Abstract
Oleogelation based on nutritionally valuable ingredients enables liquid oils to target various food and material applications where fat-like functionality is required. The present study offers a novel approach to design oleogels with high oil content (up to 98 %) structured by food protein amyloids at low concentrations. This was achieved by Pickering stabilization of oil-in-water emulsions with different food amyloid fibrils from both animal and plant proteins, followed by dehydration of finely dispersed high internal phase emulsions. The hydrophobic phase was constituted by either rapeseed or medium-chain triglyceride (MCT) oils, whereas whey, soy and potato amyloid fibrils were formed via heat-induced acidic hydrolysis. First, we studied how animal and plant proteins self-assemble into amyloid fibrils with tuneable morphological and surface properties. The emulsifying performance of the amyloid fibrils was then investigated by light microscopy, particle size distribution and viscosity. Finally, we characterised the composition, microscopical and rheological properties of amyloid fibril-templated oleogels, emphasising oil encapsulation efficiency. We demonstrate that the ensued solid-like edible oleogels have a shear elastic modulus of the order of 104–105 Pa. Mesoscopic characteristics and surface properties of the amyloid fibrils allow tuning the rheological characteristics, encapsulation efficiency and interfacial protein concentration in the oleogels. Fibrillization offers a robust strategy for solidifying liquid oil with high efficiency and stability of the oleogels. The present approach opens multifaceted applications of amyloid fibril-based oleogels in food, biomedical, and pharmaceutical fields, such as plant-based shortenings, ointments, tissue scaffolds, and controlled release drug delivery systems.
Highlights
- Amyloid fibrils structure liquid oils at low concentration
- Whey, soy, and potato amyloid fibrils at pH 2 have high oleogelation capacity
- Plant-based amyloid fibrils encapsulate oil up to 98 %
- Oleogels structured by amyloids entrap oil into polyhedral droplets of ∼2 μm
- Amyloid fibrils morphology tunes the structuring of liquid oils
- Oleogels show solid-like behaviour with shear moduli of 104–105 Pa
Introduction
Structuring liquid oils into fat-like solid materials by oleogelation is crucial for mimicking the techno-functional benefits of fats in food materials and to ensure good spreadability, firmness and stability. However, for targeting real food applications, oleogelators must be food-grade, economically affordable and capable to percolate through the liquid oil as a gel network at low concentrations, thus providing to the resulting oleogels the required solid-like structure and properties. High nutritional value and good sustainability footprint urge researchers to seek for new solutions in oleogelation, thus meeting the high consumer expectation of healthy and environmentally friendly foods (Co & Marangoni, 2012).
The amphiphilic nature of proteins allows them to interact with hydrophobic compounds while remaining water-soluble and thus behave as ideal surfactants. Nevertheless, the versatile functionality of proteins is usually constrained when forming a protein network in a hydrophobic environment. The dispersibility of proteins in oil is limited due to their prevalent hydrophilicity (Scholten, 2018). Romoscanu and Mezzenga (2006) proposed an emulsion-templated approach to incorporate proteins into hydrophobic environments. This approach provides the formation of a continuous, viscoelastic protein interfacial network embedding oil droplets. However, in that study, cross-linking of whey proteins was achieved either thermally by holding the concentrated emulsion at 80 °C for 10 min or chemically with glutaraldehyde. Oil heating is usually a crucial step to form oleogels employing waxes, lecithin, monoglycerides or ethylcellulose, which promotes lipid degradation through thermally induced auto-oxidation (Gravelle et al., 2018). Another route leading to protein-based oleogelation is the transfer of protein building blocks from an aqueous to a hydrophobic environment by stepwise solvent exchange, which requires the use of acetone or tetrahydrofuran (Vries et al., 2015), which may significantly limit large-scale food applications.
Protein self-assembly into amyloid fibrils represents a new approach for enhancing protein techno-functionality by exposing different functional amino acid groups to the environment. In this way, the above-mentioned tendency of many proteins to prefer hydrophilic environments to hydrophobic ones is bypassed through unfolding, hydrolyzation and self-assembly, thus allowing the amino acids with hydrophobic nature (naturally folded in the interior of the native proteins) to be exposed on the surface and to effectively interact with the surrounding environment. Fibrillization of proteins by heat-induced acidic hydrolysis results in the formation of nanofibrils with a high aspect ratio, Young’s modulus and stability. The paramount potential of using amyloid fibrils as food ingredients has been recently demonstrated by in-vitro and in-vivo studies (Xu, Zhou, et al., 2023), removing most of the previous health concerns about the nutritional use of food amyloid fibrils. The superior emulsification properties of amyloid fibrils are promoted by high interfacial modulus and high surface activity of the oil-water interfaces formed by amyloid fibrils (Huyst et al., 2021; Wu et al., 2022; Yu et al., 2024). Moreover, amyloid fibrils facilitate emulsion stability through irreversible interfacial absorption and anti-aggregation properties, forming a dense absorption layer in emulsions stabilised by amyloid fibrils (Peng et al., 2016; Xu, Zhou, et al., 2023). Plant proteins, recognised as more sustainable food ingredients than animal counterparts, can also be fibrillized into amyloid fibrils by heat-induced acidic hydrolysis. For example, amyloids, produced from soy, kidney bean, cowpea, and mung bean (Li et al., 2023; Mykolenko & Mezzenga, 2025) have demonstrated high gelling capacity and formed hydrogels based solely on amyloid fibrils in low ionic strength aqueous conditions.
Given their specific mesoscopic characteristics and surface activity, amyloid fibrils facilitate emulsification and promote the inhibition of transport and collision of droplets in the emulsions. However, the oleogelating capacity of amyloid fibrils has not yet been investigated to the best of our knowledge. This study introduces a three-stepwise approach to structure liquid oils by exploring the oleogelating capacity of amyloid fibrils produced from both animal and plant proteins. The carbon footprint of 1 kg of animal protein can be as high as 750 kg of CO2 eq per kg of protein when extracted from meat, while plant proteins have a massively lower footprint, varying from 4 to 17 kg of CO2 eq per kg of protein. Dairy proteins are those with the lowest carbon footprint (28–68 kg of CO2 eq per kg protein) among animal proteins (Peydayesh et al., 2023). Considering this, whey protein isolate was included as the only animal protein with high fibrillization propensity (Cao & Mezzenga, 2019) to explore the oleogelating capacity of amyloid fibrils along with plant proteins from potato and soybeans. First, whey (WAF), soy (SAF), and potato (PAF) amyloid fibrils are produced by heat-unfolding and pH hydrolysis, and their morphological characteristics are studied by AFM image statistical analysis. Then, oil-in-water Pickering emulsions stabilised by the amyloid fibrils are produced by ultrasonication of rapeseed oil (RSO) and medium-chain triglyceride oil (MCT) droplets and characterised by optical microscopy, particle size distribution and viscosity. Finally, oleogels are structured by water removal via centrifugation, and their structure is analysed by confocal laser scanning microscopy and cryo scanning electron microscopy. We demonstrate the oleogelation capacity of the amyloid fibrils by the oil encapsulation efficiency and the rheological behaviour of the oleogels in oscillatory sweeping tests. This study demonstrates for the first time the oleogelation capacity of amyloid fibrils as a generic feature of these protein self-assemblies and may open new possibilities in the applications of this strategy in food, pharma and cosmetic industry.
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Materials
Whey protein isolate (WPI, 98 % protein content (N × 6.38), w/w) was kindly supplied by Fonterra (New Zealand). Soy protein isolate (SPI, 80 % protein content (N × 5.71), w/w) and rapeseed oil (RSO) were kindly provided by Pacovis AG (Switzerland). Potato protein isolate (PPI, Solanic 200, 91 % protein content (N × 6.25), w/w) produced by Royal Avebe U.A. (Netherlands) was donated by BAVA Baumann & Cie (Switzerland). Medium-chain-triglyceride oil (MCT) was purchased from Shaanxi Haibo Biotechnology Co., Ltd. (China). 1-aniline-8-naphthalenesulfonate (ANS) and rhodamine B was purchased from Sigma-Aldrich/Merck AG (Switzerland). All solutions were prepared using Milli-Q water (18.2 MΩ cm−1; Millipore, USA).
Svitlana Mykolenko, Mattia Usuelli, Andrin Lustgarten, Peter Fischer, Raffaele Mezzenga, Amyloid fibril-templated oleogels, Food Hydrocolloids, Volume 168, 2025, 111491, ISSN 0268-005X, https://doi.org/10.1016/j.foodhyd.2025.111491.
