RU2203947C1 - Strain of bacterium bacillus licheniformis used for preparing probiotic preparation designated for prophylaxis and treatment of gastroenteric diseases in animals, poultry and fishes - Google Patents

Abstract

FIELD: biotechnology, veterinary science. SUBSTANCE: strain of bacterium Bacillus licheniformis VKM B-2252D is isolated from soil, it cells are gram-positive with aerobic respiration type. The strain hydrolyzes glucose, maltose, sucrose and shows activity of oxidase and catalase and utilizes acetate and citrate in aerobic culturing. The strain inhibits growth of staphylococci, salmonellae, proteus, candidomycosis, pyocyanic bacterium and other pathogenic microorganisms. The strain is used in industrial microbiology for preparing probiotic preparation "Subtilis-L". Invention provides rapid normalization of activity of gastroenteric tract in animals, poultry and fishes and exhibits promising in treatment and prophylaxis of bacterial infections. Invention can be used for preparing the preparation of probiotic group. EFFECT: valuable veterinary and medicinal properties of strain. 1 tbl

Description

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

 A known bacterial strain Bacillus licheniformis 31 (Academician DZ Zabolotny Institute of Microbiology and Virology NAS of Ukraine), used as one of the components of the bacterial preparation biosporin, intended for the treatment of purulent-septic diseases (SU 1722502 A1, class A 61 K 39/02, 1989).

 A known bacterial strain Bacillus licheniformis 1C-1 (VKPM B-7038) is used as a component of a bacterial preparation with a wide spectrum of antiviral and antibacterial activity (RU 2142287 C1, class A 61 K 35/74, 12/10/1999).

 However, the disadvantage of this strain can be considered a special manifestation of its activity as part of a complex bacterial preparation.

 The objective of the invention is to identify a new bacterial strain of Bacillus licheniformis, which has antibacterial activity against a wide range of Gr + and Gr-microorganisms, producing a broad-spectrum antibiotic, its use as a bacterial probiotic preparation for veterinary purposes and fisheries.

 The technical result is achieved by the implementation of the invention is to increase the effectiveness of treatment, digestibility of feed, increase productivity and weight gain of animals, birds, fish.

 The bacterial strain Bacillus licheniformis B-1602 is deposited in the All-Russian collection of microorganisms IBPM them. K.S. Scriabin RAS under V-2252D.

 The strain is derived from a natural isolate (soil). It was selected to suppress the growth of pathogenic strains: staphylococcus, Pseudomonas aeruginosa, Salmonella, trichophytes, Proteus, epidermitis, candidomycoses, etc.

 Cultural, morphological and biochemical properties of the strain Bacillus licheniformis BKM B-2252D.

 Gram-positive bacillus capable of forming spores. The size of vegetative cells is 2-5 microns. Disputes are single, central or oval, mobile. Flagellation - peritrich.

Vegetative cells of Bacillus licheniformis BKM B-2252D grow well on rich nutrient media - MPA, MPB and PGA at temperatures of 50 ^o C, pH 5.0-8.0. On MPA (meat-peptone agar) forms white colonies. The pigment does not emit on Wednesday. There is an increase to 7% NaCl.

 Hydrolyzes glucose, maltose, sucrose, oxidase-and catalase-positive.

 Non-fermentable carbon compounds: lactose, mannitol, sucrose.

 Disposes of acetate and citrate during aerobic growth.

 The strain is non-toxic to animals, humans and plants.

 The strain Bacillus licheniformis BKM B-2252D produces a broad-spectrum antibiotic that suppresses staphylococci, fungi and Pseudomonas aeruginosa.

 It is characterized by growth retardation test culture, mm (method of perpendicular strokes) presented in the table.

 For the cultivation of the strain of Bacillus licheniformis VKM B-2252D, a liquid nutrient medium based on 0.3% FGMS, pH 6.8-7.0, containing 0.2% glucose (dry matter), 0.005% aminopeptidase or 0.02% blood serum.

 Dry biomass of microorganisms is previously plated in a test tube with meat and peptone broth (MPB). When the appearance of visible growth, the colonies are subcultured on meat peptone agar in test tubes.

Typical colonies are selected and transplanted onto the MPB liquid medium in vials. After a day, the grown biomass is transferred to a 15-liter bottle with 9 liters of culture medium and cultivated for 22 hours at 37-40 ^o C, receiving seed.

 FGMS-based nutrient medium is placed in a reactor, sterilized, cooled and inoculated with the resulting material. Aerobic cultivation is carried out at a pH of 6.8-7.2 with the support of a 40% glucose solution.

 When a concentration of 20 billion bacterial cells is reached, the glucose supply is stopped and the air supply is turned off when the pH reaches 4.6.

The resulting culture fluid with biomass is cooled to 20 ^o C, pumped into containers or packaged in vials.

 With this cultivation method, the preparation is obtained in the form of a liquid dosage form called "Subtilis-L", containing 85-90% of the spores of Bacillus licheniformis VKM B-2252D bacteria.

 The drug "Subtilis-L" is harmless and does not contain extraneous microflora. The safety was tested on white mice weighing 18-20 g and on year-old carps, which were administered orally with 1.0 ml of the drug.

 The probiotic activity of the drug "Subtilis-L" in a single dose (15-20) billion cells / ml.

 The proposed strain of Bacillus licheniformis VKM B-2252D can be used as a production of a preparation of probiotic action, recommended for the treatment of gastrointestinal diseases of animals, birds and fish.

 The invention is illustrated by examples.

 Example 1. Bacterial hemorrhagic carp septicemia.

 A fish with clinical signs of bacterial hemorrhagic septicemia fell ill in a pond farm. Laboratory analysis of the material showed the presence of pathogenic microflora in the internal organs of fish. The number of sick fish (two-year-old carp) reached 30% of the total. The treatment was prescribed with the addition of 3.5 million microbial cells (spores) Subtilis-L to the feed by wetting it per 1 kg of live body weight of the fish. Feed with "Subtilis-L" was given to fish 1 time per day for 7 days. 14 days after the end of the course of treatment, it was found that the number of diseased fish decreased to 10%, and only individual specimens remained signs of acute inflammation.

 Example 2. The safety of chickens.

 Subtilis-L production tests were carried out on the safety of chickens in the first two weeks after planting. "Subtilis-L" was administered orally in liquid form by evaporation with a 4% glucose solution and in freeze-dried form by adding to food. The waste of chickens in the control group was 4.3%. The average waste in the experimental groups amounted to 2.1% when giving lyophilically dried Subtilis-L with food, and 0.6% when drinking liquid Subtilis-L. In the experimental groups, the chickens gained weight more intensively: after the first three days for the control group, the average weight was 58 grams, for the experimental group - 69 grams.

 Example 3. The safety of piglets.

 Significant waste of piglets was observed at the farm due to diseases of the gastrointestinal tract. Conducted microbiological and pathological studies revealed several types of gastrointestinal diseases of piglets of bacterial etiology. The course of treatment was prescribed: the use of "Subtilis-L" orally immediately after birth at the rate of 1 dose in 3 piglets on 4% glucose for 5-7 days. The course of prevention: the use of "Subtilis-L" inside at the rate of 1 dose per 10 newborn piglets on 10% glucose for 3 days. The safety of piglets rose by 60% and reached stabilization at 94%, which is maintained at this level by prevention.

 Studies have shown the effectiveness of the bacterial drug "Subtilis-L" based on the proposed strain of Bacillus licheni-formis BKM B-2252D.

Claims (1)

 The bacterial strain Bacillus licheniformis VKM B-2252 D used to obtain a probiotic preparation intended for the prevention and treatment of gastrointestinal diseases of animals, birds and fish.

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