Method for extracting IgY (γ-livetin) from egg yolk

Abstract

A method for extracting IgY (-livetin) from yolk is disclosed, which comprises the following steps: (A) providing a buffer solution, a yolk sample, and an inorganic salt solution; (B) diluting the yolk sample with the buffer solution to obtain a mixture, stirring the mixture for a predetermined time, and performing a centrifugation on the mixture to obtain a supernatant; and (C) adding the inorganic salt solution into the supernatant to salt out IgY, wherein a pH value of the buffer solution is in a range from 4.6 to 5.4, a salt concentration of the buffer solution is in a range from 0.05 M to 0.15 M, and a saturation degree of the inorganic salt solution is in a range from 30% to 60%.

Claims

1. A method for extracting -livetin (IgY) from yolk, consisting of the following steps: (A) providing a buffer solution, a yolk sample, and an inorganic salt solution; (B) diluting the yolk sample with the buffer solution to obtain a mixture, stirring the mixture for a predetermined time, and performing a centrifugation on the mixture to obtain a supernatant; and (C) adding the inorganic salt solution into the supernatant to salt out IgY, wherein a pH value of the buffer solution is in a range from 4.6 to 5.4, a salt concentration of the buffer solution is in a range from 0.05 M to 0.15 M, the predetermined time for stirring the mixture is 25 min or more, and a saturation degree of the inorganic salt solution is in a range from 30% to 60%.

2. The method as claimed in claim 1, wherein the yolk sample is yolk of a chicken egg.

3. The method as claimed in claim 1, wherein the salt concentration of the buffer solution is in a range from 0.08 M to 0.12 M.

4. The method as claimed in claim 1, wherein the buffer solution is an acetate-based buffer solution or a citrate-based buffer solution.

5. The method as claimed in claim 4, wherein the buffer solution is a sodium acetate buffer solution.

6. The method as claimed in claim 1, wherein the yolk sample is diluted with the buffer solution in 8-10 folds in the step (B).

7. The method as claimed in claim 1, wherein the inorganic salt solution is an ammonium sulfate solution.

8. The method as claimed in claim 1, wherein the IgY obtained in the step (C) has a purity of 50% to 95%.

9. The method as claimed in claim 1, wherein the IgY obtained in the step (C) has a yield of 45% to 98%.

10. The method as claimed in claim 1, wherein the yolk sample is yolk of a chicken egg; the salt concentration of the buffer solution is in a range from 0.08 M to 0.12 M; the buffer solution is a sodium acetate buffer solution; the yolk sample is diluted with the buffer solution in 8-10 folds in the step (B); the predetermined time for stirring the mixture is 25 min or more; and the inorganic salt solution is an ammonium sulfate solution.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is an analysis result of SDS-PAGE in Example 1 of the present invention;

(2) FIG. 2 is a photo showing crude extractions of yolk in Example 2 of the present invention;

(3) FIG. 3 is an analysis result of SDS gel electrophoresis in Example 2 of the present invention;

(4) FIG. 4 is a diagram showing the pH changes of the samples in Example 3 of the present invention; and

(5) FIG. 5 is an analysis result of SDS gel electrophoresis in Example 4 of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(6) The present invention has been described in an illustrative manner, and it is to be understood that the terminology used is intended to be in the nature of description rather than of limitation. Many modifications and variations of the present invention are possible in light of the above teachings. Therefore, it is to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

(7) Chicken eggs used in the following examples are eggs of White Leghorn laying hens fed in the inventor's lab. Before extracting the IgY, the eggshell was cracked, and the yolk was separated with and placed in a stainless filter. Next, the stainless filter with yolk placed therein was immersed in de-ionized water to remove the remaining egg white on the surface of the yolk. Then, the residual water on the surface of the yolk was absorbed with paper tissues, the yolk membrane was pricked with a dissecting needle, and the yolk liquid was collected in a volumetric cylinder. The volume of the liquid yolk used in the following examples was about 13-15 mL.

(8) In the following examples, Student's T-test was used to determine if results of two different experiments are significantly different from each other. In addition, Analysis Of Variance (ANOVA) was used to determine the difference between multiple experiments. Herein, p<0.05 indicates that the observed result has significance.

Example 1Evaluation the Time Required to Fully Remove Lipid from Yolk to Extract IgY

(9) [Crude Extraction of IgY from Yolk]

(10) Yolk liquid with a predetermined volume was diluted with 0.1 M sodium acetate buffer solution in 9 folds to obtain a mixture. After mixing the mixture well, the mixture was stirred for a predetermined time (stirring time=0.5 h, 1 h, 3 h or 6 h), and solids contained in the mixture were separated therefrom via a centrifugal force at 4 C. (12,000g, 30 min) to obtain a supernatant, which was a crude extract of the yolk. Then, the volume of the crude extract was measured, a NaN.sub.3 solution was added therein to a final concentration of 0.02% (w/v), and the final solution was stored at 4 C. for the subsequent analysis.

(11) [Quantitative Analysis of IgY]

(12) An enzyme-linked immune-sorbent assay (ELSA) was performed to determine the IgY content in the aforementioned crude extract. Briefly, an IgY standard (Jackson, 003-000-003) and the crude extract were diluted with a borate solution (0.2 M) in a serial dilution, and 50 L of each dilution sample was respectively placed into each well of the ELISA plate (Costar EW-01959-20). After the sample was incubated at room temperature for 6 h or 4 C. overnight, the sample in each well was washed with a wash buffer three times, and then a blocking buffer (180 L) was added into each well. After the sample was incubated at room temperature for 2 h or 4 C. overnight, the sample in each well was washed with the wash buffer three times, and then rabbit anti-chicken IgY antibodies (Jackson 303-005-00, 1 g/ml, adding amount=50 L) was added into each well. After 2 h, the sample in each well was washed with the wash buffer three times, and goat anti-rabbit antibody-alkaline phosphatase (Sigma A3687, 10,000 fold dilution, adding amount=50 L) was added into each well. After incubated at room temperature for 2 hr, the sample in each well was washed with the wash buffer three times, and then pNPP substrate (adding amount=50 L) was added into each well. After 30-60 min, the ELISA plate was examined with an ELISA reader (BIO-TEK MQX220) under a wavelength of 405-490 nm to obtain the absorption of each sample (OD.sub.405-490). Herein, the content of IgY in the crude extract was calculated by comparing the OD.sub.405-490 thereof with that of the serial diluted IgY standard.

(13) [Quantitative Analysis of Total Proteins]

(14) The total protein content in the crude extract was estimated by Bradford protein assay, and BSA (Sigma A7906) was used as a protein standard herein. Briefly, BSA (2 mg/mL) and the crude extract were serially diluted, and 50 L of each dilution sample was respectively placed into the wells of a 96-well plate. 200 L of Bradford reagent was added into each well and incubated for 10 min. Then, the 96-well plate was examined with an ELISA reader (BIO-TEK MQX220) under a wavelength of 595 nm to obtain the absorption of each sample (OD.sub.595). Herein, the content of total protein in the crude extract was calculated according to the curve obtained from the absorption of the serial diluted BSA standard.

(15) The IgY content and the total protein content extracted from per milliliter of the yolk for four different predetermined times (stirring time=0.5 h, 1 h, 3 h or 6 h) are shown in the following Table 1, in which the IgY contents extracted therefrom were about 6.0-7.2 mg, and the total protein contents extracted therefrom were about 21.7-24.9 mg. After analyzing with ANOVA, there was no statistical difference among the four samples (p>0.05), and this result indicates that only 0.5 h is required to remove lipid completely.

(16) TABLE-US-00001 TABLE 1 Stirring time 0.5 h 1 h 3 h 6 h IgY (mg)/mL yolk 6.9 2.0 7.2 1.7 7.2 1.8 6.0 1.1 Total protein (mg)/mL 24.9 1.1 22.7 0.9 21.7 1.6 23.1 1.4 yolk
[Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)]

(17) Proteins (8 g) was separated by SDS-PAGE with 4% of stacking gel and 12% of separating gel under a provided constant voltage 120V for 2.5 h. After the electrophoresis, the gel was stained with Coomassie blue reagent for at least 1 h, destained with a primary destain buffer for 10-20 min, and then destained with a secondary destain buffer until the protein bands were clearly observed and the background of the gel was transparent.

(18) FIG. 1 is an analysis result of SDS-PAGE, in which the arrows respectively indicate the positions of the H chain (70 kDa) and the L chain (26 kDa) of the IgY protein, and the symbol MW represents the protein marker (Bioman, PL01425). As shown in FIG. 1, the species and the contents of the proteins in the four different samples with different stirring time were almost the same. These results indicate that only 0.5 h is required to completely extract proteins from yolk while using the sodium acetate buffer solution as the extraction reagent.

Example 2Evaluation the pH Value for Extracting IgY from Yolk

(19) In the present example, the process for extracting IgY from yolk is similar to that described in [Crude extraction of IgY from yolk] of Example 1, except that the stirring time used in the present example was 30 min, and 0.1 M sodium acetate buffer solution with different pH value (pH=4.2, 4.6, 5.0, or 5.4) was used in the present example. The appearances of the crude extractions after centrifugation are shown in FIG. 2. As shown in FIG. 2, the appearances of the four crude extractions extracted with the buffer solution having different pH value are significantly different from each other, wherein the crude extraction having the muddiest appearance is the crude extraction extracted with the buffer solution having pH 4.2, the one having the middle clarity is that extracted with the buffer solution having pH 5.4, and the one having the clearest appearance is that extracted with the buffer solution having pH 5.0. Since the clarity of the crude extract is resulted from the content of the water-insoluble lipid, it can be inferred that the lipid-removing effect of the sodium acetate buffer solution is pH-dependent.

(20) In addition, the crude extractions extracted with the buffer solutions having different pH value were also analyzed with the same processes described in [Quantitative analysis of IgY] and [Quantitative analysis of total proteins] of Example 1. As shown in the following Table 2, the IgY contents in the crude extractions extracted with the buffer solutions having different pH value are similar to each other, in which the IgY contents in 10 mL of the crude extractions were about 8.7-9.2 mg. However, the protein contents in the crude extractions are positively correlated with the turbidity of the crude extractions, wherein the protein content was about 63 mg in 10 mL of the muddiest crude extraction extracted with the buffer solution having pH 4.2, and that was about 27.6 mg in 10 mL of the clearest one extracted with that having pH 5.0. In the case that the ratio of the IgY content and the protein content is used to represent the IgY purity in the crude extraction, the IgY purities of the crude extractions extracted with the buffer solutions having different pH values were about 32.6% (pH 5.0), 30.6% (pH 4.6), 21.6% (pH 5.4), and 14.7% (pH 4.2). The statistical analysis results indicate that there is no significant difference between the IgY purities of the crude extractions extracted with the pH 5.0 buffer solution and the pH 4.6 buffer solution, but a significant difference is observed between the crude extractions extracted with the pH 5.4 buffer solution and the pH 4.2 buffer solution.

(21) TABLE-US-00002 TABLE 2 Sodium acetate buffer solution pH 4.2 pH 4.6 pH 5.0 pH 5.4 IgY (mg)/mL yolk 9.2 0.7 8.7 1.8 9.2 1.1 8.7 1.9 Total protein (mg)/mL 63.6 2.2 26.4 0.6 27.6 2.5 39.3 0.7 yolk IgY purity (%) 14.7 1.1 30.6 6.0 32.6 2.5 21.6 5.1

(22) Furthermore, the crude extractions extracted with the buffer solutions having different pH value were also analyzed with the same process described in [SDS-PAGE] of Example 1, wherein 12 g of the proteins from the crude extractions and 3 g of the IgY standard were respectively loaded into the gel. The results are shown in FIG. 3, wherein the symbol S indicates the IgY standard, and the arrows respectively indicate the positions of the H chain (70 kDa) and the L chain (26 kDa) of the IgY protein. On the basis of the equal amount of the loaded proteins, large amounts of different proteins were observed in the crude extraction extracted with the pH 4.2 buffer solution, but small amounts of the IgY protein were observed therein according to the staining densities of the H chain and the L chain thereof. However, decreased amounts of different proteins and increased amounts of the IgY protein were observed in the crude extractions extracted with the pH 5.0 buffer solution and the pH 4.6 buffer solution.

Example 3Evaluation the Changes of the pH Values of the Crude Extractions

(23) In the present example, three groups were used to understand why the improved efficiency of IgY extraction can be achieved by replacing the conventional acid water with the sodium acetate buffer solution. Herein, the group 1 (sodium acetate group) was prepared by diluting a predetermined amount of yolk with 0.1 M sodium acetate buffer solution (pH 5.0) in 9 folds, and the initial pH value of the diluted sample was 5.1. The group 2 (acid water group) was prepared by diluting the same amount of yolk as that used in the group 1 with deionized water in 9 folds and adding 0.1 N HCl therein (<2 v/v %), and the initial pH value of the sample was 5.0. The group 3 (comparative group) was deionized water added with a little amount of 0.1 N HCl to adjust the pH value thereof was 5.0. The prepared samples of the aforementioned three groups were stirred at 4 C., and the changes of the pH values thereof were observed.

(24) As shown in FIG. 4, the pH value of the sample of the group 1 (sodium acetate group) maintained to be 5.1 within 6 h (incubation time); that of the group 2 (acid water group) gradually increased to 5.4 within 6 h; and that of the group 3 (comparative group) rapidly decreased to 4.4 within 1 hr and maintained to be 4.4 until the experiment was ended.

(25) The rapid decrease of the pH value of the sample of the comparative group (group 3, 5.0.fwdarw.4.4) is most likely resulted from the solution of CO.sub.2 in the air. The increases of the pH values of the samples of the sodium acetate group (group 1, 5.0.fwdarw.5.1) and the acid water group (group 2, 5.0.fwdarw.5.4) are probably resulted from that the aggregation of the lipids may consume the H.sup.+ ions in the buffer solution or the acid water. Since the sodium acetate solution have buffer capacity to resupply the consumed H.sup.+ ions, only a slight increase of the pH value of the sample was observed initially (5.0.fwdarw.5.1), and the pH value thereof can be maintained to be 5.1 until the experiment was ended. However, the acid water does not have buffer capacity and cannot resupply the consumed H.sup.+ ions, so the pH value of the sample was gradually increased as the lipids of the yolk aggregated.

(26) According to the aforementioned results, the reaction for removing lipids during the IgY extraction appears to be a pH-dependent reaction, and only a narrow pH range is suitable to remove lipids completely. In addition, the aforementioned results also confirm that H.sup.+ ions in the solution are consumed during the IgY extraction. Since the sodium acetate buffer solution has excellent buffer capacity, the pH value during the IgY extraction can be maintained, and therefore the lipids in the yolk can be removed rapidly and effectively. However, the acid water demonstrates poor buffering in the pH range for the IgY extraction, so the effect of removing lipids is not good enough, and a long operation time has to be consumed in order to obtain a sufficient effect of removing lipids.

Example 4Evaluation the Effect of IgY Precipitation by Using Ammonium Sulfate

(27) [IgY Precipitation by Using Ammonium Sulfate]

(28) Ammonium sulfate (Merk 1.01217) was added into the aforementioned crude extractions from the yolk (10 mL) to 30%, 40%, 50% or 60% saturation. After stirring the mixture at 4 C. for 30 min, the pellet was separated by a centrifuge at 4 C. (12,000g, 30 min), and then the obtained pellet was re-suspended with 1 mL PBS buffer. The obtained product was analyzed with the same processes described in [Quantitative analysis of IgY] and [Quantitative analysis of total proteins] of Example 1.

(29) As shown in the following Table 3, the precipitation amounts of total proteins and IgY were increased as the saturation of the ammonium sulfate in the crude extractions elevated. When the saturation of the ammonium sulfate in the crude extraction was 30%, 4.9 mg proteins containing 4.4 mg IgY can be precipitated from 10 mL of the crude extraction. When the saturation thereof was 60%, 16.9 mg proteins containing 8.9 mg IgY can be precipitated therefrom. These results indicate that the precipitation amounts of the total proteins and IgY do not change proportionally as the saturation of the ammonium sulfate in the crude extractions elevated. Hence, the adding amount of the ammonium sulfate influences not only the IgY yield but also the IgY purity (IgY/total proteins) obtained from the crude extractions. When the saturation of the ammonium sulfate in the crude extraction was 30%, the IgY purity obtained therefrom was about 90% though the IgY yield obtained therefrom was only about 48%. When the saturation thereof was 60%, the IgY yield obtained therefrom was about 96%, but the IgY purity obtained therefrom was decreased to 53%.

(30) TABLE-US-00003 TABLE 3 Saturation of ammonium sulfate in crude extract Yolk (mL) 30% 40% 50% 60% Total 28.3 1.2 4.9 0.7 7.6 0.3 12.6 0.6 16.9 1.6 protein (100%) (17%) (27%) (45%) (60%) content (mg) (Yield, %) IgY 9.2 0.7 4.4 0.6 6.3 0.8 8.3 0.4 8.9 0.6 content (100%) (48%) (69%) (90%) (96%) (mg) (Yield, %) IgY 32.5 1.9 89.7 2.1 82.8 7.2 65.8 4.0 53.1 6.5 purity (%)

(31) Furthermore, the obtained products obtained by salting out with ammonium sulfate in different saturations were also analyzed with the same process described in [SDS-PAGE] of Example 1, wherein 3 g of the proteins from the products were respectively loaded into the gel. The results are shown in FIG. 5, in which the symbol S indicates the IgY standard (Jackson, 003-000-003), the arrows respectively indicate the positions of the H chain (70 kDa) and the L chain (26 kDa) of the IgY protein, and the symbol MW indicates the protein marker. From the result shown in FIG. 5, the highest IgY purity can be obtained when using ammonium sulfate in 30% saturation, and only few amounts of -livetin and serum albumin were observed. As the saturation of ammonium sulfate in the crude extraction elevated, the species of the proteins increased, and the obtained IgY purity decreased. This result is consistent with those shown in Table 3.

(32) In conclusion, the sodium acetate buffer solution (especially, 0.1 M and pH 5.0) shows excellent effect of removing lipids in the yolk, and only 0.5 h has to be taken to obtain a crude extract containing more than 30% IgY, compared to the conventional method in which at least 5.5 h has to be taken by using acid water. Based upon our experience, the crude extract prepared by using the present invention is good enough for various immunochemical applications such as Western Blot and ELISA. In addition, according to the IgY precipitation results, the IgY yields obtained from the crude extraction were about 48%, 69%, 90% and 96%, and the IgY purities obtained therefrom were about 90%, 83%, 66% and 53%, when the saturations of the ammonium sulfate added in the crude extractions were 30%, 40%, 50% and 60% respectively. These results can be used as a reference for the IgY yield and the IgY purity, in order to select suitable amounts of ammonium sulfate to precipitate IgY from the crude extractions for the further applications.

(33) Although the present invention has been explained in relation to its preferred embodiment, it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the invention as hereinafter claimed.