Preparation Of Egg Allergen Ovotransferrin And Lysozyme With High Purity And Process Design On Decagram Scale

Egg allergy has become one of public health issues in food safety,and it aroused people’s concern.Egg white contains four major allergens including ovomucoid,ovalbumin,ovotransferrin and lysozyme.Among them,ovotransferrin and lysozyme are widely used in food industry because of their significant biological functions.Therefore,the preparation of ovotransferrin and lysozyme will play an important role in egg allergy.The work aims to build a technology route for the separation and purification of ovotransferrin and lysozyme with high purity efficiently.The involved research include ① developing approach to purify ovotransferrin and lysozyme;②purification of ovotransferrin and lysozyme in lab-scale;③ to design a pilot-routine preparation of ovotransferrin and lysozyme.The main results and conclusions are presented as follows.1.An approach to purify ovotransferrin for laboratory preparation was achieved by using CM-Sepharose Fast Flow cation-exchange chromatography combined with Bio-Gel HT Hydroxyapatite.The SDS-PAGE analysis showed that the purity of ovotransferrin was 95%,and the yield was 0.167g.Anew strategy that CM-Sepharose Fast Flow cation-exchange chromatography combined with Sephacryl S-100 gel filtration was used to purify lysozyme.The SDS-PAGE analysis showed that the purity of lysozyme was 97%,and the yield was 0.125g.2.Based on the technical parameters obtained from the developing stage,8 times the scale of the previous preparation technique was set up for ovotransferrin purification.It was shown that the yield and the purity of OVT were 1.2 g and 97.5%,respectively.In respect to lysozyme,4 times the scale of the previous preparation technique was set up for lysozyme purification.The yield and purity of protein were 0.6g and 98%,respectively.3.The secondary structure and surface hydrophobicity of ovotransferrin and lysozyme derived from previous preparation and lab-scale purification were evaluated by CD and ANS fluorescent probe,respectively.It was shown that there are no changes in the secondary structure and surface hydrophobicity of proteins between them,and it revealed that the properties of the proteins were protected well during the purification.4.Based on the technical parameters obtained from lab-scale,a ten gram-scale preparing technical routine was designed for preparation of OVT and Lys including the selection of facilities,the plot of the charts for facilities rout.