Polypeptide as well as preparation and application thereof

Abstract

The invention discloses a polypeptide as well as preparation and application thereof, and belongs to the technical field of active peptides. According to the present invention, three active peptides are prepared from walnut meal protein, the amino acid sequences of the three polypeptides are respectively Leuu-Pro-Gln-Phe (LPQF), Leuu-Pro-Ser-Phe (LPSF) or Vla-Pro-Phe-Pro (VPFP), and the detection results show that the three polypeptides have inhibition activity on dipeptidyl peptidase-IV (DPP-IV), such that the three polypeptides can be used for preparing the blood sugar reducing drug.

Description

Technical Field

[0001] The invention relates to a polypeptide and a preparation and application thereof, belonging to the technical field of active peptides. Background Art

[0002] Diabetes is one of the most common and fatal chronic diseases in the world. It is reported that 537 million adults have diabetes in 2021, and this number is expected to increase to 643 million by 2030 and 783 million by 2045, of which type 2 diabetes accounts for about 90% of diabetes cases. Synthetic hypoglycemic drugs currently used in clinical practice (such as saxagliptin and sitagliptin, etc.) have been shown to have many negative effects, such as weight gain, cardiovascular problems, hypoglycemia and other adverse reactions. Dipeptidyl peptidase-IV (DPP-IV) inhibitors are the latest treatment for type 2 diabetes.

[0003] DPP-IV is a metabolic enzyme distributed in human tissues, responsible for the cleavage and inactivation of the intestinal insulinotropic hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), resulting in a decrease in endogenous GLP-1 and GIP levels, inhibiting the release of insulin from pancreatic β cells, while promoting the secretion of glucagon from pancreatic α cells, that is, inhibiting insulin levels and increasing blood sugar levels in the body. Therefore, inhibiting the activity of DPP-IV can prevent the degradation of GLP-1 and GIP to a certain extent, thereby better regulating blood sugar. In this context, some food-derived protein bioactive peptides with strong DPP-IV inhibitory activity are considered to be safe means to prevent and control the occurrence and development of diabetes. Therefore, the development of food-derived protein bioactive peptides to inhibit DPP-IV and prevent and control the occurrence and development of diabetes has become a global research hotspot.

[0004] Walnut is one of the four major nuts in the world. It has extremely high nutritional value and has always been considered the best food for warming the lungs and kidneys, promoting heart and brain health, and strengthening the body. Walnut kernels are rich in oil, and the content of unsaturated fatty acids in the oil is high, which is an important material basis for the economic benefits of walnut industrialization. A large amount of walnut meal is produced in the production of walnut oil. Walnut meal has a high protein content and is a rich source of bioactive peptides. A small part of it is developed into walnut protein powder, but most of it is still used as animal feed. Whether walnut meal can be effectively developed is a new economic benefit point for the walnut industry.

[0005] Peptides are defined as fragments of proteins, usually composed of 2-20 amino acid residues, and have multiple biological activities. The potential of bioactive peptides in preventing chronic diseases, especially diabetes, is particularly recognized. Bioactive peptides in food sources generally refer to two categories, one is bioactive peptides that can effectively promote body functions or states to improve human health, and the other is bioactive peptides that can give food a savory and mellow taste. Therefore, walnut meal, a by-product of walnut oil production, can be used to develop walnut meal protein peptides with stable and simple preparation technology and good blood sugar lowering effect, which not only provides new raw materials for preventing and assisting in controlling blood sugar levels in diabetic patients, but also finds new profit growth points for the walnut industry. Summary of the invention

[0006] One of the objects of the present invention is to provide a polypeptide, the sequence of which is Leu-Pro-Gln-Phe (LPQF), Leu-Pro-Ser-Phe (LPSF) or Vla-Pro-Phe-Pro (VPFP).

[0007] Preferably, the polypeptide of the present invention can be obtained not only by separation from defatted walnut meal, but also by artificial synthesis.

[0008] The second object of the present invention is to provide a method for preparing the polypeptide, wherein the polypeptide is prepared from defatted walnut meal, and the specific steps are as follows:

[0009] (1) The defatted walnut meal is enzymatically modified using food industry protease to obtain a proteolysis product.

[0010] (2) Separating the protein hydrolysis product obtained in step (1) using an ultrafiltration membrane to obtain a separated product.

[0011] (3) Determine the inhibitory activity of the separated product obtained in step (2) against DPP-IV and screen out the component with the highest activity.

[0012] (4) Purify the most active component using a reverse phase high performance liquid chromatography system to obtain a purified product.

[0013] (5) Determine the inhibitory activity of the purified product against DPP-IV, screen out the component with the highest activity, and obtain three peptides.

[0014] Preferably, the food industry protease in step (1) is bromelain.

[0015] Preferably, the ultrafiltration membranes used in step (2) are 10 kDa and 3 kDa.

[0016] Preferably, the separated product obtained in step (2) has three parts: a part with a molecular weight less than 3 kDa, a part with a molecular weight between 3-10 kDa, and a part with a molecular weight greater than 10 kDa.

[0017] Preferably, the walnut meal is a by-product of walnut oil extraction.

[0018] The third object of the present invention is to provide an application of the polypeptide, wherein the application has the following two aspects:

[0019] (1) Use of the polypeptide in the preparation of hypoglycemic drugs.

[0020] (2) Use of the polypeptide in the preparation of health products that help maintain healthy blood sugar levels.

[0021] Beneficial effects of the present invention

[0022] (1) The half inhibitory concentration (IC) of the polypeptides LPQF, LPSF and VPFP of the present invention on DPP-Ⅳ 50 ) were 50.03, 53.06, and 52.09 μg / mL (i.e. 99.34, 114.70, and 113.58 μmol / L), respectively. It has good water solubility, can be absorbed by the human intestine, and has little side effects on the human body.

[0023] (2) The polypeptide of the present invention can not only be obtained by artificial synthesis, but also be prepared from defatted walnut meal, which provides a new way for the resource utilization of defatted walnut meal. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The inhibitory activity of walnut meal protein hydrolysates hydrolyzed by proteases used in different food industries on DPP-Ⅳ.

[0025] Figure 2 The DPP-Ⅳ inhibitory activity of different components after ultrafiltration separation of bromelain hydrolysate.

[0026] Figure 3 The DPP-Ⅳ inhibitory activity of different components after purification using a liquid chromatography system.

[0027] Figure 4 is the half maximal inhibitory concentration of LPQF, LPSF and VPFP on DPP-Ⅳ. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following embodiments are only simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0029] All methods were conventional unless otherwise specified, and all materials and reagents used were obtained from commercial sources unless otherwise specified.

[0030] Example 1

[0031] Preparation of peptides from defatted walnut meal

[0032] The defatted walnut meal used in this embodiment is a byproduct of water-based ecological production of walnut oil. The specific steps are as follows

[0033] (1) The defatted walnut meal was dried at 60°C with conventional air blowing, crushed, and passed through a 40-mesh silk sieve. The sieve residue was taken for enzymatic hydrolysis.

[0034] (2) using bromelain, alkaline protease, papain, composite protease, flavor protease, neutral protease, pancreatic enzyme, trypsin, animal hydrolyzing protease, and pepsin under the optimal working conditions of each enzyme to enzymolyze the sieve obtained in step (1), and after the enzymolysis is completed, boiling water bath is used for 20 minutes to inactivate the enzymes to obtain an enzymolysis solution.

[0035] (3) After the enzymatic hydrolysate obtained in step (2) is cooled to room temperature, it is centrifuged at 5000 rpm for 20 min to obtain the supernatant to obtain an enzymatic hydrolysate. An appropriate amount of the enzymatic hydrolysate is vacuum freeze-dried to obtain a dry product, and the remaining enzymatic hydrolysate is used for later use.

[0036] (4) Determine the inhibitory activity of the dried product obtained in step (3) against DPP-IV. The determination result is as follows: Figure 1 As shown, according to Figure 1 The results of the targeted screening showed that the product of walnut meal protein hydrolyzed by bromelain had the strongest inhibitory activity against DPP-IV, so the enzymatic hydrolysis product obtained by bromelain was selected for the next experiment.

[0037] (5) The enzymatic hydrolyzate obtained by enzymatic hydrolysis of bromelain obtained in step (3) was separated using ultrafiltration membranes with molecular cutoffs of 10 kDa and 3 kDa to obtain three components with a molecular weight of less than 3 kDa, a molecular weight between 3 and 10 kDa, and a molecular weight of greater than 10 kDa. The inhibitory activities of the three components against DPP-IV were determined. The results of the determination were as follows: Figure 2 As shown, according to Figure 2 The results of the targeted screening showed that the components with a molecular weight less than 3 kDa had the strongest inhibitory activity against DPP-IV, so the components with a molecular weight less than 3 kDa were selected for the subsequent experiments.

[0038] (6) Use a reverse phase high performance liquid chromatography system to purify components with a molecular weight less than 3 kDa. The purification conditions are as follows: mobile phase A is water containing 0.1% trifluoroacetic acid, and mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid. The flow rate is 10 mL / min, and the elution gradient is 8-60% B, which lasts for 35 minutes. The detection wavelength is 220 nm. Purified component P1 is obtained at 4.0-6.4 minutes of elution, purified component P2 is obtained at 15.0-16.4 minutes, purified component P3 is obtained at 17.7-18.6 minutes, purified component P4 is obtained at 19.7-20.6 minutes, and purified component P5 is obtained at 23.9-25.4 minutes. The inhibitory activity of the five components against DPP-Ⅳ was measured, and the measurement results are as follows: Figure 3 As shown. Figure 3 The results of the targeted screening showed that component 4 (P4) had the highest inhibitory activity, and P4 was vacuum freeze-dried to obtain walnut meal protein peptide.

[0039] (7) Ultra-high performance liquid chromatography-quadrupole-orbitrap mass spectrometry (UPLC-Q-Orbitrap-MS 2 ) Mass spectrometry information of the peptide segment in P4 obtained in step (6) of high-throughput analysis. Chromatographic conditions: Infinity-lab Poroshell120EC-C18 column (1.9 μm, 2.1×100 mm), column temperature 30°C, mobile phase A is acetonitrile containing 0.1% formic acid, mobile phase B is water containing 0.1% formic acid, flow rate 0.20 mL / min. The elution gradient is as follows: 0-1 min (5% A), 1-2.5 min (5-10% A), 2.5-12.5 min (10-25% A), 12.5-20 min (25-52.5% A), 20-22 min (52.5-95% A), 22-24 min (95% A), 24-25 min (95-5% A). Mass spectrometry conditions: positive ion mode (ESI+), spray voltage 3.2 kV, capillary temperature 350 ° C, dryer temperature 350 ° C. The data acquisition range is 200-2000 m / z, and the scanning mode includes full mass spectrum and dd-MS2. The resolution of the full mass spectrum is 70000, while the resolution of dd-MS2 is 17500.

[0040] The mass spectrometry data were analyzed using Peaks Studio 8.0 software, and the P4 component was analyzed by de novo sequencing. 57 peptides with an average confidence level (ALC%) greater than 85% were screened, and the peptide components in P4 are shown in Table 1.

[0041] Table 1 Information on peptides identified in P4 component

[0042]

[0043]

[0044] (8) The Peptide Ranker program (http: / / disilldeep.ucd.ie / PeptideRankert) was used to evaluate the biological activity of the 57 peptides in Table 4. According to the activity scores, 7 peptides with potential biological activity were screened out, and the results are shown in Table 2. The BIOPEP-UWM platform (https: / / biochemia.u wm.edu.pl / biopep-uwm / ) was used to evaluate the inhibitory activity of the 7 peptides on DPP-IV, and 3 peptides with inhibitory activity were screened out, namely LPQF, LPSF, and VPFP, and the amino acid sites of the three peptides that inhibited DPP-IV were predicted. The results are shown in Table 3. According to the prediction in Table 3, the three peptides are rich in amino acid sites that inhibit DPP-IV.

[0045] Table 2 Peptides with potential biological activity in P4 components

[0046] Serial number Peptide sequence Bioactivity prediction score 1 WPLR 0.95708 2 TFFHFR 0.938165 3 WVAF 0.912957 4 LPQF 0.905117 5 LPSF 0.897727 6 VPFP 0.897371 7 HFR 0.883585

[0047] Table 3 Three peptides with inhibitory activity against DPP-IV in walnut meal protein peptides screened using computer bioinformatics tools and their amino acid sites of inhibitory effect on DPP-IV

[0048]

[0049] Example 2

[0050] Prediction of the absorption, distribution, metabolism, excretion and toxicity properties (ADMET) of the three peptides obtained in Example 1

[0051] First, the amino acid sequences of the peptides shown in Table 3 were converted into the Simplified Molecular Input Line Entry Standard System (SMILES). AdmetSAR (http: / / lmmd.ecust.edu.cn / admetsar1) was used to predict the ADMET properties of the three peptides screened above, and the peptides with good solubility, easy absorption, good metabolic performance, and low toxicity (or non-toxic) were further subjected to molecular docking research and analysis, and the screening results are shown in Table 4. According to Table 4, the three peptides screened above all have good solubility, can be absorbed by the human intestine, have good metabolic performance and are non-toxic.

[0052] Table 4 Prediction of ADMET properties of screened peptides using SMILES code

[0053]

[0054] Example 3

[0055] Molecular docking of the three peptides prepared in Example 1 with DPP-IV

[0056] As a template for molecular docking studies, the crystal structure of DPP-IV was obtained from the RCSB Protein Data Bank (http: / / www.rcsb.org) with the PDB ID 4PNZ. The three-dimensional structure of each peptide was constructed using the “Construct aProtein” tool provided by the SYBYL-X 2.0 software. Specifically, molecular docking was performed using the Surflex-Dock tool in the SYBYL-X 2.0 software. The optimal binding conformation of the peptide with DPP-IV was predicted based on the consistency score (C-score) and the total score (T-score), and the interaction between the peptide and the active site of DPP-IV was constructed to obtain the peptides LPQF, LPSF, and VPFP that bind stably to DPP-IV (PDB ID: 4PNZ).

[0057] DPP-IV contains three active pockets, namely S1, S2 and the key S3. The S1 pocket is composed of amino acid residues such as Ser630, Asn710, His740, Tyr631, Val656, Trp629, Tyr666 and Val711; the S2 pocket is composed of Arg125, Glu205, Glu206 and Ser209; the S3 pocket is composed of Tyr547, Arg358 and Phe357. The results are shown in Table 5. All three peptides interacted with the S1, S2 or key S3 pockets of DPP-IV. LPQF, VPFP and LPSF established 7 (Tyr666, Trp629, Tyr666 and Phe357) pockets with DPP-IV, respectively. Glu205 His126 Arg125 Tyr547 Pro550 ), 4 (Tyr666 Arg669 Glu206 ) and 6 (Tyr662 Glu206 Tyr547 Arg125 ) hydrogen bond interactions. The three peptides also formed 5, 9 and 7 hydrophobic interactions with DPP-IV, respectively. Interestingly, two key amino acid residues, Phe357 and Ser209, are present in all three peptides, suggesting that this may be one of the important mechanisms by which they exert DPP-IV inhibitory activity. In summary, the three peptides can change the DPP-IV conformation to exert DPP-IV inhibitory activity.

[0058] Table 5 Interactions of LPQF, LPSF and VPFP with DPP-IV (PDB ID: 4PNZ)

[0059]

[0060] Example 4

[0061] The half inhibition rate of the three-segment polypeptide prepared in Example 1 in inhibiting DPP-IV activity

[0062] The walnut meal protein active peptides LPQF, LPSF and VPFP prepared in Example 1 were synthesized at Shanghai Jie Peptide Biotechnology Co., Ltd. to obtain products with a purity greater than 98%. DPP analysis buffer (No. 700211; Cayman Chemical, USA) was used to prepare active peptide solutions with concentrations of 250, 125, 62.5, 31.25 and 15.62 μg / mL. The DPP-IV inhibitor activity detection kit (No. 700210; Cayman Chemical, USA) was used to determine the ability of the three peptides to inhibit DPP-IV. The experimental operation was carried out according to the instructions of the kit, and the peptide concentration that achieved the half inhibition rate (IC 50 The result is Figure 4 As shown, the IC of peptide LPQF 50 is 99.34μmol / L (50.03μg / mL); the IC 50 is 114.70μmol / L (53.06μg / mL); the IC 50 It is 113.58μmol / L (52.09μg / mL).

Claims

1. A polypeptide, characterized in that: The amino acid sequence of the polypeptide is Leu-Pro-Gln-Phe, Leu-Pro-Ser-Phe or Vla-Pro-Phe-Pro.

2. The method for preparing the polypeptide according to claim 1, characterized in that: The polypeptide is prepared from defatted walnut meal.

3. Use of the polypeptide according to claim 1 in the preparation of hypoglycemic drugs.

4. Use of the polypeptide according to claim 1 in the preparation of a health product that helps maintain a healthy blood sugar level.

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