Protein/polysaccharide complexes at air/water interfaces

KEYWORDS:protein, polysaccharide,b‑lactoglobulin, pectin, electrostatic interaction, complex coacervation, adsorption, air/water interface, oil/water interface, surface pressure, surface rheology, spectroscopyProteins are often used to create and stabilise foams and emulsions and therefore their adsorption behaviour to air/water and oil/water interfaces is extensively studied. Interaction of protein and polysaccharides in bulk solution can lead to the formation of soluble or insoluble complexes. The aim of this thesis was to understand the influence of (attractive and non-covalent) protein/polysaccharide interaction on adsorption behaviour at air/water interfaces (and oil/water interfaces) in terms of adsorption kinetics, and rheological and spectroscopic characterisation of the adsorbed layers. The approach was to first identify the relevant parameters (like charge density, charge distribution or molecular weight of the ingredients) in the mixed protein/polysaccharide adsorption process. Subsequently, for each parameter a range of ingredients was selected/prepared allowing variation of only this single parameter. After investigation of the phase behaviour in bulk solution of the different protein/polysaccharide mixtures to be used, the role of each parameter in mixed protein/polysaccharide adsorption was studied. The parameters most thoroughly assessed were: protein/polysaccharide mixing ratio, polysaccharide charge density and molecular weight and the sequence of adsorption. The majority of the measurements were performed withb‑lactoglobulin (in combination with various polysaccharides e.g. pectin or carboxylated pullulan) at air/water interfaces, at standard conditions of pH 4.5 and low ionic strength (< 10 mM). In addition, experiments were performed at higher ionic strengths, different pH's, with different proteins or at an oil/water interface, to extend the insight in mixed protein/polysaccharide adsorption. This results obtained lead to a generic mechanistic model of mixed protein/polysaccharide adsorption.In conclusion, protein/polysaccharide interaction can be exploited to control protein adsorption at air/water interfaces. Any parameter affecting protein/polysaccharide interaction (e.g. ingredient parameters like polysaccharide molecular weight, charge density and distribution or system parameters like charge ratio, pH and ionic strength) may be varied to obtain the desired adsorption kinetics, surface rheological behaviour, or net charge of the surface layer. The choice of simultaneous protein/polysaccharide adsorption (in the form of complexes) versus sequential adsorption (first the protein, than the polysaccharide) provides an extra control parameter regarding the functionality of mixed adsorbed layers

On the link between surface rheology and foam disproportionation in mixed hydrophobin HFBII and whey protein systems

Here we study the surface dilational properties of spread and adsorbed layers of whey protein isolate (WPI) and hydrophobin HFBII at air/water interface using Langmuir trough and relate them to foam ability and stability. In spread and adsorbed systems, a gradual increase in modulus with the fraction of HFBII on the surface or in the bulk is observed and we can identify distinct regimes of WPI-dominated and HFBII-dominated behaviour. The dominance of HFBII is further substantiated by visual observation of microscopic wrinkles appearing on the surface of the trough. When comparing spread to adsorbed systems, it was found that a higher HFBII fraction is needed to obtain HFBII dominant behaviour in spread layers than in case of adsorbing layers (f(HFBII) = 0.6 and 0.2 respectively). Furthermore, our results indicate that the HFBII-contribution to the interfacial behaviour becomes more pronounced upon sequential large scale compression/expansion cycles. In order to explain this non-trivial behaviour, we propose that there is multi-layer formation at the interface, having a top layer enriched in HFBII and bottom layer enriched in WPI. It is also concluded that coarsening stability in foams corresponds more closely to the surface dilational properties measured in adsorbing layers than those in spread layers. Finally, it was observed that in mixed systems of HFBII and WPI, the coarsening process levels off, which corresponds to the increased dominance of HFBII in mixed WPI:HFBII-layers upon large surface deformation that is occurring at the surface of shrinking bubbles. (C) 2013 Elsevier B.V. All rights reserved

Use of polysaccharides to control protein adsorption to the air-water interface

In order to understand foaming behaviour of mixed protein/anionic polysaccharide solutions, we investigated the effect of beta-lactoglobulin/pectin interaction in the bulk on beta-lactoglobulin adsorption to the air-water interface. Adsorption kinetics were evaluated by following surface pressure development in time of several pure protein solutions and of mixed protein/polysaccharide solutions using an automated drop tensiometer (ADT). It was found that complexation of proteins with polysaccharides can slow down the kinetics of surface pressure development by at least a factor 100; and greatly diminish foam formation. In contrast, a five times acceleration in the increase of surface pressure was observed in other cases. We propose a mechanism for protein adsorption from mixed protein/polysaccharide solutions. Effects of ionic strength, pH and mixing ratio on this mechanism were studied for mixtures of beta-lactoglobulin and low methoxyl pectin, whereas other proteins and anionic polysaccharides were used to explore the role of protein and polysaccharide charge density and distribution. Whereas the possibilities to change system parameters like ionic strength or pH are limited in food related systems, selecting a suitable combination of protein and polysaccharide offers a broad opportunity to control protein adsorption kinetics and with that foam formation

Structure of mixed β-lactoglobulin/pectin adsorbed layers at air/water interfaces; a spectroscopy study

Based on earlier reported surface rheological behaviour two factors appeared to be important for the functional behaviour of mixed protein/polysaccharide adsorbed layers at air/water interfaces: (1) protein/polysaccharide mixing ratio and (2) formation history of the layers. In this study complexes of β-lactoglobulin (positively charged at pH 4.5) and low methoxyl pectin (negatively charged) were formed at two mixing ratios, resulting in negatively charged and nearly neutral complexes. Neutron reflection showed that adsorption of negative complexes leads to more diffuse layers at the air/water interface than adsorption of neutral complexes. Besides (simultaneous) adsorption of protein/polysaccharide complexes, a mixed layer can also be formed by adsorption of (protein/)polysaccharide (complexes) to a pre-formed protein layer (sequential adsorption). Despite similar bulk concentrations, adsorbed layer density profiles of simultaneously and sequentially formed layers were persistently different, as illustrated by neutron reflection analysis. Time resolved fluorescence anisotropy showed that the mobility of protein molecules at an air/water interface is hampered by the presence of pectin. This hampered mobility of protein through a complex layer could account for differences observed in density profiles of simultaneously and sequentially formed layers. These insights substantiated the previously proposed organisations of the different adsorbed layers based on surface rheological data. © 2007 Elsevier Inc. All rights reserved. Chemicals / CAS: beta lactoglobulin, 9045-23-2; Lactoglobulins; Membranes, Artificial; pectin, 9000-69-5; Pectins; Water, 7732-18-

Polysaccharide charge density regulating protein adsorption to air/water interfaces by protein/polysaccharide complex formation

Because the formation of protein/polysaccharide complexes is dominated by electrostatic interaction, polysaccharide charge density is expected to play a major role in the adsorption behavior of the complexes. In this study, pullulan (a non-charged polysaccharide) carboxylated to four different charge densities (fraction of carboxylated subunits: 0.1, 0.26, 0.51, and 0.56) was used to investigate the effect of charge density on the properties of mixed protein/polysaccharide adsorbed layers at air/water interfaces. With all pullulan samples, soluble complexes with -lactoglobulin could be formed at low ionic strength, pH 4.5. It was shown that the higher was the pullulan charge density, the more the increase of surface pressure in time was retarded as compared to that for pure -lactoglobulin. The retardation was even more pronounced for the development of the dilatational modulus. The lower dilatational modulus can be explained by the ability of the polysaccharides to prevent the formation of a compact protein layer at the air/water interface due to electrostatic repulsion. This ability of the polysaccharides to prevent "layer compactness" increases with the net negative charge of the complexes. If charge density is sufficient (0.26), polysaccharides may enhance the cohesion between complexes within the adsorbed layer. The charge density of polysaccharides is shown to be a dominant regulator of both the adsorption kinetics as well as the resulting surface rheological behavior of the mixed layers formed. These findings have significant value for the application of complex protein-polysaccharide systems

Mesoscopic structure and viscoelastic properties of ß-lactoglobulin gels at low pH and low ionic strength

Abstract In this paper, we have investigated the mesoscopic structure and rheological properties of heat-set -lactoglobulin gels at low pH and low ionic strength. We have determined the scaling of the elastic or storage modulus of the gel with protein concentration. Four batches with a pH of 2 and an ionic strength of 13 mM showed a wide range of scaling behavior. In two of the batches, two concentration regimes with distinctive exponents could be detected. We constructed the phase diagram for this system as a function of pH and ionic strength, and were able to show that the wide spread in scaling behavior between the batches is the result of differences in the mescopic structure of the gels. These differences in mesoscopic structure were the result of small variations in pH and ionic strength between the batches. Our experiments show that when characterizing gel properties of -lactoglobulin solutions at very acidic conditions and low ionic strength, extreme care has to be taken to stabilize the pH of these systems. Small variations in pH have a drastic effect on the structure of the gels, and on the macroscopic viscoelastic properties

Mesoscopic structure and viscoelastic properties of ß-lactoglobulin gels at low pH and low ionic strength

Abstract In this paper, we have investigated the mesoscopic structure and rheological properties of heat-set -lactoglobulin gels at low pH and low ionic strength. We have determined the scaling of the elastic or storage modulus of the gel with protein concentration. Four batches with a pH of 2 and an ionic strength of 13 mM showed a wide range of scaling behavior. In two of the batches, two concentration regimes with distinctive exponents could be detected. We constructed the phase diagram for this system as a function of pH and ionic strength, and were able to show that the wide spread in scaling behavior between the batches is the result of differences in the mescopic structure of the gels. These differences in mesoscopic structure were the result of small variations in pH and ionic strength between the batches. Our experiments show that when characterizing gel properties of -lactoglobulin solutions at very acidic conditions and low ionic strength, extreme care has to be taken to stabilize the pH of these systems. Small variations in pH have a drastic effect on the structure of the gels, and on the macroscopic viscoelastic properties