Cross-linking behavior and foaming properties of bovine α-lactalbumin after glycation with various saccharides.

α-Lactalbumin was glycated via the Maillard reaction in the dry state using various mono- and oligosaccharides. The reaction resulted not only in coupling of the saccharides to α-lactalbumin but also in cross-linked proteins. The glycation rate and the extent of cross-link formation were highly dependent on the saccharide used. Glycation by arabinose and xylose led to a very fast protein cross-link formation, whereas glucose showed a relatively low protein cross-linking ability. The stability of foams, created using the various glycated protein samples, depended on the type of saccharide used, the extent of glycation, and possibly the amount of cross-linked protein. Compared to nonmodified α-lactalbumin, glycation with rhamnose and fucose improved foam stability, whereas application of glucose, galacturonic acid, and their oligosaccharides did not exert a clear effect. Mass spectrometric analysis revealed that dehydration of the Amadori products is an indicator of the formation of protein cross-links.

Effect of saccharide structure and size on the degree of substitution and product dispersity of α-lactalbumin glycated via the Maillard reaction.

The course of the Maillard reaction between α-lactalbumin and various mono- and oligosaccharides in the solid state was studied using UPLC-ESI-TOF-MS. Individual reaction products were monitored for their degree of substitution per protein molecule (DSP). The Maillard reaction rate depended on the saccharide type and decreased when the saccharide size increased. Conjugation with charged saccharides was hindered when a specific average DSP was reached, probably resulting from electrostatic repulsion. The DSP varied between 0 and 15, and the standard deviation of the average DSP, which is a measure for product dispersity, increased to 1.9. Similar experiments were performed with a dipeptide. Relative reaction rates in these experiments were 1 for glucose, 0.28 for maltose, and 0.16 for maltotriose. Comparison of the results obtained using α-lactalbumin and the dipeptide made clear that the Maillard reaction rate is determined by a number of factors, including saccharide reactivity and lysine accessibility.

Proof of principle for the synthesis of hydroxy-aryl esters of glycosidic polyols and non-reducing oligosaccharides with subsequent enzymatic coupling to a tyrosine-containing tripeptide.

To enable enzymatic coupling of saccharides to proteins, several di- and trisaccharides were hydroxy-arylated using anhydrous transesterification with methyl 3-(4-hydroxyphenyl)propionate, catalyzed by potassium carbonate. This transesterification resulted in the attachment of up to 3 hydroxy-aryl units per oligosaccharide molecule, with the monosubstituted product being by far the most abundant. The alkaline reaction conditions, however, resulted in a partial breakdown of reducing sugars. This breakdown could easily be bypassed by a preceding sugar reduction step converting them to polyols. Hydroxy-arylated products were purified by using solid phase extraction, based on the number of hydroxy-aryl moieties attached. Monohydroxy-arylated saccharose was subsequently linked to a tyrosine-containing tripeptide using horseradish peroxidase, as monitored by LC-MS(n). This proof of principle for peptide and protein glycation with a range of possible saccharides and glycosidic polyols can lead to products with unique new properties.