KR101767837B1 - Method for producing mixture extract of sea cucumber and sea mustard with enhanced immunity activity - Google Patents

Abstract

The present invention relates to a production method of a mixed extract of sea cucumber and sea mustard having enhanced immunological activity. The mixed extract of sea cucumber and sea mustard of the present invention have excellent immunity, and can be used as a food, a medicine or a feed additive having an excellent effect in preventing, inhibiting or treating decline of immunization and hypoimmunity impairing an immune system.

Description

TECHNICAL FIELD The present invention relates to a method for producing sea cucumber and sea mustard mixed immune activity,

The present invention relates to a method for producing sea cucumbers and wakame mixed extracts having enhanced immunological activity.

Sea cucumbers are the 8 most important marine products of Korea, as well as ginseng. They are not only the best health and well-being food called sea ginseng, but also a clean aquatic product that lives in a clean place because it contains a lot of active ingredients such as saponin. Sea cucumbers are known to be a herbaceous form as much as wild ginseng in China, and have excellent effects on diabetes and asthma in China and other Asian countries. They have a great effect in summer and a lot of sweat due to temperature rise. It is known as a drug that can regain energy when you fall into it. Also, according to modern science, it is known that sea cucumbers are abundant in protein, vitamin B, iodine, and chondroitin, which are used as raw materials for various foods and medicines. The major components of sea cucumber are glycoprotein and chondroitin sulphate, and glycoproteins are mainly polysaccharides such as fucose, glucose, mannuronic acid and N-acetylglucosamine. .

Seaweeds refer to all the birds in the sea, and these seaweeds are divided into three types: brown algae such as kelp, seaweed, and algae, red algae such as seaweed, mugwort, and green algae. Of the 500 species of seaweeds in Korea, about 50 species are used for food. The seaweeds enjoyed by Koreans include seaweed, kelp, kim, paraya, mackerel, mackerel, and hearing. On the other hand, Korea is in contact with the sea on three sides, and seaweeds such as seaweed, kelp, and seaweed are the eighth largest producer in the world. Seaweeds are composed of elements such as Ca, P, Mg, Fe, Na, S and minerals, and vitamins A, B and C. Recently, as health functional effects are revealed,

Korean Patent Publication No. 2012-0063709 discloses a composition for enhancing immunity comprising an extract of sea cucumber as an active ingredient. Korean Patent Laid-Open Publication No. 2005-0006533 discloses a method for extracting an anti-cancer and immunostimulatory extract from seaweed . However, the preparation methods of the sea cucumber and mixed marine wax extracts with improved immunological activity of the present invention have not yet been disclosed.

The present invention has been made in view of the above-mentioned needs. In the present invention, mixed sea cucumber and sea mustard extracts were prepared using cell membrane degrading enzymes and proteolytic enzymes, and the sea cucumber and sea mustard mixed extracts of the present invention had immunological activity The present inventors have completed the present invention by confirming that the sea cucumber extract and the seaweed extract are superior to each other in immunity.

In order to solve the above-described problems, the present invention provides a method for preparing a sea cucumber, comprising the steps of: 1) removing the viscera and drying the sea cucumber washed with boiled sea cucumber at a temperature of 100 ° C or higher; 2) mixing the dried sea cucumber and the seaweed in step 1), pulverizing and adding water to prepare a mixed solution; 3) adding a cell membrane-degrading enzyme and a proteolytic enzyme to the mixed solution of step 2); And 4) adding the cell membrane lyase and protease-added mixed solution to an enzyme reactor, sterilizing the mixture at 100 to 140 ° C for 15 to 35 minutes, and then maintaining the mixture at 40 to 65 ° C for 15 to 30 hours The present invention provides a method for preparing sea cucumbers and seaweed mixed extracts with enhanced activity.

The present invention also provides sea cucumber and wakame mixed extracts prepared by the above method.

In addition, the present invention provides a health functional food composition for immune enhancement comprising the sea cucumber and the seaweed mixed extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for enhancing immunity, comprising the sea cucumber and the seaweed mixed extract as an active ingredient.

In addition, the present invention provides a feed additive composition for immunity enhancement comprising the sea cucumbers and the seaweed mixed extract as an active ingredient.

Since the sea cucumber and the seaweed mixed extract of the present invention have excellent immunity, they can be used as foods, medicines or feed additives having immunity enhancing effect for preventing, inhibiting or treating immunosuppression and immunosuppressed immunosuppression.

FIG. 1 shows the effect of increasing the secretion of NO production (A) and TNF-α (B) in RAW264.7 cells when the sea cucumber and the seaweed mixed extract of the present invention were treated. C is a negative control without any treatment, and LPS (Lipopolysaccharide) is a positive control.
FIG. 2 shows the effect of increasing the production of IFN-? (A) and TNF-? (B) in splenocytes when the sea cucumber and the seaweed mixed extract of the present invention were treated. The media is a negative control that processed the bait.

In order to accomplish the present invention, the present invention provides a method for producing sea cucumbers, comprising the steps of: 1) removing the viscera and drying seaweed washed with boiled sea cucumber at a temperature of 100 ° C or higher;

2) mixing the dried sea cucumber and the seaweed in step 1), pulverizing and adding water to prepare a mixed solution;

3) adding a cell membrane-degrading enzyme and a proteolytic enzyme to the mixed solution of step 2); And

4) immersing the mixed solution containing the cell membrane lyase and protease into an enzyme reactor, sterilizing the mixture at 100 to 140 ° C for 15 to 35 minutes, and maintaining the mixture at 40 to 65 ° C for 15 to 30 hours And a method for producing the enhanced sea cucumber and the seaweed mixed extract.

In the production method according to an embodiment of the present invention, the temperature at which the sea cucumber is boiled in the step 1) may be 100 ° C. or higher, preferably 100 to 130 ° C., but is not limited thereto.

In the production method according to an embodiment of the present invention, the cell membrane-degrading enzyme in step 3) may be tunicase, the protease may be alcalase or esperase, Preferably, the proteolytic enzyme may be, but not limited to, an alcalase.

In addition, the mixed solution of step 4) may be sterilized at 100 to 140 ° C for 15 to 35 minutes and then maintained at 40 to 65 ° C for 15 to 30 hours, preferably at 130 ° C for 30 minutes Followed by maintaining at 60 ° C for about 24 hours after sterilization, but the present invention is not limited thereto.

The present invention also provides sea cucumber and wakame mixed extracts prepared by the above method.

The sea cucumber and the seaweed mixed extract of the present invention include any one of an extract obtained by an extraction treatment, a diluted or concentrated liquid of an extract, a dried product obtained by drying an extract, a preparation to be treated, or a purified product.

In addition, the present invention provides a health functional food composition for immune enhancement comprising the sea cucumber and the seaweed mixed extract as an active ingredient.

The immunocompromising health functional food composition of the present invention may be prepared in any form of powder, granule, ring, tablet, capsule, candy, syrup and beverage, but is not limited thereto.

When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is, or may be used together with other food or food ingredients, and suitably used according to a conventional method. The active ingredient may be suitably used depending on its intended use (prevention or improvement). Generally, in the production of food or beverage, it is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the health functional food composition of the present invention. However, in the case of long-term intake for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the foods to which the health functional food composition can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, soups, Drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

In addition, the health functional food composition of the present invention can be produced as a food, particularly a functional food. The functional food of the present invention includes components that are ordinarily added in food production, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, in the case of a drink, a natural carbohydrate or a flavoring agent may be included as an additional ingredient in addition to the active ingredient. The natural carbohydrate may be selected from the group consisting of monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, , Xylitol, sorbitol, erythritol, etc.). The flavoring agent may be a natural flavoring agent (e.g., tau Martin, stevia extract, etc.) and a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).

In addition to the above-mentioned health functional food composition, it is possible to use various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, A carbonating agent used in beverages, and the like.

In addition, the present invention provides a pharmaceutical composition for enhancing immunity, comprising the sea cucumber and the seaweed mixed extract as an active ingredient.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The pharmaceutical dosage forms of the compositions according to the invention may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method . Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition including the sea cucumber and the sea mustard mixed extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, etc. . In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art.

The sea cucumber and marrow mixed extract of the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In addition, the present invention provides a feed additive composition for immunity enhancement comprising the sea cucumbers and the seaweed mixed extract as an active ingredient.

The feed additive of the present invention is added to the base feed at a certain ratio. The basic diet may be composed of corn, soybean meal, whey, fish meal, molasses, salt, vitamin premix, and mineral premix. The vitamin premix can be composed of vitamin A, vitamin D, vitamin E, riboflavin and niacin, and the mineral premix can be composed of manganese, iron, zinc, calcium, copper, cobalt and selenium.

The feed composition comprising the immunostimulating feed additive of the present invention can be prepared according to a feed production method known in the art. For example, various feed ingredients or compound feeds can be mixed with sea cucumber and sea weed mixed extract of the present invention Followed by further processing, for example, pelleting, or cutting in the form of granules, and the like.

It is apparent to those skilled in the art that the composition, composition, manufacturing method, feeding method and the like of the immunostimulating feed composition of the present invention can be suitably selected from conventional techniques known in the art.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Manufacturing example  1. Preparation of sea cucumber extracts and sea mustard extracts

Dried seaweed 40g and sea mustard 40g were crushed to a size of 5mm or less, respectively, and to each were added 58ml of purified water, 1g of tunicase, and 1g of alkalase ( alcalase) was added thereto, and the resulting mixture was placed in an enzyme reactor and sterilized at 110 ° C for 20 minutes. Then, the mixture was kept at 45 ° C for 24 hours to obtain a sea cucumber extract and a sea mustard extract. Solid components were filtered through a 100 mesh sieve, For example.

Manufacturing example  2. Preparation of sea cucumber and sea mustard mixed extract 1

After removing the viscera, sea cucumbers washed with boiled sea cucumber were dried at a temperature of 100 ° C or higher. 20 g of dried sea cucumber and 20 g of seaweed were mixed and pulverized to 5 mm or less, and 58 ml of purified water was added to prepare a mixed solution. 1 g of tunicase and 1 g of alcalase were added thereto and sterilized at 110 ° C for 20 minutes in an enzyme reactor. The mixture was kept at 45 ° C for 24 hours to obtain a mixed extract of sea cucumber and sea mustard, A solid of 100 mesh was sieved and used in the following examples.

Manufacturing example  3. Preparation of sea cucumber and sea mustard mixed extract 2

After removing the viscera, the seaweed washed with boiled sea cucumber was dried at a temperature of 100 ° C or higher. 25 g of dried sea cucumber and 20 g of seaweed were mixed and pulverized to 5 mm or less, and 51 ml of purified water was added to prepare a mixed solution. 2 g of tunicase and 2 g of alcalase were added thereto and sterilized at 110 ° C. for 20 minutes in an enzyme reactor. The mixture was kept at 45 ° C. for 24 hours to obtain a mixed extract of sea cucumber and sea mustard, A solid of 100 mesh was sieved and used in the following examples.

Manufacturing example  4. Preparation of sea cucumber and sea mustard mixed extract 3

After removing the viscera, the seaweed washed with boiled sea cucumber was dried at a temperature of 100 ° C or higher. 25 g of dried sea cucumber and 15 g of seaweed were mixed and pulverized to 5 mm or less, and 50 ml of purified water was added to prepare a mixed solution. 5 g of tunicase and 5 g of alcalase were added thereto and sterilized at 130 ° C. for 30 minutes in an enzyme reactor. The mixture was kept at 60 ° C. for 24 hours to obtain a mixed extract of sea cucumber and sea mustard, A solid of 100 mesh was sieved and used in the following examples.

Example  1. NO secretion of sea cucumber and marine mixed extract and TNF Increased effect of -α secretion

In order to measure NO secretion effect, the concentration of nitrite ion (NO ₂ ^- ) was measured by using a grease reagent based on NaNO ₂ as follows. RAW264.7 cells (ATCC The Global Bioresource Center ^TM) for the manufacture of 96-well micro-plate per well in 1 division, and by × 10 ^5, respectively 100㎍ / ㎖ concentration of sea cucumber extract, seaweed extract, and 100㎍ / ㎖ concentration Yes After culturing the mixture of sea cucumber and sea mustard extracts obtained in 2 to 4 at 37 ° C and 5% CO ₂ for 24 hours, the supernatant was taken, and 100 μl of the cell culture supernatant was mixed with the same amount of grease 1% (w / v) sulfanilamide in 5% (v / v) phosphoric acid) was added to each well and incubated at room temperature for 10 minutes. , And then absorbance was measured at 550 nm using UltroSpect 4000 spectrophotometer (Pharmacia Biotech, Uppsala, Sweden). In addition, 50 μl of the cell culture supernatant was taken under the same conditions and subjected to an ELISA test for TNF-α (inflammatory mediator) as tumor necrosis factor-α.

As a result, as shown in FIG. 1, the concentration of nitric oxide (NO) in the RAW 264.7 cells treated with the sea cucumber and the marine mixed extract of the present invention increased about 1.7 times as compared with the control group treated with sea cucumber and sea mustard extracts, In addition, TNF-α, an inflammatory mediator in the treatment of mixed sea cucumber and sea mustard extracts of RAW264.7 cells, was found to inhibit the secretion of sea cucumber and sea mustard extracts And the increase in the secretion of TNF-α and NO in the sea cucumbers and seaweed mixed extracts was increased by the similar increase of the LPS-treated group to that of the control group.

Example  2. A mixture of sea cucumber and sea mustard mixed extract Splenocyte  Proliferation effect

To determine whether the sea cucumber and sea mustard mixed extracts affected cell proliferation, splenocytes were isolated from C57BL / 6N mice to separate non-sorted spleen cells, and then 1 × 10 ⁶ spleen cells were dispensed into 96-well microplates. The sea cucumber extracts and the seaweed extracts at a concentration of 100 μg / ml and the seaweed extracts obtained in the above Preparation Examples 2 to 4 were treated for 24 hours at a concentration of 100 μg / ml, respectively, and cultured for 24 hours using the MTS method (CellTiter96 ™ AQueous Assay) Proliferation was measured. As a result, as shown in the following Table 1, the cell proliferation effect was excellent in the group treated with the sea cucumber and sea mustard mixed extracts (Production Examples 2 to 4) than the control group (the group treated with the sea cucumber and the sea mustard extracts respectively) It was confirmed that the sea cucumber and the seaweed mixed extract induce the proliferation of spleen cells.

Splenocyte proliferation effect by treatment of mixed sea cucumber and sea mustard extracts in mouse splenocytes
contorl (%)
Sea cucumber extract
Seaweed extract Sea cucumber and seaweed mixed extract Production Example 2 Production Example 3 Production Example 4 100 ± 0.95 201.56 ± 0.45 180.7 ± 10.66 219.56 ± 14.77 233.11 + - 12.52 240.05 ± 0.66

Example  3. A mixture of sea cucumber and sea mustard mixed extract In splenocytes  Increased effect of cytokine expression

To determine whether the sea cucumbers and seaweed mixed extracts affect the production of T _H 1 cytokines secreted from splenocytes, spleens were taken from the mice to separate non-sorted spleen cells and then seeded in 96-well microplates Lt; ⁶ > splenocytes. The sea cucumber extracts and the seaweed extracts at a concentration of 100 μg / ml and the seaweed extracts obtained in the above Preparation Examples 2 to 4 were treated for 24 hours at a concentration of 100 μg / ml, respectively, and IFN-γ ELISA kit (mouse IFN- The cytokine secretion level was measured using an ELISA kit (BD) and a TNF-α ELISA kit (Quantikine Mouse TNF-α / TNFSF1A kit, R & D Systems). As a result, it was confirmed that IFN-y and TNF-a secretion potency was further increased in the group treated with sea cucumber and sea mustard mixed extracts as compared with the control group treated with sea cucumber and sea mustard extracts, respectively, as shown in Fig.

Claims (6)

1) removing the viscera and drying the sea cucumber and the washed seaweed at a temperature of 100 ° C or higher;
2) mixing the dried sea cucumber and the seaweed in step 1), pulverizing and adding water to prepare a mixed solution;
3) adding a cell membrane-degrading enzyme and a proteolytic enzyme to the mixed solution of step 2); And
4) immersing the mixed solution containing the cell membrane lyase and protease into an enzyme reactor, sterilizing the mixture at 100 to 140 ° C for 15 to 35 minutes, and maintaining the mixture at 40 to 65 ° C for 15 to 30 hours And a method for producing the sea cucumber and marrow mixed extract. The method according to claim 1, wherein the cell membrane-degrading enzyme is tunicase in step 3), and the protease is alcalase or esperase. A method for preparing sea cucumbers and seaweed mixed extracts. A sea cucumber and a seaweed mixed extract prepared by the manufacturing method of claim 1 or 2. 3. The health functional food composition for immunomodulating as claimed in claim 3, which contains the sea cucumber and the seaweed mixed extract as an active ingredient. A pharmaceutical composition for immunostaining comprising the sea cucumber and the seaweed mixed extract of claim 3 as an active ingredient. A composition for feed additive for immunity enhancement comprising the sea cucumber and the seaweed mixed extract of claim 3 as an active ingredient.

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