Haliotis discus hannai viscera-derived antioxidant peptide and ACE inhibitory peptide as well as preparation method and application thereof

Abstract

The invention discloses a haliotis discus hannai viscera-derived antioxidant peptide and ACE inhibitory peptide as well as a preparation method and application thereof. DPPH free radical scavenging activity and ACE (angiotensin converting enzyme) inhibitory activity are taken as guidance, zymolyte is separated and purified by a series of methods such as ultrafiltration, sephadex chromatography and high performance liquid chromatography, one DPPH free radical scavenging active peptide and three ACE inhibitory peptides are obtained by screening in combination with bioinformatics, amino acid sequences are shown as SEQ ID NO.1-4, IC50 values are 3.05 mg / mL, 77.43 mu M, 143.93 mu M and 124.10 mu M respectively, and the ACE inhibitory peptides are used for inhibiting ACE activity. The four polypeptides have great application value. According to the method, high-value utilization of the abalone viscera is realized, and a reliable basis is provided for application of abalone byproducts.

Description

Technical Field

[0001] The invention belongs to the technical field of antioxidant peptides and ACE inhibitory peptides, and particularly relates to antioxidant peptides and ACE inhibitory peptides derived from viscera of Haliotis discus hannai and preparation methods and applications thereof. Background Art

[0002] Many natural antioxidants have been identified as having free radical scavenging or reactive oxygen scavenging functions. In order to reduce the potential side effects of synthetic antioxidants, people are increasingly interested in finding new natural antioxidants from food or medicinal materials to replace synthetic antioxidants. Protein peptides, as natural antioxidants, are non-toxic and harmless, can provide nutrition, and can effectively prevent body damage caused by chronic inflammation or oxidative stress, and have great development potential.

[0003] Hypertension is a common chronic cardiovascular disease. The mortality rate of hypertension ranks first in the world, as high as 12.8%. ACE plays an important physiological role in regulating blood pressure by converting angiotensin I into angiotensin II, which eventually causes vasoconstriction and increased blood pressure. Therefore, inhibiting ACE activity has become the main target for preventing the pathophysiology of hypertension. Although studies have applied synthetic ACE inhibitors such as captopril, acapril, enalapril, and lisinopril to treat primary hypertension and heart failure in humans, these synthetic drugs have side effects such as cough, rash, taste disorders, and angioedema.

[0004] Finding new, efficient, and low-toxic alternatives to chemical drugs has become a current research hotspot. Marine bioactive peptides have attracted great attention in the field of medicine and health due to their rich resources, unique structure, and safe and effective characteristics. my country has abundant abalone resources, and people mainly eat the gastropod of abalone. In the process of abalone processing, abalone viscera are produced as byproducts, accounting for 15% to 25% of the total mass of abalone. Abalone byproducts are usually discarded or made into fish meal of lower value, which not only does not make full use of the resources, but also causes environmental pollution. In the process of abalone food processing, although a large number of byproducts are produced with low commercial value, discovering natural active compounds with ACE inhibition or antioxidant activity can significantly improve environmental benefits and cost-effectiveness. Summary of the invention

[0005] The invention aims to provide abalone (Halion discus hannai) viscera-derived antioxidant peptides and ACE inhibitory peptides, which have high safety and industrial application value.

[0006] The first object of the present invention is to provide a DPPH free radical scavenging active peptide, whose amino acid sequence is YNKDSTASK.

[0007] The second object of the present invention is to provide an ACE inhibitory peptide, whose amino acid sequence is VYINF, FQPSF or YSLFLL.

[0008] The invention separates and purifies the collagen enzymatic hydrolysate of the visceral connective tissue of abalone discus hannai for multiple times to screen out high-activity DPPH free radical scavenging active peptides and ACE inhibitory peptides, wherein the amino acid sequence of the DPPH free radical scavenging active peptide is Tyr-Asn-Lys-Asp-Ser-Thr-Ala-Ser-Lys (YNKDSTASK); and the amino acid sequences of three ACE inhibitory peptides are Val-Tyr-Ile-Asn-Phe (VYINF), Phe-Gln-Pro-Ser-Phe (FQPSF), and Tyr-Ser-Leu-Phe-Leu-Leu (YSLFLL).

[0009] With the antioxidant peptide and ACE inhibitory peptide as the core, any corresponding adjustment or modification thereof falls within the protection scope of the present invention.

[0010] The third object of the present invention is to provide a method for preparing the above-mentioned DPPH free radical scavenging active peptide or the above-mentioned ACE inhibitory peptide, which comprises the following steps:

[0011] (1) Preparation of collagen hydrolysate of visceral connective tissue of Haliotis discus hannai: taking the visceral connective tissue collagen of Haliotis discus hannai and adding protease to hydrolyze, the hydrolysis conditions range as follows: enzyme substrate ratio 6000-8000U / g, hydrolysis 5-7h, hydrolysis temperature 30-40°C, pH 1.0-2.0, inactivating the enzyme after hydrolysis, centrifuging and filtering, and taking the supernatant;

[0012] (2) Separation and purification of enzymatic hydrolysates: The supernatant was preliminarily separated, and the components with molecular weight less than 3 KDa were separated and purified by dextran gel separation, and the DPPH free radical scavenging rate and ACE inhibition rate of each component were determined respectively. Finally, the components with higher DPPH free radical scavenging activity and ACE inhibition activity after elution from the gel column were separated on a RP-HPLC column, and the DPPH free radical scavenging activity and ACE inhibition activity of each component were determined;

[0013] (3) Peptide identification: The components with higher activity after HPLC separation are identified, and the high-purity active peptides are identified by MAIDL-TOF / TOF mass spectrometry. The secondary mass spectrometry data are obtained by ESI / LC-MS / MS, and the peptide sequences in the peptide mixture are obtained by denovo sequencing;

[0014] (4) Screening the identified peptides based on the ALC value and binding energy of the binding sequence;

[0015] (5) 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 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 described in step (3) uses a chromatographic column YMC-Pack ODS-A for analysis, and the elution conditions are: eluent A is 0.1% formic acid water by volume, and eluent B is methanol; the elution gradient is 100%-100% A 0-5min, 100%-0% A 5-55min; the flow rate is 1mL / min, the injection volume is 20μL, and then LC-ESI-MS-MS is used for mass spectrometry identification, using positive ion mode, and the m / z range is 200-2000.

[0019] Preferably, the ESI / LC-MS / MS described in step (3) uses high performance liquid chromatography-mass spectrometry to perform electrospray ionization analysis on the purified polypeptide, and the analytical molecular weight range is 200-2000. The chromatographic conditions used for LC-MS / MS are: flow rate of 1 mL / min, injection volume of 30 μL, mobile phase A is 0.1% formic acid water, mobile phase B is methanol, and elution conditions are: 99%-99% A0-5min, 99%-0% A 5-55min.

[0020] The fourth object of the present invention is to provide the use 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 comprising 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 drugs for treating hypertension.

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

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

[0025] 1. The collagen of the visceral connective tissue of Abalone discus haudiorum was enzymatically hydrolyzed, and the hydrolyzate with high antioxidant activity and ACE inhibitory activity was obtained after enzymatic hydrolysis with pepsin. The DPPH free radical scavenging activity and ACE inhibitory activity were relatively high.

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

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

[0028] 4. The present invention realizes the efficient preparation and screening of small molecule active peptides. The method is simple to operate and increases the probability of successful screening of highly active single peptides.

[0029] 5. The present invention realizes the high-value utilization of the internal organs of Haliotis discus hannai, and provides a reliable basis for the deep processing of Haliotis discus hannai by-products. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The present invention is a flow chart for separation, purification, identification and screening of antioxidant active peptides and ACE inhibitory peptides.

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

[0032] Figure 3 The graph shows the DPPH free radical scavenging activity and ACE inhibitory activity of each component separated by gel chromatography.

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

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

[0035] Figure 6 The active fraction SGⅢ separated components and their ACEI activity of the present invention.

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

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

[0038] Fig. 9 This is a graph showing the inhibitory activity of the ACE inhibitory peptide YSLFLL of the present invention. DETAILED DESCRIPTION

[0039] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0040] Example:

[0041] like Figure 1 As shown, the separation, purification and screening method of antioxidant peptides and ACE inhibitory peptides derived from abalone viscera comprises the following steps:

[0042] S1. Preparation of collagen enzymatic hydrolysate from visceral connective tissue of Haliotis discus hannai

[0043] Thaw the abalone viscera with water, separate the connective tissue and gonads of the abalone viscera, and wash the connective tissue and drain. Soak the abalone viscera connective tissue for 6 hours in a NaOH solution with a solid-liquid ratio of 1:8 (w / vg / ml), wash until neutral and drain. Add a 0.5mol / L acetic acid solution at a solid-liquid ratio of 1:10 (w / vg / ml), soak for 48 hours, take the supernatant after centrifugation (4000rpm, 30min), and freeze-dry to obtain acid-soluble collagen. Take the above centrifugal precipitation, add a 0.5mol / L acetic acid solution at a solid-liquid ratio of 1:8 (w / vg / ml), add pepsin at 240U / g, soak for 48 hours, take the supernatant after centrifugation (4000rpm, 30min), and freeze-dry to obtain enzyme-soluble collagen. The acid-soluble collagen 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-substrate 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 centrifuged at 4°C and 4000 r / min for 20 min. The supernatant was taken to obtain the collagen hydrolysate HVCH of the visceral connective tissue of the wrinkled abalone, which was freeze-dried and stored at -20°C.

[0044] S2. Separation of enzymatic hydrolysate

[0045] (1) The specific steps are as follows: ultrafiltration of HVCH was performed using ultrafiltration tubes with molecular weight cutoff (MWCO) of 3 kDa and 10 kDa, respectively, to obtain three components: UF1 (<3 kDa), UF2 (3-10 kDa) and UF3 (>10 kDa). Among them, UF1 had the highest ACE inhibition rate at 1 mg / mL (27.64±2.61%), which was significantly (P<0.05) higher than UF2 (21.14±4.53%) and UF3 (12.74±2.05%). UF1 was further separated and purified using a Sephadex G25 column (column inner diameter 3.5 cm, column length 30 cm). The UF1 ultrafiltration component was prepared into 50 mg / mL, and 4 mL was injected after passing through a 0.45 μm water filter membrane. The fractions were eluted with distilled water at a mobile phase flow rate of 5 mL / min. The fractions were automatically collected, each with 30 mL. The detection wavelength was 220 nm, and a total of 15 fractions were obtained. The same components were further combined according to the attribution of the chromatographic peaks. After freeze-drying, SGⅠ, SGⅡ and SGⅢ were obtained, a total of 3 fractions, such as 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 scavenging rates of the three fractions on DPPH free radicals were 37.77±0.75% (SGⅠ), 17.98±1.85% (SGⅡ) and 93.80±0.02% (SGⅢ), respectively. The ACEI activity of the three fractions at a concentration of 1 mg / mL was determined, with SGⅠ being 39±0.56%, SGII being 18±2.79%, and SGⅢ being 86.12±4.35% ( Figure 3 ). Further analysis was conducted on SGⅠ and SGⅢ to find out the active ingredients.

[0046] S3,

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

[0048] SGⅠ was prepared into a 100 mg / mL solution, passed through a 0.45 μm water filter membrane, and separated using an Agilent liquid chromatography system on 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, and the elution conditions used were that the mobile phase A was 0.1% trifluoroacetic acid (TFA) by volume, the 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 component high-purity peptide segment CM-4 was separated and prepared from SGⅠ by semi-preparative RP-HPLC separation.

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

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

[0051] SGⅢ was prepared into a 100 mg / mL solution, passed through a 0.45 μm water filter membrane, and separated using an Agilent liquid chromatography system on 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, the mobile phase A was 0.1% trifluoroacetic acid (TFA), the mobile phase B was acetonitrile, and the elution conditions were: 99%-99% A (0-5 min), 99%-0% A (5-55 min). After semi-preparative RP-HPLC separation, SGⅢ was divided into seven different fractions from PG1 to PG7, and the DPPH free radical scavenging activity and ACEI activity of each fraction were determined. Figure 6 .

[0052] LC-ESI-MS / MS technology was used to separate and identify the peptides and their amino acid sequences in the most active PG6 fraction. The HPLC separation conditions were as follows: SGⅢ was prepared into a 100 mg / mL solution, passed through a 0.45 μm water filter membrane, and separated using an Agilent liquid chromatography system on 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, the mobile phase A was 0.1% trifluoroacetic acid (TFA), the mobile phase B was acetonitrile, and 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; the flow rate of drying gas was 4.0 L / min. The mass spectrometry data were analyzed using PEAKS Studio software. The de novo algorithm was used to confirm the amino acid sequence of each component in the flow fraction, and a total of 54 peptides with ALC values ​​higher than 95% were obtained (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 docking target, and the protein crystal structure was obtained from the Protein Data Bank (https: / / www.rcsb.org / ). Pymol 2.1 software was used to delete water and original ligands in the ACE protein molecule, and after adding polar hydrogen atoms, the protein molecule (receptor) was imported into AutoDockTools 1.5.6 software and finally saved as a pdbqt file. The target peptide segment selected for analysis of SGIII was energy minimized by Chem3D and converted into mol2 format. The mol2 file containing the compound was imported into AutoDock Tools-1.5.6 software, and atomic charges and atomic types were added. All flexible bonds were rotatable by default, and finally saved as a pdbqt file. The treated molecule was used as a small molecule ligand, and the five protein targets were used as receptors. The center position of the GridBox determined by the interaction between the small molecule and the target (x_center=40.673, y_center=37.376, z_center=43.33) and the length, width and height were all set to 80×80×80. Finally, batch molecular docking was performed by AutoDock, and the molecular docking results were analyzed. The binding interaction between the compound and the protein was visualized using Pymol 2.1 software. During the calculation process, the Lamarckian genetic algorithm was used for molecular docking calculation. The algorithm was as follows: a population of 150, a maximum of 25 million energy evaluations, a maximum 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 law. The LibDock scores of FQPSF (score: 205.606), VYLNF (score: 202.838), and YSLFLL (score: 222.984) ranked in the top three, indicating 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 hydrolyzate (see S3 for details). The peptides FQPSF, VYLNF, and YSLPLL were commissioned by Guangzhou Tongdaxing Technology Co., Ltd. to prepare them using solid phase synthesis. The purity of all samples was greater than 98%, and their molecular weight was verified by MS and their purity was determined by HPLC.

[0060] (2) Antioxidant activity verification

[0061] The DPPH free radical scavenging activity determination method refers to the method for determining the DPPH free radical scavenging activity of the enzymatic hydrolyzate by Zheng et al. (2019). DPPH free radical (0.2mM, dissolved in ethanol) and sample (0-6.4mg / mL gradient concentration) were mixed in equal volumes and placed in the dark at room temperature for 30min. The absorbance of the solution was measured at 517nm and recorded as As. Ethanol was used instead of DPPH as the sample control group, recorded as Ac; de-distilled water was used instead of the sample as the blank control group, recorded as A0.

[0062]

[0063] The above method was used to determine the DPPH radical scavenging activity of the peptides YNKDSTASK, FQPSF, VYLNF, and YSLPLL. The test results of YNKDSTASK are shown in (Attachment Figure 5 ). Regression-Probit regression analysis was used to obtain IC 50 The clearance rate of peptides FQPSF, VYLNF, and YSLLL was not more than 50% when the dose was increased to 10 mg / mL.

[0064] (3) ACEI activity verification

[0065] The ACE inhibitory activity was determined by mixing 40 μL of ACE solution (0.1 U / ml), 200 μL of FAPGG solution (1.0 mmol / mL) and 160 μL of sample, incubating at 37°C for 30 min, and measuring the absorbance 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] Activity test results of ACEI peptide amino acids VYINF, FQPSF or YSLFLL are as follows Figure 7-Figure 9 As shown. Regression-Probit regression analysis was used to calculate the ACE inhibitory IC 50 The values ​​were 77.43 μg, 143.93 μg and 124.10 μg respectively.

[0068] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, 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 regarded as the protection scope of the present invention.

Claims

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

2. An ACE inhibitory peptide, characterized in that: The amino acid sequence is VYLNF, FQPSF or YSLFLL.

3. A method for preparing the DPPH free radical scavenging active peptide according to claim 1 or the ACE inhibitory peptide according to claim 2, characterized in that: The following steps are involved: (1) Preparation of collagen hydrolysate of visceral connective tissue of Haliotis discus hannai: taking the visceral connective tissue collagen of Haliotis discus hannai and adding protease to hydrolyze, the hydrolysis conditions range as follows: enzyme substrate ratio 6000-8000U / g, hydrolysis 5-7h, hydrolysis temperature 30-40°C, pH 1.0-2.0, inactivating the enzyme after hydrolysis, centrifuging and filtering, and taking the supernatant; (2) Separation and purification of enzymatic hydrolysates: The supernatant was preliminarily separated, and the components with molecular weight less than 3 KDa were separated and purified by dextran gel separation, and the DPPH free radical scavenging rate and ACE inhibition rate of each component were determined respectively. Finally, the components with higher DPPH free radical scavenging activity and ACE inhibition activity after elution from the gel column were separated on a RP-HPLC column, and the DPPH free radical scavenging activity and ACE inhibition activity of each component were determined; (3) Peptide identification: The most active fraction after HPLC separation is identified, and the active peptide is identified by MAIDL-TOF / TOF mass spectrometry, or the secondary mass spectrometry data is obtained by ESI / LC-MS / MS, and the peptide sequence is obtained by denovo sequencing; (4) Screening the identified peptides based on the ALC value and binding energy of the binding sequence; (5) Peptide activity verification: Synthesize peptides and measure their DPPH free radical scavenging activity or ACE inhibitory activity to verify the activity of the synthetic peptides.

4. The preparation method according to claim 3, characterized in that: The protease described in step (1) is pepsin.

5. The preparation method according to claim 3, characterized in that: The dextran gel described in step (2) is a Sephadex G25 column with an inner diameter of 3.5 cm and a column 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 column length of 250 mm.

6. The preparation method according to claim 3, wherein the peptide sequence identification is characterized in that the MALDI-TOF / TOF described in step (3) uses MALDI-TOF / TOF mass spectrometry technology to identify high-purity peptide segments, positive ion detection is performed in reflectance mode, the mass scanning range of the primary mass spectrometer is 800 to 4000 Da, and a nitrogen laser with a wavelength of 337 nm is used. The primary mass spectrometry signal is accumulated 600-800 times in a single scan, and the secondary mass spectrometry signal is accumulated 900-1200 times in a single scan. In the analysis of active peptide mixtures, LC-ESI-MS-MS is used for amino acid sequence separation and identification. The chromatographic column is YMC-Pack ODS-A for analysis. The elution conditions are: eluent A is 0.1% formic acid water by volume, and eluent B is methanol; the elution gradient is 99%-99% A0-5min, 100%-0% A 5-55min; the flow rate is 1mL / min, and the injection volume is 20μL. The mass spectrometry adopts positive ion mode, and the m / z range is 200-2000; the ESI / LC-MS / MS uses high performance liquid chromatography-mass spectrometry technology to perform electrospray ionization analysis on the purified polypeptide, and the analytical molecular weight range is 200-2000Da.

7. An antioxidant, characterized in that The invention contains the DPPH free radical scavenging active peptide according to claim 1 as an active ingredient.

8. Use of the ACE inhibitory peptide according to claim 2 in the preparation of angiotensin converting enzyme inhibitors or drugs for treating hypertension.

9. A pharmaceutical composition, characterized in that Contains the ACE inhibitory peptide according to claim 2 as an active ingredient.

Images