Identification and quantification of bovine protein lactosylation sites in different milk products

The microbiological safety of milk is typically guaranteed by thermal treatments, such as pasteurization and ultra high temperature (UHT) treatment, whereas infant formula (IF) is often produced at even harsher conditions including a drying process. Thermal treatments have raised concerns, as they may denature proteins and initiate protein modifications. Previous studies identified already many lactosylation sites in milk and showed that the lactosylation degree of some proteins correlates to thermal treatment conditions. Here, we studied the glycation degrees of 124 lactosylation sites in 28 bovine milk proteins in raw milk, three brands of pasteurized milk, three brands of UHT milk, and five brands of IF. Whereas, the glycation degree of many lactosylation sites increased from raw milk, to pasteurized milk, UHT milk, and IF, several modification sites showed a different behavior indicating that global measures do not correctly reflect the reactivity of distinct sites. Interestingly, the glycation degrees varied considerably among the brands of UHT milk and IF indicating that specific production processes of a company have to be considered and not only the classification of milk as pasteurized or UHT. Thus, proper adjustments of the technical processes should allow reducing the lactosylation levels in both UHT milk and IF.

It is well established that thermal treatment of milk triggers protein modifications, such as lactosylation of lysine residues in several proteins, although the extent of lactosylation has not been quantitatively compared for a broad panel of protein lactosylation sites among different commercial products. The current study extends previous reports by relatively quantifying 124 confirmed lactosylation sites in 28 bovine milk proteins including several low abundant proteins. Whereas, glycation is generally assumed to be an unspecific chemical reaction with the modification degrees depending on the protein and sugar concentrations, we could show that each protein and even each lactosylation site in a given protein is differently affected by thermal processes indicating that the global lactosylation degrees will not allow predicting the influence of a technical process on individual proteins and lactosylation sites. Additionally, we could show that brands of each milk product differ significantly in their glycation degrees with UHT milk brands for example spanning the whole range from the relatively low lactosylation degree of pasteurized milk to the rather high lactosylation degree of IF. Similar differences were obtained for IF that generally showed the highest glycation degree. The targeted quantification approach established and validated here will be useful to reveal technical processing steps that trigger individual lactosylation sites and thus can help to prevent such unwanted reactions. Even slight changes of the technical processes might allow reducing the lactosylation degree of milk proteins significantly without challenging the microbiological safety or affecting consumer behavior.