Antithrombotic and Antioxidant Activity of Amaranth Hydrolysate Obtained by Activation of an Endogenous Protease.

Ingestion of diets with antithrombotic and antioxidant components offer a convenient and effective way to prevent and reduce the incidence of cardiovascular diseases. The aim of the present work was to obtain an amaranth hydrolysate by the activation of an endogenous aspartic protease, to establish adequate experimental conditions, and to evaluate its antithrombotic and antioxidant activity in order to assess its potential application as an ingredient in functional foods. The results obtained not only confirmed the presence of an endogenous protease in the amaranth isolate, but also allowed us to select an adequate incubation conditions (pH 2, 40 °C, 16 h). The hydrolysate obtained (degree of hydrolysis 5.3 ± 0.4 %) showed potential antithrombotic activity (IC50 = 5.9 ± 0.1 mg soluble protein/mL) and had more antioxidant activity than the isolate, indicating that the activation of the protease released bioactive peptides from amaranth proteins. Decreasing the pH is a simple and cheap process and is another way to obtain potential functional ingredients with bioactive compounds.

Physicochemical, functional and angiotensin converting enzyme inhibitory properties of amaranth (Amaranthus hypochondriacus) 7S globulin.

BACKGROUND: Amaranth 7S globulin is a minor globulin component and its impact on the properties of an amaranth protein ingredient depends on its proportion in the variety of amaranth being considered. Some physicochemical, functional and angiotesin I-converting enzyme (ACE) inhibitory properties of amaranth vicilin were studied in this work and compared with the 11S globulin. RESULTS: Fluorescence spectroscopy results indicated that 7S globulin tryptophans were more exposed to the solvent and, by calorimetry, the 7S globulin denaturation temperature (T(d) ) was found lower than the 11S globulin T(d) , suggesting a more flexible structure. The 7S globulin surface hydrophobicity was higher than that of the 11S globulin, which is in agreement with the better emulsifying properties of the 7S globulin. The solubility in neutral buffer of the 7S globulin (851 ± 25 g kg(-1) ) was also higher than that of the 11S globulin (195 ± 6 g kg(-1) ). Bioinformatic analyses showed the presence of ACE inhibitory peptides encrypted in 7S tryptic sequences and peptides released after in vitro gastrointestinal digestion showed a high ACE-inhibitory capacity (IC(50) = 0.17 g L(-1) ), similar to that of 11S globulin peptides. CONCLUSION: Compared with the 11S globulin, the 7S globulin presents similar ACE inhibitory activity and some functional advantages, better solubility and emulsifying activity, which suits some food requirements. The functional behavior has been related with the structural properties.

Amaranth (Amaranthus hypochondriacus) vicilin subunit structure.

The 7S-globulin fraction is a minor component of the amaranth storage proteins. The present work provides new information about this protein. The amaranth 7S-globulin or vicilin presented a sedimentation coefficient of 8.6 ± 0.6 S and was composed of main subunits of 66, 52, 38, and 16 kDa. On the basis of mass spectrometry (MS) analysis of tryptic fragments, the 52, 38, and 16 kDa subunits presented sequence homology with sesame vicilin, whereas the 66 kDa subunit showed sequence similarity with a putative vicilin. Several characteristics of the 66 kDa subunit were similar to members of the convicilin family. Results support the hypothesis that the 7S-globulin molecules are composed of subunits coming from at least two gene families with primary products of 66 and 52 kDa, respectively. According to the present information, amaranth vicilin may be classified into the vicilin group that includes pea, broad bean, and sesame vicilins, among others.

Globulin-p and 11S-globulin from amaranthus Hypochondriacus: are two isoforms of the 11S-globulin.

Amaranth is an ancient crop with a high content of good quality proteins. Globulins are some of the most abundant storage proteins of amaranth grain. They contain two fractions distinguishable according to their different solubility: the salt-soluble 7S and 11S-globulins and the globulin-p soluble in mild-alkaline, low-ionic-strength solutions. As part of the amaranth proteins characterization, in this work we investigated the structural characteristics responsible for the different physicochemical properties of these globulins. We studied certain conformational parameters of the purified aggregates (AMGp) and individual molecules (IMGp) of globulin-p and of the partially purified globulin (ppGb) and compared the AMGp polypeptide sequences with the sequence of the 11S-globulin propolypeptide from Amaranthus (gi|122726601). The results indicated that the AMGp aggregates are responsible for the different solubility of globulin-p. Subtle conformational differences as determined by fluorescence spectroscopy and urea sensitivity were found between the molecules studied: The AMGp showed some surface differences from the IMGp and the ppGb; the AMGp also had a lower affinity for the hydrophobic fluorescent probe 1,8-aniline-naphthalene-sulfonate and a higher ionic charge than the ppGb and the IMGp, characteristics that might cause their lower solubility. In addition, we have demonstrated differences between the AMGp polypeptide sequences and that reported for amaranth 11S-globulin. These differences suggest that the globulin-p and 11S-globulin are two 11S-globulin isoforms comprised of polypeptides coming from different legumin-gene subfamilies.

Structural modifications of Amaranth proteins during germination.

Amaranth storage proteins begin to be hydrolyzed immediately following the completion of germination. Albumins and globulins (7S globulin, 11S-globulin and globulin-p) were formerly modified, and glutelins, the most aggregated fraction, later. Globulins mobilization starts with the proteolysis of the 7S like-globulin polypeptides and the propolypeptide and acid (A) polypeptides of 11S-globulin and globulin-p. This pattern of 11S-globulin mobilization is accounted by the structural model with propolypeptide and A polypeptides exposed to the outside. Amaranth globulin molecules showed minor changes in their sizes in spite of having some of their polypeptides cleaved. Although globulin-p is more aggregated than 11S-globulin, it showed greater conformational changes. Considering the high susceptibility of the propolypeptide to enzymatic hydrolysis, the higher content of this polypeptide in globulin-p molecules might explain their higher structural changes. According to the results, the order of mobilization of storage proteins depends on the combination of two structural characteristics, the state of aggregation and the presence on the surface of polypeptides susceptible to cleavage.

Amaranth globulin polypeptide heterogeneity.

The polypeptides integrating amaranth globulin-p and 11S-globulin were characterized by two-dimensional electrophoresis, ion-exchange chromatography and RP-HPLC. All polypeptides exhibited charge and hydrophobic heterogeneity. Almost all acid (A, pI 5-7) and basic (B, pI 9-10) polypeptides were present in both globulins, and the same happened with the unprocessed M polypeptides with pI in the range of 7-7.5 which fits well with a sequence containing both the A and B polypeptides. There were other polypeptides only present in 11S-globulin, like some of 41 and 16 kDa, which might come from another precursor or be the products of a different processing of the propolypeptide. These results suggested that, although amaranth subunits from different subfamilies are interchangeable in different oligomers, some structural differences between them might affect the assembly of globulin molecules. Structural differences arising from this behavior could account for the different physicochemical properties of globulin molecules.

Surface physicochemical properties of globulin-P amaranth protein.

Globulin-P, the polymerized 11S amaranth globulin, is composed of 280 kDa unitary molecules (UM, 23%) and aggregates larger than 500 kDa (A, 70%). Antibodies against these proteins were prepared to study their surface characteristics and to assess their homology with other storage proteins. Results showed that globulin-P unitary molecules and aggregates had similar reactive surfaces. A polypeptide of 56 kDa was found to be the most reactive to the antibodies assayed, followed by the acidic polypeptides. Such results support previous information, according to which these polypeptides appeared to be the most exposed on the molecule surface. Globulin-P fraction presented cross-reactivity with the remaining amaranth protein fractions: 11S-globulin, glutelins, and albumins. Globulin-P and 11S-globulin showed similar reactive surfaces whereas glutelin and albumins presented a lower cross-reactivity. The reactivity of the glutelin fraction depended on its sequence. Globulin-P fraction presented cross-reactivity with quinoa globulins, and to a lesser extent with globulins of sunflower and rice. Moreover, the anti-Gp serum was unable to detect either conformational or sequence epitopes in globulins of soybean, wheat, buckwheat, rice, and rye.

Influence of the extracting solvent upon the structural properties of Amaranth (Amaranthus hypochondriacus) glutelin.

Two amaranth glutelin preparations, Gt-bo extracted with borate buffer at pH 10 and Gt-na extracted with 0.1 N NaOH, were characterized and compared with the amaranth polymerized 11S globulin (Gp, globulin-P). Gt-bo and Gt-na presented very similar polypeptidic composition and a similar reactivity against an anti-Gp polyclonal antibody, although lower than that of Gp. It is demonstrated that Gt-na is composed of denatured and dissociated molecules, whereas Gt-bo consists of folded molecules. The size, polypeptidic composition, thermal stability, and denaturation enthalpy of Gt-bo molecules were similar to those of Gp subjected to a borate treatment at pH 10. The Gp immunoreactivity decreased to the level of Gt reactivity when subjected to alkaline treatment; this could be due to conformational changes. Results suggest that, like Gp, amaranth Gt molecules may be hexameric oligomers of approximately 300 kDa. They would be partially unfolded during the alkaline extraction.