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Keywords:

  • dry-heating;
  • egg white protein;
  • foaming properties;
  • foaming stability;
  • phosphorylation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgment
  8. References

ABSTRACT:  Egg white protein (EWP) was phosphorylated by dry-heating in the presence of pyrophosphate at pH 4 and 85 °C for 1 d, and the foaming properties of phosphorylated EWP (PP-EWP) were investigated. The phosphorus content of EWP increased to 0.71% as a result of phosphorylation. To estimate the foaming properties of EWP, the foams were prepared by 2 methods: bubbling of the 0.1% (w/v) protein solution and whipping of the 10% (w/w) protein solution with an electric mixer. The foaming power, which was defined as an initial conductivity of foam from 0.1% (w/v) protein solution, was a little higher in PP-EWP than in native EWP (N-EWP), and the foaming stability of PP-EWP was much higher than that of dry-heated EWP (DH-EWP) and N-EWP. The microscopic observation of foams from the 10% (w/w) solution showed that the foams of PP-EWP were finer and more uniform than those of N- and DH-EWP. Although there were no significant differences in the specific gravity and overrun of the foams between PP- and DH-EWP (P < 0.05), the specific gravity and overrun of the foams from PP-EWP were smaller and higher, respectively, than that of the foams from N-EWP. The drainage volume was smaller in the foams from PP-EWP than in those from N- and DH-EWP. These results demonstrated that phosphorylation of EWP by dry-heating in the presence of pyrophosphate improved the foaming properties, and that it was more effective for the foam stability than for the foam formation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgment
  8. References

Egg white protein (EWP) is extensively utilized as a functional food ingredient in the food industry (Nakamura and Doi 2000). For further industrial uses, it is desirable to improve their functional properties. Phsophorylation is one of the methods used for improving the functional properties of food proteins. Chemical and enzymatic methods were developed for the phosphorylation of food proteins. Since violent reagents such as phosphorus oxychloride and phosphorus pentoxide have been used in chemical phosphorylation (Matheis and Whitaker 1984; Matheis 1991), side reactions such as deterioration of amino acids and polymerization of proteins occur. Furthermore, food proteins phosphorylated by violent chemical reagents are not readily accepted by consumers due to the intense reaction and the difficulty in the removal of the remaining chemicals. Although enzymatic phosphorylation is the most desirable method for food proteins with respect to food safety (Seguro and Motoki 1989; Campbell and others 1992), it brings in too few phosphate groups for the specificity of the substrate. Such a low level phosphorylation is not enough to improve the functional properties of food proteins, and this method does not seem to fit the needs of industrial scale of production due to the high cost of enzymes.

We have succeeded in phosphorylating EWP by dry-heating in the presence of orthophosphate (Li and others 2003). Furthermore, phosphorylation of EWP by dry-heating in the presence of pyrophosphate markedly improved its functional properties such as heat-stability against insolubility, emulsifying property, gelling property, and calcium phosphate-solubilizing property with slight conformational changes (Li and others 2004, 2005; Hayashi and others 2008). This phosphorylation method is applicable for practical use, because pyrophosphate is permitted as a food additive in the food industry in Japan.

Foaming property is an important functionality of food proteins. Excellent foaming properties of proteins are needed for food products, such as angel cakes, meringue, and mousse. In the present study, we examined the foaming property of phosphorylated EWP (PP-EWP) prepared by dry-heating in the presence of pyrophosphate.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgment
  8. References

Materials

EWP was prepared according to the method of Li and others (2004) as follows: egg white, separated from infertile eggs purchased from Marui Agricultural Cooperative Assn. (Kagoshima, Japan), was homogenized, acidified to pH 5.5 with 1 N HCl, and then centrifuged. The supernatant obtained was diluted with an equal volume of water, dialyzed, and then lyophilized. All reagents used were of analytical grade.

Preparation of PP-EWP and dry-heated EWP (DH-EWP)

PP-EWP was prepared according to the method given in a previous article (Li and others 2004). Native EWP (N-EWP) was dissolved at 2% in 0.1 M sodium pyrophosphate buffer at pH 4, adjusting the pH with 1 N HCl, and the solution was lyophilized. Lyophilized samples were incubated at 85 °C for 1 d. Dry-heated samples were dissolved and dialyzed using a dialysis membrane (molecular mass cut-off, 14000) to remove free pyrophosphate for 3 d at 5 °C against 5 changes of 50 times of Milli-Q water, and then lyophilized. In comparison with PP-EWP, dry-heated EWP (DH-EWP) was prepared as follows: EWP was dissolved at a concentration of 2% in Milli-Q water and the pH of the solution adjusted to 4 with 1 N HCl; the mixture was then lyophilized and dry-heated under the same conditions as those of PP-EWP. Finally, dry-heated samples were dissolved and dialyzed as described previously.

Determination of phosphorus content of PP-EWP

Protein samples (20 mg) were digested in 2 mL of perchloric acid by heating on an electronic heater. Phosphorus in the digest was regarded as the total phosphorus of PP-EWP. For the determination of inorganic phosphorus (Pi), 5 mL of 10% trichloroacetic acid was added to the same volume of 10 g/L PP-EWP solution, and the solution was centrifugated at 1000 ×g for 20 min. The phosphorus in the supernatant was regarded as Pi. The phosphorus content was determined according to the method of Chen and others (1956). The amount of phosphorus bound to proteins was estimated by the difference between the total phosphorus and Pi content.

Measurements of the conductivity of foams prepared with EWP

The conductivity of foams prepared with EWP was measured by the method of Kato and others (1983) with a minor modification. The foams were produced when air at a constant flow rate of 50 mL/min using a flowmeter (KOFLOC Co., Kyoto, Japan) was introduced for 15 s into 5 mL of 0.1% (w/v) in 0.1 M phosphate buffer (pH 7.4) in a glass column (2.4 × 30 cm) with a glass filter (G-4) by the method of Kato and others (1983). The cell was fixed in a glass column at 1-cm interval in a distance of 2.4 cm from a glass filter, connecting with an Orion 3-Star conductivity meter (Thermo Fisher Scientific K.K., Kanagawa, Japan). The conductivity reading was recorded automatically using a communication cable with conductivity meter. After the air was introduced into the protein solution for 15 s, the conductivity of foams measured by an electrode showed the maximal value and then the changes in the conductivity of foams were measured over time. The foaming power of the protein was expressed as the conductivity of foam produced immediately after passing air through the solution for 15 s. The foam stability was estimated by measuring the half time of the conductivity immediately after foam formation (Kato and others 1983; Ibrahim and others 1993).

Preparation of foams from 10% (w/w) EWP solution

Protein samples were dissolved at a concentration of 10% (w/w) in 0.9% normal saline solution at pH 7. The protein solution (5 mL) was stirred with a single-winged electric mixer (MK-H3, National, Japan) at 650 rpm/min for 90 s in a 500-mL plastic beaker, ensuring that the stirring fan was engulfing the foaming solution by tilting the beaker. Thus prepared foams were used for microscopic observation, measurement of specific gravity, overrun, and the volume of drainage.

Microscopic observation of the foams

Samples of the foam were gently scooped out with a spatula and placed flat in a cylinder (53 mm in diameter and 12 mm in depth). Micrographs of the foams were obtained immediately and in 60 min using a Leica MZ16 stereomicroscope (Leica Microsystems, Tokyo, Japan). To compare the size distribution of foams, diameter of about 1000 pieces of foams on 3 pictures in each sample was measured by micrograph immediately after preparation from 10% (w/w) of N-, DH-, and PP-EWP. The distribution of the foam diameter was calculated by the following equation:

  • image

Measurement of the specific gravity and overrun of the foams

Protein samples were dissolved at a concentration of 10% (w/w) in 0.9% normal saline solution and the pH of the solution adjusted to 7. Protein solutions were beaten with a single-winged electric mixer at 650 rpm/min for 90 s in a 500-mL plastic beaker, and samples of the foam were gently scooped out with a spatula and placed flat in a cylinder (27 mm in diameter and 14 mm in depth); specific gravity and overrun of foams and foam stability were measured. Specific gravity was calculated by the following equation:

  • image

To make accurate overrun measurements, the protein solution must be completely incorporated into foam (Phillips and others 1987). The overrun was calculated by the following equation:

  • image

Measurement of the foam stability of foams prepared from EWP

Foam stability was measured by monitoring separate liquid from foams at ambient temperature. Protein samples were dissolved at a concentration of 10% (w/w) in 0.9% normal saline solution and the pH of the solution adjusted to 7. Protein solutions were beaten with a single-winged electric mixer at 650 rpm/min for 90 s in a 500-mL plastic beaker. The foams were placed in the infundibulum of handle length of 2 cm setting up on the graduated cylinder of 10 mL volume. Foaming stability was determined by measuring the volume of drainage from foams every 5 min for 90 min.

Statistical analysis

The data were expressed as mean values of 4 replicated determinations with standard deviation (SD). Statistical analysis was done by t-test at 5% level probability.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgment
  8. References

EWP was phosphorylated by dry-heating at pH 4 and 85 °C for 1 d in the presence of pyrophosphate. The bound phosphorus content was 0.07% in both N- and DH-EWP, and 0.71% in PP-EWP. This value was comparable to that reported previously (Li and others 2004).

To estimate the foaming properties of PP-EWP, the foams of EWP samples were prepared by 2 methods. One is by introducing air to a glass column, in which 0.1% (w/v) protein solution was poured, with conductivity cell. The other is by whipping 10% (w/w) protein solutions with an electric mixer.

Figure 1 shows the changes in conductivity of the foams of N-, DH-, and PP-EWP prepared by introducing air to the column into which the 0.1% (w/v) protein solution was poured. Foaming power was demonstrated as the conductivity immediately after the foam was produced, and foaming stability was defined as half time of the initial conductivity. As shown in Table 1, the foaming power of PP-EWP was significantly a little higher (P < 0.05) than that of N-EWP, but there was almost no difference in the foaming power between DH- and PP-EWP. On the other hand, the foaming stability of PP-EWP was much longer (P < 0.05) than that of N- and DH-EWP; it was 4.8 times that of N-EWP and 2.9 times that of DH-EWP.

image

Figure 1—. Changes in the conductivity of foams prepared with N-, DH-, and PP-EWP. The protein concentration of the sample was 0.1% (w/v) in 0.1 M phosphate buffer (pH 7.4) and foams were made by blowing air (50 mL/min) into a glass column for 15 s. N-EWP = native EWP; DH-EWP = EWP dry-heated at pH 4 and 85 °C for 1 d in the absence of pyrophosphate; PP-EWP = EWP dry-heated at pH 4 and 85 °C for 1 d in the presence of pyrophosphate. Each value is the mean with its SD (n = 4).

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Table 1—.  Foaming power and foaming stability of foams prepared from 0.1% (w/v) of N-, DH-, and PP-EWP solutions.
SampleaFoaming powerb (×103, μS/cm)Foaming stabilityb (s)
  1. Foams were made by blowing air (50 mL/min) into a glass column for 15 s.

  2. aN-EWP= native EWP; DH-EWP = EWP dry-heated at pH 4 and 85 °C for 1 d in the absence of pyrophosphate; PP-EWP = EWP dry-heated at pH 4 and 85 °C for 1 d in the presence of pyrophosphate.

  3. bEach value is the mean with its SD (n = 4); means in same column with different letters are significantly different (P < 0.05).

N-EWP1.47 ± 0.10a18.4 ± 2.01a
DH-EWP1.79 ± 0.03b31.1 ± 8.14a
PP-EWP1.76 ± 0.05b89.2 ± 4.68b

Figure 2 shows the stereo micrographs of foams immediately after and 60 min after whipping 10% (w/w) N-, DH-, and PP-EWP solutions. The foams of PP-EWP were fine and uniform in their size compared with those of N- and DH-EWP. The foams of all samples became large after 60 min. To estimate the size distribution of foams, the diameter of about 1000 pieces of foams in each sample was measured. The results of the size distribution of foams were shown in Figure 3. The number of the pieces of foam less than 0.3 μm in diameter was 3.3% for N-EWP, 17.7% for DH-EWP, and 26.2% for PP-EWP. On the other hand, the number of the foams larger than 0.5 μm in diameter was 73.7% for N-EWP, 20.1% for DH-EWP, and 3.4% for PP-EWP.

image

Figure 2—. Micrographs of foams from 10% (w/w) of N-, DH-, and PP-EWP solutions. The foams were made by mixing the EWP solutions with a mixer for 90 s, and micrographs were taken immediately (0 min) and at 60 min after preparing foams. N-, DH-, and PP-EWP: see Figure 1.

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image

Figure 3—. Size distribution of foams immediately after preparation from 10% (w/w) of N-, DH-, and PP-EWP solutions. N-, DH-, and PP-EWP: see Figure 1.

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The specific gravity and overrun were measured on the foams prepared by whipping 10% (w/w) N-, DH-, and PP-EWP solutions. As shown in Table 2, specific gravity of foams from PP-EWP was significantly smaller (P < 0.05) than that of the foams from N-EWP, but the difference was small. There was almost no difference in the specific gravity between PP- and DH-EWP. The overrun of PP-EWP was significantly larger (P < 0.05) than that of N-EWP, and did not differ significantly from that of DH-EWP.

Table 2—.  Specific gravity and overrun of foams prepared from 10% (w/w) of N-, DH-, and PP-EWP solutions.
SampleaSpecific gravitybOverrunb (%)
  1. aN-, DH-, and PP-EWP: see Table 1.

  2. bEach value is the mean with its SD (n = 4); means in same column with different letters are significantly different (P < 0.05).

N-EWP0.155 ± 0.006a 563.47 ± 25.47a 
DH-EWP0.144 ± 0.006ab611.59 ± 30.22ab
PP-EWP0.138 ± 0.005bc643.25 ± 27.69bc

The drainage is also an indicator of foaming stability. The drainage was determined by measuring the volume of the separated liquid from foams at 5-min intervals. Figure 4 shows the changes in drainage volume from foams prepared by whipping 10% (w/w) N-, DH-, and PP-EWP solutions. The drainage volume from foams of PP-EWP was 0.3 times that of N-EWP and 0.35 times that of DH-EWP after 5 min. Although the drainage of PP-EWP increased during holding time, it was significantly less (P < 0.05) than that of DH-EWP till 35 min, and than that of N-EWP even after 90 min.

image

Figure 4—. Changes in the drainage volume from foams prepared from 10% (w/w) of N-, DH-, and PP-EWP solutions during their standing. N-, DH-, and PP-EWP: see Figure 1. Each value is the mean with its SD (n = 4).

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Phosphorylation is one of the useful methods to improve the functional properties of food proteins. We succeeded in phosphorylating EWP without serious conformational changes by dry-heating in the presence of pyrophosphate (Li and others 2004). The solubility of EWP was maintained after phosphorylation, and phosphorylation improved the heat stability and the emulsifying and gelling properties. Furthermore, the calcium phosphate-solubilizing ability of EWP was effectively enhanced by phosphorylation, suggesting that calcium bioavailability might be increased by phosphorylation of EWP.

Foaming property is one of the important functionalities of EWP, and EWP is the most widely used foaming or whipping agent for foam-based foods such as angel cakes, meringue, and mousse. In foaming of protein solution, initially soluble globular proteins diffuse to the air–water interface, concentrate and reduce interfacial tension, denature to orient hydrophilic regions toward the water and hydrophobic regions toward air, and finally interactions between denatured proteins occur to a form continuous film (Kinsella 1981). Thus foams are surrounded and stabilized with denatured protein films. There are many factors affecting foaming power and stability of proteins, such as solubility, rate of unfolding, reorientation of polypeptides, association of polypeptides to facilitate formation of a viscous surface film with elasticity, tendency to aggregate without excessive surface coagulation, surface charge, and hydration (Kinsella 1981).

According to Kato and others (1985), the most important structural factor affecting emulsifying and foaming properties of proteins is the surface hydrophobicity. In addition to the surface hydrophobicity, there are good correlations between the foaming power and the digestion velocity of proteins, suggesting that flexibility of protein structure is also an important structural factor governing the foam formation. Foaming power of PP-EWP for foams produced by introducing air to the 0.1% (w/v) protein solutions, and overrun of PP-EWP for foams prepared by whipping 10% (w/w) protein solution were higher than those of N-EWP, although there were only small significant differences (P < 0.05) between PP- and DH-EWP (Table 1 and 2). It was suggested that proteins in PP-EWP might more easily diffuse to the air–water interface, reduce interfacial tension, and undergo unfolding than those in N-EWP because the surface hydrophobicity and digestibility of EWP were increased by phosphorylation as reported in the previous article (Li and others 2004).

Foams prepared by whipping 10% (w/w) PP-EWP solution were finer and more uniform in sizes than those from N- and DH-EWP (Figure 2). Phosphorylation of EWP markedly increased the foaming stability estimated by both conductivity measurement of foams prepared from 0.1% (w/v) protein solution and drainage from foams prepared by whipping 10% (w/w) proteins solution (Table 1 and Figure 4). These results indicated that phosphorylation of EWP was more effective for the foam stability than for the foam formation. Stabilization of protein foams requires the following factors: the interfacial film should be structurally stable and relatively impermeable to the entrapped air; at a critical distance contiguous bubbles should be repelled to minimize coalescence; the outward projecting polar polypeptide loops or segments should retain the lamellar liquid against gravity; and the protein in the film should possess both rigidity and flexibility to withstand local shocks (Kinsella 1981). Thus, the films surrounding bubbles must possess intermolecular cohesiveness and a certain degree of elasticity to permit localized contact deformation. Repulsion of negative charge of introduced phosphate groups may make PP-EWP relatively more unfolded compared with N- and DH-EWP. In the case of denatured ovalbumin, the electrostatic-repulsive force is important in helping to prevent random aggregation, and the balance of attractive and repulsive forces between protein molecules is needed for the formation of linear polymers (Kitabatake and others 1988). As reported in the previous article (Li and others 2004), heat-induced gel of PP-EWP was transparent while those of N- and DH-EWP were turbid. Furthermore, the PP-EWP gel was firmer and higher in water-holding capacity than those of N- and DH-EWP. In the transparent gel of PP-EWP, a network of linear polymers of denatured proteins might be formed. Accordingly, it was suggested that the linear aggregation of PP-EWP might be formed in the film of bubbles, resulting in the formation of strong film compared with that of N- and DH-EWP.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgment
  8. References

The foams from the PP-EWP solution were finer and more uniform in their sizes than those from the N- and DH-EWP solutions. The foam stability of PP-EWP was much better than that of N- and DH-EWP, although there were only small differences in foaming power, specific gravity, and overrun of the foams among N-, DH-, and PP-EWP. Thus, phosphorylation of EWP by dry-heating in the presence of pyrophosphate improved the foaming properties, and it was more effective for the foam stability than for the foam formation.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgment
  8. References

This study was supported by Grant-in-Aid 14560226 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgment
  8. References