CN103804471A - Method for preparing metal chelated peptide by enzyme method - Google Patents
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- CN103804471A CN103804471A CN201410079703.8A CN201410079703A CN103804471A CN 103804471 A CN103804471 A CN 103804471A CN 201410079703 A CN201410079703 A CN 201410079703A CN 103804471 A CN103804471 A CN 103804471A
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Abstract
The invention provides a method for preparing a metal chelated peptide by an enzyme method. The method comprises the steps of carrying out enzymolysis on raw materials, namely marine-sourced fish skin and fish scale protein, by adopting a complex of protamex and flavorzyme, separating to purify, and carrying out freeze drying, thereby obtaining the metal chelated peptide. The amino acid sequence of the peptide is vgpsgpa. According to the method, amino acids and small peptides play a role in promoting zinc absorption, and the amino acids or metal complexes of the peptide, e.g. Fe and Zn, have higher biological utilization ratio than that of inorganic salts and are free from toxic and side effects, so that the method has a very important significance in the research and development of biological-state zinc-protein hydrolysate chelated zinc.
Description
Technical field
The present invention relates to a kind of metal (Ca, Fe, Zn) chelating peptide, related more specifically to a kind of method that enzyme process is prepared metal chelating peptide, belong to biological technical field.
Background technology
Marine protein source biologically active peptides has high nutritive value, edible safety and physiological hygiene function, all play an important role at the aspect such as growth, growth and diseases prevention and treatment that promotes body, can be used as functional foodstuff or foodstuff additive and even pharmaceutical applications in people's daily life.
Calcium deficiency is global nutrition problem, and China people are due to take vegetable diet as main, and the phenomenon of calcium deficiency is more serious, therefore replenishes the calcium and becomes the important topic in China's dietary nutrition research.Traditional inorganic calcium salt, as calcium carbonate, calcium phosphate, calcium chloride etc., have certain destruction to supporting one's family, biological value is lower; Organic calcium salt, as citrate of lime, calcium lactate, calisanin etc., although calcium absorptivity increase, the molten mistake of its solubleness great Yi, and price is high.Research shows, polypeptide chelate calcium is due to its unique chelating system and transporting mechanism, is easily absorbed, safety non-toxic, price are low, can supplement amino acid and calcium simultaneously, and becomes the first-selection of replenishing the calcium.
Iron particularly plays an important role to growing of infant and children to human health.Although macro-molecular protein also can be combined with iron ion, these macro-molecular proteins itself also exist relative molecular mass compared with large and very difficult by the problem of intestinal mucosa.And research is found, amino acid, polypeptide chelate iron can improve the specific absorption of iron ion greatly.
Although occurring in nature zinc source is very abundant, the Absorption And Metabolism of zinc in human body is subject to the restriction of all factors, and zn deficiencie has become the public health problem of China and many developing countries.Research finds that amino acid and little peptide have the effect that promotes that zinc absorbs, and the composite metal of amino acid or peptide is as higher and have no side effect than inorganic salt in the biology utilization ratio of Fe, Zn.Therefore, research and develop this biological state zinc-protein zymolyte chelated zinc, tool is of great significance.
Therefore, how to obtain the peptide with metal-chelating activity, just become and prepare the urgent research direction of novel metal component extender.
Summary of the invention
In order to address the above problem, the invention provides a kind of metal chelating peptide that utilizes compound protease and flavor protease enzymolysis ocean source protein to prepare, metal (Ca, Fe, Zn) sequestering activity is realized efficiently.
For achieving the above object, the present invention adopts following technical scheme:
A kind of metal chelating peptide, is the peptide being made up of 7 amino acid, and the aminoacid sequence of described peptide is: vgpsgpa.
Described metal is Ca, Fe or Zn.
A preparation method for metal chelating peptide, take source, ocean fish-skin, fish scale albumen as raw material, adopts compound protease and flavor protease is composite that it is carried out to enzymolysis, and separation and purification, lyophilize obtain metal chelating peptide.
Enzymatic hydrolysis condition is: concentration of substrate 5%, and enzymolysis pH is 7.0,49 ℃ of temperature, enzymolysis time are that 7 hours, enzyme-substrate weight proportion are 1:25; Described enzyme is compound protease and flavor protease, and the composite ratio of weight of two kinds of enzymes is compound protease: flavor protease=2:1.
The concrete steps of described separation and purification are: first enzymolysis product utilizes TOYOPEARL DEAE-650M anion-exchange chromatography to separate, elutriant is that concentration gradient is 0.02 mol/L that contains 0-0.5 mol/L NaCl, the phosphoric acid buffer of pH 9.0, flow velocity is 0.5 mL/min, and elution peak is measured under 214 nm; Collection has the peak of the highest metal-chelating activity, then separates by Sephadex G-25 gel filtration chromatography, and elutriant is deionized water, and flow velocity is 0.3 mL/min, and elution peak is measured under 214 nm; Collection has the peak of the highest metal-chelating activity, utilize preparation RP-HPLC-C18 RPLC further to separate again, the separation condition of reversed-phase HPLC is as gradient eluent with 10-90% acetonitrile solution, flow velocity is 1mL/min, the self-contained volume ratio of elutriant is that the mixed solution of 10% acetonitrile and 90% water starts, and the mixed solution that is 90% acetonitrile and 10% water to volume ratio finishes, and carries out gradient elution, collected volume, than the elution peak that is 38% acetonitrile and 52% water place, obtains metal chelating peptide.
The present invention is based on polypeptide to be possessed and the action site of metal ion-chelant, compound that can be stable with its formation, and polypeptide-metallo-chelate has unique chelating system and transporting mechanism, the theoretical basis that is easily absorbed, can supplements amino acid and metal simultaneously, to come from fish-skin, fish scale albumen as starting material, by the cutting condition control of compound protease and flavor protease, cutting preparation has the peptide of high metal (Ca, Fe, Zn) sequestering activity, and metal-chelating activity is realized efficiently.The present invention provides new approaches for the application of ocean source protein.
Accompanying drawing explanation
The CLC-HPLC-C18 color atlas of Fig. 1 purifying ocean source protein metal chelating peptide.
Embodiment
embodiment 1
Preparation method is as follows:
(1) optimization of ocean source protein enzymatic hydrolysis condition
The ocean source protein that this technology adopts comes from laboratory self-control, and enzyme is believed Bioisystech Co., Ltd (Chinese Tianjin) purchased from Novi.Adopt experiment of single factor, respectively four enzymolysis factors are investigated, be respectively concentration of substrate (1%, 3%, 5% and 7%), hydrolysis temperature (40,50,55 and 60 ℃), enzyme composite than (compound protease: flavor protease=1:1,1:2,1:3 and 2:1 w/w), enzyme-substrate proportioning (1:100,1:50,1:25 and 1:20 w/w) and enzymolysis time (1,3,5,7,9 hours).Take certain mass protein dissolution in distilled water, then use 2mol/L NaOH by its pH regulator to 7.0.First this solution water-bath is heated to and needs temperature, then add again the enzyme of respective amount by different enzyme-substrate proportionings, start to react according to the predetermined reaction times.Then go out in boiling water bath again enzyme 10 minutes, cooling after centrifugal 10 minutes of 10000rpm again.Supernatant liquor is measured metal (Ca, Fe, Zn) sequestering activity respectively, to determine optimum enzymolysis condition after collecting.The enzymatic hydrolysis condition that obtains the enzymolysis solution with maximum metal sequestering activity is: concentration of substrate 5%, enzymolysis pH is 7.0,49 ℃ of temperature, enzymolysis time be that 7 hours, enzyme-substrate proportioning are 1:25(w/w); Described enzyme is compound protease and flavor protease, and the composite ratio of two kinds of enzymes is compound protease: flavor protease=2:1(w/w).
Take 5.0 grams of ocean source proteins and be dissolved in 100ml distilled water, then use 2mol/L NaOH by its pH regulator to 7.0.First this solution water-bath is heated to 49 ℃, the ratio that is then 1:25 according to enzyme-substrate proportioning again adds the enzyme of respective amount, and the mass ratio of compound protease and flavor protease is 2:1, and enzymolysis time is 7 hours.Then go out in boiling water bath enzyme 10 minutes, cooling after centrifugal 10 minutes of 10000rpm again, collect supernatant liquor for subsequent use.
(2) separation of enzymolysis product, purifying
By TOYOPEARL DEAE-650M anion-exchange chromatography (long 50 cm for supernatant liquor, external diameter 1.6cm) separate, elutriant is the phosphoric acid buffer of the 0.02 mol/L pH 9.0 of the NaCl of concentration gradient 0-0.5M, flow velocity is 0.5 mL/min, collects each peak sample and measures metal (Ca, Fe, Zn) sequestering activity.
What TOYOPEARL DEAE-650M anion-exchange chromatography was separated has a highest metal (Ca, Fe, Zn) elution peak of sequestering activity carries out next step separation again, collect and there is the highest metal (Ca by Sepadex G-25 gel filtration chromatography, Fe, Zn) peak of sequestering activity, recycling preparative RP-HPLC-C18 RPLC further separates again, the separation condition of reversed-phase HPLC is with 10-90%(v/v) acetonitrile solution is as gradient eluent, flow velocity is 1 mL/min, collect each peak and carry out metal (Ca, Fe, Zn) chelating vitality test, 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) finishes, carry out gradient elution, collect the elution peak that 38% acetonitrile and 52% water (v/v) are located, be that elution time is the elution peak of locating for 22.076 minutes, obtain highly purified metal chelating peptide.
Lyophilize obtains metal of the present invention (Ca, Fe, Zn) chelating peptide.
(3) test of metal-chelating activity
Adopt o-cresolphthalein colorimetry, measure the sequestering action of metal chelating peptide to calcium ion.By the CaCl of 1 mL 5 mmol/L
2add in tool plug test tube with the phosphate buffered saline buffer (pH 8.0) of 2 mL 0.2 mol/L, then add 1 mL white protein peptide solution, be placed in 37 ℃ of incubation 2h of thermostatically heating shaking bath, centrifugal 10 min of 10000 r/min normal temperature after taking out.Get 1 mL supernatant liquor, add o-cresolphthalein nitrite ion 5 mL, shake up.After placing 10 min, measure light absorption value in spectrophotometer 570 nm places, will in numerical value substitution typical curve, calculate solubility calcium binding capacity.
The making of typical curve: the accurate Ca working fluid of label taking (10 ug/ mL) 0,0.2,0.4 respectively, 0.6,0.8,1.0 mL are in 10 mL test tubes, add respectively deionized water 1.0,0.8,0.6,0.4,0.2,0 mL, adds o-cresolphthalein nitrite ion 5 mL, shake up, after placement 10 min, measure light absorption values in spectrophotometer 570 nm places.Take solubility calcium content (ug/ mL) as X-coordinate, light absorption value is that ordinate zou is figure, obtains typical curve formula and is: y=0.0974x-0.0402, R
20.9996.
Adopt phenanthroline colorimetry, measure the sequestering action of metal chelating peptide to iron ion.0.05 g sample is placed in to l00 mL beaker, adds 2 mL concentrated hydrochloric acids, after sample dissolves completely, be settled in l00 mL volumetric flask with distilled water.Accurately draw 5 mL sample liquid in 50 mL volumetric flasks, add HCl solution 1 mL of l mol/L, 10% oxammonium hydrochloride l mL, 0.12% phenanthroline 1 mL, then adds 10% sodium-acetate 5 mL, is diluted with water to scale, shakes up.Make reference liquid with the blank reagent solution that does not add iron, measure absorbancy at 510 nm wavelength places, will in numerical value substitution typical curve, calculate iron binding capacity.
The making of typical curve: standardized solution 0,2.0,4.0,6.0,8.0,10.0 mL that draw 10 ug/mL iron, be placed in respectively 50 mL volumetric flasks, add HCl solution 1 mL of l mol/L, 10% oxammonium hydrochloride l mL, 0.12% phenanthroline 1 mL, then add 10% sodium-acetate 5 mL, be diluted with water to scale, shake up.Make reference liquid with the blank reagent solution that does not add iron, measure absorbancys at 510 nm wavelength places, drawing standard curve, obtains typical curve formula and is: y=0.1717x+0.003, R
2=0.9994.
Adopt EDTA volumetry, measure the sequestering action of metal chelating peptide to zine ion.Weigh inner complex 100 mg in 100 mL small beakers, 50 mL that add water, add 6 mol/L salt acid numbers to drip.After shaking up, in water-bath, heating makes it to dissolve completely, is settled to l00 mL after cooling, therefrom draws l0 mL in triangular flask, flat 3 parts, adds NH
3-NH
4cI damping fluid (pH l0) l0 mL, chromium black T indicator is appropriate, then uses 0.0l mol/L Na
2eDTA drop is to blue; The milliliter number that record consumes EDTA, calculates inner complex zinc content.
Application TOYOPEARL DEAE-650M anion-exchange chromatography, Sephadex G-25 molecular sieve, RP-HPLC RPLC etc. separate means of purification, realize the high efficiency separation purifying of the metal-chelating protein peptide of remarkable activity.
Utilize protein sequencer (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) to measure the amino acid complete sequence of metal chelating peptide.
The specificity metal chelating peptide that purifying obtains has very high metal (Ca, Fe, Zn) sequestering activity, and as can be seen from Table 1, compared with enzymolysis solution, the metal chelating of chelating peptide makes a concerted effort to have had large increase.
The metal chelating of the specificity metal chelating peptide of table 1 purifying is made a concerted effort
Utilize protein sequencer (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) to measure the aminoacid sequence of specificity metal chelating peptide to the specificity metal chelating peptide of purifying.The aminoacid sequence of described metal chelating peptide is: vgpsgpa.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing 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> enzyme process is prepared the method for metal chelating peptide
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 7
<212> PRT
<213> metal chelating peptide
<400> 1
Val Gly Pro Ser Gly Pro Ala
1 5
Claims (5)
1. a metal chelating peptide, is characterized in that: the aminoacid sequence of described peptide is: vgpsgpa.
2. a kind of metal chelating peptide according to claim 1, is characterized in that: described metal is Ca, Fe or Zn.
3. the preparation method of an a kind of metal chelating peptide as claimed in claim 1, it is characterized in that: take source, ocean fish-skin, fish scale albumen as raw material, adopt compound protease and flavor protease is composite that it is carried out to enzymolysis, separation and purification, lyophilize obtain metal chelating peptide.
4. the preparation method of a kind of metal chelating peptide according to claim 3, is characterized in that: the condition of described enzymolysis is: concentration of substrate 5%, and enzymolysis pH is 7.0,49 ℃ of temperature, enzymolysis time are that 7 hours, enzyme-substrate weight proportion are 1:25; Described enzyme is compound protease and flavor protease, and the composite ratio of weight of two kinds of enzymes is compound protease: flavor protease=2:1.
5. the preparation method of a kind of metal chelating peptide according to claim 3, it is characterized in that: the concrete steps of described separation and purification are: first enzymolysis product utilizes TOYOPEARL DEAE-650M anion-exchange chromatography to separate, elutriant is that concentration gradient is 0.02 M that contains 0-0.5 M NaCl, the phosphoric acid buffer of pH 9.0, flow velocity is 0.5 mL/min, and elution peak is measured under 214 nm; Collection has the peak of the highest metal-chelating activity, then separates by Sephadex G-25 gel filtration chromatography, and elutriant is deionized water, and flow velocity is 0.3 mL/min, and elution peak is measured under 214 nm; Collection has the peak of the highest metal-chelating activity, utilize RP-HPLC-C18 RPLC further to separate again, the separation condition of reversed-phase HPLC is as gradient eluent with 10-90% acetonitrile solution, flow velocity is 1mL/min, the self-contained volume ratio of elutriant is that the mixed solution of 10% acetonitrile and 90% water starts, and the mixed solution that is 90% acetonitrile and 10% water to volume ratio finishes, and carries out gradient elution, collected volume, than the elution peak that is 38% acetonitrile and 52% water place, obtains metal chelating peptide.
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Cited By (6)
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| CN103960699A (en) * | 2014-05-23 | 2014-08-06 | 中国海洋大学 | Euphausia superba peptide replenisher containing calcium, iron and zinc |
| CN104725474A (en) * | 2015-03-18 | 2015-06-24 | 浙江海洋学院 | Calcium-binding peptide of tuna liver protein source and preparation method and application thereof |
| CN104928337A (en) * | 2015-04-15 | 2015-09-23 | 浙江海洋学院 | Navodon fishskin zinc chelating peptide |
| CN105273051A (en) * | 2015-11-27 | 2016-01-27 | 东山博广天兴食品股份有限公司 | Method for preparing octopus calcium chelating protein peptide by utilizing double enzymes for synergetic hydrolysis |
| CN106632597A (en) * | 2017-02-08 | 2017-05-10 | 福州大学 | Marine-sourced calcium chelating peptide and preparation method thereof |
| CN105273059B (en) * | 2015-11-27 | 2018-07-20 | 福州大学 | A kind of octopus calcium chelating protein peptides and preparation method thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103960699A (en) * | 2014-05-23 | 2014-08-06 | 中国海洋大学 | Euphausia superba peptide replenisher containing calcium, iron and zinc |
| CN103960699B (en) * | 2014-05-23 | 2015-12-02 | 中国海洋大学 | A kind of krill peptide replenishers containing calcium iron zinc |
| CN104725474A (en) * | 2015-03-18 | 2015-06-24 | 浙江海洋学院 | Calcium-binding peptide of tuna liver protein source and preparation method and application thereof |
| CN104928337A (en) * | 2015-04-15 | 2015-09-23 | 浙江海洋学院 | Navodon fishskin zinc chelating peptide |
| CN104928337B (en) * | 2015-04-15 | 2020-08-04 | 浙江海洋学院 | Navodon septentrionalis fish skin zinc chelating peptide |
| CN105273051A (en) * | 2015-11-27 | 2016-01-27 | 东山博广天兴食品股份有限公司 | Method for preparing octopus calcium chelating protein peptide by utilizing double enzymes for synergetic hydrolysis |
| CN105273059B (en) * | 2015-11-27 | 2018-07-20 | 福州大学 | A kind of octopus calcium chelating protein peptides and preparation method thereof |
| CN106632597A (en) * | 2017-02-08 | 2017-05-10 | 福州大学 | Marine-sourced calcium chelating peptide and preparation method thereof |
| CN106632597B (en) * | 2017-02-08 | 2020-09-01 | 福州大学 | Marine source calcium chelating peptide and preparation method thereof |
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