Identification and Site-Specific Relative Quantification of β-Lactoglobulin Modifications in Heated Milk and Dairy Products

Abstract

During milk processing, proteins can be severely modified by oxidation, condensation, and Maillard reaction, leading to changes in their nutritional and technological properties. In this study, major modifications of beta-lactoglobulin, formed during the heating and processing of milk, were screened by mass spectrometry. For this purpose, beta-lactoglobulin was isolated from the milk samples by gel electrophoresis and analyzed by matrix-assisted laser desorption/ionization mass spectrometry after in-gel digestion with endoproteinase AspN. In heated milk, lactulosyllysine was detected at lysine 47 and 138 or 141 as well as methionine sulfoxide at methionine 7, 24, and 145. All these modifications increased gradually when raw milk was heated for 20, 40, and 60 min at 120 degrees C. The major modifications were also relatively quantified in dairy products, such as raw, high-temperature, ultra-high-temperature, sterilized, and condensed milk as well as infant formulas. The highest contents of lactulosyllysine at Lys47 were detected in powdered infant formulas, whereas lactulosyllysine at Lys138/141 was predominant in condensed milk samples. Methionine sulfoxide at Met7 and Met24 showed a trend toward higher modification rates in more severely processed products.

Assessment of heat treatment of dairy products by MALDI‐TOF‐MS

“…MALDI-TOF-MS has been used successfully to analyze the protein profile of milk or to detect lactosylation of whey proteins in milk models [8,26]. When applied after partial enzymatic digestion, MALDI-TOF-MS can be efficiently used to analyze structure and binding site of protein modifications formed during the heat treatment of milk [7,25]. MALDI-TOF-MS, however, has also been applied to detect protein lactosylation in heated milk.…”

“…This method was applied, for example, to record productand site-specific glycation kinetics. Furthermore, site-specific relative quantification was applied according to a similar protocol to commercial dairy products [7]. In the present study, the relative quantification of lactulosylysine modifications of intact proteins in heated milk was performed as a very fast and easy method to monitor the formation of early glycation products.…”

“…During prolonged heating, further glycation products can be formed such as N e -carboxymethyllysine, oxalic acid monolysinylamide, pyrraline, or pentosidine [3][4][5][6]. Furthermore, heating in the presence of lactose can induce protein oxidation leading to the formation of methionine sulfoxide, aminoadipic semialdehyde, or dityrosine [7][8][9][10]. Finally, protein modifications can occur, which are formed independently from the presence of lactose, such as lysinoalanine, histidinoalanine, N-terminal pyrrolidone, or partial protein hydrolysis [11][12][13].…”

Identification of aminoreductones as active components in Maillard reaction mixtures inducing nuclear NF-κB translocation in macrophages

“…DFLys was chosen to represent Amadori products, which are the first relatively stable reaction products of sugars with amines. In model mixtures of proteins and sugars, as well as in processed protein rich food, the Amadori products are usually the adducts which can be detected in highest concentrations [28][29][30]. Furthermore, Amadori products are already present in lightly processed diet, so that their consumption seems to be inevitable [31].…”

Proteomics in Food Science

“…The structure and binding sites of six major protein modifications formed in milk and dairy products caused by glycation, oxidation, and condensation reactions were characterized using electrophoresis and MALDI-TOF PMF (Meltretter et al, 2008). Johnson et al (2011) developed a method that couples size exclusion chromatography with ESI-MS to characterize processing-induced changes such as aggregation and Maillard glycation in milk proteins.…”