KR101704123B1 - Method for seperating bee venom containing active amines and food composition thereof - Google Patents

Abstract

I) dissolving the tablet bee venom in sterile distilled water; ii) centrifuging the dissolved bee venom to separate proteins of greater than 3 kDa; iii) lyophilizing the separated purified bee venom sub-layer after centrifugation; iv) separating and purifying melittin, afamin, phospholipase A2 and MCD peptides from the lyophilized bee venom using liquid chromatography to obtain the remaining fraction; And v) lyophilizing the remaining fraction. The present invention also relates to a method for separating bee venom from which proteins and peptides are removed. In addition, the present invention relates to bee venom from which proteins and peptides isolated by the above method are removed, and a food composition containing the same.

Description

TECHNICAL FIELD The present invention relates to a method for separating active amine-containing bee venom,

The present invention relates to a method for separating active bean-containing bee venom characterized by the removal of proteins and peptides and a food composition comprising active amine-containing bee venom.

Bee venom (bee venom) is a poisonous liquid from a honey bee's ovipositor with a specific gravity of 1.3, a pH of 5.2, bitter taste, and a weak aroma. When evaporated at room temperature, about 30% of the dried material remains and its composition is complicated. Bee venom is 75% protein and its main ingredients are potassium, sodium, magnesium, phosphorus, alanine, valine, arginine, tryptophan, methionine and histidine. The bee venom has been known to control rheumatoid arthritis and have anti-cancer, anti-inflammatory and antinociceptive effects. The efficacy of bee venom has been known for a long time. There is a record of the use of Hippocrates in the 4th century BC, and the fluid in the stomach of a bee is being introduced as a medicine good for humans. Since ancient times, it has been applied to the treatment as a kind of folk medicine in Korea. Bee venom is a poisonous bee that is extracted from harmful substances in the body and injected into the acupuncture points where the pain or acupuncture points are placed, thereby strengthening the immune system of the human body, thereby being used as a therapeutic treatment to make it possible to overcome inflammation and foreign substances by oneself. The main components of bee venom are melittin, apamin, adolapin and mast cell degranulation peptide (MCD-peptide), which are proteins. Bee venom enters the body once activated metabolism, maximizes the immune function to overcome the bacteria invaded from the outside. However, the exact mechanism of activation of immune function, the main pharmacological action of bee venom, has not yet been elucidated. Traditionally, bee venom has been applied to arthritis, connective tissues and other inflammatory and painful diseases. Rheumatoid arthritis, acute chronic arthritis, cervical vertebra, dysplasia of the intervertebral disc, spinal stenosis, fibromyalgia, postpartum, osteoporosis, and chronic inflammation. In particular, many studies have reported that the anti-inflammatory action of bee venom inhibits the expression of iNOS and COX-2 as well as the activity of TNF-a and NF-B and inhibits the production of prostaglandin E2 . In recent years, attempts have been made to apply bean sprouts to multiple sclerosis in western countries. There are cases where the body becomes lighter and muscle mobility improves after treatment. In Brazil, it is used to treat asthma, and in China, it is used to treat musculoskeletal diseases.

Bee venom is composed of more than 40 substances including peptides and proteins (eg enzymes) and active amines. These ingredients are used to stimulate the secretion of pituitary hormones, promote blood circulation (Table 1).

As can be seen from Table 1, anti-inflammatory and immunological effects of melonin and apamin among constituents of bee venom are known. However, the utility value of bee venom containing the active amines remaining after removal of the main proteins and peptides of bee venom has not been known to date.

Thus, the present inventors have developed a method for removing proteins and peptides from bee venom, separating bee venom containing active amine, and a food composition containing the active amine-containing bee venom.

Korean Patent No. 10-0758814

It is an object of the present invention to provide a method for separating active bean-containing bee venom, in which proteins and peptides are removed.

Another object of the present invention is to provide an active amine-containing bee venom isolated by the above method.

Yet another object of the present invention is to provide a food composition comprising said active amine-containing bee venom.

In order to achieve the above object,

i) dissolving the tablet bee venom in sterile distilled water;

ii) centrifuging the dissolved bee venom to separate proteins of greater than 3 kDa;

iii) lyophilizing the separated purified bee venom sub-layer after centrifugation;

iv) separating and purifying melittin, afamin, phospholipase A2 and MCD peptides from the lyophilized bee venom using liquid chromatography to obtain the remaining fraction; And

v) lyophilizing the remaining fraction;

And a method for separating a bee venom from which a protein and a peptide are removed.

As used herein, the term "bee venom (BV)" is a mixture of acidic and basic secretions produced in the abdomen of a bee (Apis mellifera), which is in the form of a colorless bitter liquid, the main ingredient of which is melittin such as melittin, apamin, and mast cell degranulating (MCD) peptides and enzymes, phospholipase A2 (PLA2), histamine and dopamine, and others And contains trace amounts of components. In embodiments of the present invention, it refers to the major components isolated from domestic beekeeping products.

As used herein, the term " purified bee-isotope " can be obtained by dissolving bee venom collected from bees or the like with water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, filtering and removing impurities, and freeze-drying.

As used herein, the term " active amine " includes 6 kinds of histamine, dopamine, norepinephrine, putrescine, spermidine and spermine, which are components of bee venom, and is involved in antioxidant, anti- (Tom Piek (1986), Venoms of the Hymenoptera, Academic press). The " active amine-containing bee-tingling " of the present invention refers to a component of bee venom from which proteins, peptides and amino acids have been removed through the purification method of the present invention, preferably comprising melitin, anaphthalene, MCD peptide and phospholipase A2 It means no tablets.

In an embodiment of the present invention, centrifugation in step ii) can be performed at 3,000 rpm for 1 hour to isolate proteins of 3 kDa or more.

In an embodiment of the present invention, after the step iv), it may be subjected to a decontaminating step, further comprising a step of removing the salt component of the buffer used in the liquid chromatography, and the removal of the salt component may be carried out using a decontamination filter Can be performed.

The present invention also provides a bee keeping composition characterized in that the separated proteins and peptides are removed by the above method. The enamel composition may contain an active amine.

In an embodiment of the present invention, the protein may be one in which all components other than the active amine are removed, preferably phospholipase A2, and the peptide is preferably selected from the group consisting of melittin, . ≪ / RTI >

The present invention also provides a food composition comprising said active amine-containing bee venom.

In the embodiment of the present invention, the bee keeping composition characterized in that proteins and peptides isolated by the above method are removed has a high antioxidant and anti-inflammatory effect and confirmed that allergen-inducing proteins have been removed. In addition, its safety was confirmed in the cytotoxicity results, confirming that it could be used as a food material.

The food composition of the present invention includes forms such as pill, powder, granule, infusion, tablet, capsule or liquid, and the food to which the composition of the present invention can be added includes, for example, various foods, Beverages, gum, tea, vitamin complex, and health supplement foods. There are no particular restrictions on other components other than the food composition containing at least one selected from the group consisting of bee venom, melitin and apamin as an active ingredient, which can be contained in the food composition of the present invention. Herb extract, food-aid additive or natural carbohydrate. In addition, the food-aid additive may further include food-aid additives. Examples of the food-aid additive include food-aid additives customary in the art, for example, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers and the like. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above . In addition to the above, the food composition of the present invention may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, etc. In addition, the manufacture of natural fruit juice and fruit juice drinks and vegetable drinks These ingredients may be used independently or in combination.

The food composition of the present invention may be in any form of meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, drink, alcoholic beverage, . The present invention may also be provided as a cosmetic composition comprising at least one selected from the group consisting of bee venom, melitin and apamin as an active ingredient. The cosmetic composition according to the present invention may be formulated into a cosmetic, a gel, a water-soluble powder, a fat-soluble powder, a water-soluble liquid, a cream or an essence by a conventional method for producing a cosmetic by adding a pH adjusting agent, a flavoring agent, an emulsifier, have.

The composition of the present invention may further comprise an appropriate carrier, excipient and diluent which can be prepared into eyedrops or eyedrops and subcutaneous injections and can be inoculated into a living body and routinely used in the preparation of eye drops or injections.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The present invention provides a method for effectively separating allergen-inducing components of bee venom and active bean-containing bee venom from which proteins and peptides are removed. In addition, the active amine-containing bee venom having the proteins and peptides removed by the separation method of the present invention has a high antioxidant and anti-inflammatory effect and can be used as a food material because of no cytotoxicity.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of a separation process of active amine-containing bee venom according to an embodiment of the present invention. FIG.
2 is a graph showing the separation of apamin and melitin using liquid chromatography according to an embodiment of the present invention (PBV: purified bee venom, ABV; active amine-containing bee venom of the present invention).
FIG. 3 is a graph showing the presence of an active amine by silver stain HPLC analysis according to an embodiment of the present invention (M; size marker, PBV, purified bee venom, ABV; bean poison containing active amine of the present invention).
4 is a graph showing the antioxidative effect of active amine-containing bee venom according to concentration according to an embodiment of the present invention (active amine-containing bee venom (ABV), purified bee venom (PBV), melitin (Mel); Phospholipase A2 (PLA2)).
FIG. 5 is a result of evaluating the antioxidant efficacy after treatment of acupuncture and artificial gastric juice with active amine-containing bee venom according to an embodiment of the present invention.
6 is a graph showing cytotoxicity of active amine-containing bee venom in RBL-2H3 cells according to an embodiment of the present invention.
7 is a graph showing concentration-dependent inhibitory effects of β-hexosaminidase secretion of active amine-containing bee venom according to an embodiment of the present invention.
FIG. 8 is a graph showing concentration-dependent effects of IL-4 secretion of active amine-containing bee venom according to an embodiment of the present invention.
FIG. 9 is a graph showing concentration-dependent effects of TNF-.alpha. Secretion of active amine-containing bee venom according to an embodiment of the present invention.

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 construed as being limited by these embodiments.

Example  One. From the tablets Melitin , Apamin , MCD Peptides  And enzyme component separation

First, 1 g of purified bee venom purchased from Chungjin Biotech Co., Ltd. was dissolved in 10 ml of sterilized distilled water. The fully dissolved tablet bee venom was placed in a 3 K centrifugal filter and centrifuged at 3,000 rpm for 1 hour. By this method, a bee venom solution having a protein having a molecular weight of 3 kDa or more removed was obtained.

The bee venom solution obtained after the centrifugation was lyophilized and pulverized.

Thereafter, the powdered bee venom was solubilized in a developing mortar for liquid chromatography to separate the bee venom containing active amine using liquid chromatography.

The liquid chromatographic conditions are as follows:

- Device: AKTA explorer

- Column: Sephadex TM200, 75, Source 15RPC ST, superdex peptide

- Experimental conditions: Ammonium formate (Sigma), acetonitrile (Sigma), trifluoracetic acid (Sigma) and water were used as analytical solvents.

Liquid chromatography was used to separate and purify melittin, afamin and MCD peptides, and the protein and peptide components were removed and the remaining fractions were obtained.

All of the remaining fractions were subjected to a decontamination filter to remove the base component of the buffer used for liquid chromatography. After removing the salt components in the buffer, the remaining fractions were lyophilized to obtain bee venom containing active amine (FIG. 1).

Example  2. Melitin , Apamin , Phospholipase A2 ( PLA2 ) Confirm separation

The proteins and peptides of melitin, afamin and PLA2 were separated by the separation method of Example 1 through liquid chromatography and protein electrophoresis (FIGS. 2 and 3).

Electrophoresis and silver stain analysis were performed as follows.

- Electrophoresis and silver stain: SDS-polyacrylamide gel prepared according to the method of Laemmli (1970) with 15% separation gel and 5% staking gel. The cells were stained with a silver staining kit (Bio-Rad Laboratories, USA) for 1 hour and 30 minutes at 100 V and 400 mA using a mini-protein electrophoresis assay unit (Bio-Rad Laboratories, USA).

Example  3. Identification of antioxidant effect

DPPH (2,2-Dihenyl-1-picrylhydrazyl) radical scavenging activity of the active amine-containing bee venom prepared in Example 1 was tested to confirm the antioxidant effect. The results are shown in Fig. Active amine containing bee venom (ABV) prepared in Example 1; Tablet bee venom (PBV); Melitin (Mel); Apamin (Apa); DPPH antioxidant experiments were carried out by setting phospholipase A2 (PLA2) to concentration (0.1 mg / Ml, 0.2 mg / Ml, 0.5 mg / Ml) Vitamin C was used as a positive control. The formula for obtaining antioxidant ability is as follows.

[100 - (A / B x 100)

A: absorbance of the sample at 517 nm

B: Absorbance of Blank at 517 nm]

As a result, it was confirmed that the antioxidant ability was more effective than the bee venom of the present invention in the active amine-containing bee venom isolated in Example 1, and that the active amine-containing bee venom of the present invention had a higher antioxidative capacity in a concentration-dependent manner (FIG.

Example  4. Identification of component changes by needle and gastric juice

It is generally known that protein and peptide components are degraded by saliva or gastric juice. Antioxidant activity of the active amine-containing bee venom was examined after treatment with saline and artificial gastric juice to determine whether the active amine-containing bee venom of the present invention and the peptide of the present invention could be used for edible purposes. The needle and artificial gastric juice were treated with bee venom, allowed to stand at room temperature and then lyophilized and used for the assay.

At this time, the artificial gastric juice was prepared by using 2 g of NaCl, 3.2 g of pepsin (800-2500 unit / mg protein) and 7 ml of HCL, and 10 mg of active amine bee venom was dissolved in 500 μl of artificial gastric juice. The antioxidant activity was analyzed by collecting 100 μl of each sample for a period of time.

As a result, it was confirmed that the active amine-containing bee venom of the present invention maintained antioxidative ability regardless of acupuncture and gastric juice treatment. This is due to the removal of the protein and peptide components from the active amine-containing bee venom of the present invention (FIG. 5).

Example  5. Confirm cytotoxicity and anti-inflammatory effect

And to confirm whether IL-4 and TNF-a secretion amount which are inflammatory cytokines are inhibited in mast cell (R2) activated beeon with activated amine according to the present invention. Tablet bee venom is known to have a strong anti-inflammatory effect, and it is known that melitin, a peptide component, plays a major role. Therefore, in this study, we tried to use the active amine bee venom as a food composition by isolating major components such as melitin and apamin and confirming whether they have such effects.

1) Cell culture: RBL-2H3 cells, which are Rat basophilic leukemia cells, were purchased from Korea Cell Line Bank (KCLB) and cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin (Dulbecco's modified Eagle Medium) medium at 37 ° C and 5% CO ₂ .

Cell viability was measured by MTT [3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyl-tetrazolium bromide] reduction method. 21) Rat basophilic Leukemia cell line (RBL-2H3) cells were plated in 96-well plates at a concentration of 1 × 10 ⁶ cells / mL in 100 μl aliquots and cultured for 24 hours. FBS and 1% Penicillin / Streptomycin (0, 100, 250, 500, 1,000, 2,000 mg / L), and the cells were cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. After that, MTT solution (5 mg / mL) corresponding to one tenth of DMEM was added and cultured at 37 ° C for 4 hours. The MTT was reduced and the medium was carefully removed so that the generated formazan did not follow the medium. Then, after drying for 30 minutes under the dark condition, 100 μL of dimethyl sulfoxide (DMSO) was added, and the mixture was stirred for 1 hour and absorbance was measured at 570 nm.

3) Degranulation assay: Degranulation assay from RBL-2H3 cells was performed by measuring the β-hexosaminidase secretion inhibitory activity of degranulation biomarker. RBL-2H3 cells were suspended in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycine in 24-well plates, and 2 × 10 ⁵ cells / mL cells and dinitropheny-immunoglobulin E (DNP-IgE , 0.5 μg / mL), and cultured at 37 ° C in a 5% CO ₂ incubator for 12 hours. Each cell was washed twice with Siraganian buffer (119 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.4 mM MgCl ₂ , 25 mM PIPES, 1 mM CaCl ₂ , 0.1% BSA, pH 7.2) _The cells were incubated at 37 ° C in a 5% CO 2 incubator at 0, 100, 250 μg / mL. The cells were incubated in a 2% incubator for 10 minutes and then diluted to a concentration of 0, 100 and 250 μg / mL in FBS and 1% penicillin-streptomycin- And reacted for 30 minutes. The reaction was terminated by adding dinitropheny-human serum albumin (DNP-HSA, 10 μg / mL) at 37 ° C in a 5% CO ₂ incubator for 2 hours and then in an ice bath for 10 minutes. Transfer 40 μL of the supernatant to a 96-well plate, add 40 μL of substrate buffer (2 mM of 4-p-nitrophenyl-N-acetyl-β-D-glucosaminide, sodium citrate, 0.05 M, pH 4.5) . After incubation, 200 μL of stop solution was added to each well. The reaction was terminated and absorbance was measured at 405 nm using a microplate reader (EL 808 series, BioTek, Vermont, USA).

Secretion of β-Hexosaminidase (%) =

Absorbance of control - Absorbance of sample added / Absorbance of control × 100

4) IL-4, TNF- α secretion measurement: 24-well plate in 2 × 10 ⁵ cells / mL of cells and DNP-IgE (0.5 μg / mL ) a back 37 ℃, 12 in a 5% CO ₂ incubator for dividing the After incubation, samples were treated with fresh DMEM medium (0, 100, 250 μg / mL). After incubation for 30 minutes, DNP-HAS (10 μg / mL) was added, incubated at 37 ° C in a 5% CO ₂ incubator for 2 hours, and incubated in an ice bath for 10 minutes. The cultured supernatant was transferred to a 96-well ELISA plate and the concentrations of IL-4, TNF-αIL-4 and TNF-α were measured.

Example  5-1. Cytotoxicity check

As a result of the cytotoxicity, the cytotoxicity of active amine bee venom was not confirmed even at a high concentration of 10 ug, and it was confirmed to be very safe for cells (Fig. 6).

Example  5-2. β- hexosaminidase Secretory function  Confirm

To investigate the antiinflammatory effects of active amine bee venom, the effect of β-hexosaminidase secretion on secretory granules of mast cells and basophil cells was measured in RBL-2H3 basophilic cell line. The result is shown in FIG. The secretion of β-hexosaminidase was decreased in a concentration-dependent manner on the basis of the control group, and the secretion of β-hexosaminidase was significantly inhibited at 10 μg / mL (p <0.05) (FIG. 7). From these results, it was confirmed that the active amine-containing bee venom of the present invention can inhibit the secretion of β-hexosaminidase, and thus can act as an anti-inflammatory agent.

Example  5-3. Inhibition of cytokine secretion Confirm

In order to investigate the effects of the active amine-containing bee venom of the present invention on cytokine IL-4 and TNF-α production in RBL-2H3 cells induced by DNP-IgE and HSA, the amount of cytokine secretion was measured by pretreating the sample . The results are shown in Figures 8 and 9. The amount of IL-4 secretion was significantly increased to 2.68 ± 0.1 pg / mL in the control cells induced with inflammation compared with the stable cells (without IgE-HSA), which did not induce inflammation with the antigen-antibody. However, when the active amine-containing bee venom was treated at the concentrations of 1 and 10 μg / mL, the concentrations were 2.13 pg / mL and 1.96 pg / mL, respectively (p <0.001). TNF-α was 26.1 ± 1.6 pg / mL in cells not treated with DNP-IgE and HSA, which was significantly lower than that in the control group (37.8 ± 1.00 pg / mL). However, when the active amine-containing bee venom of the present invention was treated at 0.1, 1, and 10 μg / mL, it was 33.3 ± 0.3, 32.3 ± 0.4 and 31.5 ± 0.5 pg / mL, respectively (p < 0.05, p < 0.05, p < 0.01).

Through the above experiments, it was confirmed that the active amine-containing bee venom of the present invention can exhibit anti-inflammatory action by decreasing IL-4 and TNF-α production.

< Manufacturing example  1> Manufacturing of food

<1-1> Manufacture of flour food

0.01-5.0 parts by weight of bee venom, from which the protein and peptide of the present invention have been removed, was added to wheat flour, and bread, cake, cookies, crackers and noodles were prepared using this mixture.

<1-2> Manufacture of soups and gravies

0.01-5.0 parts by weight of bee venom of bee venom removed from the protein and peptide of the present invention was added to the soup and juice to prepare health promotion meat product, noodle soup and juice.

<1-3> Preparation of ground beef

1 part by weight of bee venom from which the protein and peptide of the present invention were removed was added to ground beef to prepare ground beef for health promotion.

<1-4> Manufacture of dairy products

1 to 5 parts by weight of bee venom from which the proteins and peptides of the present invention have been removed were added to milk and various dairy products such as butter and ice cream were prepared using the milk.

<1-5> Manufacturing of Sunshield

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

A bee venom powder with the protein and peptide of the present invention removed was prepared.

Grains, seeds, and beeswax powder from which the protein and peptide of the present invention had been removed were blended in the following proportions.

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

(3 parts by weight) of the protein and peptide of the present invention,

(0.5 part by weight),

(0.5 parts by weight)

< Manufacturing example  2> Manufacturing of beverages

<2-1> Preparation of health drinks

(0.5%) in which the proteins and peptides of the present invention have been removed are homogenized in a homogenous mixture such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5% And sterilized at the moment, and then packaged in small containers such as glass bottles and plastic bottles.

<2-2> Preparation of vegetable juice

Vegetable juice was prepared by adding 5 g of bee venom with the protein and peptide of the present invention removed to 1,000 ml of tomato or carrot juice.

<2-3> Preparation of fruit juice

A fruit juice was prepared by adding 1 g of bee venom from which protein and peptide of the present invention had been removed to 1,000 ml of apple or grape juice.

Claims (6)

i) dissolving the tablet bee venom in sterile distilled water;
ii) centrifuging the dissolved bee venom to separate proteins of greater than 3 kDa;
iii) lyophilizing the separated purified bee venom sub-layer after centrifugation;
iv) separating and purifying melittin, afamin, phospholipase A2 and MCD peptides from the lyophilized bee venom using liquid chromatography to obtain the remaining fraction; And
v) lyophilizing the remaining fraction;
And an amine which is a physiologically active substance. The present invention provides a method for producing an antioxidant tablet bee venom,
Wherein the concentration of the bee venom is 0.1 to 0.2 mg / mL.
The method according to claim 1,
Wherein the centrifugation in step ii) is carried out at 3,000 rpm for 1 hour, wherein the protein and the peptide are removed and the amine is a physiologically active substance.
The method according to claim 1,
And removing the salt component of the buffer used in the liquid chromatography after the step iv), wherein the protein and the peptide are removed and the amine is a physiologically active substance.
An antioxidant tablet enamel composition comprising an amine which is a physiologically active substance from which proteins and peptides produced by the method of claim 1 are removed,
Wherein the concentration of the bee venom is 0.1 to 0.2 mg / mL.
The antioxidative tablet preserving composition according to claim 4, wherein the protein and the peptide are melittin, anaphlamin, a phospholipase A2 and an MCD peptide.
A food composition comprising the antioxidant tablet enrichment composition of claim 4.

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