Manufacturing method and application of bird's nest peptide that promotes cell repair and has high moisturizing and whitening effects

Abstract

The present application relates to a method for producing bird's nest peptides, the method including the steps of: mixing bird's nest with water and stewing to prepare stewed bird's nest; homogenizing the stewed bird's nest to prepare a bird's nest paste; enzymatically hydrolyzing the bird's nest paste with alkaline protease to inactivate the enzyme and prepare an enzymatic hydrolyzate; centrifuging the enzymatic hydrolyzate, recovering the supernatant, and preparing a crude sample solution of bird's nest peptides; and ultrafiltrating the crude sample solution of bird's nest peptides and recovering the ultrafiltrate, wherein the centrifugation conditions include a speed of about 10,000-15,000 rpm and a time of about 10-30 minutes, and the pore size of the membrane used for ultrafiltration is about 100-200 nm.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to a Chinese patent application filed on November 9, 2022, bearing application number 2022113980483 and entitled "Preparation and application of bird's nest peptide with promoting cell repair and high moisturizing and whitening effects," the entire contents of which are incorporated herein by reference. [Technical field]

[0002] The present application relates to the technical field of food processing, and in particular to a method for producing and application of bird's nest peptides which promote cell repair and have high moisturizing and whitening effects. [Background technology]

[0003] Researchers have found that bird's nest peptides, which are produced after bird's nest is hydrolyzed by protease, have excellent effects such as lowering blood pressure and anti-virus. In particular, bird's nest peptides are superior to bird's nest in some functions. In addition, bird's nest peptides have a relatively low molecular weight, so they are easily absorbed by the human body and are believed to have the ability to be absorbed transdermally. Based on the above advantages, bird's nest peptides are expected by consumers, manufacturers, and researchers in the fields of food, medicine, skin care products, etc., and are considered to be an active substance with a wide range of future applications.

[0004] A conventional method for producing bird's nest peptides is, for example, as follows.

[0005] CN107668312A describes a complex defoaming method for bird nest protein liquid, which includes the steps of first adjusting the pH value of the bird nest liquid obtained after boiling bird nests in water to 8 with 0.1 mol / L NaHCO3 solution, then adding alkaline protease to partially enzymatically decompose the liquid, inactivating the enzyme in boiling water for 3 minutes, and cooling the liquid, and further repeating vacuum high-low speed whipping and high-low temperature defoaming three times.

[0006] CN111528332A includes a soaking and sorting step (1) of soaking dried bird's nest in purified water, removing impurities, crushing, and drying to prepare for use; a boiling and mashing step (2) of adding purified water to the bird's nest soaked in step (1), boiling it, and finely crushing it to obtain bird's nest paste; a first biological enzymatic hydrolysis step (3) of adjusting the concentration and pH of the bird's nest paste produced in step (2), adding neutral protease to the prepared bird's nest paste, stirring to enzymatically hydrolyze it, inactivating the enzyme, cooling, and sieving it through a 100-200 mesh sieve to obtain a first enzymatic hydrolysis solution; and a second biological enzymatic hydrolysis step (4). The present invention describes a method for producing bird's nest peptides, comprising: a homogenization and filtration step (4) of homogenizing the first enzymatic hydrolysis solution produced in step (3) and roughly filtering it to obtain an enzymatic hydrolysis homogenized solution; a second biological enzymatic hydrolysis step (5) of adjusting the concentration and pH of the enzymatic hydrolysis homogenized solution produced in step (4), adding a complex protease to the prepared enzymatic hydrolysis homogenized solution, stirring to perform enzymatic hydrolysis, inactivating the enzymes, and cooling to obtain a second enzymatic hydrolysis solution; and a purification and spray drying step (6) of microfiltrating the second enzymatic hydrolysis solution produced in step (5), concentrating it by reverse osmosis using an RO membrane, and spray drying it to obtain bird's nest peptides.

[0007] CN112006276A includes a step (1) of soaking and sorting the dried bird nests in purified water, removing impurities, crushing, drying, and sieving them for use, and a step (2) of adding an arginine solution to the sieved bird nests in step (1) to prepare bird nest liquid. The method for producing instant bird's nest powder reconstituted in hot water includes steps (2) of preparing bird's nest liquid and stewing the solution, the solution being boiled in hot water and then cooled; (3) of enzymatic decomposition, in which the pH value of the solution treated in step (2) is adjusted to 8-10, alkaline protease is added to partially decompose the solution, and the enzyme is inactivated in high-temperature boiling water to obtain an enzymatic decomposition liquid; (4) of ultrasonic degassing and high-pressure homogenization, in which the enzymatic decomposition liquid prepared in step (3) is whipped in a low-temperature vacuum, degassed with ultrasound, and subjected to a high-pressure homogenization treatment to obtain an enzymatic decomposition homogenized treatment liquid; (5) of spray drying, in which rock sugar and citric acid are added to the enzymatic decomposition homogenized treatment liquid prepared in step (4), and the resulting mixture is spray-dried in a vacuum to obtain bird's nest powder; and (6) of phospholipid spraying, in which phospholipid is sprayed on the surface of the bird's nest powder prepared in step (5) to obtain a hot water reconstituted instant bird's nest powder.

[0008] CN113564218A describes a method for producing whitening active small molecule bird's nest peptides, which includes a raw material pretreatment step (1) of heat-treating dried bird's nest in hot water to obtain a raw material liquid in preparation for use, an enzymatic hydrolysis step (2) of placing the raw material liquid in an enzymatic hydrolysis tank, adjusting the pH, heating it to an elevated temperature, and adding a protease to perform enzymatic hydrolysis to obtain an enzymatic hydrolysis liquid, an enzyme inactivation and ultrafiltration step (3) of inactivating the enzymatic hydrolysis liquid in a boiling water bath, followed by ultrafiltration and recovering the filtrate, and a step (4) of spray-drying the filtrate to obtain the whitening active small molecule bird's nest peptide powder.

[0009] CN111019984A includes a pretreatment step I of drying and pulverizing the bird's nest raw material to obtain pretreated bird's nest, and a step II of adding water 10-30 times the volume of the pretreated bird's nest to the pretreated bird's nest, stirring uniformly, and then ultrasonicating at 30-45°C for 15-40 minutes to obtain bird's nest paste, and then adding trypsin, alkaline protease, flavorzyme, pepsin, and an enzyme stabilizer to the bird's nest paste, and then oxidizing the trypsin, alkaline protease, flavorzyme, pepsin, and an enzyme stabilizer to the bird's nest paste. The present invention describes an efficient method for extracting sialic acid from bird's nest, comprising: an extraction step II of enzymatically hydrolyzing the enzyme at 40-60°C for 2-7 hours, inactivating the enzyme at 100-105°C for 5-10 minutes, cooling, and obtaining an enzymatic hydrolysis solution; and a filtration and drying step III of centrifuging the enzymatic hydrolysis solution, filtering the obtained supernatant, and subjecting it to vacuum freeze-drying to obtain a product.

[0010] CN115226886A describes a method for preparing a high sialic acid bird's nest product by the extract re-addition method, which includes the steps of (1) preparing a sialic acid extract, which includes soaking crushed bird's nest in water, wet grinding until it becomes millimeter-order granules, and then ultrasonically crushing the crushed bird's nest, followed by adding alkaline protease for enzymatic decomposition, inactivating the enzyme, centrifuging, and filtering to obtain an extract rich in sialic acid, and (2) re-adding the extract, which includes soaking the re-added dried bird's nest in the extract, then putting it into an ultrasonic device for ultrasonic swelling, and finally whipping it into threads to remove impurities, dehydrating, and stewing to obtain a high sialic acid bird's nest product.

[0011] CN111096462B includes a step S1 of soaking the bird's nest, in which water is added to the bird's nest in a ratio of 1:40 and the bird's nest is soaked at room temperature for 5 to 12 hours; a step S2 of stewing the soaked bird's nest in a water bath at 95°C to 100°C for 1 to 2 hours; a step S3 of homogenization, in which the stewing bird's nest aqueous solution is cooled and then homogenized 1 to 2 times with a homogenizer; and a step S4 of homogenization, in which alkaline protease, neutral protease, and glyceryl stearate are added to the homogenization solution. The alkaline protease contains 45-60% water, 5-10% protease A, 35-45% propylene glycol, and 0.2-0.4% calcium chloride, and the neutral protease contains 35-50% water, 5-10% protease B, 33-45% sorbitol, 7-10% propylene glycol, and 0.2-0.4% calcium chloride. The enzyme hydrolysis step S4 contains ethylene glycol and 5% sodium chloride, protease A is a product of Bacillus licheniformis, and protease B is a product of Bacillus amyloliquefaciens; the enzyme deactivation step S5 inactivates the obtained enzyme hydrolysis solution at 80-90°C for 15-20 minutes; the membrane filtration step S6 in which the temperature of the deactivated supernatant is lowered and then the supernatant is filtered through a membrane filtration, the filtration range being 100-120 mm; and the supernatant after filtration is centrifuged to remove residue, the centrifugation speed being set at 100-120 mm. The present invention describes a non-polluting bird's nest polypeptide that has been bioenzyme-degraded and purified, and a method for removing bitterness thereof, the method including a centrifugation step S7 at 3000 to 5000 rpm, a supernatant collection step S8 of recovering the supernatant after centrifugation to obtain a supernatant, a microencapsulation step S9 of preparing a β-cyclodextrin solution with a mass ratio of bird's nest supernatant to β-cyclodextrin of 1:4 to 6, and a freeze-drying step S10 of stabilizing the solution at -35°C to -36°C, freeze-drying the solution, and pulverizing the solution to obtain a microencapsulated bird's nest peptide product.

[0012] CN109852656A includes the steps of: (1) crushing bird's nest into bird's nest powder, soaking the bird's nest powder in water at a solid-liquid ratio of 1:15, and crushing the bird's nest paste with a French press; (2) adding 3% of the bird's nest powder's weight of protease to the bird's nest paste, enzymatically hydrolyzing the mixture for 3 hours, inactivating the enzyme in a boiling water bath, and centrifuging the mixture to obtain a supernatant Y1 and a precipitate Y2 after enzymatic hydrolysis; and (3) taking the precipitate Y2 and resuspending it in water at a solid-liquid ratio of 1:20 to prepare a bird's nest resuspension, and then adding 1% of the bird's nest powder's weight of protease to the bird's nest resuspension. The present invention relates to a method for producing bird's nest polypeptide powder, the method including the steps of: (1) enzymatically hydrolyzing the bird's nest polypeptide for 2 hours, inactivating the enzyme in a boiling water bath, and centrifuging to obtain a post-enzymatic hydrolysis supernatant Y3 and a precipitate Y4; (2) mixing the post-enzymatic hydrolysis supernatant Y1 and the post-enzymatic hydrolysis supernatant Y3 to obtain a post-enzymatic hydrolysis supernatant Y5; (3) desalting the post-enzymatic hydrolysis supernatant Y5 using a reverse osmosis membrane and filtering it using a membrane filtration device to obtain a bird's nest polypeptide purified solution; and (4) freeze-drying the post-enzymatic hydrolysis supernatant to obtain bird's nest polypeptide powder.

[0013] However, the bird's nest peptides produced by the conventional production methods and bird's nest raw materials are often not identical in terms of components, and their functional properties and application fields have not been characterized or clarified. The present application has been proposed in light of such circumstances. Summary of the Invention [Problem to be solved by the invention]

[0014] Therefore, according to various embodiments of the present application, a method for producing and applying bird's nest peptide that promotes cell repair and has high moisturizing and whitening effects is provided, and the technical means are as follows: [Means for solving the problem]

[0015] In a first aspect of the present application, mixing and stewing bird nests with water to prepare stewing bird nests; homogenizing the boiled bird's nest to prepare a bird's nest paste; The swallow nest paste is enzymatically decomposed with alkaline protease to inactivate the enzyme, thereby preparing an enzymatic decomposition product; Centrifugation of the enzymatic digestion product and recovery of the supernatant to prepare a crude sample solution of bird's nest peptides; and performing ultrafiltration on the crude sample solution of bird's nest peptides and recovering the ultrafiltrate to prepare bird's nest peptides. The centrifugation conditions include a speed of about 10,000 rpm to 15,000 rpm and a time of about 10 min to 30 min. The pore size of the membrane used for ultrafiltration is about 100 nm to 200 nm. A method for manufacturing the same is provided.

[0016] In some embodiments of the present application, the pore size of the membrane used for ultrafiltration is about 150 nm to 200 nm.

[0017] In some embodiments of the present application, the amount of the bird's nest used per 100 mL of water is 10 g to 50 g.

[0018] In some embodiments of the present application, the amount of bird's nest used per 100 mL of water is 25 g to 35 g.

[0019] In some embodiments of the present application, the simmering time is 0.5 to 2 hours, and preferably, the simmering time is 1.75 to 2 hours.

[0020] In some embodiments of the present application, the homogenization conditions include a power of 10 MPa to 30 MPa and a time of 10 min to 30 min, and preferably, the homogenization conditions include a power of 25 MPa to 30 MPa and a time of 20 min to 30 min.

[0021] In some embodiments of the present application, the conditions for enzymatic hydrolysis include a temperature of 50°C to 60°C, a pH value of 5.0 to 9.0, a time of 10 min to 30 min, and an amount of the alkaline protease used per gram of bird's nest of 6 U to 9 U.

[0022] In some embodiments of the present application, the method of enzyme inactivation is enzyme inactivation by heating, and the conditions for enzyme inactivation by heating include heating the reaction system obtained by enzymatic decomposition to 80°C to 100°C and maintaining it for 10 min to 30 min.

[0023] In some embodiments of the present application, the method further comprises removing the solvent in the ultrafiltrate.

[0024] In some embodiments of the present application, the step of removing the solvent comprises concentrating the ultrafiltrate and spray drying the resulting concentrate.

[0025] In some embodiments of the present application, the step of removing the solvent comprises: (1) the volume of the concentrate is 1 / 3 to about 1 / 8 of the volume of the ultrafiltrate; (2) The spray drying conditions have one or more of the following technical features: the inlet air temperature is about 150°C to 200°C, and the exhaust air temperature is > about 70°C.

[0026] In some embodiments of the present application, the spray drying conditions include an inlet air temperature of 165°C-175°C and an exhaust air temperature >90°C.

[0027] In some embodiments of the present application, the bird's nest peptide has a total amino acid content of 66.75 wt% to 68.89 wt%, a sialic acid content of 6.14 wt% to 6.64 wt%, and a water content of 11.01 wt% to 12.01 wt%.

[0028] In a second aspect of the present application, there is provided a bird's nest peptide produced by the method according to the first aspect.

[0029] In a third aspect of the present application there is provided the use of the bird's nest peptide according to the second aspect in the manufacture of a pharmaceutical or food or skin care product having moisturising, whitening, repairing and / or antioxidant effects.

[0030] The details of one or more embodiments of the application are set forth in the description that follows, and other features, objects and advantages of the application will become apparent from the description and the claims. [Brief description of the drawings]

[0031] In order to more clearly describe the technical means in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following briefly describes the drawings that need to be used in the description of the embodiments. It is obvious that the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative labor.

[0032] [Figure 1] 1 is a GPC retention spectrum of bird's nest peptide in Example 1. [Diagram 2] FIG. 1 shows the chemical antioxidant capacity of bird's nest peptides in Example 1, where FIG. A is the DPPH radical scavenging rate of bird's nest peptides at different concentrations, FIG. B is the ABTS radical scavenging rate of bird's nest peptides at different concentrations, FIG. C is the hydroxyl radical scavenging rate of bird's nest peptides at different concentrations, and FIG. D is the total antioxidant capacity value of bird's nest peptides at different concentrations. [Diagram 3] The toxicity of bird nest peptides to HaCat cells, B16 cells and MRC-5 cells in Example 1. Figure A shows the viability of HaCat cells at different bird nest peptide concentrations; Figure B shows the viability of B16 cells at different bird nest peptide concentrations; Figure C shows the viability of MRC-5 cells at different bird nest peptide concentrations; Figure D shows the effect of bird nest peptides on the viability of HaCat cells injured by H2O2; Figure E shows the effect of bird nest peptides on the hyaluronic acid content of HaCat cells; and Figure F shows the inhibition rate of bird nest peptides on melanin production in B16 cells. [Figure 4]1 shows the repair effect of bird's nest peptide on scars in MRC-5 cells in Example 1. [Diagram 5] 1 is a GPC retention spectrum of bird's nest peptide in Example 2. [Figure 6] FIG. 1 shows the chemical antioxidant capacity of bird's nest peptides in Example 2, where FIG. A is the DPPH radical scavenging rate of bird's nest peptides at different concentrations, FIG. B is the ABTS radical scavenging rate of bird's nest peptides at different concentrations, FIG. C is the hydroxyl radical scavenging rate of bird's nest peptides at different concentrations, and FIG. D is the total antioxidant capacity value of bird's nest peptides at different concentrations. [Figure 7] The toxicity of bird nest peptides to HaCat cells, B16 cells and MRC-5 cells in Example 2. Figure A shows the viability of HaCat cells at different bird nest peptide concentrations; Figure B shows the viability of B16 cells at different bird nest peptide concentrations; Figure C shows the viability of MRC-5 cells at different bird nest peptide concentrations; Figure D shows the effect of bird nest peptides on the viability of HaCat cells injured by H2O2; Figure E shows the effect of bird nest peptides on the hyaluronic acid content of HaCat cells; and Figure F shows the inhibition rate of bird nest peptides on melanin production in B16 cells. [Figure 8] 1 shows the repair effect of bird's nest peptide on scars in MRC-5 cells in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Hereinafter, the present application will be described in more detail with reference to the drawings, embodiments and examples. These embodiments and examples are merely for the purpose of explaining the present application and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the disclosure of the present application more complete and complete. Furthermore, the present application should not be construed as being limited to the embodiments and examples described herein, and can be realized in many different forms, and it should be understood that those skilled in the art can make various changes and modifications without departing from the spirit of the present application, and that equivalent forms thereof are also included in the scope of protection of the present application. In addition, in the following description, many specific details are set forth in order to provide a complete understanding of the present application. It should be understood that the present application can be implemented without some or all of these specific details.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In describing this application, the terms used herein are for the purpose of describing the embodiments and examples only, and are not intended to be limiting of this application. term

[0035] Terms or expressions used in this specification have the following meanings unless otherwise stated or contradicted:

[0036] The term "and / or", "or / and", and "and / or" as used herein includes any one item among two or more related listed items, and further includes any and all combinations of related listed items, including any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by a combination of at least two conjunctions selected from "and / or", "or / and", and "and / or", it is understood that in this application, the technical means include all technical means connected by "logical AND" and further include all technical means connected by "logical OR". For example, "A and / or B" includes three meanings: A, B, and A+B. For example, a technical means of "A and / or B and / or C and / or D" includes any one of A, B, C, and D (i.e., all technical means connected by a "logical OR"), includes any and all combinations of A, B, C, and D, i.e., includes any combination of two or three of A, B, C, and D, and further includes four combinations of A, B, C, and D (i.e., all technical means connected by a "logical AND").

[0037] In this application, the terms "plurality," "multiple kinds," "multiple times," "diverse," and the like refer to a quantity of two or more, unless otherwise specified. For example, "one or more" refers to one or two or more.

[0038] As used herein, "combinations thereof," "any combination thereof," "any manner of combination thereof," and the like include all suitable combinations of any two or more of the listed items.

[0039] In this specification, the word "appropriate" in terms of "appropriate combination method," "appropriate method," "any appropriate method," etc., is based on the criterion that the technical means of the present application can be implemented, the technical problem of the present application can be solved, and the desired technical effect of the present application can be achieved.

[0040] It should be understood that in this specification, the terms "preferred", "more preferred", "even more preferred" and "most preferred" are merely intended to describe more effective embodiments or examples, and are not intended to limit the scope of protection of the present application.

[0041] In this application, the terms "further", "also further", "particularly" and the like are for explanatory purposes and indicate the difference in content, but should not be understood as limiting the scope of protection of the application.

[0042] In this application, "optionally", "optional", and "optional" may or may not exist, that is, they refer to either one of the two parallel options "existing" or "not existing". In one technical means, unless otherwise specified, and unless there is a contradiction or mutual constraint, when there are multiple "options", each "option" is independent of each other.

[0043] In the present application, the terms "first," "second," "third," "fourth," etc. in "first embodiment," "second embodiment," "third embodiment," "fourth embodiment," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implying the importance or quantity of the technical features indicated. It should also be understood that "first," "second," "third," and "fourth," etc., serve the purpose of a non-exhaustive enumeration description only and do not constitute closed-type limitations on the quantities.

[0044] In this application, technical features described in an open format include both closed technical means consisting of the recited features and open technical means comprising the recited features.

[0045] In the present application, for a numerical interval (i.e., a numerical range), unless otherwise specified, the selectable numerical distribution is considered to be continuous within the numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the integers at the two endpoints of the numerical range, and each integer between the two endpoints, and in this specification, it is equivalent to directly listing each integer, for example, t being an integer selected from 1 to 10 means that t is any one integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when there are multiple ranges describing a feature or characteristic, they may be combined. In other words, unless otherwise specified, all ranges disclosed in this specification should be understood to include any and all subranges contained therein.

[0046] Unless otherwise specified, the temperature parameters in this application may be constant temperature treatments or may vary within a certain temperature range. It should be understood that the constant temperature treatments allow the temperature to vary within the precision range of the equipment control. For example, variations within the ranges of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0047] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass volume percentage.

[0048] All documents mentioned in this application are incorporated herein by reference in their entirety, as if each document were individually incorporated by reference. Unless inconsistent with the purpose and / or technical means of this application, the cited documents in this application are incorporated herein in their entirety and for all purposes. In this application, when a cited document is mentioned, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In this application, when a cited document is mentioned, examples of relevant technical features incorporated, preferred embodiments may also be incorporated into this application by reference, but are limited to those that can implement this application. In the event that the cited content contradicts the description of this application, it should be understood that the content is adaptively modified based on or in accordance with the description of this application. First aspect of the present application

[0049] The present application, mixing and stewing bird nests with water to prepare stewing bird nests; homogenizing the boiled bird's nest to prepare a bird's nest paste; The swallow nest paste is enzymatically decomposed with alkaline protease to inactivate the enzyme, thereby preparing an enzymatic decomposition product; Centrifugation of the enzymatic digestion product and recovery of the supernatant to prepare a crude sample solution of bird's nest peptides; and performing ultrafiltration on the crude sample solution of bird's nest peptides and recovering the ultrafiltrate to prepare bird's nest peptides. The conditions for centrifugation were a speed of about 10,000 rpm to 15,000 rpm and a time of about 10 min. Including that n~30min. The pore size of the membrane used for ultrafiltration is about 100 nm to 200 nm.

[0050] The centrifugation conditions in this application are, for example, speeds (rpm) of 10,000, 11,000, 12,000, 13,000, 14,000, and 15,000, and times (min) of 10, 12, 15, 17, 20, 22, 25, 28, and 30.

[0051] The pore size (nm) of the membrane used for ultrafiltration in the present application is, for example, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200. Preferably, the pore size of the membrane used for ultrafiltration is 150 nm to 200 nm.

[0052] Preferably, the amount of the bird's nest used per 100 mL of the water is 10 g to 50 g (for example, 10 g, 15 g, 20 g, 25 g, 30 g, 35 g, 40 g, 45 g, 50 g).

[0053] Preferably, the amount of the bird's nest used per 100 mL of the water is 25 g to 35 g (for example, 25 g, 26 g, 27 g, 28 g, 29 g, 30 g, 31 g, 32 g, 33 g, 34 g, 35 g).

[0054] Preferably, the simmering time is 0.5h to 2h (for example, 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, 1.6h, 1.7h, 1.8h, 1.9, or 2h), and preferably, the simmering time is 1.75h to 2h.

[0055] Preferably, the homogenization conditions include a power of 10 MPa to 30 MPa (e.g., 10 MPa, 11 MPa, 12 MPa, 13 MPa, 14 MPa, 15 MPa, 16 MPa, 17 MPa, 18 MPa, 19 MPa, 20 MPa, 21 MPa, 22 MPa, 23 MPa, 24 MPa, 25 MPa, 26 MPa, 27 MPa, 28 MPa, 29 MPa, 30 MPa) and a time of 10 min to 30 min (e.g., 10 min, 12 min, 15 min, 17 min, 20 min, 22 min, 25 min, 28 min, 30 min), and preferably, the homogenization conditions include a power of 25 MPa to 30 MPa and a time of 20 min to 30 min.

[0056] Preferably, the conditions for enzymatic hydrolysis include a temperature of 50°C to 60°C (e.g., 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C), a pH value of 8.0 to 10.0 (e.g., 8, 8.5, 9, 9.5, 10), a time of 5h to 6h (e.g., 5h, 5.1h, 5.2h, 5.3h, 5.4h, 5.5h, 5.6h, 5.7h, 5.8h, 5.9h, 6h), and an amount of the alkaline protease used per gram of bird's nest of 6U to 9U (e.g., 6U, 6.5U, 7U, 7.5U, 8U, 8.5U, 9U).

[0057] Preferably, the method of enzyme inactivation is enzyme inactivation by heating, and the conditions for enzyme inactivation by heating include heating the reaction system obtained by enzymatic decomposition to 80°C to 100°C (e.g., 80°C, 82°C, 84°C, 86°C, 88°C, 90°C, 92°C, 94°C, 96°C, 98°C, 100°C) and holding it for 10 min to 30 min (e.g., 10 min, 12 min, 15 min, 17 min, 20 min, 22 min, 25 min, 28 min, 30 min).

[0058] Preferably, the production method further comprises the step of removing the solvent in the ultrafiltrate.

[0059] Preferably, the step of removing the solvent comprises concentrating the ultrafiltrate and spray drying the resulting concentrate.

[0060] Preferably, the step of removing the solvent comprises: (1) The volume of the concentrate is 1 / 3 to 1 / 8 (e.g., 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8) of the volume of the ultrafiltrate; (2) The spray drying conditions include an intake air temperature of 150°C to 200°C (e.g., 150°C, 160°C, 170°C, 180°C, 190°C, 200°C) and an exhaust air temperature >70°C (e.g., 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, 110°C, 115°C, 120°C), and preferably, the spray drying conditions include an intake air temperature of 165°C to 175°C and an exhaust air temperature >90°C.

[0061] Preferably, the total amino acid content is 66.75 wt% to 68.89 wt%, the sialic acid content is 6.14 wt% to 6.64 wt%, and the water content is 11.01 wt% to 12.01 wt%.

[0062] Preferably, the bird's nest peptide contains 66.75% to 68.89% amino acids per 100 g, and the mass ratio of the amino acids is as follows: essential amino acids are 39.34% to 44.89%, semi-essential amino acids are 11.19% to 11.34%, non-essential amino acids are 43.99% to 49.31%, non-polar amino acids are 34.83% to 37.49%, polar amino acids are 25.29% to 30.40%, acidic amino acids are 13.06% to 17.02%, basic amino acids are 16.55% to 24.23%, and aromatic amino acids are 11.42% to 18.78%; In terms of mass ratios of the amino acids, the lysine content in the essential amino acids is 5.21% to 13.03%, the phenylalanine content is 4.75% to 7.76%, the leucine content is 3.90% to 7.67%, and the tyrosine content in the non-essential amino acids is 6.67% to 9.66%; In terms of mass percentage in the polypeptides, the content of polypeptides having a number average molecular weight of 2914 to 2919 Da is 6.08% to 8.07%, the content of polypeptides having a number average molecular weight of 1576 to 1589 Da is 11.36% to 11.60%, the content of polypeptides having a number average molecular weight of 710 to 715 Da is 34.11% to 34.69%, and the content of polypeptides having a number average molecular weight of 207 to 210 Da is 46.22% to 47.87%. Second aspect of the present application

[0063] The present application provides a bird's nest peptide produced by the method according to the first aspect. Third aspect of the present application

[0064] The present application provides the application of the bird's nest peptide according to the second aspect in the manufacture of a pharmaceutical or food or skin care product having moisturizing, whitening, repairing and / or antioxidant effects. Specific Examples

[0065] Hereinafter, the embodiments of the present application will be described in detail with reference to examples. Note that these examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present application. In the following examples, for experimental methods for which specific conditions are not specified, the guidance provided by the present application should be used as a guide. The experimental methods may be performed according to experimental manuals or usual conditions in the field, according to conditions recommended by the manufacturer, or by reference to experimental methods known in the field. stomach.

[0066] In the following specific examples, unless otherwise specified, the dosage parameters of the raw ingredients may have slight deviations within the weighing accuracy range. For temperature and time parameters, allowable deviations due to the testing accuracy or operating accuracy of the equipment are allowed. EXAMPLES

[0067] 1. Materials and Methods 1.1 Materials HaCat cells and MRC-5 cells were purchased from Wuhan Procell Life Science & Technology Co., Ltd., China. CCK-8 was purchased from APExBIO, USA. TAC kit (ab65329) was purchased from Abcam, UK. Hyaluronic acid ELISA kit (EU2556) was purchased from FineTest, China. Sialic acid standards and tryptophan standards were purchased from Sigma-Aldrich, USA, and amino acid standards were purchased from Beijing Century Aoba Biotechnology Co., Ltd. Other reagents were of purity equal to or higher than analytical grade and were purchased from Sigma-Aldrich, USA.

[0068] 1.2 Production of bird nest peptides The production process of bird's nest peptides includes raw material washing, boiling, grinding, homogenization, pH adjustment, enzymatic decomposition, enzyme inactivation, cooling, centrifugation, ultrafiltration, concentration, drying, and subdivision. Specifically, the edible bird's nest raw material (30% w / v) was washed, then placed in a double boiler and boiled in boiling water for 2 hours. The boiled edible bird's nest was then homogenized for 30 minutes under a condition of 30 MPa. The homogenized edible bird's nest paste was placed in an enzymatic decomposition tank, and the pH of the reaction system was stabilized in the range of 9.0 with 0.01% sodium carbonate. 6U / g of alkaline protease was added to the enzymatic decomposition tank, and the enzymatic decomposition temperature was maintained at 51°C for 6 hours to carry out the enzymatic decomposition reaction. After the completion of the enzymatic decomposition, the temperature of the reaction system was adjusted to 85°C and held for 20 minutes to fully deactivate the enzyme. After enzyme deactivation is completed, the sample is centrifuged under the condition of 13000 rpm for 30 min to obtain edible bird nest peptide crude sample solution.The edible bird nest peptide solution is filtered through 200 nm ultrafiltration membrane to obtain edible bird nest peptide solution.Finally, the solution is spray-dried to obtain edible bird nest peptide powder, and the inlet temperature of spray drying is 170°C, and the exhaust temperature is >90°C.

[0069] 1.3 Characterization of bird nest peptide components 1.3.1 Characterization of sialic acids in bird nest peptides The sialic acid in bird's nest peptides was characterized by liquid chromatography, and the characterization method was based on the Chinese national standard (GB 30636-2014).

[0070] 1.3.2 Characterization of amino acids in bird's nest peptides The amino acids in bird's nest peptides were characterized by liquid chromatography-mass spectrometry (LCMS 8050, Shimadzu). After pretreatment of the amino acid standard sample, they were detected by the instrument as a control. Specifically, the bird's nest peptide sample (100 mg / mL) was prepared in the mobile phase, filtered through a 0.22 μm polyethersulfone resin film, and then detected by the instrument. The liquid chromatography conditions were: column: Shimadzu C18-AQ, 2.1 mm × 100 mm, 1.9 μm; flow rate: 300 μL / min; column temperature: 40 °C; sample injection volume: 5 μL; analysis time: 16 min; mobile phase A: 0.05% formic acid solution; mobile phase B: methanol; elution procedure: as shown in Table 1. The mass spectrometry conditions were as follows: ion source: electrospray ion source ESI, monitoring method: positive ion multiple reaction monitoring (MRM), interface voltage: 2.5 kV, nebulization gas flow rate: 3 L / min, heating gas flow rate: 10 L / min, interface temperature: 300 °C, DL temperature: 250 °C. The heating block temperature was 400°C, the drying gas flow rate was 10 L / min, and the CID gas was 270 kPa. [Table 1]

[0071] 1.3.3 Characterization of water in bird nest peptides The moisture content of bird's nest peptides was characterized using the direct drying method, and the characterization method was based on the Chinese national standard (GB 5009.3-2016).

[0072] 1.3.4 Molecular weight characterization of bird's nest peptides The molecular weight of bird's nest peptides was characterized by gel permeation chromatography (GPC), and the characterization method was based on the Chinese national standard (GB 31645-2018).

[0073] 1.4 Evaluation of chemical antioxidant activity of bird's nest peptides 1.4.1 Evaluation of DPPH radical scavenging ability DPPH radical solution preparation: DPPH was prepared in 0.04 mg / mL DPPH solution with absolute ethanol. Sample preparation: Bird's nest peptide samples were prepared in ultrapure water to different concentrations of bird's nest peptide solutions (50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL, 2500 μg / mL). Measurement target solution preparation: A0) 2 mL of absolute ethanol and 2 mL of DPPH radical solution were added to a test tube, A1) 2 mL of bird's nest peptide sample solution and 2 mL of DPPH radical solution were added to a test tube, and A2) 2 mL of bird's nest peptide sample solution and 2 mL of absolute ethanol were added to a test tube. The above measurement target solutions were mixed uniformly and left at room temperature for 30 min. Finally, the mixed solution was centrifuged at 5000 r / min for 10 min. The absorbance of the supernatant after centrifugation was measured at 517 nm, and the DPPH radical scavenging rate of the sample was calculated using the following formula:

number

[0074] 1.4.2 Evaluation of ABTS radical scavenging ability ABTS radical solution preparation: 7 mmol / L ABTS aqueous solution and 2.45 mmol / L potassium persulfate were mixed at 1:1 (v / v) and reacted at room temperature for 12-16 hours under light-shielded conditions. Then, the solution was diluted with 10 mmol / L PBS to an absorbance value of 0.70 ± 0.02 at 734 nm. Sample preparation: Bird's nest peptide samples were prepared in ultrapure water to different concentrations of bird's nest peptide solutions (50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL, 2500 μg / mL). Preparation of the solution to be measured: A0) 0.4 mL of ultrapure water and 4 mL of ABTS radical solution were added to a test tube, A1) 0.4 mL of the bird's nest peptide sample solution and 4 mL of ABTS radical solution were added to a test tube, and A2) 0.4 mL of the bird's nest peptide sample solution and 4 mL of ultrapure water were added to a test tube. The above solutions were mixed uniformly and reacted for 6 minutes under light shielding, after which the absorbance value was measured at 734 nm to calculate the ABTS radical scavenging rate of the sample. The ABTS radical scavenging rate of the sample was calculated by the following formula.

number

[0075] 1.4.3 Evaluation of hydroxyl radical scavenging ability Sample preparation: The bird's nest peptide sample was prepared into bird's nest peptide solutions of different concentrations (50μg / mL, 100μg / mL, 250μg / mL, 500μg / mL, 1000μg / mL, 2500μg / mL) with ultrapure water. Preparation of the solution to be measured: A0) 1mL of ferrous sulfate, 1mL of salicylic acid, 11mL of ultrapure water and 1mL of hydrogen peroxide solution were added to a test tube, A1) 1mL of the bird's nest peptide sample solution, 1mL of ferrous sulfate, 1mL of salicylic acid, 10mL of ultrapure water and 1mL of hydrogen peroxide solution were added to a test tube, A2) 1mL of the bird's nest peptide sample solution, 1mL of ferrous sulfate, 1mL of salicylic acid and 11mL of ultrapure water were added to a test tube. The above solutions were mixed uniformly and then placed in a water bath at 37°C for 60 minutes. The absorbance was measured at 510 nm, and the scavenging rate of the sample against hydroxyl radicals was calculated using the following formula: OH

number

[0076] 1.4.4 Evaluation of total antioxidant capacity Total antioxidant capacity (TAC) was measured using a commercially available detection kit (Abcam ab65329).

[0077] 1.5 Evaluation of intracellular effects of bird's nest peptides 1.5.1 Evaluation of the toxicity of bird nest peptides to HaCat cells, B16 cells and MRC-5 cells The toxicity of the bird nest peptides to HaCat cells, B16 cells and MRC-5 cells was measured by CCK-8, and the intracellular safe concentration range of the bird nest peptides was determined. 5 The cells were suspended at 100 μL / mL and inoculated into a 96-well plate, and 100 μL was inoculated into each well. The wells were then placed in an incubator (CCL-170B-8, Singapore ESCO) at 37°C and 5% CO2 for 24 hours to allow the cells to adhere completely. The medium was removed, washed twice with PBS buffer, and sample solutions of bird's nest peptides at different concentrations (50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL) prepared with MEM were added, and the wells were placed in an incubator at 37°C and 5% CO2 for 24 hours. After the incubation, 10 μL of CCK-8 was added to each well and incubated at 37°C for 1 hour. Finally, the absorbance value was measured at 450 nm to calculate the cell viability. The cell viability was calculated using the following formula:

number

[0078] 1.5.2 Evaluation of the protective effect of bird nest peptides against H2O2-induced HaCat cell injury First, we measured the toxicity of H2O2 to HaCat cells and determined the concentration of H2O2 that induces cell damage. 5The cells were suspended at 100 μL / mL and inoculated into a 96-well plate, and 100 μL was inoculated into each well. The wells were then placed in an incubator at 37°C and 5% CO2 for 24 hours to allow the cells to adhere completely. After that, the medium was removed, washed twice with PBS buffer, and different concentrations of H2O2 solutions (50 μmol / mL, 100 μmol / mL, 200 μmol / mL, 300 μmol / mL, 500 μmol / mL) prepared with MEM were added, and the wells were placed in an incubator at 37°C and 5% CO2 for 2 hours of incubation. After the incubation, 10 μL of CCK-8 was added to each well and incubated at 37°C for 1 hour. Finally, the absorbance value was measured at 450 nm to calculate the cell viability. In this example, it was experimentally determined that the concentration of H2O2 that induces HaCat cell damage is 300 μmol / L.

[0079] The protective effect of the bird nest peptide against H2O2-induced HaCat cell injury was then measured. 5 The cells were suspended at 100 μL / mL and inoculated into a 96-well plate, and 100 μL was inoculated into each well. The wells were then placed in an incubator at 37°C and 5% CO2 for 24 hours to allow the cells to adhere completely. After that, the medium was removed, washed twice with PBS buffer, and sample solutions of different concentrations of bird's nest peptides (50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL) prepared with MEM were added, and the wells were placed in an incubator at 37°C and 5% CO2 for 2 hours of incubation. After the incubation, 10 μL of CCK-8 was added to each well and incubated at 37°C for 1 hour. Finally, the absorbance value was measured at 450 nm to calculate the cell viability. The calculation formula is the same as that described in section 1.5.1.

[0080] 1.5.3 Evaluation of the effect of bird nest peptides on the hyaluronic acid content in HaCat cells The hyaluronic acid content in HaCat cells was measured using a commercially available hyaluronic acid ELISA kit (EU 2556).

[0081] 1.5.4 Evaluation of the inhibitory effect of swallow nest peptides on B16 cell melanin production The B16 cell melanin production inhibition test was measured with reference to the association standard (T / SHRH 027-2019).

[0082] 1.5.5 Evaluation of the repair effect of bird's nest peptides on MRC-5 cell scars 5 × 10 log-phase MRC-5 cells 4 The cells were suspended at 100 μg / mL, inoculated into a 6-well plate, 2 mL per well, and cultured in an incubator at 37°C and 5% CO2 for 24 hours to allow the cells to completely adhere. After that, a cell scar was left in the cell culture plate using a pipette tip, the medium was removed, washed twice with PBS buffer, and sample solutions of bird's nest peptides at different concentrations (50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL) prepared in MEM were added and photographed. After that, the cells were incubated in an incubator at 37°C and 5% CO2, and periodically removed and photographed (12 hours, 24 hours).

[0083] 1.6 Data Analysis All experiments were repeated at least twice using freshly prepared samples. Statistical analysis was performed using SPSS (Version 26.0, SPSS Inc., USA). Mean values ​​were compared using Tukey's test and univariate analysis (ANOVA), and significant differences were recognized at the 5% level.

[0084] 2. Results and Discussion 2.1 Components of bird's nest peptides [Table 2] The basic components of bird's nest peptide are shown in Table 2. The bird's nest peptide prepared by the process of this example has a total amino acid content of 69.10%, a sialic acid content of 6.64%, and a water content of 11.01%.

[0085] [Table 3] Note: Nonpolar amino acids are Ala, Val, Met, Phe, Ile, Leu, Pro, Try; (* denotes uncharged) polar amino acids are Ser, Thr, Tyr, Cys, Gly; acidic amino acids are Asp, Glu; basic amino acids are His, Arg, Lys; aromatic amino acids are Tyr, Phe, Try.

[0086] Table 3 shows the composition and content of 18 kinds of amino acids in bird's nest peptide. First, the amino acid content in bird's nest peptide is 68.89 g / 100 g. Lysine is It is the most abundant amino acid in the diet, accounting for 13.03% of the total amino acids (TAA), followed by tyrosine (9.66%), phenylalanine (7.76%), and leucine (7.67%). In particular, the total essential amino acids in bird's nest peptides is 30.90g / 100g, accounting for 44.89% of the total amino acids, which is obviously higher than other protein-rich foods such as chicken eggs (4.7-7.0g / 100g) and milk (1.1g / 100g). In addition, the histidine required for infant growth in bird's nest peptides is 2.62g / 100g. According to the amino acid nutritional standards recommended by FAO / WHO, a protein with an essential amino acid (EAA) to TAA ratio of about 0.4 and an EAA to nonessential amino acid (NEAA) ratio of 0.6 or more is a good quality protein. As can be seen from Table 3, the EAA / TAA ratio in bird's nest peptides is 0.44, and the EAA / NEAA ratio is 1.02, which is far superior to the FAO / WHO standard. Therefore, bird's nest peptides are considered to be derived from high-quality essential amino acids, even from the perspective of simply supplementing essential amino acids.

[0087] 2.2 Molecular weight of bird's nest peptides [Table 4] The GPC retention spectrum of the bird's nest peptide obtained in this example is shown in Figure 1, and the corresponding relative molecular mass distribution is shown in Table 4. The number average molecular weight of the bird's nest peptide as a whole obtained by the production process used in this example is 355Da. It can be further subdivided into four components, the number average molecular weight of F1 is 2914Da, accounting for 8.07%, the number average molecular weight of F2 is 1576Da, accounting for 11.60%, the number average molecular weight of F3 is 710Da, accounting for 34.11%, and the number average molecular weight of F4 is 207Da, accounting for 46.22%. Enzymatic hydrolysis products of proteins are classified according to their molecular weight into oligopeptides (<1000 Da), mesopeptides (1000-5000 Da), macropeptides (5000-10000 Da), and proteins (>10000 Da) (10.3390 / gels8010024). Therefore, the results showed that the bird's nest peptides obtained by the production process used in this example were mainly oligopeptides.

[0088] 2.3 Chemical antioxidant activity of bird's nest peptides The detection results are shown in Figure 2, where Figure A is the DPPH radical scavenging rate of bird's nest peptides at different concentrations, Figure B is the ABTS radical scavenging rate of bird's nest peptides at different concentrations, Figure C is the hydroxyl radical scavenging rate of bird's nest peptides at different concentrations, and Figure D is the total antioxidant capacity value of bird's nest peptides at different concentrations.

[0089] Antioxidation is considered to be the basis of anti-aging ability. Therefore, the antioxidant ability of EBNP has been investigated at the biochemical and cellular levels. + and -OH, 3 These are two well-known radicals, which, when quenched by antioxidants, cause the characteristic color to disappear. Therefore, the change in absorbance indicates the presence of the antioxidants DPPH-, ABTS-, and + The scavenging ability of EBNP against DPPH-, ABTS- + The scavenging ability of EBNP against DPPH-, ABTS-, and -OH is shown in Fig. 2, A, B, and C, respectively. +The scavenging ability of EBNP against DPPH-, ABTS- and -OH increased with increasing concentration in the concentration range of 50 to 2500 μg / mL. + and the ability to scavenge -OH.

[0090] To express the antioxidant capacity of EBNP more intuitively, a quantitative characterization was further performed on the total antioxidant capacity of EBNP, targeting Trolox (a water-soluble α-tocopherol analogue, a known antioxidant). The total antioxidant capacity is the sum of the scavenging activities of different active ingredients against various radicals. The results are shown in Figure 2D, and the units of the results are mmol Trolox equivalents / L. Consistent with the trends of the other three radical scavenging capacities, the results demonstrated that EBNP has antioxidant capacity and that the total antioxidant capacity of EBNP increases with increasing concentration between 50 and 2500 μg / mL. When the concentration of EBNP is 2500 μg / mL, its total antioxidant capacity is equivalent to 28.41 mmol / L Trolox.

[0091] 2.4 Intracellular effects 2.4.1 Toxicity of bird nest peptides to HaCat cells, B16 cells and MRC-5 cells The detection results are shown in Figure 3, where Figure A is the viability of HaCat cells at different bird nest peptide concentrations, Figure B is the viability of B16 cells at different bird nest peptide concentrations, Figure C is the viability of MRC-5 cells at different bird nest peptide concentrations, Figure D is the effect of bird nest peptide on the viability of HaCat cells injured by H2O2, Figure E is the effect of bird nest peptide on the hyaluronic acid content of HaCat cells, and Figure F is the inhibition rate of bird nest peptide on melanin production in B16 cells.

[0092] In cell experiments, the viability of cells affects the results of the experiment, so it is necessary to determine the safe concentration of each substance added in the cell experiment in advance through toxicity experiments. The results of the safe concentration of bird's nest peptide in each cell determined by the experiment are shown in Figure 3, A, B, and C. In the concentration range of 50 to 1000 μg / mL, the viability of each cell exceeded 90%. These results showed that in the concentration range of 50 to 1000 μg / mL, bird's nest peptide is not cytotoxic to any of the cells, and is a safe concentration for conducting cell experiments.

[0093] 2.4.2 Protective effect of bird nest peptide against H2O2-induced HaCat cell injury The cell oxidative damage experiment induced by H2O2 is a widely used cell experimental protocol to evaluate the intracellular antioxidant capacity of interventions. The viability of HaCat cells induced by H2O2 under the protection of bird nest peptide is shown in Figure 3D. The results showed that the higher the viability of HaCat cells under the protection of bird nest peptide, the higher the antioxidant activity of bird nest peptide in HaCat cells. Therefore, the results showed that bird nest peptide has antioxidant capacity in HaCat cells, and in the concentration range of 50-1000μg / mL, the intracellular antioxidant capacity of bird nest peptide increases with increasing concentration, and at concentrations of 250μg / mL or more, it has significant intracellular antioxidant activity (p<0.05).

[0094] 2.4.3 Effect of bird nest peptides on hyaluronic acid content in HaCat cells Hyaluronic acid is a natural moisturizing factor, which is involved in skin moisturization and is an important component of the extracellular matrix. The content of hyaluronic acid in human cells decreases with age. Human cells actively synthesize hyaluronic acid to ensure cellular moisture, and an important example is HaCat cells, which produce hyaluronic acid on the inner surface of the cell membrane under the catalysis of hyaluronidase. Therefore, the measurement of intracellular hyaluronic acid content can reflect the water-retaining ability of cells to some extent and can evaluate the involved cellular moisturizing ability.

[0095] The hyaluronic acid content in HaCat cells under the influence of bird nest peptides is shown in Figure 3E. The results showed that bird nest peptides below 100μg / mL could not significantly increase the hyaluronic acid content in HaCat cells (p>0.05), while bird nest peptides above 250μg / mL could significantly increase the hyaluronic acid content in HaCat cells (p<0.05). In addition, the higher the concentration of bird nest peptides, the higher the content of hyaluronic acid in HaCat cells. Therefore, the results showed that bird nest peptides have cell moisturizing ability, further proving that bird nest peptides may be used in cosmetics with moisturizing function.

[0096] 2.4.4 Inhibitory effect of bird nest peptide on B16 cell melanin production The B16 cell melanin production inhibition test can provide the most direct evidence for the whitening effect of bird's nest peptide, and the inhibition rate of bird's nest peptide on B16 cell melanin production is shown in Figure 3F. The results showed that bird's nest peptide has the ability to inhibit B16 cell melanin production, and in the concentration range of 50-1000μg / mL, the ability of bird's nest peptide to inhibit melanin production in cells increases with increasing concentration, and at concentrations of 50μg / mL or more, it has significant intracellular melanin inhibition activity (p<0.05).

[0097] 2.4.5 Repair effect of bird's nest peptide on MRC-5 cell scar The results are shown in FIG. 4. In this example, the cell repair and proliferation promoting ability of bird's nest peptide was evaluated by the MRC-5 cell scar test, and the results are shown in FIG. 4. Obviously, without the intervention of bird's nest peptide, MRC-5 cells had obvious scars even after 24 hours of culture. Conversely, under the intervention of bird's nest peptide, MRC-5 cells had a smaller scar area after 24 hours of culture. In particular, under the intervention of bird's nest peptide at a concentration of 250 μg / mL or more, MRC-5 cells did not have obvious scars after 24 hours of culture. Therefore, the results showed that bird's nest peptide has the ability to repair and promote proliferation in MRC-5 cells, and the repair and proliferation promoting ability of bird's nest peptide in cells increases with increasing concentration. The cell repair and proliferation promoting ability can give the intervention imagination in terms of application, for example, functional products such as medicines and cosmetics that promote skin wound healing and tissue repair can be developed. Therefore, based on the results of the scar test, it is further believed that bird's nest peptides may have potential applications in the fields of medicine, cosmetics, and the like.

[0098] 3 Conclusion In summary, this embodiment provides a process for producing mature bird's nest peptides, and systematic evaluation is carried out on the bird's nest peptides produced by this process. In terms of basic properties, bird's nest peptides contain 68.89% total amino acids and 6.64% sialic acid. In particular, bird's nest peptides contain 30.90g / 100g essential amino acids, accounting for 44.89% of its total amino acid content, which is derived from high-quality essential amino acids. In terms of functional properties, bird's nest peptides have the ability of antioxidants (including DPPH radicals, ABTS radicals, and hydroxyl radical scavenging). Most importantly, bird's nest peptides have the functions of cell antioxidants, cell moisturizing, cell whitening, cell scar repair, and proliferation promotion. From the above results, the bird's nest peptides may be applied in the fields of food, medicine, cosmetics, etc. The development and use of bird's nest peptides based on the process of this embodiment is favorable to the further high-value development of the bird's nest industry. EXAMPLES

[0099] 1. Materials and Methods 1.1 Materials The materials used are the same as in Example 1.

[0100] 1.2 Production of bird nest peptides The production process of bird's nest peptides includes raw material washing, boiling, grinding, homogenization, pH adjustment, enzymatic decomposition, enzyme inactivation, cooling, centrifugation, ultrafiltration, concentration, drying, and subdivision. Specifically, the edible bird's nest raw material (30% w / v) was washed, then placed in a double boiler and boiled in boiling water for 1.75 hours. The boiled edible bird's nest was then homogenized for 20 minutes under a condition of 25 MPa. The homogenized edible bird's nest paste was placed in an enzymatic decomposition tank, and the pH of the reaction system was stabilized at 10.0 with 0.01% sodium carbonate. Alkaline protease (the amount of alkaline protease used per 1 g of bird's nest is 9 U) was added to the enzymatic decomposition tank, and the enzymatic decomposition temperature was maintained at 60°C for 5 hours to carry out the enzymatic decomposition reaction. After the completion of the enzymatic decomposition, the temperature of the reaction system was adjusted to 85°C and held for 20 minutes to fully deactivate the enzyme. After enzyme deactivation is completed, the sample is centrifuged under the condition of 15000 rpm for 10 min to obtain edible bird nest peptide crude sample solution.The edible bird nest peptide solution is filtered through 150 nm ultrafiltration membrane to obtain edible bird nest peptide solution.Finally, the solution is spray-dried to obtain edible bird nest peptide powder, and the inlet temperature of spray drying is 175°C, and the exhaust temperature is >90°C.

[0101] 1.3 Characterization of bird nest peptides' components and functions The specific method is the same as in the first embodiment.

[0102] 1.4 Data Analysis The data analysis method was the same as in Example 1.

[0103] 2. Results and Discussion 2.1 Components of bird's nest peptides [Table 5] The basic components of bird's nest peptide are shown in Table 4. The bird's nest peptide prototyped in this study has a total amino acid content of 69.10%, a sialic acid content of 6.14%, and a moisture content of 12.01%.

[0104] [Table 6] Note: Nonpolar amino acids are Ala, Val, Met, Phe, Ile, Leu, Pro, Try; (* denotes uncharged) polar amino acids are Ser, Thr, Tyr, Cys, Gly; acidic amino acids are Asp, Glu; basic amino acids are His, Arg, Lys; aromatic amino acids are Tyr, Phe, Try.

[0105] Table 5 shows the composition and content of 18 kinds of amino acids in bird's nest peptide. First, the amino acid content in bird's nest peptide is 66.75 g / 100 g. Serine is 1. It is the most abundant amino acid in the diet, accounting for 9.68% of the total amino acids (TAA), followed by glutamic acid (9.30%), threonine (9.17%), and arginine (8.12%). As can be seen from Table 5, the EAA / TAA ratio in bird's nest peptides is 0.39, and the EAA / NEAA ratio is 0.80, which is far superior to the FAO / WHO standard. Therefore, bird's nest peptides are considered to be derived from high-quality essential amino acids, even from the perspective of simply supplementing essential amino acids.

[0106] 2.2 Molecular weight of bird's nest peptides [Table 7] The GPC retention spectrum of the bird's nest peptide obtained in this example is shown in Figure 5, and the corresponding relative molecular mass distribution is shown in Table 7. The number average molecular weight of the bird's nest peptide as a whole obtained by the production process used in this example is 350 Da. It can be further subdivided into four components, the number average molecular weight of F1 is 2919 Da, accounting for 6.08%, the number average molecular weight of F2 is 1589 Da, accounting for 11.36%, the number average molecular weight of F3 is 715 Da, accounting for 34.69%, and the number average molecular weight of F4 is 210 Da, accounting for 47.87%. Enzymatic hydrolysis products of proteins are classified according to their molecular weight into oligopeptides (<1000 Da), mesopeptides (1000-5000 Da), macropeptides (5000-10000 Da), and proteins (>10000 Da) (10.3390 / gels8010024). Therefore, the results showed that the bird's nest peptides obtained by the production process used in this example were mainly oligopeptides.

[0107] 2.3 Chemical antioxidant activity of bird's nest peptides The detection results are shown in Figure 6, where Figure A is the DPPH radical scavenging rate of bird's nest peptides at different concentrations, Figure B is the ABTS radical scavenging rate of bird's nest peptides at different concentrations, Figure C is the hydroxyl radical scavenging rate of bird's nest peptides at different concentrations, and Figure D is the total antioxidant capacity value of bird's nest peptides at different concentrations.

[0108] DPPH-, ABTS + The scavenging ability of the bird nest peptides against DPPH-, ABTS- and -OH- is shown in Fig. 6A, B and C, respectively. + The scavenging ability of the bird nest peptide against DPPH-, ABTS-, and -OH increased with increasing concentration in the concentration range of 50-2500 μg / mL. + and the ability to scavenge -OH.

[0109] To express the antioxidant capacity of bird's nest peptides more intuitively, Trolox (a water-soluble α-tocopherol analogue, a known antioxidant) was used as a target to further quantitatively characterize the total antioxidant capacity of bird's nest peptides. The total antioxidant capacity is the sum of the scavenging activities of different active ingredients against various radicals. The results are shown in Figure 6D, and the units of the results are mmo. l Trolox equivalent / L. Consistent with the trends of the other three radical scavenging abilities, this result demonstrated that bird nest peptides have antioxidant ability, and that the total antioxidant ability of bird nest peptides increases with increasing concentration between 50 and 2500μg / mL. When the concentration of bird nest peptides is 2500μg / mL, its total antioxidant ability is equivalent to 26.41mmol / L Trolox.

[0110] 2.4 Intracellular effects 2.4.1 Toxicity of bird nest peptides against HaCat, B16 and MRC-5 cells The detection results are shown in Figure 7, where Figure A is the viability of HaCat cells at different bird's nest peptide concentrations, Figure B is the viability of B16 cells at different bird's nest peptide concentrations, Figure C is the viability of MRC-5 cells at different bird's nest peptide concentrations, Figure D is the effect of bird's nest peptide on the viability of HaCat cells injured by H2O2, Figure E is the effect of bird's nest peptide on the hyaluronic acid content of HaCat cells, and Figure F is the inhibition rate of bird's nest peptide on melanin production in B16 cells.

[0111] In cell experiments, the viability of cells affects the results of the experiment, so it is necessary to determine the safe concentration of each substance added in the cell experiment in advance through toxicity experiments. The results of the safe concentration of bird's nest peptide in each cell determined by the experiment are shown in Figure 7, A, B, and C. In the concentration range of 50 to 1000 μg / mL, the viability of each cell exceeded 90%. These results showed that in the concentration range of 50 to 1000 μg / mL, bird's nest peptide is not cytotoxic to any of the cells, and is a safe concentration for conducting cell experiments.

[0112] 2.4.2 Protective effect of bird nest peptide against H2O2-induced HaCat cell injury The cell oxidative damage experiment induced by H2O2 is a widely used cell experimental protocol to evaluate the intracellular antioxidant capacity of interventions. The viability of HaCat cells induced by H2O2 under the protection of bird nest peptide is shown in Figure 7D. The results showed that the higher the viability of HaCat cells under the protection of bird nest peptide, the higher the antioxidant activity of bird nest peptide in HaCat cells. Therefore, the results showed that bird nest peptide has antioxidant capacity in HaCat cells, and in the concentration range of 50-1000μg / mL, the intracellular antioxidant capacity of bird nest peptide increases with increasing concentration, and at concentrations of 250μg / mL or more, it has significant intracellular antioxidant activity (p<0.05).

[0113] 2.4.3 Effect of bird nest peptides on hyaluronic acid content in HaCat cells Hyaluronic acid is a natural moisturizing factor, which is involved in skin moisturization and is an important component of the extracellular matrix. The content of hyaluronic acid in human cells decreases with age. Human cells actively synthesize hyaluronic acid to ensure cellular moisture, and an important example is that HaCat cells produce hyaluronic acid on the inner surface of the cell membrane under the catalysis of hyaluronidase. Therefore, the measurement of the content of hyaluronic acid in cells can reflect the water-retaining ability of cells to some extent and evaluate the involved cell moisturizing ability.

[0114] The hyaluronic acid content in HaCat cells under the influence of bird nest peptides is shown in Figure 7E. The results show that bird nest peptides below 100μg / mL cannot significantly increase the hyaluronic acid content in HaCat cells (p>0.05), while bird nest peptides above 250μg / mL can significantly increase the hyaluronic acid content in HaCat cells (p<0.05). In addition, the higher the concentration of bird nest peptides, the higher the content of hyaluronic acid in HaCat cells. Therefore, the results show that bird nest peptides have cell moisturizing ability, further proving that bird nest peptides may be used in cosmetics with moisturizing function.

[0115] 2.4.4 Inhibitory effect of bird nest peptide on B16 cell melanin production The B16 cell melanin production inhibition test can provide the most direct evidence for the whitening effect of bird's nest peptide, and the inhibition rate of bird's nest peptide on B16 cell melanin production is shown in Figure 7F. The results showed that bird's nest peptide has the ability to inhibit B16 cell melanin production, and in the concentration range of 50-1000μg / mL, the ability of bird's nest peptide to inhibit melanin production in cells increases with increasing concentration, and at concentrations of 50μg / mL or more, it has significant intracellular melanin inhibition activity (p<0.05).

[0116] 2.4.5 Repair effect of bird's nest peptide on MRC-5 cell scar The results are shown in Figure 8. In this study, the cell repair and proliferation promotion ability of bird's nest peptide was evaluated by the MRC-5 cell scar test, and the results are shown in Figure 8. Obviously, without the intervention of bird's nest peptide, MRC-5 cells had obvious scars even after 24 hours of culture. Conversely, under the intervention of bird's nest peptide, MRC-5 cells had a smaller scar area after 24 hours of culture. In particular, under the intervention of bird's nest peptide at a concentration of 250 μg / mL or more, MRC-5 cells did not have obvious scars after 24 hours of culture. Therefore, the results showed that bird's nest peptide has the ability to repair and promote proliferation in MRC-5 cells, and the repair and proliferation promotion ability of bird's nest peptide in cells increases with increasing concentration. The cell repair and proliferation promotion ability can give the intervention imagination in terms of application, for example, functional products such as medicines and cosmetics that promote skin wound healing and tissue repair can be developed. Therefore, based on the results of the scar test, it is further believed that bird's nest peptides may have potential applications in the fields of medicine, cosmetics, and the like.

[0117] 3 Conclusion In summary, this embodiment provides a production process for mature bird's nest peptide, and systematic evaluation is carried out on the bird's nest peptide produced by this process. In terms of basic properties, bird's nest peptide contains 66.75% total amino acids and 6.14% sialic acid. In particular, bird's nest peptide contains 26.26g / 100g essential amino acids, accounting for 39.34% of its total amino acid content, which is derived from high-quality essential amino acids. In terms of functional properties, bird's nest peptide has the ability of antioxidant (including DPPH radical, ABTS radical, and hydroxyl radical scavenging). Most importantly, bird's nest peptide has the functions of cell antioxidant, cell moisturizing, cell whitening, cell scar repair, and proliferation promotion. From the above results, the bird's nest peptide has the potential for application in the fields of food, medicine, cosmetics, etc. The development and use of bird's nest peptide based on the process of this embodiment is favorable to the further high-value development of the bird's nest industry.

[0118] The technical features of the above embodiments and examples can be combined in any combination. For the sake of brevity, not all possible combinations of the technical features in the above embodiments and examples are described, but as long as there is no contradiction in the combinations of these technical features, all of them should be considered within the scope of this specification.

[0119] The above examples only describe some embodiments of the present application, and facilitate a specific and detailed understanding of the technical means of the present application, but should not be construed as limiting the scope of the claims of the present application. It should be noted that those skilled in the art can make some modifications and improvements without departing from the spirit of the present application, and all of these fall within the scope of protection of the present application. It can also be understood that after reading the above description of the present application, those skilled in the art can make various changes and modifications to the present application, and these equivalent forms are also included in the scope of protection of the present application. It should be further understood that any technical means obtained by those skilled in the art through logical analysis, reasoning, or limited experiments based on the technical means provided by the present application are all within the scope of the claims attached to the present application. Therefore, the scope of protection of the patent of the present application depends on the content of the appended claims, and the specification and drawings are for explaining the content of the claims. do.

Claims

1. mixing and stewing bird nests with water to prepare stewing bird nests; homogenizing the boiled bird's nest to prepare a bird's nest paste; The swallow nest paste is enzymatically decomposed with alkaline protease to inactivate the enzyme, thereby preparing an enzymatic decomposition product; Centrifugation of the enzymatic digestion product and recovery of the supernatant to prepare a crude sample solution of bird's nest peptides; and performing ultrafiltration on the crude sample solution of bird's nest peptides and recovering the ultrafiltrate to prepare bird's nest peptides. Centrifugation conditions include a speed of about 10,000 rpm to 15,000 rpm and a time of about 10 min to 30 min; A method for producing bird fossil peptides, characterized in that the pore size of the membrane used for ultrafiltration is about 100 nm to 200 nm, preferably about 150 nm to 200 nm.

2. The method for producing bird's nest peptides according to claim 1, characterized in that the amount of bird's nest used per 100 mL of water is about 10 g to 50 g, preferably, the amount of bird's nest used per 100 mL of water is about 25 g to 35 g.

3. The method for producing bird's nest peptides according to claim 1 or 2, characterized in that the simmering time is about 0.5 to 2 hours, preferably about 1.75 to 2 hours.

4. The method for producing bird's nest peptides described in any one of claims 1 to 3, characterized in that the homogenization treatment conditions include a power of about 10 MPa to 30 MPa and a time of about 10 min to 30 min, preferably the homogenization treatment conditions include a power of about 25 MPa to 30 MPa and a time of about 20 min to 30 min.

5. The method for producing bird's nest peptides according to any one of claims 1 to 4, characterized in that the enzymatic hydrolysis conditions include a temperature of about 50°C to 60°C, a pH value of about 8.0 to 10.0, a time of about 10 min to 30 min, and an amount of the alkaline protease used per gram of bird's nest of about 6 U to 9 U.

6. A method for producing bird's nest peptides described in any one of claims 1 to 5, characterized in that the method of enzyme inactivation is enzyme inactivation by heating, and the conditions for enzyme inactivation by heating include heating the reaction system obtained by enzymatic decomposition to about 80°C to 100°C and maintaining it for about 10 min to 30 min.

7. The method for producing bird's nest peptide according to any one of claims 1 to 6, further comprising a step of removing a solvent in the ultrafiltrate.

8. The method for producing bird's nest peptides according to claim 7, characterized in that the step of removing the solvent comprises concentrating the ultrafiltrate and spray-drying the resulting concentrate.

9. The step of removing the solvent comprises: (1) the volume of the concentrate is about 1 / 3 to about 1 / 8 of the volume of the ultrafiltrate; (2) The method for producing bird's nest peptides described in claim 8, characterized in that it has one or more of the following technical features: the spray drying conditions include an inlet air temperature of about 150°C to 200°C and an exhaust air temperature > about 70°C, and preferably the spray drying conditions include an inlet air temperature of about 165°C to 175°C and an exhaust air temperature > about 90°C.

10. The method for producing bird's nest peptide described in any one of claims 1 to 9, characterized in that the bird's nest peptide has a total amino acid content of about 66.75 wt% to 68.89 wt%, a sialic acid content of about 6.14 wt% to 6.64 wt%, and a water content of about 11.01 wt% to 12.01 wt%.

11. A bird's nest peptide, which is produced by the method according to any one of claims 1 to 10.

12. Use of the bird's nest peptide according to claim 11 in the manufacture of a pharmaceutical or food or skin care product having moisturising, whitening, repairing and / or antioxidant effects.

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