Effect of Phycocyanin–Rosmarinic Acid Conjugate-Stabilized Pickering Emulsions on the Gel Properties of Surimi

TL;DR

This study shows that a special emulsion made from phycocyanin and rosmarinic acid improves the quality of surimi gels, making them better textured and more stable.

Contribution

A new PC-Ra conjugate emulsion is introduced as a potential fat replacer that enhances surimi gel properties.

Findings

The PC-Ra 30 emulsion significantly improved surimi gel texture and water-holding capacity.

The emulsion reduced cooking loss and delayed lipid oxidation in surimi gels.

Optimal grafting and covalent bond formation were observed at 30 μmol/L rosmarinic acid.

Abstract

This study aimed to investigate the effects of the phycocyanin–rosmarinic acid (PC-Ra) emulsion on the gel quality of surimi. PC-Ra conjugates were synthesized firstly via laccase-catalyzed oxidation at different Ra concentrations, and their physicochemical properties—including grafting degree, sulfhydryl group content, free amino group content, and surface hydrophobicity—were characterized. The results demonstrated that Ra addition effectively reduced free amino groups, achieving optimal grafting at 30 μmol/L along with peak disulfide bond content and surface hydrophobicity in the PC-Ra conjugate. This was thus attributed to covalent bond formation, as confirmed by FTIR spectroscopy. The PC-Ra emulsion was then incorporated into surimi gels and compared with gels containing directly added corn oil. The results indicated that the PC-Ra emulsion significantly improved the textural properties of surimi gels. Furthermore, it enhanced water-holding capacity, reduced cooking loss, and delayed lipid oxidation. Among all formulations, the PC-Ra 30 emulsion exhibited the most pronounced effects and shows potential as a fat replacer in surimi gel preparation, yielding products with superior quality. This study provides a theoretical basis and technical support for developing novel surimi gel products.

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Full text

1. Introduction

Surimi, a concentrate of myofibrillar protein of fish, is usually made from repeatedly washed minced fish [1]. Washing is a critical step in surimi production, effectively removing water-soluble proteins, pigments, odors, and lipids to significantly enhance the gel quality and shelf stability of the final product. Yet, this process presents a nutritional paradox: the removal of lipids, especially unsaturated fatty acids, often results in a surimi gel with a bland taste, a weakened flavor profile, and reduced nutritional value [2]. While adding oil back into surimi is a common strategy to improve texture and nutritional profile, it introduces a technical conundrum. Replacing solid animal fat with liquid vegetable oil alone could diminish product quality, because the former contributes a solid elastic structure at room temperature, while the latter lacks this functional property [3]. Directly adding liquid oil disrupts the thermal gelation of myofibrillar proteins by impairing cross-linking, leading to a coarser, more porous three-dimensional gel network that facilitates oil accumulation and even phase separation [4]. This ultimately results in gel softening, diminished water-holding capacity, and accelerated lipid oxidation.

High internal phase Pickering emulsion (HIPPE), defined by an internal phase volume of ≥74% and stabilization by solid particles in O/W or W/O forms, offers a novel platform for functional surimi development through the embedding of bioactive ingredients [5]. This approach enables the replacement of animal fats with unsaturated-rich vegetable oils (e.g., olive oil, flaxseed oil), significantly reducing saturated fat content and enhancing the product’s health benefits [6]. Furthermore, PC additives could simultaneously improve the gel properties, nutritional value, and different colors of surimi. For instance, incorporating specific oils and proteins could enhance flavor, achieve amino acid complementarity, and promote nutritional balance [4]. Certain active substances might also confer specific health benefits to consumers.

Rosmarinic acid (Ra) is an important natural phenolic compound known for its diverse biological activities, including anti-inflammatory, antiaging, antioxidant, antibacterial, hepatoprotective, and cardioprotective effects [7]. Its chemical structure features multiple phenolic hydroxyl groups, which readily oxidize to form quinones. These quinone structures engage in covalent cross-linking with proteins, enhancing binding stability [8]. Phycocyanin (PC) is a natural phycobiliprotein widely valued as a safe and non-toxic source of vivid light-blue pigment for food applications. Beyond its color, PC is a high-quality protein containing 17 essential and non-essential amino acids (except tryptophan), conforming to the FAO/WHO ideal protein pattern. It also possesses notable bioactivities such as immunomodulatory, anticancer, antioxidant, and anti-inflammatory capacities. Hence, Ra and PC were chosen for emulsion formulation in surimi products, capitalizing on cross-linking capability of Ra alongside nutritional, coloring, and bioactive benefits of PC [9].

The stability of Pickering emulsions could be improved by modifying proteins with phenolic acids, which interact via covalent or non-covalent bonds [10]. Furthermore, interactions between proteins and phenolic acids are typically non-covalent, such as hydrogen bonds and hydrophobic interactions. Processing might lead to the disruption of these relatively weak interactions, thereby weakening the binding strength between proteins and phenolic acids [11]. During oxidation, phenolic acids are converted into free radicals and quinone intermediates, which react with amino acid side chains of proteins to form covalent cross-links, thereby constructing a robust protein–phenolic acid network. This process enables the development of highly stable complexes that significantly improve emulsion stability [12]. These covalent cross-links also exhibit upgraded, physicochemical and functional properties, such as improved antioxidant activity and emulsifying properties, indicating their potential as an effective HIPPE stabilizer. However, the impact of protein-based HIPPEs, particularly phycocyanin, on surimi gel quality remains insufficiently explained. While the direct addition of liquid oil to fish paste can lead to gel deterioration, incorporating it as an emulsion might mitigate this effect. Therefore, this study aimed to counteract such deterioration by adding a PC-RA emulsion and systematically analyzed how different concentrations of a rosmarinic acid–cross-linked phycocyanin emulsion affected the gel properties of silver carp surimi. Although previous studies have explored the interaction between proteins and phenolic acids, most have focused on non-covalent binding, which often suffers from low stability. The novelty of this work lies in the synthesis of a PC-RA conjugate via a covalent reaction. This conjugate is used to prepare an emulsion that improves the quality of surimi products, enhances their nutritional value, and imparts a distinctive color. These findings provide a theoretical basis and reference for enhancing the quality of freshwater fish surimi products, especially from silver carp.

2. Materials and Methods

2.1. Materials

Frozen surimi: Honghu Jingli Aquatic Products and Food Co., Ltd. (Honghu, China); arowana corn germ oil: Yihai Kerry Grain and Oil Industry Co., Ltd. (Wuhan, China). Sodium chloride (analytically pure): Maclean’s Biochemical Co., Ltd. (Shanghai, China). Phycocyanin: Mufan Biotechnology Co., Ltd. (Shanghai, China). Rosmarinic acid: Shanxi Guanchen Biotechnology Co., Ltd. (Jincheng, China). Laccase: Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). All the remaining chemicals utilized in the study were of analytical grade.

2.2. Preparation of PC-Ra Conjugates

The concentration range of Ra (20–40 μmol/L) used in this study was determined based on preliminary experiments. Pre-tests indicated that concentrations below 20 μmol/L had negligible effects on the gel properties of surimi. Conversely, concentrations exceeding 40 μmol/L resulted in excessive cross-linking and a decline in gel quality. Therefore, this specific concentration gradient was selected to effectively investigate the dose-dependent effects of rosmarinic acid on the physicochemical properties of the surimi gel. The synthesis of the PC-Ra conjugate was carried out as follows [13]: First, Ra was dissolved in acetate buffer (pH 5.5) to create a series of solutions with concentrations of 20, 25, 30, 35, and 40 μmol/L. Next, laccase (2 U/mL) was added to the Ra solution to catalyze the oxidation reaction, which was conducted under continuous stirring at 45 °C for 6 h. The oxidized Ra solution was then mixed with PC at a 10% (w/w) loading relative to the aqueous phase, and the mixture was incubated at 30 °C for another 6 h to facilitate conjugation, yielding the final PC-Ra conjugate solution. Then, the obtained conjugates were named PC-Ra 20, PC-Ra25, PC-Ra 30, PC-Ra35, and PC-Ra 40, respectively.

2.2.1. Bound Phenol Assay of PC-Ra Conjugates

The grafting efficiency of the conjugates was determined as follows: First, the different PC-Ra samples were precipitated with a solution of trichloroacetic acid and 0.4% phosphotungstic acid [14]. Following incubation, the samples were centrifuged. The supernatant was then carefully discarded, and the pellet was resuspended in NaOH. Subsequently, an aliquot of this protein solution was mixed with Folin–Ciocalteu reagent and incubated for 30 min. After terminating the reaction, the absorbance was measured, and the amount of unreacted phenolic acid was quantified using a standard curve generated with free Ra. The grafting efficiency was finally calculated according to the following formula:

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2.2.2. Disulfide Bond Content of PC-Ra Conjugates

The total and reactive sulfhydryl group contents were determined using Ellman’s assay. Sample concentrations were first adjusted to be consistent. For total sulfhydryl measurement, 100 μL of the sample was mixed with 1 mL of phosphate buffer (0.6 M NaCl, 20 mM sodium phosphate, 10 mM EDTA, pH 7.0) and 400 μL of 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB, 10 mM, pH 7.0). The mixture was incubated at 37 °C for 30 min with thorough mixing, followed by centrifugation at 20,000× g for 5 min. The absorbance of the supernatant was measured at 412 nm, using a blank prepared with deionized water. The procedure for measuring free sulfhydryl content was identical, except that 8 M urea was included in the 1 mL phosphate buffer [15].

2.2.3. Free Amino Acid Content of PC-Ra Conjugates

The quantity of free amino groups in the protein and its complexes was determined using the o-phthaldialdehyde (OPA) method. The OPA reagent was prepared by combining 1.0 mL of OPA in methanol (40 mg/mL), 2.5 mL of SDS (20% w/v), 25 mL of borax (0.1 M), and 100 μL of β-mercaptoethanol and then diluting the mixture to a final volume of 50 mL with deionized water. For the assay, 200 μL of the sample solution (3 mg/mL) was mixed with 4.0 mL of the OPA reagent and incubated at 35 °C for 2 min. The absorbance of the resulting mixture was subsequently measured at 340 nm [16].

2.2.4. Fourier Transform Infrared Spectroscopy (FTIR) of PC-Ra Conjugates

The lyophilized PC-Ra conjugate was analyzed by FTIR spectroscopy using the potassium bromide (KBr) pellet method. The mixture was thoroughly ground into a fine, homogeneous powder using an agate mortar and pestle under continuous infrared lamp irradiation to minimize moisture absorption. The pellet was then prepared from this powder. FTIR spectra were recorded at 25 °C by scanning over a wavenumber range of 4000 to 400 cm^−1^ with a resolution of 2 cm^−1^. The functional groups and structural composition of the PC-Ra conjugate were determined based on the characteristic absorption bands within the analyzed spectral range [17].

2.2.5. Determination of Surface Hydrophobicity of PC-Ra Conjugates

The surface hydrophobicity of the samples was determined using 8-anilino-1-naphthalenesulfonic acid (ANS) as a fluorescent probe [18]. The sample solutions were first diluted to a series of concentrations (0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL). Then, 4 mL of each diluted sample was mixed with 20 μL of an 8 mmol/L ANS solution. Fluorescence intensity was subsequently measured with a fluorescence spectrophotometer at excitation and emission wavelengths of 390 nm and 470 nm, respectively, using a slit width of 5.0 nm and a scan rate of 1200 nm/min. The initial surface hydrophobicity index (H_0_) was calculated as the slope of the linear regression curve, where the x-axis represents protein concentration and the y-axis represents the corresponding fluorescence intensity.

2.2.6. Three-Phase Contact Angle Measurement of PC-Ra Conjugates

The hydrophobicity of the sample particles was quantitatively evaluated using the sessile drop technique, with the contact angle denoted as (θ) [19]. Small quantities of PC and PC-Ra conjugates with varying Ra content were weighed and compressed into smooth, round pellets (2 mm thick, 13 mm diameter) using a hydraulic press. Images were captured after the droplet stabilized, and the contact angle was automatically calculated by software through fitting the droplet profile.

2.3. HIPPE Emulsion

Corn oil is rich in nutrients and contains a high proportion of unsaturated fatty acids, including linoleic acid, which has been shown to reduce low-density lipoprotein (LDL) cholesterol and support cardiovascular health [20]. Additionally, the presence of phytosterols in corn oil contributes to the inhibition of cholesterol absorption, further promoting heart health [21]. Based on these beneficial properties, corn oil was selected as the oil phase.

2.3.1. Preparation of HIPPE Emulsion

The prepared PC-Ra solution with different Ra concentrations was mixed with corn oil (φ = 0.80), with methodological adaptations based on previously reported methods with some modifications [22]. The mixture was homogenized at 10,000 rpm for 2 min using a homogenizer equipped with a 14 mm dispersion head. This process resulted in the preparation of five freshly formed HIPPEs, which were subsequently subjected to analysis.

2.3.2. Particle Size Measurements of the Emulsion

The particle size distribution of the emulsion was determined using the method reported by Feng et al. [19]. The droplet size of the HIPPEs was determined using a laser particle sizer (LS13 320, Beckman Coulter, Brea, CA, USA). The refractive indices were set at 1.520 for the sample and 1.333 for the dispersant (water). For analysis, an appropriate volume of the PC-Ra emulsion was introduced into the measurement chamber, and the standard measurement program was initiated to obtain the distribution profile.

2.3.3. Microstructural Analysis of the Emulsion

The morphology of the emulsion was observed under bright-field mode [23]. The emulsion was placed directly onto a glass slide for observation and photography at 100× magnification. The scale bar represents 20 μm.

2.3.4. Analysis of the Dynamic Rheological Properties of the Emulsion

The rheological properties of the emulsions were measured according to the method of Pei et al. [24]. The emulsion properties were measured with a rheometer (Anton Paar, Graz, Austria). The parallel plate gap was set to 1 mm. All measurements were conducted at 25 °C with the following preset conditions: amplitude sweeps (0.001–10) were measured for all samples at a frequency of 1.0 Hz, frequency sweeps were conducted from 0.1 to 10 Hz (strain: 0.1%), and shear rate sweeps were performed over a range of 0.1 to 50 s^−1^.

2.4. Application of Emulsion to Surimi

2.4.1. Preparation of Surimi

The surimi, stored frozen at −20 °C, was thawed at 4 °C prior to use. A 300 g aliquot was comminuted in a chopper for 10 s. Following the addition of 2.5% (w/w) NaCl, the mixture was chopped for an additional 2 min [23]. Designated quantities of a PC-Ra-based high internal phase emulsion (HIPE, φ = 0.80) were then incorporated to achieve a final added lipid content of 20% (w/w). After blending for 3 min, the paste was vacuum-sealed in casings and subjected to a two-stage heat treatment. The resulting gels were cooled in an ice-water bath and stored at 4 °C. The entire comminution process was conducted with the temperature controlled below 10 °C. For comparative analysis, two control gels were prepared simultaneously: one by directly adding 20% (w/w) corn oil to the surimi gel and the other consisting of surimi gel without any additional oil; the amount of added oil was kept consistent across all groups.

2.4.2. Texture Profile Analysis (TPA) and Gel Strength Measurements of the Surimi

Determined according to the method described by Gao et al., the TPA and gel strength of surimi gels were determined using a texture analyzer equipped with P/0.5 and P/0.5S probes [25]. Cubic gel specimens (25 × 25 × 25 mm) were prepared for the measurement. The probe was pressed vertically onto the gel surface at a speed of 1 mm/s with the following parameters: a sample height of 25 mm, a trigger force of 0.2 N, and a strain of 60%. Determined according to the method described by Jiao et al., the gel strength was evaluated based on the breaking strength and deformation depth, which were directly measured by the instrument [26]. Textural parameters, including hardness, cohesiveness, adhesiveness, springiness, chewiness, and gel strength, were analyzed and calculated using the accompanying software.

2.4.3. Determination of PC-Ra Emulsion on the Color of the Surimi

Determined according to the method described by Pei et al., the prepared surimi gel was first equilibrated at room temperature for 1 h and then sliced into approximately 5 mm thick sections [24]. The whiteness index of gel slices was measured using a colorimeter (chroma meter CR-5, Konica Minolta, Osaka, Japan) to characterize their color properties, which recorded the L* (lightness), a* (red-green), and b* (yellow-blue) values. It was calibrated with a standard white tile. The whiteness (W) was subsequently calculated using the following formula:

$$[eqn]$$

2.4.4. Determination of Water-Holding Capacity (WHC) of the Surimi

The water-holding capacity (WHC) of the gel was determined according to the method described by Lv et al. [27]. The gel was first cut into 5 mm × 5 mm × 5 mm cubes, and the initial mass was recorded (W_T_). These cubes were then wrapped in two layers of filter paper and placed into a centrifuge tube. The tube was centrifuged at 8000× g (RCF) for 10 min. After centrifugation, the gel was removed and weighed again to obtain the final mass (W_F_). The WHC was calculated using the following formula:

$$[eqn]$$

2.4.5. Determination of the Cooking Loss of the Surimi

The cooking loss of the surimi was determined according to the method described by Lv et al. [27]. Gel samples were cut into cylindrical specimens (approx. 15 mm × 15 mm × 5 mm) and weighed to obtain the initial mass (G_1_). The specimens were sealed in retort pouches and subjected to heat treatment in a 90 °C water bath for 20 min. Subsequent to retorting, the samples were conditioned at 4 °C for 2 h, after which the surface moisture was blotted with filter paper and the samples were reweighed (G_2_). The retorting loss rate was calculated according to the following equation:

$$[eqn]$$

2.4.6. Low-Field Nuclear Magnetic Resonance (NMR) of the Surimi

The transverse relaxation time (T_2_) of the gel was characterized using low-field NMR. Cylindrical specimens (15 mm × 15 mm × 20 mm) were loaded into 40 mm NMR tubes for analysis. The instrument was configured with these acquisition parameters: a spectral width (SW) of 200 kHz, receiver gain (RG_1_) of 20, P_1_ at 18.00 µs, DRG_1_ at 3, time domain (TD) of 399,950, PRG at 2, repetition time (TW) of 2500 ms, and 8 scans (NS).

2.4.7. Thiobarbituric Acid Reactive Substances (TBARSs) of the Surimi

The TBARS determination of the surimi was determined according to the method described by Glišić’s et al. [28]. Surimi gels were measured after storage at 4 °C under aerobic conditions and protection from light for one week. A trichloroacetic acid (TCA) mixed solution, containing 37.50 g of TCA and 0.50 g of EDTA-2 Na in a final volume of 500 mL, and thiobarbituric acid (TBA) aqueous solution (0.288 g TBA in 100 mL, with ultrasonication if necessary for dissolution) were prepared. A 5.00 g sample was accurately weighed and extracted with 50.00 mL of the TCA solution by shaking at 50 °C for 30 min. After cooling and filtration, the filtrate was collected. Subsequently, 5 mL aliquots of this filtrate, standard series solutions, and the TCA solution (serving as the sample blank) were precisely pipetted into separate tubes. Each aliquot was mixed of the TBA solution and heated at 90 °C in a water bath for 30 min. After cooling, the absorbance at 532 nm was measured against the sample blank, and a standard curve was constructed.

2.5. Statistical Analysis

All experiments were conducted in triplicate (n = 3), with data expressed as mean ± standard error (SE). Prior to analysis, data were tested for normality and homogeneity of variances. Statistical significance was determined by one-way analysis of variance (ANOVA) using SPSS 13.0 (SPSS Inc., Chicago, IL, USA), with a p-value of less than 0.05 considered statistically significant.

3. Results and Discussion

3.1. Conjugate Characterization

3.1.1. Grafting Rate of PC-Ra Conjugates

Grafting rate reflects the covalent bonding efficiency between PC and Ra. Figure 1A shows that the coupling rate first increases and then decreases as the acid concentration rises [29]. At low concentrations of phenolic acids, protein molecules provided sufficient binding sites. However, when the phenolic acid concentration reached a certain threshold, the protein binding sites tended towards saturation, causing the binding rate to cease increasing or even decline [30]. High concentrations of phenolic acid could induce protein conformational changes, rendering regions previously suitable for binding unstable or inaccessible, thereby affecting the binding rate. Furthermore, they might induce excessive protein aggregation or denaturation, leading to active site entrapment (e.g., disulfide bond rearrangement or exposure of hydrophobic cores), thereby reducing accessible binding sites.

3.1.2. Disulfide Bond Content of PC-Ra Conjugates

Determination of disulfide bond indirectly reflects the occurrence of deep interactions between PC and Ra, primarily mediated by covalent cross-linking. Figure 1B shows the disulfide bond content in the PC-Ra conjugates; it initially increased and then decreased with the increase in phenolic acid addition. At lower concentrations, quinone derivatives derived from oxidized Ra promote conversion of sulfhydryl groups into disulfide bonds, which subsequently cross-link with nucleophilic amino acid side chains [31]. In contrast, at higher concentrations, excessive phenolic acid molecules competitively bind to the protein surface and sterically shield key residues, particularly free cysteine thiols. This molecular crowding impedes the close proximity between free sulfhydryl groups, thereby physically inhibiting their oxidation and the consequent formation of disulfide bonds.

3.1.3. Free Amino Acid Content of PC-Ra Conjugates

Covalent cross-linking leads to a reduction in free amino groups, reflecting the formation of a denser, mechanically stronger protein adsorption layer at the oil–water interface [32]. As shown in Figure 1C, the addition of Ra significantly reduced the free amino content in the PC-Ra conjugates. The carboxyl group (-COOH) or hydroxyl group (-OH) in phenolic acid could form covalent bonds with free amino groups (-NH_2_) in proteins under oxidative conditions, creating amide or ester bonds [33]. Under oxidative stress, the sulfhydryl groups to disulfide bonds might be accompanied by alterations in the chemical environment of adjacent amino groups, thereby affecting their reactivity. The electrostatic attraction between amino groups and -COOH contributes to the structural stability of protein [34]. Non-covalent interactions between phenolic acids and proteins, such as hydrogen bonds and hydrophobic interactions, could induce conformational changes in the proteins, which mask or sequester free amino groups on the protein surface, thereby reducing free amino content [35]. Within the OPA reagent system, SDS disrupts pre-existing non-covalent interactions [36]. Based on this phenomenon, it is deduced that covalent bonding occurred between Ra and PC in the experimental system constructed in this study. Reduced free amino group reactivity might indicate protein aggregation or structural compaction. In the PC-Ra conjugates, the phenolic hydroxyl group of phenolic acids reacted with amino or sulfhydryl groups in proteins to form stable covalent bonds, thereby this method was used to describe the strengthening of the protein network structure.

3.1.4. FTIR of PC-Ra Conjugates

FTIR spectroscopy reveals key molecular interactions in the laccase-synthesized PC-Ra conjugate [17]. As shown in Figure 2A, the laccase-mediated formation of the PC-Ra conjugate involved multiple molecular interactions. Compared with phycocyanin, the broad absorption peak of PC-Ra conjugate at 3200–3600 cm^−1^, corresponding to O-H and N-H stretching vibrations, exhibited a distinct redshift, indicating the establishment of an extensive hydrogen-bonding network between phenolic hydroxyl groups and protein amino/amide groups. The blueshift in amide I band (1650–1660 cm^−1^) is associated with C=O stretching, reflecting a rearrangement from α-helix to β-sheet in the protein. As shown in Figure 2B, the observed increase in β-sheet content is likely to influence surface hydrophobicity, reflecting changes in the distribution and exposure of hydrophobic groups critical for surface-related functionality. This transition enhances intermolecular interactions among proteins and facilitates the formation of a stable protein-bound interfacial film. Changes in the amide II band (1540–1550 cm^−1^) revealed alterations in N-H bending and C-N stretching vibrations within the peptide backbone, reflecting molecular interactions between Ra and the peptide chains of phycocyanin. Furthermore, the appearance of the C-O and S=O absorption band at 1050–1200 cm^−1^ makes the formation of thioether bond via Michael addition between Ra and phycocyanin plausible, which might contribute to improving the structural stability and antioxidant activity of the conjugate.

3.1.5. Surface Hydrophobicity of PC-Ra Conjugates

ANS, as a widely employed fluorescent probe, is frequently utilized for assessing surface hydrophobicity in proteins [37]. As shown in Figure 3A, the surface hydrophobicity of the PC-Ra conjugates increased firstly and then decreased as the Ra concentrations increased from 20 μmol/L to 40 μmol/L. Among them, the PC-Ra 30 (Ra is 30 μmol/L) group exhibited the highest surface hydrophobicity, suggesting that the structure or composition of the conjugate under these conditions might be most conducive to forming a hydrophobic surface. This trend of change could be attributed to the effects of covalent conjugation. The covalent conjugation of phenolic acids to proteins primarily occurs through reactions between their phenolic hydroxyl groups and amino or sulfhydryl groups on the protein, forming stable covalent bonds. This modification alters the molecular structure and surface properties of the proteins, thereby modulating their surface hydrophobicity. At high concentrations, phenolic acids could induce protein folding or aggregation. These conformational changes might sequester hydrophobic regions within the protein interior, consequently diminishing surface hydrophobicity.

3.1.6. Three-Phase Contact Angle Measurement of PC-Ra Conjugates

The three-phase contact angle is a key indicator of the adsorption capacity of solid particles at oil–water interfaces, with an angle approaching 90° signifying optimal adsorption due to a balance of interactions with both phases [38]. As shown in Figure 3B, the PC-Ra conjugate achieved a contact angle of 87.20° ± 0.2° at a Ra concentration of 30 μmol/L, demonstrating a near-ideal hydrophilic/hydrophobic balance that maximizes interface stability [39]. However, further increasing the Ra content reduced the contact angle, shifting the particles toward a more hydrophilic state. Thus, the PC-Ra 30 conjugate facilitated the formation of a dense interfacial layer, providing excellent stability for Pickering emulsions [40].

3.2. Emulsion Characterization

3.2.1. Microstructural Analysis of the Emulsion

Figure 4A shows the microstructure of the emulsion; the incorporation of PC-Ra conjugates enabled the formation of a homogeneous HIPPE with a well-defined structure, whereas the control failed to produce an emulsion. Microscope images most directly reveal the droplet morphology, size, and distribution of the emulsion (Figure 4B). The droplet size of the emulsion initially decreased and then increased with the addition of the PC-Ra conjugate, consistent with the laser particle size analysis results (Figure 4C). All emulsions were confirmed as oil-in-water (O/W) type. These findings reflect the role of PC-Ra conjugates in adsorbing at the oil–water interface and structuring the continuous phase into a gel network, which initially hinders droplet coalescence and refines emulsion morphology [41]. A low Ra content led to irregular and loosely droplet arrangement with weak stability and a coalescence tendency, indicating suboptimal interfacial coverage. As the Ra concentration increased, droplet aggregation and coalescence became more pronounced, indicating that high concentrations of stabilizer disrupt interfacial properties or promote interparticle interactions, thereby reducing emulsion stability.

3.2.2. Particle Size Measurements of the Emulsion

Particle size is a key indicator for predicting destabilization processes in emulsions, such as phase separation, flocculation, and coalescence [42]. Figure 4C demonstrates the variations in emulsion droplet diameter resulting from the addition of conjugates prepared using distinct RA concentrations. The PC-Ra 30 sample yielded the smallest emulsion droplet size, while both higher and lower PC-Ra ratios resulted in larger droplets. Variations in the PC-RA dosage led to substantial shifts in emulsion droplet diameter, demonstrating a significant regulatory impact (p < 0.05). The covalent conjugation of phenolic acids with proteins modifies protein surface hydrophobicity, which initially promoted a more uniform protein distribution within the emulsion and effectively reduced droplet size [43]. However, the magnitude of this effect is correlated with phenolic acid concentration. As the concentration increased, the available binding sites on the protein surface progressively became saturated [44]. When exceeding a certain critical threshold, excess phenolic acids and their oxidized derivatives, quinones, triggered intermolecular cross-linking reactions. These reactions were driven by rapid interactions between quinones and nucleophilic sites (e.g., sulfhydryl and amino groups) on proteins, resulting in extensive protein aggregation [45]. This aggregation, in turn, led to an increase in emulsion droplet size. The resulting large protein aggregates exhibited reduced interfacial dispersion and adsorption efficiency, a seemingly contradictory phenomenon that ultimately disrupted emulsion stability and increased overall particle size [46].

The results from the laser particle size analyzer aligned with the microscopic observations, indicating that the smallest emulsion droplet size was achieved under PC-Ra 30 conditions.

3.2.3. Analysis of the Dynamic Rheological Properties of the Emulsion

The storage modulus (G′) and loss modulus (G″) are core parameters for measuring gel elasticity and viscosity in rheology [47]. Strain sweep tests revealed that the control sample exhibited relatively low values for both G′ and G″, characteristic of a dilute solution (Figure 5A). In contrast, the addition of PC-Ra conjugates significantly increased both modulus values, indicating substantially enhanced internal structural strength and the formation of a robust gel network within the emulsion. The PC-Ra30 emulsion exhibited the highest strain value at the G′ and G″ crossover point, reflecting its most compact and robust network structure alongside the most pronounced gel strength [48]. Frequency sweep results showed that G′ of all samples displayed slight frequency dependence, increasing slowly and steadily with frequency. This confirmed the integrity and high stability of the gel networks under different deformation rates (Figure 5B,C). The PC-Ra 30 group formed the strongest network, as evidenced by the smallest increase in G′ over the frequency range. In terms of viscosity, these emulsions exhibited typical non-Newtonian fluid characteristics, demonstrating pronounced shear thinning behavior (Figure 5D). The PC-Ra 30 group exhibited the highest viscosity under static or low-shear conditions, indicating that phenolic acid significantly enhanced the internal structural stability, dispersion uniformity, and interfacial film strength of the emulsion. This notable significant increase in viscosity indicated that phenolic acid molecules act as bridging or cross-linking agents between droplets. By adsorbing at the droplet interface and interconnecting, they formed a three-dimensional network structure that restricted flow and enhanced viscosity, thereby improving long-term storage stability and effectively suppressing crystallization and phase separation. The increase in the G′ value likely stemmed from dynamic reinforcement within the internal network, such as increased polymer chain cross-linking density, a slowed gelation rate, or enhanced interfacial film stability. However, excessive phenolic acid could lead to molecular self-aggregation, which interferes with interface interactions and might weaken protein–protein bonds, reducing interfacial film elasticity [49]. Therefore, phenolic acids at optimal concentrations (such as PC-Ra 30) connected emulsion droplets through a “bridging flocculation” mechanism, forming a robust and highly elastic three-dimensional network structure. This makes it suitable for applications in cosmetics, ointments, or food systems requiring long-term stability and specific textures [50]. Overall, PC-Ra 30 emerged as the optimal condition for forming the most robust gel network under the tested parameters.

3.3. Surimi Gel Characterization

3.3.1. TPA and Gel Strength of the Surimi

TPA and gel strength comprehensively reflect the microstructure, intermolecular forces, moisture state, and final eating quality of surimi gel [51]. Table 1 demonstrates that PC-Ra emulsion significantly altered the textural properties of surimi gel, producing marked effects on key parameters including hardness, springiness, cohesiveness, chewiness, and gel strength, while surimi with directly added oil exhibited inferior textural properties [52]. The experimental findings unequivocally demonstrated that the presence of liberated lipids interfered with the cross-linking process essential for establishing a stable myofibrillar protein gel matrix. In contrast, both the oil-free group and the PC-Ra 30 group showed higher hardness. However, excessive emulsion addition (e.g., PC-Ra 40) resulted in decreased hardness, suggesting the presence of an optimal concentration for improving texture. All emulsion-treated groups demonstrated higher cohesiveness than the control, affirming that emulsified oil could reinforce the gel structure. Notably, the PC-Ra 30 group achieved peak values in adhesiveness and chewiness, signifying its effectiveness in improving the chewy and viscoelastic texture of surimi. The improvement was attributable to the emulsion promoting covalent cross-linking and hydrogen bond formation, thereby refining the myofibrillar protein network into a more compact and uniform structure [53]. The introduction of emulsion also promoted β-sheet formation, enhanced protein network elasticity, and improved mechanical strength. At 30 μmol/L, Ra optimally enhanced protein cross-linking through disulfide bonds and hydrophobic interactions, increasing network density and water-holding capacity. This network structure facilitated the uniform distribution of tiny oil droplets and minimized potential structural damage. Conversely, at a higher concentration of 40 μmol/L, excessive Ra induced protein aggregation, resulting in a coarse and brittle gel structure, which consequently reduced hardness, adhesiveness, and water-holding capacity. In summary, the PC-Ra 30 group significantly improved the hardness, elasticity, adhesiveness, chewiness, and gel strength of surimi gel, while deviations from the 30 μmol/L Ra concentration negatively affected these texture properties.

3.3.2. Color of the Surimi Gel

The color intensity of surimi is a key parameter for assessing its quality grade [54]. The incorporation of the PC-Ra emulsion exerted a significant influence on the color characteristics of surimi gel (Table 2 and Figure 6). Across all samples, the surimi gel with PC-Ra 30 exhibited the lowest lightness, lowest whiteness, and most pronounced blue tint. Its b* value was −2.64 ± 0.02, and the lightness was relatively low (L* = 62.52 ± 0.01). This phenomenon was attributed to the blue pigment in phycocyanin covering and diminishing the inherent bright white color of the surimi matrix, leading to a significant decrease in both L* value and whiteness. The deeper the blue, the more the lightness and whiteness were lost. The addition of the PC-Ra 30 emulsion contributed to the formation of a conjugate structure in the gel with optimal light-scattering properties, characterized by small particle size and strong scattering ability. This might be attributed to the higher stability of phycocyanin within this conjugate structure [55]. Therefore, color changes could be regarded as a visual indicator of the structural stability of the gel system. With the growing consumer demand for health, natural blue derived from phycocyanin not only meets health aspirations but also possesses unique sensory appeal. The development of surimi gel characterized by a blue hue offers a significant commercial opportunity for product differentiation.

3.3.3. WHC and Cooking Loss of the Surimi Gel

The WHC and cooking loss were measured as key parameters for determining surimi gel stability. These values directly reflect the effectiveness of the gel’s three-dimensional network structure in retaining water [56]. High water-holding capacity and low cooking loss rate indicate that water is firmly bound within the gel, minimizing moisture loss during heating. Figure 7A reveals a markedly reduced WHC in the oil-added group compared with the control and others. This finding clearly demonstrates that free oil adversely affected the WHC of the surimi gel. The direct addition of free oil resulted in a significant decrease in the WHC compared with the oil-free group. Furthermore, most PC-Ra groups exhibited a superior WHC compared with the oil-free group, indicating that the addition of PC-Ra emulsion effectively improved the water-holding capacity of surimi gel. The concentration of phenolic acid significantly influenced the WHC of surimi gel. Compared with the control, moderate Ra concentrations (e.g., PC-Ra 30) significantly enhanced the WHC, while concentrations that were too high or too low could impair its WHC.

The cooking loss represents the proportion of mass lost during cooking due to the migration of easily lost substances such as water and fat from the surimi gel [57]. There were significant differences in cooking loss among different treatment groups (p < 0.05). Compared with the control, the cooking loss was highest in the surimi gel without oil. This might be because added oil interferes with protein cross-linking in surimi, weakening the gel network structure. Additionally, the poor miscibility between oil and water prevented effective water retention within the gel network, ultimately compromising its structural integrity [58]. Compared with the control, surimi gel with PC-Ra emulsion all exhibited lower cooking loss. Cooking loss initially decreased and then increased with rising phenolic acid concentration. Surimi gel with PC-Ra 30 exhibited the lowest cooking loss. Subsequently, cooking loss rose again as the phenolic acid concentration further increased to PC-Ra 35 and PC-Ra 40. The compactness of the surimi gel network might influence water distribution; a denser gel network corresponds to lower free water content, which leads to improved water-holding capacity. Consequently, changes in water-holding capacity affect cooking loss, with higher water-holding capacity resulting in lower cooking loss [59].

Thus, Ra might induce cross-linking among myofibrillar proteins in the emulsion, forming a stronger network that enhanced water and oil droplet binding. However, as the Ra concentration reached 35–40 μmol/L, excessive polymerization occurred, resulting in a coarse gel network structure. This enlarged its pores and reduced its retention capacity for water and oil droplets, thereby increasing cooking loss. In summary, the addition of PC-Ra emulsion effectively reduced moisture loss during cooking, thereby improving the WHC of surimi gel. The results indicate that adding different emulsions significantly influenced the cooking loss in surimi gel. In particular, emulsions complexed with an optimal level of phenolic acid significantly reduced cooking loss. However, excess phenolic acid had negative effects, so the dosage had to be strictly controlled. Under PC-Ra 30 conditions, the high WHC and low cooking loss indicated that water was firmly bound within the surimi gel, minimizing moisture expulsion during heating.

3.3.4. Low-Field Nuclear Magnetic Resonance of the Surimi

NMR is the most direct and effective method for determining the water distribution and composition in surimi gel [60]. The state of water within the gel directly influences its water-holding capacity, which in turn affects the quality of the surimi gel. T_2_ relaxation time is related to the mobility of water protons and their interactions with the surrounding matrix. A longer T_2_ relaxation time indicates weaker hydrogen proton confinement, signifying poorer water stability and increased fluidity. The experimental data revealed that bound water represented the major component in the gel system. Because this specific population is inextricably linked to the protein network architecture, it acts as a decisive factor governing the water-retention properties of the final gel. The distribution and proportion of different water fractions were calculated from the relative peak areas, and the results are presented in Figure 7C and Table 3. Compared with the control group, the addition of emulsion reduced the proportion of free water in the surimi gel (p < 0.05) while increasing the proportion of immobilized water (p < 0.05). This was because the emulsion dispersed uniformly within the surimi gel matrix, participating in the formation of a dense three-dimensional gel network structure that alters water mobility. The addition of corn oil might have disrupted protein–protein interactions, producing a less compact and uniform three-dimensional network. This weakening increased water mobility and released previously bound water, thereby diminishing the surimi gel’s water-holding capacity. This finding was consistent with measurements of WHC and cooking loss.

3.3.5. The TBARSs of the Surimi

TBARSs are widely used to evaluate lipid oxidation in foods [61]. As shown in Figure 7D, the PC-Ra emulsion significantly reduced the MDA content compared with the control (p < 0.05). However, this effect was highly dependent on the Ra concentration. The PC-Ra 30 emulsion achieved the lowest MDA content, indicating optimal inhibition of oil oxidation. In contrast, both excessively high and low Ra concentrations led to a significant increase in MDA, demonstrating reduced antioxidant efficacy. This diminished effect might be attributed to the pro-oxidant activity of Ra at suboptimal concentrations. Specifically, at non-optimal levels, Ra itself could be prone to oxidation, generating unstable intermediates (e.g., semiquinone radicals) that catalyze chain reactions and increase MDA production. Furthermore, it might promote lipid peroxidation by interacting with compounds such as iron in the surimi matrix [28].

4. Conclusions

In summary, protein-based fat emulsions are significantly more beneficial than the direct addition of fats for optimizing surimi gel quality. By optimizing the density and uniformity of the gel network, the emulsion system improved the gel properties and water-holding capacity while effectively inhibiting lipid oxidation. This is likely because covalent and non-covalent bonds between PC-Ra complexes and myofibrillar proteins create additional cross-linking points. This enables more myofibrillar proteins to participate in the self-assembly of the three-dimensional network, forming a denser, continuous gel matrix. Therefore, the use of the PC-Ra 30 emulsion is recommended as a viable solution to prevent the quality defect caused by liquid oil and to serve as a functional fat substitute, ultimately yielding surimi gel products with superior texture and structural integrity.