Bird's nest peptide I with skin elasticity protection and anti-inflammatory effects and its application

Abstract

The present invention discloses a bird's nest peptide I with skin elasticity protection and anti-inflammatory effects and its application. The amino acid sequence of bird's nest peptide I is DARFEDLPVY. Bird's nest peptide I has a high elastase inhibition rate, can inhibit the degradation of elastin in the skin, and delay skin aging. Bird's nest peptide I also has the effect of improving inflammation, which is of great significance for improving the economic value and utilization value of bird's nest.

Description

Technical Field

[0001] The invention belongs to the technical field of protein engineering, and specifically relates to a bird's nest peptide I having skin elasticity protection and anti-inflammatory effects and an application thereof. Background Art

[0002] Bird's nest is a nest made by the swiftlet and other swallows of the same genus, using saliva and down feathers. It is mainly produced in Southeast Asian countries and the South my country Sea islands. The "Supplement to Compendium of Materia Medica" states: "Bird's nest tastes sweet and mild, nourishes the lung yin, resolves phlegm and stops cough, nourishes and clears, and is a holy medicine for regulating deficiency and fatigue. All diseases caused by lung deficiency that cannot clear and descend can be cured with this." The "New Compendium of Materia Medica" states: "Bird's nest greatly replenishes vitality, moisturizes the lungs and nourishes yin, cures cough due to deficiency, hemoptysis, vomiting blood, guides fire back to its source, moisturizes the intestines and stimulates appetite." Edible bird's nest contains a variety of nutrients, the main nutrients of which include protein, carbohydrates, lipids, vitamins, amino acids and a variety of important inorganic elements, of which the protein content is as high as about 50%. Its main functional components are sialic acid and epidermal growth factor, which have physiological functions such as anti-oxidation, anti-aging, and immunity enhancement. Therefore, bird's nest has been used as a health food for nourishment, beauty and health preservation.

[0003] Elastin is an insoluble, macromolecular fibrous protein in the extracellular matrix. It and microfibrils together form elastic fibers in connective tissue. Elastin accounts for 2-4% of the total protein content in the skin. It has a high degree of extensibility and toughness, can give the skin strong elasticity and recoil, and is responsible for restoring the deformed skin to its normal form. Skin aging is a topic of great concern in today's society. The main characteristics of skin aging are thinning of the epidermis, flattening of the dermal junction, and overall degradation of the dermal extracellular matrix (ECM). Elastic fibers play an important role in skin aging. If elastin is degraded and destroyed, it will cause skin elasticity loss, sagging, and various wrinkles. The high expression of elastase will cause enzymatic hydrolysis of elastin and destroy elastin. Therefore, the elastase inhibition rate is one of the indicators for examining the anti-skin aging ability of the product.

[0004] Although the protein content in bird's nest is high, the glycoprotein digestion-resistant components account for a high proportion, and the digestion products include insoluble matter and high molecular weight proteins, which cannot be effectively absorbed and utilized. In addition, the solubility of total sugars in bird's nest polypeptides is less than 40%, and the mass fraction of undissolved sialic acid is about 56%. Caco-2 cell simulated absorption also shows that the absorption rate of bird's nest digestion products is limited. There are many studies on the preparation methods of existing bird's nest peptides. For example, patent CN110468176A discloses a preparation method for obtaining bird's nest small molecule peptides by enzymatic hydrolysis of bromelain, patent CN115807049A discloses a preparation method for bird's nest peptides with high antioxidant activity, and patent CN115261433A discloses a processing method for highly active anti-fatigue peptides. However, the bird's nest peptides prepared by these methods have a large molecular weight, high peptide content, and low sugar content and sialic acid content. At present, there are few studies on the inhibition of elastase activity and the anti-inflammatory effect of bird's nest peptides. Summary of the invention

[0005] In order to solve the above technical problems, make full use of the efficacy of polypeptides in bird's nests, promote digestion and absorption of bird's nests in the human body, improve people's health level and the application value of bird's nests, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a bird's nest peptide I having skin elasticity protection and anti-inflammatory effects, wherein the amino acid sequence thereof is DARFEDLPVY (SEQ ID NO: 1).

[0007] In a second aspect, the present invention provides a polypeptide mixture comprising the bird's nest peptide I described in the first aspect.

[0008] Preferably, the glycopeptide ratio in the polypeptide mixture is 0.2-0.8, for example: 0.20, 0.40, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80.

[0009] Preferably, the sialic acid content in the polypeptide mixture is 9-18wt%, for example: 9.0wt%, 11.0wt%, 13.0%, 15.0wt%, 16.25wt%, 17.50wt%, 18.0wt%.

[0010] Preferably, the content of polypeptides with a relative molecular mass of 4000-6000Da in the polypeptide mixture is ≥60%, for example: 60%, 60.25%, 65.38%, 68.31%, 70%, 75%, 80%.

[0011] Preferably, the content of sugar chains with a relative molecular mass of 3000-4000 Da in the polypeptide mixture is ≥55%, for example: 55%, 56.28%, 58.30%, 60.25%, 63.15%, 66.37%, 70.25%.

[0012] Preferably, the content of peptide chains with a relative molecular mass of 1000-2000 Da in the polypeptide mixture is ≥ 65%, for example: 65%, 66.35%, 68.25%, 70.24%, 70.26%, 72.35%, 75.16%.

[0013] In a third aspect, the present invention provides a method for preparing the polypeptide mixture according to the second aspect, which specifically comprises the following steps:

[0014] (1) soaking and crushing the bird's nest to obtain bird's nest slurry;

[0015] (2) adding protease to the bird's nest slurry, performing enzymatic hydrolysis under a hydrostatic pressure of 100 to 300 MPa, then inactivating the enzyme, and centrifuging to obtain a supernatant;

[0016] (3) The supernatant is subjected to alcohol precipitation, centrifugation, rotary evaporation, and drying to obtain the polypeptide mixture.

[0017] Preferably, the soaking in step (1) is performed by adding the bird's nest into water and then mixing it thoroughly.

[0018] Furthermore, the mass of the water is 20 to 50 times the dry weight of the bird's nest, for example, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, or 50 times.

[0019] Preferably, the expanded bird's nest is crushed by a colloid mill, and the grinding gap of the colloid mill is 80-120 μm, so that the particles in the bird's nest slurry reach the micron level.

[0020] Preferably, the grinding time of the colloid mill is 20 to 40 min, for example: 20 min, 25 min, 30 min, 35 min, 40 min.

[0021] Preferably, the protease in step (2) is selected from any one or more combinations of flavor protease, papain, and alkaline protease.

[0022] Furthermore, when the protease is a flavor protease, the pH of the bird's nest slurry is adjusted to 7.0-8.0, and the flavor protease is added at an enzyme-substrate ratio of 5000-7000 U / mg (enzyme activity / bird's nest dry weight). After mixing, the mixture is vacuum-packed in a polyethylene bag and immediately placed in a high hydrostatic pressure processing (HPP) equipment and treated at 45-55° C. and a hydrostatic pressure of 100-300 MPa for 10-14 hours.

[0023] Furthermore, when the protease is papain, the pH of the bird's nest slurry is adjusted to 5.5-7.5, and papain is added at an enzyme-substrate ratio of 5000-7000 U / mg (enzyme activity / bird's nest dry weight). After mixing, the mixture is vacuum-packed in a polyethylene bag and immediately placed in a high hydrostatic pressure processing (HPP) equipment and treated at 37-60° C. and a hydrostatic pressure of 100-300 MPa for 10-14 hours.

[0024] Furthermore, when the protease is alkaline protease, the pH of the bird's nest slurry is adjusted to 9.5-10.5, and alkaline protease is added at an enzyme-substrate ratio of 5000-7000 U / mg (enzyme activity / dry weight of the bird's nest). After mixing, the mixture is vacuum-packed in a polyethylene bag and immediately placed in a high hydrostatic pressure treatment (HPP) equipment and treated for 10-15 hours at 35-50°C and a hydrostatic pressure of 100-300 MPa.

[0025] Preferably, the enzyme inactivation temperature in step (2) is 90-100° C., for example, 90° C., 92° C., 95° C., 97° C., 100° C. The enzyme inactivation time is 10-30 min, for example, 10 min, 15 min, 20 min, 25 min, 30 min.

[0026] Preferably, the centrifugal speed in step (2) is 5000-10000 r / min, for example: 5000 r / min, 6000 r / min, 7000 r / min, 8000 r / min, 9000 r / min, 10000 r / min.

[0027] Preferably, the centrifugation time in step (2) is 15 to 30 min, for example: 15 min, 20 min, 25 min, 30 min.

[0028] Preferably, the process of alcohol precipitation, centrifugation and rotary evaporation in step (3) is repeated at least once.

[0029] Preferably, during the alcohol precipitation in step (3), the volume fraction of anhydrous ethanol is 40-60% of the total system, for example: 40%, 45%, 50%, 55%, 60%.

[0030] Preferably, the alcohol precipitation time in step (3) is 10 to 30 min, for example: 10 min, 15 min, 20 min, 25 min, 30 min.

[0031] Preferably, the centrifugal speed in step (3) is 3000-5000 r / min, for example: 3000 r / min, 3500 r / min, 4000 r / min, 4500 r / min, 5000 r / min.

[0032] Preferably, the centrifugation time in step (3) is 15 to 30 min, for example: 10 min, 15 min, 20 min, 25 min, 30 min.

[0033] Preferably, the rotary evaporation speed in step (3) is 100-200 r / min, for example: 100 r / min, 120 r / min, 150 r / min, 180 r / min, 200 r / min.

[0034] Preferably, the rotary evaporation temperature in step (3) is 30-50°C, for example: 30°C, 35°C, 40°C, 45°C, 50°C.

[0035] Preferably, the drying in step (3) is performed by freeze drying or low-temperature drying, and freeze drying is more preferably performed.

[0036] Furthermore, the freeze-drying is to firstly quick-freeze the concentrated liquid after rotary evaporation and then freeze-dry it.

[0037] Furthermore, the freeze-drying temperature is -90 to -60°C, for example: -90°C, -85°C, -80°C, -75°C, -70°C, -65°C, -60°C.

[0038] Furthermore, the quick freezing time is 10 to 12 hours, for example: 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours.

[0039] Furthermore, the freeze-drying time is 12 to 24 hours, for example: 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours.

[0040] In a fourth aspect, the present invention provides a composition comprising the bird's nest peptide I described in the first aspect or the polypeptide mixture described in the second aspect.

[0041] Preferably, the composition comprises food, health care product, cosmetic or medicine.

[0042] Preferably, the composition comprises auxiliary materials allowed to be added to food, health products, cosmetics or medicines.

[0043] In a fifth aspect, the present invention provides the use of the bird's nest peptide I described in the first aspect or the polypeptide mixture described in the second aspect in the preparation of a product having the function of inhibiting elastase.

[0044] Preferably, the product is food, health care product, cosmetic or medicine.

[0045] Preferably, the product contains auxiliary materials that are allowed to be added to food, health products, cosmetics or medicines.

[0046] Furthermore, the dosage of the bird's nest peptide I is 10 mg / kg body weight / day to 30 mg / kg body weight / day, for example: 10 mg / kg body weight / day, 15 mg / kg body weight / day, 20 mg / kg body weight / day, 25 mg / kg body weight / day, 30 mg / kg body weight / day.

[0047] In a sixth aspect, the present invention provides the use of the bird's nest peptide I described in the first aspect or the polypeptide mixture described in the second aspect in the preparation of an anti-inflammatory product.

[0048] Preferably, the anti-inflammatory product is a cosmetic or a medicine.

[0049] Preferably, the anti-inflammatory product contains auxiliary materials that are allowed to be added to cosmetics or medicines.

[0050] Furthermore, the dosage of the bird's nest peptide I is 10 mg / kg body weight / day to 30 mg / kg body weight / day, for example: 10 mg / kg body weight / day, 15 mg / kg body weight / day, 20 mg / kg body weight / day, 25 mg / kg body weight / day, 30 mg / kg body weight / day.

[0051] Beneficial effects of the present invention:

[0052] The bird's nest peptide I in the present invention has the dual effects of inhibiting elastase and anti-inflammatory, and its elastase inhibition rate is 96.60%, which can inhibit the degradation of elastin in the skin and delay skin aging. The polypeptide mixture containing the bird's nest peptide has broad application prospects in the fields of cosmetics, food, medicine, etc., promotes the further development of bird's nest products, and improves the economic value and utilization value of bird's nest. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Shown is a schematic diagram of the process of separation, identification and functional research of bird's nest peptides in the present invention. DETAILED DESCRIPTION

[0054] The technical scheme of the present invention is described clearly and completely below in conjunction with the embodiments and drawings. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0055] It should be noted that, unless otherwise specified, the experimental methods and reagents used in the embodiments of the present invention are conventional experimental methods and reagents in the art.

[0056] The main reagents used in the examples are as follows:

[0057] Flavor protease (S10153, Shanghai Yuanye Biotechnology Co., Ltd.), papain (S10011, Shanghai Yuanye Biotechnology Co., Ltd.), alkaline protease (S10154, Shanghai Yuanye Biotechnology Co., Ltd.).

[0058] Fmoc-Tyr(tBu)-Wang Resin (substitution degree 0.4 mmol / g, cross-linking degree 1%, particle size 100-200 mesh), Fmoc-Lys(Boc)-Wang Resin (substitution degree 0.4 mmol / g, cross-linking degree 1%, particle size 100-200 mesh), Fmoc-Asp-OH, Fmoc-Ala-OH, Fmoc-Arg-OH, Fmoc-Phe-OH, Fmoc-Glu-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Val-OH, Fmoc-Tyr-OH, Fmoc-Gly-OH, Fmoc-Thr-OH, Fmoc-Ser-OH, and Fmoc-Cys-OH were all purchased from Nanjing Peptide Biotechnology Co., Ltd.

[0059] Example 1 Preparation of polypeptide mixture

[0060] 1.1 Preparation and application of peptide mixture

[0061] (1) Soaking the bird's nest: Soak the bird's nest (by dry weight) in 20 times the amount of water at 25°C for 2 hours.

[0062] (2) Colloid mill grinding: The colloid mill tooth gap is set to 100 μm, and the slurry obtained in step (1) is subjected to colloid mill treatment for 30 min.

[0063] (3) High hydrostatic pressure assisted enzymatic hydrolysis: The pH value of the solution obtained in step (2) was adjusted to 7.5 using HCL and NaOH, and flavor protease (enzyme dosage 7000 U / mg) was added. After mixing, the mixture was vacuum packed in a polyethylene bag and immediately placed in an HPP equipment for treatment at 50°C and a hydrostatic pressure of 200 MPa for 12 h.

[0064] (4) Inactivation of enzyme and centrifugation: The enzymatic solution obtained in step (3) was inactivated at 95°C for 20 min, cooled to room temperature, centrifuged at 5000 rpm for 20 min, and the supernatant was collected.

[0065] (5) Secondary alcohol precipitation and rotary evaporation concentration: Add anhydrous ethanol to the supernatant obtained in step (4) so ​​that the ethanol volume fraction in the final system reaches 60%, and stir to mix. Let it stand for more than 10 minutes, centrifuge at 3500r / min for 20 minutes to separate the precipitate and the supernatant, transfer all the supernatant to a rotary evaporator, set the water bath temperature to 40°C, and rotary evaporate at 100r / min to remove ethanol until the volume in the bottle remains basically unchanged. Pour out the concentrated solution, add a small amount of water to rinse the rotary evaporator for recovery, and measure the total volume of the concentrated solution. According to the measured volume of the concentrated solution, add anhydrous ethanol again to make the ethanol volume fraction in the final system reach 60%, and stir to mix. Let it stand for more than 10 minutes, centrifuge at 3500r / min for 20 minutes to separate the precipitate and the supernatant. Take the supernatant again and transfer it to a rotary evaporator for rotary evaporation to remove ethanol.

[0066] (6) Separation and preparation: The concentrated solution obtained in step (5) was dispensed into glass plates, with each plate containing about 25 to 30 mL. After quick freezing at -80°C for 12 h, the mixture was placed in a freeze dryer and freeze-dried for 24 h to obtain a powdered polypeptide mixture.

[0067] (7) Determination of sialic acid content: The enzymatic hydrolysis product powder was added with 1% phosphoric acid solution in a boiling water bath for hydrolysis, and the supernatant was obtained by centrifugation. An equal volume of o-phenylenediamine hydrochloride solution was added and mixed with the supernatant, and the mixture was derivatized in a dark water bath at 80°C for 40 min. After cooling, the mixture was filtered through a 0.45 mm filter membrane, and the sialic acid content of the enzymatic hydrolysis product was determined to be 16.25 wt%.

[0068] (8) Glycopeptide ratio determination: The total sugar content was determined by the phenol-sulfuric acid method. The enzymatic hydrolysis product powder was weighed, 1 mL of 5% phenol and 5 mL of concentrated sulfuric acid were added, vortexed and mixed, and placed at room temperature for 20 min. The absorbance was measured at 490 nm, and the total sugar content of the enzymatic hydrolysis product was 35.46%. The total peptide content was determined by the biuret method. After the enzymatic hydrolysis sample was prepared to an appropriate concentration, biuret (sample to be tested: biuret reagent = 3:2, v / v) was added and mixed evenly on a vortex mixer. The supernatant was taken and the absorbance was measured at 540 nm. The total peptide content of the enzymatic hydrolysis product was 47.28%, and the glycopeptide ratio of the enzymatic hydrolysis product was 0.75.

[0069] (9) Determination of molecular weight distribution of polypeptides: The molecular weight distribution of the polypeptide mixture was first determined by high performance liquid chromatography, and then the deglycosylation enzyme PNCase F was used to specifically break the glycopeptide bonds to completely separate the sugar chains in the polypeptides. Then, a Sephadex G-100 polysaccharide gel column was used to collect the components of the bird's nest polysaccharide and determine the molecular weight of the sugar chains in the polypeptide mixture. The molecular weight distribution of the peptide chains in the polypeptide mixture was determined by high performance liquid chromatography. The results are shown in Table 1.

[0070] Table 1 Relative molecular weight distribution of peptide mixture

[0071]

[0072] As can be seen from Table 1, the relative molecular weight distribution of the polypeptide mixture is mainly concentrated in 4000-6000Da, and the percentage of peptides with this molecular weight reaches 68.31%, the relative molecular weight distribution of sugar chains is mainly concentrated in 3000-4000Da, and the percentage of sugar chains with this molecular weight reaches 66.37%, and the relative molecular weight distribution of peptide chains is mainly concentrated in 1000-2000Da, and the percentage of peptide chains with this molecular weight reaches 70.26%.

[0073] Example 2 Amino Acid Sequence Analysis of Peptide Mixture

[0074] Ultra-high performance liquid chromatography ionization tandem mass spectrometry (UPLC-ESI-MS / MS) was used to sequence and identify the peptide chain amino acid sequence in the peptide mixture. Specific chromatographic conditions: Acclaim PepMap C 18 Chromatographic 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 volume 5.0 μL, elution flow rate 300.0 nL / min. Mass spectrometer positive charge spray voltage 2.0 kV.

[0075] The Mascot Server online system is then used to compare the database and select the samples with high matching degree and relative intensity greater than 10 9 De novo analysis was performed on the 5 peptides, and the results are shown in Table 2.

[0076] Table 2 Peptide chain amino acid sequences in peptide mixtures

[0077]

[0078]

[0079] Example 3 Synthesis of bird's nest peptide

[0080] 3.1 Synthesis of bird's nest peptide I (SEQ ID NO: 1)

[0081] The bird's nest peptide (Asp-Ala-Arg-Phe-Glu-Asp-Leu-Pro-Val-Tyr, SEQ ID NO: 1) was synthesized using a solid phase synthesis process in the direction from C-terminus to N-terminus. The specific process is as follows:

[0082] Weigh 4.0g Fmoc-Tyr(tBu)-Wang Resin with a substitution degree of 0.4mmol / g, add it to the solid phase reaction column, add 20mL DMF to wash twice, remove the solvent, add 60mL DMF to swell for 30min. Wash twice with DMF, add piperidine-DMF mixed solution (volume ratio 1:3) and stir for 20min, and monitor the reaction completion by ninhydrin colorimetry. Wash with DMF and DCM 5 times each, dissolve 2.17g (6.40mmol) Fmoc-Val-OH and 1.04g (7.60mmol) HOBt in DMF, add 1.20mL (7.6mmol) DIC under ice bath conditions, stir for 8min in dark, then add it to the solid phase reaction column from which the solvent has been removed, add 0.08g (0.64mmol) DMAP, stir and react for 3h under nitrogen protection, and monitor the reaction completion by ninhydrin colorimetry. The solvent was removed and the resin was washed with DMF for 5 times to obtain Fmoc-Val-Tyr-Wang Resin. According to the above coupling method, the corresponding Fmoc protected amino acids were added in sequence according to the peptide sequence to condense and couple to extend the peptide chain. After the last coupling reaction, the resin was washed with DCM, DMF, and MeOH for 4 times each.

[0083] The resin was dried with nitrogen and transferred to a round-bottom flask. It was cut off three times with 209 mL of acetic acid (HOAc)-trifluoroethanol (TFE)-DCM mixed solution (volume ratio 1:3:6), and the reaction time was 30 min, 15 min, and 5 min, respectively. Filter by suction, and when the filtrate is concentrated to one-fourth of the original volume, the concentrate is added to 10 times the amount of ether for precipitation, and allowed to stand in a refrigerator overnight. Filter by suction, wash the filter cake with a small amount of ether 6 times, and vacuum dry to obtain the fully protected crude bird's nest peptide with a yield of 90.60%.

[0084] The synthesized bird's nest peptide was analyzed and identified according to the method of Example 2: sequencing and identification methods were used.

[0085] The crude bird's nest peptide was dissolved in an appropriate amount of DMSO and separated and purified by high performance liquid chromatography, the chromatographic column was a C18 reverse phase column, the eluent: liquid A was a 0.1% TFA aqueous solution, liquid B was an acetonitrile aqueous solution containing 0.1% TFA, and the detection wavelength was 220nm. The purified liquid was freeze-dried to obtain a finished bird's nest peptide with a purity of 99.2%.

[0086] 3.2 Synthesis of bird's nest peptides II-V

[0087] According to the method in 3.1, four peptide chains with amino acid sequences as shown in SEQ ID NOs: 2 to 5 were synthesized.

[0088] Example 4 Determination of the Elastase Inhibition Rate of Bird's Nest Peptides

[0089] The elastase inhibition rate reaction system was prepared according to Table 3, and the elastase inhibition rate was calculated according to formula (1). The specific process is as follows:

[0090] Take 2 mL of 2 mg / mL elastase PPE solution prepared with 37°C preheated borate solution, add 2 mL of flavor protease hydrolyzate solution of different concentrations, vortex mix thoroughly, and shake on a shaker at 37°C and 400 r / min for 20 min. Immediately add 5 mL of 0.5 mol / L phosphate buffer (pH 6.0), vortex mix, centrifuge at 5000 r / min for 15 min, take the supernatant and measure its absorbance at 495 nm. Calculate the elastase inhibition rate of bird's nest peptides, the results are shown in Table 4.

[0091]

[0092] Table 3 Elastase inhibition reaction system composition and volume

[0093]

[0094] Table 4 Elastase inhibition rate of bird's nest peptides

[0095]

[0096] Note: Bird's nest peptides I to V represent sequence 1 (SEQ ID NO: 1) to sequence 5 (SEQ ID NO: 5) respectively

[0097] As can be seen from Table 4, the elastase inhibition rate of bird's nest peptides Ⅰ to Ⅲ is significantly higher than that of bird's nest peptides Ⅳ to Ⅴ and unhydrolyzed bird's nest, all higher than 70%. Compared with unhydrolyzed bird's nest, the elastase inhibition rates of bird's nest peptides Ⅰ to Ⅲ increased by 48.21%, 32.36% and 21.79%, respectively, indicating that bird's nest peptides Ⅰ, Ⅱ and Ⅲ in the bird's nest polypeptide mixture prepared by high hydrostatic pressure assisted enzymatic hydrolysis combined with secondary alcohol precipitation have a significant inhibitory effect on elastase. Bird's nest peptides Ⅰ to Ⅴ and unhydrolyzed bird's nest can both delay the degradation of skin elastin and the loss of skin elasticity, among which bird's nest peptides Ⅰ to Ⅲ have stronger effects and are more suitable for the development of beauty, health care or pharmaceutical products related to anti-skin aging.

[0098] Example 5: Improvement of blood inflammation in mice by bird's nest peptide

[0099] 5.1 Establishment of animal model: 32 8-week-old C57BL / 6J male mice were selected. According to the dosing design, they were divided into blank control group, model group, bird's nest group and bird's nest peptide group, with 8 mice in each group. The adaptation period for all mice was 1 week. From the second week, the control group and the model group were gavaged with 0.2 mL of 0.9% saline every day, the bird's nest group was gavaged with an equal volume of bird's nest, and the bird's nest peptide group was gavaged with an equal volume of bird's nest peptide (200 mg / kg / d) for 7 consecutive weeks. In the last week of feeding, the control group was intraperitoneally injected with 0.2 mL of 0.9% saline every day, and the other groups were intraperitoneally injected with an equal volume of LPS solution (2 mg / kg) for 1 week, fasted for 12 hours, and the mice were killed.

[0100] 5.2 Determination of blood indexes: Use 50 μL of fresh whole blood to count blood cells using a fully automatic blood cell analyzer, including inflammatory cells such as white blood cells, neutrophils, and lymphocytes. Take an appropriate amount of fresh blood, let it stand at room temperature for 30 minutes, centrifuge it at 4°C and 3000r / min for 15 minutes, take the supernatant to prepare serum, and use a commercial Elisa kit to determine the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in serum. The operating steps follow the instructions of the kit, measure the absorbance of the 96-well plate at 490nm, and quantify the inflammatory factors according to the standard curve. The effects of bird's nest peptides on mouse blood indicators are shown in Table 5, and the effects on mouse blood inflammatory factors are shown in Table 6.

[0101] Table 5 Effects of bird's nest peptide on blood indexes of mice

[0102]

[0103] Note: Bird's nest peptide group I to group V represent sequence 1 (SEQ ID NO: 1) group to sequence 5 (SEQ ID NO: 5) group respectively.

[0104] As can be seen from Table 5, there are significant differences in the content of leukocytes, neutrophils, monocytes, and lymphocytes between the model group and the normal group (p<0.05), indicating that the modeling was successful. After the intervention of mice, each intervention group (bird's nest group, bird's nest peptide group) had a significant correction effect on the content of leukocytes, neutrophils, monocytes, and lymphocytes in the blood of mice compared with the model group. There was no significant difference between the bird's nest peptide group I to III and the normal group. However, compared with the bird's nest group, the correction effect of the blood index of mice taking bird's nest peptides was more obvious, and the effect of bird's nest peptides I to III was better.

[0105] Table 6 Effects of bird's nest peptides on inflammatory factors in mouse blood

[0106]

[0107] Note: Bird's nest peptide group I to group V represent sequence 1 (SEQ ID NO: 1) group to sequence 5 (SEQ ID NO: 5) group respectively.

[0108] As can be seen from Table 6, compared with the normal group, the three inflammatory factors (TNF-α, IL-1β, IL-6) in the model group were significantly different (p<0.05), indicating that the modeling was successful. After the intervention in the mice, each intervention group (bird's nest group, bird's nest peptide group) had a significant correction effect on the TNF-α, IL-1β, and IL-6 content in the mouse blood compared with the model group. There was no significant difference between the bird's nest peptide group I to III and the normal group. However, compared with the bird's nest group, the mice taking bird's nest peptides I to III had a more obvious correction effect on inflammatory factors in the blood. It shows that the bird's nest peptide prepared by the present invention has a significant improvement effect on mouse blood inflammation, and the effect of bird's nest peptides I to III is better.

[0109] Example 6: Improvement of brain inflammation in mice by bird's nest peptide

[0110] 6.1 Animal model establishment: The animal model establishment method is the same as that in 5.1 of Example 5.

[0111] 6.2 Determination of brain inflammatory factors: After obtaining the mouse cerebral cortex, the cerebral cortex was homogenized and the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the serum were determined using a commercial Elisa kit. The operation steps were guided by the instructions of the kit, and the absorbance of the 96-well plate was measured at 490 nm. The inflammatory factors were quantified according to the standard curve. The results are shown in Table 7.

[0112] Table 7 Effects of bird's nest peptides on inflammatory factors in the brain of mice

[0113]

[0114] Note: Bird's nest peptide group I to group V represent sequence 1 (SEQ ID NO: 1) group to sequence 5 (SEQ ID NO: 5) group respectively.

[0115] As can be seen from Table 7, compared with the normal group, the three inflammatory factors (TNF-α, IL-1β, IL-6) in the model group were significantly different (p<0.05), indicating that the modeling was successful. After the intervention of the mice, the intervention group (bird's nest group, bird's nest peptide group) had a significant correction effect on the TNF-α, IL-1β, and IL-6 content in the cerebral cortex of the mice compared with the model group. There was no significant difference between the bird's nest peptide group I to III and the normal group. However, compared with the bird's nest group, the mice taking bird's nest peptides I to III had a more obvious correction effect on the inflammatory factors in the cerebral cortex. It shows that the bird's nest peptide prepared by the present invention has a significant improvement effect on mouse brain inflammation, and the effect of bird's nest peptides I to III is better.

[0116] Example 7: Improvement of liver inflammation in mice by bird's nest peptide

[0117] 7.1 Animal model establishment: The animal model establishment method is the same as that in 5.1 of Example 5.

[0118] 7.2 Determination of liver inflammatory factors: After obtaining the mouse liver, the liver was homogenized and the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the serum were determined using a commercial Elisa kit. The operation steps were guided by the instructions of the kit, and the absorbance of the 96-well plate was measured at 490 nm. The inflammatory factors were quantified according to the standard curve. The results are shown in Table 8.

[0119] Table 8 Effects of bird's nest peptides on inflammatory factors in mouse liver

[0120]

[0121]

[0122] Note: Bird's nest peptide group I to group V represent sequence 1 (SEQ ID NO: 1) group to sequence 5 (SEQ ID NO: 5) group respectively.

[0123] As can be seen from Table 8, compared with the normal group, the three inflammatory factors (TNF-α, IL-1β, IL-6) in the model group were significantly different (p<0.05), indicating that the modeling was successful. After the intervention in the mice, each intervention group (bird's nest group, bird's nest peptide group) had a significant correction effect on the content of TNF-α, IL-1β, and IL-6 in the mouse liver compared with the model group. There was no significant difference between the bird's nest peptide group I to III and the normal group. However, compared with the bird's nest group, the mice taking bird's nest peptides I to III had a more obvious correction effect on inflammatory factors in the liver. It shows that the bird's nest peptide prepared by the present invention has a significant improvement effect on mouse liver inflammation, and the effect of bird's nest peptides I to III is better.

[0124] Example 8: Improvement of lung inflammation in mice by bird's nest peptide

[0125] 8.1 Animal model establishment: The animal model establishment method is the same as that in Example 5, Section 5.1.

[0126] 8.2 Determination of pulmonary inflammatory factors: After obtaining the mouse lungs, the lungs were homogenized and the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the serum were determined using a commercial Elisa kit. The operating steps were guided by the instructions of the kit, and the absorbance of the 96-well plate was measured at 490 nm. The inflammatory factors were quantified according to the standard curve. The results are shown in Table 9.

[0127] Table 9 Effects of bird's nest peptides on inflammatory factors in the lungs of mice

[0128]

[0129] Note: Bird's nest peptide group I to group V represent sequence 1 (SEQ ID NO: 1) group to sequence 5 (SEQ ID NO: 5) group respectively.

[0130] As can be seen from Table 9, compared with the normal group, the three inflammatory factors (TNF-α, IL-1β, IL-6) in the model group were significantly different (p<0.05), indicating that the modeling was successful. After the intervention of the mice, each intervention group (bird's nest group, bird's nest peptide group) had a significant correction effect on the TNF-α, IL-1β, and IL-6 content in the mouse lung tissue compared with the model group. There was no significant difference between the bird's nest peptide group I to III and the normal group. However, compared with the bird's nest group, the mice taking the bird's nest peptide had a more obvious correction effect on the inflammatory factors in the lung tissue. It shows that the bird's nest peptide prepared by the present invention has a significant improvement effect on lung inflammation, and the effect of bird's nest peptide I to III is better.

[0131] The above experiments show that the bird's nest peptide prepared by the method of high hydrostatic pressure-assisted enzymatic hydrolysis combined with secondary alcohol precipitation has a significant improvement effect on inflammation in the blood, brain, liver, and lungs of mice, and the improvement effect is higher than that of the group taking bird's nest, indicating that decomposing bird's nest into polypeptides with smaller molecular weight can better exert its anti-inflammatory effect, and it can be applied to the production of food, health products, and medicines.

[0132] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A bird's nest peptide I with skin elasticity protection and anti-inflammatory effects, characterized in that: The amino acid sequence of the bird's nest peptide I is DARFEDLPVY.

2. A composition, characterized in that: The composition comprises the bird's nest peptide I as claimed in claim 1, and the composition further comprises auxiliary materials allowed to be added to food, health products, cosmetics or medicines.

3. Use of the bird's nest peptide I according to claim 1 in the preparation of food, health products or cosmetics having the ability to inhibit elastase activity.

4. The use according to claim 3, characterized in that: The dosage of the bird's nest peptide I is 10 mg / kg body weight / day to 30 mg / kg body weight / day.

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