Mechanistic insight into the regulation of gelatin hydrogels structure and gel-forming properties by reactive species in plasma-activated water

Abstract

This study established a refined plasma activated water (PAW) system to distinguish the effects of acidification and H₂O₂ from the broader effects of PAW-derived reactive species on gelatin gelation. Acidification altered surface charge and resulting in an increased in β-turn content (38.90%) and ionic bonding (15.23%). H₂O₂ treatment induced oxidative modification, leading to sulfhydryl oxidation, increased hydrophobic interactions and decreased gelation temperature, but partially disrupted triple-helix-based crosslinking. Notably, PAW exerted unique enhanced effects, significantly enhancing gelling properties by strengthening hydrogen bonding and lowering the eutectic point. Molecular docking further suggested that PAW-derived reactive species promoted hydrogen bonding and hydrophobic interactions with gelatin. These improvements highlight the potential of PAW-modified gelatin for functional gelled foods and gelatin-based packaging requiring enhanced structural and moisture stability, although validation in complex food matrices and practical processing systems remains necessary.

Graphical abstract

Highlights

• •Gelatin was modified by distinct plasma-generated reactive species.
• •pH modulation improved gel properties by altering surface charge distribution.
• •H₂O₂ enhanced hydrophobic interactions via oxidative modification of gelatin.
• •PAW treatment exerted synergistic effects beyond pH and H₂O₂.
• •PAW₁₀ treatment enhanced gelatin gelation and structural properties.

1. Introduction

Hydrogels are three-dimensional polymer networks capable of absorbing large amounts of water and are widely used in food, cosmetics, biomedical engineering, tissue scaffolds, and drug delivery (Ge et al., 2018). They require high mechanical strength, flexibility, and reversible deformation under stress. However, conventional chemically cross-linked hydrogels are often brittle because of their heterogeneous cross-linked structures (Hong et al., 2015). Consequently, natural polymers have attracted increasing attention owing to their excellent energy dissipation capacity and mechanical performance and have been widely employed to fabricate high-strength hydrogels (Liu et al., 2024). Gelatin, one of the most widely used natural hydrogel materials, is obtained from the skin and connective tissues of mammals and aquatic animals, including fish (Fu et al., 2025). During cooling, disordered gelatin chains undergo a helix-to-coil transition and partially reassemble into collagen-like triple-helix that served as junction zones stabilized by intermolecular hydrogen bonds, thereby forming a three-dimensional gel network (Liu et al., 2026). Owing to its biocompatibility, non-immunogenicity, and biodegradability, gelatin hydrogels have been extensively applied in functional food gels, edible films, and biodegradable food packaging. These applications require excellent gel strength, water holding capacity (WHC), and structural stability, highlighting the importance of developing effective strategies to regulate gelatin gelation (Alipal et al., 2021; Parvez, Gat, Chavan, & Annapure, 2025).

Various physical modification strategies have been developed to regulate gelatin gelation. High-pressure micro-fluidization converts ordered structures into disordered conformations, thereby enhancing functional properties (Sha et al., 2018). Microwave irradiation induces protein aggregation, degradation, and cross-linking through free radicals, leading to structural rearrangement and altered hydrophobicity (Feng et al., 2021). However, these techniques generally require specialized equipment and high energy consumption. As a residue-free, low-temperature, and environmentally friendly technology, cold plasma has attracted considerable attention in food processing (Li et al., 2024). Plasma-treated gelatin has exhibited enhanced tensile strength and mechanical properties, demonstrating its potential in packaging films (Parvez et al., 2025). Plasma activated water (PAW), produced by exposing water to cold plasma, contains a variety of reactive oxygen and nitrogen species (RONS), including reactive oxygen species (ROS, H₂O₂, ·OH, and O₃) and reactive nitrogen species (RNS, NO₂⁻, NO₃⁻, and N₂O₅), which collectively induce acidification and oxidative modification (Wang et al., 2025). Previous studies have demonstrated that PAW can effectively regulate protein structure through the combined action of these reactive species (Bu, Nayak, Bruggeman, Annor, & Ismail, 2022; Wang, Zeng, Deng, Wang, & Zhang, 2025). Our previous systematically compared PAW prepared for 5, 10, 20 min and identified PAW₁₀ as the optimal treatment for improving gelatin viscosity and gel network uniformity (Gao et al., 2025). Although PAW contains numerous reactive oxygen and nitrogen species, reproducing the complete reactive environment of PAW under controlled experimental conditions remains challenging because the concentrations and reactivities of individual species are dynamic and interdependent. Therefore, acidification and oxidation systems reproducing the major physicochemical characteristics of PAW provide a practical approach for evaluating whether the effects of PAW can be explained by acidification and H₂O₂ alone.

Based on this rationale, the scientific hypothesis of this study was that the enhanced gelatin gelation induced by PAW could not be fully explained solely by acidification and H₂O₂-induced oxidation alone. To test this hypothesis, acidification, H₂O₂, and their combined effects were established and systematically compared with the complete PAW treatment to distinguish their respective contributions from the overall effects of PAW. The findings provide mechanistic insights into PAW-assisted protein modification and offer a theoretical basis for developing gelatin hydrogels with improved gel strength, WHC, and structural stability for food applications.

2. Material and methods

2.1. Materials

Gelatin (from bovine bone) was obtained from Zhongchen Biotechnology Co., Ltd. (Henan, China), and Cobalt Blue was obtained from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). All other chemicals of analytical grade were acquired from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

2.2. Preparation of PAW

PAW was prepared using a high-performance digital plasma generator (PG-1000 ZD, Suman Plasma Technology Co., Ltd., Nanjing, China) following the method described by Li et al. (2024). The plasma device operated at 800 W using air as the working gas. Based on our pervious comparative study of PAW prepared for 5, 10, and 20 min, PAW₁₀ was selected because it produced gelatin with the highest apparent viscosity and the most homogeneous and ordered network structure among the tested treatment times (Gao et al., 2025). Therefore, PAW₁₀ was used as a representative condition for the subsequent mechanistic experiments. A plasma jet was applied for 10 min at a distance of 10 cm from 300 mL of deionized water to produce PAW₁₀, which was used within 30 min of preparation.

2.3. Preparation of gelatin solution

Gelatin (2 g) was dispersed in ultrapure water and allowed to swell for 30 min at room temperature, followed by dissolution in water bath at 60 °C. The volume of each treatment solution was adjusted to ensure a final gelatin concentration of 2% (w/v) in all groups. Five gelatin systems were prepared under different treatment solutions: 1) ultrapure water (G); 2) ultrapure water was adjusted to pH 2.95 with HNO₃ prior to gelatin dissolution to reproduce the initial acidification of PAW₁₀ (G-pH); 3) ultrapure water containing 160 μmol/L H₂O₂, corresponding to the concentration in PAW₁₀ (G-H₂O₂); 4) ultrapure water containing 160 μmol/L H₂O₂, followed by adjustment to pH 2.95 using HNO₃ (G-pH+H₂O₂); and 5) PAW₁₀ (G-PAW₁₀). The G-pH, G-H₂O₂, and G-pH+H₂O₂ treatments were established as acidification and oxidation systems to reproduce two major physicochemical characteristics of PAW, namely acidification and H₂O₂, and to evaluate whether these two factors could account for the effects of PAW on gelatin gelation. Because gelatin possesses intrinsic buffering capacity, the pH treatment represented the initial acidification introduced by PAW rather than the final equilibrium pH of the gelatin system. After standing at room temperature for 30 min, all gelatin solutions were stored at 4 °C. Samples collected after 0, 30, and 60 min of storage were defined as the Original, Intermediate, and Final stages, respectively.

2.4. Gel strength

Gel strength was measured using a TAXT Plus texture analyzer (Stable Micro Systems Co., Surrey, UK) as described by Wang, Zeng, et al. (2025). The gelatin solution was transferred to a polyethylene tube, and after complete gelation, a 10 mm-high gelatin gel was obtained. Gel strength was tested with a P/5S spherical indenter under the following parameters: post-test speed of 2.00 mm/s, compression distance of 15 mm, and trigger force of 0.5 g. Six replicate samples were prepared for each gel group, with each sample tested once. Gel strength was calculated using the formula:

$$\mathrm{Gel}\text{strength}\left(\mathrm{g}\bullet \mathrm{mm}\right)=\text{Breaking force}\left(\mathrm{g}\right)\times \text{Deformation}\left(\mathrm{mm}\right)$$ (1)

2.5. Water-holding capacity (WHC)

The WHC of gelatin gels was determined using the method described by Liu et al. (2025) with slight modifications. Approximately 10 g sample of gelatin gel was weighed (M₁) into a pre-weighted 50 mL centrifuge tube and centrifuged at 8500g and 4 °C for 15 min. After centrifugation, the released supernatant was carefully discarded, and any residual surface liquid to the tube wall was gently removed using absorbent paper without disturbing the gel. The net weight of the gel (M₂) was obtained by subtracting the weight of the empty centrifuge tube. The WHC was calculated using the following formula:

$$\mathrm{WHC}\left(\%\right)=\frac{{\mathrm{M}}_2}{{\mathrm{M}}_1}\times 100$$ (2)

2.6. Scanning electron microscope (SEM)

Gelatin gels were frozen at −40 °C for 12 h and freeze-dried for 48 h. The samples were gold-coated and observed using a scanning electron microscopy (SEM) (S-3400 N, Hitachi, Ltd., Japan) at 15 kV and the magnification was 100×.

2.7. Rheological behavior

The rheological properties of gelatin solutions were measured using a Discovery HR-1 rheometer (TA Instruments Co., Ltd., New Castle DE, USA) with a 40 mm flat plate model. Samples were cooled from 60 °C to 5 °C at a rate of 1 °C/min. The storage modulus (G′) and loss modulus (G″) were measured at a strain of 2% and a frequency of 1 Hz.

2.8. Eutectic point

The eutectic point of gelatin was determined using a DSC 3500 (NETZSCH, German) following the method of Guan et al. (2024) with minor modifications. Approximately 60 ± 2 mg of gelatin was sealed in an aluminum pan, while an empty pan was used as the reference. The temperature was decreased from 10 to −40 °C at a rate of 5 °C /min under a nitrogen atmosphere (60 mL/min), and the heat flow was continuously recorded.

2.9. pH

After the sample was heated in a 60 °C water bath for 30 min and cooled to room temperature, the pH of the gel solution at different gelation stages was determined according to the method described by Li et al. (2022).

2.10. Particle size and zeta potential

The average zeta potential of gelatin solutions was measured across pH 3–10 to determine the isoelectric point. Gelatin samples were prepared by heating gelatin at 60 °C for 30 min after different gelation states. Particle size and zeta potential were determined using a Zetasizer Lab (Malvern Instruments Ltd., Worcestershire, UK), as described by Wang, Zeng, et al. (2025).

2.11. Raman spectroscopy

The secondary structure of gelatin gels was analyzed by Laser Raman microscopy (XDR, Thermo Co., Waltham, MA, USA) following the method of Li et al. (2025) with slight modifications. Gels were freeze-dried for 48 h before analysis. Raman spectra were recorded from 400 to 3000 cm⁻¹, with three accumulations, an exposure time of 60 s, and data collected at 1 cm⁻¹ intervals. Prior to spectral analysis, baseline correction was performed to remove fluorescence background, and the spectra were normalized using the integrated area of the amide I band (1600–1700 cm⁻¹). Spectra were subjected to baseline correction and Savitzky–Golay smoothing before normalization. The relative contents of α-helix, β-sheet, β-turn and random coil structures were calculated using PeakFit 4.0.

2.12. Total sulfhydryl content

Total sulfhydryl content was determined according to Xu et al. (2024). Gelatin solutions (2 mL) were mixed with 6 mL of 0.1 M Tris-HCl buffer (pH 8.0) containing 5% SDS (w/v), shaken, and heated at 80 °C for 30 min. After cooling, 250 μL of 10 mM 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) was added. Absorbance at 412 nm was recorded before and after addition of DTNB. Total sulfhydryl content was calculated as follows:

$$\text{Total sulfhydryl content}\left(\text{mmol}/\mathrm{mg}\text{protein}\right)=\frac{\left({\mathrm{A}}_{412\mathrm{a}}-{\mathrm{A}}_{412\mathrm{b}}\right)\times {\mathrm{A}}_{280}}{14.150}\times {10}^3$$ (3)

where, A₂₈₀ and A_412b represent absorbance of the sample before DTNB addition at 280 and 412 nm, respectively, and A_412a represents absorbance of the sample after DTNB addition at 412 nm. The constant 14.150 is the conversion coefficient derived from the molar extinction coefficient of the DTNB reaction product (TNB²⁻) at 412 nm and is used to convert the absorbance values into sulfhydryl content (mmol/mg protein).

2.13. Carbonyl content

Carbonyl content was measured following the method of Li et al. (2024). Gelatin solutions (2 mL) were incubated with 3.2 mL of 0.15% 2,4-dinitrophenylhydrazine (DNPH) in 3 mol/L HCl for 1 h in the dark. The samples were then treated with 1.6 mL of 40% trichloroacetic acid (TCA) and centrifuged at 8000 g for 5 min. The precipitates were washed three times with ethanol:ethyl acetate (1:1, v/v) and dissolved in 6 mL of guanidine hydrochloride (6 mol/L). Absorbance was measured at 370 nm and 280 nm, and carbonyl content was calculated as described by the formula:

$$\text{Carbonyl content}\left(\text{nmol}/\mathrm{mg}\text{protein}\right)=\frac{{\mathrm{A}}_{370}-{\mathrm{A}}_{370\mathrm{c}}}{22000\times \left[{\mathrm{A}}_{280}-\left({\mathrm{A}}_{370}-{\mathrm{A}}_{370\mathrm{c}}\right)\times 0.43\right]}\times {10}^6$$ (4)

where, 0.43 represents conversion factor, A_370c represents absorbance of the control group at 370 nm, A₂₈₀ represents absorbance of the sample group at 280 nm, A₃₇₀ represents absorbance of the sample group at 370 nm, and 22,000 represents Mohs extinction coefficient, M⁻¹·cm⁻¹.

2.14. Intermolecular forces

The intermolecular forces involved in gelatin gel formation were analyzed following the method described by Li et al. (2021) with slight modifications. Prior to analysis, the total protein content of each gelatin gel sample was determined using the Coomassie Brilliant Blue method to confirm comparable protein levels among treatments. Gel samples were dispersed in three solvents (described below) at a 1:10 (m:v) ratio and homogenized using a T25 Ultra-Turrax homogenizer (IKA Labortechnik, Staufen, Germany) at 10,000 r/min for 1 min. The homogenates were incubated at room temperature for 2 h, then centrifuged at 10,000g for 15 min. Protein concentration in the supernatants was determined using the Coomassie Brilliant Blue method, and the protein recovery rate was calculated based on the extracted protein content relative to the total protein content of the corresponding sample. The solvents were 0.6 M NaCl (S1), 0.6 M NaCl +1.5 M urea (S2), 0.6 M NaCl +8.0 M urea (S3). These solvents were selected to assess the presence of ionic bonds (S1), hydrogen bonds (difference in protein solubility between S1 and S2), and hydrophobic interactions (difference in protein solubility between S2 and S3).

2.15. Molecular docking

Molecular docking was used to model the binding of gelatin to H₂O₂, NO₃⁻, and NO₂⁻. The crystal structures of the gelatin triple helix and its ligands (H₂O₂, NO₃⁻, and NO₂⁻) were obtained from the RCSB Protein Database (https://www.rcsb.org/) and the PubChem database (https://pubchem.ncbi). These structures were imported into AutoDock 4.0 (Molecular Graphics Laboratory, USA). Conformations were optimized using the prepare protein and prepare ligand modules, applying hydrogenation and the CHARMM force field. Docking was performed using the CDOCKER module with a PoseCluster radius of 0.5, and the lowest-energy conformation was selected for further analysis of amino acid residue interactions and chemical forces.

2.16. Statistical analysis

All experiments were performed in triplicate, and the results were expressed as the mean ± standard deviation (SD). Statistical analyses were conducted using SPSS 26.0 (SPSS Inc., Chicago, IL, USA). Before statistical analyses, the assumption of normality and homogeneity of variance were evaluated, with homogeneity of variance assessed using Levene's test. When these assumptions were met, differences among groups were analyzed by one-way analysis of variance (ANOVA) followed by Ducan's multiple range test for post hoc comparisons. Statistical significance was defined as P < 0.05. Figures were generated using Origin 2025 (OriginLab, Massachusetts, MA, USA) and GraphPad 9.5 (GraphPad Software, San Diego, CA).

3. Results and discussion

3.1. Gel strength

Gel strength, a key indicator of gelatin quality, reflected the structural integrity and texture of the gel (Wang, Zeng, et al., 2025). As illustrated in Fig. 1A, the gel strength of the control group was 31.98 g·mm. After pH, H₂O₂, pH+H₂O₂, and PAW₁₀ treatments, the gel strength significantly increased (P < 0.05) to 79.30, 64.22, 64.54 and 83.14 g·mm, respectively. Among all treatments, PAW₁₀ resulted in the highest gel strength, indicating that the enhancement induced by PAW was not only attributable to its low pH or the presence of H₂O₂. The increase in gel strength after pH treatment may be associated with acid-induced conformational changes that exposed additional charged amino acid residues and promoted intermolecular interactions (Yao et al., 2024). Similarly, the enhanced gel strength observed in the G-H₂O₂ may have attributed to moderate oxidative, which increased hydrophobic interactions and facilitated molecular association through partial oxidation of amino acid side chains (Xu et al., 2024). Notably, no significant difference was observed between the G-H₂O₂ and G-pH+H₂O₂ (P > 0.05), suggesting that simply combining acidification with H₂O₂ did not further enhance gel strength under the present experimental conditions. This phenomenon indicated that the effects of acidification and H₂O₂ were not additive and that the contribution of acidification to gel strengthening was likely limited once oxidative modification had occurred (Xu, Yu, et al., 2024). In contrast, G-PAW₁₀ produced a significantly higher gel strength than the G-pH+H₂O₂ treatment (P < 0.05), demonstrating that the effects of PAW could not be reproduced by acidification and H₂O₂ alone. The highest gel strength of G-PAW₁₀ samples may have attributed to the presence of overall chemically complex PAW reactive environment, which promoted the conversion of sulfhydryl group to disulfide bonds, enhanced hydrophobic interactions, and a denser and more highly cross-linked gel network (Fig. 1C) (Bu et al., 2022).

Fig. 1.

Effects of pH, H₂O₂, pH+H₂O₂, and plasma activated water prepared for 10 min (PAW₁₀) on the gelatin gel strength (A), water-holding capacity (WHC) (B), and microstructure (C). Different letters represent the significant differences (P < 0.05).

3.2. WHC

WHC reflects the ability of a gel network to retain water and is closely associated with the integrity and compactness of its three-dimensional structure (Fu et al., 2025). As shown in Fig. 1B, the WHC of the control gelatin gel was 77.81%, which consistent with previous reports (Xu, Yu, et al., 2024). Then, it was significantly (P < 0.05) increased to 91.33%, 91.36%, and 98.98% after pH, pH+H₂O₂, and PAW₁₀ treatments, respectively, whereas no significant improvement was observed after H₂O₂ treatment. The enhanced WHC following the pH treatment was likely due to acid-induced conformational rearrangement and enhanced intermolecular interactions, which promoted the formation of a more compact gel network capable of entrapping a greater amount of water (You, Chen, Zhang, & Huang, 2025). In contrast, H₂O₂ treatment did not significantly enhance WHC, possibly because oxidation increased the lateral relaxation time of bound and immobilized water, resulting in higher water mobility and reduced WHC (Li, Tang, Lei, Wang, & Liu, 2021; Xu, Yu, et al., 2024). Similar observations were reported by Xia et al. (2022), who found that oxidation altered the distribution of immobilized and free water, thereby influencing the water-retention properties of protein gels.

Under combined pH and H₂O₂ treatment, the gel exhibited a WHC comparable to that of the pH-treated group and significantly higher than those of the control and H₂O₂-treated groups, suggesting that gel WHC were predominantly governed by pH. Wang et al. (2024) reported that at pH 3.0, starch solutions carried a positive charge, promoting stronger associations between bound water and the gel through electrostatic interactions. Accordingly, the enhanced WHC after pH+H₂O₂ modification was attributed to the positive charge during gelation, which facilitated tighter binding with water molecules (Fig. 3F–H). However, the further enhanced WHC following PAW modification was most likely attributed to PAW reactive environment, which altered the physical properties and hydrophobic interactions of gelatin, thereby increasing its hydrophobically bound water (Rout & Srivastav, 2024). Consistently, (Parvez et al., 2025) reported that plasma treatment induced surface etching, resulting in marginal yet significant increases in WHC compared with untreated gelatin gels.

Fig. 3.

Gelation stages of gelatin (A), zeta potential at different pH values (E), and effect of pH, H₂O₂, pH+H₂O₂, and PAW₁₀ on particle size and zeta potential of gelatin gel at different stages: original stage (B and F), intermediate stage (C and G), and final stage (D and H). Different letters represent the significant differences (P < 0.05).

3.3. Microstructure

The microstructure of gelatin gels was characterized by SEM (Fig. 1C). The control gel exhibited a rough and heterogeneous surface with evident etching, irregular morphology, locally increased protein aggregates, and reduced interprotein spacing. In contrast, the pH-treated gel displayed a more uniform cross-linked network with relatively larger and well-distributed pores. Gel treated with H₂O₂ alone or combined with pH adjustment showed fewer interconnected pores, a less heterogeneous network, and rougher surfaces. Among all treatments, the PAW₁₀ gel exhibited the smoothest surface, the smallest and most uniformly distributed pores, and the most compact network structure, suggesting that PAW-derived reactive species effectively improved gel microstructure. These observations were consistent with the WHC and gel strength results (Fig. 1A and B), where a denser and more homogeneous network was associated with superior gel properties (Wang, Zeng, et al., 2025).

The control group contained large and irregular protein aggregates, indicating limited network organization during gelation (Fu et al., 2025). This microstructure coincided with its relatively low gel strength and WHC (Fig. 1A, B), suggesting that the poorly organized network contributed to reduced mechanical stability and water retention. pH treatment partially restored surface hydrophobicity and protein particle size, thereby promoting the formation of a more interconnected gel network (You et al., 2025). In contrast, H₂O₂-treated gels exhibited a looser network, which was likely associated with reduced triple-helix-based crosslinking and consequently lower WHC after oxidative modification (Fig. 1B) (Xu et al., 2023). This observation agree with Xu, Jiang, et al. (2024), who found that oxidized gelatin gels typically displayed looser network structures with larger pore sizes. Compared with the pH and H₂O₂ treatments, PAW₁₀ produced the most compact and homogeneous microstructure, which may have resulted from enhanced intermolecular crosslinking together with improved molecular flexibility induced by PAW-derived reactive species (Parvez et al., 2025).Notably, the combined pH+H₂O₂ treatment failed to reproduce the microstructure observed in the PAW₁₀ gel, indicating that the PAW-induced improvement could not be explained solely by acidification and H₂O₂-mediated oxidation. This interpretation was further supported by Wang, Zeng, et al. (2025), who demonstrated that PAW-induced oxidative promoted disulfide bond formation and facilitated the transformation of protein aggregates into fibrous network structures.

3.4. Rheological behavior

Due to the gelatin's gelling ability at low temperatures, a temperature sweep was performed to monitor the evolution of the G′ and G″. As shown in Fig. 2A and B, G′ and G″ gradually increased with decreasing temperature. The gelation temperature was defined as the crossover point where G′ = G″, representing the transition from a viscous solution to an elastic gel network (Liang et al., 2025). Below the gelation temperature, G′ exceeded G″ in all samples, indicating the establishment of a continuous three-dimensional gelatin network. As shown in Fig. 2A, the gelation temperatures of the control group and gelatin solutions modified by pH, H₂O₂, pH + H₂O₂, and PAW₁₀ were 6.868 °C, 6.868 °C, 6.691 °C, 6.691 °C, and 6.507 °C, respectively. This indicated that H₂O₂ and PAW₁₀ modifications altered gelatin's rheological properties during gelation. Among the treatments, PAW exerted the strongest modifying effect, as G-PAW₁₀ exhibited the lowest gelation temperature. In the control group, both G′ and G″ increased as the temperature decreased, suggesting that molecular helices aggregated and a gelatin gel formed (Cheng et al., 2022). However, oxidative modification by H₂O₂ and PAW₁₀ reduced the gelation temperature, likely due to oxidation-induced exposure of hydrophobic residues, enhancing hydrophobic interactions (Fig. 4I) (Xu, Jiang, et al., 2024). Combined pH and H₂O₂ modification also decreased gelation temperature, suggesting oxidative modification's primarily role in determining gelation temperature. The exposure of hydrophobic groups affected protein alignment during gelation, strengthening the gel network through enhanced hydrophobic interactions (Wang, Zeng, et al. (2025)). Additionally, PAW-modified gelatin exhibited the lowest melting temperature, attributed to stronger hydrogen bonding and hydrophobic interactions within its structure. Temperature altered water molecules flow velocity through nanopores, inducing mobility among protein chains (Jing & Zhang, 2022). Increasing the gel temperature enhanced the system's deformability, while decreasing it strengthened the solid-like properties (Ji et al., 2021). Thus, the lowest gelation temperature after PAW modification may contributed to the densest and most ordered network structure in gelatin (Fig. 1C).

Fig. 2.

Effects of pH, H₂O₂, pH + H₂O₂, and PAW₁₀ on the rheological properties of gelatin solutions: storage modulus (G′) (A), loss modulus (G″) (B), differential scanning calorimetry (C), and eutectic point (D). Different letters represent the significant differences (P < 0.05).

Fig. 4.

Effects of pH, H₂O₂, pH+H₂O₂, and PAW₁₀ on the Raman spectrum and secondary structures of gelatin gels at different stages: original stage (A and D), intermediate stage (B and E), and final stage (C and F), total sulfhydryl (G), carbonyl (H) content, and intermolecular forces (I) in gelatin gels. Different letters represent the significant differences (P < 0.05).

3.5. Eutectic point

The pronounced exothermic peak observed during freezing corresponded to ice crystals formation as water crystallized at the eutectic point, and the temperature at the peak was defined as the eutectic point of the gel (Xu, Jiang, et al., 2024). The heat flux-temperature profiles and the corresponding eutectic points are presented in Fig. 2C and D. The control gel exhibited a eutectic point −15.84 °C. Compared with the control, the eutectic point of the G-PAW₁₀ gel decreased significantly (P < 0.05) to −19.34 °C, whereas no significant differences were observed in the G-pH, G-H₂O₂ and G-pH+H₂O₂ groups (P > 0.05). These results indicated that the acidic properties of PAW significantly influenced ice crystal formation during gelatin freezing, whereas its oxidative properties had a limited effect. The lower eutectic point of the G-PAW₁₀ gel may have been associated with its lower initial pH relative to the isoelectric point of gelatin (Table 1), which could influence water mobility and ice crystal nucleation during freezing. Previous studies reported that delayed ice crystallization and the formation of smaller ice crystals reduced structural damage in hydrogels and food matrices, thereby improving water retention and textural stability (Anacleto & Silva, 2026; Tan, Mei, & Xie, 2021). According, the reduced eutectic point of the G-PAW₁₀ gel was associated with its higher WHC and denser gel network (Fig. 1A-C), although additional studies are required to establish a direct causal relationship. In contrast, H₂O₂ treatment did not significantly alter the eutectic point (Fig. 2D), likely due to the absence of a significant pH difference relative to the control (Table 1), consistent with the findings of Xu, Jiang, et al. (2024). Notably, the G-PAW₁₀ gel simultaneously exhibited the lowest pH, the lowest eutectic point, and the highest WHC, suggesting that PAW-derived reactive species, together with acidification, collectively regulated freezing behavior and gel network formation. Similarly findings were reported by Parvez et al. (2025), who demonstrated that plasma treatment modified gelatin microstructure by reducing the eutectic point, thereby increasing porosity, specific surface area, and water retention.

Table 1.

The effects of five dissolution methods on the pH values of gelatin solutions at different gelation stages. Different letters represent the significant differences (P < 0.05), where lowercase letters indicate the significance of different samples at the same stage, and capital letters indicate the significance of the same sample at different stages.

Gelation stage	G	G-pH	G-H2O2	G-pH+H2O2	G-PAW10
Original	5.58 ± 0.01Aa	5.01 ± 0.01Ad	5.55 ± 0.01Ab	5.04 ± 0.01Ac	4.93 ± 0.01Ae
Intermediate	5.59 ± 0.01 Aa	5.02 ± 0.01 Ad	5.56 ± 0.00 Ab	5.06 ± 0.01 Ac	4.94 ± 0.01 Ae
Final	5.57 ± 0.01 Aa	5.00 ± 0.01 Ad	5.55 ± 0.02 Ab	5.04 ± 0.00 Ac	4.93 ± 0.00 Ae

Note: G represents gelatin dissolved in ultrapure water; G-pH represents gelatin dissolved in acidified water; G-H₂O₂ represents gelatin dissolved in H₂O₂-containing water; G-pH+H₂O₂ represents gelatin dissolved in H₂O₂-containing acidified water; and G-PAW₁₀ represents gelatin dissolved in plasma activated water prepared for 10 min.

3.6. pH

As shown in Fig. 3A, the gelatin gelation process was divided into three stages: corresponding to refrigeration for 0, 30, and 60 min, which represented the initial, intermediate, and final stage, respectively. The pH values at each stage were summarized in Table 1. Although pH variations within each gelatin group across the three stages were negligible (P > 0.05), significant differences (P < 0.05) were observed among the five gelatin groups. During gelation, the pH values followed the order: G > G-H₂O₂ > G-pH+H₂O₂ > G-pH > G-PAW₁₀. At all stages, the G-PAW₁₀ group exhibited the lowest pH values (4.93, 4.94, and 4.93), which were significantly lower (P < 0.05) than those of G-pH (5.01, 5.02, and 5.00) and G-pH+H₂O₂ (5.04, 5.06, and 5.04) (Table 1), indicating that PAW-treated gelatin exhibited the lowest pH value. This phenomenon may have attributed to the continuous transformation of reactive species in PAW, where short-lived nitrogen oxides reacted with water to form nitric acid (HNO₃) and nitrous acid (HNO₂) (Bu et al., 2022). At equivalent pH levels, the pH of G-pH+H₂O₂ was consistently higher than that of G-pH, likely because gelatin in the G-pH+H₂O₂ reacted with both HNO₃ and H₂O₂, whereas gelatin in the G-pH group reacted only with HNO₃ (Xu, Jiang, et al., 2024). In addition, after pH modification, the net surface charge of gelatin shifted, which was considered a key factor contributing to structural alterations in the modified gelatin (Yao et al., 2024).

3.7. Particle size

The particle size distributions of gelatin solutions at the three stages were shown in Fig. 3B–D. All samples exhibited bimodal distributions, indicating the coexistence of polymeric aggregates and monomeric species (Wang, Zeng, et al., 2025). At the original stage, the control group showed particle sizes of 90–300 nm and 3000–9000 nm, whereas all treated groups exhibited a leftward shift in distribution, indicating reduced particle size after modification. Specifically, G-pH, G-pH + H₂O₂, and G-PAW₁₀ exhibited particle size of 40–90 nm and 300–900 nm, while G-H₂O₂ showed ranges of 40–90 nm and 90–1000 nm. The control group formed larger aggregates, likely because its pH (5.58) was close to the pI (5.4), resulting in a near-zero net charge (Table 1, Fig. 3E). Under these conditions, reduced electrostatic repulsion promoted protein-protein interactions, leading to increased particle size (Wang et al., 2023). In contrast, pH and PAW modified samples exhibited smaller particle sizes due to decreased pH, which increased the positive surface charge of gelatin molecules (Table 1) and enhanced electrostatic repulsion, thereby inhibiting aggregation (You et al., 2025).

At the intermediate stage, the G and G-H₂O₂ groups exhibited a single peak, whereas pH-modified samples (G-pH and G-pH+H₂O₂) maintained bimodal distributions (Fig. 3C), indicating the persistence of small aggregates during gelatin gelation. This behavior was likely associated with pH-shifting treatments that promoted the exposure of hydrophobic groups on the protein surface, consistent with previous reports (Yao et al., 2024). Similarly, PAW-modified gelatin also exhibited bimodal distributions, possibly due to conformational disruption, enhanced intermolecular interactions, and partial protein repolymerization induced by reactive species in PAW (Wang, Zeng, et al., 2025). In addition, plasma treatment was reported to induce physical and chemical processes leading to fragmentation or breakdown of gelatin particles (Parvez et al., 2025). Such pre-aggregation likely contributed to the enhanced gelling properties observed after PAW treatment (Fig. 1C).

At the final stage, the particle size distributions remained relatively stable. G-pH, G-pH + H₂O₂, and G-PAW₁₀ predominantly exhibited particle sizes of 30–100 nm and 400–850 nm, whereas G and G-H₂O₂ showed broader distributions ranging from 100 to 1000 nm. This difference was likely attributed to the pH of modified groups being further from the isoelectric point (pI), which sustained electrostatic repulsion and stabilized smaller particle populations, while unmodified samples aggregated more readily (Yao et al., 2024). Excessively large particles hinder early-stage thermal aggregation and subsequent gel formation (Li et al., 2024). Therefore, the smaller initial particle size in the G-PAW₁₀ group likely facilitated later aggregation and contributed to improved gel properties.

3.8. Zeta potential

Fig. 3E presents the average zeta potential of a 2% gelatin solution across a pH range of 3–10, showing the variation in surface charge with pH. At acidic pH, gelatin molecules exhibited a positive charge, peaking at +14.60 mV, while at alkaline pH, they displayed a negative charge, reaching a minimum of −11.30 mV. The pI of gelatin, where positive and negative charges balance, was reported by Xiao, Zhong, Zhao, Guo, and Song (2025) at a potential of 0, corresponding to a pH of 5.4. Zeta potential, a key indicator of protein surface charge, reflects gelatin stability and electrostatic interactions among charged biopolymers, with larger absolute values indicating greater stability (Xu, Yu, et al., 2024). As an amphoteric protein, gelatin's properties are influenced by its charge distribution, higher absolute zeta potential values correspond to increased system stability (Yao et al., 2024). At the original stage (Fig. 3F), the control group exhibited an absolute zeta potential of 1.26 mV, while the experimental groups showed significant increases (P < 0.05), with values of 1.98 mV (G-pH), 1.46 mV (G-H₂O₂), 1.72 mV (G-pH+H₂O₂), and 4.53 mV (G-PAW₁₀). During gelation, zeta potential values all followed the order: G-PAW₁₀ > G-pH > G-pH+H₂O₂ > G-H₂O₂ > G. After pH modification, gelatin exhibited a positive potential, likely due to the pH deviation from the pI (Table 1, Fig. 3E), which promoted protonation of gelatin functional groups (Liu et al., 2024). This increased positive surface charges and higher absolute zeta potential values, consistent with findings by Yao et al. (2024). Among the groups, the zeta potential of G-pH was significantly greater than that of the G-pH+H₂O₂ group in the final stage (Fig. 3H). This was likely due to the lower pH, which reduced surface negative charges and increased positive charges, suggesting a change in surface charge patterns or the formation of the adsorption layer (Li et al., 2025; Liu et al., 2024). After PAW modification, the absolute zeta potential further increased, likely due to PAW-induced gelatin self-assembly, which exposed charged groups (Tan et al., 2026). Therefore, the enhanced intermolecular electrostatic repulsion and steric hindrance induced by PAW contributed to the improved stability of the gelatin gel system (Liang et al., 2025). Similar results have been reported in studies on PAW-induced promotion of soybean isolate protein self-assembly (Tan et al., 2026).

3.9. Secondary structures analysis

Raman spectroscopy was applied to investigate conformational changes in the gelatin backbone during gel formation (Li, Zhang, et al., 2025). As shown in Fig. 4A-C, the Raman spectra displayed several prominent characteristic bands, primarily located at approximately 1450 cm⁻¹ (CH₃ and CH₂ bending vibrations), 1650 cm⁻¹ (amide I, dominated by C O stretching with minor contributions from C—N stretching, C_α—C—N bending, and N—H in-plane bending), and 2930 cm⁻¹ (C—H stretching vibrations of aromatic, aliphatic, and charged amino acid residues) (Parvez et al., 2025). At the original stage (Fig. 4A), the modified gelatin samples showed pronounced spectral differences from the control. Treatments with pH, H₂O₂, pH+H₂O₂, and PAW produced distinct bands at approximately 1460, 1627, and 2972 cm⁻¹, indicating treatment-dependent changes in gelatin conformation. The band at 2972 cm⁻¹, observed in all treatment groups, was associated with C—H stretching vibrations and changes in the local molecular environment, potentially involving hydrogen-bond-mediated conformational rearrangement (Parvez et al., 2025). Additional bands at 1460 and 1627 cm⁻¹ following pH+H₂O₂ and PAW treatments were attributed to methyl group bending vibrations and the stabilization of the gelatin helical structure, respectively (Parvez et al., 2025). These spectral changes suggested that acidification and oxidative modification altered the local chemical environment and conformational organization of gelatin chains during the original stage of gelatin, consistent with previous observations of plasma-induced gelatin modification (Tan et al., 2026). At the intermediate stage, characteristic peaks gradually became more pronounced in the control group, indicating progressive conformational rearrangement during cooling (Fig. 4B). At the final stage, distinct spectral differences remained among the treatments, reflecting treatment-dependent variations in the final gelatin conformation (Fig. 4C). The G-pH+H₂O₂ and G-PAW₁₀ exhibited an additional band at 1385 cm⁻¹, likely attributable to the formation of carboxyl groups via oxidative modification of amino acid side chains (Tan et al., 2026). The higher intensity observed after PAW treatment was likely due to nitration of amino acid side chains induced by RNS in PAW (Tan et al., 2026). Notably, all characteristic bands of G-PAW exhibited the highest intensity, suggesting that PAW induced gelatin conformational rearrangements beyond simple acidification and oxidation, thereby favoring the formation of a dense and ordered gel network.

The secondary structure of gelatin at three gelation stages was further analyzed to elucidate the conformational rearrangements underlying gel network formation (Fig. 4D-F). Changes in the relative proportions of α-helix, β-sheet, β-turn, and random coil reflected the conformational rearrangement and degree of structural ordering of gelatin chains during cooling-induced gelation. These structural changes may affect chain association and network organization by modulating intermolecular interactions (Zhang et al., 2024). At the original stage (Fig. 4D), the α-helix content decreased significantly (P < 0.05) from 22.06% in the control to 19.26%, 18.17%, and 13.08% in G-pH, G-pH+H₂O₂, and G-PAW₁₀ groups, respectively, whereas the β-sheet content increased from 24.24% to 39.33%, 29.62%, and 36.15%, respectively. Meanwhile, the β-turn content decreased significantly (P < 0.05) in G-pH and G-H₂O₂, whereas the random coil content decreased significantly (P < 0.05) in G-pH and G-PAW₁₀. These results indicated that, at the original stage, pH reduction was associated with a redistribution of gelatin secondary structures, characterized primarily by decreased α-helix and increased β-sheet contents. The lower random coil content observed in G-pH at this stage represented an initial structural response to acidification rather than a persistent suppression of disordered conformations throughout gelation (Li et al., 2022).

At the intermediate stage (Fig. 4E), H₂O₂ treatment significantly reduced (P < 0.05) the α-helix content. Compared with the control, the α-helix content in G-H₂O₂ and G-pH+H₂O₂ decreased significantly (P < 0.05) by 10.60% and 9.56%, respectively, consistent with previous reports on H₂O₂-induced gelatin oxidation (Xu, Yu, et al., 2024). Notably, the random coil content of the G-pH group increased significantly from the original to the intermediate stage. This stage-dependent increase did not contradict the lower random coil content observed for G-pH relative to the control at the original stage, because the two observations involved different comparison bases. Instead, these results suggested that acidification dynamically modulated gelatin conformation during gelation (Bu et al., 2022). The subsequent increase in random coil content may reflect further conformational rearrangement of gelatin chains during cooling, which could facilitate chain reassociation and changes in intermolecular interactions (Fig. 4I). At the final stage, the control exhibited a pronounced redistribution from of α-helix to β-sheet, indicating gel formation (Fig. 4F). However, the control retained the highest random coils content (17.80%), suggesting a relatively disordered gel network (Fig. 1C). In contrast, the increased α-helix content in G-pH and G-H₂O₂ groups may have reflected enhanced local conformational ordering and intramolecular hydrogen bond stabilization during the later stage of gelation (Fu et al., 2025). Overall, active species in PAW (e.g., NO, NO₂⁻, H₂O₂, and ROS) rearranged the gelatin secondary structure through ionic interactions, hydrogen bonding, and hydrophobic interactions (Sun et al., 2026). Specifically, NO₃⁻ and NO₂⁻ mainly regulated α-helix, β-sheet and random coil contents, whereas H₂O₂ primarily affected α-helix and β-turn structures (Xu, Yu, et al., 2024). In G-PAW₁₀, the greater effects of multiple reactive species consistently reduced α-helix and random coil contents while increasing β-turn content, ultimately promoting the formation of a more compact and stable gelatin gel network (Fig. 1C).

3.10. Oxidation of gelatin solution

Sulfhydryl groups are highly susceptible to oxidation and readily form disulfide bonds under oxidative conditions, therefore, total sulfhydryl content is widely used as an indicator of protein oxidation (Zhao et al., 2019). As shown in Fig. 4G, the total sulfhydryl content of the G-pH group (2.17 mmol/mg protein) did not differ significantly from that of the control (2.04 mmol/mg protein), indicating that acidification alone did not induce sulfhydryl oxidation. In contrast, H₂O₂ and PAW treatment markedly reduced sulfhydryl content to 1.77 (G-H₂O₂), 1.23 (G-pH+H₂O₂), and 1.47 mmol/mg protein (G-PAW₁₀) (P < 0.05), demonstrating enhanced oxidation of cysteine residues. The depletion of sulfhydryl groups suggested the potential formation of disulfide bonds, which may contribute to intermolecular association and gel network stabilization (Rao et al., 2024; Xu, Yu, et al., 2024). However, sulfhydryl oxidation alone could not fully explain the variation in gel properties among treatments. Notably, although G-pH+H₂O₂ exhibited a lower sulfhydryl content than G-PAW₁₀, indicating a higher degree of cysteine oxidation, its gel strength was inferior to that of G-PAW₁₀ (Fig. 1A). This suggested that disulfide crosslinking was not the sole factor governing gelatin gel formation. Instead, the superior gel properties of PAW-treated gelatin likely resulted from the combined regulation of covalent and non-covalent interactions, including hydrogen bonding, hydrophobic interactions, ionic interactions, and conformational rearrangement induced by the complex PAW environment (Li et al., 2024).

Carbonyl groups are well-established biomarkers of protein oxidation and are generated through peptide backbone cleavage, amino acid side-chain oxidation, and oxidative crosslinking (Zhao et al., 2019). As shown in Fig. 4H, carbonyl content increased from 87.24 nmol/mg protein in the control to 101.60, 97.78, and 109.93 nmol/mg protein in the G- H₂O₂, G-pH+H₂O₂, G-PAW₁₀ groups, respectively (P < 0.05), whereas no significant difference was observed in the G-pH group. These findings confirmed that acidification alone did not promote gelatin oxidation and that oxidative species were primarily responsible for carbonyl formation, consistent with previous reports (Xia et al., 2022; Xu, Yu, et al., 2024). Moreover, the G-PAW₁₀ group exhibited significantly higher (P < 0.05) carbonyl content than the H₂O₂-treated groups, suggesting that additional reactive species in PAW further enhanced gelatin oxidation. Therefore, the balanced oxidative modification induced by PAW was likely associated with the enhanced gel strength, WHC, and homogeneous gel network observed in this study (Fig. 1A-C), consistent with previous reports on plasma-modified proteins (Li et al., 2024).

3.11. Intermolecular forces analysis

The formation of gelatin films was associated with the denaturation of the protein's triple-helix structure, primarily stabilized by ionic bonds, hydrogen bonds, and hydrophobic interactions (Xu, Yu, et al., 2024). As shown in Fig. 4I, the solubility of gelatin in S1, S2 and S3 reflected the contributions of ionic bonds, hydrogen bonds, and hydrophobic interactions during gelation. The control group exhibited a solubility of 8.98% in S1. After pH modification, solubility in S1 significantly increased to 15.23% (G-pH), 13.17% (G-pH+H₂O₂), and 19.52% (G-PAW₁₀) (P < 0.05), whereas G-H₂O₂ showed no significant difference from the control. The lower pH of G-pH compared to G-pH+H₂O₂ resulted in higher solubility in S1, indicating that pH adjustment enhanced ionic bond formation. The solubility of G-PAW₁₀ in S2 significantly increased from 18.50% to 23.02% (P < 0.05), while G-pH, G-H₂O₂, and G-pH+H₂O₂ showed no significant changes, implying that reactive species in PAW promoted hydrogen bond formation. In S3, the solubility of G-H₂O₂ and G-PAW₁₀ increased to 20.46% and 21.16%, respectively, significantly higher (P < 0.05) than the 17.22% in the control, demonstrating that oxidation strengthened hydrophobic interactions during gelation. pH modification enhanced ionic bonds in gelatin, likely due to the pH deviation from the pI (Table 1), where ionic interactions dominate, consistent with the findings of Ni et al. (2014). Similarly, the increased contribution of hydrophobic interactions in G-H₂O₂ may be associated with oxidative modification, which promoted exposure of hydrophobic regions on gelatin molecules (Xu, Yu, et al., 2024). Compared with acidification and H₂O₂ treatment, G-PAW₁₀ exhibited increased protein solubility in all three extraction systems, suggesting that PAW treatment was associated with simultaneous enhancement of multiple intermolecular interactions. Because the simplified model treatments reproduced only the acidification and H₂O₂ characteristics of PAW, this additional effect could not be fully explained by these two physicochemical factors alone. Instead, it was likely associated with the overall chemically complex reactive environment of PAW, which facilitated molecular rearrangement during gelation (Wang, Zeng, et al., 2025). Since hydrogen bonding was the primary force governing gel formation, PAW-modified gelatin exhibited superior gel properties and microstructure (Fig. 1).

3.12. Molecular docking

To further explore the potential molecular interactions between representative PAW-derived reactive species and gelatin, molecular docking was performed using H₂O₂, NO₃⁻, and NO₂⁻ as representative ligands. The predicted interactions between these ligands and gelatin were shown in Fig. 5A. H₂O₂ interacted with Pro66(C), Leu67(C), Ala60(C), and Ala57(C), forming hydrophobic interactions with the first three residues and one hydrogen bond with Ala57(C) (Fig. 5A(a)). NO₃⁻ interacted with Arg65(C) and Lys64(B) through hydrophobic contacts and hydrogen bonding (Fig. 5A(b)), whereas NO₂⁻ formed a hydrogen bond with Arg37(B) (Fig. 5A(c)). These docking results indicated that the representative PAW-derived reactive species were capable of interacting with gelatin through hydrogen bonding and hydrophobic interactions, which consistent with the intermolecular interaction analysis (Fig. 4I). Among the three ligands, NO₃⁻ and NO₂⁻ exhibited lower predicted binding energies (−3.86 and −3.36 kcal/mol, respectively) than H₂O₂ (−2.21 kcal/mol), indicating stronger theoretical binding affinities in the docking simulation. However, the actual contribution of individual PAW-derived species is also influenced by their concentration, lifetime, and accessibility within the PAW-gelatin system.

Fig. 5.

Enlarged views of gelatin docking modes (A) with H₂O₂ (a), NO₃⁻ (b), and NO₂⁻ (c), correlation coefficients plots between different indicators (B), and schematic model illustrating the effect of pH, H₂O₂ and PAW on gelatin during gelation (C). In the correlation analysis (B), the ellipses represent Pearson correlation coefficients between two variables. The color intensity and ellipse orientation indicate the strength and direction of the correlation, respectively. Asterisks indicate statistically significant correlations (P ≤ 0.05).

3.13. Schematic mechanism

Pearson correlation analysis was performed to evaluate the relationships among gelatin gel properties, physicochemical characteristics, secondary structures, oxidation degree, and intermolecular forces (Fig. 5B). The asterisks indicate statistically significant correlations (P ≤ 0.05). WHC exhibited a significant negative correlation with gel pH but positive correlations with zeta potential and ionic bonds, suggesting that enhanced surface charge and ionic interactions contributed to improved water retention. Conversely, gel pH was negatively correlated with zeta potential and ionic bonds, indicating that acidification promoted charge redistribution and ionic interactions during gel formation. The random coil content showed negative correlations with gel strength, hydrogen bonds, and hydrophobic interactions, suggesting that reduced disordered structures favored intermolecular association and the formation of a stronger gel network. In addition, oxidation-related parameters (total sulfhydryl and carbonyl contents) were associated with gel properties and intermolecular interactions, highlighting the contribution of oxidative modification to gelatin network formation. Based on these results, a schematic mechanism was proposed to summarize the distinct effects of the acidification and oxidation treatments and PAW on the gelatin gelation (Fig. 5C). For G-pH, acidification promoted conformational rearrangement, increased ionic interactions, and facilitated molecular rearrangement during cooling. Lowering the pH below the pI increased protonation of ionizable groups (Fig. 3E, Table 1), thereby enhanced electrostatic repulsion and reduced excessive molecular aggregation, which resulted in smaller initial particle sizes (Fig. 3B–D). In contrast, H₂O₂ treatment primarily induced oxidative modifications, as evidenced by decreased sulfhydryl content and increased carbonyl formation (Fig. 4G and H). These changes were associated with partial disruption of the triple-helix-based network and the formation of a relatively loose and heterogeneous gel structure (Fig. 1C). The G-pH+H₂O₂ did not produce additional improvements beyond those observed in the individual treatments, indicating that combining acidification with H₂O₂ was insufficient to reproduce the full effects of PAW. Compared with these treatments, G-PAW₁₀ exhibited a distinct gelation behavior that could not be fully explained by acidification and H₂O₂ alone. The additional enhancement observed after PAW treatment was attributed to the overall chemically complex reactive environment of PAW rather than to any individual reactive species. The complex reactive environment of PAW regulated surface charge, promoted α-helix to β-sheet and β-turn transitions, enhanced hydrogen bonding, hydrophobic interactions (Fig. 4I), and disulfide bond formation (Fig. 4G), and reduced initial particle size (Fig. 3B). Moreover, G-PAW₁₀ exhibited the lowest eutectic point (Fig. 2C), which may have contributed to improved structural stability during cooling and enhanced water retention. Collectively, these results suggest that PAW promoted gelatin gelation through multiple concurrent physicochemical effects that facilitated structural reorganization and network formation, resulting in superior gel properties compared with the acidification and oxidation treatments.

4. Conclusion

This study established a refined PAW system to distinguish the contributions of acidification and on H₂O₂ from the overall effects of PAW on gelatin gelation. Compared with the control, PAW treatment increased gel strength by 159.97%, enhanced WHC by 21.17%, and reduced the eutectic point from −15.84 °C to −19.34 °C. These improvements could not be fully reproduced by the combined acidification and H₂O₂ treatment, indicating that the effects of PAW could not be explained by these two physicochemical factors alone and may also be associated with the chemically complex reactive environment of PAW, which promoted favorable intermolecular interactions and structural reorganization. Molecular docking suggested potential interactions between representative PAW-derived reactive species and gelatin at the molecular level, providing complementary evidence for the proposed mechanism. Because the present model systems reproduced only the acidification and H₂O₂ characteristics of PAW, the individual contributions of representative reactive nitrogen species were not resolved. Future studies should quantify representative reactive oxygen and nitrogen species, establish dedicated model systems together with final equilibrium pH-matched controls, and further elucidate their respective roles in gelatin modification and practical food applications.

CRediT authorship contribution statement

Mengzhe Li: Writing – review & editing, Writing – original draft, Methodology, Investigation, Data curation. Ruichang Gao: Project administration, Funding acquisition, Conceptualization. Xin Wang: Software, Methodology, Formal analysis, Data curation. Wengang Jin: Validation, Data curation. Xiaoli Zhang: Validation, Project administration. Li Yuan: Supervision, Conceptualization. Tong Shi: Supervision, Resources, Methodology, Data curation.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.