3.3. Characterization of Raw Material

The moisture content analysis was performed gravimetrically, following the standard method developed by the Association of Official Analytical Chemists (AOAC, 1999, method 1) [66]. The ground sample was kept in an oven for 48 h at 105 ± 2 °C (P Theroven, JP Selecta, Barcelona, Spain). For the determination of the ash content, the sample was kept in a muffle furnace for 6 h at 575 °C (ELF, Carbolite, UK); it was then measured gravimetrically. The protein content was estimated using the Kjeldalh method, using the conversion factor (5.38) to determine the total nitrogen content [67].

The mineral content of the raw material was determined using several methods developed elsewhere [68]. In brief, 0.3 g of ash, 10 mL of nitric acid and 1 mL of hydrogen peroxide were mixed to perform an acidic digestion in a microwave (Marsxpress, CEM), operating at 200 W for 15 min before holding at 200 °C for 10 min. Ca, Fe, Cu and Mg were analyzed by atomic absorption spectroscopy, while Na and K were analyzed using atomic emission spectroscopy (SpectrAA-220 Fast Sequential from Varian, Palo Alto, CA, USA). Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the content of Cd (X Series, Thermo Scientific, Waltham, MA, USA).

The oligosaccharide content of the raw material was analyzed based on a quantitative acid hydrolysis with sulfuric acid at 72%. Firstly, in a test tube, the sample and the sulfuric acid were mixed and kept in a water bath at 30 °C for 1 h (with manual stirring). Next, the sulfuric acid was diluted to 4% (w/w) concentration with distilled water, and the procedure continued in an autoclave (P-Selecta, Barcelona, Spain) at 121 °C for 1 h at 2 atm. The mixture was separated by filtration (using a vacuum pump) and the liquid phase was analyzed by high performance liquid chromatography (HPLC, 1260 Infinity, Agilent Technologies, Santa Clara, CA, USA), according to the method described elsewhere [53]. The solid phase (after filtration) was studied as an acid insoluble residue (AIR) and quantified gravimetrically.