CN105648004B - Preparation method of dasyatis akajei cartilage antioxidative collagen peptide - Google Patents
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- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- 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
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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Abstract
The invention discloses a preparation method of dasyatis akajei cartilage antioxidative collagen peptide. The method comprises the steps of taking stingray cartilage as a raw material, extracting collagen, carrying out enzymolysis by trypsin and neutral protease to obtain an enzymolysis solution, separating and purifying the enzymolysis solution by ultrafiltration, ion exchange resin chromatography, gel column chromatography and reversed-phase high performance liquid chromatography to obtain antioxidant collagen peptide Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp, and measuring the molecular weight of the stingray cartilage by ESI-MS to be 875.85 Da; the prepared high-activity antioxidant collagen peptide has good scavenging effect on DPPH free radicals, hydroxyl free radicals, ABTS free radicals and superoxide anion free radicals; and simultaneously shows good lipid peroxidation inhibition effect, and can be developed as a medicine, a health food or a food additive.
Description
Technical Field
The invention relates to a preparation method of animal cartilage collagen peptide, and in particular relates to a preparation method of dasyatis akajei cartilage antioxidant collagen peptide.
Background
Collagen peptide is the product of collagen degradation by protease, with molecular weight of about 180-3000 Da, and is considered more easily digested and absorbed by human body than common collagen. Meanwhile, the existing research proves that the collagen peptide has various biological activities, such as angiotensin converting enzyme activity inhibition, antioxidant activity, pain reduction of osteoarthritis patients such as knee joints or hip joints, bone density enhancement, bone metabolism balance maintenance and the like.
Dasyatis akajei (Chiyashi)Dasyatis akajei) Belongs to the class chondroiridae, order Basidioidea, order Davidae, family stingray and genus stingray. Stingrays are common cartilaginous fishes and are distributed in coastal areas of China. Research shows that the dasyatis akajei cartilage contains a large amount of collagen and can be used as a raw material of collagen peptide for development and utilization.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of dasyatis akajei cartilage antioxidative collagen peptide capable of removing free radicals and inhibiting lipid peroxidation.
The technical scheme adopted by the invention for solving the technical problems is as follows: a preparation method of dasyatis akajei cartilage antioxidative collagen peptide comprises the following steps:
1) preparing dasyatis akajei cartilage collagen: drying and crushing dasyatis akajei cartilage, adding the dasyatis akajei cartilage into NaOH solution (0.1 mol/L) according to the feed liquid ratio of 1g: 15-20 mL, soaking at 1-4 ℃ for 2-4 h, and removing non-collagen; repeatedly washing the treated dasyatis akajei cartilage with distilled water to be neutral, draining, adding an EDTA solution (0.5 mol/L) with the pH value of 7.4 into the dasyatis akajei cartilage according to the material-liquid ratio of 1g: 5-8 mL, soaking the dasyatis akajei cartilage for 2-3 days at the temperature of 1-4 ℃ (the EDTA solution is replaced 1 time per day), washing the dasyatis akajei cartilage with distilled water for 2-3 times, drying and crushing the dasyatis akajei cartilage to obtain the decalcified das. Adding decalcified dasyatis powder into 0.5mol/L acetic acid solution according to the material-liquid ratio of 1g: 5-8 mL, dynamically soaking for 3d at 1-4 ℃, centrifuging for 20-25 min at 10000g, taking supernatant, adding NaCl until the final concentration of the solution is 1.0mol/L, standing for 60-90 min, centrifuging for 25-30 min at 12000g, and freeze-drying to obtain dasyatis collagen.
2) Enzymolysis of dasyatis akajei cartilage collagen: adding dasyatis akajei cartilage collagen into a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH 7.5, 0.2 mol/L) according to a material-liquid ratio of 1g: 15-20 mL, and mixing according to the collagenAdding trypsin (1.9 × 10) to the solution in an amount of 1.0-1.5%4U/g), performing enzymolysis at the temperature of 45-50 ℃ for 3-4 h, heating the solution to 90-95 ℃, keeping the temperature for 8-10 min, cooling to 45-50 ℃, adding neutral protease (1.0 × 10) into the solution according to the mass percent of 1.5-1.8 percent of the collagen5U/g), performing enzymolysis for 6-8 h at the temperature of 45-50 ℃, heating the solution to 90-95 ℃, keeping the temperature for 15-20 min, centrifuging for 20-25 min at 10000g, and taking supernatant, namely an enzymolysis product;
3) preparing dasyatis akajei cartilage antioxidative collagen peptide: performing ultrafiltration treatment on the enzymolysis product by using a 3.5kDa ultrafiltration membrane, collecting a part with the molecular weight less than 3.5kDa, and freeze-drying to obtain an ultrafiltration zymolyte; purifying the ultrafiltration zymolyte by ion exchange resin chromatography, gel column chromatography and reversed phase high performance liquid chromatography (RP-HPLC) to obtain the antioxidant collagen peptide.
Preferably, the dasyatis akajei in the step 1) is dasyatis akajei (Dasyatis akajei)Dasyatis akajei）。
Preferably, the specific processes of ion exchange resin chromatography, gel column chromatography and RP-HPLC purification in the step 4) are as follows:
ion exchange resin chromatography: dissolving the ultrafiltration zymolyte in double distilled water to prepare a solution with the concentration of 25-30 mg/mL, passing through a DEAE-52 ion exchange resin chromatographic column, eluting with water, 0.5mol/L and 1.0mol/L NaCl solution at the flow rate of 0.9-1.2 mL/min, collecting one tube of the eluate every 3min, detecting at 225nm, merging the solutions in the test tube according to peaks, comparing the removal activity of hydroxyl radicals of each peak, and selecting a component with the strongest activity for freeze-drying to obtain the ion exchange chromatography zymolyte;
gel column chromatography: dissolving the ion exchange chromatography zymolyte in double distilled water to prepare a solution with the concentration of 25-30 mg/mL, separating by Sephadex G-25 column chromatography, eluting with double distilled water at the flow rate of 0.8-1.0 mL/min, collecting one tube of the eluted solution every 3min, detecting at 225nm, merging the solutions in the test tube according to peaks, comparing the removal activity of hydroxyl radicals of each peak, selecting the component with the strongest activity, and freeze-drying to obtain the gel chromatography zymolyte;
RP-HPLC purification: preparing the gel chromatography zymolyte into a solution of 40-50 mu g/mL by using double distilled water, purifying by using RP-HPLC, and obtaining 1 high-activity antioxidant collagen peptide Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW) according to the scavenging activity of hydroxyl free radicals, wherein the molecular weight is 875.85Da by ESI-MS (electronic signature verification).
More preferably, the RP-HPLC conditions are: the sample injection amount is 15-20 mu L; chromatographic column Zorbax SB-C18(250 mm × 4.6.6 mm, 5 μm), mobile phase of 40% acetonitrile, elution speed of 0.8-1.0 mL/min, ultraviolet detection wavelength of 225 nm.
Compared with the prior art, the dasyatis akajei cartilage antioxidative collagen peptide provided by the invention has a good effect of removing DPPH free radicals, hydroxyl free radicals and superoxide anion free radicals; meanwhile, Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW) also shows good lipid peroxidation inhibition effect; Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW) has the advantages of safety, no toxic or side effect, strong antioxidant activity, easy digestion and absorption and the like, and can be used as a medicine, a health food and a food additive.
Drawings
FIG. 1 is a chromatogram of DEAE-52 ion exchange resin of the present invention.
FIG. 2 is a Sephadex G-25 chromatogram of a Sephadex gel of the invention.
FIG. 3 RP-HPLC analysis of the zymolyte prepared by Sephadex G-25.
FIG. 4 is a mass spectrum of Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW).
FIG. 5 shows the anti-lipid oxidation ability of Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW).
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Example (b):
a preparation method of dasyatis akajei cartilage antioxidative collagen peptide comprises the following preparation process flow: stingray cartilage → cartilage of cartilage → enzymolysis of trypsin and neutral protease → zymolyte → ultrafiltration → ion exchange chromatography → gel filtration chromatography → preparation of high performance liquid chromatography → anti-oxidation collagen peptide.
1) Preparing dasyatis akajei cartilage collagen: will be Dasyatis akajei (Chiyashi)Dasyatis akajei) Drying and crushing cartilage, adding the cartilage into NaOH solution (0.1 mol/L) according to the feed liquid ratio of 1g: 18 mL, soaking for 3 hours at the temperature of 3 ℃, and removing non-collagen; repeatedly washing the treated dasyatis akajei cartilage with distilled water to be neutral and draining, adding EDTA solution (0.5 mol/L) with the pH value of 7.4 according to the material-liquid ratio of 1g: 6 mL, soaking for 3 days at the temperature of 4 ℃ (the EDTA solution is replaced for 1 time per day), washing for 3 times with distilled water, drying and crushing to obtain the decalcified dasyatis akajei cartilage powder. Adding decalcified dasyatis powder into 0.5mol/L acetic acid solution according to the material-liquid ratio of 1g: 6 mL, dynamically soaking for 3d at 3 ℃, centrifuging for 20min at 10000g, taking supernatant, adding NaCl until the final concentration of the solution is 1.0mol/L, standing for 90min, centrifuging for 25min at 12000g to obtain precipitate, and freeze-drying to obtain dasyatis collagen.
2) Enzymolysis of Dasyatis akajei cartilage collagen, adding Dasyatis akajei cartilage collagen into disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH 7.5, 0.2 mol/L) according to the ratio of raw materials to liquid of 1g:15 mL, adding trypsin (1.9 × 10) into the solution according to the mass of 1.3 percent of the collagen4U/g), performing enzymolysis at 48 deg.C for 3.5 hr, heating to 90 deg.C, maintaining at the temperature for 10min, cooling to 45 deg.C, adding neutral protease (1.0 × 10) to the solution at 1.6% of collagen mass5U/g), performing enzymolysis at 45 ℃ for 6 h, heating the solution to 90 ℃, keeping the temperature for 15 min, centrifuging 10000g for 20min, and taking supernatant, namely an enzymolysis product;
3) preparing dasyatis akajei cartilage antioxidative collagen peptide: performing ultrafiltration treatment on the enzymolysis product by using a 3.5kDa ultrafiltration membrane, collecting a part with the molecular weight less than 3.5kDa, and freeze-drying to obtain an ultrafiltration zymolyte; purifying the ultrafiltration zymolyte by ion exchange resin chromatography, gel column chromatography and reversed phase high performance liquid chromatography (RP-HPLC) to obtain the antioxidant collagen peptide.
Ion exchange resin chromatography: dissolving the ultrafiltration zymolyte in double distilled water to prepare a solution with the concentration of 28 mg/mL, passing through a DEAE-52 ion exchange resin chromatographic column, eluting with water, 0.5mol/L and 1.0mol/L NaCl solution at the flow rate of 1.0-1.2 mL/min, collecting one tube of the eluate every 3min, detecting at 225nm, combining the solutions in the test tube according to peaks, comparing the removal activity of hydroxyl radicals of each peak, selecting the component with the strongest activity, and freeze-drying to obtain the ion exchange chromatography zymolyte DCP-III (figure 1);
② gel column chromatography: dissolving the ion exchange chromatography zymolyte in double distilled water to prepare a solution with the concentration of 25 mg/mL, separating by Sephadex G-25 column chromatography, eluting with double distilled water at the flow rate of 1.0mL/min, collecting one tube of the eluted solution every 3min, detecting at 225nm, merging the solutions in the test tube according to peaks, comparing the removal activity of hydroxyl radicals of each peak, selecting the component with the strongest activity, and freeze-drying to obtain the gel chromatography zymolyte DCP-III-3 (figure 2);
③ RP-HPLC purification comprises preparing 40 μ g/mL solution of the gel chromatography zymolyte with double distilled water, and purifying by RP-HPLC (RP-HPLC condition: sample size is 18 μ L; chromatographic column Zorbax SB-C)18(250 mm × 4.6.6 mm, 5 μm), mobile phase of 40% acetonitrile, elution speed of 0.8 mL/min, ultraviolet detection wavelength of 225 nm), and 1 highly active antioxidant collagen peptide Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW) according to the hydroxyl radical scavenging activity.
④ structure detection comprises collecting antioxidant collagen peptide with highest DPPH free radical and hydroxyl free radical scavenging activity, detecting by RP-HPLC to obtain single peak, determining amino acid sequence by protein/polypeptide sequence analyzer to be Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW), and determining molecular weight by ESI-MS to be 875.85Da ([ M + H ]]+876.81 Da) (FIG. 4).
The prepared dasyatis akajei cartilage antioxidative collagen peptide Gly-Ile-Glu-Gly-Trp (GIEGEEGW) is subjected to a free radical scavenging experiment and a lipid peroxidation inhibition experiment, and the experiment results show that: Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW) to DPPH free radical (EC)501.93 mg/mL), hydroxyl radical (EC)500.35 mg/mL), ABTS free radical (EC)500.16 mg/mL) and superoxide anion radical (EC)500.24 mg/mL) has good scavenging effect; meanwhile, Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW) also shows good lipid peroxidation inhibition effect (figure 5).
Finally, it should also be noted that the above-mentioned list is only one specific embodiment of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
SEQUENCE LISTING
<110> Zhejiang ocean academy
Preparation method of dasyatis akajei cartilage antioxidative collagen peptide
<130>zjou-wb-201511-302
<160>1
<170>PatentIn version 3.5
<210>1
<211>8
<212>PRT
<213> Artificial Synthesis
<400>1
Gly Ile Glu Gly Glu Glu Gly Trp
1 5
Claims (1)
1. The preparation method of dasyatis akajei cartilage antioxidative collagen peptide comprises the following steps:
1) preparing dasyatis akajei cartilage collagen: drying and crushing dasyatis akajei cartilage, adding the dasyatis akajei cartilage into NaOH solution according to the feed liquid ratio of 1g to 15-20 mL, soaking at 1-4 ℃ for 2-4 h, and removing non-collagen; repeatedly washing the treated dasyatis akajei cartilage with distilled water to be neutral and draining, adding an EDTA solution with the pH value of 7.4 according to the material-liquid ratio of 1g: 5-8 mL, soaking at 1-4 ℃ for 2-3 days, washing with distilled water for 2-3 times, drying and crushing to obtain decalcified dasyatis akajei cartilage powder; adding decalcified dasyatis akajei powder into 0.5mol/L acetic acid solution according to the material-liquid ratio of 1g: 5-8 mL, dynamically soaking for 3d at 1-4 ℃, centrifuging for 20-25 min at 10000g, taking supernatant, adding NaCl until the final concentration of the solution is 1.0mol/L, standing for 60-90 min, centrifuging for 25-30 min at 12000g, and freeze-drying to obtain dasyatis akajei cartilage collagen;
2) enzymolysis of dasyatis akajei cartilage collagen: adding dasyatis akajei cartilage collagen into a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution according to the material-liquid ratio of 1g: 15-20 mL, adding trypsin into the solution according to 1.0-1.5% of the mass of the collagen, performing enzymolysis for 3-4 h at the temperature of 45-50 ℃, heating the solution to 90-95 ℃, keeping the temperature for 8-10 min, and cooling to 45-50 ℃; adding neutral protease into the solution according to the mass of 1.5-1.8% of the collagen, performing enzymolysis for 6-8 hours at the temperature of 45-50 ℃, heating the solution to 90-95 ℃, keeping the temperature for 15-20 min, centrifuging for 20-25 min at 10000g, and taking supernatant, namely an enzymolysis product;
3) preparing dasyatis akajei cartilage antioxidative collagen peptide: performing ultrafiltration treatment on the enzymolysis product by using a 3.5kDa ultrafiltration membrane, collecting a part with the molecular weight less than 3.5kDa, and freeze-drying to obtain an ultrafiltration zymolyte; purifying the ultrafiltration zymolyte by ion exchange resin chromatography, gel column chromatography and reversed-phase high performance liquid chromatography to obtain antioxidant collagen peptide, wherein the prepared antioxidant collagen peptide Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp is octapeptide compound, and the molecular weight is 875.85Da in ESI-MS measurement;
wherein the dasyatis akajei in the step 1) is dasyatis akajeiDasyatis akajeiNaOH solution is 0.1 mol/L; the EDTA solution is 0.5mol/L and is replaced for 1 time every day;
the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution in the step 2) has a value of 0.2mol/L, pH of 7.5 and the trypsin has a value of 1.9 × 104U/g, neutral protease 1.0 × 105U/g；
The specific processes of ion exchange resin chromatography, gel column chromatography and reversed-phase high performance liquid chromatography purification in the step 3) are as follows:
ion exchange resin chromatography: dissolving the ultrafiltration zymolyte in double distilled water to prepare a solution with the concentration of 25-30 mg/mL, passing through a DEAE-52 ion exchange resin chromatographic column, eluting with water, 0.5mol/L and 1.0mol/LNaCl solutions at the flow rate of 0.9-1.2 mL/min, collecting one tube of the eluate every 3min, detecting at 225nm, merging the solutions in the test tube according to peaks, comparing the removal activity of hydroxyl radicals of each peak, and selecting the component with the strongest activity for freeze-drying to obtain the ion exchange chromatography zymolyte;
gel column chromatography: dissolving the ion exchange chromatography zymolyte in double distilled water to prepare a solution with the concentration of 25-30 mg/mL, separating by sephadex G-25 column chromatography, eluting with double distilled water at the flow rate of 0.8-1.0 mL/min, collecting one tube of the eluted solution every 3min, detecting at 225nm, merging the solutions in the test tube according to peaks, comparing the removal activity of hydroxyl radicals of each peak, selecting the component with the strongest activity, and freeze-drying to obtain the gel chromatography zymolyte;
purifying by reverse phase high performance liquid chromatography, namely preparing the gel chromatography zymolyte into a solution of 40-50 mu g/mL by using double distilled water, and purifying by using the reverse phase high performance liquid chromatography, wherein the conditions of the reverse phase high performance liquid chromatography are that the sample injection amount is 15-20 mu L, and a chromatographic column is ZorbaxSB-C with the specification of 250mm × 4.6.6 mm and 5 mu m18(ii) a The mobile phase is 40% acetonitrile; the elution speed is 0.8-1.0 mL/min; ultraviolet detecting wavelength is 225nm, and molecular weight is 875.85Da when detecting 1 high-activity antioxidant collagen peptide Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp according to hydroxyl radical scavenging activity and ESI-MS.
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