Peptide for preventing collagen loss and preparation method thereof

Abstract

The invention belongs to the technical field of protein, and relates to a peptide for preventing collagen loss and a preparation method thereof. The amino acid sequence of the peptide is KSYELPDGQVITIG, and the key active fragment of the peptide is ELPDGQVIT. The peptide is obtained by performing enzymolysis on degreased maggot powder with alkaline protease, trypsin, papain, neutral protease and flavourzyme. The peptide can inhibit the expression of MMP1 and MMP9 in L929 cells after UVA irradiation, and alleviate the loss of collagen.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of proteins, and relates to a collagen loss prevention peptide and a preparation method thereof. BACKGROUND

[0002] Skin, as the largest organ of the human body, can affect the dynamic balance of the body, and also plays a role in protecting the internal environment from harmful external factors. Studies have shown that long-term exposure to ultraviolet (UV) radiation can cause skin photoaging, which is an important cause of skin damage. It can reduce the skin's defense ability, which is not conducive to the skin's resistance to harmful external stimuli. At the same time, it can also cause various skin diseases, including solar keratosis, photoelastic fiberization, melanoma, basal cell carcinoma, etc. This chronic skin damage not only affects the appearance of the human body, causing a certain psychological burden, but also has an adverse effect on physical health.

[0003] UV can cause excessive expression of matrix metalloproteinases (MMPs), leading to collagen loss. This is the main histological feature of photoaged skin. Type I collagen (COL1) is the most abundant structural protein in the skin and plays an important role in maintaining skin elasticity, tightness, and moisture content. Among the MMP family, interstitial collagenase (MMP1) is a specific COL1-degrading protease and is one of the key MMPs that cause COL1 breakdown; gelatinase (MMP9), as another key protease, not only can degrade elastin, leading to the breaking of skin elastic fibers, but also can further enzymatically degrade the products formed by MMP1-degrading COL1 into small fragments, ultimately adversely affecting the collagen homeostasis of the skin.

[0004] Studies have shown that specific protein hydrolysates and peptide substances, such as silver carp peptides, tuna peptides, tilapia peptides, and Chaetopteris sp. peptides from Zhanjiang, have been shown to be able to inhibit MMP expression and promote collagen synthesis. Therefore, in-depth research and development of peptide substances that can down-regulate the expression of MMP1 and MMP9 and up-regulate the expression of COL1 may provide a new strategy for reducing collagen loss, which in turn can help to alleviate skin photoaging.

[0005] In the clinical practice of alleviating skin photoaging, drug therapy is still the main way, mainly including topical application of vitamin A, 5-fluorouracil cream and ointment containing antioxidants or alpha-hydroxy acid. These drugs have been proved to have good effect in alleviating skin photoaging, but at the same time, they are accompanied by certain side effects, such as skin dryness, peeling, erythema, itching, increased skin photosensitivity, etc. If long-term or improper use, it may aggravate these adverse reactions, including skin irritation symptoms, pigment changes, capillary dilation, increased risk of secondary infection, etc. In view of the potential risks of drug and surgical treatment, nutritional intervention as a complementary treatment strategy is increasingly favored due to its lower side effects and higher patient compliance. In the field of nutritional intervention, food-derived bioactive peptides have become the focus of research due to their potential benefits in dietary supplements. In particular, studies have suggested that pentatomidae peptides may have a positive impact on skin photoaging by promoting cell directional migration to the skin damage area. However, the preparation process of pentatomidae peptides and the mechanism of their role in alleviating collagen loss are not yet well understood, and there is a lack of effective screening methods to identify peptides with potential anti-collagen loss, which limits their application in improving skin photoaging. SUMMARY

[0006] Therefore, the purpose of the present application is to provide a peptide with the effect of preventing collagen loss and a preparation method thereof, providing an option for alleviating skin photoaging.

[0007] In one aspect, the present application provides a peptide for preventing collagen loss, the amino acid sequence of which is KSYELPDGQVITIG.

[0008] In an embodiment of the present application, the key active fragment of the peptide for preventing collagen loss is ELPDGQVIT.

[0009] In another aspect, the present application provides a composition for preventing collagen loss, comprising the peptide for preventing collagen loss KSYELPDGQVITIG.

[0010] In a third aspect, the present application provides the use of the peptide for preventing collagen loss in the preparation of a drug for preventing collagen loss.

[0011] In a fourth aspect, the present application provides a method for preparing the peptide for preventing collagen loss KSYELPDGQVITIG, comprising the following steps:

[0012] 1) Dissolve defatted pentatomidae powder in water, incubate at 80-90°C for 10-20 min;

[0013] 2) adjusting the pH of the defatted five-instar larva powder solution obtained in step 1) to 8.0, adding alkaline protease and carrying out enzymolysis at 45-55°C for 1.5-2.5h to obtain an enzymolysis solution;

[0014] 3) adjusting the pH of the enzymolysis solution of step 2) to 8.0, adding trypsin and carrying out enzymolysis at 35-42°C for 1h; or adjusting the pH of the enzymolysis solution to 7.5, adding papain, and carrying out enzymolysis at 45-55°C for 0.5-1.5h; or adjusting the pH of the enzymolysis solution to 7.0, adding neutral protease, and carrying out enzymolysis at 45-55°C for 0.5-1.5h;

[0015] 4) adjusting the pH of the enzymolysis solution obtained in step 3) to 7.5, adding flavor protease and carrying out enzymolysis at 45-55°C for 0.5-1.5h;

[0016] 5) identifying the peptide segment in the enzymolysis solution of step 4) to obtain the collagen loss prevention peptide KSYELPDGQVITIG.

[0017] In step 1), the incubation temperature can be 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, or 80°C, and the incubation time can be 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min, or 20min.

[0018] In step 2), the enzymolysis temperature can be 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C, and the enzymolysis time can be 1.5h, 1.6h, 1.7h, 1.8h, 1.9h, 2.0h, 2.1h, 2.2h, 2.3h, 2.4h, or 2.5h.

[0019] In step 3), for trypsin, the enzymolysis temperature can be 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C; for papain or neutral protease, the enzymolysis temperature can be 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C; and the enzymolysis time can be 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1.0h, 1.1h, 1.2h, 1.3h, 1.4h, or 1.5h.

[0020] In step 4), the enzymatic hydrolysis temperature may be 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C or 55°C, and the enzymatic hydrolysis time may be 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1.0 h, 1.1 h, 1.2 h, 1.3 h, 1.4 or 1.5 h.

[0021] In an embodiment of the present invention, the mass ratio of defatted grain insect powder to alkaline protease is 50:1.

[0022] In an embodiment of the present invention, the mass ratio of defatted grain insect powder to trypsin is 125:1.

[0023] In an embodiment of the present invention, the mass ratio of defatted grain insect powder to papain is 390:1.

[0024] In an embodiment of the present invention, the mass ratio of defatted grain insect powder to neutral protease is 50:1.

[0025] In an embodiment of the present invention, the mass ratio of defatted grain insect powder to flavor protease is 10:1.

[0026] In an embodiment of the present invention, after each enzymatic hydrolysis reaction is completed, the step of placing the enzymatic hydrolysis solution in boiling water and heating it to terminate the reaction may be included. In a specific embodiment, the enzymatic hydrolysis reaction may be terminated by boiling the enzymatic hydrolysis solution in boiling water.

[0027] The method of the fourth aspect of the present invention can be referred to as a stepwise enzymatic hydrolysis method.

[0028] In a fifth aspect, the present invention provides a method for preparing a peptide for preventing collagen loss, comprising the following steps:

[0029] 1) Dissolve defatted grain insect powder in water and incubate at 80-90°C for 10-20 minutes;

[0030] 2) adjusting the pH of the solution obtained in step 1) to 7.5, adding alkaline protease, trypsin, papain or neutral protease, and flavor protease, and performing enzymatic hydrolysis at 40-50° C. for 3-5 hours;

[0031] 3) The peptide fragments in the enzymatic hydrolysate obtained in step 2) were identified to obtain the collagen loss-preventing peptide KSYELPDGQVITIG.

[0032] In an embodiment of the present application, in step 1), the incubation temperature can be 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃ or 80℃, and the incubation time can be 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min.

[0033] In an embodiment of the present application, in step 2), the enzymolysis temperature can be 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃ or 50℃, and the enzymolysis time can be 3h, 3.1h, 3.2h, 3.5h, 3.6h, 3.7h, 3.8h, 3.9h, 4.0h, 4.1h, 4.2h, 4.3h, 4.4h, 4.5h, 4.6h, 4.7h, 4.8h, 4.9h or 5.0h.

[0034] In an embodiment of the present application, the mass ratio of defatted five-grain worm powder to alkaline protease is 50:1, and the mass ratio to trypsin, papain and neutral protease is 125:1, 390:1 and 50:1 respectively, and the mass ratio to flavor protease is 10:1.

[0035] In an embodiment of the present application, the mass ratio of alkaline protease:trypsin:flavor protease is 5:1:12.5, the mass ratio of alkaline protease:papain:flavor protease is 15.625:1:39.0625, and the mass ratio of alkaline protease:neutral protease:flavor protease is 2:1:5.

[0036] In an embodiment of the present application, after step 2), a step of heating the enzymolysis solution in boiling water to terminate the reaction can be included.

[0037] The method of the fifth aspect of the present application can be referred to as a simultaneous enzymolysis method.

[0038] The collagen loss prevention peptide KSYELPDGQVITIG of the present application can have the ability to alleviate collagen loss, which can be derived from the active fragment ELPDGQVIT. The efficacy thereof mainly manifests in the inhibition of the expression of MMP1 and MMP9 in L929 cells after UVA irradiation and the improvement of the COL1 level in cells. At the same time, the collagen loss prevention peptide can spontaneously bind to the MMP1 receptor, change the structural flexibility thereof to make the active pocket of MMP1 more relaxed, thereby facilitating the docking of the peptide segment with the active site and enhancing the inhibition of the activity of MMP1 by the peptide segment, and ultimately alleviating collagen loss. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1Process flow chart for stepwise enzymatic digestion (A) and simultaneous enzymatic digestion (B).

[0040] Figure 2 The figure shows the amino acid species and their content constituting the anti-collagen loss peptide. Figure 2 A represents the total content of different characteristic amino acids in the peptide chain; Figure 2 B represents the content of different characteristic amino acids at the 1st and 2nd positions of the C-terminal and the 1st and 2nd positions of the N-terminal of the peptide chain; Figure 2 C represents the content of 20 kinds of amino acid residues at the 1st and 2nd positions of the C-terminal and the 1st and 2nd positions of the N-terminal of the peptide chain.

[0041] Figure 3 The figure shows the affinity energy diagram of different peptide segments docking with MMP1 receptor molecules.

[0042] Figure 4 The figure shows the effect of A→T→F group of Pentatomidae peptides (A), P1 peptide segment (B) and P2 peptide segment (C) on the viability of L929 cells.

[0043] Figure 5 The figure shows the effect of A→T→F group of Pentatomidae peptides, P1 peptide segment and P2 peptide segment on the protein expression level of MMP1, MMP9 and COL1 in L929 cells. Figure 5 A represents the Western Blot development diagram of MMP1, MMP9, COL1 and internal reference glyceraldehyde-3-phosphate dehydrogenase (GAPDH); Figure 5 B represents the protein expression level of MMP1; Figure 5 C represents the protein expression level of MMP9; Figure 5 D represents the protein expression level of COL1.

[0044] Figure 6 The figure shows the visualization diagram of P1 peptide segment (A) and P2 peptide segment (B) and MMP1 receptor protein after molecular docking to relieve collagen loss.

[0045] Figure 7 The figure shows the result diagram of P1 and P2 peptide segments and MMP1 receptor protein during the molecular dynamics simulation binding process. Figure 7 A represents the root mean square deviation of MMP1 receptor protein, MMP1-P1 and MMP1-P2 complex; Figure 7 B represents the root mean square fluctuation of MMP1 receptor protein, MMP1-P1 and MMP1-P2 complex; Figure 7 C represents the gyration radius of MMP1-P1 and MMP1-P2 complex; Figure 7 D represents the solvent accessible surface area of MMP1-P1 and MMP1-P2 complex. DETAILED DESCRIPTION

[0046] The application will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments described below or technical features thereof can be combined with each other to form new embodiments without conflict.

[0047] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained through commercial channels unless otherwise specified.

[0048] The medium formula used in the following examples is as follows:

[0049] 1) Basic medium: purchased from Gibco Company, USA.

[0050] 2) Complete medium: 10 mL of heat-inactivated horse serum, 5 mL of penicillin-streptomycin, 5 mL of non-essential amino acids, 5 mL of sodium pyruvate and 475 mL of basic medium were thoroughly mixed together to obtain the complete medium. The reagents used in the complete medium were purchased from Gibco Company, USA.

[0051] 3) Medium containing A→T→F group, A→P→F group, A→N→F group, A+T+F group, A+P+F group and A+N+F group of five grain worm peptides: 5 mg of five grain worm peptides were weighed in a 10 mL centrifuge tube, 5 mL of basic medium was added thereto and the five grain worm peptides were thoroughly dissolved. The medium containing the five grain worm peptides was obtained.

[0052] 4) Medium containing peptide segments P1 and P2: 2 mg of peptide segments P1 and P2 were weighed in a 5 mL centrifuge tube, 4 mL of basic medium was added thereto and the peptide segments P1 and P2 were thoroughly dissolved. The medium containing the peptide segments P1 and P2 was obtained.

[0053] Example 1 Preparation method of collagen loss prevention peptide KSYELPDGQVITIG (stepwise enzymatic hydrolysis method)

[0054] In this embodiment, the collagen loss prevention peptide is prepared by a stepwise enzymatic hydrolysis method, and the specific operation is as follows:

[0055] In the step-by-step enzymatic hydrolysis method, the sample was divided into 3 groups, each group weighed 3.4 g of defatted mealworm powder (the protein content in the defatted mealworm powder was about 2.5 g), and 50 mL of distilled water was added. The solution was incubated at 85°C for 15 min. Then, the pH of the solution was adjusted to 8.0, 0.025 g of alkaline protease was added and incubated at 50°C for 2 h. After the treatment, the enzymatic solution was placed in boiling water for 10 min to terminate the enzymatic reaction. Then the pH of the solution was adjusted to 8.0, 7.5, 7.0, and 0.005 g of trypsin, 0.0016 g of papain, 0.0125 g of neutral protease were added, respectively, and enzymolysis was carried out at 37°C, 50°C, 50°C for 1 h, respectively. After that, the enzymatic solution was placed in boiling water for 10 min to terminate the reaction. Subsequently, the pH of the solution was adjusted to 7.5, and 0.0625 g of flavor protease was added, and the reaction was carried out at 50°C for 1 h. After the reaction was completed, the enzymatic solution was placed in boiling water for 10 min to end the reaction. Finally, the enzymatic solution was placed at -40°C and vacuum freeze-dried for 48 h to obtain the step-by-step enzymatic hydrolysis method of mealworm peptide. They were step-by-step enzymatic hydrolysis group 1: alkaline protease→trypsin→flavor protease (A→T→F); step-by-step enzymatic hydrolysis group 2: alkaline protease→papain→flavor protease (A→P→F); step-by-step enzymatic hydrolysis group 3: alkaline protease→neutral protease→flavor protease (A→N→F). The proteases used in this method were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (China, Shanghai), and the enzyme activities of the five proteases were as follows: the enzyme activity of alkaline protease was 2×10 5 U / g (S10154), the enzyme activity of trypsin was 2.5×10 5 U / g (S10032), the enzyme activity of papain was 8×10 5 U / g (S10011), the enzyme activity of neutral protease was 1×10 5 U / g (S10013), and the enzyme activity of flavor protease was 2×10 4 U / g (S10153).

[0056] The specific process is shown as A in Figure 1 .

[0057] Example 2. Preparation method of collagen loss prevention peptide KSYELPDGQVITIG (simultaneous enzymatic hydrolysis method)

[0058] In this example, the collagen loss prevention peptide was prepared by simultaneous enzymatic hydrolysis method, and the specific operation was as follows:

[0059] In the simultaneous enzymatic method, the sample was divided into 3 groups, 3.4 g of defatted mealworm powder (protein content in defatted mealworm powder is about 2.5 g) was weighed, 50 mL of distilled water was added, and the solution was incubated at 85°C for 15 min. Then, the pH of the solution was adjusted to 7.5, and 0.025 g of alkaline protease, 0.005 g of trypsin, 0.0625 g of flavor protease; 0.025 g of alkaline protease, 0.0016 g of papain, 0.0625 g of flavor protease; 0.025 g of alkaline protease, 0.0125 g of neutral protease, 0.0625 g of flavor protease were added to each group, respectively, and enzymolysis was carried out at 45°C for 4 h. After incubation was completed, the enzymolysis solution was heated in boiling water for 10 min to terminate the reaction. Finally, the enzymolysis solution was placed at -40°C and vacuum freeze-dried for 48 h to obtain the mealworm peptides by simultaneous enzymatic method. They were simultaneous enzymolysis group 1: alkaline protease + trypsin + flavor protease (A + T + F); simultaneous enzymolysis group 2: alkaline protease + papain + flavor protease (A + P + F); simultaneous enzymolysis group 3: alkaline protease + neutral protease + flavor protease (A + N + F). The proteases used in this method were purchased from Shanghai Yuan Ye Biotechnology Co., Ltd. (China, Shanghai), and the enzyme activities of the 5 proteases were as follows: the enzyme activity of alkaline protease was 2 x 10 5 U / g (S10154), the enzyme activity of trypsin was 2.5 x 10 5 U / g (S10032), the enzyme activity of papain was 8 x 10 5 U / g (S10011), the enzyme activity of neutral protease was 1 x 10 5 U / g (S10013), and the enzyme activity of flavor protease was 2 x 10 4 U / g (S10153).

[0060] The specific process is shown in B of Figure 1 .

[0061] Example 3. Screening of key active fragments of collagen loss prevention peptide

[0062] In this example, the mealworm anti-collagen loss peptide and its key active fragments were screened by a photoaging L929 cell model. The L929 mouse fibroblasts used in this example were provided by the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (China, Beijing).

[0063] In this example, the process of screening anti-photoaging peptides is as follows:

[0064] First, the effect of mealworm peptide on the migration ability of photoaged cells was determined according to the method of Ribeiro et al. [1] . L929 cells were seeded at 1.9 x 10 5The cells were inoculated in a 6-well plate containing complete medium (containing 10% heat-inactivated horse serum (Gibco, USA), 1% penicillin-streptomycin (Gibco, USA), 1% non-essential amino acids (Gibco, USA), 1% sodium pyruvate (Gibco, USA), and 87% basal medium (Gibco, USA)) at a density of 1.8 x 10

[0065] Then, the effect of the Wuguying peptide on the level of intracellular reactive oxygen species (ROS) free radicals was determined using the fluorescent probe 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (Beijing Gaoke Biological Technology Co., Ltd.). L929 cells were inoculated in a 6-well plate containing complete medium at a density of 1.8 x 10 5 The cells were inoculated in a 6-well plate containing complete medium at a density of 1.8 x 10

[0066] Then, the effect of the Wuguying peptide on the level of intracellular reactive oxygen species (ROS) free radicals was determined using the fluorescent probe 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (Beijing Gaoke Biological Technology Co., Ltd.). L929 cells were inoculated in a 6-well plate containing complete medium at a density of 1.8 x 10 5The cells were seeded at a density of 100 cells / mL in a 6-well plate containing complete medium and incubated in an incubator for 24 hours. When the cell density reached 80%, the complete medium was removed and 1 mL of PBS solution was added, followed by UVA irradiation for 50 minutes. After irradiation, different groups were added with basal medium and basal medium containing five-grain insect peptides and incubated for another 24 hours. After the incubation was completed, the solution was removed and 4°C RIPA lysis buffer (Beijing Solebold Technology Co., Ltd.) was added to lyse the cells at 4°C for 1 hour. After the end, the cells were centrifuged at 12,000 g for 5 minutes at 4°C and the supernatant was collected. Finally, the protein concentration was determined using a BCA protein concentration detection kit (Beijing Solebold Technology Co., Ltd.), and the SOD activity and MDA content were determined using a SOD assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.) and an MDA assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.).

[0067] Finally, referring to Liu et al. [2] The effect of five-grain insect peptide on the collagen metabolism level of L929 cells was determined by the method of 5 The cells were seeded at a density of 10 cells / mL in a 6-well plate containing complete medium and incubated in an incubator for 24 hours. When the cell density reached 80%, the complete medium was removed and 1 mL of PBS solution was added, followed by UVA irradiation for 50 minutes. After irradiation, the basal medium and the basal medium containing five-grain insect peptide were added to different groups and incubated for another 24 hours. [2]The sample solution was collected and the protein concentration was unified. Then, the protein (25 μg) in the sample solution was separated on a 10% separation gel and then transferred to a polyvinylidene fluoride (PVDF) (Millipore, USA) membrane. The PVDF membrane was blocked with 5% skim milk (BD-Difco, USA) at room temperature for 2 h, and then incubated with diluted specific first antibodies, including matrix metalloproteinases (MMP) 1 / 3 / 9, collagen type I (COL1) and GAPDH overnight at 4°C (MMP1 rabbit polyclonal antibody and MMP9 rabbit polyclonal antibody were purchased from Wuhan Sanying Biotechnology Co., Ltd.; MMP3 rabbit polyclonal antibody, COL1A1 rabbit polyclonal antibody and GAPDH monoclonal antibody were purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.). The volume ratio of MMP1 to Western primary antibody diluent (Shanghai Biyun Tian Biotechnology Co., Ltd.) was 1:4000; the volume ratio of MMP3 to Western primary antibody diluent was 1:2000; the volume ratio of MMP9 to Western primary antibody diluent was 1:3000; the volume ratio of COL1 to Western primary antibody diluent was 1:1000; and the volume ratio of GAPDH to Western primary antibody diluent was 1:5000. Then, the PVDF membrane was incubated with diluted secondary antibody horseradish peroxidase-labeled goat anti-rabbit (IgG (H+L)) at room temperature for 2 h. The volume ratio of IgG (H+L) to Western secondary antibody diluent (Shanghai Biyun Tian Biotechnology Co., Ltd.) was 1:2000. Finally, the expression amount of the target protein was determined according to the method of the literature [2] .

[0068] It was found that the A→T→F group of the stepwise enzymatic hydrolysis group had a strong potential to prevent collagen loss. It showed the strongest effect in promoting L929 cell migration and removing ROS free radicals, and also had a strong effect in inhibiting MDA production, MMP1 / 3 / 9 expression and promoting COL1 expression and SOD activity. Therefore, the A→N→F group was selected as the five-legged insect anti-collagen loss peptide for peptide identification.

[0069] From Figure 2 A-C, it can be seen that the C-terminal and N-terminal of the A→N→F group of the five-legged insect anti-photoaging peptide are rich in hydrophobic and charged amino acids, respectively. Studies have shown that [3] this amino acid distribution feature can greatly improve the effect of active peptides in relieving collagen loss. Therefore, this structural feature of the five-legged insect peptide may also provide it with high biological activity.

[0070] Next, the key active fragments were screened from the anti-collagen loss peptides of five grain insects by mass spectrometry. As shown in Table 1, among the identified peptide segments, a repeatedly occurring parent peptide segment ELPDGQVIT was found, which had amino acid composition and distribution characteristics consistent with the structural characteristics of collagen loss relief peptides. Therefore, it is believed that the peptide ELPDGQVIT may be the key parent fragment for playing a role in relieving collagen loss. The identified peptide segments were subjected to molecular docking with MMP1 receptor. The structure of MMP1 (PDB ID: 966C) was derived from RCSB PDB protein database (https: / / www.rcsb.org / ), and the structure of the peptide was derived from PEP-FOLD4 polypeptide structure prediction website (https: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD4 / ). The MMP1 and peptide segments were subjected to water removal and hydrogenation treatment using AutoDockTools 1.5.6. The center coordinates of MMP1 were set as: x = 6.6, y = -9.4, z = 38.5; and the docking box size was: Subsequently, molecular docking was performed using AutoDockVina to obtain the binding energy of different peptide segments with MMP1, and the results are shown in Figure 3 The affinity energy of 17 peptide segments with MMP1 receptor was negative, indicating that the two can spontaneously bind. Among them, the affinity energy of GIPPAPR, YLPGSAPCR, GFAGDDAPR, GIGTVPVGR, KSYELPDGQVIT, EAPLNPK, KSYELPDGQVITI, SYELPDGQVITIG was lower, which was -7.8, -7.8, -7.6, -7.3, -7.2, -7.2, -7.0, -7.0 kcal / mol, respectively. At the same time, the molecular weight, isoelectric point, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, total average hydrophilic / hydrophobic (GRAVY) and other physicochemical properties of the peptide segments were analyzed by ExpasyProtParam online database (https: / / web.expasy.org / protparam / ). The results are shown in Table 1. Finally, the peptide segments were screened in combination with physicochemical properties, structural characteristics, affinity energy size and content, and the peptides KSYELPDGQVITIG (referred to as P1) and the potential active fragment ELPDGQVIT (referred to as P2) that appeared multiple times were selected for subsequent verification.

[0071] Table 1. Physicochemical property analysis of different peptide segments

[0072]

[0073]

[0074] Example 4. Evaluation of the activity of collagen loss prevention peptide KSYELPDGQVITIG

[0075] 4.1 Evaluation of the effect of A→T→F group of five insects peptides and P1, P2 peptide segments on the activity of L929 cells

[0076] In this example, the effect of A→T→F group of five insects peptides and P1, P2 peptide segments on the activity of L929 cells will be evaluated by cell counting kit-8 (CCK-8). CCK-8 was purchased from Beijing High Bridge Biotechnology Co., Ltd.

[0077] 100 μL of L929 cells (provided by the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were inoculated in a 96-well plate containing complete culture medium (containing 10% heat-inactivated horse serum, 1% penicillin-streptomycin, 1% non-essential amino acids, 1% sodium pyruvate and 87% basal medium) at a density of 1.5 x 10 5 The cells were incubated in an incubator for 24 h. When the cell density was 80%, the complete culture medium was discarded and different concentrations of basal medium containing A→T→F group of five insects peptides (0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 1 mg / mL, 3 mg / mL, 5 mg / mL, 10 mg / mL) or P1, P2 peptide segments (0.03125 mg / mL, 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL) were added for further incubation for 24 h. After incubation, the culture medium was removed and the cells were washed with PBS solution, 100 μL of 10% CCK-8 solution was added, and after incubation for 1 h, the absorbance was measured at 450 nm using a microplate reader. The results are shown in Figures A-C, which show that A→T→F group of five insects peptides had no significant damage to L929 cells in the range of 0.1-10 mg / mL, and the cell viability was greater than 80%. When the concentration of A→T→F group of five insects peptides was 1 mg / mL, the cell viability was higher than that of the blank group (P>0.05), indicating that the five insects peptides at this concentration had a certain promoting effect on cell proliferation. Therefore, 1 mg / mL of A→T→F group of five insects peptides was selected for subsequent experiments. For P1 and P2 peptide segments, in the range of 0.03125-1 mg / mL, they had no toxicity to L929 cells, while when the concentration was 0.5 mg / mL, the cell viability of P1 and P2 groups was higher than that of the blank group (P>0.05), indicating that this concentration promoted cell growth. Moreover, the cell viability of each group was close to the maximum value. Therefore, 0.5 mg / mL of P1 and P2 peptide segments were selected for subsequent experiments. Figure 4

[0078] ​4.2 Western Blot (WB) test was used to detect the protein expression levels of MMP1, MMP9 and COL1

[0079] The WB test was performed according to the method of Liu et al. [2] L929 cells were seeded in 6-well plates at a density of 1.8 x 10 5 cells / mL in complete medium and incubated in an incubator for 24 h. When the cell density reached 80%, the complete medium was removed and 1 mL of PBS solution was added, followed by UVA irradiation for 50 min. After irradiation, different groups were added with basic medium and basic medium containing anti-collagen loss peptide, respectively, and incubated for 24 h. After incubation, the solution was removed and 4°C RIPA lysis buffer (Beijing Solabio Technology Co., Ltd.) was added, and the cells were lysed at 4°C for 1 h. After the end, centrifugation was performed at 4°C and 12000 g for 5 min, and the supernatant was collected. The protein concentration in the supernatant was determined by BCA protein concentration detection kit (Beijing Solabio Technology Co., Ltd.), and then the concentration was unified to 2 mg / mL. The protein (25 μg) in the supernatant was separated on a 10% separating gel, and then transferred to a PVDF membrane (USA Millipore Corporation). The PVDF membrane was blocked with 5% skim milk (USA BD-Difco Corporation) at room temperature for 2 h, and then incubated with diluted specific primary antibodies MMP1 (Wuhan Sanying Biotechnology Co., Ltd.), MMP9 (Wuhan Sanying Biotechnology Co., Ltd.), COL1 (Shanghai Biyun Tian Biotechnology Co., Ltd.) and GAPDH (Shanghai Biyun Tian Biotechnology Co., Ltd.) at 4°C overnight. Among them, the volume ratio of MMP1 to Western primary antibody diluent (Shanghai Biyun Tian Biotechnology Co., Ltd.) was 1:4000; the volume ratio of MMP9 to Western primary antibody diluent was 1:3000; the volume ratio of COL1 to Western primary antibody diluent was 1:1000; and the volume ratio of GAPDH to Western primary antibody diluent was 1:5000. Then the PVDF membrane was incubated with diluted secondary antibody IgG (H+L) at room temperature for 2 h. The volume ratio of IgG (H+L) to Western secondary antibody diluent (Shanghai Biyun Tian Biotechnology Co., Ltd.) was 1:2000. Finally, the ECL kit (Shanghai Biyun Tian Biotechnology Co., Ltd.) was used to detect the protein band, and the band gray value was analyzed by ImageJ 1.8.0 software, and the relative expression amount of the target protein was calculated.

[0080] The results are shown in Table 1 Figure 5The expression levels of MMP1 and MMP9 were significantly increased, and the expression level of COL1 was significantly decreased (P<0.05) when L929 cells were irradiated by UVA, indicating that the cells had obvious photo-damage effect. After nutritional intervention, it was found that all the peptides could significantly inhibit the expression of MMP, thereby alleviating the degradation of COL1. Among them, peptide P2 had the strongest inhibitory effect on MMP1, and the protein expression level was reduced by 22.2% compared with the model group, and there was no significant difference compared with the blank group (P>0.05), indicating that the peptide segment ELPDGQVIT could restore the collagen metabolism of the cells to the normal level. The second was P1 and A→T→F group Wufuying peptide, but there was no significant difference in the expression level of MMP1 between the two groups (P>0.05). In terms of the expression of MMP9 and COL1, the A→T→F group had the best effect, followed by the P1 and P2 treatment groups. But compared with the A→T→F group, there was no significant difference in the expression levels of MMP9 and COL1 in the P2 treatment group (P>0.05). In terms of inducing the degradation of skin collagen, MMP1 is the most critical protease in the MMP family. Therefore, the above results show that the peptide KSYELPDGQVITIG has strong ability to inhibit the degradation of COL1, and the peptide ELPDGQVIT as the parent active fragment provides the ability to alleviate the degradation of collagen.

[0081] 4.3 Analysis of the binding mode of peptides P1 and P2 with MMP1 receptor protein by molecular docking

[0082] The results of docking of peptides P1 and P2 with MMP1 receptor were imported into Pymol for visualization, and the results were as follows: Figure 6The complex formed by the two was then uploaded to Protein-Ligand Interaction profiler (https: / / plip-tool.biotec.tu-dresden.de / plip-web / plip / index) to analyze the interaction between the peptide segment and the MMP1 receptor protein (including the binding mode, binding site of the receptor protein, etc.). The results are shown in Table 2. The affinity energy of P1, P2 and MMP1 is less than 0, indicating that the peptide segments P1 and P2 can spontaneously and stably bind to MMP1. In addition, the peptide segments P1 and P2 interact with the target protein through hydrophobic interaction, hydrogen bond and salt bridge, of which hydrophobic interaction and hydrogen bond are the two main binding modes. Among the intermolecular interactions, hydrogen bond is one of the stronger non-covalent interaction types, and its number has an important influence on the stability of the complex. P2 forms the most hydrogen bonds (12) with MMP1, and the number of hydrogen bonds between P1 and MMP1 is 10. Therefore, P2 may have a stronger interaction with MMP1, thereby affecting the protein expression of MMP1 in L929 cells. This is consistent with the experimental results of WB. When the peptide segments P1 and P2 interact with the active site of MMP1, the activity of MMP1 can be inhibited, thereby hindering the degradation of COL1.

[0083] Table 2. Analysis of the interaction of P1 and P2 with MMP1 receptor

[0084]

[0085]

[0086] 4.4 Molecular dynamics simulation of the binding process of peptide segments P1 and P2 with MMP1 receptor protein

[0087] The P1-MMP1 complex, P2-MMP1 complex and MMP1 receptor protein obtained by molecular docking were placed in a dodecahedron box as the initial structure using Gromacs 2023, ensuring that the distance between the complex and the box boundary was at least 1 nm. Then, water molecules (solvation) were added to the box, and Na + and Cl - were added to make the total charge of the system 0. When constructing the simulation system, the Amber99SB force field and TIP3P water molecule model were selected. Referring to An et al. [4]method, the initial simulation system was subjected to two-step energy minimization. The first step was performed using the steepest descent method for 10,000 times, and the second step was performed using the conjugate gradient for 5,000 times. After energy optimization, 200ps of NVT temperature control simulation and NPT constant pressure simulation were performed, and the temperature and pressure were balanced to 310K and below 1 bar, respectively. Then, a formal simulation of 100ns was performed, and the conformation was saved every 10ps. The temperature control algorithm used in this process was V-rescale, and the pressure control algorithm was parrinello-rahman. Finally, Gromacs related commands were used to analyze the obtained simulation results. The stability of the system was evaluated by root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg) and solvent accessible surface area (SASA). By Figure 7 As shown in Figure A, the RMSD values ​​of MMP1-P1 and MMP1-P2 stabilized after 60ns, indicating that the conformation of the complexes formed by different peptides and MMP1 gradually reached a stable equilibrium state. In addition, the average RMSD values ​​of MMP1 and the two complexes were 0.214, 0.214, and 0.306, respectively. The average RMSD value of the peptide-target protein complex was greater than 0.214 (reference conformation), suggesting that the binding of P1 and P2 to MMP1 would cause its structure to become loose, causing slight conformational changes in MMP1. Figure 7 The RMSF changes of MMP1 and the MMP1-peptide complex in Figure B also revealed that the RMSF values ​​of both complexes, especially MMP1-P2, at residue position 171, increased significantly. This may be due to the allosteric effect caused by the binding of the peptide to MMP1, which leads to increased volatility of residues at adjacent positions. Moreover, the average RMSF values ​​of both complexes are higher than those of MMP1, indicating that after the peptide binds, the overall residue volatility of the target protein increases, suggesting that the peptide can improve the flexibility of the MMP1 conformation. Figure 7 The C in the figure reflects the changes in Rg of the two complexes. The results show that the fluctuation of the curve gradually stabilizes after 60ns, which is consistent with the results presented by the RMSD value, further verifying the stability of the conformation of the peptide-target protein during the simulation. In addition, the average Rg values ​​of different complexes are 1.537 and 1.563, respectively, suggesting that the binding of P2 to MMP1 will make the overall structure more loose, which may induce the conformational expansion of the S1' and S3' substrate binding pockets, thereby increasing the accessibility of the peptide to the MMP1 active site. Figure 7The change of the surface area of the two complexes of medium D directly contacted with the solvent can be seen that the expansion of the structure of the complex is mainly due to the increase of solvent exposure in the binding area. At the same time, the average value of SASA of MMP1-P1 and MMP1-P2 is 91.82 nm 2 and 93.67 nm 2 , respectively, both of which are in the range of 50-200 nm 2 , indicating that the surface of the molecule contains a moderately exposed flexible region and a rigid region stably combined with the peptide segment. In summary, the two peptide segments can be stably combined with MMP1, and the structure of MMP1 after combination is looser. P1 and P2 expand the active pocket of MMP1 by changing the flexibility of MMP1, thereby facilitating the docking of the peptide segment with the active site, and ultimately better play the role of inhibiting the activity of MMP1.

[0088] The above embodiments are only preferred embodiments of the present application, and cannot be used to limit the scope of protection of the present application. Any non-essential changes and substitutions made by those skilled in the art on the basis of the present application are within the scope of the present application.

[0089] References

[0090] [1] RIBEIRO F M, DE OLIVEIRA M M, SINGH S, et al. Ceria Nanoparticles Decrease UVA-Induced Fibroblast Death Through Cell Redox Regulation Leading to Cell Survival,

[0091] Migration and Proliferation [J]. Frontiers in Bioengineering and Biotechnology, 2020, 8.

[0092] [2] LIU W, YU S, HAN Y, et al. Systematic sequence characterization of enzymatic-derived soybean peptides for precision enhancement of anti-inflammatory properties [J]. Food Bioscience, 2024, 60.

[0093] [3] LIU R, HE L, CHEN L, et al. Skin’s New Shield: Food-Derived Bioactive Peptides in Combating Photoaging—An Investigation Into Inhibitory Mechanisms and Structure-Activity Relationships [J]. Journal of Food Biochemistry, 2025, 2025 (1).

[0094] [4] AN J, WANG M, LIU W, et al. Unraveling amino acid sequence features that confer high calcium-binding capacity to soybean peptides and improve absorption in caco-2 cells [J].

[0095] Journal of Functional Foods, 2025, 130.

Claims

1. Peptide that prevents collagen loss, characterized by: The amino acid sequence of the peptide is KSYELPDGQVITIG.

2. The peptide according to claim 1, characterized in that The key active fragment of the peptide is ELPDGQVIT.

3. A composition for preventing collagen loss, comprising the peptide according to any one of claims 1 to 2.

4. Use of the peptide for preventing collagen loss according to claim 1 or 2 in the preparation of a drug for preventing collagen loss.

5. A method for preparing the peptide for preventing collagen loss according to claim 1 or 2, characterized in that: The following steps are involved: 1) Dissolve defatted grain insect powder in water and incubate at 80-90°C for 10-20 minutes; 2) adjusting the pH of the defatted grain insect powder solution obtained in step 1) to 8.0, adding alkaline protease and performing enzymatic hydrolysis at 45-55° C. for 1.5-2.5 hours to obtain an enzymatic hydrolyzate; 3) adjusting the pH of the enzymatic hydrolysate from step 2) to 8.0, adding trypsin, and performing enzymatic hydrolysis at 35-42° C. for 1 hour; or adjusting the pH of the enzymatic hydrolysate to 7.5, adding papain, and performing enzymatic hydrolysis at 45-55° C. for 0.5-1.5 hours; or adjusting the pH of the enzymatic hydrolysate to 7.0, adding neutral protease, and performing enzymatic hydrolysis at 45-55° C. for 0.5-1.5 hours; 4) adjusting the pH of the enzymatic hydrolyzate obtained in step 3) to 7.5, adding flavor protease, and performing enzymatic hydrolysis at 45-55° C. for 0.5-1.5 h; 5) The enzymatic hydrolysate obtained in step 4) is subjected to vacuum freeze-drying treatment to obtain a powdered enzymatic hydrolysate, and then the peptide segments in the enzymatic hydrolysate are identified to obtain the collagen loss-preventing peptide KSYELPDGQVITIG.

6. The method according to claim 5, characterized in that After each enzymatic hydrolysis reaction is completed, the method may include placing the enzymatic hydrolysis solution in boiling water to heat the solution to terminate the reaction.

7. A method for preparing the peptide for preventing collagen loss according to claim 1 or 2, characterized in that: The following steps are involved: 1) Dissolve defatted grain insect powder in water and incubate at 80-90°C for 10-20 minutes; 2) adjusting the pH of the solution obtained in step 1) to 7.5, adding alkaline protease, trypsin, papain or neutral protease, and flavor protease, and performing enzymatic hydrolysis at 40-50° C. for 3-5 hours; 3) The enzymatic hydrolysate obtained in step 2) is subjected to vacuum freeze-drying treatment to obtain a powdered enzymatic hydrolysate, and then the peptide segments in the enzymatic hydrolysate are identified to obtain the collagen loss-preventing peptide KSYELPDGQVITIG.

8. The method according to claim 7, characterized in that After step 2), the enzymatic hydrolyzate may be placed in boiling water and heated to terminate the reaction.

Images