Preparation Of Iron Binding Peptides From Mackerel Processing By-products By Enzymatic Hydrolysis

As one of the essential trace elements, iron plays very important rolein the human body. However, because of the form that iron presents in food makes itnot easy to be absorbed by body. The existence of small peptides operation systemand the low molecular peptides transport system in the human body make thecombination of iron and peptides easy to be absorbed by the body. Marine fisheries isone of the traditional and competitive industries in Zhejiang marine economyindustries, it is also the main direction of ocean development and utilization inZhejiang Province. Mackerel as a kind of high yield and low value fish, is rich inprotein and highly unsaturated fatty acids. Many processing by-products areappeared during mackerel oil processing and these by-products usually are made intofish feeds. Therefore, these by-products are not high value added utilization. Thisresearch was focused on the iron-binding peptide derived from mackerel processingby-products (MPB).First of all, the MPB was degreased, and then hydrolyzed by five kinds ofproteases (alcalase, trypsin, neutrase, protmex and flavourzyme) to obtain high ironbinding capacity peptides. The results showed that MPB hydrolysate by alcalase hashigher degree of hydrolysis and iron-binding capacity. Through the single factorexperiment of the influence factors of enzymatic hydrolysis (pH, temperature, time,enzyme addition), the center was determined as pH8.0, temperature50oC, time3h,enzyme addition7500U. Based on this result, the optimum enzymatic hydrolysispreparation studies was used by response surface methodology. The results showedthat the optimal enzymatic hydrolysis conditions for higher iron-binding capacitywere pH8.0, temperature50oC, enzyme addition7142U/mL, time2h. At thisoptimum hydrolysis conditions, the iron binding capacity and diphenyl picrylhydrazinyl (DPPH) radical scavenging activity of hydrolysate were20.15mg-EDTA/g-protein and49.75%, respectively. The optimal enzymatic hydrolysisconditions for higher DPPH radical scavenging activity were pH7.8,temperature50oC, enzyme addition7163U/mL, time2.34h, and at theseenzymatic hydrolysis conditions the iron binding capacity and DPPH radical scavenging activity of hydrolysate were19.80mg-EDTA/g-protein49.93%,respectively. The hydrolysis conditions for producing high DPPH radicalscavenging activity peptides and high iron binding peptides were surprisingsimilar. Therefore, the optimal hydrolysis conditions was chosen at pH8.0,temperature of50oC, enzyme addition of7142U/mL, time of2h.Secondly, the iron-peptide complex was prepared, and then characterized byinfrared chromatography. From the difference in infrared chromatography betweeniron-peptides complexes and free peptides, we concluded that iron binding groupmight be hydroxyl, carbonyl or carboxyl groups. The high iron binding capacityfraction was separated and purified by ultrafiltration, affinity chromatography, andRP-HPLC. Finally, the structures of the high iron binding capacity components wereidentified by mass spectrometry to two hexapeptides and one heptapeptide. The twohexapeptides were Asn-Pro-Val-Arg-Gly-Asn (molecular weight656.104Da) andAsn-Pro-Asp-Arg-Gly-Asn (molecular weight672.079Da), respectively. Theheptapeptide was Ser-Thr-Tyr-Glu-Leu-Thr-Phe (molecular weight861.104Da).The sequence was compared to the other metal-binding peptides, the bioactivemetal-binding amino acid groups was discussed.In this study, the MPB was enzymatic hydrolyzed to obtain high iron bindingability peptides, and the hydrolysate was separated and purified. And also the highiron binding ability peptides were identified the amino acid sequence by LC/MS/MS.These results will be a support to application of the iron supplement and thefunctional food additives, and also provide an intensive processing way forlow-value fish as mackerel this high-yielding fish.