Enhancing salt-induced gelation of egg yolk granules through pH-ultrasound combined treatment: A physicochemical and microstructural analysis

Graphical abstract

Highlights

• •pH5+150W treatment resulted in optimal gelation properties of EYG.
• •Significant improvements in oil exudation (2.83%) and good sandiness (62.07%) at pH5+150W.
• •The pH5+150W treatment created a more uniform matrix with heightened electrostatic repulsion between oil droplets.
• •Significant changes in the α-helix and β-sheet content of EYG after pH-HIU treatment.

Abstract

Salted duck eggs are a popular food product, but their high salt content and uneven salt distribution can reduce acceptability. This study investigated the effects of pH-high-intensity ultrasound combined treatment on the salt-induced gelation properties of egg yolk granules. The results showed that the pH5 + 150 W treatment group exhibited the best physical and gelation properties, with the smallest particle size (1597.33 nm), optimal dispersibility (PDI 0.29), and good stability. The gelation properties of this group also demonstrated excellent springiness (0.30 mm), cohesiveness (0.56), and gumminess (0.05 N). Furthermore, the pH5 + 150 W group had the highest water holding capacity of 97.42 % and relatively high hydrophobicity (173.39 μg). Notably, it also showed high oil exudation (2.83 %) and good sandiness (62.07 %). The pH5 + 150 W led to a significant redshift of the fluorescence peak at 335 nm and an enhancement of the peak intensity at 562 nm compared to the control group. Structural characterization revealed a more ordered protein structure and a uniform gel structure with enhanced electrostatic repulsion between oil droplets. Secondary structure analysis of the proteins showed a significant reduction in α-helix and β-sheet, while β-turn and random coil increased. In summary, the pH5 + 150 W treatment displayed the best gelation properties, providing theoretical guidance for enhancing the processing performance of yolk and expanding their application in the food industry.

1. Introduction

Salted duck eggs, a traditional Chinese preserved egg product, derive their characteristic quality from the gelatinization of the egg yolk during the salting [1]. Salted duck eggs are traditionally produced using two primary methods: salt-immersion method and coating with a salt-mud mixture (smearing method). The salt-immersion method has become the dominant production method, with typical processing times ranging from 30 to 40 days [2]. During the salting process, the high salt concentration of the curing solution progressively penetrates the yolk, disrupting its inherent stable sol–gel state and inducing a transformation into a firm and elastic gel [3]. Subsequently, high-temperature treatment further refines this gel structure, promoting lipid mobilization and contributing to the distinctive “loose, sandy, and oily” texture [4]. Egg yolk granules (EYG), comprising 19 %–23 % of the dry matter in egg yolk, are the precipitation of yolk components after dilution and centrifugation [5]. Recent studies revealed that the salt-induced gelation of EYG contributes to the characteristic grainy texture of salted duck egg yolks, which is a crucial indicator of the maturity and quality of salted duck eggs [6].

EYG, characterized as spherical entities with diameters ranging from 0.5 to 1.5 μm, predominantly comprise high-density lipoprotein (HDL), low-density lipoprotein (LDL), and phosvitin. Notably, the abundant phosphatidylserine within phosvitin facilitates the formation of “calcium-phosphorus bridges” via the binding with Ca²⁺. These bridges ensure a robust association between HDL and yolk hyperphosphoproteins, which are integral for stabilizing the EYG structure [7]. The high concentration of Na⁺ replaces Ca²⁺ and disrupts the “calcium-phosphorus bridge. This disruption leads to the depolymerization of EYG, facilitating the release, aggregation, and extensive cross-linking components to form EYG gels [8]. The gel properties of EYG are intricately linked to various factors, including pH, temperature, ionic strength, and others [9]. It was found that pH value significantly affected the gel properties of EYG induced by salt. Typically, EYG exhibits a stable spherical structure, which is maintained by calcium phosphate bridges. However, when subjected to fluctuations in environmental pH, the phosphocalcic bridges are disrupted, ultimately leading to the denaturation of EYG structure. The dissociated EYG contains soluble proteins and lipids, which consequently alter their gelation properties [10]. Therefore, the functional properties of EYG gels can be modulated by adjusting the pH, as this facilitates hydrophobic aggregation, consequently enhancing the structural integrity of the proteins.

High-intensity ultrasound (HIU) technology, known for its cavitational and mechanical effects, can disrupt peptide bonds, induce structural alterations, and enhance the properties of gels by disrupting non-covalent interactions [11]. Numerous studies have explored the application of HIU technology in improving the properties of egg yolk gel. For instance, the HIU treatment triggers cavitation effects that facilitate the disruption of protein aggregates, which leads to the cleavage of non-covalent and covalent bonds. This mechanical stress, induced by the continuous formation and collapse of microbubbles, may result in the depolymerization of EYG, thereby reducing their average particle size [12]. Xie et al. [13] demonstrated that the cavitation effect can alter the aggregation state of EYG by disrupting “phospho-calcium bridges,” leading to a reduction in particle size and conformational rearrangement. Concurrently, the rupture of these “calcium-phosphorus bridges” results in the partial solubilization of Ca²⁺ and the phosphoprotein fraction from the EYG, which enhances the surface charge density and the electrostatic repulsion between particles. Furthermore, the cavitation effect also promotes the release of neutral lipids from lipoproteins, which increases the surface hydrophobicity of the EYG. These findings indicate that HIU treatment can induce a degree of depolymerization of EYG, resulting in alterations to their gel properties and the release of certain substances from EYG. However, limited research has been conducted on enhancing the salt-induced gel properties of EYG through combined pH and HIU modification. Therefore, further investigation in this area is warranted.

In this experiment, pH and HIU treatments were applied to assess the salt-induced gel properties of EYG. The parameters included particle size, zeta potential, textural attributes, surface hydrophobicity, water holding capacity (WHC), cooking loss, gel strength, microstructural morphology, infrared spectroscopy analysis, intermolecular forces, and endogenous fluorescence were measured to investigate the underlying mechanisms.

2. Materials and methods

2.1. Materials

Fresh duck eggs were sourced from Jiajiali Supermarket (Taigu District, Jinzhong City, Shanxi Province). Sodium chloride, trimethylaminomethane, sodium hydroxide, hydrochloric acid, glycine, ethylenediaminetetraacetic acid, bromophenol blue (BPB), potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, 2-nitrobenzoic acid, Nile red, Nile blue, isopropanol, potassium bromide, and urea were all obtained from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

2.2. Preparation of EYG

The egg yolk liquid was collected by piercing the membrane with a bamboo stick after separating and discarding the egg white. The egg yolk liquid was diluted with distilled water at 1:1 and thoroughly mixed at 4°C. Subsequently, the mixture was centrifuged at 10,000 r/min for 30 min at 4°C using a centrifuge (HC-2518R, USTC Zonkia Scientific Instruments Co., Ltd, Anhui, China). The resulting precipitate was collected and the procedure was repeated to obtain the EYG.

2.3. Treatment of salted EYG solution by combined pH and HIU treatment

EYG was dissolved in a 2 % NaCl solution at a ratio of 1:10, and the pH was adjusted to 3.0, 4.0, and 5.0 using 1 mol/L HCl solution with a pH meter (PHB-4, Mettler-Toledo Instruments Co., Ltd, Shanghai, China). Ultrasonication was performed using a probe-type HIU processor at a fixed frequency of 40 kHz and 25°C, with HIU power levels of 150 W and 250 W for a fixed duration of 20 min, respectively [13]. Each treatment condition was replicated three times, and the resulting solutions were combined to form one sample group. Following treatment, samples were transferred to sealed glass jars and subjected to a 60 h salting period at 4°C. The solution was agitated for 5 min at 12-h intervals.

2.4. Determination of WHC

The EYG solution was subjected to magnetic stirring for 4 h using a magnetic suspension stirrer (79-1, Changzhou Guohua Electric Appliance Co., Ltd, Jiangsu, China), weighed and recorded as W₁ before centrifugation. The precipitate was weighed and recorded as W₂ after centrifugation at 3000 r/min for 30 min. The WHC was determined using the following formula:

$$WHC\left(\%\right)=\frac{W_2}{W_1}\times 100$$

2.5. Determination of particle size and zeta potential

The particle size of EYG solution was determined using a nanoparticle size and potential analyzer (Nano-ZS90, Malvern Instruments Co., Ltd, Malvern, UK) according to the method of Du et al. [14]. The original solution was diluted five-fold with phosphate-buffered saline solution and filtered before use. The instrument was operated with water as the dispersion medium, and the measurement temperature was set to 25°C. A 1 mL sample was drawn into the sample cell using a pipette and each sample was measured in triplicate.

2.6. Determination of free and total sulfhydryl groups

EYG (0.5 mL, 1 mg/mL) was mixed with 4.5 mL buffer solution (8 M urea, 0.01 M ethylene diamine tetraacetic acid (EDTA), 0.1 M K₂HPO₄, pH6.0) to determine free sulfhydryl groups. Subsequently, 100 µL of Ellman's reagent (0.01 M 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB), 0.01 M KH₂PO₄, pH6.0) was added to the mixture, and the solution was vortexed for 1 min to ensure thorough mixing [13]. To determine total sulfhydryl groups, 0.5 mL of EYG solution (1 mg/mL) was combined with 2.5 mL of buffer solution (12.02 g urea, diluted to 25 ml with glycine buffer). After centrifugation at 4000 r/min for 15 min, 20 µL of Ellman's reagent was added [15]. The mixtures were incubated in the dark for 30 min, and absorbance was measured at 412 nm. The free and total sulfhydryl content was calculated using the following equation:

$$Sulfhydrylcontent\left(nmol/mg\right)=\frac{A_{412}\times 1000000}{1.36\times 10000\times C}$$

Where A₄₁₂ stands for the absorbance value of EYG sample solution; C stands for solution protein concentration; 1.36 × 10000 is the molar absorption coefficient.

2.7. Determination of surface hydrophobicity

1 mL of EYG solution with a 1 % protein concentration was combined with 200 µL of BPB (1 mg/mL). After centrifugation at 2000 r/min for 15 min, the supernatant was diluted sevenfold with 2 % NaCl solution. The absorbance was then measured at 595 nm using a UV/Visible spectrophotometer (WFJT200, Shanghai Meiputa instrument Co., Ltd, Shanghai, China). A blank solution consisting of 1 mL 2 % NaCl and 200 µL BPB was used for calibration. The surface hydrophobicity is calculated according to the formula [16].

$$BPBbound\left(\mu g\right)=\left(\frac{A_{Control}\mathit{-}{A}_{Sample}}{A_{Control}}\right)\times 100$$

2.8. Preparation of thermally irreversible EYG gel

The EYG solution was subsequently transferred into a 200 mL conical flask. The flask was sealed with a plastic wrap and immersed in a 90°C thermostatically controlled water bath (HH-S8, Beijing Keweiyongxing Instrument Co., Ltd, Beijing, China) for 20 min to form the EYG gel. Subsequently, the EYG gel was lyophilized into a powder for subsequent analysis.

2.9. Texture profile analysis (TPA) of EYG gel

The TPA of EYG gel samples was evaluated using a texture analyzer (TA-XT plus, Tianjin Chuangxing Electronic Equipment Manufacturing Co., Ltd, Tianjin, China). EYG gel samples measuring 2 × 2 × 1 cm were placed on the texture analysis platform. The parameters were adjusted according to the methodology of Xu et al. [17], with an initial force of 0.1 N, probe rebound height of 15 mm, extrusion distance of 1 mm, pre-test speed of 60 mm/min, post-test speed of 200 mm/min, and a 2-second pause between compressions. Hardness, springiness, cohesiveness, and gumminess data were recorded for each sample, and three parallel measurements were conducted. The gel strength was determined using the following formula:

$$Gelstrength\left(g\times mm\right)=Ruptureforce\times Displacementdata$$

2.10. Determination of cooking loss

A 10 mL of EYG was placed into a sealed tube and weighed, with the weight recorded as m₁. The sample was heated to 90°C for 20 min in a water bath, then cooled to room temperature after excess moisture removal. It was weighed again and recorded as m₂. The cooking loss was calculated using the following formula [18]:

$$\mathit{Cooking}loss\left(\%\right)=\left(\frac{m_1-m_2}{m_1}\right)\times 100$$

2.11. Determination of oil exudation

EYG gel powder (1 g) was mixed with 5 mL distilled water and stirred for 15 min. The mixture was centrifuged at 5000 rpm for 15 min, and the supernatant was dissolved in 5 mL hexane/isopropanol (3/2, v/v). The lipid layer was separated and collected in a freeze-drying dish, and its weight (m_1, representing total lipid content) was recorded. After evaporating the solvent in a fume hood, the freeze-drying dish was removed, cooled, and dried. The weight of the remaining sample was recorded as m₂ (representing free lipid content). The oil exudation was calculated as the ratio of free lipid content to total lipid content according to the following formula [19]:

$$\mathit{Oilexudation}=\frac{m_2}{m_1}\times 100$$

2.12. Determination of sandiness

EYG gel powder (0.5 g, G₁) was dispersed in 10 mL of petroleum ether by stirring for 3 min to achieve complete dispersion. The stirred salted EYG was then sieved through a standard sieve with a diameter of 0.1875 mm, with the initial weight of the sieve recorded as G_2. After sieving, the EYG remaining on the sieve was rinsed with 20 mL of petroleum ether to ensure the collection of all residues. The rinsed standard sieve and the retained EYG were placed in a steam bath at 100°C for 6 min until a constant mass was achieved. The standard sieve was then removed and placed in a dry environment to cool down to room temperature before being weighed again, and the obtained mass was recorded as G₃. The sandiness was calculated according to the following formula [20]:

$$Sandiness\left(\%\right)=\left(\frac{G_1-\left(G_3-G_2\right)}{G_1}\right)\times 100$$

2.13. Chemical force analysis of EYG gel

Three solutions were prepared: (a) 0.05 mol/L NaCl, (b) 0.6 mol/L NaCl, and (c) 0.6 mol/L NaCl + 1.5 mol/L urea. Each solution (10 mL) was mixed with 2.0 g of EYG gel powder and homogenized at 8000 rpm for 1 min. After shaking at 4°C for 1 h, the mixtures were centrifuged at 20,000 × g for 15 min at 4°C. The supernatants were collected, and the protein concentrations were determined using the Bradford protein assay to calculate the contribution of different chemical forces. The non-specific force was determined as the protein solubility in solution (a), ionic bonding was calculated as the difference in protein solubility between solution (b) and solution (a), and hydrogen bonding was calculated as the difference in protein solubility between solution (c) and solution (b). The final results were expressed as a percentage (%) of the contribution of the respective chemical forces [21].

2.14. Endogenous fluorescence analysis

The EYG solution was diluted with 0.01 mol/L phosphate buffer (pH 7), and fluorescence was measured using a fluorescence spectrophotometer (F-7000, Hitachi Co., Ltd., Tokyo, Japan). Fluorescence spectra were recorded from 295 to 600 nm with an excitation wavelength of 280 nm. Synchronous fluorescence spectra were recorded from 250 to 600 nm with an excitation wavelength of 230 nm. Both emission and excitation slit widths were set at 5 nm. The voltage was maintained at 500 V, and each spectrum was scanned twice [22].

2.15. X-ray diffraction (XRD)

The ground EYG gel powder was placed in an XRD (Rigaku Dlmax-2500, 40 KV, 100 mA, Beijing Advance Science & Technology Co., Ltd, Beijing, China), compressed and flattened, and then scanned from 0° to 60°. The experimental parameters included a 1° divergence slit, a 0.1 mm receiving slit, and Cu/Ka radiation at a scanning speed of 3°/min. The instrument was operated at a voltage of 40 kV and a current of 40 mA [23].

2.16. Fourier transform infrared spectrometry (FTIR)

The EYG gel powder sample was mixed with potassium bromide powder, pressed into a transparent sheet, and scanned via a FTIR spectrometer (Thermo Nicolet Nexus 470, Thermo Electron Scientific Instruments Co., Ltd, Madison, Wisconsin, USA) within the spectral range of 4000 to 400 cm⁻¹. Spectral data were analyzed using OMNIC 6.0 software. Initial spectra were smoothed to obtain second-derivative infrared spectra, and all samples were analyzed in triplicate to ensure accuracy [24].

2.17. Circular dichroism spectroscopy (CD)

Secondary structure analysis of the EYG gel sample was performed using a CD (Jasco 810, JASCO, Co., Ltd, Tokyo, Japan) spectrometer. A 0.25 mg/mL sample suspension was placed in a 0.1 cm path-length quartz cuvette. Measurements were conducted at 25°C with a scanning speed of 50 nm/min, a response time of 4 s, and a bandwidth of 1.0 nm. The obtained spectra were subjected to quantitative secondary structure analysis using the Yang-Usjwr software.

2.18. Confocal laser scanning microscopy (CLSM)

0.5 mL of EYG solution was mixed with 50 μL of a mixed dye solution (prepared by combining Nile Red (1 mg/mL in ethanol) and fluorescein isothiocyanate (1 mg/mL) in a 1:1 ratio). Following a 30-s mixing period, the solution was incubated in the dark for 6 h to allow for staining. Subsequently, a droplet of the stained dispersion was placed onto a microscope slide, followed by the placement of a coverslip. The observation was performed using a CLSM (Olympus FV12000MPE, Olympus Co., Ltd, Beijing, China). For lipid analysis, the excitation wavelength was set to 568 nm and the emission wavelength to 600 nm. Conversely, for protein analysis, the excitation wavelength was set to 488 nm and the emission wavelength to 520 nm. Fluorescent images of EYG solution were acquired via CLSM, and the distribution of proteins and lipids was subsequently analyzed [25].

2.19. Scanning electron microscope (SEM)

The microstructure of the EYG gel was observed using SEM (JEM1400, JEOL Co., Ltd, Tokyo, Japan). A small amount of EYG gel powder was mounted on an aluminum stub using double-sided adhesive tape and sputter-coated with gold for conductivity. Microstructural images were captured at magnifications of 10,000 × using digital image processing software [26].

2.20. Data analysis

The experiment was replicated three times, and the mean and standard deviation of each indicator were computed using Microsoft Excel 2019. The data were expressed as mean ± standard deviation. Subsequently, a one-way ANOVA (analysis of variance) was performed on the data using SPSS 22, with a significance level set at 0.05. The results were visualized using Origin 2018.

3. Result and discussion

3.1. Effects of combined pH and HIU treatment on WHC, particle size and zeta potential value of EYG

The effect of different treatment conditions on WHC is represented in Fig. 1A. There was a significant enhancement in WHC for the pH5 + 150 W, pH4 + 300 W, and pH5 + 300 W groups compared to the control, with the pH5 + 150 W group exhibiting the highest WHC at 97.42 %. This phenomenon was likely due to the exposure of hydrophobic groups post-HIU treatment, which facilitated gel formation. This observation was consistent with previous studies, such as Zhang et al. [27], who reported a 3.66 % increase in WHC of soybean protein isolate after sonication. Their microstructural analysis revealed a denser gel network with reduced pore size, contributing to the enhanced WHC. Similarly, Zhang et al. [28] observed a positive correlation between HIU power and WHC in soybean protein isolate, with a nearly one-third increase in WHC observed at higher power levels. This effect was attributed to the loosening of the protein structure, reduction in particle size, and exposure of hydrophobic groups induced by HIU. These changes facilitated gel formation and enhanced the absorption and binding of lipid substances, ultimately leading to increased WHC. The WHC decreases with lowering pH, which was mainly due to the isoelectric point (PI) of EYG. As pH decreases, the net charge on the protein reduces, weakening electrostatic repulsion and enhancing intermolecular interactions. At a pH value close to the pI, electrostatic interactions are minimized, promoting protein aggregation and expulsion of water molecules, thereby reducing WHC. This trend suggests that the optimal pH for maximizing WHC is 5 [29].

Fig. 1.

Effects of combined pH and HIU treatment on WHC, particle size and zeta potential value of EYG. (A) WHC; (B) particle size and dispersion PDI; (C) zeta potential.

pH-assisted HIU treatment significantly reduced the average particle size of the EYG solution (Fig. 1B). Notably, the pH3 + 150 W group exhibited the smallest average particle size (1576.33 nm), significantly lower than the control group. Cavitation-induced shear forces generated by HIU contribute to this phenomenon, which disrupted the hydrogen bonds within the protein structure, consequently reducing particle size [30]. Consistent with our findings, Geng et al. reported a 37.3 % decrease in the average particle size of EYG after HIU treatment, which was accompanied by changes in the degree of depolymerization and functional characteristics of EYG subjected to varying intensities of HIU [31]. Lipid particles within EYG are typically encapsulated by protein membrane. During the gelation process, smaller fat particles disperse more uniformly, facilitating the formation of a fine, homogeneous protein network structure, thereby enhancing the overall stability and mechanical strength of the gel. This phenomenon is attributed to the increased contact points between proteins facilitated by smaller particles, which elevates the crosslinking density of the network, consequently enhancing the elasticity and stability of gel [32]. The polydispersity index (PDI) measured particle size distribution within a dispersion system, with lower PDI values indicating a more homogenous particle size distribution [33]. The pH value significantly affects the surface charge of proteins. A smaller PDI observed at neutral pH compared to acidic pH conditions can be attributed to the altered electrostatic interactions between protein molecules [29]. The PDI value of the sample treated with pH5.0 was lower than that treated with pH3.0 after 150 W treatment of HIU. Specifically, the pH5 + 150 W group exhibited the lowest PDI value of 0.29, suggesting optimal particle dispersion. Therefore, the pH5 + 150 W group demonstrated more uniform dispersion, smaller particle size, and enhanced stability.

The zeta potential reflected the stability of protein solutions and influenced the gel properties of proteins. Higher absolute zeta potential values indicated stronger electrostatic repulsion between proteins, resulting in enhanced stability of the protein solution [34]. The zeta potential of EYG solutions treated with different pH and HIU exhibited negative surface charges (Fig. 1C). At a constant HIU power, the absolute value of the zeta potential increased with rising pH values. Notably, the pH5 + 150 W group demonstrated an absolute zeta potential of 8.85 mV, significantly exceeding that of the control group of 4.17 mV. This suggests enhanced stability for the pH5 + 150 W group. Previous studies established that a higher zeta potential in protein solutions correlated with reduced intermolecular adsorption forces, increased electrostatic repulsion, and decreased aggregation propensity, all of which contributed to system stability [35]. The effective charge of the particle surface primarily determines the dispersion and aggregation of particles. Generally, a higher absolute zeta potential value indicates reduced flocculation tendency, while a lower absolute zeta potential promotes flocculation [36]. A higher absolute zeta potential value suggests decreased electrostatic repulsion, indicating that EYG was more prone to gel formation [37]. Jiang observed that HIU would make the particle size of black bean protein smaller, increase the zeta potential, and enhance the stability. HIU could increase the negative charge on the surface of the protein, enhance the electrostatic repulsion between the particles, disrupt the existing protein aggregation, and inhibit the further aggregation of the protein [38]. This study's findings aligned with Geng et al.'s observation of increased EYG microenvironment polarity, strengthened internal hydrogen bonding, and enhanced zeta potential after HIU treatment [30].

3.2. Effects of combined pH and HIU treatment on free sulfhydryl content, total sulfhydryl content and surface hydrophobicity of EYG

As shown in Fig. 2A, at constant HIU power, increasing pH led to an initial rise followed by a decrease in free sulfhydryl content. In contrast, total sulfhydryl content exhibited a gradual upward trend. Increasing HIU power at fixed pH resulted in a decline in free sulfhydryl groups and an increase in total sulfhydryl groups. Specifically, both the pH5 + 300 W and pH5 + 150 W groups exhibited significantly higher total sulfhydryl content (40.83 μmol/g and 37.64 μmol/g, respectively) compared to the control group (15.98 μmol/g). Furthermore, the pH5 + 150 W group demonstrated a higher concentration of free sulfhydryl groups (14.37 μmol/g) than the control group (7.51 μmol/g). These findings were consistent with previous research suggesting a correlation between thiol content and gelation rate. Xie et al. observed a positive correlation between total sulfhydryl content and gelation rate, particularly when the concentration of free sulfhydryl groups was reduced [13]. This implied that the pH5 + 150 W group, with its elevated total sulfhydryl content and reduced free sulfhydryl groups, was likely to exhibit a faster gelation rate. HIU-induced cavitation enhanced protein molecule interaction and cross-linking, facilitating the conversion of free sulfhydryl groups into disulfide bonds [39]. This was further supported by the findings of Fu et al., who observed a significant increase in thiol transitions under high HIU intensity (500 W), indicating the formation of a denser and more organized gel structure in ultrasonically treated soy protein isolate [40].

Fig. 2.

Effects of combined pH and HIU treatment on free sulfhydryl content, total sulfhydryl content and surface hydrophobicity of EYG. (A) Free and total sulfhydryl content; (B) surface hydrophobicity.

The combined pH and HIU treatment significantly increased surface hydrophobicity compared to the control group (Fig. 2B). A positive correlation was observed between pH and hydrophobicity at a constant HIU power. The highest hydrophobicity (173.39 µg) was observed with the pH5 + 150 W treatment, which was substantially higher than that of the control group (116.80 µg). Higher values of surface hydrophobicity imply higher degree of protein unfolding. The unfolding of proteins is promoted in acidic conditions. Especially near the protein isoelectric point, the net charge is zero, so the protein unfolded as much as possible with elevated surface hydrophobicity [41]. The pH5 + 150 W group significantly enhanced EYG surface hydrophobicity, suggesting that HIU treatment generally increased EYG surface hydrophobicity by disrupting calcium-phosphate bridges within the EYG [42]. The unique cavitation effect of HIU facilitated the rearrangement of protein conformations, forcing buried hydrophobic groups to the surface and consequently augmenting the hydrophobic characteristics of the proteins [43]. Furthermore, studies indicated that HIU treatment might induce partial unfolding of lipoproteins within the EYG, exposing polar and hydrophilic residues such as aspartic acid and glutamic acid. This exposure increased the surface charge of the lipoproteins, ultimately contributing to the enhanced hydrophobicity of the EYG [13].

3.3. Effects of combined pH and HIU treatment on gel texture of EYG

The gels subjected to pH sonication complex treatment exhibited a significant overall decrease in hardness, springiness, cohesiveness and gumminess viscosity compared to the control. This decline could be attributed to the dissociation of peptide chains in the proteins following HIU treatment, which weakened cohesive forces, exposed hydrophobic groups, and reduced hardness. The intermolecular interactions were also diminished, resulting in decreased adhesive viscosity [44]. The gelation properties of pH5 + 150 W group also demonstrated excellent springiness (0.30 mm), cohesiveness (0.56), and gumminess (0.05 N). Yu et al.'s [45] research has demonstrated that low-salt egg yolk subjected to HIU (80 W) with potassium ion substitution for sodium ions exhibited a significant reduction in hardness (minimum 1.12 N) (Fig. 3). This phenomenon was attributed to HIU-induced protein denaturation, which disrupted electrostatic interactions and consequently altered gel hardness [46]. The observed decline in springiness aligned with Xu et al.'s findings on the structural changes in lipoprotein within salted duck egg yolk. This alteration might be due to the disruption of lipoprotein structures caused by adding salt, leading to protein aggregation, which compacted the EYG. As a result, the cohesiveness, gumminess, hardness, and chewiness of the EYG gel increased, while the springiness of salted duck egg yolks decreased [17].

Fig. 3.

Effects of combined pH and HIU treatment on gel texture of EYG. (A) Hardness; (B) springiness; (C) cohesiveness; (D) gumminess.

3.4. Effects of combined pH and HIU treatment on cooking loss, gel strength, oil exudation and sandiness of EYG

As depicted in Fig. 4A, the cooking loss was significantly lower in the pH5 + 150 W group and pH3 + 300 W group compared to the control group, with the pH5 + 150 W group demonstrating the lowest cooking loss at 2.14 %. This result indicated that the combined treatment of pH and HIU effectively stabilized water within the EYG gel network. This observation was supported by Zhang et al. [47], who reported that HIU-assisted arginine treatment enhanced the WHC of myofibrillar protein under low ionic strength conditions, consequently reducing cooking loss. Gel strength, a critical parameter reflecting the cohesiveness of a gel, was directly related to the strength of intermolecular forces within the gel matrix. Higher gel strength indicated stronger intermolecular interactions and a more stable molecular structure [48]. As illustrated in Fig. 4B, gel strength positively correlated with HIU power at a constant pH. The highest gel strength (0.0883 g × mm) was observed in the pH3 + 300 W group, while the lowest (0.0334 g × mm) was recorded in the pH5 + 150 W group. This suggested that deviations from the isoelectric point led to increased electrostatic repulsion between proteins, disrupting the delicate balance within the gel system and hindering gel network formation [49]. These findings were consistent with Wang et al. [50], who demonstrated that the synergistic effect of salt ions and HIU treatment improved the heat-induced gel properties of proteins.

Fig. 4.

Effects of combined pH and HIU treatment on cooking loss, gel strength, oil exudation and sandiness of EYG. (A) Cooking loss; (B) gel strength; (C) oil exudation; (D) sandiness.

Oil exudation is a critical indicator for assessing the maturity of salted duck egg curing [51]. As shown in Fig. 4C, all treatment groups exhibited a significant increase in oil exudation rates compared to the control group. This phenomenon could be attributed to the continuous loss of water from the EYG under the influence of salt, which disrupted the internal equilibrium. Consequently, the hydrophilic and lipophilic components separated, and the uniformly dispersed fine fat globules precipitated and coalesced into oil droplets that were discernible to the naked eye, thereby contributing to a relative increase in the oil content of the egg [17]. Particularly, the pH5 + 300 W treatment group demonstrated the highest oil exudation rate of 3.47 %, which was a significant increase of 29.97 % over the control group. This phenomenon could be ascribed to the fact that HIU treatment accelerated the lipoprotein decomposition in EYG, enhanced the solubility of proteins, and facilitated the release of oil, thus increasing the oil exudation [51]. Upon examination of the microstructure of pickled egg yolks, it was revealed that in the presence of salt, EYG was densely arranged, which imparted a distinctive sandy texture to the yolks [52]. Sandiness refers to the formation of small EYG following salt-induced lipid liberation [53]. All treatment groups exhibited significantly higher sandiness than the control group (Fig. 4D). Notably, at 150 W, the sandiness initially decreased and then increased substantially with rising pH. At the same time, at 300 W, the degree of sandiness showed a decreasing trend with increasing pH. The pH5 + 150 W group demonstrated the highest sanding rate of 71.74 %. This phenomenon might be attributed to the maximum dehydration of the EYG after combined pH and HIU treatment, which led to the disintegration of EYG. The subsequent flocculation and aggregation of these disintegrated particles enhanced the sandiness of the yolk [17]. Consistent with these findings, HIU-assisted cooking of salted duck egg yolk, as reported by Wang et al., led to the collapse of the yolk gel network, facilitating lipid migration and resulting in a sandy texture [24].

3.5. Effects of combined pH and HIU treatment on chemical force, endogenous fluorescence, XRD, FTIR, and CD of EYG

The chemical forces within the gel primarily consist of non-specific forces, ionic bonds, and hydrogen bonds. Fig. 5A presents the chemical forces within EYG gels following combined HIU treatment at various pH values and the proportional distribution of each type of chemical force. The results revealed that non-specific forces comprised the most significant proportion in each group, exceeding 78 %, with no significant differences observed among groups. Notably, the pH5 + 150 W group exhibited a significant increment in ionic and hydrogen bonds relative to the control group, suggesting an enhancement of non-covalent interactions. This could be attributed to the HIU treatment, which could induce the unfolding of protein molecules, the emergence of negative charges, increased electrostatic repulsion, and elevated electronegativity, thereby increasing ionic and hydrogen bonds [54].

Fig. 5.

Effects of combined pH and HIU treatment on chemical force, endogenous fluorescence, XRD, FTIR, and CD of EYG. (A) Chemical force; (B) Synchronous fluorescence; (C) Emission fluorescence; (D) XRD; (E) FTIR; (F) CD.

The endogenous fluorescence of proteins mostly came from aromatic amino acid residues, such as tyrosine and tryptophan. These residues exbibited sensitivity to microenvironmental changes, leading to alterations in their fluorescence properties. Consequently, the intensity and emission wavelength of protein fluorescence served as valuable indicators of conformational changes [55]. As depicted in Fig. 5B, synchronous fluorescence analysis revealed a dominant emission peak at 316 nm for all groups, with a less pronounced peak near 469 nm. Notably, the fluorescence emission spectrum of the pH5 + 150 W group exhibited a redshift to 335 nm compared to the control group. The occurrence of redshift suggests conformational changes within the protein, resulting in the exposure of tryptophan residues buried inside the protein to a more hydrophilic and polar environment [56]. Furthermore, at a constant HIU power, emission fluorescence intensity demonstrated a positive correlation with pH. Conversely, at a fixed pH, increasing HIU power resulted in a slight blue shift in the emission peak (Fig. 5C). These observations were consistent with the findings of Zou et al. [57], who reported an increase in fluorescence intensity and a minor blueshift in maximum fluorescence intensity with increasing pH. These results collectively suggested that HIU treatment induced conformational changes in EYG, leading to the translocation of tryptophan residues to a more hydrophobic environment, thereby facilitating gel formation.

XRD analysis, a technique sensitive to microstructural characteristics at the micrometer scale [58], was employed to investigate the structural changes in EYG subjected to different treatment conditions (Fig. 5D). There were distinct features for both the control and treatment groups at 2θ angles of 33° and 46°, while the characteristic peak at 28° was less pronounced. Notably, the groups treated at pH5 with 150 W and 300 W exhibited significantly heightened intensity and sharper peaks at 33° and 46° compared to other groups. These observations suggested that EYG primarily adopted an amorphous state following HIU treatment [23]. Furthermore, the emergence of two new, sharper peaks in the pH5 + 150 W and pH5 + 300 W groups indicated that HIU induced changes in hydrogen bonding and hydrophobic interactions within EYG. These alterations enhanced protein activity and promoted the formation of amorphous intermolecular interaction complexes, ultimately contributing to the observed increase in gel strength [59].

As shown in Fig. 5E, the amide I band, characterized by an absorption peak at 1700-1600 cm⁻¹, provided crucial information regarding protein secondary structure. Hydrogen bonding was crucial for maintaining the α-helix and β-sheet structures of proteins, and its disruption was closely linked to protein denaturation. The presence of disulfide bonds further enhanced protein folding, leading to increased exposure of the secondary structure of EYG. While α-helices and β-sheets indicated organized protein structures, β-turns and irregular curls signify disorder [15]. Among all groups (Fig. 5F), the content of α-helix ranged from 2.61 % to 23.3 %, β-sheet from 5.22 % to 46.6 %, β-turn from 12.8 % to 44.3 %, and irregular curls from 14.6 % to 55.7 %. Compared to the control group, the pH5 + 150 W group exhibited a significant increase in α-helix and β-sheet, along with a considerable decrease in β-turn and random coil. These findings suggested that the pH5 + 150 W group displayed a more organized and structured conformation compared to the control group. This structural transformation could be attributed to HIU-induced protein unfolding, leading to looser and more disordered EYG, weakened hydrogen bonds, and the disorderly-to-orderly transformation of β-turns [15].

3.6. Analysis of CLSM and SEM results

The microstructure of EYG was investigated using laser confocal microscopy after staining with Nile blue and Nile red dyes, as shown in Fig. 6A. All EYG solutions exhibited spherical structures with distinct boundaries, comprising water droplets (green) dispersed in oil (red) at the micrometer scale. The control group exhibited a mixture of small and large particles with more extensive oil distribution. This could be attributed to the natural balance of granular proteins in egg yolks, where 48 % of proteins originated from EYG. When the interfacial environment was altered by the addition of NaCl, the stability of the liquid surface formed by granular proteins was disrupted, leading to varying degrees of EYG dissociation [60]. In contrast, the HIU-treated groups showed a significant reduction in EYG particle size, with the pH3 + 150 W group displaying the most uniform distribution of EYG, indicating enhanced stability. This suggested that proteins adsorbed at the stable oil–water interfaced increased the electrostatic repulsion between oil droplets, ensuring their uniform stability [61]. Consistent with this study, Geng et al. [31] reported that EYG dissociated at low salt concentrations, forming tight structures in a single direction. The aggregation of EYG increased with increasing HIU power, which could lead to the formation of rounded aggregates due to cavitation effects, causing the aggregated structure to relax or inducing re-aggregation of EYG.

Fig. 6.

Effects of different treatment conditions on the CLSM and SEM of EYG. (A) CLSM (From the two-channel superimposed image (first column), oils fluoresce red after staining with Nile red (second column), and proteins fluoresce green after labeling with fluorescein isothiocyanate stain (third column).); (B) SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6B shows untreated EYG exhibited a dense gel structure characterized by a stacked particle arrangement. Following pH-HIU composite treatment, the network structure transitioned to a more porous and prominent morphology, with consistent pore size distribution and noticeable cluster formation. Specifically, the pH5 + 150 W group displayed the most significant increase in pore size and cluster formation. This suggested that appropriate HIU treatment exposed hydrophobic groups, enhanced hydrophobic interactions, improved gel performance, and developed a microstructure characterized by smaller pores and denser networks [45]. Previous research by Fei et al. [62] reported that salt addition led to protein dissociation, accompanied by an increase in small particles. Consistent with our results, Yang et al. [63] utilized SEM to observe a significant amount of HDL within EYG, exhibiting a compact spherical structure. In weakly acidic environments, particles adopted a spherical morphology and form large aggregates. At the isoelectric point, LDL and HDL particles binded closely, forming distinct clusters.

3.7. Pearson correlation analysis and involved mechanism

Pearson's correlation analysis revealed key relationships between gel properties and physicochemical attributes of pH-HIU-treated EYG (Fig. 7A). The dark green color in the matrix indicated a perfect positive correlation (correlation coefficient of 1.0) between β-sheet and α-helix structures. A correlation coefficient of 0.71 indicated a significant positive correlation between hydrophobicity and WHC, as shown by the darker green color in the matrix. In contrast, a correlation coefficient of -0.81 indicated a significant negative correlation between zeta potential and WHC, as indicated by the purple color block in the matrix.

Fig. 7.

Pearson's correlation analysis of pH-HIU composite treatment (A) and the mechanism diagrams of EYG change (B).

The mechanism by which pH-HIU treatment improved the salt-induced gelation properties of EYG is illustrated in Fig. 7B. Upon treatment with NaCl solution, the influx of sodium ions led to the disruption of calcium-phosphate bridges, resulting in a reduction in particle size and partial disassembly of EYG. The HDL inside the EYG was then released from the EYG. These depolymerized HDL unfolded and aggregated into irregular large-sized aggregates, which accelerated the formation of EYG gels. The formation of HDL aggregates further led to the destabilization of the EYG structure and the release of lipids from the lipoprotein core. The process further promoted the formation of loose sandy texture of the EYG gel. As the Na⁺ concentration increased, the disassembled EYG further aggregated, gradually transforming into a composite gel structure composed of fibrous lipoproteins and non-spherical lipid particles. This structural evolution further facilitated the formation of larger molecular weight EYG aggregates. The pH-HIU combined treatment significantly improved the EYG structure by accelerating HDL disassembly, leading to a more uniform dispersion of the EYG internal structure and reduced aggregation. Simultaneously, the mechanical vibrations and cavitation effects of HIU enhanced the interactions between EYG proteins and lipid molecules, promoting the formation of a relaxed gel structure and the migration of lipids. This treatment also resulted in a more uniform and compact EYG gel structure, with the protein structure becoming more ordered, thereby endowing the gel with improved springiness, cohesiveness and adhesiveness.

4. Conclusion

In this experiment, EYG were subjected to joint treatment under varying pH levels (3.0, 4.0, 5.0) and power inputs (150 W, 300 W). Among the treatment groups, the pH5 + 150 W group exhibited superior performance, characterized by good particle dispersion and stability, rapid gelation speed, excellent elasticity, cohesiveness and adhesiveness. This group also demonstrated WHC capacity, minimal cooking loss, higher hydrophobicity, moderate gel strength, and the most uniform and dense gel structure. At the same time, there was a significant improvement in oil exudation and higher sandiness. Additionally, the EYG gel structure appeared relatively dense and uniform, indicating an ordered arrangement of α-helix and β-sheet. In conclusion, the pH5 + 150 W treatment group exhibited optimal gel properties, suggesting that pH-assisted HIU could effectively enhance the gel-forming capabilities of EYG. Future research should explore the effects of HIU duration, ionic strength, temperature, and other parameters to further optimize processing and expand the application possibilities for EYG.

CRediT authorship contribution statement

Dan Zheng: Writing – original draft, Data curation, Conceptualization. Danrong Yu: Writing – original draft, Methodology. Shuai Lin: Writing – review & editing, Data curation. Liting Ji: Data curation. Yaogui Sun: Writing – review & editing. Chunyou Liu: Writing – review & editing, Data curation. Xiaoyu Zhang: Data curation, Conceptualization. Zhihui Yu: Writing – review & editing, Supervision, Formal analysis, Data curation, Conceptualization.

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.