Extraction And Purification Of Zanthoxylum Bungeanum Seed Iron-binding Peptide And Study On The Properties Of Peptide Iron Chelate

Zanthoxylum bungeanum Maxim seeds kernel are rich in protein and they are a high-quality plant protein resources with a reasonable amino acid composition.Based on defatted seeds of Zanthoxylum bungeanum Maxim of Hancheng Dahongpao as raw material,this thesis was mainly involved in the following aspects:extraction process of iron-chelating peptides from Zanthoxylum bungeanum Maxim seed protein was optimized with response surface method,separation and purification on the peptides was conducted with macroporous resin,the antioxidant and antibacterial activity of the extract were explored.Then the peptides and iron dichloride were used as raw materials to prepare peptide-iron chelate,preparation process was optimized by orthogonal test,physicochemical properties、amino acid composition and stability were carried out in the study.The study had very important practical significance for exploitation of new iron agent,utilization of resources of Zanthoxylum bungeanum Maxim byproduct,acceleration development of the local economy and environmental protection.The main results were as followings:1.Zanthoxylum bungeanum seed protein was hydrolyzed with different proteases,and the optimal protease was screened,then iron-chelating peptides were prepared by the optimal protease.The optimal extraction conditions for the iron-chelating peptide were carried out with response surface method.The results indicated alcalase protease was the optimal protease to prepare iron-chelating peptides from Zanthoxylum bungeanum seed protein.The optimal hydrolysis conditions were as follows:hydrolysis temperature 65℃,pH 10.47,hydrolysis time 2.5 h,substrate concentration 27.70 mg/mL and enzyme concentration 0.09 mg/mL.The results of verification test showed degree of hydrolysis and iron-chelating ability were 7.23%and 585.66 mg-EDTA/g-protein respectively based on the optimal extraction condition.2.The dynamic adsorption/desorption test of DA201-C macroporous resin on Zanthoxylum bungeanum seed iron-chelating peptide showed that the optimum conditions of purification were:the mass concentration of peptide was 0.25mg/mL,adsorption velocity was 1.5 mL/min,eluting agent concentration was 75%,the pH value of sample was 5.0,elution velocity was 1.5 mL/min.The peptides in extract could be basically complete washed off using 160 mL volumn of eluent.After enrichment and purification by DA201-C macroporous resin,the purity of peptides was improved 15.29%compared with the purity of peptides before purification.3.Antioxidant activity of iron-chelating peptide from Zanthoxylum bungeanum seed was investigated by 3 kinds of antioxidant systems in vitro.The results indicated the iron-chelating peptide exhibited strong DPPH radical-scavenging ability(IC50 of 0.61 mg/mL)as well as good hydroxy radical-scavenging ability(IC50 of 1.90 mg/mL)at higher concentration,and DPPH and hydroxyl radical scavenging ability were close to Vc.The peptide had certain ferric reducing ability but the ability of Vc is much higher than the peptide.In addition,within the certain concentration,the ability for 3 kinds of antioxidant system of the iron-chelating peptide showed obvious dose effect relationship.The antibacterial activity of the iron-chelating peptide were studied by the mininum inhibitory concentration(MIC)and minimal bactericidal concentration(MBC)value of salmonella typhimurium、staphylococcus aureus、escherichia coli and bacillus subtilis.The results showed that the above 4 kinds of microorganisms were sensitve in some extent to the peptide,escherichia coli and bacillus subtilis were the stronger sensitive.4.Orthogonal test were carried out to optimize the conditions of peptide-iron chelate,the results indicated:chelating time 45 min,chelating temperature 40℃,polypeptide concentration of 30 mg/mL,pH 6 and polypeptide/iron dichloride ratio(w/w)of 2.5:1.Verification test showed the peptide-iron chelating ablity was 96.84%under the optimal condition.5.Sodium sulphide analysis、spectroscopic analysis and scanning electron microscopy observation confirmed the chelating reaction between iron and Zanthoxylum bungeanum seed polypeptides happened,and the peptide-iron chelate was a new substance different from polypeptides,which was rich amino acid.Infrared scanning showed the-NH 和 COO-of iron-chelating peptide participated in the chelating reaction.Stability test showed:the solubility of the peptide-iron chelate increased gradually with temperature rise,and it exhibited better solubility at acid condition,the solubility was 3.44 g/100g at pH 1;in addition,the storage stability of peptide-iron chelate was higher than ferrous sulfate as well as ferrous chloride,sodium chloride content had not significant influence on stability of peptide-iron chelate.