Boletus edulis sporocarp umami active peptide as well as acquisition method and application thereof

Abstract

The invention discloses a boletus edulis sporocarp umami active peptide as well as an acquisition method and application thereof, and relates to the technical field of edible mushroom production, and coding amino acid sequences are IDPVHSR, PRDNF, PGDGF and PDGGY. After the obtained umami peptides are combined pairwise, when the total concentration of an umami peptide solution is 0.25 mg / mL, the umami strength can be improved, which indicates that the umami strength can be effectively improved after the obtained umami peptides are combined pairwise without increasing the concentration, which indicates that the umami peptides have a better mutual synergistic effect.

Description

Technical Field

[0001] This application relates to the field of edible fungi production technology, and in particular to a umami-active peptide from the fruiting body of Boletus edulis, its acquisition method, and its application. Background Technology

[0002] Umami is the fifth basic taste, primarily mediated by glutamate, nucleotides (such as IMP and GMP), and certain small peptides. Currently, commercially available umami enhancers mainly consist of monosodium glutamate (MSG) and I+G (5'-inosinic acid + 5'-guanylic acid). Excessive intake can lead to health problems such as asthma, migraines, nervous system damage, and obesity. A preference for umami may unintentionally increase the intake of these enhancers, thereby exacerbating the risk of hypertension and other chronic diseases. Consumer demand for natural umami enhancers is growing. Lanmao Boletus (…) Lanmaoaasiatica Matsutake mushrooms are conditionally edible wild mushrooms, delicious when cooked, but can cause poisoning if eaten raw or undercooked. This characteristic greatly limits their safe development and widespread application. Although studies have reported that small peptides in matsutake and porcini mushrooms enhance umami flavor, systematic research on the flavor components of porcini mushrooms remains lacking.

[0003] The umami peptides in existing matsutake and porcini mushrooms have relatively high binding energy, which means that an increased amount of umami enhancer is needed to achieve the desired umami flavor. This further increases the amount of umami enhancer used, and the effectiveness of existing combinations of umami enhancers is unknown.

[0004] The information disclosed in the background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] This application addresses the aforementioned technical problems by providing a umami-active peptide from Boletus edulis fruiting body, a method for obtaining it, and its application. The binding energy of the obtained umami peptide to the hT1R1 / T1R3 umami receptor can reach -9.286 kcal / mol.

[0006] This application provides a flavor-active peptide from the fruiting body of Boletus edulis, encoding the amino acid sequence IDPVHSR, PRDNF, PGDGF, and PDGGY; when any two of the flavor-active peptides are combined, the umami intensity of the resulting aqueous solution is enhanced.

[0007] Preferably, the IDPVHSR structure is as follows:

[0008] PRDNF structure:

[0009] PGDGF structure:

[0010] PDGGY structure: ; The molecular weight of IDPVHSR is 822.90 Da; the molecular weight of PRDNF is 647.68 Da; the molecular weight of PGDGF is 491.49 Da; and the molecular weight of PDGGY is 507.49 Da.

[0011] Preferably, the binding energy of IDPVHSR to the hT1R1 / T1R3 umami receptor is -9.09 kcal / mol; The binding energy of PRDNF to the hT1R1 / T1R3 umami receptor is -9.286 kcal / mol; The binding energy of PGDGF to the hT1R1 / T1R3 umami receptor is -9.211 kcal / mol; The binding energy of PDGGY to the hT1R1 / T1R3 umami receptor is -9.175 kcal / mol.

[0012] Preferably, when the concentration of the obtained umami peptide solution is 0.25 mg / mL, the umami intensity of IDPVHSR is 3; the umami intensity of PRDNF is 3; the umami intensity of PGDGF is 3; and the umami intensity of PDGGY is 3. The taste thresholds for IDPVHSR, PRDNF, PGDGF, and PDGGY were 0.11 mmol / L, 0.36 mmol / L, 0.32 mmol / L, and 0.20 mmol / L, respectively.

[0013] Another aspect of this application provides a method for obtaining umami-active peptides from Boletus edulis fruiting bodies, comprising the following steps: Fresh Boletus edulis was dried, pulverized, and mixed with water. Neutral protease, flavor protease, and papain were added, and the mixture was enzymatically hydrolyzed to obtain an enzymatic hydrolysate. The hydrolysate was freeze-dried and subjected to peptide extraction, reductive alkylation, and desalting. After screening by LC-MS / MS mass spectrometry, the umami-active peptides of Boletus edulis fruiting bodies, as described above, with umami characteristics, good water solubility, and no toxicity were isolated from the fraction F with a fraction smaller than 1 kDa.

[0014] Preferably, the preparation of the enzymatic hydrolysate includes the following steps: the pulverized material obtained by pulverization is mixed with water at a mass ratio of 1:20 to obtain a mixed solution, and 0.60% of neutral protease, 0.90% of flavor protease, and 4.70% of papain by mass of the mixed solution are added respectively. The enzyme was hydrolyzed at pH 7.0 and 50°C for 1.5 h to obtain the enzymatic hydrolysate.

[0015] Preferably, peptide extraction includes the following steps: After the enzymatic hydrolysate is freeze-dried, it is mixed with water to obtain a freeze-dried powder suspension. 1000 μL of the freeze-dried powder suspension is added to a 5 kDa ultrafiltration tube and centrifuged at 12000 g for 10 minutes. The fraction A with a weight of less than 5 kDa is collected. Add the fraction A to a 3kDa ultrafiltration tube and centrifuge at 12000g for 10 minutes. Collect the fraction B (3-5kDa) and the fraction C (less than 3kDa). Take the fraction C (less than 3 kDa) and add it to a 1 kDa ultrafiltration tube. Centrifuge at 12000 g for 10 minutes and collect the fraction D (1-3 kDa) and the fraction F (less than 1 kDa).

[0016] Preferably, the obtained enzymatic hydrolysate has an amino acid nitrogen content of 124.01 mg / 100 mL, a total free amino acid content of 12.33 mg / g, a total flavor nucleotide content of 96.48 μg / g, a total organic acid content of 4511.59 μg / g, and an EUC value of 2.46 g MSG / 100 g.

[0017] Another aspect of this application provides an application of the above-described Boletus edulis fruiting body umami active peptide, which is added to flavored foods; the Boletus edulis fruiting body umami active peptide is prepared according to the above-described method.

[0018] Preferably, the flavored food includes seasonings and / or plant-based meat.

[0019] The beneficial effects that this application can produce include: 1) The umami-active peptides from *Boletus edulis* fruiting bodies, their acquisition method, and their applications provided in this application represent the first targeted extraction and identification of a group of small molecule peptides with strong umami characteristics from safely treated *Boletus edulis* fruiting bodies. These umami peptides have been toxicologically verified to be non-toxic and can be safely used in the development of natural seasonings. This invention not only provides the market with a natural, safe, and efficient monosodium glutamate (MSG) substitute derived from rare edible fungi, but also resolves the contradiction of *Boletus edulis* being "delicious but with safety concerns," allowing its flavor to be enjoyed safely and in a standardized manner year-round.

[0020] 2) The umami-active peptides from Boletus edulis fruiting bodies, the method for obtaining them, and their applications provided in this application show that the binding energy of the obtained umami peptides to hT1R1 / T1R3 umami receptors can reach as low as -9.286 kcal / mol; the IDPVHSR taste threshold is only 0.11 mmol / L; when the concentration of the umami peptide solution is 0.25 mg / mL, the umami intensity of PDGGY can reach 3, which is much higher than the umami intensity of MSG (0.075). This indicates that the umami peptides can efficiently express umami at a low concentration, which is beneficial to reducing the amount of umami ingested by the human body.

[0021] 3) The umami-active peptides of Boletus edulis fruiting bodies, the method of obtaining them and their applications provided in this application show that when the total concentration of the umami peptide solution is 0.25 mg / mL, the umami intensity can be improved when the two obtained umami peptides are combined in pairs. This indicates that the umami intensity can be effectively improved when the two obtained umami peptides are combined in pairs without increasing the concentration, indicating that the umami peptides have a good synergistic effect. Attached Figure Description

[0022] Figure 1 This application provides a diagram showing the molecular docking results of IDPVHSR and hT1R1 / T1R3 umami receptor obtained in Example 2; Figure 2 This application provides a diagram showing the molecular docking results of PRDNF and hT1R1 / T1R3 umami receptors obtained in Example 2; Figure 3 This application provides a diagram showing the molecular docking results of PGDGF obtained in Example 2 with the hT1R1 / T1R3 umami receptor; Figure 4 This application provides a diagram showing the molecular docking results of PDGGY obtained in Example 2 with the hT1R1 / T1R3 umami receptor; Figure 5 This application provides a secondary mass spectrum of the IDPVHSR obtained in Example 2, obtained by LC-MS / MS. Figure 6 This application provides a secondary mass spectrum of the PRDNF obtained in Example 2 using LC-MS / MS. Figure 7 This application provides a secondary mass spectrum of PGDGF obtained in Example 2 by LC-MS / MS. Figure 8 This application provides a secondary mass spectrum of PDGGY obtained in Example 2 by LC-MS / MS. Figure 9 The structural diagram of the hT1R1 / T1R3 umami receptor used in this application is provided below; Figure 10 La-plot for the evaluation of the hT1R1 / T1R3 umami receptor model used in this application; Figure 11 This application provides a mass spectrum of the IDPVHSR synthetic peptide obtained in Example 2; Figure 12 Mass spectra of the PRDNF synthetic peptide obtained in Example 2 are provided for this application; Figure 13 Mass spectra of the PGDGF synthetic peptide obtained in Example 2 are provided for this application; Figure 14 Mass spectra of the PDGGY synthetic peptide obtained in Example 2 are provided for this application; Figure 15 This application provides an electronic tongue detection result image of the umami intensity of the synthetic umami peptides and monosodium glutamate obtained in Example 4; Figure 16 This application provides an electronic tongue detection result diagram of the synergistic effect between the umami peptides obtained in Example 4. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0024] Unless otherwise specified, all materials and instruments used in the following embodiments were obtained through commercial channels; and all detection methods used are existing methods unless otherwise specified.

[0025] Example 1: Preparation of Enzymatic Hydrolysate Fresh Boletus edulis (hereinafter referred to as Boletus) were purchased from the Shuimuhua Edible Fungus Trading Market, dried, and then pulverized for 20 minutes using an ultra-micro pulverizer to obtain pulverized material. Water was added at a mass ratio of 1:20 between the pulverized material and water. Neutral protease, flavor protease, and papain were selected for enzymatic hydrolysis of Boletus edulis. The addition amounts of each enzyme (based on the mass fraction of the Boletus edulis raw material used) were 0.60%, 0.90%, and 4.70%, respectively, and were added simultaneously. Enzymatic hydrolysis was carried out at pH 7.0 and 50℃ for 1.5 hours to obtain the enzymatic hydrolysate.

[0026] The obtained enzymatic hydrolysate had an amino acid nitrogen content of 124.01 mg / 100 mL, a total free amino acid content of 12.33 mg / g, a total flavor nucleotide content of 96.48 μg / g, a total organic acid content of 4511.59 μg / g, and an EUC value of 2.46 g MSG / 100 g. After freeze-drying, the obtained enzymatic hydrolysate was freeze-dried into a powder containing Boletus edulis fruiting body enzymatic hydrolysate.

[0027] Example 2: Preparation of Enzymatic Hydrolysate The peptides in the enzymatic hydrolysate were extracted, reduced and alkylated, desalted, and then detected by LC-MS / MS mass spectrometry to identify the peptide sequence. This identification process was commissioned to Beijing Baitaipaike Biotechnology Co., Ltd., and the project number of the obtained result is BTP-SSS-20241104-02.

[0028] 1. Peptide Extraction: Accurately weigh 10 mg of the lyophilized hydrolysate of *Boletus edulis* fruiting body and dissolve it in 1 mL of ultrapure water using a pipette to obtain a lyophilized powder suspension. Add 1000 μL of the lyophilized powder suspension to a 5 kDa ultrafiltration tube and centrifuge at 12000 g for 10 minutes, collecting the fraction smaller than 5 kDa (Separate A). Add Separate A to a 3 kDa ultrafiltration tube and centrifuge at 12000 g for 10 minutes, collecting the 3-5 kDa fraction (Separate B) and the fraction smaller than 3 kDa (Separate C). Add the fraction smaller than 3 kDa (Separate C) to a 1 kDa ultrafiltration tube and centrifuge at 12000 g for 10 minutes, collecting the 1-3 kDa fraction (Separate D) and the fraction smaller than 1 kDa (Separate F).

[0029] 2. Reductive alkylation: 1) Take all collected isolates A-F and add them to a final volume of 100 μL with ultrapure water. 2) Accurately pipette 1 μL of 1M DTT solution into each sample solution to achieve a final DTT concentration of 10 mmol / L, and reduce in a 56℃ water bath for 1 h. 3) Accurately pipette 2 μL of 1M IAM solution into each sample to achieve a final IAM concentration of 20 mmol / L, and react in the dark at room temperature for 40 min. 4) Accurately pipette 2 μL of 1M DTT solution into each sample to achieve a final DTT concentration of 20 mmol / L to neutralize unreacted IAM. Desalt using a C18 stage-tip and vacuum dry at 45℃ to obtain the test samples of each isolate. Separate each test sample by liquid chromatography. Analyze the eluting components by mass spectrometry to obtain the raw data. Search the software database to obtain the identification results.

[0030] Liquid chromatography conditions: Analytical column: 150 μm × 170 mm, packing: Reprosil-Pur120C18-AQ 1.9 μm; Mobile phase A: 0.1% FA; Mobile phase B: 0.1% FA, 80% ACN; Flow rate: 600 nL / min; Analysis time for each component: 66 min; Specific chromatographic conditions are shown in Table 1. Table 1 Chromatographic conditions

[0031] Mass spectrometry conditions: Primary mass spectrometry parameters: 1) Resolution: 70,000; 2) AGC target: 3e6; 3) Maximum IT: 100 ms; 4) Scan range: 100 to 1500 m / z; Secondary mass spectrometry parameters: 1) Resolution: 17,500; 2) AGC target: 1e5; 3) Maximum IT: 50ms; 4) Top N: 20; 5) NCE / stepped NCE: 28; Raw data were obtained after mass spectrometry acquisition.

[0032] The obtained raw mass spectrometry files were searched using software databases to obtain identification results. Search parameters were: 1) Fixed modifications: Carbamidomethyl (C); 2) Variable modifications: Oxidation (M), Acetyl (Peptide N-term); 3) Enzyme: Nonspecific; 4) Database: uniprotkb_Boletus; 5) Primary mass spectrometry bias (Peptide Mass Tolerance): 20 ppm; 6) Secondary mass spectrometry bias (Fragment Mass Tolerance): 0.02 Da. The final screening revealed that IDPVHSR (CDS NO.1), PRDNF (CDS NO.2), PGDGF (CDS NO.3), and PDGGY (CDS NO.4) all originated from isolate F, whose molecular weight is less than 1 kDa. The secondary mass spectra of IDPVHSR, PRDNF, PGDGF, and PDGGY are shown below. Figures 5-8 As shown.

[0033] Models were trained based on reported peptide information and related properties, and the activity of identified peptides was predicted using the model. Umami prediction was performed using the UmamiCNN model (provided by Beijing Baitaipaike Biotechnology Co., Ltd.), which yielded 1,504 peptides with umami activity that had not been previously reported. Of these, 594 peptides had a length of 3 ≤ 10 and contained both aspartic acid and glutamic acid. The toxicity of these 594 peptides was predicted using the ToxinPred database. The results showed that 593 peptides were non-toxic. After cross-validation using UMPred-FRL (http: / / pmlabstack.pythonanywhere.com / UMPred-FRL) and TastepeptidesDM (http: / / tastepeptides-meta.com / TPDM), 252 peptides exhibited umami activity, and 238 peptides showed good water solubility. Molecular docking was then performed; lower docking energy resulted in higher binding affinity and better docking performance. Peptides with binding energies below -9.0 kcal / mol were selected, revealing that IDPVHSR, PRDNF, PGDGF, and PDGGY all possessed umami characteristics, good water solubility, and were non-toxic. The predicted results of the screened peptides are shown in Table 2. Prediction results of the screened peptides in Table 2

[0034] "1" represents the umami prediction score of the UMPED-FRL model (threshold > 0.5); "2" represents the confidence probability in the TastepeptidesDM model's judgment. In the umami judgment, the higher the Pro_Umamir value, the higher the model's confidence in that judgment.

[0035] The amino acid sequences encoding the obtained umami peptides are: IDPVHSR, PRDNF, PGDGF, and PDGGY. The structural formulas of each umami peptide are as follows: IDPVHSR:

[0036] PRDNF:

[0037] PGDGF:

[0038] PDGGY:

[0039] The molecular weights of IDPVHSR, PRDNF, PGDGF, and PDGGY were determined by mass spectrometry to be 822.90 Da, 647.68 Da, 491.49 Da, and 507.49 Da, respectively. The molecular weights of the obtained peptides were all below 1000, while peptides with molecular weights in the range of 500–1000 Da provided a more intense umami effect.

[0040] Example 3: Molecular docking technology verifies umami characteristics 1. Homology Modeling of Human Umami Receptors: Amino acid sequences of umami receptors T1R1 (ID: Q7RTX1) and T1R3 (ID: Q7RTX0) were retrieved from the UniProt database, and fish taste receptors T1R2a / T1R3 (ID: 5X2Q, 39% identity) were downloaded from the PDB database. Using T1R1 (ID: Q7RTX1), T1R3 (ID: Q7RTX0), and T1R2a / T1R3 (ID: 5X2Q) as templates, homology modeling of human umami receptors (hT1R1 / T1R3) was performed on the Swiss-Model platform. The hT1R1 / T1R3 model was evaluated on the SAVESv6.0 platform. The structure of the hT1R1 / T1R3 umami receptor is shown below. Figure 9 As shown, the Laplace plot of the model evaluation of the hT1R1 / T1R3 umami receptor is as follows: Figure 10 As shown, the optimal allowed region in the hT1R1 / T1R3 umami receptor model is 89.3%, and the additional allowed region is 9.1%. More than 90% of the amino acid residues are distributed in the optimal allowed region and the additional allowed region, indicating that the model is reasonable.

[0041] 2. Peptide Structure Construction: The IDPVHSR, PRDNF, PGDGF, and PDGGY peptides were constructed using chemDraw and chem3D software. The resulting umami peptide structures are shown below. Figures 1-4 As shown.

[0042] 3. Molecular docking: IDPVHSR, PRDNF, PGDGF, and PDGGY peptides were molecularly docked with the hT1R1 / T1R3 umami receptor to verify their flavor characteristics. The conformation with the lowest binding energy was selected for further analysis. PyMOL was used to visualize the molecular docking results. The molecular docking results are shown in Table 3. Figures 1-4 As shown.

[0043] Table 3. Amino acid residue analysis of umami peptides binding to hT1R1 / T1R3 umami receptors.

[0044] Molecular docking results show that the binding energies of the umami peptides provided by this invention to the key active sites of umami receptors are all less than -9.0 kcal / mol, which is significantly lower than that of the prior art. This means that they have higher affinity for the receptor and a more stable binding conformation, indicating that the obtained umami peptides have a lower sensory threshold and a higher flavor intensity.

[0045] The results above show that, in practical applications, the amount of each umami peptide provided in this application can achieve or even exceed the umami enhancement effect of traditional umami agents (such as monosodium glutamate) or other umami peptides with a lower addition amount. This not only reduces the cost of use, but also helps to reduce the intake of umami agents in the human body, and provides core technical support for the development of 'low-additive, all-natural, high-intensity' flavor formulas.

[0046] Example 4: Electronic tongue verification of the flavor characteristics of solid-phase synthesized umami peptides 1. Synthesis of umami peptides Umami peptides IDPVHSR, PRDNF, PGDGF, and PDGGY were synthesized using solid-phase synthesis (contracted to Sangon Biotech (Shanghai) Co., Ltd.), with a purity greater than 98% and after desalting. The synthesized peptides were identified as target peptides by mass spectrometry analysis. The mass spectra of IDPVHSR, PRDNF, PGDGF, and PDGGY are shown below. Figures 11-14 As shown. After mass spectrometry acquisition, software analysis was used to extract peptide information and obtain the molecular weight of each peptide.

[0047] 2. Umami Peptide Electronic Tongue Detection Using deionized water, the peptides obtained from solid-phase synthesis were prepared into 0.25 mg / mL solutions of *Boletus edulis* umami peptides using ultrapure water as the solvent, resulting in IDPVHSR, PRDNF, PGDGF, and PDGGY umami peptide solutions. A 0.25 mg / mL monosodium glutamate (MSG) aqueous solution was prepared using the same method as a umami control. The umami intensity of the synthetic peptide solutions and MSG aqueous solution (all sample solutions used in the experiment were 0.25 mg / mL, 20 ± 1℃) was measured using the Shanghai Baosheng cTongue electronic tongue. The results are shown below. Figure 15 And Table 4.

[0048] The umami intensity of the umami peptides obtained in Table 4

[0049] As shown above, when the concentration of the test solution is 0.25 mg / mL, the umami intensity of each synthetic peptide solution is 3 times that of monosodium glutamate (MSG).

[0050] 3. Taste threshold measurement Nine members (5 men and 4 women) with professional sensory training were selected to form an evaluation team to conduct sensory evaluation of the taste thresholds of synthetic peptides and monosodium glutamate (MSG). Using ultrapure water as the solvent, MSG and each synthetic peptide were prepared as test samples at concentrations of 0.50 mg / ml, 0.25 mg / ml, 0.125 mg / ml, and 0.0625 mg / ml. During testing, each team member numbered the test samples sequentially from lowest to highest concentration (1, 2, 3…, i-1,…i+1,…, 20), tasted each sample in turn, and drank the same volume of deionized water after each test to compare the difference between the consumed sample and the deionized water. When all team members could distinguish the i-th sample from the blank group (deionized water), but could not distinguish the i-1-th sample from the blank group, the average concentration of the i-th and i-1-th samples was used. The average concentration value was then converted into the taste threshold of the synthetic peptide or MSG sample using the following formula: .

[0051] The average molecular weight of monosodium glutamate (MSG) is 187.13 Da; the results are listed in Table 5.

[0052] Table 5. Taste threshold of umami peptides from Boletus edulis.

[0053] As shown in Table 5, the taste threshold of the obtained synthetic peptides is 0.11~0.36 mmol / L, which is lower than that of MSG (1.60 mmol / L). The taste threshold of MSG is 4~5 times that of the synthetic peptides, indicating that the peptides IDPVHSR, PRDNF, PGDGF, and PDGGY provided by this invention have a stronger umami flavor than MSG. When the human body ingests only 1 / 4 of the amount of MSG, it can obtain the taste of umami. The peptides provided by this invention are beneficial to reducing the intake of umami flavoring substances.

[0054] 4. Synergistic effect between synthetic umami peptides Four synthetic umami peptides were combined in pairs at a mass ratio of 1:1 as solutes, and deionized water was used as the solvent to prepare solutions with a total solute concentration of 0.25 mg / mL. Monosodium glutamate (MSG) was used as a solute, and a solution with a total solute concentration of 0.25 mg / mL was prepared as a control. The umami intensity of each sample solution was measured using the Shanghai Baosheng cTongue electronic tongue. The results are shown in Table 6 and... Figure 16 As shown.

[0055] Table 6. Synthetic umami peptides of pairwise combinations and umami intensity of the control group.

[0056] As shown in Table 6, the umami intensity of the synthetic umami peptide solution obtained by combining two peptides at a concentration of 0.25 mg / mL was 3 to 4 times that of the control group monosodium glutamate aqueous solution. Therefore, combining any two of the four peptides can exert a synergistic effect and effectively improve the umami intensity of the peptides.

[0057] Example 5: Flavored Food 1. Making plant-based meat patties: The ingredients and quantities are as follows: 80g soy protein isolate, 70g pea protein, 100g textured protein, 30g gluten, 10g konjac powder, 2g carrageenan, 40g coconut oil, 30g palm oil, 15g yeast extract, 5g onion powder, 3g garlic powder, 3g salt, 5g white sugar, 1g baking soda, and 200mL ice water. To make the patties, first soak the textured protein in warm water and drain. Mix the soy protein isolate and pea protein with ice water to form a paste. Then emulsify the oil and yeast extract, and mix them evenly with the protein paste and gluten. Divide the mixture into 5 portions, press each portion into a 1cm thick round patty, refrigerate for 30 minutes to set, and finally pan-fry over medium heat for 3-4 minutes per side or bake at 200℃ for 15 minutes to cook. Quick-freeze for sterilization and storage.

[0058] 2. The synthetic umami peptides obtained in Example 4 were compounded in a mass ratio of 1:1:1:1 to obtain a compound. Three equal-weight plant-based meat patties were taken, and the compound (experimental group), monosodium glutamate (control), and no additive (blank) were added respectively at a weight of 0.5% of the plant-based meat patty mass. After steaming for 30 minutes, cooked plant-based meat patties were obtained. Sensory evaluation was carried out on the three groups of samples. The evaluation results are shown in Table 7.

[0059] Table 7 Sensory Evaluation Results of Flavored Foods

[0060] As shown in the table above, the MSG group and the compound umami peptide group exhibited enhanced umami flavor compared to the control group. However, the plant-based meat patties obtained from the compound umami peptide group had a stronger umami flavor and enhanced meat aroma. The umami enhancement effect of the MSG group was not as good as that of the compound umami peptide group, and it also resulted in a bitter taste. This indicates that the umami peptide has a better umami enhancement effect.

[0061] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of umami-active peptide from the fruiting body of *Boletus edulis*, characterized in that, The encoded amino acid sequences are IDPVHSR, PRDNF, PGDGF, and PDGGY; when any two of the umami-active peptides are combined, the umami intensity of the resulting aqueous solution is enhanced.

2. The active peptide according to claim 1, characterized in that, IDPVHSR structure: PRDNF structure: ; PGDGF structure: ;PDGGY structure: ; The molecular weight of IDPVHSR is 822.90 Da; The molecular weight of PRDNF is 647.68 Da; The molecular weight of PGDGF is 491.49 Da; The molecular weight of PDGGY is 507.49 Da.

3. The active peptide according to claim 1, characterized in that, The binding energy of IDPVHSR to the hT1R1 / T1R3 umami receptor is -9.09 kcal / mol; The binding energy of PRDNF to the hT1R1 / T1R3 umami receptor is -9.286 kcal / mol; The binding energy of PGDGF to the hT1R1 / T1R3 umami receptor is -9.211 kcal / mol; The binding energy of PDGGY to the hT1R1 / T1R3 umami receptor is -9.175 kcal / mol.

4. The active peptide according to claim 1, characterized in that, When the concentration of the obtained umami peptide solution was 0.25 mg / mL, the umami intensity of IDPVHSR was 3; the umami intensity of PRDNF was 3; the umami intensity of PGDGF was 3; and the umami intensity of PDGGY was 3. The taste thresholds for IDPVHSR, PRDNF, PGDGF, and PDGGY were 0.11 mmol / L, 0.36 mmol / L, 0.32 mmol / L, and 0.20 mmol / L, respectively.

5. A method for obtaining umami-active peptides from the fruiting bodies of Boletus edulis, characterized in that, Includes the following steps: Fresh Boletus edulis was dried, pulverized, and mixed with water. Neutral protease, flavor protease, and papain were added, and the mixture was enzymatically hydrolyzed to obtain an enzymatic hydrolysate. The hydrolysate was freeze-dried and subjected to peptide extraction, reductive alkylation, and desalting. After screening by LC-MS / MS mass spectrometry, the umami-active peptides of Boletus edulis fruiting bodies as described in any one of claims 1 to 4 were isolated from the fraction F with a content less than 1 kDa.

6. The method according to claim 5, characterized in that, The preparation of the enzymatic hydrolysate includes the following steps: the pulverized material obtained by pulverization is mixed with water at a mass ratio of 1:20 to obtain a mixed solution, and 0.60% of neutral protease, 0.90% of flavor protease, and 4.70% of papain by mass of the mixed solution are added respectively. The enzyme was hydrolyzed at pH 7.0 and 50°C for 1.5 h to obtain the enzymatic hydrolysate.

7. The method according to claim 5, characterized in that, Peptide extraction includes the following steps: After the enzymatic hydrolysate is freeze-dried, it is mixed with water to obtain a freeze-dried powder suspension. 1000 μL of the freeze-dried powder suspension is added to a 5 kDa ultrafiltration tube and centrifuged at 12000 g for 10 minutes. The fraction A with a weight of less than 5 kDa is collected. Add the fraction A to a 3kDa ultrafiltration tube and centrifuge at 12000g for 10 minutes. Collect the fraction B (3-5kDa) and the fraction C (less than 3kDa). Take the fraction C (less than 3 kDa) and add it to a 1 kDa ultrafiltration tube. Centrifuge at 12000 g for 10 minutes and collect the fraction D (1-3 kDa) and the fraction F (less than 1 kDa).

8. The method according to claim 5, characterized in that, The obtained enzymatic hydrolysate had an amino acid nitrogen content of 124.01 mg / 100 mL, a total free amino acid content of 12.33 mg / g, a total flavor nucleotide content of 96.48 μg / g, a total organic acid content of 4511.59 μg / g, and an EUC value of 2.46 g MSG / 100 g.

9. The application of a flavor-active peptide from the fruiting body of *Boletus edulis* as described in any one of claims 1 to 4, characterized in that, Add umami-active peptides from Boletus edulis fruiting bodies to flavored foods; the umami-active peptides from Boletus edulis fruiting bodies are prepared according to the method described in any one of claims 5 to 8.

10. The application according to claim 9, characterized in that, The flavored foods include seasonings and / or plant-based meats.

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