2.2.2. Olive Mill Wastewater Extract

The OMW was collected in the crop season 2020/2021 from Ottobratica olive cultivar and produced according to a three-phase centrifugation process. The phenolic extract was obtained following the method previously reported by [28], with some modifications. Briefly, two liters of OMW were acidified to pH 2 with hydrochloric acid and washed three times with hexane (1:1, *v*/*v*) to remove the lipid fraction. The obtained mixture was vigorously shaken and centrifuged at 1550× *g* rpm for 3 min at 10 ◦C. The extraction procedure was carried out in separate funnels and repeated three times using ethyl acetate (1:4, *v*/*v*) as a solvent. The organic phase was then separated and filtered through a Buchner apparatus. The ethyl acetate was evaporated under vacuum in a rotary evaporator (Laborota400, Heidolph Instrument, Schwabach, Germany) at 25 ◦C. Finally, the obtained residue was dissolved in water to a final volume of 100 mL, further filtered by using a PTFE syringe filter (0.45 μm pore-size) and stored at 4 ◦C before analysis. To ensure that both hexane and ethyl acetate were removed (or were present in trace amounts) from the obtained extract, a headspace analysis using GC Thermo Trace 1310 apparatus (Waltham, MA, USA) equipped with a Single Quadrupole Mass Spectrometer ISQ LT system and a fused-silica capillary column (Thermo Scientific, Waltham, MA, USA) was carried out. It was found that the concentration of both the solvents used was below the limit of detection of the instrument.

### 2.2.3. Total Polyphenol Content and Antioxidant Activity of OL and OMW Extracts

The total polyphenol content of both OL and OMW extracts (hereinafter referred to as OLE and OMWE) was measured using the Folin-Ciocalteau reagent in a spectrophotometer (mod. Cary 3500, Agilent, Cernusco, Milan, Italy) set at 750 nm [29]. Briefly, after a proper dilution, an aliquot of 0.5 mL of extract were mixed with 0.5 mL of Folin-Ciocalteau reagent, 10 mL of sodium carbonate (7.5%) and adjusted to 25 mL with distilled water. The mixture was incubated in the dark for 1 h at room temperature before readings. Calibration curves were made using gallic acid and the results (mean of three replicates) were expressed as mg of gallic acid equivalents (GAE) per g of extract.

The free radical scavenging activity of both extracts was determined using a discoloration curve of the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). After proper dilution, an aliquot of 0.3 mL of extract was added to 2.7 mL of 0.0634 μmol mL−<sup>1</sup> DPPH methanol solution for 1 h at 515 nm and 22 ◦C. Results (mean of three replicates) are expressed as decrease in absorbance (%) per mg of extract when 0.17 μmol of DPPH are available to react.

The determination of the phenolic compounds and antioxidant activity was carried out simultaneously in both the extracts and the breadstick samples only a few days after the extraction process (specifically, no more than 15 days later).

#### *2.3. Preparation of GF Breadsticks*

GF control breadsticks were prepared using a conventional GF formulation consisting of 50% rice flour and 50% corn starch as the basic recipe. The other ingredients used, which were added as % on flour basis, were: 55% water (26 ◦C), 10% sunflower oil, 4% compressed yeast, 3% sugar, 1.8% salt, 1.5% guar gum, and 1.5% *Psyllium* fiber. For the preparation of the enriched GF samples, OLE and OMWE were singly added to the basic formulation (on flour basis) a two different level of supplementation: low (500 mg kg−1) and high (1000 mg kg−1). Breadstick sample codes were defined according to the type of extract and the level of supplementation used, as follows: Control (no extract addition), Leaf50 (low addition of OLE), Leaf100 (high addition of OLE), WasteW50 (low addition of OMWE) and WasteW100 (high addition of OMWE). For each formulation, GF breadsticks were prepared by firstly suspending extracts, yeast, salt and sugar in aliquots of warm water. Then, these dissolved ingredients and the sunflower oil were slowly added to the pre-mixed dry ingredients and kneaded using a mixer (KitchenAid Professional, Model 5KSM7990, St. Joseph, MI, USA) equipped with a dough hook at speed 1 for 5 min followed by other 8 min at speed 2. After mixing, the obtained doughs (three for each sample) were proofed in a climate chamber for 30 min (33 ◦C–90% RH), manually shaped into 28 g weighted and 30 cm length sticks, placed on rectangular baked pans, and proofed once again for 30 min (33 ◦C–90% RH). Finally, the GF breadsticks were baked in an electric oven (Europa, Malo, VI, Italy) following a two-step baking process: they were firstly baked at 180 ◦C for 13 min, allowed to cool at room temperature for 30 min and then baked once again for other 22 min at 160 ◦C. After baking, breadstick samples were cooled for 1 h, before the analysis.

### *2.4. Breadsticks Measurements*

#### 2.4.1. Moisture Content and Water Activity

The moisture content of the GF breadstick samples was determined using a moisture analyzer (Model Kern-DAB 100-3, KERN & SOHN GmbH, Balingen, Germany) equipped with a halogen quartz glass heater (400 W) and set with a standard heating profile at 105 ◦C. The results were expressed in % as the average of five repetitions.

Water activity was measured on ground and homogenized samples with an electronic hygrometer (model Aw-Win, Rotronic, Bassersdorf, Switzerland) equipped with a Karl-Fast probe previously calibrated in the range of 0.1–0.95 with solutions of lithium chloride (LiCl) of known activity. A total of five repetitions for each sample were made.
