Biomacromolecular Self-assembly Based On Food Proteins And Natural Polysaccharides

Molecular self-assembly is a process in which molecules or parts of them spontaneously form ordered aggregates. Various synthetic macromolecules have been successfully used as building blocks in self-assembly. Recently more and more researchers pay their attention to the self-assembly of biomacromolecules in vitro, which involve the bio-specified interactions such as the protein domain interaction and DNA complementarity. Among all of the biomacromolecular self-assembly systems, those between globular proteins without bio-specified interactions have rarely been reported. Although singular globular protein can self-assembly into fibril structure via electrostatic interaction and hydrophobic interaction under special denaturation conditions and protein microspheres can be prepared by employing additional crosslinker, there have not been reports concerning the self-assembly of natural globular proteins without bio-specified interactions into nanoscale hydrogel probably due to the difficulties and complexities in manipulating the noncovalent interactions between natural globular proteins. Proteins, especially food proteins, are nutritious, non-toxic, edible, biocompatible and degradable in the digestive system, which make them suitable for manufacturing oral drug carriers. Therefore the self-assembly of food proteins is not only of significance in basic research but also have bright prospect in applications.This thesis focuses on the self-assembly of natural macromolecules including globular and linear food proteins, ionic and nonionic natural polysaccharides, into biomacromolecular nanogels or micelles. For the first time we developed a convenient and effective method to prepare edible nanogels by self-assembling oppositely charged globular proteins or ionic polysaccharide and globular protein. The method was proved to be applicable to a series of assembly systems. The self-assembly process is in general different from that of liner macromolecules or the conventional self-assembly of proteins with bio-specified interactions. In the method we developed to prepare nanogels from food proteins and natural polysaccharides, no chemicals were added to the assembly systems during the preparation process except the necessary acid and alkali for adjusting solution pH. Therefore the resultant nanogels are non-toxic, edible, biocompatible, biodegradable and are suitable as drug carriers. This thesis contains following three parts:The first part is preparation of nanogels by self-assembly of two food proteins with unlike isoelectrostatic point (pI). Ovalbumin and lysozyme are both the components of egg white proteins, and pI of the former is 4.8 and the latter is 10.7. The preparation method is as follows: firstly, the two protein solutions were mixed at a weak-acid pH. Here the interactions between them were relatively weak. Secondly, the pH of the solution was adjusted to that near pI of lysozyme. During the process of adjusting pH, ovalbumin and lysozyme formed electrostatic complex at first and then molecular arrangement occurred, i.e. lysozyme molecules tended to aggregate owing to its decreasing net charges and ovalbumin molecules were inclined to separate from each other on account of the strong electrostatic repulsion. Then, the solution was heated and the two proteins denaturized so that the stable nanoparticles formed via intermolecular hydrophobic association and disulfide bonds. The preparation conditions were investigated in details and the optimal condition was acquired. The morphologies of the nanoparticles were observed by AFM and TEM. The light scattering measurements and AFM and TEM observations suggest that the resultant particles are in fact nanoscale hydrogel. Furthermore, the ζ-potential measurements, ultrafiltration and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicate that the nanogels have core-shell structure with the core composed of denatured lysozyme and a small part of ovalbumin and the shell formed by denatured ovalbumin,. The nanogels can be reversibly and steadily dispersed in the solution when pH is lower than pH 4 or higher than 6.3; the nanogels secondarily aggregate near the pI of ovalbumin due to the insufficient net charges on the nanogel surface. To resolve this problem, grafting dextran molecules onto ovalbumin via Maillard reaction which links the reducing end hydroxyl group of dextran to the amines of ovalbumin was carried out. The dextran-grafted ovalbumin and lysozyme then self-assembled into nanogels which were dispersible at the pH around the pI of ovalbumin. Based on our understanding of the mechanism that ovalbumin and lysozyme self-assembly into stable nanogels by adjusting pH and heat induced denaturation, we in fact developed a universal method to prepare nanogels from oppositely charged protein pairs and this method was successfully applied to other systems.The second part of the work is preparation of pH-sensitive nanogels by self-assembling chitosan and ovalbumin. Based on the success of the first assembly system, we chose chitosan and ovalbumin, widely applicable in biomedical field, as thebuilding blocks. Chitosan is the only natural polysaccharide with cataions and owns the appealing properties such as safety, biocompatibility, biodegradability, bioadhesiveness, anticancerogen etc. It was soluble in acid due to protonization. Chitosan and ovalbumin were mixed in a proper ratio first and the pH of the mixture solution was adjusted to an appropriate value and then the solution was heated to denature ovalbumin molecules leading to stable nanogels. Efforts were made to find a set of optimized preparation conditions. SEM and TEM observations suggest that the nanogels have core-shell structure. By considering the ζ-potential values and the solubility behavior of the nanogels, we tend to think that the shell of the nanogels is composed of the extended chitosan chains while ovalbumin and part of the chitosan chains trapped in ovalbumin gel network form the nanogel core. The dynamic light scattering measurements at various pH showed that the nanogels were pH-sensitive due to the changes in the nanogel charges. We also found that the hydrophilicity/hydrophobicity balance of the nanogels changes with pH as indicated by the fluorescent measurement using pyrene as a probe.The third part of the work is dealt with encapsulation and release of bioactive lysozyme by and from β-casein/Iysozyme polyion complex (PIC) micelles, β-casein is one of the components of milk protein. It is a linear protein with pI of 4.8. Through Maillard reaction, the dextran molecules (with Mw 35kDa) were grafted onto β-casein to yield a water-soluble graft copolymer. After lysozyme solution was added dropwise into the β-casein-g-dextran solution and the pH of the mixture solution was adjusted to a proper value, PIC micelles formed with β-casein and lysozyme electrostatic complex as the core and the grafted dextran chains on β-casein as the shell. Lysozyme was encapsulated in the micelle core and can then be released in the presence of strong acid, alkali or salt. The released lysozyme molecules have the same bioactivity as the original lysozyme molecules in terms of lysing the Micrococcus lysodeiktics bacteria. We also utilize Ca²⁺ to complex with the phosphate group linked to the serine residues in β-casein chains to stabilize the micelles. When β-casein is replaced by with casein, the micelles became more stable when acid, alkali or salt was added to the micelle solution.