Prepration Of The Certified Reference Material Of A-Lactalbumin Andβ-lactoglobulin, The Major Allergens In Bovine Milk

Milk is an popular drink liked by most people,which contains rich protein. At the same time,bovine milk has received widespread attention for its sensitization. There are almost8%of new-born babies have allergic reaction to milk proteins. To avoid milk allergy, people need to prevent themselves from contacting with milk or milk products. It is particularly important to improve the detection and diagnosis methods, when dealing with food allergy. They both require a well defined measure standard. There is no doubt that the development of food allergen certified reference material(CRM) is of great significant to solve the probelm of food allergy.In this work, we focus on the preparation of the certified reference material of a-Lactalbumin and β-lactoglobulin, the major allergens in bovine. The main studies include, small scale purification of CRM bovine a-Lactalbumin and P-lactoglobulin, large scale purification of CRM bovine a-Lactalbumin and (3-lactoglobulin, the assessment of physicochemical and immunological properties. The main research methods, results and conclusions are described as follows.1. The bovine a-lactalbumin and β-lactoglobulin were purified by Sephadex G-75gel chromatography and DEAE-Sepharose Fast Flow anion exchange chromatography. The SDS-PAGE show that purity of a-lactalbumin and P-lactoglobulin were up to97%and99%,respectively. And their purified yield was28.18%and30.76%, respectively.2.8times the scale of previous preparation protocol was set up for a-lactalbumin and β-lactoglobulin separation. The proteins were identified by SDS-PAGE, and it was show that the purity of a-lactalbumin was99%and the purified yield was28.67%. Besides, the purity and purified yield of β-lactoglobulin was99%and33.93%, respectively. A whole process can obtain0.4g of a-lactalbumin and O.87g of β-lactoglobulin. Based on the parameter obtained from this, a pilot-scale preparing technical routine was designed for preparation of ALA and BLG.3. The lyophilized preparation of α-lactalbumin and P-lactoglobulin derived from lab-scale were identified by Mass Spectrometry, and the SDS-PAGE conjunction with silver staining show that the purity of two reference material candidate were both above99%. The second structure and hydrophobic were characterized by spectroscopic methods, and the specific IgE-binding capacity was evaluated by immunological technique. Besides, homogeneity and stability of lyophilized allergens were assessed. The results demonstrated the between-bottle homogeneity of the two reference material candidate. And no obvious trend was observed during the monitoring period in two months.