CN104710525A - Tuna fishbone collagen sourced zinc chelated collagen peptide, preparation method and application thereof - Google Patents
Tuna fishbone collagen sourced zinc chelated collagen peptide, preparation method and application thereof Download PDFInfo
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 90
- 108010035532 Collagen Proteins 0.000 title claims abstract description 90
- 229920001436 collagen Polymers 0.000 title claims abstract description 74
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 53
- 239000011701 zinc Substances 0.000 title claims abstract description 51
- 229910052725 zinc Inorganic materials 0.000 title claims abstract description 35
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 238000004007 reversed phase HPLC Methods 0.000 claims abstract description 16
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 8
- 230000008569 process Effects 0.000 claims abstract description 8
- 238000004440 column chromatography Methods 0.000 claims abstract description 5
- 239000011347 resin Substances 0.000 claims abstract description 5
- 229920005989 resin Polymers 0.000 claims abstract description 5
- 241000251468 Actinopterygii Species 0.000 claims description 22
- 210000000988 bone and bone Anatomy 0.000 claims description 21
- 230000000694 effects Effects 0.000 claims description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 230000014759 maintenance of location Effects 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 13
- 239000012153 distilled water Substances 0.000 claims description 12
- 239000000047 product Substances 0.000 claims description 11
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 238000010828 elution Methods 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- 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 8
- 238000001641 gel filtration chromatography Methods 0.000 claims description 8
- 238000005227 gel permeation chromatography Methods 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 229920005654 Sephadex Polymers 0.000 claims description 5
- 239000012507 Sephadex™ Substances 0.000 claims description 5
- 238000012545 processing Methods 0.000 claims description 5
- 210000002784 stomach Anatomy 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- 235000018102 proteins Nutrition 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims description 4
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000002835 absorbance Methods 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 3
- 229910052791 calcium Inorganic materials 0.000 claims description 3
- 239000011575 calcium Substances 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 238000004587 chromatography analysis Methods 0.000 claims description 3
- 230000003203 everyday effect Effects 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 238000002203 pretreatment Methods 0.000 claims description 3
- 238000010298 pulverizing process Methods 0.000 claims description 3
- 230000003252 repetitive effect Effects 0.000 claims description 3
- 238000005201 scrubbing Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- 238000000825 ultraviolet detection Methods 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 2
- 230000009469 supplementation Effects 0.000 claims description 2
- 150000003751 zinc Chemical class 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 claims 1
- 238000001976 enzyme digestion Methods 0.000 claims 1
- 229920001184 polypeptide Polymers 0.000 abstract description 4
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 4
- 102000004142 Trypsin Human genes 0.000 abstract description 3
- 108090000631 Trypsin Proteins 0.000 abstract description 3
- 238000010521 absorption reaction Methods 0.000 abstract description 3
- 150000001413 amino acids Chemical class 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 2
- 102000057297 Pepsin A Human genes 0.000 abstract 1
- 108090000284 Pepsin A Proteins 0.000 abstract 1
- 239000002253 acid Substances 0.000 abstract 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 abstract 1
- 229940111202 pepsin Drugs 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 230000001502 supplementing effect Effects 0.000 abstract 1
- 230000007723 transport mechanism Effects 0.000 abstract 1
- 239000012588 trypsin Substances 0.000 abstract 1
- 235000013305 food Nutrition 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- FXGRXIATVXUAHO-WEDXCCLWSA-N Gly-Lys-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCCN FXGRXIATVXUAHO-WEDXCCLWSA-N 0.000 description 1
- PASHZZBXZYEXFE-LSDHHAIUSA-N Gly-Trp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CNC3=CC=CC=C32)NC(=O)CN)C(=O)O PASHZZBXZYEXFE-LSDHHAIUSA-N 0.000 description 1
- WHMDKBIGKVEYHS-IYEMJOQQSA-L Zinc gluconate Chemical compound [Zn+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O WHMDKBIGKVEYHS-IYEMJOQQSA-L 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000003277 amino acid sequence analysis Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 229910052804 chromium Inorganic materials 0.000 description 1
- 239000011651 chromium Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- WGIWBXUNRXCYRA-UHFFFAOYSA-H trizinc;2-hydroxypropane-1,2,3-tricarboxylate Chemical compound [Zn+2].[Zn+2].[Zn+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O WGIWBXUNRXCYRA-UHFFFAOYSA-H 0.000 description 1
- 239000011746 zinc citrate Substances 0.000 description 1
- 235000006076 zinc citrate Nutrition 0.000 description 1
- 229940068475 zinc citrate Drugs 0.000 description 1
- 239000011670 zinc gluconate Substances 0.000 description 1
- 229960000306 zinc gluconate Drugs 0.000 description 1
- 235000011478 zinc gluconate Nutrition 0.000 description 1
- 229940091251 zinc supplement Drugs 0.000 description 1
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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a preparation method for a zinc chelated collagen peptide of tuna fishbone collagen. The preparation method specifically comprises the following steps: using tuna fishbone as a raw material; adopting an acid process to extract collagen; performing enzymolysis on the collagens with pepsin and trypsin; adopting ultrafiltration, macroporous resin column chromatography, gel column chromatography and reversed-phase high-performance liquid chromatography to separate and purify the collagens to obtain the zinc chelated collagen peptide Gly-Lys-Thr-Gly-Trp-Pro-Gly (GKTGWPG), wherein the molecular weight of the zinc chelated collagen peptide detected by ESI-MS is 701.73 Da. The zinc chelated collagen peptide is high in absorption and utilization due to the unique chelating and transport mechanism, and meanwhile capable of supplementing polypeptides/amino acid and zinc, so that the zinc chelated collagen peptide is an ideal zinc-supplementing substance.
Description
Technical field
The present invention relates to a kind of tuna fish bone collagen protein source zinc chelating collagen peptide and its production and use, belong to fishery products intensive processing technical field.
Background technology
Collagen peptide with its unique effects in the application such as medicine, healthcare products, foods and cosmetics favor by increasing human consumer, become the focus that food service industry and health products trade, daily use chemicals industry are competitively paid close attention to.Zinc is the necessary trace element of human body, and its shortage can cause the illnesss such as upgrowth and development of children is bad, intelligent growth backwardness, immunizing power reduction.Traditional Zinc supplements (as Zinc Gluconate, zinc citrate) poor stability, absorption rate is low, and stomach stimulates large, not easily long-term taking.And the chelating salt that zinc and metal chelating peptide are formed, the absorption of trace element can be promoted, the demand of body to bioactive peptide, amino acid etc. can be met again simultaneously, obtain the extensive concern of Chinese scholars.
Tuna is the important fish of sea going fisheries, and year, quantity of the catch was more than 6,000,000 tons.In the tuna course of processing, account for gross weight 25% 30% fish-bone majority and be processed into fishbone powder or lose as waste, the fishing resources of the preciousness of waste.Through retrieval, prepare zinc chelating collagen peptide with tuna fish bone collagen albumen and have no report.Based on this, the present invention, according to the present Research of zinc chelating collagen peptide and tuna fish-bone, provides a kind of collagen peptide with metallic zinc sequestering activity, and provides the preparation method of this zinc chelating collagen peptide.
Summary of the invention
First technical problem to be solved by this invention provides a kind of zinc chelating collagen peptide coming from tuna fish bone collagen albumen for the above-mentioned state of the art.
Second technical problem to be solved by this invention is to provide a kind of preparation method coming from the zinc chelating collagen peptide of tuna fish bone collagen albumen.
The technical scheme that the present invention takes for above-mentioned first technical problem of solution is: a kind of zinc chelating collagen peptide coming from tuna fish bone collagen albumen, it is characterized in that the aminoacid sequence of this collagen peptide is Gly-Lys-Thr-Gly-Trp-Pro-Gly(GKTGWPG), ESI/MS detection molecules amount is 701.73 Da.
The technical scheme that the present invention takes for above-mentioned second technical problem of solution is: a kind of preparation method coming from the zinc chelating collagen peptide of tuna fish bone collagen albumen, is characterized in that comprising the following steps:
1)
the pre-treatment of tuna fish-bone:in tuna fish-bone, add 0.1 mol/L NaOH solution according to solid-liquid ratio 1 g:10 ~ 15 mL and soak 3 ~ 5 h at 4 DEG C, remove noncollagen protein; Process after fish-bone distilled water repetitive scrubbing to pH 6.5 ~ 7.0, drain, add 0.5 mol/L EDTA solution (pH 7.4) according to solid-liquid ratio 1 g:5 ~ 10 mL, at 4 DEG C soak 3 ~ 5 days, change 1 EDTA solution every day, remove the calcium in fish-bone, dry, pulverizing, obtains decalcification fishbone powder.
2)
the preparation of tuna fish bone collagen albumen:according to solid-liquid ratio 1 g:3 ~ 5 mL to decalcification fishbone powder add 0.5 mol/L acetic acid solution and at 4 DEG C soak 2 ~ 3 d, supernatant liquor is got after centrifugal 30 min of 12 000 g, adding NaCl to solution final concentration is 0.8 ~ 1.0 mol/L, leave standstill 30 ~ 60 min, 15 000 g are centrifugal, and 25 ~ 30 min obtain throw out, and lyophilize is fish bone collagen albumen.
) enzymolysis of tuna fish bone collagen albumen:in collagen protein, add ultrapure water according to solid-liquid ratio 1g:15 ~ 20 mL, adjust pH value of solution to 1.5 ~ 2.5 with HCl, in collagen solution, add stomach en-(enzyme activity>=1.6 × 10 according to collagen protein quality 1.5 ~ 2.0%
5u/g), after enzymolysis 3 ~ 5 h, by solution warms to 90 DEG C ~ 95 DEG C, and after this temperature keeps 10 ~ 15min, be cooled to room temperature; Adjust pH value of solution to 7.5 ~ 8.5 with NaOH, in solution, add trypsin enzyme activity>=2.5 × 10 according to collagen protein quality 1.2 ~ 1.5%
4u/g), at temperature 35 ~ 45 DEG C, enzymolysis 3 ~ 4 h, obtains enzymolysis product;
4) preparation of tuna fish-bone zinc chelating collagen peptide:3 kDa ultra-filtration membranes are adopted to carry out uf processing the collagen protein enzymolysis product of preparation, collect molecular weight and be less than 3 kDa parts, obtain ultrafiltration enzymolysis solution, ultrafiltration enzymolysis solution joins in the chromatography column that 10 ~ 15 times of D101 macroporous resins are housed according to volume ratio, with 3 ~ 5 times of column volume water elution removing impurity, then wash-out is carried out with 95% ethanol of 5 ~ 8 times of column volumes, ethanol eluate revolves in less than 50 DEG C low pressure and steams removing ethanol, lyophilize obtains collagen peptide mixture, collagen peptide mixture is successively through gel filtration chromatography and RPLC (RP-HPLC) purifying, obtain tuna fish-bone zinc chelating collagen peptide.
As preferably, the gel filtration chromatography of described step 4) and the detailed process of RP-HPLC purifying are:
Gel filtration chromatography: above-mentioned collagen peptide mixture is dissolved in distilled water and is made into the solution that concentration is 10 ~ 20 mg/mL, through sephadex G-25 column chromatography for separation, wash-out is carried out with distilled water, elution fraction is collected according to the absorbance curve under 214 nm, wherein, the peak with the highest Zn sequestering activity is gel chromatography zymolyte;
RP-HPLC purifying: the solution above-mentioned gel chromatography zymolyte distilled water being made into 45 ~ 55 μ g/mL, RP-HPLC is utilized to carry out purifying, according to the sequestering activity of Zn being obtained to 1 high Zn sequestering activity collagen peptide Gly-Lys-Thr-Gly-Trp-Pro-Gly(GKTGWPG).
Preferred again, described RP-HPLC condition is: sample size 15 ~ 20 μ L; Chromatographic column is Zorbax C18; Moving phase: 30% acetonitrile; Elution speed 0.8 ~ 1.0 mL/min; Ultraviolet detection wavelength 214 nm.
The present invention is based on the theoretical basis of polypeptide and metal ion-chelant, and the unique effects of polypeptide-Zn inner complex (simultaneously can supplement polypeptide/amino acid and Zn), with tuna fish-bone for starting material, by controlling stomach en-and tryptic enzymatic hydrolysis condition, preparation has the collagen peptide of high Zn sequestering activity.The present invention is zinc supplementation protective foods and drug development provides a kind of technical support, simultaneously also for the higher value application of tuna fish-bone provides new approaches.
Accompanying drawing explanation
Fig. 1 is sephadex G-25 tomographic map of the present invention.
Fig. 2 is the RP-HPLC analysis chart that sephadex G-25 of the present invention prepares zymolyte.
Fig. 3 is Gly-Lys-Thr-Gly-Trp-Pro-Gly(GKTGWPG of the present invention) mass spectrum.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Come from a preparation method for the zinc chelating collagen peptide of tuna fish bone collagen albumen, preparation technology's flow process is as follows: tuna fish-bone " collagen protein extraction " enzymolysis " zymolyte " ultrafiltration " macroporous resin purification " gel permeation chromatography " RP-HPLC preparation " zinc chelating collagen peptide.
Embodiment:
1)
the pre-treatment of tuna fish-bone:in tuna (stripped tuna) fish-bone, add 0.1 mol/L NaOH solution according to solid-liquid ratio 1 g:15 mL and soak 5 h at 4 DEG C, remove noncollagen protein; Process after fish-bone distilled water repetitive scrubbing to pH 7.0, drain, add 0.5 mol/L EDTA solution (pH 7.4) according to solid-liquid ratio 1 g:5 mL, at 4 DEG C soak 3 days, change 1 EDTA solution every day, remove the calcium in fish-bone, dry, pulverizing, obtains decalcification fishbone powder.
2)
the preparation of tuna fish bone collagen albumen:add 0.5 mol/L acetic acid solution according to solid-liquid ratio 1 g:3 mL to decalcification fishbone powder and soak 3 d at 4 DEG C, supernatant liquor is got after centrifugal 30 min of 12 000 g, adding NaCl to solution final concentration is 0.9 mol/L, leave standstill 60 min, 15 000 g are centrifugal, and 30 min obtain throw out, and lyophilize is fish bone collagen albumen.
) enzymolysis of tuna fish bone collagen albumen:in collagen protein, add ultrapure water according to solid-liquid ratio 1g:15 mL, adjust pH value of solution to 2.0 with HCl, in collagen solution, add stomach en-(enzyme activity>=1.6 × 10 according to collagen protein quality 1.5%
5u/g), after enzymolysis 3 ~ 5 h, by solution warms to 90 DEG C ~ 95 DEG C, and after this temperature keeps 10 min, be cooled to room temperature; Adjust pH value of solution to 8.0 with NaOH, in solution, add trypsin enzyme activity>=2.5 × 10 according to collagen protein quality 1.5%
4u/g), at temperature 37 DEG C, enzymolysis 4 h, obtains enzymolysis product;
4) preparation of tuna fish-bone zinc chelating collagen peptide:3 kDa ultra-filtration membranes are adopted to carry out uf processing the collagen protein enzymolysis product of preparation, collect molecular weight and be less than 3 kDa parts, obtain ultrafiltration enzymolysis solution, ultrafiltration enzymolysis solution joins in the chromatography column that 15 times of D101 macroporous resins are housed according to volume ratio, with 3 ~ 5 times of column volume water elution removing impurity, then wash-out is carried out with 95% ethanol of 6 times of column volumes, ethanol eluate revolves in less than 50 DEG C low pressure and steams removing ethanol, lyophilize obtains collagen peptide mixture, collagen peptide mixture is successively through gel filtration chromatography and RPLC (RP-HPLC) purifying, obtain tuna fish-bone zinc chelating collagen peptide.Utilize amino acid sequence analysis and its structure of mass spectroscopy, detailed process is:
1. gel filtration chromatography: above-mentioned collagen peptide mixture is dissolved in distilled water and is made into the solution that concentration is 10 ~ 20 mg/mL, through sephadex G-25 column chromatography for separation, wash-out is carried out with distilled water, elution fraction is collected according to the absorbance curve under 214 nm, wherein, the peak with the highest Zn sequestering activity is gel chromatography zymolyte (F3) (Fig. 1);
2. RP-HPLC purifying: the solution above-mentioned gel chromatography zymolyte distilled water being made into 45 ~ 55 μ g/mL, (described RP-HPLC condition is: sample size 15 ~ 20 μ L to utilize RP-HPLC to carry out purifying; Chromatographic column is Zorbax C18; Moving phase: 30% acetonitrile; Elution speed 0.8 mL/min; Ultraviolet detection wavelength 214 nm), according to the sequestering activity of Zn being obtained to 1 high Zn sequestering activity collagen peptide (Fig. 2).
3. structure detection: Zn chelating collagen peptide is simple spike after testing, utilizing protein/polypeptide sequenator to measure aminoacid sequence is Gly-Lys-Thr-Gly-Trp-Pro-Gly(GKTGWPG), ESI/MS detection molecules amount is 701.73 Da(Fig. 3).
Zn chelating collagen peptide adopts EDTA titration measuring to the sequestering action of zine ion.Get inner complex 100 mg in 100 mL small beakers, add water 50 mL, instillation HCl(6 mol/L) several.After shaking up, in water-bath, heating makes it to dissolve completely, is settled to l00 mL after cooling, therefrom draws l0 mL in triangular flask, parallel 3 parts, adds the NH of pH 10
3-NH
4cl damping fluid l0 mL, chromium black T indicator is appropriate, then uses 0.01 mol/L Na
2eDTA liquid is titrated to blueness; Record consumes the milliliter number of EDTA, calculates inner complex zinc content.
Measurement result shows: the Zn chelating collagen peptide Gly-Lys-Thr-Gly-Trp-Pro-Gly(GKTGWPG that purifying obtains) be 83.56 μ g/mg to the sequestering power of zine ion, it is greatly improved to the sequestering power of Zn compared with collagen protein enzymolysis product (30.12 μ g/mg).
Finally, still need it is noted that what enumerate above is only a specific embodiment of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had.All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
SEQUENCE LISTING
<110> Oceanography Institute Of Zhejiang
<120> tuna fish bone collagen protein source zinc chelating collagen peptide and its production and use
<130> zjou-wb-201507
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 7
<212> PRT
<213> synthetic
<400> 1
Gly Lys Thr Gly Trp Pro Gly
1 5
Claims (5)
1. come from a zinc chelating collagen peptide for tuna fish bone collagen albumen, it is characterized in that the aminoacid sequence of this zinc chelating collagen peptide be Gly-Lys-Thr-Gly-Trp-Pro-Gly, ESI/MS detection molecules amount is 701.73 Da.
2. a kind of preparation method coming from the zinc chelating collagen peptide of tuna fish bone collagen albumen according to claim 1, is characterized in that comprising the following steps:
1)
the pre-treatment of tuna fish-bone:in tuna fish-bone, add 0.1 mol/L NaOH solution according to solid-liquid ratio 1 g:10 ~ 15 mL and soak 3 ~ 5 h at 4 DEG C, remove noncollagen protein; Process after fish-bone distilled water repetitive scrubbing to pH 6.5 ~ 7.0, drain, add according to solid-liquid ratio 1 g:5 ~ 10 mL the EDTA solution that 0.5 mol/L, pH is 7.4, at 4 DEG C soak 3 ~ 5 days; change 1 EDTA solution every day; remove the calcium in fish-bone, dry, pulverizing, obtains decalcification fishbone powder;
2)
the preparation of tuna fish bone collagen albumen:according to solid-liquid ratio 1 g:3 ~ 5 mL to decalcification fishbone powder add 0.5 mol/L acetic acid solution and at 4 DEG C soak 2 ~ 3 d; supernatant liquor is got after centrifugal 30 min of 12 000 g; adding NaCl to solution final concentration is 0.8 ~ 1.0 mol/L; leave standstill 30 ~ 60 min; 15 000 g are centrifugal, and 25 ~ 30 min obtain throw out, and lyophilize is fish bone collagen albumen;
3) enzymolysis of tuna fish bone collagen albumen:in collagen protein, add ultrapure water according to solid-liquid ratio 1g:15 ~ 20 mL, adjust pH value of solution to 1.5 ~ 2.5 with HCl, in collagen solution, add stomach en-(enzyme activity>=1.6 × 10 according to collagen protein quality 1.5 ~ 2.0%
5u/g), after enzyme digestion reaction 3 ~ 5 h, by solution warms to 90 DEG C ~ 95 DEG C, and after this temperature keeps 10 ~ 15min, be cooled to room temperature; Adjust pH value of solution to 7.5 ~ 8.5 with NaOH, in solution, add trypsinase according to collagen protein quality 1.2 ~ 1.5%, at temperature 35 ~ 45 DEG C, enzymolysis 3 ~ 4 h, obtains enzymolysis product; Described tryptic enzyme activity>=2.5 × 10
4u/g;
4) preparation of tuna fish-bone zinc chelating collagen peptide:3 kDa ultra-filtration membranes are adopted to carry out uf processing the collagen protein enzymolysis product of preparation, collect molecular weight and be less than 3 kDa parts, obtain ultrafiltration enzymolysis solution, ultrafiltration enzymolysis solution joins in the chromatography column that 10 ~ 15 times of D101 macroporous resins are housed according to volume ratio, with 3 ~ 5 times of column volume water elution removing impurity, then wash-out is carried out with 95% ethanol of 5 ~ 8 times of column volumes, ethanol eluate revolves in less than 50 DEG C low pressure and steams removing ethanol, lyophilize obtains collagen peptide mixture, collagen peptide mixture is successively through gel filtration chromatography and RPLC (RP-HPLC) purifying, obtain tuna fish-bone zinc chelating collagen peptide.
3. preparation method according to claim 2, is characterized in that the gel filtration chromatography of described step 4) and the detailed process of RPLC purifying are:
Gel filtration chromatography: above-mentioned collagen peptide mixture is dissolved in distilled water and is made into the solution that concentration is 10 ~ 20 mg/mL, through sephadex G-25 column chromatography for separation, wash-out is carried out with distilled water, elution fraction is collected according to the absorbance curve under 214 nm, wherein, the peak with the highest Zn sequestering activity is gel chromatography zymolyte;
RP-HPLC purifying: the solution above-mentioned gel chromatography zymolyte distilled water being made into 45 ~ 55 μ g/mL, utilizes RP-HPLC to carry out purifying, according to the sequestering activity of Zn being obtained to 1 high Zn sequestering activity collagen peptide Gly-Lys-Thr-Gly-Trp-Pro-Gly.
4. preparation method according to claim 3, is characterized in that described RP-HPLC condition is: sample size 15 ~ 20 μ L; Chromatographic column is Zorbax C18; Moving phase: 30% acetonitrile; Elution speed 0.8 ~ 1.0 mL/min; Ultraviolet detection wavelength 214 nm.
5. a kind of zinc chelating collagen peptide coming from tuna fish bone collagen albumen according to claim 1 is for the preparation of the purposes of zinc supplementation medicine and healthcare products.
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