Calcium binding of phosphopeptides derived from hydrolysis of alpha s-casein or beta-casein using immobilized trypsin.

Calcium binding to casein phosphopeptides that were derived from alpha s-CN or beta-CN was studied. Purified alpha s-CN or beta-CN was prepared from fresh skim milk using an anion-exchange column. Peptides were prepared by casein hydrolysis using a fluidized bed bioreactor containing 2 ml of immobilized trypsin (activity: 49.4 U/g of beads). The disappearance of intact protein and the appearance of products of low molecular mass were monitored by SDS-PAGE. alpha s-Casein and beta-CN hydrolysates were loaded on an anion-exchange column, followed by stepwise elution with 0, 0.1, 0.2, 0.4, and 0.5 M KCl in equilibration buffer to separate the phosphopeptides from the other casein peptides. Protein and P were measured in the elution peaks. Calcium binding to each fraction was determined with a Ca-selective electrode. Electrophoresis showed that intact proteins were hydrolyzed rapidly, and peptides appeared on the gel in greater concentrations as the incubation time increased. The major products were a main band with a molecular mass of 6.2 kDa from beta-CN hydrolysates and a series of bands from 4.0 to 12.8 kDa from alpha s-CN hydrolysate. The greatest yield and concentration of phosphate from beta-CN hydrolysate were found in the peak that eluted with 0.4 M KCl in equilibration buffer and for alpha s-CN in the peak that eluted with 0.1 M KCl. The alpha s-CN phosphopeptides showed greater Ca2+ binding than the phosphopeptides from beta-CN. Separation of casein phosphopeptides using anion exchange was not specific. However, results showed that each peak containing high concentrations of phosphate had Ca(2+)-binding ability. Further characterization of these casein phosphopeptides might result in a Ca-complexing food ingredient.