CN104558115A - Antioxidant polypeptide with Raja porosa meat protein as well as preparation method and application of antioxidant polypeptide - Google Patents
Antioxidant polypeptide with Raja porosa meat protein as well as preparation method and application of antioxidant polypeptide Download PDFInfo
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Abstract
The invention discloses antioxidant polypeptide with Raja porosa meat protein as well as a preparation method and application of antioxidant polypeptide. The amino acid sequence of the antioxidant polypeptide is Asn-Trp-Asp-Met-Glu-Lys-Ile-Trp-Asp, and ESI/MS detection molecular weight is 1236.38 Da. The preparation method of the antioxidant polypeptide comprises the following steps: tissue mincing, enzymolysis, ultrafiltration, chromatography and the like. The high activity polypeptide Asn-Trp-Asp-Met-Glu-Lys-Ile-Trp-Asp prepared by the preparation method can effectively eliminate hydroxyl radicals, DPPH free radicals and superoxide anion free radicals, and can be used as drugs or health-care foods relevant to oxidation resistance.
Description
Technical field
The present invention relates to a kind of hole ray meat protein antioxidant polypeptide, the invention still further relates to the preparation method of this hole ray meat protein antioxidant polypeptide.
Background technology
Hole ray (
raja porosa) be the fish that Rajidae ray belongs to, be commonly called as labor sole, Lao Zi, Pu fish etc., be distributed in North Western Pacific, it is all produced in East China Sea, the South Sea and the Huanghai Sea, the Bohai Sea.Hole ray meat thorniness is few, and without os osseum, its meat can be eaten raw, but is more pickle to be processed into unsalted dried fish.Hole ray is dry is that the coastal resident of the province such as Liaoning, Shandong is accustomed to and the fishery products of eating.
At present, applicant studies discovery, and with hole ray meat for raw material, the technical study utilizing zymolysis technique to prepare antioxidation polypeptide is still in the blank stage, and is that high reactivity antioxidation polypeptide prepared by material and application has no report especially with enzymolysis product.
Summary of the invention
First technical problem to be solved by this invention provides a kind of hole ray meat protein antioxidant polypeptide for the above-mentioned state of the art, this polypeptide effectively can remove hydroxyl radical free radical, 1,1-phenylbenzene-2-trinitrophenyl-hydrazine (DPPH) free radical and ultra-oxygen anion free radical.
Second technical problem to be solved by this invention is to provide the preparation method of a kind of hole ray meat protein antioxidant polypeptide.
3rd technical problem to be solved by this invention is to provide a kind of hole ray meat protein antioxidant polypeptide for the preparation of the purposes in anti-oxidation medicine or protective foods.
The technical scheme that the present invention takes for above-mentioned first technical problem of solution is: a kind of hole ray meat protein antioxidant polypeptide, it is characterized in that the aminoacid sequence of this polypeptide is Asn-Trp-Asp-Met-Glu-Lys-Ile-Trp-Asp(NWDMEKIWD), ESI/MS detection molecules amount is 1236.38 Da.
The technical scheme that the present invention takes for above-mentioned second technical problem of solution is: the preparation method of a kind of hole ray meat protein antioxidant polypeptide, is characterized in that comprising the following steps:
(1) ray meat in hole rubs, and adds Virahol, after 35 ~ 40 DEG C of skimming treatment 1 ~ 2 h, in centrifugal 8 ~ 10 min of 9 000 r/min, get precipitation by solid-to-liquid ratio 1 g:4 ~ 6 mL, dry, obtains degreasing hole ray meat;
(2) degreasing hole ray meat is added phosphate buffered saline buffer (0.05 mol/L by solid-to-liquid ratio 1 g:4 ~ 6 mL, pH 7.0), neutral protease (enzyme activity >=1.5 AU/g) is added, enzymolysis 3 ~ 5 h at 60 ~ 70 DEG C of temperature according to 1.8 ~ 2.5% of degreasing hole ray meat quality;
(3) by enzymolysis solution with 10 000 r/min low-temperature centrifugation 20 ~ 25 min at 3 ~ 5 DEG C, remove precipitation, get supernatant liquor; Supernatant liquor through ultrafiltration and chromatography, obtains antioxidation polypeptide successively.
As improvement, the ultrafiltration of described step (3) and the detailed process of chromatography are:
Ultrafiltration: the ultra-filtration membrane of supernatant liquor molecular weight cut-off 1 kDa is carried out uf processing, according to the clearance rate to hydroxyl radical free radical, collects molecular weight and is less than 1 kDa part, obtain ultrafiltration zymolyte;
Chromatography: the solution above-mentioned ultrafiltration zymolyte distilled water being made into 25 ~ 30 mg/mL, through Anion exchange resin separation, wash-out is carried out by distilled water, 0.08 ~ 0.12 mol/L, 0.45 ~ 0.55 mol/L and 0.90 ~ 1.10 mol/L NaCl solution, elution fraction is collected according to the absorbance curve under 280 nm, wherein, be ion exchange chromatography zymolyte to the highest component of hydroxyl radical free radical clearance rate, above-mentioned ion exchange chromatography zymolyte distilled water is made into the solution of 20 ~ 25 mg/mL, be separated through gel filtration chromatography, wash-out is carried out with distilled water, elution fraction is collected according to the absorbance curve under 280 nm, wherein, be gel chromatography zymolyte to the highest component of hydroxyl radical free radical clearance rate, above-mentioned gel chromatography zymolyte distilled water is made into the solution of 90 ~ 100 μ g/mL, RPLC (RP-HPLC) is utilized to carry out purifying, according to the clearance rate of hydroxyl radical free radical being obtained to 1 high anti-oxidation active polypeptide Asn-Trp-Asp-Met-Glu-Lys-Ile-Trp-Asp(NWDMEKIWD).
Preferably, described anionite-exchange resin is Mierocrystalline cellulose DE-52, and described gel is sephadex G-15.
Preferred again, described RP-HPLC condition is: sample size 20 ~ 25 μ L; Chromatographic column is Zorbax SB C-18(4.6 × 250 mm); Moving phase: 30% acetonitrile; Elution speed 1.0 mL/min; Ultraviolet detection wavelength 280 nm.
The present invention for above-mentioned 3rd technical scheme that technical problem is taked of solution is: the application of a kind of hole ray meat protein antioxidant polypeptide, is characterized in that Asn-Trp-Asp-Met-Glu-Lys-Ile-Trp-Asp(NWDMEKIWD) can effectively remove hydroxyl radical free radical (EC
500.176 mg/mL), DPPH free radical (EC
500.289 mg/mL) and ultra-oxygen anion free radical (EC
500.132 mg/mL), can be used for medicine or the protective foods of anti-oxidant aspect.
Compared with prior art, the invention has the advantages that: the present invention is by biologic enzymolysis method, membrane ultrafiltration classification and chromatographic refining effective integration, and obtained polypeptide has significant free radical scavenging activity; Compared with the antioxidant of chemosynthesis, antioxidation polypeptide that the present invention obtains safety, efficient, have no side effect, can be used as medicine or the protective foods of anti-oxidant aspect.
Accompanying drawing explanation
Fig. 1 is anionite-exchange resin (Mierocrystalline cellulose DEAE-52) tomographic map of hole ray fish protein ultrafiltration zymolyte (SMH-III) of the present invention.
Fig. 2 is sephadex G-15 tomographic map of the anion-exchange chromatography separated portion (Fr.5) of hole of the present invention ray meat protein zymolyte.
Fig. 3 is the RP-HPLC analysis chart that the sephadex G-15 of hole of the present invention ray meat protein zymolyte prepares zymolyte (Fr.5-3).
Fig. 4 is mass spectrum (ESI-MS) figure and the structural formula of Asn-Trp-Asp-Met-Glu-Lys-Ile-Trp-Asp of the present invention.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
A preparation method for hole ray meat protein antioxidant polypeptide, preparation technology's flow process is as follows: hole ray meat " albumen degreasing " zymolyte " ultrafiltration " ion exchange chromatography " gel permeation chromatography " high performance liquid chromatography preparation " antioxidation polypeptide.
Embodiment:
(1) ray meat in hole rubs, and adds Virahol, after 35 DEG C of skimming treatment 2 h, in centrifugal 8 min of 9 000 r/min, get precipitation by solid-to-liquid ratio 1 g:5 mL, dry, obtains degreasing hole ray meat;
(2) degreasing hole ray meat is added phosphate buffered saline buffer (0.05 mol/L, pH 7.0) by solid-to-liquid ratio 1 g:6 mL, add neutral protease (enzyme activity >=1.5 AU/g) according to 2% of degreasing hole ray meat quality, enzymolysis 5 h at 60 DEG C of temperature;
(3) by enzymolysis solution with 10 000 r/min low-temperature centrifugation 20 min at 4 DEG C, remove precipitation, get supernatant liquor; Supernatant liquor through ultrafiltration and chromatography, obtains antioxidation polypeptide successively, and utilize amino acid sequence analysis and its structure of mass spectroscopy, detailed process is:
1. ultrafiltration: the ultra-filtration membrane of supernatant liquor molecular weight cut-off 1 kDa is carried out uf processing, according to the clearance rate to hydroxyl radical free radical, collects molecular weight and is less than 1 kDa part, obtain ultrafiltration zymolyte;
2. anion-exchange chromatography: the solution above-mentioned ultrafiltration zymolyte distilled water being made into 25 mg/mL, be separated through anionite-exchange resin Mierocrystalline cellulose DE-52, wash-out is carried out by distilled water, 0.10 mol/L, 0.50 mol/L and 1.0 mol/L NaCl solution, elution fraction is collected according to the absorbance curve under 280 nm, wherein, be ion exchange chromatography zymolyte (Fig. 1) to the highest component of hydroxyl radical free radical clearance rate.
3. gel permeation chromatography: the solution above-mentioned ion exchange chromatography zymolyte distilled water being made into 20 mg/mL, through sephadex G-15 column chromatography for separation, wash-out is carried out with distilled water, elution fraction is collected according to the absorbance curve under 280 nm, wherein, be gel chromatography zymolyte (Fig. 2) to the highest component of hydroxyl radical free radical clearance rate.
4. high performance liquid chromatography is refined: the solution above-mentioned gel chromatography zymolyte distilled water being made into 100 μ g/mL, RPLC (RP-HPLC) is utilized to carry out purifying, according to the clearance rate of hydroxyl radical free radical being obtained to 1 high anti-oxidation active polypeptide (Fig. 3).
5. structure detection: collecting active 1 the highest antioxidation polypeptide is simple spike after testing, utilizing protein/polypeptide sequenator to measure aminoacid sequence is Asn-Trp-Asp-Met-Glu-Lys-Ile-Trp-Asp(NWDMEKIWD), ESI/MS detection molecules amount is 1236.38 Da(Fig. 4).
By above-mentioned obtained hole ray meat protein antioxidant polypeptide A sn-Trp-Asp-Met-Glu-Lys-Ile-Trp-Asp(NWDMEKIWD) carry out free radical experiment.Experimental result shows: this polypeptide effectively can remove hydroxyl radical free radical, DPPH free radical and ultra-oxygen anion free radical, half elimination ratio (EC
50) be respectively 0.176 mg/mL, 0.289 mg/mL and 0.132 mg/mL.
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 and belong to protection scope of the present invention.
SEQUENCE LISTING
<110> Oceanography Institute Of Zhejiang
<120> hole ray meat protein antioxidant polypeptide and its production and use
<130> 2014
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 9
<212> PRT
<213> synthetic
<400> 1
Asn Trp Asp Met Glu Lys Ile Trp Asp
1 5
Claims (6)
1. a hole ray meat protein antioxidant polypeptide, the aminoacid sequence that it is characterized in that this hole ray meat protein antioxidant polypeptide is Asn-Trp-Asp-Met-Glu-Lys-Ile-Trp-Asp, ESI/MS detection molecules amount is 1236.38 Da.
2. a preparation method for a kind of hole according to claim 1 ray meat protein antioxidant polypeptide, is characterized in that comprising the following steps:
Pre-treatment step: hole ray meat rubs, and adds Virahol, after 35 ~ 40 DEG C of skimming treatment 1 ~ 2 h, in centrifugal 8 ~ 10 min of 9 000 r/min, get precipitation by solid-to-liquid ratio 1 g:4 ~ 6 mL, dry, obtain degreasing hole ray meat;
Ferment treatment step: degreasing hole ray meat is added phosphate buffered saline buffer by solid-to-liquid ratio 1 g:4 ~ 6 mL, adds neutral protease according to 1.8 ~ 2.5% of degreasing hole ray meat quality, enzymolysis 3 ~ 5 h at 60 ~ 70 DEG C of temperature;
Purification step: by enzymolysis solution with 10 000 r/min low-temperature centrifugation 20 ~ 25 min at 3 ~ 5 DEG C, removes precipitation, gets supernatant liquor; Supernatant liquor through ultrafiltration and chromatography, obtains antioxidation polypeptide successively;
In ferment treatment step, described neutral protease enzyme activity >=1.5 AU/g;
In ferment treatment step, described phosphate buffering liquid concentration is 0.05 mol/L, pH is 7.0.
3. preparation method according to claim 2, is characterized in that the ultrafiltration of described purification step and the detailed process of chromatography are:
Ultrafiltration: the ultra-filtration membrane of supernatant liquor molecular weight cut-off 1 kDa is carried out uf processing, according to the clearance rate to hydroxyl radical free radical, collects molecular weight and is less than 1 kDa part, obtain ultrafiltration zymolyte;
Chromatography: the solution above-mentioned ultrafiltration zymolyte distilled water being made into 25 ~ 30 mg/mL, through Anion exchange resin separation, wash-out is carried out by distilled water, 0.08 ~ 0.12 mol/L, 0.45 ~ 0.55 mol/L and 0.90 ~ 1.10 mol/L NaCl solution, elution fraction is collected according to the absorbance curve under 280 nm, wherein, be ion exchange chromatography zymolyte to the highest component of hydroxyl radical free radical clearance rate; Above-mentioned ion exchange chromatography zymolyte distilled water is made into the solution of 20 ~ 25 mg/mL, be separated through gel filtration chromatography, wash-out is carried out with distilled water, elution fraction is collected according to the absorbance curve under 280 nm, wherein, be gel chromatography zymolyte to the highest component of hydroxyl radical free radical clearance rate, above-mentioned gel chromatography zymolyte distilled water is made into the solution of 90 ~ 100 μ g/mL, RPLC is utilized to carry out purifying, according to the clearance rate of hydroxyl radical free radical being obtained to 1 high anti-oxidation active polypeptide Asn-Trp-Asp-Met-Glu-Lys-Ile-Trp-Asp.
4. preparation method according to claim 3, it is characterized in that described anionite-exchange resin is Mierocrystalline cellulose DE-52, described gel is sephadex G-15; Described RP-HPLC condition is: sample size 20 ~ 25 μ L; Chromatographic column is Zorbax SB C-18,4.6 × 250 mm; Moving phase: 30% acetonitrile; Elution speed 1.0 mL/min; Ultraviolet detection wavelength 280 nm.
5. the preparation method of a kind of hole according to claim 2 ray meat protein antioxidant polypeptide, is characterized in that prepared antioxidation polypeptide Asn-Trp-Asp-Met-Glu-Lys-Ile-Trp-Asp effectively can remove hydroxyl radical free radical, DPPH free radical and ultra-oxygen anion free radical.
6. a kind of hole according to claim 1 ray meat protein polypeptide is for the preparation of the purposes in anti-oxidation medicine or protective foods.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105193851A (en) * | 2015-09-22 | 2015-12-30 | 青岛大学 | Application of raja porosa extract in alcoholic brain damage protection medicine |
| CN106434804A (en) * | 2016-09-29 | 2017-02-22 | 国肽生物科技(北京)有限公司 | Protein peptide with antioxidant activity and preparation method thereof |
| CN106632642A (en) * | 2016-09-29 | 2017-05-10 | 安徽国肽生物科技有限公司 | Soft-shelled turtle protein peptide with ACE inhibitory and antioxidant functions and preparation method thereof |
| CN108129562A (en) * | 2017-12-29 | 2018-06-08 | 澳优乳业（中国）有限公司 | It is a kind of from the antioxidation biology active peptides MPFPK of casein and its application |
| CN111825754A (en) * | 2020-07-16 | 2020-10-27 | 河南省农业科学院 | A polypeptide obtained from sesame protein and having blood pressure lowering and blood sugar lowering activities |
| CN114793988A (en) * | 2022-04-19 | 2022-07-29 | 中国科学院海洋研究所 | Method for identifying ray porosa fertilized eggs based on morphology |
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2014
- 2014-12-29 CN CN201410831842.1A patent/CN104558115A/en active Pending
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| DAI HUNG NGO 等: "Active peptides from skate(Okamejei kenojei) skin gelatin diminish angiotensin-I converting enzyme activity and intracellular free radical-mediated oxidation", 《FOOD CHEMISTRY》 * |
| 张岩 等: "酶法制备海洋活性肽及其功能活性研究进展", 《生物技术通报》 * |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105193851A (en) * | 2015-09-22 | 2015-12-30 | 青岛大学 | Application of raja porosa extract in alcoholic brain damage protection medicine |
| CN106434804A (en) * | 2016-09-29 | 2017-02-22 | 国肽生物科技(北京)有限公司 | Protein peptide with antioxidant activity and preparation method thereof |
| CN106632642A (en) * | 2016-09-29 | 2017-05-10 | 安徽国肽生物科技有限公司 | Soft-shelled turtle protein peptide with ACE inhibitory and antioxidant functions and preparation method thereof |
| CN106632642B (en) * | 2016-09-29 | 2018-04-03 | 安徽国肽生物科技有限公司 | A kind of turtle proteins peptide suppressed with ACE with anti-oxidation function and preparation method thereof |
| CN108129562A (en) * | 2017-12-29 | 2018-06-08 | 澳优乳业（中国）有限公司 | It is a kind of from the antioxidation biology active peptides MPFPK of casein and its application |
| CN111825754A (en) * | 2020-07-16 | 2020-10-27 | 河南省农业科学院 | A polypeptide obtained from sesame protein and having blood pressure lowering and blood sugar lowering activities |
| CN111825754B (en) * | 2020-07-16 | 2022-02-11 | 河南省农业科学院 | A polypeptide obtained from sesame protein and having blood pressure lowering and blood sugar lowering activities |
| CN114793988A (en) * | 2022-04-19 | 2022-07-29 | 中国科学院海洋研究所 | Method for identifying ray porosa fertilized eggs based on morphology |
| CN114793988B (en) * | 2022-04-19 | 2023-01-17 | 中国科学院海洋研究所 | Morphology-based method for identifying fertilized eggs of raja porosa |
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Application publication date: 20150429 |