RU2797587C1 - Probiotic feed supplement for cows and calves - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: invention relates to the biologically active feed additives, in particular to a probiotic feed additive for farm animals. The objective of the invention is to develop a new probiotic feed additive for farm animals with high antagonistic activity ensuring the suppression of microorganisms that cause intestinal toxic infections. The problem is solved by creating a probiotic feed additive for farm animals, which includes a composition of microorganism cultures, consisting of strains of lactobacilli Lactobacillus buchneri BX-99, Lactobacillus plantarum PL-99 and a strain of Bacillus subtilis BS2017, and peat gel, taken in a ratio of 1:1, at the same time, the total concentration of colony-forming units CFU in the feed additive must be at least 1.0⋅10⁸ CFU/cm³. A method of producing a probiotic feed additive for farm animals includes co-cultivation in a liquid nutrient medium of lactobacilli strains Lactobacillus buchneri BX-99, Lactobacillus plantarum PL-99 without aeration for 24 hours, followed by the addition of a strain of Bacillus subtilis BS2017 and further cultivation with aeration for 24 hours, and the subsequent addition of the resulting mixture of microbial cultures in peat gel in a ratio of 1:1 and their mixing. Before adding the resulting mixture of microbial cultures to the peat gel, pasteurization of the peat gel and its subsequent cooling to the operating temperature can be carried out. As a liquid nutrient medium for cultivating microbial cultures liquid nutrient medium of the following composition, g/DM³ can be used: molasses — 20.0 cm³; MgSO₄⋅7H₂O - 1,00; KН₂РО₄ - 0,4; NaCl - 0,5; СаСО₃ - 1,5; KNO₃ - 1,0; distilled water — up to 1.0 dm³.

EFFECT: obtaining probiotic feed supplement for cows and calves.

4 cl, 6 tbl

Description

The invention relates to the field of microbiology and biologically active feed additives, in particular to a probiotic feed additive for farm animals.

Currently, infectious diseases cause great economic damage to agricultural livestock and poultry enterprises. The greatest problem is intestinal infections and dysbacteriosis, which animals most often suffer from, especially in the first days of life. Of the total number of animal diseases, gastrointestinal diseases account for 54.3-71.4%. The recommended preventive measures, including the provision of adequate feeding, proper conditions for keeping farm animals and birds, maintaining the veterinary and sanitary condition of farms and incubators at the proper level, do not give the expected result, since the etiopathogenetic factor in the development of diarrhea is not taken into account. For the treatment and prevention of gastrointestinal infections of farm animals and birds in modern veterinary practice, vaccination, antibiotic therapy and probiotic preparations are most often used.

Numerous studies show that the most effective, safe method for the treatment and prevention of dysbacteriosis and intestinal infections in farm animals is probiotic therapy. Unlike antibiotics, they do not have a negative impact on the normal microflora of the gastrointestinal tract, but, on the contrary, contribute to its qualitative and quantitative restoration, as well as inhibition of the growth of opportunistic microorganisms.

The positive effect of probiotics on the microflora of the digestive tract and the body as a whole is ensured by increasing the activity of the normal microflora inhabiting the gastrointestinal tract of farm animals, increasing colonization resistance, stimulating cellular and humoral immunity factors, and improving metabolism.

A promising approach to the treatment of disorders of the microbiocenosis of the gastrointestinal tract is the use of combined polyprobiotics, which contain several strains of bacteria with the addition of growth stimulants, which ensure the restoration of microflora in a natural way, since the body's own microflora is stimulated. There is an activation of metabolic processes, leading to an increase in the digestibility of feed, an increase in live weight is accelerated, the immunity of animals is stimulated, thereby increasing the productivity and safety of farm animals, including birds.

It is known from the prior art that in the manufacture of probiotic preparations for veterinary purposes, cultures of microorganisms belonging to the genera Bacillus, Lactobacillus, Bifidobacterium are most often used.

The composition of a biologically active prophylactic feed additive for young farm animals is known from the prior art (Patent for Invention RU 2525920 C2). Supplement composition includes: chitosan-melanin complex from dead bees; lactulose; bifidumbacterin from Bifidobacterium bifidum, strains No. 1, 791 or LVA-3; vitamin C; dry extracts of herbs: rhizomes of Potentilla erectus; chamomile flowers; oak bark; calendula officinalis; echinacea purpurea; Hypericum perforatum.

Also known feed additive with probiotic activity on a mineral basis (patent for invention RU 2569002C1). The feed additive consists of viable spores of spore-forming bacteria of the Bacillus subtilis strain with a titer of 2⋅10 ⁶ -6⋅10 ⁹ CFU/g and a filler - diatomite in the form of burnt crumbs in a mass ratio of 1:10, respectively. In this case, the feed additive in the composition of the feed was taken at a ratio of 0.001:1.

Known probiotic based on the strain of bacteria Bacillus subtilis MG-8 VKPM B-12476 (Patent for invention RU 2663720 C1), used for the prevention of gastrointestinal diseases in farm animals and birds. The method for using the probiotic involves the introduction of the culture liquid of the bacterial strain Bacillus subtilis VKPM B-12476 into the complete feed of farm animals and birds.

Known biological product with probiotic activity to optimize the absorption of feed intended for farm animals and poultry (Patent for invention RU 2493723C1). The preparation differs in that Bacillus cereus 1-79 bacteria with a titer of 3.5⋅10 ⁹ CFU/g - 4.0⋅10 ⁹ CFU/g, suspended in a nutrient medium and having a high probiotic activity, is used as a strain.

Known dry probiotic for fattening farm animals and poultry (Patent for invention RU 2711924 C1), which is a lyophilized biomass of the strain Enterococcus mundtii VKPM B-11828.

A known biological product is a probiotic based on the Streptococcus faecium TE-17 strain and a method for its production (Patent for invention RU 2217493 C2). The strain has high productivity and the ability to suppress pathogenic microflora. Probiotic is a liquid culture of S. faecium TE-17, biopolymers, urea, trace elements, stabilizer, lactic acid and tap water in the specified content of components.

The objective of the present invention is to develop a new probiotic feed additive for farm animals with high antagonistic activity, which suppresses the development of pathogenic microorganisms, promotes the formation of beneficial microflora in the digestive tract and improves the productivity of farm animals.

The problem is solved by creating a probiotic feed additive for farm animals, which includes a composition of microorganism cultures, consisting of strains of lactobacilli Lactobacillus buchneri BX-99, Lactobacillus plantarum PL-99 and a strain of Bacillus subtilis BS2017, and peat gel, taken in a ratio of 1: 1, at the same time, the total concentration of colony-forming units CFU of lactobacilli and B. subtilis BS2017 in the feed additive should be at least 1.0⋅10 ⁸ CFU/cm ³ .

To create a probiotic feed additive, strains of Lactobacillus buchneri BX-99, Lactobacillus plantarum PL-99 and strain Bacillus subtilis BS2017 were selected, which have high probiotic properties and are not related to microorganisms pathogenic to humans and animals, according to the classification given in the Sanitary Rules SP 1.3 .2322-08. At the same time, to increase the effectiveness of the proposed feed additive, peat gel is included in its composition.

Selected strains provide suppression of microorganisms-causative agents of intestinal toxicoinfections. At the same time, the combination of bacteria of the genus Lactobacillus and the species Bacillus subtilis in a bacterial preparation is effective because they can affect various parts of the pathogenetic process. In turn, humic acids contained in peat gel reduce the level of toxins, improve protein digestion and the absorption of calcium, trace elements, nutrients, in addition, they tend to form a film on the mucous membrane of the gastrointestinal tract, also protecting the body from infections and toxins. . The result is an increase in the immunity of animals to diseases, as well as an increase in weight gain and an increase in milk yield.

The preparation of a probiotic feed additive for farm animals was carried out as follows.

To prepare a mixture of microbial cultures, a liquid nutrient medium was used with the composition, g/dm ³ :

Molasses 20.0 ^cm3 MgSO ₄ ⋅7H ₂ O 1.00 KN ₂ RO ₄ 0.4 NaCl 0.5 CaCO ₃ 1.5 KNO ₃ 1.0 Distilled water up to 1.0 dm ³

The cultivation of cultures included in the composition of the probiotic feed additive was carried out in the specified liquid nutrient medium in fermenters with a capacity of 20.0 DM ³ . The volume of the liquid nutrient medium was 12.0 DM ³ .

For joint cultivation of strains of lactobacilli Lactobacillus plantarum PL-99, Lactobacillus buchneri BX-99 and strain. Bacillus subtilis BS2017 conditions were chosen under which lactobacilli are first cultivated at a temperature of (37 ± 1) ° C without aeration for 24 hours, then the culture of B. subtilis BS2017 is added and further cultivation is carried out at a temperature of (28 ± 1) ° C aeration for 24 hours. After obtaining a mixture of microbial cultures, it is added to peat gel in a ratio of 1:1 and mixed.

At the same time, before adding the resulting mixture of microbial cultures to the peat gel, in order to reduce the amount of foreign microflora in it, pasteurization of the peat gel and its subsequent cooling to the operating temperature can be carried out.

To determine the concentration of lactobacilli in the finished product, seeding was carried out on a dense nutrient medium MRS-4, to determine the concentration of B. subtilis - on FT-arap. The concentration of lactobacilli strains in the finished product was 8.3 ⋅ 10 ⁸ , the concentration of B. subtilis BS2017 was 1.0 ⋅ 10 ⁹ . The control of the finished preparation for the presence of mesophilic aerobic and facultative anaerobic microorganisms, the number of which should not exceed 1.0⋅10 ³ , was carried out by inoculation on a dense nutrient medium FT-agar. The presence of mesophilic aerobic and facultative anaerobic microorganisms is due to the fact that a small amount of extraneous microflora remains in the peat gel after pasteurization.

The characteristics of the final preparation are presented in Table 1.

The antagonistic activity of the biological product was also evaluated. The evaluation of the antagonistic activity of lactobacilli was carried out using a two-layer method according to Frederic.

The following types of microorganisms were used as indicator cultures for assessing the antagonistic activity of the studied strains: Escherichia coli C600, Proteus vulgaris, Salmonella sp, Klebsiella ozaenae, Serratia odorifera biogrup 1, Morganella morgani, isolated from the intestinal contents of piglets and stored at the Department of Microbiology of Vyatka State University.

The indicator test strains, against which the antagonistic activity was evaluated, were grown during the day in meat-peptone broth, then added 0.5 cm ³ to 2.5 cm ³ of semi-liquid meat-peptone broth agar, quickly mixed and poured onto the surface of the cups with colonies of lactobacilli and bacilli. By tilting the dish from side to side, the semi-liquid agar was evenly distributed over the surface of the Petri dish. Then the crops were incubated for 24 hours at a temperature of (37±1)°C. Antagonistic activity was manifested by the formation of zones of inhibition of the growth of test strains around the colonies of lactobacilli and bacilli.

The level of antagonistic activity was assessed according to the following criteria: zero - with the width of the zone of no growth up to 1.0 mm, low - (1.1-4.9) mm, medium - (5.0-8.9) mm, high - 9 .0 mm and more.

Antagonistic activity of the studied strains of microorganisms was assessed in vitro.

The data obtained are presented in table 2.

When assessing the growth inhibition of Bacillus subtilis BS2017 by lactobacilli strains, there were no growth inhibition zones, which indicates that the Bacillus subtilis strain and lactobacilli strains: Lactobacillus plantarum PL-99 and Lactobacillus buchneri BX-99 do not inhibit each other's growth and can be used to prepare a complex probiotic drug.

At the same time, the assessment of antagonistic activity against indicator cultures allows us to conclude that the studied strains of microorganisms are able to actively inhibit the growth and reproduction of enterobacteria, potential pathogens of infectious diseases of the gastrointestinal tract of animals. Strains Lactobacillus plantarum PL-99, Lactobacillus buchneri BX-99 and Bacillus subtilis BS2017 have high antagonistic activity against all indicator strains under study.

The sensitivity of the studied strains of microorganisms to antibiotics was also analyzed using the paper disk method. The sensitivity of the studied strains was determined to the following antibiotics: azithromycin, amikacin, ampicillin, levomycetin, netilmicin, norfloxacin, rifampicin, sparfloxacin, ceftriaxone, tobramycin.

The results obtained are presented in table 3.

Based on the results obtained, it can be concluded that the strains: Lactobacillus plantarum PL-99 and Bacillus subtilis BS2017 are moderately sensitive to most of the studied antibiotics; strain Lactobacillus buchneri BX-99 is resistant to 50% of the studied antibiotics.

The specific safety of the proposed probiotic feed additive was determined on white mice in in vivo tests. For this, outbred white mice of the same sex, quarantined and not previously used in experiments, weighing (20 ± 2) ^g , were used. observations were carried out for 5 days.

The results obtained are presented in table 4.

The results obtained indicate that the claimed probiotic supplement passed the test for specific safety according to GOST 31926-2013 “Medicines for veterinary use. Methods for determining harmlessness. Also, studies have shown that the developed probiotic preparation does not form toxic compounds.

The assessment of the impact of the proposed probiotic supplement on the treatment of intestinal infections in calves was carried out in the production agricultural cooperative "Kolkhoz named after Sverdlov", located in the Kirov region.

The experiment involved 14 calves aged 2.5 to 3 months with diarrheal syndrome of unknown etiology. The probiotic supplement was given to calves individually in accordance with the "Instruction for the use of a probiotic supplement for farm animals".

The test results of the proposed probiotic supplement have established its therapeutic and prophylactic efficacy in diarrheal syndrome in calves.

The assessment of the effect of probiotic supplements on the weight gain of calves was carried out in the production agricultural cooperative "ISTOBENSKY" for breeding work, located in the Kirov region.

The results of the tests performed are presented in table 5.

The live weight of the calves of the experimental group that received the drug increased by 2.8% on the 20th day of the experiment compared to the control group that did not receive the drug. On the 40th and 80th day of the experiment, the weight of the calves increased by 6.1% and 14.5%, respectively. From the 21st to the 40th day of the experiment in the experimental group, the average daily weight gain of calves increased by 14.94% compared to the control, and from the 41st to the 80th day of the experiment by 36.59%.

Thus, as a result of the experiment, the positive effect of the proposed probiotic supplement on the increase in weight gain of calves under the conditions of the experiment was shown.

The influence of the probiotic additive on milk productivity and the chemical composition of milk was assessed at Stremilovskoye Department of the Domestic Meat and Dairy Production Association LLC (Stremilovskoye OOMMPO LLC) of the Chekhov district of the Moscow region on highly productive dairy cows of black-and-white Holsteinized breed during the milking period, 17 heads per each, an average of 58 days after calving, divided according to the principle of analogues into two groups: control and experimental. Cows of the 1st control group received a diet balanced in terms of all nutrients and energy, which met the requirements of daily milk yield at the level of 26-30 kg of milk. Animals of the 2nd experimental group received the same diet with the addition of 4 cm ³ /head/day of a new probiotic feed additive diluted in 1 dm ³ of water. The duration of the scientific and economic experiment was 90 days. Animals of the control and experimental groups were placed in the same room, where they were created the same conditions for feeding and keeping.

Data on milk productivity and chemical composition of milk from animals of the control group and treated with a probiotic feed additive as part of the diet are presented in Table 6.

As can be seen from the data in Table 3, despite almost the same feed intake, the average daily milk yield in the experimental group of cows was higher compared to the control. In particular, the average daily milk yield of natural milk in cows of the 2nd experimental group, who received 4.0 cm ³ / head / day of a new probiotic feed additive diluted in 1.0 dm ³ of water, amounted to 25.58 kg or 4.4% higher compared to animals in the control group. At the same time, the differences, both in terms of gross and average daily milk yield, between the cows of the control and the 2nd experimental group of cows were statistically significant.

The main indicator characterizing the quality of milk is the amount of fat, protein, lactose, urea and somatic cells in milk, which affects its sales value.

The presence of a large number of somatic cells in milk leads to a serious decrease in its quality indicators: biological usefulness is lost, technological properties deteriorate during processing, the acidity of milk decreases, fat, casein, and lactose are lost. Milk becomes less heat-resistant, coagulates worse with rennet, and the development of beneficial lactic acid bacteria slows down. It is impossible to make high-quality products from such milk (cheese, cottage cheese, butter, kefir, etc.). Somatic cells affect not only the assessment of milk quality, but also the assessment of the health of cows, their productivity.

In the milk of highly productive cows of the experimental group, after four months after calving, receiving 4.0 cm ³ / head / day of a new probiotic feed additive, the number of somatic cells amounted to 73.67 thousand per 1 cm ³ , which is 62.76 lower thousand or 1.85 times compared with the control.

On the basis of the conducted studies, it can be concluded that the enrichment of the diets of highly productive cows during the milking period with a probiotic feed additive had an advantage over the diet of cows in the control group in terms of the effect on milk productivity and milk quality.

Considering the foregoing, it is obvious that the claimed new probiotic feed additive for farm animals has a high antagonistic activity, which suppresses the development of pathogenic microorganisms, promotes the formation of beneficial microflora in the digestive tract and increases the productivity of farm animals.

Claims (5)

1. Probiotic feed additive for cows and calves, which includes a composition of cultures of microorganisms, consisting of strains of lactobacilli Lactobacillus buchneri BX-99, Lactobacillus plantarum PL-99 and a strain of Bacillus subtil is BS 2017, and peat gel, taken in a ratio of 1:1, at the same time, the total concentration of colony-forming units CFU in the feed additive must be at least 1.0⋅10 ⁸ CFU/cm ³ . 2. A method for producing a probiotic feed additive for cows and calves according to claim 1, including co-cultivation in a liquid nutrient medium of lactobacillus strains Lactobacillus buchneri BX-99, Lactobacillus plantarum PL-99 without aeration for 24 hours, followed by the addition of a strain of Bacillus subtilis BS2017 and further cultivation with aeration for 24 hours, and the subsequent addition of the resulting mixture of microbial cultures to peat gel in a ratio of 1:1 and their mixing. 3. A method for producing a probiotic feed additive for cows and calves according to claim 2, characterized in that before adding the resulting mixture of microbial cultures to the peat gel, pasteurization of the peat gel is carried out. 4. A method for producing a probiotic feed additive for cows and calves according to claim 2, characterized in that a liquid nutrient medium of the following composition, g/dm ^{3 ,} can be used as a liquid nutrient medium for cultivating microbial cultures: Molasses 20.0 ^cm3 MgSO ₄ ⋅ 7H ₂ O 1.00 _{KH2PO4 _} 0.4 NaCl 0.5 CaCO ₃ 1.5 KNO ₃ 1.0 Distilled water up to 1.0 dm ³