Preparation And Properties Of Collagen ACE Inhibitory Peptides From Bone Of Eel

Eel is a nutritious aquatic product.It occupies an important place in my country’s aquaculture.In 2020,eel has a breeding output of 250,700 tons in China.Grilled eel is the main processed product of eel.Protein-rich by-products such as fish heads and bones will be produced during the processing of grilled eel,which are generally used as raw materials for feed or discarded,and are less processed in depth.Fish bones are rich in collagen,making full use of it can increase the added value of by-products of eel processing.In this paper,collagen was extracted from eel bone by enzymatic method,after directional enzymatic hydrolysis of collagen,the enzymatic hydrolysate with better angiotensin-converting enzyme（ACE）inhibitory activity was obtained,and then the enzymatic hydrolyzed product was separated and purified by ultrafiltration method.Mass spectrometry（LC-MS/MS）was used to identify amino acid sequences,to clarify the interaction between amino acid sequences and ACE molecules through molecular docking,and to investigate their thermal stability and inhibition kinetics.The effect on the regulatory factors of human umbilical vein endothelial cells reveals its active mechanism.The main the main research results were as follows:Study the collagen extraction process with eel bone as the materials.The physicochemical properties of collagen were analyzed by amino acid composition analysis,infrared absorption,differential scanning calorimetry,thermogravimetric analysis.The results showed that the extraction conditions were as follows:the temperature of 37℃,the p H of 2.0,the enzyme dosage of 1%,the extraction time of 2 h,the solid-liquid ratio of 1:40,the extraction rates at 24.17%;The amino composition analysis showed that the contents of Gly,Pro and Hyp in collagen were higher,which were 20.11%,10.72%and 7.48%respectively.Infrared spectrum showed that the extracted collagen had characteristic absorption peaks of amide A,amide B,amideⅠband,amideⅡband and amideⅢband.Differential scanning calorimetry showed that the denaturation temperature of collagen was80.29℃.Thermogravimetric analysis showed that the maximum weight loss rate was at329.6℃.Scanning electron microscope showed that structure were porous and multilayer aggregates.The angiotensin-converting enzyme inhibitory peptide was prepared from eel bone collagen by enzymatic hydrolysis.The ACE inhibitory activity and degree of hydrolysis were used as evaluation index,the optimum hydrolysis conditions were determined by single factor and response surface experiments.Furthermore,the amino acid composition and molecular mass distribution of the hydrolysate prepared under the optimized conditions were determined.The results showed that alkaline protease was the optimal enzyme,and the optimum hydrolysis conditions were as follows:the temperature of 50℃,the mass concentration of 1.5 g·（100 m L）^–1,the hydrolysis time of 5.25 h,the enzyme dosage of 3.1%,and the p H of 9.2.Under these conditions,the ACE inhibitory activity were 70.33%.The molecular weight of peptides below 1 000 Da and 1 000～3 000 Da in enzymatic hydrolysate accounted for 57.02%and 36.55%;Amino acid composition analysis shows that the content of hydrophobic amino acids related to ACE inhibition activity（such as Pro、Val、Ile、Leu、Phe）could increase.ACE inhibitory activity were used as evaluation index,the hydrolyzed product of eel bone collagen was separated by ultrafiltration,and its amino acid sequence and molecular mass were analyzed by LC-MS/MS,molecular docking of peptides.The results showed that the enzymatic hydrolysis product was separated to obtain three components（F1,F2 and F3）,of which the F2 component had the highest ACE inhibitory activity,which was 79.33%.Eight amino acid sequences GPIGPPGPR,PMGPR,GPAGPAGPR,GPPGPPGL,GPMGPR,GPAGPR,GPSGAPGPR and GGPGPSGPR with potential ACE inhibitory activities were identified from the F2 fraction,with IC₅₀ of 1.441 mg·m L^-1 and 1.47 mg·m L^-1 respectively,0.627 8 mg·m L^-1,0.370 mg·m L^-1,2.247 mg·m L^-1,1.246 mg·m L^-1,1.022 mg·m L^-1 and 0.7582 mg·m L^-1,all were nontoxic.Based on GPAGPAGPR and GPPGPPGL as raw materials to study the stability of p H,temperature,and simulation pepsin and trypsin digestion.Human umbilical vein endothelial cells were as models,the effects of intracellular regulatory factors（NO,ET-1,ROS）were investigated.The results showed that the peptides GPAGPAGPR and GPPGPPGL have good temperature,GPPGPPGL have good p H stability.GPAGPAGPR have good p H stability at p H 2～8,and p H 10～12 of activities was decline.The activities of the two peptides remained basically stable,after pepsin and trypsin digestion.The MTT experiments were found that low concentrations of peptides had no obvious side effects on cells,and the cell production morphology of peptides GPAGPAGPR and GPPGPPGL under different concentrations of treatment had no obvious changes compared with the blank group,and which could promote the release of the relaxation factor nitric oxide（NO）and inhibit the endogenous contraction factor endothelin（ET-1）,and have the effect of lowering blood pressure at the cellular level.