Dephosphorylated ovalbumin composition and method for producing same

Abstract

The present invention relates to: a dephosphorylated ovalbumin composition in which the content ratio of an ovalbumin (P2) having two phosphate groups is reduced with respect to 100% of the sum of the contents of the ovalbumin (P2) having two phosphate groups, an ovalbumin (P1) having one phosphate group, and an ovalbumin (P0) having no phosphate group; and a food composition containing the dephosphorylated ovalbumin composition.　

Description

Dephosphorylated ovalbumin composition and method for producing same

[0001] The present invention relates to a dephosphorylated ovalbumin composition and a method for producing the same.

[0002] Egg white is the white part of an egg and is eaten as is or after cooking, or used as meringue in a variety of dishes, cakes, and sweets. The main protein that makes up egg white is ovalbumin, and the properties of egg white are determined by the properties of ovalbumin. Ovalbumin is used as a research material in various fields, but little research has been done on its use as a food ingredient.

[0003] Yang et al., Anal. Chem. 2013, 85(24), 12037-12045

[0004] An object of the present invention is to develop a new means for utilizing ovalbumin in the food industry.

[0005] Ovalbumin is a glycoprotein with a total of 385 residues and a molecular weight of 45,000, containing phosphorylated Ser residues (Ser68 and Ser344) within the molecule. Electrophoretically, ovalbumin is found to have two phosphate groups (P2), one phosphate group (P1), and no phosphate group (P3), with an abundance ratio of P2:P1:P0 approximately 77:22:1 (Non-Patent Document 1). The present inventors focused on the phosphorylated Ser residues in ovalbumin and discovered that a dephosphorylated ovalbumin composition, in which some phosphate groups have been removed from natural ovalbumin by the action of protein phosphatase, can be obtained. They also discovered that this composition has properties different from those of natural albumin, making it useful as a novel food ingredient, and thus completed the present invention.

[0006] That is, the present invention provides the following [1] to

[10] . [1] A dephosphorylated ovalbumin composition in which the content of ovalbumin having two phosphate groups (P2) is reduced, when the sum of ovalbumin having two phosphate groups (P2): ovalbumin having one phosphate group (P1): ovalbumin having zero phosphate groups (P0) is taken as 100%. [2] The dephosphorylated ovalbumin composition according to [1], in which the content of P2 is 60% or less. [3] The dephosphorylated ovalbumin composition according to [1] or [2], in which the content of P2 is 50% or less. [4] The dephosphorylated ovalbumin composition according to any of [1] to [3], in which the content of P2 is 40% or less. [5] The dephosphorylated ovalbumin composition according to any one of [1] to [4], wherein the content ratios of (P2):(P1):(P0) are 15% to 60%:8% to 53%:6% to 77%. [6] The dephosphorylated ovalbumin composition according to any one of [1] to [5], wherein the content ratios of (P2):(P1):(P0) are 15% to 50%:8% to 18%:35% to 77%. [7] The dephosphorylated ovalbumin composition according to any one of [1] to [3] and [5], wherein the content ratios of (P2):(P1):(P0) are 50% to 60%:30% to 40%:5% to 20%. [8] The dephosphorylated ovalbumin composition according to any one of [1] to [7], wherein the dephosphorylated ovalbumin composition is a product of treating ovalbumin with protein phosphatase. [9] A food composition containing the dephosphorylated ovalbumin composition according to any one of [1] to [8] above.

[10] A method for producing the dephosphorylated ovalbumin composition according to any one of [1] to [8] above, which comprises the step of treating ovalbumin with a protein phosphatase.

[0007] The dephosphorylated ovalbumin composition of the present invention is a composition containing ovalbumin from which some of the phosphate groups have been removed, and has higher foaming properties than natural ovalbumin, the resulting foam is more stable, and the resulting meringue has excellent properties, making it useful as a new food composition.

[0008]

[0033] Figure 1 shows an electrophoretic image of SDS-PAGE of a dephosphorylated ovalbumin composition (egg white, not desalted). Figure 2 shows an electrophoretic image of SDS-PAGE (Phos-tag) of a dephosphorylated ovalbumin composition (egg white, desalted) under reducing conditions. Figure 3 shows the foamability (foam hardness) of a dephosphorylated ovalbumin composition when mixed with a tabletop mixer. Figure 4 shows the foamability (foam height) of a dephosphorylated ovalbumin composition when mixed with a tabletop mixer. Figure 5 shows electrophoretic images of a dephosphorylated ovalbumin composition (egg white) under non-reducing conditions (left) and reducing conditions (right). Figure 6 shows an example of an electrophoretic image of SDS-PAGE (Phos-tag) of a dephosphorylated ovalbumin composition (egg white) obtained by protein phosphatase treatment in the presence of EDTA. 1 shows an example of a Native-PAGE electrophoretic image of a dephosphorylated ovalbumin composition (egg white). 2 shows an electrophoretic image of a Native-PAGE electrophoretic image of a dephosphorylated ovalbumin composition (whole egg, not desalted).

[0009] Terms used in this specification are used in the sense commonly used in the art unless otherwise specified.

[0010] As used herein, ovalbumin is the major protein constituting egg white. It is a glycoprotein with a total of 385 residues and a molecular weight of 45,000, and contains phosphorylated Ser residues (Ser68, Ser344) within the molecule. Electrophoretically, these phosphate groups are found in two forms (P2), one form (P1) cleaved by phosphatase, and zero forms (P0), with the abundance ratio of P2:P1:P0 being 77:22:1 (Non-Patent Document 1). A dephosphorylated albumin composition is a composition containing albumin from which phosphate groups have been removed. Since the dephosphorylated albumin composition is obtained by removing the phosphate groups from the phosphorylated Ser residues within the ovalbumin molecule, the P2:P1:P0 ratio is decreased, and at least one of P1 and P0 is increased.

[0011] One aspect of the present invention is a dephosphorylated ovalbumin composition in which the content of ovalbumin having two phosphate groups (P2) is reduced, when the sum of ovalbumin having two phosphate groups (P2), ovalbumin having one phosphate group (P1), and ovalbumin having zero phosphate groups (P0) is taken as 100%. The dephosphorylated ovalbumin composition of the present invention includes an egg white-derived dephosphorylated ovalbumin composition and a whole egg-derived dephosphorylated ovalbumin composition. The egg white-derived dephosphorylated ovalbumin composition and the whole egg-derived dephosphorylated ovalbumin composition differ in the content of (P2) in the dephosphorylated ovalbumin composition. The content of (P2) in both the egg white-derived dephosphorylated ovalbumin composition and the whole egg-derived dephosphorylated ovalbumin composition is preferably 60% or less.

[0012] The content of (P2) in the egg white-derived dephosphorylated ovalbumin composition is preferably 50% or less, even more preferably 40% or less, particularly preferably 30% or less, and most preferably 20% or less. The content of (P1) is preferably within the range of 1% to 60%, more preferably 5% to 55%, particularly preferably 10% to 50%, and most preferably 15% to 45%. The content of (P0) is preferably within the range of 10% to 100%, more preferably 15% to 95%, particularly preferably 20% to 90%, and most preferably 30% to 85%. The content ratio of (P2):(P1):(P0) is more preferably 15% to 50%, 8% to 18%, and 35% to 77%. That is, a more preferred embodiment of the present invention is a dephosphorylated ovalbumin composition derived from egg white, in which the content ratio of ovalbumin having two phosphate groups (P2): ovalbumin having one phosphate group (P1): ovalbumin having no phosphate groups (P0) is 15% to 50%: 8% to 18%: 35% to 77%. The P2:P1:P0 content ratio in natural ovalbumin is 77:22:1, i.e., 77%:22%:1%, whereas the P2:P1:P0 content ratio in a dephosphorylated ovalbumin composition derived from egg white in a preferred embodiment of the present invention is preferably 15%-50%:8%-18%:35%-77%, more preferably 15%-50%:9%-17%:35%-76%, and even more preferably 16%-50%:9%-17%:35%-75%.

[0013] On the other hand, the content of (P2) in the whole egg-derived dephosphorylated ovalbumin composition is preferably 60% or less, more preferably 10% to 60%, and even more preferably 20% to 60%. It may be 30% to 60%, 40% to 60%, or 50% to 60%. The content of (P1) may be 25% to 55%, preferably within the range of 30% to 50%, and even more preferably within the range of 30% to 40%. The content of (P0) may be within the range of 2% to 25%, preferably within the range of 5% to 20%, and even more preferably within the range of 6% to 15%. The content ratio of (P2):(P1):(P0) may be 10% to 60%:25% to 55%:2% to 25%, or may be 20% to 60%:30% to 50%:5% to 20%, or may be 30% to 60%:30% to 50%:5% to 20%, or may be 40% to 60%:30% to 50%:5% to 20%, or more preferably 50% to 60%:30% to 40%:5% to 20%, or even more preferably 55% to 60%:30% to 38%:6% to 15%. A whole egg-derived dephosphorylated ovalbumin composition tends to have a higher P2 content than an egg white-derived dephosphorylated ovalbumin composition. Methods for increasing the content ratio of P2 in a whole egg-derived dephosphorylated ovalbumin composition include, for example, desalting the whole egg composition or performing an enzymatic reaction in the presence of a metal chelating agent such as EDTA. More specifically, a whole egg is separated into an egg white composition and an egg yolk composition, and at least one of the compositions is subjected to a desalting treatment or an enzymatic reaction in the presence of a metal chelating agent such as EDTA, and the egg white composition and the egg yolk composition are then mixed to obtain a whole egg-derived dephosphorylated ovalbumin composition. By performing this method once or multiple times, the content ratios of P2, P1, and P0 in the whole egg-derived dephosphorylated ovalbumin composition can be made to be similar to or close to the content ratios of P2, P1, and P0 in the egg white-derived dephosphorylated ovalbumin composition. The content of (P2) in the whole egg-derived dephosphorylated ovalbumin composition is more preferably 60% or less, and may be 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less.The content ratio of (P1) is preferably in the range of 1% to 60%, more preferably in the range of 5% to 60%, particularly preferably in the range of 10% to 60%, and most preferably in the range of 15% to 60%. The content ratio of (P0) is preferably in the range of 5% to 90%, more preferably in the range of 5% to 50%, particularly preferably in the range of 5% to 40%, and most preferably in the range of 5% to 30%. The content ratio of (P2):(P1):(P0) is more preferably 5% to 60%:5% to 60%:5% to 90%. That is, another preferred embodiment of the present invention is a dephosphorylated ovalbumin composition derived from whole eggs, in which the content ratio of ovalbumin having two phosphate groups (P2): ovalbumin having one phosphate group (P1): ovalbumin having no phosphate groups (P0) is preferably within the ranges of 15% to 60%:8% to 40%:5% to 77%, 20% to 60%:15% to 40%:5% to 65%, 30% to 60%:20% to 40%:5% to 50%, 40% to 60%:25% to 40%:5% to 35%, more preferably 50% to 60%:30% to 40%:5% to 20%, and more preferably 55% to 60%:30% to 38%:6% to 15%. The P2:P1:P0 content ratio in natural ovalbumin is 77:22:1, i.e., 77%:22%:1%, whereas the P2:P1:P0 content ratio in a whole egg-derived dephosphorylated ovalbumin composition according to a preferred embodiment of the present invention is more preferably 50%-60%:30%-40%:5%-20%, and even more preferably 55%-60%:30%-38%:6%-15%.

[0014] Here, the content ratio of P2:P1:P0 refers to the value obtained by separating a dephosphorylated ovalbumin composition or a food composition containing the dephosphorylated ovalbumin composition into P2, P1, and P0 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or native-PAGE, and quantifying each fraction. There is no significant difference between SDS-PAGE and native-PAGE in terms of confirming each ovalbumin band. Electrophoresis using Phos-tag is not preferred because it tends to result in unclear bands.

[0015] The dephosphorylated ovalbumin composition of the present invention can be produced, for example, by treating ovalbumin with protein phosphatase. As the raw material ovalbumin, ovalbumin derived from chicken eggs is preferred, and whole eggs or egg whites derived from chicken eggs may also be used as they are.

[0016] Protein phosphatase is an enzyme that dephosphorylates phosphorylated proteins, and in the present specification, any enzyme capable of removing phosphate from phosphorylated serine residues in the starting ovalbumin is acceptable. The optimal (optimal) pH of the protein phosphatase used in the present invention is preferably in the range of 4.0 to 7.5, more preferably 4.5 to 7.0, and even more preferably 5.0 to 6.0. The optimal temperature of the protein phosphatase is preferably in the range of 1°C to 60°C, and more preferably 10°C to 55°C. The protein phosphatase used in the present invention preferably has the above-mentioned optimal pH and optimal temperature. The type and origin of the protein phosphatase are not important, but preferably it is a protein phosphatase derived from a microorganism belonging to the genus Trichoderma, Aspergillus, Saccharomyces, Bacillus, or Streptomyces, more preferably a protein phosphatase derived from Trichoderma virens, and even more preferably a hypothetical protein derived from Trichoderma virens Gv29-8 described in WO 2021 / 210539 (XP_013951069.1 hypothetical protein TRIVIDRAFT_87714). The disclosure of International Publication No. 2021 / 210539 is hereby incorporated by reference as part of this specification.

[0017] The amount of protein phosphatase used may be a concentration sufficient to release phosphate from phosphorylated serine in the starting ovalbumin. For example, 0.1 to 25 U per 1 g of starting egg white is preferably 0.5 to 15 U, more preferably 1 to 10 U. Herein, 1 U refers to the amount of enzyme that liberates 1 μmol of phosphate per minute under the following conditions: a substrate solution containing 1 mg of bovine milk casein (Casein, Bovine Milk, Carbohydrate and Fatty Acid Free (Merck Millipore)) dissolved in 1 mL of 20 mM MES-NaOH buffer containing 10 mM Tris-HCl at pH 6.0 is added in a 1 / 10 volume of enzyme solution, the reaction is allowed to proceed at 37°C, and an equal volume of reaction stop solution is added. Protein phosphatase treatment of ovalbumin is preferably carried out at the optimal pH and temperature for the protein phosphatase, and a treatment time of approximately 1 to 30 minutes is sufficient. When the protein phosphatase used is a metal ion-dependent protein phosphatase, it is preferable to desalt the starting ovalbumin-containing solution, e.g., liquid egg white, before use, or to carry out the reaction in the presence of a metal chelating agent such as EDTA. The final concentration of the metal chelating agent, e.g., EDTA, is preferably in the range of 0.1 mM to 100 mM, more preferably 0.5 mM to 50 mM, and even more preferably 1 mM to 10 mM, per dephosphorylated ovalbumin composition. It is more preferable to desalt the starting ovalbumin-containing solution before use. Furthermore, dialysis is a preferred desalting method. After the reaction is complete, it is preferable to inactivate the protein phosphatase by heating at 60°C for 16.5 hours or more, alkali treatment at pH 8.0 or higher, or addition of 10 mM or more of a manganese salt, a trivalent iron salt, a cobalt salt, or a chelating agent such as EDTA or citric acid.

[0018] In the obtained dephosphorylated ovalbumin composition, the phosphate group on the Ser residue has been eliminated by the action of protein phosphatase, and the P2:P1:P0 content ratio is within the above-mentioned range. The dephosphorylated ovalbumin composition of the present invention is sufficient as long as the proportion of phosphorylated ovalbumin is reduced, and it is not necessary that the phosphorylated ovalbumin content be below the lower detection limit. The dephosphorylated ovalbumin composition of the present invention may contain ovalbumin with a lower phosphorylation level than conventional. When ovalbumin is used as a raw material, the composition will contain only dephosphorylated ovalbumin and phosphorylated ovalbumin with a lower phosphorylation level than conventional. However, when egg white is used as a raw material, the composition will contain dephosphorylated ovalbumin and phosphorylated ovalbumin with a lower phosphorylation level than conventional, and such dephosphorylated ovalbumin-containing egg white compositions are also included in the present invention. Furthermore, when whole eggs are used as a raw material, the whole egg composition contains dephosphorylated ovalbumin and ovalbumin with a reduced amount of phosphorylated ovalbumin compared to conventional products, and this whole egg composition containing dephosphorylated ovalbumin is also included in the present invention.

[0019] The dephosphorylated ovalbumin composition of the present invention has the same protein function as normal ovalbumin, but has improved foaming properties. Specifically, when whipped, the foam is firm and stable, and the amount of foam (foam height) is increased. Therefore, the use of the dephosphorylated ovalbumin composition of the present invention allows for the rapid production of good meringue, and the dephosphorylated ovalbumin composition of the present invention is useful as a food composition.

[0020] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0021] Example 1: 60 g of egg white was homogenized by stirring using a homogenizer (IKA Ultra Turrax). 1 M hydrochloric acid was added to 50 g of the resulting homogenized egg white, and the pH was adjusted to 6.0. Distilled water or a predetermined concentration of EDTA was added to the egg white solution and stirred for 10 minutes. 0.067 g of 750 U / g protein phosphatase (final concentration: 1 U / g egg white) was added to the solution, and the mixture was reacted at 50°C for 60 minutes to obtain a dephosphorylated ovalbumin composition. Sampling was performed every 15 minutes. Each enzyme-reacted sample was diluted with purified water as needed, mixed 1:1 with SDS-PAGE sample buffer, and heated at 95°C for 5 minutes to prepare a sample for electrophoresis. The electrophoresis samples were loaded onto a 10% polyacrylamide gel (e-PAGEL HR, manufactured by ATTO) and run at a constant current of 20 mA. After electrophoresis, the gel was stained for proteins using CBB staining solution (APRO SP-4010). Figure 1 shows the destained gel after electrophoresis. The electrophoresis pattern was graphed using image processing software (Image J). Next, regions that varied depending on the presence or absence of phosphate modification were defined, and the area of ​​each area was calculated. The sum of ovalbumin with two phosphate groups (P2), ovalbumin with one phosphate group (P1), and ovalbumin with zero phosphate groups (P0) at each enzyme reaction time was taken as 100%, and the proportions of P2, P1, and P0 were calculated. The results are shown in Table 1. The EDTA concentration in Table 1 is the final concentration. In addition, because percentages in Table 1 are rounded to one decimal place, the total of P2, P1, and P0 may not necessarily equal 100%. OT in Figure 1 stands for ovotransferrin. Under the conditions using the e-PAGEL HR gel described above, the components, in descending order of molecular weight (top of Figure 1), were ovotransferrin, dephosphorylated ovalbumin (P0), and phosphorylated ovalbumin (P1, P2).

[0022]

[0023] The results for lanes 1 to 4, where no EDTA was added, confirmed that the reaction proceeded up to 15 minutes. After 30 minutes, P2 and P1 increased, while P0 decreased. The results for lanes 5 to 13, where a specified amount of EDTA was added, showed that the enzyme reactivity was lower compared to when no EDTA was added.

[0024] Example 2 Egg white solution obtained in the same manner as in Example 1 was placed in a cellulose dialysis tube, and the Milli-Q water was exchanged three times to obtain a dialyzed desalted egg white solution (EDTA-free). 0.067 g of 750 U / g protein phosphatase was added to the solution, and the mixture was reacted at 50°C for 16 minutes to obtain a dephosphorylated ovalbumin composition. Sampling was performed every 2 minutes. Thereafter, electrophoresis and analysis were performed in the same manner as in Example 1. Figure 2 shows the destained gel after electrophoresis. Table 2 shows the analysis results.

[0025]

[0026] 2 and Table 2 suggest that dephosphorylation of P2 was almost complete in about 10 minutes. P1 gradually decreased until 16 minutes, and P0 gradually increased.

[0027] Example 3 The foaming ability of the dephosphorylated ovalbumin composition (100% sugar added) obtained in Example 2 was evaluated using a tabletop mixer (Kenmix Aiko Chef PRO (Aikosha Seisakusho)). As a result, as shown in Figure 3 (foam hardness) and Figure 4 (foam height), it was found that the dephosphorylated ovalbumin composition of the present invention can produce a large amount of hard, stable foam by mixing.

[0028] Reference Example 1: A sample for electrophoresis of a dephosphorylated ovalbumin composition was prepared under non-reducing or reducing conditions, and the gel was coated with SuperSep. ＴＭ Phos-tag ＴＭ Electrophoretic images were obtained in the same manner as in Example 2, except that SuperSep (50 μmol / L) manufactured by Fujifilm was used and the enzyme reaction time was changed. The results are shown in Figure 5. The left side of Figure 5 shows the electrophoresis results under non-reducing conditions, and the right side shows the electrophoresis results under reducing conditions. On both the left and right sides, the enzyme reaction times are 0, 2, 4, 6, and 8 minutes from the left lane. ＴＭ Phos-tag ＴＭWhen using a pre-made molecular weight marker, it can be difficult to distinguish protein bands using a pre-made molecular weight marker. Under the conditions using the Phos-tag gel described above, the proteins were ovotransferrin, ovalbumin, and dephosphorylated ovalbumin in descending order of molecular weight (top of Figure 5).

[0029] Reference Example 2 SuperSep in gel ＴＭ Phos-tag ＴＭ An electrophoretic image under reducing conditions was obtained in the same manner as in Example 1, except that SuperSep (50 μmol / L) manufactured by Fujifilm was used. The results are shown in Figure 6. ＴＭ Phos-tag ＴＭ When using a protein band analysis kit, it may be difficult to identify protein bands using ready-made molecular weight markers. OVA in Figure 6 is ovalbumin.

[0030] Reference Example 3 An electrophoretic image (Native Page) was obtained in the same manner as in Example 2, except that the electrophoresis sample was not denatured. The results are shown in Figure 7. Note that in the case of Native Page, it may be difficult to distinguish protein bands using ready-made molecular weight markers as indicators.

[0031] Example 4: 60 g of whole eggs were homogenized by stirring using a homogenizer (IKA Ultra Turrax). 1 M hydrochloric acid was added to 50 g of the resulting homogenized whole eggs to adjust the pH to 6.0. 0.067 g of 750 U / g protein phosphatase was added (final concentration: 1 U / g egg white), and the mixture was reacted at 50°C for 60 minutes to obtain a dephosphorylated ovalbumin composition (whole egg). Sampling was performed every 2 minutes. Each enzymatically reacted sample was diluted with purified water as needed, and electrophoresis images were obtained (native-PAGE) in the same manner as in Example 2, except that the electrophoresis samples were not denatured. After electrophoresis, the gel was stained for protein using CBB staining solution (APRO SP-4010). An electrophoresis image of the resulting dephosphorylated ovalbumin composition (whole egg) is shown in Figure 8. Furthermore, in the same manner as in Example 1, the proportions of P2, P1, and P0 were calculated at each enzyme reaction time, assuming the sum of ovalbumin having two phosphate groups (P2), ovalbumin having one phosphate group (P1), and ovalbumin having zero phosphate groups (P0) to be 100%. The results are shown in Table 3. In Fig. 8 and Table 3, A1 represents P2, A2 represents P1, and A3 represents P0.

[0032]

Claims

1. A dephosphorylated ovalbumin composition in which the content of ovalbumin having two phosphate groups (P2) is reduced, when the sum of ovalbumin having two phosphate groups (P2): ovalbumin having one phosphate group (P1): ovalbumin having zero phosphate groups (P0) is taken as 100%.

2. The dephosphorylated ovalbumin composition according to claim 1, wherein the content of P2 is 60% or less.

3. The dephosphorylated ovalbumin composition according to claim 1, wherein the content of P2 is 50% or less.

4. The dephosphorylated ovalbumin composition according to claim 1, wherein the content of P2 is 40% or less.

5. The dephosphorylated ovalbumin composition according to claim 1, wherein the content ratio of (P2):(P1):(P0) is 15% to 60%:8% to 53%:6% to 77%.

6. The dephosphorylated ovalbumin composition according to claim 1, wherein the content ratio of (P2):(P1):(P0) is 15% to 50%:8% to 18%:35% to 77%.

7. The dephosphorylated ovalbumin composition according to claim 1, wherein the content ratio of (P2):(P1):(P0) is 50% to 60%:30% to 38%:2% to 20%.

8. The dephosphorylated ovalbumin composition according to claim 1, wherein the dephosphorylated ovalbumin composition is a product of treating ovalbumin with a protein phosphatase.

9. The dephosphorylated ovalbumin composition of claim 1, wherein the ovalbumin is derived from chicken eggs.

10. A food composition containing the dephosphorylated ovalbumin composition according to any one of claims 1 to 9.

11. A method for producing a dephosphorylated ovalbumin composition according to any one of claims 1 to 9, which comprises the step of treating ovalbumin with a protein phosphatase.

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