RU2406516C1 - Medication for prevention of gastrointestinal tract infections in agricultural animals and poultry and method of its obtaining - Google Patents

Abstract

FIELD: medicine, veterinary. ^ SUBSTANCE: invention relates to veterinary, namely to obtaining from yeast biomass Saccharomyces cerevisiae of antibacterial medication for prevention infections of gastrointestinal tract in animal breeding and poultry farming. Medication includes biologically available mannanoproteins, integrated in partly hydrolysed cell wall of yeasts, water-soluble mannanoproteins of cell wall, water-soluble proteins and water-soluble glucosaccharides of yeast biomass, enzymatic complex, which possesses depolymerase activity with respect to -glucan. Method of medication obtaining lies in the following: yeast mass is mixed with enzymatic complex, subjected to mechanical activation, obtained product is tabletted and warmed at temperature of enzymes action. ^ EFFECT: invention ensures high preventive activity of medication. ^ 5 cl, 7 ex, 4 dwg

Description

The invention relates to veterinary medicine, it is possible to obtain a preparation from yeast biomass Saccharomyces cerevisiae, the active ingredients of which are mannanoproteins integrated into a partially hydrolyzed yeast cell wall, and water-soluble mannanoproteins of the yeast cell wall. The drug can be used to prevent digestive tract infections in animal husbandry and poultry farming.

It is known that for the development of most diseases of the gastrointestinal tract, it is necessary that the pathogenic bacterium is fixed on the mucous membrane of the digestive tract [1, 2]. Bacteria are attached by binding pectins (specific proteins on the surface of bacteria) with mannanoligosaccharides on the surface of the epithelium. The addition of biologically available mannanooligosaccharides to animal feed prevents the development of infection, since mannanooligosaccharides block bacterial pectins.

Both water-soluble mannanooligosaccharides and mannanoproteins, as well as mannanoproteins available for communication with pectins, integrated into a partially hydrolyzed cell wall, have this effect [2, 3].

The cell wall of S. cerevisiae yeast is a layered structure (FIG. 1). The middle layer, responsible for maintaining the strength of the cell wall, consists mainly of β-glucan [4-7]. From the outer and inner sides, this layer is covered with mannanoproteins. The lipid membrane separates the cell wall and the liquid contents of the cell.

β-Glucan is a polymer consisting of glucose monomers connected in series by a β-1,3-bond. β-1,3-Glucan provides rigidity and strength of the cell wall, its resistance to environmental influences. Most of it is not extracted by either acids or alkalis. This type of glucan can be selectively hydrolyzed only by enzymes exhibiting 1,3-glucanase activity.

Mannanoproteins are distinguished by the type of binding of the mannan oligosaccharide moiety to protein [7]. Two types of mannanoproteins are most common: O-linked, in which the mannan oligosaccharide moiety is attached to the protein via an O-glycosidic link between the mannose residue and the hydroxyl group of serine or threonine, and N-linked mannanoproteins with an N-glycosidic link between the N-acetylglucosamine residue and β -amide nitrogen asparagine.

N-linked mannano-oligosaride can be cleaved from a protein molecule by the action of specific enzymes - EndoH-EndoF-glycosidases. The treatment of biomass with alkali results in the extraction of mannanol-oligosaccharide as part of mannanoprotein without breaking the bond with the protein. The degree of extraction depends on the degree of disordering of the structure of the cell wall. N-mannanoproteins are poorly extracted from the native cell wall.

The O-glycosidic bond of mannanoproteins, in contrast, is easily destroyed by the action of weak alkali (β-elimination). In most cases, treatment with 0.1-0.5 M sodium hydroxide for 12 hours (or at 0-5 ° C for several days) is enough to split the carbohydrate component from the protein [8]. It can be assumed that in case of violation of the cell wall structure, O-linked mannanoproteins are also extracted with alkali, but due to the instability of the O-bond, the mannanol-oligosaccharide component is rapidly cleaved.

Thus, mannanoproteins are largely integrated into the glucan layer, in order to convert them into a bioavailable form, it is necessary to break the connection of mannanoproteins with glucan. Various methods are used for this, for example, induced autolysis, enzymatic or acid hydrolysis. The result is a product containing bioavailable mannanoproteins integrated into a partially hydrolyzed cell wall.

A known method for the isolation of mannanoproteins from baker's yeast [9]. For this, the yeast is crushed and heated in an autoclave. In this case, part of the cell wall proteins is hydrolyzed by cell wall peptidases to amino acids and short peptides and passes into a water-soluble form. Due to the partial hydrolysis of proteins, water-soluble mannanoproteins of the yeast cell wall partially pass into solution. After cooling and separating the precipitate in a centrifuge, it is again heated in an autoclave. The supernatants are combined and concentrated in vacuo. Acetic acid was added to the resulting concentrated solution, the brown mucus that was released was washed with a small amount of diluted acetic acid and discarded. The washing solutions were combined and concentrated in vacuo. Ethanol is added to precipitate mannanoproteins. Mannanoproteins are filtered off and washed twice with 60% alcohol.

The intermediate is purified by repeated washing with hot water, treatment with Feling's reagent, alcohol and acetic acid. Using this method, water-soluble mannanoproteins are obtained, resulting from the autolysis of yeast biomass, in an amount of 4% (in terms of dry yeast). The disadvantages of the method, limiting the industrial production of mannanoproteins, are its complexity, multi-stage and low yield of the target product. The product has a reduced feed value, since in the process of obtaining and purifying mannanoproteins most of the nutrient components contained in the yeast biomass are discarded.

To obtain free mannanooligosaccharides from mannanoproteins containing O-linked mannanooligosaccharides, the authors of [10] used the insoluble part of the autolysate obtained as a result of toluene-induced autolysis [11]. Part of the cell wall proteins during autolysis is hydrolyzed to amino acids and short peptides by the peptidases contained in the cell wall. Insoluble substances of the autolysate, namely whole cells and partially hydrolyzed cell walls, are separated from the solution by centrifugation and heated in an autoclave in a NaOH solution. In this case, mannanoproteins and proteins pass into solution. Also, under the action of alkali, the mannanol-oligosaccharide part is cleaved from O-linked mannanoproteins. The supernatant is neutralized with hydrochloric acid and centrifuged. A mixture of O-mannanol-oligosaccharides, N-mannanoproteins and proteins is precipitated from the supernatant with alcohol. To remove proteins and mannanoproteins, the intermediate is treated with sodium hydroxide, Feling liquid and washed from proteins and mannanoproteins with water and acetic acid. The resulting product is the O-mannanoligosaccharides of the insoluble part of the autolysate and consists of 98% mannose.

The disadvantages of this method are its complexity, the use of autoclaving alkaline solutions, which requires special resistant equipment, energy consumption for maintaining pressure and high temperature.

As raw materials, yeast autolysate is used, which is associated with difficulties in its production. Working with alkaline solutions is a danger to personnel. Since mannanoproteins, water-soluble proteins and water-soluble glucosaccharides of yeast biomass are discarded during the preparation of O-mannanol-oligosaccharides, the antibacterial activity and feed value of the product are reduced.

The closest technical solution selected for the prototype is the drug obtained by the method [12]. It is proposed to use a preparation containing mannanoproteins integrated into a partially hydrolyzed cell wall as a remedy for coccidiosis. The active principle of the preparation is the repeatedly washed yeast cell wall, partially hydrolyzed by proteases, containing mannanoproteins integrated into it.

The preparation according to the prototype has lower antibacterial activity and feed value due to the fact that the process of its preparation involves discarding a water-soluble fraction containing water-soluble mannanoproteins.

The problem solved by the claimed technical solution is to create a drug containing mannanoproteins and having high antibacterial activity for livestock and poultry, as well as the development of an environmentally friendly, simple way to obtain this drug, which allows the use of available raw materials, such as spent yeast biomass, post-alcohol bard and etc.

The problem is solved due to the fact that the claimed preparation for the prevention of digestive tract infections in farm animals and birds, including mannanoproteins of the yeast biomass Saccharomyces cerevisiae, integrated into the cell wall of the yeast, additionally contains water-soluble mannanoproteins of the yeast cell wall in the following ratio,% wt .:

- mannanoproteins integrated into the cell wall of yeast - 2.5-4.5%;

- water-soluble mannanoproteins of the yeast cell wall - 0.8-2.5%;

- water-soluble proteins of yeast biomass - 10-40%;

- water-soluble glucosaccharides of yeast biomass - 10-20%,

- enzymatic complex with depolymerase activity with respect to β-glucan - 0.5-10%;

- mechanically activated yeast biomass - the rest.

Significant differences of the claimed drug is its composition.

The problem is also solved by the claimed method, characterized in that it involves pre-mixing the yeast biomass with an enzymatic complex having depolymerase activity against β-glucan, if necessary, with the addition of water in an amount of 0.5-5% by weight of the above components, the resulting mixture is subjected to mechanical activation in activators of the planetary, or vibrational, or vibrocentrifugal types, or roller mills, providing acceleration of grinding media 60-400 m / s ² and time My stay in the treatment zone is 0.5-10 minutes, after mechanical activation, the intermediate is subjected to tabletting and heating at an enzymatic complex action temperature of 40-60 ° C.

Preferably, tableting is carried out at a pressure of 10-20 kg / cm ² .

Preferably, the heating of the tabletted intermediate is carried out for 25-35 hours.

The patent search did not reveal drugs of a similar composition, therefore, it was concluded that the claimed technical solution meets the criterion of "novelty." The set of essential features of the proposed method is also not identified, which allows us to conclude that the claimed drug and its production method meet the criterion of "inventive step".

The technical result consists in creating a preparation containing mannanoproteins both integrated into a partially hydrolyzed cell wall and in a water-soluble form, which allows it to exhibit sorbent properties due to the presence of accessible mannanoproteins on the surface of partially hydrolyzed cell walls, and also serve as a source of water-soluble mannanoproteins.

In addition, thanks to the claimed method, the preparation contains water-soluble proteins and water-soluble glucosaccharides of yeast biomass, which increase the feed value of the drug.

During mechanical activation of a mixture of yeast biomass with an enzymatic complex, partial mechanical destruction of the cells occurs, disordering of the supramolecular structure of the cell walls (Fig. 2), an increase in their reactivity with respect to enzymatic hydrolysis, formation of a mechanocomposite, a product with increased reactivity, in which reduced diffusion pathways, structural layers of the cell wall are disordered and are diffuse.

Tableting the resulting intermediate allows to increase the volume concentration of the enzyme, facilitate mass transfer between the particles of the composite, and also prevent the loss of water, which is necessary for enzymatic hydrolysis.

The heating of the preformed tablet product at the optimum temperature of the enzyme leads to the hydrolysis of β-glucan, a structural element of the cell wall. Due to this, a significant part of mannanoproteins passes into a water-soluble form. The availability of mannanoproteins integrated into the cell wall is also increasing.

The thermal transformation of the tabletted intermediate takes place due to the formation of local hydrolysis zones and their distribution to the entire preparation. Destroyed at the stage of mechanical activation of the cells serve as centers of localization of enzymatic hydrolysis (figure 3). Residual moisture promotes the advancement of the reaction front, an increase in temperature additionally removes diffusion difficulties, and increases the mobility and hydrolytic activity of enzymes.

The enzymatic hydrolysis of tablets in the presence of natural moisture of the biomass allows you to get the final product in the form of tablets, which increases the shelf life of the drug.

Aqueous and alkaline extraction were used as a method for determining the bioavailability of mannanoproteins of the preparation [8, 9]. Extraction with water makes it possible to determine the yield of mannanoproteins in contact with biological media or water. As a result of mechanical activation and subsequent enzymatic hydrolysis, the amount of water-soluble mannanoproteins increases, which is associated with the hydrolysis of glucan-mannanoproteins bonds.

Extraction with a solution of caustic soda makes it possible to determine the amount of mannanoproteins available for reactions with solutions integrated into the cell wall. As a result of mechanical activation and subsequent enzymatic hydrolysis due to partial hydrolysis of glucan, there is an increase in the availability of mannanoproteins integrated into the cell wall. Figure 4 presents a micrograph of yeast biomass after mechanical activation and enzymatic hydrolysis. The presence in the cell wall of electron-dense particles formed on reactive defects in the structure of mannanoproteins when treated with a fixing reagent indicates an increase in the availability of wall mannanoproteins for reactions with solutions.

The invention is illustrated by the following examples.

Example 1

GOST 171-81 pressed baking yeast, dried to a moisture content of 12%, is mixed with the enzyme complex “Cellolux-A” (manufactured by LLC SIBBIOFARM Berdsk), which has depolymerase activity with respect to β-glucan, in the ratio 99.5-0 ,5. The mixture is mechanically activated in a flow activator of the type CEM or VCM, providing acceleration of grinding media 60 m / s ² and a residence time of 0.5 minutes in the treatment zone. The intermediate product is tabletted at a pressure of 10 kg / cm ² and incubated at a temperature of 40 ° C for 25 hours.

When accelerating grinding media less than 60 m / s ² , the introduction of particles of the enzymatic complex into the particles of yeast biomass is not ensured, which reduces the efficiency of subsequent enzymatic hydrolysis. The use of processing less than 0.5 minutes does not provide the necessary accumulation of free energy and practically does not lead to the formation of a mechanocomposite. Lowering the temperature of heating below 40 ° C negatively affects the rate of hydrolysis, and carrying out heating in less than 25 hours does not provide the optimal degree of conversion.

The resulting product contains available mannanoproteins of the yeast biomass Saccharomyces cerevisiae, integrated into the cell wall of the yeast, as well as other components in the following ratio, wt.%:

mannanoproteins integrated into the cell wall of yeast - 2.5%,

water-soluble mannanoproteins of the yeast cell wall - 0.8%,

water-soluble proteins of yeast biomass - 10%,

water-soluble glucosaccharides of yeast biomass - 10%,

enzymatic complex with depolymerase activity with respect to β-glucan - 0.5%,

mechanically activated biomass of yeast - the rest.

To determine the water-soluble cell wall mannanoproteins, the resulting product is suspended in water at a water module of 10. The resulting suspension is centrifuged for 15 minutes at 7000 rpm. The precipitate contains partially hydrolyzed cell walls with mannanoproteins integrated into them. The supernatant contains water-soluble mannanoproteins, water-soluble proteins and glucosaccharides, an enzymatic complex possessing depolymerase activity with respect to β-glucan. It was shown that the yield of water-soluble mannanoproteins as a result of mechanoenzymatic treatment increased by 1.4 times in comparison with the initial yeast.

To determine mannanoproteins integrated into the cell wall of the yeast, the resulting product is extracted with a weak alkali solution at a hydromodule equal to 10. Under conditions of alkaline extraction, O-mannano-oligosaccharides and N-mannanoproteins integrated into a partially hydrolyzed cell wall go into a soluble form. It was shown that the yield of mannanoproteins integrated into a partially hydrolyzed cell wall as a result of mechanoenzymatic treatment increased by 2.9 times in comparison with the initial yeast.

Example 2

GOST 171-81 pressed baking yeast, dried to a moisture content of 12%, is mixed with the enzyme complex “Cellolux-A” (manufactured by PO SIBBIOFARM LLC, Berdsk), which has depolymerase activity in relation to β-glucan in the ratio 9-1. The mixture is mechanically activated in a flow activator of the type CEM or VCM, providing acceleration of grinding media 400 m / s ² and a residence time of 10 minutes in the treatment zone. The intermediate product is tableted at a pressure of 10 kg / cm ² and incubated at a temperature of 50 ° C for 30 hours.

When accelerating more than 400 m / s ² , the processed mixture is heated, which can lead to the destruction of biologically active substances, namely mannanoproteins and the enzymatic complex. The use of treatment over 10 minutes can lead to mechanical destruction of the target components and the enzymatic complex. An increase in temperature above 50 ° C leads to denaturation of the enzymatic complex. An increase in the warm-up time over 30 hours does not significantly affect the degree of conversion due to a gradual decrease in the reaction rate over time.

The resulting product contains available mannanoproteins of the yeast biomass Saccharomyces cerevisiae, integrated into the cell wall of the yeast, as well as other components in the following ratio, wt.%:

mannanoproteins integrated into the cell wall of yeast - 4.5%,

water-soluble mannanoproteins of the yeast cell wall - 2.5%,

water soluble proteins of yeast biomass - 40%,

water-soluble glucosaccharides of yeast biomass - 20%,

enzymatic complex with depolymerase activity in

relative to β-glucan - 10%,

mechanically activated biomass of yeast - the rest.

The determination of mannanoproteins integrated into the cell wall of yeast and water-soluble mannanoproteins is carried out analogously to example 1.

Example 3

It is carried out under the conditions of example 2. Obtained as a result of mechanical activation, the intermediate is tabletted at a pressure of 20 kg / cm ² .

Tableting the resulting intermediate allows to increase the volume concentration of the enzyme, facilitate mass transfer between the particles of the composite, and also prevent the loss of water, which is necessary for enzymatic hydrolysis. The use of pressures less than 10 kg / cm ² impractical due to the fact that at low pressures the tablets are loose and porous, which complicates mass transfer between the particles of the composite. The use of pressures above 20 kg / cm ^{2 is} impractical because it does not significantly affect the properties and structure of the tablet and can lead to undesirable separation of the liquid phase.

Example 4

It is carried out in the conditions of example 2. Dried baker's yeast GOST 28483-90 with a moisture content of less than 12% is mixed with the enzyme complex "Cellolux-A", in a ratio of 9-1 and water is added 0.5-5% by weight of the dry mixture (to the mixture moisture 12%).

The moisture content of the initial yeast biomass is due to the presence of bound water inaccessible to the reaction in the cells; therefore, adding the indicated amount of water to the mixture allows increasing the rate of enzymatic hydrolysis due to faster advancement of the reaction front and also improving the quality of pressed tablets. Adding water to a moisture content of less than 12% is impractical due to the small effect on the reaction rate, and adding water to a moisture content of more than 12% is not recommended in order to avoid powder sticking to grinding media and walls of mechanical activators.

Example 5

It is carried out under the conditions of example 2. As an enzymatic complex, a mixture of the enzyme complex “Cellolux A” with depolymerase activity with respect to β-glucan and the enzymatic complex “Protosubtilin g3x” (produced by LLC PO SIBBIOFARM Berdsk) with depolymerase is used activity against cell wall proteins. The optimum temperature for enzymatic hydrolysis for this enzymatic complex is 60 ° C. The reaction time is 25 hours.

The use of a mixture of enzymatic complexes with depolymerase activity with respect to β-glucan and proteins of the cell wall allows increasing the rate of the enzymatic reaction of cell wall hydrolysis due to the complex effect on all its structural components.

Example 6

It is carried out under the conditions of example 2. As a raw material, the yeast biomass of the post-alcohol stillage stillage is used (TU 9296-302-00008064-98), dried to a moisture content of 12%. Due to the presence in the yeast biomass of the post-alcohol stillage stillage of compounds capable of inactivating the enzymatic complex, hindering the progress of enzymatic hydrolysis, it is advisable to use yeast biomass and an enzymatic complex with depolymerase activity with respect to β-glucan in a ratio of 90-10. The duration of the reaction is in this case 35 hours. Involving in the production of cheap and substandard raw materials allows you to rationally dispose of the listed waste, as well as reduce the cost of obtaining the drug.

Example 7

It is carried out in the conditions of example 2. The work was carried out at the Research Design and Technological Institute of Animal Husbandry SB RAAS. The resulting product in an amount of 1% wt. added to the diet of geese of the meat direction of productivity during the fattening period (s. Burmistrovo, Iskitimsky District, Novosibirsk Region). The duration of the experiment was 55 days. Animals of the control group in the diet received the product did not receive.

The gross growth of experimental geese was higher than that of animals from the control group, by 14.0%. The experimental geese were 15.5% less than the control spent feed per 1 g of gain in live weight.

The taken blood samples showed that the copper content in the blood of control animals exceeded the norm by 2-4 times, while in the animals of the experimental group the copper content was within the normal range. This can be explained by the fact that the amount of copper in the blood increases with infectious diseases, due to increased production of antibodies. Therefore, the geese from the control group were susceptible to infectious diseases, and the geese of the experimental group were protected from infections by the resulting product.

The inventive preparation in comparison with the prototype has a higher antibacterial activity and feed value, has been tested and therefore meets the criterion of "industrial applicability".

Information sources

1. The future of growth stimulants. Alltech European Lecture Tour, February 13 - March 5, 2006 http://www.alltech.com.

2. A.E. Wold, J. Mestecky, M. Tomana, A. Kobata, H. Ohbayashi et al. Secretory immunoglobulin a carries oligosaccharide receptors for Escherihia coli Type 1 fimbrial lectin // Infection and Immunity 58: 9 (1990 ) 3073-3077.

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5. D.J.Manners, A.J. Masson, J.C. Patterson, The structure of a β- (1-3) -D-glucan from yeast cell walls // Biochem. J., 135 (1973) 19-30.

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7.T.S. Kalebina, I.S. Kunaev, Advances in Biological Chemistry, 41 (2001) 105-130.

8. Glycoproteins, ed. A. Gottschalk, T1. / M .: Mir, 1969 .-- 304 p.

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11. V.K. Latov, T.L. Babayan, S.V. Gordienko, A.S. Kogan, V.A. Tsyryapkin et al., Complex processing of yeast biomass // Biotechnology, 3 (1990) 14-18 .

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Claims (5)

1. The drug for the prevention of digestive tract infections in farm animals and poultry, including biologically available mannanoproteins of the yeast biomass Saccharomyces cerevisiae integrated into the cell wall of the yeast, characterized in that it additionally contains water-soluble mannanoproteins of the yeast cell wall in the following ratio, wt.%:
mannanoproteins integrated into the cell wall of yeast 2.5-4.5 water soluble mannanoproteins yeast cell wall 0.8-2.5 water soluble yeast biomass proteins 10-40 water soluble glucosaccharides of yeast biomass 10-20 enzymatic complex possessing depolymerase activity in relation to β-glucan 0.5-10 mechanically activated yeast biomass rest 2. The method of obtaining the drug according to claim 1, characterized in that it involves pre-mixing the yeast biomass with an enzymatic complex having depolymerase activity with respect to β-glucan, the resulting mixture is subjected to mechanical activation in activators of the planetary, or vibrational, or vibrocentrifugal types, or roller mills, milling bodies providing acceleration 60-400 m / s ² and the residence time in the processing zone 0.5-10 minutes, after mechanical activation interm subjected to tableting and warming When the optimum temperature of action of the enzymatic complex. 3. The method according to claim 2, characterized in that the tableting is carried out at a pressure of 10-20 kg / cm ² . 4. The method according to claim 2, characterized in that the heating of the tableted intermediate is carried out for 25-35 hours 5. The method of obtaining the drug according to claim 2, characterized in that the heating of the tabletted intermediate is carried out at a temperature of 40-60 ° C, depending on the optimal temperature of the selected enzymatic complex.

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