2.2.3. Macro- and Microelements

Macro- and microelements were determined by atomic absorption spectrophotometry (AAS), according to the methods described by Păucean et al. [40] and Chi¸s et al. [41]. Briefly, 3 g of biscuits were burned at 550 ◦C in the furnace for 10 h (Nabertherm B150, Lilienthal, Germany). The ash was dissolved in HCl 20% and made up to a final volume of 20 mL in a volumetric flask. The macroelements (K, Ca, Mg) and microelements (Fe, Cu, Zn and Mn) were determined by AAS (Varian 220 FAA instrument, Mulgrave, Victoria, Australia). Mixed standard solutions with P, K, Cu, Zn, Fe, Mn, Ca, Mg (ICP Multielement Standard solution IV CertiPUR) were purchased from Merck (Merck KGaA, Darmstadt, Germany). All chemicals and solvents used in this study were of analytical grade. The results were expressed related to the weight of the fresh biscuits.

#### 2.2.4. Physical Evaluation of Biscuits

The diameter of six biscuits placed edge to edge was measured with a Vernier caliper (0.01 mm accuracy). The average diameter in centimetres was calculated by dividing [42] the value by six [4]. Similarly, the mean thickness of the biscuits was measured by stacking 6 biscuits on top of each other and dividing the value by six. For the spread factor the diameter was divided by thickness. The weight of the biscuits was measured on an analytical balance and calculated as the mean value of the weight of four individual biscuits.

#### 2.2.5. Total Phenolics Content

Total polyphenols content and antioxidant activity were determined as described by Bunea et al. [43] with slight modifications as proposed by Chis, et al. [44]. Briefly, one gram of biscuit sample was extracted three times with 100 mL acidified methanol (85:15 *v*/*v*, MeOH:HCl) by maceration under continuous stirring (magnetic stirrer, Velp Scientifica, Usmate Velate, Italy) for 24 h. Total phenolics content of the extracts was determined spectrophotometrically (Folin–Ciocalteu method). Thus, in a 100 mL volumetric flask, 10 mL of methanolic extract was mixed with 5 mL of Folin-Ciocalteu's reagent; 5 mL of 7.5% sodium carbonate solution was added. Distilled water was used to fill up the flask until the graduation marking. Samples were kept in the dark for 90 min, and then, absorbance was read at 760 nm on a model 1700UV/VIS spectrophotometer (Shimadzu Scientific Instruments, Kyoto, Japan).
