CN102219829A - Antioxidant polypeptide prepared by enzymatic hydrolysis of sharkskin collagen with acid protease - Google Patents
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Abstract
The invention provides an antioxidant polypeptide prepared by enzymatic hydrolysis of sharkskin collagen with acid protease. According to the invention, sharkskin collagen is used as the raw material; after enzymatic hydrolysis with acid protease, the sharkskin collagen is subjected to isolation and purification so as to obtain a specific antioxidant polypeptide, the complete amino acid sequence of which is as follows: Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Ala-Gly. The invention enables the elimination of shortcomings of natural anti-oxidants and of public worry about synthetic anti-oxidants and lays a theoretical basis for the development of food derived antioxidant polypeptides and for the exploration of their application in foodstuff and medical science.
Description
Technical field
The invention provides a kind of antioxidation polypeptide and preparation method thereof, related more specifically to a kind of antioxidation polypeptide that utilizes the preparation of aspartic protease enzymolysis shark skin collagen protein, belong to biological technical field.
Background technology
The oxidation of biomolecules is the process of a free radical mediated, and it can cause many adverse influences to food and biosystem.In aerobic organ, with arteriosclerosis, cancer etc. many in the free free radical of disease-relateds can produce along with the process of oxygen metabolism inevitably.In food, the oxidation meeting of food nutrient composition produces superoxide, and it not only can influence Nutritive value of food, causes that Food Quality descends serious even health generation disease that also can cause absorption person.Therefore, seek safe antioxidant is the research focus that biochemistry nutrition is learned to suppress the superoxide generation always.Because chemosynthesis antioxidants such as BHT, TBHQ have better effect and more cheap price than natural antioxidants, so it is widely used in the food service industry.But, discovers that at present synthetized oxidation preventive agent has the property of accumulating carcinogenesis to organs such as human liver, spleen, lungs, thereby caused the worry of people, and beginning limits its use in food gradually its security.So people turn to natural antioxidants to sight.Alpha-tocopherol is the natural antioxidants that a kind of quilt the most generally uses, and it can effectively keep greasy stability in the food, but but is unfavorable for Food preservation.Therefore, we are necessary to seek a kind of natural antioxidants of safety of other source.
Existing discovering, the protolysate of a lot of different sourcess all has certain resistance of oxidation, all has certain oxidation-resistance as the hydrolyzate of casein, soybean protein, bovine serum albumin, Protalbinic acid, oil seed protein, wheat gliadin and zein etc.Though the mechanism of polypeptide performance anti-oxidant activity also is not clear especially, reports that some die aromatischen Aminosaeuren and Histidine have played vital role.Domestic and international research finds that the peptide in collagen protein source has more high efficiency oxidation-resistance than other protein source peptide.And collagen protein accounts for more than 80% of gross protein in the fish-skin, and from obtaining the efficient of collagen protein, fish-skin is the important source that obtains collagen protein.Be rich in hydrophobic amino acid and other and lipid in the gelatin and have than high-affinity and can form the amino acid of stable emulsion system, so be considered to prepare the good raw material of antioxidation polypeptide.Therefore, the high-efficiency antioxidant polypeptide that how obtains to have the specific amino acids sequence from gelatin just becomes urgent research direction.
Summary of the invention
In order to address the above problem, the invention provides a kind of utilize aspartic protease enzymolysis shark skin collagen protein preparation antioxidation polypeptide, anti-oxidant activity is realized efficiently.
A kind of antioxidation polypeptide of the present invention, aminoacid sequence are Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Ala-Gly.
The present invention also provides a kind of preparation method of antioxidation polypeptide, is that raw material extracts collagen protein with the shark skin, adopts aspartic protease that it is carried out enzymolysis then, and separation and purification, lyophilize obtain antioxidation polypeptide; Described enzymatic hydrolysis condition is: pH is 3.0,50 ℃ of temperature, enzymolysis time are that 5h, enzyme-substrate proportioning are 3000U/g; Described enzyme is an aspartic protease.The means of described separation and purification comprise ultrafiltration, SP-Sephadex C-25 ion-exchange chromatography, Sephadex G-50 molecular sieve and RP-HPLC RPLC.
The concrete steps of described separation and purification are:
(1) enzymolysis product at first utilizes molecular weight to hold back scope for the ultra-filtration membrane of 8000Da and 3500Da enzymolysis product to be carried out ultra-filtration and separation, it is divided into 3 components that molecular weight ranges is different, be respectively molecular weight greater than the component of 8000Da, molecular weight between the component of 3500Da and 8000Da, molecular weight component less than 3500Da; (2) collection has the component of best anti-oxidant activity, separate with SP-Sephadex C-25 cation-exchange chromatography again, flush away absorbed component not behind the last sample, carry out linear gradient elution with 0.02mol/L, the pH4.0 acetate-sodium acetate buffer that contains 0～0.35mol/L NaCl again, flow velocity is 0.5ml/min, under 225nm, measure, measure the anti-oxidant activity of the elution fraction of each absorption peak correspondence; (3) collect the peak with best anti-oxidant activity, separate with Sephadex G-50 gel chromatography, elutriant is a deionized water, and flow velocity is 0.5ml/min, measures under 225nm, measures the anti-oxidant activity of the elution fraction of each absorption peak correspondence; (4) collect peak with best anti-oxidant activity, sharp again RP-HPLC RPLC further separates, the separation of reversed-phase HPLC is to contain the 0.1%(volume) concentration gradient of trifluoroacetic acid is that 5%～40% acetonitrile solution carries out linear elution as elutriant, used chromatographic column is Gemini 5 μ C18, applied sample amount is 100 μ L, flow velocity is 1mL/min, the detection wavelength is 215nm, measure the anti-oxidant activity of the elutriant of each absorption peak correspondence, collect and obtain three the highest components of anti-oxidant activity; (5) utilize the RP-HPLC RPLC that three components are identified, with the concentration gradient that contains the 10mM ammonium acetate is that 5%～27.5% acetonitrile solution carries out linear elution as elutriant, used chromatographic column is Gemini 5 μ C18, applied sample amount is 20 μ L, flow velocity is 1ml/min, the detection wavelength is 215nm, and three components are separated respectively and obtained single peak, promptly obtain three pure active ingredients; Utilize proteinaceous solid facies sequence analysis instrument to identify the aminoacid sequence of three peptides, the aminoacid sequence that obtains antioxidation polypeptide of the present invention is: Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Ala-Gly.
In order to realize such scheme, the concrete steps that the present invention takes are as follows:
(1) extraction of collagen protein
Shark skin of the present invention is commercially available.
Shark skin is smashed (length＜1cm), use the NaOH solution (solid-liquid ratio w/v=1:6) of 0.1mol/L to soak 24h down at 4 ℃ again to pieces then through washing 3 times with removal of impurities.Drain away the water after being washed to neutrality, add HCl solution (the solid-liquid ratio w/v=1:6), be washed to neutrality again behind the immersion 4h down of 0.03w% again at 4 ℃.At last, add distilled water (solid-liquid ratio w/v=1:6), 60 ℃ of water-bath 6h, after filtration, concentrate and lyophilize after obtain collagen protein.
(2) enzymolysis of collagen protein
Enzyme is available from Foochow good fortune big hundred special biological reagent companies (Chinese Foochow).
Adopt aspartic protease enzymolysis shark skin collagen protein, protein concentration is 30mg/ml, enzymatic hydrolysis condition gets that pH is 3.0,50 ℃ of temperature, enzymolysis time are that 5h, enzyme-to-substrate are than being (3000U/g), it is stable to regulate pH with 2M HCl, and behind the hydrolysis 5h, enzyme 15min goes out in the boiling water bath, be cooled to room temperature then rapidly, place whizzer, with the centrifugal 15min of 4000r/min, it is standby to get supernatant liquor.
(3) separation of enzymolysis product, purifying
Utilize molecular weight to hold back scope the enzymolysis product that obtains and enzymolysis product is carried out ultra-filtration and separation for the ultra-filtration membrane of 8000Da and 3500Da, it is divided into 3 components that molecular weight ranges is different, be respectively molecular weight greater than the component of 8000Da, molecular weight between the component of 3500Da and 8000Da, molecular weight component less than 3500Da; Collection has the component of best anti-oxidant activity, pass through SP-Sephadex C-25 cation-exchange chromatography (long 20cm again, diameter 1.6cm) separates, carry out linear gradient elution with 0.02mol/L, the pH4.0 acetate-sodium acetate buffer that contains 0～0.35mol/L NaCl, flow velocity is 0.5ml/min; Collection has the peak of best anti-oxidant activity, again with Sephadex G-50(long 100cm, diameter 2.6cm) gel chromatography separates, and elutriant is a deionized water, and flow velocity is 0.5ml/min, and elution peak is measured under 225nm; Collection has the peak of best anti-oxidant activity, utilize the RP-HPLC RPLC further to separate again, the separation of reversed-phase HPLC is that 5%～40% acetonitrile solution carries out gradient elution with the concentration gradient that contains 0.1% trifluoroacetic acid (volume), used chromatographic column is Gemini 5 μ C18, applied sample amount is 100 μ L, flow velocity is 1ml/min, and the detection wavelength is 215nm, collects to obtain three the highest components of anti-oxidant activity.
(4) evaluation of antioxidation polypeptide
Utilize the RP-HPLC RPLC that three components are identified, with the concentration gradient that contains the 10mM ammonium acetate is that 5%～27.5% acetonitrile solution carries out linear elution as elutriant, used chromatographic column is Gemini 5 μ C18, applied sample amount is 20 μ L, flow velocity is 1ml/min, and the detection wavelength is 215nm, and three components are separated respectively and obtained single peak, illustrate to obtain three pure active ingredients, further identify the aminoacid sequence of peptide.
(5) test of anti-oxidant activity
Utilize DPPH(1,1-Diphenyl-2-picryl-hydrazyl) free radical scavenging activity assay method research antioxidation polypeptide.Compound concentration is 1 * 10
-5The DPPH ethanolic soln of mol/L keeps in Dark Place.With 2ml, the DPPH ethanol solution of 0.1mM joins in the clean tube that contains the different enzymolysis samples of 2mL, mixing.After placing 30min under the room temperature, measure absorbancy in 517 nm places, light absorption value is more little, shows that radical scavenging activity is strong more.
Clearance rate (%)=(1-(A
i-A
j)/A
0) * 100%
In the formula, A
0Be i.e. 2 mL of the light absorption value of the DPPH solution that do not add sample, the sample solvent of DPPH ethanol solution+2ml of 0.1mM, the light absorption value of blank; A
iFor the light absorption value that adds the DPPH solution behind the sample is 2ml, the light absorption value of the sample of DPPH ethanol solution+2mL of 0.1mM; A
jThe light absorption value of the sample of dehydrated alcohol+2 ml that are 2ml for the solvent and the light absorption value after the sample mix of DPPH solution.
(6) determined amino acid sequence
Utilize proteinaceous solid facies sequence analysis instrument (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) the amino acid total order of measuring antioxidation polypeptide of the present invention is classified Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Ala-Gly as, and molecular weight is 724 Da.
The present invention sees through the defective of present natural antioxidants and the public worry to the synthetic antioxidant, be based on seeking a kind of antioxidant of efficiency natural, with the collagen protein that comes from shark skin is starting point, be conceived to cutting condition control by aspartic protease, be cut into active polypeptide, and anti-oxidant activity is realized efficiently with specific peptide chain length and structural domain composition.
The present invention changed the thinking and the method for the extraction and the utilization of existing antioxidant, eliminated the synthetic antioxidant the side effect that may cause, be a kind of natural antioxidants, can replace traditional synthetic antioxidants.And the present invention has also improved China to collagen protein utilization ratio situation on the low side in the aquatic animal skin, what both can solve a large amount of aquatic resources utilizes problem again, can remove again the human consumer to antioxidant in the misgivings aspect the food safety, will have profound significance to the development of science and technology, economy and grocery trade.
Description of drawings
Fig. 1 is the removing effect of the DPPH free radical of collagen protein enzymolysis thing after ultra-filtration and separation;
Fig. 2 is the SP-Sephadex C-25 elution profile of collagen protein enzymolysis thing;
Fig. 3 is the removing effects of four chromatographic separation components of SP-Sephadex C-25 to the DPPH free radical;
Fig. 4 is the Sepadex G-50 elution profile of B component;
Fig. 5 is the removing effect of Sepadex G-50 elution fraction to the DPPH free radical;
Fig. 6 is the RP-HPLC elution profile of B component-III;
Fig. 7 is the DPPH free radical scavenging activity of the RP-HPLC purified components of B-III;
Fig. 8 is that the RP-HPLC of antioxidation polypeptide B-III a identifies figure;
Fig. 9 is that the RP-HPLC of antioxidation polypeptide B-III c identifies figure;
Figure 10 is that the RP-HPLC of antioxidation polypeptide B-III d identifies figure;
Figure 11 is the amount-result relation curve that the antioxidation polypeptide B-III a of purifying removes the DPPH free radical.
Figure 12 is the amount-result relation curve that the antioxidation polypeptide B-III a of purifying removes the ABTS free radical.
Figure 13 is that the antioxidation polypeptide B-III a of purifying suppresses Fe
2+Induce the amount-result relation curve of lipovitellinin polyunsaturated fatty acid peroxidation.
Embodiment
The aminoacid sequence of antioxidation polypeptide of the present invention is Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Ala-Gly.
The preparation method is as follows:
Is that raw material extracts collagen protein with the shark skin, uses aspartic protease that it is carried out enzymolysis then, protein concentration is 30mg/ml, and enzymatic hydrolysis condition is: pH is 3.0, temperature is that 50 ℃, enzymolysis time are that 5 hours, enzyme-substrate proportioning are 3000U/g; Utilize molecular weight to hold back scope the enzymolysis product that obtains and enzymolysis product is carried out ultra-filtration and separation for the ultra-filtration membrane of 8000Da and 3500Da, it is divided into 3 components that molecular weight ranges is different, be respectively molecular weight greater than the component of 8000Da, molecular weight between the component of 3500Da and 8000Da, molecular weight component less than 3500Da; Collection has the component of best anti-oxidant activity, pass through SP-Sephadex C-25 cation-exchange chromatography (long 20cm again, diameter 1.6cm) separates, carry out linear gradient elution with 0.02mol/L, the pH4.0 acetate-sodium acetate buffer that contains 0～0.35mol/L NaCl, flow velocity is 0.5ml/min, measures under 225nm; Collection has the peak of best anti-oxidant activity, again with Sephadex G-50(long 100cm, diameter 2.6cm) gel chromatography separates, and elutriant is a deionized water, and flow velocity is 0.5ml/min, and elution peak is measured under 225nm; Collection has the peak of best anti-oxidant activity, utilize the RP-HPLC RPLC further to separate again, the separation of reversed-phase HPLC is that 5%～40% acetonitrile solution carries out gradient elution with the concentration gradient that contains 0.1% trifluoroacetic acid (volume), used chromatographic column is Gemini 5 μ C18, applied sample amount is 100 μ L, flow velocity is 1ml/min, and the detection wavelength is 215nm, collects to obtain three the highest components of anti-oxidant activity.Further identify through the RP-HPLC RPLC, obtain three pure active ingredients.
Instrument, detection means that the present invention adopts are as follows:
The present invention uses multiple separation and purification means such as ultrafiltration, SP-Sephadex C-25 ion-exchange chromatography, Sephadex G-50 gel filtration chromatography, RP-HPLC RPLC, and realization has the high efficiency separation purifying of the antioxidation polypeptide of remarkable activity.
Utilize protein solid phase sequenator (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) aminoacid sequence of the antioxidation polypeptide behind the mensuration purifying.
In order further to understand content of the present invention, characteristics and effect, exemplify following examples now:
Embodiment 1
Take by weighing 7.5 gram shark skin collagen proteins with deionized water dissolving and be settled to 250ml, use 2mol/L HCl then its pH regulator to 3.0.Earlier this solution water-bath being heated to 50 ℃, is the enzyme of the ratio adding respective amount of 3000U/g according to the enzyme-substrate proportioning more then, and enzymolysis time is 5 hours.Then in boiling water bath, go out enzyme 15 minutes, centrifugal 15 minutes of 4000rpm again after the cooling.The collection supernatant liquor is standby.
It is that the ultra-filtration membrane of 8000Da and 3500Da carries out ultra-filtration and separation that supernatant liquor is held back scope with molecular weight, it is divided into 3 components that molecular weight ranges is different, be respectively molecular weight greater than the component of 8000Da, molecular weight between the component of 3500Da and 8000Da, molecular weight component less than 3500Da, and measure its anti-oxidant activity (as shown in Figure 1).As can be seen, molecular weight has best anti-oxidant activity less than the component of 3500Da.
Collect the component of molecular weight less than 3500Da, with SP-Sephadex C-25 cation-exchange chromatography (long 20cm, diameter 1.6cm) further separates again, carry out linear gradient elution with 0.02mol/L, the pH4.0 acetate-sodium acetate buffer that contains 0～0.35mol/L NaCl, flow velocity is 0.5ml/min, elution peak is measured under 225nm, sees Fig. 2.Collect each peak and measure anti-oxidant activity (as shown in Figure 3).As can be seen, B component has best anti-oxidant activity.
To separate B component and separate with Sepadex G-50 gel filtration chromatography (long 20cm, diameter 1.6cm) again, elutriant is a deionized water, and flow velocity is 0.5ml/min, and elution peak is measured under 225nm, sees Fig. 4.Collect each peak and measure anti-oxidant activity (as shown in Figure 5).As can be seen, B component-III has best anti-oxidant activity.
Utilize the RP-HPLC RPLC further to separate (as shown in Figure 6) again B component-III of separating, elutriant is 5%～40% acetonitrile solution for the concentration gradient that contains 0.1% trifluoroacetic acid (volume), used chromatographic column is Gemini 5 μ C18, applied sample amount is 100 μ L, flow velocity is 1ml/min, the detection wavelength is 215nm, measure the anti-oxidant activity (as shown in Figure 7) of the elutriant of each absorption peak correspondence, collection obtains three the highest components of anti-oxidant activity, is respectively B-III a, B-III c and B-III d.
Utilize the purity of three antioxidation polypeptides that the RP-HPLC RPLC obtains separation to identify, with the concentration gradient that contains the 10mM/L ammonium acetate is that 5%～27.5% acetonitrile solution carries out linear gradient elution as elutriant, used chromatographic column is Gemini 5 μ C18, applied sample amount is 20 μ L, flow velocity is 1ml/min, and the detection wavelength is 215nm, and the result shows, it is single peak, illustrates that it has reached higher degree (as Fig. 8, Fig. 9 and shown in Figure 10).
Antioxidation polypeptide B-III a behind the purifying has very strong resistance of oxidation, by Figure 11, Figure 12 and Figure 13 as can be seen, its 503nhibiting concentration of removing DPPH free radical, ABTS free radical and suppressing peroxidatic reaction of lipid is respectively 63.56mg/ml, 82.34mg/ml and 183.13mg/ml.
Utilize protein solid phase sequenator (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) aminoacid sequence of the antioxidation polypeptide B-III a behind the mensuration purifying.Obtaining its amino acid total order classifies as: the Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Ala-Gly(molecular weight is 724 Da).
＜110〉University of Fuzhou
＜120〉utilize aspartic protease enzymolysis shark skin collagen protein to prepare antioxidation polypeptide
<160>?1
<210>?1
<211>?10
<212>?PRT
＜213〉artificial sequence
<220>
<400>?1
Gly?Pro?Ala?Gly?Pro?Ala?Gly?Ala?Ala?Gly
1 5 10
Claims (4)
1. antioxidation polypeptide, it is characterized in that: the aminoacid sequence of described antioxidation polypeptide is Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Ala-Gly.
2. the preparation method of an antioxidation polypeptide is characterized in that: be that raw material extracts collagen protein with the shark skin, adopt aspartic protease that it is carried out enzymolysis then, separation and purification, lyophilize obtain antioxidation polypeptide; Described enzymatic hydrolysis condition is: pH is 3.0,50 ℃ of temperature, enzymolysis time are that 5h, enzyme-substrate proportioning are 3000U/g; Described enzyme is an aspartic protease.
3. the preparation method of antioxidation polypeptide according to claim 2, it is characterized in that: the means of described separation and purification comprise ultrafiltration, SP-Sephadex C-25 ion-exchange chromatography, Sephadex G-50 molecular sieve and RP-HPLC RPLC.
4. require the preparation method of described antioxidation polypeptide according to right 3, it is characterized in that: the concrete steps of described separation and purification are: (1) enzymolysis product at first utilizes molecular weight to hold back scope for the ultra-filtration membrane of 8000Da and 3500Da enzymolysis product to be carried out ultra-filtration and separation, it is divided into 3 components that molecular weight ranges is different, be respectively molecular weight greater than the component of 8000Da, molecular weight between the component of 3500Da and 8000Da, molecular weight component less than 3500Da; (2) collection has the component of best anti-oxidant activity, separate with SP-Sephadex C-25 cation-exchange chromatography again, flush away absorbed component not behind the last sample, carry out linear gradient elution with 0.02mol/L, the pH4.0 acetate-sodium acetate buffer that contains 0～0.35mol/L NaCl again, flow velocity is 0.5ml/min, under 225nm, measure, measure the anti-oxidant activity of the elution fraction of each absorption peak correspondence; (3) collect the peak with best anti-oxidant activity, separate with Sephadex G-50 gel chromatography, elutriant is a deionized water, and flow velocity is 0.5ml/min, measures under 225nm, measures the anti-oxidant activity of the elution fraction of each absorption peak correspondence; (4) collect peak with best anti-oxidant activity, sharp again RP-HPLC RPLC further separates, the separation of reversed-phase HPLC is to contain the 0.1%(volume) concentration gradient of trifluoroacetic acid is that 5%～40% acetonitrile solution carries out linear elution as elutriant, used chromatographic column is Gemini 5 μ C18, applied sample amount is 100 μ L, flow velocity is 1mL/min, the detection wavelength is 215nm, measure the anti-oxidant activity of the elutriant of each absorption peak correspondence, collect and obtain three the highest components of anti-oxidant activity; (5) utilize the RP-HPLC RPLC that three components are identified, with the concentration gradient that contains the 10mM ammonium acetate is that 5%～27.5% acetonitrile solution carries out linear elution as elutriant, used chromatographic column is Gemini 5 μ C18, applied sample amount is 20 μ L, flow velocity is 1ml/min, the detection wavelength is 215nm, and three components are separated respectively and obtained single peak, promptly obtain three pure active ingredients; Utilize proteinaceous solid facies sequence analysis instrument to identify the aminoacid sequence of three peptides, the aminoacid sequence that obtains antioxidation polypeptide as claimed in claim 1 is: Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Ala-Gly.
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