Characterization, Bioaccesibility and Antioxidant Activities of Phenolic Compounds Recovered from Yellow pea (Pisum sativum) Flour and Protein Isolate.

This study focused on studying the bioaccesible phenolic compounds (PCs) from yellow pea flour (F) and protein isolate (I). Total phenolic contents (TPC), PCs composition and antioxidant activities were analysed in ethanol 60% extracts obtained by applying ultrasound assisted extraction (UAE, 15 min/40% amplitude). The preparation of I under alkaline conditions and the elimination of some soluble components at lower pH produced a change of PCs profile and antioxidant activity. After simulated gastrointestinal digestion (SGID) of both ingredients to obtain the digests FD and ID, notable changes in the PCs concentration and profiles could be demonstrated. FD presented a higher ORAC activity than ID (IC50 = 0.022 and 0.039 mg GAE/g dm, respectively), but lower ABTS•+ activity (IC50 = 0.8 and 0.3 mg GAE/g dm, respectively). After treatment with cholestyramine of extracts from FD and ID in order to eliminate bile salts and obtain the bioaccesible fractions FDb and IDb, ROS scavenging in H2O2-induced Caco2-TC7 cells was evaluated, registering a greater activity for ID respect to FD (IC50 = 0.042 and 0.017 mg GAE/mL, respectively). These activities could be attributed to the major bioaccesible PCs: OH-tyrosol, polydatin, trans-resveratrol, rutin, (-)-epicatechin and (-)-gallocatechin gallate for FD; syringic (the most concentrated) and ellagic acids, trans-resveratrol, and (-)-gallocatechin gallate for ID, but probably other compounds such as peptides or amino acids can also contribute.

Autochthonous Fermentation as a Means to Improve the Bioaccessibility and Antioxidant Activity of Proteins and Phenolic Compounds of Yellow Pea Flour.

This study focused on evaluating the potential of the natural fermentation of pea flour to improve the release of antioxidant compounds. Preliminary fermentations of 36.4% w/w flour dispersions were performed in tubes under different conditions (24 and 48 h, 30 and 37 °C). Finally, fermented flours (FFs) were obtained in a bioreactor under two conditions: 1: 36.4% w/w, 24 h, 30 °C (FF1); 2: 14.3% w/w, 24 h, 37 °C (FF2). The pH values decreased to 4.4-4.7, with a predominance of lactic acid bacteria. As in the fermentations in tubes, an increment in the proteolysis degree (TNBS method) (greater for FF2), polypeptide aggregation and a decrease in their solubility, an increase in <2 kDa peptides, and an increase in the Oxygen Radical Absorption Capacity (ORAC) potency of PBS-soluble fractions after fermentation were demonstrated. Also, fermentation increased the proteolysis degree after simulated gastrointestinal digestion (SGID, COST-INFOGEST) with respect to the non-fermented flour digests, with some differences in the molecular composition of the different digests. ORAC and Hydroxyl Radical Averting Capacity (HORAC) potencies increased in all cases. The digest of FF2 (FF2D) presented the greater ORAC value, with higher activities for >4 kDa, as well as for some fractions in the ranges 2-0.3 kDa and <0.10 kDa. Fermentation also increased the 60%-ethanol-extracted phenolic compounds, mainly flavonoids, and the ORAC activity. After SGID, the flavan-3-ols disappeared, but some phenolic acids increased with respect to the flour. Fermentation in condition 2 was considered the most appropriate to obtain a functional antioxidant ingredient.

Exploration of grape pomace peels and amaranth flours as functional ingredients in the elaboration of breads: phenolic composition, bioaccessibility, and antioxidant activity.

This study evaluated the incorporation of two ingredients as a source of bioactive compounds: amaranth flour (AF) and grape pomace peels flour (GP) to improve the nutritional qualities and functional properties of a wheat bread, emphasising the revalorisation of agricultural residues from grape winemaking as an ethical and economically viable source of bioactive compounds. Specifically, wheat flour (WF) substitutions were carried out for the individual ingredients, replacing 20% WF (A20 bread) or 5% GP (GP5 bread) and a mixture of both ingredients 20% WF and 5% GP (A20GP5 bread), and the antioxidant potential of the breads was analysed. The effect of simulated gastrointestinal digestion (SGID) on the phenolic profile and antioxidant activity of the fortified breads was also investigated. The substitution of WF by AF or GP introduced several phenolic compounds, digestion increased the bioaccessibility of phenolic compounds and reshaped their phenolic composition profiles. The combined presence of AF and GP in the breads modified the phenolic compounds composition and improved their antioxidant activity after SGID. Interactions between the phenolic compounds and other AF components (possibly proteins) were observed, which could protect the phenols from degradation during SGID, allowing them to be released after SGID.

Chemical and cell antioxidant activity of amaranth flour and beverage after simulated gastrointestinal digestion. Role of peptides.

The potential of peptides generated by simulated gastrointestinal digestion (SGID) of two products derived from Amaranthus manteggazianus seeds, flour (F) and beverage (B), to exert peroxyl scavenging activity (ORAC) and antioxidant action on intestinal cells was studied. B was prepared by solubilisation of seed proteins, with the addition of gums and the application of a pasteurization treatment. The gastrointestinal digests FD and BD showed some differences in the peptide/polypeptide composition. The SGID produced increased ORAC activity for both samples, with some differences in the ORAC of the whole digests BD and FD and of some gel filtration fractions. Bioaccessible fractions (FDdbs and BDdbs) were obtained after treatment with cholestyramine resin to remove bile salts due to their cytotoxicity and oxidative effect. BDdbs presented a greater ORAC potency (IC50: 0.05 ± 0.01 and 0.008 ± 0.004 mg protein/ml for FDdbs and BDdbs, respectively). These fractions showed low cytotoxicity values (measured by LDH release) and produced high intracellular ROS inhibition (around 80 %), increased the SOD activity and the GSH content, with no effect on GPx activity in Caco2-TC7 cells exposed to H2O2. Several fractions with MM < 2.2 kDa presented also these cellular actions; fractions from FD induced higher increases in GSH concentration. Amaranth flour and a processed matrix like the beverage are shown as sources of bioactive peptides with potential cell antioxidant activity.

Antioxidant effect of amaranth flour or protein isolate incorporated in high-fat diets fed to Wistar rats. Influence of dose and administration duration.

This study evaluated the effect on Wistar rat's oxidative status of incorporating amaranth flour (AF) and protein isolate (AI) in increased-fat diets. Five of the groups were fed for 4 weeks with either BD (basal diet), Chol+F (2% cholesterol, 10% porcine fat), Chol+F+E (0.005% α-tocopherol), Chol+F+AF1 or Chol+F+AI1 (25% of protein replacement) diets. The other two groups were fed for 4 weeks with Chol+F and then 1 week with Chol+F+AF2 or Chol+F+AI2 diet (50% of protein replacement). Various effects on the oxidative stress biomarkers in tissues (intestine and liver) were observed. These effects were dependent on the ingredients, dose, and administration time. In the intestinal cells, Chol+F+AF1 and Chol+F+AI2 produced an increment in the reduced glutathione (GSH) content (56% and 39%, respectively), while Chol+F+AF2 induced an increment in the superoxide dismutase (SOD) (25%) and glutathione peroxidase (GPx) (46%) activities. The presence of certain components in flour (e.g., fiber, polyphenols, squalene) could explain the higher activity recorded for AF. In the liver, Chol+F+AF2 produced a decrease in SOD (19%) and GSH (36%), as well as an increase in GPx (255%); Chol+F+AI1 and Chol+F+AI2 also produced a decrease in GSH (36% and 24%, respectively) and important increments in GPx activity (273% for Chol+F+AI1 and 2,900% for Chol+F+AI2 ). These effects were dependent on the AI dose and were probably produced by absorbed peptides. PRACTICAL APPLICATIONS: It is known that redox imbalances are involved in the genesis of many chronic diseases. Therefore, it is possible to prevent them or limit their severity by improving the body's antioxidant defense mechanisms through dietary incorporation of antioxidant substances. The results suggest that amaranth protein isolate and amaranth flour have the potential for regulating intestinal and liver cells redox balance; effects were more evident when they contributed 50% of the diet's protein content and were administered for 1 week. Both amaranth ingredients could be used as ingredients in the development of functional foods with beneficial antioxidant properties.

Simulated gastrointestinal digestion of amaranth flour and protein isolate: Comparison of methodologies and release of antioxidant peptides.

The effect of both the simulated gastrointestinal digestion conditions and the matrix over protein hydrolysis and antioxidant peptides generation was evaluated by comparing an in-house method with COST INFOGEST-based SGD protocols. The in-house protocol was used to digest amaranth protein isolate I (Id1), while the standardized method and a modified version (similar enzyme/substrate ratio than in our lab) were used to digest I and amaranth flour F (Id3 and Fd3, Id2 and Fd2). Protein hydrolysis degree (TNBS method) was similar for the three I digested (about 60%), but lower for F digested (45 and 34% for Fd2 and Fd3, respectively). The five digested obtained presented comparable protein solubility and only small differences in the polypeptide/peptide composition (SDS-PAGE, tricine-SDS-PAGE, gel filtration FPLC), similar antioxidant activity by the ORAC assay (IC50 values between 0.023 and 0.034 mg.mL-1) and some mild differences by the HORAC assay (IC50 values between 1.13 and 1.30 mg.mL-1 for Id1, Fd2, and Fd3; 1.50 mg.mL-1 for Id2; 1.61 mg.mL-1 for Id3). All the FPLC fractions presented high ORAC activity, while only fractions between 0.43 and 3.5 kDa showed HORAC activity (due to peptide concentration). Differences in activity and potency among fractions were registered, especially for F digested. The modification of digestion conditions produced only small differences in the molecular composition but did not affect the proteolysis degree and the antioxidant activity in the case of digested from protein isolate. The presence of other components and changes in the digestion method had an impact on proteolysis, composition and antioxidant activity of flour digested.

Polyphenols in amaranth (A. manteggazianus) flour and protein isolate: Interaction with other components and effect of the gastrointestinal digestion.

In this work, there were analysed the interaction between phenolics present in amaranth flour (F) and amaranth protein isolate (I) with other components, as well as the effect of the gastrointestinal digestion on them (Fd and Id). Extractions were performed under different conditions (temperature, acid, organic solvent, alkali). Methanol/water extracts (25 °C and 80 °C) from F showed the presence of isoquercetin/rutin, quercetin, kaempferol and two unidentified peaks (II and III). In the presence of acid (much more evident at 80 °C), the extraction of some components increased: catechin, 4-hydroxibenzoic acid, isoquercetin/rutin, II, III. When methanol/acetone/water extraction was performed, p-coumaric acid and a new unidentified peak (IV) were observed. About 15% of the total phenol -namely; p-coumaric, rutin/isoquercetin, and kaempferol- were linked to the protein fraction. After the proteins were isolated (I), the amount of some of the compounds which were originally present in a soluble form (e. g. catechin) and in the protein-bound fraction were decreased. Simulated gastrointestinal digestion of flour released some phenolics (catechin, phenolic acids) that were ligated to proteins, and they significantly incremented the ORAC and ABTS activity of most of the extracts. Isoquercetin/rutin, quercetin and kaempferol remained after digestion. Extracts from the digested protein isolate presented differences in the composition and lower ORAC and/or ABTS activities for some of them. The study of the effect of the simulated gastrointestinal digestion process on bioaccessibility and on antioxidant activity (an aspect that, to our knowledge, has not been previously studied on amaranth polyphenols) yielded promising results, which suggest that amaranth flour is a potential antioxidant functional ingredient.

Identification and characterization of antioxidant peptides obtained by gastrointestinal digestion of amaranth proteins.

The objective of the present work was to separate and identify antioxidant peptides from a simulated gastrointestinal digest (Id) from Amaranthus mantegazzianus proteins (I), which has previously been demonstrated to have this activity. I and Id were separated by preparative RP-HPLC. Fractions were evaluated by the ORAC method and the more active ones were analyzed by LC-MS/MS. Each fraction presented diverse peptides from different proteins, most of them from the 11S globulin. After grouping the peptides from 11S globulin according to their overlapping sequences, and based on previous information about structure-activity relationships, ten sequences were synthesized, in order to evaluate their antioxidant activity. Four peptides presented interesting activity: AWEEREQGSR>YLAGKPQQEH∼IYIEQGNGITGM∼TEVWDSNEQ. They exhibited some of the structural characteristics already known to demonstrate this activity, all of them containing at least one bulky aromatic residue. All belonged to little structured, internal or exposed regions of the acid subunit of the 11S globulin.

Effects of the Dietary Addition of Amaranth (Amaranthus mantegazzianus) Protein Isolate on Antioxidant Status, Lipid Profiles and Blood Pressure of Rats.

The effects of the dietary addition of 2.5% (w/w) Amaranthus mantegazzianus protein isolate (AI) on blood pressure, lipid profiles and antioxidative status of Wistar rats were evaluated. Six diets were used to feed animals during 28 days: (base (AIN93G), Chol (cholesterol 1%, w/w), CE (α-tocopherol 0.005%, w/w), CholE (cholesterol 1% (w/w) + α-tocopherol 0.005%, w/w), CAI (AI 2.5% w/w), CholAI (cholesterol 1% (w/w) + AI 2.5%, w/w). Lipid profiles of plasma and liver and faecal cholesterol content were analyzed. Antioxidant status was evaluated by the ferric reducing activity of plasma (FRAP), the 2-thiobarbituric acid (TBA) assay and superoxide dismutase (SOD) activity in plasma and liver. Blood pressure was measured in the tail artery of rats. CholA group presented a significant (α < 0.05) reduction (16%) in the plasma total cholesterol. In liver, the intake of cholesterol (Chol group) induced a significant increment in cholesterol and triglycerides (2.5 and 2.3 times, respectively), which could be decreased (18% and 47%, respectively) by the addition of AI (CholA group). This last group also showed an increased faecal cholesterol excretion (20%). Increment (50%) in FRAP values, diminution of TBA value in plasma and liver (70% and 38%, respectively) and diminution of SOD activity (20%) in plasma of CholA group suggest an antioxidant effect because of the intake of AI. In addition, CA and CholA groups presented a diminution (18%) of blood pressure after 28 days.

Amaranth peptides from simulated gastrointestinal digestion: antioxidant activity against reactive species.

We evaluated the capacity of simulated gastrointestinal digests or alcalase hydrolysates of protein isolates from amaranth to scavenge diverse physiologically relevant reactive species. The more active hydrolysate was obtained with the former method. Moreover, a prior alcalase treatment of the isolate followed by the same simulated gastrointestinal digestion did not improve the antioxidant capacity in any of the assays performed and even produced a negative effect under some conditions. Gastrointestinal digestion produced a strong increment in the scavenging capacity against peroxyl radicals (ORAC assay), hydroxyl radicals (ESR-OH assay), and peroxynitrites; thus decreasing the IC50 values to approximately 20, 25, and 20%, respectively, of the levels attained with the nonhydrolyzed proteins. Metal chelation (HORAC assay) also enhanced respect to isolate levels, but to a lesser extent (decreasing IC50 values to only 50%). The nitric-oxide- and superoxide-scavenging capacities of the digests were not relevant with respect to the methodologies used. The gastrointestinal digests from amaranth proteins acted against reactive species by different mechanisms, thus indicating the protein isolate to be a potential polyfunctional antioxidant ingredient.