Exploring the influences of ultrasound-assisted glycosylated modification on chickpea protein isolate: insights into structural, functional, and interfacial properties

Graphical abstract

Abstract

Chickpea protein isolate (CPI) offers advantages such as low allergenicity, high digestibility, and a balanced essential amino acid profile, making it a promising dietary protein. However, its relatively poor emulsifying properties in its natural form limit its application in the food industry. Therefore, this study prepared protein-polysaccharide conjugates by heating CPI and citrus pectin (CP) aqueous dispersion under different ultrasonic pre-treatment (UP) at 200  W, 400  W, and 600  W. Results confirmed that the formation of CPI-CP covalent conjugates through analyses of grafting degree, intermediates, browning intensity, and SDS-PAGE. Ultrasonic treatment at 400  W enhanced the conjugates’ surface hydrophobicity (H₀), free sulfhydryl (−SH) content, particle size, and emulsifying properties. Notably, moderate ultrasonic pre-treatment (400  W) could enhance interfacial adsorption capacity and interfacial interactions of prepared CPI-CP conjugate products at the oil–water interface. Collectively, these findings elucidated the positive role of ultrasound pretreatment in the preparation of CPI-CP conjugates with high interfacial performances, highlighting the potential application value of UP in the preparation of CPI-CP glycosylation products.

1. Introduction

In recent years, driven by plant-based diet concept, consumer preferences have increasingly shifted toward plant-based proteins. This trend has led to a significant rise in the development and utilization of plant proteins. Chickpeas rank among the world's top three edible legumes, widely cultivated in regions such as India, Americas, and partial regions of China. Rich in nutrients with a protein content of 25% [1], [2], their amino acid profile closely matches human requirements and is easily absorbed. Consequently, they serve as a vital protein source for vegetarians and individuals with lactose intolerance. However, despite these advantages, chickpea protein isolate (CPI) also has some drawbacks, such as poor emulsifying stability [3], [4], which greatly limit its broader application in the food industry.

To address this situation, researchers have employed physical or chemical methods to enhance protein functionality. They also utilize proteases for hydrolysis, aiming to broaden their applications in food and other products [5]. Glycosylation—the attachment of polysaccharides to proteins—can improve emulsifying and foaming properties while reducing allergenicity [6]. The glycosylation reaction between protein amino groups and the carbonyl group of reducing sugars represents a simple, green, and promising method for enhancing the functional properties of plant proteins. Compared to many other chemical modification approaches, glycosylation can proceed spontaneously at elevated temperatures. This process does not require the use of potentially hazardous solvents or reagents. However, careful control of the reaction remains essential, as some advanced glycation end products (AGEs) may pose health risks to humans [7]. Glycosylation is commonly employed to modify proteins for enhanced functional properties. Studies indicate that polysaccharide-modified proteins exhibit greater advantages than monosaccharide- or oligosaccharide-modified proteins in improving the quality attributes of emulsions, such as color, flavor, and stability [8]. Upon polysaccharide incorporation, protein-polysaccharide covalent complexes provide stronger electrostatic repulsion and steric hindrance between droplets, thereby enhancing emulsion stability [9]. Citrus pectin (CP) is an acidic heteropolysaccharide composed of galacturonic acid and neutral sugars. Currently, the primary commercial sources of pectin include citrus peel (accounting for 85.5%) and apple processing residues, which are commonly used as stabilizers, emulsifiers, and thickeners in the food industry. CP is also found extensive applications in other fields, including wound healing, encapsulation systems, and gut microbiome modulation [10]. Furthermore, CP addition has been demonstrated enhanced emulsifying properties for food proteins such as whey protein isolate and pea protein isolate [11]. Collectively, these studies highlight the significant potential of glycosylation in enhancing the emulsifying and interfacial properties of proteins.

Under the Maillard reaction conditions, covalent bonds are formed between the free amino groups of proteins and the reducing carbonyl groups of carbohydrate substances. Therefore, protein-polysaccharide conjugates can be prepared by dry heating or wet heating methods based on the Maillard reaction mechanism. There is a limitation in the grafting degree during protein glycosylation modification. For proteins, both dry heat and wet heat modification methods have the disadvantage of a relatively low yield of protein-polysaccharide conjugates. Therefore, new helper methods should be explored to improve the speed and degree of protein glycosylation reactions [12]. Some results have found that the structure of proteins and polysaccharides become looser and more stretched after ultrasonic treatment [13], [14], [15], [16]. The structural changes of proteins/polysaccharides expose some free amino groups/reducing carbonyl groups embedded inside the molecules, which theoretically has the potential to promote Maillard covalent crosslinking between proteins and polysaccharides. Furthermore, ultrasonic pretreatment before glycosylated reaction of protein may also affect the functional properties of its conjugates. Cui et al. observed that ultrasonic pretreatment of CPI could significantly promote its glycosylated covalent crosslinking with dextran. Moreover, the obtained CPI conjugates showed improved ability to stabilize Pickering emulsions [15]. However, current research on the application of CP with better emulsifying properties in the ultrasound-assisted glycosylation modification of CPI remains limited. Furthermore, there is a lack of systematic investigation into the interfacial behavior characteristics of CPI after this treatment.

We prepared CPI-CP covalent conjugates by the method of ultrasonic-assisted wet heating. The effects of varying ultrasonic pretreatment power on the physicochemical properties and interfacial characteristics of the subsequent prepared CPI-CP conjugates were investigated. This study is beneficial for deepening our understanding of the interaction between protein and pectin molecules, which provides a new approach for us to prepare glycosylated protein with better functional properties.

2. Materials and methods

2.1. Materials

Chickpeas were supplied by Xinjiang Mulei Yingge Biotechnology Co., Ltd. (China). Citrus pectin (P9135; galacturonic acid content >74%), o-phthalaldehyde (OPA), sodium dodecyl sulfate (SDS), and 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS-NH₄) were obtained from Sigma-Aldrich. The n-Hexane was purchased from Tianjin Fuyu Fine Chemical Co., Ltd. Medium-chain triglycerides (MCT) were acquired from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). All other chemicals used in this study were of analytical grade.

2.2. Extraction of CPI

Chickpea protein isolate (CPI) was prepared according to our previously reported method with minor modifications [17]. Chickpeas were mixed with deionized water at a ratio of 1:2 (w/v), soaked for 12 h, and subsequently dehulled. The hulled chickpeas were dried in an oven at 45 °C for 12 h, ground, and sieved through an 80-mesh screen. The resulting chickpea flour was defatted by extraction with n-hexane at a flour-to-solvent ratio of 1:3 (w/v) under continuous stirring at room temperature for 4 h. This defatting procedure was repeated three times to obtain defatted chickpea flour. The defatted flour was dispersed in deionized water at a ratio of 1:10 (w/v) and magnetically stirred for 30 min. The pH of the suspension was adjusted to 9.0 using 1 M NaOH and maintained under stirring at room temperature for 2 h. The mixture was then centrifuged at 6000 rpm for 20 min at 4 °C, and the supernatant was collected. Protein precipitation was induced by adjusting the pH of the supernatant to 4.5 with 1 M HCl, followed by stirring for 1 h and centrifugation under the same conditions. The resulting precipitate was washed three times with deionized water, redispersed in deionized water, and neutralized to pH 7.0 using NaOH solution. Finally, the protein solution was freeze-dried for 48 h, ground it, and stored it at -20 °C until use. The protein content of the CPI was determined by the Kjeldahl method to be 89.24 ± 0.13%.

2.3. CPI-CP conjugates preparation

The preparation of CPI-CP conjugates was slightly modified according to previous methods [18], [19], [20]. To prepare the covalent conjugates, a 2% (w/v) CPI: CP solution (1:1) was prepared. The mixture was stirred overnight using a magnetic stirrer. This ensured the complete dissolution of all components. The pH of the mixed solution was adjusted to 7.0. The mixed solution (100 mL) was subsequently transferred into a 250 mL glass beaker, and subjected to sonication using an ultrasonic disperser (JY92-IIDN, Ningbo, China) fitted with a 6 mm diameter probe (titanium alloy). During sonication, the probe tip was immersed 2.0 cm below the liquid surface. Ultrasonication was performed at 20–25 kHz, pulse mode (2 s on, 2 s off), for 10 minutes in an ice-water bath maintained below 10 °C. The selected ultrasonic power was 0 W, 200 W, 400 W, or 600 W. These powers corresponded to power densities of 0, 2, 4, and 6 W/mL, respectively. The mixed solution after ultrasonic treatment was heated at 80 ℃ for 30 minutes, and then rapidly cooled to room temperature under ice bath conditions. The solutions of different CPI-CP conjugates (with different ultrasonic pre-treatment power) was then freeze-dried for subsequent analysis. The CPI without glycosylated modification was set as control samples.

2.4. Measurement of the degree of grafting (DG)

The degree of glycation (DG) was determined following a previously reported method by quantifying changes in free amino group content [21]. The o-phthalaldehyde (OPA) reagent was freshly prepared prior to use by dissolving 40 mg of OPA in 1 mL of methanol, followed by the addition of 25 mL of 0.1 M sodium tetraborate buffer (pH 9.5), 100 μL of β-mercaptoethanol, and 2.5 mL of 20% (w/v) SDS, and then diluting the mixture to a final volume of 50 mL with deionized water. For the assay, 4 mL of OPA reagent was added to 200 μL of sample solution (2 mg/mL), and the resulting mixture was incubated at 35 °C for 2 min. The absorbance was immediately recorded at 340 nm using a UV-2600 spectrophotometer, and the DG was calculated according to Eq. (1):

$$\begin{array}{c}DG\left(\%\right)=\frac{A_0-A_t}{A_0}\times 100\end{array}$$ (1)

where A₀ and A_t represent the absorbance values of the samples before and after glycosylation modification, respectively.

• Measurement of browning degree and intermediate products

As previously described [22], the samples were diluted with deionized water to a final concentration of 5 mg/mL, which was also used as the blank control. The degree of browning was evaluated by measuring the absorbance at 420 nm. Maillard reaction intermediates were characterized by recording the absorbance at 294 nm. For this purpose, the samples were further diluted to 0.5 mg/mL, and the corresponding A₂₉₄ values were used to quantify the intermediate products.

2.5. Zeta potential and particle size

The sample was dissolved in phosphate buffer (0.01 M, pH 7.0). Then the particle size and zeta potential were measured by Zetasizer (Nano-ZS, Malvern Instrument Ltd., UK). All samples were equilibrated at 25 °C for 2 minutes before testing. For particle size measurement, the DTS0012 particle size cell was used, maintaining a sample volume of 1 mL with a backscatter detection angle of 173°. For potential measurement, the DTS1070 U-shaped potential cell was employed.

2.6. Endogenous fluorescence spectroscopy

The intrinsic fluorescence spectra of the samples (0.1 mg/mL protein) were recorded in the range of 300–500 nm using a fluorescence spectrophotometer with an excitation wavelength of 280 nm [23]. Both the excitation and emission slit widths were set to 5 nm, and the scanning speed was maintained at 1200 nm/min.

2.7. Ultraviolet spectrum analysis

The ultraviolet (UV) spectra of the differently glycosylated CPI samples were recorded using a UV–visible spectrophotometer (UV-2600, Shimadzu, Japan). The samples were completely dissolved in deionized water and diluted to a final concentration of 1 mg/mL, and the absorbance was measured over the wavelength range of 200–600 nm.

2.8. Fourier transform infrared spectra (FT-IR)

FT-IR spectra of the samples were recorded at room temperature using an FT-IR spectrophotometer. The sample powders were thoroughly mixed with KBr at a ratio of 1:100 (w/w) and pressed into pellets, followed by 64 scans over the wavenumber range of 4000–400 cm⁻¹ at a resolution of 4 cm⁻¹. Finally, changes in the secondary structure of the conjugates were analyzed using OMNIC 9.2 and PeakFit 4.12 software.

2.9. SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

The protein sample (2 mg/mL) was mixed with 2×dissolving buffer (5% β-mercaptoethanol, 20% glycerol, 20% SDS, 0.1% bromophenol blue, and 1 M Tris) , and then heated at 100 °C for 5 min. Subsequently, 10 μL of the mixed solution were loaded onto polyacrylamide gels consisting of a 5% stacking gel and a 12% separating gel. Electrophoresis was performed at 80 V for the stacking gel and 120 V for the separating gel, followed by staining with Coomassie Brilliant Blue R250 [24].

2.10. Scanning electron microscope (SEM)

The sample powder was adhered to a thin layer of aluminum, followed by coating all sample surfaces with a thin layer of gold via vacuum deposition. The microstructure of the samples was observed using a scanning electron microscope (SU8010, Hitachi, Tokyo, Japan) at different magnifications and an acceleration voltage of 5 kV.

2.11. Surface hydrophobicity

As previously described [25], the complex solutions were diluted with deionized water to a protein concentration of 1 mg/mL, followed by the addition of 20 μL of 8 mM 8-anilino-1-naphthalenesulfonic acid (ANS) solution to 4 mL of the diluted protein solution. After gentle mixing, the reaction mixtures were protected from light for 10 min. The fluorescence intensity was then measured using a fluorescence spectrophotometer with an excitation wavelength of 390 nm and an emission wavelength range of 400–600 nm. The surface hydrophobicity (H₀) was calculated according to Eq. (2):

$$\begin{array}{c}{H}_0=Are{a}_2-Are{a}_1\end{array}$$ (2)

where Area₂ represents the integrated fluorescence spectral area of the protein sample, and Area₁ corresponds to that of the blank control.

2.12. Free sulfhydryl content

According to a previously reported method [26], Tris–glycine buffer (pH 8.0) was prepared using 0.086 mol/L Tris, 0.09 mol/L glycine, and 0.4 mmol/L EDTA. The Ellman’s reagent solution was prepared by dissolving 120 mg of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in 30 μL of the buffer. The sample solution (1 mg/mL) was mixed with 50 μL of Ellman’s reagent, incubated at room temperature for 40 min, and subsequently centrifuged at 10,000 × g for 8 min. The absorbance of the resulting supernatant was measured at 412 nm, and the free sulfhydryl (SH) content was calculated according to Eq. (3):

$$\begin{array}{c}FreeSH\left(\mu mol/g\right)=\frac{73.53\times A\times D}{C}\end{array}$$ (3)

where A the absorbance at 412 nm, C is the sample concentration (mg/mL), and D is the dilution factor.

2.13. Solubility analysis

Solubility was determined according to the method described in the literature [27] with some modifications. A sample solution at a concentration of 3 mg/mL was mixed with 3 mL of Bradford ready-to-use dye reagent (Biorad, USA). After reacting for 10 minutes, the absorbance of the sample was measured at 595 nm using a spectrophotometer (UV-2600, Shimadzu, Japan). Protein solubility was calculated using formula (4):

$$\begin{array}{c}\text{P}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n s}\text{o}\text{l}\text{u}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y}\left(\%\right)=\frac{\text{Soluble protein content}}{\text{Initial protein content}}\times 100\end{array}$$ (4)

2.14. Determination of emulsifying capacity

The emulsifying activity index (EAI) and emulsifying stability index (ESI) of the protein samples were determined according to the method reported by Ma et al. [28]. The samples were dissolved in phosphate buffer (0.01 M, pH 7.0) to obtain a protein concentration of 0.2% (w/v). The protein solutions were then mixed with sunflower oil at a volume ratio of 3:1 and homogenized at 20,000 r/min for 2 min. Subsequently, 50 μL of the emulsion was withdrawn at 0 and 10 min and immediately dispersed into 5 mL of 0.1% (w/v) SDS solution. The absorbance was measured promptly at 500 nm, and the EAI and ESI were calculated according to Eqs. (5), (6):

$$\begin{array}{c}EAI\left(m^2/g\right)=\frac{2\times 2.303\times A_0\times N}{10000\times \theta \times L\times C}\end{array}$$ (5)

$$\begin{array}{c}ESI\left(min\right)=\frac{A_0\times 10}{A_0-A_{10}}\end{array}$$ (6)

where N denotes the dilution factor, θ oil phase volume fraction, L is the optical path length of the cuvette (cm), C denotes the protein concentration (mg/mL), and A₀ and A₁₀ represent the absorbance values measured at 0 and 10 min, respectively, with 10 indicating the time interval between the two measurements (min).

2.15. Interfacial tension and interfacial adsorption kinetics

Dynamic interfacial tension was measured using an optical contact angle meter. The initial droplet volume was set to 60 μL for the dynamic interfacial tension measurements, and all experiments were conducted at 25 °C. After loading the solution into the syringe, an equilibration period of 30 min was allowed to ensure temperature stabilization, followed by 180 min of interfacial adsorption. The interfacial pressure (π) was calculated according to the following equation [29]:

$$\begin{array}{c}\pi ={\gamma }_0-\gamma \end{array}$$ (7)

Here, γ₀ and γ denote the interfacial tensions between the buffer solution and MCT, and between the sample solution and MCT, respectively.

The diffusion rate (K_diff) of proteins at the oil-water interface is determined by a mathematical formula [29]:

$$\begin{array}{c}\pi =2C_{0}KT{(Dt/3.14)}^{1/2}\end{array}$$ (8)

Here, C₀ represents the concentration of the continuous phase, while K, T, t, and D denote the Boltzmann constant, absolute temperature, adsorption time, and diffusion coefficient, respectively. In addition, first-order kinetic equations were applied to analyze the protein penetration rate (Kp) and reorganization rate (Kr) at the interface [30]:

$$ln\left[\left({\pi }_{10800}-{\pi }_t\right)/\left({\pi }_{10800}-{\pi }_0\right)\right]=-k_{i}t$$ (9)

Among these, π₁₀₈₀₀, π₀, and π_t represent the interfacial pressures at the final adsorption time (10,800 s), the initial time, and time t, respectively.

2.16. Interfacial dilational rheology

Time-scan experiments on interfacial extensional rheology were conducted at 25 ± 0.5 °C using an optical contact angle measurement system. A sinusoidal oscillation with an amplitude of 10% (da/A) and a frequency of 0.1 Hz was applied. The time scan consisted of 30 iteration steps, and for each measurement, five sinusoidal cycles were performed with a 200 s interval maintained between consecutive scans. The total experimental duration was 10,800 s. Interfacial viscoelastic parameters were calculated using the reference method described previously [31]. After completion of the time-scan measurement (10,800 s), the oil–water interface was allowed to equilibrate for 4 min, after which a frequency sweep test was performed. The oscillation frequency was varied from 0.005 to 0.1 Hz at a constant amplitude of 10%.

2.17. Statistical analysis

All experiments were repeated three times. Results were expressed as mean ± standard deviation, with error bars indicating standard deviation. Data processing, graphing, and mathematical fitting were performed using Origin 2023b. One-way analysis of variance (ANOVA) was conducted using SPSS 25.0 (p < 0.05), followed by Tukey's post hoc test.

3. Results and discussion

3.1. Degree of glycosylation

Under Maillard reaction conditions, proteins can form covalent linkages with polysaccharides, resulting in the consumption of free amino groups in protein molecules. Therefore, the degree of grafting can be evaluated by monitoring changes in the free amino group content of proteins [32]. As shown in Fig. 1A, the grafting degrees of CPI–CP conjugates subjected to ultrasonic pretreatment (200, 400, or 600 W) prior to glycosylation were significantly higher than those of the conjugates prepared without ultrasonic pretreatment (0 W). This phenomenon may be attributed to the cavitation and mechanical effects generated by ultrasound, which promote the unfolding and dissociation of the compact molecular structures of CPI and CP. As a result, a greater number of free amino groups are exposed from within the protein molecules, while the amount of reducing carbonyl groups embedded in the pectin molecules is reduced, thereby facilitating the covalent cross-linking between CPI and CP via the Maillard reaction [14], [33]. With increasing ultrasonic pretreatment power, the grafting degree of the CPI–CP conjugates initially increased and then decreased. Moderate ultrasonic intensity facilitates the unfolding and structural expansion of proteins and polysaccharides, increasing the exposure of reactive groups and thereby accelerating the Maillard reaction. However, excessive ultrasonic pretreatment power induces aggregation of the protein, thereby reducing the efficiency of the Maillard reaction between CPI and CP [34]. Results have found that a pretreatment power of 400 W for ultrasound is more favorable for subsequent glycosylation modification of CPI.

Fig. 1.

Degree of glycosylation (A), Absorbance at 294 and 420 nanometers (B), Particle size (C), Zeta potential (D). Note: Different letters indicate significant differences (p < 0.05). CPI-CP conjugate prepared without ultrasonic pretreatment (0 W), CPI-CP conjugate prepared with 200 W ultrasonic pretreatment (200 W), CPI-CP conjugate prepared with 400 W ultrasonic pretreatment (400 W), CPI-CP conjugate prepared with 600 W ultrasonic pretreatment (600 W).

3.2. Browning degree and intermediate products

The UV absorbance value at 294 nm represents the content of intermediates—Schiff base and Amadori rearrangement products in the early Maillard reaction. The UV absorbance value at 420 nm indicates the amount of brown substances generated in the Maillard reaction [22]. As shown in Fig. 1B, the introduction of ultrasound assisted methods has led to varying degrees of improvement in the subsequent Maillard reaction between CPI and CP. At low ultrasound power (200 W), there was only a slight change in the absorbance of intermediate products and brown substances, which may be due to insufficient ultrasound intensity to alter protein structure. However, when ultrasonic power increased to 400 W and 600 W, the absorbance values of intermediate products first increased and then decreased [35]. Under ultrasonic assistance at 400 W, cavitation promotes moderate unfolding of the protein, leading to substantial exposure of free amino groups. Meanwhile, the pectin molecular chains become stretched, improving the accessibility of reducing carbonyl groups, thereby significantly increasing the effective collision frequency between amino and carbonyl groups. In contrast, the decrease in intermediate product absorbance observed at 600 W may be attributed to excessive protein aggregation induced by high-intensity ultrasound, which reduces the availability of free amino groups for the reaction [35].

3.3. Particle size

The particle size distributions of CPI and CPI–CP conjugates are presented in Fig. 1C. Compared with native CPI, all CPI–CP conjugate samples exhibited increased particle sizes, whereas the conjugates subjected to ultrasonic pretreatment showed smaller particle sizes than those prepared without ultrasound (0 W). This effect can be attributed to ultrasonic treatment effects, which not only promotes the unfolding of compact protein and polysaccharide structures but may also induce partial cleavage of peptide and glycosidic bonds, leading to fragmentation of molecular chains [36]. With increasing ultrasonic pretreatment power, the particle size of the CPI–CP conjugates initially decreased and subsequently increased. At 400 W, the protein conformation was adjusted to an optimal state, with sufficient exposure of free amino groups, resulting in complete reaction and the formation of compact conjugates, thereby yielding the smallest particle size. In contrast, the increase in particle size at 600 W is primarily attributed to the enhanced exposure and cross-linking of hydrophobic groups induced by high-intensity ultrasound, leading to aggregate formation [37].

3.4. Zeta potential

Zeta potential is a key parameter reflecting the net surface charge of complex particles and is widely used to evaluate the stability of colloidal systems. A higher absolute value of zeta potential indicates stronger electrostatic repulsion between particles, thereby reducing the likelihood of aggregation and enhancing system stability [38]. As shown in Fig. 1D, native CPI exhibited a zeta potential of −22.15 ± 1.60 mV. After glycosylation, the zeta potential of CPI decreased markedly to −47.82 ± 0.43 mV (0 W), suggesting that the introduction of negatively charged pectin significantly increased the surface negative charge of the CPI–CP conjugates. With increasing ultrasonic pretreatment power, the zeta potential further decreased from −51.16 ± 1.60 mV at 200 W to −53.34 ± 0.50 mV at 400 W. Moderate ultrasound (400 W) could induce partial unfolding of the protein through cavitation effects, exposing more carboxyl groups. Meanwhile, cavitation effects could also promote the stretching of pectin chains, exposing additional carboxyl groups, leading to an increase in the absolute value of zeta potential. These results further indicate that ultrasonic pretreatment at 400 W was more favorable for promoting effective glycosylation of CPI.

3.5. Endogenous fluorescence spectroscopy

Aromatic amino acids in proteins, such as tryptophan, tyrosine, and phenylalanine, are intrinsic fluorophores, and their fluorescence characteristics are widely used to probe protein conformational changes [39]. As shown in Fig. 2A, native CPI exhibited the highest fluorescence intensity, followed by ultrasonically assisted glycosylated CPI, whereas conventionally glycosylated CPI showed the lowest fluorescence intensity. This is because the covalent crosslinking between CP and CPI weakened the interactions among hydrophobic amino acid residues in the protein, leading to fluorescence quenching of these residues [40]. With increasing ultrasonic power, the fluorescence intensity gradually increased, which may be associated with ultrasound-induced conformational alterations that promote protein unfolding and expose a greater number of surface chromophores [41]. Furthermore, ultrasonication and glycosylation combined treatment further enhanced protein conformation unfolding.

Fig. 2.

Endogenous fluorescence spectroscopy (A), Ultraviolet spectrum (B), Fourier transform infrared spectra (C), Secondary structures (D). CPI-CP conjugate prepared without ultrasonic pretreatment (0 W), CPI-CP conjugate prepared with 200 W ultrasonic pretreatment (200 W), CPI-CP conjugate prepared with 400 W ultrasonic pretreatment (400 W), CPI-CP conjugate prepared with 600 W ultrasonic pretreatment (600 W).

3.6. Ultraviolet spectrum

In the UV spectrum, two peaks appear at 220 nm and 280 nm, respectively. The first peak represents the intensity of peptide bonds in proteins, while the second peak corresponds to the absorption of tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe) residues in proteins [42]. In Fig. 2B, for the first peak, most CPI-CP conjugate samples exhibited higher absorbance value compared to untreated CPI. The maximum absorption peak shifted slightly from 210 nm to 220 nm, indicating that peptide bonds originally buried within the hydrophobic core became exposed to the aqueous solution, enhancing the solution's polar environment. The second peak exhibited red-shifting. Compared to untreated CPI, ultrasonic pre-treatment significantly enhanced the absorption peak intensity of CPI-CP conjugates in the UV-visible spectrum, with a pronounced red shift in the maximum absorption wavelength. This result could be attributed to the cavitation effect and mechanical shear generated by ultrasonication, which exposed internally hidden hydrophobic groups within the protein to the solvent environment [43].

3.7. FT-IR

FT-IR spectroscopy is a rapid and effective technique for analyzing molecular interactions and structural changes in CPI–CP conjugates. The appearance of new absorption bands or variations in the intensity and position of existing peaks can reflect functional group interactions at the molecular level [44]. As shown in Fig. 2C, native CPI exhibited a characteristic amide I band at 1657 cm⁻¹, corresponding to C=O stretching vibrations. Following glycosylation, the amide I band of CPI showed a noticeable blue shift, which may be attributed to covalent crosslinking between CPI and CP [45]. The amide II band of CPI appeared at approximately 1545 cm⁻¹, and its intensity decreased significantly after glycosylation. These spectral changes are associated with the formation of glycosidic bonds between CP and CPI during the Maillard reaction, which consumes C=O and N–H groups and generates glycosylation products. In addition, the formation of reaction intermediates such as Amadori compounds, Schiff bases, and pyrazines can further induce shifts in C=O stretching and N–H bending vibrations [46].

The amide A region (3000–3600 cm⁻¹), mainly attributed to O–H and N–H stretching vibrations, exhibited broader and more intense absorption bands in ultrasonic-assisted glycosylated CPI compared with native CPI, indicating increased hydroxyl content due to enhanced CP grafting. Moreover, the characteristic absorption bands in the 1180–950 cm⁻¹ region, commonly referred to as the sugar region, are indicative of covalent bonding between polysaccharides and proteins [47]. The absorption intensity in this region was higher for the CPI–CP conjugate prepared without ultrasound (0 W) than for native CPI, confirming successful glycosylation. Notably, these spectral changes in both the 3500–3000 cm⁻¹ and 1180–950 cm⁻¹ regions became more pronounced in ultrasonic-assisted glycosylated CPI samples, suggesting that ultrasonic pretreatment effectively promoted covalent crosslinking between CPI and CP [46]. These observations aligned with DG results, further confirming that early ultrasonic processing could enhance the subsequent glycosylation reaction.

As shown in Fig. 2D. CPI exhibited a relatively ordered structure, consisting of α-helix, β-sheet, random coil, and β-turn, indicating a compact protein conformation stabilized by intramolecular interactions. After glycosylation without ultrasound pretreatment (0 W), the α-helix content markedly decreased to 19.32%, while β-sheet (32.58%) and random coil (32.03%) increased, suggesting that the Maillard reaction disrupted the native helical structure and induced partial unfolding of the protein molecules [48]. With the introduction of ultrasound pretreatment, further structural rearrangement occurred. At 200 W, the random coil content increased to 33.22%, accompanied by a slight increase in α-helix (20.09%). A more pronounced transition was observed at 400 W, where the α-helix content further decreased to 15.35%, while the random coil fraction increased to 35.70%, suggesting significant disruption of ordered structures and formation of a more disordered and flexible conformation. This is because the interaction between pectin and protein leads to the unfolding of protein structure, which may facilitate the exposure of reactive groups [17]. When the ultrasonic power increased to 600 W, the random coil content slightly increased further, indicating that excessive ultrasonic energy may induce additional unfolding and structural reorganization of the protein molecules. The glycosylation modification of CPI significantly alters the secondary structure under the influence of ultrasound pretreatment, which provides a structural basis for altering the functional properties of the protein.

3.8. SEM

The microstructure of different CPI samples treated with various modification methods was observed using scanning electron microscopy. Fig. 3 shows the microstructures of the different CPI samples. Native CPI exhibited a flat and smooth surface structure characterized by larger agglomerates with fewer interconnections. In contrast, the glycosylated sample (0 W) exhibited irregular flaky structures, partly due to freeze-drying and the glycosylation reaction. In addition, the partially fragmented structures in the 0 W sample indicated that covalent crosslinking between CPI and CP had formed. As shown in Fig. 3, the microstructures of the samples modified by ultrasound-assisted glycosylation (200 W, 400 W, 600 W) became rougher and more irregular, and some fragmented aggregates appeared. This observation once again confirmed the promoting effect of ultrasonic pretreatment on CPI glycosylation modification [49], [50]. The CPI–CP conjugate sample pretreated with 600 W ultrasound exhibited a certain degree of aggregation in its microstructure, thereby affecting its solution properties.

Fig. 3.

SEM images of CPI and different CPI-CP conjugates

3.9. Surface hydrophobicity

Hydrophobic amino acid residues are generally buried within the interior of protein molecules, and surface hydrophobicity (H₀) reflects the distribution of hydrophobic amino acid residues exposed on the protein surface, serving as an indicator of protein conformational changes [51]. During emulsification, the surface hydrophobicity of proteins is directly correlated with their interfacial activity. As shown in Fig. 4C, the H₀ value of CPI increased significantly after glycosylation modification, mainly because heat treatment during the Maillard reaction partially unfolded the compact globular structure of CPI, thereby exposing more hydrophobic amino acid residues on the protein surface [36]. Consequently, the H₀ value of CPI–CP conjugates was higher than that of native CPI. In addition, the H₀ value of ultrasonic-assisted glycosylated CPI was higher than that of glycosylated CPI without ultrasonic pretreatment, indicating that ultrasonic pretreatment further promoted protein unfolding and enhanced the exposure of hydrophobic amino acid residues. With increasing ultrasonic pretreatment power, the H₀ value of CPI–CP conjugates first increased and then decreased. Among these, the 400 W ultrasound-assisted glycosylation product exhibited the highest H₀ value. This may be attributed to the dissociation of protein aggregates by ultrasonic cavitation, which releases hydrophobic groups [52], combined with the glycosylation-induced extension of peptide chains that prevents the refolding of hydrophobic regions [53]. Excessively high ultrasonic power (600 W) may induce aggregation of CPI molecules, restricting the exposure of hydrophobic groups and thereby reducing the H₀ value [54]. Therefore, the CPI–CP conjugates pretreated with ultrasound at 400 W exhibited the highest surface hydrophobicity.

Fig. 4.

SDS-PAGE images (A), Determination of EAI and ESI (B), Surface hydrophobicity (C), Free sulfhydryl content (D), Solubility analysis (E). Note: Different letters indicate significant differences (p < 0.05). CPI-CP conjugate prepared without ultrasonic pretreatment (0 W), CPI-CP conjugate prepared with 200 W ultrasonic pretreatment (200 W), CPI-CP conjugate prepared with 400 W ultrasonic pretreatment (400 W), CPI-CP conjugate prepared with 600 W ultrasonic pretreatment (600 W).

3.10. Free sulfhydryl content

Free sulfhydryl groups are prone to oxidation and can form intermolecular or intramolecular disulfide bonds, thereby reflecting the interactions among protein molecules. As shown in Fig. 4D, the free sulfhydryl content of glycosylated CPI without ultrasonic pretreatment (0 W) was significantly higher than that of native CPI, indicating that glycosylation modification disrupted disulfide bonds within or between CPI molecules [55]. With increasing ultrasonic pretreatment power, the free sulfhydryl content of the subsequently formed CPI–CP conjugates gradually increased, which can be attributed to the turbulence, shear forces, and cavitation effects induced by ultrasonication. These effects promote protein unfolding and facilitate the cleavage of disulfide bonds within or between CPI molecules, thereby increasing the content of free sulfhydryl groups in the glycosylated products [56]. However, when the ultrasonic pretreatment power exceeded 400 W, the free sulfhydryl content of the CPI–CP conjugates remained essentially unchanged, suggesting that further increases in ultrasonic intensity no longer significantly enhanced disulfide bond disruption.

3.11. Solubility analysis

As shown in Fig. 4E, under the condition without ultrasonic pretreatment (0 W), the solubility of CPI significantly increased after glycosylation with CP, reaching 88.6%. This improvement could be attributed to the heat treatment during the glycosylation reaction, which disrupts secondary interactions within protein molecules, such as hydrogen bonds, disulfide bonds, hydrophobic interactions etc, thereby reducing intermolecular and intramolecular aggregation and enhancing protein solubility [17]. In contrast, the solubility of CPI–CP conjugates subjected to 200 W ultrasonic pretreatment (76.2%) was lower than that of the conjugates prepared without ultrasonication. Moreover, with the increasing of ultrasonic pretreatment power, the solubility of the CPI–CP conjugates gradually decreased. Compared to conjugates pretreated at 200 W, the solubility of conjugates pretreated at 400 W and 600 W decreased by approximately 9% and 14%, respectively. This decrease may be attributed to the reaggregation of proteins induced by excessive ultrasonic treatment [57], [58], [59]. Higher ultrasonic pre-treatment power could significantly disrupt the hydrophobic and hydrogen bonds within protein molecules, promoting greater degrees of unfolding and exposing more hydrophobic amino acid residues buried inside the molecules to their surface. The subsequent heat treatment at 80 °C further increased the exposure of hydrophobic amino acid residues embedded in protein molecules. When the hydrophobic amino acid residues of protein molecules were exposed excessively, these hydrophobic residues were also easy to associate with each other to form insoluble aggregates, resulting in the decrease of protein solubility. In addition, the increase in the rate and degree of protein glycosylation reaction caused by high-power ultrasound pretreatment was another factor leading to the decrease in protein solubility [60], [61], [62].

3.12. SDS-PAGE

The SDS-PAGE was performed to further confirm whether covalent bonding occurred between CPI and CP. The major subunits of CPI were 7S vicilin (∼50 kDa), 11S legumin (acidic subunit, ∼40 kDa; basic subunit, ∼20 kDa) [15], [63]. The protein band located at approximately 70 kDa corresponded to convicilin [63]. As shown in Fig. 4A, the subunit electrophoretic profile of CPI underwent significant alterations after ultrasound-assisted glycation treatment. The glycosylated CPI exhibited narrower bands and fading of the corresponding characteristic bands of CPI. With increasing ultrasonic power (200 W to 600 W), the band intensity in the high-molecular-weight region continued to strengthen, with particularly evident smear-like bands formed under 400 W and 600 W conditions. This indicates that the mechanical shear and cavitation effects generated by ultrasonic cavitation induce partial unfolding of CPI, exposing more free amino groups which further promoted covalent cross-linking between the protein and polysaccharide via the Maillard reaction and consequently leaded to the formation of a greater number of covalently cross-linked molecules. Moreover, the covalent complex formed between CPI and CP molecules accumulated in the loading wells of the stacking gel due to its excessive molecular weight. This observation further confirmed the occurrence of covalent binding between CPI and CP. In summary, ultrasonic pretreatment promoted the glycosylation reaction, resulting in the formation of glycosylated products with higher molecular weight.

3.13. EAI and ESI

Emulsifying properties are commonly assessed using the emulsifying activity index (EAI) and emulsifying stability index (ESI), which reflect a protein’s ability to rapidly adsorb at the oil–water interface and to maintain emulsion stability against phase separation, respectively. As shown in Fig. 4B, CPI exhibited a lower EAI (172.3 m²/g) and ESI (12.4 min) than that of CPI–CP conjugates prepared without ultrasonic pretreatment (205.5 m²/g, 28.1 min), indicating that glycosylated modification significantly enhanced both the emulsifying activity and stability of CPI. The covalent attachment of CP increased steric hindrance and electrostatic repulsion in the CPI–CP conjugates, thereby effectively inhibiting oil droplet aggregation and coalescence [64], [65]. Compared with the CPI–CP conjugates without ultrasonic pretreatment, samples subjected to ultrasonic pretreatment showed higher EAI and ESI values. With the increase of ultrasonic power, the EAI and ESI of glycosylated CPI initially increased and then decreased. As shown in Fig. 4B, the EAI and ESI values of CPI conjugates pretreated with 400 W ultrasound were 264.2 m²/g and 56.5 min, respectively, which indicated the optimal emulsifying properties. This enhanced emulsifying activity might be mainly related to the DG of CPI. In addition, the solubility and H₀ of CPI-CP conjugate sample also affected its emulsifying properties. Relative to the 0 W and 200 W pretreated samples, the 400 W pretreated sample yields improved emulsifying properties, even with reduced solubility. A possible explanation for this observation was that the 400 W pretreated sample possessed higher H₀, which could facilitate the adsorption of its molecules at the oil-water interface and promote the formation of a dense interfacial film. When compared with the 400 W pretreated sample, the 600 W pretreated sample exhibited lower solubility and H₀, which accounted for its relatively inferior emulsifying properties. The relevant results indicated that although both solubility and H₀ exerted influences on the emulsifying properties of glycosylated CPI, H₀ was the dominant governing factor for these performance attributes. Therefore, the emulsifying properties of the CPI-CP conjugate sample pretreated with 600 W was less than that of the CPI-CP conjugate sample pretreated with 400 W.

3.14. Interfacial tension

Lower interfacial tension is widely regarded as a key factor in stabilizing oil–water interfaces and facilitating emulsion formation [66]. After approximately 10,800 s of dynamic adsorption (Fig. 5A), the interfacial tension of the samples followed the order: CPI > 0 W > 200 W > 600 W > 400 W. Compared with native CPI, the CPI–CP conjugates subjected to ultrasonic pretreatment exhibited markedly lower interfacial tension, indicating that ultrasonic-assisted glycosylation enhanced the surface activity and interfacial adsorption capacity of the CPI [67]. However, the interfacial adsorption performance of the CPI–CP conjugate pretreated at 600 W was inferior to that of the sample pretreated at 400 W, which is mainly attributed to both the lower degree of glycosylation and poorer solubility of the conjugate prepared with 600 W ultrasonic pretreatment [68]. Therefore, considering the combined effects of protein structural changes, degree of glycosylation, and solubility, the CPI–CP conjugate prepared with 400 W ultrasonic pretreatment exhibited more favorable interfacial adsorption capacity.

Fig. 5.

Interfacial Tension (A), plots of surface pressure (π) vs square root of time^1/2 (s^1/2) (B), ln [(π₁₀₈₀₀ - π_t)/(π₁₀₈₀₀ - π₀)] of CPI, 0 W, 200 W, 400 W and 600 W as a function of time (s) (C). Note: CPI-CP conjugate prepared without ultrasonic pretreatment (0 W), CPI-CP conjugate prepared with 200 W ultrasonic pretreatment (200 W), CPI-CP conjugate prepared with 400 W ultrasonic pretreatment (400 W), CPI-CP conjugate prepared with 600 W ultrasonic pretreatment (600 W).

3.15. Adsorption kinetics

The adsorption behavior of molecules at interfaces generally involves three sequential processes: diffusion from the bulk phase to the interface, adsorption at the interface, and rearrangement [69]. To more comprehensively elucidate the influence of ultrasonic-assisted modification on the dynamic interfacial adsorption behavior of CPI, the diffusion rate constant (K_diff), penetration rate constant (K_p), and rearrangement rate constant (K_r) were determined. As shown in Fig. 5B, the curve illustrated the correlation between the interfacial pressure (π) and the square root of time (t^1/2). When the adsorption process of emulsifiers at the oil–water interface was diffusion-controlled, π exhibited a linear correlation with t^1/2, where the slope represented the diffusion rate, denoted as K_diff (Table 1). As shown in Table 1, The K_diff values increased with the increase of ultrasonic pretreatment power, indicating that the effect of ultrasonication enhanced molecular diffusion ability toward the interface, likely due to cavitation-induced changes in molecular structure. The functional relationship of ln[(π₁₀₈₀₀–π_t)/(π₁₀₈₀₀–π₀)] for CPI and CPI-CP conjugates as a function of time was shown in Fig. 5C. The curve exhibited two linear regions, with the slopes corresponding to the penetration rate (K_p) and reorganization rate (K_r), respectively. The variation in K_p exhibited a non-monotonic trend: it reached its maximum value at 400 W , which was significantly higher than that of the other CPI samples. This suggests that moderate ultrasonic treatment facilitated the formation of CPI-CP conjugates with more flexible structures and faster interface adsorption. Notably, the K_p value of the conjugate sample treated at 600 W was lower than that of the sample treated at 400 W, suggesting that excessive ultrasonic pretreatment may induce protein aggregation and result in suboptimal glycosylation modification, thereby inhibiting its interfacial adsorption kinetics [70]. Furthermore, all CPI-CP conjugate samples exhibited higher K_r values. When the ultrasonic pretreatment power was 400 W, the CPI-CP conjugate sample showed maximum Kr value. This indicates that appropriate ultrasonic pretreatment combined with glycosylation modification can effectively enhance the interfacial interaction capability of CPI at the oil–water interface [71]. This improvement of interfacial adsorption properties could be attribute to the dual effects of ultrasound and glycosylation on CPI which made the structure of CPI more unfolded and exposed more hydrophobic amino acid residues[72].

Table 1.

Color values of different ultrasonic pretreatment mixtures and their glycosylation products

Sample	π10800 (mN/m)	Kdiff (mN/m/s1/2)	Kp × 10-4	Kr × 10-4
CPI	15.37 ± 0.00 d	0.08 ± 0.01a	2.72 ± 0.01 d	5.68 ± 0.16a
0 W	13.22 ± 0.00a	0.10 ± 0.01b	2.76 ± 0.01c	6.91 ± 0.12b
200 W	12.41 ± 0.00b	0.12 ± 0.01c	2.73 ± 0.01b	6.93 ± 0.10c
400 W	11.33 ± 0.00c	0.13 ± 0.02c	2.83 ± 0.01a	7.68 ± 0.10e
600 W	11.79 ± 0.00e	0.13 ± 0.02c	2.75 ± 0.06c	7.38 ± 0.12 d

3.16. Interfacial dilational rheology

In order to further understand the mechanical properties and structural characteristics of the interface film formed by CPI-CP conjugates, we evaluated its dilatational rheological properties. As shown in Fig. 6A, the viscoelastic modulus (E) of all samples increased progressively with time, which can be attributed to the continuous adsorption and accumulation of protein molecules at the oil–water interface. Among all samples, the CPI-CP conjugate sample prepared with 400 W ultrasonic pretreatment showed the highest E value, indicating that it had formed a stronger and more cohesive interfacial film with superior mechanical. This enhancement could be attributed to a higher surface load-bearing capacity of interface film, which ensured sufficient interfacial viscoelastic response [73]. The time-dependent phase angle (tan φ) was analyzed to evaluate the interfacial viscoelasticity of CPI and its glycosylated conjugates (Fig. 6B). All samples maintained tan φ values below 1.0, confirming that the interfacial films were dominated by elastic behavior. The tanφ value gradually decreased with the increase of adsorption time, which indicated that its elastic contribution to the interfacial film gradually increased during the adsorption process of CPI-CP conjugates. The CPI-CP conjugate sample prepared with 400 W ultrasonic pretreatment exhibited the lower tanφ value, demonstrating superior interfacial elasticity which could be attributed to the formation of a well-structured protein-polysaccharide network at the interface. In contrast, the 600 W sample exhibited a relatively higher tan φ values, indicating a more viscous and less elastic interfacial film, which may result from excessive structural disruption and partial aggregation caused by high-intensity ultrasonic pretreatment. As shown in Fig. 6C (E–π plot), the slopes of the E–π curves for all samples were greater than 1 (a slope of 1 would reflect an ideal adsorption behavior solely dependent on the number of adsorbed molecules). This indicates that the oil/water interfacial film exhibits non-ideal gas-like behavior and the ability to form a gel-like interfacial structure.

Fig. 6.

Time evolution of the interfacial viscoelastic modulus (A), tanφ (B), Interfacial viscoelastic modulus as a function of the interfacial pressure (C), Frequency sweeps (D). Note: CPI-CP conjugate prepared without ultrasonic pretreatment (0 W), CPI-CP conjugate prepared with 200 W ultrasonic pretreatment (200 W), CPI-CP conjugate prepared with 400 W ultrasonic pretreatment (400 W), CPI-CP conjugate prepared with 600 W ultrasonic pretreatment (600 W).

Fig. 6D showed the relationship between E and oscillation frequency. As shown in Fig. 6D, the E value of all samples increased with the increasing of frequency in the range of 0.005–0.1 Hz, demonstrating clear frequency-dependent behavior. This response could attribute to interfacial relaxation mechanisms, including molecular exchange between the bulk phase and the interface, as well as structural rearrangement within the interfacial layer. At low frequencies, the interfacial film has sufficient time to adapt to deformation, resulting in relatively low viscoelastic modulus. As frequency increased, the E values enhanced, which could be explained by reduced adaptation time for molecules at the interface at high frequencies, limiting diffusion relaxation [74]. As shown in figure 6D, the interface layer stabilized by CPI-CP conjugates prepared with 400 W ultrasonic pretreatment showed the highest E value at the frequency of 0.1 Hz, surpassing the E values of interface layers stabilized by the other CPI samples. This phenomenon indicated that the CPI-CP conjugates prepared via ultrasonic-assisted glycosylation way exhibited higher dilatational viscoelasticity, resulting in a more stable emulsion system [67].

4. Conclusion

The effects of different ultrasonic pretreatment power on the chemical structures, interfacial behaviors and emulsifying properties of CPI were investigated in this study. The results indicated that ultrasonic pretreatment leads to a more stretched molecular structure of CPI, thereby effectively enhancing its subsequent glycosylated modification effects. When the ultrasonic pretreatment way was introduced in glycosylation modification process, the particle size of CPI-CP conjugates decreased but the absolute value of its zeta potential significantly increased. Ultrasonic assisted glycosylation modification effectively improved the surface hydrophobicity of CPI-CP conjugates. Compared with CPI-CP conjugates without ultrasonic pretreatment, glycosylated CPI pretreated with ultrasound possessed higher free sulfhydryl content and lower solubility. The ultrasonic assisted glycosylation modification also markedly improved the EAI and ESI of CPI. However, it should be noted that excessive ultrasonic pretreatment power could expose excess hydrophobic amino acid residues inside CPI molecules. In the subsequent glycosylated reaction, the high-intensity hydrophobic interactions between protein molecules could easily lead to the formation of insoluble aggregates, which could have a negative impact on the H₀ and solubility of CPI. A reduction in solubility and H₀ would result in the weakened emulsifying properties of CPI-CP conjugates. Moreover, it should also be noted that the reduction in H₀ induced by aggregation exerted a greater negative effect on the emulsifying properties of CPI-CP conjugates. Therefore, the CPI-CP conjugates exhibited the optimal emulsifying properties when the ultrasonic pretreatment power was set to 400 W. The analyses of CPI's interface adsorption behavior showed that ultrasonic assisted glycosylation modification significantly improved the adsorption and interaction ability of CPI at the oil-water interface, and thus formed a thicker and more robust interfacial film with better mechanical properties. These observations could be attributed to the fact that the higher values of H₀ and DG of the CPI-CP conjugates played a significant role in increasing their adsorption, diffusion, penetration, and reorganization rates, as well as facilitating the formation of an interfacial film with enhanced mechanical strength. The related results showed that the optimal composite glycosylation modification effect on CPI was achieved when the ultrasonic pretreatment power was 400 W. These findings provided new insights for further improving the emulsifying properties of glycosylated CPI. In addition, relevant research results could provide theoretical and technical references for the potential applications of CPI in the food industry.

CRediT authorship contribution statement

Changsheng Chen: Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation. Kangyu Xie: Visualization, Investigation. Yating Zhao: Software, Investigation. Xiaobing Guo: Writing – review & editing, Validation, Supervision, Resources, Investigation, Funding acquisition. Xiaoying Mao: Methodology, Data curation. Jian Zhang: Validation, Supervision, Resources, Investigation, Formal analysis, 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.