RU2437563C1 - Probiotic feed additive - Google Patents

Abstract

FIELD: food industry. ^ SUBSTANCE: invention relates to a biotechnology, in particular, to genetic engineering and may be used in medicine and veterinary science for production of a preparation against virus and bacterial infections. The probiotic feed additive includes a mixture of Bacillus subtilis All-Russian collection of industrial microorganisms 10172 and Bacillus licheniformis All-Russian collection of industrial microorganisms 10135 bacteria strains biomass at equitable ratios in the spore form, their total amount being no less than 2ù108 spores/g, and a carbohydrate protector. ^ EFFECT: invention allows to recover gut microflora. ^ 5 cl, 2 tbl, 8 ex

Description

The invention relates to biotechnology and can be used in medicine and veterinary medicine to obtain drugs against bacterial infections.

A naturally-known strain does not have antiviral activity, has insufficient antagonistic activity. In this regard, preparations made on the basis of this strain will also have low antagonistic activity.

A known preparation comprising a mixture of biomass of bacterial strains of Bacillus subtilis VKPM B-7092, VKPM B-7048 and Bacillus licheniformis VKPM B-7038 in equal proportions in spore form with a titer of at least 3 × 10 ¹⁰ spores / g containing starch and sugar (patent RF №2142287).

A disadvantage of the known solution is the presence of genetically modified components in its composition.

The problem to which the claimed invention is directed is to create a probiotic feed additive that does not contain genetically modified components, antibiotics that have an inhibitory effect against pathogenic microorganisms, without suppressing the intestinal microflora, which activates the intestines.

The technical result from the use of the invention:

1) the creation of a probiotic drug used for the treatment and prevention of diseases of animals, birds and fish caused by enteropathogenic Escherichia coli, Staphylococcus aureus, Proteus, Shigella, Klebsiella, Salmonella;

2) the creation of a probiotic drug used to normalize microbiocenosis, restore normal intestinal microflora after the use of antibiotics, anthelmintics, coccidiostats;

3) the creation of a probiotic drug used to reduce the negative effects on the body of mycotoxins;

4) the creation of a probiotic preparation with maximum survival of bacterial spores and the maximum rate of transition of bacterial spores to the vegetative form.

The specified technical result is achieved by the fact that the probiotic feed additive includes a mixture of biomass of bacteria of the strains of Bacillus subtilis VKPM 10172 (Deposit 23.12.2008) and Bacillus licheniformis VKMP 10135 (Deposit VKPM 10.20.2008) in equal proportions in spore form with a total amount of at least 2 × 10 ⁸ spores / g and tread. As a protector, saccharides are used, preferably in a mixture with starch. Preferably, the saccharides are selected from sucrose, glucose, fructose, lactose, maltose, or mixtures thereof. The use of starch in the protector provides additional protection to bacterial spores, ensures a more uniform distribution in the protector mass, and promotes more active germination of bacterial spores. The starch content in the preparation does not exceed 10% wt., Preferably 2-6% wt. Saccharides are not only a sorbent filler, but also have a stabilizing and preserving effect on the drug.

Bacteria of the Bacillus subtilis strain VKPM 10172 (name of strain B-11) were deposited in the All-Russian Collection of Industrial Microorganisms on December 23, 2008. The method of obtaining the strain: found in vivo, isolate, Kirov region, Black Kholunitsa, August 2008, Kulakov G.V. Cultural and morphological features of the strain: cells are straight, rod-shaped, 2-3 microns in length, motile. Aerobically form spores of an oval tuyere, which are located centrally in the cell. Gram stain is gram-positive. The strain grows well on meat-peptone agar (MPA), wort agar, Gromyko medium, potato agar, as well as in these media with the addition of 10 mg / ml kanamycin, forming shiny cremated colonies of a creamy color with uneven edges. On meat and peptone broth (BCH), the culture forms a uniform turbidity. The strain synthesizes a probiotic. Activity (productivity) of the strain: ferments lactose, glucose, arabinose, xylose with the formation of acid without gas. Reduces nitrates, provides methylene blue. Coagulase, hyaluronidase, lecithinase activity does not possess. It has lipase activity. The strain is not zoopathogenic. The strain is not pathogenic.

The strain is maintained on MPA and potato agar with the addition of kanamycin (50 μg / ml) with reseeding at least 1 time per month. Store on MPA and potato agar with the addition of kanamycin (50 μg / ml).

Optimum conditions and composition of the medium for propagation: Optimum growth temperature of 37 ° C, pH 7.0. The composition of the medium is peptone 5 g / l; yeast extract 3 g / l; NaCl 5 g / l; tap water up to 1 liter.

Optimum conditions and composition of the medium for fermentation: Optimum temperature 37 ° C, pH 7.0. The composition of the medium: K ₂ HPO ₄ × 3H ₂ O - 18 g / l; KH ₂ PO ₄ -6.0 g / l; (NH ₄ ) SO ₄ -2 g / l; Na ₃ C ₆ H ₂ O ₇ × 5.5H ₂ O-1.2 g / L.

Bacteria of the strain Bacillus subtilis VKPM 10172 can be obtained by any known method, but preferably by sowing and growing the original seed in a nutrient medium including peptone, glucose, corn extract, sodium chloride, calcium chloride, magnesium, manganese and iron sulfates at pH 7 4-7.5.

Bacteria of the strain Bacillus licheniformis VKPM 10135 (name of strain B-12) were deposited in the All-Russian Collection of Industrial Microorganisms on 10/20/2008. The method of obtaining the strain: found in vivo, isolate, Kirov region. Black Kholunitsa, August 2008, Kulakov G.V. Cultural and morphological features of the strain: cells are straight, rod-shaped, 3 μm long, motile. They form aerobic spores of an oval shape, which are located centrally in the cell. Gram stain is gram-positive. The strain grows well on meat-peptone agar (MPA), wort agar, Gromyko medium, potato agar, as well as in these media with the addition of 10 mg / ml kanamycin, forming shiny cremated colonies of a creamy color with uneven edges. On meat and peptone broth (BCH), the culture forms a uniform turbidity. The strain synthesizes a probiotic. Activity (productivity) of the strain: ferments lactose, glucose, arabinose, xylose with the formation of acid without gas. Reduces nitrates, provides methylene blue. Coagulase, hyaluronidase, lecithinase activity does not possess. It has lipase activity. The strain is not zoopathogenic. The strain is not pathogenic.

The strain is maintained on MPA and potato agar with the addition of kanamycin (50 μg / ml) with reseeding at least 1 time per month. Store on MPA and potato agar with the addition of kanamycin (50 μg / ml).

Optimum conditions and composition of the medium for propagation: Optimum growth temperature of 37 ° C, pH 7.0. The composition of the medium is peptone 5 g / l; yeast extract 3 g / l; NaCl 5 g / l; tap water up to 1 liter.

Optimum conditions and composition of the medium for fermentation: Optimum temperature 37 ° C, pH 7.0. The composition of the medium: K ₂ HPO ₄ × 3H ₂ O - 18 g / l; KH ₂ PO ₄ -6.0 g / l; (NH ₄ ) SO ₄ -2 g / l; Na ₃ C ₆ H ₂ O ₇ × 5.5H ₂ O-1.2 g / L.

Bacteria of the strain Bacillus licheniformis VKPM 10135 can be obtained by any known method, but preferably by sowing and growing the seed in a nutrient medium including peptone, glucose, corn extract, sodium chloride, calcium chloride, magnesium sulfates, manganese and iron at pH 7 4-7.5.

EXAMPLES

Getting the drug

Bacteria of the Bacillus subtilis strain VKPM 10172 were obtained by inoculating and growing the initial seed in a nutrient medium including peptone, glucose, corn extract, sodium chloride, calcium chloride, magnesium sulfates, manganese and iron at a pH of 7.4-7.5. Further, the biomass of bacteria was separated from the nutrient medium by centrifugation and freeze-dried.

Bacteria of the Bacillus licheniformis strain VKPM 10135 were obtained by inoculating and growing the original seed in a nutrient medium, including peptone, glucose, corn extract, sodium chloride, calcium chloride, magnesium, manganese and iron sulfates, at a pH of 7.4-7.5. Further, the biomass of bacteria was separated from the nutrient medium by centrifugation and freeze-dried.

Next, the biomass of each type of bacteria was mixed separately with a carbohydrate protector in the ratio of 1 part of the biomass of bacterial spores to 5 parts of the carbohydrate protector and mixed. In this case, immobilization of bacterial spores on the tread particles occurred. Next, the carbohydrate protector with Bacillus subtilis VKPM 10172 spores immobilized on it and the carbohydrate protector with Bacillus licheniformis VKPM 10135 spores immobilized on it were mixed in a 1: 1 ratio. The resulting immobilized bacterial cultures were mixed with a carbohydrate protector. In the manufacture of the additive, no genetically modified components were used.

Example 1

Study of the effectiveness of treatment of chicken lesions with mitoxin fusarochromanon.

The experiment was carried out on 600 laying hens infected with fusarochromanon, divided into three separately contained batches of 200 animals each: control, 1st experimental and 2nd experimental. Feed infection with F.moniliforme fungi. Infection duration 2 weeks. The control group was not treated. The 1st experimental group was treated with the drug according to the patent of the Russian Federation No. 2142287 with a titer of bacterial spores of 10 ¹⁰ spores / g per head per day. The 2nd experimental one was treated with the preparation according to the claimed invention with a bacterial spore titer of 2 × 10 spores / g per head per day, a mixture of 6% wt. Was used as a protector. starch and a mixture of sucrose, glucose and fructose, taken in a ratio of 3: 1: 1. The introduction of the drug was carried out in all cases by drinking with water through nipple drinkers.

Control was carried out by the percentage of mortality of chicken embryos from the moment of treatment initiation.

Table 1. 1 day 5 day 10 day 20 day 25 day Counter, group 67% 66% 68% 67% 69% 1 group 58% 41% 19% 16% fourteen% 2 group 27% 16% eleven% eleven% 10%

Such a sharp drop in mortality of embryos in the 2nd experimental group is associated, in our opinion, with the maximum survival of bacterial spores and the maximum rate of transition of bacterial spores to the vegetative form.

When conducting an experiment with a sample of a preparation based on Bacillus subtilis VKPM 10172 and Bacillus licheniformis VKPM 10135 with a decrease in the concentration of bacterial spores to 10 ⁸ spores / g per head per day, the effect was significantly reduced and the embryo mortality did not drop below 17% on the 25th day of treatment.

When conducting experiments with samples of a preparation based on Bacillus subtilis VKPM 10172 and Bacillus licheniformis VKPM 10135 in a ratio different from equal, the effect also decreased. When conducting experiments in which feeding of hens was carried out only Bacillus subtilis VKPM 10172 or only Bacillus licheniformis VKPM 10135 no significant effect was observed. In experiments in which feeding of hens was carried out by Bacillus subtilis VKPM 10172 and Bacillus licheniformis VKPM B-7038, the effect was reduced and the embryo mortality did not drop below 19% on the 25th day of treatment. During experiments in which feeding of hens was carried out by Bacillus subtilis VKPM B-7092 and Bacillus licheniformis VKPM 1035, the effect was reduced and the embryo mortality did not fall below 17% on the 25th day of treatment.

Example 2

A study of the effectiveness of the prevention of calf lesions with mycotoxin patulin.

The experiment was carried out on 90 calves, divided into three separate batches: the control - 10 calves, the 1st experimental and the 2nd experimental of 40 calves. Feed infection with Aspergillus, a pathogen producing patulin. The control group was not exposed to prevention. The 1st experimental group was treated with the drug according to the patent of the Russian Federation No. 2142287 with a titer of bacterial spores of 10 ¹⁰ spores / g per 1 kg of feed. The 2nd experimental one was treated with a preparation according to the claimed invention with a bacterial spore titer of 4 × 10 ⁸ spores / g per 1 kg of feed, a mixture of sucrose, glucose and fructose taken in a ratio of 3: 1: 1 was used as a protector. The introduction of the drug was carried out in all cases by drinking with water through nipple drinkers. The introduction of the drug 10 days before the feed infection.

The control was carried out according to the percentage of deaths from the time of intake of infected food. No treatment experience has been conducted.

Table 2. 1 day 2 day 3 day 4 day 5 day Counter. Group 0% twenty% 70% - - 1 group 0% 10% 12.5% Cessation of death Cessation of death 2 group 0% 0% 0% 0% 0%

The absence of calf mortality in the 2nd experimental group is associated, in our opinion, with the maximum survival of bacterial spores and the maximum rate of transition of bacterial spores to the vegetative form.

Example 3

The study of the effectiveness of the prevention and treatment of lesions of piglets with E. coli E. coli.

The experiment was carried out on 300 piglets, divided into three separately kept batches of 100 goals: control, 1st experimental and 2nd experimental. E. coli coli feed infection. The control group was not subjected to prophylaxis, tetracycline treatment for 15 days from the onset of symptoms of poisoning. The 1st experimental group was treated with the drug according to the patent of the Russian Federation No. 2142287 with a titer of bacterial spores of 10 ¹⁰ spores / g per 1 kg of feed. The 2nd experimental one was treated with a preparation according to the claimed invention with a bacterial spore titer of 4 × 10 ⁸ spores / g per 1 kg of feed, a mixture of sucrose, glucose and fructose taken in a ratio of 3: 1: 1 and 2% wt. starch. The introduction of the drug was carried out in all cases by drinking with water through nipple drinkers. The introduction of the drug 10 days before the feed infection.

The control was carried out by the percentage of deaths. In experimental groups 1 and 2 cases were not observed. In the control group, the mortality rate was 42%.

Experience in the treatment of E. coli infected piglets with E. coli showed greater activity of the claimed drug over the drug according to the patent of the Russian Federation No. 2142287, the death rate in the treatment of experimental groups was 16% and 23%, respectively.

Example 4

A study of the effectiveness of the treatment of lesions of chickens Salmonella bongori.

The experiment was conducted on 300 Hubbard cross broilers infected with Salmonella bongori, divided into three separately kept batches of 100 goals: control, 1st experimental and 2nd experimental. The control group was not subjected to prophylaxis, treatment with tetracycline for 15 days from the onset of symptoms. The 1st experimental group was treated with the drug according to the patent of the Russian Federation No. 2142287 with a titer of bacterial spores of 10 ¹⁰ spores / g per head per day. The 2nd experimental one was treated with the preparation according to the claimed invention with a bacterial spore titer of 1 × 10 ⁹ spores / g per head per day, a mixture of sucrose, glucose and fructose taken in a ratio of 3: 1: 1 and 7% wt. starch. The introduction of the drug was carried out in all cases by drinking with water through nipple drinkers.

The control was carried out by the percentage of deaths. In the control group, the mortality rate was 72%. In the experimental group 1 case was 19%. In the experimental group 1 case was 8%.

When conducting experiments with a sample of the drug according to the patent of the Russian Federation No. 2142287 with a decrease in the concentration of bacterial spores to 1 × 10 ⁹ spores / g per head per day, the mortality rate was 27%.

When conducting experiments with samples of a preparation based on Bacillus subtilis VKPM 10172 and Bacillus licheniformis VKPM 10135 in a ratio of 1: 2 the case was 20%. When conducting experiments with samples of a preparation based on Bacillus subtilis VKPM 10172 and Bacillus licheniformis VKPM 10135 in a ratio of 2: 1, the case was 22%.

When conducting experiments in which treatment was carried out only Bacillus subtilis VKPM 10172 or only Bacillus licheniformis VKPM 10135, a significant effect was not observed, the death rate was about 60%.

When conducting experiments in which feeding of hens was carried out by Bacillus subtilis VKPM 10172 and Bacillus licheniformis VKPM B-7038 1: 1, the mortality was 31%.

When conducting experiments in which feeding of hens was carried out by Bacillus subtilis VKPM B-7092 and Bacillus licheniformis VKPM 1035 1: 1, the mortality was 37%.

Example 5

A study of the effectiveness of the prevention and treatment of lesions of piglets Shigella dysenteriae.

The experiment was carried out on 300 piglets, divided into three separately kept batches of 100 goals: control, 1st experimental and 2nd experimental. Shigella dysenteriae feed infection. The control group was not subjected to prophylaxis, tetracycline treatment for 15 days from the onset of symptoms of poisoning. The 1st experimental group was treated with the drug according to the patent of the Russian Federation No. 2142287 with a titer of bacterial spores of 10 ¹⁰ spores / g per 1 kg of feed. The 2nd experimental one was treated with the preparation according to the claimed invention with a bacterial spore titer of 4 × 10 ⁸ spores / g per 1 kg of feed, a mixture of maltose, glucose and lactose taken in a ratio of 1: 4: 1 was used as a protector. The introduction of the drug was carried out in all cases by drinking with water through nipple drinkers. The introduction of the drug 10 days before the feed infection.

The control was carried out by the percentage of deaths. In experimental groups 1 and 2 cases were not observed. In the control group, the mortality rate was 67%. The experience in treating infected Shigella dysenteriae piglets showed approximately equal activity of the claimed drug over the drug according to the patent of the Russian Federation No. 2142287, the death rate in the treatment of experimental groups was 13% and 14%, respectively.

Example 6

A study of the effectiveness of the prevention and treatment of lesions of piglets Klebsiella planticola.

The experiment was carried out on 300 piglets, divided into three separately kept batches of 100 animals each: control, 1st experimental and 2nd experimental. Klebsiella planticola feed infection. The control group was not subjected to prophylaxis, tetracycline treatment for 15 days from the onset of symptoms of poisoning. The 1st experimental group was treated with the drug according to the patent of the Russian Federation No. 2142287 with a titer of bacterial spores of 10 ¹⁰ spores / g per 1 kg of feed. The 2nd experimental one was treated with the preparation according to the claimed invention with a bacterial spore titer of 4 × 10 ⁸ spores / g per 1 kg of feed, a mixture of sucrose and 3% wt. Was used as a protector. starch. The introduction of the drug was carried out in all cases by drinking with water through nipple drinkers. The introduction of the drug 10 days before the feed infection.

The control was carried out by the percentage of deaths. In experimental groups 1 and 2 cases were not observed. In the control group, the mortality rate was 56%. Experience in the treatment of pigs infected with Klebsiella planticola showed a higher activity of the claimed drug over the drug according to the patent of the Russian Federation No. 2142287, the death rate in the treatment of experimental groups was 7% and 18%, respectively.

Example 7

A study of the effectiveness of the treatment of dyspepsia in piglets.

The experiment was carried out on patients with 200 piglets, divided into three separately kept batches of 100 goals: control, 1st experimental and 2nd experimental. The control group was not subjected to prophylaxis, treatment with tetracycline for 15 days from the onset of symptoms. The 1st experimental group was treated with the drug according to the patent of the Russian Federation No. 2142287 with a titer of bacterial spores of 10 ¹⁰ spores / g per 1 kg of feed. The 2nd experimental one was treated with the preparation according to the claimed invention with a bacterial spore titer of 2 × 10 ⁸ spores / g per 1 kg of feed, a mixture of glucose and 3% wt. Was used as a protector. starch. The introduction of the drug was carried out in all cases by drinking with water through nipple drinkers. The control was carried out by the percentage of deaths. In experimental groups 1 and 2 cases were not observed. In the control group, the mortality rate was 28%.

Example 8

A study of the effectiveness of the application to normalize microbiocenosis and restore normal intestinal microflora after antibiotic use.

The experiment was carried out on 150 pigs, divided into three separately kept batches of 50 animals each: control, 1st experimental and 2nd experimental. The control group was not exposed to prevention. All groups were treated with tetracycline for 15 days. The 1st experimental group was given the preparation according to the patent of the Russian Federation No. 2142287 with a bacterial spore titer of 10 ¹⁰ spores / g per head per day. The 2nd experimental one was treated with the preparation according to the claimed invention with a bacterial spore titer of 6 × 10 ⁸ spores / g per head per day, glucose was used as a protector. The introduction of the drug was carried out in all cases by drinking with water through nipple drinkers. Control was carried out by the appearance of piglets after 5 days of feeding. The control group: the color of the piglets is gray with a ruffled hair, the behavior is passive. Experimental groups: pink-white color, shine of hair, active behavior.

As can be seen from the presented examples, it is when using Bacillus subtilis VKPM 10172 and Bacillus licheniformis VKPM 10135 that in equal proportions in the spore form and precisely with their total amount of at least 2 × 10 ⁸ spores / g has a higher activity compared to the prototype for prevention and the treatment of diseases caused by Escherichia coli, Shigella, Klebsiella, Salmonella, with normalization and stimulation of the intestine, since it shows better or identical performance at much lower dosages.

It can be seen from the presented examples that the total activity of these bacterial strains, taken precisely in the indicated ratio and at the indicated concentration and even in the absence of genetically modified components, is significantly higher than the activity of the bacterial strains taken separately or in other ratios, or together with Bacillus subtilis bacteria and Bacillus licheniformis of other strains.

Claims (5)

1. A probiotic feed additive comprising a mixture of bacterial biomass of strains of Bacillus subtilis VKPM 10172 and Bacillus licheniformis VKPM 10135 in equal proportions in spore form with a total amount of at least 2 · 10 ⁸ spores / g and a carbohydrate protector. 2. The probiotic feed additive according to claim 1, characterized in that the tread includes starch. 3. The probiotic feed additive according to one of claims 1 or 2, characterized in that sucrose, glucose, fructose, lactose, maltose or a mixture thereof are used as a carbohydrate in the tread composition. 4. The probiotic feed additive according to claim 1, characterized in that the bacteria of the Bacillus subtilis strain VKPM 10172 are obtained by inoculating and growing the original seed in a nutrient medium including peptone, glucose, corn extract, sodium chloride, calcium chloride, magnesium sulfate, manganese and iron, at a pH of 7.4-7.5. 5. The probiotic feed additive according to claim 1, characterized in that the bacteria of the strain Bacillus licheniformis VKPM 10135 are obtained by sowing and growing the original seed in a nutrient medium including peptone, glucose, corn extract, sodium chloride, calcium chloride, magnesium sulfates, manganese and iron, at a pH of 7.4-7.5.