Preparation method of small-molecular-weight sea cucumber oligopeptide, prepared sea cucumber oligopeptide and application of small-molecular-weight sea cucumber oligopeptide

Abstract

The invention relates to a preparation method of small-molecular-weight sea cucumber oligopeptide, the prepared sea cucumber oligopeptide and application, and belongs to the technical field of skin care products. The preparation method of the small-molecular-weight sea cucumber oligopeptide comprises the following steps: preparing sea cucumber fine powder; adding the sea cucumber fine powder into ultrapure water, adding papain and EDTA (Ethylene Diamine Tetraacetic Acid), and carrying out enzymolysis under the assistance of microwaves; adding trypsin and calcium ions, and carrying out enzymolysis under the assistance of microwaves; adding neutral protease and zinc ions, and performing enzymolysis under the assistance of microwaves to obtain final sea cucumber enzymatic hydrolysate; and decolorizing, separating and purifying, and freeze-drying to obtain the powdery small-molecular-weight sea cucumber oligopeptide. According to the preparation method of the small-molecular-weight sea cucumber oligopeptide, microwave heating and enzymolysis reaction are combined, the hydrolysis degree of a peptide chain is regulated and controlled by controlling an enzyme hydrolysis sequence and enzymolysis conditions, the small-molecular-weight sea cucumber oligopeptide with relatively concentrated and moderate molecular weight is prepared, and good biological activity of the sea cucumber oligopeptide is kept.

Description

Technical Field

[0001] The present invention belongs to the technical field of skin care products, and particularly relates to a preparation method of small molecular weight sea cucumber oligopeptides, the sea cucumber oligopeptides prepared thereby, and their applications. Background Art

[0002] Sea cucumbers (Apostichopus japonicus, produced in the shallow seas along the coast of Shandong, China) are precious marine organisms rich in various bioactive substances, especially sea cucumber peptides. Due to their potential in anti-aging, immunomodulation, antioxidant, anti-inflammatory and other aspects, sea cucumber peptides have become the research focus in the fields of biomedicine and nutritional health care. The biological activity of sea cucumber peptides depends on their molecular weight. Low molecular weight peptides can be better absorbed by the human body and have stronger biological activity. At present, the preparation methods of sea cucumber peptides mainly include chemical hydrolysis and enzymatic hydrolysis methods. Compared with chemical hydrolysis, the enzymatic hydrolysis method has higher selectivity and better biological safety. However, traditional enzymatic hydrolysis methods usually require a long reaction time and high-temperature environment, which may lead to the degradation of sea cucumber peptides or the loss of their active ingredients. In addition, traditional enzymatic hydrolysis methods also have problems such as uneven molecular weight of hydrolysis products, low activity, and low hydrolysis efficiency.

[0003] Therefore, how to improve the enzymatic hydrolysis efficiency and effectively control the hydrolysis of peptide chains during enzymatic hydrolysis to prepare small molecular weight sea cucumber oligopeptides with relatively concentrated and moderate molecular weights and high biological activity at the same time is a technical problem that needs to be solved urgently at present. Summary of the Invention

[0004] In view of the above technical problems, the present invention provides a preparation method of small molecular weight sea cucumber oligopeptides, the sea cucumber oligopeptides prepared thereby, and their applications. The preparation method of small molecular weight sea cucumber oligopeptides combines microwave heating and enzymatic hydrolysis reaction, controls the order and conditions of enzymatic hydrolysis to regulate the hydrolysis degree of peptide chains, prepares small molecular weight sea cucumber oligopeptides with relatively concentrated and moderate molecular weights, and maintains the good biological activity of sea cucumber oligopeptides.

[0005] The present invention provides a preparation method of small molecular weight sea cucumber oligopeptides, comprising the following steps:

[0006] (1) Cut sea cucumbers into small pieces, perform low-temperature vacuum drying and then pulverize to obtain fine sea cucumber powder;

[0007] (2) Add the fine sea cucumber powder to ultrapure water, homogenize, add papain and EDTA, and perform enzymatic hydrolysis under microwave assistance, then inactivate the enzyme to obtain sea cucumber enzymatic hydrolysate A; wherein, the mass ratio of the fine sea cucumber powder to papain is (5-20):(0.05-0.2), the concentration of EDTA is 0.15-0.2 mmol / L, the microwave power is 340-360 W, and the enzymatic hydrolysis temperature is 50-53 °C;

[0008] (3) Add trypsin and calcium ions to the sea cucumber enzymatic hydrolysate A, enzymatically hydrolyze it under microwave assistance, and inactivate the enzyme to obtain the sea cucumber enzymatic hydrolysate B; wherein, the mass ratio of trypsin to the fine sea cucumber powder added in step (2) is (0.08 - 0.1):(5 - 20), the concentration of calcium ions is 1 - 2 mmol / L, the microwave power is 310 - 315 W, and the enzymatic hydrolysis temperature is 37 - 40 °C;

[0009] (4) Add neutral protease and zinc ions to the sea cucumber enzymatic hydrolysate B, enzymatically hydrolyze it under microwave assistance, and inactivate the enzyme to obtain the final sea cucumber enzymatic hydrolysate; wherein, the mass ratio of neutral protease to the fine sea cucumber powder added in step (2) is (0.1 - 0.2):(5 - 20), the concentration of zinc ions is 0.5 - 1 mmol / L, the microwave power is 330 - 350 W, and the enzymatic hydrolysis temperature is 45 - 50 °C;

[0010] (5) Add activated carbon to the final enzymatic hydrolysate for decolorization, and perform ultrafiltration to obtain the decolorized sea cucumber solution;

[0011] (6) Separate, purify, and concentrate the decolorized sea cucumber solution through an organic membrane system, collect the concentrated solution, and freeze-dry it to obtain powdery small-molecular-weight sea cucumber oligopeptides.

[0012] In some of the embodiments, in step (2), the enzymatic hydrolysis pH is 6 - 7; in step (3), the enzymatic hydrolysis pH is 7.5 - 8.2; in step (4), the enzymatic hydrolysis pH is 7 - 7.5.

[0013] In some of the embodiments, in step (2), the enzymatic hydrolysis time is 1 h; in step (3), the enzymatic hydrolysis time is 2 h; in step (4), the enzymatic hydrolysis time is 1.5 h.

[0014] In some of the embodiments, in step (3), calcium propionate is added to obtain calcium ions; in step (4), zinc lactate is added to obtain zinc ions.

[0015] In some of the embodiments, in step (5), the addition amount of activated carbon is 2%, the decolorization temperature is 70 °C, and the decolorization time is 50 min.

[0016] In some of the embodiments, in step (5), after decolorization, freeze centrifugation is performed, and ultrafiltration is carried out on the supernatant collected by centrifugation. The centrifugation speed is 10000 r / min, the centrifugation time is 10 min, and ultrafiltration is performed using a 0.45 μm membrane.

[0017] In some of the embodiments, in step (6), an organic membrane with a molecular weight cut-off of 600 Da is used for separation and purification. During separation, the temperature is 10 - 20 °C, the pressure is 0.25 MPa, and the flow rate is 1.6 mL / s.

[0018] The present invention also provides a small - molecular - weight sea cucumber oligopeptide, which is prepared by using the preparation method of the small - molecular - weight sea cucumber oligopeptide described in any one of the above - mentioned technical solutions.

[0019] The present invention further provides a skin - care composition, comprising the small - molecular - weight sea cucumber oligopeptide and green tea extract described in any one of the above - mentioned technical solutions, and the mass ratio of the small - molecular - weight sea cucumber oligopeptide to the green tea extract is 1:1.

[0020] In addition, the present invention also provides the application of the small - molecular - weight sea cucumber oligopeptide described in any one of the above - mentioned technical solutions in the preparation of skin - care products.

[0021] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0022] 1. The preparation method of the small - molecular - weight sea cucumber oligopeptide provided by the present invention combines microwave heating and enzymatic hydrolysis reaction. Through a specific enzymatic hydrolysis sequence and precisely controlling enzymatic hydrolysis conditions such as the microwave power, enzymatic hydrolysis temperature, enzyme concentration, and enzymatic hydrolysis factor concentration in each step of hydrolysis, the regulation of the degree of peptide chain hydrolysis is achieved, enabling the sea cucumber to be hydrolyzed into small - molecular - weight sea cucumber oligopeptides with molecular weights concentrated in the range of 400 - 600 Da within a relatively short time, effectively improving the biological activity of the sea cucumber oligopeptide, especially having significant advantages in aspects such as antioxidant, anti - aging, and anti - inflammatory.

[0023] 2. The molecular weights of the small - molecular - weight sea cucumber oligopeptides provided by the present invention are mainly concentrated in the range of 400 - 600 Da, which can be rapidly absorbed by the skin and have high biological activity, especially strong antioxidant, anti - aging, and anti - inflammatory activities.

[0024] 3. The small - molecular - weight sea cucumber oligopeptides provided by the present invention can significantly scavenge free radicals, have strong antioxidant activity, and can significantly inhibit the activity of β - galactosidase in fibroblasts and significantly inhibit the expression level of inflammatory factor (IL - 6) in fibroblasts, showing good prospects in inhibiting skin inflammatory reactions and alleviating skin aging.

[0025] 4. The skin - care composition provided by the present invention, through the synergistic effect of the small - molecular - weight sea cucumber oligopeptide and green tea extract, can promote the synthesis of type I collagen in fibroblasts and has significant advantages in aspects such as antioxidant and anti - aging.

[0026] 5. The application of the small - molecular - weight sea cucumber oligopeptide provided by the present invention in the preparation of skin - care products shows good prospects in inhibiting skin inflammatory reactions and alleviating skin aging. Brief Description of the Drawings

[0027] Figure 1 It is a flow block diagram of the preparation method of the small - molecular - weight sea cucumber oligopeptide provided by the embodiment of the present invention;

[0028] Figure 2 Results of the DPPH free radical scavenging test of the small molecular weight sea cucumber oligopeptides prepared in Example 1 of the present invention at different concentrations;

[0029] Figure 3 Results of the β-galactosidase activity test of the small molecular weight sea cucumber oligopeptides prepared in Example 1 of the present invention on fibroblasts at different concentrations;

[0030] Figure 4 Results of the detection test of the transcriptional level of inflammatory factor genes of the small molecular weight sea cucumber oligopeptides prepared in Example 1 of the present invention at different concentrations;

[0031] Figure 5 Results of the type I collagen level detection test of the skin care composition, small molecular weight sea cucumber oligopeptides and green tea extract prepared in Example 5 of the present invention. Detailed implementation manners

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0033] As Figure 1 shown, the embodiments of the present invention provide a method for preparing small molecular weight sea cucumber oligopeptides, including the following steps:

[0034] (1) Cut the sea cucumbers into small pieces, dry them under low temperature and vacuum, and then pulverize them to obtain fine sea cucumber powder;

[0035] (2) Add the fine sea cucumber powder to ultrapure water, homogenize, add papain and EDTA, and carry out enzymatic hydrolysis under microwave assistance, and then inactivate the enzyme to obtain sea cucumber enzymatic hydrolysate A; wherein, the mass ratio of the fine sea cucumber powder to papain is (5-20):(0.05-0.2), the concentration of EDTA is 0.15-0.2 mmol / L, the microwave power is 340-360 W, and the enzymatic hydrolysis temperature is 50-53 °C;

[0036] (3) Add trypsin and calcium ions to the sea cucumber enzymatic hydrolysate A, carry out enzymatic hydrolysis under microwave assistance, and then inactivate the enzyme to obtain sea cucumber enzymatic hydrolysate B; wherein, the mass ratio of trypsin to the fine sea cucumber powder added in step (2) is (0.08-0.1):(5-20), the concentration of calcium ions is 1-2 mmol / L, the microwave power is 310-315 W, and the enzymatic hydrolysis temperature is 37-40 °C;

[0037] (4) Add neutral protease and zinc ions to the sea cucumber enzymatic hydrolysate B, carry out enzymatic hydrolysis under microwave assistance, and inactivate the enzyme to obtain the final sea cucumber enzymatic hydrolysate; wherein, the mass ratio of neutral protease to the fine sea cucumber powder added in step (2) is (0.1 - 0.2):(5 - 20), the concentration of zinc ions is 0.5 - 1 mmol / L, the microwave power is 330 - 350 W, and the enzymatic hydrolysis temperature is 45 - 50 °C;

[0038] (5) Add activated carbon to the final enzymatic hydrolysate for decolorization, and carry out ultrafiltration to obtain the decolorized sea cucumber solution;

[0039] (6) Separate, purify and concentrate the decolorized sea cucumber solution through an organic membrane system, collect the concentrated solution, and freeze-dry it to obtain powdery small-molecular-weight sea cucumber oligopeptides.

[0040] For the above method for preparing small-molecular-weight sea cucumber oligopeptides, different hydrolases are used for stepwise hydrolysis, and microwave-assisted hydrolysis is adopted during stepwise hydrolysis. By controlling the microwave power at medium and low power and cooperating with a specific enzymatic hydrolysis temperature, the activity of the hydrolase is maintained to the greatest extent, and at the same time, excessive temperature is avoided from causing excessive cleavage of the sea cucumber peptide chain or side reactions, or even inactivation or degradation of sea cucumber proteins; meanwhile, on the premise of keeping the enzymatic hydrolysis reaction mild, it is ensured that microwave energy can be evenly transmitted to the reaction system, improving the hydrolysis efficiency and achieving efficient hydrolysis; in addition, segmented control is carried out for each step of hydrolysis, and different microwave powers and enzymatic hydrolysis temperatures are adopted for each step of hydrolysis, which are combined with specific dosages of hydrolases for each step, enabling each step of hydrolysis to reach the optimal hydrolysis level.

[0041] At the same time, for the above method for preparing small-molecular-weight sea cucumber oligopeptides, papain, trypsin and neutral protease are successively used for stepwise hydrolysis of sea cucumber peptides, which can first release hydrophilic groups (arginine Arg, lysine Lys), and then release hydrophobic groups (leucine Leu, phenylalanine Phe), forming a mixed peptide segment combination with moisturizing, repairing and antioxidant functions. Moreover, sequential hydrolysis can avoid competitive inhibition between enzymes, maximize the utilization of the specificity of different enzymes, generate sea cucumber oligopeptides with lower molecular weights, thereby improving the transdermal absorption rate and biological activity. Specifically, papain is used as the first-step hydrolase. Utilizing the broad substrate specificity of papain (cleaving the carboxyl-terminal peptide bonds of positively charged amino acids such as Arg, Lys, Phe), the sea cucumber peptide chain can be initially cleaved to release peptide segments containing basic amino acids such as Arg and Lys, and these amino acid peptide segments have the effects of promoting collagen synthesis, anti-inflammation and moisturizing; then trypsin is used as the second-step hydrolase to further precisely cut the carboxyl-terminal of Arg and Lys, generating smaller peptide segments and releasing more hydrophilic amino acids, enhancing the moisturizing and barrier repair functions; finally, neutral protease is used as the final hydrolase to target the peptide bonds of hydrophobic amino acids (such as Leu, Phe), exposing hydrophobic groups, which helps to enhance the skin lipid barrier function, improve firmness and antioxidant capacity.

[0042] In addition, in the above method for preparing small - molecular - weight sea cucumber oligopeptides, during step - by - step hydrolysis, different enzymatic hydrolysis factors are paired with different hydrolases (specifically: EDTA is added as an enzymatic hydrolysis factor during papain hydrolysis, calcium ions are added as an enzymatic hydrolysis factor during trypsin hydrolysis, and zinc ions are added as an enzymatic hydrolysis factor during neutral protease hydrolysis), thereby improving the hydrolysis efficiency.

[0043] In summary, the above method for preparing small - molecular - weight sea cucumber oligopeptides combines microwave heating and enzymatic hydrolysis reactions. Through a specific enzymatic hydrolysis sequence and precise control of enzymatic hydrolysis conditions such as the microwave power, enzymatic hydrolysis temperature, enzyme concentration, and enzymatic hydrolysis factor concentration in each step of hydrolysis, the regulation of the degree of peptide chain hydrolysis is achieved. The sea cucumber is hydrolyzed into small - molecular - weight sea cucumber oligopeptides with a molecular weight concentrated in the range of 400 - 600 Da within a relatively short time, effectively improving the biological activity of sea cucumber oligopeptides, especially having significant advantages in aspects such as antioxidant, anti - aging, and anti - inflammation.

[0044] In a preferred embodiment, in step (2), the enzymatic hydrolysis pH is 6 - 7; in step (3), the enzymatic hydrolysis pH is 7.5 - 8.2; in step (4), the enzymatic hydrolysis pH is 7 - 7.5. This preferred embodiment specifically defines the pH of enzymatic hydrolysis in each step, provides the optimal pH environment for each hydrolase, reduces the occurrence probability of side reactions such as non - specific hydrolysis, enables each enzyme to function in the best state, not only improves the hydrolysis efficiency, but also ensures that the peptide segment structures generated in each step meet the expectations, avoids irreversible changes in the active centers of enzymes due to too high or too low pH, and reduces enzyme inactivation.

[0045] In a preferred embodiment, in step (2), the enzymatic hydrolysis time is 1 h; in step (3), the enzymatic hydrolysis time is 2 h; in step (4), the enzymatic hydrolysis time is 1.5 h. This preferred embodiment specifically defines the enzymatic hydrolysis time in each step of enzymatic hydrolysis, thereby controlling the degree of enzymatic hydrolysis in each step, ensuring that the peptide segment structures generated in each step meet the expectations, and avoiding over - hydrolysis.

[0046] In a preferred embodiment, in step (3), calcium propionate is added to obtain calcium ions; in step (4), zinc lactate is added to obtain zinc ions. This preferred embodiment specifically defines the introduction methods of calcium ions and zinc ions. Using calcium propionate and zinc lactate to introduce calcium ions and zinc ions can reduce the impact on the system pH and avoid affecting the activity of hydrolases.

[0047] In a preferred embodiment, in step (5), the addition amount of activated carbon is 2%, the decolorization temperature is 70 °C, and the decolorization time is 50 min. This preferred embodiment specifically defines the addition amount of activated carbon, decolorization temperature, and decolorization time used for decolorization, which is beneficial to ensuring product quality.

[0048] In a preferred embodiment, in step (5), after decolorization, freeze centrifugation is carried out, and the supernatant collected by centrifugation is ultrafiltered. The centrifugation speed is 10,000 r / min, the centrifugation time is 10 min, and ultrafiltration is carried out using a 0.45 μm membrane. This preferred embodiment specifically defines the specific steps of separation and ultrafiltration after decolorization, which is beneficial to improving the purity of sea cucumber oligopeptides and ensuring product quality.

[0049] In a preferred embodiment, in step (6), an organic membrane with a molecular weight cut-off of 600 Da is used for separation and purification. During separation, the temperature is 10 - 20 °C, the pressure is 0.25 MPa, and the flow rate is 1.6 mL / s. This preferred embodiment specifically defines the specific steps of separating and purifying the decolorized sea cucumber solution, which is beneficial to ensuring that the molecular weights of the small molecular weight sea cucumber oligopeptides obtained by preparation are relatively concentrated, improving product purity, and ensuring product quality.

[0050] The embodiment of the present invention also provides a small molecular weight sea cucumber oligopeptide, which is obtained by using the above-mentioned preparation method of small molecular weight sea cucumber oligopeptides. The molecular weight of this small molecular weight sea cucumber oligopeptide is mainly concentrated in the range of 400 - 600 Da, can be rapidly absorbed by the skin, and has high biological activity, especially strong antioxidant, anti-aging, and anti-inflammatory activities.

[0051] The embodiment of the present invention further provides a skin care composition, including the above-mentioned small molecular weight sea cucumber oligopeptide and green tea extract, and the mass ratio of the small molecular weight sea cucumber oligopeptide to the green tea extract is 1:1. This skin care composition has a significant effect of promoting collagen synthesis through the compounding of the small molecular weight sea cucumber oligopeptide and the green tea extract, and has significant advantages in aspects such as antioxidant and anti-aging.

[0052] In addition, the embodiment of the present invention also provides the application of the above-mentioned small molecular weight sea cucumber oligopeptide in the preparation of skin care products. The above-mentioned small molecular weight sea cucumber oligopeptide provided by the embodiment of the present invention can be widely used in the preparation of anti-aging and repair skin care products, and can also be used in products such as sunscreen and cleansing, with great market potential and application prospects.

[0053] In order to introduce the preparation method of the small molecular weight sea cucumber oligopeptide, the sea cucumber oligopeptide prepared thereby, and the application provided by the embodiment of the present invention more clearly and in detail, the following will be described in combination with specific embodiments.

[0054] I. Specific embodiments corresponding to the preparation method of small molecular weight sea cucumber oligopeptide and the sea cucumber oligopeptide prepared thereby

[0055] Example 1

[0056] A preparation method of small molecular weight sea cucumber oligopeptide, characterized by comprising the following steps:

[0057] (1) Take 200 g of fresh sea cucumbers, soak them in ultrapure water for 30 min, wash and remove sand to remove the salt and impurities in the sea cucumbers, cut them into small pieces, carry out low-temperature vacuum drying and then crush them into coarse sea cucumber powder, and put the coarse sea cucumber powder into a grinder to grind, obtaining 28.5 g of fine sea cucumber powder;

[0058] (2) Take 20 g of the fine sea cucumber powder and add it to ultrapure water, homogenize, adjust the pH to 7, add 0.2 g of papain, add EDTA to make the EDTA concentration in the reaction solution 0.2 mmol / L, and carry out enzymatic hydrolysis for 1 h under microwave assistance, with a microwave power of 360 W and an enzymatic hydrolysis temperature of 50 °C, inactivate the enzyme, obtaining sea cucumber enzymatic hydrolysate A;

[0059] (3) Adjust the pH of sea cucumber enzymatic hydrolysate A to 7.5, add 0.1 g of trypsin, add calcium propionate to make the calcium ion concentration in the reaction solution 2 mmol / L, and carry out enzymatic hydrolysis for 2 h under microwave assistance, with a microwave power of 310 W and an enzymatic hydrolysis temperature of 40 °C, inactivate the enzyme, obtaining sea cucumber enzymatic hydrolysate B;

[0060] (4) Adjust the pH of sea cucumber enzymatic hydrolysate B to 7, add 0.2 g of neutral protease, add zinc lactate to make the zinc ion concentration in the reaction solution 1 mmol / L, and carry out enzymatic hydrolysis for 1.5 h under microwave assistance, with a microwave power of 340 W and an enzymatic hydrolysis temperature of 45 °C, inactivate the enzyme, obtaining the final sea cucumber enzymatic hydrolysate;

[0061] (5) Add 2% activated carbon to the final enzymatic hydrolysate, decolorize at 70 °C for 50 min, carry out freeze centrifugation at 10000 r / min for 10 min, collect the supernatant, and carry out ultrafiltration through a 0.45 μm membrane to obtain a decolorized sea cucumber solution;

[0062] (6) Separate, purify and concentrate the decolorized sea cucumber solution through a 600 Da organic membrane. During separation, the temperature is controlled at 10 - 20 °C, the pressure is 0.25 MPa, and the flow rate is 1.6 mL / s. Collect the concentrated solution and remove the moisture through freeze-drying technology to obtain powdery small-molecular-weight sea cucumber oligopeptides.

[0063] Example 2

[0064] A method for preparing small-molecular-weight sea cucumber oligopeptides, characterized by comprising the following steps:

[0065] (1) Take 200 g of fresh sea cucumbers, soak them in ultrapure water for 30 min, wash and remove sand to remove the salt and impurities in the sea cucumbers, cut them into small pieces, carry out low-temperature vacuum drying and then crush them into coarse sea cucumber powder, and put the coarse sea cucumber powder into a grinder to grind, obtaining 28.5 g of fine sea cucumber powder;

[0066] (2) Take 15 g of fine sea cucumber powder and add it to ultrapure water, homogenize, adjust the pH to 6.5, add 0.18 g of papain, add EDTA to make the EDTA concentration in the reaction solution 0.18 mmol / L, enzymatically hydrolyze for 1 h under microwave assistance, with a microwave power of 350 W and an enzymatic hydrolysis temperature of 50 °C, inactivate the enzyme, and obtain sea cucumber enzymatic hydrolysate A;

[0067] (3) Adjust the pH of sea cucumber enzymatic hydrolysate A to 7.5, add 0.1 g of trypsin, add calcium propionate to make the calcium ion concentration in the reaction solution 2 mmol / L, enzymatically hydrolyze for 2 h under microwave assistance, with a microwave power of 315 W and an enzymatic hydrolysis temperature of 38 °C, inactivate the enzyme, and obtain sea cucumber enzymatic hydrolysate B;

[0068] (4) Adjust the pH of sea cucumber enzymatic hydrolysate B to 7.5, add 0.18 g of neutral protease, add zinc lactate to make the zinc ion concentration in the reaction solution 0.9 mmol / L, enzymatically hydrolyze for 1.5 h under microwave assistance, with a microwave power of 350 W and an enzymatic hydrolysis temperature of 50 °C, inactivate the enzyme, and obtain the final sea cucumber enzymatic hydrolysate;

[0069] (5) Add 2% activated carbon to the final enzymatic hydrolysate, decolorize at 70 °C for 50 min, centrifuge at 10000 r / min for 10 min, collect the supernatant, and ultrafilter through a 0.45 μm membrane to obtain sea cucumber decolorized solution;

[0070] (6) Separate and purify and concentrate the sea cucumber decolorized solution through a 600 Da organic membrane. During separation, the temperature is controlled at 10 - 20 °C, the pressure is 0.25 MPa, and the flow rate is 1.6 mL / s. Collect the concentrated solution and remove the moisture through freeze-drying technology to obtain powdery small molecular weight sea cucumber oligopeptides.

[0071] Example 3

[0072] A method for preparing small molecular weight sea cucumber oligopeptides, characterized by comprising the following steps:

[0073] (1) Take 200 g of fresh sea cucumbers, soak them in ultrapure water for 30 min, wash and remove sand, remove the salt and impurities in the sea cucumbers, cut them into small sections, perform low-temperature vacuum drying and then crush them into coarse sea cucumber powder, and put the coarse sea cucumber powder into a grinder for grinding to obtain 28.5 g of fine sea cucumber powder;

[0074] (2) Take 5 g of fine sea cucumber powder and add it to ultrapure water, homogenize, adjust the pH to 6, add 0.05 g of papain, add EDTA to make the EDTA concentration in the reaction solution 0.12 mmol / L, enzymatically hydrolyze for 1 h under microwave assistance, with a microwave power of 340 W and an enzymatic hydrolysis temperature of 52 °C, inactivate the enzyme, and obtain sea cucumber enzymatic hydrolysate A;

[0075] (3) Adjust the pH of the sea cucumber enzymatic hydrolysate A to 8, add 0.08 g of trypsin, add calcium propionate to make the calcium ion concentration in the reaction solution 1.1 mmol / L, and enzymatically hydrolyze for 2 h under microwave assistance. The microwave power is 312 W, the enzymatic hydrolysis temperature is 37 °C, inactivate the enzyme, and obtain the sea cucumber enzymatic hydrolysate B;

[0076] (4) Adjust the pH of the sea cucumber enzymatic hydrolysate B to 7.5, add 0.1 g of neutral protease, add zinc lactate to make the zinc ion concentration in the reaction solution 0.5 mmol / L, and enzymatically hydrolyze for 1.5 h under microwave assistance. The microwave power is 330 W, the enzymatic hydrolysis temperature is 45 °C, inactivate the enzyme, and obtain the final sea cucumber enzymatic hydrolysate;

[0077] (5) Add 2% activated carbon to the final enzymatic hydrolysate, decolorize at 70 °C for 50 min, centrifuge at 10000 r / min for 10 min, collect the supernatant, and ultrafilter through a 0.45 μm membrane to obtain the decolorized sea cucumber solution;

[0078] (6) Separate and purify and concentrate the decolorized sea cucumber solution through a 600 Da organic membrane. During separation, the temperature is controlled at 10 - 20 °C, the pressure is 0.25 MPa, and the flow rate is 1.6 mL / s. Collect the concentrated solution, and remove the moisture through freeze-drying technology to obtain powdery small molecular weight sea cucumber oligopeptides.

[0079] Example 4

[0080] A method for preparing small molecular weight sea cucumber oligopeptides, characterized by comprising the following steps:

[0081] (1) Take 200 g of fresh sea cucumbers, soak them in ultrapure water for 30 min, wash and remove sand, remove the salt and impurities in the sea cucumbers, cut them into small sections, perform low-temperature vacuum drying and then crush them into coarse sea cucumber powder. Put the coarse sea cucumber powder into a grinder and grind it to obtain 28.5 g of fine sea cucumber powder;

[0082] (2) Take 10 g of fine sea cucumber powder and add it to ultrapure water, homogenize, adjust the pH to 6.5, add 0.1 g of papain, add EDTA to make the EDTA concentration in the reaction solution 0.15 mmol / L, and enzymatically hydrolyze for 1 h under microwave assistance. The microwave power is 355 W, the enzymatic hydrolysis temperature is 53 °C, inactivate the enzyme, and obtain the sea cucumber enzymatic hydrolysate A;

[0083] (3) Adjust the pH of the sea cucumber enzymatic hydrolysate A to 8.2, add 0.08 g of trypsin, add calcium propionate to make the calcium ion concentration in the reaction solution 1 mmol / L, and enzymatically hydrolyze for 2 h under microwave assistance. The microwave power is 313 W, the enzymatic hydrolysis temperature is 38 °C, inactivate the enzyme, and obtain the sea cucumber enzymatic hydrolysate B;

[0084] (4) Adjust the pH of the sea cucumber enzymolysis solution B to 7.5, add 0.14 g of neutral protease, add zinc lactate to make the zinc ion concentration in the reaction solution 0.7 mmol / L, and carry out enzymolysis for 1.5 h under microwave assistance. The microwave power is 335 W, the enzymolysis temperature is 47 °C, inactivate the enzyme, and obtain the final sea cucumber enzymolysis solution;

[0085] (5) Add 2% activated carbon to the final enzymolysis solution, decolorize at 70 °C for 50 min, carry out refrigerated centrifugation at 10000 r / min for 10 min, collect the supernatant, and carry out ultrafiltration through a 0.45 μm membrane to obtain the decolorized sea cucumber solution;

[0086] (6) Separate, purify and concentrate the decolorized sea cucumber solution through a 600 Da organic membrane. During separation, the temperature is controlled at 10 - 20 °C, the pressure is 0.25 MPa, and the flow rate is 1.6 mL / s. Collect the concentrated solution, and remove the moisture through freeze-drying technology to obtain powdery small molecular weight sea cucumber oligopeptides.

[0087] Performance Test

[0088] 1. Molecular Weight Detection

[0089] Analyze the molecular weight distribution of the concentrated solution obtained in step (6) of Example 1 by high performance liquid chromatography (HPLC) and mass spectrometry (MS) to characterize the molecular weight distribution of the small molecular weight sea cucumber oligopeptides prepared in Example 1. The results are shown in Table 1. As can be seen from Table 1, the molecular weights of the small molecular weight sea cucumber oligopeptides prepared in the examples of the present invention are mainly concentrated in the range of 400 - 600 Da (88.31%), and have a uniform molecular weight distribution.

[0090] Table 1 Molecular Weight Distribution of Small Molecular Weight Sea Cucumber Oligopeptides

[0091] Molecular weight (Da) Molecular weight percentage (%) ＜400 6.98 400～600 88.31 ＞600 4.71

[0092] 2. Antioxidant Activity Test

[0093] Carry out DPPH (1,1-Diphenyl-2-picrylhydrazyl, abbreviated as DPPH) free radical scavenging test on the small molecular weight sea cucumber oligopeptides prepared in Example 1 at different concentrations (0.001%, 0.01% and 0.1%) to evaluate the antioxidant activity.

[0094] The specific test groups are as follows:

[0095] Blank group: Do not add any samples;

[0096] Treatment groups include: small molecular weight sea cucumber oligopeptide group with a concentration of 0.001%, small molecular weight sea cucumber oligopeptide group with a concentration of 0.01% and small molecular weight sea cucumber oligopeptide group with a concentration of 0.1%.

[0097] Take 0.1 mL of the samples in the above test groups respectively, and add 3.9 mL of DPPH solution (concentration: 6×10 -5 mol / L) respectively. Let it stand still in the dark for 2 h at room temperature, and measure its absorbance at a wavelength of 515 nm.

[0098] DPPH free radical scavenging rate = (A0 - A b ) / A0 × 100%

[0099] A0 represents the absorbance of the DPPH solution; A b represents the absorbance of the DPPH solution in different sample groups.

[0100] The test results are as Figure 2 shown. It can be seen that the small - molecular - weight sea cucumber oligopeptides prepared in the examples of the present invention have significant free radical scavenging ability and strong antioxidant activity, and can scavenge about 75% of free radicals at a content of 0.01%.

[0101] 3. Anti - aging activity test

[0102] Detect the β - galactosidase activity of fibroblasts with small - molecular - weight sea cucumber oligopeptides prepared in Example 1 at different concentrations (0.001%, 0.01% and 0.1%) to evaluate the anti - aging ability.

[0103] The test groups are specifically as follows:

[0104] Blank group: Do not add any samples;

[0105] H2O2 model group: 10 μmol / L of H2O2;

[0106] The treatment groups include: H2O2 and 0.001% oligopeptide group (the concentration of H2O2 is 10 μmol / L, and the concentration of small - molecular - weight sea cucumber oligopeptide is 0.001%), H2O2 and 0.01% oligopeptide group (the concentration of H2O2 is 10 μmol / L, and the concentration of small - molecular - weight sea cucumber oligopeptide is 0.01%), H2O2 and 0.1% oligopeptide group (the concentration of H2O2 is 10 μmol / L, and the concentration of small - molecular - weight sea cucumber oligopeptide is 0.1%).

[0107] β - galactosidase determination method: Centrifuge to collect the fibroblasts cultured for 24 h in each of the above test groups, extract proteins according to the manufacturer's instructions of the β - galactosidase detection kit (ONPG method), and sequentially add ONPG, deionized water, solution B (buffer) and solution C (termination solution) to the proteins extracted from different test groups according to the instructions. Finally, detect with a spectrophotometer and calculate the activity.

[0108] The test results are as follows Figure 3 As shown, it can be seen that the small molecular weight sea cucumber oligopeptide prepared in Example 1 has a good inhibitory effect on the activity of β-galactosidase at a content of 0.01%, indicating that the small molecular weight sea cucumber oligopeptide provided in the embodiments of the present invention has strong anti-aging activity.

[0109] 4. Anti-inflammatory activity test

[0110] The small molecular weight sea cucumber oligopeptide prepared in Example 1 at different concentrations (0.001%, 0.01% and 0.1%) was used to detect the transcriptional level of inflammatory factor genes based on fibroblasts to evaluate the anti-inflammatory ability.

[0111] The specific test groups are as follows

[0112] Blank group: without adding any samples

[0113] LPS model group: 10 μg / mL of LPS

[0114] The drug treatment groups include: LPS and 0.001% oligopeptide group (the concentration of LPS is 10 μg / mL and the concentration of small molecular weight sea cucumber oligopeptide is 0.001%), LPS and 0.01% oligopeptide group (the concentration of LPS is 10 μg / mL and the concentration of small molecular weight sea cucumber oligopeptide is 0.01%), LPS and 0.1% oligopeptide group (the concentration of LPS is 10 μg / mL and the concentration of small molecular weight sea cucumber oligopeptide is 0.1%).

[0115] For the detection of the transcriptional level of inflammatory factor genes, primers for qPCR were designed using Primer Blast software, and all primers followed the primer design principles. Fibroblasts exposed to the above test groups for 24 h were collected by centrifugation for RNA extraction to obtain total RNA; the obtained RNA was reverse transcribed to obtain cDNA; real-time fluorescence quantitative PCR was performed according to the manufacturer's instructions, and the relative expression levels of pro-inflammatory factor (il-6) genes were statistically analyzed using the 2 －ΔΔct method.

[0116] The test results are as follows Figure 4 As shown, it can be seen that the small molecular weight sea cucumber oligopeptide prepared in Example 1 can significantly inhibit the level of pro-inflammatory factor (il-6) genes at a content of 0.01%, indicating that the small molecular weight sea cucumber oligopeptide provided in the embodiments of the present invention has strong anti-inflammatory activity.

[0117] II. Specific implementation examples of the application of small molecular weight sea cucumber oligopeptides

[0118] Example 5

[0119] A skin care composition, comprising small molecular weight sea cucumber oligopeptides obtained by preparation of Example 1 and green tea extract obtained by ethanol extraction method, and the mass ratio of the small molecular weight sea cucumber oligopeptides to the green tea extract is 1:1.

[0120] The preparation method of the above skin care composition comprises the following steps:

[0121] (1) Mix the small molecular weight sea cucumber oligopeptide powder and the green tea extract at a mass ratio of 1:1 in ultrapure water. The concentrations of both the small molecular weight sea cucumber oligopeptides and the green tea extract are 10% (w / v). Place them in an ultrasonic instrument and ultrasonicate at 30 °C for 60 min to ensure that the small molecular weight sea cucumber oligopeptide powder and the green tea extract are fully combined;

[0122] (2) Take out the complex liquid and quickly cool it to room temperature, centrifuge to remove residues, and pass through a 600 Da ultrafiltration membrane;

[0123] (3) Freeze-dry the ultrafiltered complex liquid to obtain a complex of small molecular weight sea cucumber oligopeptides and green tea extract.

[0124] Perform a detection test on the type I collagen level of fibroblasts for the small molecular weight sea cucumber oligopeptides prepared in Example 1, green tea extract, and the complex of small molecular weight sea cucumber oligopeptides and green tea extract prepared in Example 5 at the same concentration to evaluate the effect of promoting collagen synthesis.

[0125] The specific test groups are as follows:

[0126] Blank group: Without adding any samples;

[0127] The treatment groups include: a small molecular weight sea cucumber oligopeptide group with a concentration of 0.01%, a green tea extract group with a concentration of 0.01%, and a complex group with a concentration of 0.01%.

[0128] Centrifuge and collect fibroblasts cultured for 24 h in each of the above test groups, and detect the content of type I collagen according to the manufacturer's instructions of the type I collagen ELISA detection kit. The specific method is as follows: Take out the enzyme-labeled plate, add the washing solution diluted to 1-fold concentration (350 μL / well) and soak, discard the liquid in the wells after standing for 30 s, and pat dry the enzyme-labeled plate; Add 100 μL of the standard product and the sample to be tested to each well, and incubate at room temperature for 2 h; Discard the liquid in the wells, add the detection antibody, and incubate at room temperature for 2 h; Discard the liquid in the wells again, add the enzyme conjugate, and incubate at room temperature for 20 min; After the incubation is completed, discard the liquid in the wells, add the substrate solution, and incubate at room temperature in the dark for 20 min; Finally, add the stop solution and perform absorbance detection.

[0129] The detection results of the type I collagen content are as Figure 5As shown, it can be seen that the complex of small - molecular - weight sea cucumber oligopeptides and green tea extract provided in Example 5 exhibits a relatively high effect on promoting collagen synthesis after 24 hours of use, and the effect is better than that of using single small - molecular - weight sea cucumber oligopeptides or green tea extract.

[0130] Example 6

[0131] An anti - aging cream, comprising the following components by mass percentage: 0.1% of the small - molecular - weight sea cucumber oligopeptides prepared in Example 1 (with high permeability and biological activity), 5% of glycerol (as the main moisturizer, having excellent hydration and helping to maintain skin moisture balance), 0.5% of hyaluronic acid (having strong moisturizing ability, capable of helping to lock in skin moisture and increase skin elasticity), 2% of squalene (while providing a sense of comfort, having antioxidant and skin - barrier - repairing effects), 15% of polysorbate - 80 (as an emulsifier), an appropriate amount of phenoxyethanol (as a preservative), an appropriate amount of xanthan gum (as a thickener, and its dosage is adjusted according to the target viscosity state), an appropriate amount of citric acid (as a pH regulator, and its dosage is determined according to the target pH value), and the balance being deionized water.

[0132] The preparation method of the above - mentioned anti - aging cream comprises the following steps:

[0133] (1) Raw material preparation: Weigh each component according to the dosage of each component in the above - mentioned anti - aging cream and set aside.

[0134] (2) Aqueous phase preparation: Add glycerol and hyaluronic acid to deionized water, stir with a stirrer at low speed until all components are completely dissolved; add an appropriate amount of xanthan gum and continue stirring until the solution becomes uniform and slightly viscous; slowly add citric acid under stirring to adjust the pH to 5.5 to ensure its suitability for skin use.

[0135] (3) Oil phase preparation: Add polysorbate - 80 and squalene to another container, stir and heat to 60 °C until polysorbate - 80 and squalene are completely dissolved to form a transparent and uniform oil phase.

[0136] (4) Emulsification: Slowly add the prepared aqueous phase to the oil phase and perform high - efficiency emulsification using an emulsifier; during the emulsification process, maintain a constant stirring speed to ensure complete fusion of the aqueous phase and the oil phase to form a stable emulsion.

[0137] (5) Adding small - molecular - weight sea cucumber oligopeptides: After emulsification is completed, slowly add small - molecular - weight sea cucumber oligopeptides to the emulsion and stir with a high - shear stirrer for 15 min to ensure its complete dissolution and uniform dispersion in the emulsion.

[0138] (6) Cooling, adding preservatives and pH adjustment: After cooling the mixed emulsion to room temperature, add phenoxyethanol and adjust the pH value to 5.5 again to ensure that the face cream has a skin-friendly acidity and alkalinity, obtaining an anti-aging face cream;

[0139] (7) Inspection and packaging:

[0140] Product inspection: Use sensory tests (such as the texture of the cream and the absorption rate) to further adjust the comfort of the cream, ensuring that it is easy to apply, absorbs quickly and is not greasy;

[0141] Packaging and storage: Dispense the prepared anti-aging face cream into suitable containers and store it sealed.

[0142] Example 7

[0143] A facial cleanser, comprising the following components by mass percentage: 0.001% of the low-molecular-weight sea cucumber oligopeptide prepared in Example 1 (which has good hydration and skin repair functions), 3% of glycerol (as the main moisturizer to help maintain the skin's moisture balance), 0.5% of hyaluronic acid (to enhance skin moisturization and prevent water loss during the cleaning process), 1% of amino acid derivatives (such as glycine and glutamic acid, which have moisturizing and soothing effects and also help reduce the sense of skin dryness that may be caused during the cleaning process), 2% of laureth-7 (as a surfactant, which can gently remove the oil and dirt on the skin surface without irritating the skin), an appropriate amount of phenoxyethanol (as a preservative to ensure the safety and long-term use of the product), an appropriate amount of citric acid (as a pH regulator, and its dosage is determined according to the target pH value), and the balance is deionized water.

[0144] The preparation method of the above-mentioned facial cleanser comprises the following steps:

[0145] (1) Raw material preparation: Weigh each component according to the dosage of each component in the above-mentioned facial cleanser and set aside;

[0146] (2) Aqueous phase preparation: Heat the deionized water to 60 °C, add glycerol, amino acid derivatives and hyaluronic acid, and stir evenly until completely dissolved;

[0147] (3) Oil phase preparation: Add laureth-7 to another container and heat it to 60 °C until it is completely dissolved to form a clear oil phase;

[0148] (4) Emulsification: Slowly add the prepared oil phase to the aqueous phase and use an emulsifier for efficient emulsification; during the emulsification process, it is necessary to maintain a stable temperature and an appropriate stirring speed to ensure the formation of a stable emulsion;

[0149] (5) Adding low-molecular-weight sea cucumber oligopeptide: After emulsification is completed, slowly add the low-molecular-weight sea cucumber oligopeptide to the emulsion to ensure its uniform dispersion in the emulsion;

[0150] (6) Cooling, adding preservatives and pH adjustment: After cooling the mixed emulsion to room temperature, add phenoxyethanol, and use citric acid to adjust the pH value to 5.5 to ensure its suitability for skin use, obtaining a facial cleanser;

[0151] (7) Packaging: Fill the prepared facial cleanser into a bottle and seal it for storage.

[0152] Example 8

[0153] A sunscreen, comprising the following components by mass percentage: 0.01% of the low-molecular-weight sea cucumber oligopeptide prepared in Example 1 (having antioxidant effects and enhancing skin barrier function), 3% of glycerol (for moisturizing and helping to maintain skin hydration), 0.5% of hyaluronic acid (enhancing skin moisturizing function and avoiding dryness caused by ultraviolet radiation), 6% of titanium dioxide (as a physical sunscreen, capable of effectively blocking the damage of ultraviolet rays to the skin), 2% of polysorbate-60 (as an emulsifier to help stabilize the emulsion system of the sunscreen), an appropriate amount of phenoxyethanol (as a preservative for preventing product contamination), an appropriate amount of citric acid (as a pH regulator, and its dosage is determined according to the target pH value), and the balance being deionized water.

[0154] The preparation method of the above-mentioned sunscreen comprises the following steps:

[0155] (1) Raw material preparation: Weigh each component according to the dosage of each component in the above-mentioned sunscreen and set aside;

[0156] (2) Aqueous phase preparation: Heat deionized water to 60°C, add glycerol and hyaluronic acid, and stir evenly until completely dissolved;

[0157] (3) Oil phase preparation: Add polysorbate-60 to another container, heat to 60°C until it is completely dissolved to form a clear oil phase;

[0158] (4) Emulsification: Slowly add the prepared oil phase to the aqueous phase, and use an emulsifier for efficient emulsification until a stable emulsion is formed;

[0159] (5) Adding sunscreen components: After emulsification is completed, disperse titanium dioxide in the emulsion and continuously stir to ensure its uniform distribution;

[0160] (6) Adding low-molecular-weight sea cucumber oligopeptide: Slowly add the low-molecular-weight sea cucumber oligopeptide to the emulsion and stir evenly;

[0161] (7) Cooling, adding preservatives and pH adjustment: After cooling the mixed emulsion to room temperature, add phenoxyethanol, and use citric acid to adjust the pH value to 5.5 to ensure its suitability for skin use, obtaining a sunscreen;

[0162] (8) Packaging: Fill the prepared sunscreen into a bottle and store it sealed.

[0163] Example 9

[0164] An anti-aging essence, comprising the following components by mass percentage: 0.05% of the low-molecular-weight sea cucumber oligopeptide prepared in Example 1 (which has the effect of enhancing skin elasticity and promoting collagen synthesis), 4% glycerol (for moisturizing and helping to maintain skin hydration), 0.3% hyaluronic acid (to improve skin hydration and skin elasticity), 0.5% vitamin C (as a powerful antioxidant, helping to inhibit free radical generation and slow down skin aging), 2% acetyl hexapeptide-8 (which has the effect of inhibiting muscle contraction and helps to reduce the appearance of fine lines and wrinkles), 2% plant essence extract (such as rhodiola and ginkgo extract, which have anti-inflammatory and antioxidant effects and help to relieve skin aging), an appropriate amount of phenoxyethanol (as a preservative for extending the shelf life of the product), an appropriate amount of citric acid (as a pH regulator, and its dosage is determined according to the target pH value), and the balance being deionized water.

[0165] The preparation method of the above anti-aging essence comprises the following steps:

[0166] (1) Raw material preparation: Weigh each component according to the dosage of each component in the above anti-aging essence and set aside.

[0167] (2) Aqueous phase preparation: Heat the deionized water to 50 °C, add glycerol, hyaluronic acid and plant essence extract, and stir evenly until completely dissolved.

[0168] (3) Oil phase preparation: Add acetyl hexapeptide-8 to another container, heat to 50 °C until it is completely dissolved to form a clear oil phase.

[0169] (4) Emulsification: Slowly add the prepared oil phase to the aqueous phase and perform high-efficiency emulsification using an emulsifier until a stable emulsion is formed.

[0170] (5) Adding low-molecular-weight sea cucumber oligopeptide: After emulsification is completed, cool the emulsion to room temperature, slowly add the low-molecular-weight sea cucumber oligopeptide to the emulsion, and stir evenly to ensure its uniform dispersion.

[0171] (6) Adding vitamin C: Dissolve vitamin C in a small amount of the aqueous phase and add it to the emulsion in step (5), and pay attention to using citric acid to control the pH to avoid degradation of vitamin C.

[0172] (7) Cooling, adding preservative and pH adjustment: After cooling the mixed emulsion to room temperature, add phenoxyethanol, and adjust the pH value to 5.5 using citric acid to ensure its suitability for skin use, thus obtaining the anti-aging essence.

[0173] (8) Packaging: Pour the prepared anti-aging essence into a dropper bottle, and then carry out packaging and store it sealed.

[0174] Example 10

[0175] A moisturizing night cream, comprising the following components by mass percentage: 0.025% of the small molecular weight sea cucumber oligopeptide prepared in Example 1 (which has the effect of deep nourishing and repairing the skin), 3% of glycerol (as a humectant to maintain the hydration of the skin), 5% of shea butter (as a moisturizing ingredient with strong moisturizing and repairing functions), 3% of jojoba oil (as a skin care oil that can nourish the skin and soften the cutin), 0.2% of vitamin E (as an antioxidant to relieve skin aging and promote repair), 1% of witch hazel extract (which has a soothing effect and is suitable for night use), an appropriate amount of phenoxyethanol (as a preservative to prevent bacterial contamination of the product), an appropriate amount of citric acid (as a pH regulator, and its dosage is determined according to the target pH value), and the balance is deionized water.

[0176] The preparation method of the above-mentioned moisturizing night cream includes the following steps:

[0177] (1) Raw material preparation: Weigh each component according to the dosage of each component in the above-mentioned moisturizing night cream and set aside;

[0178] (2) Aqueous phase preparation: Heat the deionized water to 50 °C, add glycerol, and stir evenly;

[0179] (3) Oil phase preparation: Add shea butter and jojoba oil to another container, heat to 50 °C until it is completely dissolved to form a transparent oil phase;

[0180] (4) Emulsification: Slowly add the prepared oil phase to the aqueous phase, and use an emulsifier for efficient emulsification until a stable emulsion is formed;

[0181] (5) Add small molecular weight sea cucumber oligopeptide: After emulsification is completed, cool the emulsion to 45 °C, and slowly add the small molecular weight sea cucumber oligopeptide to the emulsion and stir evenly;

[0182] (6) Add vitamin E and witch hazel extract: After the emulsion is completely cooled to room temperature, add vitamin E and witch hazel extract;

[0183] (7) Add preservative and adjust pH: Add phenoxyethanol, and use citric acid to adjust the pH value to 5.5 to ensure that it is suitable for skin use, and obtain the moisturizing night cream;

[0184] (8) Packaging: Pour the prepared moisturizing night cream into a bottle, seal it and store it in a cool and dry place.

[0185] Under normal circumstances, the human body has a certain degree of inflammation, that is, physiological inflammation that maintains the balance of the internal environment. The human body can regulate through the protective ability of the skin barrier to prevent skin damage. As people age, the balance between skin regulation and damage is broken, gradually becoming a state where damage is greater than regulation. The damaged cells begin to release a large number of inflammatory factors, which cause the overall skin environment to become inflamed, further leading to the extracellular matrix that maintains skin elasticity and appearance changing from an orderly arrangement to a disorderly one. Such a situation will further amplify the inflammatory state, forming an intertwined vicious cycle, while inhibiting the formation of collagen and making the skin loose and wrinkled. In addition, oxidative stress caused by free radicals can increase the production of cytokines through different mechanisms, thus further stimulating the activation of inflammatory cells. Therefore, a vicious cycle of spiral upward is formed between oxidative stress and inflammatory responses. Thus, inhibiting excessive inflammatory responses and scavenging free radicals are of great significance for alleviating skin aging.

[0186] The small-molecular-weight sea cucumber oligopeptide prepared by the preparation method of the small-molecular-weight sea cucumber oligopeptide provided in the embodiment of the present invention can significantly scavenge free radicals and has strong antioxidant activity; and can significantly inhibit the activity of β-galactosidase in fibroblasts and significantly inhibit the expression level of inflammatory factor (IL-6) in fibroblasts; further adding green tea extract to synergistically enhance the effect with the sea cucumber oligopeptide can also promote the synthesis of type I collagen in fibroblasts. It can be seen from this that the small-molecular-weight sea cucumber oligopeptide and its application provided in the embodiment of the present invention have good prospects in inhibiting skin inflammatory responses and alleviating skin aging.

Claims

1. A preparation method of small molecular weight sea cucumber oligopeptides, characterized in that, It includes the following steps: (1) Cut sea cucumbers into small pieces, dry them under low-temperature vacuum and then crush them to obtain fine sea cucumber powder; (2) Add the fine sea cucumber powder to ultrapure water, homogenize it, add papain and EDTA, enzymatically hydrolyze it under microwave assistance, and inactivate the enzyme to obtain sea cucumber enzymatic hydrolysate A; wherein, the mass ratio of the fine sea cucumber powder to papain is (5-20):(0.05-0.2), the concentration of EDTA is 0.15-0.2 mmol / L, the microwave power is 340-360 W, and the enzymatic hydrolysis temperature is 50-53 °C; (3) Add trypsin and calcium ions to the sea cucumber enzymatic hydrolysate A, enzymatically hydrolyze it under microwave assistance, and inactivate the enzyme to obtain sea cucumber enzymatic hydrolysate B; wherein, the mass ratio of trypsin to the fine sea cucumber powder added in step (2) is (0.08-0.1):(5-20), the concentration of calcium ions is 1-2 mmol / L, the microwave power is 310-315 W, and the enzymatic hydrolysis temperature is 37-40 °C; (4) Add neutral protease and zinc ions to the sea cucumber enzymatic hydrolysate B, enzymatically hydrolyze it under microwave assistance, and inactivate the enzyme to obtain the final sea cucumber enzymatic hydrolysate; wherein, the mass ratio of neutral protease to the fine sea cucumber powder added in step (2) is (0.1-0.2):(5-20), the concentration of zinc ions is 0.5-1 mmol / L, the microwave power is 330-350 W, and the enzymatic hydrolysis temperature is 45-50 °C; (5) Add activated carbon to the final enzymatic hydrolysate for decolorization, and perform ultrafiltration to obtain decolorized sea cucumber solution; (6) Separate, purify and concentrate the decolorized sea cucumber solution through an organic membrane system, collect the concentrated solution, and freeze-dry it to obtain powdery small-molecular-weight sea cucumber oligopeptides.

2. The preparation method of small molecular weight sea cucumber oligopeptide according to claim 1, wherein In step (2), the enzymatic hydrolysis pH is 6-7; in step (3), the enzymatic hydrolysis pH is 7.5-8.2; in step (4), the enzymatic hydrolysis pH is 7-7.

5.

3. The preparation method of small molecular weight sea cucumber oligopeptides according to claim 1, characterized in that, In step (2), the enzymatic hydrolysis time is 1 h; in step (3), the enzymatic hydrolysis time is 2 h; in step (4), the enzymatic hydrolysis time is 1.5 h.

4. The preparation method of small molecular weight sea cucumber oligopeptide according to claim 1, wherein In step (3), calcium propionate is added to obtain calcium ions; in step (4), zinc lactate is added to obtain zinc ions.

5. The preparation method of small molecular weight sea cucumber oligopeptide according to claim 1, characterized in that, In step (5), the addition amount of activated carbon is 2%, the decolorization temperature is 70 °C, and the decolorization time is 50 min.

6. The preparation method of small molecular weight sea cucumber oligopeptides according to claim 1, wherein, In step (5), after decolorization, perform freeze centrifugation, and ultrafilter the supernatant collected by centrifugation. The centrifugation speed is 10000 r / min, the centrifugation time is 10 min, and ultrafiltration is performed using a 0.45 μm membrane.

7. The preparation method of small molecular weight sea cucumber oligopeptide according to claim 1, characterized in that, In step (6), an organic membrane with a cut-off molecular weight of 600 Da is used for separation and purification. During separation, the temperature is 10-20 °C, the pressure is 0.25 MPa, and the flow rate is 1.6 mL / s.

8. A small-molecular-weight sea cucumber oligopeptide, characterized in that, It is prepared by using the preparation method of small-molecular-weight sea cucumber oligopeptides described in any one of claims 1-7.

9. A skin care composition, characterized in that, It includes the small-molecular-weight sea cucumber oligopeptides described in claim 8 and green tea extract, and the mass ratio of the small-molecular-weight sea cucumber oligopeptides to the green tea extract is 1:

1.

10. Use of the small-molecular-weight sea cucumber oligopeptides described in claim 8 in the preparation of skin care products.

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