First, MBDFs and H-EWPGs were coated with a 10 nm gold layer. Then, the samples were scanned with a scanning electron microscope (JOLE-JMS-5700E, Tokyo, Japan). The accelerating voltage, scale bar, and magnification were 10 kV, 1 μm, and 5,000, respectively (Zheng et al., 2022).

The MBDFs and H-EWPGs were freeze-dried in a vacuum freeze-dryer (HFD-6, Heyuan Ice Equipment Co., Ltd., Zhengzhou, China) and then were scanned with a Fourier-transformed infrared (FT-IR) spectrometer (8400S, Shimadzu, Japan). PeakFit software v4.12 (Seasolve, Framingham, MA, United States) was employed to investigate the secondary structure of H-EWPGs citing the procedure from Bashash et al. (2022). When calculating the secondary structure of egg white protein, the signal peak of cellulose was subtracted from the signal of the mixture.

The centrifugation method was adopted to determine water-retention ability (WRA) of the gel samples (Khemakhem et al., 2019). Briefly, approximately 2 g (M₀) of H-EWGs was added to a centrifuge tube (50 mL), along with filter paper. After filtration at 4,000 × g at 4°C for 20 min, the gel samples were weighed again (M₁). The WRA (%) was calculated according to the Equation 2:

where M₀ and M₁ represent the gel weights before and after centrifugation, respectively.

The gel samples were loaded in a 15 mL glass centrifuge tube for syneresis measurement (Khemakhem et al., 2019). The freeze-thaw cycle contained two steps: (1) the gels in the tube were frozen at −16°C for 24 h; and then (2) the frozen gels were slowly thawed for 8 h at room temperature (approximately 25°C). The gel went through five of these cycles and then weighed (W_s). After filtration (3,500 × g, 15 min), the weight of the residues was measured and record as W_d. Dehydration rate in freeze-thaw cycle was quantified according to Equation 3:

Each gel sample was transferred to a quartz colorimetric dish, and the absorbance was determined using a JH754PC UV–Vis spectrophotometer (Shanghai Jinghua Co., Ltd., China) at 600 nm.

The textural properties of H-EWPGs, including hardness, elasticity, cohesiveness, adhesiveness, chewability, and resilience were tested with the TA Plus Texture Analyzer System (LLOYD Instrument Co., HK) with a P/36 R probe. The data were measured in TPA mode, and the test parameters are as follows: the trigger force, compression rate, and test, pre-test, and post-test speeds were 3 g, 50%, 1, 2, and 1 mm/s, respectively (Hou et al., 2022).

Figures 1A–C reflect the changes in the microstructure of MBDF after the two mixed modifications. MBDF showed a relatively smooth surface microstructure with a small amount of porosity and debris (Figure 1A). In contrast, a distinct honeycomb microstructure with more porosity or debris was observed in MBDF-HEC MBDF-HEPC and MBDF-HEA (Figures 1B,C). Increase in porosity or debris mainly resulted from the breakdown of glycosidic linkages induced by heating, dual enzymatic hydrolysis, and alkali treatment during hydroxypropylation and acrylic-grafting (Kanwar et al., 2023).

As presented in Figure 2, the untreated and modified MBDFs all had strong peaks approximately at 3340, 2920, 1,600 and 1,050 cm⁻¹, respectively, conforming to the typical FT-IR spectra of dietary fibres. Moreover, changes in several characteristic peaks revealed that heating and dual enzymolysis combined with acrylic-grafting or hydroxypropylation have affected MBDF’s structure, especially the chemical bonds and functional groups. In the spectra of MBDF-HDEH and MBDF-HDEAG, the peak at 3397 cm⁻¹ in the spectrum of MBDF, which represents the asymmetric stretching of O–H, transferred to 3,337 and 3,334 cm⁻¹ after heat and dual enzymolysis united with acrylic-grafting or hydroxypropylation, respectively, indicating that the synthesis modification have changed the hydrogen bonds in MBDF (Ma et al., 2022). The spectra of MBDF-HDEH and MBDF-HDEAG both had a new absorption peak near 910 cm⁻¹ (corresponding to the vibration of β–C–H), resulting from the breakdown of β-glycosidic linkages caused by heating and/or dual enzymes hydrolysis (Jiang et al., 2020). In the spectrum of MBDF-HDEH, the red-shift of tiny absorption peak from 1,637 cm⁻¹ (representing the vibration of C–H) to 1,604 cm⁻¹, and a new peak appeared at 1427 cm⁻¹ (indicating the bending of methyl group), both confirmed the successful grafting of hydroxypropyl group on MBDF (Jiang et al., 2020). Additionally, the new peaks at 1375 cm⁻¹ (representing the stretching of C=C bond) and 1,546 cm⁻¹ (indicating the vibration of –COO⁻) suggested that acrylic groups were grafted onto MBDF (Rani et al., 2019). Changes in these typical chemical bonds certificated that the two composite modifications have affected the structure of MBDF.

The MBDFs’ X-ray diffraction spectra and crystallinity values are presented in Figures 3A,B, respectively. MBDF, MBDF-HDEH and MBDF-HDEA exhibited a strong peak at 2θ of 20^° (Figure 3A), indicating that they all have cellulose I crystal forms (Ma et al., 2022). The crystallinity of MBDF, MBDF-HDEH and MBDF-HEDEA was not obviously different (p > 0.05) (Figure 3B), showing that heating and dual enzymolysis separately united with acrylate-grafting and hydroxypropylation did not significantly affect MBDF’s crystal structure (Kanwar et al., 2023), corresponding to their low substitution degree of hydroxypropyl and acrylic groups (4.73 and 2.75%, respectively).

The scanning electron micrographs of H-EWPG, and H-EWPGs fortified with MBDF (H-EWPG/MBDF), MBDF-HDEAG (H-EWPG/MBDF-HDEAG), and MBDF-HDH (H-EWPG/MBDF-HDEH) at addition amount of 5 g/100 g are shown in Figures 4A–D, respectively. H-EWPG had a smooth surface and relatively rough inner microstructure with many pores (Figure 4A); whereas H-EWPG/MBDF, H-EWPG/MBDF-HDEH and H-EWPG/MBDF-HDEAG all showed a denser and more granular microstructure with a large amount of tiny pores (Figures 4B–D). During the preparation of heat-induced gels, the addition of fibres can provide a skeleton on which the egg white protein can aggregate, which is helpful to the formation of a dense three-dimensional microstructure with tiny pores (Zhao et al., 2023). Moreover, the skeleton structure of fiber matrix can enhance the interactions between egg white proteins and promote them adsorbing on the surface of gels, leading to a granular surface microstructure (Ullah et al., 2019). In comparison with microstructure of H-EWPG/MBDF (Figure 4B), the microstructures of H-EWPG/MBDF-HDEH and H-EWPG/MBDF-HDEAG were all denser with smaller tiny holes (Figures 4C,D), mainly due to their higher water-expansion volume and viscosity (Table 2), which can improve the aggregation of egg white proteins and lead to the formation of a denser gel (Khemakhem et al., 2019).

The microstructures of H-EWPG fortified with MBDF-HDEH at addition amount of 5, 3 and 1 g/100 g are shown in Figures 4D–F, respectively. It is obvious that the microstructure of H-EWPG became more granular and denser, and the holes became smaller as the addition amount increased. This result demonstrated that an increase in dose of fibres can enhance the intramolecular and intermolecular actions across egg white proteins and promote the formation of a denser gel (Xu et al., 2023).

Figure 5 depicts the α-helix, β-sheet, β-turn, and random coil contents of H-EWPG and H-EWPGs fortified with MBDFs. The β-turn content of H-EWPG significantly decreased (p < 0.05), but its β-sheet content increased after the addition of MBDF, MBDF-HDEH and MBDF-HDEAG. During the formation of heat-induced gels, the structure of egg white protein is destroyed as the temperature increases; then, the stretched polypeptide chains aggregate and form a three-dimensional spatial structure through hydrogen bonds or hydrophobic forces with decreasing temperature (Lee et al., 2024). The addition of MBDFs with considerable WRA and WEA (Table 2) can enhance the interactions of egg white proteins with water molecules and make the structure of gel denser, leading to a higher content of β-sheet (Xu et al., 2023). Additionally, the hydroxypropyl and acrylate groups of MBDF-HDEH and MBDF-HDEAG can increase the hydrogen bonds between polypeptide chains, facilitating the formation of β-sheet or α-helix (Xiao et al., 2020). Moreover, the β-sheet structure is helpful to the interactions between the egg white proteins (Zang et al., 2023), which was another reason for the denser and more granular microstructure of H-EWPG/MBDF, H-EWPG/MBDF-HDEH and H-EWPG/MBDF-HDEAG (Figures 4B–D).

Table 3 depicts the effects of different addition amounts of MBDFs on the colour of H-EWPG. Overall, the L value (representative of brightness) of H-EWPG reduced as the addition of MBDF, MBDF-HDEH and MBDF-HDEA increased; in contrast, the a value (representative of redness) of H-EWPG increased with increasing addition amounts of MBDF, MBDF-HDEH and MBDF-HDEAG (p < 0.05). Moreover, the addition of MBDF and MBDF-HDEH (1–3 g/100 g) increased the b value (indicative of yellowness) of H-EWPG, but this effect was negatively dose-dependent. These results show that the addition of MBDFs darkened the colour of H-EWPG, which was mainly ascribed to two reasons: (i) the addition of MBDFs (belong to polysaccharides) caused carbonyl ammonia reaction during heating-induced gelation of egg white protein (Zang et al., 2023); and (ii) the colour of these MBDFs was relatively brown (Table 2). Compared with H-EWPG, H-EWPG/MBDF-HDEH exhibited the highest colour difference (ΔE) when the addition amount of MBDF-HDEH was 5 g/100 g; followed by H-EWPG/MBDF-HDEAG as the MBDF-HDEAG addition amount was 4–5 g/100 g. After hydroxypropylation and acrylate-grafting, the introduced hydroxypropyl and acrylic groups both can promote the Maillard reaction between the egg white protein and MBDF (Lv et al., 2022), resulting in a darker colour.

Water-holding ability (WRA) is an important property that can affect the quality of EWPG, and it is influenced by the gaps in the gels’ network structure and the attraction forces of hydrophilic polymers (Manzoor et al., 2022). The results in Figure 6A showed that the addition of MBDF, MBDF-HDEH, and MBDF-HDEAG improved the WRA of H-EWPG (p < 0.05), and the improvement effects were dose-dependent. One reason for this is that these MBDFs all have considerable SDF content and water-holding capacities (1.79–3.02 g/g, Tables 1, 2), which can enhance the affinity of egg white proteins with water (1.04 g/g, Liu et al., 2022). Another reason is that the addition of these MBDFs increased the β-sheet content of H-EWPG (Figure 5) and the number of tiny holes in the microstructure of H-EWPG (Figures 4B–D), which improved the network structure and facilitates the interaction between H-EWPG and waters (Ma et al., 2021). The increase in pores and fragment on microstructure can enhance the interactions of fibres with water molecules (Khemakhem et al., 2019). Furthermore, MBDF-HDEH and MBDF-HDEAG both exhibited greater ability in improving the WRA of H-EWPG than MBDF at 2–5 g/100 g, mainly attributed to the granular microstructures with a large amount of tiny holes (Figures 4C,D), higher SDF content and WRA (Tables 1, 2). Ullah et al. (2019) found that the network structure of tofu gel has been improved by the addition of okara dietary fiber, too.

Pondus hydrogenii (pH) is one of the factors affecting the formation of EWG, as well as its network structure, texture properties, application range, and shelf life (Manzoor et al., 2022). As shown in Figure 6B, the pH value of H-EWPG was approximately 4.5, which is near the isoelectric point of ovalbumin (the predominant constituent of egg white proteins, Wang et al., 2018). Meanwhile, the pH value of H-EWPG was significantly increased after the addition of MBDF, MBDF-HDEH, and MBDF-HDEAG at 1–5 g/100 g (p < 0.05). The gels formed was weak and had low water-holding capacity and transparency at the isoelectric point of egg white proteins, because of the interactions between egg white proteins are low (Zhao et al., 2023); while a dense gel with continuous network structure can be formed between egg white proteins at around pH 7.0 (Khemakhem et al., 2019), which was consistent with the results as shown in Figures 4A–D. Therefore, the addition of MBDFs increased the pH of H-EWPG and thus improved its microstructure. Moreover, the pH values of H-EWPG with MBDF were lower (p < 0.05) than that of H-EWPG with MBDF-HDEH or MBDF-HDEAG at 2–5 g/100 g, mainly because of the grafting of hydroxypropyl and acrylic groups, which can change the charge of egg white protein (Shaikh et al., 2019).

A decrease in dehydration rate of hydrogels under freeze-thaw cycle can help hydrogel-based products to prevent dehydration, hardening, and shrinkage (Bashash et al., 2022). Obviously, the addition of MBDF, MBDF-HDEH, or MBDF-HDEAG (4–5 g/100 g) decreased the dehydration rate of H-EWPG (p < 0.05, Figure 6C), indicating their reducing effects in the deterioration of gels during freeze-thaw cycle. The addition of these MBDFs improved the microstructure and water-holding ability of H-EWPG (Figures 4A–D, 6A), and increased the β-sheet content of H-EWPG (Figure 5), leading to a stronger affinity with water molecules under freeze-thaw cycle (Ma et al., 2021). Moreover, the reducing effect of MBDF-HDEH and MBDF-HDEAG on the dehydration rate of H-EWPG was enhanced with increasing addition amount; while the addition of MBDF on the dehydration rate of H-EWPG at 3 g/100 g was higher than that at 4–5 g/100 g. The main reason was that MBDF-HDEH and MBDF-HDEAG had higher SDF content and water-holding abilities (Tables 1, 2), the egg white proteins’ aggregation was enhanced and microstructure of H-EWPG became denser with increasing addition amount of MBDF-HDEAG and MBDF-HDEAH (Figures 4D–F), which was not conducive to water lose in freeze-thaw cycle (Lee et al., 2024). By contrast, MBDF had lower SDF content and water-retaining ability (Tables 1, 2). A large addition amount of fibres with lower polarity will reduce the interaction between egg white proteins, and loosen the structure of H-EWPG, thus induce the gel to lose water after freeze-thaw cycle (Ullah et al., 2019).

The results in Figure 6D revealed that the addition of MBDFs decreased the optical transparency of H-EWPG (p < 0.05), which was perhaps due to their effects on the structure and pH of H-EWPG. As shown in Figure 6B, the pH of H-EWPG was 4.5 (near the isoelectric point of egg white protein); at this pH value the interactions between egg white proteins were weak and the network structure of gel formed was loose (Figure 4A), thus, the transparency was high (Khemakhem et al., 2019). After the addition of MBDFs, the pH was increased (Figure 6B) and the interactions between egg white proteins and water molecules were enhanced (Figure 6A), which was conducive to the formation of molecularly homogeneous network (Figures 4B–D) and a higher light scattering (Ma et al., 2021), resulting in a lower transparency. Moreover, the hydrophobic force which was a main force to form and maintain heat-induced gels may be reduced by the addition of MBDFs, which was helpful to decreasing the transparency of gels (Han et al., 2022). Additionally, MBDF showed a greater lowering effect on the transparency of H-EWPG than MBDF-HDEH and MBDF-HDEAG; and the improving effect of MBDF was positive dose-dependent, probably due to its lower WHA (Figure 6A) and granular microstructure of H-EWPG/MBDF with a large number of pores (Figures 4B–D). A granular and porous microstructure can increase the scattering of light and thus decrease the gels’ transparency (Zhao et al., 2023). A reduction in optical transparency may benefit the applications of H-EWG in protecting photosensitive foods (Lee et al., 2024).

The addition of MBDF, MBDF-HDEH, and MBDF-HDEAG all enhanced the hardness of H-EWPG (p < 0.05) in a dose-dependent manner (Table 3). During the formation of heat-induced gel, MBDFs can provide a carbon skeleton on which the egg white proteins can adsorb and aggregate; then, the crosslink between the proteins and the gel network strength are both enhanced, resulting in a higher hardness (Zhao et al., 2023). Moreover, the more granular and denser microstructures of H-EWPG/MBDFs with higher water-holding capacity (Figures 4B–E, 6A) enhanced the hardness of gels, too (Ullah et al., 2019). The biggest hardness (288.22 ± 9.54 g) was observed on H-EWPG/MBDF-HDEAG, followed by H-EWPG/MBDF-HDEPH, which was mainly ascribed to their high WRA and denser microstructure (Figures 4C,D, 6A). Additionally, the acrylic group of MBDF-HDEAG and hydroxypropyl group of MBDF-HDEH both can improve the crosslink between hydro polymers (Rani et al., 2019), and thus increase the hardness of H-EWPG.

The gumminess of H-EWPG increased with increasing addition amounts of MBDFs (Table 3). A gel’s gumminess represents the energy needed for a semi-solid food to become stable, and it is positively correlated with its hardness, cohesiveness, and viscosity (Zang et al., 2023). It can be speculated that the increased hardness was one reason for the higher gumminess of H-EWPG after the addition of MBDFs. In addition, the higher β-sheet content (Figure 5) also contributed to the high gumminess of H-EWG with MBDFs (Liu et al., 2022). The addition of MBDF-HDEH showed the highest improvement effect on gumminess of H-EWPG, which can be attributed to its high viscosity (Table 2). DFs with high viscosity can improve the hardness and cohesiveness of gels, and enhance the cohesiveness of gels, leading to a higher gumminess (Manzoor et al., 2022).

The chewiness of gels is defined as the product of its gumminess and springiness (Xu et al., 2023); thus, the increased gumminess was mainly responsible for the improvement in the chewiness of H-EWPG after the addition of MBDFs. Additionally, the increase in pH (Figure 6B) and water-holding capacity of H-EWPG (Table 2) were helpful for the formation of a three-dimensional structure with higher chewiness (Li et al., 2023). MBDF-HDEAG showed the greatest enhancing effect on the chewiness of H-EWPG, which was in agreement with its highest WRA and WEA (Table 2) and improvement effect on the hardness of H-EWPG (Table 3). An increase in hardness, chewiness, and gumminess means that the applications of H-EWPG in food, biomedicine and cosmetics industries will be expanded (Manzoor et al., 2022).

In contrast, the springiness and cohesiveness of H-EWPG were both reduced (p < 0.05) by the addition of MBDF, MBDF-HDEH and MBDF-HDEAG at 2–5 g/100 g. It was found that dietary fibres can increase the strength of gel’s network structure via providing a carbon skeleton and/or enhancing the WRA of gels, but reduce their springiness as a consequence (Ma et al., 2022; Zhao et al., 2023). Moreover, the declined β-turn content in H-EWPG after addition of MBDFs (Figure 5) was also responsible for the decrease in springiness of H-EWPG. Protein gels with a high content of β-turn have dense network structure and high springiness (Xu et al., 2023). Cohesiveness is indicative of a gel’s tensile strength. After the addition of MBDFs, the hydrophobic forces between protein molecules were reduced (Xiao et al., 2020), and the pH, water-holding capacity, and hardness of H-EWPG were all increased (Figure 6A and Tables 2, 3), which were all disadvantageous to a tight aggregation of proteins (Lv et al., 2022), resulting in a lower cohesiveness. In addition, the addition of these MBDFs did not change the resilience of H-EWPG (p > 0.05).