KR20190060256A - A composition comprising peptide extracts of Yeonsan Ogye-egg and method thereof - Google Patents

Abstract

A composition containing a peptide extract of a Yeonsan-Ogye egg as an active component of the present invention exhibits an antioxidant effect due to excellent DPPH radical scavenging activity, hydroxy radical scavenging ability, superoxide radical scavenging ability, and Fe2+ chelating ability, and also has no toxicity to cells. Therefore, the peptide extract is expected to be usefully used as a pharmaceutical composition, a food composition, a cosmetic composition, and a feed composition for antioxidation.

Description

[0001] The present invention relates to a composition comprising a peptide extract as an active ingredient and a method for producing the peptide extract,

The present invention relates to a composition comprising a peptide extract as an active ingredient and a method for preparing the same, more specifically, to a method for producing a peptide having an amino acid sequence of SEQ ID NO: 1, wherein the protein of Yeonsan Ogye Egg is optimized through hydrolysis, The present invention relates to a pharmaceutical composition, a food composition, a cosmetic composition and a feed composition exhibiting an antioxidative effect using a protein extract.

Generally, there are no large differences in appearance between the five and the chicken, but the head is small, and crests differ from chickens in that they usually have strawberry-shaped vessels. Sometimes chambers, stamens and roses are visible, It has a red color. Beak blue or black, face and body purple or blue white, eye iris brown or black, earlobe blue or blue white, legs color or yellow. The neck is short and feathery, and the tail is also short, with soft feathers. Legs are short and fine hairs are on the ankle. Skin, light and bone are all dark purple. Chickens have three toes on the front and one on the back, while the pussy has another long toe above the back toe. Feathers are mostly white, and rarely black and white and only red in the chest. Eggs are smaller than chickens.

The pentagram of the flower is characterized by a purple strawberry shape with varying concentration depending on the season and temperature. The toes are three in the front and one in the back like a regular chicken. The color of the feathers was originally red, white, black, yellow or hybrids, but only 20% of the single colors such as black and white were the result of efforts to fix the lineage such as separation breeding in 1973. Black, 5% white and 15% other mixed colors.

An antioxidant is a substance that suppresses oxidation reaction and collectively refers to a substance that eliminates or attenuates the action of an oxide substance, including a hydroxy radical, a superoxide radical, hydrogen peroxide, etc., which is called an active oxygen or oxygen free radical . As a low molecular weight compound, glutathione, N-acetylcysteine, ascorbic acid,? -Tocopherol, butylated hydroxyanilide (BHA), catechin, quercetin, uric acid, bilirubin, glucose and flavonoid are known. , Tomatoes, blueberries and other natural antioxidants.

Therefore, it is urgently required to develop a composition which is safe in living body, stable in its effective component, and most effective than a substance having an existing antioxidative effect.

As a substance having such an antioxidative effect, Korea Patent No. 10-1631657 discloses an effect of providing a hydrolyzate of overtransferrin having an increased antioxidative function by hydrolyzing over-transferrin, an egg white protein, with TECP to produce a functional peptide have.

Accordingly, the inventors of the present invention have been continuing research to develop a substance which is safe for human body and has excellent antioxidative effect as a natural substance, and the extract of DPPH radical scavenging ability, hydroxy radical scavenging ability, superoxide radical scavenging ability, Fe 2+ chelation The present inventors have completed the present invention by discovering that they are excellent in antioxidative effect and have no cytotoxicity.

KR 10-1631657 B1, June 13, 2016

Accordingly, a technical problem to be solved by the present invention is to provide a composition which is safe as a natural edible material without cytotoxicity and has an excellent antioxidative effect.

In order to solve the above technical problems, the present invention provides an antioxidant composition comprising a peptide as an active ingredient.

In the present invention, the protease enzyme is used to produce the protein hydrolyzate, and thus the peptide can be produced by the process of the present invention. At this time, the protease is preferably used in the form of Alcalase, Bromelain, Flavourzyme, Neutrase, Papain, Protamex and the like Do.

In the present invention, fractions (or extracts) can be prepared by using an ultrafiltration membrane of a peptide (or a protease hydrolyzate) of a peptide of the present invention. At this time, the ultrafiltration membrane can be fractionated using 1 kDa, 5 kDa, 10 kDa, and 100 kDa filter paper.

In the present invention, it is possible to extract, isolate and purify peptides of hydrolyzed ovum eggs using an ion exchange resin.

The composition containing the peptide extract of the present invention can exhibit an excellent antioxidative effect owing to the excellent DPPH radical scavenging ability, hydroxy radical scavenging ability, superoxide radical scavenging ability and Fe 2+ chelating ability.

Accordingly, in the present invention, the term "computational pie" includes a pharmaceutical composition, a food composition, a cosmetic composition and a feed composition containing an extract of a peptide as an active ingredient.

The pharmaceutical composition of the present invention may contain the peptide extract in an amount of 0.01 to 80% by weight, preferably 0.1 to 60% by weight. However, this can be increased or decreased according to the need of the medicinal person, and it can be appropriately increased or decreased according to the situation such as the diet, nutritional status, disease progression, degree of inflammation.

The pharmaceutical composition of the present invention can be administered orally or parenterally and can be used in the form of a general pharmaceutical preparation. The preferred pharmaceutical preparations are those for oral administration such as tablets, hard or soft capsules, liquids, suspensions, etc. These pharmaceutical preparations can be prepared into conventional pharmaceutically acceptable carriers, for example, excipients such as excipients, Binders, disintegrators, lubricants, solubilizers, suspending agents, preservatives or extenders.

The administration amount of the pharmaceutical composition comprising the peptide extract of the present invention may be determined by a specialist depending on various factors such as the condition of the patient, age, sex, and complications, but is generally from 0.1 mg to 10 g , Preferably from 10 mg to 5 g. Also, the daily dosage of the pharmaceutical composition per unit dosage form, or a half, 1/3, or 1/4 dose thereof, may be contained, and may be administered 1 to 6 times per day.

However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and the active ingredient may be used in an amount of more than the above range since there is no problem in terms of safety.

The term antioxidative effect as used in the present invention refers to a substance which suppresses or suppresses the action of so-called oxide substances, including hydroxyl radicals, superoxide anions, hydrogen peroxide, etc., which are active oxygen or free radicals As used herein, the term " antioxidant composition ", " antioxidant ", " antioxidant composition "

According to one preferred embodiment of the present invention, the composition of the present invention provides an antioxidant food composition comprising a peptide extract as an active ingredient.

The food of the present invention may be, but is not limited to, a health supplement food, a health functional food, a functional food, etc., and includes the addition of a compound of the present invention or an acceptable salt thereof to natural foods, processed foods, .

The food composition of the present invention can be used alone or in combination with other food or food compositions, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, improvement, or therapeutic treatment).

Generally, the peptidic extract of the present invention may be added in an amount of 0.1 to 70% by weight, preferably 2 to 50% by weight, based on 100% by weight of the raw material of food or beverage in the production of food or beverage.

The effective amount of the peptide extract of the present invention can be used in accordance with the effective dose of the pharmaceutical composition. However, in the case of long-term consumption intended for health and hygiene purposes or health control purposes, , And the active ingredient can be used in an amount in the above range because there is no problem in terms of safety.

There is no particular limitation on the kind of the food. Examples of the food to which the peptide extract can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, Beverages, tea, drinks, alcoholic beverages and vitamin complexes, and other nutrients, but are not limited to these kinds of foods.

According to one preferred embodiment of the present invention, the antioxidant cosmetic composition comprising the peptide extract as an active ingredient is provided.

In the cosmetic of the present invention, the compounding ratio of the composition of the present invention can be appropriately selected depending on the type and the kind, quantity and form of other components to be compounded. Usually, with respect to the total amount of the cosmetic, To 20% by weight, preferably 0.01 to 10% by weight.

The cosmetic of the present invention may contain other various other ingredients as long as the desired effect of the present invention is not impaired. Examples of the other components include one or more selected from the group consisting of purified water, an emulsion, a surfactant, a lubricant, an alcohol, a gelling agent, a moisturizer, a buffering agent, an antiseptic, can do.

The cosmetic composition of the present invention may contain a skin penetration promoter for effective transdermal absorption. The skin penetration promoting agent is not particularly limited so long as it is a general skin penetration promoting agent used in the cosmetic composition in the art. For example, the skin penetration promoter of the present invention may be a fatty acid / ester compound such as dimethylsulfoxide, oleic acid, limonene, and the like.

The cosmetic composition of the present invention has antioxidant effect, and can be used particularly for troublesome skin, acne skin, atopic skin and the like.

The cosmetic composition of the present invention may be prepared in any of the formulations conventionally manufactured in the art and may be formulated as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleansing oil, Emulsion foundation, wax foundation, and spray, but are not limited thereto.

The cosmetic composition of the present invention may also be formulated as a softening agent, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the cosmetic composition of the present invention is used, transdermal absorption may be promoted by using physical methods such as shock waves, iontophoresis, electrical invasion, and microinfiltration.

According to one preferred embodiment of the present invention, the composition of the present invention provides an antioxidant dietary composition containing the peptide extract as an active ingredient.

The feed composition of the present invention can be seeded with conventional feeds, and the feed composition of the present invention can be added to a conventional feed composition to prepare a functional feed composition. In addition, the feed composition of the present invention may further comprise a functional ingredient other than the peptide extract. In the production of a functional food composition in which the peptide composition of the present invention is mixed with the peptide composition of the present invention, the peptide extract of the present invention is added in an amount of 0.01 to 30.00% by weight, preferably 0.01 to 20.00% % ≪ / RTI > by weight. The effective amount of the peptide extract may be used in accordance with the effective dose of the food composition, but may be less than the above range for long-term consumption for the purpose of continuous health control. There is no problem, so that it can be used in an amount exceeding the above range as necessary.

The feed composition of the present invention is intended for livestock or poultry. The livestock or poultry may be any kind of animal such as cattle, pigs, chickens, horses, sheep, donkeys, mules, boars, rabbits, quail, ducks, fowls, poultry chickens, pigeons, turkeys, dogs, cats, monkeys, hamsters, , Parrots, parakeets, canaries, and the like, but are not limited to mammals or birds other than humans capable of breeding in the home.

The composition comprising the peptide extract as an active ingredient of the present invention is excellent in antioxidative effect due to DPPH radical scavenging activity, hydroxyl radical scavenging ability, superoxide radical scavenging ability and Fe 2+ chelating ability, It is expected that it can be usefully used as a pharmaceutical composition, a food composition, a cosmetic composition and a feed composition.

FIG. 1 is a schematic view showing a process for producing a peptide of a computed egg using a protease according to the present invention.
FIG. 2 shows the antioxidant activity of peptides according to the present invention (A: DPPH radical scavenging activity, B: hydroxy radical scavenging activity, C: superoxide radical scavenging activity, D: Fe 2+ chelation ability)
FIG. 3 shows the amino acid analysis results of peptides according to the present invention (A: alcalase, B: bromelain, C: flavourzyme)
FIG. 4 shows the amino acid analysis results of peptides according to the present invention. FIG. 4 shows the result of amino acid analysis of peptides according to the present invention (A: neutrase, B: papain, C: protamex)
FIG. 5 is a graph showing the results of RF-HPLC analysis of the enzyme-specific peptides of the egg yolk according to the present invention (A: alcalase, B: bromelain, C: flavourzyme)
FIG. 6 is a graph showing the results of RF-HPLC analysis of the enzyme-specific peptides of the egg yolk according to the present invention (A: neutrase, B: papain, C: protamex)
7 is a graph showing the results of MALDI-TOF analysis of the enzyme-specific peptides of mature egg according to the present invention (A: alcalase, B: bromelain, C: flavourzyme)
FIG. 8 shows the results of analysis of the enzyme-specific peptides of mature egg according to the present invention by MALDI-TOF (A: neutrase, B: papain, C: protamex)
9 shows the distribution of the molecular weight (A) and the distribution of the sample (B) of the peptides according to the present invention.
10 shows the antioxidant activity of the ultrafiltrate extract of peptides according to the present invention (A: DPPH radical scavenging ability, B: hydroxy radical scavenging ability, C: superoxide radical scavenging ability, D: Fe2 + chelation ability)
FIG. 11 shows the result of analyzing the antioxidative activity of an ultrafiltrate extract of peptides using an ion exchange resin (Dowex 1 × 2, 200 mesh chromatography (a), and the IC 50 value (mg / ml) of each fraction was measured by DPPH. (b) Different letters indicate significant differences (P <0.05)).
FIG. 12 shows chromatogram results of peptides using a gel permeation membrane (sepadex C-25 gel chromatography (a), and IC50 value (mg / ml) of each fraction was measured by DPPH. (b). Different recipients are significantly different ( P <0.05)
FIG. 13 shows HPLC chromatogram results of an ultrafiltrate extract of peptides according to the present invention.
Fig. 14 shows the results of MALDI-TOF chromatogram of ultrafiltration extract of peptides according to the present invention.
Fig. 15 shows the results of peptide structure analysis by LC-MS of ultrafiltration extract of peptides according to 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 only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Experimental Example  One : Protease  Used operation Phenotypic peptide  Production and antioxidant properties

1. Experiment  Materials and reagents

The Folin ciocalteu`s phenol (FCP) reagent used in this experiment was purchased from Sigma Company (Seoul, Korea). Tri-chloro-acetic (TCA) acid was purchased from Sigma Company (Seoul, Korea). Protease enzymes such as alcalase, bromelain, flavourzyme, neutrase, papain and protamex were purchased from Dae Jong Sang (Seoul, Korea). The eggs used in the experiment were provided by Jisan Plant (Nonsan, Choongnam, Korea).

2. Protein Hydrolyzate  Manufacturing and Extraction

The eggs used in this experiment were provided in the field farms. The eggs were heated in a constant temperature water bath at 100 ℃ for 20 minutes to separate the egg yolks and whites. Only the whites were separated and put in a zipper bag. The sample name and date of manufacture were recorded and stored at -20 ℃. For the proteolytic cleavage, 6 species were selected for protease type (Table 1). Serine protease, Alcalase, Protamex, and aspartic protease flavourzyme were selected and processed. These proteases were selected for use in foods considering the functionality of developing antioxidant health functional foods.

Product name Enzyme class Origin optimum Optimum Temp. Optimum Temp. Time, PH ℃ ℉ min. Alcalase &Quot; Protease (Subtilisin)
serine endoprotease " Bacillus licheniformis 4
8 50
85 122
185 30
10 Flavourzyme " Aminopeptidase
exopeptidase &quot; Aspergillus  oryzae 6-8 90 194 10 Neutrase "Protease (neutral)
metallo endoprotease &quot; Bacillus amyloliquefaciens 4
7 50
80 122
176 30
5 Protamex &Quot; Protease (Subtilisin)
serine endoprotease &quot; "Bacillus licheniformis "
&Quot; B acillus amyloliquefaciens " 4
8 50
85 122
185 30
10 Bromeline protease pineapple 5.0-8.0 50-60 120-140 N / S Papain protease carica papaya 5.0-7.5 50-70 N / S N / S

As shown in FIG. 1, 10% water or PB buffer was added to the sample, and 3% of the sample was mixed using a fermentor in the enzyme treatment, and then reacted in the reactor at 50 ° C and 100 rpm for 4 hours. After the reaction was completed, it was heated at 100 占 폚 for 30 minutes for inactivation. The supernatant was recovered by centrifugation at 3,000 rpm for 10 min. The hydrolyzed supernatant was freeze-dried in a cryogenic freezer (-80 ° C) and lyophilized to powder.

The hydrolyzate was prepared by using the above six enzymes, and the yield was calculated by weight / weight ratio of extract powder for hydrolysis × 100 (Table 2). The yield of protamex hydrolyzate was the highest at 46.3% and the yield of alcalase hydrolyzate was the lowest at 17.9.

Name of enzymes Yield of hydrolysate (%) Alcalase hydrolysate 19.5 Bromelain hydrolyzate 29.7 Flavourzyme hydrolysate 39.4 Neutrase hydrolysate 35.4 Papain hydrolysate 45.7 Protamex hydrolysate 46.3

In order to compare the antioxidative inhibitory ability of the hydrolysates of the five species, the hydrolysates were evaluated for their IC _{50 values} and shown in Table 3 below.

Hydrolysates Antioxidant activities DPPH radical
scavenging
activity Superoxide anion radical scavenging activity Hydroxy radical
scavenging activity Fe ^{2 +}
chelating
activity (mg / ml) Alcalase hydrolysate 4.88 5.3 19.72 1.24 Bromelain hydrolyzate 2.46 12.4 24.76 3.99 Flavourzyme hydrolysate 3.40 10.3 22.11 1.25 Neutrase hydrolysate 3.64 6.1 19.08 6.36 Papain hydrolysate 3.82 6.5 14.37 4.58 Protamex hydrolysate 1.93 6.8 14.54 17.56

3. Protein hydrolysis degree (degree of hydrolysis)

1 ml of protein hydrolyzate of egg and 1 ml of 1 N FCP (Folin-Ciocalteu`s penol reagent) was added to 1 ml of 0.5 N NaOH, and the mixture was immediately mixed with a voltex. The mixture was incubated at 30 ° C for 15 minutes Followed by filtration. The filtered solution is measured for 578 nm Absorbance. The amount of protease hydrolysis was measured using the L-Tyrosine standard curve, and the standard curve was obtained. Y = 0.0078X + 0.0182. The degree of hydrolysis was calculated from the degree of hydrolysis DH (%).

4. DPPH  Radical Scatters

The electron donating ability of DPPH-radical was measured according to the method of Huang et al. (2006). The extracted sample was dissolved in distilled water to a constant concentration (01%), and then 2 mL of the sample-containing solution and 0.5 mL of the DPPH-radical (0.2 mM) solution were mixed. The mixture was stored at room temperature for 30 minutes in a dark room and the absorbance was measured at 517 nm. As a control, ascorbic acid was used. DPPH radical scavenging activity was calculated by the following equation.

DPPH-radical scavenging activity (%) = [(BA) / B] x 100

A: absorbance at the time of sample addition, B: absorbance at the time of adding no sample

As shown in Fig. 2 (A), DPPH antioxidant capacity analysis results of hydrolysates produced under different protease and hydrolysis conditions are shown. The DPPH antioxidant capacity of the hydrolysates of the five species was affected by the type of enzymes used. This is probably due to the different peptide types of the hydrolysates due to different sites of action of the enzymes on the peptide bonds. The DPPH antioxidative capacity of the hydrolysates was about 25% to 60%. Alkalase, Flavourzyme, neutrase, and papain were similar to each other, but bromelain and protamex hydrolysates were relatively high. According to the research results, the antioxidative capacity of peptides is known to be 1) nonpolar and aromatic amino acids in the constituent amino acids, and 2) antioxidative ability in the smaller molecular weight. These results explain why antioxidant capacity differs depending on the protease enzyme used.

5. Hydroxy  Radical Scatters

The scavenging activity for hydroxyl radicals was measured by modifying Avellar's method (Avellar et al 2004). 10 mg of ascorbic acid was mixed with 10 ml DIW (deionized water). For the control, 0.1 M sodium phosphate buffer (pH 7.4) is used, and samples are prepared at 10,000 ppm and 20000 ppm. For the radical reaction, 300 μl of the sample solution, 300 μl of 3 mM 1,10-phenanthroline, 300 μl of 3 mM FeSO _{4 ,} and 300 μl of 0.01% hydrogen peroxide were mixed and incubated at 37 ° C. for 1 hour with shaking. The absorbance was measured at 536 nm using a spectrophotometer Were measured. Hydroxyl radical scavenging activity was calculated by the following equation by comparing the absorbance of the sample to the control.

Hydroxyl radical scavenging activity (%) = {[(ΔA / min) b- (ΔA / min) s] / (ΔA / min) b} × 100

b: control, s: sample, ΔA / min: absorbance after reaction absorbance before reaction

As shown in Fig. 2 (B), the results of analysis of the antioxidant ability of hydroxy radical-scavenging antioxidant ability of the protein hydrolysates of five proteins produced using different proteases are different. Hydroxy radical scavenging antioxidant capacity of hydrolysates of the five species was affected by the type of enzymes used. The antioxidative capacity of the hydrolysates ranged from 20.0% to 30.0%, the highest in alcalase and the lowest in neutrase. 20% or more, showing similar tendency to DPPH radical scavenging activity and superoxide radical scavenging activity. These results may be due to the different types of hydrolyzate peptides, because the sites of action of the enzymes on the peptide bond are different. Research shows that the smaller the antioxidant capacity of peptides, the higher is known. In addition, researchers from other researchers have reported that the increase in hydroxy radical scavenging power is reduced by peptide size, which suggests that peptides with lower molecular weights are more effective as stronger hydroxy radical scavengers than peptides with higher molecular weights. This is consistent with the fact that the antioxidant capacity of hydrolysates is different depending on the proportions of the hydroxy radical removing peptide and the non-eliminating peptide.

6. Superoxide Scatters

The superoxide scavenging activity was measured by modifying Xie's method (Xie et al 2008). As a control, 50 mM TrisHCl buffer solution (pH 8.3) was used. The reaction was carried out by mixing 500 μl of the peptide in 500 μl of 50 mM TrisHCl buffer (pH 8.3) prepared by using a micro tube, mixing 400 μl of 1.5 mM pyrogallol solution, reacting at room temperature for 4 minutes and then using a spectrophotometer And the absorbance at 420 nm was measured. The superoxide scavenging activity was calculated by comparing the absorbance of the sample with that of the control.

Superoxide scavenging activity (%) = {[(ΔA / min) b- (ΔA / min) s] / (ΔA / min) b} × 100

b: Control. s: sample, ΔA / min: absorbance after reaction absorbance before reaction

As shown in Fig. 2 (C), the results of the analysis of superanion-cleaved antioxidant capacity of the hydrolysates of five proteins produced by using different proteases are shown. The antioxidant capacity of hydrolysates of the five species was affected by the type of enzymes used. The antioxidant capacity of the hydrolysates was about 20.0% to 60.0%, which was lower in Flavourzyme and bromelain, and similar in the rest. These results may be due to the different types of hydrolyzate peptides, because the sites of action of the enzymes on the peptide bond are different. It is also considered that the kind of radicals to be cleaved differs depending on the hydrolyzate.

7. Fe2 + Chelation ability

Iron chelation ability was measured according to the method of Le et al. (2007). The extracted samples were dissolved in distilled water at a constant concentration (0-1%). 500 μL of the extracted sample, 100 μL FeCl ₂ (0.6 mM) and 900 μL of methanol were added to the test tube and mixed. After 5 min of incubation at room temperature, 100 μL ferrozine (5 mM) was added to the mixture and reacted at room temperature for 10 min. The absorbance was measured at 562 nm and ethylenediaminetetraacetic acid (EDTA) was used as a control.

Iron chelation activity (%) = (1A / B) x 100

A: absorbance at the time of sample addition, B: absorbance at the time of adding no sample

As shown in FIG. 2 (D), the chelating ability of the hydrolyzate showed a distribution ranging from about 30.0% to 70.0%, the highest in alcalase and the lowest in protamex. Other studies have reported that metal chelating abilities are strongly influenced by protein type, enzyme type and enzyme concentration. Alcalase hydrolysis products showed significantly higher metal chelating power than pepsin-pancreation hydrolysis products, possibly due to differences in the types of peptides produced in each enzyme. The metal chelating ability of the hydrolyzate also increased with increasing enzyme concentrations from 86.43% to 93.09%, indicating that a greater number of peptides were liberated with increasing enzyme steps. Also, the metal chelation capacity decreased with increasing peptide size, suggesting that lower molecular weight peptides have a greater effect on metal chelation.

8. Peptides  Amino acid analysis

9 g of Phellinus linteus and 30 mL of PB were mixed and pulverized. Then, 1% of BM1200 was reacted at 50 MPa for 5 hours, and the supernatant was centrifuged at 3,000 rpm for 20 minutes. 0.1 mL of the supernatant sample was weighed into an 18 mL test tube, 5 mL of 6 N HCl was added, and the sample was hydrolyzed in a heating block set at 110 ° C for more than 24 hours. After hydrolysis, remove acid with rotary evaporator at 50 ℃, dilute with 10 mL of sodium dilution buffer, add 1 mL of the solution, and filter with membrane filter 0.2 μm (Millipore Co., MA, USA). The filtered samples were analyzed using an amino acid autoanalyzer (S433-H, Sykam GmbH, Eresing, Germany). Columns were quantitatively analyzed by cation separation column (LCA K06 / Na, 4.6 × 150 mm). The column temperature was 57-74 ° C. The flow rates of the buffer and o-phthalaldehyde (OPA) reagents were 0.45 mL / min and 0.25 mL / min, respectively. The pH range of the buffer solution was 3.45~10.85 and the wavelength was 440 nm And 570 nm, respectively.

As shown in FIG. 3, A represents the distribution of hydrolyzed amino acids using alcalase enzyme, B represents the distribution of hydrolyzed amino acids using bromelain enzyme, and C represents the hydrolyzed amino acid distribution using flavorzyme enzyme . A shows the distribution of constitutive amino acids and essential amino acids (valine, methionine, isoleucine, leucine, phenylalanine, lysine) when hydrolyzed with alcalase enzyme. Of the constituent amino acids, histidine was the most abundant with 31.32% and valine was the most abundant amino acid among 9.33%. B showed the distribution of constitutive amino acids and essential amino acids (valine, methionine, isoleucine, leucine, phenylalanine, lysine) when hydrolyzed with bromelain enzyme. Among the constituent amino acid distributions, cystine was the highest content with 15.82% and lysine was the highest content among the essential amino acid distributions as 9.60%. C showed the distribution of constituent amino acids and essential amino acids (valine, methionine, isoleucine, leucine, phenylalanine, lysine) when hydrolyzed with flavorzyme enzyme. Among the constituent amino acid distributions, leucine occupied the highest content of 10.76%, and it occupied the largest content among essential amino acid distributions. In addition, trytophan was not found in the amino acid distribution of alcalase, bromelain, flavorzyme, especially proline was not found in alcalase and bromelain enzyme.

As shown in FIG. 4, A represents the distribution of hydrolyzed amino acids using neutrase enzyme, B represents the distribution of amino acids hydrolyzed using papain enzyme, and C represents the distribution of hydrolyzed amino acids using protomex enzyme . A shows the distribution of constituent amino acids and essential amino acids (valine, methionine, isoleucine, leucine, phenylalanine, lysine) when hydrolyzed with neutrase enzyme. Of the constituent amino acid distributions, histidine was the highest at 34.03% and valine was the highest at 12.20%. B showed the distribution of constitutive amino acids and essential amino acids (valine, methionine, isoleucine, leucine, phenylalanine, lysine) when hydrolyzed with papain enzyme. Among the constituent amino acid distributions, histidine was the highest content of 40.32%, and leucine was the most abundant among essential amino acid distributions. C showed the distribution of constitutive amino acids and essential amino acids (valine, methionine, isoleucine, leucine, phenylalanine, lysine) when hydrolyzed with protomex enzyme. Of the constituent amino acids, histidine was the most abundant (48.37%) and valine (8.61%) was the most abundant amino acid. No trytophan was found in the amino acid composition of neutrase, papain and protamex enzyme. The highest total amino acid content was found in flavorzyme enzyme and 43.73% of total amino acids were essential amino acid.

The constituent amino acids of hydrolysates hydrolyzed with Alcalase, bromelain, flavourzyme, neutrase, papain and protamex enzymes were classified into polar amino acids, nonpolar amino acids, cation amino acids and anionic amino acids. The content of polar amino acids was highest when serine was hydrolyzed by alcalase, flavourzyme, protamex enzyme, and when cystine was hydrolyzed by bromelain or neutrase enzyme, cystine was the highest. When hydrolyzed by papain enzyme glycine is the highest content. The non - polar amino acid contents showed the highest contents of leucine when hydrolyzed in all flavourzyme and papain enzymes, valine in alcalase and protamex enzyme showed the highest contents, phenylalanine in bromelain enzyme showed the highest contents, neutrase Proline is the highest content in the enzyme. Cationic amino acid content was highest when histidine was hydrolyzed with alcalase, bromelain, neutrase, flavourzyme, papain and protamex enzyme. The amount of anionic amino acid was highest when aspartic acid was hydrolyzed by neutrase enzyme And glutamic acid is the highest content in alcalase, bromelain, flavourzyme, papain and protamex enzyme.

Amino acids Alcalase Bromeline Flavourzyme Neutrase Papain Protamex Comostion  of percent of amino acids ( % ) Nonpolar side chains Proline N.D. N.D. 1.10 14.94 1.23 N.D. Alanine 3.57 1.87 6.57 1.55 1.68 1.43 Valine 9.33 4.82 9.04 12.20 4.84 8.61 Methionine 4.94 6.53 5.00 4.29 7.60 3.35 Isoleucine 1.33 3.13 7.19 N.D. 1.17 3.92 Leucine 5.19 8.79 10.76 2.94 9.15 7.91 Phenylalanine 7.83 8.96 7.10 4.92 6.47 3.09 Polar side chains Threonine 1.88 2.78 5.93 0.92 2.72 0.85 Serine 7.98 5.90 8.54 2.64 3.48 5.44 Glycine 0.42 0.15 2.50 0.95 4.35 0.85 Cystine 4.42 15.82 1.70 4.16 1.96 4.94 Tyrosine 2.26 6.02 3.06 3.88 1.04 1.85 Charged side chains Basic Histidine 31.32 15.23 6.81 34.03 40.32 48.37 Lysine 4.29 9.60 5.69 0.62 3.99 0.44 Arginine 1.88 7.46 6.47 8.07 6.72 0.51 Acids Aspartic acid 2.05 1.31 5.08 2.17 0.94 4.22 Glutamic acid 11.29 1.64 7.46 1.69 2.33 4.23 Total 100.00 100.00 100.00 100.00 100.00 100.00

9. RF- HPLC  Analysis by

Each enzyme was hydrolyzed and the lyophilized sample was dissolved in 0.1% TFA and 0.4um filtrated. Separate each peptide using HPLC (Jasco., Co.). The column used was Protein & peptide C18, and 0.1% TFA / H ₂ O in solvent A and 0.1% TFA / Acetonitrile in solvent B were re-fractionated at a flow rate of 1 ml / min. Peptide component detection was carried out at 220 nm using a UV detector.

Gradient is _{0.1% TFA / H 2 O 70} %, 0.1% TFA / Acetonitrile 30% 1 _{minutes, 0.1% TFA / H 2 O} 55%, 0.1% TFA / Acetonitrile 30 minutes by 45%, 0.1% TFA / H 2 O solvent was changed to 70%, 0.1% TFA / Acetonitrile 30% for 1 minute, 0.1% TFA / H ₂ O 70% and 0.1% TFA / Acetonitrile 30% for 9 minutes.

As a result of HPLC analysis, the standard peptides are five standard samples of GLy-Tyr, 2. Val-Tyr-Val, 3. Met enkephalin, 4. Leu enkephalin and 5. Angiotensin II. The molecular weight of each of the standard samples is 1. Gly-Tyr 238.2, 2. Val-Tyr-Val 379.5, 3. Met enkephalin acetate 573.7, 4. Leu enkephalin 555.6, and 5. Angiotensin II acetate 1046.2.

As shown in FIG. 5, the graphs of A, B, and C were confirmed by comparing the hydrolyzed graphs by enzyme with standard peptide samples. A graph is hydrolysis with alcalase enzyme, and cleave of alcalse enzyme is glutamate, leucine, tyrosine, lysine, glutamine. The B graph is hydrolyzed with bromelain enzyme and the substrate of enzyme is lysine, alanine, tyrosine, glycine. C graph is hydrolyzed with flavorzyme enzyme. The substrate of enzyme is tryptophan, isoleucine, phenylalanine, throsine, leucine, valine, lysine, arginine, serine, threonine and alanine. All of the results of the HPLC analysis compared to the standard samples are within the standard peptide distribution. However, the distribution of peptides for each enzyme is different from that of the peak. A can be seen in three major parts. From the peak on the left, molecular weight values of 27.7, 39.3 and 19.1 mV were obtained. B showed the molecular weight values of 80.9 and 60 mV for the two peaks of the graph. You can see three peaks of graph C and see molecular weights of 34, 21.8, and 128.8 mV. As a result, the production of peptides is different for different enzymes.

As shown in FIG. 6, the graphs of A, B, and C were confirmed by comparing the hydrolyzed graphs by enzyme with standard peptide samples. A graph is hydrolyzed with neutrase enzyme, cleave is phenylalanine, throsine, leucine, valine. B graph is hydrolyzed with papain enzyme, and enzyme substrate is decomposed into phenylalanine, throsine, leucine, valine, lysine and arginine. The C graph is a tryptophan, isoleucine, proline, phenylalanine, throsine, leucine, and valine hydrolyzed with the protamex enzyme. All of the results of the HPLC analysis compared to the standard samples are within the standard peptide distribution. However, the distribution of peptides for each enzyme is different from that of the peak. A can be seen in three major parts. From the peak on the left, molecular weight values of molecular weights of 100.3, 70.5 and 12.8 mV were obtained. B showed molecular weight values of 42.9 and 70.8 mV for the two peaks of the graph. Graph C shows three large peaks and 24.3, 69.3 and 12 mV molecular weight values. As a result, the production of peptides is different for different enzymes.

10. MALDI - TOF  Analysis by

Each hydrolyzed sample was centrifuged, and the supernatant was taken, freeze-dried and lyophilized to obtain 0.002 g of the powdered sample and dissolved in 1 ml of 0.1% TFA / H ₂ O. The samples were filtered using a 0.2 μm membrane filter (Millipore Co.). For peptide molecular weight, 1 mg of alpha-cyano-4-hydroxy-cinnamic acid was dissolved in 0.1 mL of 70% acetonitrile and 0.1% formic acid. The concentration of the sample was 50 ~ 100 ppm, and the matrix sample and the sample were mixed at a ratio of 1: 1. MS plate was dried by placing 1 mL of the solution, and a yellowish sample was taken and analyzed using a mass spectrometer (MALDI-TOF, Voyager DE-STR, Applied Biosystems, Foster City, CA, USA).

MALDI-TOF-MS (Matrix Assisted Laser Desorption / Ionization-Time of Flight-Mass Spectrometry) analysis was performed to determine the molecular weight of the hydrolyzate hydrolyzed using protease. 2 μL of a hydrolyzate sample was attached to a glossy steel 384 target plate. The mixture of 10 mg / mL of α-Cyano-4-hydroxycinnamic acid (CHCA) in 0.1: 1 TFA and ACN 1: 1 was mixed and dried. Each sample was analyzed using MALDI TOF MS from a Flex Control 3.0 mass spectrometer (Bruker Daltonics, Germany). Data were collected from 300 laser shots in cationization and reflection mode at 100-4,000 m / z.

As shown in FIG. 7, MALDI-TOF mass spectrometry was used to analyze the molecular weight of a sample obtained by hydrolyzing egg yolk using alcalase, bromelain, and flavorzyme. The molecular weight range of peptides of the five eggs hydrolyzed with alcalase was about 420 - 2,050 Da, and the molecular weight range of the pentagonal peptides treated with bromelain enzyme was about 490 - 3,180 Da in the same conditions. The molecular weight of the peptide hydrolyzed with Flavourzyme was 440 - 3,430 Da. From these results, it was confirmed that the molecular weight of the egg was greatly reduced as compared with the molecular weight of the common chicken, and that the molecular weight of the egg was reduced in total as compared with the molecular weight of the egg. The result showed that the hydrolyzate obtained from the egg was swiftly introduced into the human body .

As shown in FIG. 8, the molecular weight of the hydrolyzate hydrolyzed by using neutrase, papain, and protamex was analyzed using a MALDI-TOF mass spectrometer. The molecular weight range of peptides was about 410 ~ 1,965 Da when treated with papain enzyme under the same conditions. The molecular weight of the peptide hydrolyzed with protamex was 430 - 2,050 Da. From these results, it was found that the range of molecular weight was similar to that of egg. Especially, the peptide hydrolyzed with papain enzyme showed very narrow molecular weight range, and the molecular weight range was greatly reduced.

As shown in Fig. 9 (A), the distribution of the molecular weights was confirmed as an average. In the case of peptide samples hydrolyzed with Flavourzyme, the highest molecular weight was found to be 1,914 Da, and the protease hydrolyzed peptide was found to have a small molecular weight of 958 Da. The peptide samples hydrolyzed with Alcalase enzyme showed 1,155 Da and the hydrolyzate with papain enzyme showed 1,198 Da.

As shown in Fig. 9 (B), the distribution charts of samples obtained by hydrolyzing five eggs using six enzymes are shown, respectively. The hydrolyzate hydrolyzed with egg yolk using Papain enzyme was distributed in a molecular weight less than 2,000 Da, and chromatogram showed 15 peaks. In the case of Flavourzyme showing the highest peak, 35 peaks showed a molecular weight of less than 3500 Da. In the case of bromelain, only 7 peaks were analyzed with a molecular weight of less than 3200 Da. 15 hydrolysates of Alcalase enzyme were analyzed and their molecular weights were found to be less than 2100 Da.

Experimental Example  2: Computational Phenomenon Protein Hydrolyzate Han Filter membrane  Extraction and Separation Process and Antioxidative Properties

1. Experiment  Materials and reagents

Proteolytic enzyme bromelain was purchased from Daejong Sang (Seoul, Korea). The eggs used in the experiment were provided by Jisan Plant (Nonsan, Choongnam, Korea). An ultrafilter (Amicon TCF-10; Amicon Co., USA) was used for ultrafiltration of the hydrolyzate of the five species.

2. Protein Hydrolyzate  Manufacturing and Extraction

The eggs used in this experiment were provided in the field farms. The eggs were heated in a constant temperature water bath at 100 ℃ for 20 minutes to separate egg yolks and whites. Only egg yolks were extracted and placed in a zipper bag. The sample name and date of manufacture were entered and stored frozen at -20 ℃. For the proteolytic cleavage, six protease types were selected and used (Table 5). Serine protease alcalase, protamex and aspartic protease flavourzyme were selected and processed. These proteases were selected for use in foods considering the functionality of developing antioxidant health functional foods. 10% water or PB buffer was added to the sample, and 3% of the samples were mixed using a fermentor. The mixture was reacted at 50 ° C and 100 rpm for 4 hours in the reactor. After the reaction was completed, it was heated at 100 占 폚 for 30 minutes for inactivation. The supernatant was recovered by centrifugation at 3,000 rpm for 10 min. The hydrolyzed supernatant was freeze-dried in a cryogenic freezer (-80 ° C) and lyophilized to powder. The supernatant was collected by centrifugation of the hydrolyzate, and filtered with N2 gas at 180 psi (1241 kPa) at 300 rpm at room temperature using an ultrafilter (Amicon TCF-10, Amicon Co., USA) . Samples were fractionated using 10 kDa filter paper. Samples obtained by filtration were lyophilized.

Product name Enzyme class Origin optimum Optimum Temp. Optimum Temp. Time, PH ℃ ℉ min. Alcalase 2.4L FG &Quot; Protease (Subtilisin)
serine endoprotease " Bacillus licheniformis 4
8 50
85 122
185 30
10 Flavourzyme 1000 L "Aminopeptidase
exopeptidase " Aspergillus  oryzae 6-8 90 194 10 Neutrase 0.8L "Protease (neutral)
metallo endoprotease " Bacillus amyloliquefaciens 4
7 50
80 122
176 30
5 Protamex &Quot; Protease (Subtilisin)
serine endoprotease " " Bacillus licheniformis "
&Quot; B acillus amyloliquefaciens " 4
8 50
85 122
185 30
10 Bromeline 1200 GDU protease pineapple 5.0-8.0 50-60 120-140 N / S Papain T100 protease carica papaya 5.0-7.5 50-70 N / S N / S

The hydrolyzate yields according to the five protease enzymes are shown in Table 6 below.

Name of enzymes Yield of hydrolysate (%) Alcalase 29.5 Bromelain Br1200 39.7 Flavourzyme 49.4 Neutrase 0.8L 45.4 Papain T100 55.7 Protamex 56.3 Bromelain 1200GDU 42.0

3. Protein hydrolysis degree (degree of hydrolysis)

1 ml of protein hydrolyzate of egg and 1 ml of 1 N FCP (Folin-Ciocalteu`s penol reagent) was added to 1 ml of 0.5 N NaOH, and the mixture was immediately mixed with a voltex. The mixture was incubated at 30 ° C for 15 minutes Followed by filtration. The filtered solution is measured for 578 nm Absorbance. The amount of protease hydrolysis was measured using the L-Tyrosine standard curve (Fig. 53), and the standard curve was obtained. Y = 0.0078X + 0.0182. After hydrolysis, the degree of hydrolysis was calculated to obtain the value of DH (%).

4. Han Filter membrane  Used Peptides  Extraction and fractionation

The samples requiring fractionation were prepared by using lyophilized hydrolyzate powder as a 1000 ppm solution or by centrifuging the hydrolyzate and collecting the supernatant by using an ultrafilter (Amicon TCF-10; Amicon Co., USA) at room temperature N ₂ gas was filtered at 300 rpm under a pressure of 180 psi (1241 kPa). Samples were fractionated using 1 kDa, 5 kDa, 10 kDa, and 100 kDa filter paper, respectively. Samples obtained by filtration were lyophilized.

5. DPPH  Radical Scatters

The electron donating ability of DPPH-radical was measured according to the method of Huang et al. (2006). The extracted sample was dissolved in distilled water to a constant concentration (01%), and then 2 mL of the sample-containing solution and 0.5 mL of the DPPH-radical (0.2 mM) solution were mixed. The mixture was stored at room temperature for 30 minutes in a dark room and the absorbance was measured at 517 nm. As a control, ascorbic acid was used. DPPH radical scavenging activity was calculated by the following equation.

DPPH-radical scavenging activity (%) = [(BA) / B] x 100

A: absorbance at the time of sample addition, B: absorbance at the time of adding no sample

As shown in FIG. 10 (A), DPPH antioxidant capacity analysis results of protein hydrolysates fractionated at 1 kDa, 5 kDa, 10 kDa, and 100 kDa using ultrafiltration are shown. The DPPH antioxidant capacity of protein hydrolysates was affected by the type of enzymes used. This is probably due to the different peptide types of the hydrolysates due to different sites of action of the enzymes on the peptide bonds. The DPPH antioxidant capacity of the hydrolysates was about 46.86% to 75.71%. DPPH scavenging activities were 1 kDa, 10 kDa, 5 kDa and 100 kDa. The highest activity was fractionated at 1 kDa and the highest activity was 75.71%. According to the results of research, antioxidative capacity of peptides was found to be 1) non - polar and aromatic amino acids in the constituent amino acids, and 2) These findings explain why antioxidant capacity differs depending on the size of ultrafiltration used.

6. Hydroxy  Radical Scatters

The scavenging activity for hydroxyl radicals was measured by modifying Avellar's method (Avellar et al 2004). 10 mg of ascorbic acid was mixed with 10 ml DIW (deionized water). For the control, 0.1 M sodium phosphate buffer (pH 7.4) is used, and samples are prepared at 10,000 ppm and 20000 ppm. For the radical reaction, 300 μl of the sample solution, 300 μl of 3 mM 1,10-phenanthroline, 300 μl of 3 mM FeSO _{4 ,} and 300 μl of 0.01% hydrogen peroxide were mixed and incubated at 37 ° C. for 1 hour with shaking. The absorbance was measured at 536 nm using a spectrophotometer Were measured. Hydroxyl radical scavenging activity was calculated by the following equation by comparing the absorbance of the sample to the control.

Hydroxyl radical scavenging activity (%) = {[(ΔA / min) b- (ΔA / min) s] / (ΔA / min) b} × 100

b: control, s: sample, ΔA / min: absorbance after reaction absorbance before reaction

As shown in FIG. 10 (B), the results of analysis of the hydroxy radical scavenging ability of protein hydrolysates fractionated at 1 kDa, 5 kDa, 10 kDa and 100 kDa using ultrafiltration are shown. Hydroxy radical scavenging antioxidant capacity of protein hydrolysates was ranged from 15.03% to 29.388%. The superoxide radical scavenging activity was 10 kDa, 10 kDa, 5 kDa and 1 kDa. The highest activity was fractionated to 100 kDa and the highest activity was 29.38%. Research shows that the smaller the antioxidant capacity of peptides, the higher is known. Studies by other researchers have also shown that the increase in the ability to remove hydroxy radicals has been diminished by peptide size, suggesting that lower molecular weight peptides are more effective as stronger hydroxy radical scavengers than higher molecular weight peptides. This is consistent with the fact that the antioxidant capacity of the protein hydrolyzate varies depending on the proportion of the peptide that does not remove the hydroxyl radical.

7. Superoxide  Radical Scatters

The superoxide scavenging activity was measured by modifying Xie's method (Xie et al 2008). As a control, 50 mM TrisHCl buffer solution (pH 8.3) was used. The reaction was carried out by mixing 500 μl of the peptide in 500 μl of 50 mM TrisHCl buffer (pH 8.3) prepared by using a micro tube, mixing 400 μl of 1.5 mM pyrogallol solution, reacting at room temperature for 4 minutes and then using a spectrophotometer And the absorbance at 420 nm was measured. The superoxide scavenging activity was calculated by the following equation by comparing the absorbance of the sample to the control.

Superoxide scavenging activity (%) = {[(ΔA / min) b- (ΔA / min) s] / (ΔA / min) b} × 100

b: control, s: sample, ΔA / min: absorbance after reaction absorbance before reaction

As shown in FIG. 10 (C), superoxide radical scavenging ability analysis of protein hydrolysates fractionated at 1 kDa, 5 kDa, 10 kDa, and 100 kDa using ultrafiltration is shown. The superoxide scavenging activity of protein hydrolysates was about 10.53% to 80.70%. Superoxide radical scavenging activity was similar to that of DPPH in the order of 5 kDa, 1 kDa, 10 kDa and 100 kDa. The highest activity was fractionated at 5 kDa and the highest activity was 80.70%. These results may be due to the different peptide types of hydrolysates. It is also considered that the kind of radicals to be cleaved differs depending on the hydrolyzate.

8. Fe2 + Chelation ability

Iron chelation ability was measured according to the method of Le et al. (2007). The extracted samples were dissolved in distilled water at a constant concentration (0-1%), and 500 μL of the extracted sample, 100 μL FeCl 2 (0.6 mM) and 900 μL of methanol were added to the test tube and mixed. After 5 min of incubation at room temperature, 100 μL ferrozine (5 mM) was added to the mixture and reacted at room temperature for 10 min. The absorbance was measured at 562 nm and ethylenediaminetetraacetic acid (EDTA) was used as a control.

Iron chelation activity (%) = (1A / B) x 100

A: absorbance at the time of sample addition, B: absorbance at the time of adding no sample

As shown in FIG. 10 (D), Ultrafiltration showed the ability to chelate metal ions of protein hydrolysates fractionated at 1 kDa, 5 kDa, 10 kDa, and 100 kDa. The chelating ability of the protein hydrolysates showed a distribution ranging from about 4.73% to 78.45%. The chelation capacity was in the order of 1 kDa, 5 kDa, 10 kDa and 100 kDa. The highest activity was fractionated to 1 kDa and the highest activity was observed at 78.45%. It can be seen that as the enzyme step is increased, a greater number of peptides are liberated. Also, metal chelate abilities decreased with increasing peptide size, suggesting that lower molecular weight peptides have a greater effect on metal chelation.

Experimental Example  3: Predominantly, Ultrafiltration membrane  Purification and characterization of extracted peptides using

1. Materials and reagents

Ultrafiltration was performed using an ultrafilter (Amicon TCF-10; Amicon Co., USA), and the ion exchange resin was Dowex 占 1 占 2, 200 (Sigma Co.). Sephadex G-25 gel was used for the gel filtration purification. A glass column of 2.5 × 100 cm size was purchased from Korean Chemical Society.

2. DPPH  Antioxidant measurement

The electron donating ability of DPPH-radical was measured according to the method of Huang et al. (2006). The extracted sample was dissolved in distilled water to a constant concentration (01%), and then 2 mL of the sample-containing solution and 0.5 mL of the DPPH-radical (0.2 mM) solution were mixed. The mixture was stored at room temperature for 30 minutes in a dark room and the absorbance was measured at 517 nm. As a control, ascorbic acid was used. DPPH radical scavenging activity was calculated by the following equation.

DPPH-radical scavenging activity (%) = [(BA) / B] 100

A: absorbance at the time of sample addition, B: absorbance at the time of adding no sample

3. Protein Hydrolyzate Ultrafiltration  Separation using

The samples requiring fractionation were prepared by using lyophilized hydrolyzate powder as a 1000 ppm solution or by centrifuging the hydrolyzate and collecting the supernatant by using an ultrafilter (Amicon TCF-10; Amicon Co., USA) at room temperature N ₂ gas was filtered at 300 rpm under a pressure of 180 psi (1241 kPa). Samples were fractionated using 3 kDa filter paper. Samples obtained by filtration were lyophilized.

4. Protein Hydrolyzate  Isolation using ion exchange resin

Dowex® 1 × 2, 200 resin was equilibrated in a column (2.5 × 100 cm), 1 g of the peptide was dissolved in 10 ml of tertiary distilled water, and 5 ml of the peptide was injected into the equilibrated resin. After 75 ml of the third distilled water, 0.1 M NaCl and 0.5 M NaCl were added to the eluate, and 75 ml of 1 M Nacle eluate were sequentially used. The fractions were collected by Teledyne ISCO Automatic Retriever 500 and the eluate was fractionated at 5 ml / 90 sec. The absorbance of each fraction was measured at 280 nm.

Ion exchange resins are chromatograms that separate proteins and peptides. In order to isolate the peptides showing antioxidant activity, the amount of protein in 60 fractions containing 5 ml was measured, and the fractions showing the same reaction were divided into 8 fractions. After lyophilization, the DPPH radical scavenging ability was compared and shown in FIG. 11 Respectively. The antioxidant capacity of tube E in the fraction IC ₅₀ = 1.5 mg / ml. The gel permeation chromatogram was subsequently carried out to remove the salts of the fractions.

5. Protein Hydrolyzate  Separation by gel filtration

1 g of the peptide was dissolved in 10 ml of tertiary distilled water, and 5 ml of the peptide was injected into a sephadex G-25 column (2.5 × 100 cm) which was equilibrated. The eluate was fractionated at 5 m / 90 sec. For the eluate, the third distilled water was used, and the absorbance of each fraction was measured at 280 nm. Fractions were obtained using a Teledyne ISCO automatic fractionator (Retriever 500) and fractions of 5 ml for 90 seconds.

The fraction showing the highest DPPH elimination ability in the peptide purification process using ion exchange resin was separated by peptide size and chromatogram was performed using a gel permeation membrane to remove used NaCl. In order to isolate the peptides showing the highest antioxidant ability, the amount of protein in 60 fractions containing 5 ml was measured, and the fractions showing the same reaction were divided into 6 fractions, followed by lyophilization, and compared with DPPH radical scavenging ability Respectively. The antioxidant capacity of tube D in the fractions was IC ₅₀ = 0.96 mg / ml. RP-HPLC was performed to further purify the fractions.

6. Protein Hydrolyzate  RP- HPLC  Separation using

The material was purified via HPLC. As a first step, partial purification was carried out using reversed phase HPLC. The separation conditions were as follows. Each enzyme was hydrolyzed and the lyophilized sample was dissolved in 0.1% TFA and 0.4um filtrated. Separate each peptide using HPLC (Jasco., Co.). The column used was Protein & peptide C18, and 0.1% TFA / H ₂ O in Solvent A and 0.1% TFA / Acetonitrile in solvent B were flowed at a flow rate of 1 ml / min. Peptide component detection was carried out at 220 nm using a UV detector.

Gradient is _{0.1% TFA / H 2 O 70} %, 0.1% TFA / Acetonitrile 30% 1 _{minutes, 0.1% TFA / H 2 O} 55%, 0.1% TFA / Acetonitrile 30 minutes by 45%, 0.1% TFA / H 2 O solvent was changed to 70%, 0.1% TFA / Acetonitrile 30% for 1 minute, 0.1% TFA / H ₂ O 70% and 0.1% TFA / Acetonitrile 30% for 9 minutes.

As shown in FIG. 13, fractions showing the highest DPPH elimination ability in peptide purification using a gel permeation membrane were fractionated using RF-HPLC to further purify the fractions. In order to isolate the peptides showing the highest antioxidative capacity among the fractions, the fractions corresponding to the peaks were subjected to freeze-drying.

7. Protein Hydrolyzate MALDI - TOF  Analysis by

The hydrolyzate sample was centrifuged and supernatant was taken and frozen. 0.002 g of the powdered sample was dissolved in 1 ml of 0.1% TFA / H ₂ O by using a freeze dryer. The samples were filtered using a 0.2 μm membrane filter (Millipore Co.). For peptide molecular weight, 1 mg of alpha-cyano-4-hydroxy-cinnamic acid was dissolved in 0.1 mL of 70% acetonitrile and 0.1% formic acid. The concentration of the sample was 50 ~ 100 ppm, and the matrix sample and the sample were mixed at a ratio of 1: 1. MS plate was dried by placing 1 mL of the solution, and a yellowish sample was taken and analyzed using a mass spectrometer (MALDI-TOF, Voyager DE-STR, Applied Biosystems, Foster City, CA, USA).

As shown in FIG. 14, the molecular weight of the purified oval egg with respect to the peptide was analyzed using a MALDI-TOF mass spectrometer. The molecular mass of the peptide of the egg was confirmed to be in the range of 400-1,200 Da, and the molecular weight of the hydrolyzate of the five egg group was found to be decreased in the range of 410 - 3,000 Da.

8. By LC-MS Peptides  Structure analysis

Amino acid sequences and molecular weights of the peptides showing the best DPPH eliminative activity among the samples separated and purified by RF-HPLC were measured. Ultramate 3000 chromatography (Dionex UHPLC, Thermo Fisher Scientific Inc., Seoul, Korea) was used for peptide separation. The elution solvents were deionized water (0.1% formic acid, A) and acetonitrile (0.1% formic acid, B). Gradient elution B; 1% (0 min), B; 1% (3 min), B; 50% (70 min), B; 97% (6 min), B; 10% (6.1 min), B; 100% (80 min), B; 1% (81 min) B; 1% (90 min). The flow rate was 300 μl / min and the sample injection amount was 3 μl. Amino acid sequence analysis was performed using Q-TOF 5600 (Sciex, Seoul, Korea). Positive ion mode method was used. The scan was performed at 0.6 second intervals, and the MS spectra was analyzed at m / z 100 to m / z 1800. The spray voltage was 10 kV and the temperature was 500 ° C.

The lyophilized fractions showing the highest DPPH elimination ability in repeated RF-HPLC were analyzed for amino acid structure and molecular weight using LC-MS. Figure 15A shows the chromatogram of RF-HPLC and B shows the amino acid mass chromatogram of the peak showing the highest antioxidant potential. The amino acid sequence of the study was TAAEAETEAEAETSNLT. The molecular weight was 1736.75 mV. This sequence was Thr-Ala-Ala-Glu-Ala-Glu-Thr-Glu-Ala-Glu-Ala-Glu-Thr-Ser-Asn-Leu-Thr.

Claims (4)

The antioxidant composition according to claim 1, wherein the antioxidant composition comprises a peptide as an active ingredient. The method according to claim 1,
Wherein the peptides are produced by using protease. The antioxidant composition according to claim 1, wherein the antioxidant composition comprises a fraction prepared by using an ultrafiltration membrane as a peptide. The antioxidant composition according to claim 1, wherein the antioxidant composition comprises a fraction prepared by using an ion exchange resin as a peptide.

Images