N hexanoyl dl homoserine lactone

Degradation of bacterial signal molecule by vanillin, the experiment with Chromobacterium violaceum CV026 was conducted followed by the method in our previous study [24] . N-Hexanoyl-DL-homoserine lactone (HHL; Sigma-Aldrich, USA) was used as an QS marker in this experiment. Briefly, HHL stock was solubilized in solution with 0.1% acetic acid and 100 mM ethyl acetate. HHL was then diluted with 10 mM potassium phosphate buffer to be concentration of 1 μM. The AHL indicator bacterium, C. violaceum CV026, was cultivated in Luria-Bertani (LB) medium for 24 h, centrifuged, and then rinsed three times with 0.9% NaCl. For the evaluation of QSI activity, 1 mM vanillin was supplemented in to the C. violaceum CV026 culture, and violacein was extracted by a protocol described elsewhere [25] . Finally, the supernatant in which violacien was contained was extracted, and the absorbance was measured using a UV spectrophotometer (Hach, USA) at 585 nm.

Chromobacterium violaceum strain 026 (CV026) was grown in Luria Bertani broth (LB) with 20 µg/mL Kanamycin (SIGMA) and 5 µg/mL of N-Hexanoyl-DL-Homoserine Lactone (SIGMA) for 48 h at a temperature of 32°C with 140 rpm aerobically to an estimated concentration of 10 9 cell/mL. 20 mL of CV026 were mixed with 80 mL soft LB Agar (0.8%, SIGMA) and homogenized. The mixture was poured onto hard Agar (1.5%) to have a double layer, after its solidification, wells were made in the upper layer using a sterile Pasteur pipette. 20 µL of the prepared extract of each dilution was filled in the prepared well. Negative controls consist of distilled water; the positive control was Trans-cinnamaldehyde (SIGMA Aldrich) at 0.1M. Plates were incubated overnight at 32°C, CV026 was used to detect and respond to the presence of Acyl Homoserines lactones (AHL) molecules through the synthesis of purple pigmentation (violacein), absence of the purple color indicated inhibition of violacein and degradation of AHL molecules (degradation of QS activity). The diameters of the non pigmented observed zones were measured (Noorashikin et al., 2016). To confirm the degradation of AHL another assay was performed as follows.