Dynamic digestion of an amaranth protein beverage: characterization and antihypertensive activity

Elevated blood pressure (BP) is the main metabolic risk factor that contributes to the development of cardiovascular disease. The regulation of BP is complex since it involves several intertwined metabolic pathways. The renin-angiotensin system plays a fundamental role in the regulation of blood pressure. This system is mainly regulated by two proteases: renin and ACE. Food science and technology has recognized the interest in identifying healthy foods with bioactive components for complement diets in hypertensive individuals. Among the bioactive components are antihypertensive peptides. These peptides are encrypted in protein sources. Amaranth, an ancestral crop native to Latin America, have presented a high protein quality as alternative crop. In recent decades, various research groups have analyzed their bioaccessibility during the simulated gastrointestinal digestion process (GID). The aim of this work was to identify antihypertensive peptides after a dynamic digestion of a high protein beverage based on amaranth. Amaranth beverage (AB) was subjected to a dynamic in vitro digestion in a DIDGI® system where stomach and duodenal conditions were simulated. Digesta were sampled during different times and characterization was performed. Particle size was assessed by dynamic light scattering. Protein hydrolysis was followed by SDS-PAGE, Tris-Tricine gels and liberation of primary amines by OPA. Digesta was fractionated in a Waters Sep-Pak cartridges (Vac 20 cc C18-5g) and three fractions were collected: unretained (UR), eluted 1 (E1) and eluted 2 (E2). Antihypertensive activity was followed by the inhibition of angiotensin converting enzyme (ACE). The hydrolysis degree (HD) was approximately 27% after gastric stage and in the intestinal compartment, proteolysis increased drastically (60 and 80% between 15 and 90 min). The AB, before being subjected to GID, presented eleven polypeptide species, mainly albumin and globulin fractions. After 30 min of digestion only the marked decrease of a 56.8 kDa band corresponding to P-globulin is observed. After 60 min, the disappearance of practically all the initial polypeptide species was detected except for those corresponding to 40.1 (pepsin) and 20.8 kDa (basic globulin subunit) and the appearance of low molecular mass peptide species from hydrolysis. Initial particle size (D4,3) of AB was 104.4 μm. This result suggests an incomplete solubilization of amaranth proteins and the formation of aggregates. Larger particles aggregates were formed when pH was close to the isoelectric point of amaranth proteins during gastric stage. Purified fractions presented a greater inhibitory activity of ACE than the final digest (IC50 = 0.14 ± 0.02 mg/mL), being the IC50 values: 0.21 ± 0.04 mg/mL, 0.05 ± 0.01 mg/mL, 0.06 ± 0.01 mg/mL for UR, E1, and E2, respectively. Most active fraction (E1, E2) have been sent to peptide identification by LC-MS/MS analysis. These results showed that after dynamic digestion antihypertensive peptides were released from the amaranth beverage and this drink could be a nutritive and functional food for diet complement in hypertensive subjects.