Ultrasonic-assisted composite protease hydrolyzed pea protein as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to ultrasonic-assisted composite protease hydrolyzed pea protein as well as a preparation method and application thereof. The preparation method comprises the following steps: raw material treatment, high-temperature sterilization, ultrasonic enzymolysis of a compound protease mixed solution, enzymatic hydrolysate separation, supernate separation, spray drying and obtaining of a target product pea peptide, pea protein is hydrolyzed by compound protease to obtain the pea peptide with the molecular weight of 6000u or below and an anti-wrinkle function, and enzyme deactivation treatment is performed at high temperature to obtain the pea peptide with the anti-wrinkle function. Filtering by using an ultrafiltration fiber membrane, separating from the inactivated enzymatic hydrolysate, drying by using a spray dryer to prevent oxidation in the drying process, and hydrolyzing the pea protein by using a compound enzyme to obtain the pea protein peptide; the obtained product easily and directly enters deep skin through the surface layer of the skin, is quickly absorbed by the skin of a human body to play a role, and has higher biological activity and bioavailability. The anti-wrinkle peptide is prepared from pea protein, resources can be fully utilized, waste is turned into wealth, and waste of raw materials is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to an ultrasound-assisted composite protease hydrolyzed pea protein, and a preparation method and application thereof. Background Art

[0002] In recent years, with the improvement of people's living standards and the demand for their own health, various cosmetics have received more and more attention. The application of animal protein in cosmetics has a long history, but in recent years, affected by the animal protection rights movement, its application in cosmetics has been decreasing, and people have turned their attention to plant protein. Due to the variety of plant proteins, good efficacy of active ingredients and few side effects, natural beauty products based on plant proteins are favored by more and more consumers. Pea protein is a natural plant protein extracted from peas. It has high-quality nutrients and multiple functions. It is not only a great supplement to the body's nutrition, but also has a very good nourishing effect on human skin. Pea extract has a good promoting effect on the production of collagen. Combined with its strong inhibition of elastase, it shows that pea extract has skin revitalizing and anti-aging effects. Therefore, pea protein is widely used in skin care products such as anti-wrinkle, firming, conditioning and soothing repair.

[0003] my country has abundant pea seed plant protein resources at a low price. In the field of cosmetics, the application of pea protein has been widely studied and applied. However, due to low solubility and the presence of a large number of other antioxidant factors, the biological digestibility and utilization rate are greatly reduced, which greatly limits their practical application. Therefore, it is urgent to provide a pea protein with higher utilization rate and a preparation method thereof. Summary of the invention

[0004] In order to improve the digestibility and utilization rate of pea protein in the field of cosmetics, the present invention provides an ultrasound-assisted composite protease hydrolysis of pea protein and a preparation method thereof, which has simple process operation, does not destroy amino acids, and can effectively improve the solubility, biological activity and bioavailability of pea protein.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A preparation method for ultrasonic-assisted composite protease hydrolysis of pea protein, wherein pea protein peptides with a molecular weight below 6000u are obtained from pea protein by composite protease hydrolysis; the composite protease comprises flavor protease, papain, trypsin and neutral protease.

[0007] Furthermore, the mass ratio of flavor protease, papain, trypsin and neutral protease is 1:1-4:1-5:1-6.

[0008] Furthermore, the preparation method of ultrasound-assisted composite protease hydrolysis of pea protein comprises the following steps:

[0009] The pea protein solution is mixed with deionized water and stirred evenly, and then immersed in ultrasound for wall breaking treatment; the pea protein solution after the wall breaking treatment is sterilized at high temperature instantaneously, and flavor protease, papain, trypsin and neutral protease are added in sequence after cooling, and the pea protein solution is ultrasonically hydrolyzed. After the enzymolysis is completed, the mixed protease in the enzymolysis solution is inactivated, the enzymolysis solution after the enzyme inactivation treatment is centrifuged, and the supernatant is dialyzed; the dialysis filtrate is placed in a spray dryer for airflow spray drying to obtain ultrasound-assisted composite protease hydrolyzed pea protein.

[0010] Furthermore, the preparation method of the pea protein solution includes: soaking the peas from which impurities have been removed in water for 16-20 hours, then rinsing with clean water, grinding with a grinding wheel, and repeatedly grinding the dregs for multiple times; centrifuging the slurry for 2-4 minutes using a centrifuge with a rotation speed of 1300-1700 r / min to remove the residue, acid-adjusting the supernatant to precipitate the protein, centrifuging to remove the supernatant, and adjusting the acid-precipitated protein to a pH of 7.0 with alkali to dissolve the protein to obtain a pea protein solution.

[0011] Furthermore, before the cell wall breaking treatment, the temperature of the pea protein solution was controlled at 42-50° C. and ultrasonically treated for 2-4 hours.

[0012] Furthermore, after ultrasonic hydrolysis, a dilute alkali solution is added to adjust the pH value of the solution to 7.5-8.0, the hydrolysis temperature is 60-65° C., and the ultrasonic hydrolysis power is 340-380 W; the dilute alkali solution is a NaOH solution.

[0013] Furthermore, the activity of the composite protease is 50-75u / g, the enzymatic hydrolysis time is 2-5h, and the added amount is 0.15% of the mass of the pea protein solution; after the enzymatic hydrolysis is completed, the solution is heated to 90-100°C and the enzyme is inactivated for 10-20min; the centrifugal speed of the enzymatic hydrolyzate after enzyme inactivation is 8000-10000r / min, and the centrifugal treatment time is 10-30min; the dialysis conditions are dialyzed in a 6000u dialysis bag for 20-24h.

[0014] The invention also provides an ultrasound-assisted composite protease hydrolyzed pea protein, which is prepared by the above method.

[0015] The present invention also provides the use of the ultrasound-assisted composite protease hydrolyzed pea protein in the preparation of cosmetics.

[0016] Furthermore, the application includes the following applications A1-A3:

[0017] A1. Preparation of cosmetics with anti-wrinkle and firming effects;

[0018] A2. Preparation of cosmetics with moisturizing / nourishing effects;

[0019] A3. Prepare cosmetics with soothing and repairing effects.

[0020] An ultrasound-assisted composite protease hydrolyzed pea protein and a preparation method thereof, comprising the following steps:

[0021] (1) Raw material processing: The pea protein solution obtained by extracting peas is mixed with deionized water at a mass ratio of 1:10-20, stirred evenly by a stirring device, and then the temperature is controlled at 42-50° C. The solution is immersed in ultrasound for 2-4 hours and subjected to a cell wall breaking treatment.

[0022] (2) High temperature sterilization: The pea protein solution is placed in a high pressure steam sterilizer for constant temperature treatment, and high temperature instantaneous sterilization (125-149°C, 3-8 seconds) is used to inactivate the bacteria in the protein, facilitating subsequent operations;

[0023] (3) Enzymatic hydrolysis of the mixed solution: the pea protein solution is cooled to room temperature, and a mixed protease is added thereto to perform ultrasonic hydrolysis on the pea protein solution, and a dilute alkali solution is added to adjust the pH value to 7.5-8.0, the hydrolysis temperature is 60-65° C., and the enzymatic hydrolysis time is 2-5 h;

[0024] (4) Separation of enzymatic hydrolysate: After the enzymatic hydrolysis is completed, the treated enzymatic hydrolysate is heated to 90-100°C and inactivated for 10-20 minutes to inactivate the complex protease in the enzymatic hydrolysate;

[0025] (5) Centrifugation of the enzymatic hydrolysate: centrifuge the enzymatic hydrolysate after enzyme inactivation at a speed of 8000-10000 r / min for 10-30 min, and obtain the supernatant for dialysis;

[0026] (6) Spray drying: The dialysis filtrate is placed in a spray dryer for airflow spray drying to obtain the enzymatically hydrolyzed small molecule pea polypeptide product.

[0027] Furthermore, in step (1), the method for preparing the de-pea protein solution comprises the following steps:

[0028] The peas from which impurities have been removed are soaked in water for 16-20 hours, then rinsed with clean water, and then ground with a grinding wheel, and the dregs are repeatedly ground for multiple times. The slurry is centrifuged for 3 minutes using a centrifuge with a speed of 1500r / min to remove the residue, the supernatant is acid-adjusted to precipitate the protein, the supernatant is removed by centrifugation, and the protein after acid precipitation is adjusted to pH 7.0 with alkali to dissolve the protein to obtain the pea protein solution.

[0029] Furthermore, in step (1), the cell wall breaking treatment is to break the pea cells using high pressure homogenization technology.

[0030] Furthermore, in step (3), the added mixed protease is composed of four proteases: flavor protease, papain, trypsin and neutral protease. The mass ratio of the flavor protease, papain, trypsin and neutral protease is 2-3:4-6:5-7:8-9.

[0031] Furthermore, in step (3), the dilute alkali solution is a NaOH solution.

[0032] Furthermore, in step (3), the activity of the complex enzyme added for enzymatic hydrolysis is 50-75u / g, and the added amount is 0.15%.

[0033] Furthermore, in step (3), the power of the ultrasonic hydrolysis is 360W.

[0034] Furthermore, in step (5), the dialysis condition is dialysis in a 6000u dialysis bag for 20-24h.

[0035] Beneficial Effects

[0036] The main components of pea protein hydrolysate are a mixture of polypeptides and amino acids. Under the action of microbial enzymes, pea protein macromolecules are broken into small peptides. As the peptide bonds break, the increase of -COOH and -NH2 leads to an increase in its hydrophilicity. Therefore, after hydrolysis, the protein solubility increases significantly. Moreover, when the degree of hydrolysis is high, the solubility of pea protein hydrolyzate is not affected within a fairly wide pH range. A large number of amino acids, small peptides, etc. obtained by hydrolysis can form complex amino acid complexes with metal elements under certain conditions and serve as cosmetic additives.

[0037] Enzymatically modified proteins have the advantages of not destroying amino acids, easy quality control, and few side reactions, especially in the extraction and retention of nutrients and minerals. After hydrolysis, the functionality of pea protein is greatly improved as the molecular weight decreases and some groups are exposed. Therefore, pea protein is hydrolyzed by a specific process, and its product can be used as a cosmetic additive with specific functions. Therefore, the ultrasound-assisted composite protease hydrolyzed pea protein and its preparation method provided by the present invention are of great significance to the cosmetic field.

[0038] The present invention processes pea protein raw materials, mixes protein solution with water for wall breaking treatment, dissolves protein by alkali adjustment, and performs high-temperature instant sterilization treatment by high-pressure steam autoclave. On the one hand, bacteria in protein can be inactivated, and on the other hand, the structure of pea protein can be unfolded, which is convenient for subsequent hydrolysis operation, and pea protein is hydrolyzed by composite protease to obtain pea peptide with anti-wrinkle and firming effect, and enzyme inactivation treatment is performed by high temperature, and ultrafiltration fiber membrane with interception of 6000u is used to filter it, and it is separated from the inactivated enzymatic hydrolyzate, and dried by spray dryer to prevent oxidation during drying, which affects the effect of the product. After hydrolysis, pea protein is greatly improved with the decrease of molecular weight and exposure of some active groups, so pea protein is easy to enter deep skin directly through the skin surface after hydrolysis by a specific process, and is quickly absorbed by the human body to play a role, and can be used as a cosmetic additive with specific functions, so it can make full use of resources, turn waste into treasure, and reduce the waste of raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A schematic diagram of the process for preparing the method for hydrolyzing pea protein with ultrasound-assisted composite protease provided by the present invention;

[0040] Figure 2 This is a schematic diagram of the preparation process of pea protein solution;

[0041] Figure 3 The proliferation rate of human fibroblasts was compared with that of different enzymatic protein peptides. DETAILED DESCRIPTION

[0042] Example 1

[0043] (1) Raw material processing: The pea protein solution obtained by peas was mixed with deionized water at a mass ratio of 1:15, stirred evenly by a stirring device, and then the temperature was controlled at 45° C. The solution was immersed in ultrasound for 3 h and subjected to cell wall breaking treatment;

[0044] (2) High temperature sterilization: The pea protein solution is placed in a high pressure steam sterilizer for constant temperature treatment, and high temperature instant sterilization is used to inactivate the bacteria in the protein to facilitate subsequent operations;

[0045] (3) Enzymatic hydrolysis of the mixed solution: The pea protein solution was cooled to room temperature, and flavor protease was added thereto to perform ultrasonic hydrolysis on the pea protein solution, and a dilute alkali solution was added to adjust the pH value to 7.5, the hydrolysis temperature was controlled at 60° C., and the enzymatic hydrolysis time was 3 h;

[0046] (4) Separation of enzymatic hydrolysate: After the enzymatic hydrolysis is completed, the treated enzymatic hydrolysate is heated to 100° C. and the enzyme is inactivated for 10 min to inactivate the mixed protease in the enzymatic hydrolysate;

[0047] (5) Centrifugation of the enzymatic hydrolysate: The enzymatic hydrolysate after the enzyme inactivation treatment was centrifuged at a speed of 8000 r / min for 30 min, and the supernatant was obtained for dialysis;

[0048] (6) Spray drying: The dialysis filtrate is placed in a spray dryer for airflow spray drying to obtain the enzymatically hydrolyzed small molecule pea polypeptide product.

[0049] The cell wall breaking treatment is to use high-pressure homogenization technology to break the cell wall of pea cells; the dilute alkali solution is a NaOH solution; the enzyme activity of the complex enzyme added for the enzymatic hydrolysis is 60u / g, and the addition amount is 0.15% (mass percentage); the power of the ultrasonic hydrolysis is 360W; and the dialysis condition is dialysis in a 6000u dialysis bag for 20h.

[0050] Example 2

[0051] (1) Raw material processing: The pea protein solution obtained by peas was mixed with deionized water at a mass ratio of 1:15, stirred evenly by a stirring device, and then the temperature was controlled at 45° C. The solution was immersed in ultrasound for 3 h and subjected to cell wall breaking treatment;

[0052] (2) High temperature sterilization: The pea protein solution is placed in a high pressure steam sterilizer for constant temperature treatment, and high temperature instant sterilization is used to inactivate the bacteria in the protein to facilitate subsequent operations;

[0053] (3) Enzymatic hydrolysis of the mixed solution: The pea protein solution was cooled to room temperature, and papain was added thereto for ultrasonic hydrolysis of the pea protein solution. A dilute alkali solution was added to adjust the pH value to 7.5. The hydrolysis temperature was controlled at 60° C. and the enzymatic hydrolysis time was 3 h.

[0054] (4) Separation of enzymatic hydrolysate: After the enzymatic hydrolysis is completed, the treated enzymatic hydrolysate is heated to 100° C. and the enzyme is inactivated for 10 min to inactivate the mixed protease in the enzymatic hydrolysate;

[0055] (5) Centrifugation of the enzymatic hydrolysate: The enzymatic hydrolysate after the enzyme inactivation treatment was centrifuged at a speed of 8000 r / min for 30 min, and the supernatant was obtained for dialysis;

[0056] (6) Spray drying: The dialysis filtrate is placed in a spray dryer for airflow spray drying to obtain the enzymatically hydrolyzed small molecule pea polypeptide product.

[0057] The cell wall breaking treatment is to use high-pressure homogenization technology to break the cell wall of pea cells; the dilute alkali solution is a NaOH solution; the enzyme activity of the complex enzyme added for the enzymatic hydrolysis is 60u / g, and the addition amount is 0.15% (mass percentage); the power of the ultrasonic hydrolysis is 360W; and the dialysis condition is dialysis in a 6000u dialysis bag for 20h.

[0058] Example 3

[0059] (1) Raw material processing: The pea protein solution obtained by peas was mixed with deionized water at a mass ratio of 1:15, stirred evenly by a stirring device, and then the temperature was controlled at 45° C. The solution was immersed in ultrasound for 3 h and subjected to cell wall breaking treatment;

[0060] (2) High temperature sterilization: The pea protein solution is placed in a high pressure steam sterilizer for constant temperature treatment, and high temperature instant sterilization is used to inactivate the bacteria in the protein to facilitate subsequent operations;

[0061] (3) Enzymatic hydrolysis of the mixed solution: The pea protein solution was cooled to room temperature, and trypsin was added thereto for ultrasonic hydrolysis of the pea protein solution, and a dilute alkali solution was added to adjust the pH value to 7.5, the hydrolysis temperature was controlled at 60° C., and the enzymatic hydrolysis time was 3 h;

[0062] (4) Separation of enzymatic hydrolysate: After the enzymatic hydrolysis is completed, the treated enzymatic hydrolysate is heated to 100° C. and the enzyme is inactivated for 10 min to inactivate the mixed protease in the enzymatic hydrolysate;

[0063] (5) Centrifugation of the enzymatic hydrolysate: The enzymatic hydrolysate after the enzyme inactivation treatment was centrifuged at a speed of 8000 r / min for 30 min, and the supernatant was obtained for dialysis;

[0064] (6) Spray drying: The dialysis filtrate is placed in a spray dryer for airflow spray drying to obtain the enzymatically hydrolyzed small molecule pea polypeptide product.

[0065] The cell wall breaking treatment is to use high-pressure homogenization technology to break the cell wall of pea cells; the dilute alkali solution is a NaOH solution; the enzyme activity of the complex enzyme added for the enzymatic hydrolysis is 60u / g, and the addition amount is 0.15% (mass percentage); the power of the ultrasonic hydrolysis is 360W; and the dialysis condition is dialysis in a 6000u dialysis bag for 20h.

[0066] Example 4

[0067] (1) Raw material processing: The pea protein solution obtained by peas was mixed with deionized water at a mass ratio of 1:15, stirred evenly by a stirring device, and then the temperature was controlled at 45° C. The solution was immersed in ultrasound for 3 h and subjected to cell wall breaking treatment;

[0068] (2) High temperature sterilization: The pea protein solution is placed in a high pressure steam sterilizer for constant temperature treatment, and high temperature instant sterilization is used to inactivate the bacteria in the protein to facilitate subsequent operations;

[0069] (3) Enzymatic hydrolysis of the mixed solution: The pea protein solution was cooled to room temperature, and a neutral protease was added thereto to perform ultrasonic hydrolysis on the pea protein solution, and a dilute alkali solution was added to adjust the pH value to 7.5, the hydrolysis temperature was controlled at 60° C., and the enzymatic hydrolysis time was 3 h;

[0070] (4) Separation of enzymatic hydrolysate: After the enzymatic hydrolysis is completed, the treated enzymatic hydrolysate is heated to 100° C. and the enzyme is inactivated for 10 min to inactivate the mixed protease in the enzymatic hydrolysate;

[0071] (5) Centrifugation of the enzymatic hydrolysate: The enzymatic hydrolysate after the enzyme inactivation treatment was centrifuged at a speed of 8000 r / min for 30 min, and the supernatant was obtained for dialysis;

[0072] (6) Spray drying: The dialysis filtrate is placed in a spray dryer for airflow spray drying to obtain the enzymatically hydrolyzed small molecule pea polypeptide product.

[0073] The cell wall breaking treatment is to use high-pressure homogenization technology to break the cell wall of pea cells; the dilute alkali solution is a NaOH solution; the enzyme activity of the complex enzyme added for the enzymatic hydrolysis is 60u / g, and the addition amount is 0.15% (mass percentage); the power of the ultrasonic hydrolysis is 360W; and the dialysis condition is dialysis in a 6000u dialysis bag for 20h.

[0074] Example 5

[0075] (1) Raw material processing: The pea protein solution obtained by peas was mixed with deionized water at a mass ratio of 1:15, stirred evenly by a stirring device, and then the temperature was controlled at 45° C. The solution was immersed in ultrasound for 3 h and subjected to cell wall breaking treatment;

[0076] (2) High temperature sterilization: The pea protein solution is placed in a high pressure steam sterilizer for constant temperature treatment, and high temperature instant sterilization is used to inactivate the bacteria in the protein to facilitate subsequent operations;

[0077] (3) Mixed protease hydrolysis: The pea protein solution is cooled to room temperature, and a mixed protease is added thereto to perform ultrasonic hydrolysis on the pea protein solution, and a dilute alkali solution is added to adjust the pH value to 7.5, and the hydrolysis temperature is controlled at 60°C, and the enzymatic hydrolysis time is 3 hours; the mixed protease is composed of four proteases, namely, flavor protease, papain, trypsin and neutral protease. The mass ratio of the flavor protease, papain, trypsin and neutral protease is 2-3:4-6:5-7:8-9;

[0078] (4) Separation of enzymatic hydrolysate: After the enzymatic hydrolysis is completed, the treated enzymatic hydrolysate is heated to 100° C. and the enzyme is inactivated for 10 min to inactivate the mixed protease in the enzymatic hydrolysate;

[0079] (5) Centrifugation of the enzymatic hydrolysate: The enzymatic hydrolysate after the enzyme inactivation treatment was centrifuged at a speed of 8000 r / min for 30 min, and the supernatant was obtained for dialysis;

[0080] (6) Spray drying: The dialysis filtrate is placed in a spray dryer for airflow spray drying to obtain the enzymatically hydrolyzed small molecule pea polypeptide product.

[0081] The cell wall breaking treatment is to use high-pressure homogenization technology to break the cell wall of pea cells; the dilute alkali solution is a NaOH solution; the enzyme activity of the complex enzyme added for the enzymatic hydrolysis is 60u / g, and the addition amount is 0.15% (mass percentage); the power of the ultrasonic hydrolysis is 360W; and the dialysis condition is dialysis in a 6000u dialysis bag for 20h.

[0082] Comparative Example 1

[0083] The method for preparing pea protein provided in this comparative example is different from that in Example 5 in that in step (1), the pea cells are not subjected to cell wall disruption treatment.

[0084] The pea hydrolysis degree of the comparative example is 18.3%, which is less than the hydrolysis degree of Example 5.

[0085] Comparative Example 2

[0086] The method for preparing pea protein provided in this comparative example is different from that in Example 5 in that ultrasonic hydrolysis is not used in step (3).

[0087] The pea hydrolysis degree of the comparative example is 23.8%, which is less than the hydrolysis degree of Example 5.

[0088] Comparative Example 3

[0089] The method for preparing pea protein provided in this comparative example is different from that in Example 5 in that in step (3), no dilute alkali solution is added to adjust the pH value.

[0090] The pea hydrolysis degree of the comparative example is 26.8%, which is less than the hydrolysis degree of Example 5.

[0091] Test example

[0092] 1. Effect of different proteases on the yield of pea protein peptides

[0093] Take pea enzymatic hydrolysate, dilute to a measurable concentration, take 2 mL of hydrolysate in a 25 mL colorimetric tube, add 1 mL of ninhydrin solution and mix well, heat in a boiling water bath for 15 min, cool, add 5 mL of 40% ethanol solution and react for 15 min, measure the absorbance at a wavelength of 570 nm, and calculate the protein content based on the absorbance. The results are shown in Table 1. Calculate the degree of hydrolysis according to the following formula:

[0094] DH(%)=AV1 100 / 1000MV2×100%

[0095] Wherein, A is the mass of protein (mg); M is the mass of sample (g); V1 is the total volume of hydrolyzate (mL); V2 is the volume of diluent used for color development (mL).

[0096] Table 1 Effect of different proteases on the hydrolysis degree of pea protein

[0097]

[0098] According to the data in Table 1, among the single proteases, papain has the highest hydrolysis degree of peas at 26.8. However, when the four enzymes are combined in a certain proportion to form a composite enzyme, the hydrolysis degree of the composite enzyme is higher than that of any single enzyme.

[0099] 2. Determination of Molecular Mass

[0100] The molecular weight distribution of the prepared soybean meal enzymatic hydrolysate was determined using an Agilent Technologies 1290infinity high performance liquid chromatograph (equipped with an Agilent Technologies 1200series DAD detector), as shown in Table 2. Chromatographic column: TSKgeL2000 SWXL (300mm×7.8mm); mobile phase: water (0.1% trifluoroacetic acid); detection wavelength 214nm; flow rate: 0.6mL / min; column temperature: 30°C; injection volume: 10μL.

[0101] Table 2 Molecular weight distribution of different proteases under optimal conditions

[0102]

[0103] The data in Table 2 show that the enzymatic hydrolysis products of the complex enzyme have a high content of small peptides, which can effectively improve product quality and promote skin absorption.

[0104] 3. Application of pea protein peptide in anti-aging

[0105] Skin fibroblast proliferation assay

[0106] The skin is composed of the epidermis, dermis and subcutaneous tissue. The epidermis mainly plays a protective role; the subcutaneous tissue is composed of a large number of fat cells and connective tissue, with a large number of nerves and blood vessels, which mainly provide nutrition; the dermis is mainly composed of a large number of collagen, elastic fibers, hyaluronic acid and fibroblasts. Fibroblasts account for more than 95% of all cells in the dermis, and have the function of synthesizing and secreting collagen fibers, elastic fibers and other extracellular matrix components. These components and fibroblasts work together to maintain the firmness, elasticity and moisture of the dermis. Therefore, the number, quality and activity of fibroblasts directly determine the degree of skin aging. Therefore, this experiment used fibroblasts as a proliferation test cell model, added pea protein hydrolysate, and detected its cell proliferation effect to study whether it has anti-wrinkle / firming effects, and can also be used as a basis for efficacy evaluation. In vitro, cell proliferation can be detected by the MTT method, and the results are shown in Figure 3 The MTT method can indirectly reflect the number of living cells and is a universal method for detecting cell activity.

[0107] (1) Cell inoculation: Select well-grown primary human fibroblasts, digest them and use cell culture medium to prepare about 1-2x10 4 Cell suspension of 100 μL / mL was inoculated into a 96-well plate, 200 μL per well, and 200 μL of sterile PBS was added to the edge wells. Place in a carbon dioxide incubator and culture for 24h±2h. After 24h±2h, the cell fusion should reach 20%-30%. If it is not within the range of 20%-30%, the cell inoculation density needs to be adjusted. A blank control group should be set up for each test. The blank control group is not inoculated with cells, and only the same volume of cell culture medium is added. Generally, the test is carried out after amplification to P5-7 generations, and 4 96-well plates are inoculated, which are 0h, 24h, 48h and 72h well plates respectively.

[0108] (2) Sample addition: No sample addition is required for the 0h plate, and the MTT test is performed directly; a blank control group and a test sample group are set up for the 24h, 48h, and 72h plates, and a DMEM culture medium containing 10%-20% serum is added to the positive control group, and a certain concentration of pea polypeptide solution is added to the other groups. The sample addition method and sample concentration of the 24h, 48h, and 72h plates are consistent, and the amount of liquid added to each well is 200μL. After the sample addition is completed, the plate is placed in a carbon dioxide incubator and incubated for 24h, 48h, and 72h, respectively.

[0109] (3) MTT assay: Measure the absorbance of the well plate at 0h, 24h, 48h and 72h, aspirate the culture medium in the culture plate, add 200μL of 0.5mg / mL MTT solution to each well, and incubate in a carbon dioxide incubator for 4h±15min. After the incubation, aspirate the MTT solution, add 150μL DMSO to each well, place the 96-well plate on an oscillator, and shake for 10min in the dark. After shaking, place the plate on a microplate reader and read the absorbance value (OD value) at 490nm.

[0110] (4) Calculation of relative cell viability at each time point

[0111] Relative cell viability = average OD value of each group at each incubation time point - average OD value of the corresponding blank group / average OD value at 0h - average OD value of the blank group at 0h × 100%

[0112] Figure 3 The results showed that the cell proliferation rate gradually increased with the increase of time, and the cell proliferation rate of Example 5 was the highest, indicating that the anti-aging ability of pea protein peptides obtained by compound enzyme hydrolysis was significantly higher than that of any single protease hydrolyzed protein peptide.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing ultrasound-assisted composite protease hydrolysis of pea protein, characterized in that: Pea protein peptides with a molecular weight below 6000u are obtained from pea protein by hydrolysis with a composite protease; the composite protease comprises flavor protease, papain, trypsin and neutral protease.

2. The method for preparing ultrasound-assisted composite protease hydrolysis of pea protein according to claim 1, characterized in that: The mass ratio of flavor protease, papain, trypsin and neutral protease is 1:1-4:1-5:1-6.

3. The method for preparing ultrasound-assisted composite protease hydrolysis of pea protein according to claim 1, characterized in that: The preparation method of ultrasound-assisted composite protease hydrolysis of pea protein comprises the following steps: The pea protein solution is mixed with deionized water and stirred evenly, and then immersed in ultrasound for wall breaking treatment; the pea protein solution after the wall breaking treatment is sterilized at high temperature instantaneously, and flavor protease, papain, trypsin and neutral protease are added in sequence after cooling, and the pea protein solution is ultrasonically hydrolyzed. After the enzymolysis is completed, the mixed protease in the enzymolysis solution is inactivated, the enzymolysis solution after the enzyme inactivation treatment is centrifuged, and the supernatant is dialyzed; the dialysis filtrate is placed in a spray dryer for airflow spray drying to obtain ultrasound-assisted composite protease hydrolyzed pea protein.

4. The method for preparing ultrasound-assisted composite protease hydrolysis of pea protein according to claim 3, characterized in that: The preparation method of the pea protein solution comprises: soaking peas from which impurities have been removed in water for 16-20 hours, then rinsing with clean water, grinding with a grinding wheel, and repeatedly grinding the dregs for multiple times; centrifuging the slurry for 2-4 minutes using a centrifuge with a rotation speed of 1300-1700 r / min to remove residues, acid-adjusting the supernatant to precipitate protein, centrifuging to remove the supernatant, and alkaline-adjusting the protein after acid precipitation to a pH of 7.0 to dissolve the protein to obtain a pea protein solution.

5. The method for preparing ultrasound-assisted composite protease hydrolysis of pea protein according to claim 3, characterized in that: Before the cell wall breaking treatment, the temperature of the pea protein solution was controlled at 42-50°C and ultrasonically treated for 2-4 hours.

6. The method for preparing ultrasound-assisted composite protease hydrolysis of pea protein according to claim 3, characterized in that: After ultrasonic hydrolysis, dilute alkali solution is added to adjust the pH value of the solution to 7.5-8.0, the hydrolysis temperature is 60-65° C., and the ultrasonic hydrolysis power is 340-380W; the dilute alkali solution is NaOH solution.

7. The method for preparing ultrasound-assisted composite protease hydrolysis of pea protein according to claim 3, characterized in that: The activity of the composite protease is 50-75u / g, the enzymolysis time is 2-5h, and the added amount is 0.15% of the mass of the pea protein solution; after the enzymolysis is completed, the solution is heated to 90-100°C and the enzyme is inactivated for 10-20min; the centrifugal speed of the enzymolysis solution after the enzyme inactivation treatment is 8000-10000r / min, and the centrifugal treatment time is 10-30min; the dialysis condition is dialysis in a 6000u dialysis bag for 20-24h.

8. An ultrasound-assisted composite protease hydrolysis of pea protein, characterized in that: The invention is prepared by the method according to any one of claims 1 to 7.

9. Use of the ultrasound-assisted composite protease hydrolyzed pea protein according to claim 8 in the preparation of cosmetics.

10. The use according to claim 9, characterized in that: The applications include the following applications A1-A3: A1. Preparation of cosmetics with anti-wrinkle and firming effects; A2. Preparation of cosmetics with moisturizing / nourishing effects; A3. Prepare cosmetics with soothing and repairing effects.

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