Estudo do mecanismo de ação antifúngica de quitooligossacarídeos de quitosana e prospecção de outras aplicações biotecnológicas

Abstract

Chitosan is a natural polysaccharide derived from chitin that has several biotechnological applications. However, insolubility at neutral pH values and high viscosity are factors that limit the applicability of this polymer. On the other hand, the oligosaccharides obtained from chitosan hydrolysis, called chitooligosaccharides (QOS), do not have the same physicochemical limitations and present biological activities that can have practical applications, such as antimicrobial, insecticidal, antitumor, and anti-HIV action. as well as elicitation of plant defenses. QOS can be produced by physical, chemical and enzymatic methods, however the enzymatic method is more efficient in producing molecules with a well-defined degree of polymerization and does not cause harm to the environment. The present work aimed to explore the potential of QOS produced enzymatically by chitosanase CvCsn46 from Chromobacterium violaceum against the following biological activities: antifungal action, fruit protection and insecticidal action. The investigation into the antifungal activity against different Candida species (C. albicans, C. krusei, C. parapsilosis and C. tropicalis) showed that the QOS produced presented inhibitory activity against 8 of the 10 strains used, with the MIC varying from 78 to 1,250 μg/mL and lethal activity against 6 to 10 strains, with CLM ranging from 156 to 625 μg/mL. Deepening the analyzes with two non-albicans species (C. krusei ATCC 6258 and C. parapsilosis ATCC 22019), it was observed that the death time of C. krusei when in contact with QOS at the lethal concentration (312 μg/mL) is 4 hours and for C. parapsilosis (CLM = 312 μg/mL) it is only 2 hours. Results of tests related to biofilm formation and degradation showed that although QOS had no effect on biofilm formation in the two species tested, these molecules were able to degrade 41.5% of the C. krusei biofilm and 22.5% of the C. parapsilosis biofilm when twice the MIC was used. Analysis of the mode of action of QOS showed that these molecules severely affect the cell membrane of these yeasts, in addition to stimulating the production of reactive oxygen species and causing morphological damage, evidenced by flow cytometry, fluorescence microscopy and electron microscopy analyses. scan. In the analyzes on the ability of QOS to protect fruits in the post-harvest phase, minimally processed Cantaloupe melon was used. Fruits treated with QOS (1 g/L) were able to maintain their firmness, pH and vitamin C content when compared to the control, in addition to demonstrating better total antioxidant and enzymatic antioxidant capacity. Regarding the insecticidal effect, although QOS did not demonstrate larvicidal activity against Aedes aegypti larvae when these molecules were used alone, QOS was able to enhance the larvicidal activity of Bt toxins. The joint action of LC20 (11.01 ng/mL) of Bt toxins with QOS in different

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concentrations (from 500 to 32 μg/mL) caused severe damage to the midgut of treated larvae. The present work demonstrated the activity of chitooligosaccharides produced by C. violaceum chitosanase against fungi, in the protection of post-harvest fruits and against insects, highlighting the potential of QOS in several areas.