CN104402972A - Sea cucumber antioxidant polypeptide and preparation method thereof - Google Patents
Sea cucumber antioxidant polypeptide and preparation method thereof Download PDFInfo
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Abstract
The invention provides a sea cucumber antioxidant polypeptide and a preparation method thereof. The preparation method comprises the following steps: taking sea cucumber meat or sea cucumber internal organ protein as the raw materials, performing enzyme hydrolysis on the raw materials by acidic protease, and carrying out separation and purification so as to obtain the specific sea cucumber antioxidant polypeptide, wherein the complete sequence of the amino acid of the polypeptide is malsvl. The prepared sea cucumber antioxidant polypeptide can overcome the shortages of natural antioxidants, moreover can eliminate the anxieties of people on artificial antioxidant, and provides foundations for development on food-originated antioxidant polypeptide, and wide applications of the food-originated antioxidant polypeptide on food and medicine.
Description
Technical field
The invention belongs to biological technical field, specifically provide a kind of sea cucumber antioxidation polypeptide and preparation method thereof.
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, the free free radical relevant to the various diseases such as arteriosclerosis, cancer can inevitably produce along with the process of oxygen metabolism.In food, the oxidation of nutritive ingredient can produce superoxide, and it not only can affect Nutritive value of food, causes Food Quality to decline, the serious health generation disease that even also can cause absorption person.Therefore, the antioxidant of safety is found to suppress superoxide generation to be the study hotspot of biochemistry nutrition always.Because the chemosynthesis antioxidants such as BHT, TBHQ have better effect and more cheap price than natural antioxidants, therefore it has been widely used in food service industry.But, studies have found that synthetized oxidation preventive agent has accumulative carcinogenesis to organs such as human liver, spleen, lungs at present, thus cause the worry of people to its security, and start to limit its use in food gradually.So people turn to natural antioxidants sight.Alpha-tocopherol is a kind of natural antioxidants be commonly used, and it effectively can keep the stability of grease in food, but is but unfavorable for Food preservation.Therefore, we are necessary the natural antioxidants of the safety finding other source a kind of.
Polypeptide is the compounds of molecular structure between amino acid and protein, makes protein have certain physiological function.In the vital movement of the mankind, peptide in vivo digest and assimilate the amino acid being better than dissociating, and be different from delivery system in amino acid whose body.Some small peptide, while providing growth in humans, growing necessary nutritive substance, can also be prevented and cured diseases, and regulate function of human body, these have bioactive polypeptide and are called as biologically active peptides.Research finds, the polypeptide of a lot of different sources all has resistance of oxidation, and the polypeptide obtained as caseinhydrolysate, soybean protein, bovine serum albumin, Protalbinic acid, oil seed protein, wheat gliadin and zein etc. all has certain oxidation-resistance.
Domestic Holothurian machining rests on the elementary process segment at present mostly, and because resource utilization is low, the reasons such as the tankage waste that the course of processing produces cause the wasting of resources, or even environmental pollution.A large amount of protein is contained in sea cucumber meat or sea cucumber internal organ, and its composition is balanced, utilizing modern biotechnology to carry out deep processing to protein resource wherein, reasonably select enzyme etc., by making the research of antioxidation active peptides, there is more wide prospect by process optimization.
Summary of the invention
The object of the invention is to cause anxiety for the deficiency of natural antioxidants and the security of synthetic antioxidant, a kind of sea cucumber antioxidation polypeptide and preparation method thereof is provided.The sea cucumber antioxidation polypeptide anti-oxidant activity that the present invention obtains is high, compensate for the defect of natural antioxidants, eliminates the worry of people to the security of synthetic antioxidant.
In order to realize the object of the invention, the present invention adopts following technical scheme:
A kind of sea cucumber antioxidation polypeptide, the aminoacid sequence of described sea cucumber antioxidation polypeptide is: malsvl.
Prepare the method for sea cucumber antioxidation polypeptide as above: extract an albumen with sea cucumber meat or sea cucumber internal organ for raw material, then enzymolysis is carried out to it; Enzymolysis product obtains sea cucumber antioxidation polypeptide through concentrating and impurity removing, separation and purification, lyophilize.
Described enzymatic hydrolysis condition is: pH is 3.5, temperature 50 C, enzymolysis time are 6 h, enzyme-substrate proportioning is 3000U/g; Described enzyme is aspartic protease.
The method of described separation and purification comprises 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) first utilize membrane filtration system to carry out ultra-filtration and separation to enzymolysis product, obtain the polypeptide fraction of different molecular weight;
(2) component with best anti-oxidant activity is collected, be separated with SP-Sephadex C-25 cation-exchange chromatography again, non-absorbed component is washed away after loading, linear gradient elution is carried out again with acetic acid-sodium acetate buffer solution, flow velocity is 0.5ml/min, measure under 215nm, measure the anti-oxidant activity of elution fraction corresponding to each absorption peak;
(3) collect the component with best anti-oxidant activity, then be separated with Sephadex G-50 gel chromatography, elutriant is deionized water, and flow velocity is 0.5ml/min, measures under 215nm, measures the anti-oxidant activity of elution fraction corresponding to each absorption peak;
(4) component with best anti-oxidant activity is collected, sharp RP-HPLC RPLC is further separated again, then adopt the mixed solution of acetonitrile and water to carry out gradient elution, collected volume, than the elution peak being 42 % acetonitriles and 58 % water places, obtains sea cucumber antioxidation polypeptide;
(5) proteinaceous solid facies sequence analysis instrument is utilized to identify the aminoacid sequence of polypeptide.
Polypeptide fraction described in step (1) comprises molecular weight >=3000 Da, 1500-3000 Da, the component of≤1500 Da.
Acetic acid-sodium acetate buffer solution concentration described in step (2) is 0.02mol/L, pH4.5, containing 0 ~ 0.35mol/L NaCl.
When being separated in step (4), chromatographic column used is Gemini 5 μ C18, and applied sample amount is 100 μ L, and flow velocity is 1ml/min, and determined wavelength is 215nm.
In step (4), gradient elution is: the self-contained volume ratio of elutriant is that the mixed solution of 10% acetonitrile and 90% water starts, and the mixed solution being 90% acetonitrile and 10% water to volume ratio terminates.
The present invention is based on the antioxidant finding a kind of efficiency natural, with sea cucumber albumen for starting point, controlled by the cutting condition of aspartic protease, be cut into the active polypeptide with specific peptide chain length and structural domain composition, and make anti-oxidant activity be able to effective implemention.
Beneficial effect of the present invention is:
The present invention changes the extraction of existing antioxidant and the thinking and countermeasure of utilization, eliminates the side effect that synthetic antioxidant may cause, be separated the sea cucumber antioxidation polypeptide obtained and can replace traditional synthetized oxidation preventive agent; And present invention also improves China to sea cucumber protein utilization rate situation on the low side, both the efficiency utilization problem of a large amount of Holothurian Resources can have been solved, human consumer can be removed again to the misgivings of antioxidant in food safety, to the development of science and technology, economy and foodstuffs industry, there is far reaching significance.
Accompanying drawing explanation
Fig. 1 is the RP-HPLC collection of illustrative plates of sea cucumber antioxidation polypeptide, and B peak is the chromatographic peak of sea cucumber antioxidation polypeptide;
Fig. 2 is " amount-effect " relation curve of the sea cucumber antioxidation polypeptide removing DPPH free radical of purifying.
Fig. 3 is " amount-effect " relation curve of the sea cucumber antioxidation polypeptide removing ABTS free radical of purifying.
Fig. 4 is that the sea cucumber antioxidation polypeptide of purifying suppresses Fe
2+" amount-effect " relation curve of induction lipovitellinin polyunsaturated fatty acid peroxidation.
Embodiment
The preparation method of sea cucumber antioxidation polypeptide is as follows:
(1) extraction of sea cucumber albumen
Sea cucumber proteins extraction processing condition are:
Sea cucumber or sea cucumber internal organ are cleaned 3-5 time, lixiviate pH is 7.0, and Extracting temperature is 40-80 DEG C, solid-liquid ratio is 1:4-1:8 (weight ratio), and extraction time is 2-6 h, centrifugal 10000g 20 minutes, collect supernatant liquor, after filtration, concentrate and obtain sea cucumber albumen after lyophilize.
(2) enzymolysis of sea cucumber albumen
Enzyme is purchased from Shanghai biological reagent company (Chinese Shanghai).
Adopt aspartic protease enzymolysis sea cucumber albumen, protein concentration is 30mg/ml, enzymatic hydrolysis condition gets that pH is 3.5, temperature 50 C, enzymolysis time are 6h, enzyme-to-substrate is than being (3000U/g), regulate pH to stablize with 2M HCl, after hydrolysis 6h, go out in boiling water bath enzyme 15min, then room temperature is cooled to rapidly, be placed in whizzer, with the centrifugal 15min of 4000r/min, get supernatant liquor for subsequent use.
(3) concentrated, the removal of impurities of enzymolysis product
Utilize nanofiltration system to protein enzymatic hydrolyzate carry out concentrated after, add 4 times of volume ethanol sedimentation macro-molecular proteins, the centrifugal 20min of 12000r/min obtains supernatant liquor and is sea cucumber polypeptide solution.
(4) separation of enzymolysis product
Utilize membrane filtration system to carry out ultra-filtration and separation to sea cucumber polypeptide solution, the ultra-filtration membrane utilizing molecular weight to retain scope different is separated enzymolysis product, obtains the polypeptide of different molecular weight, comprises >=3000 Da, 1500-3000 Da, the components such as≤1500 Da.
(5) purifying of enzymolysis product
The component being divided into 3 molecular weight ranges different the enzymolysis product obtained, is respectively molecular weight and is greater than the component of 3000Da, molecular weight be less than 1500Da component between the component of 1500Da and 3000Da, molecular weight; Collect the component with best anti-oxidant activity, again through SP-Sephadex C-25 cation-exchange chromatography (long 20cm, diameter 1.6cm) be separated, carry out linear gradient elution with 0.02mol/L, pH4.5 acetic acid-sodium acetate buffer solution containing 0 ~ 0.35mol/L NaCl, flow velocity is 0.5ml/min; Collect and there is the component of best anti-oxidant activity, then use the long 100cm of Sephadex G-50(, diameter 2.6cm) gel chromatography is separated, and elutriant is deionized water, and flow velocity is 0.5ml/min, and elution peak is measured under 215nm; Collect the component with best anti-oxidant activity, utilize RP-HPLC RPLC to be further separated, chromatographic column used is Gemini 5 μ C18, and applied sample amount is 100 μ L, and flow velocity is 1ml/min, and determined wavelength is 215nm.The mixed solution of self-contained 10% acetonitrile of elutriant and 90% water (v/v) starts, mixed solution to 90% acetonitrile and 10% water (v/v) terminates, carry out gradient elution, collect the elution peak at 42 % acetonitriles and 58 % water (v/v) places, obtain highly purified sea cucumber antioxidation polypeptide of the present invention.
(6) determined amino acid sequence of antioxidation polypeptide
The overall amino acid sequence utilizing proteinaceous solid facies sequence analysis instrument (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) to measure antioxidation polypeptide of the present invention is malsvl.
(7) test of anti-oxidant activity
The mensuration of I DPPH radical scavenging 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.The DPPH ethanol solution of 2mL, 0.1mM is joined in the clean tube containing the different enzymolysis sample of 2mL, mixing.After ambient temperatare puts 30min, measure absorbancy in 517 nm places, light absorption value is less, shows that radical scavenging activity is stronger.
In formula, A
0be the sample solvent of the DPPH ethanol solution+2mL of 2 mL, 0.1mM, blank; A
ifor the sample of the DPPH ethanol solution+2mL of 2mL, 0.1mM; A
jfor the sample of dehydrated alcohol+2 mL of 2mL.
The mensuration of II ABTS free radical scavenging activity
With deionized water, ABTS is dissolved, make ABTS concentration reach 7mmol/L, add Potassium Persulphate, make the concentration of Potassium Persulphate be 2.45 mmol/L.Afterwards this solution is at room temperature placed in dark place to spend the night 12 ~ 16h.By ABTS free-atom aqueous solution phosphoric acid buffer (PBS, 0.2 mol/L, the pH 7.4) dilution generated, its light absorption value under 734nm is made to be 0.70.Get 0.1ml enzymolysis solution to mix with the free base fluid of 2.9ml ABTS, shake up 30s, 10 min are reacted in dark place, then the light absorption value of assaying reaction liquid under 734nm.Hydrolyzed solution is replaced to do with distilled water blank.
In formula, A
0it is the light absorption value of 2.9 mL ABTS reagent and 0.1 mL distilled water mixed solution; A
jit is the light absorption value of 2.9 mL ABTS reagent and 0.1 mL enzymolysis solution mixed solution.
The mensuration of III anti-peroxidation activity
The egg yolk fresh with equal-volume and phosphoric acid buffer (pH7.4,0.1mol/L) mixing, dilution 25 times before using also stirs with magnetic stirrer.2.4ml yolk diluent, 2.4ml 25mmol/L FeSO is added respectively in test tube
4h
2o, 200 μ L hydrolyzed solution samples, after mixing at 37 DEG C water-bath 4h, add 0.8ml 50% trichoroacetic acid(TCA) and 2ml TBA, at 95 DEG C of constant temperature 30min after mixing.After being cooled to room temperature, the centrifugal 20min of 8000r/min, gets supernatant liquor and measure light absorption value under 532nm.Replace hydrolyzed solution as blank using distilled water.
In formula, A
0for sky is from the absorbancy of control group; A
jfor the absorbancy of sample sets.
In order to understand content of the present invention, Characteristic further, hereby exemplify following examples, but the invention is not restricted to following examples.
embodiment 1
Take 5.0 grams of sea cucumber albumen deionized water dissolvings and be settled to 250ml, then using 2mol/L HCl by its pH regulator to 3.0.First this solution water-bath is heated to 50 DEG C, the ratio being then 3000U/g according to enzyme-substrate proportioning again adds the enzyme of respective amount, and enzymolysis time is 6 hours.Then go out enzyme 15 minutes in boiling water bath, centrifugal 15 minutes of 4000rpm again after cooling.Collection supernatant liquor is for subsequent use.
Utilized by supernatant liquor membrane filtration system to carry out ultra-filtration and separation to sea cucumber polypeptide solution, the ultra-filtration membrane utilizing molecular weight to retain scope different is separated enzymolysis product, obtains the polypeptide of different molecular weight, >=3000Da, 1500-3000Da, the component of≤1500 Da, and measure its anti-oxidant activity.The component that molecular weight is less than 1500Da has best anti-oxidant activity.
Collect the component that molecular weight is less than 1500Da, with SP-Sephadex C-25 cation-exchange chromatography (long 20cm, diameter 1.6cm) be further separated, linear gradient elution is carried out with 0.02mol/L, pH4.5 acetic acid-sodium acetate buffer solution containing 0.2mol/L NaCl, flow velocity is 0.5ml/min, elution peak is measured under 215nm, collects each peak and measures anti-oxidant activity.
The anti-oxidant activity the most obvious component peaks Sepadex G-50 gel filtration chromatography (long 20cm, diameter 1.6cm) separated be separated, elutriant is deionized water, and flow velocity is 0.5ml/min, and elution peak is measured under 215nm again.Collect each peak and measure anti-oxidant activity.
Utilize RP-HPLC RPLC to be further separated the best anti-oxidant activity component separated, chromatographic column used is Gemini 5 μ C18, and applied sample amount is 100 μ L, and flow velocity is 1ml/min, and determined wavelength is 215nm.The mixed solution of self-contained 10% acetonitrile of elutriant and 90% water (v/v) starts, mixed solution to 90% acetonitrile and 10% water (v/v) terminates, carry out gradient elution, collect the elution peak at 42% acetonitrile and 58 % water (v/v) places, obtain highly purified specificity sea cucumber antioxidation polypeptide of the present invention, as shown in Figure 1.B peak is the chromatographic peak of this antioxidation polypeptide, obtains highly purified sea cucumber antioxidation polypeptide.
Sea cucumber antioxidation polypeptide after purifying has very strong resistance of oxidation, and as can be seen from Fig. 2, Fig. 3 and Fig. 4, it has the ability of stronger removing DPPH free radical, ABTS free radical and anti-lipid peroxidation reaction.
Protein solid-phase sequencer (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) is utilized to measure the aminoacid sequence of the antioxidation polypeptide after purifying.Obtaining its overall amino acid sequence is: malsvl.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCE LISTING
<110> University of Fuzhou
<120> sea cucumber antioxidation polypeptide and preparation method thereof
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 6
<212> PRT
<213> aminoacid sequence
<400> 1
Met Ala Leu Ser Val Leu
1 5
Claims (9)
1. a sea cucumber antioxidation polypeptide, is characterized in that: the aminoacid sequence of described sea cucumber antioxidation polypeptide is:
malsvl。
2. prepare a method for sea cucumber antioxidation polypeptide as claimed in claim 1, it is characterized in that: extract albumen with sea cucumber meat or sea cucumber internal organ for raw material, then enzymolysis is carried out to it; Enzymolysis product obtains sea cucumber antioxidation polypeptide through concentrating and impurity removing, separation and purification, lyophilize.
3. the preparation method of sea cucumber antioxidation polypeptide according to claim 2, is characterized in that: described enzymatic hydrolysis condition is: pH is 3.5, temperature 50 C, enzymolysis time are 6 h, enzyme-substrate proportioning is 3000U/g; Described enzyme is aspartic protease.
4. the preparation method of sea cucumber antioxidation polypeptide according to claim 2, is characterized in that: the method for described separation and purification comprises ultrafiltration, SP-Sephadex C-25 ion-exchange chromatography, Sephadex G-50 molecular sieve and RP-HPLC RPLC.
5. the preparation method of sea cucumber antioxidation polypeptide according to claim 2, is characterized in that: the concrete steps of described separation and purification are:
(1) first utilize membrane filtration system to carry out ultra-filtration and separation to enzymolysis product, obtain the polypeptide fraction of different molecular weight;
(2) component with best anti-oxidant activity is collected, be separated with SP-Sephadex C-25 cation-exchange chromatography again, non-absorbed component is washed away after loading, linear gradient elution is carried out again with acetic acid-sodium acetate buffer solution, flow velocity is 0.5ml/min, measure under 215nm, measure the anti-oxidant activity of elution fraction corresponding to each absorption peak;
(3) collect the component with best anti-oxidant activity, then be separated with Sephadex G-50 gel chromatography, elutriant is deionized water, and flow velocity is 0.5ml/min, measures under 215nm, measures the anti-oxidant activity of elution fraction corresponding to each absorption peak;
(4) component with best anti-oxidant activity is collected, sharp RP-HPLC RPLC is further separated again, then adopt the mixed solution of acetonitrile and water to carry out gradient elution, collected volume, than the elution peak being 42 % acetonitriles and 58 % water places, obtains sea cucumber antioxidation polypeptide;
(5) proteinaceous solid facies sequence analysis instrument is utilized to identify the aminoacid sequence of polypeptide.
6. the preparation method of sea cucumber antioxidation polypeptide according to claim 5, is characterized in that: the polypeptide fraction described in step (1) comprises molecular weight >=3000 Da, 1500-3000 Da, the component of≤1500 Da.
7. the preparation method of sea cucumber antioxidation polypeptide according to claim 5, is characterized in that: the acetic acid-sodium acetate buffer solution concentration described in step (2) is 0.02mol/L, pH4.5, containing 0 ~ 0.35mol/L NaCl.
8. the preparation method of sea cucumber antioxidation polypeptide according to claim 5, is characterized in that: when being separated in step (4), chromatographic column used is Gemini 5 μ C18, and applied sample amount is 100 μ L, and flow velocity is 1ml/min, and determined wavelength is 215nm.
9. the preparation method of sea cucumber antioxidation polypeptide according to claim 5, it is characterized in that: in step (4), gradient elution is: the self-contained volume ratio of elutriant is that the mixed solution of 10% acetonitrile and 90% water starts, and the mixed solution being 90% acetonitrile and 10% water to volume ratio terminates.
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Cited By (9)
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| CN106589068A (en) * | 2017-02-08 | 2017-04-26 | 福州大学 | Snapper anti-oxidation polypeptide and preparation method thereof |
| CN108129552A (en) * | 2017-12-22 | 2018-06-08 | 大连深蓝肽科技研发有限公司 | The antioxidant activity peptide fragment and extracting method in a kind of sea cucumber source |
| CN110195090A (en) * | 2019-05-15 | 2019-09-03 | 池州市月亮湾生物科技有限公司 | The preparation method and gained sea cucumber intestine ovum polypeptide powder of sea cucumber intestine ovum polypeptide powder |
| CN111592582A (en) * | 2020-06-29 | 2020-08-28 | 中食都庆（山东）生物技术有限公司 | Sea cucumber intestinal peptide and preparation method and application thereof |
| CN113186243A (en) * | 2021-06-23 | 2021-07-30 | 大洲新燕(厦门)生物科技有限公司 | Method for extracting sea cucumber polypeptide from sea cucumber viscera |
| CN113416231A (en) * | 2021-05-11 | 2021-09-21 | 四川大学 | Sea squirt antioxidant polypeptide and preparation method thereof |
| CN113861272A (en) * | 2021-11-08 | 2021-12-31 | 时代生物科技（深圳）有限公司 | Sea cucumber active peptide and preparation method thereof |
| CN115651066A (en) * | 2022-12-20 | 2023-01-31 | 烟台参福元海洋科技有限公司 | Sea cucumber polypeptide, and preparation process and application thereof |
| CN118496315A (en) * | 2024-07-17 | 2024-08-16 | 威海海洋职业学院 | Preparation containing sea cucumber polypeptide and preparation method thereof |
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| CN106589068A (en) * | 2017-02-08 | 2017-04-26 | 福州大学 | Snapper anti-oxidation polypeptide and preparation method thereof |
| CN106589068B (en) * | 2017-02-08 | 2020-09-01 | 福州大学 | Sea bream antioxidant polypeptide and preparation method thereof |
| CN108129552A (en) * | 2017-12-22 | 2018-06-08 | 大连深蓝肽科技研发有限公司 | The antioxidant activity peptide fragment and extracting method in a kind of sea cucumber source |
| CN108129552B (en) * | 2017-12-22 | 2023-07-07 | 大连深蓝肽科技研发有限公司 | Sea cucumber-derived antioxidant active peptide and extraction method |
| CN110195090A (en) * | 2019-05-15 | 2019-09-03 | 池州市月亮湾生物科技有限公司 | The preparation method and gained sea cucumber intestine ovum polypeptide powder of sea cucumber intestine ovum polypeptide powder |
| CN111592582A (en) * | 2020-06-29 | 2020-08-28 | 中食都庆（山东）生物技术有限公司 | Sea cucumber intestinal peptide and preparation method and application thereof |
| CN113416231A (en) * | 2021-05-11 | 2021-09-21 | 四川大学 | Sea squirt antioxidant polypeptide and preparation method thereof |
| CN113186243A (en) * | 2021-06-23 | 2021-07-30 | 大洲新燕(厦门)生物科技有限公司 | Method for extracting sea cucumber polypeptide from sea cucumber viscera |
| CN113861272A (en) * | 2021-11-08 | 2021-12-31 | 时代生物科技（深圳）有限公司 | Sea cucumber active peptide and preparation method thereof |
| CN115651066A (en) * | 2022-12-20 | 2023-01-31 | 烟台参福元海洋科技有限公司 | Sea cucumber polypeptide, and preparation process and application thereof |
| CN115651066B (en) * | 2022-12-20 | 2024-04-30 | 烟台参福元海洋科技有限公司 | Sea cucumber polypeptide, preparation process and application thereof |
| CN118496315A (en) * | 2024-07-17 | 2024-08-16 | 威海海洋职业学院 | Preparation containing sea cucumber polypeptide and preparation method thereof |
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