Amaranth Protein Research Articles

Amaranth proteins foaming properties: Adsorption kinetics and foam formation—Part 1

This work has focused on the study of the relationships between the structural changes in proteins of amaranth under different conditions of pH and ionic strength and the ability to form foam, also taking into consideration the kinetics of adsorption of proteins at the interface. Results showed that treatment at pH 2.0 significantly improves the foaming properties of amaranth proteins. The structural studies performed indicate that amaranth proteins at acidic pH are denatured, dissociated and undergo partial hydrolysis due to the existence of an endoprotease. They also present a lower content of β-sheet and random coil secondary structures. Diffusion–adsorption studies of proteins at the air:water interface allowed to determine that the acidic pH favors adsorption thereof (higher values of kdiff and ka) and reduces the need for a rearrangement (higher values of γr). The interfacial behavior of amaranth proteins is a direct consequence of the structural changes they undergo at acidic pH, changes that also were reflected on the increased foaming capacity (higher vo) thus forming more dense and homogeneous foams. The behavior of the soluble proteins as surfactants was not altered by the presence of protein aggregates and insoluble proteins.

Development of novel ultrathin structures based in amaranth (Amaranthus hypochondriacus) protein isolate through electrospinning

Amaranth protein isolate (API) ultrathin structures have been developed using the electrospinning technique. The effects of pH, type of solvent and surfactant addition on the spinnability, morphology and molecular organization of the obtained structures have been studied. Regarding the effect of pH on API electrospinning, capsule morphologies were only obtained at extreme pH values (i.e. pH 2 and pH 12), which allowed the solubilisation of the proteins, and the process was favoured when the solutions were previously heated to induce protein denaturation. Fibre-like morphologies were only obtained when the solvent used for electrospinning was hexafluoro-2-propanol, as this organic solvent promotes the formation of random coil structures and, thus, an increase in the biopolymer entanglements.Capsule morphologies were obtained from the API-containing formic acid solutions and this solvent was better for electrospraying than the acetic acid, probably due to the higher viscosity and lower surface tension of the solutions thereof. Addition of 20 wt.% of Tween 80 considerably improved the formation of capsule-like structures from the formic acid solution, as this surfactant contributed to the formation of alpha helical structures. Similar results were obtained when combining the surfactant with the reducing agent 2-mercaptoethanol. However, denaturation of the protein structure was not sufficient for fibre formation through electrospinning, as the solution properties play a fundamental role in determining the morphology of the electrospun structures.

In vitro inhibition of dipeptidyl peptidase IV by peptides derived from the hydrolysis of amaranth (Amaranthus hypochondriacus L.) proteins

Bioactive compounds present in foods could potentially have beneficial effects on human health. In this study we report the in vitro inhibitory capacity of peptides released from amaranth seed proteins after enzymatic digestion, against dipeptidyl peptidase IV (DPPIV); an enzyme known to deactivate incretins, hormones involved in insulin secretion. Other seeds, such as soybean, black bean, and wheat were also tested. The highest inhibition of DPPIV was observed with amaranth peptides released after simulated gastrointestinal digestion, showing an IC50 of 1.1mg/mL in a dose-dependent manner. In silico tryptic digestion of amaranth globulins was carried out releasing peptides larger than 13 residues. Some of these peptides were used for the in silico prediction of their binding modes with DPPIV. Docking models showed that the possible mechanism of globulin peptides to inhibit DPPIV was through blocking the active dimer formation. Peptides were also found inside the major cavity where the natural substrates reach the catalytic site of the enzyme. This is the first report of the identification of inhibitory DPPIV peptides from amaranth hydrolysates and the prediction of their binding modes at the molecular level, leading to their possible use as functional food ingredients in the prevention of diabetes.

Mitigating effect of amaranth (Amarantus hypochondriacus) protein on acrylamide formation in foods

The effect of addition of amaranth flour and amaranth protein isolate to both a glucose/asparagine model system and real foods (cookies, fried tortilla chips, and baked tortilla chips) was studied to analyse the acrylamide mitigating potential of this underexploited plant with attractive nutraceutical properties. Addition of amaranth flour, with a relatively low protein content (16.45%), did not mitigate acrylamide in either the model system or the studied foods. On the contrary, addition of amaranth protein isolate decreased acrylamide content by 35–40% in the model system, 89% in cookies, 51% in fried tortilla chips, and 62% in baked tortilla chips. This acrylamide reduction was obtained without change in the colour or the texture of the cookies. On the contrary, colour remained unchanged in tortilla chips, but the addition of amaranth protein isolate increased the hardness (16–36%) of the produced tortillas. Although a much more detailed sensory evaluation of cookies and tortilla chips prepared using amaranth protein isolate is needed, the above results suggest that the use of amaranth protein may be an interesting way to both mitigate acrylamide formation and improve nutritional properties of foods.

Effect of acid treatment on structural and foaming properties of soy amaranth protein mixtures

To obtain a food ingredient composed of soybean and amaranth proteins with better functionality, the proteins were subjected to an acid treatment followed by neutralization. The native and treated proteins, amaranth (A and TA), soybean (S and TS) and the 1:1 mixture (M and TM) were studied. The structural characteristics and surface tension and foaming properties of the proteins were analyzed.The acid-neutralization treatment caused structural modifications on all the proteins. The soybean proteins suffered some conformational changes and dissociation whereas the amaranth proteins were partially hydrolyzed by an endogenous aspartic protease, activated at acid pH. M showed the equivalent presence of S and A proteins, but TM, presented characteristics more similar to the TA proteins suggesting that the amaranth protease acted on the soybean proteins.The acid treatment did not modify the S tensioactivity while TA and TM increased their tensioactivity compared to A and M. Amaranth proteins showed to be faster and more efficient than S in decreasing the surface tension, and present the higher velocity of foam formation. The acid treatment improved the foam formation capacity of all samples. Foam stabilization was also enhanced by the acid treatment, though in this case S proteins were better foam stabilizers than A. Although M showed an intermediate behavior between S and A, the TM showed a foam stability nearer the TS.The mixture of amaranth and soybean proteins subjected to acid treatment make up an ingredient with improved surface and foam properties compared with its non treated components.

Molecular rearrangements in extrusion processes for the production of amaranth-enriched, gluten-free rice pasta

Gluten-free pasta represents a challenge for food technologists and nutritionists since gluten-free materials used in conventional formulations have poor functional and nutritional properties. A novel extrusion-cooking process was set up to improve the textural characteristics of rice-based pasta, and to enrich it with amaranth. Mineral and fiber content, and protein digestibility were improved by amaranth enrichment. Extrusion-cooking of a 75/25 mixture of rice flour and amaranth prior to pasta-making gave the best results as for the textural characteristics of the final product. The firmness of cooked pasta increased due to the extrusion-cooking process, that also decreased protein solubility in the amaranth-enriched pasta. The content in accessible thiols also decreased in amaranth-enriched pastas, indicating that amaranth proteins may be involved in forming disulphide bonds during the pasta-making process. Our results suggest that starch in rice flour interacts best with amaranth proteins when starch gelatinization occurs simultaneously to protein denaturation in the extrusion-cooking process.

Physicochemical and structural properties of amaranth protein isolates treated with high pressure

Abstract The effects of high pressure (HP) (200, 400 and 600 MPa) on physicochemical and structural properties of amaranth proteins treated at different protein concentrations (1, 5 and 10% w/v) were studied. HP provoked denaturation of amaranth proteins which were very sensitive to HP treatment, achieving almost complete denaturation (93%) at 400 MPa. After HP, the resistant structures from glutenins, globulin-11S and globulin-P exhibited an increased thermal stability while those from albumins and globulin-7S exhibited a decrease of thermal stability. Increasing intensities of HP treatments provoked the disappearance of electrophoretic bands with molecular mass higher than 45 kDa, together with changes in the polypeptides fractions of low molecular weight. HP treatments induced the formation of insoluble aggregates and the dissociation of soluble aggregates. Protein concentration modulated the effects of HP on amaranth proteins. These modified proteins could present improved functional properties. Industrial relevance The important effects of high pressure on structural properties of amaranth proteins and their consequences in their functionality may be useful in the handling of these proteins as food ingredients or in the formulation of novel foods.

Biochemical and Immunochemical Characterization of Different Varieties of Amaranth (Amaranthus L. ssp.) as a Safe Ingredient for Gluten-free Products

Celiac disease is a food intolerance triggered by the ingestion of gluten-containing cereals; the only therapy is a strict gluten-free diet for life. In recent years, amaranth flour has received considerable attention as an interesting source for the formulation of gluten-free products due to its high nutritional value and low content of prolamins, the toxic proteins for celiacs. The aim of this study was to characterize 40 amaranth varieties using both SDS-PAGE/immunoblotting and ELISA to assess their possible tolerance by celiac subjects. All of the amaranth samples studied showed similar binding affinities for both specific anti-gliadin antibodies and human IgAs. In most amaranth grains, the content of gluten-like proteins measured by ELISA was <20 ppm. The molecular characterization of amaranth proteins suggests that amaranth is safe for celiacs to consume. It is recommended that the most suitable amaranth varieties are those having the lowest content of proteins cross-reacting with anti-gliadin antibodies.

Chemical Composition and Bile Acid Binding Activity of Products Obtained from Amaranth (Amaranthus cruentus) Seeds

Cardiovascular diseases are currently the greatest cause of mortality in the world, and dislipidemia is appearing as one of the most important risk factors. The binding of bile acids (BAs) has been hypothesized as a possible mechanism by which dietary fibers lower blood cholesterol levels. Besides the fibers, other components in the amaranth seeds may be related to this hypocholesterolemic effect. The objective of the present study was to evaluate the BA binding capacity of some products obtained from defatted amaranth flour (DAF) and from the amaranth protein concentrate (APC). The alkaline residue, rich in fibers (8.6%), presented the lowest binding activity for the BAs tested, with the exception of glycocholic acid. The DAF showed intermediary binding activity for all the BAs tested, although similar to that of the APC for deoxycholic acid, and to that of the amaranth protein hydrolysate (APH) for taurocholic acid. The DAF and APC showed binding activity for secondary bile acids toxic to the intestinal mucus. From the results, amaranth products were shown to have the ability to bind BAs, but it was not possible to affirm whether the main component responsible for this activity was the proteins, fibers or eventually some other non-evaluated component.

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

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. 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. 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.

Interfacial and emulsifying properties of amaranth (Amaranthus hypochondriacus) protein isolates under different conditions of pH

Amaranth proteins have adequate amino acid balance for substituting either partly or completely animal proteins in human nutrition. However, they present poor emulsifying properties in basic conditions corresponding to their extraction medium. Consequently their use in acidic conditions could be envisaged to better exploit their potentialities. To better understand their emulsifying properties we have studied their interfacial activities at pHs 2.0 and 8.0 and tried to make the link between 2D and 3D properties.Our results clearly demonstrate the better properties of AI at pH 2.0 than at pH 8 in terms of protein solubility, spreading, adsorption, viscoelastic properties of interfaces and emulsion stability. These results are in relation with the denaturated state of proteins at pH 2.0 where proteins form a harder interfacial film, as compared to pH 8.0. Thus the potential use of amaranth proteins in emulsifying applications should be oriented towards acidic applications.

Antioxidant activity of amaranth protein or their hydrolysates under simulated gastrointestinal digestion

Amaranth proteins were subjected to a simulated gastrointestinal digestion to evaluate the antioxidant activity of the products. A protein isolate (I) was first hydrolyzed with pepsin (Pe) (pH 2, 37 °C) and then with pancreatin (Pa) (pH 6, 37 °C). Different hydrolysis conditions were assayed and control reactions (without enzymes) were performed. Hydrolysis degree (HD) determined by TNBS method ranged from 13 to 37%. Soluble fractions in 35 mmol/L phosphate buffer, pH = 7.8 were obtained from freeze-dried samples, and antioxidant activity was evaluated by the ABTS+·scavenging and the ORAC assays. Antioxidant activity increased significantly (p < 0.05) after simulated gastrointestinal digestion. According to the results, digestion conditions (Pe/protein: 1:10, 60 min; and Pa/protein: 1:10, 60 min) were selected and applied to an amaranth protein alcalase-hydrolysate (H) (HD = 29.2 ± 1.3). After pepsin and pancreatin action (Hpepa), HD was 42.0 ± 2.6, slightly higher than that of the digested isolate (Ipepa) (36.9 ± 0.5). The corresponding soluble fractions exhibited different electrophoretic profiles (tricine-SDS-PAGE) and gel filtration chromatograms, evidencing the presence of different molecular species. Previous hydrolysis with alcalase did not improve the antioxidant activity after simulated gastrointestinal digestion according to the methodologies assayed. Both the protein isolate and the alcalase-hydrolysate showed a potential capacity to scavenge free radicals after gastrointestinal digestion, appearing as promising ingredients to formulate functional foods with antioxidant activity.

Influence of pH on Structure and Function of Amaranth (Amaranthus hypochondriacus) Protein Isolates

ABSTRACTStructural and functional properties of two amaranth protein isolates as a function of pH were studied. Isolates, A9 and A11, were obtained by alkaline extraction at pH 9 and 11, respectively. Gel filtration chromatograms of A9 and A11 showed similar profiles. The A11 isolate contained mainly albumins and globulins, and a small proportion of globulin‐P aggregates, suggesting the presence of species with a higher degree of denaturation compared to A9. Differential scanning calorimetry (DSC) showed that A9 was characterized by two thermal transitions (65.8 and 98°C); A11 exhibited only a small endotherm (66.6°C) and a second, less defined one. DSC analysis of A9 at pH 2–4 did not show endotherms, but at pH 5, some protein structures were observed. A11 showed a greater degree of denaturation. FPLC results showed that the proteins in A9 are more folded and their conformation is closer to the native state than those in A11, which are more unfolded due to pH‐mediated denaturation, mainly in acid media. The surface hydrophobicity of the isolates in acid media was lower than in alkaline media. The fluorescence emission spectra of the isolates showed differences in acidic pH conditions. As expected, the highest solubility was at alkaline pH. The water‐holding capacity was similar for both isolates. The water‐imbibing capacity and speed of foaming was higher for A11 than for A9. In summary, intense pH treatment of amaranth isolates generated partial or total protein denaturation and differences in the functional properties.

Tryptic amaranth glutelin digests induce endothelial nitric oxide production through inhibition of ACE: Antihypertensive role of amaranth peptides

Amaranth seed proteins have a better balance of essential amino acids than cereals and legumes. In addition, the tryptic hydrolysis of amaranth proteins generates, among other peptides, angiotensin converting enzyme (ACE) inhibitory (ACEi) peptides. ACE converts angiotensin I (Ang I) into Ang II, but is also responsible for the degradation of bradykinin (BK). In contrast to Ang II, BK stimulates vasodilation modulated through endothelial nitric oxide (NO) production. The aim of the present study was to characterize the ACEi activity of amaranth trypsin-digested glutelins (TDGs) and their ability to induce endothelial NO production. An IC 50 value of 200 μg ml −1 was measured for TDG inhibition of ACE. TDGs stimulated endothelial NO production in coronary endothelial cells (CEC) by 52% compared to control. The effects of TDGs were comparable to those of BK and Captopril, both used as positive controls of NO production. Consistent with these effects, TDGs induced, in a dose-dependent manner, endothelial NO-dependent vasodilation in isolated rat aortic rings. These results suggest that TDGs induce endothelial NO production and consequent vasodilation through their ACEi activity. Amaranth TDGs have a high potential as a nutraceutical food in prevention of cardiovascular diseases. Further molecular, cellular and physiological studies are currently under way and the results may contribute to a better understanding and control of cardiovascular disorders.

Structural and rheological properties of amaranth protein concentrate gels obtained by different processes

The heat-induced gelation of amaranth protein concentrates (APCs) by three processes was studied. The first was the conventional process for isolating protein (standard process-st), the second included an acid washing step prior to protein extraction (acid washing process-aw) and the third included heating (50 °C) during the alkaline extraction step (heat process-ht). The dispersions (12%, w/v) were heated to 55–90 °C and assessed by rheological measurements made under small deformations, whereas the gels obtained by heating at 70, 80 or 90 °C/30 min were subjected to uniaxial compression measurements (TPA and mechanical properties). The rheological parameters associated with the network structure, elasticity modulus (E) and storage modulus ( G′), increased with increasing gelation temperature. For the APCst and APCht gels, protein aggregation occurred in two steps, whereas for APCaw, gelation occurred in a single step. The APCht gels showed the highest fracturability, hardness and adhesiveness, followed by the APCst and APCaw gels ( p < 0.05). Similar results were obtained for the mechanical properties at fracture. Increasing the heat treatment temperature from 80 to 90 °C resulted in a more structured matrix with greater water-holding capacity as compared to gels obtained at 70 °C, and these properties were influenced by the extraction processes used to obtain the APCs. Heat extraction (APCht) improved the gelation and water-holding properties, whereas the acid treatment had the opposite effect.

Stability of oil: Water emulsions of amaranth proteins. Effect of hydrolysis and pH

Abstract In this contribution we have determined the effect of limited enzymatic hydrolysis on the emulsifying capacity of amaranth proteins. The action of enzyme (alcalase and trypsin) and the pH of the continuous phase of the oil/water emulsion (pH 2.0, 6.3 and 8.0) were the variables analyzed. The results obtained show that amaranth protein isolates, AI, contain proteins species capable of forming and stabilizing emulsions, mainly at acidic pH (2.0) and to a lesser extent at pH 8.0. While the emulsions obtained are sensitive to creaming and flocculation, they do not undergo destabilization by coalescence. The emulsions prepared from proteins subjected to low grade trypsin hydrolysis (TH2.2) are sensitive to creaming - flocculation, whereas alcalase-hydrolyzed proteins (AH1.7 and AH9.5) exhibited a significant destabilization by creaming, flocculation and coalescence, mainly at pH 6.3. The effect of the pH of the aqueous phase was determining on the emulsion stability beside the structural and physicochemical characteristics of protein species utilized as tensioactive. At acidic pH (pH 2.0) the unfolding and charge of polypeptides and the capacity of form a viscoelastic film at the interface were essential while at alkaline pH (pH 8.0) the balance among high and low molecular mass protein species and flexibility of the molecule fixed the emulsions properties.

Effect of alternative processes on the yield and physicochemical characterization of protein concentrates from Amaranthus cruentus

Three different processes for the preparation of amaranth protein concentrates (APCs) were investigated with respect to the yield and physicochemical characteristics. The first was the conventional process for isolating protein (standard process), the second included an acid washing step prior to protein extraction (acid washing process) and the third included heating (50 °C) during the alkaline extraction step (thermal process). The protein yields were 17.8 and 25.6% for the acid washing and thermal processes, respectively. The concentrates were analyzed by SDS-PAGE, free sulfhydryl groups (SH), differential scanning calorimetry (DSC), size-exclusion high performance liquid chromatography (SE-HPLC), solubility in water and in 0.05 mol L −1 NaCl at pH 3.0, 4.5 and 7.0, phenolic compounds content and color parameters. The results indicated that the acid washing process had positive effects on the composition, color parameters, protein solubility and thermal stability of the concentrates. The thermal process also brought about positive effects on the characteristics of the proteins and color parameters although it negatively affected solubility due to the formation of high molar mass aggregates. The conventional process, when compared with the thermal and acid washing processes, adversely affected the properties evaluated.

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.

Amaranth proteins as a source of antioxidant peptides: Effect of proteolysis

Abstract The antioxidant activity of peptides present in the phosphate buffer-soluble fraction of: – Amaranthus mantegazzianus protein isolates ( Is ), – protein fractions ( Albs , Globs , GlobPs and Gluts ), alcalase hydrolysates of isolates (hydrolysis degree –HD-: 2.4% ( IH ls ) and 30% ( IH hs ) and protein fractions ( AlbHs , GlobHs , GlobPHs , and GlutHs) was investigated. Fractions separated by molecular exclusion chromatography were also analyzed. ABTS +. scavenging method showed the presence of antioxidant peptides in Is , Albs , Globs , and Gluts , being the last the one with the highest activity. No activity was detected in the GlobPs . After hydrolysis, the scavenging activity of all samples increased, especially at high HD. The GlobPs fraction presented the highest scavenging capacity after hydrolysis. Naturally-occurring peptides and polypeptides presented also the capacity to inhibit the linoleic acid oxidation, which was partially lost after hydrolysis. FPLC fractionation evidenced the appearance of Is and IH hs of several peptides and polypeptides which can act as antioxidants by different mechanisms.

Characterization and ACE‐Inhibitory Activity of Amaranth Proteins

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, respectively). 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.