Preparation And Application Of Functional Magnetic Nanoparticles In Food System

Since it possesses the magnetic responsively, as well as the functionality, the functional magnetic nanoparticles can be convenient, fast separation and purification of the target biomolecules from reaction system with the help of magnets, it has extensive application prospect in biotechnology, biomedicine, food industry, and so on. At present, exploring the functionalization of magnetic nanoparticles and its practical application is the important research direction for magnetic nanoparticles. This dissertation aimed at finding out new techniques in protein separation and enzyme immobilization which using the prepared novel magnetic nanoparticles as Nano carriers, the practical bio-system was chosen as the application system, and obtained some innovative research results. The main research results are as follows:1. Using polyethylene glycol 6000 (PEG 6000) as dispersant, the functional superparamagnetic nanoparticles which conjugated with carboxymethyl chitosan (CM-CTS) were developed (shorted as Fe₃O₄ (PEG + CM-CTS)); the obtained Fe₃O₄ (PEG + CM-CTS) with uniform size, its diameter was about 15 nm, furthermore, the dispersing behavior of Fe₃O₄ (PEG+CM-CTS) nanoparticles has obviously been improved in comparison with the naked Fe₃O₄; On this basis, through electrostatic interaction, the functional Fe₃O₄ (PEG+CM-CTS) nanoparticles was used as a novel magnetic absorbing carrier for the separation and purification of lysozyme from the aqueous solution and chicken egg white respectively, the purity of lysozyme which purified from chicken egg white was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS?PAGE), it indicated that: The lysozyme which purified from chicken egg white in a single step had higher purity; Considering that the Fe₃O₄ (PEG+CM-CTS) nanoparticles possesses the advantages of high efficiency, cost-effectiveness and excellent binding of a larger amount of lysozyme and easier separation from the reaction system, thus this type of superparamagnetic nanoparticles would bring advantages to the conventional separation techniques of lysozyme from chicken egg white.2. An fundamental understanding of the conformational behaviors of lysozyme during the process of adsorption and desorption has been studied using Fourier transform infrared spectroscopy (FTIR), circular dichroic (CD) and fluorescence spectroscopy, and it indicated that: on the surface of Fe₃O₄ (PEG+CM-CTS) nanoparticles, the lysozyme would adopt a more compact conformation state; a complex between lysozyme and the Fe₃O₄ (PEG+CM-CTS) nanoparticles has been formed, Trp 62 and Trp108 mol/Light be the possible site of binding of lysozyme to Fe₃O₄ (PEG+CM-CTS) nanoparticles; Lysozyme which desorbed by PBS (20 mmol/L, pH 8.0, 0.2 mol/L NaCl) and PBS (20 mmol/L, pH 8.0, 0.5 mol/L NaCl) retain high fraction of its native structure with negligible effect on its activity, and about 92.4% and 89.5% activity were retained upon desorption from nanoparticles, however, lysozyme desorbed by acetic acid solution (0.2 mol/L, pH 4.0) showed significant conformational changes, only about 24.7% activity were retained; Due to the small diameter, the Fe₃O₄ (PEG+CM-CTS) magnetic nanoparticles has favorable properties to ensure highest level of conformational stabilization of protein compared to the larger particles of the same material, so it may find useful applications in biotechnology ranging from enzyme immobilization to protein purification.3. The immobilized trypsin which absorbed on the surface of the Fe₃O₄ (PEG+CM-CTS) nanoparticles was prepared and applied for fast and efficient proteolysis. FTIR and fluorescence spectroscopy data demonstrated that the magnetic Fe₃O₄ (PEG+CM-CTS) nanoparticles were capable of improving conformation stability of trypsin and preventing the trypsin unfolding; the immobilized trypsin, compared with native trypsin, has less affinity to the substrate, however, the immobilized trypsin could be used for fast and efficient Bovine Serum Albumin (BSA) digestion under very facile processes, thanks to the easy manipulation of the magnetic nanoparticles, as well as the greatly reduced digestion time (from 12 hr to 15 min); These results are expected to open up a new possible application of the trypsin-Fe₃O₄ (PEG+CM-CTS) conjugates in proteome research as well as a great potential use of such Fe₃O₄ (PEG+CM-CTS) nanoparticles as a superior nanosupport for the immobilization of trypsin.4. In the research, Fe₃O₄ (PEG+CM-CTS) nanoparticles were treated with Cu (II), Fe (III) and Zn (II) ions solutions to obtain immobilized metal affinity magnetic nanoparticles (IMANs) (short as IMAN @ Cu (II), IMAN @ Fe (III) and IMAN @ Zn (II)). The as-prepared IMANs were conveniently applied for lysozyme adsorption; The maximum equilibrium adsorption capacity of IMAN @ Cu (II), IMAN @ Fe (III) and IMAN @ Zn (II) nanoparticles to lysozyme were calculated to be 185.19 mg/g, 232.56 mg/g and 200 mg/g, respectively; Fluorescence analysis demonstrated that IMAN @ Zn (II) and IMAN @ Fe (III) nanoparticles may not cause the conformational change and denaturation for the lysozyme, but the lysozyme which desorbed from IMAN @ Cu (II) underwent some subtle change in comparison with the original lysozyme.5. IMAN @ Fe (III) were conveniently applied for phosvitin phosphopeptides (PPPs) enrichment for the first time, the research indicated that: IMAN @ Fe (III) nanoparticles showed greater specificity for PPPs enrichment (N/P can be reduced from 39.49 to 5.52); Since the separation process is convenient, economic and efficient, this type of IMAN @ Fe (III) nanoparticles would bring advantages to the conventional separation techniques of PPPs from chicken egg yolk; In addition, the chosen IMAN @ Fe (III) nanoparticles not only showing potential application in PPPs separation from chicken egg yolk, but also in phosphopeptides enrichment in proteomics research.In summary, the main contribution of the dissertation is that we initially synthesized the functional magnetic nanoparticles and successfully utilized them for protein separation and enzyme immobiliztation; we aimed at exploring and finding out new techniques in lysozyme separation from chicken egg white, PPPs enrichment from chicken egg yolk, trypsin immobilization and its efficient proteolysis.