Preparation Of Mercapto-rich Flaxseed Peptide And Its Protective Effect On Chronic Lead Poisoning In Mice

Lead is a stable,non-degradable,non-essential heavy metal,which is widely used in various industries because of its good application characteristics,but it has also become a great threat to the environment and human health.Therefore,the search for safe and effective natural products is of great significance for the prevention and treatment of lead poisoning.Some studies have shown that natural proteins and peptides containing sulfhydryl groups have strong reactivity and can chelate with lead ions to reduce the harm of lead to human body.As a dominant plant protein resource in Inner Mongolia,flaxseed protein is a highquality plant protein with balanced amino acid composition in accordance with the WHO/FAO standard,and its amino acid composition has a relatively high proportion of cysteine containing sulfhydryl groups,so it has the potential to prepare sulfhydryl-rich flaxseed peptides and prevent lead poisoning.However,it is not clear whether the sulfhydrylrich flaxseed peptide with the effect of preventing and treating lead poisoning can be prepared.therefore,using low-temperature defatted flaxseed cake as raw material,the preparation technology of sulfhydryl-rich flaxseed peptide was discussed in this thesis.the chelating ability of flaxseed peptide with lead ion was preliminarily explored,and its structure was characterized,and then the chelating mechanism between flaxseed peptide and lead ion was analyzed by molecular docking method.Finally,the protective effect of sulfhydryl flaxseed peptide on chronic lead poisoning in mice was explored.The purpose of this study is to provide basic theoretical basis for flaxseed peptide in the prevention and treatment of lead poisoning.The main results are as follows:1.Firstly,using low temperature defatted flaxseed cake as raw material,flaxseed protein was prepared by alkali dissolution and acid precipitation.The enzymatic hydrolysis temperature,pH,enzymatic hydrolysis time,enzyme addition and ratio of material to liquid were taken as the factors,and the sulfhydryl content was taken as the evaluation index.The enzymatic hydrolysis process was optimized by single factor experiment and orthogonal experiment.The results showed that the optimum conditions of enzymatic hydrolysis were as follows: papain was used to hydrolyze 4.0 h with papain at 1:60(g/m L),pH7.0,55 ℃ and3% enzyme.Under these conditions,the sulfhydryl content of flaxseed protease hydrolysate was the highest,which was 16.69 μmol/g.The sulfhydryl-containing peptides in the hydrolysate of flaxseed protease were selectively adsorbed and eluted by Thiopropyl Crystarose6 Fast Flow covalent chromatography,and the sulfhydryl-rich flaxseed peptide(TCPs)with sulfhydryl content of 71.13 μmol/g was obtained.the lead chelating ability of TCPs was discussed,and the results showed that the lead chelating capacity of TCPs was 23.90mg/g.Amino acid analysis showed that the content of sulfhydryl-containing cysteine in TCPs increased by 2.96 times compared with flaxseed protein.Four kinds of flaxseed peptides containing sulfhydryl groups were identified from TCPs by MALDI-TOFMS/MS.Their amino acid sequence is peptide Ⅰ: GQQSQHFDSCCDDLKQLR;peptide Ⅱ:QGGQGQQQQCEKQIQEQDYLR;peptide Ⅲ: EVERWVQQAKQVARDLPGQC;peptideⅣ: SCQQFLWEKVQKGGR,in which peptide Ⅰ contains two cysteine residues,and peptideⅡ ～ peptide Ⅳ contains one cysteine residue,that is,one sulfhydryl group,indicating that flaxseed peptides rich in sulfhydryl groups have been prepared.2.The lead chelation characteristics of peptide Ⅰ ～ peptide Ⅲ were analyzed by molecular docking technique.The results show that the Cys11 site provided by peptide Ⅰ,the Cys20 site provided by peptide Ⅱ and the Cys10 site provided by peptide Ⅲ can all provide sulfur atoms to chelate with lead ions,indicating that sulfhydryl groups play an important role in the chelation of lead ions with peptide Ⅰ ～ peptide Ⅲ.In addition,Asp8,Ser4 and Phe7 sites in peptide I can provide oxygen atoms,His6 sites can provide nitrogen atoms as lead ion chelating sites,Leu16 and Pro17 sites in peptide Ⅱ can provide oxygen atoms as lead ion chelating sites,Glu11,Gln17 and Arg21 in peptide Ⅲ can provide oxygen atoms,and Glu16 sites can also provide oxygen atoms and nitrogen atoms as lead ion chelating sites.3.Furthermore,the protective effect of TCPs on chronic lead poisoning mice was explored by chronic lead poisoning mice.The results showed that TCPs could promote the growth and development of chronic lead poisoning mice and significantly promote the excretion of lead in the feces of mice;TCPs could effectively reduce the levels of renal function indexes BUN,CR and UA and liver function indexes ALT and AST;TCPs could also significantly increase the activities of antioxidant enzymes SOD and CAT and reduce the excessive release of MDA,indicating that TCPs also played a certain role in antioxidation.The histopathological results of liver and kidney also showed the protective effect of TCPs on liver and kidney in mice.In short,TCPs can promote the excretion of chronic lead poisoning mice,but also play a certain role in antioxidation,thus reducing the toxicity induced by lead.This study provides some basic theoretical basis for the prevention and treatment of lead poisoning by TCPs.