Antioxidant peptide and ace inhibitory peptide from the viscera of haliotis discus hannai and preparation method and application thereof

Abstract

The application discloses wrinkle abalone visceral-derived antioxidant peptides and ACE inhibiting peptides, and a preparation method and application thereof. 50 With DPPH free radical scavenging activity and ACE inhibiting activity as the guidance, the enzymatic hydrolysate is separated and purified through a series of methods such as ultrafiltration, dextran gel chromatography and high performance liquid chromatography, and is combined with bioinformatics screening to obtain one DPPH free radical scavenging activity peptide and three ACE inhibiting peptides, the amino acid sequences of which are shown as SEQ ID NO. 1-4, and the IC 50 values are 3.05 mg / mL, 77.43 μM, 143.93 μM and 124.10 μM respectively, and the four polypeptides have great application value. The application realizes high-value utilization of abalone viscera, and provides a reliable basis for application of abalone by-products.

Description

Technical Field

[0001] This invention belongs to the field of antioxidant peptides and ACE inhibitory peptides, specifically relating to antioxidant peptides and ACE inhibitory peptides derived from the viscera of the wrinkled abalone, their preparation methods, and applications. Background Technology

[0002] Many natural antioxidants have been identified as having free radical scavenging or reactive oxygen species scavenging functions. To reduce the potential side effects of synthetic antioxidants, there is growing interest in finding new natural antioxidants from food or medicinal materials to replace them. Protein peptides, as natural antioxidants, are non-toxic, harmless, and provide nutrition, effectively preventing damage to the body caused by chronic inflammation or oxidative stress, and thus possess great development potential.

[0003] Hypertension is a common chronic cardiovascular disease. It has the highest mortality rate in the world, reaching 12.8%. ACE plays a crucial physiological role in regulating blood pressure by converting angiotensin I to angiotensin II, which ultimately leads to vasoconstriction and elevated blood pressure. Therefore, inhibiting ACE activity is a major target in the prevention of hypertension pathophysiology. Although studies have shown that ACE synthesis inhibitors such as captopril, acarbose, enalapril, and lisinopril are used to treat essential hypertension and heart failure in humans, these synthetic drugs have side effects such as cough, rash, taste disturbance, and angioedema.

[0004] The search for novel, highly effective, and low-toxicity alternatives to chemical drugs has become a current research hotspot. Marine bioactive peptides, due to their abundant resources, unique structures, and safe and effective properties, have attracted significant attention in the pharmaceutical and health fields. my country is rich in abalone resources, and people mainly consume the abalone's foot. However, during abalone processing, abalone viscera, accounting for 15%–25% of the total abalone weight, are generated as a byproduct. These byproducts are usually discarded or processed into low-value fishmeal, which not only fails to fully utilize this resource but also causes environmental pollution. Although the large amount of byproducts generated during abalone food processing has low commercial value, identifying natural bioactive compounds with ACE inhibitory or antioxidant activities from them can significantly improve environmental and cost-effectiveness. Summary of the Invention

[0005] The purpose of this invention is to provide antioxidant peptides and ACE inhibitory peptides derived from the viscera of abalone (wrinkled abalone), which are highly safe and have industrial application value.

[0006] The first objective of this invention is to provide a DPPH free radical scavenging active peptide with the amino acid sequence YNKDSTASK.

[0007] A second objective of this invention is to provide an ACE inhibitory peptide having the amino acid sequence VYINF, FQPSF, or YSLFLL.

[0008] This invention involves multiple separations and purifications of collagen protein hydrolysates from the visceral connective tissue of the wrinkled abalone to screen for highly active DPPH free radical scavenging peptides and ACE inhibitory peptides. The DPPH free radical scavenging peptide has the amino acid sequence Tyr-Asn-Lys-Asp-Ser-Thr-Ala-Ser-Lys (YNKDSTASK); and three ACE inhibitory peptides have the amino acid sequences Val-Tyr-Ile-Asn-Phe (VYINF), Phe-Gln-Pro-Ser-Phe (FQPSF), and Tyr-Ser-Leu-Phe-Leu-Leu (YSLFLL).

[0009] Based on the aforementioned antioxidant peptides and ACE inhibitory peptides, any corresponding adjustments or modifications thereof fall within the scope of protection of this invention.

[0010] A third objective of this invention is to provide a method for preparing the aforementioned DPPH free radical scavenging active peptide or the aforementioned ACE inhibitory peptide, comprising the following steps:

[0011] (1) Preparation of collagen hydrolysate from visceral connective tissue of abalone: ​​Collagen from visceral connective tissue of abalone was added to a protease for enzymatic hydrolysis. The enzymatic hydrolysis conditions were: enzyme-to-protein ratio 6000-8000 U / g, hydrolysis time 5-7 h, hydrolysis temperature 30-40℃, pH 1.0-2.0. After hydrolysis, the enzyme was inactivated, centrifuged and filtered, and the supernatant was collected.

[0012] (2) Separation and purification of enzymatic hydrolysate: The supernatant was initially separated, and the components with a molecular weight less than 3 kDa were separated and purified by dextran gel. The DPPH free radical scavenging rate and ACE inhibition rate of each component were measured. Finally, the components with high DPPH free radical scavenging activity and ACE inhibition activity after gel column elution were separated on RP-HPLC column, and the DPPH free radical scavenging activity and ACE inhibition activity of each component were measured.

[0013] (3) Identification of peptides: The highly active components separated by high performance liquid chromatography were identified. The high-purity active peptides were identified by MAIDL-TOF / TOF mass spectrometry. The secondary mass spectrometry data were obtained by ESI / LC-MS / MS. The peptide sequences in the peptide mixture were obtained by de novo sequencing.

[0014] (4) Screening of identified peptides by combining ALC values ​​and binding energies of the sequences;

[0015] (5) and activity verification: synthesize peptides and measure their DPPH free radical scavenging activity or ACE inhibitory activity to verify the activity of the target peptide.

[0016] Preferably, the protease mentioned in step (1) is pepsin.

[0017] Preferably, the dextran gel in step (2) is a Sephadex G25 column with an inner diameter of 3.5 cm and a length of 30 cm; the RP-HPLC column is a YMC-Pack ODS-A C18 semi-preparative chromatography column with a particle size of 5 μm, an inner diameter of 10 mm, and a length of 250 mm.

[0018] Preferably, the MAIDL-TOF / TOF in step (3) is analyzed using a YMC-Pack ODS-A chromatographic column. The elution conditions are as follows: eluent A is 0.1% formic acid water (v / v), and eluent B is methanol; the elution gradient is 100%-100% A for 0-5 min, 100%-0% A for 5-55 min; the flow rate is 1 mL / min, and the injection volume is 20 μL. Then, mass spectrometry identification is performed using LC-ESI-MS-MS in positive ion mode with an m / z range of 200-2000.

[0019] Preferably, in step (3), the purified peptide is analyzed by electrospray ionization using high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) with a molecular weight range of 200-2000. The chromatographic conditions for LC-MS / MS are as follows: flow rate of 1 mL / min, injection volume of 30 μL, mobile phase A of 0.1% formic acid water, mobile phase B of methanol, and elution conditions of 99%-99% A for 0-5 min and 99%-0% A for 5-55 min.

[0020] A fourth objective of this invention is to provide the application of the above-mentioned DPPH free radical scavenging active peptide in the preparation of antioxidants.

[0021] A fifth object of the present invention is to provide an antioxidant containing the above-mentioned DPPH free radical scavenging active peptide as an active ingredient.

[0022] The sixth object of the present invention is to provide the use of the above-mentioned ACE inhibitory peptide in the preparation of angiotensin-converting enzyme inhibitors or in the preparation of drugs for treating hypertension.

[0023] A seventh object of the present invention is to provide a pharmaceutical composition containing the above-mentioned ACE inhibitory peptide as an active ingredient.

[0024] The beneficial effects of this invention are as follows:

[0025] 1. Collagen from the connective tissue of the wrinkled abalone viscera was enzymatically hydrolyzed. After hydrolysis with pepsin, the hydrolysate was obtained with high antioxidant and ACE inhibitory activity. It also showed high DPPH free radical scavenging and ACE inhibitory activity.

[0026] 2. Guided by DPPH free radical scavenging activity, the enzymatic hydrolysate was separated and purified by a series of methods including ultrafiltration, dextran gel chromatography, and high-performance liquid chromatography to obtain a peptide with DPPH free radical scavenging activity. Its amino acid sequence is: Tyr-Asn-Lys-Asp-Ser-Thr-Ala-Ser-Lys, IC50. 50 The concentration was 3.05 mg / mL.

[0027] 3. Guided by ACE inhibitory activity, the enzymatic hydrolysate was separated and purified using a series of methods including ultrafiltration, dextran gel chromatography, and high-performance liquid chromatography. Combined with bioinformatics screening, three ACE-inhibiting peptides were obtained with the following amino acid sequences: VYINF (MW: 655.3433 Da), FQPSF (MW: 625.3051 Da), and YSLFLL (MW: 704.85 Da). These three peptides were biosynthesized using a solid-phase synthesis method, and their activity was determined. The results showed that their ACE-inhibiting IC50 values ​​were [value missing]. 50 The values ​​were 77.43 μg, 143.93 μg and 124.10 μg, respectively, which have extremely high application value.

[0028] 4. This invention achieves efficient preparation and screening of small molecule bioactive peptides. The method is simple to operate and increases the success rate of screening highly active single peptides.

[0029] 5. This invention realizes the high-value utilization of the viscera of wrinkled abalone, and provides a reliable basis for the deep processing of wrinkled abalone by-products. Attached Figure Description

[0030] Figure 1 This is a flowchart illustrating the separation, purification, identification, and screening of antioxidant active peptides and ACE inhibitory peptides of the present invention.

[0031] Figure 2 This is a gel chromatography elution curve of Sephadex G25.

[0032] Figure 3 This is a graph showing the DPPH radical scavenging activity and ACE inhibitory activity of each separated component by gel chromatography.

[0033] Figure 4 This is a secondary mass spectrum of the DPPH radical scavenging active peptide YNKDSTASK of the present invention.

[0034] Figure 5 The diagram shows the inhibitory activity of the DPPH free radical scavenging peptide YNKDSTASK of the present invention.

[0035] Figure 6 The active fraction SGⅢ of this invention is used to separate the components and their ACEI activity.

[0036] Figure 7 This is a diagram showing the inhibitory activity of the ACE inhibitory peptide VYINF of the present invention.

[0037] Figure 8 This is a diagram showing the inhibitory activity of the ACE inhibitory peptide FQPSF of the present invention.

[0038] Figure 9 This is a diagram showing the inhibitory activity of the ACE inhibitory peptide YSLFLL of the present invention. Detailed Implementation

[0039] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0040] Example:

[0041] like Figure 1 The method for isolating, purifying, and screening antioxidant peptides and ACE inhibitory peptides derived from abalone viscera includes the following steps:

[0042] S1. Preparation of collagen protein hydrolysate from the visceral connective tissue of Abalone fasciatus.

[0043] Thaw abalone viscera with water, separate the connective tissue and gonads from the viscera, and wash and drain the connective tissue. Soak the abalone viscera connective tissue in a 0.10 mol / L NaOH solution at a material-to-liquid ratio of 1:8 (w / vg / ml) for 6 hours, then wash until neutral and drain. Add 0.5 mol / L acetic acid solution at a material-to-liquid ratio of 1:10 (w / vg / ml), soak for 48 hours, centrifuge (4000 rpm, 30 min), collect the supernatant, and freeze-dry to obtain acid-soluble collagen. Take the precipitate obtained from the centrifugation, add it to a 0.5 mol / L acetic acid solution at a material-to-liquid ratio of 1:8 (w / vg / ml), add pepsin at 240 U / g, soak for 48 hours, centrifuge (4000 rpm, 30 min), collect the supernatant, and freeze-dry to obtain enzyme-soluble collagen. The acid-soluble and enzyme-soluble collagen solutions were combined and preheated at 37°C for 10 min. The pH of the solution was adjusted to 1.0 with 1 mol / L HCl. Pepsin was added at an enzyme-to-protein ratio of 7000 U / g. The enzymatic hydrolysis temperature was 37°C. After reacting for 7 h, the enzyme was inactivated by heating at 100°C for 10 min. The solution was then centrifuged at 4°C and 4000 r / min for 20 min. The supernatant was collected to obtain HVCH, a collagen hydrolysate of the visceral connective tissue of the wrinkled abalone. The hydrolysate was lyophilized and stored at -20°C.

[0044] S2. Separation of enzymatic hydrolysates

[0045] (1) The specific steps were as follows: HVCH was ultrafiltered using ultrafiltration tubes with molecular weight cutoffs (MWCO) of 3 kDa and 10 kDa, respectively, to obtain three fractions: UF1 (<3 kDa), UF2 (3-10 kDa), and UF3 (>10 kDa). Among them, UF1 showed the highest ACE inhibition rate (27.64±2.61%) at 1 mg / mL, which was significantly higher (P<0.05) than UF2 (21.14±4.53%) and UF3 (12.74±2.05%). UF1 was further separated and purified using a Sephadex G25 dextran gel column (3.5 cm inner diameter, 30 cm length). The UF1 ultrafiltration unit was prepared at a concentration of 50 mg / mL, filtered through a 0.45 μm aqueous membrane, and 4 mL was injected. Elution was performed with distilled water at a mobile phase flow rate of 5 mL / min. Fractions were automatically collected in 30 mL portions at a detection wavelength of 220 nm, yielding a total of 15 fractions. These fractions were further combined according to peak assignment to obtain identical components. After lyophilization, three fractions (SGⅠ, SGⅡ, and SGⅢ) were obtained. Figure 2 As shown. The DPPH free radical scavenging rates of the three fractions SGⅠ, SGⅡ, and SGⅢ were determined. At a concentration of 1 mg / mL, the DPPH free radical scavenging rates of the three fractions were 37.77±0.75% (SGⅠ), 17.98±1.85% (SGⅡ), and 93.80±0.02% (SGⅢ), respectively. The ACEI activities of the three fractions at a concentration of 1 mg / mL were determined: SGⅠ was 39±0.56%, SGⅡ was 18±2.79%, and SGⅢ was 86.12±4.35%. Figure 3 Further analysis of SGⅠ and SGⅢ will be conducted to identify the effective components.

[0046] S3

[0047] (1) Isolation and identification of active peptides in SGI

[0048] SGⅠ was prepared into a 100 mg / mL solution, filtered through a 0.45 μm aqueous membrane, and separated using an Agilent liquid chromatography system via a YMC-Pack ODS-A C18 semi-preparative column (250 × 10.0 mm, 5 μm). The flow rate was 2 mL / min, and the injection volume was 100 μL. The elution conditions were: mobile phase A was 0.1% trifluoroacetic acid (TFA) by volume, mobile phase B was acetonitrile, and the gradient elution program was: 99%-99% A (0-5 min), 99%-74% A (5-30 min). A single, high-purity peptide, CM-4, was isolated from SGⅠ by semi-preparative RP-HPLC.

[0049] The amino acid sequence of CM-4 was identified using MALDI-TOF / TOF mass spectrometry. Positive ion detection was performed in reflectance mode. The mass scan range of the primary mass spectrometer was 800–4000 Da, using a nitrogen laser with a wavelength of 337 nm. The primary mass spectrometer signal was accumulated 600–800 times per scan, and the secondary mass spectrometer signal was accumulated 900–1200 times per scan. The mass spectrometry data were analyzed using the de novo algorithm, and the peptide was identified as YNKDSTASK (YK-9, MW: 1013.05 Da). Figure 4 The DPPH free radical scavenging ability was determined, and the results are shown in [the table below]. Figure 5 .

[0050] (2) Isolation and identification of active peptides in SGIII

[0051] SGⅢ was prepared into a 100 mg / mL solution, filtered through a 0.45 μm aqueous filter membrane, and separated using an Agilent liquid chromatography system via a YMC-Pack ODS-A C18 semi-preparative column (250 × 10.0 mm, 5 μm). The flow rate was 2 mL / min, the injection volume was 100 μL, mobile phase A was 0.1% trifluoroacetic acid (TFA), and mobile phase B was acetonitrile. The elution conditions were: 99%–99% A (0–5 min), 99%–0% A (5–55 min). After semi-preparative RP-HPLC separation, SGⅢ was separated into seven different fractions, PG1–PG7. The DPPH radical scavenging activity and ACEI activity of each fraction were determined. Figure 6 .

[0052] The peptides and their amino acid sequences in the most active PG6 fraction were separated and identified using LC-ESI-MS / MS. The HPLC separation conditions were as follows: SGⅢ was prepared into a 100 mg / mL solution, filtered through a 0.45 μm aqueous membrane, and separated using an Agilent liquid chromatography system via a YMC-Pack ODS-AC18 semi-preparative column (250 × 10.0 mm, 5 μm). The flow rate was 2 mL / min, the injection volume was 100 μL, mobile phase A was 0.1% trifluoroacetic acid (TFA), and mobile phase B was acetonitrile. The elution conditions were: 99%–99% A (0–5 min), 99%–0% A (5–55 min). The ESI mass spectrometry conditions were as follows: positive ion mode, m / z range 200–3000, drying temperature: 180 °C; electrospray capillary voltage: 4.5 kV; drying gas flow rate: 4.0 L / min. Mass spectrometry data were analyzed using PEAKS Studio software. The de novo algorithm was used to confirm the amino acid sequences of each fraction in the stream, resulting in 54 peptides with ALC values ​​higher than 95% (Table 1).

[0053] Table 1

[0054]

[0055]

[0056] Molecular docking was performed using AutoDock Tools 1.5.6 software. ACE (PDB ID: 1O8A) was used as the target site, and the protein crystal structure was obtained from the protein database (https: / / www.rcsb.org / ). Water and the original ligand in the ACE protein molecule were removed using Pymol 2.1 software, and polar hydrogen atoms were added. The protein molecule (receptor) was then imported into AutoDock Tools 1.5.6 and saved as a pdbqt file. Energy minimization was performed on the target peptide selected for SGIII analysis using Chem3D, and the result was converted to mol2 format. The mol2 file containing the compound was imported into AutoDock Tools 1.5.6 software, atomic charges were added, atom types were assigned, and all flexible bonds were rotatable by default. Finally, it was saved as a pdbqt file. The processed molecules were used as small molecule ligands, and five protein targets were used as receptors. The center position of the GridBox (x_center = 40.673, y_center = 37.376, z_center = 43.33) and the dimensions (length, width, and height) were all set to 80×80×80, determined based on the interaction between the small molecules and the targets. Batch molecular docking was performed using AutoDock, and the docking results were analyzed. The binding interaction between the compounds and proteins was visualized using Pymol 2.1 software. During the calculation, the Lamarckian genetic algorithm was used for molecular docking calculations. The algorithm was as follows: a population of 150, a maximum energy evaluation of 25 million, a maximum number of iterations of 2000, a crossover rate of 0.8, a mutation rate of 0.02, and 10 independent docking runs. The LibDock score of each ligand peptide was determined according to the prescribed method. The top three LibDock scores for FQPSF (score: 205.606), VYLNF (score: 202.838), and YSLFLL (score: 222.984) suggest that these three peptides may have good ACEI activity.

[0057] S4, Activity Verification

[0058] (1) Sample

[0059] The peptide YNKDSTASK (CM-4) sample was prepared from abalone viscera hydrolysate (see Section S3 for details). Peptides FQPSF, VYLNF, and YSLPLL were prepared by Guangzhou Tongdaxing Technology Co., Ltd. using solid-phase synthesis. All samples had a purity greater than 98%, and their molecular weight was verified by MS, while purity was determined by HPLC.

[0060] (2) Antioxidant activity verification

[0061] The method for determining DPPH radical scavenging activity was based on the method described by Zheng et al. (2019). Equal volumes of DPPH radicals (0.2 mM, dissolved in ethanol) and samples (0-6.4 mg / mL gradient concentrations) were mixed and incubated at room temperature in the dark for 30 min. The absorbance of the solution was measured at 517 nm and denoted as As. Ethanol was used as the sample control group instead of DPPH, denoted as Ac; distilled water was used as the blank control group instead of the sample, denoted as A0.

[0062]

[0063] The DPPH radical scavenging activities of the peptides YNKDSTASK, FQPSF, VYLNF, and YSLPLL were determined using the above methods. The YNKDSTASK test results are shown in the appendix. Figure 5 Regression-Probit regression analysis was used to obtain IC. 50 The concentration was 3.05 mg / mL. For peptides FQPSF, VYLNF, and YSLLL, the dose was increased to 10 mg / mL, but the clearance rate did not exceed 50%.

[0064] (3) ACEI activity verification

[0065] The ACE inhibitory activity was determined as follows: 40 μL of ACE solution (0.1 U / mL), 200 μL of FAPGG solution (1.0 mmol / mL), and 160 μL of sample were mixed and incubated at 37 °C for 30 min. The absorbance was then measured at 340 nm. The ACE inhibitory activity was determined by the following formula:

[0066] ACE inhibition rate = (OD control group - OD sample group) / (OD control group - OD blank group) × 100%

[0067] The activity test results of ACEI peptide amino acids VYINF, FQPSF, or YSLFLL are as follows: Figures 7-9 As shown. Regression-Probit regression analysis was used to determine the ACE inhibition IC50. 50 The values ​​were 77.43 μg, 143.93 μg, and 124.10 μg, respectively.

[0068] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A DPPH free radical scavenging active peptide, characterized in that, The amino acid sequence is YNKDSTASK.

2. The method for preparing the DPPH free radical scavenging active peptide according to claim 1, characterized in that, Includes the following steps: (1) Preparation of collagen hydrolysate from visceral connective tissue of abalone: ​​Collagen from visceral connective tissue of abalone was added to a protease for enzymatic hydrolysis. The enzymatic hydrolysis conditions were: enzyme-to-protein ratio 6000-8000 U / g, hydrolysis time 5-7 h, hydrolysis temperature 30-40℃, pH 1.0-2.

0. After hydrolysis, the enzyme was inactivated, centrifuged and filtered, and the supernatant was collected. (2) Separation and purification of enzymatic hydrolysate: The supernatant was initially separated, and the components with a molecular weight less than 3 kDa were separated and purified by dextran gel. The DPPH free radical scavenging rate of each component was measured. Finally, the gel column with high DPPH free radical scavenging activity was separated on an RP-HPLC column, and the DPPH free radical scavenging activity and ACE inhibition activity of each component were measured. (3) Identification of peptides: The most active component after separation by high performance liquid chromatography is identified. The active peptides are identified by MAIDL-TOF / TOF mass spectrometry, or by ESI / LC-MS / MS to obtain secondary mass spectrometry data, and the peptide sequence is obtained by de novo sequencing. (4) Screening of identified peptides by combining ALC values ​​and binding energies of the sequences; (5) Peptide activity verification: Synthesize peptides and measure their DPPH free radical scavenging activity to verify the activity of the synthesized peptides.

3. The preparation method according to claim 2, characterized in that, The protease mentioned in step (1) is pepsin.

4. The preparation method according to claim 2, characterized in that, The dextran gel mentioned in step (2) is a Sephadex G25 column with an inner diameter of 3.5 cm and a length of 30 cm; the RP-HPLC column is a YMC-Pack ODS-AC18 semi-preparative chromatography column with a particle size of 5 μm, an inner diameter of 10 mm, and a length of 250 mm.

5. The preparation method according to claim 2, characterized in that the peptide sequence identification in step (3) uses MALDI-TOF / TOF mass spectrometry to identify high-purity peptides, performs positive ion detection in reflectance mode, the mass scan range of the primary mass spectrometry is 800-4000 Da, a nitrogen laser with a wavelength of 337 nm is used, the primary mass spectrometry signal is accumulated 600-800 times per scan, the secondary mass spectrometry signal is accumulated 900-1200 times per scan, and in the analysis of active peptide mixtures, amino acid sequences are separated and identified using LC-ESI-MS-MS, with a YMC-Pack column. ODS-A analysis was performed under the following elution conditions: eluent A was 0.1% formic acid water (v / v), and eluent B was methanol; elution gradient: 99%-99% A for 0-5 min, 100%-0% A for 5-55 min; flow rate: 1 mL / min; injection volume: 20 μL; mass spectrometry was performed in positive ion mode, with an m / z range of 200-2000; the purified peptides were analyzed by electrospray ionization using high performance liquid chromatography-mass spectrometry (ESI / LC-MS / MS), with an analytical molecular weight range of 200-2000 Da.

6. An antioxidant, characterized in that, It contains the DPPH free radical scavenging active peptide as described in claim 1 as an active ingredient.

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