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 β(1-3)[N-acetyl-neuraminic acid α(2-6)]N-acetyl galactose} or trisaccharides {N-acetyl-neuraminic acid α(2-3)galactose β(1-3)N-acetyl galactose and galactose β(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.

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 β-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.

The initial stage of the Maillard reaction, protein lactosylation, occurs during heat treatment of milk and continues during subsequent storage. We compared the initial lactosylation as well as the rate of lactosylation of milk proteins during storage in UHT milk subjected to direct or indirect heat treatment using liquid chromatography (LC) coupled with electrospray injection mass spectrometry (ESI-MS). Furosine content was used as an overall marker to allow for a quantitative correlation of lactosylation measured by LC-ESI-MS in the UHT milks. Protein lactosylation increased during the storage period of 6 months at 20°C. Both the initial extent and the rate of lactosylation positively correlated with the number of lysine residues in the different proteins. An exponential or linear correlation with furosine concentration could be established for major and minor lactosylated proteins, respectively.

Milk that does not coagulate after rennet addition, also called noncoagulating (NC) milk, is unwanted in cheese production due to prolonged processing time. Amounts of whey and casein proteins, genetic variants, as well as posttranslational modifications (PTM) of proteins are all contributing factors in rennet-induced coagulation of milk. In this study, we conducted a wideranging investigation of milk proteins in milk samples from 616 Swedish Red dairy cattle using liquid chromatography-high resolution mass spectrometry. Relative concentration of proteins, genetic variants, and PTM were compared between NC milk and coagulating milk. The PTM investigated were phosphorylation of caseins and glycosylation of κ-casein. Several genetic variants and PTM were found, including rare phosphorylation variants of the α S -caseins. Genetic variants were found to effect the expressed amount of different proteins. Further, the effect of protein amounts and PTM on a binary NC milk trait was modeled using a generalized linear model. The model showed that NC milk significantly correlated with higher relative concentrations of α-lactalbumin and β-casein and lower relative concentrations of β-lactoglobulin and κ-casein. Regarding PTM of caseins, an effect on NC milk from a lower relative concentration of α S1 -casein with 8 phosphate groups were found, even though an effect from total relative concentration of α S1 -casein was not found. This study has provided insights into protein variants and PTM important for NC milk to improve this undesirable property.

Previous standards in the area of effect of heat treatment processes on milk protein denaturation were based primarily on laboratory-scale analysis and determination of denaturation degrees by, for example, electrophoresis. In this study, whey protein denaturation was revisited by pilot-scale heating strategies and liquid chromatography quadrupole time-of-flight mass spectrometer (LC/MC Q-TOF) analysis. Skim milk was heat treated by the use of 3 heating strategies, namely plate heat exchanger (PHE), tubular heat exchanger (THE), and direct steam injection (DSI), under various heating temperatures (T) and holding times. The effect of heating strategy on the degree of denaturation of β-lactoglobulin and α-lactalbumin was determined using LC/MC Q-TOF of pH 4.5-soluble whey proteins. Furthermore, effect of heating strategy on the rennet-induced coagulation properties was studied by oscillatory rheometry. In addition, rennet-induced coagulation of heat-treated micellar casein concentrate subjected to PHE was studied. For skim milk, the whey protein denaturation increased significantly as T and holding time increased, regardless of heating method. High denaturation degrees were obtained for T >100°C using PHE and THE, whereas DSI resulted in significantly lower denaturation degrees, compared with PHE and THE. Rennet coagulation properties were impaired by increased T and holding time regardless of heating method, although DSI resulted in less impairment compared with PHE and THE. No significant difference was found between THE and PHE for effect on rennet coagulation time, whereas the curd firming rate was significantly larger for THE compared with PHE. Micellar casein concentrate possessed improved rennet coagulation properties compared with skim milk receiving equal heat treatment.