RU2652836C1 - Fodder additive with probiotic activity for farm animals, birds, horses and fish - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: invention relates to agriculture and fodder production, namely to a dry feed additive with probiotic activity for farm animals, birds, horses, fish. Additive with probiotic activity includes a filler and a dried biomass of a complex of bacteria of two strains: a strain of bacteria Enterococcus faecium 1-35 with the titer of living bacteria of 3.8×10⁷–2.2×10⁸ CFU and a strain of bacteria Bacillus megaterium B-4801 with the titer of living bacteria of 3.8×10⁷–3.3×10⁸ CFU. Dried biomass of these strains is applied in a mixture in equal amounts onto the filler, in the first variant – in the form of bran containing live bacteria Enterococcus faecium 1-35 3.8×10⁷ CFU and living bacteria Bacillus megaterium B-4801 3.8×10⁷ CFU in 1 g of the fodder additive; in the second variant – in the form of sunflower meal containing live bacteria Enterococcus faecium 1-35 1.8×10⁸ CFU and living bacteria Bacillus megaterium B-4801 1.0×10⁸ CFU in 1 g of the fodder additive; in the third variant – in the form of burnt diatomite crumb containing living bacteria Enterococcus faecium 1-35 2.2×10⁸ CFU and living bacteria Bacillus megaterium B-4801 2.1×10⁸ CFU in 1 g of the fodder additive; in the fourth variant – in the form of kizelgur containing live bacteria Enterococcus faecium 1-35 1.3×10⁸ CFU and living bacteria Bacillus megaterium B-4801 3.3×10⁸ CFU in 1 g of the fodder additive.

EFFECT: implementation of the invention provides normalizing the digestion processes, increasing the digestibility and accessibility of nutrients in the diet, improving the indicators of growth and development of farm animals, birds, horses and fish.

4 cl, 8 tbl, 5 ex

Description

The invention relates to agricultural biotechnology, in particular to a Profort dry feed additive with probiotic activity, containing viable bacteria of the Enterococcus faecium 1-35 strain and viable bacteria of the Bacillus megaterium B4801 strain and used as feed for farm animals, birds, horses, fish.

Known drugs with probiotic activity used in the feeding of farm animals and poultry and containing microorganisms, including bacteria Enterococcus faecium.

Known synbiotic composition for the correction of dysbiotic disorders of the microbiocenosis of the gastrointestinal tract, including the prebiotic component and the probiotic component in the form of a combination of bacterial strains Lactobacillus delbrueckii TS1-06 and Lactobacillus fermentum TS3-06, as well as dried culture liquids with metabolites of the probiotic bacterial strain Bacillus Bacillus bact -2335 and metabolites of the probiotic bacterial strain Enterococcus faecium L-3, RU No. 2592988 C1, A61K 35/741, A61K 36/899, A61K 47/02, 07.27.2016.

A known bacterial strain Enterococcus faecium Ef79OSAU with antagonistic activity against bacteria of the genus Listeria and the species Enterococcus faecalis, deposited under registration number-1251 and used for the preparation of probiotic preparations, RU No. 2571852 C2, C12N 1/20, A61K 35/744, 20.12. 2015.

Known feed additive with probiotic activity on a mineral basis, used in feed for farm animals and poultry, consisting of viable spores of spore-forming bacteria of the strain Bacillus subtilis 111 with a titer of 2 × 10 ⁶ -6 × 10 ⁹ CFU / g and filler diatomite in the form burnt crumbs, RU No. 2569002 C1, A23K 1/16, A23K 1/175, 11/20/2015.

Known means for the prevention of mastitis in cows during lactation, containing probiotic strains having a composition in the following ratio of components, wt. %: Bacillus subtilis - B-5225 - 4% at a concentration of 1 × 10 ⁸ CFU / ml, Enterococcus faecium STF 1/56 - 4% at a concentration of 1 × 10 ⁸ CFU / ml, glycerin - 2%, sterile water - the rest - 90%, RU No. 2560277 C1, A61K 35/741, A61K 35/744, 08.20.2015.

The Enterococcus faecium 8G-1 strain (NRRL B-50173), the Enterococcus faecium 8G-73 strain (NRRL B-50172) and the Bacillus pumilus 8G-134 strain (NRRL B-50174) are known to improve the health of ruminants based on any from these strains or a combination thereof in combination with monensin, a composition is obtained that provides treatment or prevention of acidosis, improvement of health indicators of ruminants and / or increase of their productivity, RU No. 2528859 C2, C12N 1/20, C12P 1/04, A61K 35/74 09/20/2014.

Known drug for the treatment and prevention of intestinal dysbacterial conditions in birds, containing inulin and fermentation of live microorganisms Lactobacillus acidophilus, Enterococcus faecium, No. 2410105 C1, A61K 35/66, 01/27/2011.

A known bacterial strain Bacillus licheniformis BKM B-2414D is used to produce a component of a probiotic feed additive intended for the production of high-quality feeds that increase productivity and reduce the risk of gastrointestinal diseases of animals, birds and fish, RU No. 2398872 C1, C12N 1/20, A23K 1/165, 09/10/2010.

Known probiotic feed additive in the form of a mixture of microorganisms selected from the group Enterococcus faecium, DSM 16211, Lactobacillus reuteri, DSM 16350, Lactobacillus salivarius ssp. salivarius, DSM 16351, Pediococcus acidilactici, DSM 16210, Bifidobacterium animalis, DSM 16284, which prevents the harmful effects of microbes in the digestive system of farm birds, RU No. 2372788 C2, A23K 1/00, A23K 1/18, 11/20/2009.

Known feed additive for young pigs containing dry beet pulp and an additional probiotic, which includes bacteria cultures of Bifidobacterium globosum, Enterococcus faecium and Bacillus subtilis, fumaric acid and zeolite, RU No. 2359465 C1, A23K 1/00, 06/27/2009.

Known probiotic additive used in the manufacture of animal feed containing probiotics for farm animals, poultry and fish, and containing the biomass of spore-forming bacteria Bacillus subtilis B-2250 and / or Bacillus licheniformis B-2252, and a carrier sorbent containing hydrophilic aerosil brand A and hydrophobic brand AM, as well as a moisture-intensive filler, RU No. 2252956 C2, C12N 1/20, A23K 1/165, 05/27/2005; RU No. 2203947 C1, C12N 1/20, 05/10/2003.

Known methods for producing drugs with probiotic activity for farm animals, poultry and fish containing microorganisms:

A known method of obtaining a comprehensive feed preparation for farm animals, poultry and fish, characterized in that a liquid culture of the strain Corynebacterium glutamicum VKPM B-1959 and liquid cultures of the strains of Bacillus subtilis VKPM B-8130, Bacillus subtilis VKPM B-2984, Bacillus subtilis VKPM B -4099 and Bacillus licheniformis VKPM B-4162, then 30 l of the liquid culture of the strain Corynebacterium glutamicum VKPM B-1959 is added to 35 l of the mixture consisting of liquid cultures of Bacillus subtilis VKPM B-8130, Bacillus subtilis VKPM B-2984 and Bacillus subtilis VKPM B-2984 -4099 taken in a ratio of 6: 6: 1, respectively, and the resulting total mixture of liquid tur in an amount of 65 l is applied to 200 kg of previously prepared support - sterile beet pulp enriched fermentative yeast Saccharomyces cerevisiae, and solid state fermentation is carried out, then added 65 L of a liquid culture of Bacillus licheniformis VKPM B-4162, containing not less than 5,6 × ¹⁰ August CFU / g, the mixture is thoroughly mixed and dried to a moisture content of 8-10%, after which dry grass powders are added, mixed and crushed to obtain a homogeneous mass, RU No. 2499829 C1, C12N 1/20, A23K 1/00, 11/27/2013 ; RU No. 2477614 C1, A23K 1/16, A23K 1/14, 03.20.2013.

Known methods for producing drugs with probiotic activity for farm animals and poultry containing Enterococcus faecium bacteria:

A known method of producing a biological product with probiotic properties for feeding farm animals and poultry, comprising a mixture of essential oils of garlic, eucalyptus, rosemary and thyme, a bacterial strain Enterococcus faecium 1-35 and a filler, RU No. 2458527 C1, A23K 1/165, 20.08 .2012.

It is known that uterine culture is prepared by co-growing Enterococcus faecium B 3491, Lactobacillus plantarum B 3242, Bifidobacterium bifidum AC 1247 taken in a 1: 1: 1 ratio to a predetermined biotiter of each microorganism of at least 5-10 ⁸ CFU under deep cultivation of lactic acid microorganisms providing a probiotic preparation for the prevention of gastrointestinal diseases, increase the safety and increase in live weight of young farm animals, RU No. 2380107 C1, A61K 35/66, A23K 1/165, 01/27/2010.

The use of microorganisms in known preparations with probiotic activity, including in feed additives, of Enterococcus faecium bacteria is very diverse and requires individual selection of their introduction into the feed in each case.

Known bacterial strain Bacillus megaterium and its use:

A known bacterial strain Bacillus megaterium var. phosphaticum BKM B-2357D, mobilizing phosphorus and silicon from lithosphere objects, resistant to polyhexamethylene guanidine and promising for the production of a bacterial preparation, RU No. 2327737 C2, C12N 1/20, C05F 11/08, 06/27/2008.

The known strain of bacteria Bacillus megaterium is promising for bacterial preparations.

In addition, the well-known bacterial strain Bacillus megaterium is used for biological treatment of wastewater from pollution, for the restoration of soil contaminated with oil, for the microbiological protection of plants, RU No. 2185339 C2, C02F 3/34, C12N 1/20, 07.20.2002; RU No. 2037472 C1, C02F 3/34, C12N 1/20, 06/19/1995; RU No. 2571219 C2, C12N 11/14, B09C 1/10, C02F 3/34, 12.20.2015; RU No. 2421291 C2, B09C 1/10, C12N 1/26, 06/20/2011; RU No. 2558291 C2, C12N 1/20, A01N 63/00, 07.27.2015; RU No. 2539025 C1, A01N 63/00, C12N 1/20, 01/10/2015; RU No. 2518252 C1, A01N 63/00, 06/10/2014; RU No. 2454075 C2, A01N 63/00, C12N 1/20, June 27, 2012; RU No. 2147181 C1, A01N 63/00, C05F 11/08, 10.04.2000.

Known feed additives based on mineral carriers:

Known feed additive for pigs, which is a natural mineral of the Vodinsky deposit of the Krasnoyarsk region of the Samara region, containing 47.37% sulfur and 21.4% calcium, RU No. 2480025 C2, A23K 1/175, 04/27/2013.

Known feed additive for young cattle, including bentonite Donguz deposits, sunflower fuse and inorganic sodium selenite, and as a filler - wheat bran, RU No. 2497382 C2, A23K 1/16, A23K 1/175, 10.11.2013.

Known feed additive for animals and birds, including natural zeolite, RU No. 2484640 C1, A23K 1/00, 06/20/2013; RU No. 2467591 C1, A23K1 / 16, A23K 1/00, 11/27/2012; RU No. 2448472 C2, A23K 1/175, 04/27/2012; RU No. 2390254 C1, A23K 1/00, A23K 1/16, A23K 1/175, 05.27.2010; RU No. 2385623 C2, A23K 1/16, A23K 1/175, 04/10/2010; RU No. 2374896 C1, A23K 1/00, A23K 1/175, 12/10/2009; RU No. 2262863 C2, A23K1 / 16, A23K 1/175, 10.27.2005.

Known feed additive for cattle, including vegetable and grain components and a mineral additive in the form of blue clay, which is a natural sorbent and a cheap source of vital macro- and micronutrients and has high adsorption and ion exchange properties, RU No. 2238659 C2, A23K 1/16 A23K 1/175, 10.27.2004.

Known organic-mineral feed additive containing a filler and a siliceous material, the additive of which contains diatomite from the Inza deposit of the Ulyanovsk region with a silica content of 81.30-85.60% or flask of the Dubinsky deposit of the Ulyanovsk region with a silica content of 84.10-89.62%, having high porosity and hygroscopicity with the ability to accumulate physiologically active substances from the filler, RU No. 2199883 C1, A23K 1/16, A23K 1/175, 03/10/2003.

Known feed additive with phytoprobiotic activity on a mineral basis, used in feed for farm animals and poultry and consisting of viable bacteria of the strain Enterococcus faecium 1-35 with a bacterium titer of at least 10 ⁶ CFU / mg, plant material and diatomite in the form of burnt chips, RU No. 2574689 C1, A23K 1/16, 02/10/2016.

Known feed additives contain in their composition natural materials based on clay, or peat, or natural zeolite (the most common), which in each case have individual processing technologies.

Known feed product for farm animals and birds, containing in its composition a filler and dried biomass of a complex of bacteria of microorganisms, RU No. 2552872 C1, A23K 1/16, 06/10/2015.

This technical solution is made as the closest analogue of the present invention.

The feed product of the closest analogue contains sorption material in the form of a mixture of vermiculite and bentonite clay, lactic acid, succinic acid, citric acid and additionally dried biomass of Lactobacillus acidophilus, Enterococcus faecium, Bacillus subtilis, Bacillus polymyxa.

In the feed product the closest analogue of another composition is not provided.

The present invention is based on the solution of a problem that allows you to create a new feed supplement "Profort", to increase the quantitative and qualitative component of probiotic activity and to expand the functional applicability and its use.

The technical result of the present invention is to create a new nutritional supplement "Profort" based on the Enterococcus faecium 1-35 and Bacillus megaterium B4801 strains, to obtain the combined effect of two bacterial strains with high probiotic activity and, as possible, applying dry biomass of these strains to various carriers: bran (Option 1), sunflower meal (Option 2), burnt crumbs of diatomaceous earth (Option 3), tripoli (Option 4), and its use.

According to the invention, this problem is solved due to the fact that the feed additive with probiotic activity for farm animals, birds, horses and fish contains a filler and dried biomass of a complex of bacteria of microorganisms.

The feed additive contains dried biomass of two strains: a bacterial strain Enterococcus faecium 1-35 with a titer of live bacteria 3.8 × 10 ⁷ -2.2 × 10 ⁸ CFU and a bacterial strain Bacillus megaterium B-4801 with a titer of live bacteria 3.8 × 10 ⁷ -3.3 × 10 ⁸ CFU.

Option 1

The dried biomass of these strains is applied in equal amounts to the filler in the form of bran containing live bacteria Enterococcus faecium 1-35 3.8 × 10 ⁷ CFU and live bacteria Bacillus megaterium B-4801 3.8 × 10 ⁷ CFU in 1 g of feed additives.

Option 2

The dried biomass of these strains is applied in equal amounts to the filler in the form of a sunflower meal containing live bacteria Enterococcus faecium 1-35 1.8 × 10 ⁸ CFU and live bacteria Bacillus megaterium B-4801 1.0 × 10 ⁸ CFU in 1 g feed additives.

Option 3

The dried biomass of these strains is applied in equal amounts to the filler in the form of a burnt crumb of diatomaceous earth containing live bacteria Enterococcus faecium 1-35 2.2 × 10 ⁸ CFU and live bacteria Bacillus megaterium B-4801 2.1 × 10 ⁸ CFU in 1 g of feed additive.

Option 4

The dried biomass of these strains is applied in equal amounts to the filler in the form of tripoli containing live bacteria Enterococcus faecium 1-35 1.3 × 10 ⁸ CFU and live bacteria Bacillus megaterium B-4801 3.3 × 10 ⁸ CFU in 1 g of feed additives.

The applicant has not identified sources containing information about technical solutions identical to this group of inventions, which allows us to conclude that it meets the criterion of "novelty."

Due to the implementation of the distinguishing features of the group of inventions (together with the features indicated in the restrictive part of the formula), important new properties of the object are achieved.

The feed supplement "Profort" normalizes digestion processes.

Living bacteria Enterococcus faecium 1-35 contribute to the creation of normal microflora, increase digestibility and assimilation of nutrients in the diet.

Living bacteria Bacillus megaterium B-4801 when used together with living bacteria Enterococcus faecium 1-35 increase probiotic activity.

Using the proposed excipients Options 1-4 when interacting with the dried biomass of bacteria Enterococcus faecium 1-35 and Bacillus megaterium B-4801 improves the growth and development of farm animals, birds, horses and fish and enhances functional applicability.

The applicant is not aware of any publications that would contain information on the influence of the distinguishing features of the group of inventions on the achieved technical result. In this regard, according to the applicant, it can be concluded that the claimed technical solutions meet the criterion of "inventive step".

The present group of inventions is as follows.

Profort feed additive with probiotic activity is used as feed for farm animals, birds, horses and fish.

The feed additive contains dried biomass of two strains: the bacterial strain Enterococcus faecium 1-35 and the bacterial strain Bacillus megaterium B-4801.

The bacterial strain Enterococcus faecium 1-35 was deposited in the collection of the All-Russian State Research Institute for the Control, Standardization and Certification of Veterinary Drugs (VGNKI) on March 20, 2008 under the registration number Enterococcus faecium VGNKI 08.03.53-DEP and is stored in the collection of microorganisms of BIOTROF LLC.

The bacterial strain Bacillus megaterium B-4801 was deposited in the All-Russian Collection of Industrial Microorganisms of FSUE GosNII Genetika under registration number VKPM: B-4801 and is stored in the collection of microorganisms of BIOTROF LLC.

Preparation of dry biomass of the bacterial strains Enterococcus faecium 1-35 and Bacillus megaterium B-4801 and the feed additive "Profort".

Experimental samples of cultures of bacteria strains Enterococcus faecium 1-35 and Bacillus megaterium B-4801 are produced in fermenters.

To obtain the “Profort” feed additive, take the dried biomass of the Enterococcus faecium 1-35 bacterial strain with titers of live bacteria within 3.8 × 10 ⁷ -2.2 × 10 ⁸ CFU and the Bacillus megaterium B-4801 bacterial strain with titers of live bacteria in within 3.8 × 10 ⁷ -3.3 × 10 ⁸ CFU.

The dried biomass of these strains is applied in a mixture in equal amounts to the filler.

Bran containing live bacteria Enterococcus faecium 1-35 3.8 × 10 ⁷ CFU and live bacteria Bacillus megaterium B-4801 3.8 × 10 ⁷ CFU in 1 g of feed additive is used as a filler (Option 1).

Bran comply with GOST 7169-66.

Sunflower meal containing live bacteria Enterococcus faecium 1-35 1.8 × 10 ⁸ CFU and live bacteria Bacillus megaterium B-4801 1.0 × 10 ⁸ CFU in 1 g of feed additive is used as filler (Option 2).

Meal sunflower corresponds to GOST 11246-96.

As a filler, burnt diatomaceous crumb containing live bacteria Enterococcus faecium 1-35 2.2 × 10 ⁸ CFU and live bacteria Bacillus megaterium B-4801 2.1 × 10 ⁸ CFU in 1 g of feed additive is used (1).

Diatomite - sedimentary rock loose or slightly cemented, consisting of the remains of diatoms and having a gray or yellow color of weak tones.

Chemically, diatomite is more than 80% composed of aqueous silica.

Diatomite in the form of burnt chips is supplied by the "Diatom Plant" in Inza, Ulyanovsk Region.

Tripoli containing live bacteria Enterococcus faecium 1-35 1.3 × 10 ⁸ CFU and live bacteria Bacillus megaterium B-4801 3.3 × 10 ⁸ CFU in 1 g of feed additive is used as a filler (Option 4).

Tripoli corresponds to TU 5761-001-59266087-2005.

"The safety of bacteria on different types of media" are shown in table 1.

The quantitative content of live bacteria is determined experimentally and is optimal.

A mixture of live bacteria Enterococcus faecium 1-35 and Bacillus megaterium B-4801 is applied to the fillers according to Options 1-4, respectively, and the wet components of the “Profort” feed additive are dried.

The drying of the components of the “Profort” feed additive is carried out in RT-ShS drying ovens by the air-heat method at a temperature from 55 ° C to 60 ° C for 9 hours to a final moisture content of 7.0-7.2%.

After drying, the components of the “Profort” feed additive are sent for grinding to the feed mill DKR-3.

Profort feed additive is ground to a powder state.

Organoleptic, physico-chemical and microbiological indicators of the feed additive "Profort"

1. Definition of appearance and color

The determination of appearance and color is carried out visually in daylight diffused light or under illumination with incandescent or fluorescent lamps.

The Profort feed additive is a loose powder, from light gray to brick in color and has a characteristic odor.

2. Determination of moisture content (GOST R 54951)

Mass fraction of moisture is determined in two parallel samples. Two clean dried containers are removed from the desiccator and weighed with an error of no more than 0.01 g. A sample of the average sample is thoroughly mixed and placed in each weighed container weighing 5.0 g, after which the boxes are closed with lids and put into the desiccator. The cabinet is heated to 140 ° C and quickly placed in it open boxes with a sample of the product. The sample is dried for 2 hours, counting from the moment the temperature reaches 140 ° C. After drying, the boxes are removed from the cabinet with crucible forceps, closed with lids and transferred to a desiccator for complete cooling for about 20 minutes, but not more than 2 hours. Cooled boxes are weighed with an error of not more than 0.01 g and placed in a desiccator until the results are processed analysis.

Mass fraction of moisture in percent (X) is calculated by the formula:

X = 100 (m _1- m ₂ ) / m ₁ ,

where m ₁ is the mass of the sample additives before drying, g;

m ₂ - weight of the sample additives after drying, g.

The permissible discrepancy between the results of two parallel determinations shall not exceed 0.2%. The arithmetic mean of the results of two parallel determinations is taken as the final analysis result.

The moisture content should not exceed 12.0%.

To determine the moisture content can be used thermogravimetric method.

3. Determination of the number of live bacteria (CFU / cm ³ )

Determination of the concentration of microbial cells of Bacillus megaterium B-4801 (CFU / g)

To determine the content of bacteria Bacillus megaterium B-4801 in the feed additive "Profort", three samples of each batch are examined. From the samples taken for analysis, 10 g are taken and transferred to a flask with 90 cm ^{3 of a} 0.9% sterile sodium chloride solution, thus obtaining an initial dilution of 10 ^-1 . After five minutes of shaking from it, dilutions from 10 ^-2 to 10 ^{-8 are} prepared using serial ten-fold dilutions, in each case using a separate sterile pipette. 20 cm ^{3 of} molten meat and peptone agar are poured into sterile Petri dishes and left to solidify the nutrient medium. From dilutions 10 ^-5 , 10 ^-6 , 10 ^-7 and 10 ^-8 do the seeding of 0.1 cm ³ in three Petri dishes for each dilution, grinding the material with a sterile glass spatula on the surface of the medium. Then the Petri dishes are incubated at a temperature of 37 (± 1) ° C for 24 hours.

Count the number of colonies of Bacillus megaterium B-4801.

The number of living cells (CFU) of bacteria in 1 g of the drug (X) is calculated by the formula:

where X is the number of colony forming units;

a ₁ ; a ₂ ; and ₃ - the number of colonies in the considered breeding;

10n - breeding ratio, which takes into account growth.

Allowable discrepancies between the results of two parallel determinations should not exceed 20% with respect to a larger value. Otherwise, the measurements are repeated.

The arithmetic mean of the results of two parallel measurements is taken as the final test result.

Determination of the concentration of microbial cells of Enterococcus faecium 1-35 (CFU / g)

An agar medium is prepared.

To prepare the medium, take pancreatic hydrolyzate of fish meal 10.0 g, baker's yeast extract - 5.0 g, glucose - 2.0 g; K ₂ NRA ₄ - 2.0 g; NaHCO ₃ - 1 g; tween-80 - 0.5 ml; sodium azide - 0.5 g; 2,3,5-triphenyltetrazolium chloride - 0.1 g; crystalline violet 0.001 g; agar - 10 g, drinking water up to 1000 cm ³ .

Preparation: all the listed components, except agar, are added to a sterile conical flask, water is added and the salts introduced are dissolved in it when heated to boiling. Then microbiological agar is introduced and it is melted by heating the medium. The acidity of the medium is adjusted with sodium bicarbonate, or hydrochloric acid to 6.9-7.3. Sterilize the medium at 1 atm for 10 minutes.

From dilutions 10 ^-6 , 10 ^-7 , 10 ^-8 prepared above with a sterile pipette, 1 cm ^{3 of} solution is transferred into 3 Petri dishes, and then they are poured with sterile molten medium cooled to 55 ° C. In a circular motion of the Petri dishes, the medium is mixed in them and left on the table until the agar solidifies. Cups with media are placed in a thermostat and incubated at 37 ° C for 1-2 days. Then, the grown colonies are counted using a colony counting device.

The tests are carried out in two parallel replicates.

The number of colonies of Enterococcus faecium 1-35 was counted on plates.

The number of living cells (CFU) of bacteria in 1 g of the drug (X) is calculated by the formula 1.

The arithmetic mean of the results of two parallel determinations is taken as the final test result, the permissible difference between them should not exceed 10%.

4. The determination of microbiological purity is carried out according to GOST R 55291

The Profort feed additive must not contain bacteria of the Enterobacteriaceae family, Pseudomonas aeruginosa, Staphyloccocus aureus. Contamination by mesophilic aerobic, optionally anaerobic microorganisms, yeast and molds should not exceed 300 thousand bacteria per 1 g of the additive.

5. Determination of harmlessness

Preparing a working solution of the Profort feed additive. To do this, 0.25 g of the biological product is diluted in 5.0 cm ^{3 of a} sterile 0.9% sodium chloride solution. Within five days, five outbred mice weighing 18-20 g, using a needle at the end of which an olive with a diameter of 1.0-2.0 mm is fused, enter intragastrically 1.0 cm ^{3 of the} prepared working solution. Parallel to five control mice, 1.0 cm ^{3 of a} sterile 0.9% sodium chloride solution used to prepare the working solution was intragastrically administered. Mice are monitored for 7 days.

The Profort feed additive is harmless if during the observation period all the mice from the experimental and control groups remain alive and clinically healthy. If one or more mice die, the test is repeated at twice their number. If at least one mouse dies in a repeated experiment, the Profort feed additive is rejected.

"Organoleptic, physico-chemical and microbiological indicators of the feed additive" Profort "" are presented in table 2.

Studies on the use of the proposed feed additives "Profort" are presented in examples 1-5.

In the study of the feed additive "Profort", fillers were used according to Options 1-4.

Example 1

The use of the feed additive "Profort" with probiotic activity in poultry farming

The experiment was carried out on the basis of the FSBI SGC "Zagorsk EPH" VNITIP Sergiev Posad, Moscow Region from August 1 to September 06, 2016 on broilers of the cross "Cobb-500"

2 groups of broiler chickens were formed (control and experimental) with 1000 goals each.

Broiler chickens were grown in Big Dutcmen cell batteries under the same conditions of feeding and feeding according to the Guidelines for feeding poultry, 2015.

Planting density, light and temperature conditions were carried out in accordance with the Methodological Recommendations for the Technological Design of Poultry Enterprises, 2013.

Broiler chickens of both groups received complete feed with composition and nutrition according to the “Guidelines for feeding poultry”, 2015.

The first control group received the main diet (RR) - full feed in accordance with the “Methodological Guide for Feeding Poultry”, 2015.

The second experimental group received the main diet (RR) - full feed in accordance with the Methodological Guide for the Feed of Poultry, 2015 with the feed additive "Profort" at the rate of 500 g / t.

The duration of the experiment is 37 days.

During the experiment, the main zootechnical indicators were taken into account: live weight at 7, 14, 21, 28 days and at the end of cultivation, livestock safety, average daily gain in live weight and feed costs per 1 kg of live weight gain.

The results of the experiment are presented in table 3 "The influence of the feed additive" Profort "on the main zootechnical indicators of broiler chickens"

From table 3 it is seen that when introducing the feed additive "Profort" in the diet of broiler chickens from the first day of life and throughout the entire feeding period, an increase in live weight of poultry of the experimental group was noted by 4.2%.

A positive result on the safety of birds is obvious, which in the experimental group amounted to 98.9%, which exceeds the indicator of the control group by 3.8% in absolute terms.

From table 3 it is seen that the use of the feed additive "Profort" allowed to reduce the cost of feed per 1 kg of gain in live weight by 0.4 kg.

As a filler, we used burnt diatomaceous crumb containing 1 g of the Profort feed additive of live bacteria Enterococcus faecium 1-35 2.2 × 10 ⁸ CFU and live bacteria Bacillus megaterium B-4801 2.1 × 10 ⁸ CFU.

Example 2

The use of feed additives "Profort" with probiotic activity in pig farming.

The experiment was carried out on the basis of AO PZ "Flame" of the Leningrad region of the Gatchinsky district from March 2 to August 1, 2016 on piglets of weaning and feeding groups.

Two groups of analogues of piglets-weaners of 20 animals each and two groups of analogues of piglets of the fattening group of 15 animals each were formed by return and live weight. Group content - 10 goals in a cage of piglets of weaning groups and 15 goals in a cage of piglets of a feeding group.

Two-time feedings.

The main diet (RR) of weaned piglets (kg):

compound feed - 1.1; rolled grain - 0.5; silo - 0.6; milk powder - 35 g; serum - 3.0 l; fish oil - 0.2 g; Ministry of Internal Affairs - 300 g.

The main diet (RR) of young pigs of the fattening group: May, June (kg): compound feed - 1.2; flattened grain, crushed - 1.8; silo - 2.5; serum - 8.0; grain waste - 0.5; meat broth - 0.1; Ministry of Internal Affairs - 0.150.

July (kg): compound feed - 1.5; silo - 2.5; serum - 8.0; grain waste - 0.5; meat broth - 0.1; Crusher - 1.5.

The first control group received the main diet (RR).

The second experimental group received the main ration (OR) with the Profort feed additive at the rate of 500 g / t of feed.

The results of the experiment are presented in table 4 "The influence of the feed additive" Profort "on the main zootechnical indicators of the cultivation of piglets of the weaning and feeding group."

From table 4 it is seen that weaned piglets fed with the “Profort” feed additive showed better results compared to the control group: the average daily gain was 36% higher, the live weight of one head at the end of the experiment was 16.8% higher than the control group. At the same time, feed costs per 1 kg of gain of experimental piglets were lower than the control by 7.4%.

Bran was used as a filler, containing 1 g of the Profort feed additive of live bacteria Enterococcus faecium 1-35 3.8 × 10 ⁷ CFU and live bacteria Bacillus megaterium B-4801 3.8 × 10 ⁷ CFU.

Example 3

The use of feed additives "Profort" with probiotic activity in animal husbandry.

The experiment was carried out on the basis of CJSC PZ Bolshevik of the Leningrad Region of the Gatchinsky District from April 18 to June 20, 2016 on dairy cows.

2 groups of dairy cows of the black-motley breed of the galstinized second lactation were formed (control and experimental).

Animals were in tethered keeping.

The structure of the main diet (RR) of dairy cows (kg):

compound feed - 8, flattened grain - 3, sunflower meal (38% SP) - 1, corn - 0.5, clover-timothy hay - 1.0, silage (cereal-bean) - 37.5, dry beet pulp - 1 , 0, molasses from beets - 1.0, Ministry of Internal Affairs - 0.235.

The first control group received the main diet (RR).

The second experimental group received the main diet (OR) with the Profort feed additive at the rate of 25.0 g per head per day.

The duration of the experiment is 63 days.

During the experiment, the main zootechnical indicators were taken into account: milk yield, milk quality, the number of somatic cells, a biochemical blood test, and economic indicators.

The results of the experiment are presented in table 5 "The influence of the feed additive" Profort "on the productivity of cows and milk quality."

Table 5 shows that the average daily milk yield of natural fat in the experimental group was 2.1 kg higher, the fat content in milk was 0.11% higher, which allowed to obtain 34.0 kg of milk of 4% fat per one head per day against 31.2 kg in control or 2.8 kg more.

The resulting difference in the amount of milk fat and protein in the cows of the experimental group (85.84 and 75 kg, respectively) compared with animals in the control group (78.48 and 69.7 kg) is due to a change in the direction of the interstitial metabolism.

A comparable analysis between the groups shows that a higher indicator of milk fat in animals of the experimental group was obtained both due to an increase in milk yield and an increase in the percentage of fat and protein in milk. The number of somatic cells in the milk of cows of the experimental group decreased by 28.4% compared with animals of the control group.

Tripoli was used as a filler, containing 1 g of Profort feed additive of live Enterococcus faecium bacteria 1-35 1.3 × 10 ⁸ CFU and live bacteria Bacillus megaterium B-4801 3.3 × 10 ⁸ CFU.

Example 4

The use of feed additives "Profort" with probiotic activity in fish farming.

The experiment was carried out on the basis of Sumy Salmon-Whitefish Nursery LLC

Kingisepp district of the Leningrad region in the period from January 10 to April 10, 2016 on fry and biennials of rainbow trout.

2 groups (control and experimental) of fry (500 animals) were grown, which were grown in indoor swimming pools, and 2 groups (control and experimental) of two-year-old rainbow trout (300 animals) weighing 1200-1400 g in an open pond in cages.

Fish care and feeding were carried out according to FSUE VNIRO.

The daily feed dose was determined according to the standards developed by FSUE VNIRO, depending on the weight of the fish and the temperature of the water.

The composition of the feed of the main diet (OR): fish meal, fish oil, soy flour, wheat, vitamins, mineral supplements.

The first control group received the main diet (RR).

The second experimental group received the main ration (OR) with the Profort feed additive at the rate of 2.5 kg per 1 ton of feed.

The duration of the experiment is 91 days.

During the experiment, indicators were taken into account: the safety of the livestock, the average daily gain and feed costs.

The results of the experiment are presented in table 6 "The effect of the feed additive" Profort "on fish breeding indicators of fry and two-year-old rainbow trout."

From table 6 it is seen that in the experimental groups the safety of the livestock increased by 3.4% in fry and by 1.4% in adult fish, the average daily gain in fry of the experimental group exceeded the control by 14.4% and by 40.4% in biennials rainbow trout, feed costs decreased by 31.6% and 25.0%, respectively.

Sunflower meal was used as a filler, containing 1 g of Profort feed additive of live Enterococcus faecium bacteria 1-35 1.8 × 10 ⁸ CFU and live bacteria Bacillus megaterium B-4801 1.0 × 10 ⁸ CFU.

Example 5

The use of the feed supplement "Profort" with probiotic activity in the diet of horses.

The experiment was carried out on the basis of the ProfiZOO public group uniting private owners of sports horses in the village of Nizino, Lomonosov district, Leningrad region from August 1 to September 20, 2016 on sports horses.

The experiment involved 6 goals of adult horses aged 3 to 17 years and 5 goals of young animals aged 5-12 months.

The duration of the experiment is 50 days.

The “Profort” feed additive was introduced into the main diet of horses by mixing 50 g per head per day for adults and 30 g per head per day for young animals in dry granulated feed.

After 30 days of feeding horses with the “Profort” feed additive, there was a slight increase in the fatness of the animals, improvement of endurance, less fatigue and sweating of adult animals, and young animals began to consume freshly cut grass and hay more actively.

By the end of the experiment, after 50 days, all the positive aspects were preserved. The “Profort” feed additive has improved the digestibility of the feed and the digestibility of nutrients, which has reduced the concentrate load of the diet.

Bran was used as a filler, containing 1 g of the Profort feed additive of live bacteria Enterococcus faecium 1-35 3.8 × 10 ⁷ CFU and live bacteria Bacillus megaterium B-4801 3.8 × 10 ⁷ CFU.

The introduction of the feed supplement “Profort” had a positive effect on the health of animals and contributed to an increase in physical endurance while maintaining sports loads of varying intensity.

The results obtained on the basis of Examples 1-5 confirm the feasibility of using the new Profort feed additive with probiotic activity in the preparation of feed for farm animals and birds, horses and fish.

The effect of all the studies in Examples 1-5 above is probably also related to the ability of the bacterial strain Bacillus megaterium B-4801 to carry out biodegradation of mycotoxins, and the use of this strain with Enterococcus faecium 1-35 strain summarizes this effect.

Studies of the potential of bacterial strains of the genus Bacillus, conducted in March 2017 at BIOTROF LLC, to carry out biodegradation of mycotoxins are confirmed experimentally, but they do not meet the solution of the problem and cannot be used when considering this application.

Species identification of Enterococcus faecium 1-35 strain (using molecular genetic analysis of sequencing)

DNA isolation.

DNA was isolated from a microorganism culture according to the protocol of the “Kit for the isolation of genomic DNA from various sources” (Fermentas).

PCR amplification.

Primers for the production of conserved fragments of the 16S rDNA gene - 27F AGAGTTTGATCMTGGCTCAG and 1525r AAGGAGGTGWTCCARCC

PCR reaction mode: 1) 95 ° С - 3 min, 2) 35 cycles (95 ° С - 30 sec, 55 ° С - 30 sec, 72 ° С - 1 min), 3) 72 ° С - 20 min.

Cloning of PCR fragments was carried out in pTZ-57R vector into competent cells of E. coli strain DH5A.

Transformants were screened using the PCR colony method using primers M13: fM13: 5'-GCCAGGGTTTTCCCAGTCACGA-3 ', rM13: 5'-GAGCGGATAACAATTTSAC ACAGG-3.

PCR reaction mode: 1) 95 ° С - 3 min, 2) 35 cycles (95 ° С - 40 sec, 50 ° С - 40 sec, 72 ° С - 1 min), 3) 72 ° С - 20 min.

The nucleotide sequence of the PCR fragments was determined using a reagent kit from Beckman (USA) in accordance with the manufacturer's recommendations. Separation of fragments and their registration was carried out using an automatic sequencing device CEQ8000, Beckman Coulter, USA.

Determination of the phylogenetic affiliation of the strain was carried out in the NCBI BLAST database (http://blast.ncbi.nlm.nih.gov/Blast.cgi)

Analysis results

Sequenced DNA fragment of the deposited strain (648 bp).

This DNA fragment sequence encoding the 16S rRNA gene has a 100% similarity to the sequence Enterococcus faecium 1-35 (Bacteria; Firmicutes; Bacilli; Lactobacillales; Enterococcaceae; Enterococcus) according to the GenBank database (http: //blast.ncbi.nlm .nih.gov / Blast.cgi).

A comparative sequence analysis of the DNA fragment encoding the 16S rRNA gene with the closest species according to the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) is presented in table 7.

Species identification of a strain of Bacillus megaterium B-4801 (using molecular genetic analysis of sequencing)

DNA isolation.

DNA was isolated from a microorganism culture according to the protocol of the “Kit for the isolation of genomic DNA from various sources” (Fermentas).

PCR amplification.

Primers for the production of conserved fragments of the 16S rDNA gene - 27F AGAGTTTGATCMTGGCTCAG and 1525r AAGGAGGTGWTCCARCC.

PCR reaction mode: 1) 95 ° С - 3 min, 2) 35 cycles (95 ° С - 30 sec, 55 ° С - 30 sec, 72 ° С - 1 min), 3) 72 ° С - 20 min.

Cloning of PCR fragments was carried out in pTZ-57R vector into competent cells of E. coli strain DH5A.

Transformants were screened using the PCR colony method using primers M13: fM13: 5'-GCCAGGGTTTTCCCAGTCACGA-3 ', rM13: 5'-GAGCGGATAACAATTTCAC ACAGG-3.

PCR reaction mode: 1) 95 ° С - 3 min, 2) 35 cycles (95 ° С - 40 sec, 50 ° С - 40 sec, 72 ° С - 1 min), 3) 72 ° С - 20 min.

The nucleotide sequence of the PCR fragments was determined using a reagent kit from Beckman (USA) in accordance with the manufacturer's recommendations. Separation of fragments and their registration was carried out using an automatic sequencing device CEQ8000, Beckman Coulter, USA.

Determination of the phylogenetic affiliation of the strain was carried out in the NCBI BLAST database (http://blast.ncbi.nlm.nih.gov/Blast.cgi)

Analysis results

Sequenced DNA fragment of the deposited strain (583 bp).

This DNA fragment sequence encoding the 16S rRNA gene has a 99% similarity with the sequence of Bacillus megaterium B-4801 (Bacteria; Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus) according to the GenBank database (http: //blast.ncbi.nlm .nih.gov / Blast.cgi).

A comparative sequence analysis of the DNA fragment encoding the 16S rRNA gene with the closest species according to the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) is presented in table 8.

The proposed “Feed additive with probiotic activity for farm animals, birds, horses and fish” was obtained using well-known biotechnologies that are widely used in agriculture, and studies have been carried out on the use of the feed supplement “Profort” on the basis of FSBI SGC “Zagorsk EPH” VNITIP, g Sergiev Posad, Moscow Region, on the basis of AO PZ “Flame” of the Leningrad Region of the Gatchinsky District, on the basis of CJSC PZ “Bolshevik” of the Leningrad Region of the Gatchinsky District, on the basis of LLC Sumy Salmon and Whitefish Nursery eppskogo district of Leningrad region, on the basis of the social group of owners of sport horses "ProfiZOO" d. Nizino Lomonosov district of the Leningrad region are responsible, according to the applicant, its compliance with the criterion of "industrial applicability".

The proposed “Feed supplement with probiotic activity for farm animals, birds, horses and fish” is a new broad-spectrum feed supplement with high probiotic activity.

Preservation of bacteria on different types of carriers

Organoleptic, physico-chemical and microbiological indicators of the feed additive "Profort"

The influence of the feed additive "Profort" on the main zootechnical indicators of broiler chickens

The influence of the feed additive "Profort" on the main zootechnical indicators of the cultivation of piglets of the weaning and feeding group

The effect of the feed supplement “Profort” on cow productivity and milk quality

The effect of the feed supplement “Profort” on fish breeding indicators of fry and biennial rainbow trout

Homology of the sequenced DNA fragment of the Enterococcus faecium 1-35 strain according to the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi)

Homology of the sequenced DNA fragment of the strain Bacillus megaterium B4801 in accordance with the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi)

Claims (4)

1. A feed additive with probiotic activity for farm animals, birds, horses and fish, containing in its composition a filler and dried biomass of a complex of bacteria of microorganisms, characterized in that the feed additive contains dried biomass of two strains: Enterococcus faecium 1-35 bacterial strain with a titer live bacteria 3.8 × 10 ⁷ -2.2 × 10 ⁸ CFU and a strain of bacteria Bacillus megaterium B-4801 with a titer of live bacteria 3.8 × 10 ⁷ -3.3 × 10 ⁸ CFU, applied in equal amounts in a mixture a filler in the form of bran, containing living bacteria Enterococcus faecium 1-35 3,8 × ¹⁰ July OE and live bacteria Bacillus megaterium B-4801 July ¹⁰ 3,8 × CFU per 1 g of the feed additive. 2. A feed additive with probiotic activity for farm animals, birds, horses and fish, containing a filler and dried biomass of a complex of bacteria of microorganisms, characterized in that the feed additive contains dried biomass of two strains: Enterococcus faecium 1-35 bacterial strain with a titer live bacteria 3.8 × 10 ⁷ -2.2 × 10 ⁸ CFU and a strain of bacteria Bacillus megaterium B-4801 with a titer of live bacteria 3.8 × 10 ⁷ -3.3 × 10 ⁸ CFU, applied in equal amounts in a mixture filler in the form of sunflower meal containing live bacteria Enterococcus faecium 1-35 1.8 × 10 ⁸ CFU and live bacteria Bacillus megaterium B-4801 1.0 × 10 ⁸ CFU in 1 g of feed additive. 3. A feed additive with probiotic activity for farm animals, birds, horses and fish, containing a filler and dried biomass of a complex of bacteria of microorganisms, characterized in that the feed additive contains dried biomass of two strains: Enterococcus faecium 1-35 bacterial strain with a titer live bacteria 3.8 × 10 ⁷ -2.2 × 10 ⁸ CFU and a strain of bacteria Bacillus megaterium B-4801 with a titer of live bacteria 3.8 × 10 ⁷ -3.3 × 10 ⁸ CFU, applied in equal amounts in a mixture filler in the form of burnt crumbs of diatomite containing live bacteria Enterococcus faecium 1-35 2.2 × 10 ⁸ CFU and live bacteria Bacillus megaterium B-4801 2.1 × 10 ⁸ CFU in 1 g of feed additive. 4. A feed additive with probiotic activity for farm animals, birds, horses and fish, containing a filler and dried biomass of a complex of bacteria of microorganisms, characterized in that the feed additive contains dried biomass of two strains: Enterococcus faecium 1-35 bacterial strain with a titer live bacteria 3.8 × 10 ⁷ -2.2 × 10 ⁸ CFU and a strain of bacteria Bacillus megaterium B-4801 with a titer of live bacteria 3.8 × 10 ⁷ -3.3 × 10 ⁸ CFU, applied in equal amounts in a mixture filler in the form of tripoli containing live bacteria Enterococcus faecium 1-35 1.3 × 10 ⁸ CFU and live bacteria Bacillus megaterium B-4801 3.3 × 10 ⁸ CFU in 1 g of feed additive.