A method of manufacturing a vaccine against salmonellosis farm animals

 

(57) Abstract:

The invention relates to biotechnology and can be used in the industrial production of dry live vaccine against salmonellosis farm animals. For this deep cultivation should be performed within 6-8 h at (371)oWith decreasing redox potential of the medium after planting, maintaining the partial pressure of dissolved in the culture liquid oxygen and dosed supply of glucose as the limiting growth of Salmonella glucose. The resulting culture concentrate, diluted, Saharsa gelatin with thiourea protective environment for califorina buffer solution, Packed into vials and freeze-dried. The use of this mode of cultivation of Salmonella and protective environment can reduce the cost of vaccine and to increase the accumulation of viable bacteria, the quality, stability and standardization of the biological properties of the vaccine. table 1.

The invention relates to biotechnology and can be used in industrial production of dry live vaccine against salmonellosis farm animals.

A method of obtaining dry live vaccine PA vaccine against salmonellosis farm animals has a number of disadvantages: the cultivation process is quite long (12-14 h), does not allow to use modern equipment and devices, protective environment for freeze drying does not provide stability of the vaccine during long-term storage, which affects the quality of the drug, as well as on the efficiency of production.

In the analysis of patent documents and scientific literature we have not found quite effective way of making living dry vaccines against salmonellosis farm animals approaching the current level of production of biological products.

The aim of the invention is the reduction of time of cultivation, reducing the cost of the drug, as well as improving the quality, stability during storage and standard biological properties of the vaccine.

This goal is achieved due to the fact that periodical deep cultivation of Salmonella is carried out in a nutrient medium for 6-8 h in accordance with the developed control algorithm process, concentrating the obtained culture, bacterial mass resuspending developed in a protective atmosphere and dried.

The method is as follows.

In sterile farms is entire seeded with a culture of Salmonella (Salmonella choleralsuis or Salmonella dublin), grown in liquid nutrient medium of the same composition, and is grown at a temperature (371)oC for 6-8 hours.

After inoculation of the fermenter redox potential of the culture liquid is reduced to (-110) - (-130) mV by keeping culture without air for aeration and minimum speed mixer, then to the end of the cultivation process with changes in the flow rate of air for aeration and the rotation speed of the mixer to maintain the partial pressure of dissolved in the culture liquid oxygen at the level of 15-25% of the oxygen saturation of the air, the pH of the culture fluid regulate 10% NaOH solution, supported at the level of 7.6 to 7.8 pH units, the fractional flow of glucose implement doses to a concentration of 0.05 to 0.15% when limiting the growth of Salmonella glucose, characterized by a sharp increase pO2at constant air flow rate and the speed of the stirrer and the termination of reducing the pH of the culture fluid. The resulting culture concentrate, diluted, Saharsa gelatin with thiourea protective environment for potassium-phosphate buffer solution, Packed in vials and freeze dried.

Example 1. A method of manufacturing a vaccine with a minimum of Zn is ve parivara of Hottinger.

The environment is seeded with a culture of Salmonella grown in liquid nutrient medium of the same composition. Cultivated at (371)owithin 6 hours. After inoculation of the fermenter redox potential of the culture liquid is reduced to -110 mV by keeping culture without air for aeration and turned off the mixer, then to the end of the cultivation process with changes in the flow rate of air for aeration and the rotation speed of the mixer to maintain the partial pressure of dissolved in the culture liquid oxygen at the level of 15% of the oxygen saturation of the air, the pH of the culture fluid at 7.6 pH units with delivery of 10% NaOH solution, and the fractional flow of glucose is performed by doses up to concentrations of 0.05%, while limiting the growth of Salmonella glucose, characterized by a sharp increase pO2at a constant air flow and the speed of the stirrer and the termination of reducing the pH of the culture fluid. The accumulation of biomass in 1 cm3is 22.6 billion Obtained culture concentrate and dilute saharso gelatin with thiourea protective environment for califorina buffer solution, prepared according to the following recipe, wt.%:

Sucrose - 8,0

Gelatin - 0,4

4

Distilled water - Up to 100.0

Diluted culture Packed into vials and freeze-dried.

The number of living cells in the vaccine prior to drying in 1 cm3is of 15.0 billion, after drying, of $ 7.3 billion, after 12 months. storage - 2.9 billion

Example 2. A method of manufacturing a vaccine with optimal parameter values.

For the cultivation of Salmonella using liquid nutrient medium on the basis of parivara of Hottinger.

The environment is seeded with a culture of Salmonella grown in a nutrient medium of the same composition. Cultivated at (371)oC for 7 hours After inoculation of the fermenter redox potential of the culture liquid is reduced to -120 mV by keeping culture without air for aeration and turned off the mixer, then to the end of the cultivation process with changes in the flow rate of air for aeration and the rotation speed of the mixer to maintain the partial pressure of dissolved in the culture liquid oxygen at 20% of saturation with oxygen, the pH of the culture fluid is maintained at 7.7 pH units with delivery of 10% NaOH solution, and the fractional flow of glucose is performed by doses up to a concentration of 0.1% when limityou and speed stirrer and termination of reducing the pH of the culture fluid. The accumulation of biomass in 1 cm3the environment is of 40.0 billion Obtained culture concentrate and dilute saharso gelatin with thiourea protective environment for califorina buffer solution, prepared according to the following recipe, wt.%:

Sucrose - 10,0

Gelatin - 0,5

Thiourea - 05

Potassium phosphate disubstituted - 0,5

Potassium phosphate one-deputizing - 0,17

Distilled water - Up to 100.0

Diluted culture Packed into vials and freeze-dried.

The number of living cells in the vaccine prior to drying in 1 cm3is 30,0 billion, after drying of 28.0 billion in 12 months. storage - 24.0 billion

Example 3. A method of manufacturing a vaccine with the maximum values of the parameters.

For the cultivation of Salmonella using liquid nutrient medium on the basis of parivara of Hottinger.

The environment is seeded with a culture of Salmonella grown in liquid nutrient medium of the same composition. Cultivated at (371)oC within 8 hours After inoculation of the fermenter redox potential of the culture liquid is reduced to -130 mV by keeping culture without air for aeration and turned off the mixer, then to the end about the Ute partial pressure dissolved in the culture liquid oxygen at 25% of saturation by oxygen, the pH of the culture fluid is maintained at the level of 7.8 pH units with delivery of 10% NaOH solution, and the fractional flow of glucose implement doses to a concentration of 0.15%, while limiting the growth of Salmonella glucose, characterized by a sharp increase pO2at a constant air flow and the speed of the stirrer and the termination of reducing the pH of the culture fluid. The accumulation of biomass in 1 cm3the environment was 25.0 billion Obtained culture concentrate and dilute saharso gelatin with thiourea protective environment for califorina buffer solution, prepared according to the following recipe, wt.%:

Sucrose - 12,0

Gelatin - 0,6

Thiourea - 0,6

Potassium phosphate disubstituted - 0,6

Potassium phosphate one-deputizing - 0,2

Distilled water - Up to 100.0

Diluted culture Packed into vials and freeze-dried.

The number of living cells in the vaccine prior to drying in 1 cm3is 20.0 billion, after drying 14.0 billion in 12 months. storage - 8.0 billion

Example 4. A method of manufacturing a vaccine with values below the minimum.

For the cultivation of Salmonella using liquid nutrient medium on the basis of parivara of Hottinger.

The number of living cells in the vaccine prior to drying in 1 cm3is 2.0 billion, after drying 0.9 billion, after 12 months. storage - 0.03 bln

The environment is seeded with a culture of Salmonella grown in liquid nutrient medium of the same composition. Cultivated at a temperature of (371)oC for 9 h After inoculation of the fermenter redox potential of the culture liquid is reduced to -140 mV by keeping culture without air for aeration and turned off the mixer, then to the end of the cultivation process with changes in air flow and the speed of rotation of the agitator support partial pressure dissolved in the culture liquid oxygen at 30% of saturation with oxygen, the pH of the culture fluid is maintained at the level of 7.9 pH units using 10% NaOH solution, and the fractional flow of glucose implement doses to a concentration of 0.20% when limiting the growth of Salmonella glucose, characterized by a sharp increase pO2at a constant air flow and the speed of the stirrer and the termination of reducing the pH of the culture fluid. The accumulation of biomass in 1 cm3environment is 10.0 billion Obtained culture concentrate and dilute saharso gelatin with thiourea environment califorina buffer solution, prepared according to the following recipe, wt.%:

Sucrose - 14,0

Gelatin - 0,7

Distilled water - Up to 100.0

Diluted culture Packed into vials and freeze-dried.

The number of living cells in the vaccine prior to drying in 1 cm3is 5.0 billion, after drying 1.8 billion, after 12 months. Storage - 0.2 billion

Technological parameters of manufacturing dry live vaccine against salmonellosis farm animals specified in the examples in the table.

Advantages of the claimed method known before:

1. The intensified use of the method of cultivation can improve the accumulation of viable bacteria while reducing time growing from 12 to 14 up to 6-8 hours.

2. The vaccine obtained in this way, more stable in biological properties, including immunogenicity.

A method of manufacturing a vaccine against salmonellosis farm animals, including seeding vaccine strain of Salmonella, culturing in a liquid nutrient medium on the basis of parivara of Hottinger with fractional flow of glucose, followed by concentration and lyophilization of the obtained bacterial suspension in a protective medium containing sucrose and gelatin, wherein the cultivation is carried out is to the end of the cultivation process support partial pressure dissolved in the culture liquid oxygen level 15 - 25% of the oxygen saturation of the air, the pH of the culture fluid is maintained at the level of 7.6 to 7.8 pH units, and the fractional flow of glucose implement doses to a concentration of 0.05 to 0.15% at limiting the growth of Salmonella glucose, freeze drying are in a protective environment, optionally containing thiourea, potassium phosphate and one-deputizing potassium phosphate disubstituted, in the following ratio, wt.%:

Sucrose - 8,0 - 12,0

Gelatin - 0,40 - 0,60

Thiourea - 0,40 - 0,60

Potassium phosphate disubstituted - 0,40 - 0,60

Potassium phosphate one-deputizing - 0,14 - 0,20

Distilled water Up to 100.0

 

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