*2.5. Breads Characterization*

Prior to characterization, the breads were stored for 60 min at room temperature.

The specific volume of the bread samples was measured through the rapeseed displacement method [19].

Crumb firmness was determined using the MLFTA apparatus (Guss, Strand, South Africa) and a probe with diameter of 7.9 mm. Three distinct measurements were performed on two bread slices originating from the center of the bread samples. The following parameters were considered when measuring the crumb firmness: test speed of 5 mm/s, trigger threshold force of 1.96 N and bread slices penetration wide of 25 mm.

In order to determine the total phenolic content, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, trolox equivalent antioxidant capacity and iron reducing antioxidant power, the extraction procedure described by Aprodu and Banu [25] was used.

The total phenolic content, the DPPH-radical scavenging activity, the Trolox equivalent antioxidant capacity (TEAC) and the ferric reducing antioxidant power (FRAP) were determined using the procedures described by Aprodu and Banu [25]. The total phenolic content was reported as mg ferulic acid equivalent (FAE) per 100 g d.w. bread sample, the TEAC was expressed as μmols Trolox/g d.w., while results of the FRAP were expressed in μmol Fe2+/g d.w.

The content of resistant starch was determined through the AACC Method 32–40.01 [20], using the Megazyne assay kit (Megazyne International Ireland Ltd. Wicklow, Ireland).

Rapidly and slowly digestible starch contents were determined using the procedure described by Miao et al. [26]. The glucose content of the samples was determined using the glucose oxidase/peroxidase (GOPOD) assay kits (Wicklow, Ireland). The amount of hydrolyzed starch was determined by multiplying the assayed glucose content by a factor of 0.9. Finally, the rapidly and slowly digestible starch values were obtained by considering the contents of glucose released after 20 min and 120 min (G20 and G120, respectively) and free glucose (FG).
