Glycation Reactions of Casein Micelles

Abstract

After suspensions of micellar casein or nonmicellar sodium caseinate had been heated, respectively, in the presence and absence of glucose for 0-4 h at 100 °C, glycation compounds were quantitated. The formation of Amadori products as indicators for the "early" Maillard reaction were in the same range for both micellar and nonmicellar caseins, indicating that reactive amino acid side chains within the micelles are accessible for glucose in a comparable way as in nonmicellar casein. Significant differences, however, were observed concerning the formation of the advanced glycation end products (AGEs), namely, N(ε)-carboxymethyllysine (CML), pyrraline, pentosidine, and glyoxal-lysine dimer (GOLD). CML could be observerd in higher amounts in nonmicellar casein, whereas in the micelles the pyrraline formation was increased. Pentosidine and GOLD were formed in comparable amounts. Furthermore, the extent of protein cross-linking was significantly higher in the glycated casein micelles than in the nonmicellar casein samples. Dynamic light scattering and scanning electron microscopy showed that glycation has no influence on the size of the casein micelles, indicating that cross-linking occurs only in the interior of the micelles, but altered the surface morphology. Studies on glycation and nonenzymatic cross-linking can contribute to the understanding of the structure of casein micelles.

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

“…Alkaline treatment, especially in combination with heating, transforms particular amino acid residues into reactive intermediates, which form cross-links by reactions with other functional groups. Typical products are lysinoalanine, histidinoalanine and lanthionine, which can be determined by chromatographic techniques, e.g., [43][44][45][46][47][48]. Severe heat treatment may even induce the formation of isopeptide bonds between lysine and glutamine (N-ε-(γ-glutamyl)-lysine) or asparagine (N-ε-(β-aspartyl)-lysine), however, these reactions are competed by the Maillard reaction and therefore restricted in the presence of reducing carbohydrates [23,42,49,50].…”

“…SEC column materials differ in composition and pore volume. The column most frequently used for studying cross-linked casein is Superdex 200, e.g., [30,44,47,48,54,100,115,131,[134][135][136][137][138][139][140][141][142][143][144][145][146][147][148], which is provided by GE Healthcare (Uppsala, Sweden). The column material is based on agarose/dextran with a ratio of pore volume to void volume of V i /V o = 1.7 and exhibits a fractionation range from 10 to 600 kg/mol for globular proteins [128].…”

“…The use of globular proteins for molar mass calibration results in overestimation because caseins exhibit higher hydrodynamic radii and thus elute earlier than globular proteins with similar molar mass [150]. Moeckel et al [48] used instead polymers of polystyrene sulphonate in the range from 6.4 to 152 kg/mol to assign the position of casein monomers, dimers, trimers, and oligomers in the chromatograms.…”

“…For instance, the highest number of casein dimers in TGase-catalysed polymerisation was observed in moderately cross-linked samples, i.e., at high levels of residual monomers (>50%), indicating that dimers as well as smaller oligomers are good substrates for further cross-linking by TGase [32,78,146]. Moreover, the polymerisation degree (sometimes also referred to as oligomerisation degree) was calculated in several studies from size exclusion chromatograms by relating peak areas of cross-linked caseins to the entire sample area [30,32,44,47,48,53,78,[134][135][136][146][147][148]. Because reference casein samples often reveal a low level of polymerised casein (see Figures 9 and 10), the polymerisation degree after cross-linking was also expressed as the difference between cross-linked samples and reference [137][138][139]144,145].…”

“…From studies on non-enzymatic cross-linking it was concluded that [CLAA] min higher than experimental data (e.g., for lysinoalanine or histidinoalanine) implies the occurrence of unknown cross-links, and that lower [CLAA] min is an indicator for intramolecular cross-linking [44,45,48]. Higher [CLAA] min may also indicate underestimation by insufficient protein hydrolysis prior to amino acid analysis.…”