An economic and simple purification procedure for the large-scale production of ovotransferrin from egg white.

The objective of this study was to develop a simple and economical protocol for separating ovotransferrin from egg white. Egg white was separated from the yolk and diluted with the same volume of distilled water. To prevent denaturation during the separation process, ovotransferrin in 2x-diluted egg white was converted to its holo-form by adding 20 mM FeCl3.6H2O solution (0.25 to 3 mL/100 mL). The pH of egg white was adjusted to pH 7.0, 8.0, or 9.0, and NaHCO3 and NaCl were added to 50 mM and 0.15 M, respectively (final concentrations) to facilitate iron binding to ovotransferrin. The iron-bound ovotransferrin was separated from the egg white using different concentrations of ethanol (30 to 50%). Ethanol at 43% (final concentration) and pH at 9.0 were the best conditions for separating iron-bound ovotransferrin from 2x-diluted egg white solution. Almost all egg white proteins including ovalbumin were precipitated at 43% ethanol, but most of the iron-bound ovotransferrin remained in the supernatant. Holo-ovotransferrin in the 43% ethanol solution started to precipitate as the concentration of ethanol increased, but the optimal condition for precipitating ovotransferrin was when the ethanol concentration reached 59% (final). The precipitated holo-ovotransferrin was dissolved with distilled water, and AG1-X(2) ion exchange resin (at 3x iron content in ovotransferrin) was used to remove iron bound to ovotransferrin after pH adjustment to 4.7 using 500 mM citric acid. The apo-ovotransferrin obtained using this protocol was >80% in purity and around 99% in yield. The protocol developed is simple, economical, and appropriate for a large-scale production of ovotransferrin from egg white. Also, the isolated ovotransferrin can be applied in human foods, because the only solvent used in this process is ethanol. Furthermore, the AG1-X2 ion exchange resin and ethanol used in this process can be regenerated and recovered.