Antimicrobial Photodynamic Inactivation Mediated by Rose Bengal and Erythrosine Is Effective in the Control of Food-Related Bacteria in Planktonic and Biofilm States

The thermal and chemical-based methods applied for microbial control in the food industry are not always environmentally friendly and may change the nutritional and organoleptic characteristics of the final products. Moreover, the efficacy of sanitizing agents may be reduced when microbial cells are enclosed in biofilms. The objective of this study was to investigate the effect of photodynamic inactivation, using two xanthene dyes (rose bengal and erythrosine) as photosensitizing agents and green LED as a light source, against Staphylococcus aureus, Listeria innocua, Enterococcus hirae and Escherichia coli in both planktonic and biofilm states. Both photosensitizing agents were able to control planktonic cells of all bacteria tested. The treatments altered the physicochemical properties of cells surface and also induced potassium leakage, indicating damage of cell membranes. Although higher concentrations of the photosensitizing agents (ranging from 0.01 to 50.0 μmol/L) were needed to be applied, the culturability of biofilm cells was reduced to undetectable levels. This finding was confirmed by the live/dead staining, where propidium iodide-labeled bacteria numbers reached up to 100%. The overall results demonstrated that photoinactivation by rose bengal and erythrosine may be a powerful candidate for the control of planktonic cells and biofilms in the food sector.


Light doses calculations
The absolute irradiance of the LEDs was evaluated in a Spectroradiometer obtained by Ocean Optics model USB2000 +. The power absorbed (PAbs) and number of absorbed photons (NAbs) were performed following the methodology described previously [19,20].
The number of photons emitted (NEm) by a monochromatic light source (LASER), was calculated by Equation S1, corresponding to a given energy inherent to the photon frequency: where E is the energy (in J), h is the Planck constant (6.626×10 -34 , in J s) and υ is the frequency (in s -1 ). Equation S1 can be rearranged to provide Equation S2, which includes Avogadro's number (Na: 6.022×10 23 / mol) to represent the equation in number of photon moles emitted (equivalent to Einsteins): gives the number of photon moles (Einsteins) emitted by a monochromatic light source. When using polychromatic light sources (such as LEDs), light irradiation must be considered throughout the spectral region. Thus, the emitted power (PEm) can be obtained by Equation S3: Where λi and λf are the initial and final wavelengths of the LED irradiation spectrum, respectively. Substituting the value of Pem in Equation S3 it is obtained: The fraction of light absorbed by a given sample can be defined as: can be used only for a specific λ, it means, monochromatic irradiation sources. For polychromatic sources, such as LEDs, it is necessary to consider the value of XAbs over the entire spectrum of electronic absorption of PS. For that Equation S6 considers the light fraction throughout the spectral region.
The absorbed power (PAbs) results from the product between the total power emitted and the absorbed light fraction (Equation S7).

= (Equation S7)
For a polychromatic light source PAbs is defined by Equation S8.
Thus, these terms can be inserted in Equation S4, number of photons emitted (NEm) to obtain the equation of the number of absorbed photons (NAbs). Therefore, to determine the number of photons absorbed by a PS employing a monochromatic irradiation source, the term Pem is replaced by the term PAbs in Equation S4, obtaining Equation S9 (in Einsteins): For polychromatic irradiation sources, it is necessary to consider the power absorbed by the entire LED/PS overlap spectral region: Finally, it is possible to obtain the actual light dose (J/cm 2 ) absorbed by the PS from Equation S11: in which A is the irradiated area, t is the illumination time, PAbs is the power absorbed by the PS.  Figure S1. Survival of (a,b) E. coli, (c,d) S. aureus, (e,f) E. hirae and (g,h) L. innocua planktonic cells exposed only to the light source during 20 min or only to RB (left) and ERY (right). Values are shown as medians, including 25 and 75% quantiles of at least three independent experiments. Figure S2. Survival of (a,b) E. coli, (c,d) S. aureus, (e,f) E. hirae and (g,h) L. innocua biofilms cells exposed only to the light source during 60 min or only to RB (left) and ERY (right) for 60 min. Values are shown as medians, including 25 and 75% quantiles of at least three independent experiments.