A high-activity, low-viscosity sea cucumber peptide and its bidirectional enzymatic preparation method and application

Abstract

The present invention provides a high-activity, low-viscosity sea cucumber peptide and its bidirectional enzymatic preparation method and application. The steps of the bidirectional enzymatic preparation method include: taking fresh sea cucumbers, washing, cutting, and homogenizing to obtain sea cucumber homogenate; adjusting the pH of the sea cucumber homogenate, adding protease and mixed carbohydrase, inactivating the enzyme after enzymatic hydrolysis, and obtaining sea cucumber hydrolyzate; adding activated carbon to the sea cucumber hydrolyzate for decolorization, and obtaining sea cucumber decolorization liquid after ultrafiltration; separating and purifying the sea cucumber decolorization liquid through an organic membrane system, and concentrating it, collecting the concentrated liquid and drying it to obtain sea cucumber peptide. The sea cucumber peptide prepared by the method of the present invention has high biological activity, low viscosity, good quality, simple preparation, no skin irritation, and exhibits good skin repair activity. It can be used in the preparation of cosmetics and skin care products and has a wide range of application value.

Description

Technical Field

[0001] The invention relates to the technical field of biological enzymolysis, and in particular to a high-activity and low-viscosity sea cucumber peptide and a bidirectional enzymolysis preparation method and application thereof. Background Art

[0002] Sea cucumber is a traditional tonic in my country. With the prosperity of my country's sea cucumber farming industry, the requirements for deep processing of sea cucumbers are gradually increasing. Collagen in sea cucumbers is rich in a variety of non-essential amino acids, and has biological activities such as anti-fatigue, anti-oxidation, enhancing immunity, lowering blood lipids, and anti-thrombosis. The biological activity of sea cucumber peptides is closely related to the molecular weight. The smaller the molecular weight, the better the antioxidant activity. When sea cucumbers are enzymatically hydrolyzed to obtain sea cucumber peptides composed of 2-10 amino acids, they have the advantages of small molecular weight, low viscosity, good solubility, high stability, and high bioavailability.

[0003] Enzymatic hydrolysis is the main processing form of sea cucumber peptides, which can significantly improve the bioavailability of sea cucumber peptides. Enzymatic hydrolysis technology is used in the market to deeply process sea cucumbers to form a series of sea cucumber extended products, but the sea cucumber hydrolysate usually has long hydrolysis time, incomplete hydrolysis, and high viscosity of the hydrolysate, which will seriously affect the product's later operation process, such as decolorization, concentration, filtration, etc., and will also seriously affect the product's later stability, bringing a series of problems. In addition to highly active proteins, the sea cucumber body wall also contains a large amount of fucoidan. Fucoidan will cause the viscosity of the sea cucumber hydrolysate to be high, affect the hydrolysis efficiency of the protein, and make the molecular weight of the sea cucumber peptide larger. Therefore, whether the sugar component is completely removed during the processing process also seriously affects the quality of the sea cucumber peptide.

[0004] The synergistic effect of traditional carbohydrase and protease is mostly achieved by adding a single carbohydrase, and the addition method is mostly step-by-step. Although the interference of sugar components is solved, the operation steps are increased, which makes the operation cumbersome and the processing technology more complicated while improving the extraction rate of sea cucumber peptides. The present invention adopts a mixed carbohydrase and protease simultaneous enzymolysis method, which not only improves the proteolysis rate, but also avoids increasing the complexity of the operation process, and solves the practical problem of preparing sea cucumber peptides in production. Summary of the invention

[0005] The present invention provides a high-activity, low-viscosity sea cucumber peptide and a bidirectional enzymatic preparation method and application thereof. The present invention solves the technical problem of high viscosity in the preparation process of sea cucumber antioxidant active peptides, and prepares sea cucumber peptides with high antioxidant activity, high extraction rate and good quality.

[0006] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for preparing a high-activity and low-viscosity sea cucumber peptide by bidirectional enzymatic hydrolysis, which comprises the following steps:

[0008] (1) taking fresh sea cucumber, cleaning, cutting and homogenizing to obtain sea cucumber homogenate;

[0009] (2) adjusting the pH of the sea cucumber homogenate, adding protease and mixed carbohydrase, inactivating the enzyme after enzymolysis, and obtaining a sea cucumber enzymolysis solution;

[0010] (3) adding activated carbon to the sea cucumber enzymatic hydrolyzate for decolorization, and obtaining a sea cucumber decolorization solution after ultrafiltration;

[0011] (4) separating and purifying the sea cucumber decolorized liquid through an organic membrane system, and concentrating the liquid, collecting and drying the concentrated liquid, and obtaining sea cucumber peptides.

[0012] Furthermore, the protease in step (2) is at least one of flavor protease and bromelain.

[0013] Furthermore, the solid content of the protease in step (2) is 3000-8000 U / g.

[0014] Furthermore, in step (2), the mixed carbohydrase is composed of two of cellulase, pectinase and β-glucanase, and the mixing mass ratio thereof is 1:0-5:1.

[0015] Furthermore, in the step (2), the mixed carbohydrase is composed of cellulase and pectinase in a mass ratio of 1:2 to 1:5.

[0016] Furthermore, the amount of the mixed carbohydrase added in step (2) is 0.02-0.15% of the volume of the sea cucumber homogenate.

[0017] Furthermore, in the step (2), the pH of the sea cucumber homogenate is adjusted to 4-8; the enzymolysis temperature is 35-60° C., and the enzymolysis time is 2-6 hours.

[0018] Furthermore, the amount of activated carbon added in step (3) is 0.5-1.5% of the volume of the sea cucumber enzymatic hydrolysate.

[0019] Furthermore, in step (3), the decolorization temperature is 50-70° C., and the decolorization time is 20-60 min.

[0020] The present invention also provides sea cucumber peptides prepared by the bidirectional enzymatic hydrolysis preparation method, the total protein content of which is greater than 90% and the molecular weight is 524Da-680Da.

[0021] The present invention also provides the use of the sea cucumber peptide in preparing skin care products and / or cosmetics for repairing skin barrier damage and / or moisturizing.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] The invention provides a method for preparing sea cucumber peptides by adding a mixed carbohydrate enzyme and a protease to a sea cucumber homogenate for simultaneous enzymolysis. When the sea cucumber polysaccharide is degraded, the protein in the sea cucumber is simultaneously degraded. When the sea cucumber body wall releases macromolecular sugars, the macromolecular proteins are continuously decomposed to release active peptides, which not only improves the release rate of active peptides, but also improves the clarity of the enzymolysis solution and reduces the viscosity of the enzymolysis solution. After subsequent decolorization and filtration by a graded membrane system, a clear and transparent sea cucumber antioxidant peptide solution can be obtained. The enzymolysis solution is purified and graded to obtain the sea cucumber peptide with high biological activity, low viscosity, good quality and simple preparation.

[0024] In addition, the present invention has been experimentally confirmed that the sea cucumber peptide obtained by synchronous enzymatic hydrolysis has no skin irritation, and can upregulate the expression of FLG and AQP genes in 3D skin cells, thereby achieving the effects of moisturizing and skin repairing, and can therefore be used in the preparation of cosmetics and skin care products, and has a wide range of application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Screening of conditions for the preparation of sea cucumber peptides.

[0026] Figure 2 This is the response surface diagram for the interaction of each factor.

[0027] Figure 3 The effect of sea cucumber peptide on the expression of skin repair genes. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described in detail with reference to the following specific examples.

[0029] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0030] Example 1

[0031] Take fresh sea cucumbers, wash and remove sand, cut into small pieces, weigh, add ultrapure water at a ratio of 1:4 (m / w), homogenize, and prepare sea cucumber homogenate. Adjust the pH of the sea cucumber homogenate to neutral, add flavor protease at a ratio of 4200U / g solid content, 45℃, enzymatic hydrolysis for 6h, inactivate the enzyme, and obtain sea cucumber hydrolysate. Sea cucumber polysaccharides will affect the viscosity of the hydrolysate, so different glycosidases need to be selected for enzymatic hydrolysis to decompose the saponins in the sea cucumber into secondary glycosides. Filter the crude hydrolysate, adjust the pH to 6, add mixed saccharide enzymes (the mass ratio of cellulase to pectinase is 1:2) at 0.12%, 50℃, enzymatic hydrolysis for 3.6h, inactivate the enzyme for 20min, adjust the pH to neutral, filter, and obtain sea cucumber peptide hydrolysate. Add 1.2% activated carbon to the above enzymatic hydrolysate, decolorize for 50 minutes at 70°C, centrifuge at 10000r / min for 10 minutes, collect the supernatant, and ultrafilter through a 0.45μm membrane to obtain a sea cucumber decolorized solution. Use an organic membrane system for separation, purification and concentration, pass the sea cucumber decolorized solution through a 1000 / 700 / 500 / 200Da organic membrane in sequence, control the temperature at 10-20°C, the pressure at 0.25MPa, and the flow rate at 1.6mL / s, collect the concentrated solution, and obtain the final enzymatic hydrolysate of the sea cucumber peptide. The above liquid is frozen in a plate and further vacuum freeze-dried to obtain the refined sea cucumber peptide SCOP1.

[0032] Example 2

[0033] Take fresh sea cucumber, wash and remove sand, cut into small pieces, weigh, add ultrapure water at a ratio of 1:4 (m / w), homogenize, and prepare sea cucumber homogenate. Adjust the pH of sea cucumber homogenate to neutral, add bromelain at a ratio of 5700U / g solid content, 50℃, enzymolysis for 5h, inactivate the enzyme, and obtain sea cucumber enzymolysis solution. Filter the crude enzymolysis solution, adjust the pH to 5, add 0.12% mixed carbohydrase (the mass ratio of cellulase to pectinase is 1:4), 57℃, enzymolysis for 4.0h, inactivate the enzyme for 15min, adjust the pH to neutral, filter, and obtain sea cucumber peptide enzymolysis solution. Add 0.5-1.5% activated carbon to the above enzymolysis solution, 70℃, decolorize for 50min, 10000r / min refrigerated centrifugation for 10min, collect the supernatant, and ultrafilter through a 0.45μm membrane to obtain sea cucumber decolorization solution. The organic membrane system was used for separation, purification and concentration. The sea cucumber decolorization solution was sequentially passed through 1000 / 700 / 500 / 200Da organic membranes, the temperature was controlled at 10-15°C, the pressure was 0.22MPa, and the flow rate was 1.4mL / s. The concentrated solution was collected to obtain the final enzymatic hydrolyzate of sea cucumber peptide. The above liquid was frozen in a plate and further vacuum-freeze-dried to obtain the refined sea cucumber peptide SCOP2.

[0034] Example 3

[0035] Take fresh sea cucumbers, wash and remove sand, cut into small pieces, weigh, add ultrapure water at a ratio of 1:4 (m / w), homogenize, and prepare sea cucumber homogenate. Adjust the pH of the sea cucumber homogenate to 5.6, add flavor protease at a ratio of 4750U / g solid content, and add mixed carbohydrase at 0.12% (cellulase and pectinase mass ratio is 1:5), 60℃, enzymolysis for 2.7h, enzyme inactivation for 20min, and obtain sea cucumber enzymolysis solution. Add 1.5% activated carbon to the above enzymolysis solution, 60℃, decolorize for 40min, 10000r / min refrigerated centrifugation for 10min, collect the supernatant, and ultrafilter through a 0.45μm membrane to obtain sea cucumber decolorization solution. The organic membrane system was used for separation, purification and concentration. The sea cucumber decolorization solution was sequentially passed through 1000 / 700 / 500 / 200Da organic membranes, the temperature was controlled at 18°C, the pressure was 0.25MPa, and the flow rate was 1.5mL / s. The concentrated solution was collected to obtain the final enzymatic hydrolyzate of sea cucumber peptide. The above liquid was frozen in a plate and further vacuum freeze-dried to obtain the refined sea cucumber peptide SCOP3.

[0036] Example 4

[0037] Take fresh sea cucumbers, wash and remove sand, cut into small pieces, weigh, add ultrapure water at a ratio of 1:4 (m / w), homogenize, and prepare sea cucumber homogenate. Adjust the pH of the sea cucumber homogenate to 6.8, add bromelain at a ratio of 4100U / g solid content, and add mixed carbohydrase at 0.12% (the mass ratio of cellulase to pectinase is 1:3), 45℃, enzymolysis for 3.25h, enzyme inactivation for 15min, and obtain sea cucumber enzymolysis solution. Add 1.5% activated carbon to the above enzymolysis solution, 60℃, decolorization for min, 10000r / min refrigerated centrifugation for 10min, collect the supernatant, and ultrafilter through a 0.45μm membrane to obtain sea cucumber decolorization solution. An organic membrane system was used for separation, purification and concentration. The sea cucumber decolorization liquid was sequentially passed through 1000 / 700 / 500 / 200Da organic membranes, the temperature was controlled at 10-20°C, the pressure was 0.21MPa-0.26MPa, and the flow rate was 0.2-1.8mL / s. The concentrated liquid was collected to obtain the final enzymatic hydrolyzate of the sea cucumber peptide; the above liquid was frozen in a plate, and further vacuum freeze-dried to obtain the refined sea cucumber peptide SCOP4.

[0038] The conditions in the above four embodiments are the preparation schemes of high-activity sea cucumber peptides obtained by screening based on antioxidant indicators. The specific screening process is as follows: first, the glycosidase is optimized and the most suitable ratio scheme is experimentally screened, and β-glucanase, pectinase, and cellulase are selected and added in different proportions, and the total addition amount is controlled to be 0.05% of the substrate mass. The enzymatic hydrolysis test is carried out under the condition that the sea cucumber is used as the raw material and the superoxide anion free radical scavenging rate is used as an indicator to determine the optimal mixed sugar enzyme preparation addition amount and ratio. The results are as follows Figure 1As shown in the figure, when pectinase and cellulase are combined, their antioxidant activity is better, and their activity is highest when the ratio is 2:1. Then, with this mixed carbohydrase ratio, the optimal matching scheme of carbohydrase and protease is further analyzed by response surface analysis. The response surface design uses the Box-Behnken central combination experimental design principle of Design-Expert 11.0 software to design a three-factor three-level response surface optimization experiment, and the superoxide anion test box is used for detection. The experimental results are shown in Table 1.

[0039] Table 1 Response surface analysis experimental design and results

[0040] Sorting Enzyme hydrolysis time Addition amount of mixed glycosidase Protease activity Superoxide anion clearance rate % 1 2.00 0.02 5000.00 30.946 6 6.00 0.07 2000.00 54.1723 8 6.00 0.07 8000.00 58.1936 3 2.00 0.12 5000.00 68.4921 15 4.00 0.07 5000.00 58.3864 14 4.00 0.07 5000.00 59.0943 9 4.00 0.02 2000.00 57.8962 5 2.00 0.07 2000.00 53.9836 4 6.00 0.12 5000.00 63.9332 11 4.00 0.02 8000.00 46.6261 13 4.00 0.07 5000.00 57.8833 7 2.00 0.07 8000.00 53.8745 10 4.00 0.12 2000.00 67.3452 16 4.00 0.07 5000.00 63.9257 17 4.00 0.07 5000.00 59.2236 2 6.00 0.02 5000.00 52.7489 12 4.00 0.12 8000.00 69.5487

[0041] The data in Table 1 were analyzed by Design-Expert software, and the results are shown in Table 2. Taking the superoxide radical scavenging rate as the response value, after regression fitting, the variance analysis results showed that the model P<0.01, indicating that the regression model is significant, and the lack of fit term P=0.0981, which is not significant, indicating that the model fits the actual experiment well. Therefore, this regression equation can be used to determine the multi-enzyme hydrolysis process of high-activity sea cucumber peptides. The response surface diagram of the interaction of various factors is shown in Figure 2 .

[0042] Table 2 Analysis of variance of regression model

[0043]

[0044] According to the response surface test results, the top four optimal conditions determined by software analysis are shown in Table 3. Therefore, Examples 1-4 were obtained according to the response surface screening conditions.

[0045] Table 3 Sea cucumber peptide preparation solution

[0046]

[0047] Example 5

[0048] The basic components of the sea cucumber peptides in Examples 1-4 were determined according to the national standards GB / T 5009.5-2016, GB / T9695.31-2008, GB / T 5009.6-2016, GB 5009.3-2016, and GB5009.4-2016, respectively.

[0049] The molecular weight of the sea cucumber peptides in Examples 1-4 was determined using the HPLC method under the following conditions:

[0050] Chromatograph: Agilent 1260, equipped with UV detector and GPC software processing system;

[0051] Chromatographic column: TSKgel G2000SWXL (7.8mm*30cm, particle size 5μm);

[0052] Mobile phase: acetonitrile: water (pH 2.0) 3:7; flow rate: 1.0 mL / min; injection volume: 10 μL;

[0053] Column temperature: 30°C; detection wavelength: 220nm.

[0054] Take 1 mg of each sample and standard substance used for relative molecular weight calibration curve: uracil (MW112), glutathione (MW307), bacitracin (MW1422), aprotinin (MW6511), cytochrome C (MW12384), bovine serum albumin V (MW68000), respectively, dissolve in 1 mL of pure water, filter with 0.45 nm microporous membrane and inject. The results are analyzed by GPC software.

[0055] The basic composition and molecular weight of sea cucumber peptide are shown in Table 4. Sea cucumber peptide is composed of 90.57% total protein, 2.80% water, 4.12% ash, 0.19% lipid, and 2.05% total sugar, and its molecular weight is 524Da. It shows that the sea cucumber peptide prepared by the present invention has high purity and small molecular weight.

[0056] Table 4 Analysis of basic components of sea cucumber peptides

[0057] sample Total Protein Moisture Ash Lipids Total Sugar Molecular weight SCOP1 90.57±4.17 2.80±0.25 4.12±0.31 0.19±0.09 2.05±0.24 524Da SCOP2 89.65±2.05 3.60±0.48 3.67±0.33 0.10±0.02 1.55±0.44 674Da SCOP3 90.25±3.56 3.00±0.35 4.02±0.30 0.18±0.04 1.80±0.20 582Da SCOP4 87.45±1.33 4.50±0.54 3.82±0.55 0.30±0.12 1.75±0.30 625Da

[0058] Example 6

[0059] According to SN / T 4577-2016 "In vitro test method for skin irritation detection of cosmetics using a reconstructed human epidermal model", the sea cucumber peptides of Examples 1-4 were evaluated for skin irritation / corrosion of the active ingredients.

[0060] 1. Use the skin irritation (SIT) test. According to the standard method, purchase the 3D recombinant skin cell model EpiKutis from Boxi Company and test it according to the SIT-Skin Irritation Test process. After receiving the 3D reconstructed skin, it was placed in a 6-well plate, and 0.5ml and 1ml of EpiRecovery medium were added to the upper and lower wells respectively. It was incubated in a carbon dioxide incubator for 18±3h, and then 25μL PBS was added to moisten the surface of the model. 25mg of each sea cucumber peptide sample and positive control (SDS) were added to each well. The negative control was a sample with only culture medium added. After being placed on a clean bench for accurate timing of 30±1min, the surface of the model was washed with 1ml PBS, and repeated 15 times. Then, the model was taken out with tweezers, placed in a new 6-well plate, and 0.9mL EpiRecovery was added. After incubation for 42h, the model was transferred to a new 24-well plate, 0.3mL of 5mg / ml MTT working solution was added, and incubated at 37℃ in a carbon dioxide incubator for 3h±5min. The MTT solution was discarded and 200μL was added to each well. Wash with PBS, repeat 3 times, then transfer to a new 24-well plate again, add 2 mL of isopropanol to the inner surface of the 3D reconstructed skin cell model EpiKutis, seal it with a film, place it at 4°C for 18 hours, pierce the model with the tip of the gun, mix it in the lower chamber, aspirate 200 μL of isopropanol extract, and detect its OD value at 570 nm.

[0061] The skin irritation results of the four sea cucumber peptides are analyzed as shown in Table 5. The sea cucumber peptides of Examples 1-4 all promoted the proliferation of recombinant 3D skin cells in vitro and had no cytotoxicity. The cell survival rates were all higher than 50%, indicating that the sea cucumber peptides prepared by the present invention were non-irritating.

[0062] Table 5 Skin irritation results of ginseng peptides

[0063] Relative tissue vitality average Judgment (≤50% irritating) SCOP1 128.43％±31.20％* Non-irritating (NI) SCOP2 92.40％±13.62％* Non-irritating (NI) SCOP3 105.25％±10.50％ Non-irritating (NI) SCOP4 112.35％±16.20％ Non-irritating (NI) Negative control 100.00％±22.41％ Non-irritating (NI) Positive Control 8.59％±1.47％ Irritation (I)

[0064] Example 7 Evaluation of the skin repairing effect of sea cucumber peptide

[0065] This example tests the skin repairing effect of the sea cucumber peptides prepared in Examples 1-4. When the skin is exposed to irritating factors, it will cause clinical acute damage to the skin barrier, leading to dry skin and erythema. This example uses the commonly used surfactant sodium dodecyl sulfate (SLS) to simulate irritating skin damage, and evaluates the repairing effect of the sea cucumber peptides by the tissue morphology after active substance treatment and barrier-related protein indicators.

[0066] The revived model was transferred to a 6-well plate containing 0.9 mL EpiGrowth medium and divided into a model control group, sea cucumber peptide group 1 (200 μg / mL), sea cucumber peptide group 2 (200 μg / mL) and a negative control group, with 3 parallels in each group. 200 μL SLS solution (4 mg / mL) and 200 μL sample were mixed, and 25 μL SLS-sample mixture was slowly dripped onto the model surface as the sample group; 25 μL SLS solution (2 mg / mL) was used as the model control group, and the negative control group (sterile PBS) was not treated with medication. After the administration, the cells were cultured in an incubator (37 ° C, 5% CO2) for 24 hours. After the incubation, the model culture fluid was collected, and the reconstructed skin model was removed by circumcision. The subsequent RNA extraction and protein expression analysis were performed to detect the transcription levels of skin repair-related genes FLG and AQP3 in the cell model, and then the skin repair efficacy of sea cucumber peptide was evaluated.

[0067] The results are as follows Figure 3 As shown, sea cucumber peptides (SCOP1 and SCOP2) can upregulate the expression of FLG and AQP genes in 3D skin cells. Filaggrin (FLG) is a key component in the CE assembly process. In addition to being a structural component of the skin barrier, FLG can also be hydrolyzed by Caspase-14 to form a natural moisturizing factor, thus also playing a role in moisturizing and skin repair.

[0068] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A method for preparing a high-activity and low-viscosity sea cucumber peptide by bidirectional enzymatic hydrolysis, characterized in that: The following steps are involved: (1) taking fresh sea cucumbers, washing, cutting and homogenizing them to obtain sea cucumber homogenate; (2) adjusting the pH of the sea cucumber homogenate, adding protease and mixed carbohydrase, inactivating the enzymes after enzymolysis, and obtaining a sea cucumber enzymolysis solution; the protease is flavor protease or bromelain, and the solid content of the protease is 3000-8000 U / g; the mixed carbohydrase is composed of cellulase and pectinase; the mass ratio of cellulase to pectinase is 1:5-1:3; the pH of the sea cucumber homogenate is 4-8; the enzymolysis temperature is 35-60°C, and the enzymolysis time is 2-6 h; the amount of the mixed carbohydrase added is 0.02-0.15% of the volume of the sea cucumber homogenate; (3) adding activated carbon to the sea cucumber enzymatic hydrolyzate for decolorization, and obtaining a sea cucumber decolorized solution after ultrafiltration; (4) Separating and purifying the decolorized sea cucumber liquid through an organic membrane system, and concentrating it, collecting and drying the concentrated liquid to obtain sea cucumber peptides; the organic membrane system separation and purification is to pass the decolorized sea cucumber liquid through 1000 / 700 / 500 / 200 Da organic membranes in sequence, with the temperature controlled at 10~20°C, the pressure at 0.21~0.26MPa, and the flow rate at 0.2~1.8 mL / s.

2. The bidirectional enzymatic hydrolysis preparation method according to claim 1, characterized in that: The amount of activated carbon added in step (3) is 0.5-1.5% of the volume of the sea cucumber enzymatic hydrolysate.

3. The bidirectional enzymatic hydrolysis preparation method according to claim 1, characterized in that: In the step (3), the decolorization temperature is 50-70°C and the decolorization time is 20-60 min.

4. The sea cucumber peptide prepared by the bidirectional enzymatic hydrolysis preparation method according to any one of claims 1 to 3, characterized in that: The total protein content of the sea cucumber peptide is greater than 90%, and the molecular weight is 524 Da-680 Da.

5. Use of the sea cucumber peptide according to claim 4 in the preparation of cosmetics for repairing skin barrier damage.

6. The use according to claim 5, characterized in that: The cosmetics are skin care products.

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