*3.1. Fabrication of Nanosuspensions*

During the fabrication of thymol nanosuspensions through the pH-driven method, thymol must be dissolved in the alkaline solutions for at least 5 min. When thymol was dissolved in alkaline solutions, the phenolic hydroxyl group that was thymol ionized and

the potential charge of the thymol molecule became negative, which caused an enhancement in its hydrophilic properties and water solubility. It is well known that phenols are unstable under alkaline conditions [26,27]. Therefore, the chemical stability of thymol was firstly investigated via measuring the retention concentration at different pH conditions. As displayed in Figure 1, the retention rate of thymol at a pH range of 7.0 to 10.0 slightly decreased to 93.0% after 24 h, while thymol at pH 11.0 and 12.0 was ultra-stable without any degradation. During the pH-driven process, the thymol was dissolved in alkaline solutions (around pH 12.5), which meant the loss of thymol during the pH-driven process was negligible.

**Figure 1.** The influence of pH values on the stability of thymol.

Thymol showed relatively high chemical stability compared with other phenols. Our previous study showed that the chemical stability of phenols negatively corresponded to the number of hydroxyl groups [28]. Thymol molecules have only one hydroxyl group, which could explain why they showed extremely high stability. On the other hand, thymol showed higher stability at pH 11.0 and 12.0 compared to lower pH values (7.0 to 10.0), which was similar to curcumin [29]. The chemical stability of quercetin and resveratrol, however, negatively corresponded to the pH values (i.e., the higher the pH, the poorer the chemical stability). The difference could be assigned to the presence of the −O−CH3 group or the −CH−(CH3)2 group in curcumin and thymol molecules, respectively. There are no other groups in the quercetin and resveratrol except the hydroxyl group.

Initially, the influence of caseinate concentration on the encapsulation efficiency and loading capacity was investigated (Figure 2A). The EE increased with the increase of caseinate concentration from 2.5 to 5 mg/mL and then became steady. The initial low EE of samples could be mainly due to the insufficient caseinate that could not form enough particles for the encapsulation of free thymol, and the free part of thymol was removed by centrifugation. At high and sufficient caseinate concentration, most of the thymol nanosuspensions could be completely coated by caseinate during the change of pH values, and the EE and amount of encapsulated thymol increased. Since part of thymol could be dissolved in water, even the extra protein and nanosuspensions were present with caseinate increasing; no significant increase of EE could be observed. To further investigate the encapsulation level of thymol into nanosuspensions, the influence of caseinate concentration on the LC was also evaluated (Figure 2A). The LC of thymol

nanosuspensions decreased with the increase of caseinate concentration, which could again be due to the hypothesis that most of the thymol had been encapsulated. The largest LC of thymol nanosuspensions coated by caseinate was nearly 60%; this phenomenon and result proved that the nanosuspensions coated by caseinate are extremely suitable encapsulation systems for thymol via the pH-driven method.

**Figure 2.** The influence of casein concentration on encapsulation efficiency and loading capacity (**A**), and average diameter and zeta potential (**B**) of thymol nanosuspensions.

The influence of caseinate on the average particle size and zeta potential of nanosuspensions are shown in Figure 2B. It is obvious that the size of nanosuspensions dramatically decreased with the caseinate concentration increasing from 2.5 to 10 mg/mL. The decrease of nanosuspension size could be mainly attributed to the behavior and function of caseinate molecules, which are adsorbed at the surface of thymol particles via their hydrophobic regions to prevent the aggregation of nanosuspensions. When the concentration of caseinate increased from 10 to 25 mg/mL, the size of nanosuspensions increased gently, which could be due to an accumulation of caseinate at the nanosuspension surface. Conversely, there was no significate difference between the zeta potential of nanosuspensions fabricated with different concentration caseinate; all of the nanosuspensions presented a moderately negative charge. The charge of nanosuspensions could be mainly due to the properties of caseinate by which the pH values of water were higher than its isoelectric point, and the surface charge of nanosuspensions was dominated by caseinate, which covered and was present at the surface of nanosuspensions.

The ideal function and behavior of caseinate during the pH-driven method could be related to the emulsifying and binding ability of this kind of protein. It has been reported that amphiphilic biopolymers (such as whey protein isolate) can adsorb to the surface of the lipophilic nanoparticle, prevent contact, and aggregate between them [17]. These data and phenomena showed that the water solubility and dispersibility of thymol could be enhanced by encapsulation via the pH-driven method with caseinate, and this information could promote the development and application of thymol in food and cosmetic productions.
