*4.5. Determination of Minimum E*ff*ective Concentration (MEC10)*

The assay has been designed to determine the killing effects of the test compounds for a certain period of time. In brief, a wide concentration range (0.03–80 μg/mL) of the test samples were used to treat ~106 cells/mL for one hour. One milliliter of treated and untreated samples were taken and were diluted 10<sup>5</sup> times followed by spreading 50 μL samples onto 20 mL nutrient agar media and incubated for 24 h at 35 ◦C (bacteria) and 30 ◦C (fungi) for colony-forming unit (CFU/mL) quantification. The data were compared with growth control and positive control (Van for bacteria and Cas for fungi) for percentage mortality (~10% death) determination. It is noteworthy that in the MEC10 experiments, the inoculated microbial cell number was 10<sup>3</sup> times higher than was used in the MIC90 determinations (10<sup>6</sup> vs. 103). However, in both cases, the same formula was used (see Section 4.7). All the measurements were performed by applying three technical replicates in six independent experiments.

#### *4.6. Determination of Microbial Oxidative Generation and Killing Activity*

#### 4.6.1. Quantification of Total ROS Generation

Total ROS generation was assayed according to previously published protocols [27,28,34,36]. Briefly, ~106 cells/mL were collected and centrifuged at 1500 *g* for 5 min and were suspended in PBS. The cells were stained with a 20 mM stock solution of DCFDA in PBS (pH 7.4) to achieve an end concentration of 25 μM, and were incubated at 35 ◦C (for bacteria) and 30 ◦C (for fungi) for 30 min in the dark with mild shaking. The cells were centrifuged (Hettich Rotina 420R, Auro-Science Consulting Ltd., Budapest, Hungary) and suspended in RPMI media. The cells were treated with CPe, CT80, CEt, Van (bacteria), and Cas (fungi) at their respective MEC10 concentrations for one hour. The fluorescence signals were recorded at Ex/Em = 485/535 nm wavelengths by a Hitachi F-7000 fluorescence spectrophotometer/plate reader (Auro-Science Consulting Ltd., Budapest, Hungary). The percentage increase in oxidative balance was measured by comparing the signals to those of the growth controls (Gc). Six independent experiments were done with three technical replicates for each treatment.

#### 4.6.2. Detection of Peroxide (O2 <sup>2</sup><sup>−</sup>) and Superoxide Anion (O2 •−) Generation

The previously described protocol was adapted for peroxide [36,37] and superoxide anion radicals [34,38] with modifications. A positive control, Me (0.5 mmol/L as end concentration), CPe, CT80, CEt, Van (bacteria), and Cas (fungi) at their respective MEC10 concentrations were used to treat the cell suspensions (~106 cells/mL) for an hour at 35 ◦C (bacteria) and 30 ◦C (fungi) in RPMI media. Thereafter, the cells were centrifuged at 1500 *g* for 5 min at room temperature followed by resuspension of pellets in PBS of the same volume. DHR 123 (10 μmol/L, end concentration) and DHE (15 μmol/L, end concentration) were added separately to the cell samples for peroxide and superoxide determination. The stained cells were further incubated at 35 ◦C (bacteria) and 30 ◦C (fungi) in the dark with mild shaking. The samples were centrifuged and resuspended in PBS followed by the distribution of the samples into the wells of 96-well microplates. The fluorescence was measured at excitation/emission wavelengths of 500/536 nm for peroxides and 473/521 nm for superoxide detection by a Hitachi F-7000 fluorescence spectrophotometer/plate reader (Auro-Science Consulting Ltd., Budapest, Hungary). The percentage increase in oxidative stress was measured by comparing the signals to those of the growth controls (Gc). Six independent experiments were done with three technical replicates for each treatment.

#### 4.6.3. Time–Kill Kinetics Assay

We followed a protocol previously published by T. Appiah et. al., with modifications [39]. In brief, CPe, CT80, CEt, Van (bacteria), and Cas (fungi) at their respective MEC10 concentrations were used to treat the microbial population of ~10<sup>6</sup> CFU/mL and were incubated at 35 ◦C (bacteria) and 30 ◦C (fungi). One milliliter of the treated and untreated samples was pipetted at time intervals of 0, 2, 6, 8, 16, and 24 h for bacteria, and 0, 6, 12, 30, 36, and 48 h for fungi, and were diluted 10<sup>5</sup> times followed by spreading 50 μL onto 20 mL nutrient agar media using a cell spreader and incubated at 35 ◦C (bacteria) and 30 ◦C (fungi) for 24 h. Van and Cas were used as reference controls for bacteria and fungi. Control without treatment was considered as growth control (Gc). The colony-forming unit (CFU/mL) of the microorganisms were determined, performed in triplicate and was plotted against time (h). Six independent experiments were done with three technical replicates for each treatment.

#### 4.6.4. Live/dead Discrimination of Microbial Cells

For live/dead cell discrimination, we followed the protocol published previously [35]. In brief, the cell population of ~10<sup>6</sup> cells/mL were treated with CPe, CT80, CEt, Van (bacteria), and Cas (fungi) at their respective MEC10 concentrations and were incubated at 35 ◦C (bacteria) and 30 ◦C (fungi). Treated and untreated samples were pipetted at time intervals of 0, 2, 6, 8, 16, and 24 h for bacteria, and 0, 6, 12, 30, 36 and 48 h for fungi followed by centrifugation at 1000 *g* for 5 min, washed, and resuspended in PBS (100 μL/well). One hundred microliters of freshly prepared working dye solution in PBS (using 20 μL SYBR green I and 4 μL propidium iodide diluted solutions as described earlier) were added to the samples. The plate was incubated at room temperature for 15 min in the dark with mild shaking. A Hitachi F-7000 fluorescence spectrophotometer/plate reader (Auro-Science, Consulting Ltd., Budapest, Hungary) was used to measure the fluorescence intensities of SYBR green I (excitation/emission wavelengths: 490/525 nm) and propidium iodide (excitation/emission wavelengths: 530/620 nm), respectively. A green to red fluorescence ratio for each sample and for each dose was achieved and the % of dead cells with the response to the applied dose was plotted against the applied test compound doses using a previously published formula [35]. All treatments were done in triplicates and six independent experiments were performed.

#### *4.7. Statistical Analysis of Microbiological Experiments*

All data were given as mean ± SD. Graphs and statistical analyses were conducted using OriginPro 2016 (OriginLab Corp., Northampton, MA, USA). All experiments were performed independently six times and data were analyzed by one-way ANOVA test. *P* < 0.01 was considered statistically significant. The growth inhibition concentration (MIC90) and minimum effective concentration (MEC10) were calculated using a nonlinear dose–response sigmoidal curve function as follows:

$$y = A\_1 + \frac{A\_2 - A\_1}{1 + 10^{(\log\_x 0 - x)p}}$$

where A1, A2, LOGx0 and p as the bottom asymptote, top asymptote, center, and hill slope of the curve have been considered.

### *4.8. Interaction Study between the Cell Model (Unilamellar Liposomes) and Di*ff*erent Formulations of Chamomile EO*

Unilamellar liposomes (ULs) have been prepared from phosphatidylcholine (Phospolipon 90G, Phospholipid GmbH, Berlin, Germany) by the modified method described before by Alexander Moscho et al. [40]. Phosphatidylcholine was dissolved in chloroform (≥98% stabilized, VWR Chemicals, Debrecen, Hungary) in 0.1 M concentration, and 150 μL of this solution was diluted in a mixture of 6 mL chloroform and 1 mL of methanol. This solution was added dropwise to 40 mL of PBS buffer while stirring on a magnetic stirrer at 600 rpm (VELP Scientifica Microstirrer, Magnetic Stirrer, Usmate Velate MB, Italy). The solvents were removed on a rotational evaporator at 40 ◦C (Heidolph Laborota 4000, Heidolph Instruments GmbH & CO. KG, Germany). The resulting suspension volume was set to 25 mL with PBS buffer and stored in the refrigerator at 8 ◦C until further use. A 5 mL suspension of ULs was mixed with 3 mL Pickering nanoemulsion, conventional emulsion, or ethanolic solution, and the chamomile EO concentration was 100 μg/mL for the different formulations. The mixture was stirred at 600 rpm for 24 h at 35 ◦C, and 1 mL aliquots were taken after 1, 2, and 24 h. The samples were centrifuged at 3000 rpm and 20 ◦C for 5 min, and the ULs were collected and dissolved in absolute ethanol. The chamomile–EO content of samples was determined with UV/VIS Spectroscopy at 290 nm (Jasco V-550 UV/VIS Spectrophotometer; Jasco Inc., Easton, MD, USA). For UV/VIS measurements we have prepared samples without chamomile–EOs, i.e., ULs with SNP suspension, Tween 80 solution, or ethanol were also mixed and centrifuged and were used as blanks.

#### *4.9. GC-MS Analysis of Chamomile EO*

Gas chromatography and mass spectrometry (GC–MS) analyses were carried out on an Agilent Technologies (Palo Alto, CA, USA) gas chromatograph model 7890A with 5975C mass detector. Operating conditions were as follows: column HP-5MS (5% phenylmethyl polysiloxane), 30 m × 0.25 mm i.d., 0.25 μm coating thickness. Helium was used as the carrier gas at 1 mL/min, injector temperature was 250 ◦C. HP-5MS column temperature was programmed at 70 ◦C isothermal for 2 min, and then increased to 200 ◦C at a rate of 3 ◦C/min and held isothermal for 18 min. The split ratio was 1:50, ionization voltage 70 eV; ion source temperature 230 ◦C; mass scan range: 45–450 mass units. The percentage composition was calculated from the GC peak areas using the normalization method (without correction factors). The component percentages were calculated as mean values from duplicate GC–MS analyses of the oil sample. The results of GC–MS analysis can be seen in Supplementary Materials.

**Supplementary Materials:** The followings are available online: Composition of chamomile essential oil analyzed by GC-MS method. Results can be seen in Supplementary Table S1 and Figure S1.

**Author Contributions:** Conceptualization T.K., and A.S.; methodology S.D., B.H., T.K. and A.S.; software, S.D.; formal analysis, S.D. and B.H.; investigation, S.D. and B.H.; GC analysis S.Š. and S.J. resources, A.S.; data curation, S.D., B.H.; writing—original draft preparation, S.D. and B.H.; writing—review and editing, T.K. and A.S.; visualization, S.D. and B.H.; supervision, T.K. and A.S.; funding acquisition A.S. and T.K.

**Funding:** This work was supported by EFOP 3.6.1-16-2016-00004 project (Comprehensive Development for Implementing Smart Specialization Strategies at the University of Pécs), University of Pécs, Medical School, KA-2018-17 and NKFI-EPR K/115394/2015 grants.

**Conflicts of Interest:** The authors declare no conflict of interest.
