Association of Enzymatically and Nonenzymatically Functionalized Caseins Analyzed by Size-Exclusion Chromatography and Light-Scattering Techniques

Abstract

The influence of covalent protein modifications resulting from the Maillard reaction (glycation) of casein and lactose on the noncovalent association behavior of the protein was studied. Nonenzymatic cross-linking with methylglyoxal (MGO) and glutaraldehyde (GTA) as well as enzymatic cross-linking with microbial transglutaminase (mTG) was investigated in comparison. Molar mass, particle size, and conformational characteristics of nonmicellar casein associates as well as the extent of intraparticle protein cross-linking were examined utilizing size-exclusion chromatography (SEC) combined with UV detection and static and dynamic light scattering. Cross-linking resulted in the stabilization of a certain fraction of casein associates, with particle sizes of approximately 30 nm in radius of gyration (R g), and promoted an incorporation of further casein molecules into those particles, yielding molar masses (M w) of 1.0–1.2 × 106 g/mol. When caseins were additionally conjugated with lactose during the early Maillard reaction, a further growth of the associates up to approximately 50 nm in R g with a M w of 2.1 × 106 g/mol was observed. Furthermore, glycation reactions induced a transition from slightly elongated, random-coil structures toward more anisotropic conformations. Associates consisting of caseins cross-linked with GTA appeared to preserve the original particle conformation.

Size Modulation of Enzymatically Cross-Linked Sodium Caseinate Nanoparticles via Ionic Strength Variation Affects the Properties of Acid-Induced Gels

“…When using only a Superose 6 column for separation of Cn-PB under denaturing and reducing conditions, the two polymeric species eluted at~7-9 mL and~9-13 mL, and monomeric casein at 15-17 mL [36]. Other researchers used the same column under non-denaturing and nonreducing conditions for characterising native, uncross-linked NaCn and observed three peaks at very similar elution volumes (Table 2) [25,44]. Based on multi-angle light scattering results, these research groups concluded that the last eluting fraction corresponded to small casein aggregates as well as some monomeric caseins at the end of the peak.…”

“…In case of excessively cross-linked NaCn (≥24 h), rearrangements are impaired on the molecular level due to the fixation of casein molecules. In combination with the increased internal density and higher sphericity of the casein particles [24][25][26], this might increase their mobility in the gel network, as was also indicated by the higher tan δ (see Figure 3). Thus, rearrangements of casein clusters on the microstructure level might be facilitated, leading to higher forced syneresis.…”

“…With regard to the first peak in the chromatograms, Hannß et al [25] reported that it contained only low amounts of protein, but also triglycerides and phospholipids, suggesting a co-elution of large casein particles and fat droplets. Nevertheless, the peak intensity and the protein content of this fraction increased with different, non-enzymatic cross-linking treatments [25], and the present study demonstrated a remarkable increase in this fraction with casein cross-linking by mTGase at increased ionic strength.…”

“…Casein Monomers [36] Denaturing and reducing~7 -9~9-13~15-17 [44] Native~6-8.5~8.5-12~12-17 [25] Native~7.5-10~10-15~15-20…”

“…It was reported that the cross-linking of casein-bound glutamine and lysine residues by mTGase [23] occurs predominantly between casein molecules within the same NaCn nanoparticle, resulting in a change from elliptical to spherical shape [24,25] as well as in a transition from a soft particle character towards a hard sphere-like behaviour [26]. The size and aggregation number of native NaCn nanoparticles varied with casein concentration, temperature, pH and ionic strength due to modulation of hydrophobic and electrostatic interactions [27,28].…”