Preparation And Chelating Properties Of Egg Yolk Protein Ferrous Chelating Peptide

Iron is the most important mineral elements in the human body.However,iron deficiency anemia has become one of the major global nutritional deficiency problems.Peptide-ferrous chelate can be used as an effective way to solve this problem owing to its high bioavailability,safety and non toxicity.Egg yolk,a part of eggs,is rich in nutritious.At present,the deep processing of egg yolk was not enough,and its products had lower additional value.Free-lecithin egg yolk protein powder was used to prepare egg yolk peptides according to the ferrous chelating ability.And the effect of ethanol stepwise separation on the ferrous chelation of peptide was studied.Then,the absorption and utilization of peptide-ferrous chelate in Caco-2were investigated,so as to provide a theoretical guidance for the application of peptide-ferrous chelate in the iron nutrition fortifier.The specific conclusions were as follows:(1)The egg yolk peptide with high ferrous chelating activity was prepared by enzymatic hydrolysis method:the result of three metal ions and enzyme type selection showed that the Fe²⁺was best chelating metal ion and Alcalase 37071 was the best enzyme;through one-factor experiment and the response surface optimization experiment,the optimal enzymatic hydrolysis process was obtained as follows:concentrate was fixed at 10%,initial pH 10.0,temperature55.59°C,enzyme dosage 1.08%,,and 3.22 h of hydrolysis time.Under these conditions,the ferrous chelating activity was 87.32%,which was close to the theoretical value 88.57%,and the DH of protein hydrolysis was 15.41%.(2)Egg yolk hydrolysates were isolated and enriched by ethanol precipitation,and there were four components were obtained.In addition,the ferrous chelating activity of the four components and the hydrolysates was determined.Results showed that:the ferrous chelating activity of each component was E3>E2>E1>E4,and the total nitrogen content in each component was E4>E1>E2>E3.E3 showed the highest ferrous chelating activity(89.93%)and the lowest total nitrogen content(4.48%).The E3 component with molecular weight distribution of 1000-5000 Da had the largest con tent(33.44%).And the amino acids composition analysis showed that the content of S er,Asp,Lys,Arg and His in E3 were significantly higher than those in hydrolysates.In addition,the five amino acids were related to ferrous ion chelation.Six peptideswe re identified from E3 by LC-MS/MS,DDSSS*S*S*S*S*SVLSK,ARIITEVNPES*EEE DE and AGSRAAARIITEVNPES*EEEDES*SPYE were derived from egg yolk phosph oprotein;NIRDRQS*VEDVS*SGNSF,TDNIRDRQS*VEDVS*SGNSF and RQS*VVED VS*SGNSF come from vitellogenin,and most serines in this peptide were phosphoryla ted;The peptide-ferrous chelate(E3-Fe)showed higher stability at different pH(5-9)a nd temperatures.(3)The in vitro antioxidant activity,retention rate of iron in simulated digestion and the absorption and transportation of iron in Caco-2 cells were investigated.These experiments demonstrated that E3-Fe could effectively scavenge DPPH free radical,·OH and ABTS⁺,and its antioxidant activity was higher than E3.In addition,E3-Fe expressed stability in simulated gastrointestinal treatment,and the iron retention rate was 66.89%when digested in a simulated intestinal fluid for 120 min.Compared with the traditional ion supplement ferrous sulfate,peptide-ferrous chelate before ethanol fractionation and E3-Fe promoted the absorption of ferrous ions in Caco-2 cells more effectively,In terms of bioavailability testing we identified that iron uptake from E3-Fe was 3.2 times of treatment with ferrous sulfate.