Separation and Identification of Highly Efficient Antioxidant Peptides from Eggshell Membrane

Qian-Cheng Zhao; Jie-Yuan Zhao; Dong Uk Ahn; Yong-Guo Jin; Xi Huang

doi:10.3390/antiox8100495

Separation and Identification of Highly Efficient Antioxidant Peptides from Eggshell Membrane

Antioxidants (Basel). 2019 Oct 18;8(10):495. doi: 10.3390/antiox8100495.

Authors

Qian-Cheng Zhao¹, Jie-Yuan Zhao², Dong Uk Ahn³, Yong-Guo Jin⁴, Xi Huang⁵

Affiliations

¹ College of Food Science and Technology, National Research and Development Centre for Egg Processing, Huazhong Agricultural University, No 1 Shizishan Street, Wuhan 430070, China. zhaoqiancheng5696@163.com.
² College of Food Science and Technology, National Research and Development Centre for Egg Processing, Huazhong Agricultural University, No 1 Shizishan Street, Wuhan 430070, China. m18202782622_1@163.com.
³ Department of Animal Science, Iowa State University, Ames, IA 50011, USA. duahn@iastate.edu.
⁴ College of Food Science and Technology, National Research and Development Centre for Egg Processing, Huazhong Agricultural University, No 1 Shizishan Street, Wuhan 430070, China. jinyongguo@mail.hzau.edu.cn.
⁵ College of Food Science and Technology, National Research and Development Centre for Egg Processing, Huazhong Agricultural University, No 1 Shizishan Street, Wuhan 430070, China. huangxi@mail.hzau.edu.cn.

Abstract

The enzymatic hydrolysates (EHs) of the eggshell membrane (ESM) were obtained after incubating eggshell membrane in solutions prepared with Na₂SO₃ and alkaline protease combinations. The effects of enzyme species, enzyme dosage, Na₂SO₃ concentration, and hydrolysis time on the antioxidant activity of the ESM-EH were determined. Also, the correlation between the degree of hydrolysis (DH) and the antioxidant activity of ESM-EH was analyzed. The DH of ESM-EH showed a highly positive correlation with the reducing power (R² = 0.857) and total antioxidant activity (TAA) (R² = 0.876) and performed negative correlation with the Fe²⁺-chelating ability (R² = -0.529). The molecular weight distribution of the ESM-EH was determined by MALDI-TOF/MS. Cation exchange chromatography (Sephadex C-25) was used to isolate the ESM-EH and then the enzymatic hydrolysis fragment (EHF) was obtained. Among the five isolated fragments (F1~F5), fragment 3 (F3), which was composed of 28 polypeptides, showed the highest ability to quench ABTS• (2,2-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid) (90.44%) and also displayed stronger TBARS (thiobarbituric acid- reactive substances) (58.17%) and TAA (303.82 µg /mL) than the ESM-EH. Further analysis of the 28 peptides in F3 identified using LC-MS/MS indicated that five peptides (ESYHLPR, NVIDPPIYAR, MFAEWQPR, LLFAMTKPK, MLKMLPFK) showed high water-solubility, biological activities, and antioxidant characteristics. Finally, the TAA of the synthetic peptide was verified, the synthetic peptides ESYHLPR and MFAEWQPR performed the best activity and have high potentials to be used as antioxidant agents in functional foods, pharmaceuticals, or cosmetics.

Keywords: Na2SO3-assisted enzymatic hydrolysis; antioxidant peptide; eggshell membrane; inhibition of liposome peroxidation; mass spectrometry; total antioxidant activity.

Abstract

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