The thermal aggregation behavior and functional properties of SPI-catechin complexes under spray-drying conditions

Abstract

Commercial soy protein isolates (SPI) often suffer from poor solubility and diminished functional properties due to the harsh conditions of spray-drying. This study demonstrated that catechin complexation effectively modulated SPI thermal aggregation behavior during spray-drying conditions. Structural characterization revealed that incorporating catechin (0.25%-1.75%, w/w) promoted a conformational transition in SPI from predominantly random coil structures to more ordered α-helical configurations. Fluorescence spectroscopy and electrophoresis results suggested hydrophobic interactions dominated between SPI and catechin. Notably, at 1% catechin, thermal aggregation was notably mitigated, transforming insoluble aggregates into soluble ones stabilized by electrostatic forces. At this optimal concentration, SPI solubility improved by 181.09% compared to commercial SPI, while emulsifying activity, thermal stability, and foaming stability were also markedly enhanced. Moreover, at 0.25% catechin, gel hardness reached 1.58 ± 0.02 N, higher than in other groups. Disulfide bonds and hydrophobic interactions were found to be key in forming the gel network.

Highlights

• •Soy protein isolate-catechin (SPI-C) complexes were prepared to inhibit thermal aggregation of SPI during commercial spray-drying processes.
• •With an optimal catechin concentration, the SPI-C complexes exhibited significantly improved solubility, emulsifying activity, and gel strength.
• •Structural modifications, interaction mechanisms, and thermal aggregation behavior of the SPI-C complexes were systematically investigated.

1. Introduction

Soy protein isolate (SPI) is a readily available and cost-effective plant-based protein source, offering high nutritional value. It possesses biodegradable, thermally stable, non-polluting, and environmentally friendly characteristics. Commercial SPI production typically involves high-temperature spray-drying to achieve increased protein concentrations, extended shelf life, and enhanced convenience compared to native SPI. However, food processing operations such as high-temperature sterilization and spray-drying can significantly impact the structural and functional properties of proteins (He et al., 2015). These processes may result in denaturation and aggregation of SPI proteins, leading to reduced solubility and limiting their application in the food industry (Li et al., 2023).

To mitigate the negative impacts of spray-drying on the functional properties of SPI, physical regulation (Li et al., 2023), chemical modification (Liu et al., 2023), or enzymatic treatment can be employed to intervene in the thermal denaturation process (Yan et al., 2025). It has been shown that the addition of polyphenols can prevent the thermal aggregation of lactoferrin without the use of organic solvents (Liu et al., 2016). Yan et al. (2022) investigated the effect of preheating treatment on the structural and functional properties of polyphenol-camphorated isolated proteins and found that the addition of polyphenols reduced the aggregation of proteins and enhanced emulsifying properties. Liu, Sun, Wang, et al. (2015) also discovered that the hydroxyl groups in phenolic compounds could interact with free amino groups and tryptophan (Trp) residues in proteins, increasing the denaturation temperature and thereby inhibiting protein aggregation during heat treatment.

Catechin, a flavonoid polyphenolic compound characterized by its high hydroxyl content and multiple reactive sites suitable for protein binding, has been shown to interact with proteins in various ways (Muntaha et al., 2025). Wang et al. (2014) demonstrated that the complexation of catechins with heat-treated α-lactalbumin not only elevated the denaturation temperature of α-lactalbumin but also enhanced its antioxidant and bioactive properties. Chen et al. (2024) revealed that the conjugation of ferulic acid with SPI led to improvements in both the emulsifying properties and solubility of proteins.

While numerous studies have examined the interactions between proteins and polyphenols, most investigations have centered on freeze-dried proteins prepared under controlled laboratory conditions. In contrast, industrial SPI production commonly employs spray-drying technology at temperatures surpassing 140 °C, markedly higher than the preheating temperatures used in laboratories. Under these conditions, native globular proteins may undergo unfolding and aggregation due to the interplay of attractive and repulsive forces during heating, adversely affecting their structural and functional attributes (Cornacchia et al., 2014). However, limited research has explored the mitigation of thermal aggregation behavior in proteins, particularly soy proteins, by incorporating exogenous substances during spray-drying. The influence of catechins on the thermal degradation and aggregation behavior of SPI, along with the underlying mechanisms, remains underexplored. Therefore, this study systematically investigates the thermal aggregation behavior and mechanisms of catechin-SPI complexes under spray-drying conditions, as well as their functional characteristics. This study introduces an effective method to mitigate the heat-induced aggregation of SPI during commercial spray-drying, thereby enhancing the application potential of SPI.

2. Materials and methods

2.1. Materials

Catechin (≥98%) was purchased from Hefei Bomei Biological Reagent Co., Ltd. (Hefei, China). Defatted soy flake was obtained from Yu King Plant Protein Co., Ltd. (Harbin, China). 1-Anilinonaphtha-lene-8-sulfonate was purchased from Sigma Chemical Co., Ltd. (St. Louis, USA). Tris (hydroxymethyl) aminomethane, sodium dodecyl sulfate (SDS) and other chemicals were sourced from Sinopharm Co., Ltd. (Shanghai, China). All chemicals and reagents were of analytical grade.

2.2. Sample preparation

2.2.1. Preparation of soy protein isolate (SPI) and soy protein isolate-catechin (SPI-C) complexes

The extraction of SPI was based on the method reported by our previous study (Zhao et al., 2024), and the protein concentration of the obtained SPI was determined to be 91.83% in this work. According to our previous research (Zhao et al., 2024), catechins were incorporated into SPI at varying concentrations of 0.25, 0.5, 0.75, 1, 1.25, 1.5, and 1.75% (w/w) to form SPI-C complexes. Resultant mixtures underwent dialysis in 8–14 kDa molecular weight cutoff bags at 4 °C for 48 h to remove unbound catechins. To simulate commercial thermal processing, dialyzed samples were spray-dried under conditions adapted from Chen et al. (2020), with inlet air temperature fixed at 170 °C and flow rate maintained at 3.5 mL/min. Untreated native SPI and commercial SPI subjected only to spray-drying without catechin conjugation, served as the control groups.

2.2.2. Preparation of different soluble/insoluble protein aggregate dispersions

Each SPI-C complex sample was prepared as a solution with a protein concentration of 1% according to Zhao et al. (2024) with some modifications. The soluble (SAs) and insoluble (SAi) SPI aggregate dispersions were prepared as follows: (1) Each SPI-C complex dispersion (with or without catechin conjugation) was centrifuged at 8000 rpm for 30 min at 4 °C. The precipitate collected from the lower phase was designated as SAi (2) After centrifugation, the supernatant was separated from the insoluble protein macro-particulate material using a 0.45 μm microfiltration membrane and then dialyzed with a 100 kDa dialysis bag to retain soluble aggregates with a molecular weight (MW) less than 100 kDa, forming SAs. The protein contents of both SAs and SAi were determined using the Biuret's method (Gornall et al., 1949).

2.3. Structural characteristics of SPI-C complexes and their aggregates

2.3.1. Circular dichroism (CD) spectroscopy

CD spectra of 0.1 mg/mL SPI-C complex samples incorporating catechins at varying concentrations were recorded in the spectral range of 190 to 260 nm using a spectropolarimeter (J-1500, Jasco International Co., Ltd., Japan) (Lei et al., 2022). The path length was 0.1 cm, and the scanning speed was 100 nm/min. Each sample was measured with three scans. The secondary structure content was analyzed using CD Pro software (Bio-Logic, France).

2.3.2. UV–vis spectrophotometry

The UV–vis absorption spectra of SPI-C samples (1 mg/mL, SPI concentration) were recorded using a UV–vis spectrophotometer (UV-8000, Shanghai Metash Instruments Co., Ltd., China). The measurements were conducted in 10 mM phosphate buffer at pH 7.0, with a scanning wavelength range of 200–400 nm and a scan rate of 100 nm/min.

2.3.3. Intrinsic fluorescence spectroscopy

The SPI-C complex samples were diluted to a concentration of 0.1 mg/mL using phosphate buffer solution (10 mM, pH 7.0). Intrinsic fluorescence spectroscopy (LS55, PerkinElmer Inc., USA) measurements were conducted with an excitation wavelength of 295 nm and a scanning speed of 500 nm/min. The fluorescence intensity was recorded between 300 and 500 nm at temperatures of 293 K and 310 K, respectively (Yan et al., 2023). The fluorescence quenching mechanism was analyzed using the Stern-Volmer equation:

$$\frac{{\mathrm{F}}_0}{\mathrm{F}}=1+{\mathrm{K}}_{\mathrm{q}}{\mathrm{\tau }}_0\left[\mathrm{Q}\right]=1+{\mathrm{K}}_{\mathrm{SV}}\left[\mathrm{Q}\right]\#$$ (1)

where, F₀ represents the fluorescence intensity of SPI in the absence of catechin. F is the fluorescence intensity of SPI after adding catechin. K_q is the molecular quenching constant. K_sv is the Stern-Volmer quenching constant. τ₀ is the lifetime of the fluorophore in the absence of the quencher (= 10⁻⁸ s). Q is the concentration of catechin.

For static quenching, the number of binding sites (n) and the binding constant K_a between catechin and SPI were calculated using the double logarithmic Stern-Volmer equation (Yan et al., 2023).

$$\begin{array}{c}\log \frac{\left({\mathrm{F}}_0–\mathrm{F}\right)}{\mathrm{F}}=\log {\mathrm{K}}_{\mathrm{a}}+\text{nlog}\left[\mathrm{Q}\right]\end{array}$$ (2)

where, K_a is the binding constant and n is the number of binding sites. Plotting the double logarithmic Stern-Volmer data yields the number of binding sites from the slope and the binding constant from the intercept.

Thermodynamic parameters were obtained according to the Van't Hoff equations:

$$\begin{array}{c}\ln {\mathrm{K}}_{\mathrm{a}}=–\frac{\mathrm{\Delta H}}{\mathrm{RT}}+\frac{\mathrm{\Delta S}}{\mathrm{R}}\#\end{array}$$ (3)

$$\begin{array}{c}\mathrm{\Delta G}=\mathrm{\Delta H}–\mathrm{T\Delta S}\#\end{array}$$ (4)

where, K_a represents the binding constant; R represents the gas constant (8.314 J·mol⁻¹·K⁻¹); T represents the absolute temperature, and ΔH, ΔS, and ΔG represent the changes in enthalpy, entropy, and free energy, respectively.

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

SDS-PAGE was carried out according to the method of Wu et al. (2024) with slight modifications. Samples containing proteins at a concentration of 2 mg/mL were denatured by boiling for 5 min, cooled, and loaded at 15 μL per well. Separation occurred in a 5% stacking gel and 15% separating gel, with an initial voltage of 80 V, increased to 120 V upon entry into the separating gel. Post-electrophoresis, gels were stained for 30 min and decolorized for 24 h.

2.3.5. Differential scanning calorimetry (DSC)

Based on (Parolia et al., 2022), the thermostability of SPI and SPI-C complexes was analyzed using a differential scanning calorimeter (DSC 4000, PerkinElmer Inc., USA). Each sample weighing 5 mg was sealed in an aluminum hermetic pan and heated from 30 °C to 180 °C at 10 °C/min under 20 mL/min nitrogen flow. Denaturation peak temperature and enthalpy changes were determined from the resulting thermal curves.

2.3.6. Soluble/insoluble aggregate content

The SAs and SAi were prepared using the methodology outlined in section 2.2.2. The SAs and SAi content was quantified using the classical protein content method reported by Biuret's method (Gornall et al., 1949).

2.3.7. UV–vis absorbance of soluble aggregates

The UV–Vis spectra of SAs were determined using the methodology outlined in section 2.3.2.

2.3.8. Intrinsic fluorescence spectroscopy of soluble aggregates

The Fluorescence spectroscopy of SAs was determined using the methodology outlined in section 2.3.3.

2.4. Functional characterization of SPI-C complexes

2.4.1. Solubility and turbidity

The solubility was determined using the method of Stübler et al. (2020). The samples were diluted to 2 mg/mL with PBS (10 mM, pH 7.0) and then centrifuged (6800 rpm, 15 min, 4 °C). Then, the protein content of the supernatant was measured using a UV-8000 at an absorbance of 540 nm. Protein solubility was calculated based on the following formula:

$$\begin{array}{c}\text{Protein solubility}\left(\%\right)=\frac{\text{Supernatant protein content}}{\text{Total protein content before centrifugation}}\times 100\%\#\end{array}$$ (5)

The turbidity was determined using the method of Chen et al. (2025). The concentration of the sample was prepared as 2 mg/mL, and the absorbance of the UV–Vis spectrophotometer was measured at 600 nm, with OD₆₀₀ nm as the turbidity value.

2.4.2. Emulsifying properties

The emulsion activity index (EAI) and emulsion stability index (ESI) of the samples were determined according to a previous report (Lin et al., 2022). Briefly, 3 mL of soybean oil was mixed with 9 mL of sample solution (10 mg/mL), and the mixture was homogenized at 10000 rpm for 2 min. After adding SDS solution (0.1%, w/v), the absorbance of each emulsion sample was measured at 500 nm. EAI and ESI were calculated as follows:

$$\begin{array}{c}\mathrm{EAI}\left(\frac{{\mathrm{m}}^2}{\mathrm{g}}\right)=\frac{2\times 2.303}{\mathrm{C}\times \left(1-\mathrm{\phi }\right)\times {10}^4}\times {\mathrm{A}}_0\times \mathrm{N}\end{array}$$ (6)

$$\begin{array}{c}\mathrm{ESI}\left(\%\right)=\frac{{\mathrm{A}}_{10}}{{\mathrm{A}}_0}\times 100\%\#\end{array}$$ (7)

where, C is sample concentration (g/mL); φ is the proportion of oil phase in the emulsion, 0.25; N is the dilution ratio (100); A₀ represents the absorbance of the emulsion at 0 min and A₁₀ represents the absorbance of the emulsion at 10 min.

2.4.3. Foaming properties

The foaming properties of the conjugates are based on a previous report (Flores-Jiménez et al., 2019). Briefly, the foams were generated by stirring sample solution (10 mL, 3 mg/mL) for 2 min at a speed of 17,500 rpm. Foaming capacity (FC) and Foaming stability (FS) were calculated as follows:

$$\begin{array}{c}\mathrm{FC}\left(\%\right)=\frac{{\mathrm{V}}_0–\mathrm{V}}{\mathrm{V}}\times 100\%\#\end{array}$$ (8)

$$\begin{array}{c}\mathrm{FS}\left(\%\right)=\frac{{\mathrm{V}}_{30}–\mathrm{V}}{{\mathrm{V}}_0–\mathrm{V}}\times 100\%\#\end{array}$$ (9)

where, V is the foam volume before homogenization (mL), and V₀ and V₃₀ are the foam volume at 0 min and 30 min after homogenization (mL).

2.4.4. Gel strength

Gel strength was measured following Zhao et al. (2017). Sample solutions with 12% protein concentration were stirred at 20 °C for 30 min in 2-cm-diameter glass weighing bottles. After heating at 90 °C for 30 min in a water bath, samples were refrigerated at 4 °C for 12 h. Gels were equilibrated to room temperature for 30 min before texture analysis (TMS-Touch 250 N, Food Technology Inc., USA). Using a P/0.5 cylinder probe, gels were penetrated at 2.0 mm/s with a 0.045 N trigger load and 4 mm compression depth. Gel strength was defined as the peak hardness force during the first compression cycle.

2.4.5. Rheology measurements

The rheological properties of SPI and SPI-C were analyzed using a rheometer (H-PTD20, Anton Paar (Shanghai) Trading Co., Ltd., China) based on Xu et al. (2021) with modifications. A 40-mm-diameter parallel plate at 1 mm gap was employed in oscillatory temperature sweep mode at 2% strain and 0.2 Hz frequency. Samples were heated from 30 °C to 90 °C at 4 °C/min, held at 90 °C for 10 min with mineral oil coverage to prevent evaporation, and storage modulus G′ was recorded.

2.4.6. Intermolecular forces

The intermolecular forces of gels were determined by treating them with protein denaturing solvents, following the method described by Yang, Su, and Li (2020), with appropriate modifications. Specifically, gel samples weighing 1 g each were dispersed in solutions containing 0.7 mol/L NaCl, 6 mol/L GuHCl, 10 mol/L DTT, and a mixture of 10 mol/L DTT and 6 mol/L GuHCl. After homogenization at a speed of 15,000 rpm for 2 min, the mixtures were reacted at 4 °C for a duration 1 h and subsequently centrifuged at a speed of 9700 rpm for 15 min at the same temperature. The protein content in supernatants was quantified using Biuret's method (Gornall et al., 1949).

2.4.7. Atomic force microscope (AFM)

Following the method described by Wang et al. (2024) with certain modifications, the SPI and SPI-C solutions were diluted to a concentration of 20 μg/mL. Subsequently, 50 μL of the SPI and SPI-C suspension was meticulously deposited onto a freshly cleaved mica surface in a culture dish, followed by subjecting the samples to gelation through heating at 90 °C for 30 min to induce gelation. After each sample was blow-dried using clean air, AFM (Dimension Icon PT, Bruker Inc., Malaysia) observation was performed utilizing BioScope (BS3–02, Bruker Inc., Malaysia) tapping mode.

2.5. Statistical analysis

Three parallel samples were set up in each group, and the results were expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 26.0 software (IBM, USA) to determine significant differences (P < 0.05). Origin 2021 software (OriginLab, USA) was utilized for data visualization.

3. Results and discussion

3.1. Structural changes of SPI/catechin complexes under spray-drying conditions

3.1.1. CD spectrum analysis

Far-UV CD can be used to analyze the changes in the secondary structure content of proteins in solution (Lei et al., 2022). The CD spectra results are shown in Table 1. Compared to native SPI, commercial SPI showed a decrease in α-helix content from 22.80 ± 0.02% to 21.58 ± 0.04%, along with an increase in random coil content from 29.58 ± 0.13% to 29.96 ± 0.20%. This phenomenon could be attributed to the disruption of hydrogen bonds within protein molecules during spray-drying treatment, which destabilized the α-helical structure and led to a more disordered conformation (Chen et al., 2016). After the addition of catechins, the ellipticity of the SPI-C complex at 206 nm decreased significantly compared to the control group (Fig. S1). This reduction might result from the formation of polypeptide-catechin complexes, where catechin's phenolic hydroxyl groups interact with N-H/C-N/C=O groups within SPI molecules, thereby inducing an extension in SPI's conformational arrangement (Zhang et al., 2018). As illustrated in Table 1, the α-helical content progressively increased while the random coil content correspondingly decreased with the rise in catechin concentration. These findings suggest that the interaction between catechins and SPI promotes the transition from a predominantly random coil to a more α-helical conformation, indicating a shift towards a more ordered structural arrangement of SPI (Cui et al., 2023), which generally enhanced thermal stability by reducing the exposure of hydrophobic regions and minimizing aggregation-prone unfolded states. Notably, compared to the other groups, the 1% treatment group exhibited the highest α-helix content (27.26 ± 0.03%), suggesting that the SPI-C complex achieved optimal conformational stability at this concentration. These results are consistent with those shown in the DSC data (section 3.1.5, Table 3), which directly associate the maximum α-helix content observed at 1% catechin with the highest thermal denaturation temperature (T_p).

Table 1.

Secondary structures of SPI and SPI-C non-covalent complexes.

Samples	a-helix (%)	β-sheet (%)	β-turn (%)	random coil (%)
Native SPI	22.80 ± 0.02e	24.90 ± 0.03a	22.72 ± 0.08c	29.58 ± 0.13b
Commercial SPI (0%)	21.58 ± 0.04f	24.97 ± 0.04a	23.49 ± 0.12a	29.96 ± 0.20a
0.25%	22.84 ± 0.03e	24.66 ± 0.13b	23.08 ± 0.06b	29.42 ± 0.12b
0.5%	23.91 ± 0.01d	24.74 ± 0.03a	22.81 ± 0.02c	28.54 ± 0.03d
0.75%	24.67 ± 0.05c	24.09 ± 0.05c	22.09 ± 0.17d	29.15 ± 0.26c
1%	27.26 ± 0.03a	24.74 ± 0.04a	22.27 ± 0.08d	25.73 ± 0.13f
1.25%	25.61 ± 0.02b	24.24 ± 0.03c	21.62 ± 0.04f	28.53 ± 0.07d
1.5%	25.50 ± 0.05b	24.52 ± 0.04b	21.90 ± 0.03e	28.08 ± 0.12e
1.75%	24.59 ± 0.02c	24.49 ± 0.01b	21.29 ± 0.05g	29.63 ± 0.06b

Note: Different letters represent significant differences (P < 0.05).

Table 3.

Effect of catechin addition on SPI-C denaturation temperature.

Samples	Tp (°C)	ΔH (J/g)
Native SPI	69.41 ± 0.53e	288.20 ± 1.33f
Commercial SPI (0%)	68.75 ± 1.05g	248.00 ± 2.78g
0.25%	70.21 ± 1.09de	331.86 ± 1.58e
0.50%	72.24 ± 0.91cd	332.32 ± 1.67e
0.75%	80.90 ± 0.81a	389.57 ± 0.61d
1.00%	81.90 ± 0.63a	443.67 ± 0.80a
1.25%	76.62 ± 1.40b	435.87 ± 2.52b
1.50%	74.17 ± 1.05c	431.96 ± 1.48b
1.75%	71.70 ± 0.99cd	423.98 ± 0.47c

Note: Different letters represent significant differences (P < 0.05).

3.1.2. UV–vis spectrum analysis of SPI-C complex

UV–vis absorption spectra of SPI-C complexes were recorded, and the absorption peaks in the wavelength ranges of 200–214 nm and 260–280 nm indicated the absorption of polypeptide backbone structure C O and amino acid residues, respectively (Chen et al., 2019). As shown in Fig. 1, compared with the native SPI, the 0% group showed a decrease in UV absorption intensity at both 220 and 260 nm; and the absorption peaks at 220 and 260 nm were red-shifted, which can be attributed to the thermally induced aggregation of the SPI after the spray-drying treatment, and an increase in the polarity of the microenvironment of the aromatic amino acids. Interestingly, the UV absorption intensity of the SPI-C complexes at both 220 and 260 nm wavelengths increased compared to native SPI and commercial SPI, likely attributed to unfolding of the tertiary structure caused by catechins introduction, enhancing flexibility and exposing previously buried hydrophobic regions. When the amount of catechin added was low (less than 1.25%), the UV spectra of both SPI-C treated groups were slightly red-shifted at 220 nm compared to the 0% control group, suggesting that binding of proteins to polyphenols resulted in changes in the microenvironment around Tyr and Trp residues. When the catechin addition was higher (more than 1.25%), the UV spectra of SPI-C were blue-shifted compared to the low catechin treatment group, indicating that the high catechin addition caused an increase in the non-polar nature of the environment in which the aromatic amino acids were located. Similar results were reported by Chen et al. (2024), who found that the addition of ferulic acid disrupted the natural structure of proteins by forming new hydrogen bonds and altering the hydrophobic core, resulting in a shift of aromatic amino acid residues in proteins to a more hydrophilic microenvironment, indicating that the microenvironment of the Trp chromophore became more hydrophilic. (See Fig. 2.)

Fig. 1.

UV–vis spectrum of SPI-C complex.

Fig. 2.

(A) and (B) show the fluorescence spectra of SPI at various catechin concentrations at 293 and 310 K, respectively.

3.1.3. Fluorescence spectrum analysis

The aromatic amino acid Trp, which produces intrinsic fluorescence sensitive to its microenvironment, is widely used in studying protein-polyphenol interactions and detecting changes in protein tertiary structure (Jiang et al., 2018). Fig. 2 A and 2B show the fluorescence spectra of native SPI and SPI-C complexes. Compared with native SPI, the fluorescence intensity at both 293 K and 310 K for all spray-dried SPI groups decreased, and this effect was dose-dependent. According to Vivian and Callis (2001), Trp residues were assigned in a polar environment when λ_max was obtained in the range greater than 330 nm. Moreover, compared with commercial SPI, the fluorescence intensity of all SPI-C complexes (0.25%-1.75%) was consistently lower, and it gradually decreased with increasing catechin addition. The observed results suggested that the addition of catechins further altered the protein structure. Quinones in the catechin structure, acting as potent electrophilic intermediates, reacted with nucleophilic amino acid residues (such as Trp) in the protein chain, leading to the formation of protein-polyphenol conjugates (Meng & Li, 2021). This binding formed non-fluorescent SPI-C complexes, leading to static quenching of Trp fluorescence (Dai et al., 2019). Consequently, the fluorescence intensity of the protein decreased. These findings aligned with our previous studies (Zhao et al., 2024), where the addition of catechins increased protein unfolding, exposing Trp residues, and resulting in reduced fluorescence intensity. Additionally, compared with native SPI, each treatment group exhibited a significant redshift in the maximum emission wavelengths (Fig. 2 A-B), indicating that hydrophobic groups buried within the protein were exposed to a more hydrophilic, polar environment after spray-drying.

To clarify the binding mechanism with the quencher, the quenching and binding constants were calculated using the Stern-Volmer Eqs. (1) and (2) and displayed in Table 2. These results demonstrated a highly linear relationship at both temperatures of 293 and 310 K (R² > 0.99). The fluorescence quenching of SPI by catechin is likely due to static quenching, as evidenced by the decrease in the quenching constant K_SV when the temperature increased from 293 to 310 K. Moreover, the bimolecular quenching constant K_q was significantly higher than the maximum diffusion collision rate constant (2 × 10¹⁰ L·mol⁻¹·s⁻¹), indicating that a static mechanism is appropriate for analyzing the binding interactions between catechin and SPI (Dai et al., 2019). Similar results have been reported for complexes of SPI with other polyphenols (Ji et al., 2023). By examining the relationship between log[(F₀—F)/F] and Q, we obtained the binding constant K_a and the number of binding sites n. Our calculations revealed that K_a increased with rising temperature. Additionally, the n value of approximately 1 suggested that there is one binding site between SPI and catechin. This observation aligned with the study by Chen, Jiang, et al. (2019), which reported respective K_a values of 1.60 × 10³ L·mol⁻¹·s⁻¹ for the interaction between SPI and cyanidin-3-O-glucoside at 297 K, with a binding site value of 1.

Table 2.

Quenching constants and thermodynamic parameters of SPI-C complex.

T (K)	Kq (1011 L·mol−1·s−1)	Ksv (103 L·mol−1)	Ka (103 L·mol−1)	n	ΔG (kJ·mol−1)	ΔS (J·mol-1·K-1)	ΔH (kJ·mol−1)
293	2.91	2.91	3.9994	1.0297	−20.20	178.36	32.06
310	2.60	2.60	8.2300	1.1125	−23.23

Hydrogen bonds, hydrophobic interactions, and electrostatic forces play crucial roles in protein-polyphenol interactions. As shown in Table 2, ΔH = 32.06 kJ·mol⁻¹, ΔS = 178.36 J·‍mol⁻¹·K^-‍1. Previous studies have indicated that ΔH > 0 and ΔS > 0 suggested that the interaction between SPI and catechin was primarily driven by hydrophobic interactions (Feng et al., 2025). Le Bourvellec and Renard (2012) reported that heating at a specific temperature caused partial denaturation and conformational changes in proteins, leading to increased exposure of amino acid binding sites. Consequently, after spray-drying treatment, heat-induced aggregation of SPI particles was disrupted, forming non-fluorescent complexes along with exposed hydrophobic regions which interact with the nonpolar aromatic rings of catechins through hydrophobic interactions. This result is in accord with the research of Ren et al. (2018), who reported that loose peptides generated by SPI following high-temperature treatment exhibit thermo-dynamic instability and tend to associate with the aromatic rings of black bean seed coat extracts through hydrophobic interactions.

3.1.4. SDS-PAGE analysis

An electropherogram of SPI-C conjugation is shown in Fig. 3. In the lane of SPI, α, α', and β subunits were identified as 7S subunits, while the A and B subunits represented the 11S subunits (Guo, Bao, Sun, Chang, & Liu, 2021). Under reducing conditions, non-covalent and disulfide bonds were disrupted by the addition of SDS and β-mercaptoethanol (Wang et al., 2024). As shown in Fig. 3A and B, all SPI-C complex samples exhibited a significant upward migration of electrophoretic bands compared to the control. Additionally, the intensity of the β-subunit band gradually increased relative to the control group. Notably, a new band with an approximate MW of 180 kDa appeared at the top of the gel in all conjugate samples, likely due to oligomer formation during the complexation of catechins with SPI subunits following spray-drying treatment. In contrast, the control group did not exhibit any prominent bands with MW exceeding 180 kDa, suggesting that heat-induced aggregates formed by commercial SPI were primarily insoluble and were removed during the centrifugation step of sample preparation for SDS-PAGE analysis. Interestingly, the α and α' subunit bands (with MW of 76 and 72 kDa, respectively) faded in the control samples, indicating that these subunits might have contributed to the formation of insoluble aggregates. However, under reducing conditions, these bands reappeared in the SPI-C complex samples (with catechin concentrations ranging from 1% to 1.75%), possibly due to catechin inhibiting β-conglycinin aggregation during thermal treatment. Yan et al. (2022) reported similar findings, observing that after the protein isolate of the cereal seed kernel was complexed with purified polyphenol extracts, two higher-molecular-weight bands appeared under non-reducing conditions, whereas these bands lightened under reducing conditions. These findings suggested that polyphenols induce cross-linking of the protein and the formation of soluble oligomers. Furthermore, with increasing catechin concentration, the bands of acidic subunits in Native-PAGE tended to become less intense, while those in SDS-PAGE became more pronounced. This might indicate that the interaction sites between catechins and SPI were located on the acidic subunits of SPI, and that this interaction promoted the formation of high-MW polymers through disulfide bonds between the acidic subunits.

Fig. 3.

Native-PAGE (A) and SDS-PAGE (B) patterns of control (labeled as Commercial SPI) and SPI-C complexes (with the catechin addition from 0.25% to 1.75%).

3.1.5. DSC analysis

DSC is used to measure the transition of proteins from their folded form to their unfolded state during thermal denaturation (Parolia et al., 2022). As shown in Table 3, compared with the native SPI, the T_p and ΔH values of the control group decreased to 68.75 ± 1.05 °C and 248.00 ± 2.78 J/g, respectively. This suggested that the native SPI maintained its original structure, whereas the commercial group experienced partial structural destruction due to the high-temperature spray-drying conditions, which led to a reduction in both T_p and ΔH values (Guo et al., 2012). It can be observed from Table 3 that with the increase in catechin addition, the T_p and ΔH of the complex peak gradually increase. The denaturation temperature of the 1% group was 81.90 ± 0.63 °C, which increased by 13.15% compared with that of native SPI. Notably, the superior thermal stability of the 1% catechin group, with the highest T_p, is closely associated with its secondary structure. Specifically, the increased α-helix content and decreased random coil proportion (as illustrated in section 3.1.1) indicated a more ordered and compact protein conformation, which enhanced resistance to thermal unfolding and thereby accounts for the maximum T_p observed. These findings align with those of Huang et al. (2025), who reported that the soybean lipophilic protein–epicatechin complex exhibited peak thermal stability when intramolecular hydrogen bonds within the α-helix were preserved, while destabilization occurred upon disruption of the α-helix and loss of hydrogen bonding. In comparison to the control SPI, all the SPI-C complex groups demonstrated a higher transition temperature, indicating that the interaction of catechin with proteins altered the conformational properties (consistent with the results of CD and fluorescence spectrum analysis) and improved the thermal stability of SPI. These findings aligned with the study by Ren et al. (2022), who demonstrated that combining zein and resveratrol could improve protein thermal stability. When catechin concentration exceeded 1%, the T_p value exhibited a significant downward trend (Table 3). This phenomenon might be attributed to the disruption of secondary structural balance, as excessive catechin interfered with the intramolecular hydrogen bonds that stabilized the α-helical conformation (as shown in Table 1). Such structural rearrangement led to a looser protein conformation, rendering the protein more susceptible to thermal denaturation and thus contributing to a marked decrease in T_p (Cui et al., 2023).

3.2. Structural characteristics analysis of SPI-C aggregates

3.2.1. Soluble/insoluble protein aggregates content

The contents of SAs and SAi within the control and SPI-C complexes are presented in Fig. 4. The precipitated fraction after centrifugation represented the insoluble aggregates, while a smaller residual solution indicated higher solubility of the complexes (Tang, Wang, Yang, & Li, 2009). The content of SAs (> 100 kDa) increased with the addition of 1% catechin, whereas the content of SAi decreased (P < 0.05). Notably, the highest amount of SAs was observed at 1% catechin, suggesting that an optimal concentration of catechin could promote the conversion of insoluble aggregates into soluble aggregates, thereby mitigating the thermal aggregation behavior of SPI induced by spray-drying. This effect could be attributed to the hydrophobic interactions between catechin and SPI, which prevented excessive protein aggregation during spray-drying. This not only stabilized the protein structure and enhanced SPI's thermal stability but also promoted the conversion of insoluble aggregates into soluble aggregates (Cui et al., 2023; Huang et al., 2025).

Fig. 4.

The SAs/SAi content of SPI-C dispersions. Different letters represent significant differences (P < 0.05).

3.2.2. UV–vis of soluble aggregates spectrum analysis

The UV spectra of the soluble aggregates in the SPI-C complexes with different concentrations of catechins are shown in Fig. 5. The intensity of the UV absorption peaks of all soluble aggregates decreased compared to the peak intensities of the UV spectra of the SPI-C complexes, which were in the wavelength range from 200 to 300 nm (Fig. 1). This suggests that after high-temperature thermal induction, aromatic amino acids are more buried in the hydrophobic nonpolar environment of the protein aggregates, leading to a decrease in the UV absorption intensity (Lee et al., 2024; Zhao et al., 2024). Compared with native soluble aggregates, the soluble aggregates in commercial SPI showed a decrease in the UV intensity at 260 nm and a blue-shift of the absorbance peak at 260 nm, suggesting that the spray-drying treatment led to the burying of aromatic amino acids inside the soluble aggregates, again demonstrating that thermally induced aggregation occurred in the SPI. In addition, the intensity of the absorption peaks in the UV region at 220 and 260 nm increased with increasing catechin concentration in all treatment groups, suggesting that the addition of catechin induced the unfolding of the structure of the soluble aggregates, exposing the aromatic amino acids inside. Moreover, the UV absorbance of the soluble aggregates was related to the amount of catechin added to the SPI-C complexes. When the amount of catechin added was low (less than 1.25%), the UV spectra of the soluble aggregates of all SPI-C treatment groups were slightly red-shifted at 280 nm compared to those of the 0% group, indicating that the polarity of the environment to which the aromatic amino acids were transferred was increased at this time and the structure of the soluble aggregates was unfolded. However, when the amount added was higher (more than 1.25%), the UV spectra of the soluble aggregates were blue-shifted compared to those of the group treated with low concentrations of catechin, indicating that the aromatic amino acids within the soluble aggregates were buried back into the proteins. From these results, it could be concluded that the addition of a moderate amount of catechin helps to unfold the structure of the soluble aggregates and intervenes to some extent in the degree of aggregation of the SPI structure caused by spray-drying. Similar results were obtained by Ren et al. (2018), who found that after the addition of black soybean seed coat extract (BE), the UV maximum absorption peak of SPI was red-shifted at 280 nm, and the formation of SPI/BE complexes inhibited the aggregation of SPI during heating. (See Fig. 6.)

Fig. 5.

UV–vis spectrum of soluble aggregates in SPI-C complex.

Fig. 6.

(A) and (B) show the fluorescence spectra of SPI-C soluble aggregates at 293 and 310 K respectively.

3.2.3. Intrinsic fluorescence spectrum analysis of soluble aggregates

The fluorescence spectra of soluble aggregates in the SPI-C complex are shown in Fig. 6. In comparison with the SPI-C complex (Fig. 2), the fluorescence intensity of these soluble aggregates exhibited a substantial decrease. This reduction could be attributed to the embedding of fluorophores within the soluble aggregates due to the spray-drying process, which results in endogenous fluorescence quenching. Additionally, the fluorescence intensity of soluble aggregates decreased with increasing catechin concentration, indicating that catechin exerted a significant fluorescence quenching effect on soluble aggregates. No notable redshift was observed in the peak value of SPI-C soluble aggregates at 293 K, suggesting that the local polar environment surrounding the aromatic amino acid residues of soluble aggregates remained relatively stable. These observations aligned with those reported by Rawel et al. (2002), who noted a reduction in fluorescence intensity upon the interaction of soy glycinin with flavonoids and did not observe a corresponding redshift due to Trp residue exposure. Furthermore, as the catechin concentration increased, the maximum emission wavelength of the soluble aggregates shifted from 338 nm to 340 nm at 310 K, suggesting a transition of Trp and Tyr residues from a hydrophobic to a more hydrophilic environment, indicative of protein unfolding (Dai et al., 2022). This might imply that an increase in temperature is more conducive to the inhibitory effect of catechin on the heat-induced aggregation of SPI.

According to the Stern-Volmer formula, F₀/F is plotted on the Y-axis and [Q] on the X-axis, resulting in a linear relationship. In Fig. S3, K_sv represents the slope of this linear relationship. As shown in Fig. S3 and Table 4, the slope of the linear relationship gradually decreased with increasing temperature, indicating a decrease in the quenching constant. This suggested that the fluorescence quenching mechanism of soluble aggregate SPI-C was static quenching. From Table 4, it could be observed that the K_sv of soluble aggregates were 2.22 × 10¹² L·mol⁻¹ at 293 K and 1.59 × 10¹² L·mol^-‍1 at 310 K, both significantly higher than 2 × 10¹⁰ L·mol⁻¹. This further confirmed that catechin induced static quenching in SPI within soluble aggregates. Additionally, compared to the K_sv of the SPI-C complex, those of soluble aggregates were notably higher, indicating that a more pronounced quenching effect of catechin on soluble aggregates.

Table 4.

Quenching constants (K_sv, K_q, and K_a), number of binding sites (n), and thermodynamic parameters (ΔH, ΔS, and ΔG) at 293 and 310 K of the soluble aggregates.

T (K)	Kq (1012 L·mol−1·s−1)	Ksv (104 L·mol−1)	Ka (104 L·mol−1)	n	ΔG (kJ·mol−1)	ΔS (J·mol-1·K-1)	ΔH (kJ·mol−1)
293	2.22	2.22	6.3674	1.0243	−26.95	80.59	−3.34
310	1.59	1.59	5.9114	1.0422	−28.32

The ΔH, ΔS, and ΔG were calculated using the Van't Hoff equation to determine the interaction forces between the soluble aggregates (Fig. S4). The observed ΔG was negative, suggesting that the binding interaction between soluble aggregates and catechin was spontaneous (Yang et al., 2020). In addition, a negative value of ΔH shows that the reaction is an exothermic process (Zhang et al., 2023). This is consistent with the result that as the temperature increases, K_a decreases and the reaction is exothermic. From the thermodynamic parameters results of the SPI-C complexes described in section 3.1.3, it could be seen that the hydrophobic interactions between the SPI and catechin were dominant, whereas the interaction between soluble aggregates and catechin was mainly electrostatic interaction (as evidenced by ΔH < 0 and ΔS > 0). Compared with the SPI-C complex, the hydrophobic groups in the soluble aggregates were partially reduced. This phenomenon might be attributed to the fact that part of the hydrophobic groups inside SPI were buried during the formation of aggregates, resulting in some positively charged areas on the surface of the soluble aggregates interacting with negatively charged catechins through electrostatic attraction (Ren et al., 2018).

3.3. Functional characterization analysis of SPI-C complex

3.3.1. Solubility analysis

The solubility of proteins plays a crucial role in determining their applicability in the food industry, as higher solubility can enhance processing properties and broaden application scopes (Dong et al., 2019). As illustrated in Fig. 7, the solubility of native SPI was measured at 75.29%, whereas after commercial spray-drying, this value decreased to 69.90%. This reduction could be attributed to the elevated processing temperature during spray-drying, which not only exposed charged residues initially buried within the protein molecules but also induced denaturation and unfolding of the protein structure. Additionally, the formation of a highly moisture-resistant film on the protein surface hindered water molecule penetration, thereby decreasing solubility. Notably, the addition of 1% catechin significantly increased protein solubility by 36.4% compared to commercial SPI. According to (Jia et al., 2022), this enhancement was due to the binding of exposed hydrophobic groups from 7S to 11S proteins with the polyphenol component. However, further increases in catechin concentration led to excessive catechin presence, occupying most of the protein's binding sites and causing cross-linking between protein molecules, ultimately reducing solubility. These findings align with those of Hu et al. (2023), who found that excess vitexin enveloped mung bean protein, shielding hydrophilic amino acids and weakening protein-water interactions, thereby reducing the solubility of the complex. Therefore, it was evident that moderate catechin additions could improve the solubility of SPI while inhibiting the thermal denaturing aggregation of SPI.

Fig. 7.

Effect of catechin addition on solubility of SPI. Different letters represent significant differences (P < 0.05).

3.3.2. Turbidity analysis

As illustrated in Fig. 8, commercial SPI exhibited higher turbidity compared to native SPI, which might be due to the high temperatures experienced during spray-drying, leading to protein denaturation and excessive molecular aggregation, thereby destabilizing SPI in solution and causing eventual precipitation (Gao et al., 2018). Compared with both native and commercial SPI, a decrease in turbidity was observed when catechin concentrations ranged from 0.25% to 1%, suggesting that catechins formed soluble complexes with SPI, thereby reducing light scattering. However, as catechin exceeded 1%, a significant increase in turbidity was observed for the SPI-C complex (P < 0.05). This phenomenon is likely due to heat-induced oxidation of catechins into quinones (Pan et al., 2019), which subsequently interacted with SPI to form large, insoluble aggregates with high MW (as evidenced by SDS-PAGE analysis in section 3.1.4). Consequently, this led to an increase in turbidity. Furthermore, this finding aligns with the observations of Chen, Jiang, et al. (2019), who reported a marked increase in the turbidity of porcine plasma protein hydrolysates at elevated concentrations of oxidized tannic acid and chlorogenic acid. Therefore, the inhibitory effect of catechin on heat-induced aggregation of SPI is closely related to the concentration of added catechin.

Fig. 8.

Impact of varying catechin supplementation levels on the turbidity of the SPI-C complex. Different letters within each group denote statistically significant differences (P < 0.05).

3.3.3. Emulsifying properties analysis

The EAI reflects the protein's ability to form an oil-water interface, while the ESI indicates the strain resistance of emulsion droplets formed by the protein (Pan et al., 2019). As shown in Fig. 9, compared with the native SPI, both the EAI and ESI of the commercial SPI decreased. According to Liu et al. (2017), protein conformation is a critical factor influencing emulsifying properties. The high temperature during spray-drying induced conformational changes in SPI, leading to reduced emulsifying properties. Additionally, as catechin concentration increased from 0.25% to 1%, the EAI of SPI-C complexes was significantly enhanced relative to the control SPI. Notably, when the catechin concentration reached 1%, the EAI was 181.09% higher than that of commercial SPI. This enhancement can be attributed to two primary factors. Firstly, hydrophobic interactions between catechins and soy proteins facilitated the exposure of more hydrophobic amino acid residues on the surface of SPI, as evidenced by fluorescence spectrum analysis (section 3.1.3). This increased conformational flexibility and improved the protein's ability to adsorb at the oil-water interface, thereby enhancing SPI's emulsifying capacity (W. He et al., 2019). Secondly, at appropriate concentrations, catechins may inhibit the aggregation of soy protein molecules, maintaining a higher concentration of free protein molecules available for effective emulsification. Similar findings were reported by Yan et al. (2021), who observed that the addition of EGCG could improve the emulsification properties of SPI.

Fig. 9.

Effect of catechin addition on EAI and ESI of catechin/SPI coincidence system. Different letters represent significant differences (P < 0.05).

3.3.4. Foaming properties analysis

Protein foaming properties are fundamental and critical functional attributes in the food industry, particularly in products like ice cream, beer, and cakes. As shown in Fig. 10, commercial SPI exhibited lower FA and FS compared to native SPI. This reduction could be attributed to the elevated temperature during spray-drying, which induced protein aggregation and consequently reduced the number of proteins available to stabilize the gas-liquid interface, thereby diminishing both foaming and foam stability. Furthermore, as the catechin concentration increased from 0.25% to 1%, the FA and FS of SPI-C complex gradually improved. In contrast, when the catechin concentration further increased (exceeded 1%), the FA and FS of SPI-C complex decreased. This phenomenon might be explained by the fact that at moderate concentrations, polyphenols acted as bridges between proteins, enhancing molecular interactions and forming more robust protein membranes at the interface, thus improving FS (Princen, 1983). Conversely, excessive polyphenols could block protein interaction sites, hinder interfacial protein adsorption, promote greater protein aggregation, and create steric hindrance that prevents the formation of tightly packed interfacial layers (Chang et al., 2021).

Fig. 10.

Effect of catechin addition on foaming and foaming stability of SPI. Different letters represent significant differences (P < 0.05).

3.3.5. Evaluation of gel properties

Gel hardness is an important parameter influencing the quality characteristics of gels, usually higher gel hardness typically correlates with superior gel product quality (Ren et al., 2023; Shen et al., 2024). As illustrated in Fig. 11A, compared to native SPI, the gel hardness of commercial SPI significantly decreased (P < 0.05). This could be attributed to the high temperature experienced during spray-drying, which induced secondary and tertiary conformational changes in soy proteins, as evidenced by CD, UV, and fluorescence spectroscopy analyses presented in 3.1.1, 3.1.2, 3.1.3, leading to irreversible denaturation. Additionally, spray-drying facilitated the formation of insoluble aggregates, as demonstrated in section 3.2.1. These aggregates were less effective in forming a continuous network structure necessary for optimal gel properties. Notably, when the catechin concentration was 0.25%, the gel hardness of SPI reached the maximum value compared to other treatment groups (P < 0.05), and the highest G′ values were also observed in the 0.25% SPI-C complex group (Fig. 11B). According to the fact that G' represents the storage modulus, and a higher G' value suggests a stronger gel network and better resistance to deformation. The enhancement of hardness and G′ value was due to catechin facilitating protein intermolecular interactions by bridging polypeptide chains through non-covalent interactions such as hydrophobic, electrostatic, and hydrogen bonds, as well as interactions mediated by quinone-derived cross-linking (Cao et al., 2024). Similar enhancements in gel strength have been observed in the results of Jia, Lin, et al. (2022), who found that the G′ value of myofibrillar protein increased with adding appropriate concentration (50 μmol/g) of catechin. However, with increasing catechin concentrations, the gel hardness of SPI exhibited a downward trend. This decline was attributed to excessive catechin-induced cross-linking of the sulfhydryl groups of SPI with catechin, leading to the oxidation of catechin's hydroxyl groups into o-quinone. The subsequent reaction between the protein sulfhydryl group and quinone formed thiol-quinone adducts that inhibited the formation of stabilizing disulfide bonds, resulting in reduced gel strength (Tang et al., 2017). Jongberg, Terkelsen, Miklos, & Lund (2015) also reported that high-dose (1500 ppm) green tea extract disrupted protein gel strength due to thiol-quinone adducts inhibiting the formation of structure-stabilizing disulfide bonds.

Fig. 11.

(A) Effect of catechin addition on SPI gel strength. (B) Temperature sweep curves of SPI gels. (C) AFM images of protein gels. Note: A refers to native SPI and B—I refers to spray-dried SPI-C complexes at 0%-1.75% concentration. Different letters represent significant differences (P < 0.05).

AFM can be utilized to characterize the apparent morphological features of protein samples. Fig. 11C presents the AFM images of the sample gels. As illustrated in Fig. 11C, larger, irregular, cluster-like aggregates were observed in the control SPI gel compared to the native SPI gel. This observation corroborated the previous inference that spray-drying promoted the formation of insoluble aggregates and reduced the hardness and rheological properties of protein gels. The average surface roughness (Ra) of SPI can reflect both the surface morphology and aggregation state of the samples (Wang et al., 2024). As shown in Fig. 11C, the control SPI gel exhibited disordered aggregation and uneven distribution. In contrast, the surfaces of most SPI-C complex gels (0.25%-1.5% groups) displayed regular, uniform, and dense structures. Notably, the Ra value of the sample supplemented with 0.25% catechin was significantly lower compared to other treatment groups, which was consistent with the findings from gel hardness and rheology analysis. These observations suggested that an appropriate addition of catechins could enhance the hardness and rheological properties of commercial SPI gels while improving their surface morphology, making the gels more uniform, dense, and smooth. However, as the catechin concentration increased, the Ra of SPI-C complex gels also increased. This might be attributed to the higher catechin concentration leading to increased aggregation between complexes, forming larger clusters and thus increasing the Ra of SPI-C complex gels. This observation is consistent with the findings of Hu et al. (2022), who found that high concentrations of tea polyphenols result in greater attachment of tea polyphenols molecules to myofibrillar proteins, thereby increasing the roughness and complexity of the myofibrillar gel surface.

3.3.6. Analysis of intermolecular forces driving protein gel formation

Hydrogen bonds, hydrophobic interactions, disulfide bonds, and electrostatic interactions are the primary forces that contribute to the formation of a protein gel network. The composition of these forces determines the characteristics of the protein gel (Cheng et al., 2021). These chemical interactions can be disrupted by specific chemical agents, and the solubility of mixed protein gels in different chemical agents characterizes the changes in these interactions. Since DTT can break intermolecular or intramolecular disulfide bonds, a higher solubility of protein gels in DTT indicates a greater contribution of disulfide bonds to gel formation. Hydrophobic interactions and hydrogen bonds can be disrupted by GuHCl. NaCl is used to assess electrostatic interactions and other weaker molecular forces during the formation of gel network structures. As shown in Fig. 12, the solubility in the three chemical reagents for commercial SPI was significantly lower compared to the native SPI (P < 0.05), suggesting that spray-drying-induced insoluble aggregates adversely affected gel properties. Additionally, all SPI-C sample gels exhibited significantly higher solubility than both the native and control SPI, indicating that catechin addition enhanced intermolecular forces within the gel. Notably, when the addition of catechin was 0.25%, the gel solubility in the three reagents was significantly higher than in other treatment groups, suggesting that appropriate concentrations of catechins enhanced the intermolecular forces of SPI gel formation. Furthermore, as catechin concentration increased, the solubility in all three chemical agents exhibited a declining trend. This phenomenon could be attributed to the fact that at higher concentrations of catechins, the quinones of catechins reacted with the sulfhydryl groups of proteins to form mercaptoquinone covalent adducts, thereby weakening the interactions within the stable gel network structure.

Fig. 12.

Solubility of sample gels in DTT, GuHCl, and NaCl reagents. Note: “Native” refers to native SPI, whereas 0%-1.75% indicates spray-dried SPI-C complexes with varying catechin concentrations. Different letters represent significant differences (P < 0.05).

As depicted in Fig. 12, the solubility of the gel in DTT was significantly higher compared to that in the other two chemical reagents, suggesting that disulfide bonds were primarily responsible for maintaining the gel network structure. Moreover, as the catechin concentration increased, the solubility of protein gels in GuHCl exhibited a decreasing trend. This reduction could be attributed to excessive catechin, causing structural unfolding of SPI and interfering with normal protein-protein interactions. An overabundance of catechin molecules might induce steric hindrance or even aggregation, thereby weakening the overall gel structure and reducing its stability and solubility in GuHCl. These findings are similar to those reported by Xue et al. (2022), who found that excessive addition of tea polyphenols causes aggregation and rearrangement of egg white protein gels, resulting in reduced hydrophobic interactions and thus lower solubility in GuHCl. Furthermore, when catechin concentration exceeded 1%, gel solubility in NaCl decreased compared to other SPI-C gel samples. This might be attributed to excessive catechins promoting protein aggregation and precipitation, which reduced the electrostatic interactions within the gel network. Similar observations were obtained by Cheng et al. (2023), who found that increasing quercetin concentration to 160 μmol/g protein weakened electrostatic repulsion between particles, resulting in larger psizes.

4. Conclusions

This study demonstrates that conjugating catechin with soy protein to form SPI-catechin (SPI-C) complexes during spray drying treatment can effectively intervene in the thermal aggregation behavior of SPI. The incorporation of catechins into SPI facilitated a conformational transition of SPI under spray drying conditions, leading to a more ordered structural arrangement and partial unfolding of the tertiary structure post-treatment, which correlates with enhanced thermal stability. Moreover, the interaction between SPI and catechin was primarily driven by hydrophobic interactions. Furthermore, at an optimal concentration of catechin (1%, w/w), the thermal aggregation behavior of SPI induced by spray drying was mitigated, reducing insoluble aggregates content by 71.7% while increasing soluble aggregates (> 100 kDa). Notably, at this catechin addition level, several functional properties of SPI were significantly improved, including solubility, emulsifying properties, and foaming properties. Additionally, at a catechin concentration of 0.25% (w/w), gel strength, storage modulus, and surface smoothness reached their maximum values compared to other groups, attributed to reinforced intermolecular disulfide bonds and enhanced hydrophobic interactions within the gel network. These findings elucidate catechin-mediated inhibition of SPI thermal aggregation and provide a novel strategy to mitigate functional degradation in spray-dried plant proteins for food applications.

CRediT authorship contribution statement

Juyang Zhao: Writing – review & editing, Writing – original draft, Funding acquisition. Xuwei Fang: Writing – review & editing, Writing – original draft. Jing Liu: Software, Formal analysis. Feiran Yang: Data curation. Ting Wang: Software. Jiangbei Wang: Validation. Jiangjiang Yang: Data curation. Liya Gu: Funding acquisition.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: This document is the results of the research project funded by the China Postdoctoral Science Foundation, No. 18 Special Fund (2025 T180823), Natural Science Foundation of Heilongjiang Province (LH2024C068), Heilongjiang Provincial Postdoctoral Science Foundation (LBH-Z2204).

Appendix A. Supplementary data

Supplementary material: Supplementary figures of spectral and thermodynamic characterization for soy protein isolate-catechin (SPI-C) composite system and its soluble aggregates.

Data availability

Data will be made available on request.