Biotransformação fúngica da hesperetina e sua aplicação na produção de metabólitos ativos

Abstract

Hesperetin belongs to the flavanone class of flavonoids, its human metabolites (i.e. hesperetin-3’-sulfate, hesperetin-7-sulfate, hesperetin-3’-glucoronide and hesperetin -7-glucoronide), and its glycosylated derivative has potential pharmacological properties. The use of microorganisms, such as filamentous fungi, is an alternative in derivatives production and a useful tool in scale up process. Therefore, the goal of the work was to apply and evaluate different bioprocesses for the production of active metabolites of hesperetin by fungal biotransformation. Application of microbioreactors, use of different carbon sources, substrate concentration, fungal immobilization and monitored reactions by liquid chromatography coupled to high resolution mass spectrometry were some of evaluated bioprocesses. From 15 tested fungi, 12 strains were able to biotransform hesperetin. In silico metabolite prediction modeling with MetaPrint 2D software, indicated positions 7 e 3’ as energetically favored for glycosylation, glucuronidation and sulfation reactions. Mortierella isabellina NRRL 1757 and Beauveria bassiana ATCC 7159 produced only sulfate and glucuronide hesperitins, respectively. Cunninghamella echinulata ATCC 9244 strain produced the glycosylated metabolite. A morphological difference was observed in C. echinulata ATCC 9245 growth using different microbioreactors. Square microwell plates resulted in an amorphous growth whereas round bottom microwell plates favored pellets growth. The latest, with volume well of 2.2 ml led to a higher content of fungi and 48 hours was found to be the ideal time to substract addition. All four strands of Cunninghamella tested for biotransformation in a microscale were able to produce the glycosylated metabolite. Analyzes of the biotransformation kinetics by HPLC-HRMS showed that Cunninghamella echinulata ATCC 9244 produced four microplate derivatives: eriodictiol, sulfated hesperetin, glycosylated hesperetin and glucuronidated hesperetin. Such analyzes have also shown that high concentrations of hesperetin inhibit these derivatives formation. Stainless steel sponge proved to be efficient for Cunninghamella echinulata and Cunninghamella elegans strains immobilization. The immobilization process achieved biofilm in 48 hours. ESI-IT-MS and ESI-IT-MS/MS analysis indicated production of a glycosylated and a hydroxylated-sulfonated derivative. Thus, studies demonstrated that different bioprocesses can be applied to optimize scale up reactions and produce hesperetin derivatives using filamentous fungi