RU2758880C1 - Method for producing biological preparation for correcting metabolism in pre-nursery pigs - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology in animal husbandry, namely, to a method for producing a biological preparation for correcting the metabolism in pre-nursery pigs. A method for producing a biological preparation for correcting the metabolism in pre-nursery pigs, including introduction of a bacterial concentrate of a day-old culture of probiotic microorganisms into a nutrient medium, wherein cellobacterin containing probiotic microorganisms Clostridium thermocellulociticus, Ruminococcus olbus, Clostridium lochheadii, is used as a culture of probiotic microorganisms, and sterilised beet molasses is used as a nutrient medium, wherein in order to prepare the nutrient medium, 15.0 g of beet molasses is weighed, the volume is brought to 100.0 ml with distilled water, the mixture is sterilised in a water bath for 35 to 45 minutes at a temperature of 85 to 90°C, filtered, 1 ml of a bacterial concentrate of a day-old culture of probiotic microorganisms, pre-standardized to 1×10⁶ CFU/cm³ is added, held for 8 days in a thermostat at a temperature of 37°C and pasteurised at a temperature of 90°C to 22°C for 35 minutes.

EFFECT: above method provides a possibility of creating an effective and stable preparation for forming gastrointestinal microbiocenosis and increasing the non-specific immunity in pre-nursery pigs by introducing biologically active substances (metabiotics) into the body, formed in the process of vital activity of microorganisms inhibiting the development of pathogenic microbiota and stimulating the development of beneficial microorganisms.

1 cl, 1 dwg, 4 tbl, 2 ex

Description

The invention relates to biotechnology in animal husbandry and can be used in feeding farm animals to correct metabolism and indices of nonspecific immunity due to the introduction into the body of biologically active substances (metabiotics) formed during the life of microorganisms that inhibit the development of pathogenic microflora and stimulate the development of beneficial microorganisms.

Currently, in the scientific literature there is an insufficient number of publications devoted to the study and biotechnological production of various types of biologically active substances, produced mainly by spore-forming microorganisms used in animal feeding. At the same time, the problem of the systemic study of metabolites of probiotic bacteria, due to its complexity, is still quite far from a final solution. This concerns the issues of the synthesis of biologically active substances under various conditions of cultivation of probiotic microorganisms, the qualitative and quantitative composition of the components of biological products, their relationships, improving the methods of isolation and purification. The cultivation of probiotic organisms takes place mainly on cereal nutrient media. A distinctive feature of our research is the cultivation of probiotic microorganisms on 5-25% molasses medium.

The use of molasses as a nutrient medium is known when creating a feed additive "EM-Vita", which consists of a mixture of strains Lactobacillus plantarum 376, Lactobacillus casei MDP-1, Saccharomyces cerevisiae, molasses and water. 1 cm ³ contained at least 1 × 107 CFU (colony-forming units) of lactic acid bacteria and 1 × 104 CFU of yeast (ZAO "Bioprogress") [Krapivina E.V. Productivity and immune status of cows' organism when using the EM-Vita feed additive / E.V. Krapivina, D.S. Zhuk, D.I. Ivanov and A.I. Albulov, Yu.N. Fedorov // Young Scientist. Kazan. - 2015. - No. 8.3 (88.3). - S. 30-33.], However, the feed additive "EM-Vita" does not reflect the qualitative and quantitative composition of metabolites of lactobacilli. Known feed additive based on molasses with trace elements in the form of iron sulfate and succinic acid (RF patent No. 2554498), while molasses is used as a feed and is not supplemented with probiotic microorganisms. The disadvantage of such drugs is their relatively low biological activity associated with the process of deactivation of living microorganisms during their passage through the upper gastrointestinal tract (stomach, duodenum), as well as the lack of stability of their biological properties during long-term storage, which leads to a decrease in the effectiveness of use. Known drug used in medical practice - Bactistatin, including (wt.%): Sterilized culture fluid containing metabolites of Bacillus subtilis - 0.1-2.0%; zeolite - 68-85%; soy flour hydrolyzate - 15-30%; calcium stearate - 0.5-5.0%. [RF patent No. 2287335]. At the same time, obtaining the compositions of these preparations has laborious technical difficulties and their receipt is associated with the cultivation of probiotic cultures on special media.

The closest in purpose and set of essential features is "A method of obtaining a biologically active feed additive for farm animals and poultry with probiotic and insect protein" (RU 2576200, 02/27/2016). The feed additive was prepared by mixing part of the biomass of Hermetia illucens larvae with a part of the raw probiotic product obtained by solid-phase fermentation of the Bacillus subtilis strain on a sterile carrier, which is beet pulp soaked in a nutrient medium containing molasses, potassium and magnesium salts, and autolysate of yeast and water. The method provides an increase in the productivity and safety of the livestock of animals and birds, stimulation of metabolic and immune processes, however, it is technologically complex, the components are multicomponent, and the process itself consists of several stages.

The aim of the invention is to obtain a dietary supplement based on components of biologically active substances of probiotic cultures for the correction of metabolism in farm animals by influencing the state of the microbiocenosis of the gastrointestinal tract and indices of nonspecific immunity.

The task solved by the authors was the creation of an effective and stable biological product for the formation of microbiocenosis of the gastrointestinal tract and increasing nonspecific immunity due to the introduction into the body of biologically active substances (metabiotics) formed during the life of microorganisms that inhibit the development of pathogenic microflora and stimulate the development of beneficial microorganisms. This problem was solved by creating a biological product, which includes sterilized culture liquid of beet molasses (SCF), containing metabolites obtained as a result of cultivation of strains: Clostridium thermocellulociticus, Ruminococcus olbus, Clostridium lochheadii in 5-10-15-25% nutrient medium from molasses , with the introduction of which in the gastrointestinal tract, the inhibition of pathogenic microflora occurs.

The essence of the invention is illustrated by examples.

Example 1. The object of research was sugar beet molasses 5% -10% -15% -20% -25%. For fermentation of molasses, we used probiotic microorganisms Clostridium thermocellulociticus, Ruminococcus olbus, Clostridium lochheadii, which are part of the enzymatic probiotic Cellobacterin. To obtain a bacterial concentrate, a daily culture of microorganisms was used, which was standardized to 1 × 10 ⁶ CFU / cm ³ .

The indicators of the chemical composition of sugar beet molasses, taken as the basis of the nutrient medium due to the sufficient content of carbohydrate components for the nutrition of microorganisms, are shown in Table 1.

Based on the results of testing the fermentation activity, the variability of the mass fraction of the sum of fermentable sugars was established in the range of 53.70% -33.79%, the pH value from 6.70 to 3.87 in experimental samples of cultured microorganisms, which indicates the activity of microorganisms in the nutrient medium.

To prepare the culture liquid, the mass fraction of beet molasses (5.0 g, 10.0 g, 15.0 g, 20.0 g, 25.0 g) was weighed corresponding to the percentage concentration in a 0.5 L flask. The volume was brought to 100.0 ml with distilled water. Sterilized in a water bath for 35-45 minutes at a temperature of 85-90 ° C. After sterilization, the culture medium was filtered and 1 ml of bacterial concentrate of a daily culture of microorganisms was added, which was standardized to 1 × 10 ⁶ CFU / cm ³ . The cultivation was carried out in a thermostat for 10 days at a temperature of 37 ° C. When receiving a biologically active additive, the constancy of the biochemical characteristics of the composition is ensured, the subsequent appearance of living cells of the producer in the final product is excluded due to pasteurization at a temperature of 90 ° C to 22 ° C for 35 minutes.

The studies carried out allow us to note the variability of the dynamics of colony-forming units (CFU 10 ⁶ ) of probiotic microorganisms in relation to the nutrient medium, which was molasses in various concentrations (Fig.).

When fermentation of molasses by a complex of probiotic microorganisms Cellobacterin, the maximum CFU value of 9.5 × 10 ⁶ CFU / cm ³ in 20% molasses is reached on days 2-3, the minimum 5 × 10 ⁶ CFU / cm ³ in 25% molasses, this is due to a certain type microorganism and the specificity of its metabolism: there is a sharp increase and then a decrease in the number. When using Cellobacterin, a repeated increase in the CFU number was noted on the fourth day (1.3-3 × 10 ⁶ CFU / cm ³ ) and reached a maximum on the eighth day (9.9 × 10 ⁶ CFU / cm ³ ) at a molasses concentration of 15%. During fermentation with this probiotic, the most optimal concentration of molasses, in our opinion, is 15%, since it provided the largest CFU number due to fermentation of less available nutrients from 4 to 8 days.

Thus, it was found that the number of probiotic microorganisms that make up the enzymatic probiotic Cellobacterin on the eighth day in a 15% nutrient medium based on molasses is 9.9 × 10 ⁶ CFU / cm ³ .

Analysis of the indicators given in Table 2 allows us to note the production of metabiotics in all cultured microorganisms. It is known that amino acids are 16% nitrogen, which is their main chemical difference from the other two essential nutrients - carbohydrates and fats. Each protein in the body is unique and exists for a specific purpose. Proteins are not interchangeable. They are synthesized in the body from amino acids, which are formed as a result of the breakdown of proteins found in food.

Thus, it is amino acids, and not proteins themselves, that are the most valuable nutrients, which is especially important in the development of biologically active additives for the formation of microbiocenosis and nonspecific immunity in animals. In this case, the relationship between metabolism and the formation of immunity in general should be taken into account. Studies have established a significant activity of the complex of microorganisms Clostridium thermocellulociticus, Ruminococcus olbus, Clostridium lochheadii. Attention is drawn to the fact of a significant increase in the synthesis of amino acids by the complex of probiotic microorganisms included in the enzymatic probiotic Cellobacterin, in relation to the control. It should be noted that amino acids are transferred to molasses from beets by 50-60%. The studies found a significant excess of indicators for Arginine by 2.8 times, Tyrosine by 57.7 times, Leucine + Isoleucine 3.9 times, and other essential and conditionally nonessential amino acids, which indicates a significant activity of probiotic microorganisms in the nutrient medium (table. 2).

It should be noted that the culture liquid of the nutrient medium from molasses is adequate for the synthesis of amino acids, including essential ones, some vitamins and organic acids of cultivated species of probiotic microorganisms, which is the basis for the creation of a biologically active supplement.

As a result of the studies carried out, the possibility of obtaining metabiotics of probiotic microorganisms by cultivating them on a nutrient medium from beet molasses has been established. Cultivation of probiotic microorganisms that make up the enzymatic probiotic Cellobacterin is most effective in a 15% molasses medium. According to the test results, the variability of the mass fraction of the sum of fermented sugars was established within the range of 53.70% -33.79%, the pH value from 6.70 to 3.87 in experimental samples of cultured microorganisms, which indicates an active metabolism of microorganisms in the nutrient medium.

Example 2

Clinical studies of the biological product were carried out on 30 healthy suckling piglets of 23-25 days of age during the period of preparation for weaning and at 60 days of age when using compound feed SK-5. Two groups of piglets were formed, 15 heads each. The first group is a control group, piglets of group 2 were additionally fed once every 7 days the claimed biological product based on Clostridium thermocellulociticus, Ruminococcus olbus, Clostridium lochheadii in an amount of at least 10 ⁹ CFU / head. The selection of the contents of the large intestine from three pigs from each group for bacteriological studies in compliance with sterility was carried out at 60 days of age.

Early weaning is known to be a powerful stressor for the body. At the same time, a complete transfer of weaned piglets to plant compound feed at 60 days of age often causes the activation of opportunistic microflora and various diseases of the digestive system, leading to a violation in the qualitative composition and quantitative ratio of the intestinal microflora. During the period of transfer of weaning piglets to plant compound feed, it is most expedient to use probiotic feed additives, as well as biologically active components (metabiotics) of probiotic microorganisms. The studies carried out at 60 days of age (Table 3) allow us to note that there is a tendency to change the intestinal microflora with the phenomena of relative disadvantage of the microbiocenosis. There was a decrease in CFU / g of lactobacilli 10 ³ , an increase in staphylococci and clostridia to 10 ⁶ and 10 ⁷ , a slight increase in other opportunistic microflora. At the same time, the content of bifidobacteria 105 remains at a sufficient level. At the same time, in the experimental group, there is also a natural decrease in bifidum and lactobacilli associated with a change in diet, but their number> 10 ⁷ and 10 ⁵ is significantly higher than in the control. Also, in the experimental group, a smaller amount of conditionally pathogenic microflora was noted than in the control.

The role of normal microflora as an antigenic stimulator of the immune system is no less important, the absence of a normal microbial biocenosis causes numerous dysfunctions, and underdevelopment of the main immunocompetent organs is noted in suckling pigs. During normal colonization, numerous microbes induce low antibody titers that are unlikely to target specific species. These antibodies are mainly Ig of class A, secreted on the surface of the mucous membranes.

Considering separately the dynamics of T- and B-lymphocytes (Table 4), it should be noted that in the first days of life of piglets, the bulk of the lymphocytes were T-cells. As the age of the piglets increases, there is a significant increase in the absolute number of B-lymphocytes and a positive trend towards an increase in T-helper cells. At the same time, cellular factors of immunity give an objective idea of the relationship between intestinal microbiocenosis and indices of nonspecific immunity of the animal organism.

Claims (1)

A method of obtaining a biological product for the correction of metabolism in suckling pigs, including the introduction of a bacterial concentrate of a daily culture of probiotic microorganisms into a nutrient medium, characterized in that cellobacterin is used as a culture of probiotic microorganisms, containing probiotic microorganisms Clostridium thermocellulocii Clostridium, Ruminococcus lohead, as nutrient medium sterilized beet molasses, and for the preparation of the nutrient medium 15.0 g of beet molasses are weighed, the volume is adjusted to 100.0 ml with distilled water, sterilized in a water bath for 35-45 minutes at a temperature of 85-90 ° C, filtered, added 1 ml of a bacterial concentrate of a daily culture of probiotic microorganisms, which is pre-standardized to 1 × 10 ⁶ CFU / cm ³ , is kept for 8 days in a thermostat at 37 ° C and pasteurized at a temperature of 90 ° C to 22 ° C for 35 minutes.

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