## 2 Materials and Methods
### Raw Material  
The seeds used in this study were brown seeded, belonging to Sideral variety. They were produced organically in an on-farm trial, carried out in the 2017-2018 growing season in the lowland area (latitude 43 deg40'48''N, longitude 10 deg30'1''E) of the Pisa Province (northern Tuscany, Italy). Flaxseed cake was obtained after oil extraction by cold pressing, ground and stored at \(-20\lx@math@degree\)C in a sealed vacuum container until the analysis and processing.  
The sourdough utilized during the study was supplied by Dolcezze Savini Srl (Via S.Aleramo, 24/26-50063-Figline Valdarno (FI)) while the wheat flours used for the refreshment procedure (hard wheat flour type 0) and the bread making (weak wheat flour type 0) were provided by Molino F.Ili Giambastiani Srl (Via Nazionale del Brennero, 798-55029-Ponte a Moriano (LU)).  
Both flour type 0 and flaxseed cake were chemically characterized in terms of dry matter percentage, water activity, free acidity, and phytochemical properties. The results obtained from these assays are reported in Table 1.  
Refreshment procedure of starter dough, as well as baking protocol and operating conditions (time and temperature) adopted in the storage of starter dough, in bulk fermentation and in cooking phases, were performed as described in a previous paper [3]. Bread making tests were conducted at the Food Technology laboratory of the Department of Agriculture Food and Environment of Pisa University; moreover, for each formulation, three replications were performed.  
Different formulations of sourdough bread (water 32%; sourdough 16%, flour 52%) were produced using flaxseed cake flour at different percentages (Table 2).  
\begin{table}
\begin{tabular}{c c c} \hline \hline  & **Flaxseed Cake Flour** & **Weak Wheat Flour Type 0** \\ \hline Dry matter (dm, \%) & 90.61 \(\pm\) 0.09 & 91.55 \(\pm\) 0.10 \\ Water activity & 0.54 \(\pm\) 0.01 & 0.26 \(\pm\) 0.01 \\ Protein (g/100 g) & 29.20 \(\pm\) 0.93 & 10.00 \(\pm\) 0.44 \\ Fat (g/100 g) & 4.41 \(\pm\) 0.55 & 1.00 \(\pm\) 0.12 \\ Total phenols (mg GAE/g dm) & 7.40 \(\pm\) 0.41 & 1.56 \(\pm\) 0.16 \\ Total flavonoids (mg GAE/g dm) & 0.90 \(\pm\) 0.03 & n.d. \\ Anti-radical activity (\(\mu\)umol TE/g dm) & 17.49 \(\pm\) 0.77 & n.d. \\ TEAC (\(\mu\)mol TE/g DW) & 8.60 \(\pm\) 0.02 & 0.30 \(\pm\) 0.06 \\ C16:0 & 7.68 \(\pm\) 0.24 & 3.13 \(\pm\) 0.18 \\ C16:1c9 & 0.10 \(\pm\) 0.03 & n.d. \\ C17:0 & 0.14 \(\pm\) 0.02 & n.d. \\ C18:0 & 3.65 \(\pm\) 0.13 & 0.45 \(\pm\) 0.05 \\ C18:1c9 & 20.44 \(\pm\) 0.89 & 6.65 \(\pm\) 0.56 \\ C18:1c11 & 0.88 \(\pm\) 0.05 & 0.87 \(\pm\) 0.04 \\ C18:2n6 & 16.32 \(\pm\) 1.10 & 52.64 \(\pm\) 2.43 \\ C20:0 & 0.19 \(\pm\) 0.02 & n.d. \\ C18:3n6 & 0.22 \(\pm\) 0.04 & n.d. \\ C18-3n3 & 49.96 \(\pm\) 3.63 & 2.89 \(\pm\) 0.10 \\ C22:0 & 0.20 \(\pm\) 0.03 & n.d. \\ C24:0 & 0.23 \(\pm\) 0.03 & n.d. \\ SFA & 12.07 \(\pm\) 0.95 & 34.71 \(\pm\) 1.89 \\ MUFA & 21.43 \(\pm\) 0.84 & 7.70 \(\pm\) 0.82 \\ PUFA & 66.50 \(\pm\) 4.22 & 55.53 \(\pm\) 3.12 \\ \hline \hline \end{tabular}  
* **Notes**: n.d. = not detectable; GAE= gallic acid equivalents; TE= Trolox equivalents; TEAC=Trolox equivalent antioxidant capacity; SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids.  
\end{table}
Table 1: Physical and chemical characterization of flaxseed cake flour and weak wheat flour: dry matter (dm %), water activity (aw), protein (%), fat (%), total phenols, total flavonoids, anti-radical activity, TEAC, and most representative fatty acids (relative %). Data presented are the mean \(\pm\) SD (\(n=3\)).