CN113930471A

CN113930471A - Bird's nest active peptide and preparation method and application thereof

Info

Publication number: CN113930471A
Application number: CN202111285269.5A
Authority: CN
Inventors: 何志伟; 姚媛; 盖永强; 朴美子
Original assignee: Qingdao Zhengdian Biotechnology Co ltd
Current assignee: Qingdao Zhengdian Biotechnology Co ltd
Priority date: 2021-11-01
Filing date: 2021-11-01
Publication date: 2022-01-14

Abstract

The invention provides an edible bird's nest active peptide, a preparation method and application thereof. The preparation method provided by the invention is characterized in that the degradation is carried out by a biological enzyme method, the degradation condition is mild, the process is controllable, no harmful byproducts such as chemicals are generated in the degradation process, and the obtained cubilose active peptide is safe and green and is easy to be absorbed by human bodies. The cubilose active peptide can inhibit the photoaging of human immortalized keratinocytes, has the activity of resisting photoaging, can effectively reduce the generation of active oxygen, and simultaneously can effectively reduce the contents of matrix metalloproteinase-1, matrix metalloproteinase-3 and matrix metalloproteinase-9, remove DPPH free radicals, inhibit photodamage and resist photoaging, thereby effectively protecting the skin.

Description

Bird's nest active peptide and preparation method and application thereof

Technical Field

The invention belongs to the technical field of cubilose active peptides, and particularly relates to a cubilose active peptide with anti-photoaging activity, and a preparation method and application thereof.

Background

Bird's Nest (EBN) refers to Bird Nest formed by mixing and coagulating saliva secreted by swiftlet (Collocalia) of Potiria of Apodidae and swiftlets of the same genus with their down feather. The bird's nest is recorded and described in detail in Ben Cao gang mu written by Ming Dynasty Li Shizhen, and has the effects of nourishing yin, moistening lung, benefiting qi, strengthening middle warmer, invigorating spleen, invigorating stomach, relieving cough, eliminating phlegm, etc. Modern researches show that the cubilose has active components such as epidermal growth factors, sialic acid, glycoprotein and the like, has the functions of promoting cell growth and reproductive capacity, promoting the regeneration of human tissues and cells, improving the cellular immune function, enhancing the virus resistance of human bodies and helping the human bodies to resist cold symptoms such as cough, cold and the like.

Ultraviolet (UV) radiation is believed to be a major contributory factor to the extrinsic aging of the skin. According to the wavelength, the solar ultraviolet has 3 main parts, namely long-wave ultraviolet (320-400nm), medium-wave ultraviolet (280-320nm) and short-wave ultraviolet (<280 nm). UVA and UVB can pass through the atmospheric layer to reach the ground, and in the action of sunlight on the skin, the UVB accounts for 75-85%. With increasing wavelength, the penetration depth of UV into the skin increases, and the available tissue goes from the superficial to the deep epidermis, and even the dermis.

The skin covering the whole body is the largest organ of the human body, can protect other tissues and organs in the body from physical, chemical and pathogenic microorganism invasion, and has important physiological functions. Skin aging due to repeated exposure to ultraviolet light is also known as photo aging (Photoaging) of the skin. It is clinically characterized by loss of elasticity and relaxation of the skin, roughness and atrophy of the exposed part and thickening of the epidermis, deepening and thickening of wrinkles, appearance of irregular pigmentation and freckles, canceration, etc. Photo-aging of skin not only causes aging, affects appearance and beauty, but also is closely related to many clinical skin diseases, such as solar lentigo, skin cancer, actinic stretch fiber disease, melanoma, actinic keratosis, and the like.

Peptides (peptides) are compounds formed by the polymerization of two or more amino acids through amide bonds, have a smaller relative molecular mass than proteins, and have a structure between amino acids and proteins. Among them, a polypeptide that exerts an important physiological regulation function in a human body is called an active peptide, which is also called a bioactive peptide or bioactive polypeptide. The bioactive peptide has high efficiency, and can still exert remarkable physiological activity under the condition of low content in vivo. The active peptide also has higher edible safety, can be used as a functional component to be added into various products, is a hot point direction for the research in the field of food at present, and has wide market prospect.

The bird's nest contains 50-60% of high-quality protein and is a good source for preparing active peptide. Although many related whitening products of bird's nest are successively developed, intensive research on bird's nest peptides is deficient.

Disclosure of Invention

The invention provides a cubilose active peptide with anti-photoaging activity, a preparation method and application thereof, wherein the preparation method utilizes protease enzymolysis to prepare the cubilose active peptide; the obtained nidus Collocaliae bioactive peptide has anti-photoaging activity. The applicability of the prepared cubilose active peptide in the field of inhibiting the photoaging of human immortalized keratinocytes is proved by cell experiments.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the invention provides a preparation method of cubilose active peptide, which comprises the following steps: pretreating bird's nest, adding dry bird's nest into purified water and fully soaking; after stewing, the stewed cubilose is frozen and dried to obtain cubilose powder; an enzymolysis step, namely adding water to the obtained cubilose powder for dissolving, and carrying out full enzymolysis by using protease; a step of obtaining the bird's nest protein enzymolysis liquid, which is to adjust the pH value of the liquid after enzymolysis to 6-7.5, and centrifugate to obtain supernatant fluid to obtain the bird's nest protein enzymolysis liquid; and (3) preparing the cubilose active peptide, namely grading the obtained cubilose protease hydrolysate to obtain a degradation product with the molecular weight of less than 1kDa, and freeze-drying the degradation product to obtain freeze-dried powder, namely the cubilose active peptide.

Further, in the step of pretreating the cubilose, the temperature for stewing the cubilose is 90-110 ℃, the time is 60-120 min, and the pressure is less than 0.1 MPa.

Further, in the step of pretreating the cubilose, adding the dry cubilose into purified water 15-50 times of the weight of the cubilose, and soaking for 5-20 hours.

Further, in the enzymolysis step, the ratio of the cubilose powder to the water is 1: 50-1: 30.

Further, in the step of enzymolysis, the protease is a neutral protease.

Further, the enzyme activity of the neutral protease is 100-500U/gprot.

Further, in the enzymolysis step, the enzyme adding amount of protease added into the cubilose powder is 0.2-0.8% of the weight of the cubilose powder; carrying out enzymolysis for 2-6 h by using protease.

The application also provides the cubilose active peptide which is prepared by adopting the preparation method.

Use of an active peptide of bird's nest for inhibiting photoaging of human immortalized keratinocytes.

Further, the method for inhibiting photoaging of human immortalized keratinocytes by the cubilose active peptide comprises the following steps: inhibiting the generation of active oxygen in human immortalized keratinocytes, reducing the contents of MMP-1, MMP-3 and MMP-9 secreted by the human immortalized keratinocytes, and eliminating DPPH free radicals.

Compared with the prior art, the invention has the advantages and beneficial effects that: the preparation method provided by the invention is characterized in that the degradation is carried out by a biological enzyme method, the degradation condition is mild, the process is controllable, no harmful byproducts such as chemicals are generated in the degradation process, and the obtained cubilose active peptide is safe and green and is easy to be absorbed by human bodies. The cubilose active peptide has anti-photoaging activity, can effectively reduce the generation of Reactive Oxygen Species (ROS), and simultaneously can effectively reduce the contents of matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-3 (MMP-3) and matrix metalloproteinase-9 (MMP-9), and remove DPPH free radicals, thereby effectively protecting the skin. Therefore, the compound can be applied to preparing products for inhibiting photodamage and resisting photoaging, such as cosmetics and foods. And sufficient theoretical support and experimental proof are provided for the edible bird's nest applied to the food field, and the edible bird's nest has good market application prospect.

Drawings

FIG. 1 shows the morphology of normal HaCaT cells, photoaged cells and bird's nest active peptide (neutrase) treated cells under the microscope provided in this example;

FIG. 2A is a blank control in Table 2 showing the observation of the fluorescence intensity of ROS in HaCaT cells as provided in this example;

FIG. 2B is a model corresponding to Table 2 for the fluorescence intensity observation of ROS in HaCaT cells provided in this example;

FIG. 2C is a graph showing the fluorescence intensity of ROS in HaCaT cells according to this example, corresponding to the neutral proteases of Table 2;

FIG. 2D is an observation of the fluorescence intensity of ROS in HaCaT cells provided in this example, corresponding to alkaline protease of Table 2;

FIG. 2E is an observation of the fluorescence intensity of ROS in HaCaT cells as provided in this example, corresponding to the flavourzyme of Table 2;

FIG. 2F is a graph showing the fluorescence intensity of ROS in HaCaT cells as provided in this example, versus papain in Table 2;

FIG. 2G is an observation of the fluorescence intensity of ROS in HaCaT cells as provided in this example, corresponding to trypsin in Table 2;

FIG. 3 shows the toxic effect of the < 1kDa bird's nest active peptide on HaCaT cells after enzymolysis by five proteases with different concentrations provided in this example

FIG. 4 is a MMP-1 standard curve provided in this example;

FIG. 5 is a result of detecting MMP-1 content secreted by < 1kDa bird's nest active peptide intervening HaCaT cells after five proteases are subjected to enzymolysis;

FIG. 6 is a MMP-3 standard curve provided in this example;

FIG. 7 is a result of detecting the content of MMP-3 secreted by < 1kDa bird's nest active peptide intervening HaCaT cells after enzymolysis by five proteases provided in this example;

FIG. 8 is a MMP-9 standard curve provided in this example;

FIG. 9 is a result of detecting the content of MMP-1 secreted by < 1kDa bird's nest active peptide intervening HaCaT cells after enzymolysis by five proteases provided in this example;

FIG. 10 is a graph showing the effect of the feed liquid ratio provided in this example on the DPPH radical scavenging ability of the bird's nest active peptide after neutral protease enzymolysis;

FIG. 11 is a graph showing the effect of pH on DPPH radical scavenging ability of active peptide from bird's nest after neutral protease enzymolysis;

FIG. 12 is a graph showing the effect of the enzyme addition amount on DPPH free radical scavenging ability of bird's nest active peptide after neutral protease enzymolysis;

FIG. 13 is a graph showing the effect of the time provided in this example on the DPPH radical scavenging ability of the active peptide from bird's nest after neutral protease digestion.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. It is to be understood that the embodiments described are only a few embodiments of the present application and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The invention provides a preparation method of cubilose active peptide, which comprises the following steps of S1-S4: s1, pretreatment of the edible bird 'S nest, namely adding the dry edible bird' S nest into purified water for fully soaking; after stewing, the stewed cubilose is frozen and dried to obtain cubilose powder; s2, an enzymolysis step, namely dissolving the obtained cubilose powder in water, and carrying out full enzymolysis by using protease; s3, obtaining the bird 'S nest protein enzymatic hydrolysate, namely adjusting the pH value of the liquid after enzymolysis to 6-7.5, and centrifuging to obtain supernatant fluid to obtain the bird' S nest protein enzymatic hydrolysate; s4, preparing the cubilose active peptide, namely, carrying out grading treatment on the obtained cubilose protease hydrolysate to obtain a degradation product with the molecular weight of less than 1kDa, and then carrying out freeze drying on the degradation product to obtain freeze-dried powder, namely the cubilose active peptide.

In the pretreatment step of the cubilose, the temperature for stewing the cubilose is 90-110 ℃, the time is 60-120 min, and the pressure is less than 0.1 MPa.

In the step of pretreating the cubilose, adding the dry cubilose into purified water 15-50 times of the weight of the cubilose, and soaking for 5-20 hours.

In the enzymolysis step, the ratio of the cubilose powder to the water is 1: 50-1: 30.

In the enzymolysis step, the protease is neutral protease, and the enzymatic activity of the neutral protease is 100-500U/gprot.

In the enzymolysis step, adding protease into the cubilose powder in an amount which is 0.2-0.8% of the weight of the cubilose powder; carrying out enzymolysis for 2-6 h by using protease.

Wherein, the method for inhibiting the photoaging of the human immortalized keratinocyte by the cubilose bioactive peptide comprises the following steps: inhibiting the generation of active oxygen in human immortalized keratinocytes, reducing the contents of MMP-1, MMP-3 and MMP-9 secreted by the human immortalized keratinocytes, and eliminating DPPH free radicals.

The following embodiments provide a method for preparing an active peptide from bird's nest to better illustrate the present invention. However, the present invention is not limited to the following examples.

The preparation method of the active peptide from the bird's nest source comprises the following specific steps:

(1) pretreatment of the cubilose: weighing a certain amount of dry edible bird's nest, adding 20 times of purified water, stirring uniformly to ensure that the raw materials can absorb water, and soaking for 14 h. Stirring again after soaking is finished, and no large cubilose, hard core and the like are ensured;

stewing is carried out under the following stewing conditions: stewing temperature: 101-103 ℃; stewing pressure: 0 pa; stewing time: 60min-90 min; and (4) freeze-drying the stewed cubilose to obtain cubilose powder.

(2) Enzymolysis: respectively selecting five proteases to carry out enzymolysis on the cubilose powder, wherein the proteases are respectively neutral protease, alkaline protease, flavourzyme, papain and trypsin; dissolving the cubilose powder obtained in the step (1) in water according to a feed-liquid ratio of 1:50, and performing enzymolysis for 4 hours by using five kinds of protease according to the action conditions and the dosage provided in the table 1.

TABLE 1 conditions and amounts of the five proteases

(3) Adjusting the pH of the liquid after enzymolysis in the step (2) to 7.0, and centrifuging at 10000rpm and 4 ℃ for 15min to obtain supernatant as an enzymolysis liquid.

(4) And (3) respectively carrying out grading treatment on the five enzymolysis solutions obtained in the step (3) by using an organic membrane separator to obtain degradation products with the molecular weight of less than 1kDa, and respectively carrying out freeze drying on the degradation products to respectively obtain five freeze-dried powders, namely the cubilose active peptide.

The bird's nest active peptide has an inhibiting effect on photoaging human immortalized keratinocyte (HaCaT) cells. The effect of five bird's nest active peptides prepared in the embodiment on inhibiting HaCaT cell photoaging comprises the following contents:

1. method for screening effective and safe concentration of cubilose active peptide

When the cell growth density reached about 80% to 90%, the cells were digested, centrifuged, suspended in 1mL of MEM, and suspended at 1X 10⁴The cells are inoculated to a 96-well plate with the density of each well, each group is provided with 6 multiple wells, and the mixture is placed at 37 ℃ and 5 percentCO₂The incubator of (a) is to have the cells fused to about 80%.

Cells in 96-well plates were divided into blank control and (0.01, 0.05, 0.1) mg/mL bird's nest active peptide groups:

(1) blank control group: and (3) sucking out the old solution, adding 100 mu L of new culture solution, putting the new culture solution into an incubator for incubation for 24h, sucking out the old solution, adding 100 mu L of new culture solution again, and measuring the cell proliferation rate after continuing incubation for 24 h.

(2) (0.01, 0.05, 0.1) mg/mL bird's nest active peptide group:

and (3) sucking out the old solution, adding 100 mu L of culture solution containing (0.01, 0.05 and 0.1) mg/mL of cubilose bioactive peptide, putting the culture solution into an incubator, incubating for 24h, sucking out the old solution, adding 100 mu L of cubilose bioactive peptide culture solution with corresponding concentration again, and continuously incubating for 24h to measure the cell proliferation rate.

The experimental results are shown in figure 3, and it can be known that the < 1kDa cubilose active peptide after enzymolysis by 0.01, 0.05 and 0.1mg/mL of five proteases has no toxic effect on the growth of HaCaT cells.

2. UVB irradiation establishment of photoaging HaCaT cell model

When the cell growth density reached about 80% to 90%, the cells were digested, centrifuged, suspended in 1mL of MEM, and suspended at 1X 10⁴Cell/well Density cells were seeded into 96-well plates, each set was 6 multiple wells, placed at 37 ℃ with 5% CO₂When the cells fused to about 80%, the old solution was aspirated off, the cells were covered with PBS, and the cells were incubated at 0, 15, 30, 60, 90mJ/cm²After different doses of UVB irradiate HaCaT cells, PBS is discarded, 100 mu L of new culture solution is added again, and the cell proliferation rate is determined after incubation is continued for 24 h.

3. MTT method for detecting cell proliferation rate

(1) Adding 200 μ L of 0.5mg/mL MTT solution in dark place, and placing at 37 deg.C with 5% CO₂Incubate for 4 h.

(2) The solution in the 96-well plate was discarded, and then 150. mu.L of DMSO solution was added.

(3) Then placing in a constant temperature culture shaker, shaking for 10min, and finally measuring absorbance value (OD) of each well at 490nm wavelength of an enzyme labeling instrument₄₉₀)。

Cell proliferation rate (%) ═ OD₄₉₀Administration group/OD₄₉₀Blank control x 100%.

4. Five bird's nest active peptides with 0.1mg/mL interfere with photoaging cells

When the cell growth density reached about 80% to 90%, the cells were digested, centrifuged, suspended in 1mL of MEM, and suspended at 1X 10⁵Cell/well Density cells were seeded into 24-well plates, each set was plated with 4 multiple wells, placed at 37 ℃ with 5% CO₂When the cells fused to about 80%, the cells were cultured with a culture solution containing 0.1mg/mL of the bird's nest active peptide for 24 hours. The cells are divided into:

(1) blank control group: 0mg/mL cubilose active peptide without UVB irradiation;

(2) model group: 0mg/mL bird's nest active peptide, 60mJ/cm²UVB irradiation;

(3) five protease groups: 0.1mg/mL bird's nest active peptide, 60mJ/cm²UVB irradiation.

Washing with PBS before irradiation, covering cells with a small amount of PBS, removing PBS after irradiation, adding culture solution containing the same concentration of cubilose bioactive peptide, and culturing for 24 h. Collecting supernatant for detecting MMP-1, MMP-3 and MMP-9 contents; collecting cells, detecting the fluorescence intensity of ROS in HaCaT cells by adopting flow cytometry, and observing the fluorescence intensity by adopting a laser confocal microscope.

5. Detection of ROS in HaCaT cells

Groups of HaCaT cells were washed with PBS, treated with 10mM DCFH-DA and incubated at 37 ℃ in the dark for 30 min. DCFH-DA enters HaCaT cells and is hydrolyzed into DCFH by esterase, and DCFH is oxidized into a strong green fluorescent substance DCF which cannot penetrate through cell membranes by ROS. Cells were collected and the fluorescence intensity of ROS was observed under a confocal laser microscope and detected by flow cytometry.

TABLE 2 flow cytometry results for ROS fluorescence intensity detection

Table 2 shows that all of the bird's nest active peptides obtained by five protease enzymatic hydrolysis have the effect of inhibiting the production of ROS in HaCaT cells, wherein the < 1kDa bird's nest active peptide after the neutral protease enzymatic hydrolysis has the best effect of inhibiting the production of ROS in HaCaT cells, and the content of ROS produced in photoaging HaCaT cells can be reduced to 52.90%, as can be seen by observing the comparison of ROS fluorescence intensity through a laser confocal microscope in combination with fig. 2A-2G.

6. Determination of MMP-1, MMP-3, MMP-9 content

The MMP-1, MMP-3 and MMP-9 contents are measured according to the instruction of a matrix metalloproteinase enzyme-linked immunosorbent assay (ELISA) kit. The kit adopts a double-antibody sandwich method, a microporous plate is coated by a purified matrix metalloproteinase antibody to prepare a solid-phase antibody, the matrix metalloproteinase (sample) and an enzyme-labeled matrix metalloproteinase antibody are sequentially added into micropores coated with a monoclonal antibody to form an antibody-antigen-enzyme-labeled antibody compound, a substrate is added for color development after thorough washing, and the color shade is positively correlated with the matrix metalloproteinase in the sample. Absorbance was measured at a wavelength of 450nm and calculated by using a standard curve, and FIG. 4, FIG. 6 and FIG. 8 show the standard curves for MMP-1, MMP-3 and MMP-9, respectively.

The results in FIG. 5 show that the effect of < 1kDa cubilose active peptide on the MMP-1 level secretion of photoaging HaCaT cells is obviously reduced after the enzymolysis of neutral protease and trypsin; the results in FIG. 7 show that the MMP-3 level secreted by < 1kDa cubilose active peptide to photoaging HaCaT cells is obviously reduced after the enzymolysis of neutral protease, flavourzyme and trypsin; the results in FIG. 9 show that the effect of < 1kDa bird's nest active peptide on the MMP-9 secretion level of photoaging HaCaT cells is obviously reduced after the neutral protease and the flavourzyme are subjected to enzymolysis.

In conclusion, after the neutral protease is subjected to enzymolysis, the < 1kDa cubilose active peptide has obvious effects of reducing the levels of MMP-1, MMP-3 and MMP-9 secreted by photoaging HaCaT cells, and the levels are respectively reduced by 9.69%, 13.10% and 12.48% compared with the secretion levels of the photoaging HaCaT cells. As can be seen from FIG. 1, when the cell morphology is observed under an inverted microscope, the normal HaCaT cells are closely arranged, full and cellular; the photoaging HaCaT cells irradiated by UVB shrink, are loosely and irregularly arranged, and have more cell death; photoaging HaCaT cell atrophy degree and cell death number of bird's nest active peptide with less than 1kDa obtained after neutral protease enzymolysis are reduced after treatment. Therefore, the cubilose active peptide can inhibit photodamage and resist photoaging.

Wherein, according to the effect of the neutral protease enzymolysis liquid on removing DPPH free radicals, the enzymolysis condition of the neutral protease is optimized by single factor experiment and orthogonal experiment design.

1. Evaluation index DPPH radical scavenging ability measurement

Taking 4 groups of test tubes, adding corresponding reagents according to the sequence in the table 3, carrying out reaction according to the reaction condition sequence in the table 3 to determine DPPH and scavenging capacity, fully and uniformly mixing at room temperature, standing for 30min in a dark place, determining the light absorption value at 517nm, carrying out three groups of parallel tests each time, and repeating the tests for 3 times.

TABLE 3 determination of DPPH radical scavenging Capacity of enzymatic hydrolysate

The calculation is performed according to the following formula:

DPPH radical clearance (%) (1- (sample a-ginseng a)/control a) × 100.

2. Neutral protease enzymolysis single factor experiment

(1) Influence of feed-liquid ratio on enzymolysis effect of neutral protease

The influence of the feed liquor ratio on the enzymolysis effect of the neutral protease is analyzed under the conditions that the enzymolysis temperature is 55 ℃, the pH value is 7.0, the enzyme adding amount is 0.3 percent, and the enzymolysis time is 4 hours. The result is shown in FIG. 10, when the feed liquid ratio is higher than 1:50, the DPPH free radical scavenging capacity of the neutral protease enzymolysis liquid is rapidly reduced, but the bird's nest solution is too thick due to the low feed liquid ratio and is not beneficial to experimental operation, so that the feed liquid ratio is 1:50, and subsequent experiments are carried out.

(2) Influence of pH value on enzymolysis effect of neutral protease

The influence of pH value on the enzymolysis effect of neutral protease is analyzed under the conditions that the ratio of material to liquid is 1:50, the enzymolysis temperature is 55 ℃, the enzyme adding amount is 0.3%, and the enzymolysis time is 4 h. As a result, as shown in FIG. 11, when the pH was more than 7.5, the ability of the neutral protease enzymatic hydrolysate to scavenge DPPH free radicals was rapidly decreased, and the pH of the bird's nest solution itself was selected to be about 7.0, and the pH of the bird's nest solution was selected to be used in the subsequent experiments.

(3) Influence of enzyme addition amount on enzymolysis effect of neutral protease

And analyzing the influence of the enzyme addition amount on the enzymolysis effect of the neutral protease under the conditions of a feed-liquid ratio of 1:50, an enzymolysis temperature of 55 ℃, a pH value of 7.0 and an enzymolysis time of 4 hours. The results are shown in FIG. 12, the DPPH free radical scavenging ability of the neutral protease enzymolysis liquid is gradually increased along with the increase of the enzyme adding amount, but when the enzyme adding amount exceeds 0.3%, the DPPH free radical scavenging ability is not greatly changed, and in order to save cost, the enzyme adding amount of 0.3% is selected for subsequent experiments.

(4) Influence of enzymolysis time on enzymolysis effect of neutral protease

The influence of the enzymolysis time on the enzymolysis effect of the neutral protease is analyzed under the conditions that the ratio of the feed to the liquid is 1:50, the enzymolysis temperature is 55 ℃, the pH value is 7.0 and the enzyme adding amount is 0.3%. The results are shown in fig. 13, the DPPH free radical scavenging capacity of the neutral protease enzymolysis liquid is gradually increased along with the increase of the enzymolysis time, but the DPPH free radical scavenging capacity is not changed greatly when the enzymolysis time exceeds 4 hours, and the enzymolysis time is selected to be 4 hours for subsequent experiments in order to save time.

3. Neutral protease enzyme hydrolysis orthogonal assay design

On the basis of a single-factor experiment, a 4-factor 3 horizontal orthogonal experiment is adopted to optimize the extraction process, and the horizontal design of the orthogonal experiment factors is shown in table 4.

TABLE 4 orthogonal test factor horizon

4. Design and results of orthogonal experiments

The research takes enzyme adding amount, pH value, feed-liquid ratio and time as factors, takes DPPH free radical clearance as an index, and adopts 4-factor 3 horizontal orthogonal test optimization. Orthogonal experiment design and results are shown in table 5, and according to the range R value, the influence of various factors on DPPH free radical clearance is in the order of enzyme addition amount > pH > feed-liquid ratio > time, the optimal enzymolysis condition combination is A2B2C2D2, namely enzyme addition amount is 0.3%, pH is 7.0, enzymolysis is carried out for 4 hours at 55 ℃, and DPPH free radical clearance is 17.21% under the optimal enzymolysis condition. The better the enzymolysis effect, the higher the yield of the low molecular especially < 1kDa bird's nest active peptide in the obtained fermentation liquor, and the better the effect of removing DPPH free radicals.

TABLE 5 enzyme conditions optimization orthogonal test design and results

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Claims

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

pretreating bird's nest, adding dry bird's nest into purified water and fully soaking; after stewing, the stewed cubilose is frozen and dried to obtain cubilose powder;

an enzymolysis step, namely adding water to the obtained cubilose powder for dissolving, and carrying out full enzymolysis by using protease;

a step of obtaining the bird's nest protein enzymolysis liquid, which is to adjust the pH value of the liquid after enzymolysis to 6-7.5, and centrifugate to obtain supernatant fluid to obtain the bird's nest protein enzymolysis liquid;

and (3) preparing the cubilose active peptide, namely grading the obtained cubilose protease hydrolysate to obtain a degradation product with the molecular weight of less than 1kDa, and freeze-drying the degradation product to obtain freeze-dried powder, namely the cubilose active peptide.

2. The preparation method according to claim 1, wherein in the step of pretreating the edible bird's nest, the edible bird's nest is stewed at 90-110 ℃ for 60-120 min, and under the pressure of less than 0.1 MPa.

3. The preparation method according to claim 1, wherein in the step of pretreating the bird's nest, the dry bird's nest is added into purified water 15-50 times the weight of the bird's nest and soaked for 5-20 h.

4. The preparation method according to claim 1, wherein in the enzymolysis step, the ratio of the cubilose powder dissolved by water to the feed liquid is 1: 50-1: 30.

5. The method according to claim 1, wherein in the step of subjecting to enzymolysis, the protease is a neutral protease.

6. The method according to claim 5, wherein the neutral protease has an enzymatic activity of 100 to 500U/gprot.

7. The preparation method according to claim 1, wherein in the enzymolysis step, the protease is added into the cubilose powder in an amount of 0.2-0.8% of the weight of the cubilose powder; carrying out enzymolysis for 2-6 h by using protease.

8. An active peptide of bird's nest, which is prepared by the preparation method of any one of claims 1-7.

9. Use of the bird's nest active peptide of claim 7, wherein the bird's nest active peptide is used to inhibit photoaging of human immortalized keratinocytes.

10. Use of an active bird's nest peptide according to claim 9, wherein the active bird's nest peptide inhibits the photoaging mode of human immortalized keratinocytes by: inhibiting the generation of active oxygen in human immortalized keratinocytes, reducing the contents of MMP-1, MMP-3 and MMP-9 secreted by the human immortalized keratinocytes, and eliminating DPPH free radicals.