Lactosylated casein phosphopeptides as specific indicators of heated milks

Abstract

Casein phosphopeptides (CPP) were identified in small amounts in milks heated at various intensities by using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. CPP selectively concentrated on hydroxyapatite (HA) were regenerated using phosphoric acid mixed in the matrix. Unphosphorylated peptides not retained by HA were removed by buffer washing. This procedure enhanced the MALDI signals of CPP that are ordinarily suppressed by the co-occurrence of unphosphorylated peptides. CPP, belonging to the β-casein (CN) family, i.e., (f1-29) 4P, (f1-28) 4P, and (f1-27) 4P, and the α(s2)-CN family, i.e., (f1-21) 4P and (f1-24) 4P, were observed in liquid and powder milk. The lactosylated counterparts were specific to intensely heated milks, but absent in raw and thermized/pasteurized milk. Most CPP with C-terminal lysines probably arose from the activity of plasmin; an enzyme most active in casein hydrolysis. A CPP analogue was used as the internal standard. The raw milk signature peptide β-CN (f1-28) 4P constituted ~4.3% of the total β-CN. Small amounts of lactosylated peptides, which varied with heat treatment intensity, were detected in the milk samples. The limit of detection of ultra-high-temperature milk adjunction in raw or pasteurized milk was ~10%.

Bioactive Casein Phosphopeptides in Dairy Products as Nutraceuticals for Functional Foods

“…Native and lactosylated forms of -CN (f1-28)4P, (f1-27)4P and s2-CN (f1-24)4P, although typical of UHT milk and milk powder, are missing in raw, pasteurized milk (71.7 °C for 15 s) and intensely pasteurized milk. The lactosylated peptides that varied with heat treatment characterize UHT milk added in amounts not lower than 10% to raw and pasteurized milk [102]. Milk delactosed with microbial -galactosidase did not suppress the Maillard reaction; indeed, the furosine concentration increased to 35-400 mg/100 g of protein [103].…”

“…Few CPP were less phosphorylated than the native peptides due to the presence of milk phosphatase, which was denatured in all pasteurized cheese-milks. The presence of lactosylated -CN (f1-28)4P CPP was indicative of yogurt made with high-heat treated or milk fortified with milk protein powder [102]. Proteolysis of milk proteins in model yogurt systems has shown a similar set of primary CPP (Table 5).…”

“…In yogurt, enzymes could give rise to the liberation of a particularly high number of bioactive peptides, among them CPP, which could be partly due to LAB proteolytic activity. Comparison of CPP in raw, pasteurized and intensely heated milks has previously shown that there is a plethora of milk peptides among which a few were glycosylated CPP [102]. Yogurt is prepared from intensely heated milk instead of low-pasteurized drinking milk.…”

“…As reported above, a number of available techniques allow the separation of CPP and non-CPP. To reduce the large dynamic range of non-CPP, HA was used for CPP enrichment [90,94,102]. CPP included multiply phosphorylated peptides (up to 4 phosphorylation residues).…”

Farm animal milk proteomics

“…Pinto in a recent work showed that casein lactosylation is a function of the heating intensity [148]. Holland described temperature dependent molecular changes in milk proteins (non-disulfide cross-linking, deamidation and lactosylation) during storage of UHT-treated milk using 2-DE coupled to MALDI-TOF MS [149].…”