EV [kcal/100 g] = 4 × (P + C) + 2 × TDF + 9 × F

For mineral content determination, about 1 g of each sample was weighted into Teflon vessels and 7 mL of HNO<sup>3</sup> (65%) and 1 mL of H2O<sup>2</sup> (30%) were added [77]. The digestion was carried out according to the conditions: temperature of 210 ◦C, ramp time of 15 min, hold time of 15 min, pressure of 800 psi and power of 900 to 1050 W (CEM 6, Mars, CEM Corporation, Matthews, NC, USA). After cooling, the digests were transferred to polymethylopropylene flasks and diluted to 50 mL with demineralized water. For each bread, three digestions were prepared.

The concentrations of the most common minerals—Ca, Cu, Fe, K, Mg, Mn, and Zn—were determined by microwave plasma atomic emission spectrometry using Agilent MP-AES 4210 (Agilent Technologies, Melbourne, Australia) [78]. At least two calibration curves were prepared for the measurement. The content of P was determined using the molybdate-ascorbic acid colorimetric method AOAC 995.11 [79] transformed into microwell plate measurements (PowerWave XS2, Biotek Instruments, Winooski, VT, USA). For each digest, three reactions were performed. The values of population reference intake (PRI) and adequate intake (AI) were established at the level of EFSA recommendations [80]: PRIs for Ca, Cu, Fe, Mg, Mn, and Zn, and AIs for K and Na.

The fatty acid composition of the lipids in the bread was determined according to the AOCS Official Method Ce 1 h-05 [81]. The lipids were extracted by the traditional Folch method [82] and then fatty acid methyl esters (FAME) were analyzed with an Agilent 7820A GC (Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector (FID) and SLB-IL111 capillary column (Supelco, Bellefonte, PA, USA) (100 m, 0.25 mm, 0.20 µm). The detailed parameters of the analysis were described earlier [83]. The results are presented as a percentage of total fatty acids.
