Study On Separation And Purification Of ACE Inhibitory Peptides From Konjac And Its Antihypertensive Activity In Vitro

Angiotensin Converting Enzyme inhibitory peptides are a kind of polypeptides that can inhibit ACE activity.ACE inhibitory peptides competitively bind to ACE that can block the production of angiotensin II which play a role in elevating blood pressure,and promote the production of bradykinins and enkephalins which play a role in antihypertensive activity,so it can lower blood pressure.ACE inhibitory peptides are small molecular with high bioavailability and no allergenicity to human body.Currently,more and more people pay attention to ACE inhibitory peptides,source and preparative methods at home and abroad.It was reported that chinese annual production of konjac fine powder were more than 5,000 tons,light weight and small particles of powder were produced when the konjac flour was processed,which are known as konjac flying powder,which occupy for 30% to 40% of konjac fine powder.Most of Konjac flying powder is directly discarded,only a very small part is exploited.Studies shows that Konjac flying powder contains abundant protein,ACE inhibitory peptides were acquired by direct enzymolysis Konjac protein.Therefore,Konjac flying powder may be a new source of natural medicine for the preparation of ACE inhibitory peptides.Here,konjac flying powder was selected as the raw material,Konjac Protein was extracted by alkaline?solution and acid?isolation method.ACE inhibitory peptides were prepared by direct enzymolysis konjac protein.The enzymatic solution was purified by ultrafiltration,SephadexG?15 column chromatography and Reversed?phase High Performance Liquid Chromatography to obtain highly purity konjac ACE inhibitory peptides.The antihypertensive activity in vitro and structure analysis of konjac ACE inhibitory peptides were investifated.The main contents and results are as follows:(1)Konjac protein was extracted by alkaline-solution and acid-isolation m-ethod.ACE inhibition rate and degree of hydrolysis were determined as the indexs,ACE inhibitory peptides were prepared by enzymolysis of konjac protein with Bromelain,Alkaline Protease,Complex Protease,Flavor Protease,Collagenase.Enzymatic hydrolysis of konjac ACE inhibitory peptides was optimized throught single factor test and Orthogonal test.The results showed that the best hydrolytic enzymes was selected as alkaline protease.The effect of enzymatic p H was significant,the effect of enzymatic temperature,substrate concentration and enzyme dosage on ACE inhibition rate were non-significant under the given enzymatic hydrolysis conditions.The optimum enzymolysis conditions were as follows:the enzymatic pH was 10,the substrate concentration was 3%,the enzymolysis temperature was 46 ?,the enzyme dosage was 5000 U/g,the enzymolysis time was 3 h,the ACE inhibition rate of the enzymolysis solution was 82.02%.(2)Separation and purification of Konjac ACE inhibitory peptides.The membrane flux and ACE inhibition rate were determined as the indexs,ultrafiltration membrane with size of the 10,5 and 1 KDa was used for Ultrafiltration,the results showed that the separation efficency of 1KDa was the best.Its ultrafiltration conditions were optimized,and the optimum conditions for the separation were as follows: the pH value was 9,the ultrafiltration pressure was 0.07 Mpa,the ultrafitration time was 15 min,the ACE inhibition rate of the separated components was 85.30%,and the membrane flux was 38 L/m2·h.The ACE inhibition rate was determined as the index.Using Sephadex G?15 column chromatography separated crude product of konjac ACE inhibitory peptides that derived from ultrafiltration separated,the optimum conditions for Sephadex G?15 column chromatography were as follows:sample concentration was 120 mg/mL,the flow rate was 0.8 mL/min,the sampling amount was 1.0 mL,and ACE inhibition rate of the separated components was 90.20%.Using Reversed-phase High Performance Liquid Chromatography purified components that separated by Sephadex G?15 column chromatography,and the two components of peak?and peak? were obtained,The ACE inhibition rate of peak?was 24.35%,and ACE inhibition rate of peak?was 92.85%.The content of konjac ACE inhibitory peptides in sample was 77.20% by UV spectrophotom-etry.(3)The structural analysis of konjac ACE inhibitory peptides.A total of 16 amino acids were detected in the ACE inhibitory peptides from konjac using an amino acid analyzer,in which the contents of arginine,glutamic acid and aspartic acid were higher.The maximum absorption wavelength of konjac ACE inhibitory peptides was 218.0 nm,which was consistent with the UV scan of ACE inhibitor peptides standard.Konjac ACE inhibitory peptides samples was tested by IR,the results show that:infrared spectrum characteristics of samples and ACE inhibitory peptide standard were consistent,where exist amido dond,C-C,C-H,O-H,C-N,C=O and so on in their structure.The molecular weight and molecular weight distribution of konjac ACE inhibitory peptides were determined by gel permeation chromatography,The results showed that the peak molecular were 262 Da and 634 Da,and the molecular weight distribution range was 200 to 700 Da.(4)Antihypertensive activity in vitro of konjac ACE inhibitory peptides was detected.High Performance Liquid Chromatography test results show that the inhibitory activity of konjac ACE inhibitory peptides was 58.90%.The half inhibitory concentration of IC50 was 11.09 ?g/mL according to UV spectrophotometry.