Investigation on the Formation and the Determination of γ-Glutamyl-β-alanylhistidine and Related Isopeptide in the Macromolecular Fraction of Beef Soup Stock

To confirm the formation of γ-glutamyl-β-alanylhistidine and related peptide, a model solution (amide-containing amino acids and carnosine) has been heated, and the products are investigated. Spectroscopical analysis indicates that the major product from asparagine and carnosine is β-aspartyl-β-alanylhistidine, and that from glutamine and carnosine is γ-glutamyl-β-alanylhistidine. Furthermore, to confirm the increase of the above peptides during the heating process of food, an HPLC method for the determination of these isopeptides in food protein is constructed. The isopeptides are liberated by proteolytic digestion and fractionated by solid-phase extraction using Toyopack IC-SP cartridges. The fraction containing the isopeptides is derivatized with phenylisothiocyanate (PITC) and separated and quantified by HPLC using an octadecyl−silica column. As a result of quantification, an increase of the γ-glutamyl-β-alanylhistidine isopeptide in the macromolecular fraction of heated beef soup stock solution has b...