*3.2. Characterization of Nanosuspensions*

A caseinate concentration of 10 mg/mL was used in the following studies since it led to relatively high EE and LC values with a small nanosuspension size distribution. As discussed above, the nanosuspensions with final formal were relatively small (79.4 nm) and negatively charged (−19.4 mV). At the same time, they had high EE (85.2%) and LC (30.0%), and the free thymol was dissolved in water instead of sediment. These results were relatively similar to caseinate-coated curcumin nanoparticles [17]. Previous researchers had also applied some delivery systems to encapsulate thymol and investigated the LC. For example, Shi et al. prepared thymol-loaded solid lipid nanoparticles with a maximum LC of 16% [30], and the LC of thymol/soy protein nano-complexation was 10.36% [2]. All

of the data and experiments indicated that the thymol was suitable for the construction of nanosuspensions coated with caseinate via the pH-driven method.

To further prove the fabrication of nanosuspensions, their morphology was observed and detected by AFM and TEM (Figure 3). The AFM results demonstrated that the thymol nanosuspensions are spherical and uniformly distributed in the systems. As expected, the height and size of nanosuspensions were consistent with results provided by dynamic light scattering. In the TEM image, most of the particles were uniformly distributed. The sizes of nanosuspensions were in the range of 50–150 nm, which agrees with the data obtained by dynamic light scattering.

**Figure 3.** Atom force microscopy (**A**) and transmission electron microscopy (**B**) image of thymol nanosuspension.

The physical states of the thymol in caseinate-coated nanosuspensions were investigated via X-ray diffraction. The diffraction peak of thymol was obtained at 2θ values from 5◦ to 35◦ (Figure 4), which indicates the high crystal state of thymol. The relatively smooth curve of caseinate indicated that it was not present in a crystalline structure. As expected, the diffraction peak of the nanosuspensions could not be observed, indicating that all of the thymol are present in an amorphous state in the core of nanosuspensions. These phenomena and data sugges<sup>t</sup> that the incorporation of thymol into the caseinate-coated nanosuspensions could prevent its crystallization, which is consistent with previous results and may be beneficial for the application in commercial products, such as food colloids.

**Figure 4.** XRD spectra of thymol, casein, and powdered thymol nanosuspension.
