KR102541470B1 - Drinking additive for companion animals using mushroom-derived vegetable chitosan and its manufacturing method - Google Patents

Abstract

The present invention comprises the steps of a) mixing and stirring mushroom-derived chitosan in distilled water and adding an acid to create a first mixture in which the mushroom-derived chitosan is dissolved; b) mixing a basic aqueous solution with the first mixture to produce a second mixture having a pH adjusted to 5.0 to 7.0; c) mixing a functional additive with the second mixture to produce a third mixture; it relates to a method for manufacturing a negative additive for livestock and companion animals using chitosan derived from mushrooms, characterized in that it is produced including.

Description

Drinking additive for companion animals using mushroom-derived vegetable chitosan and its manufacturing method}

The present invention provides drinking water for livestock and companion animals using mushroom-derived chitosan, which can prevent viral and bacterial disease infections and create a deodorizing effect by feeding livestock and companion animals a negative water additive prepared using mushroom-derived chitosan. It relates to additives and their manufacturing methods.

Chitosan is the only natural product that has an active amino group and exhibits positive ions, so it can easily attach or adsorb bacteria and viruses that have negative ions.

Chitosan is obtained by deacetylating chitin obtained by immersing keratin of crustaceans such as crab, shrimp, and squid in acid to remove calcium components and then immersing in caustic soda to remove proteins. This is called animal chitosan.

Korean Patent Publication No. 10-0314958, Korean Patent Publication No. 10-2000139, and Korean Patent Registration Publication No. 10-2000139 have devised a technique for producing nutrients or supplementary feeds for laying hens and broilers using the animal chitosan. .

However, the above technology has the disadvantage of inducing indigestion of livestock and companion animals due to the characteristics of animal chitosan, which dissolves in acid and is swelled by a large molecular weight ranging from 20,000 to 500,000 kDa. , there was a problem that led to poor fattening.

In addition, when animal-based chitosan is used to produce nutrients for laying hens and broilers, virus inactivation tests must be performed in the nutrient processing step due to concerns about zoonotic virus infection or animal-animal infection. trouble caused

In addition, there was a problem in that the quality of nutrients for laying hens and broilers fluctuated according to the collection time of fish resources, which are raw materials of animal chitosan.

Accordingly, while fundamentally solving the problems of animal chitosan, which can be used as a nutritional supplement and supplementary feed for livestock and companion animals, it improves the suitability of chitosan as a feed additive and additive for drinking water, safely and effectively exhibiting various physical properties of chitosan. There is a need to develop technologies that can be utilized.

[Prior Document 1] Korean Registered Patent Publication No. 10-0314958 (2001.11.05) [Prior Document 2] Korean Patent Publication No. 10-2002-0031144 (2002.04.26.) [Prior Document 3] Korea Patent Registration No. 10-2000139 (2019.07.09)

The present invention provides a negative additive for livestock and companion animals using mushroom-derived chitosan, which enables prevention of viral and bacterial disease infection and deodorizing effect by feeding a negative additive prepared using mushroom-derived chitosan, and a method for manufacturing the same is intended to provide

The method for producing a negative additive for livestock and companion animals of the present invention includes the steps of a) mixing and stirring mushroom-derived chitosan in distilled water, and then adding an acid to produce a first mixture in which the mushroom-derived chitosan is dissolved; b) mixing a basic aqueous solution with the first mixture to produce a second mixture having a pH adjusted to 5.0 to 7.0; c) generating a third mixture by mixing a functional additive with the second mixture; characterized in that it comprises a.

In addition, the manufacturing method of the negative additive for livestock and companion animals of the present invention is characterized in that it further comprises; d) freeze-drying the third mixture to prepare a sponge-like dry product.

In addition, the manufacturing method of the negative additive for livestock and companion animals of the present invention is characterized in that it further comprises; e) after crushing the dry matter, mixing the sub-materials and tableting them in the form of tablets.

The above mushrooms are stone ear mushrooms, shiitake mushrooms, chaga mushrooms, wood ear mushrooms, enoki mushrooms, bare mushrooms, Agaricus mushrooms, Ganoderma lucidum mushrooms, king oyster mushrooms, situation mushrooms, cauliflower mushrooms, jujube mushrooms, maitake mushrooms, fingering mushrooms, ericium erinaceus mushrooms, It is characterized in that it includes at least one selected from the group consisting of longevity mushrooms, oyster mushrooms, spirit mushrooms, mulberry mushrooms, and tile mushrooms.

The chitosan of step a) is carboxymethyl chitosan, carboxymethyl chitosan, carboxyethyl chitosan, cyanoethyl chitosan, hydroxyethyl chitosan, hydroxypropyl chitosan, hydroxypropyl chitosan, glyceryl chitosan, glycol chitosan, succinyl chitosan, It is characterized in that it comprises at least one selected from the group consisting of carboxymethyl chitosan succinimide.

The functional additives in step c) include 5 to 15% by weight of xylitol, 3 to 15% by weight of bovine extract, 3 to 15% by weight of propolis, 2 to 10% by weight of mushroom extract, 5 to 15% by weight of Schisandra chinensis extract, 40% by weight of complex minerals It is characterized in that it contains ~ 50% by weight as an active ingredient.

The mushroom extract is characterized by mixing a stone mushroom extract and a zinnia mushroom extract in a weight ratio of 1:1.

It is characterized by providing a negative additive for livestock and companion animals using chitosan derived from mushrooms prepared by the above-mentioned manufacturing method.

The present invention has the effect of creating a deodorizing effect and preventing viral and bacterial disease infections by feeding a negative additive prepared using mushroom-derived chitosan.

1 shows a method for producing a negative additive for livestock and companion animals using chitosan derived from mushrooms according to the present invention.
Figure 2 shows an example of the use of a negative additive for livestock and companion animals prepared in the form of a sponge, which is another embodiment of the present invention.
Figure 3 shows an antibacterial activity test of mushroom-derived chitosan used in a negative additive for livestock and companion animals using the mushroom-derived chitosan of the present invention.

In order to fully understand the present invention, it will be described with reference to preferred embodiments of the present invention. Embodiments of the present invention may be modified in various forms, and the scope of the present invention should not be construed as being limited to the examples described in detail below. This embodiment is provided to more completely explain the present invention to those skilled in the art.

The present invention comprises the steps of a) mixing and stirring mushroom-derived chitosan in distilled water and adding an acid to create a first mixture in which the mushroom-derived chitosan is dissolved; b) mixing a basic aqueous solution with the first mixture to produce a second mixture having a pH adjusted to 5.0 to 7.0; c) mixing a functional additive with the second mixture to produce a third mixture; provides a method for manufacturing a negative additive for livestock and companion animals using chitosan derived from mushrooms, characterized in that produced including.

In step a), the mushroom-derived chitosan corresponds to vegetable chitosan, which is different from animal chitosan obtained from crustaceans such as crabs and shrimps. In other words, it is a kind of aminopolysaccharide obtained by deacetylating chitin that exists in nature, such as mushroom mycelium and cell walls of microorganisms, and contains β-glucan with immunity enhancement and anticancer activity, and has excellent antibacterial and antiviral properties. In addition to exhibiting antifungal, antifungal and deodorizing properties, it is easily decomposed in the body compared to animal-derived chitosan, leaving no residue, so it is less likely to cause allergies and side effects, so it can be safely used as drinking water for livestock and companion animals.

The mushroom-derived chitosan can be obtained from mushrooms or fungi by a method known in the art, and more specifically, it can be prepared by heating an alkaline aqueous solution of mushrooms, followed by separation, washing, and drying. At this time, the mushrooms are stone ear mushrooms, shiitake mushrooms, chaga mushrooms, wood ears mushrooms, enoki mushrooms, bare strand mushrooms, Agaricus mushrooms, Ganoderma lucidum mushrooms, king oyster mushrooms, situation mushrooms, cauliflower mushrooms, jujube mushrooms, maitake mushrooms, fingernail mushrooms, erinaceus It may be mushrooms, longevity mushrooms, oyster mushrooms, spirit mushrooms, mulberry mushrooms, and tile mushrooms, but is not limited thereto.

The molecular weight of the mushroom-derived chitosan in step a) may be 200 to 30,000 kDa. Since the vegetable chitosan exists in nature in the form of a polymer, it is difficult to obtain a molecular weight of less than 200 kDa in the manufacturing process, and when the molecular weight exceeds 30,000 kDa, it is not easily decomposed and may remain as a residue in the body. It is undesirable because it causes allergies and side effects. Accordingly, in detail, the molecular weight of vegetable chitosan may be 300 to 15,000 kDa, and more specifically, 300 to 2,000 kDa.

The chitosan of step a) is carboxymethyl chitosan, carboxymethyl chitosan, carboxyethyl chitosan, cyanoethyl chitosan, hydroxyethyl chitosan, hydroxypropyl chitosan, hydroxypropyl chitosan, glyceryl chitosan, glycol chitosan, succinyl chitosan, It is characterized in that it comprises at least one selected from the group consisting of carboxymethyl chitosan succinimide. More preferably, carboxymethyl chitosan may be used. The carboxymethylchitosan can be dissolved in distilled water in a wide pH range, and the aggregation characteristics of chitosan can be minimized, so that the aggregation rate can be alleviated even after the basic aqueous solution is added after step a).

The acid of step a) is at least one selected from the group consisting of hydrochloric acid, nitric acid, sulfuric acid, acetic acid, tartaric acid, salicylic acid, citric acid, hydroxy acetic acid, lingic acid, propionic acid, lactic acid, glyceric acid, ascorbic acid, adipic acid, and malic acid The above solution may be diluted in distilled water, but most preferably, it may be ascorbic acid exhibiting immunity-inducing activity.

The first mixture prepared in step a) may be produced by mixing 0.003 to 3.0 parts by weight of acid and 0.003 to 3.0 parts by weight of mushroom-derived chitosan based on 100 parts by weight of distilled water. If the mushroom-derived chitosan is less than 0.003 based on 100 parts by weight of the distilled water, it is difficult to obtain the desired effects in the present invention, such as antibacterial, antiviral, immune and deodorizing, and if it exceeds 3 parts by weight, the growth of beneficial bacteria such as lactic acid bacteria as well as harmful bacteria And it may have a negative effect on survival, difficulty in handling may be caused due to high viscosity, and problems such as increase in manufacturing cost may occur, which is undesirable.

In addition, relative to 100 parts by weight of distilled water, if the acid is less than 0.003 parts by weight, there is a concern that the mushroom-derived chitosan may not be sufficiently dissolved, and if the acid exceeds 0.003 parts by weight, it may cause allergies and side effects, which is not preferable.

On the other hand, since mushroom-derived chitosan has poor solubility in distilled water, acid is added to the dispersion in which the distilled water and mushroom-derived chitosan are mixed and stirred.

However, when mushroom-derived chitosan is added after mixing the acid with distilled water, the mushroom-derived chitosan is instantaneously gelled and the solubility decreases, so that the preparation time for the first mixture in which the mushroom-derived chitosan is dissolved increases. there is. Accordingly, it is preferable to first mix and stir the mushroom-derived chitosan in distilled water, and then add an acid to produce a first mixture in which the mushroom-derived chitosan is dissolved.

More preferably, the first mixture is mixed and stirred for 2 to 10 minutes after adding 0.003 to 3.0 parts by weight of mushroom-derived chitosan based on 100 parts by weight of the distilled water at a temperature of 20 to 30 ° C. After relatively uniformly dispersing in distilled water, 0.003 to 3.0 parts by weight of an acid is added and stirred for 10 minutes to 30 minutes. Outside of the above temperature and time conditions, acid or mushroom-derived chitosan may not be sufficiently dissolved or efficiency may decrease in the manufacturing process, which is not preferable.

Meanwhile, in step b), a basic aqueous solution may be added in an appropriate amount to adjust the pH of the first mixture.

In this case, the basic aqueous solution of step b) may be a solution obtained by diluting at least one solution selected from the group consisting of sodium hydroxide, ammonium hydroxide, potassium hydroxide, and hydroxyphosphate in distilled water. It is preferable to adjust the pH level of the second mixture to be 5.0 to 7.0 so that mushroom-derived chitosan can exhibit the most suitable activity effect. More preferably, 0.01 to 0.5 parts by weight of hydroxyphosphate is added to 100 parts by weight of the distilled water to adjust the pH to 5.5 to 7.0.

It is preferred that the first mixture produced by step a) is mixed with an aqueous basic solution to produce a second mixture whose pH is adjusted to between 5.0 and 7.0.

On the other hand, the functional additives in step c) include 5 to 15% by weight of xylitol, 3 to 15% by weight of bovine extract, 3 to 15% by weight of propolis, 2 to 10% by weight of mushroom extract, 5 to 15% by weight of Schisandra chinensis extract, complex 40 to 50% by weight of minerals may be included as active ingredients.

The xylitol can be used as a natural sweetener, has a high pellicle formation inhibitory effect, and can create a calculus formation inhibitory effect. When the amount of xylitol is less than 5% by weight, pharmacological activity by xylitol may be insignificant, and when it is contained in more than 15% by weight, there is no benefit due to the excess content, and thus economic feasibility may be reduced.

The bovine extract and propolis not only have antibacterial, antiviral and immune effects, but can also act as natural antibiotics, solving residual problems and resistance problems that may occur during feeding or drinking water containing antibiotics. , stable growth of livestock or companion animals can be expected. In addition, in the case of livestock, there is an effect of improving meat quality compared to livestock fed with feed or drinking water mixed with general negative additives. When the subgye (Jobaengi) extract and propolis are contained at less than 5% by weight, there is a concern that the antibacterial, antiviral and immune effects of the subgye (Jobaengi) extract and propolis may be insignificant, and the content in excess of 15% by weight In this case, there is no benefit according to the excess content, so economic efficiency may be lowered.

The mushroom extract can be absorbed through oral mucosa such as micro lesions and gums to activate dendritic cells and macrophages in the oral cavity to locally enhance oral immunity without acting on systemic immunity. That is, the mushroom extract has an effect of preventing oral diseases such as periodontitis by inducing oral immunity enhancement of livestock or companion animals. When the mushroom extract is contained in less than 2% by weight, there is a concern that the pharmacological activity effect by xylitol may be insignificant, and when it is contained in more than 10% by weight, economic efficiency may be reduced due to no benefit due to excess content.

More preferably, the mushroom extract not only has a significant effect on improving oral diseases, but also suppresses bad breath that may occur after livestock or companion animals consume feed or food, and can expect fecal odor relief and odor removal A zinnia mushroom extract or a stone mushroom extract may be used. More preferably, the mushroom extract may use a mixture of a zinnia mushroom extract and a stone mushroom extract in a weight ratio of 1:1.

The Schizandra chinensis extract has antibacterial activity against oral pathogens that cause tooth decay and periodontal disease in livestock and companion animals, and can significantly inhibit the expression of hydrogen sulfide, methyl mercaptan, and dimethyl sulfide, which are the main components of bad breath. More specifically, the Schisandra chinensis extract is Streptococcus mutans, Streptococcus sanguis, Streptococcus sanguinis, Streptococcus salivaris subsp. It has an antibacterial effect against Porphyromonas gingivalis or Candida albicans, and is effective in improving the oral health of livestock and companion animals that are difficult to brush their teeth from oral diseases such as tooth decay and periodontitis. In addition, the extract of Schisandra chinensis contains ingredients such as chizandrin, gomisin, citral, malic acid, and citric acid, which have an effect of improving immunity in the body.

When the Schizandra chinensis extract is contained in less than 5% by weight, the antibacterial activity of dental caries-related strains and the inhibitory effect of halitosis inducing factors may be insignificant, and when it is contained in excess of 15% by weight, there is no benefit due to excess content Economic efficiency may decrease.

The complex mineral is intended to synergistically induce an immune enhancing effect, and when the complex mineral is contained, a chelate reaction with the mushroom-derived chitosan, which has not completed the reaction in the second mixture, proceeds, creating a complex synergistic effect. . In the case of complex minerals, bioavailability can be maximized. This is because the complex minerals are organically passivated by the buffering action of mushroom-derived chitosan, and are uniformly dissolved in drinking water in an activated state and supplied to the body of animals, thereby improving the quality of livestock and animals. It has the effect of increasing immunity. In addition, since chitosan derived from mushrooms that have not completed the reaction in the second mixture can be used as a factor capable of increasing the immunity of livestock or animals by the complex mineral, the use efficiency of the raw material can be significantly increased.

When the complex mineral is contained in less than 40% by weight, the degree of chelation reaction with mushroom-derived chitosan is insignificant, and it may be difficult to create a complex synergistic effect. Failure to do so may result in reduced economic efficiency.

More specifically, the composite mineral may be composed of a mixture of 35 to 45% by weight of illite, 20 to 30% by weight of zeolite, 5 to 10% by weight of selenium, and 20 to 30% by weight of vanadium. It is desirable to allow

By adding the functional additive, there is an effect of improving fecal odor, urine odor and oral odor of livestock or companion animals, and preventing tooth decay or periodontal disease.

The negative additive for livestock and companion animals using mushroom-derived chitosan according to the present invention can be used in various forms by treating and processing the third mixture, and can be prepared in any one of liquid, solid, and granular formulations. .

d) freezing the third mixture and then lyophilizing it to produce a sponge-shaped dried product; characterized in that it is prepared further comprising.

More specifically, when the third mixture is rapidly frozen at -40 to 60 ° C and solidified, it becomes like an ice mass because it contains more than 50 wt% of water. Then, in order to remove moisture from the frozen third mixture in an ice-like state, the moisture content is about 3 to 6 wt% for 15 to 25 hours, preferably 18 to 22 hours, at -30 ° C or lower and 400 mbar or higher. freeze-dried to At this time, reducing the moisture content to about 3 wt% only by freeze-drying may reduce the efficiency in terms of time efficiency, and in the case of hot air drying in a direct heating method, there is a concern that the product may be denatured by direct heat, and when the moisture content is 6wt When % is exceeded, there is a possibility that the dried material may be brittle. Most preferably, freeze-drying such that the water content is 5 wt% shows the optimal effect.

When the lyophilization is completed, an empty space (hole) in which the grains of moisture are located can be produced, and a porous sponge-like dry product can be produced. Depending on the method, it can be prepared by adding sodium bicarbonate, talc, carboxymethyl cellulose, etc. in order to ensure the cohesiveness of the dried material and to come out properly during the freeze-drying process. At this time, it is possible to determine the shape of a building manufactured in the form of a sponge using a mold having a predetermined shape. That is, buildings having various shapes can be manufactured. More preferably, a self-manufactured frame may be used so that the sponge-shaped dried product can be accommodated in a stopper of a drinking container usually mounted on a companion animal's tableware. In a preliminary experiment in which the sponge-shaped dried product is prepared in various sizes to confirm solubility, when the size of the sponge-shaped dried product is prepared in a size of 2 cm in diameter and 2 cm in height, the components of mushroom-derived chitosan and functional additives at a constant concentration This was eluted and it was confirmed that it was effective. However, the size of the sponge-shaped building can be varied and manufactured by a person skilled in the art, so the specific size is not limited.

In addition, depending on the method, the third mixture may be immersed in a processed fabric, nonwoven fabric, fabric, etc. and freeze-dried to prepare a molded article coated with a sponge-type dried material on the surface of the fiber.

Since the dried product prepared in the form of a sponge by step d) can be used by dissolving it in drinking water of animals, storage and convenience can be improved.

In addition, by using the porous characteristics of the sponge-shaped dried product, it can be easily applied to a drinking container that allows a certain amount of drinking water to flow into a companion animal's tableware. For example, as shown in FIG. 2 , the tableware 10 of the companion animal may continuously receive a certain amount of negative water from the negative water supply unit 20 . More specifically, a negative additive 22 is provided between the spout of the drinking container 21 and the stopper 23. Since the negative additive 22 is in the form of a porous sponge, the negative water in which the active ingredient is dissolved is watered in the tableware 10 ) has the effect of continuously supplying the tableware. Depending on the method, the negative additive 22 may be configured to be accommodated inside the stopper 23.

On the other hand, the step e) is a step of crushing the dried product, mixing auxiliary materials and tableting them in a tablet form. That is, in the step e), after crushing the dry matter, mixing the auxiliary materials, and introducing them into a tableting machine, tableting is performed.

More preferably, the auxiliary material is added with one or more first auxiliary materials selected from the group consisting of excipients, sweeteners, powdered milk, flavoring agents, coloring agents, wetting agents, and preservatives, including a second auxiliary material composed of sodium bicarbonate and magnesium stearate. Most preferably, the first auxiliary material is mixed with the pulverized dried material at 20 to 30 ° C to prepare a first auxiliary material mixture, and the second auxiliary material is mixed with the first auxiliary material mixture at 0 to 5 ° C. Thus, the second auxiliary material mixture can be prepared. Thereafter, by introducing the second auxiliary material mixture into a tableting machine, it may be tableted into tablets. At this time, it is possible to obtain an optimal effect by preparing the first auxiliary material mixture at 20 to 30 ° C and preparing the second auxiliary material mixture at 0 to 5 ° C. This minimizes the reaction between sodium bicarbonate and magnesium stearate by adding sodium bicarbonate and magnesium stearate at a low temperature to prevent denaturation, and minimizes disintegration due to internal moisture during the mixing process and after the mixing process. When sodium bicarbonate and magnesium stearate are added together in the process of preparing the first auxiliary material mixture and mixed, hardening or denaturation may occur due to other components or moisture.

Depending on the method, between the step d) and the step e), the freeze-dried product is indirectly heated to a surface temperature of 50 to 80 ° C. and a moisture content of 3 to 4% by weight, followed by hot air drying for 2 to 4 hours. It may further include steps to do.

To prevent deterioration of the product due to high temperature, it is recommended to repeat hot air drying for 40 minutes and stop for 20 minutes. Drying with hot air so that the water content in the powder is about 3% by weight shows the optimal effect. Hot air can be dried by blowing hot air maintained at a temperature of 60 to 120 ° C. at a wind speed of 1 to 2 m / sec.

The hot air drying method as described above has an effect of preventing loss of active ingredients due to excessive hot air drying.

Provided is a negative additive for livestock and companion animals using chitosan derived from mushrooms prepared by the manufacturing method of claim 1.

Hereinafter, the environmentally friendly sound-absorbing non-combustible foam for construction according to a preferred embodiment of the present invention will be described in more detail. However, the scope of the present invention is not limited by the following examples.

■ Experimental Example 1 - Preparation of mushroom-derived chitosan and antibacterial activity test

(1) Experiment method

Mushroom-derived chitosan was used, but 0.5 g of mushroom-derived chitosan powder having a molecular weight of 200 to 30,000 kDa was prepared.

Then, with respect to 100 ml of distilled water, 20 μl of the sample prepared by mixing and stirring 0.5 g of chitosan powder having a molecular weight of 200 to 30,000 kDa derived from mushrooms at a temperature of 25 ° C was spread on a test piece of 5 cm x 5 cm dimension, Chitosan test pieces were prepared. Thereafter, the entire surface of the mushroom-derived chitosan test piece was sterilized with ethanol, and then inoculated with Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, respectively, covered with a film of 50 ± 2 mm width, and left at 37 ± 0.2 ° C. for 24 hours. Thereafter, the number of viable cells cultured on each test piece film was measured, and the results are shown in FIG. 3 and Table 1. For reference, a 5 cm x 5 cm Stomacher film was used as a control, and the control is indicated as "BLANK" in Table 1 below.

Test Items Experiment result initial concentration
(CFU/mL) Concentration after 24 hours
(CFU/mL) bacterial reduction rate
(%) Escherichia coli BLANK

3.0 ×

1.3 ×

- Mushroom-derived chitosan test piece 3.0 ×

<10 99.9 Staphylococcus aureus BLANK 1.1 ×

4.8 ×

- Mushroom-derived chitosan test piece 1.1 ×

<10 99.9 Pseudomonas aeruginosa BLANK 3.6 ×

3.2 ×

- Mushroom-derived chitosan test piece 3.6 ×

<10 99.9

CFU/mL의 초기 농도를 가지는 반면, 24시간 경과 후, “BLANK”로 표시된 대조편은 1.3 ×

CFU/mL의 현저히 증가된 대장균 생균수를 보이는 것을 확인할 수 있었다.황색포도상구균 시험에서는 대조편과 버섯유래키토산 시험편 모두 1.1 ×

CFU/mL의 초기 농도를 가지는 반면, 24시간 경과 후 “BLANK”로 표시된 대조편은 4.8 ×

CFU/mL의 대장균 생균수를 보이는 것을 확인할 수 있었다.In the E. coli test, both the control sample marked “BLANK” and the mushroom-derived chitosan test sample were 3.0 ×

While having an initial concentration of CFU/mL, after 24 hours, the control labeled “BLANK” was 1.3 ×

It was confirmed that the number of Escherichia coli viable cells increased significantly in CFU/mL. In the Staphylococcus aureus test, both the control and mushroom-derived chitosan test samples were 1.1 ×

While having an initial concentration of CFU/mL, the control labeled “BLANK” after 24 hours was 4.8 ×

It was confirmed that the number of E. coli viable cells of CFU/mL was shown.

CFU/mL의 초기 농도를 가지는 반면, 24시간 경과 후, “BLANK”로 표시된 대조편은 3.2 ×

CFU/mL의 현저히 증가된 대장균 생균수를 보이는 것을 확인할 수 있었다.In the Pseudomonas aeruginosa test, both the control and mushroom-derived chitosan test pieces were 3.6 ×

While having an initial concentration of CFU/mL, after 24 hours, the control labeled “BLANK” was 3.2 ×

It was confirmed that the number of E. coli viable cells significantly increased in CFU/mL.

However, in experiments with Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, it was confirmed that the mushroom-derived chitosan test piece showed a bacterial reduction rate of about 99.9%.

That is, in the mushroom-derived chitosan test piece, when compared to the control piece, the number of residual viable cells corresponds to about 1 ppm (part per million), and even when compared to the control piece, the number of remaining viable cells corresponds to about 0.01%. That is, it can be confirmed that the excellent antibacterial properties of the mushroom-derived chitosan test piece are exhibited.

■ Experimental Example 2- Preparation of negative additives for livestock and companion animals containing mushroom-derived chitosan and experiment using the same

<Example 1> - Preparation of liquid negative additive

According to the manufacturing method described in Table 2 below, the present invention, a negative additive for livestock and companion animals (liquid) was prepared.

division Example 1 a For 100 ml of distilled water, 0.5 g of chitosan powder with a molecular weight of 200 to 30,000 kDa derived from mushrooms was mixed and stirred at 25 ° C for 3 minutes, and then 0.5 g of ascorbic acid was added at 25 ° C for 20 minutes. By stirring, a first mixture in which the mushroom-derived chitosan is dissolved is produced b Mixing 0.1 g of a basic aqueous solution in which sodium hydroxide is dissolved in the first mixture to produce a second mixture having a pH adjusted to 6.0 c By mixing 1 g of functional additives with the second mixture
create a third mixture

In step c, the functional additives include 5 to 15% by weight of xylitol, 3 to 15% by weight of bovine extract, 3 to 15% by weight of propolis, 2 to 10% by weight of mushroom extract, 5 to 15% by weight of Schisandra chinensis extract, 40% by weight of complex minerals ~ 50% by weight was used as an active ingredient.

<Example 2> - Preparation of negative additives in sponge form

In Example 1, the third mixture was frozen and then lyophilized to prepare a sponge-shaped dry product; further comprising, a sponge-shaped negative additive was prepared, and Example 2 is shown in Table 3 below. (Steps a to c were prepared in the same way as in Example 1.)

division Example 2 a For 100 ml of distilled water, 0.5 g of chitosan powder with a molecular weight of 200 to 30,000 kDa derived from mushrooms was mixed and stirred at 25 ° C for 3 minutes, and then 0.5 g of ascorbic acid was added at 25 ° C for 20 minutes. By stirring, a first mixture in which the mushroom-derived chitosan is dissolved is produced b Mixing 0.1 g of a basic aqueous solution in which sodium hydroxide is dissolved in the first mixture to produce a second mixture having a pH adjusted to 6.0 c By mixing 1 g of functional additives with the second mixture
create a third mixture d Freeze-drying the third mixture after freezing to prepare a sponge-shaped dry product

In step d, lyophilization was performed by putting the third mixture into a self-made cylindrical mold and rapidly freezing at -40 to 60 ° C, and the frozen third mixture at -30 ° C or less and 400 mbar or more for 20 hours. By freeze-drying, a sponge-like dried product having a water content of 5 wt%, a height of 2 cm, and a diameter of 2 cm was prepared.

<Example 3> - Preparation of negative additive 1 in tablet form

In Example 2, after crushing the dry matter, mixing the sub-materials and tableting them in tablet form; further comprising, a tablet-type negative additive was prepared, and Example 3 is shown in Table 4 below. .(Steps a to d were prepared in the same way as in Example 2.)

division Example 3 a For 100 ml of distilled water, 0.5 g of chitosan powder with a molecular weight of 200 to 30,000 kDa derived from mushrooms was mixed and stirred at 25 ° C for 3 minutes, and then 0.5 g of ascorbic acid was added at 25 ° C for 20 minutes. By stirring, a first mixture in which the mushroom-derived chitosan is dissolved is produced b Mixing 0.1 g of a basic aqueous solution in which sodium hydroxide is dissolved in the first mixture to produce a second mixture having a pH adjusted to 6.0 c By mixing 1 g of functional additives with the second mixture
create a third mixture d Freeze-drying the third mixture after freezing to prepare a sponge-like dried product; e After pulverizing the dried material, the auxiliary materials are mixed and tableted.

At this time, in step e, 3% by weight of hydroxymethylcellulose, 2% by weight of dextrin, 3% by weight of sucralose, 1% by weight of fragrance and 2% by weight of pigment were added to 63% by weight of the dried material, and first mixed at 23 ° C. After preparing the first sub-material mixture, 22% sodium bicarbonate and 4% by weight of magnesium stearate were added to the first sub-material mixture at 3° C., followed by secondary mixing to prepare a second sub-material mixture. Thereafter, the second auxiliary material mixture was introduced into a tablet press and tableted at a temperature of about 23 ° C. and a relative humidity of about 25% at high pressure and low speed to prepare a tablet having a weight of 1 g, a diameter of 1.2 cm, and a thickness of 4 mm.

<Example 4> - Preparation of negative additive 2 in tablet form

In Example 3, hot air drying the freeze-dried product between step d) and step e); was further included to prepare a tablet-type negative additive, and Example 5 is shown in Table 5 below. .(Steps a to d, e were prepared in the same way as in Example 3.)

division Example 5 a For 100 ml of distilled water, 0.5 g of chitosan powder with a molecular weight of 200 to 30,000 kDa derived from mushrooms was mixed and stirred at 25 ° C for 3 minutes, and then 0.5 g of ascorbic acid was added at 25 ° C for 20 minutes. By stirring, a first mixture in which the mushroom-derived chitosan is dissolved is produced b Mixing 0.1 g of a basic aqueous solution in which sodium hydroxide is dissolved in the first mixture to produce a second mixture having a pH adjusted to 6.0 c By mixing 1 g of functional additives with the second mixture
create a third mixture d Freeze-drying the third mixture after freezing to prepare a sponge-shaped dry product sirocco
dry The freeze-dried product was dried in hot air for 3 hours using an indirect heating method to achieve a surface temperature of 50 to 80 °C and a moisture content of 3% by weight. e After pulverizing the dried material, the auxiliary materials are mixed and tableted.

At this time, in the hot air drying step, in order to prevent product deterioration due to high temperature, hot air was applied for 40 minutes and stopped for 20 minutes, and the hot air was frozen at a temperature of 60 to 120 ° C. and a wind speed of 1 to 2 m / sec. It is applied to dry matter.

■ Test Example 1: Toxicity test

Experimental solution prepared by using 5 ICR-based mice with a body weight of 20 ± 3g as a sample for toxicity testing, and adding each of the negative additives prepared in Examples 1 to 4 to water at a weight ratio of 1:400. After oral administration of 50 mg, changes in motor activity, convulsions, and reflex activities were observed for 14 days, but all were confirmed to be normal.

■ Test Example 2: Antiviral test on porcine epidemic diarrhea virus (PEDV)

TCID50/㎖의 역가로 3㎖씩을 하기 표 6에서와 같은 시험군 I, II, III, Ⅳ, Ⅴ 군의 동물들에 경구 접종하였고 II군의 동물에는 DMEM 배지를 3㎖ 경구 접종하였다. 또한, 처치 물질로서, 상기 실시예 1 내지 실시예 4에 의하여 제조된 음수첨가제 각각을 물에 1:400 중량비로 첨가하여 제조된 실험용 용액 1 내지 4를 500mg을 이용하였다. Pigs at 1 week of age were purchased and used from a place where PEDV vaccine unvaccinated mothers gave birth and viral diarrhea did not occur recently. Pigs with no disease and apparently healthy pigs were classified as having similar weight averages when the groups were composed, and milk powder for newborn pigs was fed. In addition, PED virus culture is performed by culturing PEDV into Vero cells.

3 ml each at a titer of TCID 50 / ml was orally inoculated into animals in groups I, II, III, IV, and V as shown in Table 6 below, and animals in group II were orally inoculated with 3 ml of DMEM medium. In addition, as a treatment material, 500 mg of experimental solutions 1 to 4 prepared by adding each of the negative additives prepared in Examples 1 to 4 to water at a weight ratio of 1:400 was used.

army process two numbers PEDV matter I 0 menstruum 3 II 0 Lab solution 1 3 III 0 Lab solution 2 3 IV 0 Lab solution 3 3 V 0 Lab solution 4 3

2. Observation of fecal condition

(1) Experiment method

During the course of the test, the fecal condition was observed every day, the fecal score of each individual was recorded, and the mean and standard deviation of each group were obtained. The fecal score was "3; normal, 2; soft stool, 1; diarrhea, 0; dead". The average and standard deviation of the fecal scores of each group after PEDV inoculation are shown in Table 7 below.

(2) Experimental results

After PEDV inoculation, subjects in group I showed severe diarrhea. On the other hand, on the 7th day after PEDV inoculation, the fecal scores of groups II to V, groups administered with the extract according to the present invention, were significantly higher than those of group I (p<0.05). Therefore, it was confirmed that the extract of the present invention relieves diarrhea caused by PEDV.

army evaluation time 1 day 2 days 3 days 4 days 5 days 6 days 7 days I 2.0 1.3 2.0 1.6 1.6 1.3 1.0 II 2.0 1.6 2.0 2.3 2.3 2.3 2.3 III 2.0 1.6 2.0 2.3 2.3 2.3 2.3 IV 2.0 1.6 2.0 2.3 2.3 2.3 2.3 V 2.0 1.6 2.0 2.3 2.3 2.3 2.3

3. Plaque inhibition effect analysis

(1) Experiment method

80-90% confluent Vero cells cultured at 7°C were cultured in a virus culture medium (MEM, 0.3% tryptosephosphate broth [sigma], 10 μg/ml trypsin, penicillin and streptomycin). After washing once with streptomycin), they were infected with PEDV. At this time, the experimental solutions 1 to 4 prepared by adding each of the negative additives prepared in Examples 1 to 4 to water at a weight ratio of 1:400 were treated together with 200 μl, and the concentration of the experimental solution for each experimental group during the infection period was kept.

After infection, the cells were incubated at 37°C for two hours with rocking every 15 minutes, and then virus culture medium was added. After a further 10 hours, the virus released into the virus culture medium was obtained, and whether the virus release was reduced by treatment with the experimental solution was examined by plaque assay.

(2) Experimental results

The experimental results of counting and comparing the number of plaques in the experimental group treated with 200 μl of the experimental solution 1 to 4 and the untreated control group (PEDV plaque measurement method: Veterinary Microbiology, 20, pp 131-142, 1989) are shown in Table 8 below.

administration group plaque forming unit PEDV control: no treatment

2.0X

Experimental solution 1 1.9X

Experimental solution 2 1.8x

Experimental solution 3 1.8x

Experimental solution 4 1.9X

As shown in the table above, when the experimental solutions 1 to 4 prepared in Examples 1 to 4 were administered, the effect was reduced by about 10 to 100 times compared to the untreated control group, resulting in a strong antiviral effect against porcine epidemic diarrhea virus. was able to confirm that the

■ Test Example 3: Immunity enhancing effect

(1) Experimental crime prevention

Piglets with an average weight of 26.409 were experimented on 24 pigs, 6 pigs each, and reared in the following way. After adding each of the negative additives of Examples 1 to 4 to water in a weight ratio of 1:400, it was made to drink instead of drinking water. The feed was fed using a commercially available basic general feed for pigs. As a control group (CON), only basal feed and bottled water were used. Lighting and other specification management were performed according to general practices.

Thereafter, blood analysis was performed to confirm the effect of increasing immunity. After the end of the experiment, 1 μl of blood was collected using a syringe from 24 heads of 6 heads in each experimental group, put in an EDTA tube and immediately stored at 4 ° C, analyzed whole blood for CBC test, and antigen-antibody reaction For serum separation, centrifugation was performed at 3000 rpm for 10 minutes, and serum was immediately frozen at -70 ° C.

The CBC Test includes red blood cell (RBC), white blood cell (WBC), hemoglobin (HGB), hematocrit (HCT), mean cell volume (MCV), mean hematocrit (MCH), It is a test showing clinical and pathological values such as mean hemoglobin concentration (MCHC), platelet (PLT), and IgG, and collected whole blood was analyzed using an automatic blood analyzer.

For comparison of immunity, IgG analysis using serum isolated from whole blood was performed by ELISA. As a result, it was shown in Table 9.

Item Control group (bottled water) experimental group Example 1 Example 2 Example 3 Example 4

/ul)WBC
(

/ul) 21.43 24.96 25.45 25.61 25.64 RBC
(

/ul) 6.58 6.89 6.93 6.95 6.97 HGB
(g/dl) 9.75 10.51 10.55 10.60 10.68 HCT
(%) 30.64 32.15 32.31 32.40 32.45 MCV
(fl) 46.23 48.22 48.35 48.43 48.45 MCH
(pg) 14.61 16.01 16.11 16.15 16.17 MCHC
(g/dl) 32.45 32.69 32.75 32.86 32.88 PLT
(

/ul) 327.1 410.5 418.1 420.3 425.4 IgG
(mg/dl) 224 275.5 280.4 284.6 285.6

As shown in Table 9, when the experimental solutions 1 to 4 prepared in Examples 1 to 4 were administered, the effect was reduced by about 10 to 100 times compared to the untreated control group, resulting in a strong antiviral against porcine epidemic diarrhea virus. It was confirmed that the effect was visible.

■ Test Example 4: Deodorization Test

(1) Experiment method

In order to examine the deodorizing effect of the negative additives prepared in Examples 1 to 4, first, negative water for experiments was prepared by adding each of the negative additives of Example 1 to water in a weight ratio of 1:400. Thereafter, a plurality of sealed deodorization test containers (40 cm × 40 cm × 60 cm) were prepared, and each test container was divided into an ammonia-injected test container and a trimethylamine-injected test container. At this time, the initial concentration of each test device was adjusted to be about 50 ppm. Thereafter, 20 ml of the negative water for the experiment was sprayed into the test container, and the odor concentration after 0 minutes, 1 hour, 2 hours, and 4 hours was measured using a gas stack. In the same manner, odor concentration was measured for the negative water to which the negative additives of Examples 2 to 4 were added and the sterilized water (control group). The results are shown in Table 10 below.

(2) Experimental results

item unit result Early 1 hour later 2 hours later 4 hours later Example 1
negative ammonia ppm 50.0 0.5 or less 0.5 or less 0.5 or less trimethylamine ppm 50.0 0.5 or less 0.5 or less 0.5 or less Example 2
negative ammonia ppm 50.0 0.5 or less 0.5 or less 0.5 or less trimethylamine ppm 50.0 0.5 or less 0.5 or less 0.5 or less Example 3
negative ammonia ppm 50.0 0.5 or less 0.5 or less 0.5 or less trimethylamine ppm 50.0 0.5 or less 0.5 or less 0.5 or less Example 4
negative ammonia ppm 50.0 0.5 or less 0.5 or less 0.5 or less trimethylamine ppm 50.0 0.5 or less 0.5 or less 0.5 or less control group ammonia ppm 50.0 49 49 48 trimethylamine ppm 50.0 50 49 49

As a result, it was confirmed that the concentrations of ammonia and trimethylamine in the test vessel injected with negative water added with the negative additives of Examples 1 to 4 were significantly reduced compared to the control group.

■ Test Example 5: Manure odor reduction experiment

(1) Experiment method

According to the addition of the negative additives of Examples 1 to 4, changes in ammonia levels, which are odorous components of urine of pets, were investigated. First, negative water for experiments was prepared by adding each of the negative additives of Example 1 to water in a weight ratio of 1:400. Thereafter, while maintaining the breeding temperature at 25 ± 2.0, KW-100 of Example 1 and each experimental negative water were fed 50 mL every day for 10 days, and then, urine was extracted from the bladder of the experimental animal with a syringe, and the urine contained in the urine The level of ammonia (ppm) was analyzed. Sterile water was administered to the control group in the same dose. The ammonia reduction rate (%) represents the relative difference in ammonia levels before and after administration of drinking water or sterilized water for each experiment.

(2) Experimental results

item Example 1
negative Example 2
negative Example 3
negative Example 4
negative control group ammonia reduction rate 45.4% 48.8% 49.1% 53.2% 2%

As a result, it was confirmed that the reduction rate of ammonia contained in the urine of the experimental animals fed the negative water to which the negative additives of Examples 1 to 4 were added was significantly higher than that of the control group. These results confirm that the composition of the present invention effectively removes ammonia contained in manure.

■ Test Example 6: Antibacterial evaluation of oral disease causative bacteria

(1) Experimental crime prevention

It was investigated whether the negative additive of the present invention has an antibacterial effect on oral disease-causing bacteria. Thereafter, the strains in the table below were lyophilized and suspended in BHI liquid medium, incubated at 37° C. for 24 hours to activate the bacteria, and then plated again on BHI solid medium and cultured for 24 hours. Antibacterial properties were evaluated using the disk test method, and each of the negative additives of Examples 1 to 4 was diluted in water and treated to a concentration of 10 mg/ml. As a control group, experiments were conducted using a 5% phenol solution and BHI liquid medium. Accordingly, after culturing for 24 hours under optimal culture conditions, the size (mm) of the growth arrest ring was measured, and based on this, the antibacterial activity was verified, which is shown in the table below.

(2) Experimental results

division test bacteria Detail One Streptococcus mutans
(Streptococcus mutans) caries-causing bacteria 2 Streptococcus sanguis
(Streptococcus sanguis) gingivitis-causing bacteria 3 Streptococcus sanguinis
(Streptococcus sanguinis) gingivitis-causing bacteria 4 Porphyromonas gingivalis
(Porphyromonas gingivalis) periodontal disease causing bacteria

division Example 1
10 mg/ml Example 2
10 mg/ml Example 3
10 mg/ml Example 4
10 mg/ml BHI badge
10 mg/ml 5% phenol
10 mg/ml growth
low cost
(mm) Streptococcus mutans
(Streptococcus mutans) 5 4.5 3 3 - 11 Streptococcus sanguis
(Streptococcus sanguis) 4 4 4 3 - 10 Streptococcus sanguinis
(Streptococcus sanguinis) 3 3 3 3 - 14.5 Porphyromonas gingivalis
(Porphyromonas gingivalis) 4 4 4 4 - 11

As a result, the treatment groups of Examples 1 to 4 were also investigated to increase the size of growth arrest rings for oral disease-causing strains (Streptococcus mutans, Streptococcus sanguis, Streptococcus sanguinis, Porphyromonas gingivalis), confirming that they had antibacterial effects.

■ Test Example 7: Examination of acid-producing bacteria in the oral cavity

(1) Experiment method

Thirty pet dogs weighing 7 to 8 kg were divided into 5 groups of 6 dogs each and bred in the following manner. In 4 groups of 5 groups, each of the negative additives of Examples 1 to 4 was added to water at a weight ratio of 1:400, and then they were made to drink instead of drinking water. The feed was fed using commercially available basic pet food. In addition, only basic feed and bottled water were fed to the remaining 1 group as a control group (CON). Lighting and other specification management were performed according to general practices.

Then, based on the fact that bacteria contained in dental plaque or saliva form acid, the activity level of acid-producing bacteria in saliva was measured by a colorimetric method. The test method is to rub the teeth of the pet dog 4 times (2 months), once every 2 weeks, on the buccal surface of the upper and lower posterior teeth to collect the tooth surface bacterial film, put the collected cotton ball into the prepared medium, close the stopper, and then remove the medium. Incubated for 48 hours at 37 ° C. in an incubator, and the average color change of the medium was compared and judged.

The decision criteria are blue for inactive (-), green for mildly active (+), yellow green for moderately active (++), and yellow for highly active (+++). ) means. The experimental results for this are shown in the table below.

(2) Experimental results

division Example 1 Example 2 Example 3 Example 4 control group
(bottled water) acid-producing bacteria
situation
+ + + + +++

According to the above table, in the case of pets fed only negative water, it was confirmed that acid-producing bacteria in the oral cavity showed high activity. In the case of pets fed negative water to which the negative additive of the present invention was added, it was confirmed through experiments that there was a composition showing inactivity or mild activity.

10: tableware
20: negative water supply unit
21: drinking container
22: negative additive
23: stopper

Claims (8)

a) mixing and stirring mushroom-derived chitosan in distilled water and then adding an acid to produce a first mixture in which the mushroom-derived chitosan is dissolved;
b) mixing a basic aqueous solution with the first mixture to produce a second mixture having a pH adjusted to 5.0 to 7.0;
c) In the second mixture, 5 to 15 wt% of xylitol, 3 to 15 wt% of bovine extract, 3 to 15 wt% of propolis, 2 to 10 wt% of mushroom extract, 5 to 15 wt% of Schisandra chinensis extract, 40 to 100% of complex minerals A method for producing a negative additive for livestock and companion animals using chitosan derived from mushrooms, characterized in that it is produced including; mixing a functional additive with 50% by weight as an active ingredient to produce a third mixture. According to claim 1,
d) freezing and then freeze-drying the third mixture to produce a sponge-shaped dried product; method for producing a negative additive for livestock and companion animals using chitosan derived from mushrooms, characterized in that produced by further comprising. According to claim 2,
e) after crushing the dry matter, mixing the sub-materials and tableting them in the form of a tablet; Method for producing a negative additive for livestock and companion animals using chitosan derived from mushrooms further comprising.
According to any one of claims 1 to 3,
The above mushrooms are stone ear mushrooms, shiitake mushrooms, chaga mushrooms, wood ear mushrooms, enoki mushrooms, bare mushrooms, Agaricus mushrooms, Ganoderma lucidum mushrooms, king oyster mushrooms, situation mushrooms, cauliflower mushrooms, jujube mushrooms, maitake mushrooms, fingering mushrooms, ericium erinaceus mushrooms, Method for producing a negative additive for livestock and companion animals using chitosan derived from mushrooms, characterized in that it comprises at least one selected from the group consisting of longevity mushrooms, oyster mushrooms, spirit mushrooms, mulberry mushrooms, and tile mushrooms. According to claim 1,
The chitosan of step a) is carboxymethyl chitosan, carboxymethyl chitosan, carboxyethyl chitosan, cyanoethyl chitosan, hydroxyethyl chitosan, hydroxypropyl chitosan, hydroxypropyl chitosan, glyceryl chitosan, glycol chitosan, succinyl chitosan, Method for producing a negative additive for livestock and companion animals using chitosan derived from mushrooms, characterized in that it comprises at least one selected from the group consisting of carboxymethyl chitosan succinimide. delete According to claim 1,
The mushroom extract is a method for producing a negative additive for livestock and companion animals using mushroom-derived chitosan, characterized in that the mushroom extract and the zinnia mushroom extract are mixed in a weight ratio of 1: 1. Negative additives for livestock and companion animals using chitosan derived from mushrooms prepared by the manufacturing method of claim 1

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