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

Wang, J., Liao, W., Nimalaratne, C. Chakrabarti, S., Wu. J. Purification and characterization of antioxidant peptides from cooked eggs using a dynamic in vitro gastrointestinal model in vascular smooth muscle A7r5 cells. npj Sci Food 2, 7 (2018). https://doi.org/10.1038/s41538-018-0015-7

antioxidants, egg, peer-reviewed, Publication

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Eggs are an inexpensive source of high-quality proteins. Egg proteins are widely used in the food and nutraceutical industry due to their unique nutritional and functional properties. Peptides derived from egg white proteins were reported to have good antioxidant properties. Our previous research suggested that egg yolk is also a rich source of antioxidants including free aromatic amino acids. However, cooking of eggs reduced the antioxidant activity. Given such a background, objectives of this study were to collect the cooked egg digests for a series of fractionation by liquid column chromatography techniques and antioxidant activity of the fraction was screened based on A7r5 cells. The digested egg samples collected were fractionated using a series of liquid chromatography and screened for antioxidant activity on A7r5 cells (VSMC line). Peptide sequences in the most active fractions were determined using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Antioxidant activity of the identified peptides were synthesized and the activity was verified using A7r5 cells from rat aorta.

Approach

Fresh eggs (n = 60) were obtained, boiled, vacuum packed, frozen immediately at −20 °C, and then packed in a Styrofoam box with dry ice for shipment to the Institute of Nutrition and Functional Foods at Laval University (Quebec City, QC, Canada) for digestion. The eggs were then 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 from rat aorta). Further identification of peptides from peptide fractions with the highest antioxidant activity was carried out using LC-MS/MS. Four identified peptides with sequences of DSTRTQ, DKLPG, DVYSF, and ESKPV were synthesized by “Gen Script” with a purity >98% for antioxidant activity validation. The cells were treated with 50 μM of each peptide for 2 h.

Analysis of Results

Current study used cell-based assays via dihydroethidium (DHE) staining to evaluate the antioxidant activity of the digests. The whole egg digests significantly reduced the superoxide level in VSMCs, confirming egg as a potential antioxidant commodity. Antioxidant fractions were subjected to LC-MS/MS and the results were analyzed by a combination of Mascot searching and MassLynx V4.1 software. Two peptides were identified from F1-3-4 with the amino acid sequence of ESKPV and DSTRTQ, respectively. The other two peptides with the sequence of DKLPG and DVYSF were identified from fraction F1-3-5. Most identified antioxidant peptides were derived from ovalbumin, suggesting ovalbumin is a rich source of antioxidant peptides that can be released by gastrointestinal proteases. The above four peptides were then synthesized for activity validation; of which DKLPG did not show antioxidant activity, while ESKPV and DVYSF showed the highest (p

Application

Although cooking of eggs can decrease the antioxidant activity, results from this study suggested that gastrointestinal digestion of cooked eggs generate peptides with more potent antioxidant activity. Overall, this study provides interesting evidence on the formation of beneficial antioxidant peptides after digestion, indicating the potential benefits of egg consumption against oxidative stress in human body.

Abstract

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.