PLE was carried out using an accelerated solvent extractor system (ASE 150, Dionex, Sunnyvale, CA, USA). Sample was prepared by mixing 1.5 g of milled malt rootlets and 9 g of sand (used as dispersive agent) which was displayed in a stainless-steel extraction cell (10 mL). A cellulose filter (2.5 cm diameter, Whatman) was fixed at the cell bottom to avoid particles filtration. Initial equipment conditioning involved preheating at 1500 psi for 6 min followed by 100 s of nitrogen purge. Solvents were previously degassed in an ultrasonic bath for 30 min. Optimization of extraction conditions (extraction time, percentage of EtOH, and extraction temperature) was performed by a Box-Behnken incomplete factorial experimental design using the protein content as response variable and fitting the data to a quadratic equation similar to the previously shown for the optimization of UAE variables. As previously, the fitting of the regression model was assessed by ANOVA and the determination of R2.
All extracts were evaporated in a centrifugal concentrator (Eppendorf AG, Hamburg, Germany) and stored at −20 °C until use. Before use, solid samples were solubilized in a 5 mM phosphate buffer (pH 8).
All extracts were evaporated in a centrifugal concentrator (Eppendorf AG, Hamburg, Germany) and stored at −20 °C until use. Before use, solid samples were solubilized in a 5 mM phosphate buffer (pH 8).