Model 8300

Fourier-transform infrared (FT-IR) spectrophotometry was done on B. subtilis cells in a FT-IR spectrophotometer (Shimadzu, model 8300, Shimadzu, Kyoto, Japan), as described by [14 (link)]. Cells were cultivated in LB medium (29 °C) up to the concentration of 10⁵ cells/mL, then treated with BC1 (50 µg/mL), T9A (40 µg/mL), nisin (5 µg/mL), or a negative control without the addition of any compound for 30 min. Aliquots of 1.5 mL of cells were then centrifuged at 10.000 x g for 2 min, washed with water, and centrifuged two more times to remove traces of medium. After that, the samples were air-dried, and mixed with 150 mg of KBr. Next, samples were compressed at 40 kN for 5 min. Absorbance was measured from 400 cm⁻¹ to 4000 cm⁻¹, with 32 scans at a resolution of 4 cm⁻¹. Data treatment and analyses were performed using the software Origin 8.00 (OriginLab, Northampton, MA, USA).

FT‐IR spectroscopy was performed according Zeroual, Manfait, and Choisy (1995) with some modifications. Cells were analyzed before and after treatment with the compounds using the FT‐IR spectrophotometer Shimadzu, Model 8300. Briefly, bacteria were cultivated in liquid medium up to the concentration of 10⁵ cells/mL; after growth, cells were centrifuged at 10,000 g for 2 min in order to form pellets at the bottom of 1.5 mL microcentrifuge tubes. Cells were dissolved in deionized water, centrifuged and then washed two more times in water to remove traces of medium. After that, cell mass was mixed together with 149 mg of KBr. To obtain the FT‐IR spectra of treated cells, compounds were added to the cultures at MIC90 and allowed to react for 30 min before the centrifugation steps. Nisin was added as a control for membrane pore formation at 5 μg/mL. The dry samples with KBr were homogenized and compressed at 40 kN for 5 min in preparation for the FT‐IR readings. Absorbance was analyzed over a range of 400 to 4000 cm⁻¹, with 32 scans at a resolution of 4 cm⁻¹. Data treatment and analyses were performed using the software Origin 8.00.

One gram of carvacrol (Sigma-Aldrich^® St. Louis, MO, USA—W224502) was added to acetic anhydride (15 mL) and sodium acetate (1.5 g). Acetic anhydride acted as an acetylating agent, and sodium acetate acted as a catalyst. The mixture was refluxed for 1 h. The room temperature solution was added to water (20 mL) and neutralized to pH 7.0 with 5% sodium bicarbonate. The reaction mixture was transferred to a separatory funnel and washed three times with chloroform (100 mL). The chloroform layer containing acetylated material was washed with water and then dried over sodium sulfate. The solvent was evaporated [14 ]. The product was subjected to thin-layer chromatography and characterized by infrared spectroscopy (FTIR) (Model 8300 Shimadzu Corporation, Kyoto, Japan).