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

BACKGROUND: ß-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.

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.

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.

Decomposition of the interaction energy of several flavonoids with Escherichia coli DNA Gyr using the SAPT (DFT) method: The relation between the interaction energy components, ligand structure, and biological activity.

In this study, the SAPT (DFT) method is used to determine the components of the electronic interaction energies (electrostatic (Eele), exchange (Eex), induction (Eind), exchange-induction (Eex-ind), dispersion (Edisp), and exchange-dispersion (Eex-disp)) between the several selected flavonoids and the important residues of the active site of Escherichia coli DNA Gyr determined by molecular docking. A significant linear correlation between the calculated SAPT (DFT) interaction energies of flavonoids and their experimental pIC50 values is found, which is not observed for the free binding energies (ΔGb) of flavonoids obtained from molecular docking. The variation of the interaction energy components of flavonoids with their structural differences is investigated to find the relation between the flavonoids structures and their biological activity based on the interaction energy components.

Interaction between the Human OX2 Orexin Receptor and Suvorexant and Some of Its Analogues: SAPT (DFT) Interaction Energy Decomposition Analysis.

In this study, the interaction energy (Eint) of suvorexant (as an orexin receptor antagonist) and some of its analogues with the important residues of the human OX2 orexin receptor, determined by molecular docking, is calculated using the symmetry-adapted perturbation theory-density functional theory (SAPT (DFT)) method. Also, the important residues with the dominant interaction with each ligand are determined based on the obtained SAPT (DFT) interaction energies. To analyze the interaction of the receptor with each ligand, the decomposition of Eint to its constituent components including electrostatic (Eele), exchange (Eex), induction (Eind), and exchange-induction (Eex-ind), dispersion (Edisp), and exchange-dispersion (Eex-disp) is performed. The change of interaction energy components with the replacement of the benzoxazole part of suvorexant by pyrimidine containing different functional groups, thieno pyrimidine, and furo pyrimidine is also investigated, separately. It is found that the change in Eint, due to these replacements, is controlled more by the variation of the electrostatic interaction energy component of Eint than by the other interaction energy components. A linear correlation (R2 = 0.91) is found for the variation of Eint versus experimental ligand-binding affinities. Also, the existence of the linear correlation for the variation of the interaction energy components with experimental ligand-binding affinities is investigated. The variation of the electrostatic component versus experimental ligand-binding affinities shows a more linear correlation compared to the other interaction energy components.

Chaperone-like food components: from basic concepts to food applications.

The significance of chaperones in preventing protein aggregation including amyloid fibril formation has been extensively documented in the biological field, but there is limited research on the potential effect of chaperone-like molecules on food protein functionality and food quality. This review is intended to extend the potential of chaperone and chaperone-like molecules in the prevention of food protein aggregation and amyloid fibril formation. The common features of chaperone molecules responsible for suppressing aggregation and amyloid fibril formation are firstly presented. Then, the function and applicability of chaperone and chaperone-like molecules in food products, in particular high-protein beverages where aggregation and fibril formation are undesirable, are extensively discussed. Protein aggregation in high-protein beverages can be prevented by chaperone or chaperone-like components, such as caseins, heme-containing proteins, specific peptide fragments, phospholipids and polyphenols. The diversity of chaperone-like components and mechanisms might provide the flexibility in taking advantage of their potential applications in different food products.

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

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 µM 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.

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-1ß) 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.

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.

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.

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, ß-turn, and ß-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.