Static and dynamic properties of proteins adsorbed at liquid interfaces

The aim of the investigation described in this thesis was to increase the level of understanding of the role that proteins play in the preparation and subsequent stabilisation of foams and emulsions. One aspect of this role is facilitation of break-up, due to surface tension lowering. A second aspect is the formation of a viscoelastic interfacial layer, which affects both the short-term and long-term stability of the dispersion. Therefore, a systematic study of the changes in static and dynamic interfacial properties induced by proteins was carried out.For part of this study, dealing with the interfacial rheology, several experimental techniques were used. These techniques were either properly modified existing techniques (Chapter 3, modified longitudinal wave set-up) or newly developed (Chapter 4, Dynamic Drop Tensiometer; Chapter 5, Concentric Ring Surface Shear Rheometer) to meet the requirements for measuring the rheology of adsorbed protein layers at liquid/liquid interfaces. These requirements are (i) isotropic deformation, without leakage of the interfacial layer, for the dilational modulus measurements at air/water and oil/water interfaces and (ii) shear modulus measurements at small oscillatory deformation.The proteins chosen for this study wereβ-casein,β-lactoglobulin (BLG), bovine serum albumin (BSA), ovalbumin and lysozyme. This set of proteins was chosen, because they differ considerably in relevant aspects, such as molecular weight, molecular structure and iso-electric point.In Chapter 1 the scope and context of this study are given including a brief introduction into (i) the molecular properties of these proteins, that are relevant to the adsorption, (ii) protein adsorption and interfacial rheology, and (iii) the relation between interfacial properties and the properties of emulsions and foams.Chapter 2 deals with the adsorption of proteins at the air/water interface. The adsorption was determined by ellipsometry, a method by which not only the adsorbed amount but also the layer thickness and protein concentration in the adsorbed layer could be determined. The ellipsometric studies were combined with surface tension measurements at the same surface.All proteins examined show high affinity adsorption, i.e. strong adsorption at low concentration in solution. The initial rate of adsorption of all proteins is well described by a simple diffusion equation. For all proteins examined, the value of the surface pressure (Π) are protein-specific, but otherwise unique, time-independent functions of the adsorption (Γ). Time independence of theΠ(Γ) curve was concluded from the finding thatΠandΓpairs measured at different bulk concentrations and at different stages of adsorption, all collapse into one single curve. In other words, each protein has a unique surface equation of state indicated by its measuredΠ(Γ) curve. This curve reflects the relative rigidity of the protein molecule. For flexible molecules likeβ-casein and PVA ,Γ min (=ΓwhereΠstarts to deviate measurably from zero) is low and from this point onward the surface pressure increases gradually with increasingΓ. For rigid globular proteins (BSA, ovalbumin and lysozyme)Γ min is higher and with further increase of the surface concentration the surface pressure increases steeply. At high protein concentration and long adsorption times, for most proteins multilayer adsorption takes place.For ovalbumin, in the pH range 4-8 the effect of pH on theΠ-Γcurve is small, which indicates that electrostatic intermolecular forces do not contribute much to the surface pressure.In Chapter 3 a longitudinal wave technique, modified to ensure isotropic surface deformation, was used to determine the dilational modulus,ε, of adsorbed protein layers, at the air/water interface. This modification fully eliminated the complicating shear effects that became apparent in dilational modulus measurements with adsorbed layers of proteins in a conventional set-up.For all proteins examined at frequencies in the range from 0.01 to 1 rad/s, the initial part of theε(Π) plot is a straight line through the origin. The slope of this initial part ranges between +4 and +12 . No clear relationship between the slope and the rigidity of the protein molecule was found. However, the extent of this linear range is smaller for the flexible molecules (β-casein and PVA). From the fact that this slope significantly exceeds the ideal value of +1, it must be concluded that the behaviour of the adsorbed layer is far from ideal. In the linear range, the measured moduli coincide with the limiting moduli,ε 0 , calculated from theΠ(Γ) curve. This indicates that the surface pressure adjusts "instantaneously" to the changing adsorption during a compression-expansion cycle in time-scales ranging from 1 to 100 s. This also means that the modulus is purely elastic, i.e. the effect of relaxation processes is negligible. In this elastic range, differences between individual proteins are related to different degrees of non-ideality, reflected in the surface equation of state.At higher surface concentrations a relaxation mechanism becomes operative, which is most probably not caused by diffusional exchange between surface and solution. This conclusion is based on calculations of the diffusional transport rate and the theoretical frequency spectrum of the modulus. Relaxation due to conformational changes is plausible. In the visco-elastic regionε≥ε 0 for all proteins examined. This is an extra argument against diffusional exchange.The modulus increases in the order: PVA 1g/l), during the first minutes after adsorption a situation exists that differs from the equilibriumΠ(Γ) curve. At low interfacial pressures, during a modulus measurement, the adaptation of the conformation is faster (< 1 s.). Non-ideality of the adsorbed layer increases in the sequence TAG-oil < tetradecane < air, which is probably related to a decrease of solution quality for the more hydrophobic amino acids, which decreases in the same sequence. At each of the different interfaces non-ideality increases with increasing rigidity of the protein molecule (β-casein