Technical paper
Effects of pH on Foam Formation by Whipping in Spray-Dried Egg White Dispersion
2018 年 24 巻 2 号 p. 355-361
詳細
2018 年 24 巻 2 号 p. 355-361
Three types of spray-dried egg white powders, of which dispersion in water showed pH of 7.4 (EW7.4), 8.4 (EW8.4) and 10 (EW10), respectively, were adjusted to pH 4.0, 7.4 and 9.0. The obtained egg white dispersions were separately whipped to afford foams similar to meringue. Foaminess (specific volume) was highest at pH 4.0 for EW8.4 and EW10, but lowest for EW7.4. From stress-strain curves, three parameters (maximum stress, breakdown and apparent elasticity) were calculated and compared among pH levels adjusted just before whipping. All parameters were lowest at pH 4.0 and showed inhibited foam formation in contrast with foaminess. Thus, EW7.4-whipped foam showed a different pH-dependency from EW8.4 and EW10. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis of insoluble fractions derived from whipped foams, lysozyme was washed out by KCl solution for EW7.4, differing from EW8.4 and EW10, in which lysozyme remained in the insoluble fraction after washing with KCl or water. From the above results, pH adjustment for egg white before spray drying is considered to modify the interaction between egg proteins and to change the physical properties of whipped foam.
Chicken egg white is widely utilized in various types of food processing, including angel cake, surimi gel, and noodles because of properties such as heat coagulation, foaming and emulsifying (Mine, 1995; Alleoni, 2006). Particularly with regard to the foaming properties of egg white, many researchers have investigated the individual proteins present, namely ovalbumin, ovotransferrin, ovomucin, ovomucoid and lysozyme (Mine, 1995; Alleoni, 2006; Abdou et al., 2013). MacDonnell et al. (1955), Nakamura and Sato (1961) and Nakamura et al. (1964) reported that ovoglobulin, which is one constituent of egg white and has yet to be characterized, is responsible for its foaminess. Furthermore, foam stability was reported to be mainly caused by ovomucin (MacDonnell et al. (1955); Nakamura and Sato, 1964). These results also suggested that major constituents, such as ovalbumin and ovotransferrin contributed in lesser amounts of foam formation and stabilization. Denaturation (Nakamura, 1964; Relkin et al., 1999) and addition of copper ions (Shimofuji et al., 2010) were reported to increase the foaming activity of ovalbumin. pH shifting of egg albumin was also reported to change the rheological properties of foam through pH-induced protein unfolding and refolding (Mleko et al., 2007).
Egg white is frequently supplied and utilized as frozen egg white and spray-dried egg white. Spray-dried egg white is produced from raw egg white by spray drying after removing glucose. During spray drying, egg white is heated to some extent, so some functionality, such as gel properties, are known to be changed. Hammershøj et al. (2004) reported increased overrun and decreased stability of foam by spray drying. On the other hand, Ayadi et al. (2008) reported that the foaming capacity and stability of foam were increased by spray drying of egg white under moderate operating conditions, as well as its gel strength, water-holding capacity and emulsifying properties. This difference may be dependent on spray-drying conditions, e.g., temperature and flow rate. Thus, spray drying appears to have some potential to change the functional properties of egg white. Egg white powder spray dried after pH adjustment was reported to show different foaming abilities depending on the pH, where pH 6.5 led to the highest foaming ability (Kim et al., 2006). The aim of this study was to estimate the effects of pH (4–9) at whipping on foaming properties of egg white powder, which was prepared by spray drying after adjusting to the desired pH levels. To assess the whipped foam of spray-dried egg white, stress–strain curves were measured, because foam hardness is thought to be an important factor in foam qualities, such as texture. Furthermore, the proteins that participated in foam structure were also analyzed and compared.
Spray-dried egg white and its foaming Three types of spray-dried egg white powder that were all prepared without pasteurization were supplied by Kewpie Egg Corporation (Chofu, Japan). Inlet and outlet temperatures of spray drying were 150–160°C and 60°C, respectively. Each egg white powder was independently added to water at 10% (w/w) and dispersed by gently mixing with a glass rod. The first dispersion was pH 7.4, so the spray-dried egg white powder was named EW7.4, the second was pH 8.4, named EW8.4, and the third was pH 10, named EW10. The three types of egg white dispersion were divided into 3 or 4 parts, and adjusted to pH 4, 7.4, and 9 with 1 M HCl or 1 M NaOH. The pH-adjusted egg white dispersions were then separately whipped with a stand mixer (Kenmix KM-300; Aicohsha Manufacturing Co., Ltd., Toda, Japan) in a stainless bowl at 440 rpm for 5 min.
Measurement of specific volume of whipped foam prepared from spray-dried egg white Whipped foam formed from spray-dried egg white was added to a disposable petri dish (49 mm i.d., height, 5 mm) and weighed.
Measurement of hardness of whipped foam prepared from spray-dried egg white The foam in the disposable petri dish (49 mm i.d., height, 5 mm) was set on the measuring stage of a creep meter (RE-3305; Yamaden Co., Ltd., Tokyo, Japan). A cylindrical plunger (40 mm o.d.) was used and the sample was compressed at 1 mm/s to 90% strain.
Analysis of insoluble fraction from whipped foam prepared from spray-dried egg white An aliquot of the meringue-like foam (8 g) was placed on a funnel with filter paper, and was washed with 200 g of distilled water or 2% KCl solution and left to stand. The resulting precipitate was dissolved in a 3 mol/L urea solution containing 1% sodium dodecyl sulfate (SDS), mixed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (EzApply; Atto Co., Tokyo, Japan), and applied to the gel.
Electrophoresis SDS-PAGE was performed using 12.5% gels, according to the method reported by Laemmli (1970). The gels were stained with commercial Coomassie Brilliant Blue solution (EzStain Aqua; Atto Co., Tokyo, Japan).
Statistics All data obtained were subjected to one-way ANOVA. Significant differences among averages were determined by Bonferroni multiple comparison.
Specific volume of whipped foam prepared from spray-dried egg white Dispersions of spray-dried egg white (EW7.4, EW8.4, and EW10) were separately whipped with a stand mixer for 5 min after pH adjustment. The specific volumes of foams obtained are shown in Fig. 1. The specific volumes of EW8.4 and EW10 were higher at pH 4 than at alkaline pH, which coincided with previous reports (Nakamura and Sato, 1964). However, EW7.4 behaved differently from the other samples. The specific volume of foam derived from EW7.4 was highest at pH 7 and the value at pH 9 was almost equivalent. The specific volume at pH 4 was significantly lower than at other pH levels, differing from EW8.4 and EW10. The foam prepared from EW7.4 at pH 4 appeared to be thin. From these data, neutralization of egg white before spray drying inhibited whipped foam formation at pH 4, although spray drying is generally reported to accelerate foam formation (Hammershøj et al., 2004; Ayadi et al., 2008). Kim et al. (2006) reported that overrun of foam prepared from the egg white spray dried after pH adjustment was high in order of adjustment at pH levels 6.5, 7 and 8. The reasons for this difference are not clear, but conditions for spray drying may have some effect on the foaminess of spray-dried egg white.
Effect of pH on specific volume of whipped foam from spray-dried egg white. Different letters mean significance ( p < 0.05).
Stress-strain curves of whipped meringue-like foams and comparison of three parameters calculated from the curves In many papers, foaming activity was estimated using volume of foam, namely overrun or specific volume. Considering the utilization of meringue, texture is also an important factor, so the stress–strain curve of whipped foam was measured to estimate some parameters for hardness. Figure 2 shows the stress–strain curves of EW7.4 foams. There was a peak at about 20% strain at pH 7 and pH 9, but no peak at pH 4. With the increase in strain after the peak, the curve dropped and then increased again. The drop in stress appears to be different between pH 7 and pH 9. Therefore, two parameters, namely maximum stress at the peak and breakdown, which refers to the stress drop after maximum stress, were selected to assess the foams. Furthermore, the stress–strain curves were seen to increase linearly at the early stages of strain, so the slope of the curve at the linear increase was designated the apparent elasticity.
Typical stress-strain curves of whipped foams derived from spray-dried egg white, which was previously neutralized to pH 7.4 before spray-drying. pH 4, spray-dried egg white dispersion was whipped after adjustment of pH to 4; pH 9, after adjustment of pH to 9; pH 7.4, no adjustment of pH.
Maximum stress of EW8.4 (Fig. 3B) was highest at pH 7.4 and the values decreased with increasing pH until pH 9. At pH 4, there was no peak detected, so values at the flat region of the stress-strain curves were expressed as the maximum stress. As a result, the value at pH 4 decreased markedly. EW10 (Fig. 3C) showed almost equivalent results as EW8.4, but the maximum stress at pH 10 was equivalent to that at pH 9. These data suggest that the structure of foam is strong at neutral pH, but weak under acidic and alkaline conditions. At pH 4, specific volume of the foam increased, but the foam structure appeared to be weakened. Considering these data, specific volume, which is considered to be equivalent to overrun, is not thought to be enough to express the quality of whipped foam derived from spray-dried egg white dispersion. EW7.4 showed significantly different behavior at the maximum stress of foams whipped at various pH levels, as compared with EW8.4 and EW10 (Fig. 3A). It was reported that pH adjustment before spray drying affects the functionality of egg white by changing the structure of egg white proteins (Kim et al., 2006). The data presented in this study also suggest that pH adjustment before spray drying also modifies the dependence of egg white dispersion on pH of whipping as well as its foaming property.
Effect of pH on maximum stress of whipped foams from spray-dried egg white. Different letters mean significant differences ( p < 0.05).
Breakdowns, which were assigned to drops in stress after the maximum stress, were almost negligible at pH 4 for all samples (Fig. 4), thus acidic foam may be considered to show viscous flow without a clear breaking point. The breakdowns of EW8.4 and EW10 (Fig. 4B and C) were together the largest at pH 7.4, similar to the maximum stress. For EW7.4 (Fig. 4A), however, the breakdowns were small and increased with increasing in pH. Considering the breakdown of whipped foam, interactions among individual foams at pH 4 were thought to be very weak and individual bubbles appeared to exist separately.
Effect of pH on breakdown of whipped foam from spray-dried egg white. Different letters mean significant differences ( p < 0.05). n.d.: not detected.
Further, the apparent elasticities of all samples (EW7.4, EW8.4, and EW10) were low and almost the same as at pH 4 (Fig. 5), similar to the results of the maximum stress (Fig. 3). At neutral and alkaline pH, the apparent elasticity was higher than at pH 4 and was also nearly equivalent. Although strict comparison among EW7.4, EW8.4, and EW10 was not possible in this study, EW7.4 showed much less apparent elasticity, maximum stress (Fig. 3) and breakdown (Fig. 4) than EW8.4 and EW10. Neutralization of egg white until about pH 7 before spray drying was thought to decrease hardness of whipped foam, differing from the foaminess.
Effect of pH on apparent elasticity of whipped foam from spray-dried egg white. Different letters mean significant differences ( p < 0.05).
Protein constituents in insoluble fraction derived from meringue-like foam With regard to foam preparation by whipping of egg white, such as for meringue, it was reported that egg white proteins are denatured by beating, leading to adsorption on the interface between liquid and air, and to be further denatured and re-arranged to form an insoluble membrane-like structure (Nakamura and Sato, 1963; Mine, 1995). Therefore, the insoluble fraction obtained by washing foam with water or 2% KCl was subjected to SDS-PAGE. As a large amount of liquid (200 g of water or KCl solution to 8 g of the whipped foam) was used to wash out proteins that were not adsorbed onto the air–liquid interface, and residual proteins were considered to construct a foam membrane-like structure. The residual insoluble fraction from EW7.4 after washing with water was shown in Fig. 6A. The insoluble fractions without dithiothreitol (DTT) (lanes 2 and 4) showed smear patterns and a clear band assigned to lysozyme. Almost all proteins in non-reducing buffer were considered to be aggregated through disulfide bonds, except for lysozyme. The sample with DTT (lanes 3 and 5) contained mainly ovoglobulin (∼49,000) and lysozyme (∼14,000), and smaller amounts of ovomucin (>100,000), ovotransferrin (∼70,000) and ovalbumin (∼45,000) were also detected (Fig. 6A, lanes 3 and 5). It was previously reported that ovoglobulin mainly contributes to foaming and ovomucin to stability (MacDonnell et al., 1955; Nakamura & Sato, 1961; 1964). The detection of a large amount of ovoglobulin supports these previous papers. A small amount of ovomucin, however, was detected in the insoluble fraction. Considering the protein composition of spray-dried egg white (Fig. 6A, lanes 6 and 7), there was little ovomucin detected. Thus, a small amount of ovomucin in spray-dried egg white was thought to be rather concentrated in the insoluble fraction by interacting with the membrane-like structure of the foam. On the other hand, ovalbumin was also reported to be adsorbed on the air–liquid interface to a similar extent as ovoglobulin (Damodaran et al., 1998). Considering the data in this study, ovalbumin was thought to be adsorbed so weakly on the interface that most of the ovalbumin was washed out by water. From the SDS-PAGE patterns of the KCl insoluble fraction, little lysozyme was detected (Fig. 6B, lanes 2 to 5), differing from the results in Fig. 6A. Thus, lysozyme was considered to bind other proteins such as ovoglobulin and ovomucin through mainly electrostatic interaction and to be washed out with KCl solution, coinciding with previous results (Damodaran et al., 1998).
SDS-PAGE patterns of insoluble fraction from whipped foam of egg white powder spray-dried at pH 7.4.
A, Insoluble fractions after washed with water; B, washed with 2% KCl. Lanes 1 and 8, marker; lanes 2 and 4, insoluble fraction without DTT; lanes 3 and 5, insoluble fraction with DTT; lane 6, egg white powder without DTT; lane 7, with DTT; lanes 4 and 5, applied about two-fold volume of lanes 2 and 3, respectively. OM, ovomucin; OT, ovotransferrin; OG, ovoglobulin; OA, ovalbumin; LZ, lysozyme.
The insoluble fraction of the foam derived from EW10, which was whipped after adjusting to pH 7.4, was then analyzed by SDS-PAGE (Fig. 7A). In the case of washing with water, SDS-PAGE patterns of the insoluble fractions without DTT (lanes 2 and 4) were similar to the results from EW7.4 (Fig. 6A, lanes 2 and 4). Furthermore, the sample with DTT (lanes 3 and 5) also showed equivalent results to EW7.4 with DTT (Fig. 6A, lanes 3 and 5). Thus, after washing with water, protein composition of the insoluble fraction derived from EW10 coincided with the results of EW7.4. However, after washing with the KCl solution, an intense band assigned to lysozyme was detected in the insoluble fraction (Fig. 7B, lanes 2 to 5), in contrast with EW7.4 (Fig. 6B, lanes 2 to 5). Furthermore, the sample without pH adjustment, namely the insoluble fraction from the foam whipped at pH 10 was subjected to SDS-PAGE. As a result, similar patterns were obtained (data not shown), showing that lysozyme may be more dependent on pH under spray drying than under whipping. In the case of EW7.4, lysozyme was considered to bind other proteins through electrostatic interaction because lysozyme was washed out with the KCl solution. On the other hand, in the case of EW10, lysozyme was considered to bind other proteins through some additional interactions. The results in Fig. 7B showed that lysozyme was equivalently detected both in lanes 2 and 4 (without DTT), and lanes 3 and 5 (with DTT), supporting the minimal participation of disulfide bonds between lysozyme and other proteins. Thus, the interaction was probably a hydrophobic interaction. EW8.4 also showed equivalent results (data not shown). Mild heating under spray drying made it possible to modify the main interactions among proteins from electrostatic interaction (Damodaran et al., 1998) to others such as hydrophobic interaction.
SDS-PAGE patterns of insoluble fraction from whipped foam of egg white powder spray-dried at pH 10.
Whipping was done at pH 7.4. A, Insoluble fractions washed with water; B, washed with 2% KCl. Lanes 1 and 8, marker; lanes 2 and 4, insoluble fraction without DTT; lanes 3 and 5, insoluble fraction with DTT; lane 6, egg white powder without DTT; lane 7, with DTT; lanes 3 and 5, applied about two-fold volume of lanes 2 and 3, respectively. OM, ovomucin; OT, ovotransferrin; OG, ovoglobulin; OA, ovalbumin; LZ, lysozyme.
Based on the results in Figs. 1, 3, 4 and 5, EW7.4 showed unusual behaviors when compared with EW8.4 and EW10; namely, lower foaming activity at pH 4, lower maximum stress at pH 7.4, and lower breakdown at pH 7.4. Considering the SDS-PAGE patterns, lysozyme in EW7.4 is thought to bind to other proteins through electrostatic interaction (Fig. 6). However, lysozyme in EW10 binds through hydrophobic interaction as well as electrostatic interaction (Fig. 7). As for EW7.4, a lack of hydrophobic interaction of lysozyme may weaken the membrane-like structures of whipped foam to result in decreased foam properties. Interactions among protein molecules are considered to be affected by pH before spray-drying and the changes in the interactions are likely to change the insoluble membrane-like structures of whipped foam.
In this study, there were no marked differences in proteins other than lysozyme among the different pH treatments before spray drying; however, considering the significance of the unusual behaviors in EW7.4, further detailed study may clarify the changes in the interactions between these proteins. The results are expected to improve the quality of meringue derived from spray-dried egg white powder.
Whipped foam made from spray-dried egg white was evaluated using three hardness parameters, which were calculated from the stress-strain curve. The results showed that pH adjustment of egg white before spray drying affected the hardness of whipped foam, as well as foaminess. Neutralization of egg white before spray drying (EW7.4) greatly decreased maximum stress, breakdown and apparent elasticity, and specific volume, which was related to foaminess. The specific volume of foams derived from EW8.4 and EW10, namely alkaline egg white powders, was higher when pH levels were adjusted to pH 4 before whipping. However, maximum stress, breakdown and apparent elasticity decreased, differing from the specific volume. These changes in foam properties were thought to be caused by modification of protein-protein interactions. In particular, the decrease in hardness of foam in EW7.4 was thought to be related to a lack of hydrophobic interaction between lysozyme and other proteins.
Acknowledgment The authors are grateful to Professor E. Arai and Dr. S. Ito at the University of Shizuoka for use of a stand mixer. The authors would also like to thank Enago for the English language review.
