CN116731112A - Soy bean curd antioxidation active peptide and preparation method thereof - Google Patents
Soy bean curd antioxidation active peptide and preparation method thereof Download PDFInfo
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- CN116731112A CN116731112A CN202310909363.6A CN202310909363A CN116731112A CN 116731112 A CN116731112 A CN 116731112A CN 202310909363 A CN202310909363 A CN 202310909363A CN 116731112 A CN116731112 A CN 116731112A
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- 239000003963 antioxidant agent Substances 0.000 claims abstract description 28
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Molecular Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Dermatology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a green soy bean curd antioxidation active peptide and a preparation method thereof, wherein the amino acid sequence of the green soy bean curd antioxidation active peptide is VFEEDDK (Val-Phe-Glu-Glu-Asp-Asp-Lys, and the molecular weight is 880.38 Da). The invention obtains the antioxidant active peptide in the raw tofu, has good oxidation resistance and no toxic or harmful effect on human cells. Meanwhile, the obtained green soy bean curd antioxidant active peptide is used as an additive to prepare a green soy bean curd active peptide mask, and experimental detection results show that the green soy bean curd active peptide mask has good antioxidant activity and good whitening effect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a green soy bean curd antioxidation active peptide and a preparation method thereof.
Background
The food-borne active peptide is a functional factor which is directly or indirectly derived from food protein hydrolysis and has specific physiological activity, has the advantages of high safety, high absorbability, multiple and high physiological activity, and has great development prospect. The soybean fermented food is food formed by taking soybean or soybean product as fermentation matrix and performing microorganism effect. It has stronger biological activity than non-fermented soybean food because of the rich active peptide produced in the fermentation process. The Hui Zhou Canon's green soy bean curd is a typical soy fermented food, and in the fermentation process, the Mucor breaks down soy protein of the bean curd into a mixture of various amino acids and peptides, so that the fresh taste is increased, and the green soy bean curd has various important health care functions of helping digestion, preventing and treating arteriosclerosis, hypertension, constipation, apoplexy, coronary heart disease, inhibiting cholesterol and the like.
The antioxidant peptide in the active peptide has important functions in scavenging free radicals, inhibiting lipid peroxidation, improving body resistance to aging and diseases, and has extremely high application value in the fields of food, cosmetics, medicine, etc.
The current research on soybean fermented food raw tofu mainly focuses on the aspects of fermentation technology and analysis of chemical components, and the research on the preparation, functions and action mechanisms of active peptide is deficient. The Huizhou present candelas Luo Shimao bean curd is a local non-heritage product, is a typical soybean fermented food, and has better biochemical indexes of nutrient components than common raw bean curd. According to the invention, the Huizhou present bank Luo Shimao bean curd is taken as a raw material, the antioxidant active peptide with a specific amino acid sequence is obtained from the raw bean curd, and functional product development is carried out to improve the added value of the raw bean curd, so that new power is injected for promoting transformation of the traditional industry.
Disclosure of Invention
The invention aims to provide a green soy bean curd antioxidation active peptide and a preparation method thereof, solve the problems that the added value of the traditional green soy bean curd industry is low and no deep research on the antioxidation active peptide in the green soy bean curd is performed, and lay a foundation for developing the antioxidation active peptide based on food sources and exploring the wide application of the antioxidation active peptide in foods, cosmetics and medicine.
The technical scheme adopted for solving the technical problems is as follows: the amino acid sequence of the green soy bean curd antioxidant peptide is VFEEDDK (Val-Phe-Glu-Glu-Asp-Asp-Lys, and the molecular weight is 880.38 Da).
The preparation method of the green soy bean curd antioxidant peptide comprises the following steps:
1) Mixing raw bean curd and deionized water according to the ratio of 1:10g/mL, and grinding;
2) Crushing the ground mixed solution in ice bath for 1h by ultrasonic waves, centrifuging in a high-speed refrigerated centrifuge at 4 ℃ and 12000rpm for 15min, and standing for separation to obtain a supernatant serving as a protein extract;
3) Adding pancreatin into the obtained protein extract, performing enzymolysis at 37deg.C for 2 hr, adding pancreatin and protein extract at a ratio of 1:50g/mL, adjusting pH value of the solution to 10, and inactivating enzyme by boiling water bath for 5min after enzymolysis;
4) Centrifuging the protein extract after enzymolysis in a high-speed refrigerated centrifuge at 4deg.C and 12000rpm for 10min, standing, collecting supernatant, adjusting pH to 7.2 to obtain mixed peptide solution, lyophilizing, and concentrating;
5) Re-dissolving the freeze-dried concentrated mixed peptide solution, separating by using a Sephadex G-25 gel chromatographic column (1 cm multiplied by 15 cm), eluting with deionized water at a flow rate of 0.3mL/min, collecting components corresponding to each absorption peak, freeze-drying and preserving at-20 ℃;
6) Measuring the antioxidant activity (ABTS, DPPH and OH free radical scavenging activity and total antioxidant capacity T-AOC) of each component collected in the step 5 to obtain the component with the comprehensively optimal antioxidant activity;
7) Desalting and purifying the component with the comprehensively optimal antioxidant activity collected in the step 6) by adopting a C18 solid phase extraction column, separating the component with the comprehensively optimal antioxidant activity by adopting reverse phase high performance liquid chromatography (RP-HPLC), collecting each peak-outlet substance according to a peak diagram by using a full gradient method, and measuring the antioxidant activity (ABTS, DPPH and OH free radical scavenging activity and total antioxidant capacity T-AOC) of each component after spin drying to obtain three peak components (peak 3, peak 4 and peak 5) with the comprehensively optimal antioxidant activity;
8) Determining the amino acid sequence of the active peptide by using a triple tandem liquid chromatography-mass spectrometry (LC-MS/MS) for the peak 3, the peak 4 and the peak 5 in the step 7) to obtain 9 active peptides with strong antioxidant activity;
9) According to the amino acid sequence of the active peptide obtained in the step 8), the corresponding active peptide is obtained by adopting an artificial synthesis mode, 293T cells are used for antioxidant activity test, MDA, SOD and POD are used as evaluation indexes, and the active peptide with the strongest antioxidant activity is obtained, and the sequence is VFEEDDK (Val-Phe-Glu-Glu-Asp-Asp-Lys, and the molecular weight is 880.38 Da).
The invention also discloses a mask which is characterized by containing the green soy bean curd antioxidant active peptide with an amino acid sequence of VFEEDDK (Val-Phe-Glu-Asp-Asp-Lys, and a molecular weight of 880.38 Da).
The invention also discloses application of the green soy bean curd antioxidant active peptide with the amino acid sequence of VFEEDDK (Val-Phe-Glu-Asp-Asp-Lys, and the molecular weight of 880.38 Da) in preparing a mask.
The invention has the beneficial effects that: the invention obtains the antioxidant active peptide from the raw tofu, and has the advantages of high antioxidant activity, small molecular weight, safety and easy absorption. As an antioxidant, the antioxidant active peptide obtained by the invention is applied to a facial mask product, can improve the antioxidant performance of the facial mask product, slow down aging, can well inhibit the activity of tyrosinase, and has a certain whitening effect.
The invention will be described in more detail below with reference to the drawings and examples.
Drawings
FIG. 1 is a graph showing comparison of antioxidant activity of 7 active peptides prepared by enzymatic hydrolysis.
FIG. 2 is a graph of Sephadex G-25 gel chromatograms.
FIG. 3 is a reverse phase high performance liquid chromatography (RP-HPLC) profile.
FIG. 4a shows the MDA content change of 293T cells treated with three peptides 1 to 3.
FIG. 4b shows MDA content change of 293T cells treated with three peptides 4 to 6.
FIG. 4c shows MDA content variation of 293T cells treated with three peptides Peptides 7 to 9.
FIG. 5a shows the SOD content change of 293T cells treated with three active peptides Peptides 1 to 3.
FIG. 5b shows the SOD content change of 293T cells treated with three peptides 4 to 6.
FIG. 5c shows the SOD content change of 293T cells treated with three active peptides Peptides 7 to 9.
FIG. 6a shows the variation of POD content of 293T cells treated with three peptides 1 to 3.
FIG. 6b shows the variation of POD content of 293T cells treated with three peptides 4 to 6.
FIG. 6c shows the variation in POD content of 293T cells treated with three peptides Peptides 7 to 9.
Detailed Description
The invention provides a green soy bean curd antioxidant active peptide, which is obtained from the following raw materials: raw tofu was purchased from Canon products Inc. of Canon, huang Shanhui. The alkaline enzymes, pepsin, pancreatin, papain, neutral protease, alpha-chymotrypsin and ficin were all purchased from Shanghai Meilin Biochemical technologies Co. 293T cell line (American culture Collection, manassas, va.). ABTS (2, 2' -thiazoline-6-ammonium sulfonate salt) free radical, DPPH (2, 2-diphenyl-1-trinitrohydrazinium salt) free radical, hydroxyl free radical, total antioxidant capacity (T-AOC), MDA (malondialdehyde), SOD (superoxide dismutase) and POD (peroxidase) detection kit (beijing solibao technologies ltd, china). DPPH, tyrosinase, vitamin C, vitamin E, methyl parahydroxybenzoate, levodopa, adenosine, absolute ethyl alcohol, sodium dihydrogen phosphate, disodium hydrogen phosphate and the like are all analytically pure (China national medicine group chemical reagent Co., ltd.).
And (3) enzyme selection and determination:
1) Enzymatic hydrolysis and peptide extraction
10g of green soy bean curd samples were weighed and mixed with 100mL deionized water. The mixture was ground in a mortar and transferred to a 250mL beaker. Ultrasonic disruption was performed in an ice bath for 1 hour, and then centrifuged at 12000rpm at 4℃for 15 minutes, and the resulting supernatant was collected as a protein extract.
Equal amount of protein extract is taken and added into 7 centrifuge tubes respectively, and alkaline enzyme, pepsin, pancreatin, papain, neutral protease, alpha-chymotrypsin and ficin are added into the 7 centrifuge tubes respectively according to the proportion of 1:50 g/mL. And (3) regulating the pH value and the temperature of each centrifuge tube to enable the protein extract to carry out enzymolysis under the optimal pH value, the optimal temperature and the optimal time of each enzyme. After the enzymolysis is completed, the centrifuge tube is put into boiling water for boiling water bath for 5min, and enzyme is inactivated. And then placing the inactivated enzymolysis liquid into a high-speed refrigerated centrifuge, centrifuging at 4 ℃ and 12000rpm for 10min, collecting supernatant, and marking the supernatant as polypeptide solution, wherein the total number of the polypeptide solutions is 7.
2) Evaluation of antioxidant Activity of polypeptide solutions
In order to screen the optimal protease for the hydrolysis of green soy bean curd, the scavenging activity and total reducing power (T-AOC) of each protease hydrolysis peptide on free radicals (ABTS, DPPH and OH) were determined by a kit method, and all the kits used were detection kits corresponding to Beijing Soy Bao technology Co., ltd. As a result, as shown in FIG. 1, the peptide solution obtained by pancreatin hydrolysis was comprehensively compared to be optimal in oxidation resistance, and thus pancreatin was selected as the enzyme used for preparing the antioxidant active peptide by soybean rot.
Example 1: the preparation method of the green soy bean curd antioxidant peptide comprises the following steps:
1) Mixing raw bean curd and deionized water according to the ratio of 1:10g/mL, and grinding;
2) Crushing the ground mixed solution in ice bath for 1h by ultrasonic waves, centrifuging in a high-speed refrigerated centrifuge at 4 ℃ and 12000rpm for 15min, and standing to separate the obtained supernatant as protein extract;
3) Adding pancreatin into the obtained protein extract, carrying out enzymolysis for 2 hours at 37 ℃, adding pancreatin and the protein extract according to the proportion of 1:50g/mL, regulating the pH value of the solution to 10 for enzymolysis, and inactivating the enzyme by boiling water bath for 5 minutes after the enzymolysis is completed;
4) Centrifuging the protein extract after enzymolysis in a high-speed refrigerated centrifuge at 4deg.C and 12000rpm for 10min, standing, collecting supernatant, adjusting pH to 7.2 to obtain mixed peptide solution, lyophilizing, and concentrating;
5) Re-dissolving the freeze-dried concentrated mixed peptide solution, separating by using a Sephadex G-25 gel chromatographic column (1 cm multiplied by 15 cm), eluting with deionized water at a flow rate of 0.3mL/min, wherein the curve of the obtained gel chromatogram is shown in figure 2; collecting components corresponding to each absorption peak in the gel chromatogram curve, lyophilizing, and preserving at-20deg.C;
6) And 5) measuring the antioxidant activity of each component collected in the step 5), including the scavenging activity of ABTS, DPPH and OH free radicals and the total antioxidant capacity T-AOC, wherein the detection is carried out by a detection kit corresponding to Beijing-Soy-Bao technology Co., ltd.m. of China, and the specific operation is carried out according to the specification of the kit, and the detection result is shown in the following table 1, so that the component 3 with the comprehensively optimal antioxidant activity is obtained.
Evaluation of in vitro antioxidant Activity of Each component of Table 1
7) Desalting and purifying the component 3 collected in the step 6) by using a C18 solid phase extraction column, and then performing reversed phase chromatographic separation on the component 3 by using reversed phase high performance liquid chromatography (RP-HPLC), wherein the reversed phase chromatographic separation result is shown in figure 3. The peak substances are collected according to the figure 3 by using a full gradient method, the antioxidant activity of each component is measured after spin drying, the measurement of the scavenging activity of ABTS, DPPH and OH free radicals and the total antioxidant capacity T-AOC are included, the detection is performed by a detection kit corresponding to Beijing Soy Bao technology Co., ltd., china, and the specific operation is performed according to the specification of the kit. The detection results are shown in the following table 2, and peak components with better antioxidant activity are obtained, namely peak 3, peak 4 and peak 5;
table 2 evaluation of in vitro antioxidant Activity of each component peak
8) Determining the amino acid sequences of the contained active peptides by liquid chromatography mass spectrometry (LC-MS/MS) on the peak 3, the peak 4 and the peak 5 in the step 7) to obtain 9 active peptide amino acid sequences;
9) According to the amino acid sequence of the active peptide obtained in the step 8), the corresponding active peptide is obtained by adopting an artificial synthesis mode, and then 293T cells are used for performing an antioxidant activity test. In cell culture, DMEM complete medium is used as control group, and 160. Mu. Mol/L H is added into DMEM complete medium 2 O 2 As H 2 O 2 Model group to add 160. Mu. Mol/L H to DMEM complete medium 2 O 2 Then 2, 10 and 50. Mu.g/mL of active peptide were added as experimental groups, respectively. The experiment is carried out by dividing 9 active peptides into three groups, detecting the content and activity of MDA, SOD and POD according to 3 active peptides as one group, detecting MDA, SOD and POD by detection kit corresponding to Beijing Soy Bao technology Co., ltd. The results are shown in FIGS. 4 to 6, and the results obtained after treatment with active peptides of different concentrations are compared with each other in the control group and the model group, so that the active Peptide with the optimal antioxidant activity, which has the reduced MDA content and the increased SOD and POD activities, is Peptide6, the sequence of which is VFEEDDK (Val-Phe-Glu-Asp-Asp-Lys, and the molecular weight of which is 880.38 Da).
Example 2: application of Mao Doufu antioxidant active peptide in facial mask:
mao Doufu preparation of antioxidant active peptide facial mask, preparing facial mask stock solution, and thenAdding different proportions of the green soy bean curd antioxidant active peptide into the mask stock solution according to the volume ratio, and adding the green soy bean curd antioxidant active peptide into the mask stock solution according to the volume ratio of V C For a control group experiment, the antioxidant effect of the raw tofu antioxidant active peptide mask is tested.
The preparation process of the mask stock solution comprises the following steps: taking preparation of 2L mask stock solution as an example:
(1) Firstly preparing the solution C, measuring 400mL of distilled water, placing in a beaker, setting the temperature to 60 ℃, weighing 0.15% of xanthan gum, slowly adding into the beaker, and stirring to fully dissolve the xanthan gum.
(2) Preparing solution B, weighing 400mL of distilled water, placing in a beaker, weighing 0.05% EDTA (ethylenediamine tetraacetic acid) and adding into the beaker, stirring uniformly, setting the temperature to 60 ℃, weighing 0.1% hyaluronic acid, slowly adding and stirring continuously to dissolve the hyaluronic acid.
(3) Preparing preservative D liquid, measuring 12mL of ethanol, placing the mixture in a beaker, weighing 0.2% of methyl parahydroxybenzoate, adding the mixture, stirring the mixture by using a glass rod to fully dissolve the mixture, and then transferring the D liquid by using a pipette to add the D liquid into the B liquid.
(4) Preparing solution A, weighing 800mL of distilled water, placing in a beaker, and weighing 8% of glycerol, 2% of 2, 6-glycerol alcohol, 1% of betaine, 2% of 1,2 propylene glycol and 0.05% of V respectively E (vitamin E) and 0.02% of adenosine were added to the beaker and dissolved thoroughly with constant stirring on a glass rod.
(5) Sequentially adding the prepared solution C, solution B, solution D and solution A into a reaction kettle, opening the reaction kettle, and rotating to 600-800rpm/min, homogenizing for 1min each time at intervals of 5min for 30min to obtain the mask stock solution.
The antioxidant active peptide obtained in the step 9 of the example 1 is redissolved by deionized water to obtain 180mg/g antioxidant active peptide solution, and the solution is added into mask stock solution according to the volume ratio (0%, 10%, 20%, 30%, 40% and 50%) to obtain 6 groups of mask essence containing antioxidant active peptide of the raw tofu with different proportions, and the antioxidant performance is evaluated.
Determining the DPPH free radical scavenging capacity of the active peptide mask of the hair tofu:
(1) Measurement of DPPH free radical scavenging ability of the active peptide mask of Mao Bean curd: 2mL of mask essence is taken and put into a test tube, 3.0mL of DPPH absolute ethanol solution with the concentration of 0.08mmol/L is added, after the mask essence is uniformly mixed, the mask essence reacts for 30 minutes in a dark place, distilled water is used as a blank control, and the light absorption value is measured at the wavelength of 517 nm; three replicates were run for each sample and the results are expressed as averages. The clearance of DPPH free radical is calculated according to the formula as follows.
Clearance (%) = [1- (As-Ac)/(a ] ×100%
Wherein: a is the absorbance value of distilled water+DPPH solution.
Ac is the absorbance value of the sample solution+distilled water.
As is the absorbance value of the sample solution+DPPH solution.
(2) Measurement of Vc ability to scavenge DPPH free radical: preparing a Vc solution with the concentration of 50 mug/mL by deionized water, and then gradually diluting the Vc solution into Vc solutions with the concentration of 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0 mug/mL, and determining the scavenging capacity of Vc on DPPH free radicals by referring to the step (1);
(3) Vc is used as a control to evaluate the DPPH free radical scavenging capacity of the active peptide mask.
The test results are shown in table 3:
TABLE 3 capability of Green soy bean curd active peptide mask to scavenge DPPH free radical
As can be seen from table 3: the active peptide mask of the hair bean curd has a certain scavenging capability to DPPH free radical, and is enhanced along with the increase of the addition amount of the active peptide of the hair bean curd, when the addition amount of the active peptide is 50%, the scavenging rate to the DPPH free radical is up to 68.46%, which is equivalent to the scavenging capability of Vc of 8.69 mug/mL.
Determination of tyrosinase inhibition effect of the active peptide mask of the hair tofu:
tyrosinase is a key enzyme for synthesizing melanin by human body, and proper inhibition of tyrosinase activity can realize healthy whitening.
The specific experimental method is that the experiment is directly reacted in a 96-well ELISA plate. The tyrosinase inhibition rate was calculated by sequentially adding the above-described reagents in the following order and measuring the absorbance.
1) 50. Mu.L of buffer;
2) 80 μL of 10 mM levodopa;
3) 50 μl sample;
4) Blank group + 20. Mu.L buffer, experimental group + 20. Mu.L tyrosinase (125 units/mL);
5) Vibrating and reacting for 15min;
6) Absorbance was measured under irradiation at 475nm wavelength. The calculation method refers to the following formula:
wherein: i represents tyrosinase inhibition rate,%; a is that S Absorbance for the sample; a is that SB Absorbance was 50 μl of extract and 20 μl of buffer; ac is the absorbance of the solvent of 50. Mu.L of the extract with 20. Mu.L of tyrosinase; a is that CB The absorbance was 50. Mu.L of the solvent of the extract and 20. Mu.L of the buffer. The test results are shown in Table 4.
TABLE 4 inhibition ratio of Soy bean curd active peptide mask to tyrosinase
As can be seen from table 4: the green soy bean curd active peptide has a certain inhibition effect on tyrosinase in the mask essence, and the inhibition effect on tyrosinase is enhanced with the increase of the addition amount of the green soy bean curd active peptide, which reaches 57.11% at most, so that the green soy bean curd active peptide mask prepared by the experiment has a good whitening effect.
The experiment shows that the green soy bean curd active peptide mask has good antioxidant activity and whitening effect.
The invention is described above by way of example with reference to the accompanying drawings. It will be clear that the invention is not limited to the embodiments described above. As long as various insubstantial improvements are made using the method concepts and technical solutions of the present invention; or the invention is not improved, and the conception and the technical scheme are directly applied to other occasions and are all within the protection scope of the invention.
Claims (4)
1. A green soy bean curd antioxidation active peptide is characterized in that: the amino acid sequence of the green soy bean curd antioxidation active peptide is VFEEDDK.
2. The preparation method of the green soy bean curd antioxidant active peptide with the amino acid sequence of VFEEDDK is characterized by comprising the following steps:
1) Mixing raw bean curd and deionized water according to the ratio of 1:10g/mL, and grinding;
2) Crushing the ground mixed solution in ice bath for 1h by ultrasonic waves, centrifuging in a high-speed refrigerated centrifuge at 4 ℃ and 12000rpm for 15min, and standing to separate the obtained supernatant as protein extract;
3) Adding pancreatin into the obtained protein extract, performing enzymolysis at 37deg.C for 2 hr, adding pancreatin and protein extract at a ratio of 1:50g/mL, adjusting pH value of the solution to 10, and inactivating enzyme by boiling water bath for 5min after enzymolysis;
4) Centrifuging the protein extract after enzymolysis in a high-speed refrigerated centrifuge at 4deg.C and 12000rpm for 10min, standing, collecting supernatant, adjusting pH to 7.2 to obtain mixed peptide solution, lyophilizing, and concentrating;
5) Re-dissolving the freeze-dried concentrated mixed peptide solution, separating by using a Sephadex G-25 gel chromatographic column, eluting by using deionized water, collecting components corresponding to each absorption peak, freeze-drying and preserving at the temperature of minus 20 ℃;
6) Measuring the antioxidant activity of each component in the step 5 to obtain a component with the comprehensively optimal antioxidant activity;
7) Desalting and purifying the component with the comprehensively optimal antioxidant activity collected in the step 6) by adopting a C18 solid-phase extraction column, separating the component with the comprehensively optimal antioxidant activity by adopting reversed-phase high-performance liquid chromatography, collecting each peak-emitting substance according to a peak diagram by using a full gradient method, and measuring the antioxidant activity of each component after spin drying to obtain the peak component with the comprehensively optimal antioxidant activity;
8) Carrying out LC-MS/MS triple tandem liquid chromatography-mass spectrometry on the peak component with the comprehensive optimal antioxidant activity in the step 7) to determine the amino acid sequence of the active peptide;
9) According to the amino acid sequence of the active peptide obtained in the step 8), the corresponding active peptide is obtained by adopting an artificial synthesis mode, 293T cells are used for antioxidant activity test, MDA, SOD and POD are used as evaluation indexes, and the active peptide with the strongest antioxidant activity is obtained, and the sequence is VFEEDDK.
3. A facial mask comprising the antioxidant active peptide of raw tofu according to claim 1.
4. The use of the antioxidant active peptide of raw tofu in the preparation of a mask according to claim 1.
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