*3.3. Stability of Nanosuspensions*

In food products, most nanosuspensions and food colloids are applied by incorporating them into different products and are exposed to different environmental conditions. Therefore, the thermal and storage stability of nanosuspensions were investigated in this section (Figure 5), which could provide information for the application of thymol in commercial products.

It has been reported that the volatile properties of thymol limit its application in the food industry, and the decrease of thymol mass begins from 50 ◦C [31]. Thus, the impact of high temperature on the stability of thymol nanosuspensions was investigated at 80 ◦C. The retention rate of both pure thymol and nanosuspensions decreased with time, and the retention rate of nanosuspensions was slightly higher than pure thymol. These results and phenomena indicated that the coating of caseinate at the surface of nanosuspensions could enhance its thermal stability. Similar results were also reported in nano-complexation of thymol-soy protein isolate, and Chen et al. attributed the enhancement of thermal stability to the shift of thymol evaporation temperature point from 50 to 90 ◦C and the inclusion complex fabrication [2,32]. Meanwhile, the influence of heat on the average size of nanosuspensions was also investigated, and it is obvious that there are no significate changes. The results of particle size indicate that there are no aggregation and dissociation of caseinate at the surface of the nanosuspensions at elevated temperature, which further proved that caseinate is suitable for the encapsulation of thymol nanosuspensions via the pH-driven method under high temperature. Meanwhile, in the visual figures (Figure 5c), all of the samples remained clear, which indicated there was no significant recrystallization and dissolution of thymol at high temperature. However, in previous research, the curcumin nanoparticles coated by caseinate via a pH-driven shift exhibited a large leakage of curcumin and a slight increase in turbidity [17]. These differences of nanoparticles are mainly due to the difference between thymol and curcumin in their molecular weight or hydrophobicity.

**Figure 5.** The influence of heating on the thymol retention rate (**A**), average diameter (**B**), and visual appearance (**C**) of nanosuspensions.

To further investigate the stability of thymol nanosuspensions, their storage stability at room temperature was examined via investigating the retention rate and average diameter during 28 days of storage. While the results showed that the retention of thymol nanosuspensions decreased slowly, the value was higher than 90% during the storage period (Figure 6). Meanwhile, there were no significant changes in the average diameter of thymol nanosuspensions, which indicated that the caseinate-coated nanosuspensions via the pH-driven method were stable at longterm storage. However, it has been reported that the retention rate of curcumin nanoparticles coated by caseinate by the pH-driven method decreased dramatically during storage at room temperature [17]. This difference is the same as the thermal stability, which could be mainly due to the difference between the encapsulated lipophilic nutrients.

**Figure 6.** Changes of thymol retention rate and nanosuspension diameter during storage for four weeks.

#### *3.4. MIC and MBC of Thymol*

As a natural antibacterial agent, thymol has been applied in many products to extend shelf life, and its antibacterial activity is better than many other plant oil components, such as carvacrol, diacetyl, and eugenol [2,13]. Moreover, it has been reported that the antibacterial activity of thymol could be mainly attributed to the thymol's capacity to change the structure and properties of the outer and cytoplasmic membrane of bacteria [33]. Therefore, to investigate the antibacterial activity of thymol nanosuspensions, the MICs and MBCs of nanosuspensions against four common pathogenic bacteria (*Salmomella enterca*, *Staphlococcus aureus*, *Escherichia coli*, and *Listeria monocytogenes*) were investigated and calculated (Table 1).


**Table 1.** Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of nanosuspensions and free thymol for inhibition and inactivation of *L. monocytogenes*, *S. aureusd*, *S. typhimurium*, and *E. coli*.

The MICs of free thymol against four kinds of bacteria were the same as 142 mg/mL, and the values of all MBCs were the same as MICs. These results and phenomena indicated that thymol could be used as antibacterial and bacteriostatic agents for all of the four kinds of bacteria and there was no significant influence of bacteria kinds on antibacterial activity. However, all of the MICs and MBCs of thymol nanosuspensions were higher than the free thymol, which showed that there was no enhancement in antimicrobial activity of thymol by encapsulating with caseinate via pH-driven methods. These results could be due to the

bonds between thymol and caseinate, which prevent the release of thymol from coated nanosuspensions. Thus, the amount of thymol in contact with bacteria decreased at the same concentration, the same antibacterial effect required a higher concentration of thymol. Similar results have also been reported by Wattanasatcha et al., that there was no difference between free thymol and thymol encapsulated in the ethylcellulose/methylcellulose sphere on its antimicrobial activity [34]. Moreover, the MIC/MBC values of thymol encapsulated into liposomes were also lower than free thymol, which has been mainly attributed to the strong binding between thymol/liposomes and the lower release of encapsulated thymol [13]. Therefore, in this work, the decrease in the amount of encapsulated thymol release could be due to the hydrophobic interaction force between thymol and caseinate, which influenced the antibacterial properties of thymol.
