Screening Of Multifunctional Umami Peptides From Stropharia Rugosoannulata Based On Computer Simulation And SPR Sensing Strategy And Analysis Of Its Active Molecular Mechanism

As a new flavoring agent,umami peptide is in line with the food development concept of’natural,nutritional and safe’’.In addition to good taste,umami peptides also have certain functional properties.Therefore,the discovery and identification of novel umami peptides from food-derived proteins has attracted more and more attention.Stropharia rugosoannulata is a kind of edible fungus for both medicine and food.It has delicious taste,unique flavor and rich nutrition.In the previous experiment,we found that the free peptide in the dry basis of S.rugosoannulata could reach 148.65～167.32 mg/g,and the high quality peptide content provided a reliable raw material for the excavation of umami peptides.Based on the preliminary work of the laboratory,this study identified the mass spectrometry structure of the umami-enriched components of the water extract from S.rugosoannulata prepared by previous separation and purification,and constructed the polypeptide database of S.rugosoannulata.Based on this database,umami peptides with potential Angiotensin-converting enzyme and Dipeptidyl peptidase-Ⅳ inhibitory activities were screened by computer virtual screening technology.Chemical synthesis to identify umami peptides and systematically study their taste properties;the molecular mechanism of umami peptides and hT1R1/T1R3 umami receptors was explored by molecular docking to reveal the umami mechanism of synthetic peptides.Based on the hT1R3 receptor module,a bionic taste sensor was constructed to explore the targeted interaction mode between synthetic peptides and hT1R3,so as to realize the rapid identification of umami peptides.The inhibitory activity and mechanism of ACE and DPP-Ⅳ of synthetic peptides were further explored.The experimental results provide a new strategy for the rapid screening and identification of multifunctional umami peptides,and also provide a theoretical basis of the multi-dimensional development and utilization of S.rugosoannulata deep processing products.The specific research results are as follows:1.Establishment of polypeptide library of S.rugosoannulata and obtaining potential multifunctional umami peptides were screened by silico simulation.By UHPLC-Q-Orbitrap-MS/MS,123 peptides were identified from the water extract umami-enriched components of Stropharia rugosoannulata,and a basic database of Stropharia rugosoannulata peptides was constructed.The peptide library was composed of 19 amino acids,among which Leu,Pro,Ser,Glu and Ala appeared frequently,which provided favorable conditions for the umami and functional activity of peptides.A high-quality human umami receptor hT1R1/T1R3 protein model was successfully constructed based on homology modeling.Through online prediction tools and molecular docking screening,23 umami peptides with both ACE and DPP-Ⅳ inhibitory activities were obtained.According to the lowest molecular docking energy and similar amino acid sequence,12 most promising peptides were selected from peptides with different peptide chain lengths for solid-phase synthesis.2.The analysis of the taste properties of synthetic peptides and the mechanism of their interaction with hT1R1/T1R3 molecules of umami receptors.Twelve identified umami peptides were synthesized by solid phase synthesis.The results of sensory evaluation combined with electronic tongue showed that the umami threshold range of 12 synthetic peptides was 0.105 m M～0.728 m M,which was higher than that of traditional umami agent MSG,showing strong umami peptide.Twelve kinds of synthetic umami peptides showed different degrees of freshening effect on MSG,and the freshening rate was 7.50～35.31%.MGPENGV had the strongest freshening effect.In the 12.5%salt reduction model,compared with the same mass concentration of Na Cl（0.4%）solution,the umami and salty intensity of the mixed solution of 12 synthetic peptides and Na Cl increased,and the umami intensity increased by 1.91～3.19 times.DGFASD had the strongest umami effect,the salty intensity increased by 1.29～2.27 times,and EPQDC had the strongest salty effect.There were significant differences in the taste characteristics of synthetic peptides and amino acid mixtures with the same mass ratio,indicating that the umami intensity of umami peptides was not formed by the simple superposition of the taste of a single amino acid,but determined by the specific sequence and structure of amino acids formed by peptide bonds.The presence of umami amino acids,sweet amino acids and hydrophobic amino acids can promote the expression of umami,and when the C-terminal is umami amino acid,the umami peptide usually has a lower umami threshold.The results of molecular docking showed that hT1R3 was the main recognition region of umami peptides,Ala302,Glu45,Ser146 and His145 were the key binding sites of umami peptides and hT1R3,and hydrogen bonding and electrostatic interaction could promote the presentation of umami.3.The interaction mode between synthetic peptides and hT1R3 was explored based on surface plasmon resonance.In order to facilitate the efficient screening of umami peptides,hT1R3 biosensor chip was constructed.Through SPR targeting interaction analysis,the synthetic peptide binds to hT1R3 in a’fast-on/fast-off’mode.All 12 were confirmed to have strong affinity with hT1R3,and the KD value ranged from 2.283×10^-6M to 8.066×10^-5M.The obtained affinity showed good consistency with the umami threshold,which further confirmed the molecular docking results.hT1R3 subunit plays a major role in human recognition of umami perception.Through kinetic parameters,it was found that synthetic peptides have a more lasting advantage than MSG umami.This method provides a new strategy for the efficient identification of umami peptides.4.Determination of ACE and DPP-Ⅳ inhibitory activity of synthetic peptides in vitro and analysis of inhibitory mechanism.The results of in vitro activity assay showed that all 12 peptides had ACE inhibitory activity,and the IC₅₀value ranged from 5.73±0.01μM～2111.49±5.77μM.EAF,EPQDC,EPQCD,SNGNE and MGPENGV also showed good DPP-Ⅳ inhibitory activity,and the IC₅₀values were 1099.03±7.59μM～2763.77±9.55μM.Among them,EAF showed prominent dual activity of ACE inhibition and DPP-Ⅳ inhibition.According to the structural characteristics of peptides,it was found that peptides with higher hydrophobicity or negative charge at the end showed stronger ACE inhibitory activity.EAF,EPQDC and EPQCD with Ala or Pro structural characteristics of the second amino acid at the N-terminal showed stronger DPP-Ⅳ inhibitory activity than SNGNE and MGPENGV.The results of molecular docking showed that the inhibitory peptides mainly combined with ACE key sites Ala354,Tyr523,Lys511,His513 and Zn²⁺through hydrogen bond,metal bond and electrostatic interaction to exert their ACE inhibitory effect.The inhibitory activity of DPP-Ⅳ is mainly through hydrogen bonding,electrostatic interaction and hydrophobic interaction with DPP-Ⅳ key sites His740,Tyr547,Glu205 and Tyr662.