Calcium Binding of Phosphopeptides Derived from Hydrolysis of a _s-Casein or ß-Casein Using Immobilized Trypsin

¹ Southeast Dairy Foods Research Center, Department of Food Science, North Carolina State University, Raleigh 27695-7624

Calcium binding to casein phosphopeptides that were derived from _s-CN or ß-CN was studied. Purified _s-CN or ß-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. _s-Casein and ß-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 ß-CN hydrolysates and a series of bands from 4.0 to 12.8 kDa from a _s-CN hydrolysate. The greatest yield and concentration of phosphate from ß-CN hydrolysate were found in the peak that eluted with 0.4 M KCl in equilibration buffer and for _s-CN in the peak that eluted with 0.1 M KCl. The _s-CN phosphopeptides showed greater Ca²⁺ binding than the phosphopeptides from ß-CN. Separation of casein phosphopeptides using anion exchange was not specific. However, results showed that each peak containing high concentrations of phosphate had Ca²⁺-binding ability. Further characterization of these casein phosphopeptides might result in a Ca-complexing food ingredient.

Key Words: casein • immobilized trypsin • casein phosphopeptide • calcium binding

Submitted on August 4, 1997
Accepted on July 13, 1998

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