Preparation And Purification Of Iron Binding Peptides From Soybean Isolate Protein Hydrolysate

Iron is one of the essential trace elements and is the essential componentof hemoglobin a variety of biochemical metabolism in human body participating invarious human biochemical metabolism.While the content of inorganic iron in foodis high, but the absorption rate is low, and it is difficult to make full use of the humanbody. Absorption rate of iron of biological peptide iron (such as peptide) is muchhigher than that of inorganic iron, remarkable curative effect in the treatment of irondeficiency anemia. China is rich in soybean resources and the protein hydrolysistechnology is maturing. The enzymatic hydrolysis iron chelating peptide made bysoybean protein not only improve the absorption rate of iron and other trace elementsbut also provide a high-quality nitrogen source for the human body.In this papertaked dephytated soybean protein isolation (SPI) as a raw material, pretreatment ofmaterial, choice of hydrolase, the optimal conditions for enzymatic hydrolysis werestudied to produce hydrolysate adopting enzymolysis technique. Iron binding abilitywas detected to be the evaluation guide. Amino acid composition, molecular weightdistribution, iron binding ability in different assay systems and the stability of ironbinding ability were analized to understand the properties of soybean proteinhydrolysate (SPH). The hydrolysate was further isolated and purified by several stepsincluding ultrafiltration step by step, affinity chromatography and a two-stepreverse-phase high-performance liquid chromatography (RP-HPLC) in order topurify a peptide. The purified iron-chelating peptide was identified by electrosprayionization tandem mass spectrometry (ESI/MS/MS).The main findings are asfollows:(1) Anion exchange resin was effective for dephytatinise from SPI, whicheliminated about80%phytate in the protein.(2)Phosphorylation by Sodium Trimetaphosphate(STMP) can increasesignificantly the iron binding ability, but did not change the degree of hydrolysis(DH).The results of opyimization conditions of phosphorylated soy protein isolatepeptides after hydrolysis were as follows: STMP2.0%(w/v), pH11, temperature30℃, time duration30min. The iron binding ability was18.45mg EDTA/g-protein。(3)SPI was independently hydrolyzed by Trypsin, Pepsin, Protamex,Flavourzyme, Neutrase, and Alcalase. Results showed that the Trypsin hydrolysatetook on the highest iron binding ability and also gived the highest DH of all. Themolecular weight distribution of each hydrolysate was not that big of a difference. (4) The single-factor was used to product peptides with high iron binding ability.The results showed that the optimal enzymatic hydrolysis conditions were substrateconcentration40mg/mL, enzyme concentration1000U/L, temperature37℃, timeduration120min, pH8.0. DH and iron binding ability of peptides were21.8%and2.85mgEDTA/g-protein.(5) The results of opyimization conditions of the preparation of iron-peptidecomplex were as follows: pH5, the ratio of peptide and iron being3:1, substrateconcentration of40mg/mL, temperature of25℃, time duration of20min.(6) The hydrolysate produced under optimized phosphorylated soy protein isolatepeptides with high iron binding ability was choosed for properties research. Theresults were as follows: The fraction of molecular size less than3kDa exhibitedhighest iron binding ability, but there were no significant different in amino acidcompositions between the four ultrafiteration fractions.(7) The hydrolysate was isolated and purified by several steps and iron bindingability was used to evaluate the biological activity of fractions. The purified peptidewas identified as Met-Pro-Val-Asn-Cys (563.3Da), and the iron binding ability was805.64mgEDTA/g-protein, which was a4-fold higher compared with the first stepseparation by affinity chromatography.