Emulsifying Capacity of Cowpea Protein Isolates. Effect of Thermal and Hydrolytic Treatment.

In this work, modifications due to the effect of thermal treatments (TT 70 and 90 °C) and partial hydrolysis by digestion with alcalase (LH) on the emulsifying properties of cowpea protein isolates (CPIs) extracted at pH 8 and 10 were analyzed. In addition, the influence of protein concentration [0.1 and 1% (w/v)] was evaluated. Emulsions (O:W) were prepared and particle size, stability, interfacial composition, and microstructure were studied. Fresh emulsions formulated with TT CPIs presented lower values of volume-weighted mean droplet size (D4.3), with the increase in temperature and treatment time, compared to the untreated CPIs. After seven days of storage, D4.3 and the indexes of flocculation (FI) and coalescence (CI) increased, mainly at 90 °C. On the other hand, the emulsions with LH CPIs presented lower D4.3 values compared to all the conditions tested, remaining unchanged during the storage time. The destabilization process in the TT CPIs emulsions revealed coalescence at 0.1% (w/v) and cremated-flocculation at 1% (w/v). The presence of polypeptides of low molecular mass (MM) at the interface would be responsible for the better stability found in emulsions with LH CPIs, compared to those formulated with untreated and TT CPIs. Increasing the protein concentration resulted in a significant improvement of all emulsifying properties.

Spray-Dried Infant Formula Emulsion Stability as Affected by Pre-Heat Treatment and Total Solids Content of the Feed.

Pre-spray-drying processing may affect stability after reconstitution of emulsion-based powders, such as infant formulas. This study aimed to evaluate the effects of pasteurization temperature and total solids (TS) of the feed on the stability of the emulsions obtained from the reconstituted powders. Four infant formula powders (50%-75 °C, 50%-100 °C, 60%-75 °C, and 60%-100 °C) were produced at pilot scale, from emulsions with 50 or 60% TS pasteurized at 75 or 100 °C for 18 s. Both the emulsion feeds and the emulsions from the reconstituted powders (12.5% TS) were analyzed. The results showed that feeds with 60% TS were flocculated, as indicated by the large particle size and viscosity and the pseudoplastic behavior. Light microscopy revealed that, during spray drying, the flocs were disrupted in 60%-100 °C, while the 60%-75 °C emulsion remained flocculated, reducing its stability post-reconstitution. Although all four emulsions were mainly stabilized by caseins, the presence of ß-lactoglobulin was also detected at the oil-water interface, in native state in the formulas preheated at 75 °C and aggregated in the formulas preheated at 100 °C. In conclusion, both the degree of whey protein denaturation (resulting from pasteurization) and the TS of the concentrates during infant formula production affected the emulsion stability of the reconstituted powders.

Identification and characterization of antioxidant peptides obtained from the bioaccessible fraction of α-lactalbumin hydrolysate.

Whey is an abundantand sustainable source of bioactive peptides obtained from cheese making process. Whey proteins such as α-lactalbumin can be biologically active when the bioactive peptides encrypted in the amino acid sequence of the native protein are released by enzymatic hydrolysis. In the present work, the identification, sequence analysis, and antioxidant activity of bioaccessible peptides from α-lactalbumin alcalase-hydrolysate was assessed. Antioxidant activity (ABTS, ORAC, and HORAC) of α-lactalbumin showed a significant increase (p < 0.05) after the enzymatic treatment with alcalase and this capacity increased even more after the simulation of the gastrointestinal digestion process. Peptides contained in the gastrointestinal digest of α-lactalbumin hydrolysate were separated by preparative RP-HPLC (55 fractions), and three peptides were identified by LC-MS/MS analysis from selected fractions: IWCKDDQNPH (MW: 1254.54 Da) f(59-68), KFLDDDLTDDIM (MW: 1439.64 Da) f(79-90), DKFLDDDLTDDIM (MW: 1554.67 Da) f(78-90). Among the chemically synthesized peptides, IWCKDDQNPH showed the highest antioxidant capacity determined by ORAC, ABTS, and HORAC assays (IC50 0.015 ± 0.002, 0.45 ± 0.02, and 1.30 ± 0.05 mg/ml, respectively) and this activity may be related to the amino acid sequence. This is the first report where these bioaccessible peptides from α-lactalbumin hydrolysate were identified. The α-lactalbumin hydrolysate could be employed as a functional antioxidant ingredient. PRACTICAL APPLICATION: The present work studied the bioaccessibility of antioxidant peptides from an α-lactalbumin alcalase-hydrolysate by identifying three novel bioaccessible peptides responsible for the antioxidant capacity, providing evidence of the hydrolysate potential as an antioxidant ingredient in the formulations of functional foods and/or food supplements.

Broken Rice as a Potential Functional Ingredient with Inhibitory Activity of Renin and Angiotensin-Converting Enzyme(ACE).

The aim of this work was to evaluate the ability of broken rice, an underutilized industrial by-product, as a potential functional and health promoting ingredient. With this purpose, the ability to inhibit the angiotensin converting enzyme and renin of a rice protein hydrolyzate (RPH) obtained from a high-protein variety of broken rice (var. Nutriar FCAyF) was analyzed (IC50 = 0.87 and 2.7 mg/mL, respectively). RPH was separated by gel permeation chromatography and in a second purification step by RP-HPLC. The sequence of antihypertensive peptides presented in two RP-HPLC fractions was analyzed. Peptides capable of interacting with the active sites of both enzymes were identified. In this study, we demonstrate that the hydrolysis treatment improves functional and biological properties of rice proteins. Protein preparations obtained from a by-product of rice industry, such as broken rice, are a promising ingredient with potentially good biological properties.

In Vitro Modulation of Renin-Angiotensin System Enzymes by Amaranth (Amaranthus hypochondriacus) Protein-Derived Peptides: Alternative Mechanisms Different from ACE Inhibition.

Among the factors affecting the development of cardiovascular diseases, hypertension is one of the most important. Research done on amaranth proteins has demonstrated their hypotensive capacity in vivo and in vitro; nevertheless, the mechanism underlying this effect remains unclear. The aim of this study was to analyze in vitro the inhibition of peptides derived from an amaranth hydrolysate (AHH) on other RAS enzymes other than ACE. The chymase and renin activities were studied. AHH was not able to inhibit chymase activity, although a dose-response effect was found on renin activity (IC50 0.6 mg/mL). To provide an approach to the renin inhibition mechanism, we analyzed AHH renin inhibition kinetics and performed a structural characterization of the peptides involved in the effect in terms of molecular size and hydrophobicity. Results suggest that amaranth peptides exhibit renin competitive inhibition behavior. Renin inhibition potency was directly related to peptide hydrophobicity. RP-HPLC separation of AHH and subsequent analysis of the peptide sequences showed 6 peptides belonging to 11S globulin (that can be grouped into 3 families) that would be responsible for renin inhibition. These results demonstrate that Amaranthus hypochondriacus seeds are an adequate source of peptides with renin inhibitory properties that could be used in functional food formulations.

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.

Amaranth proteins foaming properties: Film rheology and foam stability - Part 2.

In this work the influence of pH and ionic strength on the stability of foams prepared with amaranth protein isolate was analyzed. The behaviour observed was related to the physico-chemical and structural changes undergone by amaranth protein as a result of those treatments. The results obtained show that foams prepared at acidic pH were more stable than the corresponding to alkaline pH. At pH 2.0 the foams presented higher times and more volumes of drainage. This behaviour is consistent with the characteristics of the interfacial film, which showed a higher viscoelasticity and a greater flexibility at acidic pH than alkaline pH value, which in turn increased by increasing the concentration of proteins in the foaming solution. It is also important to note that the presence of insoluble protein is not necessarily detrimental to the properties of the foam. Detected changes in the characteristics of the interfacial film as in the foam stability have been attributed to the increased unfolding, greater flexibility and net charge of amaranth proteins at acidic conditions.

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.

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 ß-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.

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.

Structural changes in gluten protein structure after addition of emulsifier. A Raman spectroscopy study.

Food protein product, gluten protein, was chemically modified by varying levels of sodium stearoyl lactylate (SSL); and the extent of modifications (secondary and tertiary structures) of this protein was analyzed by using Raman spectroscopy. Analysis of the Amide I band showed an increase in its intensity mainly after the addition of the 0.25% of SSL to wheat flour to produced modified gluten protein, pointing the formation of a more ordered structure. Side chain vibrations also confirmed the observed changes.

ß-Xylosidase in strawberry fruit: Isolation of a full-length gene and analysis of its expression and enzymatic activity in cultivars with contrasting firmness.

Strawberry is a non-climateric fleshy fruit, which softens quickly and has short post-harvest life. Ripening is associated with an increment of pectin solubility and a reduction of the content of hemicelluloses. In this work, we have cloned the full-length cDNA encoding a ß-xylosidase (FaXyl1) from Fragaria×ananassa and we have characterized its expression in two strawberry cultivars with contrasting fruit firmness. The analysis of the predicted protein showed that FaXyl1 is closely related to other ß-xylosidases from higher plants. The recombinant protein obtained by over-expressing FaXyl1 in Escherichia coli had ß-xylosidase activity against the artificial substrate p-nitrophenyl ß-d-xilopyranoside. Differently from other bifunctional xylosidases, no α-l-arabinofuranosidase activity was detected in the recombinant enzyme. The expression of FaXyl1 gene was analyzed by northern-blot in Camarosa and Toyonaka strawberry cultivars, and compared with the corresponding protein data obtained by Western-blot and with the ß-xylosidase activity during ripening. The softest cultivar (Toyonaka) showed an early accumulation of FaXyl1 transcript and a higher expression of the corresponding protein during ripening, which correlates with a higher ß-xylosidase activity in all ripening stages analyzed.

A Method of Screening for Highly Inhibitory Lactic Acid Bacteria.

A rapid method of screening for lactic acid bacteria with high inhibitory power was developed. The methodology employed was the agar-diffusion assay, which was standardized for the indicator strain, medium composition, and incubation conditions. The assay was performed in nutrient agar at 30°C with 108 spores of Bacillus subtilis per plate as indicator strain. The inhibition produced by supernatants of lactic acid bacteria cultures harvested at stationary phase was determined. The inhibitory powers of different strains were compared with a standard curve obtained with racemic lactic acid. Results obtained with lactic acid and supernatants of bacterial cultures demonstrate that the diameter of the inhibition zone (d) was related to the pH by the exponential relation d = a exp (- b pH). Results obtained with strains that produced inhibitory substances other than lactic acid could not be fitted into the standard linear curves obtained in the plot of ln d versus pH of lactic acid.