Protein Nanofibrils as Emerging Biomaterials for Sustainable Food Packaging: Advances, Challenges, and Future Opportunities

ABSTRACT

The transition toward sustainable food packaging has driven intensified research into bio‐based materials capable of reducing dependence on conventional plastics. Functional amyloid protein nanofibrils (PNFs) have emerged as a novel class of supramolecular biomaterials owing to their highly ordered β‐sheet–rich structures. PNFs can be derived from multiple protein sources, including dairy, plant, egg, and underutilized agro‐industrial protein sources, thereby supporting resource valorization and integration into the circular bioeconomy. PNFs exhibit great mechanical strength, barrier properties, and intrinsic biocompatibility. This review systematically analyzes recent advances in PNF‐based materials for food packaging applications. It covers fundamental aspects of PNF molecular structure, self‐assembly mechanisms, and fibrillation kinetics, along with fabrication and film‐forming strategies. The influence of processing routes on mechanical, thermal, and barrier properties is critically discussed. In addition, emerging approaches for imparting active and smart functionalities are evaluated. It also identifies and covers the core challenges to scaling up production, safety, regulation, and public perception, and map out pathways for industrial adoption. PNF‐based films exhibit outstanding mechanical performance and gas barrier properties, often exceeding those of conventional biopolymer packaging materials, while maintaining biodegradability. Despite these advantages, industrial translation is limited by processing efficiency and regulatory uncertainties. Future research should focus on scalable continuous fibrillation, low‐cost protein side streams, hybrid composite systems, bioactive/smart functionalities, pilot‐scale validation, and circular economy approaches for next‐generation edible and biodegradable packaging solutions.

1. Introduction

Globally, plastic packaging is essential for protecting food products from spoilage and damage during transport, most of which is single‐use plastic, revealing that the food industry is responsible for millions of tons of plastic waste each year, usually from single‐use synthetic packaging and petroleum‐based plastics. Currently plastics are classified based on their biodegradability and source of material used, as shown in Figure 1. A large portion of this food packaging plastic waste contributes to social and ecological pollution, landfilling, and microplastic creation and accumulation in ecosystems (Fredi and Dorigato 2021). These microplastics are infiltrating the food supply chain, raising concerns about potential health implications (Ncube et al. 2021). So, there is a growing focus on transitioning to sustainable packaging methods necessary to achieve circular economic outcomes while complying with ever more stringent regulatory frameworks. Potentially viable sustainable materials, such as bio‐based and biodegradable materials have been reported in the literature for their applications (Thapliyal et al. 2024). Biopolymers in food packaging, such as cellulose and chitosan, can be sustainable alternatives to petroleum‐based materials. Despite their advantages, these starch‐based packaging materials lack functionality compared with conventional plastics. These functional drawbacks can be rectified by advanced biomaterials sourced from renewable proteins, polysaccharides, and lipids, which show promising biodegradable properties and are also considered the safest option for food contact. An additional point of significance lies in the integration of nanotechnology, which can provide bio‐derived packaging with improved strength and stability while also enhancing functionality (Ghosh et al. 2025).

FIGURE 1.

Types of plastic based on source and biodegradability.

Although traditional protein‐ and polysaccharide‐based films are natural, they have key issues involving low mechanical strength, low moisture barrier, and low thermal stability. These films have poor barrier properties against gases such as oxygen and carbon dioxide, limiting their use in high moisture or long‐shelf‐life applications. Scaling these materials is also a challenge owing to inconsistent processing methods and incompatibility with industrial manufacturing systems (G. Wei et al. 2017). Moreover, achieving functional characteristics of these films, such as antimicrobial or sensing activity, often relies on chemically modifying the material, which could affect their biodegradability and safety. The above issues indicate the need for new, innovative nanostructured materials that are mechanically robust, functionalized, and sustainable. These shortcomings have spurred research to engineering nanostructured biomaterials, such as functional amyloid protein nanofibrils (PNFs), engineered to narrow the performance gap between natural polymers and synthetic plastics (Knowles and Mezzenga 2016).

Functional amyloid PNFs have become an innovative platform for applications across industries, including biomedicine, nanotechnology, and now sustainable food packaging. This holds promise as a biomaterial due to its perfectly ordered β‐sheet structure, mechanical properties, and the structural stability of these structures under diverse environmental conditions (Zhao and Yang 2020). PNFs are formed by controlling the denaturation and self‐assembly of naturally derived proteins of various origins, including β‐lactoglobulin (lg), soy, or pea proteins, and PNFs exhibit high surface area, rigidity, and tunable functional properties (Reynolds 2019). Good compatibility with bioactive compounds has facilitated the production of active and intelligent packaging films that can demonstrate antimicrobial, antioxidant, or pH‐responsiveness functionality. Recently, studies have shown that PNFs can be produced at larger scales and incorporated into composite matrices, providing significant barrier and mechanical improvements for packaging food products (Kumar et al. 2021).

This review evaluates the feasibility of PNFs as an edible, biodegradable biomaterial for next‐generation food packaging by critically examining their formation pathways, structure, and functional properties of PNFs, and the modes of combining PNFs with other biopolymers for composite films. Primarily focusing on applications for active and smart packaging concepts, where the PNF can add functionality along with safety and sustainability credentials. The analysis encompasses industrial up‐scaling issues, environmental ramifications, regulations, and pinpoints existing research gaps. Highlighting the latest developments and opportunities for commercialization, the review also outlines the further assistive development of utilizing PNFs to deliver safer, functional, and sustainable food packaging systems on a global scale.

2. Fundamentals of Functional Amyloid PNFs

Amyloid PNFs are highly ordered, self‐assembled protein nanostructure composed of cross‐β‐sheets rich assemblies formed through the aggregation of proteins or peptides into elongated fibrillar networks with diameters of a few nanometers and length extending to several micrometers. Unlike pathological amyloid fibrils, whose formation occurs in vivo through protein misfolding and aggregation within extracellular matrix or intracellular compartments such as neurons, pathogenic and food‐derived fibrils are biologically disconnected; ingestible amyloid‐like structure do not include pathogenic aggregation. Food‐derived functional amyloid nanofibrils (ANFs) are non‐pathogenic biomaterials fabricated via controlled in vitro thermal‐acid denaturation followed by self‐assembly via molecular attraction to form nanofibrils of safe, globular, non‐prion dietary proteins, they are often referred to as functional amyloids. They lack self‐replicating toxicity, offering large aspect ratio, precise viscoelasticity, functional delivery, and superior structural stability, represent a new class of materials that can serve as alternatives to food structuring materials or for use in sustainable packaging systems.

2.1. Structural Characteristics of Functional ANFs

Function amyloid PNFs are made of a canonical cross‐β topology, in which β strands are roughly perpendicular to the axis of the fibril and stack via backbone H‐bonds to create β‐sheets in 8 different arrangements from the fibril's axis. These form spacing values of inter‐strand ∼4.7 Å and inter‐sheet ∼10 Å, along with its eight different β‐sheet formation namely four antiparallel β‐sheet and four parallel β‐sheet as clearly shown in Figure 2, may be observed by diffraction and cryo‐EM/SSNMR, and account for extensive stiffness, solvent, and protease/container resistance (Wilkinson et al. 2023). Recent advances in cryo‐EM enable visualization of polymorphic filament folds, structural breaks, and protofilament interfaces, providing structural evidence that multiple topologies can coexist within a single fibril and control the mechanics and surface chemistry of interfacial films and coatings (Frieg et al. 2024). Many of the solved structures contain parallel in‐register β‐sheets; use side‐chain ladders and steric‐zipper interfaces tuned surface hydrophobicity and adsorption, which is important for barrier films.

FIGURE 2.

(A) Schematic representation of parallel and antiparallel β‐sheet arrangements formed during amyloid assembly (B) Characteristic cross‐β architecture of amyloid fibrils (C) Functional Amyloid protein nanofibrils formation kinetics.

Lipid‐bound fibril structures revealed a continuous lipid‐fibril complex, suggesting that amphiphilic environments, such as fatty food matrices, can stabilize alternative fibril morphologies in solution and influence film permeability and aroma retention in interfacial films (Das et al. 2018). Polymorphism (i.e., different cross‐sections, twist pitches, and protofilament numbers) enables tunable properties via process parameters (e.g., pH, ionic strength, cosolvents). Importantly, advanced resolution maps have recently solidified the cross‐β paradigm while revealing structural heterogeneity that materials engineers can exploit for film design, adhesion, and controlled release in packaging applications (B. Liu, Zhang, et al. 2025).

2.2. Formation Mechanisms From Food Proteins

Food proteins such as β‐lactoglobulin, serum albumins, and plant globulins typically assemble into PNFs in two steps via a nucleation‐polymerization mechanism with sigmoidal kinetics, characterized by a lag, growth, and plateau phase. The model for the formation of fibrils is expressed as (Equation 1) (J. Wei et al. 2025):

$$\frac{dM}{dt}=-2k_{+}MP-n_c{k}_n{M}^{n_c}-n_2{k}_2{M}^{n_2}$$ (1)

where M is monomer concentration, P is fibril number, $k_{+}$ the elongation rate constant, and $k_n$, $k_2$ denote primary and secondary nucleation rates respectively. Primary nucleation initiates fibril formation during the lag phase, elongation drives fibril growth, while secondary nucleation accelerates fibrillation by generating new nuclei on existing fibril surfaces, producing the characteristic sigmoidal growth curve observed in functional amyloid PNF formation. Proteins exhibiting higher hydrophobic amino acid content and greater exposure of amyloidogenic peptide sequences generally possess larger values of k₊ and k₂ , resulting in faster fibrillation kinetics and the formation of more rigid β‐sheet‐rich nanofibrillar networks. Modeling with programs like AmyloFit allows rate constants and nucleation orders to be extracted at different pH, temperatures, and ionic strengths (Sárkány et al. 2024).

Studies indicate that secondary nucleation is frequently the dominant fibrillation mechanism at mildly acidic, heated conditions similar to those in food processing. In general, pH (∼1.8–3), temperature (75°C–95°C), ionic strength, and buffer composition are important modulators of monomer unfolding and peptide fragment generation, which can initially act as seeds to enhance fibril formation (Pang et al. 2025). Peptide fragments generated through hydrolysis (∼12–20 residues) significantly shorten nucleation lag and prompt fibril morphology as shown in Figure 3. Air–water or oil–water interfaces promote oligomer formation and anisotropic fibril growth, both of which are desirable for even film casting (Jaklin et al. 2022). Recent reviews highlight that modifications to the fibrils after fibrillation, such as cation bridging or acylation, allow for additional modulation of their dispersibility and interfacial characteristics relevant to coatings (Ouyang et al. 2025).

FIGURE 3.

Hierarchical assembly and structural organization of functional amyloid protein nanofibrils. (a) Formation of amyloid fibrils from unfolded polypeptide chains. (b) Assembly of polymorphic fibrils, association of different protofilament types. (c) Cross‐sectional views of peptide residue packing within β‐sheet structures. (d) Side views illustrating intermolecular hydrogen bonding and β‐sheet organization. (e) Cross‐sectional arrangement and packing of protofilaments within fibrils. (f) Mature amyloid fibrils showing protofilament twisting and polymorphism, with enlarged views of the cross‐β‐sheet architecture.

3. Protein Sources for Nanofibril Formation

Many proteins, both from animal and vegetable sources, can be made to self‐assemble in a controlled fashion to produce ANFs. The self‐assembly of protein into a fibrillar structure depends upon two separate but related properties: how many amino acids there are in a polymer and how stable the three‐dimensional conformation of the polymer is when it is allowed to partially denature. The most frequently used proteins for the study of self‐assembly into fibril structures are, among other proteins, dairy‐derived proteins such as β‐lactoglobulin and casein—and egg‐derived proteins such as lysozyme and ovalbumin and plant‐derived proteins such as soy and pea. These proteins can form stable nanofibrillar structures under certain conditions, including low pH and high temperature, which makes them an excellent source for developing functional biomaterials.

3.1. Dairy Proteins

Milk‐derived biomacromolecules include a variety of dairy proteins (whey proteins and caseins) that are widely used in the food and pharmaceutical industries. Dairy proteins possess high nutritional value, structural integrity, and the potential to provide some of the best functional qualities of any protein source. β‐Lactoglobulin (β‐Lg) is the primary driver for producing food‐grade PNFs because it unfolds and hydrolyses under acid‐heat conditions (pH 2.0, 80°C) to peptide fragments that quickly self‐assemble to cross‐β fibrils with sigmoidal kinetics, leading to the promise of scalable and manageable feedstocks for films and coating (Lendel and Solin 2021).

Once seed formation occurs, the kinetics becomes dominated by elongation and secondary nucleation which has a shortening of lag phase and creates high aspect‐ratio fibrils within the biopolymer matrices (Rathod and Amamcharla 2024). For instance, the superior packaging performance of whey protein PNFs is directly linked to their highly ordered hydrogen‐bonded β‐sheet network. Yang et al. (2024) reported that incorporation of 15 wt% of whey PNFs significantly increased film elastic modulus from 19 to 54 MPa while maintaining ductility, confirming that fibril‐induced reinforcement originates from rigid β‐sheet domains acting as nanoscale load‐bearing structures within the protein matrix.

Caseins, with their historic characterization as intrinsically disordered micelles, can also be used to form functional amyloid fibrils with mild heating (∼65°C) and the pH shift, opening possibilities of exposing more dairy‐sourced compounds (beyond β‐Lg) to yield interfacial layers that may exhibit desirable rheology for coatings (Rafikova et al. 2025). Due to their low molecular weight (19–25 kDa) and self‐aggregation, casein exhibits poor electrospinning ability. However, dispersions containing 1 wt% κ‐carrageenan prepared at 10 pH, 50% ethanol and 60°C produces electrospun mats with minimal bead defect (2.0 × 10⁻³ µm⁻²), tensile strength of 0.2 MPa and modulus of 12 MPa. After 48 h at 100% RH, water uptake reached 140%, increasing strength and modulus by more than 10‐fold with 2‐fold decrease in elongation at break, highlighting the potential of casein PNF films for biodegradable packaging applications (Sharma et al. 2025). These reviews have highlighted dairy PNFs as sustainable building blocks for “smart” packaging, combined with natural pigments, to create colorimetric freshness indicators for fish and meat (Peydayesh et al. 2025; Holt and Carver 2024).

3.2. Egg Proteins

Another class of highly promising fibril forming proteins are egg proteins. Lysozyme an egg white protein is among the earliest proteins used to study functional amyloid fibrillation. Lysozyme nanofibril (LNFs) have shown excellent potential for active food packaging. Silva et al. (2018) developed transparent pullulan/LNF films containing up to 15 wt% LNFs, exhibiting high mechanical strength; young's modulus ranges from 1.91–2.50 GPa, high thermal stability up to 225°C, 77% DPPH scavenging antioxidant activity. Antimicrobial activity against Staphylococcus aureus increased with nanofibril concentration, demonstrating that β‐sheet rich lysozyme fibrils can simultaneously enhance mechanical, thermal, antioxidant, and antimicrobial properties of edible packaging film.

The study on ovalbumin nanofibrils (OVNFs) based antibacterial edible film showed significantly improved performance compared with native ovalbumin films. OVNFs increased tensile strength by 3.15 MPa and elongation at break by 17.71%, while reducing water absorption and solubility by ∼30%. Similarly, oxygen and water vapor permeability (WVP) decreased to 5.94 g m⁻² d⁻¹ and 2.58 g mm m⁻² h⁻¹ kPa⁻¹. Further incorporation of 0.5% bioactive glass provides nearly 100% antibacterial inhibition demonstrating the potential of egg PNFs for developing mechanically robust, barrier enhanced, and active packaging materials (Han et al. 2026; Ye et al. 2026). Ovotransferrin (OVT), a major egg‐white protein, can be converted into β‐sheet‐rich ovotransferrin fibrils (OVTNFs) through thermal polymerization at 90°C, 2.0 pH for 16 h. Structural characterization confirmed successful fibrillation. OVTF exhibited strong antibacterial activity with MIC values of 64 µg mL⁻¹ against S. aureus and 128 µg mL⁻¹ against E. coli, attributed to membrane disruption, electrostatic interactions, hydrophobic binding, and iron sequestration. These findings highlight the potential of egg‐PNFs as multifunctional antimicrobial materials for active food packaging applications.

3.3. Plant Proteins

Plant proteins are renewable and biodegradable source for making functional amyloid PNFs has recently attracted considerable interest as a sustainable approach. Plant sources of protein including soy, pea, and wheat gluten can produce controlled denaturation assemblies (self‐assembled) to create fibrillar structures. Moreover, the nanofibrils generated from crops provide a sustainable (renewable and biodegradable) biomaterial for designing sustainable food packaging systems. Plant proteins form PNFs through acid‐heat, or enzyme‐assisted routes providing fossil‐free reinforcements for edible films and coatings (Liu, Chen, et al. 2025). Soy protein amyloid fibrils (SAFs) can be produced by acidic heating and used to create electrospun pullulan/Soy‐PNF films with significantly improved mechanical attributes and similar morphologies suitable for packaging applications (J. Zhao et al. 2025).

Pea protein forms amyloid PNFs under controlled profiles of pH and temperature, and recent work has focused on developing PNF–polyphenol co‐networks that reinforce hydrogels and could extend to moisture‐stable coatings. Rice‐protein‐derived PNF stabilizes mixed‐gel composites, which suggests the potential for accompanying toughness in mixed‐protein films bedecked with similarly derived PNF (Ma et al. 2025).

Zein is traditionally processed in ethanol, but is now also producing nanofibrils via protease‐mediated means in aqueous phases to overcome previous solvent barriers, enabling greener methods for producing corn‐protein PNF coatings (M. Li et al. 2025). PNF coats, across a diversity of plant systems, lower WVP, and OTR through a densification of the hydrogen bonded PNF network and thus increase tortuosity while also allowing dispersal of active payloads for controlled release of actives typically antioxidants/antimicrobials controlled by Fickian diffusion (Karabulut 2025). Recent reviews enclosing motivating commentary that maps plant protein sequence motifs, fibrillation windows, and multi‐scale morphologies providing researchers with a means to make rational selections from the source protein based upon their specific barrier or active‐function objectives (Miao et al. 2025).

3.4. Underutilized and Agro‐Industrial By‐Product Proteins

Underutilized plant sources are coming to the forefront like coconut endosperm proteins that can be made into functional amyloid fibrils used to stabilize Pickering emulsions, demonstrating feasibility for packaging‐relevant interfacial films and maybe edible coatings after peer‐reviewed scale‐up evidence is available (Song et al. 2026). Potato proteins present in the side stream of the starch industry have been shown recently to self‐assemble into PNFs with tunable morphology opening pathways to valorization for circular packaging materials (J. Zhang et al. 2025). Broader surveys have also shown that many food proteins, beyond dairy, can form PNFs with variability in unfolding and proteolysis, leading to a large design space for residue‐dependent fibrillation engineering (D. Xu et al. 2023).

Notable agro‐industrial waste valorization is the production of soy fibril from tofu wastewater. The resulting fibrils demonstrate a persistence length of 1.2 µm and β‐sheet rich structure, effectively reinforcing starch‐pectin bioplastics. Incorporation of fibrils increased elongation at break from 11.7% to 34.5% and toughness from 411 to 1141 MJ/m³, representing nearly a three‐fold improvement. Furthermore, the films displayed oxygen and water vapor barrier properties comparable to polyamide 6 and polylactic acid (Roy Goswami et al. 2025). The ideal sustainability model should include insect proteins given their high yields and protein quality. At the same time, there is a lack of direct packaging demonstrations of insect protein PNFs; however, the functional chemistry (gelation/emulsification) and their amino‐acid profile suggest that they will be amenable to fibrillation as soon as suitable denaturation and hydrolysis conditions are established. Nutrition and safety based surveys identify the processing routes that minimize allergens and microbiological concerns, which would be required to make edible PNF packaging from insects (Andrade et al. 2025). As summarized, these novel substrates offer the potential for diversifying supply chains, added valuations of by‐products, and new interfacial and mechanical behaviors that can be engineered for moisture sensitive and active‐release packaging concepts (Ouyang et al. 2025).

4. Fabrication Techniques of Functional Amyloid PNFs

Functional amyloid PNFs are fabricated using multiple advanced techniques, as summarized in Table 1, each enabling distinct structural and functional outcomes. For example, coaxial electrospinning of 12% (w/v) amyloid BSA yielded a bi‐layered coating that allowed for a controlled drug release pattern with biphasic release kinetics (Kabay et al. 2018). Superfast β‐sheet assembly with tris(2‐carboxyethyl) phosphine produced stiff bio‐composite sheet materials with elastic moduli up to 30 GPa and improved interfacial adhesion in a flexible, ultrathin work sensor (Wu et al. 2024). Artificial induction and electrospinning of PNFs resulted in zipper‐like cross‐β structures that can be formed into speciality multifunctional nanomaterials for a variety of uses (B. Liu, Zhang, et al. 2025). Scalable acid‐heat hydrolysis and enzymatic fibrillation were also used to produce edible, biodegradable packaging tobacco films with good barrier properties and embedding. The production of functional amyloid PNFs involves protein extraction, controlled fibrillation, and various physical or chemical modification strategies, as illustrated in Figure 4. Together, these structures allow us to adapt to and understand the amorphous, semi‐crystalline, and crystalline PNF structures found in nature, and to apply them to study, develop, and implement PNF technologies in environmental, biomedical, and electronic devices, including sustainable packaging.

TABLE 1.

Critical comparison of fabrication strategies for food‐grade protein nanofibrils (PNFs) with significant outcomes.

Methods	Technique	Experimental condition	Fibril length (µm) and diameter (nm)	Structural heterogeneity	Reference
Chemical method	Acid hydrolysis + heat‐induced self‐assembly	pH 2.0, 85°C, 10 h; whey protein isolate	Fibril length exceeds 4 µm and height distribution was around 4 nm periodicity ranged from 60 to 100 nm	Elongated and unbranched flexible amyloid fibril	Cheng et al. (2026)
		pH 2.7, 65°C; egg white lysozyme	Fibril length of 1 µm	Thin, long and unbranched fibrils	Poniková et al. (2015)
		pH ∼ 2–2.5, 80°C–90°C, 5–24 h; β‐lactoglobulin	β‐lactoglobulin fibrils typically 1–10 µm length; 2–10 nm diameter	Low; produces long, linear fibrils with high β‐sheet ordering	Ouyang et al. (2025)
	Glycation	1:100 of β‐Lg and sugar; pH adjusted to 7; duration of 8, 16, or 36 h; dialyzed for 48 h at 4°C	Process of fibrillation was inhibited, large polar sugar residue on the glycated fibrillogenic peptides	Impose steric restrictions and disrupting hydrophobic interaction	Dave et al. (2014)
		Whey protein isolate and saccharide were taken in equal ratio; hydrated in H2O by mild stirring overnight at 20°C	Fibril length was shortened above 75%	Fibrils with more heterogenous branches and irregular	G. Liu et al. (2020)
	Ethanol	Protein solution containing six range of absolute ethanol 0%–50%, incubated at 37°C and pH 2.0	Multiple properties of strands were denatured in the new fibril formed	Flexible fibrils with worm‐like morphology	Jordens et al. (2011)
		pH 2.0; containing 0%, 10%, 30%, and 50% ethanol; stirred at 400 rpm	Observation of reduction in fibril length	Exclusive worm‐like structure; increased oxidative stability	G. Liu et al. (2021)
	pH‐mediated enzymatic hydrolysis (protease/pepsin)	Mild enzyme AspN endoproteinase pre‐treatment (pH 7–2), 60°C for 8–12 h	Fibril length 1–10 µm; thickness ∼4 nm	Long, unbranched fibril	Hense and Strube (2023)
Physical/novel method	High pressure (HP)	Microfluidized at 50—170 MPa, adjusted to 2.0 pH, heated at 80°C for 20 h in water bath	Fibril height of 2–40 nm; length up to 15 µm; thickness (∼6 nm)	Exhibited linear morphology	Oboroceanu et al. (2011)
		Nanofibril solutions were passed for one cycle through a high pressure microfluidizer at 20,000 psi	Fibril length declined to range from 97 to 350 nm	Short fibrils influenced by high viscosity	Koo et al. (2018)
		pH adjusted from 7.3 to 2; Microwave heating at 80°C for 2 or 16 h at 4 W	Fibril width 7 nm and a length varying 0.1 to few µm	MH fibrils composed large peptides and higher surface hydrophobicity	Hettiarachchi et al. (2012)
	Shear‐ and ultrasonication‐assisted fibrillation	Ultrasonic probe 20 kHz/30 min or high‐shear mixing (1000–2000 rpm) during heating at 85°C	> 1 µm fibril length	Reduction of total α‐helix and β‐sheet.	Pathak et al. (2022)
		Ultrasonic frequency 20 kHz, 250 W power; 0–60 cycle sonication with 30 s sonication and 30 s pause; solution temperature at 25°C using	Fibril length range 50–200 nm, > 1 µm long fibrils; diameter range; 203.2 to 259.3 nm	Accelerated fibrillation process	D. Zhao et al. (2018)

FIGURE 4.

Schematic representation of protein fibrillation pathways and modification strategies to produce functional amyloid protein nanofibrils.

4.1. Acid Hydrolysis and Heat‐Induced Self‐Assembly

Innovation in the controlled formation of PNFs occurs under conditions of acid hydrolysis and spontaneous thermal self‐assembly. The acid hydrolysis of native proteins at low pH (2.0–2.2) permits controlled unfolding of native secondary structure, followed by fibrillation into β‐sheet‐rich structures. At a pH of 2.0, the proteolytic effect of acid generates partial peptide bond cleavage, potentially exposing amyloidogenic sequences which provide nucleation sites for protofilaments (Ouyang et al. 2025). Fibril self‐assembly is usually accelerated by incubating at 80°C–90°C for 5–24 h, producing fibrils < 10 nm in diameter and > 1 µm in length that are stabilized not only by intermolecular hydrogen bonding.

Prior research concluded that phase anisotropic nanostructures were produced through acid–heat treatment of food proteins, such as, β‐lactoglobulin and soy and consequently improved functional stability for use in edible packaging, emulsifiers, and hydrogels (T. Li et al. 2024). Waste valorization approaches such as working with tofu wastewater proteins can produce PNFs at scale under acid–heat conditions, with abundant yields of up to 75% conversion efficiency. Thermal‐induced fibrillation can produce transferable interfacial films with mechanical moduli of 2–3 GPa by potential use as gas–liquid filters or bioplastics (Mykolenko and Mezzenga 2025). For silk fibroin systems, generating the nanofibril dispersions via sulfuric acid degradation (40 wt% H₂SO₄, 60°C, 2 h) proceeded to self‐assembly through thermal evaporation (gas–liquid interface) producing transferable films with moduli of 2–3 GPa (Y. Liu et al. 2025). Overall, acid hydrolysis and thermal self‐assembly are both effective and inexpensive pathways for synthesizing functional PNFs with tunable morphology and mechanical properties for applications in food, biomedical, or sustainable materials.

4.2. Enzymatic and pH‐Mediated Methods

The formation of functional amyloid PNFs can be achieved via enzymatic and pH‐mediated pathways, exploiting the self‐assembly capability of proteins and peptides. Enzymatic mechanism uses a proteolytic or crosslinking reaction to induce fibrillation. For example, fibrinogen is converted into fibrin nanofibrils by thrombin cleaving fibrinopeptides and forming protofibrils. introducing calcium ions (Ca²⁺) promotes all phases of fibrillogenesis and stabilization through interactions with the binding sites of fibrinogen, allowing enzyme‐free gelation resembling pseudo‐fibrin (Hense and Strube 2023). Resilin‐like polypeptides (RLPs), inspired by the insect protein resilin, are synthesized to behave like resilin and its elasticity and resilience. During RLP processing, pro‐resilins are secreted as uncross linked proteins that are enzymatically crosslinked through di tyrosine and tri‐tyrosine bonds to give amorphous nanofibrillar hydrogels. The mechanical properties of RLPs include an impressive resilience up to 97% which is higher than natural elastin (90%) or synthetic rubbers (Balu et al. 2021).

The pH‐mediated approach utilizes the conformational transitions of amyloidogenic proteins. As with the self‐assembly of all amyloidogenic proteins, the self‐assembly of β‐lactoglobulin, amyloid‐β fragments, and diphenylalanine motifs in nanofibrils is strongly pH‐dependent because protonation reduces charge repulsion and favors β‐sheet stacking (G. Wei et al. 2017). The bacterial biofilm‐associated protein (Bap) of S. aureus has two behaviors: at an acidic pH, it forms amyloid fibrils that are rich in β‐sheet content that stabilize the matrix, while at a neutral pH it exists in a soluble adhesin state (Matilla‐Cuenca et al. 2022). Amyloid fibrils are influenced by external factors such as ionic strength, pH, and co‐factors for the enzyme that catalyze the crosslinking. Tuning these factors can directly influence the assembly of β‐sheet stacking of amyloid fibrils, fibril diameter, and rigidity of the fibrillar network. In food packaging, tuning these parameters helps design protein nanostructures with enhanced barrier, mechanical, and intrinsic antimicrobial capabilities to create sustainable alternatives to synthetic plastic (L. Wang, Gong, et al. 2019).

4.3. Shear and Ultrasonication Assisted Fibrillation

Shear flow and ultrasonication‐assisted techniques are becoming highly effective methods for synthesizing PNFs that resemble natural amyloid, as they provide the energy needed to overcome aggregation constraints. Shear‐driven fibrillation mimics the natural process of spinning silk, and, instead of forming a linear structure, hierarchical assembly occurs because shear flow induces preferential alignment of proteins (i.e., fibroin) into β‐sheet nanofibrils. The formed structures are stable fibrillar morphologies that are accomplished almost 90% greater energy efficiency than programming, and were originally assembled, in a quiescent state with rotational symmetry (Mu et al. 2020). Ultrasonication typically occurs at 20 kHz with 20–30 W/cm³, and the associated acoustic cavitation produces localized hotspots (> 5000 K, > 100 atm) and generate shear microjets (∼100 m/s). In addition to extreme conditions that suggest time scales for protein unfolding, nucleation, and β‐sheet stacking, sonication drives proteins into ordered amyloid fibrils or crystals with mean lengths of ∼22 µm.

Shear forces help induce helical amyloid assembly in β‐lactoglobulin and lysozyme by orienting unfolded intermediates, decreasing nucleation barriers, and increasing the rates of β‐sheet stacking, thereby providing a degree of directed molecular organization to form stable fibrils with controllable morphology and improved mechanical properties (Pathak et al. 2022). Arachin fibrillation by ultrasonication is achieved through cavitation‐driven nanofibrils. Sonication‐induced arachin fibrillation processes enable rapid nucleation and growth, yielding stable amyloid fibrils that demonstrate increased interfacial activity and tunable functional properties for both food and biomedical applications (Yang, Wang, et al. 2024). Low to moderate‐intensity sonication disrupts metastable states, promoting nucleation and the development of proteins such as insulin, α‐synuclein, and β2‐microglobulin; with extended sonication, mature fibrils fragment into shorter, uniform nanostructures that can repeat the process as controlled fibrillation seeds (Heyn 2020).

5. Film and Coating Formation From Functional Amyloid PNFs

Nanofibrils derived from amyloids are of great interest as materials for biodegradable films and coatings because of their special structural and functional properties. The combination of a high aspect ratio for mechanical strength, strong intermolecular forces, and a rich β‐sheet structure gives rise to the ability to form dense, highly solid networks. When dispersed in water and processed through either solvent casting or coating methods, nanofibrils form continuous films with improved mechanical and barrier properties. These properties of nanofibrils make them an excellent source for creating sustainable and edible food packaging materials. Functional amyloid PNFs can be converted into standalone films or directly applied as edible coatings using various processing techniques, as illustrated in Figure 5.

FIGURE 5.

Preparation and application routes of functional amyloid protein nanofibril (PNF)‐based films and coatings for food packaging.

5.1. Casting and Solvent Evaporation Methods

Solvent‐evaporation casting is the most popular laboratory method to produce continuous PNF films using casting, which is where a homogenous PNF/polymer dispersion is poured onto a supportive medium and the solvent is removed under controlled conditions (Pires et al. 2024). Leveling, Marangoni flows, and the levelling and final uniformity of thickness (or lack thereof) depend on the initial dispersion rheology (η and G′, G″) which must be modified based on solids content and plasticizer fraction. The evaporation rate (J, g·m⁻²·s⁻¹) and solvent‐vapor gradient created and controlled by temperature and relative humidity, will determine the film formation front and can create fibril alignment or coffee‐ring effects that can cause non‐uniformity from flow‐driven movements during drying (Davoodi et al. 2025; X. Zhang et al. 2023).

The solvent employed (water, water/alcohol mixtures, or other mixtures) will affect the extent of inter‐fibril hydrogen bonding, film fogging, and residual stress; aqueous systems would be preferred in food applications. The degree of post‐casting annealing (mild heating or exposure to controlled humidity) could enhance inter‐chain hydrogen bonding between β‐sheets and lower the film's free volume, thereby reducing the WVP, as predicted by solution–diffusion theory (P = D·S) (Liu et al. 2023). When using additives such as glycerol or sorbitol as plasticizers, the added dimension is that WVP increases relative to gradient films without plasticizers. Therefore, it is possible to optimize plasticizer use to achieve the desired flexibility and barrier performance for a specific food product.

5.2. Extrusion and Compression Molding Approaches

The processing of thermoplastics (through extrusion and compression molding) is adaptable for mini‐scale production of bioplastics based on PNF when fibrils are incorporated into thermoplastic biopolymers or plasticized protein food matrices (Peydayesh et al. 2021). Effective thermoplasticization requires that the fibril feedstocks be in a form that disperses in the mix, that appropriate levels of plasticizers be used to lower the glass transition temperature (T _g), and that the thermoplastic processing temperatures be below decomposition yet sufficient to maintain melt viscosity. In the case of twin‐screw extruders, the screws allow for distributive and dispersive mixing which facilitates the distribution of PNFs in the melt. The screw speed and the L/D ratio control the shear history of the melt and the potential loss of fibril length (Bettelli et al. 2022).

The residence time distribution is a key factor in balancing elongation of fibrils (monomer/fibril interactions) against mechanical breakage; Population balance models incorporating fragmentation rate (𝑘𝑓) maintain sequences of particle/length distribution to predict fibril or fiber length spectra. PNF loaded sheets are compression molded into a defined thickness and densification under pressure provides stresses at the interstices to improve inter‐fibril contacts, and unique barrier properties of the composites (Lingling et al. 2023). One caution is to maintain moisture control, as moisture loss will cause porosity in the final product. Numerous additives can be added, including reactive compatibilizers or enzymatic crosslinkers, which can improve interphase adhesion between the (fibril) PNFs and polymer matrix to increase the tensile strength and prevent creasing at service temperature (Bettelli et al. 2022).

5.3. Blending With Other Biopolymers

Combining functional amyloid proteins (nanofibrils) with other polymer‐based compounds—that is polysaccharides or starch, chitosan, pectin—creates better structural and functional properties of biomaterials. Enhanced mechanical strength, barrier performance, and stability are achieved in the hybrid systems due to intermolecular interactions between the different materials. Researchers continue to explore composite matrices to develop functional, biodegradable packaging materials with improved performance and environmental sustainability. Blending form interpenetrating networks through hydrogen bonding and electrostatic interactions, which control mechanical and barrier properties. Polysaccharide–PHA coacervation or electrostatic complexation can yield layered microstructures that increase the tortuosity of diffusing gas molecules, thereby reducing diffusivity (𝐷) in the solution‐diffusion permeability equation (Peydayesh et al. 2023).

The processing route selected strongly influences dispersion, dispersed phase size and interfacial area, including co‐casting emulsions (with occupation of dispersed phase by PHA's Pickering stabilization), layer‐by‐layer (by adjusting the orientation of the phases to improve dispersibility), and extrusion compounding of thermoplastic blends (Wardana et al. 2025). Interfacial engineering, including surfactant addition, pH adjustments, or enzymatic grafting, can improve compatibility of the constituent materials to prevent accumulation of large phase separation that could generate defects and raise OTR in the blend film (decreasing performance) (Simin Feng et al. 2024). Similar component loading (e.g., formative toxins; oxidants) can be accomplished relatively easily by combining PNFs and using their high surface area and sites available for surface adsorption; release kinetics from blended films (L. Liu et al. 2023).

5.4. Surface Coating on Edible Products and Food‐Contact Materials

The application of edible coatings (e.g., thin layers) to food extends its shelf life, improving product quality and safety. All coatings are designed to protect foods from physical changes caused by moisture and oxygen exposure, as well as from microbial spoilage, while preserving the sensory attributes of the food product. Protein‐based coatings can be used to increase the stability of a product and to deliver functional additives. Polysaccharide‐based or lipid‐based coatings can also be used to improve product stability or add functional additive functionality. PNF coatings are water‐based, aqueous coatings that can be applied to fresh produce, meat, and baked goods to form edible barrier films that slow respiration and moisture loss.

Dipping, spraying, roller coating, and electrostatic deposition are introduced as methods of application, with the dispersion rheology and wetting (contact angle θ) on the droplet/substrate highly relevant to uniformity of coverage and film adhesion to the substrate. For instance, phase transitioned functional amyloid lysozyme coating were prepared from lysozyme (10 mg/mL), sodium alginate (10 mg/mL), glycerol 0.3%, cellulose nanocrystals (0.1 wt%) and cysteine (10 mg/mL, pH 8). Applied and validated on 17 fruit varieties, by spray coating and dip coating method with 2 min immersion and air drying, that showed 2–5‐fold shelf‐life extension, 60%–98% nutrient retention, 90% reduction of CO₂ emission in comparison with refrigerated and achieved 2.5 fold longer preservation through microbial inhibition, moisture‐loss reduction, and rot suppression (N. Feng et al. 2025). All interfacial adhesion in PNF films is based upon (predominantly) hydrogen bonding and Van der Waals interactions between the PNF film and the substrate cuticle or the interface between substrate surface proteins and PNF. Pre‐treatments of the substrate surface (mild plasma, enzymatic etching, etc.) provided improvements over PNF barefoot hydrophobic skins by reducing the contact angle and thereby increasing adhesion potential (Peydayesh et al. 2025).

Active coatings, such as Q. Wang et al. (2020) incorporating carvacrol as an antimicrobials agent in 5% whey PNF with glycerol for the preparation of edible coating of salted duck egg yolk and noted this coating significantly reduced hardness increase rate by 18.22% that prevent weight loss and texture deterioration during storage or pH indicators (e.g., anthocyanins for intelligent freshness sensing), are intended to be edible and to enable controlled release based on film hydrophilicity and degree of crosslinking. Industrial PNF‐implementation requires a food‐grade formulation, food‐grade spray stability where the nozzles do not clog, and regulatory clearance for food contact materials if there's end‐use food contact required, and taste tests to assess smell/taste flavor changes, therefore, no visual appearance changes as well (N. Feng et al. 2025).

6. Functional Properties of Nanofibril‐Based Packaging Films

The exceptional performance of functional amyloid PNF‐based packaging materials originates from their extensive property. Unlike conventional protein films, PNFs form dense percolating nanostructures that effectively reinforce polymer matrices, resulting in significant improvements in mechanical, barrier, thermal, and biodegradable properties.

6.1. Mechanical Properties

Mechanical reinforcement is among the most extensively reported advantages of PNFs. Generally, native protein exhibits low tensile strength not more than 10 MPa; however, incorporation of PNFs substantially increases intermolecular interactions and load transfer efficiency. Loveday et al. (2011)reported that whey PNF film exhibited tensile strength of 18–25 MPa and Young's modulus approaching 1.512.3 GPa. Similarly, X. Xu et al. (2012) demonstrated that electro spun soy PNF/pullulan composite films achieved tensile strength of 32.8 MPa, representing a threefold increase compared with non‐fibrillated soy protein films tensile strength 11.2 MPa. The enhancement is attributed to fibril entanglement and crystallinity, which restrict polymer chain mobility and promote stress distribution throughout the matrix. Nevertheless, excessive fibril loading > 10 wt% often increases the aggregation, leading to brittleness and reduced elongation at break (Zhang et al. 2024).

6.2. Barrier Properties

Barrier performance is critical for food packaging applications. Their highly aligned fibrillar network creates a tortuous diffusion pathway that prevents oxygen and moisture transport. Bi et al. (2025) observed that 5 wt% incorporation of β‐lactoglobulin PNFs reduced oxygen permeability approximately by 60%–75% in comparison with native protein films. Oxygen permeability values decreased from 4.8 × 10⁻¹⁸ to 1.3 × 10⁻¹⁸. Similarly, Lal and Mhaske (2019) reported a reduction in WVP from 5.4 × 10⁻¹⁰ to 2.8 × 10⁻¹⁰ g m/m²s Pa after incorporation of whey PNFs into starch matrices. The improvement is attributed to a higher hydrogen‐bond density and reduced free volume within the film matrix. However, under high relative humidity (> 75%), plasticization remains significantly limited due to the hydrophilic nature of protein‐based materials.

6.3. Thermal Stability

The β‐sheet ‐rich structure of PNFs exhibits remarkable thermal resistance. Differential scanning calorimetry and thermogravimetric analysis have shown that fibrillated protein exhibit degradation temperature between 25°C and 320°C, significantly higher than those of the corresponding native proteins, which typically degrade at 180°C–220°C. Farrokhi et al. (2018) reported that β‐lactoglobulin nanofibrils exhibited a thermal degradation onset temperature of approximately 287°C compared with 218°C for native β‐lactoglobulin. The improved thermal stability results from extensive intermolecular hydrogen bonding and highly ordered crystalline domains within the fibrillar network. Such ability broadens the processing window for packaging fabrication methods including extrusion, compression molding and multilayer lamination.

6.4. Optical and Surface Properties

PNF‐based films generally maintain excellent transparency due to fibril diameters below the wavelength of visible light. Aydogdu et al. (2019) reported light transmittance values exceeding 85% at 600 nm for soy PNF composite films. Furthermore, the structure of fibril increases surface roughness and hydrophobicity, often raising water contact angles from approximately 52°C to 85°C, thereby improving moisture resistance.

6.5. Biodegradation Performance

PNF films exhibit rapid biodegradation under composting conditions in comparison with petroleum‐based plastics, Dong et al. (2023) reported PNFs films degradation level exceeding 70% within 30 days under controlled composting conditions. Recent studies involving whey and soy PNF films demonstrated complete degradation within 45–60 days, whereas PLA films required more than 6 months under the same conditions. A detailed comparison of functional properties between PNF‐based films and conventional packaging materials is provided in Table 2. The biodegradation mechanism involves enzymatic hydrolysis of peptide bonds followed by microbial mineralization into CO₂, H₂O, and biomass. The degradation rate depends strongly on fibril density, crosslinking degree, environmental moisture, and microbial activity.

TABLE 2.

Quantitative comparison of functional properties of PNF‐based films and conventional packaging materials.

Source	Tensile strength (MPa)	Oxygen permeability (×10−18 m3 m m−2 s−1 Pa−1)	WVP (×10−1 0 g m/m2 s Pa)	Thermal stability (°C)	Biodegradable time	Reference
β‐Lactoglobulin PNF film	18–25	1.3–1.8	2.5–3.2	280–290	30–45 days	Basheva et al. (2006)
Whey PNF composite	20–30	1.5–2.0	2.8–3.5	270–300	45–60 days	Samadani et al. (2019)
Soy PNF film	25–33	2.0–2.8	3.0–4.0	260–290	40–60 days	González et al. (2019)
Pea PNF film	18–28	2.5–3.2	3.5–4.8	250–280	45–65 days	Jia et al. (2022)
Cellulose film	50–90	0.5–1.5	5–10	280–320	60–90 days	Gao et al. (2022)
Chitosan film	15–60	10–50	3–12	250–300	30–60 days	Mujtaba et al. (2019)
PLA film	50–70	15–25	1–2	320–350	180–365 days	Malek et al. (2021)
LDPE film	8–20	150–650	0.2–0.5	350–400	> 10 years	Arvanitoyannis et al. (1998)

7. Active and Smart Functionalities

Functional amyloid protein fibrous nanostructures have many potential uses in the development of intelligent and active food packaging systems due to their high surface area, strong intermolecular interactions, and ability to form robust films. Nanofibril‐based films enable the incorporation of bioactive compounds (e.g., antimicrobial agents, antioxidants, and natural extracts) to create controlled‐release systems for food products. In addition, due to their active characteristics, PNFs can also be combined with indicator elements using responsive technologies, enabling the incorporation of sensing devices. Table 3 provides a detailed summary of the performance of PNFs‐based active and smart packaging systems. Therefore, PNFs are being studied for use in multifunctional food packaging systems that improve food safety, extend the shelf life of food products, and provide immediate quality assurance.

TABLE 3.

Quantitative Performance of PNFs based active and smart packaging system.

Functionality	PNF source	Active compound	Efficiency (%)	Qualitative performance	Release/sensing mechanism	Food application	Key limitation	Reference
Antimicrobial	Whey protein nanofibril TiO2 coating	TiO2 nanotubes	NR	Total visible count reduced by 2.3 log CFU g−1; TBARS reduced from 1.72 to 0.82 mg MDA kg−1 after 12 days storage	Diffusion‐controlled release and ROS‐mediated bacterial inactivation	Chilled meat	Nanoparticle migration and regulatory approval	Z. Feng et al. (2019)
	Electrospun PVA/β‐CD/CEO/LYS nanofilm	Cinnamon essential oil (CEO) + Lysozyme (LYS)	CEO: 2% (w/w); LYS: 0.25% (w/w)	MIC against L. monocytogenes and S. enteritidis: 0.8–1.0 mg mL−1 (CEO: 7.6–9.5 µg mL−1, LYS: 36–45 U mL−1); showed excellent antifungal activity against Aspergillus niger and Penicillium spp.	Synergistic antimicrobial action of CEO and lysozyme; controlled release from electrospun nanofiber matrix;	Active food packaging	Potential sensory impact from cinnamon oil; migration and long‐term storage studies required	K. Feng et al. (2017)
	WPNF‐Nisin film	Nisin	82%–89%	Reduction of Listeria monocytogenes by 2.8–3.5 log CFU g−1 for 14 days refrigerated storage	Fickian‐diffusion controlled release	Cheese and meat products	Activity decreases at high moisture	Shiroodi et al. (2016)
Antioxidant	WPNF‐PGA nanoparticle film	Anthocyanins	95.34%	DPPH scavenging increased from 1.47% to 18.92%; WVP decreased from 15.76 × 10−7 to 8.40 × 10−7 g·m−1·Pa−1·h−1	Hydrogen bonding and diffusion‐controlled release	Fish freshness packaging	Pigment degradation during prolonged storage	Lv et al. (2024)
	β‐Lactoglobulin nanofibrils	EGCG	88.5%–93.2%	DPPH scavenging improved by 41%–56%; antioxidant retention > 75% after 30 days	Hydrogen bonding and π–π interactions	Active packaging films	Release affected by humidity	Mao et al. (2024)
	WPNF pickering emulsion film	Curcumin	91.6%	Controlled release reduced from 78% to 43% after crosslinking; oxidative stability improved by 35%	Diffusion through Pickering emulsion droplets	Lipid‐rich foods	Curcumin photodegradation	X. Xu et al. (2022)
Smart packaging	WPNF‐PGA Film	Anthocyanins	95.34%	Distinct color transition across pH 2–12; stable for 14 days at 4°C and 25°C	pH‐responsive structural transformation of anthocyanins	Fish spoilage monitoring	Humidity sensitive	Huang et al. (2025)
	WPNF‐anthocyanin film	Red cabbage anthocyanins	89%–94%	ΔE increased from 5.3 to 32.7 during fish spoilage; response time < 20 min	pH‐responsive color change	Fish freshness indicator	Photostability concerns	Abedi‐Firoozjah et al. (2022)
	Soy protein nanofibril film	Purple sweet potato anthocyanins	87%–91%	pH response from 3–11; correlation with TVB‐N R 2 = 0.94	Colorimetric response to spoilage metabolites	Shrimp freshness monitoring	Anthocyanin degradation	Xiao et al. (2021)
Nutraceutical delivery	β‐Lactoglobulin nanofibrils	Vitamin D3	94.8%	Bioaccessibility increased from 42% to 81% after INFOGEST digestion	Controlled release during gastrointestinal digestion	Functional edible films	Regulatory approval required	Berino et al. (2019)
	Rice bran PNF Pickering emulsion	β‐Carotene	92.3%	Storage retention 84.5% after 30 days; bioaccessibility improved by 1.9‐fold	Pickering‐emulsion‐mediated delivery	Functional packaging	Sensitivity to oxidation	M. Zhao et al. (2024)
	β‐Lactoglobulin nanofibrils	Curcumin	90%–95%	Sustained release for 48–72 h; bio accessibility improved by 2.1‐fold	Hydrophobic interaction within fibrillar network	Nutraceutical films	Scale‐up challenges	Zhu et al. (2022)

7.1. Antimicrobial Activity

Whey and other food‐protein PNF films and coatings reduce the colony counts of E. coli and S. aureus, experimental studies have confirmed that these food proteins effectively reduce bacterial counts (Hasan et al. 2022). In the study on the development of whey PNF‐containing TiO₂ nanotubes for chilled meat preservation, the coating reduced total viable counts by approximately 2.3 log CFU g⁻¹ during refrigeration and delayed lipid oxidation, lowering TBARS values from 1.78 to 0.82 mg MDA kg⁻¹ after 12 days. The treated meat maintained acceptable microbiological quality for nearly twice the storage duration of the untreated control (Z. Feng et al. 2019). High loading levels of bioactive agents (essential oils, phenolics, and metal nanoparticles) can be achieved using PNFs because of the high surface area/nature of the fibrils as well as the number of adsorbent sites on fibrils to achieve higher payloads and longer release (Mei et al. 2023).

In a study acid‐heat treatment (5 h) converted soy/whey proteins into robust amyloid‐fibril films. Incorporating vanillin conferred strong bacteriostatic activity with greater inhibitory effect on P. expansum followed by E. coli and B. subtilis with the values of IZ was 726.6 ± 148.0, 671.3 ± 208.3, and 378.0 ± 78.8 mm² respectively. These flexible films demonstrate enhanced elongation from 33.50% to 100.57% for sustainable food packaging (L. Liu et al. 2023). Another study reported a contemporary strategy for developing konjac glucomannan/zein/curcumin composite nanofibril film that exhibits an exceptional antibacterial effect on E. coli and S. aureus with a major halo inhibition zone of 12–22 mm (L. Wang et al. 2019).

Mechanistic studies suggest that PNFs can concentrate reactive oxygen species (if a photosensitizer is present) locally around bacterial membranes, increasing oxidative damage to the microbes. Before food‐grade formulations can enter the marketplace they need to be assessed for antimicrobial efficacy, migration, organoleptic effects, and regulatory compliance (Herneke et al. 2021).

7.2. Antioxidant Incorporation and Release Mechanisms

PNF matrices can be loaded with antioxidants such as polyphenols, vitamin E, or carotenoids via adsorption, hydrogen bonding, and π–π interactions, thereby enhancing oxidation stability. Loaded antioxidants occupy a specific volume and the ride of the fibrils provides a greater surface area ratio than bulk protein films, therefore at a similar volume occupied, PNF films can provide a greater loading capacity, which translates to lower concentrations of active (Rezagholizade‐shirvan et al. 2024). The amount of loaded antioxidant and its eventual release depend on the microstructure of the film, and how hydrophilic the PNF (polypeptide nano‐fibril) scaffolding is, and if there is cross‐linking, and generally, release in food simulants follows either Fickian or anomalous transport as determined by the interactions between the polymer and the antioxidant itself (Xu, Yan et al. 2024).

Rashidi et al. (2021) developed novel bioactive ethyl cellulose/soy protein isolate (EC/SPI) nanofibrils via electrospinning. The optimal 1:1 ratio (ES11) provided superior morphology, thermal stability and 78% porosity. Incorporating 20 wt% bitter orange peel extract (BOPE) significantly enhanced antioxidant activity by 64.7%. One remarkable study on shelf‐life enhancement of cherry using zein/gelatin‐proanthocyanidins‐zinc oxide nanoparticles (ZE/GE‐PC‐ZnO) and ZE/GE‐gallic acid‐ZnO nanofiber films inhibited B. cinerea, while composite films improved contact angles and antioxidant activity 5–9 folds. This reduced weight and firmness loss by 20% and 60%, delayed respiration by 5 days and halved ethylene release, supporting sustainable packaging (Yuan et al. 2023)

If the antioxidant is encapsulated into the PNF stabilized Pickering emulsions this can allow for a practical delivery of lipophilic antioxidants during aqueous film‐forming dispersions have better stability than emulsions alone and some of the kinetics (diffusivity [D] and partition coefficient [S]) fit the solution–diffusion model that govern release can be obtained, from the release fit and used in shelf‐life modelling of food that is sensitive to oxidative degradation/antioxidant activity distraction of antioxidants (Lingling et al. 2023). The thermal and light stability of loaded antioxidants is significantly enhanced when bound to the PNF, as the fibrillar morphology limits the mobility of potential active compounds and provides some shielding of the labile components from reactive species. Antioxidant activity of the films is represented as radical scavenging by way of radical scavenging assays (DPPH, ABTS), determined by measuring the added oxidative markers in packaged food products (TBARS, peroxide value), which offers a better representation of the protective capacity of the film in practical settings (Ma et al. 2024).

7.3. Intelligent pH‐Responsive Packaging

Among smart packaging technologies, indicators with pH‐responsive properties change colors in response to acidity changes associated with food spoilage, and natural pigments (anthocyanins, betalains, curcumin) have been commonly used. PNF films supply a mechanically strong, edible matrix to immobilize (but not leach) the pigments (D. Liu et al. 2022). Anthocyanins extracted from red cabbage or berries provide noticeable transitions in color across pH ranges (4–9) that are applicable in monitoring meat and fish spoilage making them suitable for monitoring food freshness. H‐bonding and electrostatic interactions embed the pigments into the PNF networks such that it limits pigments' susceptibility to photobleaching and lessen migration into the food (Abedi‐Firoozjah et al. 2022).

In a study, whey PNF‐red radish anthocyanin (AM/RRA) smart film displayed reversible pH responsiveness across pH 2–12, changing from red to yellow/blue depending on alkalinity. The film of 52 1.7 m thickness successfully monitored shrimp spoilage, showing visible change in color within 24 h at room temperature and after 7 days in 4°C. The sensor remained functional for 4 pH cycles, responded to 40% NH₃/N₂ gas and demonstrated superior mechanical, antioxidant and antimicrobial properties in comparison with monomer‐based films (Peydayesh et al. 2025). Amjadi et al. (2024) developed a double‐layer intelligent packaging system comprising salep film incorporated with 8% β‐cyclodextrin/black chickpea peel anthocyanin complex and electrospun nanofibrils of black chickpea isolate showed contemporary pH‐ responsive behavior. It showed distinct color transitions across pH 2–12, changing from red (acidic) to olive green (alkaline) and successfully monitored spoilage of shrimp and fish fillets through color changes corresponding to total volatile basic nitrogen (TVB‐N), microbial growth and lipid oxidation during storage.

Key performance metrics for the sensor system include: the colorimetric response range, sensitivity (ΔE; color change), response time, and reversibility, which are measured under controlled temperature and humidity to mimic supply‐chain logistical conditions. Some state‐of‐the‐art indicators spatially/temporally combine pH dyes with antimicrobial or antioxidant properties consisting of PNF to generate, ultimately, a multifunctional film that can sense and slow spoilage. If the indicator were to be embedded in packaging, immobilization processes (layer‐by‐layer deposition, microencapsulation, covalent grafting) are selected such that the dye will not leach into the matrix and reagents will retain their responsive nature (Qin et al. 2024).

7.4. Nutraceuticals Encapsulated Edible Packaging Using PNFs

PNF networks can encapsulate nutraceuticals (polyphenols, vitamins, peptides, lipophilic vitamins), providing edible wrappers with barrier and nutrient‐delivery capabilities. Mechanisms of entrapment include adsorption to fibril surfaces, entrapment in fibril gel meshes, and, when used for lipophilic actives, stabilization in PNF‐stabilized emulsions (Trajkovska Petkoska et al. 2021). The release profiles can be controlled by fibril concentration, crosslink density, plasticizer concentration, and even using triggerable bonds (pH‐sensitive, enzyme‐cleavable links) that could target the release in the gastrointestinal tract (Xu, Wu et al. 2024).

Bioavailability improvements come from protecting labile actives during storage and from mucoadhesion and nano‐delivery within plasma protein networks, which can provide a means for better dissolution and uptake. When assessing, we can quantify encapsulation efficiency and loading capacity via extraction and HPLC/UPLC analysis, and use in vitro digestion models (INFOGEST) to evaluate release under simulated gastric and intestinal conditions (Priyanto et al. 2022). For nutraceutical delivery application, Alehosseini et al. (2019) successfully encapsulated curcumin within electrospun zein and gelatin PNF mats, achieving encapsulation efficiency close to 90%. Particularly, the coating developed using gelatin fibril protein displayed 89 ± 2% encapsulation efficiency without green tea extract, providing superior protection against curcumin degradation and slower release kinetics in food stimulants, while the nanofibril architecture improved curcumin stability compared to free curcumin, demonstrating the potential of PNF‐based coatings as edible nutraceutical delivery systems for active food packaging.

The safety side of the assessment includes evaluating potential interactions between nutraceuticals and matrices; assessing total migration limits; and mitigating adverse effects on the organoleptic qualities of an edible film. Product ideas made during prototyping include ready‐to‐eat meals with vitamin‐enhanced edible wrappers, and liners that release antioxidant properties to fresh produce. Techno‐economic analysis supports scaling up opportunities (Senthilkumar et al. 2022).

8. Safety, Toxicology, and Regulatory Aspects

The increasing interest in functional amyloid PNFs for food packaging applications necessitates a comprehensive evaluation of their safety, toxicological profile and regulatory compliance. Although PNFs are derived from food‐grade proteins and generally considered safe, biodegradable, and digestible. Their nanoscale dimensions, rich β‐sheet structure, and altered physicochemical properties may influence biological interactions compared with those of native proteins. Therefore, understanding these aspects is critical for successful commercialization of PNF‐based food contact and edible packaging materials (Lozano‐Ojalvo and Benedé 2023).

To evaluate the potential systemic absorption of fibril degradation products following oral exposure and to determine whether prolonged consumption could induce amyloid deposition in peripheral tissues, an in vivo histopathological assessment was conducted in mouse tissues. As a control, amyloid plaques were observed in brain tissue sections from the Alzheimer's disease mouse group. However, no observation was found in the brain tissues or other main organs of the mouse group fed with a normal diet (control) and dietary β‐lactoglobulin fibrils and lysozyme fibrils at both high and low dosages after 30 and 60 days, indicating no trace of cytotoxicity of digested fibrils (D. Xu et al. 2023).

In vitro gastrointestinal digestion revealed that HSAFs‐2C (heated soy amyloid fibrils, 2% w/v), HSAFs‐8C, and USPIs‐2C (unheated soy protein isolate, 2% w/v) exhibited distinct hydrolysis behaviors. During simulated gastric digestion, HSAFs‐8C showed greater resistance to proteolysis, with total hydrolysis being 3.65% lower than HSAFs‐2C and a prolonged initial digestion phase of 90 min characterized by a slower digestion rate (G‐k1; R ² > 0.90). This enhanced resistance was attributed to the higher β‐sheet ordering and larger fibrillar aggregates formed at 8% protein concentration. During the intestinal phase, differences in digestion rates became negligible, with both fibrils exhibiting similar rates over the final 60 min (I‐k4 = 0.18 ± 0.03; R ² > 0.90). However, HSAFs‐8C maintained lower overall hydrolysis after complete digestion, indicating that higher protein concentration enhances fibril resistance to enzymatic degradation (Zheng et al. 2023). Similarly, risk assessment of PNF ingredients should take into consideration immunoassays (IgE binding), in‐silico epitope mapping, and, if indicated, clinical‐grade assays to demonstrate that fibrillation or formulation does not additionally increase allergenic potential (Pereira et al. 2023).

From a regulatory standpoint, PNFs that are intended to be consumed directly as edible films or incorporated into food‐contact materials are covered by existing food contact substance (FCS) regulations in the United States. Premarket notification or threshold of regulation pathways would apply, depending on usage, migration potential, and novelty of products made with PNFs. In the EU, one of the requirements of the EFSA for novel food or food‐contact applications is that the PNF manufacturers submit data on the composition, production process, migration, toxicology results, and (for nano‐sized) particle characterization relevant to their technical guidance (More et al. 2021).

In India, FSSAI specifies that manufacturers ensure the safety of food products, accurate labelling and only apply with applicable notifications or novel food assessments on food‐contact materials. Ultimately, consumer acceptance and sensory are the main reasons for why companies would use edible PNFs for packaging and industry studies show that transparency, specified naturalness, safety confidence, and sensory neutrality (that can ensure no off‐flavor's/musty odor's) will be significant drivers of consumer purchase intent (X. Zhang et al. 2024). Therefore, companies should ensure that they conduct sensory evaluation (trained panels + consumer tests), migrate tests for sensory compounds, clearly provide identification, and ensure communication on degradation, recycling/compostability to ensure consumer confidence and use in the market (Taylor et al. 2024; Khin et al. 2024).

9. Scaling‐Up Strategies for Industrial Production

Scaling up methods for the industrial production of functional amyloid PNFs requires optimal bioengineering, processing, and functionalization methods. Synthetic biology and recombinant protein expression are sustainable avenues for cheap and green routes to protein production, with downstream purification as the bottleneck to scale up PNF production to industrial‐scale (Miserez et al. 2023). The possibility of constructing complexes from whey‐ and crystallin‐derived protein fibrils, demonstrated through cross‐linking (e.g., with glutaraldehyde), or via surface assembly, which would enable easier PNF enzyme immobilization and biosensing capabilities, providing a modular construction pathway. This study identified its optimized shear rates (325 s⁻¹, 10 h, −90°C) consistently generate reproducible whey/soy PNFs, and identified the importance of specific hydrodynamic shear conditions for scalability, iron‐delivery mode (Barrera 2020).

A notable example of functional amyloid PNFs is soy PNFs, starch and pectin blends, that can be extruded as filaments or casted to form film, exploring the application of such bioplastics beyond just another flexible packaging while also maintaining the production cost of $4–$6/kg similar to other biodegradable bioplastics but with a significant amount of reduction in carbon footprints. This provides a sustainable strategy for conversion of food waste into bioplastics that outperforms EVOH, PVDC, PET, PA6, PEF, PGA in terms of better water vapor permeability (WPV) (Roy Goswami et al. 2025).

Hemoglobin fibrils, a promising scale‐up approach involves the valorization of livestock waste obtained as a major slaughterhouse by‐product, for plasma amyloid fibril production. Following 20 h incubation, mature semiflexible fibrils with an average length of 0.65 µm and persistence length of 261 nm were obtained. This shows enhanced performance over PVA and MC based bioplastics, with elongation at break up to 300%, enhanced WVP and smooth surface morphology. In comparison with petroleum‐based packaging material, plasma PNF reinforced PVA films achieved a sustainable footprint improvement of up to 92% and also estimated production cost per kg of plasma PNFs‐based film was $3.6, demonstrating strong potential for industrial‐scale circular bioeconomy applications (Ding et al. 2025).

10. Future Research Directions

ANFs show great potential as sustainable bio‐nanomaterials. However, more work is needed before ANFs can be fully realized as usable materials. Future research should shift towards structural design and development to ensure fine‐tunable surface chemistry, mechanical properties, and polymorphs through dynamic binding, molecular dynamics, and AI‐assisted simulations, and build pre‐designed fibrils to perform designated functions. In biomedical applications, it's critical that researchers address biocompatibility, immunogenicity, and other barriers to ensure safety in areas such as drug delivery and hydrogel wound healing. Innovative and sustainable cryogel scaffold based packaging material development with tunable properties (Priyadharshini et al. 2025). ANFs also show promise in their integration into environmental and energy technologies as additives to hybrid membranes, absorbers and catalytic reactors to improve water purification processes, to aid in the sequestration of heavy metals and increase the capacity of bio‐electrochemical energy generation systems (Soon 2023).

Equally critical is to focus research on circular‐economy applications in which industrial or food protein waste is converted into functional fibrils, reducing emissions, and expanding sustainable production. Changes in scalability and novel processing methods, such as continuous flow fibrillation, the use of sustainable solvents and additive manufacturing, are also essential for taking researchers from academic objectives to industry success (Isogai 2020). Finally, life‐cycle studies and biodegradability assessments should be prioritized as part of technical development to ensure environmental safety. It is important to close loops between molecular design, scalability, and functional optimization as a means to transmute ANFs from being niche biomaterials into components of commonplace biomedicine, environmental regeneration, and renewable energy (Mollahosseini et al. 2025).

The techno‐economic assessments indicate that packaging based on PNF has significant scale‐up potential, as costs can be reduced by integration of the process, use of renewable energy, and valorization of protein‐rich residues like whey and plant‐based by‐products (Chalermthai et al. 2021). Continuous‐flow fibrillation and heat‐recovery systems may be able to lower production costs more than 30% compared to batch systems (Rabbi and Amin 2024). A combination of life‐cycle and economic frameworks will allow concurrent optimization of environmental and financial outcomes (Miserez et al. 2023). Future work should establish cost–performance models as a standard and validate the economic feasibility of PNF‐based packaging utilizing diverse feedstock and energy scenarios (Baishya et al. 2025). Hence ANFs offer significant potential; future directions may highlight scalable, sustainable processing, structural engineering, and safe applications in biomedicine, environmental remediation, and renewable energy applications which is illustrated in Figure 6.

FIGURE 6.

Potential future direction and emerging research avenue for functional amyloid protein nanofibril.

11. Conclusion

Functional amyloid PNFs symbolize an exciting development that crosses the boundaries of nanotechnology and sustainable food packaging. Its unique β‐sheet‐rich structure and adjustable self‐assembly confer remarkable mechanical strength, moisture and gas barriers, and high thermal stability, making it a great candidate for edible, biodegradable films. By testing a variety of protein sources, including dairy byproducts, plant isolates, egg protein, and novel substrates, yield variability and sustainability have also improved. Recent advances in fabrication methods such as microwave assisted, electrospinning, enzymatic and shear‐assisted self‐assembly have also led to improvements in the scalability and uniformity of materials. The production cost of functional amyloid PNFs based film was estimated to be $3–$6/kg which is similar to any other degradable bioplastics.

Despite these advances, significant hurdles remain in achieving industrial‐scale production. Energy‐intensive processes, yield variability, and purification challenges are barriers to commercial translation. Regulatory frameworks developed by the FDA, EFSA, and FSSAI require that, before industry stakeholders can introduce food products to market, extensive safety, allergenicity, and digestibility assessments be conducted. Finally, how stakeholders communicate and address limitations related to public perception, sensory attributes, and cost competitiveness may ultimately determine acceptance.

In future studies, it is important to focus on hybrid nanofibril composites with polysaccharides, lipids, and bioactive agents towards multifunctional applications. The functional space will also grow with the incorporation of smart packaging technologies, including freshness indicators and pH‐responsive sensors. Including PNF development into circular economy models, by upcycling protein‐rich waste from industry, and developing films that are fully compostable or recyclable, will positively impact the ecological footprint and market value. Through an interdisciplinary approach to overcome techno‐economic, safety, and regulatory challenges, amyloid‐based edible packaging can be rapidly advanced from laboratory novelty to a viable commercial solution, accelerating the transformation towards sustainability in the global food industry.

Author Contributions

Priyadharshini S R: conceptualization, investigation, writing – original draft, visualization, methodology. R. Mahendran: conceptualization, investigation, writing – review and editing, visualization, validation, project administration, resources, supervision, data curation.

Conflicts of Interest

The authors declare no conflicts of interest.