutrient media, cooked in a dry form. Dry hydrolyzed conveniently transported and stored for long periods at room temperature while maintaining its quality, unlike the liquid form stored in (6 yo 2)C. Liquid form of enzymatic hydrolysate of fibrin concentrated in 6-7 times by evaporation on the installation of vacuum evaporation UVV-50 and dried convection way to the spray drying plant type KAUL 101325.002 in pseudocyesis layer".
In the aggregate state of dried enzymatic hydrolysate of fibrin was a fine powder with a pale yellow hue. At the analysis of physical-chemical parameters defined: the content of total nitrogen (0,76 ħ 0.03)%; amine nitrogen (0,39±0,03%; percentage of protein cleavage - (50,7±1,7%; content peptone (on a scale of Difco) - (53,7 ą1.5)%; traces of undigested protein was absent; dry residue (4,7±0,2%; chlorides - (0,22±0,01)%; humidity - (2,4±of 0.2%; pH (6,9±0,2)that meets the requirements applicable to dry the basics of nutrient media. Control the dry form the basis of the nutrient medium was carried out in accordance with the regulations [bacteriological Methods of control of nutrient media: guidelines MUK 4.2.2316-08. - M: Rospotrebnadzor. - 2008. - 67 C.; Methods of control of medical immunobiological preparations entered people: HOWTO MUK 4.1/4.2.588-96. - M: Information and publishing center of the Russian Ministry of health. - 1998. - 128 S.].
Nutrient medium with a basis of enzymatic hydrolysate of fibrin for growing tularemia pathogen is prepared in the following way. The broth is cooked in the reactor: under vacuum pump the water purified by distillation, adding 10% on its boiling, add 50-55 g/l dry enzymatic hydrolysate of fibrin in number, ensuring that the environment is not less than 0,32% amino nitrogen. Then make glucose concentrations up to 1%. All mix and set in the environment pH (7,2+0,2) using a 20% solution of sodium hydroxide and boil for 30 minutes the Soup is filtered through a filter Salnikova at temperature (75±5)OC C under pressure (2,5±0,5) MPa. Liquid environment of enzymatic hydrolysate of fibrin sterilized water and saturated steam at (110+2)OC C for 30 min, pressure (0,05 of 0.02) MPa. Before making inoculum sown culture in a sterile environment add a sterile solution of calcium Pantothenate at a concentration of 0.005% (0.05 g/l).
Possibility of realization of the claimed invention is confirmed by the following example.
Example 1. Deep cultivation of tularemia pathogen in a liquid nutrient medium.
Prepared culture F. tularensis vaccine strain 15 NIIEG grown using submerged culture in the bioreactor with automatic keeping of cultivation parameters: aeration 0.5-0.7 liters of air per 1 l of nutrient media, stirring speed of 500 rpm for 20 yo 2 h liquid nutrient medium of the following composition: hydrolyzed fibrin 5%, glucose 1%, calcium Pantothenate 0,005% pH of 7.2 at a temperature of 37 C. After the process of cultivation of native culture F. tularensis vaccine strain 15 NIIEG has the following characteristics: pH 7.0±0,1, the concentration of microbial cells 35±0,5 mlrd in ml environment (industry standard sample turbidity CCA turbidity 42-28-85-P (10 ME) fgbu ncesmp Ministry of Russia, the equivalent concentration of 5 mltl/ml).
Biomass tularemia pathogen, obtained by the claimed nutrient medium on the basis of the hydrolysate Tiberina, has morphology (shape and size of the cells) and cultural (form colonies) properties characteristic of tularemia. The obtained biomass tularemia pathogen has the following characteristics: coefficient of viability 62±0,5%, the degree of dissociation 97 of + / -1% SR (white) immunogenic colonies from the total amount raised. Cultural morphological traits typical of the tularemia pathogen: small coccoid sticks, motionless, gram-negative. Culture agglutinated tularemia serum to titer 1:3200.
Thus, the claimed nutrient medium for submerged culture of tularemia pathogen based on enzymatic hydrolysate of fibrin allows to accumulate and to get within 20 yo 2 h high concentration (up to 35±0,5 mlrd in ml of medium) viable biomass F. tularensis with a low degree of dissociation, in excess result in similar environments, which can significantly reduce the cost of the medium and therefore the ultimate drug mibp. Performance indicator environments, based on hydrolyzed fibrin, 2 times higher in comparison with the environment, based on pankreaticheskoi hydrolyzate of rabotoy flour. The increase of biomass of microorganisms due to presence in a nutrient medium for a balanced and effective team.
The invention extends the range of nutrient medium for bacterial accumulation mass F. tularensis and for the first time allows the accumulation of biomass in liquid medium in conditions of deep cultivation. Using the structure of the liquid environment of hydrolyzed fibrin, which is a waste of serum-vaccine production, allows to dispose of waste and reduces pollution of the external environment, and increases the profitability of production of the vaccine through the use of economically beneficial nutrient medium, and the scale of serum-vaccine production allow to get a considerable number of such raw materials.
Nutrient medium for accumulation of biomass tularemia pathogen that contains nutritional basis, glucose notable as nutrition contains dry enzymatic hydrolysate fibrin - waste serum-vaccine production, and additionally contains calcium Pantothenate, in the following ratio, g/l:
enzymatic hydrolysate of fibrin
50-55(content no less than 0,32% amino nitrogen)
glucose 10calcium Pantothenate
0,05distilled water
else pH 7,0-7,2