Rethinking the interfacial behaviour of surfactants and their interactions with proteins. A structure, temperature and time-dependent approach.

Surfactants are widely used as emulsifiers in food and related industries due to their low molecular weight and high interfacial activity. While surfactants are traditionally described as forming simple interfacial films, some surfactants, such as certain phospholipids, have been shown to form viscoelastic interfacial networks. When combined with proteins, interactions between the two species can either improve or impair emulsion stability. Clearly, the molecular structure of the surfactant (including the pH-dependent headgroup charge) influences their interfacial behaviour in oil-water emulsions, hence also their interactions with proteins. However, the findings remain contradictory and require further attention. Additionally, in industrial emulsion production, emulsions are typically subjected to thermal stress either before or after emulsification, which can induce a phase transition of the surfactant’s fatty acyl (FA) chain (and vice versa upon cooling). Nevertheless, the impact of these thermal cycles is often neglected in academic work, although the rheological properties of such a film may be markedly different from an emulsifier interface produced at the commonly studied room temperature. To address these knowledge gaps, this thesis aims to systematically investigate how the molecular structure of surfactants, in combination with environmental factors such as pH and temperature, influence their interfacial behaviour and interactions with proteins in oil-water emulsions over time (milliseconds to hours) using a stepwise model system approach. Firstly, interfacial techniques (drop tensiometry, interfacial shear rheology, CLSM) were used to characterise the adsorption kinetics, viscoelastic properties and composition of the interface (short to long timescales). These were complemented by microfluidic experiments to study adsorption kinetics and coalescence stability on (very) short time scales, i.e., during emulsification. Furthermore, the thermal and structural properties, as well as potential interactions between the species, were investigated using DSC, SAXS and FT-IR spectroscopy (intermediate to long timescale). On short timescales, surfactants showed similar adsorption kinetics and coalescence stabilities, regardless of the molecular structure. Simultaneous adsorption of protein and surfactant led to faster interfacial stabilisation, making the interfacial layer less prone to rupture (drop tensiometry, microfluidics). Working with elevated temperatures during emulsification accelerated emulsifier adsorption and led to faster interfacial stabilisation as long as the temperature was below the phase transition temperature of the surfactant. This is due to increased molecular mobility and decreased oil viscosity, which together enhanced the rate at which molecules were incorporated into the interface. However, once the chain melting point of the surfactant was reached (T > 50°C), process stability decreased rapidly because the high mobility and low adsorption energy of the surfactant made it prone to desorb from the interface when two droplets approached each other, favouring oil droplet coalescence (microfluidics). In the same way, above the denaturation temperature of the protein, protein denaturation favoured oil droplet aggregation via a bridging effect (microfluidics). On intermediate timescales, surfactants with small headgroups and saturated FA chains were able to pack tightly at the oil-water interface (high CMC, low area per molecule), unlike surfactants with large/ highly charged headgroups or unsaturated FA chains (drop tensiometry). Notably, unsaturated surfactants such as PC 18:1 contain cis-double bonds that introduce bends into the FA chains, thereby reducing van der Waals packing efficiency while increasing lateral mobility and interfacial activity. In blending with proteins, saturated surfactants interacted with the proteins and the degree of interaction increased with increasing headgroup hydrophobicity due to additional hydrophobic interactions between the surfactant’s headgroup and hydrophobic groups within the protein (FT-IR). Protein-surfactant interactions further increased with decreasing pH due to reduced electrostatic repulsion and were the strongest at a pH below 5, where the two species carried the opposite charge. Unsaturated surfactants, on the other hand, did not interact with the protein, regardless of the pH, because the bend in the FA chain sterically restricted binding to the protein (FT-IR). In all cases, increasing the temperature decreased lateral interactions and with it interfacial viscoelasticity (interfacial rheology). On long timescales, surfactants with saturated FA chains formed stiff interfacial networks due to surfactant-surfactant interactions, unless the headgroup size or charge prevented dense packing (interfacial rheology). If a cooling step was involved, chain crystallisation further increased interfacial viscoelasticity and, consequently, improved the process stability of the emulsion. In blending with proteins, surfactants with saturated FA chains formed a heterogeneous interfacial network over time due to protein-surfactant interactions, improving both the process and the kinetic stability of the emulsion (interfacial rheology, CLSM). Interfacial stiffness increased with decreasing headgroup size and charge (low pH). These interactions enhanced protein unfolding, thus favouring a more cross-linked interfacial network, but were not strong enough to hinder interfacial protein-bulk protein interactions. When heating the emulsion above the protein denaturation temperature, strong interfacial protein-protein interactions led to protein aggregation at the interface, although this process took time and only occurred after holding the temperature for several minutes (CLSM, SAXS). In the case of unsaturated surfactants, the resulting high interfacial pressure led to protein displacement over time, thereby lowering the process and kinetic stability of the emulsion. This effect became more pronounced with increasing surfactant headgroup size (interfacial rheology, CLSM). To conclude, for the industrial production of emulsions, the surfactant should be carefully chosen, depending on the molecular structure (headgroup, FA chain) and the temperature treatment of the emulsion during/ after emulsification.