# Antibacterial Activity of Crude Extract and Purified Acetogenins from _Amona muricata_ Seeds
## 2 Materials and Methods
### Antibacterial Assays of Crude Extract and Purified Acetogenins  
The antibacterial assays were performed using the agar disk diffusion method [23] with modifications. Gram-negative strains (_A. hydrophilla, Escherichia coli, Klebsiella pneumoniae, Salmonella paratyupli, Salmonella abatetuba, Salmonella cholerasius, and Burkholderia encocapacia_) and Gram-positive strains (_Listeria monocytogenes, S. aureus, E. faecalis, S. salivaris, S. mutans_, and _Streptococcus mitis_) were used during the experiment. The strains were aerobically cultivated for 24 h at 37 \({}^{\circ}\)C in a nourishing broth (8 g/L, pH 7.0 \(\pm\) 0.1) until the bacterial suspensions reached 1 \(\times\) 10\({}^{6}\) CFU/mL according to McFarland standard (0.5 OD). Sterile forceps were used to insert the discs of sterile filter paper (7 mm in diameter) into Petri dishes with nutrient agar (23 g/L, pH 6.8 \(\pm\) 0.1). The filter paper discs were impregnated according to treatment: 200 uL sterile distilled water (negative control), ampicillin at 500 ug/mL and dimethyl sulfoxide (DMSO) (a positive control), and solutions of CE or P-ACGs at different concentrations (12.5, 25, 50, 100, 200, 400, 800, 1000, 2000, and 4000 ug/mL). The inoculated Petri dishes were incubated for 24 h at 37 \({}^{\circ}\)C, and the inhibition halo (IH) in mm\({}^{2}\) formed around the discs was measured using a vernier. Minimum Inhibitory Concentration (MIC) of the Crude Extract and Purified Acetogenins against Gram-Positive and Gram-Segative Bacteria  
MIC was calculated according to Parhusip and Sitanggang [24]. CE and P-ACGs concentrations (12.5-4000 ug/mL) were converted to values of natural logarithm (Ln), and data of IH were converted to values of squared millimeters (mm\({}^{2}\)). Then, a plot was constructed (squared millimeters of the IH vs. Ln concentrations of CE or P-ACGs). A linear regression model (R\({}^{2}\)) was realized to obtain Equation (1). Equation (1) was used to obtain Equation (2), where \"X\" is the Ln concentration, \"y\" is the minimum value of the IH in mm\({}^{2}\), b is the intercept of the _X_-axis, and \"m\" is the slope. Subsequently, Equation (3) was used to calculate MIC. \[\mathrm{y} = \mathrm{mx}+\mathrm{b} \tag{1}\] \[\mathrm{X} = \frac{\mathrm{y}-\mathrm{b}}{\mathrm{m}}\] (2) \[\mathrm{MIC} = \mathrm{e}^{\mathrm{x}}\times 0.25 \tag{3}\]  
Inhibition Percentage of the Most Prevalent Harnful Bacteria Found in Foods: Enterococcus faecalis, Salmonella paratyphi, Escherichia coli, and Listeria monocytogenes by the Crude Extract and Purified Acetogenins  
According to results in the antibacterial activity, the inhibition percentage among harmful bacteria prevalent in food was calculated based on the procedure outlined by [PERSON] et al. [25] (Equation (4)). \[\mathrm{Inhibition\ percentage(\%)}=\frac{\mathrm{TS}-\mathrm{NC}}{\mathrm{PC}} \times 100 \tag{4}\]  
Tested sample (TS), negative control (NC) and positive control (PC) were calculated in millimeters (mm). All procedures described above were performed in triplicate for each bacterial strain using both CE and P-ACGs. Measurement of the Membrane Potential of Enterococcus faecalis, Listeria monocytogenes, Salmonella paratyphi, and Escherichia coli Treated with the Crude Extract and Purified Acetogenins  
Foodborne pathogens were also selected to evaluate the membrane potential. The bacterial strains (10 times diluted in saline) were dyed using Molecular Probes LIVE/DEAD BacLight kit, distinguishing between bacteria with intact and damaged plasma membranes [26]. Briefly, pathogenic strains were cultivated for 24 h at 37 \({}^{\circ}\)C in nutrient broth, and the biomass was recovered by centrifugation (11,624\(\times\) g, 1 min). Subsequently, the pellets were washed twice with sterile phosphate-buffered saline (PBS) solution and resuspended (1 \(\times\) 10\({}^{6}\) CFU/mL) in a sterile PBS.