CN114671937A - Polypeptide with tyrosinase inhibitory activity and preparation method thereof - Google Patents
Polypeptide with tyrosinase inhibitory activity and preparation method thereof Download PDFInfo
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- 229920001184 polypeptide Polymers 0.000 title claims abstract description 96
- 102000003425 Tyrosinase Human genes 0.000 title claims abstract description 42
- 108060008724 Tyrosinase Proteins 0.000 title claims abstract description 42
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 26
- 241000276707 Tilapia Species 0.000 claims abstract description 22
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- 229910021645 metal ion Inorganic materials 0.000 claims description 18
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 14
- 229910052698 phosphorus Inorganic materials 0.000 claims description 14
- 239000011574 phosphorus Substances 0.000 claims description 14
- 238000004108 freeze drying Methods 0.000 claims description 12
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 11
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 11
- 238000011068 loading method Methods 0.000 claims description 10
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 8
- 238000002390 rotary evaporation Methods 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 4
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 3
- 229930195725 Mannitol Natural products 0.000 claims description 3
- 235000010355 mannitol Nutrition 0.000 claims description 3
- 239000000594 mannitol Substances 0.000 claims description 3
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- 230000005764 inhibitory process Effects 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 230000009920 chelation Effects 0.000 description 9
- 235000002595 Solanum tuberosum Nutrition 0.000 description 8
- 244000061456 Solanum tuberosum Species 0.000 description 8
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- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Natural products O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 7
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- 230000002829 reductive effect Effects 0.000 description 6
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- 238000003786 synthesis reaction Methods 0.000 description 2
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- 206010014970 Ephelides Diseases 0.000 description 1
- 208000003351 Melanosis Diseases 0.000 description 1
- BJRNKVDFDLYUGJ-ZIQFBCGOSA-N alpha-Arbutin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-ZIQFBCGOSA-N 0.000 description 1
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- VJNCICVKUHKIIV-UHFFFAOYSA-N dopachrome Chemical compound O=C1C(=O)C=C2NC(C(=O)O)CC2=C1 VJNCICVKUHKIIV-UHFFFAOYSA-N 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- -1 gold ion Chemical class 0.000 description 1
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- SWGJCIMEBVHMTA-UHFFFAOYSA-K trisodium;6-oxido-4-sulfo-5-[(4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2-sulfonate Chemical compound [Na+].[Na+].[Na+].C1=CC=C2C(N=NC3=C4C(=CC(=CC4=CC=C3O)S([O-])(=O)=O)S([O-])(=O)=O)=CC=C(S([O-])(=O)=O)C2=C1 SWGJCIMEBVHMTA-UHFFFAOYSA-K 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/40—Colouring or decolouring of foods
- A23L5/41—Retaining or modifying natural colour by use of additives, e.g. optical brighteners
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Toxicology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Nutrition Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Food Science & Technology (AREA)
- Birds (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a polypeptide with tyrosinase inhibitory activity and a preparation method thereof. The method of the invention comprises the following steps: the polypeptide with high purity and ion chelating ability is obtained by taking polypeptide from tilapia scales as a raw material and performing enzymolysis, column chromatography and EDTA elution. The invention can not only verify whether the sample has ion chelating ability, but also elute the sample chelated with ions; the method has the advantages of simple operation and strong condition controllability, can determine whether the sample has the ion chelating capacity, can separate and elute chelate ions from the chelated ions, and has good application prospect.
Description
Technical Field
The invention relates to a polypeptide with tyrosinase inhibitory activity and a preparation method thereof, belonging to the technical field of food processing.
Background
Tilapia is originally produced in Africa and belongs to tropical fishes, the breeding of the Tilapia extends over 140 countries and regions, and the Tilapia is the second largest bred fish variety in the world. The annual yield of the cultivated Chinese tilapia is 162.5 ten thousand tons, which accounts for 1/4 of the total annual yield of the world, wherein the annual yield of the scales of the tilapia is about 30 ten thousand tons, and the utilization of the scales is mainly focused on the aspect of collagen, so that the scales are used as a packaging preservative to prolong the shelf life of food. However, except for the effective utilization of collagen in fish scales, the utilization rate of other components in the fish scales, especially fish scale peptide, is not high, and the overall utilization rate of the fish scales is only 20%, so that the great resource waste is caused.
The tyrosinase is the key for controlling the activity of the melanin cells, determines the synthesis rate of the melanin, and screens tyrosinase inhibitors to develop products for whitening, removing freckles and the like. For the polypeptide inhibitor of tyrosinase, the currently known preparation method is less, and the application is more common to carry out enzymolysis on a sample and then determine the inhibitory activity of the sample on tyrosinase (CN111748593A), or to carry out molecular docking on a substance with a known structure and an active center of the tyrosinase by adopting Autodock or Discovery Studio software in advance, and to screen out a substance capable of being effectively combined with the active center of the tyrosinase for chemical synthesis. The polypeptide inhibitor of tyrosinase was isolated and purified by ultrafiltration membrane (CN108379550A, CN112321701A) and identified by RP-HPLC (CN 110218240A). The peptide components prepared by the known methods are complex and the separation of the components is not thorough. Meanwhile, the methods are very complicated and tedious to operate, and are not suitable for mass production.
Disclosure of Invention
[ problem ] to
The technical problem to be solved by the invention is that tilapia mossambica phosphorus resource is wasted, and the existing method for screening and preparing the tyrosinase polypeptide inhibitor has low efficiency and complicated steps and is not suitable for large-scale production.
[ solution ]
The invention aims to provide a polypeptide composition with tyrosinase inhibitory activity and a preparation method thereof. The polypeptide composition with tyrosinase inhibitory activity is obtained by the methods of preparation of chelating liquid, sample loading and elution, rotary evaporation, freeze drying and the like, and the application range of the mixed polypeptide powder is expanded. The polypeptide composition with tyrosinase inhibitory activity is obtained by eluting and separating polypeptide components capable of successfully chelating copper ions in the polypeptide powder derived from tilapia phosphorus for the first time and verifying through biochemical and cell experiments, the generation of melanin can be effectively reduced, the polypeptide composition is expected to be applied to preventing browning of fruits and vegetables, developing cosmetics with whitening efficacy, treating melanoma diseases and the like, and has good research and development values.
The invention provides a preparation method of a polypeptide composition with tyrosinase inhibitory activity, which comprises the following steps:
s1, preparing mixed polypeptide liquid from tilapia phosphorus polypeptide powder and water according to a certain mass ratio, adding anhydrous copper sulfate into the mixed polypeptide liquid, and chelating at a proper pH value, water bath temperature and time to obtain polypeptide-ion chelating solution; loading the polypeptide-ion chelating solution to a C18 column;
S2, competitively binding metal ions in the polypeptide-ion chelating solution by using an EDTA solution, an EDTA-2Na solution or a mixed solution of EDTA and EDTA-2Na to elute the metal ions, and then dissolving and eluting the polypeptide left on the C18 column by using water to obtain a polypeptide solution;
s3, carrying out rotary evaporation and freeze-drying on the polypeptide solution obtained in the step S2 to obtain the polypeptide with ion chelation capacity.
In certain embodiments, the tilapia phosphorus polypeptide meal of step S1 is extracted from tilapia phosphorus.
In certain embodiments, the mass ratio of the tilapia phosphorus polypeptide powder to water in step S1 is 1: 20-1: 25.
In certain embodiments, the mass ratio of the tilapia phosphorus polypeptide powder to the anhydrous copper sulfate in the step S1 is 4: 1-7: 1.
In some embodiments, the pH of step S1 is 5-7.
In some embodiments, the chelating time of step S1 is 50-70 min.
In certain embodiments, the loading of the polypeptide-ion chelating solution peptide in step S1 is no more than 3 times the packing volume of the C18 column.
In certain embodiments, the EDTA elution described in step S2 is directed to removing ions from the polypeptide-ion chelating solution.
In some embodiments, the concentration of the EDTA solution, the EDTA-2Na solution or the mixed solution of EDTA and EDTA-2Na in step S2 is 10% to 20%.
In some embodiments, the rotary evaporation temperature in step S3 is 60-65 ℃.
In certain embodiments, the lyophilization temperature of step S3 is between-30 and-40 ℃.
In some embodiments, mannitol is added as a protective agent during the lyophilization process of step S3, so that the lyophilization effect is better, and the re-solubility of the sample is better. The dosage of the freeze-drying protective agent is 5 percent.
The invention provides a polypeptide composition with tyrosinase inhibitory activity, which is prepared according to the method.
The invention also provides application of the polypeptide composition in inhibiting tyrosinase activity.
The invention also provides application of the polypeptide composition in inhibiting browning of fruits and vegetables.
[ advantageous effects ]
1. The invention separates polypeptide components capable of chelating copper ions from tilapia phosphorus polypeptide powder for the first time, and the components have stable physiological activity and are easy to store.
2. The polypeptide composition separated from tilapia phosphorus polypeptide powder has the capability of chelating copper ions, and results of biochemical experiments and cell experiments show that the composition can effectively inhibit the activity of tyrosinase and reduce the generation of melanin, and the inhibition activity of the composition is superior to that of arbutin.
3. The composition capable of inhibiting tyrosinase is obtained by purifying tilapia scale polypeptide powder through a C18 column, so that fish scale peptide resources are fully utilized, the economic value of the composition is improved, and the composition is convenient, fast and efficient by adopting a column chromatography for sample loading and elution. A large amount of ion chelates can be obtained by sampling on a large scale, so that the raw material cost in the chelation preparation process is greatly reduced, the chelation rate is greatly improved, the manpower and resources are greatly saved, and the large-scale production is realized.
4. The method adopts a chemical screening mode to screen the composition capable of inhibiting tyrosinase from the tilapia mossambica scale polypeptide powder, does not need molecular docking, realizes quick and efficient screening, and greatly saves manpower and resources.
Drawings
FIG. 1 shows the results of liquid chromatography analyses performed on polypeptide samples.
FIG. 2 shows the results of the verification of the inhibition of tyrosinase by the polypeptide in biochemical experiments. (A) The method comprises the following steps Blank group, (B): negative control group, (C): positive control group, (D): a sample set; (a) the method comprises the following steps When the potato slices are just smeared with corresponding medicines, (b): the potato slices were coated with the corresponding drug and left for two days.
FIG. 3 is a result of measurement of tyrosinase inhibitory activity of a sample by a microcolometry method.
FIG. 4 is a morphological observation of the effect of the polypeptide on mouse melanoma cells. (A) The method comprises the following steps Blank group, (B): 50. mu.g/mL-1Arbutin, (C): 20. mu.g/mL-1Polypeptide sample, (D): 50. mu.g/mL-1A polypeptide sample.
FIG. 5 shows the effect of tilapia scale polypeptide powder on tyrosinase activity without treatment according to the method of the present invention.
Detailed Description
The tilapia scale polypeptide powder used in the following examples was purchased from Qingdao Yihexing food Co.
The C18 column packing used in the examples described below was purchased from Suzhou Nami Microscience Inc. and the experiments were carried out by packing C18 packing in a sand-lined flash column.
The following examples used the following methods for determining the gold ion chelation rate: PAN indicator titration. Distilling to dissolve chelate, adding PAN indicator, titrating with EDTA, when the solution color changes from purple red to light yellow, the end point of titration is determined, and the volume of EDTA consumed at the time is recorded as V1And ml. The same procedure was used to directly titrate the chelated ion solution (containing the chelated and unchelated peptides, metal ions) and the volume of EDTA consumed at this time was recorded as V0，ml。
Chelating ratio of metal ion ═ V1/V0×100％。
Example 1
S1, preparing polypeptide liquid from tilapia mossambica scale polypeptide powder and water according to the mass ratio of 1:25 to obtain mixed polypeptide liquid, adding 1/6 of anhydrous copper sulfate solid in the mass of the polypeptide powder, adjusting the pH value to 6, and incubating for 50min at the water bath temperature of 50 ℃ to obtain polypeptide-ion chelating solution. And then, measuring the metal ion chelation rate by a PAN indicator titration method, wherein the ion chelation rate is 37-40% by measurement.
S2, the polypeptide-ion chelating solution prepared in the step S1 is subjected to sample loading and elution by adopting a C18 column. Specifically, the C18 column is balanced by using methanol, then the sample is loaded, the sample loading amount of the polypeptide-ion chelating solution is 3 times of the column volume, an EDTA solution with the concentration of 0.2mol/L is added to elute the metal ions in the components (the EDTA competitively binds the metal ions in the polypeptide-ion chelate so that the copper ions originally chelated with the polypeptide are chelated with the EDTA and flow out along with the EDTA solution), the elution volume is 3 times of the column volume, and the residual polypeptide components on the C18 column are dissolved and collected by using water, so that the polypeptide solution with the metal ion chelating capacity is obtained.
S3, carrying out rotary evaporation on the sample solution eluted by the EDTA at 60 ℃, and then carrying out freeze-drying at-30 to-40 ℃ to obtain the polypeptide with the ion chelating capacity.
Comparative example 1 was used directly for elution on a C18 column without being chelated with anhydrous copper sulfate
S1, preparing polypeptide liquid from tilapia mossambica scale polypeptide powder and water according to the mass ratio of 1:25 to obtain mixed polypeptide liquid. The product is directly used for elution of a C18 column without being chelated by anhydrous copper sulfate.
S2, the polypeptide-ion chelating solution prepared in the step S1 is subjected to sample loading and elution by adopting a C18 column. Specifically, the C18 column was equilibrated with methanol, and then loaded with a polypeptide solution in an amount 3 times the column volume, and the polypeptide fraction on the C18 column was collected by dissolution in water to obtain a polypeptide solution eluted through the C18 column.
And then chelating the polypeptide solution eluted by the C18 column with anhydrous copper sulfate, specifically, adding anhydrous copper sulfate solid with the mass ratio of 1:6 to tilapia mossambica scale polypeptide powder, adjusting the pH value to 6, and incubating for 50min at the water bath temperature of 50 ℃ to obtain the polypeptide-ion chelating solution. Through determination, the ion chelation rate of the polypeptide-ion chelation solution is 23-30%. That is, the chelating rate of the metal ions by the eluate can be reduced by directly eluting the sample by using the C18 column without chelating by using anhydrous copper sulfate, so that the chelating capacity of the polypeptide sample and the metal ions is reduced, the probability of effectively combining the polypeptide sample and the metal ions at the activity center of tyrosinase is reduced, and the inhibition activity of tyrosinase is reduced.
Example 2
S1, preparing polypeptide liquid from tilapia mossambica scale polypeptide powder and water according to the mass ratio of 1:25 to obtain mixed polypeptide liquid, adding anhydrous copper sulfate solid with the polypeptide powder according to the mass ratio of 1:6, adjusting the pH value to 6, and incubating at the water bath temperature of 50 ℃ for 50min to obtain the polypeptide-ion chelating solution.
S2, loading and eluting the polypeptide-ion chelating solution prepared in the step S1 by using a C18 column. Specifically, the C18 column is balanced by methanol, then the sample is loaded, the loading amount of the polypeptide-ion chelating solution is 3 times of the column volume, EDTA solution with the concentration of 0.2mol/L is added to elute the metal ions in the components, the elution volume is 3 times of the column volume, and the rest polypeptide components on the C18 column are dissolved and collected by water to obtain the polypeptide solution with the metal ion chelating capacity.
S3, carrying out rotary evaporation on the sample solution eluted by EDTA at 60 ℃, then carrying out freeze-drying at-30 to-40 ℃, and adding 5% of mannitol as a protective agent in the freeze-drying process to obtain the polypeptide with ion chelating ability. The obtained sample has uniform particles, good freeze-drying effect and better re-solubility, and does not generate layering.
Comparative example 2
The difference from the example 2 is that no protective agent is added in the S3 freeze-drying process. The obtained sample particles are rough and not uniform enough, and the sample is redissolved, so that the sample and the solution are layered.
Example 1 inhibition of browning of potatoes by the polypeptide obtained in step 3:
coating corresponding medicines on the surfaces of potato chips with uniform sizes and thicknesses, wherein the group A is a blank group, and the surfaces of the potato chips are coated with deionized water; the group B is a negative control group, and the surface of the group B is coated with 5mg/mL L-Tyr solution; group C is positive control group, and 5mg/mL arbutin solution is smeared on the surface; group D is a polypeptide sample group, the polypeptide sample solution with metal ion chelating ability prepared in example 1 is coated on the surface (the concentration of the sample coated with B, C, D is the same, and is 5mg/mL), and the browning condition of the potato slices after 48 hours is observed. Judging the inhibition effect of the potato on tyrosinase according to the browning degree of the potato. The experimental result is shown in fig. 2, and the result shows that the polypeptide component with ion chelation capacity can obviously inhibit the browning condition of the potatoes, and according to the observation of the browning condition of the potatoes, the inhibition effect of the polypeptide sample on tyrosinase is superior to that of the positive control group arbutin.
Example 1 inhibition of tyrosinase Activity by Polypeptides obtained in step 3
After 20 μ g of the polypeptide sample or the standard arbutin of example 1 was mixed well with tyrosinase and the substrate tyrosine, respectively, the absorbance at 475nm was measured immediately at 10s and recorded as A1, and then it was quickly placed in a 37 ℃ water bath for 3 min. Then, the absorbance at 190 seconds after the wiper was quickly removed was measured and recorded as A2. Δ a is calculated as a2-a 1. Tyrosinase activity (U/mL) ═ delta A/di (epsilon x d) x V anti-total x 10 was calculated 9V-sample/T
Epsilon: molar extinction coefficient of dopachrome;
d: the optical path of the cuvette is in cm
V inverse Total: total reaction volume, unit: mL
And V sample: volume of sample added in mL
T: reaction time in units of min
The experimental result is shown in fig. 3, and the polypeptide with metal ion chelating ability prepared by the invention has obvious inhibition effect on tyrosinase. Compared with the positive control arbutin with the same concentration, the polypeptide sample with the metal ion chelating capacity has stronger inhibition effect on tyrosinase. Subsequently, sample solutions with different concentrations are selected for tyrosinase activity determination, and the concentrations are respectively 2mg/mL and 5mg/mL, and the results show that the inhibition effect on tyrosinase is enhanced along with the increase of the sample concentration.
Example 1 action of the polypeptide obtained in step 3 on mouse melanoma B16F10 cells
B16-F10 cells were passaged and were mostly found in two-stage morphology.
Blank group: only the cell culture solution is added, and the cell growth state is better and has no obvious change in shape through microscope observation.
Control group: adding arbutin solution with concentration of 50 μ g/mL after filtration sterilization.
Experimental groups: solutions of the polypeptide sample of example 1 were added at concentrations of 20 and 50. mu.g/mL, respectively, after they had been filter sterilized.
Through microscope observation, when alpha-Arbutin and polypeptide samples with different concentrations are added, the cell morphology changes in different degrees, the cell distribution begins to become sparse along with the increase of the administration concentration, a dense reticular structure is not formed any more, the cells obviously deform, the growth of the cells is obviously inhibited by an experimental group and a control group, and the effect of inhibiting the growth of the cells by the experimental group is more obvious. Meanwhile, the experimental result also shows that the B16-F10 cells have concentration dependence on the sample. The results of the experiment are shown in FIG. 4.
Comparative example 3
Directly detecting the inhibition effect of tilapia mossambica scale polypeptide powder purchased from Qingdaoyi Yihexing food company Limited on the tyrosinase activity, wherein the detection method is the same as the detection method of the inhibition effect of the polypeptide obtained in the step 3 of the example 1 on the tyrosinase activity, and no polypeptide is added in a blank group. The results of the experiments are shown in fig. 5, and the results of the enzyme activity assay show that the untreated polypeptide powder has no inhibitory effect on tyrosinase activity, but rather stimulates tyrosinase activity.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method of preparing a polypeptide composition having tyrosinase inhibitory activity, comprising the steps of:
s1, preparing mixed polypeptide liquid from tilapia phosphorus polypeptide powder and water according to a certain mass ratio, adding anhydrous copper sulfate into the mixed polypeptide liquid, and chelating at a proper pH value, water bath temperature and time to obtain polypeptide-ion chelating solution; loading the polypeptide-ion chelating solution to a C18 column;
s2, competitively binding metal ions in the polypeptide-ion chelating solution by using an EDTA solution, an EDTA-2Na solution or a mixed solution of EDTA and EDTA-2Na to elute the metal ions, and then dissolving and eluting the polypeptide left on the C18 column by using water to obtain a polypeptide solution;
s3, performing rotary evaporation and freeze-drying on the polypeptide solution obtained in the step S2 to obtain the polypeptide composition with ion chelating ability.
2. The method of claim 1, wherein the tilapia phosphorus polypeptide meal of step S1 is extracted from tilapia phosphorus.
3. The method according to claim 1, wherein the mass ratio of the tilapia phosphorus polypeptide powder to water in step S1 is 1: 20-1: 25.
4. The method according to claim 1, wherein the mass ratio of the tilapia phosphorus polypeptide powder to the anhydrous copper sulfate in the step S1 is 4: 1-7: 1.
5. The method of claim 1, wherein the pH value of step S1 is 5-7, and the chelating time is 50-70 min.
6. The method according to claim 1, wherein the concentration of the EDTA solution, the EDTA-2Na solution or the mixed solution of EDTA and EDTA-2Na in step S2 is 10-20%.
7. The method according to claim 1, wherein the rotary evaporation temperature of step S3 is 60-65 ℃, and the freeze-drying temperature is-30-40 ℃.
8. The method according to claim 1, wherein a lyoprotectant, such as mannitol, is added during the lyophilization process of step S3.
9. A polypeptide composition having tyrosinase inhibitory activity prepared by the method of any one of claims 1-8.
10. The use of the polypeptide composition of claim 9 for inhibiting tyrosinase activity or inhibiting browning of fruit or vegetable.
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