Liquid medium of drying for stabilisation of biomass of secondary harvesting of plague microbe of vaccine strain ev

FIELD: biotechnology.

SUBSTANCE: invention is a liquid medium of drying for stabilisation of biomass of secondary harvesting of plague microbe of vaccine strain EV. The medium comprises 8.0-12.0 g/l medical gelatine; 80.0-120.0 g/l sucrose; 8.0-12.0 g/l thiourea; 2-3 ml of 20% sodium hydroxide solution in flakes, and water for injection up to 1 litre.

EFFECT: invention enables to obtain high-quality liquid medium of drying for stabilisation of biomass of secondary harvesting of plague microbe of vaccine strain EV with enhanced stabilising properties and can be used to prepare suspension for injections, cutaneous scarification application in ampoules in the volume of 1 ml.

3 ex

 

The invention relates to biotechnology and Microbiology, may be used for preparation of suspension for injection and skin scratch application.

Known nutrient medium for drying the biomass of the plague microbe, which is used for making, g/l: sucrose - 0.4 kg; gelatin - 0.04 kg; sodium glutinously - 0.06 kg; the thiourea - 0,02 kg; peptone is an enzymatic - 0,002 kg; distilled water to 1 liter Distilled water, pH 7.0, and 7.1 is heated to a temperature of (50±2)°C, dissolve in her sucrose; lactose; sodium glutinously; thiourea 0,02; peptone is an enzymatic - 0,002; check pH and, if necessary, correct 20% solution of caustic soda to pH of 7.1 to 7.2. After complete dissolution of the ingredients of the medium is filtered through a cloth filter, pour it into bottles, and then sterilized. After sterilization, the bottles allow to cool down to a temperature of (22±3)°C, then placed in a thermostat at a temperature of (37±1)°C (48±2) hours In the absence of the gains of the environment drying bottles of its use in production. (Regulation of Production No. 368-92. The plague vaccine live dry. 1997 238 C.).

The disadvantage of this nutrient medium drying is insufficient stabilizing properties.

Closest to the present invention on the technological nature of nutritive medium is vissian what I biomass plague vaccine strain EV, which is used for making the sucrose - 1 kg; gelatin - 0.1 kg; the thiourea - 0.1 kg; sodium glutinously - 0.15 kg; peptone is an enzymatic - 0.05 kg; sodium hydroxide (20±1)% solution - 2.5 ml; distilled water to 1l Distilled water pH 7.0, and 7.1 is heated to a temperature of (50±2)°C, dissolve gelatin in it, check the pH and, if necessary, correct 20% solution of caustic soda to a pH of 7.1 to 7.2, add saccharose, thiourea, peptone, sodium and glutinously gelatin medical. After complete dissolution of the ingredients of the medium is filtered through a cloth filter, pour it into bottles, and then sterilized. After sterilization, the bottles allow to cool down to a temperature of (22±3)°C, then placed in a thermostat at a temperature of (37±1)°C (48±2) hours In the absence of the gains of the environment drying it is used in production. (Regulation of Production No. 702-97. The plague vaccine live dry. 2002 262 S.).

The disadvantage of this nutrient medium drying is the large number of ingredients in its composition and the fact that biomass harvesting is done in ampoules of 5 ml 2 ml and insufficient number of vaccinated remains vaccines have to destroy that unprofitable.

The technological results of the present invention to provide a environment drying liquid as a stabilizing medium new the second composition to obtain a high-quality vaccine plague microbe in sufficient quantities for secondary biomass harvesting, obtained at a lower temperature -(21±1)°C and poured into ampoules of 1 ml

The specified effect is achieved by the fact that the environment of the drying liquid stabilizing contains sucrose, gelatin medical, thiourea and purified water with the following ingredients, g/l:

Gelatin medical8,0-12,0
Sucrose80,0-120,0
Thiourea8,0-12,0
Sodium hydroxide in flake solution 20%2-3 ml
Water for injectionto 1 l

Gelatin - raw materials for the medical industry GOST 23058-89. Indicators required for verification: appearance - grains light yellow color; flavor sweet; authenticity - withstand test on GF X, article 309; duration of dissolution, min, not more than 25, pH of 5.6±0,4; odor - specific, without putrefaction; microorganisms (in the absence of bacteria of the family Enterobacteriacea, Staphylococcus aureus, Pseudomonas aemginosa). To obtain the vaccine.

Sucrose GOST 5833-75 analytical grade. Indicators required for control: appearance crystalline powder; authenticity - remove the supports test X State Pharmacopoeia (GF - X); solubility - soluble in water, insoluble in absolute alcohol, chloroform; the color - withstand test according to GOST 5833-75; acidity as CH3COOH, % standard 0,005; invert sugar 0,05. To obtain the vaccine.

Thiourea GOST 6344-73 analytical grade, reagent-grade. Indicators required for control: appearance colourless shiny crystals; solubility - soluble in water, dissolved by heating in alcohol; mass fraction of basic substance,%, not less than 99.

Sodium hydroxide in flake GOST - 4328-77, (analytical grade) is used to establish a pH of 7.6±0,2.

Water for injection - performance standard FS 42-2619-97. Colorless transparent liquid, odourless and taste: specific conductance, MK/cm) - not more than 0.5; pH - 5,0-7,0; reducing substances on FS - 2619-97; chlorides, mg/ml not more than 0,0001; sulfites, mg/ml not more than 0,003; calcium, mg/ml - 0,0035; heavy metals, mg/ml not more than 0,0005; microorganisms units/ml not more than 100 in the absence of bacteria of the family Enterobacteriacea, Staphilococcus aureus, Pseudomonas aemginosa.

Experiments to simulate temperature conditions of cultivation of the plague microbe in a gradient of 21°C in comparison with regulation 27°C showed that the temperature of the growing significantly affect the number of microbial cells. The temperature acts as a factor influencing biosynthetic about Essy in cytoplasmic membranes of the cells by increasing the content of unsaturated fatty acids, accumulation of which in the membranes of cells contributes to their greater resistance to freeze-drying (Ivanova GF, Boudica D.A., Abzaeva NV Collection of scientific papers. - Stavropol, 2007. - S-188; Tinker A.I., Boudica D.A., Verkhovtseva G.N., Pechnikov E.N. Topical prevention of especially dangerous infectious diseases. Kirov, 1991. - Ñ.38-40).

Unlike the prototype of the proposed environment provides high stabilizing properties, simple to use to obtain secondary biomass harvesting plague vaccine live dry.

Upon receipt of secondary biomass harvesting plague vaccine strain EV it is necessary to count the number of microbial cells in a nutrient medium dense for the cultivation of plague vaccine strain EV prepared by enzymic hydrolysate beef.

The method of counting the number of microbial cells

Nutrient agars from pancreatic parivara meat (agar of Hottinger for plague microbe in FS 42-3204-98)used to determine the amount of live microbial cells must provide, without added stimulants, growth of microbes vaccine strain EV at all Petri dishes with agar, seeded 10, M.K. on the TOC turbidity 42-28-P 10 IU. As a growth promoter in the nutritional environments before pouring into Petri dishes add 1 ml/l demolizione blood or 0.25 g/l fresh is cooked boiled sodium semitecolo. Tubes with 0.9% sodium chloride solution and pipette used to determine the content of living microbial biomass, must be cooled to a temperature (4±2)°C. of each tube with a nutrient liquid content in an amount of 0.1 ml diluted with 0.9% solution of sodium chloride in the amount of 0.9 ml (main breeding 10-1). Add sequentially saline solution to produce 1 billion M.K./ml. Then pipette make a serial tenfold dilution of the resulting suspension in 0.9% solution of sodium chloride ranging from 10-1(0,5±0,01) ml suspension from 4.5±0.05 ml of 0.9% solution of sodium chloride and ending 10-5. Divorced biomass of the last two test tubes (10-5sow the pipette (0,1±0,01) ml 2 Petri dishes with nutrient agar (pH of 7.2) and incubated at a temperature (21±1)°C for 2-3 days.

Preparation of enzymatic hydrolysate of beef.

In the reactor pour drinking water from the calculation of available meat (1 kg of meat 1.5 liters of water). Meat without fat and sinew, cut into pieces of size 2.5 2.5 see Cook the meat for 15 minutes, then remove. Cool and mince. The broth has cooled to 50°C alkalinized Na2Co3based 3.0 g soda to 1 L. Add the pancreas based 80,0-100.0 g per 1 kg of meat, depending on the activity of the pancreas. The activity must be not less than the 5 thousand units of Fold-gross. Add chloroform to 2% of the total volume of the contents of the reactor. The digestion is performed at 37°C. the First day of the hydrolysate is stirred with a mechanical stirrer, which include every 2 h for 5 minutes Daily to determine the amine nitrogen in the hydrolysate. With halting amine nitrogen, which is 7-10 days, the stirrer is switched off, the heating is stopped. Allow to settle for 2 days, then the liquid is filtered from the precipitate at a press filter, poured into bottles with addition of 1% chloroform, zapressovyvajut and stored at a temperature of 4-6°C.

Characterization of the enzymatic hydrolysate beef - pH 7.0; amine nitrogen 1100 mg %; amine nitrogen 878 mg %; the percentage of cleavage 78,0%; sodium chloride 0,087; peptone 1,2; calcium 4,2 mg %; magnesium to 5.21 mg %; phosphorus total of 6.5 mg %; inorganic phosphorus to 57.1 mg %; dry residue 10,0-1,5%; reducing agents 367 mg %.

Preparation of enzymatic hydrolysate of beef (MUK 4.1/2.588-96, p.45)

Enzymatic hydrolysate of beef decanted from the container, then filtered through a cloth. Determine the number of amine nitrogen and its content calculate the quantity of hydrolysate necessary for preparation of the nutrient medium. If you want clarification charcoal (dark hydrolysate), you take 10% more, considering the adsorption of amino acids when processing the coal. If the hydrolyzate after breeding on the amine nitrogen has a straw-yellow color, it does not lighten. Diluted with purified water 2 times, heated to boiling and add 2% activated charcoal brand OS (in the calculation of the diluted hydrolysate). Boil for 15-20 minutes while stirring. Allow to settle for 20-30 minutes and filtered first through cotton wool, and then through the filter cloth.

Preparation of nutrient media for cultivation of plague vaccine strain EV.

Nutrient agars from pancreatic parivara meat (agar of Hottinger for plague microbe in FS 42-3204-98)used to determine the amount of live microbial cells, should provide (without added stimulants) microbial growth vaccine strain EV at all Petri dishes with agar, seeded 10, M.K. on the TOC turbidity 42-28-P ME. As a growth promoter in the nutritional environments before pouring into Petri dishes add 1 ml/l demolizione blood or 0.25 g/l of freshly boiled sodium semitecolo. Tubes with 0.9% sodium chloride solution and pipette used to determine the content of living microbial biomass, must be cooled to a temperature (4±2)°C. of each tube with a nutrient liquid content in an amount of 0.1 ml diluted with 0.9% solution of sodium chloride in the amount of 0.9 ml (main breeding 10-1. Add sequentially saline solution to produce 1 billion MCL/ml. Then pipette make a serial tenfold dilution of the resulting suspension in 0.9% solution of sodium chloride ranging from 10-1(0,5±0,01) ml suspension from 4.5±0.05 ml of 0.9% solution of sodium chloride and up to 10-7. Divorced biomass of the last two test tubes (10-6and 10-7sow the pipette (0,1±0,01) ml 2 Petri dishes with nutrient agar pH of 7.2±0.2 and maintained at a temperature (21±1)°C for 2-3 days.

Cooking environment drying liquid to stabilize the biomass of secondary collection plague vaccine strain EV.

Water for injection is heated to a temperature of (50±2)°C, dissolve gelatin in it, set the pH of a 20% sodium hydroxide solution to (7,6±0,2), add sucrose and thiourea, bring to a boil. After complete dissolution of the ingredients of the medium is filtered through a filter cloth and filter paper, poured into a graduated flask and siphons. In the latter case, a funnel with a filter insert directly into the bottle, pre-sterilized, together with installation, and pour the environment about. Then in the bottle again insert the installation, tie it with twine and throat bottles wrapped with cheesecloth, folded into several layers. The medium is sterilized at 121°C for 30 minutes After sterilization, allow the bottles to cool to the fact the temperature (22±3)°C. Then remove the gauze and then the throat of the bottle thoroughly smeared in hot mixture of wax with paraffin in a ratio of (3:2) or pour paraffin. Bottles with the medium placed in a thermostat at (37±1)°C (48±2) hours In the absence of the gains of the environment it is used in production.

As an example, experienced a plague culture vaccine strain EV grown on plates, 2% agar of Hottinger pH (7,2±0,1) at a temperature (27±1)°C within 24 hours Of daily culture was prepared a suspension of 1 billion MCL/ml vaccine strain of the plague microbe EV, equal to 10 units on the optical standard turbidity (CCA 42-28-85 P), equivalent to 1.0×109M.K./ml in sterile 0.9% sodium chloride solution. Serial 10-multiple dilutions in physiological solution of 4.5 ml conveyed to the content in 1 ml of 1.03·106living microbial cells. From this breeding suspension culture were sown 0.1 ml to 5 ml of liquid nutrient medium. Crops were incubated at (21±0,1)°C. analysis was performed every 3 hours for 48 h to determine the number of microbial cells.

Example 1. When the secondary biomass harvesting plague vaccine strain EV grown on solid nutrient medium, the application of the stabilizing liquid environment containing, g/l: medical gelatin - 8,0; sucrose - 80,0; thiourea - 8,0; sodium hydroxide in flake solution 20% - 2 ml; water for injects the th to 1 L. With this ratio of ingredients, the number of microbial cells at a temperature of 21±1°C after 48 h of cultivation amounted to 11 billion M.K./ml

Example 2. When the secondary biomass harvesting plague vaccine strain EV grown on solid nutrient medium, the application of the stabilizing liquid environment containing, g/l: medical gelatin - 10,0; sucrose - 100,0; thiourea - 10,0; sodium hydroxide in flake solution 20% - 2.5 ml; water for injection up to 1 L. this ratio of ingredients, the number of microbial cells at a temperature of 21±1°C after 48 h of cultivation amounted to 30 billion M.K./ml

Example 3. When the secondary biomass harvesting plague vaccine strain EV grown on solid nutrient medium, the application of the stabilizing liquid environment containing, g/l: medical gelatin - 10,0; sucrose - 100,0; thiourea - 10,0; sodium hydroxide in flake solution 20% - 2.5 ml; water for injection up to 1 L. this ratio of ingredients, the number of microbial cells at a temperature of 21±1°C after 48 h of cultivation amounted to 13 billion M.K./ml

Thus, the claimed Wednesday drying liquid to stabilize the biomass of secondary collection plague vaccine strain EV (sample No. 2) is the optimal number of carefully selected ingredients, which together allows to obtain a sufficient number of microbial cells at t is mperature 21±1°C after 48 h of cultivation. When the secondary biomass harvesting plague vaccine strain profitability vaccine is increased by 5%.

Wednesday drying liquid to stabilize the biomass of secondary collection plague vaccine strain EV, consisting, g/l:

Gelatin medical8,0-12,0
Sucrose80,0-120,0
Thiourea8,0-12,0
Sodium hydroxide in flake solution 20%2-3 ml
Water for injectionto 1 l



 

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