CN103992384A - Pseudosciaena crocea fish bone collagen peptide, and preparation method and application thereof - Google Patents
Pseudosciaena crocea fish bone collagen peptide, and preparation method and application thereof Download PDFInfo
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- CN103992384A CN103992384A CN201410218174.5A CN201410218174A CN103992384A CN 103992384 A CN103992384 A CN 103992384A CN 201410218174 A CN201410218174 A CN 201410218174A CN 103992384 A CN103992384 A CN 103992384A
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 40
- 241001596950 Larimichthys crocea Species 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 210000000988 bone and bone Anatomy 0.000 title abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000012153 distilled water Substances 0.000 claims abstract description 19
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- 238000004587 chromatography analysis Methods 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 9
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- 239000011780 sodium chloride Substances 0.000 claims abstract description 7
- 239000003814 drug Substances 0.000 claims abstract description 5
- 230000008569 process Effects 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 4
- 231100000331 toxic Toxicity 0.000 claims abstract description 4
- 230000002588 toxic effect Effects 0.000 claims abstract description 4
- 238000000108 ultra-filtration Methods 0.000 claims description 20
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 claims description 16
- 230000007760 free radical scavenging Effects 0.000 claims description 16
- 229920005654 Sephadex Polymers 0.000 claims description 12
- 239000012507 Sephadex™ Substances 0.000 claims description 12
- 238000010828 elution Methods 0.000 claims description 12
- 238000012545 processing Methods 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 102000035195 Peptidases Human genes 0.000 claims description 8
- 108091005804 Peptidases Proteins 0.000 claims description 8
- -1 DPPH free radical Chemical class 0.000 claims description 7
- 230000003078 antioxidant effect Effects 0.000 claims description 7
- 238000005227 gel permeation chromatography Methods 0.000 claims description 7
- 238000004255 ion exchange chromatography Methods 0.000 claims description 7
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 6
- 238000009413 insulation Methods 0.000 claims description 6
- 235000018102 proteins Nutrition 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims description 5
- 238000004007 reversed phase HPLC Methods 0.000 claims description 5
- 235000013305 food Nutrition 0.000 claims description 4
- 230000003859 lipid peroxidation Effects 0.000 claims description 4
- 229910052760 oxygen Inorganic materials 0.000 claims description 4
- 239000001301 oxygen Substances 0.000 claims description 4
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- 210000002784 stomach Anatomy 0.000 claims description 4
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- 239000000654 additive Substances 0.000 claims description 3
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- 102000057297 Pepsin A Human genes 0.000 abstract 1
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- 235000013373 food additive Nutrition 0.000 abstract 1
- 239000002778 food additive Substances 0.000 abstract 1
- 235000013402 health food Nutrition 0.000 abstract 1
- 230000000415 inactivating effect Effects 0.000 abstract 1
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a Pseudosciaena crocea fish bone collagen peptide, and a preparation method and application thereof. The amino acid sequence of the collagen peptide is Gly-Phe-Pro-Gly-Ser-Phe-Arg (SEQ ID No:1), and the molecular weight by ESI-MS detection is 823.92Da ([M+H]+824.77Da). The method comprises the following steps: chopping Pseudosciaena crocea fish bone, removing non-collagen substances, carrying out decalcification and degreasing treatment, and preparing fish bone collagen by acid extraction and NaCl salting-out; adding the prepared fish bone collagen into distilled water according to the material-to-liquid ratio of 1g:(15-20mL), regulating the pH value to 1.2-2.0, keeping the temperature at 35-40 DEG C for 5-10 minutes, adding pepsin which accounts for 1.0-1.5 wt% of the collagen, and carrying out enzymolysis for 4-6 hours; regulating the temperature of the enzymolysis solution to 40-50 DEG C, regulating the pH value to 9.0-10.0, keeping the temperature for 5-10 minutes, adding alkaline proteinase which accounts for 1.0-1.5 wt% of the collagen, and carrying out enzymolysis for 3-5 hours; and inactivating the enzyme of the enzymolysis solution by high temperature, centrifuging, ultrafiltering, and carrying out chromatography to obtain the collagen peptide. The preparation technique is scientific and reasonable; the enzymolysis process is easy to control; and the prepared fish bone collagen peptide has the advantages of high oxidation-resistant activity, high safety and no toxic or side effect, and can be used as a medicine, health food or food additive.
Description
Technical field
The present invention relates to a kind of fish Bone gillg and its production and use, relate in particular to a kind of large yellow croaker fish-bone collagen peptide and its production and use.
Background technology
Collagen protein is ubiquitous a kind of high molecular weight protein in animal body, be mainly present in the reticular tissue (bone, cartilage, skin, tendon, tough etc.) of animal, to body and internal organs play support, protection, in conjunction with and form boundary every etc. effect.Collagen protein is because having good biocompatibility, biodegradable and biological activity, such as low antigenicity, promotion cell survive and grow, promote that thrombocyte condenses, obtain application widely in fields such as food, medicine, organizational project, makeup.Collagen protein is hydrolyzed under the effect of acid, alkali, heat, enzyme, can produce collagen peptide, compared with collagen protein, due to reduction and the water miscible raising of molecular weight, collagen peptide is more easily absorbed by the body and brings into play effect with utilization, meanwhile, the collagen peptide that Collagen Hydrolysate produces, also there is more biological activity compared with collagen protein, as anti-oxidant activity, antihypertensive activity, anti-tumor activity, raising immunizing power etc.
Along with the development of domestic fish processing industry, the tankage that aquatic products processing enterprise produces are as increase year after years such as fish head, fish-skin, fish scale, fish-bone, fins.But the utilization of aquatic products processing tankage is also insufficient, causes the wasting of resources.Therefore, make full use of aquatic products processing tankage, to improve overall economic efficiency, become the problem of aquatic products processing enterprise growing interest.At present, existing data proves that aquatic products processing tankage fish-skin, fish scale, fish-bone etc. are the high quality raw material that extract collagen protein, and has successfully utilized fish-skin to carry out collagen protein or collagen peptide exploitation.
But, applicant studies discovery, taking large yellow croaker fish-bone as raw material, utilize technical study that zymolysis technique prepares large yellow croaker fish-bone collagen protein and collagen peptide in the blank stage, and prepare anti-oxidant activity collagen peptide taking fish-bone collagen protein enzymolysis product as material and application has no report especially.
Summary of the invention
First technical problem to be solved by this invention is to provide a kind of large yellow croaker fish-bone collagen peptide for the above-mentioned state of the art, this collagen peptide safe without toxic side effect, and there is stronger antioxygenation.
Second technical problem to be solved by this invention is to provide a kind of preparation method of large yellow croaker fish-bone collagen peptide, and this craft science is reasonable, easy handling.
The 3rd technical problem to be solved by this invention is to provide a kind of application of large yellow croaker fish-bone collagen peptide.
The technical scheme that the present invention takes for above-mentioned first technical problem of solution is: a kind of large yellow croaker fish-bone collagen peptide, the aminoacid sequence that it is characterized in that this collagen peptide is Gly-Phe-Pro-Gly-Ser-Phe-Arg(SEQ ID No:1), ESI-MS detects and provides molecular weight is m/z 823.92 Da([M+H]
+824.77 Da).
The technical scheme that the present invention takes for above-mentioned second technical problem of solution is: a kind of preparation method of large yellow croaker fish-bone collagen peptide, is characterized in that comprising the following steps:
1) after large yellow croaker fish-bone minces, add 0.1 mol/L NaOH solution according to solid-liquid ratio 1 g:15 ~ 25 mL and at 4 DEG C, soak 3 ~ 5 h, remove noncollagen protein; Fish-bone distilled water repetitive scrubbing after processing, adds 0.5mol/L EDTA solution (pH7.4) according to solid-liquid ratio 1 g:5 ~ 10 mL after fully draining, and soaks 3 ~ 5 days at 4 DEG C, changes EDTA solution every day 1 time, removes the calcium in fish-bone; After decalcification fish-bone drains, add 10% aqueous isopropanol according to solid-liquid ratio 1 g:15 ~ 20 mL, at 4 DEG C, soak 18 ~ 24 h, remove fat, dry, obtain degreasing fish-bone.
2) degreasing fish-bone is added to 0.5mol/L acetic acid solution and at 4 DEG C, soaks 3 ~ 4 days according to solid-liquid ratio 1 g:10 ~ 15 mL.In 4 DEG C, centrifugal 25 ~ 30 min of 20000 r/min, obtain supernatant liquor and be thick collagen protein; Get appropriate thick collagen protein add NaCl to solution final concentration be 0.8 ~ 1.0 mol/L, leave standstill 3 ~ 4 h to separating out flocks, in 4 DEG C, 12000 r/min, centrifugal 15 ~ 20 min obtain throw out, lyophilize, is the collagen protein after saltouing.
3) collagen protein of preparation is joined in distilled water according to solid-liquid ratio 1 g:15 ~ 20 mL, adjust pH to 1.2 ~ 2.0, in 35 ~ 40 DEG C of insulation 5 ~ 10 min, add the first proteolytic enzyme according to 1.0 ~ 1.5% of collagen protein quality, enzymolysis 4 ~ 6 h; Enzymolysis solution temperature is adjusted to 40 ~ 50 DEG C, and pH is adjusted to 9.0 ~ 10.0, and insulation 5 ~ 10 min, add the second proteolytic enzyme according to 1.0 ~ 1.5% of collagen protein amount, enzymolysis 3 ~ 5 h;
4) enzymolysis solution is heated to 90 ~ 95 DEG C of deactivation 10 ~ 15 min, centrifugal 10 ~ 15 min of 9000 ~ 10000 r/min, get supernatant liquor; Supernatant liquor through ultrafiltration and chromatography, obtains collagen peptide successively.
As preferably, the first proteolytic enzyme in described step 3) is stomach en-, enzyme activity>=1.5 × 10
5u/g.
As preferably, the second proteolytic enzyme in described step 3) is Sumizyme MP, enzyme activity>=1.9 × 10
5u/g.
As improvement, the ultrafiltration of described step 4) and the detailed process of chromatography are:
Ultrafiltration: supernatant liquor is adjusted to pH 6.5 ~ 7.5, under the working temperature of the operating pressure of 0.12 ~ 0.15 MPa and 25 ~ 30 DEG C, adopt the successively ultra-filtration membrane of 5 kDa and 1 kDa to carry out uf processing, collect molecular weight and be greater than 5 kDa components, molecular weight is less than 5 kDa and is greater than 1 kDa component, molecular weight is less than 1 kDa component, wherein, the highest component of DPPH free radical scavenging activity is ultrafiltration enzymolysis solution;
Chromatography: by above-mentioned ultrafiltration enzymolysis solution NaH
2pO
4-Na
2hPO
4(0.2mol/L, pH 7.0) damping fluid is made into the solution of 20 ~ 25 mg/mL, through anion-exchange resin column chromatographic separation, carry out wash-out with distilled water, 0.45 ~ 0.55 mol/L and 0.90 ~ 1.10 mol/L NaCl solution, collect elution fraction according to the absorbancy curve under 280 nm, wherein, the highest component of DPPH free radical scavenging activity is ion exchange chromatography enzymolysis solution, above-mentioned ion exchange chromatography enzymolysis solution is made into the solution of 10 ~ 15 mg/mL with distilled water, separate through gel filtration chromatography, carry out wash-out with distilled water, collect elution fraction according to the absorbancy curve under 280 nm, wherein, the highest component of DPPH free radical scavenging activity is gel chromatography zymolyte, above-mentioned gel chromatography zymolyte is made into the solution of 45 ~ 50 μ g/mL with distilled water, utilize RPLC (RP-HPLC) to carry out purifying, obtain 1 high reactivity anti-oxidation peptide Gly-Phe-Pro-Gly-Ser-Phe-Arg(SEQ ID No:1 according to DPPH free radical scavenging activity).
Preferably, described anionite-exchange resin is SP-sephadex C25, and described gel is sephadex G-25.
Preferred again, described RPLC condition is: sample size 10 ~ 15 μ L; Chromatographic column is Kromasil C18; Column temperature is 25 ~ 30 DEG C; Moving phase: A water (containing 0.1% trifluoroacetic acid) and B acetonitrile; Gradient elution: 0 ~ 40 min acetonitrile concentration from 0 to 50%; Elution speed 1.0 mL/min; Ultraviolet detection wavelength 280 nm.
The present invention for above-mentioned the 3rd technical scheme that technical problem is taked of solution is: a kind of application of large yellow croaker fish-bone collagen peptide, is characterized in that large yellow croaker fish-bone collagen peptide Gly-Phe-Pro-Gly-Ser-Phe-Arg(SEQ ID No:1) DPPH free radical, hydroxyl radical free radical and ultra-oxygen anion free radical are had to good scavenging(action); Meanwhile, Gly-Phe-Pro-Gly-Ser-Phe-Arg(SEQ ID No:1) also demonstrate good Lipid peroxidation; Gly-Phe-Pro-Gly-Ser-Phe-Arg(SEQ ID No:1) there is the advantages such as the strong and safe without toxic side effect of anti-oxidant activity, can be used as medicine, protective foods and foodstuff additive.
Compared with prior art, the invention has the advantages that: the present invention selects stomach en-and Sumizyme MP as enzymolysis enzyme, merge ultrafiltration classification and chromatographic refining by biologic enzymolysis method, enzymolysis process is easily monitored simultaneously, and antioxidant collagen peptide is farthest discharged; The antioxidant collagen peptide of preparation is that large yellow croaker fish-bone collagen protein makes through enzymic hydrolysis, and safely, have no side effect, and antioxygenation is remarkable, can be used as medicine, protective foods and foodstuff additive.
Brief description of the drawings
Fig. 1 is the DPPH free radical scavenging activity figure of ultrafiltration component of the present invention;
Fig. 2 is anionite-exchange resin SP-sephadex C25 tomographic map of the present invention;
Fig. 3 is the DPPH free radical scavenging activity figure of anionite-exchange resin SP-sephadex C25 each component that chromatography separates of the present invention;
Fig. 4 is sephadex G-25 of the present invention tomographic maps;
Fig. 5 is the DPPH free radical scavenging activity figure of sephadex G-25 of the present invention each component that chromatography separates;
Fig. 6 is the RP-HPLC figure that zymolyte (F3-3-2) is prepared in sephadex G-25 of the present invention;
Fig. 7 is antioxidant collagen peptide Gly-Phe-Pro-Gly-Ser-Phe-Arg(SEQ ID No:1 of the present invention) RP-HPLC figure;
Fig. 8 is Gly-Phe-Pro-Gly-Ser-Phe-Arg(SEQ ID No:1 of the present invention) mass spectrum (ESI-MS) figure and structural formula.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
A preparation method for large yellow croaker fish-bone collagen peptide, preparation technology's flow process is as follows: large yellow croaker fish-bone " de-noncollagen protein, decalcification, degreasing " extracts collagen protein " two enzyme enzymolysis " zymolyte " ultrafiltration " ion exchange chromatography " gel permeation chromatography " high performance liquid chromatography preparation " collagen peptide.
Embodiment 1:
1) after large yellow croaker fish-bone minces, add 0.1 mol/L NaOH solution according to solid-liquid ratio 1 g:15 mL and at 4 DEG C, soak 3 ~ 5 h, remove noncollagen protein; Fish-bone distilled water repetitive scrubbing after processing, adds 0.5 mol/L EDTA solution (pH7.4) according to solid-liquid ratio 1 g:10 mL after fully draining, and soaks 4 days at 4 DEG C, changes EDTA solution every day 1 time, removes the calcium in fish-bone; After decalcification fish-bone drains, add 10% aqueous isopropanol according to solid-liquid ratio 1 g:15 mL, at 4 DEG C, soak 24 h, remove fat, dry, obtain degreasing fish-bone.
2) degreasing fish-bone added to 0.5 mol/L acetic acid solution and at 4 DEG C, soaks 4 days according to solid-liquid ratio 1 g:15 mL, in 4 DEG C, centrifugal 25 min of 20000 r/min, obtaining supernatant liquor and be thick collagen protein; Get appropriate thick collagen protein add NaCl to solution final concentration be 0.9 mol/L, leave standstill 3 h to separating out flocks, in 4 DEG C, 12000 r/min, centrifugal 20 min obtain throw out, lyophilize, is the collagen protein after saltouing.
3) collagen protein of preparation is joined in distilled water according to solid-liquid ratio 1 g:20 mL, adjust pH to 1.2, in 37 DEG C of insulation 10 min, add stomach en-(enzyme activity>=1.5 × 10 according to 1.0 % of collagen protein quality
5u/g), enzymolysis 4 h; Enzymolysis solution temperature is adjusted to 45 DEG C, and pH is adjusted to 9.5, and insulation 5 min, add Sumizyme MP (enzyme activity>=1.9 × 105 U/g), enzymolysis 5 h according to 1.5% of collagen protein amount;
4) enzymolysis solution is heated to 90 DEG C of deactivation 10 min, centrifugal 10 min of 10000 r/min, get supernatant liquor; Supernatant liquor through ultrafiltration and chromatography, obtains collagen peptide successively.
1. ultrafiltration: supernatant liquor is adjusted to pH 7.0, under the working temperature of the operating pressure of 0.15 MPa and 25 DEG C, adopt the ultra-filtration membrane of 5 kDa and 1 kDa to carry out uf processing, collect molecular weight and be greater than 5 kDa components, molecular weight is less than 5 kDa and is greater than 1 kDa component, molecular weight is less than 1 kDa component, wherein, the highest component of DPPH free radical scavenging activity be ultrafiltration enzymolysis solution (F3) (Fig. 1);
2. SP-sephadex C25 anion-exchange chromatography: above-mentioned ultrafiltration enzymolysis solution (F3) is used to NaH
2pO
4-Na
2hPO
4(0.2mol/L, pH 7.0) damping fluid is made into the solution of 20 mg/mL, through SP-sephadex C25 anion-exchange resin column chromatographic separation, carry out wash-out with distilled water, 0.5 mol/L and 1.0 mol/L NaCl solution, collect elution fraction according to the absorbancy curve under 280 nm, wherein, the highest component of DPPH free radical scavenging activity be ion exchange chromatography enzymolysis solution (F3-3) (Fig. 2);
3. gel chromatography: the solution that above-mentioned ion exchange chromatography enzymolysis solution (F3-3) is made into 15 mg/mL with distilled water, through sephadex G-25 column chromatography for separation, carry out wash-out with distilled water, collect elution fraction according to the absorbancy curve under 280 nm, wherein, the highest component of DPPH free radical scavenging activity be gel chromatography zymolyte (F3-3-2) (Fig. 3)
4. high performance liquid chromatography is refining: above-mentioned gel is prepared to zymolyte (F3-3-2) and is made into distilled water the solution of 40 μ g/mL, utilize RPLC (RP-HPLC) to carry out purifying (condition: sample size 15 μ L; Chromatographic column is Kromasil C18; 30 DEG C of column temperatures; Moving phase: A water (containing 0.1% trifluoroacetic acid) and B acetonitrile; Gradient elution: 0 ~ 40 min acetonitrile concentration from 0 to 50%; Elution speed 1.0 mL/min; Ultraviolet detection wavelength 280 nm(are shown in Fig. 4).
5. structure detection: collecting 1 the highest anti-oxidation peptide of DPPH free radical scavenging activity is simple spike (Fig. 5) after testing, utilizing protein/polypeptide sequenator to measure aminoacid sequence is Gly-Phe-Pro-Gly-Ser-Phe-Arg(SEQ ID No:1), ESI-MS detects and provides molecular weight is m/z 823.92 Da([M+H]
+824.77 Da) (Fig. 6).
By the above-mentioned large yellow croaker fish-bone collagen peptide Gly-Phe-Pro-Gly-Ser-Phe-Arg(SEQ ID No:1 making) carry out that experiment is removed in DPPH free radical scavenging experiment, hydroxyl radical free radical, ultra-oxygen anion free radical removes experiment and lipid peroxidation suppresses experiment.Experimental result shows: Gly-Phe-Pro-Gly-Ser-Phe-Arg(SEQ ID No:1) to DPPH free radical (EC
500.29 mg/mL), hydroxyl radical free radical (EC
500.36 mg/mL) and ultra-oxygen anion free radical (EC
500.51 mg/mL) there is good scavenging(action); Meanwhile, Gly-Phe-Pro-Gly-Ser-Phe-Arg(SEQ ID No:1) also demonstrate good Lipid peroxidation.
Finally, still should be noted, what more than enumerate is only a specific embodiment of the present invention.Obviously, the invention is not restricted to above embodiment, can also have many distortion.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
SEQUENCE LISTING
<110> Oceanography Institute Of Zhejiang
<120> large yellow croaker fish-bone collagen peptide and its production and use
<130> 2014
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 7
<212> PRT
<213> artificial sequence
<400> 1
Gly Phe Pro Gly Ser Phe Arg
1 5
Claims (7)
1. a large yellow croaker fish-bone collagen peptide, the aminoacid sequence that it is characterized in that this collagen peptide is Gly-Phe-Pro-Gly-Ser-Phe-Arg(SEQ ID No:1), molecular weight is m/z 823.92 Da.
2. a preparation method for large yellow croaker fish-bone collagen peptide claimed in claim 1, is characterized in that comprising the following steps:
1) after large yellow croaker fish-bone minces, add 0.1 mol/L NaOH solution according to solid-liquid ratio 1 g:15 ~ 25 mL and at 4 DEG C, soak 3 ~ 5 h, remove noncollagen protein; Fish-bone distilled water repetitive scrubbing after processing, adds 0.5mol/L EDTA solution (pH7.4) according to solid-liquid ratio 1 g:5 ~ 10 mL after fully draining, and soaks 3 ~ 5 days at 4 DEG C, changes EDTA solution every day 1 time, removes the calcium in fish-bone; After decalcification fish-bone drains, add 10% aqueous isopropanol according to solid-liquid ratio 1 g:15 ~ 20 mL, at 4 DEG C, soak 18 ~ 24 h, to remove fat, dry, obtain degreasing fish-bone;
2) degreasing fish-bone is added to 0.5mol/L acetic acid solution and at 4 DEG C, soaks 3 ~ 4 days according to solid-liquid ratio 1 g:10 ~ 15 mL;
In 4 DEG C, centrifugal 25 ~ 30 min of 20000 r/min, obtain supernatant liquor and be thick collagen protein; Get appropriate thick collagen protein add NaCl to solution final concentration be 0.8 ~ 1.0 mol/L, leave standstill 3 ~ 4 h to separating out flocks, in 4 DEG C, 12000 r/min, centrifugal 15 ~ 20 min obtain throw out, lyophilize, is the collagen protein after saltouing;
3) collagen protein of preparation is joined in distilled water according to solid-liquid ratio 1 g:15 ~ 20 mL, adjust pH to 1.2 ~ 2.0, in 35 ~ 40 DEG C of insulation 5 ~ 10 min, add the first proteolytic enzyme according to 1.0 ~ 1.5% of collagen protein quality, enzymolysis 4 ~ 6 h; Enzymolysis solution temperature is adjusted to 40 ~ 50 DEG C, and pH is adjusted to 9.0 ~ 10.0, and insulation 5 ~ 10 min, add the second proteolytic enzyme according to 1.0 ~ 1.5% of collagen protein amount, enzymolysis 3 ~ 5 h;
4) enzymolysis solution is heated to 90 ~ 95 DEG C of deactivation 10 ~ 15 min, centrifugal 10 ~ 15 min of 9000 ~ 10000 r/min, get supernatant liquor; Supernatant liquor through ultrafiltration and chromatography, obtains collagen peptide successively.
3. preparation method according to claim 2, is characterized in that the first proteolytic enzyme in described step 3) is stomach en-, enzyme activity>=1.5 × 10
5u/g.
4. preparation method according to claim 2, is characterized in that the second proteolytic enzyme in described step 3) is Sumizyme MP, enzyme activity>=1.5 × 10
5u/g.
5. preparation method according to claim 2, is characterized in that the ultrafiltration of described step 4) and the detailed process of chromatography are:
Ultrafiltration: supernatant liquor is adjusted to pH 6.5 ~ 7.5, under the working temperature of the operating pressure of 0.12 ~ 0.15 MPa and 25 ~ 30 DEG C, adopt the ultra-filtration membrane of 5 kDa and 1 kDa to carry out uf processing, collect molecular weight and be greater than 5 kDa components, molecular weight is less than 5 kDa and is greater than 1 kDa component, molecular weight is less than 1 kDa component, wherein, the highest component of DPPH free radical scavenging activity is ultrafiltration enzymolysis solution;
Chromatography: by above-mentioned ultrafiltration enzymolysis solution NaH
2pO
4-Na
2hPO
4(0.2mol/L, pH 7.0) damping fluid is made into the solution of 20 ~ 25 mg/mL, through anion-exchange resin column chromatographic separation, carry out wash-out with distilled water, 0.45 ~ 0.55 mol/L and 0.90 ~ 1.10 mol/L NaCl solution, collect elution fraction according to the absorbancy curve under 280 nm, wherein, the highest component of DPPH free radical scavenging activity is ion exchange chromatography enzymolysis solution, above-mentioned ion exchange chromatography enzymolysis solution is made into the solution of 10 ~ 15 mg/mL with distilled water, separate through gel filtration chromatography, carry out wash-out with distilled water, collect elution fraction according to the absorbancy curve under 280 nm, wherein, the highest component of DPPH free radical scavenging activity is gel chromatography zymolyte, above-mentioned gel chromatography zymolyte is made into the solution of 45 ~ 50 μ g/mL with distilled water, utilize RPLC (RP-HPLC) to carry out purifying, obtain 1 high reactivity anti-oxidation peptide Gly-Phe-Pro-Gly-Ser-Phe-Arg(SEQ ID No:1 according to DPPH free radical scavenging activity).
6. preparation method according to claim 5, is characterized in that described anionite-exchange resin is SP-sephadex C25, and described gel is sephadex G-25; Described RPLC condition is: sample size 10 ~ 15 μ L; Chromatographic column is Kromasil C18; Column temperature is 25 ~ 30 DEG C; Moving phase: A water (containing 0.1% trifluoroacetic acid) and B acetonitrile; Gradient elution: 0 ~ 40 min acetonitrile concentration from 0 to 50%; Elution speed 1.0 mL/min; Ultraviolet detection wavelength 280 nm.
7. an application for large yellow croaker fish-bone collagen peptide claimed in claim 1, is characterized in that Gly-Phe-Pro-Gly-Ser-Phe-Arg(SEQ ID No:1) DPPH free radical, hydroxyl radical free radical and ultra-oxygen anion free radical are had to good scavenging(action); Meanwhile, Gly-Phe-Pro-Gly-Ser-Phe-Arg(SEQ ID No:1) also demonstrate good Lipid peroxidation; Gly-Phe-Pro-Gly-Ser-Phe-Arg(SEQ ID No:1) there is anti-oxidant activity by force and safe without toxic side effect, can be used as medicine, protective foods and foodstuff additive.
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