Microwave Pretreatment of Peanuts Modulates Oil Body Emulsion Stability: Mechanism and Application as a Source Modification Strategy for Efficient Demulsification

Abstract

This study investigated microwave pretreatment (0–900 W) of peanuts as a source modification strategy to reduce the stability of peanut oil body emulsions (POBEs) and improve aqueous enzymatic extraction. Results indicated that higher power treatment (≥540 W) significantly destabilized POBE. The optimal condition at 720 W increased POBE extraction yield and demulsification rate by 16.82% and 46.32%, respectively, compared with the control. This destabilization was attributed to marked changes in interfacial properties, including decreased apparent viscosity, lowered absolute ζ-potential (from 35.93 mV to 27.09 mV), increased particle size (from 1177.16 nm to 1976.98 nm), and the microstructure of droplet aggregation. Compositional analysis revealed that microwave treatment induced POBE reorganization, characterized by increased lipid and phospholipid contents alongside reduced moisture, solid, and protein levels. Further interfacial protein analysis revealed that exposure triggered protein conformational unfolding, hydrophobic group exposure, and subsequent aggregation, which weakened protein adsorption at the interface and reduced the mechanical strength of the interfacial film. These findings elucidate the mechanism of microwave-induced emulsion instability, providing a theoretical basis for enhancing oil extraction efficiency through raw material pretreatment.

1. Introduction

Aqueous enzyme extraction (AEE) is an environmentally friendly extraction technique that promotes oil release by enzymatically breaking down the cell walls and the interface barrier of the oil body [1]. However, a persistent challenge in AEE is the formation of stable peanut oil body emulsion (POBE), which hinders efficient oil recovery [2]. During this process, the released oil bodies can adsorb co-extracted surface-active components such as storage proteins and phospholipids, thereby forming a complex oil-in-water (O/W) colloidal system that contributes to emulsion stability [3]. Previous research has mainly focused on post-demulsification techniques, but the strategic reduction in POBE stability through targeted pretreatment is also crucial for improving AEE efficiency.

Several studies have shown that environmental factors and pretreatment can alter emulsion stability [4]. For example, Liao et al. (2025) demonstrated that SDS can replace the interfacial protein of the soybean oil body emulsion to disrupt the stability [5]. In addition, Chen et al. (2025) reported that soybean oil body emulsion is more easily aggregated at an extraction pH of 6.0 [6]. Similarly, Lu et al. (2023) found that combining isopropanol ultrasonic pretreatments with Ca²⁺ flow additions effectively destabilized camellia oil body emulsion [7]. Li et al. (2016) proposed that roasting pretreatment could reduce emulsion formation by influencing the functional properties of peanut proteins [8]. Microwave pretreatment, an efficient and eco-friendly technology, has been shown to modulate the interfacial structure and component migration of plant oil bodies [9,10]. As demonstrated by Yu et al. (2023) [11], microwave pretreatment (700 W, 1–5 min) can destabilize the body emulsions of flaxseeds by disrupting the integrity of the film [11]. In contrast, Zheng et al. (2024) revealed that microwave pretreatment (560 W, 1–3 min) could enhance the interaction between flaxseed oil droplets and proteins, leading to the formation of a stable interfacial layer [12]. These outcomes underscore that the impact of pretreatment on oil body emulsion stability is complex and species dependent.

Peanuts represent a distinct high-protein, high-oil matrix where oil body stability is critically influenced by membrane proteins and abundant storage proteins. Although the microwave pretreatment (700 W, 4 min) of peanuts could assist with AEE to improve the extraction yield of the POBE [13], it remains unclear how the microwave affects the specific interfacial reorganization of the POBE, and this is a key determinant of the stability and demulsification efficiency. To address the research gap, our study shifts the focus to elucidating the multi-scale destabilization mechanism. We systematically examined the extraction yield and stability (freeze–thaw demulsification rate, storage, centrifugation, and thermal stability) of the POBE following microwave pretreatment at various powers (0–900 W). These macro-scale changes were then linked to their colloidal properties (rheological behavior, particle size distribution, ζ-potential, and microstructure) and chemical composition. Furthermore, the underlying mechanism was revealed by analyzing the composition, structure, and functional characteristics of the interfacial proteins. These insights contribute to a predictable and controllable preprocessing strategy for enhancing oil extraction through targeted interfacial engineering.

2. Materials and Methods

2.1. Materials and Chemicals

Peanut kernels (YuHua 37 cultivar) were purchased from QiuLe Seed Industry Technology Co., Ltd. (Zhengzhou, China). Food-grade enzyme Viscozyme^®L was obtained from Novozymes Biotechnology Co., Ltd. (Tianjin, China). The peanut oil was provided by Luhua Co., Ltd. (Laiyang, China). Analytical grade chemicals and solvents were obtained from Shanghai Macklin Biochemical (Shanghai, China).

2.2. Characterization of POBE

2.2.1. POBE Yield and Freeze–Thaw Demulsification Rate Analysis

The POBE was extracted by the aqueous enzymatic extraction (AEE) method reported by Guo et al., (2025a) with slight modification [14]. Peanut kernels were microwaved for 3 min at different power levels (0, 90, 270, 540, 720, and 900 W), followed by a drying and grinding process in a high-speed blender (FW-100, Tianjin Taiste Instrument Co Ltd., Tianjin, China) for 10 s. Subsequently, the microwaved powder was well mixed with deionized water at a solid–liquid ratio of 1:2 (w/v). An amount of 1.5% of the Viscozyme^® L was incorporated, and the mixture was stirred at a constant temperature for 2 h in a water bath at 50 °C. The mixture was stirred at a constant temperature for 5 min at 90 °C to inactivate the enzyme. The emulsion layer was obtained by means of centrifugation at 5000× g for 15 min. Finally, the obtained emulsion was frozen at −20 °C for 24 h and thawed in a 50 °C water bath for 2 h to obtain peanut oil. The following formulas were employed to determine the oil body yield and demulsification rate:

oil body yield (%) = M_o/M_p × 100%

(1)

demulsification rate (%) = M_f/(M_o × C_o)× 100%

(2)

where M_o, M_p and M_f were the mass of the emulsion layer, peanut, and free oil after demulsification, respectively. C_o was the oil content of the oil body.

2.2.2. Stability Assessment

Storage Stability

Referring to the method of Lu et al. (2023) [7], POBE was transferred into a glass bottle and stored at room temperature away from light for 7 d. The creaming index (CI) was measured to assess physical stability under static conditions.

Centrifugal Stability

To evaluate resistance to mechanical separation, POBE samples in centrifuge tubes were subjected to centrifugation at 5000× g for 30 min. The CI was then determined following centrifugation.

Thermal Stability

Emulsion samples were placed in a centrifuge tube and heated in a water bath at 90 °C for 30 min and subsequently cooled to 25 °C. The emulsions were subsequently subjected to centrifugation at 5000× g for 10 min, after which the CI was measured.

The CI of the POBE under each condition was calculated using the following formula:

CI (%) = Hs/Hp × 100%

(3)

where Hs and Hp were the height of the separation layer (cm) and the height of the POBE (cm), respectively.

2.2.3. Rheological Behaviors Measurement

Rheological tests of POBE were carried out on a rheometer (DHR-3, TA Instruments, New Castle, DE, USA) using a flat plate rheometer with a diameter of 40 mm and a detection gap of 0.5 mm. The shear viscosity of the emulsion was collected in the shear rate range of 1–100 s⁻¹. At a testing temperature of 25 °C, an oscillation frequency sweep was performed in the range of 0.1–10 Hz at a strain amplitude value of 0.5%. All dynamic measurements employed a 0.5% strain amplitude, as they fall within the LVR range for all samples (Figure S1).

2.2.4. Particle Size Distribution and ζ-Potential Measurement

Referring to the method of Liao et al. (2025) [5], a 100-fold dilution of the POBE was measured using the Bettersize 2600 laser particle size analyser (Dandong Bettersize Instruments Ltd., Dandong, China). The particle size distribution was measured at a refractive index of the substance of 1.46, a refractive index of the medium of 1.33, and a final shade of between 8.00 and 10.00%. ζ-potential measurements were performed using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) equipped with a universal folded capillary cell (DTS1070). All samples were diluted under identical conditions using the same deionized water, and the background environment (including pH) remained consistent across all measurements. The ζ-potential was calculated using the Smoluchowski model. All tests were performed at 25 °C with a pre-equilibration time of 120 s. Reported values represent the average of at least three consecutive measurements.

2.2.5. Optical Imaging Observation

The microstructure of the POBE was observed using an optical microscope (BWM–410C, Shanghai Hui Tong Optical Instrument Co., Ltd., Shanghai, China). The specimen was then observed at 100× magnification. The diluted POBE was then transferred onto a microscope slide and covered with a coverslip that was tilted at a 30° angle from the side, to ensure that no air bubbles were present. Images were captured using the microscope’s built-in digital camera and processed with the accompanying image acquisition software. For each sample, at least five fields of view were examined, and representative images were selected to illustrate the typical microstructure.

2.2.6. Confocal Laser Scanning Microscopy (CLSM) Observation

The microstructure of the POBE was visualized by the CLSM (Olympus Fluoview FV3000, Tokyo, Japan) according to Tu et al. (2024a) [15]. A quantity of 2 mL of emulsion was diluted 10-fold with deionized water. Subsequently, 10 μL of 0.1% fluorescein-5-isothiocyanate (FITC) and 10 μL of 0.01% Nile red were added and thoroughly mixed. The mixture was then left to incubate for 30 min away from light. Then, 10 μL of the sample was deposited on a fluted slide, and the coverslip was meticulously sealed to preclude the formation of air bubbles. The distribution of oil (red color) and proteins (green color) in the emulsion was observed using CLSM at 488 nm and 545 nm excitation wavelengths, respectively. At least five fields of view were captured per sample, and representative images were selected.

2.2.7. Size Distribution of Oil Droplets

The particle size distribution of oil droplets in the slurries was analyzed by Image J software (Version 2.14.0, National Institutes of Health, Bethesda, MD, USA) with slight modifications according to Tu’s report [16]. The particle size distribution data of the oil droplets were then frequency analyzed using Prism 10.0 to obtain the relative frequency distribution of the particles.

2.2.8. Determination of the Main Composition

Components of POBE can be divided into moisture, lipid, and solid according to our previous research. Briefly, the moisture content of the emulsion was determined according to the direct drying method. The solid mixtures were purified by Soxhlet extraction (GB/T5009.6-2016 [17]) to remove the residual oil. The lipid and solid contents were calculated by a weighing method. In addition, the content of protein and phospholipid was determined by the Kjeldahl method (GB/T5009.5-2016 [18]) and the molybdenum blue colorimetry method (GB/T5537-2008 [19]), respectively.

2.2.9. Interfacial Protein Concentration

The calculation method of interfacial protein concentration (Γpr) referred to the following equations:

Γpr (mg/m²) = M_pr/o/SSA

(4)

$$\mathrm{SSA} ({\mathrm{m}}^2/\mathrm{g})={\displaystyle \frac{6}{{\mathrm{D}}_{\mathrm{3,2}}}}\times {\displaystyle \frac{1}{{\mathsf{\rho }}_{\mathrm{o}\mathrm{i}\mathrm{l}}}}$$

(5)

where M_pr/o was expressed, respectively, as the mass ratio of protein to lipid (mg/g); the surface area of the POBE (SSA) was expressed in square meters per gram (m²/g); the area-averaged particle size of the POBE (D_3,2) was expressed in micrometers (μm); and the density of peanut oil (ρ_oil) was expressed in grams per cubic centimeter (g/cm³).

2.3. Structural and Functional Properties of Interfacial Proteins

2.3.1. Extraction of Interface Proteins

The peanut interfacial proteins were studied using the methodology outlined by Li et al. (2023) [20]. The POBE was mixed with a chloroform/methanol (2,1, v/v) solution in a proportion of 1:3 (w/v), stirred for 4 h, and subsequently subjected to a centrifuge at 5000× g for 10 min. The process was repeated three times, after which nitrogen blowing was performed to remove the residual organic solvent in the precipitation and to obtain the interface proteins.

2.3.2. Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Equal amounts of protein were dissolved in a loading buffer at a concentration of 5 mg/mL and then heated in a water bath. The samples were separated by a 12% stacking gel and a 5% separating gel in a PowerPac Basic (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The gel was stained with a Coomassie brilliant blue solution for 1 h, after which it was washed with a decolorizing solution to remove the stain.

2.3.3. FTIR Spectroscopy

Fourier transform infrared (FTIR) spectroscopy was performed according to the method of Chen et al. (2025) [6]. The secondary structure of interfacial proteins was conducted through FTIR spectroscopy (NICOLET 6700, Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, 2 mg of freeze-dried interfacial proteins were mixed and ground thoroughly with 200 mg of dried KBr pellet, respectively. The samples were then compressed into semi-transparent sheets using a tablet press. The instrument was calibrated with a resolution of 4 cm⁻¹, and 256 scans were conducted. The infrared absorption of the samples was recorded in the wavelength range of 400–4000 cm⁻¹. The secondary structure of the protein was analyzed by fitting the amide bands of its IR spectra to the second-order derivative IR deconvolution spectra using Peakfit software (Version 4.12, Systat Software Inc., San Jose, CA, USA).

2.3.4. Intrinsic Fluorescence Spectroscopy

The intrinsic fluorescence of the proteins was measured with a FL970Plus fluorescence spectrophotometer (Tianmei Yito Laboratory Equipment Co., Ltd., Shanghai, China). The samples were dispersed in the phosphate-buffered solution (PBS, 10 mM, pH 7.0) to a concentration of 0.1 mg/mL. The detection was carried out at an excitation wavelength of 280 nm, and the fluorescence spectra were obtained in the wavelength range of 300–450 nm with the emission and excitation slit widths of 5 nm.

2.3.5. Surface Hydrophobicity Measurement

Protein samples were prepared at a concentration of 1 mg/mL by mixing with PBS (10 mM, pH 7.0) using the fluorescent detector 8-anilinonaphthalenesulfonic-1-acid (ANS) method. Then, the protein solution was diluted with PBS to prepare a series of samples with a concentration range of 0.2–1.0 mg/mL. The fluorescence intensity was measured at an excitation wavelength of 390 nm and an emission wavelength of 470 nm after the addition of 10 μL of an 8 mmol/L ANS solution to the samples. The surface hydrophobicity was determined by the slope of the relationship between fluorescence intensity and protein concentration.

2.4. Statistical Analysis

All experimental results were expressed as mean ± standard deviations, and all tests were done in triplicate. The data were subjected to a one-way analysis of variance (ANOVA) and a Tukey’s test using the statistical software GraphPad Prism 10.0 to assess differences between samples. Significant differences in the comparisons were defined as p ≤ 0.05. In addition, the graphical representation of the data was plotted using Prism 10.0 software.

3. Results

3.1. Effect of Microwave Pretreatment on the Characterization of the POBE

3.1.1. The POBE Yield and Freeze–Thaw Demulsification Rate

In the aqueous enzyme extraction method (AEE), oil body yield and demulsification efficiency serve as indicators of extraction efficiency [20]. As shown in Figure 1, the oil body yield rate and demulsification rate reached the lowest value (45.54 ± 0.50% and 62.30 ± 0.99%) in the untreated sample. The extraction efficiency increased significantly with rising microwave power (p ≤ 0.05), particularly under higher-power microwave pretreatment conditions (≥540 W). Specifically, it was observed that when the microwave power reached 720 W, both oil body yield and demulsification rate peaked at 53.20 ± 0.11% and 91.15 ± 0.10%, respectively. Further increases in microwave power to 900 W did not result in significant changes. The microwave pretreatment may increase the yield of oil by improving the accessibility of enzymes to substrates and encouraging the enzymatic breakdown of peanut cell walls [13]. During AEE, the POBE remains stable due to the combined effects of electrostatic repulsion and steric hindrance [21]. The higher demulsification rate suggested that the emulsion stability was lower after higher-power microwave pretreatment.

3.1.2. Visual Appearance and Creaming Index (CI)

The visual appearance and creaming index (CI) of the emulsion following different external stress treatments served as direct indicators of its stability [22]. As depicted in Figure 2A, the untreated POBE showed no visible changes after storage, centrifugation, or heat treatment, indicating a superior physical stability. When peanuts were pretreated with low-power microwave irradiation (90–270 W), the extracted emulsion displayed relatively slight phase separation under centrifugal stress. However, as the microwave power was increased to 540–900 W, phase separation became markedly more pronounced, accompanied by a significant increase in CI (p ≤ 0.05). These results clearly indicated that the microwave pretreatment of peanut kernels compromises the macroscopic stability of the extracted POBE, with the degree of destabilization showing a distinct power-dependent trend.

3.1.3. Rheological Characteristics

Apparent viscosity serves as a crucial indicator for assessing emulsion stability, with its value reflecting the synergistic effect between the interfacial adsorption capacity of interfacial substances and internal network structure [23]. Figure 2B illustrates that all the POBE exhibited significant non-Newtonian fluid characteristics, with their apparent viscosity decreasing with an increasing shear rate. This obvious shear-thinning behavior was due to the disruption of intermolecular cross-linking structures when a stable emulsion system was subjected to shear forces [24]. Notably, the apparent viscosity of the POBE decreased substantially with increasing microwave power relative to the control. A reduction in apparent viscosity usually suggests a weakened mutual adhesion and a collision of molecules, resulting in decreased stability [25].

Frequency scanning was characterized within the linear viscoelastic region of the emulsion, and the results are shown in Figure 2C. Interfacial films with high elasticity can resist the strain stresses generated by oil droplet deformation, thereby preventing droplet rupture and coalescence [1]. Across the tested frequency range (0.1–10 Hz), the storage modulus (G′) was consistently higher than the loss modulus (G″) for all samples, indicating the dominant elastic behavior. Both G′ and G″ values of the POBE treated with microwaves were lower than those of the control group, suggesting weakened viscoelastic properties and a more fragile, loosened network structure between interface components. The adsorption of emulsifiers at the oil–water interface has been demonstrated to induce elasticity in the emulsion, thereby enhancing its overall integrity and strengthening the three-dimensional elastic network. Consequently, the observed decreases in G′ and G″ directly reflect that the microwave pretreatment (particularly at power levels of 540 W and above) reduced the intermolecular interactions at the POBE interface, thereby reducing emulsion stability and promoting subsequent demulsification.

3.1.4. Particle Size Distribution and ζ-Potential

The particle size distribution of emulsions has been demonstrated to be a reliable indicator of their instability [20]. As demonstrated in Figure 3A, the particle size of all samples exhibited a single-peaked distribution. However, following the microwave at varying power levels, the main peak of the POBE shifted markedly towards larger droplet sizes, suggesting droplet aggregation or coalescence.

ζ-potential is also a key parameter for evaluating the instability of the POBE [7]. In comparison with the untreated group (35.93 mV), the absolute value of the surface potential of the samples treated by microwave irradiation decreased significantly (p ≤ 0.05), further declining to 25.57 mV at 900 W (Figure 3B). The initially higher ζ-potential absolute value reflected stronger electrostatic repulsion between droplets in the untreated emulsion, which helps maintain a uniformly dispersed system [26]. Microwaves have been demonstrated to disrupt the original interfacial membrane structure, thereby promoting the rearrangement of charged amino acid residues at the interface [5]. This reorganization reduces interfacial charge density and weakens electrostatic stabilization. The observed increase in particle size and decrease in the absolute value of the ζ-potential implied a reduction in emulsion stability. Thus, the microwave pretreatment of peanuts significantly reduced the physical stability of the extracted POBE (p ≤ 0.05), with the effect being more pronounced at higher power levels (≥540 W). These findings were consistent with macroscopic stability observations.

3.1.5. Microstructure and Size Distribution of Oil Droplets

Optical microscopy revealed that all emulsion droplets exhibited a regular, nearly spherical morphology with distinct interfacial edges (Figure 3C), consistent with the report of Tu et al. (2024a) [15]. However, after treatment at higher microwave powers (≥540 W), a substantial increase in mean droplet size was observed, accompanied by noticeable droplet aggregation. These observations aligned well with the increased emulsion particle size, decreased ζ-potential absolute value, and rheological properties. This phenomenon can be ascribed to microwave-induced denaturation of interfacial proteins, which weakens the mechanical strength and electrostatic stabilization provided by the interfacial film. Consequently, droplet aggregation occurred, leading to the destabilization of the emulsion system. These results indicated that high-power microwave pretreatment (≥540 W) significantly promotes the overall disruption of emulsion stability.

Further microstructural analysis of the emulsions extracted from pretreatments with different microwave powers was performed using confocal laser scanning microscopy (CLSM). As illustrated in Figure 4, the untreated POBE was filled with red fluorescent triglycerides and surrounded by a green fluorescent protein membrane, thus exhibiting the characteristic structure of emulsio [27]. It was observed that as the microwave power increased (particularly ≥540 W), there were many large aggregates. The observed microstructural changes could be attributed to the fact that the electrostatic repulsive force of the POBE after microwave treatments was insufficient to overcome the attractive forces, which led to an unstable aggregation phenomenon. This agreed with the findings of Zaaboul et al. (2019) in their study on destabilization of the interfacial structure of roasting-induced peanut oil bodies [9].

The oil droplet size distribution analysis based on oil images obtained from CLSM provided additional evidence of emulsion instability (Figure 4). Under low power conditions (≤270 W), 99% of the oil droplets have a particle size of ≤20 μm. However, as the microwave power increased, larger oil droplets emerged, suggesting an oil droplet coalescence [14]. Moreover, the proportion of droplets ≤ 10 μm decreased to 59.52% (540 W), 54.68% (720 W), and 50.39% (900 W), confirming that microwave pretreatment reduces the population of small droplets while promoting the formation of larger ones. These results further supported the conclusion that the microwave pretreatment of peanuts compromises emulsion stability.

3.2. Effect of Microwave Pretreatment on the Composition of POBE

Microwave pretreatment significantly altered the composition of the peanut oil body emulsion during aqueous enzyme extraction (

Table 1. Main composition of the POBE with various pretreatments.

POBE	0 W	90 W	270 W	540 W	720 W	900 W
Moisture (%)	13.24 ± 0.60 a	12.16 ± 0.25 ab	11.74 ± 0.40 b	7.98 ± 0.56 c	4.80 ± 0.21 d	5.14 ± 0.15 d
Lipid (%)	79.50 ± 0.61 d	80.61 ± 0.49 cd	81.88 ± 0.38 c	86.91 ± 0.35 b	90.33 ± 0.72 a	89.74 ± 0.36 a
Solid (%)	7.77 ± 0.07 a	7.78 ± 0.12 a	6.88 ± 0.10 b	5.73 ± 0.13 c	5.36 ± 0.06 c	5.66 ± 0.54 c
Protein (%)	3.83 ± 0.26 a	3.58 ± 0.37 a	2.99 ± 0.13 b	1.90 ± 0.13 c	1.42 ± 0.05 d	1.47 ± 0.08 d
Phospholipid (%)	0.19 ± 0.01 de	0.18 ± 0.01 e	0.21± 0.01 d	0.25 ± 0.00 c	0.35 ± 0.01 b	0.39 ± 0.02 a
Γpr (mg/m2)	7.78 ± 0.13 a	7.64 ± 0.19 a	7.05 ± 0.22 a	5.74 ± 0.14 b	4.27 ± 0.43 c	4.33 ± 0.18 c

Note: Values are averages of three determinations. Different lowercase letters (a–e) in the same row indicate significant differences among the values at the 95% confidence level (p ≤ 0.05).

). In this study, the oil content of the emulsion increased as the microwave power increased, rising from 72.50% (0 W) to 90.33% (720 W). Conversely, moisture content, solid content and protein content all decreased compared to those of the control. This could be attributed to the fact that disruption of the POBE structure by heat treatment enhances the extractability of lipophilic components [28]. No significant changes occurred in the emulsion components as the microwave power was further increased to 900 W, indicating that the effect of the microwave treatment was approaching saturation. It has been demonstrated that emulsions with a higher protein content and lower lipid content exhibit smaller sizes and stronger mechanical properties and are also more stable [6].

The variation in the content of the key emulsifiers at the oil–water interface of the POBE increased with the power of the microwave pretreatment (Table 1). Surface protein concentration decreased from 7.78 mg/m² in the untreated group to 4.27 mg/m² in the 720 W microwave group, indicating a reduction in the amount of protein covering the surface of oil droplets [29]. Concurrently, phospholipid content increased from 0.19% (0 W) to 0.39% (720 W), which agreed with the results of the flaxseed oil body emulsion reported by Yu et al. (2023) [11]. This change was most likely due to microwave-induced protein denaturation and aggregation, which led to the protein detaching from the interface [13]. Furthermore, disruption of the cell membrane structure by microwaves facilitated the release of internal phospholipids. A decrease in the interfacial protein concentration caused the interfacial film to thin, which weakened the spatial stabilizing effect and the electrostatic shielding effect of the POBE [30]. As the proteins degraded, the released phospholipids adsorb at the interface, promoting greater oil dispersion into the emulsion droplets and ultimately forming a high-oil-content emulsion system [31]. The above results suggested that microwave exposure can effectively modulate the adsorption behaviors of proteins and phospholipids at the oil–water interface during AEE, thereby altering the final composition of the interfacial membranes. This was manifested as an increase in lipid and phospholipid content and a decrease in water, solids, and protein content.

3.3. Effect of Microwave Pretreatment on the Interfacial Proteins Structure

3.3.1. SDS-PAGE Analysis

SDS-PAGE was used to analyze the effect of microwave pretreatments on the major protein composition at the interface in POBE. As illustrated in Figure 5A, the main membrane proteins in emulsions were adsorbed proteins and intrinsic proteins. The adsorbed proteins mainly comprised lipoxygenase, which has a molecular weight of around 95 kDa, and the 58 kDa arachin subunit. Intrinsic proteins were primarily composed of steroleosin (36–43 kDa), caleosin (27–35 kDa) and oleosin (14–26 kDa). Overall, microwave pretreatment did not alter the primary band composition of the interfacial proteins. However, higher-power microwave treatment (≥540 W) resulted in a reduction in the characteristic protein band at 95 kDa, accompanied by the emergence of high-molecular-weight aggregated bands at the top of the gel. This behavior might be related to the degradation and aggregation of protein induced by microwaves [32]. Oxidative processes catalyzed by microwave thermal effects and Maillard reactions could also induce these alterations [30]. Similar to the phenomenon reported by Waszkowiak et al. (2020) [33], steaming and baking caused degradation of the flaxseed oil body’s interficial protein components. From the SDS-PAGE results, higher-power microwave (≥540 W) irradiation induced protein degradation, aggregation, and denaturation, leading to the formation of high-molecular-weight insoluble polymers. This process reduced the protein content at the interface, ultimately compromising the emulsion’s interfacial stability.

3.3.2. FTIR Spectroscopy Analysis

As described in Figure 5B, the FTIR spectra of the interfacial proteins of the POBE were obtained following the microwave treatment of peanuts with varying powers. The results showed that the application of diverse power microwave treatments did not induce the generation of new characteristic absorption peaks in the interfacial proteins, indicating that the protein functional groups were not essentially changed. The broad peak at 3282 cm⁻¹ could be attributed to the O-H stretching vibration; 3005 and 2925 cm⁻¹ peaks were the stretching vibration peaks of the C-H and 1648 and 1535 cm⁻¹ were the characteristic peaks of the amide I band and the amide II band of the proteins, from the C=O stretching vibration, N-H bending vibration and C-N [23,34]. However, the intensity of the hydroxyl absorption peak increased significantly, while the intensity of the characteristic absorption peak at 1749 cm⁻¹, which was attributed to the stretching vibration of the carbonyl (C=O) bond, diminished markedly.

Similar changes were demonstrated in studies by Wang and Liu. (2024) [30] and Yang et al. (2025) [35]. It was hypothesized that microwave pretreatment effectively disrupts the integrity of the oil bodies and the binding structure between interface proteins and lipids. This process exposed the lipids, making them easier to remove during subsequent degreasing steps using organic solvents [36]. In general, the FTIR spectroscopy results suggested that most secondary structures persist following pretreatment.

The effect of pretreatment on the secondary structure was further quantified by the alterations in the proportion of the four kinds of structures of the amide I band, namely α-helix, β-sheet, β-turn, and random coil, and the results were displayed in Figure 5C. Within the control group, the interfacial proteins were predominantly composed of α-helix (21.04%) and β-sheet (34.69%), which imparted a compact conformation and enhanced stability [37]. In comparison with the control, the content of α-helix and β-turn in the interfacial proteins decreased with the application of microwave pretreatments of ≥540 W, and the β-sheet and random coil increased (p ≤ 0.05). This conformational change may weaken the intrinsic intra- and inter-chain interactions, disrupting their ordered conformation [38]. Microwave treatment disrupted the original hydrophilic and hydrophobic equilibrium between interfacial protein molecules, thereby promoting the formation of β-sheet secondary protein structures. This suggested that microwave pretreatment may result in a process of reconfiguration denaturation.

3.3.3. Intrinsic Fluorescence Analysis

The intrinsic fluorescence of the interfacial proteins was found to be predominantly reflective of alterations of the microenvironment in which the tryptophan (Trp) residues were located [39]. The maximum emission wavelength (λ-max) of the interfacial proteins in all treatments underwent a red shift, demonstrating that the Trp residues were in a higher polarity microenvironment, implying that they were exposed to the hydrophilic interfacial region. It was noteworthy that the fluorescence intensity of the absorption peak increased with rising microwave power, indicating that the tertiary structure of the interfacial proteins was significantly altered. This phenomenon was consistent with the effect of short-term microwave exposure (1–3 min) observed by Yu et al. (2023) [11] in flax OB. Generally, microwave pretreatment resulted in the unfolding of the molecular structure of interfacial proteins, thereby prompting the exposure of a greater number of hydrophobic amino acids to the oil–water interface. This, in turn, results in alterations to their functional properties.

3.3.4. Surface Hydrophobicity Analysis

The hydrophobicity of the interfacial proteins was displayed in Figure 5E, where the surface hydrophobicity exhibited a significant increase with the increase in microwave power (p ≤ 0.05). This was consistent with the results obtained from the endogenous fluorescence assay. The hydrophobic groups of natural proteins spontaneously cluster together towards the interior of the protein structure to form a hydrophobic core [40]. The increase in surface hydrophobicity might be attributable to the thermal effect of microwaves, which have been shown to result in an increased degree of protein de-folding at the POBE interface [41]. This process exposed a greater number of hydrophobic groups, thereby leading to elevated hydrophobicity. Furthermore, the exposure of hydrophobic regions significantly enhanced the propensity for protein intermolecular interactions, resulting in aggregation and precipitation [42].

3.4. Mechanism of Microwave Pretreatment Reducing the Stability of POBE

Figure 6 showed a mechanistic diagram illustrating the mechanism based on the results of the above research, providing a clearer understanding of how microwave pretreatment of peanut kernels promoted the decline in emulsion stability during AEE. This destabilization process of the POBE fundamentally stemmed from alterations in both the composition and properties of the interface. In untreated systems, emulsion stability primarily relied on the phospholipid membrane and the interfacial proteins embedded within it, with the adsorption of exogenous proteins also contributing to stability to a certain extent.

Following microwave pretreatment, the composition and conformation of interfacial proteins underwent significant alterations, with the α-helix and β-turn structures decreasing and transitioning to disordered random coil structures. Microwave pretreatment also promoted intermolecular hydrophobic interactions, leading to partial protein aggregation due to increased hydrophobicity. These conformational changes and aggregation behaviors resulted in a significant reduction in the protein content at the POBE interface. Ultimately, this weakened the electrostatic repulsion and steric hindrance effects within the interface, resulting in reduced mechanical strength and stability of the POBE.

This destabilization effect exhibited an increasing trend with rising microwave power, stabilizing beyond 720 W. At lower power (90 W and 270 W) pretreatments, preliminary alterations in interfacial composition occur, with a slight decline in emulsion stability. As power further elevated to 540 W and above, the synergistic action of microwave thermal effects intensifies protein denaturation and hydrophobic aggregation, thereby precipitating rapid destabilization of the emulsion system.

4. Conclusions

Microwave pretreatment of peanut kernels, particularly at power levels ≥ 540 W, effectively induced the destabilization of peanut oil body emulsions (POBEs) by altering the interfacial properties. Following higher-power microwave pretreatment, emulsions showed decreased apparent viscosity and viscoelasticity, increased particle size, lower absolute ζ-potential values, and visible droplet aggregation in microstructure observation. Additionally, the main compositional analysis further revealed that increasing microwave power led to increased lipid and phospholipid content, while reducing levels of moisture, solids, and protein. Interface protein analysis revealed that microwave treatment triggered conformational changes in the interfacial protein, including the unfolding of molecular chains and exposure of hydrophobic groups. This led to partial protein aggregation and sedimentation, which significantly reduced the availability of interface proteins. Consequently, the mechanical strength and stabilizing capacity of the interfacial film were weakened. In summary, the conformation of interfacial proteins disrupts the main composition of the POBE, thereby significantly enhancing the demulsification efficiency of the aqueous enzymatic extraction (AEE). These findings provide a theoretical basis for optimizing AEE processes with controlled pretreatment strategies. While three-phase distribution experiments have clarified the primary fate of proteins, an equally in-depth quantitative tracking of the dynamic distribution of phospholipids within the interfacial film would further complete the mechanistic understanding.