Currently, plant proteins are fractionated to ingredients with high purities, but an often ignored point is the impact of the extraction and fractionation process on protein functionality. To allow a fair and effective comparison, it is key to understand the changes in protein's aggregated state occurring in the extracted ingredients during processing. We review conventional and upcoming plant protein extraction and fractionation processes (on pulses and oilseeds) and focus on how the processing history influences the macroscopic functional properties of the proteins. To establish this link, we dive into seed morphology and give an overview of the plant seed composition. In addition, we explain the essence of each process step and how it impacts the protein's aggregated state. The latter is linked to the macroscopic functionality (foaming, emulsification, and gelation). We identified three major protein structure-changing steps in the conventional protein extraction process: defatting, alkaline extraction, and isoelectric point precipitation. These steps lead to large, insoluble aggregated structures, which strongly impacts the protein macroscopic functionality. Milder extraction methods reduce these alterations, but a potential consequence is the presence of non-proteinaceous components, which could give challenges in sensory and nutritional aspects and affect the techno-functional properties of the ingredient. The take-home-message is that we need to consider the process-induced change of the protein aggregated structures, which are likely to dominate the functionality over the protein's molecular parameters.

It has been reported that lipid droplets (LDs), called oleosomes, have an inherent ability to inflate or shrink when absorbing or fueling lipids in the cells, showing that their phospholipid/protein membrane is dilatable. This property is not that common for membranes stabilizing oil droplets and when well understood, it could be exploited for the design of responsive and metastable droplets. To investigate the nature of the dilatable properties of the oleosomes, we extracted them from rapeseeds to obtain an oil-in-water emulsion. Initially, we added an excess of rapeseed oil in the dispersion and applied high-pressure homogenization, resulting in a stable oil-in-water emulsion, showing the ability of the molecules on the oleosome membrane to rearrange and reach a new equilibrium when more surface was available. To confirm the rearrangement of the phospholipids on the droplet surface, we used molecular dynamics simulations and showed that the fatty acids of the phospholipids are solubilized in the oil core and are homogeneously spread on the liquid-like membrane, avoiding clustering with neighbouring phospholipids. The weak lateral interactions on the oleosome membrane were also confirmed experimentally, using interfacial rheology. Finally, to investigate whether the weak lateral interactions on the oleosome membrane can be used to have a triggered change of conformation by an external force, we placed the oleosomes on a solid hydrophobic surface and found that they destabilise, allowing the oil to leak out, probably due to a reorganisation of the membrane phospholipids after their interaction with the hydrophobic surface. The weak lateral interactions on the LD membrane and their triggered destabilisation present a unique property that can be used for a targeted release in foods, pharmaceuticals and cosmetics.

Protein blends are used to stabilise many traditional and emerging emulsion products, resulting in complex, non-equilibrated interfacial structures. The interface composition just after emulsification is dependent on the competitive adsorption between proteins. Over time, non-adsorbed proteins are capable of displacing the initially adsorbed ones. Such rearrangements are important to consider, since the integrity of the interfacial film could be compromised after partial displacement, which may result in the physical destabilisation of emulsions. In the present review, we critically describe various experimental techniques to assess the interfacial composition, properties and mechanisms of protein displacement. The type of information that can be obtained from the different techniques is described, from which we comment on their suitability for displacement studies. Comparative studies between model interfaces and emulsions allow for evaluating the impact of minor components and the different fluid dynamics during interface formation. We extensively discuss available mechanistic physical models that describe interfacial properties and the dynamics of complex mixed systems, with a focus on protein in-plane and bulk-interface interactions. The potential of Brownian dynamic simulations to describe the parameters that govern interfacial displacement is also addressed. This review thus provides ample information for characterising the interfacial properties over time in protein blend-stabilised emulsions, based on both experimental and modelling approaches.

There is a growing interest in replacing dairy proteins with their plant-based counterparts in food emulsions. Plant proteins generally contain a substantial insoluble protein fraction, of which the properties may differ from the soluble proteins. Therefore, the use of a commercial pea protein isolate, its insoluble fraction and whey protein isolate to stabilize oil-in-water (O/W) emulsions is explored. In 100 g/kg O/W emulsions, the use of full pea protein isolate led to physically instable emulsions that showed droplet flocculation and coalescence, whereas its insoluble fraction and whey protein formed physically stable emulsions. The insoluble pea protein fraction was also able to physically stabilize high internal phase O/W emulsions (HIPEs) containing 700 g/kg oil, giving ~10 times higher viscosity than whey protein-based HIPEs. Under oxidative conditions, whey protein-stabilized emulsions showed extensive coalescence, and fast formation of lipid oxidation products. Insoluble pea protein-stabilized emulsions, showed fast lipid oxidation, but this did not affect the physical stability. In contrast, full pea proteins-based emulsions were physically instable in oxidative conditions but showed the lowest accumulation of oxidation products. These results suggest that the constituents of commercial pea protein isolate have specific functionalities, which is important knowledge for the design of stable plant protein-based emulsions.

Hypothesis: Many traditional or emergent emulsion products contain mixtures of proteins, resulting in complex, non-equilibrated interfacial structures. It is expected that protein displacement at oil-water interfaces depends on the sequence in which proteins are introduced during emulsion preparation, and on its initial interfacial composition. Experiments: We produced emulsions with whey, pea or a whey-pea protein blend and added extra protein post-emulsification. The surface load was measured indirectly via the continuous phase, or directly via the creamed phase. The interfacial composition was monitored over a three-day period using SDS-PAGE densitometry. We compared these findings with results obtained using an automated drop tensiometer with bulk-phase exchange to highlight the effect of sequential protein adsorption on interfacial tension and dilatational rheology. Findings: Addition of a second protein increased the surface load; especially pea proteins adsorbed to pre-adsorbed whey proteins, leading to thick interfacial layers. The addition of whey proteins to a pea protein- or whey-pea protein blend-stabilized emulsion led to significant displacement of the pea proteins by β-lactoglobulin. We determined that protein-protein interactions were the driving force for this displacement, rather than a decrease in interfacial tension. These outcomes could be instrumental in defining new strategies for plant-animal protein hybrid products.

In conventional emulsification devices, interface formation and stabilisation occur within milliseconds. Protein network formation at liquid-liquid interfaces starts at time scales similar to those of droplet formation in conventional emulsification devices (i.e., in milliseconds). Classical methods, like drop tensiometry, do not allow measurements at these time scales. Using a tailor-made microchip, we probed droplet deformation to study the interfacial rheological properties of droplets, within time scales ranging from 0.16 to 1 s. We further investigated the coalescence stability of droplets at the same time scales. Whey protein isolate (WPI), pea protein isolate (PPI), or their blends were used as emulsifiers at 0.01–1 g/L. The rheological properties of the protein-interfaces showed that early network formation takes place (<1 s). WPI-stabilised interfaces were mechanically stronger compared to PPI-stabilised interfaces, and WPI-stabilised droplets were much less prone to coalescence than their PPI counterparts. Although the blend-stabilised films showed high interconnectivity, this did not prevent droplet coalescence, probably due to structural heterogeneity. The insights obtained with the tailor-made microfluidic devices help to capture effects at short time scales and are relevant to unravel phenomena occurring in large scale processing.

The use of plant proteins to design colloidal food systems is a hot topic in the current context of the protein transition. However, replacing animal-derived proteins (in particular, dairy proteins) that have been traditionally used for this purpose by plant proteins is a challenge from various perspectives, and in particular, because of drastically different solubility and functionality. A possible route to mitigate these issues is to combine plant and dairy proteins, providing that their interactions can be understood from the molecular to the macroscopic scale. This review addresses the major advances that have occurred in the field of such blend-based systems, all the way from their behaviour in aqueous dispersions to their potential applications in gels, foams and emulsions.

Recent work suggests that using blends of dairy and plant proteins could be a promising way to mitigate sustainability and functionality concerns. Many proteins form viscoelastic layers at fluid interfaces and provide physical stabilization to emulsion droplets; yet, the interfacial behavior of animal-plant protein blends is greatly underexplored. In the present work, we considered pea protein isolate (PPI) as a model legume protein, which was blended with well-studied dairy proteins (whey protein isolate (WPI) or sodium caseinate (SC)). We performed dilatational rheology at the air-water and oil-water interface using an automated drop tensiometer to chart the behavior and structure of the interfacial films, and to highlight differences between films made with either blends, or their constituting components only. The rheological response of the blend-stabilized interfaces deviated from what could be expected from averaging those of the individual proteins and depended on the proteins used; e.g. at the air-water interface, the response of the caseinate-pea protein blend was similar to that of PPI only. At the oil-water interface, the PPI and WPI-PPI interfaces gave comparable responses upon deformation and formed less elastic layers compared to the WPI-stabilized interface. Blending SC with PPI gave stronger interfacial layers compared to SC alone, but the layers were less stiff compared to the layers formed with WPI, PPI and WPI-PPI. In general, higher elastic moduli and more rigid interfacial layers were formed at the air-water interface, compared to the oil-water interface, except for PPI.

Proteins are used to stabilise oil-in-water (O/W) emulsions, and plant proteins are gaining interest as functional ingredients due to their higher sustainability potential compared to e.g., dairy proteins. However, their emulsifying properties are not that well understood, and depend on how their production process affects their physicochemical status. In the present work, we use the soluble fraction of commercial pea protein isolate to stabilise O/W emulsion droplets formed in a microfluidic device, and record coalescence stability after droplet formation (11–173 ms) for different protein concentrations (0.1–1 g/L). For the shortest adsorption times (11–65 ms) droplets were unstable, whereas for longer adsorption times differences in coalescence stability could be charted. Metal-catalysed oxidation of pea proteins performed for up to 24-h, prior to emulsion formation and analysis, increased the coalescence stability of the droplets, compared to fresh pea proteins. This may be explained by oxidation-induced protein fragmentation, leading to low molecular weight products. The Langmuir-Blodgett films looked highly heterogeneous for films prepared with fresh or mildly oxidised (3-h) proteins, and was more homogenous for 24-h oxidised proteins. This could be the cause for the observed differences in emulsion coalescence stability, structurally heterogeneous films being more prone to rupture. From this work, it is clear that the emulsifying properties of pea are strongly dependent on their chemical status, and associated structural properties at the molecular and supramolecular levels. The present microfluidic device is an efficient tool to capture such effects, at time scales that are relevant to industrial emulsification.

Complex interfaces stabilized by proteins, polymers or nanoparticles, have a much richer dynamics than those stabilized by simple surfactants. By subjecting fluid-fluid interfaces to step extension-compression deformations, we show that in general these complex interfaces have dynamic heterogeneity in their relaxation response that is well described by a Kohlrausch-Williams-Watts function, with stretch exponent β between 0.4–0.6 for extension, and 0.6–1.0 for compression. The difference in β between expansion and compression points to an asymmetry in the dynamics. Using atomic force microscopy and simulations we prove that the dynamic heterogeneity is intimately related to interfacial structural heterogeneity and show that the dominant mode for stretched exponential relaxation is momentum transfer between bulk and interface, a mechanism which has so far largely been ignored in experimental surface rheology. We describe how its rate constant can be determined using molecular dynamics simulations. These interfaces clearly behave like disordered viscoelastic solids and need to be described substantially different from the 2d homogeneous viscoelastic fluids typically formed by simple surfactants.

Proteins from animal and plant sources are known to be able to physically stabilise emulsions, whereas much less is known about emulsions prepared with blends of proteins of different origin. Here we use blends of pea protein isolate (PPI) with whey protein isolate (WPI) or with sodium caseinate (SC) to physically stabilise emulsions prepared by high pressure homogenisation. For both the blends and the individual proteins, droplet size, emulsion stability, surface load and interfacial compositions were determined. The d_3,2 and surface load (measured over a concentration range 0.2–1.6 wt% protein in the starting aqueous solution) were the lowest for SC- and WPI-stabilised emulsions, and the highest for PPI-stabilised emulsions, whereas emulsions stabilised by the blends (1:1 ratio) had intermediate d_3,2 values and surface loads. PPI- and SC-stabilised emulsions showed some physical destabilisation (e.g., flocculation and coalescence, respectively) over 14 days of storage, whereas the WPI-PPI or SC-PPI blends formed emulsions that remained stable, suggesting synergistic effects.When used in blends, both dairy and plant proteins adsorbed at the oil-water interface, but compositional rearrangements at the interface occurred within days. More specifically, whey proteins were able to partly displace pea proteins from the interface, which were themselves able to displace SC. However, such a displacement was only possible when the displacing protein was present in sufficiently high excess. Such considerations are usually not taken into account in food emulsion formulation, even though they are very relevant, as the interfacial layer protects emulsions droplets against physical destabilisation.