Preparation and Stability Study of High Internal Phase Emulsion of Peanut Protein Isolate and Corn Silk Polysaccharide

Abstract

This work sought to explore a new method for using corn silk polysaccharide (CSP) and peanut protein isolate (PPI) to stabilize high internal phase emulsions (HIPEs). An ultrasound-assisted hydrothermal technique was used to make the PPI-CSP covalent complexes and HIPEs. Particle size analysis, rheological studies, and multiple light scattering techniques were used to analyze the stability and attributes of the emulsion. Microscopic studies reveal that the PPI-CSP complex encapsulates oil droplets at the interface, forming a typical oil-in-water (O/W) emulsion. The stability of the HIPEs is notably improved by the inclusion of CSP; the smallest particle size was recorded at a 2:1 PPI to CSP ratio (12.91 ± 0.13 μm). According to rheological evaluations, all HIPEs behave in a shear-thinning manner and have solid-like properties. Furthermore, this emulsion exhibits excellent stability during thermal treatment, changes in ion concentration, different pH values, and storage.

1. Introduction

The primary by-product of peanut oil extraction, peanut protein isolate (PPI), has a high nutritional value and is abundant in essential amino acids [1,2]. During emulsion formation, PPI can adsorb at the oil–water interface, creating a viscoelastic layer and maintaining stability through electrostatic and steric stabilization. In complex food systems or tough production conditions, though, PPI’s capacity to emulsify is limited, which makes the emulsion less stable and less useful [3]. According to Zhang et al. [4], peanut protein modified by ultrasound and pH adjustment greatly enhanced its solubility, emulsification, foaming performance, and foam stability. During ultrasound-assisted glycosylation, peanut protein’s emulsifying properties, antioxidant activity, and in vitro digestibility all significantly improved [5].

Corn silk, a popular traditional Chinese medicine and functional food, contains polysaccharides with a range of biological effects, including antioxidant, blood sugar-lowering, immunomodulatory, anti-cancer, anti-fatigue, hemostatic, and anti-obesity properties [6]. These polysaccharides, which are characterized by a specific spatial structure and macromolecular chains, can form physical barriers at the oil–water interface and reduce the adsorption forces there through electrostatic repulsion, thereby improving the stability of the emulsion [7,8]. Furthermore, polysaccharides have the capacity to enhance protein functional properties through glycosylation processes [9]. Recent research by [10] suggests that the loading levels of polysaccharides also play a role in influencing protein characteristics. PPI’s functional qualities, including its solubility and thermal stability, are improved when combined with corn silk polysaccharides (CSP), according to research by Han et al. [11]. Nevertheless, high internal emulsions have not been treated with this method.

Specialized emulsion systems known as High Internal Phase Emulsions (HIPEs) are distinguished by a dispersed phase volume percentage that usually surpasses 74%. Due to their high viscosity, substantial loading capacity, and distinctive three-dimensional network structure, HIPEs demonstrate significant potential applications in the food industry. In this context, HIPEs can serve as effective fat substitutes, significantly reducing the levels of trans fatty acids and saturated fatty acids in products such as butter and margarine. They are capable of simulating the texture and flavor of traditional fats and oils while concurrently lowering caloric intake [12]. Furthermore, HIPEs function as carriers for functional ingredients, encapsulating fat-soluble vitamins (such as vitamins A, D, and E), plant polyphenols, and probiotics. They safeguard these components from environmental degradation by forming dense interfacial films, thereby enhancing their stability and bioavailability during food processing and storage [13]. Han et al. [11] investigated the effect of CSP addition levels on the functional properties of PPI. The riboflavin loading capacity of corn silk polysaccharide–peanut isolate protein composite gels was enhanced, demonstrating certain sustained-release and protective effects for riboflavin. However, no studies have been conducted on the preparation and stability of HIPE using this composite.

Current research on the application of the PPI-CSP glycosylation complex system in the preparation of high internal phase emulsions (HIPEs) remains insufficient. This study aims to prepare high internal phase emulsions by using proteoglycan complexes and investigate their stability mechanisms. By systematically exploring how preparation conditions affect HIPEs’ stability and revealing their stabilization mechanism, this study is expected to provide a solid theoretical basis and feasible technical support for developing novel, efficient, safe, and stable plant-based emulsifiers and highly stable HIPEs—facilitating their wide application in food, cosmetics, medicine, and other fields.

2. Materials and Methods

2.1. Materials

Corn silk polysaccharide (CSP), characterized by a polysaccharide purity of 80%, was sourced from Lanzhou Waterless Biotechnology Co., Ltd. in Lanzhou, China. Peanut protein isolate (PPI) with a protein purity of 98% was sourced from Beijing Global Sci-Tech Bio-Technology Development Co., Ltd. in Beijing, China. (China Sales Agent for the Life Sciences Department of Bellancom in the USA). Soybean oil was obtained from Yihai Kerry Golden Dragon Fish Grain and Oil Food Co., Ltd. in Shanghai, China. Fluorescein isothiocyanate (FITC) and Nile Red were procured from Shanghai Aladdin Biochemical Technology Co., Ltd. in Shanghai, China.

2.2. Fourier Transform Infrared (FTIR) Spectroscopy

Glycosylation reactions in CSP, PPI, and PPI-CSP complex samples were characterized using FTIR spectroscopy. Following the method of Hu et al. [14], 2 mg of the sample was ground uniformly with 0.2 g of potassium bromide in an agate mortar under an infrared lamp. The mixture was then pressed into a thin film for full-wavelength scanning (4000–400 cm⁻¹) at 64 scans, 4 cm⁻¹ resolution, and 25 °C to obtain the FTIR spectrum curve.

2.3. Preparation of PPI-CSP Complexes and HIPE

The preparation of PPI-CSP glycosylation products was conducted based on the protocol outlined by Han et al. [11] with slight adjustments. To initiate the process, 2 g of PPI was dissolved in 0.1 L of 0.1 mol/L phosphate buffer at pH 7. Subsequently, the solution was magnetically stirred at 300 rpm for 2 h to yield a 2% (w/v) PPI solution. Take 0.2 g, 0.4 g, 1 g, 2 g, and 4 g of CSP samples of different masses and mix them separately with the PPI solution to achieve PPI-to-CSP mass ratios of 10:1, 5:1, 2:1, 1:1, and 1:2. Composite solutions with varying mass ratios were denoted as PC1, PC2, PC3, PC4, and PC5, respectively. The untreated peanut protein isolate PPI was used as a control and labeled as PPI. The resulting PPI-CSP complexes were stirred magnetically at 300 rpm for 2 h at ambient temperature. The mixture was stored overnight at 4 °C for thorough blending. Subsequently, the ultrasonic power and time settings were adjusted to 300 W and 40 min (consisting of 2 s ultrasonication followed by a 2 s interval), respectively, while the entire procedure was carried out in an ice bath. The ultrasonicated solution was then subjected to a 30 min heat treatment in a water bath at 95 °C. Following heat treatment, rapid cooling to room temperature was achieved by immersing the sample in an ice-water bath, yielding covalent complexes (hereinafter referred to as complexes) with varying proportions.

A composite solution comprising 25% PPI-CSP was blended with 75% soybean oil and subjected to high-speed shear mixing at 18,000 rpm for 1 min to generate a high internal phase emulsion containing over 74% oil phase.

2.4. Particle Size and Zeta Potential Analysis of Emulsions

Particle size and size distribution were measured using a laser analyzer for particle size (BT-9300HT, Dandong Instruments, Dandong, China). The zeta potential of the emulsions was determined using a Malvern Laser Particle Size Analyze (Zeta sizer Advance, Malvern Instruments, Malvern, UK). Particle size is a critical factor in evaluating emulsion stability. Smaller particle sizes result in slower emulsion separation and greater stability [15,16]. Emulsion stability is primarily governed by particle size, with smaller particles enhancing stability [17]. The magnitude of absolute zeta potential reflects the interaction strength between droplets; a higher value indicates greater repulsive forces between droplets and a more stable emulsion system.

2.5. Microscopic Structure Observation of the Emulsion

Fluorescent staining of emulsions was performed with minor modifications based on the protocol reported by [18]. First, two fluorescent dye stock solutions were prepared separately: Nile Red (2 mg/mL) and fluorescein isothiocyanate (FITC, 5 mg/mL) were each dissolved in anhydrous ethanol, and the solutions were stored in light-protected centrifuge tubes to prevent photodegradation. For co-staining of soybean oil (targeted by Nile Red) and PPI (targeted by FITC) in the emulsion, 20 μL of Nile Red stock solution and 20 μL of FITC stock solution were added to 1 g of emulsion sample. The mixture was left to stand in the dark for 1 h to ensure thorough staining. The stained sample was then observed using a STELLARIS 5 super-resolution fluorescence lifetime white laser confocal microscope (Leica, Wetzlar, Germany). Fluorescence detection parameters were set as follows: excitation wavelengths of 488 nm and 561 nm were used for FITC and Nile Red, respectively. Images were acquired at an objective magnification of 20×, and post-acquisition processing was conducted with a 4× digital magnification. The final images were adjusted to a consistent pixel resolution of 1024 × 1024 pixels for subsequent analysis.

2.6. Determination of Protein Adsorption Rate at the Interface

First, 5 mL of high internal phase emulsions (HIPEs) was centrifuged at 7826× g for 10 min. After centrifugation, the supernatant was carefully collected with a pipette to avoid disturbing the lower phase. Protein contents of the original PPI-CSP complex solution and collected supernatant were determined via bicinchoninic acid (BCA) assay following the manufacturer’s standard protocol. Herein, C_t refers to the protein concentration in the original solution, and C_s denotes that in the supernatant. The interfacial protein adsorption rate was calculated using the following formula:

$$\mathrm{I}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{f}\mathrm{a}\mathrm{c}\mathrm{e} \mathrm{P}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{i}\mathrm{n} \mathrm{A}\mathrm{d}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{p}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{R}\mathrm{a}\mathrm{t}\mathrm{e} (\%)={\displaystyle \frac{C_t-C_s}{C_t}}\times 100$$

(1)

2.7. Determination of Water Distribution in Emulsions

The prepared emulsion was transferred into a cylindrical glass vessel with a diameter of 15 mm. The water distribution within the emulsion was quantified using a low-field nuclear magnetic resonance (low-field NMR) analyzer. The analysis was conducted in the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence mode to accurately determine the transverse relaxation time (T₂) value.

The experimental parameters were configured as follows: spectral width (SW) = 200 kHz, waiting time (TW) = 3500 ms, number of scans (NS) = 8, echo time (TE) = 0.40 ms, and number of echoes (NECH) = 7000.

2.8. Rheological Characterization of Emulsion

The rheological properties of the emulsion were investigated, including shear-induced and temperature-induced changes in viscosity, as well as variations in modulus (storage modulus, G′; and loss modulus, G″). All measurements were performed using an MCR302 rheometer (Anton Paar, Graz, Austria) equipped with a steel plate of 25.0 mm in diameter, and the gap between the plate and the lower fixture was set to 1.0 mm. All experiments were conducted at a constant temperature of 25 °C. Dynamic viscoelastic analysis was conducted with a fixed strain amplitude of 1% within the linear viscoelastic region and an angular frequency scan range of 0.1–100 rad/s to determine the variation patterns of G′ and G″ with respect to angular frequency. For viscosity characterization, shear tests were performed for a duration of 20–100 s, and the corresponding viscosity curves were recorded.

2.9. Physical Stability of the Emulsion

The physical stability of the PPI-CSP high internal phase emulsion (HIPE) was systematically characterized using a multi-light-scattering instrument (Turbiscan Tower, Formulaction, Toulouse, France). Following the instrument’s standardized operating procedures and the methodology reported by Du et al. [19], the vertical scanning mode was employed for the analysis. Specifically, equal volumes of the emulsion samples were carefully transferred into specialized glass sample vials, and the scanning process was conducted using a pulsed near-infrared light source with a wavelength of λ = 880 nm. At a constant temperature of 25 °C, light signals were collected at 40 μm intervals during each scan. Each individual scan cycle was set to a duration of 30 min, and the total detection period spanned 24 h. The Turbiscan Stability Index (TSI) was employed to assess the stability of the emulsion. This index quantifies the variations in signal intensity between transmitted and scattered light as the light beam traverses the emulsion; an elevated TSI value signifies a decline in sample stability [20]. The change in backscattering intensity (ΔBS) was determined by calculating the difference between the current BS value and the initial BS value obtained from the first scan. All TSI and BS data were systematically recorded and acquired using the Turbiscan Stability Analysis software (v1.3.0.0) (Formulation, Toulouse, France). TSI is computed using the formula in which $x_i$ signifies the backscatter intensity of the i-th scan, $x_{BS}$ represents the mean backscatter intensity, and n indicates the total number of scans in the measurement.

$$TSI=\sqrt{{\displaystyle \frac{{\sum }_{i=1}^n{(x_i-x_{BS})}^2}{n-1}}}$$

(2)

2.10. Ionic Stability of Emulsion

To further evaluate the stability of PPI-CSP HIPEs, PPI-CSP solutions at different concentrations were prepared. The NaCl concentration in each solution was adjusted to 0 mmol/L, 100 mmol/L, 200 mmol/L, 300 mmol/L, and 400 mmol/L. The emulsion preparation method was the same as described in Section 2.2. All prepared HIPEs were stored at 4 °C for 24 h for subsequent characterization.

After storage, the particle size and zeta potential of the HIPEs were measured using the corresponding instruments.

2.11. Acid-Base Stability of Emulsions

Using 1 mol/L HCl and 1 mol/L NaOH solutions, the produced PPI-CSP composite solution was brought to various pH levels (2, 5, 7, 9, and 11). To produce HIPEs with an oil phase volume fraction of 75%, incorporate 30 mL of soybean oil for every 10 mL of pH-adjusted composite solution. Homogenize the mixture for one minute, utilizing a high-shear apparatus functioning at 18,000 rpm. For further examination, all produced HIPEs were kept at 4 °C for 24 h. Following storage, HIPEs’ visual changes were noted and documented, and the appropriate tools were used to assess the particles’ size and zeta potential. Each sample was analyzed in triplicate, and results are expressed as the mean ± standard deviation.

2.12. Heating Thermal Stability of Emulsions

Thermal treatment was applied to freshly made PPI-CSP high internal phase emulsion (HIPE) samples, which were heated for 30 min at a steady 90 °C (preparation method as described in Section 2.2). The samples were allowed to naturally cool to ambient temperature (25 °C) following the thermal treatment, and then kept for 24 h at 4 °C to evaluate the stability of post-thermal storage. The emulsions’ appearance and microstructural properties were recorded using photography after the 24 h storage period. Additionally, the same particle size analyzer used in the previous particle size measurement tests was used to determine the emulsions’ particle sizes at 25 °C.

2.13. Freeze–Thaw Stability of Emulsion

Freshly manufactured emulsions (preparation method as described in Section 2.2) were exposed to a freeze–thaw cycle protocol: first frozen at −20 °C for 20 h, then thawed at ambient temperature for 2 h, followed by refreezing at −20 °C for another 20 h. For every emulsion sample, this freeze–thaw cycle was carried out three times.

Photographs of the emulsion appearance were captured before the first cycle and after each completed freeze–thaw cycle to visually track changes. For the oil–water mixtures that exhibited phase separation after freeze–thawing, high-speed shearing was performed at 18,000 rpm for 1 min to evaluate their re-emulsification capability. HIPEs that could be re-emulsified following freeze–thaw-induced separation were considered to have excellent temperature responsiveness. Additionally, the particle size and rheological properties of the emulsions were measured both before the first freeze-thaw cycle and after the third cycle.

2.14. Storage Stability of Emulsion

Freshly prepared emulsions (preparation method as described in Section 2.2) were stored under normal room conditions. During the storage period, photographs of the emulsion appearance were captured on day 1, day 10, day 20, and day 30, respectively, to visually monitor changes in appearance. Furthermore, the particle size and rheological properties of the emulsions were measured at identical time intervals.

2.15. Statistical Analysis

All experiments were conducted in triplicate, with the resultant data expressed as the mean ± standard deviation. Graphs were made utilizing Origin 2021 (Origin Lab, Northampton, MA, USA). For statistical analysis, one-way ANOVA was conducted with SPSS Statistics 27 software (SPSS Institute Inc., Chicago, IL, USA), and the significance level among all samples was set as p < 0.05.

3. Results and Discussion

3.1. FTIR Analysis

FTIR spectroscopy reflects protein secondary structure and hydrogen bonding forces, and is widely used to analyze protein glycosylation modifications. To validate the formation of PPI and CSP glycosylation, Fourier Transform Infrared Spectroscopy (FTIR) was employed to characterize structural changes in PPI, CSP, and their glycosylated complexes PC1–PC5. The FTIR spectrum (Figure 1) reveals significant shifts in characteristic peaks of PPI and CSP following ultrasonic-assisted moist heat treatment. All samples exhibit distinctive absorption peaks at 3600–3200 cm⁻¹ associated with intermolecular hydrogen bonding, attributed to O–H and N–H stretching vibrations within the proteins. Following glycosylation, the absorption intensity of the PPI-CSP complex increased at peaks within the 3600–3200 cm⁻¹ range, with peak shifts toward lower wavenumbers. A plausible explanation is the introduction of CSP hydroxyl groups, leading to increased hydrogen bond density between the complex and water molecules. This may reduce electron density, elongate O–H and N–H bonds, thereby decreasing the energy required for hydrogen substitution [21]. The absorption peak of the PPI-CSP complex is enhanced at 1650 cm⁻¹. This change relates to C–N stretching and N–H bending vibrations in the amide I band induced by the glycosylation process. This finding aligns with the glycosylation results reported by Sun et al. [22] for soybean protein isolates with sodium carboxymethyl cellulose.

3.2. Particle Size and Zeta Potential Analysis of the Emulsion

Average size is a critical determinant in assessing emulsion stability. A smaller particle size results in a reduced separation rate and enhances emulsion stability. Therefore, the stability and fineness of the PPI-CSP composite emulsion were evaluated based on particle size, with the corresponding results presented in Figure 2A, the volume-averaged median particle size (D₅₀) of the control emulsion (PPI—without CSP addition) was 17.07 ± 1.13 μm. As the concentration of CSP rises, the D₅₀ of the composite emulsion initially diminishes and thereafter ascends. Notably, the D₅₀ values of the emulsion groups supplemented with CSP were significantly smaller than that of the control group ($p<0.05)$, indicating that CSP addition effectively improved the emulsion’s fineness. When the mass ratio of PPI to CSP reached 2:1 (referred to as the PC3 group), the emulsion’s D₅₀ reached a minimum value of 12.91 ± 0.16 μm. This indicates that complex formation is beneficial for emulsion stability. CSP addition increases protein adsorption at the oil–water interface, effectively preventing droplet coalescence. When the mass ratio of PPI to CSP reaches 1:1, the D₅₀ value rebounds, and the particle size subsequently increases. This phenomenon is most likely caused by the aggregation of the polysaccharides themselves [1,23].

The magnitude of the absolute value of the zeta potential represents the strength of interactions between droplets. A higher strength indicates greater repulsive forces between droplets, resulting in a more stable emulsion system. As shown in Figure 2B, the absolute zeta potential of the control emulsion (without CSP) was 38.8 mV. After adding CSP, the absolute zeta potential of the experimental group was consistently higher than that of the control group (p < 0.05). The absolute zeta potential first increased and then decreased: at PC3 (PPI/CSP mass ratio = 2:1), it reached the maximum of 48.91 mV, consistent with the trend observed in the average particle size results. These results indicate that CSP altered the protein surface charge distribution, enhancing inter-particle electrostatic repulsion and thus increasing the emulsion’s absolute zeta potential [24].

3.3. Rheological Characterization

In addition to revealing a sample’s rheological type, shear rheology studies show minute variations across different samples. The apparent viscosity of the emulsions decreases rapidly with increasing shear rate (Figure 2C), exhibiting shear-thinning behavior—a characteristic of non-Newtonian fluids. At a constant shear rate, the apparent viscosity gradually increases with rising CSP concentration. This may be attributed to CSP’s high molecular weight, strong hydrophilicity, and interchain interactions, which collectively endow the polymer itself with high viscosity [25].

As shown in Figure 2D, within the frequency range of 0.1–100 rad/s, the storage modulus (G′) of all emulsions exceeded the loss modulus (G″). Both G′ and G″ gradually increased with rising frequency, indicating that emulsions stabilized by PPI-CSP composites (all CSP-to-PPI ratios) exhibited elastic, frequency-dependent behavior. With increasing CSP concentration (PPI/CSP = 10:1, 5:1, 2:1, 1:1,1:2), both G′ and G″ of the emulsion first increased but began to decrease at PPI/CSP = 1:1. This indicates the emulsion had the strongest gel strength at PC3 (PPI/CSP = 2:1).

3.4. Microstructure

HIPES (φ = 74%, v/v) prepared at different PPI-CSP concentrations were characterized via CLSM. The fluorescent signals of oil and water overlapped: soybean oil appeared green in images (stained by Nile Red), while PPI-CSP composite particles appeared red (stained by FITC). As shown in Figure 3, from the overlay images, all emulsion systems exhibit a typical oil-in-water (O/W) structure, with the oil phase distributed inside the droplets and the complexes on the droplet surface to form a dense shell. Among these, the PPI emulsion exhibits uneven droplet distribution, with some droplets aggregating and a loosely structured shell layer, resulting in limited interfacial stability. As the polysaccharide content increases, HIPE droplets exhibit a more uniform distribution. At PC3 (PPI-CSP ratio = 2:1), droplets became smaller and more uniformly distributed. This result may stem from increased CSP concentration, which enhances emulsion stability: it promotes protein adsorption on oil droplet surfaces via increased negative charge repulsion and emulsion viscosity, thereby inhibiting droplet aggregation [26].

3.5. Interfacial Protein Adsorption Rate

The adsorption rate of interface proteins is a key parameter for evaluating the effect of CSP dosage on the interface properties of the emulsion [27]. As shown in Figure 4A, in the determination of interfacial protein adsorption rates, the control group without CSP addition exhibited the lowest adsorption rate of 76.23%. In contrast, the adsorption rates of the CSP-added PC1–PC5 groups all significantly increased (p < 0.05). The PC3 group reached a peak adsorption rate of 83.29%, while the PC4 and PC5 groups showed slight decreases to 81.19% and 80.87%, respectively, exhibiting an overall trend of “initial increase followed by decline.” Despite this decline, the interfacial protein content remained higher than that of the PPI group, suggesting successful adsorption of the PPI-CSP complex at the oil–water interface. Particularly under PC3 treatment conditions, over 80% of the proteins were effectively adsorbed at the oil–water interface, playing a crucial role in reducing emulsion particle size.

3.6. Water Distribution

Low-field nuclear magnetic resonance (NMR) techniques are frequently used to investigate the physical state and distribution of water in emulsion gels. These properties are closely related to the emulsion’s internal structure and interactions between water, proteins and polysaccharides [7,28]. Based on hydrogen proton relaxation time, water in HIPES can be classified into three forms: bound water (0.01 ms < T₂₁ < 10 ms), semi-bound water (10 ms < T₂₂ < 200 ms), and free water (200 ms < T₂₃ < 1000 ms). Bound water is defined as water molecules that are securely attached to the sample matrix by hydrogen bonds and van der Waals forces, resulting in highly restricted movement and the inability to freely diffuse. In contrast, free water is made up of molecules that are either unattached or weakly bound to the matrix. It can be found in the emulsion’s continuous phase, huge holes, or between droplets. These molecules are very mobile and can spread easily. The higher the bound water content, the more strongly the water molecules bind to macromolecules. The denser the network structure within the emulsion’s aqueous phase, the more readily water is trapped in the network [29]. The water distribution in HIPEs at different CSP concentrations and their respective change rates are shown in Figure 4B,C. The bound water content first increased and then decreased—this increase reflects modifications in protein tertiary and quaternary structures, promoting gel network transformation. As demonstrated by Han et al. [11], the covalent binding of high-concentration polysaccharides stretches the spatial structure of peanut isolate protein, unfolds peptide chains, and increases flexibility, thereby enhancing water retention. These modifications contribute to strengthening the overall network structure of the emulsion. As shown in Figure 4B, most water in all samples is semi-bound water, trapped in HIPEs’ 3D network. With increasing CSP addition, the emulsion’s bound water content increased from 61.5% to 64.71% then decreased to 55.01%, while free water content decreased from 38.5% to 35.29% then increased to 44.59%. Owing to CSP’s thickening effect, higher concentrations enhanced HIPEs’ three-dimensional network, thereby binding more free water. Thus, a small amount of CSP addition promoted the aggregation of PPIs and the formation of 3D network structures, but too much CSP addition hindered the aggregation of PPIs and led to the formation of loose 3D networks.

3.7. Physical Stability

Emulsion instability manifests itself in the form of flocculation, emulsification, sedimentation, coalescence, Ostwald ripening, and phase separation [30]. Continuous 24 h scanning of HIPE preparations using different concentrations of CSP and PPI, combined with analysis of changes in reflected light, allows for the tracking of emulsion particle movement and prediction of stability. TSI and backscattered light intensity changes (ΔBS) dynamically reflect the system’s global and local stability variations [31]. In solution systems, larger solute particles lead to higher backscattered light intensity. When instability occurs (with gradual precipitation), backscattered light intensity increases at the solution bottom, while the top becomes clearer with decreasing intensity. In stable solutions, backscattered light intensity remains relatively constant over time [32]. Backward scattering (ΔBS) is a reliable indicator of the internal distribution of emulsions and their microstructural state [33]. An increase in ΔBS values indicates significant changes in internal microstructure, thereby reducing emulsion stability. Conversely, lower ΔBS values mean greater stability and consistent microstructure. As shown in Figure 5A–F, PPI-CSP emulsions containing CSP had lower ΔBS values, especially in the 2:1 PPI-CSP group. The curve in this group was smoother, with consistently lower ΔBS values, indicating superior physical stability compared to other groups. This may be due to the formation of a strong barrier between CSP and PPI at high concentrations, which promotes the formation of smaller droplets and inhibits aggregation. These droplets are less sensitive to gravitational forces, which promotes emulsion stability [30]. Particle flotation at the vessel base was shown by PPI, PC3, and PC4, which only showed negative BS values near the bottom. Among these, PC3 eventually reverted to a BS value close to zero, indicating a slow redistribution of particles. PC5 displayed negative results at different heights, suggesting poor stability and varying particle distribution.

The emulsions’ TSI values were studied to further evaluate their overall stability. As shown in Figure 4C, during storage, all sample emulsions’ TSI values initially increased gradually with prolonged time. After 14 h, the TSI values of PPI, PC4, and PC5 rapidly increased. This may be attributed to droplet aggregation within the HIPE solution over time, causing the oil phase to rise. Meanwhile, PC3 consistently maintained the lowest TSI value with the smallest slope over time—indicating its optimal stability. PC5 exhibits a low TSI value prior to 20 h, followed by a rapid increase thereafter, indicating significant instability beginning at the 20 h mark. The TSI of PPI shows a trend of first increasing, then decreasing, and subsequently increasing again, demonstrating poorer stability compared to PC3 and PC5. This may be attributed to CSP-PPI covalent bonding stretching PPI’s spatial structure, unfolding peptide chains, and exposing originally enclosed hydrophobic groups. This facilitates composite protein adsorption at the O/W interface, enhancing stability. PC3 (PPI/CSP = 2:1) exhibited higher stability, consistent with ΔBS analysis results.

3.8. Ion Stability

To investigate ions’ effect on emulsion stability, emulsions with different salt ion concentrations were prepared. Their D₅₀ and zeta potential values were measured (Figure 6A,B). CSP-stabilized emulsions showed good stability across within the range of 0–300 mmol/L salt ion concentrations, with the average particle size decreasing as salt concentration increased—typically attributed to salt-reduced electrostatic repulsion. Contributing factors are, first, salt reduces electrostatic repulsion between polysaccharide molecules, promoting tighter packing and smaller effective radii. Second, it diminishes attraction between polysaccharide negative groups and protein-coated droplet positive groups, inducing polysaccharide rearrangement on droplets to form thinner, denser coatings [34,35]. When the ionic concentration further increased to 400 mmol/L, the average particle size increased to varying degrees. This may be due to NaCl affecting CSP-PPI interaction, impairing PPI-CSP complex adsorption at oil/water interfaces; continuous-phase CSP caused repulsive flocculation, leading to droplet aggregation and larger particle size.

As NaCl concentration increased from 0 to 400 mmol/L, the zeta potential values of emulsions PPI and PC1–PC5 increased by 13.13 ± 1.74 mV, 12.52 ± 2.02 mV, 10.62 ± 1.29 mV, 12.84 ± 1.58 mV, 15.54 ± 1.50 mV, and 9.39 ± 1.07 mV, respectively, compared to the initial salt ion concentration (decreasing in absolute value). This may result from NaCl’s strong electrostatic shielding, reducing the emulsion droplet surface charge.

3.9. Acid-Base Stability

Figure 7A shows macroscopic emulsion images at pH 2, 5, 7, 9, and 11. All samples except those at pH 5 exhibit a uniform milky-white color. HIPE_S have a pale-yellow shading at a pH of 5. Superior fluidity when compared to emulsions under different pH values is visible upon visual inspection. When the emulsion reaches equilibrium at room temperature, it exhibits gravitational diffusion at a pH of 5. At this point, the emulsion’s fluidity remains stable, indicating weak droplet-to-droplet interactions that confer greater fluidity. Conversely, emulsions at different pH levels (pH 2, 7, 9, and 11) exhibit a semi-solid state. This macroscopic manifestation of significantly reduced fluidity suggests the formation of a robust solid network structure. Figure 7B illustrates HIPEs groups’ particle size changes under different pH conditions. All samples showed the smallest particle size at pH 7, suggesting that the emulsion was in a reasonably stable state at this pH. The average particle sizes at pH 2, 5, 9, and 11 all significantly increased (p < 0.05) in comparison to pH 7. At pH 5, PPI and PC1–PC5 increased by 10.76 ± 0.50 μm, 7.28 ± 0.40 μm, 7.06 ± 0.40 μm, 6.00 ± 0.18 μm, 5.97 ± 0.36 μm, and 5.40 ± 0.67 μm, respectively, compared to pH 7. According to Bengoechea et al. [36], this might be because the system is getting closer to the PPI’s isoelectric point, which reduces electrostatic repulsion and encourages aggregation. The CSP-added experimental groups’ D₅₀ values at pH 2, 9, and 11 were significantly lower than those of the control group (PPI—without CSP addition), suggesting that CSP inclusion can somewhat increase the emulsion’s resilience to acidic and alkaline conditions. HIPES’ potential changes at different pH values are shown in Figure 7C. At pH 2, the zeta potential of this system exhibits a positive value. For PPI, since pH 2 is significantly below its isoelectric point (pI), the amino groups on the protein surface become fully protonated, thereby conferring a strong positive charge to PPI. For CSP, its carboxyl groups are partially protonated at pH 2. This protonation neutralizes the original negative charge of the carboxyl groups, reducing the absolute value of its surface charge. Consequently, the system’s zeta potential remains positive. As the pH increases, the absolute value of the system’s zeta potential first rises and then stabilizes. When the pH exceeds PPI’s isoelectric point, amino groups on PPI lose protons and become negatively charged. Simultaneously, carboxyl groups on CSP gradually dissociate to form negatively charged groups. The synergistic interaction between PPI and CSP’s negative charges enhances electrostatic repulsion between emulsion droplets, thereby improving HIPEs’ stability.

3.10. Heating Stability

Heat treatment (e.g., pasteurization) is a common food-industry process [37], that maintains good texture and extends shelf life. Thus, the thermal stability of HIPE was evaluated (90 °C, 30 min), and visual appearance as well as particle size after the heating of heated samples were assessed. After heating, emulsions showed volume expansion and formed pores, as shown in Figure 8A. The phenomenon may be caused by bubble formation and rupture as the mixture approaches water’s boiling point. Alternatively, air could have been incorporated during mechanical stirring in emulsification. Upon heating, the protein structure is relaxed and gas bubbles will emerge; thus, bubbles can be clearly seen [38]. As polysaccharide content increased, the surface of HIPE darkened, possibly due to Maillard reactions between CSP and PPI after heating [39].

Particle sizes before and after heating were also determined. All the emulsions had larger D₅₀ values after heating. The largest increase appeared for the emulsion without CSP (an increase of 10.26 ± 1.47 μm). These findings may show that PPI was degraded under high temperatures of 90 °C. Since the PPI is the only emulsifier used in the formula without CSP, its breakdown weakened the stability, resulting in more oil droplets aggregating into clusters. Emulsions supplemented with CSP (PC1–PC5) showed larger particle sizes than their respective control counterparts upon heating (Figure 8B). The particle size increased by 8.37 ± 0.73 μm, 4.82 ± 0.64 μm, 5.42 ± 0.63 μm, 6.34 ± 0.49 μm, 7.02 ± 0.70 μm after heating. An increase in particle size smaller than those observed in PPI emulsions in absence of CSP appeared. As PPI-CSP acts as composite emulsifier, high temperature induced PPI decomposition will cause the immediate adhesion of CSP to the surface of oil droplets, preventing their aggregation as well as minimizing an increase in the overall particle size.

3.11. Freeze–Thaw Stability

Figure 9A illustrates the macroscopic states pre- and post-freeze–thaw cycles. After three cycles in which the samples were frozen for 20 h at −20 °C and then thawed for 2 h at room temperature, all HIPES samples exhibited increased sensitivity to the freeze–thaw process, leading to oil phase separation. This indicates that HIPE exhibits poor stability following freeze–thaw cycles. However, after undergoing high-speed shear mixing at 1800 rpm for one minute, the separated samples re-formed into uniform HIPEs with an appearance highly similar to the untreated samples. This demonstrates a certain degree of temperature responsiveness and re-emulsification capability. Note that this capability is not equivalent to freeze–thaw stability itself. This observation highlights the dominant characteristic of HIPEs, namely their extreme reversibility in destabilization and re-emulsification processes [40].

Figure 9B shows the changes in D₅₀ for samples with different PPI-CSP ratios before and after freeze–thaw cycles without re-shearing. The figure indicates that particle size significantly increased in all groups after freeze–thaw cycles ($p<0.05$). The D₅₀ value of the single PPI emulsion without CSP addition showed the largest increase, rising from 17.07 ± 0.13 μm to 46.67 ± 0.23 μm—an increase of 29.6 ± 0.27 μm. Among the samples with CSP addition, PC3 (PPI/CSP = 2:1) exhibited the smallest D₅₀ value, though it still increased by 18.22 ± 0.20 μm. This indicates that ice crystals disrupted the interfacial membrane, leading to droplet coalescence, demonstrating that the emulsions prepared in this study possess poor freeze–thaw stability. This phenomenon can be attributed to alterations in the hydrogen bond- and hydrophobic bond strengths of proteins during the freeze–thaw process, resulting in structural modifications within the PPI. As a consequence, certain PPI molecules precipitated and formed aggregates, consequently compromising their ability to uphold efficient interfacial protection [41].

Figure 9C,D show the frequency scan changes in HIPEs formed by re-shearing before and after freeze–thaw cycles for stable PPI and PPI-CSP complexes. Across the entire frequency range, G′ values consistently exceeded G″ values in all emulsion models, indicating the formation of a gel-like network within the emulsion [42]. Freeze–thaw cycles lead to decreases in both G′ and G″, possibly because of the significant disruption of interfaces caused by crystals forming in the aqueous and oil phases during freezing. Moreover, these cycles result in thermal expansion and contraction of the aqueous and oil phases, causing polymer desorption from the interfaces, ultimately resulting in reductions in G′ and G″. Comparing frequency scans of fresh emulsions with re-sheared emulsions revealed that the G′ and G″ values of the re-sheared emulsion were slightly lower than those of the fresh emulsion. However, these values remained stable across the frequency range assessed. This observation suggests that HIPE_S demonstrates exceptional freeze–thaw recovery capability [43].

3.12. Storage Stability

Figure 10A displays macroscopic images of the emulsion at different storage durations: 1, 10, 20, and 30 days. The emulsion prepared on-site, obtained by high-speed shearing, has a homogeneous appearance, with no apparent separation of the emulsified layers. Even after 30 days of storage, no separation between the oil and water phases is observed; only minimal bubble formation is observed in the emulsion, indicating long-term stability.

One important consideration when evaluating the stability of an emulsion is the particle size [44]. To evaluate the stability of the PPI-CSP emulsion mixture, changes in the average particle size (D₅₀) were recorded over a 30-day storage period. Figure 10B illustrates how the average particle size (D₅₀) of the control coup (PPI-without CSP added) increased to 25.1 ± 0.22 μm after 30 days of storage, representing an increase of 8.03 ± 0.26 μm compared to the pre-storage value. The polysaccharide-added groups (PC1-PC5), on the other hand, experienced average increases in particle size of 4.28 ± 0.18 μm, 6.51 ± 0.57 μm, 5.64 ± 0.49 μm, 6.17 ± 0.30 μm, and 6.48 ± 0.68 μm, respectively. These findings suggest that while the presence of CSP led to a slight increase in emulsion particle size, the magnitude of this increase was smaller compared to emulsions without CSP. This observation implies that CSP has the potential to enhance the storage stability of PPI emulsions. This phenomenon arises due to the competitive adsorption of CSP with PPI at the oil–water interface, thereby impeding droplet aggregation [45] and leading to a lesser increment in particle size in the test emulsions. Compared to other components, the optimal CSP concentration in the PC3 group simultaneously inhibits excessive aggregation of PPI molecules at the interface through competitive adsorption, thereby limiting droplet flocculation caused by excessive interfacial layer thickness. Droplet coalescence and aggregation are simultaneously suppressed by the composite interfacial film’s electrostatic repulsion and steric hindrance effect. As a result, this sample’s D₅₀ increase is ultimately much lower than that of other groups. The system’s long-term stability is improved by the decreased growth in particle size, which directly lowers the emulsion droplets’ gravitational separation rate [46]. PC3 has significant resistance to gravitational separation, hence reducing the growth in particle size.

The emulsion test group with the addition of CSP exhibited reduced variation in particle size distribution compared to the emulsion without CSP. This trend was similarly reflected in rheological tests, where the viscosity changes in the CSP-added emulsion were less significant after heating, under both identical shear rates (Figure 10C) and identical angular frequencies (Figure 10D,E). As shown in Figure 10D, the dynamic equilibrium of droplet coalescence–decoalescence and the conformational rearrangement of interfacial film molecules are linked to the irregular fluctuations of G″ at low angular frequencies. While the adsorption–desorption process of interfacial layer stabilizer molecules becomes active at low frequencies, causing non-steady-state variations in viscous dissipation, the extended shear cycle permits the slow dissociation and re-agglomeration of small droplet aggregates. The shear cycle shortens, and the emulsion structure reacts more synchronously as the angular frequency rises, with G″ eventually stabilizing. This illustrates how the system moves from a state of non-equilibrium to one of dynamic equilibrium. As shown in Figure 10E at frequencies higher than 40 rad/s, the G′ values for samples PC1, PC2, and PC3 show a decrease. A total of 30 days of storage is considered long-term aging. It can be deduced that small droplets dissolved and merged into larger droplets, resulting in a rise in the average particle size of the emulsion, as the average particle size rose across all samples. Large droplets have substantially less shear resistance than small droplets: by closely packing, tiny droplets create a network that resembles a solid and may diffuse stress at high frequencies through molecular entanglement. On the other hand, bigger droplets cause more voids, which breaks the continuity of the network. The modulus decreases as a result of this concentration of stress.

4. Conclusions

This study analyzed the influence of different concentrations of CSP on the stability of PPI emulsions and composites under different treatment conditions. We found that the emulsion particle size first reduced and subsequently increased as the CSP content increased, reaching a minimum size of 12.91 ± 0.16 μm at PC3. Nonetheless, the particle size continuously stayed lower than that of the protein emulsion made from a single peanut isolate, suggesting that the addition of CSP efficiently improves emulsion stability. The gelatinous structure, rheological behavior, and visual characteristics of HIPE confirm this. These HIPEs have demonstrated exceptional stability at temperatures as high as 90 °C and ionic strengths ranging from 0 to 400 mmol/L. The addition of CSP enhanced the acid and alkali resistance of the PPI emulsion. After 30 days of storage, the D₅₀ showed minimal change, indicating excellent storage stability. Emulsions that undergo demulsification following freeze–thaw treatment re-form into uniform, non-flowing emulsions after homogenization by a high-speed shear homogenizer, demonstrating HIPE’s excellent temperature-responsive properties. The storage modulus (G′) continuously outperformed the loss modulus (G″) across the frequency spectrum examined, suggesting that HIPEs maintained their gel-like structure under a range of processing circumstances. The HIPE prepared using food-grade stabilizers in this study enhances the quality of protein-polysaccharide emulsions and holds broad application prospects in food systems, such as nutrient delivery and encapsulation technologies.

The emulsion can be homogenized with a high-speed shear homogenizer to re-form a uniform, non-flowing emulsion when inverted after demulsification after freeze–thaw cycles. This illustrates how well the HIPE responds to temperature changes.