Study On The Preparation,identification And Mechanism Of Sturgeon Anti-inflammatory Peptides

Sturgeon is a kind of fish with great biological and economic importance.Sturgeon meat has the best dietary protein and essential amino acids.It is a good source of bioactive peptides and can be used in the development of functional foods.Currently,a few research report on the biological activity of sturgeon peptides.In this paper,sturgeon hydrolysates were prepared by controllable enzymatic hydrolysis technology,and then,isolation and purification,structure identification,and activity verification.The anti-inflammatory mechanism of sturgeon peptides was preliminarily explored by LPS-stimulated RAW264.7 macrophages inflammatory model.The main research results are as follows:?1?The preparation of sturgeon peptides was carried out by controlled enzymatic hydrolysis?single enzyme hydrolysis?,membrane separation technology and Sephadex G-15 gel chromatography.The NO production was selected as an evaluation index.The best enzyme for preparing sturgeon anti-inflammatory peptides was pepsin,and the<3 kDa ultrafiltration component obtained had better anti-inflammatory activity.The NO inhibitory rate of sturgeon peptides purified by further gel chromatography was significantly higher than that of sturgeon hydrolysates?p<0.05?.The method of gel chromatography was as follows:column size was 1.2 cm×70 cm,the flow rate was1.0 mL/min,sample loading was 75 mg,6 min/tube were collected and detected on 280nm.The amino acid analysis showed that sturgeon peptides were rich in hydrophobic amino acids and Phe,Tyr,etc.,aromatic amino acids.?2?The antioxidant potential of sturgeon peptides was evaluated by the chemical method in vitro.Sturgeon anti-inflammatory peptides had strong antioxidant ability,and the IC₅₀ values of two free radical scavenging abilities?DPPH and ABTS?were 293.0and 282.2?g/mL,respectively.To further explore the anti-inflammatory mechanism of sturgeon peptides,an inflammation model of RAW264.7 macrophage stimulated by LPS was established.The results showed that sturgeon peptides decreased the secretion of inflammatory factors IL-6,IL-1?,TNF-?in the LPS-stimulated RAW264.7macrophages in a dose-dependent manner with the concentration range of 0.125⁰.5mg/mL.Western blot analysis showed that sturgeon peptides could decrease the phosphorylation of ERK,JNK,p38,I?B?and inhibit the transfer of p65 in the cytoplasm to the nucleus,suggesting that sturgeon peptides played an anti-inflammatory role by down-regulating MAPK and NF-?B signaling pathways.?3?The sturgeon peptides were further purified by ion exchange chromatography?SP-650M?and hydrophobic chromatography?ODS?,simultaneously.The high active peptides were identified by LC-MS/MS.The peptide sequences were synthesized and the activity was verified.Finally,three peptides with anti-inflammatory potential were obtained.Their amino acid sequences were:Lys-Ile-Trp-His-His-Thr-Phe?KIWHHTF?;Val-His-Tyr-Ala-Gly-Thr-Val-Asp-Tyr?VHYAGTVDY?;His-Leu-Asp-Asp-Ala-Leu-Arg-Gly-Gln-Glu?HLDDALRGQE?,which is derived from actin,myosin,and myosin,respectively.These peptides were the first reported.These three peptides could significantly improve the SOD activity in the LPS-stimulated RAW264.7 macrophages,and effectively inhibit the secretion of NO,IL-1?and IL-6 in the inflammatory model at a concentration of 50?M.Besides,three peptides could down-regulate the MAPK signaling pathway to exert anti-inflammatory influence.