Separation,Purification And Identification Of Angiotensin Converting Enzyme Inhibitory Peptide From Porphyra Haitanensis And Application On Beverage

Grinding and protease hydrolysis condition was investigated to prepare peptide extracts from Porphyra haitanensis.Three pure constituents were identified by NMR and mass spectrum after using a series of process including salting-out method,gelatin filtration,reversed phase column chromatography.A kind of peptide beverage was developd after deodorization,sterilization and formulating of recipe.Sensory,physicochemical,microbial indicators showed the qualification of the product in pilot scale production.Finally,animal toxicological experiment validated the innocuity of laver extracts and beverage.The content and extraction rate of protein were determined to calculate optimal parameter showing that material-water ratio was 1:15 and time needed 80 mins.Subsequently,ACE inhibitory activity were determined to evaluate the effect of material-water ratio and protease on enzymolysis.Final orthogonal optimization showed that the first enzymolysis time needed 2 h,material-water ratio was 1:15,trypsin dosage was 2% and the second enzymolysis time needed 3h,material-water ratio was 1:10,alkaline protease dosage was 1%,both at optimum temperature and pH.Under this condition,content and extraction rate of protein were 1.25% and 85.22%respectively,and ACE inhibitory activity reached 93.51%.The laver extracts was separated into seven components by ammonium sulfate gradient salting-out method,and subsequently the seven components above were separated into twenty-six components by Sephadex G-15 columniation.High ACE inhibitory activity components were analyzed by RP-HPLC and separated and purified by RP-C18 columniation,gaining five pure fractions.Three of the five pure fractions were identified by NMR and ESI-MS,namely phenylalanine,isoleucine and leucine.1.5% ?-cyclodextrin showed the best deodorization effect of the laver extracts according to sensory evaluation.The optimal sterilization parameter was 120? for 3 mins according to sensory evaluation and microbial indicators.What's more,it remains good ACE inhibitory activity of extracts treated by sterilization,which indicated that it has good thermal stability.The sensory evaluation was used to adjust beverage formula.Subsequently the indicator of food hygiene quality including three sensory indexes,sixteen physicochemical indexes,sevenmicrobial indexes showed it is qualified of the product in pilot scale production.Finally,animal toxicological experiment validated the innocuity of the laver extracts and beverage.