CN103936823A - Metal chelating peptide and preparation method thereof - Google Patents
Metal chelating peptide and preparation method thereof Download PDFInfo
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- CN103936823A CN103936823A CN201410078823.6A CN201410078823A CN103936823A CN 103936823 A CN103936823 A CN 103936823A CN 201410078823 A CN201410078823 A CN 201410078823A CN 103936823 A CN103936823 A CN 103936823A
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Abstract
The present invention provides a metal (Ca, Fe, Zn) chelating peptide prepared through whey protein enzymolysis by using a compound protease and a flavor protease, wherein the whey protein is adopted as the raw material and is subjected to enzymolysis by using the compound protease and the flavor protease, separation purification is performed to obtain the purified specific metal (Ca, Fe, Zn) chelating peptide, and the whole sequence of the amino acid is: eg. The prepared metal chelating peptide can be used for production of novel metal supplement agents such as peptide chelated calcium, peptide chelated iron and peptide chelated zinc, has characteristics of unique chelation mechanism, unique transport mechanism, easy absorption, safety, no toxicity and low price, can be provided for concurrently supplementing amino acids and metal ions, and can be adopted as the first choice for calcium, iron, zinc supplement agents. According to the present invention, a new thought is provided for whey protein application.
Description
Technical field
The present invention relates to a kind of metal (Ca, Fe, Zn) chelating peptide, related more specifically to a kind of metal chelating peptide that utilizes compound protease and flavor protease enzymolysis lactoalbumin to prepare, belong to biological technical field.
Background technology
Along with the research of newborn source activity peptide and the rise of product development, find to contain in whey-protein there is immunomodulatory, hypotensive, hypoglycemic, decreasing cholesterol, the bioactive potential peptide section such as anti-oxidant, antibacterial and antiviral.Whey 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.
At present, calcium deficiency is global nutrition problem, and China people are due to taking 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.
Trace elements 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 that 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
The object of the present invention is to provide a kind of metal chelating peptide that utilizes compound protease and flavor protease enzymolysis lactoalbumin 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, the aminoacid sequence of described peptide is: eg.
Described metal is Ca, Fe or Zn.
A preparation method for metal chelating peptide, taking whey-protein 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.
Described enzymatic hydrolysis condition is: concentration of substrate 5%, enzymolysis pH is 7.0,49 DEG C 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 the phosphoric acid buffer of the 0.02 mol/L pH 9.0 that contains 0-0.5 M NaCl, 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 to be that 0-30% acetonitrile solution is as elutriant gradient elution by volume ratio, flow velocity is 4 mL/min, collected volume, than the elution peak that is 20% acetonitrile and 80% water place, obtains described 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, taking the whey-protein that comes from whey 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 application that the present invention is whey-protein provides new approaches.
Brief description of the drawings
The RP-HPLC-C18 color atlas of Fig. 1 purifying whey protein source metal chelating peptide; Wherein WPH-1 is the chromatographic peak of this specificity metal chelating peptide.
Embodiment
embodiment 1
The instrument, the detection means that adopt are as follows:
The whey-protein (WPC 80) that this technology adopts, buys in HILMAR company (Texas ,Usa), 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 DEG C), 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 whey-protein and be dissolved 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 DEG C 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).
The calcium sequestering activity measuring method of preparing metal chelating peptide of the present invention, adopts o-cresolphthalein colorimetry, measures 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 DEG C 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.Taking 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
2=0.9996.
The iron sequestering activity measuring method of preparing metal chelating peptide of the present invention, adopts phenanthroline colorimetry, measures 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.
The zinc sequestering activity measuring method of preparing metal chelating peptide of the present invention, adopts EDTA volumetry, measures 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 whey-protein metal chelating peptide of remarkable activity.
Take 5.0 grams of whey-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 DEG C, 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.
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.
The elution peak with the highest metal (Ca, Fe, Zn) sequestering activity that TOYOPEARL DEAE-650M anion-exchange chromatography is separated carries out next step separation again, with Sepadex G-25 gel filtration chromatography (long 100 cm, external diameter 2.0 cm) separate, the sample that obtains best metal (Ca, Fe, Zn) sequestering activity utilizes RP-HPLC-C18 RPLC to carry out further separation and purification again.The mixed solution of self-contained 100% water of elutriant (v/v) starts, mixed solution to 30% acetonitrile and 70% water (v/v) finishes, flow velocity is that 4 ml/min carry out gradient elution, collect the elution peak that 20% acetonitrile and 80% water (v/v) are located, obtain highly purified specificity metal of the present invention (Ca, Fe, Zn) chelating peptide, as shown in Figure 1.WPH-1 peak is the chromatographic peak of this specificity 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 WPH-1 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
The specificity metal chelating peptide of purifying is utilized to ESI mass spectrograph, and ((WATERS MALDI SYNAPT Q-TOF MS, Waters Co., U.S.A) measures the aminoacid sequence of specificity metal chelating peptide.The aminoacid sequence of described metal chelating peptide is: eg.
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> metal chelating peptide and preparation method thereof
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 2
<212> PRT
<213> metal chelating peptide
<400> 1
Glu Gly
1
Claims (6)
1. a metal chelating peptide, is characterized in that: the aminoacid sequence of described peptide is: eg.
2. a kind of metal chelating peptide according to claim 1, is characterized in that: described metal is Ca, Fe or Zn.
3. a preparation method for a kind of metal chelating peptide as claimed in claim 1, is characterized in that: taking whey-protein 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: described enzymatic hydrolysis condition is: concentration of substrate 5%, enzymolysis pH is 7.0,49 DEG C of temperature, enzymolysis time are that 7 hours, enzyme-substrate weight proportion are 1:25.
5. the preparation method of a kind of metal chelating peptide according to claim 3, is characterized in that: 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.
6. 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 the phosphoric acid buffer of the 0.02 mol/L pH 9.0 that contains 0-0.5 M NaCl, 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 to be that 0-30% acetonitrile solution is as elutriant gradient elution by volume ratio, flow velocity is 4 mL/min, collected volume, than the elution peak that is 20% acetonitrile and 80% water place, obtains the described whey protein peptide that metal chelating is made a concerted effort that has.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108047307A (en) * | 2018-02-13 | 2018-05-18 | 福建农林大学 | A kind of edible medicinal bacterium source selenium chelating peptide and preparation method thereof |
| CN108047310A (en) * | 2018-02-13 | 2018-05-18 | 福建农林大学 | A kind of grifola frondosus selenium chelating pentapeptide and preparation method thereof |
| CN108276474A (en) * | 2018-02-13 | 2018-07-13 | 福建农林大学 | One species specificity selenium element chelating peptide and preparation method thereof |
| CN114671937A (en) * | 2022-03-18 | 2022-06-28 | 大连工业大学 | Polypeptide with tyrosinase inhibitory activity and preparation method thereof |
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| EP0705105A1 (en) * | 1993-05-14 | 1996-04-10 | Mallinckrodt Medical, Inc. | Metal chelates as spacer compounds in biologically active peptides |
| US8106020B2 (en) * | 2005-11-09 | 2012-01-31 | Ajinomoto Co., Inc. | Calcium receptor activator |
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| EP0705105A1 (en) * | 1993-05-14 | 1996-04-10 | Mallinckrodt Medical, Inc. | Metal chelates as spacer compounds in biologically active peptides |
| EP0705105A4 (en) * | 1993-05-14 | 1998-12-30 | Mallinckrodt Medical Inc | Metal chelates as spacer compounds in biologically active peptides |
| US8106020B2 (en) * | 2005-11-09 | 2012-01-31 | Ajinomoto Co., Inc. | Calcium receptor activator |
Non-Patent Citations (1)
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| 邢颖: "氨基酸金属离子螯合物合成条件及测定方法的研究", 《中国优秀硕士学位论文全文数据库》, 15 May 2012 (2012-05-15) * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108047307A (en) * | 2018-02-13 | 2018-05-18 | 福建农林大学 | A kind of edible medicinal bacterium source selenium chelating peptide and preparation method thereof |
| CN108047310A (en) * | 2018-02-13 | 2018-05-18 | 福建农林大学 | A kind of grifola frondosus selenium chelating pentapeptide and preparation method thereof |
| CN108276474A (en) * | 2018-02-13 | 2018-07-13 | 福建农林大学 | One species specificity selenium element chelating peptide and preparation method thereof |
| CN108276474B (en) * | 2018-02-13 | 2019-09-20 | 福建农林大学 | One species specificity selenium element chelating peptide and preparation method thereof |
| CN108047310B (en) * | 2018-02-13 | 2019-10-18 | 福建农林大学 | A kind of grifola frondosus selenium chelating pentapeptide and preparation method thereof |
| CN108047307B (en) * | 2018-02-13 | 2020-01-10 | 福建农林大学 | Edible and medicinal fungus source selenium chelating peptide and preparation method thereof |
| CN114671937A (en) * | 2022-03-18 | 2022-06-28 | 大连工业大学 | Polypeptide with tyrosinase inhibitory activity and preparation method thereof |
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