Enzymatic Hydrolysis of Heated Whey: Iron-Binding Ability of Peptides and Antigenic Protein Fractions

Mean (±SD) hydrolysis of heated whey protein concentrate (WPC) by different enzymes. Heated (for 10min at 100°C) WPC (2% protein solution) was incubated at 50°C for 30, 60, 90, 120, 150, 180, and 240min with 2% (on a protein-equivalent basis) Alcalase (♦), Flavourzyme (■), trypsin (▴), and papain (•)

Mean (±SD) NPN fractions in enzymatic hydrolysates of heated whey protein concentrate (WPC). Heated (for 10min at 100°C) WPC (2% protein solution) was incubated at 50°C for 30, 60, 90, 120, 150, 180, and 240min with 2% (on protein-equivalent basis) Alcalase (♦), Flavourzyme (■), trypsin (▴), and papain (•)

Sodium dodecyl sulfate-PAGE patterns of enzymatic hydrolysates of heated whey protein concentrate (WPC). Heated WPC (2% protein solution) was incubated at 50°C for 30, 60, 90, 120, 150, 180, and 240min with 2% (on protein-equivalent basis) Alcalase (a), Flavourzyme (b), trypsin (c), and papain (d). A = standard broad-range marker (Bio-Rad, Hercules, CA): myosin (209 kDa), β-galactosidase (124 kDa), BSA (80 kDa), ovalbumin (49.1 kDa), carbonic anhydrase (34.8 kDa), soybean trypsin inhibitor (28.9 kDa), lysozyme (20.6 kDa), and aprotinin (7.1 kDa); B = WPC; C = heated WPC; lanes 1 to 7 = heated WPC hydrolysates produced at 30, 60, 90, 120, 150, 180, and 240min of incubation, respectively, with different enzymes.

Reversed-phase HPLC chromatograms of enzymatic hydrolysates of whey protein concentrate (WPC). Heated WPC (2% protein solution) was incubated at 50°C for 240min with 2% (on a protein-equivalent basis) Alcalase (A), Flavourzyme (B), trypsin (C), and papain (D). Hydrolysates were applied to the column. The columns were equilibrated with solvent A (0.1% trifluoroacetic acid in H₂O) and eluted with a linear gradient of solvent B (0.1% trifluoroacetic acid in acetonitrile). The flow rate was 1 mL/min, injection volume was 10μL, and detection was at 214nm.

Diethylaminoethylcellulose ion-exchange column chromatogram of heated whey protein concentrate hydrolysates derived from Alcalase (A), Flavourzyme (B), trypsin (C), and papain (D) treatments, respectively. Enzymatic hydrolysate dissolved in 20mM Tris-HCl buffer (pH 7.8) was applied to the column. The column, packed with diethylaminoethylcellulose, was washed with the same buffer and then eluted with a step gradient of NaCl as indicated. The flow rate was 3 mL/min, fraction volume was 15mL per tube, and elution was monitored at 280nm.

Mean iron contents (mg/kg) of fraction 1 and fraction 2 of enzymatic hydrolysates of heated whey protein concentrate.

Reversed-phase HPLC chromatogram of fraction 1 of Alcalase hydrolysates (Figure 5). Heated whey protein concentrate (2% protein solution) was incubated at 50°C for 240min with 2% (on a protein-equivalent basis) Alcalase. Fraction 1 was applied to the column. The columns were equilibrated with solvent A (0.1% trifluoroacetic acid in H₂O) and eluted with a linear gradient of solvent B (0.1% trifluoroacetic acid in acetonitrile). The flow rate was 1 mL/min, injection volume was 10μL, and detection was at 214nm.