Preparation Of Cow's Milk Allergen β-Lactoglobulin With High Purity On Gram Scale

Milk and milk products are nutritious food containing numerous essential nutrients which are consumed by many people. However, milk proteins belong to one of the common food allergens. Milk allergy affects as high as 8% of children and 1% of adult population, resulting in severe impact on quality of life. Therefore, the investigation on milk allergy may help to better understand it.Milk contains various allergens, mainly including casein, a-lactalbumin andβ-lactoglobulin. Here we tookβ-lactoglobulin as an example, and design a preparing technical route to purify it at a high-purity and a high yield. Based on the purification parameters from laboratory work, a large design for pilot-scale preparation of P-lactoglobulin was performed. The purified P-lactoglobulin at pilot-scale was characterized by physiochemical and immunological methods. The main research results and conclusions are presented as follows.1. A simple approach to purifyβ-lactoglobulin for laboratory preparation was achieved using Sephadex G-75 gel filtration combined with DEAE-Sepharose Fast Flow anion-exchange chromatography. The analysis of SDS-PAGE shows that the purity of P-lactoglobulin was 99%,and the recovery-ratio was 35.14%。2. On the basis of the preparing parameters forβ-lactoglobulin in small scale,using Sephadex G-75 gel filtration and DEAE-Sepharose Fast Flow anion-exchange chromatography, we carried out a design to enlarge the purification progress including facility choosing, flow chart for processing technology and equipment.3. The progress technology for pilot-scale of preparingβ-lactoglobulin was tested. The test has been done for two times to verify the stability of preparing parameters, and make this technique reliable.4. The characterizations of purified P-lactoglobulin at pilot-scale were defined by SDS-PAGE, CD and ELISA. The analysis of SDS-PAGE shows that the purity of P-lactoglobulin was 99.7%, and the recovery-ratio was 38.02%, and the CD chromatograph shows that the second structure is almost the same between the purifiedβ-lactoglobulin from both the laboratory and pilot-scale preparing. The inhibition ELISA test was similar between both of this two purifiedβ-lactoglobulin, indicating pilot-scale preparing; can preserve antigenicity well.5.The pilot-scale preparing designing is successfully completed, and it can meet the need of preparingβ-lactoglobulin with high purity on gram-scale.