Preparation Of Chickpea Peptide By Enzymolysis And Study On Its Biological Activity

Chickpea is an excellent source of plant-based protein and produce high-quality bioactive peptides.However,there are problems such as low proteolysis efficiency,insufficient research on biological activity,and active peptide sequences to be discovered.Therefore,in order to solve these problems,this paper takes chickpea protein hydrolysates（CPHs）as the research object,screened a variety of biological activities;optimized the enzymatic hydrolysis scheme;evaluated the ability of CPHs to resist in vitro simulated gastrointestinal digestion;identified,screened,synthesized and verified a number of ultra-high activity peptides;evaluated the activities of CPHs and peptides in vitro and at the cellular level,respectively,in order to prepare bioactive peptides derived from chickpea protein with high yield.The main research content and results are as follows:First,the extraction and enzymatic hydrolysis of chickpea protein and the determination and screening of the biological activity of CPHs were completed:The chickpea protein obtained by washing the protein precipitate with acid water to remove the starch and removing the fat with n-hexane had a purity of 91.12%.Four proteases were screened using the degree of hydrolysis（DH）and 9 biological activities as indicators.CPHs were found to be prominent in DPP-IV inhibitory（Neutrase,p H 7.0,45℃）and ADH activating（Alcalase,p H8.5,55℃）activities.It was verified that chickpea-derived protein hydrolysates has the ability to activate ADH.The quality analysis of CPHs showed that it was rich in amino acids,and small molecular peptides account for 68.70%.Secondly,the DPP-IV inhibitory peptides were identified,synthesized and verified based on CPHs:Through the optimization of enzymatic hydrolysis,it was determined that the CPHs obtained by Neutrase under the optimal enzymatic hydrolysis time of 60 min（CPHs-Neutrase-60）had the strongest inhibitory ability to DPP-IV,and double enzymatic hydrolysis was not required.The IC₅₀value of CPHs-Neutrase-60 for DPP-IV inhibition was0.86±0.05 mg/m L.The in vitro simulated digestion showed that the DPP-IV inhibition rate of CPHs could be maintained at 60.36%after digestion,which had a strong anti-digestion ability.Four DPP-IV inhibitory peptides were identified and screened from CPHs:IAIPPGIPYW,PPGIPYW,AAWPGHPEF and LAFP.Molecular docking verification showed that all of them could tightly combine with the active center of DPP-IV.After synthesizing and verifying the peptide sequence,it was found that IAIPPGIPYW(IC₅₀:12.43μM)was a competitive-noncompetitive mixed inhibitor,and PPGIPYW,AAWPGHPEF and LAFP(IC₅₀:25.75±2.23μM,57.99±3.70μM and 110.50±2.47μM)were competitive inhibition.At the same time,both IAIPPGIPYW and PPGIPYW showed good DPP-IV inhibitory activity in Caco-2 cells,and both were safe at the optimal concentration for inhibiting DPP-IV activity(IC₅₀:138.63±14.98μM and 270.56±13.92μM).Finally,the ADH activating peptides were screened,synthesized and verified based on CPHs:through the optimization of enzymatic hydrolysis,it was determined that the CPHs obtained by Alcalase under the optimal enzymatic hydrolysis time of 30 min（CPHs-Alcalase-30）had the strongest activation ability on ADH.The EC₅₀value of CPHs-Alcalase-30 on ADH activation was 0.0569±0.0083 mg/m L.The in vitro simulated digestion showed that the activation rate of ADH after digestion of CPHs can still be as high as 82.65%,which has outstanding resistance to digestion.A new set of methods for rapid screening of ADH activating peptides was summarized,and four strong ADH activating peptides were finally verified from CPHs through identification,screening,and synthesis:ILPHF,MFPHLPSF,LMLPHF,and FDLPALRF(EC₅₀:1.56±0.07μM,1.62±0.23μM,1.76±0.03μM,and 9.11±0.11μM).Through molecular docking screening,it was found that all of them can combine with ADH through hydrogen bonding to form a stable complex to activate ADH.