Enhancement of gel properties of low‐salt Scomberomorus niphonius surimi by egg white protein: Structural refinement and intermolecular interaction analysis

Abstract

BACKGROUND

High‐quality gelation of Scomberomorus niphonius (SN) surimi under low‐salt conditions remains challenging for the seafood industry. This study evaluated egg white protein (EWP) substitution (1–9%) to enhance the gel properties and water retention of low‐salt (1% sodium chloride) SN surimi under reduced ionic strength.

RESULTS

The results demonstrated that the optimal performance was achieved at 5% EWP substitution, where the breaking force significantly increased by 58.9% compared to the low‐salt control (LC) group, and the water‐holding capacity (WHC) concurrently improved to 73.03%. Quantitative cryo‐scanning electron microscopy (cryo‐SEM) analysis revealed substantial network densification, characterized by a decrease in porosity from 55.67% (LC group) to 34.85% and a minimum mean pore size of 0.25 ± 0.16 μm. Pearson correlation and heat map visualization confirmed that this microstructural refinement (r = −0.921 with WHC) and the enhanced formation of disulfide bonds (r = 0.993 with breaking force) were the primary drivers for the improved water retention and gel strength, respectively. Mechanistically, at moderate substitution levels (≤ 5%), EWP was partially compatible with SN myofibrillar proteins, functioning as a co‐gellant and crosslinker via disulfide‐related interactions that transformed the loose, porous matrix into a compact honeycomb structure; however, excessive substitution (> 5%) promoted preferential EWP self‐aggregation and microphase separation, thereby undermining network continuity and integrity.

CONCLUSION

Optimal EWP substitution (5%) effectively overcomes the gelation defects of low‐salt SN surimi by forming a co‐gel network driven by disulfide crosslinking and structural densification. These findings elucidate the quantitative structure–function relationship of EWP in low‐ionic‐strength systems, providing a solid theoretical foundation for the development of healthy, salt‐reduced surimi products. © 2026 Society of Chemical Industry.

INTRODUCTION

Surimi‐based products, such as fish cakes and imitation crab sticks, are extensively consumed gel‐forming foods recognized for their distinctive textural qualities and nutritional content. ¹ Traditional surimi production processes typically incorporate 2–3% (w/w) sodium chloride (NaCl) to facilitate myofibrillar protein solubilization, thereby achieving optimal thermal gelation and textural characteristics. ² However, excessive dietary sodium intake has been associated with an increased risk of chronic diseases, including hypertension and cardiovascular disorders, ³ prompting the World Health Organization (WHO) to recommend a 30% reduction in global salt intake. ⁴ Consequently, developing reduced‐sodium surimi products that preserve their gel functionality properties remains a critical challenge for the food industry.

For surimi manufacturers, reducing salt content to 1% (w/w) or lower commonly results in deterioration of functional properties, including diminished water‐holding capacity (WHC), decreased gel strength, and structural disorganization of the gel network. ² Scomberomorus niphonius (SN) represents an economically significant marine species characterized by elevated myofibrillar protein concentration (about 12% wet weight) and low bone‐to‐meat ratio. ⁵ However, the myofibrillar proteins from SN provide low solubility under reduced‐salt conditions, which has established low‐salt surimi gelation as an industrial challenge despite its commercial importance.

Exogenous proteins have emerged as a promising strategy to mitigate the quality deterioration in low‐salt surimi gels by functioning as filler molecules to reinforce network continuity or as crosslinking mediators to enhance protein–protein interactions.6, 7 Among potential candidates, egg white protein (EWP) is attractive due to its nutritional value and favorable gelling functionality. ⁸ EWP exhibits rapid gelation at low temperatures and concentrations following short‐term heating, attributed to its unique protein structure. ⁹ Unlike soy or whey proteins, EWP contains distinct sulfhydryl groups and structural features that enable rapid thermal gelation and intermolecular crosslinking even at low temperatures and concentrations. ¹⁰ Research has shown that EWP can improve the gel properties in surimi gels from giant squid (Dosidicus gigas), ¹¹ blue round scad (Decapterus maruadsi), ¹² and grass carp (Ctenopharyngodon idellus). ¹³ However, the performance of EWP in low‐salt SN surimi systems and the interaction mechanisms between EWP and SN myofibrillar proteins under restricted‐NaCl conditions remain insufficiently elucidated. At the typical surimi pH range (approximately pH 6.5–7.0), both EWP (isoelectric point ~4.5–4.8) and membrane protein (MP) possess net negative surface charges. The low ionic strength associated with low‐salt conditions reduces electrostatic screening, increasing intermolecular repulsion that prevents random aggregation during processing and promotes uniform distribution of EWP within the protein matrix. Heating unfolds proteins to overcome the electrostatic barrier and expose hydrophobic domains and sulfhydryl groups for specific intermolecular crosslinking via hydrophobic interactions and disulfide bonds. Meanwhile, reduced ionic strength affects MP solubilization, which plays an important role in co‐gelation, WHC, and the balance between covalent and non‐covalent interactions in gel network formation. Therefore, understanding how EWP addition controls the microstructure and moisture distribution in low‐salt SN surimi gel is essential to rationally design reduced‐sodium surimi products with desirable textural and functional properties.

This study aimed to elucidate the mechanism through which EWP enhances the gel properties of low‐salt (1% NaCl) SN surimi. Specifically, composite gels were prepared by substituting EWP for SN surimi protein at levels ranging from 1% to 9% (protein‐equivalent basis), and their rheological behavior, water migration dynamics, and microstructural evolution were systematically investigated. Molecular‐level mechanisms were explored through analyses of intermolecular interactions, protein secondary structures, and electrophoretic profiles. These findings are expected to provide fundamental insights into heteroprotein interactions in low‐salt gel systems and to offer practical guidelines for the industrial production of high‐quality reduced‐sodium SN surimi products.

MATERIALS AND METHODS

Materials and chemicals

The SN samples were obtained from Qianhe seafood market (Dalian, China). EWP (purity = 80%) was purchased from Jiangsu Kangde Egg Industry Company (Jiangsu, China). NaCl was sourced from Macklin Biochemical Co., Ltd (Shanghai, China). All other reagents (analytical grade) were purchased from Aladdin Biochemical Technology Co., Ltd (Shanghai, China).

Preparation of SN surimi gels supplemented with EWP

The fish meat was separated and rinsed with distilled water, then chopped for 1 min using a chopper, NaCl (1%, w/w) was initially added to promote myofibrillar protein extraction, followed by EWP addition during continued chopping. The EWP substitution levels were defined on a protein‐equivalent basis. Specifically, 1%, 3%, 5%, 7%, and 9% of the target total protein were supplied by EWP in place of SN protein. Because the commercial EWP powder contained 80% protein, the actual powder addition level was calculated according to its protein purity. The total protein content (SN protein + EWP) in each sample was standardized to 18 g per 100 g of batter, while the moisture content of the mixed surimi was adjusted to approximately 78%. After the intense mechanical shearing during the subsequent 2 min chopping provided effective in situ hydration, ensuring uniform dispersion without localized agglomeration. Air bubbles were removed via centrifugation (5000 × g, 5 min) without significant water loss. ⁴ The mixed surimi samples were subjected to a two‐stage thermal processing through water bath incubation (40 °C for 30 min followed by 90 °C for 20 min), subsequently quenched in ice water to form surimi gel, and stored at 4 °C prior to analysis. Surimi gels containing 1% and 2.5% NaCl served as low‐salt control (LC) and high‐salt control (HC) groups, respectively. To minimize formulation bias, all batters were adjusted to the same total protein content and moisture content; therefore, the observed differences were primarily attributed to protein substitution type and interaction behavior rather than uncontrolled changes in total solids.

Mechanical properties

The breaking force and texture properties were determined using a Texture Analyzer (TA.XT.plus; Stable Micro Systems, Godalming, UK) according to the method of Zhang et al. ¹⁴ with minor modifications. Cylindrical gel samples (diameter: 20 mm; height:10 mm) were prepared for texture analysis. Breaking force measurements and texture profile analysis were performed using P/0.5 and P/50 probes, respectively, under standardized parameters: pre‐test speed of 1.5 mm s⁻¹, test speed of 1 mm s⁻¹, post‐test speed of 1 mm s⁻¹, trigger force of 5 g, and 40% compression ratio.

Moisture properties

Cooking loss

The cooking loss was determined with minor modifications as described in Jiao et al. ¹⁵ Prior to thermal gelation, the raw mixed surimi paste was weighed (m ₁). Following heat treatment and subsequent cooling to ambient temperature, the surface moisture was removed by gentle blotting, and the final mass (m ₂) was recorded. Cooking loss was quantified using Eqn (1):

$$\text{Cooking loss}\left(\%\right)=\left(m_1-m_2\right)/m_1\times 100$$ (1)

Water‐holding capacity (WHC)

The WHC was determined using a modified method described by Liu et al. ⁴ Gel samples (approximately 1 g, designated as W ₁) were centrifuged at 10 000 × g for 10 min at 4 °C after being wrapped in filter paper, and the residual gel sample was reweighed to obtain W ₂. The WHC value was quantified through Eqn (2):

$$\mathrm{WHC}\left(\%\right)=\left(W_2/W_1\right)\times 100$$ (2)

where W ₁ and W ₂ denote the sample weights before and after centrifugation, respectively.

Low‐field nuclear magnetic resonance (LF‐NMR)

Low‐field nuclear magnetic resonance (LF‐NMR) analyzer (MesoMR23‐060V‐1; Niumag Analytical Instrument Co., Ltd, Shanghai, China) was used to measure the transverse spin–spin relaxation (T ₂) data. Cylindrical sample specimens measuring 10 mm in height and 20 mm in diameter were placed within NMR tubes for analysis. The Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence parameters were configured as follows: P1 = 16 μs; P2 = 32 μs; echo time (TE) = 0.5 ms; repetition time (TW) = 5000 ms; number of echoes (NECH) = 5000; and number of scans (NS) = 8. The relative proportions of bound, immobilized, and free water were expressed as normalized peak area percentages, with the sum of all T ₂ peak areas set to 100%.

The method for obtaining proton density images was modified based on Yan et al. ¹⁶ Single‐slice spin‐echo pulse sequences were employed with imaging parameters set as follows: repetition time (TR) = 2000 ms; echo time (TE) = 20 ms; three contiguous slices with a thickness of 2.2 mm and an interslice gap of 1.0 mm. Subsequent image processing of the grayscale datasets was performed using OsiriX (Niumag Analytical Instruments Co., Ltd) to generate false‐color representations of proton‐density‐weighted images.

Rheological properties

The viscoelastic properties of SN surimi gels were characterized using a DHR‐1 rotational rheometer (TA Instruments, Dallas, TX, USA) equipped with a 20 mm parallel‐plate geometry and a 1 mm gap, following the method of Liu et al. ¹ with minor modifications. Frequency sweep tests were performed over the range of 0.1–10 Hz at 25 °C and 0.3% strain to assess the frequency dependence of the storage modulus (G') and loss modulus (G"). Subsequently, temperature‐sweep tests were conducted from 20 to 90 °C at a heating rate of 5 °C min⁻¹ under a fixed frequency of 1 Hz and 0.3% strain, with continuous monitoring of G' for subsequent analysis.

Cryo‐scanning electron microscopy (cryo‐SEM)

All samples were snap‐frozen in liquid nitrogen and subsequently imaged using a SU8000 scanning electron microscope (Hitachi Co., Ltd, Tokyo, Japan). Following imaging, specimens were transferred to a vacuum‐sealed cryo‐preparation chamber where sequential processing included freeze‐fracture, sublimation at −60 °C for 40 min, and platinum sputter coating. The prepared samples were then examined under an accelerating voltage of 5.0 kV. Quantitative analysis of the cryo‐scanning electron microscopy (cryo‐SEM) micrographs was performed using Angitool64 software.

Intermolecular interaction

To quantify specific protein interactions, samples were subjected to sequential extraction using distinct solvent systems (Sa: 0.6 mol L⁻¹ NaCl; Sb: 1.5 mol L⁻¹ urea/0.6 mol L⁻¹ NaCl; Sc: 8 mol L⁻¹ urea/0.6 mol L⁻¹ NaCl; Sd: 0.5 mol L⁻¹ β‐mercaptoethanol/8 mol L⁻¹ urea/0.6 mol L⁻¹ NaCl, pH 7) following the protocol of Yu et al. ¹⁷ with minor modifications. Each extraction step involved homogenizing 4 g of sample with 20 mL solvent, followed by mechanical shaking, centrifugation (10 000 × g, 20 min), and supernatant collection; notably, the Sc extraction was performed twice consecutively. The residual pellet was solubilized in 1 mol L⁻¹ sodium hydroxide (NaOH). Protein concentrations across all extracts were determined via the Bradford assay. The sequential extraction fractions obtained by Sa, Sb, Sc, and Sd treatments specifically solubilized proteins associated with ionic, hydrogen, hydrophobic, and disulfide bonds, respectively.

Determination of Fourier‐transform infrared (FTIR) spectroscopy

Fourier‐transform infrared (FTIR) spectra were obtained using a Frontier spectrometer (PerkinElmer, Waltham, MA, USA) to identify potential shifts in characteristic chemical bonds. Prior to analysis, samples were homogenized with potassium bromide (KBr) powder at a 1:100 mass ratio and compressed into translucent pellets. Following this preparation, interferograms were acquired over the spectral range of 4000–400 cm⁻¹.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE)

The protein profile analysis was performed according to the method described by Song et al. ¹⁸ with minor modifications. All samples were lyophilized, resuspended in loading buffer (1 mg mL⁻¹), and denatured by boiling for 10 min. The samples were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) using a 5% stacking gel and a 10% separating gel. Electrophoresis was conducted at 15 mA for stacking and 30 mA for separation, each lasting approximately 1 h, employing the MiniUnit AE‐8135 system (ATTO Corp., Tokyo, Japan). Subsequent to electrophoresis, the gel was stained with Coomassie Brilliant Blue and de‐stained following standard protocols.

Statistical analysis

All measurements were performed using at least three experimental replicates, except for texture analysis, which utilized a minimum of six replicates per sample. Data values are expressed as the mean ± standard deviation (SD). Statistical analyses were conducted using one‐way analysis of variance (ANOVA) followed by Duncan's multiple‐range test in IBM SPSS Statistics (version 26.0), with statistical significance defined as P < 0.05. Graphical representations were created using Origin 2021.

RESULTS AND DISCUSSIONS

Mechanical properties of low‐salt EWP–SN composite surimi gels

Mechanical performance serves as a critical parameter for evaluating surimi gel quality. As shown in Fig. 1(A), the incorporation of EWP into low‐salt (1% NaCl) SN surimi significantly increased breaking force (P < 0.05). Notably, the breaking force of low‐salt EWP–SN composite surimi gels reached its peak at a 5% substitution level, exhibiting a 58.9% increase compared to the LC group and a statistically significant 22.75% improvement over the HC group (P < 0.05). While further increases in EWP substitution beyond 5% reduced breaking force, these values remained superior to those of the LC group. A comparable bell‐shaped pattern was observed for hardness, cohesiveness, and chewiness (Fig. 1(B)–(D)), with optimal improvements consistently occurring at the 5% EWP substitution level. Specifically, the low‐salt EWP–SN composite surimi gels exhibited enhancements of 60.18% in hardness, 17.74% in cohesiveness, and 100.19% in chewiness relative to the LC group (P < 0.05). From a fracture mechanics perspective, the substantial increase in chewiness was primarily driven by the enhanced hardness. Crucially, because this was accompanied by stable/increased cohesiveness, the gel did not become brittle or undergo irreversible plastic deformation under compression. Instead, the EWP‐reinforced network exhibited an enhanced elastic energy storage capacity, shifting toward a more rubbery and resilient characteristic that effectively restricts premature crack propagation.

Figure 1.

Mechanical properties of low‐salt EWP–SN (egg white protein–Scomberomorus niphonius) composite surimi gels. (A) Breaking force; (B) hardness; (C) cohesiveness; (D) chewiness. Different letters indicated significant differences (P < 0.05). (LC, low‐salt control; HC, high‐salt control.)

Under low‐salt conditions, limited extraction and dispersibility of myofibrillar proteins typically results in the formation of a weak and discontinuous gel network, ¹⁹ which consequently leads to suboptimal mechanical properties (Fig. 1). The observed improvements at moderate EWP levels (≤ 5%) indicate that EWP contributes to the reinforcement of the network, likely functioning as an active co‐gelling agent and a microstructural ‘filler’ that improves network continuity. ²⁰ During thermal processing, EWP undergoes unfolding, exposing reactive functional groups that facilitate crosslinking with myofibrillar proteins and strengthen the continuous gel phase. ¹³ Considering that 1% NaCl primarily induces the swelling rather than complete solubilization of myofibrillar proteins, EWP is hypothesized to exert a dual role: it co‐assembles with the limited fraction of solubilized myosin, while its relatively small molecular size allows it to penetrate the interstitial spaces of swollen, un‐solubilized myofibrils, effectively bridging these fragmented structural domains. However, when the EWP substitution exceeds 5%, the mechanical properties deteriorate. This decline is attributed to thermodynamic incompatibility between the two proteins at higher concentrations. At supraoptimal concentrations, EWP tends to self‐aggregate preferentially rather than interacting with myofibrillar proteins, thereby introducing structural defects that disrupt the integrity of the primary actomyosin network and reduce fracture resistance. ²¹ Comparable enhancement effects have been documented in EWP–grass carp composite surimi gel systems. ¹³ These findings demonstrate that 5% EWP substitution achieves optimal improvement in the mechanical properties of low‐salt SN surimi gels.

Moisture properties of low‐salt EWP–SN composite surimi gels

Moisture retention plays a pivotal role in determining surimi gel quality and sensory attributes. Cooking loss, defined as water released during thermal gelation, contrasts with WHC, which reflects the gel network's ability to retain water under external pressure. As presented in Fig. 2(A), the LC group exhibited the highest cooking loss among all samples. EWP substitution significantly reduced cooking loss across all substitution levels (P < 0.05), achieving the lowest value at 5% EWP incorporation, reducing from 7.56% (LC group) to 4.03%. This reduction exceeded that of the HC group. Consistent with this, the incorporation of EWP notably enhanced WHC (Fig. 2(B)). The peak WHC was attained at 5% substitution, increasing from 60.26% (LC group) to 73.03%.

Figure 2.

Moisture characteristics of EWP–SN (egg white protein–Scomberomorus niphonius) composite surimi gels. (A) Cooking loss; (B) water‐holding capacity (WHC); (C) T ₂ relaxation times; (D) pseudo‐color picture of water protons. The color bars ranged from red (high hydrogen proton density) to blue (low hydrogen proton density). Different letters indicated significant differences (P < 0.05). (LC, low‐salt control; HC, high‐salt control.)

The simultaneous reduction in cooking loss and enhancement of WHC suggest improved water entrapment within the composite gel matrices. During thermal processing, EWP functions as a water‐binding agent that strengthens interactions with water molecules, thereby mitigating cooking loss in surimi gels. ²² Zhang et al. ²³ similarly demonstrated that EWP incorporation effectively reduced cooking loss in clam (Meretrix meretrix) surimi gels. Furthermore, EWP is frequently used as a network enhancer, ²¹ promoting the formation of a more uniform and dense gel network structure. This structural improvement enables greater retention of water molecules, consequently enhancing WHC in low‐salt EWP–SN composite surimi gels (Fig. 2(B)). Corroborative findings have been reported in EWP–blue round scad surimi composite gels. ¹² This structural modification provides more effective capillary confinement while restricting water migration pathways during thermal processing and centrifugation.

Water mobility and distribution in low‐salt EWP–SN composite surimi gels

LF‐NMR was employed to characterize water mobility within the gel matrices. The T ₂ relaxation spectra (Fig. 2(C) and Table 1) displayed three distinct peaks corresponding to different water states: T ₂₁ (0–10 ms) for bound water, T ₂₂ (10–100 ms) for immobilized water, and T ₂₃ (1000 ms) for free water. ²⁴ As illustrated in Fig. 2(C), T ₂₂ constituted the predominant water population across all samples. Following EWP incorporation, T ₂₂ shifted marginally towards shorter relaxation times (from 47.68 ms to 44.49 ms) compared to the LC group, indicating more constrained water mobility. Additionally, the T ₂₁ relaxation time decreased from 0.79 ms to 0.45 ms. For quantitative analysis, the relative peak area percentage of T ₂ was calculated. As demonstrated in Table 1, the relative proportion of bound water (A ₂₁) increased from 1.28% (LC group) to 4.44% at 5% EWP substitution level, accompanied by corresponding reductions in both immobilized and free water fractions. This trend correlates well with the enhanced WHC and decreased cooking loss. To complement these findings, spatial moisture distribution patterns were visualized using pseudo‐color proton‐density maps generated from high‐resolution, non‐destructive magnetic resonance imaging (MRI). (Fig. 2(D)). Higher hydrogen proton concentrations produce brighter signals and correspondingly redder pseudo‐color representations. ²⁵ Compared with the LC group, EWP‐containing gels exhibited higher, more uniform signal intensities, with the 5% substitution group showing the most pronounced enhancement. These imaging data indicate an enhanced WHC and a macroscopically uniform moisture distribution following EWP incorporation. Crucially, these uniform signal intensities indicate an optimized macroscopic water partition. During thermal volumetric contraction, myofibrillar gels naturally tend to expel water (micro‐syneresis). The EWP‐reinforced compact honeycomb microstructure effectively resisted this contraction via enhanced capillary forces, mitigating micro‐syneresis and thereby immobilizing water uniformly throughout the matrix.

Table 1.

Water distribution of low‐salt EWP–SN (egg white protein–Scomberomorus niphonius) composite surimi gels

Samples	Peak area ratio (%)
	Bound water	Immobile water	Free water
LC	1.28 ± 0.03f	97.29 ± 0.01a	1.43 ± 0.02a
HC	1.74 ± 0.03e	97.08 ± 0.17a	1.17 ± 0.14ab
1%	2.82 ± 0.05d	96.22 ± 0.16b	0.96 ± 0.11bc
3%	3.86 ± 0.02b	95.89 ± 0.14bc	0.62 ± 0.13cd
5%	4.44 ± 0.32a	95.51 ± 0.28cd	0.36 ± 0.05d
7%	3.92 ± 0.15b	95.20 ± 0.05d	0.87 ± 0.10bc
9%	3.42 ± 0.16c	95.20 ± 0.55d	0.69 ± 0.39cd

Note: Peak area ratios were obtained from normalized relative integration of the T ₂ relaxation peaks. Different letters in the same column indicate a significant difference. P < 0.05. LC, low‐salt control; HC, high‐salt control.

The increase in bound water and the shortened T ₂₁ relaxation time observed in low‐salt SN surimi gels could be attributed to the interactions between water molecules and the polar groups of EWP. ²⁶ The reduced T ₂ relaxation times indicate restricted water mobility, suggesting enhanced protein–water interactions. This phenomenon was similarly reported by Huang et al. ²¹ who demonstrated that increasing EWP concentration elevated the bound water content while decreasing free water content in eel myofibrillar protein gels. Additionally, EWP may facilitate the unfolding of myofibrillar proteins, exposing internal hydrophobic sulfhydryl groups and thereby strengthening protein–water binding capacity. Supporting this mechanism, Luo et al. ²⁷ found that higher proportions correlated with stronger water–protein interactions, resulting in increased hydrogen proton counts within surimi gel matrices. These findings demonstrate that the substitution of EWP promotes the transformation of immobile water and free water into bound water fractions in low‐salt SN surimi gels, enhancing water–protein interactions under sodium‐reduced conditions.

Rheological properties of low‐salt EWP–SN composite surimi gels

Frequency sweep tests offer valuable insights into the viscoelastic properties of surimi gels. ²³ In the mechanical spectra derived from these tests, the G' and G" correspond to the elastic and viscosity components of the gel matrix, respectively. ²⁸ As presented in Fig. 3(A), all experimental groups exhibited G' values consistently higher than G", which confirms their predominantly elastic, solid‐like rheological behavior. ²⁹ Notably, the incorporation of EWP increased both G' and G″ compared to the LC group, indicating reinforcement of the gel network elasticity and viscosity. The 5% EWP substitution showed optimal reinforcement effects, exhibiting 0.61‐fold and 3.37‐fold increases in G' and G″ values at 1 Hz relative to the LC group, respectively. However, further increases in EWP substitution beyond this critical threshold led to lower moduli for both parameters.

Figure 3.

Rheological behavior of low‐salt EWP–SN (egg white protein–Scomberomorus niphonius) composite surimi gels. (A) Frequency sweep profiles; (B) temperature ramp profile. (LC, low‐salt control; HC, high‐salt control.)

The observed increase in G' and G" is likely attributable to strengthened intermolecular interactions and crosslinking between EWP and myofibrillar proteins, which increase the rigidity of the composite network and thereby elevate both viscoelastic moduli. A similar reinforcement effect was reported by Yi et al. ³⁰ who found that EWP addition significantly elevated the G' of chicken myofibrillar protein gel. Specifically, the incorporation of EWP facilitated the formation of a more continuous gel matrix, resulting in concurrent increases in viscoelastic parameters. In this study, moderate EWP substitution enhanced the formation of a more cohesive viscoelastic network, consequently improving the rheological properties and corresponding mechanical performance of the LC group (Fig. 1; Mechanical properties of low‐salt EWP–SN composite surimi gels section). In contrast, when the EWP substitution rate exceeded 5%, G' decreased, potentially because the relatively high solubility of EWP increased system fluidity and disrupted effective network connectivity under low‐salt conditions. ³¹ This trend aligns with the mechanical property results (Mechanical properties of low‐salt EWP–SN composite surimi gels section), indicating that moderate EWP substitution reinforces the viscoelastic network. In contrast, excessive EWP compromises overall network integrity in low‐salt SN surimi gels.

Gelation process of low‐salt EWP–SN composite surimi gels

The temperature–sweep tests (Fig. 3(B)) revealed a characteristic thermal gelation pattern typical of myofibrillar protein systems. The G' value initially decreased to a minimum value at approximately 57 °C, followed by a significant increase with further heating, indicating the onset of protein denaturation, aggregation, and network formation. ³² This reduction may be further exacerbated by the activation of endogenous proteolytic enzymes, which partially degrade myofibrillar proteins prior to thermal inactivation. ³³ Additionally, the biochemical presence of ovomucoid (~11% of EWP), a known serine protease inhibitor, likely suppressed these endogenous proteolytic enzymes. This mitigated the enzymatic degradation of the myosin network, synergistically contributing to the subsequent elevation of G′. During continued heating, G' increased and plateaued at 90 °C, demonstrating that SN myofibrillar proteins formed a relatively stable gel network. This transition is likely driven by heat‐induced actomyosin aggregation and disulfide bond‐mediated crosslinking, which collectively stabilize the gel structure. ³⁴ The LC group exhibited the lowest G' values throughout the heating process, consistent with limited myofibrillar protein solubilization and, consequently, restricted network development under low ionic strength conditions. As the EWP substitution levels increased, G' increased progressively and reached a maximum at 5% substitution, indicating more effective network formation during thermal gelation. Consistently, the 5% EWP sample exhibited the highest final G' value at 90 °C, correlating with its superior mechanical performance (Mechanical properties of low‐salt EWP–SN composite surimi gels section). However, when the substitution exceeded 5%, the G' of the samples decreased, implying that excessive EWP may interfere with continuous SN‐dominated network formation under low‐salt conditions. The elevated pre‐heating G' observed with EWP incorporation may be attributed to enhanced viscoelastic network formation and increased system rigidity contributed by EWP. ³⁵ The continuous G' increase during the gelation phase can be attributed to a ‘heteroprotein solubilization effect.’

Furthermore, the HC group exhibited a higher G' value than the LC group, likely attributable to enhanced myofibrillar protein extraction/solubilization and improved gel network formation at elevated NaCl concentrations. ³⁶ It is postulated that EWP‐facilitated solubilization of SN myofibrillar proteins through heterologous associations, thereby promoting denser network formation analogous to observations in scallop–soy protein systems. ³⁷ Additionally, thermal processing promoted sulfhydryl group exposure, enabling disulfide bond formation between myofibrillar proteins and EWP. These covalent crosslinks reinforce the gel matrix structure, which correspondingly increases G'. ³⁸ However, a distinct structural threshold was observed, with G' peaking at 5% substitution before declining upon further increases in EWP concentration beyond this critical level. These findings indicate that while moderate EWP incorporation functions as an effective crosslinking agent to strengthen the network architecture, excessive globular protein content may introduce steric hindrance that disrupts the continuous SN myofibrillar network framework, ultimately compromising overall structural integrity.

Appearance and microstructure of low‐salt EWP–SN composite surimi gels

Macroscopic appearance serves as a direct indicator of the integrity and continuity of the gel network. To evaluate the gelation capacity of EWP under the designated thermal treatment, EWP aqueous dispersions at formulation‐relevant concentrations were prepared and heated in parallel (Fig. 4(A)). The results demonstrated that EWP dispersions exhibited weak gelation behavior at a concentration corresponding to the 5% substitution level, while forming self‐supporting gels at higher concentrations, indicating self‐association propensity with increased substitution levels. In EWP–SN composite surimi gels, EWP incorporation significantly altered both visual and microstructural characteristics. The LC group exhibited a coarse cross‐sectional morphology characterized by irregular, large pores (Fig. 4(B)), typical of surimi processed under suboptimal ionic‐strength conditions, in which actomyosin solubilization is restricted. In contrast, the HC group exhibited a finer matrix, confirming the role of salt in facilitating protein unfolding. As EWP substitution progressively increased from 1% to 5%, the gel surface became notably smoother, accompanied by a microstructural transition to a more compact, honeycomb‐like network with a uniform pore size. Notably, the 5% EWP substitution sample exhibited the most densely packed and integrated three‐dimensional architecture. This structural refinement corroborates the improved mechanical properties and WHC observed in Mechanical properties of low‐salt EWP–SN composite surimi gels section and Water mobility and distribution in low‐salt EWP–SN composite surimi gels section, suggesting that moderate EWP effectively occupies interstitial voids within the loose SN protein matrix. However, excessive substitution exceeding 5% led to the reappearance of larger, irregular voids and reduced network uniformity, indicating compromised network continuity.

Figure 4.

(A) Appearances of egg white protein (EWP) solutions and low‐salt EWP–SN (egg white protein–Scomberomorus niphonius) composite surimi gels; (B) cryo‐scanning electron microscopy (cryo‐SEM) images (×10 000) of low‐salt EWP–SN composite surimi gels. (LC, low‐salt control; HC, high‐salt control.)

To quantitatively evaluate the microstructural evolution, pore metrics were analyzed using AngioTool64 (Table 2). The LC group exhibited a loose network with a large mean pore size of 1.04 ± 0.44 μm and a high porosity of 55.67%. Upon adding EWP, the network became progressively denser. The 5% EWP group achieved the most compact structure, characterized by the smallest mean pore size (0.25 ± 0.16 μm) and the lowest porosity (34.85%). This quantitative reduction in pore dimension significantly increases the capillary forces within the gel matrix. However, when EWP substitution was increased to 9%, the mean pore size rebounded to 0.65 ± 0.62 μm and porosity increased to 57.28%. This ‘U‐shaped’ trend in porosity and pore size quantitatively supports the hypothesis of thermodynamic incompatibility. At moderate levels (< 5%), EWP acts as a filler to reduce void volume; however, at excessive levels (> 5%), the segregation of EWP aggregates creates large interstitial spaces, returning the overall porosity to levels comparable to the control.

Table 2.

Quantitative microstructural parameters (mean pore size and porosity) of low‐salt EWP–SN (egg white protein–Scomberomorus niphonius) composite surimi gels derived from cryo‐scanning electron microscopy (cryo‐SEM) image analysis

Group	Mean pore size (μm)	Porosity (%)
LC	1.04 ± 0.44a	55.67 ± 5.04bc
HC	0.80 ± 0.34b	54.49 ± 3.76c
1%	0.47 ± 0.30cd	51.01 ± 1.30d
3%	0.46 ± 0.36cd	60.55 ± 2.42a
5%	0.25 ± 0.16d	34.85 ± 3.82e
7%	0.35 ± 0.29d	36.03 ± 3.69e
9%	0.65 ± 0.62bc	57.28 ± 0.89b

Note: Different letters in the same column indicate a significant difference. P < 0.05. LC, low‐salt control; HC, high‐salt control.

These structural modifications can be mechanistically explained through the phase behavior of the binary protein system. At moderate substitution levels below its strong self‐gelling threshold (< 5%), EWP can function as an interacting filler that associates with myofibrillar proteins and bridges structural discontinuities within the SN matrix, thereby forming a denser, more coherent network. ⁶ However, at elevated substitution levels exceeding this critical concentration (> 5%), thermodynamic incompatibility between coexisting protein phases becomes evident. When EWP concentration surpasses its self‐gelling threshold, the composite microstructure is determined by the relative phase behavior of the protein components. This phase behavior is predominantly driven by ovalbumin, the major constituent (~54%) of EWP. Excessive EWP tends to undergo self‐aggregation and phase‐separate from the SN‐rich matrix rather than achieve molecular integration. ³⁹ These phase‐separated domains introduce structural heterogeneities that disrupt network continuity, resulting in measurable reductions in gel mechanical properties. This phase separation‐induced mechanical degradation mechanism has been similarly documented in eel myofibrillar protein gel systems. ²¹

Intermolecular interaction of low‐salt EWP–SN composite surimi gels

The formation of gel networks depends on a delicate balance of multiple intermolecular interactions between biological molecules. ² To elucidate the stabilization mechanisms governing composite gel structures, protein solubility in selective dissociating media was measured as an operational proxy for specific molecular interactions. ⁴⁰ As shown in Fig. 5(A), disulfide bonds were identified as the predominant contributors to the gel network architecture. With increasing EWP substitution levels, the proportion of protein dissolved in β‐mercaptoethanol‐containing medium (indicating disulfide bond disruption) progressively increased, reaching maximum values at 5% substitution (1.31‐fold relative to LC group), while the hydrophobically associated fraction correspondingly decreased. This observation was further supported by the significant increase in disulfide bond‐related indices with EWP incorporation, which peaked at 5% substitution, thereby results are consistent with the hypothesis that disulfide bonds represent the dominant covalent crosslinking mechanism, reinforcing the gel network in this system. Additionally, EWP substitution elevated the insoluble protein fraction, which may be attributed to enhanced formation of non‐dissociable crosslinks such as ε‐(γ‐glutamyl)lysine isopeptide bonds when endogenous transglutaminase activity occurs, particularly given the high lysine and glutamine contents in EWP. ⁴¹

Figure 5.

(A) Intermolecular interaction; (B) Fourier‐transform infrared (FTIR) spectroscopy curves; (C) protein secondary structure; (D) electrophoresis images of low‐salt EWP–SN (egg white protein–Scomberomorus niphonius) composite surimi gels. Different uppercase letters within the same sample indicated significant differences. Different lowercase letters within the same intermolecular forces indicated significant differences (P < 0.05). (LC, low‐salt control; HC, high‐salt control; MHC, myosin heavy chain; ‘actin + ovalbumin’ indicates the co‐migration of surimi actin and egg white ovalbumin.)

EWP exhibits a high content of sulfur‐containing amino acids, which provide reactive sulfhydryl/disulfide groups that can undergo conversion during thermal gelation. ⁴² Upon thermal treatment, these reactive groups can participate in sulfhydryl oxidation and sulfhydryl–disulfide interchange reactions with cysteine residues in SN myofibrillar proteins (e.g., myosin), forming intermolecular covalent bridges that reinforce the gel network. This mechanism aligns with previous observations demonstrating EWP‐enhanced disulfide bond formation in chicken myofibrillar protein gels, presumably through heat‐induced sulfhydryl oxidation. ³¹ Moreover, while conventional solubility assays cannot definitively differentiate between intra‐EWP and EWP–myosin disulfide bonds, the increased prevalence of disulfide‐bond‐mediated interactions combined with the preservation of the myosin‐dominated rheological profile (Fig. 3(B)) strongly suggests heteroprotein co‐construction/co‐assembly. If homologous interactions completely dominated at the 5% level, EWP would likely form isolated interpenetrating clusters, failing to produce the uniform, densified network observed in cryo‐SEM (Fig. 4(B)). This deduction is supported by similar demonstrated co‐assembly between scallop muscle proteins and soy proteins, ³⁷ which lends support to our hypothesis that EWP can co‐assemble with SN proteins (below the pronounced EWP self‐gelling regime) to facilitate covalent crosslinking. However, excessive crosslinking or the inherent hydrophilicity of EWP might obscure surface hydrophobic patches on myofibrillar proteins. Furthermore, at elevated substitution levels, excessive exogenous protein incorporation could introduce steric hindrance, ⁴³ thereby inhibiting exposure of buried hydrophobic domains that typically drive non‐specific aggregation. These findings collectively indicate that optimal EWP substitution promotes a shift from predominantly physical aggregation toward enhanced covalent crosslinking, yielding a more compact and mechanically stable network architecture.

FTIR spectra of low‐salt EWP–SN composite surimi gels

FTIR spectroscopy was conducted to assess molecular‐level changes, including protein conformation and microenvironmental shifts, in the composite gel systems. ⁴⁴ As presented in Fig. 5(B), the broad absorption band spanning 3000–3600 cm⁻¹, attributed to O–H and N–H stretching vibrations, ²⁹ demonstrated significant spectral migration. With increasing EWP substitution from 1% to 5%, the characteristic peak underwent a blue shift from 3417 cm⁻¹ to 3433 cm⁻¹. A shift to higher‐frequency suggest a tendency toward altered hydrogen‐bonding interactions or altered hydration environments within the protein network. ⁴⁵ Notably, further elevation of the substitution level to 7% resulted in partial reversion of the peak position to 3427 cm⁻¹, suggesting a complex modulation of hydrogen‐bonding dynamics at excessive EWP concentrations. Concurrently, distinct spectral shifts were observed in the amide I (1600–1700 cm⁻¹) and amide II (1530–1550 cm⁻¹) regions, which serve as key indicators for monitoring protein backbone conformational changes and intermolecular hydrogen‐bonding networks. ⁴⁶ To quantitatively assess these structural transformations, deconvolution analysis of the amide I band was performed to determine secondary‐structure compositions (Fig. 5(C)). With increasing EWP substitution, α‐helices decreased, whereas β‐sheets and β‐turns increased, indicating partial unfolding and a transition toward β‐structures during gelation. Mechanistically, this conformational conversion likely proceeded through partially unfolded intermediate states, in which helix melting generated more disordered chain segments before their rearrangement into intramolecular and/or intermolecular β‐structures during thermal aggregation. Notably, at a 5% substitution level, β‐sheets and β‐turns increased by 9.15% and 23.28%, respectively, relative to the LC group. Such an α‐helix‐to‐β‐structure transition has been associated with altered hydrogen‐bonding patterns. ⁴⁷ It has also been observed in EWP‐modified blue round scad (Decapterus maruadsi) protein gels, ¹² which supports the notion that EWP promotes conformational reorganization of SN proteins and modulates the interaction landscape of the composite gel network.

SDS‐PAGE of SN surimi gels of low‐salt EWP–SN composite surimi gels

SDS‐PAGE was employed to characterize protein profiles in heat‐induced gels (Fig. 5(D)). The profiles of SN myofibrillar proteins dominated by characteristic bands corresponding to myosin heavy chain (MHC, 245 kDa), paramyosin (100 kDa) and actin (48 kDa), identifying them as the primary structural components of the surimi gel network. ² Consequently, MHC and actin were identified as the primary contributors to the SN surimi gel matrix. In the EWP‐containing samples, new protein bands characteristic of egg white appeared. Notably, a distinct band corresponding to ovomucoid (~28 kDa) was observed. Furthermore, the band intensity in the 42–45 kDa region increased significantly. This is attributed to the presence of ovalbumin (the most abundant protein in EWP, ~45 kDa), which co‐migrated with the actin band (~42 kDa), resulting in a merged ‘actin + ovalbumin’ band. Ovotransferrin (~70 kDa) was also observed, confirming the effective incorporation of EWP into the composite matrix. With increasing EWP substitution levels, the fundamental SN myofibrillar banding pattern remained intact, with no detectable low‐molecular‐weight degradation products, indicating minimal proteolytic activity under these experimental conditions. Notably, densitometric analysis revealed progressive increases in MHC band intensity from 11.23% to 14.63%, indicating improved extractability of cytoskeletal proteins. Additionally, the band intensity at the actin + ovalbumin position increased markedly from 13.42% to 16.43%. It is important to note that this increase is primarily driven by the superposition of the exogenous ovalbumin on the endogenous actin band, confirming the successful incorporation of EWP into the gel network. However, no further improvement was observed beyond the 5% substitution level (Table 3). It is important to note that SDS‐PAGE is performed under denaturing and reducing conditions. Therefore, these intensity increases do not represent native solubility in the low‐salt buffer, but rather enhanced extractability from the gel matrix. In the LC group, the low ionic strength likely promotes the formation of tight, non‐specific hydrophobic aggregates that are difficult to resolubilize even in SDS loading buffer. The presence of EWP appears to interfere with this excessive aggregation, possibly in a chaperone‐like or interfacial‐protective manner, thereby reducing hydrophobic collapse of myofibrillar proteins during heating and preserving a larger fraction of MHC in an SDS‐extractable state. Thus, the observed band increase should be interpreted as improved SDS‐extractable recoverability rather than direct evidence of enhanced native solubility.

Table 3.

Myosin heavy chain, paramyosin and actin bands intensity proportion of low‐salt EWP–SN (egg white protein–Scomberomorus niphonius) composite surimi gels

Samples	Band intensity proportion (%)
	Myosin heavy chain	Paramyosin	Actin + ovalbumin
LC	11.23 ± 0.04b	3.28 ± 0.02c	13.42 ± 0.03b
HC	11.42 ± 0.02b	3.30 ± 0.01c	13.53 ± 0.02b
1%	13.49 ± 0.02ab	3.41 ± 0.02b	16.22 ± 0.04a
3%	14.56 ± 0.07a	3.51 ± 0.00a	16.24 ± 0.03a
5%	14.63 ± 0.02a	3.55 ± 0.01a	16.43 ± 0.02a
7%	14.34 ± 0.04a	3.44 ± 0.01b	16.12 ± 0.02a
9%	14.44 ± 0.05a	3.40 ± 0.01b	15.93 ± 0.03a

Note: Different letters in the same column indicate significant difference (P < 0.05). LC, low‐salt control; HC, high‐salt control; actin + ovalbumin indicates the co‐migration of surimi actin and egg white ovalbumin.

Based on these observations, Fig. 6 demonstrates that moderate EWP substitution (1–5%) enhances SN gelation by reinforcing protein–protein interactions, improving extractability and dispersion of SN proteins under low‐salt conditions, and promoting covalent network stabilization. The incorporation of EWP at ≤ 5% concentration addresses functional limitations via dual pathways. First, the heteroprotein solubilization effect increases SN protein solubility, thereby making additional actomyosin available for gel formation. Second, heat‐induced sulfhydryl oxidation enables the formation of heterologous disulfide bonds between EWP and SN proteins, creating crosslinking bridges that reinforce the composite network. However, a critical threshold exists at approximately 5% substitution. Exceeding this optimal level (> 5%) induces thermodynamic incompatibility between the self‐gelling EWP‐rich phase and the SN matrix, resulting in microphase separation. These self‐aggregated EWP domains exhibit weak interfacial adhesion with the myofibrillar network, generating structural discontinuities that ultimately compromise gel integrity and mechanical properties.

Figure 6.

Schematic representation of the proposed gelation mechanism of low‐salt EWP–SN (egg white protein–Scomberomorus niphonius) composite surimi gels. (LC, low‐salt control; HC, high‐salt control.)

Quantitative structure–function relationship analysis

To elucidate the underlying mechanism, Pearson correlation analysis was performed to quantify the relationships between multiscale structural parameters and macroscopic functional properties, with the global correlation landscape visualized in a heatmap (Fig. 7). As illustrated in Fig. 7, macroscopic strengthening was strongly driven by molecular‐level interactions. The breaking force exhibited a significant positive correlation with disulfide bond content (r = 0.993, P < 0.01) and β‐sheet content (r = 0.999, P < 0.01). This indicates that the heat‐induced exposure of sulfhydryl groups and subsequent disulfide crosslinking are the primary molecular drivers for gel strength. Furthermore, microstructural refinement played a decisive role in moisture retention. WHC was found to be negatively correlated with the mean pore size (r = −0.921, P < 0.01) derived from quantitative SEM analysis. This statistical evidence supports the capillary condensation theory: the addition of 5% EWP refined the pore structure (to 0.25 μm), thereby increasing capillary forces. Additionally, a perfect positive correlation was observed between WHC and the bound water proportion (A ₂₁) (r = 1.000, P < 0.01), attributing enhanced water retention to both physical entrapment and molecular binding. However, excessive EWP substitution (> 5%) disrupted these correlations. The recurrence of larger voids and increased porosity at 9% substitution (Appearance and microstructure of low‐salt EWP–SN composite surimi gels section), which positively correlated with the decline in gel strength, quantitatively supports the ‘micro‐phase separation’ mechanism. The heatmap (Fig. 7) clearly distinguishes two clusters of variables: strictly structural metrics (pore size, porosity) and chemical interaction metrics (disulfide bonds), both of which converge optimally at the 5% substitution level.

Figure 7.

Pearson correlation heatmap summarizing the relationships between structural parameters (secondary structure, intermolecular forces, microstructure metrics) and functional properties (texture, water‐holding capacity (WHC), water distribution) of low‐salt EWP‐SN (egg white protein–Scomberomorus niphonius) composite surimi gels. Red indicates positive correlation, while blue indicates negative correlation. (*P < 0.05, **P < 0.01).

CONCLUSION

This study elucidates the mechanistic role of EWP in modulating the gelation behavior and microstructural evolution of low‐salt SN surimi. Optimal functional performance was observed at 5% protein‐equivalent substitution, yielding maximal breaking force and enhanced water retention, as evidenced by decreased cooking loss and improved WHC. Multi‐scale characterization demonstrated that EWP functioned as an active filler and crosslinking agent at substitution levels ≤ 5%, promoting myofibrillar protein unfolding and intermolecular disulfide bond formation, which is the primary driver of gel strengthening, to counteract weakened protein interactions inherent to low ionic strength environments. This molecular intervention transformed the gel matrix from a coarse‐porous architecture to a compact honeycomb morphology, reducing the average pore size to one‐third of the control value and thereby restricting water mobility. Critically, the quantitative analysis of pore size explains the water retention data, confirming that microstructural refinement (smaller pores) directly dictates the capillary holding of water. Conversely, excessive substitution (> 5%) caused thermodynamic incompatibility and phase separation between the EWP aggregates and the myofibrillar matrix. These phase‐separated domains acted as structural defects, disrupting the continuity of the gel network and consequently diminishing its mechanical integrity. These findings establish a theoretical framework for modulating heteroprotein interactions in low‐salt food matrices and present a viable strategy for engineering salt‐reduced surimi products with customized textural attributes.

CONFLICT OF 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 article.

AUTHOR CONTRIBUTIONS

Yi‐Xiao Tang: writing – original draft, methodology, validation, investigation, formal analysis, data curation, conceptualization; Lin‐Da Zhang: validation, methodology, investigation, formal analysis, data curation, conceptualization; Xinyan Man: investigation; Yin‐Yin Lv: investigation; Jia‐Nan Yan: investigation; Ce Wang: investigation; Bin Lai: conceptualization, methodology, writing – review and editing, supervision, funding acquisition; Hai‐Tao Wu: conceptualization, resources, supervision, project administration, funding acquisition.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.