3.1 Foaming ability and foam stability
HIUS treatment can significantly enhance the foaming ability of EWP, which has been found in previous reports. For example, Sheng et al. Found that the foaming ability of fresh EWP increased by 4.9 times after sonication with 360 W (20 kHz) for 10 min [11]. However, Arzeni et al. found that after EWP was sonicated (frequency: 20 kHz; amplitude: 20%) for 20 minutes, the foam overrun value decreased from 205–127% [9]. This diversity should be attributed to the difference of ultrasonic processing parameters and protein sources. In present work, the foaming ability of fresh EWP increased by 36% after HIUS treatment (Fig. 1A). On the contrary, freezing led to the continuous decline of the foaming capacity of EWP from 110% to less than 80% (day 21). It should be pointed out that freeze-thaw cycle treatment could significantly increase the foamability and foaming stability of fresh EWP, which was mainly due to the repeated effect of ice crystals on protein conformation [22].
It is worth noting that the foaming ability of EWP with different frozen storage time after HIUS treatment is increased by 40%-60% compared with that of protein without sonication (Fig. 1A). Especially for EWP stored for 21 days, its foaming capacity was more than doubled after HIUS treatment. A similar improvement was also observed in the report on the foaming ability of EWP treated with ultrasound at different cold times [1]. On the other hand, there was almost no significant difference in foam stability among all samples (Fig. 1B). These results show that short-term (5 min) ultrasonic treatment has a prominent effect on improving the foam characteristics of frozen EWP. In fact, we also tried a longer ultrasonic treatment time (10 and 15min), because compared with 5min sonication, the foaming ability of EWP was not significantly improved (p > 0.05), so the results were not shown.
3.2 Solubility and average size of EWP
The solubility of fresh EWP is about 57%, while that of sonicated EWP is increased to 73% (Fig. 2A). Sheng et al. observed that HIUS treatment could increase the solubility of fresh EWP from less than 60–90%, and this improvement did not depend on ultrasonic power [11]. However, Yu et al. found that the solubility of EWP decreased significantly after ultrasonic treatment [12]. These discrepancies should be related to the various experimental conditions. It is worth noting that the solubility of EWP continues to decline to about 30% with the extension of frozen storage time (Fig. 2A). This may be due to the enhanced freezing induced protein aggregation [7], which was easier to be separated into the precipitation in the process of solubility determination, thus showing a low solubility. After sonication, the solubility of all frozen samples increased significantly to about 70%. In addition to EWP, the same phenomenon was also observed in the reports of using HIUS treatment to improve the interface and physicochemical properties of soybean protein and whey protein isolate [23, 24].
From the average size shown in Fig. 2B, it can be found that the average diameter of fresh EWP increases from about 225 nm to 400 nm after ultrasonic treatment. Conversely, the poly diffusivity index (PDI) decreased significantly to 0.55, suggesting that the size distribution of protein aggregates induced by ultrasound was more concentrated. This result is consistent with past findings [1, 9, 10]. The increased solubility of EWP after ultrasonic treatment indicates that these particles should be soluble aggregates [12]. Similarly, the size of EWP with different freezing time increased significantly after HIUS treatment. Nevertheless, compared with fresh samples (232 nm), the average size of frozen EWP (150–225 nm) showed a significant reduction. In addition, the size of aggregates increased monotonously depending on the extension of freezing time, while the PDI value (0.5–0.6) did not change significantly. This phenomenon should be due to the destruction of ovomucoid and ovomucin in EWP by ice crystals due to short-term freezing (3 days), resulting in a significant reduction in size. With the increase of freezing time, the extrusion of ice crystals leads to protein aggregation and shows an increase in size [8, 22].
3.3 Apparent viscosity
The viscosity of protein solution will affect the adsorption rate of protein molecules to the air-water interface, and then impact the foam characteristics of protein. Obviously, fresh EWP shows a high constant viscosity (about 0.5 Pa.s) in the lower shear rate range (< 6 1/s), and a shear thinning non-Newtonian fluid behavior in the higher shear rate range (Fig. 3A). This phenomenon can be explained by the fact that ovomucoid and ovomucin contained in fresh EWP cause a large number of protein molecules to tangle together. The disruption and formation of these entanglements is balanced at low shear rates, ensuing higher constant viscosity (η0) [25]. The mechanical energy produced by freezing and HIUS treatment destroyed the aggregation and structure of protein [22, 26], which significantly reduced the apparent viscosity of EWP. Therefore, the viscosity of samples at different freezing periods decreased after further ultrasonic treatment (Fig. 3B). The same phenomenon was also observed in the treatment of fresh EWP with different power ultrasound [11].
Furthermore, in order to quantitatively compare the effects of freezing and HIUS treatment on EWP fluid behavior, the cross model was used to fit the data of apparent viscosity versus shear rate. The fitting parameter results are shown in Table 1, and the regression coefficient (R2) shows that the model has a good fitting. For the samples without sonication, both infinite viscosity (η∞) at high shear rate and zero shear limiting viscosity (η0) at low shear rate showed a gradual decrease depending on the extension of freezing time. Similarly, the η∞ and η0 of the samples treated by HIUS were lower than that before ultrasonic treatment. On the other hand, compared with fresh EWP, the decrease of m value indicated that the strength of shear-thinning behavior was less obvious for all treated samples. In addition, the increase of k parameter implies that shear rate at which transition from Newtonian to shear-thinning behavior occurs shifts to lower values with the extension of freezing time. Also, HIUS treatment induced a remarkable increase in the k value of EWP in fresh and frozen samples for 3 days, but had no significant effect on frozen samples for 7 and 14 days. In particular, it is worth noting that the k value of EWP frozen for 21 days after acoustic energy treatment shows a sharp decrease, indicating that the increase of shear-thinning behavior rate. This result can also be obtained by comparing the fitting curve profile of samples frozen for 21 days before and after HIUS treatment (Fig. 3). This result may be related to the enhanced protein aggregation (size increase, Fig. 2B) caused by ultrasonic mechanical effect. These effects of freezing and subsequent HIUS treatment on EWP fluid behavior should be closely related to the diffusion rate of protein during whipping, especially the reduction of viscosity.
Table 1
Parameters of the Cross’ model for the samples investaged.
| η0(Pa.s) | ηꝏ(Pa.s) | k(s− 1) | m | R2 |
E0 | 0.506 ± 0.015 | 0.255 ± 0.023 | 0.103 ± 0.007 | 4.925 ± 1.610 | 0.926 |
E3 | 0.094 ± 0.003 | 0.014 ± 0.003 | 0.170 ± 0.007 | 1.956 ± 0.195 | 0.994 |
E7 | 0.084 ± 0.002 | 0.011 ± 0.001 | 0.267 ± 0.008 | 2.633 ± 0.170 | 0.996 |
E14 | 0.045 ± 0.001 | 0.009 ± 0.004 | 0.254 ± 0.007 | 1.985 ± 0.117 | 0.998 |
E21 | 0.043 ± 0.003 | 0.006 ± 0.002 | 0.631 ± 0.063 | 2.607 ± 0.298 | 0.994 |
E0-U | 0.292 ± 0.016 | 0.012 ± 0.001 | 0.253 ± 0.028 | 1.322 ± 0.246 | 0.989 |
E3-U | 0.092 ± 0.002 | 0.004 ± 0.001 | 0.235 ± 0.006 | 2.000 ± 0.116 | 0.999 |
E7-U | 0.064 ± 0.003 | 0.002 ± 0.001 | 0.219 ± 0.014 | 1.647 ± 0.219 | 0.991 |
E14-U | 0.014 ± 0.001 | 0.003 ± 0.001 | 0.206 ± 0.005 | 2.893 ± 0.174 | 0.996 |
E21-U | 0.007 ± 0.001 | 0.003 ± 0.001 | 0.305 ± 0.024 | 1.932 ± 0.255 | 0.986 |
3.4 Zeta potential, surface hydrophobicity and intrinsic fluorescence
The surface potential of protein can reflect the changes of its conformational, and indicating the exposure of charged groups in the side chain of protein molecules. It can be seen from Fig. 4 that compared with fresh EWP, the zeta potential of most other samples changes slightly (P > 0.05), regardless frozen or ultrasonic treatment. This phenomenon is consistent with the performance of EWP in the long-term cold storage [1]. In addition, the absolute potential of EWP increased after 21 days of freezing, while HIUS treatment had no effect. This implied that freezing was the main factor inducing protein conformational transition, and the effect of HIUS was relatively weak.
Furthermore, the surface hydrophobicity (H0) of fresh and frozen EWP for 21 days before and after ultrasonic treatment was characterized (Fig. 5A). It could be found that HIUS treatment has no significant impact on the surface hydrophobicity of fresh EWP. However, after 21 days of freezing, the H0 value of egg white increased remarkably, and sonication further induced its enhancement. This result agreed with the findings of freeze-thaw cycle treatment[22]. For frozen EWP, the increase of surface hydrophobicity after ultrasonic treatment should be attributed to the cavitation, which leaded to protein aggregates to expose more hydrophobic micro regions. Moreover, intrinic fluorescence spectra were used to further confirm these results (Fig. 5B). As expected, ultrasonic treatment has little effect on the emission fluorescence curve of fresh EWP. This was different from most previous reports on EWP treatment with high-intensity ultrasound [1, 13, 27]. It was inferred that this discrepancy mainly derived from the diversification of ultrasonic treatment conditions. Also, a strong fluorescence emission peak can be observed at 335 nm for all samples after excitation at 280 nm. However, freezing and subsequent HIUS treatment resulted in a significant decrease in the fluorescence intensity of EWP compared with fresh samples. This phenomenon is similar to the result of ultrasonic treatment of OVA [10]. Since tyrosine and tryptophan residues can be excited simultaneously at 280 nm [28], the reduction of fluorescence intensity means that the chromophores of EWP become exposed to solvent and the transformation of tertiary conformation due to HIUS treatment. Besides, owing to the indole group of tryptophan is more hydrophobic than the phenol group of tyrosine and the enhanced results of surface hydrophobicity, it is speculated that freezing and subsequent ultrasonic treatment mainly induce the exposure of more tryptophan residues.
3.5 Interfacial adsorption behavior
Indeed, the improvement of foaming ability of frozen EWP after ultrasonic treatment was mainly due to the changes of the above physicochemical properties, which has an impact on the air-water interface adsorption behavior of protein. The plots of surface pressure (π) versus time (t1/2) exhibited the same profile, and π value showed a continuous increase due to the absorption of proteins (Fig. 6A). It can be found that within 400s of the initial stage of adsorption, the π values of all samples show a linear rapid increase trend, and then gradually reach a platform region. The slope of the linear region could be fitted by a modified ward and Tordai equation (the fitting curves are the red dotted lines in Fig. 6A), and was used as the diffusion rate of protein to the air-water interface (Kdiff, Fig. 6B). Compared with fresh EWP, ultrasonic treatment alone led to a significant increase in the diffusion rate. Nevertheless, long-term freezing and subsequent cavitation can not remarkably affect its Kdiff compared with sonicated fresh EWP (Fig. 6C). Additionally, the surface pressure of the selected samples at the beginning (π0) and end point (π10800) of adsorption has been significantly increased after ultrasonic treatment compared with fresh samples (Fig. 6C). On the other hand, after ultrasonic and freezing treatment, the amount of protein adsorption at the air-water interface are significantly higher than fresh EWP (Fig. 6D). The difference of these interface behavior parameters should be attributed to the discrepancy of physicochemical properties of continuous phase and protein.
Firstly, the π0 value of protein is weakly correlated with the properties of bulk phase (such as viscosity), which is mainly determined by the physicochemical properties of aggregates [29, 30]. Generally, the increase of surface hydrophobicity and the decrease of average size of proteins are conducive to driving their shifting to the interface, while the increase of zeta potential will hinder the diffusion due to the enhancement of intermolecular repulsion force [6, 31–34]. Therefore, the increase of π0 of EWP (E0-U and E21-U) after HIUS treatment was mainly contributed by the surface hydrophobicity. Secondly, with the continuous adsorption, the diffusion of protein to the interface depends on its own physicochemical properties and bulk viscosity. Obviously, the decrease of viscosity and the enhancement of surface hydrophobicity dominated the migration of proteins to the interface, resulting in a significant increase in the diffusion rate of samples (E0-U, E21 and E21-U), although the average size and zeta potential were increased by ultrasound and freezing treatment. Finally, the increase of surface pressure (π10800) and interfacial adsorption capacity of the ultrasonic treated samples at the end of adsorption should also be mainly attributed to the enhancement of surface hydrophobicity. Therefore, although freezing and ultrasonic treatment leaded to the increase of the average size and surface potential strength of EWP, which was not conducive to the diffusion of protein to the interface. Instead, the decrease of bulk phase viscosity and the enhancement of surface hydrophobicity play a more important role, thereby EWP showed stronger foamability.