Proteomic Analysis of Temperature-Dependent Changes in Stored UHT Milk

Abstract

Molecular changes in milk proteins during storage of UHT-treated milk have been investigated using two-dimensional electrophoresis (2-DE) coupled to MALDI-TOF mass spectrometry. UHT-treated samples were stored at three different temperatures, 4 °C, 28 °C, and 40 °C, for two months. Three main changes could be observed on 2-DE gels following storage. They were (1) the appearance of diffuse staining regions above the position of the monomeric caseins caused by nondisulfide cross-linking of α and β-caseins; (2) the appearance of additional acidic forms of proteins, predominantly of α(S1)-casein, caused by deamidation; and (3) the appearance of "stacked spots" caused by lactosylation of whey proteins. The extent of the changes increased with increased storage temperature. Mass spectrometric analysis of in-gel tryptic digests showed that the cross-linked proteins were dominated by α(S1)-casein, but a heterogeneous population of cross-linked forms with α(S2)-casein and β-casein was also observed. Tandem MS analysis was used to confirm deamidation of N(129) in α(S1)-casein. MS analysis of the stacked spots revealed lactosylation of 9/15 lysines in β-lactoglobulin and 8/12 lysines in α-lactalbumin. More extensive analysis will be required to confirm the nature of the cross-links and additional deamidation sites in α(S1)-casein as the highly phosphorylated nature of the caseins makes them challenging prospects for MS analysis.

Size Separation Techniques for the Characterisation of Cross-Linked Casein: A Review of Methods and Their Applications

“…Additionally, a high molar mass fraction appeared even under reducing conditions after heat treatment at 90 • C for 30 min, which was assigned to a trimer composed of α S1 -, α S2 -, and κ-casein formed as a result of non-disulphide cross-linking. This roughly corresponds to the findings of Holland et al [124] who, however, observed the formation of non-disulphide cross-links between α S1 -, α S2 -, and β-casein during storage of heat treated milk and attributed this to lysinoalanine formation. In the study of Le et al [125], polymer formation during storage of milk protein powders was correlated with the formation of particular Maillard reaction products.…”

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].…”

Electrophoretic characterization of protein interactions suggesting limited feasibility of accelerated shelf-life testing of ultra-high temperature milk

“…Production of ultra high temperature (UHT) milk involves heating of milk at a high temperature (usually 130-140 °C) for 3-5 seconds (s) followed by aseptic packaging to produce a 'commercially sterile' product with minimal changes in quality 1 . Thermal treatment enables storage of UHT milk at room temperature up to 9 months, thus, eliminating the requirement for cold chain and refrigeration facilities in the distribution chain.…”

“…Maillard products, dehydroalanine and deamidated proteins 1,[7][8][9] . However, the exact sequence and crosslinking mechanisms that lead to build up of sediment or gel is not known.…”

“…Besides increasing concentration of advanced Maillard products, higher storage temperatures also accelerate other protein crosslinks through lipid oxidation intermediates and end products (in case of WM), dehydroalanine, deamidated proteins and hydrophobic or other non-covalent interactions 22,23 . Dehydroalanine is produced by heat-induced elimination of phosphate from phosphoserine residues in caseins.…”