CN115896219A

CN115896219A - Preparation method and application of cubilose peptide with effects of promoting cell repair, and high moisturizing and whitening effects

Info

Publication number: CN115896219A
Application number: CN202211398048.3A
Authority: CN
Inventors: 范群艳; 柳训才; 郭宝忠; 白伟娟; 连建梅; 李有泉
Original assignee: Xiamen Yanzhiwu Silken Food Co ltd
Current assignee: Xiamen Yanzhiwu Silken Food Co ltd
Priority date: 2022-11-09
Filing date: 2022-11-09
Publication date: 2023-04-04
Anticipated expiration: 2042-11-09
Also published as: CN115896219B; WO2024098848A1

Abstract

The invention relates to a preparation method and application of cubilose peptide with the effects of promoting cell repair and high moisturizing and whitening, wherein the preparation method comprises the following steps: mixing nidus Collocaliae and water, stewing, and preparing nidus Collocaliae clinker; homogenizing the cubilose clinker to prepare cubilose slurry; carrying out enzymolysis on the cubilose pulp by using alkaline protease, inactivating enzyme, and preparing an zymolyte; centrifuging the zymolyte, collecting supernatant, and preparing a bird's nest peptide crude sample solution; carrying out ultrafiltration on the cubilose peptide crude sample solution, collecting ultrafiltrate, and preparing cubilose peptide; the conditions of centrifugation include: the speed is 10000 rpm-15000 rpm, and the time is 10 min-30 min; the aperture of the membrane adopted by ultrafiltration is 100 nm-200 nm. The bird's nest peptide prepared by the preparation process is basically consistent with bird's nest raw materials in components, inherits and maintains the excellent characteristics of the bird's nest raw materials, and has the functions of cell oxidation resistance, cell moisture preservation, cell scratch repair and proliferation in functional properties.

Description

Preparation method and application of cubilose peptide with effects of promoting cell repair, and high moisturizing and whitening effects

Technical Field

The invention relates to the technical field of food processing, and particularly relates to a preparation method and application of cubilose peptide with the effects of promoting cell repair and high moisturizing and whitening.

Background

Researchers find that the bird's nest peptide generated by hydrolyzing bird's nest with protease has excellent effects of reducing blood pressure, resisting virus and the like. In particular, the bird's nest peptide is superior to bird's nest in some functions. In addition, because of its low molecular weight, it is believed that it is readily absorbed by the body and has the ability to be absorbed through the epidermis. Based on the above advantages, the bird's nest peptide is expected by consumers, manufacturers and researchers in the fields of food, medicine, skin care products and the like, and is considered to be an active substance with wide application prospect.

The conventional preparation method of the cubilose peptide comprises the following steps:

CN107668312A describes an integrated defoaming method for cubilose protein liquid, comprising: will be provided withAdding water into cubilose, stewing to obtain cubilose liquid, and adding 0.1mol/L NaHCO ₃ Regulating pH value of the solution to 8, adding alkaline protease for partial enzymolysis, inactivating enzyme with boiling water for 3min, cooling, and circularly processing for 3 times by vacuum high-low speed beating and high-low temperature defoaming.

CN111528332A describes a preparation method of cubilose peptide, which comprises the following steps: soaking and picking bird's nest: soaking dried nidus Collocaliae in purified water, removing impurities, mincing, and draining; (2) stewing and grinding: adding purified water into the cubilose soaked in the step (1) for stewing, and then finely grinding to obtain cubilose pulp; (3) first biological enzymolysis: adjusting the concentration and pH of the cubilose pulp prepared in the step (2), adding neutral protease into the prepared cubilose pulp, stirring for enzymolysis, inactivating enzyme, cooling, and sieving with a 100-200-mesh sieve to obtain a first enzymolysis liquid; (4) homogenizing and filtering: homogenizing and roughly filtering the first enzymolysis liquid prepared in the step (3) to obtain enzymolysis homogenized liquid; (5) carrying out secondary biological enzymolysis: adjusting the concentration and pH of the enzymatic hydrolysis homogeneous liquid prepared in the step (4), then adding compound protease into the prepared enzymatic hydrolysis homogeneous liquid, stirring for enzymatic hydrolysis, inactivating enzyme, and cooling to obtain a second enzymatic hydrolysis liquid; (6) refining and spray drying: and (4) carrying out fine filtration and RO membrane reverse osmosis concentration on the secondary enzymolysis liquid prepared in the step (5), and then carrying out spray drying to obtain the cubilose peptide.

CN112006276A describes a preparation method of brewing type instant cubilose powder, which comprises the following steps: soaking and picking bird's nest: soaking dried nidus Collocaliae in purified water, removing impurities, pulverizing, draining, and sieving; preparing cubilose liquid and stewing: adding the arginine solution into the screened cubilose obtained in the step (1) to prepare cubilose liquid, stewing in a water-proof way, and cooling; (3) enzymolysis: adjusting the pH value of the solution treated in the step (2) to 8-10, adding alkaline protease for partial enzymolysis, and then inactivating the enzyme by high-temperature boiling water to obtain an enzymolysis solution; (4) ultrasonic defoaming and high-pressure homogenizing: performing low-temperature vacuum whipping on the enzymolysis liquid prepared in the step (3), performing ultrasonic defoaming, and performing high-pressure homogenization to obtain an enzymolysis homogeneous liquid; (5) spray drying: adding crystal sugar and citric acid into the enzymatic hydrolysis homogenized liquid prepared in the step (4), and then carrying out vacuum spray drying to obtain cubilose powder; (6) spraying phospholipid: and (5) spraying phospholipid on the surface of the cubilose powder prepared in the step (5) to obtain the brewing type instant cubilose powder.

CN113564218A describes a preparation method of a whitening active small molecular cubilose peptide, which comprises the following steps: (1) pretreatment of raw materials: heating dried nidus Collocaliae in hot water to obtain feed liquid; (2) enzymolysis: pouring the feed liquid into an enzymolysis tank, adjusting the pH, heating, and adding protease for enzymolysis to obtain an enzymolysis liquid; (3) enzyme deactivation and ultrafiltration: carrying out boiling water bath enzyme deactivation on the enzymolysis liquid, then carrying out ultrafiltration, and collecting filtrate; (4) And spray-drying the filtrate to obtain the whitening active small-molecule bird's nest peptide powder.

CN111019984A records an efficient method for extracting sialic acid in cubilose, which comprises the following steps: i, pretreatment: drying and crushing the cubilose raw material to obtain pretreated cubilose; II, extraction: adding water with the volume 10-30 times of that of the pretreated cubilose into the pretreated cubilose, uniformly stirring, and then performing ultrasonic treatment at 30-45 ℃ for 15-40 min to obtain cubilose slurry; and then adding trypsin, alkaline protease, flavourzyme, pepsin and an enzyme stabilizer into the cubilose pulp, wherein the mass ratio of the cubilose pulp to the trypsin to the alkaline protease to the flavourzyme to the pepsin to the enzyme stabilizer is 100: (0.5-1): (0.5-1): (0.5-1): (0.5-1): (0.01-0.05), carrying out enzymolysis for 2-7 h at 40-60 ℃, inactivating enzyme for 5-10 min at 100-105 ℃, and cooling to obtain enzymolysis liquid; and III, filtering and drying: centrifuging the enzymolysis liquid, filtering the obtained supernatant, and freeze-drying in vacuum to obtain the product.

CN115226886A describes a method for preparing a finished product of high-sialic acid cubilose by an extract back-adding method, which comprises the following steps: (1) preparation of sialic acid extract: soaking crushed swallow horn in water, grinding by a wet method to form millimeter-sized particles, then carrying out ultrasonic crushing, adding alkaline protease for enzymolysis, carrying out enzyme deactivation, and carrying out centrifugal filtration to obtain an extract rich in sialic acid; (3) adding back the extracting solution: soaking the extract and adding the dry bird's nest material, placing the soaked material in an ultrasonic device for ultrasonic swelling, and finally stirring the soaked material into shreds, removing impurities, dehydrating and stewing to obtain the high-sialic acid bird's nest finished product.

CN 111096462B describes a green biological enzymolysis purification method of bird's nest polypeptide and debitterizing method thereof, comprising the following steps: s1, soaking bird' S nest: the edible bird's nest is soaked and foamed for 5 to 12 hours at normal temperature according to the ratio of edible bird's nest to water being 1; s2, cooking: boiling the soaked bird's nest in water bath at 95-100 ℃ for 1-2 h; s3, homogenizing: after the temperature of the boiled cubilose aqueous solution is reduced, homogenizing for 1 to 2 times by a homogenizer; s4, enzymolysis: adding alkaline protease, neutral protease and flavor enzyme in sequence into the homogenate and homogenization solution, and carrying out enzymolysis for 4-8 h at 50-65 ℃, wherein the alkaline protease comprises 45-60% of water, 5-10% of protease A, 35-45% of propylene glycol and 0.2-0.4% of calcium chloride, and the neutral protease comprises 35-50% of water, 5-10% of protease B, 33-45% of sorbitol, 7-10% of propylene glycol and 5% of sodium chloride; protease A is a product of Bacillus licheniformis; the protease B is a product of bacillus amyloliquefaciens; s5, inactivation: inactivating enzyme of the obtained enzymolysis liquid for 15-20 min at 80-90 ℃; s6, filtering with a filter membrane: cooling the inactivated supernatant, and filtering by a filter membrane, wherein the filtering range is 100-120 mm; s7, centrifugation: centrifuging the filtered supernatant to remove residues, wherein the centrifugal speed is 3000-5000 rpm; s8, taking a supernatant: collecting the centrifuged supernatant to obtain a supernatant; s9, microencapsulation: according to the supernatant of the bird's nest: preparing a beta-cyclodextrin solution according to the mass ratio of 1; s10, freeze-drying: freeze-drying to-35-36 deg.c, freeze-drying and grinding to obtain microcapsule bird's nest peptide product.

CN 109852656A describes a preparation method of cubilose polypeptide powder, comprising the following steps: (1) Crushing the cubilose into cubilose powder, soaking the cubilose powder in water according to the material-liquid ratio of 1; (2) Adding protease in an amount which is 3% of the weight of the cubilose powder into the cubilose homogenate, performing enzymolysis for 3 hours, inactivating the enzyme in a boiling water bath, and performing centrifugal treatment to obtain an enzymolysis supernatant Y1 and a precipitate Y2; (3) Taking the precipitate Y2 and water to be resuspended into bird's nest heavy suspension according to the feed liquid ratio of 1; (4) Combining the enzymolysis supernatant Y1 and the enzymolysis supernatant Y3 to obtain an enzymolysis supernatant Y5; (5) Desalting the enzymolysis supernatant Y5 by using a reverse osmosis membrane, and filtering by using membrane filtering equipment to obtain a bird's nest polypeptide purified solution; (6) And freeze-drying the cubilose polypeptide purified solution to obtain cubilose polypeptide powder.

However, the bird's nest peptide prepared by the traditional preparation method and bird's nest raw materials are often inconsistent in components, and the functional characteristics and the application field are not characterized and clear. In view of this, the present application is specifically made.

Disclosure of Invention

The object of the application comprises providing a preparation method of the cubilose peptide, and the cubilose peptide prepared by the preparation method is basically consistent with cubilose raw materials in terms of components.

In a first aspect of the present application, there is provided a method for preparing an nidus Collocaliae peptide, comprising the steps of:

mixing nidus Collocaliae and water, stewing, and preparing nidus Collocaliae clinker;

homogenizing the cubilose clinker to prepare cubilose slurry;

carrying out enzymolysis on the cubilose pulp by using alkaline protease, and inactivating enzyme to prepare an zymolyte;

centrifuging the zymolyte, collecting supernatant, and preparing a bird's nest peptide crude sample solution;

ultrafiltering the coarse cubilose peptide sample solution, collecting ultrafiltrate, and preparing cubilose peptide;

the conditions of centrifugation include: the speed is 10000 rpm-15000 rpm, and the time is 10 min-30 min;

the aperture of the membrane adopted by ultrafiltration is 100 nm-200 nm.

In some embodiments of the present application, ultrafiltration is carried out using membranes having a pore size of 150nm to 200nm.

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

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

In some embodiments of the present application, the stewing time is 0.5h to 2h; optionally, the stewing time is 1.75-2 h.

In some embodiments of the present application, the conditions of homogenization include: the power is 10MPa to 30MPa, and the time is 10min to 30min; optionally, the conditions of homogenization include: the power is 25MPa to 30MPa, and the time is 20min to 30min.

In some embodiments of the present application, the conditions of enzymatic hydrolysis include: the temperature is 50-60 ℃, the pH value is 5.0-9.0, the time is 10-30 min, and the dosage of the alkaline protease corresponding to each 1g of cubilose is 6-9U.

In some embodiments of the present application, the enzyme is inactivated by heating, and the conditions for heating to inactivate the enzyme include: heating the system obtained by enzymolysis to 80-100 ℃ and keeping for 10-30 min.

In some embodiments of the present application, the preparation method further comprises the step of removing the solvent from the ultrafiltrate.

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

In some embodiments of the present application, the step of removing the solvent has one or more of the following technical features:

(1) The volume of the concentrate is 1/3-1/8 of the volume of the ultrafiltrate; and,

(2) The conditions for spray drying include: the air inlet temperature is 150-200 ℃, and the air outlet temperature is more than 70 ℃.

In some embodiments of the present application, the conditions of spray drying include: the air inlet temperature is 165-175 ℃, and the air outlet temperature is more than 90 ℃.

In some embodiments of the present application, the cubilose peptide has a total amino acid content of 66.75wt% to 68.89wt%, a sialic acid content of 6.14wt% to 6.64wt%, and a moisture content of 11.01wt% to 12.01wt%.

In a second aspect of the present application, there is provided a bird's nest peptide prepared by the preparation method of the first aspect.

In a third aspect of the application, there is provided a use of the bird's nest peptide of the second aspect in preparing a medicament or a food or skin care product with moisturizing, whitening, repairing and/or antioxidant effects.

Compared with the prior art, the beneficial effects of the application include:

according to the preparation process, the bird's nest raw material is stewed by water, the obtained bird's nest clinker is homogenized, then the obtained bird's nest slurry is subjected to alkaline protease enzymolysis, then the obtained zymolyte is subjected to moderate centrifugation, the supernatant obtained by the centrifugation is subjected to moderate ultrafiltration, and the obtained ultrafiltrate is collected to prepare the bird's nest peptide, so that the specific bird's nest peptide is finally formed. The bird's nest peptide prepared by the preparation process is basically consistent with bird's nest raw materials in components, inherits and maintains the excellent characteristics (including high content of sialic acid and polypeptide and essential amino acid) of the bird's nest raw materials, and has the functions of cell antioxidation, cell moisture retention, cell scratch repair and proliferation in functional properties. Based on this, the cubilose peptide prepared by the preparation process has the potential of being applied in the fields of food, medicines, skin care products and the like, and is beneficial to further high-valued development of the cubilose industry.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and the advantages thereof, the drawings used in the description of the embodiments will be briefly introduced below. It is obvious that the drawings in the following description are only some embodiments of the application, and that for a person skilled in the art, other drawings can also be derived from them without inventive effort.

FIG. 1 is a bird's nest peptide GPC retention map of example one;

FIG. 2 is a chemical antioxidant capacity diagram of the bird's nest peptide in the first example; wherein, the graph A is the DPPH free radical clearance rate of the cubilose peptide with different concentrations, the graph B is the ABTS free radical clearance rate of the cubilose peptide with different concentrations, the graph C is the hydroxyl free radical clearance rate of the cubilose peptide with different concentrations, and the graph D is the total antioxidant capacity value of the cubilose peptide with different concentrations;

FIG. 3 shows B16 cell and MRC-5 cytotoxicity of the bird's nest peptide HaCat cells in example one; wherein, the graph A is the survival rate of HaCat cells under different concentrations of the bird's nest peptide, the graph B is the survival rate of B16 cells under different concentrations of the bird's nest peptide, the graph C is the survival rate of MRC-5 cells under different concentrations of the bird's nest peptide, and the graph D is the survival rate of the bird's nest peptide to H ₂ O ₂ The effect of damaged HaCat cell survival rate, panel E is the effect of bird's nest peptide on HaCat cell hyaluronic acid content, panel F is the inhibition rate of bird's nest peptide on B16 cell melanin production;

FIG. 4 shows the repairing effect of bird's nest peptide on MRC-5 cell scratch in the first embodiment;

FIG. 5 is a bird's nest peptide GPC retention map of example two;

FIG. 6 is a chemical antioxidant capacity diagram of the bird's nest peptide of example two; wherein, the graph A is the DPPH free radical clearance rate of the cubilose peptide with different concentrations, the graph B is the ABTS free radical clearance rate of the cubilose peptide with different concentrations, the graph C is the hydroxyl free radical clearance rate of the cubilose peptide with different concentrations, and the graph D is the total antioxidant capacity value of the cubilose peptide with different concentrations;

FIG. 7 shows B16 cell and MRC-5 cytotoxicity of the bird's nest peptide HaCat cells in example two; wherein, the graph A is the survival rate of HaCat cells under different concentrations of the cubilose peptide, the graph B is the survival rate of B16 cells under different concentrations of the cubilose peptide, the graph C is the survival rate of MRC-5 cells under different concentrations of the cubilose peptide, and the graph D is the survival rate of the cubilose peptide to H ₂ O ₂ The effect of damaged HaCat cell survival rate, panel E is the effect of bird's nest peptide on HaCat cell hyaluronic acid content, panel F is the inhibition rate of bird's nest peptide on B16 cell melanin production;

FIG. 8 shows the repairing effect of bird's nest peptide on MRC-5 cell scratch in example two.

Detailed Description

The present invention will be described in further detail with reference to the drawings, embodiments and examples. It should be understood that these embodiments and examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention, which is provided for the purpose of making the present disclosure more thorough and complete. It is also understood that the present invention may be embodied in many different forms and is not limited to the embodiments and examples described herein, and that various changes and modifications may be effected therein by one of ordinary skill in the art without departing from the spirit and scope of the invention and the resulting equivalents are within the scope and range of equivalents of the present application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention, and it is to be understood that the present invention may be practiced without one or more of these details.

Unless defined otherwise, 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 invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments and examples only and is not intended to be limiting of the invention.

Term(s)

Unless otherwise stated or contradicted, terms or phrases used herein have the following meanings:

the term "and/or", "and/or" as used herein is intended to be inclusive of any one of the two or more items listed in association, and also to include any and all combinations of the items listed in association, including any two or more of the items listed in association, any more of the items listed in association, or all combinations of the items listed in association. It should be noted that when at least three items are connected by at least two conjunctive combinations selected from "and/or", "or/and", "and/or", it should be understood that, in the present application, the technical solutions definitely include the technical solutions all connected by "logic and", and also the technical solutions all connected by "logic or". For example, "A and/or B" includes three parallel schemes of A, B and A + B. For example, a reference to "a, and/or, B, and/or, C, and/or, D" includes any one of a, B, C, and D (i.e., all references connected by "logical or"), any and all combinations of a, B, C, and D (i.e., any two or any three of a, B, C, and D), and four combinations of a, B, C, and D (i.e., all references connected by "logical and").

The term "plural", and the like in the present invention mean, unless otherwise specifically defined, 2 or more in number or 2 or less. For example, "one or more" means one or two or more.

As used herein, "a combination thereof," "any combination thereof," and the like, includes all suitable combinations of any two or more of the listed items.

In the present specification, the term "suitable" in "a suitable combination, a suitable manner," any suitable manner "and the like shall be construed to mean that the technical solution of the present invention can be implemented, the technical problem of the present invention can be solved, and the technical effect of the present invention can be achieved.

The terms "preferably", "better" and "suitable" are used herein only to describe preferred embodiments or examples, and it should be understood that the scope of the present invention is not limited by these terms.

In the present invention, "further", "still", "specifically", etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of the present invention.

In the present invention, "optionally", "optional" and "optional" refer to the presence or absence, i.e., to any one of two juxtapositions selected from "present" and "absent". If multiple optional parts appear in one technical scheme, if no special description exists, and no contradiction or mutual constraint relation exists, each optional part is independent.

In the present invention, the terms "first", "second", "third", "fourth", etc. in the terms of "first aspect", "second aspect", "third aspect", "fourth aspect", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying that importance or quantity indicating the technical feature being indicated. Also, "first," "second," "third," "fourth," etc. are used for non-exhaustive enumeration of description purposes only and should not be construed as a closed limitation to the number.

In the present invention, the technical features described in the open type include a closed technical solution including the listed features, and also include an open technical solution including the listed features.

In the present invention, where a range of values (i.e., a numerical range) is recited, unless otherwise specified, alternative distributions of values within the range are considered to be continuous, and include both the numerical endpoints of the range (i.e., the minimum and maximum values), and each numerical value between the numerical endpoints. Unless otherwise specified, when a numerical range refers to integers only within the numerical range, both endpoints of the numerical range and each integer between the two endpoints are included, and in this document, it is equivalent to reciting each integer directly, for example, t is an integer selected from 1 to 10, meaning t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. Further, when multiple range-describing features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or a variation within a certain temperature range. It will be appreciated that the described thermostatic process allows the temperature to fluctuate within the accuracy of the instrument control. Allowing fluctuations in the range of, for example,. + -. 5 deg.C,. + -. 4 deg.C,. + -. 3 deg.C,. + -. 2 deg.C, + -. 1 deg.C.

In the present invention,% (w/w) and wt% each represent a weight percentage,% (v/v) means a volume percentage, and% (w/v) means a mass volume percentage.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. The citation of the present application is incorporated by reference herein in its entirety for all purposes unless otherwise in conflict with the present objectives and/or technical disclosure of the present application. Where a citation is referred to herein, the definition of a reference in the document, including features, terms, nouns, phrases, etc., that is relevant, is also incorporated by reference. In the present invention, when the citation is referred to, the cited examples and preferred embodiments of the related art features are also incorporated by reference into the present application, but the present invention is not limited to the embodiments. It should be understood that where a reference conflicts with the description herein, the application is controlling or adaptively modified in accordance with the description herein.

First aspect of the present application

The application provides a preparation method of cubilose peptide, which comprises the following steps:

homogenizing the cubilose clinker to prepare cubilose slurry;

the aperture of the membrane used for ultrafiltration is 100 nm-200 nm.

The conditions of centrifugation in the present application include, for example, 10000, 11000, 12000, 13000, 14000, and 15000 in speed (rpm) and 10, 12, 15, 17, 20, 22, 25, 28, and 30 in time (min).

Ultrafiltration in the present application uses membrane pore sizes (nm) of, for example, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200. Optionally, ultrafiltration is carried out using membranes having pore sizes of 150nm to 200nm.

Optionally, the edible bird's nest is used in an amount of 10g to 50g (e.g., 10g, 15g, 20g, 25g, 30g, 35g, 40g, 45g, 50 g) per 100mL of the water.

Optionally, the edible bird's nest is used in an amount of 25g to 35g (e.g., 25g, 26g, 27g, 28g, 29g, 30g, 31g, 32g, 33g, 34g, 35 g) per 100mL of the water.

Optionally, the stewing 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 h, 2 h); optionally, the stewing time is 1.75-2 h.

Optionally, the conditions of homogenization include: the power is 10MPa to 30MPa (for example, 10MPa, 11MPa, 12MPa, 13MPa, 14MPa, 15MPa, 16MPa, 17MPa, 18MPa, 19MPa, 20MPa, 21MPa, 22MPa, 23MPa, 24MPa, 25MPa, 26MPa, 27MPa, 28MPa, 29MPa, 30 MPa), and the time is 10min to 30min (for example, 10min, 12min, 15min, 17min, 20min, 22min, 25min, 28min, 30 min); optionally, the conditions of homogenization include: the power is 25MPa to 30MPa, and the time is 20min to 30min.

Optionally, the conditions of enzymatic hydrolysis include: the alkaline protease is prepared at 50-60 deg.C (such as 50 deg.C, 51 deg.C, 52 deg.C, 53 deg.C, 54 deg.C, 55 deg.C, 56 deg.C, 57 deg.C, 58 deg.C, 59 deg.C, 60 deg.C) and pH of 8.0-10.0 (such as 8, 8.5, 9, 9.5, 10) for 5h-6h (such as 5h, 5.1h, 5.2h, 5.3h, 5.4h, 5.5h, 5.6h, 5.7h, 5.8h, 5.9h, 6 h), and the alkaline protease is used in an amount of 6-9U (such as 6U, 6.5U, 7U, 7.5U, 8U, 8.5U, 9U) per 1g nidus Collocaliae.

Optionally, the enzyme is inactivated by heating, and the conditions for inactivating enzyme by heating include: heating the system obtained by enzymolysis to 80-100 deg.C (such as 80 deg.C, 82 deg.C, 84 deg.C, 86 deg.C, 88 deg.C, 90 deg.C, 92 deg.C, 94 deg.C, 96 deg.C, 98 deg.C, 100 deg.C) for 10-30 min (such as 10min, 12min, 15min, 17min, 20min, 22min, 25min, 28min, 30 min).

Optionally, the preparation method further comprises the step of removing the solvent from the ultrafiltrate.

Optionally, the step of removing the solvent comprises: concentrating the ultrafiltrate and spray drying the resulting concentrate.

Optionally, the step of removing the solvent has one or more of the following technical features:

(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; and,

(2) The conditions for spray drying include: the inlet air temperature is 150-200 deg.C (e.g. 150 deg.C, 160 deg.C, 170 deg.C, 180 deg.C, 190 deg.C, 200 deg.C), and the outlet air temperature is more than 70 deg.C (e.g. 75 deg.C, 80 deg.C, 85 deg.C, 90 deg.C, 95 deg.C, 100 deg.C, 105 deg.C, 110 deg.C, 115 deg.C, 120 deg.C). Optionally, the conditions of spray drying include: the air inlet temperature is 165-175 ℃, and the air outlet temperature is more than 90 ℃.

Optionally, the total amino acid content is 66.75wt% to 68.89wt%, the sialic acid content is 6.14wt% to 6.64wt%, and the moisture content is 11.01wt% to 12.01wt%.

Optionally, every 100g of the cubilose peptide contains 66.75-68.89% of amino acid, and by mass percentage in the amino acid, the content of essential amino acid is 39.34-44.89%, the content of semi-essential amino acid is 11.19-11.34%, the content of non-essential amino acid is 43.99-49.31%, the content of nonpolar amino acid is 34.83-37.49%, the content of polar amino acid is 25.29-30.40%, the content of acidic amino acid is 13.06-17.02%, the content of basic amino acid is 16.55-24.23%, and the content of aromatic amino acid is 11.42-18.78%;

the content of lysine in the essential amino acid is 5.21-13.03%, the content of phenylalanine is 4.75-7.76%, the content of leucine is 3.90-7.67%, and the content of tyrosine in the non-essential amino acid is 6.67-9.66%;

based on the mass ratio in the polypeptide, the content of the polypeptide with the number average molecular mass of 2914-2919 Da is 6.08% -8.07%, the content of the polypeptide with the number average molecular mass of 1576-1589 Da is 11.36% -11.60%, the content of the polypeptide with the number average molecular mass of 710-715 Da is 34.11% -34.69%, and the content of the polypeptide with the number average molecular mass of 207-210 Da is 46.22% -47.87%.

Second aspect of the present application

The application provides an edible bird's nest peptide, which is prepared by the preparation method of the first aspect.

Third party of the inventionNoodle

The bird's nest peptide provided by the second aspect is applied to preparation of medicines or food or skin care products with moisturizing, whitening, repairing and/or antioxidant effects.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Embodiments of the present invention will be described in detail with reference to examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for the conditions not specified in the following examples, preferably with reference to the guidelines given in the present invention, may also be performed according to the experimental manual or the conventional conditions in the art, may also be performed according to the conditions suggested by the manufacturer, or may be performed according to the experimental procedures known in the art.

In the following specific examples, the measurement parameters relating to the components of the raw materials may have slight deviations within the weighing accuracy, unless otherwise specified. Temperature and time parameters are involved to allow for acceptable deviation due to instrument test accuracy or operational accuracy.

The first embodiment is as follows:

1 materials and methods

1.1 materials

HaCat cells and MRC-5 cells were purchased from Wuhan Punuoise Life technologies, inc. (Procell Life Science & Technology Co., ltd.). CCK-8, available from APExBIO, USA. TAC kit (ab 65329) available from Abcam, UK. The hyaluronic acid ELISA kit (EU 2556) was purchased from FineTest, china. Sialic acid standards, tryptophan standards were purchased from Sigma-Aldrich, usa; the amino acid standard was purchased from Olympic Biotechnology, inc. of Beijing century. Other reagents were analytically pure and above and purchased from Sigma-Aldrich, USA.

1.2 production of bird's nest peptide

The production process flow of the cubilose peptide comprises the following steps: cleaning raw materials, stewing, grinding into slurry, homogenizing, adjusting pH, performing enzymolysis, inactivating enzyme, cooling, centrifuging, ultrafiltering, concentrating, drying, and packaging. Specifically, edible bird's nest raw material (30% w/v) was washed and then stewed in boiling water in a jacketed kettle for 2h. Then homogenizing the cooked edible bird's nest under 30MPa for 30min. The homogenized edible bird's nest pulp was poured into an enzymatic tank and the pH was stabilized in the 9.0 range using 0.01% sodium carbonate. Adding 6U/g alkaline protease into an enzymolysis tank for enzymolysis for 6h, wherein the enzymolysis temperature is kept at 51 ℃. And (3) after the enzymolysis is finished, adjusting the temperature of the system to 85 ℃ and maintaining for 20min to fully inactivate the enzyme. And after enzyme inactivation is finished, centrifuging the sample for 30min at 13000rpm to obtain the edible bird's nest peptide crude sample solution. Filtering the edible bird's nest peptide solution by using a 200nm ultrafiltration membrane to obtain the edible bird's nest peptide solution. And finally, spray drying the solution to obtain edible bird's nest peptide powder, wherein the air inlet temperature of the spray drying is 170 ℃, and the air outlet temperature is more than 90 ℃.

1.3 characterization of peptide component of bird's nest

1.3.1 characterization of the peptide sialic acid of bird's nest

The cubilose peptide sialic acid is characterized by liquid chromatography, and the characterization method refers to Chinese national standard (GB 30636-2014).

1.3.2 bird's nest peptide amino acid characterization

Amino acids in the bird's nest peptide were characterized by liquid chromatography-mass spectrometry (LCMS 8050, shimadzu). The amino acid standard sample is pre-treated and then is tested on a computer to be used as a reference. Specifically, a bird's nest peptide sample (100 mg/mL) was prepared using a mobile phase, filtered through a 0.22 μm polyethersulfone resin membrane, and then subjected to machine detection. Liquid chromatography conditions: the chromatographic column is Shimadzu C18-AQ,2.1mm × 100mm,1.9 μm; the flow rate is 300 mu L/min; the column temperature is 40 ℃; the sample injection amount is 5 mu L; the analysis time is 16min; the mobile phase A is 0.05% formic acid solution, and the mobile phase B is methanol; the elution procedure is as in table 1. Mass spectrum conditions: the ion source is an electrospray ionization source ESI; the monitoring mode is positive ion Multiple Reaction Monitoring (MRM). The interface voltage is 2.5kV; the flow rate of the atomized gas is 3L/min; the flow rate of the heating gas is 10L/min; the interface temperature is 300 ℃; the DL temperature is 250 ℃; the temperature of the heating block is 400 ℃; the flow rate of the drying gas is 10L/min; CID gas was 270kPa.

TABLE 1 elution procedure

1.3.3 characterization of bird's nest peptide Water content

The water content of the cubilose peptide is characterized by a direct drying method, and the characterization method refers to the Chinese national standard (GB 5009.3-2016).

1.3.4 molecular weight characterization of bird's nest peptide

The molecular weight of the cubilose peptide is characterized by Gel Permeation Chromatography (GPC), and the characterization method refers to Chinese national standard (GB 31645-2018).

1.4 evaluation of chemical antioxidant ability of bird's nest peptide

1.4.1 Evaluation of DPPH radical scavenging ability

Preparing a DPPH free radical solution: DPPH was prepared as a 0.04mg/mL solution of DPPH in absolute ethanol. Sample preparation: the bird's nest peptide samples were prepared into bird's nest peptide solutions (50. Mu.g/mL, 100. Mu.g/mL, 250. Mu.g/mL, 500. Mu.g/mL, 1000. Mu.g/mL, 2500. Mu.g/mL) of different concentrations using ultrapure water. Preparing a solution to be tested: a0 2mL of absolute ethanol and 2mL of DPPH free radical solution are added to the test tube, A1) 2mL of cubilose peptide sample solution and 2mL of DPPH free radical solution are added to the test tube, and A2) 2mL of cubilose peptide sample solution and 2mL of absolute ethanol are added to the test tube. The above solutions to be tested are mixed uniformly and left at room temperature for 30min. Finally, the mixed solution was centrifuged at 5000r/min for 10min. And measuring the absorbance value of the centrifuged supernatant at 517nm to calculate the DPPH free radical clearance rate of the sample. The DPPH radical clearance rate of the sample is calculated by the following formula:

wherein,

a0: absorbance values of 2mL absolute ethanol +2mL DPPH free radical solution;

a1: absorbance values of 2mL of the bird's nest peptide sample solution +2mL of the DPPH radical solution;

a2: absorbance values of 2mL of the bird's nest peptide sample solution +2mL of absolute ethanol.

1.4.2 Evaluation of ABTS radical scavenging ability

ABTS free radical solution preparation: a7 mmol/L ABTS aqueous solution and 2.45mmol/L potassium persulfate are mixed at 1 (v/v), and the mixture is protected from light and reacted at room temperature for 12-16 h. Then, the mixture was diluted with 10mmol/L PBS to give an absorbance at 734nm of 0.70. + -. 0.02. Sample preparation: the bird's nest peptide samples were prepared into bird's nest peptide solutions (50. Mu.g/mL, 100. Mu.g/mL, 250. Mu.g/mL, 500. Mu.g/mL, 1000. Mu.g/mL, 2500. Mu.g/mL) of different concentrations using ultrapure water. Preparing a solution to be tested: a0 0.4mL of ultrapure water and 4mL of ABTS free radical solution were added to the test tube, A1) 0.4mL of the peptide sample solution and 4mL of the ABTS free radical solution were added to the test tube, and A2) 0.4mL of the peptide sample solution and 4mL of ultrapure water were added to the test tube. The solutions are uniformly mixed, and after the light-shielding reaction is carried out for 6min, the absorbance value is measured at 734nm to calculate the clearance rate of the ABTS free radicals by the sample. The clearance of the sample to ABTS free radicals is calculated by the following formula:

wherein, A0: absorbance values of 0.4mL ultrapure water +4mL ABTS free radical solution;

a1: absorbance values of 0.4mL of the bird's nest peptide sample solution +4mL of the ABTS free radical solution;

a2: absorbance values of 0.4mL of cubilose peptide sample solution +4mL of ultrapure water.

1.4.3 evaluation of hydroxyl radical scavenging ability

Sample preparation: the bird's nest peptide samples were prepared into bird's nest peptide solutions (50. Mu.g/mL, 100. Mu.g/mL, 250. Mu.g/mL, 500. Mu.g/mL, 1000. Mu.g/mL, 2500. Mu.g/mL) of different concentrations using ultrapure water. Preparing a solution to be tested: a0 1mL ferrous sulfate, 1mL salicylic acid, 11mL ultrapure water, and 1mL hydrogen peroxide solution) were added to the test tube, A1) 1mL cubilose peptide sample solution, 1mL ferrous sulfate, 1mL salicylic acid, 10mL ultrapure water, and 1mL hydrogen peroxide solution were added to the test tube, A2) 1mL cubilose peptide sample solution, 1mL ferrous sulfate, 1mL salicylic acid, and 11mL ultrapure water were added to the test tube. The solutions are mixed uniformly, and the absorbance value is measured at 510nm after the solution is bathed for 60min at 37 ℃ so as to calculate the scavenging rate of the sample on the hydroxyl radical. The hydroxyl radical scavenging rate of the sample is calculated by the following formula:

wherein,

a0:1mL of ferrous sulfate, 1mL of salicylic acid, 11mL of ultrapure water and 1mL of hydrogen peroxide solution;

a1:1mL of cubilose peptide sample solution, 1mL of ferrous sulfate, 1mL of salicylic acid, 10mL of ultrapure water and 1mL of hydrogen peroxide solution;

a2:1mL of cubilose peptide sample solution +1mL of ferrous sulfate +1mL of salicylic acid +11mL of ultrapure water.

1.4.4 evaluation of Total antioxidant Capacity

Total Antioxidant Capacity (TAC) was determined using a commercially available assay kit (Abcam ab 65329).

1.5 evaluation of intracellular Effect of bird's nest peptide

1.5.1 evaluation of toxicity of cubilose peptide on HaCat cells, B16 cells and MRC-5 cells

Toxicity of bird's nest peptide on HaCat cells, B16 cells and MRC-5 cells was measured by CCK-8 to determine the safe intracellular concentration range of bird's nest peptide. Specifically, haCat cells, B16 cells and MRC-5 cells in logarithmic growth phase were cultured at 5X 10 ⁵ suspension/mL, and plating on 96-well plates, 100. Mu.L/well, at 37 ℃ and 5% CO ₂ The cells are cultured in a culture box (CCL-170B-8, singapore ESCO) for 24 hours to ensure that the cells are completely attached to the wall. The medium was removed, washed 2 times with PBS buffer, and different concentrations of bird's nest peptide sample solutions (50. Mu.g/mL, 100. Mu.g/mL, 250. Mu.g/mL, 500. Mu.g/mL, 1000. Mu.g/mL) prepared from MEM were added at 37 ℃ and 5% CO ₂ Incubate in incubator for 24h. After incubation, 10. Mu.LCCK-8 was added to each well and incubated at 37 ℃ for 1h. Finally, the absorbance value was divided at 450nm to calculate the cell viability. Cell viability was calculated using the following formula:

wherein, the sample group: bird's nest peptide sample + cells;

control group: MEM medium + cells;

blank group: MEM medium.

1.5.2 bird's nest peptide pair H ₂ O ₂ Evaluation of protective Effect of induced HaCat cell Damage

First, H is measured ₂ O ₂ Cytotoxicity to HaCat to determine H ₂ O ₂ Concentration to induce cell damage. Specifically, haCat cells in logarithmic growth phase are cultured at the speed of 5 multiplied by 10 ⁵ suspension/mL, plating on 96-well plates, plating 100. Mu.L/well, standing at 37 ℃ and 5% CO ₂ The cells are cultured in the incubator for 24 hours to ensure that the cells are completely attached to the wall. The medium was then removed, washed 2 times with PBS buffer, and different concentrations of H formulated in MEM were added ₂ O ₂ Solution (50. Mu. Mol/mL, 100. Mu. Mol/mL, 200. Mu. Mol/mL, 300. Mu. Mol/mL, 500. Mu. Mol/mL) at 37 ℃ 5% ₂ Incubate in incubator for 2h. After incubation, 10. Mu.LCCK-8 was added to each well and incubated at 37 ℃ for 1h. Finally, the absorbance value was divided at 450nm to calculate the cell viability. This example was tested to determine H ₂ O ₂ The concentration inducing HaCat damage was 300. Mu. Mol/L.

Subsequently, the bird's nest peptide pair H was determined ₂ O ₂ Induced HaCat cell damage protection. Specifically, haCat cells in logarithmic growth phase are treated at 5 × 10 ⁵ Suspending each/mL, inoculating 100 μ L of the suspension in a 96-well plate, standing at 37 deg.C and 5% CO ₂ The cells are cultured in the incubator for 24 hours to ensure that the cells are completely attached to the wall. Then, the medium was removed, washed 2 times with PBS buffer, and various concentrations of bird's nest peptide sample solutions (50. Mu.g/mL, 100. Mu.g/mL, 250. Mu.g/mL, 500. Mu.g/mL, 1000. Mu.g/mL) prepared from MEM were added at 37 ℃ with 5% CO ₂ Incubate in incubator for 2h. After incubation, 10. Mu.LCCK-8 was added to each well and incubated at 37 ℃ for 1h. Finally, the absorbance value was measured at 450nm to calculate the cell viability. The calculation formula is described in the same formula as in reference 1.5.1.

1.5.3 evaluation of Effect of bird's nest peptide on hyaluronic acid content of HaCat cells

HaCat cell hyaluronic acid content was determined using a commercially available hyaluronic acid ELISA kit (EU 2556).

1.5.4 evaluation of inhibitory Effect of bird's nest peptide on melanin synthesis of B16 cell

B16 cell melanin synthesis inhibition assay reference group standard (T/SHRH 027-2019).

1.5.5 evaluation of bird's nest peptide on repairing MRC-5 cell scratch

MRC-5 cells in logarithmic growth phase were cultured at 5X 10 ⁴ suspension/mL, plating on 6-well plates, plating 2mL per well, standing at 37 ℃ and 5% CO ₂ The cells are cultured in the incubator for 24 hours to ensure that the cells are completely attached to the wall. Subsequently, cell scratches were made in the cell culture plate using a pipette tip, the medium was removed, washed 2 times with PBS buffer, and various concentrations of bird's nest peptide sample solutions (50. Mu.g/mL, 100. Mu.g/mL, 250. Mu.g/mL, 500. Mu.g/mL, 1000. Mu.g/mL) prepared from MEM were added and photographed. Subsequently it was placed at 37 ℃ and 5% CO ₂ Incubate in the incubator and take pictures periodically (12 h, 24 h).

1.6 data analysis

All experiments were repeated at least twice with freshly prepared samples. Statistical analysis was performed using SPSS (version 26.0, SPSS corporation, usa). Mean comparisons were performed using Tukey's test, one-way analysis (ANOVA), confirming significant differences at the 5% level.

2 results and discussion

2.1 bird's nest peptide composition

TABLE 2 bird's nest peptide basic Components

	Total amino acids	Sialic acid	Moisture content
				Content (c) of	68.89％±1.21％	6.64％±0.06％	11.01％±0.31％

The basic components of the bird's nest peptide are shown in table 2. The bird's nest peptide produced by the process of the present example had a total amino acid content of 69.10%, a sialic acid content of 6.64%, and a moisture content of 11.01%.

TABLE 3 bird's nest peptide amino acid composition and content (dry weight)

Note: the nonpolar amino acids Ala, val, met, phe, ile, leu, pro, try; (. About. Represents uncharged) polar amino acids Ser, thr, tyr, cys, gly; acidic amino acids Asp, glu; basic amino acids His, arg, lys; the aromatic amino acids are Tyr, phe and Try.

Table 3 shows the composition and content of 18 amino acids in the bird's nest peptide. First, the amino acid content in cubilose peptide was 68.89g/100g, with lysine being the most abundant amino acid in cubilose peptide, accounting for 13.03% of Total Amino Acids (TAA), and second, tyrosine (9.66%), phenylalanine (7.76%), leucine (7.67%). Particularly, the total essential amino acids in the cubilose peptide reach 30.90g/100g, account for 44.89% of the total amino acids, and are obviously higher than other foods rich in protein such as eggs (4.7-7.0 g/100 g) and milk (1.1 g/100 g). It is noteworthy that histidine in the bird's nest peptide, which is essential for infant growth, reached 2.62g/100g. According to the amino acid nutrition standard recommended by FAO/WHO, the ratio of Essential Amino Acid (EAA) to TAA is about 0.4, and the ratio of EAA to non-essential amino acid (NEAA) is above 0.6, so that the protein is a high-quality protein. As can be seen from Table 3, the peptide of bird's nest has EAA/TAA of 0.44 and EAA/NEAA of 1.02, which is far superior to the FAO/WHO standard. Therefore, it is considered that the bird's nest peptide is a high-quality essential amino acid source even from the viewpoint of simply supplementing essential amino acids.

2.2 molecular weight of bird's nest peptide

TABLE 4 molecular weight parameters of bird's nest peptides

The GPC retention profile of the bird's nest peptide obtained in this example is shown in fig. 1, with the corresponding relative molecular mass distribution shown in table 4. The bird's nest peptide obtained from the bird's nest by the production process used in this example had an overall number average molecular mass of 355Da. By further subdividing, the bird's nest peptide can be subdivided into four components, wherein the number average molecular mass of F1 is 2914Da, 8.07%, the number average molecular mass of F2 is 1576Da, 11.60%, the number average molecular mass of F3 is 710Da, 34.11%, and the number average molecular mass of F4 is 207Da, 46.22%. Enzymatic hydrolysates of proteins can be classified according to their molecular weight into oligopeptides (< 1000 Da), mesopeptides (1000-5000 Da), macropeptides (5000-10000 Da), and proteins (> 10000 Da) (10.3390/gels 8010024). Therefore, the results show that the peptide of the bird's nest obtained from the bird's nest by the production process used in this example is mainly oligopeptide.

2.3 chemical antioxidant capacity of bird's nest peptide

The detection result is shown in figure 2, wherein a graph A shows the DPPH free radical clearance rate of the cubilose peptide with different concentrations, a graph B shows the ABTS free radical clearance rate of the cubilose peptide with different concentrations, a graph C shows the hydroxyl free radical clearance rate of the cubilose peptide with different concentrations, and a graph D shows the total antioxidant capacity value of the cubilose peptide with different concentrations.

Antioxidant is considered to be the basis of anti-aging ability. Thus, the antioxidant capacity of EBNP was studied from biochemical and cellular levels. DPPH-, ABTS + and-OH are three well-known free radicals that can be scavenged by antioxidants, resulting in the disappearance of characteristic colors. Thus, the DPPH-, ABTS + and-OH scavenging ability of an antioxidant can be determined by the change in absorbance. The DPPH-, ABTS + and-OH clearance capabilities of EBNP are shown in Panel A, panel B and Panel C, respectively, of FIG. 2. DPPH-, ABTS-of EBNP in the concentration range of 50-2500. Mu.g/mL ⁺ and-OH scavenging ability increases with increasing concentration. Thus, the results indicate that EBNP has the ability to clear DPPH-, ABTS ⁺ and-OH.

To more intuitively express the antioxidant capacity of EBNP, trolox (a water-soluble α -tocopherol analog, a known antioxidant) was targeted for further quantitative characterization of the total antioxidant capacity of EBNP. The total antioxidant capacity is the sum of the various free radical scavenging actions of the different active ingredients. The results are shown in FIG. 2, panel D, in units of mmol Trolox equivalents/L. Consistent with the trend of the scavenging ability of the other three free radicals, the results confirmed that EBNP has antioxidant ability, and the total antioxidant ability of EBNP increases with increasing concentration between 50-2500. Mu.g/mL. When the concentration of EBNP is 2500 mu g/mL, the total antioxidant capacity of the EBNP is equivalent to 28.41mmol/L Trolox.

2.4 intracellular Effect

2.4.1 bird's nest peptide HaCat cells B16 cells and MRC-5 cytotoxicity

The detection results are shown in FIG. 3, wherein, the graph A: survival of HaCat cells at different concentrations of cubilose peptide, panel B: survival of B16 cells at different concentrations of cubilose peptide, panel C: viability of MRC-5 cells at different concentrations of cubilose peptide, panel D: bird's nest peptide pair H ₂ O ₂ Effect of injured HaCat cell survival, panel E: effect of cubilose peptide on hyaluronic acid content of HaCat cells, fig. F: the inhibition rate of cubilose peptide on the generation of melanin of B16 cells.

In the cell experiment, the survival rate of cells influences the experiment result, so that the safe adding concentration of each substance in the cell experiment needs to be determined in advance through a toxicity experiment. The results of the safe concentrations of bird's nest peptide in each cell determined by the experiment are shown in fig. 3, panels a, B, and C. The survival rate of each cell was over 90% in the concentration range of 50-1000. Mu.g/mL. These results indicate that the bird's nest peptide is not cytotoxic to each cell in the concentration range of 50-1000. Mu.g/mL, and is a safe concentration for performing cell experiments.

2.4.2 bird's nest peptide pair H ₂ O ₂ Induced HaCat cell damage protection

H ₂ O ₂ The induced cell oxidative damage assay is a cell assay protocol widely used to evaluate the intracellular antioxidant capacity of an intervention substance. Bird's nest peptide protected H ₂ O ₂ The induced cell viability of HaCat is shown in panel D of fig. 3. In the result, the higher the survival rate of the HaCat cells under the protection of the bird's nest peptide indicates that the oxidation resistance activity of the bird's nest peptide in the HaCat cells is stronger. Therefore, the results show that the bird's nest peptide has the antioxidant capacity in HaCat cells, the intracellular antioxidant capacity of the bird's nest peptide is enhanced along with the increase of the concentration in the concentration range of 50-1000 mu g/mL, and the bird's nest peptide has obvious intracellular antioxidant activity (p) at the concentration of 250 mu g/mL and above (p)<0.05)。

2.4.3 Effect of cubilose peptide on hyaluronic acid content of HaCat cells

Hyaluronic acid is a natural moisturizing factor, participates in skin moisturizing, and is an important component of extracellular matrix. Its content in human cells decreases with age. The human cells actively synthesize hyaluronic acid to ensure cell moisture, and an important example is that HaCat cells generate hyaluronic acid on the inner surface of cell membranes under the catalysis of hyaluronidase. Therefore, the determination of the hyaluronic acid content in the cells can reflect the water-retaining capacity of the cells to a certain extent, so as to evaluate the intervening cell water-retaining capacity.

The hyaluronic acid content of HaCat cells under the influence of bird's nest peptide is shown in fig. 3, panel E. The results showed that bird's nest peptides of 100. Mu.g/mL and below could not significantly increase the hyaluronic acid content in HaCat cells (p > 0.05), while bird's nest peptides of 250. Mu.g/mL and above could significantly increase the hyaluronic acid content in HaCat cells (p < 0.05). Meanwhile, the higher the concentration of the bird's nest peptide, the higher the content of hyaluronic acid in the HaCat cells. Therefore, the result shows that the cubilose peptide has the cell moisturizing capability, and further proves that the cubilose peptide has the potential of being used for cosmetics with the moisturizing function.

2.4.4 inhibition of B16 cell melanin synthesis by bird's nest peptide

The experiments of inhibition of B16 melanin synthesis may provide the most direct evidence for the whitening effect of cubilose peptide, the inhibition rate of cubilose peptide on the melanin production of B16 cells is shown in fig. 3, panel F. The results show that the cubilose peptide has the inhibition capacity on the melanin synthesis of B16 cells, the inhibition capacity of the cubilose peptide on the melanin synthesis in the cells is enhanced along with the increase of the concentration in the concentration range of 50-1000 mug/mL, and the cubilose peptide has obvious inhibition activity on the melanin in the cells at the concentration of 50 mug/mL or more (p is less than 0.05).

2.4.5 bird's nest peptide repairing effect on MRC-5 cell scratch

The results are shown in FIG. 4. In this example, the scratch test of MRC-5 cells was performed to evaluate the cell repair and proliferation ability of the bird's nest peptide, and the results are shown in FIG. 4. Clearly, MRC-5 cells retained significant streaking after 24h of culture without bird's nest peptide intervention. In contrast, MRC-5 cells had reduced scratch area after 24h culture with bird's nest peptide intervention. In particular, MRC-5 cells have no obvious scratch and remain after 24h culture under the intervention of the cubilose peptide with the concentration of 250 mu g/mL and above. Therefore, the results show that the bird's nest peptide has the repairing and proliferating capacity in MRC-5 cells, and the repairing and proliferating capacity in the cells of the bird's nest peptide is enhanced along with the increase of the concentration. The ability to repair and proliferate cells can provide imagination to an agent for intervention in application, and for example, functional products such as drugs and cosmetics for promoting skin wound healing and tissue repair can be developed. Therefore, based on the results of the scratch test, it can be further considered that the bird's nest peptide has a potential for application in the fields of medicines, cosmetics, and the like.

3 conclusion

In summary, this example provides a mature bird's nest peptide production process, and systematically evaluates the bird's nest peptide produced by this process. The bird's nest peptide basically contains 68.89% of total amino acids and 6.64% of sialic acid, and particularly contains 30.90g/100g of essential amino acids accounting for 44.89% of the total amino acids, so that the bird's nest peptide is a high-quality essential amino acid source. In functional properties, the bird's nest peptide has antioxidant (including scavenging DPPH free radicals, ABTS free radicals, hydroxyl free radicals) ability. Most importantly, the cubilose peptide has the functions of cell antioxidation, cell moisturizing, cell whitening, cell scratch repair and proliferation. Based on the above results, the bird's nest peptide has potential application in the fields of food, medicine, cosmetics and the like. The development and utilization of the cubilose peptide based on the process of the embodiment are beneficial to further high-valued development of the cubilose industry.

Example two:

1 materials and methods

1.1 materials

The materials are the same as in the first embodiment.

1.2 production of bird's nest peptide

The production process flow of the cubilose peptide comprises the following steps: cleaning raw materials, stewing, grinding into slurry, homogenizing, adjusting pH, performing enzymolysis, inactivating enzyme, cooling, centrifuging, ultrafiltering, concentrating, drying, and packaging. Specifically, edible bird's nest raw material (30%, w/v) is stewed in boiling water for 1.75h in a jacketed kettle after being cleaned. The cooked edible bird's nest is then homogenized at 25MPa for 20min. The homogenized edible bird's nest slurry was poured into an enzymatic tank and the system pH was stabilized at 10.0 using 0.01% sodium carbonate. Adding alkaline protease (the dosage of the alkaline protease is 9U per 1g of cubilose) into an enzymolysis tank for enzymolysis reaction for 5h, wherein the enzymolysis temperature is kept at 60 ℃. And after the enzymolysis is finished, adjusting the temperature of the system to 85 ℃ and maintaining for 20min to fully inactivate the enzyme. And after enzyme deactivation, centrifuging the sample at 15000rpm for 10min to obtain edible bird's nest peptide crude sample solution. Filtering the edible bird's nest peptide solution by using a 150nm ultrafiltration membrane to obtain the edible bird's nest peptide solution. And finally, carrying out spray drying on the solution to obtain edible bird's nest peptide powder, wherein the air inlet temperature of the spray drying is 175 ℃, and the air outlet temperature is more than 90 ℃.

1.3 bird's nest peptide composition and functional characterization

The specific method is the same as the first embodiment.

1.4 data analysis

The data analysis method is the same as the first embodiment.

2 results and discussion

2.1 bird's nest peptide composition

TABLE 5 bird's nest peptide basic Components

	Total amino acids	Sialic acid	Moisture content
				Content (c) of	66.75.89％±1.21％	6.14％±0.04％	12.01％±0.21％

The basic ingredients of the bird's nest peptide are shown in table 4. The bird's nest peptide produced by the pilot plant of the research technology has the total amino acid content of 69.10 percent, the sialic acid content of 6.14 percent and the water content of 12.01 percent. .

TABLE 6 bird's nest peptide amino acid composition and content (dry weight)

Table 5 shows the composition and content of 18 amino acids in the bird's nest peptide. First, the amino acid content in the bird's nest peptide was 66.75g/100g, with serine being the most abundant amino acid in bird's nest peptide, accounting for 9.68% of Total Amino Acids (TAA), and second, glutamic acid (9.30%), threonine (9.17%), arginine (8.12%). As shown in Table 5, the peptide of bird's nest has EAA/TAA of 0.39 and EAA/NEAA of 0.80, which are far superior to FAO/WHO standard. Therefore, it is considered that, even from the viewpoint of simply supplementing essential amino acids, bird's nest peptide is a high-quality essential amino acid source.

2.2 molecular weight of bird's nest peptide

TABLE 7 molecular weight parameters of bird's nest peptides

Fraction (b) of	Number average molecular weight (Mn)	Weight average molecular weight (Mw)	Mw/Mn	Area of
					F1	2919	3080	1.05	6.08％
F2	1589	1630	1.03	11.36％
					F3	715	745	1.04	34.69％
F4	210	217	1.04	47.87％
					General of	350	735	2.10	100％

The GPC retention profile of the bird's nest peptide obtained in this example is shown in fig. 5, with the corresponding relative molecular mass distribution shown in table 7. The bird's nest peptide obtained from the bird's nest by the production process used in this example had an overall number average molecular mass of 350Da. By further subdividing this, the bird's nest peptide can be subdivided into four fractions, wherein the number average molecular mass of F1 is 2919Da, and is 6.08%, the number average molecular mass of F2 is 1589Da, and is 11.36%, the number average molecular mass of F3 is 715Da, and is 34.69%, and the number average molecular mass of F4 is 210Da, and is 47.87%. Enzymatic hydrolysates of proteins can be classified according to their molecular weight into oligopeptides (< 1000 Da), mesopeptides (1000-5000 Da), macropeptides (5000-10000 Da), and proteins (> 10000 Da) (10.3390/gels 8010024). Therefore, the result shows that the bird's nest peptide obtained by the production process in the embodiment of the bird's nest is mainly oligopeptide.

2.3 chemical antioxidant capacity of bird's nest peptide

The detection result is shown in fig. 6, wherein a graph A shows the DPPH free radical clearance rate of the cubilose peptide with different concentrations, a graph B shows the ABTS free radical clearance rate of the cubilose peptide with different concentrations, a graph C shows the hydroxyl free radical clearance rate of the cubilose peptide with different concentrations, and a graph D shows the total antioxidant capacity value of the cubilose peptide with different concentrations.

The DPPH-, ABTS + and-OH-scavenging abilities of bird's nest peptide are shown in panels A, B and C, respectively, in FIG. 6. In the concentration range of 50-2500 mug/mL, DPPH-, ABTS + and-OH removal capacity of the cubilose peptide is increased along with the increase of the concentration. Thus, the results indicate that the bird's nest peptide has the ability to scavenge DPPH-, ABTS + and-OH.

In order to express the antioxidant capacity of the cubilose peptide more intuitively, trolox (a water-soluble alpha-tocopherol analogue, a known antioxidant) is taken as a target, and the total antioxidant capacity of the cubilose peptide is further quantitatively characterized. The total antioxidant capacity is the sum of the various free radical scavenging actions of the different active ingredients. The results are shown in FIG. 6, panel D, with the units of the results being mmol of Trolox equivalents/L. Consistent with the trend of the scavenging capacity of other three free radicals, the result proves that the cubilose peptide has the antioxidant capacity, and the total antioxidant capacity of the cubilose peptide is increased with the increase of concentration between 50 and 2500 mu g/mL. When the concentration of the cubilose peptide is 2500 mu g/mL, the total antioxidant capacity of the cubilose peptide is equivalent to 26.41mmol/L Trolox.

2.4 intracellular Effect

2.4.1 bird's nest peptide HaCat cell B16 cell and MRC-5 cytotoxicity

The detection results are shown in FIG. 7, wherein, the graph A: survival of HaCat cells at different concentrations of cubilose peptide, panel B: survival of B16 cells at different concentrations of cubilose peptide, panel C: viability of MRC-5 cells at different concentrations of cubilose peptide, panel D: bird's nest peptide pair H ₂ O ₂ Effect of injured HaCat cell survival, panel E: effect of cubilose peptide on hyaluronic acid content of HaCat cells, fig. F: the inhibition rate of cubilose peptide on the generation of B16 cell melanin.

In the cell experiment, the survival rate of cells influences the experiment result, so that the safe adding concentration of each substance in the cell experiment needs to be determined in advance through a toxicity experiment. The results of the safe concentrations of bird's nest peptide in each cell determined by the experiment are shown in fig. 7, panels a, B and C. The survival rate of each cell was over 90% in the concentration range of 50-1000. Mu.g/mL. These results indicate that the bird's nest peptide is not cytotoxic to each cell in the concentration range of 50-1000. Mu.g/mL, and is a safe concentration for performing cell experiments.

2.4.2 bird's nest peptide pair H ₂ O ₂ Induced HaCat cell damage protection

H ₂ O ₂ The induced cell oxidative damage assay is a widely used cell assay protocol for assessing the intracellular antioxidant capacity of an intervention. Bird's nest peptide protected H ₂ O ₂ The induced cell viability of HaCat is shown in fig. 7, panel D. In the result, the higher the survival rate of the HaCat cells under the protection of the bird's nest peptide indicates that the oxidation resistance activity of the bird's nest peptide in the HaCat cells is stronger. Therefore, the result shows that the bird's nest peptide has the antioxidant capacity in HaCat cells, the intracellular antioxidant capacity of the bird's nest peptide is enhanced along with the increase of the concentration in the concentration range of 50-1000 mu g/mL, and the bird's nest peptide has obvious intracellular antioxidant activity (p) at the concentration of 250 mu g/mL or more<0.05)。

2.4.3 Effect of cubilose peptide on hyaluronic acid content of HaCat cells

Hyaluronic acid is a natural moisturizing factor, participates in skin moisturizing, and is an important component of extracellular matrix. Its content in human cells decreases with age. The human cells actively synthesize hyaluronic acid to ensure cell moisture, and an important example is that HaCat cells generate hyaluronic acid on the inner surface of cell membranes under the catalysis of hyaluronidase. Therefore, the determination of the hyaluronic acid content in the cell can reflect the water retention capacity of the cell to some extent, thereby evaluating the intervened cell water retention capacity.

The hyaluronic acid content of HaCat cells under the influence of cubilose peptide is shown in fig. 7, panel E. The results showed that bird's nest peptides of 100. Mu.g/mL and below could not significantly increase the hyaluronic acid content in HaCat cells (p > 0.05), while bird's nest peptides of 250. Mu.g/mL and above could significantly increase the hyaluronic acid content in HaCat cells (p < 0.05). Meanwhile, the higher the concentration of the bird's nest peptide, the higher the content of hyaluronic acid in the HaCat cells. Therefore, the result shows that the cubilose peptide has the cell moisturizing capability, and further proves that the cubilose peptide has the potential of being used for cosmetics with moisturizing functions.

2.4.4 inhibition of B16 cell melanin synthesis by bird's nest peptide

The experiments on the inhibition of B16 melanin synthesis may provide the most direct evidence for the whitening effect of cubilose peptide, the inhibition rate of cubilose peptide on the melanin production of B16 cells is shown in fig. 7, panel F. The results show that the cubilose peptide has the inhibition capacity on the melanin synthesis of B16 cells, the inhibition capacity of the cubilose peptide on the melanin synthesis in the cells is enhanced along with the increase of the concentration in the concentration range of 50-1000 mug/mL, and the cubilose peptide has obvious inhibition activity on the melanin in the cells at the concentration of 50 mug/mL or more (p is less than 0.05).

2.4.5 bird's nest peptide repairing effect on MRC-5 cell scratch

The results are shown in FIG. 8. In this study, the scratch test of MRC-5 cells was used to evaluate the cell repair and proliferation ability of nidus Collocaliae peptide, and the results are shown in FIG. 8. Clearly, MRC-5 cells retained significant streaking after 24h of culture without bird's nest peptide intervention. In contrast, MRC-5 cells had reduced scratch area after 24h culture with bird's nest peptide intervention. In particular, MRC-5 cells have no obvious scratch and remain after 24h culture under the intervention of the cubilose peptide with the concentration of 250 mu g/mL and above. Therefore, the results show that the bird's nest peptide has the repairing and proliferating capacity in MRC-5 cells, and the repairing and proliferating capacity in the cells of the bird's nest peptide is enhanced along with the increase of the concentration. The ability to repair and proliferate cells can provide imagination to an agent for intervention in application, and for example, functional products such as drugs and cosmetics for promoting skin wound healing and tissue repair can be developed. Therefore, based on the results of the scratch test, it can be further considered that the bird's nest peptide has a potential to be applied in the fields of medicines, cosmetics, and the like.

3 conclusion

In summary, this example provides a mature peptide production process, and systematic evaluation is performed on the peptide produced by the process. The bird's nest peptide basically contains 66.75% of total amino acids and 6.14% of sialic acid, and particularly contains 26.26g/100g of essential amino acids, accounting for 39.34% of the total amino acids, and is a high-quality essential amino acid source. In functional properties, the bird's nest peptide has the ability to resist oxidation (including scavenging DPPH free radicals, ABTS free radicals, hydroxyl free radicals). Most importantly, the bird's nest peptide has the functions of cell antioxidation, cell moisturizing, cell whitening, cell scratch repair and proliferation. Based on the above results, the bird's nest peptide has potential application in the fields of food, medicine, cosmetics and the like. The development and utilization of the bird's nest peptide based on the process of the embodiment are beneficial to further high-valued development of the bird's nest industry.

The technical features of the embodiments and examples described above can be combined in any suitable manner, and for the sake of brevity, all possible combinations of the technical features of the embodiments and examples described above are not described, but should be considered within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Furthermore, it should be understood that after reading the above teachings of the present invention, various changes or modifications may be made to the invention by those skilled in the art, and equivalents may be obtained and still fall within the scope of the present application. It should also be understood that technical solutions obtained by logical analysis, reasoning or limited experiments based on the technical solutions provided by the present invention by those skilled in the art are all within the scope of the present invention as set forth in the appended claims. Therefore, the protection scope of the present invention should be subject to the content of the appended claims, and the description and the drawings can be used for explaining the content of the claims.

Claims

1. The preparation method of the cubilose peptide is characterized by comprising the following steps of:

homogenizing the cubilose clinker to prepare cubilose slurry;

carrying out ultrafiltration on the cubilose peptide crude sample solution, collecting ultrafiltrate, and preparing cubilose peptide;

the aperture of the membrane used for ultrafiltration is 100 nm-200 nm; optionally, ultrafiltration is carried out using membranes having pore sizes of 150nm to 200nm.

2. The method for preparing cubilose peptide according to claim 1, wherein the amount of cubilose used is 10-50 g per 100mL of water; optionally, the amount of the cubilose used for every 100mL of water is 25 g-35 g.

3. The method for preparing cubilose peptide according to claim 1, wherein the stewing time is 0.5-2 h; optionally, the stewing time is 1.75-2 h.

4. The method for preparing cubilose peptide according to claim 1, wherein the homogenizing conditions comprise: the power is 10MPa to 30MPa, and the time is 10min to 30min; optionally, the conditions of homogenization include: the power is 25MPa to 30MPa, and the time is 20min to 30min.

5. The method for preparing cubilose peptide according to claim 1, wherein the enzymolysis conditions comprise: the temperature is 50-60 ℃, the pH value is 8.0-10.0, the time is 10-30 min, and the dosage of the alkaline protease corresponding to each 1g of cubilose is 6-9U.

6. The method for preparing cubilose peptide according to claim 1, wherein the enzyme is inactivated by heating, and the conditions for heating to inactivate the enzyme comprise: heating the system obtained by enzymolysis to 80-100 ℃ and keeping for 10-30 min.

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

8. The method of claim 7, wherein the step of removing the solvent comprises: concentrating the ultrafiltrate and spray drying the resulting concentrate.

9. The method for preparing cubilose peptide according to claim 8, characterized in that the step of removing the solvent has one or more of the following technical characteristics:

(2) The conditions for spray drying include: the air inlet temperature is 150-200 ℃, and the air outlet temperature is more than 70 ℃; optionally, the conditions of spray drying include: the air inlet temperature is 165-175 ℃, and the air outlet temperature is more than 90 ℃.

10. The method for producing the bird's nest peptide according to any one of claims 1 to 6, 8, and 9, characterized in that the bird's nest peptide has a total amino acid content of 66.75wt% to 68.89wt%, a sialic acid content of 6.14wt% to 6.64wt%, and a moisture content of 11.01wt% to 12.01wt%.

11. An avian nidus Collocaliae peptide prepared by the preparation method of any one of claims 1 to 10.

12. Use of the bird's nest peptide of claim 11 in the preparation of a medicament or a food or skin care product with moisturizing, whitening, repairing and/or antioxidant effects.