Study On The Preparation And Hypertensive Effect Of Angiotensin-I Converting Enzyme Inhibitory Peptides From Pumpkin Seed Protein

As a by-product of the processing of pumpkin seed oil,the protein content of pumpkin seed meal is more than 40%,but most of the pumpkin seed meal is treated as cheap feed or directly discarded,resulting in a great waste of resources.The prevalence of cardiovascular diseases in China is still on the rise.Hypertension is a high-risk factor for cardiovascular and cerebrovascular diseases.The production of natural and safe antihypertensive peptides from plant protein sources has received widespread attention.In recent years,polypeptides with angiotensin converting enzyme(ACE)inhibitory activity have been successfully isolated from a variety of meal protein sources.In this study,ACE inhibitory peptide was prepared from pumpkin seed meal by enzymatic hydrolysis.Ball-milling pretreatment and Plastein reaction were used to improve the ACE inhibitory activity of pumpkin seed peptides and the mechanism of ball milling to promote proteolysis of pumpkin seeds was analyzed.The ACE inhibitory peptide was separated and purified by ultrafiltration and sephadex chromatography,and the amino acid sequence of the most active component of ACE inhibitory peptide from pumpkin seed meal was identified by mass spectrometry.Finally,the stability of ACE inhibitory peptide was studied.The main conclusions are as follows:Ball-milling assisted enzymatic hydrolysis of pumpkin seed meal protein for preparing angiotensin-I converting enzyme(ACE)inhibitory peptides was investigated.Alcalase was found to be the best enzyme for the enzymatic preparation of antihypertensive peptides with angiotensin converting enzyme(ACE)inhibitory activity.The operating parameters ball-milling time,enzymolysis time,substrate concentration,p H and temperature were evaluated for their effects on the protein hydrolysis and inhibition rate of ACE.Based on the results of single factor experiment,the hydrolysis conditions were optimized by the response surface methodology.The results showed that the pretreatment of ball milling can significantly improve the enzymatic hydrolysis efficiency of pumpkin seed protein,the optimum conditions were determined as follows:the ball-milling time was 6 min,enzymolysis time was 10 h,substrate concentration was 0.08g/m L,p H 8.5 and the temperature was 55?.The ACE inhibition rate of enzymatic hydrolysates could reach at 86.65±0.55%under these conditions.The structure of pumpkin seed protein treated with different ball-milling time was determined to explore the mechanism of ball-milling affecting the ACE inhibitory activity of enzymatic hydrolysates.The ball-milling treatment caused the peak intensity of the characteristic peaks in the infrared spectrum to be significantly weakened,the position of the characteristic peaks also shifted,breaking the hydrogen bonds between or within the protein peptide chain and changing the secondary structure of the pumpkin seed protein.The results of fluorescence spectroscopy and ultraviolet spectroscopy showed that more aromatic amino acid residues of pumpkin seed protein after ball-milling are gradually exposed to the external hydrophilic environment,the tertiary structure of pumpkin seed protein becomes more dispersed.DSC results showed that the denaturation temperature of pumpkin seed protein decreased and the particle size distribution and scanning electron micrograph showed that the particle size of pumpkin seed protein decreased after ball milling.The ball-milling process affected the structure of pumpkin seed protein,so that the alkaline protease was easily combined with the enzyme cleavage site,thereby promoting enzymatic hydrolysis and improving the ACE inhibitory activity of the pumpkin seed protein hydrolysate.The ACE inhibitory peptide of pumpkin seed protein was modified by Plastein reaction with alkaline protease.Taking the decrease of free amino acids and the ACE inhibition rate as indicators,the optimal conditions obtained were:substrate mass fraction45%,reaction temperature 20?,and reaction time 3h.The above optimized conditions were used to carry out the plastein reaction with the addition of exogenous amino acids.The optimal addition amount of leucine,phenylalanine and glycine was 0.5 mmol/g,0.7mmol/g and 0.4 mmol/g,respectively.Plastein reaction can effectively improve the ACE inhibitory activity of the modified product.The Plastein reaction modified product with addition of leucine has the highest ACE inhibition rate,which is 24.50%higher than that of ACE inhibitory peptide without Plastein reaction modification,and 14.57%higher than the modified product without adding amino acids under the same preparation conditions.ACE inhibitory peptides from pumpkin seed protein were separated and purified by ultrafiltration and Sephadex G-25 and the amino acid sequences of the most active components were identified by LC-MS/MS analysis.A total of 76 kinds of peptides were obtained,and four kinds of peptides were synthesized from them by solid phase,their amino acid sequences were IFH,IFF,LAAF and DFHPR,and their IC₅₀ values were 1.55mmol/L,2.24 mmol/L,3.79 mmol/L,and 7.86 mmol/L respectively,which were the first discovered ACE inhibitory peptides from pumpkin seed protein.The stability of ACE inhibitory peptides from pumpkin seed protein was studied.When the temperature is 20?-80?,it had good thermal stability.When the p H is 2-8,it had good acid-base stability;K⁺and Mg²⁺had little effect on the ACE inhibitory activity of pumpkin seed peptides,while Cu²⁺,Fe³⁺and Ca²⁺could significantly reduce the ACE inhibitory activity,and Fe³⁺had the greatest effect on the ACE inhibitory rate.When the mass fraction of food ingredients was 8%,sucrose and citric acid had no significant effect on the stability of ACE inhibitory peptide of pumpkin seed meal,while sodium chloride and glucose significantly reduced ACE inhibitory rate.In the process of simulating gastrointestinal digestion,the ACE inhibitory peptide from pumpkin seed protein still maintained high ACE inhibitory activity after digestion with pepsin and trypsin.