Tilapia skin DPP-IV inhibitory peptide as well as preparation method and application thereof

Abstract

The invention discloses a tilapia skin DPP-IV inhibitory peptide as well as a preparation method and application thereof, and belongs to the technical field of food-borne active peptide development. The preparation method comprises the following steps: taking tilapia skin as a raw material, extracting gelatin from the tilapia skin through an acid-alkali treatment method, and then performing simulated gastrointestinal digestion on the gelatin by utilizing simulated digestive juice prepared from pepsin, trypsin and chymotrypsin to obtain a digestion product with DPP-IV inhibitory activity; the tilapia skin active peptide with relatively high DPP-IV inhibitory activity is obtained through further ultrafiltration and identification, amino acid sequences of the tilapia skin active peptide are AKVELLK, VPGPMGPMGPR and DGIR respectively, and the three inhibitory peptides have good DPP-IV inhibitory activity and can be used for developing food, health care products or medicines for assisting in regulating blood sugar.

Description

Technical Field

[0001] The invention belongs to the technical field of food-derived active peptide development, and specifically relates to a tilapia skin DPP-IV inhibitory peptide and a preparation method and application thereof. Background Art

[0002] Diabetes can lead to severe damage, dysfunction and failure of multiple organs and tissues, accompanied by progressive metabolic complications such as neuropathy, retinopathy, kidney disease and cardiovascular disease. About 80% of diabetes is type 2 diabetes (T2DM), which is treated with drugs to increase the action of insulin or inhibit carbohydrate digestion enzymes such as glycosidase inhibitors, thereby reducing blood glucose concentrations. Therefore, controlling blood glucose is a key step in the management and treatment of diabetes.

[0003] The most effective treatment strategy for T2DM is to maintain normal blood sugar levels. One of the effective ways to inhibit postprandial hyperglycemia is to control the release of glucose by inhibiting α-glucosidase. α-glucosidase inhibitors can reduce the digestibility of sugars. Two α-glucosidase inhibitors, acarbose and miglitol, are clinically used to control postprandial hyperglycemia, but these drugs usually have many side effects, such as bloating, flatulence, diarrhea, dizziness and vomiting. Therefore, it is necessary to find low-cost and relatively safe animal foods or supplements as alternatives to α-glucosidase inhibitors. A large number of studies have confirmed that some food-derived bioactive ingredients (peptides, phenols, terpenes, etc.) have excellent α-glucosidase inhibitory potential. Among them, α-glucosidase inhibitory peptides prepared from food-derived proteins have nutritional properties and have attracted widespread attention in the research community due to their high bioavailability, significant T2DM treatment effect, and low side effects compared with traditional α-glucosidase inhibitors.

[0004] Recent studies have found that some food protein-derived hydrolysates and peptides have significant inhibitory effects on DPP-IV activity in vitro and in vivo in experimental animals. Therefore, developing a food-derived hydrolysate or peptide with DPP-IV inhibitory properties has important application value. Summary of the invention

[0005] The invention provides a tilapia skin DPP-IV inhibitory peptide and a preparation method and application thereof. The invention uses pepsin, trypsin and chymotrypsin to hydrolyze tilapia skin gelatin to prepare the DPP-IV inhibitory peptide, which exhibits good DPP-IV inhibition. The method of the invention adopts acid-base pretreatment, warm water extraction, simulated gastrointestinal digestion, separation, purification and identification to obtain a DPP-IV inhibitory peptide with high activity.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a tilapia skin DPP-IV inhibitory peptide D1A7, characterized in that the amino acid sequence of the inhibitory peptide is shown in SEQ ID NO:1.

[0008] SEQ ID NO: 1: AKVELLK.

[0009] The second object of the present invention is to provide a tilapia skin DPP-IV inhibitory peptide D2V11, characterized in that the amino acid sequence of the inhibitory peptide is shown in SEQ ID NO:2.

[0010] SEQ ID NO:2: VPGPMGPMGPR.

[0011] The third object of the present invention is to provide a tilapia skin DPP-IV inhibitory peptide D3D4, characterized in that the amino acid sequence of the inhibitory peptide is shown in SEQ ID NO:3.

[0012] SEQ ID NO:3: DGIR.

[0013] The present invention also provides the use of the three inhibitory peptides or their complexes in the preparation of DPP-IV protease activity inhibitor products.

[0014] The present invention also provides the use of the three inhibitory peptides or their complexes in the preparation of blood sugar lowering drugs or health products with auxiliary blood sugar lowering function.

[0015] The present invention also provides a method for preparing a tilapia skin DPP-IV inhibitory peptide, comprising the following steps:

[0016] S1. Preparation of tilapia skin gelatin: treating tilapia skin with an acid-base treatment method, placing the acid-base treated fish skin in distilled water and shaking for 12 hours, and taking the supernatant and freeze-drying it to obtain tilapia skin gelatin;

[0017] S2. Preparation of tilapia skin gelatin solution: dissolving the tilapia skin gelatin described in step S1 in distilled water to prepare a 40 mg / mL solution;

[0018] S3. Preparation of simulated gastric fluid and simulated intestinal fluid: simulated gastric fluid contained 5.51 mg / mL NaCl, 1.65 mg / mL KCl, 0.53 mg / mL NaH2PO4, 0.60 mg / mL CaCl2, 0.61 mg / mL NH4Cl and 2000 U / mL pepsin, and the pH value was adjusted to 1.4 with 1.0 M HCl. The simulated intestinal fluid contained 14.02 mg / mL NaCl, 1.13 mg / mL KCl, 6.78 mg / mL NaHCO3, 0.16 mg / mL KH2PO4, 0.10 mg / mL MgCl2, 100 U / mL trypsin and 25 U / mL chymotrypsin;

[0019] S4. Preparation of tilapia skin gelatin gastric digestion hydrolysate: The tilapia skin gelatin solution prepared in step S2 was subjected to simulated gastric digestion for 2 h using the DIVH-IV system, the digestion temperature was maintained at 37 ° C, the gastric motility frequency was set to 3 times / min, the gastric juice injection rate was 1.2-1.5 mL / min, the digestion solution was removed after gastric digestion, the enzyme was heated and inactivated, and the supernatant was taken after centrifugation and freeze-dried to obtain the tilapia skin gelatin gastric digestion hydrolysate;

[0020] S5. Preparation of tilapia skin gelatin intestinal digestion hydrolysate: The tilapia skin gelatin solution was subjected to simulated intestinal digestion for 4 h using the DIVH-IV system, the digestion temperature was maintained at 37 ° C, the pyloric opening size was 0-2 mm, the intestinal peristalsis frequency was 6 times / min, the simulated intestinal fluid injection rate was 0.8 mL / min, the digestion solution was removed after the gastric digestion, the enzyme was heated and inactivated, and the supernatant was taken after centrifugation and freeze-dried to obtain the tilapia skin gelatin intestinal digestion hydrolysate;

[0021] S6. Separation and purification of tilapia skin active peptides with DPP-IV inhibitory activity: ultrafiltration separation of the enzymatic hydrolysate after digestion in step S4 and step S5, separation and purification of the ultrafiltration fraction with the strongest DPP IV inhibitory activity by a dextran gel column, and collection of the fraction with the strongest DPP IV inhibitory activity again to obtain a polypeptide mixture;

[0022] S7. Identification, screening and synthesis of tilapia skin active peptides with DPP-IV inhibitory activity: Liquid chromatography-mass spectrometry was used to identify and analyze the peptide mixture, and then the peptides with biological activity were screened. They were then molecularly docked with the DPP IV enzyme and the peptides with the lowest binding bond energy were selected. The selected peptides were synthesized by solid phase chemical synthesis to obtain DPP-IV inhibitory peptides.

[0023] The present invention utilizes pepsin, trypsin and chymotrypsin to prepare DPP-IV inhibitory peptides, and the principle is as follows:

[0024] 1. Use acid-base treatment method to extract fish skin gelatin under appropriate temperature conditions to produce the required tilapia skin gelatin and reduce protein impurities.

[0025] 2. Use a complex enzyme system to hydrolyze large molecular proteins into small molecular polypeptides. Under the action of proteases, the peptide groups are cut and broken to obtain bioactive peptides and amino acids, which may produce certain physiologically active factors and improve the nutritional value and health functions of polypeptide products.

[0026] 3. Composite proteases are widely used in the preparation of active peptides by enzymatic hydrolysis because they can cut different sites of proteins during the enzymatic hydrolysis process and have the characteristics of low price, simple operation, safety and harmlessness.

[0027] The effective effects of the present invention are as follows:

[0028] The raw material used in the present invention is tilapia skin, which is a byproduct in the processing of tilapia, has a wide source and is low in price. Using it as a raw material to develop a polypeptide with the ability to inhibit DPP-IV enzyme activity can significantly improve the added value of tilapia skin. The tilapia skin gelatin is hydrolyzed by simulating gastrointestinal digestion to release a polypeptide with the ability to inhibit DPP-IV enzyme activity, and the DPP-IV inhibitory peptide with high inhibitory activity is separated, purified and identified. The active peptide prepared by the present invention has a good effect of inhibiting DPP-IV enzyme activity and can effectively inhibit the increase of postprandial blood sugar in patients in the early stage of T2DM. The enzymatic hydrolysis technology used in the present invention is simple and feasible, the equipment is easy to obtain, and the active ingredients in the fish skin can be released to the greatest extent. The active peptide obtained by the present invention has the characteristics of reasonable amino acid composition, no allergy, safety and reliability, etc., can be added to health products as a functional ingredient, and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described below in conjunction with embodiments with reference to the accompanying drawings.

[0030] Figure 1 This is the secondary mass spectrum for identification of the polypeptide D1A7 of Example 1 of the present invention.

[0031] Figure 2 This is the secondary mass spectrum for identification of the polypeptide D2V11 of Example 1 of the present invention.

[0032] Figure 3 This is the secondary mass spectrum for identification of the polypeptide D3D4 of Example 1 of the present invention.

[0033] Figure 4 This is a graph showing the inhibition rates of three DPP-IV inhibitory peptides in Example 2 of the present invention. DETAILED DESCRIPTION

[0034] In order to better understand the present invention, the present invention is further described in detail below in conjunction with the embodiments and drawings. However, those skilled in the art understand that the following embodiments are not limitations on the scope of protection of the present invention, and any changes and modifications made on the basis of the present invention are within the scope of protection of the present invention.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] Example 1 Preparation of DPP-IV inhibitory peptide

[0038] S1. Preparation of tilapia skin gelatin: The tilapia skin was treated with an acid-base method, that is, first treated with 0.1 M NaOH for 1 hour, then rinsed with distilled water until the pH value was about 7.0, and then mixed with 0.05 M acetic acid for 3 hours under the shaking conditions of 37° C. and 120 rpm.

[0039] The fish skin after acid and alkali treatment is placed in distilled water and shaken for 12 hours, and the supernatant is freeze-dried to obtain tilapia skin gelatin;

[0040] S2. Preparation of tilapia skin gelatin solution: tilapia skin gelatin was dissolved in distilled water to prepare a 40 mg / mL solution;

[0041] S3. Preparation of simulated gastric fluid and simulated intestinal fluid: simulated gastric fluid contained NaCl (5.51 mg / mL), KCl (1.65 mg / mL), NaH2PO4 (0.53 mg / mL), CaCl2 (0.60 mg / mL), NH4Cl (0.61 mg / mL) and pepsin (2000 U / mL), and the pH value was adjusted to 1.4 with 1.0 M HCl. The simulated intestinal fluid (pH 7.8) contained NaCl (14.02 mg / mL), KCl (1.13 mg / mL), NaHCO3 (6.78 mg / mL), KH2PO4 (0.16 mg / mL), MgCl2 (0.10 mg / mL), trypsin (100 U / mL) and chymotrypsin (25 U / mL);

[0042] S4. Preparation of tilapia skin gelatin gastric digestion hydrolysate: The tilapia skin gelatin solution was subjected to simulated gastric digestion for 2 h using the DIVH-IV system (Suzhou Xiaodong Yijian Instrument Co., Ltd., China), the digestion temperature was maintained at 37 ° C, the gastric motility frequency was set to 3 times / min, and the gastric juice injection rate was 1.2-1.5 mL / min. The tilapia skin gelatin gastric digestion hydrolysate was taken: the digestion solution was removed after gastric digestion, and the enzyme was inactivated by heating in a 100 ° C water bath for 10 minutes, the centrifugation speed was 5000 × g, the time was 15 min, and the supernatant was taken after centrifugation and freeze-dried to obtain the tilapia skin gelatin gastric digestion hydrolysate;

[0043] S5. Preparation of tilapia skin gelatin intestinal digestion hydrolysate: The tilapia skin gelatin solution was subjected to simulated intestinal digestion for 4 h using the DIVH-IV system (Suzhou Xiaodong Yijian Instrument Co., Ltd., China). The digestion temperature was maintained at 37 ° C. The pyloric opening size was 0-2 mm, the intestinal peristalsis frequency was 6 times / min, and the simulated intestinal fluid injection rate was 0.8 mL / min. After the intestinal digestion, the digestion solution was removed, and the enzyme was inactivated by heating in a 100 ° C water bath for 10 minutes. The centrifugation speed was 5000 × g for 15 min. After centrifugation, the supernatant was freeze-dried to obtain the tilapia skin gelatin intestinal digestion hydrolysate.

[0044] S6. Isolation, purification and screening of tilapia skin active peptides with DPP-IV inhibitory activity: ultrafiltration separation is performed on the digested enzymatic hydrolysate, and the ultrafiltration component with the strongest DPP IV inhibitory activity is separated and purified by a dextran gel column, the semi-dextran gel column includes a Sephadex G 25 gel column, the separation and purification conditions are a sample volume of 2 mL, a sample concentration of 4 mg / mL, a flow rate of 1.2 mL / min, and separation and purification is performed at 280 nm. The ultrafiltration separation molecular retention amounts are 10 kDa, 5 kDa and 3 kDa, respectively. Nano LC ESIMS / MS liquid chromatography-mass spectrometry is used to identify and analyze the polypeptide mixture, and the component with the strongest DPP-IV inhibitory activity is collected again to obtain a polypeptide mixture; tilapia skin active peptides with DPP-IV inhibitory activity are screened, and the screening is performed by a server, the server is a Peptide Ranker server, and the polypeptides predicted by the server to have a score greater than 0.5 have biological activity. Then, the peptide was molecularly docked with the DPP-IV enzyme, and the peptide with the lowest binding bond energy was selected. The selected peptide was synthesized by solid phase chemical synthesis to obtain tilapia skin active peptide with DPP-IV inhibitory activity.

[0045] The DPP-IV inhibitory peptide D1A7 was obtained, and its amino acid sequence was Ala-Lys-Val-Glu-Leu-Leu-Lys (its amino acid sequence is shown in SEQ ID NO: 1, and its secondary mass spectrum is shown in Figure 1); DPP-IV inhibitory peptide D2V11, whose amino acid sequence is Val-Pro-Gly-Pro-Met-Gly-Pro-Met-Gly-Pro-Arg (whose amino acid sequence is shown in SEQ ID NO: 2, and the secondary mass spectrum is shown in Figure 2 ), DPP-IV inhibitory peptide D3D4, whose amino acid sequence is Asp-Glu-Ile-Arg (whose amino acid sequence is shown in SEQ ID NO: 3, and the secondary mass spectrum is shown in Figure 3 shown).

[0046] Example 2 Determination of DPP-IV inhibitory peptide activity

[0047] The three DPP-IV inhibitory peptides were re-dissolved in ultrapure water (concentration 10 mg / mL), and then 40 μL of substrate Gly-Pro-pNA (dissolved in 100 mM Tris-HCl buffer, 0.5 mM), 50 μL of sample and 10 μL of DPP-IV enzyme (dissolved in 100 mM Tris-HCl buffer, 0.05 U / mL) were added to a 96-well plate, respectively, and reacted at 37°C in the dark for 60 min. Finally, the absorbance value was measured at 405 nm, and the DPP-IV inhibition rate was calculated according to the following formula:

[0048] DPP-IV inhibition rate = (positive control - sample absorbance) * 100% / positive control

[0049] Graphpad Prism was used to draw a bar graph of DPP-IV inhibition rate, with the three DPP-IV inhibitory peptides as the abscissa and the DPP-IV inhibition rate as the ordinate. Figure 4 As shown. Figure 4 It can be seen that the inhibition rates of DPP-IV inhibitory peptide D1A7, DPP-IV inhibitory peptide D2V11 and DPP-IV inhibitory peptide D3D4 are 56.75%, 51.88% and 53.73%, respectively, indicating that these three peptides have a strong inhibitory effect on DPP-IV enzyme activity.

[0050] In summary, the active peptide prepared by the present invention has a good effect of inhibiting DPP-IV enzyme activity, and can effectively inhibit the increase of postprandial blood sugar in patients in the early stage of T2DM. The enzymatic hydrolysis technology used in the present invention is simple and feasible, the production is fast, the equipment is easy to obtain, and the active ingredients in the fish skin can be released to the greatest extent. The active peptide obtained by the present invention has the characteristics of reasonable amino acid composition, no allergy, safety and reliability, and can be used to develop foods, health products or medicines that assist in regulating blood sugar.

[0051] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A tilapia skin DPP-IV inhibitory peptide D1A7, characterized in that: The amino acid sequence of the inhibitory peptide is as shown in SEQ ID NO:

1.

2. A tilapia skin DPP-IV inhibitory peptide D2V11, characterized in that: The amino acid sequence of the inhibitory peptide is as shown in SEQ ID NO:

2.

3. A tilapia skin DPP-IV inhibitory peptide D3D4, characterized in that The amino acid sequence of the inhibitory peptide is as shown in SEQ ID NO:

3.

4. Use of the inhibitory peptide D1A7 or a complex thereof as claimed in claim 1 in the preparation of a DPP-IV protease activity inhibitor product.

5. Use of the inhibitory peptide D2V11 or a complex thereof as claimed in claim 2 in the preparation of a DPP-IV protease activity inhibitor product.

6. Use of the inhibitory peptide D3D4 or a complex thereof as claimed in claim 3 in the preparation of a DPP-IV protease activity inhibitor product.

7. Use of the inhibitory peptide D1A7 or a complex thereof as claimed in claim 1 in the preparation of hypoglycemic drugs or health products with auxiliary hypoglycemic function.

8. Use of the inhibitory peptide D2V11 or a complex thereof as claimed in claim 2 in the preparation of hypoglycemic drugs or health products with auxiliary hypoglycemic function.

9. Use of the inhibitory peptide D3D4 or a complex thereof as claimed in claim 3 in the preparation of hypoglycemic drugs or health products with auxiliary hypoglycemic function.

10. A method for preparing the tilapia skin DPP-IV inhibitory peptide according to claims 1 to 3, comprising the following steps: S1. Preparation of tilapia skin gelatin: treating tilapia skin with an acid-base treatment method, placing the acid-base treated fish skin in distilled water and shaking for 12 hours, and taking the supernatant and freeze-drying it to obtain tilapia skin gelatin; S2. Preparation of tilapia skin gelatin solution: dissolving the tilapia skin gelatin described in step S1 in distilled water to prepare a 40 mg / mL solution; S3. Preparation of simulated gastric fluid and simulated intestinal fluid: simulated gastric fluid contained 5.51 mg / mL NaCl, 1.65 mg / mL KCl, 0.53 mg / mL NaH2PO4, 0.60 mg / mL CaCl2, 0.61 mg / mL NH4Cl and 2000 U / mL pepsin, and the pH value was adjusted to 1.4 with 1.0 M HCl. The simulated intestinal fluid contained 14.02 mg / mL NaCl, 1.13 mg / mL KCl, 6.78 mg / mL NaHCO3, 0.16 mg / mL KH2PO4, 0.10 mg / mL MgCl2, 100 U / mL trypsin and 25 U / mL chymotrypsin; S4. Preparation of tilapia skin gelatin gastric digestion hydrolysate: The tilapia skin gelatin solution prepared in step S2 was subjected to simulated gastric digestion for 2 h using the DIVH-IV system, the digestion temperature was maintained at 37 ° C, the gastric motility frequency was set to 3 times / min, the gastric juice injection rate was 1.2-1.5 mL / min, the digestion solution was removed after gastric digestion, the enzyme was heated and inactivated, and the supernatant was taken after centrifugation and freeze-dried to obtain the tilapia skin gelatin gastric digestion hydrolysate; S5. Preparation of tilapia skin gelatin intestinal digestion hydrolysate: The tilapia skin gelatin solution was subjected to simulated intestinal digestion for 4 h using the DIVH-IV system, the digestion temperature was maintained at 37 ° C, the pyloric opening size was 0-2 mm, the intestinal peristalsis frequency was 6 times / min, the simulated intestinal fluid injection rate was 0.8 mL / min, the digestion solution was removed after the gastric digestion, the enzyme was heated and inactivated, and the supernatant was taken after centrifugation and freeze-dried to obtain the tilapia skin gelatin intestinal digestion hydrolysate; S6. Separation and purification of tilapia skin active peptides with DPP-IV inhibitory activity: ultrafiltration separation of the enzymatic hydrolysate after digestion in step S4 and step S5, separation and purification of the ultrafiltration fraction with the strongest DPP IV inhibitory activity by a dextran gel column, and collection of the fraction with the strongest DPP IV inhibitory activity again to obtain a polypeptide mixture; S7. Identification, screening and synthesis of tilapia skin active peptides with DPP-IV inhibitory activity: Liquid chromatography-mass spectrometry was used to identify and analyze the peptide mixture, and then the peptides with biological activity were screened. They were then molecularly docked with the DPP IV enzyme and the peptides with the lowest binding bond energy were selected. The selected peptides were synthesized by solid phase chemical synthesis to obtain DPP-IV inhibitory peptides.

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