LUANA CAROLINA MARTINS ROSA

Título da Dissertação: INATIVAÇÃO FOTODINÂMICA MEDIADA POR CURCUMINA SOLÚVEL EM ÁGUA CONTRA PATÓGENOS DE ORIGEM ALIMENTAR

Orientadora: Prof.ª Dr.ª Jane Martha Graton Mikcha

Data da Defesa: 18/02/2022

 GENERAL ABSTRACT
INTRODUCTION. Safe and nutritious foods are essential to ensure a good quality of life, however, the presence of pathogenic microorganisms in food represents a serious public health problem. In addition, to having health consequences, it affects social, environmental, and economic development. Food can be contaminated at any stage of production and the methodologies currently used are not always effective for microbial control; often limited to the type of food as they result in changes in sensory and nutritional characteristics. A promising methodology for microbial control is the antimicrobial photodynamic therapy – aPDT. This technique consists of the use of a photosensitive compound that, in the presence of light, produces reactive oxygen species that result in damage that causes the death of microorganisms. Curcumin is a natural yellow colorant widely used by the food industry that, when applied in photodynamic therapy, has shown to be effective and promising against microorganisms. However, the use of curcumin is restricted as it is extremely hydrophobic. To overcome this problem, water-soluble formulations are a viable alternative. In addition, studies indicate better action of curcumin at low pH, potentiating antimicrobial activity. AIMS. The present study aimed to evaluate the in vitro susceptibility of Staphylococcus aureus and Escherichia coli to water-soluble curcumin (WSC) and the compound exposed to visible blue light at acidic pH with organic acids. Furthermore, this work purposed to apply an experimental design and surface response methodology to evaluate the interaction of WSC concentration at pH 5.0 and illumination time against Staphylococcus aureus, Salmonella Typhimurium, and Escherichia coli.
MATERIAL AND METHODS. The WSC was obtained in partnership with the Federal University of Technology – Paraná (Campo Mourão campus). The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined using the broth microdilution method in 96-well microplates. Based on the results obtained, conditions for aPDT were established at acidic pH with concentrations of curcumin ranging from 3.90 to 125 μg/mL. The samples were submitted to treatments with different illumination times of 2.5, 5, and 10 min with a blue LED light system built to illuminate a 96-well plate, with an irradiance of 16 mW/cm² and a wavelength of 450 nm. The results found were expressed in log CFU/mL to determine the reduction in cell viability. In addition, an experimental model was proposed to determine the optimal conditions for the application of photodynamic therapy, using a Rotable Central Composite Design. The design proposed eight experiments with four replications of the center point to evaluate the combined effects of lighting time and curcumin concentration.
RESULTS AND DISCUSSION. For S. aureus, the combination of WSC with lactic acid and 2.5 min of illumination reduced the MIC from 500 μg/mL to 15.62 μg/mL, and WSC with citric acid and 5 min of exposure to light reduced the MIC from 125 μg/mL to 7.81 μg/mL. While for E. coli, curcumin alone did not inhibit bacterial growth, however, when exposed to 5 min of illumination with a concentration of 62.5 μg/mL curcumin with lactic acid and curcumin at 7.81 μg/mL with citric acid, no viable cells were recovered. The models sowed predictive capacity (R2adj) of 64% for S. aureus, 91% for E. coli, and 81% for S. Typhimurium. The models demonstrated that there was no significant interaction between exposure time and photosensitizer concentration for S. aureus and E. coli. The greatest reductions observed for S. aureus were approximately 5.16 log CFU/mL at 4.41 μg/mL and 6.77 min of illumination time, while higher concentrations and illumination times were able to inhibit E. coli and S. Typhimurium growth.
CONCLUSIONS. The results demonstrated that WSC when combined with blue LED light was more effective against foodborne pathogens than to non-irradiated compounds. Low illumination times and photosensitizer concentrations were sufficient to eradicate Staphylococcus aureus and Escherichia coli. The application of experimental design was effective for the optimization of photodynamic therapy, providing information on photodynamic activity against microorganisms in different combinations of photosensitizer concentration and lighting time, reducing the number of experiments and costs.
Keywords: Curcumin, Foodborne pathogens, Photodynamic therapy

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