# Repurposing Olive Oil Mill Wastewater into a Valuable Ingredi for Functional Bread Production
## 2 Materials and Methods
### Determination of Functional Properties of Breads  
Two grams of bread samples were homogenized using a Waring blender (Waring, New Hartford, CT, USA) equipped with a semi-micro stainless-steel jar in 10 mL of a pH 6.8 buffered solution designed to replicate the chemical composition of human saliva according to European Pharmacopoeia. The aqueous suspension was submitted to in vitro digestion following the standardized INFOGEST 2.0 method [22]. To separate the bioaccessible fraction from the undigested material, the post-intestinal mixture was subjected to ultracentrifugation at a force of 167,000\(\times\)\(g\) for 35 min at 4 \({}^{\circ}\)C, using a Beckman Optima TLX ultracentrifuge (Beckman Instruments, Inc., Palo Alto, CA, USA). This process yielded a supernatant consisting of the bioaccessible fraction isolated from the residual nonabsorbable particulate material. Bread samples were subjected to in vitro digestion following a previously established protocol. During the intestinal phase, 1 mL aliquots of the digesta were collected at intervals of 0, 20, 40, 60, 80, and 120 min after the addition of pancreatin. These aliquots were immediately transferred to tubes placed in boiling water for 4 min to stop enzymatic activity. Following ultracentrifugation, the glucose content in the supernatant was measured spectrophotometrically at 510 nm using a D-glucose assay kit (GOPOD Format, Megazyme International, Ltd., Bray, Ireland). The starch digestibility was expressed as mg of glucose per gram of initial starch. One milliliter of the aqueous suspension of bread, both before digestion and from the bio-accessible fractions obtained post in vitro digestion, was centrifuged at 1000\(\times\)\(g\) for 15 min at 4 \({}^{\circ}\)C. This process effectively separated the soluble components from any particulate matter. The resulting soluble fractions were then processed to determine total phenolic content and assess radical scavenging activity. For total phenol content determination, aliquots of 100 \(\upmu\)L from each sample, prepared at three different dilutions to ensure accuracy across a range of concentrations, were mixed with 3 mL of 2% sodium carbonate (Na\({}_{2}\)CO\({}_{3}\)) solution [30]. Then, 100 \(\upmu\)L of the Folin-Ciocalteu reagent, pre-diluted in a 1:1 ratio with distilled water, was added to initiate the reaction. This mixture was left to incubate in the dark at room temperature for 60 min to allow color development. The absorbance of the resulting solutions was measured at 765 nm using a Beckman DU 640 spectrophotometer (Beckman Instruments, Milan, Italy). A blank solution was used as a reference to zero the instrument. The phenolic content in each sample was determined by measuring absorbance and comparing it to calibration curves created using gallic acid standards, with concentrations ranging from 5 to 100 \(\upmu\)g/mL. The results were expressed as milligrams of gallic acid equivalents per gram of bread. The radical-scavenging capacity of the bread was assessed through the decolorization assays of the 2,2\({}^{\prime}\)-azino-bis (3-ethylbenzothazoline-6-sulfonic acid) (ABTS) radical cation and of the 2,2-diphenyl-1-picrylhydrazyl (DPPPH) radical. The ABTS**+** radical was generated by oxidizing ABTS with potassium persulfate and diluted with 5 mM phosphate saline buffer to obtain an absorbance of 0.700 \(\pm\) 0.020 units at 734 nm [31]. Aliquots of properly diluted bread solution were added, and the absorbance registered again after 15 min. The 2,2-diphenyl-1-picrylhydrazyl (DPPPH) free radical scavenging activity was determined following the method described by [PERSON] et al. [32]. For the assay, 10 \(\upmu\)L of bread solution was mixed with 1 mL of DPPH ethanol solution at a concentration of 1 \(\times\) 10\({}^{-4}\) mol/L. The mixture was incubated at room temperature in the dark for 30 min, after which the absorbance was measured at 515 nm. To ensure precision, each sample was analyzed in duplicate and at three different dilutions within the assay's linear range.