KR101998583B1 - A composition comprising activated minerals for enhancing immunity of poultry - Google Patents

Abstract

The present invention relates to a composition for enhancing immunity comprising minerals activated by a chelating method, and more particularly, to a composition for enhancing immunity which is obtained by mixing a specific mineral combination with chitosan or amino acid at a predetermined ratio, To a composition for enhancing immunity.
Since the composition of the present invention maintains the solubility and stability of the active mineral at a constant level compared with the conventional immunostimulant, the water absorption of the active mineral is increased more than twice as much as that of the feed supplement by adding it to the drinking water, , Exhibits an immunostimulatory activity through the enhancement of secretion of immunocytokines, and has an excellent effect of improving the productivity by enhancing the growth factors.

Description

[0001] The present invention relates to a composition for enhancing immunity of poultry containing active minerals,

The present invention relates to a composition for immunizing a poultry containing minerals activated by the chelate method.

In the livestock industry, the production efficiency has improved to some extent due to genetic improvement of animals and development of excellent feeds, but various metabolic diseases of animals are increasing, which is a great obstacle to the competitiveness of animal husbandry industry. As the production of livestock becomes a commercialized mass production system in a narrow space, the stress and immune deficiency of animals are also increasing, and the increase in the amount of synthetic diets is also a cause of various diseases as well as immunological degradation of animals . In order to prevent and cure these diseases, animal farmers have been abusing antibiotics. As a result, problems such as the appearance of antibiotic resistant strains and the remaining antibiotics are emerging as social issues. Therefore, environmentally friendly therapeutic agents containing no antibiotics Feed products are constantly being demanded.

In Korea, the effect of copper sources on the productivity of broiler chickens, the effects of copper sources in feed on the productivity of laying hens, microorganisms in the small intestine and the immune system, and the relationship between natural minerals and poultry development and immunity enhancement Has been consistently conducted, and studies on various major diseases and products of various breeds have been continuously conducted overseas.

However, most commercial livestock immune enhancers have a powder form such as vitamins, minerals, and probiotics, and are used for feed addition. Therefore, they have a low water absorption rate compared to the drinking water administration. When they are dissolved in water for drinking water administration, they have low solubility and non- Deterioration, nipple clogging, and the like.

On the other hand, physicochemical and biological studies on minerals, which are one of the five nutrients absolutely necessary for livestock, have been extensively studied in recent years, focusing on livestock feed research. In the past, inorganic minerals have been widely used as animal feed additives However, the utilization rate in the animal body was only 20 ~ 30%, and the rest was excreted in manure, resulting in resource waste and environmental pollution problems. Chelated minerals chelated with organic compounds such as proteins or carbohydrates have been used, and their absorption capacity has been reported to be extremely high (70-80%) (Kratzer and Vohra, 1986). These chelated minerals Various production methods are known (US2877253, US5061815, US5504055, US6458981 B1, US7087775 B2, US7375243 B2, US2008 / 0248583 A1).

Chitosan, a natural substance in nature, has been proven effective as a useful resource by many researchers since its discovery in crustacea. Chitosan is much stronger than dietary fiber in its fat-binding capacity, reducing animal fat absorption and lowering serum cholesterol levels in animal studies to prevent and cure hypercholesterolemia and atherosclerosis (Vahouny et al., 1984; Sugano et al., 1980). Antimicrobial activity, moisturizing and emulsifying stability, and physiological functions of dietary fiber have been attempted for application to high value-added products (Jeon and Kim et al., 2001). However, since chitosan is insoluble in water, it is insoluble in water, and when used in a powder state, it is not easily absorbed in a living body of an animal or human, and thus there is a problem that the effect on the physiological activity of chitosan is insufficient. Therefore, it is required to use chitosan in water for animal feed or food.

In the related art, Patent Document 0001 (KR 10-0470763) discloses a method for producing an amino acid chelate which is electrically neutral and does not generate interfering ions and by-products by reacting a metal carbonate and an acidic amino acid in an aqueous solution , Patent Document 0002 (KR 10-2001-0066982) proposes a method of synthesizing a chitosan-metal chelate substance by chelating zinc, copper or chromium by adding a solution of low molecular weight chitosan dissolved in an organic acid. However, the solidified formulations prepared by the above-mentioned Patent Documents 0001 and 0002 are disadvantageous in that a dispersant, a suspending agent or a stabilizer is additionally required due to low solubility when applied by drinking water administration method. Japanese Patent Application Laid-Open No. 10-0814012 discloses a method for producing chitosan-selenium complex in the form of chitooligosaccharide by binding selenium with chitosan and then decomposing the chitosan with a chitosanase, but this is not applicable to various mineral elements, Patent Document 0004 (KR 10-1150583) discloses a method for producing vanadium water containing an organic vanadium compound at a concentration that is known to be effective for treating diabetes or the like by producing an organic vanadium compound by chelation by reacting an inorganic vanadium compound with an amino acid However, low pH and warming conditions are required in the preparation of the solution, which complicates the manufacturing process and requires a lot of cost and effort.

Korean Patent Registration No. 10-0470763 Korean Patent Publication No. 10-2001-0066982 Korean Patent Publication No. 10-0814012 Korean Patent Registration No. 10-1150583

As a result of continuous efforts to develop an immune enhancer that can reduce the use of antibiotics and improve the therapeutic and / or preventive effects against diseases of poultry such as necrotic diseases, the inventors have found that the combination of certain kinds of minerals Or amino acid, it has high solubility and stability in an aqueous solution, and inhibits the disease of the livestock without promoting cytotoxicity and promotes the growth, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a composition for enhancing immunity which comprises a chitosan-mineral chelate composition and an amino acid-mineral chelate composition as an active ingredient.

Another object of the present invention is to provide a method for producing the immunoconjugate composition.

It is still another object of the present invention to provide a method for using the composition for enhancing immunity in animal husbandry.

In order to achieve the above object, the present invention provides a composition for immunomodulating a chitosan-mineral chelate composition and an amino acid-mineral chelate composition as an effective ingredient.

(A) preparing a chitosan-mineral chelate composition; (b) preparing a lysine-mineral chelate composition; and mixing the respective compositions prepared in said steps (a) and (b) And a step (c) for preparing an immunostimulating composition.

In addition, the present invention provides a method for using the immunoconjugate composition for increasing the body weight gain, increasing the productivity, increasing the immunity, preventing the disease of the poultry, or reducing mortality in the livestock industry.

The composition for enhancing immunity comprising seven minerals and chitosan according to the present invention has the effect of enhancing the immunity of poultry to prevent diseases, reduce mortality and improve productivity by promoting growth factors.

The present invention provides a synergistic effect by synergistically inducing an immune enhancing effect while lowering cytotoxicity by selecting minerals having good chelate reactivity with chitosan and / or a specific amino acid and mixing them at a specific ratio.

Further, by selectively using chitosan having a specific molecular weight, it is possible to maximize the reactivity of the chelate in the aqueous solution and maximize the immune enhancing effect.

According to the preparation method of the present invention, the chelate reactivity and solubility can be increased only by the combination of the two kinds of polymers and the seven kinds of minerals bound thereto and the content ratio specification while maintaining the temperature at a constant temperature.

In addition, the present invention can be applied to the housing continuously and conveniently without the nipple clogging even when added to the drinking water by keeping the solubility and uniformity of the activated mineral complex constant. .

FIG. 1 is a graph showing the results of analysis of cytotoxicity by MTT assay after preparation of different kinds of mineral chelates in order to confirm the combination of mineral components that mitigate cytotoxicity.
FIGS. 2A and 2B are graphs showing the results of measurement of immunocytochemical (IL-2, IL-4, IL-12, IFN-?) Expression in real time PCR to analyze the immunological activity of five samples, .
FIG. 3 shows an automatic administration device necessary for applying the immunoconjugation composition of the present invention to potable water by adding it to potable water.
FIG. 4 is a graph showing the effect of the composition of the present invention on the immunity enhancement of the rabbits by diluting with drinking water at low concentration and high concentration for 33 days, / CD8 ratio. 4A is a control group, FIG. 4B is a low concentration group, and FIG. 4C is a high concentration group.
FIG. 5 is a graph showing the effect of the composition of the present invention on the immunity and cell proliferation of the rabbits by diluting them with drinking water at a low concentration and a high concentration for 33 days, And the concentration of the protein was measured.

The present invention overcomes the conventional conjecture that solid formulations such as vitamins, minerals, plant extracts, and probiotics are used for the purpose of feed addition, and adds a highly concentrated liquid preparation in which organically activated complex minerals are stably and uniformly dissolved to the drinking water Thereby providing a new concept of an auxiliary feed composition for immune augmentation that shows a water absorption rate twice as high as that of water through conventional feeds and has a high stability (solubility, uniformity, etc.) and can maximize application convenience and disease defense efficacy will be.

Further, the present invention is characterized by using the chelate method. When inorganic mineral minerals are surrounded by polymer organic materials and chelated, cells recognize inorganic minerals as organic and increase the absorption rate in the body. The present inventors have unexpectedly found a chelating agent for a low molecular weight buffer and a single metal. In contrast, the present invention provides a new concept of an auxiliary feed composition containing a complex micro-minerals in a polymer to provide a uniform mineral in the body as a drinking water preparation.

On the other hand, the polymer buffer should take into consideration the solubility for the cytotoxicity and the chelate reaction and the bioabsorbability after the reaction, in particular, the solubility and the water absorption depending on the ratio of the mineral and the buffer. Therefore, the present inventors chose chitosan, a natural substance, as a polymer buffer for the chelating reaction, selected minerals having excellent chelate reactivity and solubility with chitosan, selected lysine as an amino acid having excellent reactivity and solubility with minerals, And the combination and blending ratio of the mineral-chelating agent having excellent immunity were examined to complete the present invention.

Accordingly, the present invention relates to a composition containing as an active ingredient a chitosan-mineral chelate composition and an amino acid-mineral chelate composition.

According to a preferred embodiment of the present invention, it is preferable that the chitosan-mineral chelate composition comprises chitosan complexed with minerals composed of cobalt and vanadium.

As used herein, "chitosan" is a polyglucosamine obtained by deacetylation of chitin present in crab shells and the like, which does not cause rejection in the body, and is effective in growth promotion, immunity enhancement, physiological activity, inhibition of pathogenic bacteria growth, It is possible to solve the problem of cytotoxicity of polymer because it has properties of digestive function and detoxification, and can be used together with minerals to synergistically induce the immune enhancement effect.

Generally, the effect of chitosan varies greatly depending on the molecular weight, degree of deacetylation, and degree of water solubilization. In the present invention, the chitosan has a molecular weight of 5,000 to 50,000 Kda. When the molecular weight is small, the immunostimulating effect is lowered and the chelate reactivity with the mineral is lowered. When the molecular weight is too low, the water solubility is limited even when the chitosan is too high. Most preferably, the case of having a molecular weight of 5,000 to 10,000 maximizes the absorption rate and utilization rate in the body. The chitosan of the present invention preferably has a degree of deacetylation of 60 to 80% and most preferably 70% of deacetylation.

Meanwhile, since the composition of the present invention is in a liquid form and is an aqueous solution, the charge or polar functional group is required for the chitosan to dissolve in water in an aqueous solution. However, when absorbed into the body, the chitosan must have lipophilic property to be absorbed well. Specifically, in the case of chitosan, the number of functional groups capable of chelating with minerals is about 30-40. Therefore, it is necessary to control the solubility in water and the absorbability of water by setting a suitable ratio without using all functional groups in consideration of water solubility and fat solubility.

Therefore, in order to select minerals that can enhance the immunity enhancing effect of chitosan in consideration of the solubility and the absorbability of chitosan, zinc, copper, selenium, iron, magnesium, manganese, phosphorus, potassium, calcium, sodium, Molybdenum, cobalt, and so on, in order to search for and select immunopotentiating substances.

In order to determine the amount of minerals synthesized to chitosan, various concentrations of chitosan and chelate reacted with each other. As a result, the content of minerals that could be reacted with chitosan was low, so that vanadium and cobalt Chitosan was chelated and the remaining minerals were further examined for chelate reactivity with amino acids.

According to a preferred embodiment of the present invention, it is preferable that the amino acid-mineral chelate composition of the present invention contains chelated complex minerals including copper, selenium, molybdenum, manganese and zinc in amino acid.

Since the amino acid-mineral chelate differs depending on the structure of the amino acid and the type of the central metal, the conditions such as the optimum reaction temperature, pH, and reaction time are different, and the solubility of the prepared solidifying agent in water also varies depending on the kinds of amino acids and minerals. When the organic minerals are simply mixed, they are not sufficiently dissolved in water, and precipitates may be generated or returned to the inorganic form, which requires the use of a large amount of the mineral preparation or the absorption rate is reduced.

According to a preferred embodiment of the present invention, the chelate reactivity according to the kind of selected minerals (copper, zinc, manganese, iron, molybdenum and selenium) and amino acids (lysine, arginine, methionine, aspartic acid, glycine) From the viewpoint of solubility and the like, the amino acid of the present invention is preferably lysine or glycine, more preferably 5-minerals at room temperature, and lysine having excellent chelate reactivity and solubility.

According to a preferred embodiment of the present invention, the immunopotentiating composition of the present invention preferably comprises a chitosan-mineral chelate composition and a lysine-mineral chelate composition in a weight ratio of 7: 3.

If the mixing ratio of the chitosan-mineral chelate composition and the lysine-mineral chelate composition is out of the above range, the productivity of the livestock animal may be lowered due to the cytotoxicity of the minerals and the polymer, and the synergistic effect of the immunity enhancement due to the combination of chitosan and minerals is lowered There is a problem.

According to a preferred embodiment of the present invention, the immunomodulating composition of the present invention comprises 0.05 to 0.1 parts by weight of chitosan, 0.006 to 0.02 parts by weight of cobalt, 0.003 to 0.01 parts by weight of vanadium, 10 to 15 parts by weight of lysine, 0.8 to 1.5 parts by weight of copper sulfate 5 to 10 parts by weight of zinc sulfate, 0.7 to 1.3 parts by weight of manganese sulfate, 0.01 to 0.025 parts by weight of sodium molybdate and 0.001 to 0.015 parts by weight of selenium oxide.

When the amount of lysine is excessive, the mineral metal ions are not chelated and precipitated. When the amount of lysine is too small, the amount of activated minerals is decreased, and the efficiency of the auxiliary feed is decreased. If the amount of chitosan is excessive, the absorption rate of chitosan is decreased. If the amount of chitosan is too small, the amount of activated minerals is decreased and the bioavailability of minerals is decreased. In addition, when the content ratio of the composition for immunomodulation is out of the above range, the productivity of the animal can be lowered due to the cytotoxicity of minerals and polymers, and the synergistic effect of immunity enhancement due to the combination of chitosan and minerals is lowered.

According to a preferred embodiment of the present invention, it is preferable that the composition for immunostaining of the present invention is in a liquid phase.

According to the present invention, since the solubility, uniformity and stability of the active ingredient are kept constant by using the solution itself in which the complex ion mineral is chelated with chitosan or lysine as a liquid preparation, Can be conveniently applied to the poultry house, and not only is it uniformly mixed with drinking water, but also has a water absorption rate that is twice as high as that of the feed added.

The present invention provides a method for preparing a chitosan-mineral chelate composition comprising the steps of (a) preparing a chitosan-mineral chelate composition, (b) preparing a lysine-mineral chelate composition, and mixing the respective compositions prepared in steps (a) and (c) a method for producing an immunoconjugate composition.

In the method for preparing an immunoconjugate composition of the present invention, the chitosan-mineral chelate composition of the step (a) may include a first step of preparing a chitosan solution by adding chitosan while stirring purified water; A second step of reacting the chitosan solution obtained in the first step with vanadium sulfate and cobalt sulfate sequentially and reacting for 2 to 3 hours to induce metal chelate reaction between the complex ion mineral and chitosan at room temperature; And a third step of taking the upper layer of the solution in which the second step is performed.

According to a preferred production method of the present invention, the organic acid may be added after the second step reaction.

In the case of preparing the chitosan-mineral chelate by further comprising the step of adding the organic acid, the organic acid is added to the chelating reaction-enhanced second-stage solution unlike the conventional case where the organic acid is added in the step of preparing the chitosan solution It is characterized by.

Generally, the reason for adding the organic acid to the chitosan solution is to completely dissolve the chitosan. In the present invention, by reacting the chitosan solution not containing the organic acid and the mineral first, the chitosan can react with the mineral at 20 to 30 The water solubility and the water absorptivity of the water can be controlled by controlling the ratio of the complex mineral and the buffer by using only a moderate degree of functional groups.

According to a preferred embodiment of the present invention, the organic acid may be acetic acid, citric acid or oxalic acid, and citric acid is preferred in terms of dissolving chitosan rapidly and completely.

In the method for preparing an immunoconjugate composition of the present invention, the lysine-mineral chelate composition of step (b) is prepared by adding sodium molybdate, selenium oxide, zinc sulfate, manganese sulfate and copper sulfate sequentially to purified water, A first step of preparing a solution; A second step of introducing lysine into the mineral solution obtained in the first step and then reacting for 2-3 hours to induce metal chelate reaction of the complex ion mineral and lysine; And the third step of taking the upper layer of the solution in which the second step is performed.

In the method for producing an immunoconjugate composition of the present invention, it is preferable that the steps (a), (b) and (c) proceed at room temperature of 24 ± 1 ° C. In particular, when the temperature of the chelate reaction is not maintained at room temperature, the chelate reaction may be reversed or a precipitate may be formed when the liquid composition is applied to drinking water at room temperature. That is, the method of increasing the reactivity of the chelate includes controlling the temperature and the pH, adding the amino acid, and the chelating reaction is preferably carried out at room temperature in order to enhance the stability and solubility of the synthesized chelate in an aqueous solution.

The present invention relates to a method for using the immunoconjugate composition for an increase in weight gain, an increase in feed efficiency, an increase in immunity, and prevention or reduction of mortality of livestock in an animal husbandry industry.

The composition for enhancing immunity according to the present invention may be added to drinking water, or may be added to a feed composition according to each animal, a feed composition commercially available for each animal, or a feed.

The amount of the composition for immune enhancement according to the present invention added to the drinking water is not particularly limited, but is preferably 30,000 to 90,000: 1 per 1 ton of the water supply, and it is preferably added at a rate of 33,000 to 60,000: It is more preferable in terms of improving immunity and productivity without toxicity.

There is no particular limitation on an animal to which the immunostimulant according to the present invention can be fed, for example, a unit animal such as a pig, a chicken, or a rumen such as a cow. However, it is easy to manufacture and supply the immunostimulant to the poultry rather than the other livestock, and in particular, to the chicken such as the broiler or laying hens, is effective in mass production of poultry products in domestic poultry industry, In view of the inevitability of use, it is more preferable in terms of improving the immunity of livestock without using antibiotics and reducing the production cost.

In the EU, since January 2006, the use of antibiotics for growth promotion has been totally prohibited, and thus environmentally friendly feed additives having the same effect as antibiotics have been actively developed. However, most domestic animal immune enhancers (Lactic acid bacteria) or plant extracts. Mineral components are mainly used as a minor component as an auxiliary component of immunity enhancer due to the cytotoxicity problem, or mostly added to feed as a solid preparation, so that the effect of the mineral on immune enhancement is insignificant , And the bioavailability is lowered because the feed is not uniformly compounded.

However, since the present invention has low cytotoxicity, it can be continuously used at a high concentration and at the same time has excellent immunity enhancing effect. In particular, the immune enhancement effect of minerals and chitosan is synergistically induced by harmonizing the reactivity between chitosan and minerals and the bio- And the activated minerals dissolved in the liquid composition are uniformly dissolved in the drinking water and continuously supplied to the poultry, so that they can be effectively used to enhance the immunity of the livestock.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Example 1. Selection of Buffer-Mineral Combinations

(1) Preparation of amino acid-mineral chelate composition

Minerals were added to the purified water sequentially at room temperature and stirred to prepare a mineral solution. Then, lysine was added to induce metal chelating reaction between the complex ion mineral and the amino acid for 2 to 3 hours. After 2 to 3 hours of reaction, the upper layer of the solution was taken to prepare an aqueous solution in which the amino acid-complex mineral chelate was dissolved.

(2) Preparation of chitosan-mineral chelate composition

The chitosan solution was prepared by adding chitosan to the chitosan solution while stirring the purified water at room temperature. The mineral chelate solution was added to the chitosan solution for 2 to 3 hours to induce metal chelate reaction. After 2 to 3 hours of reaction, the upper part of the solution was taken to prepare a brown transparent aqueous solution containing the chitosan-complex mineral chelate.

(3) Preparation of a liquid immune enhancer

The lysine-complex mineral chelate composition and the chitosan-complex mineral chelate composition were mixed at a predetermined ratio at room temperature. After 2 to 3 hours, the upper layer was taken to prepare a liquid immunostimulating composition containing 7 kinds of minerals and chitosan.

ingredient Comparative Example 1-1  Cu-Lysine Comparative Example 1-2  Mn-Lysine Comparative Example 1-3  Zn-Lysine Comparative Example 1-4  (V, Co) - chitosan Comparative Example 1-5  (Cu, Se) -Lysine, (V, Co) - chitosan Comparative Example 1-6  (Mn, Se) -Lysine, (V, Co) - chitosan Comparative Example 1-7  (Zn, Se) -Lysine, (V, Co) - chitosan Comparative Example 1-8  (Cu, Mn, Zn, Se) -Lysine, (V, Co) Comparative Example 1-9  (Cu, Mn, Mo, Zn) -Lysine, (V, Co, Se) Example 1  (Cu, Mn, Mo, Zn, Se) -Lysine, (V, Co)

EXPERIMENTAL EXAMPLE 1. Cytotoxicity Mitigation Experiment of Multicomponent Mineral-Chelate (FIG. 1)

In order to confirm the combination of minerals that mitigate cytotoxicity, MTT assays were performed on a total of 24 samples prepared by different kinds of mineral chelates, and in Comparative Examples 1-1 to 1-9 of Table 1 The results for Example 1 are shown in Fig.

EXPERIMENTAL EXAMPLE 2 Experiments for Enhancing Immunity of Multicomponent Mineral-Chelates (FIGS. 2 (a) and 2 (b)

Comparative examples 3, 6, 8, 9, and 1, which showed effectiveness in cell viability, were analyzed for their immunity by real time PCR. As a result, it was toxic at the concentration of 10 ^-4 , decreased the cytokine expression ability, increased at the concentration of 5 × 10 ^-6 , and showed the highest effect at ¹ × 10 ^-7 . (IL-2, IL-4, IL-12, IFN-γ) expression was increased in comparison with Control at 5 × 10 ^-6 and ¹ × 10 ^-7 concentrations (Fig. 2).

Meanwhile, in order to increase the specific gravity of chitosan, which is an important element of immunity enhancement, the minerals combination of Example 1, in which chitosan-mineral chelate and lysine mineral chelate were mixed at a ratio of 7: 3, Chitosan chelated with selenium was chelated with lysine in Example 1, showing better immunity than in Comparative Example 9, which resulted in the final selection of the mineral combination of Example 1.

Example 2. Preparation of a liquid immune enhancer

A chitosan-complex mineral chelate aqueous solution and a lysine-complex mineral chelate aqueous solution were prepared by the composition shown in the following Table 2 and the method of Example 1, and then mixed at a ratio of 7: 3 to prepare a liquid immune enhancer. The properties of the immunostimulant prepared with the composition of Example 3 are shown in Table 3.

ingredient Example
2-1 Example
2-2 Example
2-3 Example
2-4 Example
2-5 Comparative Example
2-1 Comparative Example
2-2 Comparative Example
2-3 Comparative Example
2-4  Chitosan (chitooligosaccharide) 0.05 0.06 0.07 0.08 0.1 0.04 0.12 0.1 0.1  Lysine hydrochloride (Lysine) 10 10 12 15 15 15 15 9 16 Copper sulfate (CuSO ₄ .5H ₂ O) 15 17 20 20 22 22 22 22 22 Zinc sulfate (ZnSO ₄ .7H ₂ O) 5.0 7.0 7.0 7.0 10 10 10 10 10 Manganese sulfate (MnSO ₄ .H ₂ O) 0.7 1.0 1.0 1.3 1.3 1.3 1.3 1.3 1.3 Cobalt sulfate (CoSO ₄ .7H ₂ O) 0.006 0.01 0.015 0.02 0.02 0.02 0.02 0.02 0.02 Vanadium sulfate (VOSO ₄ .H ₂ O) 0.003 0.005 0.006 0.01 0.01 0.01 0.01 0.01 0.01 Selenium oxide (Na ₂ SeO ₃₎ 0.007 0.01 0.01 0.015 0.015 0.015 0.015 0.015 0.015 Sodium molybdate
(Na ₂ MoO ₄ .2H ₂ O) 0.01 0.02 0.02 0.025 0.025 0.025 0.025 0.025 0.025  Purified water Remainder Remainder Remainder Remainder Remainder Remainder Remainder Remainder Remainder Sum 100 100 100 100 100 100 100 100 100

(Unit: parts by weight)

Immune Enhancer Temperature p H importance Stability 22.6 1.12 1.28 Clear blue liquid

Experimental Example 3. Solubility and stability test

The results of Examples 2-1 to 2-5 and Comparative Examples 2-1 to 2-4 were visually confirmed, and as a result, in Examples 2-1 to 2-5, it was found that, immediately after production and after the elapse of time, But the precipitates were produced in Comparative Examples 2-1 to 2-4.

Example
2-1 Example
2-2 Example
2-3 Example
2-4 Example
2-5 Comparative Example
2-1 Comparative Example
2-2 Comparative Example
2-3 Comparative Example
2-4  color clear
blue clear
blue clear
blue clear
blue clear
blue blue blue blue blue  Appearance Liquid phase Liquid phase Liquid phase Liquid phase Liquid phase Liquid phase Liquid phase Liquid phase Liquid phase  Presence or absence of crystal formation radish radish radish radish radish U U U U

Production Examples. Preparation of Drinking Water with Immunostimulant

Using the automatic dispenser shown in FIG. 3, a drinking water was prepared by adding a liquid immune enhancer per 1 ton of water to a ratio of 60: 1, and for comparison, a drinking water mixed with conventional powder type minerals was prepared Each drinking water was directly applied to the house, and the results were compared and evaluated, and it is shown in Table 5 below.

division Mineral or solidified chelating agents Activated mineral-containing liquid formulation Depending on the type of benefit
Absorption rate - The majority of feed diets
- Low Absorption Rate  - Maximization of absorption rate by applying drinking water Ease of Use and Persistence - Intermittent drinking water administration
- Nipple clogging, low persistence -365 days continuous administration
- Pay in proportion to water usage
- Easy and quick convenience of applying whole house For water
Solubility - low solubility in water
- Cause excessive use  - Excellent solubility and homogeneity at room temperature using liquid formulations in which activated minerals are dissolved.
Activation - Mostly deposited on the walls and floors to be treated.
- Jonny phenomenon occurred  - High solubility and storage stability in water and high activity of minerals Uniformity  - Mix uniformly to feed  Maintain uniformity regardless of -pH and temperature conditions

Bioabsorption rate - In the case of inorganic minerals, the bioavailability is low due to absorption method, absorption site, intestinal pH, and binding with anion materials
- Organic form of minerals has low solubility in water and low bioavailability - active absorption, diffusion absorption, absorption rate is high
- After passing through the stomach without extra ionization process, it is absorbed in the small intestine and absorbed into the plasma through the cell membrane.
- Less affected by intestinal pH or other obstructing substances

From the above results, the immune enhancer prepared from the activated mineral as a liquid has a water absorption rate twice as high as the water absorption rate through the use of drinking water, and shows a higher absorption rate than the conventional mineral powder when applied to drinking water, Absorption power and convenience of application.

Experimental Example 4 Breeding and Specification of Experimental Animals

The efficacy of the immune enhancer was evaluated in the actual broiler farm (3 breeding, more than 2.5 million breeding). In order to evaluate the efficacy accurately, all the factors (dietary habitat, breeding condition, chick quality, etc.) were controlled in the same way as the control diet, low density diet and high density diet, The animals were housed for 33 days, and disease incidence and health status were checked at 10-day intervals on the basis of the autopsy, and the body weight of each group was measured. (IGF-I, IL-2, IL-4, IL-12, and IFN-γ) and cytokine PBMC assays (CD3, CD4, and CD8) were used to evaluate productivity and immunogenicity.

Group Dosing substance Route of administration Duration of administration Inspection day 1 group Control group Drinking water administration 33 days Shipping 2 group Low concentration group (60,000: 1 dilution) 3 group High concentration group (33,000: 1 dilution)

The cultivar used in this study was broiler broiler, and the experimental farm was a broiler farm of 150,000 scale and experimented with 3 cultivars. As a result of continuous monitoring of the farm until the age of 33 weeks before the experiment, there were no abnormal findings in the mortality and autopsy findings of the chicken.

(1) specification management

The cage operation system was irradiated with the same three consenting unchanged cages, and the ventilation system and humidity were also applied in the same way, and the same products of the same feed company were fed. The salary system was managed by putting it in a large feed box.

(2) Sampling

Since serum enzymes (AST, LDH, CK, etc.) are affected by storage conditions, the blood collected from the farm is separated directly from the farm and transferred to the laboratory on the day to measure serum enzymes. Blood was randomly selected from each group and 30 mice were sampled from 10:00 am to 11:00 am. The serum was centrifuged at 2,500 rpm for 20 min. The separated serum was carried in an ice box and the temperature at the time of blood sampling was 25 ° C.

Experimental Example 5. Identification of efficacy as an auxiliary feed

The results of evaluating the efficacy of the composition of the present invention in the breeding farms (three dogs, breeding of more than 2.5 million) are shown in Table 6 below. In the low concentration group, the composition of Example 3 was added at a ratio of 60,000: 1 per 1 ton of feed water, and the high concentration group was added at a ratio of 33,000: 1 for 33 days in broiler chickens, Production rate, and growth rate.

division Control group
(No additives) Low concentration group High concentration group Remarks  Number of pieces (grain) 32,000 27,000 31,000  Number of shipment (horses) 29,230 23,580 28,408  Average weight (g) 1,376 1,406 1,562  Weight gain (g) 1,339 1,369 1,525 Shipment Average Weight - Average Weight Received  Total drinking water (L) 88,803 61,492 94,580  Average drinking water per cattle (L) 2.78 2.28 3.05  Feed intake (kg) 63,359.6 51,112.5 61,577.8  Feed rate 1.62 1.58 1.42 Feed intake ÷ Weight gain  Fostering rate (%) 91.34 87.33 91.64 (Number of shipment ÷ quantity) × 100  Production index 258.81 258.5 335.68 [(Shipment rate (%) x average weight (g)) / (age x feed demand rate)] / 10

Compared with the control group (non - additive group), the weight gain and the amount of water increased and the feed conversion ratio increased and the production index increased. Especially, the highest effect was obtained in the high concentration added group.

Experimental Example 6. Growth Rate Analysis according to Breeding Period

The body weight was measured at 2, 11, 19, 28, and 32 days of age for the growth rate by administering the immune enhancer-added drinking water at different concentrations for 33 days.

Weight (g) Breeding day 2 11 19 28 32 Control group 35.90 + - 6.76 172.67 + 49.87 516.12 + - 79.86 1,446.19 ± 215.25 1,970.34 ± 291.98 Low concentration group 35.45 ± 6.34 199.34 ± .52.98 504.198 + - 80.24 1,576.78 ± 245.56 2,009.85 ± 313.98 High concentration group 35.02 + - 6.23 214.98 ± 60.67 586.119 ± 57.90 1,710.62 ± 264.76 2,100.09 + - 319.56

As shown in Table 8, no significant difference in body weight gain was observed at 2 and 11 days of age, but the high concentration group started to increase significantly at the 19th day of broth compared with the control group. At 28 and 32 days of age, (P <0.05) compared to the control group.

EXPERIMENTAL EXAMPLE 7. Confirmation of Enhancement of Immune Enhancement and Growth Factor

(1) PBMC analysis (Figs. 4A to 4C)

Peripheral blood mononuclear cells (PBMCs) are cells with spherical nuclei present in the blood, including T cells, B cells, NK cells, monocytes and DCs. The method of measuring the proliferative capacity of such PBMCs is the most widely used method of evaluating the immune system. B cells and T cells of the lymphocytes directly involved in immunity have antibodies (CD19, CD3, CD4 and CD8) on their surface (C.H. Chen, 1994; Fair, Taylor McCabe, Shou, & Marrone, 2008). By analyzing the number and ratio of these antibodies, the immunological ability of the chicken can be clarified more clearly. Therefore, in the present invention, broiler broilers were divided into three groups, and then 10 blood, spleen and thymus were collected at randomly at 33 days, PBMC were separated from them, and CD3, CD4 and CD8 , CD8 and CD4 / CD8 ratios were analyzed. Analysis was performed using a flow cytometer (BD Accuri C6, MI, USA) and the results were expressed as% in FSC / SSC.

CD3, CD4, CD8 and CD4 / CD8 ratios of PBMCs isolated from the blood are shown in Table 9 below. In the blood, CD3, CD4 and CD8 in the control group were 28.72 ± 17.12, 45.8 ± 21.09 and 23.92 ± 12.91% And 24.12 ± 12.68% in the low concentration group, and 27.78 ± 19.60, 64.14 ± 25.99 and 24.01 ± 14.18% in the high concentration group, respectively. According to the results, the ratio of CD4 / CD8 in the blood was 1.91 ± 0.61 in the control group, 2.16 ± 0.54 in the low concentration group and 2.67 ± 0.61 in the high concentration group, and the concentration of CD4 / CD8 in the blood was increased depending on the concentration. Especially, (P <0.05), respectively.

division CD4 and CD8 defined subset (%) of CD3 PBMC CD3 CD4 CD8 CD4 / CD8 Control 28.72 ± 17.12 45.8 ± 21.09 23.92 ± 12.91 1.91 + - 0.61 Low concentration group 24.6 ± 18.6 52.14 + - 22.85 24.12 ± 12.68 2.16 ± 0.54 High concentration group 27.78 ± 19.6 64.14 ± 25.99 24.01 + - 14.18 2.67 ± 0.61

(2) Measurement of serum IgG, IGF-I, GH protein (FIG. 5)

Serum samples were collected at the 33rd day of broiler chickens and analyzed for IgG (IgG) using IgG Elisa kit (Bethyl laboratories Inc.TX, USA). IGF-I and GH hormone) was measured using a commercially available ELISA kit (Elabscience Bioscience Inc, USA). After 100 μl of the sample was added to the well plate coated with the antibody by sandwich-ELISA assay, 100 μl of the supernatant was removed, and the biotinylated detection antibody and HRP conjugate sol. After addition, 450 nm was measured using an ELISA reader (BioTek synergy 2, VT, USA). Intra-extra variation coefficient was <10%, and each sample was repeated 3 times. B-cell and T-cell activity and proliferative activity have a direct influence on chicken immunity enhancement and inhibition. These cells are the cells responsible for humoral immunity and cellular immunity. First, the concentration of IgG (Immunoglolubin G) in blood was measured to confirm the change of humoral immune activity. As a result, serum IgG concentration was significantly increased in the low concentration group (3.88 ± 0.56 mg / ml) and the high concentration group (3.97 ± 0.57 mg / ml) as compared with the control group (3.48 ± 0.65 mg / ml). The IGF-I and GH levels in the plasma were increased in the low and high concentration groups compared to the control group, and the concentration of IGF-I and GH in the high concentration group was significantly increased (Fig. 5).

Control group Low concentration group High concentration group IgG (mg / ml) 3.48 ± 0.65 3.88 ± 0.56 * 3.97 ± 0.57 ** IGF-I mRNA espression
(arbitrary unit) 3,412.92 + 1.047.9 4,377.03 ± 1,760.16 * 6,509.48 ± 1,902.69 ** GH (ng / ml) 2.61 0.70 2.78 ± 0.89 3.10 ± 0.96 *

* The means were significantly different form those of control (p <0.05),

** The means are significantly different forms of control (p <0.01), values represent the mean ± SD, n = 30.

(3) Immunocytocin analysis in blood

In order to analyze the immune cytokines in blood, 33-day-old broiler blood was collected and primers for each cytokine were prepared (Wang et al., 2006). Although the immunological activity of poultry is directly affected by the activities and proliferative activities of B cells and T cells, secretion of IL-2, IL-4, INF-γ and IL-12 is also essential for proliferation of these cells.

IL-2 (Interleukin-2) is actually synthesized during T cell activation and is known to have a profound effect on the activity of active T cells (Lillehoj et al., 2001). IL-2 is a T cell growth factor that converts T cells from G1 to S phase, and is produced by CD4-T cells. It is a 14-17 kDa glycoprotein that functions as an autocrine or paracrine factor. It acts on NK cells to promote growth, enhance the killing ability, and act on lymphokine activated killer (LAK) and B cell to promote its growth Adair, 2000). Serum IL-2 concentrations were 40.96 ± 18.69, 53.26 ± 27.99, and 60.67 ± 22.49 pg / ml, respectively, which were higher than the control group.

IFN-γ acts on T cells, B cells, neutrophils, NK cells, and vascular endothelial cells to activate them and acts as a macrophage activating factor, resulting in Class I and II Increase MHC expression and inhibit virus proliferation like other interferons (Peter Staehell, 2001). Serum IFN-γ concentrations were 230.35 ± 132.83, 525.35 ± 198.70 and 642.12 ± 207.24 pg / ml, respectively, which were higher than the control group.

IL-4 (interleukin 4) is a cytokine that induces Th2 cell differentiation of naive helper T cells, stimulates cell growth of activated B cells and T cells, and differentiates B cells into plasma cells It has many biological functions (Brown, 1997). In addition, IL-4 is a 20 kDa protein produced by CD4 T cells and activated mast cells. It acts as a B cell growth factor and can act as a differentiation factor involved in class switching of immunoglobulins of B cells. CD4 T cell, mast cell, and macrophage (Lumeng, Bodzin, & Saltiel, 2007). The serum levels of IL-4 were 138.54 ± 58.94, 171.54 ± 66.39 and 181.63 ± 93.97 pg / ml, respectively, which was significantly higher than the control group (Table 9, p <0.05).

IL-12 is a cytokine composed of p35 and p40 proteins and has a very important immunological function that links innate immunity and adaptive immunity (Degen, van Daal, van Zuilekom, Burnside, & Schijns, 2004). The serum levels of IL-12 were 63.04 ± 27.31, 82.24 ± 35.68, and 103.17 ± 37.14 pg / ml, respectively (Table 10, p <0.01).

Control group Low concentration group High concentration group IL-2 (pg / ml) 40.96 ± 18.69 53.25 ± 27.99 60.67 + - 22.49 * IFN-gamma (pg / ml) 230.35 + 132.83 525.35 ± 198.70 * 642.12 + - 207.24 ** IL-4 (pg / ml) 138.54 ± 58.94 171.54 + - 66.39 181.63 + - 93.97 * IL-12 (pg / ml) 63.04 ± 27.31 82.24 ± 35.68 103.17 ± 37.14 **

* The means were significantly different form those of control (p <0.05),

** The means are significantly different forms of control (p <0.01), values represent the mean ± SD, n = 30.

(4) Statistical processing

All statistical analyzes were performed using SPSS. Student t-test was used to compare the mean values of the groups with a significance level of 0.01 or less (p <0.05). In particular, all samples were run in triplicate.

Experimental Example 8. Confirmation of chelate reactivity according to kinds of amino acids

Chelates of minerals (Cu, Zn, Mn, Mo, Se) and amino acids (lysine, arginine, methionine, aspartic acid and glycine) were selected to make highly concentrated organic minerals. Reactivity, and solubility in water. When the chelate formation was not good, the concentration of aqueous solution was investigated by addition of purified water or by controlling the temperature. Finally, the presence of the extract was confirmed by pH and filtration. Mineral standard 2 equivalents of amino acids were added, but 2.01 equivalent was added to compensate for minerals. The information on the minerals and amino acids used are shown in Table 12 below. As a result of performing various chelating reactions, the maximum concentrations of amino acids and various trace minerals capable of water-soluble states are summarized in Table 13 below.

division Molecular formula Molecular Weight Mineral atomic weight mineral Na ₂ MoO ₄ 2H ₂ O 241.95 95.94 ZnSO ₄ 7H ₂ O 287.56 65.38  ZnO 81.38 65.38 CuSO ₄ 5H ₂ O 249.18 63.54 MnSO ₄ H ₂ O 169.01 54.9 Na ₂ SeO ₃ 172.94 78.96 amino acid  Lysine 182.65 -  Arginine 174.2 -  Methionine 149.21 -  Aspartic acid 133.1 -  Glycine 75.07 -

Cu
(ppm) Zn
(ppm) Mn
(ppm) Mo
(ppm) Se
(ppm) Lysine 100,000 160,000 130,000 100,000 25,000 pH  1.80  3.70  4.36  7.58  7 to 8 Arginine 3,300 X X 50,000 50,000 pH 7 to 8 7 to 8 -  7 to 8 7 Methionine 5,000 6,000 5,000 7,500 pH -  3 to 4  4 to 5  7 to 8  7 to 8 Aspartic acid X X X 2,500 2,500 pH - - - 3 to 4 3 to 4 Glycine 100,000 100,000 25,000 50,000 50,000 pH 2 to 3 2 to 3 4 to 5 7 to 8 7 to 8

X: 1000ppm or less

As a result of the experiment using 5 kinds of amino acids and 5 kinds of minerals, arginine showed weak chelate reactivity with Zn and Mn, methionine showed little chelate reactivity with copper, and aspartic acid chelated only with Mo and Se . According to the experimental results, the order of the water soluble chelates of minerals and amino acids was best in order of lysine> glycine> methionine> arginine> aspartic acid, and lysine was most reactive with five mineral combinations selected at room temperature.

Claims (11)

delete delete delete delete delete delete delete (A) preparing a chitosan-mineral chelate composition;
(B) and (c) of preparing a lysine-mineral chelate composition,
A method for preparing an immunoconjugate composition, comprising the step (c) of mixing the respective compositions prepared in steps (a) and (b)
The step (a)
A first step of preparing a chitosan solution by adding chitosan while stirring the purified water;
A second step of sequentially introducing vanadium sulfate and cobalt sulfate into the chitosan solution obtained in the first step to induce metal chelate reaction between the complex ionic mineral and chitosan at room temperature; And
And a third step of taking the upper layer of the solution in which the second step is performed. (A) preparing a chitosan-mineral chelate composition;
(B) and (c) of preparing a lysine-mineral chelate composition,
A method for preparing an immunoconjugate composition, comprising the step (c) of mixing the respective compositions prepared in steps (a) and (b)
The step (b)
A first step of adding sodium molybdate, copper sulfate, zinc sulfate, manganese sulfate, and selenium oxide to the purified water sequentially and stirring to prepare a mineral solution;
A second step of introducing lysine into the mineral solution obtained in the first step to induce metal chelate reaction of the complex ion mineral and lysine and
And a third step of taking the upper layer of the solution in which the second step is performed. The method according to claim 8 or 9, wherein the steps (a), (b) and (c) proceed at a temperature of 24 ± 1 ° C. delete

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