Tuning the Gel Network Structure and Rheology of Acid-Induced Casein Gels via Thiol Blocking

Abstract

This study systematically investigates how thiol-disulfide interactions influence the structure and mechanical properties of casein gels. Acid gels were prepared from suspensions of micellar casein (MC) powder that were heat-treated at 70 °C. Thiol groups were variably blocked with N-ethylmaleimide (NEM). The gels were characterized using stress-strain measurements, rheological analyses, and confocal microscopy. The stress-strain curves exhibited a biphasic behavior, with an initial linear elastic phase followed by a linear plastic region and a nonlinear failure zone. Compared to control samples, the addition of 100 mM NEM reduced the gel strength by 50%, while G' and G″ increased by around 100%, unexpectedly. NEM-treated gels consist of uniformly sized building blocks coated with a whey protein layer. Strong physical interactions and dense packing enhance viscoelasticity under short deformations but reduce the compressive strength during prolonged loading. In contrast, control samples without NEM demonstrate weak viscoelasticity and increased compressive strength. The former is attributed to a broader particle size distribution from lower acid stability in the untreated gels, while the particularly high compressive strength of heat-treated gels additionally results from disulfide cross-links. The results show that thiol blocking and heating enable the targeted formation of acid casein gels with high shear stability but a low compressive strength.

Keywords: confocal fluorescence microscopy; gels; mechanical characterization; micellar casein powder; milk proteins.

Figure 1

Thiol exchange reaction after temperature activation and NEM’s effect on milk proteins.

Figure 2

Relative numbers of reducible disulfide bonds in suspensions of micellar casein (MC) after different treatments, as determined with Ellman’s reagent. (a) Total system (whey protein + MC), (b) supernatant after centrifugation (mainly whey protein).

Figure 3

Confocal fluorescence micrographs of casein acid gels from casein micelles (a) without temperature treatment, (b) with temperature treatment, (c) with temperature treatment and 40 mM added NEM, and (d) with temperature treatment and 100 mM added NEM. In each image, insets (1 µm × 1 µm and 5 µm × 5 µm) highlight the typical microstructure at higher magnification, allowing a detailed visualization of the aggregate morphology and substructure.

Figure 4

Changes in viscoelastic behavior due to addition of NEM before gel formation. (a) Storage modulus G′ for elastic behavior and (b) loss modulus G″ for viscous behavior.

Figure 5

Pressure versus penetration depth measurements (a) and material parameters obtained (b) for gels of temperature-pretreated casein micelles mixed with different concentrations of NEM compared to the reference without the temperature pre-treatment shown as stars.

Figure 6

Values of the parameters of model 1 (a–d) obtained by fitting Equation (1) to the individual measured curves in Figure 5a. The simultaneous fit is shown as a hypersurface of all force–displacement curves as a function of the NEM concentration (e) using Equations (3)–(5), with simulated curves also shown as lines in a-d. Calculated transition functions for selected NEM concentrations are shown (f).

Figure 7

Schematic representation of the protein states before and after the acid-induced gelation of micellar casein-based systems. The upper row illustrates the structural organization of micellar casein (MC), denatured whey proteins, and their interactions prior to gelation under different treatments. NEM blocks free thiol groups as indicated by red stars. The lower row shows the resulting gel microstructures after acidification.