Orientadora: Prof. Dra. Jane Martha Grathon Mikcha

Data da Defesa: 21/02/2017



INTRODUCTION: Microbial contamination of food can cause foodborne illnesses to generate economic, social and environmental impact. Photodynamic therapy is an emerging technology that has been successfully applied in several areas, is effective in controlling microorganisms and can be a promising alternative in the food industry. This treatment is based on the use of a nontoxic photosensitizer activated by visible light. Among the available photosensitizers, Eosin Y is a xanthene dye, with properties of generating singlet oxygen species with effective action in the inactivation of microorganisms.
AIM: The aim of the present study was to evaluate the effect of photodynamic therapy using Eosin Y in foodborne and food spoilage bacteria.
MATERIAL AND METHODS: In this study, Gram-negative bacteria: Salmonella enterica serotype Typhimurium ATCC 14028, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 and the Gram-positive bacteria: Staphylococcus aureus ATCC 25923 and Bacillus cereus ATCC 11778 were evaluated. Eosin Y was applied as a photosensitizer in the photodynamic inactivation experiments at concentrations of 0.5, 5 and 10 μM for Gram-negative bacteria and 0.1, 0.25, 0.5, 1, 2.5, 5 and 7.5 μM for the Gram-positive. The light source used was green LED (λ max 495-570 nm) and the light doses were calculated according to the concentration of the photosensitizer at ilumination times of 5, 10 and 15 min. The bacterial inoculum of each isolate was standardized to contain approximately 108 CFU (Colony Forming Unit)/mL and diluted to 107 CFU/mL for use in photodynamic inactivation tests. For the photoinactivation assay, three control groups were used: positive control (bacterial suspension), photosensitizer control (bacteria and eosin without light), and light control (bacteria exposed to LED without the photosensitizer). Aliquots of 50 μL of each standard bacterial suspensions were incubated with 950 μL of eosin Y solution at different concentrations and kept in the dark for 10 min. After incubation, 500 μL of the sample was LED illuminated for 5, 10 and 15 min. After, serial dilutions of the treated and control samples were plated on Trypticase Soy Agar (TSA) and incubated at 37 ° C for 24h. The colony counts were performed and the cell viability results were expressed in log CFU/ml. Analysis of photoinativated S. aureus with 0.5 and 1 μM eosin was performed at 5, 10 and 15 min by flow cytometry using the LIVE/DEAD Kit with Syto 9 and propidium iodide. Morphological changes of S. aureus treated with 1.0 μM eosin for 15 min were examined by scanning electron microscopy.
RESULTS AND DISCUSSION: For all species evaluated, the photosensitizer without light application (photosensitizer control) and the LED without the photosensitizer (light control) had no effect on bacterial viability. Among the Gram-negative bacteria, P. aeruginosa was the most sensitive presenting total inactivation when treated with 10 μM eosin and illuminated for 10 and 15 min (106.2 and 159.3 J/cm2, respectively). Significant reductions were observed in the other treatments when compared to the control, except with treatment of 0.5 μM illuminated for 5 min (5.4 J/cm2). The antimicrobial activity was dependent on the photosensitizer concentration and the illumination time. E. coli and S. Typhimurium did not show significant reductions when compared to control. All treatments were able to significantly reduce the number of CFU/mL of B. cereus compared to the control group, where mean reductions of 2.7 log/UFC were observed. S. aureus was the most sensitive bacteria to photodynamic inactivation mediated by Eosin Y. Treatment with 5 μM photosensitizer caused total photoinitiation of the bacteria in the shortest time of illumination (5 min; 38.2 J/cm2). The highest reductions were obtained with the combination of 15 min of illumination and eosin at 0.5 and 1 μM, where reductions of 2.5 and 3 log/UFC, respectively, were obtained. The effectiveness of the photodynamic inactivation was dependent on the photosensitizer concentration and the illumination time for S. aureus. Although the efficacy of several photosensitizers in the control of microorganisms has been described, few studies have evaluated the use of eosin in microbial inactivation. Johnson et al. (2013) evaluated the photodynamic activity of conjugated Eosin Y with the peptide (KLAKLAK)2 against P. aeruginosa, E. coli and S. aureus. Eosin Y at 10 μM illuminated for 30 min had no effect on bacterial viability. According to our knowledge, no studies were found with eosin in the photodynamic inactivation of Salmonella spp. and B. cereus. The integrity of the S. aureus membrane treated with eosin at 0.5 and 1 μM and with illumination times of 5, 10 and 15 min was analyzed by flow cytometry. The positive control (bacterium with PBS) revealed that 96.2% of the labeled cells exhibited strong green fluorescence, positive for Syto 9 and 1.6% showed strong red fluorescence, stained with propidium iodide. When S. aureus was exposed to 0.5 μM eosin and 5 min illumination, 79.1% viable cells and 16% cells with membrane damage were obtained. At 10 min of exposure, 71.7% of viable cells and 16.9% dead cells were obtained. Treatment with eosin at the maximum illumination time (15 min) revealed 59% viable cells and 30.9% dead cells. By increasing the concentration of the photosensitizer to 1 μM and maintaining the illumination times were obtained 74.1% viable cells and 16.6% dead cells (5 min), 68.7% viable cells and 17% dead cells (10 min) and 48.1% viable cells and 33.9% dead cells (15 min). The results of flow cytometry confirm the results obtained in the photoinactivation of S. aureus. The morphological changes of S. aureus induced by photodynamic inactivation with 1 μM eosin and 15 min of illumination were visualized by scanning electron microscopy. Bacteria without treatment (positive control) presented a smooth cell surface, with uniform and characteristic morphology, while the photoinactivated cells presented distortions in the membrane and in the morphology, appearing wrinkled and wilted.
CONCLUSIONS: Photodynamic inactivation mediated by eosin Y and green LED light was effective in inactivate foodborne bacteria. S. aureus was the most susceptible to photodynamic treatment; on the other hand, E. coli and S. Typhimurium were the most resistant. Photodynamic inactivation could be a promising alternative for bacterial control in food industry.
Key words: Photodynamic inactivation; Pathogenic bacteria; Spoilage bacteria; Eosin Y; LED.

 Artigos Publicados Vinculados a Dissertação:

doi: 10.1111/jam.13727