Edible fungus glycopeptide for activating salty taste and umami receptors and application of edible fungus glycopeptide in low-sodium healthy food

Abstract

The invention provides edible mushroom glycopeptide for activating salty and umami receptors and application of the edible mushroom glycopeptide in low-sodium healthy food, and belongs to the technical field of deep processing of edible mushrooms. The molecular structural formula of the edible fungus glycopeptide is shown in the formula I. The edible fungus glycopeptide has the dual flavor development characteristic of activating salty receptors TRPV1, ENaC and TMC4 and umami receptors T1R1 and T1R3, can replace or reduce the use of traditional salt (sodium chloride) and monosodium glutamate (sodium glutamate), and is suitable for development of low-sodium healthy food, low-sodium salty enhancer, composite flavor enhancer and functional food.

Description

Technical Field

[0001] The present invention belongs to the technical field of deep processing of edible fungi, and particularly relates to an edible fungus glycopeptide that activates salty and umami taste receptors and an application thereof in low-sodium healthy foods. Background Art

[0002] Taste receptors include epithelial cell Na + Channels (epithelial Na+ channels, ENaC), transient receptor potential (TRP) channel receptor member TRPV1, transmembrane channel-like protein 4 (TMC4), umami receptor G protein-coupled receptors (Tastereceptor type 1 member 1 and Taste receptor type 1 member 3, T1R1 / T1R3). ENaC is one of the main salty taste receptors. On taste bud cells, ENaC mainly senses low concentrations of sodium ions, and by stimulating the regulatory sites in the receptor domain in taste cells, it increases the frequency of sodium ion-induced reactions, thereby enhancing the brain's perception of salty taste. TRPV1 receptors nonspecifically respond to Na + , K + and NH 4+ Cations such as cations enter cells through TRP ion channels, polarizing calcium ions. This influx of calcium ions triggers neurotransmitter release and signal transduction, resulting in the perception of salty taste. The TMC4 receptor is a chloride ion channel that responds to high concentrations of sodium chloride. Certain salt-enhancing peptides can bind to the TMC4 receptor, thereby enhancing salty taste perception. T1R1 and T1R3, as umami receptors, are primarily responsible for sensing umami components in food, such as amino acids and flavor nucleotides.

[0003] Traditional salty taste relies on high sodium salt, and excessive intake leads to health risks such as high blood pressure. Umami enhancers may cause adverse reactions in sensitive people. Moreover, seasonings such as salt and MSG all achieve taste sensing by activating a single taste receptor. It is difficult for seasonings to simulate the complex taste levels of natural food, and the multi-target synergistic activation mechanism of taste receptors has not yet been effectively utilized. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an edible fungus glycopeptide that activates salty and umami receptors and its application in low-sodium health foods. The edible fungus glycopeptide can activate salty and umami receptors, replace or reduce the use of traditional salt (sodium chloride) and MSG (monosodium glutamate), and is suitable for the development of low-sodium health foods, low-sodium salty enhancers or compound umami enhancers and functional foods.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides an edible fungus glycopeptide, the molecular structure of which is shown in Formula I.

[0007]

[0008] The invention provides a preparation method of the edible fungus glycopeptide, comprising the following steps: mixing Stropharia rugosa with water, ultrasonicating, centrifuging to collect the supernatant, and freeze-drying; desalting the Stropharia rugosa freeze-dried powder using a microchromatographic column, and drying; and performing LC-MS / MS identification and PEAKS software analysis on the desalted dry powder.

[0009] Preferably, the mass volume ratio of the Stropharia rugosa to water is 1 g: 15-25 mL.

[0010] Preferably, the ultrasound is slit triple-frequency ultrasound, the frequency sequence of the ultrasound is 22-24kHz, 24-26kHz, and 27-29kHz, the total time of the ultrasound is 20-40min, and the power density of the ultrasound is 90-110W / L.

[0011] Preferably, the working time and the intermittent time of the ultrasonic three-frequency are 3-9s: 1-3s, and the alternating working time of each frequency in the slit three-frequency is 1-3s.

[0012] Preferably, the centrifugal speed is 7500-8500 rpm, and the centrifugal time is 10-20 min.

[0013] Preferably, the microchromatography column is a ZipTip C18 microchromatography column.

[0014] Preferably, the eluent used in the microchromatographic column is an aqueous solution containing 50-70 v / v% acetonitrile (ACN) and 0.05-0.15 v / v% trifluoroacetic acid (TFA).

[0015] Preferably, the mobile phase A used in the LC-MS / MS identification is a formic acid aqueous solution, and the mobile phase B is an acetonitrile solution containing formic acid.

[0016] The present invention provides the use of the edible fungus glycopeptide or the edible fungus glycopeptide obtained by the preparation method in preparing condiments.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The glycopeptide EEET(Fuc)S(Glc)LHQ provided by the present invention can activate the salty taste receptors TRPV1, ENaC, TMC4 and the umami taste receptors T1R1, T1R3, and has the dual taste characteristics of salty and umami. It is suitable for the development of low-sodium health foods, low-sodium salty taste enhancers, compound umami enhancers and functional foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the mass spectrum of the glycopeptide molecule EEET(Fuc)S(Glc)LHQ.

[0020] Figure 2 The structure of the glycopeptide molecule EEET(Fuc)S(Glc)LHQ is shown. The left image shows the 2D structure of the glycopeptide molecule, and the right image shows the 3D structure of the glycopeptide molecule. In both the 2D and 3D structures, green represents fucose and red represents glucose.

[0021] Figure 3 Flowchart for receptor construction, molecular docking and sensory evaluation.

[0022] Figure 4 Evaluation of the Ramachandran plot and transmembrane structure diagram for the TRPV1 receptor model. The left image is the Ramachandran plot, and the right image is the transmembrane structure diagram.

[0023] Figure 5 Evaluation of the Ramachandran plot and subunit transmembrane structure diagram for the ENaC receptor model. The upper left figure is the Ramachandran plot, the upper right figure is the transmembrane structure diagram of the α subunit, the lower left figure is the transmembrane structure diagram of the β subunit, and the lower right figure is the transmembrane structure diagram of the γ subunit.

[0024] Figure 6 The Ramachandran plot and transmembrane structure diagram for the TMC4 receptor model were evaluated. The left image is the Ramachandran plot, and the right image is the transmembrane structure diagram.

[0025] Figure 7 Evaluation of the Ramachandran plot and transmembrane structure diagram for the T1R1 receptor model. The left image is the Ramachandran plot, and the right image is the transmembrane structure diagram.

[0026] Figure 8 Evaluation of the Ramachandran plot and transmembrane structure diagram for the T1R3 receptor model. The left image is the Ramachandran plot, and the right image is the transmembrane structure diagram.

[0027] Figure 9 This is the molecular docking diagram of EEET(Fuc)S(Glc)LHQ and TRPV1.

[0028] Figure 10 This is the molecular docking diagram of EEET(Fuc)S(Glc)LHQ and ENaC.

[0029] Figure 11This is the molecular docking diagram of EEET(Fuc)S(Glc)LHQ and TMC4.

[0030] Figure 12 This is the molecular docking diagram of EEET(Fuc)S(Glc)LHQ and T1R1.

[0031] Figure 13 This is the molecular docking diagram of EEET(Fuc)S(Glc)LHQ and T1R3. DETAILED DESCRIPTION

[0032] The present invention provides an edible fungus glycopeptide. The molecular structure of the edible fungus glycopeptide is shown in Formula I.

[0033]

[0034] In the present invention, the glycopeptide sequence of the edible fungus glycopeptide is EEET(Fuc)S(Glc)LHQ, with a molecular weight of 1280Da. The sugar chain in the glycopeptide molecule is composed of fucose (Fuc) and glucose (Glc). Fucose is O-linked glycosylated and linked to the threonine (Thr) hydroxyl group of the peptide chain via an α-glycosidic bond, while glucose is O-linked glycosylated and linked to the serine (Ser) hydroxyl group of the peptide chain via an α-glycosidic bond. The peptide segment contains characteristic savory amino acid sequences (glutamic acid and glutamine) and sweet amino acid sequences (serine and threonine), accounting for 50% and 25% respectively.

[0035] The present invention also provides a method for preparing the edible fungus glycopeptide, comprising the following steps: mixing Stropharia capillaris with water, sonicating, centrifuging to collect the supernatant, and freeze-drying; desalting the freeze-dried Stropharia capillaris powder using a microchromatographic column and drying; and subjecting the desalted dry powder to LC-MS / MS identification and PEAKS software analysis. The Stropharia capillaris mixed with water in the present invention is Stropharia capillaris powder. The preparation of the Stropharia capillaris powder comprises the following steps: selecting dried Stropharia capillaris as a raw material, subjecting the raw material to a pretreatment step of crushing and sieving to obtain Stropharia capillaris powder with a mesh size of less than 100.

[0036] In the present invention, the mass volume ratio of the Stropharia rugosa to water is 1 g:15-25 mL, preferably 1 g:18-23 mL, and more preferably 1 g:20 mL.

[0037] In the present invention, the ultrasound is a slit triple-frequency ultrasound, and the order of the ultrasound frequencies is 22-24kHz, 24-26kHz, 27-29kHz, preferably 23kHz, 25kHz, and 28kHz; the total ultrasound time is 20-40min, preferably 25-35min, and more preferably 30min; the ultrasound power density is 90-110W / L, preferably 95-105W / L, and more preferably 100W / L. The ratio of the triple-frequency working time to the intermittent time in the ultrasound of the present invention is 3-9s:1-3s (the triple-frequency ultrasound works for 3-9s and stops for 1-3s), preferably 6s:2s; the alternating working time of each frequency in the slit triple-frequency is 1-3s, preferably 2s.

[0038] In the present invention, the centrifugal speed is 7500-8500 rpm, preferably 7700-8300 rpm, more preferably 8000 rpm; the centrifugal time is 10-20 min, preferably 12-18 min, more preferably 15 min.

[0039] In the present invention, the microchromatography column is a ZipTip C18 microchromatography column (Merck-Millipore), purchased from Shanghai Anpu Laboratory Technology Co., Ltd. The eluent used in the microchromatography column of the present invention is an aqueous solution containing 50-70 v / v% ACN and 0.05-0.15 v / v% TFA, preferably an aqueous solution containing 60% ACN and 0.1% TFA.

[0040] In the present invention, the mobile phase A used in the LC-MS / MS analysis and identification is an aqueous formic acid solution, and the mobile phase B is an acetonitrile solution containing formic acid. The aqueous formic acid solution of the present invention is a 0.05-0.15 v / v% formic acid aqueous solution, more preferably a 0.1% formic acid aqueous solution. The formic acid acetonitrile solution of the present invention is an acetonitrile solution containing 0.05-0.15% formic acid, more preferably an acetonitrile solution containing 0.1% formic acid.

[0041] The present invention also provides the use of the edible fungus glycopeptide or the edible fungus glycopeptide obtained by the preparation method in the preparation of condiments. The edible fungus glycopeptide of the present invention can be used in the development of low-sodium condiments (salt substitutes), vegetarian meat flavor enhancers, and flavor enhancers for elderly food.

[0042] In the present invention, unless otherwise specified, all components or reagents are commercially available products well known to those skilled in the art.

[0043] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0044] Example 1

[0045] 1. Dried Stropharia officinalis was used as the raw material and pre-processed by crushing and sieving to obtain Stropharia officinalis powder with a particle size less than 100 mesh. The powder was mixed with water at a ratio of 1 g powder to 20 mL. Glycopeptides were extracted using a slit triple-frequency ultrasonic method: the ultrasonic frequency combinations were 23 kHz, 25 kHz, and 28 kHz, with an ultrasonic power density of 100 W / L for a total of 30 minutes. The ratio of the three-frequency on-time to the rest-time was 6 s: 2 s (23 kHz, 25 kHz, and 28 kHz, respectively, for a total of 6 s, followed by a 2 s pause, and this cycle was repeated). The alternating on-time of each frequency was 2 s. After ultrasonic treatment, the sample was centrifuged at 8000 rpm for 15 minutes. The supernatant was collected and freeze-dried at -70°C for 48 hours to obtain Stropharia officinalis freeze-dried powder.

[0046] 2. Identification of glycopeptide molecules in freeze-dried powder of Stropharia rugosa using mass spectrometry

[0047] (1) The lyophilized powder was pretreated by desalting using a ZipTip C18 microchromatographic column (Merck-Millipore, Shanghai Anpu Laboratory Technology Co., Ltd.). The desalting method was as follows:

[0048] (1.1) Accurately weigh 1.0 mg of Stropharia rugosa freeze-dried powder and dissolve it in 10 μL of 0.1% (v / v) TFA to obtain a Stropharia rugosa freeze-dried powder solution.

[0049] (1.2) Rinse the column 10 times with 50 μL of a solution containing 60% (v / v) ACN and 0.1% (v / v) TFA in pure water; rinse the column 10 times with 10 μL of 0.1% TFA;

[0050] (1.3) The dissolved solution of the Stropharia rugosa freeze-dried powder was aspirated and discharged through the chromatography column 20 times; the chromatography column was rinsed 5 times with 10 μL of 0.1% TFA; the chromatography column was eluted with 10 μL of a solution containing 60% (v / v) ACN and 0.1% (v / v) TFA in purified water. The eluate was collected and transferred to a polypropylene centrifuge tube and vacuum dried to obtain the desalted dry powder.

[0051] (2) The desalted dry powder was dissolved in 20 μL of 0.1% (v / v) formic acid aqueous solution, vortexed, and centrifuged at 17,000 rpm and 4°C for 20 min. The supernatant was collected and transferred to a sample tube with an injection volume of 3 μL for LC-MS / MS sequence analysis. The liquid chromatography mobile phase A was 0.1% (v / v) formic acid aqueous solution, and the mobile phase B was acetonitrile containing 0.1% (v / v) formic acid. The LC-MS / MS parameters are shown in Table 1. The LC-MS / MS identification data were searched in the identification sequence database using PEAKS software.

[0052] Table 1 LC-MS / MS parameter settings

[0053]

[0054] After LC-MS / MS identification and PEAKS software processing, a glycopeptide molecule with high mass spectrum abundance was obtained in the freeze-dried powder sample of Pleurotus ostreatus. The glycopeptide sequence is EEET(Fuc)S(Glc)LHQ, with a molecular weight of 1280Da. In the glycopeptide molecule, the sugar chain is composed of fucose and glucose. Fucose is linked to the threonine (Thr) hydroxyl group of the peptide chain through an α-glycosidic bond in an O-linked glycosylation manner, and glucose is linked to the serine (Ser) hydroxyl group of the peptide chain through an α-glycosidic bond in an O-linked glycosylation manner. The peptide segment contains characteristic savory amino acid (glutamic acid, glutamine) and sweet amino acid (serine, threonine) sequences, accounting for 50% and 25% respectively. The mass spectrum of the glycopeptide molecule EEET(Fuc)S(Glc)LHQ is shown below. Figure 1 As shown in the figure, and the molecular structure of glycopeptide is as shown in the figure Figure 2 shown.

[0055] At the same time, another glycopeptide molecule was identified using the above method. The glycopeptide sequence was AVS(GalNAc)VPLQ. The sugar chain composition of the glycopeptide molecule was N-acetylgalactosamine, which was connected to the serine hydroxyl group of the peptide chain through an α-glycosidic bond in an O-linked glycosylation manner.

[0056] Example 2

[0057] The taste sensory evaluation of EEET(Fuc)S(Glc)LHQ and AVS(GalNAc)VPLQ obtained in Example 1 was performed

[0058] 1. Prepare glycopeptide solutions, NaCl solutions, and monosodium glutamate (MSG) solutions with water at concentrations of 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%, respectively. As shown in Table 2, set sensory scores corresponding to each concentration of NaCl and MSG solutions. Using the scores for each concentration of NaCl and MSG as the standard, evaluate the concentration of glycopeptide solution that is equivalent in saltiness intensity to that of NaCl solution and the concentration of glycopeptide solution that is equivalent in umami intensity to that of MSG solution.

[0059] Table 2 Sensory scores of NaCl solution and MSG solution at different concentrations

[0060] Fraction 2 4 6 8 10 NaCl solution concentration (%) 0.1 0.2 0.3 0.4 0.5 MSG solution concentration (%) 0.1 0.2 0.3 0.4 0.5

[0061] The sensory evaluation experiment was conducted in a sensory analysis room at 25°C. The samples were prepared and placed in 30mL tasting cups, randomly numbered with three digits. The assessors randomly selected samples and tasted at least 5mL. They stirred the samples with their tongues for 10 seconds and then spat them out. To avoid fatigue and carryover effects, the assessors rinsed their mouths with ultrapure water and rested for 2 minutes between tastings. Ten trained sensory assessors (5 women and 5 men, aged 22-30 years) blindly tested the glycopeptide solutions at various concentrations, comparing the saltiness and umami intensity of each concentration of NaCl and MSG, and scoring the glycopeptide solutions at each concentration. The results are shown in Tables 3 and 4.

[0062] Table 3 The scores of salty taste intensity of EEET(Fuc)S(G1c)LHQ glycopeptide solutions at different concentrations

[0063] Glycopeptide solution concentration (%) 0.1 0.2 0.3 0.4 0.5 Fraction 2.8 6.1 9.8 11-3 11-3

[0064] Table 4 Scores of umami intensity obtained for EEET(Fuc)S(Glc)LHQ glycopeptide solutions at different concentrations

[0065] Glycopeptide solution concentration (%) 0.1 0.2 0.3 0.4 0.5 Fraction 1.5 3.4 5.1 10.4 11.1

[0066] The scores in Tables 3 and 4 were compared with the scores specified in Table 2. Sensory evaluation results showed that the saltiness intensity of a 0.3% EEET(Fuc)S(Glc)LHQ glycopeptide solution was equivalent to that of a 0.5% NaCl solution, and the umami intensity of a 0.4% EEET(Fuc)S(Glc)LHQ glycopeptide solution was equivalent to that of a 0.5% MSG solution. AVS(GalNAc)VPLQ glycopeptide, on the other hand, lacked saltiness or umami.

[0067] Sensory evaluation results indicate that the glycopeptide EEET(Fuc)S(G1c)LHQ can reduce the amount of NaCl by 40% without reducing saltiness, and the amount of MSG by 20% without reducing umami. Here, the amount of NaCl reduced = (NaCl solution concentration equivalent to saltiness intensity - glycopeptide solution concentration equivalent to saltiness intensity) / NaCl solution concentration equivalent to saltiness intensity, and the amount of MSG reduced = (MSG solution concentration equivalent to umami intensity - glycopeptide solution concentration equivalent to umami intensity) / MSG solution concentration equivalent to umami intensity.

[0068] 2. Based on the above salty taste evaluation process, 0.2% EEET(Fuc)S(Glc)LHQ glycopeptide solution is the lowest concentration with salty taste intensity. The 0.2% EEET(Fuc)S(Glc)LHQ glycopeptide solution was diluted to concentrations of 0.18%, 0.16%, 0.14%, 0.12%, and 0.10%. Using the evaluation experiment described in step 1 above, the lowest solution concentration at which the assessors tasted salty was 0.12%, that is, the salty taste threshold of the EEET(Fuc)S(Glc)LHQ glycopeptide was 0.12%.

[0069] Based on the above umami evaluation process, 0.3% EEET(Fuc)S(Glc)LHQ glycopeptide solution is the lowest concentration with umami intensity. The 0.3% EEET(Fuc)S(Glc)LHQ glycopeptide solution was diluted to concentrations of 0.29%, 0.28%, 0.27%, 0.26%, 0.25%, 0.24%, 0.23%, 0.22%, and 0.21%. Using the evaluation experiment described in step 1 above, the lowest solution concentration at which the assessors tasted umami was 0.27%, that is, the umami threshold of the EEET(Fuc)S(Glc)LHQ glycopeptide was 0.27%.

[0070] Example 3 Molecular Docking Glycopeptide Taste Analysis

[0071] 1. Receptor modeling and evaluation

[0072] Alphafold (https: / / alphafold.ebi.ac.uk / ) was used to model the TRPV1 receptor (UniProt ID: Q8NER1), ENaC receptor (PDB ID: 6WTH), TMC4 receptor (UniProt Accession: A0A0G2JP95), T1R1 receptor (UniProt ID: Q7RTX1) and T1R3 receptor (UniProt ID: Q7RTX0), SAVES v6.1 (https: / / saves.mbi.ucla.edu / ) was used to verify the rationality of the receptor structure, and TMHMM-2.0 (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ) was used to analyze the transmembrane region of the receptor.

[0073] The SAVES server is a commonly used structure detection server used to verify the accuracy of protein structures obtained through calculations. Access the server through http: / / services.mbi.ucla.edu / SAVES / , upload the PDB file of the receptor structure to the server, use PROCHECK to generate a Ramachandran plot, use ERRAT to evaluate the overall structure quality, and use VERIFY_3D to check the match between the sequence and the structure. Based on the output results of the above tools, evaluate whether the constructed receptor structure model is reasonable. The receptor construction, molecular docking and sensory evaluation process is as follows: Figure 3 shown.

[0074] The SAVES Ramachandran plot results for evaluating the rationality and accuracy of the TRPV1 receptor model showed that 100% of the amino acid residues were located in the allowed region of the Ramachandran plot, of which 86.2%, 13.6% and 0.2% were located in the fully allowed region, acceptable region and generally allowed region of the Φ angle and Ψ angle, respectively. The dihedral angles of the amino acid residues in the model structure were reasonable and the predicted model was usable. The TRPV1 receptor predicted by TMHMM contains 6 transmembrane regions, with the N-terminus and C-terminus located on the inner side of the membrane ( Figure 4 , Table 5).

[0075] Table 5 TRPV1 receptor structure sequence information

[0076]

[0077]

[0078] SAVES evaluated the rationality and accuracy of the ENaC receptor model using a Ramachandran plot. The results showed that 100% of the amino acid residues were located within the permitted region of the Ramachandran plot, of which 83.1% and 16.9% were located within the fully permitted region and acceptable region of the Φ and Ψ angles, respectively. The dihedral angles of the amino acid residues in the model structure were reasonable, and the predicted model was acceptable. TMHMM predicted that the N-termini of the three ENaC receptor subunits are all located on the inner side of the membrane, with the α and β subunits each containing one transmembrane helix, and the γ subunit containing two transmembrane helices ( Figure 5 , Table 6).

[0079] Table 6 ENaC receptor transmembrane structure sequence information

[0080]

[0081] The SAVES Ramachandran plot results for evaluating the rationality and accuracy of the TMC4 receptor model showed that 100% of the amino acid residues were located in the allowed region of the Ramachandran plot, of which 93.8%, 4.4% and 1.0% were located in the fully allowed region, acceptable region and generally allowed region of the Φ angle and Ψ angle, respectively. The dihedral angles of the amino acid residues in the model structure were reasonable and the predicted model was usable. The TMHMM-predicted TMC4 receptor contains 8 transmembrane regions, with the N-terminus and C-terminus located on the inner side of the membrane ( Figure 6 , Table 7).

[0082] Table 7 TMC4 receptor transmembrane structure sequence information

[0083] area Starting amino acid number End amino acid number intracellular 1 169 transmembrane helix 170 192 Extracellular 193 247 transmembrane helix 248 270 intracellular 271 348 transmembrane helix 349 371 Extracellular 372 390 transmembrane helix 391 413 intracellular 414 425 transmembrane helix 426 448 Extracellular 449 542 transmembrane helix 543 565 intracellular 566 585 transmembrane helix 586 608 Extracellular 609 654 transmembrane helix 655 677 intracellular 678 712

[0084] SAVES evaluated the rationality and accuracy of the T1R1 receptor model using a Ramachandran plot. The results showed that 100% of the amino acid residues were located within the permitted region of the Ramachandran plot, of which 93% and 7% were located within the fully permitted region and acceptable region of the Φ and Ψ angles, respectively. The dihedral angles of the amino acid residues in the model structure were reasonable, and the predicted model was acceptable. TMHMM predicted that the T1R1 receptor N-terminus was located on the inner side of the membrane, and the C-terminus was located on the outer side of the membrane, containing a total of 7 transmembrane helices ( Figure 7 , Table 8).

[0085] Table 8 T1R1 receptor transmembrane structure sequence information

[0086]

[0087]

[0088] The SAVES Ramachandran plot results for evaluating the rationality and accuracy of the T1R3 receptor model showed that 100% of the amino acid residues were located in the allowed region of the Ramachandran plot, of which 91.8%, 7.6%, and 0.5% were located in the fully allowed region, acceptable region, and generally allowed region of the Φ angle and Ψ angle, respectively. The dihedral angles of the amino acid residues in the model structure were reasonable, and the predicted model was usable. The TMHMM predicted that the N-terminus of the T1R1 receptor is located on the outside of the membrane and the C-terminus is located on the inside of the membrane, containing a total of 7 transmembrane helices ( Figure 8 , Table 9).

[0089] Table 9 T1R3 receptor transmembrane structure sequence information

[0090]

[0091]

[0092] 2. Molecular docking

[0093] The MOE 2019 molecular docking software (Chemical Computing Group ULC, Montreal, Canada) was used to optimize the crystal structure of the receptor protein. The water molecules in the receptor protein sequence were deleted, and the hydrogen atoms were completed by clicking the "QuickPrep" of the MOE software. Click the "Protein Builder" module under the "Protein" tab of the MOE software to construct the 3D structure of the glycopeptide molecule according to the glycopeptide molecule sequence, and click the "Minimize" module to perform molecular energy minimization. The "Site Finder" module under the "Protein" tab of MOE was used to determine the active site of the amino acid residues of the receptor protein. Click the "Dock" module under the "Compute" tab of the MOE software to perform molecular docking of the receptor molecule and the glycopeptide molecule in the software. In the "Dock" panel, select the active site determined by "Site Finder" in "Site" and select the glycopeptide molecule in "Ligand". In the molecular docking result list (Ligand Interaction Report), the number of bonds and the binding energy between the glycopeptide and the receptor protein are used as screening indicators to select the tightly bound complex between the glycopeptide and the receptor protein. The MOE software is used to analyze the binding sites and interaction modes between the glycopeptide molecules and the receptor protein in the complex.

[0094] The glycopeptide molecule EEET(Fuc)S(Glc)LHQ formed 17 hydrogen bonds, 8 ionic bonds and 1 pi-H interaction force with the TRPV1 molecule, with a binding energy of -57.9kcal / mol, of which the binding energy of the peptide molecule binding to TRPV1 was -49.6kcal / mol, and the umami amino acid glutamic acid residue contributed a binding energy of -46.4kcal / mol; the binding energy of fucose binding to TRPV1 was -3.4kcal / mol, and the binding energy of glucose binding to TRPV1 was -4.9kcal / mol. The oxygen atoms in fucose and glucose formed hydrogen bond interactions with the TRPV1 molecule respectively. In summary, the glycopeptide molecule EEET(Fuc)S(Glc)LHQ achieved strong binding to the TRPV1 receptor with multiple binding bonds and low binding energy ( Figure 9 , Table 10).

[0095] Table 10 Molecular docking results of EEET(Fuc)S(Glc)LHQ and TRPV1

[0096]

[0097]

[0098] The glycopeptide molecule EEET(Fuc)S(Glc)LHQ forms 21 hydrogen bonds and 2 ionic bond interactions with the ENaC molecule (α subunit, γ subunit), with a binding energy of -62.1kcal / mol, of which the binding energy of the peptide molecule binding to ENaC is -45.4kcal / mol, the umami amino acid glutamic acid residue contributes a binding energy of -14.4kcal / mol, and the glutamine residue contributes a binding energy of -23.7kcal / mol; the binding energy of fucose binding to ENaC is -5.5kcal / mol, and the binding energy of glucose binding to ENaC is -11.2kcal / mol. Hydrogen bond interactions are formed between the oxygen atoms in fucose and glucose and the ENaC molecule. In summary, the glycopeptide molecule EEET(Fuc)S(Glc)LHQ achieves strong binding to the ENaC receptor with multiple binding bonds and low binding energy ( Figure 10 , Table 11).

[0099] Table 11 Molecular docking results of EEET(Fuc)S(Glc)LHQ and ENaC

[0100]

[0101]

[0102] The glycopeptide molecule EEET(Fuc)S(Glc)LHQ forms 15 hydrogen bonds and 13 ionic bond interactions with the TMC4 molecule, with a binding energy of -88.4kcal / mol, of which the binding energy of the peptide molecule binding to TMC4 is -76.9kcal / mol, the umami amino acid glutamic acid residue contributes a binding energy of -66.8kcal / mol, and the glutamine residue contributes a binding energy of -10.1kcal / mol; the binding energy of fucose binding to TMC4 is -5kcal / mol, and the binding energy of glucose binding to ENaC is -6.5kcal / mol. Hydrogen bond interactions are formed between the oxygen atoms in fucose and glucose and the TMC4 molecule. In summary, the glycopeptide molecule EEET(Fuc)S(Glc)LHQ achieves strong binding to the TMC4 receptor with multiple binding bonds and low binding energy ( Figure 11 , Table 12).

[0103] Table 12 Molecular docking results of EEET(Fuc)S(Glc)LHQ and TMC4

[0104]

[0105]

[0106] The glycopeptide molecule EEET(Fuc)S(Glc)LHQ formed 9 hydrogen bonds, 2 ionic bonds and 1 H-pi interaction force with the T1R1 molecule, with a binding energy of -37.8kcal / mol. The binding energy of the peptide molecule binding to T1R1 was -30kcal / mol, and the umami amino acid glutamic acid residue contributed -25.1kcal / mol of the binding energy; the binding energy of fucose binding to T1R1 was -4.6kcal / mol, and the binding energy of glucose binding to T1R1 was -3.2kcal / mol. Hydrogen bonds and H-pi interactions were formed between the oxygen atoms in fucose and glucose and the T1R1 molecule. Compared with the binding strength of glycopeptide molecules to salty taste receptors TRPV1, ENaC, and TMC4, the binding strength of glycopeptide molecule EEET(Fuc)S(Glc)LHQ to T1R1 receptor was above medium level ( Figure 12 , Table 13).

[0107] Table 13 Molecular docking results of EEET(Fuc)S(Glc)LHQ and T1R1

[0108]

[0109]

[0110] The glycopeptide molecule EEET (Fuc) S (Glc) LHQ formed 15 hydrogen bonds and 1 ionic bond interaction force with the T1R3 molecule, with a binding energy of -42.5 kcal / mol, of which the binding energy of the peptide molecule binding to T1R3 was -29.9 kcal / mol, and the umami amino acid glutamic acid residue contributed a binding energy of -20.1 kcal / mol; the binding energy of fucose binding to T1R3 was -5.5 kcal / mol, and the binding energy of glucose binding to T1R3 was -7.1 kcal / mol. Hydrogen bond interaction forces were formed between the oxygen atoms in fucose and glucose and the T1R3 molecule. Compared with the binding strength of glycopeptide molecules to salty taste receptors TRPV1, ENaC, and TMC4, the binding strength of glycopeptide molecule EEET (Fuc) S (Glc) LHQ to T1R3 receptor was above medium level ( Figure 13 , Table 14).

[0111] Table 14 Molecular docking results of EEET(Fuc)S(Glc)LHQ and T1R3

[0112]

[0113]

[0114] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An edible fungus glycopeptide, characterized in that The molecular structural formula of the edible fungus glycopeptide is shown in Formula I.

2. The method for preparing the edible fungus glycopeptide according to claim 1, wherein: The steps include: Stropharia rugosus was mixed with water, sonicated, centrifuged, and the supernatant was collected and freeze-dried; The freeze-dried powder of Stropharia rugosa was desalted and dried using a microchromatographic column; The desalted dry powder was subjected to LC-MS / MS identification and PEAKS software analysis.

3. The preparation method according to claim 2, wherein The mass volume ratio of the Stropharia rugosa to water is 1g:15-25mL.

4. The preparation method according to claim 2, wherein The ultrasound is slit triple-frequency ultrasound, the frequency sequence of the ultrasound is 22-24 kHz, 24-26 kHz, and 27-29 kHz, the total time of the ultrasound is 20-40 min, and the power density of the ultrasound is 90-110 W / L.

5. The preparation method according to claim 4, wherein The ratio of the triple-frequency working time to the intermittent time in the ultrasound is 3-9s:1-3s, and the alternating working time of each frequency in the triple-frequency slit is 1-3s.

6. The preparation method according to claim 2, wherein The centrifugal speed is 7500-8500 rpm, and the centrifugal time is 10-20 min.

7. The preparation method according to claim 2, wherein The microchromatography column is a ZipTip C18 microchromatography column.

8. The preparation method according to claim 2, wherein The eluent used in the microchromatographic column is an aqueous solution containing 50-70 v / v% ACN and 0.05-0.15 v / v% TFA.

9. The preparation method according to claim 2, wherein The mobile phase A used in the LC-MS / MS identification is a formic acid aqueous solution, and the mobile phase B is an acetonitrile solution containing formic acid.

10. Use of the edible fungus glycopeptide according to claim 1 or the edible fungus glycopeptide obtained by the preparation method according to any one of claims 2 to 9 in preparing condiments.

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