A Magnetic Nanoparticle Based Method For Lactoferrin Separation

Lactoferrin (Lf) is an active natural glycoprotein with high iron affinity potentials whichmainly exists in colostrum and whey. The protein has many biological functions such asbroad-spectrum antibacterial acitivity, antiviral acitivity, anticarcinogenic activity andimmunomodulatory activity. Lf is in high demand because of its importance for infantformula milk powder. The traditional separation/purification methods, however, are complexand high-cost. Therefore, it is of great immportance to develop a simple and low-cost methodfor lactoferrin separation/purification. The magnetic nanoparticle basedseparation/purification method is a facile technique for the separation/purification of differentvarieties of proteins. In this study, a novel magnetic adsorbant was developed for Lfseparation/purification. The adsorption performance of the magnetic adsorbants was evaluatedusing bovine serum albumin (BSA) as a model protein. The magnetic adsorbants were finallyutilized for the separation of Lf from aqueous solution and from bovine whey. A magneticparticle based separation method with high purity of Lf was achieved. The main results fromthe study were as follows.1. Reacitive Red120was utilized to functionize the magnetic chitosan microspheres todevelop a novel magnetic adsorbants. The magnetic adsorbants were spherical and presented asmooth surface. The particle size was in the range of10~100μm. The immobilization amountof Reactive Red120on the surface of the magnetic adsorbant was19.99mg/g. The isoelectricpoint of the adsorbants was~6.5.2. The adsorption performance of the as-prepared magnetic adsorbants was evaluatedusing bovine serum albumin (BSA) as the model protein. The resusts showed that the mediumpH, ionic strength, adsorption time and initial concentration of BSA showed significant effecton the adsorbed amount of BSA: the maximum adsorbed amount of BSA appeared at pH5.0,the adsorbed amount of BSA decreased with the increase of ionic strength, the adsorbed BSAamount of the Reactive Red120modified magnetic chitosan microspheres was about5timeshigher than the unmodified microspheres, the adsorption behavior of magnetic adsorbant forBSA fitted pseudo second–order kinetics and Langmuir thermaldynamic model,0.5mol/LNaCl (pH8.0) was used as eluant and the eluted rate was99.03%. The adsorbed amount ofBSA on the magnetic adsorbant did not decrease significantly after5adsorption–desorptioncycles. 3. The magnetic adsorbants were utilized for the separation of Lf from aqueous solutionand from bovine whey. The results showed that the medium pH, ionic strength, adsorptiontime and initial concentration of Lf showed significant effect on the adsorbed amount of Lf inaqueous solutions. The maximum adsorbed amount of Lf appeared at pH7.0. The adsorbedamount of Lf decreased with the increase of ionic strength. The adsorption behavior ofmagnetic adsorbant for Lf fitted pseudo second–order kinetics and Langmuir thermaldynamicmodels.1mol/L NaCl (pH7.0) was used as eluant and the eluted rate was92.37%. Theadsorption process of the magnetic adsorbant for Lf was mainly based on physical adsorptionwith a rapid and endothermic reaction. The adsorbed amount of Lf on the magnetic adsorbantdid not decrease significantly after8adsorption–desorption cycles. The absorbants wasthereafter used for the separation of Lf from bovine whey. SDS–PAGE electrophoresis testshowed that the purity of the obtained Lf was higher than the standard Lf reagent (~90%) andthat the yield was~34.3%, indicating that the as-prepared magnetic adsorbant showed highselectivity for Lf in bovine whey, which can be used as a feasible method to separate Lf frombovine whey.