RU2468079C1 - Bacterium lactobacillus reuteri okt strain, used for production of probiotic preparations for poultry - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to biotechnology. Strain of bacterium Lactobacillus reuteri okt is isolated from natural material of gastrointestinal tract of chicken as a result of purposeful systemic search, deposited in Russian National Collection of Industrial Microorganisms (VKPM) under registration number VKPM B-10609 and can be used for breeding poultry, as well as prevention and treatment of poultry diseases, simultaneously with carrying out antibiotic therapy courses.

EFFECT: invention makes it possible to increase efficiency of poultry breeding.

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Description

The invention relates to microbiology and biotechnology and can be used in the microbiological industry in the production of probiotic preparations used in poultry farming for the treatment and prevention of diseases of farm birds.

In recent years, probiotics, feed additives based on strains of living microorganisms, are found in poultry farming that have a positive effect on the physiological, biochemical and immune responses of the host through the stabilization and optimization of the function of its normal microflora. They are used for dysbacterioses, for regulating microbiological processes in the digestive tract, for the prevention and treatment of certain digestive disorders of an infectious and alimentary etiology, as substitutes for antibiotics, etc. The effectiveness of probiotics is not inferior to some antibiotics and chemotherapeutic agents, but they do not have a detrimental effect on the microflora of the digestive tract, they do not pollute animal products and the environment, that is, they are environmentally friendly.

To date, various strains of lactobacilli are known that are part of the probiotic preparations used for the treatment and prevention of diseases of farm birds.

Known recombinant strain of Lactobacillus species 8 TIMES (pLF-SL2) expressing somatoliberin in the digestive tract (application RU 2002111437/13, publ. 20.06.2004). The strain is used as part of a probiotic preparation to increase the productivity of farm animals and improve product quality. However, the achievement of the result of using the strain — increasing productivity, is achieved due to the genetic modification of the strain, which provides the ability to express somatoliberin in the digestive tract.

A known tool for the treatment of diseases of the gastrointestinal tract of chickens containing a symbiotic system of strains of lactic acid bacteria Lactobacillus acidophilus D-75 and D-76 of the drug "Vitaflor", enrofloxacin and tween-80 in the following ratio of ingredients (wt.%): Symbiotic microorganisms of the drug Vitaflor "- 10-20; enrofloxacin - 75-85; tween-80 - 1-2 (patent RU 2371190, publ. 27.10.2009). The tool reduces the mortality of young birds and increases the average daily gain of chickens by 10-15%.

A disadvantage of the known agent is the risk of developing antibiotic resistance to enrofloxacin, since the use of this drug is accompanied by the obligatory administration of the antibiotic-enrofloxacin, while the use of the symbiotic microorganisms of the Vitaflor drug is accompanied by the simultaneous use of only one antibiotic, which narrows the range of infectious agents to those that are sensitive only to enrofloxacin.

Known probiotic agent for farm animals and poultry containing a mixture of suspensions of bacterial strains and a protective environment in the following ratio of components, wt.%: Sucrose 15, gelatin 5.9% sodium chloride solution 80. As a mixture of suspensions of strains, the product contains suspensions of the following strains, wt.%: Azomonas agilisc B-2586-8, Azotobacter chroococcum B-3162-7, Azotobacter vinelandii B-2587-7, Bacillus subtilis B-5225-7, Bacillus subtilis B-4828-9, Bifidum bacterium globosum B-2584 - 10, Escherichia coli B-4412 - 8, Enterococcus faecium B-2898 - 7, Lactobacillus acidophilus B-3488 - 7, Lactobacillus acidophilus B-2585 - 7, Propionibacterium freudenreichii B-6561 - 7, Pseudomonas sp.B-3908 - 7, Pseudomonas sp.B-2589-7, Saccharomyces cerevisiae Y-365-2 (patent RU 2326692, publ. 06/20/2008). The tool allows you to normalize the intestinal microbiocenosis of healthy animals and birds, reduce the incidence of purulent-inflammatory and dyspepsia, reduce mortality and increase body weight gain in young animals.

A disadvantage of the known agent is the use in the composition of the preparation of microorganisms having antagonistic activity towards each other (for example, E. coli and Lactobacillus acidophilus), as well as having mutually opposite biological effects on the body, for example, lactobacilli and propionic acid bacteria more often inhibit inflammatory processes, while Bacillus subtilis, Escherichia coli, as a rule, have anti-inflammatory activity and increase the reactivity of the immune system.

The known probiotic lactomycrocycol for growing and feeding broiler chickens containing the microbial biomass of bacterial strains Lactobacillus amylovorus BT-24/88 (VKPM, B-6253) and Escherichia coli S 5/98 (VKPM, B-8564), taken in the ratio 1: 1 at a colony concentration of the forming units (cfu) of each strain in the range of 5 × 10 ⁸ -5 × 10 ⁹ in 1 ml of liquid or 1 g of dry preparation. Lactobacillus strain has a wide spectrum of antagonistic activity and multiple antibiotic resistance, and Escherichia and Salmonella generates microcin B 5/98, which suppresses opportunistic and pathogenic bacteria.

A disadvantage of the known agent is that both strains are not representative of the normal microflora of the intestine of the bird and, accordingly, are not able to multiply and accumulate in the intestine for a long time.

An urgent task today is the search for new strains of lactic acid bacteria, which are representatives of the normal microflora of the gastrointestinal tract of birds, which are resistant to antibiotics and can reduce the incidence of poultry.

The most promising for use are strains of bacteria Lactobacillus reuteri, which are natural inhabitants of the gastrointestinal tract of animals and birds and have antibacterial and anti-inflammatory activities. When L. reuteri cells are grown under anaerobic conditions in the presence of glycerol, they produce an antimicrobial substance known as reiterin (beta-hydroxypropionaldehyde). Since strains of Lactobacillus reuteri are different in different animal species, strains isolated from the gastrointestinal tract of the corresponding bird species should be used for treating poultry (Casas IA, Dobrogosz WJ Lactobacillus reuteri: An overview of a new probiotic for animals and animals. Microecol Therap, 1997, 25, 221-231).

Closest to the claimed strain, the prototype is a strain of Lactobacillus reuteri Pg4 producing β-gluconase used in the probiotic preparation for chickens (B. Yu, JRLiu, FSHsiao and PWSChiou Animal Feed Science and Technology, V.141, Issues 1 -2, 1 March 2008, P.82-91). Strain Pg4 is a recombinant superproducer of the enzyme β-gluconase and is designed to increase the efficiency of assimilation of plant foods rich in non-starch polysaccharides.

A disadvantage of the known strain is that it is not intended for use simultaneously with courses of antibiotic therapy and prophylaxis, as well as the fact that this strain is genetically modified, which limits its use.

An object of the invention is to obtain a bacterial strain, which is a natural inhabitant of the gastrointestinal tract of poultry and is resistant to antibiotics.

The technical task is achieved by obtaining a strain of Lactobacillus reuteri okt, which is resistant to a wide range of antibiotics, in particular colistin, enrofloxacin, norfloxacin, tetracycline, furazolidone, as well as an enzyme producing esterase.

The proposed strain is isolated from natural material of the gastrointestinal tract of chicken as a result of a targeted systematic search.

The obtained strain of Lactobacillus reuteri okt was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) of the Federal State Unitary Enterprise of the State Research Institute of Genetics and Breeding of Industrial Microorganisms (GosNIIgenetika) under registration number VKPM B-10609.

The strain of Lactobacillus reuteri okt VKPM B-10609 is characterized by the following features.

Cultural and morphological characteristics: gram positive short rods and / or coccobacteria grow on MRS medium. The colonies are grayish-white with a flat surface with a smooth edge.

Physiological and biochemical characteristics: anaerobic facultative, non-pathogenic for poultry and laboratory animals. Toxigenicity during inoculation of daily culture intraperitoneally to white mice at a concentration of 1 × 10 ⁶ cfu / ml was not detected. The strain produces in the culture medium the enzyme esterase, resistant to heat at a temperature of 90 ° C for 10 minutes.

The strain is characterized by resistance to therapeutic concentrations of colistin, enrofloxacin, norfloxacin, tetracycline, furazolidone. It is sensitive to amoxicillin, chloramphenicol, rifampicin.

The strain is identified on the basis of the analysis of morphological and biochemical properties according to the determinant [Determinant of bacteria Bergie. / Ed. J. Houlta et al .: (9th edition in 2 volumes: Translated from English under the editorship of Acad. RAS G.A.Zavarzin). - M., 1997], as well as on the basis of sequencing of a 16S gene fragment of ribosomal RNA.

The strain is stored in a freeze-dried state or in a solution of 30% glycerol at a temperature of -60 ° C.

For the cultivation of the strain, the MPC medium (Man, Rogosa, Sharp) of the following composition (g / l) is used: hydrolyzed skim milk of enzymatic dry milk (GOM) in the form of a fine-dispersed hygroscopic white-gray powder 33.0; manganese sulfate 0.1; magnesium sulfate 0.4; potassium phosphate disubstituted 4.0; cysteine 0.4; glucose 40.0; peptone 20.0;

yeast hydrolyzate 0.01; sodium acetate 10.0; ammonium citrate disubstituted 4.0; microbiological agar 2.0. The biomass yield is 5 g / l of medium.

The defining difference of the proposed strain from the prototype strain is its resistance to antibiotics - tetracycline, colistin, enrofloxacin, furazolidone, as well as the ability to produce thermostable esterase in the culture fluid, resistant to heating to 90 ° C for 10 minutes and able to hydrolyze the substrate with the formation of fluorescein fluorescein, which allows the use of this trait as a strain-specific, as well as the use of esterase activity to control the stability of characteristics to ultures during production and storage.

Since the proposed strain was obtained for the first time and it is characterized by a unique spectrum of antibiotic resistance, we can conclude that the proposed strain meets the criteria of the invention of “novelty” and “inventive step”.

The invention is illustrated by examples of specific performance.

Example 1. Growing a strain of Lactobacillus reuteri okt and isolation of the esterase enzyme.

To obtain biomass, cells of the producer strain were transferred onto agarized MPC medium in a Petri dish and incubated overnight at 37 ° C. Freshly grown colonies were transferred with a sterile bacteriological loop to flasks containing liquid MPC medium and cultured at 37 ° C until a stationary growth phase was reached. The biomass yield was 5 g / l of medium. Isolation and purification of the enzyme was performed as follows: 1 g of crude biomass was resuspended in 50 ml of 50 mM TrisHCl buffer pH 7.2-7.4, containing 20 mM EDTA. The cell suspension was cooled on ice and destroyed by ultrasound for 3 minutes at a frequency of 20 kHz. The resulting suspension was centrifuged at a speed of 9 thousand rpm, for 15 minutes at + 5 ° C. The supernatant was transferred to other tubes and the pellet was discarded. Lipids and fat-soluble components were removed by adding cold chloroform to the extract in a ratio of 1: 5, the tube with the extract was thoroughly shaken and left for 3 hours at + 5 ° C. The tube was centrifuged at a speed of 9 thousand rpm, for 15 minutes at + 5 ° C, the aqueous phase was transferred to another tube. Ammonium sulfate was added to the protein solution to 70% saturation and incubated at + 5 ° C for 16 hours. Salted proteins were precipitated by centrifugation at a speed of 9 thousand rpm, for 15 minutes at + 5 ° C. The supernatant was transferred to a dialysis bag and ammonium sulfate was removed by dialysis at + 5 ° C against a 10-fold volume of 50 mM TrisHCl buffer pH 7.2-7.4 containing 20 mM EDTA for 3 hours on a magnetic stirrer. The dialysis procedure was repeated three times, changing the buffer solution. To remove proteins larger than 20 kDa, the enzyme preparation was ultrafiltered through a membrane with a throughput of more than 20 kDa. The resulting enzyme preparation was tested for activity with a substrate for alkaline phosphatase and esterase. The output of the enzyme was at least 1000 units. per 1 ml.

Example 2. The study of resistance to antibiotics of the strain Lactobacillus reuteri okt.

Antibiotic resistance of the inventive strain of lactobacilli was determined by the disk diffusion method on blood agar with a selective additive for isolation of lactobacilli from the company "Biolot". For this, a lactobacillus culture was scattered on the surface of blood agar with a continuous lawn. Disks with various antibiotics were placed on agar. The calculation was carried out in areas of absence or inhibition of antibiotic growth around the disk with cells of the strain. The results are shown in table 1.

Table 1 Name of antibiotic test result, mm ampicillin 0 polodoxin 0 metronidazole 0 amoxiclav 32 doxycycline thirty chloramphenicol thirty rifampicin 22 ofloxacin 0 enrofloxacin 0 tetracycline 0 spectinomycin 16 enrocoli twenty furazolidone 10 chloramphenicol 24 penicillin 10 erythromycin 28 florfenicol 35 colistin 0 clindamycin 0 clubactin 0 gentamicin fourteen

Table 1 shows that the new strain of Lactobacillus reuteri okt VKPM B-10609 shows resistance to a wide range of antibiotics: tetracycline, colistin, enrofloxacin, furazolidone.

Example 3. The study of the toxicity of the bacterial strain Lactobacillus reuteri okt.

The acute and chronic toxicity of the bacterial strain Lactobacillus reuteri okt VKPM B-10609 was studied on white mice weighing 20-22 g according to the approved methodology (L.P. Malanin and others. "Guidelines for determining the toxic properties of drugs used in veterinary medicine and animal husbandry", M., 1988). To determine acute toxicity, 6 animal groups were formed with 6 animals each. Before the start of the experiment, the mice were quarantined for 14 days. For testing, we used a cell culture of the claimed strain in the form of a suspension of cells with a bacterial concentration of at least 1 × 10 ⁶ cfu / ml. The test culture was injected directly into the stomach using a syringe with a blunt needle, at the end of which tin was deposited in an amount of 0.8-2 ml per animal. Observation of the animals was carried out for 14 days, noting signs of intoxication and the number of dead animals.

To study the chronic toxicity of the claimed strain, 4 groups of white mice were formed, 10 animals each with a live weight of 20-22 g. The mice were kept on a normal diet in the vivarium. Every day for 2.5 months the test culture was added to the mice in drinking water: in the first group - at a dose of 0.5 ml per head, in the second group - 0.8 ml; in the third - 1.0 ml. The fourth group served as a control; mice were given drinking water without additives. During the experiment, the following indicators were taken into account: the appearance and behavior of animals, the condition of the coat and visible mucous membranes, mobility, attitude to food, and death.

To study the long-term effects of the claimed bacterial strain Lactobacillus reuteri okt VKPM B-10609, two groups of white mice were formed with a live weight of 22-25 g, 6 animals in each group (4 females and 2 males). In the experimental group, for 2 months, the test culture was added to mice daily in drinking water at doses of 0.5 ml per animal. The second group served as a control and was kept on the usual diet of vivarium. Observation was conducted for 2 months. We took into account the general condition, the number of females that gave birth, the number of mice in the litter, their mobility, the time of ear detachment, the appearance of hair, the proportionality of the development of the body and tail, and deviations in development and behavior. Received offspring were monitored for a month. No statistically significant differences between the experimental and control groups were found, which indicates the absence of toxicity of the bacterial strain Lactobacillus reuteri okt.

Example 4. The use of a new strain for growing and fattening broiler chickens.

The bacterial strain Lactobacillus reuteri okt VKPM B-10609 in the form of a suspension of cells with a bacterial concentration of 2 × 10 ⁶ cfu / ml was fed to the broilers of the experimental group (40,000 heads) at a rate of 0.5 ml per head per day for 5 days, starting with a ten-day age, simultaneously with the administration of the antibiotic enrofloxacin in dosages recommended by the instructions for use. The control group received the same antibiotic, but did not receive the L.reuteri okt culture. The average daily gain in broiler chickens of the experimental group at the age of 35 days was 53.7 g., And in the control group 51.4 g. Within 35 days, 4% of chickens died in the experimental group and 7% of chickens died in the control group. Thus, the use of a new strain of bacteria Lactobacillus reuteri okt VKPM B-10609 simultaneously with an antibiotic allowed to increase the average daily gain and reduce the death of chickens during the growing process.

The use of the proposed bacterial strain Lactobacillus reuteri okt VKPM B-10609 will increase the efficiency of growing agricultural poultry, as well as apply the latter simultaneously with courses of antibiotic therapy and prevention of bird diseases.

Claims (1)

The bacterial strain Lactobacillus reuteri okt, deposited under the registration number VKPM B-10609, used for the production of probiotic preparations for poultry.