Study On The Chelating Mechanism And Promoting Iron Absorption Characteristics Of Peanut Peptide Ferrous Chelate

As one of the fourth generation of new iron supplements,protein peptide ferrous chelates have the advantages of high safety,high iron absorption promoting efficiency,and strong stability.Metal chelating peptides not only can supplement the trace elements that are lacking in the human body,but also have the nutritional function of peptides,which have dual effects.When metal chelated peptides are transported in the digestive tract,they can directly pass through the peptide transport system,consuming less energy and being fast.So,they have the high bioavailability.Peanut meal is a low-cost source of high protein in food processing.After peanut protein is extracted and enzymatically hydrolyzed,the peanut peptides are produced with various biological activities,such as antioxidant,bacteriostatic,ACE inhibitory and metal ion chelating properties.However,at present,there is still a lack of the in-depth research on the ferrous chelating effect and iron absorption promoting mechanism of peanut peptides,which limits the development of peanut peptide iron supplements.Therefore,this article will use defatted peanut meal as raw material to prepare peanut peptides.Firstly,the optimal chelating conditions for peanut peptides and ferrous ions are determined;Then,the peanut protein hydrolysate was isolated and purified,and the peptide fragment with the highest chelation rate was selected.The chelation mechanism was explored through mass spectrometry analysis and molecular dynamics simulation.The structure of peanut peptide ferrous chelate and its stability were characterized by modern analytical techniques.Finally,the digestion and absorption characteristics of peanut peptide ferrous chelate were investigated through in vitro digestion system and Caco-2 cell experiments.The main contents and results are as follows:(1)The defatted peanut meal was used as raw material,and peanut peptide ferrous chelate was prepared by complex enzymatic hydrolysis.Firstly,using the chelation rate of ferrous ions as an indicator,the effects of protease types,enzymatic hydrolysis time and enzymatic hydrolysis mode on the chelation rate were investigated.The results showed that the peanut peptide solution obtained by mixing alkaline protease and flavoring protease under ultrasound assisted conditions had the highest chelating rate(58%).Through single factor and response surface experiments,the chelating conditions were further optimized,and the optimal conditions were obtained:p H 8.0,peptide iron mass ratio 3.2:1,temperature38℃,and time 30 minutes.Under these conditions,the iron chelating rate of peanut peptide reached 67.1%,which is close to the predicted value of 67.8%.(2)The peanut protein hydrolysate was isolated and purified,and the peptides with high chelation rate were analyzed and identified.The peanut peptide was separated by ultrafiltration,gel filtration chromatography and ion exchange chromatography,and F-122component with the highest chelation rate was obtained.F-122 components were analyzed by mass spectrometry and their amino acid sequences were determined.Six peptide fragments were selected for solid phase synthesis.It was found that four peptides(ESTHLVLR,WGTPGSEVR,ISGLIYEETR,and LSGSFLPK)had high chelating activity,with chelating rates of 91.1%,93.9%,92.6%,and 91.7%.The molecular dynamics simulation study of four peptides showed that glutamic acid,glycine,arginine,threonine,and phenylalanine had strong chelating ability with ferrous ions.In addition,the WGTPGSEVR peptide segment with the highest chelation rate was selected for chelation reaction to prepare a chelate,which will be used for subsequent research on iron absorption promoting properties.(3)The structural characteristics and stability of peanut peptide ferrous chelate were studied.The results of ultraviolet spectrum analysis and infrared spectrum analysis showed that ferrous ions mainly undergo chelation reactions with the carboxyl and hydroxyl O atoms and amino N atoms of amino acid groups.Fluorescence spectrum analysis showed that ferrous ions can chelate with chromogenic groups in peanut peptides,resulting in a decrease in fluorescence intensity and fluorescence quenching;The ~1H NMR spectra showed that the carboxyl,hydroxyl and amino groups in the peptide chain all participated in the chelation reaction.Zeta potential showed that the carboxyl group contained in the peanut peptide fraggment was a chelating site.The microstructure of peanut peptide ferrous chelate was observed using scanning electron microscopy.The results showed that after the reaction between peanut peptide and ferrous ions,a dense spherical structure appeared on the surface,which was significantly different from the smooth surface structure of peanut peptide,In addition,the results of DSC,TG,and thermal stability analysis showed that the thermal stability of the peanut peptide ferrous chelate was superior to the peanut peptide.Under neutral and weak alkaline solution systems,low ethanol concentration,and low PBS concentration conditions,the peanut peptide ferrous chelate had strong stability and high iron ion retention.(4)The digestive and absorption characteristics of WGTPGSEVR-ferrous chelate by simulating gastrointestinal digestion and constructing Caco-2 cell model were explored.The results showed that the iron transfer ability of the peptide chelate remained above 90%after in vitro digestion,indicating that the WGTPGSEVR ferrous chelate has strong stability.The results of cell experiment measurement showed that at the 21st day of cell culture,the cell monolayer membrane was complete and dense,and the differentiation was completed,indicating that the model was successfully established.The MTT method was used to determine the safe concentration of WGTPGSEVR-ferrous chelate.When the concentration of the chelate reached to 0.5 mg/m L,the cell survival rate was still above 90%,and when the concentration of inorganic iron salt was 0.1 mg/m L,the cell survival rate was about 90%;The results of iron ion transport showed that the bioavailability of WGTPGSEVR-ferrous chelate was 51.66%,and significantly higher than 32.14%of inorganic ferrous salts,indicating that peanut peptide iron chelate had higher digestion and absorption characteristics.The sturdy will provide an important theoretical basis for the development of new peptide iron.