H: Health, Nutrition, & Food JFS H: Health, Nutrition, and Food Characterization and ACE-Inhibitory Activity of Amaranth Proteins A. TIENGO, M. FARIA, AND F.M. NETTO ABSTRACT: Amaranth seeds have been considered as an excellent alternative or complementary source of food protein due to their balanced amino acid composition. However, their potential as a source of bioactive peptides has not been explored. The present study is aimed at characterizing and evaluating the activity of the angiotensin converting enzyme inhibitor of the amaranth protein concentrate and of hydrolysates produced with Alcalase. The protein concentrate, after simulated gastrointestinal digestion, showed lower angiotensin converting enzyme- inhibitory activity (IC 50 of 0.439 ± 0.018 mg protein/mL and 0.475 ± 0.021 mg protein/mL, for untreated and heat treated protein concentrate, respectively) than the hydrolysates produced with Alcalase, before and after simulated gastrointestinal digestion (IC 50 0.118 ± 0.009, 0.123 ± 0.007, 0.137 ± 0.002, and 0.176 ± 0.014 mg protein/mL, re- spectively). The simulated gastrointestinal digestion (pepsin–pancreatin) did not significantly alter the angiotensin- converting enzyme inhibiting activity of the Alcalase hydrolysates, suggesting that the peptides of the hydrolysates were resistant to gastrointestinal hydrolysis. These results highlight the angiotensin converting enzyme-inhibitory potential of amaranth proteins, which is an indication of their health-promoting potential. Keywords: Alcalase, angiotensin-converting enzyme, enzymatic digestion, inhibitory peptides, protein concen- trate, protein hydrolysates Introduction S ystemic arterial hypertension is the most common chronic health problem facing us today and without treatment it can lead to various cardiovascular complications, including coro- nary disease, peripheral arterial disease, and even coronary and kidney failure (Lo and others 2006). The angiotensin converting enzyme (ACE; EC. 3.4.15.1) plays an important physiological role in regulating blood pressure, converting an inactive form of the decapeptide angiontensin I into the octapeptide angiotensin II, a potent vasoconstrictor, and deactivating bradykinin, a vasodilator (Li and others 2005). Peptides that can inhibit ACE are receiving special attention since they are considered a nonpharmacological alternative for the prevention and control of systemic arterial hypertension (Vermeirssen and others 2004). Various ACE- inhibitory peptides have been described, including those derived from milk whey proteins (Pihlanto-Lepp¨ al¨ a and others 2000), and more recently the peptides derived from various vegetable proteins have come under investigation (Wu and Ding 2002; Yust and others 2003; Li and others 2005; Paris and others 2008). Another critical factor in bioactive peptide production is the adequate matching of the enzyme and the protein source. Alcalase has been widely used for the preparation of ACE-inhibitory peptides from vegetable protein sources (Pedroche and others 2002; Wu and Ding 2002; Chiang and others 2006). This enzyme releases peptides with greater ACE-inhibitory activity than other proteases (Chiang and others 2006; Costa and others 2007) and is also advantageous because of its low cost compared to other proteases (Li and others 2005). Amaranth seeds are an excellent alternative source of proteins since they are high in protein (17%) and have a balanced composi- MS 20080768 Submitted 10/2/2008, Accepted 2/23/2009. Authors are with Dept. of Food and Nutrition, Faculty of Food Engineering, Univ. of Campinas–Unicamp, Brazil. Direct inquiries to author Netto (E-mail: flavia@fea.unicamp.br). tion of essential amino acids, especially rich in the limiting amino acids such as lysine, methionine, and cystein (Becker and others 1981). However, amaranth proteins have been little explored as a source of bioactive peptides. The in silico analysis performed by Silva-Sanch´ ez and others (2008) revealed that amaranth proteins are a potential source of antihypertensive peptides, though, to the best of our knowledge, the ACE-inhibitory activity of amaranth pro- teins has not yet been evaluated. Thus the aims of the present study were (1) to obtain and characterize protein products from ama- ranth seeds–protein concentrate and its hydrolysates with Alcalase, (2) to study the ACE-inhibitory activity of the concentrate and hy- drolysates before and after simulated gastrointestinal digestion to study its effect on the activity, and (3) to investigate the effect of heat treatment of the protein concentrate on the release of active peptides. Materials and Methods Materials Amaranth seeds (Amaranthus cruentus, variety BR Alegria), harvested in April 2005, were obtained from producers in the municipality of Ituporanga, State of Santa Catarina, Brazil. The enzymes used for the in vitro digestion were pepsin from porcine stomach mucosa (Sigma Chemical Corp., St. Louis, Mo., U.S.A., code P-7-12, lot 120K7654) and porcine pancreatin (Sigma, code P- 1625, lot 41K1271). Alcalase 2.4 L, used for the production of the hydrolysates, was provided by Novozymes (Araucaria, PR, Brazil). ACE-inhibitory activity was determined using the angiotensin con- verting enzyme from rabbit lung (Sigma, code A6778, lot 084 K 1430) and the synthetic ACE substrate hippuryl-histidyl-leucine (HHL) (Sigma, code H1635, lot 062K1092). Amaranth flour was obtained 3 mo after harvesting by grind- ing the grains in a mill (Model MA630, Marconi, Piracicaba, SP, Brazil) with temperature control at 16 ± 3 ◦ C. The flour (particle size <250 μm) was defatted with hexane in a flour/hexane ratio of C  2009 Institute of Food Technologists R  Vol. 74, Nr. 5, 2009—JOURNAL OF FOOD SCIENCE H121 doi: 10.1111/j.1750-3841.2009.01145.x Further reproduction without permission is prohibited