Proteins and protein/surfactant mixtures at interfaces in motion

The research described in this thesis covers a number of aspects of the relation between surface properties and foaming properties of proteins, low molecular surfactants and mixtures thereof. This work is the result of a question of the industrial partners if it is possible to understand the foaming properties of protein hydrolysates. As there are many aspects of the surface properties that can be responsible for the foaming behaviour a number of problems were defined by which we can obtain a better understanding of the relation between surface properties and foam formation and stability in relation to the type of surface active substance.In this thesis an important question a priori has been: How can we understand the foaming properties from the properties of the surface active species. We presumed that the molecular properties cannot be translated forthwith to foaming properties but that a number of translation steps are necessary. First of all the molecular properties need to be translated into surface properties which manifest themselves in mechanical properties of surfaces and films. In addition there is a relation between these mechanical properties and the foaming properties.An important consideration a priori was that the way in which the surface is deformed is important for finding the relevant relation between the mechanical properties and the foaming properties. Here we made a distinction in two types of deformation being deformations caused by forces applied parallel and perpendicular to the surface. A force that is applied perpendicular to the surface (pressure) generally leads to a homogeneous deformation of the surface. The properties of the surface change with time or time scale but at the surface there are no differences with respect to surface tension. surface concentration or relative rate of expansion. Forces applied perpendicular to the surface generally lead solely to enlargement or reduction in surface area. Forces applied parallel to the surface (shear forces) can also lead to enlargement or reduction in surface area. These forces are generally due to viscous friction with the surface. In addition to a change in surface area these forces also cause a redistribution of surface active material over the surface. Hence the surface concentration. the surface tension as well as the relative rate of expansion vary over the surface. This leads to a surface tension gradient that is necessarily equal to the viscous drag at the surface. It is striking that in literature only homogeneous deformations are studied in detail. This fact can be attributed to the difficult experimental accessibility of surfaces subjected to shear forces. Nevertheless shear forces may play a role in the foaming properties such as in drainage and bubble break-up. An important part of this thesis will be devoted to the relation between viscous friction and surface motion.A device which enables the quantification of the relation between viscous drag and motion of the surface in relation to the surface properties is the overflowing cylinder. This device consists of an inner cylinder surrounded by an outer cylinder with a larger diameter. In this inner cylinder liquid is pumped up which flows over the rim into the space between the inner and outer cylinder. At the top surface we find a continuously expanding surface of which the relative expansion rate remains approximately constant over the surface in the vicinity of the centre of the cylinder. The expansion rate of the surface can be influenced by changing the length of the falling film. Hence within certain limits the expansion rate at the top surface c5n be varied. If we consider the falling film at the leading edge of the inner cylinder the falling motion of the liquid in the film causes the deformation of the surface parallel to the surface. If we would be able to measure the properties of the falling film we could learn how the surface properties vary with distance. However the surface of the falling film is experimentally not accessible. Therefore in this thesis the changes in surface properties of the falling film have been studied by measuring the surface tension of the top and bottom surface at a fixed place. This provides a reasonable measure of the surface tension difference over the falling film of the overflowing cylinder.In order to interpret this difference, the conditions at the falling film have been approximated by means of simple hydrodynamic theory. From this approximation we could conclude that there is a relation between the relative expansion rate, the length of the falling film and the surface tension difference. The surface of the falling film is propelled by the falling motion of the liquid which causes a surface tension gradient at the surface as a consequence of the viscous drag. If we compare the calculations with experimental data we can find that this approach is in agreement with the experiments. Hence the difference in surface tension over the falling film can be considered to be a measure of the resistance to deformation of the surface to forces applied parallel to the surface.The surface tension gradients which could be generated by means of different surface active species appeared to differ significantly from each other. Especially the difference between low molecular surfactants and proteins could be shown to be large. This can be ascribed to the sensitivity of the surface tension of these substances to expansion and compression of the surface. Adsorbed layers of proteins have a high surface tension in expansion. This can be explained by the time required for unfolding at a surface of these substances. In compression low surface tensions are found for proteins due to the slow desorption of proteins. The surface tension of low molecular surfactants is less sensitive to compression and expansion. In expansion the relatively short diffusion length causes the surface tension to deviate much less from the equilibrium surface tension. In compression these substances desorb easily causing the surface tension to be close to the equilibrium surface tension as well. Hence the surface tension gradient that can be generated by proteins is much larger the for low molecular surfactants. If mixtures of low molecular surfactants and proteins (Tween 20 andβ-casein) are considered it is found that in expansion the surface tension is influenced by both species. In compression however the surface tension is around the equilibrium surface tension of the low molecular surfactant. From this we can conclude that the low molecular surfactant determines the surface tension in compression. Most probably there the affinity of the surface active substance for the surface is important which causes the preferential desorption of proteins. This means that the surfaces of these mixtures have a low resistance to deformation by forces applied parallel to the surface.Unfolding behaviour of proteins at interfacesThe most important class of surface active substances which have been studied in this thesis are proteins due to the similarities in structure and properties with protein hydrolysates. Proteins consist of 20 different amino acids which vary in residual group. Despite the similarity in the basic structure of these substances the difference in foaming properties between proteins is large. In literature this difference in foaming properties is ascribed to the difference in unfolding rate during adsorption at air/water interfaces of these polymers. There are no reliable data on the unfolding rate of proteins however. The overflowing cylinder technique can be used to determine the unfolding rates of proteins since the top surface is in a steady state while the relative expansion rate is finite. The relative expansion rate can be seen as a characteristic time scale of the surface.In order to measure the unfolding rate of the proteins, at the top surface, the surface tension, the relative rate of expansion and the surface concentration were determined using the Wilhelmy plate technique, laser Doppler anemometry and ellipsometry respectively. With these three parameters the surface can be characterised completely. The reasoning behind the characterisation is as follows: If proteins need time for the unfolding at an interface, the relative expansion rate determines the mean degree of unfolding of the proteins at the interface. As a function of the relative rate of expansion and at equal adsorbed amounts the surface tension will vary due to a difference in the mean degree of unfolding.If we would know the surface tension and surface concentration for different bulk concentrations as a function of the relative rate of unfolding then we would be able to establish the influence of the unfolding rate of the protein on the surface tension. Since this is difficult to determine on the basis of the raw date, a simple model was used to express this influence of unfolding in an unfolding and a refolding parameter. In this model the transport to and the unfolding at the top surface of an overflowing cylinder has been described. The unfolding and refolding has been described by means of a first order reaction. In essence we assume that the degree of unfolding can be seen as an average over many stages of unfolding over a large number of molecules. By dividing the interface into a large number of concentric rings and by carrying out the calculations for the transport and unfolding for each concentric ring the surface properties can be calculated as a function of the distance to the centre. By varying the relative rate of expansion as a function of the distance to the centre in the same way as takes place at the top surface of the overflowing cylinder also the surface tension gradient can be determined. Since there is a fixed relation between the surface tension gradient and the maximum relative rate of expansion, the maximum relative rate of expansion can be determined by means of iteration.In this thesis the unfolding behaviour of the proteins:β-casein,β-lactoglobulin, BSA and lysozyme has been determined experimentally. In literature these proteins have been characterised well with respect to adsorption and unfolding behaviour. The unfolding rates of these proteins differ several orders of magnitude.β-Casein andβ-lactoglobulin were shown to unfold most rapid. The characteristic time scales of unfolding of these proteins is in the order of a tenth forβ-casein to a few tenths forβ-lactoglobulin. Lysozyme hardly unfolds within the time scale of the experiment which indicates that the unfolding takes more than 100 seconds. The model was shown not to apply for BSA since the change in surface tension proceeds in two steps. Nevertheless it could be calculated that the unfolding of BSA takes place in a time scale in the order of 20 seconds.The model and the overflowing cylinder technique have also been applied to mixtures of proteins and low molecular surfactants. The systemβ-casein/Tween 20 was chosen because in this system no complications are known such as aggregation in he bulk phase, or electrostatic interactions at the surface. At expanding interfaces these systems were shown to behave in a more or less additive manner. The surface tension of the mixture appeared to be lower than the addition of the decrease in surface tension of each species individually. This can be attributed to the fact that both species occupy part of the space at the interface. In static conditions it was found in literature that low molecular surfactants can displace proteins from the interface. This difference in behaviour is caused by the fact that in expansion the surface tension is controlled mainly by transport to and unfolding at the surface while in equilibrium time scale is irrelevant and the affinity of the substances to the surface determines the behaviour. Despite certain deviations the measured properties were indicated to be consistent with the calculations by the model. Higher order effects such as the presence of the surfactants in micelles and preferential adsorption were shown to have little effect of these mixtures at expanding surfaces.Foam formation and foam stabilityIn order to quantify the experimental values and techniques of this research for practical situations, foaming experiments have been performed for a number of relevant systems. In these experiments, the bubble size and the drainage rate of the foams have been determined. Subsequently the results of these experiments were related to mechanical surface properties.The experiments indicated that the surface tension difference over the falling film in the overflowing cylinder correlated with the bubble size and the rate of drainage. For the formation of foam this means that not only the amount of energy supplied is important for the bubble size but that also characteristic properties of the surface being the maximal viscous drag that can be transferred is important for the break-up of foam bubbles. The explanation that can be given for this is that the shear stress generated at the surface provides the deformation that leads to an unstable shape of the bubbles which finally leads to break-up. Surface active species that enable the generation of a high shear stress therefore promote the generation of small bubbles.In addition it was demonstrated that there is a relation between drainage and viscous friction at the bubble surface. The rate of drainage expressed in the decrease of the characteristic film thickness decreases when a higher surface tension difference between top and bottom surface in the overflowing cylinder is present at a rather arbitrarily chosen relative rate of expansion of 1 s -1 .In chapter 6 the foaming properties of protein hydrolysates were discussed. The most important reason that protein hydrolysates have good foaming properties is that in these systems the low molecular components do not displace high molecular components. This causes the surface tension difference between a compressed and expanded surface to be high. This supports the creation of small bubbles and the resistance to drainage. It is possible that the good foamability of protein hydrolysates compared to proteins is caused by the lower surface tension in equilibrium.In general it can be said that surface tension gradients play a larger role in foam formation and foam stability than the attention in literature would suggest. More attention for the properties which determine the resistance against deformation of surfaces, parallel to the surface would lead to a better insight in the reasons why different surface active substances exhibit different foaming behaviour.