## 3 Results and Discussion
### In Vitro Experiments  
#### 3.2.1 Effect of Photosensitization on _A. flavus_ Spores  
The efficiency of _A. flavus_ spores' inactivation by photosensitization was dependent on the light dose applied for the treatment. Whereas, using the same solvent (ethanol or propylene glycol), the tested concentrations of curcumin did not show a significant difference (\(p>0.05\)) in their inactivation efficiency. Photosensitization (P\({}^{+}\)L\({}^{+}\)) significantly (\(p<0.05\)) reduced the viability of _A. flavus_ spores using light doses above 34.7 J\(\cdot\)cm\({}^{-2}\) (Figure 2). Compared to the negative control (P\({}^{0}\)L\({}^{0}\)), no significant change was observed on the population of spores that were either treated with the photosensitizer (P\({}^{+}\)L\({}^{0}\)) or light (P\({}^{0}\)L\({}^{+}\)) (Figure 2), indicating a synergic effect from the combination of light and curcumin in reducing the multiplication of _A. flavus_. The efficiency of inactivation was not compared between the solvents as different concentrations of curcumin were used for each solvent. An increase in the light dose to 86.9 J\(\cdot\)cm\({}^{-2}\) enhanced photosensitization efficiency. However, further increase of light dose to 130.3 J\(\cdot\)cm\({}^{-2}\) did not significantly increase (\(p>0.05\)) the efficiency (Figure 2). The presence of curcumin in ethanolic solution resulted in the highest inactivation with 1.9 log CFU\(\cdot\)mL\({}^{-1}\) reduction at 104.2 J\(\cdot\)cm\({}^{-2}\) using 25 \(\upmu\)M curcumin. However, the same light dose combined with 40-fold higher curcumin concentration (1000 \(\upmu\)M prepared in propylene glycol) resulted in 2.3 log CFU-mL\({}^{-1}\) reduction. Since curcumin is partially soluble in propylene glycol, higher concentrations of the photosensitizer are required to achieve a significant fungal inactivation as previously reported [24]. Treatments with light doses below 34.7 J\(\cdot\)cm\({}^{-2}\) did not show any significant effects on the viability of _A. flavus_ spores (\(p>0.05\)) compared to the negative control P\({}^{0}\)L\({}^{0}\) (data not shown). Furthermore, the spores did not exhibit any susceptibility to ethanol or propylene glycol solutions, as evaluated by the inhibition zone through well-diffusion assay, suggest  
Figure 1: Temperature profile inside the light chamber upon blue LED illumination at three different irradiances (10.4, 28.9 and 36.2 mW\(\cdot\)cm\({}^{-2}\)). ing that the solvents used are non-toxic to the _A. flavus_ spores at the concentrations applied in this study. Although an increase in temperature inside the box was recorded during illumination, the microbial inactivation was achieved as a result of a photodynamic effect of curcumin in the presence of light, but not the heating effect. This was ascertained by submitting the spores to the light alone, which did not show a significant effect on spores' viability even though a temperature as high as 42.27 \({}^{\circ}\)C was achieved for 130.4 J-cm\({}^{-2}\) light dose. _Aspergillus flavus_ can grow in temperatures ranging from 12 to 48 \({}^{\circ}\)C, although the optimum growth occurs at 37 \({}^{\circ}\)C [33]. Moreover, variation of fluid temperature from 15 to 45 \({}^{\circ}\)C has been reported to have no significant impact on _A. flavus_ viability [23]. Therefore, the temperatures achieved upon LED illumination were not high enough to inactivate the spores. The phototoxicity of curcumin has been demonstrated against fungi, bacteria and virus [8; 9; 24]. Although the exact mechanism of microbial inactivation through photosensitization is still not well understood, it has been proposed as a result of ROS formation when the photosensitizer is illuminated at an appropriate wavelength [34; 35]. In turn, ROS react with cellular components such as lipids, proteins and nucleic acids, ultimately causing microbial cell death [5].