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Improvement of Foaming Property of Egg WhiteProtein by Phosphorylation through Dry-Heatingin the Presence of PyrophosphateY. HAYASHI, S. NAGANO, H. ENOMOTO, C.-P. LI, Y. SUGIMOTO, H.R. IBRAHIM, H. HATTA, C. TAKEDA, AND T. AOKI

ABSTRACT: Egg white protein (EWP) was phosphorylated by dry-heating in the presence of pyrophosphate at pH 4and 85 ◦C for 1 d, and the foaming properties of phosphorylated EWP (PP-EWP) were investigated. The phosphoruscontent of EWP increased to 0.71% as a result of phosphorylation. To estimate the foaming properties of EWP, thefoams were prepared by 2 methods: bubbling of the 0.1% (w/v) protein solution and whipping of the 10% (w/w) pro-tein solution with an electric mixer. The foaming power, which was defined as an initial conductivity of foam from0.1% (w/v) protein solution, was a little higher in PP-EWP than in native EWP (N-EWP), and the foaming stabilityof PP-EWP was much higher than that of dry-heated EWP (DH-EWP) and N-EWP. The microscopic observation offoams 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 betweenPP- 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 thanin those from N- and DH-EWP. These results demonstrated that phosphorylation of EWP by dry-heating in the pres-ence of pyrophosphate improved the foaming properties, and that it was more effective for the foam stability thanfor the foam formation.

Keywords: dry-heating, egg white protein, foaming properties, foaming stability, phosphorylation

Introduction

Egg white protein (EWP) is extensively utilized as a functionalfood ingredient in the food industry (Nakamura and Doi 2000).

For further industrial uses, it is desirable to improve their func-tional properties. Phsophorylation is one of the methods used forimproving the functional properties of food proteins. Chemicaland enzymatic methods were developed for the phosphorylationof food proteins. Since violent reagents such as phosphorus oxy-chloride and phosphorus pentoxide have been used in chemicalphosphorylation (Matheis and Whitaker 1984; Matheis 1991), sidereactions such as deterioration of amino acids and polymerizationof proteins occur. Furthermore, food proteins phosphorylated byviolent chemical reagents are not readily accepted by consumersdue to the intense reaction and the difficulty in the removal of theremaining chemicals. Although enzymatic phosphorylation is themost desirable method for food proteins with respect to food safety(Seguro and Motoki 1989; Campbell and others 1992), it brings intoo few phosphate groups for the specificity of the substrate. Such alow level phosphorylation is not enough to improve the functional

MS 20080615 Submitted 8/12/2008, Accepted 10/8/2008. Authors Hayashi,Enomoto, and Sugimoto are with United Graduate School of Agricul-tural Sciences and authors Nagano, Ibrahim, and Aoki are with Dept. ofBiochemical Science and Technology, Faculty of Agriculture, KagoshimaUniv., Kagoshima 890-0065, Japan. Author Li is with Dept. of Food andPharmacy Engineering, School of Chemistry Science and Technology, Yun-nan Univ., Kunming 650091, China. Author Hatta is with Dept. of Foodand Nutrition, Faculty of Home Economics, Kyoto Women’s Univ., 35 Ki-tahiyoshi, Imakumano, Higashiyama, Kyoto, Kyoto 605-8501, Japan. Au-thor Takeda is with Dept. of Health and Nutrition, Faculty of Nursingand Nutrition, Kagoshima Immaculate Heart Univ., 2365 Amatatsu, Sat-sumasendai, Kagoshima 859-0011, Japan. Direct inquiries to author Aoki(E-mail: aoki@chem.agri.kagoshima-u.ac.jp).

properties of food proteins, and this method does not seem to fitthe needs of industrial scale of production due to the high cost ofenzymes.

We have succeeded in phosphorylating EWP by dry-heating inthe presence of orthophosphate (Li and others 2003). Further-more, phosphorylation of EWP by dry-heating in the presence ofpyrophosphate markedly improved its functional properties suchas heat-stability against insolubility, emulsifying property, gellingproperty, and calcium phosphate-solubilizing property with slightconformational changes (Li and others 2004, 2005; Hayashi andothers 2008). This phosphorylation method is applicable for prac-tical use, because pyrophosphate is permitted as a food additive inthe food industry in Japan.

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

Materials and Methods

MaterialsEWP was prepared according to the method of Li and others

(2004) as follows: egg white, separated from infertile eggs pur-chased from Marui Agricultural Cooperative Assn. (Kagoshima,Japan), was homogenized, acidified to pH 5.5 with 1 N HCl, andthen centrifuged. The supernatant obtained was diluted with anequal volume of water, dialyzed, and then lyophilized. All reagentsused were of analytical grade.

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Preparation of PP-EWP anddry-heated EWP (DH-EWP)

PP-EWP was prepared according to the method given in a pre-vious article (Li and others 2004). Native EWP (N-EWP) was dis-solved 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-heatedsamples were dissolved and dialyzed using a dialysis membrane(molecular mass cut-off, 14000) to remove free pyrophosphate for3 d at 5 ◦C against 5 changes of 50 times of Milli-Q water, and thenlyophilized. In comparison with PP-EWP, dry-heated EWP (DH-EWP) was prepared as follows: EWP was dissolved at a concentra-tion of 2% in Milli-Q water and the pH of the solution adjusted to4 with 1 N HCl; the mixture was then lyophilized and dry-heatedunder the same conditions as those of PP-EWP. Finally, dry-heatedsamples were dissolved and dialyzed as described previously.

Determination of phosphorus content of PP-EWPProtein samples (20 mg) were digested in 2 mL of perchloric acid

by heating on an electronic heater. Phosphorus in the digest wasregarded as the total phosphorus of PP-EWP. For the determinationof inorganic phosphorus (Pi), 5 mL of 10% trichloroacetic acid wasadded to the same volume of 10 g/L PP-EWP solution, and the so-lution was centrifugated at 1000 × g for 20 min. The phosphorus inthe supernatant was regarded as Pi. The phosphorus content wasdetermined according to the method of Chen and others (1956).The amount of phosphorus bound to proteins was estimated by thedifference between the total phosphorus and Pi content.

Measurements of the conductivityof foams prepared with EWP

The conductivity of foams prepared with EWP was measuredby the method of Kato and others (1983) with a minor modifica-tion. The foams were produced when air at a constant flow rate of50 mL/min using a flowmeter (KOFLOC Co., Kyoto, Japan) was in-troduced 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 aglass column at 1-cm interval in a distance of 2.4 cm from a glassfilter, connecting with an Orion 3-Star conductivity meter (ThermoFisher Scientific K.K., Kanagawa, Japan). The conductivity read-ing was recorded automatically using a communication cable withconductivity meter. After the air was introduced into the protein so-lution for 15 s, the conductivity of foams measured by an electrodeshowed the maximal value and then the changes in the conductiv-ity of foams were measured over time. The foaming power of theprotein was expressed as the conductivity of foam produced imme-diately after passing air through the solution for 15 s. The foam sta-bility was estimated by measuring the half time of the conductivityimmediately after foam formation (Kato and others 1983; Ibrahimand others 1993).

Preparation of foams from 10% (w/w) EWP solutionProtein 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, ensuringthat the stirring fan was engulfing the foaming solution by tiltingthe beaker. Thus prepared foams were used for microscopic obser-vation, measurement of specific gravity, overrun, and the volume ofdrainage.

Microscopic observation of the foamsSamples 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 in60 min using a Leica MZ16 stereomicroscope (Leica Microsystems,Tokyo, Japan). To compare the size distribution of foams, diame-ter of about 1000 pieces of foams on 3 pictures in each sample wasmeasured by micrograph immediately after preparation from 10%(w/w) of N-, DH-, and PP-EWP. The distribution of the foam diam-eter was calculated by the following equation:

% Distribution of the foam diameter

= Nr of the foams with a certain range of diameterTotal nr of measured foams

× 100

Measurement of the specific gravityand 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 adjustedto 7. Protein solutions were beaten with a single-winged electricmixer at 650 rpm/min for 90 s in a 500-mL plastic beaker, and sam-ples of the foam were gently scooped out with a spatula and placedflat in a cylinder (27 mm in diameter and 14 mm in depth); specificgravity and overrun of foams and foam stability were measured.Specific gravity was calculated by the following equation:

Specific gravity = Weight of 1 mL foamsWeight of 1 mL water

To make accurate overrun measurements, the protein solutionmust be completely incorporated into foam (Phillips and others1987). The overrun was calculated by the following equation:

% Overrun

= (Weight 100 mL solution) − (Weight 100 mL foam)(Weight 100 mL foam)

× 100

Measurement of the foam stabilityof foams prepared from EWP

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

Statistical analysisThe data were expressed as mean values of 4 replicated determi-

nations with standard deviation (SD). Statistical analysis was doneby t-test at 5% level probability.

Results and Discussion

EWP was phosphorylated by dry-heating at pH 4 and 85 ◦C for1 d in the presence of pyrophosphate. The bound phospho-

rus 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 andothers 2004).

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

Figure 1 --- Changes in the conductivity of foams preparedwith N-, DH-, and PP-EWP. The protein concentration ofthe 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 theabsence of pyrophosphate; PP-EWP = EWP dry-heated atpH 4 and 85 ◦C for 1 d in the presence of pyrophosphate.Each value is the mean with its SD (n = 4).

Table 1 --- Foaming power and foaming stability of foamsprepared from 0.1% (w/v) of N-, DH-, and PP-EWP solu-tions.

Foaming powerb Foaming stabilityb

Samplea (×103, μS/cm) (s)

N-EWP 1.47 ± 0.10a 18.4 ± 2.01aDH-EWP 1.79 ± 0.03b 31.1 ± 8.14aPP-EWP 1.76 ± 0.05b 89.2 ± 4.68b

Foams were made by blowing air (50 mL/min) into a glass column for 15 s.aN-EWP= native EWP; DH-EWP = EWP dry-heated at pH 4 and 85 ◦C for 1 d inthe absence of pyrophosphate; PP-EWP = EWP dry-heated at pH 4 and 85 ◦Cfor 1 d in the presence of pyrophosphate.bEach value is the mean with its SD (n = 4); means in same column withdifferent letters are significantly different (P < 0.05).

Figure 2 --- Micrographs of foamsfrom 10% (w/w) of N-, DH-, andPP-EWP solutions. The foams weremade by mixing the EWP solutionswith a mixer for 90 s, andmicrographs were takenimmediately (0 min) and at 60 minafter preparing foams. N-, DH-, andPP-EWP: see Figure 1.

which the 0.1% (w/v) protein solution was poured. Foaming powerwas demonstrated as the conductivity immediately after the foamwas produced, and foaming stability was defined as half time of theinitial 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 betweenDH- 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; itwas 4.8 times that of N-EWP and 2.9 times that of DH-EWP.

Figure 2 shows the stereo micrographs of foams immediately af-ter and 60 min after whipping 10% (w/w) N-, DH-, and PP-EWP so-lutions. The foams of PP-EWP were fine and uniform in their sizecompared with those of N- and DH-EWP. The foams of all sam-ples became large after 60 min. To estimate the size distribution offoams, the diameter of about 1000 pieces of foams in each samplewas measured. The results of the size distribution of foams wereshown in Figure 3. The number of the pieces of foam less than 0.3μm 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 than0.5 μm in diameter was 73.7% for N-EWP, 20.1% for DH-EWP, and3.4% for PP-EWP.

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

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

Phosphorylation is one of the useful methods to improvethe functional properties of food proteins. We succeeded inphosphorylating EWP without serious conformational changesby dry-heating in the presence of pyrophosphate (Li and oth-ers 2004). The solubility of EWP was maintained after phospho-rylation, and phosphorylation improved the heat stability andthe emulsifying and gelling properties. Furthermore, the calciumphosphate-solubilizing ability of EWP was effectively enhanced by

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phosphorylation, suggesting that calcium bioavailability might beincreased 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 forfoam-based foods such as angel cakes, meringue, and mousse. Infoaming of protein solution, initially soluble globular proteins dif-fuse to the air–water interface, concentrate and reduce interfacialtension, denature to orient hydrophilic regions toward the waterand hydrophobic regions toward air, and finally interactions be-tween denatured proteins occur to a form continuous film (Kinsella1981). Thus foams are surrounded and stabilized with denaturedprotein films. There are many factors affecting foaming power and

Figure 3 --- Size distribution of foams immediately afterpreparation from 10% (w/w) of N-, DH-, and PP-EWP so-lutions. N-, DH-, and PP-EWP: see Figure 1.

stability of proteins, such as solubility, rate of unfolding, reori-entation of polypeptides, association of polypeptides to facilitateformation of a viscous surface film with elasticity, tendency to ag-gregate without excessive surface coagulation, surface charge, andhydration (Kinsella 1981).

According to Kato and others (1985), the most important struc-tural factor affecting emulsifying and foaming properties of pro-teins is the surface hydrophobicity. In addition to the surfacehydrophobicity, there are good correlations between the foamingpower and the digestion velocity of proteins, suggesting that flexi-bility of protein structure is also an important structural factor gov-erning the foam formation. Foaming power of PP-EWP for foamsproduced 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 therewere only small significant differences (P < 0.05) between PP- andDH-EWP (Table 1 and 2). It was suggested that proteins in PP-EWPmight more easily diffuse to the air–water interface, reduce interfa-cial tension, and undergo unfolding than those in N-EWP becausethe surface hydrophobicity and digestibility of EWP were increasedby phosphorylation as reported in the previous article (Li and oth-ers 2004).

Foams prepared by whipping 10% (w/w) PP-EWP solution werefiner and more uniform in sizes than those from N- and DH-EWP (Figure 2). Phosphorylation of EWP markedly increased thefoaming stability estimated by both conductivity measurement offoams prepared from 0.1% (w/v) protein solution and drainagefrom foams prepared by whipping 10% (w/w) proteins solution(Table 1 and Figure 4). These results indicated that phosphorylationof EWP was more effective for the foam stability than for the foamformation. Stabilization of protein foams requires the following

Table 2 --- Specific gravity and overrun of foams preparedfrom 10% (w/w) of N-, DH-, and PP-EWP solutions.

Samplea Specific gravityb Overrunb (%)

N-EWP 0.155 ± 0.006a 563.47 ± 25.47aDH-EWP 0.144 ± 0.006ab 611.59 ± 30.22abPP-EWP 0.138 ± 0.005bc 643.25 ± 27.69bcaN-, DH-, and PP-EWP: see Table 1.bEach value is the mean with its SD (n = 4); means in same column withdifferent letters are significantly different (P < 0.05).

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

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factors: the interfacial film should be structurally stable and rela-tively impermeable to the entrapped air; at a critical distance con-tiguous bubbles should be repelled to minimize coalescence; theoutward projecting polar polypeptide loops or segments shouldretain the lamellar liquid against gravity; and the protein in thefilm should possess both rigidity and flexibility to withstand localshocks (Kinsella 1981). Thus, the films surrounding bubbles mustpossess intermolecular cohesiveness and a certain degree of elas-ticity to permit localized contact deformation. Repulsion of neg-ative charge of introduced phosphate groups may make PP-EWPrelatively more unfolded compared with N- and DH-EWP. In thecase of denatured ovalbumin, the electrostatic-repulsive force isimportant in helping to prevent random aggregation, and the bal-ance of attractive and repulsive forces between protein moleculesis needed for the formation of linear polymers (Kitabatake and oth-ers 1988). As reported in the previous article (Li and others 2004),heat-induced gel of PP-EWP was transparent while those of N- andDH-EWP were turbid. Furthermore, the PP-EWP gel was firmer andhigher in water-holding capacity than those of N- and DH-EWP. Inthe transparent gel of PP-EWP, a network of linear polymers of de-natured proteins might be formed. Accordingly, it was suggestedthat the linear aggregation of PP-EWP might be formed in the filmof bubbles, resulting in the formation of strong film compared withthat of N- and DH-EWP.

Conclusions

The foams from the PP-EWP solution were finer and more uni-form in their sizes than those from the N- and DH-EWP solu-

tions. The foam stability of PP-EWP was much better than that ofN- and DH-EWP, although there were only small differences infoaming power, specific gravity, and overrun of the foams amongN-, DH-, and PP-EWP. Thus, phosphorylation of EWP by dry-heating in the presence of pyrophosphate improved the foamingproperties, and it was more effective for the foam stability than forthe foam formation.

AcknowledgmentThis study was supported by Grant-in-Aid 14560226 from the Min-istry of Education, Culture, Sports, Science, and Technology ofJapan.

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