CN103710416A - Preparation method and applications of rapeseed cake-sourced metal chelating peptide and peptide metal chelate - Google Patents
Preparation method and applications of rapeseed cake-sourced metal chelating peptide and peptide metal chelate Download PDFInfo
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Abstract
The invention belongs to the technical field of deep processing of rapeseed cake byproducts, and particularly relates to a preparation method and applications of rapeseed cake-sourced metal chelating peptide and peptide metal chelate. The preparation method comprises the steps: soaking and cleaning rapeseed cakes with ethanol and water in sequence, and performing solid-liquid separation to reserve a filter cake, thus obtaining a crude rapeseed protein product; adding water into the crude rapeseed protein product, regulating the pH value, then adding protease for hydrolysis reaction, then placing reaction liquid into boiling water for cooking and carrying out enzyme deactivation, to obtain hydrolysate; or, adding the protease for hydrolysis reaction, then placing reaction liquid into an autoclave for high-pressure treatment, to obtain the hydrolysate with higher hydrolysis degree; and centrifuging the hydrolysate, and drying supernate to obtain crude metal chelating peptide powder. The prepared metal chelating peptide prepared by the preparation method disclosed by the invention can be directly used as a natural antioxidant for retarding aging. In addition, the metal chelating peptide can also be used for preparing an excellent parent of the peptide metal chelate, and can be chelated with different microelements to prepare a micro trace chelating agent for supplementing nutrient elements which are lack by the human body.
Description
Technical field
The invention belongs to rapeseed cake by product deep process technology field, specifically relate to the Preparation method and use of a kind of rapeseed cake source metal chelating peptide, peptide metallo-chelate.
Background technology
Metal chelating peptide (Metal chelating peptide) refers to biologically active peptides class that can chelated metal ions.Metal ion is extensively present in the Organ and tissue of body as copper, zinc, iron etc.While there is excessive metal ion in body, easily cause the generation of active oxygen (Reactive oxygen species, ROS), and then the biomacromolecule in damage body, produce some degenerative diseases, as cardiovascular and cerebrovascular diseases, gastrointestinal inflammation and cancer etc.Metal chelating peptide itself can chelating body in free metal ion, play and suppress or reduce the effect that activity in vivo oxygen causes oxidative damage.In addition, many micro-metalss are important component parts of enzyme for organism metabolism, when micro-metals is not enough in body, also can produce a series of diseases.For example, while lacking zinc, can affect the synthetic and conversion of archaeal dna polymerase, RNA synthetic enzyme and superoxide-dismutase etc. in body, growing of human body had a negative impact.
Metal chelating peptide can by with metal ion generation sequestering action, preparation forms peptide metallo-chelate (Peptide-metal chelator).This inner complex be called as the 4th generation trace element replenisher, it be with respect to the first-generation with inorganic salts additive, as copper sulfate, zinc sulfate etc., the s-generation is with simple organic acid salt, as Zinc Gluconate, Ferrous Fumarate etc., the amino acid trace element chelated salt of the third generation.
Peptide metallo-chelate, can enter in body by the absorption approach transhipment of peptide.The absorption of peptide has advantages of that speed is fast, path is many and with the uncontested property of amino acid.Thereby inner complex can improve the bioavailability of micro-metals significantly, and effectively reach the object of supplementing the required trace element of body.At present, about the research of metal chelating peptide and inner complex thereof, be the focus of scientists extensive concern, especially there is the preparation of the bioactive peptide of excellent metal-chelating ability, especially the emphasis of this area research.
Rapeseed cake is the by product after Semen Brassicae campestris oil expression, wherein contain 30%～50% protein, at present, method about protein extraction is more, for example, in Chinese patent CN1962671A and CN102296099, announce the method for the molten sour formulation extraction rape seed protein of alkali, in CN200510056753.5, announced the method that priority water and ethanol are prepared rape seed protein.In addition, prepare in addition the method for third generation microelement chelate, as in CN1115311A, applicant by rapeseed cake be hydrolyzed, neutralization and chelatropic reaction, prepare amino acid and amino-acid chelate.
Visible, the protein resource of take in rapeseed cake processing byproduct is raw material, carries out intensive processing, prepares peptide metallo-chelate, can further solve the problem of higher value application rapeseed cake by product, prepares efficient trace element replenisher.
Summary of the invention
For the technical problem existing in prior art, one of object of the present invention is to provide a kind of rapeseed cake source metal chelating peptide.
In order to realize this object, the technical solution adopted in the present invention is: the preparation method of a kind of rapeseed cake source metal chelating peptide, and step is as follows:
1., rapeseed cake adopts ethanol and water soaking, cleaning successively, solid-liquid separation retains filter cake, obtains rapeseed protein crude product;
2., by feed liquid weight ratio, be in rapeseed protein crude product, to add water in 1: 2～1: 30, regulate pH value to 1.5～10.0;
3., by albumen enzyme-to-substrate weight ratio, be to add again proteolytic enzyme in 1: 200～1: 20, adjust the temperature to 37～60 ℃, the reaction 0.5～6h that is hydrolyzed, then reaction solution is placed in to boiling water boils 5～10min, the enzyme that goes out, obtains hydrolyzed solution;
Or;
By albumen enzyme-to-substrate weight ratio, be within 1: 200～1: 20, to add again proteolytic enzyme, adjust the temperature to 37～60 ℃, reaction 0.5～6h is hydrolyzed, again reaction solution is placed in to boiling water and boils 5～10min, enzyme goes out, then be placed in pressure kettle interior in 121 ℃ of autoclaving 2～20min, obtain the higher hydrolyzed solution of degree of hydrolysis;
4., by the centrifugal removal residue of hydrolyzed solution, the supernatant liquor drying of acquisition makes the crude product powder of metal chelating peptide.
Preferably, the preparation method of rapeseed protein crude product is: first rapeseed cake is ground into fine powder, under 4～60 ℃ of temperature condition, by feed liquid weight ratio, be the first aqueous ethanolic solution that is 60～90% through volume percent of this fine powder to be stirred to immersion 1～48h in 1: 5～1: 50, change at set intervals during this time liquid, until the liquid color after filtering is without brown or yellow; Remove supernatant liquor, adopt the clear water of at least 5 times of volumes to clean throw out, then filter, until the clear water after filtering is without brown or yellow, and then obtain being brown or lurid rapeseed protein crude product.
Preferably, hydrolysis reaction is one or more composite in stomach en-, trypsinase, pancreatin, flavor protease, Alcalase enzyme, papoid and bromeline with proteolytic enzyme.
Preferably, drying mode is that lyophilize, vacuum-drying or spraying are dry.
Further, the preparation method of rapeseed cake source metal chelating peptide also comprises the metal chelating peptide crude product powder making further carried out to the separated step that obtains high purity metal chelating peptide by metal-chelating filler, and metal-chelating filler adopts affine agarose filler.
Preferably, described affine agarose filler comprises chelating sepharose FF, efficient affine sepharose (IMAC sepharose high performance), agarose compatible medium (Ni-IDA QZT6FF and Ni-NTA QZT6FF), metallic nickel chelating sepharose 6FF (Ni sepharose6fast flow).
This metal chelating peptide crude product (or high purity metal chelating peptide) has chelating Ca
2+, Fe
2+, Fe
3+, Zn
2+, Cu
2+the ability of ion, thus prepare peptide metallo-chelate, and sequestering power is 4.6%～96.39%, is 0.62～10.19 times of homogenous quantities reduced glutathion.Feed liquid weight ratio during chelating between metal chelating peptide and metallic element is 1: 10～10: 1, and pH value in reaction is 1.5～10, and temperature of reaction is 4～80 ℃, reaction times 1～480min.
Ca
2+ion adopts CaCl
2or (with) CaSO
4as ion source; Fe
2+adopt FeCl
2or (with) FeSO
47H
2o is as ion source; Fe
3+adopt FeCl
36H
2o or (with) Fe
2(SO4)
3as ion source; Zn
2+adopt ZnSO
47H
2o or (with) ZnCl
2as ion source; Cu
2+ion adopts CuCl
22H
2o or (with) CuSO
45H
2o is as ion source.
Metal chelating peptide prepared by the present invention can directly be used as natural antioxidant and use, delaying senility, in addition, also can be used for preparing the good parent of peptide metallo-chelate, can be from different trace element chelated, be prepared into trace metal sequestrant, for supplementing body to nutrient deficiency symptom.
Accompanying drawing explanation
Fig. 1 is IMAC-Zn
2+separated vegetable seed proteolysate collection of illustrative plates.
Embodiment
Below with reference to embodiment, the present invention is carried out to further, detailed explanation, all raw materials are all purchased from preparing in market or according to prior art.
Embodiment 1:
Adopt high speed disintegrator, rapeseed cake carried out to pulverization process, get 500g raw material, add the aqueous ethanolic solution 3L of 75% (volume percent), stir and soak 24h, during every 6h, change liquid one time.After filtration, adopt 3L clear water to clean throw out and refilter, after being dried, obtain beige rapeseed protein crude product.The average yield of albumen crude product is 21.46%.
Embodiment 2:
Get 4g vegetable seed crude protein, add 100ml deionized water, adopt the NaOH solution of 1mol/L to regulate pH to 8.0, add 0.08g Alcalase enzyme, reaction mixture is carried out to enzyme digestion reaction in 55 ℃, every 15min, adopt the NaOH solution of 1mol/L to regulate pH during this time, making reaction system maintain pH is 8.0.Reaction times is while reaching 4h, and reaction solution, in the boiling water bath enzyme that goes out, subsequently, then is placed in protein hydrolyte in pressure kettle, in 121 ℃ of autoclaving 10min, obtains hydrolyzed solution.
The autoclaving hydrolyzed solution obtaining is processed through lyophilize, obtains metal chelating peptide crude product powder.While adopting TNBS method to detect 4h, the degree of hydrolysis of hydrolyzed solution is 22.31%.When peptide concentration is 5mg/ml, adopt EDTA volumetry to detect its Zn
2+average chelation percent is 84.36%.
Embodiment 3:
Get 4g vegetable seed crude protein, add 100ml deionized water, adopt the NaOH solution of 1mol/L to regulate pH to 7.0, add 0.08g papoid, reaction mixture is carried out to enzyme digestion reaction in 60 ℃, every 15min, adopt the NaOH solution of 1mol/L to regulate pH during this time, making reaction system maintain pH is 7.0.Reaction times, while reaching 1h, by hydrolyzed solution, in boiling water, the enzyme that goes out was processed 10min.
The hydrolyzed solution obtaining is processed through lyophilize, obtains metal chelating peptide crude product powder.While adopting TNBS method to detect 1h, the degree of hydrolysis of hydrolyzed solution is 20.82%.When peptide concentration is 5mg/ml, adopt EDTA volumetry to detect its Zn
2+average chelation percent is 29.79%.
Embodiment 4:
Get 4g vegetable seed crude protein, add 100ml deionized water, adopt the HCl solution of 1mol/L to regulate pH to 1.5, add 0.08g stomach en-, reaction mixture is carried out to enzyme digestion reaction in 37 ℃, during every 15min, adopt the HCl solution of 1mol/L to regulate pH, making reaction system maintain pH is 1.5.Reaction times, while reaching 2h, by hydrolyzed solution, in boiling water, the enzyme that goes out was processed 10min.
The hydrolyzed solution obtaining is processed through lyophilize, obtains metal chelating peptide crude product powder.While adopting TNBS method to detect 2h, the degree of hydrolysis of hydrolyzed solution is 15.77%.When peptide concentration is 5mg/ml, adopt EDTA volumetry to detect its Zn
2+average chelation percent is 43.02%.
Embodiment 5:
Get metal chelating peptide powder 4g prepared by Alcalase enzyme, add 100ml deionized water, regulate pH value to 8.0, by this peptide solution in 121 ℃ of autoclaving 10min, the degree of hydrolysis average out to 33.71% of the hydrolyzed solution of acquisition, Zn
2+average chelation percent is 91.36%.
Embodiment 6:
IMAC-Zn
2+chromatography column preparation:
(1) by after 25ml agarose compatible medium (Ni-IDA QZT6FF) filler dress post, with 100ml deionized water, carry out drip washing chromatography column, and then (contain 0.1M NaCl and 50mM EDTA with 250ml20mM PBS, pH7.4) carry out drip washing, use respectively more afterwards 250ml20mM PBS (containing 0.1M NaCl, pH7.4) and 250ml washed with de-ionized water pillar, to remove residual EDTA.
(2) adopt 0.2M ZnCl
2solution 250ml drip washing pillar, makes Zn
2+can be combined by the group on filler, use again afterwards 250ml washed with de-ionized water chromatography column, remove free Zn
2+ion, makes IMAC-Zn
2+chromatography column.
Column chromatography process: get metal chelating peptide crude product powder 200mg prepared by Alcalase enzyme, adopt the 20mM PBS (containing 0.1M NaCl, pH7.4) of 1ml to dissolve, use afterwards 0.45 μ m water system membrane filtration, by IMAC-Zn on filtered liquid
2+chromatography column carries out chromatographic separation.
Column chromatography condition is: column volume: 45ml; Flow velocity 1ml/min; Detect wavelength 220nm; 0-90min, level pad 20mM PBS (containing 0.1M NaCl, pH7.4); 90-180min, elution buffer 20mM PBS (containing 0.1M NaCl, pH3.5).
Wash-out collection of illustrative plates result refers to Fig. 1: wherein, F3 component is for having Zn
2+the high purity peptide of ion chelating ability.The Zn of this sample
2+ion chelating amount (when concentration is 2mg/ml) is 4.62 times of chelating peptide crude product (when concentration is 5mg/ml), is also 3.13 times of reduced glutathion (when concentration is 5mg/ml).
Embodiment 7:
Get Zinc Ions Chelated peptide crude product prepared by Alcalase enzyme, adopt deionized water to be mixed with the peptide solution of 5mg/ml.Get this solution of 1ml, add 4ml ethanol solution, the final concentration that makes ethanol is 80%.This mixed solution room temperature is placed to 30min, with whizzer, carries out centrifugal treating afterwards, acquisition be precipitated as peptide-Zinc Ions Chelated thing.
Above embodiment is illustrating that the preparation method of rapeseed cake of the present invention source metal chelating peptide, peptide metallo-chelate has been done, but scope of the present invention is not limited.
Claims (10)
1. a preparation method for rapeseed cake source metal chelating peptide, is characterized in that step is as follows:
1., rapeseed cake adopts ethanol and water soaking, cleaning successively, solid-liquid separation retains filter cake, obtains rapeseed protein crude product;
2., by feed liquid weight ratio, be in rapeseed protein crude product, to add water in 1: 2～1: 30, regulate pH value to 1.5～10.0;
3., by albumen enzyme-to-substrate weight ratio, be to add again proteolytic enzyme in 1: 200～1: 20, adjust the temperature to 37～60 ℃, the reaction 0.5～6h that is hydrolyzed, then reaction solution is placed in to boiling water boils 5～10min, the enzyme that goes out, obtains hydrolyzed solution;
Or;
By albumen enzyme-to-substrate weight ratio, be to add proteolytic enzyme in 1: 200～1: 20 again, adjust the temperature to 37～60 ℃, reaction 0.5～6h is hydrolyzed, again reaction solution is placed in to boiling water and boils 5～10min, the enzyme that goes out, is then placed in pressure kettle in 121 ℃ of autoclaving 2～20min, obtains hydrolyzed solution;
4., by the centrifugal removal residue of hydrolyzed solution, the supernatant liquor drying of acquisition makes the crude product powder of metal chelating peptide.
2. the preparation method of rapeseed cake according to claim 1 source metal chelating peptide, it is characterized in that: the preparation method of rapeseed protein crude product is: first rapeseed cake is ground into fine powder, under 4～60 ℃ of temperature condition, by feed liquid weight ratio, be the first aqueous ethanolic solution that is 60～90% through volume percent of this fine powder to be stirred to immersion 1～48h in 1: 5～1: 50, change at set intervals during this time liquid, until the liquid color after filtering is without brown or yellow; Remove supernatant liquor, adopt the clear water of at least 5 times of volumes to clean throw out, then filter, until the clear water after filtering is without brown or yellow, and then obtain being brown or lurid rapeseed protein crude product.
3. the preparation method of rapeseed cake according to claim 1 source metal chelating peptide, is characterized in that: hydrolysis reaction is one or more composite in stomach en-, trypsinase, pancreatin, flavor protease, Alcalase enzyme, papoid and bromeline with proteolytic enzyme.
4. the preparation method of rapeseed cake according to claim 1 source metal chelating peptide, is characterized in that: drying mode is that lyophilize, vacuum-drying or spraying are dry.
5. according to the preparation method of the rapeseed cake source metal chelating peptide described in claim 1～4 any one, it is characterized in that: also include the metal chelating peptide crude product powder making is further carried out to the separated step that obtains high purity metal chelating peptide by metal-chelating filler, metal-chelating filler adopts affine agarose filler.
6. the preparation method of rapeseed cake according to claim 5 source metal chelating peptide, is characterized in that: described affine agarose filler comprises chelating sepharose FF, efficient affine sepharose, agarose compatible medium, metallic nickel chelating sepharose 6FF.
7. the rapeseed cake source metal chelating peptide that as described in claim 1～6 any one prepared by method is in the purposes of the antioxidant for delaying senility.
8. a peptide metallo-chelate, is by rapeseed cake source metal chelating peptide and Ca that as described in claim 1～6 any one prepared by method
2+, Fe
2+, Fe
3+, Zn
2+or Cu
2+ion carries out chelating and makes.
9. peptide metallo-chelate according to claim 8, is characterized in that: feed liquid weight ratio during chelating between metal chelating peptide and metallic element is 1: 10～10: 1, and pH value in reaction is 1.5～10, and temperature of reaction is 4～80 ℃, reaction times 1～480min.
One kind as described in claim 8 or 9 peptide metallo-chelate making trace metal sequestrant and for supplementing the purposes of body to the shortage symptom of nutritive element.
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Cited By (4)
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| CN104232719A (en) * | 2014-09-30 | 2014-12-24 | 中国海洋大学 | Method for preparing calcium chelated peptide |
| CN107156856A (en) * | 2017-05-09 | 2017-09-15 | 安徽珠峰生物科技有限公司 | A kind of soybean source zinc chelating peptide, the preparation method and its usage of peptide chelates of zinc |
| CN105273059B (en) * | 2015-11-27 | 2018-07-20 | 福州大学 | A kind of octopus calcium chelating protein peptides and preparation method thereof |
| CN111411139A (en) * | 2020-03-02 | 2020-07-14 | 安徽省农业科学院农产品加工研究所 | Cysteine-containing peptide of mandarin fish protein source and preparation method thereof, cysteine-containing peptide-zinc chelate and preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104232719A (en) * | 2014-09-30 | 2014-12-24 | 中国海洋大学 | Method for preparing calcium chelated peptide |
| CN105273059B (en) * | 2015-11-27 | 2018-07-20 | 福州大学 | A kind of octopus calcium chelating protein peptides and preparation method thereof |
| CN107156856A (en) * | 2017-05-09 | 2017-09-15 | 安徽珠峰生物科技有限公司 | A kind of soybean source zinc chelating peptide, the preparation method and its usage of peptide chelates of zinc |
| CN111411139A (en) * | 2020-03-02 | 2020-07-14 | 安徽省农业科学院农产品加工研究所 | Cysteine-containing peptide of mandarin fish protein source and preparation method thereof, cysteine-containing peptide-zinc chelate and preparation method and application thereof |
| CN111411139B (en) * | 2020-03-02 | 2022-04-29 | 安徽省农业科学院农产品加工研究所 | Cysteine-containing peptide of mandarin fish protein source and preparation method thereof, cysteine-containing peptide-zinc chelate and preparation method and application thereof |
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