CN109609480B - Method for extracting protein from egg white - Google Patents
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Abstract
The invention provides a method for extracting protein from egg white, which mainly adopts a salting-out precipitation combined resin adsorption method to separate six proteins including ovomucin, ovotransferrin, ovoalbumin, an ovo inhibitor, ovomucoid and lysozyme from the egg white at one time. The invention greatly improves the added value of egg processing and generates obvious economic and social benefits.
Description
Technical Field
The invention relates to the technical field of protein extraction, and particularly relates to a method for extracting protein from egg white.
Background
China is a big poultry raising country, the yield of eggs accounts for the first place in the world, and eggs are used as one of the most common animal foods in human life and are also important sources for acquiring nutrition for human beings. In addition, eggs have various industrial properties such as foamability, emulsifiability, and gel property, and are widely used in various fields such as medicine, food industry, and daily chemical industry. The characteristics are closely related to various proteins rich in eggs, for example, the egg white protein with the highest abundance in egg white accounts for about 54 percent of the total protein content of egg white, and has better foamability, gel property and emulsibility; the ovotransferrin with the content of 12 percent has strong iron ion chelating capacity and has the potential of being developed into iron supplement products; although the content of the lysozyme is only 3.4 percent, the lysozyme has stronger antibacterial action and is widely applied to the corrosion prevention of food; the same content is only 3.5% of ovomucin, and research shows that the ovomucin has various biological activities of resisting virus, resisting bacteria, regulating immunity, reducing cholesterol and the like. Therefore, there is a great demand for proteins with different functional properties, both in the market and in research.
With the intensive research on the physiological and biochemical activities of various proteins in eggs, more and more researches are focused on separating different kinds of proteins from eggs in sequence to realize the development of physiologically active substances with higher added values. However, as for the current research situation, the research on extracting single protein is the most common, and the research on the combined extraction of multiple proteins in eggs is not common. The main method for extracting egg white protein is to precipitate the protein by pH or salting out, and then to purify the protein by chromatography. The patent No. 201210083362.2 adopts polyethylene glycol 8000 to fractionate and precipitate protein, and then Q Sepharose FF anion exchange chromatography is used to further separate lysozyme, ovalbumin, ovotransferrin and flavin-bound protein. In 2010, wu et al disclosed a two-step NaCl salting-out method to precipitate ovomucin, combined with anion cation exchange chromatography to separate ovalbumin, ovotransferrin, lysozyme and riboflavin binding proteins. In 2013 BingFu et al use ammonium sulfate precipitation and ion exchange chromatography to separate three proteins of ovalbumin, lysozyme and ovotransferrin.
It can be seen from the above patents and documents that the chromatographic separation technique adopted for separating proteins makes the application of the protein in industrial production very limited, and the establishment of the method for extracting a plurality of proteins from egg white on a large scale is still immature at present, firstly, toxic reagents such as trichloroacetic acid are added in the extraction process, so that the protein is not only easily irreversibly denatured, but also the application of the protein in food industry is not facilitated due to the safety problem caused by the residual toxic reagents. Secondly, most researchers need to adopt different chromatographic devices to improve the purity of various proteins when jointly separating various proteins, but the protein preparation cost is too high due to low sample loading amount and low production efficiency of the chromatographic devices, and the protein preparation cost is not beneficial to industrial production.
Aiming at the problems of toxic reagents introduced in the process of extracting various proteins from eggs, low extraction efficiency, cost of industrial production and the like, an efficient, green and economic extraction method is needed to be developed.
Disclosure of Invention
In view of the defects of the prior art, the invention provides a method for extracting protein from egg white, which is characterized by comprising the following steps:
(1) and (3) lysozyme extraction: adding distilled water into 3000mL of egg white, adjusting the pH value, adding resin, stirring, standing, centrifuging to obtain a first precipitate and a first supernatant, washing the first precipitate with distilled water and a buffer solution, eluting an eluent, and centrifuging to obtain lysozyme;
(2) extracting ovomucin: adding a calcium chloride solution into the first supernatant obtained in the step (1), adjusting the pH, centrifuging to obtain a second precipitate and a second supernatant, adding distilled water into the second precipitate, homogenizing, adjusting the pH, and centrifuging to obtain ovomucin;
(3) extracting ovotransferrin: adding ammonium sulfate and citric acid into the second supernatant obtained in the step (2), regulating the pH value for the first time, standing and centrifuging to obtain a third precipitate and a third supernatant, adding the third precipitate into a sodium dodecyl sulfate solution, centrifuging to obtain a fourth precipitate and a fourth supernatant, pouring the fourth precipitate into distilled water, regulating the pH value for the second time, standing, regulating the pH value for the third time, and centrifuging to obtain ovotransferrin;
(4) extracting ovomucoid: taking the third supernatant and the fourth supernatant in the step (3), adding ammonium sulfate, standing and centrifuging to obtain a fifth supernatant, heating the fifth supernatant, centrifuging to obtain a supernatant, namely a sixth supernatant, adding absolute ethyl alcohol, and centrifuging to obtain a fifth precipitate and ovomucoid;
(5) extracting ovalbumin: adding distilled water into the fifth precipitate obtained in the step (4), adjusting the pH value, and centrifuging to obtain ovalbumin and a seventh supernatant;
(6) and (3) extracting an egg inhibitor: and (4) taking the seventh supernatant obtained in the step (5), concentrating, desalting, adjusting the pH value, adding an ammonium sulfate solution, stirring and centrifuging to obtain a supernatant which is ovomucoid, and obtaining a precipitate which is an ovostatin.
The proportion of the egg white, the distilled water and the resin in the step (1) is 50 mL: 50mL of: 5-20g, wherein the resin is FPC3500 type resin; the stirring speed is 3500r/min, and the stirring time is 10-15 min. The buffer solution is a glycine sodium hydroxide solution with the pH value of 9.3, and the eluent is a mixed solution of glycine sodium hydroxide and sodium chloride; the glycine sodium hydroxide solution is 0.05-0.2mol, and the sodium chloride solution is 0.1-1.0 mol.
The concentration of the calcium chloride solution in the step (2) is 0.2-0.5 mol/L; the volume ratio of the calcium chloride solution to the first supernatant is 1: 1.
the mass of the ammonium sulfate in the step (3) is 137-275g, and the mass of the citric acid is 82.5-137.5 g; the mass amount of the sodium dodecyl sulfate solution is 1-30mmol, and the volume is 800-1200 mL.
The mass of the ammonium sulfate in the step (4) is 93-155g, and the volume of the absolute ethyl alcohol is 1500-4500 mL; the heating temperature is 50-70 ℃, preferably, the heating temperature is 55 ℃, and the heating time is 15-30 min.
And (6) the ammonium sulfate solution in the step (6) is a saturated solution.
Adjusting the pH value to 9.0-9.5 in the step (1);
adjusting the pH value to 6.0 in the step (2);
in the step (3), the pH value is adjusted to be 4.5 for the first time, 8.0-10.0 for the second time and 4.0-6.0 for the third time;
adjusting the pH value to 7.0 in the step (5);
and (6) adjusting the pH value to 4.0.
The standing temperature is 4 ℃, and the standing time is 12 hours.
The method for extracting the proteins can separate six proteins including ovomucin, ovotransferrin, ovoalbumin, an ovomucoid inhibitor, ovomucoid and lysozyme from egg white at one time, compared with the existing method for extracting the proteins from the egg white, the method has the advantages of simple reagents, low cost, simple and convenient process flow and low time cost, can realize large-scale industrial production of a plurality of proteins in the egg white, greatly improves the utilization efficiency of raw materials, and can obviously increase economic benefits; the extraction process has no corrosive substance and toxic and harmful substance, and the proposed six proteins have good safety; the method is to use a combined extraction method to separate the egg inhibitor protein for the first time. Because the content of the egg inhibitor is less and only accounts for 1.5 percent of the egg white, and a small amount of the egg inhibitor can be extracted only by affinity chromatography, the method has good application value in the experimental field and the actual production.
Drawings
FIG. 1 is a graph showing the comparison of the yield and purity of six proteins in egg white extracted by the present invention in combination with the prior art;
FIG. 2 is an electrophoretogram of egg white and various extracts of the present invention, wherein 1 is the object of detection of egg white, 2 is lysozyme, 3 is ovomucin, 4 is ovotransferrin, 5 is ovomucin, 6 is ovalbumin, and 7 is an egg inhibitor.
FIG. 3 is a schematic view of the process flow of the combined extraction of six proteins from egg white according to the present invention;
Detailed Description
The preparation method comprises the steps of egg white pretreatment and then six kinds of protein are separated through different extraction steps, and is characterized by comprising the following steps, as shown in figure 3:
(1) taking 3000mL of egg white of a fresh egg, mixing with distilled water with the same volume, homogenizing, uniformly mixing the egg white at a rotating speed of 3500r/min, homogenizing for 15min, adjusting the pH of the mixed solution to 9.3, adding 1200g of resin, stirring for 12h at 4 ℃, centrifuging to obtain a first precipitate and a first supernatant, washing off the egg white on the surface of the first precipitate by using distilled water, washing off surface impurity proteins by using 0.1mol of glycine-sodium hydroxide buffer solution with the pH of 9.30, and washing off lysozyme in the resin by using 16L of 0.1mol of glycine-sodium hydroxide (containing 0.5mol of sodium chloride) eluent and collecting.
(2) Diluting the separated fresh egg white without lysozyme with an equal volume of 0.5mol calcium chloride solution, stirring for 15min, adjusting the pH value to 6.0, standing for 12h, centrifuging to obtain a second precipitate and a second supernatant, adding the second precipitate into 1500mL distilled water, homogenizing, adjusting the pH value to 6.0, and centrifuging again to obtain a precipitate, namely the ovomucin.
(3) Adding 165g of ammonium sulfate and 110g of citric acid solid into the second supernatant, stirring, adjusting the pH of the solution to 4.5, standing at 4 ℃ for 12 hours, and centrifuging to obtain a third precipitate and a third supernatant; taking the third precipitate in 200mL of 20mmol sodium dodecyl sulfate solution, fully stirring and centrifuging, suspending the obtained new precipitate in 800mL of 20mmol sodium dodecyl sulfate solution again and centrifuging, mixing the supernatants obtained by two times of centrifugation for later use, and marking as a fourth supernatant; adding the fourth precipitate into distilled water, stirring, adjusting pH to 8.0, standing at 4 deg.C for 12 hr, adjusting pH to 6.0, and centrifuging to obtain ovotransferrin;
(4) taking the third supernatant and the fourth supernatant, adding 105g of ammonium sulfate, standing at 4 ℃ for 12 hours, centrifuging to obtain a fifth supernatant, heating the fifth supernatant in a water bath for 30min (at 55 ℃), centrifuging, discarding the precipitate, continuously and slowly adding about 1800mL of absolute ethyl alcohol into the sixth supernatant until the final concentration of the ethyl alcohol reaches 20% by volume, centrifuging the obtained suspension, suspending the precipitate in 1500mL of 20% ethyl alcohol solution again, centrifuging, and obtaining a supernatant which is ovomucoid protein by twice centrifugation; adding the fifth precipitate into 2000mL of distilled water, stirring uniformly, centrifuging (repeating the step once), collecting precipitate as ovalbumin, combining the supernatants obtained by two times of centrifugation, adjusting the pH value to 7.0, and centrifuging to obtain a seventh supernatant, wherein the precipitate is the ovalbumin;
(5) taking the seventh supernatant, desalting, concentrating, adjusting the pH value of the solution to 4.0, adding 20% saturated ammonium sulfate solution, stirring, centrifuging, and obtaining a precipitate as an egg inhibitor, wherein the obtained supernatant is ovomucoid;
the six extracted proteins of the invention (as shown in figure 1 and figure 2) have the following yields in sequence, wherein the yields of lysozyme 95.40%, ovomucin 90.31%, ovotransferrin 92.06%, ovomucoid 91.76%, ovomucin 92.18% and ovodepressor 51%, and the purities of the extracted proteins are respectively 97.15% of lysozyme, 91.50% of ovomucin, 92.3% of ovomucoid, 96.7% of ovomucoid, 95.9% of ovoalbumin and 91.9% of ovodepressor by using a Berkele GEL imaging system (model: GEL-EQ) for analysis and determination.
The extraction method with the application number of CN201210083362.2 obtains the lysozyme, the ovomucin, the ovotransferrin and the ovomucin with the purity of 91.84%, 82.40%, 94.55% and 96.45% in sequence, and the obtained yield is 29.97-88.63%, but the extraction method can obtain six proteins, and the yield of the corresponding protein is improved by 15.3-152.13%, the purity of the lysozyme is improved by 5.78% and the purity of the ovomucin is improved by 11.04%.
The method mainly adopts a salting-out precipitation combined resin adsorption method to separate six proteins in the egg white, has simple process and short extraction period, not only avoids the problem of toxic reagent residue, but also greatly improves the production efficiency and is easy to realize industrialization. The invention greatly improves the added value of egg processing and generates obvious economic and social benefits.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. The method for extracting the protein from the egg white is characterized by comprising the following steps:
(1) and (3) lysozyme extraction: adding distilled water into 3000mL of egg white, adjusting the pH value, adding resin, stirring, standing, centrifuging to obtain a first precipitate and a first supernatant, washing the first precipitate with distilled water and a buffer solution, eluting an eluent, and centrifuging to obtain lysozyme;
(2) extracting ovomucin: adding a calcium chloride solution into the first supernatant obtained in the step (1), adjusting the pH, centrifuging to obtain a second precipitate and a second supernatant, adding distilled water into the second precipitate, homogenizing, adjusting the pH, and centrifuging to obtain ovomucin;
(3) extracting ovotransferrin: adding ammonium sulfate and citric acid into the second supernatant obtained in the step (2), regulating the pH value for the first time, standing and centrifuging to obtain a third precipitate and a third supernatant, adding the third precipitate into a sodium dodecyl sulfate solution, centrifuging to obtain a fourth precipitate and a fourth supernatant, pouring the fourth precipitate into distilled water, regulating the pH value for the second time, standing, regulating the pH value for the third time, and centrifuging to obtain the ovotransferrin, wherein the mass of the sodium dodecyl sulfate solution is 1-30mmol, and the volume is 1200 mL;
(4) extracting ovomucoid: adding ammonium sulfate into the third supernatant and the fourth supernatant obtained in the step (3), standing and centrifuging to obtain a fifth supernatant, heating the fifth supernatant, centrifuging to obtain a supernatant, namely a sixth supernatant, adding absolute ethyl alcohol, and centrifuging to obtain a fifth precipitate and ovomucoid, wherein the heating temperature is 50-70 ℃;
(5) extracting ovalbumin: adding distilled water into the fifth precipitate obtained in the step (4), adjusting the pH value, and centrifuging to obtain ovalbumin and a seventh supernatant;
(6) and (3) extracting an egg inhibitor: concentrating and desalting the seventh supernatant obtained in the step (5), adjusting the pH value, adding an ammonium sulfate solution, stirring and centrifuging to obtain a supernatant which is ovomucoid, and a precipitate which is an ovostatin;
adjusting the pH value to 9.0-9.5 in the step (1);
adjusting the pH value to 6.0 in the step (2);
in the step (3), the pH value is adjusted to be 4.5 for the first time, 8.0-10.0 for the second time and 4.0-6.0 for the third time;
adjusting the pH value to 7.0 in the step (5);
and (6) adjusting the pH value to 4.0.
2. The method for extracting protein from egg white according to claim 1, wherein the ratio of the egg white, distilled water and resin in step (1) is 50 mL: 50mL of: 5-20 g.
3. The method for extracting protein from egg white according to claim 1,
the resin in the step (1) is FPC3500 type resin;
the stirring speed is 3500r/min, and the stirring time is 10-15 min.
4. The method for extracting protein from egg white according to claim 1, wherein the buffer solution is a sodium glycine hydroxide solution having a pH of 9.3, the eluent is a mixture of sodium glycine hydroxide and sodium chloride,
the glycine sodium hydroxide solution is 0.05-0.2mol, and the sodium chloride solution is 0.1-1.0 mol.
5. The method for extracting protein from egg white according to claim 1, wherein the concentration of the calcium chloride solution in the step (2) is 0.2-0.5 mol/L;
the volume ratio of the calcium chloride solution to the first supernatant is 1: 1.
6. the method for extracting protein from egg white as claimed in claim 1, wherein the mass of the ammonium sulfate in step (3) is 137-275g, and the mass of the citric acid is 82.5-137.5 g.
7. The method for extracting protein from egg white as claimed in claim 1, wherein the mass of the ammonium sulfate in step (4) is 93-155g, and the volume of the absolute ethanol is 1500-; the heating temperature is 55 ℃, and the heating time is 15-30 min.
8. The method for extracting protein from egg white according to claim 1, wherein the ammonium sulfate solution in step (6) is a saturated solution.
9. The method for extracting protein from egg white according to claim 1, wherein the temperature of the standing is 4 ℃ and the time of the standing is 12 hours.
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| CN110204609B (en) * | 2019-05-31 | 2022-01-07 | 华中农业大学 | Industrial extraction method of ovotransferrin and protein iron product thereof |
| CN110563837B (en) * | 2019-08-01 | 2021-07-30 | 华中农业大学 | Method for efficiently extracting ovomucoid from egg white |
| CN111848721A (en) * | 2020-07-29 | 2020-10-30 | 中国人民解放军联勤保障部队第九二〇医院 | Special extracting solution for chicken protein and extracting method thereof |
| CN112898408B (en) * | 2021-01-19 | 2022-12-20 | 南开大学 | Method and device for separating ovalbumin from albumen serum |
| CN113214385A (en) * | 2021-05-26 | 2021-08-06 | 杭州佰倍优生物科技有限公司 | Enzymolysis ovalbumin composition for improving hypoalbuminemia and immunity |
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| CN1727475A (en) * | 2004-07-26 | 2006-02-01 | 涂小燕 | Method for picking-up lysozyme from eggs |
| CN102604915B (en) * | 2012-03-27 | 2013-08-21 | 华中农业大学 | Method for jointly extracting a variety of proteins from egg white |
| CN102796193A (en) * | 2012-07-23 | 2012-11-28 | 华中农业大学 | Method for extracting ovotransferrin from egg white |
| CN102796190A (en) * | 2012-07-26 | 2012-11-28 | 华中农业大学 | Method for extracting riboflavin binding protein from egg white |
| CN102875669B (en) * | 2012-10-29 | 2014-06-18 | 天津商业大学 | Method for separating and extracting ovotransferrin |
| CN103160482B (en) * | 2013-03-14 | 2015-03-04 | 江苏大学 | Method for preparing egg white lysozyme and active protein by adopting coseparation |
| CN105602918A (en) * | 2016-03-22 | 2016-05-25 | 烟台大学 | Method for extracting lysozyme from egg white or whole egg juice |
| CN105949300A (en) * | 2016-05-23 | 2016-09-21 | 吉林厚德食品有限公司 | Method for extracting and separating and purifying protein |
| CN106496318B (en) * | 2016-12-01 | 2019-10-01 | 成都大学 | A method of combined extracting cluster protein and ovomacroglobulin from fowls albumen |
| 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 |
| CN107434825A (en) * | 2017-08-28 | 2017-12-05 | 湖州师范学院 | A kind of method that oralbumin is separated from egg |
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