Development of a Gel Assay for Analysis of Bovine Milk Casein

The main purpose of this research was to develop a gel assay for analysis of casein samples for ultimate application for studies of in vitro models for stomach and intestinal processing of dairy products in adults and infants. It is proposed that casein completely dissolves and is digestible in simulated gastric, and is somewhat soluble and digestible in simulated intestinal environments of adults and infants. The following experimental methods were devised to thoroughly examine behavior and properties of casein originating from different sources in range of solvents and varying pH values. First, casein was enriched in comparison to soluble materials by centrifugation from skim and whole milk samples, and then SDS-PAGE was utilized to analyze and compare skim milk casein, enzymatic hydrolysate casein, and whole and skim milk samples in a range of solvents to validate casein extraction. We found that skim and whole milk caseins are structurally the same and not influenced by fat content in milk, and that both experimental and commercial caseins dissolve well in urea binding buffer, SDS, and sodium hydroxide, and not well in acetic acid. Casein bands corresponded to values in the literature. We found that the SDS-PAGE results were highly dependent upon the initial solubility of casein. In acidic solutions, the casein formed curds, intractable material for the purpose of gel analysis. Subsequently, SDS-PAGE analysis and comparison was done on skim and whole milk caseins and casein standard solutions which were dissolved in sodium hydroxide to determine resolution of the gel in highly basic solution, a condition which would quench protease activity in our in vitro assay of pepsin and trypsin activity