Development of alginate microparticles as efficient adsorption matrix for protein recovery

Highlights

• The alginate microparticles were successfully obtained by the spray drying technique.

• The crosslinked process was carried out by a covalent reaction with Epichlorohydrin.

• The crosslinked alginate microparticles were characterized by SEM, Zeta potential, TGA/DSC and Raman spectroscopy.

• The adsorption capacity of two model proteins were studied.

• The capacity of selection and recovery of crosslinked alginate microparticles was assayed.

Abstract

Alginate microparticles were prepared by spray drying technique and chemically cross-linked with epichlorohydrin. Microparticles were characterized by size, surface morphology (SEM), zeta potential, thermal analysis (TGA/DSC) and Raman spectroscopy. Microparticles cross-linked with epichlorohydrin were found to have averaged sizes of 648 nm, and zeta potential value of −84 mV. The adsorption capacity of the matrix was studied using Lysozyme and Chymotrypsinogen as model proteins. The equilibrium isotherms were described by the Langmuir and Hill models, respectively. The maximum adsorption capacities for Lysozyme and Chymotrypsinogen were 1880 and 3034 mg/g of Alg microparticle, respectively, which represent excellent results compared to the adsorption capacities showed by commercial resins and macro spheres of alginate. At the end, the capacity of selection and recovery of cross-linked alginate microparticles was assayed for both proteins.

Introduction

The adsorption process is frequently applied in the recovery of enzymes at macro scale level, under the methodology of stirred tank, packed bed or expanded bed [[1], [2], [3]]. The use of commercial beds is usually expensive, so it is becoming more common the use of beds obtained by environmentally friendly materials, such is the case of polysaccharides which are used as adsorption matrices. They, in turn, have the advantage to possess electrically charged groups which allow to increase the affinity of the bed to the enzyme of interest. In many cases, natural polyelectrolytes such as alginate (COOH), chitosan (NH₂), pectin (COOH), carrageenan (SO₃H), are used in as adsorption tolls because they can act as ion exchangers for themselves. Some advantages of using these matrices are: the ease to prepare and dispose of them into the environment without no negative impact as well as being cost-efficient. Generally, the beds used are spheres of 1–3 mm in diameter [4,5] obtained by ionotropic gelification, as is the case of sodium alginate, which has the property to form an insoluble complex in the presence of calcium ion [6,7]. Alginate, guar gum or chitosan beds have been obtained and used as affinity matrices for the isolation of enzymes due to their simplicity of preparation and easy handling [8,9]. In some cases, porous epichlorohydrin cross-linked alginate beads were used like adsorbent with high protein adsorption capacity [10,11]. Nevertheless, these authors present the capacity of adsorption per g of dry bed, and, the correct way to word this capacity would be by mass of hydrated gel. In that way, the real adsorption capacity would be minor that the adsorption capacity expressed in that works. In addition, it is known that when the particle size of the bead is reduced 100–500 times, its physical and chemical properties significantly change, increasing its adsorption capacity [12].

Microparticles are commonly described as solid colloidal particles, ranging in size above 1 μm [13]. For the generation of sodium alginate microparticles the spray drying technology has been used, that produces submicron particles from a solution. A traditional spray dryer is generally used to transform liquid substances into powders rapidly and efficiently. The speed of the process and the consequently short drying time enables the drying of even temperature sensitive products without degradation [14]. This spray drying process is particularly used to improve product conservation in dried solid form. With the development of active compound and emulsion encapsulation used in pharmaceutics, cosmetics and functional food preparation, this method has also been used for encapsulation purposes. The powder thus generated is a matrix system in the form of microparticles, exhibiting a spherical or hollowed morphology depending on the nature of the wall material used and the operational drying conditions such as the inlet temperature, solid concentration, gas flow rate or feed rate. The powder samples are generally heterogeneous and amorphous [15].

The aim of this study is to characterize physically the alginate microparticles obtained by spray drying technique before and after crosslinking them in a covalent way with epichlorohydrin. Then, to evaluate their adsorption capacity, two enzymes with biotechnology importance were adsorbed. To obtain this last track we have selected two proteins with positive electrical charge, Lysozyme and Chymotrypsinogen, as models. Finally, the efficiency of the cross-linked Alginate microparticles in the recovery of both model proteins from a mixture of protein was studied.