2.3.1. Proximate Composition

Moisture and ash contents were evaluated following the Association of O fficial Analytical Chemists (AOAC) [19] methods. Protein content was measured using a LECO FP-2000 Nitrogen Determinator (Leco Corporation, St Joseph, MI, USA). Fat content was evaluated according to Bligh and Dyer [20]. Fatty acid profiles were carried out in freeze-dried samples (Lyophilizer Telstar Cryodos Equipment, Tarrasa, Barcelona, Spain) by gas chromatography as reported by Pintado, et al. [21] and results were expressed as g of fatty acid per 100 g of product. All the measurements were performed in triplicate.

#### 2.3.2. Dietary Fibre Content

Dietary fibre was determined in SUN and, due to the stability of this food constituent, dietary fibre content in the derived frankfurters was calculated according to the proportion of the ingredient. In particular, dietary fibre was measured by the indigestible fraction method [22], where the sample was subjected to several enzymatic treatments (pepsin, pancreatin, α-amylase, and amyloglucosidase) and dialysis in order to remove the digestible components of the sample and to separate soluble dietary fibre from insoluble dietary fibre. In the soluble dietary fibre, nonstarch polysaccharides were hydrolyzed with sulfuric acid and spectrophotometrically quantitated after alkalinization and reaction with dinitrosalicylic acid [23]. Insoluble dietary fibre was also subjected to hydrolysis, and nonstarch polysaccharides were measured in the supernatant by the same method, while the residue (klason lignin) was determined gravimetrically after overnight drying at 105 ◦C. Total dietary fibre was determined as the sum of the soluble and the insoluble dietary fibre.

### 2.3.3. Mineral Content

For mineral content determination, samples were prepared by acid digestion with nitric acid in a microwave digestion system (ETHOS 1, Milestone, Srl, Sorisole, Italy) [24]. The minerals were quantified on a ContrAA 700 High-Resolution Continuum Source spectrophotometer (Analytik Jena AG, Jena, Germany) equipped with a Xenon short-arc lamp (GLE, Berlin, Germany). Three determinations were carried out per sample to measure Ca, Mg, Na, K, P, Fe, Zn, Cu and Mn and results were expressed as mg/100 g product.

#### 2.3.4. Polyphenol Content and Profile

Total extractable polyphenols in SUN and the frankfurters were extracted by means of double aqueous–organic extraction, following the method of Nardoia, et al. [25]. Extractable polyphenols were determined in the corresponding supernatants by the Folin–Ciocalteu procedure [26], using gallic acid (Sigma-Aldrich, St. Louis, MO, USA) as a standard. Results were expressed as gallic acid equivalents (mg GAE/kg of the sample).

Polyphenol profile was additionally evaluated in SUN by HPLC-ESI-QTOF MS analysis in the same extract, after concentration (6:1) with a N2 stream, according to procedures previously described [27]. For separation, the HPLC apparatus (Agilent 1200, Agilent Technologies, Santa Clara, CA, USA) was coupled with a diode array detector (DAD) (Agilent G1315B) and a quadrupole time-of-flight (QTOF) mass analyser (Agilent G6530A) with an atmospheric pressure electrospray ionization (ESI). The column used was a 50 mm × 42.1 mm i.d., 3.5 μm, Luna C18 (Phenomenex, Torrance, CA, USA).

Gradient elution was performed with a binary system consisting of 0.1% aqueous formic acid (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The following gradient was applied at a flow rate of 0.4 mL/min: 0 min, 8% B; 10 min, 323% B; 15 min, 50% B; 20 min, 50% B; 23 min, 100% B, followed by a re-equilibration step. The injection volume was 10 μL, and the column temperature was 25 ◦C. Data were acquired using negative ion mode with a mass range of 100−1200 Da and using a source temperature of 325 ◦C and a gas flow of 10 L/h. Peak identity was established by comparison with the retention times of commercial standards when available. In addition, the molecular formula proposed by the MassHunter Workstation software version 4.0 for the different signals obtained in the MS experiments were compared with previously reported phenolic compounds, especially in sunflower, and a maximum error of 10 ppm was accepted.
