2021 Volume 27 Issue 3 Pages 491-496
In this study, we investigated the role of ovalbumin (OVA), ovotransferrin (OVT), and lysozyme (LYZ) as the major proteins involved in heat-induced gelation of egg white. OVT and LYZ deficient egg white (OLdEW) solutions were prepared from raw egg white (REW) subjected to heat treatment at 64 °C and centrifugation. OLdEW had almost the same native-OVA protein as in REW, confirmed by gel electrophoresis, differential scanning calorimetry, and surface hydrophobicity measurement. Texture profile analysis, syneresis rate measurement, and observation by scanning electron microscopy were performed for the heat-induced gels. The OLdEW gel had more porous and fibrous networks, resulting in less hardness, more resilience and compatible water holding capacity compared to the REW gel. These results suggest that OVA contributes to the water holding capacity and resilience in egg white gel, while OVT and LYZ contribute to enhanced gel hardness.
Hen egg is one of the most widely used foods worldwide. The egg white holds significant importance in the food processing industry given its excellent processing functions, such as heat-induced gelling and foaming properties. The gelling properties are utilized as a raw material for meat and surimi processed products (Alleoni, 2006; Mine, 1995). Egg white contains 9.7–12 % (w/w) protein, of which globular proteins account for the majority. Intermolecular interactions following heat denaturation of proteins form the egg white gel (Ma and Holme, 1982). Non-covalent bonds, such as hydrophobic and hydrogen bonds, and covalent bonds, such as disulfide and lanthionine bonds, contribute to gelation (Philips et al., 1994; Koyama et al., 2020).
Major globular proteins include ovalbumin (OVA, 54 %), ovotransferrin (OVT, 12 %), ovomucoid (OVM, 11 %), ovoglobulin G2 and G3 (OVG, 4 % each), and lysozyme (LYZ, 3.4 %) (Alleoni, 2006). Egg white proteins can be purified using various methods, such as salt or polyethylene glycol precipitation, isoelectric precipitation, and liquid chromatography (Abeyrathne et al., 2014; Geng et al., 2012; Omana et al., 2010). Since OVA can be readily purified, many tests with respect to gel properties have been conducted (Doi, 1993; Hatta and Kitabatake, 1986) as models for studying gelforming mechanisms. In addition, for OVT and LYZ, studies focusing on thermal aggregation properties have been conducted using their low-concentration solutions (Iwashita et al., 2019; Matsudomi et al., 1991). Johnson and Zabik (1981) compared the gel properties of purified egg white proteins in terms of their contribution to gel formation, either singly or in combination.
However, it is not clear how each protein contributes to the physical properties with the protein ratio in chicken egg white. In addition, the purification process could affect the intermolecular interaction and gel network formation.
In this study, we aim to elucidate the roles of OVA, OVT, and LYZ in the different gelling properties between egg white and OVA alone. As described above, changes in intermolecular interaction and gelling properties could occur while purifying the egg white proteins. Therefore, we first prepared OVT and LYZ deficient egg white (OLdEW) solutions, containing OVA as the main component, without using any reagents and column purification, and then the gel properties were compared with those of raw egg white (REW).
Reagents 8-Anilino-1-naphthalenesulfonic acid magnesium (II) salt (ANS) and bromophenol blue were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Glutaraldehyde (electron microscopy grade) was purchased from TAAB Laboratories Equipment Ltd. (Aldermaston, Berks, UK). Other reagents were purchased from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan).
Sample preparation Egg white was separated from 2-day-old hen eggs purchased from a local supermarket. The egg white was homogenized for 30 s in a homogenizer (Hirosawa Tekkojo, Tokyo, Japan) and centrifuged (10 000 × g, 15 min, 25 °C). As per the report by Koyama et al. (2020), the supernatant was subjected to heat treatment (64 °C, 10 min) and cooled with ice water. The supernatant obtained by centrifuging the heated egg white (10 000 × g, 30 min, 25 °C) was used as OLdEW. The sample kept at 25 °C for 10 min in place of heat treatment was used as a control (REW).
The protein content of each sample was determined by the Dumas method using rapid MAX N (Elementar Analysensysteme GmbH, Langenselbold, Germany). The pH values were measured using a D-72 LAQUA (HORIBA, Ltd., Kyoto, Japan).
Polyacrylamide gel electrophoresis (PAGE) analysis
Sodium dodecyl sulfate (SDS)-PAGE was performed using the method proposed by Laemmli (1970) with slight modifications. Protein solutions were mixed with a half volume of 60.6 mM Tris-hydrochloride (HCl) (pH 6.8) containing 8 M urea, 60 % glycerol, 3 % SDS, 50 mM dithiothreitol and 0.01 % bromophenol blue, and allowed to stand at 20 °C for 24 h. A molecular weight marker (XL-ladder broad; AproScience Co., Tokushima, Japan) and 10–20 % e-PAGEL (ATTO Co., Tokyo, Japan) were used for electrophoresis.
For native-PAGE analysis, protein solutions were mixed with a half volume of 60.6 mM Tris-HCl buffer (pH 6.8) containing 60 % glycerol and 0.01 % bromophenol blue. The mixtures were subjected to 5–20 % e-PAGEL (ATTO Co.).
One-step Coomassie Brilliant Blue (CBB) (AproScience, Tokushima, Japan) was used for gel staining. Band intensity was analyzed using ChemiDoc and Quantity One (Bio-Rad Laboratories, Inc., Hercules, CA).
Differential scanning calorimetry (DSC) Thermal properties of the egg white proteins were analyzed using a differential scanning calorimeter, DSC 1 (Mettler-Toledo GmbH, Greifensee, Switzerland). A 20 µL sample was sealed in an aluminum pan and a reference was made in another pan with water. The thermogram peaks were analyzed at a temperature range of 30.0–93.0 °C at 2.0 °C/min with nitrogen gas (flow rate 100 mL/min). Native OVA (N-OVA) is known to convert to more heat stable types, stable OVA (S-) and/or their intermediate (I-) OVA due to heat treatment or storage (Hegg et al., 1979). The composition of N-OVA, I-OVA, and S-OVA were calculated from the area rate of endothermic peaks at 78.3, 83.3, and 87.2 °C, respectively, according to the method proposed by De Groot and De Jongh (2003).
Surface hydrophobicity Protein surface hydrophobicity was determined according to a previous report with slight modifications (Hayakawa and Nakai, 1985). Samples were adjusted to 0.2 mg/mL protein content with 0.1 M sodium phosphate buffer (pH 7.0) and mixed with 2 mM ANS solution. Fluorescence intensities at an excitation wavelength of 390 nm and an emission wavelength of 470 nm were measured using a RF-5000 (Shimadzu, Kyoto, Japan).
Gel preparation The sample was placed into a 2 mL microtube (diameter, 8.7 mm), heated in a water bath (90 °C, 30 min), and then allowed to stand at room temperature (approximately 25 °C) for 2 h. The gel was then taken from the tube and cut into a cylindrical shape with a height of 7.0 mm.
Color measurement of the gel The color difference of the gel pieces was measured using the report by Handa et al. (1998). The reflectance L* value was measured using a spectrophotometer, CM-5 (Konica Minolta Co., Ltd., Tokyo, Japan).
Scanning electron microscopy (SEM) The gel was immersed in a 2 % glutaraldehyde solution at 25 °C for 24 h. The gel pieces were dehydrated stepwise with 50–100 % ethanol and fractured in liquid nitrogen. Samples immersed in t-butyl alcohol were lyophilized in an ES-2030 (Hitachi High-Tech Corp., Tokyo, Japan). Platinum palladium coating was performed using a MC1000 (Hitachi High-Tech Corp., Tokyo, Japan) and the microstructure of the gel was observed with a S-4800 SEM (Hitachi High-Tech Corp., Tokyo, Japan).
Texture profile analysis (TPA) A double compression test of the gel pieces was performed using a Tensipresser My Boy II system (Taketomo Electric Co., Tokyo, Japan) equipped with a 30 mm disk-shaped plunger. The 7.0 mm-thick gel pieces were compressed twice to 50 % of their thickness at a rate of 1.5 mm/s. Referring to the report by Bourne (1978), texture profiles of hardness, cohesiveness, resilience, and springiness were calculated using the above data.
Syneresis rate A piece of gel was wrapped in a nylon membrane, placed in a microtube laid with glass beads (φ3 mm), and centrifuged (1,000 × g, 10 min, 25 °C). The syneresis rate of the gel was calculated from the weight of the gel before and after centrifugation.
Statistical analysis The data are expressed as mean value ± standard deviation (SD). Statistical analysis was performed using Microsoft Excel 2016 and an unpaired t-test. Statistical significance was defined at p < 0.05 (*) and p < 0.01 (**).
Preparation and property evaluation of OLdEW The REW and OLdEW samples exhibited the pH value of 9.12 ± 0.07 and 9.13 ± 0.06, respectively. Compared to REW, the protein concentration in OLdEW was reduced from 10.5 ± 0.2 to 8.6 ± 0.1. SDS-PAGE was performed to determine the protein composition of REW and OLdEW (Fig. 1A). Clear protein bands of OVA, OVT, OVM, and LYZ were observed in REW, whereas the OVT and LYZ bands disappeared in OLdEW. Simultaneously, the bands (approximately 250 and 150 kDa) with low intensity were also faint. OVA and OVM were observed as major bands in OLdEW; the OVA band intensities were similar for REW (1.00 ± 0.04) and OLdEW (1.02 ± 0.05). It has been reported that OVM does not participate in the formation of heat-induced gel networks (Handa et al., 1998). These results suggested that OLdEW is depleted of OVT and LYZ and mainly contains OVA as the protein that contributes to heat-induced gel formation.
Electrophoretic patterns of REW and OLdEW proteins separated by reducing SDS-PAGE (A) and Native-PAGE (B). The bands corresponding to each protein (OVT, OVA, OVG, OVM, and LYZ) were displayed. OVT, ovotransferrin; OVA, ovalbumin; OVG, ovoglobulin; OVM, ovomucoid; LYZ, lysozyme.
Subsequently, the degree of denaturation of the proteins in OLdEW was evaluated. Native-PAGE showed that the band intensity of OVA did not differ between REW and OLdEW, suggesting the OVA was not aggregated during the preparation process of OLdEW (Fig. 1B, Table 1). DSC analysis in REW showed major peaks corresponding to OVT and OVA at 65.7 °C and 78.9 °C, respectively (Fig. 2). In contrast, DSC analysis in OLdEW showed a peak corresponding to OVA only, in which the endothermic peak area was comparable to REW (Table 1). It is also reported that S-OVA affects the gel network formation (Shitamori et al., 1984). Therefore, the percentage of N-OVA, I-OVA, and S-OVA was confirmed (Fig. 2, Table 1). Peaks corresponding to N-OVA and I-OVA were observed at 78.9 °C and 83.9 °C, respectively, whereas no S-OVA peak was observed in both the samples. The abundance of I-OVA determined from the area ratio was slightly higher in OLdEW than in REW. Surface hydrophobicity was slightly higher in OLdEW than in REW (Table 1). It has been reported that the surface hydrophobicity of an egg white solution was increased approximately 5.8-fold due to heating at 65 °C (Mine et al., 1990). Whereas, no significant changes in the surface-sulfhydryl (SH) groups occurred below 70 °C, indicating the absence of any structural changes in OVA (Van Der Plancken et al., 2005). Therefore, the surface hydrophobicity of both samples was inferred to be comparable. From these results, it was assumed that OVA in OLdEW maintains a structure close to REW, thus rendering negligible effects on the gelling properties.
| REW | OLdEW | ||
|---|---|---|---|
| Native-PAGE | OVA band intensity | 1.00 ± 0.03 | 0.99 ± 0.05 |
| DSC | Peak area of OVA (J/mL) | 0.60 ± 0.02 | 0.63 ± 0.01 |
| I-OVA rate | 0.103 ± 0.021 | 0.150 ± 0.003** | |
| Surface hydrophobicity | 29.1 ± 0.6 | 35.3 ± 0.8** | |
Data are shown as mean value ± standard deviation (SD) (n = 3). Band intensities of protein separated by SDS- and native-PAGE are shown as relative values.
DSC thermograms of REW (A) and OLdEW (B) proteins.
Color and microstructure of the gel Heat-induced REW and OLdEW gels were prepared and their properties were compared. The L* value, corresponding to gel opacity, was lower in OLdEW than in REW (Fig. 3).
Opacity of REW and OLdEW gels were displayed as L* value. Data are shown as mean value ± standard deviation (n = 6).
The fracture section of the gel observed by SEM (Fig. 4) revealed spherical particles with dense and homogenous networks in the REW gel. In contrast, the OLdEW gel had a less dense network with a large ratio of thin fibrous structures. It has been reported that when globular proteins are heated, the surface charge of protein molecules away from the isoelectric point is stronger, resulting in greater electrostatic repulsion and formation of a network that is mainly comprised of a fibrous polymer (Doi, 1993). The isoelectric point for OVA, OVT, and LYZ is 4.5, 6.1 and 10.7, respectively (Alleoni, 2006). Previous studies have revealed that the incorporation of a positively charged LYZ into negatively charged OVA and OVT results in electrostatic attraction, which increases the size of aggregates produced by heating (Iwashita et al., 2017, 2019). Thus, it was inferred that in OLdEW, despite the strong electrostatic repulsive force during network formation, the limited number of interaction sites resulted in a large number of thin and fibrous networks. In REW, OVT and LYZ with a lower denaturation temperature aggregated earlier than OVA with increasing temperature, which seemingly influenced the formation of fibrous aggregates of OVA. The higher ratio of fibrous networks resulted in the higher transparency of the OLdEW gel by affecting the suppression of light scattering.
Microstructure of gel networks of REW and OLdEW observed by SEM at a magnification of 50,000.
TPA and syneresis rate The physical properties of the REW and OLdEW gels were evaluated (Fig. 5). The REW gel was harder than the OLdEW gel. This could be attributed to the higher total protein content of REW. LYZ has been reported to contribute to the hardness of heat-induced gels using purified egg white proteins alone or in combination with a certain amount of proteins (Johnson and Zabbik, 1981). Therefore, LYZ might contribute to the enhancement of gel hardness. In contrast, higher cohesiveness, resilience, and springiness in the OLdEW gel compared to the REW gel suggested that the network formed by OVA alone has a strong inner structure and excellent resilience. The presence of OVT and LYZ seemingly affected the formation of the fibrous network of OVA and reduced the resilience.
Texture profile analysis and syneresis rate of REW and OLdEW gels. Data are shown as mean value ± standard deviation (n = 12).
In addition, although the OLdEW gel had a lower protein concentration, the syneresis rate after centrifugation was comparable to that of the REW gel (Fig. 5). This suggests that OVA contributes to water retention in egg white gels. Since OLdEW forms a fibrous network, the increased surface area seemingly contributed to water retention despite the smaller amount of total protein. These results suggested that OVA forms a gel mainly composed of a fibrous structure and was responsible for the gel resilience, whereas OVT and LYZ along with OVA form a hard and less resilient gel with spherical and dense networks.
In the present study, we attempted to elucidate the role of OVA, OVT, and LYZ in the gelling properties of egg white.
OLdEW prepared by heat treatment (64 °C for 10 min) and centrifugation maintained OVA in almost its native state. Comparing the gel properties of REW and OLdEW, it was observed that the OLdEW gel was transparent and comprised of fibrous networks, while the REW gel contained dense networks. In terms of physical properties, the OLdEW gel was less hard and highly resilient compared to the REW gel. In addition, both gels had equivalent water retention ability. These results suggest that OVA formed fibrous networks, exhibiting excellent resilience and water retention. In contrast, OVT and LYZ together with OVA formed dense networks with spherical units, exhibiting hardness and less resilience.
Acknowledgements The authors are grateful to Dr. Yukio Yaguchi for his cooperation in SEM observation. We also thank Ms. Megumi Kubo and Ms. Yuka Nakamoto for their help with the experiments.
Declaration of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
