CN106632597A - Marine-sourced calcium chelating peptide and preparation method thereof - Google Patents
Marine-sourced calcium chelating peptide and preparation method thereof Download PDFInfo
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- CN106632597A CN106632597A CN201710068502.1A CN201710068502A CN106632597A CN 106632597 A CN106632597 A CN 106632597A CN 201710068502 A CN201710068502 A CN 201710068502A CN 106632597 A CN106632597 A CN 106632597A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The invention provides a marine-sourced calcium chelating peptide and a preparation method thereof; the preparation method comprises: enzymatically hydrolyzing Schizochytrium limacinum meal protein as a raw material through an alkaline proteinase and a complex flavor enzyme, and separating and purifying to obtain purified specific metallic calcium chelating peptide with an amino acid complete sequence of SSV. The metallic chelating peptide prepared herein is applicable to the production of novel a calcium supplement preparation, to be specific, calcium chelating peptide; since the calcium chelating peptide has unique chelating mechanism and transport mechanism and is easy to absorb, good in safety, free of side effects, low in price and capable of supplementing amino acids and trace metal elements in the same time, the calcium chelating peptide is sure to become a first choice among calcium supplements. A brand-new idea to apply Schizochytrium limacinum meal protein is provided herein.
Description
Technical field
The present invention relates to a kind of calcium chelating peptide, has related more specifically to a kind of ocean source calcium chelating peptide and preparation method thereof,
Belong to biological technical field.
Background technology
At present, calciprivia is global nutrition problem, and calcium deficiency causes the various diseases of body, is made one in inferior health shape
State.In recent years, with reagent fast development of replenishing the calcium so that its people's consciousness of replenishing the calcium also is being continuously increased.Although these calcium tonics
The pressure of calciprivia can to a certain extent be alleviated, but but also without the situation for fundamentally improving national calcium deficiency, its main original
Because be calcium bioavilability it is relatively low.The meals composition of China is plant food, and the oxalic acid, phosphoric acid and plant in plant
Easily calcium binding forms the oxalates of slightly solubility, phosphate and phytate to the compositions such as acid in enteron aisle, reduces in body
The bioavilability of calcium.Therefore, in addition to developing calcium supplementing product, the bioavilability for improving calcium is also the key for solving calciprivia
One of method.With going deep into for scientific research, it is found that polypeptide-calcium chelate has the chelating system and transporting mechanism of uniqueness, more
Easily be absorbed by the body transhipment, peptide calcium chelate Stability Analysis of Structures.Absorption of the body to peptide calcium chelate is the absorbing path by peptide,
Such that it is able to avoid being competed with the antagonism of other absorbed metal ions, the absorptivity of calcium ion is improved, improve its biological profit
Expenditure.As can be seen here, the absorptivity of polypeptide-metallo-chelate is high, and good stability is preferable health products additive.
With ocean source activity thing Quality Research and the rise of product development, microalgae biology is determined by FAO (Food and Agriculture Organization of the United Nation)
For 21 century green health food, microalgae not only rich in nutrition content, and have excellent medical care effect.Schizochytrium limacinum is made
For the microalgae source of high-quality bait, inherently containing higher protein, and rich in can with the amino acid residue of calcium binding, because
This, extracts albumen as development of raw materials calcium chelating peptide from the schizochytrium limacinum dregs of rice, can provide a kind of effectively load for calcium preparation
Body, both increased the added value of schizochytrium limacinum protein product, and can provide new thinking for supplementation product；
Therefore, the peptide with calcium sequestering activity how is obtained, just becomes grinding of preparing that novel micro metallic element replenishers need badly
Study carefully direction.
The content of the invention
Object of the present invention is to provide a kind of utilize alkali protease and compound fertilizer production stepwise discretization fragmentation
Metal chelating peptide prepared by chytrid cake protein, enables calcium sequestering activity efficiently to realize.
For achieving the above object, the present invention is adopted the following technical scheme that：
A kind of metal chelating peptide, the amino acid sequence of the peptide is：SSV.
The metal is Ca.
A kind of preparation method of metal chelating peptide, with schizochytrium limacinum cake protein as raw material, using alkali protease and compound
Flavor protease carries out stepwise discretization to it, isolates and purifies, freeze-drying obtains metal chelating peptide.
Described first step enzymatic hydrolysis condition is：Most suitable enzyme is alkali protease, and the time is 8 h, and pH is 9, and enzyme bottom ratio is
10%, concentration of substrate is 1%.Described second step enzymatic hydrolysis condition is：Most suitable enzyme is compound fertilizer production, and enzyme bottom ratio is 10%, temperature
Spend for 40 DEG C, pH is 6, the time is 3 h50 min.
It is described isolate and purify concretely comprise the following steps：Enzymolysis product enters first with Sephadex G-25 gel filtration chromatographies
Row is separated, and eluent is deionized water, and flow velocity is 0.3 mL/min, and eluting peak is measured under 214 nm；Collect and have most
The peak of high metal sequestering activity, is further separated again using RP-HPLC-C18 RPLCs, reversed-phase HPLC
It with volume ratio is 0-40% acetonitrile solutions as eluent gradient elution that separation condition is, flow velocity is 2 mL/min, collects wash-out
Peak, obtains described metal chelating peptide.
The present invention possesses the action site with metal ion-chelant based on polypeptide, being capable of formed stable chemical combination
Thing, and polypeptide-metallo-chelate has unique chelating system and transporting mechanism, easily by absorption and transport and can supplement amino acid
With the theoretical foundation of metal, with the schizochytrium limacinum cake protein from Yu Haiyang source as raw material, through alkali protease and compound
Flavor protease stepwise discretization is optimized, and to prepare the peptide with high calcium sequestering activity, and enables metal chelating activity height
Realize on effect ground.The present invention provides a brand-new thinking for the application of schizochytrium limacinum cake protein.
Description of the drawings
The RP-HPLC-C18 chromatograms of Fig. 1 purifying schizochytrium limacinum cake proteins source metal chelating peptide；Wherein SPH-2A is the spy
The chromatographic peak of different in nature metal chelating peptide.
Specific embodiment
Embodiment 1
Using instrument, detection means it is as follows：
The schizochytrium limacinum dregs of rice that this technology is adopted, are provided by Fujian Prov. Inst. of Aquatic Products, and enzyme is limited purchased from Novi's letter biotechnology
Company（Chinese Tianjin）.The extraction of schizochytrium limacinum cake protein is carried out first, and using medicinal herb grinder the schizochytrium limacinum dregs of rice, mistake are processed
60 mesh sieves, obtain experiment raw material.The protein in the schizochytrium limacinum dregs of rice is extracted using the heavy method of alkali carries acid.Compound concentration is
The NaOH solution of 0.39mol/L, by solid-liquid ratio 1:100（w/v）30min is extracted at 90 DEG C.By the sample liquid for being obtained at 4 DEG C
20min is centrifuged with the rotating speed of 10000r/min, supernatant is taken and is abandoned precipitation.Again the pH value in supernatant is adjusted with 6mol/L HCl, adjusted
Section pH value carries out acid to protein heavy to 3.0.After standing 30min, 20min is centrifuged with the rotating speed of 10000r/min at 4 DEG C.
Finally take precipitation and abandon supernatant, then freeze-drying further digests to it.
Enzymolysis process adopts experiment of single factor, first step enzymolysis experiment to investigate to five enzymolysis factors respectively, respectively
For most suitable enzyme（Neutral proteinase, alkali protease, papain, trypsase）, the time（0.0h、0.5h、1.0h、1.5h、
2.0h、2.5h、3.0h、3.5h、4.0h、6.0h、8.0h、10.0h、12.0h）, pH（7.0、8.0、9.0、10.0、11.0、
12.0, enzyme bottom ratio（2%th, 4%, 6%, 8%, 10%, 12%, w/w）, concentration of substrate（1%th, 3%, 5%, 7%, 9%, w/v）.In first step enzyme
On the basis of solution, second step enzymolysis experiment is investigated respectively to five enzymolysis factors, most suitable enzyme（Compound fertilizer production, in
Property protease, papain, trypsase）, the time（0h、0.5h、1h、2h、3h、4h、6h、8h）, enzyme bottom ratio（4%、6%、
8%th, 10%, 12%, w/w）, pH（5.0、6.0、7.0、8.0、9.0）, temperature（40℃、50℃、60℃、70℃、80℃）.Weigh one
Quality schizochytrium limacinum protein dissolution is determined in distilled water, then its pH is adjusted to optimal pH with 2mol/L NaOH.It is first that this is molten
Liquid heating water bath, then again by different enzyme bottoms than the enzyme of addition respective amount, is opened to temperature is needed according to the predetermined reaction time
Begin to react.Then go out in boiling water bath again enzyme 10 minutes, and again 10000rpm is centrifuged 10 minutes after cooling.After supernatant collection, point
It is other that calcium metal sequestering activity is measured, to determine optimum enzymolysis condition.Obtain that there is maximum metal chela in second step enzymolysis
Closing the enzymatic hydrolysis condition of the enzymolysis liquid of activity is：Enzyme bottom is than 40 DEG C of 10%, temperature, pH6, time 3h50min.
The calcium sequestering activity assay method for preparing metal chelating peptide of the present invention, using o-cresol phthalein colorimetric method, determines gold
Chelation of the category chelating peptide to calcium ion.By the CaCl of the mmol/L of 1 mL 52With the phosphate-buffered of the mol/L of 2 mL 0.2
Liquid（pH 8.0）In adding tool plug test tube, 1 mL albumin peptide solutions are added, be placed in 37 DEG C of temperature in heated at constant temperature shaking bath
2h is educated, 10000 r/min normal temperature are centrifuged 10 min after taking-up.1 mL supernatants are taken, the mL of o-cresol phthalein nitrite ion 5 is added, is shaken
It is even.Place 10 min and light absorption value is determined at the nm of spectrophotometer 570, it is solvable by calculating in numerical value substitution calibration curve
Property calcium binding capacity.
The making of calibration curve：Standard Ca working solution is taken respectively（10 ug/ mL）0,0.2,0.4,0.6,0.8,1.0 mL
In 10 mL test tubes, the mL of deionized water 1.0,0.8,0.6,0.4,0.2,0 is added respectively, adds the mL of o-cresol phthalein nitrite ion 5,
Shake up, place 10 min and light absorption value is determined at the nm of spectrophotometer 570.With solubility calcium content（ug/mL）For horizontal seat
Mark, light absorption value does figure for ordinate, and obtaining calibration curve formula is：Y=0.0992x -0.0887, R2=0.9988.
Means of purification is separated using Sephadex G-25 molecular sieves, RP-HPLC RPLCs etc., is realized aobvious
Write active schizochytrium limacinum cake protein metal chelating peptide efficiently separates purifying.
Weigh 1.0 grams of schizochytrium limacinum cake proteins and be dissolved in 100ml distilled water → use 2mol/L NaOH regulation pH to 9.0
→ plus alkali protease → be immediately placed in 50 DEG C of shaking bath react 8h → boiling water bath go out enzyme 10min → cooling immediately →
10000r/min is centrifuged 10min, takes supernatant → thick enzymolysis liquid → adjust pH to 6.0 → plus composite flavor egg with 2mol/L NaOH
White enzyme → be immediately placed in 40 DEG C of shaking bath reacts 3h50min → boiling water bath and goes out the cooling → 10000r/ of enzyme 10min → immediately
Min is centrifuged 10min, takes supernatant standby.
By supernatant Sepadex G-25 gel filtration chromatographies（Long 100 cm, the cm of external diameter 2.0）Separated, obtained
The sample of best calcium metal sequestering activity is further separated again pure using RP-HPLC-C18 RPLCs
Change.Self-contained 100% deionized water of eluent（v/v）Mixed liquor start, to 40% acetonitrile and 60% water（v/v）Mixed liquor terminate,
Flow velocity carries out gradient elution for 2 ml/min, collects eluting peak, obtains the highly purified Specific metal calcium chelating peptide of the present invention,
As shown in Figure 1.SPH-1A peaks are the chromatographic peak of the Specific metal chelating peptide.
The Specific metal chelating peptide that purifying is obtained has very high calcium metal sequestering activity, as can be seen from Table 1, with enzyme
Solution liquid phase ratio, the metal chelating of SPH-2A makes a concerted effort had large increase.
The metal chelating of the Specific metal chelating peptide of the purifying of table 1 is made a concerted effort
To purify Specific metal chelating peptide using ESI mass spectrographs ((WATERS MALDI SYNAPT Q-TOF MS,
Waters Co., U.S.A) determine Specific metal chelating peptide amino acid sequence.The amino acid sequence of the metal chelating peptide
For：SSV.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modification, should all belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>University of Fuzhou
<120>A kind of ocean source calcium chelating peptide and preparation method thereof
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 3
<212> PRT
<213>Artificial sequence
<400> 1
Ser Ser Val
1
Claims (6)
1. a kind of ocean source metal chelating peptide, it is characterised in that：The amino acid sequence of the peptide is：SSV.
2. a kind of metal chelating peptide according to claim 1, it is characterised in that：The metal is Ca.
3. a kind of a kind of preparation method of metal chelating peptide as claimed in claim 1, it is characterised in that：With ocean schizochytrium limacinum
Cake protein is raw material, and stepwise discretization is carried out to it using alkali protease and compound fertilizer production, is isolated and purified, freeze-drying
Obtain metal chelating peptide.
4. the preparation method of a kind of metal chelating peptide according to claim 3, it is characterised in that：Described first step enzymolysis
Condition is：Most suitable enzyme is alkali protease, and the time is 8 h, and pH is 9, and enzyme bottom ratio is 10%, and concentration of substrate is 1%；Described second
Walking enzymatic hydrolysis condition is：Most suitable enzyme is compound fertilizer production, and enzyme bottom ratio is 10%, and temperature is 40 DEG C, and pH is 6, and the time is 3 h50
min。
5. the preparation method of a kind of calcium metal chelating peptide according to claim 3, it is characterised in that：The enzyme is alkaline egg
White enzyme and compound fertilizer production, two kinds of enzymes carry out stepwise discretization to schizochytrium limacinum cake protein.
6. the preparation method of a kind of metal chelating peptide according to claim 3, it is characterised in that：The tool for isolating and purifying
Body step is：Enzymolysis product is separated first with Sephadex G-25 gel filtration chromatographies, and eluent is deionized water,
Flow velocity is 0.3 mL/min, and eluting peak is measured under 214 nm；The peak with highest metal chelating activity is collected, is utilized
RP-HPLC-C18 RPLCs are further separated again, and it with volume ratio is 0- that the separation condition of reversed-phase HPLC is
Used as eluent gradient elution, flow velocity is 2 mL/min to 40% acetonitrile solution, collects eluting peak, obtains described with metal chelating
Schizochytrium limacinum cake protein peptide with joint efforts.
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| CN201710068502.1A CN106632597B (en) | 2017-02-08 | 2017-02-08 | Marine source calcium chelating peptide and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108866134A (en) * | 2018-07-13 | 2018-11-23 | 广东省农业科学院蚕业与农产品加工研究所 | A kind of chelated calcium preparation method of silkworm pupa protein polypeptide |
| CN109371084A (en) * | 2017-11-16 | 2019-02-22 | 中国水产科学研究院南海水产研究所 | It is a kind of to split the chelated calcium preparation method of pot algae peptide with antioxidant activity |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103804471A (en) * | 2014-03-06 | 2014-05-21 | 福州大学 | Method for preparing metal chelated peptide by enzyme method |
| CN103880920A (en) * | 2014-03-06 | 2014-06-25 | 福州大学 | Preparation method of metal chelating peptide |
| CN105273059A (en) * | 2015-11-27 | 2016-01-27 | 福州大学 | Octopus calcium chelating protein peptide and preparation method thereof |
| 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 |
-
2017
- 2017-02-08 CN CN201710068502.1A patent/CN106632597B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103804471A (en) * | 2014-03-06 | 2014-05-21 | 福州大学 | Method for preparing metal chelated peptide by enzyme method |
| CN103880920A (en) * | 2014-03-06 | 2014-06-25 | 福州大学 | Preparation method of metal chelating peptide |
| CN105273059A (en) * | 2015-11-27 | 2016-01-27 | 福州大学 | Octopus calcium chelating protein peptide and preparation method thereof |
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 林佳萍 等: "裂殖壶菌蛋白水解产物-钙纳米复合物的制备与表征", 《中国食品科学技术学会第十二届年会暨第八届中美食品业高层论坛论文摘要集》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109371084A (en) * | 2017-11-16 | 2019-02-22 | 中国水产科学研究院南海水产研究所 | It is a kind of to split the chelated calcium preparation method of pot algae peptide with antioxidant activity |
| CN108866134A (en) * | 2018-07-13 | 2018-11-23 | 广东省农业科学院蚕业与农产品加工研究所 | A kind of chelated calcium preparation method of silkworm pupa protein polypeptide |
| CN108866134B (en) * | 2018-07-13 | 2021-12-21 | 广东省农业科学院蚕业与农产品加工研究所 | Preparation method of silkworm pupa protein polypeptide chelated calcium |
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