| Iron, one of the microelements, is necessary to human and animal.Involved in various reactions in body metabolism and because of its low absorption,iron deficiency can cause many symptoms that affects our health. Bioactive peptidesextended its physiological activity while belonging proteins lacked, peptide becomingthe popular research direction in recent years. At the same time, peptides derivedfrom marine natural products also gets more and more attention. Zhejiang province isa coastal marine processing industrial province, a large amount of by-products weregenerated in every minute. It’s a innegligible problem that we should try to furtherstudy its potential economical value and increasing the additional value on theseby-products. The protein content of shrimp processing by-products wasapproximately equaled to shrimp meat and contain several minerals, recent studiesreported that nutritious bioactive substances can be obtained by protease enzymatichydrolysis method, these substances tend to be digested and absorbed easily whencompared to its protein in vivo test. The research was focused on the iron-bindingpeptide derived from shrimp processing by-products.First of all, the optimum enzymatic hydrolysis preparation was studies usingresponse surface methodology (RSM). Six kinds of protease (trypsin, pepsin,protmex, flavourzyme, neutrase and alcalase) were used to hydrolysis the shrimpprocessing by-products protein, trypsin was found to be the best protease forproducing high iron-binding peptides. Fractional experiment showed time, enzymeaddition, pH, amount of pH, temperature, substance concentration, enzyme addtionand hydrolysis time, were the remarkable fractions effecting the iron-binding activityof peptides. The trypsin hydrolysis condition were optimized by RSM using centralcomposition design for producing highest iron-binding active peptides. The pH8.18,enzyme concentration of500U/mL and a hydrolysis time of120min were found to bethe optimum conditions to obtain a higher iron-binding activity of 6.75μg-EDTA/mg-protein. Three certificated replicated experiments showed theactual iron-binding activity equivalent to6.73μg-EDTA/mg-protein.The iron-binding peptides hydrolysate was obtained using the optimumcondition after the RSM result was settled, then the iron-peptide complex wasprepared with ferrous chloride. The iron-peptide complex was mainly composed of11.1%iron,71.5%protein and19.3%ash. The content of iron was approached to thecurrent iron intensifier, EDTAFeNa. The amino acid composition of the iron-peptidescomplex were determined by the amino acid analyzer, the results was similar to theFood and Agriculture Organization (FAO) suggestion. The peptides bound with ironwere certified by infrared chromatography, the binding function group may behydroxyl, carbonyl and carboxyl groups.Finally the iron-binding peptide was separated and purified and its amino acidssequence was determined by mass spectrometry. The molecular weight (MW)distribution on gel permeation chromatography (GPC) showed that it was rangingfrom1to6kDa. Through SP-Sepharose Fast Flow ion-exchanged chromatography,Sephadex G-25molecular sieve chromatography, semi preparative high performanceliquid chromatography (HPLC) and analytic HPLC, the fractions were collected andconcentrated, the pure peptide with relative highest iron-binding activity was chosen.After scanned by the Electrospray ionization mass spectrometry (MALDI-TOF-TOF)with multiple cleavage and alignment of figure in the peptide database, the peptidewas found out a hepta peptide: LPTGPKS, MW=699.274Da. The sequence wascompared to the other metal-binding peptides, the bioactive metal-binding amino acidgroups was discussed.This study was focused on the iron-binding bioactive peptide derived fromshrimp processing by-products hydrolysates, the peptide amino acid structure will bea support to application to the iron supplement and the functional food additives,uplifting the added-value of the low-valued marine food processing by-products. |
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