The determination of casein micelle size distribution in skim milk by chromatography and photon correlation spectroscopy

Abstract Exclusion chromatography on a controlled-pore glass column was used to fractionate the casein micelles in shim milk according to size. To fix the size distribution, skim milk was treated with glutaraldehyde before being loaded on the column. Column profiles from absorbance measurements at 280 nm and 340 nm showed three peaks: a1) material excluded by the column, which was identified as membrane material plus very large micelles, (b) smaller micelles, and (c) whey proteins plus the smallest casein micelles and soluble casein. The average size of the particles in each casein micelle fraction was determined by photon correlation spectroscopy; for the purposes of comparison measurements were obtained from two photon correlation spectrometers of very different levels of sophistication. The relative concentration of protein was obtained from the absorbance at 280 nm, after digestion with pronase to clarify the turbid casein micelle suspension. The combination of size and concentration data gave a casein micelle size distribution. This approach would be useful in the identification of individual proteins separated by other chromatographic methods.