Mesoscale structure and techno-functional properties of enzymatically cross-linked a-lactalbumin nanoparticles

Abstract

The aim of this thesis is to understand the connection between molecular, meso and macroscales of enzymatically cross-linked proteins. It was hypothesised that the techno-functional properties at macroscale, such as bulk rheology and foam stability, are affected by the structure of nanoparticles at mesoscale. The approach was to make α-lactalbumin (α-LA) nanoparticles by using two different enzymes, horseradish peroxidase (HRP) or microbial transglutaminase (mTG), to produce an open and compact mesoscale structure, respectively. In addition to the control over the mesoscale structure, the size of the nanoparticles can be independently controlled by varying the dosage of hydrogen peroxide in the case of HRP and by thermal inactivation in the case of mTG. The other important parameters determining the size are protein concentration and ionic strength. The size (radius of gyration) range that could be achieved by varying the above mentioned control parameters is 20 – 200 nm. The polydispersed nanoparticles were separated by asymmetrical flow field flow fractionation (AF4) and characterised inline with multi angle light scattering (MALS). Polymerization of apo α-LA with HRP and mTG proceeds in a step growth way i.e. first monomers react to form oligomers and the oligomers are cross-linked to form polymers (nanoparticles). Extensive cross-linking of α-LA with HRP gives rise to not only di-tyrosine cross-links, but also tri–octa tyrosine cross-links, which was hitherto unknown. The two different mesoscale structures result in gels of different storage moduli. The storage modulus of gels made by concentrating the α-LA/mTG nanoparticles was around ten times higher than that made with open nanoparticles. The half-life time (t0.5) of the foam made with α-LA nanoparticles was two to six times higher than that of the monomeric α-LA. The higher foam-stability of the α-LA nanoparticles as compared to the monomeric α-LA is due to their higher thickness of the interfacial layer and thin films. In conclusion, it is shown that the techno-functional properties of α-LA are directly correlated to the size and meso-scale structures of the nanoparticles and enzymatic cross-linking is an effective way to control them.