Preparation method of bird's nest peptide and application thereof

Abstract

The application discloses a preparation method of bird's nest peptide and application of the bird's nest peptide. The amino acid sequence of the bird's nest peptide is KPAPPKPEPK. The bird's nest peptide prepared by the application has strong anti-ultraviolet damage ability, can improve cell survival rate, improve the level of antioxidant enzymes in serum, remove free radicals and reduce the level of inflammatory factors in skin tissue. The bird's nest peptide is used for development of anti-ultraviolet damage products, so that the efficacy of the bird's nest can be deeply explored, and the quality and economic value of the bird's nest products are improved.

Description

Technical Field

[0001] This invention belongs to the field of protein engineering technology, specifically relating to a method for preparing bird's nest peptides and their applications. Background Technology

[0002] Bird's nest is the nest built by swiftlets (Apodidae) and other related species using a mixture of saliva and down feathers. The *Compendium of Materia Medica Supplement* states: "Bird's nest is sweet, bland, and neutral in nature. It greatly nourishes lung yin, resolves phlegm and stops coughs, and is both nourishing and clearing, making it a holy medicine for treating deficiency and fatigue. It can treat all diseases caused by lung deficiency and impaired downward flow of qi." The *New Compilation of Materia Medica* states: "Bird's nest greatly replenishes vital energy, moistens the lungs and nourishes yin, treats consumptive coughs, hemoptysis, and hematemesis, guides fire back to its source, and moistens the intestines and stimulates the appetite." Edible bird's nest contains various nutrients, including proteins, carbohydrates, lipids, vitamins, amino acids, and various important inorganic elements. Studies have shown that the active ingredients in bird's nest have the ability to promote the growth of epidermal keratinocytes.

[0003] One of the fundamental characteristics of photoaging is the alteration of the skin's cellular matrix composition. Continuous and repeated proteolytic degradation of the extracellular matrix leads to an imbalance between its synthesis and degradation in the dermis, hindering collagen synthesis and altering dermal connective tissue. Ultraviolet (UV) radiation can cause abnormal changes in collagen fibers and elastin, resulting in photoaging characteristics. UV radiation acts on various photosensitive substances or chromophores in the skin, inducing the production of large amounts of free radicals, damaging the skin's own antioxidant system, reducing the activity of antioxidant enzymes, and causing the accumulation of lipid peroxidation products. Furthermore, UV radiation also causes cells to produce more cytokines such as interleukins, tumor necrosis factor, and intercellular adhesion molecules.

[0004] Currently, there is limited research on bird's nest peptides that have anti-UV damage effects. Therefore, in-depth research on bird's nest and the isolation of bird's nest peptides with anti-UV damage effects are of great significance for delaying skin photoaging, deep processing of bird's nest, and enriching anti-UV damage products. Summary of the Invention

[0005] In order to fully explore the efficacy of bird's nest, conduct in-depth processing of bird's nest, and enrich products for preventing ultraviolet damage, this invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides the application of bird's nest peptide in the preparation of products that resist ultraviolet damage, wherein the amino acid sequence of the bird's nest peptide is KPAPPKPEPK (SEQ ID NO.1).

[0007] Secondly, the present invention provides the application of bird's nest peptide in the preparation of products that can improve cell survival rate under ultraviolet damage, wherein the amino acid sequence of the bird's nest peptide is KPAPPKPEPK.

[0008] Thirdly, the present invention provides the application of bird's nest peptide in the preparation of products that can improve the level of antioxidant enzymes in serum under ultraviolet damage, wherein the amino acid sequence of the bird's nest peptide is KPAPPKPEPK.

[0009] Fourthly, the present invention provides the application of bird's nest peptide in the preparation of products that can reduce the level of inflammatory factors in skin tissue under ultraviolet damage, wherein the amino acid sequence of the bird's nest peptide is KPAPPKPEPK.

[0010] Preferably, the products described in the first to fourth aspects are pharmaceuticals, health foods, animal feed, or cosmetics.

[0011] Furthermore, the product also contains excipients permitted to be added to pharmaceuticals, health foods, animal feed, or cosmetics.

[0012] Preferably, the preparation process of the bird's nest peptide includes the following steps:

[0013] S1. Soak the bird's nest, sort and dry it, then stew it.

[0014] S2, heat the stewed bird's nest liquid to 60~85℃ and keep it warm for 1~3 hours;

[0015] S3, add a mixture of alkaline protease and papain to the stewed bird's nest, hydrolyze and inactivate the enzyme, and centrifuge to obtain the supernatant;

[0016] S4, the supernatant is subjected to alcohol precipitation, centrifugation, rotary evaporation and drying to obtain a polypeptide mixture containing the bird's nest peptide.

[0017] Preferably, the stewing temperature in step S1 is 110~120℃, for example: 110℃, 115℃, 120℃.

[0018] Preferably, the stewing time in step S1 is 10 to 20 minutes, for example: 10 minutes, 15 minutes, or 20 minutes.

[0019] Preferably, the temperature in step S2 is 60~85℃, for example 60℃, 65℃, 70℃, 75℃, 80℃, 85℃.

[0020] Preferably, the heat preservation time in step S2 is 1 to 3 hours, for example: 1 hour, 1.5 hours, 2 hours, 2.5 hours, or 3 hours.

[0021] Preferably, the mixing ratio of alkaline protease and papain in step S3 is 1~5:1~5, for example 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5.

[0022] Preferably, the enzymatic hydrolysis temperature in step S3 is 40~60℃, for example: 40℃, 45℃, 50℃, 55℃, 60℃.

[0023] Preferably, the enzymatic hydrolysis time in step S3 is 2 to 6 hours, for example: 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, and 6 hours.

[0024] Preferably, the enzyme inactivation temperature in step S3 is ≥90℃, for example: 90℃, 92℃, 95℃, 97℃, 100℃.

[0025] Fifthly, the present invention provides a method for preparing anti-ultraviolet damage bird's nest peptide, the preparation method comprising the following steps:

[0026] S1. Soak the bird's nest, sort and dry it, then stew it.

[0027] S2, heat the stewed bird's nest liquid to 60~85℃ and keep it warm for 1~3 hours;

[0028] S3, add a mixture of alkaline protease and papain to the stewed bird's nest, hydrolyze and inactivate the enzyme, and centrifuge to obtain the supernatant;

[0029] S4, the supernatant is subjected to alcohol precipitation, centrifugation, rotary evaporation and drying to obtain a polypeptide mixture containing the bird's nest peptide.

[0030] Preferably, the stewing temperature in step S1 is 110~120℃, for example: 110℃, 115℃, 120℃.

[0031] Preferably, the stewing time in step S1 is 10 to 20 minutes, for example: 10 minutes, 15 minutes, or 20 minutes.

[0032] Preferably, the temperature in step S2 is 60~85℃, for example 60℃, 65℃, 70℃, 75℃, 80℃, 85℃.

[0033] Preferably, the heat preservation time in step S2 is 1 to 3 hours, for example: 1 hour, 1.5 hours, 2 hours, 2.5 hours, or 3 hours.

[0034] Preferably, the mixing ratio of alkaline protease and papain in step S3 is 1~5:1~5, for example 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5.

[0035] Preferably, the enzymatic hydrolysis temperature in step S3 is 40~60℃, for example: 40℃, 45℃, 50℃, 55℃, 60℃.

[0036] Preferably, the enzymatic hydrolysis time in step S3 is 2 to 6 hours, for example: 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, and 6 hours.

[0037] Preferably, the enzyme inactivation temperature in step S3 is ≥90℃, for example: 90℃, 92℃, 95℃, 97℃, 100℃.

[0038] Preferably, the amino acid sequence of the bird's nest peptide is KPAPPKPEPK.

[0039] Preferably, the preparation method may further include the following steps:

[0040] S5, perform amino acid sequence analysis on the polypeptide mixture to synthesize the bird's nest peptide.

[0041] In a sixth aspect, the present invention provides a cosmetic product containing bird's nest peptide prepared according to the method described in the fifth aspect.

[0042] Preferably, the cosmetic also contains excipients permitted for use in cosmetics.

[0043] In a seventh aspect, the present invention provides a food product containing bird's nest peptide prepared according to the method described in the fifth aspect.

[0044] Eighthly, the present invention provides a medicine containing bird's nest peptide prepared according to the method described in the fifth aspect.

[0045] Preferably, the medicine further contains excipients that are permitted to be added to the medicine.

[0046] In a ninth aspect, the present invention provides a feed containing bird's nest peptides prepared according to the method described in the fifth aspect.

[0047] Preferably, the feed further contains additives that are permitted to be added to the feed.

[0048] The beneficial effects of this invention are:

[0049] This invention utilizes a bird's nest peptide with the amino acid sequence KPAPPKPEPK in anti-UV damage products, which can improve cell survival rate, increase serum antioxidant enzyme levels, scavenge free radicals, and reduce inflammatory factor levels in skin tissue. This bird's nest peptide and products containing it have broad application prospects in cosmetics, food, pharmaceuticals, and animal feed. This invention improves the quality of bird's nest products and enhances the economic value of bird's nest. Attached Figure Description

[0050] Figure 1 The figure shows the effects of bird's nest peptides on SOD activity, CAT activity, GSH-Px activity and MDA content in the serum of mice irradiated with ultraviolet light.

[0051] Figure 2 The image shows the effect of bird's nest peptides on the levels of inflammatory factors in the skin tissue of mice irradiated with ultraviolet light. Detailed Implementation

[0052] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] It should be noted that, unless otherwise specified, the experimental methods and reagents used in the embodiments of the present invention are all conventional experimental methods and reagents in the art.

[0054] Papain (Shanghai Yuanye Biotechnology Co., Ltd., S10011), alkaline protease (Shanghai Yuanye Biotechnology Co., Ltd., S10154).

[0055] Fmoc-Lys(Boc)-Wang Resin (substitution degree 0.4 mmol / g, cross-linking degree 1%, particle size 100-200 mesh), Fmoc-Arg(Pbf)-Wang Resin (substitution degree 0.4 mmol / g, cross-linking degree 1%, particle size 100-200 mesh), Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Lys-OH, Fmoc-Glu-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Thr-OH, Fmoc-Gly-OH, Fmoc-Tyr-OH, and Fmoc-Phe-OH were all purchased from Nanjing Peptide Biotechnology Co., Ltd.

[0056] Example 1 Preparation of polypeptide mixtures

[0057] 1.1 Preparation and Application of Peptide Mixtures

[0058] (1) Soak the bird's nest, pick and dry it, and then stew it at 115℃ for 10-20 minutes.

[0059] (2) Heat the stewed bird's nest liquid to 60~85℃ and keep it warm for 2 hours;

[0060] (3) Add a mixture of alkaline protease and papain in a mass ratio of 1:1 to the bird's nest, with an addition amount of 1-5%, and enzymatically hydrolyze at 55℃ for 3 h. After enzymatic hydrolysis, inactivate the enzyme at 95℃ for 15 min, and then centrifuge to obtain the supernatant.

[0061] (4) Add anhydrous ethanol to the supernatant obtained in step (3) to make the ethanol volume fraction reach 60%. After stirring and mixing, let stand for 15 min, centrifuge at 3500 r / min for 20 min, transfer the supernatant to a rotary evaporator, and remove the ethanol by rotary evaporation at 40℃ and 100 r / min. Pour out the concentrate, add a small amount of water to rinse the rotary evaporator and recover the concentrate, and measure the total volume of the concentrate. Repeat the rotary evaporation once, dispense the concentrate into glass petri dishes, about 25~30 mL per plate, freeze quickly at -80℃ for 12 h, and freeze dry in a freeze dryer for 24 h to obtain a powdered polypeptide mixture.

[0062] Example 2 Amino acid sequence analysis of a polypeptide mixture

[0063] The amino acid sequences of peptide chains in a polypeptide mixture were sequenced and identified using ultra-high performance liquid chromatography-electrolysis tandem mass spectrometry (UPLC-ESI-MS / MS). Specific chromatographic conditions included: an Acclaim PepMap C18 column (75 μm × 25 cm); mobile phase A: aqueous solution containing 0.1% formic acid; mobile phase B: acetonitrile solution containing 0.1% formic acid; sample loading volume 5.0 μL; elution flow rate 300.0 nL / min; and a positive charge spray voltage of 2.0 kV.

[0064] Then, the Mascot Server online system is used to compare data against the database and select those with high matching scores and relative strength greater than 10. 9 Two peptide segments.

[0065] The identified peptide amino acid sequence is as follows:

[0066] KPAPPKPEPK(SEQ ID NO.1);

[0067] DARFEDLPVY (SEQ ID NO. 2).

[0068] Example 3 Synthesis of Bird's Nest Peptides

[0069] Bird's nest peptide (KPAPPKPEPK, SEQ ID NO:1) was synthesized using a solid-phase synthesis process, following the direction from the C-terminus to the N-terminus. The specific process is as follows:

[0070] Weigh 4.0 g of Fmoc-Lys(Boc)-Wang Resin with a substitution degree of 0.4 mmol / g, add it to a solid-phase reaction column, wash twice with 20 mL of DMF to remove the solvent, and add 60 mL of DMF to swell for 30 min. Wash twice with DMF, add a piperidine-DMF mixed solution (volume ratio 1:3), and stir for 20 min. Monitor the completion of the reaction using the ninhydrin colorimetric method. Wash five times each with DMF and DCM, dissolve 2.17 g (6.40 mmol) of Fmoc-Pro-OH and 1.04 g (7.60 mmol) of HOBt in DMF, add 1.20 mL (7.6 mmol) of DIC under ice bath conditions, stir for 8 min in the dark, and then add it to the above-mentioned solid-phase reaction column with the solvent removed. Add 0.08 g (0.64 mmol) of DMAP, stir under nitrogen protection for 3 h, and monitor the completion of the reaction using the ninhydrin colorimetric method. The solvent was removed, and the resin was washed five times with DMF to obtain Fmoc-Pro-Lys-Wang Resin. Following the coupling method described above, the corresponding Fmoc protecting amino acids were added sequentially according to the peptide sequence to elongate the peptide chain. After the final coupling reaction, the resin was washed four times each with DCM, DMF, and MeOH.

[0071] After drying the resin with nitrogen, it was transferred to a round-bottom flask and subjected to three cycles of 209 mL of a HOAc-TFE-DCM mixed solution (volume ratio 1:3:6) for 30 min, 15 min, and 5 min respectively. The mixture was filtered, and the filtrate was concentrated to one-quarter of its original volume. The concentrate was then added to 10 times its volume of diethyl ether for precipitation, and the mixture was allowed to stand overnight in a refrigerator. The filter cake was then filtered again, washed six times with a small amount of diethyl ether, and vacuum dried to obtain fully protected crude bird's nest peptides with a yield of 90.85%.

[0072] The synthesized bird's nest peptides were analyzed and identified according to the method in Example 2: sequencing and identification methods were used.

[0073] The crude bird's nest peptides were dissolved in an appropriate amount of DMSO and purified by high-performance liquid chromatography (HPLC). A C18 reversed-phase column was used as the chromatographic column. The eluents were: solution A, an aqueous solution of 0.1% TFA; and solution B, an aqueous solution of acetonitrile containing 0.1% TFA. The detection wavelength was 220 nm. The purified liquid was lyophilized to obtain the bird's nest peptide product with a purity of 99.3%.

[0074] Example 4: Effects of bird's nest peptide (SEQ ID NO:1) on cellular UV damage

[0075] Cultured Hacat cells were trypsinized for 7 min, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Cells were diluted with 4 mL of DMEM high-glucose medium containing 10% serum, and 20 μL was used for cell counting. Cells were diluted to a final concentration of 4 × 10⁻⁶ cells in each group. 4 Cells were mixed at a concentration of 100 μL / mL and placed in a 96-well plate. The control group received only culture medium. Each group was repeated in triplicate. Cells were incubated at 37°C and 5% CO2 for 24 h. All solutions were aspirated from the wells. 100 μL of PBS was added to each group. The wells were capped and placed in a UV catalytic analyzer. The control group was covered with aluminum foil. The other groups were irradiated at 308 nm for 30–120 min. For human HDF cells, UVB lamp irradiation for 30–120 min was used for modeling. The remaining experimental procedures were the same as for Hacat cells. After aspirating the PBS from each well, 10 mg / mL of bird's nest peptide (SEQ ID NO.1) was added to the example group, and the same concentration of bird's nest peptide (SEQ ID NO.2) was added to the control and control wells. Cells were incubated at 37°C and 5% CO2 for 24 h. Remove all solution from the wells and add 100 μL of mixed medium (DMEM high glucose culture medium: CCK-8 = 10:1). Incubate at 37℃ and 5% CO2 for 1-2 h. When the color of the wells turns brown, the absorbance can be detected at 450 nm. Calculate the cell viability according to the following formula.

[0076] Cell viability = [(As-Ab) / (Ac-Ab)] × 100%

[0077] In the formula: As is the absorbance of the experimental well (containing cell culture medium, CCK-8 and the treatment); Ac is the absorbance of the control well (containing cell culture medium and CCK-8); Ab is the absorbance of the blank well (containing no cells and the test substance, and no CCK-8).

[0078] Table 1. Effects of bird's nest peptides on the UV damage resistance of HaCaT and human HDF cells.

[0079]

[0080] Table 1 shows that the survival rate of HaCaT cells in the model group after UV damage treatment was only 48.66%, while that in the control group was 68.97%. After treatment with bird's nest peptide (SEQ ID NO.1), the cell survival rate was 80.02%, significantly higher than both the model and control groups (p < 0.05). For human HDF cells, the survival rate of cells in the model group after UV damage treatment was only 56.95%, while that in the control group was 60.05%, with no significant difference between the two groups. After treatment with bird's nest peptide (SEQ ID NO.1), the cell survival rate was 77.81%, significantly higher than both the model and control groups (p < 0.05). These experimental results for both cell types indicate that bird's nest peptide (SEQ ID NO.1) has a certain anti-UV damage effect.

[0081] Example 5: Effects of bird's nest peptides on resistance to ultraviolet damage in mice

[0082] Female KM mice (18±0.5 g) were selected as the experimental system. Sixty mice were divided into five groups: a blank control group, a model group, a positive control group (sialic acid, 40 mg / kg), an example group (bird's nest peptide SEQ ID NO.1, 20 mg / kg), and a comparative group (bird's nest peptide SEQ ID NO.2 synthesized according to the method in Example 3, 20 mg / kg). After anesthesia, the mice's entire backs were waxed. Three days after waxing, except for the blank control, the other waxed mice were anesthetized and placed in a UV radiation chamber. The exposed backs of the mice were irradiated with UVA+UVB+UVC (254nm+302nm+365nm) UV lamps for 30 minutes daily for eight consecutive days to establish the model. Skin samples from the irradiated areas were analyzed using a skin analyzer. After 28 days of continuous drug administration, the mice were euthanized, and skin tissue samples from the irradiated areas on the backs, cecal contents, and abdominal aortic blood were collected and preserved for later use.

[0083] (1) Determination of superoxide dismutase (SOD) activity, catalase (CAT) activity, glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) content in serum.

[0084] Methods: Blood samples were collected from the abdominal aorta and centrifuged at 3000 r / min and 4℃ for 10 min. The supernatant was collected to detect SOD activity, CAT activity, GSH-Px activity and MDA content in the serum.

[0085] like Figure 1As shown, compared with the blank control group, the activities of SOD, CAT, and GSH-Px in the serum of mice in the model group were significantly decreased, and the MDA content in the serum of mice in the model group was significantly increased, indicating that the model was successfully established. Compared with the model group, the SOD enzyme activity in the serum of mice in the positive control group, the example group, and the comparative group was significantly increased (p<0.05); compared with the model group, the CAT enzyme activity in the serum of mice in each treatment group was significantly increased (p<0.05), especially the example group showed a highly significant increase, with CAT enzyme activity significantly higher than other groups; compared with the model group, the GSH-Px enzyme activity in the serum of mice in each treatment group increased highly significantly (p<0.05), and the example group and the comparative group were significantly higher than the enzyme activity level in normal mice, but there was no significant difference between the two groups; for MDA, compared with the model group, the MDA content in the serum of mice in the example group was significantly lower than that in the model group (p<0.01), indicating that bird's nest peptide (SEQ ID NO.1) can alleviate oxidative damage caused by ultraviolet radiation in mice and has a strong anti-ultraviolet damage function.

[0086] (2) Determination of the levels of IL-1β, IL-6 and TNF-α in skin tissue.

[0087] Inflammation and the immune process are involved in the development and progression of certain diseases, leading to a variety of health problems. Inflammation is a self-protective mechanism of the body, and various cytokines are involved in the regulatory process. Abnormal secretion of some major pro-inflammatory cytokines (such as IL-1β, IL-6, and TNF-α) and anti-inflammatory cytokines (such as IL-4, IL-10, and TGF-β) is an important cause of chronic inflammation.

[0088] Methods: A small amount of back skin sample was taken, and the skin tissue was homogenized. The supernatant was collected by centrifugation, and the contents of IL-1β, IL-6, and TNF-α in the skin tissue homogenate were detected by ELISA kit.

[0089] like Figure 2 As shown, compared with the blank control group, the levels of IL-1β, IL-6, and TNF-α in the model group were significantly increased (p<0.05), indicating that ultraviolet radiation caused skin damage in mice, increased the level of inflammatory factors in the skin tissue, and induced an inflammatory response, thus successfully establishing the model. Compared with the model group, the levels of IL-1β, IL-6, and TNF-α in the skin of mice in the positive control group, the example group, and the comparative group were significantly reduced (p<0.05). In particular, the levels of IL-1β and TNF-α in the example group were significantly reduced (p<0.01), showing better effects than the positive control group and the comparative group. This indicates that bird's nest peptide (SEQ ID NO.1) can alleviate the inflammatory response of skin damage caused by ultraviolet radiation in mice and has a strong anti-ultraviolet damage function.

[0090] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. The application of bird's nest peptides in the preparation of products that resist ultraviolet damage, characterized in that, The amino acid sequence of the bird's nest peptide is KPAPPKPEPK.

2. The application of bird's nest peptides in the preparation of products that can improve cell survival rate under ultraviolet damage, characterized in that, The amino acid sequence of the bird's nest peptide is KPAPPKPEPK.

3. The application of bird's nest peptides in the preparation of products that can increase the level of antioxidant enzymes in serum under ultraviolet damage, characterized in that, The amino acid sequence of the bird's nest peptide is KPAPPKPEPK.

4. The application of bird's nest peptides in the preparation of products capable of reducing the level of inflammatory factors in skin tissue under ultraviolet damage, characterized in that, The amino acid sequence of the bird's nest peptide is KPAPPKPEPK.

5. The application according to any one of claims 1-4, characterized in that, The products mentioned are pharmaceuticals, health foods, animal feed, or cosmetics.

6. The application according to claim 5, characterized in that, The product also contains excipients that are permitted to be added to pharmaceuticals, health foods, animal feed, or cosmetics.

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