RU2493723C1 - Biopreparation with probiotic activity for optimisation of assimilation of fodders intended for farm animals and birds - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: invention relates to farm biotechnology and may be applied during preparation of fodders for farm animals and birds. The biopreparation contains microbial mass of Bacillus cereus 1-79 bacteria strains. The strain consists of microbial mass of Bacillus cereus 1-79 spore-forming bacteria with titre equal to 3.5×10⁹ CFU/g - 4.0×10⁹ CFU/g, suspended in a nutrient medium. The strain is deposited in the All-Russian State Collection of Microorganism Strains under Registration Number of Strain Bacillus cereus 10.09.63 DEP and stored in microorganisms collection LLC BIOTROF.

EFFECT: invention allows to enhance fodders assimilation due to usage of a new biopreparation with high probiotic activity.

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Description

The invention relates to agricultural biotechnology and can be used in the preparation of feed for farm animals and poultry.

A known strain of Bacillus firmus VGNKI "SK" used for the manufacture of a probiotic preparation intended for the prevention and treatment of bacterial intestinal infections of young farm animals, RU No. 2190016 C1, C12N 1/20, A23K 35/74, 09/27/2002.

A known bacterial strain Bacillus licheniformis BKM B-2414D is used to obtain 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 35/74, A23K 1/165, 09/10/2010.

Known strain Bifidobacterium gallinarum GB used to obtain bacterial preparations, biologically active feed additives, fermented and non-fermented feed intended for the prevention and treatment of intestinal diseases of young farm birds, RU No. 2301828 C1, C12N 1/20, A61K 35/74, A23K 1/165, 06/27/2007.

Known probiotic strain Bacillus subtilis 8130, on the basis of which a probiotic feed additive "Probiocel" was created, which affects the intestinal microflora, secretory and enzymatic activity of the gastrointestinal tract of animals and generally activates functional activity, improving metabolism, dissercat. com / content / izuchenie ..... RU No. 2346463 C2, A23K 1/165, 02.20.2009.

Known probiotic preparation intended for the treatment and prevention of gastroenteritis of weaned pigs, based on the strain of Bacillus subtilis TPI and strain Bacillus lichemformis, which is a mixture of culture fluids containing products of microbial synthesis of these strains, with a concentration of microbial cells of 5 billion MK / cm ³ in a volume ratio of the indicated strains 1: 1, to which an equal weight mixture of bran and dicalcium phosphate mixture, RU No. 2399662 C1, C12N 1/20, A23K 1/16, 09/20/2010; RU No. 2399661 C1, C12N 1/20, A23K 1/16, 09/20/2010.

Known probiotic composition for animals and birds, containing bacteria Rhuminococcus albus. Bacillus subtilis, taken in equal amounts, and a method for producing a probiotic composition, RU No. 2266747 C1, A61K 35/66, A23K 1/165, 12/27/2005; RU No. 2266126 C1, A61K 35/66, A23K 1/165, 12.20.2005.

Known probiotic additive containing the biomass of spore-forming bacteria Bacillus subtilis and a hydrophilic carrier brand A and hydrophobic brand AM, RU No. 2252956 C2, C12N 1/20, A23K 1/165, A23K 35/66, 05/27/2005.

Known probiotic feed additive comprising a mixture of bacterial biomass of strains of Bacillus subtilis VKPM 10172 and Bacillus licheniformis VKPM 10135 in equal proportions in spore form with a total amount of at least 2-10 ⁸ spores / g, and carbohydrate tread, RU No. 2437563 C1, A23K 1 / 00, 12/27/2011.

Known drugs are individual in which the strains are cultured in various environments and under certain conditions.

Known biological product for the prevention and treatment of infectious diseases and dysbiosis of various etiologies, containing the microbial mass of a bacterial strain of the genus Bacillus, taken in an effective amount, RU No. 2264454 C2, C12N 1/20, A23K 35/66, 20.11.2005.

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

The biological product in the closest analogue contains the microbial mass of strains of Bacillus subtilis VKPM No. B-8611, Bacillus licheniformis VKPM No. B-8610, characterized by a wide spectrum of antagonistic activity, high proteolytic and amylase activity.

However, the use of the closest analogue of two strains in a biological product complicates the biotechnological process of its preparation.

The present invention is based on the solution of a problem that allows to identify a new biological product with high probiotic activity in a simple biotechnological process of its production.

The technical result of the present invention is the use of a bacterial strain Bacillus cereus 1-79 as the basis of a biological product with high probiotic activity.

According to the invention, this problem is solved due to the fact that a biological product with probiotic activity to optimize the assimilation of feed intended for farm animals and poultry contains the microbial mass of a bacterial strain of the genus Bacillus, taken in an effective amount.

The strain of bacteria Bacillus cereus 1-79 was taken as a strain.

The bacterial strain Bacillus cereus 1-79 was deposited in the All-Russian state collection of microorganism strains under registration number Bacillus cereus strain 10.09.63 DEPT and is stored in the microorganism collection of BIOTROF LLC.

The bacterial strain Bacillus cereus 1-79 consists of the microbial mass of the spore-forming bacteria Bacillus cereus 1-79 with a titer of 3.5 × 10 ⁹ CFU / g -4.0 × 10 ⁹ CFU / g and suspended in a nutrient medium.

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

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

Identification of a new strain of bacteria Bacillus cereus 1-79 allows you to get a biological product with high probiotic activity.

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

The present invention is carried out as follows.

Species identification of microorganism strains

Samples of biological products were analyzed by molecular genetic sequencing.

DNA from biologics was isolated according to the standard protocol of the “Kit for the isolation of genomic DNA from various sources”.

The key part of the DNA sequence of the bacterial strain Bacillus cereus has been established.

The bacterial strain Bacillus cereus is the basis of a biological product with probiotic activity to optimize the assimilation of animal feed.

Bacillus cereus partial 16S rRNA gene, strain B 203 (100%).

Selection of bacterial strains for use as a basis for biological products with probiotic activity

The selection criterion for the bacterial strain Bacillus cereus as the basis of a biological product with probiotic activity is a high antagonistic activity to test cultures of putrefactive bacteria and molds.

The results of the selection of bacterial strains of Bacillus cereus by antagonistic activity are presented in Table 1.

From Table 1 it can be seen that the bacterial strain Bacillus cereus 1-79 has a high antagonistic activity against putrefactive bacteria, molds, and yeast.

The bacterial strain Bacillus cereus 1-79 was tested for pathogenic properties.

In accordance with GOST 12.1.007-76, the bacterial strain Bacillus cereus 1-79 was studied for pathogenic properties: virulence, toxicity, toxigenicity, and the ability to cause desimination in the internal organs of warm-blooded animals.

Tests were conducted on outbred white rats and outbred white mice.

The virulence and dissemination of the bacterial strain Bacillus cereus 1-79 was studied with a single administration of daily agar culture in physiological solution in the stomach of white mice and white rats in doses of 10 ⁶ , 10 ⁷ , 10 ⁸ and 10 ⁹ and intraperitoneal 10 ⁶ , 10 ⁷ , 10 ⁸ and 10 ⁹ microbial cells (MK) per animal. In the experiment, 12 animals were used per dose (6 males and 6 females). During the observation of clinical symptoms of the disease in animals was not observed, death was absent. 30 days after the introduction of the culture of microorganisms, the animals were sacrificed by inhalation of CO ₂ and the method of imprints was inoculated from blood and internal organs (lungs, liver, kidneys and spleen) onto Petri dishes with agar medium. The culture growth in seeding from animal organs with both methods of administration was not detected.

The toxicity of the bacterial strain Bacillus cereus 1-79 was evaluated by intraperitoneal administration to mice of a suspension of an agar culture of microorganisms prepared in sterile physiological saline and inactivated by heating at 60 ° C for 30 minutes at concentrations of 10 and 10 (m.c.) per animal (6 individuals per dose). During the observation period - the first two days - there was no death of mice.

The toxicity of the bacterial strain Bacillus cereus 1-79 was studied in mice by intraperitoneal and intragastric administration of a sterile filtrate of culture fluid (filtration through Millipor filter with a pore size of 0.45 μm) of 3 and 7-day-old cultures at doses of 0.3 ml, 0, 6 ml and 1.0 ml (6 animals per dose). The animals of the control groups were injected with sterile liquid nutrient medium in the same volumes. The death of mice was not. LD50 has not been established, with both methods of administration it exceeded 1.0 ml per animal for 3 and 7 day old culture fluids.

The results are presented in Table 2.

Table 2 shows that the bacterial strain Bacillus cereus 1-79 in terms of virulence, dissemination, toxicity and toxigenicity is not pathogenic for warm-blooded animals and belongs to hazard class 4 (low hazard), which meets the requirements for industrial microorganisms.

Additional studies revealed a high cellulolytic activity in the bacterial strain Bacillus cereus 1-79. According to this criterion, the specified strain was made the basis for a biological product with probiotic activity.

The bacterial strain Bacillus cereus 1-79 is the basis of a biological product with probiotic activity.

Determination of optimal conditions for the growth of a strain of the bacterium Bacillus cereus 1-79 biological product with probiotic activity.

The selection results are shown in Table 3, where

- Wednesday 1 - molasses 1.5%; corn extract 0.9%; fodder yeast 2%.

- Wednesday 2 - corn extract 5%; lactose 1%.

- Medium 3 - soy flour 3%, sodium nitrate 0.3%, potassium phosphate disubstituted 0.1%, potassium phosphate monosubstituted 0.1%, magnesium sulfate 0.02%, potassium chloride 0.02%.

Table 3 shows that the best conditions for the growth of the strain of the bacterium Bacillus cereus 1-79 correspond to medium 3.

In medium 3, the spore-forming bacteria Bacillus cereus 1-79 have a titer of 3.5 × 10 ⁹ CFU / g - 4.0 × 10 ⁹ KOE / g

Preparation of a biological product based on the bacterial strain Bacillus cereus 1-79

The bacterial strain Bacillus cereus 1-79 is used to obtain a uterine culture.

Uterine culture is grown from 2 to 5 days in a thermostat on a rocking platform at a temperature of 32 ° C.

The composition of the production environment is shown in Table 4.

The nutrient medium is prepared in a fermenter, thoroughly mixing and sterilizing with hot steam at a pressure of 1.6 atm and a temperature of 128 ° C for 1 hour. After sterilization, the medium is cooled to a temperature of 38-40 ° C, uterine culture is sterilely introduced with stirring, and cultivation is carried out for 24-48 hours.

The culture fluid from the fermenter is mixed with meal and dried in drying ovens RT-ShS at a temperature of 40 to 60 ° C for 9 hours to a final moisture content of 7.0-7.2%.

Next, the dried feed additive is crushed to a particle size of not more than 2 mm with a bacterium titer of 10 ⁶ -10 ⁹ cells / g of feed additive.

Taking into account the optimal growth conditions, a biological product based on the bacterial strain Bacillus cereus 1-79 with high probiotic activity was developed.

The characteristics of the biological product are established:

- appearance and color: nibs of light brown color;

- the biological product consists of a strain of microorganisms Bacillus cereus 1-79, not subjected to genetic engineering modifications;

- the titer of bacteria that are not lactic acid bacteria is 3.5 × 10 ⁹ CFU / g - 4.0 × 10 ⁹ KOE / g;

- marked cellulolytic activity - 11.9% according to the method of Henderson, Horvath and Blok in the modification of Churlis;

- extraneous microflora is absent.

A bacterial strain Bacillus cereus 1-79 with probiotic activity was identified from the collection of production strains to optimize the assimilation of feed for farm animals and poultry.

The strain Bacillus cereus 1-79 was deposited on April 18, 2011 in the All-Russian state collection of strains of microorganisms used in veterinary medicine and animal husbandry (123022, Moscow, Zvenigorodskoye Shosse, 5).

The strain Bacillus cereus 1-79 was deposited under the registration number of the strain Bacillus cereus 10.09.63 DEPT.

The strain Bacillus cereus 1-79 proposed for use in the composition of feed additives and medicines for animals.

The strain Bacillus cereus 1-79 is stored in the collection of microorganisms of BIOTROF LLC (196608, St. Petersburg, Pushkin, 9, Podbelsky sh.).

Experimental studies on the use of a biological product based on the bacterial strain Bacillus cereus 1-79 confirm examples 1-3.

Example 1. The use of a biological product - an enzymatic probiotic based on the bacterial strain Bacillus cereus 1-79 in poultry farming

The experiment was carried out from April to July 2010 on the basis of the FSUE Zagorsk EPH VNITIP of the Russian Agricultural Academy on broilers of the Cobb Avian 48 cross-country from daily to 35 days old according to the scheme shown in Table 5.

The bird was kept in Big Dutchman cell batteries (dimensions H × W × H - 150 × 120 × 50 cm). During the experiment, the main zootechnical indicators were taken into account, balance experiments were carried out, at the end of the cultivation, the poultry was slaughtered, followed by anatomical cutting. The livestock in groups was 35 goals.

From Table 5 it is seen that in the 3rd experimental group, a biological product based on the bacterial strain Bacillus cereus 1-79 was introduced into the main diet for the birds.

The results of the experiment are presented in Table 6.

The results indicate that the live weight of the birds of the experimental groups was close to control with a slight upward trend.

Given that the groups had a different number of hens and males, the analysis of the obtained data on live weight was carried out according to the arithmetic mean value between the mass of hens and males.

Due to the addition of the drug based on the bacterial strain Bacillus cereus l-79 in the 3rd experimental group, the average live weight of the bird exceeded the control by 1.53%.

In the 2nd control group (with cellobacterin), the average live weight of the bird exceeded the control by 0.8%.

Against the background of cheaper animal feed, the differences in live weight are significant.

Example 2. The use of a biological product - an enzymatic probiotic based on the bacterial strain Bacillus cereus 1-79 in pig fattening

Scientific and industrial experience was carried out at the pig farm OJSC PZ "Flame" of the Leningrad region. 86 pigs of 120-day-old pig meat were selected, which were distributed according to the principle of analogues (taking into account age, live weight and average daily weight gain in the preparatory period) into two groups.

The content of pigs was a group of 15 goals in the machine. Feeding animals was two times. The duration of the experiment was 92 days before reaching a live weight of 90-100 kg.

In the experimental group, a biological product based on the bacterial strain Bacillus cereus 1-79 was introduced into the feed (at the rate of 2 kg per 1 ton of feed).

During the experiment, monthly weighing was carried out, and the state of healthy animals and the digestion process were monitored.

The results obtained are presented in Table 7, which shows that the introduction of a biological product based on the bacterial strain Bacillus cereus 1-79 probiotic into the pig diet favorably affects animal health, reduces feed costs per unit of production, increases live weight, increases the safety of the number of pigs, and decreases the use of antibiotics and other drugs.

Example 3. The use of a biological product - an enzymatic probiotic based on the bacterial strain Bacillus cereus 1-79 for piglet feeding

Scientific and industrial experience was carried out on weaning pigs at the pig farm OJSC PZ "Flame" of the Leningrad Region. during the period from March 1 to April 30, 2011. The duration of the experiment is 60 days.

In the experimental group, a biological product based on the bacterial strain Bacillus cereus 1-79 was introduced into the feed (at the rate of 1 kg per 1 ton of feed). Animals willingly ate such compound feed.

The results are presented in Table 8, which shows that the introduction of a biological product based on the bacterial strain Bacillus cereus 1-79 probiotic into the diet contributed to increased immunity, normalized digestion processes, increased digestibility and assimilation of nutrients in the diet, piglets were always alert and with eaten the proposed food with appetite. In the experimental group, the average daily gain in weaned piglets was 6.3% higher.

On the basis of Examples 1-3, it was found that a dry biological product based on the bacterial strain Bacillus cereus 1-79 with probiotic activity corresponds to the assimilation of feed in poultry and during the feeding of pigs and weaned piglets.

The obtained results confirm the feasibility of using a new biological product based on the bacterial strain Bacillus cereus 1-79, which has high probiotic activity.

The proposed biological product with probiotic activity to optimize the assimilation of animal feed was obtained using well-known biotechnologies that are widely used in agriculture, and the experimental work carried out on the basis of the FSUE Zagorsk EPH VNITIP of the Russian Agricultural Academy, at the pig farm PZ Flame of the Leningrad Region, is determined, according to the applicant, , its compliance with the criterion of "industrial applicability".

The proposed biological product has a high probiotic activity and helps to optimize the assimilation of animal feed. The biotechnology for producing a biological product based on the bacterial strain Bacillus cereus 1-79 is simple.

Selection of bacterial strains of Bacillus cereus by antagonistic activity

Table 1 Strains Antagonistic activity (in the zone of inhibition of growth of the test culture), mm Fusarium oxisporum Aspergillus niger Penicillum sp. Sacharomices cerevisia Bacillus megaterium E.coli L.pl-1 5 0 0 0 fifteen 10 L.pl-2 10 10 5 0 5 5 L.pl-3 0 5 0 5 10 10 L.pl-60 28 25 23 17 10 10 L.buch.-1 0 0 3 0 5 5 L.buch.-2 5 3 3 0 fifteen 10 L.buch.-3 10 12 5 0 0 0 L.buch 600 27 35 12 10 fifteen 10 B.cereus 1 fifteen twenty 22 16 5 thirty B.cereus 2 10 12 12 5 5 5 B.cereus 3 fourteen 21 5 0 0 0 B.cereus 4 10 fifteen fifteen 12 21 23 B.cereus 1-79 24 fifteen 5 25 twenty 38 L.pl - Lactobacillus plantarum; L.buch - Lactobacillus buhneri

The results of the study of the bacterial strain Bacillus cereus 1-79 on pathogenic properties

table 2 Indicator Bacillus cereus 1-79 Virulence Not detected Toxicity Not detected Toxigenicity Not detected Desimination in the internal organs of warm-blooded animals Not detected

Selection of optimal growth conditions for strains of bacteria Bacillus cereus 1-79 biological product with probiotic activity (growth after 24 hours, CFU / ml)

Table 3 Wednesday T ° C Bacillus cereus 5 Wednesday 1 27 ° C 8.2 × 10 ⁸ 35 ° C 9.0 × 10 ⁸ Wednesday 2 27 ° C 5.6 × 10 ⁸ 35 ° C 1.0 × 10 ⁹ Wednesday 3 27 ° C 3.5 × 10 ⁹ 35 ° C 4.0 × 10 ⁹

The composition of the production environment for obtaining a biological product based on the bacterial strain Bacillus cereus 1-79

Table 4 Component Name Amount kg (L) Content% The volume of the uterine environment is 15 l Volume of production environment Fodder yeast 0.005 1,5 0.05 Corn extract 0,035 2,5 0.3 Beet molasses 0,075 7.5 0.5 Antifoam (sunflower oil) 2 0.4 Tap water Up to 15 Up to 500 l Up to 100% The acidity of the medium is adjusted with caustic soda 20-40% to a pH of 8.0-8.2

Experimental scheme for the use of a probiotic biological product in poultry based on the bacterial strain Bacillus cereus 1-79

Table 5 Group Diet from the 6th to the 21st day Diet from the 22nd to the 38th day 1 control OR - the main diet OR - the main diet 2 control RR - 2nd group + Cellobacterin (1 kg / t) RR - 2nd group + Cellobacterin (1 kg / t) 3 experienced RR - with a high fiber content (by 30%) + biological product based on Bacillus cereus 1-79 (1 kg / t) RR - with a high fiber content (40%) + biological product based on Bacillus cereus 1-79 (1 kg / t)

Zootechnical indicators for the use of a probiotic biological product in poultry based on the bacterial strain Bacillus cereus 1-79

Table 6 Indicator 1-control 2-control 3 experienced Initial stock, goal. 35 35 35 The final stock, goal. 35 35 35 Preservation,% 100.0 100.0 100.0 Live weight in 21 days, g 736.8 ± 15.0 729.8 ± 11.0 728.7 ± 10.7 Live weight at 35 days average statistical, g 1819.2 ± 31.8 1811.0 ± 34.2 1832.6 ± 36.4 including the mass of chickens, g 1695.6 1705.9 1723.5 including the mass of males, g 1950,2 1968.6 1978,1 Arithmetic average weight, g 1822.9 1837.2 1850.8 The number of chickens, goal. eighteen 21 twenty The number of males, goal. 17 fourteen fifteen The average daily increase (arithmetic mean), g 52,2 52.0 52.6 Feed costs per 1 head per day, g 89.6 90.8 90.0 per 1 kg of growth, kg 1.77 1.80 1.76

Indicators for the use of a biological product based on the bacterial strain Bacillus cereus 1-79 probiotic for fattening pigs

Table 7 Indicators, unit Group The control Experienced Number of goals 42 44 Total live weight at the beginning of the experiment, kg 1642.2 1711.6 Weight of 1 head at the beginning of the experiment, kg 39.1 38.9 Total live weight at the end of the experiment, kg 3547.5 4238.9 Weight of 1 head at the end of the experiment, kg 93.3 98.6 The total increase in live weight per experience, kg 1905.3 2527.3 The gain in live weight of 1 head per experience 54,2 59.7 The average daily gain per experience, g 518.1 629.1 % to control - 21.3 Spent units per 1 kg gain 6.5 5.62 % to control - 86.5 Number of feeddays 3678 4017 % safety 90,4 97.7 The number of goals at the end of the experiment, pcs. 38 43

Indicators for the use of a biological product based on the bacterial strain Bacillus cereus 1-79 probiotic for fattening piglets

Table 8 Indicators The control Experienced The number of pigs in the experience, goal. fifty fifty The duration of the experiment, days 60 60 Average live weight 1 goal. at the beginning of the experiment, kg 16.6 15.7 Weight gain for 60 days of experience, kg 1468 1560 Average live weight 1 goal. at the end of the experiment, kg 46.0 46.9 Number of feeddays 3000 3000 Daily gain, g 489 520 Feed consumption for the entire experiment period per 1 kg of weight gain, unit 8.28 7.80

Claims (1)

Biological product with probiotic activity to optimize the assimilation of feed intended for farm animals and poultry, containing the microbial mass of a strain of bacteria of the genus Bacillus, taken in an effective amount, characterized in that the strain of bacteria Bacillus cereus 1-79, deposited in the All-Russian State Collection, is taken as a strain microorganism strains under registration number Bacillus cereus strain 10.09.63 DEPT stored in the microorganism collection of BIOTROF LLC, consisting of the microbial mass of spore-forming bacteria Bacillus cereus 1-79 with a titer of 3.5 × 10 ⁹ CFU / g - 4.0 × 10 ⁹ CFU / g suspended in a nutrient medium.