Preparation Of Magnetic Chitosan Microspheres And Adsorption On Angiotensin Converting Enzyme Inhibitory Peptides

Angiotensin converting enzyme inhibitory peptides(ACEI) can decrease the blood pressure via inhibiting the angiotensin-converting enzyme activity. It is a very tedious and time-consuming process to isolation and purification ACEI from natural products. In this paper, magnetic chitosan microspheres were chelated by the Cu2+to be affinity media because of having good biocompatibility. The VSLPEW which come from the a-lactalbumin hydrolyzate system was used as model molecules to study the adsorption of three affinity media for VSLPEW, in order to provide a theoretical basis for the separation and purification of ACEI.Three kinds of magnetic chitosan microspheres in different sizes were prepared by emulsion cross-linking method, they were Small size Magnetic Chitosan Microspheres(S-MCS), Middle size Magnetic Chitosan Microspheres(M-MCS) and Large size Magnetic Chitosan microspheres(L-MCS) respectively. The optimal preparation conditions for preparation of S-MCS were:mass ratio of chitosan and Fe3O4was1:1.5, glutaraldehyde amount was1mL, stirring rate was800r·min-1, reaction time was4h. The morphology and properties of the three magnetic chitosan microspheres of different particle sizes were characterized, it tells that the average particle size of S-MCS was283nm, M-MCS was1.3μm, L-MCS was15μm. Three kinds of magnetic chitosan microspheres are all showing with good superparamagnetic, Fe3O4contents of three kinds of magnetic chitosan microspheres were39.8%,29.6%,40.6%respectively, saturation magnetization were31.35emu·g-1,25.50emu·g-1,42.06emu·g-1.Three kinds of magnetic chitosan microspheres were modified epoxy groups by epichlorohydrin, and the optimal conditions for activation were: temperature of activation was40℃, volume fraction of epichlorohydrin was50%, time of activation was4h, concentration of NaOH solution was0.5mol·L-1. Under these condition, epoxy-density of three kinds of magnetic chitosan microspheres can reach251.51μmol·g-1,275.83μmol·g-1,294.53umol·g-1respectively. Three kinds of magnetic chitosan microspheres were modified by iminodiacetic acid (IDA), and the optimal conditions for reaction were:IDA amount was0.1g, time of chelation was8h, the temperature of chelation was40℃. Under these conditions, the density of Cu2+of three kinds of magnetic chitosan microspheres were211.03μmol·g-1,236.63μmol·g-1and271.51μmol·g-1respectively.VSLPEW which was founded from milk-derived protein was used as model molecules to study the adsorption of three affinity media for VSLPEW, the optimal conditions of adsorption for VSLPEW were:the initial concentration of VSLPEW was2.0mg·mL-1, time of adsorption for VSLPEW was40min, temperature of adsorption for VSLPEW was30℃, pH of buffer was7.5. Under the optimal conditions, the saturated adsorption of three kinds of magnetic affinity media were36.38mg·g-1,51.28mg·g-1,58.56mg·g-1. The adsorption model of three kinds of affinity media for VSLPEW were also investigated. The results showed that:The adsorption of VSLPEW on three kinds of magnetic affinity media accorded with Langmuir model description; The adsorption kinetics of VSLPEW on three kinds of magnetic affinity media consistent with Pseudo second-order kinetic adsorption kinetics.Finally, the adsorption of L-MCS affinity media for VSLPEW in mixed system conteined BSA and VSLPEW were researched. The results showed that the adsorption of L-MCS affinity media for VSLPEW were influenced by macromolecules and adsorption amount were decreasing with concentration decreased. It indicated that the application of affinity media for ACEI should take the interference of impurity molecules into account.