3.5.2. Macronutrients and Minerals

Moisture, crude protein, crude fat and ash contents were determined following the methods of the Association of Official Analytical Chemists [63]. The crude protein content was evaluated by the Kjeldahl procedure, using 6.25 as a nitrogen-to-protein conversion factor. Nonprotein nitrogen (NPN) was determined by the Kjeldahl method after protein precipitation with 10% (w/v) trichloroacetic acid and filtration. The crude fat content was determined by Soxhlet extraction. The ash content was determined gravimetrically after incineration in a muffle furnace at 550 °C. Total dietary fiber and soluble and insoluble fiber fractions were determined according to Prosky et al. [64]. Carbohydrates were calculated by difference. All macronutrients analyses were performed in triplicate.

Macro-minerals and trace elements (Ca, Mg, Na, K, P, Fe, Zn, Cu, Mn) were quantified by inductively coupled plasma optical emission spectrometry (Optima 8000^™ ICP-OES, Perkin-Elmer, Waltham, MA, USA) after liquid ashing in a microwave digestion system (1200 Mega, Milestone srl, Sorisole, Italy). Mineral analyses were performed in quadruplicate.