*2.4. Characterization of the Extract and of Microencapsulated Powders*

Both the extract and the powders were characterized for flavonoids, total phenolic compound contents and antioxidant activity using the aluminum chloride method, Folin– Ciocâlteu and DPPH method, respectively, as described by Milea et al. [12]. The total flavonoid contents are expressed in mg quercetin equivalents (QE)/g dry weight (DW), whereas the total polyphenol contents are expressed in mg gallic acid equivalents (GAE)/g DW. In each case, the concentrations of bioactives and antioxidant activity were expressed through selected standard calibration curves.

The encapsulation efficiency of the powders was calculated as described by Saénz et al. [15]. In brief, the microencapsulation efficiency was determined by assessing the surface flavonoid contents (SFC) and total flavonoid contents (TFC) of the powders, expressed as mg QE/g DW. In order to quantify the SFC, 50 mg of powders was mixed with 5 mL of ethanol and methanol (ratio 1:1, *v*/*v*). These dispersions were stirred at room temperature for 1 min and then centrifuged at 4000× *g* and 4 ◦C for 10 min. For TFC, 50 mg of powder was accurately weighed and dispersed in 5 mL of a mixture of ethanol, acetic acid, and water (50:8:42, *v*/*v*/*v*). The resulting dispersion was vortexed (1 min), followed by ultrasonication for 30 min at 40 ± 1.0 ◦C, to break the microcapsules. The supernatant was centrifuged at 14,000× *g* for 10 min and then filtered. The content of flavonoids in the resulting supernatants was measured by the aluminum chloride method, as explained by Milea et al. [12]. The microencapsulation efficiency (ME, %) was calculated using Equation (1):

$$\text{ME} \left( \% \right) = \frac{\text{TFC} - \text{SFC}}{\text{TFC}} \times 100 \tag{1}$$

The antioxidant activity was assessed using the DPPH method. Briefly, 0.1 mL of supernatant resulted from TFC determination was added to 3.9 mL of DPPH stock solution. The DPPH stock solution was prepared mixing 3 g of DPPH with 100 mL of methanol. Simultaneously, a control sample was prepared by adding 0.1 mL methanol to 3.9 mL

DPPH. The absorbance for both samples was read at 515 nm after 1.5 h. The results were calculated using a calibration curve and are expressed in mMol Trolox/g DW.
