RU2382051C1 - Method for making chitosan-melanin complex of dead bees - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: chitosan-melanin complex of dead bees can be used in veterinary science for making treatment-and-prophylactic preparations, in medicine for prevention and treatment of intestinal infectious diseases of various origin, and also wound healing ointments and as a radio protector of organism and skin. The method involves preliminary drying of chitin-melanin raw materials to humidity 4-6%, providing its chopping, then the dried mass is powdered, deproteinized with 10% alkali NaOH at temperature 78-82°C within 1.5-2.0 hours. Then 50% NaOH is added to the reaction mixture thereby reducing its concentration to 30% from the initial. The prepared mass is deacetylated at temperature 90-95°C within 1.5-2.0 hours, filtered. The filtered mass representing chitosan is prepared by washing the mass with distilled water to make pH=7. Melanin is precipitated from the solution remained after filtering by to concentrated hydrochloric acid to ensure changing pH of processed solution medium to sour area and flocculation representing melanin that is separated from a liquid phase by centrifugation, washed out; chitosan and melanin are dried separately at temperature 50-60°C to humidity of 8…10 % and mixed to prepare an end product of chitosan-melanin complex.

EFFECT: invention allows simplifying method with higher yield of chitosan and melanin.

1 dwg, 5 tbl, 5 ex

Description

FIELD OF THE INVENTION

The invention relates to the field of biotechnology and, in particular, to methods for producing a chitosan-melanin complex from the death of bees, which can be used in veterinary medicine for the production of drugs for preventive purposes, in medicine for the prevention and treatment of enteric infections of various origins, and wound healing ointments and as a radioprotector of the body and skin.

State of the art

The raw material for obtaining the chitosan-melanin complex is the death of bees, as well as beetles, flies, which are grown artificially, and other insects containing chitin, which is the basis of the cuticle, and melanin, as well as cocoons and pupae of insects.

Known extraction method for producing chitosan, which consists in the sequential destruction of the structure of the raw material in solutions of caustic soda NaOH and hydrochloric acid HCl concentration of 4% according to the following scheme.

After autofermentolysis, the dried raw material is sequentially subjected to the following processing:

Stage 1 Demineralization with HCl solution.

2 stage. Rinse with distilled water until neutral.

3 stage. Deproteination with NaOH solution.

4 stage. Rinse with distilled water until neutral.

5 stage. Demineralization with HCl solution.

6th stage. Rinse with distilled water until neutral.

7th stage. Drying with warm air t = 55 ... 60 ° С.

The dried mass is chitin from which to obtain chitosan.

8 stage. Deacetylation with NaOH.

9th stage. Drying with warm air t = 55 ... 60 ° С. The dried mass is chitosan.

At the stages of demineralization, deproteination, deacetylation, the processes last up to two hours. The drying process lasts up to 48 hours (see O.Ya. Mezenova, A.S. Lysova, E.V. Grigoryeva, S.M. Vilt. Technology for producing chitin / chitosan from dried gammarus crustacean using autofermentolysis. // Izvestiya Kaliningradskogo GTU. - 2004. - No. 5).

The above method is universal for obtaining chitin / chitosan from chitin-containing raw materials. The use of this technology to obtain chitin / chthiosan from bee subpestilence is undesirable for the following reasons:

firstly, because of its double demineralization (stages 1 and 5), since bee subpestilence is practically free of mineral substances and this treatment is not needed;

secondly, due to unnecessary washing stages (stages 2 and 6), because demineralization stages 1 and 5 are excluded.

It is also irrational to wash the processed raw materials with distilled water (stage 4) after deproteination (stage 3), since melanin dissolved in an alkaline medium is washed out with water. In addition, in the removed NaOH solution, there is melanin, which is not used by this method, and the yield of chitosan according to our experimental data decreases to 6% by weight of the initial subpestilence, which is unacceptable when obtaining a chitosan-melanin complex from bee subpestilence.

Varieties of the basic extraction method are methods for producing chitosan from raw materials with a high content of mineral substances, for example, from crustacean shell (see Patents of the Russian Federation No. 1336291 A1, 6 A23L 1/33; No. 2000066 C, A23L 1/33; No. 211163333 C1, 6 А23L 1/33; No. 2147590 С1, 7 С08В 37/08.), Which also contain processing steps that worsen the properties of chitosan even when it is obtained from crustaceans, and are completely unsuitable for producing chitosan and melanin from dead bees, since they reduce the content of these substances in the final product - chitosan-chalk Ninove complex.

The results of the verification treatment of bee subpestilence according to the basic method and its variants showed that the chitosan content in the final product is more than halved compared to the proposed method, and only trace amounts of no practical value remain in melanin.

A known method of producing chitosan directly from the dry dead bees. Upon a detailed examination of the scorpion, it shows whole, not destroyed bees and various parts of the bees (head, chest, legs, abdomen, wings, etc.). To obtain chitin, a single-stage treatment with 10% alkali solution of NaOH is used, which allows you to separate up to 80% of protein and a large amount of melanins. After removal of the liquid part - alkaline hydrolyzate, the remaining mass - chitin - is washed with plenty of water to pH 7 and dried at a temperature of 55 ± 5 ° C. Dry chitin from dead bees has the appearance of a dark brown mass with a specific smell. Articulate segments of the insect (head, chest, legs and wings in the form of clumped lumps and in the form of separate fragmented parts), as well as individual segments of the abdomen are clearly visible in it. In the bee cuticle, chitin is associated with melanin, and in the process of deproteination, some of the melanin is lost with protein. Of course, some of the melanin remains with chitin, so the resulting product can be considered a chitin-melanin complex. Subsequent treatment - bees chitin deacetylation - is carried out in a 50% NaOH solution at a temperature of 125 ± 5 ° C. In this case, the dissolution of melanins occurs, which color the alkali in black. After treatment of chitin, the solution is drained, and the remainder is washed to pH 7 with wash water. The resulting residue is chitosan. Its brown color indicates a residual melanin content. The resulting product is used to obtain the drug "Pchelosan" (see Nemtsev S.V., Zueva O.Yu., Khismatullin R.G., Khismatullin M.R., Varlamov V.P. Bee as a potential source of chitosan. // Materials 6th International Conference (Schelkovo, Moscow Region, October 22-24, 2001), Center for Bioengineering RAS, 398 pp.).

The disadvantage of this technology is that the processing is performed to kill the bees without their preliminary grinding, which is necessary to destroy parts of the bees and increase the active contact surface of the particles with reagents. As a result of the absence of grinding, parts of the bee are preserved at all stages of processing, and this indicates a shallow processing of raw materials and incomplete separation of protein and other substances. Therefore, to judge the completeness of the processing of raw materials is not possible, which is confirmed by the data given in tables 1, 2, 3 (see link). It is also not possible to judge the quantitative yield of chitosan from bee death by the data given in these tables, since they are reduced to different initial masses.

Another significant drawback is that after separation of the NaOH solution, the valuable melanin product is not released from it. In addition, the use of CO ₂ extraction requires additional equipment, which significantly increases the cost of the process, but does not give the desired result.

Also known is a method of producing chitosan from cuticular shells of insects, which are crushed to particles with a size of 0.2 ... 0.3 mm, treated with a 40% alkali solution at a temperature of 100 ... 110 ° C, a liquid module of 1:20 for 3 ... 3.5 hours with simultaneous exposure to ultrasound with a frequency of 15 ... 35 kHz. The use of ultrasound is due to the fact that the structure of chitin from the external skeleton of insects that do not have mineral salts is notable for its dense packaging. When treated with chemicals, this structure prevents the penetration of reagents deep into the particles of chitin (see Russian Federation Patent No. 2067588 C1, 6 C08B 37/08. Method for producing chitosan).

The disadvantage of this method is that the grinding of raw materials, including cuticle of insects, silkworm pupae without preliminary drying to a grain size of 0.2 ... 0.3 mm is practically not feasible for the following reasons:

firstly, the feedstock, depending on its type, has a certain equilibrium humidity (it is 8-10% for dead bees), which does not allow grinding using conventional methods. The initial mass is not crushed, but smeared, that is, it turns into some soft mass with the preservation of particles of the skeleton of bees, insects, and silkworm cocoons are crushed, but not divided into particles;

secondly, the density of the initial mass is very low, so it is not crushed under the influence of the impact forces of the knives of laboratory mills.

These reasons do not allow grinding material not only to the recommended particle sizes of 0.2 ... 0.3 mm, but also large sizes. The result is some raw, unmilled mass, which, due to its viscosity, reacts poorly to the use of ultrasound, i.e. mass is not destroyed. To solve this problem, an appropriate preliminary drying of the feedstock and an appropriate grinding method as applied to bee death are necessary, which will exclude the use of ultrasound and increase the yield of chitosan. It should be noted that this technology does not provide for the extraction of melanin, which is present in the death of bees.

Closest to the claimed method is a method for producing chitin and a method for producing chitosan from synanthropic flies cultivated on solid organic waste, as well as from the forms of their gradual development. The chitin-containing fraction is obtained by crushing chitin-containing raw materials with separation of the latter into hydrophilic and hydrophobic liquid fractions, followed by obtaining chitin-containing mass. This preparatory part of the method of obtaining the feedstock is characteristic and necessary for the technology for producing chitin-containing feedstock from flies, and when using the feedstock from the death of bees, it is not required.

Common in this technology with the proposed method is that the initial mass of flies has high humidity and requires drying to humidity, allowing for fine grinding. Dead bees also have an initial moisture content and require drying.

Further, according to the technology of the prototype, the obtained dried mass is subjected to fine grinding, loaded into a flask, filled with a 40-50% NaOH solution, preheated to 85-90 ° C, to the ratio of solid and liquid phases T: W = 1: (3- 5) by weight and maintained for 1.5-2 hours at a temperature of 100-110 ° C, stirring, lack of air in the flask to the reaction medium and exposure to ultrasound at a frequency of 20-30 kHz. Then the solution is aspirated, and the precipitate is washed with distilled water until neutral. The washed precipitate is reprocessed in the same way. After treatment, the solution is removed, and the precipitate is washed with distilled water until neutral. The solutions obtained in the treatment of chitin-containing mass are further processed in order to isolate protein, crude fat, sodium chloride (see Russian Federation Patent No. 2139887 C1, 6 C08B 37/08).

Consideration of the method described in the patent, and analysis of its shortcomings, it is rational to begin with the stage of drying and obtaining chitosan.

The first step in producing chitosan is to dry the wet mass. How, under what mode and to what humidity it was carried out in the prototype is not indicated, although this is important, since the second grinding stage indicates a fine grinding. What is the size of the grains or what the ground mass is, is not said again. Further, in the prototype it is indicated that there are 0.5 mm particles in the residue, and this is after repeated treatment with alkali using ultrasound. The conclusion follows: in the prototype, the chitin-containing mass was not dried enough, at least not to the stage of crumbling, and in this case it is impossible to finely grind the mass, you can only grind it a little, but this was not enough during processing and was required for the solution to penetrate apply ultrasound into particles of the mass with its expensive equipment and technology. Unfinished material will not be grinded, but will be spread in paste form over the working bodies of the grinder. And since fine grinding does not occur, further complications arise when exposed to a paste-like mass with reagents, and ultrasound with its expensive equipment is forced to use to intensify the process.

At the stages of processing the chitin-containing mass, one can also note disadvantages, namely: in the method under consideration, the mass was sequentially twice treated with an alkaline solution of the same (45-50%) concentration, although it is known that the reaction first occurs in the surface layer and does not require either an increase in the concentration of the reagent ultrasound applications. With repeated in-depth processing, an increased concentration of the reagent is justified, and the use of ultrasound is forced due to low-quality fine grinding.

A very serious drawback is that in the prototype, as in other methods, absolutely no attention is paid to the allocation of spent solutions of melanin, which is a valuable drug.

It should also be noted that at the stages of obtaining chitosan from a chitin-containing mass, a double treatment with a 45-50% alkali solution is not required, because protein removal (deproteination) was carried out in the preparation stage of the chitin-containing raw material. The use of ultrasound indicates insufficient preliminary drying of the mass and its insufficient grinding, that is, a small active contact surface. In addition, it should be noted that during the preparation, the chitin-containing mass in the method under consideration was treated with various organic solvents: chloroform, ethyl alcohol, etc., which is unacceptable in the preparation of preparations for medical and veterinary purposes, since when using these solvents, the drug acquires a specific smell and taste, which humans and animals react negatively.

Disclosure of invention

The technical result that can be obtained using the present invention is to increase the yield of chitosan obtained from the death of bees, 1.5 ... 2 times and melanin up to 85 ... 92% of its content in bee subpestilence, as well as simplifying the method of producing chitosan -melanine complex due to more rational technology.

The technical result is achieved using a method for producing a chitosan-melanin complex, including drying, grinding of chitin-melanin raw materials, processing the crushed mass with an aqueous alkali solution when heated, washing to pH 7 with wash water, characterized in that the chitin-melanin mass is pre-dried to a moisture content of 4 -6%, ensuring its crumbling, the dried mass is ground to a powder state, deproteinated with 10% NaOH alkali solution at a temperature of 78-82 ° C for 1.5 ... 2.0 hours, after which it is added to the reaction 50% NaOH solution and thus bring its concentration to 30% of the initial one, deacetylate the resulting mass at a temperature of 90 ... 95 ° C for 1.5 ... 2.0 hours, filter, precipitate melanin from the filtered solution by exposure to concentrated hydrochloric acid until the pH of the medium of the treated solution changes to the acidic region and a flocculent precipitate, which is melanin, precipitates, the melanin is separated from the liquid phase by centrifugation, washed, chitosan and melanin are separately dried at a temperature of 50 ... 60 ° С to a humidity of 8 ... 10% and cm Shiva them to produce the final product, melanin chitosan complex.

The essence of the method for producing chitosan-melanin complex from the death of bees is as follows.

The technological process consists of the following successive stages of processing (see drawing):

Stage 1 Preliminary drying of bee death by a stream of warm air (60-82 ° C) to a moisture content of 4-6%, ensuring its crumbling. The degree of drying of dead bees is determined by the organoleptic method, i.e. by compressing subpestilence in the palm of the hand. In this case, the death must crumble.

2 stage. Grinding the dried mass to a size of 0.2-0.3 mm by grinding it to a powdery state in order to increase the contact surface with the reagent.

3 stage. Deproteination of the mass with a 10% NaOH solution at a ratio of 1:10, with constant stirring, t = 78-82 ° C, without air for 1.5-2.0 hours.

4 stage. Deacetylation of the reaction mass is carried out with a 30% NaOH solution, obtained by adding a 50% NaOH solution to the original, at t = 90-95 ° C for 1.5-2.0 hours.

5 stage. Filtering the reacted mass with preservation of the used solution for subsequent precipitation of melanin from it.

6th stage. Rinse the obtained chitosan with distilled water until the washings are neutral.

7th stage. Precipitation of melanin from the solution stored after stage 5 with concentrated hydrochloric acid until the pH of the medium changes to the acidic region and a flocculent precipitate forms.

8 stage. Separation of a flocculent precipitate, which is melanin, by centrifugation followed by washing to a neutral wash water reaction.

9th stage. Separate drying of chitosan and melanin at t = 50-60 ° C to a moisture content of 8-10%.

10 stage. Mixing the obtained chitosan and melanin to obtain the final product of the chitosan-melanin complex.

Thus, these drawbacks that occur in the above methods for producing chitosan from dried crustacean of gammarus, flies, beetles and other chitin-containing raw materials were taken into account when developing a method for producing chitosan-melanin complex from bee death, namely, that:

1. Upon receipt of the chitosan-melanin complex, the feedstock — dead bees — is practically free of minerals. Therefore, there is no need to treat it with a solution of hydrochloric acid Hcl.

2. Drying of dead bees should be carried out to the stage of its crumbling, which is established by a known organoleptic method. The under-mass is not ground. It is smeared on the wall of grinding devices. Therefore, grinding to the stage of crumbling the corpses of bees and their parts is necessary and necessary.

3. Dead bees are light, has a low bulk density. Therefore, the shock method, that is, in grinders with knives, can not be crushed. As a result, in a crushed mass remain in a large number of parts of the corpses of bees. Therefore, dried up to the stage of crumbling dead bees must be crushed in a roller or drum grinder to a powder state. Moreover, the powdered mass ground in this way does not contain distinguishable parts of the corpses of bees and has a large active contact surface with reagents and does not require the use of ultrasound.

Thus, the technology of preparing bee subpestilence for exposure to chemical reagents is greatly simplified, and the quality of the subsequent processing stages is improved, which contributes to a higher yield of both chitosan and melanin from the feedstock.

4. Bee subpestilence, dried to the extent of its grinding and crushed to a powder state, allows you to limit yourself to only one treatment with a solution of 10% alkali NaOH, which also simplifies the process.

5. In the claimed method, in contrast to the prototype and other methods, the NaOH solution is not removed from the treated mass, and the remaining mass is not washed with distilled water, since the next treatment (deacetylation) is also carried out with alkali, only with its concentration of 30% to obtain chitosan, which simplifies technology.

6. In the claimed method, at the end of the deacetylation, the filtered NaOH solution is retained, since it contains melanin.

7. In the inventive method, melanin is precipitated from the stored solution by adding concentrated hydrochloric acid HCl and subsequent precipitation of the precipitate formed by centrifugation.

Thus, the sequential performance in the above order of the above operations not only simplifies the process of obtaining chitosan-melanin complex, but also allows you to increase its output from the initial mass from 12% chitosan to 21 ... 24%, and melanin to 85 ... 92%, as well as more rational technology for its industrial production.

The implementation of the invention

Examples of specific performance of the method for producing chitosan-melanin complex from the death of bees

Example 1

Obtaining a chitosan-melanin complex from raw materials not dried to the degree of crumbling with the separation of the stages of deproteination and deacetylation

Stage 1 Bee morbidity is taken, consisting of whole bees and their parts, trunk, wings, individual parts, when selected from hives and subsequent storage, it has a moisture content of 12 ... 18%, and is crushed.

2 stage. To carry out deproteination, a sample of 5 g of ground mass is taken and poured into a flask with a ground stopper, filled with 10% NaOH alkali solution with a mass ratio of 1:10 and heated to t = 78 ... 82 ° C with constant stirring with a magnetic stirrer for 1.5 ... 2.0 hours.

3 stage. The resulting mass is filtered on a Buchner funnel, the filtered solution is stored for further isolation of melanin from it. The filtered mass is chitin with some melanin in it, as evidenced by its dark brown color.

4 stage. The obtained chitin is washed with distilled water until the pH of the washings is neutral.

5 stage. To carry out deacetylation, the mass of the obtained chitin is poured with a 30% NaOH solution in a ratio of 1:10. The resulting mass is heated to t = 90 ... 95 ° C without access of air with stirring for 1.5 ... 2.0 hours.

6th stage. The mass cooled after deacetylation is filtered on a Buchner funnel, and the filtered solution is preserved.

7th stage. The obtained chitosan with some melanin content in it is washed with distilled water until the pH of the washings is neutral.

8 stage. Precipitation of melanin from the stored solution (p. 3 and p. 6) is carried out with concentrated hydrochloric acid until the pH of the medium changes to the acidic region and a dark flocculent precipitate forms.

9th stage. Separation of precipitated melanin from the solution is carried out by centrifugation, followed by washing to a neutral pH 7 wash water reaction.

10 stage. Drying of chitosan and melanin is carried out simultaneously without mixing at t = 50 ... 60 ° C to a moisture content of 8 ... 10%, which is equilibrium.

11 stage. To obtain the final product of the chitosan-melanin complex, chitosan and melanin are mixed.

The yield of chitosan in this case is 6 ... 8% of the initial mass of the subpestilence, and melanin - 62 ... 69% of its content in the subpestilence.

The yield of chitosan can be increased by applying the initial mass to a degree of crumbling, followed by its finer grinding.

Example 2

Obtaining a chitosan-melanin complex from raw materials, previously dried to the degree of crumbling, followed by finer grinding and separation of the stages of deproteination and deacetylation

Bee subpestilence, consisting of whole bees and their parts, trunk, wings, individual parts, when selected from hives and subsequent storage, has a moisture content of 12 ... 18%.

With such humidity, bee subpestilence is poorly crushed and requires drying up to 4-6% humidity, at which it becomes brittle and easily crushed. Drying is carried out in one of the ways.

Stage 1 The 1st method consists in drying the initial mass, distributed by a layer of 5 ... 10 mm on a baking sheet in an oven at t = 60 ... 82 ° C. The drying time is 2.0 ... 2.5 hours.

According to the 2nd method, the initial mass is placed in a bag of cotton cloth, laid on a grid and distributed in the bag with a layer 2-4 cm thick and blown with an air stream at t = 60 ... 82 ° C. The drying process ends in 15 ... 20 minutes. The degree of drying of the mass is determined by the organoleptic method, that is, by compressing subpestilence in the palm of the hand. In this case, the death must crumble.

Having a low density, bee subpestilence is poorly crushed in mills with knives. Therefore, in laboratory conditions, with a small mass, it is necessary to grind by grinding in a laboratory mortar, and with a large mass - in a drum or cone mill to a powder state with a grain size of 0.2 ... 0.3 mm. The ground mass has a very large active contact surface with reagents, which significantly accelerates the flow of chemical reactions and increases the completeness of processing.

2 stage. To carry out deproteination, a powdered sample weighing 5 g is taken and poured into a flask with a ground stopper, filled with a 10% NaOH alkali solution with a ratio of 1:10 by weight and heated to t = 78-82 ° C with constant stirring with a magnetic stirrer for 1.5 ... 2.0 hours.

3rd and subsequent stages are performed similarly to the corresponding stages from example 1.

In example 2, compared with example 1, the yield of chitosan increased from 6 ... 8% to 13 ... 15% of the initial mass of death, and the yield of melanin remained within 62 ... 69%. The increase in the yield of chitosan in the 2nd example is explained by its more complete extraction from the dead mass, which indicates the necessity of drying the dead dead to the stage of crumbling, providing its fine grinding, which increases the active surface of the mass coming into contact with the reagents.

It is possible to increase the yield of chitosan and melanin by eliminating the intermediate stage 3 washing and combining (in examples 1 and 2) of stages 2 and 4.

Example 3

Obtaining a chitosan-melanin complex from raw materials previously dried to the degree of crumbling, followed by finer grinding and without separation of the stages of deproteination and deacetylation

Stage 1 Drying and grinding of the initial mass is carried out analogously to stage 1 of example 2.

2 stage. Deproteination is carried out similarly to stage 2 of example 2.

Unlike examples 1 and 2, the deacetylation of the mass is carried out without filtering and separating the solution containing melanin in stage 3, as follows.

3 stage. To carry out deacetylation, 50 ml of a 50% NaOH solution are poured into the resulting flask into the same flask, and thus its concentration in the reaction mixture is brought to 30% of the initial one. The resulting mass is heated to t = 90 ... 95 ° C without access of air with stirring for 1.5 ... 2.0 hours.

4 stage. The resulting mass is filtered on a Buchner funnel, the filtered solution is stored for further isolation of melanin from it. The filtered mass is chitosan with some melanin in it, as evidenced by its dark brown color.

5 stage. The resulting chitosan is washed with distilled water until a neutral reaction of wash water pH 7.

6th stage. Precipitation of melanin from the stored solution is carried out with concentrated hydrochloric acid until the pH of the medium changes to the acidic region and a dark flocculent precipitate forms.

7th stage. Separation of precipitated melanin from the solution is carried out by centrifugation, followed by washing to a neutral pH 7 wash water reaction.

8 stage. Drying of chitosan and melanin is carried out simultaneously without mixing at t = 50 ... 60 ° C to a moisture content of 8 ... 10%, which is equilibrium.

9th stage. To obtain the final product of the chitosan-melanin complex, chitosan and melanin are mixed.

Subsequent treatment of chitin to obtain chitosan is carried out according to items 2-8 of Example 1. The ratio of chitin: NaOH solution is 1:10. Precipitation of melanin is carried out from an alkaline hydrolyzate left after receiving chitin and from a solution separated after deacetylation, according to claim 3.

As a result of obtaining by this method, the yield of chitosan is 21-24% of the initial mass of the taken morbidity, and melanin is 85-92% of its initial amount in bee subpestilence.

The increase in the yield of chitosan in the 3rd example from 13 ... 15% to 21 ... 24% and melanin from 62 ... 69% to 85 ... 92% was due to the exclusion of their loss when washing the filtered mass of chitin used in examples 1 and 2 of stage 3 Thus, the claimed technology for producing chitosan-melanin complex, shown in example 3, can be considered the most optimal.

Example 4

Testing chitosan-melanin complex on newborn calves

Drinking newborn calves in the first seven days of life of the chitosan-melanin complex in an amount of 10 (group I) - 15 (group II) mg per 1 kg of live weight contributed to a 28.6% reduction in the incidence of gastrointestinal infections with diarrhea syndrome, and an increase in rates nonspecific resistance - hemoglobin, gamma-globulin levels, lysozyme and bactericidal activity, an average of 16.4% (p <0.01). An analysis of the growth and development of calves revealed that drinking water had a positive effect on the increase in live weight and daily weight gain. The animals of the experimental groups already after the 29-day period had more live weight in relation to the animals of the control group by 3.05 kg (5.6%). In the period from 29 to 61 and from 61 to 120 days, the difference, respectively, was 3.0 kg (4.6%), 5.85 kg (7.6%, p <0.05). Moreover, the average daily gain in the first and third periods was significantly higher (by 24.1 and 21.4%, p <0.01, respectively) in animals of the II experimental group in comparison with the control.

The use of the complex led to a significant decrease in the number of microorganisms of the genus E. Coli, Proteus, Citrobacter by 48.0, 12.1, and 8.3 abs. percent. The results of the tests of the action of the chitosan-melanin complex on the biochemical parameters of blood and intestinal microflora of calves are presented in tables 1 and 2.

table 2
The effect of the use of chitosan-melanin complex on the intestinal microflora of calves Microorganisms of the genus, 10 ⁵ CFU * / g Control Experienced groups at the beginning of the experiment after 29 days I II at the beginning of the experiment after 29 days at the beginning of the experiment after 29 days E.Coli 9.8 7.8 8.7 2.7 10,4 5.9 Proteus 5.2 3.8 5.9 3.6 4.8 2.7 Citrobacter 2.7 1.9 2.9 1.8 2.1 1,6

Example 5

Testing chitosan-melanin complex on pigs during weaning

In order to stimulate the immune processes and prevent dysbacteriosis in piglets during weaning, the chitosan-melanin complex was dosed at a dose of 10 and 15 mg per 1 kg of live weight (I and II and experimental groups, respectively). Analysis of the data revealed that the use of the complex reduced the incidence of piglets from gastrointestinal infections. So, if 31.2 and 25.0% of animals with signs of gastrointestinal tract dysfunction were registered in the experimental groups, then among the piglets of the control group there were 37.5% of them. Moreover, in the experimental groups, the best piglet safety was observed at 6.3 and 12.5% (table 3).

In addition, it was found that the use of the complex contributed to an increase in the protective functions of the body. In individuals of the experimental groups, the level of total protein, gamma globulins, bactericidal and lysozyme activity was higher by an average of 21.8% (p <0.01); 26.3% (p <0.01); 18.2% (p <0.05) and 12.9% (p <0.05) (table 4).

The use of chitosan-melanin complex also had a positive effect on the dynamics of live weight and daily weight gain of piglets. Young animals of the experimental groups on the 30th day after stimulation, compared with the analogues from the control group, had a greater live weight of 3.1% (p <0.05). The trend continued in subsequent periods of ontogenesis: the difference in their benefit at 2 and 3 months of age averaged 2.3 and 4.0% (p <0.05), which, apparently, is due to the large average daily weight gain by 8.0 and 3.3% (p <0.05). The results of this effect are shown in table 5.

The positive effect of chitosan in the prevention of gastrointestinal diseases of young farm animals, apparently, is due to its structural feature, which allows you to create a kind of protective film-substrate on the intestinal mucosa, which prevents the attachment of pathogens to the intestinal villi, absorbs monopolysaccharides, thereby stopping the growth of pathogenic microorganisms, exhibiting bactericidal properties.

Table 3
The effectiveness of chitosan for the prevention of diarrhea in piglets Indicators Groups control I experienced II experienced Ill, goal. 6 5 four % 37.5 31,2 25.0 of them fell, a goal. 3 2 2 Preservation,% 81.2 87.5 93.7 Preventive efficacy, abs. - 6.3 12.5

Table 5
Dynamics of live weight and daily weight gain of piglets using chitosan-melanin complex Group Live weight, kg / Average daily gain, g 30 days 60 days 90 days control 6.37 ± 0.048
401.0 ± 2.16 18.4 ± 0.067
349.5 ± 5.21 28.89 ± 0.17
371.5 ± 4.71 I experienced 6.57 ± 0.056 **
401.1 ± 4.28 18.61 ± 0.13
380.6 ± 8.40 ** 30.17 ± 0.33 **
393.2 ± 5.22 * II experienced 6.56 ± 0.065 *
407.2 ± 2.74 18.69 ± 0.073 **
374.5 ± 6.81 ** 29.92 ± 0.24 **
374.5 ± 5.81 * P <0.05; ** P≤0.01 with respect to control

Thus, summarizing the data of our studies, we can conclude that the use of chitosan-melanin complex at a dose of 10-15 mg per 1 kg of live weight helps to normalize the gastrointestinal microflora, activate immune reactions, and increase the protective properties of the body, which, in its in turn, it has a beneficial effect on the growth and development of young animals.

Claims (1)

A method of producing a chitosan-melanin complex, characterized in that it involves pre-drying the chitin-melanin raw material to a moisture content of 4-6%, ensuring its crumbling, then the dried mass is ground to a powder state, deproteinated with 10% NaOH alkali solution at a temperature of 78- 82 ° C for 1.5-2.0 hours, after which a 50% NaOH solution is added to the reaction mixture and its concentration is thus adjusted to 30% of the initial one, the resulting mass is deacetylated at a temperature of 90-95 ° C for 1.5-2.0 h, filtered, with fil Melted chitosan is obtained by washing the mass with distilled water until pH 7 is reached, and melanin is precipitated from the solution remaining after filtration by exposure to concentrated hydrochloric acid until the pH of the medium being treated is changed into an acidic region and melanin is precipitated in a flocculent precipitate. from the liquid phase by centrifugation, washed, separately dried chitosan and melanin at a temperature of 50-60 ° C to a moisture content of 8-10% and mixed them to obtain the final product THE PRODUCT chitosan-melanin complex.