Heat-Induced Covalent Complex between Casein Micelles and β-Lactoglobulin from Goat's Milk: Identification of an Involved Disulfide Bond

Goat milk is characterized by a very low heat stability that could be attributed, in part, to the covalent interaction between whey proteins and casein micelles. However, the formation of such a complex in goat milk has never been evidenced. This study was designed to assess whether heat-induced covalent interaction occurs between purified casein micelles and β-lactoglobulin. We used a multiple approach of ultracentrifugation of heated mixture, chromatographic fractionation of resuspended pellets, sequential enzyme digestion of disulfide-linked oligomers, and identification of disulfide-linked peptides by on-line liquid chromatography−electrospray ionization mass spectrometry (LC−ESI/MS), and tandem MS. We identified three different types of disulfide links: (1) expected intermolecular bridges between β-Lg molecules; (2) disulfide bond involving two κ-casein molecules; and (3) a disulfide bond between two peptides, one from β-Lg and the other from κ-casein. The involved sites in this last bond were Cys160 of β-Lg and Cys88 of κ-casein. Although the identified heterolinkage is possibly only one of several different types, the results of this study constitute the first direct evidence of the formation of a covalent complex between casein micelles and β-lactoglobulin derived from goat milk. Keywords: Goat milk; β-lactoglobulin/κ-casein complex; enzyme digestion; disulfide bond; mass spectrometry