Enzymatic Preparation And Characterization Of Angiotensin Converting Enzyme Inhibitory Peptides Derived From Duck Meat Protein Hydrolysate

With the improvement of standard of living,the morbidity of hypertension which carries a high risk factor for stroke,hemangioma,kidney failure and heart attack has been increasing in China.Angiotensin-converting enzyme(ACE)is an exopeptidase distributed in the blood and tissues of the body.It can cause an increase in blood pressure through the renin-angiotensin system(RAS),and therefore the ACE inhibitory peptides are valued as the effective substance to alleviate hypertension.In recent years,considerable attention has been focused on the food-derived ACE inhibitory peptide due to their easier absorption,lower cost and higher safety.In this study,the ACE inhibitory peptides were prepared from duck meat protein by controlled enzymatic technique.The ACE inhibitory activity of hydrolysate was evaluated through chemical models in vitro.Then the effect of processing conditions and simulated gastrointestinal digestion on the activity of ACE inhibitory peptide were studied.In addition,the ACE inhibitory peptides were purified by using consecutive chromatographic methods and the amino acid sequences of the peptides were identified by Nano-LC-ESI-LTQ-Orbitrap MS/MS.The detailed contents and results are shown as follows:1.Optimization of ACE inhibitory peptides from duck meat protein using response surface methodologyThree enzymes(Alcalase 2.4 L,Protamex,Pepsin)were selected to prepare the duck meat protein hydrolysates.Considering the degree of hydrolysis(DH)and ACE inhibition rate,Alcalase 2.4 L was the optimal protease to hydrolyze duck meat protein.Furthermore,the response surface methodology(RSM)was applied to optimize the hydrolysis conditions,including hydrolysis time,enzyme dosage,pH and hydrolysis temperature.The optimal hydrolysis conditions were as following:hydrolysis time 3.74 h,enzyme dosage 1904.31 U/g,pH 9.82 and hydrolysis temperature 63.59?.Under the actual optimal conditions,the ACE inhibition rate was 67.54%which has no significant difference with the predicted value by RSM.2.The activity stability of ACE inhibitory peptides from duck meat protein hydrolysateThe effect of different processing conditions(heating,NaCl,glucose,pH)and simulated gastrointestinal digestion on the activity of peptide were investigated.The heating temperatures(up to 100?),heating times(20-120 min)and contents of NaCl and glucose had no significant effect on the activity of ACE inhibitory peptide.The peptide attained highest activity at pH=7,while decreased at acid and alkaline condition,rapidly declined in alkaline environment.During the gastrointestinal digestion,the activity of ACE inhibitory peptides increased with pepsin treatment but then declined with further trypsin digestion.Peptides were cleaved into smaller fragments after pepsin digestion and leading to increased exposure of internal hydrophobic residues.With further treatment of trypsin,more free amino acids were released and surface hydrophobicity was declined.3.Purification and identification of ACE inhibitory peptides from duck meat protein hydrolysateThe ACE inhibitory peptides derived from duck meat were isolated and purified by ultrafiltration and serial chromatographic techniques including size-exclusion chromatography and reversed-phased high performance liquid chromatographic method.In total seven peptides were sequenced and identified by Nano-LC-ESI-LTQ-Orbitrap MS/MS.These peptides were synthesized,and their activities were determined.Among this peptides,the peptide TLVP exhibited highest ACE inhibitory activity.