Preparation Of Angiotensin I-Converting Enzyme(ACE)Inhibitory Peptides Derived From Tilapia Skin And Sea Cucumber

Collagen can be hydrolyzed by proteases to produce peptides,which have special biological activity.Collagen peptides have smaller molecular weight which can be easily digested and absorbed.Moreover collagen peptides show excellent antihypertensive effect,antioxidant activity,scavenging free radicals and antitumor activity.Therefore,they have a broad application prospect in bio-medicine,functional foods and beauty areas.An acid-solubilized collagen(ASC)was purified from the skin of tilapia(Oreochromis sp).Using the trypsin which was purified to homogeneity from the hepatopancreas of Pacific white shrimp(Litopenaeus vannamei)as the tool enzyme,ASC was hydrolyzed.The molecular weight distribution and angiotensin I-converting enzyme inhibitory activity of enzymatic hydrolysates were used to explore the optimal hydrolysis condition.Results showed that the optimized parameters of preparation were: enzyme dosage of 17,000 U/g,at 40 °C,pH 8.0,for 90 minutes.Gel filtration chromatography analysis showed that small-size peptides(< 3 kDa)occupied 99.7 % in the trypsin hydrolysates,suggesting that enzymolysis by trypsin can be used initially to break down collagen into peptides.A novel peptide(NPARTCR)with high ACE inhibition potential was isolated by series of column chromatographies and revealed ACE inhibitory activity with IC50 of 61.4 ?mol/L.Further digestion of this peptide by a proline specific endopeptidase(PSEP),produced a pentapeptide ARTCR with ACE inhibitory activity(IC50)of 77.0 ?mol/L.Both NPARTCR and ARTCR inhibited ACE in a non-competitive manner.NPARTCR and ARTCR were stable against high temperature,acidic or alkaline pH,and ARTCR revealed no obvious change after in vitro incubation with gastrointestinal enzymes.But NPARTCR could be further cleaved by trypsin to form smaller peptides and lose its ACE inhibitory activity completely.Meanwhile,NPARTCR and ARTCR have no toxic effect on Caco-2 cells.Administration of NPARTCR,however,also did not cause a decrease of the SBP during the investigation period.This suggested that gastrointestinal enzymes had further digested NPARTCR in the digestive tract producing smaller peptides or amino acids which had no antihypertensive effect.On the other hand,after oral administration of ARTCR,SBP clearly decreased,and the activities were maintained for about 12 h.In the edible portions of sea cucumbers,the highly insoluble collagen fibers account for about 70 % of total proteins,thus,it can be used ideal raw materials to produce bioactive peptides.The body wall of sea cucumber(Stichopus japonicus)was hydrolyzed by protamex proteinase to produce angiotensin I-converting enzyme(ACE)inhibitory peptides.Tricine-SDS-PAGE and ACE inhibitory activity of the enzymatic hydrolysates were analyzed to explore the optimal hydrolysis condition.Our results showed that the optimized parameters of preparation were: solid to liquid ratio of 1:6(w/v),proteinase dosage of 0.8 %(w/w),6 h,pH 7.0,respectively.After membrane filtration,fractions with molecular weight below 3 kDa revealed higher ACE inhibitory activity with IC50 of 0.8 mg/mL,and its scavenging activities of hydroxyl radical(EC50,7.03 mg/m L),DPPH radical(EC50,3.47 mg/mL)and ABTS radical(EC50,2.25 mg/m L)were obtained.Kinetic analysis showed that the inhibitory pattern of peptides was non-competitive manner against ACE.Meanwhile,ACE inhibitory peptides were stable against high temperature,acidic or alkaline conditions and were tolerant against gastrointestinal digestion in vitro.Moreover,sea cucumber peptides have no toxic effect on Caco-2 cells.Furthermore,antihypertensive effects were analyzed using spontaneously hypertensive rats(SHRs)and the results exhibited that oral administration of sea cucumber peptides could significantly decrease systolic blood pressure,and the maximal decrement in SBP after oral administration of sea cucumber peptides(300 mg/kg)was 26 mmHg at 4 h.Our present study suggested that ACE inhibitory active peptides derived from collagen and sea cucumber hydrolysates may be used as an ideal nutrient for exploitation of functional foods.