Purification and characterization of antioxidant peptides from cooked eggs using a dynamic in vitro gastrointestinal model in vascular smooth muscle A7r5 cells.

Antioxidant peptides derived from food sources are considered as safer alternatives to commercially available antioxidant drugs. As one of the most abundant protein sources, hen's egg proteins were extensively used to produce antioxidant peptides by enzymatic hydrolysis. Our previous work indicated that gastrointestinal digestion of cooked eggs significantly increased the antioxidant activity due to hydrolysis of egg proteins. To characterize the responsible antioxidant peptides, cooked eggs were digested in a simulated in vitro model of human gastro-intestinal digestion. Prepared digests were fractionated with FPLC (Fast Protein Liquid Chromatography) and RP-HPLC (Reverse-Phase High-Performance Liquid Chromatography) and the antioxidant activity was determined in A7r5 cells (vascular smooth muscle cell line). Further identification of peptides from peptide fractions with the highest antioxidant activity was carried out using LC-MS/MS. Four peptides derived from ovalbumin, DSTRTQ (48-53), DKLPG (61-65), DVYSF (96-100), and ESKPV (205-209), were identified; of which DKLPG did not show antioxidant activity in cells. Enzyme cleave analysis suggested that these four peptides were likely released from ovalbumin only by pepsin non-specific cleaves. It is postulated that egg consumption may exert protection against oxidative stress on human health due to release of antioxidant peptides during digestion.

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.

Effects of storage and cooking on the antioxidant capacity of laying hen eggs.

The aromatic amino acids and carotenoids are the major contributors to the antioxidant properties of egg yolk. This study aimed to evaluate the effect of simulated retail storage and domestic cooking on the antioxidant activity as well as on the aromatic amino acid and carotenoid contents in ordinary table eggs, omega 3/lutein (n-3/lutein) enriched eggs, and eggs from heritage chicken breeds. The oxygen radical scavenging capacity (ORAC) was the highest in n-3/lutein enriched eggs (161.4µmolTE/gsample), while eggs from heritage white leghorns (HW) showed the lowest levels (127.6µmolTE/gsample). Six weeks of storage at refrigerated temperature did not change the ORAC values, as well as the contents of free amino acid, carotenoid, and malondialdehyde (MDA) in egg yolk. Boiling and frying however, significantly reduced the ORAC value, and the contents of free amino acid, lutein and zeaxanthin, and increased the MDA content in eggs. Our results showed that the antioxidant activity is stable during six weeks of simulated retail storage.

A fast isocratic liquid chromatography method for the quantification of xanthophylls and their stereoisomers.

A fast isocratic liquid chromatography method was developed for the simultaneous quantification of eight xanthophylls (13-Z-lutein, 13'-Z-lutein, 13-Z-zeaxanthin, all-E-lutein, all-E-zeaxanthin, all-E-canthaxanthin, all-E-ß-apo-8'-carotenoic acid ethyl ester and all-E-ß-apo-8'-carotenal) within 12 min, compared to 90 min by the conventional high-performance liquid chromatography method. The separation was achieved on a YMC C30 reversed-phase column (100 mm x 2.0 mm; 3 µm) operated at 20°C using a methanol/tert-butyl methyl ether/water solvent system at a flow rate of 0.8 mL/min. The method was successfully applied to quantify lutein and zeaxanthin stereoisomers in egg yolk, raw and cooked spinach, and a dietary supplement. The method can be used for the rapid analysis of xanthophyll isomers in different food products and for quality control purposes.

Hen Egg as an Antioxidant Food Commodity: A Review.

Intake of antioxidants through diet is known to be important in reducing oxidative damage in cells and improving human health. Although eggs are known for their exceptional, nutritional quality, they are not generally considered as antioxidant foods. This review aims to establish the importance of eggs as an antioxidant food by summarizing the current knowledge on egg-derived antioxidants. Eggs have various natural occurring compounds including the proteins ovalbumin, ovotransferrin and lysozyme in egg white, as well as phosvitin, carotenoids and free aromatic amino acids in egg yolk. Some lipophilic antioxidants such as vitamin E, carotenoids, selenium, iodine and others can be transferred from feed into egg yolk to produce antioxidant-enriched eggs. The bioactivity of egg antioxidants can be affected by food processing, storage and gastrointestinal digestion. Generally thermal processing methods can promote loss of antioxidant properties in eggs due to oxidation and degradation, whereas gastrointestinal digestion enhances the antioxidant properties, due to the formation of new antioxidants (free amino acids and peptides). In summary, in addition to its well-known nutritional contribution to our diet, this review emphasizes the role of eggs as an important antioxidant food.

Purification and characterization of antioxidant peptides from enzymatically hydrolyzed chicken egg white.

Egg white is considered as a rich source of high quality proteins with various bioactive peptide fractions. Enzymatic hydrolysis of proteins can be used to release bioactive fractions and different enzymes have different abilities in releasing such bioactive fractions depending on the enzyme's site of activity on a protein. In this study, several proteases were examined for their ability to release antioxidant peptides from hen egg white and protease P was selected based on the antioxidant activity and the digestion yield of the crude protein hydrolysate. A combination of several purification steps including ultrafiltration with low molecular weight cut-off membranes, cation exchange chromatography and reversed phase high performance liquid chromatography was used to purify 'protease P egg white hydrolysate'. Sixteen antioxidant peptides, which were derived from ovalbumin, ovotransferrin and cystatin were isolated from the most active fractions. Amino acid sequences of those peptides were determined using LC-MS/MS. Oxygen radical absorbance capacity (ORAC) values of selected short chain peptides were determined using synthetic peptides. Two peptides AEERYP and DEDTQAMP (Ala-Glu-Glu-Arg-Tyr-Pro and Asp-Glu-Asp-Thr-Gln-Ala-Met-Pro) showed the highest ORAC values. The results from this study indicate that egg white is rich in antioxidant peptides which can be used as a potential source for preparing bioactive ingredients using enzymatic hydrolysis followed by purification techniques.

Bioaccessibility and digestive stability of carotenoids in cooked eggs studied using a dynamic in vitro gastrointestinal model.

Among dietary carotenoids, lutein and zeaxanthin are known to protect against age-related macular degeneration, a leading cause of irreversible vision loss in the elderly. Egg yolk is rich in lutein and zeaxanthin, however, the effect of cooking and gastrointestinal digestion on yolk carotenoids is poorly understood. An in vitro dynamic gastrointestinal model (TIM-1) was used to investigate the digestive stability and bioaccessibility of carotenoids from boiled, fried, and scrambled eggs. Bioaccessibility but not digestive stability was significantly affected by the method of cooking. The main egg carotenoids, all-E-lutein and all-E-zeaxanthin, were stable during the digestion with average recoveries of 90 and 88%, respectively. No trans-cis isomerization of carotenoids was observed during digestion. Both all-E-lutein and all-E-zeaxanthin from scrambled eggs showed significantly lower bioaccessibility compared to boiled eggs. The results indicate that the bioaccessibility of egg carotenoids can be affected by different food preparation methods.

Effect of domestic cooking methods on egg yolk xanthophylls.

Xanthophylls are a class of bioactive compounds known to play an important role in preventing age-related macular degeneration. Egg yolk is a rich source of highly bioavailable xanthophylls including lutein and zeaxanthin. The effects of domestic cooking methods (boiling, frying, microwaving) on egg yolk xanthophyll content were investigated. A LC-(APCI)-MS/MS method was used to identify and quantify all-E- and Z-isomers of lutein, zeaxanthin, canthaxanthin, and ß-apo-8'-carotenoic acid ethyl ester in fresh and cooked egg yolks. Both fresh and cooked yolks showed similar xanthophyll profiles but with higher contents of Z-isomers in cooked samples. All-E-lutein was the most affected, with 22.5%, 16.7%, and 19.3% reductions in boiled, microwaved, and fried yolk extracts, respectively. Total xanthophyll losses ranged from 6% to 18%. The results presented here could be useful in calculating the dietary intake of xanthophylls and also in assessing the xanthophyll profiles and contents of egg-containing products.

Effects of addition of egg ovotransferrin-derived peptides on the oxygen radical absorbance capacity of different teas.

Ovotransferrin-derived peptides showed synergistic effects with vitamin C, epigallocatechin gallate (EGCG), and caffeic acid, but not quercetin in our previous report. In this study, we further investigated the interactions between ovotransferrin-derived peptides and teas, based on the oxygen radical absorbance capacity (ORAC) assay. Our results showed that there was no significant difference in ORAC values among green, oolong, and black teas. For all the samples, 80% methanol extracts possessed better antioxidant capacity than hot water extracts. The antioxidant capacity of teas were improved by adding either ovotransferrin hydrolysate or its purified peptide IRW; however, adding hydrolysate did not improve antioxidant stability of teas. ORAC values of both teas and hydrolysate added teas were decreased during 22weeks of storage, while samples stored at 4°C exhibited higher antioxidant capacity than those stored at room temperature. This suggested that ovotransferrin hydrolysate could be used as functional food ingredients in enhancing antioxidant capacities of foods, which would benefit human nutrition and health.

Characterization and quantification of polyphenols in Amazon grape (Pourouma cecropiifolia Martius).

The phenolic profile of Amazon grape fruit (Pourouma cecropiifolia Martius) was investigated by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS/MS). For this purpose, suitable extraction and liquid chromatographic methods were developed. Anthocyanins, flavonols and chlorogenic acids were found mainly in the peel. Besides the main anthocyanins, i.e. delphinidin 3-glucoside, cyanidin 3-glucoside and cyanidin 3-(6"-malonyl)glucoside, several minor anthocyanins were identified in the peel. Among these, cyanidin 3,5-diglucoside, delphinidin 3-galactoside, cyanidin 3-rutinoside, cyanidin 3-(3"-malonyl)glucoside, malvidin 3-glucoside, pelargonidin 3-glucoside, peonidin 3-glucoside and petunidin 3-glucoside were characterized on the basis of their fragmentation patterns in MS/MS experiments. The total anthocyanin content in the peel was 420.26±3.07 mg kg(-1) fresh weight. The pulp contained mainly 5-O-caffeoylquinic acid (210.39±3.43 mg kg(-1) fresh weight). Rutin was the predominant flavonol found in Amazon grape (peel 155.45 ± 2.06 mg kg(-1) fresh weight and pulp 2.64±1.21 mg kg(-1) fresh weight). Total polyphenols content was higher in the peel than in the pulp.