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Changes in physicochemical properties of egg white and yolk proteins from duck shell eggs due to hydrostatic pressure treatment
Changes in physicochemical properties of egg white and yolk proteins from duck shell eggs due to hydrostatic pressure treatment K. M. Lai,*† Y. S. Chuang,‡ Y. C. Chou,‡ Y. C. Hsu,‡ Y. C. Cheng,‡ C. Y. Shi,* H. Y. Chi,* and K. C. Hsu‡1 *Department of Health Diet and Industry Management, and †Department of Nutrition, Chung Shan Medical University Hospital, No. 110 sec. 1, Jianguo N. Road, Taichung 40242, Taiwan, Republic of China; and ‡Department of Nutrition, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan, Republic of China insignificant changes in the physicochemical properties. On the other hand, pressure treatments at 400 and 500 MPa significantly reduced the solubility and residual denaturation enthalpy of egg yolk proteins. However, the native PAGE result showed that pressure treatment up to 500 MPa did not affect the protein components of egg white and yolk. The results showed that the application of pressure treatment on duck shell egg may induce reversible denaturation of both egg white and yolk proteins. The egg white and yolk proteins may be prevented from denaturation after pressure treatment in the presence of the eggshell compared with the absence of the eggshell. As reported in the literature, pressure treatments at 300 to 500 MPa and 25°C would be efficient for decontamination of duck shell eggs. Therefore, based on the consideration for food safety and functional properties, pressure processing can be a good preservation technique for duck shell eggs.
Key words: duck egg, hydrostatic pressure, egg white, yolk, physicochemical property 2010 Poultry Science 89:729–737 doi:10.3382/ps.2009-00244
INTRODUCTION
et al., 2003, 2004, 2005a, 2006). In these studies, the changes in structural properties of ovalbumin or the other egg white proteins, induced by heating, resulted in the change of the functional properties. Heating of egg white solutions at 50 to 85°C results in significant unfolding of the proteins accompanied by an exposure of hydrophobic groups and sulfhydryl (SH) groups, originally buried in the protein core. The decrease in denaturation enthalpy indicates loss of secondary protein structures (Ma and Harwalkar, 1991). During heating, the protein solubility decreased and turbidity increased due to hydrophobic interactions and the formation of disulfide (SS) bonds through SH-SS exchange reactions and SH oxidation (Van der Plancken et al., 2005b). Hen egg yolk is a complex association of lipids (33% by weight), proteins (17%), and water (50%) in which several types of solids such as spheres, granules, low-
Eggs contain proteins of high biological value as compared with other dietary proteins. Egg proteins possess desirable functional and nutritional properties and therefore are widely used in many food products. Egg white proteins are well known for their gelling, foaming, and emulsifying characteristics, and the structural properties of egg white proteins correlated to their functional characteristics have been reported by many studies, with an emphasis on the contribution of the major egg white protein, ovalbumin (Van der Plancken ©2010 Poultry Science Association Inc. Received May 18, 2009. Accepted January 7, 2010. 1 Corresponding author: [email protected]
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ABSTRACT The shell of the duck egg did not crack after pressure treatments (300 to 500 MPa; 25°C; 10 min) in this study; therefore, the changes of physicochemical properties of egg white and yolk proteins from the intact shell egg by pressure treatment were first investigated and compared with those of pressurized hen liquid eggs. Although the proximate compositions of duck eggs and hen eggs were similar, the moisture and protein contents of hen whole eggs were higher than those of duck whole eggs. The protein contents of duck egg white and yolk were slightly lower than those of hen eggs, and the moisture content of duck egg white was equal to that of hen egg white, whereas that of duck egg yolk was lower than that of hen egg yolk. After pressure treatment at 500 MPa, the results of solubility, sulfhydryl content, surface hydrophobicity, and residual denaturation enthalpy showed that egg white proteins underwent slight but significant unfolding and aggregation, whereas pressure treatments below 500 MPa induced
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yolk compositions of duck eggs are similar to those of hen eggs (Ricklefs, 1977); therefore, duck egg white and yolk are suggested to possess functional properties similar to hen eggs. Furthermore, intact shell eggs were not used in the previous studies, which reported the effect of pressure treatment on the physicochemical properties of egg white and yolk proteins, probably due to the cracking of the eggshells under pressure. According to the observation of our preliminary experiment, no cracks were found in the duck eggshell, which might be attributed to the greater thickness (0.5 to 0.6 mm) of the duck eggshell than that (0.3 to 0.4 mm) of the hen eggshell. The thickness and composition would determine the breaking strength of the eggshell. Further research is needed to study the composition and breaking strength of the duck eggshell to determine the mechanism by which the shell withstands pressure. The aim of this study was to investigate the effect of pressure treatment (300 to 500 MPa) at ambient temperature (25°C) on the physicochemical properties of egg white and yolk proteins from duck shell eggs. The changes in secondary structures of the proteins were determined by differential scanning calorimetry (DSC); the native protein components of egg white and yolk proteins were also studied in more detail by electrophoresis.
MATERIALS AND METHODS Materials Collected duck eggs were 1 d old and produced by a genetically closed flock of Anas platyrhynchos (Tsaiya). The eggs, which weighed 67 to 74 g, were purchased from a local market in Taichung City and all cracked or thin-shelled eggs were discarded. Eggs were washed by tap water, left to dry, and then stored at 4°C within 5 d of purchase before experiments. 1-Anilinonaphthalene-8-sulfonic acid (ANS) and 5′,5′-dithiobis(2nitrobenzoic acid) (DTNB) was obtained from Sigma (St. Louis, MO). Reagent-grade chemicals were used to prepare the following: Tris-glycine buffer [0.1 M Tris(hydroxymethyl)-aminomethane (Tris), 0.1 M glycine, and 4 mM EDTA disodium salt, pH 8.0], 5% sodium dodecyl sulfate in Tris-glycine buffer (denoted SDSTris-glycine), Ellman’s reagent (4 mg/mL of DTNB in Tris-glycine buffer), Tris-HCl buffer (0.2 M Tris-HCl, pH 7.4), and sodium phosphate buffer (0.1 M, pH 7.4). The yolk protein content reagent Coomassie Brilliant Blue G-250 (100 mg) was dissolved in 50 mL of 95% ethanol. To this solution, 100 mL of 85% (wt/vol) phosphoric acid was added and then diluted to a final volume of 1 L.
Proximate Composition and pH For determining the proximate compositions of a duck egg, the egg white was separated from the egg yolk and the chalazae were removed. The yolk was care-
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density lipoproteins (LDL), and myelin figures are suspended in a protein solution or plasma (McCully et al., 1962). Almost 68% of yolk DM is LDL, with 16% being high-density lipoproteins, 10% livetins, and 4% phosvitins. Egg yolk is widely used in the food industry due to the excellent functional properties of its lipoproteins, such as flavor, aroma, color, viscosity, emulsifying, and foaming (Fennema, 1993). Denaturation of egg yolk proteins resulted in functional modifications, such as emulsifying and rheological properties (Ahmed et al., 2003; Speroni et al., 2005). High-pressure processing, a nonthermal processing technique, is now suggested to be an interesting alternative to heat processing and results in a better balance between food safety and food quality. Pressure can, however, also induce protein denaturation, depending on the protein concentration, pressure level, temperature, and pH (Balny and Masson, 1993). Similar to heat treatment, pressure treatment of egg white proteins above 450 MPa results in a loss of secondary structure (Hayakawa et al., 1996). The pressure-induced structural changes in egg white proteins can also be demonstrated by the exposure of previously buried hydrophobic and SH groups (Iametti et al., 1998, 1999; Van der Plancken et al., 2004, 2005b, 2006). A strong increase in surface hydrophobicity was observed between 400 and 700 MPa (Van der Plancken et al., 2005a). Furthermore, the decrease and increase of total and exposed SH groups, respectively, were enhanced by pressure above 500 MPa. On the other hand, pressure treatment at 410 MPa induced proteins in egg yolk dispersions to aggregate and undergo a sol-gel transition (Aguilar et al., 2007), whereas that at 600 MPa resulted in the modification of emulsifying properties with no effect on the protein solubility of LDL solutions (Speroni et al., 2005). For application in food products, pasteurization of the whole egg by thermal treatment is often required to ensure microbial safety, principally to control Salmonella species (USDA, 1969). Previous research has shown that pressure treatments (300 to 450 MPa) at various temperatures (15, 20, or 50°C) for 5 to 15 min efficiently inactivated Salmonella Enteritidis inoculated in liquid whole egg (Ponce et al., 1995, 1999). However, the inactivation of Salmonella species contaminating the eggshell, resulting from pressure treatment, has not been investigated. According to the observations of previous studies, pressure treatments of 300 to 400 MPa at ambient temperature were effective in reducing Salmonella Enteritidis or Salmonella Typhimurium populations in cheese (De Lamo-Castellví et al., 2007), tryptone soy broth (Erkmen, 2009b), and raw milk (Erkmen, 2009a). Therefore, pressure treatments at 300 to 500 MPa and 25°C should be efficient for decontamination of duck shell eggs. The choice of decontamination technique, however, would affect not only the physicochemical characteristics of egg white and yolk proteins but also their functional properties. In fact, the egg white and
PRESSURE TREATMENT OF DUCK SHELL EGG
fully rolled on filter paper (Whatman No. 4, Whatman International Ltd., Maidstone, UK) to remove chalazae and traces of albumen adhering to the vitelline membrane. The vitelline membrane was then disrupted with a scalpel blade and yolk was collected in a beaker. The whole egg was obtained by manually breaking an egg. Then the egg white, yolk, and whole egg were gently mixed by stirring. The proximate composition (moisture, ash, protein, and lipid content) of whole egg, egg white, and yolk was analyzed following the AOAC methods (AOAC, 2000). The pH values of egg white and yolk were determined by a pH meter (SP-2200, Suntex Instruments Co. Ltd., Taipei, Taiwan).
For each experiment, 1 shell duck egg was packed in a Cryovac polyethylene pouch (10 × 15 cm; Sealed Air Corporation, Taoyuan, Taiwan) and vacuumsealed. The packaged samples were submerged in the high-pressure vessel that had water as the hydrostatic fluid medium in the press. Then pressure treatments of 300 to 500 MPa at 25°C for 10 min were applied. After pressure release, the samples were immediately cooled in ice water to stop further heat denaturation and analyzed within 6 h of storage at 4°C. Experiments at atmosphere pressure (0.1 MPa), set under the same conditions of holding time (10 min) and temperature (25°C), constituted controls. The intact shell eggs, after pressure treatment, were carefully checked and then used for the following experiments. For confirming that the applied pressure was transmitted through the shell to egg white and yolk, the albumen index [albumen height/(long diameter of albumen + short diameter of albumen)/2 × 100] and yolk index (yolk height/yolk diameter × 100) were both determined. Although the albumen index was not changed after all pressure treatments, the yolk index significantly increased from 37.88 (control) to 45.14 after the 500-MPa treatment (data not shown). A high-pressure apparatus (CIP UNIT, Mitsubishi Heavy Industries Ltd., Shinagawa, Japan) with an oilpressure generator and a compressing vessel, in which the internal portion (diameter: 100 mm; height: 180 mm) was a flat-bottomed cylindrical shape, was employed. During pressure treatments, the vessel of the pressure unit was thermostatically controlled by a circulator. The pressure-raising rate was 200 MPa/min, and the pressure-decreasing rate was 400 MPa/min. Due to compressive heating, an increase of the temperature of the pressurizing fluid, by up to a maximum of 16°C at 500 MPa, was observed when the pressure reached 500 MPa. During the holding time, the fluid temperature decreased rapidly to 25 ± 2°C at 6 min and then maintained this temperature until the end of treatment. After the following instantaneous decompression, the temperature of the fluid decreased to 18 ± 2°C. According to the results of previous studies (Mine
et al., 1990; Cordobés et al., 2004), denaturation of hen egg white and yolk proteins was not found within this temperature range. Therefore, the effect of adiabatic heating during pressurization on protein denaturation was disregarded.
Solubility The solubility of egg white protein was determined according to the method of Van der Plancken et al. (2005a) with some modifications. After pressure treatments, the egg white was immediately separated from the egg yolk and the chalazae were removed. The egg white was then gently mixed and diluted with 10-fold volume-distilled water (vol/vol). The egg white solution was then centrifuged at 18,000 × g and 4°C for 15 min. Protein content of the supernatant was determined using Sigma Procedure No. TRPO-562. This method of protein quantification is based on the reduction of Cu2+ by protein in an alkaline environment. Bicinchoninic acid forms a colored complex with the resulting Cu+. The absorbance of this complex was measured at 562 nm. The content was determined by comparison with a standard curve using BSA. The solubility of 100% was assigned to samples containing the same amount of protein as the control sample. The protein solubility of egg yolk was determined according to the method of Bradford (1976). The yolk was diluted 5-fold (vol/vol) with 0.05 M Tris-HCl buffer (pH 6.5) and equilibrated for 1 h at room temperature under continuous stirring. A 4-mL aliquot was taken for the determination of the initial protein content. The remaining solution was centrifuged at 10,000 × g and 4°C for 20 min. Then, 4 mL of the supernatant was used for protein content determination. Protein content of samples was determined according to the method of Bradford (1976). Briefly, 5 mL of reagent solution [0.01% (wt/vol) Coomassie Brilliant Blue G-250, 4.7% (wt/ vol) ethanol, and 8.5% (wt/vol) phosphoric acid] was added to the protein solution and mixed by vortexing. The absorbance of the mixture was measured at 595 nm after 2 min. Calibration of the assay was performed with standard BSA. Protein solubility was calculated as (mg of protein in the supernatant/mg protein in the initial solution) × 100.
SH Groups The concentration of SH groups of egg white was determined using Ellman’s reagent, which is based on the reaction at neutral and alkaline pH between protein SH groups and DTNB, resulting in the formation of the thionitrophenylated protein and a yellow thionitrophenylate anion (Kalab, 1970). The content of total, exposed, and buried SH groups of the total (1:10 dilution of the treated sample in Tris-HCl buffer, vol/ vol) or soluble protein fraction (supernatant obtained as described previously) was measured in duplicate us-
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High-Pressure Treatment
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Surface Hydrophobicity To 3-mL aliquots of egg white solution (concentration ranging from 0 to 0.02% protein in sodium phosphate buffer), 15 μL of 8 mM ANS in sodium phosphate buffer was added and mixed well by vortexing (Alizadeh-Pasdar and Li-Chan, 2000). After 15 min in the dark, the fluorescence intensity (FI) of each solution was measured by a fluorescence spectrofluorometer (Hitachi F-2000, Tokyo, Japan) with an excitation at 390 nm and an emission at 470 nm. For eliminating the effect of turbidity of egg white solution on FI, the FI values of each concentration of protein without fluorescence probe were also determined (Van der Plancken et al., 2006). The slope of the relative corrected FI versus protein concentration plot was used as an index of surface hydrophobicity.
Enthalpy of Denaturation The enthalpies of denaturation of egg white and yolk were determined using the DSC (Speroni et al., 2005; Van der Plancken et al., 2006). Pressure-treated samples were directly and qualitatively transferred to stainless steel pans and hermetically sealed. About 4 mg of proteins was used in each assay. An empty pan was used as a reference. The egg white samples were analyzed from 20 to 100°C at a heating rate of 10°C/ min, and the egg yolk samples were analyzed from 30 to 110°C at a heating rate of 1°C/min in a Nano DSC (CSC 6300, Calorimeter Sciences Corp., Linden, UT). The enthalpy of denaturation was calculated from the peak area of the thermogram, using CpCalc (version 2.2.10.10) software (Calorimeter Sciences Corp.).
Electrophoresis Native PAGE was performed to demonstrate the effects of pressure treatment on the protein components of egg white and yolk as per the method described by Alomirah et al. (1998) for meat protein. The egg white was diluted 10-fold in 0.2 M Tris-HCl buffer (pH 6.8) to ensure complete solubilization of proteins, the egg yolk was diluted with a 10-fold volume of 10% NaCl solution (wt/vol), and then all samples were stored at −20°C. The resolving gel of native PAGE was 12.5% acrylamide (wt/vol). Electrophoretic migration was performed at 20 mA for 2 h. The proteins were stained with a solution containing 0.27% (wt/vol) Coomassie Brilliant Blue R250, 45% (vol/vol) methanol, and 45% (vol/vol) acetic acid and then destained in the solution containing 10% (vol/vol) acetic acid and 20% (vol/vol) methanol.
Statistical Analysis The Statistical Analysis System Version 8.2 (SAS Institute Inc., Cary, NC) was adapted to perform data analysis and statistical computations for ANOVA and Duncan’s test. Significance of differences was defined at P < 0.05. The differences among treatments were verified by their least significant difference. Experiments were conducted in triplicate.
RESULTS AND DISCUSSION Proximate Composition and pH Duck egg composition is shown in Table 1 and confirmed the report by Ricklefs (1977). The albumen ratio (calculated as albumen weight/egg weight) is 55.76%. The moisture, protein, lipid, and ash contents of duck egg white are 87.23, 8.56, 0, and 0.72%, respectively; on the other hand, those of yolk are 44.53, 14.05, 35.81, and 2.23%, respectively. The pH values of egg white and yolk were 7.42 and 6.51, respectively. As compared with the proximate compositions of hen eggs (Mine, 2007), the albumen ratio of a duck egg is lower, whereas the yolk ratio is higher. The moisture and protein contents of hen whole eggs are higher than those of duck whole eggs, whereas the lipid and ash contents of both whole eggs are similar. The protein contents of duck egg white and yolk are slightly lower than those of hen eggs, and the lipid content of duck egg yolk is higher than hen egg yolk. The moisture content of duck egg white is equal to that of hen egg white, whereas that of duck egg yolk is lower than that of hen egg yolk. Furthermore, the shell ratio is higher in a duck egg than a hen egg.
Solubility Due to pressure treatment, egg white proteins may unfold; therefore, hydrophobic residues and SH groups
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ing both of the following procedures (Van der Plancken et al., 2005b). For determination of exposed SH groups, 2 mL of the total or soluble protein fraction was added to 2 mL of Tris-glycine buffer and 40 μL of Ellman’s reagent. The mixture was allowed to stand at ambient temperature for 15 min and then either centrifuged at 18,000 × g and 4°C for 15 min (soluble protein fraction) or maintained at 4°C for 15 min (total protein fraction). Finally, the absorbance was measured at 412 nm against a reagent blank. For determination of total SH groups, 2 mL of the total or soluble protein fraction was added to 2 mL of SDS-Tris-glycine buffer and 40 μL of Ellman’s reagent. The mixture was maintained at 40°C in a water bath for 15 min to allow the protein to unfold and all SH groups to be accessible to DTNB. Finally, the absorbance was also measured at 412 nm against a reagent blank. A molar extinction coefficient of 13,600 M−1·cm−1 for the thionitrophenylate anion at 412 nm was used to calculate the amount of exposed or surface and total SH groups (Beveridge et al., 1974). The amount of buried SH groups (inaccessible to DTNB) was calculated by subtracting the amount of exposed SH groups from the total amount of the SH groups.
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PRESSURE TREATMENT OF DUCK SHELL EGG Table 1. The comparison of proximate compositions of duck eggs and hen eggs Hen egg2
Duck egg Item Weight (g) Moisture (%) Protein (%) Lipid (%) Ash (%) pH 1n
Whole egg
Egg white
Yolk
n1
70.0 ± 2.91 62.9 ± 0.62 9.3 ± 1.64 11.4 ± 1.43 1.1 ± 0.12 7.14
39.0 ± 1.58 87.2 ± 0.13 8.6 ± 1.31 0.00 ± 0.0 0.7 ± 0.11 7.42
22.4 ± 1.29 44.5 ± 0.13 14.1 ± 1.48 35.8 ± 1.69 2.2 ± 0.23 6.51
80 10 10 10 10 10
Whole egg
Egg white
Yolk
55 66.1 12.8–13.4 10.5–11.8 0.8–1.0
33–35 87.6 9.7–10.6 0.03 0.5–0.6
15–16 48.7 15.7–16.6 31.8–35.5 1.1
= number of eggs analyzed. data were from Mine (2007).
2The
SH Groups Ovalbumin is the only egg white protein to contain 4 free SH groups, which are originally buried in the protein core, and the protein contains 1 SS bond (Powrie and Nakai, 1986). All of these groups are relevant to thermal aggregation, and the exposure of both SS and SH groups in this protein has been considered an index of protein denaturation (Tani et al., 1997). Heatinduced denaturation of ovalbumin results in exposure of these SH groups, accompanied by a decrease in total SH content, due to oxidation of SH groups to SS bonds (Rumbo et al., 1996). The contribution of both the soluble and the insoluble protein fractions of pressurized egg white to the SH content was investigated (Figure 1). In the soluble protein fraction, pressure treatment resulted in a tendency to pressure-dependent exposure of buried SH groups (P > 0.05), and significant (P < 0.05) decreases of total and buried SH content were observed at 500 MPa. In the total protein fraction, only buried SH content decreased significantly (P > 0.05) as the result of pressure treatment at 500 MPa. Essentially, all free SH groups remained in the soluble protein fraction, as could be expected based on the high residual protein solubility (96.3% after 10 min at 500 MPa, as shown in Table 2). This indicates that most of the ovalbumin molecules remained soluble, even though they might be involved in aggregation through SH-SS exchange reactions or the formation of SS bonds (Van der Plancken et al., 2005b).
Table 2. Effect of pressure treatment on the protein solubility of egg white and yolk from shell duck eggs1 Solubility (%) Pressure treatment (MPa) 300 400 500
Egg white
Yolk
99.9 ± 1.34a 99.4 ± 0.98a 96.3 ± 1.43b
97.6 ± 1.67a 93.9 ± 2.38b 84.3 ± 3.44c
a–cDifferent letters in the same row indicate significant differences (P < 0.05). 1All values are means ± SD of data from 3 independent experiments.
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are exposed and aggregates are formed and stabilized by hydrophobic interactions and SS bonds (Van der Plancken et al., 2005a,b). The aggregates may be soluble or insoluble according to their sizes and the insoluble fractions can be removed by centrifugation. The protein content loss of the soluble fractions represents protein aggregation attributed to pressure treatment. As shown in Table 2, pressure treatments at 300 to 500 MPa induced slight decreases in protein solubility of egg white. However, this decrease (3.7%) in solubility of egg white was significant (P < 0.05) only at 500 MPa. On the other hand, the solubility of yolk decreased with elevated pressure, whereas the solubility decreased to 84.3% with the 500-MPa treatment. A previous study reported that the protein solubility of egg white solution (10% in Tris-HCl buffer, pH 7.6, vol/vol) treated with 100 to 500 MPa at 25°C decreased less than 10%, whereas that treated with 550 to 700 MPa declined dramatically from 60 to 40% (Van der Plancken et al., 2005a,b). However, another previous study reported that fresh egg white turned opaque with a partial coagulation by the 500-MPa treatment (25°C) and formed hard gels that stood under their own weight above 600 MPa (Okamoto et al., 1990). The different results obtained in these studies might be attributed to protein concentrations and confirmed the report by Iametti et al. (1998). The latter study showed that ovalbumin solution (0.5, 2, and 5 mg/mL in 50 mM phosphate buffer, pH 6.8) at higher concentrations formed more pronounced aggregates under pressure treatment. Furthermore, pressure treatment at 450 MPa did not induce ovalbumin solution at the concentrations of 0.5 and 2 mg/mL to form aggregates, whereas the solubility of ovalbumin solution at the concentration of 5 mg/mL decreased to about 50%. In the present study, however, pressure treatment slightly decreased the solubility of duck egg white at the concentration of 84.6 mg/mL. Okamoto et al. (1990) observed that hen egg yolk was transformed to a gel structure by the 400-MPa treatment. The result showed that the egg white and yolk proteins may be prevented from forming aggregates in the whole egg treated by pressure treatments at 300 to 500 MPa, as compared with egg white or yolk individually pressurized at the same pressure levels.
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Lai et al. Table 3. Effect of pressure treatment on the surface hydrophobicity of egg white from shell duck eggs1 Pressure (MPa) Surface hydrophobicity S0
0.1 (control) 188 ±
16.1a
300 197 ±
18.3a
400 209 ±
16.9a
500 238 ± 11.5b
a,bDifferent 1All
letters indicate significant differences (P < 0.05). values are means ± SD of data from 3 independent experiments.
Surface Hydrophobicity
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The effect of pressure on the surface hydrophobicity of egg white proteins is shown in Table 3. Pressure treatment at 500 MPa resulted in a significant (P < 0.05) pressure-dependent increase (26%) in surface hydrophobicity, and slight but insignificant increases in surface hydrophobicity were observed below 500 MPa. This indicates that pressure treatment of egg white proteins causes the hydrophobic residues to be exposed and thus more readily available for binding with ANS (Smith et al., 2000). The previous study reported that ovalbumin (0.25 mg/mL, pH 6.5) showed little increase in ANS fluorescence after the 400-MPa treatment. However, a large enhancement of the fluorescence was observed after higher pressure (500 to 800 MPa) treatments. Pressure treatment affects protein internal interactions solely by volume changes. Consequently, covalent bonds are not disrupted by pressure, except for the oxidation of SH groups or SH-SS exchange reactions. Further, pressure mainly disrupts or induces hydrophobic and electrostatic interactions (Silva and Weber, 1993). Previous studies reported that, however, a pronounced increase in surface hydrophobicity of the hen egg white protein solution or albumen by pressure treatment was observed between 400 and 700 MPa (Smith et al., 2000; Van der Plancken et al., 2006). The result showed that the egg white proteins may be prevented from exposing hydrophobic residues in the whole egg treated by pressure treatments at 300 to 500 MPa, as compared with the pressurized egg white solution at the same pressure levels.
proteins (Van der Plancken et al., 2007). The endothermic reactions reflect the disruption of hydrogen bonds, and the exothermic processes show the break up of hydrophobic interactions and protein aggregation (Ma
Enthalpy of Denaturation Secondary structure of egg white proteins is altered by pressure treatment, as shown by Fourier transform infrared spectroscopy, circular dichroism spectroscopy, DSC, and intrinsic fluorescence measurements (Hayakawa et al., 1992; Smith et al., 2000). Hayakawa et al. (1992, 1996) reported that the DSC endothermic enthalpy and the α-helical content of the pressure-denatured ovalbumin decreased together, and they also demonstrated that pressure treatment caused the breakdown of secondary structures of ovalbumin. In addition to the changes in secondary structure, pressure does not cause extensive denaturation (Smith et al., 2000). Therefore, the residual enthalpy is correlated with the content of ordered secondary structure of the pressure-treated
Figure 1. Effect of pressure treatment on the (A) exposed, (B) total, and (C) buried sulfhydryl (SH) content of egg white from shell duck eggs. Mean value of SD is less than 1.2%.
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and Harwalkar, 1991). Therefore, DSC thermograms of the pressure-treated egg white and yolk proteins show the subsequent thermal denaturation of the proteins remaining as the native conformation. Thus, a reduction in residual enthalpy is an indication for a partial loss of protein structure during pressure processing. In Figure 2, the thermograms of egg white and yolk after pressure treatment are shown. According to the DSC thermogram of egg white, there are 3 endothermic peaks (65.8, 72.8, and 84.1°C) observed in egg white. The left-hand peak corresponds to the denaturation of ovotransferrin, the middle peak to that of lysozyme, and the peak at 84.1°C to that of ovalbumin (Van der Plancken et al., 2005a). On the other hand, there was a major endothermic peak (80.9°C) corresponding to the denaturation of phosvitin in egg yolk (Chung and Ferrier, 1995). In Figure 3, the residual denaturation enthalpies of egg white and yolk after pressure treatment are shown. The residual enthalpies of egg white did not significantly (P > 0.05) decrease after 300- and 400-MPa pressure treatments but decreased significantly (P < 0.05) to 96.3% after the 500-MPa treatment, whereas those of egg yolk decreased significantly (P < 0.05) to 84.3% after the 500-MPa treatment. The results may confirm the observations of the changes of solubility, SH groups, and surface hydrophobicity as described above (Tables
2 and 3, Figure 1). A previous study reported that an apparent decrease of residual denaturation enthalpy of egg white solution (9.64 mg/mL in 0.2 M Tris-HCl at pH 7.6) was observed after pressure treatment at 25°C and 400 MPa, and no residual enthalpy could be detected at 700 MPa (Van der Plancken et al., 2005a). On the other hand, hen egg yolk showed a progressive decrease in thermal denaturation enthalpy, without any significant change in denaturation temperature, as the pressure level was raised from 250 to 440 MPa (Aguilar et al., 2007).
Figure 2. Differential scanning calorimetry thermograms of egg white and yolk from shell duck eggs by pressure treatment. Arrows indicate endothermic peaks.
Figure 3. Effect of pressure treatment on the residual denaturation enthalpy (ΔHdenaturation) of egg white and yolk from shell duck eggs. Mean value and error bars represent SD of triplicate measurement.
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The native PAGE (Figure 4) result showed that pressure did not affect the protein components of egg white and yolk, although the solubility and residual denaturation enthalpy of egg yolk slightly but significantly decreased after the 500-MPa treatment. Pressure treatment can induce protein denaturation of hen egg white and yolk solutions, which has been reported in previous studies (Van der Plancken et al., 2005a; Aguilar et al., 2007). Smith et al. (2000) observed that pressure treatment at 400 MPa induced limited irreversible changes in the secondary structure of ovalbumin. Van der Plancken et al. (2005b) demonstrated that ovalbumin aggregates engaged in the SS bonds were present in the water-soluble fraction, demonstrated by SDS-PAGE patterns. This work demonstrated that the denaturation of duck egg white and yolk proteins was prevented in shell eggs under pressure up to 500 MPa, as compared with that of the pressurized hen egg white and yolk solutions at the same pressure levels. However, slight decreases in the solubility and the residual denaturation enthalpies of both egg white and yolk proteins were observed after pressure treatment, especially at 500 MPa. Native PAGE patterns showed that no aggregates of egg white or yolk proteins were formed after
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pressure treatments, probably due to the renaturation of the proteins during storage at −20°C. The results may indicate that pressure treatment causes reversible denaturation of these proteins in shell eggs. Furthermore, the sensitivities of egg white and yolk proteins in the intact shell egg to pressure treatment seemed to be different from those in the liquid form. We suggested that the egg white and yolk in the liquid form have been mixed and represented as the well-distributed state; therefore, pressure treatment would directly induce their protein denaturation. On the other hand, the effect of pressure treatment on the egg white and yolk represented as the native state in the shell egg may not be equal to those in the liquid form. Based on the consideration for food safety and functional properties, therefore, pressure processing can be a good preservation technique for duck shell eggs.
ACKNOWLEDGMENTS This study was financially supported by the National Science Council, Republic of China, no. NSC 97-2313B-039-006. We thank Chin-Shuh Chen and Po-Yuan Chiang (Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, Taiwan, Republic of China) for supplying the high-pressure equipment.
REFERENCES Aguilar, J. M., F. Cordobés, and A. Jerez. 2007. Influence of high pressure processing on the linear viscoelastic properties of egg yolk dispersions. Rheol. Acta 46:731–740. Ahmed, J., H. S. Ramaswamy, I. Alli, and M. Ngadi. 2003. Effect of high pressure on rheological characteristics of liquid egg. Lebensm. Wiss. Technol. 36:517–524. Alizadeh-Pasdar, N., and E. C. Y. Li-Chan. 2000. Comparison of protein surface hydrophobicity measured at various pH values using three different fluorescent probes. J. Agric. Food Chem. 48:328–334. Alomirah, H. F., I. Alli, B. F. Gibbs, and Y. Konishi. 1998. Identification of proteolytic products as indicators of quality in ground and whole meat. J. Food Qual. 21:299–316. AOAC. 2000. Official Methods of Analytical Chemists. 17th ed. Association of Official Analytical Chemists Inc., Arlington, VA. Balny, C., and P. Masson. 1993. Effects of high pressure on proteins. Food Rev. Int. 9:611–628. Beveridge, T., S. J. Toma, and S. Nakai. 1974. Determination of SHand SS-groups in some food proteins using Ellman’s reagent. J. Food Sci. 39:49–51. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254. Chung, S. L., and L. K. Ferrier. 1995. Heat denaturation and emulsifying properties of egg yolk phosvitin. J. Food Sci. 60:906–908, 916. Cordobés, F., P. Partal, and A. Guerrero. 2004. Rheology and microstructure of heat-induced egg yolk gels. Rheol. Acta 43:184– 195. De Lamo-Castellví, S., A. X. Roig-Sagués, T. López-Pedemonte, M. M. Hernández-Herrero, B. Guamis, and M. Capellas. 2007. Response of two Salmonella enterica strains inoculated in mod-
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Figure 4. Native PAGE analysis of (A) egg white and (B) yolk from shell duck eggs by pressure treatment. Std = native standard; C = control; 300 to 500 = 300 to 500 MPa, respectively. Color version available in the online PDF.
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Powrie, W. D., and S. Nakai. 1986. The chemistry of eggs and egg products. Pages 97–139 in Egg Science and Technology. O. J. Cotterill, ed. AVI Publishing, New York, NY. Ricklefs, R. E. 1977. Composition of eggs of several bird species. Auk 94:350–356. Rumbo, M., F. G. Chirdo, C. A. Fossati, and M. C. Anon. 1996. Analysis of structural properties and immunochemical reactivity of heat-treated ovalbumin. J. Agric. Food Chem. 44:3793– 3798. Silva, J. L., and G. Weber. 1993. Pressure stability of proteins. Annu. Rev. Phys. Chem. 44:89–113. Smith, D., V. B. Galazka, N. Wellner, and I. G. Sumner. 2000. High pressure unfolding of ovalbumin. Int. J. Food Sci. Technol. 35:361–370. Speroni, F., M. C. Puppo, N. Chapleau, M. De Lamballerie, O. Castellani, M. C. Aňón, and M. Anton. 2005. High-pressure induced physicochemical and functional modifications of lowdensity lipoproteins from hen egg yolk. J. Agric. Food Chem. 53:5719–5725. Tani, F., N. Shirai, T. Onishi, F. Venelle, K. Yasumoto, and E. Doi. 1997. Temperature control for kinetic refolding of heat-denatured ovalbumin. Protein Sci. 6:1491–1502. USDA. 1969. Egg pasteurization manual. ARS 74–48. Poultry Laboratory, Department of Agriculture, Agriculture Research Service, Albany, CA. Van der Plancken, I., M. Delattre, A. Indrawati, Van Loey, and M. E. G. Hendrickx. 2004. Kinetics study on the changes in the susceptibility of egg white proteins to enzymatic hydrolysis induced by heat and high hydrostatic pressure pretreatment. J. Agric. Food Chem. 52:5621–5626. Van der Plancken, I., A. Van Loey, and M. E. G. Hendrickx. 2005a. Combined effect of high pressure and temperature on selected properties of egg white proteins. Innov. Food Sci. Emerg. Technol. 6:11–20. Van der Plancken, I., A. Van Loey, and M. E. G. Hendrickx. 2005b. Changes in sulfhydryl content of egg white proteins due to heat and pressure treatment. J. Agric. Food Chem. 53:5726–5733. Van der Plancken, I., A. Van Loey, and M. E. G. Hendrickx. 2006. Effect of heat-treatment on the physico-chemical properties of egg white proteins: A kinetic study. J. Food Eng. 75:316–326. Van der Plancken, I., A. Van Loey, and M. E. G. Hendrickx. 2007. Kinetic study on the combined effect of high pressure and temperature on the physico-chemical properties of egg white proteins. J. Food Eng. 78:206–216. Van der Plancken, I., and M. Van Remoortere., IIndrawati, A., Van Loey, and M. E. G. Hendrickx. 2003. Heat-induced changes in the susceptibility of egg white proteins to enzymatic hydrolysis: A kinetic study. J. Agric. Food Chem. 51:3819–3823.
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el cheese treated with high hydrostatic pressure. J. Dairy Sci. 90:99–109. Erkmen, O. 2009a. High hydrostatic pressure inactivation kinetics of Salmonella Typhimurium. High Press. Res. 29:129–140. Erkmen, O. 2009b. Mathematical modeling of Salmonella Typhimurium inactivation under high hydrostatic pressure at different temperatures. Food Bioprod. Process. 87:68–73. Fennema, O. R. 1993. Principles of Food Science: Food Chemistry. Marcel Dekker, New York, NY. Hayakawa, I., J. Kajihara, K. Morikawa, M. Oda, and Y. Fujio. 1992. Denaturation of bovine serum albumin (BSA) and ovalbumin by high pressure, heat and chemicals. J. Food Sci. 57:288–292. Hayakawa, I., Y. Y. Linko, and P. Linko. 1996. Mechanism of high pressure denaturation of proteins. Lebensn. Wiss. Technol. 29:756–762. Iametti, S., E. Donnizzelli, P. Pittia, P. P. Rovere, N. Squarcina, and F. Bonomi. 1999. Characterization of high-pressure-treated egg albumen. J. Agric. Food Chem. 47:3611–3616. Iametti, S., E. Donnizzelli, G. Vecchio, P. P. Rovere, S. Gola, and F. Bonomi. 1998. Macroscopic and structural consequences of high-pressure treatment of ovalbumin solutions. J. Agric. Food Chem. 46:3521–3527. Kalab, M. 1970. Factors affecting the Ellman determination of sulfhydryl groups in skimmilk powder and gels. J. Dairy Sci. 53:711–718. Ma, C., and V. R. Harwalkar. 1991. Thermal analysis of food proteins. Adv. Food Nutr. Res. 35:317–366. McCully, K. A., C. C. Mok, and R. H. Common. 1962. Paper electrophoresis characterization of proteins and lipoproteins of hen’s egg yolk. Can. J. Biochem. Physiol. 40:937–952. Mine, Y. 2007. Structure and chemical composition of eggs. Page 15 in Egg Bioscience and Biotechnology. E. C. Y. Li-Chan and H. O. Kim, ed. John Wiley & Sons, Hoboken, NJ. Mine, Y., T. Noutomi, and N. Haga. 1990. Thermally induced changes in egg white proteins. J. Agric. Food Chem. 38:2122–2125. Okamoto, M., Y. Kawamura, and R. Hayashi. 1990. Application of high pressure to food processing: Textural comparison of pressure- and heat-induced gels of food proteins. Agric. Biol. Chem. 54:183–189. Ponce, E., R. Pla, M. Capellas, E. Sendra, and M. Mor-Mur. 1995. Effect of high hydrostatic pressure treatments on egg’s functional properties. Pages 223–229 in Proceedings of the VI European Symposium on the Quality of Eggs and Egg Products. R. Cepero, ed. Facultad Veterinaria de Zaragoza, Zaragoza, Spain. Ponce, E., R. Pla, E. Sendra, B. Guamis, and M. Mor-Mur. 1999. Destruction of Salmonella Enteritidis inoculated in liquid whole egg by high hydrostatic pressure: Comparative study in selective and non-selective media. Food Microbiol. 16:357–365.
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Key words: duck egg, hydrostatic pressure, egg white, yolk, physicochemical property 2010 Poultry Science 89:729–737 doi:10.3382/ps.2009-00244
INTRODUCTION
et al., 2003, 2004, 2005a, 2006). In these studies, the changes in structural properties of ovalbumin or the other egg white proteins, induced by heating, resulted in the change of the functional properties. Heating of egg white solutions at 50 to 85°C results in significant unfolding of the proteins accompanied by an exposure of hydrophobic groups and sulfhydryl (SH) groups, originally buried in the protein core. The decrease in denaturation enthalpy indicates loss of secondary protein structures (Ma and Harwalkar, 1991). During heating, the protein solubility decreased and turbidity increased due to hydrophobic interactions and the formation of disulfide (SS) bonds through SH-SS exchange reactions and SH oxidation (Van der Plancken et al., 2005b). Hen egg yolk is a complex association of lipids (33% by weight), proteins (17%), and water (50%) in which several types of solids such as spheres, granules, low-
Eggs contain proteins of high biological value as compared with other dietary proteins. Egg proteins possess desirable functional and nutritional properties and therefore are widely used in many food products. Egg white proteins are well known for their gelling, foaming, and emulsifying characteristics, and the structural properties of egg white proteins correlated to their functional characteristics have been reported by many studies, with an emphasis on the contribution of the major egg white protein, ovalbumin (Van der Plancken ©2010 Poultry Science Association Inc. Received May 18, 2009. Accepted January 7, 2010. 1 Corresponding author: [email protected]
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ABSTRACT The shell of the duck egg did not crack after pressure treatments (300 to 500 MPa; 25°C; 10 min) in this study; therefore, the changes of physicochemical properties of egg white and yolk proteins from the intact shell egg by pressure treatment were first investigated and compared with those of pressurized hen liquid eggs. Although the proximate compositions of duck eggs and hen eggs were similar, the moisture and protein contents of hen whole eggs were higher than those of duck whole eggs. The protein contents of duck egg white and yolk were slightly lower than those of hen eggs, and the moisture content of duck egg white was equal to that of hen egg white, whereas that of duck egg yolk was lower than that of hen egg yolk. After pressure treatment at 500 MPa, the results of solubility, sulfhydryl content, surface hydrophobicity, and residual denaturation enthalpy showed that egg white proteins underwent slight but significant unfolding and aggregation, whereas pressure treatments below 500 MPa induced
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yolk compositions of duck eggs are similar to those of hen eggs (Ricklefs, 1977); therefore, duck egg white and yolk are suggested to possess functional properties similar to hen eggs. Furthermore, intact shell eggs were not used in the previous studies, which reported the effect of pressure treatment on the physicochemical properties of egg white and yolk proteins, probably due to the cracking of the eggshells under pressure. According to the observation of our preliminary experiment, no cracks were found in the duck eggshell, which might be attributed to the greater thickness (0.5 to 0.6 mm) of the duck eggshell than that (0.3 to 0.4 mm) of the hen eggshell. The thickness and composition would determine the breaking strength of the eggshell. Further research is needed to study the composition and breaking strength of the duck eggshell to determine the mechanism by which the shell withstands pressure. The aim of this study was to investigate the effect of pressure treatment (300 to 500 MPa) at ambient temperature (25°C) on the physicochemical properties of egg white and yolk proteins from duck shell eggs. The changes in secondary structures of the proteins were determined by differential scanning calorimetry (DSC); the native protein components of egg white and yolk proteins were also studied in more detail by electrophoresis.
MATERIALS AND METHODS Materials Collected duck eggs were 1 d old and produced by a genetically closed flock of Anas platyrhynchos (Tsaiya). The eggs, which weighed 67 to 74 g, were purchased from a local market in Taichung City and all cracked or thin-shelled eggs were discarded. Eggs were washed by tap water, left to dry, and then stored at 4°C within 5 d of purchase before experiments. 1-Anilinonaphthalene-8-sulfonic acid (ANS) and 5′,5′-dithiobis(2nitrobenzoic acid) (DTNB) was obtained from Sigma (St. Louis, MO). Reagent-grade chemicals were used to prepare the following: Tris-glycine buffer [0.1 M Tris(hydroxymethyl)-aminomethane (Tris), 0.1 M glycine, and 4 mM EDTA disodium salt, pH 8.0], 5% sodium dodecyl sulfate in Tris-glycine buffer (denoted SDSTris-glycine), Ellman’s reagent (4 mg/mL of DTNB in Tris-glycine buffer), Tris-HCl buffer (0.2 M Tris-HCl, pH 7.4), and sodium phosphate buffer (0.1 M, pH 7.4). The yolk protein content reagent Coomassie Brilliant Blue G-250 (100 mg) was dissolved in 50 mL of 95% ethanol. To this solution, 100 mL of 85% (wt/vol) phosphoric acid was added and then diluted to a final volume of 1 L.
Proximate Composition and pH For determining the proximate compositions of a duck egg, the egg white was separated from the egg yolk and the chalazae were removed. The yolk was care-
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density lipoproteins (LDL), and myelin figures are suspended in a protein solution or plasma (McCully et al., 1962). Almost 68% of yolk DM is LDL, with 16% being high-density lipoproteins, 10% livetins, and 4% phosvitins. Egg yolk is widely used in the food industry due to the excellent functional properties of its lipoproteins, such as flavor, aroma, color, viscosity, emulsifying, and foaming (Fennema, 1993). Denaturation of egg yolk proteins resulted in functional modifications, such as emulsifying and rheological properties (Ahmed et al., 2003; Speroni et al., 2005). High-pressure processing, a nonthermal processing technique, is now suggested to be an interesting alternative to heat processing and results in a better balance between food safety and food quality. Pressure can, however, also induce protein denaturation, depending on the protein concentration, pressure level, temperature, and pH (Balny and Masson, 1993). Similar to heat treatment, pressure treatment of egg white proteins above 450 MPa results in a loss of secondary structure (Hayakawa et al., 1996). The pressure-induced structural changes in egg white proteins can also be demonstrated by the exposure of previously buried hydrophobic and SH groups (Iametti et al., 1998, 1999; Van der Plancken et al., 2004, 2005b, 2006). A strong increase in surface hydrophobicity was observed between 400 and 700 MPa (Van der Plancken et al., 2005a). Furthermore, the decrease and increase of total and exposed SH groups, respectively, were enhanced by pressure above 500 MPa. On the other hand, pressure treatment at 410 MPa induced proteins in egg yolk dispersions to aggregate and undergo a sol-gel transition (Aguilar et al., 2007), whereas that at 600 MPa resulted in the modification of emulsifying properties with no effect on the protein solubility of LDL solutions (Speroni et al., 2005). For application in food products, pasteurization of the whole egg by thermal treatment is often required to ensure microbial safety, principally to control Salmonella species (USDA, 1969). Previous research has shown that pressure treatments (300 to 450 MPa) at various temperatures (15, 20, or 50°C) for 5 to 15 min efficiently inactivated Salmonella Enteritidis inoculated in liquid whole egg (Ponce et al., 1995, 1999). However, the inactivation of Salmonella species contaminating the eggshell, resulting from pressure treatment, has not been investigated. According to the observations of previous studies, pressure treatments of 300 to 400 MPa at ambient temperature were effective in reducing Salmonella Enteritidis or Salmonella Typhimurium populations in cheese (De Lamo-Castellví et al., 2007), tryptone soy broth (Erkmen, 2009b), and raw milk (Erkmen, 2009a). Therefore, pressure treatments at 300 to 500 MPa and 25°C should be efficient for decontamination of duck shell eggs. The choice of decontamination technique, however, would affect not only the physicochemical characteristics of egg white and yolk proteins but also their functional properties. In fact, the egg white and
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fully rolled on filter paper (Whatman No. 4, Whatman International Ltd., Maidstone, UK) to remove chalazae and traces of albumen adhering to the vitelline membrane. The vitelline membrane was then disrupted with a scalpel blade and yolk was collected in a beaker. The whole egg was obtained by manually breaking an egg. Then the egg white, yolk, and whole egg were gently mixed by stirring. The proximate composition (moisture, ash, protein, and lipid content) of whole egg, egg white, and yolk was analyzed following the AOAC methods (AOAC, 2000). The pH values of egg white and yolk were determined by a pH meter (SP-2200, Suntex Instruments Co. Ltd., Taipei, Taiwan).
For each experiment, 1 shell duck egg was packed in a Cryovac polyethylene pouch (10 × 15 cm; Sealed Air Corporation, Taoyuan, Taiwan) and vacuumsealed. The packaged samples were submerged in the high-pressure vessel that had water as the hydrostatic fluid medium in the press. Then pressure treatments of 300 to 500 MPa at 25°C for 10 min were applied. After pressure release, the samples were immediately cooled in ice water to stop further heat denaturation and analyzed within 6 h of storage at 4°C. Experiments at atmosphere pressure (0.1 MPa), set under the same conditions of holding time (10 min) and temperature (25°C), constituted controls. The intact shell eggs, after pressure treatment, were carefully checked and then used for the following experiments. For confirming that the applied pressure was transmitted through the shell to egg white and yolk, the albumen index [albumen height/(long diameter of albumen + short diameter of albumen)/2 × 100] and yolk index (yolk height/yolk diameter × 100) were both determined. Although the albumen index was not changed after all pressure treatments, the yolk index significantly increased from 37.88 (control) to 45.14 after the 500-MPa treatment (data not shown). A high-pressure apparatus (CIP UNIT, Mitsubishi Heavy Industries Ltd., Shinagawa, Japan) with an oilpressure generator and a compressing vessel, in which the internal portion (diameter: 100 mm; height: 180 mm) was a flat-bottomed cylindrical shape, was employed. During pressure treatments, the vessel of the pressure unit was thermostatically controlled by a circulator. The pressure-raising rate was 200 MPa/min, and the pressure-decreasing rate was 400 MPa/min. Due to compressive heating, an increase of the temperature of the pressurizing fluid, by up to a maximum of 16°C at 500 MPa, was observed when the pressure reached 500 MPa. During the holding time, the fluid temperature decreased rapidly to 25 ± 2°C at 6 min and then maintained this temperature until the end of treatment. After the following instantaneous decompression, the temperature of the fluid decreased to 18 ± 2°C. According to the results of previous studies (Mine
et al., 1990; Cordobés et al., 2004), denaturation of hen egg white and yolk proteins was not found within this temperature range. Therefore, the effect of adiabatic heating during pressurization on protein denaturation was disregarded.
Solubility The solubility of egg white protein was determined according to the method of Van der Plancken et al. (2005a) with some modifications. After pressure treatments, the egg white was immediately separated from the egg yolk and the chalazae were removed. The egg white was then gently mixed and diluted with 10-fold volume-distilled water (vol/vol). The egg white solution was then centrifuged at 18,000 × g and 4°C for 15 min. Protein content of the supernatant was determined using Sigma Procedure No. TRPO-562. This method of protein quantification is based on the reduction of Cu2+ by protein in an alkaline environment. Bicinchoninic acid forms a colored complex with the resulting Cu+. The absorbance of this complex was measured at 562 nm. The content was determined by comparison with a standard curve using BSA. The solubility of 100% was assigned to samples containing the same amount of protein as the control sample. The protein solubility of egg yolk was determined according to the method of Bradford (1976). The yolk was diluted 5-fold (vol/vol) with 0.05 M Tris-HCl buffer (pH 6.5) and equilibrated for 1 h at room temperature under continuous stirring. A 4-mL aliquot was taken for the determination of the initial protein content. The remaining solution was centrifuged at 10,000 × g and 4°C for 20 min. Then, 4 mL of the supernatant was used for protein content determination. Protein content of samples was determined according to the method of Bradford (1976). Briefly, 5 mL of reagent solution [0.01% (wt/vol) Coomassie Brilliant Blue G-250, 4.7% (wt/ vol) ethanol, and 8.5% (wt/vol) phosphoric acid] was added to the protein solution and mixed by vortexing. The absorbance of the mixture was measured at 595 nm after 2 min. Calibration of the assay was performed with standard BSA. Protein solubility was calculated as (mg of protein in the supernatant/mg protein in the initial solution) × 100.
SH Groups The concentration of SH groups of egg white was determined using Ellman’s reagent, which is based on the reaction at neutral and alkaline pH between protein SH groups and DTNB, resulting in the formation of the thionitrophenylated protein and a yellow thionitrophenylate anion (Kalab, 1970). The content of total, exposed, and buried SH groups of the total (1:10 dilution of the treated sample in Tris-HCl buffer, vol/ vol) or soluble protein fraction (supernatant obtained as described previously) was measured in duplicate us-
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High-Pressure Treatment
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Surface Hydrophobicity To 3-mL aliquots of egg white solution (concentration ranging from 0 to 0.02% protein in sodium phosphate buffer), 15 μL of 8 mM ANS in sodium phosphate buffer was added and mixed well by vortexing (Alizadeh-Pasdar and Li-Chan, 2000). After 15 min in the dark, the fluorescence intensity (FI) of each solution was measured by a fluorescence spectrofluorometer (Hitachi F-2000, Tokyo, Japan) with an excitation at 390 nm and an emission at 470 nm. For eliminating the effect of turbidity of egg white solution on FI, the FI values of each concentration of protein without fluorescence probe were also determined (Van der Plancken et al., 2006). The slope of the relative corrected FI versus protein concentration plot was used as an index of surface hydrophobicity.
Enthalpy of Denaturation The enthalpies of denaturation of egg white and yolk were determined using the DSC (Speroni et al., 2005; Van der Plancken et al., 2006). Pressure-treated samples were directly and qualitatively transferred to stainless steel pans and hermetically sealed. About 4 mg of proteins was used in each assay. An empty pan was used as a reference. The egg white samples were analyzed from 20 to 100°C at a heating rate of 10°C/ min, and the egg yolk samples were analyzed from 30 to 110°C at a heating rate of 1°C/min in a Nano DSC (CSC 6300, Calorimeter Sciences Corp., Linden, UT). The enthalpy of denaturation was calculated from the peak area of the thermogram, using CpCalc (version 2.2.10.10) software (Calorimeter Sciences Corp.).
Electrophoresis Native PAGE was performed to demonstrate the effects of pressure treatment on the protein components of egg white and yolk as per the method described by Alomirah et al. (1998) for meat protein. The egg white was diluted 10-fold in 0.2 M Tris-HCl buffer (pH 6.8) to ensure complete solubilization of proteins, the egg yolk was diluted with a 10-fold volume of 10% NaCl solution (wt/vol), and then all samples were stored at −20°C. The resolving gel of native PAGE was 12.5% acrylamide (wt/vol). Electrophoretic migration was performed at 20 mA for 2 h. The proteins were stained with a solution containing 0.27% (wt/vol) Coomassie Brilliant Blue R250, 45% (vol/vol) methanol, and 45% (vol/vol) acetic acid and then destained in the solution containing 10% (vol/vol) acetic acid and 20% (vol/vol) methanol.
Statistical Analysis The Statistical Analysis System Version 8.2 (SAS Institute Inc., Cary, NC) was adapted to perform data analysis and statistical computations for ANOVA and Duncan’s test. Significance of differences was defined at P < 0.05. The differences among treatments were verified by their least significant difference. Experiments were conducted in triplicate.
RESULTS AND DISCUSSION Proximate Composition and pH Duck egg composition is shown in Table 1 and confirmed the report by Ricklefs (1977). The albumen ratio (calculated as albumen weight/egg weight) is 55.76%. The moisture, protein, lipid, and ash contents of duck egg white are 87.23, 8.56, 0, and 0.72%, respectively; on the other hand, those of yolk are 44.53, 14.05, 35.81, and 2.23%, respectively. The pH values of egg white and yolk were 7.42 and 6.51, respectively. As compared with the proximate compositions of hen eggs (Mine, 2007), the albumen ratio of a duck egg is lower, whereas the yolk ratio is higher. The moisture and protein contents of hen whole eggs are higher than those of duck whole eggs, whereas the lipid and ash contents of both whole eggs are similar. The protein contents of duck egg white and yolk are slightly lower than those of hen eggs, and the lipid content of duck egg yolk is higher than hen egg yolk. The moisture content of duck egg white is equal to that of hen egg white, whereas that of duck egg yolk is lower than that of hen egg yolk. Furthermore, the shell ratio is higher in a duck egg than a hen egg.
Solubility Due to pressure treatment, egg white proteins may unfold; therefore, hydrophobic residues and SH groups
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ing both of the following procedures (Van der Plancken et al., 2005b). For determination of exposed SH groups, 2 mL of the total or soluble protein fraction was added to 2 mL of Tris-glycine buffer and 40 μL of Ellman’s reagent. The mixture was allowed to stand at ambient temperature for 15 min and then either centrifuged at 18,000 × g and 4°C for 15 min (soluble protein fraction) or maintained at 4°C for 15 min (total protein fraction). Finally, the absorbance was measured at 412 nm against a reagent blank. For determination of total SH groups, 2 mL of the total or soluble protein fraction was added to 2 mL of SDS-Tris-glycine buffer and 40 μL of Ellman’s reagent. The mixture was maintained at 40°C in a water bath for 15 min to allow the protein to unfold and all SH groups to be accessible to DTNB. Finally, the absorbance was also measured at 412 nm against a reagent blank. A molar extinction coefficient of 13,600 M−1·cm−1 for the thionitrophenylate anion at 412 nm was used to calculate the amount of exposed or surface and total SH groups (Beveridge et al., 1974). The amount of buried SH groups (inaccessible to DTNB) was calculated by subtracting the amount of exposed SH groups from the total amount of the SH groups.
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PRESSURE TREATMENT OF DUCK SHELL EGG Table 1. The comparison of proximate compositions of duck eggs and hen eggs Hen egg2
Duck egg Item Weight (g) Moisture (%) Protein (%) Lipid (%) Ash (%) pH 1n
Whole egg
Egg white
Yolk
n1
70.0 ± 2.91 62.9 ± 0.62 9.3 ± 1.64 11.4 ± 1.43 1.1 ± 0.12 7.14
39.0 ± 1.58 87.2 ± 0.13 8.6 ± 1.31 0.00 ± 0.0 0.7 ± 0.11 7.42
22.4 ± 1.29 44.5 ± 0.13 14.1 ± 1.48 35.8 ± 1.69 2.2 ± 0.23 6.51
80 10 10 10 10 10
Whole egg
Egg white
Yolk
55 66.1 12.8–13.4 10.5–11.8 0.8–1.0
33–35 87.6 9.7–10.6 0.03 0.5–0.6
15–16 48.7 15.7–16.6 31.8–35.5 1.1
= number of eggs analyzed. data were from Mine (2007).
2The
SH Groups Ovalbumin is the only egg white protein to contain 4 free SH groups, which are originally buried in the protein core, and the protein contains 1 SS bond (Powrie and Nakai, 1986). All of these groups are relevant to thermal aggregation, and the exposure of both SS and SH groups in this protein has been considered an index of protein denaturation (Tani et al., 1997). Heatinduced denaturation of ovalbumin results in exposure of these SH groups, accompanied by a decrease in total SH content, due to oxidation of SH groups to SS bonds (Rumbo et al., 1996). The contribution of both the soluble and the insoluble protein fractions of pressurized egg white to the SH content was investigated (Figure 1). In the soluble protein fraction, pressure treatment resulted in a tendency to pressure-dependent exposure of buried SH groups (P > 0.05), and significant (P < 0.05) decreases of total and buried SH content were observed at 500 MPa. In the total protein fraction, only buried SH content decreased significantly (P > 0.05) as the result of pressure treatment at 500 MPa. Essentially, all free SH groups remained in the soluble protein fraction, as could be expected based on the high residual protein solubility (96.3% after 10 min at 500 MPa, as shown in Table 2). This indicates that most of the ovalbumin molecules remained soluble, even though they might be involved in aggregation through SH-SS exchange reactions or the formation of SS bonds (Van der Plancken et al., 2005b).
Table 2. Effect of pressure treatment on the protein solubility of egg white and yolk from shell duck eggs1 Solubility (%) Pressure treatment (MPa) 300 400 500
Egg white
Yolk
99.9 ± 1.34a 99.4 ± 0.98a 96.3 ± 1.43b
97.6 ± 1.67a 93.9 ± 2.38b 84.3 ± 3.44c
a–cDifferent letters in the same row indicate significant differences (P < 0.05). 1All values are means ± SD of data from 3 independent experiments.
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are exposed and aggregates are formed and stabilized by hydrophobic interactions and SS bonds (Van der Plancken et al., 2005a,b). The aggregates may be soluble or insoluble according to their sizes and the insoluble fractions can be removed by centrifugation. The protein content loss of the soluble fractions represents protein aggregation attributed to pressure treatment. As shown in Table 2, pressure treatments at 300 to 500 MPa induced slight decreases in protein solubility of egg white. However, this decrease (3.7%) in solubility of egg white was significant (P < 0.05) only at 500 MPa. On the other hand, the solubility of yolk decreased with elevated pressure, whereas the solubility decreased to 84.3% with the 500-MPa treatment. A previous study reported that the protein solubility of egg white solution (10% in Tris-HCl buffer, pH 7.6, vol/vol) treated with 100 to 500 MPa at 25°C decreased less than 10%, whereas that treated with 550 to 700 MPa declined dramatically from 60 to 40% (Van der Plancken et al., 2005a,b). However, another previous study reported that fresh egg white turned opaque with a partial coagulation by the 500-MPa treatment (25°C) and formed hard gels that stood under their own weight above 600 MPa (Okamoto et al., 1990). The different results obtained in these studies might be attributed to protein concentrations and confirmed the report by Iametti et al. (1998). The latter study showed that ovalbumin solution (0.5, 2, and 5 mg/mL in 50 mM phosphate buffer, pH 6.8) at higher concentrations formed more pronounced aggregates under pressure treatment. Furthermore, pressure treatment at 450 MPa did not induce ovalbumin solution at the concentrations of 0.5 and 2 mg/mL to form aggregates, whereas the solubility of ovalbumin solution at the concentration of 5 mg/mL decreased to about 50%. In the present study, however, pressure treatment slightly decreased the solubility of duck egg white at the concentration of 84.6 mg/mL. Okamoto et al. (1990) observed that hen egg yolk was transformed to a gel structure by the 400-MPa treatment. The result showed that the egg white and yolk proteins may be prevented from forming aggregates in the whole egg treated by pressure treatments at 300 to 500 MPa, as compared with egg white or yolk individually pressurized at the same pressure levels.
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Lai et al. Table 3. Effect of pressure treatment on the surface hydrophobicity of egg white from shell duck eggs1 Pressure (MPa) Surface hydrophobicity S0
0.1 (control) 188 ±
16.1a
300 197 ±
18.3a
400 209 ±
16.9a
500 238 ± 11.5b
a,bDifferent 1All
letters indicate significant differences (P < 0.05). values are means ± SD of data from 3 independent experiments.
Surface Hydrophobicity
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The effect of pressure on the surface hydrophobicity of egg white proteins is shown in Table 3. Pressure treatment at 500 MPa resulted in a significant (P < 0.05) pressure-dependent increase (26%) in surface hydrophobicity, and slight but insignificant increases in surface hydrophobicity were observed below 500 MPa. This indicates that pressure treatment of egg white proteins causes the hydrophobic residues to be exposed and thus more readily available for binding with ANS (Smith et al., 2000). The previous study reported that ovalbumin (0.25 mg/mL, pH 6.5) showed little increase in ANS fluorescence after the 400-MPa treatment. However, a large enhancement of the fluorescence was observed after higher pressure (500 to 800 MPa) treatments. Pressure treatment affects protein internal interactions solely by volume changes. Consequently, covalent bonds are not disrupted by pressure, except for the oxidation of SH groups or SH-SS exchange reactions. Further, pressure mainly disrupts or induces hydrophobic and electrostatic interactions (Silva and Weber, 1993). Previous studies reported that, however, a pronounced increase in surface hydrophobicity of the hen egg white protein solution or albumen by pressure treatment was observed between 400 and 700 MPa (Smith et al., 2000; Van der Plancken et al., 2006). The result showed that the egg white proteins may be prevented from exposing hydrophobic residues in the whole egg treated by pressure treatments at 300 to 500 MPa, as compared with the pressurized egg white solution at the same pressure levels.
proteins (Van der Plancken et al., 2007). The endothermic reactions reflect the disruption of hydrogen bonds, and the exothermic processes show the break up of hydrophobic interactions and protein aggregation (Ma
Enthalpy of Denaturation Secondary structure of egg white proteins is altered by pressure treatment, as shown by Fourier transform infrared spectroscopy, circular dichroism spectroscopy, DSC, and intrinsic fluorescence measurements (Hayakawa et al., 1992; Smith et al., 2000). Hayakawa et al. (1992, 1996) reported that the DSC endothermic enthalpy and the α-helical content of the pressure-denatured ovalbumin decreased together, and they also demonstrated that pressure treatment caused the breakdown of secondary structures of ovalbumin. In addition to the changes in secondary structure, pressure does not cause extensive denaturation (Smith et al., 2000). Therefore, the residual enthalpy is correlated with the content of ordered secondary structure of the pressure-treated
Figure 1. Effect of pressure treatment on the (A) exposed, (B) total, and (C) buried sulfhydryl (SH) content of egg white from shell duck eggs. Mean value of SD is less than 1.2%.
PRESSURE TREATMENT OF DUCK SHELL EGG
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and Harwalkar, 1991). Therefore, DSC thermograms of the pressure-treated egg white and yolk proteins show the subsequent thermal denaturation of the proteins remaining as the native conformation. Thus, a reduction in residual enthalpy is an indication for a partial loss of protein structure during pressure processing. In Figure 2, the thermograms of egg white and yolk after pressure treatment are shown. According to the DSC thermogram of egg white, there are 3 endothermic peaks (65.8, 72.8, and 84.1°C) observed in egg white. The left-hand peak corresponds to the denaturation of ovotransferrin, the middle peak to that of lysozyme, and the peak at 84.1°C to that of ovalbumin (Van der Plancken et al., 2005a). On the other hand, there was a major endothermic peak (80.9°C) corresponding to the denaturation of phosvitin in egg yolk (Chung and Ferrier, 1995). In Figure 3, the residual denaturation enthalpies of egg white and yolk after pressure treatment are shown. The residual enthalpies of egg white did not significantly (P > 0.05) decrease after 300- and 400-MPa pressure treatments but decreased significantly (P < 0.05) to 96.3% after the 500-MPa treatment, whereas those of egg yolk decreased significantly (P < 0.05) to 84.3% after the 500-MPa treatment. The results may confirm the observations of the changes of solubility, SH groups, and surface hydrophobicity as described above (Tables
2 and 3, Figure 1). A previous study reported that an apparent decrease of residual denaturation enthalpy of egg white solution (9.64 mg/mL in 0.2 M Tris-HCl at pH 7.6) was observed after pressure treatment at 25°C and 400 MPa, and no residual enthalpy could be detected at 700 MPa (Van der Plancken et al., 2005a). On the other hand, hen egg yolk showed a progressive decrease in thermal denaturation enthalpy, without any significant change in denaturation temperature, as the pressure level was raised from 250 to 440 MPa (Aguilar et al., 2007).
Figure 2. Differential scanning calorimetry thermograms of egg white and yolk from shell duck eggs by pressure treatment. Arrows indicate endothermic peaks.
Figure 3. Effect of pressure treatment on the residual denaturation enthalpy (ΔHdenaturation) of egg white and yolk from shell duck eggs. Mean value and error bars represent SD of triplicate measurement.
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The native PAGE (Figure 4) result showed that pressure did not affect the protein components of egg white and yolk, although the solubility and residual denaturation enthalpy of egg yolk slightly but significantly decreased after the 500-MPa treatment. Pressure treatment can induce protein denaturation of hen egg white and yolk solutions, which has been reported in previous studies (Van der Plancken et al., 2005a; Aguilar et al., 2007). Smith et al. (2000) observed that pressure treatment at 400 MPa induced limited irreversible changes in the secondary structure of ovalbumin. Van der Plancken et al. (2005b) demonstrated that ovalbumin aggregates engaged in the SS bonds were present in the water-soluble fraction, demonstrated by SDS-PAGE patterns. This work demonstrated that the denaturation of duck egg white and yolk proteins was prevented in shell eggs under pressure up to 500 MPa, as compared with that of the pressurized hen egg white and yolk solutions at the same pressure levels. However, slight decreases in the solubility and the residual denaturation enthalpies of both egg white and yolk proteins were observed after pressure treatment, especially at 500 MPa. Native PAGE patterns showed that no aggregates of egg white or yolk proteins were formed after
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pressure treatments, probably due to the renaturation of the proteins during storage at −20°C. The results may indicate that pressure treatment causes reversible denaturation of these proteins in shell eggs. Furthermore, the sensitivities of egg white and yolk proteins in the intact shell egg to pressure treatment seemed to be different from those in the liquid form. We suggested that the egg white and yolk in the liquid form have been mixed and represented as the well-distributed state; therefore, pressure treatment would directly induce their protein denaturation. On the other hand, the effect of pressure treatment on the egg white and yolk represented as the native state in the shell egg may not be equal to those in the liquid form. Based on the consideration for food safety and functional properties, therefore, pressure processing can be a good preservation technique for duck shell eggs.
ACKNOWLEDGMENTS This study was financially supported by the National Science Council, Republic of China, no. NSC 97-2313B-039-006. We thank Chin-Shuh Chen and Po-Yuan Chiang (Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, Taiwan, Republic of China) for supplying the high-pressure equipment.
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Figure 4. Native PAGE analysis of (A) egg white and (B) yolk from shell duck eggs by pressure treatment. Std = native standard; C = control; 300 to 500 = 300 to 500 MPa, respectively. Color version available in the online PDF.
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