Amaranth grain flour and protein hydrolysates

Amaranthus hypochondriacus grain cultivar Revancha obtained from INIFAP-Campus Montecillo, México was used in this research. The whole grain was milled using a Udy mill (Udy Corporation Fort Collins, CO, USA) until a 60-mesh screen defatted flour was obtained. Then albumin 1 (Alb1) and globulin (Glo) were extracted from defatted flour according to the method described by Tovar-Pérez et al. (2009). Briefly, defatted flour (50 g) was mixed with 300 ml 0.04 Na₂SO₄ containing 20 mM β-mercaptoethanol and stirred for 30 min, centrifuged for 20 min at 13,000g; the supernatant was mixed with 50, 70 and 100% (sat.) (NH₄)₂SO₄. Alb 1 was separated from Glob when supernatant was dialysed for 24 h against distilled water. These crude proteins were lyophilised and stored at 5 °C.

The residue, after Alb1 and Glo were obtained, was treated with 70% ethanol to discard prolamins. Thus glutelins (Glu) were extracted following the method reported by Barba de la Rosa et al. (2010), using 0.1 M Tris buffer at pH 8.0, in a residue/buffer ratio 1:10. Protein content was assessed by the Kjeldahl method (AOAC 2010). Protein extracts were lyophilized and stored at 5 °C until alcalase hydrolysis.

Alcalase hydrolysis of proteins was carried out following the method reported by Tovar-Pérez et al. (2009) with some modifications: 0.5 M phosphate buffer (pH 7.4) was added to a 5 mg/ml of protein solution. The solution was incubated for 5 min at 50 °C; then 2.4 UA/ml of alcalase solution in 0.5 M phosphate buffer was added to each test tube to reach a final ratio E/S = 0.8 UA/g protein. The reaction, at the appropriate time was stopped by adding 100 μl phenylmethylsufonyl fluoride in ethanol (2 mg/ml). Then several amaranth grain protein hydrolysates were obtained at different conditions.

The degree of hydrolysis (DH) was conducted according to the method reported by Condés et al. (2009). Free amino groups, released by alcalase hydrolysis, were assessed by their reaction with 2,4,6-trinitrobenzenesulfonic acid (TNBS). l-Leucine was used as a standard. The DH was calculated as reported by the previous authors.

Molecular weight characterization of amaranth grain storage protein hydrolysates was carried out in agreement with the method used by Tovar-Pérez et al. (2009), using a molecular exclusion column Sephadex G-15 (1.4 × 29 cm; Pharmacia, Uppsala, Sweden) and a Pharmacia LKB FPLC System (Uppsala, Sweden) for peptides separation. 200 μl of hydrolyzed proteins (15 mg/ml) disolved in 32.5 mMK₂HPO₄–2.6 mM KH₂PO₄, pH 7.5, which contained 0.4 M NaCl and 20 mM 2-mercaptoethanol, were injected and eluted with the same buffer at 0.2 ml/min. Absorbance at 214 nm was monitored and 0.5 ml fractions were collected. An ultra-low range molecular weight marker (Sigma-Aldrich, St. Louis, MO, USA), containing triose phosphate isomerase 26.6 kDa; myoglobin 17 kDa; α-lactalbumin 14.2 kDa; aprotinin 6.5 kDa; insulin 3.5 kDa; bradykinin 1.06 kDa, was used. All experiments throughout this study were performed in triplicate.