RU2398872C1 - Bacillus licheniformis BACTERIA STRAIN USED FOR MAKING PROBIOTIC SUPPLEMENT FEED USED FOR PRODUCING HIGH-QUALITY FODDER IMPROVING PERFORMANCE AND REDUCING RISK OF GASTROINTESTINAL DISTURBANCES IN ANIMALS, BIRDS AND FISHES - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: Bacillus licheniformis bacteria strain, Russian National Collection of Microorganisms No. V-2414D is recovered from soil. The cells are gram-positive, capable for spore formation, aerobic breathing. The strain hydrolyses glucose, maltose, sucrose; it is oxidase- and catalase-positive. In aerobic cultivation, it utilises acetate and citrate. It inhibits the growth of staphylococci, salmonellae, Proteus, candidamycosis, Pseudomonas aeruginosa and other pathogenic microorganisms. The strain is used as industrial for producing a component of a probiotic supplement feed named "Provagen" by the authors.

EFFECT: higher performance and reduced risk of gastrointestinal disturbances in animals, birds, fishes.

1 tbl, 3 ex

Description

The invention relates to biotechnology and can be used in the microbiological industry for the production of probiotic feed additives intended for the production of high-quality feeds that increase productivity and reduce the risk of gastrointestinal diseases of animals, birds and fish.

The known bacterial strain Bacillus licheniformis No. 31 (Institute of Microbiology and Virology named after academician D.Z. Zabolotny of the National Academy of Sciences of Ukraine), used as one of the components of the biosporin preparation intended for the treatment of purulent-septic diseases (SU 1722502, 03.30.1992).

A known bacterial strain Bacillus licheniformis VKPM B-7038, used as a component of a feed additive with a wide range of antiviral and antibacterial activity (RU 2142287, 10.12.1999).

However, the disadvantage of this strain can be considered the features of the manifestation of its activity in the complex bacterial feed additive, because due to the presence of recombinant properties, its use in animal feed production requires special permissions.

The known strain of Bacillus licheniformis VKM B-2252D, intended for the prevention and treatment of gastrointestinal diseases of animals, birds, fish (RU 2203947, 05/10/2003). However, this strain does not have sufficient activity, which limits its use as a component of a feed additive.

The objective of the invention is the selection of a new strain of Bacillus licheniformis, which has antibacterial activity against a wide range of Gr + and Gr-microorganisms producing broad-spectrum antibiotic substances, for use as a component of a probiotic feed additive.

The technical result achieved by the implementation of the invention is to increase productivity and reduce the risk of gastrointestinal diseases of animals, birds, fish.

The specified technical result is achieved in the bacterial strain Bacillus licheniformis VKM V-2414D, used to obtain a component of a probiotic feed additive intended for the production of high-quality feeds that increase productivity and reduce the risk of gastrointestinal diseases of animals, birds and fish.

The bacterial strain Bacillus licheniformis BL-03 is deposited in the All-Russian collection of microorganisms IBPM them. K.S.Skryabin RAS, reg. VKM number V-2414D.

The strain is obtained from a natural isolate (soil). Was subjected to selection to suppress the growth of pathogenic strains: staphylococcus, Pseudomonas aeruginosa, Salmonella, trichophytes, Proteus, epidermis, candidiasis, etc.

Cultural, morphological and biochemical properties of the strain Bacillus licheniformis VKM V-2414D.

Gram-positive bacillus capable of forming spores. The size of vegetative cells is 3-5 microns. Disputes are single, central or oval, mobile. Flagellation - peritrich.

Vegetative cells of Bacillus licheniformis VKM B-2414D grow well on rich nutrient media - meat-peptone agar (MPA), meat-peptone broth (MPB) and semi-liquid agar (PFA) at temperatures of 50 ° C, pH 5.0-8.0. On the MPA it forms white colonies. The pigment does not emit on Wednesday. There is an increase to 8% 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 VKM B-2414D produces a broad-spectrum antibiotic that suppresses staphylococci, fungi and Pseudomonas aeruginosa.

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

For the cultivation of the strain of Bacillus licheniformis VKM B-2414D, a liquid nutrient medium is used based on 0.3% dry muscle enzymatic hydrolysates (FGMS), pH 6.0, containing 0.2% glucose (dry matter), 0.005% aminopeptide, or 0, 02% blood serum.

Dry biomass of microorganisms is pre-seeded in a test tube with BCH. With the appearance of visible growth, the colonies are transferred to meat peptone agar in test tubes.

Typical colonies are selected and transferred to a liquid medium M 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 ° C, receiving seed.

FGMS-based nutrient medium is placed in a bioreactor, 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 ° C, pumped into containers or packaged in vials.

With this cultivation method, a strain is obtained for a component of a feed additive called Provagen with a content of 85-90% of Bacillus licheniformis VKM B-2414D bacteria spores.

A feed additive with a component based on the proposed strain is harmless and does not contain extraneous microflora. The safety was tested on white mice weighing 18-20 g and on yearlings of carps, which were administered orally 1.0 ml of feed additive.

The probiotic activity of the Provagen feed additive in a single dose (10-12) billion cells / ml.

The proposed strain of Bacillus licheniformis VKM B-2414D can be used as a production component of a feed additive of probiotic action, recommended for obtaining high-quality feeds that increase productivity and reduce the risk 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 (rubella) 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 25% of the total. The treatment was prescribed with the addition of 3.0 million microbial cells (spores) of Provagen per 1 kg of live body weight of the fish to the feed by wetting it. Food with Provagen 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 9%, and only a few specimens left signs of acute inflammation.

Example 2. The safety of chickens.

Production tests of Provagen for the safety of chickens were carried out in the first two weeks after their landing. "Provagen" was used orally in liquid form by evaporation with 5% glucose solution and in freeze-dried form by adding to food. The waste of chickens in the control group was 3.8%. The average waste in the experimental groups with the introduction of dry “Provagen” into the feed is 2.1%, with the evaporation of liquid “Provagen” - 0.5%. In the experimental groups, the chickens gained weight more intensively: after the first three days for the control group, the average weight was 57 g, for the experimental group - 71 g.

Example 3. The safety of piglets.

Significant waste of piglets was observed on the farm due to diseases of the gastrointestinal tract (GIT). 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 "Provagen" orally immediately after birth at the rate of 1 dose for 3 piglets on 8% glucose for 5 days. The course of prevention: the use of "Provagen" inside at the rate of 1 dose per 10 newborn piglets on 8% glucose for 3 days. The safety of piglets rose by 65% and reached stabilization at 95%, which is maintained at this level by prevention.

The studies showed the effectiveness of the Provagen feed additive with a component based on the proposed Bacillus licheniformis strain BKM B-2414D.

The spectrum of antagonistic activity of the strain Bacillus licheniformis BKM B-2414D.

Table Culture test Zones of growth inhibition, mm Staphylococcus aureus 30-35 Pseudomonas aeragenose 15-16 Salmonella typhimarium 28-33 S.stenly 20-25 S.derby 20-24 Klebsiella pneumoniae 14-18 Proteus vulgaris 18-22 Candida albicans 31-35 C.tropicalis 28-31 Shigella sonnei 25-29

Claims (1)

The bacterial strain Bacillus licheniformis BKM B-2414D, used to produce a component of a probiotic feed additive, is intended for the production of high-quality feeds that increase productivity and reduce the risk of gastrointestinal diseases of animals, birds and fish.