Complex coacervates of pH shift-modified walnut protein isolate and sugar beet pectin: Characterization and formation mechanism
JiangjuanYuan1
YifanCheng1
ZiyueWang2
FengJin1✉EmailJinfeng502@bjfu.edu.cn
FengjunWang1✉Emailwangfengjun@bjfu.edu.cn
1State Key Laboratory of Efficient Production of Forest Resources, Beijing Key Laboratory of Forest Processing and Safety, Hebei Province Key Laboratory of Sustainable Utilization and Development of Forest Food Resources, School of Biological Sciences and TechnologyBeijing Forestry University100083BeijingChina
2Beijing Bayi School100080BeijingChina
Jiangjuan Yuan a, 1, Yifan Cheng a, 1, Ziyue Wang b, Feng Jin a, *, Fengjun Wang a, *
Author affiliation
a State Key Laboratory of Efficient Production of Forest Resources, Beijing Key Laboratory of Forest Processing and Safety, Hebei Province Key Laboratory of Sustainable Utilization and Development of Forest Food Resources, School of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China.
b Beijing Bayi School, Beijing 100080, China.
1 These authors contributed equally to the work and should be regarded as co-first authors.
*Corresponding authors
Feng Jin
E-mail addresses: Jinfeng502@bjfu.edu.cn
Fengjun Wang
E-mail addresses: wangfengjun@bjfu.edu.cn
Abstract
The application of walnut protein isolate (WPI) in food processing is limited by its poor solubility. This study explored the characterization and formation mechanism of complex coacervates formed by pH shift-modified walnut protein isolate (HWPI) in combination with sugar beet pectin (SBP). The results showed that pH shift modification enhances the molecular flexibility and functional properties of the protein compared to the WPI without modifications. The optimal conditions for complex coacervate formation were found at pH 5 with a HWPI to SBP ratio of 7:1, where the strongest electrostatic interactions and highest turbidity were observed. The binding process was spontaneous under these conditions, influenced by thermodynamic factors. Complex coacervates demonstrated the highest turbidity at pH 5, 65°C, and NaCl concentration of 150 mmol/L, while the emulsifying activity (EA) and emulsifying stability (ES) were optimal at 25°C. This study introduces a novel approach for utilizing WPI in food applications.
Keywords:
Walnut protein isolate
Sugar beet pectin
Electrostatic interactions
Complex coacervates
1. Introduction
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Walnuts (Juglans regia L.) are one of the important nuts in the world, valued for their high content of unsaturated fatty acids, polyphenols, and walnut protein isolate (WPI). These nutrients confer multifunctional benefits to walnuts, making them a valuable natural functional food (Shen et al., 2024). WPI, the primary nutritional component extracted from walnut meal, is rich in essential amino acids and maintains a balanced amino acid profile. Moreover, WPI demonstrates excellent emulsifying properties, which enable it to effectively stabilize bioactive compounds, enhancing its potential applications in various food systems (Li et al., 2024). However, its high glutenin content (over 70%) and compact structure result in poor solubility, limiting its applications in the food industry (Yang et al., 2024). To address these challenges, physical, chemical, and enzymatic modification methods have been explored to enhance WPI's functionality. Among these, pH-shift modification is a simple and environmentally friendly physical technique by dissolving proteins at extremely acidic or alkaline pH to induce subunit separation and enhance molecular flexibility (Liu et al., 2025). Therefore, the unfolding refolding process adjusted by pH can obtain a looser and randomly arranged structure, thereby enhancing the functional properties of proteins (Liu et al., 2022). It has been successfully used to regulate the functional properties of legumes and other plant proteins. Zhou et al. (2024) found that pH treatment significantly enhanced the surface wettability of glutenin. Similarly, Li et al. (2020) reported that treating peanut protein isolates (PPI) at pH 10 significantly improved solubility, reduced particle size, increased free sulfhydryl content, and enhanced surface hydrophobicity, leading to greater breaking force and improved water-holding capacity of PPI gels.Recently, the complex coacervates formed by proteins and polysaccharides have garnered significant attention due to their excellent flexibility and self-assembly ability. Complex coacervates are phase-separated materials formed by the interaction of two or more distinct molecules in a liquid (X. Zhang et al., 2025). Their formation involves the redistribution and concentration of different components in solution, leading to the separation of condensed matter and the emergence of a high-concentration phase (Zhang et al., 2024). The primary mechanism governing complex coacervate formation relies on electrostatic interactions between charged molecules (Huang et al., 2012). Variations in the solution’s pH or ionic strength can alter these interactions, promoting the aggregation of macromolecules and the formation of stable complexes (Zhang et al., 2020). Research on the complex coacervates has been gained increasing attention, as they not only can be used as a novel materials pharmaceutical and food applications but also to gain a deeper understanding of molecular assembly and the origins of neurodegenerative diseases (Ghorbani et al., 2025). The most popular application of complex coacervates in food industries is encapsulating and delivering bioactive compounds, including vitamins (Santos et al., 2021), antioxidants (Yuan et al., 2017), and probiotics (Zhao et al., 2020), enhancing their stability during food processing and storage. Additionally, coacervate-based coatings have been shown to enhance food texture and taste, contributing to a better sensory experience for consumers (Oliveira et al., 2020).
Sugar beet pectin (SBP), an amphiphilic block copolymer composed of protein-polysaccharide conjugates, contains 1–10% protein (Guo et al., 2025). Its rich side-chain structure, high acetyl content, and significant protein fraction endow it with excellent emulsifying properties, making it widely used as a gelling agent in food processing (Sun et al., 2024). Recently, the potential applications of SBP in various food products have been emphasized, particularly its role in forming complex coacervates with proteins, such as pea protein isolate (Lan et al., 2020) and whey protein isolate (Wang et al., 2018), which have attracted considerable attention.
To the best of our knowledge, no research has been conducted on the complex coacervation of WPI and SBP. While prior studies have investigated the preparation, characterization and bio-functional activity of microencapsulation of carvacrol by complex coacervation of walnut meal protein isolate and gum Arabic (Sun et al., 2022), there is still a lack of specialized research on WPI and SBP complexes to gain a deeper understanding of their molecular interactions. Therefore, this study aims to systematically investigate the effect of pH-shift modification on the formation process of HWPI-SBP complex coacervates and to elucidate their formation mechanism. First, the emulsifying activity (EAI) and emulsifying stability (ESI) of WPI, HWPI, and HWPI-SBP complex coacervates at different ratios were evaluated. Changes in secondary structure were analyzed to determine the effects of pH-shift modification on WPI structure and complex coacervate formation. Additionally, the turbidity of HWPI, SBP, and their complex coacervates at varying ratios and functional pH levels was measured. The rheological properties of the complex coacervates were also assessed. Under optimal conditions, the turbidity and yield of the complex coacervates were examined. Furthermore, fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), microstructural analysis, and fluorescence quenching mechanisms were used to elucidate the potential formation mechanism of HWPI-SBP complex coacervates. This study provides a theoretical foundation and technical support for the application of plant protein-polysaccharide complex coacervates in the food industry.
2. Materials and methods
2.1. Materials
Commercial walnuts were obtained from the Akesu Industrial Base (Xinjiang, China). Sugar beet pectin (SBP) was purchased from Nanjing Sijiqianshun Biotechnology Co., Ltd. Hydrochloric acid (HCl) was sourced from Beijing Chemical Plant, while sodium hydroxide (NaOH) was supplied by Xilong Chemical Co., Ltd. Sodium chloride (NaCl) and all other reagents were of analytical grade.
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2.2. Extraction and pH-shift treatment of WPI2.2.1. Extraction of WPI
The extraction of walnut protein isolate (WPI) was performed as described by Wang et al. (2024). Defatted walnut powder was dissolved in deionized water at a 1:20 (w/v) ratio, and the pH was adjusted to 11.0 using NaOH solution. The mixture was magnetically stirred at room temperature for 3.5 h, followed by centrifugation at 8000 r/min for 15 min to collect the supernatant. The pH of the supernatant was subsequently adjusted to 4.5 with HCl solution, and the mixture was left overnight at 4°C to facilitate protein precipitation. The precipitated protein was collected by centrifugation under the same conditions, followed by pH neutralization using NaOH solution. The final product was vacuum freeze-dried and stored at 4°C for further analysis.
2.2.2. pH-shift modification of WPI
WPI was dissolved in deionized water, and the solution pH was adjusted to 11.0 with 1M NaOH solution. The mixture was continuously stirred at room temperature for 3 h. The pH was then readjusted to 7.0 with 1 M HCl solution, followed by centrifugation at 3000 r/min for 10 min to remove insoluble components. The resulting solution was freeze-dried to obtain HWPI (Plati et al., 2021).
2.3. Preparation of HWPI and SBP coacervates solution
The SBP solution was prepared by dissolving SBP in deionized water and stirring magnetically at room temperature for 2 h to ensure complete dissolution. The composite solution was then formulated by mixing 1% (w/v) HWPI and 1% (w/v) SBP solutions at various ratios (1:1, 3:1, 5:1, 7:1, and 9:1), with deionized water added to achieve a final concentration of 0.1% (w/v). The pH of the composite solutions was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, or 9.0 using 1M NaOH to facilitate complex coacervate formation. The preparation conditions were optimized by evaluating turbidity, zeta potential. The ζ-potential of the mixtures was measured using a laser nanoparticle analyzer (ZS90, Malvern Instruments Limited, UK), with each sample analyzed in triplicate. Turbidity was assessed using a method adapted from Qiu et al. (2022). WPI, HWPI, and SBP were individually dissolved in deionized water to prepare 0.1% (w/v) solutions, with the pH adjusted in increments of 0.5 units from 2.0 to 7.0. Turbidity was recorded at OD600 using a UV-Vis spectrophotometer (L60, Yidian Analysis Instrument Co., Ltd, Shanghai, China).
2.4. Determination of emulsifying capacity (EA) and emulsifying stability (ES)
The emulsifying capacity (EA) and emulsifying stability (ES) of WPI, HWPI, and the complex coacervates formed by HWPI and SBP at different ratios were evaluated using the method described by Wang et al. (2024). The HWPI-SBP composite was mixed with WO using a high-speed shear mixer (FM-200A, Fluke Technology Development Co., Ltd, Shanghai, China) at 10,000 r/min for 2 min. A 5 µL sample was taken from the bottom of the beaker at both the initial time and after 10 min. A 1% SDS solution was then added at a lotion-to-SDS volume ratio of 1:500. The absorbance of the sample at 500 nm was recorded. EA and ES were calculated using the following formulas:
Where A0 represents the absorbance value of the initial sample; A10 is the absorbance value of the sample at 10 min; DF is the dilution factor; Φ represents the optical path (1 cm); θ is the dispersion coefficient of oil phase in lotion (0.25).
2.5. Rheological properties
The rheological properties of HWPI-SBP complex coacervates was measured according to Yu et al. (2024) with slight modifications. The viscosity was measured using a Physical MCR30 rheometer (Anton Paar GmbH) with the temperature maintained at 25°C and an equilibrium time of 2 min, within a shear rate range of 0.1–100 s⁻¹. The storage modulus (G′) and loss modulus (G″) as a function of frequency were determined at a fixed strain of 1% and a shear rate of 0-100 Hz. Each sample was measured in triplicate.
2.6. Determination of yield
For yield determination, an HWPI-SBP solution with an optimal blending ratio of 7:1 (w/w) and a total concentration of 0.1% (w/v) was prepared. The pH of the solution was adjusted to 3.5, 4.0, 4.5, 5, and 5.5, respectively, using acetic acid, and the solution was stirred magnetically at 600 rpm to ensure complete reaction. The precipitate was then collected via centrifugation and dried in an oven. The yield of the complex coacervates was calculated using the following formula:
Where m1 and m2 respectively represent the mass of dried complex coacervates, initial mass of complex coacervates, (g).
2.7. The analysis of interaction
2.7.1. Intrinsic fluorescence spectroscopy (IF)
A 0.01% (w/v) HWPI-SBP composite solution was prepared, and its intrinsic fluorescence intensity was measured at different pH values using a fluorescence spectrometer (F-7100, Hitachi Ltd., Japan). The excitation wavelength was set to 295 nm, with a scanning range of 300–450 nm. The results were expressed as the average of three scans.
2.7.2. Fourier transform infrared spectroscopy (FTIR) analysis
Following freeze-drying, 2 mg of HWPI, SBP powder, and HWPI-SBP complexes coacervates with varying ratios were analyzed using a Nicolet iS5 infrared spectrometer (Thermo Fisher Scientific Co., Ltd). Each sample was scanned 32 times within a spectral range of 400–4000 cm⁻¹. Background spectra were recorded under identical conditions and used as controls.
2.7.3. Secondary structure
Fourier Transform Infrared Spectroscopy (FTIR) was used to analyze the secondary structure composition of WPI, HWPI, and HWPI-SBP complex coacervates at different compounding ratios. The amide I band (1600–1700 cm⁻¹) was recorded and processed using second derivative techniques. Peak fitting and deconvolution were performed using Peakfit software to determine the relative content of each secondary structure present in the samples.
2.8. Microstructure
Freeze-dried WPI, HWPI, and HWPI-SBP condensed layer powders were mounted onto circular aluminum stubs using double-sided tape and coated with a 15 nm gold layer. Scanning electron microscopy (SEM) images were then captured at magnifications of 200× and 500×.
2.9. Thermodynamic properties
The interaction between HWPI and SBP was evaluated based on intrinsic fluorescence (IF) analysis, following the method of Lin et al. (2022) with slight modifications. A series of test solutions was prepared by adding 0.2 mL of SBP solutions at varying concentrations to 3 mL of HWPI solution, resulting in final SBP concentrations of 0, 0.1, 0.2, 0.3, and 0.5 mmol/mL. The solution pH was adjusted to 5.0. The excitation wavelength was set at 295 nm, with both excitation and emission slit widths maintained at 2.5 nm.
The intrinsic fluorescence of HWPI as a function of SBP concentration was measured at different temperatures (298, 308, and 318 K). The Stern-Volmer equation was applied to elucidate the fluorescence quenching mechanism, while the double logarithmic equation was used to determine the binding constant and number of binding sites (n) of SBP to HWPI. The Van't Hoff equation and thermodynamic equations were employed to analyze the IF data across different temperatures and derive key thermodynamic parameters. Based on these parameters, the interaction forces and thermodynamic properties governing the HWPI-SBP system were inferred.
Where F and F0 respectively represent the fluorescence intensity of HWPI solution in the presence and absence of SB; KSV is the Stern Volmer quenching constant, given by the slope of the linear S-V curve of Eq. (5); [Q] It is the concentration of polysaccharides, measured in mol/L. Kq is the quenching rate, and τ 0 = 10 − 8 (represents the average fluorescence lifetime). Ka is the binding constant between HWPI and SBP, n represents the number of binding sites between each polysaccharide and protein, and R is the gas constant (8.314 J. K− 1. mol− 1). Calculate the number of binding sites and binding constants based on the slope and intercept of the double logarithm of the parameter obtained from Eq. (6). ∆G is Gibbs free energy change; ∆H is enthalpy change; T is temperature; ∆S is entropy change and represents the ideal gas constant. Calculate the changes in enthalpy and entropy based on the slope and intercept of the lnKa curve relative to 1/T.
2.10. Effect of temperature and NaCl concentration and on the turbidity of complex coacervates
SBP solution was prepared by dissolving SBP in deionized water and stirring magnetically at room temperature for 2 h to ensure complete dissolution. The composite solution was then obtained by mixing 1% (w/v) HWPI and 1% (w/v) SBP solutions at a 7:1 ratio, followed by the addition of NaCl solution at varying concentrations (50, 150, 300, and 500 mmol/L) to achieve a final solution concentration of 0.1% (w/v). The composite solutions were subsequently heated at different temperatures (25, 45, 65, and 85ºC) for 30 min, and the pH was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, or 9.0 using NaOH to facilitate the formation of complex coacervates. Following the method described in Section 2.4, the effects of ionic strength (0–500 mmol/L) and temperature (25–85ºC) on the formation of complex coacervates were investigated while maintaining all other conditions constant, with a fixed HWPI-SBP ratio of 7:1 (w/w).
2.11. Effect of pH, NaCl concentration and temperature on the EA and ES stability of complex coacervates
The effects of pH, NaCl concentration, and temperature on the EA and ES of complex coacervates were evaluated according to the method described in Section 2.4. The solution pH was adjusted to 3.0, 4.0, 5.0, 6.0, and 7.0, while the water bath temperature was set at 25, 45, 65, and 85ºC. Additionally, NaCl concentrations of 0, 50, 150, 300, and 500 mmol/L were used to assess their impact on the EA and ES of complex coacervates.
2.12. Statistical analysis
All experiments were conducted in triplicate, and results are expressed as the mean ± standard deviation (SD). Data analysis was performed using Origin 2021 software, and statistical significance was assessed using IBM SPSS Statistics 22.0. A p-value of < 0.05 was considered statistically significant.
3. Results and discussion
3.1. The optimization of formation conditions for complex coacervates
3.1.1. EA and ES of WPI, HWPI and complex coacervates
Protein molecules, due to their hydrophilic and lipophilic properties, can adsorb onto the oil-water interface, forming a protective layer on the surface and thereby exhibiting enhanced emulsifying ability (Yu et al., 2024). The emulsifying properties of proteins are typically assessed using the emulsifying activity index (EAI) and the emulsifying stability index (ESI) (Alavi et al., 2021)As shown in Fig. 1A, EA and ES of HWPI increased after pH shift modification. The EA reflects the emulsifier's ability to rapidly adsorb at the oil-water interface. The pH shift modification increased the exposure of hydrophobic groups (Ma et al., 2025), and this structural alteration enhanced protein-oil droplet interactions, promoting protein adsorption at the oil-water interface (Ma et al., 2025). Consequently, the EA and ES of HWPI improved. When the ratio of HWPI to SBP exceeded 7:1 (w/w), a decline in EA and ES was observed. Compared to WPI and HWPI, the enhanced EA and ES of the HWPI-SBP complex coacervates can be attributed to the increased exposure of aromatic residues following the protein-polysaccharide interaction. This exposure of hydrophobic groups facilitates interactions between the complex coacervates and the oil phase, promoting the formation of a stable network structure (Zhang et al., 2021).
3.1.2. Secondary structure analysis
The changes in the protein secondary structure are depicted in Fig. 1B. After pH modified, the content of a-helix from 26% decreased to 23.2% while the random coil from 15% increased to 17%. Compared to WPI, no significant changes were observed in the β-sheet and β-turn contents of the HWPI. The α-helix and β-sheet represent the tightly packed arrangements of the protein structure, while β-turns and random coils reflect its looseness (Yang et al., 2024). This suggests that after treatment with pH shift, WPI transitions from an ordered, stable structure to a disordered, flexible. With the introduce of SBP, the content of α-helix decreased while β-sheet, β-turns and random coils were increased. This can be attributed to the introduction of SBP, which may interact with WPI through hydrogen bonding, electrostatic interactions, or hydrophobic interactions, thereby promoting further structural rearrangement. These interactions likely destabilize the α-helix and favor the formation of other secondary structures, such as β-sheets, β-turns, and random coils. β-sheets and β-turns are typically associated with less ordered, more flexible regions in proteins, thus increased content in the presence of SBP (Hou et al., 2017). The higher proportion of random coils indicates further disorder and increased flexibility in the protein structure, resulting from the combined effects of the pH shift and SBP introduction.
3.1.3. Range of HWPI-SBP ratios and pH for turbidity determination
The ratio of protein to polysaccharide and pH conditions can significantly affect the formation of complex coacervates by altering the charge density of charged particles (Chen et al., 2025). The turbidity changes during the formation process of the complex coacervates, induced by variations in the HWPI to SBP ratio and pH, are shown in Fig. 1C. The alteration of the HWPI to SBP ratio significantly affected the turbidity of the system, which was closely correlated with the number of proteins attached to the polysaccharide branches, thereby influencing the assembly of the micelles (J. Zhang et al., 2025). The complex coacervates exhibit maximum turbidity within the pH range of 4.5–5.0. As the ratio of HWPI to SBP increases from 1:1 to 7:1, turbidity initially increases, then decreases, with the highest turbidity value (0.97) observed at a HWPI to SBP ratio of 7:1. Generally, the maximum turbidity indicates the optimal conditions for complex coacervate formation, attributed to the charge equilibrium between the protein and polysaccharide (Zheng et al., 2022). The variation in peak values at different ratios is due to the distinct molecular structures and charge levels of HWPI and SBP, resulting in different internal charge densities of the insoluble complexes. When the HWPI to SBP ratio exceeds 7:1, the turbidity of the complex coacervates decreases. This is because higher ratios of HWPI or SBP limit the electrostatic interactions between the two biopolymers, leading to electrostatic repulsion and self-aggregation (Cui et al., 2025).
3.1.4. Rheological characterization of the complex coacervates
In the food industry, proteins and polysaccharides are used as food thickeners to enhance water-binding capacity, modify structural properties, and alter the flow behavior of food systems (Fu et al., 2025). Figure 1D shows the viscosity changes of HWPI and SBP composite aggregates with different ratios at pH 5 as the shear rate increases. Within the shear rate range of 0-100 s− 1, the viscosity of all samples sharply decreases with increasing shear rate, exhibiting a shear thinning phenomenon unique to non-Newtonian fluids. In the HWPI-SBP complex coacervate system, viscosity decreases as the SBP content decreases. The reduction in SBP concentration weakens intermolecular interactions, leading to a decrease in shear viscosity. Higher viscosity suppresses molecular movement, thereby delaying the phase separation process and contributing to system stability. A moderate amount of SBP plays a crucial role in enhancing the stability of the HWPI-SBP complex coacervates system.
Figure 1E shows the influence of different blending ratios of HWPI-SBP complex coacervates on their viscoelastic properties. The storage modulus reflects the elastic characteristics of the aggregates, while the loss modulus reflects the viscous characteristics. Throughout the measurement process, the storage modulus consistently remains higher than the loss modulus, indicating that the aggregates exhibit greater elasticity than viscosity in terms of rheological properties. As the frequency increases, the viscoelastic moduli of all aggregates show a significant upward trend, suggesting the formation of a more compact network structure by the copolymers (Fu et al., 2025). When the polysaccharide content becomes saturated, the polysaccharides are unable to form a coiled structure due to steric hindrance. Therefore, as the SBP ratio increases (from a HWPI-SBP ratio of 9:1 to 1:1), both the storage modulus and loss modulus of the complex coacervates decrease.
Fig. 1
EA and ES (A) and secondary structure content (B) of WPI, HWPI and different ratio of HWPI to SBP; turbidity of HWPI, SBP and coacervate formed by different HWPI and SBP mixing ratios (C); Rheological sweeps (D) and frequency sweeps (E) of HWPI-SBP coacervates formed by different HWPI and SBP mixing ratios.
3.2. Effect of pH on the zeta potential, turbidity and complex coacervates yield
3.2.1. ζ-potentials of HWPI-SBP complex coacervates as a function of pH
The ζ potential can be used to assess the stability of coacervates and the interactions between different complex. Under the same charge, a higher absolute ζ potential value (typically greater than 30 mV) indicates strong electrostatic repulsion between particles, which helps maintain the stability of the dispersion system. Conversely, lower ζ potential values make flocculation or sedimentation more likely to occur (Souza et al., 2023). The zeta potential of WPI, HWPI, and SBP molecules was shown in Fig. 2A. The isoelectric point of WPI is approximately 4.6, while that of HWPI is around 5.2. As the pH decreased from 6.0 to 3.0, the zeta potential of HWPI increased from − 19.1 mV to + 36.7 mV. The isoelectric point of SBP is approximately 3.9, and within the pH range of 6.0 to 3.0, its zeta potential continuously increased as the pH decreased, becoming positive when the pH reached 3.9. Thus, when HWPI and SBP are within the pH range of 3.9 to 5.2, HWPI carries a positive charge while SBP carries a negative charge. The opposite charges of the two components facilitate electrostatic interactions. The optimal pH for complex aggregation, determined by turbidity titration, is 5.0, where electrostatic interactions are strongest. Pillai et al. found that the complex aggregation reaction between mung bean protein isolate and apricot peel pectin occurs only at pH values below 4.8. This is because mung bean protein isolate becomes positively charged at pH values below 4.8, while apricot peel pectin remains negatively charged across the tested pH range (Souza et al., 2023). The opposite charges enable electrostatic interactions, promoting the complex aggregation reaction.
3.2.2. Turbidity of complex coacervates
A HWPI to SBP ratio of 7:1 was selected to further investigate the turbidity variations resulting from changes in solution pH (Fig. 2B). The turbidity of HWPI and SBP alone was significantly lower than that of the complex coacervates, further indicating that the formation of complex coacervates depends on the interaction between HWPI and SBP. When pH < pHφ2, the turbidity values of HWPI-SBP were close to zero, which can be attributed to the strong electrostatic repulsion between the negatively charged HWPI and SBP, resulting in minimal aggregation. When pHφ2< pH < pHₒₚₜ, turbidity increased, indicating that the complex grew in both size and number, causing the solution to transition from transparent to cloudy (Pillai et al., 2019). The maximum complex formation (highest turbidity) occurred at pHₒₚₜ (pH = 5), primarily due to the neutralization of the charges on HWPI and SBP, achieving charge equilibrium (Zheng et al., 2022) When pHₒₚₜ < pH < pHc, turbidity gradually decreased until the critical point pHc (pH = 7.5), indicating the dissociation of the HWPI-SBP complex coacervates. In conclusion, the turbidity change in HWPI-SBP complex coacervates is primarily due to the protonation/deprotonation of amino and carboxyl groups on the protein side chains caused by changes in solution pH (X. Zhang et al., 2025). The HWPI-SBP aggregation at pH 4.5–5.0 exhibits excellent complexation cohesion.
As shown in Fig. 2C, the 7:1 ratio is confirmed again as the optimal ratio for complex coacervate formation, as the highest coacervate yield was observed at this ratio. This further supports the finding that the electrostatic interaction between HWPI and SBP is most pronounced under pH 5.0 conditions (X. Zhou et al., 2024). Therefore, the optimal conditions for preparing HWPI-SBP complex coacervates are a HWPI to SBP ratio of 7:1 at pH 5.0.
Fig. 2
The zeta potential of WPI, HWPI and SBP (A), turbidity of HWPI, SBP and HWPI:SBP (7:1) mixed solution (B) and coacervates yield of HWPI:SBP (7:1) mixed solution as a function of pH (C).
3.3. The formation and properties of HWPI-SBP
3.3.1. Intrinsic fluorescence (IF) spectroscopy
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IF was used to demonstrate the successful complexation and aggregation between HWPI and SBP. As shown in Fig. 3A, the fluorescence intensity of HWPI was higher than that of WPI. This increase in fluorescence intensity is attributed to the disruption of hydrophobic groups within HWPI, which exposes aromatic residues. However, the fluorescence intensity of HWPI-SBP complex coacervates significantly decreased. This decrease is due to the entanglement of polysaccharide molecules with protein molecules, which causes some of the tryptophan residues in the protein to be encapsulated within the side chains of the polysaccharide macromolecules (Li et al., 2021). Consequently, the fluorescence intensity is reduced due to the quenching effect of the polysaccharide. At pH 5.0, under the influence of electrostatic interactions, complexes form between SBP and HWPI molecules. The side chains of SBP molecules exhibit a certain binding affinity when interacting with HWPI, and HWPI molecules may also attract one or more side chains of SBP molecules. These electrostatic interactions, along with the repulsive forces between proteins, affect the microenvironment of amino acids within the protein, causing some tryptophan residues to shift to a more hydrophobic environment (Cui et al., 2025). This shift further reduces the fluorescence intensity of the HWPI-SBP coacervates.3.3.2. Fourier transform infrared (FTIR) spectroscopy
FTIR spectroscopy was used to confirm the formation of HWPI-SBP complex coacervates and analyze the interactions between the biopolymers during the aggregation process (Fig. 4D). The FTIR spectrum of proteins exhibits three characteristic peaks: Amide I, Amide II, and Amide III. The Amide I band (1700–1600 cm⁻¹) corresponds to the C = O stretching vibration of the amide group, the Amide II band (1550–1450 cm⁻¹) is primarily associated with the N-H bending vibration, and the Amide III band (1229–1301 cm⁻¹) represents the N-H bending and C-N stretching vibrations, respectively. As shown in Fig. 3B, the SBP spectrum displays typical polysaccharide absorption bands at 3316.04 cm⁻¹, 2923.56 cm⁻¹ (C-H stretching vibration), 1637.12 cm⁻¹ (COO⁻ symmetric stretching vibration), and 1011.85 cm⁻¹ (C-O-C stretching vibration) (Synytsya, 2003). The FTIR spectrum of the HWPI-SBP complex coacervates exhibits absorption peaks at 3273.78 cm⁻¹, 1630.18 cm⁻¹, and 1248 cm⁻¹, which are similar to those in the HWPI spectrum; however, the peak at 1448.15 cm⁻¹ disappears. In comparison to the SBP spectrum, the absorption peak at 3316.04 cm⁻¹ vanishes, and a distinct peak appears at 1017.24 cm⁻¹. Additionally, the characteristic peak at 1637.12 cm⁻¹ is absent. The changes observed in the carbonyl-amide region of the HWPI-SBP complex coacervates suggest electrostatic interactions between the NH₃⁺ group in HWPI (1448.15 cm⁻¹) and the -COO⁻ group in SBP (1637.12 cm⁻¹). The shift in the Amide I region of the HWPI-SBP complex coacervates, compared to SBP, indicates the involvement of hydrogen bonding in the reaction. This shift is attributed to the introduction of SBP, which enhances hydrogen bonding within the composite aggregation layer (Huang et al., 2012).
3.3.3 Microstructure of the complex coacervates
SEM was used to examine the surface morphology and microstructural characteristics of the coacervates. The variation in coacervate morphology at different pH levels was observed (Fig. 3C). A comparison of the surface morphology of WPI and HWPI revealed that the surface of WPI exhibits a smooth, flat, and irregular block-like structure with slightly noticeable angular features. In contrast, the surface of HWPI shows a degree of unevenness with folds, and plate-like structures similar to those of WPI can be observed. Although these plate structures are much smaller than those of WPI, they tend to form large aggregates. This behavior is attributed to the protein structure unfolding during the pH shift modified process, which promotes protein denaturation and exposes hydrophobic groups (Yingying Ma et al., 2025). For the HWPI-SBP coacervates (formed at pH 7.0), the addition of SBP results in aggregates with irregular plate-like and rod-like structures that exhibit a loose morphology and varying sizes. SEM images (Fig. 3C) revealed that HWPI-SBP coacervates at pH 5.0 formed dense plate-like structures, whereas at pH 7.0, irregular aggregates predominated. This morphology difference correlates with turbidity data (Fig. 2B) and suggests that charge neutrality at pH 5.0 promotes ordered coacervate assembly (X. Zhang et al., 2025). The neutralization of effective charges between the biopolymers facilitates the formation of a denser network structure in the aggregation layer, which further enhances the stability of the aggregation layer and improves the aggregation performance of the complex coacervates. 
Figure 3 IF of WPI, HWPI and HWPI-SBP coacervates (A) and FTIR of SBP, HWPI and HWPI-SBP coacervates (B); SEM images (C) of WPI (a) and (d); HWPI (b) and (e); HWPI-SBP coacervates at pH5.0 (c) and (f); a schematic diagram of the structural mechanism of the HWPI-SBP network (g).
3.4. Fluorescence quenching mechanism and binding constant
Fluorescence spectroscopy can be employed to investigate the molecular interactions of polymers in small molecule mixtures (Lan et al., 2020). The fluorescence quenching spectra at different temperatures, shown in Fig. 4A-C, reveal that increasing the SBP concentration at three different temperatures leads to a gradual reduction in fluorescence intensity at 332 nm. This indicates that SBP induces fluorescence quenching in HWPI. Fluorescence quenching mechanisms are generally categorized into static and dynamic quenching, with the quenching mechanism determined by the quenching rate constant (Kq). To further investigate the quenching mechanism between HWPI and SBP, the Stern-Volmer equation was applied. The relationship between F₀/F and polysaccharide concentration at 288 K (14.85°C), 298 K (24.85°C), and 310 K (36.85°C) is shown in Fig. 5D. The dynamic quenching constant (Kₛv) was calculated using the formula, with the results presented in Table 1. The curve is linear, indicating that there is only one type of fluorophore on HWPI, and all of them can be equally quenched by the polysaccharide (Qiu et al., 2022). The decrease in the KSV value with increasing temperature suggests that the quenching process is likely due to static quenching resulting from the binding of HWPI and SBP (Joye et al., 2015). The binding constant and binding sites of the HWPI-SBP complex coacervates were determined using the double logarithmic equation, with results shown in Fig. 4E and Table 1. At room temperature, the number of binding sites between HWPI and SBP is approximately 1. The binding constants at 298 K, 308 K, and 318 K are 5.7387, 2.5676, and 0.8943, respectively. As the temperature increases, the binding constants decrease, indicating that the binding strength of the complex coacervates weakens with increasing temperature, which suggests thermal instability. The thermodynamic parameters of the HWPI-SBP interaction were calculated using the van 't Hoff equation. According to Table 1, the negative ∆G for the HWPI-SBP complex coacervates binding process indicates that the binding is spontaneous under the measured conditions and driven by thermodynamic factors. Additionally, the negative values for both ∆S and ∆H suggest that the binding is primarily influenced by hydrogen bonding interactions and involves an exothermic reaction (Wang et al., 2022).
Fig. 4
Effect of SBP on intrinsic fluorescence of HWPI at 298 K (A) 308 K (B) and 318 K (C); Stern–Volmer plots of HWPI-SBP coacervates at different temperatures (D); Double logarithmic plots of HWPI-SBP coacervates at different temperatures (E); Van’t Hoff plot of HWPI interacting with SBP (F)
Complex coacervates | Temperature (K) | KSV | Ka (M− 1) | n | ∆S (kJ/mol.K) | ∆H (kJ/mol) | ∆G (kJ/mol) |
|---|---|---|---|---|---|---|---|
HWPI-SBP | 298 | 23.7742 | 5.7387 | 1.4612 | -0.3729 | -123.8308 | -12.7139×104 |
308 | 29.1630 | 2.5676 | 0.8593 | -8.9851×104 | |||
318 | 39.6440 | 0.8943 | 0.5043 | -5.2564×104 |
3.5. Different effects on the turbidity of complex coacervates
3.5.1. The influence of temperature on the turbidity of complex coacervates
The effect of temperature on the HWPI-SBP complex coacervates was examined within the range of 25–85ºC. As shown in Fig. 5A, the turbidity value peaked at 65ºC, the interaction between oppositely charged proteins and polysaccharides involves both hydrogen bonding and hydrophobic interactions (Lim & Blocher McTigue, 2024). At lower temperatures, hydrogen bonding predominates, while at higher temperatures, increased molecular motion shifts the balance toward hydrophobic interactions as the dominant force. This suggesting that hydrogen bonding plays a crucial role in stabilizing the complex aggregates at this temperature (Maude Girard, 2002). Xiong et al. (2021) found that as temperature increased, the binding constant of the gelatin-pectin complex decreased, indicating a weakening of the binding strength between gelatin-pectin aggregates with heating. They proposed that the gelatin-pectin complexation involved both electrostatic interactions and hydrogen bonding
3.5.2. The influence of salt ion concentration on the formation of complex coacervates
The effect of NaCl concentration on coacervate formation was investigated (Fig. 5B). As the NaCl concentration increased, turbidity initially rose, then decreased at pH 5.0. The highest turbidity value was observed at a NaCl concentration of 150 mmol/L. Further increases in NaCl concentration led to a decline in turbidity. Excessive NaCl addition results in competition between Na+ ions and the positive charge groups of HWPI, affecting its adsorption to the side chains of SBP. Similarly, Cl− ions compete with the negatively charged groups on SBP side chains, reducing the adsorption efficiency of HWPI molecules. This competitive effect weakens the binding between the protein and polysaccharide molecules, diminishing the electrostatic interactions between them (Liu et al., 2023). Additionally, high salt concentrations reduce the entropic driving force for coacervate formation, ultimately destabilizing the liquid-liquid phase separation (Meng et al., 2017). In contrast to the inhibitory effect of high salt concentrations, the presence of a small amount of salt can enhance coacervate formation by softening electrostatic interactions, promoting more accessible polymer conformations, and facilitating chain rearrangements (Gul et al., 2023).
Fig. 5
Effect of temperature (A) and NaCl concentration (B) on the turbidity of HWPI:SBP (7:1) mixed solution as a function of pH
3.6. Different effect on the EA and ES of complex coacervates formed by HWPI-SBP
3.6.1. Effect of pH on the EA and ES of complex coacervates
Figure 6A illustrates the effect of pH on the EA and ES of HWPI-SBP complex coacervates. As pH increases, both the EA and ES of the coacervates also increase. However, under acidic conditions, particularly near the isoelectric point of HWPI, significant aggregation and sedimentation occur, leading to the formation of large, structurally dense molecular particles in the solution, which results in a lower EA. When pH > 5.2, both HWPI and SBP carry negative charges. SBP then interacts with HWPI through hydrophobic or hydrogen bonding interactions, promoting the exposure of hydrophobic groups on the protein surface. This facilitates protein dissociation and diffusion at the interface, leading to a significant increase in EA (Lim & Blocher McTigue, 2024).
3.6.2. Effect of NaCl concentration on the EA and ES of complex coacervates
Figure 6B illustrates the EA and ES of HWPI-SBP complex coacervates as a function of NaCl concentration, ranging from 0 to 500 mmol/L. The addition of NaCl influences the composite aggregation process. At optimal ionic strengths, salt ions shield electrostatic repulsions, creating a favorable environment for protein aggregation. This enhances protein interactions and promotes aggregate formation, leading to the highest emulsion stability at a NaCl concentration of 150 mmol/L. As the salt concentration increases further, SBP molecules gradually desorb from the droplet surface, causing the polysaccharide molecular layer to thin, which in turn affects ES. The initial addition of ions to the protein suspension may improve the hydrophilic/lipophilic balance by increasing surface hydrophobicity. However, as the ion concentration increases, ions begin to screen the net charge of the proteins and emulsion droplets, reducing electrostatic repulsion and impairing emulsifying properties. At high ionic strengths, the reduction in net charge promotes protein aggregation (decreasing solubility), leading to flocculation and instability of the emulsion (Gul et al., 2023). Therefore, the significant decrease in EA and ES under high ionic strength is likely due to the electrostatic shielding effect of the ions.
3.6.3. Effect of temperature on the EA and ES of complex coacervates
Figure 6C examines the EA and ES of HWPI-SBP complex coacervates within the temperature range of 25–85ºC. The results show that the thermal stability of HWPI-SBP coacervates is low, with ES continuously decreasing upon heating. Elevated temperatures likely disrupt the equilibrium of interaction forces between the composite condensates, influencing their phase separation (Ma et al., 2025). As the temperature increases, the protein-polysaccharide composite is prone to bridge flocculation, leading to a rapid decline in ES. Huang et al. found that with the temperature increases, the absolute value of the zeta potential of the emulsion gradually decreases. This reduction in the absolute value of the zeta potential weakens the electrostatic repulsion between the emulsion droplets, which is detrimental to the stability of the emulsion (Huang et al., 2023). Therefore, high temperatures are detrimental to maintaining the EA and ES of complex coacervates.
Fig. 6
Effect of pH (A); NaCl concentration (B); temperature (C) on the EA and ES of HWPI-SBP coacervates
4. Conclusion
This study investigated the complex coacervation between HWPI and SBP. At a fixed HWPI:SBP ratio of 7:1 and pH 5, the system exhibited optimal EA and ES, along with the highest yield. The SBP chains form a network around the HWPI molecules, imparting viscoelasticity to the condensed layer, with elasticity being the dominant characteristic. Rheological measurements indicated that the condensed layer shows strong gel like behavior and high mechanical strength, particularly at pH 5. Additionally, both excessively high and low pH values disrupt the electrostatic interactions between polymers or enhance electrostatic repulsion, thereby weakening the interactions between the polymers. Electrostatic interactions and hydrogen bonding are the primary forces driving the formation of complex coacervates. The binding process of the condensed layer occurs spontaneously under the tested conditions, suggesting potential applications in the food industry. This study provides valuable insights into the complexation mechanism between plant proteins and polysaccharides, which could inform the development of new food formulations.
CRediT authorship contribution statement
Jiangjuan Yuan: Writing –original draft, Methodology, Investigation, Conceptualization. Yifan Cheng: Data curation, Software, Formal analysis. Ziyue Wang: Software, Formal analysis, Data curation. Feng Jin: Validation, Formal analysis, Data curation. Fengjun Wang: Resources, Conceptualization, Supervision, Writing - review & editing, Funding acquisition, Validation.
Declaration of competing interest
The authors declared that there is no conflict of interest with the work reported in this paper.
A
Acknowledgement
This research was supported by the Fundamental Research Funds for the Central Universities (BLX202322), the Fundamental Research Funds for the Central Universities (QNTD202509) and the Fundamental Research Funds for the Central Universities (BFUKF202515).
A
Funding
This research was supported by the Fundamental Research Funds for the Central Universities (BLX202322), the Fundamental Research Funds for the Central Universities (QNTD202509) and the Fundamental Research Funds for the Central Universities (BFUKF202515).
A
Data Availability
Data will be made available on request.
A
Author Contribution
Jiangjuan Yuan: Writing –original draft, Methodology, Investigation, Conceptualization. Yifan Cheng: Data curation, Software, Formal analysis. Ziyue Wang: Software, Formal analysis, Data curation. Feng Jin: Validation, Formal analysis, Data curation. Fengjun Wang: Resources, Conceptualization, Supervision, Writing - review & editing, Funding acquisition, Validation.
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