2.2.6. DPPH Radical Scavenging Activity Assay

The radical scavenging activity was determined spectrophotometrically by the 2,2 diphenyl-1-picrylhydrazyl (DPPH) method as described by Păucean et al. [40]. The phenolic extracts (0.1 mL) were mixed with DPPH solution (3.9 mL), kept in the dark at ambient temperature for 30 min. The absorbance of the mixtures was recorded at 515 nm (1700 UV/VIS spectrophotometer Shimadzu) against a methanol blank. Negative control was prepared using 0.1 mL methanol and 3.90 mL of DPPH. The radical scavenging activity was calculated according to the following Equation (2):

$$RSA[\%] = \frac{Abs\_{DPP} - Abs\_{sample}}{Abs\_{DPP}} \times 100\tag{2}$$

where *AbsDPPH* = absorbance of DPPH solution and *AbsSample* = absorbance of the sample.

#### 2.2.7. Volatile Compounds

Qualitative analyses of the volatile compounds were achieved using "in-tube extraction" coupled with gas-chromatography mass spectrometry (ITEX/GC-MS, Shimadzu GC-MS model QP-2010) [45]. Three grams of each sample were weighed into a 20 mL sealed-cap headspace vial and incubated for 30 min at 70 ◦C with continuous agitation. The volatile compounds from the gaseous headspace of the sample were repeatedly adsorbed onto a microtrap (ITEX-2TrapTXTA, TA 80/100 mesh, Tenax, Zwingen, Switzerland) using

a CombiPAL AOC-5000 autosampler (CTC Analytics, Zwingen, Switzerland). The volatile compounds were further thermally desorbed into the injection port of the GC-MS QP2010 system and separated on a ZB—5 ms column of 30 m × 0.25 mm i.d. and 0.25 μm film thickness (Phenomenex, Torrance, CA, USA). The column oven temperature program was set initially at 40 ◦C (kept at this temperature for 6 min) and then increased to 50 ◦C at 2 ◦C/min, followed by an increase to 240 ◦C at 7 ◦C/min (kept at this temperature for 5 min). The temperatures of the ion source, injector, and interface were set at 250 ◦C. The carrier gas was helium at a flow rate of 1 mL/min. The split ratio was 1:2. The MS detector was operated in the EI mode, using a scan acquisition mode in the range of 40–400 *m/z.* Mass spectra was identified by comparison with a series of standard alkanes—alkane standard solution.

The identification of volatile compounds was performed by comparing their mass spectra with those in the NIST27 and NIST147 libraries and by retention indices drawn from [46,47] (for columns with a similar stationary phase to ZB-5 ms). Afterwards, the mass spectra values were compared and verified with the retention indices drawn from Pherobase [46] and Flavornet [47] databases. The results were expressed as relative percentage of the total peaks area.

#### 2.2.8. Instrumental Analysis of Texture

The textural properties of the biscuits were measured with a CT 3 Texture Analyzer (Brookfield Engineering Labs, Middleboro, MA, USA), equipped with a 10 kg load cell and the 6 mm cylindrical probe (TA41). The textural properties were determined as described by Pop et al. [48] with slight modifications. A compression test was applied to all samples (5 mm target distance, 3 mm/s test and post-test speed, trigger load 5 g). The Brookfield Engineering Labs Texture Pro CT V1.6 software was used to calculate the specific texture parameters.
