Flavor Characteristics Of Umami Peptides From Meretrix Meretrix (Linnaeus) And Aloididae Aloidi And Their Interactions With Umami Receptor T1R1/T1R3

Meretrix meretrix?Linnaeus?and Aloididae aloidi are the major economic marine-cultured bivalve species in north China.They are popular edible shellfish with abundant nutrition and flavor substances.However,there are few studies on umami peptide in thses two clams.Four kinds of flavoring bases were made by boiling water extraction and enzymatic hydrolysis from Meretrix meretrix?Linnaeus?and Aloididae aloidi.To investigate the differences in flavor components of four kinds of flavoring bases,contents of flavoring substances and volatile substances in four kinds of flavoring bases were determined.Methods of separation and identification were used to separate the small molecular peptides from flavoring bases.Sensory evaluation analysis and electronic tongue sensor were employed to screen the peptide fractions which showed the umami taste.After that,the peptide fractions were further identified by MALDI-TOF/TOF MS to determine their amino acid sequences.The identified peptides were synthesized by solid-phase method and the flavor characteristics of peptides were determined.In order to reveal umami mechanism of umami peptide,three-dimensional structure of umami receptor T1R1/T1R3 was made by homology simulation,and molecular docking was used to study the interactions between umami peptides?ligands?and umami receptor T1R1/T1R3.1.Four kinds of flavoring bases,including cooking liquor of Meretrix meretrix?Linnaeus?,and cooking liquor of Aloididae aloidi,enzymatic hydrolysate of Meretrix meretrix?Linnaeus?and enzymatic hydrolysate of Aloididae aloidi,were prepared by traditional water boiling and enzymatic hydrolysis.The flavoring substances and volatile substances in four kinds of flavoring bases were analyzed.The results showed that there were some differences between flavor substances and volatile substances in the four kinds of flavoring bases,which were mainly manifested in the following aspects.The contents of sweet and umami substances?sweet amino acids,5?-nucleotides,succinic acid?in cooking liquor was higher than that in the corresponding enzymatic hydrolysate,among which alanine,succinic acid and chloride ion played an important role in taste.The contents of soluble peptides in cooking liquor was significantly lower than that in the corresponding enzymatic hydrolysate.The contents of other flavor substances(such as umami amino acids,PO₄^3-)had no significant difference.In addition,the contents of umami substances in cooking liquor of Meretrix meretrix?Linnaeus?were generally higher than that of in cooking liquor of Aloididae aloidi,which explained the reason why Meretrix meretrix?Linnaeus?was more delicious than Aloididae aloidi.The number of volatile components identified in cooking liquor of Meretrix meretrix?Linnaeus?,and cooking liquor of Aloididae aloidi,enzymatic hydrolysate of Meretrix meretrix?Linnaeus?and enzymatic hydrolysate of Aloididae aloidi were 37,37,36 and 44,respectively,including aldehydes,alcohols,ketones,hydrocarbons and esters.Among them,aldehydes and N/O/S compounds were the most abundant.The difference of flavor?aroma?was mainly reflected in N/O/S compounds.The electronic tongue results showed that the overall flavor profile of these four flavoring basees were similar,except for slight differences in saltiness and richness,and there were no significant difference in other tastes.2.Four kinds of flavoring bases were separated by ultrafiltration and gel chromatography and obtained the umami fractions WS-5,LS-3,WM-4 and LM-3,respectively.The umami components were further purified by RP-HPLC and the main peak components WS-5-I,LS-3-II,WM-4-II and LM-3-???in the umami fractions were subjected to MALDI-TOF/TOF MS to identify sequence.Seven novel umami peptides from WS-5-I were identified with following sequences:GLRPDGTPR?WS-5-I-a?,RPNPFENR?WS-5-I-b?,STMLLESER?WS-5-I-c?,ANPGPVRDLR?WS-5-I-d?,QVAIAHRDAK?WS-5-I-e?,VLPTDQNFILR?WS-5-I-f?,VTADESQQDVLK?WS-5-I-g?.No umami peptides were identified in other flavoring bases.3.Low-molecular weight peptides were synthesized by solid-phase peptide synthesis according to identification of peptide in the umami fraction WS-5-I.Their flavor characteristics were further verified and analyzed by sensory analysis combined with electronic tongue.The result showed that these synthetic seven peptides had umami taste and had been proved to be umami peptides,and their umami thresholds were 0.125 mg/mL;after adding to the model broth,they had the effects of improving the umami and salty taste,and the umami-enhancing effect is better than the salty-enhancing effect.The three-dimensional structure model of umami receptor T1R1/T1R3 was constructed by SWISS-MODEL server.Discovery Studio software optimized the model and Ramachandran plot further evaluated the optimized model.It was shown that 94.29%of residues were in allowed region and 5.71%of residues were in disallowed region.This result showed that the homology simulation result of umami receptor T1R1/T1R3 was reasonably accurate.The molecular docking between umami peptides and receptor T1R1/T1R3 was verified using the Discovery Studio software.It could be found that umami peptides obviously inserted into the so-called Venus flytrap domain?VFTD?of T1R3 cavity.The result of molecular docking also showed that the interactions between these seven umami peptides with umami receptor T1R1/T1R3 were mainly hydrogen bonding,hydrophobic interaction,electrostatic interaction and van der Waals.The total docking energy of these umami peptides interacted with umami receptor T1R1/T1R3 had no correlation with the length of umami peptide.The key amino acid residues of umami receptor T1R1/T1R3 interacted with these seven umami peptide were Asp196,Glu128,His125,His368,Glu281 And Arg34.