CN106866785A - A kind of calcium chelating peptide and preparation method thereof - Google Patents
A kind of calcium chelating peptide and preparation method thereof Download PDFInfo
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- CN106866785A CN106866785A CN201710246306.9A CN201710246306A CN106866785A CN 106866785 A CN106866785 A CN 106866785A CN 201710246306 A CN201710246306 A CN 201710246306A CN 106866785 A CN106866785 A CN 106866785A
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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
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- 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 kind of calcium chelating peptide and preparation method thereof, with schizochytrium limacinum cake protein as raw material, using alkali protease and compound fertilizer production stepwise discretization, then by isolating and purifying the specific calcium calcium chelating peptide for being purified, overall amino acid sequence is：yl.Calcium chelating peptide prepared by the present invention can be used to produce calcium nutritious supplementary pharmaceutical, there is the chelating system and transporting mechanism of uniqueness because of it, and easily absorbed, safety non-toxic has no side effect, first-selection that is cheap, while supplement amino acid and calcium ion, can will turning into calcium complement agent.The present invention provides a brand-new thinking for the application of schizochytrium limacinum cake protein.
Description
Technical field
The present invention relates to a kind of calcium chelating peptide, a kind of utilization alkali protease and composite flavor albumen are related more specifically to
Calcium chelating peptide prepared by enzyme stepwise discretization schizochytrium limacinum cake protein, belongs to biological technical field.
Background technology
Microalgae biology is defined as 21 century green health food by FAO (Food and Agriculture Organization of the United Nation), and not only nutritional ingredient is rich for microalgae
Richness, and have excellent medical care effect.Schizochytrium limacinum belongs to microalgae source biology, and it contains rich in protein, at these
Rich in the glutamic acid and aspartic acid that can provide coordinate bond in protein, can be effectively as calcium ion part and calcium constituent shape
Into chelate, while microelement-supplementing, the schizochytrium limacinum albumen of high nutritive value can be made full use of again.
At present, calciprivia is global nutrition problem.In recent years, with reagent fast development of replenishing the calcium, sales volume is year by year
Rise, but the calcium deficiency situation of people is not improved fundamentally, its main cause be calcium bioavilability
It is relatively low.As long as the meals composition plant food of China, and the composition such as oxalic acid, phosphoric acid and phytic acid in plant is easily in enteron aisle
Middle calcium binding forms oxalates, phosphate and the phytate of slightly solubility, so as to reduce the bioavilability of calcium in body.This
Outward, some inorganic calciums need to rely on hydrochloric acid in gastric juice decomposition, and the bioavilability of this calcium for the abnormal crowd of some gastric acid secretions is then
It is just lower.Therefore, except develop calcium supplementing product in addition to, improve calcium bioavilability be also solve calciprivia key method it
One.With going deep into for scientific research, have been reported that and show, polypeptide-calcium chelate has unique chelating system and transporting mechanism, than
Play amino acid chelated calcium and be easier to the transhipment that is absorbed by the body.Peptide calcium chelate Stability Analysis of Structures, into intestines and stomach after can avoid receiving
The precipitation and absorption of phytic acid and oxalic acid in food to calcium ion；Absorption of the body to peptide calcium chelate simultaneously is by the suction of peptide
Passage is received rather than Calcium-ion absorption passage, therefore can avoid being competed with the antagonism of other absorbed calcium ions, so as to carry
The absorptivity of high-calcium ionic, improves its bioavilability.As can be seen here, the absorptivity of polypeptide-calcium chelate is high, good stability,
It is preferable health products additive.
Therefore, the peptide with calcium sequestering activity how is obtained, is just turned into and is prepared what novel micro calcium constituent replenishers were needed badly
Research direction.
The content of the invention
Alkali protease and compound fertilizer production stepwise discretization fragmentation are utilized object of the present invention is to provide one kind
Calcium chelating peptide prepared by chytrid cake protein, enables calcium sequestering activity efficiently to realize.
To achieve the above object, the present invention is adopted the following technical scheme that：
A kind of calcium chelating peptide, the amino acid sequence of the peptide is：YL.
A kind of preparation method of calcium chelating peptide, with schizochytrium limacinum cake protein as raw material, using alkali protease and compound wind
Taste protease carries out stepwise discretization to it, isolates and purifies, freeze-drying obtains calcium 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
10wt.%, concentration of substrate is 1wt.%.Described second step enzymatic hydrolysis condition is：Most suitable enzyme is compound fertilizer production, and enzyme bottom ratio is
10 wt.%, temperature is 40 DEG C, and pH is 6, and the time is the min of 3 h 50.
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；Collecting has most
The peak of high calcium sequestering activity, is further separated again using RP-HPLC-C18 RPLCs, reversed-phase HPLC point
It with volume fraction is 0-40% acetonitrile solutions as eluent gradient elution to be from condition, and flow velocity is 2 mL/min, collects wash-out
Peak, freeze-drying obtains described calcium chelating peptide.
The present invention possesses the action site chelated with calcium ion based on polypeptide, is capable of the compound of formed stabilization,
And polypeptide-calcium chelate has unique chelating system and transporting mechanism, is easily absorbed, can be while supplementing the reason of amino acid and calcium
It is raw material with the schizochytrium limacinum cake protein from Yu Haiyang source, by alkali protease and compound fertilizer production by basis
The cutting condition control of stepwise discretization, cutting prepares the peptide with high calcium sequestering activity, and enables calcium sequestering activity efficiently
Realize.The present invention provides a brand-new thinking for the application of schizochytrium limacinum cake protein.
Brief description of the drawings
The RP-HPLC-C18 chromatograms of Fig. 1 purifying schizochytrium limacinum cake proteins source calcium chelating peptide；Wherein SPH-1A is the spy
The chromatographic peak of different in nature calcium chelating peptide.
Specific embodiment
Embodiment 1
The instrument of use, detection means are as follows：
The schizochytrium limacinum dregs of rice that this technology is used, 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 the schizochytrium limacinum dregs of rice, mistake are processed using medicinal herb grinder
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.The sample liquid that will be obtained is at 4 DEG C
20min is centrifuged with the rotating speed of 10000r/min, supernatant is taken and is abandoned precipitation.The pH value in supernatant is adjusted with 6mol/L HCl again, is 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 taking precipitation abandons supernatant, and then freeze-drying further digests to it.
Enzymolysis process uses experiment of single factor, and first step enzymolysis experiment is investigated to five enzymolysis factors respectively, respectively
It is 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 to five enzymolysis factors respectively, 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, is then adjusted to optimal pH its pH with 2mol/L NaOH.It is first that this is molten
To temperature is needed, then enzyme again by different enzyme bottoms than addition respective amount, opens liquid heating water bath according to the predetermined reaction time
Begin to react.Then go out in boiling water bath enzyme 10 minutes again, and 10000rpm is centrifuged 10 minutes again after cooling.After supernatant collection, point
It is other that calcium sequestering activity is measured, to determine optimum enzymolysis condition.Obtain that there is maximum calcium sequestering activity in second step enzymolysis
The enzymatic hydrolysis condition of enzymolysis liquid be：Enzyme bottom is than 40 DEG C of 10%, temperature, pH6, time 3h50min.
The calcium sequestering activity assay method for preparing calcium chelating peptide of the invention, using o-cresol phthalein colorimetric method, determines calcium chela
Close chelation of the peptide to calcium ion.By the CaCl of the mmol/L of 1 mL 52With the phosphate buffer of the mol/L of 2 mL 0.2
（pH 8.0）Add in tool plug test tube, add 1 mL albumin peptide solutions, be placed in 37 DEG C of incubations in heated at constant temperature shaking bath
2h, 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, shaken up.
Place 10 min and light absorption value is determined at the nm of spectrophotometer 570, solubility calcium is calculated during numerical value is substituted into standard curve
Binding capacity.
The making of standard curve：Standard Ca working solutions are 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）It is 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 calcium chelating peptide efficiently separates purifying.
Weigh 1.0 grams of schizochytrium limacinum cake proteins to be dissolved in 100ml distilled water, pH adjusted to 9.0 with 2mol/L NaOH,
Plus alkali protease, it is immediately placed in 50 DEG C of shaking bath and reacts 8h, boiling water bath goes out after enzyme 10min, cools down immediately,
10000r/min is centrifuged 10min, takes supernatant and obtains thick enzymolysis liquid, and pH to 6.0, plus composite flavor albumen are adjusted with 2mol/L NaOH
Enzyme, is immediately placed in 40 DEG C of shaking bath and reacts 3h50min, and boiling water bath goes out enzyme 10min, cools down immediately, 10000r/min from
Heart 10min, takes supernatant standby.
By supernatant Sepadex G-25 gel filtration chromatographies（100 cm, the cm of external diameter 2.0 long）Separated, obtained
The sample of best calcium sequestering activity is further isolated and purified again using RP-HPLC-C18 RPLCs.
Self-contained 100% deionized water of eluent（v/v）Mixed liquor start, to 40% acetonitrile and 60% water（v/v）Mixed liquor terminate, flow
Speed carries out gradient elution for 2 ml/min, collects eluting peak, and freeze-drying obtains the specific calcium calcium chela of high-purity of the invention
Peptide is closed, as shown in Figure 1.SPH-1A peaks are the chromatographic peak of the specific calcium chelating peptide.
The specific calcium chelating peptide that purifying is obtained has calcium calcium sequestering activity very high, as can be seen from Table 1, with enzymolysis liquid
Compare, the calcium chelating ability of SPH-1A has large increase.
The calcium chelating ability of the specific calcium chelating peptide of the purifying of table 1
Specific calcium chelating peptide to purifying utilizes ESI mass spectrographs ((WATERS MALDI SYNAPT Q-TOF MS, Waters
Co., U.S.A) determine the amino acid sequence of specific calcium chelating peptide.The amino acid sequence of the calcium chelating peptide is：YL.
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 covering scope of the invention.
SEQUENCE LISTING
<110>University of Fuzhou
<120>A kind of calcium chelating peptide and preparation method thereof
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 2
<212> PRT
<213>Calcium chelating peptide
<400> 1
Tyr Leu
1
Claims (5)
1. a kind of calcium chelating peptide, it is characterised in that：The amino acid sequence of the peptide is：YL.
2. a kind of calcium chelating peptide according to claim 1, it is characterised in that：The peptide is chelated calcium protein peptides.
3. a kind of preparation method of calcium chelating peptide as claimed in claim 1, it is characterised in that：With schizochytrium limacinum cake protein as former
Material, stepwise discretization is carried out to it using alkali protease and compound fertilizer production, isolate and purify, freeze-drying obtain calcium chelating
Peptide.
4. the preparation method of a kind of calcium chelating peptide according to claim 3, it is characterised in that：First step enzymatic hydrolysis condition is：
Enzyme is alkali protease, and the time is 8 h, and pH is 9, and enzyme bottom ratio is 10wt.%, and concentration of substrate is 1wt.%；Second step enzymatic hydrolysis condition
For：Enzyme is compound fertilizer production, and enzyme bottom ratio is 10wt.%, and temperature is 40 DEG C, and pH is 6, and the time is the min of 3 h 50.
5. the preparation method of a kind of calcium chelating peptide according to claim 3, it is characterised in that：It is described isolate and purify it is specific
Step is：Enzymolysis product is separated first with Sephadex G-25 gel filtration chromatographies, and eluent is deionized water, stream
Speed is 0.3 mL/min, and eluting peak is measured under 214 nm；The peak with most high calcium sequestering activity is collected, using RP-
HPLC-C18 RPLCs are further separated again, and it with volume fraction 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 calcium chelating
The schizochytrium limacinum cake protein peptide of power.
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Cited By (4)
| 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 |
| CN110627896A (en) * | 2019-09-03 | 2019-12-31 | 华南农业大学 | Calcium chelating peptide and preparation method and application thereof |
| CN110669127A (en) * | 2019-09-03 | 2020-01-10 | 华南农业大学 | Novel calcium chelating peptide and preparation method and application thereof |
| CN113397034A (en) * | 2021-05-31 | 2021-09-17 | 济南大学 | Sesame peptide-calcium chelate as well as preparation method and application thereof |
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| 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 |
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| CN110669127A (en) * | 2019-09-03 | 2020-01-10 | 华南农业大学 | Novel calcium chelating peptide and preparation method and application thereof |
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| CN113397034A (en) * | 2021-05-31 | 2021-09-17 | 济南大学 | Sesame peptide-calcium chelate as well as preparation method and application thereof |
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