Mechanistic insights into ultrasound‑improved foaming properties of freeze‑thawed egg white: From macroscopic rheology to interfacial proteomics

Graphical abstract

Abstract

Repeated freeze-thawed (F-T) cycles critically damaged the foaming properties of egg white, thereby constraining its application in the food industry. This study investigated the effect of ultrasonic pretreatment in restoring the foaming property of egg white protein (EWP) subjected to repeated F-T cycles. A comprehensive analysis was conducted from the perspectives of foam behavior, physicochemical and interfacial properties, protein structure and interfacial proteomics. The results demonstrated that ultrasonic pretreatment (360 W, 10 min) effectively maintained foaming properties of egg white that underwent eight F-T cycles (EWP8-FTC) compared with those of fresh EWP. Moreover, ultrasonic pretreatment reduced the foaming ability (FA) from 41.35% to 18.56%, while concomitantly enhancing foam stability (FS) by 32.69%. Furthermore, there was no significant difference (p > 0.05) in the FA of ultrasonically pretreated egg white that underwent eight F-T cycles (UEWP8-FTC) compared with those of EWP. Ultrasound reduced the particle size of EWP, enhanced molecular flexibility, increased the content of free sulfhydryl groups and surface hydrophobicity, as well as optimized secondary structure. In addition, it regulated the adsorption behavior of interfacial proteins such as lysozyme C, mucin-5B, clusterin and Ovodefensin A1 at the air–water interface. This study elucidated the molecular mechanism by which ultrasound modulates interfacial protein adsorption, providing a theoretical reference for improving the quality of frozen egg white and promoting its high‑value industrial applications.

1. Introduction

Egg white protein (EWP) is universally acknowledged as a premium protein source, distinguished by its exceptional nutritional profile and versatile techno-functional properties. In the industry, frozen storage is commonly adopted to prolong the shelf-life of fresh egg white. The egg products made from fresh egg white, after processes such as peeling, processing and freezing, are called frozen egg white. Foaming property is the core functional characteristic of EWP. As protein molecules rapidly adsorbed at the air‑water interface, their subsequent conformational rearrangement and formation of a viscoelastic interfacial film, this property directly determined the lightness, microstructure and flavor profile of aerated products, and is a key parameter for assessing the suitability of egg white for processing [1]. However, in the practical processes of cold-chain storage and transport, warehousing and transhipment, and factory thawing and pre-processing, temperature fluctuations are difficult to avoid and can easily lead to multiple freeze-thawed (F-T) cycles. The impact of F-T cycles on food quality, particularly that of meat products, has been extensively studied. Repeated F‑T exposure triggers quality deterioration including protein oxidation, textural degradation and moisture loss [2]. During freezing, the growth of ice crystals inflicts irreversible damage to protein spatial structure, triggering partial denaturation, abnormal hydrophobic‑group aggregation and insoluble‑aggregate generation, consequently lowering solubility and surface hydrophobicity. Meanwhile, the damaged proteins exhibit poorer migration and adsorption properties at the air–water interface, and their ability to form an interface film is inhibited, thereby overall reducing the foaming properties [3]. Previous research has demonstrated that F‑T cycles expedited protein oxidation and further trigger protein denaturation [4]. A study by Li et al. [5] pointed out that the decrease in diffusion and adsorption rates at the air–water interface was the key rate-limiting step in the decline of the foaming properties of frozen egg white. Zhang et al. [6] investigated the effects of various cryoprotectants on the physicochemical properties and structural changes in EWP induced by F-T cycles. At present, the mechanisms by which F-T cycles caused damage to proteins have been reported. Although the current approached for mitigating the adverse effects of F-T cycles on protein quality largely involve the addition of F-T protectants, the results have been less than satisfactory.

Ultrasound, as an environmentally friendly and highly efficient physical processing technique, can modify protein structure via cavitation, thereby enhancing their functional performance [7]. Prior work indicated that moderate ultrasonication can significantly improve the foaming properties of proteins [8]. Research by Zhao et al. [9] indicated that ultrasound significantly enhanced the solubility and foaming properties of proteins at a pH of 11, while reducing particle size and turbidity and optimising surface hydrophobicity. Yang et al. [10] demonstrated that ultrasound can alter the secondary structure of proteins, consequently enhancing their foaming properties. In addition, Chen et al. [11] explored the influences of ultrasonic treatment on the foaming properties and physicochemical characteristics of fresh egg white over storage, demonstrating that appropriate ultrasonic treatment can improve the foaming functional properties of EWP. Ultrasonic treatment elevated free sulfhydryl content and surface hydrophobicity in egg white, facilitating the adsorption and reorganisation of protein molecules at the air–water interface and enhancing the flexibility of interfacial film, thus effectively enhancing foaming properties. Dong et al. [12] explored the influence of repeated F-T cycles under four distinct thawing methods on foaming properties and underlying regulatory mechanisms of EWP. Their findings illustrated that ultrasonic thawing exhibited the best foaming performance. In summary, ultrasonic technology suggested significant advantages and promising prospects for enhancing the foaming properties of proteins. However, there are still few reports on the effects of ultrasonic treatment on proteins during F-T cycles and the underlying mechanisms.

To address this critical gap, this study aims to systematically investigate and explore the deterioration of EWP foaming properties induced by repeated F-T cycles. We employed ultrasonic pretreatment to optimize the foaming properties of EWP and counteract the quality degradation resulting from repeated F-T cycles. Combining the macroscopic functional characteristics with the microscopic molecular structural characteristics, interfacial adsorption kinetics and proteomics, we systematically analyzed the regulatory pathways and molecular mechanisms underlying ultrasonic pretreatment. The outcomes of this research are expected to provide a theoretical foundation for improving cold-chain management, quality preservation, and processing of frozen egg white. Furthermore, the insights garnered may offer a valuable paradigm for cryo-modification and cryo-protection of other animal and plant-based proteins in the food industry.

2. Materials and methods

2.1. Materials and reagents

Fresh hen eggs were procured from a local market. Potassium bromide and Nile Blue A were analytical grade and were purchased from Shanghai McLean Biotechnology Co., Ltd. (Shanghai, China). All other chemicals were of analytical grade.

2.2. Sample preparation

Egg whites were carefully separated from yolks and divided into five equal portions. Four portions were subjected to ultrasonic treatment at different power levels (120 W, 240 W, 360 W and 480 W) for 10 min, whereas the remaining portion was kept untreated as the control. Dilute the egg white to 25% of its original concentration; take 40 mL of the diluted egg white solution and homogenize at 8,000 r/min for 1 min, then transfer to a 100 mL graduated cylinder to measure the volume and determine the foam volume. The experiment revealed that the foaming properties of the egg white were greatest following ultrasonic treatment at 360 W for 10 min; further increases in ultrasonic power resulted in a decrease in foaming properties. Therefore, the experimental conditions selected were 360 W for 10 min (3 s on, 3 s off).

F-T cycles method: One F-T cycle consists of 24 h at −18 °C followed by 24 h at 2–8 °C. Egg whites subjected to 0, 2, 4, 6 and 8F-T cycles were selected for parameter measurement. Preliminary experiments revealed that egg white subjected to ultrasonic pretreatment exhibited foaming properties comparable to those of fresh egg white after undergoing 8F-T cycles; and this cycle number can partially simulate repeated temperature fluctuations that frozen egg white products may experience during cold‑chain logistics and household storage. Consequently, 8F-T cycles were used as the experimental conditions.

2.3. Foam characteristics

2.3.1. Foaming ability (FA) and foam stability (FS)

Egg white was diluted to 25% of its original volume. 40 mL of the diluted egg white solution (denoted as V₀) was homogenized at 8,000 r/min for 1 min to ensure the solution was thoroughly foamed. The solution was transferred to a 100 mL graduated cylinder for volume measurement. Record the total volumes of liquid and foam at 0 min and 30 min as V₁ and V₃₀, respectively. The formulas for calculating FA and FS are as follows:

$$\text{FA=}\frac{\text{(}{\text{V}}_{\text{1}}\text{-}{\text{V}}_{\text{0}}\text{)}}{{\text{V}}_{\text{0}}}\text{×100\%}$$ (1)

$$\text{FS=}\frac{{\text{V}}_{\text{30}}\text{-}{\text{V}}_{\text{1}}}{{\text{V}}_{\text{1}}\text{-}{\text{V}}_{\text{0}}}\text{×100\%}$$ (2)

2.3.2. Microscopic observation

The microscopic foam morphology of various samples was visualized via an optical microscope (DMZL‑431088, Xiamen Jingyi Xingye Technology Co., Ltd., Xiamen, China) under 5 × magnification.

2.4. Rheological properties

2.4.1. Foam rheology

A high-speed shear rheometer (HR20, TA Instruments, USA) was used to investigate the rheological properties of the foam system. Following the method described by Zhan et al. [13], fresh foam prepared by a high-shear mixer (T25, IKA Works GmbH & Co. KG, Staufen, Germany), then it was immediately placed at the centre of the lower plate, with the gap between the two discs set at 2 mm. Measurements were taken after the foam had stabilized for 30 s. A steady-state shear test was conducted for 1,000 s at shear rates ranging from 0.05 s⁻1 to 1 s⁻1, and changes in shear stress (Pa) were recorded. Dynamic scanning was performed within the frequency range of 0.1 to 100 rad/s, and changes in the foam’s storage modulus (G') and loss modulus (G“) were recorded.

2.4.2. Large amplitudes oscillatory shear (LAOS) test

The EWP samples were subjected to LAOS testing. Prior to testing, the samples were placed between two plates and left to equilibrate for 2 min to eliminate any mechanical effects caused by loading. Silicone oil was used to reduce water evaporation during the measurement process. The LAOS tests were conducted at 25 °C, at a strain range of 1% to 1,000%, within a frequency of 1 Hz [14].

2.5. Particle size and ζ-potential

The EWP sample concentration was diluted to 0.1 mg/mL, followed by 120 s equilibration inside the chamber of a Malvern laser particle size analyser (ZS90, Malvern Instruments, Worcestershire, UK) for particle size and ζ‑potential measurement [15].

2.6. Protein molecular flexibility

Molecular flexibility is characterized by the sensitivity of protein to trypsin. Following the method described by Zhang et al. [16] with slight modifications, trypsin was dissolved in Tris‑HCl buffer (0.05 mol/L, pH > 8.0) to obtain a 1 mg/mL trypsin solution. Then, the 250 μL of trypsin stock solution was fully mixed with 4 mL of the 5 mg/mL protein solution, and the mixture was incubated at 37 °C for 15 min in a thermostatic water bath. The enzymatic reaction was terminated by the addition of 4 mL of 5% (w/v) trichloroacetic acid solution. Following centrifugation, the absorbance of the supernatant was measured at 280 nm using a microplate reader (Synergy H1, Bio Tek Instruments, USA). The obtained absorbance value (A) was used to characterize the molecular flexibility of the protein.

2.7. Surface hydrophobicity tests

Surface hydrophobicity was determined using the bromophenol blue method [16]. Thoroughly mix 400 μL of bromophenol blue solution (1 mg/mL) with 2 mL of the 5 mg/mL protein sample solution. Then centrifuged at 8,000 × g and 4 °C for 20 min. After dilution of the resulting supernatant, absorbance at 595 nm was measured and defined as A. Deionized water served as the blank control, with its absorbance recorded as A₁. The surface hydrophobicity of EWPs was calculated according to Formula (3).

$$\text{Surface hydrophobicity (}\mathrm{\mu }\text{g) = 200×}\frac{{\text{A}}_{\text{1}}\text{-A}}{{\text{A}}_{\text{1}}}$$ (3)

2.8. Free sulfhydryl content

Based on the method by Xie et al [17]. Ellman’s reagent was prepared by dissolving 40 mg 5,5′-Dithiobis (2-nitrobenzoic acid) (DTNB) in 10 mL Tris-Gly buffer (10.4 g Tris, 6.9 g glycine and 1.2 g ethylenediaminetetraacetic acid (EDTA), pH 8.0). The protein samples were dissolved in Tris-Gly-SDS buffer (45 mL Tris-Gly buffer mixed with 5 mL of 2.5% (m/v) aqueous SDS solution) to yield a final protein concentration of 10 mg/mL. Then 4 mL of protein sample were mixed with 0.04 mL Ellman’s reagent. The mixture was shaken and incubated at room temperature in the dark for 10 min. Absorbance of the supernatant was measured at 412 nm, with a blank without Ellman’s reagent or protein sample serving as the control. Free sulfhydryl contents were calculated from absorbance values based on a standard calibration curve established with reduced glutathione.

2.9. Interfacial adsorption kinetics measurement

The measurement of interfacial adsorption kinetics was carried out using an optical contact angle meter (OCA-25, Dataphysics, Germany) to determine the air–water interfacial tension of sample solutions [18]. The interfacial pressure is calculated using the Young-Laplace equation.

$$\pi ={\gamma }_0-\gamma$$ (4)

In this equation, γ₀ stands for the interfacial tension of pure water at the air–water interface, γ denotes the interfacial tension of the sample solution at the air–water interface, and π represents the interfacial pressure of the sample solution at the air–water interface.The Ward and Tordai equation was adopted to analyze the diffusion rate of sample molecules at the air‑water interface.

$$\pi ={\text{2C}}_0\text{KT}{\left(\text{Dt}/\text{3}.\text{14}\right)}^{\text{1}/2}$$ (5)

In the equation, C₀ represents the solution concentration of the bulk phase, K stands for the Boltzmann constant, T denotes the absolute temperature, D is the diffusion coefficient, and t refers to the adsorption time. The diffusion rate (K_diff) was calculated from the slope of the π-t^1/2curve.

$$\text{ln}\left[\left({\pi }_{\text{f}}-{\pi }_{\text{t}}\right)/\left({\pi }_{\text{f}}-{\pi }_0\right)\right]=-{\text{K}}_{\text{i}}\text{t}$$ (6)

In this equation, π_f is the final interfacial pressure at the air–water interface, π_t represents the instantaneous interfacial pressure at the air–water interface, π₀ stands for the initial interfacial pressure at the air–water interface, K_i and t represent the first-order reaction rate constant and time respectively. The rate constant was derived from the slope of the ln [(π_f – π_t)/(π_f – π₀)] versus t plot. Two distinct linear regions can be identified: the first arises from molecular permeation at the interface, yielding the permeation rate constant (K_P), while the second reflects interfacial molecular rearrangement, giving the rearrangement rate constant (K_R).

2.10. Interface proteomics

2.10.1. Preprocessing

The EWP, EWP8-FTC and UEWP8-FTC were mixed with distilled water in a ratio of 1:9 (v/v) and stirred at low speed (150 r/min) for 1 h to minimise foaming and protein denaturation. Subsequently, the mixture was agitated for 1 min at 8,000 r/min using a homogeniser to generate sufficient foam. A portion of the newly formed foam was immediately collected with a small spoon and stored in a centrifuge tube; this was placed in a foam box and rapidly frozen in liquid nitrogen for 30 min before being stored in a freezer at −80 °C for further analysis.

2.10.2. LC-MS/MS and database retrieval analysis

Enzymatic hydrolysates and digestion products were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Orbitrap Exploris 480 mass spectrometer coupled to an EASY-nLC 1200 liquid chromatography system. Peptide samples were reconstituted in loading buffer and loaded via autosampler onto a C18 analytical column (75 μm × 25 cm, 1.9 μm particle size, 100 Å pore size) for separation. Chromatographic separation was performed using a binary mobile phase system: mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in 80% acetonitrile, ACN), with a constant flow rate of 300 nL/min. Mass spectrometric data were acquired in data-dependent acquisition (DDA) mode, where each scan cycle consisted of one full MS survey scan followed by 20 MS/MS scans. Higher-energy collisional dissociation (HCD) was set at a collision energy of 30 eV. The quadrupole isolation window was 1.6 Da, and the dynamic exclusion time for repeated ion sampling was set to 35 s. Raw MS data were processed with MaxQuant software (v1.6.6) equipped with the Andromeda search algorithm, against the Uniprot.Proteome.Chicken.20201018.fasta reference proteome database. Trypsin/P was specified as the proteolytic enzyme with up to two missed cleavages allowed. For MS scans, the mass tolerance was 20 ppm for the initial search and 4.5 ppm for the main search; for MS/MS scans, the mass tolerance was set to 20 ppm. Carbamidomethylation of cysteine (C) was set as a fixed modification, while oxidation of methionine (M), protein N-terminal acetylation, and deamidation of asparagine/glutamine (NQ) were selected as variable modifications. All search results were filtered to achieve a false discovery rate (FDR) of ≤ 1% at both protein and peptide levels.

2.11. Characterization of protein structure

2.11.1. Circular dichroism (CD) spectrum

The J-810 circular dichroism spectrometer (JASCO Corporation, Japan) fitted with a quartz cuvette with an optical path length of 0.1 cm, the EWP solution was scanned in the far-ultraviolet region (wavelength range 190–250 nm) to obtain far-ultraviolet circular dichroism spectra; the content of α-helix, β-sheet, β-turn and random coil was subsequently simulated using BestSEL.

2.11.2. Fluorescence spectroscopy

The fluorescence intensity of EWP samples was used a fluorescence spectrophotometer (RF-5301, Shimadzu Corporation, Kyoto, Japan) at a concentration of 0.1 mg/mL. The excitation wavelength was fixed at 280 nm, with a slit width of 5 nm; the emission spectrum was scanned over the wavelength range of 300–500 nm, and the fluorescence intensity was recorded [16].

2.12. Microstructure observation

2.12.1. Scanning electron microscope (SEM)

The microstructure of the EWP samples was examined using an MSP-IS SEM (Hitachi Hi Tech Co., Ltd., Tokyo, Japan) [19]. A specified quantity of freeze-dried egg white powder was deposited onto the sample stage; after applying a thin layer of gold, images of the EWP samples were captured at an acceleration voltage of 20 kV.

2.12.2. Confocal laser scanning microscope (CLSM)

The microstructure of the samples was observed using CLSM (Leica TCS SP8X DLS, Leica Microsystems, Wetzlar, Germany). Mix 1 mL of the sample with a protein concentration of 0.1 mg/mL and 40 μ L of Nile blue solution (0.1% w/w), and stain them in the dark for 30 min [16].

2.13. Statistical analysis

All the experiments were executed in triplicate and data were presented as mean ± standard deviation. To assess the statistical significance of the means, a one-way analysis of variance (ANOVA) was conducted at a significance level of p < 0.05, followed by the Duncan test using IBM SPSS Statistics 27. Graphs were plotted with Origin 2022.

3. Results and discussion

3.1. Foaming properties analysis

3.1.1. FA and FS

FA and FS are core evaluation indicators for interfacial functional properties of EWP, where FA refers to the adsorption capacity of proteins at the air–water interface [20]. As shown in Fig. 1A, the FA of EWP8-FTC decreased by 41.35% after 8F-T cycles. The significant decline in FA of fresh egg white following F-T treatment can be attributed to impaired interfacial adsorption caused by protein aggregation. The large protein aggregates induced by F-T have a slow diffusion rate and poor interfacial spreading ability, and unable to rapidly form a continuous film at the air–water interface, thereby reducing the FA [21]. The FA of UEWP was remarkably higher than that of EWP. The growth in FA by ultrasonic treatment was attributed to the synergistic optimization of structural and physicochemical properties: ultrasonic treatment reduced particle size, thereby accelerating the diffusion rate of proteins to the interface. Moderate surface hydrophobicity and molecular flexibility strengthened the interfacial adsorption driving force and diffusion ability of interface adsorption, facilitating the rapid formation of foam [22]. The FA of UEWP8‑FTC was only 18.56% lower than that of UEWP. Moreover, it was significantly higher than that of EWP8‑FTC. This indicated that ultrasonic treatment can obviously mitigate the deterioration in the foaming properties of EWP during F-T cycles, suppress the large-scale aggregation and maintain the great interfacial adsorption and film-forming capabilities of the proteins, thereby making its foaming properties close to that of fresh egg white.

Fig. 1.

Effects of ultrasonic treatment on FA (A), FS (B), actual foam photographs (C) and micrographs (D) of freeze‑thawed egg white. (All photographs were acquired under identical light source, shooting distance and camera settings for visual comparison.).

FS refers to the capacity of a stable foam to resist gravitational drainage; it is affected by protein concentration, hydration status and molecular interactions [20]. As shown in Fig. 1B, EWP exhibited the lowest FS at 30.76%, while the FS of EWP8-FTC showed a slight improvement. It might be due to formation of a dense, rigid interfacial film resulting from excessive aggregation. Although membrane inhibited formation of foam, it also delayed the merging of bubbles and discharge of liquid [12]. After ultrasonic treatment, FS was further improved. The ultrasonic cavitation effect can disrupt large protein aggregates, reduce particle size and accelerate interfacial diffusion rates, enabling proteins to rapidly adsorb at the air–water interface and form a dense and continuous interfacial film, thereby significantly improving FS [5]. The FS of UEWP8-FTC was 32.69% higher than that of EWP8-FTC, illustrating that ultrasonic treatment simultaneously enhanced both the FA and FS of freeze-thawed EWP.

3.1.2. Microscopic foam

The optical microscope images of EWPs foam under different treatment conditions (Fig. 1C, D) clearly illustrated the differences in bubble size distribution, morphological integrity and uniformity within the foam system. The bubble size distribution in fresh EWP foam was uneven. At low magnification, both large and tiny bubbles were visible and the bubble distribution was dispersed. Under high magnification, the bubble walls were thin and irregular in shape. Some bubbles showed signs of coalescence and deformation. This reflected the interfacial film-forming ability of natural EWP was relatively weak, making it difficult to form a uniform and stable foam system. In comparison with EWP, the structural integrity of EWP8-FTC foam was obviously compromised. Under low magnification, numerous air bubbles can be observed, with the bubbles being sparse and unevenly distributed. At high magnification, the size of bubbles was significantly increased, and their shapes were highly irregular. There were obvious signs of rupture and fusion on the bubble walls. This was directly related to irreversible protein aggregation induced by F-T cycles and weakening the elasticity and toughness of interfacial film. This result was consistent with the conclusion in rheology that F-T cycles reduced the stability of foam structures. The microscopic morphology of UEWP foam has been significantly improved. Under low magnification, the bubbles sizes were generally small and size distribution was relatively uniform. When magnified at high magnification, the bubbles were regular, nearly circular, with uniform wall thickness and no evidence of bubble fusion or deformation. This indicated that ultrasonic cavitation effect enhanced the interfacial adsorption and film-forming capabilities of protein molecules, contributing to formation of a finer and more uniform foam system [23]. The bubbles morphology of UEWP8-FTC located between EWP and UEWP. It was mainly composed of medium-sized bubbles, with a relatively uniform size distribution. Under high magnification, the bubbles were regular in shape with moderate wall thickness. Although traces of protein aggregation were visible, no bubble rupture or large-scale fusion was observed, suggesting that ultrasonic treatment effectively mitigated the destructive effects of F-T cycles on the foam structure, thereby maintaining great foam structure stability throughout the F-T process.

3.1.3. Foam rheology analysis

As shown in Fig. 2A, the frequency sweep behavior of the foams indicated that the storage modulus (G') of all sample foams was consistently remarkably higher than the loss modulus (G“) within the angular frequency range of 0.1–100 rad/s, and both increase monotonically with rising of angular frequency. This suggested that behavior of each foam system was predominantly elastic, forming a three-dimensional protein network with a certain structural strength [24]. The G' of a foam was positively correlated with liquid film strength and inter-bubble interactions. Moreover, the higher the G', the denser the foam structure and the greater its deformation resistance [25]. As presented in Fig. 2, the order of magnitude of G' was UEWP > EWP > UEWP8-FTC > EWP8-FTC. The values of G' and G” for EWP were at an intermediate level. Due to the lower degree of cross-linking of its natural protein network compared to the ultrasonic treatment system, its elasticity and structural strength were consequently reduced.

Fig. 2.

Effects of ultrasonic treatment on frequency sweep (A) and shear stress (B) of freeze‑thawed egg white foam.

F-T cycles had a pronounced adverse effect on the rheological behavior of foams. The G' and G“ values of EWP8-FTC foam were the lowest among all systems, and the difference between G' and G” was the smallest. It indicated that formation and growth of ice crystals during F-T cycles severely disrupted the continuity of protein network, leading to irreversible protein aggregation, a marked decline in elastic properties, and a rise in the contribution of viscous behavior [26]. The G' and G“ values of UEWP foam were the highest across entire frequency range and exhibited the greatest increase with rising frequency, indicating that ultrasonic treatment obviously enhanced the elasticity and structural strength of foam network. This reflected ultrasonic cavitation caused degradation and unfolding of proteins, generating more hydrophobic groups and cross-linking sites, thereby constructing a denser and more uniform physical cross-linking network [14]. In contrast, the G' and G” values of UEWP8-FTC foam were similar to those of EWP, and were significantly higher than those of EWP8-FTC. This indicated that the ultrasound degraded protein network and effectively suppressed formation of large protein aggregates during F-T cycles, thereby maintaining the continuity of network. This endowed the foam with both great elasticity and a more smoother rheological response, which was in good agreement with its excellent FS.

The curve showing the variation of shear stress in the foam over time, as shown in Fig. 2B, further characterized the strength of its interfacial film. A higher shear stress indicates a denser interfacial film, a more uniform distribution of air bubbles, and a more stable foam structure. It can be seen that the shear stress of UEWP was consistently higher than that of EWP. Research by Chen et al. [18] demonstrated that ultrasound caused EWP molecules to denature and unfold; the exposed sulfhydryl groups facilitated protein adsorption and aggregation at the air‑water interface, forming a denser and more robust interfacial film, thereby enhancing the shear stress. Furthermore, UEWP8-FTC exhibited a higher shear stress than EWP, demonstrating that ultrasonic treatment conferred greater interfacial film strength to the protein foam.

3.2. Physicochemical property analysis

3.2.1. Rheological analysis of egg white

Fig. 3A presented the strain sweep rheological curves. The range of linear viscoelastic region (LVR) and structural resistance to deformation in different egg white foam systems are key indicators for evaluating the stability and mechanical strength of the protein network [14]. In the low-strain region, G' for all systems was remarkably higher than G“ and remained essentially stable, illustrating that they were within the linear viscoelastic region. When the strain exceeded a critical value, both G' and G” declined rapidly with strain, and the protein network disintegrated and ruptured under external force. Furthermore, although the G' value of EWP8-FTC egg white increased, the critical strain significantly reduced, indicating that protein network structure in the EWP8-FTC solution was fragile and prone to destabilization under external force [27]. Compared with EWP, the G' plateau value of UEWP solution increased, while the range of linear viscoelastic region narrows, and the modulus decayed more rapidly at high strains. This reflected that the dense network formed by ultrasound was highly elastic but lacked resilience to disturbances. This was also the underlying reason for its good initial foaming properties but poor long-term stability. UEWP8-FTC exhibited the highest G' plateau value and the broadest linear viscoelastic region, implying that the EWP after ultrasonic treatment and F-T cycling treatment, formed a stable protein network with both elasticity and toughness, possessing stronger resistance to deformation. This was consistent with its subsequent great interfacial film stability and strong resistance to the expulsion of liquid from the foam.

Fig. 3.

Effects of ultrasonic treatment on frequency sweep (A), particle size (B), ζ-potential (C) and molecular flexibility (D) of freeze‑thawed egg white. (**is p < 0.01, *is p < 0.05).

3.2.2. Particle size and ζ- potential analysis

Particle size is commonly used to characterize the aggregation and dispersion of protein molecules. A smaller particle size corresponds to better protein dispersion, which is conducive to the foaming properties of the protein [28]. As shown in Fig. 3B, the particle size of untreated EWP was approximately 488.5 nm. In contrast to EWP, the particle size of EWP8-FTC increased significantly to approximately 520 nm. This phenomenon can be explained by the fact that mechanical shear stress generated during F‑T cycles process by the formation and melting of ice crystals, which disrupted natural conformation of protein molecules and exposed the originally hidden hydrophobic groups. These exposed hydrophobic groups promoted irreversible protein aggregation via hydrophobic interactions, giving rise to larger protein assemblies and consequently increased particle size [6]. The particle size of UEWP was significantly reduced to 202.5 nm. It might be attributed to the fact that appropriate ultrasound disrupted the spatial conformation of protein molecules, while the turbulence and shear forces induced by cavitation intensifies intermolecular collisions, thereby effectively reduced particle size. Compared with UEWP, UEWP8‑FTC exhibited a slightly smaller particle size. This illustrated that ultrasonic treatment altered natural conformation of EWP, modifying the excessive aggregation behavior of proteins during F-T cycles. By keeping protein particle size at a relatively low level, it facilitated the protein to distributed rapidly at the air–water interface, thereby enhancing the foaming properties.

The ζ-potential is a key indicator of the stability of colloidal systems, and its absolute magnitude reflects the strength of electrostatic repulsion among protein aggregates [29]. In Fig. 3C, the EWPs in all treatment groups exhibited a negative ζ-potential, indicating the surface of EWPs were rich in amino acid residues with negative charges (such as carboxyl groups), which are consistent in their characteristics. Compared to EWP, the absolute value of ζ-potential in the EWP8‑FTC group was remarkably reduced. This change was primarily attributable to the protein aggregation induced by F-T, causing some surface charged groups to embedded within the aggregates. At the same time, the hydrophobic aggregation between protein molecules reduced the charge density per unit area, leading to a weakening of electrostatic repulsive forces within the system and a decline in colloidal stability [6]. The absolute value of the ζ-potential for UEWP was 6.7% higher than that for EWP. This might be due to the ultrasonic treatment induced full unfolding of protein molecules, thereby exposing abundant charged groups that are originally embedded within molecules. The surface charge density of protein significantly enhanced, and the electrostatic repulsion between particles strengthened, effectively preventing protein aggregation. This finding was corroborated by the significant reduction in the particle size of UEWP. The absolute ζ‑potential magnitude of UEWP8-FTC was approximately 22.8 mV. Although it was slightly lower than that of UEWP, it was still significantly higher than the value for EWP8-FTC. This indicated that ultrasonic treatment can productively counteracted the negative impact of F-T cycles on the surface charge of proteins, maintaining a high electrostatic repulsion ability of the system, thereby enhancing the colloidal stability of the freeze-thawed EWP. A study by [30] indicated that a moderate absolute value of the ζ-potential can inhibit bubble coalescence through electrostatic repulsion, thereby improving FS.

3.2.3. Molecular flexibility

The flexibility of protein molecules directly mirrors the capacity of to extend and move along their chains. Moderate increase in molecular flexibility can accelerate the diffusion rate at the interface, which is a key structural feature for improving the foaming performance [31]. Highly flexible proteins are able to enhance their own interfacial properties, promoting their assembly into interfacial films, thereby enhancing the interfacial properties of proteins [32].

As illustrated in Fig. 3D, the molecular flexibility of EWP8-FTC is significantly reduced compared to EWP, falling from 0.35 to 0.33. This might be correlated to the compressive force exerted by ice crystal growth throughout the F-T cycles, which disrupted the natural conformation of protein molecules and induced irreversible intermolecular cross-linking, ultimately leading to reduction in conformational flexibility of the polypeptide chains. The molecular flexibility of UEWP8-FTC obviously increased by 15.91%. This phenomenon derived from the mechanical forces induced by ultrasonic cavitation, which effectively disrupted protein aggregates and break secondary bonds. The molecular chains adopted a more extended conformation, thereby enhancing conformational adjustability [16]. Compared with EWP8‑FTC, UEWP8‑FTC displayed markedly greater molecular flexibility. This demonstrated that ultrasonic treatment optimized the initial conformation and aggregation state of protein, effectively counteracted the rigidification effect caused by the F-T cycle, and maintained great molecular flexibility of system.

3.2.4. Surface hydrophobicity

Surface hydrophobicity is widely adopted to assess the exposure extent of internal hydrophobic groups within proteins; it mirrors protein conformational alterations and governs their functional performance [33]. As shown in Fig. 4A, EWP8-FTC is the lowest among all groups. This phenomenon might be attributed to irreversible aggregation of protein molecules caused by growth of ice crystals and mechanical compression during repeated F-T treatment. As a result, hydrophobic residues were encapsulated within the aggregates. Compared with the EWP, the surface hydrophobicity of UEWP has significantly increased. It might be related to the mechanical shear force generated by ultrasonic cavitation, which disrupted the natural spherical folding structure of EWPs molecules and exposing a large number of hydrophobic amino acid residues that were originally hidden within the protein[16]. It was worth noting that the surface hydrophobicity of UEWP8-FTC was remarkably higher than that of EWP8-FTC. This can be explained by the fact that ultrasonic treatment causes moderate depolymerization of proteins and exposed of hydrophobic groups. During the subsequent F-T cycles, more uniform protein particles reduced excessive ice crystal growth and inhibited large-scale protein aggregation. It has confirmed that a moderate surface hydrophobicity, while ensuring interfacial adsorption driving forces, inhibited excessive protein aggregation and was an essential structural feature for optimizing the foaming properties [18]. Combining the experimental results of particle size and ζ-potential: ultrasonic treatment can reduce particle size and increase the absolute value of ζ-potential, thereby making the system more stable [34]; these conditions promoted rapid diffusion of protein molecules at the air–water interface [35].

Fig. 4.

Effects of ultrasonic treatment on the surface hydrophobicity (A) and free sulfydryl content (B) of freeze‑thawed egg white.

3.2.5. Free sulfhydryl content

Free sulfhydryl groups are a key structural indicator of the flexibility of EWPs molecules and their interfacial film-forming ability. Changes in their content directly reflect the degree of protein conformational unfolding, the breaking and formation of disulfide bonds, and are closely related to the covalent cross-linking strength of interfacial films [36]. As shown in Fig. 4B, the free sulfhydryl content of EWP8-FTC is remarkably decreased to 45.44 μmol/g compared with EWP. This might be attributed to the mechanical stress induced by ice crystal formation and thawing during freeze‑thaw cycles, which resulted in protein aggregation, sulfhydryl groups being buried or formation of disulfide bonds, thereby reducing the content of free sulfhydryl contents.[27]. Furthermore, oxidative reactions can also cause free sulfhydryl groups to form disulfide bonds [37]. The content of free sulfhydryl in UEWP increased significantly by 51.96% compared with EWP. This was primarily attributed to the shear forces and cavitation effect generated by ultrasonic treatment, which led to the breakage of disulfide bonds in protein molecules, converting bound sulfhydryl into free sulfhydryl, while the unfolding of the protein conformation further exposed internal sulfhydryl. Furthermore, UEWP8‑FTC exhibited markedly higher free sulfhydryl content compared with EWP8‑FTC, implying ultrasonic treatment of EWP can effectively regulate the oxidation process and cross-linking behavior of protein sulfhydryl during F-T cycles; this trend corroborated the findings regarding molecular flexibility.

3.3. Analysis of adsorption behavior at air–water interface

3.3.1. Kinetics of protein adsorption at the interface

As illustrated in Fig. 5A–B, the surface tension of EWP decreases by 31.65 mN/m, while that of UEWP decreases by 21.84 mN/m. This indicated that ultrasonic modification can significantly enhance the surface activity [1]. The interfacial pressure rose rapidly during the initial adsorption phase, indicating that all samples were capable of rapid adsorption at the interface. With increasing adsorption time, the quantity of protein adsorbed onto the interface nears saturation, thereby causing a gradual reduction in the rate of interfacial pressure rise. Among the groups, EWP8-FTC presented the minimum interfacial pressure magnitude, suggesting poorer surface activity, which was consistent with the results regarding foaming properties. Compared with EWP8-FTC, UEWP8-FTC presented a higher interfacial pressure and superior surface activity, demonstrating that ultrasonic treatment can mitigate the deterioration in surface activity among the F-T cycles of EWP.

Fig. 5.

The effect of ultrasonic treatment on the surface tension of proteins at the freeze-thawed egg white interface (A) and the interfacial pressure (π) of four samples at the air–water interface as a function of time (where K_diff represents the slope of the diffusion rate and t^1/2 is the square root of the adsorption time) (B-C); typical profiles of the penetration and rearrangement steps of samples at air–water interface (D).

The adsorption of protein at the air–water interface primarily involved three processes: diffusion, permeation and re-arrangement [18]. In the diffusion stage, protein molecules diffused from the bulk solution to the interfacial region. Proteins with a greater diffusion rate constant (K_diff) exhibited faster adsorption kinetics, thereby enhancing the FA. As shown in Fig. 5C, EWP had the highest diffusion rate (K_diff). Although the diffusion rate of UEWP is lower, its higher interfacial pressure resulted in better foaming property [38]. In the initial stage, the diffusion rate of UEWP8-FTC was greater than that of EWP8-FTC, and its interfacial pressure is higher, thereby demonstrating stronger FA. As time progressed, the diffusion rate of EWP8-FTC began to increase, indicating that its diffusion rate might exhibit a time delay, which was disadvantageous for rapid foaming process.

As illustrated in Fig. 5D, the slope of interfacial pressure–time curve during initial adsorption phase denoted the penetration rate constant (K_P), while during the second adsorption phase corresponded to the rearrangement rate constant (K_R). K_P reflects the rate of protein molecules penetrating into the air–water interface, while K_R represents the rate of molecular rearrangement after interfacial adsorption. Furthermore, the higher K_R facilitated protein molecular rearrangement and cross-linking at the interface, contributing to formation of compact and viscoelastic interfacial films [18]. The absolute values of K_P and K_R were highest for UEWP, suggesting that UEWP exhibited a stronger propensity toward interfacial penetration and rearrangement, which was crucial for FS. The interfacial activity is critical for FA, while the correlation between interfacial properties and FS warrants further exploration.

3.3.2. Interfacial proteomic analysis

Proteomics analysis revealed that identification of 2,213 peptides, corresponding to 310 proteins, of which 309 could be quantifiable (Fig. 6). The results of principal component analysis (PCA) clearly illustrated the overall differences and repeatability of different interface protein groups [39]. In the two‑dimensional PCA score plot, PC1 and PC2 account for 44.93% and 13.96% of total variance, with a cumulative explanatory power of 58.89%, effectively reflecting the primary variation characteristics of the interfacial proteome. The biological replicates within each group were closely clustered within the same confidence ellipse, indicating good experimental reproducibility, minimal intra-group error and reliable data. In terms of inter-group differences, the interfacial proteome profiles of different treatment groups exhibited varying degrees of separation: the confidence ellipses for the EWP8-FTC, EWP and UEWP8-FTC were completely separated with no overlap. This indicated that F-T treatment significantly altered the composition of the egg white interfacial proteins, with differences far exceeding intra-group repeatability error and thus being statistically significant. In contrast, the confidence ellipses of EWP and UEWP8-FTC groups showed partial overlap, indicating that the two groups were similar, and the overall differences in the protein profiles of the two groups were weaker than those of the group that only received F-T cycles.

Fig. 6.

Quantitative proteomic analysis results of interfacial proteins (identification results (A); principal component analysis (PCA) (B); interfacial proteins whose abundance changes are consistent with the variation trends of FA (C-F) and FS (G, H)).

The results of quantitative analysis of the top 20 proteins by abundance were presented in Table 1. Compared to EWP, the abundance of several key proteins was significantly reduced in the EWP8-FTC group, including Lysozyme C, Orosomucoid 1, Ovotransferrin, Protein TENP, Ovoinhibitor, Lipid-binding serum glycoprotein C-terminal domain-containing protein, Mucin-5B, Ovostatin, Ovodefensin A1 and Cilia and flagella associated protein 92. These proteins might be associated with the reduction in FA of EWP during F-T cycles. Compared with the EWP8-FTC, the abundance of several key proteins was elevated in UEWP8-FTC group, which was attributed to the ultrasonic treatment. The most significant change occurred in Lysozyme C. The content in the UEWP8-FTC was restored and significantly increased to 1.64 × 10⁸, which was 43.9% higher than that in the EWP8-FTC group, indicating that ultrasonic treatment effectively mitigated the loss of Lysozyme C at the interface caused by F-T cycles. Ovalbumin, a major foaming protein, was further enriched in the foam phase of the UEWP8-FTC group, with its abundance increasing significantly from 3.69 × 10⁸ in the EWP8-FTC group to 3.80 × 10⁸. Furthermore, Mucin-5B, Clusterin, Ovodefensin A1, Cilia and flagella associated protein 92 and Alpha-2-macroglobulin-like protein 1 also showed an upward trend. Ma et al. [40] found that ovalbumin contributed most significantly to FA, while ovotransferrin and ovomucoid also participated in the regulation of FA, with both acting synergistically with ovalbumin to enhanced foam formation efficiency. Lysozyme was the key regulatory protein for FS, with clusterin and ovoglobulin playing auxiliary stabilizing roles, which aligned with the results obtained in this study.

Table 1.

Comparison of differences in high-abundance proteins identified by interface proteomics in egg white samples from different groups.

NO.	Protein	EWP	EWP8-FTC	UEWP8-FTC
1	Ovalbumin	3.66 × 108b	3.69 × 108b	3.80 × 108a
2	Ovotransferrin	2.38 × 108a	2.33 × 108b	2.22 × 108c
3	Ovomucoid	2.07 × 108a	1.95 × 108b	1.92 × 108b
4	Lysozyme C	1.36 × 108b	1.14 × 108c	1.64 × 108a
5	Orosomucoid 1 (ovoglycoprotein)	6.65 × 107a	6.17 × 107b	6.29 × 107b
6	Ovotransferrin	6.94 × 107a	6.56 × 107b	6.65 × 107b
7	Ovalbumin-related protein Y	6.06 × 107a	6.00 × 107a	5.69 × 107b
8	Ovalbumin-related protein X (Fragment)	4.07 × 107a	4.12 × 107a	3.89 × 107b
9	Ovalbumin-related protein X (SERPINB14C)	3.49 × 107a	3.47 × 107a	3.38 × 107b
10	Protein TENP	3.28 × 107a	2.93 × 107b	2.67 × 107c
11	Ovoinhibitor	2.59 × 107a	2.46 × 107b	2.43 × 107b
12	Riboflavin-binding protein	1.74 × 107a	1.70 × 107a	1.57 × 107b
13	Lipid-binding serum glycoprotein C-terminal domain-containing protein	1.20 × 107a	1.12 × 107b	1.16 × 107ab
14	Mucin-5B	1.06 × 107b	1.05 × 107c	1.20 × 107a
15	Clusterin	1.01 × 107c	1.14 × 107b	1.15 × 107a
16	Ovostatin	9.56 × 106a	9.26 × 106b	9.01 × 106c
17	Ovodefensin A1	7.82 × 106a	7.08 × 106b	7.93 × 106a
18	Cilia and flagella associated protein 92 (putative)	7.58 × 106a	6.58 × 106b	7.33 × 106a
19	HEP21 sreted LY6/PLAUR domain containing, MHCB region	5.78 × 106b	7.60 × 106a	7.63 × 106a
20	Alpha-2-macroglobulin-like protein 1	5.30 × 106c	5.56 × 106b	6.10 × 106a

Different lowercase letters indicate significant differences between sample groups (p < 0.05).

Furthermore, the trends in the abundance of the four proteins: Lysozyme C, Mucin-5B, Ovodefensin A1 and Cilia and flagella associated protein 92, correspond clearly with the trends in FA. There was a downward trend from EWP to EWP8-FTC, indicating a reduction after a simple F-T cycles. Compared to EWP8-FTC, UEWP8-FTC showed an increase. This suggested that ultrasonic treatment was strongly associated with the regulation of adsorption behavior of these four key proteins at the interface, which might contribute to EWP change the foaming properties during F‑T cycles. It was reported that lysozyme has a certain promoting effect on the foaming properties of egg white [41]. It was also confirmed that the abundance of Mucin-5B was significantly correlated with the foam drainage rate[42]. Mucin-5B regulated protein aggregation, thereby altering foam properties. Currently, there is no reference reporting a direct association between Ovodefensin A1, Cilia, flagella associated proteins and foaming properties. Whether they exert a regulatory effect on foaming properties requires further confirmation.

Similarly, the trends in the abundance of two proteins: Clusterin and Alpha-2-macroglobulin-like protein 1, clearly corresponded to the trends in FS. The F-T cycles promoted increase in FS, and the FS further rose after ultrasound treatment. This suggested that ultrasonic treatment might regulate the FS of EWP during F-T cycles by modulating the adsorption behavior of these two key proteins at the interface. It was also confirmed that Clusterin and Alpha-2-macroglobulin-like protein 1 are key interfacial proteins responsible for maintaining long-term FS [43].

3.4. Structural analysis of protein

3.4.1. Protein secondary structure analysis

The secondary structure of protein is a key indicator of its molecular conformation, directly influence the surface activity, rheological behavior and functional properties of proteins [44]. Circular dichroism (CD) spectroscopy and secondary structure analysis systematically can reveal the regulatory effects of different physical treatment methods on the conformation of protein. CD spectral results were shown in Fig. 7A, indicating all samples exhibited strong positive absorption peak at 192 nm and a typical double negative absorption feature in the 210–220 nm region. These are characteristic signals of α-helix and β-sheet structures in proteins, suggesting that all groups retain an intact secondary structure framework. Compared with EWP, there were significant differences across the treatment groups in the intensity and shape of absorption peaks: UEWP8-FTC exhibited the highest intensity of characteristic absorption peaks, followed by UEWP and EWP, while EWP8-FTC showed the weakest absorption peak intensity. This indicated that UEWP8-FTC exerted the most significant regulatory effect on the secondary structure of protein, whereas the impact of pure F-T treatment was relatively limited. Fig. 7B presented the secondary structure of EWP consists of random coil (42%) and β-sheet (30%), along with 18% α-helix and 11% β-turn. In EWP8-FTC, the relative content of α‑helix decreased from 18% to 15%, while the β-sheet rose from 30% to 32%. There was no significant change in the random coil and β-turn contents. It indicated that growth of ice crystals throughout F-T cycles induced a transition of part of ordered α-helix structure to a β-sheet structure. The finding was ascribed to the gradual unfolding of protein conformations during freezing, which weakened intramolecular hydrogen bonding and reduced the stability of the α-helix structure. At the same time, it promoted the transition to other structures, thereby decreasing its proportion [6]. The α-helix content of UEWP increases from 18% to 19%, while the random coil content decreased from 42% to 41%, illustrating that the ultrasonic cavitation effect can induce moderate unfolding of the protein molecules, promote the transition of a small fraction of random coil into ordered α-helix. In contrast, the changes in the secondary structure of UEWP8-FTC were most pronounced: the α-helix content rose from 18% to 21%, the β-sheet content decreased from 30% to 27%, and the random coil content dropped to 41%. This indicated that ultrasonic treatment first promotes the depolymerization and conformational unfolding of protein molecules. Subsequently, the F-T cycles caused the protein molecules to rearrange and form a more ordered α-helical structure, while inhibiting excessive β-sheet aggregation. These conformational features conferred greater surface activity and structural stability [11]. However, this refolding of α-helix structures is not a typical phenomenon of the F-T cycles. Under certain specific conditions (such as an increase in temperature), a brief α-helix re-folding may occur in the short term [45].

Fig. 7.

Effects of ultrasonic treatment on circular dichroism spectra (A), secondary structure (B) and intrinsic fluorescence spectra (C) of freeze‑thawed egg white protein.

3.4.2. Fluorescence spectroscopy analysis

Fluorescence spectroscopy is a sensitive technique for probing tertiary structural alterations in EWP induced by various modification treatments. The intrinsic fluorescence intensity of proteins is predominantly determined by the polarity of local microenvironment around aromatic amino acid residues and their specific intramolecular interactions [46]. As shown in Fig. 7C, all samples exhibited characteristic emission peaks near 320–330 nm, which was consistent with the fluorescence properties of Trp residues embedded in the hydrophobic core of native EWP. Compared with EWP, EWP8-FTC exhibited a tendency for the peak position to shift towards the blue end of the spectrum, and its fluorescence intensity was significantly reduced. This might be ascribed to protein denaturation induced by F-T cycles, where the exposure and aggregation of hydrophobic groups, thereby leading to an irreversible change in the tertiary structure. The fluorescence intensity of UEWP was markedly lower than EWP. It might be related to the fact that ultrasonic treatment facilitated the unfolding of protein, exposing previously hidden tryptophan residues to the polar solution, thereby leading to a decrease in fluorescence intensity [47]. The fluorescence intensity of UEWP8-FTC was slightly lower than that of UEWP, but significantly higher than that of EWP8-FTC. It indicated ultrasonic treatment can significantly mitigate the structural stiffening and aggregation caused by F-T cycles, preserving a relatively extended conformation and high molecular flexibility, thereby suppressing the attenuation of egg white fluorescence intensity after F-T cycles and reducing damage to the protein tertiary structure during F-T cycles [6].

3.5. Microstructural analysis

Confocal laser scanning microscopy (CLSM) provides a clear visual representation of microscopic aggregation state of protein across various treatment regimes. As shown in Fig. 8A, the fluorescent signal in the EWP is sparse and dispersed, with only a few isolated small-sized aggregates, denoting that the native EWP was predominantly uniformly dispersed in a low-aggregation state [16], with relatively weak intermolecular interactions. However, multiple large and dense red fluorescent aggregates were observed in EWP8-FTC, which directly corroborated the destructive influence of F-T cycles on protein structure. The compression caused by ice crystals made the proteins denatured and exposed hydrophobic groups, thereby promoting irreversible intermolecular aggregation. The fluorescence signal distribution of UEWP was the most uniformly, with virtually no discernible large aggregates and only a small number of minute fluorescent spots, demonstrating the ultrasonic cavitation effect can effectively break down the original aggregation structure of protein and significantly improve the dispersion uniformity of system [48]. After a single F-T cycle, the fluorescence signal of UEWP8-FTC between that of UEWP and EWP8-FTC, with a small number of medium-sized aggregates and fine particles. However, the overall distribution remained relatively uniform, and no large and dense aggregates similar to those in EWP8-FTC were observed. This indicated that protein can partially mitigated from F-T-induced protein aggregation through ultrasonic treatment and maintain a relatively stable dispersion state.

Fig. 8.

Effects of ultrasonic treatment on the microstructure of freeze‑thawed Egg White Proteins (confocal laser scanning microscopy: A and scanning electron microscopy: B).

Scanning electron microscope images of protein foams under different treatment are illustrated in Fig. 8B. The microstructures of samples are characterized at magnifications of 100× and 500×, respectively. The results clearly illustrated the regulatory effects of ultrasonic cavitation and F-T cycles on the three-dimensional network architecture of protein foams. The microstructure of EWP foam exhibited characteristics of oriented, elongated macropores. At low magnification, the pores were arranged in an irregular, elongated pattern, with a non-uniform pore size distribution, poor pore connectivity and obvious structural fissures. Under high magnification, numerous irregular folds and fracture marks were visible, with significant variations in pore wall thickness [49]. After F-T cycles, the morphology of EWP8-FTC foam significantly deteriorated, exhibiting a loose and porous structure consisting of fragmented aggregates. Additionally, the repeated growth and melting of ice crystals continuously exerted mechanical pressure on the protein interface membrane, disrupting the natural conformation of protein molecules and inducing irreversible intermolecular aggregation. During the drying process, this structure collapsed and fractured, eventually forming a loose and fragmented structure. By comparison, the microstructure of the UEWP foam was significantly improved, exhibiting a uniform honeycomb-like porous structure [16]. The pore walls were smooth and of uniform thickness, with no obvious fractures or wrinkles. The microjets and shock waves generated by the ultrasonic cavitation effect, acting as the core acoustic chemical driving force, effectively disrupted the original aggregates of protein, enhancing the flexibility and dispersion uniformity of protein molecules. The microstructure of UEWP8-FTC exhibited a superior microstructure to EWP8-FTC, displaying a finer and sponge-like porous structure. The pore walls were rough, with the wall being the thinnest and having good uniformity, and no cracks or gaps were observed. This result demonstrated that ultrasonic treatment achieved the dispersion of protein molecules through the cavitation effect and enhances their flexibility, laying the foundation for the formation of a continuous interface membrane. Although the subsequent F-T cycles may cause some damage to the protein structure, since the protein molecules have been uniformly dispersed. The molecular cross-linking caused by F-T cycles was limited to local areas and will not lead to the formation of large aggregates or layered breakage.

4. Conclusion

This study investigated the detrimental effects of F‑T cycles on the foaming properties of EWP and clarified the protective mechanisms of ultrasonic treatment via physicochemical, rheological, structural, microstructural and interfacial proteomic analyses. Ultrasonic treatment mitigated the degradation of EWP foaming properties caused by F-T cycles. Ultrasonic cavitation optimized particle size distribution, enhanced molecular flexibility and free sulfhydryl content, and modified protein secondary structure. Interfacial proteomics further revealed that ultrasound selectively promoted the interfacial enrichment of key functional proteins (Lysozyme C, Mucin-5B, etc.). Collectively, ultrasonic treatment improved the foaming properties of freeze‑thawed EWP through synergistic effects: modulating protein spatial conformation, restraining aggregation‑denaturation, and tuning the interfacial distribution of critical proteins. This study elucidated the molecular regulatory mechanisms underlying the interfacial adsorption of EWPs It provides important theoretical support and technical guidance for maintaining the storage quality of frozen egg white, modifying proteins to resist freeze damage, and the high‑value processing of egg products, and holds promising prospects for industrial application. Although the laboratory‑scale ultrasonic treatment (360 W, 10 min) holds potential for batch and continuous liquid egg white processing, practical challenges including localized overheating and heterogeneous energy distribution in large‑volume systems. These issues need to be addressed through equipment optimization and process improvement to achieve industrialized production of egg products.

CRediT authorship contribution statement

Qian Zhao: Writing – original draft, Investigation, Data curation. Chaoya Zhao: Visualization, Supervision. Xun Huang: Resources. Xiaole Xiang: Software. Yingmei Wu: Software. Yingqun Wu: Supervision. Xiaojuan Song: Software. Kai Dong: Supervision. Lan Liu: Resources. Shaoxiao Zeng: Software, Methodology. Qun Huang: Resources.

Declaration of competing 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.