Preparation Of Magnetic Chitosan Microspheres And Its Application On Acei Purification

Saurida elongate was one of the main economic fishes in our country with large annual production, and it had been reported that its protein hydrolysate contained multiple in vitro angiotensin converting enzyme inhibitory peptides(ACEI). However it was a complex process to separate ACEI from the hydrolysate. In this paper, magnetic chitosan microspheres coupling pig lung ACE were applied to Saurida elongate hydrolysate for ACEI separation and purification through affinity adsorption, aiming to provide a certain technical support and theoretical basis for ACEI purification.Magnetic chitosan microspheres were prepared via suspension cross-linking method, and factors influencing the preparation were investigated, finally the optimal conditions for preparation were:HAc concentration was3%(V/V), chitosan concentration was10mg·mL-1, ratio of W/O was1:6, stirring rate was800rpm. Also, the properties of magnetic chitosan microspheres were characterized, the results showed that the average diameter of microspheres was901nm with homogeneous size, Fe3O4magnetic nanoparticles were successfully wrapped up in the composite microspheres and its content was28%. Magnetic chitosan microspheres showed up to be superparamagnetism with good magnetic responsivenesscan, which could be separated fast from solution.Magnetic chitosan microspheres were modified with epoxy groups by epichlorohydrin, and factors influencing the modification were investigated, the optimal conditions were:NaOH concentration was0.9mol·L-1, epichlorohydrin concentration was40%(V/V), NaBH4concentration was0.5g·L-1, reaction temperature was40℃, reaction time was4h. Under the optimal condition, density of epoxy groups can reach159.23μmol·g-1, which indicated that magnetic chitosan microspheres could be used for enzyme immobilization. Magnetic chitosan microspheres modified with epoxy were used for ACE immobilization, and the optimal conditions were investigated to be as follow: pH was8.3, reaction temperature was50℃, reaction time was1.5h, protein concentration was6mg·mL-1, under these conditions, immobilized enzyme had a large activity of0.048U·g-1. Compared with free ACE, the pH stability, thermal stability of immobilized ACE were improved, with good repeated operation stability and storage stability.Applying Immobilized ACE magnetic chitosan microspheres as affinity carriers, adsorption experiments for ACEI of Saurida elongate hydrolysate was carried out. Research results showed that after adsorption, the ACE inhibitory activity of hydrolysate increased from71.5%to82.06%. High performance liquid chromatography(HPLC) analysis showed that chromatographic results were in accordance with the ACE inhibitory activity test results, indicating that magnetic chitosan affinity carriers can be applied to ACEI rapid separation and purification of Saurida elongate hydrolysate.