CN102796193A - Method for extracting ovotransferrin from egg white - Google Patents
Method for extracting ovotransferrin from egg white Download PDFInfo
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- CN102796193A CN102796193A CN201210254812XA CN201210254812A CN102796193A CN 102796193 A CN102796193 A CN 102796193A CN 201210254812X A CN201210254812X A CN 201210254812XA CN 201210254812 A CN201210254812 A CN 201210254812A CN 102796193 A CN102796193 A CN 102796193A
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Abstract
The invention discloses a method for extracting ovotransferrin from egg white, and the method comprises the following steps of: saturating an egg white diluent and iron; and combining ethanol precipitation and QSepharoseFF anion exchange chromatography. As transferrin saturation is carried out on the ovotransferrin in the egg white, the stability of the ovotransferrin is improved, the ovotransferrin can keep a natural structure in an ethanol solution with a certain concentration, and further the ovotransferrin can be extracted by using ethanol. The method for extracting the ovotransferrin from the egg white is simple in process flow and short in extraction cycle, and the obtained ovotransferrin product is high in purity and good in recovery rate.
Description
Technical field
The present invention relates to method for extracting protein, be specifically related to a kind of method of from Ovum Gallus domesticus album, extracting ovotransferrin.
Background technology
Lf lactoferrin in ovotransferrin in the Ovum Gallus domesticus album and the Mammals breast milk is similar, is bringing into play the raising immunologic function in vivo, is resisting bacterium and virus infection, keeps the vital role that the body normal growth is grown.Especially with the high affinity of iron ion, make it take on the vital task of transporting iron in vivo, Mammals iron has been absorbed crucial effects.
At present existing several different methods is separated the preparation ovotransferrin, but all has some shortcomings.Early stage main salting-out process or the organic solvent precipitation method of adopting, the ovotransferrin purity of acquisition is not high, and after too high salt and organic solvent processing, proteic natural structure and activity are destroyed.At present, mainly adopt liquid chromatography to obtain ovotransferrin, but present chromatography product purity and yield are not high; And the method that generally needs the employing ion exchange chromatography to combine with gel permeation chromatography or affinity chromatography, the multistep chromatography often needs exchange buffering liquid or solution is concentrated complicated operation between each step chromatography; Extraction process is consuming time very long; Can reach 3 ~ 5 days, in addition, the gel permeation chromatography treatment capacity is very little; And affinity chromatography filler cost is expensive, and these problems are seriously restricting the extraction of ovotransferrin.
Summary of the invention
The purpose of this invention is to provide a kind of method of from Ovum Gallus domesticus album, extracting ovotransferrin; The present invention is directed to the deficiency that exists in the prior art; The technical scheme that adopts ethanol precipitation to combine with Q Sepharose FF anion-exchange chromatography; And confirmed suitable processing parameter, overcome that complicated operation in the prior art, treatment capacity are little, product purity and the low problem of yield.
Above-mentioned purpose realizes through following technical scheme:
A kind of method of from Ovum Gallus domesticus album, extracting ovotransferrin may further comprise the steps:
(1) egg white pre-treatment: in the egg white diluent, add NaHCO
3And NaCl, make concentration be respectively 10 ~ 50mmol/L and 20 ~ 100mmol/L, adding concentration then is that 0.01mol/L, volume are the FeCl of egg white diluent 2%
3, fully stir, obtain the egg white diluent of saturated iron;
(2) extract ovotransferrin: in the egg white diluent of saturated iron, add ethanol, make the alcoholic acid volumetric concentration reach 35-45%, spinning gets supernatant, and freeze-drying is the ovotransferrin bullion;
(3) separation and purification: is in the Tris-HCl damping fluid of 8.0-8.5 with the ovotransferrin dissolving crude product in pH; Separate with Q Sepharose FF chromatographic column; PH with containing 0,0.06 ~ 0.12,0.25 ~ 0.5 and 2 mol/L sodium-chlor is the Tris-HCl buffer solution for gradient elution of 8.0-8.5; Collect the elutriant of 0.06 ~ 0.12 mol/L sodium-chlor, water dialysis postlyophilization obtains finished product.
In the step (1), said egg white diluent is mixed by Ovum Gallus domesticus album and equal-volume water.
In the step (1), add NaHCO
3And NaCl, preferably make concentration be respectively 20mmol/L and 50mmol/L.
In the step (2), preferably make the alcoholic acid volumetric concentration reach 40%.
The beneficial effect of present method is:
1, after the present invention carries out the saturated pre-treatment of iron to ovotransferrin in the Ovum Gallus domesticus album; Improved the stability of ovotransferrin; Make it in certain density ethanolic soln, keep natural structure, thereby promptly obtain to be rich in the bullion of ovotransferrin with a step ethanol sedimentation.Compare with salting-out process commonly used at present, present method can reduce the dialysis desalting step of the preceding sample of chromatography, has simplified technical process greatly, has shortened extracting cycle;
2, the ovotransferrin product purity that obtains is high, and the recovery is good.Select Q Sepharose FF as the ion exchange chromatography filler, this filler has advantages such as production cost is low, stable in properties, suitable large-scale industrial production.
Description of drawings
Fig. 1 is the SDS-polyacrylamide gel electrophoresis figure of the centrifugal back of different concentration ethanol deposition egg white supernatant.
Fig. 2 is the anion-exchange chromatography collection of illustrative plates of ovotransferrin bullion.
Fig. 3 is the SDS-polyacrylamide gel electrophoresis figure of product (3) and ovotransferrin standard substance (4) behind egg white (1), the thick product of ethanol sedimentation (2), the chromatography.
Embodiment
Below in conjunction with accompanying drawing and specific embodiment the present invention is described further.
A kind of method of from Ovum Gallus domesticus album, extracting ovotransferrin may further comprise the steps:
1, egg white pre-treatment:
(1) fresh egg is beaten eggs, use yolk separator separates egg white and yolk, collect egg white;
(2) get 1000mL egg white, in egg white, add isopyknic zero(ppm) water while stirring, mix, promptly get the egg white diluent;
(3) in the egg white diluent, add 3 ~ 4g sodium hydrogencarbonate while stirring; The volumetric molar concentration that makes sodium hydrogencarbonate is about 20mM, adds about 6 ~ 9g sodium-chlor simultaneously, and the volumetric molar concentration that makes sodium-chlor is about 50mM; Fully stir, add the FeCl of 40mL 0.01mol/L then
3Solution, fully stirring makes the abundant iron of ovotransferrin in the egg white saturated.
2, extract ovotransferrin:
Add absolute ethyl alcohol, making the alcoholic acid final concentration is 40%, stirs 1min, and produce deposition in the solution this moment, is under 3000 rev/mins the condition centrifugal 20 minutes at 4 ℃, rotating speed then, obtains supernatant; Freeze-drying gets the ovotransferrin bullion.
3, separation and purification:
(1) with Q Sepharose FF anion exchange filler be packed into chromatography column (in 50 * 5cm), with the Tris-HCl damping fluid (pH8.0,50mM), with the flow velocity balance chromatography column of 2mL/min 2 hours;
(2) with the ovotransferrin bullion 5g that obtains be dissolved in 250mL Tris-HCl damping fluid (pH8.0,50mM) in, 4 ℃ down 10000 * g be injected into then in the anion-exchange chromatography post to remove a small amount of insolubles in centrifugal 10 minutes;
(3) earlier with Tris-HCl damping fluid (pH8.0; 50mM) flow velocity with 2mL/min washes chromatography column; Subsequently respectively with contain 0.08,0.3 and the Tris-HCl damping fluid of 2M sodium-chlor (pH8.0 50mM) carries out wash-out with identical flow velocity, collects the elutriant of 0.08M sodium-chlor; With distill water dialysis four times, lyophilize.
4, SDS-gathers propionic acid amide gel electrophoresis evaluation:
Adopt the SDS-polyacrylamide gel of 12% concentration to carry out electrophoresis.The lyophilized powder of sample to be analyzed is taken by weighing about 10 milligrams; Be dissolved in 5 milliliters the distilled water, fully get 80 microlitre solution after the dissolving, add the albumen sample-loading buffer (5 times of concentration) of 20 microlitres; Boiling water bath heated about 5 minutes behind the mixing; Get 5 microlitres after the cooling and be injected in the point sample hole, initial concentration voltage 80V, separation gel voltage are that 120V carries out electrophoresis.After electrophoresis is accomplished; At first use stationary liquid (50% ethanol; 10% acetate) gel is fixed 30 minutes, use Xylene Brilliant Cyanine G R-250 solution (R-250 content 0.1% contains 25% ethanol, 8% acetate simultaneously) to dye 30 minutes down then at 45 ℃; Be transferred to decolouring in the destainer (25% ethanol and 8% acetate) after dyeing is accomplished, to background color take off, band clear till.
The electrophoresis result of the centrifugal back of different concentration ethanol deposition egg white supernatant is as shown in Figure 1.Increase along with alcohol concn; The band of ovalbumin in the supernatant (between 40 ~ 55 kDa) shoals gradually; When alcohol concn was 40%, the ovalbumin band disappeared basically, was mainly ovotransferrin in the supernatant; Explain that 40% alcohol concn can precipitate the overwhelming majority's ovalbumin, thereby obtain to be rich in the supernatant of ovotransferrin.
The ovotransferrin bullion that ethanol sedimentation is obtained separates through anion-exchange chromatography, and the result is as shown in Figure 2." elution peak 2 " carried out electrophoretic analysis; The result sees Fig. 3, and swimming lane 1 ~ 4 is followed successively by sample and ovotransferrin standard substance behind egg white, ovotransferrin bullion, the ovotransferrin chromatography, can be known by figure; Through behind the chromatography; The ovotransferrin sample purity is very high, and the article that are near the mark show and adopt technology of the present invention can obtain highly purified ovotransferrin.
5, the purity testing and the recovery are calculated:
(1) proteinaceous product purity testing: the ovotransferrin product gas purity is measured with the SDS-polyacrylamide gel electrophoresis.Adopt electrophoretic band 2 and band 3 in software (the Gel-pro analyzer Version 4.0) analysis 3, the purity that obtains band 2 is 56.23%, and the purity of band 3 is 87.54%.Bullion purity after promptly handling through 40% ethanol sedimentation is 56.23%, and purity is 87.54% behind the chromatography purification.
(2) recovery is calculated
According to quality, protein content and the purity of purifying gained range protein product from the 1000g Ovum Gallus domesticus album, use each recovery of protein rate of following formula calculating gained,
According to bibliographical information, protein contnt calculates with 10.2% in the fresh albumen.Egg white 1000g use in experiment, must thick product 19.56g behind the ethanol sedimentation, with thick product divide 5 times, about 5g is dissolved in the 250mL Tris-HCl damping fluid at every turn, obtaining the ovotransferrin total amount behind the anion-exchange chromatography altogether is 9.53g; The theoretical content of ovotransferrin is about 12% in the egg white, and the ovotransferrin total amount is 12.24g in the 1000g egg white, so the total yield of ovotransferrin is 77.9%.
Claims (4)
1. a method of from Ovum Gallus domesticus album, extracting ovotransferrin is characterized in that, may further comprise the steps:
(1) egg white pre-treatment: in the egg white diluent, add NaHCO3 and NaCl, make concentration be respectively 10 ~ 50mmol/L and 20 ~ 100mmol/L, adding concentration then is that 0.01mol/L, volume are the FeCl of egg white diluent 2%
3Solution fully stirs, and obtains the egg white diluent of saturated iron;
(2) extract ovotransferrin: in the egg white diluent of saturated iron, add ethanol, make the alcoholic acid volumetric concentration reach 35-45%, the spinning supernatant, freeze-drying gets the ovotransferrin bullion;
(3) separation and purification: is in the Tris-HCl damping fluid of 8-8.5 with the ovotransferrin dissolving crude product in pH; Separate with Q Sepharose FF chromatographic column; PH with containing 0,0.06 ~ 0.12,0.25 ~ 0.5 and 2 mol/L sodium-chlor is the Tris-HCl buffer solution elution of 8-8.5; Collect the elutriant of 0.06 ~ 0.12 mol/L sodium-chlor, water dialysis postlyophilization obtains finished product.
2. the method for from Ovum Gallus domesticus album, extracting ovotransferrin as claimed in claim 1 is characterized in that said egg white diluent is made up of Ovum Gallus domesticus album and equal-volume water.
3. the method for from Ovum Gallus domesticus album, extracting ovotransferrin as claimed in claim 1 is characterized in that, in the step (1), adds NaHCO
3And NaCl, make concentration be respectively 20mmol/L and 50mmol/L.
4. the method for from Ovum Gallus domesticus album, extracting ovotransferrin as claimed in claim 1 is characterized in that, in the step (2), makes the alcoholic acid volumetric concentration reach 40%.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105998059A (en) * | 2016-06-22 | 2016-10-12 | 华中农业大学 | Preparation method of transferrin and Fe combined Fe supplementing agent |
| CN107253990A (en) * | 2017-08-11 | 2017-10-17 | 山东阿斯可来生物技术有限公司 | A kind of preparation method that ovotransferrins and its freeze-dried powder are extracted from egg |
| CN109609480A (en) * | 2019-01-21 | 2019-04-12 | 华中农业大学 | The extracting method of protein in a kind of egg white |
| CN110204609A (en) * | 2019-05-31 | 2019-09-06 | 华中农业大学 | The method and its albumen iron product of ovotransferrins are extracted in a kind of industrialization |
| CN111153986A (en) * | 2020-02-26 | 2020-05-15 | 仲恺农业工程学院 | Method for extracting ovotransferrin from pigeon eggs |
-
2012
- 2012-07-23 CN CN201210254812XA patent/CN102796193A/en active Pending
Non-Patent Citations (3)
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| 佟平等: "一步层析法分离高纯度蛋清过敏原卵转铁蛋白", 《食品工业科技》 * |
| 袁小军等: "两步层析法分离纯化鸡蛋清中的卵转铁蛋白", 《第九届中国蛋品科技大会论文集》 * |
| 袁小军等: "两步超滤法分离鸡蛋清中卵转铁蛋白的研究", 《食品与机械》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105998059A (en) * | 2016-06-22 | 2016-10-12 | 华中农业大学 | Preparation method of transferrin and Fe combined Fe supplementing agent |
| CN105998059B (en) * | 2016-06-22 | 2018-12-25 | 华中农业大学 | A kind of preparation method of albumen iron iron supplementary |
| CN107253990A (en) * | 2017-08-11 | 2017-10-17 | 山东阿斯可来生物技术有限公司 | A kind of preparation method that ovotransferrins and its freeze-dried powder are extracted from egg |
| CN109609480A (en) * | 2019-01-21 | 2019-04-12 | 华中农业大学 | The extracting method of protein in a kind of egg white |
| CN110204609A (en) * | 2019-05-31 | 2019-09-06 | 华中农业大学 | The method and its albumen iron product of ovotransferrins are extracted in a kind of industrialization |
| CN110204609B (en) * | 2019-05-31 | 2022-01-07 | 华中农业大学 | Industrial extraction method of ovotransferrin and protein iron product thereof |
| CN111153986A (en) * | 2020-02-26 | 2020-05-15 | 仲恺农业工程学院 | Method for extracting ovotransferrin from pigeon eggs |
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Application publication date: 20121128 |