Freeze dryer 4

The extraction of proteins from legumes was conducted using a previously established methodology [24 (link)]. Common black bean seeds were soaked in water at room temperature for 16 h. Black bean hulls were manually removed; then, bean cotyledons, green pea, chickpea, lentil and fava bean were ground separately in a commercial blender in a 1:10 grain/water ratio. The pH of the supernatant was adjusted to 8.0 with 1 M sodium hydroxide, and protein extraction was carried out at 35 °C with stirring for 1 h. The mixture was centrifuged at 5000× g for 15 min at 25 °C. Then, the pH was adjusted to the isoelectric point of the different legumes with 1 M hydrochloric acid to precipitate proteins, followed by centrifugation at 10,000× g for 20 min at 4 °C. The supernatant was discarded and the pellet was freeze-dried in a Lab Conco Freeze Dryer 4.5 (Kansas, MO, USA). Legume protein isolates (LPI) were stored at −20 °C until further analysis.

To determine the LCFA profile in feces, we used 40 mg of previously lyophilized feces during 48 h (Labconco, freeze dryer 4.5). Forty microliter of C19 were added as extraction standard for homogenization with methanol:chloroform (1:2 V/V) by brief sonication in ice (Labsonic U, B. Braun). Homogenates were then filtered with Whatman filters n°1 (10 μm of porosity). Filters were rinsed with 2 ml of chloroform and 1 ml of methanol. Homogenates were purified with KCl 0.88% and KCl 0.88%: methanol (1:1 V/V). After centrifugation (1500 g, 5 min), the chloroform phase was collected in new tubes and evaporated under nitrogen flux until samples were completely dry. The esterified fatty acids were then subjected to alkaline hydrolysis (saponification) and free fatty acids were methylated and quantified by gas chromatography with flame ionization detector as previously described^{50 (link)}.

The hydrolysis was carried out according to the methodology reported by Megías et al.
[52 (link)] with some modifications as reported in Montoya-Rodriguez et al.
[26 (link)]. Briefly, amaranth flour (2.5 g) was suspended in water (1:20 w/v) and a sequential enzyme digestion was carried out with pepsin [EC 3.4.23.1, 662 units/mg; enzyme/substrate, 1:20 (w/w); pH 2.0] and pancreatin [8x USP (a mixture of several digestive enzymes produced by the exocrine cells of the porcine pancreas, EC 232-468-9, Sigma-Aldrich P7545); enzyme/substrate, 1:20 (w/w); pH 7.5] at 37°C for 120 min for each enzyme. The final hydrolysis was stopped by heating at 75°C for 20 min, and the resulting hydrolysate was centrifuged at 20,000 g for 15 min at 4°C. The hydrolysates were desalted using 500 Da cellulose acetate membranes (The Nest Group, Inc.), and freeze dried in a Labconco (Kansas, MO) Freeze Dryer 4.5.