KR101437259B1 - Sequential Separation of Lysozyme and Ovalbumin from Chicken Egg White - Google Patents

Abstract

The present invention relates to an extraction method for successively separating and extracting lysozyme and ovalbumin from egg whites, wherein a continuous separation and separation of lysozyme and ovalbumin from eggs is carried out by mixing the egg white protein with an ion exchange resin and stirring the mixture A second step of allowing the ion exchange resin stirred in the first step to stand at 2 to 7 ° C for 12 to 24 hours and filtering the ion exchange resin through a reduced pressure filtration apparatus; A washing step (step 3) of washing the ion-exchange resin filtered in the second step with distilled water and washing with 0.01 to 0.5 M glycine-NaOH buffer solution, and a step of washing the ion- M NaCl in a buffer solution containing 0.01-0.5 M glycine-NaOH, removing the salt, concentrating with a filter, freeze-drying the lysozyme, A first separation step (step 5) in which citric acid and ammonium sulfate are mixed with the egg white protein remaining after separating lysozyme in the fourth step, and a first separation step (fifth step) in the fifth step; A second separation step (sixth step) of mixing and distilling the precipitated precipitate with distilled water, followed by secondary separation by mixing citric acid and ammonium sulfate, and a second separation step (step 6) (Step 7) of collecting the supernatant by centrifugation, removing the salt by a filter, and separating and extracting the ovalbumin by heat-treating the supernatant after removing the salt in the seventh step, followed by lyophilization And an ovo albumin separation and extraction step (eighth step).
The extraction method for successively separating and extracting lysozyme and ovalbumin from egg whites according to the present invention establishes an efficient method for separating high yield and high purity lysozyme and ovalbumin from egg whites and establishes a continuous separation process Thereby enabling mass production and increasing the value added of the poultry industry.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an extraction method for continuously separating and extracting lysozyme and ovalbumin from egg white,

The present invention relates to an extraction method for continuously separating and extracting lysozyme and ovalbumin from egg white, and more particularly, to an extraction method for mixing and stirring an egg white protein with an ion exchange resin (first step) Filtration step (second step) of allowing the ion-exchange resin stirred at 2 ~ 7 ° C for 12-24 hours to filter the ion-exchange resin through a low-pressure filtration apparatus, and a step (second step) Washing step (step 3) of washing the exchange resin with distilled water and washing with 0.01 to 0.5 M glycine-NaOH buffer solution; and washing the ion exchange resin washed in the third step with 0.01 to 0.5 M glycine-NaOH buffer solution, removing the salt, concentrating with a filter, and lyophilizing the lysozyme to separate and extract the lysozyme. In the fourth step, the remaining lysozyme is separated from the lysozyme, A first separation step (first step) of first mixing the egg white protein with citric acid and ammonium sulfate, and a first separation step (step 5) in which citric acid and ammonium sulfate are mixed with each other; and a fifth step of dissolving distilled water in the precipitate precipitated after the first separation, After the secondary separation in the sixth step, the mixture is centrifuged at 2 to 7 ° C for 12 to 24 hours, and then the supernatant is collected and the filtrate is removed with a filter (Step 7); and a step of separating and extracting ovo albumin by heat-treating the supernatant from which the salt has been removed in step 7 and lyophilizing it (step 8).

Lysozyme and ovo albumin are known to be proteins that have various functions as major proteins in egg white proteins. Ovo albumin is a glycoprotein with phosphate groups attached and a molecular weight of 45 KDa and an isoelectric point of 4.5. Half of the 385 amino acids of ovo albumin are hydrophobic, with one in every three minutes charged. Obovalbumin also has six cystein residues and one single disulfide bond and oligosaccharide. Although ovo albumin is widely used for animal cell culture and functional peptide production, the research on the nutritional value of ovo albumin is still insufficient.

Lysosyme, called N-Acetyl-muramic-hydrolase, is a lytic enzyme that acts to prevent bacterial infection by destroying the cell walls of bacterial infiltration into animal fluids and algae eggs. Egg white lysozyme is a single peptide chain consisting of 129 amino acids. It is a basic protein with a molecular weight of 14.4 KDa and an isoelectric point of 11.1. It has 8 molecules of cysteine and has 4 S-S bonds made by it and therefore is a very stable enzyme. Also, it has antibacterial activity without cofactor, unlike many other enzymes in nature. Although lysozyme is present in many forms in nature, most lysozyme in egg white is mostly soluble and stable. Currently, the World Health Organization (WHO) and many other countries are using lysozyme as a preservative in foods and are widely used for kimuchi pickles, sushi, Chinese noodles, cheese and wine in Japan.

Studies on the separation of ovo albumin and lysozyme from eggs have been actively conducted. In the case of ovalbumin, it was separated using ammonium sulfate and acetic acid in the 1900's, but it has problems such as low purity, low yield and difficulty in mass production. Since Datta et al . (PES) flat disk membranes, which can be separated by high purity, have been studied. However, this method also has many drawbacks such as difficulty in mass production. In addition, the separation of ovo albumin has been studied by many methods such as electrophoretic method, foam fractionation method and liquid chromatographic method. However, these methods also exhibit high purity and yield and can not be efficiently produced. Therefore, we aim to establish a separation process that can efficiently produce high purity and high yield of ovalbumin from egg white.

Separation studies of lysozyme have also been carried out steadily since the 1900s. The initial lysozyme was separated by ammonium sulfate precipitation method, but it was difficult to control the pH of lysozyme and the separation by ion exchange chromatography was studied. In particular, Carboxymethyl cellulose (CMC) resin is a resin capable of separating lysozyme from egg white under basic conditions, but it has disadvantages that CMC resin itself is difficult to handle and its particle size is very small and its separation speed is slow. Recently, CMC magnetic macroporous cellulose was used. In this resin, it is possible to separate by a single method and its purity is also high as 96%, but the resin itself is so expensive that mass production is impossible. β-mercaptoethanol and various chemicals, but this method also has a disadvantage that it can not be used in human body. Although there have been a lot of studies on lysozyme isolation in egg white so far, separation methods capable of high purity, high yield and mass production have not yet been established.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide an efficient method for separating high-purity lysozyme and ovalbumin from egg white eggs, It is possible to make mass production by establishing the separation process and to increase the value added of the poultry industry.

In order to achieve the above object, there is provided an extraction method for continuously separating and extracting lysozyme and ovalbumin from egg white, according to the present invention comprises: stirring step (step 1) of mixing and stirring an egg white protein with an ion exchange resin; A filtration step (second step) of allowing the ion-exchange resin stirred in the first step to stand at 2 to 7 ° C for 12 to 24 hours, filtering the ion-exchange resin through a reduced pressure filtration device (second step) (Step 3) of washing the ion-exchange resin with a 0.01 to 0.5 M glycine-NaOH buffer solution, washing the ion-exchange resin washed with distilled water and 0.01 to 0.5 M glycine-NaOH buffer solution, A lysozyme isolation extraction step (step 4) of mixing 0.5 M glycine-NaOH buffer solution, removing salt, concentrating with a filter and freeze-drying to separate and extract lysozyme, (Step 5) in which citric acid and ammonium sulfate are mixed with the egg white protein remaining after the first separation step (step 5), and the precipitate precipitated after the first separation in the fifth step is mixed with distilled water, (Step 6), which is followed by secondary separation by mixing ammonium sulfate. After the second separation in the sixth step, the mixture is allowed to stand at 2 to 7 ° C for 12 to 24 hours, centrifuged, and the supernatant is collected and filtered (Step 7) of removing the salt, and a step of separating and extracting the ovalbumin by heat-treating the supernatant from which the salt has been removed in the seventh step and lyophilizing it (step 8) .

The ion exchange resin is an Amberlite FPC 3500 cation exchange resin maintained at a pH of 8 to 10. [

In the first separation step of the fifth step, the concentration of citric acid is 1 to 5%, and the concentration of ammonium sulfate is 2 to 10%.

The concentration of citric acid in the secondary separation step of the sixth step is 1 to 3%, and the concentration of ammonium sulfate is 2 to 5%.

Also, in the salt removing step of the seventh step, the salt-free supernatant is heat-treated at 60 to 80 ° C for 10 to 20 minutes and then lyophilized to separate and extract ovo albumin.

The extraction method for successively separating and extracting lysozyme and ovalbumin from egg whites according to the present invention establishes an efficient method for separating high yield and high purity lysozyme and ovalbumin from egg whites and establishes a continuous separation process Thereby enabling mass production and increasing the value added of the poultry industry.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart schematically showing an extraction method for successively separating and extracting lysozyme and ovalbumin from egg white eggs according to the present invention.
Fig. 2 shows the separation rate according to the resin in separating lysozyme from egg white of the present invention.
FIG. 3 shows the results of observing the higher separation rate among the two acids (acetic acid, citric acid) treated with ammonium sulfate in the separation of ovo albumin from the egg white of the present invention.
FIG. 4 is a graph showing the results of observing the separation rate of ovo albumin from the egg white of the present invention while adjusting the concentration of citric acid to 5% and the concentration of ammonium sulfate to 2.5, 5, 7.5 and 10%.
FIG. 5 shows the result of observing the separation rate by adjusting the ammonium sulfate concentration to 5% and the citric acid concentration to 2.0, 2.5, 3.0, and 3.5%, respectively, at the time of separating obovalbumin from the egg white of the present invention.
FIG. 6 shows the results of observing the optimum concentrations of ammonium sulfate and citric acid when the ammonium sulfate and citric acid are mixed with the precipitate at the time of separation of obovalbumin from the egg white of the present invention.
FIG. 7 shows the results of observing the temperature condition and time of the heat treatment at the time of separating obovalbumin from the egg white of the present invention.
Fig. 8 shows SDS-PAGE of lysozyme and ovalbumin separated from egg white of the present invention.
FIG. 9 shows Western blotting of ovo albumin isolated from egg white of the present invention.
FIG. 10 shows Western blot analysis of lysozyme from egg white of the present invention.

Hereinafter, the extraction method of the present invention will be described in detail with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart schematically showing an extraction method for successively separating and extracting lysozyme and ovalbumin from egg white eggs according to the present invention.

1. Stirring step (first step)

In the stirring step, the egg white protein is mixed with an ion exchange resin,

Amberlite FPC 3500 cation exchange resin maintained at pH 8 ~ 10 is mixed with egg white protein and stirred.

Here, Amberlite FPC 3500 cation exchange resin maintained at a pH of 8 to 10 was mixed with the egg white protein, and it was found through repeated experiments that Amberlite FPC 3500 cation exchange resin maintained at the above pH 8-10 among various ion exchange resins This is because the lysozyme separation rate was the highest.

The above-mentioned isolated yields are shown in Fig.

2. Filtration step (second step)

In the filtration step, the ion exchange resin stirred in the first step is left at 2 to 7 ° C, and the ion exchange resin is filtered through a filtration device.

The ion exchange resin stirred in the first step is allowed to stand at 2 to 7 ° C for 12 to 24 hours, and then the ion exchange resin is filtered through a reduced pressure filtration device.

In order to separate the lysozyme of high purity and high purity by stabilizing the ion exchange resin stirred in the first step, the stirring of the ion exchange resin in the first step is allowed to be carried out at 2 to 7 ° C for 12 to 24 hours.

3. Cleaning step (third step)

The washing step is a step of washing the ion exchange resin filtered in the second step,

The ion-exchange resin filtered in the second step is washed with distilled water three to five times and further washed with 0.01 to 0.5 M glycine-NaOH buffer solution.

In this case, the ion-exchange resin filtered in the second step is washed with distilled water three to five times, and further washed with 0.01 to 0.5 M glycine-NaOH buffer solution, which is not bound to the ion-exchange resin filtered in the second step It is to remove protein.

At this time, it is preferable that the 0.01 to 0.5 M glycine-NaOH buffer solution is also pH 8 to 10.

4. Isolation and extraction step of lysozyme (step 4)

In the lysozyme separation step, the ion exchange resin washed in the third step is mixed with 0.01 to 0.5 M glycine-NaOH buffer solution containing 0.5 M NaCl, and the salt is removed, concentrated, and lyophilized.

The ion exchange resin washed in the third step is mixed with 0.01-0.5 M glycine-NaOH buffer solution containing 0.5 M NaCl. The salt is removed, concentrated by a filter, and lyophilized to separate and extract lysozyme.

Here, the ion exchange resin washed in the third step is mixed with 0.01-0.5 M glycine-NaOH buffer solution containing 0.5 M NaCl in order to separate lysozyme from the ion exchange resin washed in the third step.

At this time, it is preferable to use the buffer solution of 0.01 ~ 0.5 M glycine-NaOH containing 0.5 M NaCl at pH 8 ~ 10.

The ion exchange resin washed in the third step is mixed with a 0.01 to 0.5 M glycine-NaOH buffer solution containing 0.5 M NaCl, and then the salt is removed, concentrated by filtration and freeze-dried to remove unnecessary salts from lysozyme To facilitate storage and storage, as well as to increase the yield and purity of lysozyme.

Here, the method of concentration and freeze-drying is not limited to the method of concentrating and freeze-drying using a filter and a freeze-dryer commonly used in the art.

5. Primary separation step (step 5)

In the first separation step, the lysozyme is separated in the fourth step, and the remaining egg egg white protein is firstly separated by mixing citric acid and ammonium sulfate.

In the fourth step, the lysozyme is separated, and the remaining egg white egg protein is firstly separated by mixing citric acid and ammonium sulfate.

The mixing of citric acid and ammonium sulfate in the remaining egg white protein after separating lysozyme in the fourth step separates lysozyme from the remaining egg lysozyme protein in the fourth step and precipitates other egg white proteins except ovo albumin, To obtain a supernatant containing albumin.

Here, it is preferable that the concentration of citric acid is 1 to 5% and the concentration of ammonium sulfate is 2 to 10%. Concentrations of citric acid and ammonium sulfate derived optimal concentrations for isolating ovoalbumin through repeated experiments, the results of which are shown in Figures 3, 4 and 5.

6. Second Separation Step (Step 6)

In the second separation step, distilled water is mixed and dissolved in the precipitate precipitated after the first separation in the fifth step, and then citric acid and ammonium sulfate are mixed.

In the fifth step, distilled water is mixed and dissolved in the sediment precipitated after the separation, and then citric acid and ammonium sulfate are mixed to separate them secondarily.

In the fifth step, distilled water is mixed and dissolved in the precipitate precipitated after the separation. Secondary separation of citric acid and ammonium sulfate is performed by separating the ovo albumin contained in the precipitate after the separation in the fifth step It is for bet.

It is preferable that the concentration of citric acid is 1 to 3% and the concentration of ammonium sulfate is 2 to 5%.

Here, the use of the citric acid concentration of 1 to 3% and the ammonium sulfate concentration of 2 to 5% resulted in an optimum concentration of citric acid and ammonium sulfate for separating ovoalbumin through repeated experiments, 6.

7. Salt Removal Step (Step 7)

In the salt removal step, after the second separation in the sixth step, the mixture is allowed to stand at 2 to 7 ° C, centrifuged, and the supernatant is collected and removed with a filter.

After the secondary separation in the sixth step, the mixture is allowed to stand at 2 to 7 ° C for 12 to 24 hours, centrifuged at 4.500 x g for 20 minutes, collect the supernatant, and remove the salt with a filter.

Here, in the sixth step, the second separation is carried out at 2 ~ 7 ° C for 12 ~ 24 hours, so that the separated material separated in the sixth step is stabilized to separate the ovo albumin with high yield and high purity.

After the second separation in the sixth step, the mixture is allowed to stand at 2 to 7 ° C for 12 to 24 hours. After centrifugation, the supernatant is collected and the salt is removed by a filter. The supernatant containing ovo albumin is obtained through centrifugation And to remove unnecessary salts from ovo albumin.

8. Extraction of ovo albumin (step 8)

The seventh step is a step of heat-treating the supernatant from which the salt has been removed, followed by freeze-drying to separate and extract the ovalbumin,

In the seventh step, the salt-free supernatant is heat-treated at 60 to 80 ° C for 10 to 20 minutes and lyophilized to separate and extract ovo albumin.

The heat treatment of the supernatant having the salt removed in the seventh step at 60 to 80 ° C for 10 to 20 minutes is performed in order to remove other proteins except ovalbumin in the salt-removed supernatant in the seventh step The temperature and time for the optimal separation rate were derived from Fig. 7.

The freeze-drying method is freeze-drying using a freeze dryer generally used in the art, and the method is not limited.

Hereinafter, the present invention will be described more specifically by way of examples. It will be apparent to those skilled in the art, however, that the following examples are illustrative of the present invention only and do not limit the scope of the present invention. That is, a simple modification or change of the present invention can be easily performed by those skilled in the art, and all such modifications and alterations can be considered to be included in the scope of the present invention.

Example: Extraction of lysozyme and ovalbumin from eggs egg white protein

First, Amberlite FPC 3500 cation exchange resin maintained at pH 9.3 was mixed with egg white protein, and the mixture was allowed to stand at 4 ° C for 18 hours. Then, the ion exchange resin was filtered through a reduced pressure filtration apparatus and washed with distilled water After washing with 0.1 M glycine-NaOH buffer solution, 0.1 M glycine-NaOH buffer solution containing 0.5 M NaCl was added, and the salt was removed. The solution was concentrated by filtration and lyophilized to separate and extract lysozyme.

After isolating the lysozyme, the residual egg white protein was mixed with citric acid having a concentration of 2.5% and ammonium sulfate having a concentration of 5%, and the mixture was subjected to primary separation. Distilled water was mixed and dissolved in the precipitate precipitated after the primary separation, And 2.5% ammonium citrate, and the mixture was centrifuged at 4.500 × g for 20 minutes. The supernatant was collected, and the filtrate was removed with a filter And the salt-free supernatant is heat-treated at 70 ° C for 15 minutes and then lyophilized to separate and extract ovo albumin.

Experiment 1: Isolating lysozyme

Among the various ion exchange resins, the degree of lysozyme separation was compared by selecting Carbozymethyl cellulose (CMC) and Amberlite FPC 3500 cation exchange resin.

As shown in FIG. 2, amberlite FPC 3500 resin of the above two resins showed better separation than CMC resin and higher purity. In the case of lysozyme isolated with Amberlite FPC 3500 resin, the separation efficiency was 89% or more when compared with the theoretical yield.

Experiment 2: Isolation of Ob Albumin

Figure 3 shows that citric acid among the two acids (acetic acid, citric acid) treated with ammonium sulphate exhibits a higher separation rate. In the next step of the experiment, optimal separation conditions were investigated while varying the concentrations of ammonium sulphate and citric acid. FIG. 4 and Table 1 show that the separation rate of ammonium sulfate concentration was 5%, which was high in the isolation rate of ovo albumin, after adjusting the concentration of citric acid to 5% and then changing the concentration of ammonium sulfate to 2.5, 5, 7.5 and 10% .

FIG. 5 and Table 1 show that the highest concentration of ovo albumin was selected as 2.5%, which was obtained by adjusting the ammonium sulfate concentration to 5%, and adjusting the citric acid concentration to 2.0, 2.5, 3.0 and 3.5%. In the ovo albumin isolation experiment, 5% ammonium sulfate and 2.5% citric acid were treated together.

As shown in FIG. 5, it can be seen that ovo albumin still remains in the precipitate even when protein is precipitated in addition to ovo albumin by treatment with ammonium sulfate and citric acid in egg white. The remaining ovo albumin was also subjected to a secondary treatment with ammonium sulfate and citric acid to remove the proteins other than ovo albumin. In this step, the optimum concentration was also selected after varying the concentrations of ammonium sulfate and citric acid. The results are shown in FIG. 6, and it was found that the ovo albumin having the concentration of 2% ammonium sulfate and the concentration of 1.5% citric acid remaining in the first supernatant can be optimally separated.

Finally, to remove pure ovo albumin, other proteins were removed from the supernatant by heat treatment. FIG. 7 shows that the separation rate of ovo albumin was highest when it was treated at 70 ° C for 15 minutes.

≪ Isolation of obovalbumin according to citric acid and ammonium sulfate concentration >

CA: Citric acid, AS: Ammonium sulfate. + - Level of precipitation obtained after treating with different concentrations. n = 3.

Experiment 3: SDS-PAGE

Lobozyme isolated using cation exchange resin and ovo albumin separated by treatment with citric acid and ammonium sulphate at optimum concentration were confirmed by SDS-PAGE. Each sample was injected into a 10% SDS-polyacrylamide gel prepared using a mini-protein II cell, and then electrophoresed at 100 volts, stained with coomassie Brilliant Blue R-250, and decolorized to identify the protein bands.

In order to carry out the two previous separation processes as a continuous process rather than a separate separation process, only lysozyme is separated from the egg white protein using Amberlite FPC 3500 cation exchange resin, and the residual protein is treated with ammonium sulfate and citric acid to separate ovo albumin Respectively. The results are shown in Fig.

Experiment 4: Western-blot

After transferring the electrophoresed gel to the nitrocellulose membrane by SDS-PAGE in Experiment 3, the transferred membrane was blocked with phosphate-buffered saline (PBS) in a solution containing 5% skim milk for 1 hour.

In the case of lysozyme, the membrane was reacted with the primary antibody rabbit polyclonal antibody overnight at 4 ° C, and the membrane was washed three times with PBST solution at intervals of 10 minutes. ECL substrate solution and exposed to x-ray film to confirm the result.

In the case of ovo albumin, the membrane was incubated overnight at 4 ° C with anti-ovalbumin antibody, which was the primary antibody, and the membrane was washed three times with PBST solution every 10 minutes. The reaction membrane in primary antibody was for 1 hour in a solution of AP ¹² is attached to the Rabbit anti-mouse IgG (H + L) secondary antibodies was added and then washed three times in PBST, and then the reaction solution with ECL substrate solution and x- ray film to confirm the result.

Finally, ovo albumin (FIG. 9) and lysozyme (FIG. 10) were confirmed by Western blot.

Experiment 5: Yield Experiment

As shown in Table 2 below, the yields of lysozyme and ovalbumin obtained through the continuous process of the examples were as high as 89% and 97%, respectively.

Sample Weight (g) Yield (%) Egg white
Ovalbumin ¹
Lysozyme ¹
16.63
1.08
100
100 Final ovalbumin ² 16.24 ± 0.75 97.70 + - 4.47 Final lysozyme ² 0.96 + 0.02 88.90 ± 1.93

<Yield of lysozyme and ovo albumin>

Based on the above results, it shows high yield and purity, and since the separation process is also simplified, it can be easily used for mass production and industrialization in the future.

Claims (7)

The lysozyme was separated and extracted from the egg white protein using an ion exchange resin. The lysozyme was separated and the remaining egg white egg protein was firstly separated by mixing citric acid and ammonium sulfate in the egg white protein,
A method for continuously separating and extracting lysozyme and ovalbumin from eggshell egg white, characterized in that distilled water is mixed and dissolved in the precipitate precipitated after the primary separation, and then citric acid and ammonium sulfate are mixed and then separated.
An agitation step (first step) in which an egg white protein is mixed with an ion exchange resin and stirred;
A filtration step (second step) of allowing the ion-exchange resin stirred at the first step to stand at 2 to 7 ° C for 12 to 24 hours and then filtering the ion-exchange resin through a vacuum filtration device;
Washing the ion-exchange resin filtered in the second step with distilled water and washing with 0.01-0.5 M glycine-NaOH buffer (step 3);
The ion exchange resin washed in the third step was mixed with 0.01-0.5 M glycine-NaOH buffer solution containing 0.5 M NaCl, and the salt was removed, concentrated by filtration, and lyophilized to separate lysozyme Extraction step (fourth step);
A first separation step (step 5) of separating lysozyme from the lysozyme in the fourth step and primary separation by mixing citric acid and ammonium sulfate in the remaining egg white protein;
A second separation step (step 6) in which distilled water is mixed and dissolved in the sediment precipitated after the primary separation in the fifth step, and then the secondary separation is performed by mixing citric acid and ammonium sulfate;
A step of separating the supernatant by centrifuging and then collecting the supernatant and removing the salt by a filter (step 7);
(Step 8) of separating and extracting ovo albumin by heat-treating the supernatant from which the salt has been removed in step 7, followed by lyophilization to isolate and extract the ovo alumina (step 8). The ovo- Extraction method to extract.
3. The method of claim 2,
Wherein the ion exchange resin is an Amberlite FPC 3500 cation exchange resin maintained at a pH of 8 to 10. The extraction method of continuously separating and extracting lysozyme and ovalbumin from egg white.
3. The method of claim 2,
Wherein the concentration of citric acid is 1 to 5% and the concentration of ammonium sulfate is 2 to 10% in the first separation step of the fifth step. Continuous separation and extraction of lysozyme and ovalbumin from egg white .
3. The method of claim 2,
Wherein the concentration of the citric acid is 1 to 3% and the concentration of the ammonium sulfate is 2 to 5% in the secondary separation step of the sixth step, and the extraction is performed to continuously separate and extract lysozyme and ovalbumin from egg white Way.
3. The method of claim 2,
The salt removing step of the eighth step is a step of heat-treating the supernatant from which the salt has been removed in the seventh step at 60 to 80 ° C for 10 to 20 minutes, followed by freeze-drying to separate and extract the ovalbumin. An extraction method in which ovo albumin is continuously separated and extracted.
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