RU2378000C2 - Method for production of therapeutic additive - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention is related to biotechnology and may be used in agriculture, in particular in fodder production. Method includes preparation of nutrient medium, which contains meat fermentative peptone, glucose, yeast extract, corn-lactose nutrient medium, cobalt sulfate, ammonium sulfate, agar-agar and distilled water with specified ratio of components. Introduction of bacterial strains Lactobacillus plantarum VKPM V-2347 and Propionibacterium freudenreichii VKPM V-6561 into prepared nutrient medium with their further incubation in prepared nutrient medium untik they achieve titre of at least 1×10⁶ CFU per 1 cm³.

EFFECT: invention makes it possible to normalise microflora of gastrointestinal tract, to increase efficiency and resistance to stress-factors of animals and birds.

15 tbl, 1 dwg, 5 ex

Description

The invention relates to a technology for producing feed biologically active additives and can be used in biotechnology, microbiology and agriculture, in particular in feed production.

A known method of producing a feed product, including making wort, fermenting it to obtain mash, distilling the mash to produce ethyl alcohol and post-alcohol stillage, cultivating microorganisms in the post-alcohol distillery stillage to obtain their biomass, separating biomass into solid and liquid fractions, and using the solid fraction as a feed product, characterized in that on post-alcohol bard grow pairs of strains of lactic acid and propionic acid bacteria: Lactobacillus plantarum B578 / 25 and Propionibacterium freudenreichii subsp.shermanii Ac103 / 12 or Lactobacillus plantarum B 578/25 and Propionibacterium acnes Ac 1450/28 (RF patent No. 2250265, IPC С12Р 7/06, published on 04/20/05).

The closest analogue (prototype) is a method of producing protein-vitamin feed, which consists in incubating microorganisms in a nutrient medium containing mineral salts and a carbon source in the form of waste from the processing of natural raw materials or as a fermentolizate waste to obtain the target product in the form of biomass of incubated microorganisms while cobalt salt is used as mineral salts, grain, flour waste, starch waste, post-alcohol are used as production waste vinasse bard, beer pellet, fruit squeezed or whey, fermentolizate is prepared from grain, flour waste, starch waste, beer pellet or fruit squeezed, and incubated in a culture medium of lactic acid and propionic acid bacteria (Lactobacillus plantarum 578/25 and Propionibacterium freudenreichii subsp.shermanii 103/12), used in pairs: jointly lactic acid and propionic acid bacteria (RF patent No. 2243678, IPC A23K 1/06, C12N 1/16, publ. January 10, 2005).

The disadvantage of these analogues is the use of non-standardized nutrient media (waste from the production of natural raw materials), which reduces the quality of the finished product in terms of the biological activity of probiotic bacteria, increases the time of cultivation of bacteria (up to 48.5 hours). However, the titer of bacteria in the final product is not indicated. However, in example 1, it was noted that the cultivation process ceases in the exponential growth phase, which indirectly confirms the low titer of the specified consortium of bacteria in the final product. In addition, known feed additives are mainly tested for nutritional properties and do not contain data on their therapeutic and prophylactic properties.

The technical result of the invention is the creation of such a method that would make it possible to obtain an additive in food with a wider range of preventive and therapeutic properties and in a shorter time, as well as increasing the productivity and stress resistance of animals.

The specified technical result is achieved by the fact that in the method for producing a therapeutic and prophylactic supplement, which involves preparing a nutrient medium, administering inoculated doses of the bacterial strains Lactobacillus plantarum and Propionibacterium freudenreichii with subsequent cultivation to obtain the finished product, the bacterial strain Lactobacillus plantarum VKPM B is used according to the invention -2347 and a strain of bacteria Propionibacterium freudenreichii VKPM B-6561, the nutrient medium contains meat enzymatic peptone, glucose, yeast extract, corn-lactose nutrient medium food, cobalt sulfate, ammonium sulfate, agar-agar and distilled water, with the following components, g / l of distilled water:

Peptone meat enzymatic 9-11 Glucose 29-31 Yeast extract 4,5-5,5 Corn Lactose Culture Medium 1.9-2.1 Cobalt sulfate 0.24-0.26 Ammonium sulfate 4,5-5,5 Agar agar 0.9-1.1 Distilled water 1000 ml

and the incubation of bacteria in a nutrient medium is carried out until they reach a titer of at least 1 × 10 ⁶ CFU in 1 cm ³ .

Powdered yeast extract was obtained by autolysis of Sacchoromyces cerevisiae yeast strains grown on molasses media. Physico-chemical characteristics: dry matter, nitrogen, amino nitrogen, sodium chloride, protein, carbohydrates, pH 7.0. The average amino acid composition: alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylanine, proline, serine, theonine, tyrosine, tryptophan, valine. The average vitamin composition: B1, B2, B5, B6, B8, B9, B12, PP. Mineral composition: sodium, potassium, phosphorus, calcium, magnesium, selenium, zinc.

The chemical composition of meat peptone enzymatic in the form of a powder: pH 6.5-7.0; insoluble impurities in%, <7.0; sulfonated ash,% <5.0; true peptone,% <70.0; total nitrogen,%> 14.0; amine nitrogen,%> 3.0; free protein, optical density <0.25.

Characterization of strains. Lactobacillus plantarum strain VKPM B-2347. Morphological and tinctorial properties: gram-positive non-spore forming sticks. Colonies with a folded surface are formed on agarized nutrient media (MPA). Cell size, microns: width 0.6-0.8; length 1.5-3.0. Cultural properties: grows on 7% NaCl. Virulent and toxigenic properties: non-virulent, non-toxic. Conditions for the preservation of the strain at the enterprise: on sloping dense nutrient agar at (6 ± 2) ° С. Strain passage conditions: on solid nutrient agar in Petri dishes at (37 ± 2) ° С. Fermentation of fructose, lactose, galactose, glucose (acid), maltose, mannitol, ribose, sucrose, cellobiose. Forms DL-forms of lactic acid. The optimum pH is 5.8-6.0. The optimum temperature is 37 ° C. The method, conditions and composition of the medium for the propagation of the strain: distilled water - 600 ml, meat water - 400 ml, yeast extract - 5.0 g, sodium acetate - 5.0 g, glucose - 2.5 g, ammonium citrate - 2, 0 g, K ₂ НРО ₄ - 2.0 g, MgSO ₄ · 7Н ₂ О - 0.2 g, MgO ₄ · 4Н ₂ O - 0.05 g, Tween 80 - 1 ml, agar - 5.0 g. The strain is not zoopathogenic, phytopathogenic and is not dangerous for other reasons. The strain was deposited in the Central Museum of Industrial Microorganisms of the State Research Institute of Genetics (Moscow) under the number VKPM V-2347 and in the NPF Research Center (Koltsovo) under the number IC-762-2-3 (see Tables 1 and 2).

Table 1
Characterization of the cultural and morphological properties of the strains No. p / p Sign Propionibacterium freudenreichii strain IC-763-3-4, VKPM B-6561 Lactobacillus plantarum strain IC-762-2-3, VKPM B-2347 one. Common colony color Not determined * White 2. Form and size of colonies on solid nutrient medium Not determined * Convex, with a solid edge, opaque with a diameter of (2-5) mm 3. The form of colonies on a semi-liquid nutrient medium Comet tail-like colonies Carnation Colonies four. Gram stain Positive Positive 5. Mobility Motionless Motionless 6. Cell size Not determined Not determined 7. Cell shape Cocciform sticks, sometimes forked. They are located singly, in pairs, in short chains. Sticks of the correct form, in short chains 8. Spore formation Absent Absent 9. Attitude towards oxygen Facultative anaerobes Optional anaerobes grow weakly under aerobic conditions 10. Optimum temperature for growth 37 ° C 30 ° C Note: "+" - the reaction is positive, "-" - the reaction is negative, * - the parameters are not determined due to the lack of conditions necessary for growth Propionibacterium freudenreichii strain VKPM B-6561. The strain is deposited in the Central Museum of Industrial Microorganisms of the State Research Institute of Genetics. Cultural and morphological features of the strain: fixed rods of 0.5-1.5 microns in size, sometimes forming short curved chains, sometimes look like cocci. Gram-positive. Non-spore forming. Aerial Tolerance. Fermentation of fructose, lactose, galactose, glucose, mannose. The main product of metabolism is propionic acid. In the cell wall, the main component is meso-DAN. The optimum pH is 7.0-7.2. The optimum temperature is 37 ° C.

table 2
Characterization of the biochemical properties of the strains No. p / p Sign Propionibacterium freudenreichii strain IC-763-3-4, VKPM B-6561 Lactobacillus plantarum strain IC-762-2-3, VKPM B-2347 one. Growth under anaerobic conditions + + 2. Aerobic growth - + 3. Tolerance to different concentrations of sodium chloride (presence of growth): 0.5% + + one% + + 2% + + 3% + + four% + + 5% + + 6% + + 7% + + 8% + + 9% + + 10% + + eleven% + + 12% - - four. Catalase activity - - 5. Amylase activity + + 6. Protease activity - - 7. Ammonia formation - - 8. Education - - hydrogen sulfide 9. Indole formation - - 10. Sugar Disposal and - alcohols: - glucose + + - maltose - + - ramnose + + - lactose - + - mannitol + + - dulcite + + - arabinose + + - sorbitol + + eleven. Lecithinase activity + + 12. Esculin hydrolysis + - 13. B - hemolysis - - Note: "+" is a positive reaction, "-" is a negative reaction.

The method, conditions and composition of media for long-term storage of the strain is lyophilization in separated milk under conditions devoid of oxygen and 10% sugar gelatin agar. The method, conditions and composition of the medium for the propagation of the strain - distilled water - 1000 ml, yeast extract - 10.0 g, K ₂ NRA ₄ - 1.0, Na ₂ HPO ₄ · 2H ₂ O - 3.0 g, Na lactate ( 70%) - 40 ml. Dissolve all ingredients and add sodium lactate. The optimum pH is 7.0. The strain is not zoopathogenic, phytopathogenic and is not dangerous for other reasons.

More detailed results of the study of the cultural-morphological and biochemical properties of bacteria Propionibacterium freudenreichii strain IC-763-3-4, VKPM B-6561 and Lactobacillus plantarum strain IC-762-2-3, VKPM B-2347 are presented in tables 1 and 2.

A method of obtaining a feed biologically active additives.

The technological process for the production of Veles biologically active food additives includes the following stages: propagation of producer strains on selective nutrient media, seed production, fermentation, preparation of the final formulation and packaging.

The cultivation of microorganisms. Microbial strains obtained from the Central Museum of Industrial Microorganisms of the All-Russian Research Institute of Genetics or from the Research Center Research and Production Museum Museum, in the isotope cabinet (SHI-11) of the box room, are suspended in sterile physiological saline, and then selective nutrient media are seeded in the ratio 0.1 cm ³ suspension per 10 cm ³ nutrient medium. MRS medium is used as a selective nutrient medium for lactobacilli, and a medium based on sodium lactate and ammonium sulfate is used for propionic acid ones. Incubation of lactobacilli is carried out at 32 ° C for 2-3 days, propionic acid at 37 ° C for 3-5 days. From 2 to 5 passages of cultures are carried out on selective nutrient media with a gradual increase in the volume of the nutrient medium.

The nutrient medium for lactobacilli consists of the following components (per 1000 cm ³ ):

Peptone enzymatic dry GOST 13805 10.0 Sigma Yeast Extract 20.0 cm ³ Anhydrous glucose GOST 6038 20.0 g Twin-80 TU 6-14-938 1.0 cm ³ Potassium phosphate disubstituted GOST 2493 2.0 g Sodium acetic acid GOST 2080 5.0 g Triammonium Citrate 2.0 g Magnesium sulphate 0.2 g Manganese four-water sulfate 50.0 mg Microbiological agar GOST 17206 950.0 mg Meat water 1000.0 cm ³

The nutrient medium for propionic acid bacteria consists of the following components (per 1000 cm ³ ):

Sodium Lactate TU 9199-060-00334557 40.0 g Ammonium sulfate "h" GOST 3769 10.0 g Sigma Yeast Extract 5.0 g Microbiological agar GOST 17206 950.0 mg

At this stage, microbiological and microscopic analysis of the strains is carried out, that is, the correspondence of their cultural and morphological characters to the passport data.

The strains of lactic and propionic acid bacteria in liquid nutrient media are stored in a refrigerator at 4-6 ° C. For the production of feed additives, they can be used within a month.

Next, produce a feed additive in the form of a consortium of biomass Lactobacillus plantarum strain VKPM B-2347 and Propionibacterium freudenreichii strain VKPM B-6561:

To do this, prepare the nutrient medium of the following composition

Distilled water according to GOST 6709-72 1000 Glucose thirty Peptone meat enzymatic 10 Yeast extract 5 Nutrient medium MMC 2 Cobalt sulfate 0.25 Ammonium sulfate 5 Agar agar one.

To obtain a feed additive in bottles, the medium is poured into graduated bottles with a capacity of 500 ml and 300 ml each. From the test tubes with microorganisms (Lactobacillus plantarum strain VKPM B-2347 and Propionibacterium freudenreichii strain VKPM B-6561) stored for storage, 9 ml of culture is transferred over the burner flame to each vial with culture medium. Vials are placed on a rack in a thermal room and cells are grown at a temperature of (37 ± 1) ° C for 18-20 hours until a titer of lactic and propionic acid bacteria is obtained in an amount of at least 1 × 10 ⁶ CFU in 1 cm ³ .

To obtain a feed additive in bottles, a nutrient medium is prepared above the given composition by 40,000 cm ³ . The finished medium is poured into sterile glass bottles of (5000 ± 0.1) cm ³ . After sterilization, the bottles with the medium are cooled to a temperature of (37 ± 2) ° C. Each bottle is seeded with 300 cm ³ (each strain in 1 bottle) of a suspension from bottles of Lactobacillus plantarum strain VKPM B-2347 and Propionibacterium freudenreichii strain VKPM B-6561. Incubated at a temperature of (37.0 ± 0.5) ° C for 20-24 hours to obtain a titer of lactic and propionic acid bacteria in an amount of at least 1 × 10 ⁶ CFU in 1 cm ³ .

Lactobacilli, in particular Lactobacillus plantarum, synthesize a wide range of substances that inhibit the growth of other bacteria. These substances include end products of metabolism, such as organic acids (lactic and acetic), hydrogen peroxide and compounds known as bacteriocins: lysozin, lectrolin, nisin, lactocidin, acidophilus. Bacteriocins are proteins that are produced by certain microorganisms and have a detrimental effect on closely related microorganisms. In general, bacteriocins have a lower spectrum of activity than antibiotics, but their effect is more pronounced.

Propionic acid bacteria, in particular Propionibacterium freudenreichii, belong to the resident species of the symbiotic microflora of the intestinal tract of animals. Most of them live in the rumen of ruminants. In the process of their life, they are able to reduce the excess acidity of silos, thereby preventing the occurrence of acidosis in animals. Propionic acid and metabolic products are propionic and acetic acid, which are well absorbed by animals. In addition, propionic acid has pronounced bactericidal and fungicidal properties.

Feeding cultures of propionic acid bacteria or preparations based on them leads to the elimination of pathogenic microflora and an increase in the number of lacto-, bifido- and propionic-acid bacteria. For example, giving the drug propiacin to piglets for 7-10 days 1 time per day at the rate of 0.5% by weight of the feed led to a decrease in the content of Escherichia coli by 17.1-20.1%, of other types of opportunistic microorganisms by 20, 1-24.1%. At the same time, an increase in the number of lactic and propionic acid bacteria was noted. The trend towards a decrease in conditionally pathogenic and putrefactive microflora persisted on days 28 and 61 of the development of piglets. The animals had a good appetite, their growth rate was higher compared to the control.

One of the main distinguishing features of propionic acid bacteria is their ability to produce a high amount of B vitamins, mainly B12. The use of propionic bacteria biomass as a source of vitamin B12 is not inferior to synthetic analogues in effectiveness. An increase in daily weight gain and a decrease in feed costs of 1 kg of growth of piglets were noted. The most optimal dosages of propionic bacteria biomass in terms of vitamin B12 were 10-40 μg per 1 kg of live weight. Similar results on the use of bacterial biomass as a source of vitamin B12 were obtained in chickens. At the same time, the gain in live weight increased (1.1-6.9%) compared with the commercial drug B12.

The participation of propionic bacteria in digestion is also associated with their high enzymatic activity. For example, some strains have amylase, proteolytic activity; able to absorb a wide range of carbohydrates.

Example 1. The effect of the Veles feed additive on the resident microflora of the intestinal tract of animals, the effectiveness of the feed additive in the treatment of dysbiosis

The influence of the feed additive on the species composition of the microflora of the gastrointestinal tract of animals was studied in laboratory conditions on clinically healthy white outbred mice (3 series of experiments, 100 animals each); Cobb cross broilers: 3 series of experiments - 100 chicks each; in domestic and clinical conditions on dogs: the total number of animals of various breeds and sexes is 120 individuals; calves: in conditions of livestock complexes, farms and farmsteads - 312 animals; piglets: the total number of animals is 450 individuals. The feed additive was fed individually in the calculation of 0.5 cm ³ per 1 kg of live weight 3 times a day.

In the course of the work, it was shown that feeding the Veles feed additive normalizes the species composition of the microflora of the gastrointestinal tract of animals. So, in experiments on white outbred mice, it was found that feeding a feed additive leads to a 10-100-fold increase in the number of lactobacilli and bifidobacteria. However, there was no change in the number of strains of Escherichia coli, Proteus, fungi, staphylococci and streptococci. The effect of the use of the feed additive remained for a month after the cessation of its feeding, which probably indicates the ability of the lacto- and propionic acid bacteria that make up the drug to take root in the gastrointestinal tract of animals. Similar results were obtained when feeding the feed additive to other animal species: piglets, chickens, calves, dogs (Table 3).

Table 3.
The effect of feed additives on the resident gastrointestinal microflora Microorganism groups The number of microorganisms, depending on the duration of feeding the feed additive, CFU / g excrement Before the beginning In 5 days After 10 days In 20 days After 40 days Mice Pathogenic enterobacteria 0 0 0 0 0 E. coli with normal enzymatic activity 9.1 × 10 ⁵ 8.7 × 10 ⁵ 9.0 × 10 ⁵ 8.6 × 10 ⁵ 8.3 × 10 ⁵ E. coli with reduced enzymatic activity (lactose-negative) 8.8 × 10 ² 1.3 × 10 ³ 0.9 × 10 ³ 7.2 × 10 ² 0.8 × 10 ³ E. coli with hemolytic activity 0 0 0 0 0 Protea 5.4 × 10 ² 4.2 × 10 ² 6.5 × 10 ² 5.6 × 10 ² 8.3 × 10 ² Other opportunistic bacteria 7.0 × 10 ² 3.0 × 10 ² 8.0 × 10 ² 6.0 × 10 ² 4.0 × 10 ² Plasma-negative staphylococci 8.1 × 10 ² 7.4 × 10 ² 9.0 × 10 ² 8.8 × 10 ² 7.3 × 10 ² Hemolytic streptococci 1.2 × 10 ⁶ 0.9 × 10 ⁶ 0.7 × 10 ⁶ 0.6 × 10 ⁶ 1.0 × 10 ⁶ Mushrooms 9.2 × 10 ² 8.3 × 10 ² 8.2 × 10 ² 7.9 × 10 ² 7.3 × 10 ² Clostridia 0 0 0 0 0 Lactobacilli 0.2 × 10 ⁷ 3.0 × 10 ⁷ 5.1 × 10 ⁷ 4,5 × 10 ⁷ 3.5 × 10 ⁷ Bifidobacteria 1 × 10 ⁷ 100.0 × 10 ⁷ 200.4 × 10 ⁷ 250.7 × 10 ⁷ 220.5 × 10 ⁷ Calves Pathogenic enterobacteria 0 0 0 0 0 E. coli with normal enzymatic activity 5.1 × 10 ⁶ 8.1 × 10 ⁶ 6.2 × 10 ⁶ 6.5 × 10 ⁶ 4.1 × 10 ⁶ E. coli with reduced enzymatic activity (lactose-negative) 3.3 × 10 ⁶ 5.8 × 10 ⁵ 5.6 × 10 ⁵ 4.9 × 10 ⁵ 3.7 × 10 ⁵ E. coli with hemolytic activity 0 0 0 0 0 Protea 0 0 0 0 0 Other opportunistic bacteria 0 0 0 0 0 Plasma-negative staphylococci 0 0 0 0 0 Hemolytic streptococci 5.0 × 10 ⁴ 6.1 × 10 ⁴ 4.8 × 10 ⁴ 4.7 × 10 ⁴ 4.2 × 10 ⁴ Mushrooms 0 0 0 0 0 Clostridia 0 0 0 0 0 Lactobacilli 1.2 × 10 ⁷ 8.4 × 10 ⁷ 8.1 × 10 ⁷ 7.9 × 10 ⁷ 8.2 × 10 ⁷ Bifidobacteria 8.3 × 10 ⁷ 98.0 × 10 ⁷ 111.1 × 10 ⁷ 114.2 × 10 ⁷ 122.6 × 10 ⁷ Piglets Pathogenic enterobacteria 0 0 0 0 0 E. coli with normal enzymatic activity 3.6 × 10 ⁵ 3.4 × 10 ⁵ 4.1 × 10 ⁵ 3.8 × 10 ⁵ 4.2 × 10 ⁵ E. coli with reduced enzymatic activity (lactose-negative) 3,5 × 10 ³ 3.6 × 10 ³ 3.7 × 10 ³ 3.8 × 10 ³ 4.1 × 10 ³ E. coli with hemolytic activity 0 0 0 0 0 Protea 6.5 × 10 ³ 0 0 0 0 Other opportunistic bacteria 0 0 0 0 0 Plasma-negative staphylococci 0 0 0 0 0 Hemolytic streptococci 4.4 × 10 ⁴ 4.9 × 10 ⁴ 4.7 × 10 ⁴ 4.6 × 10 ⁴ 3.9 × 10 Mushrooms 0 0 0 0 0 Clostridia 0 0 0 0 0 Lactobacilli 0.8 × 10 ⁶ 6.0 × 10 ⁷ 8.0 × 10 ⁷ 8.1 × 10 ⁷ 9.0 × 10 ⁷ Bifidobacteria 0.8 × 10 ⁸ 9.0 × 10 ⁸ 62.1 × 10 ⁸ 50.2 × 10 ⁸ 32.6 × l0 ⁸ Chickens Pathogenic enterobacteria 0 0 0 0 0 E. coli with normal enzymatic activity 1.1 × 10 ⁵ 2.1 × 10 ⁵ 1.5 × 10 ⁵ 2.3 × 10 ⁵ 2.7 × 10 ⁵ E. coli with reduced enzymatic activity (lactose-negative) 2.9 × 10 ⁶ 3.1 × 10 ⁶ 2.8 × 10 ⁶ 3.2 × 10 ⁶ 2.9 × 10 ⁶ E. coli with hemolytic activity 0 0 0 0 0 Protea 4.8 × 10 ⁴ 3.9 × 10 ² 4.7 × 10 5.1 × 10 ² 5.1 × 10 ² Other opportunistic bacteria 0 0 0 0 0 Plasma-negative staphylococci 0 0 0 0 0 Hemolytic streptococci 1.1 × 10 ⁶ 1.9 × 10 ⁶ 2.0 × 10 ⁶ 1.7 × 10 ⁶ 1.6 × 10 ⁶ Mushrooms 0 0 0 0 0 Clostridia 0 0 0 0 0 Lactobacilli 0.8 × 10 ⁶ 6.0 × 10 ⁷ 8.0 × 10 ⁷ 8.1 × 10 ⁷ 9.0 × 10 ⁷ Bifidobacteria 0.8 × 10 ⁸ 9.0 × 10 ⁸ 62.1 × 10 ⁸ 50.2 × 10 ⁸ 32.6 × 10 ⁸

Dogs Pathogenic enterobacteria 0 0 0 0 0 E. coli with normal enzymatic activity 2.7 × 10 ⁵ 2.1 × 10 ⁵ 3.1 × 10 ⁵ 3.0 × 10 ⁵ 3.2 × 10 ⁵ E. coli with reduced enzymatic activity (lactose-negative) 3.1 × 10 ⁴ 3.0 × 10 ⁴ 2.8 × 10 ⁴ 2.9 × 10 ⁴ 3.0 × 10 ⁴ E. coli with hemolytic activity 0 0 0 0 0 Protea 0 0 0 0 0 Other opportunistic bacteria 0 0 0 0 0 Plasma-negative staphylococci 0 0 0 0 0 Hemolytic streptococci 0 0 0 0 0 Mushrooms 0 0 0 0 0 Clostridia 0 0 0 0 0 Lactobacilli 0.5 × 10 ⁷ 5.5 × 10 ⁷ 6.7 × 10 ⁷ 6.0 × 10 ⁷ 6.9 × 10 ⁷ Bifidobacteria 1.2 × 10 ⁸ 11.2 × 10 ⁸ 12.3 × 10 ⁸ 14.8 × 10 ⁸ 15.7 × 10 ⁸

The effect of the feed additive on the species composition of the microflora of animals was studied, depending on the consumption rate. The experiments were performed on chickens of various breeds in 5 repetitions. In each group, at least 1000 birds.

It was established that the effect of the feed additive on the species composition of microflora is manifested in dosages ranging from 0.1 cm ³ per 1 kg of live weight in the group method of giving. An increase in the consumption rate of more than 0.5 cm ³ does not increase the effect (Table 3).

The drawing shows a graph of the effect of the Veles feed additive on the number of lacto-, bifido- and propionic bacteria in chickens, depending on its consumption rate.

In the course of the work, the effectiveness of the feed additive in the treatment of animal dysbacteriosis was studied. For this purpose, animals were selected in livestock farms and poultry farms that showed a reduced content of lactobacilli and bifidobacteria.

The experiments involved 3200 chickens of various crosses and 410 calves of various breeds, 450 piglets of various breeds. The feed additive was given with drinking water at the rate of 0.1, 0.2, 0.3, 0.4 and 0.5 cm ³ per 1 kg of live weight. The duration of giving the drug is 10 days, the frequency is from 1 to 3 times a day.

It was found (Tables 4 and 5) that feeding a feed additive leads to a sharp increase in the number of lactobacilli and bifidobacteria. The effectiveness of the feed additive is directly dependent on the dosage and frequency of use. Maximum efficiency was obtained with a 3-fold use of a feed additive of 0.5 cm ³ per 1 kg of live weight. A decrease in the number of lactobacilli and bifidobacteria was not registered within 2 months after cessation of feeding with the feed additive, which indicates the ability of the strains included in the feed additive to take root in the gastrointestinal tract of animals.

Table 4.
The effect of Veles feed additive on the content of lactobacilli in the gastrointestinal tract of chickens, depending on the frequency and rate of consumption of the drug (Number of lactobacilli, CFU / g of excrement) Multiplicity, once a day Doses of the drug, cm ³ per kg of live weight 0.1 0.2 0.3 0.4 0.5 Chickens one 90.3 × 10 ⁴ 110.1 × 10 ⁴ 930.2 × 10 ⁴ 2550.6 × 10 ⁴ 3560.3 × 0 ⁴ 2 100.7 × 10 ⁴ 150.1 × 10 ⁴ 3500.3 × 10 ⁴ 1950.7 × 10 ⁴ 9700.5 × 10 ⁴ 3 250.8 × 10 ⁴ 1070.3 × 10 ⁴ 1480.8 × 10 ⁴ 3207.3 × 10 ⁴ 10500.7 × 10 ⁴ Piglets one 10.5 × 10 ⁴ 524.3 × 10 ⁴ 327.6 × 10 ⁴ 2590.1 × 10 ⁴ 4000.4 × 10 ⁴ 2 50.3 × 10 ⁴ 375.2 × 10 ⁴ 2567.1 × 10 ⁴ 5240.3 × 10 ⁴ 6500.5 × 10 ⁴ 3 150.7 × 10 ⁴ 1800.7 × 10 ⁴ 3195.5 × 10 ⁴ 6700.7 × 10 ⁴ 8500.6 × 10 ⁴ Calves one 150.2 × 10 ⁴ 54.4 × 10 ⁴ 274.4 × 10 ⁴ 3500.3 × 10 ⁴ 4000.2 × 10 ⁴ 2 300.5 × 10 ⁴ 250.5 × 10 ⁴ 2579.6 × 10 ⁴ 4500.2 × 10 ⁴ 4980.7 × 10 ⁴ 3 900.4 × 10 ⁴ 1980.4 × 10 ⁴ 3256.7 × 10 ⁴ 5439.1 × 10 ⁴ 5400.1 × 10 ⁴

Table 5.
The effect of Veles feed additive on the content of bifidobacteria in the gastrointestinal tract of chickens, depending on the frequency and rate of consumption of the drug (Number of bifidobacteria, CFU / g of excrement) Multiplicity, once a day Doses of the drug, cm ³ per kg of live weight 0.1 0.2 0.3 0.4 0.5 Chickens one 142.2 × 10 ⁵ 306.3 × 10 ⁵ 92.1 × 10 ⁶ 306.2 × 10 ⁶ 2090.3 × 10 ⁶ 2 546.3 × 10 ⁵ 905.4 × 10 ⁵ 450.2 × 10 ⁶ 1500.3 × 10 ⁶ 5999.1 × 10 ⁶ 3 1650.2 × 10 ⁵ 550.1 × 10 ⁶ 2780.2 × 10 ⁶ 7754.2 × 10 ⁶ 9006.3 × 10 ⁶ Calves one 120.1 × 10 ⁶ 127.3 × 10 ⁶ 92.5 × 10 ⁶ 306.4 × 10 ⁶ 809.3 × 10 ⁶ 2 37.3 × 10 ⁶ 305.4 × 10 ⁶ 450.3 × 10 ⁶ 1500.3 × 10 ⁶ 5999.2 × 10 ⁶ 3 212.5 × 10 ⁶ 455.6 × 10 ⁶ 2780.2 × 10 ⁶ 7754.1 × 10 ⁶ 9006.6 × 10 ⁶ Piglets one 164.1 × 10 ⁶ 274.2 × 10 ⁶ 587.7 × 10 ⁶ 1200.4 × 10 ⁶ 3100.3 × 10 ⁶ 2 247.3 × 10 ⁶ 684.3 × 10 ⁶ 950.5 × 10 ⁶ 1700.5 × 10 ⁶ 6600.6 × 10 ⁶ 3 594.4 × 10 ⁶ 1050.1 × 10 ⁶ 1587.3 × 10 ⁶ 2788.3 × 10 ⁶ 8500.7 × 10 ⁶

Example 2. The effectiveness of the feed additives Veles when feeding lactating cows

The effect of the feed supplement on the milk production of cows was studied. Tests of the drug were carried out under various conditions of keeping cows: livestock complexes, farms and private farmsteads. Breeds of animals were various, including not purebred. The total number of animals involved in the experiment was 940 cows of various ages. The drug was administered with water at the rate of 0.1, 0.2, 0.3, 0.4 and 0.5 cm ³ per 1 kg of live weight 2 times a day for one cow.

Tests have shown that giving the drug for 10 days leads to a significant increase in milk yield. In general, the milk yield in the experimental group of animals was 1.5-2.8 liters more compared to the milk yield in the control group. At the same time, milk fat content increased from 3.31-3.48% to 3.69-4.1%. The experimental results are presented in table.6.

Table 6.
The effect of feed additives on milk yield in cows. Indicators The control Experience, drug consumption per kg of live weight 0.1 0.2 0.3 0.4 0.51 Start of experience The average daily milk yield of actual fat content, kg 32,2 31.3 30.9 31.8 32,4 32,0 Fat content,% 3.46 3.31 3.43 3.34 3.48 3.40 End of experience The average daily milk yield of actual fat content, kg 31.3 33.7 33.1 34.0 34.2 33.9 Fat content,% 3,50 3.69 3.72 3.82 4.10 3.72

In another series of experiments, the effect of the feed additive on milk yield, as well as feed consumption in the production of 1 kg of milk was studied (Table 7). The experiments were also carried out on a black-and-white breed of cows in industrial conditions. The experimental and control groups of animals consisted of 25 animals. The experiment is repeated 4 times. The conditions of keeping and feeding animals were the same in the control and experimental groups. The drug was administered with drinking water at the rate of 0.1 cm ³ per 1 kg of live weight 1 and 2 times a day for 10 days.

Table 7.
The effect of feed additives on milk yield, as well as feed costs in the production of 1 kg of milk Indicators The control Experience, the frequency of giving the drug per day 1 time 2 times The average daily dry matter intake, kg 21.9 21.8 21,4 Average daily feed intake 18.9 18.7 18.6 Daily consumption of exchange energy, MJ 234 235 231 Milk yield of natural milk, kg 2490 2629 2532 The protein content in milk,% 4.10 4.11 4.09 Daily milk yield of 4% fat 25.7 27.1 26.1 Costs per 1 kg of milk 4% fat dry matter, g 0.84 0.80 0.82 Costs per 1 kg of milk of 4% fat content of feed units 0.74 0.69 0.71 Costs per 1 kg of milk of 4% fat exchange energy, MJ 9.11 8.67 8.85

Thus, the use of the Veles feed additive helps to increase the productive indicators of cows during lactation. At the same time, under the influence of the feed additive, there is a significant reduction in the cost of producing a unit of milk. The increase in milk yield and fat content in milk is probably due to the positive effect of the feed additive on the digestion process and, as a result, on the metabolism in the body of cows.

Example 3. The effect of the Veles feed additive on the immunological parameters of the animal organism

The experiments were carried out in vivarium NPF IC. The object of the study was broiler chickens 7 days old cross "Change". For the experiment, 400 chickens were selected and kept under the same conditions. At the age of 20 days, the chickens were transferred to the feed of wheat-barley type. Chickens were divided into four groups of 100 individuals each. The first group did not receive a feed supplement (control), the second and third received a feed supplement at the rate of 0.1, 0.3 and 0.5 cm ³ per 1 kg of live weight. The duration of feeding the drug was 10 days.

In the course of the work, it was found that feeding the feed additive leads to a statistically significant increase in the level of lysozyme, beta-lysines and bactericidal activity of the blood of chickens. After stopping the feeding of the feed additive, a gradual decrease in the level of the studied parameters to the control level was recorded. The maximum effect of the feed additive on the immunological parameters of the blood of chickens was observed at a rate of 0.5 cm ³ per 1 kg of live weight. It should also be noted that the effect of the feed additive persists for about a month after cessation of its feeding. The experimental results are presented in table.8.

Table 8.
The effect of feed additives on the immunological parameters of the body of chickens Indicators The control Experience, dose, ml per kg of live weight 0.1 0.3 0.5 Before feeding the drug Lysozyme, mcg / ml 3.2 3.3 3,1 3.3 Beta lysines,% 25,4 26.2 25,2 26.3 Bactericidalness,% 52.7 53.1 52.8 52.7 10 days after the start of feeding the drug Lysozyme, mcg / ml 3.3 3,5 3,7 4.0 Beta lysines,% 27.1 34.1 36,2 38.8 Bactericidalness,% 53.1 58.4 59.8 61.9 30 days after the start of feeding the drug Lysozyme, mcg / ml 3.2 3.6 3,5 3,5 Beta lysines,% 26,4 31.8 32.9 32,4 Bactericidalness,% 52.8 59.7 58.4 57.9 50 days after the start of feeding the drug Lysozyme, mcg / ml 3.3 3.4 3.2 3.3 Beta lysines,% 25.9 27.9 27.8 28.1 Bactericidalness,% 52.7 54,4 53.7 53.6

Thus, the use of a feed additive increases the nonspecific resistance of the animal organism. A similar effect is probably associated with an increase in the antigenic load on the animal organism when feeding a feed additive.

Example 4. The effect of Veles feed additive on the growth, development, preservation of animals and the spread of diseases of the gastrointestinal tract (GIT) of diarrheal symptoms (Table 9).

The experiments were carried out on broilers of the Cobb cross. The experiment included 4 options of 10,000 goals. The first option served as control (animals did not receive the drug), the other three options were experimental, animals from these options received the feed additive as a group method at the rate of 0.1, 0.2, 0.3, 0.4 and 0.5 cm ³ per 1 kg of live weight. The duration of the feeding period was 10 days, 1 time per day. The experiment was carried out in two versions with the use of antibiotics and without the use of antibiotics. In the first case, the feed supplement was started to be fed on the 5th day after antibiotic withdrawal, in the second case, the feed supplement was started from 1 day of life of the chickens. The duration of the feeding period in both cases was 10 days, 1 time per day.

From the data presented in table 10 it is seen that feeding the feed additive leads to a significant increase in the safety of chickens and a decrease in the spread of diseases of the gastrointestinal tract of diarrheal symptoms. When feeding a feed additive, feed conversion increases, animal weight gain increases. The positive effect of the feed additive is probably due to the fact that when it is used, the species composition of the microflora of the gastrointestinal tract is optimized and the digestion process is normalized.

Table 9. The effect of feed additives on the growth, development, preservation of animals and the spread of diseases of the gastrointestinal tract (GIT) of diarrheal symptoms

Indicators The control Experience, the consumption rate of the drug, ml per kg of live weight 0.1 0.2 0.3 0.4 0.5 Without antibiotics Live weight kg 2,276 2,316 2,325 2,34 2,37 2,38 Preservation,% 96.1 96.3 97.1 97.5 98.9 99.0 Feed conversion, kg / kg 1,870 1,830 1,840 1.81 1.79 1.78 The spread of diseases of the gastrointestinal tract diarrheal symptoms,% 15.6 13,2 12,2 5.1 1.85 1.65 After antibiotic use Live weight kg 2,199 2,218 2,305 2,31 2,34 2,350 Preservation,% 95.5 96.4 97.7 98.2 99.1 99.1 Feed conversion, kg / kg 1,870 1,830 1,840 1.81 1,790 1,780 The spread of diseases of the gastrointestinal tract diarrheal symptoms,% 3.2 2.95 1,54 0.85 0.75 0.55

The effect of feed additives on the safety of piglets was studied. For the experiment, 200 farrowing sows were selected. After giving birth, sows with pigs were divided into an experimental and control group. Pigs of the experimental group received the drug at the rate of 0.1 cm ³ 3 times a day. Piglets of the control group did not receive the drug.

Table 10.
The effect of Veles 6.59 feed additive on the safety of piglets Indicators Experience The control The average number of pigs per sow 10.1 11,2 Preservation,% 90 78 Birth weight, g 1200 1250 Weight at weaning, g 6900 5800

It was found that giving the drug significantly (more than 10%) increases the safety of young animals and increases the gain by 18.9%.

The effect of the feed additive on the safety and increase in weight gain in animals compared to feed antibiotics was studied (Table 11). The experiments were carried out on sprouts and chickens. The feed additive was fed to animals 3 times a day at the rate of 0.1 cm ³ per 1 kg of live weight for 10 days. Animals of the control group did not receive drugs. The development of animals was determined 30 days after the use of the drug.

Table 11.
The effect of Veles feed additive on the safety and development of animals in comparison with feed antibiotics Indicators Veles 6.59 Bacitracin Biovit The control Piglets Number of animals 1500 1300 1300 1300 The initial mass, g 7120 7250 7070 7089 Final mass 22700 20258 20182 19174 Daily increase, g 389.5 325,2 327.8 302.1 Preservation,% 96.4 96.2 95.5 85.0 Chicken Boilers Number of animals 22000 25,000 23000 22000 The initial mass, g 42 45 40 43 Final mass 2100 1987 1900 1750 Daily increase, g 51.5 48.6 46.5 42.6 Preservation,% 98.7 99.0 98.0 87.0

Thus, feeding a feed additive increases the safety of animals and stimulates their development. In addition, the feed additive can be used as an effective tool for the treatment and prevention of gastrointestinal infections of the gastrointestinal tract. Moreover, the feed additive is not inferior to feed antibiotics in terms of effectiveness.

The effect of the feed additive as a therapeutic agent for gastroenteritis of fur-bearing animals was studied (Table 12). The experiments were carried out on the basis of the experimental farm SB RASX. As experimental animals, silver-black foxes aged 4-5 months with a weight of 4.0-4.3 kg were used. The feed additive was given to animals at the rate of 0.5 cm ³ 3 times a day. The foxes of the control group were treated with furolysidone according to the standard regimen. The feed additive was given with drinking water. In animals, hematological status was studied before and after the experiment — hematological parameters. The change in weight of the animals was carried out 30 days after the start of the experiment.

It was found that 2-3 days after feeding the fodder supplement to foxes, their general condition improves, signs of oppression, dehydration, anemia and diarrhea are eliminated, and appetite and feed intake are increased. Recovery of foxes occurred on day 4-5. Fecal matter acquired a shaped and dense consistency. On palpation, the walls of the abdominal cavity were painless, the animals were mobile and had a good appetite. Similar results were obtained in the control using furolysidone.

Table 12.
The effectiveness of feed additives in the treatment of fox gastroenteritis Indicators The control Experience The beginning of recovery, days 2-3 2-3 Duration of treatment, days 5-7 4-5-7 The average weight before treatment, g 4190 4140 The average weight 30 days after treatment, g 4520 4620 The average daily gain, g

Table 13.
Hematological indicators of foxes when feeding Veles 6.59 Indicators Before treatment After treatment The control Experience The control Experience Red blood cells, 10 ¹² / L 8.56 ± 0.02 8.50 ± 0.05 9.02 ± 0.1 9.83 ± 0.2 Hemoglobin, g% 11.0 ± 0.32 10.8 ± 0.30 11.3 ± 0.25 13.0 ± 0.37 White blood cells, 10 ⁹ / L 5.64 ± 0.68 5.78 ± 0.51 6.32 ± 0.04 6.76 ± 0.04 Total protein, g / l 61.2 ± 0.29 60.8 ± 0.4 62.5 ± 0.65 65.5 ± 0.58

It was found that the use of a feed additive leads to an increase in the number of red blood cells, white blood cells, hemoglobin, and total protein (Table 13).

Thus, Veles feed additive is an effective treatment for fox gastroenteritis. In addition, it can be used as an immunostimulating agent.

Example 5. The effectiveness of the use of feed additives Veles in stress

In industrial animal husbandry, animals are exposed to various stress factors. As a result, their development is significantly impaired, productivity and resistance to infectious diseases are reduced. We have studied the possibility of using the Veles feed additive as an adaptogenic agent to increase the resistance of animals to abiotic stress factors (Table 14).

The experiments were performed on chickens of the cross "Smena" under conditions of heat stress. The feed additive was used according to the previously described scheme. The number of chickens in the control and experimental groups was 15,000 heads.

Table 14.
The effectiveness of feed additives as an anti-stress drug Indicators The control Experience Live weight kg 1587 1750 Preservation,% 75.6 92.1 Feed conversion, kg / kg 2.2 1.8

The duration of the experiment is 42 days.

In another series of experiments, the effect of feed additives was studied as a means of preventing vaccine stress in chickens and forming immunity to the Newcastle disease virus. For this, a control and experimental group of 120 goals were formed. The experiments were carried out on broiler cross broilers "Broiler-6". For the experiment, animals with the same mass and zero titers of specific antibodies to the Newcastle disease virus were used. Determination of the titer of the virus was carried out in the reaction of delayed hemagglutination according to the conventional method 15 and 35 days after vaccination. The drug was administered with feed at the rate of 0.5 cm ³ per 1 kg of live weight. Feeding the drug began 3 days before vaccination and continued for 7 days after vaccination. The control group did not receive a feed supplement.

Table 15.
The influence of Veles feed additive on the formation of immunity to the Newcastle disease virus Indicators The control Experience Antibody titer, 15 days after vaccination, log 5.7 6.4 Antibody titer 35 days after vaccination, log 0.4 0.4 Mass g 1850 2020 Preservation,% 97.2 98.9

It was shown that under the influence of the feed additive there is a significant increase in the number of antibodies to the Newcastle disease virus. In addition, feeding a feed additive has a positive effect on the safety and weight gain of animals (Table 15).

Thus, based on the data obtained as a result of experimental testing of the Veles feed additive, it was found that this animal feed additive is effective when used to normalize the species composition of the microflora of the gastrointestinal tract, increase safety and productivity, and increase resistance to stress factors, infectious and non-infectious diseases, stimulating the growth and development of young animals: agricultural (cattle and small cattle, pigs, horses), domestic (cats, dogs, etc.), etc. FIR, including birds and fur-bearing animals.

The drug can be prescribed to animals inside individually or in a group method with drinking (milk, colostrum, not containing aseptic preparations or boiled cooled water) and food (grain mix, animal feed, etc.), as well as rectally after a cleansing enema.

The optimal consumption rate of Veles when it is assigned as a group method in the process of complex growing of farm animals and birds is at least 1500 cm ³ per 1 ton of feed or water.

The optimal consumption rate of Veles for individual oral and rectal administration is from 0.1 to 0.5 cm ³ per 1 kg of live weight.

It is established that the effect of Veles application is directly dependent on the frequency of its application.

In recommended doses, Veles feed additive has no side effects and does not cause complications.

Side effects and complications when using Veles in recommended doses have not been identified. Contraindications for use have not been established.

Livestock products after the use of the drug can be used for food purposes without restrictions.

Claims (1)

A method of obtaining a therapeutic and prophylactic additive, comprising the preparation of a nutrient medium, the introduction of inoculated doses of the bacterial strains Lactobacillus plantarum and Propionibacterium freudenreichii with their subsequent cultivation to obtain the finished product, characterized in that they use the Lactobacillus plantarum VKPM B-2347 strain and the Propionibacteri fre bacteria bacteria VKPM B-6561, the growth medium contains enzyme meat peptone, glucose, yeast extract, corn-lactose growth medium, cobalt sulfuric acid, ammonium sulfuric acid, agar-agar and distill distilled water with the following components, g / l distilled water:
Peptone meat enzymatic 9-11 Glucose 29-31 Yeast extract 4,5-5,5 Corn Lactose Culture Medium 1.9-2.1 Cobalt sulfate 0.24-0.26 Ammonium sulfate 4,5-5,5 Agar agar 0.9-1.1 Distilled water 1000 ml
and the incubation of bacteria in a nutrient medium is carried out to
they achieve a titer of at least 1 · 10 ⁶ CFU in 1 cm ³ .