CN1301264C - Oxidation resistant bioactive peptide of halobios and preparation method - Google Patents

Abstract

The invention relates to a novel marine biological antioxidant active peptide and a preparation method thereof. The marine biological antioxidant active peptide is characterized by a small molecular weight polypeptide substance with a molecular weight of 794Da1; it is composed of 7 kinds of amino acids, namely aspartic acid, Glycine, threonine, alanine, proline, valine and lysine have a material ratio of 1:1:1:1:2:1:1. Contains 8 amino acid residues. Among them, the relative content of acidic amino acids is 9.22%, the relative content of basic amino acids is 12.62%, and the relative content of polar amino acids is 44.8%. The marine biological antioxidant active peptide provided by the present invention has the following obvious advantages: strong antioxidant activity, good water solubility, safety, non-toxicity, and no side effects, etc., and is suitable for use in the fields of food, cosmetics, and medicine.

Description

Marine biological antioxidant active peptide and preparation method thereof

technical field

The invention relates to the field of biological peptides, in particular to marine biological antioxidant active peptides extracted from marine animal shellfish and a preparation method thereof.

Background technique

As we all know, marine biological products are an important source of protein in people's daily life, which shows that marine products contain a variety of rich proteins. According to reports, proteins with antioxidant activity from marine shellfish are a class of proteins containing acidic heteropolysaccharides and have strong heat resistance. The viscera of marine shellfish also contains a large amount of glycogen and other polysaccharides, as well as rich in protein. In the past, salting out and acetone precipitation were often used to separate the polysaccharide components from the extract.

As post-processing products of proteins, enzymatically active polypeptides are mostly small molecule polypeptides with the same normal amino acid structure as protein molecules. Most of these polypeptides play an important role in regulating functions in the process of life activities. Therefore, once the structure is clarified, analogs such as antagonists and active center fragments can be synthesized by means of chemical pathways. Simultaneously study the cleavage sites of active polypeptides in the process of degradation and metabolism, the necessary conditions for maintaining their conformation, the main sites where they bind to specific receptors, and the active centers that initiate or transmit information, so that the entire molecule can be consciously changed, Drugs with higher potency and stronger selectivity than natural peptides are prepared.

At present, there have been extensive studies on the separation and purification of peptide proteins at home and abroad. Generally, HPLC is generally used for the purification of peptides, and the activity recovery rate can reach more than 80%. Domestic Ma Jiannong et al. used CM-52, Bio-Gel P-6, DEAE-52 and R-HPLC chromatography to obtain pure insulin antagonistic peptide from the acid-alcohol extract of porcine pancreas; Dong Wenyu et al. The high-performance liquid chromatography was used to analyze the small molecule peptides synthesized by combinatorial chemistry, and the results successfully separated 10 kinds of target peptides from other impurities, providing a conventional analysis method. However, the separation and purification of antioxidant active peptides from marine animal shellfish is still blank.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art. After the inventor has been engaged in the development and research of marine organisms, microorganisms and their subsequent processing for a long time, a new marine shellfish viscera mass is provided. Biological antioxidant active peptide and preparation method thereof.

The marine biological antioxidant active peptide provided by the present invention is characterized by a small molecular weight polypeptide substance with a molecular weight of 794Dal; it is composed of 7 kinds of amino acids, which are respectively aspartic acid (Asp), glycine (Gly), threonine ( Thr), alanine (Ala), proline (Pro), valine (Val) and lysine (Lys), the ratio of its substance amount is Asp: Gly: Thr: Ala: Pro: Val: Lys=1:1:1:1:2:1:1. Contains 8 amino acid residues. Wherein the relative content of acidic amino acids is 9.22%, the relative content of basic amino acids is 12.62%, and the relative content of polar amino acids is 44.8%. The relative content of each amino acid is shown in Table 1,

Table 1. amino acid Relative content (mol%) AspGlyThrAlaProValLys 9.210.312.614.927.612.612.6

The content of the polypeptide acidic amino acid and basic amino acid is relatively balanced, so the acidity and alkalinity are not strong, and the relative content of polar amino acid and non-polar amino acid is relatively balanced, so it does not show strong or weak hydrophobic effect.

The ninhydrin reaction was positive, indicating that the N-terminus of the peptide was not blocked and was a chain polypeptide.

The scanning results of the absorption spectrum showed that the maximum absorption of the peptide was 280nm, which was the same as the typical protein absorption peak.

Purity identification The purity is greater than 96.42%, reaching HPLC purity.

Antioxidant Biological Activity

Ferrous ions are used to catalyze hydrogen peroxide to produce hydroxyl radicals (Fenton reaction), and the hydroxyl radicals produced by this reaction can fade the color of saffron.

Get 0.025M, pH74 phosphate buffer 1ml, 40ug/ml saffron 1ml, test drug 0.5ml, 3% hydrogen peroxide 1ml (freshly prepared), 0.945mM EDTA-Fe(II) 1ml (freshly prepared ), mixed and reacted in a water bath at 37°C for 30 minutes, then measured the absorbance at 520nm. In the blank group, 0.5ml of distilled water was used instead of the test sample, and in the control group, 1.5ml of distilled water was used instead of EDTA-Fe(II) and the test sample. And calculate the clearance rate according to the following formula:

In the Fenton reaction, ferrous ions react with hydrogen peroxide to generate hydroxyl radicals, which can decolorize saffron. After adding scallop antioxidant active peptide, it can inhibit the fading of saffron by eliminating hydroxyl radicals. The ability to scavenge hydroxyl free radicals was calculated according to the degree of inhibition of fading.

The results showed that the antioxidant active peptides of marine organisms had a strong scavenging effect on hydroxyl radicals within the test concentration range (0.1-1.0 mg/mL), and the scavenging rate continued to increase with the increase of the concentration. And when the concentration is low, the antioxidant activity increases rapidly, and when the concentration increases, the increase rate of the activity does not decrease significantly. Logarithmic regression was performed according to the obtained curve, and the calculated IC ₅₀ was 0.39 mg/mL.

Through a series of specific chemical experiments on antioxidant activity, it was found that the peptide has an inhibitory effect on the fading reaction of saffron red, but has no obvious effect on other antioxidant reactions, but whether the peptide really has anti-hydroxyl free radical activity has not yet been determined . Because there are several possibilities that the polypeptide has an inhibitory effect on the fading reaction: 1. The polypeptide can capture hydroxyl radicals, thereby inhibiting the fading of saffron; 2. The polypeptide and H ₂ O ₂ or EDTA-Fe(II) The combination destroys the occurrence of Fenton's hydroxyl radical reaction, causing saffron to fail to fade; 3. The polypeptide has a strong absorption at 513nm, which offsets the degree of saffron's fading during the reaction; 4. Peptide and saffron Saffron is combined into a chemically stable substance, thus shielding the reaction between hydroxyl radicals and saffron. In view of the above four possibilities, we conducted some targeted experiments, and the results are shown in Table 2 below:

Table 2 saffron red fade mechanism determination result Sample volume A _513nm A _{513nm average} 1ml EDTA-Fe(II)1ml 1 EDTA-Fe(II)1mlH ₂ O1mlH ₂ O 1mlH ₂ O1mlH ₂ O1mlH ₂ O ₂ 1mlH ₂ O ₂ 0.1ml peptide 0.1mlH ₂ O0.1ml peptide 0.1mlH ₂ O 0.709; 0.700; 0.680; 0.620; 0.710; 0.704; 0.683; 0.624; 0.7110.6970.6840.626 0.7100.7000.6820.623

From the data in Table 2 above, it can be found that: 1. The fading of saffron is indeed caused by the hydroxyl radicals produced by the Fenton reaction, because EDTA-Fe(II) or H ₂ O ₂ cannot make saffron The bonus fades; the addition of the polypeptide basically does not cause a large increase in the absorbance of the reactant. Therefore, the saffron color fading reaction caused by the Fenton reaction can be used to detect the activity of the anti-hydroxyl free radical polypeptide.

At the same time, the antioxidant activity of scallop antioxidant polypeptide was studied by ESR spin trapping technology and chemiluminescence method. It was found that the small peptide has a very strong ability to scavenge hydroxyl radicals generated by the Fenton reaction, further confirming the peptide's capture effect on hydroxyl radicals.

The preparation method of the marine biological antioxidant active peptide provided by the present invention comprises:

(1) Preparation of oxidation active protein:

① Mince the viscera of shellfish such as fresh scallops, then add 1.0% NaCl solution (containing 0.2% Triton-100; 0.1mmol/L EDTA) according to the wet weight 1:1 (w/w) to homogenize the tissue Make a homogenate in the machine;

② Adjust the pH to 5.6, heat in a water bath at 70°C for 5 minutes, and cool down rapidly. The cooling solution is centrifuged at 15000rpm for 10min, the supernatant is collected, the pH is adjusted to 6.7, and the (NH ₄ ) ₂ SO ₄ precipitation with 60% to 90% saturation is collected, and the precipitation is 5mmol/L Tris hydroxymethyl aminomethane (Tris) Dissolve in -HCl (pH 7.5) buffer and dialyze.

③ Take the supernatant from the centrifugal dialysate and add 1.5 times pre-cooled acetone. The acetone precipitate was redissolved in 5mmol/L Tris-HCl (pH 7.5) buffer solution and fully dialyzed to obtain crude protein solution;

④Concentrate the crude protein solution with polyethylene glycol 20000, centrifuge to remove insoluble matter, and take the supernatant and put it on a DEAE-Sephadex A-50 column (20×400mm). Fully equilibrate with 5mmol/L Tris-HCl buffer (pH 7.5) first, and then use gradient elution with the above buffer containing 0-0.2M sodium chloride at a flow rate of 20ml/h. Collect the fractions with absorption at 280nm, combine the fractions with SOD activity, concentrate, fully dialyze with double distilled water, freeze-dry and store (the above test temperature conditions are 4°C unless otherwise specified).

(2) Preparation of Antioxidant Active Peptides

①According to the orthogonal experiment, the optimal conditions for enzymatic hydrolysis of shellfish such as scallop viscera to prepare antioxidant peptides are as follows: add 0.016g of low-temperature alkaline protease to the antioxidant protein, the weight ratio of protein to enzyme is 33:1.6, and the pH is adjusted to 9.0, enzymatic hydrolysis in a constant temperature water bath at 30°C for 24 hours; place the enzymatic hydrolysis solution in a boiling water bath and heat for 10 minutes to inactivate the enzyme, then cool. The denatured macromolecular protein was removed by centrifugation, and the supernatant was freeze-dried.

② After enzymatic hydrolysis, the crude product was dissolved in deionized water, and further removed by Sephadex G-25 gel column chromatography to further remove macromolecular proteins: equilibrate with Tris-HCl buffer (pH 7.2), pack into a column (18×1000mm), and load the sample Afterwards, it was eluted with equilibration buffer at a flow rate of 3ml/min and detected at 280nm.

(3) Purification

①CM Sepharose cation exchange column chromatography:

The cation exchanger CM Sepharose Fast Flow, after pretreatment, equilibrated with 2mM disodium hydrogen phosphate-citrate buffer solution (pH3.0) and loaded the column (30×160mm), and eluted with the buffer solution until the baseline appeared after loading the sample. Then use the gradient elution of the above buffer solution containing 0-0.5M sodium chloride. Collect active peaks.

②Reversed phase high performance liquid chromatography (RP-HPLC) analysis:

Waters 2690 high-performance liquid chromatography system, Symmetry C ₁₈ analytical chromatographic column (5um, 3.9 × 150mm), the sample solution is membrane-passed deionized water, and the eluent is 30% acetonitrile-water solution. 0% ~ 100% eluent elution 15ml. The samples were vacuum freeze-dried after collection to obtain pure marine biological antioxidant active peptides.

According to the marine biological antioxidant active peptide and the preparation method thereof provided by the present invention, the shellfish viscera include various shellfish viscera, such as clams, ball nuts, clams, meretrix meretrix, scallops, etc., especially scallops such as combs Hole scallops usually have all the remaining viscera after peeling off the scallop column, so as to be economical and practical. Fresh shellfish such as fresh scallops are preferably used to remove all the remaining viscera after peeling off the scallop column. In addition, leftover scraps after processing various fish species can also be used.

The animal physiological activity of the marine biological antioxidant active peptide (PCF) provided by the present invention:

①Antioxidant activity Dexamethasone (DEX) was used to co-culture spleen and thymus lymphocytes, and a lymphocyte immunosuppression test model was established. The experimental results show that DEX can significantly reduce the activity of spleen and thymus lymphocytes, while PCF can not only significantly reduce its inhibitory effect on immune cells, but also promote the activity of immune cells, indicating that PCF has protection against lymphocyte inhibition caused by DEX. effect.

②The protective effect of shellfish such as scallop polypeptide on thymocytes under the condition of ⁶⁰ Co radiation damage and the effect on the repair ability of thymocytes damaged by ⁶⁰ Co radiation were investigated by using thiazole salt colorimetry. The results suggest that the scallop polypeptide can resist ⁶⁰ Co radiation damage to thymocytes in a dose-dependent manner, and can promote the repair of thymocytes damaged by radiation within a certain time range.

③Explored the protective effect of scallop polypeptide on neurons after rat middle cerebral artery ischemia-reperfusion (MCAO) injury, and concluded that scallop polypeptide has a protective effect on neurons after middle cerebral artery ischemia-reperfusion injury in rats, The mechanism is related to the fact that scallop polypeptide can increase the content of antioxidant enzymes and inhibit lipid peroxidation.

④ The marine peptide can significantly increase the average thickness of the epidermis and the number of fibroblasts of aging skin. Increase the content of elastic fibers in the dermis, which has a significant anti-aging effect on the skin. And the skin toxicology experiment showed that the marine peptide did not cause acute skin toxic reaction, was non-irritating, and was a mild sensitizer.

⑤The scallop polypeptide has the effect of anti-ultraviolet UVA on the skin oxidative damage of hairless mice. The mechanism is related to the scallop polypeptide up-regulating the expression of Bcl-2 protein, down-regulating the expression of NOS protein, increasing the content of antioxidant enzymes, and inhibiting lipid peroxidation.

The obtained polypeptide is determined to be an antioxidant activity polypeptide through biological and animal physiological activity assays.

In the preparation of antioxidant active protein, a commercially available homogenizer such as a tissue homogenizer is usually used for homogenization (such as Seagull YQ-3 homogenizer, sold by Jiangyin Xinbei Hardware Leather Plastic Factory).

The scallop protein with antioxidant activity is collected as the target of enzymatic hydrolysis to prepare antioxidant polypeptides, which improves the targeting and feasibility of subsequent operations.

In this experiment, the step of heating for a long time was used to denature and precipitate unnecessary miscellaneous proteins, thereby improving the purity of the product, which is beneficial to the subsequent operation steps (Figure 1).

The viscera of seafood shellfish contains a large amount of glycogen and other polysaccharides. The presence of polysaccharides increases the hygroscopicity and viscosity of the ingredients, making it difficult to freeze-dry and store. The polysaccharides are separated from the extract by salting out and acetone precipitation. .

Compared with terrestrial protease, marine alkaline low-temperature protease has particularity in enzyme properties because it comes from low-temperature marine environment. When preparing polypeptide active substances by enzymatic hydrolysis, the reaction conditions of various enzymes are different, which affect the conformational changes of the substrate protein, and the specificity of various enzymes causes the amino acid composition and arrangement of the N-terminal and C-terminal of the product polypeptide to be different . Marine low-temperature alkaline protease was selected for enzymatic hydrolysis of scallops, which has a strong target.

The crude antioxidant peptide obtained by enzymatic hydrolysis was analyzed by Sephadex G-25, and the record showed two peaks (Figure 2).

According to the working principle of molecular sieves, the peak time of substances is negatively correlated with the molecular weight. Compared with the anti-oxidant protein without enzymatic hydrolysis, it shows that the first peak of the protein absorption curve and the first peak of the peptide absorption curve in the figure are the same substance, while the second peak of the peptide absorption curve is more Substances of lower molecular weight. Therefore, the second peak of the protein absorption curve in the figure is the product of scallop antioxidant protein hydrolyzed by alkaline protease, which is a small peptide substance. The peptide peaks were collected and stored in lyophilized form.

This step of the experiment is mainly to separate the miscellaneous protein and polypeptide after enzymolysis, and the molecular weight difference between the protein and the polypeptide after enzymolysis is large, so when performing molecular sieve purification, a larger flow rate is selected to make the protein flow out faster. . After this step of purification, rapid separation of proteins and peptides is achieved. The target components are concentrated for further operation.

The results after putting the marine peptide on the CM Sepharose FF cation exchange chromatography column are shown in Figure 3.

A lower pH value can ensure that most of the polypeptide molecules have better adsorption with the cation exchange resin, but considering the tolerance pH range of the resin and to avoid hydrolysis of the polypeptide, the pH value of the loading buffer is determined to be 3.0 , as the eluent, it should be a buffer with high salt concentration to ensure that all the adsorbed polypeptide molecules are eluted.

The second peak obtained by cation exchange was analyzed by RP-HPLC, the result is shown in Figure 4, and the peak with a retention time of 20.919 min was the active peak. Because the peak is close to the miscellaneous peak and partially crossed, only the peak part was collected and lyophilized for storage.

The reversed-phase high-performance liquid phase method is currently one of the most commonly used methods for the separation of small molecule peptides.

The crude marine antioxidant peptide was purified by 35.88 times through CM Sepharose cationic column chromatography and RP-HPLC column chromatography. The results are shown in Table 3.

Table 3 purification results step Total protein content (mg) _EC50 (mg/mL) Purification factor Enzymatic solution CM sepharose cationic column chromatography RP-HPLC 1.7020.7360.016 13.991.7940.39 1.007.835.88

The invention provides a marine biological antioxidant active peptide and its preparation method is a fast, convenient and reliable method, which has a very strong ability to scavenge the hydroxyl radicals produced by the Fenton reaction, and its scavenging EC ₅₀ = about 0.39Mg/ml has a significant antioxidant effect. Lipid peroxidation induced by oxygen free radicals is closely related to the occurrence of many diseases. Among them, hydroxyl free radical is the strongest known oxidant, which can react with almost all cell components, and is extremely harmful to the body. Therefore, marine antioxidant peptides may be used as natural food antioxidants to inhibit lipid autooxidation. Hydroxyl free radicals can depolymerize hyaluronic acid, which can lead to dry skin and wrinkles due to the decline in water strength. Marine antioxidant peptides can also be considered as an effective anti-aging marine active substance. In addition, studies have shown that peptides with strong antioxidant effects generally have a small molecular weight, so they are easily absorbed by the intestinal tract and directly act on the body, and are safe, non-toxic, and have no side effects. At present, the theory of free radicals and the determination of related indicators have become a general method for explaining the mechanism of action of anti-aging substances. In addition, after enzymatic hydrolysis, marine protein resources generally have the advantages of good solubility, strong emulsification, and increased fluidity. Therefore, as a natural antioxidant, the antioxidant peptide has broad prospects for development and utilization in medicine, daily chemical industry, food, etc.

Description of drawings

Figure 1 is the DEAE-Sephadex A-50 column chromatography curve of antioxidant protein.

Fig. 2 is the gel chromatography curve of scallop antioxidant protein and peptide.

Fig. 3 is the cationic column chromatography curve of antioxidant peptide CM Sepharose.

Figure 4 shows the results of RP-HPLC analysis of antioxidant peptides.

Detailed ways

The present invention further illustrates the present invention with following examples, but protection scope of the present invention is not limited to examples.

Example 1

Preparation of antioxidant active protein

(1) Mince 600 grams of fresh scallop viscera, then add 1.0% NaCl solution (containing 0.2% Triton-100; 0.1mmol/L EDTA) according to the wet weight 1:1 (w/w) and homogenize the tissue Homogenized in a machine. Adjust the pH to 5.6, heat in a water bath at 70°C for 5 minutes, and cool down rapidly. The cooling solution was centrifuged at 15000rpm for 10min, the supernatant was collected, and the pH was adjusted to 6.7, and the (NH ₄ ) ₂ SO ₄ precipitate with a saturation of 60-90% was collected, and the precipitate was dissolved in 5 mmol/L Tris-HCl (pH 7.5) buffer , Dialysis. Take the supernatant from the centrifugal dialysate, and slowly add 1.5 times pre-cooled acetone. The acetone precipitate was redissolved in 5 mmol/L Tris-HCl (pH 7.5) buffer solution and fully dialyzed to obtain a crude protein solution, which was lyophilized and stored (sample I).

(2) The crude protein solution was concentrated with polyethylene glycol 20,000, centrifuged to remove insoluble matter, and the supernatant was put on a DEAE-Sephadex A-50 column (20×400mm). Fully equilibrate with 5mmol/L Tris-HCl buffer (pH 7.5) first, and then use gradient elution with the above buffer containing 0-0.2M sodium chloride at a flow rate of 20ml/h. The fractions with absorption at 280nm were collected, and the fractions with SOD activity were combined, concentrated, fully dialyzed with double distilled water, and lyophilized for storage (sample II).

Enzymolysis

Take 90ml of sample II (containing 0.33g of protein), add 0.016g of marine low-temperature alkaline protease (product of Yellow Sea Fisheries Research Institute), adjust pH=9.0, and enzymolyze in a constant temperature water bath at 30°C for 24 hours. After the enzymatic hydrolysis is completed, place the enzymatic hydrolysis solution in a boiling water bath and heat for 10 minutes to inactivate the enzyme. Cool and centrifuge to remove denatured macromolecular proteins. The supernatant was freeze-dried and sealed in vacuum (sample III).

separate

After enzymatic hydrolysis, the crude product was dissolved in deionized water, and the macromolecular protein was further removed by Sephadex G-25 gel column chromatography: equilibrated with Tris-HCl buffer (pH7.2) and packed into a column (18×1000mm). Elute with equilibration buffer, collect the polypeptide peak, and freeze-dry for storage (sample IV).

purify

(1) The cation exchanger CM Sepharose Fast Flow, after pretreatment, equilibrate with 2mM disodium hydrogen phosphate-citrate buffer solution (pH3.0) and load the column (30×160mm), after loading the sample, it is eluted with the buffer solution to When the baseline appears, it is then eluted with a gradient of the above-mentioned buffer solution containing 0-0.5M sodium chloride. The active peak (sample V) was collected.

(2) Waters 2690 high-performance liquid chromatography system, Symmetry C ₁₈ analytical chromatographic column (5um, 3.9 × 150mm), the sample solution is membrane-passed deionized water, and the eluent is 30% acetonitrile-water solution. 0% ~ 100% eluent elution 15ml. Vacuum freeze-drying (sample VI) after the samples were collected to obtain pure marine biological antioxidant active peptides.

Claims (1)

1. A preparation method of marine biological antioxidant active peptide, comprising (1) Preparation of antioxidant active protein: ① Grind shellfish viscera, then add 1.0% NaCl solution containing 0.2% Triton-100; 0.1mmol/L EDTA according to the wet weight 1:1 (w/w), put it in a tissue homogenizer to make a homogenate ; ② Adjust the pH to 5.6, heat in a water bath at 70°C for 5 minutes, cool down rapidly, centrifuge the cooling liquid for 10 minutes, collect the supernatant, adjust the pH to 6.7, collect (NH ₄ ) ₂ SO ₄ precipitation with 60-90% saturation, and use it for precipitation 5mmol/L Tris-HCl buffer solution with pH 7.5 was dissolved and dialyzed; ③Take the supernatant from the centrifugal dialysate, add 1.5 times pre-cooled acetone, redissolve the acetone precipitate in 5mmol/L tris-HCl buffer solution with pH 7.5, and fully dialyze to obtain the crude protein solution; ④Concentrate the crude protein solution with polyethylene glycol 20,000, centrifuge to remove insoluble matter, take the supernatant and put it on a DEAE-Sephadex A-50 20×400mm column, and first buffer it with 5mmol/L tris-HCl at pH 7.5 The liquid is fully balanced, and then gradient eluted with the above buffer solution containing 0-0.2M sodium chloride, the flow rate is 20ml/h, the fractions with absorption at 280nm are collected, and the fractions with SOD activity are combined, concentrated and fully diluted with double distilled water. Dialysis, freeze-dried storage; (2) Preparation of Antioxidant Active Peptides ①Add marine low-temperature alkaline protease to the antioxidant protein, the weight ratio of protein to enzyme is 33:1.6, adjust the pH to 9.0, and enzymolyze it in a constant temperature water bath at 30°C for 24 hours. After the enzymolysis is completed, put the enzymolysis solution in boiling water Heat in the bath to inactivate the enzyme, cool down, centrifuge to remove denatured macromolecular protein, and take the supernatant to freeze-dry; ② After enzymatic hydrolysis, the crude product was dissolved in deionized water, and further removed by Sephadex G-25 gel column chromatography to further remove macromolecular proteins: equilibrate with pH 7.2 tris-HCl buffer, install on a 18×1000mm column, and put on After the sample is eluted with equilibration buffer, the flow rate is 3ml/min, and the detection is at 280nm; (3) Purification ①CM Sepharose cation exchange column chromatography: The cation exchanger CM Sepharose Fast Flow, after pretreatment, equilibrated with 2mM disodium hydrogen phosphate-citric acid buffer solution of pH 3.0, then installed on a 30×160mm column, and eluted with buffer solution until the baseline appeared after loading the sample, and then used Gradient elution with the above-mentioned buffer solution of 0-0.5M sodium chloride, and collect the active peak; ②Reversed phase high performance liquid chromatography (RP-HPLC) Waters 2690 high performance liquid chromatography system, Symmetry C18 analytical chromatography 5um, 3.9×150mm column, the sample solution is membrane deionized water, the eluent is 30% acetonitrile-water solution, 0% to 100% eluent elution 15ml, vacuum freeze-dried after sample collection to obtain pure marine biological antioxidant active peptides.

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