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Typically, heat-induced aggregation of proteins is studied using a single protein under various conditions (e.g., temperature). Because different studies use different conditions and methods, a mechanistic relationship between molecular properties and the aggregation behavior of proteins has not been identified. Therefore, this study investigates the kinetics of heat-induced aggregation and the size/density of formed aggregates for three different proteins (ovalbumin, β-lactoglobulin, and patatin) under various conditions (pH, ionic strength, concentration, and temperature). The aggregation rate of β-lactoglobulin was slower (>10 times) than that of ovalbumin and patatin. Moreover, the conditions (pH, ionic strength, and concentration) affected the aggregation kinetics of β-lactoglobulin more strongly than for ovalbumin and patatin. In contrast to the kinetics, for all proteins the aggregate size/density increased with decreasing electrostatic repulsion. By comparing these proteins under these conditions, it became clear that the aggregation behavior cannot easily be correlated to the molecular properties (e.g., charge and exposed hydrophobicity).

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DLVO theory is often considered to be applicable to the description of flocculation of protein-stabilized oil-in-water emulsions. To test this, emulsions made with different globular proteins (β-lactoglobulin, ovalbumin, patatin, and two variants of ovalbumin) were compared under different conditions (pH and electrolyte concentration). As expected, flocculation was observed under conditions in which the zeta potential is decreased (around the isoelectric point and at high ionic strength). However, the extent of flocculation at higher ionic strength (>50 mM NaCl) decreased with increasing protein-exposed hydrophobicity. A higher exposed hydrophobicity resulted in a higher zeta potential of the emulsion droplets and consequently increased stability against flocculation. Furthermore, the addition of excess protein strongly increased the stability against salt-induced flocculation, which is not described by DLVO theory. In the protein-poor regime, emulsions showed flocculation at high ionic strength (>100 mM NaCl), whereas emulsions were stable against flocculation if excess protein was present. This research shows that the exposed hydrophobicity of the proteins and the presence of excess protein affect the flocculation behavior.

10The protein concentration is known to determine the stability against coalescence during 11 formation of emulsions. Recently, it was observed that the protein concentration also influences 12 the stability of formed emulsions against flocculation as a result of changes in the ionic strength. 13In both cases, the stability was postulated to be the result of a complete (i.e. saturated) coverage 14 of the interface. By combining the current views on emulsion stability against coalescence and 15 flocculation with new experimental data, an empiric model is established to predict emulsion 16 stability based on protein molecular properties such as exposed hydrophobicity and charge. It 17 was shown that besides protein concentration, the adsorbed layer (i.e. maximum adsorbed 18 amount and interfacial area) dominates emulsion stability against coalescence and flocculation. 19 Surprisingly, the emulsion stability was also affected by the adsorption rate. From these 20 observations, it was concluded that a completely covered interface indeed ensures the stability of 21 an emulsion against coalescence and flocculation. The contribution of adsorption rate and 22 2 adsorbed amount on the stability of emulsions was combined in a surface coverage model. For 23 this model, the adsorbed amount was predicted from the protein radius, surface charge and ionic 24 strength. Moreover, the adsorption rate, which depends on the protein charge and exposed 25 hydrophobicity, was approximated by the relative exposed hydrophobicity (Q H ). The model in 26 the current state already showed good correspondence with the experimental data, and was 27 furthermore shown to be applicable to describe data obtained from literature. 28 29 KEYWORDS 30Coalescence, flocculation, model, adsorption rate, adsorbed amount, surface coverage 31 32 CONTENTS 33