Ultrasound-assisted multiscale structural engineering of plant proteins: mechanisms and structure–property relationships

1 Introduction

Plant-based proteins have emerged as key functional ingredients in the transition toward sustainable, resilient, and health-oriented food systems. Their increasing relevance is driven not only by environmental considerations, but also by their versatility in food formulation and their potential to replace animal-derived proteins across a broad range of applications (Jafarzadeh et al., 2024). Despite these advantages, the broader industrial utilization of plant proteins remains fundamentally constrained by their intrinsic molecular and supramolecular organization. Most plant proteins exhibit compact globular conformations, strong hydrophobic interactions, and extensive intermolecular associations, which collectively reduce solubility, impair interfacial activity, and limit their ability to form stable foams and structured gel networks (Yildiz et al., 2017). As a consequence, their techno-functional performance is often suboptimal in complex food matrices.

In recent years, non-thermal and clean-label processing technologies have gained increasing attention as sustainable alternatives to conventional thermal and chemical modification approaches. Among these, ultrasound-assisted processing has emerged as one of the most promising physical strategies for tailoring plant protein functionality. The efficiency of ultrasound originates from acoustic cavitation phenomena, in which the formation, growth, and collapse of microbubbles generate highly localized energy hotspots characterized by intense shear forces, microjets, and transient pressure and temperature gradients (Lee et al., 2016). These extreme microenvironmental conditions are capable of inducing profound physicochemical transformations in protein systems without the need for chemical additives, making ultrasound particularly attractive for sustainable food processing applications.

At the molecular level, cavitation-induced effects disrupt hydrogen bonding networks, weaken hydrophobic interactions, and alter disulfide bond configurations, leading to partial unfolding of protein structures and exposure of previously buried functional groups (Yildiz, 2025a). At the supramolecular level, these molecular perturbations result in dissociation of protein aggregates, modifications in secondary structural elements, and reorganization of protein–protein interaction networks (Thirunavookarasu et al., 2022). At the colloidal scale, these changes manifest as reduced particle size, improved dispersion stability, and altered surface charge properties, which collectively govern interfacial adsorption and stability in multiphase food systems (Jang et al., 2025). Importantly, these hierarchical transformations are not independent but are strongly coupled, forming a continuous multiscale pathway that links energy input to macroscopic functional outcomes.

Within this framework, ultrasound should not be considered merely a processing aid, but rather a multiscale structural engineering tool capable of systematically modulating structure–function relationships in plant proteins. This perspective aligns with the emerging view of food systems as soft matter systems, in which functional performance arises from hierarchical organization and dynamic structural rearrangements rather than static composition alone. Therefore, a comprehensive understanding of ultrasound-induced transformations across multiple length scales is essential for the rational design of next-generation plant-based food systems with tailored techno-functional and nutritional properties.

2 Fundamentals of ultrasound processing in plant protein systems

Ultrasound treatment in food systems is fundamentally based on the phenomenon of acoustic cavitation, which involves the formation, growth, and subsequent violent collapse of microbubbles in a liquid medium. This collapse generates highly localized and transient energy zones, often referred to as “hot spots,” characterized by extreme temperature and pressure gradients, as well as intense shear forces, shock waves, and microjets (Chavan et al., 2022). Although these effects occur at the microscale and within extremely short time intervals, they collectively generate a highly energetic environment capable of inducing significant physicochemical perturbations in biopolymer systems (Zhu et al., 2024). Importantly, because these effects are localized, ultrasound enables structural modification of proteins without substantially increasing the bulk temperature of the system, which is a key advantage for preserving heat-sensitive nutritional and functional attributes.

From a plant protein perspective, aqueous protein dispersions behave as dynamic soft matter systems, where macromolecular organization is governed by a delicate balance of intermolecular interactions, including hydrogen bonding, hydrophobic interactions, electrostatic forces, and van der Waals interactions (Kusova et al., 2022). In their native state, many plant proteins exist as compact globular structures or aggregated assemblies stabilized by these interactions. The introduction of ultrasound energy disrupts this equilibrium by imposing external mechanical stress on the system, thereby triggering progressive structural rearrangements across multiple hierarchical levels (Quarato et al., 2023). In this sense, ultrasound acts not only as a physical processing method, but also as an external field that reshapes the energy landscape of protein assemblies.

At the molecular level, cavitation-induced forces disrupt non-covalent interactions stabilizing native protein conformations. Hydrogen bonding networks are weakened, hydrophobic cores become partially exposed, and disulfide bond arrangements may be altered through mechanical perturbation and localized redox microenvironments (Hua et al., 2024). These changes promote partial unfolding of protein chains, increasing molecular flexibility and exposing functional groups that were previously buried within the protein interior. As a result, proteins transition from compact structures toward more open and reactive conformations, which are more favorable for interfacial and enzymatic interactions (Zhang J. et al., 2025).

At the supramolecular level, ultrasound induces the dissociation of protein aggregates and the reorganization of higher-order assemblies. Large protein clusters are broken down into smaller entities due to shear-induced fragmentation and cavitation-driven collisions (Pathak et al., 2022). Concurrently, partial unfolding enhances intermolecular interactions in a more controlled manner, allowing for the formation of new protein–protein interaction networks. Depending on processing intensity, this can either lead to improved dispersion stability or, under excessive energy input, promote reaggregation due to exposure of hydrophobic regions (Lee et al., 2025). Therefore, this stage represents a critical transition zone where the balance between dispersion and aggregation is determined.

At the colloidal scale, these molecular and supramolecular transformations manifest as significant changes in particle size distribution, surface charge properties, and dispersion stability. Reduction in particle size increases the specific surface area of protein particles, enhancing their ability to interact with surrounding phases (Yildiz, 2025b). Simultaneously, changes in surface hydrophobicity and ζ-potential influence electrostatic repulsion and colloidal stability, which are key determinants of protein behavior in complex food systems (Jiao et al., 2025). These colloidal modifications directly govern macroscopic properties such as solubility, emulsification capacity, foaming stability, and gel formation behavior.

The efficiency and direction of these ultrasound-induced modifications are highly dependent on processing parameters such as frequency, amplitude, power density, and treatment time. Higher amplitudes generally increase cavitation intensity, resulting in stronger structural disruption (Sesis et al., 2013). In addition to ultrasound intensity, treatment time plays a critical mechanistic role because cavitation events occur as a sequence of repeated energy pulses rather than as a continuous perturbation. The collapse of individual cavitation bubbles releases energy over microsecond timescales, whereas protein relaxation, conformational rearrangement, and partial refolding generally occur over substantially longer time intervals (Cui et al., 2025). Consequently, the structural evolution of plant proteins depends on the competition between the frequency of cavitation events and the available off-time during which partially unfolded molecules may reorganize (Jadhav et al., 2024). When sufficient relaxation time exists between successive cavitation events, proteins may partially recover stable conformations or establish favorable intermolecular interactions. Conversely, prolonged or continuous sonication repeatedly perturbs proteins before structural relaxation is completed, promoting cumulative unfolding, increased exposure of hydrophobic domains, and ultimately reaggregation under excessive energy input (Lee et al., 2025). This temporal interplay between cavitation dynamics and protein structural kinetics provides an additional mechanistic explanation for the existence of an optimal cavitation window, in which controlled unfolding enhances functional properties while avoiding irreversible structural deterioration. In addition, system-specific factors such as protein concentration, pH, ionic strength, and molecular composition significantly influence cavitation dynamics and energy distribution within the protein matrix (Abd Rahim et al., 2024). These factors collectively determine whether ultrasound acts predominantly as a dispersing, unfolding, or aggregating force within the system. Therefore, ultrasound processing should be understood as a complex, energy-driven structuring tool whose effects emerge from the interplay between process conditions and intrinsic protein susceptibility. Rather than inducing uniform changes, ultrasound generates hierarchical and system-dependent structural transformations that propagate from molecular to macroscopic scales (Poluri et al., 2026). This multiscale nature is fundamental to understanding and controlling protein functionality in plant-based food systems, as conceptually illustrated in Figure 1, which integrates energy input, hierarchical structural transitions, and macroscopic functional outcomes.

3 Ultrasound-induced multiscale structural modifications and mechanisms

3.1 Modulation of solubility and dispersion stability

Ultrasound is an effective physical strategy for enhancing the solubility and dispersion stability of plant proteins through acoustic cavitation, which generates localized shear forces, shock waves, and microstreaming (Das M. et al., 2022). These phenomena disrupt protein aggregates, reduce particle size, and induce partial unfolding of native structures. As a result, hydrophilic residues become more exposed, strengthening protein–water interactions and improving colloidal stability (Hoo et al., 2022). Across plant protein systems, cavitation-induced structural modifications generally enhance hydration and dispersibility; however, the response is inherently non-linear. Moderate ultrasound promotes aggregate dissociation and the formation of smaller, more flexible protein assemblies, thereby improving solubility and emulsifying performance (Qian et al., 2023). In contrast, excessive energy input promotes hydrophobic exposure and intermolecular reaggregation, leading to partial loss of functionality (Tay et al., 2026). This balance is governed by processing intensity, treatment duration, and medium properties. System-dependent differences are evident. Legume proteins are generally more susceptible to cavitation-induced unfolding, whereas structurally compact proteins such as rice protein require combined treatments (e.g., alkaline or thermal assistance) to achieve comparable improvements (Kim et al., 2024). These trends are summarized in Table 1.

TABLE 1. Summary of ultrasound-induced structural modifications and solubility enhancement mechanisms in plant protein systems.

Although plant protein isolates are commonly discussed as single ingredients, they are inherently heterogeneous systems composed of multiple protein fractions possessing distinct molecular architectures, quaternary organizations, and physicochemical behaviors. For example, soy protein primarily consists of 7S β-conglycinin and 11S glycinin, whereas pea protein contains vicilin, legumin, and convicilin fractions, each exhibiting different structural stability and susceptibility to cavitation-induced unfolding. Similarly, cereal proteins comprise structurally diverse glutelins, prolamins, and albumin–globulin fractions that respond differently to ultrasound treatment. Consequently, the overall functional response observed in a protein isolate represents the combined contribution of these individual fractions rather than a uniform structural modification of a single protein species.

3.2 Ultrasound-induced conformational rearrangements and structural transitions

Ultrasound induces multiscale conformational rearrangements in plant proteins via cavitation-generated mechanical forces and localized thermal effects. These perturb hydrogen bonding, hydrophobic interactions, electrostatic forces, and disulfide linkages, resulting in partial unfolding and structural reorganization at secondary and tertiary levels (Zhu et al., 2026). A consistent trend across protein systems is a reduction in α-helix content accompanied by increases in β-sheet and random coil structures, indicating a transition toward more flexible conformations with enhanced interfacial functionality (Table 2). These changes are also associated with increased surface hydrophobicity, altered intrinsic fluorescence, and shifts in zeta potential (Zheng et al., 2026). The extent of structural modification is strongly dependent on ultrasound intensity. Moderate conditions favor controlled unfolding and functional enhancement, whereas excessive treatment promotes reaggregation or structural compaction (Antunez-Medina et al., 2025). Synergistic treatments such as pH shifting, thermal processing, or enzymatic modification further stabilize partially unfolded intermediates and enhance functional outcomes.

TABLE 2. Ultrasound-induced conformational rearrangements and secondary/tertiary structural transitions in plant protein systems and their functional consequences.

3.3 Emulsifying and interfacial functionalities

Ultrasound enhances the emulsifying and interfacial properties of plant proteins by reducing aggregate size, increasing molecular flexibility, and exposing hydrophobic domains. These modifications improve adsorption kinetics at oil–water and air–water interfaces and facilitate the formation of viscoelastic interfacial films (Yildiz et al., 2017). Moderate ultrasound promotes rapid interfacial rearrangement, resulting in improved stability of emulsions, foams, and high internal phase emulsions (HIPEs) (Dong X. et al., 2026). Conversely, excessive cavitation can induce protein–protein reassociation at interfaces, reducing interfacial stability and functional performance (Manzoor et al., 2026). While these trends are broadly consistent across protein systems, structurally rigid proteins such as rice protein typically require synergistic treatments (pH adjustment, thermal processing, or enzymatic modification) to achieve comparable functionality (Yin et al., 2026; Table 3).

TABLE 3. Effect of ultrasound treatment on emulsifying and interfacial functionalities of plant proteins.

3.4 Gelation, rheology, and network formation

Ultrasound significantly influences gelation behavior and rheological properties by inducing partial unfolding and exposing reactive groups such as hydrophobic residues and sulfhydryl moieties. These changes enhance protein–protein interactions and promote the formation of three-dimensional networks with improved mechanical strength and water-holding capacity (Dong Z. et al., 2026). Moderate cavitation facilitates the formation of dense and elastic gel networks stabilized by hydrophobic interactions, hydrogen bonding, and disulfide linkages. In contrast, excessive treatment disrupts network continuity, resulting in weaker gel structures and reduced viscoelastic properties (Yildiz et al., 2025). Protein-specific responses are evident. Legume proteins generally exhibit pronounced gelation enhancement, whereas rice proteins require combined treatments for effective network formation (Náthia-Neves et al., 2025). These trends are summarized in Table 4.

TABLE 4. Effect of ultrasound on gelation, rheology, and network formation of plant proteins.

3.5 Protein–polysaccharide and protein–polyphenol interactions

Ultrasound enhances protein–biopolymer interactions by increasing molecular flexibility and exposing reactive residues, thereby facilitating hydrogen bonding, electrostatic interactions, hydrophobic associations, and, in some cases, covalent bonding (Ye et al., 2025). Cavitation-induced unfolding improves compatibility with polysaccharides and polyphenols, leading to enhanced emulsification, encapsulation efficiency, and colloidal stability. However, excessive ultrasound may reduce interaction specificity due to overexposure of hydrophobic domains and partial destabilization of preformed complexes (Jiang et al., 2019). Soy, hemp, and lentil proteins show strong improvements in complex formation, whereas structurally rigid proteins such as rice protein require synergistic treatments to achieve similar performance (Table 5).

TABLE 5. Effect of ultrasound on protein–polysaccharide and protein–polyphenol interactions in plant proteins.

3.6 Digestibility, bioactivity, and nutritional enhancement

Ultrasound improves protein digestibility and bioactivity by increasing enzymatic accessibility through structural loosening and exposure of cleavage sites. This facilitates gastrointestinal hydrolysis and promotes the release of bioactive peptides with antioxidant, antihypertensive, and other functional properties (Aghababaei et al., 2024). Moderate ultrasound enhances enzymatic efficiency, whereas excessive treatment may induce reaggregation, limiting substrate accessibility. Synergistic approaches, including germination, enzymatic hydrolysis, and thermal or alkaline treatments, further amplify bioactive outcomes (Guo et al., 2026). Across protein systems, improved antioxidant capacity, ACE-inhibitory activity, and metabolic enzyme inhibition are consistently reported (Table 6).

TABLE 6. Ultrasound effects on digestibility, bioactivity, and nutritional enhancement of plant proteins.

3.7 Ultrasound-assisted extraction and sustainable protein recovery

Ultrasound-assisted extraction (UAE) enhances protein recovery through cavitation-driven cell disruption, improved mass transfer, and increased solvent penetration into protein-rich matrices. Compared with conventional methods, UAE reduces extraction time, solvent usage, and energy consumption (Kumar et al., 2021). Moderate ultrasound improves yield while preserving functional integrity, whereas excessive cavitation may induce aggregation, oxidation, or loss of nutritional quality. Rice and oilseed proteins exhibit strong yield enhancement, while legumes respond effectively under milder conditions (Table 7). Synergistic combinations with alkali, heat, or pulsed electric fields further enhance extraction efficiency and functional performance. These effects are summarized in Table 7.

TABLE 7. Ultrasound-assisted extraction (UAE) and sustainable protein recovery from plant sources.

3.8 Mechanistic integration: aggregation–disaggregation equilibrium and the optimal cavitation window

Across plant protein systems (Tables 1–7), ultrasound acts as a cavitation-driven physicochemical stimulus that governs protein structural transitions through a balance between destabilization and reassembly. Cavitation events generate extreme localized conditions that transiently disrupt hydrogen bonding, hydrophobic interactions, electrostatic forces, and disulfide linkages, enabling partial unfolding and exposure of buried residues (Hua et al., 2024).

Proteins do not follow a linear unfolding pathway but instead occupy a dynamic ensemble of conformational states, including native aggregates, partially unfolded intermediates, and re-associated supramolecular assemblies. Functional properties arise from the relative distribution of these states rather than a single dominant structure. At intermediate ultrasound intensities, proteins predominantly adopt partially unfolded conformations characterized by increased flexibility and surface accessibility (Zhou et al., 2025). These states promote enhanced interfacial adsorption, improved hydration, and the formation of viscoelastic networks, leading to improved solubility, emulsification, and digestibility. At higher intensities, the system exceeds this functional window, where excessive hydrophobic exposure promotes uncontrolled intermolecular association and aggregation. This results in kinetically trapped structures with reduced colloidal and interfacial functionality (Frolova et al., 2022).

The optimal cavitation window is strongly protein-dependent, reflecting differences in molecular architecture, protein composition, oligomeric organization, and structural stability. Cereal proteins such as rice protein are characterized by relatively compact and structurally resistant assemblies, which can limit ultrasound-induced unfolding and may necessitate complementary treatments such as alkaline or thermal processing. In contrast, legume proteins, including chickpea, pea, lentil, and faba bean proteins, contain predominantly globulin fractions with distinct oligomeric organizations that can undergo aggregate disruption, partial unfolding, and increased exposure of hydrophobic or sulfhydryl groups under suitable ultrasound conditions. Oilseed proteins, such as pumpkin seed protein, exhibit yet another response pattern, with ultrasound-associated fragmentation, molecular-weight reduction, increased flexibility, and sulfhydryl exposure being reported (Li et al., 2025). These differences demonstrate that the same nominal ultrasound conditions cannot be assumed to produce equivalent molecular responses across plant protein systems.

Importantly, the optimal cavitation window should also be interpreted in relation to quantitative ultrasound processing conditions rather than as a purely descriptive concept. Frequency, acoustic power density, and treatment duration collectively influence the effective cavitation environment and, consequently, the extent of protein structural modification. Specific energy input (J/g), when sufficient information is available, can provide an additional basis for comparing treatments performed under different processing configurations. However, these parameters should not be considered independently, because the effective energy delivered to the protein suspension also depends on sample mass, protein concentration, medium properties, and ultrasonic system characteristics. Thus, the optimal cavitation window should be regarded as a system-dependent operating region rather than a universal numerical threshold.

4 Limitations and future perspectives

From an application standpoint, ultrasound-assisted processing represents a scalable, energy-efficient, and environmentally friendly technology aligned with current trends in clean-label formulation, sustainable food processing, and the valorization of plant-based protein resources (Prempeh et al., 2025). Its capacity to enhance extraction efficiency, improve functional properties, and reduce reliance on harsh chemical treatments makes it highly attractive for industrial implementation. However, several limitations still hinder its full-scale adoption. These include the lack of standardization of processing parameters across different ultrasonic systems, incomplete understanding of energy efficiency and heat dissipation at industrial scale, potential variability in product functionality arising from matrix-dependent responses, and limited real-time monitoring of protein structural transitions during processing (Yao et al., 2020; Thirumdas and Mudgil, 2025; Liu X. et al., 2025; Patil et al., 2025). In addition, scale-up often results in non-uniform energy distribution, which may compromise the reproducibility of functional outcomes.

Future research should therefore focus on bridging the gap between laboratory-scale optimization and industrial implementation. Key directions include the integration of ultrasound with complementary emerging technologies such as pulsed electric fields, cold plasma, and high-pressure processing to achieve synergistic and controllable structural modifications. The development of in situ and real-time monitoring tools capable of tracking protein unfolding, aggregation, and interfacial dynamics during sonication will be essential for precise process control. Furthermore, multiscale computational modeling combined with advanced spectroscopic and imaging techniques should be expanded to better elucidate ultrasound-induced structural transformations across molecular, supramolecular, and colloidal scales. Finally, the design and optimization of continuous-flow ultrasound reactors with improved acoustic uniformity, energy efficiency, and scalability will be critical for industrial translation. Overall, advancing these research directions will enable more precise control over structure–function relationships in plant protein systems, facilitating the rational design of next-generation functional ingredients and sustainable plant protein-based food formulations.

5 Conclusion

Ultrasound processing has emerged as a versatile and sustainable green technology for tailoring the structural and functional properties of plant proteins across multiple hierarchical levels. This review demonstrates that acoustic cavitation drives key physicochemical modifications, including partial unfolding, disruption of non-covalent interactions, reduction in aggregate size, and reorganization of supramolecular structures. Collectively, these changes lead to improved techno-functional properties such as solubility, emulsification, gelation, interfacial activity, digestibility, and bioactivity across diverse plant protein systems.

A central finding of this review is the highly non-linear relationship between ultrasound intensity and functional performance. While moderate cavitation enhances molecular flexibility and functional properties, excessive treatment may induce reaggregation, structural degradation, and loss of functionality. Therefore, defining an optimal cavitation window for each protein system is critical for controlled structural engineering. Future advances should focus on process standardization, scale-up of continuous-flow systems, and integration of ultrasound with complementary non-thermal technologies to enable precise control over structure–function relationships in sustainable plant protein-based food systems.

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Footnotes

Edited by: Daniela Lamas, Institute of Marine and Coastal Research (IIMyC), Argentina

Reviewed by: Riquelme, N., University of Chile, Chile

Giulia D'Alessio, Università degli studi di Teramo, Italy