## 2 Materials and Methods
### Methods
#### 2.2.4 Analytical Determinations in Bread  
Nutritional composition was estimated from the information reported on the packaging of flours, starches and other ingredients used in the formulations. This information was completed with fiber, protein and mineral analyses in APP, performed in the laboratory.  
Physicochemical determinations on bread samples were performed 2 h after elaboration, once breads were at room temperature. These were the determinations performed specifically in bread samples:  
1. Specific volume: Breads were weighed with a precision balance (Mettler Toledo) and the volume was measured on a volumetric measuring cylinder by water displacement [32], covering each piece of bread with a plastic film, finely adapted to the loaf shape. As bread is a porous material, the plastic film prevented the samples from getting wet. Specific volume was calculated according to the formula: \[\text{Specific volume (cm}^{3}/\text{g)}=\text{Volume (cm}^{3})/\text{weight (g)}.\]
2. Moisture: This was determined by weight difference [25]; crumb samples (cylinders 1 cm diameter) were weighed and maintained at 105 \(\pm\) 2 \({}^{\circ}\)C (J.P. Selecta) overnight until constant weight. The water content of each sample was calculated according to the formula: \[\text{Moisture (\%)}=\text{(M - m) \times 100/M}\] where, M = Initial weight of the sample (g); m = final weight of the sample (g).  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Ingredients**} & \multicolumn{5}{c}{**Formulations**} \\ \cline{2-6}  & **Ctrl** & **CP** & **GFB5** & **GFB6** & **GFB8** \\ \hline Water (\%) & 44.0 & 44.0 & 47.8 & 47.8 & 47.8 \\ GF flour (\%) & 40.8 & 37.0 & 28.3 & 27.3 & 25.3 \\ Rice Flour (\%) & 4.8 & 4.8 & 4.8 & 4.8 & 4.8 \\ Corn starch (\%) & 4.8 & 4.8 & 4.8 & 4.8 & 4.8 \\ Whey protein (\%) & 0.0 & 3.8 & 3.8 & 3.8 & 3.8 \\ APP (\%) & 0.0 & 0.0 & 5.0 & 6.0 & 8.0 \\ Oil (\%) & 2.9 & 2.9 & 2.9 & 2.9 & 2.9 \\ Salt (\%) & 1.5 & 1.5 & 1.5 & 1.5 & 1.5 \\ Yeast 1 (\%) & 1.2 & 1.2 & 1.1 & 1.1 & 1.1 \\ \hline \hline \end{tabular}  
* Ctrl, control batch; CP, control with protein added; GFB5, GFB6, and GFB8, gluten-free bread with protein and apple pomace powder added at 5, 6 or 8%. 1 Exact yeast amount for controls and formulations containing APP was 8.2 g per load of bread or 24 g per gram of solid ingredients.  
\end{table}
Table 1: List of ingredients of each sample batch and their relative amounts, expressed as percentage of the complete dough mixture.  
Crumb texture: This was assessed instrumentally by texture profile analysis with a TA.XT2 plus texture analyzer (Stable Micro System, Godalming, UK). Longitudinal cylinders (1 cm diameter) were extracted from the central part of each bread roll, with the aid of a cylindrical core sampler. The crumb sample was recovered from inside the cylinder by pushing it with a plunger. Samples' lengths were standardized before the assay at 4 cm. Texture profile analysis (50%) was performed twice on 4 pieces of bread (n = 8), perpendicular to the axis of the cylindrical samples. Graphics were analyzed as described by other authors [33,34] and the following parameters were obtained:  
* Hardness: The highest force peak of the first compression cycle (N).
* Cohesiveness: Ratio of the areas under the curve of the second compression cycle to the first compression cycle.
* Resilience: Ratio between the areas under the curve of the withdrawal divided by the area under the curve in the downstroke, both in the first compression cycle.
* Springiness: Ratio between the distances the sample is compressed in the second downstroke divided by the first downstroke.
* Chewiness is calculated as cohesiveness \(\times\) hardness \(\times\) springiness (N).