A salty peptide from enoki mushroom, its preparation method and application

Abstract

This invention belongs to the field of biotechnology, specifically relating to a salty peptide from *Flammulina velutipes*, its preparation method, and its application. Water-soluble substances were extracted from different varieties of *Flammulina velutipes* using a high-pressure cooking method. The salty-umami peptides in *Flammulina velutipes* were then separated stepwise using ultrafiltration, gel chromatography, sensory evaluation, and electronic tongue analysis. Seventy-two peptides were identified using reversed-phase liquid chromatography-mass spectrometry. Two key peptides were selected for solid-phase synthesis through molecular docking with transmembrane channel-like protein 4. Electronic tongue and sensory evaluation results showed that the flavor thresholds of both synthesized peptides were higher than those of NaCl, exhibiting excellent saltiness-enhancing effects. Molecular dynamics simulation analysis of the flavor mechanism between the salty peptides and the TMC4 receptor revealed that the interaction between the peptides and the TMC4 receptor is mainly driven by hydrophobic interactions.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a salty peptide from enoki mushrooms, its preparation method, and its application. Background Technology

[0002] Saltiness is one of the five basic tastes besides sour, sweet, bitter, umami, and salty. Salt makes a significant contribution to the flavor and quality of food and regulates the osmotic balance between cells and blood, as well as normal water and electrolyte metabolism. However, excessive salt intake is closely related to high blood pressure and can increase the risk of other cardiovascular diseases. According to the World Health Organization (WHO), most people worldwide consume an average of 9-11 grams of salt per day, significantly exceeding the recommended daily intake of 5 grams. Therefore, many alternative solutions have emerged to reduce the NaCl content in food without altering the original saltiness. For example, other metal salts such as magnesium chloride (MgCl2) and calcium chloride (CaCl2) can be used as substitutes. However, increasing the amount of metal salts added can lead to side effects such as bitterness, metallic taste, and texture degradation. In addition, flavor enhancers can be used to replace NaCl, such as monosodium glutamate (MSG), nucleotide phosphates (IMP / GMP), herbal and spice blends, certain basic amino acids, salty peptides, and salty-enhancing peptides. Salty peptides are small peptide molecules with inherent saltiness, while saltiness-enhancing peptides possess saltiness-enhancing activity. These peptides do not contain sodium. + It has a salty taste comparable to or even stronger than sodium chloride, and is more easily absorbed by the human body, thus attracting widespread attention.

[0003] Humans and other mammals perceive taste through taste receptor cells on the tongue and soft palate. When taste compounds interact with these receptor cells, taste signals are generated and transmitted to the central nervous system via the glossopharyngeal and medullary nerves. The transduction of saltiness involves three mechanisms: epithelial sodium channels (ENaC), TRPV1 (transient receptor potential vanillin 1), and transmembrane channel-like 4 (TMC4). TMC4 is a voltage-dependent chloride ion channel expressed in the posterior part of taste buds and plays a crucial role in saltiness perception and signal transduction. Shen et al. [Identification of novel saltiness-enhancing peptides from yeast extract and their mechanism of action for transmembrane channel-like 4 (TMC4) protein through experimental and integrated computational modeling] constructed a three-dimensional model of the saltiness receptor TMC4 protein. Through molecular docking, they discovered that saltiness peptides from yeast extract have a strong saltiness-enhancing effect, and molecular simulations revealed that specific active sites play a key role in the interaction between the peptide and the TMC4 receptor. Wang et al. [Novel salty peptides derived from bovine bone: Identification, taste characteristic, and salt-enhancing mechanism] obtained five salty peptides from bovine bone and found that all synthesized peptides had both salty and umami effects. Molecular docking results showed that Arg plays a crucial role in the binding of TMC4 to the salty peptides. Therefore, TMC4, as a specific salt receptor, provides a new approach for exploring flavor-enhancing effects.

[0004] Edible fungi are recognized worldwide as healthy foods, possessing unique flavors, rich nutrients, and extremely high nutritional and medicinal value. In recent years, the extraction of flavor peptides and bioactive peptides from edible fungi has become a hot research topic. Li [Molecular characteristics and thermal stability of salty / saltiness-enhancing peptides from enzymatic hydrolysates of Agaricus bisporus] identified peptides with salty and saltiness-enhancing properties from Agaricus bisporus, which exhibited high saltiness-enhancing effects at low NaCl concentrations and good thermal stability. Salty peptides derived from Coprinus comatus were isolated and identified, among which GDNVGF functions as both a salty peptide and a saltiness-enhancing peptide. Its interaction with the TMC4 receptor revealed that Asn588, Ser165, Asp5, and Arg168 are key amino acid residues. By analyzing the binding characteristics and interaction mechanism between mushroom savory peptides and the TMC4 receptor, it was found that the TMC4 receptor spontaneously binds to peptide molecules via hydrogen bonds. The amino acid residues in the intracellular pocket 1 of the receptor primarily recognize KSWDFTR, while the amino acid residues in the extracellular pocket 4 primarily bind to RIEDNLVIIR. *Flammulina velutipes* is a widely accepted edible and medicinal fungus, belonging to the phylum Basidiomycota, class Agaricales, order Agaricales, family Pleuronectiaceae, and genus *Flammulina*. It is recognized as one of the world's four major cultivated edible fungi. *Flammulina velutipes* has high nutritional value, with a protein content of approximately 15.32-23.81 g / 100g and a total essential amino acid content of 50-87 mg / g, higher than the FAO / WHO model and the egg model. However, the isolation and extraction of savory peptides from *Flammulina velutipes* and their interaction mechanism with TMC4 have not yet been investigated. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0006] As one aspect of the present invention, the present invention provides a salty peptide from enoki mushrooms, characterized in that the amino acid sequence of the salty peptide is ECYECG or PPPPPP.

[0007] This invention also provides a method for preparing the aforementioned savory peptides from *Flammulina velutipes*, comprising,

[0008] The salty peptide was obtained by extraction, separation and purification from the fruiting body of enoki mushroom (Flammulina filiformis); or by chemical synthesis.

[0009] As a preferred embodiment of the method for preparing the salty peptides from *Flammulina velutipes* according to the present invention, the steps of extraction, separation, and purification from the fruiting body of *Flammulina velutipes* include:

[0010] (1) Extraction: The fruiting bodies of enoki mushrooms were steamed, solid-liquid separation was performed, and the supernatant was collected to obtain enoki mushroom extract;

[0011] (2) Ultrafiltration: The extract obtained in step (1) is subjected to ultrafiltration, and ultrafiltration fractions with a molecular weight of less than 1 kDa are collected;

[0012] (3) Gel chromatography: The ultrafiltration components obtained in step (2) are separated by gel chromatography, and the components with salty activity are collected.

[0013] As a preferred embodiment of the preparation method of the salty peptide from enoki mushrooms according to the present invention, the cooking includes boiling at a pressure of 70-90 Pa for 1-2 hours.

[0014] As a preferred embodiment of the preparation method of the salty peptides of enoki mushrooms described in this invention, the steaming or boiling process involves steaming the fruiting bodies of enoki mushrooms in water at a mass ratio of 1:1.5 to water, and boiling at a pressure of 70 Pa for 1.5 hours.

[0015] In a preferred embodiment of the preparation method of the salty peptide from enoki mushrooms according to the present invention, the gel chromatography medium is Sephadex G-15 and the eluent is water.

[0016] As a preferred embodiment of the preparation method of the salty peptide of enoki mushroom described in this invention, the gel chromatography includes dissolving the component obtained in step (2) in water at a concentration of 20-40 mg / mL, separating it using a gel chromatography column packed with Sephadex G-15, and eluting at a rate of 0.5-0.75 mL / min.

[0017] The present invention also provides the application of the aforementioned enoki mushroom salty peptide in food or condiments.

[0018] The enoki mushroom salty peptide is used to enhance the saltiness of food, reduce the sodium content in food, or prepare low-sodium foods.

[0019] The food products mentioned include condiments, meat products, convenience foods, puffed foods, sauces, soup bases, beverages, health foods, or special medical foods.

[0020] The beneficial effects of this invention are as follows: This invention uses a high-pressure cooking method to extract water-soluble substances from different varieties of *Flammulina velutipes*. Then, the salty-umami peptides in *Flammulina velutipes* are separated stepwise by ultrafiltration, gel chromatography, sensory evaluation, and electronic tongue analysis. Reversed-phase liquid chromatography-mass spectrometry (RPLC-MS / MS) identifies 72 peptides. Through molecular docking with transmembrane channel-like protein 4 (TMC4), nine peptides with potential salt-reducing functions are screened. Two key peptides, ECYECG and PPPPPP, are selected for solid-phase synthesis. Electronic tongue and sensory evaluation experiments show that the taste thresholds of both synthesized peptides are higher than that of NaCl, exhibiting excellent salt-enhancing effects. Molecular dynamics simulation analysis of the taste mechanism between the salty peptides and the TMC4 receptor in *Flammulina velutipes* reveals that the interaction between the peptides and the TMC4 receptor is mainly driven by hydrophobic interactions. Compared to ECYECG, the complex formed by PPPPPP and the TMC4 receptor has higher structural stability, with Val366, Gly365, and Tyr364 being the key amino acid residues. This invention provides a scientific basis for the basic research on flavor substances in enoki mushrooms and the development of industrialized production processes for flavor peptides. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein:

[0022] Figure 1 This is an analytical diagram of water-soluble substances in enoki mushrooms, in which... Figure 1 A is the taste radar map detected by the electronic tongue. Figure 1 B is a taste radar chart obtained through sensory taste detection. Figure 1 C represents peptide content. Figure 1 D represents the molecular weight distribution of the peptide. "W", "Y", and "YL" refer to white enoki mushroom, yellow enoki mushroom, and large-capped yellow enoki mushroom, respectively.

[0023] Figure 2 The images show the ultrafiltration and gel chromatography component analysis of *Flammulina velutipes*, where... Figure 2 A is a taste radar map of ultrafiltration components detected by the electronic tongue. Figure 2 B is a sensory taste radar diagram of the ultrafiltration components. Figure 3 C represents the gel chromatography separation spectrum of component U1. Figure 2 D is the electronic radar image of the gel component on the tongue. Figure 2 E is a sensory taste radar diagram of the gel components.

[0024] Figure 3 The image shows the molecular docking results between the peptide and TMC4. Figure 3 A is a schematic diagram of the 3D structure of the TMC4 receptor. Figure 3 B is the Ramachandran diagram of the TMC4 receptor after modeling. Figure 3 C is a 3D structural diagram of the interaction between the peptide and TMC4.

[0025] Figure 4 This is a sensory analysis diagram of peptides, where, Figure 4 A is a taste radar diagram detected by an electronic tongue for two synthetic peptides. Figure 4 B is a radar chart showing the sensory taste of the two synthetic peptides. Figure 4 C is the dose-response relationship diagram between sodium chloride solution and the two peptides.

[0026] Figure 5 Figures showing the molecular dynamics simulation results for the TMC4 / ECYECG and TMC4 / PPPPPP systems are provided. Figure 5 A and Figure 5 B is a graph showing the change of RMSD values ​​over the simulation period. Figure 5 C is a graph showing the changes in RMSF values. Figure 5 D is a graph showing the change of Rg value over simulation time. Figure 5 E is a graph showing the change in the number of hydrogen bonds over the simulation period. Figure 5 F is a graph showing the change in the protein solvent-accessible region over simulation time.

[0027] Figure 6 This is a molecular dynamics simulation diagram of ECYECG, PPPPPP, and TMC4. Figure 6 A is the conformational diagram of the polypeptide ECYECG before molecular dynamics simulation. Figure 6 B is the conformation diagram of the polypeptide ECYECG after molecular dynamics simulation. Figure 6 C represents the conformational diagram of the polypeptide PPPPPP before molecular dynamics simulation. Figure 6 D is the conformation diagram of the polypeptide PPPPPP after molecular dynamics simulation. Figure 6 E is a schematic diagram of the combination mode between ECYECG and TMC4. Figure 6 F is a schematic diagram of the combination mode between PPPPPP and TMC4. Detailed Implementation

[0028] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0029] Experimental materials:

[0030] White enoki mushroom (W), yellow enoki mushroom (Y), and large-capped yellow enoki mushroom (YL). The synthetic peptides were synthesized by Beijing Research Dog Technology Co., Ltd. using SPPS technology, with a purity exceeding 95%.

[0031] Example 1:

[0032] Ultrafiltration and gel chromatography of enoki mushroom hydrolysate:

[0033] The extraction of water-soluble substances from enoki mushrooms was performed using a high-pressure cooking method, an improvement upon the method described by Zhang et al. [Novel umamipeptides from two Termitomyces mushrooms and molecular docking to the tastereceptor T1R1 / T1R3]. Fresh enoki mushroom fruiting bodies were washed, chopped, and added to ultrapure water at a ratio of 1:1.5 (m / m). They were boiled at 70 Pa for 1.5 h. After cooling to room temperature, they were centrifuged at 5000 rpm for 30 min at 4 °C, and the supernatant was collected. An appropriate amount of deionized water was added again, and the precipitate was soaked and dissolved for 2 h. After centrifugation twice, the supernatant was collected. The combined supernatants were then subjected to rotary evaporation and cooling.

[0034] Following the method described in the literature "Research progress of flavor peptide components in foods," water-soluble extracts of *Flammulina velutipes* were separated using an MSM2013 ultrafiltration system equipped with 1 and 3 kDa ultrafiltration membranes. Three ultrafiltration fractions (UFs) with different molecular weights (MW) were collected, freeze-dried, and named UI, U2, and U3 (MW <1, 1-3, and >3 kDa, respectively). Sensory evaluation and electronic tongue analysis were subsequently performed.

[0035] Gel chromatography was performed according to Yang's method [Isolation and analysis of flavor-presenting substances and umami peptides from soybean and chicken peptides by consequential chromatography and UPLC-MS / MS]. The fractions obtained from ultrafiltration were dissolved in ultrapure water at a concentration of 20 mg / mL, and then separated using a gel filtration column (ID 2.6 × 60 cm) packed with Sephadex G-15. 3 mL of the solution was added to the gel filtration column, and ultrapure water was used as the mobile medium, eluting at a rate of 0.75 mL / min. The detection sensitivity was adjusted to 1.0, and the detection wavelength was adjusted to 220 nm. During collection, one tube of eluent was collected every 5 min. Each gel-chromatographically separated fraction was precisely collected based on the peak time of the chromatogram. The collected components were then concentrated and freeze-dried, and their flavor characteristics were studied through sensory evaluation and electronic tongue analysis.

[0036] Example 2:

[0037] Determination of characteristic indicators of enoki mushroom hydrolysate

[0038] Determination of free amino acids: Free amino acids were analyzed using UHPLC-MS / MS. Sample pretreatment involved mixing an equal volume of 10% trichloroacetic acid with the sample at 4°C, homogenizing in an ice-water bath for 30 min to precipitate large protein molecules. The mixture was then filtered through a 0.22 μm microporous membrane, centrifuged at 12000 r / min for 10 min, and 50 μL of the suspension was collected and transferred to a 1.5 mL EP container. 200 μL of acetonitrile:methanol (1:1 mixture containing isotopically labeled internal standards) was added, and the mixture was stirred for 30 s. Finally, the sample was ultrasonically treated in an ice-water bath for 15 min. After cooling to -40°C, the sample was subjected to ion treatment at 12000 rpm for 15 min. The target compounds were separated using an Agilent 1290 Infinity II ultra-high performance liquid chromatograph with a Waters ACQUITY UPLC BEH Amide column (100 × 2.1 mm, 1.7 μm, Waters). A and B were separated using 1% formic acid and acetonitrile as mobile phases, respectively. Mass spectrometry analysis was performed using an Agilent 6460 triple quadrupole mass spectrometer equipped with an AJS-ESI ion source and MRM (multiple reaction monitoring) technology.

[0039] Peptide content analysis: The absorbance of the peptide extract at 562 nm was measured using a peptide content kit. Based on the measured absorbance, the absorbance was calculated using the following formula:

[0040]

[0041] Peptide molecular weight distribution determination: The molecular weight distribution of peptides in different *Flammulina velutipes* hydrolysates was determined using a Waters 2695 high-performance liquid chromatograph equipped with a TSKgel 2000SWxl column (300 mm × 7.8 mm) and a UV detector at 220 nm. The mobile phase was acetonitrile / water / trifluoroacetic acid (40 / 60 / 0.1, V / V), the flow rate was 0.5 mL / min, and the column temperature was 30 °C.

[0042] Peptide identification using reversed-phase liquid chromatography-mass spectrometry (RPLC-MS / MS): A Thermo EASY nLC 1000 liquid chromatograph coupled with a Thermo Scientific QE mass spectrometer was used to identify and analyze peptides in the gel chromatography fractions. 200 μg of lyophilized powder was thoroughly dissolved in 0.1% trifluoroacetic acid (TFA) solution and purified by desalting using a ZipTip C18 column. The peptide was dissolved in 0.1% formic acid, and 4 μL of the sample was loaded onto a PepMap RSLC C18 column (75 μm x 150 mm). Separation was performed using gradient elution over 70 min, starting with 5% solvent B (0.1% formic acid ACN), gradually increasing to 95% over 60 min, and maintaining this level for 5 min. The column flow rate was maintained at 250 nL / min, and the column temperature at 40°C.

[0043] Example 3:

[0044] Homology Modeling and Molecular Docking of the TMC4 Salty Taste Receptor: The obtained peptide sequences were used to construct 3D taste structures using ChemDraw3D, which were then docked with the 3D structure of the TMC4 receptor established through homology modeling. The TMC4 receptor sequence (ID: Q7Z404) was retrieved from the UniProt database. Homology modeling was performed using the open-source software AlphaFold. PyMOL 3.10.0 was used. The homology model and ligand peptides were imported into Auto Dock Tools 1.5.6 for hydrogenation, charge calculation, and charge distribution, and then used for molecular docking.

[0045] The structures of peptide ligands and protein receptors were preprocessed using Autodock Tools software. Then, a grid file was generated using the AutoGrid program. Finally, AutoDock software was used to achieve efficient peptide-protein molecule linkage, resulting in more accurate results. Ligand binding sites were defined with reference to the spatial positions of Trp residues. The number of grid points in each of the three directions was adjusted to 80 to form an 80×80×80 three-dimensional space, and the distance between grid points was adjusted to 0.375 Å. Furthermore, the number of docking runs was adjusted to 100, while other unmentioned parameters remained at their default values. The conformation most likely to bind the peptide to the protein has the lowest degree of freedom.

[0046] Example 4:

[0047] Electronic tongue analysis: The taste characteristics of the samples were analyzed using an SA402B electronic tongue. The concentrations of UF1, UF2, UF3, and peptides were adjusted to 1.5 mg / mL. 30 mM potassium chloride and 0.3 mM tartaric acid were used as reference solutions. The five-taste sensor (C00, AE1, CA0, CTO, AAE) was used to measure each sample four times. The data from the first cycle was discarded, and the average of the last three measurements was taken as the final result. The GL1 sweetness sensor was used to measure each sample five times. The data from the first and last measurements were discarded, and the average of the three measurements was taken to obtain an accurate conclusion. The response value was obtained by recording the changes in lipid membrane potential detected by the sensor.

[0048] Sensory evaluation: Quantitative descriptive analysis (QDA) was used to evaluate the taste characteristics of different samples (1 mg / mL). The panel consisted of 10 experienced evaluators, 5 men and 5 women, aged 20-28 years. All evaluators underwent systematic training and rigorous assessment before the sensory evaluation. They were able to quantify and evaluate the intensity of various taste qualities such as sourness, sweetness, bitterness, saltiness, and umami, and signed informed consent forms before participating. This invention uses standardized reference solutions for systematic training. Sourness was represented by citric acid solution, sweetness by sucrose solution, bitterness by quinine sulfate aqueous solution, and saltiness and umami by sodium chloride solution and monosodium glutamate solution, respectively. A 9-point intensity rating method (representing no taste, 9 indicating the strongest taste) was used for sensory evaluation. During the evaluation, evaluators placed 5 mL of the sample in their mouths and scored it within 5 seconds. Rinsing the mouth thoroughly with distilled water was required before and after the evaluation, and a rest interval was provided after each evaluation to prevent sensory fatigue. Ultimately, 10 evaluators each conducted 3 experiments, and the average of their results was used as the taste intensity value of the sample to ensure the reliability and repeatability of the data.

[0049] Taste threshold determination and dose-sensory feedback test of synthetic peptides:

[0050] The taste threshold of synthetic peptides in ultrapure water was determined using taste dilution analysis (TDA). First, two synthetic peptide powders were mixed thoroughly and prepared as a 0.4% stock solution at a 1:1 ratio. The stock solution was then diluted at a specific ratio to form a complete mixture with the ultrapure water standard. This diluted solution was simultaneously submitted to sensory evaluators so they could accurately distinguish between the diluted sample and the standard, and this evaluation process was repeated. The concentration of the diluted sample solution was taken as the taste threshold of the synthetic peptide. Finally, the results measured by all evaluators were summarized and used as the taste threshold of the synthetic peptide.

[0051] Two synthetic peptide powders were separately dissolved in ultrapure water to prepare 0.4% stock solutions, which were then gradually diluted at a 1:1 ratio. Each time, the sample solution and ultrapure water were submitted simultaneously to sensory evaluators, repeating this evaluation process until they could accurately distinguish between the diluted sample and the control. The concentration of the diluted sample solution was used as the taste threshold of the synthetic peptide. Finally, the results measured by all evaluators were summarized and used as the taste threshold for that synthetic peptide.

[0052] Two synthetic peptides were added to a 1.5 g / L sodium chloride salty solution at concentration gradients of 0%, 0.05%, 0.1%, 0.2%, 0.5%, and 1.0%. A 9-point scale sensory evaluation method was used to assess the salty, umami, and sweet taste characteristics of the prepared solutions, where 0 points indicated no perceptible taste and 9 points represented the strongest taste intensity. This method was used to explore the effect of different concentrations of synthetic peptides on the flavor of the solution.

[0053] Molecular simulation: Small molecule pretreatment was performed using AmberTools 22 to add a GAFF force field to the small molecules. Simultaneously, Gaussian 16W was used to perform hydrogenation and calculate the RESP potential; the potential data were added to the molecular dynamics system topology file. Simulation conditions were conducted at a static temperature of 300 K and atmospheric pressure (1 Bar). The force field used was Amber99sb-ildn, and the solvent was water molecules (Tip3p water model). An appropriate amount of Na was added to further refine the simulation. + Ions were used to neutralize the total charge of the simulated system. The molecular dynamics simulation system first employed the steepest descent method for energy minimization, followed by 100,000 steps of isothermal-isochoric (NVT) and isothermal-isobaric (NPT) ensemble equilibrium, with a coupling constant of 0.1 ps and a duration of 100 ps. Finally, a free molecular dynamics simulation was run, consisting of 5,000,000 steps at a step size of 2 fs, for a total duration of 100 ns. After the simulation, the software's built-in tools were used to analyze the trajectories and calculate the root mean square variance (RMSD), root mean square fluctuation (RMSF), protein cyclotron radius (Rg), and binding free energy (MM / GBSA) for each amino acid trajectory.

[0054] Data analysis: Statistical analysis of the experimental results was performed using SPSS 27. Data from three replicate experiments are expressed as mean ± standard deviation. Duncan's test was used to assess the significance of multiple comparisons.

[0055] Experimental results:

[0056] Electronic tongue analysis and sensory characterization of enoki mushroom extract:

[0057] Extracts of *Flammulina velutipes*, *Flammulina velutipes*, and *Flammulina floribunda* were obtained by high-pressure cooking. The water-soluble substances were evaluated using electronic tongue and sensory methods. Figure 1 (A and B) The results showed that all three samples exhibited high levels of umami, saltiness, and sweetness. The yellow and white enoki mushrooms showed some differences in perceived saltiness and abundance, with saltiness and umami being the most noticeable. The large-capped yellow enoki mushroom, however, had a more prominent sweetness. Sensory experiments revealed that, overall, the yellow enoki mushroom had slightly higher levels of saltiness and umami than the other varieties. The taste description table showed that the yellow enoki mushroom possessed umami, saltiness, and a slightly acidic flavor. All three extracts still retained a distinct mushroom flavor. Interviews with the sensory panel members revealed that they preferred the yellow enoki mushroom extract, likely due to its lower acidity, higher umami and saltiness, and a certain degree of sweetness.

[0058] Analysis of free amino acid and peptide content and peptide molecular weight in enoki mushroom extract:

[0059] Free amino acids are one of the important components affecting the flavor of mushrooms. Sweetness is related to the content of alanine, glycine, proline, serine, and threonine. Table 1 shows that the content of alanine in white enoki mushrooms is much higher than that of other sweet amino acids, and also much higher than that in yellow and large-capped yellow enoki mushrooms. Lysine is the sweet amino acid with the lowest content in white enoki mushrooms, while proline is the sweet amino acid with the lowest content in yellow and large-capped yellow enoki mushrooms.

[0060] Table 1. Free amino acids in crude peptide solution of *Flammulina velutipes*

[0061]

[0062] In white enoki mushrooms, phenylalanine is the most abundant, while methionine is relatively low. The content of phenylalanine, isoleucine, leucine, and methionine is related to bitterness. In yellow enoki mushrooms, valine is the most abundant bitter amino acid, and methionine is the least abundant. In the large-capped yellow enoki mushroom, valine is the most abundant bitter amino acid, and tyrosine is the least abundant. Glutamic acid and aspartic acid are related to umami flavor. Among the three umami amino acids, glutamic acid is the most abundant in enoki mushrooms, with the white variety having significantly higher levels than the other two. L-glutamine is the least abundant in both white and yellow varieties. Aspartic acid is the least abundant in the large-capped yellow enoki mushroom.

[0063] The composition and content of free amino acids also affect taste. The table shows the highest proportion of sweet amino acids, while the proportion of umami amino acids is relatively low, and the proportion of bitter amino acids is even lower. This may be because bitter amino acids participate in peptide synthesis to form specific peptides, which may be precursor peptides of flavor peptides.

[0064] Figure 1 C indicates that peptides with a molecular weight less than 3000 Da accounted for 99.69%. Gao et al. obtained approximately 71.56%-71.7% of peptides less than 3000 Da through enzymatic hydrolysis, significantly lower than the peptides obtained after autoclaving, and 76.67% of the peptides had a molecular weight less than 180 Da. This demonstrates that autoclaving is a relatively simple and efficient method for extracting flavor peptides from *Flammulina velutipes*, effectively breaking down long peptides or proteins into smaller peptide fragments. Furthermore, the peptide content of three commercial *Flammulina velutipes* species was determined using a peptide content kit. Figure 1 D clearly shows that the crude peptide liquid from *Enoki mushroom with yellow cap* has the highest peptide content, followed by *Enoki mushroom with yellow cap*. However, since *Enoki mushroom with yellow cap* has a weaker umami and salty flavor, *Enoki mushroom with yellow cap*, which has a stronger umami and salty flavor, was selected for subsequent separation and purification.

[0065] Isolation and purification of peptides from *Flammulina velutipes* – ultrafiltration and gel chromatography fraction analysis:

[0066] Ultrafiltration of *Enoki mushrooms* yielded three components: U1, U2, and U3 (MW <1, 1-3, and >3 kDa, respectively). Electronic tongue analysis and sensory analysis of the three components (U1, U2, and U3) revealed that with decreasing molecular weight, umami intensity gradually increased, while sweetness intensity gradually decreased, and the flavor scores were relatively high. Figure 2 A). In sensory evaluation ( Figure 2 In B), it was found that the saltiness intensity increased with decreasing molecular weight, consistent with the finding by Zhao et al. that peptides smaller than 1 kDa extracted from soy protein isolate exhibited strong umami and saltiness. This may be due to the higher charge density, greater water solubility, and easier dissociation of smaller molecular weight peptides. Simultaneously, the higher diffusion rate makes it easier for their charged groups to exchange with ions in solution, thus increasing salinity. The electronic tongue results showed a correlation with sensory evaluation scores. Component U3 showed a significant sweetness, while component U1 showed a significant salty-umami taste. Since this invention investigates salty peptides from *Flammulina velutipes*, component U1 was selected for further separation and purification. Figure 2 C shows the purification results of fraction U1 separated by Sephadex G-15 gel chromatography. Eight fractions were obtained: F1, F2, F3, F4, F5, F6, F7, and F8. The results were displayed by electronic tongue and sensory evaluation. Figure 2 Components F3 and F4 exhibited a stronger umami and richer flavor. Therefore, component F3 was selected for further research.

[0067] RPLC-MS / MS Identification of Peptide Sequences: RPLC-MS / MS (Reversed-Phase Liquid Chromatography-Mass Spectrometry) was used to separate and identify the F3 fraction, which exhibited the strongest thickness in gel chromatography. Subsequent de novo sequencing analysis using PEAKS software was performed to retrieve peptides. Peptide information is shown in Table 2. A total of 72 target peptides, ranging in length from 4 to 7 amino acids, were identified in the hydrolysis products. The flavor characteristics of flavor peptides are influenced by the type, number, sequence, spatial structure, and chain length of amino acids. Generally, hydrophilic amino acids impart pleasant tastes to peptides, such as umami and sweetness, while hydrophobic amino acids may produce bitterness, affecting the mouthfeel of food. The flavor intensity of a peptide is affected by its chain length; the longer the chain, the stronger the flavor. Previous studies have found a positive correlation between positively charged R groups and some umami amino acids in the perception of saltiness and umami. Therefore, subsequent analysis focused on peptides containing these amino acid residues.

[0068] Table 2. Identification results of F3 components using RPLC-MS

[0069]

[0070] Molecular docking: The TMC4 receptor on taste buds plays a crucial role in the transduction of salty taste and is frequently used in studies to identify and detect salty compounds. This invention establishes a TMC4 receptor protein homology model (…). Figure 3 A), and docked with 72 identified potential flavor peptides to determine their binding sites and binding energies with TMC4. The initial structure of TMC4 was obtained from the UniProt database and homology modeling was performed using AlphaFold. Raphaelite diagrams are mainly used to detail the conformational distribution of amino acids in proteins or peptides. Figure 3 As shown in B, the most common amino acid configurations account for 92.5%, while the remaining less common amino acid configurations account for only 0%. Most amino acids are within the error bars, indicating the high accuracy of the model.

[0071] The active groups of flavor peptides bind to receptors at key binding sites. 3D docking diagrams can reveal the optimal docking posture between the receptor and ligand, highlighting important active sites for binding. Further analysis of the binding sites and interactions between flavor peptides and TMC4 is needed. Figure 3As shown in Figure C, the flavor peptide interacts with key residues surrounding the TMC4 active site. Hydrogen bonds lead to the binding of the flavor peptide to the TMC4 receptor, consistent with previous findings. Hydrogen bonds are formed between Glu384, Glu371, Glu367, Tyr364, Glu418, Gly419, and Ala415. Tyr364 is likely the key binding site, and Glu, Gly, and Tyr are found to play crucial roles in the binding of TMC4 to the salty peptide. This is similar to previous findings, where Arg, Thr, and Tyr are considered to play important roles in the binding of salty-enhancing peptides to the TMC4 receptor. Furthermore, Wang et al. obtained peptides with unique salty and umami flavors from bovine bone, and molecular docking experiments also confirmed that Arg plays an important role in the binding of TMC4 to the salty peptide, similar to the findings in this invention. Additionally, the differences in binding sites may be related to the amino acid composition of the peptide and the docking active site (Table 3). In conclusion, Glu, Gly, and Tyr in TMC4 may significantly influence the binding of phytidines.

[0072] Table 3 Scoring of docking results for nine polypeptide molecules

[0073]

[0074] A binding energy less than 0 kcal / mol was considered suitable for natural docking, while less than -1.2 kcal / mol was considered a good docking result. Based on this, nine potential umami peptides with the lowest binding energies were screened: GSPT, AARAP, AKGKVP, EPPVRP, GTEahW, ECYECG, PPPPPP, GKKTVP, and AAKLGSP. Among them, we discovered a polypeptide PPPPPP composed of six identical amino acids, which may be a novel peptide for enhancing saltiness.

[0075] Sensory and electronic tongue experimental results of synthetic peptides:

[0076] Quantitative descriptive sensory evaluation of synthetic peptides: To verify the flavor characteristics of the selected peptides with salt-enhancing effects, two peptides with the highest scores in molecular docking, ECYECG (EG6) and PPPPPP (PP6), were selected for solid-phase synthesis. Their synthetic purity was greater than 98% and met edible standards. Sensory evaluation and electronic tongue testing were then conducted. Figure 4Analysis (A and B) revealed that the two synthetic peptides primarily exhibited salty and bitter tastes, followed by sweet and sour tastes, with a particularly significant enhancement in saltiness. The enhanced sourness may be due to the carboxyl groups in the peptide chain dissociating more strongly than the amino groups, resulting in an acidic solution. The formation of sourness may involve two mechanisms: one is that the carboxyl groups in the peptide chain dissociate more strongly than the amino groups, thus making the solution acidic; the other is that residual chemicals from the synthesis process impart a unique sour taste to the peptide. The bitterness, on the other hand, is directly related to the presence of hydrophobic amino acids—the higher the content of hydrophobic amino acids in the peptide, the easier it is for them to bind to taste receptors, thereby enhancing the perceived intensity of bitterness.

[0077] Taste threshold determination of synthetic peptides: In the sodium chloride threshold test, the critical value of sodium chloride in water was found to be 0.03 mmol / L, while the taste threshold of the synthetic peptide PPPPPP in aqueous solution was 0.181 mmol / L, and the taste threshold of ECYECG in aqueous solution was 0.635 mmol / L. Sodium chloride has the lowest threshold in water, while the taste thresholds of both synthetic peptides are higher than those of sodium chloride. However, the threshold of the synthetic peptide PPPPPP is closer to that of sodium chloride, indicating that the synthetic peptides obtained in this experiment have salt-substituting effects, and PPPPPP is more effective.

[0078] Dosage feedback sensory testing: such as Figure 4 As shown in Figure C, the saltiness intensity of the two synthetic peptides at different concentrations exhibited similar trends, but differences existed. With increasing synthetic peptide concentration, the saltiness intensity gradually increased; however, once the concentration exceeded a certain threshold, the saltiness intensity stopped increasing and instead showed a decreasing trend. Both ECYECG and PPPPPP reached their maximum saltiness intensity at an addition level of 0.2%. Comparison revealed that, at the same concentration, the saltiness intensity of the PPPPPP solution was significantly higher than that of the ECYECG solution. When the amount of synthetic peptide added is too high, its inherent sweet, bitter, and astringent taste characteristics gradually become more prominent, thus masking the perception of saltiness.

[0079] Molecular dynamics simulations: To further evaluate the interaction between the salty-enhancing peptide and TMC4, molecular dynamics simulations were performed over 100 ns. The root mean square deviation (RMSD) represents the conformational fluctuation of the protein; it is the sum of all atoms deviating from the target conformation over a certain period of time and is an important indicator of system stability. Figure 5 A represents the RMSD values ​​of the TMC4 protein over time in the TMC4 / ECYECG and TMC4 / PPPPPP systems, respectively. The TMC4 / PPPPPP system initially stabilized after 7 ns. The RMSD value of the TMC4 / ECYECG system gradually increased to 60 ns, then increased sharply between 60 and 100 ns, indicating that the structures began to loosen. That is, the TMC4 / PPPPPP system showed a small increase, but the magnitude of the increase was relatively small. Figure 5B represents the fluctuation curves of the RMSD values ​​of the peptides in the TMC4 / ECYECG and TMC4 / PPPPPP systems over time. Both the TMC4 / ECYECG and TMC4 / PPPPPP systems tend to stabilize after 5 ns. This means the RMSD value of the TMC4 / ECYECG system is higher than that of the TMC4 / PPPPPP system, and the TMC4 / PPPPPP system exhibits relatively smaller fluctuations during the simulation, indicating greater stability. The results suggest that the peptide binds to the receptor protein, causing a slight conformational change. Ultimately, the peptide and protein form a tight binding complex.

[0080] The root mean square fluctuation (RMSF) value reflects the flexibility of a protein. Figure 5 As shown in Figure C, the root mean square (RMS) values ​​of the TMC4 / ECYECG and TMC4 / PPPPPP systems are relatively low. Most of the RMS values ​​on the protein surface of the TMC4 / ECYECG system are below 1.2 nm, while most of the RMS values ​​on the protein surface of the TMC4 / PPPPPP system are below 0.5 nm, indicating that the TMC4 / PPPPPP system is relatively stable. Both TMC4 / ECYECG and TMC4 / PPPPPP exhibit high elasticity in the 385-390 nm and 425-429 nm regions, presumably representing the cyclic regions of the catalytic substrate or adjacent catalytic centers. The high elasticity of these regions may facilitate ligand interactions, thereby promoting functional adaptation. The low RMSF values ​​in other regions indicate structurally stable regions within PP6, maintaining the integrity of the peptide structure in the complex.

[0081] The radius of gyration (Rg) is an important indicator of the overall structural density of a protein. If a protein's folded state is stable, its Rg remains relatively constant, indicating that the protein possesses high spatial conformation and stability, and will not exhibit expansion or contraction. Changes in Rg indicate that the system has high unfoldability and that protein structure unwinding may occur. Figure 5 As shown in D, the Rg of the TMC4 / ECYECG system protein decreases rapidly after 60 ns, indicating possible structural unwinding, and its Rg fluctuates within a relatively large range, between 1.28 nm (12.8 Å) and 1.69 nm (16.9 Å). The Rg of the TMC4 / PPPPPP system protein, on the other hand, ranges from 1.35 nm (13.5 Å) to 1.56 nm (15.6 Å), with a fluctuation range of only 2.1 Å, indicating that this system has better stability than TMC4 / ECYECG.

[0082] Hydrogen bonds play a crucial role in maintaining the integrity of ligand-peptide complexes and may affect binding affinity and specificity. Figure 5E illustrates the changes in the number of hydrogen bonds between the two peptides and the TMC4 protein during molecular dynamics simulations. The number of hydrogen bonds between peptide ECYECG and the protein gradually decreased as the simulation progressed, remaining relatively constant at 3-4 before 50 ns, then stabilizing at around 2 after 50 ns, with an overall variation of 0-12. However, the number of hydrogen bonds between PPPPPP and the protein remained relatively stable throughout the simulation, varying between 0-5 and stabilizing at around 3. The number of hydrogen bonds formed between PPPPPP and the protein was significantly higher than that between ECYECG, consistent with the molecular docking results, indicating that hydrogen bonding is one of the key driving forces for peptide-protein molecular recognition.

[0083] SASA diagram ( Figure 5 F) shows the solubility of the TMC4 / ECYECG and TMC4 / PPPPPP complexes in solution. The results show that the SASA values ​​of the systems were relatively stable in the molecular dynamics simulations, with slight fluctuations. In the TMC4 / PPPPPP system, the protein's SASA gradually decreased throughout the simulation, reaching a relatively stable value at 50 ns. This is because as the simulation progressed, the interaction between the protein and the small molecule became more robust, and the stability of the SASA increased accordingly. The protein-peptide system maintains high stability through peptide linkage. In the TMC4 / ECYECG system, the SASA exhibited fluctuating characteristics throughout the simulation, indicating that the interaction between the small molecule and the protein in this system was not stable. Stability was achieved at 60 ns, indicating that the TMC4 / ECYECG system disintegrated at this point.

[0084] Based on the MM / GBSA equation, the free energy of the interaction between the peptide and the protein was calculated, as shown in Table 4. A negative free energy indicates that there is a binding affinity between the peptide and the target protein. The results showed that the total binding free energy (ΔG) of ECYECG and PPPPPP was... bind The energy values ​​were -22.00±4.43 kcal / mol and -27.49±3.23 kcal / mol, respectively, indicating good binding ability, and the binding strength of PPPPPP to proteins was greater than that of ECYECG. The van der Waals forces (ΔE) of ECYECG and PPPPPP were... vdw The electrostatic energies (ΔE) are -39.54±1.97 kcal / mol and -37.73±0.15 kcal / mol, respectively, and the electrostatic energy (ΔE) is... elec The energy values ​​were -79.57±3.52 kcal / mol and -25.73±3.10 kcal / mol, respectively. The van der Waals forces and electrostatic forces of ECYECG / TMC4 were lower than those of PPPPPP / TMC4, indicating that this system may have more hydrogen bond bridges and salt bridge structures, and greater hydrophobicity.

[0085] Table 4. Binding free energy and energy composition (kcal / mol) of the interactions between ECYECG, PPPPPP and TMC4 proteins.

[0086]

[0087] Figure 6 The interaction modes of ECYECG and PPPPPP with TMC4 are shown after molecular dynamics simulations. ECYECG binds within a groove of the TMC4 receptor. Figure 6 As shown in AB, a change in hydrogen bond binding sites occurred in the TMC4 / ECYECG system. Figure 6 E indicates that the integrity of the active pocket has been compromised. Combined with previous findings, RMSD ( Figure 5 The number of hydrogen bonds in AB decreased significantly and failed to recover until the end of the simulation. Figure 5 E), indicating that the TMC4 / ECYECG system is relatively unstable. PPPPPP binds to a groove in the TMC4 receptor, and the two exhibit good shape complementarity. Figure 6 As shown in CD, in the TMC4 / PPPPPP system, the charged amino acid residues in the peptide include hydrogen bonds Val366, Gly365, and Tyr364. The number of hydrogen bonds and the number of binding sites remained unchanged before and after binding, indicating that the TMC4 / PPPPPP system formed a relatively stable binding conformation. Furthermore, at the simulated endpoint, the conformation of the small molecule ligand was more compact, allowing for a closer binding to the receptor, reflecting spatial complementarity. A compact conformation can improve binding stability and reduce ligand energy, thus minimizing conformational fluctuations. In summary, the TMC4 / PPPPPP system is more stable than the TMC4 / ECYECG system. Moreover, the main binding forces between ECYECG and PPPPPP and the TMC4 receptor are hydrophobic interactions, with other key forces being hydrogen bonds and salt bridges. This suggests that the system tends to reduce free energy through hydrophobic aggregation rather than maintaining polar interactions or a hydrated state through hydrogen bonds. This also explains why small molecule ligands in the PPPPPP / TMC4 system curl up. Nonpolar groups in the ligands spontaneously move together to form folded or curled conformations; while polar groups may be wrapped up due to hydrophobic aggregation, resulting in a more compact overall conformation.

[0088] In summary, this invention employs high-pressure cooking, ultrafiltration, gel chromatography, and RPLC-MS / MS techniques to separate, purify, and identify savory peptides from *Flammulina velutipes*, yielding novel savory peptides ECYECG and PPPPPP. Electronic tongue and sensory evaluation results show that ECYECG and PPPPPP have taste thresholds higher than NaCl, exhibiting excellent saltiness-enhancing effects. Molecular dynamics simulation analysis indicates that the interaction between the savory peptide PPPPPP and the TMC4 receptor is primarily driven by hydrophobic interactions, with Val366, Gly365, and Tyr364 playing key roles. This invention supplements research on savory peptides from *Flammulina velutipes* and provides a basis for the industrial application of savory peptides ECYECG or PPPPPP.

[0089] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A salty peptide from enoki mushrooms, characterized in that, The amino acid sequence of the salty peptide is ECYECG or PPPPPP.

2. The method for preparing savory peptides from *Flammulina velutipes* according to claim 1, characterized in that, include, The salty peptide was obtained by extraction, separation and purification from the fruiting body of enoki mushroom (Flammulina filiformis); or by chemical synthesis.

3. The method for preparing savory peptides from *Flammulina velutipes* according to claim 2, characterized in that, The steps for extraction, separation, and purification from the fruiting bodies of *Flammulina velutipes* include: (1) Extraction: The fruiting bodies of enoki mushrooms were steamed, solid-liquid separation was performed, and the supernatant was collected to obtain enoki mushroom extract; (2) Ultrafiltration: The extract obtained in step (1) is subjected to ultrafiltration, and ultrafiltration fractions with a molecular weight of less than 1 kDa are collected; (3) Gel chromatography: The ultrafiltration components obtained in step (2) are separated by gel chromatography, and the components with salty activity are collected.

4. The method for preparing savory peptides from *Flammulina velutipes* according to claim 3, characterized in that, The cooking process includes boiling at a pressure of 70-90 Pa for 1-2 hours.

5. The method for preparing savory peptides from *Flammulina velutipes* according to claim 4, characterized in that, The steaming process involves steaming the fruiting bodies of enoki mushrooms in water at a mass ratio of 1:1.5, and boiling at 70 Pa pressure for 1.5 hours.

6. The method for preparing savory peptides from *Flammulina velutipes* according to any one of claims 2-5, characterized in that, The gel chromatography medium was Sephadex G-15, and the eluent was water.

7. The method for preparing savory peptides from *Flammulina velutipes* according to claim 6, characterized in that, The gel chromatography includes dissolving the component obtained in step (2) in water at a concentration of 20-40 mg / mL, separating it using a gel chromatography column packed with Sephadex G-15, and eluting at a rate of 0.5-0.75 mL / min.

8. The application of the enoki mushroom salty peptide according to claim 1 in food or condiments.

9. The application according to claim 8, characterized in that, The enoki mushroom salty peptides are used to enhance the saltiness of food, reduce the sodium content of food, or prepare low-sodium foods.

10. The application according to claim 8, characterized in that, The food products include condiments, meat products, convenience foods, puffed foods, sauces, soup bases, beverages, health foods, or special medical foods.

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