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 pre pared from fresh skim milk using an anion-exchange column. Peptides we re prepared by casein hydrolysis using a fluidized bed bioreactor cont aining 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 ste pwise elution with 0, 0.1, 0.2, 0.4, and 0.5 M KCl in equilibration bu ffer to separate the phosphopeptides from the other casein peptides. P rotein and P were measured in the elution peaks. Calcium binding to ea ch fraction was determined with a Ca-selective electrode. Electrophore sis showed that intact proteins were hydrolyzed rapidly, and peptides appeared on the gel in greater concentrations as the incubation time i ncreased. 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 1 2.8 kDa from alpha(S)-CN hydrolysate. The greatest yield and concentra tion 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 s howed greater Ca2+ binding than the phosphopeptides from beta-CN. Sepa ration of casein phosphopeptides using anion exchange was not specific . However, results showed that each peak containing high concentration s of phosphate had Ca2+- binding ability. Further characterization of these casein phosphopeptides might result in a Ca-complexing food ingr edient.