Bicontinuous aerogels constructed with protein fibrils: A template for multiscale biomaterial customization

Bicontinuous aerogels constructed with protein fibrils: A template for multiscale biomaterial customization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bicontinuous aerogels constructed with protein fibrils: A template for multiscale biomaterial customization Kefan Ouyang, Yuanyuan Feng, Songyu Wang, Zihang Yan, Qin Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7596478/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract This study elucidates the structural regulation of whey protein fibrils (WPF) via shear modification, revealing how shear rates govern their hierarchical organization. Shear forces induce a rheological shift from elastic gels (tanδ 1), with particle size reduced to ~ 273 nm at 20,000 rpm. Molecularly, X-ray diffraction reveals that shear compacts form Cross-β sheets at 9.1 Å, thereby enhancing structural density. Mesoscopically, controlled shear rates (5,000–10,000 rpm) enable tunable porosity in bicontinuous emulsions. Lower rates (5,000 rpm) yield uniform, small pores, while higher rates (10,000 rpm) enlarge pores while maintaining connectivity. This approach produces aerogels with multiscale porosity. Notably, WPF-5 aerogels—characterized by dense microporous networks—exhibit a 4,512% increase in soybean oil adsorption, highlighting their potential for biomaterials. By integrating microscopic, mesoscopic, and molecular insights, this work provides a framework for precision-engineering multiscale protein-based materials, bridging fundamental science and functional applications. Whey protein fibrils Shearing treatment Bicontinuous intra-phase emulsions Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Emulsions, as a paradigmatic example of multiphase dispersed systems, have long been a focal point in colloid and interface science due to the delicate balance between their thermodynamic metastability and kinetic stability (Ortiz et al., 2020 ). Among these, Bicontinuous intra-phase emulsions distinguish themselves with their unique interpenetrating network structure, transcending the conventional binary division into "dispersed phase" and "continuous phase." This distinctive feature offers novel perspectives for developing functional materials. By enabling continuous networks between two phases, this system markedly enhances interfacial mass transfer efficiency, showcasing significant potential in applications such as catalytic reactors and tissue engineering scaffolds (Shen et al., 2024). The breakthrough in stabilizing bicontinuous emulsions was initiated by the development of bijel (Bicontinuous interfacially jammed emulsion gels) systems, which leverage nanoparticles' self-assembly during phase separation to form a mechanical barrier at the interface through spinodal decomposition kinetics, effectively locking the bicontinuous structure (Di Vitantonio et al., 2021 ). However, traditional bijel systems impose stringent requirements on the physicochemical properties of stabilizing particles: neutral wettability (contact angle ≈ 90°) and rapid interfacial adsorption kinetics. These constraints limit suitable particle types primarily to inorganic nanomaterials like silica (Sprockel et al., 2024 ), severely restricting the application scope of bicontinuous emulsion systems, especially in biomedicine. To overcome this limitation, researchers have developed innovative stabilization strategies, including bijels and SeedGel (Di Vitantonio et al., 2021 ). These approaches extend stabilization mechanisms beyond mere interfacial adsorption to three-dimensional spatial confinement by modulating particle interactions within the bulk phase, significantly broadening the range of acceptable particle surface properties. In this context, biocolloidal materials are emerging as promising candidates for stabilizing bicontinuous emulsions due to their excellent biocompatibility and sustainability. Chitin nanocrystals (ChNC), among other biological nanomaterials, have already demonstrated successful applications in bicontinuous emulsion systems (Lu et al., 2023 ), providing valuable insights into the use of other biomacromolecular materials in such systems. Protein fibrils, as emerging bio-colloids, exhibit unique advantages: first, their fibrous structure provides high aspect ratios, facilitating the construction of three-dimensional networks; second, abundant functional groups on their surfaces offer chemical sites for interfacial modification; third, shear-induced fibrillization enables precise tuning of fibril size and flexibility (Ouyang et al., 2025 ; Xu et al., 2023 ; Zhao et al., 2022 ). Notably, current research predominantly focuses on protein fibrils in conventional emulsion systems, leaving the structure-function relationship in Bicontinuous intra-phase emulsions largely unexplored. Specifically, the regulatory mechanism of shear-induced structural evolution of protein fibrils on interfacial behavior and bulk network formation remains to be investigated. Therefore, this study aims to explore the stabilization mechanism of Bicontinuous intra-phase emulsions through shear-induced protein fibril modifications. The findings not only provide theoretical support for expanding the application of proteins in complex emulsion systems but also open new avenues for developing environmentally friendly multiscale emulsion materials. 2. Materials and methods 2.1. Materials Whey protein isolate (Hilmar 9410) was purchased from Hilmar Co., Ltd (California, USA). 2,6-Lutidine was sourced from Macklin Biochemical Co., Ltd (Shanghai, China). Thioflavin T was obtained from Solarbio Co., Ltd (Beijing, China). All other chemical reagents used were of analytical grade. 2.2. Preparation and treatments of proteins In this study, 2 M phosphoric acid was employed to unidirectionally adjust the pH of whey protein isolate (WPI) solutions containing various protein concentrations, ensuring that variations in ionic strength would not interfere with the experimental outcomes (Ouyang et al., 2023b ). The whey protein solutions were stirred at room temperature for 2 h and subsequently stored overnight at 4°C for hydration. Following this, the solutions were incubated under the following conditions: pH 2.5, WPI concentration of 4.0% (w/v), temperature of 90°C, and incubation duration of 12 h. Shear treatment was performed using a shear mixer (IKA T10, Germany) at 5,000, 10,000, and 20,000 rpm for 2 min to prepare shear-modified protein fibrils, which were designated as WPF, WPF-5, WPF-10, and WPF-20, respectively. Additionally, protein fibrils underwent ultrasonic treatment for 2 min, labeled as WPF-U. To obtain purified protein fibril samples for further structural analysis, dialysis was carried out using a dialysis membrane (Spectra/Por® Dialysis Membrane, MWCO 100 kDa) against ultrapure water (pH 2.5) at 4°C for 72 h to remove non-fibrillar proteins/peptides from the samples. 2.3. Thioflavin T (Th T) Fluorescence Assay First, 8 mg of Thioflavin T (Th T) was dissolved in 10 mL of PBS buffer. The solution was then filtered using a 0.22 µm aqueous filter. The filtered solution was diluted 50-fold with PBS to prepare the Th T working solution. Subsequently, protein samples were diluted 20-fold with ultrapure water adjusted to the corresponding pH values. Each sample (50 µL) was mixed with 5 mL of the Th T working solution and allowed to react in the dark for 1 min. Fluorescence spectra were measured using a fluorometer with an excitation wavelength of 440 nm and emission wavelengths ranging from 460 to 560 nm (both emission and excitation slit widths were set to 10 nm). For each sample, measurements were performed using Th T working solution without protein as a blank control, and background correction was applied accordingly (Ouyang et al., 2023a ). 2.4. Intrinsic Fluorescence Intrinsic fluorescence spectra were measured using an excitation wavelength of 280 nm and an emission wavelength range of 300 to 400 nm. Both excitation and emission slit widths were set to 5 nm, with a scan speed of 1200 nm/min. 2.5. ζ-Potential and Particle Size Distribution Measurement The ζ-potential and particle size distribution were measured using a Zetasizer Nano instrument (Mastersizer 2000). This method allows for precise characterization of the electrokinetic potential and size distribution of particles within the sample. 2.6. Far-Ultraviolet Circular Dichroism (CD) Spectroscopy Far-ultraviolet circular dichroism (CD) spectra of WPF were characterized using a far-UV CD spectrometer (MOS-450/AF-CD, Bio-Logic, France). The measurements were conducted in the wavelength range of 200 to 250 nm to assess the secondary structure content of the protein fibrils. 2.7. X-ray Diffraction (XRD) XRD measurements were conducted using an X-ray diffractometer (Bruker AXS D8 ADVANCE) with Cu-Kα radiation (λ = 1.5418 Å) operated at 40 kV and 40 mA. Measurements and diffraction patterns were recorded over a 2θ range of 5 to 50° at a scan rate of 3° 2θ/min. Data processing was performed using Jade 6.0 software. 2.8. Rheological Properties Prepared protein solutions were transferred to the testing plate. Rheological measurements were carried out using a DHR-2 rheometer (TA Instruments Inc., USA) equipped with a parallel plate geometry of 40 mm diameter and a gap setting of 0.5 mm at 25°C. Dynamic frequency sweeps were performed within an angular frequency range of 0.1–10 rad/s to determine the storage modulus (G′) and loss modulus (G″) of the samples within the linear viscoelastic region at a strain of 0.5%. Steady shear rheology tests were conducted in the shear rate range of 0.1–100 s⁻¹ to analyze the relationship between shear rate and apparent viscosity. 2.9. Transmission Electron Microscopy (TEM) To observe the morphological structure of the samples, a Hitachi HT7800 transmission electron microscope (Tokyo, Japan) was used, operating at an accelerating voltage of 80 kV. Negative staining of samples was performed as follows: First, 5 µL of the sample solution (protein concentration of 0.20% w/v) was placed onto a carbon-coated copper grid. After a 1-min adsorption time, excess liquid was carefully removed using filter paper. Subsequently, the samples were stained with a 2% phosphotungstic acid aqueous solution for 10 min before drying again with filter paper. TEM images were processed using FiberApp (Usov et al., 2015) to calculate two-dimensional distribution parameters (S 2D ) of the protein fibrils. 2.10. Preparation of Bicontinuous Intra-Phase Emulsions and Aerogels To prepare the miscible phase solution, WPF suspensions were mixed with 2,6-lutidine at a ratio of O/W = 28.4%, with the WPF concentration calculated based on the entire system. The mixture was first vortexed for 1 min at room temperature to ensure thorough mixing. Subsequently, the mixture was placed in a preheated water bath at 60°C for 5 min to initiate phase separation between water and lutidine, thereby forming a WPF-jammed bicontinuous intra-phase emulsion. Finally, the emulsion was incubated in an oven at 60°C for at least 24 h (Lu et al., 2023 ). To remove lutidine as much as possible, the WPF-jammed emulsion was inverted in the oven at 60°C for 24 h. The emulsion was then transferred to a freezer at -80°C to freeze its structure. Lastly, the frozen emulsion jammed hydrogel was freeze-dried to obtain protein fibril aerogels. 2.11. Optical Microscopy After vortexing the miscible phase solution for 1 min, it was heated on a hot stage at 60°C for 20 min and immediately transferred to the microscope stage for imaging. Microscopic images were captured every 20 s to document the phase transition process of the bicontinuous intra-phase system as it cooled to room temperature (25°C). 2.12. Oil Adsorption Performance The oil adsorption performance of the aerogels was evaluated by measuring their weight gain (%) after exposure to cyclohexane and soybean oil. Briefly, aerogels were immersed in the oil/organic solvent for 20 s until adsorption equilibrium was reached, defined as no further liquid dripping from the aerogel. The aerogels were then immediately weighed (Bi et al., 2013 ). The weight gain (Wg) of the aerogel after adsorption was calculated using the following formula: \(\:{W}_{g}\:=\:\frac{{m}_{c}-{m}_{0}}{{m}_{0}}\times\:100\) %(1) Where m c and m 0 represent the weights (g) of the aerogel after and before adsorption, respectively. 2.13. Scanning Electron Microscopy (SEM) Scanning electron microscopy (SEM) images were acquired using a Regulus 8100 microscope (Hitachi, Tokyo, Japan). Freeze-dried samples were sputter-coated with gold prior to imaging. The accelerating voltage of the electron beam was set to 5.0 kV. 2.14. Statistical analysis Data are presented as mean ± standard deviation. All experiments were performed at least in triplicate to ensure reliability and accuracy. Statistical analysis was conducted using Origin 2024 software (OriginLab Corporation, USA). Analysis of variance (ANOVA) was used to determine significant differences between means, followed by Tukey’s test for pairwise comparisons. The significance level was set at p ≤ 0.05. 3. Results and discussion 3.1. Macroscopic Effects of Shear Modification on Protein Fibrils Shear modification exerts a significant regulatory effect on the macroscopic structure of protein fibrils. Rheological tests and polarization imaging were employed to investigate the macroscopic changes in protein fibrils after treatment at varying shear rates. Rheological profile indicate that protein fibril solutions exhibit typical shear-thinning behavior, likely due to the disruption of fibril entanglements as shear rate increases ( Figure. 1a-d ). At low shear rates, extensive fibril entanglements result in higher viscosity; at high shear rates, some fibrils align along the flow direction, reducing entanglements and lowering viscosity (Loveday et al., 2012 ). As the shear processing speed increases, the apparent viscosity of protein fibril solutions decreases, accompanied by reductions in both storage modulus (G') and loss modulus (G''). Similarly, with increasing shear processing speed, the entangled network of fibrils is progressively disrupted, leading to decreased aggregation within the system and ultimately resulting in lower viscosity (Zhao et al., 2022 ). The value of tanδ can indicate the state of a material within a certain range. When tanδ 1, the material's viscosity dominates, showing liquid-like properties; and when tanδ ≈ 1, the material exhibits typical viscoelastic behavior with comparable elasticity and viscosity. Overall, protein fibril solutions during the shearing process have tanδ < 1, indicating that fibril entanglements form a gel-like network structure, making the system more inclined toward solid characteristics (WPF, WPF-5, WPF-10, WPF-U). The fibrillization process of proteins, such as β-lactoglobulin, can lead to entanglement phenomena. Under specific conditions, they can assemble into fibrous hydrogels. This process involves the fibrillization and cross-linking of protein monomers, during which semi-flexible long fibrils intermingle. The number and distribution of entanglements directly affect the elastic behavior of the protein fibril network (Cao et al., 2018 ). Compared to untreated solutions, WPF-5 and WPF-10 exhibit tanδ values closer to 1. This phenomenon may be attributed to the destruction of fibril entanglement structures by shear forces, leading to a reduction in the number of cross-linking points and a decrease in material elasticity, shifting its rheological behavior towards the viscoelastic transition zone. However, when the shear rate is increased to 20,000 rpm, WPF-20 solutions display typical fluid flow characteristics, with tanδ values exceeding 1 at frequencies above 1 Hz. This suggests that extreme shear conditions may cut semi-flexible long fibrils into rigid short fibrils, significantly reducing the number of entanglements compared to long fibril systems, thus altering their rheological properties. In addition to changes in flow characteristics, birefringence under polarized light also shows significant differences ( Figure. 1e ). Birefringence indicates anisotropic optical properties in the medium, where refractive indices differ in various directions (Bolisetty et al., 2011 ). Experimental results reveal that birefringence is observed in WPF solutions, suggesting a transition from isotropic to nematic liquid crystal phases. As shear rates gradually increase, birefringence in the solution diminishes and eventually disappears under high shear rates (20,000 rpm). This phenomenon implies that high shear forces disrupt the ordered arrangement of fibril aggregates. When shear intensity reaches a critical value, the originally oriented fibril aggregates completely dissociate, leading to the disappearance of macroscopic birefringence (Mezzenga et al., 2010 ). Notably, ultrasonic treatment does not affect the birefringence characteristics of the solution. To further investigate the mechanism, we used polarization microscopy and potential measurements to analyze the effects of shear treatment on the microscopic structure of protein fibrils. 3.2. Microscopic Effects of Shear Modification on Protein Fibrils To explore the impact of shear treatment on whey protein fibrils at the microscopic level, we employed polarization microscopy and ζ-potential measurements to systematically observe the evolution of microscopic morphology in sheared whey protein fibrils. Polarization microscopy revealed that although shear significantly disrupts the macroscopic structure of protein aggregates ( Figure. 1f ), under the microscope, sheared fibril aggregates still exhibit characteristic yellow birefringence, a hallmark of amyloid fibrils (Yang et al., 2023a ). This phenomenon indicates that while the macroscopic structure may be compromised, the fibrils retain an ordered arrangement at the microscopic level, maintaining their anisotropic characteristics. In studying the dispersion characteristics of protein fibril solutions, we comprehensively evaluated how high-speed shear treatment affects their particle size distribution and ζ-potential ( Figure. 1g-i ). The shear treatment markedly alters the dispersion behavior of fibril solutions, primarily manifesting as a significant reduction in particle size. Untreated WPF solutions had an average particle size of 439.50 ± 12.52 nm with a polydispersity index (PDI) of 0.56 ± 0.04, indicating a poorly dispersed polydisperse system. When shear rates reached 10,000 rpm and 20,000 rpm, the particle size distribution shifted from an initial bimodal distribution to a uniform unimodal distribution (WPF-10: 300.83 ± 20.53 nm, WPF-20: 273.10 ± 14.09 nm), with a concurrent decrease in PDI (WPF-10: 0.46 ± 0.07, WPF-20: 0.34 ± 0.02). This suggests that high-speed shear effectively shortens and homogenizes fibril lengths by disrupting inter-fibril aggregation and entanglement (Zhao et al., 2022 ). Notably, ultrasonic treatment reduced the average particle size of WPF to 293.77 ± 23.45 nm but did not significantly change the PDI (WPF-U: 0.62 ± 0.04), indicating that it only dissociated large aggregates without improving dispersion uniformity. The surface potential (ζ-potential) of colloidal particles is a key parameter for assessing the stability of colloidal dispersions. Experimental data indicate that shear treatment generally has a limited effect on the ζ-potential of WPF (WPF: 50.87 ± 1.03 mV, WPF-5: 48.80 ± 3.30 mV, WPF-10: 48.57 ± 2.87 mV). However, under high-speed shear conditions of 20,000 rpm, the ζ-potential of WPF-20 significantly decreased to 43.17 ± 1.19 mV. This phenomenon may be related to changes in the surface characteristics of fibril particles; under high shear stress, some functional groups on the fiber surface may rearrange or detach, leading to a reorganization of surface charges and a slight decrease in ζ-potential (Ren et al., 2018 ). These results demonstrate that high-speed shear treatment not only significantly alters the particle size distribution of protein fibril solutions, making them more uniform and smaller, but achieves this effect by disrupting physical entanglements between fibrils rather than altering surface charge characteristics. This provides a new technical approach for optimizing dispersion systems while maintaining surface electrical stability, with relatively minor impacts on ζ-potential. These findings offer experimental evidence for understanding the dispersion behavior of complex fluids under high shear environments. 3.3. Mesoscopic Effects of Shear Modification on Protein Fibrils At the mesoscopic scale, we employed transmission electron microscopy (TEM) to image the microstructure of fibril samples and used FiberApp software to calculate the two-dimensional orientation parameter (S 2D ), thereby characterizing the morphological and alignment changes of the fibrils. TEM imaging can clearly reveal the microscopic structural features of fibrils, while the S 2D parameter provides a quantitative analysis tool for understanding the orientation patterns and dynamic changes of fibrous polymers under different conditions (Usov et al., 2015). TEM images show that under 12-h thermal induction, whey protein isolate undergoes fibrillization to form semi-flexible protein fibrils (WPF) with significant aspect ratios ( Figure. 2a ). Compared to the evident entanglement in WPF, ultrasonically treated fibrils (WPF-U) exhibit a more uniform distribution. This may be attributed to the cavitation effect of ultrasound disrupting the microscopic structure of fibril aggregates, thereby improving fiber entanglements. Additionally, larger gel regions are visible in WPF micrographs, which transform into uniformly dispersed smaller gel particles after ultrasonic treatment (WPF-U). Notably, no local gel formation was observed in shear-treated samples, correlating with changes in solution rheological properties. Compared to WPF-U, shear treatment more effectively truncates long fibrils into short ones, significantly reducing physical entanglements between fibrils while maintaining their linear structures (Zhao et al., 2022 ). Specifically, at a shear rate of 20,000 rpm, WPF-20 samples exhibit highly uniform fibril sizes (approximately 500 nm) and, due to the shortened length, display characteristics closer to rigid short fibrils (Cao et al., 2018 ). In two-dimensional orientation analysis, TEM images were processed using FiberApp software to calculate the S 2D parameter, defined as S 2D = 2 -1 (Usov et al., 2015). Compared to untreated WPF (S 2D = 0.12), ultrasonically treated fibrils showed a more isotropic alignment (S 2D = 0.064), indicating a trend toward isotropic distribution ( Figure. 2b-d ). This change likely results from ultrasound-induced cavitation leading to random redistribution of fibrils, thus weakening nematic order (Adamcik et al., 2010 ). On the other hand, the higher initial bulk concentration (C init = 0.20%, w/v) might also contribute to the overall tendency towards isotropy (Jordens et al., 2013 ). However, after shear treatment at 20,000 rpm, the two-dimensional distribution of protein fibrils became more nematic (S 2D = 0.24). This change could be due to truncated semi-flexible fibrils aligning into a nematic phase when they come close together, minimizing orientation entropy by straightening out ( Figure. 2e ). From an energetic perspective, this arrangement lowers the total free energy of the system, making the fibrils more inclined to exist in a nematic phase (Jordens et al., 2013 ). These findings indicate that shear treatment significantly enhances the uniformity and alignment order of fibril sizes, whereas ultrasonic treatment reinforces the uniformity and isotropy of fibril distribution. The TEM observations are highly consistent with particle size analysis data, providing critical experimental evidence for elucidating the behavior mechanisms of protein fibrils under various processing conditions. 3.4. Molecular-Level Effects of Shear Modification on Protein Fibrils At the molecular scale, the regulatory effect of shear treatment on protein conformation is equally crucial (Huyst et al., 2021 ). To comprehensively assess the impact of shear treatment on protein conformation and characteristic fibril structures, we employed various detection methods, including intrinsic fluorescence spectroscopy, Th T fluorescence spectroscopy, far-UV circular dichroism (CD), and X-ray diffraction (XRD). Intrinsic fluorescence spectroscopy is commonly used to track the conformational evolution of protein tertiary structure ( Figure. 3a ). Results show that after 12 h of heating, the maximum emission wavelength of WPI shifts from 341 nm to 349 nm. This redshift may be due to changes in the internal microenvironment of the protein, making the microenvironment of the Trp chromophore more hydrophilic (Jones et al., 2012). Notably, the intrinsic fluorescence intensity of sheared fibrils shows only a slight decrease, and similar characteristics are observed in ultrasonically treated WPF-U samples, indicating that shear and ultrasound treatments have limited effects on the tertiary structure of proteins. Their primary influence may be at the secondary structure or aggregation state level. Th T dye, as a specific probe for amyloid fibrils, can be used to evaluate the content of characteristic structures (Cross-β) in protein fibrils (Arad et al., 2020 ). Through Th T-specific fluorescence spectroscopy, it was found that WPI exhibited high fluorescence intensity after 12 h of thermal treatment, confirming the formation of amyloid fibril structures ( Figure. 3b ). Notably, shear treatment reduced Th T fluorescence intensity, particularly under shear conditions of 10,000 rpm and 20,000 rpm (WPF-10/20), where fluorescence intensity significantly decreased. This result suggests that shear treatment may partially disrupt Cross-β structures. In contrast, ultrasonic treatment did not significantly affect Th T fluorescence ( Figure. 3c ). To further investigate this phenomenon, we measured the CD spectra of protein solutions ( Figure. 3d ). The results showed that shear treatment had a minimal effect on secondary structures, but under high-speed shear conditions of 20,000 rpm, the β-sheet content decreased (at 215 nm) (Cao et al., 2021 ), which may be related to local conformational changes induced by mechanical stress. Cross-β structures ( Figure. 3f ), as the core feature of amyloid fibrils, consist of parallel-aligned β-sheets. β-chains are perpendicular to the long axis of the fibrils, forming layered structures with inter-chain distances (d 1 ) of approximately 4.8 Å, maintained by hydrogen bonds between peptide bonds; inter-layer distances (d 2 ) range from 10–12 Å, depending on the stacking mode of sheets (Cao et al., 2019). The distance between β-sheets in Cross-β structures has a decisive impact on the nucleation ability of fibril core structures (Yang et al., 2023b ). To further explore the effect of shear on the Cross-β structure of protein fibrils, we conducted XRD tests ( Figure. 3d ). The results showed that both sheared and ultrasonically treated protein fibrils exhibited two characteristic peaks representing the distances between β-sheets and β-chains, indicating that these fibrils still maintained complete Cross-β structures. Notably, high-speed shear treatment significantly reduced the inter-layer distances of Cross-β structures in fibrils. Compared to untreated WPF samples (9.70 Å), the inter-layer distances of Cross-β structures in sheared fibrils decreased to around 9.1 Å (WPF-5: 9.11 Å, WPF-10: 9.09 Å, WPF-20: 9.16 Å). This result indicates that, unlike hydrolysis treatment which makes Cross-β structures looser, high-speed shear treatment makes them more compact. This denser structure may be less conducive to the nucleation ability of sheared fibrils. Additionally, ultrasonically treated protein fibrils showed no significant changes in inter-sheet distances and β-chain spacings compared to untreated samples. Overall, shear treatment not only affects the macroscopic, microscopic, and mesoscopic states of protein fibrils but also exerts regulatory effects at the molecular level. These experimental results reveal how shear treatment alters the behavior of protein fibrils across different scales: from macroscopic rheological properties and birefringence phenomena to microscopic size and distribution, to mesoscopic two-dimensional orientation parameters (S 2D ), and to molecular-level secondary and tertiary structural changes. These findings highlight the multiscale impact of shear treatment on whey protein fibrils. Importantly, these insights provide theoretical foundations for applying such treatments in Bicontinuous intra-phase emulsions. For instance, when designing protein fibril-based materials with multiscale structures and functional properties, shear treatment conditions can be adjusted to control fibril morphology, alignment, and molecular conformation, thereby optimizing material performance. 3.5. Performance of Proteins in Bicontinuous Intra-Phase Emulsions Bicontinuous intra-phase (BC) emulsions exhibit application potential in functional materials due to their three-dimensional continuous network structure formed by interpenetrating aqueous and oil phases. Particle-mediated jamming behavior is considered a key mechanism for regulating the structural stability of these systems. This part of the experiment investigates the impact of thermal treatment on phase jamming phenomena in protein solutions, aiming to reveal the potential of protein particles in stabilizing the formation of bicontinuous structures. The experiments are based on the phase separation properties of the water/Lutidine miscible system: at room temperature (25°C), water and Lutidine form a homogeneous mixed solution. Upon heating to the critical temperature (34.1°C, corresponding to a 28.4% Lutidine concentration), the system undergoes spinodal decomposition, leading to the formation of a bicontinuous phase structure through jamming (Grattoni et al., 1993 ; Stratford et al., 2005 ). During this process, by regulating the dynamic adsorption behavior of protein particles during phase separation, it may be possible to achieve morphological locking of non-equilibrium bicontinuous structures. The mixed system was heated to 60°C to trigger phase separation. It is hypothesized that WPF particles might inhibit the rapid expansion of the Lutidine phase through interfacial adsorption, thereby hindering the transition of the system towards a thermodynamic equilibrium state (dispersed matrix phase), ultimately forming a non-equilibrium bicontinuous structure ( Figure. 4 ). Firstly, WPF solution was mixed with Lutidine at a volume ratio of 71.6:28.4 (v/v) and vortexed for 60 s to ensure uniform dispersion, resulting in a miscible solution. Prior to heat-induced phase separation, the optical characteristics of the solution were observed using polarization microscopy. Results showed that untreated WPF miscible phase solutions exhibited significant birefringence, indicating that WPF aggregates displayed an ordered orientation distribution within the solution, possessing optical anisotropy (Bolisetty et al., 2011 ). However, as the solution underwent high-speed shear treatment, the intensity of birefringence gradually decreased with increasing shear rate, suggesting that shear forces disrupted the ordered arrangement of WPF aggregates. Notably, ultrasonic treatment did not significantly alter the birefringence characteristics of the miscible phase. These observations are highly consistent with the behavior of WPF in pure aqueous solutions—where the ordered structure of WPF in the aqueous phase is similarly sensitive to shear treatment. Therefore, it can be inferred that the distribution pattern of WPF in the water/Lutidine miscible phase does not fundamentally differ from that in pure water, indicating a universal structural response mechanism across solvent systems. Upon heating to 60°C to trigger phase separation in the water/Lutidine miscible system, it is hypothesized that WPF particles can regulate phase separation kinetics through interfacial adsorption. Specifically, WPF particles might inhibit the rapid expansion of the Lutidine phase by adsorbing at the phase interface, thus preventing the system from transitioning to a thermodynamic equilibrium state (dispersed matrix phase), ultimately locking the non-equilibrium bicontinuous phase structure (Lu et al., 2023 ). Experimental results confirmed this hypothesis: when the miscible phase solution (WPF concentration 2.0% w/v) was heated to 60°C and maintained for 20 min, untreated WPI solutions failed to form bicontinuous intra-phase emulsions due to the lack of effective interfacial regulation mechanisms. Conversely, WPF and its sheared (WPF-5/10/20) or ultrasonically treated (WPF-U) samples successfully constructed stable bicontinuous structures through jamming effects. Notably, high shear rates (WPF-20) led to a decrease in emulsion thermal stability, likely due to excessive shear disrupting the ordered arrangement of WPF aggregates, weakening their jamming capabilities. After being left at room temperature for 12 h, all systems except WPF-20, which collapsed completely due to initial structural instability, maintained a stable gel-like emulsion state, further validating the formation of bicontinuous intra-phase emulsion gels (Lu et al., 2023 ). On the other hand, the appearance of bicontinuous emulsions gradually changed from initially white to translucent, possibly due to enhanced miscibility between Lutidine and water during cooling. As the temperature dropped below the critical temperature, the mutual solubility of Lutidine and water increased, reducing interfacial tension and causing partial mixing, leading to protein particle sedimentation or aggregation, decreasing interfacial coverage, and altering light scattering paths, thus shifting the system from highly scattering white to less scattering translucent states. Despite this, the specific mechanisms still require validation through microscopic characterization using optical microscopy. 3.6. Microscopic Structure of Bicontinuous Jammed Emulsions To analyze the microscopic structure of Bicontinuous Intra-Phase (BC) Emulsions, this experiment employed optical microscopy to observe the micro-morphology of solutions after heating at 60°C for 5 min and documented dynamic changes during the cooling process at room temperature (25°C) ( Figure. 5 ). At a WPI concentration of 2.0%, the biphasic system only briefly formed an emulsion structure post-heating, which demulsified within 20 s due to imbalanced interfacial tension, leading to rapid merging into a homogeneous phase. When the WPI concentration was increased to 3.0%, the system formed a bicontinuous structure through thermal induction but became unstable after 40 s due to insufficient interfacial coverage, ultimately reverting to a single phase. This observation suggests that WPI needs to reach a relatively high concentration (3.0%) to initially form an interpenetrating network of bicontinuous structures, yet its stability remains limited by the strength of interfacial interactions. After regulating fibril dispersion via shear treatment, the effect of shear rate on the pore size of the bicontinuous phase became pronounced. Untreated WPF, due to severe entanglement and aggregation of fibrils within the system, resulted in highly uneven phase size distribution in the bicontinuous structure formed after heating. Insufficient local interfacial adsorption led to structural defects such as abnormally enlarged pores or localized occlusions. Despite these defects, the robust bicontinuous network could maintain topological connectivity through tight packing, remaining stable for up to 60 s. At a shear rate of 5,000 rpm (WPF-5), moderate shear effectively improved fibril aggregation, alleviating long fibril entanglements, allowing fibrils to disperse uniformly in the system in slightly shorter yet still flexible forms. This dispersion supported the formation of a stable three-dimensional interpenetrating network, with more uniform phase size distribution and significantly reduced pore size variation, maintaining structural stability for 60 s. Increasing the shear rate to 10,000 rpm (WPF-10) further enhanced the synergistic effects of shear cutting and fibril dispersion, significantly increasing pore sizes (nearly doubling compared to WPF-5), while fibril alignment increased interfacial coverage density, thus maintaining structural stability for 60 s. However, at a shear rate of 20,000 rpm (WPF-20), excessive shear forces drastically shortened the aspect ratio of fibrils, converting them into rigid short fibrils, with significantly reduced interfacial adsorption capabilities due to structural damage. Consequently, the support capacity of the continuous phase network weakened, leading to rapid destabilization after 40 s due to localized pore closure. These results suggest that shear rates should be controlled within a reasonable range of 5,000–10,000 rpm: 5,000 rpm can prepare bicontinuous structures with small pore sizes and high uniformity by improving fibril dispersion, whereas 10,000 rpm can moderately enlarge pore sizes to form large-pore networks while maintaining structural integrity, thereby achieving precise control over pore size. After ultrasonic treatment (WPF-U), fibrils formed a uniform and dense bicontinuous network during heating. Compared to the untreated group (which destabilized after 40 s), the ultrasonically treated group remained stable for 60 s, indicating that ultrasound may enhance interfacial stability in the bicontinuous phase system by reducing fibril aggregation. Experimental results show that ultrasonic pretreatment is an effective method to improve the uniformity of the bicontinuous phase structure. 3.7. Mechanism of Protein-Induced Phase Jamming Phase jamming mechanisms are complex processes involving the synergistic effects of particle concentration, interfacial adsorption, and steric hindrance. In binary solvent systems approaching phase separation critical points, fluctuations in solvent concentration intensify. If particles selectively adsorb one component of the solvent, an adsorption layer forms on their surfaces (Wang et al., 2018 ). As the temperature approaches the critical point, the correlation length of the solvent increases, leading to thicker adsorption layers, which in turn alters the interaction energy between particles, ultimately resulting in solvent-mediated critical Casimir forces that promote particle attraction (Bertrand et al., 2015 ). However, steric hindrance limits excessive aggregation by physically impeding particle clustering. This dynamic equilibrium among multiple factors is central to the stabilization of bicontinuous intra-phase (BC) emulsions by bio-based particles, directly influencing particle distribution, interfacial properties, and emulsion stability, thus serving as a key factor in regulating the structure of BC emulsion materials ( Figure. 6 ). At a WPI concentration of 2.0%, the system fails to form BC emulsions due to insufficient particle numbers. With large particle spacings and weak interactions, these particles cannot form continuous networks through tight packing, leading to free droplet coalescence and eventual demulsification into a single phase. However, when the WPI concentration is increased to 3.0%, the number of particles significantly increases, reducing spacing and enhancing inter-particle interactions. These particles form physical jams through tight packing, restricting droplet mobility and successfully constructing a bicontinuous phase structure. Additionally, proteins, as bio-based particles, form dense protective layers at interfaces through adsorption, further regulating interfacial tension and modifying interfacial rheological properties. Reduced interfacial tension decreases droplet coalescence tendencies, while improved interfacial rheology enables uniform dispersion of droplets within the continuous phase, synergizing with particle crowding effects to stabilize BC emulsions. Compared to traditional silica particles, which require high concentrations (24.3%) to form stable networks due to weaker inter-particle interactions (Xi et al., 2021 ), protein-based particles can construct dense networks at lower concentrations via strong interfacial adsorption and intermolecular forces, providing an efficient pathway for designing functional materials. Importantly, the rich structural variations of proteins offer additional topological control for the formation and construction of BC emulsions. Furthermore, whey protein fibrils (WPF), owing to their elongated aspect ratios, can stabilize BC emulsions even at a concentration of 2.0%. This highlights the significant impact of particle shape on stabilizing emulsions, with elongated particles showing clear advantages in forming and stabilizing bicontinuous emulsions. However, entanglement issues with semi-flexible long fibrils lead to uneven fiber distribution in some regions, preventing uniform jamming structures and causing defects. Such structural imperfections may affect the stability and performance of BC emulsions. Notably, Lu et al. ( 2023 ) attempted to use cellulose nanofibers (ChNF) to construct BC emulsions but failed to achieve stable jamming structures, possibly due to ChNF's strong flexibility and insufficient intrinsic rigidity. However, WPF shares similar structural characteristics (e.g., flexibility and low stiffness) yet successfully stabilizes BC emulsions, suggesting that the aforementioned hypothesis might not be the root cause. Based on experimental results, it can be inferred that ChNF's failure is more likely due to insufficient concentration (0.6%) failing to reach the threshold required for effective three-dimensional network support. Increasing the concentration could potentially enable ChNF to stabilize BC emulsions. Moderate shear treatment of WPF (e.g., 5,000 rpm or 10,000 rpm) transforms fibril morphology from semi-flexible long fibrils to rigid short fibrils. This transformation leads to: 1) Fibril Morphology Regulation: Short fibril networks formed by breaking long fibrils achieve more uniform dispersion and tighter packing, reducing local defects caused by long fibril entanglements. Shear flow induces nematic ordering along the flow direction, forming oriented aggregate structures. This arrangement reduces random aggregation, providing efficient channels for phase separation and enlarging pore size distributions; 2) Structural Stability Maintenance: Studies by Huyst et al. ( 2021 ) show that low-speed shear does not significantly alter protein structure due to unchanged interfacial contact areas, thus retaining the original mechanical support capability of protein fibrils. Retaining linear structures ensures short fibril networks possess both rigidity for maintaining pore connectivity and reduced interface defects for enhanced stability. Therefore, by adjusting shear rates, precise control over pore size distribution (e.g., 5,000 rpm maintains small pores, 10,000 rpm enlarges pores) can be achieved, balancing structural stability and functional tunability, offering effective strategies for customized material design. However, in the WPF-20 system, excessively high shear rates (20,000 rpm) introduce dual defects: 1) Interface Interaction Imbalance: High surface charge density on protein fibrils leads to strong electrostatic repulsion (dominated by net repulsion), hindering sufficient aggregation and coverage at interfaces (Xi et al., 2021 ); 2) Morphological Degradation and Support Failure: Shearing drastically reduces the aspect ratio of fibrils, compromising their mechanical support capabilities needed for BC emulsion networks. Despite high particle concentrations, shortened fibrils fail to achieve the critical threshold for "crowding effects" through physical jamming or interfacial adsorption, rendering the BC emulsion structure unstable. On the other hand, ultrasonic treatment improves the uniformity of WPF in BC emulsions through multi-scale actions, involving structural regulation and defect suppression. On a macro scale, ultrasonic treatment maintains the nematic ordering of protein fibrils (birefringence phenomenon), providing a critical framework for the topological connectivity of BC emulsions. On a mesoscopic scale, high-frequency vibrations and cavitation bubble collapse induce random reorientation of fibrils within two-dimensional planes, reducing inherent anisotropy and achieving a more isotropic distribution (lower S 2D ). This "macro-order-meso-disorder" synergy retains long-range orientation along the flow direction to maintain pore connectivity in BC emulsions while suppressing fibril entanglement and localized aggregation through uniform dispersion at the mesoscopic scale, avoiding droplet coalescence or phase separation due to uneven interface coverage. Therefore, ultrasonic pretreatment represents a viable approach for preparing biomaterial networks. Thus, by uniformly mixing WPF solutions with Lutidine and heating at 60°C to trigger phase separation, BC emulsions can be formed. This heating step drives solvent systems close to phase separation critical points, prompting WPF fibrils to self-assemble into three-dimensional interpenetrating networks under thermodynamic driving forces, thereby stabilizing BC emulsion structures. Subsequently, inverting the emulsion container and incubating at 60°C for 12 h leverages gravity to further drive phase separation and solidification of the solvent-nonsolvent system (Lu et al., 2023 ). During this process, inversion facilitates the outflow of the Lutidine phase from continuous pore structures, promoting solvent component separation and uniform gelation, ultimately forming structurally stable BC hydrogels. Finally, freeze-drying techniques transform water in the BC hydrogel into ice crystals, which are then removed by vacuum sublimation. This process avoids structural collapse associated with solvent evaporation during traditional drying methods, with ice crystal sublimation preserving the original fibril network topology within the gel, ultimately yielding BC cryogels with multiscale pore skeletons. The entire process achieves precise structural transfer from liquid to solid phases through coordinated temperature control, phase jamming processes, and freeze-drying techniques, providing a controllable path for preparing bio-based porous materials. Based on experimental results, WPF-U, WPF-5, and WPF-10 samples were selected for further preparation of BC cryogels. 3.8. Construction of Protein-Based Aerogels Using Bicontinuous Emulsions By subjecting uniform bicontinuous intra-phase (BC) emulsions to inverted incubation and subsequent freeze-drying, we successfully prepared three types of cryogels (WPF-U cryogel, WPF-5 cryogel, and WPF-10 cryogel). Due to their ultra-light weight, the fabricated aerogels could be stably placed on top of a single dandelion seed ( Figure. 7b ) and lifted by the static electricity of a plastic measuring spoon ( Figure. 7c ). To further characterize these aerogels, their micro-morphology was observed using scanning electron microscopy (SEM), and their oil adsorption capacity was evaluated by measuring the weight gain after oil absorption (Bi et al., 2013 ). SEM observations revealed that the WPF-U aerogel exhibited a highly uniform lamellar structure with large pores and thin, dense layers ( Figure. 7d ). This structural feature directly reflects the initial three-dimensional network formed during the curing process of the bicontinuous phase emulsion (Jia et al., 2021 ). However, at high magnifications, the impact of the freeze-drying process on the microstructure can be observed: sparse distributions of micropores formed during freeze-drying. Although the WPF-U aerogel maintained uniformity and density of the bicontinuous structure at the macro scale, its micro-porosity was not further refined, failing to form an improved multiscale pore network. Its structural characteristics are more akin to traditional porous shell-like materials prepared via the bicontinuous gel template method (Lee et al., 2010). This structure may result from the entanglement of semi-flexible long protein fibrils—when fibers are not sufficiently dispersed, their higher aspect ratios tend to form local entanglements in solution, hindering uniform micropore generation and resulting in thicker layers and insufficient micropore density. Despite its excellent performance in soybean oil adsorption tests (weight gain of 2,766%), the adsorption capacity of WPF-U is significantly lower than that of WPF-5, likely due to the limitations of larger pore sizes: while larger pores can accommodate soybean oil molecules, insufficient micropore density reduces the number of adsorption sites, limiting specific surface area and adsorption efficiency. In contrast, the WPF-5 aerogel exhibits a remarkable multiscale pore size structure ( Figure. 7e ). Overall, its macroporous structure is similar to that of WPF-U, but it features densely distributed and highly interconnected micropores on the fibril scaffold during freeze-drying, forming a channel network. The formation of this micropore network closely aligns with the initial structure of the BC emulsion: moderate shear (5,000 rpm) ensures even dispersion of fibrils, avoiding entanglements, while the semi-flexibility of fibrils allows them to aggregate into stable interpenetrating networks during phase jamming. During freeze-drying, ice crystal structures produced by water sublimation further refine the microstructure of hydrogel layers, ultimately forming fine and uniform multiscale micropores. This dual advantage of structural design lies in the macropores ensuring mechanical strength and permeability, while the micropore network significantly enhances specific surface area, providing abundant adsorption sites for molecules. Oil adsorption tests confirmed this advantage, with WPF-5 achieving a soybean oil adsorption capacity of 4,512% weight gain, significantly surpassing WPF-U (2,766%) and WPF-10 (3,071%). This result validates the improvement effect of moderate shear on fibril dispersibility—it avoids structural defects caused by entanglements and retains the integrity of the fibril network through freeze-drying, thus optimizing the distribution of multiscale pore sizes. Although the WPF-10 aerogel also displays macroporous structures, its microstructure shows significant heterogeneity ( Figure. 7f ). In high-magnification SEM images, most regions lose their bicontinuous characteristics due to collapse during freeze-drying, leaving only a few micropores. This phenomenon indicates that excessively high shear rates (10,000 rpm) shorten fibril lengths and alter aggregation morphology but weaken the support capability of the fibril network. During freeze-drying, insufficiently supported fibril networks cannot withstand the stress from ice crystal sublimation, leading to localized collapses and ultimately forming heterogeneous microstructures with poor connectivity. Despite maintaining continuous phase-formed macroporous structures on the surface, inadequate micropore density and accumulated structural defects result in lower soybean oil adsorption capacity (weight gain of 3,071%) compared to WPF-5. The adsorption capacities of the three aerogels for n-hexane (weight gain of 1,637%–2,064%) show no significant differences, possibly due to the small size of n-hexane molecules, which can freely diffuse into all pores. However, soybean oil molecules are larger, making their adsorption capacity more dependent on pore size structure. The multiscale pore network of WPF-5 therefore demonstrates superior performance. This comparison indicates that the regulation of pore size distribution by shear rate directly affects the functional properties of aerogels, while the synergistic effect of multiscale structures is crucial for enhancing specific surface area and adsorption capacity. 4. Conclusions This study investigates the multi-scale structural regulation of whey protein fibrils (WPF) via shear modification and their application in bicontinuous intra-phase emulsions. Macroscopically, shear disrupts fibril entanglements, altering rheological properties from elasticity-dominated to liquid-like behavior, while maintaining microscopic order even as birefringence disappears at high shear rates (20,000 rpm). Microscopically, shear reduces fibril size to 273 nm (PDI = 0.34), enhancing dispersion but decreasing ζ-potential due to surface group rearrangement. Mesoscopically, TEM and S 2D analysis show that high shear cuts long fibers into shorter ones (500 nm) with nematic ordering (S 2D = 0.064), whereas ultrasonic treatment increases isotropy (S 2D = 0.24). Molecularly, XRD indicates no disruption of the Cross-β structure but a decrease in Cross-β sheet spacing to 9.1 Å, suggesting a more compact structure. In BC emulsions, WPF stabilizes structures at 2.0% concentration, far lower than silica particles (24.3%), due to semi-flexible long fibrils. Controlled shear rate optimizes pore size: 5,000 rpm minimizes entanglement for uniform small pores, 10,000 rpm enlarges pores while maintaining connectivity, whereas 20,000 rpm causes structural collapse. Ultrasonic pretreatment enhances uniformity through macroscopic order-mesoscopic disorder regulation. Using heating-induced phase separation and freeze-drying, we fabricated BC cryogels with multiscale pores. The WPF-5 cryogel, with its dense micropore network, showed superior soybean oil adsorption (4,512% weight gain) compared to WPF-U (2,766%) and WPF-10 (3,071%). N-hexane adsorption was similar across samples (1,637%–2,064%), highlighting the importance of multiscale porosity and shear control in functional material design. These results emphasize the significance of multi-scale optimization and controlled shear processing for advanced functional materials. Declarations Ethics declarations Competing interests The authors declare no competing interests. Funding This research was financially supported by the National Natural Science Foundation of China (32460588), the Natural Science Foundation of Jiangxi Province (20232BAB205075), and the Nanchang University Jiangxi Province Financial Science and Technology Special Project (ZBG20230418037) Author Contribution Kefan Ouyang: Methodology, Investigation, Visualization, Writing - Original Draft. 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21:05:08","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127354,"visible":true,"origin":"","legend":"","description":"","filename":"a0250c3e648b4ef89ac6b8ed6fe83bf61structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7596478/v1/6e9dfc5bf85cb04afbca1a70.xml"},{"id":92749826,"identity":"6b2215a2-3947-4820-8c57-6820d35cebc9","added_by":"auto","created_at":"2025-10-03 21:05:08","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":135181,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7596478/v1/608f88f926a29a324975e1e9.html"},{"id":92749802,"identity":"e50e7869-7bb4-47b6-a5dd-142f6a5bf001","added_by":"auto","created_at":"2025-10-03 21:05:07","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":90875,"visible":true,"origin":"","legend":"\u003cp\u003eRheology (a-d), polarized light imaging (e), polarized light microscopy imaging (f), particle size distribution (g), average particle size (h), and ζ-potential of protein fibrils (i); The different letters respectively represent significant differences at the \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 level\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7596478/v1/1c83fc7b084384c205fbd6b4.jpg"},{"id":92749806,"identity":"a102dbab-28ef-4eec-818f-1be4eb62e447","added_by":"auto","created_at":"2025-10-03 21:05:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":113859,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission electron microscopy imaging (a), 2D parameter distributions (WPF: b, WPF-20: c, WPF-U: d), and sketch of protein fibrils (f)\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7596478/v1/119bacebce142a8708c45a12.jpg"},{"id":92749803,"identity":"79972a02-0caf-4df6-9e48-04d4caa65c5f","added_by":"auto","created_at":"2025-10-03 21:05:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":38563,"visible":true,"origin":"","legend":"\u003cp\u003eIntrinsic fluorescence spectra of protein fibrils (a), Th T characteristic fluorescence (b-c), far-ultraviolet circular dichroism spectra (d), XRD (e), Cross-β structure (f); Different letters represent significant differences at the \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 level\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7596478/v1/40888886bb6840b9486c9b2e.jpg"},{"id":92750237,"identity":"095a216e-c2fb-43e9-a27e-0e88f89215d7","added_by":"auto","created_at":"2025-10-03 21:21:07","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":89679,"visible":true,"origin":"","legend":"\u003cp\u003ePhotos of the miscible solution and the phase-jammed emulsion of protein fibrils\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7596478/v1/42d4d5bb38495ff000ecbb01.jpg"},{"id":92750121,"identity":"5a1971f7-4176-4d66-92a8-843b301b5e96","added_by":"auto","created_at":"2025-10-03 21:13:07","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":127224,"visible":true,"origin":"","legend":"\u003cp\u003eThe microstructure of whey protein and different treated whey protein fibrils under phase-jammed state\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7596478/v1/b340922313afced0dc2eed90.jpg"},{"id":92749809,"identity":"94752a7f-d548-4063-89e3-794504ac9baa","added_by":"auto","created_at":"2025-10-03 21:05:07","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":294230,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the mechanism of protein-induced phase-jamming\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7596478/v1/4a2fd71dfa08b642a0ac91f2.jpg"},{"id":92749814,"identity":"f0ff63ff-6dca-4d8a-bd0b-ef3a3493985e","added_by":"auto","created_at":"2025-10-03 21:05:07","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":71620,"visible":true,"origin":"","legend":"\u003cp\u003eOil adsorption capacity of aerogels (a); Appearance images of aerogels (b-c); Scanning electron microscopy image of WPF-U (d), WPF-5 (e) and WPF-10 (f); Different letters represent significant differences at the \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 level\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7596478/v1/fb36d253b8ff48e5b0016543.jpg"},{"id":92750557,"identity":"e7bfa171-4b94-4773-8782-651cc4feb167","added_by":"auto","created_at":"2025-10-03 21:29:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1955531,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7596478/v1/daf5ab6b-0bc4-46af-a843-24965bb20b89.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bicontinuous aerogels constructed with protein fibrils: A template for multiscale biomaterial customization","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEmulsions, as a paradigmatic example of multiphase dispersed systems, have long been a focal point in colloid and interface science due to the delicate balance between their thermodynamic metastability and kinetic stability (Ortiz et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Among these, Bicontinuous intra-phase emulsions distinguish themselves with their unique interpenetrating network structure, transcending the conventional binary division into \"dispersed phase\" and \"continuous phase.\" This distinctive feature offers novel perspectives for developing functional materials. By enabling continuous networks between two phases, this system markedly enhances interfacial mass transfer efficiency, showcasing significant potential in applications such as catalytic reactors and tissue engineering scaffolds (Shen et al., 2024).\u003c/p\u003e\u003cp\u003eThe breakthrough in stabilizing bicontinuous emulsions was initiated by the development of bijel (Bicontinuous interfacially jammed emulsion gels) systems, which leverage nanoparticles' self-assembly during phase separation to form a mechanical barrier at the interface through spinodal decomposition kinetics, effectively locking the bicontinuous structure (Di Vitantonio et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, traditional bijel systems impose stringent requirements on the physicochemical properties of stabilizing particles: neutral wettability (contact angle\u0026thinsp;\u0026asymp;\u0026thinsp;90\u0026deg;) and rapid interfacial adsorption kinetics. These constraints limit suitable particle types primarily to inorganic nanomaterials like silica (Sprockel et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), severely restricting the application scope of bicontinuous emulsion systems, especially in biomedicine.\u003c/p\u003e\u003cp\u003eTo overcome this limitation, researchers have developed innovative stabilization strategies, including bijels and SeedGel (Di Vitantonio et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These approaches extend stabilization mechanisms beyond mere interfacial adsorption to three-dimensional spatial confinement by modulating particle interactions within the bulk phase, significantly broadening the range of acceptable particle surface properties. In this context, biocolloidal materials are emerging as promising candidates for stabilizing bicontinuous emulsions due to their excellent biocompatibility and sustainability. Chitin nanocrystals (ChNC), among other biological nanomaterials, have already demonstrated successful applications in bicontinuous emulsion systems (Lu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), providing valuable insights into the use of other biomacromolecular materials in such systems.\u003c/p\u003e\u003cp\u003eProtein fibrils, as emerging bio-colloids, exhibit unique advantages: first, their fibrous structure provides high aspect ratios, facilitating the construction of three-dimensional networks; second, abundant functional groups on their surfaces offer chemical sites for interfacial modification; third, shear-induced fibrillization enables precise tuning of fibril size and flexibility (Ouyang et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Notably, current research predominantly focuses on protein fibrils in conventional emulsion systems, leaving the structure-function relationship in Bicontinuous intra-phase emulsions largely unexplored. Specifically, the regulatory mechanism of shear-induced structural evolution of protein fibrils on interfacial behavior and bulk network formation remains to be investigated. Therefore, this study aims to explore the stabilization mechanism of Bicontinuous intra-phase emulsions through shear-induced protein fibril modifications. The findings not only provide theoretical support for expanding the application of proteins in complex emulsion systems but also open new avenues for developing environmentally friendly multiscale emulsion materials.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials\u003c/h2\u003e\u003cp\u003eWhey protein isolate (Hilmar 9410) was purchased from Hilmar Co., Ltd (California, USA). 2,6-Lutidine was sourced from Macklin Biochemical Co., Ltd (Shanghai, China). Thioflavin T was obtained from Solarbio Co., Ltd (Beijing, China). All other chemical reagents used were of analytical grade.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Preparation and treatments of proteins\u003c/h2\u003e\u003cp\u003eIn this study, 2 M phosphoric acid was employed to unidirectionally adjust the pH of whey protein isolate (WPI) solutions containing various protein concentrations, ensuring that variations in ionic strength would not interfere with the experimental outcomes (Ouyang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). The whey protein solutions were stirred at room temperature for 2 h and subsequently stored overnight at 4\u0026deg;C for hydration. Following this, the solutions were incubated under the following conditions: pH 2.5, WPI concentration of 4.0% (w/v), temperature of 90\u0026deg;C, and incubation duration of 12 h.\u003c/p\u003e\u003cp\u003eShear treatment was performed using a shear mixer (IKA T10, Germany) at 5,000, 10,000, and 20,000 rpm for 2 min to prepare shear-modified protein fibrils, which were designated as WPF, WPF-5, WPF-10, and WPF-20, respectively. Additionally, protein fibrils underwent ultrasonic treatment for 2 min, labeled as WPF-U. To obtain purified protein fibril samples for further structural analysis, dialysis was carried out using a dialysis membrane (Spectra/Por\u0026reg; Dialysis Membrane, MWCO 100 kDa) against ultrapure water (pH 2.5) at 4\u0026deg;C for 72 h to remove non-fibrillar proteins/peptides from the samples.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Thioflavin T (Th T) Fluorescence Assay\u003c/h2\u003e\u003cp\u003eFirst, 8 mg of Thioflavin T (Th T) was dissolved in 10 mL of PBS buffer. The solution was then filtered using a 0.22 \u0026micro;m aqueous filter. The filtered solution was diluted 50-fold with PBS to prepare the Th T working solution. Subsequently, protein samples were diluted 20-fold with ultrapure water adjusted to the corresponding pH values. Each sample (50 \u0026micro;L) was mixed with 5 mL of the Th T working solution and allowed to react in the dark for 1 min.\u003c/p\u003e\u003cp\u003eFluorescence spectra were measured using a fluorometer with an excitation wavelength of 440 nm and emission wavelengths ranging from 460 to 560 nm (both emission and excitation slit widths were set to 10 nm). For each sample, measurements were performed using Th T working solution without protein as a blank control, and background correction was applied accordingly (Ouyang et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Intrinsic Fluorescence\u003c/h2\u003e\u003cp\u003eIntrinsic fluorescence spectra were measured using an excitation wavelength of 280 nm and an emission wavelength range of 300 to 400 nm. Both excitation and emission slit widths were set to 5 nm, with a scan speed of 1200 nm/min.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. ζ-Potential and Particle Size Distribution Measurement\u003c/h2\u003e\u003cp\u003eThe ζ-potential and particle size distribution were measured using a Zetasizer Nano instrument (Mastersizer 2000). This method allows for precise characterization of the electrokinetic potential and size distribution of particles within the sample.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Far-Ultraviolet Circular Dichroism (CD) Spectroscopy\u003c/h2\u003e\u003cp\u003eFar-ultraviolet circular dichroism (CD) spectra of WPF were characterized using a far-UV CD spectrometer (MOS-450/AF-CD, Bio-Logic, France). The measurements were conducted in the wavelength range of 200 to 250 nm to assess the secondary structure content of the protein fibrils.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. X-ray Diffraction (XRD)\u003c/h2\u003e\u003cp\u003eXRD measurements were conducted using an X-ray diffractometer (Bruker AXS D8 ADVANCE) with Cu-Kα radiation (λ\u0026thinsp;=\u0026thinsp;1.5418 \u0026Aring;) operated at 40 kV and 40 mA. Measurements and diffraction patterns were recorded over a 2θ range of 5 to 50\u0026deg; at a scan rate of 3\u0026deg; 2θ/min. Data processing was performed using Jade 6.0 software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.8. Rheological Properties\u003c/b\u003e\u003c/h2\u003e\u003cp\u003ePrepared protein solutions were transferred to the testing plate. Rheological measurements were carried out using a DHR-2 rheometer (TA Instruments Inc., USA) equipped with a parallel plate geometry of 40 mm diameter and a gap setting of 0.5 mm at 25\u0026deg;C. Dynamic frequency sweeps were performed within an angular frequency range of 0.1\u0026ndash;10 rad/s to determine the storage modulus (G\u0026prime;) and loss modulus (G\u0026Prime;) of the samples within the linear viscoelastic region at a strain of 0.5%. Steady shear rheology tests were conducted in the shear rate range of 0.1\u0026ndash;100 s⁻\u0026sup1; to analyze the relationship between shear rate and apparent viscosity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Transmission Electron Microscopy (TEM)\u003c/h2\u003e\u003cp\u003eTo observe the morphological structure of the samples, a Hitachi HT7800 transmission electron microscope (Tokyo, Japan) was used, operating at an accelerating voltage of 80 kV. Negative staining of samples was performed as follows: First, 5 \u0026micro;L of the sample solution (protein concentration of 0.20% w/v) was placed onto a carbon-coated copper grid. After a 1-min adsorption time, excess liquid was carefully removed using filter paper. Subsequently, the samples were stained with a 2% phosphotungstic acid aqueous solution for 10 min before drying again with filter paper.\u003c/p\u003e\u003cp\u003eTEM images were processed using FiberApp (Usov et al., 2015) to calculate two-dimensional distribution parameters (S\u003csub\u003e2D\u003c/sub\u003e) of the protein fibrils.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Preparation of Bicontinuous Intra-Phase Emulsions and Aerogels\u003c/h2\u003e\u003cp\u003eTo prepare the miscible phase solution, WPF suspensions were mixed with 2,6-lutidine at a ratio of O/W\u0026thinsp;=\u0026thinsp;28.4%, with the WPF concentration calculated based on the entire system. The mixture was first vortexed for 1 min at room temperature to ensure thorough mixing. Subsequently, the mixture was placed in a preheated water bath at 60\u0026deg;C for 5 min to initiate phase separation between water and lutidine, thereby forming a WPF-jammed bicontinuous intra-phase emulsion. Finally, the emulsion was incubated in an oven at 60\u0026deg;C for at least 24 h (Lu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo remove lutidine as much as possible, the WPF-jammed emulsion was inverted in the oven at 60\u0026deg;C for 24 h. The emulsion was then transferred to a freezer at -80\u0026deg;C to freeze its structure. Lastly, the frozen emulsion jammed hydrogel was freeze-dried to obtain protein fibril aerogels.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11. Optical Microscopy\u003c/h2\u003e\u003cp\u003eAfter vortexing the miscible phase solution for 1 min, it was heated on a hot stage at 60\u0026deg;C for 20 min and immediately transferred to the microscope stage for imaging. Microscopic images were captured every 20 s to document the phase transition process of the bicontinuous intra-phase system as it cooled to room temperature (25\u0026deg;C).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12. Oil Adsorption Performance\u003c/h2\u003e\u003cp\u003eThe oil adsorption performance of the aerogels was evaluated by measuring their weight gain (%) after exposure to cyclohexane and soybean oil. Briefly, aerogels were immersed in the oil/organic solvent for 20 s until adsorption equilibrium was reached, defined as no further liquid dripping from the aerogel. The aerogels were then immediately weighed (Bi et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The weight gain (Wg) of the aerogel after adsorption was calculated using the following formula:\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{W}_{g}\\:=\\:\\frac{{m}_{c}-{m}_{0}}{{m}_{0}}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e%(1)\u003c/p\u003e\u003cp\u003eWhere m\u003csub\u003ec\u003c/sub\u003e and m\u003csub\u003e0\u003c/sub\u003e represent the weights (g) of the aerogel after and before adsorption, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13. Scanning Electron Microscopy (SEM)\u003c/h2\u003e\u003cp\u003eScanning electron microscopy (SEM) images were acquired using a Regulus 8100 microscope (Hitachi, Tokyo, Japan). Freeze-dried samples were sputter-coated with gold prior to imaging. The accelerating voltage of the electron beam was set to 5.0 kV.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14. Statistical analysis\u003c/h2\u003e\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. All experiments were performed at least in triplicate to ensure reliability and accuracy. Statistical analysis was conducted using Origin 2024 software (OriginLab Corporation, USA). Analysis of variance (ANOVA) was used to determine significant differences between means, followed by Tukey\u0026rsquo;s test for pairwise comparisons. The significance level was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Macroscopic Effects of Shear Modification on Protein Fibrils\u003c/h2\u003e\u003cp\u003eShear modification exerts a significant regulatory effect on the macroscopic structure of protein fibrils. Rheological tests and polarization imaging were employed to investigate the macroscopic changes in protein fibrils after treatment at varying shear rates.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRheological profile indicate that protein fibril solutions exhibit typical shear-thinning behavior, likely due to the disruption of fibril entanglements as shear rate increases (\u003cb\u003eFigure. 1a-d\u003c/b\u003e). At low shear rates, extensive fibril entanglements result in higher viscosity; at high shear rates, some fibrils align along the flow direction, reducing entanglements and lowering viscosity (Loveday et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). As the shear processing speed increases, the apparent viscosity of protein fibril solutions decreases, accompanied by reductions in both storage modulus (G') and loss modulus (G''). Similarly, with increasing shear processing speed, the entangled network of fibrils is progressively disrupted, leading to decreased aggregation within the system and ultimately resulting in lower viscosity (Zhao et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe value of tanδ can indicate the state of a material within a certain range. When tanδ\u0026thinsp;\u0026lt;\u0026thinsp;1, the material's elasticity dominates, exhibiting solid-like properties; when tanδ\u0026thinsp;\u0026gt;\u0026thinsp;1, the material's viscosity dominates, showing liquid-like properties; and when tanδ\u0026thinsp;\u0026asymp;\u0026thinsp;1, the material exhibits typical viscoelastic behavior with comparable elasticity and viscosity. Overall, protein fibril solutions during the shearing process have tanδ\u0026thinsp;\u0026lt;\u0026thinsp;1, indicating that fibril entanglements form a gel-like network structure, making the system more inclined toward solid characteristics (WPF, WPF-5, WPF-10, WPF-U). The fibrillization process of proteins, such as β-lactoglobulin, can lead to entanglement phenomena. Under specific conditions, they can assemble into fibrous hydrogels. This process involves the fibrillization and cross-linking of protein monomers, during which semi-flexible long fibrils intermingle. The number and distribution of entanglements directly affect the elastic behavior of the protein fibril network (Cao et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Compared to untreated solutions, WPF-5 and WPF-10 exhibit tanδ values closer to 1. This phenomenon may be attributed to the destruction of fibril entanglement structures by shear forces, leading to a reduction in the number of cross-linking points and a decrease in material elasticity, shifting its rheological behavior towards the viscoelastic transition zone. However, when the shear rate is increased to 20,000 rpm, WPF-20 solutions display typical fluid flow characteristics, with tanδ values exceeding 1 at frequencies above 1 Hz. This suggests that extreme shear conditions may cut semi-flexible long fibrils into rigid short fibrils, significantly reducing the number of entanglements compared to long fibril systems, thus altering their rheological properties.\u003c/p\u003e\u003cp\u003eIn addition to changes in flow characteristics, birefringence under polarized light also shows significant differences (\u003cb\u003eFigure. 1e\u003c/b\u003e). Birefringence indicates anisotropic optical properties in the medium, where refractive indices differ in various directions (Bolisetty et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Experimental results reveal that birefringence is observed in WPF solutions, suggesting a transition from isotropic to nematic liquid crystal phases. As shear rates gradually increase, birefringence in the solution diminishes and eventually disappears under high shear rates (20,000 rpm). This phenomenon implies that high shear forces disrupt the ordered arrangement of fibril aggregates. When shear intensity reaches a critical value, the originally oriented fibril aggregates completely dissociate, leading to the disappearance of macroscopic birefringence (Mezzenga et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Notably, ultrasonic treatment does not affect the birefringence characteristics of the solution. To further investigate the mechanism, we used polarization microscopy and potential measurements to analyze the effects of shear treatment on the microscopic structure of protein fibrils.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Microscopic Effects of Shear Modification on Protein Fibrils\u003c/h2\u003e\u003cp\u003eTo explore the impact of shear treatment on whey protein fibrils at the microscopic level, we employed polarization microscopy and ζ-potential measurements to systematically observe the evolution of microscopic morphology in sheared whey protein fibrils.\u003c/p\u003e\u003cp\u003ePolarization microscopy revealed that although shear significantly disrupts the macroscopic structure of protein aggregates (\u003cb\u003eFigure. 1f\u003c/b\u003e), under the microscope, sheared fibril aggregates still exhibit characteristic yellow birefringence, a hallmark of amyloid fibrils (Yang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). This phenomenon indicates that while the macroscopic structure may be compromised, the fibrils retain an ordered arrangement at the microscopic level, maintaining their anisotropic characteristics.\u003c/p\u003e\u003cp\u003eIn studying the dispersion characteristics of protein fibril solutions, we comprehensively evaluated how high-speed shear treatment affects their particle size distribution and ζ-potential (\u003cb\u003eFigure. 1g-i\u003c/b\u003e). The shear treatment markedly alters the dispersion behavior of fibril solutions, primarily manifesting as a significant reduction in particle size. Untreated WPF solutions had an average particle size of 439.50\u0026thinsp;\u0026plusmn;\u0026thinsp;12.52 nm with a polydispersity index (PDI) of 0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, indicating a poorly dispersed polydisperse system. When shear rates reached 10,000 rpm and 20,000 rpm, the particle size distribution shifted from an initial bimodal distribution to a uniform unimodal distribution (WPF-10: 300.83\u0026thinsp;\u0026plusmn;\u0026thinsp;20.53 nm, WPF-20: 273.10\u0026thinsp;\u0026plusmn;\u0026thinsp;14.09 nm), with a concurrent decrease in PDI (WPF-10: 0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, WPF-20: 0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02). This suggests that high-speed shear effectively shortens and homogenizes fibril lengths by disrupting inter-fibril aggregation and entanglement (Zhao et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Notably, ultrasonic treatment reduced the average particle size of WPF to 293.77\u0026thinsp;\u0026plusmn;\u0026thinsp;23.45 nm but did not significantly change the PDI (WPF-U: 0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04), indicating that it only dissociated large aggregates without improving dispersion uniformity.\u003c/p\u003e\u003cp\u003eThe surface potential (ζ-potential) of colloidal particles is a key parameter for assessing the stability of colloidal dispersions. Experimental data indicate that shear treatment generally has a limited effect on the ζ-potential of WPF (WPF: 50.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03 mV, WPF-5: 48.80\u0026thinsp;\u0026plusmn;\u0026thinsp;3.30 mV, WPF-10: 48.57\u0026thinsp;\u0026plusmn;\u0026thinsp;2.87 mV). However, under high-speed shear conditions of 20,000 rpm, the ζ-potential of WPF-20 significantly decreased to 43.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19 mV. This phenomenon may be related to changes in the surface characteristics of fibril particles; under high shear stress, some functional groups on the fiber surface may rearrange or detach, leading to a reorganization of surface charges and a slight decrease in ζ-potential (Ren et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThese results demonstrate that high-speed shear treatment not only significantly alters the particle size distribution of protein fibril solutions, making them more uniform and smaller, but achieves this effect by disrupting physical entanglements between fibrils rather than altering surface charge characteristics. This provides a new technical approach for optimizing dispersion systems while maintaining surface electrical stability, with relatively minor impacts on ζ-potential. These findings offer experimental evidence for understanding the dispersion behavior of complex fluids under high shear environments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Mesoscopic Effects of Shear Modification on Protein Fibrils\u003c/h2\u003e\u003cp\u003eAt the mesoscopic scale, we employed transmission electron microscopy (TEM) to image the microstructure of fibril samples and used FiberApp software to calculate the two-dimensional orientation parameter (S\u003csub\u003e2D\u003c/sub\u003e), thereby characterizing the morphological and alignment changes of the fibrils. TEM imaging can clearly reveal the microscopic structural features of fibrils, while the S\u003csub\u003e2D\u003c/sub\u003e parameter provides a quantitative analysis tool for understanding the orientation patterns and dynamic changes of fibrous polymers under different conditions (Usov et al., 2015).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTEM images show that under 12-h thermal induction, whey protein isolate undergoes fibrillization to form semi-flexible protein fibrils (WPF) with significant aspect ratios (\u003cb\u003eFigure. 2a\u003c/b\u003e). Compared to the evident entanglement in WPF, ultrasonically treated fibrils (WPF-U) exhibit a more uniform distribution. This may be attributed to the cavitation effect of ultrasound disrupting the microscopic structure of fibril aggregates, thereby improving fiber entanglements. Additionally, larger gel regions are visible in WPF micrographs, which transform into uniformly dispersed smaller gel particles after ultrasonic treatment (WPF-U). Notably, no local gel formation was observed in shear-treated samples, correlating with changes in solution rheological properties. Compared to WPF-U, shear treatment more effectively truncates long fibrils into short ones, significantly reducing physical entanglements between fibrils while maintaining their linear structures (Zhao et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Specifically, at a shear rate of 20,000 rpm, WPF-20 samples exhibit highly uniform fibril sizes (approximately 500 nm) and, due to the shortened length, display characteristics closer to rigid short fibrils (Cao et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn two-dimensional orientation analysis, TEM images were processed using FiberApp software to calculate the S\u003csub\u003e2D\u003c/sub\u003e parameter, defined as S\u003csub\u003e2D\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2\u0026thinsp;\u0026lt;\u0026thinsp;cos\u0026sup2;θ\u003csub\u003en\u003c/sub\u003e \u0026gt;-1 (Usov et al., 2015). Compared to untreated WPF (S\u003csub\u003e2D\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.12), ultrasonically treated fibrils showed a more isotropic alignment (S\u003csub\u003e2D\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.064), indicating a trend toward isotropic distribution (\u003cb\u003eFigure. 2b-d\u003c/b\u003e). This change likely results from ultrasound-induced cavitation leading to random redistribution of fibrils, thus weakening nematic order (Adamcik et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). On the other hand, the higher initial bulk concentration (C\u003csub\u003einit\u003c/sub\u003e = 0.20%, w/v) might also contribute to the overall tendency towards isotropy (Jordens et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, after shear treatment at 20,000 rpm, the two-dimensional distribution of protein fibrils became more nematic (S\u003csub\u003e2D\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.24). This change could be due to truncated semi-flexible fibrils aligning into a nematic phase when they come close together, minimizing orientation entropy by straightening out (\u003cb\u003eFigure. 2e\u003c/b\u003e). From an energetic perspective, this arrangement lowers the total free energy of the system, making the fibrils more inclined to exist in a nematic phase (Jordens et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThese findings indicate that shear treatment significantly enhances the uniformity and alignment order of fibril sizes, whereas ultrasonic treatment reinforces the uniformity and isotropy of fibril distribution. The TEM observations are highly consistent with particle size analysis data, providing critical experimental evidence for elucidating the behavior mechanisms of protein fibrils under various processing conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Molecular-Level Effects of Shear Modification on Protein Fibrils\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAt the molecular scale, the regulatory effect of shear treatment on protein conformation is equally crucial (Huyst et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To comprehensively assess the impact of shear treatment on protein conformation and characteristic fibril structures, we employed various detection methods, including intrinsic fluorescence spectroscopy, Th T fluorescence spectroscopy, far-UV circular dichroism (CD), and X-ray diffraction (XRD).\u003c/p\u003e\u003cp\u003eIntrinsic fluorescence spectroscopy is commonly used to track the conformational evolution of protein tertiary structure (\u003cb\u003eFigure. 3a\u003c/b\u003e). Results show that after 12 h of heating, the maximum emission wavelength of WPI shifts from 341 nm to 349 nm. This redshift may be due to changes in the internal microenvironment of the protein, making the microenvironment of the Trp chromophore more hydrophilic (Jones et al., 2012). Notably, the intrinsic fluorescence intensity of sheared fibrils shows only a slight decrease, and similar characteristics are observed in ultrasonically treated WPF-U samples, indicating that shear and ultrasound treatments have limited effects on the tertiary structure of proteins. Their primary influence may be at the secondary structure or aggregation state level.\u003c/p\u003e\u003cp\u003eTh T dye, as a specific probe for amyloid fibrils, can be used to evaluate the content of characteristic structures (Cross-β) in protein fibrils (Arad et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Through Th T-specific fluorescence spectroscopy, it was found that WPI exhibited high fluorescence intensity after 12 h of thermal treatment, confirming the formation of amyloid fibril structures (\u003cb\u003eFigure. 3b\u003c/b\u003e). Notably, shear treatment reduced Th T fluorescence intensity, particularly under shear conditions of 10,000 rpm and 20,000 rpm (WPF-10/20), where fluorescence intensity significantly decreased. This result suggests that shear treatment may partially disrupt Cross-β structures. In contrast, ultrasonic treatment did not significantly affect Th T fluorescence (\u003cb\u003eFigure. 3c\u003c/b\u003e). To further investigate this phenomenon, we measured the CD spectra of protein solutions (\u003cb\u003eFigure. 3d\u003c/b\u003e). The results showed that shear treatment had a minimal effect on secondary structures, but under high-speed shear conditions of 20,000 rpm, the β-sheet content decreased (at 215 nm) (Cao et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which may be related to local conformational changes induced by mechanical stress.\u003c/p\u003e\u003cp\u003eCross-β structures (\u003cb\u003eFigure. 3f\u003c/b\u003e), as the core feature of amyloid fibrils, consist of parallel-aligned β-sheets. β-chains are perpendicular to the long axis of the fibrils, forming layered structures with inter-chain distances (d\u003csub\u003e1\u003c/sub\u003e) of approximately 4.8 \u0026Aring;, maintained by hydrogen bonds between peptide bonds; inter-layer distances (d\u003csub\u003e2\u003c/sub\u003e) range from 10\u0026ndash;12 \u0026Aring;, depending on the stacking mode of sheets (Cao et al., 2019). The distance between β-sheets in Cross-β structures has a decisive impact on the nucleation ability of fibril core structures (Yang et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). To further explore the effect of shear on the Cross-β structure of protein fibrils, we conducted XRD tests (\u003cb\u003eFigure. 3d\u003c/b\u003e). The results showed that both sheared and ultrasonically treated protein fibrils exhibited two characteristic peaks representing the distances between β-sheets and β-chains, indicating that these fibrils still maintained complete Cross-β structures. Notably, high-speed shear treatment significantly reduced the inter-layer distances of Cross-β structures in fibrils. Compared to untreated WPF samples (9.70 \u0026Aring;), the inter-layer distances of Cross-β structures in sheared fibrils decreased to around 9.1 \u0026Aring; (WPF-5: 9.11 \u0026Aring;, WPF-10: 9.09 \u0026Aring;, WPF-20: 9.16 \u0026Aring;). This result indicates that, unlike hydrolysis treatment which makes Cross-β structures looser, high-speed shear treatment makes them more compact. This denser structure may be less conducive to the nucleation ability of sheared fibrils. Additionally, ultrasonically treated protein fibrils showed no significant changes in inter-sheet distances and β-chain spacings compared to untreated samples.\u003c/p\u003e\u003cp\u003eOverall, shear treatment not only affects the macroscopic, microscopic, and mesoscopic states of protein fibrils but also exerts regulatory effects at the molecular level. These experimental results reveal how shear treatment alters the behavior of protein fibrils across different scales: from macroscopic rheological properties and birefringence phenomena to microscopic size and distribution, to mesoscopic two-dimensional orientation parameters (S\u003csub\u003e2D\u003c/sub\u003e), and to molecular-level secondary and tertiary structural changes. These findings highlight the multiscale impact of shear treatment on whey protein fibrils. Importantly, these insights provide theoretical foundations for applying such treatments in Bicontinuous intra-phase emulsions. For instance, when designing protein fibril-based materials with multiscale structures and functional properties, shear treatment conditions can be adjusted to control fibril morphology, alignment, and molecular conformation, thereby optimizing material performance.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Performance of Proteins in Bicontinuous Intra-Phase Emulsions\u003c/h2\u003e\u003cp\u003eBicontinuous intra-phase (BC) emulsions exhibit application potential in functional materials due to their three-dimensional continuous network structure formed by interpenetrating aqueous and oil phases. Particle-mediated jamming behavior is considered a key mechanism for regulating the structural stability of these systems. This part of the experiment investigates the impact of thermal treatment on phase jamming phenomena in protein solutions, aiming to reveal the potential of protein particles in stabilizing the formation of bicontinuous structures. The experiments are based on the phase separation properties of the water/Lutidine miscible system: at room temperature (25\u0026deg;C), water and Lutidine form a homogeneous mixed solution. Upon heating to the critical temperature (34.1\u0026deg;C, corresponding to a 28.4% Lutidine concentration), the system undergoes spinodal decomposition, leading to the formation of a bicontinuous phase structure through jamming (Grattoni et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Stratford et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). During this process, by regulating the dynamic adsorption behavior of protein particles during phase separation, it may be possible to achieve morphological locking of non-equilibrium bicontinuous structures. The mixed system was heated to 60\u0026deg;C to trigger phase separation. It is hypothesized that WPF particles might inhibit the rapid expansion of the Lutidine phase through interfacial adsorption, thereby hindering the transition of the system towards a thermodynamic equilibrium state (dispersed matrix phase), ultimately forming a non-equilibrium bicontinuous structure (\u003cb\u003eFigure. 4\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eFirstly, WPF solution was mixed with Lutidine at a volume ratio of 71.6:28.4 (v/v) and vortexed for 60 s to ensure uniform dispersion, resulting in a miscible solution. Prior to heat-induced phase separation, the optical characteristics of the solution were observed using polarization microscopy. Results showed that untreated WPF miscible phase solutions exhibited significant birefringence, indicating that WPF aggregates displayed an ordered orientation distribution within the solution, possessing optical anisotropy (Bolisetty et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, as the solution underwent high-speed shear treatment, the intensity of birefringence gradually decreased with increasing shear rate, suggesting that shear forces disrupted the ordered arrangement of WPF aggregates. Notably, ultrasonic treatment did not significantly alter the birefringence characteristics of the miscible phase. These observations are highly consistent with the behavior of WPF in pure aqueous solutions\u0026mdash;where the ordered structure of WPF in the aqueous phase is similarly sensitive to shear treatment. Therefore, it can be inferred that the distribution pattern of WPF in the water/Lutidine miscible phase does not fundamentally differ from that in pure water, indicating a universal structural response mechanism across solvent systems.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUpon heating to 60\u0026deg;C to trigger phase separation in the water/Lutidine miscible system, it is hypothesized that WPF particles can regulate phase separation kinetics through interfacial adsorption. Specifically, WPF particles might inhibit the rapid expansion of the Lutidine phase by adsorbing at the phase interface, thus preventing the system from transitioning to a thermodynamic equilibrium state (dispersed matrix phase), ultimately locking the non-equilibrium bicontinuous phase structure (Lu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Experimental results confirmed this hypothesis: when the miscible phase solution (WPF concentration 2.0% w/v) was heated to 60\u0026deg;C and maintained for 20 min, untreated WPI solutions failed to form bicontinuous intra-phase emulsions due to the lack of effective interfacial regulation mechanisms. Conversely, WPF and its sheared (WPF-5/10/20) or ultrasonically treated (WPF-U) samples successfully constructed stable bicontinuous structures through jamming effects. Notably, high shear rates (WPF-20) led to a decrease in emulsion thermal stability, likely due to excessive shear disrupting the ordered arrangement of WPF aggregates, weakening their jamming capabilities. After being left at room temperature for 12 h, all systems except WPF-20, which collapsed completely due to initial structural instability, maintained a stable gel-like emulsion state, further validating the formation of bicontinuous intra-phase emulsion gels (Lu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). On the other hand, the appearance of bicontinuous emulsions gradually changed from initially white to translucent, possibly due to enhanced miscibility between Lutidine and water during cooling. As the temperature dropped below the critical temperature, the mutual solubility of Lutidine and water increased, reducing interfacial tension and causing partial mixing, leading to protein particle sedimentation or aggregation, decreasing interfacial coverage, and altering light scattering paths, thus shifting the system from highly scattering white to less scattering translucent states. Despite this, the specific mechanisms still require validation through microscopic characterization using optical microscopy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Microscopic Structure of Bicontinuous Jammed Emulsions\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo analyze the microscopic structure of Bicontinuous Intra-Phase (BC) Emulsions, this experiment employed optical microscopy to observe the micro-morphology of solutions after heating at 60\u0026deg;C for 5 min and documented dynamic changes during the cooling process at room temperature (25\u0026deg;C) (\u003cb\u003eFigure. 5\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eAt a WPI concentration of 2.0%, the biphasic system only briefly formed an emulsion structure post-heating, which demulsified within 20 s due to imbalanced interfacial tension, leading to rapid merging into a homogeneous phase. When the WPI concentration was increased to 3.0%, the system formed a bicontinuous structure through thermal induction but became unstable after 40 s due to insufficient interfacial coverage, ultimately reverting to a single phase. This observation suggests that WPI needs to reach a relatively high concentration (3.0%) to initially form an interpenetrating network of bicontinuous structures, yet its stability remains limited by the strength of interfacial interactions.\u003c/p\u003e\u003cp\u003eAfter regulating fibril dispersion via shear treatment, the effect of shear rate on the pore size of the bicontinuous phase became pronounced. Untreated WPF, due to severe entanglement and aggregation of fibrils within the system, resulted in highly uneven phase size distribution in the bicontinuous structure formed after heating. Insufficient local interfacial adsorption led to structural defects such as abnormally enlarged pores or localized occlusions. Despite these defects, the robust bicontinuous network could maintain topological connectivity through tight packing, remaining stable for up to 60 s. At a shear rate of 5,000 rpm (WPF-5), moderate shear effectively improved fibril aggregation, alleviating long fibril entanglements, allowing fibrils to disperse uniformly in the system in slightly shorter yet still flexible forms. This dispersion supported the formation of a stable three-dimensional interpenetrating network, with more uniform phase size distribution and significantly reduced pore size variation, maintaining structural stability for 60 s. Increasing the shear rate to 10,000 rpm (WPF-10) further enhanced the synergistic effects of shear cutting and fibril dispersion, significantly increasing pore sizes (nearly doubling compared to WPF-5), while fibril alignment increased interfacial coverage density, thus maintaining structural stability for 60 s. However, at a shear rate of 20,000 rpm (WPF-20), excessive shear forces drastically shortened the aspect ratio of fibrils, converting them into rigid short fibrils, with significantly reduced interfacial adsorption capabilities due to structural damage. Consequently, the support capacity of the continuous phase network weakened, leading to rapid destabilization after 40 s due to localized pore closure. These results suggest that shear rates should be controlled within a reasonable range of 5,000\u0026ndash;10,000 rpm: 5,000 rpm can prepare bicontinuous structures with small pore sizes and high uniformity by improving fibril dispersion, whereas 10,000 rpm can moderately enlarge pore sizes to form large-pore networks while maintaining structural integrity, thereby achieving precise control over pore size.\u003c/p\u003e\u003cp\u003eAfter ultrasonic treatment (WPF-U), fibrils formed a uniform and dense bicontinuous network during heating. Compared to the untreated group (which destabilized after 40 s), the ultrasonically treated group remained stable for 60 s, indicating that ultrasound may enhance interfacial stability in the bicontinuous phase system by reducing fibril aggregation. Experimental results show that ultrasonic pretreatment is an effective method to improve the uniformity of the bicontinuous phase structure.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.7. Mechanism of Protein-Induced Phase Jamming\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePhase jamming mechanisms are complex processes involving the synergistic effects of particle concentration, interfacial adsorption, and steric hindrance. In binary solvent systems approaching phase separation critical points, fluctuations in solvent concentration intensify. If particles selectively adsorb one component of the solvent, an adsorption layer forms on their surfaces (Wang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). As the temperature approaches the critical point, the correlation length of the solvent increases, leading to thicker adsorption layers, which in turn alters the interaction energy between particles, ultimately resulting in solvent-mediated critical Casimir forces that promote particle attraction (Bertrand et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, steric hindrance limits excessive aggregation by physically impeding particle clustering. This dynamic equilibrium among multiple factors is central to the stabilization of bicontinuous intra-phase (BC) emulsions by bio-based particles, directly influencing particle distribution, interfacial properties, and emulsion stability, thus serving as a key factor in regulating the structure of BC emulsion materials (\u003cb\u003eFigure. 6\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eAt a WPI concentration of 2.0%, the system fails to form BC emulsions due to insufficient particle numbers. With large particle spacings and weak interactions, these particles cannot form continuous networks through tight packing, leading to free droplet coalescence and eventual demulsification into a single phase. However, when the WPI concentration is increased to 3.0%, the number of particles significantly increases, reducing spacing and enhancing inter-particle interactions. These particles form physical jams through tight packing, restricting droplet mobility and successfully constructing a bicontinuous phase structure. Additionally, proteins, as bio-based particles, form dense protective layers at interfaces through adsorption, further regulating interfacial tension and modifying interfacial rheological properties. Reduced interfacial tension decreases droplet coalescence tendencies, while improved interfacial rheology enables uniform dispersion of droplets within the continuous phase, synergizing with particle crowding effects to stabilize BC emulsions. Compared to traditional silica particles, which require high concentrations (24.3%) to form stable networks due to weaker inter-particle interactions (Xi et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), protein-based particles can construct dense networks at lower concentrations via strong interfacial adsorption and intermolecular forces, providing an efficient pathway for designing functional materials. Importantly, the rich structural variations of proteins offer additional topological control for the formation and construction of BC emulsions.\u003c/p\u003e\u003cp\u003eFurthermore, whey protein fibrils (WPF), owing to their elongated aspect ratios, can stabilize BC emulsions even at a concentration of 2.0%. This highlights the significant impact of particle shape on stabilizing emulsions, with elongated particles showing clear advantages in forming and stabilizing bicontinuous emulsions. However, entanglement issues with semi-flexible long fibrils lead to uneven fiber distribution in some regions, preventing uniform jamming structures and causing defects. Such structural imperfections may affect the stability and performance of BC emulsions. Notably, Lu et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) attempted to use cellulose nanofibers (ChNF) to construct BC emulsions but failed to achieve stable jamming structures, possibly due to ChNF's strong flexibility and insufficient intrinsic rigidity. However, WPF shares similar structural characteristics (e.g., flexibility and low stiffness) yet successfully stabilizes BC emulsions, suggesting that the aforementioned hypothesis might not be the root cause. Based on experimental results, it can be inferred that ChNF's failure is more likely due to insufficient concentration (0.6%) failing to reach the threshold required for effective three-dimensional network support. Increasing the concentration could potentially enable ChNF to stabilize BC emulsions.\u003c/p\u003e\u003cp\u003eModerate shear treatment of WPF (e.g., 5,000 rpm or 10,000 rpm) transforms fibril morphology from semi-flexible long fibrils to rigid short fibrils. This transformation leads to: 1) Fibril Morphology Regulation: Short fibril networks formed by breaking long fibrils achieve more uniform dispersion and tighter packing, reducing local defects caused by long fibril entanglements. Shear flow induces nematic ordering along the flow direction, forming oriented aggregate structures. This arrangement reduces random aggregation, providing efficient channels for phase separation and enlarging pore size distributions; 2) Structural Stability Maintenance: Studies by Huyst et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) show that low-speed shear does not significantly alter protein structure due to unchanged interfacial contact areas, thus retaining the original mechanical support capability of protein fibrils. Retaining linear structures ensures short fibril networks possess both rigidity for maintaining pore connectivity and reduced interface defects for enhanced stability. Therefore, by adjusting shear rates, precise control over pore size distribution (e.g., 5,000 rpm maintains small pores, 10,000 rpm enlarges pores) can be achieved, balancing structural stability and functional tunability, offering effective strategies for customized material design.\u003c/p\u003e\u003cp\u003eHowever, in the WPF-20 system, excessively high shear rates (20,000 rpm) introduce dual defects: 1) Interface Interaction Imbalance: High surface charge density on protein fibrils leads to strong electrostatic repulsion (dominated by net repulsion), hindering sufficient aggregation and coverage at interfaces (Xi et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e); 2) Morphological Degradation and Support Failure: Shearing drastically reduces the aspect ratio of fibrils, compromising their mechanical support capabilities needed for BC emulsion networks. Despite high particle concentrations, shortened fibrils fail to achieve the critical threshold for \"crowding effects\" through physical jamming or interfacial adsorption, rendering the BC emulsion structure unstable.\u003c/p\u003e\u003cp\u003eOn the other hand, ultrasonic treatment improves the uniformity of WPF in BC emulsions through multi-scale actions, involving structural regulation and defect suppression. On a macro scale, ultrasonic treatment maintains the nematic ordering of protein fibrils (birefringence phenomenon), providing a critical framework for the topological connectivity of BC emulsions. On a mesoscopic scale, high-frequency vibrations and cavitation bubble collapse induce random reorientation of fibrils within two-dimensional planes, reducing inherent anisotropy and achieving a more isotropic distribution (lower S\u003csub\u003e2D\u003c/sub\u003e). This \"macro-order-meso-disorder\" synergy retains long-range orientation along the flow direction to maintain pore connectivity in BC emulsions while suppressing fibril entanglement and localized aggregation through uniform dispersion at the mesoscopic scale, avoiding droplet coalescence or phase separation due to uneven interface coverage. Therefore, ultrasonic pretreatment represents a viable approach for preparing biomaterial networks.\u003c/p\u003e\u003cp\u003eThus, by uniformly mixing WPF solutions with Lutidine and heating at 60\u0026deg;C to trigger phase separation, BC emulsions can be formed. This heating step drives solvent systems close to phase separation critical points, prompting WPF fibrils to self-assemble into three-dimensional interpenetrating networks under thermodynamic driving forces, thereby stabilizing BC emulsion structures. Subsequently, inverting the emulsion container and incubating at 60\u0026deg;C for 12 h leverages gravity to further drive phase separation and solidification of the solvent-nonsolvent system (Lu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). During this process, inversion facilitates the outflow of the Lutidine phase from continuous pore structures, promoting solvent component separation and uniform gelation, ultimately forming structurally stable BC hydrogels. Finally, freeze-drying techniques transform water in the BC hydrogel into ice crystals, which are then removed by vacuum sublimation. This process avoids structural collapse associated with solvent evaporation during traditional drying methods, with ice crystal sublimation preserving the original fibril network topology within the gel, ultimately yielding BC cryogels with multiscale pore skeletons. The entire process achieves precise structural transfer from liquid to solid phases through coordinated temperature control, phase jamming processes, and freeze-drying techniques, providing a controllable path for preparing bio-based porous materials. Based on experimental results, WPF-U, WPF-5, and WPF-10 samples were selected for further preparation of BC cryogels.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.8. Construction of Protein-Based Aerogels Using Bicontinuous Emulsions\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBy subjecting uniform bicontinuous intra-phase (BC) emulsions to inverted incubation and subsequent freeze-drying, we successfully prepared three types of cryogels (WPF-U cryogel, WPF-5 cryogel, and WPF-10 cryogel). Due to their ultra-light weight, the fabricated aerogels could be stably placed on top of a single dandelion seed (\u003cb\u003eFigure. 7b\u003c/b\u003e) and lifted by the static electricity of a plastic measuring spoon (\u003cb\u003eFigure. 7c\u003c/b\u003e). To further characterize these aerogels, their micro-morphology was observed using scanning electron microscopy (SEM), and their oil adsorption capacity was evaluated by measuring the weight gain after oil absorption (Bi et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSEM observations revealed that the WPF-U aerogel exhibited a highly uniform lamellar structure with large pores and thin, dense layers (\u003cb\u003eFigure. 7d\u003c/b\u003e). This structural feature directly reflects the initial three-dimensional network formed during the curing process of the bicontinuous phase emulsion (Jia et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, at high magnifications, the impact of the freeze-drying process on the microstructure can be observed: sparse distributions of micropores formed during freeze-drying. Although the WPF-U aerogel maintained uniformity and density of the bicontinuous structure at the macro scale, its micro-porosity was not further refined, failing to form an improved multiscale pore network. Its structural characteristics are more akin to traditional porous shell-like materials prepared via the bicontinuous gel template method (Lee et al., 2010). This structure may result from the entanglement of semi-flexible long protein fibrils\u0026mdash;when fibers are not sufficiently dispersed, their higher aspect ratios tend to form local entanglements in solution, hindering uniform micropore generation and resulting in thicker layers and insufficient micropore density. Despite its excellent performance in soybean oil adsorption tests (weight gain of 2,766%), the adsorption capacity of WPF-U is significantly lower than that of WPF-5, likely due to the limitations of larger pore sizes: while larger pores can accommodate soybean oil molecules, insufficient micropore density reduces the number of adsorption sites, limiting specific surface area and adsorption efficiency.\u003c/p\u003e\u003cp\u003eIn contrast, the WPF-5 aerogel exhibits a remarkable multiscale pore size structure (\u003cb\u003eFigure. 7e\u003c/b\u003e). Overall, its macroporous structure is similar to that of WPF-U, but it features densely distributed and highly interconnected micropores on the fibril scaffold during freeze-drying, forming a channel network. The formation of this micropore network closely aligns with the initial structure of the BC emulsion: moderate shear (5,000 rpm) ensures even dispersion of fibrils, avoiding entanglements, while the semi-flexibility of fibrils allows them to aggregate into stable interpenetrating networks during phase jamming. During freeze-drying, ice crystal structures produced by water sublimation further refine the microstructure of hydrogel layers, ultimately forming fine and uniform multiscale micropores. This dual advantage of structural design lies in the macropores ensuring mechanical strength and permeability, while the micropore network significantly enhances specific surface area, providing abundant adsorption sites for molecules. Oil adsorption tests confirmed this advantage, with WPF-5 achieving a soybean oil adsorption capacity of 4,512% weight gain, significantly surpassing WPF-U (2,766%) and WPF-10 (3,071%). This result validates the improvement effect of moderate shear on fibril dispersibility\u0026mdash;it avoids structural defects caused by entanglements and retains the integrity of the fibril network through freeze-drying, thus optimizing the distribution of multiscale pore sizes.\u003c/p\u003e\u003cp\u003eAlthough the WPF-10 aerogel also displays macroporous structures, its microstructure shows significant heterogeneity (\u003cb\u003eFigure. 7f\u003c/b\u003e). In high-magnification SEM images, most regions lose their bicontinuous characteristics due to collapse during freeze-drying, leaving only a few micropores. This phenomenon indicates that excessively high shear rates (10,000 rpm) shorten fibril lengths and alter aggregation morphology but weaken the support capability of the fibril network. During freeze-drying, insufficiently supported fibril networks cannot withstand the stress from ice crystal sublimation, leading to localized collapses and ultimately forming heterogeneous microstructures with poor connectivity. Despite maintaining continuous phase-formed macroporous structures on the surface, inadequate micropore density and accumulated structural defects result in lower soybean oil adsorption capacity (weight gain of 3,071%) compared to WPF-5.\u003c/p\u003e\u003cp\u003eThe adsorption capacities of the three aerogels for n-hexane (weight gain of 1,637%\u0026ndash;2,064%) show no significant differences, possibly due to the small size of n-hexane molecules, which can freely diffuse into all pores. However, soybean oil molecules are larger, making their adsorption capacity more dependent on pore size structure. The multiscale pore network of WPF-5 therefore demonstrates superior performance. This comparison indicates that the regulation of pore size distribution by shear rate directly affects the functional properties of aerogels, while the synergistic effect of multiscale structures is crucial for enhancing specific surface area and adsorption capacity.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis study investigates the multi-scale structural regulation of whey protein fibrils (WPF) via shear modification and their application in bicontinuous intra-phase emulsions. Macroscopically, shear disrupts fibril entanglements, altering rheological properties from elasticity-dominated to liquid-like behavior, while maintaining microscopic order even as birefringence disappears at high shear rates (20,000 rpm). Microscopically, shear reduces fibril size to 273 nm (PDI\u0026thinsp;=\u0026thinsp;0.34), enhancing dispersion but decreasing ζ-potential due to surface group rearrangement. Mesoscopically, TEM and S\u003csub\u003e2D\u003c/sub\u003e analysis show that high shear cuts long fibers into shorter ones (500 nm) with nematic ordering (S\u003csub\u003e2D\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.064), whereas ultrasonic treatment increases isotropy (S\u003csub\u003e2D\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.24). Molecularly, XRD indicates no disruption of the Cross-β structure but a decrease in Cross-β sheet spacing to 9.1 \u0026Aring;, suggesting a more compact structure.\u003c/p\u003e\u003cp\u003eIn BC emulsions, WPF stabilizes structures at 2.0% concentration, far lower than silica particles (24.3%), due to semi-flexible long fibrils. Controlled shear rate optimizes pore size: 5,000 rpm minimizes entanglement for uniform small pores, 10,000 rpm enlarges pores while maintaining connectivity, whereas 20,000 rpm causes structural collapse. Ultrasonic pretreatment enhances uniformity through macroscopic order-mesoscopic disorder regulation.\u003c/p\u003e\u003cp\u003eUsing heating-induced phase separation and freeze-drying, we fabricated BC cryogels with multiscale pores. The WPF-5 cryogel, with its dense micropore network, showed superior soybean oil adsorption (4,512% weight gain) compared to WPF-U (2,766%) and WPF-10 (3,071%). N-hexane adsorption was similar across samples (1,637%\u0026ndash;2,064%), highlighting the importance of multiscale porosity and shear control in functional material design. These results emphasize the significance of multi-scale optimization and controlled shear processing for advanced functional materials.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics declarations\u003c/b\u003e\u003c/h2\u003e\u003ch2\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was financially supported by the National Natural Science Foundation of China (32460588), the Natural Science Foundation of Jiangxi Province (20232BAB205075), and the Nanchang University Jiangxi Province Financial Science and Technology Special Project (ZBG20230418037)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eKefan Ouyang: Methodology, Investigation, Visualization, Writing - Original Draft. Yuanyuan Feng: Investigation, Writing - Review \u0026amp; Editing. Songyu Wang: Investigation. Zihang Yan: Investigation. Qin Zhang: Writing - Review \u0026amp; Editing. Qiang Zhao: Conceptualization, Project administration, Funding acquisition, Writing - Review \u0026amp; Editing, Supervision.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdamcik, J., Jung, J. M., Flakowski, J., De Los Rios, P., Dietler, G., \u0026amp; Mezzenga, R. (2010). 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Environmental parameters-dependent rheological behaviors of whey protein fibril dispersions: Shear and extensional flow behaviors. \u003cem\u003eFood Hydrocolloids\u003c/em\u003e, \u003cem\u003e133\u003c/em\u003e, 107974. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodhyd.2022.107974\u003c/span\u003e\u003cspan address=\"10.1016/j.foodhyd.2022.107974\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Whey protein fibrils, Shearing treatment, Bicontinuous intra-phase emulsions","lastPublishedDoi":"10.21203/rs.3.rs-7596478/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7596478/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study elucidates the structural regulation of whey protein fibrils (WPF) via shear modification, revealing how shear rates govern their hierarchical organization. Shear forces induce a rheological shift from elastic gels (tanδ\u0026thinsp;\u0026lt;\u0026thinsp;1) to viscoelastic states (tanδ\u0026thinsp;\u0026gt;\u0026thinsp;1), with particle size reduced to ~\u0026thinsp;273 nm at 20,000 rpm. Molecularly, X-ray diffraction reveals that shear compacts form Cross-β sheets at 9.1 \u0026Aring;, thereby enhancing structural density. Mesoscopically, controlled shear rates (5,000\u0026ndash;10,000 rpm) enable tunable porosity in bicontinuous emulsions. Lower rates (5,000 rpm) yield uniform, small pores, while higher rates (10,000 rpm) enlarge pores while maintaining connectivity. This approach produces aerogels with multiscale porosity. Notably, WPF-5 aerogels\u0026mdash;characterized by dense microporous networks\u0026mdash;exhibit a 4,512% increase in soybean oil adsorption, highlighting their potential for biomaterials. By integrating microscopic, mesoscopic, and molecular insights, this work provides a framework for precision-engineering multiscale protein-based materials, bridging fundamental science and functional applications.\u003c/p\u003e","manuscriptTitle":"Bicontinuous aerogels constructed with protein fibrils: A template for multiscale biomaterial customization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-03 21:05:02","doi":"10.21203/rs.3.rs-7596478/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-08T23:21:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-10T11:05:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-03T08:41:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"298359695035843983394908454513860067881","date":"2025-09-27T11:08:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282838427111344931843139127347076008265","date":"2025-09-24T06:13:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"71656651088458125925432287990126491382","date":"2025-09-24T05:45:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"218274852427402951501543401553778147547","date":"2025-09-22T14:58:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-21T21:51:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-12T07:56:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-12T05:10:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food and Bioprocess Technology","date":"2025-09-12T04:39:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e8e8b243-ff9e-47a3-9f28-c60e94b6ec12","owner":[],"postedDate":"October 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T11:08:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-03 21:05:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7596478","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7596478","identity":"rs-7596478","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}