Genetic variation and posttranslational modification of bovine κ-casein: Effects on caseinomacropeptide release during renneting

Chymosin-induced cleavage of kappa-casein (kappa-CN) occurs during the first enzymatic phase in milk coagulation during cheese manufacturing, where the hydrophilic C-terminal peptide of kappa-CN, named caseino-macropeptide (CMP), is released into the whey. The CMP peptide is known for its rather heterogeneous composition with respect to both genetic variation and multiple post-translational modifications, including phosphorylation and O-linked glycosylation. An approach of liquid chromatography coupled with mass spectrometry was used to investigate (1) the overall protein profile and (2) the release of various forms of CMP after addition of chymosin to individual cow milk samples from 2 breeds, Danish Jersey (DJ) and Danish Holstein-Friesian (DH). The cows were selected to represent distinct homo- and heterozygous types of the kappa-CN genetic variants A, B, and E (i.e., genotypes AA, BB, AB, EE, and AE). Initially, investigation of the protein profile showed milk with kappa-CN BB exhibited the highest relative content of kappa-CN, whereas AE milk exhibited the lowest, and after 40 min of renneting >90% of intact kappa-CN was hydrolyzed by chymosin in milk representing all kappa-CN genotype. By in-depth analysis of the CMP chromatographic profile, multiple CMP isoforms with 1 to 3 O-linked glycans (1-3 G) and 1 to 3 phosphate groups (1-3 P) were identified, as well as nonmodified CMP isoforms. The number of identified CMP isoforms varied to some extent between breeds (21 CMP isoforms identified in DJ, 26 CMP isoforms in DH) and between kappa-CN genetic variants (CMP variant A being the most heterogeneous compared with CMP B and E), as well as between individual samples within each breed. The predominant forms of glycans attached to CMP were found to be the acidic tetrasaccharide {N-acetyl-neuraminic acid alpha(2-3)galactose beta(1-3)[N-acetyl-neuraminic acid alpha(2-6)]N-acetyl galactose} or trisaccharides {N-acetyl-neuraminic acid alpha(2-3)galactose beta(1-3)N-acetyl galactose and galactose beta(1-3)[N-acetyl-neuraminic acid (alpha 2-6)]N-acetyl galactose}. The CMP release was calculated to follow first-order kinetics and was determined by the measurement of CMP content during renneting. The highest rate of release for all CMP isoforms occurred from 0 to 2 min after chymosin addition. Concurring results from both breeds showed that CMP variant A with 1-2 P had the highest reaction rate of CMP release, followed by CMP B 1-2 P and then by CMP E 1-2 P (only in DH). All the identified glycosylated CMP isoforms had lower reaction rates of release compared with that of nonglycosylated CMP, thus glycan modifications seemed to negatively influence the reaction rate of chymosin-induced hydrolysis of kappa-CN.