KR20140139366A - Composition containing anti-oxidative peptides - Google Patents

Abstract

The present invention relates to a composition including an anti-oxidant peptide having a radical removal function. According to the present invention, provided is a composition including an anti-oxidant peptide having a radical removal function to be used for developing various functional food products. Furthermore, usability and value-added of eggs can be enhanced.

Description

Composition containing anti-oxidative peptides [

The present invention relates to a composition comprising an antioxidant peptide having a radical scavenging ability.

The egg whites or albumin, which surrounds the egg yolk, account for 60% of the total weight of the egg. White contains proteins such as ovalbumin, ovotransferrin, ovomucin and lysozyme, which are often used as additives in food processing due to their functional characteristics.

Of various dietary peptides derived from the egg protein, peptides having antioxidative properties have been attracting attention. Antioxidants can protect the body from damage by free radicals such as reactive oxygen species (ROS) produced by the oxidative reaction and thus have a beneficial effect on human health. That is, antioxidants cause oxidative damage to all important cellular components and may be associated with the treatment of the onset of various diseases including hypertension, neurodegenerative diseases, inflammation, diabetes, cancer and aging.

Ovomucin, which is a glycoprotein that accounts for about 2 to 4% of the albumin protein in the egg protein, is an essential material for maintaining egg white viscosity. Ovomucin is composed of? -Units and? -Units linked by disulfide bonds, and these units contain carbohydrates of 10-15% and 50-65% as 220 kDa and 400 kDa, respectively. Conventional ovomucin proteins have been reported to have functional effects such as bubble generation and emulsifying activity, and have been reported to be effective in relieving low cholesterol levels by inhibiting cholesterol absorption and reabsorption of bile acid in the small intestine. Ovomucin is also known as an antimicrobial agent against Helicobacter pylori . However, no peptides having antioxidative activity derived from ovomucin or its hydrolyzate have been reported.

Currently, antioxidant peptides have been identified from egg white lysozyme (non-patent document 1) or hydrolyzate of obotransferrin (non-patent document 2) by papain, trypsin and combinations thereof.

 Memarpoor-Yazdi, M .; Asoodeh, A .; Chamani, J. A novel antioxidant and antimicrobial peptide from hen egg white lysozyme hydrolysates. J. Funct. Foods. 2012, 4, 278 - 286.  Shen, S .; Chahal, B .; Majumder, K .; You, S. J .; Wu, J. Identification of novel antioxidative peptides derived from a thermolytic hydrolyzate of ovotransferrin by LC-MS / MS. J. Agric. Food. Chem. 2010, 58, 7664 - 7672.

Under such technical background, the present inventors have made an effort to develop antioxidant peptides having radical scavenging ability and compositions containing them.

As a result, it is an object of the present invention to provide an antioxidative composition comprising an antioxidant peptide as an active ingredient.

In order to solve the above technical problem,

According to one aspect of the present invention, there can be provided an antioxidant composition comprising, as an active ingredient, an antioxidant peptide having an amino acid sequence represented by SEQ ID NO: 2.

In one embodiment, the composition may further comprise a hydrolyzate of ovomucin.

In one embodiment, the hydrolyzate of the ovomucin may have the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the hydrolyzate of the ovomucin is hydrolyzed by at least one enzyme selected from Alcalase, Flavourzyme, Neutrase and Protamex. .

According to the present invention, it is possible to provide a composition comprising an antioxidant peptide having a radical scavenging ability so as to utilize it in the development of various functional foods, and further to improve the availability and the added value of eggs.

Figure 1 shows the location of ovomucin (SDS-PAGE) isolated from whole egg protein. M represents the standard protein, 1 represents the ovomucin isolated in the present invention, and 2 represents the ovomucin of the present invention (Omana, DA; Wu, J. A New Method of Separating Ovomucin from Egg White, J. Agric. , 3596-3603).

Hereinafter, the present invention will be described in more detail.

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention. The contents of all publications cited herein are hereby incorporated by reference in their entirety.

Justice

In the present invention, the term "peptide" means a linear molecule formed by combining amino acids with each other by a peptide bond. The peptides of the present invention can be prepared according to synthetic methods known in the art, for example, the solid phase synthesis technique (Merrifield, J. Amer. Chem. Soc. 85: 2149-54 (1963 , Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co .: Rockford, 111 (1984)).

In the present invention, the term "antioxidant" means prevention of oxidation and prevents denaturation or destruction of cells due to an oxidation reaction caused by free radicals such as active oxygen species. The antioxidants can be made in a variety of mechanisms, and most antioxidants act by eliminating or eliminating radicals.

In the present invention, the term "antioxidant composition" means a composition comprising 20 to 50% by weight, preferably 30 to 40% by weight, of the composition or hydrolyzate containing the antioxidant peptide, based on the total weight of the composition, Acceptable excipients, carriers, excipients or diluents, which are well known to those of ordinary skill in the art.

According to one aspect of the present invention, there can be provided an antioxidant composition comprising, as an active ingredient, an antioxidative peptide having an amino acid sequence represented by the following general formula I or SEQ ID NO: 2.

[General Formula I]

Leu-Asp-Glu-Pro-Asp-Pro-Leu (LDEPDPL)

It is to be appreciated by those skilled in the art that the above-described general formula representing the peptide according to the present invention has been described in order to express the peptide structure of the present invention for convenience and modifications thereof belong to the present invention. For example, it is apparent to those skilled in the art that variants of the ovomucin-derived sequence in the general formula I belong to the scope of the present invention.

In one embodiment, the N-terminal or C-terminal of the peptide of the present invention is an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, a polyethylene glycol (PEG) A protecting group selected from the group consisting of amino acids can be additionally combined.

In one embodiment, the N-terminal or C-terminal of the peptide of the present invention may be modified with a hydroxy group (-OH) or an amino group (-NH ₂ ), and more preferably a hydroxyl group. Modification of the amino acid can greatly improve the stability of the peptide of the present invention. Here, the term "stability" means in vivo stability and storage stability (e.g., room temperature storage stability). The protecting group may function to protect the peptide according to the present invention from attack of a protein cleaving enzyme in vivo.

In one embodiment, the peptide is derived from ovomucin, which is one of the albumin proteins. The peptide has higher DPPH scavenging activity than the natural ovomucin protein, and has a short sequence (up to 9 amino acids The peptide of the present invention contains a radical scavenging ability and can be used for the development of functional foods having antioxidant activity.

Thus, a composition comprising an antioxidant peptide according to the present invention may be provided in the form of a composition for preventing or treating oxidative damage by free radicals in cells, tissues or organs by eliminating free radicals. In addition, the antioxidant composition may be provided as a feed having a radical scavenging ability, a feed additive or a food additive.

In one embodiment, the composition may further comprise a hydrolyzate of ovomucin. When the antioxidant composition according to the present invention is used as a feed, a feed additive or a food additive having a radical scavenging ability, other antioxidant peptides may be further added to improve the radical scavenging ability. In this case, the other antioxidant peptides may be peptides themselves or peptides ≪ / RTI >

In one embodiment, such other antioxidant peptides as described above may be further included in the antioxidant composition according to the present invention as a hydrolyzate of ovomucin. In addition, the hydrolyzate of the ovomucin may have the amino acid sequence represented by the following general formula II or SEQ ID NO: 1.

[Formula II]

Asn-Ile-Gln-Thr-Asp-Asp-Phe-Arg-Thr (NIQTDDFRT)

In one embodiment, the hydrolyzate of the ovomucin may be an enzyme hydrolyzate and the enzyme is selected from the group consisting of Alcalase, Flavourzyme, Neutrase and Protamex ), ≪ / RTI > but is not necessarily limited thereto.

Hereinafter, the present invention will be described in more detail by way of examples. However, these embodiments are not intended to limit the scope of the present invention.

Experimental Method

1. Ovomucin preparation step

Egg white protein ovomucin was isolated from whole egg protein according to a conventionally known method (Omana, DA; Wu, J. A New Method of Separating Ovomucin from Egg White, J. Agric. 3603).

2. Electrophoresis step

SDS-PAGE (sodium dodecyl sulfate polyacryamide gel electrophoresis) was performed by a conventionally known method using 5% stacking gel and 15% separating gel (Chang, OK; Perrin, C .; Galia, W .; Saulnier, F.; Miclo, L .; Roux, E .; Driou, A .; Humbert, G .; Dary, A. Release of the cell-envelope protease PrtS in the growth medium of Streptococcus thermophilus 4F44. Int. Dairy J. 2012 , 23, 91 - 98 / Egito , A .; Miclo, L .; Lopez, C .; Adam, A., Girardet, JM; Gaillard, JL Separation and 398 characterization of mares' milk α s1 -, β-, κ -caseins, gamma-casein-like, and protease peptone component 5-like peptides, J. Dairy Sci . 2002, 85, 697-706).

3. Hydrolysis step of ovomucin

The purified ovomucin was hydrolyzed by direct treatment with the microbial protein hydrolyzing enzymes Alcalase, Flavourzyme, Neutrase or Protamex. Ovomucin solutions of 2 mg / ml or 5 mg / ml were prepared in 10 mM sodium phosphate buffer, and Flavourzyme, Neutrase and Protamex were treated at pH 7 and Alcalase at pH 8. The ratio of enzyme and substrate was 2.5: 1. The reaction mixture was incubated at 50 < 0 > C. Enzymatic hydrolysis was terminated by heating in boiling water for 5 minutes and partial samples were immediately recovered at each incubation or treatment time (2, 4, 6 and 24 hours). Samples were filtered through a 0.45 μm filter and stored at -20 ° C.

4. Antioxidant activity measurement step: ABTS and DPPH radical scavenging assay

The free radical scavenging ability of seven species (Holo-Ovotransferrin, Apo-Ovotransferrin, IgY, Ovalbumin, Lysozyme, Phosvitin and Ovomucin) isolated from eggs, hydrolysates thereof and synthetic peptides of the present invention can be determined by 2,2'- bis (3-ehtylbenzothiazoline-6-sulfonic acid) (ABTS ⁺ ) radical cation (Sigma-Aldrich, St. Louis, Mo., USA) and 2,2-di (4-tert-octylphenyl) , Sigma-Aldrich) (Re, R .; Pellegrini, N .; Proteggente, A.; Pannala, A .; Yang, M .; Rice-Evans, C. Antioxidant activity using an improved ABTS radical cation .. decolorization assay Free Radic Biol Med 1999, 26, 1231 -..... 1237 / Madhujith, T .; Izydorczyk, M .; Shahidi, F. Antioxidant properties of pearled barley fractions J. Agric Food Chem 2006, 54, 3283 - 3289).

The ratio (%) of radical scavenging activity was calculated according to the following general formula III (ABTS) and general formula IV (DPPH).

[Formula III]

(%) = {(A _control - A _sample ) / A _control ]} x 100

Here, A _control represents the absorbance of the first ABTS ⁺ (distilled water instead of the sample).

[General Formula IV]

(%) = {1 - [(A _sample - A _blank ) / A _control ]} x 100

Here, A _control represents the absorbance of the initial DPPH (distilled water instead of the sample), and A _blank represents the absorbance of methanol.

Example

1. Antioxidant activity of egg protein

(1) DPPH (2,2-di (4-tert-octylphenyl) -1-picrylhydrazyl) radical scavenging ability

The DPPH (2.2 Diphenyl-1-picrylhydrazyl) radical scavenging activity was measured by separating proteins of seven species (Holo-Ovotransferrin, Apo-Ovotransferrin, IgY, Ovalbumin, Lysozyme, Phosvitin, Ovomucin) from eggs. As shown, the DPPH radical scavenging ability of the ovomucin protein was 31.524%.

5 mg / ml Control group Holo-Ovotransferrin Apo-ovotransferrin IgY Ovalbumin Lysozyme Phosvitin Ovomucin Protein + DPPH
OD 0.351 0.403 0.268 1.264 0.195 0.710 0.259 0.308 OD 0.350 0.423 0.271 1.312 0.243 0.557 0.254 0.312 OD 0.349 0.418 0.273 1.253 0.248 0.549 0.256 0.306 Average 0.350 0.415 0.271 1.276 0.229 0.605 0.256 0.309 Standard Deviation 0.001 0.010 0.003 0.031 0.029 0.091 0.003 0.003 Ref
OD 0.038 0.219 0.057 0.973 0.242 0.334 0.081 0.064 OD 0.037 0.239 0.054 0.967 0.116 0.615 0.080 0.074 Average 0.038 0.229 0.056 0.970 0.179 0.475 0.081 0.069 Standard Deviation 0.001 0.014 0.002 0.004 0.089 0.199 0.001 0.007 Scavenging ability (%) 46.952 38.524 12.476 85.810 62.619 49.762 31.524

* OD = optical density

(2) ABTS (2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging ability

The DPPH (2.2 Diphenyl-1-picrylhydrazyl) radical scavenging activity was measured by separating proteins of seven species (Holo-Ovotransferrin, Apo-Ovotransferrin, IgY, Ovalbumin, Lysozyme, Phosvitin, Ovomucin) from eggs. As described above, the ABTS radical scavenging activity of the ovomucin protein was 71.7%.

5 mg / ml Control group Holo-Ovotransferrin Apo-ovotransferrin IgY Ovalbumin Lysozyme Phosvitin Ovomucin Protein + ABTS
OD 0.699 0.461 0.404 0.598 0.597 0.546 0.664 0.246 OD 0.702 0.451 0.388 0.601 0.600 0.565 0.665 0.261 OD 0.701 0.458 0.380 0.601 0.592 0.552 0.660 0.266 Average 0.701 0.457 0.391 0.600 0.596 0.554 0.663 0.258 Standard Deviation 0.002 0.005 0.012 0.002 0.004 0.010 0.003 0.010 Ref
OD 0.038 0.080 0.063 0.084 0.048 0.053 0.055 0.059 OD 0.038 0.080 0.066 0.079 0.046 0.055 0.056 0.060 Average 0.038 0.080 0.065 0.082 0.047 0.054 0.056 0.060 Standard Deviation 0.000 0.000 0.002 0.004 0.001 0.001 0.001 0.001 Scavenging ability (%) 46.242 53.449 25.999 21.598 28.592 13.297 71.717

* OD = optical density

2. DPPH radical scavenging activity (antioxidative activity) measurement of enzymatic hydrolysates of albumin protein containing ovomucin

(1) Enzymatic hydrolyzate by Alcalase

Proteins of seven species (Holo-Ovotransferrin, Apo-Ovotransferrin, IgY, Ovalbumin, Lysozyme, Phosvitin, Ovomucin) were isolated from eggs and hydrolyzed with Alcalase, a hydrolytic enzyme, at 50 ° C and pH 8 for 4 hours. As a result of measurement of DPPH (2.2 Diphenyl-1-picrylhydrazyl) radical scavenging activity for each hydrolysis product, the DPPH radical scavenging ability of ovomucin hydrolyzate was 51.815% as shown in Table 3 below, The highest.

5 mg / ml Control group Holo-Ovotransferrin Apo-ovotransferrin IgY Ovalbumin Lysozyme Phosvitin Ovomucin Protein + DPPH
OD 0.542 0.439 0.434 0.604 0.456 0.403 0.510 0.296 OD 0.551 0.438 0.433 0.604 0.455 0.401 0.510 0.298 OD 0.550 0.436 0.434 0.603 0.457 0.400 0.512 0.292 Average 0.548 0.438 0.434 0.604 0.456 0.401 0.510 0.296 Standard Deviation 0.005 0.001 0.000 0.000 0.001 0.002 0.001 0.003 Ref
OD 0.039 0.056 0.043 0.204 0.041 0.049 0.103 0.051 OD 0.038 0.054 0.041 0.200 0.043 0.048 0.103 0.050 Average 0.038 0.055 0.042 0.202 0.042 0.048 0.103 0.050 Standard Deviation 0.000 0.001 0.002 0.003 0.001 0.001 0.000 0.001 Scavenging ability (%) 24.767 23.101 21.069 18.716 30.631 19.982 51.815

* OD = optical density

(2) Enzyme hydrolyzate by Flavourzyme

Proteins of seven species (Holo-Ovotransferrin, Apo-Ovotransferrin, IgY, Ovalbumin, Lysozyme, Phosvitin and Ovomucin) were isolated from eggs and hydrolyzed with Flavourzyme, a hydrolytic enzyme, at 50 ° C and pH 7 for 4 hours. As a result of measurement of DPPH (2.2 Diphenyl-1-picrylhydrazyl) radical scavenging activity of each hydrolysis product, the DPPH radical scavenging ability of ovomucin hydrolyzate was 52.535% as shown in Table 4 below, The highest.

5 mg / ml Control group Holo-Ovotransferrin Apo-ovotransferrin IgY Ovalbumin Lysozyme Phosvitin Ovomucin Protein + DPPH
OD 0.473 0.483 0.433 0.681 0.499 0.428 0.756 0.356 OD 0.496 0.495 0.441 0.674 0.496 0.398 0.720 0.329 OD 0.501 0.441 0.688 0.524 0.408 0.732 0.317 Average 0.484 0.493 0.438 0.681 0.506 0.411 0.736 0.334 Standard Deviation 0.016 0.009 0.004 0.007 0.015 0.015 0.018 0.020 Ref
OD 0.039 0.146 0.078 0.290 0.254 0.070 0.359 0.090 OD 0.038 0.138 0.078 0.228 0.225 0.119 0.371 0.095 Average 0.039 0.142 0.078 0.259 0.239 0.094 0.365 0.092 Standard Deviation 0.000 0.006 0.000 0.044 0.020 0.035 0.009 0.003 Scavenging ability (%) 31.116 29.198 17.112 47.645 37.788 27.175 52.535

* OD = optical density

(3) Enzymatic hydrolyzate by Neutrase

Proteins of seven species (Holo-Ovotransferrin, Apo-Ovotransferrin, IgY, Ovalbumin, Lysozyme, Phosvitin and Ovomucin) were isolated from eggs and hydrolyzed with Neutrase, a hydrolytic enzyme, at 50 ° C and pH 7 for 4 hours. As a result of measurement of DPPH (2.2 Diphenyl-1-picrylhydrazyl) radical scavenging activity for each hydrolysis product, DPPH radical scavenging ability of ovomucin hydrolyzate was 46.379% as shown in the following Table 5, The second highest.

5 mg / ml Control group Holo-Ovotransferrin Apo-ovotransferrin IgY Ovalbumin Lysozyme Phosvitin Ovomucin Protein + DPPH
OD 0.477 0.567 0.459 0.845 0.322 0.638 0.525 0.380 OD 0.475 0.566 0.465 0.834 0.309 0.602 0.512 0.355 OD 0.475 0.568 0.463 0.829 0.297 0.619 0.520 0.347 Average 0.476 0.567 0.462 0.836 0.309 0.620 0.519 0.361 Standard Deviation 0.001 0.001 0.003 0.008 0.012 0.018 0.006 0.017 Ref
OD 0.037 0.132 0.042 0.407 0.052 0.207 0.091 0.090 OD 0.039 0.128 0.042 0.394 0.049 0.202 0.089 0.086 Average 0.038 0.130 0.042 0.400 0.050 0.204 0.090 0.088 Standard Deviation 0.001 0.003 0.000 0.010 0.020 0.003 0.001 0.003 Scavenging ability (%) 14.308 17.459 14.455 49.108 18.493 15.754 46.379

* OD = optical density

(4) Enzyme hydrolyzate by Protamex

Proteins of seven species (Holo-Ovotransferrin, Apo-Ovotransferrin, IgY, Ovalbumin, Lysozyme, Phosvitin and Ovomucin) were isolated from eggs and then hydrolyzed with Protamex, a hydrolytic enzyme, at 50 ° C and pH 7 for 4 hours. As a result of measurement of DPPH (2.2 Diphenyl-1-picrylhydrazyl) radical scavenging activity for each hydrolysis product, DPPH radical scavenging ability of ovomucin hydrolyzate was 53.225% as shown in Table 6 below, The highest.

5 mg / ml Control group Holo-Ovotransferrin Apo-ovotransferrin IgY Ovalbumin Lysozyme Phosvitin Ovomucin Protein + DPPH
OD 0.539 0.438 0.473 0.725 0.497 0.401 0.755 0.334 OD 0.542 0.429 0.503 0.733 0.503 0.387 0.745 0.312 OD 0.547 0.437 0.466 0.723 0.520 0.403 0.699 0.263 Average 0.543 0.435 0.481 0.727 0.507 0.397 0.733 0.303 Standard Deviation 0.004 0.005 0.019 0.006 0.012 0.009 0.030 0.036 Ref
OD 0.038 0.089 0.097 0.321 0.233 0.057 0.319 0.067 OD 0.039 0.138 0.098 0.314 0.252 0.059 0.326 0.063 Average 0.039 0.113 0.097 0.318 0.242 0.058 0.322 0.065 Standard Deviation 0.001 0.035 0.001 0.005 0.013 0.001 0.005 0.002 Scavenging ability (%) 36.846 24.737 19.639 48.087 33.452 19.393 53.225

* OD = optical density

3. Results of ABTS radical scavenging (antioxidant activity) measurement of enzymatic hydrolysates of ovomucin

(1) Enzyme hydrolyzate by Protamex

Ovomucin protein was isolated from eggs and treated with the hydrolytic enzyme Protamex at 50 ° C, pH 7 for 2 hours, 4 hours, 6 hours and 24 hours. Gallic acid was selected as a positive control for creating standard curves. The IC ₅₀ value of gallic acid calculated from the standard curve equation was 62 μM.

As a result of measuring the ABTS radical scavenging activity according to each treatment time, as shown in the following Table 7, the ABTS radical scavenging ability of the enzyme hydrolyzate by Protamex of ovomucin was 85 ~ 92% and showed a remarkably high radical scavenging ability .

2 mg / ml Control group 0 hours 2 hours 4 hours 6 hours 24 hours Hydrolyzate + ABTS
OD 0.701 0.284 0.140 0.121 0.119 0.108 OD 0.700 0.282 0.185 0.133 0.114 0.115 OD 0.701 Average 0.701 0.283 0.163 0.127 0.117 0.112 Standard Deviation 0.001 0.001 0.032 0.008 0.004 0.005 Ref
OD 0.060 0.060 0.060 0.060 0.060 0.060 OD 0.060 0.060 0.060 0.060 0.060 0.060 Average 0.060 0.060 0.060 0.060 0.060 0.060 Standard Deviation 0.000 0.000 0.000 0.000 0.000 0.000 Scavenging ability (%) 68.173 85.371 90.438 91.936 92.650

* OD = optical density

(2) Enzyme hydrolyzate by Flavourzyme

Ovomucin protein was isolated from eggs and treated with hydrolytic enzyme Flavourzyme at 50 ℃, pH 7 for 2 hours, 4 hours, 6 hours and 24 hours. Gallic acid was chosen as a positive control for creating standard curves.

As a result of measuring the ABTS radical scavenging activity according to each treatment time, as shown in Table 8 below, the ABTS radical scavenging ability of the enzyme hydrolyzate by the flavorzyme of ovomucin was 85 ~ 90% and showed a high radical scavenging ability .

2 mg / ml Control group 0 hours 2 hours 4 hours 6 hours 24 hours Hydrolyzate + ABTS
OD 0.701 0.359 0.153 0.129 0.127 0.129 OD 0.700 0.367 0.161 0.135 0.123 0.129 OD 0.701 Average 0.701 0.363 0.157 0.132 0.125 0.129 Standard Deviation 0.001 0.006 0.006 0.004 0.003 0.000 Ref
OD 0.058 0.058 0.058 0.058 0.058 0.058 OD 0.058 0.058 0.058 0.058 0.058 0.058 Average 0.058 0.058 0.058 0.058 0.058 0.058 Standard Deviation 0.000 0.000 0.000 0.000 0.000 0.000 Scavenging ability (%) 56.470 85.871 89.439 90.438 89.867

* OD = optical density

4. Antioxidant activity of synthetic peptides

(1) ABTS radical scavenging activity (antioxidative activity) measurement results of the peptide fraction contained in the enzyme hydrolyzate of ovomucin

From the above results, it was also confirmed that the ovomucin protein that was not treated with the enzyme had an antioxidative activity, but the hydrolysis thereof further increased the antioxidative activity. Therefore, it has been confirmed that the ovomucin protein or its hydrolyzate can be used as a natural antioxidant as a feed, feed additive or food additive.

To search for peptides having antioxidative activity from hydrolysates of ovomucin, ovomucin was hydrolyzed with protamex and ultrafiltration was performed to collect peptides of 3 kDa or less. Then, the obtained peptides were fractionated by high performance liquid chromatography (HPLC). As shown in Table 9 below, antioxidative activity was observed in all fractions, but significant antioxidative activity was confirmed especially in fraction No. 2 .

2 mg / ml Control group One 2 3 4 5 6 7 8 9 10 Fraction + ABTS
OD 0.698 0.626 0.405 0.502 0.610 0.619 0.632 0.636 0.630 0.630 0.616 OD 0.697 0.636 0.405 0.515 0.610 0.628 0.639 0.637 0.632 0.632 0.630 OD 0.698 Average 0.698 0.631 0.405 0.509 0.610 0.624 0.636 0.637 0.631 0.631 0.623 Standard Deviation 0.001 0.007 0.000 0.009 0.000 0.006 0.005 0.001 0.001 0.001 0.010 Ref
OD 0.061 0.061 0.061 0.061 0.061 0.061 0.061 0.061 0.061 0.061 0.061 OD 0.060 0.060 0.060 0.060 0.060 0.060 0.060 0.060 0.060 0.060 0.060 Average 0.061 0.061 0.061 0.061 0.061 0.061 0.061 0.061 0.061 0.061 0.061 Standard Deviation 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 Scavenging ability (%) 18.23 50.62 35.77 21.24 19.30 17.58 17.44 18.23 18.23 19.37

Analysis of the second fraction with the strongest ABTS radical scavenging activity (antioxidant activity) by liquid chromatography mass spectrometry (LC / MS / MS) revealed that the second fraction contained various peptides with antioxidant ability (Including peptides NIQTDDFRT, SKGDPGEWR, SGLYIIVEIPELEVYVSY, DWKAPV, NIQTDDFR, FYKPGET, SKGDPGEW CLPFEESNCVPGTVDVTSDGC, IKILSSAGVQIRVQMKPVMQLS, VVPPQPY, VCLPFEESNCVPGTVDV, QFGNFHKVEDPSE INMTDLCADQPFKSALGQKH, LDEPDPL etc.).

The present inventors selected antioxidant peptides having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 2 represented by the following general formula I or II, solid-phase synthesis technique.

[General Formula I]

Asn-Ile-Gln-Thr-Asp-Asp-Phe-Arg-Thr (NIQTDDFRT)

[Formula II]

Leu-Asp-Glu-Pro-Asp-Pro-Leu (LDEPDPL)

(2) DPPH radical scavenging ability of synthetic peptide

An antioxidant peptide having the amino acid sequence of SEQ ID NO: 1 represented by the general formula I or SEQ ID NO: 2 represented by the general formula II was synthesized and 1, 2.5 and 5 mg / mL were added to 0.2 mM DPPH solution, respectively, Were measured.

As a result, as shown in Table 10 below, the peptide NIQTDDFRT showed a DPPH radical scavenging ability of 51.877% when using 5 mg / ml of 24.78% and 5 mg / ml of peptide LEDPDPL, It was confirmed that the antioxidant activity can be maintained even with the synthesized peptide having a short length.

DPPH
Control group
NIQTDDFRT LDEPDPL 1 mg / ml 2.5 mg / ml 5 mg / ml 1 mg / ml 2.5 mg / ml 5 mg / ml OD 0.462 0.448 0.417 0.382 0.391 0.321 0.262 0.507 0.425 0.402 0.372 0.360 0.324 0.255 0.521 0.452 0.429 0.396 0.387 0.320 0.261 Average 0.497 0.442 0.416 0.383 0.379 0.322 0.259 Scatters
(%) 0.000 12.033 17.644 24.780 25.625 38.292 51.877

(3) ABTS radical scavenging ability of synthetic peptides

An antioxidant peptide having the amino acid sequence of SEQ ID NO: 1 represented by the general formula I or SEQ ID NO: 2 represented by the general formula II was synthesized and 1, 2.5 and 5 mg / mL were added to 0.2 mM of the ABTS solution to obtain radical scavenging ability Were measured.

As a result, as shown in Table 11 below, the peptide NIQTDDFRT showed an ABTS radical scavenging ability of 19.721% when using 5 mg / ml of 0.987% and 5 mg / ml of peptide LEDPDPL, It was confirmed that the antioxidant activity can be maintained even with the synthesized short-length peptide LEDPDPL.

ABTS
Control group
NIQTDDFRT LDEPDPL 1 mg / ml 2.5 mg / ml 5 mg / ml 1 mg / ml 2.5 mg / ml 5 mg / ml OD 0.664 0.657 0.661 0.661 0.638 0.598 0.536 0.665 0.662 0.662 0.658 0.641 0.597 0.533 0.666 0.663 0.661 0.657 0.639 0.596 0.533 Average 0.665 0.660 0.661 0.659 0.639 0.597 0.534 Scatters
(%) 0.000 0.722 0.596 0.987 3.864 10.289 19.721

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, It will be obvious that it is not. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> RURAL DEVELOPMENT ADMINISTRATION <120> Anti-oxidative peptide and composition containing the same <130> NPF-23171 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> anti-oxidative peptide <400> 1 Asn Ile Gln Thr Asp Asp Phe Arg Thr   1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> anti-oxidative peptide <400> 2 Leu Asp Glu Pro Asp Pro Leu   1 5

Claims (4)

An antioxidative composition comprising an antioxidant peptide having an amino acid sequence represented by SEQ ID NO: 2 as an active ingredient.
The method according to claim 1,
Wherein the composition further comprises a hydrolyzate of ovomucin.
3. The method of claim 2,
Wherein the hydrolyzate of the ovomucin comprises an antioxidant peptide having an amino acid sequence represented by SEQ ID NO: 1.
3. The method of claim 2,
Wherein the hydrolyzate of ovomucin is a hydrolyzate of at least one enzyme selected from Alcalase, Flavourzyme, Neutrase and Protamex.

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