RESUMO
Chymosin-induced cleavage of κ-casein (κ-CN) occurs during the first enzymatic phase in milk coagulation during cheese manufacturing, where the hydrophilic C-terminal peptide of κ-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 posttranslational 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 κ-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 κ-CN BB exhibited the highest relative content of κ-CN, whereas AE milk exhibited the lowest, and after 40min of renneting >90% of intact κ-CN was hydrolyzed by chymosin in milk representing all κ-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 (21CMP isoforms identified in DJ, 26CMP isoforms in DH) and between κ-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 α(2-3)galactose ß(1-3)[N-acetyl-neuraminic acid α(2-6)]N-acetyl galactose} or trisaccharides {N-acetyl-neuraminic acid α(2-3)galactose ß(1-3)N-acetyl galactose and galactose ß(1-3)[N-acetyl-neuraminic acid (α2-6)]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 2min 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 κ-CN.
Assuntos
Caseínas/genética , Bovinos/fisiologia , Quimosina/metabolismo , Variação Genética , Leite/química , Processamento de Proteína Pós-Traducional , Animais , Cruzamento , Caseínas/metabolismo , Bovinos/genética , Cromatografia Líquida , Feminino , Genótipo , Hidrólise , Espectrometria de Massas , Leite/enzimologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Isoformas de ProteínasRESUMO
Plasmin, the major indigenous protease in milk, is linked to quality defects in dairy products. The specificity of plasmin on caseins has previously been studied using purified caseins and in the indigenous peptide profile of milk. We investigated the specificity and proteolytic pathway of plasmin in directly heated UHT milk (>150 °C for <0.2 s) during 14 weeks of storage at 20 °C in relation to age gelation and bitter peptides. Sixty-six peptides from αS- and ß-caseins could be attributed to plasmin activity during the storage period, of which 23 were potentially bitter. Plasmin exhibited the highest affinity for the hydrophilic regions in the caseins that most probably were exposed to the serum phase and the least affinity for hydrophobic or phosphorylated regions. The proteolytic pattern observed suggests that plasmin destabilizes the casein micelle by hydrolyzing casein-casein and casein-calcium phosphate interaction sites, which may subsequently cause age gelation in UHT milk.