CN116693616A - Soy bean curd antioxidation active peptide and application thereof - Google Patents
Soy bean curd antioxidation active peptide and application thereof Download PDFInfo
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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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Dermatology (AREA)
- Peptides Or Proteins (AREA)
- Cosmetics (AREA)
Abstract
The invention discloses a green soy bean curd antioxidant active peptide and a preparation method thereof, wherein the amino acid sequence of the green soy bean curd antioxidant active peptide is NLQGENEWDQK. 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 antibacterial property, so that the antioxidant active peptide has antibacterial property.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a green soy bean curd antioxidation active peptide and application 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 application 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 carried out, 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: a green soy bean curd antioxidant active peptide, wherein the amino acid sequence of the green soy bean curd antioxidant active peptide is NLQGENEWDQK.
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 sequence of the active peptide with better comprehensive antioxidant activity is NLQGENEWDQK.
The invention also discloses a mask which is characterized by containing the green soy bean curd antioxidant active peptide with the amino acid sequence of NLQGENEWDQK.
The invention also discloses application of the green soy bean curd antioxidant active peptide with the amino acid sequence of NLQGENEWDQK 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, 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 and has good antibacterial activity.
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.
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). ABTS, betaine, vitamin C, vitamin E, levodopa, adenosine, absolute ethanol, sodium dihydrogen phosphate, disodium hydrogen phosphate, propyl hydroxybenzoate, potassium persulfate, physiological saline, etc. are all analytically pure (chinese national pharmaceutical group chemical reagent limited). Pseudomonas aeruginosa, escherichia coli, staphylococcus aureus and bacillus subtilis are provided by the yellow mountain academy of health food and functional skin care product innovation technology research and development center.
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 1h, followed by centrifugation at 12,000 rpm for 15min at 4 ℃. 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 active 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, and obtaining a gel chromatographic curve shown in figure 2; collecting the components corresponding to each absorption peak, lyophilizing, and preserving at-20deg.C;
6) And 5) measuring the antioxidant activity of each component collected in the step 5), including ABTS, DPPH, OH free radical scavenging activity and total antioxidant capacity T-AOC, wherein the detection is carried out by a detection kit corresponding to Beijing Soy Bao technology Co., ltd., 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 performing reversed phase chromatographic separation on the component 3 by using reversed phase high performance liquid chromatography (RP-HPLC), wherein the curve of the reversed phase high performance liquid chromatography is shown in figure 3. Collecting each peak-emitting substance according to a peak diagram by using a full gradient method, performing antioxidant activity measurement of each component after spin drying, including measurement of the scavenging activity of ABTS, DPPH and OH free radicals and the total antioxidant capacity T-AOC, wherein the detection is performed by a detection kit corresponding to Beijing Soy Bao technology Co., ltd. 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 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 respectively adding 2, 10 and 50 mug/mL of active peptide as an experimental group, carrying out experiments on 9 active peptides in three groups, and detecting the content and activity of MDA, SOD and POD according to 3 active peptides as one group, wherein the content and activity of the MDA, SOD and P are detectedOD detection was performed by a detection kit corresponding to beijing solebao technologies, ltd, china, and specific operations were performed according to the specifications of the kit, and the results are shown in table 3:
TABLE 3 MDA, SOD and POD content and Activity changes of the Peptide1-Peptide9 treated T cells
As can be seen from table 3: the comparison is made between the control group and the model group, and after the treatment of active Peptide with different concentrations, the active Peptide with better antioxidant activity, which has reduced MDA content and increased SOD and POD activity, is Peptide4 with sequence NLQGENEWDQK.
Example 2: application of Mao Doufu antioxidant active peptide in mask
Mao Doufu preparation of an antioxidant active peptide facial mask, firstly preparing a facial mask stock solution, then adding different proportions of the green soy bean curd antioxidant active peptide into the facial mask stock solution according to the volume ratio, and adding V C For a control group experiment, the antioxidant effect of the hair tofu antioxidant peptide mask is tested.
The preparation process of the mask stock solution comprises the following steps:
(1) Phase a (oil phase) preparation: 400mL of distilled water, 80mL of glycerol, 20mL of 2, 6-glycerol, 10g of betaine, 20mL of 1, 2-propanediol, V E 0.5mL and 0.2g of adenosine are mixed and stirred uniformly;
(2) Phase B (aqueous phase) preparation: 200mL of distilled water and 1g of hyaluronic acid were taken and stirred at 60℃until the hyaluronic acid was completely dissolved.
(3) And C, preparing a phase C: 200mL of distilled water and 1.5g of xanthan gum were taken and stirred at 60℃until the xanthan gum was completely dissolved.
(4) Sequentially adding the phase C, the phase B and the phase A into a reaction kettle, opening the reaction kettle, and carrying out rotating speed adjustment to 600-800rpm/min, homogenizing for 1min each time, and homogenizing for 30min at intervals of 5min to obtain the mask essence.
And (3) re-dissolving the antioxidant active peptide with the amino acid sequence of NLQGENEWDQK obtained in the step 9) of the example 1 by deionized water to obtain 180mg/g antioxidant active peptide solution, and adding the solution into mask stock solution according to the volume ratio (0%, 10%, 20%, 30%, 40% and 50%) to obtain 6 groups of mask essence containing the antioxidant active peptides of the hair tofu with different proportions.
Determination of ABTS free radical scavenging ability of active peptide mask of Mao Bean curd:
(1) Configuration of ABTS solution: 0.192g of 1, 1-diphenyl-2-trinitrophenylhydrazine (ABTS) and 0.033g of potassium persulfate were accurately weighed and 50mL of deionized water was added to prepare 7 mmol/LABSS solution and 2.45mmol/L potassium persulfate solution, respectively. Mixing uniformly according to the volume ratio of 1:2, and placing in a dark environment for 16h. The ABTS solution was diluted with absolute ethanol prior to use to give a absorbance at 734nm of 0.70±0.02.
(2) Determination of ABTS free radical scavenging ability of active peptide mask of raw tofu: taking 0.6mL of the raw tofu active peptide mask solution, adding 2.4mL of ABTS solution, carrying out light-shielding reaction for 12min, measuring a light absorption value at a wavelength of 734nm, and calculating the clearance of the ABTS free radicals according to the following formula (three groups of parallel experiments are arranged). The same experimental procedure was performed using absolute ethanol as a control.
ABTS clearance% = (1-a) S /Ac)*100％
Note that: as is the absorbance of ABTS and the sample solution, ac is the absorbance of ABTS and the ethanol solution, and the blank is absolute ethanol.
(3) Determination of Vc ability to scavenge ABTS free radicals: first, the V of 20 mug/mL is prepared by deionized water C The solution was then gradually diluted to a concentration of 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 14.0, 16.0. Mu.g/mL VC solution, and the ability of Vc to scavenge ABTS free radicals was determined with reference to step (2).
(4) The Vc concentration is used as a control to evaluate the ability of the active peptide mask to remove ABTS free radicals. The test results are shown in table 4:
TABLE 4 ability of Soy bean curd active peptide mask to scavenge ABTS free radical
As can be seen from table 4: the active peptide mask of the hair bean curd has a certain scavenging capability to the ABTS 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 ABTS free radical is up to 87.75%, which is equivalent to the scavenging capability of Vc of 22.53 mug/mL.
Determination of bacteriostatic activity of the raw tofu active peptide skin care product mask:
(1) Preparation of experimental and control groups: the antioxidant active peptide with the amino acid sequence of NLQGENEWDQK obtained in the step 9) of the example 1 is redissolved by deionized water to obtain 180mg/g antioxidant active peptide solution. And (3) sequentially adding antioxidant active peptide solutions according to 0%, 10%, 20%, 30%, 40% and 50% of the volume of the facial mask essence to prepare 6 groups of facial mask essence containing antioxidant active peptides of the raw tofu with different proportions for experimental use (the same applies below). Sterile distilled water was used as a blank control group, and 1mg/mL propyl hydroxybenzoate was used as a positive control group.
(2) Strain activation and strain suspension preparation: inoculating four mode bacterial strains of pseudomonas aeruginosa, escherichia coli, staphylococcus aureus and bacillus subtilis to a fresh slant culture medium for activation, and culturing at a constant temperature of 37 ℃ for 24 hours. Preparing activated four species of strain with physiological saline into a concentration of 10 6 ～10 7 The CFU/mL bacterial suspension is shaken and evenly shaken, and is preserved at the temperature of 4 ℃ for experimental use.
(3) The inhibition zone is measured by adopting a filter paper diffusion method: pouring the sterilized culture medium into a culture dish in an ultra-clean workbench, sucking 100 mu L of prepared pseudomonas aeruginosa, escherichia coli, staphylococcus aureus and bacillus subtilis bacterial suspension on the culture dish after cooling and solidifying, and uniformly coating. A pipetting gun is used for adding 10 mu L of mask essence with the content of the active peptide extract of the hair tofu with different proportions into the center of a filter paper sheet with the sterile diameter of 6 mm. The treated petri dishes are inverted and cultured in a constant temperature incubator at 37 ℃ for 24 hours, and after the culture, the diameter (mm) of the inhibition zone is measured by a vernier caliper by adopting a crisscross method. Each set of experiments was repeated 3 times and the measurement results were expressed as mean ± standard deviation. The test results are shown in Table 5:
TABLE 5 inhibition of Soy bean curd active peptide mask to 4 experimental bacteria
As can be seen from table 5: the skin care product mask containing the active peptides of the hair tofu in different proportions has inhibition effect on pseudomonas aeruginosa, escherichia coli, staphylococcus aureus and bacillus subtilis, and the higher the content of the active peptides of the hair tofu is, the more obvious the inhibition effect is, and the inhibition strength on 4 experimental bacteria is pseudomonas aeruginosa, bacillus subtilis, staphylococcus aureus and escherichia coli in sequence from strong to weak.
The experiment shows that the active peptide with the amino acid sequence NLQGENEWDQK obtained in the step 9) of the embodiment 1 has better antioxidation and antibacterial activity.
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 (3)
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 NLQGENEWDQK.
2. The use of the antioxidant active peptide of raw tofu in the preparation of a mask according to claim 1.
3. A facial mask comprising the antioxidant active peptide of raw tofu according to claim 1.
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