Bacterial preparation, method for its producing, nutrient medium for culturing cells escherichia coli vkm cr-322d and method for prophylaxis and treatment of gastroenteric disease in agricultural and domestic animal and poultry

FIELD: biotechnology, microbiology, veterinary science.

SUBSTANCE: for preparing a preparation cells of microorganism Escherichia coli VKM CR-322D is cultured in nutrient medium containing Hottinger's broth, glucose, yeast extract, manganese sulfate, potassium hydrophosphate, sodium chloride and tap water in the content of amine nitrogen 125-155 mg%. Glucose is added by batch portions in the process of culturing cells that is carried out at temperature 30-31oC at stirring and aeration for 10-12 h. Prepared cultural fluid containing 3 x 109 bacterial cells/ml is mixed with protective sucrose-gelatin medium and subjected for lyophilic drying. Dried mass is stored under nitrogen that enhances safety of viable cells in the preparation. Applying the preparation for prophylaxis and treatment of agricultural and domestic animals and poultries with gastroenteric diseases provides its effectiveness.

EFFECT: improved preparing method, valuable veterinary properties of preparation.

17 cl, 8 ex

 

The invention relates to biotechnology and may find application in veterinary medicine.

The invention can be used for the prevention and treatment of gastrointestinal diseases in livestock and domestic animals and birds. In most cases, these diseases are contagious and are caused by the enterobacteria of different types, and some types of viruses. Often these diseases occur in the form of mixed viral-bacterial infections. Severe gastrointestinal diseases are also a dysbacteriosis.

A known method for the production of bacterial drug for prevention and treatment of gastrointestinal diseases (in particular, colibacillosis) of farm animals, including growing Escherichia coli cells BKM CR-322D at pH 7.0-7.5 on a nutrient medium containing broth of Hottinger, glucose, mineral salts and water, to obtain the bacterial cells, followed by mixing the resulting cells with sharethelove environment and freeze dried prepared mixture (Patent USSR No. 1819428, C 12 N 15/30,1990) [1] is the prototype of the method according to claim 1.

However, this known method are the small biomass of the cells and made from them the drug during storage loses its biological activity.

Known nutrient medium for cultivation of E. coli cells BKM CR-322D-containing broth, Hotti the Gera, glucose, MgS4salt of phosphoric acid and water [1] is the prototype of the nutrient medium.

However, the yield of cells when grown on this known environment is low.

Known bacterial preparation containing a lyophilized mixture of E.coli cells BKM CR-322D and sharethelove environment [1] is the prototype of the bacterial preparation.

However, the preservation of viable cells during storage of this well-known drug is low.

A known method of prevention and treatment of gastrointestinal diseases in animals, including the imposition of oral administration of Escherichia coli cells BKM CR-322D [1] is the prototype of the method according to claim 10.

However, this known method has been tested only on the calves, and the prescribed dose of the bacterial preparation is necessary for the prevention and treatment of gastrointestinal diseases in other animals and birds, is not specified.

The technical result achieved during the implementation of the present group of inventions is to increase the output of E.coli cells BKM CR-322D at their cultivation, improve the safety of viable cells during storage made with them of the bacterial preparation, empowerment of bacterial drug for prevention and treatment of gastrointestinal diseases in farm and domestic animals and birds.

This technical result is achieved by the in the way of getting a bacterial drug for the prevention and treatment of gastrointestinal diseases in livestock and domestic animals and birds, providing for the growing cells of Escherichia coli CR-322D on a nutrient medium containing broth of Gattinara, glucose, manganese sulfate, salt of phosphoric acid, water at pH 7.0-7.5, the mixing of the received biomass sharethelove environment and freeze drying, the distinguishing feature is that the composition of the nutrient medium further added yeast extract, sodium chloride as a salt of phosphoric acid it contains potassium phosphate in water quality - water, water, glucose is injected explored in the process of growing cells, which is conducted at a temperature of 30-31°and accompany him stabilization level pH, constant stirring and aeration, complete the process in 10-12 hours, and lyophilized subjecting the mixture obtained culture liquid with sharethelove environment in the ratio of 3:1.

Preferably use zacharopoulou a medium containing sucrose, gelatin and distilled water when the following ratio, wt.%:

sucrose 39-41

gelatin 5-6

distilled water the rest

Aeration of the nutrient medium is recommended by filing it sterile air at 1-2 l/min to 1 l of medium.

Mix the nutrient medium of a suitable stirrer with a speed of 300-500 rpm./minutes

Complete the process of growing cells in achieving them is the actual content in the culture fluid not below 30· 109cells/ml,

Freeze drying is recommended before the moisture content in the resulting product is not above 3%.

A container of dried product is preferably filled with nitrogen.

Achieved the specified technical result is also the fact that a nutrient medium for growing cells of Escherichia coli CR-322D containing broth of Hottinger, glucose, manganese sulfate, salt of phosphoric acid and water, additionally contains yeast extract, sodium chloride as a salt of phosphoric acid - phosphate of potassium, as a water - tap water in the following ratio, wt.%:

the broth of Hottinger 18-23

glucose 0,35-0,45

yeast extract 0,45-0,55

manganese sulfate 0,055-0,065

phosphate potassium 0,15-0,16

water water the rest

This technical result is also achieved by the fact that bacterial drug for profilaktiki and treatment of gastrointestinal diseases in livestock and domestic animals and birds characterized by the fact that is a freeze-dried biomass of Escherichia coli cells CR-322D obtained by the method according to claim 1 of the claims.

This technical result is also achieved by the fact that the method of prevention and treatment of gastrointestinal diseases in livestock and domestic animals and birds, including the appointment of them peroral the CSOs receiving cells of Escherichia coli CR-322D, has the distinctive feature that the cells of Escherichia coli CR-322D used in the form of the bacterial preparation according to claim 9 of the formula of the invention at a dose of 0.25-42 conventional units at the reception, taking 1 unit 1 billion microbial cells.

This technical result is also achieved due to the fact that newborn calves appointed reception of Escherichia coli cells CR-322D in the first 1.5 to 2.5 hours, and then 4-5 days of life in the dose 38-42 conventional units.

Calves when the disease is prescribed daily dose of cells of Escherichia cli CR-322D 2 times a day dose 38-42 conventional units until clinical recovery.

Newborn piglets appointed reception of Escherichia coli cells CR-322D in the first 4-6 hours. life, then for 6-7 days at a dose of 5-7 conventional units, and at the age of 2-3 weeks at a dose of 6-8 conventional units.

The piglets to weaning appointed reception of Escherichia coli cells CR-322D for 4-5 days prior to weaning, on the day of weaning and 4-5 days after weaning dose of 18-22 conventional units.

Dogs at dysbacteriosis appointed reception of Escherichia coli cells CR-322D dose 6-40 conventional units 2 times a day for 1.5-2.0 hours before food intake.

Cats in dysbacterioses appointed reception of Escherichia coli cells CR-322D at a dose of 3-5 units, 2 times a day.

Chiplatham in 1-5 days of life prescribed daily dose of Escherichia coli cells CR-322D dose of 0,25-0,35 conventional units 2 times a day, then 20-25 and 40-45 days of life - 1,8-,2 conventional units.

Our own observations have shown that the output cells of Escherichia coli CR-322D can be increased, if in a nutrient medium for cultivation to introduce yeast extract, change the qualitative and the quantitative ratio of its constituent salts, and glucose to enter into it gradually, as the incubation, which prevents the acidification of the environment and contributes to the intensification of increasing the biomass of cells.

It is established that growing cells of E. coli BKM CR-322D on the new composition of the nutrient medium with constant mixing and aeration, the incubation temperature should be 30-31°and pH of the medium should be maintained at around 7.0-7.5 by periodically adding 40%aqueous NaOH solution.

Preservation of viable E.coli cells BKM CR-322D subjected to freeze drying, can be improved by mixing with a protective sharethelove environment not E. coli cells BKM CR-D separated from the nutrient medium, as in the known method-prototype [1], and containing culture fluid. This allows to enrich the composition of the lyophilized drug is biologically active components remaining in the medium after incubation of the cells of E.coli BKM CR-322D. Preservation of viable cells in the drug also increases due to its storage in an atmosphere of nitrogen.

Below are examples of the implementation group izobreteny is.

Example 1.

To prepare the drug used Escherichia coli M17(R)obtained in the Institute of molecular genetics RAS [1], which was deposited under the number BKM CR-322D. The ampoule with liofilizovannyh culture of E.coli BKM CR-322D opened under aseptic conditions, the contents of the ampoule added 1-2 ml mycopathologia broth obtained cell suspension was inoculated into vials with mesopartner broth and incubated at 30C for 18 h.

The obtained culture was inoculated in a test tube with a beveled mesopartner agar with kanamycin and incubated for 18 h at 30°C. the Grown cells were perestal in a flask with 100 ml mycopathologia broth and incubated for 7 h on a shaker at 30°C. the resulting stock culture were used to produce inoculum. In bottles of 2 l mycopathologia broth sowed uterine culture at 2% of the volume of the nutrient medium in the flask. Seeded bottles were incubated at 30°on the rocking chair for 10-16 hours. The grown culture was introduced into the fermenter in the amount of 2.5% of the volume contained in the nutrient medium of the following composition, wt.%:

the broth of Hottinger 18

glucose 0,35

yeast extract 0,45

MgSO40,055

To2NRA40,15

NaCl 0,45

water water remainder (to 100%).

Medium containing 125 mg % amino nitrogen. The culture is incubated at a temperature of 30� C, under stirring with a stirrer at a speed of 300 rpm and aeration carried out by feeding the culture medium was sterile air at a rate of 1 l/min to 1 l of medium.

Glucose was injected into the environment in the form of a 40%aqueous solution. Just added 1% of the volume of the culture fluid of a 40%aqueous solution of sterile glucose.

One hour after the beginning of cultivation was added 0.05% glucose, 3 h - 0,1% glucose over 4 hours with 0.1% (final concentration), after 4-5 hours of cultivation glucose was added to a final concentration of from 0.8 to 1.2%.

During the culture pH was maintained at 7.0 and 7.5, occasionally giving it a 40%NaOH solution. After 12 hours. incubation was observed cessation of population growth. The number of bacterial cells in the obtained culture liquid was 30·109Bakal/ml optical density, measured by the FEC. For comparison: when growing E. coli cells ACM SC-D in accordance with the method of the prototype [1] was obtained culture fluid containing 7-10·109Bakal/ml

At the end of the cultivation process the temperature of the culture liquid has reduced and maintained at the level of 16°by submitting a shirt fermenter circulating water with a temperature of 9-10°C.

Then in the fermenter with the cooled suspension when operating the mixer filed protective sakh resoulation Wednesday, prepared in the following way: with 20%NaOH solution was made pH of distilled water to 7.0, and 7.1. Water brought to boil, added a 5% solution of gelatin, using a 20%NaOH solution brought the pH of the resulting solution to 8.5 and boiled for 15 minutes and Then to the resulting solution was added 40% sucrose. The resulting solution contained 5% of gelatin and 40% sucrose. After filtration and sterilization obtained zacharopoulou environment used as a protective environment. The culture fluid and protective environment were mixed in a ratio of 3:1. After 15 min stirring the resulting mixture was passed through sublimation (freeze) drying.

The mixture was poured into 4-10 ml in sterile bottles with a capacity of 20 ml, was subjected to freezing at -35°C for 20 hours.

The lyophilization was carried out in a vacuum drying apparatus, after sealing from which it was pumped out the air to vacuum 5·10-2mm Hg

The dried mass was humidity of 3%. After drying turned off the vacuum pump and to the tank through the filters filed purified nitrogen.

Before capping the vials filled with nitrogen, closed sterile rubber tubes, which are then fixed with metal caps.

The product was a dry porous mass of yellowish-white color.

Example 2.

According to the method of example 1 poluchiliss of E.coli cells BKM CR-322D, which is introduced into the nutrient medium of the following composition, wt.%:

the broth of Hottinger 23

glucose 0,45

yeast extract 0,55

MgSO40,065

To2NRA40,16

NaCl 0,55

water water the rest,

the content of amine nitrogen in the environment 155 mg %.

Incubation was conducted at a temperature of 31°C. the culture medium is metered filed glucose in the form of a 40%aqueous solution (see example 1). The vessel was continuously stirred with a stirrer at a speed of 500 rpm and continually introducing sterile air at 2 l/min to 1 l of medium. The process was completed after 10 hours when the content in the culture fluid 40·109Bakal/ml

The obtained culture liquid (3 parts) was mixed with 1 part of the protective sharethelove medium of the following composition, wt.%, so that the final concentration in the raw material was:

sucrose 10,5

gelatin 2

water remainder (to 100%).

Humidity lyophilized preparation was 2.5%.

Example 3. For the prevention of gastrointestinal diseases calves them with the first feeding of colostrum, but not later than within the first 2 hours of life, and then on the 4th day of life was administered at 40 conventional units suspension of Escherichia coli cells BKM CR-322D in the form of the bacterial preparation prepared by the method of example 1. Cases calves were observed.

Example 4. When detecting in the household of cases, infection is a rule-intestinal diseases of calves they had an appointment 2 times a day for 40 conventional units of the bacterial preparation prepared in accordance with example 1. The treatment was carried out for 3 days before full clinical recovery. Cases of recurrence was not detected.

Example 5. The piglets in the first 4-6 hours of life and then on the 7th day of life was drinking over 6 units, the bacterial preparation, prepared in accordance with example 1. To prevent edema disease to pigs, nursery pigs fed 20 conventional units of the preparation prepared according to example 1, at least 5 days prior to weaning, on the day of weaning and 5 days after weaning - to 20 conventional units. Cases of gastrointestinal diseases and edema disease of pigs was not detected.

Example 6. For the prevention of gastrointestinal diseases chickens 1-5 days of life with food or water was given to 0.3 conventional units of the bacterial preparation prepared according to example 1, on a daily basis. Then give 2 units of periods of 20-25 days of life and 40-45 days of life. Gastrointestinal diseases were not observed.

Example 7. Dogs with dysbacteriosis was drinking bacterial preparation prepared according to example 1 to 2 times a day for 1.5-2 hours before food intake in the number 6-40 conventional units to full clinical recovery (2-5 days).

Example 8. Cats with dysbacteriosis was drinking bacterial preparation prepared according to example 1 to 2 times a day for 1.5-2 hours before food intake for 3-5 from the course units to complete clinical recovery (2-5 days).

Thus, the group of inventions can improve the output of E.coli cells BKM CR-D when grown on a nutrient medium, to prepare a freeze-dried bacterial preparation, to improve its safety during storage and to improve the effectiveness of prevention and treatment of gastrointestinal diseases in livestock and domestic animals and birds.

1. A method of obtaining a bacterial preparation for the prevention and treatment of gastrointestinal diseases in livestock and domestic animals and birds, providing for the growing cells of Escherichia coli CR-322D on a nutrient medium containing broth of Hottinger, glucose, manganese sulfate, salt of phosphoric acid, water at pH 7.0-7.5, the mixing of the received biomass sharethelove environment and freeze drying, characterized in that the composition of the nutrient medium further added yeast extract, sodium chloride as a salt of phosphoric acid it contains potassium phosphate in water quality - water, water, glucose is injected explored in the process of growing cells, which is conducted at a temperature of 30-31°and accompany him stabilization of pH, constant stirring and aeration, complete the process after 10-12 h, and lyophilized subjecting the mixture obtained culture liquid with sharethelove environment in the ratio of 3:1.

2. SPO is about according to claim 1, characterized in that use zacharopoulou a medium containing sucrose, gelatin and distilled water when the following ratio, wt.%:

Sucrose 39-41

Gelatin 5-6

Distilled water the Rest

3. The method according to any one of claims 1 and 2, characterized in that the aeration of the nutrient medium is carried out by feeding it sterile air at 1-2 l/min, 1 l of medium.

4. The method according to any one of claims 1 to 3, characterized in that the mixed culture medium stirrer with a speed of 300-500 rpm./minutes

5. The method according to any one of claims 1 to 4, characterized in that the complete process of growing cells in the achievement of their content in the culture fluid not below 30·109CL/ml

6. The method according to any one of claims 1 to 5, characterized in that the freeze drying is carried out before the moisture content of the resulting product is not above 3%.

7. The method according to any one of claims 1 to 6, characterized in that the container with the dried product is filled with nitrogen.

8. Nutrient medium for growing cells of Escherichia coli CR-322D containing broth of Hottinger, glucose, manganese sulfate, salt of phosphoric acid and water, characterized in that it additionally contains yeast extract, sodium chloride as a salt of phosphoric acid - phosphate of potassium, as a water - tap water in the following ratio, wt.%:

Broth of Hottinger 18-23

Glucose 0,35-0,45

Yeast extract 0,45-0,55

Manganese sulfate 0,055-0,065

Phosphate potassium 0,15-0,16

Water water the Rest

9. Bacterial drug for prevention and treatment of gastrointestinal diseases in livestock and domestic animals and birds, characterized by the fact that is libfilesystem biomass cells of Escherichia coli CR-322D obtained by the method according to claim 1.

10. The method of prevention and treatment of gastrointestinal diseases in livestock and domestic animals and birds, including the imposition of oral administration of Escherichia coli cells CR-322D, characterized in that the cells of Escherichia coli CR-322D used in the form of the bacterial preparation according to claim 9 in a dose of 0.25-42 conventional units at the reception, taking 1 unit 1 billion microbial cells.

11. The method according to claim 10, characterized in that the newborn calf appointed reception of Escherichia coli cells CR-322D in the first 1.5 to 2.5 hours of life, and then 4-5 days of life in the dose 38-42 conventional units.

12. The method according to claim 10, characterized in that the calves when the disease is prescribed a daily dose of Escherichia coli cells CR-322D 2 times a day dose 38-42 conventional units until clinical recovery.

13. The method according to claim 10, characterized in that newborn piglets appointed reception of Escherichia coli cells CR-322D in the first 4-6 h of life, then 6-7 days of life in the village is ze 5-7 conventional units, and at the age of 2-3 weeks at a dose of 6-8 conventional units.

14. The method according to claim 10, characterized in that the piglets to weaning appointed reception of Escherichia coli cells CR-322D for 4-5 days prior to weaning, on the day of weaning and 4-5 days after weaning dose of 18-22 conventional units.

15. The method according to claim 10, characterized in that the dogs at dysbacteriosis appointed reception of Escherichia coli cells CR-322D dose 6-40 conventional units 2 times a day for 1.5-2.0 hours before receiving food.

16. The method according to claim 10, characterized in that the cats at dysbacteriosis appointed reception of Escherichia coli cells CR-322D at a dose of 3-5 units, 2 times a day.

17. The method according to claim 10, characterized in that the chickens in 1-5 days of life prescribed daily dose of Escherichia coli cells CR-322D dose of 0,25-0,35 conventional units 2 times a day, then 20-25 and 40-45 days of life - 1,8-2,2 conventional units.



 

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