Fatemeh Bamdad

Fractionation and characterization of antioxidant peptides derived from barley glutelin by enzymatic hydrolysis.

The antioxidant properties of the barley glutelin hydrolysates were evaluated based on their radical scavenging capacity (DPPH/O₂(·-)/OH(·)), Fe(2+)-chelating effect and reducing power. Alcalase hydrolysates (AH) demonstrated significantly higher antioxidant capacity than those treated by flavourzyme in most of the assays. The AH was separated using ultra-filtration and reversed-phase chromatography, and assessment of the fractions indicated that the large-sized peptides (Mw>10 kDa) possessed stronger DPPH scavenging activity and reducing power, whereas small-sized peptides (Mw<1 kDa) were more effective in Fe(2+)-chelating and OH(·) scavenging effect. The hydrophobic fraction contributed more to Fe(2+)-chelating and OH(·) scavenging activity. Four peptides contributing to antioxidant activities were identified using LC-MS/MS: Gln-Lys-Pro-Phe-Pro-Gln-Gln-Pro-Pro-Phe, Pro-Gln-Ile-Pro-Glu-Gln-Phe, Leu-Arg-Thr-Leu-Pro-Met and Ser-Val-Asn-Val-Pro-Leu. Compared to the positive controls, AH exhibited excellent Fe(2+)-chelating activity and strong DPPH/OH scavenging effect. Thus hydrolyzed barley glutelin is a potential source of antioxidant peptides for food and nutraceutical applications.

Antioxidant capacities of fractionated barley hordein hydrolysates in relation to peptide structures.

SCOPE This study has revealed strong antioxidant activities of barley hordein hydrolysate fractions and investigated the relationships between antioxidant properties and structural features of hordein peptides. METHODS AND RESULTS Barley hordein fractions separated based on molecular weight and surface hydrophobicity demonstrated strong antioxidant bioactivities, especially ferrous ion chelating capacity. Structural studies revealed the poly-l-proline helix, β-turn, and β-sheet as the dominant structural elements in the large molecular weight fraction, while most of the small-sized peptides assumed unordered random coil conformation accompanied by a large amount of ionized carboxyl groups produced as a result of partial deamidation of glutamine residues during hydrolysis. A study of antioxidant capacity in relation to peptide structure indicated the contribution of hydrophobic residues in radical scavenging and the positive effect of ionized carboxyl groups together with Lys and Met in metal chelation activity. QPYPQ was identified as the most repeated sequence in potent RP-HPLC fractions, which might be a particular structural motif that plays a key role in scavenging free radicals. CONCLUSION Barley hordein with its unique sequence partitioning has potential to produce peptides with strong antioxidant bioactivities.

Metal solubility enhancing peptides derived from barley protein.

Mineral supplements are required to be soluble as their bioavailability is highly correlated to their solubility in body fluids. In this study, metal binding capacity of barley protein hydrolysates and their purified fractions was investigated and expressed as increase in solubility of metal ions. Metal ions in the presence of hydrolysates exhibited a remarkable increase in solubility: 118, 32, 10, 29 and 35-fold for Fe(2+), Fe(3+), Ca(2+), Cu(2+) and Zn(2+), respectively. A mixture of low molecular weight peptides possesses a synergistic combination of both charged and hydrophobic residues and achieves the best binding metal ions. Electrostatic interactions via charged side chains and coordination binding with His and Cys, initially attract the metal ions and, afterward, hydrophobic interactions and aromatic ring stacking stabilize the positioning of metal ions in the structure of the peptide. Barley hordein hydrolysates show potential as dietary supplements that enhance both mineral solubility and bioavailability.

Hydrogel particles and other novel protein-based methods for food ingredient and nutraceutical delivery systems

Abstract: Biopolymer hydrogels have gained considerable attention in recent years as one of the most promising delivery systems for food ingredients and nutraceuticals. In this chapter, an attempt is made to review the state of the art of food grade polysaccharide- and protein-based hydrogels over past decades. Several approaches have been used in the way to prepare and engineer the hydrogel matrix, which can be classified in two major groups of physical and chemical crosslinking. Regardless of the type of biopolymer composed, the release mechanism of the loaded compounds from hydrogels is complex, while mainly resulting from three vectors: via diffusion, swelling followed by diffusion, and degradation. Various stimuli-responsive hydrogels developed from polysaccharides, proteins and their derivatives are also introduced, which can deform quickly and reversibly under environmental stimuli. Finally, the applications of biopolymer hydrogels in food science, i.e. delivery of antioxidants, bioactive peptides and probiotics, are highlighted.

Anti-Inflammatory and Antioxidant Properties of Casein Hydrolysate Produced Using High Hydrostatic Pressure Combined with Proteolytic Enzymes

Casein-derived peptides are shown to possess radical scavenging and metal chelating properties. The objective of this study was to evaluate novel anti-inflammatory properties of casein hydrolysates (CH) produced by an eco-friendly process that combines high hydrostatic pressure with enzymatic hydrolysis (HHP-EH). Casein was hydrolysed by different proteases, including flavourzyme (Fla), savinase (Sav), thermolysin (Ther), trypsin (Try), and elastase (Ela) at 0.1, 50, 100, and 200 MPa pressure levels under various enzyme-to-substrate ratios and incubation times. Casein hydrolysates were evaluated for the degree of hydrolysis (DH), molecular weight distribution patterns, and anti-inflammatory properties in chemical and cellular models. Hydrolysates produced using HHP-EH exhibited higher DH values and proportions of smaller peptides compared to atmospheric pressure-enzymatic hydrolysis (AP-EH). Among five enzymes, Fla-digested HHP-EH-CH (HHP-Fla-CH) showed significantly higher antioxidant properties than AP-Fla-CH. The anti-inflammatory properties of HHP-Fla-CH were also observed by significantly reduced nitric oxide and by the suppression of the synthesis of pro-inflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) revealed that 59% of the amino acids of the peptides in HHP-Fla-CH were composed of proline, valine, and leucine, indicating the potential anti-inflammatory properties. In conclusion, the HHP-EH method provides a promising technology to produce bioactive peptides from casein in an eco-friendly process.

Anti-Inflammatory and Antioxidant Properties of Peptides Released from β-Lactoglobulin by High Hydrostatic Pressure-Assisted Enzymatic Hydrolysis

Background: β-lactoglobulin hydrolysates (BLGH) have shown antioxidant, antihypertensive, antimicrobial, and opioid activity. In the current study, an innovative combination of high hydrostatic pressure and enzymatic hydrolysis (HHP–EH) was used to increase the yield of short bioactive peptides, and evaluate the anti-inflammatory and antioxidant properties of the BLGH produced by the HHP–EH process. Method: BLG was enzymatically hydrolyzed by different proteases at an enzyme-to-substrate ratio of 1:100 under HHP (100 MPa) and compared with hydrolysates obtained under atmospheric pressure (AP-EH at 0.1 MPa). The degree of hydrolysis (DH), molecular weight distribution, and the antioxidant and anti-inflammatory properties of hydrolysates in chemical and cellular models were evaluated. Results: BLGH obtained under HHP–EH showed higher DH than the hydrolysates obtained under AP-EH. Free radical scavenging and the reducing capacity were also significantly stronger in HHP-BLGH compared to AP-BLGH. The BLGH produced by alcalase (Alc) (BLG-Alc) showed significantly higher antioxidant properties among the six enzymes examined in this study. The anti-inflammatory properties of BLG-HHP-Alc were observed in lipopolysaccharide-stimulated macrophage cells by a lower level of nitric oxide production and the suppression of the synthesis of pro-inflammatory cytokines. Peptide sequencing revealed that 38% of the amino acids in BLG-HHP-Alc are hydrophobic and aromatic residues, which contribute to its anti-inflammatory properties. Conclusions: Enzymatic hydrolysis of BLG under HHP produces a higher yield of short bioactive peptides with potential antioxidant and anti-inflammatory effects.

High Hydrostatic Pressure-Assisted Enzymatic Treatment Improves Antioxidant and Anti-inflammatory Properties of Phosvitin

BACKGROUND Phosvitin (PV) is a highly-phosphorylated metal-binding protein in egg yolk. Phosphoserine clusters make PV resistant to enzymatic digestion, which might be nutritionally undesirable. OBJECTIVE This study was designed to determine the effects of high hydrostatic pressure and enzymatic hydrolysis (HHP-EH) on the antioxidant and anti-inflammatory properties of PV hydrolysates (PVHs). METHODS PV was hydrolyzed by alcalase, elastase, savinase, thermolysin, and trypsin at 0.1, 50, and 100 MPa pressure levels. PVHs were evaluated for degree of hydrolysis, molecular weight distribution patterns, antioxidant and anti-inflammatory properties in chemical and cellular models. The effect of PVH on gene expression of pro-inflammatory cytokines (TNF-α and IL-1β) was also evaluated using real time-PCR. The hydrolysate with most potent antioxidant and anti-inflammatory properties was subjected to LC-MS/MS analysis to identify the peptide sequence. RESULTS Hydrolysates produced at 100 MPa exhibited higher degree of hydrolysis and greater reducing power and free radical scavenging activity compared to those obtained at atmospheric pressure. After adjusting the phosphate content, alcalase- and trypsin-digested PVHs showed superior iron chelation capacity (69-73%), regardless of pressure. Both alcalase- and trypsin-digested PVHs significantly inhibited nitric oxide production by RAW264.7 macrophage cells. LPS-stimulated up-regulation of proinflammatory cytokines was also suppressed by alcalase-digested PVH. CONCLUSION The HHP-EH method could play a promising role in the production of bioactive peptides from hydrolysis-resistant proteins. HHP-assisted PVH may be useful in preparing a potential pharmaceutical with antioxidant and anti-inflammatory properties.

Sensitive double antibody sandwich ELISA for the quantification of phosvitin

ABSTRACT An effective double antibody sandwich ELISA (DAS-ELISA) method based on monoclonal (mAb) and chicken egg yolk IgY antibodies was developed to determine phosvitin (PV) content in therapeutic and functional products. Leghorn laying hens were immunized with purified PV to produce anti-PV IgY antibody in the egg yolk. High anti-PV IgY titer obtained from the egg yolks collected during 4–10 weeks of the immunization period contained approximately 6.2% of specific anti-PV IgY in total IgY. The PV detection range of the DAS-ELISA and biotinylated DAS-ELISA was 16.8–90 and 7.5–40 ng/mL, respectively. However, biotinylated DAS-ELISA was the better method for PV quantification in terms of accuracy and sensitivity. This highly efficient PV detection method may recuperate the performance of the existing protein assay methods as well as facilitate future research on PV bioactivities and applications.

Antioxidant and anti-inflammatory properties of chicken egg vitelline membrane hydrolysates

ABSTRACT Vitelline membrane (VM) is a multilayered structure that surrounds the egg yolk serving to separate the yolk and the white. Due to its poor solubility in aqueous‐based media, VM proteins and their biological properties have not been fully defined. In the current study, VM was hydrolyzed using different enzymes under the optimum hydrolysis conditions. Antioxidant and anti‐inflammatory properties were evaluated in chemical and cellular models. Flavourzyme‐ and trypsin‐treated samples showed the highest radical scavenging and ferric ion reducing effect (31% and 20 &mgr;M of Trolox equivalents/mg, respectively). In cellular studies, all VM hydrolysates were cyto‐compatible and inhibited nitric oxide production by RAW264.7 macrophage cells significantly. Lipopolysaccharide‐stimulated up‐regulation of pro‐inflammatory cytokines in RAW264.7 cells was suppressed by flavourzyme‐treated VM. These results revealed that enzymatic hydrolysis of VM is a promising approach to produce peptides with several bioactivities (free radical scavenging, metal chelation, and anti‐inflammatory) as valuable ingredients for cosmeceuticals and nutraceuticals.

A Combination of Egg Yolk IgY and Phosvitin Inhibits the Growth of Enterotoxigenic Escherichia coli K88 and K99

BACKGROUND Enterotoxigenic Escherichia coli (ETEC) is the main cause of fatal diarrhea in piglets during the first week of life and over the time of weaning. Pathogenesis of ETEC-causing diarrhea involves intestinal colonization mediated by fimbriae. Although, both IgY and egg yolk phosvitin (PV) possess antimicrobial activity, their combined activity has not been explored. A combination of IgY specific for ETEC and metal-chelating PV may show synergistic effect in reducing the growth of ETEC by inhibiting bacterial proliferation and stipulating protection against ETEC infection. OBJECTIVE The goal of this study was to determine the effects of anti-ETEC IgY and PV on in vitro growth inhibition of ETEC strains possessing K88 and K99 fimbriae prevalent in the porcine population. METHODS Anti-K88 and -K99 IgY antibodies were obtained from egg yolks of 23-week-old Single- Comb White Leghorn hens immunized with K88 and K99 fimbriae of ETEC, respectively, with high titres sustained over 6 to 8 weeks of the immunization period. Specific IgY, PV, and PV-hydrolysate from alcalase-hydrolysis under high hydrostatic pressure (PVH-Alc-HHP) alone or in combination, were used to treat ETEC K88 and K99 cultures at optimal concentrations of 100 μg/mL, 1 mg/mL, and 1 mg/mL, respectively, for 24 h. RESULTS PVH-Alc-HHP demonstrated the highest degree of hydrolysis, 38.9%. Combined use of IgY and PVH-Alc-HHP showed the highest bactericidal effect resulting in ETEC K88 and K99 growth inhibition of 2.8 and 2.67 log CFU/mL, respectively. CONCLUSION Combined IgY-PVH effectively control ETEC, therefore holds a great potential for microbial control in veterinary pharmaceutical industry.