Sea cucumber peptide, preparation method and application thereof

Abstract

The invention discloses a sea cucumber peptide, a preparation method and an application thereof, and belongs to the field of biotechnology. The sea cucumber peptide has an anti-photoaging effect and is composed of any one or two of the following polypeptides: the amino acid sequence of polypeptide one is SPQTPGSP, and the amino acid sequence of polypeptide two is DLVVVK. Studies have shown that the sea cucumber peptide has high activity, good biocompatibility, safety and no toxic side effects. The sea cucumber peptide has good anti-photoaging activity and can be used to prepare anti-photoaging products, especially for cosmetics.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a sea cucumber peptide, a preparation method and an application thereof. Background Art

[0002] Skin is the largest organ in the human body. With the increase of age and changes in the environment, skin aging problems are increasing. Generally, there are two forms of skin aging: natural aging and exogenous aging. Among them, exogenous aging can be prevented artificially, mainly due to environmental factors such as ultraviolet (UV) radiation, smoking, physical and chemical pollution, etc. Skin aging caused by photoaging. Studies have shown that 80% of skin aging is caused by photoaging, especially excessive exposure to ultraviolet B (UVB). Skin photoaging is mainly manifested as dryness and roughness, reduced elasticity, and coarse and deep wrinkles.

[0003] Collagen is the most abundant structural protein in the skin of humans and other mammals, of which type I collagen accounts for the largest proportion. When the skin is exposed to ultraviolet rays for a long time, a large number of reactive oxygen free radicals will be generated, inducing the degradation of collagen. A large amount of collagen in the human body will be lost, causing the skin to lose elasticity, become loose, dry and rough, etc. Photoaging not only gives people an aging appearance, but also causes photorelated skin diseases. It is closely related to the occurrence and development of a variety of photorelated inflammatory and immune skin diseases. With the improvement of people's living standards and the demand for skin rejuvenation, the prevention and treatment of photoaging are receiving more and more attention.

[0004] At present, there are various methods for anti-photoaging, mainly antioxidants, physical barriers and cell repair. Although these methods provide multi-level protection for delaying photoaging, long-term use of antioxidants or chemical sunscreens may cause skin irritation or allergic reactions, increase skin sensitivity, and cause harm to the skin; while the research or application of cell repair technology is expensive, involves high-end equipment, and is difficult to promote on a large scale. Therefore, it is necessary to develop a more natural and safe anti-photoaging product that can not only meet market demand, but also provide important support for improving people's quality of life.

[0005] Sea cucumbers are echinoderms and contain a wealth of active substances, including active polysaccharides, saponins and collagen. The collagen contained in them has been proven to improve and reduce skin aging damage, but collagen has a stable structure and a large molecular weight (300kDa), and the human body has a very low direct absorption and utilization rate. Therefore, it is necessary to prepare sea cucumber peptides with a smaller molecular weight so that they can be better absorbed and utilized by the skin.

[0006] However, the composition of the polypeptide products after enzymatic hydrolysis of sea cucumber proteins is very complex, including a large number of polypeptides with unknown sequences and unknown functions. At present, it is known that sea cucumber peptides have the functions of enhancing immunity, moisturizing, lowering uric acid, and anti-oxidation, but most of them are mixed peptides, and the specific polypeptide sequence has not been truly identified, and there is no report on the amino acid sequence composition of sea cucumber peptides with anti-photoaging. Summary of the invention

[0007] In view of the deficiencies in the prior art and actual needs, the present invention provides a sea cucumber peptide, a preparation method and an application thereof, thereby achieving efficient and full utilization of sea cucumbers.

[0008] The object of the present invention is achieved through the following technical solutions:

[0009] A sea cucumber peptide has an anti-photoaging effect and is composed of any one or two of the following polypeptides: the amino acid sequence of polypeptide one is SPQTPGSP, and the amino acid sequence of polypeptide two is DLVVVK.

[0010] The present invention also provides a method for preparing the above-mentioned sea cucumber peptide, comprising the following steps:

[0011] S1, after the sea cucumber is soaked, put it into a homogenizer, add purified water twice the mass of the sea cucumber, 6000-8000 rpm, homogenize for 5 minutes to obtain a sea cucumber homogenate;

[0012] S2, adding protease to the sea cucumber homogenate for enzymolysis, and inactivating the enzyme after the enzymolysis is completed;

[0013] S3, using a centrifuge and an ultrafiltration membrane to filter the sea cucumber peptide hydrolysate in turn, and retaining the hydrolysate of the required molecular weight;

[0014] S4, using RP-HPLC reverse phase high performance liquid chromatography for separation and purification, and separating and obtaining sea cucumber peptides under C18 column reverse phase high performance liquid chromatography, acetonitrile mobile phase, and 210 nm wavelength;

[0015] S5. Concentrating and freeze-drying the separated and purified eluate to obtain the anti-photoaging active sea cucumber peptide.

[0016] Preferably, the specific process of step S2 is: adding 0.5%-0.7% by weight of flavor protease and 0.1%-0.2% by weight of aminopeptidase to the sea cucumber homogenate, stirring and enzymolyzing for 4-5 hours at a pH of 7.3-7.6 and a temperature of 50-55°C.

[0017] Preferably, in step S3, the sea cucumber peptide hydrolysate is filtered through an ultrafiltration membrane to retain the retentate of the desired molecular weight, the speed of the centrifuge is 12000-14000 rpm / min, the time is 5-10 min, the ultrafiltration membrane uses an ultrafiltration membrane with a pore size of 1000 Daltons, and the collected retentate has a molecular weight of less than 1000.

[0018] The present invention also provides an application of a sea cucumber peptide, that is, an application of the sea cucumber peptide in the preparation of an anti-photoaging product.

[0019] Preferably, the anti-photoaging products include essence and sunscreen.

[0020] The present invention also provides an anti-photoaging essence, comprising the following components by mass: 1-2 parts of the above-mentioned sea cucumber peptide, 1-3 parts of oyster polypeptide, 1-2 parts of vitamin C, 1-2 parts of niacinamide, 0.1-0.2 parts of aloe vera, 0.1-0.2 parts of grape seed extract and 90-98 parts of water.

[0021] The present invention also provides an anti-photoaging sunscreen lotion, comprising the following components in parts by mass: 1-2 parts of the above-mentioned sea cucumber peptide, 2-4 parts of collagen peptide, 1.5-2.5 parts of natural moisturizing factor, 0.1-0.3 parts of matrine, 0.1-0.3 parts of taurine, 0.05-0.2 parts of North American witch hazel extract, 0.05-0.1 parts of retinol (A alcohol) and 90-96 parts of water.

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

[0023] The present invention prepares an anti-photoaging sea cucumber peptide by enzymatic hydrolysis of sea cucumbers, and the sea cucumber peptide is composed of any one or two of the following polypeptides: the amino acid sequence of polypeptide one is SPQTPGSP, and the amino acid sequence of polypeptide two is DLVVVK. Studies have shown that the sea cucumber peptide has high activity, good biocompatibility, safety and no toxic side effects. The sea cucumber peptide has good anti-photoaging activity and can be used to prepare anti-photoaging products, especially for cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a mass spectrum of the polypeptide of the present invention;

[0025] Figure 2 This is the mass spectrum of the polypeptide of the present invention;

[0026] Figure 3 is the moisture content of mouse skin, where A is polypeptide group 1 and B is polypeptide group 2;

[0027] Figure 4 is the hyaluronic acid content of mouse skin, where A is polypeptide group 1 and B is polypeptide group 2;

[0028] Figure 5 is the hydroxyproline content of mouse skin, where A is polypeptide group 1 and B is polypeptide group 2;

[0029] Figure 6 is the content of type I collagen in mouse skin, where A is polypeptide group 1 and B is polypeptide group 2;

[0030] Figure 7 is the content of type III collagen in mouse skin, where A is polypeptide group 1 and B is polypeptide group 2;

[0031] Figure 8 is the content of superoxide dismutase (SOD) in mouse skin, where A is polypeptide group 1 and B is polypeptide group 2;

[0032] Fig. 9 is the catalase (CAT) content in mouse skin, where A is polypeptide group 1 and B is polypeptide group 2;

[0033] Fig.10 is the content of glutathione peroxidase (GSH-Px) in mouse skin, where A is polypeptide group 1 and B is polypeptide group 2;

[0034] Fig.11 is the malondialdehyde (MDA) content of mouse skin, where A is polypeptide group 1 and B is polypeptide group 2;

[0035] Fig.12 These are the results of mouse skin tissue morphology, where A is the negative control group, B is the model group, C is the low-dose group of polypeptide 1, D is the high-dose group of polypeptide 1, E is the low-dose group of polypeptide 2, and F is the high-dose group of polypeptide 2. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0037] Example 1

[0038] A sea cucumber peptide has an anti-photoaging effect and is composed of any one or two of the following polypeptides: the amino acid sequence of polypeptide one is SPQTPGSP, see sequence list SEQ ID NO: 1, and the amino acid sequence of polypeptide two is DLVVVK, see sequence list SEQ ID NO: 2.

[0039] Example 2

[0040] A method for preparing the sea cucumber peptide described in the embodiment comprises the following steps:

[0041] S1, after the sea cucumber is soaked, it is put into a homogenizer, and purified water twice the mass of the sea cucumber is added, and the homogenization speed is 6000-8000 rpm for 5 minutes to obtain the sea cucumber homogenate. The homogenization speed of this embodiment is 6500 rpm, and any speed in 6000-8000 rpm can be selected in practical applications to achieve the purpose.

[0042] S2, add 0.5%-0.7% flavor protease and 0.1%-0.2% aminopeptidase by mass percentage to the sea cucumber homogenate, stir and hydrolyze for 4-5 hours at pH 7.3-7.6 and temperature 50-55°C, and inactivate the enzyme after the hydrolysis is completed. The specific parameters of this embodiment are: add 0.6% flavor protease and 0.1% aminopeptidase by mass percentage to the sea cucumber homogenate, stir and hydrolyze for 4 hours at pH 7.4 and temperature 50°C. In practical applications, "add 0.5%-0.7% flavor protease and 0.1%-0.2% aminopeptidase by mass percentage to the sea cucumber homogenate, stir and hydrolyze for 4-5 hours at pH 7.3-7.6 and temperature 50-55°C," and any parameter within the parameter range can achieve the purpose.

[0043] S3, the sea cucumber peptide hydrolysate is filtered by ultrafiltration membrane, and in the step of intercepting the retentate of the desired molecular weight, the speed of the centrifuge is 12000-14000rpm / min, the time is 5-10min, the ultrafiltration membrane uses an ultrafiltration membrane with a pore size of 1000 Daltons, and the collected retentate is a retentate with a molecular weight less than 1000. The specific parameters of this embodiment are: the speed of the centrifuge is 12000rpm / min, the time is 5min, and in actual applications, "the speed of the centrifuge is 12000-14000rpm / min, and the time is 5-10min," and any parameters within the parameter range can achieve the purpose.

[0044] S4, using RP-HPLC reverse phase high performance liquid chromatography for separation and purification, and separating and obtaining sea cucumber peptides under C18 column reverse phase high performance liquid chromatography, acetonitrile mobile phase, and 210 nm wavelength;

[0045] S5) The separated and purified eluate is concentrated and freeze-dried to obtain the anti-photoaging active sea cucumber peptide.

[0046] The mass spectrum of peptide 1 is shown in Figure 1 The mass spectrum of peptide 2 is shown in Figure 2 .

[0047] Conclusion: The present method obtained polypeptide 1 with the amino acid sequence of SPQTPGSP, see sequence listing SEQ ID NO: 1, and polypeptide 2 with the amino acid sequence of DLVVVK, see sequence listing SEQ ID NO: 2.

[0048] Example 3

[0049] Evaluation of anti-photoaging activity

[0050] 80 healthy adult SPF female mice weighing 24-28g were selected and divided into 8 groups, namely negative control group, model group, and experimental group. The experimental groups included low-dose group of polypeptide 1, medium-dose group of polypeptide 1, high-dose group of polypeptide 1, low-dose group of polypeptide 2, medium-dose group of polypeptide 2, and high-dose group of polypeptide 2. The negative control group was not irradiated with ultraviolet rays. The model group and the experimental group were irradiated with 1 40W UVB lamp 308nm and 2 UVA lamps 340nm. The irradiation height was 30cm, and the irradiation was performed every other day. Each time for 1h in the 3rd to 6th week; each time for 2h in the 7th to 10th week, and the irradiation lasted for 8 weeks. The mice in each experimental group were smeared for 2 weeks at the beginning of the experiment, and hair removal (6% sodium sulfide alcohol solution, hair removal area of ​​2cm×2cm) was started in the third week, and ultraviolet rays were irradiated 3 times a week, and smeared once 30 minutes before irradiation until the end of the experiment. The concentration of the low-dose group was 1% peptide solution, the concentration of the medium-dose group was 2% peptide solution, and the concentration of the high-dose group was 4% peptide solution. 0.5MED / time in the first week, 1.0MED / time in the second week, and the dose was increased by 0.5MED / time per week. From the fourth week, the irradiation dose was maintained at 2MED / time until the end of the eighth week. After the mice were fed for 10 weeks, they were fasted for 12 hours and then killed by cervical dislocation. The skin tissue blocks were taken from the back after hair removal. The moisture content, hyaluronic acid, and hydroxyproline content of the mouse skin were determined using a detection kit. The content of type I collagen and type III collagen in the skin was determined. The activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) content in the skin tissue were determined. The results are shown in Figure 3-Figure 11 .

[0051] Depend on Figure 3-Figure 11It can be seen that compared with the negative control group, the water content, hydroxyproline, hyaluronic acid, type I collagen and type III collagen content of the model group mice skin were significantly reduced (P < 0.05 or P < 0.01); the activities of superoxide dismutase, catalase and glutathione peroxidase were significantly reduced (P < 0.01), while the malondialdehyde content was significantly increased (P < 0.01). Compared with the model group, both polypeptide 1 and polypeptide 2 can significantly increase the water content, hydroxyproline, hyaluronic acid, type I collagen and type III collagen content in the skin of photoaging model mice (P < 0.05 or P < 0.01), and can significantly increase the activities of superoxide dismutase, catalase and glutathione peroxidase (P < 0.05 or P < 0.01), and significantly reduce the malondialdehyde content in the skin (P < 0.01). The results show that both polypeptide 1 and polypeptide 2 can significantly improve the skin condition of photoaging model mice, and both have anti-photoaging effects.

[0052] Example 4

[0053] General morphological changes of skin tissue

[0054] The mouse skin tissue of Example 3 was taken for general morphological observation, and the results are shown in Fig.12 .

[0055] Conclusion: The skin tissue structure of mice in the negative control group was intact, the skin tissue stratification was clear, the epidermis was of normal thickness, and the fibrous tissue of the dermis was wavy and orderly arranged; the skin tissue layer of mice in the model group showed obvious local thickening of the epidermis, uneven thickness, and the collagen fibers were clumped, with obvious fractures, and the skin stratification was not obvious; compared with the model group, the skin structure of the high and low dose groups of the sea cucumber peptide in the test group was intact, and although the collagen fibers were partially fractured, there was a significant improvement. Therefore, the polypeptide 1 and polypeptide 2 of the present invention have the function of resisting photoaging.

[0056] Example 5

[0057] An anti-photoaging essence comprises the following components by mass: 1-2 parts of the sea cucumber peptide described in Example 1, 1-3 parts of oyster polypeptide, 1-2 parts of vitamin C, 1-2 parts of niacinamide, 0.1-0.2 parts of aloe vera, 0.1-0.2 parts of grape seed extract and 90-98 parts of water.

[0058] Example 6

[0059] An anti-photoaging sunscreen lotion comprises the following components by mass: 1-2 parts of the sea cucumber peptide described in Example 1, 2-4 parts of collagen peptide, 1.5-2.5 parts of natural moisturizing factor, 0.1-0.3 parts of matrine, 0.1-0.3 parts of taurine, 0.05-0.2 parts of witch hazel extract, 0.05-0.1 parts of retinol and 90-96 parts of water.

[0060] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. Sea cucumber peptide, characterized in that The sea cucumber peptide has an anti-photoaging effect. The sea cucumber peptide comprises polypeptide one and polypeptide two. The amino acid sequence of polypeptide one is SPQTPGSP, and the amino acid sequence of polypeptide two is DLVVVK.

2. A method for preparing the sea cucumber peptide according to claim 1, characterized in that: The following steps are involved: S1, after the sea cucumber is soaked, put it into a homogenizer, add purified water twice the mass of the sea cucumber, 6000-8000 rpm, homogenize for 5 minutes to obtain a sea cucumber homogenate; S2, adding protease to the sea cucumber homogenate for enzymolysis, and inactivating the enzyme after the enzymolysis is completed; the specific process is: adding 0.5%-0.7% flavor protease and 0.1%-0.2% aminopeptidase by weight to the sea cucumber homogenate, stirring and enzymolyzing for 4-5 hours at a pH of 7.3-7.6 and a temperature of 50-55°C; S3, using centrifuge and ultrafiltration membrane to filter the sea cucumber peptide hydrolysate in turn, intercepting the enzymatic solution of the required molecular weight; the speed of the centrifuge is 12000-14000rpm / min, the time is 5-10min, the ultrafiltration membrane adopts an ultrafiltration membrane with a pore size of 1000 Dalton, and the retentate collected is a retentate with a molecular weight less than 1000; S4, using RP-HPLC reverse phase high performance liquid chromatography to separate and purify, and separating and obtaining sea cucumber peptide under C18 column reverse phase high performance liquid chromatography, acetonitrile mobile phase, and 210nm wavelength; S5) The separated and purified eluate is concentrated and freeze-dried to obtain the anti-photoaging active sea cucumber peptide.

3. An application of sea cucumber peptide, characterized in that: Use of the sea cucumber peptide described in claim 1 in the preparation of anti-photoaging products.

4. The use of a sea cucumber peptide according to claim 3, characterized in that: The anti-photoaging products include essence and sunscreen.

5. An anti-photoaging essence, characterized in that: It includes the following components by mass: 1-2 parts of the sea cucumber peptide according to claim 1, 1-3 parts of oyster polypeptide, 1-2 parts of vitamin C, 1-2 parts of niacinamide, 0.1-0.2 parts of aloe vera, 0.1-0.2 parts of grape seed extract and 90-98 parts of water.

6. An anti-photoaging sunscreen lotion, characterized in that: It includes the following components by mass: 1-2 parts of the sea cucumber peptide according to claim 1, 2-4 parts of collagen peptide, 1.5-2.5 parts of natural moisturizing factor, 0.1-0.3 parts of matrine, 0.1-0.3 parts of taurine, 0.05-0.2 parts of witch hazel extract, 0.05-0.1 parts of retinol and 90-96 parts of water.

Images