Mechanism study on the improvement of egg white emulsifying characteristic by ultrasound synergized citral: Physicochemical properties, molecular flexibility, protein structure

Abstract

As a natural emulsifier, egg white protein (EWP) has great interfacial characteristics and high security, and has broad development prospects. This study explored the impact of ultrasound synergized citral (CI) treatment on the microstructure, molecular flexibility and emulsifying property of EWP, and predicted the interaction between CI and ovalbumin (the main protein in EWP) through molecular docking. The decrease in free amino content and the growth in molecular weight of EWP suggested that CI and proteins were successfully grafted. The results of physicochemical properties revealed that UCEWP (ultrasound synergized citral-treated EWP) had smaller particle size and larger ζ-potential absolute value, which meant that the stability of UCEWP system was enhanced. From the perspective of interfacial characteristics, UCEWP had lower interfacial tension, which remarkably improved its emulsifying property. The emulsifying activity index (EAI) and emulsifying stability index (ESI) of UCEWP were 1.99 times and 3.19 times higher than that of natural EWP (NEWP). Analysis of Fourier transform infrared spectroscopy (FT-IR) and fluorescence spectroscopy illustrated that the secondary and tertiary structures of UCEWP were more disordered and stretched than those of EWPs. Protein microstructure demonstrated that UCEWP presented loose small particle distribution, and correlation analysis reflected that the improvement of molecular flexibility was positively correlated with the enhancement of emulsifying property. These results elucidated that ultrasound synergized CI treatment is an effective mean to improve the molecular flexibility and emulsifying property of EWP, which provides a valuable reference for further application of EWP.

1. Introduction

The protein content in egg white is about 13–15 %, of which the content of ovalbumin is the highest. Egg white protein (EWP) has high nutritional value and excellent functional properties, including its emulsifying property. Due to its amphiphilic nature, EWP can be adsorbed on the oil–water interface and rapidly diffused to form a stable interfacial protein film, which is an effective emulsifier and can enhance the emulsifying effect of emulsion [1], [2]. Generally, molecular flexibility is closely related to protein structure, flexible proteins adsorbed more quickly than rigid ones and are more prone to change in protein conformation [3]. Zhu et al. [4] observed that proteins with lower content of α-helix structure exhibited greater molecular flexibility. Yan et al. [5] found that ultrasound altered the tertiary structure of proteins, which became loose and flexible. In addition, molecular flexibility of protein had a good correlation with emulsifying property, which was considered an important factor affecting emulsifying property. Cui et al. [6] believed that the improvement of molecular flexibility of soy isolate protein-glucose complex was beneficial for enhancing its emulsifying property. Shen et al. [7] proposed that bovine serum albumin with higher molecular flexibility was more likely to form a more viscoelastic protein film at the interface, thereby exhibiting superior emulsifying property. Currently, food emulsifiers constructed by EWP were sensitive to environmental conditions such as temperature, ionic strength and pH, which limited their effectiveness in the food industry. The emulsifying and interfacial properties of EWP were particularly critical for its application in food processing [8]. Therefore, the emulsifying property of EWP still deserve further extensive research.

The working principle of ultrasound primarily involves disrupting the chemical bonds of protein through cavitation and mechanical forces, and exposing additional hydrophobic groups at the interface. This enhances molecular flexibility of proteins, promotes formation of a stable protein film at the oil–water interface, and thereby improving its emulsifying property. Consequently, ultrasound is regarded as a green, efficient and simple physical treatment technology, which has been widely studied and applied [5]. O'Sullivan et al. [9] reported that ultrasound facilitated the rapid adsorption of proteins at the oil-in-water interface, leading to a decrease in interfacial tension and an improvement in the emulsifying property of EWP. Xie et al. [10] assumed that ultrasonic modification of egg yolk altered the conformation of low-density lipoprotein, increased the interface flexibility, and enhanced the emulsifying property of egg yolk. Wu et al. [11] believed that ultrasound treatment unfolded the structure of EWP, and effectively improved the solubility and emulsifying property. These studies demonstrated that ultrasound treatment can enhance the emulsifying property by modifying protein structures.

Citral (CI) is an unsaturated aldehyde, containing unsaturated C = C double bond and C = O group, which is the main active ingredient of citronella oil [12]. What’s more, it has a strong lemon smell, excellent antibacterial, antioxidant and free radical scavenging properties [13], which leads to its widespread application in the manufacture of food, essence, spices and cosmetics. In the food industry, flavor compounds are commonly used as food additives to increase consumer acceptance of food. Aldehydes play a crucial role in the flavor formation of food, and some aldehydes can interact with various functional amino acid residues, such as the ε-amino group of lysine, thiol group of cysteine, imidazole ring of histidine and phenolic group of tyrosine [14]. Studies have shown that aldehydes could bind to amino acid residues in proteins through irreversible covalent bonding, hydrophobic interactions or hydrogen bonding, depending on the specific chemical structure of the interacting molecules [15]. Furthermore, some aldehydes contain free carbonyl groups that can react with free amino groups on protein molecules through Schiff base formation, thereby improving protein functional properties. Liao et al. [15] argued that cinnamaldehyde and CI could reduce the interfacial tension of sodium caseinate and enhance its interfacial properties. Zhao et al. [16] declared that the heating reduced the interfacial tension of the whey protein-cinnamaldehyde conjugate, resulting in better emulsifying ability and effectively stabilized emulsions. These studies suggested that CI could improve the emulsifying property of proteins by altering the protein interface properties. Therefore, CI was selected to modify EWP to improve its emulsifying characteristic in this study.

However, there are still few reports on the effects of ultrasound synergized CI on structural and functional properties of proteins. In this paper, EWP was used as raw material and treated with ultrasound synergized CI. This research offered new insights into the relationship between functional properties and structure from the perspective of molecular flexibility, further elucidating the structure-flexibility-function relationship between proteins. This study also focused on exploring the mechanism of ultrasound synergized CI to improve the emulsifying property of EWP. Ultimately, this work seeks to provide a theoretical foundation for composite modification of proteins and to expand the application of EWP emulsifying property in the food industry.

2. Materials and methods

2.1. Materials

Fresh eggs were purchased from Fujian Guangyang Egg Industry Co., Ltd. (Fuzhou, China). CI (purity ≥ 97 %) and trichloroacetic acid were bought from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). The BCA protein quantitative kit was from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Glycine standard (purity ≥ 99 %) purchased from Sinopharm chemical Reagent Co., Ltd. (Shanghai, China). The other reagents used in this study were all analytical grade.

2.2. Preparation of samples

Fresh eggs were washed, and the egg white and egg yolk were separated. The collected egg white was removed from the chalaza and whisked with a magnetic stirrer (HO5-1, Shanghai Meiyingpu Instrument Manufacturing Co., Ltd., Shanghai, China) to a homogeneous state. This sample was designated as natural egg white protein (NEWP). The egg white was treated with a probe ultrasonic instrument (VOSHIN-1500C, Wuxi Voshin Instrument Manufacturing Co., Ltd., Wuxi, China) at 120 W for 15 min, and the sample was named as ultrasound-treated EWP (UEWP). CI (0.4 wt%) was added to egg white and stirred gently for 3 h, allowing the Schiff base reaction between CI and the protein to develop adequately, resulting in a gradual yellowing of the system (depicted in Fig. 1) [17]. The sample was named as citral-treated EWP (CEWP). The egg white containing 0.4 % CI was subjected to ultrasonic treatment under the same condition, and the sample was designated as ultrasound synergized citral-treated EWP (UCEWP). All ultrasonic treatments in these experiments were conducted in an ice water bath.

Fig. 1.

Simplified schematic diagram of EWP and CI reaction.

2.3. Measurement of free amino content

Free amino content was determined by method of o-phthalaldehyde (OPA) [11]. The OPA reagent was prepared by dissolving 80 mg of OPA in 2 mL ethanol, 1 mL of sodium dodecyl sulfate (SDS, 10 %), 200 μL of β-mercaptoethanol, and 50 mL of sodium borate buffer (0.1 mol/L) with deionized water to 100 mL. A 400 µL of sample solution was mixed with 8 mL of OPA reagent and reacted in the dark at 37 °C for 3 min. The absorbance of mixtures was measured at 340 nm. Glycine was utilized to construct a standard curve for calculating the free amino content of in samples.

2.4. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)

Reducing SDS-PAGE was performed according to the method of described by Liu et al. [18] with slight modifications. The concentrations of stacking gel and separation gel were 5 % and 12 %, respectively. A total of 60 μL of protein solution at a concentration of 1 mg/mL was mixed with 20 μL of loading buffer, heated in boiling water for 10 min, and then centrifuged at 10000 × g for 10 min using a centrifuge (H1650R, Hunan Xiangyi Laboratory Instrument Development Co., Ltd., Changsha, China) before electrophoresis (JY600E, Beijing JUNYI Electrophoresis Co., Ltd., Beijing, China). A 10 μL sample was loaded into the gel well and electrophoresed at 80 V for 20 min, followed by 130 V for 1 h. After electrophoresis, the gel was stained with 0.05 % Coomassie Brilliant Blue G-250 for 4 h, then decolorized with a decolorizing solution and imaged using a gel imager.

2.5. Measurement of physicochemical properties

2.5.1. Solubility

The solubility was determined by the method proposed by Tao et al. [19]. The protein samples were dissolved in deionized water at a concentration of 2.5 mg/mL and centrifuged at 10000 × g for 15 min at 4 °C. The protein concentration was determined by the BCA method, and the solubility was calculated by equation (1):

$$\text{So}\text{lubility=}\frac{{\text{m}}_{\text{2}}}{{\text{m}}_{\text{1}}}\times \text{100\%}$$ (1)

In this equation, m₂ was the protein content of the supernatant after centrifugation (mg/mL); m₁ was the protein content of the solution before centrifugation (mg/mL).

2.5.2. Surface hydrophobicity

The surface hydrophobicity of EWP was measured according to the method of Huang et al. [20] with slightly modified. The 200 μL of bromophenol blue solution (1 mg/mL) was mixed thoroughly with 1 mL of protein sample solution (5 mg/mL), then the mixture was centrifuged at 8000 × g and 4°C for 15 min. After diluting the obtained supernatant, the absorbance value at wavelength 595 nm was measured and denoted as A. The deionized water was used as a blank, and its absorbance was recorded as A₁. Surface hydrophobicity of EWPs was calculated by the following formula (2):

$$\text{Surface hydrophobicity ([TxErr]μg) =200}\times \frac{{\text{A}}_{\text{1}}\text{-A}}{{\text{A}}_{\text{1}}}$$ (2)

2.5.3. Particle size, polydispersity index (PDI) and ζ-potential

The particle size, PDI and ζ-potential of samples were measured by Zetasizer Nano analyzer (ZS90, Malvern Instruments, Worcestershire, UK) [21], [22]. Protein concentration for measuring ζ-potential was 10 mg/mL, the protein concentration for particle size and PDI was 0.1 mg/mL. The equilibrium and was maintained at 25°C for after 120 s before determination. Each protein sample was tested three times.

2.6. Determination of interface characteristics

2.6.1. Molecular flexibility

Molecular flexibility can be characterized by the sensitivity of proteins to trypsin. Following the method of Sun et al. [23] with appropriate modifications, trypsin was dissolved in Tris HCl buffer (0.05 mol/L, pH 8.0) to prepare the trypsin solution (1 mg/mL). Then, 250 μL of trypsin solution was thoroughly mixed with 4 mL protein solution (5 mg/mL), and incubated in a 37 °C water bath for 15 min. The reaction was terminated by adding 4 mL of 5 % trichloroacetic acid solution. The absorbance of supernatant at 280 nm was measured after centrifugation. The absorbance value A was used to characterize the molecular flexibility of proteins.

2.6.2. Interfacial tension

The video optical contact meter (OCA25, Dataphysics Instruments, Filderstadt, Germany) was employed to monitor the changes in interfacial tension [24]. During testing, the aqueous phase (egg white) and oil phase (corn oil) were placed in the syringe and cuvette, respectively. Each sample formed a 10 μL droplet at the tip of the syringe, and the instantaneous interfacial tension within 160 s was monitored. The interfacial tension values were calculated by computer based on the Young-Laplace equation and changes in droplet area.

2.6.3. Emulsifying property

Following the method of Huang et al. [25] with appropriate modification to determine the emulsifying property of proteins, egg white was dispersed in deionized water to prepare a protein solution with a concentration of 10 mg/mL. Corn oil was added to the solution with a protein-to-oil volume ratio of 3:1. The mixture was then homogenized using a high-speed homogenizer (T25, IKA, Germany) at 10,000 r/min for 2 min. At 0 min and 30 min, 50 μL samples of the emulsion were taken from the bottom and mixed with 10 mL of 1 mg/mL SDS solution, followed by thorough shaking. The absorbance at 500 nm was measured and recorded as A₀ and A₃₀, respectively. The emulsifying activity index (EAI) and emulsifying stability index (ESI) were calculated by equations (3), (4):

$$\text{EAI/(}{\text{m}}^{\text{2}}\text{/g) =}{\text{A}}_{\text{0}}\times \text{4.606}\times \frac{\text{N}}{\text{C}\times \text{F}\times {\text{10}}^{\text{4}}}$$ (3)

$$\text{ESI(min) =}\frac{{\text{A}}_{\text{0}}\times \text{30}}{{\text{A}}_{\text{0}}\text{-}{\text{A}}_{\text{30}}}$$ (4)

where N is the dilution factor (201); C is the protein concentration (10 mg/mL); F is the volume ratio of oil (0.25).

2.7. Characterization of protein structure

2.7.1. Fourier transform infrared spectroscopy (FT-IR)

The method proposed by Li et al. [26] was adopted and slightly modified. The 4 mg of freeze-dried EWP powder was mixed with 400 mg of KBr, and then pressed into thin sheets after full grinding. FT-IR analysis (VERTEX70, Bruker Instruments Co., Ltd., Saarbrucken, Germany) was employed for spectral scanning with a scanning range of 4000–400 cm⁻¹ and a scanning number of 32 times.

2.7.2. Fluorescence spectroscopy

Fluorescence spectrophotometer (RF-5301, Shimadzu Corporation, Kyoto, Japan) was applied to determine the fluorescence intensity of protein samples (deionized water was used to dilute the protein concentration to 0.1 mg/mL). The measurement conditions were as follows: an excitation wavelength of 280 nm, an emission wavelength range of 300–480 nm, and a slit width of 5 nm [27].

2.7.3. Molecular docking

The three-dimensional structure of OVA was obtained from the RCSB PDB protein database (PDB ID: 1UHG), the three-dimensional structure of CI (CID: 638011) was obtained from the PubChem database. The molecular docking of this experiment was performed on YASARA v17.4.17 software in Autodock Vina program (v18.3.23). OVA was placed in a three-dimensional spatial grid box with the size of 119.3 × 119.3 × 119.3 Å for multiple docking simulations to identify the binding sites and select the optimal docking conformation. The optimal docking conformation was generally considered a more stable conformation, and the more stable conformation meant the greater docking energy [28]. Lamarck genetic algorithm (LGA) defined docking energy as: docking energy = receptor energy + ligand energy − complex energy [28]. The optimal conformation obtained from docking of CI and OVA was used as the subsequent analysis, and the Discovery Studio 4.5 Visualizer program was employed to analyze the interactions between the receptor and ligand, generating both two-dimensional and three-dimensional structural diagrams.

2.7.4. Observation by confocal laser scanning microscope (CLSM)

The sample was observed with a CLSM (Leica TCS SP8X DLS, Leica Microsystems, Wetzlar, Germany). The excitation wavelength was 560 nm and the emission wavelength was 633 nm. Prior to observation, the samples were pre-treated by mixing 500 μL of the diluted sample (0.1 mg/mL) with 20 μL of Nile Blue solution (0.1 %) and staining for 30 min [29].

2.7.5. Scanning electron microscopy (SEM)

MSP-IS SEM (Hitachi Hi Tech Co., Ltd., Tokyo, Japan) was applied to observe the surface morphology of samples [30]. A certain amount of egg white powder was adhered to the sample stage, and a gold conductive layer was sputter-coated to secure the samples. The samples were examined at magnifications of 100 × and 500 × using an accelerating voltage of 5 kV.

2.8. Statistical analysis

All experiments were conducted in triplicate, and the results were displayed as mean ± standard deviation. IBM SPSS Statistics 27 was used for data analysis by Duncan test and one-way analysis of variance (ANOVA). The significant level was p < 0.05. Origin 2023 was used for plotting and correlation analysis.

3. Results and analysis

3.1. Analysis of free amino content

The grafting degree between CI and EWP was evaluated by measuring the free amino content using the OPA method. As shown in Fig. 2A, the content of free amino in the modified samples shows a falling trend. The reduction of free amino content in UEWP might be attributed to the mechanical and cavitation effects of ultrasound, which disrupted amino acid sequences and facilitated the formation of water-soluble small molecular amino acids [31]. Additionally, the free amino content of CEWP and UCEWP was significantly lower than that in NEWP, which might be associated with the reduction in free amino groups resulting from CI grafting. Notably, Fig. 2A shows that the free amino content of UCEWP was remarkably lower than that of CEWP, further demonstrating that ultrasound changed the structure of EWP, facilitating Schiff base reaction between CI and EWP, thus effectively reducing the free amino content.

Fig. 2.

Free amino content (A) and SDS-PAGE (B) of modified EWPs. (NEWP: native egg white protein; UEWP: ultrasound-treated EWP; CEWP: citral-treated EWP; UCEWP: ultrasound synergistic citral-treated EWP.)

3.2. SDS-PAGE analysis

To determine whether CI and EWP were successfully grafted, SDS-PAGE was performed and the results were shown in Fig. 2B. Compared with NEWP and UEWP, CEWP and UCEWP exhibited a slight upward shift in the bands of ovotransferrin, ovalbumin, and lysozyme. This shift reflected that CI was successfully grafted onto these proteins, resulting in an upward trend in their molecular weight. The reduced intensity of the protein bands might be correlated to the formation of large molecular aggregates that could not enter the gel lanes. Furthermore, UCEWP exhibited a greater ascending shift in the bands of ovotransferrin, ovalbumin and lysozyme compared to CEWP, meaning that ultrasonic treatment facilitated the reaction between protein and CI, which was consistent with the results of free amino content. Zhao et al. [32] illustrated that proteins reacted with aldehydes, resulting in a decrease in free amino content.

3.3. Analysis of physicochemical characteristics

3.3.1. Solubility

Protein solubility is a prerequisite for its application in food processing and obviously impacts protein functionality [23]. Fig. 3A displays the solubility of various EWPs after different treatments. It was evident that solubility of UEWP, CEWP and UCEWP higher than NEWP. Ultrasonic treatment disrupted the hydrogen bonds and hydrophobic interactions in protein molecules, thereby transforming insoluble aggregates into soluble and uniform monomers [33]. Additionally, ultrasonic treatment enhanced the surface charge of proteins, leading to stronger electrostatic repulsion between protein molecules. The solubility of CEWP and UCEWP was outstandingly improved, which might be associated to the fact that CI could enhance the surface charge of NEWP and reduce the particle size of NEWP, thereby accelerating electrostatic repulsion and reducing aggregation. Moreover, the addition of CI unfolded the protein structure, exposing more hydrophilic groups, which was also one of the reasons for improvement of solubility. The solubility of UCEWP was significantly higher than that of CEWP, illustrating that ultrasound synergized CI treatment could substantially improve the solubility of EWP. The growth of protein solubility could facilitate dispersion of protein in water and oil, which might be the basis for improving emulsifying property.

Fig. 3.

Solubility (A) and surface hydrophobicity (B) of modified EWPs.

3.3.2. Surface hydrophobicity

Surface hydrophobicity can be utilized to evaluate the degree of exposure of hydrophobic groups inside proteins, reflecting changes in protein structure and affecting protein functional properties [34]. As shown in Fig. 3B, surface hydrophobicity gradually increased under different modification methods. Individual ultrasonic treatment promoted the development of protein structure and exposed hydrophobic groups buried inside protein molecules to the surface, resulting in an augment in the surface hydrophobicity of proteins [19]. The addition of CI rised surface hydrophobicity of NEWP, which might be correlated to the fact that CI boosted exposure of some hydrophobic groups from inside protein molecules to polar solutions. In addition, there was still some controversy about the relationship between solubility and surface hydrophobicity. Generally, there was a balance relationship between the growth in surface hydrophobicity and the decline of protein solubility [35]. While some studies found that modification of proteins can simultaneously expose both hydrophilic and hydrophobic groups, leading to enhancement of solubility and surface hydrophobicity [36]. The exposure of hydrophobic groups could promote adsorption of EWP at the oil–water interface, potentially improving emulsifying property. Cui et al. [6] also confirmed that increasing surface hydrophobicity was beneficial for improving emulsifying property.

3.3.3. Particle size, polydispersity index (PDI), and ζ-potential

The particle size can describe the aggregation and dispersion of protein molecules. The smaller particle size, the better dispersibility of protein molecules [37]. As shown in Table 1, the particle size of NEWP declined after ultrasonic treatment. This might be due to the spatial structure destruction of proteins by appropriate ultrasonic treatment. The turbulence and shear caused by cavitation aggravated the collision between molecules, thus effectively reducing the particle size. The particle size of CEWP (236.10 nm) and UCEWP (184.73 nm) was remarkably smaller than that of NEWP (381.90 nm). The main reason might be that the combination of CI and EWP inhibited protein aggregation. Similar results were obtained by Zhao et al. [38] using cinnamaldehyde modified whey protein isolate. EWP was decomposed into smaller molecules after modification, which might be one of the reasons for the increase in protein solubility.

Table 1.

Particle size, polydispersity index, and ζ-potential of modified EWPs.

Samples	particle size (nm)	PDI	ζ-potential (mV)
NEWP	381.900 ± 3.204a	0.996 ± 0.007a	−17.767 ± 0.153c
UEWP	317.533 ± 5.499b	0.547 ± 0.006b	−15.167 ± 0.115d
CEWP	236.100 ± 2.751c	0.415 ± 0.005c	–23.567 ± 0.231b
UCEWP	184.733 ± 3.921d	0.327 ± 0.004d	−26.633 ± 0.153a

(NEWP: native egg white protein; UEWP: ultrasound-treated EWP; CEWP: citral-treated EWP; UCEWP: ultrasound synergistic citral-treated EWP.).

The polydispersity index (PDI) is an indicator for evaluating particle size distribution. The smaller value, the more uniform particle size and distribution [39]. The PDI of NEWP was 0.996 and that of UEWP was 0.547, meaning that ultrasonic treatment could promote a more uniform protein distribution. The PDI of CEWP was 0.415, which was remarkably lower than that of NEWP and UEWP. This reduction was attributed to the addition of CI, which increased spatial hindrance and electrostatic repulsion, resulting in smaller and more uniformly distributed particles in CEWP. Compared with CEWP, UCEWP continuously broke down large particles into small particles under the action of ultrasound, and the presence of CI enhanced the electrostatic repulsion of these small particles, making the protein solution system more stable.

The ζ-potential characterizes the stability of dispersion system and is closely related to the electrostatic forces between protein molecules. The higher absolute value of ζ-potential indicates greater stability [40]. Table 1 presents the ζ-potential results for different modified samples. The absolute value of ζ-potential of UEWP obviously declined from 17.77 mV to 15.17 mV, while that of CEWP and UCEWP prominently increased to 23.57 mV and 26.63 mV, respectively. Ultrasonic treatment disrupted the spatial structure of proteins, exposing positive charges and neutralizing some negative charges [41], thereby reducing the electronegativity of UEWP. The addition of CI reacted with EWP to form Schiff bases, consuming the positive charged amino groups, thus increasing the electronegativity of CEWP system. Zhao et al. [38] observed an increase in negative charge of whey protein isolate with the addition of cinnamaldehyde. Compared with NEWP, UEWP and CEWP, UCEWP had the highest absolute ζ-potential value. This might be associated with moderate ultrasonic treatment altered the structure of EWP and promoted the Schiff base reaction between CI and EWP. The results revealed that EWP solutions treated with ultrasound synergized CI were more stable than CEWP.

3.4. Interface characteristics

3.4.1. Molecular flexibility

The molecular flexibility of proteins refers to the flexibility of molecular structure, reflecting the extent of protein extension and is closely related to the function of protein [23]. Fig. 4A illustrates the impact of different modifications on the molecular flexibility of EWPs, elucidating the trend was in keeping with changes in solubility and surface hydrophobicity. The molecular flexibility of proteins was affected by non-covalent bonds such as hydrogen bond, van der Waals force, electrostatic attraction and hydrophobic interaction. Ultrasonic treatment could destroy these non-covalent bonds, thus expanding the protein structure and damaging the rigid structure of proteins, resulting in a rise in molecular flexibility. NEWP had a compact structure with low molecular flexibility. With the addition of CI, the protein structure of CEWP expanded, forming more flexible intervals and enhancing molecular flexibility. The molecular flexibility of UCEWP also notably rised, which might be related to the disruption of protein rigid structure to form a smaller molecular structure. In general, flexible proteins were more prone to structural rearrangement and adsorption at interfaces, thereby improving the emulsifying property of EWP [42]. Cui et al. [6] also confirmed that highly flexible proteins can promote the enhancement of their interfacial property, making it easier for proteins to form interface films, thus improving emulsifying property.

Fig. 4.

Molecular flexibility (A) and interfacial tension (B) of modified EWPs.

3.4.2. Interfacial tension

The interfacial tension reflectes that amphiphilicity of polymers and their ability to stabilize the oil/water interfaces [21]. Fig. 4B displays the interfacial tension under different modification conditions. Compared with NEWP, the interfacial tension of modified EWP declined, among which UCEWP had the lowest interfacial tension (10.83 mN/m). On the one hand, ultrasound synergized CI treatment weakened the aggregation of proteins, which facilitated rapid adsorption of proteins to the oil–water interface, forming a denser interfacial film and thereby reducing interfacial tension. On the other hand, the increased protein molecular flexibility in UCEWP enhanced the structural unfolding and rearrangement of protein molecules at the interface, further lowering the interfacial tension. Generally, the ability of protein molecules to reduce interfacial tension of adjacent liquids was closely related to the physicochemical and structural properties of proteins. Therefore, changes in protein structure would alter the adsorption rate and interfacial properties of proteins. The improvement of protein molecular flexibility would increase the adsorption of protein on the interface [43]. The preliminary results also demonstrated that the solubility, surface hydrophobicity and molecular flexibility of EWPs modified by different methods were markedly improved, which led to an increase in the adsorption rate of EWP at the interface and a decrease in interfacial tension.

3.4.3. Emulsifying property

Emulsifying property refers to the ability of oil and water to form emulsion, including emulsifying activity index (EAI) and emulsifying stability index (ESI). EWP, as a natural emulsifier, possesses amphiphilicity (hydrophilic and hydrophobic characteristics), which allows it to reduce the interfacial tension between two phases [44]. Fig. 5 illustrates the variation trends of EAI and ESI for different modified samples. The EAI of UEWP was significantly improved compared with NEWP (p < 0.05). This might be correlated to the fact that ultrasound treatment disrupted the protein structure, exposing additional internal groups. The flexible region of protein changed, and the shape of molecule changed from a dense structure to a loose structure, which was conducive to the adsorption and expansion of protein on the oil–water interface. The addition of CI changed the spatial conformation of protein, making protein structure looser and molecular flexibility further improved, which was more conducive to the adsorption of protein at the oil–water interface and increased the EAI of CEWP. It was worth noting that the EAI of UCEWP (25.48 m²/g) was remarkably higher than that of CEWP (21.67 m²/g) and UEWP (15.62 m²/g), which might be associated to the greater structural extension of EWP caused by ultrasound synergized CI treatment altering the flexible region of proteins. It was beneficial for protein rearrangement at the oil–water interface and further improved the EAI of EWP, which echoed the results of solubility and surface hydrophobicity mentioned above.

Fig. 5.

Emulsifying property of modified EWPs.

ESI presents an upgrade trend with modification. Studies have shown that ESI is largely influenced by the electrostatic repulsion between proteins adsorbed on the interfacial protein film [45]. As shown in Fig. 5, the ESI of CEWP and UCEWP ascending obviously, rising from 41.70 min for NEWP to 90.49 min and 133.15 min, respectively. The enhancement in ESI can be attributed to the addition of CI, which generated greater electrostatic repulsion between droplets, thereby reducing their aggregation and increasing stability. The ESI of UCEWP was higher than that of CEWP. It might be related to ultrasonic treatment facilitated protein interactions, and further prevented protein aggregation. Additionally, proper ultrasonic treatment increased the flexibility of protein molecules, thereby enhancing the stability of the oil–water interface and contributing to improving ESI.

3.5. Analysis of protein structure changes

3.5.1. FT-IR analysis

FT-IR is one of the methods for determining changes in protein secondary structure [46]. Fig. 6A displays the FT-IR of different samples. Two bands were observed in the spectrum of NEWP at 1651 and 1542 cm⁻¹, corresponding to amide I band (1700–1600 cm⁻¹, C-O stretching vibration) and the amide II band (1600–1500 cm⁻¹, N-H bending and C-N stretching vibration) [47]. Compared with NEWP, the absorption peak of CEWP and UCEWP at 1239 cm⁻¹ shifted to 1241 cm⁻¹, and the peak at 1542 cm⁻¹ shifted to 1545 cm⁻¹ and 1548 cm⁻¹, respectively. These shifts were attributed to the stretching vibrations of C-N bonds and the bending vibrations of N-H bonds. The reduction of peak intensity at the amide II band in CEWP and UCEWP suggested a decline in the number of N-H bonds involved in the reaction, and the Schiff base reaction might have occurred. The absorption peak of UCEWP at the amide I band shifted from 1651 cm⁻¹ (NEWP) to 1658 cm⁻¹, indicating the generation of Schiff bases (containing C = N structure), which was consistent with the results of Wu et al. [48]. Additionally, the absorption peaks of CEWP and UCEWP at 3419 cm⁻¹ shifted to 3414 cm⁻¹ and 3416 cm⁻¹, respectively, reflecting that hydrogen bonds might be formed between proteins and CI. Furthermore, the absorption peak of UCEWP at 3077 cm⁻¹ shifted to 3080 cm⁻¹ compared with CEWP, which might be owning to different degrees of reaction between the proteins and CI.

Fig. 6.

FT-IR spectrum (A) and secondary structure (B) of modified EWPs.

Further analysis of amide I band revealed specific changes in the protein secondary structure (Fig. 6B). Compared with NEWP, the α-helix content in UEWP, CEWP and UCEWP was evidently reduced (p < 0.05), impling that ultrasonic treatment and addition of CI made the protein molecules unfold and the structure become loose. The α-helix content of UCEWP was lower than that of CEWP, which might be correlated to the fact that ultrasonic treatment was conducive to combination of CI and EWP, transforming the tight α-helix structure into a loose structure and rising the molecular flexibility of protein [49]. After different modifications, the β-sheet content of EWP reduced, while the β-turn and random coil content rised, meaning that β-sheet was converted to β-turn and random coil, and the protein conformation was improved. Meanwhile the reduction in β-sheet content suggested that hydrophobic sites in the protein were exposed, leading to an increase in surface hydrophobicity [3]. The growth of random coil content revealed that molecular structure of EWP was gradually disordered, the protein structure was stretched and molecular flexibility was improved [50], suggesting that ultrasound synergized CI treatment was conducive to improvement of emulsifying property.

3.5.2. Fluorescence spectroscopy

Endogenous fluorescence spectroscopy can reflect changes in the tertiary structure of EWP under different modifications. The fluorescence intensity of proteins was mainly determined by the environmental polarity of aromatic amino acid residues and their specific interactions [51]. The endogenous fluorescence spectra of different EWPs were shown in Fig. 7. The fluorescence intensity of UEWP was prominently lower than that of NEWP, which might be attributed to ultrasound treatment accelerated unfolding of EWP structure, exposed the originally embedded tryptophan residues to a polar environment, resulting in a decrease in fluorescence intensity [52]. Compared with NEWP, the fluorescence intensity of EWP decreased after adding CI, which might indicate that the addition of CI changed the environment of EWP aromatic residues and further proving the formation of complexes between CI and the aromatic residues (mainly tryptophan) of EWP [15]. Furthermore, the fluorescence intensity obtained by UCEWP was lower than that of CEWP, indicating that UCEWP grafted more CI. During the process of generating complexes, the tryptophan residues had a strong shielding effect. The structural changes of EWP can be strengthened, which became loose from original tight structure, and the molecular flexibility was enhanced.

Fig. 7.

Fluorescence spectroscopy of modified EWPs.

3.5.3. Molecular docking analysis

Molecular docking technology provides a more intuitive and comprehensive understanding of the binding characteristics between small molecules and macromolecules [53]. In this study, the Schiff base reaction occurred between CI and protein, but free CI and protein substances still existed. In order to study the interaction between CI and protein, molecular docking was performed. The major components of EWP was ovalbumin (OVA), which accounted for approximately 54 % of EWP. Therefore, the receptor used in homology modeling was OVA. The molecular docking results at the lowest binding energy were visualized using Discovery Studio software (Fig. 8). It can be seen that 12 key amino acids were involved in the interaction between OVA and CI. CI was inserted into the hydrophobic amino acid sites of OVA (ALA37, MET40, PRO93, LEU87, LEU101, LEU124), which were buried inside proteins, forming a hydrophobic pocket. The residue SER36 on OVA bound to CI through hydrogen bonds with bond distances of 2.44 Å and 3.07 Å, respectively. These hydrogen bonds played a crucial role in maintaining protein structure, which was in keeping with FT-IR results. The residues LEU101, LEU87 and PRO93 intacted with CI through hydrophobic interactions, with bond distances of 4.51 Å, 5.18 Å, 4.40 Å and 5.06 Å, respectively. Additionally, there was van der Waals force between OVA and CI. Amino acid residues bound by van der Waals force included SER36, ALA37, MET40, ASN88, THR91, LEU124, TYR125 and ARG126. Although van der Waals force was a weak force, it played a supporting role in maintaining the stability of spatial structure of the complex. These interactions could effectively promote the formation of stable complexes between OVA and CI. The results of molecular docking reflected that the non-covalent binding forces between OVA and CI included hydrophobic interactions, hydrogen bonds and van der Waals forces. Furthermore, Omid Soltanabadi et al. [54] also declared that the interaction between cinnamaldehyde and bovine serum albumin was determined by hydrophobic interactions and hydrogen bonds, which was similar to the findings of this study.

Fig. 8.

Molecular docking between ovalbumin and CI.

3.5.4. CLSM analysis

The impact of different processing on EWP microstructure through CLSM is observed. The red area in Fig. 9A represents the protein rich region [26]. NEWP exhibited larger particle size with uneven distribution, while UEWP showed smaller particle sizes and improved dispersion. This might be responsible for the huge shear force and pressure during the ultrasonic treatment process, which had a certain dispersion effect on the original structure of EWP. The protein structure became loose and was in accordance with the results of particle size above. Ding et al. [55] also affirmed that the particle size of protein decreased after ultrasonic treatment. Compared with NEWP and UEWP, CEWP had smaller protein particle size and more uniform distribution. This might be attributed to the fact that the combination of CI and EWP hindered the aggregation among EWPs. The particle of UCEWP was smaller and more dispersed, and ultrasonic treatment could facilitate the more uniform distribution of CI and EWP complex. This was in agreement with the PDI results above.

Fig. 9.

CLSM (A) and surface morphology (B) of modified EWPs.

3.5.5. Microstructure (SEM)

Fig. 9B displays SEM images of freeze-dried EWP powders with different modifications at 100 × and 500 × magnifications. Compared with NEWP, UCEWP tended to be irregular, which was in agreement with reference [56]. The changes in the microstructure of proteins might be responsible for the impact and shear forces generated by cavitation during ultrasound, which disrupted the cross-linking between protein molecules. CEWP displayed surface pores, and the loose structure facilitated rapid adsorption of EWP at the oil–water interface, which might also contribute to the improved emulsifying property of EWP. UCEWP had a more loose structure and more pores compared with CEWP. It might be owning to the cavitation effect generated by ultrasound, which weakened non-covalent interactions between proteins, reduced aggregation in proteins, and boosted sufficient grafting between proteins and CI. The particle size of UCEWP was effectively reduced, which was beneficial to the dispersibility of protein, the solubility was improved. The stability of system was improved, and the absolute value of ζ-potential increased. This was in keeping with the results of physicochemical properties and functions described above.

3.6. Correlation analysis

Functional characteristics are closely related to protein structure. Fig. 10 presents a correlation analysis between EWP structure and functional characteristics. The red area represents positive correlations, while the blue area represents negative correlations. The solubility was strongly negatively correlated with particle size (p > 0.9), and a drop in particle size led to the breakdown of non-covalent interactions between protein molecules, and the interaction between protein and water was enhanced, which was instrumental to the improvement of solubility. There was a strongly positive correlation between the solubility and ESI (p > 0.9), which might be associated with the fact that the higher solubility improved the distribution of protein at the oil–water interface and formed a thicker interface layer, which further improved the emulsifying property. The ζ-potential showed a strong negative correlation with the EAI and ESI of EWP (p > 0.9) and negative correlation with solubility and surface hydrophobicity (p > 0.7). The higher absolute value of ζ-potential indicated greater intermolecular repulsion, making aggregation less likely, which in turn enhanced the solubility and surface hydrophobicity of protein and subsequently influenced the emulsifying property of EWP. Molecular flexibility had a strong positive correlation with protein EAI and ESI (p > 0.9), suggesting that increased molecular flexibility facilitated rapid adsorption of proteins at the interface, forming an interfacial film that effectively improved emulsifying property. Additionally, there was a strong positive correlation between surface hydrophobicity and emulsifying property. As surface hydrophobicity upgraded, the interactions between adjacent protein molecules adsorbed on the surface of oil droplets strengthened, resulting in a more stable oil–water interface and improved emulsifying property.

Fig. 10.

Correlation analysis between the protein structure and functional properties of EWP.

4. Conclusion

This study compared different modification methods for EWP and found that ultrasound synergized CI treatment significantly improved the emulsifying property of EWP. Compared with NEWP, UCEWP exhibited a more extended structure and enhanced molecular flexibility. Physicochemical properties reflected that the solubility and surface hydrophobicity of UCEWP were significantly improved. Additionally, UCEWP had a lower interfacial tension, further demonstrating that ultrasound synergized CI treatment rised the molecular flexibility of EWP. Correlation analysis suggested a positive relationship between molecular flexibility and emulsifying property, and the more flexible proteins were more readily adsorbed on the interface, thereby effectively enhancing the emulsifying property of EWP. In conclusion, ultrasound synergized CI treatment was an effective modification method to unfold EWP structure, promote molecular flexibility and improve its emulsifying property, which provides a theoretical basis for application of EWP in the food industry.

CRediT authorship contribution statement

Pei Zhang: Writing – original draft, Methodology, Investigation, Formal analysis. Lan Liu: Writing – review & editing, Investigation, Data curation. Qun Huang: Resources, Methodology. Shugang Li: Funding acquisition, Formal analysis. Fang Geng: Visualization, Investigation. Hongbo Song: Visualization, Formal analysis. Fengping An: Resources. Xin Li: Data curation, Conceptualization. Yingmei Wu: Supervision, Project administration, Funding acquisition.

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.