Study On The Preparation And Hypertensive Effect Of Angiotensin-i Converting Enzyme Inhibitory Peptides From Palm Kernel Expeller Glutelin-2

Hypertension is the major risk factor arteriosclerosis,stroke,myocardial infarction and other diseases,threating one fourth of adults in the world.Antihypertensive peptides derived from food receiving more and more attention for the stable antihypertensive effect and safe.They can paly antihypertensive effect through being added into food.The production and trade quantum of palm oil is the first among plat oils in the world.Palm kernel expeller(PKE),a byproduct of palm milling industry,has a high content of protein(14%?20%).Palm kernel expeller protein(PKP)is a good potential edible protein for its high nutritional value,functional properties,antioxidant activity and hypolipidemic ability.However,PKE is only used as fertilizer or feed ingredients,not widely used in food and other industries.Previous studies found that PKP may be a resource of antihypertensive peptides for its inhibition ability on ACE.In this paper,five protein fractions were extracted from PKE.Based the analysis on their nutritional value,physicochemical properties,functional properties and ACE inhibition activity,PKE glutelin-2 was chose to product antihypertensive peptides.Then PKE glutelin-2 was hydrolyzed by mixed enzymes assited with high pressure pretreatment,and hydrolysis conditions were optimumed by central composite rotatable design.The hydrolysates were further separated and purified by ultrafiltration,gel chromatography and PR-HPLC.Moreover,the physicochemical properties,structure characterization,in vivo and in vitro antihyperteion and functional mechanism of the ACE inhibition peptides were studied.The main results were shown as follows:The albumin,globulin,prolamine,glutelin-1 and glutelin-2 were separately obtained from PKE using sequential method.Results showed that palm kernel expeller glutelin-2(PKEG)was the predominant fraction,accounting for 33.32 ± 1.56 g/100 g total protein.The content of essential amino acid,hydrophobic amino acid,sulfur amino acid and aromatic amino acid in PKEG were all higher than other proteins.And the in vitro digestibility and biological value of PKEG were 65.87%and 63.03,respectively.This means that PKEG has high nutritional vale.Among the five protein fractions,PKEG showed the highest ACE inhibitory activity and good antioxidantive ability.Thus,PKEG was chose to produce ACE inhibitory peptides.Results showed that the DH and ACE inhibition activity of PKEG could be significantly improved by igh pressure pretreatment(300 MPa,35?,for 20 min).The highest ACE inhibition ability(61.4 4%)could be obtained when dose of enzymes was 0.97 g/100g,at 49.09?,pH6.68,for 6.45 h.Furthermore,the antioxidant activity,biological value,solubility,emulsifying and foming properties of PKEG were all imporved by the enzymatic hydrolysis.It is obvious that the the usage of PKEG was expanded by the enzymatic hydrolysis.PKEGH were separated by ultrafiltration and Sephadex G-15 gel chromatography,and PKEGH-?-E with the highest ACE inhibitory was obtained.It was found that the systolic blood pressure and diastolic blood pressure of spontaneously hypertensive rats(SHRs)were reduced markedly(P<0.05)after single and chronic oral administration of PKEGH-?-E,without significant effect on the body weight and heart rat of SHRs.PKEGH-?-E was further separated by RP-HPLC and LC-MS/MS.Finally,four novel ACE inhibitory peptides:Ala-Asp-Val-Phe-Asn-Pro-Arg,Val-Val-Leu-Tyr-Lys,Leu-Pro-Ile-Leu-Arg and Val-Ile-Glu-Pro-Arg(with molecular weight of 817.41,620.39,610.42 and 612.36 Da;IC50:0.397,0.331,0.476 and 0.387 mg/mL,respectively)were identified,of which Val-Val-Leu-Tyr-Lys showed the highest inhibition activity.It was found that peptides VVLYK?LPILR and VIEPR were the inhibitore type,while ADVFNPR was the substrated type.The inhibition modality of ADVFNPR and VIEPR is the characteristic of noncompetitive inhibition modalities;whereas VVLYK and LPILR exhibited a mixed modality of noncompetitive and uncompetitive inhibition.Furthermore,all the four peptides identified in PKEG showed relatively good stability against gastrointestinal enzymes digestion,pasteurization and metal ions like K+,Ca2+and Mg2+.This indicated that these peptides could be used as a functional food ingredient to control ACE activity.Results showed that all VVLYK,LPILR,VIEPR and ADVFNPR could effectively inhibite the proliferation of hunman vascular endothelin cells without significant cytoxicity.Moreover,these ACE inhibitory peptides could lower the expression of ACE mRAN,ET-1 mRNA,but increase the expreesion of ACE2 mRA,resulting in the decrease of intracellular ACE and ET-1 content and increase of ACE2 content.Moreover,the four peptides showed excellet scavenging ability on superoxide radical and hydroxyl radical.Then also can protect vascular endothelial cells from damage induced by H2O2.Hence,these peptides may exert antihypertensive ability through inhibiting the proliferation of vascular endothelial cells,reducing the ACE and ET-1 content,and/or through protecting vascular endothelial cells from reactive oxygen species mediated damage.