Studies Of Preparation,purification And Bioactivity Analysis Of Peptides From Bighead Carp(Hypophthalmichthys Nobilis) Muscle Hydrolysates

Hypophthalmichthys nobilis,commonly known as the bighead carp,is one of the four major domestic fish.The head taste delicious and have high nutritional value,which have attracted most of people.The utilization of fish muscle part was low because they contain too much fish bones with the inherent odour of freshwater fish.The development of deep processing of freshwater fish to increase its added value has become an important research direction.Bighead carp protein is rich in essential amino acids and the ratio of composition is close to human,which are high quality raw materials for enzymatic preparation of bioactive peptides.Therefore,in this study,fish flesh protein was used as a raw material to prepare DPP-IV inhibitory peptide,ACE inhibitory peptide and antioxidant peptide by enzymatic method and to evaluate its biological activity through cell experiment,which provided a theoretical basis for deep processing and application of bighead carp protein.In this study,pepsin,trypsin,papain and Alcalase were used to hydrolyze fish carp protein.The degree of hydrolysis of bighead carp fish protein and DPP-? inhibitory activity,ACE inhibitory activity and antioxidant activity of the hydrolysate were determined.The molecular weight distribution was also determined and the relationship between degree of hydrolysis and molecular weight distribution and biological activity was analyzed.The results showed that the pepsin hydrolysate had higher DPP-? inhibitory capacity,ACE inhibitory potency and antioxidative activity.Meanwhile,the degree of hydrolysis of pepsin hydrolysate was relatively high,and the proportion of its molecular weight which was less than 2 kDa was higher.The bighead carp protein peptides were isolated by ultrafiltration,gel filtration chromatography and reversed-phase high performance liquid chromatography(HPLC).The bioactivity of the protein was determined at each stage of isolation.Finally,DPP-? inhibitory peptide,ACE inhibitory peptide and antioxidant peptide were identified by LC-MS/MS method respectively.After being synthesized,the biological activities of DPP-? inhibitory peptide,ACE inhibitory peptide and antioxidant peptide were determined respectively.The antioxidant peptide Met-Lys-Ala-Val-Cys-Phe-Ser-Leu(MKAVCFSL)has higher DPPH scavenging ability,reducing power and ferrous ion chelating ability,in which the EC50 value of DPPH scavenging ability was 4.58 ± 0.15 ?M;Tyr-Asn-Leu-Lys-Glu-Arg-Tyr-Ala-Ala-Trp(YNLKERYAAW)and Tyr-Asn-Arg-Leu-Pro-Glu-Leu(YNRLPEL)had higher ACE inhibitory activity and their IC50 value was 1.35±0.23 ?M and 3.42±0.39 ?M,repectively;Ile-Ala-Asp-His-Phe-Leu(IADHFL)had higher DPP-? inhibitory activity,which IC50 value was 610.1 ± 82.6 ?M.In vitro simulation gastrointestinal digestion experiments of IADHFL showed that the peptide was degraded into IADHF and L,and some remained intact.The Caco-2 cell monolayer model was constructed and the transepithelial electrical resistance(TEER)was evaluated for its integrity.The Caco-2 monolayer model was established and the apparent permeability coefficient Papp of DPP-IV inhibitory peptide was determined.The results showed that the peptide could be degraded by Caco-2 cell membrane peptidase after being identified by mass spectrometry.The fragment of FL was observed in AP side of Caco-2 monolayer and new fragments of FL,DHFL,IADHF were found in BL side.However,intact absorption was still happened during the process of experiment.Absorption coefficient Papp(AP-BL)was(4.91±1.90)×10-8 cm/s,and the absorption mechanism studied show that the main pathways for the intact absorption of IADHFL was by-pass absorption.DPP-? inhibition of IADHFL at the cellular level was investigated by measuring membrane DPP-? activity and extracellular DPP-? activity in Caco-2 cells.The results showed that IADHFL can significantly reduce membrane DPP-? and extracellular DPP-? activity of Caco-2.In the meantime,the effects of IADHFL on DPP-? gene expression and protein expression were investigated by real-time quantitative PCR and western blot.The results showed that IADHFL could effectively inhibit DPP-? expression.The content of one of the DPP-? substrates,incretin(GLP-1),also increased,and its gene expression was not significantly affected.To explore the ability of IADHFL affecting insulin secretion,INS-1 cells were assayed for insulin secretion by the Caco-2/INS-1 cell double-layered cell model and direct peptide treatment of INS-1.The results showed that IADHFL could stimulate the insulin secretion in INS-1 cells at the concentration of 2.8 mM and 16.7 mM glucose stimulation.The antioxidant peptide MKAVCFSL was investigated for simulated gastrointestinal digestion.The peptide was partially degraded into small fragments of MKA,FSL,AVCFSL and MKAVCF by pepsin and trypsin determined by LC-MS/MS.Part of the MKAVCFSL still retains its integrity.Caco-2 cells were under oxidative stress after stimulated by H2O2.Cytotoxicity assay showed that MKAVCFSL could protect Caco-2 cells from H2O2-induced cell injury.To investigate the mechanism,the content of intracellular reactive oxygen species(ROS)and lipid peroxidation product malondialdehyde(MDA)were tested,and the results showed that the peptide can significantly eliminate intracellular ROS and reduce the content of MDA.In addition,MKAVCFSL enhanced the activities of cellular endogenous antioxidant enzymes superoxide dismutase(SOD),glutathione peroxidase(GPx)and glutathione reductase(GR)under oxidative stress,But no significant effect on catalase(CAT).At the same time,the contents of endogenous GSH and GSSG were significantly increased.In Caco-2 cells that were not induced by H2O2,MKAVCFSL could also promote a significant increase in GSH content without significant changes in GSSG.