*2.2. Preparation of Nanosuspensions*

Thymol nanosuspensions were prepared by a pH-driven method according to our previous study with some modification [15,22]. Thymol powder was weighed and dissolved in 0.1 M NaOH to obtain a thymol alkaline solution (10 mg/mL). Caseinate stock solutions (50 mg/mL) were prepared by dissolving caseinate in PBS (5 mM, pH 6.5) for 4 h and the solution was centrifuged at 8000 rpm for 30 min to remove the undissolved impurities. The solution was then diluted with PBS to different concentrations and stored at 4 ◦C overnight before use. Thymol alkaline solutions were then added into the caseinate solution (1:1, *v/v*) with constant stirring using a magnetic stir plate. Nanosuspensions with a thymol concentration of 5 mg/mL and different caseinate concentrations (2.5, 5, 10, 15, 20, and 25 mg/mL) were prepared by adjusting the pH value to 6.5 using 6 M HCl solutions.

#### *2.3. The Physical Properties of Nanosuspensions*

The average particle size and surface potential of nanosuspensions were determined by Zetasizer Nano ZSP (Malvern Instruments Ltd., Worcestershire, UK) at room temperature. Samples were diluted tenfold with PBS, and each sample was tested three times in parallel.

The microstructure of nanosuspensions was observed using Atomic Force Microscopy (AFM, C300, Nanosurf, Liestal, Switzerland). The nanosuspension solutions were diluted 1000-fold with distilled water and one drop of samples was placed on a freshly cleaved mica substrate. The images of the sample were obtained using the AFM operated with a silicon cantilever force constant of 0.58 N m<sup>−</sup><sup>1</sup> in tapping mode.

The morphology of the nanosuspensions was further confirmed using transmission electron microscopy (TEM). Briefly, nanosuspensions were placed onto a copper mesh grid for 4 min. The sample was then stained with 1% uranyl acetate solution for 1 min and then washed with double distilled water. The sample-loaded grid was then air dried at room temperature and imaged using a TEM (JEM-2000FX, JEOL, Ltd., Tokyo, Japan) operating at a voltage of 200 kV.

The crystalline properties of nanosuspensions were investigated using X-ray diffraction. The blank casein nanoparticles (without thymol) fabricated using the pH-driven method and thymol powder were used as control groups. The emission slit was 1◦, the accepted slit was 0.1 mm, and the scanning speed was 2◦/min.

The loading capacity (LC) and encapsulation efficiency (EE) were calculated according to the previous method [2]. Samples were extracted with n-hexane (volume ratio 1:9) and the absorbance was then measured at 263 nm using a UV–visible spectrophotometer. The thymol concentration was then calculated using a standard curve prepared. Then, the insoluble part of the thymol was removed by centrifugation (6000 rpm, 10 min), and the thymol in the supernatant fluid was extracted by n-hexane to measure the amount of dissolving thymol in the samples. The free thymol was separated and measured via an ultrafiltration method (3 kDa). The EE and LC of samples were calculated with the following formulas:

$$\rm{EE} \left( \% \right) = \left( \rm{W}\_{\rm{s}} - \rm{W}\_{\rm{f}} \right) / \rm{W}\_{\rm{t}} \text{ } ^{\ast} \text{ 100} \tag{1}$$

$$\text{LCC } \text{(\%)}=(\text{W}\_{\text{s}}-\text{W}\_{\text{f}})/\text{M} \text{ \* 100} \tag{2}$$

where Wt represents the total thymol content, Ws represents the dissolved thymol content, and Wf represents the free thymol content. M is the total mass of the loaded nanosuspensions: Thymol and caseinate.

#### *2.4. The Stability of Thymol Nanosuspensions*

Thermal stability: Nanosuspensions were treated at 80 ◦C for 1 h and sampled every 10 min to measure the properties (retention rate, average diameter, and visual pictures) according to previous methods of nanosuspension after heating.

Storage stability: Samples were stored at room temperature (25 ◦C) for one month, and their properties (retention rate and average diameter) were investigated every 7 days.

#### *2.5. The Antibacterial Properties of Nanosuspensions*

LB liquid medium: 10 g tryptone, 5 g yeas<sup>t</sup> extract, and 5 g sodium chloride were weighed and dissolved in 1000 mL distilled water. The medium was autoclaved at 121 ◦C for 20 min. The medium was placed at 4 ◦C for use after cooling.

LB solid medium: 10 g tryptone, 5 g yeas<sup>t</sup> extract, 5 g sodium chloride, and 2 g agar were weighed in 1000 mL distilled water, autoclaved at 121 ◦C for 20 min, and then poured into 90 mm Petri dishes with about 12–15 mL in each dish. After cooling and solidification, they were placed upside down at 4 ◦C.

Bacterial resuscitation and rejuvenation: First, 50 μL of glycerin bacteria were vaccinated in 5 mL of LB liquid medium and cultured in a biochemical incubator at 37 ◦C overnight to turbidity, and then the strains were inoculated from the LB liquid medium to the LB solid medium using inoculation loops to an incubator at 37 ◦C constant temperature for 10–12 h. Then, the appropriate single colony was inoculated to a liquid medium and cultured to a later growth stage. The bacterial strains were transferred to a solid medium again, sealed by film, and stored at 4 ◦C with the appropriate single colony observed.

Bacterial culture: The plate was taken out of a refrigerator at 4 ◦C; a single colony was inoculated in a 5 mL LB liquid medium and cultured in a constant temperature vibration incubator at 37 ◦C and 220 rpm. For the minimum inhibitory concentration and maximum bactericidal concentration, the bacterial solution was diluted to 10<sup>6</sup> CFU/mL.

MIC measurement: The 96-well microplates method was adopted according to the previous method [13]. The test bactericidal strains were CICC 22956, CICC 21600, CICC 10003, and CICC 21635. First, 100 μL of liquid LB medium was added to each well. Next, 100 μL samples were placed into the first row of the wells and mixed, 100 μL of the mixture was placed into the next well, and so forth. Then, 100 μL bacterial solutions were mixed into each well and incubated (37 ◦C, 24 h) and put into an incubator at 37 ◦C for culture for 24 h. The minimum sample concentration with clear culture and no substrate precipitation was the minimum inhibitory concentration (MIC).

MBC measurement: 100 μL was obtained from the MIC well and inoculated in culture, then the cultures were placed in an incubator at 37 ◦C for 12 h. The sample concentration corresponding to the plate without bacterial colony was the maximum bactericidal concentration (MBC).
