Strain of bacterium bacillus subtilis used for preparing probiotic preparation designated for prophylaxis and treatment of gastroenteric diseases in agricultural animal, poultry and fish

FIELD: biotechnology, veterinary science, microbiology.

SUBSTANCE: the strain of bacterium Bacillus subtilis VKM B-2287 is isolated from soil. Bacterial cells are gram-positive with aerobic type of respiratory, don't form capsule but form round spores. The strain hydrolyzes glucose, mannitol and lactose but it doesn't ferment sucrose, inositol, sorbitol and maltose, it doesn't form gas in fermentation process and inhibits the growth of staphylococcus, Escherichia coli, enterobacteria, citrobacteria and aeromonas. The strain is used as industrial one for preparing a preparation named by authors as "Subtilis+". The preparation normalizes function of digestive tract in agricultural animals, poultries and fishes and can be used in treatment and prophylaxis of bacterial infections. Invention can be used for preparing the probiotic preparation.

EFFECT: valuable properties of strain and preparation.

1 tbl, 3 ex

 

The invention relates to biotechnology and can be used in microbiological industry to obtain probiotic drug used in veterinary medicine for the treatment and prevention of gastrointestinal diseases of animals, birds and fishes.

A known strain Bacillus subtilis 534 producing probiotic “Sporobacterin”, which is intended for the prevention and treatment of gastro-intestinal dysbacteriosis. SU 1708350, class a 61 K 35/66.

The disadvantage is a small shelf, as it contains live bacteria that can a long time to maintain their properties, low purity of the drug that has a narrow scope as feed additive for animals. The strain is also sensitive to antibiotics except polymyxin b, which limits the scope of application of the drug.

Known strain of Bacillus subtilis 3H (GISC No. 248), bearing property of antibiotic resistance, used for receiving the probiotic preparation “Backspin”, which is used in conjunction with antibiotics for the treatment and prevention of dysbacteriosis, enzyme deficiency of the digestive system, purulent infections, food allergies. EN 2067616 C1, class a 61 K 35/74, 10.10.1996.

Known strain of Bacillus subtilis TPAXC-KM-117, exhibiting inhibitory activity against pathogenic microorganisms and having multiple Les is arctonoe resistance. The strain is resistant to tetracycline, rifampin, altillo, chloramphenicol, arctomecon. On the basis of preparing antibiotic-probiotic for the treatment and prevention of infectious diseases when the same antibiotic (EN 2118364 C1, C 12 N 1/20, 27.08.1988).

Known Bacillus subtilis strain VKM B-2250 (RU # 2184774, class a 61 K 35/74, 10.07.02), which is the basis of the preparation for veterinary purposes and fisheries.

The problem to which the invention is directed, is the identification of a new efficient producer strain probiotic product for veterinary purposes and fisheries.

Technical result achieved in the implementation of the invention is to improve the effectiveness of treatment, increase the digestibility of feed efficiency and gain of animals, birds, fish, due to the use of probiotic preparation on the basis of the proposed strain-producer, stability of the drug during storage in a wide range of ambient temperatures.

The Bacillus subtilis strain B-9 isolated from soils that were deposited in the all-Russian collection of microorganisms (ibfm them. Kgosana) number VKM B-2287.

The Bacillus subtilis strain VKM B-2287 may be stored in a lyophilized state for several years or jambs with agar medium on the basis of mycopathologia stock with mandatory re is evom at least 1 time in 2 months on the same environment.

Characteristics of the strain.

Cultural and morphological characteristics. The sticks. The size of the one-day agar culture of 3-5 µm. Cells stained Gram-positive, form round spores, single, Central diameters less than the diameter of the cell. Colonies on nutrient white pigment in the environment is not isolated.

Physiological signs. Aerobe, optimum temperature for growth is 37° C and pH 3.5 to 8.0. Growth is possible in the temperature range 4-50° C. Relationship to NaCl growth at a content of up to 3%.

Biochemical characteristics. Breaks down glucose, lactose, mannitol. Nabrasyvanie carbon compounds: sucrose, Inositol, sorbitol, maltose, lactose. Disposes of citrate and acetate. During fermentation does not form a gas. Produces oxidase, catalase.

Antagonistic traits. The Bacillus subtilis strain BKM-2287 inhibits the growth of staphylococci, Proteus, Klebsiella, Escherichia coli, Enterobacteriaceae, Citrobacter, Aeromonas, yeast fungi.

The Bacillus subtilis strain BKM-2287 is not pathogenic to plants, animals, fish and humans.

The data in table 1 show antagonistic activity of the test strains (method deferred antagonism).

For culturing Bacillus subtilis strain BKM-2287 use liquid nutrient medium containing casein hydrolysate - 5 cm3·DM-3(NaM=300 mg%); corn extract - 80 cm 3·DM-3(Nam=290 mg %), MnSO45H2On - 0,250 g-DM-3; MgSO47H2O - 0,300 g-DM-3; FeSO47H2On - 0,015 g-DM-3; CaCl22H2On - 0,052 g-DM-3; NaCl - 11,000 g-DM-3, distilled water.

Pre-dry biomass of microorganisms are sown in the tube with the broth. When visible growth colonies subcultured on mastopathy agar in a test tube.

Select typical colonies are subcultured in liquid medium in vials. After 22 hours all grown mass is transferred into a 20-liter bottle with 10 liters of nutrient medium and cultured for 26 hours at 37-39° receiving seed.

The nutrient medium on the basis of hydrolyzed casein is placed in a biological reactor, sterile 60 min at 1 ATM, cooled to 39° and sowing the seeds out of the bottle in the ratio of 1:9.

During aerobic cultivation pH maintained within the range of (6,8-7,2) pH, energising environment (10-15)% glucose to a final concentration of 0.1-0.2)%. Upon reaching biological concentration in Bq (15-20) 109clsm-3and (8-10) 109clsm-3on Kt stop adding glucose to reduce pH 4.0 and disconnect the air supply. Then turn off the heating of the reactor, the medium is cooled to (15-19)° C. Obtained chilled culture is pumped into the vessel or R is Spasojevic vials.

At the specified method of cultivation receive probiotic preparation in a liquid form with content (80-95)% spores and live vegetative cells of bacteria Bacillus subtilis strain VKM B-2287.

Proposed probiotic preparation is harmless, does not contain extraneous microflora. Safety tested on white mice body weight (18-20) g, which is orally administered the drug in a volume of 1.0 ml.

The drug has a specific activity: the number of cells in a single dose (8-20)109clsm-3, antagonistic activity of the zone of growth inhibition of the test organisms is from 10 to 38 mm

Thus, the proposed Bacillus subtilis strain VKM B-2287 can be used as a production to obtain probiotic preparation steps are recommended for the prevention and treatment of gastrointestinal diseases in animals, birds, fish.

Authors are asked to call the probiotic preparation, obtained on the basis of the proposed strain, “Subtilis+”.

The invention is illustrated by examples.

Example 1. Tests proposed probiotic drug to newborn calves and piglets.

The effectiveness of the drug on the basis of the proposed Bacillus subtilis strain VKM B-2287 was tested on newborn calves and piglets with a diagnosis of diarrhea occurring on the background of complex epizootic situation and in agriculture. The control group of calves and pigs were kept according to the farm technology. Calves and piglets in experimental groups were additionally asked the drug based on the proposed Bacillus subtilis strain BKM-2287 oral with a small amount of water for 20 minutes before feeding a single dose on the head 15 ml calves and 20 ml of pigs three times a day for three days. Observations showed that in the experimental groups, a day after giving the drug improved the General condition of all animals, diarrhea has ceased and after two days all the animals were healthy. The condition of the animals in the control groups was characterized by a continuation of the status diarrhoea, mortality was 10% in calves and 22% in piglets.

Example 2. Adding probiotic preparation “Subtilis+” to feed aquarium fish.

Conducted feeding of rising juvenile Golden fish (orange) extruded feed with the addition of probiotic preparation “Subtilis+”. The amount of feed was 10 kg, added probiotic 1 ml. Number of fish in the experimental and control groups were 250 copies Feeding was carried out 4-6 times a day. Food was eaten readily. The growth rate of juveniles in the experimental group compared to the control was 22%. The output of fish in experimental 98%, in the control of 78%. The water in the aquarium is not spoiled, turbidity was absent.

Note the p 3. The safety of the Chicks in the first week.

Tested “Subtilis+” chickens broiler poultry farm (5 poultry houses in the experimental and control groups). Waste chickens of the control group who did not receive the probiotic amounted to 4%, experienced to 0.2%. In the experimental groups of chickens more intensively gaining weight. After the first three days the average weight of the Chicks in the control group was equal to 61 g in the test - 70,

The tests have demonstrated the effectiveness of the drug “Subtilis+”, obtained on the basis of the proposed Bacillus subtilis strain BKM-2287.

Table 1
Spectrum of antagonistic activity of Bacillus subtilis strain BKM-2287.
The test cultureZone of inhibition of growth, mm
Salmonella veslaco 247/4926-30
Staphylococcus aureus 20927-33
S. aureus 102324-35
P. aerugenosa 41415-7
P. vulgaris 7216-19
Klebsiella pneumoniae T-2413-16
Escherechia coli 15733-38
Candida tropicalis26-30
C. albicans23-28

The bacterial strain Bacillus subtilis BKM-2287 used to obtain probiotic preparation, etc is naznachennogo for the prevention and treatment of gastrointestinal diseases in animals, poultry and fish.



 

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