Article pubs.acs.org/JAFC
Glycation Reactions of Casein Micelles Ulrike Moeckel, Anja Duerasch, Alexander Weiz, Michael Ruck, and Thomas Henle* Department of Chemistry and Food Chemistry, Technische Universität Dresden, 01062 Dresden, Germany 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, Nε-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. KEYWORDS: casein micelle, sodium caseinate, glycation, Maillard reaction, cross-link, scanning electron microscopy
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glycation), occur.12 However, to date the influence of micelle association on the glycation of casein has not been studied. Glycation reactions between carbonyl groups of reducing sugars and amino acid side chains have been investigated in a number of studies for nonmicellar caseinate. Solutions of sodium caseinate (NaCas), as a model for nonmicellar casein, showed a polymodal size distribution. At a pH of 6.0, three main particle populations with hydrodynamic diameters 50,000 Da). The increase of high molecular weight cross-links correlated with the increasing intensity of browning. Consequently, for the results presented here, it might be possible that a high proportion of trimers and oligomers larger than trimers (Figure 4b) contain melanoidins. Browning of glucose−sodium caseinate samples (Figure 6c) was more pronounced than browning of the micelle suspensions (Figure 6a). However, for the glucose−casein micelles it can be assumed that the refractive and light-scattering properties of the “micelle-bound” melanoidins are changed in comparison to those of free melanoidins, because the isolated 4 h casein micelle pellets (Figure 6b) are nearly black, whereas the suspensions were only weakly colored. Structural Aspects of Glycated Casein Micelles. To investigate the changes of the casein micelles in size and surface morphology during heating in the presence and absence of glucose, the micelles were characterized with DLS and SEM. Figure 7 shows the results of the DLS measurements. The
Figure 7. Particle size distributions of casein micelles after heat treatment for 0−4 h at 100 °C (a) in the absence and (b) in the presence of glucose, analyzed by dynamic light scattering. 2959
DOI: 10.1021/acs.jafc.6b00472 J. Agric. Food Chem. 2016, 64, 2953−2961
Article
Journal of Agricultural and Food Chemistry
Figure 8. Scanning electron micrographs of casein micelles after incubation for 0, 1, and 4 h at 100 °C (a) in the absence and (b) in the presence of glucose.
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ABBREVIATIONS USED CM, casein micelle; NaCas, sodium caseinate; SMUF, synthetic milk ultrafiltrate; HPLC-ESI-MS/MS, high-performance liquid chromatography with electrospray ionization and tandem mass spectrometry; MWCO, molecular weight cutoff; LAL, lysinoalanine; AGE, advanced glycation end-product; CML, Nεcarboxymethyllysine; GOLD, glyoxal-derived lysine dimer; dp, degree of polymerization; CLAAmin, minimum concentration of intermolecular cross-linked amino acids; DLS, dynamic light scattering; SEM, scanning electron microscopy
and the prestructuring promotes the polymerization. Despite the pronounced polymerization, it could be shown by means of DLS and SEM that the proteins were cross-linked exclusively intramicellar. Furthermore, scanning electron micrographs of the glycated casein micelles showed changes in the surface morphology, which appeared to be less structured and smoother, compared to the micelles heated in the absence of glucose. These results indicate that the glycation and crosslinking reactions occur across and close to the water channels and the protruding tubules. Thereby, the Maillard-induced integration of glucose leads to a higher internal micellar order. As a consequence, the micelle stability, for instance, in the case of a loss of micellar calcium or against digestive enzymes, could be improved. Such stable casein micelles could be applied as natural nanovehicles for pharmaceuticals. Furthermore, from the behavior of the casein micelles during glycation, more details can be learned about the casein micelle structure and could be incorporated in the discussions about the micelle models. Further studies are needed to investigate whether the casein micelle stability is increased due to glycation and whether the modifications form internal cross-linking structures as assumed in the present work.
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AUTHOR INFORMATION
Corresponding Author
*(T.H.) Phone: +49-351-463-34647. Fax: +49-351-463-34138. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
ACKNOWLEDGMENTS
We thank Karla Schlosser, Institute of Food Chemistry, TU Dresden, for performing the amino acid analysis; Stephen Schulz, Institute of Inorganic Chemistry, TU Dresden, for performing the elemental analysis; and Felix Hippauf, Institute of Inorganic Chemistry, TU Dresden, for performing the supercritical point drying. 2960
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