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.

Food design is often done based on a trial-and-error basis, using structure properties as an indicator of product quality. Although this has led to many good products in the market, this ‘cook and look’ approach could benefit from insights into dynamic processes as they occur during food formation, storage, and digestion. Currently microfluidic devices are being developed to allow these types of observations, and here we show the latest examples in the field of emulsions and foams, including effects that occur during digestion. We expect that these techniques will supply a stepping stone to thorough understanding at various length and timescales that are all instrumental in designing high-quality food products, and ultimately creating foods with health benefits.

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.

Proteins are widely used to stabilize emulsions, and plant proteins have raised increasing interest for this purpose. The interfacial and emulsifying properties of proteins depend largely on their molecular properties. We used fluorescence spectroscopy to characterize the conformation of food proteins from different biological origins (dairy or pea) and transformation processes (commercial or lab-made isolates) in solution and at the oil-water interface. The fourth derivative of fluorescence spectra provided insights in the local environment of tryptophan (Trp) residues and thus in the protein structure. In emulsions, whey proteins adsorbed with their Trp-rich region at the oil-water interface. Proteins in the commercial pea isolate were present as soluble aggregates, and no changes in the local environment of the Trp residues were detected upon emulsification, suggesting that these structures adsorb without conformational changes. The lab-purified pea proteins were less aggregated and a Trp-free region of the vicilin adsorbed at the oil-water interface.

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.

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.

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.

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.

Dynamic flavor release curves from chewing gum were measured using an Artificial Mouth coupled to the AFFIRM®. A flavor distribution model for chewing gum is proposed, where flavor is present as droplets in both the hydrophilic (water-soluble) and the hydrophobic (water insoluble) parts of the chewing gum and as molecularly dissolved in the hydrophobic part of the gum. During mastication, the flavor droplets in the water-soluble phase are released and responsible for an initial burst release. The flavor droplets captured in the gum-base are pushed towards the interface by mastication and are responsible for the subsequent release. The flavor molecules dissolved in the gum-base, released by diffusion, are only responsible for the release at very long time scales. It was found that the oil-water partition constant is an important parameter to explain the flavor release, where hydrophobic components show slower and longer release, while more hydrophilic components show more burst release.

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.

To understand droplet formation and stabilisation, technologies are needed to measure interfacial tension at micrometer range and millisecond scale. In this paper, microtechnology is used, and that allows us to access these ranges and derive a model for surfactant free systems. The predicting power of the model was tested, and we found that it can be used to accurately (validated with >60 experiments) describe droplet size for a wide range of flow rates, interfacial tensions, and continuous phase viscosities.The model was used next to determine interfacial tensions in a system with hexadecane and sodium dodecylsulfate (SDS) solutions, and it was found that the model can be used for droplet formation times ranging from 0.4 to 9.4 ms while using a wide range of process conditions.The method described here differs greatly from standard dynamic interfacial tension methods that use quiescent, mostly diffusion-limited situations. The effects that we measured are much faster due to enhanced mass transfer; this allows us to assess the typical time scales used in industrial emulsification devices.