Investigating the Mesoscale of β-lactoglobulin Fibril Hydrogels

The objective of this doctoral thesis was to investigate the relationship between the architecture of protein fibril networks and their macroscopic properties. This requires investigation of the mesoscale; the scale between the macroscopic and microscopic scales where fibril self-assembly processes occur resulting in structures organized in different hierarchical levels. The mesoscale of such networks is not extensively studied and this is where I want to add knowledge, in order for physical sciences to contribute to New Zealand agricultural food sectors by changing the way in which soft materials and biopolymer engineering is done and by taking biomaterials from commodities to specialties by adding knowledge. The protein selected for the current study was β-lactoglobulin which forms amyloid-like fibrils when heated at 80oC under acidic conditions. Specifically, hydrogels were formed under three pH conditions; 1) pH 2.3, 2) pH 2.0, and 3) pH 0.9. These three conditions result in three different types of hydrogels being formed. The β-lactoglobulin hydrogels formed at pH 0.9, which have not previously been reported in the literature, exhibit completely different structure and macroscopic properties compared with the standard and widely reported in the literature β-lactoglobulin hydrogels formed at pH 2.3 and pH 2.0. Cryo-scanning electron microscopy (cryo-SEM) was used to investigate the intact 3D structure of the hydrogels. On the contrary, there are a lot of studies reported in the literature using other microscopy techniques, like atomic force microscopy (AFM) or transmission electron microscopy (TEM), which allow the fibril characteristics and not the intact interior of the hydrogel structure to be investigated. Cryo-SEM showed that β-lactoglobulin fibrils formed at pH 2.3 are the most flexible fibrils with the longest end-to-end fibril lengths, while the fragmented-particles fibrils formed at pH 0.9 are the thickest and least flexible with the shortest end-to-end lengths. Determination of fibril characteristics helps in predicting the macroscopic behaviour of fibril networks. Small-angle X-ray scattering (SAXS) was used as a complementary method to cryo-SEM. SAXS allows structural investigation of fibril networks, that cryo-SEM is not able to achieve. Specifically, SAXS showed that fibril hydrogels formed at pH 2.3 exhibit the least compact structure with the least fibril surface roughness, while hydrogels formed at pH 0.9 exhibit the most compact structure and the roughest fibril surfaces. Another crucial point is that SAXS allows the thermodynamics of these systems to be probed. SAXS data confirmed that the more rod-like the network, the more favourable it is for the system to organize into a nematic phase. Rheology was used to investigate the macroscopic properties of the hydrogels. Rheology demonstrated that there are two types of behaviour exhibited by these three types of hydrogels. Although it was assumed at the start of this project that these three types of hydrogels could exhibit different macroscopic behaviour, since their end-to-end fibril length scales are different, finally, it was demonstrated experimentally that hydrogels formed at pH 2.3 and pH 2.0 exhibit roughly the same macroscopic behaviour, while hydrogels formed at pH 0.9 exhibit a completely different macroscopic behaviour. Specifically, hydrogel networks formed at pH 2.0 are slightly stiffer than those formed at pH 2.3 and exhibit fast gelation time, while hydrogels formed at pH 0.9 are the softest and exhibit slow gelation time. Considering the cryo-SEM data, the fibrils formed at pH 0.9 are the least flexible. These two sets of data appear to contradict each other, but it must be noted that the length scales being probed are different. These results indicate that the characteristics of individual fibrils do not necessarily manifest themselves directly in the macroscopic behaviour of the whole fibril network. This means that the interplay of the fibrils with each other is important in defining longer length scale behaviour. All in all, while most studies to date refer to the structural investigation of β-lactoglobulin fibril systems on a macroscopic or microscopic level, in this work it is addressed that thermodynamics, chain stiffness and thickness, electrostatic interactions, inter-fibril distances, orientation of β-sheet stacks, and the number of cross-links constitute the basic factors on the mesoscale that affect the architecture of the fibril networks and connect their architecture with their macroscopic properties. Hence, by controlling the mesoscale (self-assembly process), the manipulation of biomaterials that already exist in the market is feasible, in order to exhibit novel macroscopic properties.