An eel polypeptide with DPP-IV inhibitory activity and its preparation method

Abstract

The present invention discloses an eel polypeptide with DPP-IV inhibitory activity and a preparation method thereof, belonging to the technical field of functional food processing. The present invention for the first time obtains and determines the composition structure of the active compound from eel muscle. The amino acid sequences of the eel protein DPP-IV inhibitory polypeptides are Phe-Pro-Arg, Tyr-Pro-Tyr-Pro-Ala-Ser, and Tyr-Pro-Pro-Ser-Phe-Ser. These polypeptides have good DPP-IV inhibitory activity and can be used as drugs, or dietary supplements, or added into ordinary foods as food bases, which have a certain preventive or adjuvant therapeutic effect on type 2 diabetes, or people suffering from chronic metabolic disorders such as obesity, immune deficiency, and heart disease, and have broad application prospects.

Description

Technical Field

[0001] The present invention relates to an eel polypeptide with DPP-IV inhibitory activity and a preparation method thereof, and particularly relates to an eel protease hydrolysate with DPP-IV inhibitory activity, belonging to the technical field of functional food processing. Background Art

[0002] The latest report of the International Diabetes Federation (IDF) shows that 10% of adults globally suffer from diabetes, and the number of patients has increased by 16% in two years. In 2021, the number of adult diabetes patients globally reached 537 million. It is estimated that 44.7% of adult diabetes patients (240 million people) are undiagnosed, and more than four-fifths (81%) of them live in low- and middle-income countries. The main reason for the continuous increase in the incidence of diabetes is the surge in type II diabetes and related risk factors, including obesity, unhealthy diet, and general lack of exercise. Globally, 12.2% of deaths are caused by diabetes, and nearly half of these deaths occur in people under 60 years old. As a chronic disease, diabetes increases the risk of early death. Diabetes mainly has three types: type I, type II, and gestational diabetes. The most common form of diabetes is type II diabetes (T2DM), accounting for about 90% - 95% of all cases.

[0003] Type II diabetes is a complex endocrine and metabolic disease caused by insulin resistance or the inability of peripheral tissues to uptake glucose, resulting in the body's insufficient utilization of insulin. This disease form accounts for about 90 - 95% of all diagnosed diabetes cases. Type II diabetes has multiple causes, and GLP-1 / DPP-IV inhibitors have become a new means of treating type II diabetes. Glucagon-like peptide 1 (GLP-1) can stimulate the rapid release of insulin, stimulate insulin biosynthesis, inhibit β-cell apoptosis, and promote β-cell differentiation in the body. However, GLP-I can be rapidly cleaved and inactivated by DPP-IV. Therefore, DPP-IV inhibitors that inhibit DPP-IV activity and thus do not degrade GLP-1 have become one of the main research directions for treating type II diabetes. Currently, the most intensively studied and clinically applied DPP-4 inhibitors are Sitagliptin, Vildagliptin, and Saxagliptin. However, although these chemically synthesized drugs have the effect of alleviating type II diabetes, they have certain toxic side effects. Collagen peptides with DPP-IV inhibitory activity derived from natural proteins have attracted wide attention due to their high food safety, strong tolerance, and no toxic side effects.

[0004] Eels and their products are very popular among consumers around the world. Eel meat is tender and nutritious. The protein content in eel meat can reach more than 50%, and the fat content reaches more than 30%. It is also rich in various vitamins and nutritional components such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) known as "brain gold", making it a recognized precious edible fish species. According to the statistical data of the Food and Agriculture Organization of the United Nations (FAO), since 2000, the annual global eel production has been 22×10 4 ~30×10 4 t, and the vast majority of which comes from aquaculture. China is a major country in eel farming and export in the world, with an annual eel production of 60,000 tons. In 2020, the export volume of roasted eel in China was 39,000 tons, mainly roasted eel. As eels are increasingly recognized by consumers, the eel market has a very broad prospect. However, at present, the eel processing industry mainly stays in the research on the processing technology of roasted eel, and it is difficult to develop the comprehensive utilization value of eels. In order to better develop the edible and economic value of eels, more and more researchers are studying the functional components of eels. Proline, tyrosine, phenylalanine and other amino acids are relatively abundant in eel protein. For food-derived DPP-IV inhibitory polypeptides, their structural characteristics are that the chain length generally contains 2-14 amino acid residues, the molecular weight is less than 1000 Da, and it is considered that peptides with proline, phenylalanine, tyrosine or hydrophobic amino acids in the peptide chain at the C-terminus of the peptide chain have higher DPP-IV inhibitory activity. Therefore, developing functional peptides with DPP-IV inhibitory activity from eel protein has great economic and social benefits. It can be used as a food supplement to improve the value of eel protein, as a functional component of health foods, and can also be applied to the production of a new generation of anti-diabetic drugs, with very broad prospects. Summary of the Invention

[0005] To solve the problems existing in the prior art, the present invention provides a polypeptide with DPP-IV inhibitory activity and a preparation method thereof. The polypeptide is an enzymatic hydrolysate obtained by decomposing eel protein powder with a commercial protease, and the protease is a compound protease.

[0006] A method for preparing a polypeptide with DPP-IV inhibitory activity provided by the present invention mainly includes the following steps:

[0007] (1) Dissolve the crude eel protein powder in deionized water to make a 4% (w / v) crude eel protein solution, vortex to fully dissolve it, heat it in a boiling water bath for 10 min, and cool it to 55 °C on ice cubes.

[0008] (2) Enzymatically hydrolyze the crude eel protein solution to obtain an enzymatic hydrolysate;

[0009] (3) Inactivate the enzyme in the enzymatic hydrolysate, centrifuge to remove the precipitate, concentrate the supernatant and then freeze-dry it;

[0010] (4) Separation and purification of bioactive polypeptides.

[0011] In one embodiment, in step (1), the crude eel protein powder is prepared from eel meat through pretreatment, primary degreasing, drying, secondary degreasing, and drying to obtain the secondary degreased crude eel protein powder.

[0012] Preferably, the preparation method of the crude eel protein powder is as follows:

[0013] ① Pretreatment of eel meat: Cut the eel meat into pieces, rinse with distilled water for 5 min, then squeeze out the excess water, and then make it into minced meat and freeze at low temperature (-20 °C). Cut the frozen eel meat minced into relatively uniform small pieces and directly put them into boiling water containing 0.1% sodium bicarbonate, and cook for 10 min to make the fish meat denature into a gel state.

[0014] ② Primary degreasing: After removing the water from the denatured fish meat with a suction filter, put it into a beaker, add sufficient anhydrous ethanol, and stir on a stirrer for 1 h. Put the pre-degreased fish meat into a beaker and soak it in distilled water for 10 min, and stir constantly. Pour out the soaked water and repeat 4 - 5 times.

[0015] ③ Drying: After removing the water from the degreased fish meat with a suction filter, dry it at 70 °C for 4 h to make a dry product, and then crush it with a high-speed pulverizer.

[0016] ④ Secondary degreasing: Take an appropriate amount of the primary degreased eel protein powder, use petroleum ether as the degreasing solvent, and carry out secondary degreasing by Soxhlet extraction method.

[0017] ⑤ Drying: Dry the degreased sample to obtain the secondary degreased crude eel protein powder for standby.

[0018] In one embodiment, the preparation conditions of the enzymatic hydrolysate are pH 7.5, substrate concentration 4% (w / v), temperature 55 °C, protease addition amount 2% (w / w), hydrolysis time 2 h. After the hydrolysis is completed, inactivate the enzyme at 90 °C for 10 min and quickly cool it.

[0019] Preferably, the protease is trypsin, bromelain, flavor protease, and / or compound protease.

[0020] In one embodiment, in the preparation of the enzymatic hydrolysate in step (3), the preparation method of the enzymatic hydrolysis sample is: Centrifuge the inactivated enzymatic hydrolysate at 4 °C and 8000 r / min for 15 min, filter to remove the precipitate after centrifugation, and concentrate and then freeze-dry.

[0021] In one embodiment, the steps of the method for separating and purifying bioactive polypeptides in step (4) are as follows:

[0022] ① Dissolve the freeze-dried sample from step (3) in deionized water to make a 10 mg / ml solution. After filtering through a 0.22 μm filter membrane, use 3 kDa and 10 kDa molecular weight ultrafiltration centrifugal tubes for ultrafiltration. Collect the fractions <3 kDa, 3 - 10 kDa, and >10 kDa, and detect the DPP-IV inhibitory activity of each fraction after freeze-drying.

[0023] ② Make a 10 mg / ml solution of the freeze-dried sample from step ① using Tris-HCl buffer (0.1 M, pH = 8), with a sample loading volume of 5 ml. Separate and purify using a gel chromatography column. The specific method is as follows: Use Superdex G-15 (2 × 21.2 cm) as the separation column, and use an AKTA gel protein purification system for separation. Detect the absorption peak at 220 nm. Use Tris-HCl buffer (0.1 M, pH = 8) as the mobile phase, with a flow rate of 1 ml / min. Collect the samples according to the absorption peak at room temperature. Repeat the sample loading multiple times for product enrichment. After concentrating and freeze-drying the collected product, detect the DPP-IV inhibitory activity of each fraction.

[0024] ③ Purify the sample collected in step ② by reverse-phase liquid chromatography. The chromatographic conditions are as follows: Chromatographic column: 21.2 × 250 mm C18; Mobile phase A: Aqueous solution containing 0.1% (v / v) TFA, Mobile phase B: Acetonitrile solution containing 0.1% (v / v) TFA; Sample injection volume: 1 mL; Sample injection concentration: 10 mg / mL; Flow rate: 10 ml / min; Detection wavelength: 220 nm; Detection temperature: 30 °C; Elution program: From 0 to 40 min, the proportion of mobile phase B increases from 0% to 100%; From 40 - 50 min, the proportion of mobile phase B decreases from 100% to 0. Collect the separated fractions according to the peaks, repeat the sample loading multiple times for product enrichment, and detect the DPP-IV inhibitory activity of each fraction after freeze-drying.

[0025] ④ Identify the polypeptide sequence of the sample collected in step ③: Use MALDI-TOF / TOF MS / MS to identify the peptide sequence of the active peptide; Mobile phase A solution is 0.1% (v / v) formic acid aqueous solution, and mobile phase B solution is acetonitrile solution containing 0.1% (v / v) formic acid. The liquid chromatography column (2.1 × 150 mm, BEH C 18 ) The column temperature is 45 °C, the flow rate is 0.3 ml / min, and it is equilibrated with 100% of mobile phase A. The sample is loaded by an automatic sampler with a sample injection volume of 5 μL and separated by the liquid chromatography column. The elution program is as follows: The elution curve of mobile phase B: From 0 to 40 min, 0 - 30%; From 40 - 45 min, 30% - 80%; From 45 - 50 min, 80% - 100%; From 50 - 55 min, 100% - 0. Data processing is carried out according to the Masslynx software for sequence interpretation, comparison with the eel protein database, and structural characteristics of the DPP-IV inhibitory peptide to determine the peptide sequence.

[0026] The present invention provides DPP-IV inhibitory peptides derived from eels, with amino acid sequences FPR, YPPSFS, and YPYPAS, and IC 50 being 64.14 mM, 102.65 mM, and 68.30 mM respectively.

[0027] Advantages of the present invention:

[0028] The DPP-IV inhibitory polypeptide described in the present invention is obtained from eel muscle for the first time, and the composition structure of the active compound is determined. The amino acid sequences of the eel protein DPP-IV inhibitory polypeptide are Phe-Pro-Arg, Tyr-Pro-Tyr-Pro-Ala-Ser, and Tyr-Pro-Pro-Ser-Phe-Ser. It has good DPP-IV inhibitory activity and can be used as a drug, or a dietary supplement, or added as a food ingredient into ordinary foods, which has a certain preventive or adjuvant therapeutic effect on type 2 diabetes, or people suffering from chronic metabolic disorders such as obesity, immune deficiency, and heart disease, and has a very broad application prospect. Brief Description of the Drawings

[0029] Figure 1 For the inhibition rate and degree of hydrolysis of the product (5 mg / ml) in Specific Example 2;

[0030] Figure 2 For the inhibition rate and degree of hydrolysis of the product (5 mg / ml) in Specific Example 3;

[0031] Figure 3 For the inhibition rate and degree of hydrolysis of the product (5 mg / ml) in Specific Example 4;

[0032] Figure 4 For the inhibition rate of the ultrafiltration product (1 mg / ml) under the optimal hydrolysis conditions;

[0033] Figure 5 For the inhibition rate of the product separated by gel filtration chromatography (0.5 mg / mL);

[0034] Figure 6 For the inhibition rate of the product prepared by reverse-phase liquid chromatography (0.25 mg / mL). Detailed Description of the Invention

[0035] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention.

[0036] Example 1

[0037] (1) Preparation of active polypeptide

[0038] The crude eel protein powder was dissolved in deionized water to prepare a substrate concentration of 4% (w / v). After being fully shaken and dissolved, it was boiled in a boiling water bath for 10 min, cooled to 55 °C on ice, the pH was adjusted to 7.5, 2% (w / w) compound protease was added, and the enzymatic hydrolysis reaction was carried out with stirring at 55 °C for 2 h. After the reaction, the enzyme was inactivated at 90 °C for 10 min. At 4 °C, the supernatant was taken after centrifugation at 8000 r / min for 15 min, filtered and then freeze-dried. The degree of hydrolysis (DH) was detected by the OPA method.

[0039] (2) Isolation and purification

[0040] The above enzymatic hydrolysis sample was resuspended in deionized water to prepare a sample solution of 10 mg / ml. After filtration with a 0.22 μm filter membrane, dialysis was carried out using a 1000 Da dialysis bag. The dialysis solution was concentrated and freeze-dried.

[0041] The above freeze-dried sample was made into a 10 mg / ml solution with deionized water, and the sample loading volume was 5 ml. Gel chromatography column was used for separation and purification. The specific method was as follows: Tris-HCl buffer solution (0.1 M, pH = 8) was used as the mobile phase, the flow rate was 1 ml / min, and the sample was collected according to the absorption peak at room temperature and then freeze-dried for standby.

[0042] The fraction with high inhibitory activity in the above gel chromatography separation components was dissolved in Tris-HCl buffer solution (0.1 M, pH = 8), and then reverse-phase liquid chromatography purification was carried out. The chromatographic conditions were as follows: chromatographic column: 4.6×250 mm C18; mobile phase A: aqueous solution containing 0.1% (v / v) TFA, mobile phase B: acetonitrile solution containing 0.1% (v / v) TFA; sample injection volume: 1 mL; sample injection concentration: 10 mg / mL; flow rate: 10 ml / min; detection wavelength: 214 nm and 280 nm; detection temperature: 30 °C; elution program: from 0 to 40 min, the proportion of mobile phase B increased from 0% to 100%; from 40 - 50 min, the proportion of mobile phase B decreased from 100% to 0.

[0043] The polypeptide sequence of the fraction with the highest DPP-IV inhibitory activity collected after reversed-phase liquid chromatography separation and purification was identified: detected using an ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometer. Chromatographic conditions: Mass spectrometry conditions: Waters Platform 2MD 4000, ion source temperature 100 °C, ionization mode: EIS+, desolvation gas temperature 400 °C, capillary voltage: 3.5 kV, cone gas flow rate 50.0 L / h, cone voltage: 20 V, desolvation gas flow rate: 700.0 L / h; mass-to-charge ratio scanning range 50 - 2000 m / z. Chromatographic conditions: Mobile phase A is an aqueous solution of 0.1% (v / v) formic acid, and mobile phase B is an acetonitrile solution containing 0.1% (v / v) formic acid. Liquid chromatography column (2.1×150 mm, BEH C18), column temperature 45 °C, flow rate 0.3 ml / min, equilibrated with 100% of mobile phase A, the sample was loaded by an autosampler, and the injection volume was 5 μL and separated by the liquid chromatography column. The elution program is as follows: Elution curve of mobile phase B: 0 - 40 min, 0 - 30%; 40 - 45 min, 30% - 80%; 45 - 50 min, 80% - 100%; 50 - 55 min, 100% - 0.

[0044] (3) Sequence identification and activity detection

[0045] The mass spectrometry results were analyzed using Masslynx, and the polypeptide sequence was identified by combining with the eel protein sequence database. The selected peptide sequences were sent to Nanjing Peptide Valley Biotechnology Co., Ltd. for synthesis. The synthesis method was solid-phase peptide synthesis (SPPS), and the purity of the synthesized peptide was greater than 95%. The inhibitory activity of the synthesized polypeptide was identified.

[0046] Example 2

[0047] In step (1) of Specific Example 1, the enzymatic hydrolysis times in the enzymatic hydrolysis conditions were 1, 2, 3, 4, and 5 hours respectively, and the other conditions were the same as those in Example 1. The prepared bioactive polypeptide was separated and purified, then sequenced and identified and its activity was detected. The results are as Figure 1 shown. When the enzymatic hydrolysis time was 2 h, the polypeptide inhibition rate could reach 21.56%, and the degree of hydrolysis could reach 19.48%.

[0048] Example 3

[0049] In step (1) of Specific Example 1, the enzyme addition amounts in the enzymatic hydrolysis conditions were 1%, 2%, 3%, 4%, and 5% respectively, and the other conditions were the same as those in Specific Example 1. The prepared bioactive polypeptide was separated and purified, then sequenced and identified and its activity was detected. The results are as Figure 2As shown, when the enzyme addition amount is 2%, the polypeptide inhibition rate can reach 33.47% and the degree of hydrolysis can reach 23.47%.

[0050] Example 4

[0051] In Specific Example 1, in the enzymatic hydrolysis conditions of step (1), the substrate addition amounts are 2%, 3%, 4%, 5%, and 6% respectively, and the remaining conditions are the same as those in Specific Example 1. The prepared active polypeptide is separated and purified, then sequenced and identified and its activity is detected. The results are as Figure 1 shown. When the substrate addition amount is 3%, 4%, and 5%, the polypeptide inhibition rates can reach 26.67%, 29.14%, and 28.44% respectively, and the degrees of hydrolysis can reach 28.54%, 28.86%, and 29.17% respectively.

[0052] Example 5

[0053] In step (2) of Example 1, the separation steps are ultrafiltration, Sephadex gel chromatography separation, and reverse-phase liquid chromatography separation in sequence. The DPP-IV inhibitory activity of each component after each separation is detected, and the component with the highest inhibitory activity is selected for the subsequent separation steps. The detection results are respectively as Figure 4 、 5 、6 shown. For <3 kDa, the detection concentrations of the P4 and F4 components are 1 mg / ml, 0.5 mg / ml, and 0.25 mg / ml respectively, and the inhibition rates are 32.17%, 54.75%, and 74.51% respectively. The F4 component with the highest inhibition rate in the final preferred reverse-phase liquid chromatography separation is analyzed by liquid chromatography-mass spectrometry. The mass spectrometry results are analyzed by Masslynx, and the polypeptide sequence is identified in combination with the eel protein sequence database (as shown in Table 1). According to the structural characteristics of the DPP-IV inhibitory peptide, the selected peptide sequences are Phe-Pro-Arg, Tyr-Pro-Tyr-Pro-Ala-Ser, and Tyr-Pro-Pro-Ser-Phe-Ser. After synthesizing the three peptides, their inhibitory activities are detected. The IC 50 values are 64.14 mM, 102.65 mM, and 68.30 mM respectively.

[0054] Table 1. Mass spectrometry analysis sequence of F4 component

[0055]

[0056]

[0057] 1: The peptide sequence is abbreviated as a single-letter amino acid code.

[0058] 2: GRAVY represents the hydrophilicity of the peptide. The smaller the value, the stronger the hydrophilicity.

[0059] 3: The eel protein data source from Uniprot was used for screening and identifying peptide sequences.

[0060] Example 6: Detection of polypeptide inhibitory activity

[0061] (1) Principle

[0062] The chromogenic substrate method using glycylprolyl-p-nitroanilide (Gly-Pro-PNA) as the substrate was adopted to screen DPP-IV inhibitors. The detection principle of this method is that under alkaline conditions, DPP-IV catalyzes the hydrolysis of the substrate Gly-Pro-p-nitroanilide to generate yellow p-nitroaniline, which has a characteristic absorption peak at a wavelength of 405 nm. The absorbance measured by the microplate reader at 405 nm reflects the level of enzyme activity.

[0063] (2) Method

[0064] Sample: The sample was dissolved in 100 mM Tris-HCl buffer (pH = 8.0) to prepare a 40 mg / mL stock solution, and then the stock solution was diluted to different concentrations as the sample solution.

[0065] Substrate: Gly-Pro-PNA solution. Gly-Pro-PNA was configured into a 1.59 mM Gly-Pro-PNA solution with 100 mM Tris-HCl buffer (pH = 8.0).

[0066] Enzyme: DPP-IV solution. DPP-IV was configured into a 0.1 U DPP-IV solution with 100 mM Tris-HCl buffer (pH = 8.0).

[0067] Termination solution: 1 M acetic acid-sodium acetate buffer (pH = 4.0).

[0068] The experiment was carried out in a 96-well plate, and the absorbance was detected at 405 nm using a microplate reader. First, the enzyme, buffer, and drug were water-bathed at 37 °C for 30 min respectively. Then, the sample (or buffer) and substrate were added into the 96-well plate in sequence and incubated at 37 °C for 10 minutes. Then, the DPP-IV enzyme solution was added, and after mixing, it was incubated at 37 °C for 60 minutes. 50 μL of 1 M acetic acid-sodium acetate buffer (pH 4.0) was added to terminate the reaction. The absorbance (OD) at 405 nm was measured using a microplate reader. The total reaction volume was 150 μl.

[0069] The experiment was divided into 4 groups, with 3 replicates in each group.

[0070] Each group was as follows:

[0071] Sample group (S group): Sample + enzyme + substrate.

[0072] Sample blank group (SB group): Sample + substrate.

[0073] Negative control group (C group): Enzyme + substrate.

[0074] Blank group (B group): Substrate.

[0075] For the specific samples added to each group, see Table 1.

[0076] Table 2. Grouping and sample addition amounts for DPP-VI inhibitory activity experiment

[0077]

[0078] Note:

[0079] (1) The unit of the numbers in the table is μl.

[0080] (2) The total reaction volume is 150 μl. After adding the reactants according to the table for each group, the final volume is made up with buffer solution.

[0081] (3) Calculation of inhibition rate

[0082]

[0083] Detection results: The reaction conditions in Specific Example 1 are the optimal conditions for polypeptide preparation. The screened target polypeptides are respectively tested for DPP-IV inhibitory activity at concentrations of 12.5 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, and 400 μg / ml, and their IC 50 .

[0084] The results are as shown in Table 3 below:

[0085] Table 3. Inhibitory activity of inhibitory peptides

[0086]

[0087] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Use of a DPP-IV inhibitory polypeptide in the preparation of a medicament for treating diabetes, wherein the amino acid sequence of the DPP-IV inhibitory polypeptide is FPR, YPPSFS and / or YPYPAS.

2. A polypeptide having DPP-IV inhibitory activity, characterized in that, The amino acid sequence is YPPSFS and / or YPYPAS.

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