## 2 Results and Discussion
### Determination of the Modes of Action of EOEP on B. cereus Using Synchrotron FTIR Microspectroscopy  
#### 2.4.1 Assessment of Antimicrobial Action of EOEP Using Synchrotron FTIR Microspectroscopy  
The effect of EOEP on the cellular components of B. cereus was investigated by Synchrotron FTIR microspectroscopy. FTIR spectra of cells treated with 0.1% EOEP for 0, 6 and 12 h are shown in Figure 4A. The FTIR spectra for most bacteria were divided into four regions comprising the fatty acid in the cell (Region I; peak from 3000 to 2800 cm\({}^{-1}\)), the amide groups from proteins and peptides (Region II; peak from 1700-1500 cm\({}^{-1}\)), the mixed region governed by fatty acids, proteins and phosphate-carrying molecules (Region III; peak from 1500-1200 cm\({}^{-1}\)) and nucleic acid, DNA and RNA (Region IV; peak from 1200-1000 cm\({}^{-1}\)) [32]. The spectra obtained for B. cereus corresponded to those previously described [33; 34]. Strong absorption units obtained for all regions, demonstrating the main components of a cell. To assess the detailed structure of the FTIR spectra and characterize the related variations among bacterial FTIR spectra, the second derivative was analyzed, as shown in Figure 4B,C. The representative FTIR band assignments of biomolecule groups are shown in Table 3.  
Figure 4: FTIR spectra region of B. cereus treated with 0.1% EOEP for 0, 6 and 12 h (**A**) and second derivative transformation spectra of B. cereus treated with 0.1% EOEP for 0, 6 and 12 h (**B**,**C**). Triplicate experiments were averaged.  
The FTIR micrographs revealed the change in cellular components in the spectral intensity at the dominant peaks at 3295, 2925, 2851, 1657, 1541, 1444, 1400, 1240 and 1085 cm\({}^{-1}\). Overall, the cells show significant changes after being treated with EOEP for 6-12 h (\(p\leq 0.05\)). After cells were treated for 6 h, the most apparent change occurs at the DNA/nucleic acid region where the absorptions of bands 1240 and 1085 cm\({}^{-1}\) increase about 4- and 3-fold, respectively. The band at 1240 cm\({}^{-1}\) represented the phosphate group (P=O) asymmetric stretching of phosphodies in phospholipids and RNA [35,36] and 1085 cm\({}^{-1}\) represented a phosphate symmetric stretching in DNA, RNA and phospholipids [36,37]. We also observed a 2-fold increase in the Amide I of \(\alpha\)-helical structures (1650 cm\({}^{-1}\)), the Amide I of \(\beta\)-pleated sheet structures (1630 cm\({}^{-1}\)) and the Amide II (1541 cm\({}^{-1}\)) region after 6 and 12 h of treatment, respectively [35,37]. Moreover, we hypothesized a major disruption in the cell membrane due to the change in the two absorbance bands at 1400 cm\({}^{-1}\) and 1444 cm\({}^{-1}\), with the former attributed to the symmetric stretching of the COO-group in amino acids and fatty acids in the cell membrane [36] and the deformation of the CH\({}_{2}\) of lipids and fatty acids in the cell membrane [35]. The increase in the spectra at 1650, 1541, 1444, 1400, 1240 and 1085 cm\({}^{-1}\) suggests a leakage of cellular components. These changes generally reflect changes in the cell membrane and the leakage of the nucleic acids into the solvent. These results showed the most substantial cellular biomolecule changes in phosphodiesers in nucleic acid, including DNA and RNA, followed by cell membrane and proteinaceous substances after being treated with EOEP for 6 h.