Hexose-derived glycation sites in processed bovine milk

Abstract

Milk products are consumed by many people on a daily basis, which demands sophisticated technical processes to guarantee the microbiological safety and to retain the nutritional value. The heating during pasteurization and ultra high temperature (UHT) treatment triggers diverse chemical reactions, such as the reaction of sugars and amino groups of proteins typically termed protein glycation. The glycation by lactose as dominant sugar in milk has been recently investigated, whereas the contribution of hexoses remains open. We identified first hexose-derived glycation sites in raw milk, colostrum, three brands of pasteurized milk, three brands of UHT milk, five brands of infant formula, and one brand of lactose-free pasteurized and UHT milk using tandem mass spectrometry and electron transfer dissociation. In total, we could identify 124 hexosylated tryptic peptides in a bottom-up proteomics approach after enriching glycated peptides by boronate affinity chromatography, which corresponded to 86 glycation sites in 17 bovine milk proteins. In quantitative terms glycation increased from raw milk to pasteurized milk to UHT milk and infant formula. Lactose-free milk contained significantly higher hexosylation degrees than the corresponding regular milk product. Interestingly, the glycation degrees varied considerably among different brands with lactose-free UHT milk and infant formula showing the highest levels.

Biological significance: The established proteomics strategy enables the identification and relative quantification of different protein glycation types in diverse milk products ranging from raw milk to milk powders. This will allow detailed in vitro studies to judge positive or negative aspects when consuming differently processed milk products including lactose-free milk that is obligatory for people with lactose intolerance but is increasingly consumed by the general population assuming health benefits. The established analytics will also permit studying the influence of each technical processing step on the glycation degrees and thus offers the possibility to reduce glycation early during production, as obvious from the variation among different brands. Special attention should be given to the high hexose- and lactose-derived glycation levels found in infant formula, although it is still controversially discussed if protein glycation has a negative biological impact.

Keywords: Boronate affinity chromatography (BAC); Electron transfer dissociation (ETD); Infant formula; Lactose-free milk; Maillard reaction; Non-enzymatic glycosylation.