Maillard reaction and protein cross-linking in milk powders during storage

Thao Thi Thu Le (2011). Maillard reaction and protein cross-linking in milk powders during storage PhD Thesis, School of Agriculture and Food Sciences, The University of Queensland.

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Author Thao Thi Thu Le
Thesis Title Maillard reaction and protein cross-linking in milk powders during storage
School, Centre or Institute School of Agriculture and Food Sciences
Institution The University of Queensland
Publication date 2011-12
Thesis type PhD Thesis
Supervisor Prof. Hilton Deeth
Prof. Bhesh Bhandari
Dr. John Holland
Total pages 245
Total colour pages 31
Total black and white pages 214
Language eng
Subjects 060109 Proteomics and Intermolecular Interactions (excl. Medical Proteomics)
090899 Food Sciences not elsewhere classified
Abstract/Summary The solubility and chemical changes due to the Maillard reaction (MR) and protein cross-linking were investigated in milk powders during storage. Furosine, hydroxymethylfurfural (HMF) and brown coloration were used as indicators of different stages of the MR in the powders. Proteomic techniques were used to analyse protein changes in more detail and to investigate the mechanism of protein cross-linking in stored powders. There was an increase in the amount of MR products and a decrease in solubility of milk protein concentrate powder containing 80% protein (MPC80) during storage at different temperatures and relative humidities (RHs). The relationship between the MR and solubility loss was explored in MPC80 modified by addition of 2% glucose or lactose and adjustment of pH. MPC80 with added-glucose formed more furosine and brown pigments than MPC80 with added-lactose as glucose reacts faster than lactose with lysine in the MR. The opposite trend was clearly seen for solubility, suggesting that the MR may be a cause of solubility loss. However, altering the pH of MPC80 did not give a clear trend in solubility or MR changes. Overall, there was a positive correlation between MR indicators and solubility, suggesting that chemical changes to the proteins are closely related to solubility changes. Protein changes in relation to solubility and the MR in whole milk powder (WMP), skim milk powder (SMP) and whey protein concentrate containing 80% whey proteins (WPC80) were also investigated. The changes were compared with those observed for MPC80, under the same storage conditions, in the above study. WMP and SMP reached minimum solubility very quickly, especially at high RH. WPC80 showed no change in solubility while MPC80 solubility decreased gradually, again more rapidly at high RH. The minimum solubility closely corresponded with the casein content (and fat in the case of WMP). The effect of relative humidity on maximal MR rate differed in different types of milk powder. The high retention of solubility of WPC80 compared with the other powders which contain casein suggests that the whey proteins play a minor role, if any, in the solubility decrease in milk powders under the conditions studied. In order to analyse changes in the protein of stored milk powders at the molecular level, a proteomic technique combining two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) was used. Lactosylation, deamidation and protein cross-linking were observed on the 2-DE gels; they were time-, humidity- and storage temperature-dependent. Lactosylated whey proteins were well separated on 2-DE in vertical stacks of spots. The spots had masses which varied by multiples of 324 indicating the sequential attachment of lactose to lysine residues in the proteins. In addition, protein cross-linking was observed on 2-DE gels by the presence of high-molecular-weight protein complexes in all milk powders except WPC80. The development of these high-molecular-weight proteins was closely related to the loss of solubility in milk powders. The lactosylated forms of α-lactalbumin, which were separated as individual stacked spots by 2-DE, were quantified by image analysis. The normalised lactosylation levels showed a good correlation with furosine levels. The image analysis method was most suitable for powders stored under relatively mild conditions. In addition, a multiple reaction monitoring (MRM) method was developed from a knowledge of peptide fragmentation. The neutral losses of 162 Da (cleavage of galactose) and 216 Da (the formation of furylium ion) represent lactosylated peptides and were specifically selected as MRM transitions. Quantification of lactosylated protein was based on the peak areas of these fragmentation ions. The MRM results showed an increase in peak areas of the two transition fragments from tryptic digests of whey proteins in stored milk protein concentrate powder. A good correlation between the MRM and furosine results indicated that MRM based on tryptic digests of whole products was a feasible method for quantification of modified milk proteins. Proteomics was used to investigate whether MR-induced protein cross-linking could cause solubility loss in stored milk powders. Protein cross-linking could occur via advanced MR products (e.g., methylglyoxal) or dehydroalanine. This study was designed to determine the relevance of these pathways in protein cross-linking in milk powders. The insoluble material from reconstituted stored MPC80 was isolated and characterized by 2-DE and MALDI-TOF-MS. Large amounts of αS1-, αS2-, β-, and κ-casein and a small amount of β-lactoglobulin were present in the cross-linked protein spots on the gels of the insoluble material. αS1-casein was used in model experiments to investigate the mechanism of protein cross-linking. There was an increased amount of cross-linking of αS1-casein when incubated with added lactose or methylglyoxal. Although dephosphorylation could eliminate dehydroalanine formation, it did not prevent protein cross-linking. The possible contribution of dicarbonyls, acting as a connecting bridge between two protein molecules, like methylglyoxal to cross-linking in stored MPC80 was confirmed by comparing the protein cross-linking pattern on 2-DE gels of MPC80 incubated at 37°C for 1 day with that of a sample with added methylglyoxal. It was concluded that a possible cause of the decrease in solubility of milk powders containing micellar casein is through cross-linking of caseins by advanced MR products such as methylglyoxal.
Keyword milk powder, solubility, Maillard reaction, lactosylation, protein cross-linking, proteomics
Additional Notes Pages in colour: 49, 66, 68, 70, 117, 119, 120-121, 123, 125, 129, 137, 141-143, 145-146, 163, 166-169, 171-175, 216-218, 220. Page in landscape: 138-139, 214-219

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Created: Wed, 16 May 2012, 22:18:30 EST by Thao Thi Thu Le on behalf of Library - Information Access Service