Defective matsutake fruiting body umami peptide, acquisition method and application thereof

Abstract

This application discloses an umami peptide from defective matsutake fruiting bodies, a method for obtaining it, and its application, relating to the technical field of umami peptides. The peptide comprises LPVDP, PLTDP, PAPLDP, PGAPGGPP, and GFPGGPG; its structural formula is shown below: #imgabs0##imgabs1##imgabs2##imgabs3##imgabs4#. The umami peptide, isolated from defective matsutake fruiting bodies, binds to hT1R1 / T1R3 umami receptors, exhibits good water solubility, and is predictably non-toxic. At equivalent concentrations, its umami value is higher than that of a monosodium glutamate aqueous solution, and its umami value increases more rapidly with concentration than monosodium glutamate.

Description

Technical Field

[0001] The present application relates to the technical field of umami peptides, in particular to an umami peptide from defective matsutake fruiting bodies, an acquisition method and applications thereof. Background Art

[0002] Edible fungi are delicious and rich in protein, making them an important source of natural peptides and a current focus of umami peptide research. In recent years, condiments made with matsutake extract as a base, such as matsutake seasoning, matsutake soy sauce, and matsutake seasoning sauce, have become increasingly popular among consumers, demonstrating that matsutake is an important source of umami substances. Mature matsutake fruiting bodies have strong metabolic activity, open quickly, and are prone to aging and deterioration. Furthermore, as the degree of opening increases, their commercial value, nutritional value, and taste are all affected. Existing methods for processing matsutake mushrooms that have opened, as defective mushrooms, utilize flavor extraction technology to prepare a matsutake umami peptide base for use as a food flavoring. This approach increases the added value of defective mushrooms, reduces production costs, and minimizes resource waste.

[0003] In recent years, condiments based on matsutake extract have become increasingly popular among consumers. However, most products suffer from simple processing techniques and low quality, failing to fully and effectively utilize the natural flavor of matsutake. Furthermore, the main active ingredients in existing matsutake extracts are unclear, leading to inconsistent evaluation standards and difficulty in accurately identifying extracts with high freshness.

[0004] With the continuous development of peptidomics, many studies have extracted flavor peptides from edible fungi through peptidomics methods and applied them to the development of mushroom seasonings. However, as an important source of umami substances, there have been no research reports on related umami peptides of Matsutake.

[0005] The information disclosed in the background section is only intended to enhance understanding of the overall background of the invention and should not be considered as an admission or any form of suggestion that the information constitutes the prior art already known to a person of ordinary skill in the art. Summary of the Invention

[0006] In response to the above technical problems, the present application provides a defective matsutake fruiting body umami peptide, an acquisition method and its application.

[0007] The present application provides a defective pine mushroom fruiting body umami peptide, including: LPVDP, PLTDP, PAPLDP, PGAPGGPP, GFPGGPG; the structural formula is as follows:

[0008]

[0009]

[0010]

[0011]

[0012] .

[0013] The molecular weights of LPVDP, PLTDP, PAPLDP, and PGAPGGPP are 539.62, 541.59, 608.68, and 648.71, respectively; the molecular weight of GFPGGPG is 587.62. LPVDP, PLTDP, PAPLDP, and PGAPGGPP have low molecular weights, which are easy to perceive and easily absorbed by the body, thus helping consumers to get a stronger taste experience at the same usage amount.

[0014] Another aspect of the present application further provides a method for obtaining the above-mentioned defective matsutake fruiting body umami peptide, comprising the following steps:

[0015] The peptides in the freeze-dried powder of the enzymatic hydrolysate of the defective fruiting bodies of Tricholoma matsutake were detected by LC-MS / MS. The detection data were searched using PEAKS software, and a total of 948 Tricholoma matsutake peptides were obtained.

[0016] The UmamiCNN model was used to predict the umami properties of matsutake peptide segments, and the defective matsutake fruiting body umami peptides that could dock with the hT1R1 / T1R3 umami receptor molecules were screened.

[0017] Preferably, the preparation method of the freeze-dried powder of the enzymatic hydrolyzate of the fruiting body of the defective matsutake mushroom comprises: selecting defective matsutake mushrooms, removing impurities, drying, and then grinding into ultrafine powder with a particle size of 10 to 25 μm, dissolving the obtained ultrafine powder in water, and sequentially performing segmented enzymatic hydrolysis using a neutral protease and a flavor protease to obtain an enzymatic hydrolyzate of the fruiting body of the defective matsutake mushroom, and freeze-drying the enzymatic hydrolyzate of the fruiting body of the defective matsutake mushroom to obtain the freeze-dried powder of the enzymatic hydrolyzate of the fruiting body of the defective matsutake mushroom;

[0018] The equivalent umami concentration of the obtained enzymatic hydrolyzate of the defective matsutake mushroom fruiting body is greater than or equal to 3.110±0.046 gMSG / 100g.

[0019] Preferably, the neutral protease enzymatic hydrolysis method comprises: mixing ultrafine powder and water at a material-liquid volume ratio of 1:10, adding neutral protease to the resulting mixture, and performing enzymatic hydrolysis to obtain a first enzymatic hydrolysis solution; the amount of neutral protease added is 4000-6000 U / g; the enzymatic hydrolysis temperature is 50-60° C.; the pH of the enzymatic hydrolysis solution is 6.5-7.5; and the enzymatic hydrolysis time is 1-2 h;

[0020] Flavor protease enzymatic hydrolysis method: flavor protease is added to the mixture obtained from the first enzymatic hydrolysis solution, and enzymatic hydrolysis is performed to obtain an enzymatic hydrolysis solution of defective matsutake mushroom fruiting bodies; the amount of flavor protease added is 4000~6000 U / g; the enzymatic hydrolysis temperature is 50~60℃; the pH of the enzymatic hydrolysis solution is 6.5~7.5, and the enzymatic hydrolysis time is 4~5 hours.

[0021] Preferably, before LC-MS / MS detection, the freeze-dried powder of the enzymatic hydrolysate of the defective Matsutake mushroom fruiting body is subjected to ultrafiltration and sample pretreatment to obtain a pretreated sample.

[0022] Preferably, the ultrafiltration step is as follows: weigh 10 mg of freeze-dried powder of the enzymatic hydrolysate of defective Matsutake mushroom fruiting bodies, add 1 mL of ultrapure water and shake to dissolve for 1 min, add to a 5KD ultrafiltration tube, centrifuge at 12000× g for 10 min, and collect samples with less than 5KD;

[0023] The collected samples with a mass less than 5 KD were added to 1 mL of ultrapure water and shaken for 1 min to dissolve, then added to a 3 KD ultrafiltration tube and centrifuged at 12,000 × g for 10 min to collect samples with a mass greater than 3 KD to obtain 3-5 KD samples.

[0024] The collected samples less than 3 KD were added to 1 mL of ultrapure water and shaken for 1 min to dissolve, then added to a 1 KD ultrafiltration tube and centrifuged at 12000 × g for 10 min to collect samples greater than 1 KD and samples less than 1 KD, respectively, to obtain 1-3 KD samples and samples less than 1 KD;

[0025] The defective pine mushroom fruiting body umami peptides LPVDP, PLTDP, PAPLDP, PGAPGGPP, and GFPGGPG all come from samples with a molecular weight of less than 1KD.

[0026] Preferably, the sample pretreatment step comprises: performing reductive alkylation on a sample with a molecular weight of 3-5KD, a sample with a molecular weight of 1-3KD, and a sample with a molecular weight of less than 1KD, and performing C18 desalting followed by vacuum drying at 45° C. to obtain a pretreated sample;

[0027] The steps of reductive alkylation are as follows: take 100 μL of sample, add 1 μL of 1M DTT solution to make the final DTT concentration 10 mmol / L, and reduce in a 56°C water bath for 1 h to obtain a reducing solution;

[0028] 2 μL of 1 M IMA solution was added to the resulting reduction solution to make the final IMA concentration 20 mmol / L. The mixture was reacted in the dark at room temperature for 40 min to obtain a reaction solution.

[0029] 1 μL of 1M DTT was then added to the reaction solution to neutralize the unreacted IMA, so that the final DTT concentration was 10 mmol / L to obtain a reductive alkylation solution.

[0030] Another aspect of the present application also provides the use of the above-mentioned defective pine mushroom fruiting body umami peptide or the defective pine mushroom fruiting body umami peptide prepared by the above-mentioned acquisition method in the preparation of condiments and / or plant meat.

[0031] The specific application method is to add it according to conventional methods in the field, and the addition ratio refers to the amount used in existing flavor enhancers such as monosodium glutamate. Because this substance is derived from matsutake mushrooms, which are edible and non-toxic, it is safe and non-toxic when added to food at this concentration.

[0032] Another aspect of the present application provides a flavored food, the active ingredients of which include the above-mentioned defective pine mushroom fruiting body umami peptide or the defective pine mushroom fruiting body umami peptide prepared by the above-mentioned acquisition method; the flavored food includes seasonings and / or plant meat.

[0033] The beneficial effects of this application include:

[0034] 1) This application provides umami peptides from defective matsutake fruiting bodies, methods for obtaining them, and their applications. The umami peptides isolated from defective matsutake fruiting bodies bind to hT1R1 / T1R3 umami receptors, exhibit good water solubility, and are predictably non-toxic. At equivalent concentrations, their umami values ​​are higher than those of aqueous monosodium glutamate solutions, and their umami value increases more rapidly with increasing concentration than that of monosodium glutamate. The resulting umami peptides can be used as a monosodium glutamate substitute, as an umami base for preparing matsutake seasonings, or as a flavor enhancer added to other seasonings to enhance their umami flavor.

[0035] 2) The umami peptides from defective matsutake fruiting bodies, their extraction methods, and their applications are provided in this application. The resulting matsutake umami peptides effectively bind to hT1R1 / T1R3 umami receptors with a binding energy of up to -8.731 kcal / mol and four hydrogen bonds. They exhibit low molecular weight, good water solubility, non-toxicity, and a pronounced umami flavor. Electronic tongue testing revealed that a 1.00 mg / mL aqueous solution had an umami value of 3.123, significantly higher than that of monosodium glutamate at the same concentration. The peptides can be used in the preparation of matsutake seasonings, as a substitute for monosodium glutamate, and as a flavor enhancer for other seasonings, potentially broadly applicable to a variety of foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the mass spectrum of LPVDP obtained by screening in Example 2 provided in this application;

[0037] Figure 2 This is the mass spectrum of PLTDP obtained by screening in Example 2 provided in this application;

[0038] Figure 3 This is the mass spectrum of PAPLDP obtained by screening in Example 2 provided in this application;

[0039] Figure 4 This is the mass spectrum of PGAPGGPP obtained by screening in Example 2 provided in this application;

[0040] Figure 5 This is the mass spectrum of GFPGGPG obtained by screening in Example 2 provided in this application;

[0041] Figure 6 This is a diagram of the structure of the hT1R1 / T1R3 umami taste receptor provided in this application; the colored parts in the figure constitute the umami taste receptor;

[0042] Figure 7 A pull-through diagram for the model evaluation of the hT1R1 / T1R3 umami taste receptor provided in this application;

[0043] Figure 8 This is the structure diagram of the umami peptide in Example 2 provided in this application;

[0044] Figure 9 The molecular docking results of LPVDP, PLTDP, PAPLDP, PGAPGGPP, and GFPGGPG in Example 2 provided in this application with hT1R1 / T1R3 umami receptors, respectively, Figure 9 A is the molecular docking result of LPVDP and hT1R1 / T1R3 umami taste receptor; Figure 9 B is the molecular docking result of PLTDP and hT1R1 / T1R3 umami taste receptor; Figure 9 C is the molecular docking result of PAPLDP and hT1R1 / T1R3 umami taste receptor; Figure 9 D is the molecular docking result of PGAPGGPP and hT1R1 / T1R3 umami taste receptor; Figure 9 E is the molecular docking result of GFPGGPG and hT1R1 / T1R3 umami taste receptor;

[0045] Figure 10 This is the mass spectrum of LPVDP in Example 3 provided in this application;

[0046] Figure 11 The mass spectrum of PLTDP in Example 3 provided in this application;

[0047] Figure 12 This is the mass spectrum of PAPLDP in Example 3 provided in this application;

[0048] Figure 13 This is the mass spectrum of PGAPGGPP in Example 3 provided in this application. DETAILED DESCRIPTION

[0049] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the scope of protection of the present invention.

[0050] Unless otherwise specified, the materials and instruments used in the following examples were obtained from commercial channels; the detection methods used were all existing methods unless otherwise specified.

[0051] Example 1 Preparation of Matsutake Enzyme Hydrolysate

[0052] Defective matsutake mushrooms, such as the shimeji mushroom, were selected, cleaned, dried, and then ground into ultrafine powder with a particle size of 10-25 μm. A matsutake hydrolysate was prepared by segmented enzymatic hydrolysis using neutral protease and flavor protease. The preparation process involved mixing the ultrafine powder with water at a material-to-liquid ratio of 1:10 by volume. Neutral protease was added to the resulting mixture and enzymatic hydrolysis was performed to obtain the first hydrolysate. The amount of neutral protease added was 5000 U / g. The hydrolysis temperature was 55°C, the pH of the hydrolysate was 7.0, and the hydrolysis time was 1.5 h.

[0053] Then, flavor protease was added to the mixture obtained from the first enzymatic hydrolyzate for enzymatic hydrolysis to obtain a matsutake mushroom defective fruiting body enzymatic hydrolyzate. The amount of flavor protease added was 5000 U / g, the enzymatic hydrolysis temperature was 55°C, the pH of the enzymatic hydrolyzate solution was 7.0, and the enzymatic hydrolysis time was 4.5 h to obtain a matsutake mushroom enzymatic hydrolyzate.

[0054] The free amino acid content was determined using an automatic amino acid analyzer in accordance with the National Food Safety Standard Determination of Amino Acids in Food GB5009.124-2016.

[0055] The 5'-nucleotide content was determined by referring to the 5'-nucleotide determination method disclosed in "Dai Qiuqiong, Sun Dafeng, Hua Rong, et al. Establishment of UHPLC detection method for 6 nucleotides in edible fungi [J]. Chinese Edible Fungi, 2023, 42(05): 58-61."

[0056] The peptide content was determined using the peptide content detection kit from Suzhou Mengxi Biopharmaceutical Technology Co., Ltd.;

[0057] The peptide content, free amino acid content, and 5'-nucleotide content in the Matsutake hydrolysate were determined. Under these conditions, the peptide content, free amino acid content, and 5'-nucleotide content of the Matsutake hydrolysate obtained were 6.464±0.115 mg / mL, 8.089±0.124 mg / mL, and 126.12±1.89 ug / mL, respectively. The equivalent umami concentration ( ) value was 3.110 ± 0.046 gMSG / 100g. In the formula for equivalent umami concentration, ai is the mass of the umami amino acid (glutamic acid or aspartic acid) in g / 100g; aj is the mass of the flavor nucleotide (5'-AMP, 5'-IMP, 5'-GMP) in g / 100mL; bi is the value of the umami amino acid relative to glutamic acid (glutamic acid = 1.00, aspartic acid = 0.077); bj is the value of the flavor nucleotide relative to 5'-IMP (5'-IMP = 1.00, 5'-AMP = 0.18, 5'-GMP = 2.30); and 1218 is the synergistic effect constant.

[0058] The obtained enzymatic hydrolysate is freeze-dried to obtain freeze-dried powder, which is collected and stored for future use.

[0059] Example 2 Isolation and Identification of Umami Peptides from Matsutake Hydrolysate

[0060] (1) The specific steps of ultrafiltration are as follows: accurately weigh 10 mg of lyophilized powder of the enzymatic hydrolysate, add 1 mL of ultrapure water and shake to dissolve for 1 min, add to a 5KD ultrafiltration tube, centrifuge at 12000× g for 10 min, and collect samples with a molecular weight less than 5KD; add 1 mL of ultrapure water to dissolve the collected samples with a molecular weight less than 5KD for 1 min, add to a 3KD ultrafiltration tube, centrifuge at 12000× g for 10 min, and collect samples with a molecular weight greater than 3KD to obtain samples with a molecular weight of 3~5KD;

[0061] The collected samples less than 3 kD were added to 1 mL of ultrapure water and shaken for 1 min to dissolve. The samples were then added to a 1 kD ultrafiltration tube and centrifuged at 12,000 × g for 10 min. The samples greater than 1 kD and less than 1 kD were collected separately to obtain samples with a molecular weight of 1-3 kD and less than 1 kD, respectively. Samples with molecular weights of 3-5 kD, 1-3 kD, and less than 1 kD were collected.

[0062] (2) Sample pretreatment. Each sample was subjected to reductive alkylation and C18 desalting, then dried in vacuo at 45°C for later use. The specific steps are as follows: 100 μL of sample was added with 1 μL of 1M DTT solution to a final DTT concentration of 10 mmol / L, and reduced in a 56°C water bath for 1 h; 2 μL of 1M IMA solution was added to a final IMA concentration of 20 mmol / L, and the reaction was carried out at room temperature in the dark for 40 min; 1 μL of 1M DTT was further added to neutralize the unreacted IMA to a final DTT concentration of 10 mmol / L. After the reductive alkylation treatment, desalting was performed using a C18 column to obtain the pretreated sample.

[0063] (3) LC-MS / MS peptide sequence. After the pre-treatment sample was dissolved in 0.1% formic acid, the supernatant was taken for detection. Liquid chromatography mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid, 80% acetonitrile; flow rate: 600 nL / min, and the analysis time for each component was 66 min. The chromatographic conditions are shown in Table 1. The chromatographic conditions were used to detect samples with molecular weights of 3~5KD, 1~3KD, and less than 1KD, respectively. The detection data were searched using PEAKS software, and a total of 948 matsutake peptide segments were obtained, and the mass spectra of each matsutake peptide segment were obtained;

[0064] The protein database search parameters are shown in Table 2.

[0065] Table 1 Chromatographic conditions

[0066]

[0067] Table 2 Target protein database search parameters

[0068]

[0069] Screening of umami peptides from matsutake enzymatic hydrolysate: The UmamiCNN model was used to predict the umami taste of 948 matsutake peptides, and the potential umami peptides screened out included: LPVDP (LC-MS / MS secondary mass spectrometry as shown in Figure 2). Figure 1 As shown, the molecular formula is shown below), PLTDP (LC-MS / MS secondary mass spectrum is shown below Figure 2 , molecular formula is shown below), PAPLDP (LC-MS / MS secondary mass spectrum is shown below Figure 3 , molecular formula is shown below), PGAPGGPP (LC-MS / MS secondary mass spectrum is shown below Figure 4 , molecular formula is shown below), GFPGGPG (LC-MS / MS secondary mass spectrum is shown below Figure 5 , the molecular formula is shown below).

[0070]

[0071]

[0072]

[0073]

[0074] .

[0075] The screened LPVDP, PLTDP, PAPLDP, PGAPGGPP, and GFPGGPG all originated from samples with a molecular weight of less than 1KD.

[0076] The flavor, water solubility, and toxicity of the candidate peptides were predicted using the BIOPEP-UWM database, the Proteomics tools on the INNOVAGEN peptidomics website, and the ToxinPred website. All five peptides were found to have umami properties, good water solubility, and no toxicity.

[0077] (4) Molecular docking technology verifies the umami characteristics. The specific steps are as follows:

[0078] (4.1) Homology modeling of human umami taste receptor. The amino acid sequences of T1R1 (ID: Q7RTX1) and T1R3 (ID: Q7RTX0) were obtained from UniProtKB, and the fish taste receptor T1R2a / T1R3 (ID: 5X2Q, with a 39% identity) was downloaded from the PDB database. Using T1R1 (ID: Q7RTX1), T1R3 (ID: Q7RTX0) and T1R2a / T1R3 (ID: 5X2Q) as templates, homology modeling of human umami taste receptor (hT1R1 / T1R3) was performed on the Swiss-Model platform. The hT1R1 / T1R3 model was evaluated on the SAVES v6.0 platform. The structure of hT1R1 / T1R3 umami receptor is shown in the figure below. Figure 6 As shown, the model evaluation pull-through diagram of hT1R1 / T1R3 umami taste receptor is as follows Figure 7 The hT1R1 / T1R3 umami taste receptor model has an optimally allowed region of 89.2% and an additionally allowed region of 9.3%. More than 90% of the amino acid residues are distributed in the optimally allowed and additionally allowed regions, indicating that the model is reasonable.

[0079] (4.2) Peptide structure construction. 2D and 3D structures of five peptides, LPVDP, PLTDP, PAPLDP, PGAPGGPP, and GFPGGPG, were constructed using chemDraw and chem3D software. The resulting umami peptide structures are shown in the figure below. Figure 8 shown.

[0080] Molecular docking. The five peptides LPVDP, PLTDP, PAPLDP, PGAPGGPP, and GFPGGPG were docked with the hT1R1 / T1R3 umami receptor to verify their flavor properties. The conformation with the lowest binding energy was selected for further analysis. The molecular docking results were visualized and analyzed using PyMOL. The molecular docking results are shown in Table 3. Figure 9 The molecular docking results of LPVDP and hT1R1 / T1R3 umami receptor are shown in Figure 9 As shown in A; the molecular docking results of PLTDP and hT1R1 / T1R3 umami receptor are shown in Figure 9 As shown in B; the molecular docking results of PAPLDP and hT1R1 / T1R3 umami receptor are shown in Figure 9 C; the molecular docking results of PGAPGGPP and hT1R1 / T1R3 umami receptor are shown in Figure 9 D; the molecular docking results of GFPGGPG and hT1R1 / T1R3 umami receptor are shown in Figure 9 As shown in E.

[0081] The results showed that all five peptides could bind well to hT1R1 / T1R3 umami taste receptors. LPVDP formed five hydrogen bonds of different lengths with hT1R1 / T1R3, with a binding energy of -8.731 kcal / mol. The bound amino acid residues were Val-105, Asp-147, Ser-148, and Arg-227.

[0082] PLTDP forms three hydrogen bonds of different lengths with hT1R1 / T1R3, with a binding energy of -7.721 kcal / mol, and the bound amino acid residues are Asn-69, Leu-51, and Ser-148; PAPLDP forms five hydrogen bonds of different lengths with hT1R1 / T1R3, with a binding energy of -7.298 kcal / mol, and the bound amino acid residues are Gln-368, Ser-59, and Lys-111;

[0083] PGAPGGPP forms four hydrogen bonds of different lengths with hT1R1 / T1R3, with a binding energy of -9.353 kcal / mol, and the bound amino acid residues are Asp-218, Ser-276, and His-71;

[0084] GFPGGPG forms one hydrogen bond with hT1R1 / T1R3 with a binding energy of -6.425 kcal / mol, and the bound amino acid residue is Ser-59.

[0085] The above results indicate that the preferred peptides LPVDP, PLTDP, PAPLDP, PGAPGGPP, and GFPGGPG exhibit umami flavor properties. However, since the binding energy of GFPGGPG to the hT1R1 / T1R3 umami receptor is greater than -7 and there is only one binding site, it was not tested using the electronic tongue.

[0086] Table 3 Amino acid residue analysis of umami peptides binding to hT1R1 / T1R3 umami receptors

[0087]

[0088] Example 3 Electronic tongue verification of taste characteristics

[0089] 1. Umami peptide synthesis

[0090] Solid phase synthesis (commissioned by Sangon Biotech (Shanghai) Co., Ltd.) was used to synthesize four pine mushroom flavor peptides, LPVDP, PLTDP, PAPLDP, and PGAPGGPP, which have low binding energy and a large number of binding sites. The purity is greater than 98%. The synthesized peptides were confirmed to be target peptides by mass spectrometry analysis. The mass spectrometry analysis results are as follows Figures 10-13 shown.

[0091] 2. Preparation of umami peptide solution and electronic tongue detection

[0092] Using ultrapure water, the peptide fragments obtained by solid-phase synthesis were prepared into solutions of 0.25, 0.50, and 1.00 mg / mL of Matsutake umami peptide. Aqueous monosodium glutamate solutions were prepared using the same method at concentrations of 0.25, 0.50, and 1.00 mg / mL as umami controls.

[0093] The umami values ​​of the synthetic peptide solution and the MSG aqueous solution were measured using the Shanghai Baosheng cTongue4 electronic tongue. The results are shown in Table 4.

[0094] As can be seen from Table 4, at the same concentration, the umami values ​​of the peptide solutions of LPVDP, PLTDP, PAPLDP, and PGAPGGPP are 2.11 to 2.81 times that of the MSG aqueous solution, and within the test concentration range, the umami value increases with increasing concentration, while the increase in umami value is limited when the MSG concentration increases. This demonstrates that the peptides LPVDP, PLTDP, PAPLDP, and PGAPGGPP described in the present invention have a strong umami taste.

[0095] Table 4 Umami value of Matsutake umami peptides

[0096]

[0097] Example 4 Application of Matsutake Tasty Peptide

[0098] From the above results, it can be seen that the four umami peptides of Matsutake provided by the present invention have strong umami properties, good water solubility and non-toxicity. They can be used as substitutes for MSG, as umami bases for preparing Matsutake seasonings, or added to other seasonings to increase the umami taste.

[0099] The obtained pine mushroom flavor peptides LPVDP, PLTDP, PAPLDP, and PGAPGGPP were added to the ham sausage at an amount of 1% of the total mass of the ham sausage raw materials, and used as additives for the ham sausage to produce ham sausage containing pine mushroom flavor peptides LPVDP, PLTDP, PAPLDP, and PGAPGGPP.

[0100] The obtained pine mushroom flavor peptides LPVDP, PLTDP, PAPLDP, and PGAPGGPP were added into the soy milk, the raw material for making dried tofu, at an addition amount of 1% of the total mass of the dried tofu raw material, and used as additives for dried tofu to produce dried tofu containing pine mushroom flavor peptides LPVDP, PLTDP, PAPLDP, and PGAPGGPP.

[0101] Example 5

[0102] The difference from Example 1 is:

[0103] Neutral protease enzymatic hydrolysis method: neutral protease was added to the obtained mixture, and the amount of neutral protease added was 4000 U / g; the enzymatic hydrolysis temperature was 50°C; the pH of the enzymatic hydrolysis solution was 6.5; the enzymatic hydrolysis time was 2 h;

[0104] Flavor protease enzymatic hydrolysis method: flavor protease was added to the obtained mixture, the added amount of flavor protease was 4000 U / g; the enzymatic hydrolysis temperature was 50°C; the pH of the enzymatic hydrolysis solution was 6.5, and the enzymatic hydrolysis time was 4 h.

[0105] Example 6

[0106] The difference from Example 1 is:

[0107] Neutral protease enzymatic hydrolysis method: ultrafine powder and water were mixed at a material-liquid volume ratio of 1:10, and neutral protease was added to the resulting mixture at an amount of 6000 U / g. The enzymatic hydrolysis temperature was 60°C, the pH of the enzymatic hydrolysis solution was 7.5, and the enzymatic hydrolysis time was 1 h.

[0108] Flavor protease enzymatic hydrolysis method: flavor protease was added to the obtained mixture, the added amount of flavor protease was 6000 U / g; the enzymatic hydrolysis temperature was 60°C; the pH of the enzymatic hydrolysis solution was 7.5, and the enzymatic hydrolysis time was 5 h.

[0109] Example 7

[0110] The difference from Example 1 is:

[0111] The matsutake hydrolysate obtained by enzymatic hydrolysis with flavor protease is cooled in running water and then placed in a 4°C environment for cooling. Low temperature can inhibit enzyme activity and is not easy to destroy flavor substances such as amino acids and nucleotides, which can better preserve the flavor substances in the hydrolysate.

[0112] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A defective pine mushroom fruiting body umami peptide, characterized in that: The encoded amino acid sequence is LPVDP, PLTDP, PGAPGGPP; the structural formula is shown in the figure below: 。 2. The method for obtaining umami peptides from defective Tricholoma matsutake fruiting bodies according to claim 1, characterized in that: The following steps are involved: Select defective matsutake mushrooms, remove impurities, dry them, and grind them into ultrafine powder with a particle size of 10-25 μm. The obtained ultrafine powder is dissolved in water and then subjected to segmented enzymolysis using neutral protease and flavor protease in sequence to obtain an enzymatic hydrolyzate of the defective fruiting body of Tricholoma matsutake mushroom. The enzymatic hydrolyzate of the defective fruiting body of Tricholoma matsutake mushroom is freeze-dried to obtain freeze-dried powder of the enzymatic hydrolyzate of the defective fruiting body of Tricholoma matsutake mushroom. The equivalent umami concentration of the obtained enzymatic hydrolyzed solution of the defective matsutake mushroom fruiting body is greater than or equal to 3.110±0.046 gMSG / 100g; Neutral protease enzymatic hydrolysis method: ultrafine powder and water are mixed at a material-liquid volume ratio of 1:10, neutral protease is added to the resulting mixture, and enzymatic hydrolysis is performed to obtain a first enzymatic hydrolysis solution; the amount of neutral protease added is 4000-6000 U / g; the enzymatic hydrolysis temperature is 50-60°C; the pH of the enzymatic hydrolysis solution is 6.5-7.5; and the enzymatic hydrolysis time is 1-2 hours; Flavor protease enzymatic hydrolysis method: flavor protease is added to the first enzymatic hydrolysis solution for enzymatic hydrolysis to obtain a matsutake mushroom fruiting body enzymatic hydrolysis solution; the amount of flavor protease added is 4000-6000 U / g; the enzymatic hydrolysis temperature is 50-60°C; the pH of the enzymatic hydrolysis solution is 6.5-7.5, and the enzymatic hydrolysis time is 4-5 hours; The peptides in the freeze-dried powder of the enzymatic hydrolysate of the defective fruiting bodies of Tricholoma matsutake were detected by LC-MS / MS, and the detection data were searched using PEAKS software, and a total of 948 Tricholoma matsutake peptides were obtained; The UmamiCNN model was used to predict the umami properties of matsutake peptides, and the umami peptides of the defective matsutake fruiting bodies that could dock with the hT1R1 / T1R3 umami receptor molecules were screened; The defective mushrooms selected are umbrella mushrooms.

3. The method according to claim 2, characterized in that Before LC-MS / MS detection, the freeze-dried powder of the enzymatic hydrolysate of the defective Matsutake mushroom fruiting body was subjected to ultrafiltration and sample pretreatment to obtain a pre-treated sample.

4. The method according to claim 3, characterized in that The ultrafiltration steps are as follows: weigh 10 mg of lyophilized powder of the enzymatic hydrolysate of defective Matsutake fruiting bodies, add 1 mL of ultrapure water and shake to dissolve for 1 min, add to a 5KD ultrafiltration tube, centrifuge at 12000× g for 10 min, and collect samples with less than 5KD; The collected samples with a mass less than 5 KD were added to 1 mL of ultrapure water and shaken for 1 min to dissolve, then added to a 3 KD ultrafiltration tube and centrifuged at 12,000 × g for 10 min to collect samples with a mass greater than 3 KD to obtain 3-5 KD samples. The collected samples less than 3 KD were added to 1 mL of ultrapure water and shaken for 1 min to dissolve, then added to a 1 KD ultrafiltration tube and centrifuged at 12000 × g for 10 min to collect samples greater than 1 KD and samples less than 1 KD, respectively, to obtain 1-3 KD samples and samples less than 1 KD; The defective matsutake fruiting body umami peptides LPVDP, PLTDP, and PGAPGGPP all come from samples with a molecular weight of less than 1KD.

5. The method according to claim 3, characterized in that The sample pretreatment steps are as follows: reductive alkylation of samples with a molecular weight of 3-5 KD, samples with a molecular weight of 1-3 KD, and samples with a molecular weight of less than 1 KD, C18 desalting, and vacuum drying at 45°C to obtain pretreated samples; The steps of reductive alkylation were as follows: 100 μL of sample was taken, 1 μL of 1 M DTT solution was added to make the final DTT concentration 10 mmol / L, and the sample was reduced in a 56°C water bath for 1 h to obtain a reducing solution; 2 μL of 1 M IMA solution was added to the resulting reduction solution to make the final IMA concentration 20 mmol / L. The mixture was reacted in the dark at room temperature for 40 min to obtain a reaction solution. 1 μL of 1M DTT was then added to the reaction solution to neutralize the unreacted IMA, so that the final DTT concentration was 10 mmol / L to obtain a reductive alkylation solution.

6. Use of the defective matsutake fruiting body umami peptide according to claim 1 or the defective matsutake fruiting body umami peptide prepared by the acquisition method according to any one of claims 2 to 5 in the preparation of condiments and / or vegetable meat.

7. A flavored food, characterized in that: The active ingredients of the flavored food include: the umami peptide from the defective matsutake fruiting body according to claim 1 or the umami peptide from the defective matsutake fruiting body prepared by the acquisition method according to any one of claims 2 to 5; the flavored food includes condiments and / or plant meat.

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