Heat Treatment Modulates Structure, Functionality, and Digestion-Related Antioxidant Activity of Xanthoceras sorbifolium Seed Meal Protein

Abstract

Enhancing plant protein structure, functionality, and digestion-associated bioactivity is pivotal to advancing sustainable food applications. In this study, a controlled thermal treatment was applied to Xanthoceras sorbifolium seed meal protein (XSMP) to characterize alterations in structural features, functional performance, and digestion-related bioactivity. Structural analyses showed that moderate heating induced partial unfolding and disaggregation, leading to reduced particle size and improved colloidal stability, with optimal performance observed at 65 °C. Accordingly, foaming capacity and emulsifying activity index reached their highest values under moderate heat pretreatment (71.43% and 27.21 m²/g, respectively). Simulated in vitro gastrointestinal digestion revealed that moderate heat pretreatment enhanced protease accessibility and was associated with increased formation of low-molecular-weight fragments. As a result, digestion products from optimally treated XSMP exhibited significantly enhanced antioxidant activities during the intestinal phase, including higher reducing power, Fe²⁺-chelating capacity (up to 51.21%), and lipid peroxidation inhibition (82.83%). In contrast, insufficient unfolding at lower temperatures or excessive aggregation at higher temperatures reduced the susceptibility to digestive proteases and the associated functional performance. These findings demonstrate that controlled heat treatment provides a simple and eco-friendly strategy to enhance the functional potential of XSMP, supporting its application as a functional protein ingredient.

1. Introduction

The sustainable provision of protein has become a critical challenge in reshaping global food systems, driven by increasing nutritional demands and mounting environmental constraints [1]. Plant proteins are increasingly recognized as promising food ingredients, owing to their diverse sources, balanced nutritional profiles, and versatile functional attributes [2]. Nevertheless, conventional plant proteins such as soybeans, despite extensive utilization, face limitations including restricted cultivation areas, allergenicity, and over-reliance, which hinder their wider adoption [3]. Consequently, enhancing the functionality and broadening the utilization of plant proteins have become central tasks in protein research. From a circular economy perspective, the valorization of agricultural by-products as alternative protein resources is increasingly regarded as a key pathway toward sustainable food systems.

Xanthoceras sorbifolium is an economically valuable oilseed crop in northern China, and large quantities of seed meal are produced as a by-product during oil extraction. This seed meal is rich in proteins, representing a promising resource for nutritional and functional applications [4]. However, current studies on Xanthoceras sorbifolium have primarily focused on oils and saponins [5,6], whereas investigations into the structure, functionality, and processing adaptability of Xanthoceras sorbifolium seed meal protein (XSMP) remain scarce. Similarly, proteins from soybean and pea have shown improved solubility, emulsification, and foaming properties after physical or chemical modifications [7], suggesting that comparable strategies could expand the practical use of XSMP in food systems. Among these approaches, controlled heat treatment is particularly attractive for XSMP as a low-cost, food-grade strategy to enhance processing adaptability and enable high-value utilization.

Thermal treatment is widely used to regulate protein structures and associated functionalities in food systems. For example, studies on pea protein have demonstrated that heat treatment induces aggregation, enhances emulsifying stability, and increases emulsion viscosity in a concentration-dependent manner [8]. These effects are mainly attributed to heat-induced conformational changes, including molecular unfolding, exposure of hydrophobic regions, and controlled aggregation, which collectively enhance solubility, emulsifying capacity, and gelation [9,10]. In addition to structural and functional improvements, thermal processing can also influence digestibility and facilitate the generation of peptides exhibiting antioxidant activity, supporting their use as functional components in food formulations [11]. Rao et al. [12] reported that controlled heat treatment affected egg white protein by promoting heat-induced unfolding, gastrointestinal digestibility, and the antioxidant performance of peptides generated during digestion, highlighting the general benefits of controlled thermal processing in enhancing protein bioactivity.

However, a more systematic understanding of the relationships among heat-induced structural changes, gastrointestinal digestibility, and antioxidant bioactivity remains limited. This limitation largely arises from the strong dependence of thermal regulation mechanisms on protein composition and molecular structure [13]. For XSMP, systematic evaluations are still lacking. It is hypothesized that moderate heating may partially unfold XSMP molecules and alter intra- and intermolecular interactions, thereby enhancing solubility, emulsification, and foaming, while also increasing enzymatic accessibility during digestion. Such structural alterations may facilitate the generation of bioactive peptide fractions, ultimately improving the antioxidant potential of digestion products.

Based on this hypothesis, this study provides a systematic investigation into the effects of thermal treatment on the structural characteristics, functional properties, and antioxidant activities of XSMP and its digestion products. The specific objectives are to: (1) characterize heat-induced conformational changes using particle size analysis, zeta potential, ultraviolet-visible (UV–Vis) spectroscopy, and Fourier transform infrared (FTIR) spectroscopy; (2) evaluate the effects of thermal treatment on solubility, emulsifying capacity, and foaming properties; and (3) assess the antioxidant activities of simulated gastrointestinal digestion products to elucidate the relationship between structure and bioactivity. The findings are expected to provide mechanistic insights and technical support for the high-value utilization of XSMP, thereby promoting its value-added applications for functional ingredient development.

2. Materials and Methods

2.1. Materials and Reagents

Xanthoceras sorbifolium seed press cake, generated after oil extraction, was provided by Gaotai Hongsheng Oil Co., Ltd., Gaotai, China. Pepsin (15,000 U/g) and trypsin (>2500 U/mg) were acquired from Shanghai Macklin Biochemical Co., Ltd., Shanghai, China. Petroleum ether, sodium hydroxide (NaOH), ethanol (95%), and other analytical-grade reagents were supplied from Beijing Innochem Technology Co., Ltd., Beijing, China. Hydrochloric acid (HCl) was sourced from Beijing Chemical Factory, Beijing, China.

2.2. XSMP Preparation

The XSMP extraction procedure was adapted from commonly used alkaline extraction and isoelectric precipitation methods for plant proteins, with minor modifications [14]. Xanthoceras sorbifolium seed meal was cleaned, dried, ground using a small high-speed grinder (WK-2008, Qingzhou Fulkang Pharmaceutical Machinery Co., Ltd., Qingzhou, China), followed by sieving through an 80-mesh screen. The resulting material was subsequently defatted by reflux extraction with petroleum ether for 8 h and then dried at 40 °C for 12 h. Defatted powder (5.00 g) was dispersed in distilled water at a solid-to-liquid ratio of 1:15 (w/v). After adjusting the pH to 11.0 with 1 mol/L NaOH, the solution was stirred at 45 °C for 30 min. The mixture was subjected to ultrasound (20 kHz, 40% amplitude) for 9 min at 25 °C using an ultrasonic extractor (SFX550, Branson Ultrasonics Corporation, Danbury, CT, USA) equipped with a standard 12.7 mm probe operated in continuous mode. Afterwards, the suspension was centrifuged at 3800× g with a bench-top high-speed centrifuge (H1850, Hunan Xiangyi Laboratory Instrument Development Co., Ltd., Changsha, China) for 20 min, and the supernatant was collected. The remaining residue underwent a second extraction under identical conditions, after which the resulting supernatants were pooled, adjusted to pH 4.6 for isoelectric precipitation, and centrifuged again. The precipitate was freeze-dried with a vacuum freeze dryer (FD-1-50, Beijing Boyikang Experimental Instrument Co., Ltd., Beijing, China), affording XSMP powder. The protein content of XSMP was analyzed via the Kjeldahl method with a nitrogen-to-protein conversion factor of 6.25.

2.3. Heat Treatment of XSMP

XSMP solutions (20 mg/mL) were prepared by dispersing XSMP in distilled water. The pH of the solutions was then adjusted to 7.0 with 1 mol/L NaOH. Samples were heated at 45, 55, 65, 75, 85, or 95 °C in a water bath (BWS-10, Yiheng Scientific Instrument Co., Ltd., Shanghai, China) for 30 min under continuous stirring. The selected temperature range and heating duration were based on preliminary experiments and previous studies on plant proteins, aiming to represent mild, moderate, and severe heat treatments [13]. Heat treatments were performed in covered containers immersed in the water bath under atmospheric conditions. After heating, samples were allowed to cool rapidly and were subsequently stored at 4 °C before analysis.

2.4. Precipitation Rate of XSMP

The precipitation rate was determined according to previously reported methods [15]. A total of 10.00 mL of the heat-treated protein solution was centrifuged at 1700× g for 30 min, after which the precipitate was dried at 65 °C until constant weight. Precipitation rate (PR) was then calculated according to the equation shown below:

$$\mathrm{P}\mathrm{R}\mathrm{\%} ={\displaystyle \frac{W_p}{W_t}}\times 100$$

where W_p denotes the mass of the precipitate and W_t represents the total protein mass present in 10.00 mL of the solution.

2.5. SDS-Polyacrylamide Gel Electrophoresis of XSMP

The molecular weight distributions of XSMP were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, following established procedures [16]. The samples were dissolved in deionized water and adjusted to pH 7.0 using 1 mol/L NaOH. After adjustment to a final XSMP concentration of 2 mg/mL, each sample was mixed with loading buffer at a volume ratio of 2:1. The mixtures were heated in a boiling water bath for 4 min and then centrifuged at 1700× g for 4 min, and the supernatant was collected for electrophoretic analysis. An aliquot of 7 μL was loaded onto the gel; separation was conducted at 130 V with a DYY-6D electrophoresis apparatus (Liuyi Biotechnology Co., Ltd., Beijing, China). A commercially available three-color pre-stained protein molecular weight marker (Beckman Biotech, Shanghai, China) was used for molecular weight calibration.

2.6. Particle Size and Zeta-Potential of XSMP

The characteristics of particle size and surface charge in XSMP dispersions (0.2 mg/mL) were determined with a nanoparticle analyzer equipped for size and zeta-potential determination (BT-Zeta100, Bettersize Instruments Ltd., Dandong, China). The dispersion medium was deionized water. The refractive index values applied for the sample and the dispersion medium were 1.35 and 1.33 [17].

2.7. Structure Analysis of XSMP

2.7.1. FTIR Spectroscopy of XSMP

Freeze-dried protein powder was analyzed by FTIR with an attenuated total reflectance module (Nicolet iS50, Thermo Fisher Scientific, Waltham, MA, USA) [18]. Spectra were collected over the range of 500–4000 cm⁻¹ at 4 cm⁻¹ resolution.

2.7.2. UV–Vis Spectroscopy of XSMP

UV–Vis spectra of XSMP solutions (0.2 mg/mL) were collected using a UV–visible spectrophotometer (UV-1780, Shimadzu Corporation, Kyoto, Japan). Distilled water was used as the blank, and spectra were collected over the wavelength range of 190–400 nm.

2.8. Functional Properties Analysis of XSMP

2.8.1. Foaming Capacity and Foam Stability of XSMP

Foaming properties of XSMP were evaluated following the method reported by Zhao et al. [19]. XSMP solutions (10 mg/mL, 30 mL) were subjected to high-speed homogenization at 10,000 r/min for 1 min with a high-speed homogenizer (AD500S-H, Anne Instrument & Meter Co., Ltd., Shanghai, China). Foaming capacity (FC) and foam stability (FS) of XSMP were calculated as follows:

$$\begin{gathered} \mathrm{F}\mathrm{C}\left(\mathrm{\%}\right)=\left({\displaystyle \frac{V_1-V_0}{V_0}}\right)\times 100 \\ \mathrm{F}\mathrm{S}\left(\mathrm{\%}\right)=\left({\displaystyle \frac{V_2-V_0}{V_1-V_0}}\right)\times 100 \end{gathered}$$

V₀, V₁, and V₂ denote the volumes measured initially, immediately after homogenization, and after 30 min, respectively.

2.8.2. Emulsifying Activity and Emulsion Stability of XSMP

Emulsifying properties of XSMP were evaluated following the method reported by Wu et al. [17]. XSMP solutions (2 mg/mL, 24 mL) were combined with soybean oil (8 mL) and subjected to high-speed homogenization at 10,000 r/min for 1 min. Emulsion samples (50 μL) were pipetted from the bottom at defined time points (0, 10, and 30 min) after homogenization and immediately diluted with 5 mL of SDS solution. The absorbance of the diluted emulsions at 500 nm was recorded, and the emulsifying activity index (EAI) and emulsion stability index (ESI) values were calculated according to:

$$\begin{gathered} \mathrm{EAI}/({\mathrm{m}}^2/\mathrm{g}) = {\displaystyle \frac{2 \times 2.303 \times A_0 \times \mathrm{D}}{\mathsf{\rho } \times (1-\mathsf{\theta }) \times \mathsf{\phi } \times 1000}} \\ \mathrm{ESI}/\%= {\displaystyle \frac{{\mathrm{A}}_{10}\left({\mathrm{A}}_{30}\right)}{{\mathrm{A}}_0}} \times 100 \end{gathered}$$

Absorbance values recorded at 0, 10, and 30 min are denoted as A₀, A₁₀, and A₃₀, respectively; D, ρ, θ, and φ correspond to the fold dilution (100), XSMP concentration before emulsification (g/L), oil volume fraction (0.25), and cuvette path length (0.01 m), respectively.

2.9. In Vitro Digestion of XSMP

An established method was employed for the fresh preparation of simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) [20]. The SGF consisted of pepsin (32 g/L; 15,000 U/g) and NaCl (2 g/L), with the pH adjusted to 1.2 using 1 mol/L HCl. The SIF contained trypsin (10 g/L; >2500 U/mg), KH₂PO₄ (6.8 g/L), and pig bile salt (12 g/L), with the pH adjusted to 7.5 using NaOH solution. Heat-treated XSMP samples (25 mg/mL, 8 mL) were combined with SGF (2 mL) and incubated with constant shaking (130 r/min) at 37 °C using a thermostatic shaking incubator (THZ-98A, Yiheng Scientific Instrument Co., Ltd., Shanghai, China) for 2 h. To initiate the intestinal phase, gastric digesta were neutralized to pH 7.5, supplemented with 2 mL of SIF, and subsequently incubated for 12 h under the same conditions. At designated time points, enzyme activity was inactivated by thermal treatment in a boiling water bath for 10 min, and the samples were subsequently cooled to 25 °C before further analyses.

2.10. Antioxidant Capacity Analysis of XSMP Digestion Products

2.10.1. Reducing Power of XSMP Digestion Products

The reducing power of XSMP digestion products was assessed following a previously reported method [21]. Equal volumes (2.5 mL) of the XSMP digestion solution, potassium ferricyanide solution (1%, w/v), and phosphate buffer (0.2 mol/L, pH 6.6) were mixed, and the mixture was then incubated at 50 °C for 20 min. After the reaction, the mixture was cooled to 25 °C, 2.5 mL of trichloroacetic acid solution (10%, w/v) was added, and the mixed solution was then centrifuged at 1700× g for 10 min. A 2.5 mL aliquot of the supernatant obtained after centrifugation was taken and mixed with an equal volume of deionized water and ferric chloride solution (0.5 mL, 0.1%, w/v). After standing for 10 min, absorbance was measured at 700 nm as an indicator of reducing power.

2.10.2. Fe²⁺-Chelating Ability of XSMP Digestion Products

A mixture of 0.2 mL XSMP digestion solution, 3.65 mL deionized water, and 0.05 mL FeCl₂ solution (2 mmol/L) was allowed to react for 30 s. Subsequently, 0.1 mL of ferrozine solution (5 mmol/L) was added, and the reaction was continued for 10 min. The mixture was centrifuged at 1700× g for 5 min, and the absorbance of the supernatant was measured at 562 nm. The equation below was used to determine the Fe²⁺-chelating capacity (%):

$${\mathrm{F}\mathrm{e}}^{2+}\text{-}\mathrm{chelating} \mathrm{capacity} (\%) = (1-{\displaystyle \frac{A}{{\mathrm{A}}_0}}) \times 100$$

where A₀ and A represent the absorbance of deionized water and the tested sample, respectively [22].

2.10.3. Inhibition of Lipid Peroxidation of XSMP Digestion Products

The inhibitory effect of XSMP’s digests on lipid peroxidation was assessed using a previously reported method [22]. The XSMP digestion solution, lecithin solution, FeCl₃ solution (400 μmol/L), and ascorbic acid solution (400 μmol/L) were mixed in equal volumes (1.0 mL). The mixture was incubated at 37 °C in the dark for 1 h. Subsequently, TCA–TBA–HCl reagent (2.0 mL) was added, and the mixture was heated in boiling water for 15 min. The mixture was cooled, centrifuged at 1700× g for 10 min, and the absorbance of the supernatant was measured at 532 nm. The lipid peroxidation inhibition (%) was calculated using the equation below:

$$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{p}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e}\left(\mathrm{\%}\right)=\left(1-{\displaystyle \frac{A}{A_0}}\right)\times 100$$

where A and A₀ represent the absorbance of the sample and deionized water, respectively.

2.10.4. DPPH Radical Scavenging Capacity of XSMP Digestion Products

A 1.0 mL aliquot of XSMP digestive solution was mixed with 3.0 mL of DPPH solution (0.1 mmol/L) prepared in ethanol. The mixture was incubated in the dark for 20 min, after which the absorbance was measured at 517 nm [21]. The DPPH radical scavenging activity of the XSMP digestion products was calculated as follows:

$$\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H} \mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\left(\mathrm{\%}\right)=\left(1-{\displaystyle \frac{A_1-A_2}{A_0}}\right)\times 100$$

where A₁ represents the absorbance of the testing sample, A₂ corresponds to the absorbance of ethanol used as a blank, and A₀ denotes the absorbance of deionized water used in place of the testing sample.

2.10.5. ABTS Radical Scavenging Capacity of XSMP Digestion Products

The ABTS radical scavenging capacity was determined as previously described [20]. 1.0 mL of the XSMP digestion solution was mixed with 3.0 mL of freshly prepared ABTS working solution, and the reaction mixture was incubated at 25 °C for 6 min. The absorbance was then recorded at 734 nm. The ABTS radical scavenging capacity was calculated according to:

$$\mathrm{A}\mathrm{B}\mathrm{T}\mathrm{S} \mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\left(\mathrm{\%}\right)=\left(1-{\displaystyle \frac{A}{A_0}}\right)\times 100$$

where A and A₀ denote the absorbance of the testing sample and deionized water, respectively.

2.10.6. Hydroxyl Radical-Scavenging Capacity of XSMP Digestion Products

A 1.0 mL XSMP digestion solution was mixed with 2.0 mL FeSO₄ solution (2 mmol/L) and 2.0 mL salicylic acid solution (2 mmol/L), followed by the addition of 2.0 mL H₂O₂ (12 mmol/L) and incubation for 30 min [23]. After completion of the reaction, absorbance changes were recorded at 510 nm. The hydroxyl radical scavenging capacity was calculated using:

$$\mathrm{H}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{o}\mathrm{x}\mathrm{y}\mathrm{l} \mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\left(\mathrm{\%}\right)=\left(1-{\displaystyle \frac{A_1-A_2}{A_0}}\right)\times 100$$

where A₁ denotes the absorbance of the testing sample, A₂ corresponds to the absorbance with deionized water instead of salicylic acid solution, and A₀ represents the absorbance obtained when the testing sample was replaced with deionized water.

2.10.7. Superoxide Anion Radical Scavenging Capacity of XSMP Digestion Products

Superoxide anion radical scavenging capacity was assessed as previously described [23]. A 0.3 mL XSMP digestion solution was mixed with 4.5 mL Tris–HCl buffer (0.05 mol/L, pH 8.2) for 10 min, followed by the addition of 0.1 mL pyrogallol solution (30 mmol/L) and further reaction for 4 min. The reaction was finally terminated by adding 0.5 mL concentrated HCl. Absorbance changes were recorded at 325 nm. Superoxide anion radical scavenging activity was calculated according to:

$$\mathrm{S}\mathrm{u}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{e} \mathrm{a}\mathrm{n}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\left(\mathrm{\%}\right)=\left(1-{\displaystyle \frac{A_1-A_2}{A_0}}\right)\times 100$$

where A₁ denotes the absorbance of the testing sample, A₂ corresponds to the absorbance of deionized water instead of pyrogallol solution, and A₀ denotes the absorbance of deionized water instead of the testing sample.

2.11. Statistical Analysis

All experiments were conducted independently in triplicate, and results are expressed as mean ± standard deviation (SD). Differences among groups were evaluated by one-way analysis of variance, with Duncan’s multiple range test applied for post hoc comparisons. Statistical analyses were performed using SPSS 24.0 (IBM Corp., Armonk, NY, USA), and significance was established at p < 0.05.

3. Results and Discussion

3.1. Heat-Induced Structural Characteristics of XSMP

The protein content of the extracted XSMP was 64.95 ± 0.46%, indicating that the alkaline extraction and isoelectric precipitation process effectively enriched the protein fraction.

3.1.1. Aggregation Behavior of XSMP

As shown in Figure 1a, XSMP exhibited a pronounced temperature-dependent aggregation. The precipitation rate reached its minimum at 65 °C (15.27 ± 2.52%, p < 0.05), whereas higher temperatures led to a rapid increase, indicating irreversible aggregate formation. SDS-PAGE profiles (Figure 1b) provided additional insight into the aggregation behavior of XSMP at the molecular level. The major protein bands were mainly distributed in the molecular weight range of approximately 35–45 and 18 kDa, which is consistent with previous reports on Xanthoceras sorbifolia Bunge kernel proteins and their major fractions [24]. At 65 °C, the intensified bands of soluble proteins suggest partial unfolding of XSMP molecules, which is commonly associated with increased hydration and improved solubility, thereby reducing aggregation tendencies [25]. In contrast, excessive heating above 65 °C led to the formation of high-molecular-weight aggregates, which are speculated to be related to intermolecular disulfide bond reshuffling and enhanced hydrophobic interactions [26]. Similar heat-induced aggregation mechanisms have been reported for other plant storage proteins [27], supporting the role of thermal denaturation and intermolecular interactions in driving XSMP aggregation at elevated temperatures.

3.1.2. Particle Size and Zeta-Potential

To further evaluate temperature-induced structural transitions, particle size, polydispersity index (PDI), and zeta-potential were analyzed (Figure 2). Between samples heated at 45 °C and 65 °C, particle size remained relatively stable and reached its minimum value of 279.68 ± 0.42 nm, accompanied by the lowest PDI (0.344 ± 0.015). This phenomenon suggests that moderate heating improved XSMP dispersion and homogeneity by promoting partial disaggregation and exposing hydrophilic groups, while extensive thermal aggregation had not yet occurred [28]. At heating temperatures above 75 °C, a pronounced increase in particle size was observed, reaching 340.63 ± 7.25 nm at 95 °C, which is indicative of heat-induced aggregation [29]. The similar change trend was found for PDI, consistent with the change trend of precipitation rate (Figure 1a).

Zeta-potential measurements further revealed the dispersion behavior of XSMP under different heat treatment conditions. All samples exhibited negative surface charges, reflecting the presence of negatively charged amino acids (Figure 2b). With increasing heating temperature, the zeta-potential values gradually shifted toward more negative levels, reaching a minimum of −38.87 ± 1.70 mV at 65 °C, but showed only slight changes at higher temperatures. These variations were primarily attributed to temperature-dependent solubility alterations, which affected the exposure of surface-charged residues. Moderate heating (65 °C) therefore simultaneously reduced particle size and increasing solubility, leading to stronger electrostatic repulsion among particles and enhanced colloidal stability [30]. Taken together, the concurrent attainment of the minimum particle size and the most negative zeta-potential at 65 °C indicates that this temperature provides the most favorable balance between XSMP dispersion and colloidal stability.

3.1.3. FTIR Spectra

FTIR spectral variations are closely linked to protein solubility and aggregation behavior. No new characteristic peaks were detected in the FTIR spectra of XSMP following heat treatment (Figure 3), indicating that thermal processing did not cause covalent bond cleavage or chemical modification of XSMP. Notable temperature-dependent variations were observed in the amide I region (1600–1700 cm⁻¹), with an absorption peak at approximately 1642 cm⁻¹, which is mainly attributed to C=O stretching vibrations of the peptide backbone [31]. With increasing temperature up to 65 °C, the relative intensity of the amide I band increased, suggesting partial structural relaxation and enhanced molecular mobility. When the heating temperature exceeded 65 °C, the amide I band gradually flattened and decreased in intensity, indicating restricted backbone motion and the formation of aggregated structures. Importantly, the amide II band around 1544 cm⁻¹ exhibited a temperature-dependent trend consistent with that of the amide I region, confirming that the observed spectral variations reflect coordinated rearrangements of the peptide backbone. These FTIR results are in good agreement with the particle size and zeta-potential changes (Figure 2), collectively demonstrating that heat treatment modulates XSMP structure primarily through non-covalent interactions.

3.1.4. UV–Vis Spectra

Changes in XSMP conformation under heat treatment were evaluated by using UV–Vis spectroscopy. A distinct absorption maximum was observed at 277 nm for all samples (Figure 4), attributable to π–π* transitions of tryptophan and tyrosine residues, which is a typical spectral feature observed in plant proteins [32]. When the heating temperature was raised from 45 °C to 65 °C, the absorbance at 277 nm intensified and reached its highest absorbance at 65 °C, indicating partial unfolding of XSMP and enhanced exposure of aromatic residues [33]. However, further heating at 75 °C and above led to a marked decrease in absorption, likely due to the re-burial of chromophores within aggregated structures, which limits their contribution to UV-Vis absorption despite continued thermal input [28]. These findings are consistent with particle size (Figure 2a) and SDS-PAGE results (Figure 1b), confirming that moderate heating (~65 °C) promotes partial unfolding, dispersion, and stability of XSMP, whereas excessive heating (>75 °C) triggers aggregation.

3.2. Foaming and Emulsifying Capacities

Heat treatment markedly altered the foaming and emulsifying behavior of XSMP, exhibiting distinct temperature-dependent trends. FC and EAI increased with heating temperature and reached their maximum values at 65 °C (FC: 71.43 ± 2.62%; EAI: 27.21 ± 0.19 m²/g), followed by a decline at higher temperatures. In contrast, FS decreased at moderate temperatures and increased again upon further heating, while ESI exhibited a trend similar to EAI (Figure 5). These behaviors are closely associated with heat-induced structural transitions in XSMP. Moderate heating induced partial unfolding and aggregate dissociation of the protein [7], which facilitated interfacial adsorption and enhanced FC and EAI [34]. At the same time, the resulting reduction in bulk viscosity of protein accelerated lamella drainage, leading to decreased FS [35]. Further temperature increases promoted extensive aggregation, which increased solution viscosity and led to a partial recovery of FS but simultaneously reduced conformational flexibility and interfacial activity, ultimately impairing overall foaming and emulsifying performance [36,37]. Therefore, heating around 65 °C represents an optimal balance between protein flexibility and aggregation, consistent with the structural changes discussed in Section 3.1.

Comparable temperature-dependent responses of foaming and emulsifying behavior have been observed in other plant proteins, such as soybean, lacquer seed, and pea, where moderate heat treatment improves interfacial functionality, whereas excessive heating promotes aggregation and functional deterioration [34,38,39]. Consistent with these observations, XSMP exhibited optimal interfacial performance under moderate heating, highlighting its competitive functionality among oilseed meal proteins and its potential as a functional plant protein ingredient in food systems [40].

3.3. In Vitro Digestibility

To evaluate how heat-induced structural changes affect the gastrointestinal behavior of XSMP, SDS-PAGE was employed to monitor its digestion patterns under different pretreatment temperatures (Figure 6). During the gastric digestion stage, electrophoretic bands within each temperature group remained largely unchanged over time, indicating that XSMP exhibited strong resistance to pepsin hydrolysis. Notably, in the 55 °C and 65 °C groups, the 35 kDa band gradually diminished, accompanied by the appearance of fragments in the 14–25 kDa range, suggesting that moderate heating partially unfolded the protein, enhanced its solubility and exposed cleavage sites, thereby improving pepsin susceptibility [41]. By contrast, samples pretreated at 45 °C and 75 °C retained strong high-molecular-weight bands, likely due to either insufficient unfolding or aggregation that masked enzymatic sites, resulting in reduced susceptibility to digestive proteases. At even higher pretreatment temperatures, protein bands nearly disappeared, indicating the formation of insoluble aggregates that were removed during centrifugation, consistent with the results of precipitate rate (Figure 1a) and particle size (Figure 2a).

In the simulated intestinal phase, the 35 kDa band disappeared within 2 h across all treatments. Distinct fragments in the 25–35 kDa range were observed in the 55 °C and 65 °C groups, reflecting further hydrolysis of partially unfolded proteins into smaller peptides. With prolonged digestion, these bands progressively weakened or disappeared, consistent with continued enzymatic breakdown into low-molecular-weight peptides. Accordingly, XSMP exhibited greater susceptibility to digestive proteases following heat treatment at 65 °C, as reflected by comparative electrophoretic patterns. These digestion-derived peptides will be further evaluated for their antioxidant potential.

3.4. Antioxidant Activities of Digested XSMP

3.4.1. Reducing Power

Reducing power is commonly employed to evaluate the electron-donating capacity of proteins or peptides, thereby providing insight into their antioxidant potential [42]. As shown in Figure 7, gastric digests showed relatively low reducing power with limited differences among pretreatments, whereas intestinal digestion markedly increased activity; the 65 °C-pretreated group reached the highest value at 8 h (Figure 7b). This enhancement is consistent with improved proteolysis following moderate pretreatment (Figure 6), which facilitates the release of electron-donating peptide fragments enriched in residues such as methionine, glycine, and proline [43,44]. In contrast, insufficient unfolding at lower temperatures and aggregation at higher temperatures limited enzyme accessibility and reduced activity [45]. A slight decline at prolonged digestion may be related to further degradation of active peptides [46].

3.4.2. Fe²⁺-Chelating Ability

Fe²⁺-chelating ability, reflecting the capacity of proteins or peptides to bind pro-oxidant metal catalysts, is a widely used indicator of antioxidant potential [47]. As shown in Figure 8a, gastric digests exhibited generally low activity with only a slight time-dependent increase. In the simulated intestinal phase, the Fe²⁺-chelating ability of XSMP digests first increased and then declined with rising heat-treatment temperature at a given digestion time (Figure 8b). Prolonged digestion led to a steady increase, plateauing after 2 h, with the 65 °C-pretreated sample reaching the highest value (51.21 ± 0.22%) at 8 h. This pattern is consistent with previous research that low-molecular-weight peptides generally possess stronger Fe²⁺ affinity [48]. Moderate heat treatment improved protein solubility (Figure 1a) and structural flexibility, exposing potential metal-binding sites, including acidic amino acid residues, which may contribute to Fe²⁺ coordination, consequently further enhancing Fe²⁺ chelation [49].

3.4.3. Lipid Peroxidation Inhibition

Lipid peroxidation disrupts cellular homeostasis and is closely associated with the development of various diseases; its inhibition therefore serves as a key indicator of antioxidant capacity [50]. As shown in Figure 9a, XSMP digests exhibited weak inhibition of lipid peroxidation during gastric digestion, with only minor time-dependent changes. However, its inhibitory activity increased markedly in the simulated intestinal phase, and the 65 °C-pretreated sample reached the highest value (82.83 ± 0.59%) after 10 h (Figure 9b). In contrast, pretreatment above 65 °C resulted in a significant reduction in inhibition. The lipid peroxidation inhibitory profile paralleled the trends observed for reducing power and Fe²⁺ chelation, suggesting that peptides released during intestinal digestion contributed to terminating lipid radical chain reactions [51].

3.4.4. ABTS and DPPH Radical-Scavenging Activities

The ability of XSMP digests to quench free radicals was examined using ABTS and DPPH assays. As shown in Figure 10, ABTS and DPPH assays displayed similar temperature- and time-dependent trends. During gastric digestion, scavenging activities changed only slightly. In the simulated intestinal phase, however, pancreatin markedly accelerated degradation of XSMP, releasing smaller peptides and exposing functional residues that rapidly increased scavenging capacity, with maxima reached at 8–10 h. At later stages, further breakdown of active peptides into short fragments or free amino acids disrupted antioxidant structures, leading to a gradual decline in activity [52]. Moderate heating (65 °C) consistently yielded stronger scavenging activity, while overheating (>75 °C) weakened the response, consistent with enzymatic accessibility under aggregation-prone conditions [53].

3.4.5. Hydroxyl Radical-Scavenging Activity

Hydroxyl radicals play a key role in oxidative damage to biomacromolecules, and the ability to counteract these radicals is commonly assessed to characterize the antioxidant potential of proteins and peptides [54]. During simulated gastric digestion, the low pH environment induced low solubility and oxidative degradation of XSMP [55], thereby impairing its antioxidant function and reducing hydroxyl radical scavenging activity to below 40% (Figure 11a). In the simulated intestinal phase (Figure 11b), the scavenging activity increased substantially, and the 65 °C-pretreated sample reached the highest value (72.82 ± 0.75%) at 10 h (Figure 11b). This result indicates that peptides enriched in hydrophobic residues interacted effectively with hydroxyl radicals, leading to a marked increase in scavenging capacity [56].

3.4.6. Superoxide Anion Radical-Scavenging Activity

The superoxide anion (O₂⁻) is an important reactive oxygen species involved in oxidative damage to DNA and cellular membranes, making its scavenging crucial for cellular protection against oxidative stress [57]. As shown in Figure 12, superoxide scavenging was limited during gastric digestion but increased markedly in the intestinal phase. The 65 °C-pretreated sample exhibited the highest activity (61.64 ± 0.38%) at 10 h, which was associated with the increased release of aromatic residues, thereby improving proton-donating capacity and markedly enhancing radical scavenging activity. This trend is consistent with improved peptide release under moderate heating [58].

Overall, the antioxidant results show a clear temperature-dependent pattern, with the most pronounced enhancement observed for samples pretreated at 65 °C, particularly during the intestinal digestion stage. Among the evaluated assays, lipid peroxidation inhibition, hydroxyl radical scavenging, and superoxide anion scavenging exhibited the most sensitive responses, while reducing power, Fe²⁺ chelation, and ABTS/DPPH scavenging provided consistent trends. This pattern across multiple antioxidant indices indicates that the key outcome of moderate heating is not an isolated improvement in a single parameter, but a coordinated enhancement of antioxidant functionality following digestion.

As illustrated in Figure 13, the antioxidant advantages of XSMP digests are attributed to a coherent mechanism: moderate heat pretreatment induced partial unfolding and disaggregation of protein structures, thereby enhancing interfacial functionality and increasing protease accessibility, which in turn was associated with the appearance of antioxidant-active peptide fractions. These peptides exert their effects through multiple complementary mechanisms, including electron donation, transition-metal chelation, and chain-breaking of free radical propagation, collectively explaining the superior performance of the 65 °C-pretreated samples across antioxidant assays.

Similar observations were reported on soy proteins, where mild heating enhanced peptide-mediated antioxidant activity after digestion [41]. These parallels suggest that moderate heat pretreatment represents a generalizable strategy to enhance the functional properties of plant protein-derived peptides, providing a rationale for the use of controlled thermal conditioning in the development of antioxidant-enriched food products. Nevertheless, as the present findings are derived from simulated digestion models and comparatively evaluated experimental observations, further in vivo validation and additional quantitative characterization would help to strengthen physiological relevance and refine mechanistic interpretation for optimized processing applications.

4. Conclusions

This study systematically investigated heat pretreatment-induced changes in the structural characteristics, interfacial properties, susceptibility to digestive proteases, and antioxidant activity of XSMP. Moderate heating at approximately 65 °C induced partial unfolding and improved protein dispersion, which enhanced interfacial properties and increased enzymatic accessibility during simulated gastrointestinal digestion. As a result, the digestates generated under this condition exhibited enhanced antioxidant activities, whereas insufficient unfolding at lower temperatures or excessive aggregation at higher temperatures reduced susceptibility to digestive proteases and subsequent functional responses. These results demonstrate that heat-induced structural modulation in XSMP governs the interplay among protein unfolding, susceptibility to digestive proteases, and antioxidant activity. The findings highlight controlled heat processing as an effective and eco-friendly strategy to enhance the nutritional and functional potential of underutilized plant proteins, providing a scientific basis for their broader application in sustainable food systems and functional product development. Future studies may further strengthen its physiological relevance and refine process design through bioactive peptide profiling and in vivo validation.