Electrophoretic isolation of β-casein and optimization of a radial immunodiffusion test for bovine milk quality control

Abstract

Beta-casein (β-CN) is a major dairy protein subject to preferential degradation during storage. Our study aimed to isolate β-casein from bovine milk under dissociative conditions by the electrophoretic technique using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and optimize an immunochemical technique such as Mancini radial immunodiffusion in order to monitor the evolution of this milk protein during storage. For this purpose, a series of physicochemical and bacteriological analyses revealed conformity with international standards. This allowed us to use quality milk for more detailed studies of proteins, in particular β-casein, which was selected as a marker of milk protein quality. The total casein of mixed milk samples was isolated and then submitted to electrophoretic separation. Various combinations of acrylamide and bisacrylamide were used and the one corresponding to the ratio 8 g / 230 mg was retained. A total casein deposit of 833 µg provided an adequate b-casein concentration (300 µg) for consistent animal immunization. Thirty Wistar rats were subjected to an immunization protocol for 35 days. The obtained antisera were used to optimize the Mancini technique and to assay b-casein in our samples at different storage times ranging from 3 h to 69 h/+4°C. A decrease in b-casein content from 0.85 mg ml-1 to 0.32 mg ml-1 was detected. These results demonstrate the vulnerability of b-casein at low temperatures and provide information on the origin of milk and its application for dairy or cheese production. In addition, our study confirms the usefulness of immunochemical techniques such as the Mancini test in the determination of β-casein.