KR100207807B1 - Antioxidant peptide and method for prducing it - Google Patents

Abstract

The present invention provides a peptide having excellent antioxidant power and a method for producing the same. The peptide has an amino acid sequence of Gly-Pro-Hyp in the molecule, and is composed of 9-16 amino acids. The method is characterized by enzymatic-hydrolysis of cowhide or skin gelatin, followed by molecular weight 1.5-4.5KDa and-. It is characterized by separating and purifying the peptide as described above from the hydrolyzate in the range of 1KDa.

Description

Antioxidant Peptide and Method of Production thereof

FIELD OF THE INVENTION

The present invention relates to a peptide having excellent antioxidant activity and a method for producing the same.

Prior art in the technical field to which the invention belongs

Antioxidants are commonly referred to as substances that prevent oxidation, free radical scavengers that control the chain reaction of automatic oxidation according to their mechanism of action, peroxide decomposers that decompose and inactivate peroxides to non-radicals, and trace metals. Metal deactivators for inactivating oxidation promotion, synergists for increasing antioxidant activity when co-existing with radical inhibitors in automatic oxidation, and singlet oxygen scavengers for eliminating various active oxygen systems. Ingold (1968) divided antioxidants into two broad groups: first, as a primary or chain-reactive antioxidant, a group that reacts with lipid radicals to produce more stable products, and second, as a second or delayed antioxidant as a different mechanism of action. It was divided into groups that delayed automatic oxidation. Primary antioxidants block the prolongation of the chain reaction by providing hydrogen atoms to the lipid free radicals to make relatively stable radicals, preventing automatic oxidation. Antioxidants of this type are mostly phenolic compounds, such as tocopherol, gallic acid and derivatives thereof, quercetin, rhamnetin, camperol, rutin, quercitrin and caffeic acid. Flavonoids such as acid) are known. Secondary antioxidants do not have antioxidant effects on pure lipids but increase the effectiveness of or inhibit the effects of primary antioxidants. Antioxidants in this form include phospholipids, synergistic citric acid chelates with oxidative metal ions, and vitamin C, which are synergistic with primary antioxidants such as tocopherol. The oxidation of lipids, which is a typical oxidation reaction, is a reaction in which the formation and decomposition of peroxides are mainly caused by lipid radicals (L ·) or singlet oxygen produced by high temperature or light. In the presence of phenolic hydroxy groups act as free radical acceptors of fats and oils, the free radicals produced in the early stages of fat and oil have a stable resonance hybrid to act as an antioxidant. Such antioxidants are present in various plant extracts, flavorings, fermentation products, etc. as flavonoids or phenolic compounds. It has also been reported that oligopeptides produced by hydrolyzing proteins and plants using an acid or various enzymes also exhibit antioxidant activity (未 綱 et al., 1989; 本 井, 1989). Synthetic antioxidants include phenolic compounds, sulfur compounds, citric acid and derivatives thereof, phosphoric acid and derivatives thereof (Macrae et al., 1993), which have a large substituent indicating steric hindrance to form stable radicals and exhibit antioxidant properties. The current research direction of antioxidants is the role of scavengers of oxygen radicals and oxide radicals that cause aging and carcinogenesis in the limited study as a conventional food antioxidant (大 澤, 1985) and the in vivo lipid peroxidation reaction. Extensive research has been conducted on effects such as mutagenicity (大 澤, 1982) and cancer-induced expression suppression (大 澤, 1983), which are presumed to be caused by inhibition or radical involvement. Synthetic antioxidants affect incidence and various enzyme groups in animal experiments. In particular, BHT has been reported to be involved in teratogenic or cancerous development (Branen, 1975). Natural antioxidants have little side effects and are now widely used as food additives or drugs. However, since tocopherol, which is the most widely used natural antioxidant, has limitations in terms of high price and fat solubility, development of alternative natural antioxidants is desired.

The present inventors have obtained a high antioxidant antioxidant gelatin hydrolyzate fraction by hydrolyzing the fish gelatin in a special method (Korean Patent Publication No. 96-31582), and continue the research to have a specific amino acid sequence in the gelatin hydrolyzate fraction. It was found that the peptides exhibit particularly good antioxidant activity and have come to complete the present invention.

That is, an object of the present invention is to provide a peptide having excellent antioxidant activity.

The above object of the present invention is achieved by an antioxidant peptide having a Gly-Pro-Hyp amino acid sequence in the molecule and consisting of 9 to 16 amino acid residues.

Another object of the present invention is to provide a method for producing a peptide having excellent antioxidant activity.

Another object of the present invention is to provide a method for preventing rancidity of fats and oils using the peptide having excellent antioxidant activity.

The objects of the present invention described above, and other objects and features will be apparent to those skilled in the art by the following detailed description.

1 is a graph showing the results of comparing the antioxidant power of fish gelatin hydrolyzate. In Figure 1, F1 is the first hydrolyzate of fish gelatin, F2 is the second hydrolyzate of fish gelatin, F3 is the third hydrolyzate of fish gelatin, FG is fish gelatin, Toco is alpha-tocopherol, And BHT means butylated hydroxytoluene. Each value is the average of three replicates.

Figure 2 is a graph showing the results of comparing the antioxidant power of the commercial gelatin hydrolyzate. In Figure 2, K1 is the primary hydrolyzate of commercial gelatin, K2 is the secondary hydrolyzate of commercial gelatin, K3 is the tertiary hydrolyzate of commercial gelatin, KG is commercial gelatin, Toco is alpha-tocopherol, And BHT means butylated hydroxytoluene. Each value is the average of three replicates.

3 is a graph showing the results of comparing lipid peroxidation values of fish gelatin hydrolyzate and commercial gelatin hydrolyzate. In FIG. 3, F1, F2, F3, K1, K2, K3, Toco and BHT have the same meanings as in FIGS. 1 and 2. Each value is the average of three replicates. In Figure 3, lipid peroxidation (%) is expressed as (absorbance of hydrolyzate / absorbance of control) x 100.

4 is a graph showing the results of comparing the radical scavenging effect of the fish gelatin hydrolyzate and the commercial gelatin hydrolyzate. In FIG. 4, F1, F2, F3, K1, K2, K3, Toco and BHT have the same meanings as in FIGS. 1 and 2. Each value is the average of three replicates. In FIG. 4, the radical scavenging effect (%) is expressed as (absorbance of hydrolyzate / absorbance of control) × 100.

5 to 7 are graphs showing changes in MDA concentration, lipid peroxidation, and radical scavenging effects of linolenic acid of various concentrations of gelatin hydrolysates, respectively. 5 to 7, F2 and K2 have the same meanings as in FIGS. 1 and 2. Each value is the average of three replicates.

8 and 9 are graphs showing the synergistic effect on the change in the MDA concentration and the lipid peroxidation of linolenic acid after keeping the gelatin hydrolyzate of various concentrations for 6 hours, respectively. 8 and 9, F, F2, F3, K1, K2, and K3 have the same meanings as in FIGS. 1 and 2. Each value is the average of three replicates.

10 and 11 are Sephadex G-25 column chromatographs (gel filtration chromatographs) of fish gelatin secondary hydrolysates and commercial gelatin secondary hydrolysates, respectively. Chromatography conditions are column size 2.5 × 90 cm, eluent 0.05 M sodium phosphate buffer (pH 7.0), flow rate 0.5 ml / min, partition volume 10 ml.

12 and 13 are SP- Sephadex C-25 chromatographs (ion exchange chromatographs) of the G1 and G2 fractions of FIGS. 10 and 11, respectively. The elution condition is a 0-0.5 M concentration gradient of sodium chloride dissolved in 20 mM sodium phosphate buffer (pH 4.0), with a distribution volume of 10 ml at a flow rate of 2 ml / min.

FIG. 14 and FIG. 15 show the results of reverse phase HPLC on the Primesphere 10-C18 column of the G1-III peak fraction and the G2-III peak fraction, which are the active fractions in FIGS. 12 and 13, respectively. Prime sphere 10 C-18 (10.0 × 250 mm), mobile phase is CH3CN containing 0.1% TFA, and flow rate is 1.5 ml / min.

FIG. 16 and FIG. 17 show reverse phase HPLC results of Primesphere 10-C18 columns of G1-III-III peak fraction and G2-III-II peak fraction, which are the active fractions of FIGS. 14 and 15, respectively. Prime sphere 10 C-18 (10.0 × 250 mm), mobile phase is CH3CN containing 10 mM ammonium acetate, and flow rate is 1.5 ml / min.

18 shows the effect of antioxidant peptides on cell survival of liver cell cultures exposed to t-BHP. Cells were incubated for 150 min with 1 mM t-BHP. In Fig. 18, F2 means peptide extracted from secondary hydrolyzate of fish gelatin, and K2 means peptide extracted from secondary hydrolyzate of commercial gelatin. Each value is the mean value ± SE, n = 3, and each value is the average of two values. * P0.05, ** P0.02, *** P0.01 vs. control.

FIG. 19 shows t-when treated with peptide (F2,-▽-) extracted from secondary hydrolyzate of 1 mg / ml fish gelatin or peptide (K2,-●-) extracted from secondary hydrolyzate of commercial gelatin. It shows the effect of preventing the toxicity of BHP (tert-butyl hydroxyperoxide). The control group (-▼-) was pretreated with medium containing no peptide.

20 is a result of comparing cell viability in cells cultured for 150 minutes by adding F2, K2 or tocopherol (Toco) and 1 mM t-BHP. Each value is the SE of the mean value measured for three separate cultures.

Antioxidant peptides according to the invention are obtained from skin or cowhide gelatin. That is, the gelatin can be extracted from the skin or cowhide and then hydrolyzed to obtain peptide mixtures of various molecular weight ranges and then separated therefrom.

In the specific example of the present invention, the method described in Korean Patent Application Publication No. 96-31582 by the applicant of the present invention was used as a method of obtaining gelatin from the skin or the skin and hydrolyzing the same, but the present invention is not limited to this specific method. no. The gelatin extraction method and the hydrolysis method of gelatin described in Korean Patent Publication No. 96-31582 are also described in detail herein. Particularly, in the present invention, gelatin was extracted from the shells of pollock and terrestrial cows by hot water extraction, and the gelatin was hydrolyzed by enzymes using a three-stage membrane reactor, and then peptides having excellent antioxidant activity were separated therefrom.

Separation and purification of peptides having excellent antioxidant activity from gelatin hydrolyzate can be carried out according to conventional peptide separation and purification methods. In the present invention, for example, a series of chromatography techniques are used. Specifically, by measuring the antioxidant activity of each fraction in each step while performing gel filtration, ion exchange, and high performance liquid chromatography in sequence. The specific peptide which is especially excellent in antioxidant activity was isolate | separated, and the amino acid sequence could be identified.

The antioxidant peptide provided according to the present invention has the amino acid sequence of Gly-Pro-Hyp in the molecule and consists of 9-16 amino acids. Characteristic sequences of Gly-Pro-Hyp included in a molecule may be present one or more times in the molecule. Among them, in particular, one having Gly at the C-terminus and consisting of 10-13 amino acids, in particular one described in SEQ ID NOs: 1-5.

As the raw material of gelatin in the present invention, it is possible to use cowhide or skin, which is particularly advantageous for pollack.

The method of preventing rancidity of fats and oils using the antioxidant peptides according to the present invention is achieved by adding the antioxidant peptides of the present invention to fats and oils in an amount of 0.05-1.0% (w / w) based on the weight of fats and oils. Used.

As described above, the peptide of the present invention is excellent in antioxidant power and can be used for various purposes for anti-oxidation.

That is, when used to prevent the oxidation of the substance to be prevented from oxidation, for example, to prevent the oxidation of fats and oils, it may be used in an amount of 0.05 to 1.0% by weight based on the weight of the fat. It may be added to the material for antioxidant in an effective amount in the above range, or formulated in a separate antioxidant formulation.

According to the invention, the antioxidant peptides exhibit synergism when used with known antioxidants such as tocopherol and the like.

Hereinafter, various materials, test methods, and the like used in the present invention will be described in detail.

Materials and methods

material

Raw materials were hydrolyzed using alkalase, pronase E, and collagenase, which were extracted from Alaska pollack and commercially available gelatin (Ge Gyeonggi Co., Ltd.), and then separated and used. Sephadex G-25 (superfine), SP- Sephadex C-25, Thiobituric acid (TBA), t-butyl hydroperoxide (t-BHP), ammonium thiocyanate, diphenyl-pi Diphenyl-picrylhydrazyl (DPPH) was used as manufactured by Sigma Chemical Co., and the GPC column was produced by Alltech Co., and the ODS reversed phase column was Phenomenex Co. The product was used.

Linolenic acid (99% purity), the fats and oils used in the determination of antioxidant activity, was determined by Sigma Chemical Co. As the product, tetramethoxypropane (TMP) and the control antioxidant butylated hydroxytoluene (BHT, purity 99%) were used in Fluka Chemical AU, and α-tocopherol (purity 95%) was used as a chemical product. In addition, Donryu rat liver cell (Ac2F) was used from the Japanese cell bank was used, Dulbecco`s Modified Eagle Medium (DMEM), dimethyl sulfoxide (DMSO), (4,5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide (MTT), trypsin-EDTA solution was obtained from Sigma Chemical Co. As the product, calf serum (FBS) and antibiotic-antimycotics (S.M. 10 mg / ml, P.C. 10,000 U / ml) were used by Gibco Co. (Grand island, U.S.A.). For other reagents, an analytical special reagent was used.

Way

Gelatin hydrolysis

Hydrolysis of each gelatin was performed in a 3-step continuous ultrafiltration membrane reactor (1-step; MW 10,000 cut-off, 2-step; MW 5,000 cut-off, 3-step; MW 1,000 cut-). off) was hydrolyzed successively with hydrolases such as alcalase, pronase E, collagenase and the like and separated through membranes in each step. The first hydrolyzate passed through the membrane having a limit molecular weight of 10,000, the hydrolyzate passed through the membrane having a limit molecular weight of 5,000, and the second hydrolyzate, and the hydrolyzate passed through the membrane having a limit molecular weight of 1,000 were called tertiary hydrolysates.

Comparison of the Antioxidant Activity of Gelatin Hydrolysates with Existing Antioxidants

Antioxidant activity of each hydrolyzate in each step was added to the oil turbid solution at a concentration of 1% (W / W), and stored in a thermostat controlled at 40 ± 1 ° C daily for quantitative determination of malondialdehyde (MDA) and lipid peroxidation ( %) And radical scavenging effect (RSA) were measured and compared.

Linolenic acid water-alcohol fat suspension preparation

Linolenic acid water-alcohol fat suspension was prepared by the method of Osawa et al. (1981). That is, 0.13 ml of linolenic acid, 10 ml of ethanol, and 10 ml of 50 mM phosphate buffer (pH 7.0) were mixed, and the volume was adjusted to 25 ml with deionized water.

Malondialdehyde (MDA) Assay

Malondialdehyde was quantified according to the method of Ohkawa et al. (1978). That is, 50 µl of the oil-suspension was aliquoted, and 0.8 ml of distilled water and 0.2 ml of 8.1% sodium dodecyl sulfate (SDS), 1.5 ml of 20% acetic acid (adjusted to pH 3.5 with 10N NaOH), and 1.5 ml of 0.8% thiobarbituric acid. After mixing the mixture, the mixture was left at 5 ° C. for 1 hour and developed at 100 ° C. for 1 hour, and then the absorbance was measured at 532 nm. Malondialdehyde quantification was determined by obtaining a standard curve using tetramethoxypropane (TMP).

Lipid Peroxidation Measurement

Lipid peroxidation was performed according to the method of Mitsuda et al. (1966). That is, 0.1 ml of 75% ethanol, 0.1 ml of 30% ammonium thiocyanate, and 0.1 ml of 2 × 10-2M ferric chloride / 3.5% hydrochloric acid were added to 0.1 ml of the oil-based turbidity, and the absorbance was measured at 500 nm after 3 minutes. Compared.

Radial scanvenging activity (RSA) measurement

RSA was measured according to the method of Hatano et al. (1988). 50 mg of each hydrolyzate was dissolved in 4 ml of deionized water, mixed with 1 ml of 1.5 × 10-4 M DPPH / MeOH, shaken well, left at room temperature for 30 minutes, and the absorbance at 517 nm was measured to determine the amount of DPPH remaining. . Antioxidant activity of each hydrolyzate was shown to be lower than the absorbance of DPPH of the control.

Optimal Concentration Measurement

The optimal concentration of antioxidant peptides to prevent rancidity of fats and oils was determined by quantifying malondialdehyde and measuring lipid peroxidation values. That is, antioxidant peptides were added to each fat and oil suspension so as to be 0.5, 1.0, and 2.0% of the fat weight, and the malondialdehyde quantification and lipid peroxidation values were measured at 6 days.

Synergy measurement

The synergistic effect of the combination of known antioxidants and antioxidant peptides according to the present invention was determined by the determination of malondialdehyde and measurement of lipid peroxidation values. That is, 1% by weight of the holding weight of α-tocopherol was added to the fat and oil suspension, and 1% of the weight of the antioxidant peptide was added thereto, followed by quantification of malondialdehyde and storage in a thermostat controlled at 40 ± 1 ° C. Lipid peroxidation values were measured. At this time, a comparison was made using the α-tocopherol fat-and-oil turbid solution to which the hydrolyzate was not added.

Isolation of Antioxidant Peptides

In order to separate only peptides having excellent antioxidant properties, fractions were collected by gel filtration, ion exchange chromatography, and high performance liquid chromatography, and the antioxidant effect of each fraction was evaluated by malondialdehyde quantification method. Fractions.

First, the peptide showing the highest antioxidant capacity in the crude state of the extracted gelatin hydrolyzate was determined and injected into a Sephadex G-25 column (φ2.5 × 90 cm) equilibrated with 50 mM phosphate buffer (pH 7.0), Elution (flow rate: 100 ml / hr, partition volume: 10 ml) with the same buffer solution, fractions were measured by absorbance at 280 nm, lyophilized by fractions to measure the antioxidant properties of the fractions, and the fraction with the highest antioxidant power Was injected into an SP-Sephadex C-25 column (φ3 × 40 cm) equilibrated with 20 mM phosphate buffer (pH 4.0), and the non-adsorbed portion was eluted with 1 l of the same buffer solution, followed by 700 ml of buffer and 0.5 M. Using a 700 ml of sodium chloride, fractional elution (flow rate: 120 ml / hr, partition volume: 10 ml) by linear concentration gradient method was applied to the membrane filter device (MW 1,000 cut-off). To remove salt and dialyze against distilled water Results were dry. After determining the fractional antioxidant properties, the most antioxidant fractions were eluted using a C-18 reverse phase column (φ10.0 × 250 mm) on high performance liquid chromatography. At this time, the elution rate was 1.0 ml / min, the mobile phase was fractionated by measuring the absorbance at 230 nm using acetonitrile (0-40%, 40min) containing 0.1% trichloroacetic acid as a linear phase gradient. The fraction with the highest antioxidant capacity was refractionated by high performance liquid chromatography. The column was a C-18 reversed phase column (φ10.0 × 250 mm) and the mobile phase was flowed at 1.0 ml using a linear phase gradient using acetonitrile (0-40%, 40 min) containing 10 mM ammonium acetate. The single peptide with the highest antioxidant power was isolated by partitioning and measuring the absorbance at 230 nm.

Amino acid sequencing

Amino acid sequencing was determined by gas phase autosequencing (Applied Biosystem Co.) after digestion by Edman's method of cleaving amino acids from the N-terminus.

That is, the amino acid that inhibited dissociation under basic conditions was attached to phenylisothiocyanate (PICT) to form phenylthiocarbamyl (PTC) peptide. ATZ) derivatives and modified with the more stable derivative phenylthiohydantoin (PTH) amino acid. The sequence was determined by repeating the above procedure for the N-terminus of the remaining peptide.

Effect of Antioxidant Peptides on Cell Lipid Peroxidation

To examine the effect of peptides on cellular lipid peroxidation, the effects of toxic t-butyl hydroperoxide (t-BHP) on cells were examined by concentration. Various concentrations and concentrations of t-butyl hydroperoxide were examined by cell viability (MTT assay) and compared with the α-tocopherol addition group. The synergistic effect of the peptide in combination with α-tocopherol was also examined.

Cell culture

Normal hepatocytes (Ac2F) were cultured in DMEM medium containing 10% calf serum (FBS) at 37 ° C., 95% O 2, and 5% CO 2. That is, in a DMEM medium containing 10% calf serum inactivated (55 ° C., 15 min) in a 75-cm2 flask, 7.5 μg 150 g / ml of sodium bicarbonate, 58.4 μg / ml of glutamine and 4.4 μl / ml of antibiotics. % Carbon dioxide, and incubated at 37 ℃ conditions.

Cell quantification

Remove the culture medium supernatant into a 75-cm2 flask in which cells have grown uniformly, wash with Calcium magnesium free-phosphate buffered saline, drop the cells with trypsin / EDTA solution, and add the medium. Cell homogenate was mixed with trypan blue in a 1: 1 ratio and measured using a hemocytometer on an optical microscope.

Cytotoxicity

In the cytotoxicity experiment, the cells cultured for 18 to 20 hours were divided into 24-well plates at a ratio of 5 × 104 cells / well, and the addition and non-addition of the purified and purified antioxidant peptide were incubated for 17 hours in a CO2 incubator. After washing with isotonic phosphate buffer, treatment with 1 mM t-butyl hydroperoxide for 150 minutes on medium without calf serum, cell viability (MTT assay) was measured.

MTT analysis

MTT analysis was performed according to the method of Sladowski et al. (1993). After washing the cells of each well with isotonic phosphate buffer, MTT was added to each well in 1/10 volume, incubated for 4 hours at 37 ° C, followed by centrifugation (800 rpm, 20 min), followed by ethanol: DMSO (= 1: 1, V / V) was added to each well, and then shaken for 20 minutes, and the absorbance was measured at 570 nm using an ELISA reader.

Antioxidant Measurement by t-Butyl Hydroperoxide Concentration

The cells were cultured for 18 to 20 hours, and the antioxidant peptides were dispensed. The medium was added so that the total volume was 1 ml per well, followed by incubation for 18 to 20 hours. The cells were washed with isotonic phosphate buffer and washed on a medium without calf serum. t-butyl hydroperoxide was added to a final concentration of 0.1, 0.5 1, 2 mM, incubated for 150 minutes, followed by MTT analysis.

Comparison of Antioxidant Capacity with α-tocopherol

1 μM α-tocopherol or antioxidant peptides were added to cells incubated for 18 to 20 hours, cultured for 18 to 20 hours, washed with isotonic phosphate buffer, and washed with 1 mM t-butyl hydrogel in a medium without calf serum. The peroxide was treated for 150 minutes, followed by MTT analysis.

Results and Discussion

Antioxidant Activity and Synergistic Effect with Antioxidants of Gelatin Hydrolysates

Antioxidant Activity of Gelatin Hydrolysates

The antioxidant power of gelatin hydrolyzate was automatically oxidized in a thermostat controlled at 40 ± 1 ° C, and the antioxidative power against linolenic acid was compared to that of malondi epidermal gelatin hydrolyzate compared to the case without addition of hydrolysates at each stage. The aldehyde quantitative value decreased by 35-56% (FIG. 1), the lipid peroxidation decreased by 8-15% (FIG. 3), and the radical scavenging effect (RSA) was 12-22% (FIG. 4). The quantitative value of malondialdehyde in the wadded gelatin hydrolyzate added group was 32-48%, lipid peroxidation was reduced by 5-10.5%, and the radical scavenging effect (RSA) was 20-22% (Fig. 2, Fig. 3, FIG. 4). The quantitative values of malondialdehyde of primary hydrolysates (MW 10,000 cut-off) and tertiary hydrolysates (MW 1,000 cut-off) of pollack and gelatin are 35-46% and 32-37%, respectively. It showed a decrease, 10-15% lower antioxidant activity than α-tocopherol, and gelatin itself showed very low antioxidant activity. Pollack and gelatin secondary hydrolyzate showed 8% and 3% higher antioxidant activity than α-tocopherol, a commercial antioxidant. Among the hydrolysates at each stage, the highest antioxidants were the secondary hydrolysates. Among them, the pollack gelatine secondary hydrolysates had 56% quantitative value of malondialdehyde, 11% lipid peroxidation and 22% RSA. Indicated.

Optimal concentration

As a result of measuring the antioxidant power according to the concentration of secondary hydrolyzate with the highest antioxidant power, the concentration of hydrolyzate was 1% in the determination of malondialdehyde (MDA) (linolenic acid: hydrolyzate = 100: 1 (w / w)) was the highest antioxidant capacity, 0.5% and 2% concentration was found to decrease the antioxidant power compared to the 1% concentration (Fig. 5). Lipid peroxidation showed the same tendency at the concentration of 1.0% (w / w) as well as the malondialdehyde quantitative value (FIG. 6).

These results indicate that when the concentration of peptide is low, the degree of oxidation of the peptide itself does not significantly affect the antioxidant power of the peptide, but when the peptide is added more than 1.0% (w / w), the degree of oxidation of the peptide affects the antioxidant effect. In this study, the optimum antioxidant concentration of the hydrolyzate was 1.0% (w / w).

However, it was determined that the antioxidant activity of the hydrolyzate was related to RSA by showing the concentration-dependent value in RSA (FIG. 7).

Synergy

In order to examine the synergistic effect of hydrolyzate, the quantitative value of malondialdehyde and lipid peroxidation when combined with the natural antioxidant α-tocopherol were measured, and when linolenic acid added α-tocopherol and pollack skin gelatin and dermal gelatin hydrolyzate, The synergistic effect of malondialdehyde was 20% to 50% higher than that of the control to which only α-tocopherol was added (Fig. 8), and the synergistic effect was 5% to 65% in lipid peroxidation (Fig. 9). . As a result, it was found that the most synergistic effect with commercially available antioxidants was a secondary hydrolyzate which exhibited the highest antioxidant power by itself.

Isolation of Antioxidant Peptides

Among the hydrolysates separated in each stage of the membrane reactor, only single peptides showing the strongest antioxidant power in the secondary hydrolyzate with high antioxidant power were separated by gel filtration, ion exchange chromatography, and high performance liquid chromatography. First, the gel filtration was performed on Sephadex G-25 and the antioxidant activity of each fraction was measured. The second hydrolyzate of pollack larvae showed the highest antioxidant activity in fractions 41 to 45 (peak G1) (Fig. 10). The primary hydrolysates showed the highest antioxidant activity at fractions between 46 and 50 (peak G2) (Fig. 11), and the molecular weights ranged from 2.1 to 2.7 KDa for pollock and 1.6 to 2.0 KDa for cowhide. It was.

Only the fraction with the highest antioxidant capacity was again fractionated using SP-Sephadex C-25, and then the antioxidant properties of each fraction were measured. In each case, fraction III (named fractions G1-III and G2-III, respectively). The highest antioxidant capacity was shown in Figs. 12 and 13, and each fraction was separated on a reverse phase ODS column using high performance liquid chromatography.

As a result, Ⅲ and Ⅱ fractions were obtained respectively (Figs. 14 and 15; fractions G1-III-III and G2-III-II, respectively), which were re-fractionated on high-performance liquid chromatography, respectively. This high single peptide was obtained (FIGS. 16 and 17; named fractions G1-III-III-II and G2-III-II-II, respectively).

Fractions G1-III-III-I to P1, G1-III-III-II to P2, G2-III-II-I to P3, G2-III-II-II to P4, and G2-III- II-III were named P5 and their amino acid sequences were analyzed.

Amino Acid Sequencing of Single Peptides

The amino acid sequencing of the antioxidant peptides P1-P5 was the same as SEQ ID NO: 1-5 (SEQ ID NO 1-5), respectively.

Peptides finally isolated from bovine gelatin and pollack gelatin hydrolysates had Gly at the N-terminus and contained all Gly, Pro and Hyp residues in the sequence. P1 and P2 have high antioxidant properties when Pro is located in position 2 due to lack of three residues of Pro-Hyp-Gly in Pro-Hyp-Gly and substitution of Glu in position 2 there was. P3 and P4 also replaced Pro with Glu at position 2 and Pro with position Ala. In addition, in the comparison between P4 and P5, all sequences were identical except that the C-terminus lacked Gly, indicating that Gly at the C-terminus had a significant effect on the antioxidant properties of the peptide.

The above results indicate that the antioxidant activity of the isolated peptide is the highest when it shows the continuous arrangement of Gly-Pro-Hyp, and it shows higher antioxidant activity compared to other sequences when the C-terminus is Gly and 10 or 13 residues of amino acid. Could know.

Effect of Antioxidant Peptides on Cell Lipid Oxidation

Cytotoxicity of t-butylhydroperoxide by concentration

The viability of hepatocytes according to the concentration of t-butyl hydroperoxide was measured after 150 minutes of t-butyl hydroperoxide. As a result, 50% cell survival was observed in 1 mM t-butyl hydroperoxide treatment. Butyl hydroperoxide was defined as MTT50 (FIG. 19).

Effect of Antioxidant Peptides on the Toxicity of t-Butylhydroperoxide

As a result of measuring the effect of antioxidant peptides on the oxidative cytotoxicity of t-butylhydroperoxide, it was found that the cell addition rate was significantly increased compared to the non-addition group. In the addition group, antioxidant peptides derived from pollock showed an increase in cell viability by 15-21% at various concentrations, and increased by 5-19% in the case of antioxidant peptides derived from cowhide (Fig. 18). . In addition, as a result of measuring the viability of the hepatocytes according to the concentration of t-butyl hydroperoxide after 150 minutes of t-butyl hydroperoxide, the antioxidant peptide added group was found to have a greater effect on the toxicity of t-butylhydroperoxide than the non-added group. It showed a less sensitive response of about 5 to 15% and a survival rate of about 40% even at a high concentration of 2mM (FIG. 19).

Antioxidant Activity of α-Tocopherol and Antioxidant Peptides

In the cytotoxicity test of the antioxidant peptide and α-tocopherol on t-butyl hydroperoxide, the synergistic effect of α-tocopherol showed 25 ~ 35% increase in cell viability in the dermal and pollock gelatin hydrolysates. In the case of α-tocopherol, there was a similar tendency, but α-tocopherol showed high antioxidant activity at high concentrations of 5 mg / ml or more, depending on the concentration, whereas antioxidant peptide showed concentration independence at high concentrations (FIG. 20). .

According to the present invention as described above, a specific peptide having high antioxidant capacity is provided from hydrolyzate of cowhide or fish gelatin, which peptide is characterized by having the amino acid sequence of Gly-Pro-Hyp in the molecule.

Since the peptide is excellent in antioxidant power, it can be used in various ways for the purpose of antioxidant activity, and since it is obtained from cowhide or skin, there is also an effect that it is possible to usefully recycle these raw materials currently disposed of.

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[SEQUENCE TABLE]

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Kim three books

(B) STREET: # 105 105, Sedong Hanshin Apartment 353-1, Yeonji-dong, Busanjin-gu

(C) CITY: Busan

(E) COUNTRY: US

(F) POSTAL CODE (ZIP): 614-070

(ii) TITLE OF a: antioxidant peptide and method for producing the same

(iii) NUMBER OF SEQUENCES: 5

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Pollack (Alaska pollack)

(F) TISSUE TYPE: Skin

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Gly Glu Hyp Gly Pro Hyp Gly Pro Hyp Gly Pro Hyp Gly Pro Hyp Gly

1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Pollack (Alaska pollack)

(F) TISSUE TYPE: Shell (Skin)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Gly Pro Hyp Gly Pro Hyp Gly Pro Hyp Gly Pro Hyp Gly

1 5 10

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE: small

(F) TISSUE TYPE: Shell

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Gly Glu Hyp Gly Pro Hyp Gly Ala Hyp

1 5

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE: small

(F) TISSUE TYPE: Shell

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Gly Pro Hyp Gly Pro Hyp Gly Pro Hyp Gly

1 5 10

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE: small

(F) TISSUE TYPE: Shell

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Gly Pro Hyp Gly Pro Hyp Gly Pro Hyp

1 5

Claims (7)

A peptide having the amino acid sequence of Gly-Pro-Hyp in a molecule | numerator, consisting of 9-16 amino acids, and having antioxidant power. The peptide according to claim 1, which has a Gly at the C-terminus and consists of 10-13 amino acids. The peptide according to claim 1 or 2, wherein the peptide is one selected from the group consisting of peptides having the amino acid sequence set forth in SEQ ID NOs 1 to 5. The following steps: (a) a step of extracting gelatin by hot water immersion of cow skin or cow skin; (b) hydrolyzing the gelatin with alcalase and passing the ultrafiltration membrane having a molecular weight cut-off of 10,000 to obtain a primary hydrolyzate comprising peptides having a molecular weight of 8-10 KDa and 6.5-7.5 KDa; (c) hydrolyzing the primary hydrolyzate obtained by the above step using a pronase and passing through an ultrafiltration membrane having a molecular weight limit of 5,000 to obtain a secondary hydrolyzate comprising peptides having a molecular weight of 1.5-4.5 KDa and 1 KDa or less. ; (d) hydrolyzing the secondary hydrolyzate obtained by the above step using collagenase and passing through an ultrafiltration membrane having a molecular weight limit of 1,000 to obtain a tertiary hydrolyzate comprising a peptide having a molecular weight of 0.5-2KDa; And (e) separating and purifying peptides having an amino acid sequence of Gly-Pro-Hyp and 9-16 amino acids in the molecule from the secondary hydrolyzate obtained in the step Method for producing an antioxidant peptide comprising a. 5. The method of claim 4, wherein the peptide has a Gly at the C-terminus and is composed of 9-16 amino acids. The method for producing an antioxidant peptide according to claim 4 or 5, wherein the peptide is one selected from the group consisting of peptides having the amino acid sequence set forth in SEQ ID NOs 1 to 5. 6. A peptide having an amino acid sequence of Gly-Pro-Hyp and consisting of 9-16 amino acids in the molecule, and having an antioxidant power, 0.05 to 1.0 based on the weight of the substance with respect to a substance to be prevented from oxidation. A method for preventing oxidation of a substance, characterized in that the addition in the amount of weight percent.