Mycena macrospora pentapeptide, preparation method and application thereof, pharmaceutical composition and mycena macrospora pentapeptide food

Abstract

The present application provides a large ball cover mushroom pentapeptide and a preparation method and application thereof, a pharmaceutical composition and a large ball cover mushroom pentapeptide food, and relates to the technical field of deep processing of edible fungi.The large ball cover mushroom pentapeptide EPQCD with the structure shown in formula I and the large ball cover mushroom pentapeptide SNGNE with the structure shown in formula II provided by the present application have relatively small relative molecular mass, are easily absorbed in the human body, can not only provide the nutrition required by human growth and development, but also can regulate human physiological functions, have high inhibitory activity on angiotensin converting enzyme and dipeptidyl peptidase-4, and have a good application prospect in the preparation of medicines and foods, especially health products, for inhibiting or treating ACE and DPP-IV mediated diseases.The large ball cover mushroom pentapeptide has umami characteristics, can supplement and enhance the overall taste of food as a seasoning, and makes the flavor more harmonious.

Description

Technical Field

[0001] This invention relates to the field of edible fungi deep processing technology, specifically to Pleurotus ostreatus pentapeptide and its preparation method and application, pharmaceutical composition and Pleurotus ostreatus pentapeptide food products. Background Technology

[0002] Stropharia rugosoannulata, also known as wrinkled stropharia, wine-red stropharia, or red pine mushroom, belongs to the phylum Basidiomycota, class Agrimonycetes, order Agricaliaes, family Strophariaceae, and genus Stropharica. It is a rare saprophytic edible fungus rich in high-quality protein, with a crude protein content of 20-31 wt% in its dried fruiting body. It is an excellent raw material for developing edible fungal proteins, peptides, and their derivatives.

[0003] Angiotensin-converting enzyme (ACE) is a key enzyme regulating blood pressure levels; inhibiting ACE can control or treat hypertension. Existing technology, "Li Wen, Chen Wanchao, Ma Haile, Wu Di, Zhang Zhong, Yang Yan. Ultrasonic preparation of peptide-based material from *Agaricus bisporus* and analysis of its edible and medicinal properties [J]. Journal of Edible Fungi, 2022, 29(03):81-94," discloses FP-20-120-t20-UF, PB-20-120-t20-UF, FP-20 / 28-120-t20-UF, and FP-20 / 40-120-t20-UF. Nine types of *Agaricus bisporus* ultrafiltrate peptide-based materials, including F, FP-28 / 40-120-t20-UF, FP-20 / 28 / 40-120-t20-UF, FP-20 / 40 / 28-120-t20-UF, FP-S20 / 28-300-t20-UF, and PB-S20 / 28-300-t20-UF, exhibit inhibitory effects on angiotensin-converting enzyme (ACE), with an ACE inhibitory activity IC50. 50 The values ​​ranged from 0.037 to 0.119 mg / mL. However, the ACE inhibitory activity of the above-mentioned Peptide-based ultrafiltrate from *Stropharia macrocarpa* was not high enough. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide *Stropharia spp.* pentapeptide, its preparation method and application, pharmaceutical composition and *Stropharia spp.* pentapeptide food products. The *Stropharia spp.* pentapeptide used in the present invention has high inhibitory activity against angiotensin-converting enzyme and dipeptidyl peptidase-4.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a *Pseudomonas aeruginosa* pentapeptide, comprising *Pseudomonas aeruginosa* pentapeptide EPQCD having the structure shown in Formula I and / or *Pseudomonas aeruginosa* pentapeptide SNGNE having the structure shown in Formula II:

[0007]

[0008] This invention provides a method for preparing the *Agaricus davidii* pentapeptide described in the above technical solution, comprising the following steps:

[0009] The mushroom cap mushroom was subjected to water extraction to obtain an aqueous extract;

[0010] The aqueous extract was subjected to ultrafiltration to obtain ultrafiltration crude peptides; the molecular weight cutoff of the ultrafiltration separation was 3-5 kDa.

[0011] The ultrafiltration crude peptide was separated by a Sephadex G-15 gel filtration column to obtain gel-separated crude peptide; the eluent used in the Sephadex G-15 gel filtration column separation was water.

[0012] The crude peptides separated by gel separation were separated by high performance liquid chromatography (HPLC) to obtain the Pleurotus ostreatus pentapeptide EPQCD and the Pleurotus ostreatus pentapeptide SNGNE, respectively; the mobile phase for the HPLC separation was an aqueous methanol solution, and the volume fraction of methanol in the aqueous methanol solution was 3-5%.

[0013] Preferably, the ratio of the mass of the giant puffball mushroom to the volume of the water used for water extraction is 1g:20-40mL.

[0014] Preferably, the water extraction is a boiling water extraction, and the water extraction time is 45-60 minutes.

[0015] Preferably, the pressure difference for ultrafiltration separation is 0.58 to 0.62 bar.

[0016] Preferably, the high-performance liquid chromatography separation conditions include: the chromatographic column is an XBridge Peptide BEH C18OBDTM Prep column; the column temperature is 25℃; the elution method is isocratic elution; and the flow rate of the mobile phase is 1 mL / min.

[0017] This invention provides the application of the above-described *Agaricus bisporus* pentapeptide or the *Agaricus bisporus* pentapeptide prepared by the above-described preparation method in the preparation of drugs, health products, or seasonings;

[0018] The drugs include those for treating diseases mediated by angiotensin-converting enzyme and / or dipeptidyl peptidase-4.

[0019] Preferably, the health products include blood pressure lowering health products and / or blood sugar lowering health products.

[0020] This invention provides a pharmaceutical composition comprising *Stropharia spp.* pentapeptide and pharmaceutically acceptable excipients; wherein the *Stropharia spp.* pentapeptide is the *Stropharia spp.* pentapeptide described in the above technical solution or the *Stropharia spp.* pentapeptide prepared by the preparation method described in the above technical solution.

[0021] This invention provides a Pleurotus ostreatus pentapeptide food product, comprising Pleurotus ostreatus pentapeptide and food-grade excipients; wherein the Pleurotus ostreatus pentapeptide is the Pleurotus ostreatus pentapeptide described in the above technical solution or the Pleurotus ostreatus pentapeptide prepared by the preparation method described in the above technical solution.

[0022] The *Pseudomonas aeruginosa* pentapeptide EPQCD with the structure shown in Formula I and *Pseudomonas aeruginosa* pentapeptide SNGNE with the structure shown in Formula II provided by this invention have relatively small molecular weights and are easily absorbed by the human body. They not only provide the nutrients needed for human growth and development but also regulate human physiological functions. They exhibit high inhibitory activity against angiotensin-converting enzyme and dipeptidyl peptidase-4, showing promising applications in the preparation of drugs, health products, and seasonings that inhibit ACE and / or DPP-IV activity or treat ACE and DPP-IV mediated diseases. Traditional single flavor amino acids or flavor nucleotides used as umami agents result in a monotonous taste, while the *Pseudomonas aeruginosa* pentapeptide used in this invention possesses umami characteristics and belongs to the umami peptide category. It can be used as a seasoning to supplement and enhance the overall flavor of food, making its flavor more harmonious, soft, and full-bodied. As shown in the test results of the examples, *Pseudomonas aeruginosa* pentapeptides EPQCD and SNGNE have a high IC50 value for ACE. 50 The IC50 values ​​for DPP-IV were 441.83±0.34 μM and 425.50±3.82 μM, respectively. 50 The inhibition rates of *Pleurotus ostreatus* pentapeptide EPQCD and SNGNE at concentrations of 2 mg / mL were 89.15±0.42% and 90.51±0.21% against ACE, respectively, and 57.68±0.96% and 61.42±0.33% against DPP-IV, respectively. The umami characteristic thresholds of *Pleurotus ostreatus* pentapeptide EPQCD and SNGNE were 0.304 mmol / L and 0.214 mmol / L, respectively, and the electronic tongue test results for 0.5 mg / mL *Pleurotus ostreatus* pentapeptide EPQCD and SNGNE were 7.12 and 8.82, respectively. This indicates that the *Pleurotus ostreatus* pentapeptide EPQCD and SNGNE used in this invention have high inhibitory activity against ACE and DPP-IV and possess umami characteristics. Attached Figure Description

[0023] Figure 1 De nove mass spectra of peptides EPQCD and SNGNE;

[0024] Figure 2Electronic tongue taste profiles of Pleurotus ostreatus pentapeptide and positive control (MSG);

[0025] Figure 3 The graph shows the results of the test on the inhibitory activity of Pleurotus ostreatus pentapeptide EPQCD against ACE and DPP-IV.

[0026] Figure 4 The figure shows the results of the test on the inhibitory activity of SNGNE, a pentapeptide from Pleurotus ostreatus, against ACE and DPP-IV.

[0027] Figure 5 The graph shows the results of the ACE inhibitory activity test of the positive control Lisinopril.

[0028] Figure 6 The graph shows the results of the positive control Sitagliptin's inhibitory activity against DPP-IV. Detailed Implementation

[0029] This invention provides a *Pseudomonas aeruginosa* pentapeptide, comprising *Pseudomonas aeruginosa* pentapeptide EPQCD having the structure shown in Formula I and / or *Pseudomonas aeruginosa* pentapeptide SNGNE having the structure shown in Formula II:

[0030]

[0031] This invention provides a method for preparing the *Agaricus davidii* pentapeptide described in the above technical solution, comprising the following steps:

[0032] The mushroom cap mushroom was subjected to water extraction to obtain an aqueous extract;

[0033] The aqueous extract was subjected to ultrafiltration to obtain ultrafiltration crude peptides; the molecular weight cutoff of the ultrafiltration separation was 3-5 kDa.

[0034] The ultrafiltration crude peptide was separated by a Sephadex G-15 gel filtration column to obtain gel-separated crude peptide; the eluent used in the Sephadex G-15 gel filtration column separation was water.

[0035] The crude peptides separated by gel separation were separated by high performance liquid chromatography (HPLC) to obtain the Pleurotus ostreatus pentapeptide EPQCD and the Pleurotus ostreatus pentapeptide SNGNE, respectively; the mobile phase for the HPLC separation was an aqueous methanol solution, and the volume fraction of methanol in the aqueous methanol solution was 3-5%.

[0036] Unless otherwise specified, all raw materials used in this invention are commercially available products.

[0037] This invention involves water extraction of *Stropharia macrocarpa* to obtain an aqueous extract. In this invention, the *Stropharia macrocarpa* is preferably the fruiting body of *Stropharia macrocarpa*. The fruiting body is preferably crushed before use; however, the crushing process is not particularly limited, as long as it yields *Stropharia macrocarpa* fruiting body powder with a particle size of 60-100 mesh. In this invention, the mass ratio of the *Stropharia macrocarpa* to the volume of water used for extraction is preferably 1g:20-40mL, more preferably 1g:25-35mL, and even more preferably 1g:30mL. In this invention, the water extraction is preferably performed using boiling water, and the extraction time is preferably 45-60 minutes, more preferably 50-60 minutes.

[0038] After the water extraction is completed, the present invention preferably further includes solid-liquid separation of the obtained water extraction system to obtain water extract; the solid-liquid separation preferably includes sequentially performing filter cloth filtration, centrifugation, and microporous filtration; the temperature of the centrifugation is preferably 0-4℃, more preferably 1-4℃, and even more preferably 2-4℃; the speed of the centrifugation is preferably 9000-10000g, more preferably 9500-9900g, and even more preferably 9700-9850g; the time of the centrifugation is preferably 10-15min, more preferably 10-14min, and even more preferably 10-12min; the pore size of the microporous membrane used for microporous filtration is preferably 0.45μm.

[0039] After obtaining the aqueous extract, the present invention performs ultrafiltration separation on the aqueous extract to obtain ultrafiltration crude peptides. In the present invention, the molecular weight cutoff of the ultrafiltration separation is 3-5 kDa, preferably 3-4 kDa. In the present invention, the ultrafiltration separation is preferably performed using an ultrafiltration column, preferably a UFP-3-C-4MA column, the filtration system of the ultrafiltration column is preferably an AKETFlux 6, the ultrafiltration separation temperature is preferably room temperature (25°C), the pressure difference (ΔP) of the ultrafiltration separation is preferably 0.58-0.62 bar, more preferably 0.56-0.61 bar, and even more preferably 0.6 bar; the feed cylinder rotation speed of the ultrafiltration separation is preferably 240-260 rpm, more preferably 250 rpm.

[0040] After completing the ultrafiltration separation, the present invention preferably further includes drying the obtained ultrafiltrate to obtain crude ultrafiltration peptides. In the present invention, the drying is preferably freeze-drying, and the freeze-drying temperature is preferably -40 to -10°C, more preferably -40 to -20°C. The present invention does not have a specific limitation on the freeze-drying time; freeze-drying to constant weight is sufficient. In the present invention, the storage temperature of the crude ultrafiltration peptides is preferably -20°C.

[0041] After obtaining the ultrafiltration crude peptide, the present invention separates the ultrafiltration crude peptide using a Sephadex G-15 gel filtration column to obtain gel-separated crude peptide; the eluent used in the Sephadex G-15 gel filtration column separation is water. In the present invention, the ultrafiltration crude peptide is preferably first dissolved in water, filtered through a microporous membrane, and then the obtained ultrafiltration crude peptide solution is separated using a Sephadex G-15 gel filtration column; the concentration of the ultrafiltration crude peptide solution is preferably 50-100 mg / mL, more preferably 50-80 mg / mL, and even more preferably 50-60 mg / mL; the pore size of the microporous membrane is preferably 0.22 μm. In the present invention, the Sephadex G-15 gel filtration column is preferably an XK16 / 10 column. In the present invention, the flow rate of the ultrafiltration crude peptide solution is preferably 0.75-1 mL / min, more preferably 0.75-0.8 mL / min; the water is preferably deionized water. In this invention, the detection wavelength for separation by the Sephadex G-15 gel filter column is preferably 220 nm.

[0042] After completing the separation using the Sephadex G-15 gel filtration column, the present invention preferably further includes drying the obtained eluent to obtain crude peptides separated by gel filtration. In the present invention, the drying is preferably freeze-drying, and the freeze-drying temperature is preferably -40 to -10°C, more preferably -40 to -20°C; the present invention does not have a special limitation on the freeze-drying time, freeze-drying until constant weight is achieved.

[0043] After obtaining the crude peptides through gel separation, the present invention further separates the crude peptides through high-performance liquid chromatography (HPLC) to obtain the *S. macrocarpa* pentapeptide EPQCD and the *S. macrocarpa* pentapeptide SNGNE, respectively. In the present invention, the preferred HPLC separation conditions are: the preferred instrument is an LC 1620RP-HPLC system; the preferred column is an XBridge PeptideBEH C18 OBDTM Prep column; the preferred column temperature is 25℃; the preferred mobile phase is a methanol-water solution, wherein the volume fraction of methanol in the methanol-water solution is 3-5%, preferably 4-5%; the preferred elution method is isocratic elution; and the preferred flow rate of the mobile phase is 1 mL / min.

[0044] After completing the high-performance liquid chromatography separation, the present invention preferably further includes drying the obtained eluent to obtain the *Stropharia macrocarpa* pentapeptide EPQCD and the *Stropharia macrocarpa* pentapeptide SNGNE, respectively. In the present invention, the drying is preferably freeze-drying, and the freeze-drying temperature is preferably -40 to -10°C, more preferably -40 to -20°C; the present invention does not have a special limitation on the freeze-drying time, freeze-drying to constant weight is sufficient.

[0045] This invention provides the application of the *Stropharia macrocarpa* pentapeptide described in the above-described technical solutions, or the *Stropharia macrocarpa* pentapeptide prepared by the above-described preparation method, in the preparation of pharmaceuticals, health products, or seasonings; the pharmaceuticals include drugs for treating angiotensin-converting enzyme (ACE) and / or dipeptidyl peptidase-4 (DPP-IV) mediated diseases. In this invention, the pharmaceuticals preferably include one or more of antihypertensive drugs, lipid-lowering drugs, and hypoglycemic drugs. In this invention, the health products preferably include antihypertensive health products, hypoglycemic health products, or lipid-lowering health products; the health products preferably include solid health products or liquid health products.

[0046] This invention provides a pharmaceutical composition comprising *Stropharia spp.* pentapeptide and pharmaceutically acceptable excipients; wherein the *Stropharia spp.* pentapeptide is the *Stropharia spp.* pentapeptide described in the above technical solution or the *Stropharia spp.* pentapeptide prepared by the preparation method described in the above technical solution.

[0047] In this invention, the pharmaceutically acceptable excipients preferably include one or more of solid excipients, semi-solid excipients, and liquid excipients. In this invention, the dosage form of the pharmaceutical composition preferably includes liquid formulations, solid formulations, sprays, or aerosols. In this invention, the liquid formulation preferably includes injections, suspensions, emulsions, solutions, or syrups; when the dosage form is a liquid formulation, the pharmaceutically acceptable excipients preferably include one or more of dispersion media, suspending agents, wetting agents, flocculants, and anti-flocculating agents; this invention does not have any particular limitation on the dispersion media, suspending agents, wetting agents, flocculants, and anti-flocculating agents, and any dispersion media, suspending agents, wetting agents, flocculants, and anti-flocculating agents well known to those skilled in the art can be used. In this invention, the solid formulation preferably includes tablets, capsules, granules, or powders. In this invention, when the dosage form is a tablet, the pharmaceutically acceptable excipients preferably include one or more of fillers, adsorbents, binders, lubricants, dispersants, wetting agents, and disintegrants. This invention does not specifically limit the types of fillers, adsorbents, binders, lubricants, dispersants, wetting agents, and disintegrants; fillers, adsorbents, binders, lubricants, dispersants, wetting agents, and disintegrants well-known to those skilled in the art can be used. In this invention, when the dosage form is a capsule, the pharmaceutically acceptable excipients preferably include capsule material. This invention does not specifically limit the types of capsule material; capsule materials well-known to those skilled in the art can be used. In this invention, when the dosage form is a granule, the pharmaceutically acceptable excipients preferably include one or more of diluents, absorbents, wetting agents, binders, and flavoring agents. This invention does not specifically limit the types of diluents, absorbents, wetting agents, binders, and flavoring agents; diluents, absorbents, wetting agents, binders, and flavoring agents well-known to those skilled in the art can be used. In this invention, when the dosage form is a granule, the pharmaceutically acceptable excipients preferably include one or more of excipients and flavoring agents. This invention does not specifically limit the types of excipients and flavoring agents; any excipients and flavoring agents well-known to those skilled in the art can be used. In this invention, the preferred routes of administration for the pharmaceutical composition include injection, oral administration, sublingual administration, or mucosal dialysis; the injection preferably includes intravenous injection, intravenous infusion, intramuscular injection, or subcutaneous injection.

[0048] This invention also provides a *Stropharia macrocarpa* pentapeptide food product, comprising *Stropharia macrocarpa* pentapeptide and food-grade acceptable excipients; the *Stropharia macrocarpa* pentapeptide is the *Stropharia macrocarpa* pentapeptide described in the above-mentioned technical solution or the *Stropharia macrocarpa* pentapeptide prepared by the preparation method described in the above-mentioned technical solution. In this invention, the food-grade acceptable excipients preferably include one or more of sweeteners, acidulants, preservatives, colorants, disintegrants, fillers, lubricants, and binders. In this invention, the sweeteners preferably include one or more of granulated sugar, fructose, glucose, honey, maltose, maltitol, sorbitol, cyclamate, starch syrup, high-fructose corn syrup, and protein sugar. In this invention, the acidulants preferably include one or more of vinegar, sorbic acid, citric acid, and lactic acid. In this invention, the preservatives preferably include one or more of benzoic acid, sodium benzoate, sodium citrate, sorbic acid, sodium sorbate, vinegar, methylparaben, and ethylparaben. In this invention, the colorant preferably includes one or more of the following food colorings: amaranth, carmine, tartrazine, gardenia yellow, indigo, scarlet, brilliant blue, gardenia blue, and caramel color. In this invention, the disintegrant preferably includes one or more of the following: starch, microcrystalline cellulose, sodium carboxymethyl cellulose, sodium bicarbonate, and citric acid. In this invention, the filler preferably includes one or more of the following: pregelatinized starch, dextrin, powdered sugar, and microcrystalline cellulose. In this invention, the lubricant preferably includes one or more of the following: stearic acid, calcium stearate, magnesium stearate, talc, and polyethylene glycol. In this invention, the binder preferably includes one or more of the following: sucrose, gelatin, and pregelatinized starch.

[0049] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0050] Example 1

[0051] (1) 1.1 Isolation, purification and identification of Pentapeptide from Pleurotus ostreatus

[0052] (1.1) Preparation of Peptide from Pleurotus ostreatus: Polypeptide separation and purification were performed using ultrafiltration, gel permeation and reversed-phase chromatography.

[0053] Fresh large-cap mushroom fruiting bodies were mixed with water at a material-to-liquid ratio of 1:30 (g:mL), extracted with boiling water for 60 min, filtered through a filter cloth, and the resulting filtrate was centrifuged at 9803g at 4℃ for 10 min. The resulting supernatant was filtered through a 0.45μm microporous membrane to obtain the water extract.

[0054] The aqueous extract was separated by an ultrafiltration column (UFP-3-C-4MA, AKETFlux 6) with a relative molecular weight cutoff size of 3 kDa. The ultrafiltrate with a molecular weight less than 3 kDa was collected, freeze-dried to constant weight at -40°C, and stored at -20°C to obtain ultrafiltration crude peptides. The separation conditions of the ultrafiltration column were: 25°C, ΔP = 0.60 bar, and cylinder rotation speed of 250 rpm.

[0055] The ultrafiltration crude peptide was prepared into a 50 mg / mL ultrafiltration crude peptide solution using deionized water. The solution was filtered through a 0.22 μm microporous membrane. 2 mL of the ultrafiltration crude peptide solution was loaded onto a Sephadex G-15 gel filtration column (XK16 / 100) for separation at room temperature. The eluent was collected and freeze-dried at -40°C to constant weight to obtain the gel-separated crude peptide. The sample flow rate for separation using the Sephadex G-15 gel filtration column was 0.75 mL / min, and the detection wavelength was 220 nm. Sensory evaluation of the gel-separated crude peptide revealed that the component containing *Pleurotus ostreatus* pentapeptide exhibited the highest umami sensory intensity.

[0056] The crude peptides separated by gel electrophoresis were further separated by high performance liquid chromatography (HPLC). The eluent of the purified fraction was freeze-dried at -40°C to constant weight to obtain the Pleurotus ostreatus tripeptide CEW (retention time t). R =1.03min), Giant King Pleurotus ostreatus pentapeptide EPQCD (t R =3.97min), SNGNE (t) pentapeptide from Pleurotus ostreatus R =2.66min) and Pleurotus ostreatus tetrapeptide EEHG(t R =14.00min); where, the high performance liquid chromatography separation conditions: the instrument was an LC 1620RP-HPLC instrument, and the chromatographic column was an XBridge PeptideBEH C18 OBDTM Prep column ( The column was 5 μm (10 × 150 mm); the mobile phase was an aqueous methanol solution (5% methanol by volume), eluted at 1 mL / min; the column temperature was 25 °C, the mobile phase was deionized water containing 5% (v:v) methanol, the purified fraction was collected and lyophilized.

[0057] Pleurotus ostreatus tripeptide CEW;

[0058] Giant Pleurotus ostreatus tetrapeptide EEHG.

[0059] (1.2) Peptide identification using UHPLC-Q-Orbitrap-MS / MS

[0060] The pentapeptide of *Stropharia macrocarpa* was dissolved in deionized water to prepare a pentapeptide solution with a concentration of 5 mg / mL. The mass-to-charge ratio (m / z) of the whole molecule and its fragments was further analyzed using an Ultimate 3000U HPLC system and a Q Exactive Plus mass spectrometer (Q-Orbitrap MS / MS). The liquid chromatography conditions were as follows: the column was a ChromCore 120C18 column (2.1×100mm, 1.9μm); mobile phase A was a 0.1% formic acid aqueous solution (formic acid volume fraction 0.1%); mobile phase B was a methanol solution of formic acid (formic acid volume fraction 0.1%); the mobile phase flow rate was 0.3mL / min; gradient elution was used; the gradient elution program was as follows: 0–1 min, mobile phase B volume fraction 10%; 1–20 min, mobile phase B volume fraction linearly increased from 10% to 100%; 20–22 min, mobile phase B volume fraction 100%; 22–25 min, mobile phase B volume fraction linearly decreased from 100% to 10%; the injection volume was 4μL; and the sample was kept at 4℃ before analysis.

[0061] Mass spectrometry conditions: Spectroscopic data acquisition was performed in positron emission tomography (ESI) mode, collecting ion fragments in the mass range of 100–1000 amu. Ion source parameters were set as follows: capillary temperature 325 °C, source voltage 4.5 kV, and capillary voltage 49 V. Mass spectrometry data scanning mode was used, with an Orbitrap resolution of 70,000 for full MS and a scan range of 200–2000 m / z, and a ddMS2 resolution of 17,500. External instrument calibration was performed at the start of the analysis.

[0062] Data from tandem secondary mass spectrometry were analyzed using de novo sequencing. Peptides were automatically identified using Peaks Studio 7.5 software, and peptides with high average confidence (ALC) values ​​(>85%) were selected for further analysis.

[0063] Figure 1 The De nove mass spectra of peptides EPQCD and SNGNE are provided by [the Chinese text is incomplete and requires further context]. Figure 1 It can be seen that the structures of the pentapeptide from *Agaricus macrocarpa* identified by the De nove method are EPQCD (Formula I) and SNGNE (Formula II).

[0064] (2) Molecular docking experiment

[0065] (2.1) Molecular docking of Pleurotus ostreatus pentapeptide with umami receptor hT1R1-T1R3

[0066] Molecular docking was performed using the CDOCKER semi-flexible docking method in Discovery Studio 2019 Client. The specific steps are as follows: First, the 3D structure of the *Pleurotus ostreatus* pentapeptide was drawn using ChemDraw 18.0 and energy minimization was performed (MMFF94). The ligand was then imported into DS and a CHARMM force field was added. Next, the constructed umami receptor was dehydrated and hydrogenated in DS software, and a CHARMM force field was introduced to optimize the protein structure. The active cavity of the dimer T1R1 / T1R3 receptor protein was determined in the *From Receptor Cavities* section of the *Receptor-Ligandinteractions* module, using the ligand Glu in the template protein T1R2a-T1R3 (PDB id: 5X2P) as a reference to determine the location of the active cavity. The radius of the T1R1 cavity was [missing information]. The cavity location is (101.18, 78.96, 23.66); the cavity radius of T1R3 is... The cavity is located at (108.35, 62.16, 54.65).

[0067] (2.2) Molecular docking of Pleurotus ostreatus pentapeptide with target active protein

[0068] Regarding functional activity, the structures of *Pleurotus ostreatus* pentapeptide (PDB: 1O86) and DPP-IV protein (PDB: 1X70) were optimized using DS software in the same manner as in step (2.1). The active cavity was also designed with the ligand in the original crystal structure as the active center. The ACE cavity radius was... Cavity location (40.997, 34.127, 46.348); DPP-IV cavity radius is Cavity location (40.997, 51.230, 35.624).

[0069] The optimized Pleurotus ostreatus pentapeptide and DPP-IV protein molecules were introduced into the same window for CDOCKER docking. The successfully docked ligands retained the lowest energy (most stable) conformation. After the calculation, the docking results were sorted according to the docking energy to achieve further prediction.

[0070] (3) Verification of the taste characteristics and functional activity of Pseudolarix amabilis pentapeptide

[0071] (3.1) Electronic tongue determination of the taste characteristics of Pleurotus ostreatus pentapeptide

[0072] The taste characteristics of *Stropharia stolonifera* pentapeptide were verified using an SA402B electronic tongue (Tokyo, Japan). This device includes a taste sensor based on an artificial lipid membrane and positive and negative reference electrodes. Taste information from samples is collected using five sensors: AAE (umami), CTO (salty), CAO (sour), COO (bitter), and AE1 (astringent). Before the experiment, the sensors and electrodes were activated and the instrument calibrated. The experimental temperature was 25°C. A 0.3 mmol / L tartaric acid-30 mmol / L potassium chloride mixed aqueous solution was used as the reference solution, monosodium glutamate (MSG) as the positive control group, and a 0.5 mg / mL *Stropharia stolonifera* pentapeptide aqueous solution as the sample group.

[0073] (3.2) Determination of the umami threshold of pentapeptide from Pleurotus ostreatus

[0074] The umami threshold of the synthetic peptide was determined using taste dilution analysis (TDA). Deionized water was used as the solvent. Sample groups were prepared by serially diluting a 0.5 mg / mL solution of *Scutellaria baicalensis* pentapeptide with deionized water, resulting in concentrations of 0.25 mg / mL, 0.125 mg / mL, and 0.0625 mg / mL. Deionized water served as the blank control. At room temperature, samples were tested sequentially from low to high concentration using a three-point method (following the electronic tongue test method for the taste characteristics of *Scutellaria baicalensis* pentapeptide) at each dilution level. When the Nth group of samples could be distinguished from the blank control, but the N-1th group could not, the average concentration of the Nth and N-1th groups was considered the taste threshold of the synthetic peptide.

[0075] (3.3) Determination of the inhibitory activity of Pseudolarix amabilis pentapeptide

[0076] (3.3.1) Assay of ACE inhibitory activity of Pleurotus ostreatus pentapeptide

[0077] Sample group: HHL was dissolved in 0.05M borate buffer (containing 0.3M NaCl, pH=8.3). 12.5 μL of 1 mg / mL *Pseudomonas aeruginosa* pentapeptide aqueous solution was mixed with 50 μL of 4.7 mmol / L HHL substrate solution and incubated at 37℃ for 5 min. 25 μL of ACE (0.025 U / mL) was added, and the mixture was incubated at 37℃ for 30 min. 375 μL of 0.3 mol / L NaOH solution was added to terminate the enzyme reaction. Then, 25 μL of 2% o-phthalaldehyde (OPA) aqueous solution was added for color development, and the mixture was incubated at 25℃ in the dark for 10 min. 50 μL of 3.0 mol / L hydrochloric acid was added to stop the reaction. The fluorescence absorption intensity of the reaction solution was detected at an excitation wavelength of 340 nm and an emission wavelength of 455 nm. Blank group: The difference from the sample group was the absence of ACE. Captopril was used as a positive control. IC was calculated using the ACE activity inhibition rate curve. 50 The ACE inhibition rate (%) is calculated as shown in equation (1):

[0078]

[0079] In formula (1), A1 is the fluorescence absorption intensity of the negative control group, which uses deionized water instead of strophanthidin; A2 is the fluorescence absorption intensity of the negative control group, which does not contain strophanthidin and ACE; A3 is the fluorescence absorption intensity of the sample group; and A4 is the fluorescence absorption intensity of the blank group, which does not contain strophanthidin.

[0080] (3.3.2) Determination of DPP-IV inhibitory activity of Pleurotus ostreatus pentapeptide

[0081] Sample group: All reagents and samples were dissolved in a 100 mmol / L Tris-HCl (pH=8) buffer system. 25 μL of 1 mg / mL Pleurotus ostreatus pentapeptide solution and 25 μL of 1.6 mmol / L glycine-proline-p-nitroaniline (GP-pNA) solution were added to a 96-well plate and mixed. After incubation at 37°C for 10 min, 50 μL of DPP-IV (0.025 U / mL) was added to the reaction system and incubated at 37°C for 60 min. The reaction was terminated by adding 100 μL of sodium acetate buffer (1 mol / L, pH=4.0). The absorbance at 405 nm was detected using a microplate reader. Blank group: DPP-IV in the sample group was replaced with Tris-HCl. Sitagliptin was used as the positive control group. The DPP-IV inhibition rate / % was calculated as shown in formula (2):

[0082]

[0083] In formula (2), a is the absorbance value of the negative control group, with Tris-HCl replacing the pentapeptide of *Stropharia stropharia*; b is the absorbance value of the negative control group, with Tris-HCl replacing the pentapeptide of *Stropharia stropharia* and DPP-IV in the sample group; c is the absorbance value of the sample group; and d is the absorbance value of the blank group, with Tris-HCl replacing DPP-IV.

[0084] (4) Experimental Results

[0085] The test results are shown in Tables 1-2 and Figures 1-5 As shown, where, Figure 2 Electronic tongue taste profiles of Pleurotus ostreatus pentapeptide and the positive control (MSG). Figure 3 The image shows the results of the assay for the inhibitory activity of Pleurotus ostreatus pentapeptide EPQCD against ACE and DPP-IV. Figure 4 The image shows the results of the assay for the inhibitory activity of SNGNE, a pentapeptide from *Pleurotus ostreatus*, against ACE and DPP-IV. Figure 5 The graph shows the results of the ACE inhibitory activity assay using the positive control Lisinopril. Figure 6 The graph shows the results of the positive control Sitagliptin's inhibitory activity against DPP-IV.

[0086] The molecular docking results of the Pleurotus ostreatus pentapeptide are shown in Table 1:

[0087] Table 1. Molecular docking data of Pseudolarix amabilis pentapeptide

[0088]

[0089]

[0090]

[0091] Table 1 shows that the main forces binding peptides to ACE proteins are hydrogen bonds, metallic bonds, and electrostatic interactions. ALA354, TYR523, LYS511, HIS353, and Zn... 2+ These are key binding sites for ACE-inhibiting peptides. The main forces binding the peptide to DPP-IV protein include hydrogen bonds, electrostatic interactions, and hydrophobic interactions. SER209, GLU205, ARG125, and HIS740 are key binding sites for the peptide to DPP-IV.

[0092] Table 2. Validation experimental data of Pseudolarix amabilis pentapeptide

[0093]

[0094]

[0095] Note: In Table 2, "-" indicates that no relevant experiments were performed; " / " indicates that the IC50 of the peptide was not detected within the concentration range with a maximum concentration of 2 mg / mL. 50 Values; During the ACE inhibitory activity and DPP-IV inhibitory activity tests, the concentrations of all drugs except Lisinopril (ACE) and Sitagliptin (DPP-IV) were 2 mg / mL, with a concentration of 0.5 μg / mL.

[0096] As shown in Table 2, the pentapeptide of *Stropharia macrocarpa* prepared in this invention is a multifunctional active umami peptide that not only provides a good taste and rich flavor, but also has high inhibitory activity against ACE and DPP-IV, exhibiting good biological activity and the ability to improve bodily functions.

[0097] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A pentapeptide from *Stropharia macrocarpa*, characterized in that, The structure of Pleurotus ostreatus pentapeptide EPQCD as shown in Formula I and / or the structure of Pleurotus ostreatus pentapeptide SNGNE as shown in Formula II: Equation I; Formula II.

2. The preparation method of the *Agaricus macrocarpa* pentapeptide according to claim 1, comprising the following steps: The mushroom cap mushroom was subjected to water extraction to obtain an aqueous extract; The aqueous extract was subjected to ultrafiltration to obtain ultrafiltration crude peptides; The molecular weight cutoff for the ultrafiltration separation is 3~5 kDa; The ultrafiltration crude peptide was separated by a Sephadex G-15 gel filtration column to obtain gel-separated crude peptide; the eluent used in the Sephadex G-15 gel filtration column separation was water. The crude peptides separated by gel separation were further separated by high performance liquid chromatography (HPLC) to obtain Pleurotus ostreatus pentapeptide EPQCD and Pleurotus ostreatus pentapeptide SNGNE, respectively; the mobile phase for HPLC separation was an aqueous methanol solution with a methanol volume fraction of 3-5%; The ratio of the mass of the giant puffball mushroom to the volume of water used for water extraction is 1g: 20~40mL; The water extraction is a boiling water extraction, and the water extraction time is 45~60 minutes; The pressure difference for ultrafiltration separation is 0.58~0.62 bar; The conditions for high-performance liquid chromatography separation include: the chromatographic column is an XBridge Peptide BEH C18 OBDTM Prep column; the column temperature is 25℃; the elution method is isocratic elution; and the flow rate of the mobile phase is 1 mL / min.

3. The application of the *Stropharia macrocarpa* pentapeptide according to claim 1 or the *Stropharia macrocarpa* pentapeptide prepared by the method according to claim 2 in the preparation of seasonings.

4. A pentapeptide food product made from *Stropharia macrocarpa*, characterized in that, It includes Pleurotus ostreatus pentapeptide and food-acceptable excipients; the Pleurotus ostreatus pentapeptide is the Pleurotus ostreatus pentapeptide of claim 1 or the Pleurotus ostreatus pentapeptide prepared by the preparation method of claim 2.

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