Preparation Of Iron Binding Peptides By Enzymatic Hydrolysis Of Scad(decapterus Maruadsi)processing By-products And The Absorption In Model Cells

As a kind of low-value marine fishes,blue-round scad(Decapterus maruadsi)is rich in protein resources.At present,the development and utilization of blue-round scad is rare and there are almost no high value-added products in the market.In this paper,we optimize the hydrolysis process of blue round scad processing by-products(SPB)and separate and purify peptides with high iron binding capacity from the scad hydrolysate.The preparation of iron-binding peptide was studied in order to obtain a higher iron binding capacity of peptides,which lay the foundation for the future study of iron-binding peptide.The main results of this paper are as follows:(1)The degreasing method of SPB was studied.The results showed that the isobutanol degreasing effect was better than that of the ether and petroleum ether,the protein content was 59.62% and the fat content was only 0.6%.The SPB was hydrolyzed by four commercial enzymes,namely,trypsin,flavourzyme,protamex and alcalase,for preparing high iron-binding capacity(IBC)hydrolysate.Alcalase was the best choice for obtaining high IBC hydrolysate from SPB.Response surface methodology(RSM)using a central-composite design was employed to optimize the enzymatic hydrolysis conditions with alcalase to obtain a maximum hydrolysate yield from the SPB with high iron-binding capacity.The best alcalase hydrolysis conditions were as following by RSM: hydrolysis temperature of 46 °C,enzyme substrate ratio of 6040 U/g-protein and hydrolysis time of 66 min,respectively.Under these optimal hydrolysis conditions,the predicted iron binding capacity was 317.2 ?g/g,which was consistent with the average of three replicates of 296.2 ?g/g obtained in the validation experiments.The IBC of hydrolysate did not displayed linear relationship with antioxidative ability(DPPH free radical scavenging activity,DSA)or the degree of hydrolysis(DH).(2)Four fractions(>10 k Da,5-10 k Da,3-5 k Da,<3 k Da)were obtained from SPB hydrolysates by using ultrafiltration and then they were used for preparing the iron-peptides complexes.Qualitative testing was used to identify the complex including UV-spectroscopy and infrared spectroscopy.It was confirmed by spectroscopy that the iron peptide complex with the polypeptide was indeed formed.The amino acid compositions of the four ultrafiltration components was analyzed.The amino acid contents of the four components were basically the same,and the content of glutamic acid was the highest,followed by lysine and aspartic acid.(3)The IBC of ultrafiltration fractions was determined.Among them,the <3 k Da fraction has the highest iron binding capacity,reaching 346.46 ?g/g-protein.The components of the gastrointestinal digestion stability was studied,the results show that the digestion in the gastrointestinal still maintained the good stability.And the subsequent iron binding capacity was analyzed.The components were separated to IMAC Sepharose Fast Flow affinity chromatography.The active fraction of IMAC had high iron binding capacity of 431.54 ?g/g,and further purified by Sephacryl 100 gel column.The components iron binding capacity was 470.55 ?g/g.The final half-preparation of reversed-phase high performance liquid chromatography separation and purification,and finally the two amino acid sequences were identified: the amino acid sequence of molecular weight: 1386.63 Da The sequence is Gln-Lys-Gly-Thr-Tyr-Asp-Asp-Tyr-Val-Glu-Gly-Leu;the next amino acid sequence having the molecular weight of 1437.67 Da is Phe-Asp-Arg-Val-Gly-Asp-GlyGln-Val-Gly-Phe-Asn-Gln.By NCBI alignment,the two small peptides were the sequences of No.91 to 104 and No.31-43 of the blue-round scad light chain 3.(4)The effects of scad peptides on absorption of iron in the Caco-2 cell model was studied and the results confirmed that the scad peptides significantly enhanced iron uptake in the Caco-2 cells,and the intracellular ferritin content(261.08 ng)was significantly higher than that of the Fe Cl3 group(105.43 ng)and the positive control ascorbic acid group(159.24 ng).Thus,the iron-binding peptide has the potential to promote iron uptake,and the results of this study lay the foundation for its application as an iron supplement.