Manufacturing method and application of bird's nest peptides that promote cell repair and have a high moisturizing and whitening effect

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 "Method for producing and application of bird's nest peptides that promote cell repair and have 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 applying bird's nest peptides that promote cell repair and have a high moisturizing and whitening effect. [Background technology]

[0003] Researchers have found that bird's nest peptides, produced after bird's nest is hydrolyzed by proteases, have excellent antihypertensive and antiviral effects. In particular, bird's nest peptides are superior to bird's nest in some functions. Furthermore, bird's nest peptides have a relatively low molecular weight, making them easily absorbed by the human body and thought to have the ability to be absorbed transdermally. Based on these 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 wide 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's nest protein liquid, which includes the steps of first adjusting the pH of the bird's nest liquid obtained after boiling bird's 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 then cooling the liquid, and finally repeating vacuum high-low speed whipping and high-low temperature defoaming three times.

[0006] CN111528332A includes a step (1) of soaking and sorting dried bird's nests in purified water, removing impurities, crushing, and drying them for use; a step (2) of boiling and mashing the bird's nest paste, adding purified water to the bird's nests soaked in step (1), boiling them, and then finely crushing them; a first bioenzymatic 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 the paste, inactivating the enzyme, cooling, and sieving the paste through a 100-200 mesh sieve to obtain a first enzymatic hydrolysis solution; and a step (4) of The method for producing bird's nest peptides is described, which includes the steps of homogenization and filtration (step (4)), in which the first enzymatic hydrolysis solution produced in step (3) is homogenized and coarsely filtered to obtain an enzymatic hydrolysis homogenized solution; the step (5) of second bioenzyme hydrolysis, in which a complex protease is added to the prepared enzymatic hydrolysis homogenized solution, stirred to perform enzymatic hydrolysis, and then the enzymes are inactivated and cooled to obtain a second enzymatic hydrolysis solution; and the step (6) of purification and spray drying, in which the second enzymatic hydrolysis solution produced in step (5) is microfiltered, concentrated by reverse osmosis using an RO membrane, and then spray-dried to obtain bird's nest peptides.

[0007] CN112006276A includes a step (1) of soaking and sorting the dried bird's nests in purified water, removing impurities, crushing, drying, and sieving them for use, and a step (1) of adding an arginine solution to the sieved bird's nests to prepare bird's nest liquid. The method for producing instant bird's nest powder reconstituted in hot water includes the steps of: (1) preparing a bird's nest liquid and stewing it, followed by boiling in hot water and cooling; (2) enzymatic hydrolysis step (3) of adjusting the pH of the solution treated in step (2) to 8-10, adding alkaline protease to partially hydrolyze the solution, and then inactivating the enzyme in high-temperature boiling water to obtain an enzymatic hydrolysis liquid; (3) ultrasonic degassing and high-pressure homogenization step (4) of whipping the enzymatic hydrolysis liquid prepared in step (3) at a low temperature in a vacuum, degassing it with ultrasound, and homogenizing it at a high pressure to obtain an enzymatic hydrolysis homogenized liquid; (4) spray drying step (5) of adding rock sugar and citric acid to the enzymatic hydrolysis homogenized liquid prepared in step (4), followed by spray drying in a vacuum to obtain bird's nest powder; and (5) phospholipid spray step (6) of spraying phospholipid on the surface of the bird's nest powder prepared in step (5) to obtain instant bird's nest powder reconstituted in hot water.

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

[0009] CN111019984A includes a pretreatment step I in which bird's nest raw material is dried and crushed to obtain pretreated bird's nest; a pretreatment step II in which water 10 to 30 times the volume of the pretreated bird's nest is added to the pretreated bird's nest, and the mixture is uniformly stirred and then ultrasonically treated at 30 to 45°C for 15 to 40 minutes to obtain bird's nest paste; a pretreatment step III in which trypsin, alkaline protease, flavorzyme, pepsin, and an enzyme stabilizer are added to the bird's nest paste, and the bird's nest paste, trypsin, alkaline protease, flavorzyme, pepsin, and an enzyme stabilizer are added to the bird's nest paste, and the pretreatment step III in which the ... The present invention describes an efficient method for extracting sialic acid from bird's nest, which comprises: Step II of enzymatic hydrolysis at 40-60°C for 2-7 hours, inactivating the enzyme at 100-105°C for 5-10 minutes, and cooling to obtain an enzymatic hydrolysis solution, and Step III of filtration and drying, in which the enzymatic hydrolysis solution is centrifuged, the resulting supernatant is filtered, and the resulting product is freeze-dried to obtain a product.

[0010] CN115226886A describes a method for producing high-sialic acid bird's nest products by an extract re-addition method, which includes the following steps: (1) preparing a sialic acid extract; soaking crushed bird's nests in water, wet-grinding them to millimeter-order granules, and then ultrasonically crushing them, followed by adding alkaline protease for enzymatic hydrolysis, inactivating the enzyme, centrifuging, and filtering to obtain a sialic acid-rich extract; and (2) re-adding the extract; soaking the re-added dried bird's nests in the extract, placing them in an ultrasonic device for ultrasonic swelling, and finally whipping them into threads, removing impurities, dehydrating, and boiling them to obtain a high-sialic acid bird's nest product.

[0011] CN111096462B includes a step S1 of soaking bird's nests, in which water is added to bird's nests at a ratio of 1:40 and the nests are soaked at room temperature for 5 to 12 hours; a step S2 of boiling the soaked bird's nests in a water bath at 95 to 100 degrees Celsius for 1 to 2 hours; a step S3 of homogenizing the boiled bird's nest solution by lowering the temperature and then homogenizing it once or twice with a homogenizer; and a step S4 of adding alkaline protease, neutral protease, etc. 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, wherein protease A is a product of Bacillus licheniformis and protease B is a product of Bacillus amyloliquefaciens; the enzyme deactivation step S5 in which the obtained enzyme hydrolysis solution is deactivated 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 of which is 100-120 mm; and the supernatant after centrifugation to remove residue, the centrifugation rotation speed is 100-120 mm. The present invention describes a method for producing a non-polluting bird's nest polypeptide that has been bioenzyme-degraded and purified, and a method for removing the bitterness thereof, which method includes 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 from the bird's nest supernatant and β-cyclodextrin in a mass ratio 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 nests into bird's nest powder, soaking the bird's nest powder in water at a solid-liquid ratio of 1:15, and crushing it in a French press to obtain a bird's nest paste; (2) adding 3% of the bird's nest powder's weight of protease to the bird's nest paste, enzymatically hydrolyzing it for 3 hours, inactivating the enzyme in a boiling water bath, and centrifuging it to obtain a supernatant liquid 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 method for producing bird's nest polypeptide powder includes the steps of: (3) 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; (4) mixing the post-enzymatic hydrolysis supernatant Y1 and the post-enzymatic hydrolysis supernatant Y3 to obtain a post-enzymatic hydrolysis supernatant Y5; (5) 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 (6) freeze-drying the bird's nest polypeptide purified solution to obtain bird's nest polypeptide powder.

[0013] However, the bird's nest peptides produced by conventional production methods and bird's nest raw materials often do not match 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 these 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 peptides that promote cell repair and have 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 bird's nests with water and simmering the mixture to prepare simmered bird's nests; homogenizing the boiled bird's nest to prepare bird's nest paste; Enzymatically hydrolyzing the swallow's nest paste with alkaline protease to inactivate the enzyme, thereby preparing an enzymatic hydrolyzate; 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 minutes to 30 minutes. The pore size of the membrane used for ultrafiltration is approximately 100 nm to 200 nm. A manufacturing method 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 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 minutes to 30 minutes, and preferably, the homogenization conditions include a power of 25 MPa to 30 MPa and a time of 20 minutes to 30 minutes.

[0021] In some embodiments of the present application, the enzymatic hydrolysis conditions include a temperature of 50°C to 60°C, a pH value of 5.0 to 9.0, a time of 10 to 30 minutes, 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 minutes to 30 minutes.

[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: an intake air temperature of about 150°C to 200°C, and an exhaust air temperature of > about 70°C.

[0026] In some embodiments of the present application, the spray drying conditions include an inlet air temperature of 165°C to 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, the present application provides a bird's nest peptide produced by the production 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 claims. [Brief explanation of the drawings]

[0031] In order to more clearly describe the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following will briefly describe the drawings that need to be used in the description of the embodiments. Obviously, 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 work.

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

[0033] The present application will be described in more detail below with reference to the drawings, embodiments, and examples. These embodiments and examples are merely for the purpose of illustrating 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 provide a more complete and comprehensive understanding of the disclosure of the present application. Furthermore, the present application should not be construed as being limited to the embodiments and examples described herein. It should be understood that the present application can be realized in many different forms, and that those skilled in the art can make various changes and modifications without departing from the spirit of the present application, and that equivalents thereof are also within the scope of protection of the present application. Furthermore, in the following description, many specific details are set forth to provide a thorough understanding of the present application. It should be understood that the present application can be practiced 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 terminology used herein is for the purpose of describing embodiments and examples only and is not intended to be limiting of the application. term

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

[0036] As used herein, the term "and / or," "or / and," and "and / or" encompasses any one item among two or more related listed items, and further encompasses any and all combinations of the related listed items, where any and all combinations include any two related listed items, any more related listed items, or all combinations of the related listed items. It should be understood 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," the technical means in this application include all technical means connected by a "logical AND" and further include all technical means connected by a "logical OR." For example, "A and / or B" encompasses three meanings: A, B, and A+B. For example, a technical measure referred to as "A and / or B and / or C and / or D" includes any one of A, B, C, and D (i.e., all technical measures connected by a "logical OR"), any and all combinations of A, B, C, and D, i.e., 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 measures connected by a "logical AND").

[0037] As used herein, unless otherwise specified, terms such as "plurality," "multiple kinds," "multiple times," and "various" refer to a quantity of two or more. 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 appropriate combinations of any two or more of the listed items.

[0039] In this specification, the term "appropriate" in terms such as "appropriate combination method," "appropriate method," and "any appropriate method" is based on whether the technical means of the present application can be implemented, the technical problems of the present application can be solved, and the desired technical effects 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 used to describe more effective embodiments or examples, and do not limit the scope of protection of the present application.

[0041] In this application, the terms "further", "furthermore", "particularly" and the like are for explanatory purposes and indicate differences 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, refer to a choice between two alternatives, "exist" and "not exist." In a technical means, if there are multiple "options," each "option" is independent of the other, unless otherwise specified, and unless there is a contradiction or mutual constraint.

[0043] In this application, terms such as "first," "second," "third," and "fourth," as used 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 implicitly indicating 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 non-exhaustive enumeration description only and do not constitute closed-type limitations on quantity.

[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 including the recited features.

[0045] In this application, unless otherwise specified, with respect to a numerical interval (i.e., a numerical range), selectable numerical values ​​are considered continuous within the numerical interval, including the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range and each value between those two 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 those two endpoints. In this specification, this is equivalent to directly listing each integer. For example, when t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when there are multiple ranges describing a feature or characteristic, these ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to encompass any and all subranges subsumed therein.

[0046] Unless otherwise specified, the temperature parameters in this application may be constant temperature treatments or may fluctuate within a certain temperature range. It should be understood that the constant temperature treatments allow the temperature to fluctuate within the precision range of the equipment control. For example, fluctuations 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, to the same extent 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. When a cited document is mentioned in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. When a cited document is mentioned in this application, examples of relevant technical features and preferred embodiments incorporated may also be incorporated herein by reference, but are limited to those that enable the application to be implemented. If the cited content contradicts the description of this application, it should be understood that the present application should be used as a reference or be adaptively modified in accordance with the description of this application. First aspect of the present application

[0049] This application is mixing bird's nests with water and simmering the mixture to prepare simmered bird's nests; homogenizing the boiled bird's nest to prepare bird's nest paste; Enzymatically hydrolyzing the swallow's nest paste with alkaline protease to inactivate the enzyme, thereby preparing an enzymatic hydrolyzate; 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 approximately 10,000 rpm to 15,000 rpm and a time of approximately 10 minutes. n ~ 30 min, The pore size of the membrane used for ultrafiltration is about 100 nm to 200 nm, and the method for producing bird's nest peptides is provided.

[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 bird's nest used per 100 mL of 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 bird's nest used per 100 mL of 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 0.5 h to 2 h (for example, 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9, or 2 h), and more preferably, the simmering time is 1.75 h to 2 h.

[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 more 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 enzymatic hydrolysis conditions 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 of 8.0 to 10.0 (e.g., 8, 8.5, 9, 9.5, 10), a time of 5 hours to 6 hours (e.g., 5 hours, 5.1 hours, 5.2 hours, 5.3 hours, 5.4 hours, 5.5 hours, 5.6 hours, 5.7 hours, 5.8 hours, 5.9 hours, 6 hours), and an amount of alkaline protease used per gram of bird's nest of 6 U to 9 U (e.g., 6U, 6.5U, 7U, 7.5U, 8U, 8.5U, 9U).

[0057] Preferably, the method of enzyme inactivation is 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 minutes to 30 minutes (e.g., 10 minutes, 12 minutes, 15 minutes, 17 minutes, 20 minutes, 22 minutes, 25 minutes, 28 minutes, 30 minutes).

[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 100g, and the mass proportions of the amino acids are 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 proportions of the amino acids, the lysine content of 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 of the non-essential amino acids is 6.67% to 9.66%; In terms of mass proportion within 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 production 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 pharmaceutical or food or skin care products with 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, experimental methods for which specific conditions are not specified should be referred to the guidance provided by the present application. The experimental methods may be performed according to experimental manuals or conventional conditions in the field, according to conditions recommended by manufacturers, or by reference to experimental methods known in the field. stomach.

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

[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, Inc., USA. The TAC kit (ab65329) was purchased from Abcam, UK. The hyaluronic acid ELISA kit (EU2556) was purchased from FineTest, China. Sialic acid standards and tryptophan standards were purchased from Sigma-Aldrich, USA. Amino acid standards were purchased from Beijing Century Aoba Biotechnology Co., Ltd. Other reagents were of analytical grade or higher purity and were purchased from Sigma-Aldrich, USA.

[0068] 1.2 Production of bird's nest peptides The bird's nest peptide production process involves raw material washing, boiling, mashing, homogenization, pH adjustment, enzymatic hydrolysis, enzyme inactivation, cooling, centrifugation, ultrafiltration, concentration, drying, and portioning. Specifically, edible bird's nest raw material (30% w / v) was washed and 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 30 MPa. The homogenized edible bird's nest paste was placed in an enzymatic hydrolysis tank, and the pH of the reaction system was stabilized at 9.0 with 0.01% sodium carbonate. 6 U / g of alkaline protease was added to the enzymatic hydrolysis tank, and the enzymatic hydrolysis reaction was carried out for 6 hours at a temperature of 51°C. After enzymatic hydrolysis was completed, the reaction temperature was adjusted to 85°C and held for 20 minutes to fully inactivate the enzyme. After enzyme deactivation, the sample was centrifuged at 13,000 rpm for 30 minutes to obtain a crude sample solution of edible bird's nest peptides. The edible bird's nest peptide solution was filtered through a 200 nm ultrafiltration membrane to obtain an edible bird's nest peptide solution. Finally, the solution was spray-dried to obtain edible bird's nest peptide powder, with the inlet air temperature of the spray-drying being 170°C and the outlet air temperature being >90°C.

[0069] 1.3 Characterization of bird's nest peptide components 1.3.1 Characterization of sialic acids in bird's nest peptides The sialic acids in the bird's nest peptides were 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 using a liquid chromatography-mass spectrometer (LCMS 8050, Shimadzu). Amino acid standards were prepared and then detected by the instrument. Specifically, bird's nest peptide samples (100 mg / mL) were 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: a Shimadzu C18-AQ column (2.1 mm x 100 mm, 1.9 μm), a flow rate of 300 μL / min, a column temperature of 40 °C, a sample injection volume of 5 μL, a run time of 16 min, mobile phase A: 0.05% formic acid solution, mobile phase B: methanol, and the elution procedure is 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, atomization 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 pressure was 270 kPa. [Table 1]

[0071] 1.3.3 Characterization of water content in bird's 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 the 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 the 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 absolute ethanol to a concentration of 0.04 mg / mL. Sample preparation: Bird's nest peptide samples were prepared in ultrapure water to different concentrations (50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL, and 2500 μg / mL). Measurement 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; 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 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 value of the supernatant after centrifugation was measured at 517 nm, and the scavenging rate of the sample against the DPPH radical was calculated. The erasure rate was calculated by 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 a 1:1 (v / v) ratio and incubated at room temperature for 12-16 hours in the dark. The solution was then diluted with 10 mmol / L PBS to an absorbance of 0.70 ± 0.02 at 734 nm. Sample preparation: Bird's nest peptide samples were prepared in ultrapure water at different concentrations (50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL, and 2500 μg / mL). Preparation of the measurement solution: 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; 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 in the dark. The absorbance was measured at 734 nm, and the ABTS radical scavenging rate of the sample was calculated using the following formula:

number

[0075] 1.4.3 Evaluation of hydroxyl radical scavenging ability Sample preparation: Bird's nest peptide samples were prepared in ultrapure water to give bird's nest peptide solutions of different concentrations (50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL, 2500 μg / mL). Measurement solution preparation: A0) 1 mL of ferrous sulfate, 1 mL of salicylic acid, 11 mL of ultrapure water, and 1 mL of aqueous hydrogen peroxide solution were added to a test tube. A1) 1 mL of bird's nest peptide sample solution, 1 mL of ferrous sulfate, 1 mL of salicylic acid, 10 mL of ultrapure water, and 1 mL of aqueous hydrogen peroxide solution were added to a test tube. A2) 1 mL of bird's nest peptide solution was added to a test tube. The sample solution of foveolar peptide, 1 mL of ferrous sulfate, 1 mL of salicylic acid, and 11 mL of ultrapure water were added. The above solution was mixed uniformly and placed in a water bath at 37°C for 60 minutes. The absorbance was measured at 510 nm, and the hydroxyl radical scavenging rate of the sample 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 the intracellular effects of bird's nest peptides 1.5.1 Evaluation of the toxicity of bird's nest peptides to HaCat cells, B16 cells, and MRC-5 cells The toxicity of bird's nest peptides to HaCat, B16, and MRC-5 cells was measured by CCK-8, and the intracellular safe concentration range of bird's nest peptides was determined. Specifically, 5 × 10 logarithmic phase HaCat, B16, and MRC-5 cells were cultured at 100°C. 5The cells were suspended at 100 μL / mL and inoculated into a 96-well plate. 100 μL was added to each well and incubated in an incubator (CCL-170B-8, ESCO, Singapore) at 37°C with 5% CO2 for 24 hours to allow complete cell adhesion. The medium was removed, washed twice with PBS buffer, and various concentrations of bird's nest peptides (50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL) prepared in MEM were added and incubated in an incubator at 37°C with 5% CO2 for 24 hours. After incubation, 10 μL of CCK-8 was added to each well and the plate was incubated at 37°C for 1 hour. Finally, absorbance was measured at 450 nm to calculate cell viability. Cell viability was calculated using the following formula:

number

[0078] 1.5.2 Evaluation of the protective effect of bird's 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. Specifically, we used 5 × 10 log-phase HaCat cells. 5The cells were suspended at 100 μL / mL and inoculated into a 96-well plate. Each well was inoculated with 100 μL and incubated in an incubator at 37°C with 5% CO2 for 24 hours to allow complete cell adhesion. The medium was then removed, washed twice with PBS buffer, and various concentrations of HO2 solutions (50 μmol / mL, 100 μmol / mL, 200 μmol / mL, 300 μmol / mL, and 500 μmol / mL) prepared with MEM were added. The cells were then incubated in an incubator at 37°C with 5% CO2 for 2 hours. After incubation, 10 μL of CCK-8 was added to each well and incubated at 37°C for 1 hour. Finally, absorbance was measured at 450 nm to calculate cell viability. In this example, the concentration of HO2 required to induce HaCat cell damage was determined to be 300 μmol / L.

[0079] The protective effect of the bird's nest peptides against H2O2-induced HaCat cell injury was then measured. Specifically, 5 × 10 log-phase HaCat cells were cultured in a 5 × 10 5 The cells were suspended at 100 μL / mL and inoculated into a 96-well plate. Each well was inoculated with 100 μL of the solution and incubated in an incubator at 37°C with 5% CO2 for 24 hours to allow complete cell adhesion. The medium was then removed, the plates were washed twice with PBS buffer, and various concentrations of bird's nest peptides (50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, and 1000 μg / mL) prepared in MEM were added. The plates were then incubated in an incubator at 37°C with 5% CO2 for 2 hours. After incubation, 10 μL of CCK-8 was added to each well and the plates were incubated at 37°C for 1 hour. Finally, the absorbance at 450 nm was measured to calculate 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's 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's nest peptides on B16 cell melanin production The B16 cell melanin production inhibition test was measured in accordance with the organization's 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 and inoculated into a 6-well plate, with 2 mL in each well. The cells were then cultured in an incubator at 37°C with 5% CO2 for 24 hours to allow complete cell adhesion. A cell scar was then created in the cell culture plate using a pipette tip. The medium was removed, washed twice with PBS buffer, and various concentrations of bird's nest peptide solution (50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL) prepared in MEM were added and photographed. The cells were then incubated in an incubator at 37°C with 5% CO2 and periodically removed for photographing (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 swallow'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, and Try; polar amino acids (* indicates uncharged) are Ser, Thr, Tyr, Cys, and Gly; acidic amino acids are Asp and Glu; basic amino acids are His, Arg, and Lys; and aromatic amino acids are Tyr, Phe, and Try.

[0086] Table 3 shows the composition and content of 18 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%). The total essential amino acids in bird's nest peptides are 30.90 g / 100 g, accounting for 44.89% of the total amino acids, significantly higher than other protein-rich foods such as chicken eggs (4.7-7.0 g / 100 g) and cow's milk (1.1 g / 100 g). Furthermore, the histidine content in bird's nest peptides, necessary for infant growth, is 2.62 g / 100 g. According to the amino acid nutritional references recommended by the FAO / WHO, a protein with an essential amino acid (EAA) to TAA ratio of approximately 0.4 and an EAA to nonessential amino acid (NEAA) ratio of 0.6 or greater, is considered high-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 standards. 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 355 Da. When further subdivided, the bird's nest peptide can be subdivided into four components, with the number average molecular weight of F1 being 2914 Da, accounting for 8.07%, the number average molecular weight of F2 being 1576 Da, accounting for 11.60%, the number average molecular weight of F3 being 710 Da, accounting for 34.11%, and the number average molecular weight of F4 being 207 Da, accounting for 46.22%. Enzymatic protein hydrolysis products are classified by molecular weight into oligopeptides (<1000 Da), mesopeptides (1000-5000 Da), macropeptides (5000-10000 Da), and proteins (>10000 Da) (10.3390 / gels8010024). Therefore, the results indicated 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 shows the DPPH radical scavenging rate of bird's nest peptides at different concentrations, Figure B shows the ABTS radical scavenging rate of bird's nest peptides at different concentrations, Figure C shows the hydroxyl radical scavenging rate of bird's nest peptides at different concentrations, and Figure D shows the total antioxidant capacity value of bird's nest peptides at different concentrations.

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

[0090] To more intuitively express the antioxidant capacity of EBNP, we further quantitatively characterized its total antioxidant capacity using Trolox (a water-soluble α-tocopherol analogue, a known antioxidant). Total antioxidant capacity is the sum of the radical scavenging activities of different active ingredients. The results are shown in Figure 2D, where the units are mmol Trolox equivalents / L. Consistent with the trends of the other three radical scavenging capacities, these results demonstrate that EBNP possesses antioxidant capacity and that its total antioxidant capacity increases with increasing concentration between 50 and 2500 μg / mL. At a concentration of 2500 μg / mL, EBNP's total antioxidant capacity is equivalent to 28.41 mmol / L Trolox.

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

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

[0093] 2.4.2 Protective effect of bird's nest peptides against H2O2-induced HaCat cell injury H2O2-induced cellular oxidative damage experiments are a widely used cellular experimental protocol for evaluating the intracellular antioxidant potential of interventions. Figure 3D shows the viability of HaCat cells induced by H2O2 under the protection of bird's nest peptides. The results indicated that the higher the viability of HaCat cells under the protection of bird's nest peptides, the greater the intracellular antioxidant activity of bird's nest peptides. Therefore, the results indicated that bird's nest peptides possess antioxidant activity in HaCat cells. Over the concentration range of 50-1000 μg / mL, the intracellular antioxidant activity of bird's nest peptides increased with increasing concentration, with significant intracellular antioxidant activity at concentrations above 250 μg / mL (p<0.05).

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

[0095] The effect of bird's nest peptide on the hyaluronic acid content in HaCat cells is shown in Figure 3E. The results showed that bird's nest peptide at concentrations below 100 μg / mL failed to significantly increase the hyaluronic acid content in HaCat cells (p>0.05), whereas bird's nest peptide at concentrations above 250 μg / mL significantly increased the hyaluronic acid content in HaCat cells (p<0.05). Furthermore, the higher the concentration of bird's nest peptide, the higher the hyaluronic acid content in HaCat cells. Therefore, the results demonstrated that bird's nest peptide has the ability to moisturize cells, further demonstrating its potential for use in moisturizing cosmetics.

[0096] 2.4.4 Inhibitory effect of swallow's nest peptides on B16 cell melanin production The B16 cell melanin production inhibition test provided the most direct evidence for the whitening effect of bird's nest peptides, and the inhibitory rate of bird's nest peptides on B16 cell melanin production is shown in Figure 3F. The results showed that bird's nest peptides have the ability to inhibit B16 cell melanin production, and that in the concentration range of 50-1000 μg / mL, the ability of bird's nest peptides to inhibit intracellular melanin production increased with increasing concentration, and that at concentrations of 50 μg / mL or higher, they had significant intracellular melanin inhibition activity (p<0.05).

[0097] 2.4.5 Repair effect of bird's nest peptides on MRC-5 cell scars The results are shown in Figure 4. In this example, the cell repair and proliferation-promoting ability of bird's nest peptides was evaluated using an MRC-5 cell scar test, and the results are shown in Figure 4. Clearly, without bird's nest peptides, MRC-5 cells still had obvious scars even after 24 hours of culture. Conversely, with bird's nest peptides, MRC-5 cells showed a smaller scar area after 24 hours of culture. In particular, with bird's nest peptides at concentrations of 250 μg / mL or higher, MRC-5 cells showed no obvious scars after 24 hours of culture. Therefore, the results indicated that bird's nest peptides have the ability to promote repair and proliferation in MRC-5 cells, and that this ability increases with increasing concentration. The cell repair and proliferation-promoting ability of bird's nest peptides can provide innovative applications for interventions, such as the development of functional products such as pharmaceuticals and cosmetics that promote skin wound healing and tissue repair. Therefore, based on the results of the scar test, it is further believed that bird's nest peptides have potential applications in the fields of medicine, cosmetics, etc.

[0098] 3 Conclusion In summary, this example provides a production process for mature bird's nest peptides, and systematic evaluation of the bird's nest peptides produced by this process was performed. Regarding basic properties, bird's nest peptides consisted of 68.89% total amino acids and 6.64% sialic acid. In particular, bird's nest peptides contained 30.90 g / 100 g of essential amino acids, accounting for 44.89% of the total amino acid content, indicating that they were derived from high-quality essential amino acids. Regarding functional properties, bird's nest peptides possess antioxidant properties (including scavenging DPPH radicals, ABTS radicals, and hydroxyl radicals). Most importantly, bird's nest peptides possess the functions of cellular antioxidants, cellular moisturizing, cellular whitening, cellular scar repair, and proliferation promotion. Based on these results, bird's nest peptides may have potential applications in fields such as food, pharmaceuticals, and cosmetics. The development and use of bird's nest peptides based on the process of this example will be beneficial to further advance the value-added development of the bird's nest industry. [Example]

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

[0100] 1.2 Production of bird's nest peptides The bird's nest peptide production process involves raw material washing, boiling, mashing, homogenization, pH adjustment, enzymatic hydrolysis, enzyme inactivation, cooling, centrifugation, ultrafiltration, concentration, drying, and portioning. Specifically, edible bird's nest raw material (30% w / v) was washed and 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 at 25 MPa. The homogenized edible bird's nest paste was placed in an enzymatic hydrolysis tank, and the pH of the reaction system was stabilized at 10.0 with 0.01% sodium carbonate. Alkaline protease (9 U per gram of bird's nest) was added to the enzymatic hydrolysis tank, and the enzymatic hydrolysis reaction was carried out for 5 hours at a temperature of 60°C. After enzymatic hydrolysis, the reaction temperature was adjusted to 85°C and maintained for 20 minutes to fully inactivate the enzyme. After enzyme deactivation, the sample was centrifuged at 15,000 rpm for 10 minutes to obtain a crude sample solution of edible bird's nest peptides. The edible bird's nest peptide solution was filtered through a 150 nm ultrafiltration membrane to obtain an edible bird's nest peptide solution. Finally, the solution was spray-dried to obtain edible bird's nest peptide powder, with the inlet air temperature of the spray-drying being 175°C and the outlet air temperature being >90°C.

[0101] 1.3 Characterization of the components and functions of bird's nest peptides 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 swallow's nest peptides [Table 5] The basic components of swallow's nest peptides 5 The bird's nest peptides produced in this research process have a total amino acid content of66.75 %, the sialic acid content is 6.14% and the water content is 12.01%.

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

[0105] Table 5 shows the composition and content of 18 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 the It is the most abundant amino acid in the body, 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, 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 obtained by the production process used in this example is 350 Da. When further subdivided, the bird's nest peptide can be subdivided into four components, with the number average molecular weight of F1 being 2919 Da, accounting for 6.08%, the number average molecular weight of F2 being 1589 Da, accounting for 11.36%, the number average molecular weight of F3 being 715 Da, accounting for 34.69%, and the number average molecular weight of F4 being 210 Da, accounting for 47.87%. Enzymatic protein hydrolysis products are classified by molecular weight into oligopeptides (<1000 Da), mesopeptides (1000-5000 Da), macropeptides (5000-10000 Da), and proteins (>10000 Da) (10.3390 / gels8010024). Therefore, the results indicated 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 shows the DPPH radical scavenging rate of bird's nest peptides at different concentrations, Figure B shows the ABTS radical scavenging rate of bird's nest peptides at different concentrations, Figure C shows the hydroxyl radical scavenging rate of bird's nest peptides at different concentrations, and Figure D shows the total antioxidant capacity value of bird's nest peptides at different concentrations.

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

[0109] To more intuitively express the antioxidant capacity of bird's nest peptides, 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 components against various radicals. The results are shown in Figure 6D, and the units of the results are mmo. 1 Trolox equivalent / L. Consistent with the trends of the other three radical scavenging activities, these results demonstrate that bird's nest peptides have antioxidant properties and that the total antioxidant capacity of bird's nest peptides increases with increasing concentration between 50 and 2500 μg / mL. When the concentration of bird's nest peptides is 2500 μg / mL, the total antioxidant capacity is equivalent to 26.41 mmol / L Trolox.

[0110] 2.4 Intracellular effects 2.4.1 Toxicity of bird's nest peptides to 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 peptides on the viability of H2O2-damaged HaCat cells, Figure E is the effect of bird's nest peptides on the hyaluronic acid content of HaCat cells, and Figure F is the inhibitory rate of bird's nest peptides on melanin production in B16 cells.

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

[0112] 2.4.2 Protective effect of bird's nest peptides against H2O2-induced HaCat cell injury H2O2-induced cellular oxidative damage experiments are a widely used cellular experimental protocol for evaluating the intracellular antioxidant potential of interventions. Figure 7D shows the viability of HaCat cells induced by H2O2 under the protection of bird's nest peptides. The results indicated that the higher the viability of HaCat cells under the protection of bird's nest peptides, the greater the intracellular antioxidant activity of bird's nest peptides. Therefore, the results indicated that bird's nest peptides possess antioxidant activity in HaCat cells. Over the concentration range of 50-1000 μg / mL, the intracellular antioxidant activity of bird's nest peptides increased with increasing concentration, with significant intracellular antioxidant activity at concentrations above 250 μg / mL (p<0.05).

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

[0114] The effect of bird's nest peptide on the hyaluronic acid content in HaCat cells is shown in Figure 7E. The results showed that bird's nest peptide at concentrations below 100 μg / mL failed to significantly increase the hyaluronic acid content in HaCat cells (p>0.05), whereas bird's nest peptide at concentrations above 250 μg / mL significantly increased the hyaluronic acid content in HaCat cells (p<0.05). Furthermore, the higher the concentration of bird's nest peptide, the higher the hyaluronic acid content in HaCat cells. Therefore, the results demonstrated that bird's nest peptide has the ability to moisturize cells, further demonstrating its potential for use in moisturizing cosmetics.

[0115] 2.4.4 Inhibitory effect of swallow's nest peptides on B16 cell melanin production The B16 cell melanin production inhibition test provided the most direct evidence for the whitening effect of bird's nest peptides, and the inhibitory rate of bird's nest peptides on B16 cell melanin production is shown in Figure 7F. The results showed that bird's nest peptides have the ability to inhibit B16 cell melanin production, and that in the concentration range of 50-1000 μg / mL, the ability of bird's nest peptides to inhibit intracellular melanin production increased with increasing concentration, and that at concentrations of 50 μg / mL or higher, they had significant intracellular melanin inhibition activity (p<0.05).

[0116] 2.4.5 Repair effect of bird's nest peptides on MRC-5 cell scars The results are shown in Figure 8. In this study, we evaluated the cell repair and proliferation-promoting abilities of bird's nest peptides using an MRC-5 cell scar test. Figure 8 shows the results. Clearly, without bird's nest peptides, MRC-5 cells still had obvious scars even after 24 hours of culture. Conversely, with bird's nest peptides, MRC-5 cells showed a smaller scar area after 24 hours of culture. In particular, with bird's nest peptides at concentrations above 250 μg / mL, MRC-5 cells showed no obvious scars after 24 hours of culture. Therefore, the results demonstrate that bird's nest peptides have the ability to promote repair and proliferation in MRC-5 cells, and that their ability to promote repair and proliferation increases with increasing concentration. The cell repair and proliferation-promoting abilities of these interventions offer potential applications, such as the development of functional products such as pharmaceuticals and cosmetics that promote skin wound healing and tissue repair. Therefore, based on the results of the scar test, it is further believed that bird's nest peptides have potential applications in the fields of medicine, cosmetics, etc.

[0117] 3 Conclusion In summary, this example provides a production process for mature bird's nest peptides, and systematic evaluation of the bird's nest peptides produced by this process was performed. Regarding basic properties, bird's nest peptides consisted of 66.75% total amino acids and 6.14% sialic acid. In particular, bird's nest peptides contained 26.26 g / 100 g of essential amino acids, accounting for 39.34% of the total amino acid content, and were derived from high-quality essential amino acids. Regarding functional properties, bird's nest peptides possess antioxidant properties (including scavenging DPPH radicals, ABTS radicals, and hydroxyl radicals). Most importantly, bird's nest peptides possess the functions of cellular antioxidants, cellular moisturizing, cellular whitening, cellular scar repair, and proliferation promotion. Based on these results, bird's nest peptides may have potential applications in fields such as food, pharmaceuticals, and cosmetics. The development and use of bird's nest peptides based on this process will be beneficial to further advance the value-added development of the bird's nest industry.

[0118] The technical features of the above embodiments and examples can be combined in any manner, and 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 solutions 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 should 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 within the scope of protection of the present application. It should be further understood that any technical solutions obtained by those skilled in the art through logical analysis, reasoning, or limited experiments based on the technical solutions provided by the present application are all within the scope of the claims attached hereto. Therefore, the scope of protection of the patent of the present application depends on the content of the appended claims, and the description and drawings are intended to explain the content of the claims. do.

Claims

1. mixing bird's nests with water and simmering the mixture to prepare simmered bird's nests; homogenizing the boiled bird's nest to prepare a bird's nest paste; Enzymatically hydrolyzing the swallow's nest paste with alkaline protease to inactivate the enzyme, thereby preparing an enzymatic hydrolyzate; Centrifuging the enzymatic digest and recovering 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 13,000 rpm to 15,000 rpm and a time of 10 minutes to 30 minutes; The pore size of the membrane used for ultrafiltration is 150 nm to 200 nm, The method for producing bird's nest peptides is characterized in that the enzymatic hydrolysis conditions include a temperature of 50°C to 60°C, a pH value of 9.0 to 10.0, a time of 5 to 6 hours, and an amount of alkaline protease used per 1 g of bird's nest of 6 U to 9 U.

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

3. A method for producing bird's nest peptides as described in claim 1, characterized in that the simmering time is 1.75 to 2 hours.

4. A method for producing bird's nest peptides as described in claim 1, characterized in that the homogenization conditions include a pressure of 25 MPa to 30 MPa and a time of 20 minutes to 30 minutes.

5. The method for producing bird's nest peptides described in claim 1, 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 80°C to 100°C and holding it for 10 minutes to 30 minutes.

6. The method for producing bird's nest peptides according to claim 1, further comprising the step of removing the solvent in the ultrafiltrate.

7. The method for producing bird's nest peptides according to claim 6, wherein the step of removing the solvent comprises concentrating the ultrafiltrate and spray-drying the resulting concentrate.

8. In the step of removing the solvent, The method for producing bird's nest peptides described in claim 7, characterized in that (1) the volume of the concentrate is 1 / 3 to 1 / 8 of the volume of the ultrafiltrate, and / or (2) the spray drying conditions are an intake air temperature of 150°C to 200°C and an exhaust air temperature of >70°C.

9. A method for producing bird's nest peptides as described in claim 7, characterized in that the spray drying conditions are an intake air temperature of 165°C to 175°C and an exhaust air temperature of >90°C.

10. The method for producing bird's nest peptides according to claim 1, characterized in that the bird's nest peptides have 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%.

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