Isolation，purification And Characterization Of Zinc-chelating Peptides From Wheat Germ Protein Hydrolysates And Its Biological Activity

Wheat germ, as a by-product of wheat flour processing, contains abundant naturalprotein. It has becoming a hotspot for researchers both at home and abroad to producebiological active peptides from protein hydrolysates. The aim of this study was to isolate andidentify one or more novel zinc-chelating peptides production from wheat germ proteinhydrolysates using immobilized metal ion affinity chromatography (IMAC-Zn2+),macroporous adsorption resin, reversed phase high performance liquid and MALDI TOF/TOF.The zinc bioavailability of peptides was estimated by Caco-2cells after simulatedgastrointestinal digestion.Firstly, the wheat germ protein was hydrolyzed by Alcalase FG2.4L, Flavourzyme500MG and Papain, respectively. Degree of hydrolysis (DH) and metal chelating ability weretwo kinds of standards for screening the best hydrolysates. It indicated that the hydrolysatesprepared by Alcalase possessed higher DH than two other hydrolysates. Besides, thehydrolysates prepared by Alcalase at200min had the highest metal chelating ability of69.62±0.96%, which were selected for further separation.Secondly， The WGPH were injected into the IMAC-Zn2+column. WGPH1, thefractions unadsorbed on Zn2+, was washed off by double-distilled water. WGPH2was thecrude zinc-chelating peptides, the metal chelating ability of which was69.05%. The analysisof relative molecular weight showed that propotion of the molecular weight of WGPH1formore than2000was higher, while the molecular weight of WGPH2was centrally distributedin range of180-2000. After separated from IMAC-Zn2+, the content of Asp increased from7.95%to7.95%, and the content of Glu increased from13.67%to22.64%. IMAC-Zn2+is anefficient method to enrich peptide fragments containing Glu and Asp.Thirdly, WGPH22was separated from macroporous adsorption resin DA201-C fordesalination. WGPH22was then jumped to RP-HPLC for further purification, and the frctionWGPG22c was collected for mass analysis. Two major single charged ion with m/z at1084Da and1221Da were chosen for MS/MS analysis. The sequences of the two majorzinc-chelating peptides from N to C terminal are determined to be Asn-Ala-Pro-Leu-Pro-Pro-Pro-Leu-Lys-His and His-Asn-Ala-Pro-Asn-Pro-Gly-Leu-Pro-Try-Ala-Ala.Finally, the zinc-chelating peptides (Asn-Ala-Pro-Leu-Pro-Pro-Pro-Leu-Lys-His andHis-Asn-Ala-Pro-Asn-Pro-Gly-Leu-Pro-Try-Ala-Ala) were synthesized using solid phasepeptide synthesis method. The peptide-zinc complexes were also produced, which werecharacterized by ultraviolet spectrum and infrared spectrum. The results showed that the Zn2+may participate in the coordination function with N and O atoms of peptides. The zincchelating ability of Asn-Ala-Pro-Leu-Pro-Pro-Pro-Leu-Lys-His was just15.15±0.70%, while His-Asn-Ala-Pro-Asn-Pro-Gly-Leu-Pro-Try-Ala-Ala possessed a high zinc chelating abilityof91.67±0.81%, which indicated that peptide with His residues at the N-terminal exhibithigher binding capacity than at the C-termnal. Therefore, the zinc bioavailability ofpeptide(His-Asn-Ala-Pro-Asn-Pro-Gly-Leu-Pro-Try-Ala-Ala)-zinc complex was estimated byCaco-2cells after simulated gastrointestinal digestion. The results showed that the zincbioavailability of peptide(His-Asn-Ala-Pro-Asn-Pro-Gly-Leu-Pro-Try-Ala-Ala)-zinc complexwas51.26±0.68%, significantly higher than inorganic zinc (28.00±1.22%).