# Repurposing Olive Oil Mill Wastewater into a Valuable Ingredi for Functional Bread Production
## 2 Materials and Methods
### Functional Aspects  
It is well established that only 2% of the total phenolic content of milled olive fruit transfers to the oil phase, approximately 53% goes to the OOMW, and the remaining amount to the pomace [51]. Then, OOMW represents a valuable reservoir of organic compounds that have high radical scavenger activity and health-promoting properties. The OOMW used in this study contained 18.56 \(\pm\) 1.35 mg GAE/mL of total phenols, as determined by the Folin-Ciocalteu assay. The antioxidant potential of OOMW was higher in the ABTS test (30.68 \(\pm\) 2.56 \(\upmu\)mol TE/mL) than DPPH test (9.64 \(\pm\) 0.83 \(\upmu\)mol TE/mL). Oxidative stress concurs in the development and progression of various human diseases, such as diabetes, atherosclerosis, and even cancer. Correct nutrition is one of the most important prevention approaches because diet antioxidants, including phenolic compounds, can contribute to human health by scavenging free radicals [52]. To assess the functional properties of the fortified bakery product, we evaluated the radical-scavenging ability of breads containing OOMW produced using the \(B\) process (EXP\({}_{B}\)-1 and EXP\({}_{B}\)-2), in comparison to the CTR\({}_{B}\) bread. Given that olive oil phenols can be unstable under digestive conditions [53], we measured their quantity and anti-radical activity in bread samples both before and after simulated gastrointestinal digestion. Phenols released from EXP\({}_{B}\)-1 and EXP\({}_{B}\)-2 breads before digestion were measured at 0.32 \(\pm\) 0.04 mg GAE/g and 0.83 \(\pm\) 0.05 mg GAE/g (\(n\) = 4), respectively (Figure 3). Considering the addition of 0.2 mL/g and 0.4 mL/g of OOMW to EXP\({}_{B}\)-1 and EXP\({}_{B}\)-2, respectively, and that semolina has a content of phenolic compounds of 0.06 mg GAE/g (CTR\({}_{B}\) bread), it turns out that about 10% of the phenols can be solubilized. Indeed, as reported by [PERSON] et al. [54], interactions between the polyphenols of the olive oil and the starch-gluten matrix of the semolina greatly limit their release. Instead, post-intestinal bioaccessible fractions obtained after in vitro simulated digestion of the OOMW-enriched breads showed a tenfold increase in phenol content compared to the relevant samples before digestion (Figure 3), with an almost 100% release of the phytochemicals. The DPPH decoloration assay revealed 2.5-fold and 1.8-fold higher activities in the EXP\({}_{B}\)-1 and EXP\({}_{B}\)-2 breads vs. the CTR\({}_{B}\) bread, increasing their radical scavenging capacity by, respectively, before digestion (Figure 4A). Post-digestion values reached 1.91 \(\pm\) 0.11 and 3.16 \(\pm\) 0.28 \(\upmu\)mol TE/g in EXP\({}_{B}\)-1 and EXP\({}_{B}\)-2 breads, respectively, which is 2.4-fold and 3.9-fold higher than CTR\({}_{B}\) bread (Figure 4A). Similar results were obtained using the ABTS**+**decolorization assay (Figure 4B). These findings demonstrate that the digestion process of bread fortified with OOMW facilitates the release of active compounds in the digested fraction, thereby enhancing the antioxidant potential of the product and improving its functional health properties. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Cumpound** & \multicolumn{3}{c}{**mg/100 g**} \\ \hline  & **CTR\({}_{B}\)** & **EXP\({}_{B}\)-1** & **EXP\({}_{B}\)-2** \\ Hydroxy thyrosol & n.d. & 3.39 & 4.97 \\ Coumaric acid & n.d. & 0.74 & 0.97 \\ Luteolin & n.d. & NF & 0.57 \\ Caffeic Acid & n.d. & 1.35 & 1.69 \\ Trans Hydroxy Cynamic & n.d. & 1.62 & 2.07 \\ \(\sum\) Polyphenols & n.d. & 7.10 & 10.27 \\ \hline \hline \end{tabular} Abbreviations: CTR\({}_{B}\), control production; EXP\({}_{B}\)-1, experimental 1 production (50% OOMW in substitution of water); EXP\({}_{B}\)-2, experimental 2 production (100% OOMW in substitution of water); n.d., not detected. All the values of EXP\({}_{B}\)-1 and EXP\({}_{B}\)-2 are significantly different (at \(p\) 0.05%) from trial CTR\({}_{B}\).  
\end{table}