Influence of process factors on the formation and fragmentation of amyloid and amyloid-like aggregates from beta-lactoglobulin
Protein amyloid-aggregation is increasingly being recognized to expand, broaden and enrich protein functionalities. A large number of very different proteins, including the whey protein beta-lactoglobulin (BLG) have been shown to convert into amyloid fibrils under properly designed conditions. At high temperatures and acid conditions, BLG forms semi-flexible amyloid fibrils (at pH 2) or flexible amyloid-like aggregates (at pH 3.5). It was mentioned, that the interfacial properties of amyloid aggregates are different from those of non-aggregated BLG. For example, the application of fibrils leads to a higher physical and chemical stabilization of oil-water emulsions and foams. However, in the production of emulsions the protein structures are exposed to high shear forces and cavitation in combination with a high specific surface area of the oil-water interface. These process factors can contribute to the degradation of the amyloid aggregates. Otherwise, shear and interfaces also have a high relevance for the formation of amyloid structures. To date, no comparison of the different morphologies with respect to physico-chemical properties, as well as formation kinetics and stability as a function of mechanical stress or interfaces has been performed. This information is relevant to estimate the value of the respective aggregates for different applications. In order to obtain this information, several studies were conducted in this thesis. The investigation of the aggregate building blocks have shown that pH 2 fibrils build from peptides, while worm-like aggregates formed from unhydrolysed BLG. Analysis of the physico-chemical properties revealed a higher proportion of beta-sheets, a higher compactness, a higher zeta-potential and a lower hydrophobicity of the fibrils compared to the worm-like aggregates. Using a statistically designed experiment, it was possible to identify the temperature as the most important factor for aggregation kinetics. Interactions of temperature with pH value and stirring speed were determined. The nucleation supporting effect of mechanical energy input and surfaces on the amyloid aggregation kinetics was demonstrated by using chemically modified glass beads in the shaking incubator. The impact on the length of the aggregates was investigated in more detail in shearing experiments with rotor-stator and ultra-sonication in the presence and absence of a dispersed oil-phase. A dependence of the fibril fragmentation on the amount of shear stress and the presence of oil was found, while the worm-like aggregates remained unaffected to rotor-stator-shear. In summary, this thesis demonstrates that amyloid and amyloid-like aggregates have different characteristics with respect to structure, physico-chemical properties, formation kinetics and process stability, which makes them suitable for different technological applications.
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