Preparation Of Large Yellow Croaker ACE Inhibitory Peptide And Its Inhibitory Mechanism Study

In this paper,the meat of large yellow croaker fish was used as raw materials to prepare hydrolysates with high angiotensin I converting enzyme(ACE)inhibitory rate,and the optimal hydrolysis conditions was determined through single-factor experiments and orthgonal experiment.Then,the large yellow croaker fish hydrolysates(NHs)were further separated and purified by Sephadex G-25 to gain the target fraction which had highest ACE inhibitory rate.In addition,Nano-LCESI-Q-Orbitrap-MS/MS analysis was used to identify the amino sequence of the peptides in the separated components,and the peptides matching the characteristics of ACE inhibitory peptides was screened out to synthetic and verify the ACE inhibitory rate.Finally,the inhibitory mechanism of ACE inhibitory peptides with ACE was obtained by using molecular docking technology.(1)The degree of hydrolysis(DH)and ACE inhibitory rate was used to evaluate the activity of hydrolysates.Single-factor and orthogonal experiments were used to optimize enzymatic hydrolysis conditions to prepare hydrolysates originated from large yellow croaker fish meat.The ACE inhibitory rate and the amino acid composition of NHs were determined in vitro.The results showed that the optimal conditions were fixed as follows:the most suitable protease is a neutral protease;solid-liquid ratio,8 g/100 mL;volume of enzyme,4000 U/g;pH value,7;temperature,50?;and hydrolysis time,7 h.NHs prepared under these best conditions were rich in hydrophobic and acidic amino acids,and its ACE inhibitory rate was 83.8%,with the IC50 value of 0.27 mg/mL.(2)The relationship between the activity and the molecular weight was determined by measuring the molecular weight distribution of the NHs.Sephadex G-25 was used to separate and purify NHs,NHs was separated different components,The molecular weight of the components with highest ACE inhibitory rate was tested and its amino acid sequences were determined by Nano-LC-ESI-QOrbitrap-MS/MS,and the peptides with ACE characteristics were screened out.The results showed that NHs were mainly composed of small molecular peptides with molecular weight distribution below 4500 Da,Four fractions were obtained after purification by Sephadex G-25,among them,fraction 3(F3)had the strongest ACE inhibitory activity.The ACE inhibitory rate of F3 was 84.06%,which was 1.03 times higher than 81.60%before purification.10 peptides matching the characteristics of ACE inhibitory peptides with ALC?99%was identified through Nano-LC-ESIQ-Orbitrap-MS/MS.(3)The ACE inhibitory rates of 10 synthetic peptides were measured in vitro and the peptides with higher activity were screened out,Discovery Studio 2016 Client was used to analysis the molecular docking mechanism of ACE inhibitory peptides with ACE,the result are follows:Among the synthetic peptides,LLAGGW,YVGGW and LGYSF have better ACE inhibitory activity and are significantly higher than other peptides,the IC50 value were 0.49 mg/mL,0.39 mg/mL,0.57 mg/mL;His353,Glu384 from ACE active pockets S1 is important amino that influence the activity of ACE inhibitory peptides,and hydrogen bonding,hydrophobic is main forces to combine ACE and ACE inhibitory peptides,the Zn701 of ACE activity center is also key amino.