Research note: Preparation and stability of egg white fluidic gel induced by ultrasonic pretreatment

Abstract

Ultrasound-induced egg white fluidic gels (UEFG) were prepared through ultrasonic pretreatment followed by subsequent heating. The optimal preparation parameters for UEFG were: ultrasonic power density between 0.2 to 0.8 W/mL, ultrasonic treatment time surpassing 150 s, heating temperature within 70 to 76°C, and heating time under 16 min. The prepared UEFG is a milky white solution with a viscosity lower than that of fresh egg white, and displayed a particle size distribution primarily between 100 and 1,200 nm. Stability assessments conducted over 28 d at 4°C revealed that UEFG remained stable at pH 6-10, with exceptional stability from pH 8 to 10, while it is less stable in highly acidic (pH 2-4) or basic environments (pH 12). The UEFG also showed commendable stability in the presence of salt and sucrose solutions. We report a simple and novel method for preparing UEFG with good flowability after heating treatment. The UEFG has broad applications in the food industry, such as precooked egg white powder, high protein beverages, composite dairy products, etc.

INTRODUCTION

When the heat treatment temperature exceeds 60 to 65°C, egg white proteins rapidly denature and aggregate via hydrophobic interactions, disulfide bonds, and hydrogen bonds, forming highly ordered egg white thermal gels (Duan et al., 2018). The formation of egg white thermal gels is one of the primary changes during the cooking process of eggs, playing a critical role in shaping the texture and mouthfeel. In the food industry, the gelling properties of egg white are widely applied. However, the formation of egg white thermal gels also presents challenges and limitations for the use of eggs. For example, in the processing of liquid egg whites, the formation of thermal gels limits the temperature for pasteurization, consequently restricting the shelf-life of liquid egg whites to 2 to 4 wk under refrigeration. Furthermore, the coagulation characteristics of heated egg whites also limit their application in the production of beverages and other food processing areas. To expand the use of egg whites, a method for preparing egg white fluidic gels has been developed: egg whites are first heated to form a gel, which is then broken down and homogenized through high-speed shearing and other methods, resulting in a flowable egg white gel (Singer and Dunn, 1990). However, this “heat-breakdown-homogenize” method for forming egg white fluidic gels is relatively complex and involves high-energy processes.

In recent years, the use of new food processing methods to improve the properties of egg white gels has been extensively studied, including ultraviolet processing, high-pressure processing, cold plasma, ball-milling treatment, and microwave technology. Among them, ultrasound technology is a prominent gelling method of egg whites due to its safety, efficiency and environmental friendliness (Mirarab et al., 2023). In our previous research, we unexpectedly found that subjecting egg whites to ultrasonic pretreatment (180–360 W) followed by heating (72°C, 8 min) can result in the formation of ultrasound-induced egg white fluidic gels (UEFG). These UEFG possess good flowability, with an average particle size of 544.97 nm, and are simple and low-cost to prepare, with broad potential applications (Wang et al., 2022). However, the parameters for ultrasonic pretreatment in preparing UEFG are not yet clear, and the stability of the fluid gels during storage and processing is unknown. Therefore, this work aims to study the effects of ultrasonic power, treatment time, post-treatment heating temperature, and heating duration on the formation of UEFG to determine the optimal conditions for ultrasonic pretreatment. Subsequently, the stability of UEFG in relation to storage time, pH, salt addition, and sucrose addition was examined. This research will establish a novel method for preparing UEFG, enabling egg whites to have a wide range of applications in high-protein beverages, protein-based fat substitutes, and pickering emulsions, among others.

MATERIALS AND METHODS

Preparation of UEFG

The eggs within 24 h after laying were selected for the experiment. Eggshells were manually cracked to collect fresh egg white (FEW), which was then homogenized using magnetic stirring (500 rpm, 15 min). The method for ultrasonic treatment of the egg white was slightly modified from our previous research (Geng et al., 2018). We placed 1 L of egg white into a 2 L beaker and subjected it to ultrasonication using an ultrasonic cell crusher (JY99-IIDN, maximum power 1,000 W, 20 kHz; Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China). The sample was kept in an ice-water bath to prevent potential temperature increases. The fixed parameters for ultrasonic treatment were as follows: the ultrasonic probe had a diameter of 15 mm; it was positioned 15 mm below the surface of the egg white; and the ultrasonic pretreatment time and interval were both set to 3 s. After ultrasonic treatment, the FEW was heated in a water bath at 72°C for 10 min to form UEFG.

The effects of varying ultrasonic power density (0, 0.2, 0.4, 0.6, 0.8, 1.0 W/mL), ultrasonic processing time (0, 60, 90, 120, 150, 180 s), postultrasonication heating temperatures (68, 70, 72, 74, 76, 78, 80°C), and heating durations (0, 9, 10, 11, 12, 13, 14, 15, 16 min) on UEFG were studied.

Observation of UEFG Formation Process

FEW was placed in a 6-well plate (4 mL/well) and then heated in a 72°C water bath. Photographs were taken every 2 min to monitor the formation of UEFG.

Particle Size Distribution and Zeta-Potential Analysis

The particle size and zeta-potential of the samples were measured using a Nano particle analyzer (ZS90, Malvern Zetasizer Nano, UK) following the previous method (Ye, et al., 2022). Samples were diluted 100 times with phosphate-buffered saline (10 mM, pH 7.4) and measured at room temperature for both zeta potential and particle size.

Rheological Behavior

Flow curves were determined using advanced rotary rheometers FEW and UEFG with geometric plates. Shear rheological parameters were set: the test temperature was 25°C, the plate spacing was 1 mm, and the shear rate was 40 to 100 per s. The relationship between shear rate and shear stress was proved by fitting the data with a mathematical model. Shear rate as the abscissa and apparent viscosity (η) as the ordinate were plotted. Temperature rheological parameter setting: temperature range is 25 to 100°C.

Stability Assessment

A 28-d stability assessment was conducted for UEFG. The influence of pH on UEFG stability: 5 mL of UEFG was placed in a sample bottle, diluted with 4 mL of deionized water, the pH of the solution at this time was 8.9 ± 0.1, which was used as the control group for the experiment. Then adjusted to pH values of 2, 4, 6, 8, 10, and 12 using 3 M HCl or NaOH. The total volume was made up to 10 mL; the bottles were stored at 4°C for 28 d. Samples were taken out and evaluated on d 1, 3, 5, 7, 14, 21, and 28. The influence of salt and sucrose on UEFG stability: 5 mL of UEFG was placed in sample bottles and mixed with 5 mL of deionized water, salt solution (final concentration 1%, 2%), or sucrose solution (final concentration 5%, 10%). The sample bottles were stored at 4°C for 28 d and evaluated on designated days. Samples were observed, images were collected, and particle size distribution, zeta-potential, and turbidity were measured. All the instruments used in the UEFG stability test were sterilized in advance, and a FEW were obtained in a super-clean workbench. Deionized water and UEFG small bottles were sterilized in a high-pressure steam sterilizer at 121°C for 30 min. Finally, the bottle was sealed.

Turbidity Measurement

Samples were diluted 50 times with deionized water and then measured using a turbidimeter (WZS-188, Shanghai Precision & Scientific Instrument Co., Ltd., Shanghai, China).

Data Statistical Analysis

Each sample was measured at least 3 times. Experimental data were expressed as mean values with standard deviation (mean ± SD) and analyzed using one-way ANOVA. Duncan's multiple comparison test was implemented in GraphPad Prism 8.0 (GraphPad Software, San Diego, CA). The P values < 0.05 (*) were regarded as a significant difference.

RESULTS AND DISCUSSION

Formation of UEFG

The gelation of FEW (0 W/mL) and UEW (0.2, 0.4, 0.6, 0.8, 1.0 W/mL) was monitored and compared at 72°C over a period of 0-10 min. FEW began to gel at the container's edges at 4 min, spreading toward the center and eventually solidifying. The results indicated that: 1) Ultrasonic pretreatment facilitated the formation of a fluid gel in egg whites at 72°C; 2) UEW treated at intensities from 0.2 to 0.6 W/mL formed UEFG with desirable flowability and uniformity; 3) UEW treated at higher intensities of 0.8 to 1.0 W/mL could still form UEFG, but the texture was less even and the flowability was compromised. (Figure 1A).

Figure 1.

Influence of ultrasonic power density (A), ultrasonic treatment time (B), water bath temperature (C) and water bath heating time (D) on the formation of egg white liquid gel (UEFG); Mobility (E), particle size distribution (F), zeta potential (G), shear rate-viscosity (H), and temperature-viscosity (I) of fresh egg white (FEW), ultrasonic induced egg white (UEW), egg white solid gel (EWSG) and egg white fluidic gel (UEFG). The different letters a, b and c in Figure 1G indicate significant differences (p < 0.05).

The required ultrasonic pretreatment time for UEFG formation was studied at a constant ultrasonic power density of 0.25 W/mL, water bath temperature of 72°C, and a duration of 10 min. It was found that with less than 150 s of ultrasonication, the gel formed progressively from the edge to the center, yielding a non-fluid gel. At the 150 s, increasing heating time led to the formation of a milky suspension, culminating in a fluid gel (Figure 1B).

The range of heating temperatures required to form UEFG was examined with a fixed ultrasonic power density of 0.25 W/mL for 4 min, followed by a 10 min water bath. At 68°C, egg white proteins failed to form a thermal gel, indicating insufficient denaturation and gelation participation. Between 70 and 76°C, higher temperatures increased gelation rate but reduced UEFG fluidity. Above 78°C, the egg white protein gel shifted from fluid to solid (Figure 1C).

The formation time for UEFG was explored using a constant ultrasonic power density of 0.25 W/mL for 4 min, followed by heating at 72°C. Findings revealed that at 14 min, the egg white thermal gel started to shift, losing fluidity as it moved from a fluid to a solid state. By 16 min, the egg white thermal gel had fully solidified (Figure 1D).

Properties of UEFG

FEW (without ultrasonic pretreatment) and UEW (pretreated with ultrasonic power density of 0.25 W/mL for 5 min) were heated at 72°C for 10 min to form egg white solid gel (EWSG) and UEFG, respectively. To assess the fluidity of the gels, their respective vials were tilted. The results showed that EWSG displayed a macroscopically solid and non-flowing state. In contrast, UEFG demonstrated superior fluidity (Figure 1E).

The particle size distribution curves for FEW, UEW, and UEFG are depicted in Figure 1F, primarily distributed within the ranges of 1 to 10 nm, 10 to 100 nm, and 100 to 1000 nm. These are speculated to correspond to small molecular weight egg white proteins, large molecular weight egg white proteins, and protein aggregates from egg whites, respectively (Liu et al., 2021). The UEW curve showed a shift towards smaller particle diameters compared to FEW, indicating a reduction in egg white protein aggregate size due to ultrasonication. After heating, UEFG displayed a significant presence of protein aggregates, predominantly in the 200-400 nm range.

The zeta potential is a critical parameter for quantifying the surface charge on proteins and is indicative of colloidal dispersion stability (Li et al., 2018). The absolute zeta potential value for UEW was reduced in comparison to FEW (Figure 1G), which aligns with previous research findings (Chen et al., 2019). After heating, the absolute value of the zeta potential for UEFG significantly increased. This was attributed to the rapid unfolding of heat-sensitive proteins in egg white due to thermal treatment, leading to the exposure of charged groups within the proteins to the surface. An increased absolute value of zeta potential results in stronger electrostatic repulsion between proteins, thereby improving the stability of the egg white system.

To compare the viscosity of FEW and UEFG, their flow properties were analyzed with a rotary rheometer. As shown in Figures 1H and 1I, FEW exhibited shear-thinning behavior, indicative of a pseudoplastic fluid, with viscosity decreasing as shear rate increased. UEFG, however, maintained a consistent viscosity across different shear rates, characteristic of a Newtonian fluid where viscosity was solely temperature-dependent. Both FEW and UEFG's viscosities remained stable up to 80°C, with FEW being the more viscous. The viscosity of UEFG experienced a minor shift with a steep temperature increase, likely resulting from ultrasonic treatment altering protein interactions.

Stability of UEFG

The shelf life of UEFG was assessed by refrigerating samples at different pH levels and solvent systems at 4°C for 28 d, and monitoring their appearance, particle size, zeta potential, and turbidity.

The stability of UEFG solutions was highest within a pH range of 8 to 10, maintaining consistency over 28 d. At pH 2, phase separation commenced on d 3, while at pH 4, the solutions quickly destabilized, separating on d 1 and showing significant layering by d 3. Improved stability was observed at pH 6, with slight separation only by d 21. At pH 12, UEFG shifted in color to light yellow-green and turbidity markedly decreased (Figure 2A). These results indicated that lower pH levels led to more layering and reduced stability, likely due to nearing the isoelectric point of egg white proteins, which diminished electrostatic repulsion and promoted protein aggregation. At higher pH values, while UEFG solutions remained more homogeneous, the proteins were more susceptible to denaturation in strongly alkaline conditions. Figure 2B presents that on the first day, UEFG solutions at pH 6 to 10 had particle sizes mostly ranging from 100 to 2,000 nm, with fewer particles in the 10 to 100 nm and 2,000 to 10,000 nm ranges. As pH decreased, the main peak in particle size distribution shifted right, indicating more protein aggregation. At pH 2 and 4, particles were predominantly in the 1,000 to 10,000 nm range, with a notable drop in the absolute value of Zeta potential, leading to less electrostatic repulsion and more aggregation. The lowest absolute Zeta potential and largest protein aggregates occurred at pH 2. At pH 12, particle sizes remained in the 100 to 2,000 nm range. Turbidity decreased under extreme pH conditions, most significantly in alkaline environments, while at pH 4 and 6, turbidity increased substantially, reflecting the influence of solution electrostatic repulsion: the higher the absolute Zeta potential, the clearer the solution.

Figure 2.

Stability changes of UEFG solution in 4°C storage environment. Effects of pH on macroscopic appearance changes (A), particle size, zeta potential and turbidity (B) of UEFG solution; Effects of salt solution and sucrose solution on macroscopic appearance changes (C), particle size, zeta potential and turbidity (D) of UEFG solution. P value is defined as P < 0.05 (*), P < 0.01 (* *), P < 0.001 (* * *) and P < 0.0001 (* * * *), the same letters a said there was no significant difference.

Furthermore, when UEFG was incorporated into saline solutions at 1% and 2% w/v and sucrose solutions at 5% and 10% w/v, there was no significant layering observed (Figure 2C), and the particle size, zeta potential, and turbidity remained comparable to the control group (UEFG diluted equally with deionized water). These findings suggested that UEFG maintained its stability well in moderate saline and sucrose environments (Figure 2D).

In this study, optimal ultrasonication of UEFG formation was achieved using a power density of 0.2 to 0.6 W/mL, with a critical pretreatment time of 150 s. The ideal heating range was found to be between 70 and 76°C, with a transition to solid gelation occurring after 14 min at 72°C. The UEFG exhibited improved fluidity, smaller particle size, and consistent Newtonian viscosity, distinguishing it from traditional egg white solutions. Our previous research has reported that ovomucin is one of the key proteins involved in the initiation and formation of egg white heat-set gels (Wang et al., 2022). Ultrasonic pretreatment was hypothesized to disrupt interprotein interactions, thereby inhibiting the incorporation of ovomucin into the structural matrix of the thermal gel. This mechanism was suggested to facilitate the formation of a distinct gel state referred to as UEFG. Stability tests indicated that the UEFG is stable for up to 28 d in pH conditions between 8 and 10, and in the presence of moderate concentrations of saline and sucrose, suggesting its suitability for diverse food industry applications. Furthermore, the method for preparing UEFG was compatible with existing egg liquid production lines, offering energy and time savings compared to traditional mechanical grinding. Moreover, the fluidity of the gel obtained through this method was superior to that achieved via traditional “heat-breakdown-homogenize” method.

DISCLOURES

The authors declare no conflicts of interest.