LAUSR

Given the growing interest in phytoprostanes (PhytoPs) and phytofurans (PhytoFs) in the field of plant physiology, biotechnology, and biological functions, the present study aimed to optimize a method of enzymatic hydrolysis which utilizes bacterial and yeast esterases that allow the appropriate quantification of PhytoPs and PhytoFs. To obtain the highest concentration of PhytoPs and PhytoFs, a response surface methodology/Box-Behnken design was used to optimize the hydrolysis conditions. Based on the information available in the literature on the most critical parameters which influence the activity of esterases, the three variables selected for the study were temperature (ºC), time (min), and enzyme concentration (%). The optimal hydrolysis conditions retrieved differed between PhytoPs (21.5 ºC, 5.7 min, and 0.61 μg enzyme per reaction) and PhytoFs (20.0 ºC, 5.0 min, and 2.17 μg enzyme per reaction), and provided up to 25.1 and 1.7-fold higher contents relative to non-hydrolyzed extracts. The models were validated by comparing theoretical and experimental values for PhytoPs and PhytoFs yield (1.01 and 1.06 theoretical/experimental rates, respectively). The optimal conditions were evaluated on their relative influence on the yield of individual non-esterified PhytoPs and PhytoFs in order to define the limitation of the models for obtaining the highest concentration of most considered compounds. In conclusion, the models developed provided valuable alternatives to the currently-applied methods using unspecific alkaline hydrolysis to obtain free non-esterified PhytoPs and PhytoFs, which give rise to a more specific hydrolysis of PhytoPs and PhytoFs esters, reducing the degradation of the free compounds by classical chemical procedures.