| Synthetic polymer materials have been widely used in different fields at present. These polymers not only consume the limited petroleum resources, but also are difficult to degrade naturally. They cause serious damage to environment. Natural polymer materials are environmentally friendly materials and can degrade naturally. As a consequence, natural polymer materials became an important research area. Protein belongs to natural macromolecular polymer and has been used to produce nano materials or novel materials based on the self-assembly properties.The subject of this research mainly involves two kinds of natural protein: casein and keratin. In order to investigate the assembly laws between different proteins and expand the usage of complex micelles based on self-assembly technique. Complex micelles with controlled morphology, size and hydrophilicity were made by casein and keratin through specific method. The structure, performance and formation mechanism for casein/keratin complex micelles were studied. Preliminary studies on biofilms and drug release for complex micells were also carried out in this research. The main research work includes the following aspects:(1) Na2 S reduction method was introduced to prepare wool keratin solution containing thiol group.Wool keratin with different isoelectric point were separated and purified by acid precipitation, isoelectric focusing, ultrafiltration and dialysis method. The main factors of the p H value, time and temperature which influence precipitation were dicussed. Results showed that the best conditions for wool keratin separation were p H2.0, 5℃, 3hours. The productivity of keratin by acidic precipitation was 62.8%. Keratin with isoelectric point 5.5 and 3.0 was selected according to the requirements for assembly complex micelles. The productivity of keratin with isoelectric point 5.5 and 3.0 was 12.5% and 5.8%, and the cystine content were 21.45% and 2.57% respectively. The molecular weight of the keratin p I 3.0 and p I 5.5 components were 13355 Da and 6191 Da according to the gel chromatography measurement. Keratins acquired through isoelectric focusing method have high molecular weight and more complete structure. It also have more active groups. These factors enable keratin to assemble typical complex micelles.(2) Oxidation fixing method and enzyme fixing method were adopoted in preparing stable casein/keratin(p I3.0) complex nano-micelles and casein/keratin(p I5.5) complex micelles. Scanning electron microscope(SEM), atomic force microscope(AFM), dynamic light scattering(DLS), differential scanning calorimetry(DSC) methods were used to characterize the complex micelles. Spherical complex micelles can be prepared at 20℃ from a 4:1 mass ratio of casein to keratin either in the 1.5mg/m L concentration for casein/keratin p I3.0 complex micelles or in the 3.0 mg/m L concentration for casein/keratin p I5.5 complex micelles. The results showed that the size of the casein/keratin(p I3.0) complex nano-micelles ranged around 50~80 nm and the size of the casein/keratin(p I5.5) complex micelles ranged around 100~200nm. Thermal performance changed dramatically when complex micelles underwent fixing process. Casein/keratin(p I3.0) complex micelles prepared by oxidation fixing method have greater thermostability than casein/keratin(p I5.5) complex nano-micelles prepared by enzyme fixing method. Casein/keratin complex micelles have good dilution stability, storage stability, freeze stability and thermal stability. Polymer can be produced with molecular weight of 43~200KDa in the process of preparing casein/keratin(p I5.5) complex micelles.(3) Assembly protein concentration, mass ratio and assembly temperature were controlled manually to produce complex micelles with sphere shape, cube shape,worm-like shape, vesicle shape,sea urchin shape, coral clusters shape, and mixed shape. The nano-complex micelles and the complex micelles with size range around 100~200nm were prepared by controlling the composition of the peptide chains. Complex micelles with high hydrophobicity can be prepared by raising the protein concentration or decreasing the mass ratio of casein to keratin. The shape of the casein/keratin complex micelles changed from sphere to cube and hydrophilcity enhanced when the assembly temperature increased from 20℃ to 60℃.(4) The aggregation process of the complex micelles includes three steps: electrostatic attraction, forming of the shell-core structure through the hydrophobic interaction, and fixation of the structure. The process of preparing casein/keratin(p I3.0) nano-complex micelles involved the oxidation of the thiol group and the re-establishment of the disulfide bonds. The network of the disulfide bonds can joint the adjacent casein components together. The process of preparing casein/keratin(p I5.5) complex micelles involved the formation of amide bond network between casein and keratin. Meso Dyn simulations were also introduced to simulate the process of the aggregation of the peptide chains of these two proteins. The results showed that the casein/keratin(p I3.0) system can reach the equilibrium state rapidly. The near UV CD, far UV CD spectroscopy and raman spectroscopy were used to analyze the changes of the secondary and tertiary structure when complex micelles were assembled at different mass ratio. The influence of p H value and temperature on the secondary and tertiary structure of the protein molecule in assembled complex micelles were also been investigated. It demonstrated that the casein/keratin(p I3.0) nano-complex micelles had more α-helix and β-sheet structure when it prepared at the mass raio of 4:1 for casein and keratin. It also had stably secondary structure and tertiary structure. The structure of the complex micelles remains stable in the range of p H6.0~11.0 and temperature 20~50℃.(5) Films were made by preparing complex micelles in lower concentration and concentrating to the high concentration. The results showed that the mechanical properties of the films were enhanced after assembly. Film breaking elongation and tensile strength increased by 38% and 32% respectively at the mass ratio of 4:1 after assembly. The tensile strength and surface hydrophobicity increased a little after assembled. Water vapor permeability, gas permeability, total content of solute and light transmittance was decreased after assembled. The assembly effect lead to the orderliness and densification of the film compared with the cross-section of the films un-assembled. The structure change effect was more obviously at the mass ratio of 4:1 for casein and keratin. It can be concluded that the performance of the film have great relationship with the sphere shape of the complex micelles.(6) Casein/keratin(p I3.0) complex nano-micelles were applied in embedding the hydrophobic β-carotene in the experiment. The drug-loading properties were studied preliminary. Data showed that the nano-micelles’ drug loading capacity was 35.3% at the mass ratio of 2:1 for β-carotene to complex micelles. It has sustained release function and the accumulated amount of drug released was 12.39% in gastric fluid in 16 hours. Meanwhile,the cumulative release amount was 3.08% in intestinal fluid in 16 hours.The complex micelles embedding β-carotene have better freeze-dried stability, dilution stability, gastrointestinal p H value stability and storage stability. |
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