Preparation And Bioactivity Of Fe²⁺ Chelating Peptide And Peptide-Fe²⁺ Complexes From Tilapia Skin Collagen

Iron is a necessary micronutrient for the constitution of human tissues and the maintenance of normal physiological functions.When the body is lack of iron,it is easy to lead to anemia,growth retardation and other iron deficiency.It is reported that Fe²⁺chelating peptide is a kind of peptide with the activity of chelating iron ions.The peptide-Fe²⁺chelating complexs can improve the absorption of Fe²⁺in intestine.Therefore,in this research,tilapia skin,which is one of the by-products of tilapia processing,was used as raw material to extract tilapia skin collagen.Using Fe²⁺chelating activity as an indicator,the hydrolysate with Fe²⁺chelating ativity was prepared by enzymatic hydrolysis.The hydrolysate with higheast chelating ativity was purified by immobilized metal affinity chromatography(IMAC-Fe²⁺)to obtain Fe²⁺chelating peptides.The amino acid sequence of Fe²⁺chelating peptide was identified by liquid chromatography tandem mass spectrometry（LC-MS/MS）.In addition,the structure of Fe²⁺chelating peptide and peptide-Fe²⁺chelating complexes were characterized by spectroscopy（such as fluorescence spectrum,ultraviolet spectrum,infrared spectrum and circular dichroism）and morphology（such an particle size distribution and scanning electron microscopy）.Finally,Caco-2 cell monolayer was used as a model to explore the effects of Fe²⁺chelating peptide on the bioavailability of Fe²⁺in cells.The main results were as follows:（1）The hydrolysate obtained by trypsin 2 h（TSCH）have the highest Fe²⁺chelating rate;The best chelating conditions were p H 5,37℃,90 min;TSCH was rich in Asp,Glu,Gly,Arg and pro.（2）The hydrolysate（TSCH）with the highest Fe²⁺chelating rate was eluted by IMAC-Fe²⁺affinity chromatography to obtain the Fe²⁺chelating peptides（TSCICP）.Next,four Fe²⁺chelating peptides were identified from TSCICP by using LC-MS/MS.They were Gly-Pro-Ala-Gly-Pro-Ala-Gly-Glu-Lys（GPAGPAGEK,782.39 Da）Asp-Gly-Pro-Ser-Gly-Pro-Lys-Gly-Asp-Arg（DGPSGPKGDR,984.46 Da）Gly-Glu-Ala-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Pro-Arg（GEAGPSGPAGPR,1052 Da）and Gly-Leu-Pro-Gly-Pro-Ser-Gly-Glu-Glu-Gly-Lys-Arg（GLPGPSGEEGKR,1198.59 Da）.GLPGPSGEEGKR exhibited the highest Fe²⁺chelating rate（55.72±12.72μmol/mmol）（3）The TSCICP was used as the raw material to prepare TSCICP-Fe²⁺complexes.The structural characteristics and the Fe²⁺bio-accessibility of TSCICP and TSCICP-Fe²⁺complexes were analyzed by fluorescence spectroscopy,ultraviolet spectroscopy,infrared spectroscopy,circular dichroism,particle size distribution,Zeta potential,scanning electron microscopy and simulating gastrointestinal digestion.The results show that the TSCICP-iron complexes were formed primarily due to the interaction between carboxylic groups of Glu.After chelated with iron ions,TSCICP was folded and aggregated to form spherical particles with increased particle size.The TSCICP-iron complexes could maintain high iron solubility at different p H（including 1,3,5,7and 9）and in simulated gastrointestinal digestion.The Fe²⁺bio-accessibility of TSCICP-iron complexes was high than that of ferrous glycinate and ferrous sulfate.（4）Take Caco-2 cell monolayer membrane as a model to explore the effects of Fe²⁺chelating peptide（GLPGPSGEEGKR,Peptide G-R）on the Fe²⁺transport,retention,uptake,intracellular ferritin synthesis,and relative gene expression in the Caco-2 cell.The results showed that,compared with the Fe²⁺group,the Fe²⁺+Peptide G-R group significantly increased the content of Fe²⁺transport,retention and uptake in the cell.In addition,the content of transferrin in the cell significantly increased.Peptide G-R can reverse the inhibitory effect of phytic acid on Fe²⁺absorption.Peptide G-R had excellent Fe²⁺chelating activity,which can improve the bioavailability of Fe²⁺in Caco-2 cells.It is expected to develop into a new type of Fe²⁺ dietary supplement.