Role of protein-protein interactions on protein aggregation and emulsion flocculation

In this thesis, the effect of molecular properties on the aggregation and flocculation behaviour is studied. The aggregation behaviour was thought to be mainly affected by the structural stability of the protein. A decreased structural stability results in unfolded proteins which are more prone to aggregation. The flocculation behaviour was shown to be affected by the adsorbed amount at saturation and the adsorption rate. These parameters have been combined in a surface coverage model, which describes the stabilization of emulsions away from the iso‑electric point (pI) to be affected by excess protein in the continuous phase. In addition, a model was proposed for the prediction of the adsorbed amount at saturation. This is influenced by the protein charge and radius and system conditions (i.e. pH and ionic strength). The adsorption rate, which is a measure for the affinity of the protein towards the adsorption to the interface, was shown to increase with increasing relative exposed hydrophobicity and a decrease of the electrostatic repulsion (i.e. decrease of ionic strength or the protein charge). Close to the pI, the applicability of protein-stabilized emulsions is limited. Hence, a steric interaction was introduced to stabilize the emulsion. It was shown that glycation of the protein with a trisaccharide was sufficient to sterically stabilize the emulsions against pH-induced flocculation

Towards predicting the stability of protein-stabilized emulsions

The protein concentration is known to determine the stability against coalescence during formation of emulsions. Recently, it was observed that the protein concentration also influences the stability of formed emulsions against flocculation as a result of changes in the ionic strength. In both cases, the stability was postulated to be the result of a complete (i.e. saturated) coverage of the interface. By combining the current views on emulsion stability against coalescence and flocculation with new experimental data, an empiric model is established to predict emulsion stability based on protein molecular properties such as exposed hydrophobicity and charge. It was shown that besides protein concentration, the adsorbed layer (i.e. maximum adsorbed amount and interfacial area) dominates emulsion stability against coalescence and flocculation. Surprisingly, the emulsion stability was also affected by the adsorption rate. From these observations, it was concluded that a completely covered interface indeed ensures the stability of an emulsion against coalescence and flocculation. The contribution of adsorption rate and adsorbed amount on the stability of emulsions was combined in a surface coverage model. For this model, the adsorbed amount was predicted from the protein radius, surface charge and ionic strength. Moreover, the adsorption rate, which depends on the protein charge and exposed hydrophobicity, was approximated by the relative exposed hydrophobicity (QH). The model in the current state already showed good correspondence with the experimental data, and was furthermore shown to be applicable to describe data obtained from literature

Quantitative description of the parameters affecting the adsorption behaviour of globular proteins

The adsorption behaviour of proteins depends significantly on their molecular properties and system conditions. To study this relation, the effect of relative exposed hydrophobicity, protein concentration and ionic strength on the adsorption rate and adsorbed amount is studied using ß-lactoglobulin, ovalbumin and lysozyme. The curves of surface elastic modulus versus surface pressure of all three proteins, under different conditions (i.e. concentration and ionic strength) superimposed. This showed that the interactions between the adsorbed proteins are similar and that the adsorbed proteins retain their native state. In addition, the adsorption rate (kadsorb) was shown to scale with the relative hydrophobicity and ionic strength. Moreover, the adsorbed amount was shown to be dependent on the protein charge and the ionic strength. Based on these results, a model is proposed to predict the maximum adsorbed amount (Gmax). The model approximates the adsorbed amount as a close-packed monolayer using a hard-sphere approximation with an effective protein radius which depends on the electrostatic repulsion. The theoretical adsorbed amount was in agreement with experimental Gmax (±10%)

Comparison of Heat-Induced Aggregation of Globular Proteins

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, ß-lactoglobulin, and patatin) under various conditions (pH, ionic strength, concentration, and temperature). The aggregation rate of ß-lactoglobulin was slower (>10 times) than that of ovalbumin and patatin. Moreover, the conditions (pH, ionic strength, and concentration) affected the aggregation kinetics of ß-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)

Protein Concentration and Protein-Exposed Hydrophobicity as Dominant Parameters Determining the Flocculation of Protein-Stabilized Oil-in-Water Emulsions

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 (ß-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

Enzymatic cross-linking of a-lactalbumin to produce nanoparticles with increased foam stability

Hard colloidal nanoparticles (e.g. partly hydrophobised silica), are known to make foams with very high foam-stability. Nanoparticles can also be produced from proteins by enzymatic cross-linking. Such protein based particles are more suitable for food applications, but it is not known if they provide Pickering foam stabilisation to the same extent as hard colloidal particles. a-Lactalbumin (a-LA) was cross-linked with either microbial transglutaminase (mTG) or horseradish peroxidase (HRP) to produce a-LA/mTG and a-LA/HRP nanoparticles. With both enzymes a range of nanoparticles were produced with hydrodynamic radii ranging from 20–100 nm. The adsorption of nanoparticles to the air–water interface was probed by increase in surface pressure (¿) with time. In the beginning of the ¿ versus time curves, there was a lag time of 10–200 s, for nanoparticles with Rh of 30–100 nm, respectively. A faster increase of ¿ with time was observed by increasing the ionic strength (I = 0–125 mM). The foam-ability of the nanoparticles was also found to increase with increasing ionic strength. At a fixed I, the foam-ability of the nanoparticles decreased with increasing size while their foam-stability increased. Foams produced by low-shear whipping were found to be 2 to 6 times more stable for nanoparticles than for monomeric a-LA (Rh ˜ 2 nm). At an ionic strength of 125 mM ionic strength and protein concentration = 10 g L-1, the foam-stability of a-LA/mTG nanoparticles (Rh = 100 nm, ¿app = 21.6 kg m-3) was 2–4 times higher than a-LA/HRP nanoparticles (Rh = 90 nm, ¿app = 10.6 kg m-3). This indicated that foam-stablity of nanoparticles is determined not only by size but also by differences in mesoscale structure. So, indeed enzymatic cross-linking of proteins to make nanoparticles is moving a step towards particle like behavior e.g. slower adsorption and higher foam stability. However, the cross-link density should be further increased to obtain hard particle-like rigidity and foam-stability

The influence of the primary and secondary xanthan structure on the enzymatic hydrolysis of the xanthan backbone

Differently modified xanthans, varying in degree of acetylation and/or pyruvylation were incubated with the experimental cellulase mixture C1-G1 from Myceliophthora thermophila C1. The ionic strength and/or temperature of the xanthan solutions were varied, to obtain different xanthan conformations. The exact conformation at the selected incubation conditions was determined by circular dichroism. The xanthan degradation was analyzed by size exclusion chromatography. It was shown that at a fixed xanthan conformation, the backbone degradation by cellulases is equal for each type of xanthan. Complete backbone degradation is only obtained at a fully disordered conformation, indicating that only the secondary xanthan structure influences the final degree of hydrolysis by cellulases. It is thereby shown that, independently on the degree of substitution, xanthan can be completely hydrolyzed to oligosaccharides. These oligosaccharides can be used to further investigate the primary structure of different xanthans and to correlate the molecular structure to the xanthan functionalities

Protein Concentration and Protein-Exposed Hydrophobicity as Dominant Parameters Determining the Flocculation of Protein-Stabilized Oil-in-Water Emulsions

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 (ß-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

Effect of Glycation on the Flocculation Behavior of Protein-Stabilized Oil-in-Water Emulsions

Glycation of proteins by the Maillard reaction is often considered as a method to prevent flocculation of protein-stabilized oil-in-water emulsions. The effect has been suggested, but not proven, to be the result of steric stabilization, and to depend on the molecular mass of the carbohydrate moiety. To test this, the stabilities of emulsions of patatin glycated to the same extent with different mono- and oligosaccharides (xylose, glucose, maltotriose, and maltopentaose) were compared under different conditions (pH and electrolyte concentration). The emulsions with non-modified patatin flocculate under conditions in which the zeta potential is decreased (around the iso-electric point and at high ionic strength). The attachment of monosaccharides (i.e., glucose) did not affect the flocculation behavior. Attachment of maltotriose and maltopentaose (Mw > 500 Da), on the other hand, provided stability against flocculation at the iso-electric point. Since the zeta potential and the interfacial properties of the emulsion droplets are not affected by the attachment of the carbohydrate moieties, this is attributed to steric stabilization. Experimentally, a critical thickness of the adsorbed layer required for steric stabilization against flocculation was found to be 2.29–3.90 nm. The theoretical determination based on the DLVO interactions with an additional steric interaction coincides with the experimental data. Hence, it can be concluded that the differences in stability against pH-induced flocculation are caused by steric interactions