Preparation method and application of high-purity sea cucumber peptide

Abstract

The invention provides a high-purity sea cucumber peptide as well as a preparation method and application thereof, and belongs to the technical field of sea cucumber peptides. Comprising the following steps: S1, washing, freeze-drying and crushing viscera of sea cucumbers, and filtering with a screen to obtain freeze-dried powder; s2, adding the freeze-dried powder into water, adding a compound enzyme, adjusting the pH value and the temperature, stirring for enzymolysis, performing enzyme deactivation, performing centrifugation, and collecting supernate; s3, primarily purifying the supernate by ultrafiltration and nanofiltration methods in sequence to obtain a primarily purified solution; s4, adding modified activated carbon into the primarily purified liquid, stirring for adsorption, and separating the modified activated carbon by a magnet to obtain deeply purified liquid; and S5, adding cyclodextrin into the deeply purified liquid, stirring, embedding, centrifuging, and freeze-drying supernate to obtain the high-purity sea cucumber peptide. The prepared high-purity sea cucumber peptide has the advantages of high purity, low pigment and salt content, almost no fishy smell, and good anti-oxidation, anti-inflammatory, anti-tumor and anti-fatigue effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of sea cucumber peptide, and particularly relates to a high-purity sea cucumber peptide and a preparation method and application thereof. BACKGROUND

[0002] Proteins in sea cucumber are important nutritional components, and contain nearly 20 kinds of amino acids such as glycine, glutamic acid and aspartic acid, and most of them are essential amino acids for human body. Biological metabolism experiments have proved that most proteins are absorbed by human body in the form of polypeptide. The extraction methods of sea cucumber peptide mainly include hydrolysis method and enzymatic hydrolysis method. However, the hydrolysis method mainly uses acid or alkali to hydrolyze proteins, and the reaction condition is severe, which seriously damages the structure of sea cucumber peptide, and strong acid and strong alkali are easy to corrode equipment, so the production equipment has high requirements. Therefore, the enzymatic hydrolysis method has become the main method for preparing sea cucumber peptide in recent years. At present, sea cucumber peptide is mostly prepared by enzymatic hydrolysis of sea cucumber body wall, and sea cucumber is expensive, so the extraction cost is high. The annual output of sea cucumber in China is high, and a large amount of sea cucumber viscera is discarded in the sea cucumber processing process, which causes great waste of marine biological resources. The discarded sea cucumber viscera is a good raw material for preparing active components such as sea cucumber peptide and sea cucumber polysaccharide. The sea cucumber viscera contains rich nutrients such as protein, polysaccharide, fat and saponin, and has activities such as delaying aging, enhancing immunity, anti-tumor, anti-fatigue and anti-coagulation, but there is little report on the preparation of sea cucumber peptide from sea cucumber viscera.

[0003] At present, the main methods for preparing polypeptides include protease enzymatic hydrolysis method and hydrolysis method. The hydrolysis method generally uses acid or alkali to hydrolyze proteins, and the reaction is severe, so the equipment has high requirements. Bioactive peptides are protein fragments that have positive effects on human body functions and may ultimately affect human health, and must be prepared by safe and reliable methods. However, the alkali extraction method is easy to hydrolyze collagen in sea cucumber into D-type amino acids, which has the risk of teratogenicity, carcinogenicity and mutagenicity, so it is less used. The protease hydrolysis method is mild, safe and easy to control, and specific physiological function active peptides can be obtained by selecting the type of enzyme, controlling the reaction time, enzyme addition amount and substrate concentration, so the protease enzymatic hydrolysis of sea cucumber is the most commonly used method for preparing sea cucumber peptide at present.

[0004] In the enzymatic hydrolysis process, the optimal pH of the protease is usually maintained by continuously adding acid or alkali, so that the enzymatic hydrolysis liquid contains salt, and the pigments in sea cucumber will deposit in the enzymatic hydrolysis liquid in the enzymatic hydrolysis process, which will affect the activity and application range of the obtained polypeptide. At present, the main methods for removing salt, color and odor of bioactive peptides include ultrafiltration, dialysis and ion exchange resin method.

[0005] Traditional methods for desalination, decolorization, and deodorization are difficult to achieve simultaneously. For example, the salts (inorganic small molecule ions), pigments (which may be small molecules or bound to large molecules), and odor-causing substances (mostly volatile small molecule organic compounds) that need to be removed differ greatly in molecular weight, charge, and chemical properties. Methods based on a single physical principle are unlikely to efficiently remove all impurities at the same time. Ultrafiltration membranes separate based on molecular weight, effectively retaining large peptide molecules while allowing salt to pass through, but cannot remove pigments with small molecular weights or those bound to peptides. Ion exchange resins specialize in targeting charged ions, but are ineffective against neutral odor-causing substances and some pigments, and the process may damage peptide activity.

[0006] Therefore, developing an efficient method to improve the purity of sea cucumber peptides would have potential application value. Summary of the Invention

[0007] The purpose of this invention is to propose a high-purity sea cucumber peptide, its preparation method, and its application. It has high purity, low pigment and salt content, and almost no fishy smell. It also has good antioxidant, anti-inflammatory, anti-tumor, and anti-fatigue effects, and has broad application prospects.

[0008] The technical solution of this invention is implemented as follows:

[0009] This invention provides a method for preparing high-purity sea cucumber peptides, comprising the following steps:

[0010] S1. Wash the sea cucumber viscera, freeze-dry, pulverize, and filter through a sieve to obtain freeze-dried powder;

[0011] S2. Add the lyophilized powder to water, add the compound enzyme, adjust the pH and temperature, stir to hydrolyze the enzyme, inactivate the enzyme, centrifuge, and collect the supernatant;

[0012] S3. The supernatant is subjected to ultrafiltration and nanofiltration for preliminary purification to obtain a preliminary purified solution;

[0013] S4. Add modified activated carbon to the preliminary purified solution, stir to adsorb, and separate the modified activated carbon with a magnet to obtain a deeply purified solution;

[0014] S5. Add cyclodextrin to the deep purification solution, stir to embed, centrifuge, freeze-dry the supernatant to obtain high-purity sea cucumber peptide.

[0015] As a further improvement of the present invention, the sieve mesh size in step S1 is 60-100 mesh; the complex enzyme in step S2 includes neutral protease and papain in a mass ratio of 3-7:2-4, the pH value is adjusted to 7-8, the temperature is 50-60℃, the enzymatic hydrolysis time is 1-3h, and the mass ratio of the freeze-dried powder to the complex enzyme is 100:3-5.

[0016] As a further improvement of the present invention, the ultrafiltration temperature in step S3 is 40-50℃, the pressure is 0.1-0.5MPa, a flat plate assembly is used, and the flow rate is 3-5L / m. 2 The nanofiltration process involves a molecular weight cutoff of 1-2 kDa, a temperature of 30-40°C, a pressure of 1-2 MPa, and a flow rate of 2-4 m / s. In step S4, the amount of modified activated carbon added is 1-3 wt% of the total system mass, and the stirring and adsorption time is 30-40 min. In step S5, the cyclodextrin is β-cyclodextrin, and the amount added is 0.5-0.8 wt% of the total system mass. The stirring and encapsulation time is 30-50 min.

[0017] As a further improvement of the present invention, the method for preparing the modified activated carbon is as follows:

[0018] T1. Preliminary treatment: Coconut shell activated carbon is mixed and ground with melamine, calcined under inert gas protection, washed, and dried to obtain preliminarily modified coconut shell activated carbon;

[0019] T2. Preparation of eutectic solvent / ionic liquid: Choline chloride and zinc chloride are mixed, heated to dissolve, and then an ionic liquid is added and stirred until homogeneous to obtain the eutectic solvent / ionic liquid;

[0020] T3. Modification: The preliminarily modified coconut shell activated carbon is added to a eutectic solvent / ionic liquid, heated and impregnated, stirred once every period of time, filtered, washed, and dried to obtain eutectic solvent / ionic liquid modified activated carbon.

[0021] T4. Doping Modification: Dissolve magnesium salt, aluminum salt, ferrous salt, and ferric salt in water, add eutectic solvent / ionic liquid to modify activated carbon, heat under inert gas protection, adjust the pH value of the solution, stir the reaction, age, filter, wash, and dry to obtain doped modified activated carbon.

[0022] T5. KH560 modification: Add doped modified activated carbon to ethanol, add silane coupling agent KH560, heat and stir to react, centrifuge, wash, dry, and obtain KH560 / doped modified activated carbon.

[0023] T6. Coupling: KH560 / doped modified activated carbon was added to a mixture of acetonitrile and ethanol, followed by the addition of mPEG-NH2 and branched polyethyleneimine. The mixture was stirred at room temperature, filtered, washed, and dried to obtain modified activated carbon.

[0024] As a further improvement of the present invention, in step T1, the mass ratio of coconut shell activated carbon to melamine is 10:3-7, the grinding time is 20-40 min, the calcination temperature is 500-700℃, and the time is 1-3 h; in step T2, the mass ratio of zinc chloride, choline chloride, and ionic liquid is 25-30:12-16:8-15, the ionic liquid is selected from at least one of 1-ethyl-3-methylimidazolium chloride and 1-(2-hydroxyethyl)-3-methylimidazolium acetate, and the heating and dissolution temperature is 95-105℃.

[0025] As a further improvement of the present invention, in step T3, the mass ratio of the preliminarily modified coconut shell activated carbon to the eutectic solvent / ionic liquid is 1:10-15, the heating and impregnation temperature is 85-95℃, and the impregnation time is 2-4h; in step T4, the molar ratio of magnesium salt, aluminum salt, ferrous salt, and ferric salt is 1-3:1:1:2, the heating temperature is 75-85℃, the pH value of the solution is adjusted to 10-11, the stirring reaction time is 3-5h, and the aging temperature is 75-85℃ for 12-18h.

[0026] As a further improvement of the present invention, in step T5, the mass ratio of the doped modified activated carbon to the silane coupling agent KH560 is 100:2-3, and the temperature of the heating and stirring reaction is 40-50℃ for 1-2 hours; in step T6, the mass ratio of KH560 / doped modified activated carbon, mPEG-NH2 and branched polyethyleneimine is 10:1-2:0.5-1.5, and the time of the room temperature stirring reaction is 10-15 hours.

[0027] This invention further protects a high-purity sea cucumber peptide prepared by the above-described preparation method.

[0028] This invention further protects the application of the above-mentioned high-purity sea cucumber peptide in the preparation of pharmaceuticals with antioxidant, anti-fatigue, anti-tumor, and anti-aging properties.

[0029] This invention further protects an antioxidant sea cucumber peptide, the preparation method of which is as follows: the above-mentioned high-purity sea cucumber peptide is added to water, sodium tripolyphosphate is added, the pH value of the solution is adjusted, potassium bromide is added, the reaction is heated and stirred, dialyzed, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are added to the product, stirred and activated, hyaluronic acid is added, the reaction is stirred, dialyzed, zinc salt is added to the unpermeated liquid, stirred and chelated, dialyzed, and freeze-dried to obtain the antioxidant sea cucumber peptide.

[0030] The present invention has the following beneficial effects:

[0031] This invention uses sea cucumber viscera as raw material, greatly improving the utilization rate of waste resources. Sea cucumber peptides are separated through a compound enzyme hydrolysis method. The compound enzyme synergistically hydrolyzes various proteins such as collagen and actin in sea cucumber viscera, breaking different types of peptide bonds to generate small molecule active peptides. The conditions are mild and the efficiency is high, and the obtained active peptides have high activity. However, during the enzymatic hydrolysis process, the continuous addition of acid or alkali to maintain the optimal pH of the protease results in the presence of salt in the hydrolysate. In addition, pigments from sea cucumbers will be deposited in the hydrolysate during the enzymatic hydrolysis process. At the same time, many fishy-smelling substances will also be retained. These substances will affect the activity and application range of the obtained peptides.

[0032] This invention employs a combined ultrafiltration-nanofiltration process. Ultrafiltration first removes macromolecular impurities and partially desalinates the water, while the nanofiltration membrane, with its smaller pore size, can further remove the remaining salt and most of the pigment molecules.

[0033] This invention employs modified activated carbon to further decolorize the product from the previous step. This activated carbon, based on coconut shell activated carbon, is mixed with melamine, ground, and calcined to introduce polar functional groups onto the activated carbon surface. This enhances the interaction between the activated carbon and odor molecules such as aldehydes, thereby promoting the adsorption of odor molecules by the activated carbon. Nitrogen modification significantly enhances the surface polarity of the carbon material and generates abundant surface alkaline sites, resulting in a marked improvement in the adsorption efficiency for odor molecules (such as aldehydes, ketones, and sulfides). Further modification through impregnation with ionic liquids and eutectic solvents results in a low-toxicity, non-volatile, environmentally friendly, and safe impregnation solution. This avoids corrosion of activated carbon by strong acids and alkalis, prevents pore collapse and reduction of specific surface area, and preserves its original porous structure to the greatest extent. Simultaneously, the ionic liquid introduces "imidazolium ion sites," while the eutectic solvent specifically optimizes the polar / non-polar adsorption sites of the activated carbon, enhancing its adsorption capacity for pigments and odorous substances. Both have strong dissolving power and can quickly penetrate into the pores of the activated carbon, resulting in more uniform modification, higher surface active site density, and significantly improved adsorption capacity and rate. Their synergistic effect also regulates pore structure and pore size, enhancing the adsorption of macromolecular pigments and enabling better selective adsorption. Furthermore, the impregnation solution can be reused multiple times, reducing costs.

[0034] By doping LDHs onto the surface of impregnated activated carbon, ferrous and ferric ions are mixed in, resulting in ferromagnetic LDHs. This allows the modified activated carbon to be magnetically separated, improving its separability and simplifying the operation. Simultaneously, iron, aluminum, and magnesium can selectively bind to odor-causing substances containing N, S, and O, such as trimethylamine and methanethiol, achieving specific deodorization. Furthermore, they provide numerous exchangeable anion sites and Lewis acid sites, enhancing the capture effect.

[0035] Finally, the surface is modified with the silane coupling agent KH560 to have epoxy groups, which can couple with amino-containing mPEG-NH2 and branched polyethyleneimine, thereby forming a hydrophilic "brush-like" layer on the activated carbon surface. The steric hindrance and hydration layer repel large molecular peptides, allowing pigments and fishy odor molecules to diffuse in. At the same time, it physically blocks peptides from approaching the carbon surface, greatly reducing loss. Meanwhile, the amino groups formed generate electrostatic attraction to anionic impurity molecules and fishy odor acids.

[0036] This invention adds cyclodextrin to a deeply purified solution, which encapsulates the remaining small amount of fishy substances and pigments in its cavity, forming a physical barrier. At the same time, it does not change the activity of the peptides, and can achieve almost complete deodorization and decolorization. Thus, the high-purity active peptides have better antioxidant, anti-tumor, anti-aging, and anti-fatigue effects, and are pure white in appearance. When consumed, they have almost no off-flavor or fishy taste, and have a wider range of applications.

[0037] The present invention further modifies the high-purity sea cucumber peptide by phosphorylation and hyaluronic acid, which greatly improves its chelating ability for zinc ions and its antioxidant effect, thus further improving its quality. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 Infrared spectrum of the modified activated carbon prepared in Example 1;

[0040] Figure 2 The image shows the effect of the modified activated carbon prepared in Example 1 being adsorbed by a magnet. Detailed Implementation

[0041] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] mPEG-NH2, Mw=1000; branched polyethyleneimine, Mw=600; neutral protease, 110,000 U / g; papain, 100,000 U / g.

[0043] Preparation Example 1: Modified Activated Carbon

[0044] The preparation method is as follows:

[0045] T1. Preliminary treatment: Mix 1g of coconut shell activated carbon with 0.3g of melamine and grind for 20min. Under nitrogen protection, calcine at 500℃ for 1h, wash and dry to obtain preliminarily modified coconut shell activated carbon.

[0046] T2. Preparation of eutectic solvent / ionic liquid: 12g of choline chloride and 25g of zinc chloride were mixed and heated to 95℃ to dissolve. 8g of 1-ethyl-3-methylimidazolium chloride was added and stirred for 30min to obtain the eutectic solvent / ionic liquid.

[0047] T3. Modification: Add 1g of pre-modified coconut shell activated carbon to 10g of eutectic solvent / ionic liquid, heat to 85℃, impregnate for 2h, stir once every 20min, filter, wash, and dry to obtain eutectic solvent / ionic liquid modified activated carbon.

[0048] T4. Doping Modification: Dissolve 0.01 mol magnesium chloride, 0.01 mol aluminum nitrate, 0.01 mol ferrous chloride, and 0.02 mol ferric chloride in 100 mL of water, add 5 g of eutectic solvent / ionic liquid modified activated carbon, heat to 75 °C under nitrogen protection, adjust the pH of the solution to 10, stir for 3 h, age at 75 °C for 12 h, filter, wash, and dry to obtain doped modified activated carbon;

[0049] T5. KH560 modification: Add 1g of doped modified activated carbon to 50mL of ethanol, add 0.02g of silane coupling agent KH560, heat to 40℃, stir and react for 1h, centrifuge, wash, and dry to obtain KH560 / doped modified activated carbon.

[0050] T6. Coupling: Add 1g of KH560 / doped modified activated carbon to 100mL of a mixture of acetonitrile and ethanol (volume ratio 3:1), add 1g of mPEG-NH2 and 0.5g of branched polyethyleneimine, stir at room temperature for 10h, filter, wash, and dry to obtain modified activated carbon. Figure 1 The infrared spectrum of the modified activated carbon obtained; Figure 2 The image shows the effect of the modified activated carbon being adsorbed by a magnet.

[0051] Preparation Example 2: Modified Activated Carbon

[0052] The preparation method is as follows:

[0053] T1. Preliminary treatment: 1g of coconut shell activated carbon was mixed and ground with 0.7g of melamine for 40min, calcined at 700℃ for 3h under nitrogen protection, washed and dried to obtain preliminarily modified coconut shell activated carbon;

[0054] T2. Preparation of eutectic solvent / ionic liquid: 16g of choline chloride and 30g of zinc chloride were mixed and heated to 105℃ to dissolve. 15g of 1-(2-hydroxyethyl)-3-methylimidazolium acetate was added and stirred for 30min to obtain eutectic solvent / ionic liquid.

[0055] T3. Modification: Add 1g of pre-modified coconut shell activated carbon to 15g of eutectic solvent / ionic liquid, heat to 95℃, impregnate for 4h, stir once every 20min, filter, wash, and dry to obtain eutectic solvent / ionic liquid modified activated carbon.

[0056] T4. Doping Modification: Dissolve 0.03 mol magnesium chloride, 0.01 mol aluminum nitrate, 0.01 mol ferrous chloride, and 0.02 mol ferric chloride in 100 mL of water, add 5 g of eutectic solvent / ionic liquid modified activated carbon, heat to 85 °C under nitrogen protection, adjust the pH of the solution to 11, stir for 5 h, age at 85 °C for 18 h, filter, wash, and dry to obtain doped modified activated carbon;

[0057] T5. KH560 modification: Add 1g of doped modified activated carbon to 50mL of ethanol, add 0.03g of silane coupling agent KH560, heat to 50℃, stir and react for 2h, centrifuge, wash, and dry to obtain KH560 / doped modified activated carbon.

[0058] T6. Coupling: Add 1g of KH560 / doped modified activated carbon to 100mL of a mixture of acetonitrile and ethanol (volume ratio 3:1), add 2g of mPEG-NH2 and 1.5g of branched polyethyleneimine, stir at room temperature for 15h, filter, wash, and dry to obtain modified activated carbon.

[0059] Preparation Example 3: Modified Activated Carbon

[0060] The preparation method is as follows:

[0061] T1. Preliminary treatment: Mix 1g of coconut shell activated carbon with 0.5g of melamine and grind for 30min. Under nitrogen protection, calcine at 600℃ for 2h, wash and dry to obtain preliminarily modified coconut shell activated carbon.

[0062] T2. Preparation of eutectic solvent / ionic liquid: 14g of choline chloride and 27g of zinc chloride were mixed and heated to 100℃ to dissolve. 11g of 1-(2-hydroxyethyl)-3-methylimidazolium acetate was added and stirred for 30min to obtain eutectic solvent / ionic liquid.

[0063] T3. Modification: Add 1g of pre-modified coconut shell activated carbon to 12g of eutectic solvent / ionic liquid, heat to 90℃, soak for 3h, stir once every 20min, filter, wash, and dry to obtain eutectic solvent / ionic liquid modified activated carbon.

[0064] T4. Doping Modification: Dissolve 0.02 mol magnesium chloride, 0.01 mol aluminum nitrate, 0.01 mol ferrous chloride, and 0.02 mol ferric chloride in 100 mL of water, add 5 g of eutectic solvent / ionic liquid modified activated carbon, heat to 80 °C under nitrogen protection, adjust the pH of the solution to 10.5, stir for 4 h, age at 80 °C for 15 h, filter, wash, and dry to obtain doped modified activated carbon;

[0065] T5. KH560 modification: Add 1g of doped modified activated carbon to 50mL of ethanol, add 0.025g of silane coupling agent KH560, heat to 45℃, stir and react for 1.5h, centrifuge, wash, and dry to obtain KH560 / doped modified activated carbon.

[0066] T6. Coupling: Add 1g of KH560 / doped modified activated carbon to 100mL of a mixture of acetonitrile and ethanol (volume ratio 3:1), add 1.5g of mPEG-NH2 and 1g of branched polyethyleneimine, stir at room temperature for 12h, filter, wash, and dry to obtain modified activated carbon.

[0067] Comparative Preparation Example 1

[0068] The difference compared to Preparation Example 3 is that step T1 was not performed.

[0069] The preparation method is as follows:

[0070] T1. Preparation of eutectic solvent / ionic liquid: 14g of choline chloride and 27g of zinc chloride were mixed and heated to 100℃ to dissolve. 11g of 1-(2-hydroxyethyl)-3-methylimidazolium acetate was added and stirred for 30min to obtain eutectic solvent / ionic liquid.

[0071] T2. Modification: Add 1g of coconut shell activated carbon to 12g of eutectic solvent / ionic liquid, heat to 90℃, impregnate for 3h, stir once every 20min, filter, wash, and dry to obtain eutectic solvent / ionic liquid modified activated carbon.

[0072] T3. Doping Modification: Dissolve 0.02 mol magnesium chloride, 0.01 mol aluminum nitrate, 0.01 mol ferrous chloride, and 0.02 mol ferric chloride in 100 mL of water, add 5 g of eutectic solvent / ionic liquid modified activated carbon, heat to 80 °C under nitrogen protection, adjust the pH of the solution to 10.5, stir for 4 h, age at 80 °C for 15 h, filter, wash, and dry to obtain doped modified activated carbon;

[0073] T4. KH560 modification: Add 1g of doped modified activated carbon to 50mL of ethanol, add 0.025g of silane coupling agent KH560, heat to 45℃, stir and react for 1.5h, centrifuge, wash, and dry to obtain KH560 / doped modified activated carbon.

[0074] T5. Coupling: Add 1g of KH560 / doped modified activated carbon to 100mL of a mixture of acetonitrile and ethanol (volume ratio 3:1), add 1.5g of mPEG-NH2 and 1g of branched polyethyleneimine, stir at room temperature for 12h, filter, wash, and dry to obtain modified activated carbon.

[0075] Comparative Preparation Example 2

[0076] The difference compared to Preparation Example 3 is that steps T2 and T3 were not performed.

[0077] The preparation method is as follows:

[0078] T1. Preliminary treatment: Mix 1g of coconut shell activated carbon with 0.5g of melamine and grind for 30min. Under nitrogen protection, calcine at 600℃ for 2h, wash and dry to obtain preliminarily modified coconut shell activated carbon.

[0079] T2. Doping Modification: Dissolve 0.02 mol magnesium chloride, 0.01 mol aluminum nitrate, 0.01 mol ferrous chloride, and 0.02 mol ferric chloride in 100 mL of water, add 5 g of pre-modified coconut shell activated carbon, heat to 80 °C under nitrogen protection, adjust the pH of the solution to 10.5, stir for 4 h, age at 80 °C for 15 h, filter, wash, and dry to obtain doped modified activated carbon;

[0080] T3. KH560 modification: Add 1g of doped modified activated carbon to 50mL of ethanol, add 0.025g of silane coupling agent KH560, heat to 45℃, stir and react for 1.5h, centrifuge, wash, and dry to obtain KH560 / doped modified activated carbon.

[0081] T4. Coupling: Add 1g of KH560 / doped modified activated carbon to 100mL of a mixture of acetonitrile and ethanol (volume ratio 3:1), add 1.5g of mPEG-NH2 and 1g of branched polyethyleneimine, stir at room temperature for 12h, filter, wash, and dry to obtain modified activated carbon.

[0082] Comparative preparation example 3

[0083] The difference compared to preparation example 3 is that step T4 was not performed.

[0084] The preparation method is as follows:

[0085] T1. Preliminary treatment: Mix 1g of coconut shell activated carbon with 0.5g of melamine and grind for 30min. Under nitrogen protection, calcine at 600℃ for 2h, wash and dry to obtain preliminarily modified coconut shell activated carbon.

[0086] T2. Preparation of eutectic solvent / ionic liquid: 14g of choline chloride and 27g of zinc chloride were mixed and heated to 100℃ to dissolve. 11g of 1-(2-hydroxyethyl)-3-methylimidazolium acetate was added and stirred for 30min to obtain eutectic solvent / ionic liquid.

[0087] T3. Modification: Add 1g of pre-modified coconut shell activated carbon to 12g of eutectic solvent / ionic liquid, heat to 90℃, soak for 3h, stir once every 20min, filter, wash, and dry to obtain eutectic solvent / ionic liquid modified activated carbon.

[0088] T4. KH560 modification: 1g of eutectic solvent / ionic liquid modified activated carbon was added to 50mL of ethanol, 0.025g of silane coupling agent KH560 was added, the mixture was heated to 45℃, stirred and reacted for 1.5h, centrifuged, washed and dried to obtain KH560 / doped modified activated carbon.

[0089] T5. Coupling: Add 1g of KH560 / doped modified activated carbon to 100mL of a mixture of acetonitrile and ethanol (volume ratio 3:1), add 1.5g of mPEG-NH2 and 1g of branched polyethyleneimine, stir at room temperature for 12h, filter, wash, and dry to obtain modified activated carbon.

[0090] Comparative preparation example 4

[0091] The difference compared to preparation example 3 is that steps T5 and T6 were not performed.

[0092] The preparation method is as follows:

[0093] T1. Preliminary treatment: Mix 1g of coconut shell activated carbon with 0.5g of melamine and grind for 30min. Under nitrogen protection, calcine at 600℃ for 2h, wash and dry to obtain preliminarily modified coconut shell activated carbon.

[0094] T2. Preparation of eutectic solvent / ionic liquid: 14g of choline chloride and 27g of zinc chloride were mixed and heated to 100℃ to dissolve. 11g of 1-(2-hydroxyethyl)-3-methylimidazolium acetate was added and stirred for 30min to obtain eutectic solvent / ionic liquid.

[0095] T3. Modification: Add 1g of pre-modified coconut shell activated carbon to 12g of eutectic solvent / ionic liquid, heat to 90℃, soak for 3h, stir once every 20min, filter, wash, and dry to obtain eutectic solvent / ionic liquid modified activated carbon.

[0096] T4. Doping Modification: Dissolve 0.02 mol magnesium chloride, 0.01 mol aluminum nitrate, 0.01 mol ferrous chloride, and 0.02 mol ferric chloride in 100 mL of water, add 5 g of eutectic solvent / ionic liquid modified activated carbon, heat to 80 °C under nitrogen protection, adjust the pH of the solution to 10.5, stir for 4 h, age at 80 °C for 15 h, filter, wash, and dry to obtain doped modified activated carbon, which is the modified activated carbon.

[0097] Example 1

[0098] This embodiment provides a method for preparing high-purity sea cucumber peptides, including the following steps:

[0099] S1. Wash the sea cucumber viscera, freeze-dry, pulverize, and filter through a 60-mesh sieve to obtain freeze-dried powder;

[0100] S2. Add 10g of lyophilized powder to 200mL of water, add 0.3g of compound enzyme, adjust the pH value to 7, set the temperature to 50℃, stir and hydrolyze for 3h, inactivate the enzyme, centrifuge, and collect the supernatant.

[0101] The complex enzyme consists of neutral protease and papain in a mass ratio of 3:2.

[0102] S3. The supernatant is subjected to ultrafiltration and nanofiltration for preliminary purification to obtain a preliminary purified solution;

[0103] The ultrafiltration temperature was 40℃, the pressure was 0.1MPa, a flat panel assembly was used, and the flow rate was 3L / m³. 2 •h, with a molecular weight cutoff of 1 kDa;

[0104] The nanofiltration temperature is 30℃, the pressure is 1MPa, and the flow rate is 2m / s;

[0105] S4. Add the modified activated carbon prepared in Preparation Example 1 to the preliminary purified solution. The amount of modified activated carbon added is 1 wt% of the total system mass. Stir and adsorb for 40 min. Separate the modified activated carbon with a magnet to obtain the deep purified solution.

[0106] S5. Add β-cyclodextrin to the deep purification solution at a concentration of 0.5 wt% of the total system mass, stir and embed for 50 min, centrifuge, freeze-dry the supernatant to obtain high-purity sea cucumber peptide.

[0107] Example 2

[0108] This embodiment provides a method for preparing high-purity sea cucumber peptides, including the following steps:

[0109] S1. Wash the sea cucumber viscera, freeze-dry, pulverize, and filter through a 100-mesh sieve to obtain freeze-dried powder;

[0110] S2. Add 10g of lyophilized powder to 200mL of water, add 0.5g of compound enzyme, adjust the pH value to 8, set the temperature to 60℃, stir and hydrolyze for 3h, inactivate the enzyme, centrifuge, and collect the supernatant.

[0111] The complex enzyme consists of neutral protease and papain in a mass ratio of 7:4.

[0112] S3. The supernatant is subjected to ultrafiltration and nanofiltration for preliminary purification to obtain a preliminary purified solution;

[0113] The ultrafiltration temperature is 50℃, the pressure is 0.5MPa, a flat panel module is used, and the flow rate is 5L / m. 2 •h, with a molecular weight cutoff of 2kDa;

[0114] The nanofiltration temperature is 40℃, the pressure is 2MPa, and the flow rate is 4m / s;

[0115] S4. Add the modified activated carbon prepared in Preparation Example 2 to the preliminary purified solution. The amount of modified activated carbon added is 3 wt% of the total system mass. Stir and adsorb for 30 min. Separate the modified activated carbon with a magnet to obtain the deep purified solution.

[0116] S5. Add β-cyclodextrin to the deep purification solution at a concentration of 0.8 wt% of the total system mass, stir and embed for 30 min, centrifuge, freeze-dry the supernatant to obtain high-purity sea cucumber peptide.

[0117] Example 3

[0118] This embodiment provides a method for preparing high-purity sea cucumber peptides, including the following steps:

[0119] S1. Wash the sea cucumber viscera, freeze-dry them, pulverize them, and filter them through an 80-mesh sieve to obtain freeze-dried powder;

[0120] S2. Add 10g of lyophilized powder to 200mL of water, add 0.4g of compound enzyme, adjust the pH value to 7.5, set the temperature to 55℃, stir and hydrolyze for 2h, inactivate the enzyme, centrifuge, and collect the supernatant.

[0121] The complex enzyme consists of neutral protease and papain in a mass ratio of 5:3;

[0122] S3. The supernatant is subjected to ultrafiltration and nanofiltration for preliminary purification to obtain a preliminary purified solution;

[0123] The ultrafiltration temperature was 45℃, the pressure was 0.3MPa, a flat panel assembly was used, and the flow rate was 4L / m³. 2 •h, with a molecular weight cutoff of 1.5 kDa;

[0124] The nanofiltration temperature is 35℃, the pressure is 1.5MPa, and the flow rate is 3m / s;

[0125] S4. Add the modified activated carbon prepared in Preparation Example 3 to the preliminary purified solution. The amount of modified activated carbon added is 2 wt% of the total system mass. Stir and adsorb for 35 min. Separate the modified activated carbon with a magnet to obtain the deep purified solution.

[0126] S5. Add β-cyclodextrin to the deep purification solution at a concentration of 0.7 wt% of the total system mass, stir and embed for 40 min, centrifuge, freeze-dry the supernatant to obtain high-purity sea cucumber peptide.

[0127] Comparative Example 1

[0128] The difference from Example 3 is that the modified activated carbon was prepared by Comparative Preparation Example 1.

[0129] Comparative Example 2

[0130] The difference from Example 3 is that the modified activated carbon was prepared from Comparative Preparation Example 2.

[0131] Comparative Example 3

[0132] The difference from Example 3 is that the modified activated carbon was prepared by Comparative Preparation Example 3.

[0133] Comparative Example 4

[0134] The difference from Example 3 is that the modified activated carbon was prepared by Comparative Preparation Example 4.

[0135] Comparative Example 5

[0136] The difference from Example 3 is that step S4 was not performed.

[0137] Includes the following steps:

[0138] S1. Wash the sea cucumber viscera, freeze-dry them, pulverize them, and filter them through an 80-mesh sieve to obtain freeze-dried powder;

[0139] S2. Add 10g of lyophilized powder to 200mL of water, add 0.4g of compound enzyme, adjust the pH value to 7.5, set the temperature to 55℃, stir and hydrolyze for 2h, inactivate the enzyme, centrifuge, and collect the supernatant.

[0140] The complex enzyme consists of neutral protease and papain in a mass ratio of 5:3;

[0141] S3. The supernatant is subjected to ultrafiltration and nanofiltration for preliminary purification to obtain a preliminary purified solution;

[0142] The ultrafiltration temperature was 45℃, the pressure was 0.3MPa, a flat panel assembly was used, and the flow rate was 4L / m³. 2 •h, with a molecular weight cutoff of 1.5 kDa;

[0143] The nanofiltration temperature is 35℃, the pressure is 1.5MPa, and the flow rate is 3m / s;

[0144] S4. Add β-cyclodextrin to the preliminary purification solution at a concentration of 0.7 wt% of the total system mass, stir for 40 min for encapsulation, centrifuge, freeze-dry the supernatant to obtain sea cucumber peptide.

[0145] Comparative Example 6

[0146] The difference from Example 3 is that ordinary coconut shell activated carbon is used instead of modified activated carbon in step S4.

[0147] Includes the following steps:

[0148] S1. Wash the sea cucumber viscera, freeze-dry them, pulverize them, and filter them through an 80-mesh sieve to obtain freeze-dried powder;

[0149] S2. Add 10g of lyophilized powder to 200mL of water, add 0.4g of compound enzyme, adjust the pH value to 7.5, set the temperature to 55℃, stir and hydrolyze for 2h, inactivate the enzyme, centrifuge, and collect the supernatant.

[0150] The complex enzyme consists of neutral protease and papain in a mass ratio of 5:3;

[0151] S3. The supernatant is subjected to ultrafiltration and nanofiltration for preliminary purification to obtain a preliminary purified solution;

[0152] The ultrafiltration temperature was 45℃, the pressure was 0.3MPa, a flat panel assembly was used, and the flow rate was 4L / m³. 2 •h, with a molecular weight cutoff of 1.5 kDa;

[0153] The nanofiltration temperature is 35℃, the pressure is 1.5MPa, and the flow rate is 3m / s;

[0154] S4. Add ordinary coconut shell activated carbon to the preliminary purified solution. The amount of ordinary coconut shell activated carbon added is 2wt% of the total system mass. Stir and adsorb for 35 min. Separate the modified activated carbon with a magnet to obtain the deep purified solution.

[0155] S5. Add β-cyclodextrin to the deep purification solution at a concentration of 0.7 wt% of the total system mass, stir and embed for 40 min, centrifuge, freeze-dry the supernatant to obtain high-purity sea cucumber peptide.

[0156] Comparative Example 7

[0157] The difference from Example 3 is that step S5 was not performed.

[0158] Includes the following steps:

[0159] S1. Wash the sea cucumber viscera, freeze-dry them, pulverize them, and filter them through an 80-mesh sieve to obtain freeze-dried powder;

[0160] S2. Add 10g of lyophilized powder to 200mL of water, add 0.4g of compound enzyme, adjust the pH value to 7.5, set the temperature to 55℃, stir and hydrolyze for 2h, inactivate the enzyme, centrifuge, and collect the supernatant.

[0161] The complex enzyme consists of neutral protease and papain in a mass ratio of 5:3;

[0162] S3. The supernatant is subjected to ultrafiltration and nanofiltration for preliminary purification to obtain a preliminary purified solution;

[0163] The ultrafiltration temperature was 45℃, the pressure was 0.3MPa, a flat panel assembly was used, and the flow rate was 4L / m³. 2 •h, with a molecular weight cutoff of 1.5 kDa;

[0164] The nanofiltration temperature is 35℃, the pressure is 1.5MPa, and the flow rate is 3m / s;

[0165] S4. Add the modified activated carbon prepared in Preparation Example 3 to the preliminary purification solution. The amount of modified activated carbon added is 2 wt% of the total system mass. Stir and adsorb for 35 min. Separate the modified activated carbon with a magnet to obtain sea cucumber peptide.

[0166] Test Example 1

[0167] The products obtained by freeze-drying the deep purification solutions prepared in Examples 1-3 and Comparative Examples 1-4, step S4 of 6, and the preliminary purification solution prepared in step S3 of Comparative Example 5 were used to determine the purity of sea cucumber peptides and calculate the extraction rate. The results are shown in Table 1.

[0168] Determination of total protein content in sea cucumber viscera: The micro Kjeldahl method in GB / T 5009.5-2010 was used for determination.

[0169] Purity determination: The Folin-phenol reagent method was used. The sample was diluted to a 100 mL volumetric flask. 5 mL of the solution was taken and 5 mL of 10% trichloroacetic acid solution was added. The mixture was shaken and centrifuged. 1 mL of the supernatant was taken and 5 mL of Folin-phenol reagent A was added. The mixture was mixed and placed at 30 °C for 10 min. Then, 0.5 mL of Folin-phenol reagent B was added, and the mixture was immediately shaken and incubated at 30 °C for 30 min. Using the solution in a test tube without standard protein as a blank, the absorbance value (D(650)) was measured at 650 nm. Substituting this value into the standard curve plotted using bovine serum albumin as a standard, the mass of sea cucumber peptides could be calculated. The percentage of sea cucumber peptides in the sample was then calculated, which is the purity.

[0170] Extraction rate (%) = (Mass of extracted sea cucumber peptides / Total protein content of sea cucumber viscera) × 100%

[0171] Table 1

[0172] Group Purity (%) Extraction rate (%) Example 1 94.5 84.2 Example 2 95.2 85.6 Example 3 95.7 86.9 Comparative Example 1 92.1 83.0 Comparative Example 2 90.3 82.2 Comparative Example 3 91.4 82.0 Comparative Example 4 90.9 77.3 Comparative Example 5 82.3 74.2 Comparative Example 6 85.5 75.1

[0173] As can be seen from the table above, the product obtained by freeze-drying the deep purification solution in step S4 of the method in Examples 1-3 of the present invention has high purity, and the extraction rate of this method is relatively high.

[0174] Test Example 2

[0175] The high-purity sea cucumber peptides prepared in Examples 1-3 and Comparative Examples 1-7 were subjected to performance tests, and the results are shown in Table 3.

[0176] Evaluation of bitter and fishy odor: The high-purity sea cucumber peptides obtained in Examples 1-3 and Comparative Examples 1-7 were prepared into 10 mg / mL solutions with purified water. Five sensory evaluators evaluated each sample, using a smell-then-taste method. Each sample was scored according to a scoring standard, with a maximum score of 10 points. Purified water was used as a reference; a higher score indicated a stronger bitter and fishy odor. The scoring standard for bitter and fishy odor is shown in Table 2.

[0177] Table 2

[0178] Sensory evaluation criteria Bitter taste 8-10 Very heavy, unable to swallow 6-8 General 4-6 Weak 2-4 Slight 0-2 None

[0179] Whiteness test: Refer to GB / T 13025.2-2008 "General Test Methods for Salt Industry - Determination of Whiteness".

[0180] Table 3

[0181] Group Bitter taste score Whiteness Example 1 0.5 98.2 Example 2 0.6 98.4 Example 3 0.4 98.8 Comparative Example 1 1.2 96.3 Comparative Example 2 1.6 93.2 Comparative Example 3 2.4 95.8 Comparative Example 4 2.2 91.3 Comparative Example 5 5.5 70.3 Comparative Example 6 4.1 81.2 Comparative Example 7 5.9 88.4

[0182] As can be seen from the table above, the high-purity sea cucumber peptides obtained in Examples 1-3 of this invention have almost no bitter or fishy smell and have a high degree of whiteness.

[0183] Example 4

[0184] This embodiment provides a method for preparing antioxidant sea cucumber peptides as follows:

[0185] 1g of the high-purity sea cucumber peptide prepared in Example 3 was added to 30mL of 0.1mol / L sodium tripolyphosphate aqueous solution, the pH of the solution was adjusted to 4-4.5, stirred and mixed evenly, and then freeze-dried to obtain powder. 10g of potassium bromide was dissolved in 50mL of water, 10g of powder was added, the mixture was heated to 45℃, stirred and reacted for 12h, dialyzed through a dialysis bag with a pore size of 3000Da for 48h, and then freeze-dried. The product was added to 100mL of water, 2g of N-hydroxysuccinimide and 1g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, stirred and activated for 30min, 3g of hyaluronic acid was added, stirred and reacted for 12h, dialyzed through a dialysis bag with a pore size of 3000Da for 24h, 0.5g of zinc chloride was added to the undiluted solution, stirred and chelated for 30min, dialyzed through a dialysis bag with a pore size of 1000Da for 12h, and then freeze-dried to obtain antioxidant sea cucumber peptide.

[0186] Test Example 3: Determination of DPPH free radical scavenging ability

[0187] The experiment included a sample group, a control group, and a reference group.

[0188] Sample group: Prepare 5 mg / mL sample solutions from the products obtained in Examples 1-4 and Comparative Examples 1-7. Take 1 mL of sample solution into a test tube, add 4 mL of 0.1 mmol / L DPPH ethanol solution, vortex to mix, and let stand in the dark for 30 min. Measure the absorbance (A) at a wavelength of 517 nm.

[0189] Reference group: Replace the DPPH ethanol solution with an equal volume of anhydrous ethanol, and measure the absorbance (A0) using the same method.

[0190] Control group: The absorbance (A1) was measured by replacing the sea cucumber extract with an equal volume of ultrapure water.

[0191] All measurements were performed in triplicate, and the results were averaged.

[0192] DPPH radical scavenging rate (%) = 1 - (A - A1)A0 × 100%

[0193] In the formula: A—absorbance of the sample; A0—absorbance of the sample in place of anhydrous ethanol; A1—absorbance of the sample in place of ultrapure water.

[0194] The results are shown in Table 4.

[0195] Table 4

[0196] Group DPPH radical scavenging rate (%) Example 1 78.9 Example 2 79.2 Example 3 80.4 Example 4 87.9 Comparative Example 1 76.7 Comparative Example 2 72.1 Comparative Example 3 74.3 Comparative Example 4 69.5 Comparative Example 5 60.1 Comparative Example 6 64.2 Comparative Example 7 74.5

[0197] As can be seen from the table above, the high-purity sea cucumber peptides obtained in Examples 1-3 and the antioxidant sea cucumber peptides obtained in Example 4 of this invention have good antioxidant properties.

[0198] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing high-purity sea cucumber peptide, characterized in that, The method comprises the following steps: S1. Wash the internal organs of sea cucumber, freeze-dry, crush, and sieve to obtain freeze-dried powder; S2. Add the freeze-dried powder to water, add a composite enzyme, adjust the pH value and temperature, stir for enzyme hydrolysis, inactivate the enzyme, centrifuge, and collect the supernatant; S3. The supernatant is sequentially subjected to preliminary purification by ultrafiltration and nanofiltration to obtain a preliminary purified liquid; S4. Add modified activated carbon to the preliminary purified liquid, stir for adsorption, separate the modified activated carbon with a magnet, and obtain a deeply purified liquid; S5. Add cyclodextrin to the deeply purified liquid, stir for embedding, centrifuge, and freeze-dry the supernatant to obtain high-purity sea cucumber peptide.

2. The production method according to claim 1, characterized by, The mesh size of the sieve in step S1 is 60-100 mesh; the composite enzyme in step S2 comprises neutral protease and papain, and the mass ratio is 3-7:2-4; the pH value is adjusted to 7-8, the temperature is 50-60 DEG C, the enzyme hydrolysis time is 1-3 h, and the mass ratio of the freeze-dried powder and the composite enzyme is 100:3-5.

3. The preparation method according to claim 1, characterized in that, The temperature of the ultrafiltration in step S3 is 40-50℃, the pressure is 0.1-0.5MPa, a flat plate assembly is used, and the flow rate is 3-5L / m 2 The temperature of the nanofiltration is 30-40℃, the pressure is 1-2MPa, the flow rate is 2-4m / s, the added amount of the modified activated carbon in step S4 is 1-3wt% of the total system, the stirring adsorption time is 30-40min, the cyclodextrin in step S5 is β-cyclodextrin, the added amount is 0.5-0.8wt% of the total system, and the stirring embedding time is 30-50min.

4. The production method according to claim 3, characterized by, The preparation method of the modified activated carbon is as follows: T1. Preliminary treatment: mix and grind coconut shell activated carbon and melamine, calcine under the protection of inert gas, wash, and dry to obtain preliminarily modified coconut shell activated carbon; T2. Preparation of eutectic solvent / ionic liquid: mix and heat chlorocholine and zinc chloride to dissolve, add ionic liquid, and stir to mix uniformly to obtain eutectic solvent / ionic liquid; T3. Modification: add the preliminarily modified coconut shell activated carbon to the eutectic solvent / ionic liquid, heat for impregnation, stir every other time, filter, wash, and dry to obtain eutectic solvent / ionic liquid modified activated carbon; T4. Doping modification: dissolve magnesium salt, aluminum salt, ferrous salt, and iron salt in water, add the eutectic solvent / ionic liquid modified activated carbon, heat under the protection of inert gas, adjust the pH value of the solution, stir for reaction, age, filter, wash, and dry to obtain doped modified activated carbon; T5. KH560 modification: add the doped modified activated carbon to ethanol, add silane coupling agent KH560, heat and stir for reaction, centrifuge, wash, and dry to obtain KH560 / doped modified activated carbon; T6. Coupling: add the KH560 / doped modified activated carbon to a mixture of acetonitrile and ethanol, add mPEG-NH2 and branched polyethyleneimine, stir for reaction at room temperature, filter, wash, and dry to obtain modified activated carbon.

5. The preparation method according to claim 4, characterized in that, In step T1, the mass ratio of the coconut shell activated carbon and melamine is 10:3-7, the grinding time is 20-40 min, and the calcination temperature is 500-700 DEG C for 1-3 h; in step T2, the mass ratio of the zinc chloride, chlorocholine, and ionic liquid is 25-30:12-16:8-15, the ionic liquid is selected from at least one of 1-ethyl-3-methylimidazole chloride salt and 1-(2-hydroxyethyl)-3-methylimidazole acetate, and the heating dissolution temperature is 95-105 DEG C.

6. The preparation method according to claim 4, characterized in that, The mass ratio of the preliminary modified coconut shell activated carbon and the eutectic solvent / ionic liquid in step T3 is 1:10-15, the temperature of the heating impregnation is 85-95℃, and the impregnation time is 2-4h; the molar ratio of the magnesium salt, the aluminum salt, the ferrous salt, and the iron salt in step T4 is 1-3:1:1:2, the heating temperature is 75-85℃, the pH value of the solution is adjusted to 10-11, the stirring reaction time is 3-5h, the aging temperature is 75-85℃, and the aging time is 12-18h.

7. The preparation method according to claim 4, characterized in that, The mass ratio of the doped modified activated carbon and the silane coupling agent KH560 in step T5 is 100:2-3, the temperature of the heating stirring reaction is 40-50℃, and the time is 1-2h; the mass ratio of the KH560 / doped modified activated carbon, mPEG-NH2, and branched polyethyleneimine in step T6 is 10:1-2:0.5-1.5, and the stirring reaction time at room temperature is 10-15h.

8. A high-purity sea cucumber peptide prepared by the preparation method of any one of claims 1-7.

9. Use of the high-purity sea cucumber peptide of claim 8 in the preparation of an antioxidant, anti-fatigue, anti-tumor, and anti-aging drug.

10. An antioxidant sea cucumber peptide, characterized in that, The preparation method is as follows: the high-purity sea cucumber peptide of claim 8 is added to water, sodium tripolyphosphate is added, the pH value of the solution is adjusted, potassium bromide is added, a heating stirring reaction is performed, dialysis is performed, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole are added to the product, stirring activation is performed, hyaluronic acid is added, stirring reaction is performed, dialysis is performed, a zinc salt is added to the undialyzed liquid, stirring chelation is performed, dialysis is performed, and freeze-drying is performed to prepare an antioxidant sea cucumber peptide.

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