An ACE inhibitory peptide from tricholoma matsutake and a preparation method and application thereof

Abstract

The application discloses an ACE inhibitory peptide from Tricholoma matsutake, and a preparation method and application thereof, relates to the field of biotechnology, and establishes an integrated method of double enzymolysis-ultrafiltration fractionation-LC-MS / MS identification-bioinformatics and molecular docking simulation screening, realizes efficient and directional screening and identification of a novel ACE inhibitory peptide from Tricholoma matsutake. The ACE inhibitory peptide from Tricholoma matsutake with a clear sequence, high activity and excellent properties is screened through the method, the biological source of the ACE inhibitory peptide is expanded, the deep development of the protein resource of the precious edible fungus Tricholoma matsutake is realized, and the high-value utilization way of Tricholoma matsutake is expanded.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to an ACE-inhibiting active peptide derived from matsutake mushrooms, its preparation method, and its application. Background Technology

[0002] Hypertension is one of the leading risk factors for cardiovascular disease worldwide, seriously endangering human health. Angiotensin-converting enzyme (ACE) is a key enzyme in the renin-angiotensin system, its function being to convert angiotensin I into angiotensin II, the latter being a potent vasoconstrictor that raises blood pressure. Furthermore, ACE can degrade bradykinin, which has a hypotensive effect, thereby weakening vasodilatory function. By inhibiting ACE activity, the production level of angiotensin II can be effectively reduced, exerting a hypotensive effect. Therefore, the development of safe and effective ACE inhibitors has always been a hot topic in hypertension prevention and treatment research. Although several synthetic ACE inhibitors are currently available on the market, such as captopril and lisinopril, long-term use may cause side effects such as dry cough, rash, and thrombocytopenia. Therefore, researchers have begun to focus on finding food-derived ACE-inhibiting peptides from food or natural products. These bioactive peptides typically have advantages such as fewer side effects, good tolerability, and easy absorption by the gastrointestinal tract, and are widely used in the development of functional foods and nutritional supplements.

[0003] Edible fungi, as a natural food resource rich in protein and polysaccharides, have seen rapid development in research on their blood pressure-lowering bioactive peptides, despite a relatively late start globally. Studies have shown that edible fungi are rich in various bioactive peptides, some of which have significant inhibitory effects on ACE. The earliest report on ACE-inhibiting peptides from fungi dates back to 2005, when researchers found that aqueous extracts of edible fungi such as Grifola frondosa could inhibit ACE by up to 58.7%. Since then, research has gradually expanded to include various other edible fungi such as shiitake, oyster mushrooms, and enoki mushrooms. Currently, developing new sources of ACE-inhibiting bioactive peptides remains a key focus in this field. Summary of the Invention

[0004] The purpose of this invention is to provide an ACE-inhibiting active peptide derived from matsutake mushrooms, its preparation method, and its application. The aim is to establish an efficient and systematic screening and preparation method to extract highly active ACE-inhibiting peptides from matsutake mushroom raw materials, broaden the sources of ACE-inhibiting peptides, and improve the practical value of matsutake mushrooms.

[0005] The embodiments of the present invention are implemented as follows: An ACE-inhibiting peptide derived from matsutake mushroom, wherein the amino acid sequence of the ACE-inhibiting peptide is any one of SEQ ID NO.1 to SEQ ID NO.3.

[0006] A method for preparing the above-mentioned ACE-inhibiting active peptide, comprising: S1. The matsutake mushroom raw material is subjected to segmental enzymatic hydrolysis with neutral protease and flavor protease to obtain matsutake mushroom hydrolysate; S2. The matsutake mushroom hydrolysate is subjected to ultrafiltration separation, and the components with a molecular weight of less than 3 kDa are collected. After identification and bioinformatics screening, the ACE inhibitory active peptide is obtained.

[0007] The application of the above-mentioned ACE-inhibiting active peptide in the preparation of a product for inhibiting angiotensin-converting enzyme.

[0008] The beneficial effects of the embodiments of the present invention are: This invention provides an ACE-inhibiting peptide derived from matsutake mushrooms, its preparation method, and its applications. An integrated method was established, comprising dual enzymatic hydrolysis, ultrafiltration fractionation, LC-MS / MS identification, and bioinformatics and molecular docking simulation screening. This method enables efficient and targeted screening and identification of novel ACE-inhibiting peptides from matsutake mushrooms. This invention, through this method, screened matsutake-derived ACE-inhibiting peptides with well-defined sequences, high activity, and excellent properties. This not only expands the biological sources of ACE-inhibiting peptides but also enables the in-depth development of matsutake mushroom protein resources, a valuable edible fungus, and broadens the avenues for high-value-added utilization of matsutake mushrooms. Attached Figure Description

[0009] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0010] Figure 1 The molecular formula of PDGLP, PGLPP, and VGLP, derived from matsutake mushrooms, is provided in the embodiments of the present invention. Figure 2 The docking results of PDGLP (A), PGLPP (B), and VGLP (C) ACE inhibitory active peptides derived from matsutake mushroom with ACE receptor molecules provided in the embodiments of the present invention; Figure 3 The inhibition curve of PDGLP, an ACE-inhibiting active peptide derived from matsutake mushroom, on ACE is provided as an experimental example of the present invention. Figure 4 The inhibition curve of ACE by PGLPP, an ACE-inhibiting active peptide derived from matsutake mushroom, provided in an embodiment of the present invention; Figure 5 The curve shows the inhibition of ACE by captopril. Detailed Implementation

[0011] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0012] The following is a detailed description of an ACE-inhibiting active peptide derived from matsutake mushroom, its preparation method, and its application, according to an embodiment of the present invention.

[0013] This invention provides an ACE-inhibiting peptide derived from matsutake mushrooms, wherein the amino acid sequence of the ACE-inhibiting peptide is any one of SEQ ID NO.1 to SEQ ID NO.3.

[0014] Specifically, the details of SEQ ID NO.1 to SEQ ID NO.3 are as follows: SEQ ID NO.1: Pro-Asp-Gly-Leu-Pro; SEQ ID NO.2: Pro-Gly-Leu-Pro-Pro; SEQ ID NO. 3: Val-Gly-Leu-Pro.

[0015] The three ACE-inhibiting active peptides mentioned above were named PDGLP, PGLPP, and VGLP, respectively.

[0016] The three ACE-inhibiting peptides were constructed in 2D and 3D, and after energy minimization, their structures are as follows: Figure 1 As shown.

[0017] This invention also provides a method for preparing the above-mentioned ACE-inhibiting active peptide, comprising: S1. The matsutake mushroom raw material is subjected to segmental enzymatic hydrolysis with neutral protease and flavor protease to obtain matsutake mushroom hydrolysate.

[0018] S2. The matsutake mushroom hydrolysate was separated by ultrafiltration, and the components with a molecular weight of less than 3 kDa were collected. After identification and bioinformatics screening, ACE inhibitory active peptides were obtained.

[0019] Furthermore, the matsutake mushroom raw material is matsutake ultrafine powder obtained by pulverizing matsutake mushrooms. Before pulverizing, the matsutake mushrooms are first washed to remove impurities and dried. The particle size of the matsutake ultrafine powder is 10~25μm. Making matsutake ultrafine powder can significantly increase the specific surface area of ​​the raw material and destroy the cell structure, allowing the enzyme and substrate to come into more complete contact, thereby greatly improving the enzymatic hydrolysis efficiency and the yield of target peptides.

[0020] Optionally, step S1 includes: S11. The matsutake mushroom raw material is enzymatically hydrolyzed with neutral protease at pH 6.5~7.5 and temperature 50~60℃ for 1~2 hours to obtain a preliminary hydrolysate; S12. The preliminary enzymatic hydrolysate is enzymatically hydrolyzed with flavor protease at pH 6.5-7.5 and temperature 50-60℃ for 4-5 hours.

[0021] The above reaction conditions can achieve optimal enzymatic hydrolysis efficiency, maximize the release of target active peptides, and ensure process stability and reproducibility.

[0022] Furthermore, the addition amounts of neutral protease and flavor protease are both 4000-6000 U / g. Within this range, sufficient enzymatic hydrolysis can be ensured while avoiding incomplete hydrolysis due to insufficient enzyme dosage or cost waste due to excessive use, thus achieving the effect of optimizing process economy and ensuring a balance between product yield and activity.

[0023] Furthermore, the peptide content in the matsutake mushroom enzymatic hydrolysate is 6-7 mg / mL. By limiting the peptide content, a rapid and objective assessment can be made as to whether the aforementioned enzymatic hydrolysis process has achieved the expected results. This is also crucial for ensuring subsequent separation efficiency, batch-to-batch stability, and the final product yield.

[0024] Further, in step S2, the matsutake mushroom hydrolysate is ultrafiltered using an ultrafiltration tube with a molecular weight cutoff of 3 kDa. Subsequently, the fraction with a molecular weight less than 3 kDa is further separated and purified by LC-MS / MS to obtain the target peptide. Preferably, ultrafiltration tubes with molecular weight cutoffs of 5 kDa, 3 kDa, and 1 kDa can be used. The samples are centrifuged at a relative centrifugal force of 12000 × g for 10 min to separately collect samples greater than 5 kDa, 3–5 kDa, 1–3 kDa, and less than 1 kDa, thereby reducing the difficulty of subsequent separation. The centrifuged samples are then subjected to reductive alkylation, C18 desalting, and vacuum dried at 45°C for later use.

[0025] Furthermore, bioinformatics screening of the target peptides is performed, including: The ACE inhibitory activity of target peptides was predicted using a bioactive peptide database, and at least one of the following properties—stability, digestibility, sensitization, and toxicity—was evaluated using online tools. Peptides with good activity, high stability, non-toxicity, non-sensitization, and good digestibility were screened.

[0026] This invention also provides an application of the above-mentioned ACE-inhibiting active peptide in the preparation of a product for inhibiting angiotensin-converting enzyme.

[0027] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0028] Example

[0029] This embodiment provides an ACE-inhibiting active peptide derived from matsutake mushrooms, wherein the amino acid sequence of the ACE-inhibiting active peptide is any one of SEQ ID NO.1 to SEQ ID NO.3.

[0030] Specifically, the details of SEQ ID NO.1 to SEQ ID NO.3 are as follows: SEQ ID NO.1: Pro-Asp-Gly-Leu-Pro; SEQ ID NO.2: Pro-Gly-Leu-Pro-Pro; SEQ ID NO. 3: Val-Gly-Leu-Pro.

[0031] The three ACE-inhibiting active peptides mentioned above were named PDGLP, PGLPP, and VGLP, respectively.

[0032] Its preparation method is as follows: S1. Preparation of Matsutake mushroom enzymatic hydrolysate: After impurities are removed and the mushrooms are dried, substandard matsutake mushrooms are pulverized into ultrafine powder with a particle size of 10-25 μm. Matsutake enzymatic hydrolysate is prepared by fractional enzymatic hydrolysis using neutral protease and flavor protease. The preparation process is as follows: material-to-liquid ratio 1:10, neutral protease addition 5000 U / g, hydrolysis temperature 55℃, pH 7.0, and hydrolysis time 1.5 h.

[0033] Then, flavor protease hydrolysis was performed at a dosage of 5000 U / g, a hydrolysis temperature of 55℃, a pH of 7.0, and a hydrolysis time of 4.5 h. Under these conditions, the peptide content of the hydrolysate was 6.464 ± 0.115 mg / mL. The hydrolysate was collected and stored after freeze-drying for later use.

[0034] S2. Isolation and identification of ACE inhibitory peptides: (1) Using ultrafiltration tubes with molecular weight cutoffs of 5kDa, 3kDa, and 1kDa, centrifuge at a relative centrifugal force of 12000×g for 10 min, and collect samples with molecular weight cutoffs greater than 5kDa, 3~5kDa, 1~3kDa, and less than 1kDa separately. After reduction alkylation and C18 desalting, the samples are dried under vacuum at 45℃ for later use.

[0035] (2) Peptide sequence identification was performed using LC-MS / MS. Mobile phase A: 0.1% formic acid; Mobile phase B: 0.1% formic acid, 80% acetonitrile; Flow rate: 600 nL / min; Analysis time for each component: 66 min. The percentage of mobile phase B at different times (min) is as follows: At 0 min, mobile phase B accounted for 4%; at 2 min, mobile phase B accounted for 8%; at 45 min, mobile phase B accounted for 28%; at 55 min, mobile phase B accounted for 40%; at 56 min, mobile phase B accounted for 95%; and at 66 min, mobile phase B accounted for 95%.

[0036] The detection data was retrieved using PEAKS software, yielding a total of 908 peptide fragments.

[0037] (3) Peptides with molecular weight less than 3kDa were preferred. Based on the BIO-UWM database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php) to predict ACE inhibitory activity, 26 peptides with potential ACE activity and good activity were screened out.

[0038] For the above 26 peptides, peptide comparison was performed based on the DFBP website. Peptides that have been reported were excluded, resulting in 23 peptides with potential ACE inhibitory activity that have not been reported.

[0039] The stability, digestibility, sensitization, and toxicity of the peptides were assessed using online tools such as PLifepred (https: / / webs.iiitd.edu.in / raghava / plifepred / ), PeptideCutter (https: / / www.expasy.org / resources / peptidecutter), Algpred 2 (https: / / webs.iiitd.edu.in / raghava / algpred2 / algo.html), and Toxin Pred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html). Three peptides were ultimately selected as candidates: PDGLP (Pro-Asp-Gly-Leu-Pro), PGLPP (Pro-Gly-Leu-Pro-Pro), and VGLP (Val-Gly-Leu-Pro). Specific information on these three peptides is shown in Table 1.

[0040] Table 1. Information on candidate peptides PDGLP 497.54 835.11 non non good PGLPP 479.57 834.61 non non good VGLP 384.47 824.51 non non good In the table above, stability was assessed using the PLifepred online tool. Peptides with a half-life > 800 s were considered to have good stability in the blood. Toxicity was assessed using the Toxin Pred online tool, with "non" indicating no toxicity. Sensitization was assessed using the Algpred 2 online tool, with "non" indicating no risk of sensitization. Digestibility was assessed using the PeptideCutter online tool. If the amino acid sequence of the peptide does not contain sites that can be cleaved by pepsin, chymotrypsin, and trypsin, the peptide is considered to have the potential to resist gastrointestinal digestion.

[0041] Experimental Example 1 This experimental example uses the ACE-inhibiting peptides PDGLP, PGLPP, and VGLP provided in the examples. Molecular docking technology is used to verify the ACE-inhibiting activity of the candidate peptides. The specific steps are as follows: The receptor protein structure of angiotensin-converting enzyme (ACE) (PDB ID: 1O86) was downloaded from the PDB database (https: / / www.rcsb.org). After pretreatment with PyMOL, the receptor protein was used for molecular docking studies with three candidate peptides: PDGLP, PGLPP, and VGLP. The molecular docking results were visualized and analyzed using PyMOL and Discovery Studio software. The molecular docking results are shown in Table 2. Figure 2 As shown.

[0042] Table 2. Amino acid residue analysis of ACE inhibitory peptides binding to ACE receptors

[0043] From Table 2 and Figure 2 It can be seen that the binding energies of the three peptides PDGLP, PGLPP, and VGLP to the ACE receptor protein are all less than -7 kcal / mol, specifically -8.199 kcal / mol, -8.38 kcal / mol, and -7.623 kcal / mol, respectively, indicating that the candidate peptides have strong binding activity to the receptor protein.

[0044] Among them, PDGLP and PGLPP have relatively low binding energies, indicating stronger potential ACE inhibitory activity. PDGLP forms 5 hydrogen bonds with the ACE receptor protein, binding to amino acid residues including GLU 376, ASN 277, THR 282, and GLN 281; PGLPP forms 4 hydrogen bonds with the ACE receptor protein, binding to amino acid residues including TYR 360, ASN 66, and ALA 356; VGLP forms 5 hydrogen bonds with the ACE receptor protein, binding to amino acid residues including GLU 411, TYR 523, ALA 354, and GLU 384.

[0045] Experimental Example 2 The inhibition rate of the ACE-inhibiting active peptides PDGLP and PGLPP (purity greater than 98%) provided in the examples was detected using an angiotensin-converting enzyme inhibitor activity assay kit (ACE inhibitor activity assay kit, Beijing Solarbio Science & Technology Co., Ltd.). The specific operation is as follows: PDGLP and PGLPP powders were prepared into solutions of 2.0 mM, 1.0 mM, 0.10 mM, 0.05 mM, 0.025 mM, and 0.0125 mM. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) -7 mM, 1×10 -6 mM, 1×10 -5 mM, 1×10 -4 mM, 1×10 -3 Using captopril at mM, 0.01mM, and 0.1mM as positive controls, the results were as follows: Figures 3-5 As shown. The formula for calculating the ACE inhibition rate is as follows:

[0046] Among them: A a This represents the decrease in absorbance at 340 nm for the blank control group; A b This represents the decrease in absorbance of the sample group at 340 nm.

[0047] As shown in the figure, the IC50 value of PDGLP against ACE is 0.1750 mM, and the IC50 value of PGLPP against ACE is 0.0253 mM. Although these values ​​do not reach the inhibitory level of captopril (IC50 = 1.314 × 10⁻⁶ mM), they are still lower than the levels observed with captopril. -5 While the molecular weights of PDGLP and PGLPP are 497.54 and 479.57 respectively, their lower molecular weights make them more easily absorbed by the human body, showing promising application prospects in the preparation of products for inhibiting angiotensin-converting enzyme.

[0048] In summary, this invention provides an ACE-inhibiting peptide derived from matsutake mushrooms, its preparation method, and its applications. It establishes an integrated method encompassing dual enzymatic hydrolysis, ultrafiltration fractionation, LC-MS / MS identification, and bioinformatics and molecular docking simulation screening, enabling efficient and targeted screening and identification of novel ACE-inhibiting peptides from matsutake mushrooms. This invention, through this method, screened matsutake-derived ACE-inhibiting peptides with well-defined sequences, high activity, and excellent properties. This not only expands the biological sources of ACE-inhibiting peptides but also enables the in-depth development of matsutake mushroom protein resources, a valuable edible fungus, and broadens the avenues for high-value-added utilization of matsutake mushrooms.

[0049] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An ACE-inhibiting peptide derived from matsutake mushroom, characterized in that, The amino acid sequence of the ACE-inhibiting active peptide is SEQ ID NO.1 or SEQ ID NO.

2.

2. A method for preparing the ACE-inhibiting active peptide as described in claim 1, characterized in that, include: S1. The matsutake mushroom raw material is subjected to segmental enzymatic hydrolysis with neutral protease and flavor protease to obtain matsutake mushroom hydrolysate; S2. The matsutake mushroom hydrolysate is subjected to ultrafiltration separation, and the components with a molecular weight of less than 3 kDa are collected. After identification and bioinformatics screening, the ACE inhibitory active peptide is obtained.

3. The preparation method according to claim 2, characterized in that, Step S1 includes: S11. The matsutake mushroom raw material is enzymatically hydrolyzed with the neutral protease at pH 6.5-7.5 and temperature 50-60℃ for 1-2 hours to obtain a preliminary hydrolysate; S12. The preliminary enzymatic hydrolysate is enzymatically hydrolyzed with the flavor protease at pH 6.5-7.5 and temperature 50-60℃ for 4-5 hours.

4. The preparation method according to claim 3, characterized in that, The matsutake mushroom raw material is matsutake ultrafine powder obtained by crushing matsutake mushrooms, and the particle size of the matsutake ultrafine powder is 10~25μm.

5. The preparation method according to claim 2, characterized in that, Based on the mass of the matsutake mushroom raw material, the amount of neutral protease and flavor protease added is 4000-6000 U / g.

6. The preparation method according to claim 2, characterized in that, The peptide content in the matsutake mushroom hydrolysate is 6-7 mg / mL.

7. The preparation method according to claim 2, characterized in that, In step S2, the matsutake enzymatic hydrolysate is separated by ultrafiltration using an ultrafiltration tube with a molecular weight cutoff of 3 kDa. Subsequently, the components with a molecular weight less than 3 kDa are further separated and purified by LC-MS / MS to obtain the target peptide.

8. The preparation method according to claim 7, characterized in that, Bioinformatics screening of the target peptide includes: The ACE inhibitory activity of the target peptide was predicted using a bioactive peptide database, and the stability, digestibility, sensitization, and toxicity of the target peptide were evaluated using online tools.

9. The use of the ACE-inhibiting active peptide as described in claim 1 in the preparation of antihypertensive products.

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