RU2217493C2 - Biopreparation- probiotic, method for its preparing and strain streptococcus faecium te-17 for preparing biopreparation-probiotic for animals and poultry - Google Patents

Abstract

FIELD: biotechnology, veterinary science, microbiology. SUBSTANCE: strain Streptococcus faecium TE-17 is stored in collection ZAO "BIOFLORA" at = B-07. Strain shows high productivity and ability to inhibit pathogenic microflora. In preparing probiotic for culturing the strain nutrient medium is enriched with yeast autolyzate, aqueous solution of vitamins and unsaturated fatty acids as precursors of prostaglandins. This results to preparing probitic that represents liquid culture of S. faecium TE-17, biopolymers, carbamide, trace elements, stabilizing agent, lactic acid and tap water taken in the definite ratio of components. Use of probiotic provides weight gain for poultry, inhibition of pathogenic microflora and sanitation effect. Invention can be used in agriculture for growing animals and sanitation of young stock. EFFECT: improved preparing method, valuable properties of probiotic. 3 cl, 6 tbl, 7 ex

Description

 The invention relates to animal husbandry and veterinary medicine, can be used in biotechnology, an industrial and farm method of growing agricultural animals for the recovery of young animals, obtaining additional weight gain of animals.

 It is known that some lactic acid bacteria inhibit the growth and development of pathogenic and conditionally pathogenic intestinal microflora of animals and birds. So, the bacterial strain Streptococcus faecium M-3185 is used to combat dysbacteriosis of agricultural animals [1].

 Known biological products based on lactic acid bacteria used in animal husbandry. For example, a drug based on Lactobacillus acidophilus is known for the prevention of intestinal diseases of young animals of agricultural animals [2].

 Known biological product based on lactic acid bacteria Streptococcus and Lactobacillus galliferm, inhibiting the development of conditionally pathogenic and putrefactive intestinal microflora of animals [3].

 A nutrient medium is also known for growing a strain of Streptococcus faecium STF-1/56 upon receipt of a probiotic containing milk whey, sea water as a source of trace elements Zn, Mg, Fe, Cu, Mn, Co, glucose, monosubstituted potassium phosphate, ammonium chloride and tap water [4].

 However, preparations based on the above strains are not widely used; firstly, the strains used have low productivity, and secondly, the proposed technology requires exceptionally high energy consumption, since the form of the preparations is dry, using freeze-drying or spray drying.

 Isolates containing lactic acid bacteria were isolated from the chicken intestine, which, after a stepwise selection of productive variants, were identified as Streptococcus faecium [5].

 To isolate lactic acid bacteria, direct seeding from samples, accumulative cultures on liquid media with alcohol, and subsequent seeding on agar media with chalk were used.

 1. Cultural and morphological features.

 Streptococcus faecium belongs to the genus Streptococcus Rosenbach. It is a cocci with a size of 0.8-1.0 μm, arranged in short chains, groups or in pairs, does not form a spore, is motionless, Gram stain is positive. On agarized nutrient media form shiny dense discs of regular shape with smooth edges with a diameter of 1-2 mm. When grown on a liquid nutrient medium after 12-24 hours, a uniform turbidity forms, settling for 2-3 days.

 2. Physiological properties.

 Optional anaerobic, gram-positive, ferments mannose, galactose, arabinose, trehalose, maltose, glucose, lactose, salicin, ribose, cellobiose, amygdalin, fructose, dextrin, mannitol; does not ferment sucrose, raffinose, dulcite, inulin, inositol, sorbitol, sorbose, xylose. Does not contain cytochrome of the respiratory tract, catalase-negative, does not reduce nitrates, forms ammonia from arginine, does not thin the gelatin, ferments and weakly peptones milk, restores milk with litmus and grows on an environment containing 40% bile, 6.5% sodium chloride, 4% taurocholate. It forms lactic acid 0.1-0.15% at 7-9 days of fermentation.

 A feature of the Streptococcus faecium TE-17 strain is a high specific growth rate, homoenzymatic lactic acid fermentation, antagonism against certain pathogenic and conditionally pathogenic microorganisms: Ps aeruginosa, Str. aureus, you. mesentericus, Salmonella typhimurium, E. coli, Mycobacterium. smegmatis, Klebsiella pneumonia.

 Resistant to antibiotics: tetracycline, ampicillin, levomycin, penicillin, erythromycin.

Strain conditions for the strain: lyophilized culture grown on a medium containing 20% yeast autolysate, 3.0% glucose, dry return (or milk-under-cure) serum - 10%, water - up to 1 liter. Storage in ampoules or vials at a temperature not exceeding minus 2 ^o C.

 A comparative assessment of the productivity of the strain Streptococcus faecium TE-17 is presented in table 1.

 The strain is stored in the collection of microorganisms of CJSC "BIOFLORA", collection B-07.

 The revealed properties of the strain give reason to use it to obtain a biological product for animal husbandry as a probiotic with therapeutic and prophylactic properties.

Example 1. To obtain 1 l of a liquid culture of a strain of Streptococcus faecium TE-17 (collection of Bioflora V-07 CJSC) in order to obtain a biological product, a liquid nutrient medium was prepared: 200 g of yeast autolysate, 3.0 g of glucose were added to 1 l of whey , the medium was sterilized for 20 minutes at 0.5 kgf / cm ² . Vitamin supplements were added to chilled sterile water in the form of a sterile solution of vitamins: thiamine, para-aminobenzoic acid, riboflavin, ascorbic acid in equal amounts for a total of 0.1 g / l of medium, pH of 6.5. The medium was inoculated with the prepared seed culture of Streptococcus faecium TE-17 (collection of Bioflora V-07 CJSC) at the rate of 10% inoculum, i.e. 100 ml. Fermentation was carried out at 41 ^o C. At 6 hours the fermentation was sterile, with gentle stirring, 2 l of an alcohol solution of unsaturated fatty acids with a concentration of 0.005 M was introduced. The cultivation was carried out for 18 hours, the pH of the obtained liquid culture was 3.8, the number of CFU was 2 • 10 ¹⁰ ml ^-1 . The resulting liquid culture was used to obtain a biological product.

Example 2. The liquid culture obtained as described in example 1 was diluted with tap water in a ratio of 1: 2 and 20 g of fucus alginates and 20 ml of a liquid culture of Bacillus mucilaginosus (6) with a viscosity of 43 • 10 ³ poise were added with stirring. carbamide at a rate of 10 g / l and trace elements in the form of glycinates Zn, Mg, Mn, Fe, Cu, B, Se, at a rate of 10 mg / l, stabilizers calcium propionate, sodium benzoate, ascorbic acid in an amount of 100 mg / l in total. After stirring, the pH of the preparation was adjusted to a value of 3.5 by the addition of lactic acid. The resulting preparation was used for further experiments.

Example 3. Investigated the safety of viable cells in the preparation during long-term storage at a temperature of 7 ± 2 ^o C. The results are presented in the table. 2
Example 4. The biological product obtained as described in example 2, was introduced into the broiler feed in the amount of 3.0 and 1.5 ml per 1 kg of feed during the entire period of feeding of chickens. The results of the experiment are presented in table 3.

 Example 5. The biological product obtained as described in example 2 was introduced into the feed for chickens with signs of diarrhea at a dose of 3 and 1.5 ml per 1 kg of feed during the entire period of feeding the chickens. Observed for 7 days. The results of the experiment are presented in table 4.

 Example 6. The biological product obtained as described in example 2, was introduced into the feed of piglets with signs of diarrhea at a dose of 3 and 1.5 ml per 1 kg of feed during the entire period of feeding piglets. Observed for 14 days. The results of the experiment are presented in table 5.

 Thus, the use of the claimed biological product allows to obtain an increase in weight (weight) of the bird 11-15%, while the claimed drug inhibits the growth of pathogenic microflora, providing a healing effect.

 Example 7. The biological product obtained as described in example 2 was introduced into the feed to piglets in an amount of 1.5 ml per 1 kg of feed for 1 month. The results of the experiment are presented in table 6.

Sources of information
1. RF patent 2152433, publ. 07/10/2000

 2. L.F. Abyzova, M.S. Polonskaya, V.V. Leonovich. Proceedings of VNIIBaktpreparat, issue. 1. M., 1973, with. 97-105.

 3. Manual on the use of galliferm in veterinary medicine. M., 1990

 4. RF patent 2086647, publ. 08/10/97

 5. The determinant of bacteria Bergey. M., 1987

 6. RF patent 2081867, publ. 06/20/97

Claims (3)

1. The strain Streptococcus faecium TE-17 (collection of JSC "BIOFLORA" No. B-07) to obtain a biological product probiotic with therapeutic and prophylactic properties for animals and birds. 2. A method for producing a probiotic for animals and birds, which provides for the production of Streptococcus faecium liquid culture by culturing a microorganism to a predetermined microbial cell content on a nutrient medium containing milk serum, glucose, characterized in that the Streptococcus faecium TE-17 strain is cultivated (collection of BIOFLORA CJSC No. B-07) on a nutrient medium additionally containing yeast autolysate in an amount of 18-25% of whey, a vitamin supplement in the form of an aqueous solution of vitamins: para-aminobenzoic acid, thiamine, ascor ynoic acid, riboflavin total concentration of 0,005-0,06% of whey, and unsaturated fatty acids - precursors of prostaglandins in an amount of 10 ^-5 -10 ^-6 M. 3. A probiotic for animals and birds containing a liquid culture of Streptococcus faecium TE-17 (collection of BIOFLORA CJSC No. B-07) obtained according to claim 2, biopolymers in the form of algae or their alginates and a liquid culture of Bacillus mucilaginosus VKPM B-5987 containing the polysaccharide synthesized by it in an amount of 3-10% in total, carbamide, trace elements Zn, Mg, Mn, Fe, Cu, B, Se, in the form of complexonates based on glycine or hydroxyethylidene diphosphonic acid (HEDP), a stabilizer in the form of a mixture of calcium propionate, sodium benzoate and ascorbic acid, lactic acid and tap water in the wake containing components, wt.%: Liquid Streptococcus Culture faecium TE-17 (collection of CJSC “BIOFLORA” No. B-07) according to Claim 2 50 Biopolymers 3-10 Urea 0.2-2.0 Trace elements 0.0001-0.003 Stabilizer 0.001-0.01 Lactic acid (in the amount necessary and enough to bring the pH of the drug is up to 3.5-4.2) Tap water

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