Structural properties, rheological characteristics, and drug delivery efficiency of resveratrol-loaded soybean protein isolate and hemp protein isolate emulsion gels: Synergistic effects of carboxymethyl cellulose incorporation and ultrasound treatment
Department of Food Engineering, Provincial Engineering Laboratory of Green Food Processing and Storage, Heilongjiang Key Laboratory of Food Science and Engineering, Heilongjiang Key Laboratory of Grain Food and Comprehensive Processing, Harbin University of Commerce, Harbin 150028, Heilongjiang, China
⁎Corresponding author. xqzhuwang@163.comAbstract
Plant protein emulsion gels often show suboptimal mechanical properties. This study aimed to improve the protein secondary structure, rheological behavior, and resveratrol (RES) delivery performance of soybean protein isolate (SPI)–hemp protein isolate (HPI) emulsion gels via carboxymethyl cellulose (CMC) incorporation combined with ultrasound treatment. Initially, the degree of protein–polysaccharide grafting increased with the rise in CMC concentrations before decreasing thereafter. The partial conversion of α-helices to β-sheets exposed hydrophobic groups, facilitating the formation of an interpenetrating protein–polysaccharide network. Rheological analysis revealed that CMC decreased the ellipticity of elastic Lissajous curves and inhibited the linearization of viscous Lissajous curves, thereby improving resistance to the external environment. Moderate ultrasound treatment (400 W) further enhanced the encapsulation efficiency (71.84%), loading capacity (23.46%), and bioaccessibility (40.31%) of RES within the gels. In contrast, excessive ultrasound treatment (800 W) weakened protein–CMC interactions. Overall, combined CMC–ultrasound treatment effectively improved the mechano-structural properties and bioactive compound delivery performance of plant protein emulsion gels.
1Introduction
Plant proteins are widely used in the preparation of gels due to their excellent gelation and emulsification properties (Oliveira Júnior & Cunha, 2022). Soybean protein isolate (SPI) is highly nutritious and exhibits outstanding functional properties, including high solubility (Wang et al., 2023), emulsification capacity (Geng et al., 2020), and gelation behavior (Liu et al., 2023). However, its compact globular structure and limited molecular flexibility limit its application in food systems (Sun et al., 2023). Protein–protein complexation has emerged as an effective strategy for addressing the limitations inherent to single-protein systems and improving gel functionality (Wang et al., 2025). Hemp protein isolate (HPI), an emerging plant-based protein source, possesses favorable characteristics such as hypoallergenicity, high digestibility, a balanced amino acid profile, and high nutritional value (Wang, Tang, et al., 2024). Our previous study demonstrated that SPI and HPI can form composite emulsion gels when mixed at specific ratios, demonstrating their gelation potential. However, the resulting gels did not meet the requirements for the delivery of bioactive compounds, and their delivery potential was not explored (An et al., 2025).
Protein gels often exhibit weak mechanical strength or poor stability, which can be improved through the incorporation of plant polysaccharides. As natural macromolecules with excellent biocompatibility, polysaccharides interact with proteins via electrostatic interactions and hydrogen bonding, thereby enhancing cross-linking density and network stability in gels (Miao et al., 2021). As a result, protein–polysaccharide complexation has been widely employed to fabricate novel network structures and improve gel functionality (Nourmohammadi et al., 2024). For instance, Yu, Wang, et al. (2022) reported that incorporating guar gum into SPI-based emulsion gels improved surface smoothness and enhanced structural integrity due to polysaccharide–protein interactions. Similarly, Huang et al. (2024) demonstrated that plant polysaccharides such as inulin, κ-carrageenan, and konjac glucomannan promoted the formation of compact and well-organized gel networks, leading to more uniform gels. Carboxymethyl cellulose (CMC) is an anionic water-soluble cellulose derivative in which some hydroxyl groups in the original cellulose backbone are partially replaced with carboxymethyl groups (-CH2COOH). CMC exhibits excellent chemical stability and thickening properties (Rahman et al., 2021). Previously, Tang et al. (2022) demonstrated that CMC can improve the mechanical strength of egg white protein aerogels and enhance oil retention within these systems. In addition, studies have shown that proteins and polysaccharides can undergo glycosylation reactions, causing polysaccharide chains to be embedded within the protein network. This enhances structural stability, improves gel texture, and can protect functionally active substances (Zhou, Cai, et al., 2024) such as β-carotene (Geng et al., 2022) and quercetin (Han et al., 2024).
Various integrated processing techniques—including ultrasonication (Guo et al., 2024), microwave treatment (Cheng et al., 2024), and microjet treatment (Reiter et al., 2025)—have been developed to modify the molecular structure of proteins and thereby enhance gel properties. Among these, ultrasound is an efficient and rapid method that utilizes the high temperatures and mechanical forces generated locally by cavitation effects to functionally alter proteins (Li et al., 2024). Ultrasonic treatment exposes the hydrophobic groups within protein molecules and promotes intermolecular interactions such as disulfide bonding and hydrophobic interactions, thereby enhancing the gelation capacity of proteins (Chu et al., 2024). For example, Zhang et al. (2022) demonstrated that ultrasonication can improve the gel properties and nutrient delivery performance of SPI. Similarly, Geng et al. (2022) reported that ultrasonication can strengthen the structure of SPI emulsion gels and improve their water-holding capacity, enhancing their suitability as fat-soluble nutrient delivery systems. Emulsion gels, which show dual characteristics of both emulsions and hydrogels, exhibit stable structures and excellent functional properties. Hence, they are frequently employed as carriers for bioactive compounds (Han et al., 2024). Resveratrol (RES), a phenolic compound with antioxidant, neuroprotective, and anti-atherosclerotic activities (Wang et al., 2021), is highly sensitive to environmental factors such as UV irradiation and low pH, which reduce its bioavailability. This limitation hinders the utilization of RES in food systems, especially given the acidic environment of the gastrointestinal tract. Therefore, effective delivery systems are required to improve the stability and bioaccessibility of RES and other such compounds.
Accordingly, the present study explored whether the combined application of ultrasound treatment and CMC incorporation can improve the properties of SPI-HPI emulsion gels and enhance their ability to protect RES from UV degradation while enhancing RES bioaccessibility through controlled release. Specifically, this study evaluated the effects of ultrasonication power (0 W, 400 W, and 800 W) and CMC concentration (0%, 0.6%, 0.8%, and 1.0%) on the structural and functional properties of SPI-HPI emulsion gels and their ability to deliver RES. The protein conformation and molecular interaction profiles of the gels were characterized based on grafting degree, zeta potential, particle size, surface hydrophobicity, Fourier transform infrared (FTIR) spectroscopy, and intermolecular force analysis. In addition, gel network structure, mechanical properties, and water distribution were assessed using texture analysis, rheological measurements, water-holding capacity (WHC) measurements, low-field nuclear magnetic resonance (LF-NMR) spectroscopy, and microstructural analysis. The effects of ultrasound and CMC incorporation on RES delivery and gel digestion characteristics were evaluated by analyzing the encapsulation efficiency, loading capacity, light stability, and in vitro digestion behavior of the gels. The findings revealed the synergistic mechanisms by which ultrasound and polysaccharides regulate the properties of composite protein emulsion gels, providing a theoretical basis for fabricating efficient delivery systems for bioactive compounds. The results are also expected to offer technical support for the further development and utilization of HPI as a novel plant-based resource.
2Materials and methods
2.1Materials
Defatted hemp seed meal was purchased from Jinzhou Qiao Brand Biotechnology Company Limited (Liaoning Province, China) (protein: 70.20%; fat: 2.90%; moisture: 9.80%; ash: 11.30%). Soybean isolate protein (SPI) was sourced from Suihua Jinlong Oil & Fat Company Limited (Heilongjiang Province, China) (protein: 91.90%). Carboxymethyl cellulose (CMC, MW = 90,000 kDa, DS = 0.7) was bought from Tianjin Guangfu Technology Development Company Limited (Tianjin, China). TGase (50 u/g), Fluorescein 5-isothiocyanate (FITC), pepsin (3000 u/g), porcine bile salts, and pancreatin (250 u/mg) were sourced from Yuanye Biotechnology Co. Ltd. (Shanghai, China). Nile Blue was acquired from Aladdin Biochemical Technology Co. (Shanghai, China) Nile Red and RES were obtained from Shanghai McLean Biochemical Technology Co. Ltd. (Shanghai, China). Commercial soybean oil was obtained from a retail market in Harbin, Heilongjiang Province. Analytical-grade chemicals were supplied by Tianjin Canbi Chemical Reagent Co., Ltd. (Tianjin, China).
2.2Preparation of HPI
The extraction of HPI was achieved using the method of An et al. (An et al., 2025). The defatted hemp seed meal was ground into powder and passed through a 80-mesh screen, followed by dispersion in deionised water at a solid-to-liquid ratio of 1:20 (w/v). The pH of the resulting suspension was adjusted to 9.5 using 2.0 M NaOH. The extraction was done for 2 h at 45 °C in a water bath. The obtained hemp seed solution was centrifuged at 4000 ×g for 20 min, and the supernatant after centrifugation was adjusted to pH 4.7 with 2.0 M HCl and left for 30 min. The precipitate was collected, rinsed using deionised water and centrifuged at 4000 ×g for 15 min. The washing step was repeated three times to reduce the inorganic salts in the proteins. Add the precipitate to 100 mL of water to dissolve. After adjusting the pH of the suspension to 7.0, the sample was freeze-dried, subsequently pulverised, and finally sieved through an 80-mesh screen. The protein content HPI measured by Kjeldahl method was 89.20%).
2.3Preparation of SPI-HPI emulsion gel
According to the results of the previous study, the extracted HPI was mixed with SPI using a protein ratio of 6:4 (SPI/HPI, w/w) at room temperature and stirred at 500 rpm for 3 h to prepare a dispersion with 6% protein content (An et al., 2025). The protein dispersion was thoroughly mixed with 0%, 0.6%, 0.8% and 1.0% (w/v) CMC. Oil-in-water (O/W) emulsion was prepared by mixing soybean oil with the mixed protein solution at a water-to-oil ratio of 9:1 (v/v), dispersed at high speed at 15,000 rpm (XHF-DY, Ningbo Xin zhi Bio-technology Co. Ltd., Zhejiang, China), and homogenised for 2 min. Insert the Φ6 probe of ultrasonic cell crusher (LC-CB-1000E, Li-Chen; Shanghai, China) was inserted 1 cm from the bottom of the beaker. Based on preliminary experimental results, ultrasonic treatment was performed at 0 W, 400 W, and 800 W for 20 min (the pulse mode: 4 s on, 4 s off), with an ultrasonic frequency range of 20–25 kHz. During the ultrasound treatment, the sample is placed in an ice-water bath to ensure that the temperature does not exceed 25 °C.After ultrasonic treatment, add TGase (10 U/g) and stir thoroughly until completely dissolved to form a protein network (Zhang et al., 2022). Incubate at 55 °C for 1 h. Heat it in a 95 °C water bath for 30 min, and then quickly cool it in an ice bath for 20 min after enzyme inactivation. All sample were subsequently maintained at 4 °Covernight. Partial samples were then freeze-dried and mechanically pulverised to facilitate subsequent characterisation.
2.4Determination of the degree of grafting (DG)
To evaluate the interaction between proteins and CMC, this study determined the DG using a method adapted from Tang et al. (Tang et al., 2022). The O-phthalaldehyde (OPA) reagent (80 mg) was solubilized in 2 mL of 95% ethanol, followed by mixing with 50 mL sodium tetraborate buffer solution (0.01 M, pH 9.7). The mixture was supplemented with 5 ml of 20% w/v SDS aqueous solution and 200 μl of β-ME. The resulting solution was then brought to a final volume of 100 mL using deionised water. The sample dispersion (200 μL, 2 mg mL−1) was reacted with 4 mL of OPA reagent for 5 min at 35 °C. The absorbance was measured at 340 nm by UV–Vis spectrophotometer. The DG content was calculated according to the formula (1).where Aᵣ is the absorbance of the reference sample (without CMC) and As is the absorbance of the sample.
2.5Determination of particle size and zeta potential
The Yang et al.method was used to measure the particle size and potential of emulsion gels (Yang et al., 2025). The emulsion particle size was determined using a laser particle size analyzer (Bettersize 3000, Dandong Better Instrument Co., Ltd., China), with the laser scattering index set within the range of 8%–12%. Emulsion particle size results are expressed as the volume-average particle size D(4,3). Zeta-potential was measured using a Malvern particle size analyzer (Nano ZS, Malvern Instruments, UK).
2.6Determination of surface hydrophobicity
Surface hydrophobicity (H0), of emulsion gels was determined using the method of Li et al. (Li et al., 2019). Hydrophobic fluorescent probe ANS (20 μL) was added to 2 mL of protein sample solution and reacted for 15 min under dark conditions. Fluorescence intensity was measured at 390 nm excitation wavelength and within the spectral region of 350 to 420 nm using a fluorescence spectrophotometer (f - 7000, Hitachi Ltd., Tokyo, Japan) with a slit width of 5 nm. The initial slope of the fluorescence intensity versus the protein concentration was expressed as H0.
2.7FTIR
The freeze-dried emulsion gel powder was ground by KBr at the mass ratio of 1:50, and the absorption spectrum was measured by Fourier transform infrared spectrophotometer (PerkinElmer, USA) in the range of 4000 cm−1-400 cm−1. The scan resolution of the background spectrum was 4 cm−1, and the scanning time of each sample was about 1 min. Using Peakfit 4.12 software, changes in protein secondary structure were analysed (Lian et al., 2023).
2.8Microstructure
Microstructural observations of the emulsion gels were conducted employing a Leica SP2 confocal microscope (Leica, Wetzlar, Germany). Polysaccharides, proteins and oils were stained using FITC, Nile Blue and Nile Red. The gel samples were cut into 2 ± 0.5 mm slices and stained using a mixture of stains (0.1% FITC +0.1% Nile Red +0.1% Nile Blue) for 10 min and washed three times with deionised water. Confocal laser scanning microscopy (CLSM) was performed under a 40× objective lens. Three fluorescent stains—FITC, Nile red, and Nile blue—were excited at 490 nm, 488 nm, and 633 nm, respectively.
2.9Determination of intermolecular forces
Intermolecular forces in the emulsion gels were determined using the method of Yang et al. with minor modifications (Yang et al., 2021). Freeze-dried powder sample (0.25 g) was added to 5 mL of different dispersions (A: 0.05 M NaCl; B: 0.6 M NaCl; C: 0.6 M NaCl +1.5 M urea; D: 0.6 M NaCl +8 M urea; E: 0.6 M NaCl +8 M urea +1.5 M β-ME), and then homogenised in a high-speed disperser for 30 s. The solution was then stored at 4 °C for 2 h. The well-mixed gel suspension was centrifuged at 10000 r/min and 4 °C for 15 min and the precipitate was discarded. The concentration of protein in the sample was determined by measuring its absorbance at 595 nm using a UV–Vis spectrophotometry system (A1pHa-1506, Shanghai Spectrometer Company, China). The formula for each chemical bond: disulfide bond = SE-SD, hydrophobic interaction = SD-SC,hydrogen bond = SC-SB，ionic bond = SB-SA.
2.10Determination of textural properties
Gel strength, elasticity, cohesion and chewability of the emulsion gels were measured at room temperature using a texture analyzer (TA-XT Plus C; Stable Micro Systems, Godalming, UK). The emulsion gel sample was cut into 2 cm × 2 cm × 1 cm cubes, and TPA analysis was performed using a cylindrical p/0.5 probe (diameter 12.7 mm). Under the experimental setup, the triggering force was 5 g with dual compression phases intervals 5 s. The approach velocity, measurement velocity, and retraction velocity were uniformly maintained at 1.00 mm/s.
2.11Rheological property
The rheological behavior of the emulsion gels was analysed at room temperature (25 °C) using a MCR102 rotational rheometer (AntonPaar, Germany) equipped with a PP50 parallel plate geometry probe. Cut the emulsion gel sample into cylindrical pieces approximately 25 mm in diameter and 1 mm in height for rheological testing.
2.11.1Large-amplitude oscillatory shear (LAOS) test
A fixed frequency of 1 Hz was used in the strain sweep testing of LAOS, with a sweep range spanning from 0.01% ∼ 1000%. Raw sinewave data at 10%, 50%, 100%, 200%, 400% and 800% strain were collected and the data were analysed using the MITlaos program and Lissajous curve (Yu, Wang, et al., 2022).
2.11.2Small-amplitude oscillatory shear (SAOS) test
Within the angular frequency span of 0.1–100 rad/s, a dynamic frequency scan experiment was performed. A constant strain amplitude of 0.5% was used. This strain was within the linear viscoelastic region (LVR), and the storage modulus (G′) and loss modulus (G″) are determined.
2.11.3Creep recovery test
Constant stress creep and recovery tests were performed at 4 Pa for 120 s in the LVR range of the emulsion gel. The recovery phase lasted for 120 s.
2.11.4Three interval thixotropic test (3-ITT)
Refer to the method of Sun et al. and make some modifications (Sun et al., 2024). Changes in viscosity were recorded during a three-stage variable-speed shear-rate scans, including a low-speed scan for 120 s, a high-speed scan for 120 s and a low-speed scan for 120 s.
2.12Determination of WHC
WHC of the emulsion gels was determined following the method of Lian et al. with slight modification (Lian et al., 2023). Emulsion gel (2 g, M1) was placed in a 10 mL centrifuge tube and centrifuged at 8000 g for 10 min. After centrifugation, the supernatant was drained, and filter paper was used to absorb the surface water of the gel sample. The total weight of the test tube and gel before centrifugation (M2) and after centrifugation (M3) were recorded. WHC of the gel was calculated using formula (2).
2.13LF-NMR
A LF-NMR analyzer (NM120, Niumag, Shanghai, China) was used to perform nuclear magnetic resonance relaxation measurements. The LF-NMR parameters were set as follows: SF = 18 MHz, TW = 3000 ms, SW = 200KHz, TE = 0.224, RG1 = 20db, NS = 8. The CPMG sequence was used for inversion.
2.14Preparation of RES-loaded emulsion gels
RES was encapsulated into the emulsion gel using the method described by Yi et al. (Yi et al., 2018). A reserve solution of 40 mg mL−1 RES was configured by dissolving RES in 80% ethanol (v/v). Thoroughly mix the Res reserve solution with the oil phase, then homogenize it with the mixed protein solution. Prepare the emulsion gel according to the method described in section 2.3. Part of the emulsion gel was freeze-dried away from light and pulverised for further analysis.
2.15Determination of encapsulation efficiency (EE) and loading amount (LA)
Encapsulation efficiency and loading amount of RES in the gel were determined using Wang et al.'s method (Wang et al., 2021). RES standard solution was prepared and its standard curve (y = 0.1968× + 0.1524, R2 = 0.995) was measured using a UV–vis UV spectrophotometer. Freeze-dried gel sample powder (50 mg) was dissolved in 4.5 mL of ethanol (95% concentration) to clean the surface of RES. The RES dispersion was centrifuged at 7000 rpm for 5 min and the supernatant was discarded. The resulting precipitate was dissolved in 4 mL ethanol and ultrasonicated at 40 kHz for 30 min. The supernatant was centrifuged at 7000 rpm and passed through a 0.22 μm acetate membrane filter to remove insoluble macromolecular impurities, and the absorbance of the filtrate supernatant was subsequently determined at 306 nm. The total RES content was calculated according to the standard curve. EE and LA were calculated using (3), (4) as follows:
2.16Light stability
The RES-loaded emulsion gel was cut into 2 × 2 × 1 cm3 pieces and placed 15 cm below a 365 nm UV lamp for irradiation. Samples were collected at intervals of 0, 30, 60, 90, 120, 150, and 180 min, freeze-dried in the dark, and lyophilized. Then, 50 mg of each lyophilized gel sample was dissolved in 4.5 mL of ethanol. The absorbance of each solution was measured at 306 nm using UV–Vis spectroscopy to determine the retention rate of RES (Sun et al., 2024).where St denotes the initial concentration of RES, and S denotes the concentration of RES after UV exposure.
2.17In vitro digestion and bioaccessibility
The method of Cheng et al. (Cheng et al., 2024) was used with slight modification. Lyophilised sample powder (100 mg) was dissolved in 10 mL of gastric simulated digestive fluid (SGF, containing 0.15 M NaCl and 3 mg mL-1 pepsin), and then shake at 200 rpm in a 37 °C water bath shaker. Supernatant (1 mL) was taken at each of the following 30, 60, 90 and 120 min (replenishing 1 mL of gastric digestive fluid after each sampling) and then set aside. At the end of the second sampling the sample pH was adjusted to 7.4 and transferred to the intestinal simulated digest (SIF, containing 10 mg mL−1 bile salts and 3 mg mL−1 pancreatic enzymes and pH adjusted to 8.0 with 0.1 M NaOH) and shaken at constant temperature under the same conditions. The above operation was repeated at 180, 240, 300 and 360 min, and the supernatant was centrifuged at 3000 r/min for 5 min, and passed through 0.25 μm acetate fiber membrane and set aside. The sample (200 μL) were transferred into a 96-well microplate, and the absorbance value at 306 nm was measured with a microplate reader (Spectramax M2, Molecular Devices, USA). Rate of RES release was calculated using formula (6):
At the end of the simulated small bowel phase, samples were collected and centrifuged at 4000 ×g and 4 °C for 40 min. The resulting supernatant contained a micellar layer composed of RES, representing the “mixed micelles” fraction of the intestinal fluid, and was retained. The RES in the mixed micelles was extracted using ethanol, and vortexing ensured thorough mixing, inducing the precipitation of denatured proteins and digestive enzymes. The mixture was then centrifuged at 4000 ×g for 20 min to remove these insoluble components. Thereafter, the absorbance of the supernatant was measured at 306 nm. RES bioaccessibility was calculated as follows:
2.18Statistical analysis
All experiments were independently repeated three times under the same conditions, and the data were presented as the mean ± standard deviation. Using SPSS software (version 20.0, IBM, Chicago, IL, USA), data were statistically analysed using one-way analysis of variance (ANOVA) and Duncana multiple comparison was used to separate significant different (p < 0.05) data means. Origin 2022pro software was used to plot graphs.
3Results and discussion
3.1Degree of grafting (DG)
DG denotes the number of available free amino groups and thus can express the degree of protein-polysaccharide binding. As shown in Fig. 1a, both ultrasound power and the amount of CMC affected the DG of SPI-HPI emulsion gels. When the ultrasound power was constant, increasing the concentration of CMC initially led to a rise in the DG value since more polysaccharide molecules were grafted onto the protein chain. However, since CMC also acted as a thickener, the excessive addition of CMC increased the viscosity of the gel system, hindering the collision of protein and polysaccharide molecules and consequently causing a slight decrease in the DG value (Li, Wang, et al., 2024).
Meanwhile, as the ultrasound power was increased, the DG value showed an initial increase followed by a reduction, with the highest DG detected at a power of 400 W. This was likely because moderate sonication caused protein unfolding, exposing the ε-amino groups of protein molecules. These amino groups could covalently bind to the carbonyl group of the reducing terminus of sugar molecules, promoting the Maillard reaction (de Oliveira et al., 2014). As a result, more CMC molecules were attached to the protein chain. In contrast, excessive ultrasonication (800 W) led to the folding of protein molecules and the re-burial of free amino groups. This inhibited the carbonyl–amino condensation reaction between protein and polysaccharide molecules, reducing the DG of the emulsion gels (Zhao et al., 2021).
3.2Particle size and zeta potential
To further examine the effect of different CMC and ultrasound conditions on protein aggregates, the particle size and zeta potential of the composite emulsion gels were evaluated (Fig. 1b). The negatively charged CMC was adsorbed onto proteins via electrostatic interactions, thereby enlarging the particle size of the emulsion gel (Wang, Li, et al. 2023). For the samples containing CMC, as the ultrasonication power increased to 400 W, the particle size of the emulsion gels decreased gradually. This was due to the cavitation effect of ultrasound, which reduced the size of oil droplets, promoted protein–oil interactions, and decreased the size of agglomerates (Zhou, Kang, et al., 2024). Nevertheless, when the ultrasound power was increased further to 800 W, protein molecules underwent refolding and aggregation, leading to a rise in particle size (Pan et al., 2024).
The strength of electrostatic interactions between the particles in protein-based gel systems can be assessed based on the zeta potential, which also affects system stability. CMC, as an anionic polysaccharide, altered the zeta potential of SPI-HPI emulsion gels. Gels containing CMC all showed a negative zeta potential, and this value peaked after the incorporation of 0.8% CMC (Fig. 1c). However, further increasing the CMC concentration to 1.0% caused only a slight increase in the surface negative charge, indicating that the surface of protein aggregates was almost completely saturated. Ultrasonication at a lower power did not alter the zeta potential of the emulsion gels significantly. However, the absolute zeta potential decreased significantly when the ultrasound power was increased to 800 W. This was likely because intensive ultrasonic treatment exposed the polar groups of the protein molecules within the gel (Wang, Ma, et al., 2024).
3.3Surface hydrophobicity (H0)
The H0 value acts as the main indicator of the tertiary structural changes and intermolecular interactions of proteins during gelation. Fig. 1d shows that the H0 of the emulsion gels first increased and then decreased with the addition of CMC. A slight increase in H0 was detected at lower CMC concentrations. This was likely because the amount of CMC was insufficient to adequately cover the hydrophobic groups on the surface of the proteins, resulting in lower H0 values (Zhou et al., 2024). The H0 of the emulsion gel was significantly enhanced until the concentration of CMC reached 0.8%. This suggested that the protein structures of SPI and HPI unfolded at these concentrations, and hydrophobic groups were exposed. Another plausible explanation lied in the CMC-induced changes in protein conformation and the enhancement of intermolecular interactions, causing the exposure of more hydrophobic residues in protein molecules (Tang et al., 2022). After ultrasonic treatment, the H0 of the emulsion gel first increased and then decreased. This was partly due to ultrasonication-induced protein unfolding, which significantly exposed the hydrophobic groups of proteins, thereby increasing H0 (Ai et al., 2019). Notably, when the ultrasound power was set to 800 W, H0 dropped significantly. This was probably because high-power ultrasound treatment induced protein denaturation, causing the structural rearrangement of protein molecules and the formation of insoluble macromolecular aggregates. As a result, hydrophobic groups were buried within the aggregates, lowering the H0 of the emulsion gel (Yao et al., 2019). Interestingly, increasing the CMC to 1.0% led to another rapid rise in H0. This was attributed to the strong electrostatic interactions between CMC and protein molecules under ultrasonication conditions, promoting the exposure of hydrophobic groups. These interactions ultimately caused the gel network structure to loosen, leading to a decline in functional properties (Zhou et al., 2024).
3.4Protein secondary structure
FTIR spectroscopy can be employed to identify changes in protein secondary structures (Fig. 1e). The intensity of peaks in the amide I band indicates the proportion of different secondary structures, as follows: α-helix, 1650–1660 cm−1; β-sheet, 1600–1640 cm−1; β-turn, 1660–1700 cm−1; and random coil, 1640–1650 cm−1. In this study, the increase in CMC concentrations gradually increased the β-sheet content while significantly reducing the proportion of α-helices in the composite protein system. This could be attributed to the formation of hydrogen bonds between CMC and protein molecules, which promoted the transition of molecular chains from an ordered α-helix structure to a more extended β-sheet conformation (Taghavi et al., 2017). Overall, the incorporation of CMC facilitated the formation of hydrogen bonds between molecules, increased the β-sheet content, and enabled the development of a dense protein gel network. This structural densification was visually confirmed through subsequent microstructural analysis using CLSM.
After ultrasonic treatment, the β-sheet content of the emulsion gel significantly increased with a rise in ultrasonication power. The α-helix content showed a further decrease, and the protein conformation shifted from α-helices to β-sheets. This suggested that ultrasonication alters the protein structure of SPI-HPI emulsion gels, increases hydrogen bonding, and thus promotes polysaccharide–protein interactions (Zhao et al., 2021). The increase in β-sheet content appeared critical for gel aggregation and network formation (Han et al., 2024). Notably, the content of random coils also increased significantly as the ultrasound power was increased, peaking at 800 W. This was likely because excessive ultrasonication disrupted hydrogen bonding, caused molecular unfolding, and led to the subsequent formation of random coil structures (Li, Tao, et al., 2024). Overall, the combination of ultrasonic treatment with CMC incorporation synergistically drove the transition of protein conformations from α-helices to β-sheets and strengthened the gel network structure. Thus, this method showed promise as a technical approach for precisely regulating and improving the properties of protein gels.
3.5Microstructure
CLSM observations provided more intuitive insights into the distribution of proteins, oil droplets, and polysaccharides in SPI-HPI emulsion gels (Fig. 1f; red: oil; blue: proteins; green: CMC; pink: proteins stacked with oil droplets). In the absence of CMC, the porosity of the protein network decreased initially following a rise in ultrasound power before increasing thereafter. In particular, ultrasound treatment at 400 W caused the pores in the protein network to become significantly smaller and more densely packed. Moderate ultrasonication enabled the even distribution of droplets and produced a better gel network structure (Pan et al., 2024). At 800 W, the pores in the protein network were significantly enlarged. This was likely owing to cavitation effects, which caused excessive protein unfolding, thus exacerbating damage to oil droplets (Han et al., 2024). CMC, protein, and oil were equally distributed within the emulsion gels, especially those containing 0.8% CMC and ultrasonicated at 400 W, in which the oil droplets were uniformly attached to the gel network. These microstructural features enabled the formation of a dense gel network and were in line with the protein conformation alterations observed using FTIR. Simultaneously, these features were also structurally conducive for enhancing the mechanical properties of emulsion gels and improving their recovery behavior (Yan & Zhang, 2024).
3.6Molecular interaction forces
Figs. 2a, b, and c reveal the changes in molecular interactions during gel formation, which were primarily influenced by the CMC concentration. Hydrophobic interactions and disulfide bonds were found to be the main forces maintaining the stability of the emulsion gels. CMC significantly enhanced the hydrophobic interactions between SPI and HPI, exposing more hydrophobic groups (Wang et al., 2024). This increase in hydrophobic interactions enhanced the texture of the protein gels. Additionally, as the concentration of CMC rose, its electrostatic interactions gradually increased. This was due to the adsorption of CMC onto the surface of proteins via electrostatic interactions, creating a core–shell protein–polysaccharide structure (Wu et al., 2022). The disulfide bonds and hydrophobic interactions within the emulsion gels increased further following ultrasonic treatment. This was attributed to the ultrasonication-induced transition of α-helices to β-sheets, causing the partial unfolding of protein molecules. The exposure of hydrophobic groups and free sulfhydryl groups within the proteins promoted hydrophobic interactions and disulfide bond formation, thereby enhancing the compactness of the gel network. These findings corroborated the conclusions regarding protein secondary structure presented in section 3.4. Meanwhile, when the ultrasonication power was 800 W, the trends of hydrophobic interactions, hydrogen bond interactions, and disulfide bond interactions were similar to those observed at 400 W, but the number of interactions was reduced (Fig. 2c). This was likely because intensive ultrasound treatment caused excessive protein aggregation, leading to the uneven formation of a three-dimensional gel network (Sun et al., 2021). The results confirmed the synergistic effects of CMC incorporation and ultrasonic treatment, which collectively improved the molecular interactions within the emulsion gels.
3.7Texture properties
The effects of ultrasound power and CMC concentration on the texture of emulsion gels were evaluated using texture profile analysis (Fig. 2d, e, and f). Hardness and elasticity are important parameters for evaluating protein gels, and they are closely related to the texture and structural state of food products. The hardness of a gel depends on the interactions among its components and is an index of gel strength. In this study, the addition of CMC increased the hardness of the emulsion gels from 3.65 g to 52.38 g. This increase was associated with the formation of additional molecular interactions between proteins and polysaccharides, promoting the development of a compact gel network and enhancing gel hardness (Cheng et al., 2022). In contrast, the elasticity decreased with the incorporation of CMC, indicating that the composite emulsion gels were less elastic.
Cohesiveness represents the strength or weakness of internal bonds within a gel system, reflecting the gel's potential to resist damage due to external forces and compression (Mirarab Razi et al., 2018). Meanwhile, chewability denotes the energy required to masticate a sample into a swallowable form (Huang et al., 2007). In this study, the cohesiveness and chewiness of the emulsion gels increased gradually as the CMC concentration rose from 0% to 0.8%. Hence, CMC promoted intermolecular covalent cross-linking as well as the structural transition from α-helices to β-sheets, which enhanced the ordered structure of proteins. During the second deformation stage, the resistance of the gel network was enhanced, thus improving cohesion. However, at a 1.0% CMC concentration, the presence of polysaccharides within the gel matrix inhibited the intermolecular bonding among protein molecules. Consequently, upon exposure to a second external force, the gel network failed to keep its internal structure intact, which led to a decrease in cohesiveness (Cortez-Trejo et al., 2022).
Notably, the texture characteristics of the emulsion gels were also significantly affected by the ultrasonication power. The hardness, springiness, cohesiveness, and chewiness of the emulsion gels increased significantly as the ultrasound power increased from 0 W to 400 W. In emulsion gels containing 0.8% CMC, the hardness increased from 46.87 N to 82.95 N, the elasticity increased from 0.43 to 0.74, the cohesiveness increased from 0.46 to 0.65, and the chewiness increased from 7.40 N to 43.46 N as the ultrasound power increased from 0 W to 400 W. Thus, a tighter and more uniform network was formed within the emulsion gel as the ultrasound power was increased up to 400 W. Conversely, at 800 W ultrasound power, the gel hardness decreased to 80.33 N, springiness to 0.55, cohesiveness to 0.48, and chewiness to 25.51 N. Thus, intensive ultrasonic treatment led to excessive protein denaturation, thereby disrupting the gel network structure and compromising gel quality. In line with these findings, Wang et al. (2023) showed that 300 W ultrasound treatment can improve the texture of mung bean protein gels, while 540 W ultrasound treatment disrupts the tight, ordered gel network, leading to a significant decrease in gel hardness and chewiness.
3.8Rheological properties
3.8.1Laos
Fig. 3a shows the effect of different CMC concentrations on the LAOS behavior of SPI-HPI emulsion gels following ultrasonic treatment. The rheological behavior of protein gels under large strain amplitudes more reliably predicts structural changes during processing (Liu et al., 2024). In the LVR region, G′ was larger than G″ for all emulsion gels, indicating that they exhibited elastic responses. With the increase in strain, the G′ of the emulsion gel decreased significantly beyond the critical strain threshold, and G″ showed a local maximum. Typically, higher G′ values reflect better self-supporting properties in gels. Fig. 3a shows that the G′ of the emulsion gel increased and then decreased with an increase in CMC concentration following 400 W ultrasonic treatment, and the G′ of the emulsion gel containing 0.8% CMC was the highest. This suggested that the emulsion gel resulting from CMC–protein interactions had a network structure conducive to good self-supporting properties under a destructive mechanical force (Zhang et al., 2024). This had important developmental significance for the application of SPI-HPI emulsion gels. However, extreme hydration occurred between water molecules and excess CMC, which reduced the quality of emulsion gels by weakening their viscoelasticity.
Lissajous curves further demonstrated the LAOS rheological behavior of the emulsion gels (Figs. 4a, b, and c). In the testing strain range (10%–800%), the elastic Lissajous curves of the emulsion gels transformed from a narrow ellipse to a circle (Fig. 4b and c). This suggested that as the strain increased, the elastically dominated linear viscoelastic behavior of the gels was converted into viscous behavior, and the gels eventually formed fully collapsed structures (Zhang, Zhang, Yu,et al., 2023). Emulsion gels that did not contain CMC displayed rounder elliptical Lissajous curves under different strains, indicating that these emulsion gels were fragile and could immediately be destroyed under the dual effects of ultrasonication and shear (Fig. 4b and c). Meanwhile, the incorporation of an appropriate amount of CMC helped the gels resist deformation under both ultrasound and shear forces, confirming that CMC had a binding effect on proteins. Spatial steric hindrance was generated between proteins and CMC particles, and the three-dimensional skeleton of the emulsion gels was strengthened, causing the gel network to become denser and tighter (Duan et al., 2024). Additionally, as the ultrasonication power increased, the ellipse-shaped deformation in the Lissajous curve was attenuated, indicating that the gel could withstand stronger strain amplitudes. This was because ultrasonication enhanced the gel's ability to resist external environmental changes and enhanced its intermolecular interactions (Cao et al., 2022). However, the Lissajous curves of emulsion gels treated with 800 W ultrasound were circular under smaller strain amplitudes. This also corroborated the texture data, which showed that intensive ultrasonication degraded the quality of emulsion gels (Fig. 2).
Viscous Lissajous curves represent the changes in gel viscosity during the strain cycle. All emulsion gels exhibited good elliptical shapes under low strain conditions (Fig. 4d, e, and f). With increasing strain, the viscous Lissajous curves became more narrowly elliptical and even approached a straight line in the 0% CMC state. Thus, the incorporation of CMC prevented the viscous Lissajous curves from becoming a straight line. This indicated that CMC improved the viscoelasticity of emulsion gels and protected them against high strain by producing a denser gel network (Feng et al., 2023). Additionally, under 800% strain, the viscosity curve of the untreated sample was nearly linear, indicating that the system had approached a fully fluid state and its network structure was almost completely disrupted. In contrast, samples treated with 400 W ultrasonication (containing 0.6% and 0.8% CMC) exhibited viscous curves that maintained a distinct closed elliptical shape, indicating that the gel network showed good structural recovery and viscoelasticity under high shear conditions (Li et al., 2021). This phenomenon could be attributed to the moderate ultrasonication-induced uniform dispersion and partial unfolding of protein molecules, along with enhanced intermolecular interactions. This facilitated the formation of a more elastic and strain-tolerant three-dimensional gel network structure. Hence, the findings indicated that ultrasonication and CMC incorporation synergistically enhance the mechanical stability and dynamic rheological properties of emulsion gels. The different conditions and compositional differences among the gels also created different network structures, yielding different Lissajous curves when compared to control samples.
3.8.2SAOS
To further elucidate the structural characteristics of the emulsion gels, frequency sweep measurements were performed on SPI-HPI emulsion gels subjected to different treatments, generating G′ and G″ profiles (Fig. 3b) (Yu et al., 2022). Across all samples, G′ consistently exceeded G″, and both moduli increased with the angular frequency. This confirmed that all the gels exhibited predominantly elastic behavior, with a degree of frequency dependence. Increasing the CMC concentration led to a concurrent increase in both G′ and G″, reflecting enhanced viscoelasticity and mechanical strength of the gel network. This effect could be attributed to the progressive interaction of CMC with protein chains, resulting in the formation of a continuous protein–polysaccharide interpenetrating network structure (Sun et al., 2024). As the ultrasonic power rose, G′ initially increased and then decreased. Under moderate ultrasonic treatment (400 W), G′ increased significantly and remained substantially higher than G″, indicating the formation of a stronger and more stable gel network. This indicated that appropriate ultrasonication promotes gel network formation, consistent with previous findings (Qayum et al., 2024). In contrast, samples treated at higher ultrasonic power (800 W) exhibited reduced G′ and G″ values compared with those treated at 400 W, particularly in the absence of CMC. This decline was attributed to excessive ultrasonication-induced protein denaturation, which led to weakened gel structures. Similar observations have been reported by Wang et al. (2022), who showed that ultrasonic treatment at 800 W caused extensive myosin denaturation and resulted in the production of weaker gels.
3.8.3Creep recovery
Creep recovery experiments are the analysis of the viscoelastic behavior of SPI-HPI emulsion gels on long time scales. The larger value of the maximum strain in the creep phase (0–120 s) implied greater denaturation of the gel structure and weakened elasticity of the gel. Higher values of maximum strain during the recovery phase (120–240 s) are associated with weaker gel strength (Qu et al., 2021). The creep curves showed similar viscoelasticity but differed in the maximum strain (Fig. 3c). Without ultrasonic treatment, the emulsion gels with larger CMC concentrations had lower final strain values, indicating greater gel elasticity and strength, and the formation of a highly tightly bound and homogeneous gel network structure of polysaccharides and proteins. After ultrasonic treatment, as the CMC concentration increased, the maximum strain value of the emulsion gel first decreased and then increased, attaining a maximum at 0.8% CMC. This indicates that the gel network of samples treated with ultrasonication exhibits higher structural strength and stability, making it less prone to irreversible deformation under external stress. Simultaneously, this indicates that ultrasound and CMC content had some synergistic effect in improving the structural stability of protein-polysaccharide couplings and that ultrasound contributed to the creation of more elastic macromolecular network architectures (Spotti et al., 2017). Additionally, the samples without CMC exceeded the measurement range, further confirming the poor structural recovery capability of the 0% CMC samples.
3.8.43-ITT
The 3-ITT test shows how a food product recovers its structure after receiving strain and provides insights on the structural recovery characteristics of the product. As shown in Fig. 3d, under constant oscillatory strain, all emulsion gels recovered to varying degrees and at a faster rate, and the viscosity was more stable over time, but none of them could recover to the original complex viscosity level (Naderi et al., 2024). Among them, emulsion gel with 0.8% CMC showed better resilience and recovery. After its macromolecular entanglement was dispersed, the molecules were prone to migrate and then re-formed macromolecular entanglement under low oscillatory force due to its small spatial site resistance, resulting in a high recovery rate of the emulsion gel (Liu et al., 2019). The findings revealed that CMC improved the structural properties of the emulsion gel. Recoverability of the emulsion gel facilitated the encapsulation of the active substance, thus improving its stability and resistance to gastrointestinal digestion.
Furthermore, ultrasonic treatment further affected interfacial behavior of the emulsion gel. Structural disruption of the interface occurred during shear testing of the gel, but the interfacial structure underwent a gradual reconstruction behavior after shearing (Li et al., 2018). Emulsion gels after ultrasonic treatment had increased recoverability and higher resistance to high shear speed. Ultrasonication can enable proteins and polysaccharides to achieve better encapsulation of oils and fats, resulting in the formation of an oil-encapsulated gel network structure (da Silva & Danthine, 2022). However, the emulsion gels treated under 800 W ultrasound conditions showed a significant decrease in the ability to resist high- shear speed. It is possible that the formation of large aggregates caused by excessive ultrasound resulted in severe protein aggregation, which adversely affected the stability of the emulsion gel (Zhang et al., 2023).
3.9WHC
In studies on emulsion gel stability, WHC is used to characterize the water retention and structural integrity of gels. Fig. 5a shows that the addition of an appropriate amount of CMC significantly enhanced the WHC of the emulsion gels. CMC contains a large number of hydrophilic groups, such as hydroxyl and carboxymethyl groups, which bind to water molecules via hydrogen bonds, thereby retaining them within the network architecture of the gel system (Sun et al., 2024). However, we observed that the WHC of the gels decreased considerably following the incorporation of 1.0% CMC. This was likely because the entanglement of excess CMC with proteins produced large agglomerates, reducing the contact area and preventing interactions with water molecules (Nourmohammadi et al., 2024). Notably, the WHC tended to be higher in emulsion gels treated with ultrasound than in those not subjected to ultrasonic treatment. Ultrasound changed the conformation of proteins within the gel and promoted the exposure of active groups, increasing protein amphiphilicity and facilitating protein–protein and protein–polysaccharide interactions. This led to the formation of dense gel networks and improved the WHC of the emulsion gels. Nevertheless, the texture of the emulsion gels was destroyed when the ultrasound power exceeded 400 W. This indicated that the steric hindrance effect of proteins and the competitive adsorption of water molecules affected gel network formation (Ji et al., 2024).
3.10Water distribution and migration
LF-NMR can be used to analyze the dynamic migration characteristics and spatial distribution of water molecules in emulsion gel systems. Fig. 5 shows the relaxation time (T2) distribution of SPI-HPI emulsion gels following ultrasonic treatment. Notably, T21 (0.01–10 ms) reflects the water molecules tightly bound to protein molecules, i.e., bound water; T22 (10–100 ms) reflects the water trapped in the gel, i.e., immobilized water, which affects the properties of the gel; and T23 (100–1000 ms) reflects the free water in the gel, which has high mobility (Lei et al., 2023).
The analysis showed that emulsion gels containing CMC had a greater proportion of fixed water. This was mainly due to the CMC-induced enhancement of protein–protein and protein–polysaccharide interactions, causing the transition of free water into fixed water. In addition, CMC adsorbed water molecules and created a smooth and cylindrical multilayered three-dimensional mesh or honeycomb structure within the emulsions, which trapped water molecules and thus increased the amount of immobilized water (Rahman et al., 2021). Ultrasonic treatment further shortened the relaxation time, indicating that the water and macromolecules were more tightly bound within the system. Thus, the water had lower mobility and formed a denser gel network structure (Chu et al., 2024). In addition, SPI-HPI emulsion gels containing 0.8% CMC had the shortest relaxation time for immobilized water following ultrasonication at 400 W. These results revealed that CMC addition and ultrasonic treatment synergistically altered the protein–water interactions required for forming an emulsion gel network, thereby impacting the WHC of the gel.
3.11RES loading
3.11.1Encapsulation efficiency (EE) and loading amount (LA)
EE% and LA% are key metrics for evaluating the delivery performance of functional active ingredients. RES was embedded in SPI-HPI emulsion gels, and the corresponding EE% and LA% were determined. With the increase in CMC concentration, both EE% and LA% initially increased and then decreased, peaking at 0.8% CMC (EE%: 71.84%; LA%: 23.46%) (Fig. 6a,b). This improvement was attributed to the formation of a dense “core–shell” structure, in which CMC coated the protein surface, thereby enhancing the encapsulation and loading capacity of RES (Wu et al., 2022). At higher CMC concentrations, the EE% and LA% decreased, likely due to the intermolecular entanglement and aggregation of CMC, which disrupted the internal network structure and reduced the availability of loading sites within the emulsion gel (Liu et al., 2023). Additionally, moderate ultrasonication (400 W) significantly increased the EE% and LA% of SPI-HPI-CMC emulsion gels. Specifically, ultrasonic treatment enhanced protein solubility and promoted the unfolding of protein molecules, thereby improving the stability of encapsulated RES (Chen et al., 2024). In contrast, excessive ultrasonication (800 W) decreased the EE% and LA% of SPI-HPI-CMC emulsion gels, resulting in a more porous and weakened gel network with enlarged mesh size, which reduced the loading capacity for RES.
3.11.2Light stability
To evaluate the effects of CMC concentration and ultrasound power on the protective capacity of SPI-HPI emulsion gels for RES, RES-loaded gels were subjected to UV irradiation, and retention efficiency was measured. Under UV exposure, RES retention decreased progressively with increasing irradiation time across all gel samples (Figs. 6c, d, and e). The untreated emulsion gel (without CMC or ultrasound) exhibited the lowest RES retention (19.17%). At 0 W, the incorporation of 0.8% CMC increased RES retention to 27.26% (Fig. 6c). Upon ultrasonication, RES retention in SPI-HPI emulsion gels without CMC increased to 28.91%, and further increased to 51.72% at 0.8% CMC (Fig. 6d). This enhancement was attributed to the interaction between CMC and proteins, which led to the formation of a thicker and denser protein–polysaccharide interfacial layer surrounding RES-loaded oil droplets. This barrier reduced light penetration and limited RES degradation, thereby improving retention (Yi et al., 2018). In contrast, the RES retention rate decreased significantly following 800 W ultrasonic treatment (Fig. 6e). Excessive ultrasound likely disrupted oil droplets and promoted protein aggregation and droplet flocculation, leading to the greater exposure of RES and reducing its retention rate (Han et al., 2024). These results indicated that CMC incorporation and moderate ultrasonication can act synergistically to enhance RES retention.
3.11.3In vitro digestion and bioaccessibility
Controlled release and protection of RES are critical functions of carrier systems. Therefore, the release behavior of RES under simulated gastrointestinal conditions was evaluated (Fig. 7). During the gastric phase, the RES release rate was slow in all emulsion gels, indicating that the gel matrix effectively resisted pepsin digestion. In contrast, during the intestinal phase, RES release increased significantly due to pH changes and the continuous erosion of the gel matrix, resulting in the structural disruption of the gel and the enhanced release of RES (Han et al., 2024). The incorporation of CMC promoted protein–polysaccharide cross-linking, resulting in a denser gel network structure, which more effectively retained RES and reduced its release rate. In addition, samples treated with 400 W ultrasound treatment exhibited a lower overall RES release than untreated gels (Fig. 7b). This suggested that ultrasound enhanced the resistance of SPI-HPI-CMC emulsion gels and strengthened their ability to undergo enzymatic degradation (Li et al., 2022). This effect could be attributed to improved network integrity, which slowed down gel disintegration and limited the diffusion of RES into the external environment.
Bioaccessibility is a key parameter for evaluating the effectiveness of emulsion gels as delivery systems. At 0 W, gels containing 0.8% CMC exhibited relatively high bioaccessibility (Fig. 7d), suggesting that CMC enhances network strength, reduces gel degradation during gastric digestion, and ultimately improves RES bioavailability (Wang, Zhang, Chen, Hu, et al., 2023). At lower CMC concentrations (0.6% and 0.8%), 400 W ultrasound treatment further enhanced the bioaccessibility of RES. This improvement was likely due to the ultrasound-induced disruption of large protein–polysaccharide aggregates, which increased the number of trypsin target sites, facilitated digestion, and thus enhanced RES bioaccessibility (Mekala et al., 2024). However, at higher CMC concentrations (1.0%), ultrasonic treatment reduced the bioavailability of RES. This could be attributed to excessive protein denaturation, which hindered enzyme accessibility and activity. Overall, the impact of ultrasonication on bioaccessibility was concentration-dependent, requiring an optimal CMC level to achieve synergistic effects.
4Conclusion
This study demonstrated that ultrasound and CMC exert synergistic effects in improving the mechanical, structural, and delivery properties of emulsion gels. Increasing the CMC concentration, particularly to 0.8%, promoted glycosylation, increased the β-sheet content, and strengthened electrostatic interactions, thereby improving the quality and rheological behavior of the emulsion gels. Rheological analysis confirmed the formation of a continuous protein–polysaccharide interpenetrating network, which contributed to enhanced structural integrity. Ultrasonication further modulated protein secondary structure. Moderate treatment (400 W) increased G′ and G″, apparent viscosity, and the structural recoverability of the emulsion gels, which enhanced network stability. The strengthened gel network provided effective physical protection for RES, improving its light stability and bioaccessibility. These findings highlighted the potential of this system for delivering photosensitive functional bioactive compounds. However, excessive ultrasonication (800 W) promoted the formation of large protein–polysaccharide aggregates, leading to weakened gel properties and reduced RES retention. Overall, these findings demonstrate that the combined application of ultrasound and CMC incorporation offers an effective strategy for improving the performance of plant protein-based emulsion gels as delivery systems for functional bioactive compounds.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Data availability
The data that has been used is confidential.
Acknowledgements
This study was supported by the 10.13039/501100001809National Natural Science Foundation of China (U24A20471), for which we would like to express our gratitude.