CN102372765A - Ruditapes philippinarum enzymatic polypeptide and preparation method thereof - Google Patents
Ruditapes philippinarum enzymatic polypeptide and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a ruditapes philippinarum enzymatic polypeptide. The ruditapes philippinarum enzymatic polypeptide is characterized by comprising the amino acid sequence of Asp Trp Pro His. The invention also discloses a preparation method of the ruditapes philippinarum enzymatic polypeptide. Compared with the prior art, the preparation method of the ruditapes philippinarum enzymatic polypeptide has the advantages that the ruditapes philippinarum enzymatic polypeptide obtained by the preparation method has high inoxidizability and specifically, hydroxy radical clearance, an DPPH radical amount and a reducing capacity of the ruditapes philippinarum enzymatic polypeptide are higher than those of Vitc; through ultrafiltration, anion exchange and anti-phase high performance liquid chromatography purification, desired active peptides can be separated out; and preparation processes are simple and reliable.
Description
Technical field
The present invention relates to a kind of philippine clam whelp enzymolysis polypeptide, the invention still further relates to the separation method of this polypeptide.
Background technology
Philippine clam whelp (Ruditapes philippinarum) is under the jurisdiction of the sea life of Mollusca (Mollusca), Bivalvia (Bivalvia), Veneridae (Veneridae), is commonly called as colored clam, is one of China's four big cultivated shellfishes.Flower clam meat tissue protein content is high, and lipid content is low, and contains abundant VITAMINs and trace element, has good nutrient health-care function.The traditional Chinese medical science thinks, clam meat has that enriching yin makes eye bright, the effect of softening and eliminating sputum.Recent studies proves, the philippine clam whelp extract has the immunizing power of raising, suppress that cell micronucleus forms, antitumor, atherosclerosis, effect such as antibiotic
[1-6]Big quantity research confirms the superoxide anion (O that organism metabolism produced
2-), hydrogen peroxide (H
2O
2), peroxylradicals (ROO) and hydroxy radical qiao (OH) etc. radical can cause the damage of cytolemma, DNA and protein-active, and with multiple human disease-relateds such as arteriosclerosis, apoplexy, alzheimer's disease, obesity, cataract and hepatic diseases
[7-11]Therefore, in the last few years, became the research focus for the separation and Extraction and the mechanism of action of anti-oxidation active substance.Protein in the diet mainly is absorbed with polypeptide and amino acid form at enteron aisle, and wherein polypeptide plays an important role in the body antioxidation process
[10]Up to now; Utilize enzyme solution from terrestrial life albumen, to obtain a large amount of antioxidation active peptides; And it is less relatively to obtain the report of antioxidation polypeptide for enzymolysis sea life albumen, and wherein, the research that philippine clam whelp meat albumen is carried out the enzymolysis and extraction antioxidation polypeptide is still for appearing in the newspapers.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of anti-oxidant activity high philippine clam whelp enzymolysis polypeptide to the above-mentioned state of the art.
Another technical problem to be solved by this invention provides the high philippine clam whelp enzymolysis polypeptide preparation method of a kind of anti-oxidant activity.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of philippine clam whelp enzymolysis polypeptide is characterized in that comprising following aminoacid sequence:
Asp?Trp?Pro?His。
A kind of as above-mentioned preparation method of philippine clam whelp enzymolysis polypeptide is characterized in that comprising the steps:
1. carry out enzymolysis with trypsinase after getting the equal slurries thin up of philippine clam whelp, centrifugal behind the enzymolysis, filter, get the upper strata enzymolysis solution;
2. with upper strata enzymolysis solution process ultra-filtration membrane; Enzymolysis solution below the intercepting 3KDa molecular weight carries out wash-out with the DEAESepharoseFF anion-exchange column, former and later two peptide peaks occur; NaCL with phosphoric acid buffer and 0.1mol/L carries out wash-out respectively, and collects respectively;
3. to the propetide peak through RPLC, a main peak occurs, and this main peak collected, the sample of collecting then carries out aminoacid sequence and detects, and confirms as Asp Trp Pro His.
As preferably, the trypsinase pH value of step described in 1. is 7.8, and the enzymolysis problem is 37 ℃, and enzymolysis time is 24 hours, 90 ℃ of enzymes 15 minutes of going out, and 4 ℃ are centrifugal down.
As preferably, step 2. described in the column type of anion-exchange column be 2.6 * 50cm, last appearance volume is 3mL, elution speed is 1ml/min, carries out ultraviolet detection with 220nm.
As preferably, the chromatographic condition of the said RPLC of step in 3. is: C
18Reversed-phase column, filler: ODS; Column type: 10 * 250mm; Acetonitrile/water is a moving phase, and flow velocity is 0.8ml/min, and last appearance volume is 20 μ L, detects wavelength 220nm.
Compared with prior art; The invention has the advantages that: philippine clam whelp enzymolysis polypeptide oxidation-resistance is high; Be in particular in that hydroxy radical qiao clearance rate, DPPH radical and reducing power all can be higher than VitC; Through ultrafiltration, anionresin, and RPLC purifying, separablely go out targeted activity peptide, simple and reliable process.
Description of drawings
Fig. 1 can obtain the histogram of amino nitrogen value after to every gram philippine clam whelp for various proteolytic enzyme.
Fig. 2 is the histograms of various zymolytes to the hydroxy radical qiao clearance rate.
Fig. 3 is the peak value figure behind the DEAE SepharoseFF post wash-out.
Fig. 4 is the peak value figure behind the RT-HPLC wash-out.
Fig. 5 is a target peptide hydroxy radical qiao clearance rate coordinate diagram.
Fig. 6 is a target peptide DPPH radical scavenging activity coordinate diagram.
Fig. 7 is a target peptide reducing power tester light absorption value coordinate diagram.
Embodiment
Embodiment describes in further detail the present invention below in conjunction with accompanying drawing.
The enzymolysis process flow process:
The philippine clam whelp meat tissue of getting-20 ℃ of refrigerations is put into 30 ℃ of water-baths and is thawed, and homogenate adds 3 times of volume zero(ppm) water with homogenate, and the white enzyme pH of pawpaw is 5, and temperature is 50 ℃, and enzyme concentration is 1200u/g; Trypsinase pH is 7.8, and temperature is 37 ℃, and enzyme concentration is 1200u/g; Stomach en-pH is 2.4, and temperature is 37 ℃, and enzyme concentration is 1200u/g; Sumizyme MP pH is 8.5, and temperature is 45 ℃, and enzyme concentration is 1200u/g; Be incubated enzymolysis 24h respectively, 90 ℃ of enzyme 15min that go out, 4 ℃ of centrifugal 15min of following 8500r/min get supernatant.
Degree of hydrolysis is measured:
The mensuration of degree of hydrolysis with reference to methods such as Zhao Xinhuai (Zhao Zhihuai, Feng Zhibiao. the mensuration of protein hydrolystate degree of hydrolysis [J]. Food science, 1994,179 (11): 65-67.), and make an amendment slightly.Get 20ml enzymolysis solution and place beaker, add water 60mL, start magnetic stirring apparatus, use the titration of 0.05mol/L sodium hydroxide reference liquid to pH be 8.2.Add 10mL formaldehyde solution mixing, use again the 0.05mol/L sodium hydroxide solution continue titration to pH be 9.2, write down the milliliter number of hydrogen consuming sodium oxide standard, the 80mL that fetches water simultaneously does the reagent blank test.Calculate amino nitrogen (ANN) content by following formula, amino nitrogen content becomes positive correlation with degree of hydrolysis.
X＝(V
1-V
2)×C×0.014×100(5×V)
In the formula, X is the content (g/100mL) of amino nitrogen in the sample; V1 is that test sample adds formaldehyde dilution post consumption sodium hydroxide reference liquid (mL); V2 is the volume (mL) that the reagent blank test adds formaldehyde post consumption standard solution of sodium hydroxide;
V is the sample liquid amount of taking (mL); C is the concentration (mol/L) of sodium hydroxide reference liquid; 0.014 be the mmole quality (g/mmoL) of nitrogen.
The mensuration of anti-oxidant activity
The mensuration of hydroxy radical qiao clearance rate:
With reference to said methods such as Jin Ming (gold ring, Cai Yaxin, Li Jinrong, etc. phenanthroline-Fe2+ oxidation style detects the hydroxy radical qiao [J] that H2O2/Fe2+ produces. biological chemistry is made progress with biophysics, 1996,23 (6): 553-555.), and do suitable adjustment.Get concentration and be 0.75mmol/L phenanthroline 1mL in test tube in, add the pH value successively and be 7.4 phosphoric acid buffer 2mL, sample liquid 1mL; Abundant mixing, adding concentration then is the ferrous sulfate 1mL of 0.75mol/L, mixing; Add 0.12%H2O21mL; 37 ℃ of water-bath 90min survey its absorbancy in the 536nm place, are As; Replace 1mLH2O2 with 1mL zero(ppm) water, be Ab; Replace the 1mL sample liquid with 1mL zero(ppm) water, be Ap.
Clearance rate (%)=(As-Ap)/(Ab-Ap) * 100%
The mensuration of reducing power:
With reference to Oyaizu method (Song LY; Li TF, Yu RM, et al.Antioxidant activities of hydrolysates of Arca subcrenata prepared with three proteases [J] .Mar Drugs; 2008,6 (4): 607-619.) and slightly change.Extracting sample solution 2mL joins thorough mixing in the Tripotassium iron hexacyanide of 2mL0.2mol/L (PH6.6) phosphate buffered saline buffer and 2mL 1%; The trichoroacetic acid(TCA) that adds 2mL10% behind 50 ℃ of insulation 20min mixes; Get 2mL, add the iron trichloride of 2mL zero(ppm) water and 0.4mL0.1%, behind the reaction 10min; Measure light absorption value at the 700nm place, the light absorption value height shows that reducing power is high.The reducing power of measuring different concns Vit C simultaneously as a comparison.
The mensuration of DPPH radical scavenging activity:
Sample thief liquid 2mL and 1 * 10-4mol/L DPPH solution 2mL add in the tool plug test tube and shake up, and the 30min of black out reaction at room temperature makes reference with neat solvent, measures absorbancy down in the 517nm wavelength.Calculate clearance rate according to formula:
Clearance rate=[1-(AS-ASB)/AC] * 100%
Wherein: AS is for adding DPPH solution absorbency after the sample liquid; ASB is the absorbancy of sample liquid; ASB is the DPPH solution absorbency when not adding sample liquid.Measure inhibitor Vit C as a comparison to the clearance rate of DPPH radical.
The preparation of antioxidation polypeptide
Zymolyte is pressed the molecular weight size fractionation and is separated:
Get 400g flower clam meat through trypsin digestion; Get supernatant after centrifugal; Use ultrafiltration cup and intercepting molecular weight to be respectively the ultra-filtration membrane of 3KDa and 5KDa; With supernatant ultrafiltration 11h respectively, intercepting obtains below the 3KDa, the enzymolysis solution of 3～5KDa and the above molecular weight of 5KDa, surveys anti-oxidant activity after the lyophilize respectively in 20 ℃ of environment.
DEAE SepharoseFF anionresin:
To the strongest component of anti-oxidant activity with DEAE SepharoseFF anion-exchange column wash-out; Column type: 2.6 * 50cm, last appearance volume 3mL, the NaCL solution with phosphoric acid buffer, 0.1mol/L, 0.3mol/L, 0.5mol/L carries out stepwise elution respectively; Elution speed is 1ml/min; Carry out ultraviolet monitoring in 280nm, automatically the every pipe 4mL of Fraction Collector collects, with different peptides peak collect respectively store after the lyophilize subsequent use.
RPLC (RT-HPLC):
RT-HPLC carries out purifying.Chromatographic condition: C18 reversed-phase column, filler: ODS; Column type: 10 * 250mm; Acetonitrile/water is a moving phase, and flow velocity is 0.8ml/min, and last appearance volume is 20 μ L, detects wavelength 220nm.
Elution requirement: acetonitrile concentration is 15%, and wash-out 20min repeats appearance.
The aminoacid sequence of target peptide detects
Data processing:
Each is organized experimental data mensuration and all does 3 parallel laboratory tests, and the result gets its MV.Each is organized and adopts one-way analysis of variance between the data, checks the comparison of organizing a mean with t, uses SPSS 13.0 to carry out data analysis.
Degree of hydrolysis and anti-oxidant activity:
To behind the young meat enzymolysis of Fei Libin clam, the available amino nitrogen of every gram meat is followed successively by 6.2418mg, 6.3648mg, 5.2637mg, 7.4562mg (as shown in Figure 1) respectively for stomach en-, trypsinase, papoid and Sumizyme MP; When concentration is 2.5mg/mL; Each zymolyte is respectively 46.55%, 65.43%, 34.31% and 57.27% to the clearance rate of hydroxy radical qiao; And the hydroxy radical qiao clearance rate of classical antioxidant vitamin C is 72.04%, and each data differences is (P＜0.05=(as shown in Figure 2) significantly.Because it is the strongest that the trypsin digestion thing is removed the hydroxy radical qiao ability, so it is carried out separation and purification.
Antioxidation polypeptide separates:
Trypsin digestion liquid obtains 3 components after ultrafiltration, promptly (I1), 3～5KDa (I2) below the 3KDa, 5KDa above (I3) obtain 3.9g, 2.8g and 3.2g pale yellow powder shape sample respectively after the lyophilize, and yield is respectively 0.7%, 1.0% and 0.8%.Measure the anti-oxidant activity of I1, I2 and I3, when concentration was 1mg/mL, they were respectively 49.36%, 41.65% and 29.44% to the clearance rate of hydroxy radical qiao, had significant difference (P＜0.05).To the stronger I1 of anti-oxidant activity behind DEAE SepharoseFF post wash-out; 2 peaks appear; Be peak 1 (II1) and peak 22 (II2) (Fig. 3), be respectively the elution fraction of damping fluid and 0.1mol/L NaCL solution, the NaCL solution of 0.3mol/L and 0.5mol/L does not have tangible elution peak.
Collect II1 and II2, obtain 1.3g and 0.9g dry sample after the lyophilize respectively, yield is respectively 0.33% and 0.23%.Be provided with 0.2mg/mL, 0.4mg/mL, 0.8mg/mL, 1.6mg/mL and 3.2mg/mL totally 5 concentration group measure the hydroxy radical qiao clearance rate (table 1) of II1 and II2.Analyze according to table 1, II1 and II2 all have the ability of removing hydroxy radical qiao more by force, and along with concentration increases, the two hydroxy radical qiao clearance rate also increases.In each concentration group, II1 to the clearance rate of hydroxy radical qiao greater than II2.
Table 1:
RT-HPLC wash-out result:
II1 through RPLC post wash-out (Fig. 4), when RT is about 4.5min, a main peak (peak 4) occurred; Peak height is 1021.8; Peak area is 5190.1, collects this peak, obtains 0.37g sample III after the lyophilize; Yield is 0.1%, and the aminoacid sequence of this target peptide is: Asp-Trp-pro-His.
The target peptide anti-oxidant activity is measured:
Sample be set to 0.5mg/mL, 1.0mg/mL, 1.5mg/mL, 2.0mg/mL, 2.5mg/mL totally 5 measured hydroxy radical qiao clearance rates of concentration gradient be followed successively by 42.16%, 55.26%, 63.05%, 69.85% and 79.41%; The DPPH radical scavenging activity is 18.13%, 36.86%, 53.39%, 70.04% and 85.91%; Reducing power is measured its light absorption value and is respectively 0.129,0.294,0.429,0.601,0.736 (Fig. 5; Fig. 6; Fig. 7), has significant difference (P＜0.05) between each data.
The present invention has studied degree of hydrolysis and the hydroxy radical qiao clearance rate of philippine clam whelp tissue protein behind different protease hydrolysiss; Wherein, Sumizyme MP zymolyte degree of hydrolysis is maximum, and the trypsin digestion thing is the strongest to the removing power of hydroxy radical qiao; It is thus clear that; When different proteolytic enzyme are hydrolyzed to the philippine clam whelp tissue protein, the anti-oxidant activity of resulting enzymolysis polypeptide form with its amino acid and structure relevant, and with degree of hydrolysis between do not have the related of certainty.The component that obtains 3 different molecular weights with ultra-filtration membrane intercepting trypsin digestion liquid all has the hydroxy radical qiao of removing activity; But it is active maximum with I1; Can infer that thus the antioxidant property of zymolyte maybe be also relevant with its molecular weight size except outside the Pass having with its composition and structure.I1 finally obtains 1 antioxidant property polypeptide III preferably after DEAE-sepharoseFF anionresin and RP-HPLC separation and purification, this peptide increases and increases along with concentration the clearance rate of hydroxy radical qiao, and IC50 is about 0.8mg/mL; When concentration is 2.5mg/mL, to compare with Vit C, the hydroxy radical qiao clearance rate of III, the free clearance rate of DPPH and redox power are higher relatively, have the potential that is developed as foodstuff additive and pharmaceuticals.But the antioxidation activity in vitro of enzymolysis polypeptide has only been examined or check in this research, therefore, further studies the removing ability of enzymolysis polypeptide to various radicals in the body, and is very necessary for it being developed to the novel anti oxygenated products.
Claims (5)
1. philippine clam whelp enzymolysis polypeptide is characterized in that comprising following aminoacid sequence:
Asp?Trp?Pro?His。
2. the preparation method of philippine clam whelp enzymolysis polypeptide according to claim 1 is characterized in that comprising the steps:
1. carry out enzymolysis with trypsinase after getting the equal slurries thin up of philippine clam whelp, centrifugal behind the enzymolysis, filter, get the upper strata enzymolysis solution;
2. with upper strata enzymolysis solution process ultra-filtration membrane; Enzymolysis solution below the intercepting 3KDa molecular weight carries out wash-out with the DEAESepharoseFF anion-exchange column, former and later two peptide peaks occur; NaCL with phosphoric acid buffer and 0.1mol/L carries out wash-out respectively, and collects respectively;
3. to the propetide peak through RPLC, a main peak occurs, and this main peak collected, the sample of collecting then carries out aminoacid sequence and detects, and confirms as Asp Trp Pro His.
3. preparation method according to claim 2 is characterized in that the trypsinase pH value described in step is 1. is 7.8, and hydrolysis temperature is 37 ℃, and enzymolysis time is 24 hours, 90 ℃ of enzymes 15 minutes of going out, and 4 ℃ are centrifugal down.
4. preparation method according to claim 2, the column type that it is characterized in that anion-exchange column described in step is 2. is 2.6 * 50cm, and last appearance volume is 3mL, and elution speed is 1ml/min, carries out ultraviolet detection with 220nm.
5. preparation method according to claim 2 is characterized in that the chromatographic condition of the said RPLC during step 3. is: C
18Reversed-phase column, filler: ODS; Column type: 10 * 250mm; Acetonitrile/water is a moving phase, and flow velocity is 0.8ml/min, and last appearance volume is 20 μ L, detects wavelength 220nm.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104212861A (en) * | 2013-05-29 | 2014-12-17 | 浙江海洋学院 | Preparation method of ruditapes philippinarum oligopeptide and application in resisting prostate cancer |
| CN106191184A (en) * | 2015-05-05 | 2016-12-07 | 于荣敏 | A kind of preparation of novel Scapharca broughtonii antioxidation active peptides and application thereof |
| CN106498015A (en) * | 2016-11-21 | 2017-03-15 | 浙江海洋大学 | A kind of preparation method and application of Ruditapes philippinarum blood pressure lowering peptide |
| CN106868084A (en) * | 2017-03-23 | 2017-06-20 | 福建省水产研究所 | The preparation method and application of Ruditapes philippinarum high activity natineoplaston |
| CN113817792A (en) * | 2021-11-25 | 2021-12-21 | 山东方明药业集团股份有限公司 | Application of marine biological polypeptide in preparation of medicine for treating osteoporosis |
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| CN1994107A (en) * | 2005-12-31 | 2007-07-11 | 大连玉璘企业集团有限公司 | Method for preparing polypeptide nutrient |
| WO2009026218A2 (en) * | 2007-08-17 | 2009-02-26 | University Of Illinois | Identification and use of peptide inhibitors of protein synthesis |
| CN101548989A (en) * | 2009-05-15 | 2009-10-07 | 南京中医药大学 | Mactra veneriformis extract, preparation method and application thereof |
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2010
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Patent Citations (4)
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| CN1099582A (en) * | 1993-08-28 | 1995-03-08 | 中山大学 | Haihuang series sea foods and preparing method |
| CN1994107A (en) * | 2005-12-31 | 2007-07-11 | 大连玉璘企业集团有限公司 | Method for preparing polypeptide nutrient |
| WO2009026218A2 (en) * | 2007-08-17 | 2009-02-26 | University Of Illinois | Identification and use of peptide inhibitors of protein synthesis |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104212861A (en) * | 2013-05-29 | 2014-12-17 | 浙江海洋学院 | Preparation method of ruditapes philippinarum oligopeptide and application in resisting prostate cancer |
| CN106191184A (en) * | 2015-05-05 | 2016-12-07 | 于荣敏 | A kind of preparation of novel Scapharca broughtonii antioxidation active peptides and application thereof |
| CN106191184B (en) * | 2015-05-05 | 2022-02-25 | 于荣敏 | Preparation and application of novel arca inflata reeve antioxidant active peptide |
| CN106498015A (en) * | 2016-11-21 | 2017-03-15 | 浙江海洋大学 | A kind of preparation method and application of Ruditapes philippinarum blood pressure lowering peptide |
| CN106868084A (en) * | 2017-03-23 | 2017-06-20 | 福建省水产研究所 | The preparation method and application of Ruditapes philippinarum high activity natineoplaston |
| CN106868084B (en) * | 2017-03-23 | 2020-10-09 | 福建省水产研究所 | Preparation method and application of Ruditapes philippinarum high-activity antitumor peptide |
| CN113817792A (en) * | 2021-11-25 | 2021-12-21 | 山东方明药业集团股份有限公司 | Application of marine biological polypeptide in preparation of medicine for treating osteoporosis |
| CN113817792B (en) * | 2021-11-25 | 2022-04-01 | 山东方明药业集团股份有限公司 | Application of marine biological polypeptide in preparation of medicine for treating osteoporosis |
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