Study On Correlation Model Between Flavor Substances And Protease Activity In Fish Sauce And The Flavor Characteristics Of Umami Peptides

Engranlis japonicus is a small fish with rich resources and excellent nutritional value,and is often used as an ideal raw material for fish sauce.During the fermentation process of fish sauce,the type and quantity of taste substances will be different due to different regions and processes.This article took anchovy sauce as the research object,and analyzes the changes of flavor substances and the protease activity in anchovy sauce during fermentation,so as to explore the effect of anchovy sauce protease activity on the formation of flavor substances at different fermentation times.The umami components were separated and purified,and then the amino acid sequence of the umami peptides were identified.The taste characteristics of the identification peptide,which was synthesized by solid phase,were analyzed and verified by sensory evaluation and electronic tongue analysis.The molecular interaction between umami peptides and umami receptors T1R1 / T1R3 was used to study the umami mechanism.1.In this experiment,the properties of endogenous protease in northern(Shandong)anchovy sauce during fermentation were studied.The results showed that at least four proteases were present in the endogenous enzyme solution,and the optimum p H values were2.5,5.5,9.0,and 12.5,respectively.The maximum inhibition rate of endogenous protease was:serine proteinase inhibitor > trypsin inhibitor > aspartic proteinase inhibitor(pepsin inhibitor)>cysteine proteinase inhibitor > metalloproteinase inhibitor.At the sixth month of fermentation,fish sauce had stronger trypsin and pepsin-like activity,and at the ninth month,the cathepsin activity was stronger.2.The northern(Shandong)fish sauce under different fermentation time was selected as the raw material,and the changes of the flavor substances were analyzed.The results showed that the flavor substances in fish sauce increased at first and then decreased at different fermentation time,and there were some differences.The contents of amino acid nitrogen and polypeptide in fish sauce reached the highest at 12 months of fermentation,which were 1.39g/100 m L and 5.81 g/100 m L,respectively.The content of total free amino acids was the highest at 18 months of fermentation,while the content of nucleotides and organic acids was the highest at fermentation for 12 months.The e-tongue analysis showed that the fish sauce had the highest umami value and richness value at 12 months.Peptides and amino acids may be important compounds that affect the taste of anchovy sauce.The correlation model of protease activity change and flavor formation showed,the change of protease activity during the fermentation of anchovy sauce had an important effect on the content changes of Ala,Glu,Lys,Asp,Leu,TCA soluble peptides and succinic acid.3.The fish sauces from four different regions were separated by ultrafiltration,nanofiltration and gel chromatography,and further purifed by reversed-phase high-performance liquid chromatography(RP-HPLC).MAASGDVGK,VYETPDR,DAPYDYK,TREQMIHER,LLDAFFFDNK,EATLWDMEEK,LPLLEEAFLSR,NALKSVECYDAR,AKLTSLEEECQR,YLASCLSSVKEEK,EQLEATVQKLDESR,MEREQEESTMR,SGVVAAVNDAAKDFHG,VLSLNSGTEAVEAAIK,EGSTIGLSK,IMEALAGAGIDPRR,NQEGLFR,MTNLLEDLSFR,GFGDSCTPGKNER,YADSNIQINGTDR,AREIALQELGEQAK,IQQDDCK and QAEADMAR were successfully identified.The taste characteristics of the identification peptide,which was synthesized by solid phase,were analyzed and verified by sensory evaluation and electronic tongue analysis.The results indicated that the synthetic peptides showed different degrees of umami taste and could be defined as umami peptides after verification.The umami threshold of these peptides,except for YADSNIQINGTDR and AREIALQELGEQAK,were lower than that of MSG(0.3 mg/m L).Synthetic peptides can enhance the umami of the simulated broth,and have a certain degree of saltiness-enhancement and bitterness-reduction effects.The molecular docking results showed that umami peptides were embedded in the "Venus flytrap domain(VFTD)" of the T1R3 subunit to bind to the umami receptor T1R1/ T1R3;the interactions were mainly hydrogen bonding,hydrophobic interaction,electrostatic interaction and van der Waals;the key binding sites with T1R3 subunit included Asp196,Glu128,Glu197,Arg34,Glu25,Glu281,His125,His368,Ser126 and Ser127,among which Asp196,Glu128 and Glu197 were the most important binding amino acid residues.