Characterization of Multiply Phosphorylated Peptides Selectively Precipitated from a Pancreatic Casein Digest

Abstract

Anticariogenic phosphopeptides, released during the hydrolysis of casein with trypsin, contain the cluster sequence Ser(P)-Ser(P)Ser(P)-Glu-Glu and have commercial potential as toothpaste, mouthwash, and food additives for the prevention of dental caries. To develop a commercial-scale process for the production of these peptides, we have comprehensively characterized casein phosphopeptides that were selectively precipitated using Ca2+ and ethanol from an acid-clarified (pH 4.6) pancreatic casein hydrolysate. Casein was hydrolyzed using pancreatin at 50°C for 2 h. The precipitate contained a series of casein phosphopeptides that were slightly truncated relative to tryptic casein phosphopeptides. The major casein phosphopeptides released by pancreatin were β-CN-4P(f7-24), αs1-CN-5P(f61–78), and aS1-CN-5P(f59–78), all containing the cluster sequence. The truncation of the tryptic peptides β-CN-4P(f1–25) and αS1-CN-5P(f59–79) resulted from the chymotryptic and carboxypeptidase activities of the pancreatin. The peptides containing the cluster sequence constituted 77.8 ± 6.7 mol/100 mol of the total peptides that were selectively precipitated. This composition was not significantly different from that of casein phosphopeptides produced under identical conditions using trypsin. In conclusion, pancreatin should be a suitable enzyme preparation for the production of anticariogenic casein phosphopeptides on a commercial scale. © 1995, American Dairy Science Association. All rights reserved.