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

Abstract

The invention provides a DPP-IV activity inhibitory peptide as well as a preparation method and application thereof, and belongs to the technical field of active peptides. The DPP-IV activity inhibitory peptide provided by the invention comprises at least one of amino acid sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 4. According to the present invention, the DPP-IV activity inhibition peptide having effects of significant DPP-IV activity inhibition and blood sugar reducing is obtained through separation and purification from corn germ meal for the first time, wherein IC50 of LSPY, AGFAGDPR, ENPILQPY and IIPY on inhibition of DPP-IV are respectively 0.9385 mg / mL, 1.293 mg / mL, 3.177 mg / mL and 3.491 mg / mL. The DPP-IV activity inhibitory peptide provided by the invention can be used as an additive of products with the function of reducing blood sugar, such as food and medicines.

Description

Technical Field

[0001] The present invention belongs to the technical field of active peptides, and in particular relates to a DPP-IV activity inhibitory peptide and a preparation method and application thereof. Background Art

[0002] At present, drugs used to treat type 2 diabetes mainly regulate blood sugar levels in patients by inhibiting α-glucosidase and dipeptidyl peptidase-4 (Dipeptidyl Peptidase-IV, DPP-IV). DPP-IV is an enzyme that plays an important physiological function in the body. It is widely distributed in some tissues and organs, such as plasma, small intestine, kidney, liver and pancreatic beta cells. It can block the transport of glucagon-like peptide-1 (GLP-1) in the small intestine. GLP-1 can increase the secretion of insulin, inhibit the secretion of glucagon, and have a significant inhibitory effect on gastric emptying. DPP-IV inhibitors can inhibit the activity of DPP-IV, reduce the inactivation of GLP-1 and GIP, increase the content of GLP-1 and GIP in the body, and prolong the duration of GLP-1 and GIP. The indications of DPP-IV inhibitors are mainly to treat type 2 diabetes, but DPP-IV inhibitors generally have certain drug side effects. When using DPP-IV inhibitors, they may cause serious adverse reactions, such as headaches, elevated liver enzymes, upper respiratory tract infections and pancreatitis. Regarding the adverse reactions caused by drugs, seeking a safe and effective food-derived functional food to prevent and control the normal blood sugar level in the body has attracted widespread attention from researchers. In fact, in addition to taking drugs, diabetic patients can also control the increase of blood sugar through their daily living habits and dietary rules. In addition, DPP-IV inhibitory peptides can also be extracted from plant and animal sources, such as seaweed, amaranth, beans, milk protein, marine fish and cowhide.

[0003] The crude protein content in corn germ meal is about 25%, of which soluble protein accounts for about 80-88% of the whole corn germ protein. Compared with corn endosperm protein, whose protein components are mainly alcohol-soluble proteins, the main components of corn germ protein are soluble albumin and globulin (albumin (30%), globulin (30%), alcohol-soluble proteins (5%) and gluten (25%)). At present, corn germ meal is mainly used in agricultural production, mostly as a feed raw material, which wastes protein resources and causes great pollution to the environment. There is still a lack of relevant research in the field on further preparing DPP-IV active peptides using corn germ meal as raw materials. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a DPP-IV activity inhibitory peptide derived from corn germ meal.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a DPP-IV activity inhibitory peptide, which includes at least one of LSPY (SEQ ID NO.1), AGFAGDPR (SEQ ID NO.2), ENPILQPY (SEQ ID NO.3) and IIPY (SEQ ID NO.4).

[0007] The present invention also provides a corn germ protein hydrolysate, wherein the corn germ protein hydrolysate contains the above-mentioned DPP-IV activity inhibitory peptide.

[0008] The present invention also provides a method for preparing the corn germ protein hydrolysate, comprising the following steps: mixing corn germ meal with alkaline protease for enzymolysis, and centrifuging to obtain a supernatant as the corn germ protein hydrolysate; the enzymolysis temperature is 60°C, the enzymolysis time is 3 hours, and the enzymolysis pH is 7.5; and the amount of alkaline protease used is 1000U / g protein based on the protein mass in the corn germ meal.

[0009] The present invention does not specifically limit the specific source of corn germ meal, and any conventional commercially available product in the art can be used. In the present invention, before enzymatic hydrolysis, it is preferably further included the step of mixing the corn germ meal with water to prepare a suspension, and the mass concentration of the suspension is preferably 4% (based on protein). In the present invention, after the enzymatic hydrolysis is completed, it is preferably necessary to first perform an enzyme inactivation treatment and then centrifuge. The temperature of the enzyme inactivation treatment is preferably 100°C; the time of the enzyme inactivation treatment is preferably 10 minutes. In the present invention, the centrifugal speed is preferably 3000r / min, and the centrifugal time is preferably 15 minutes.

[0010] The present invention also provides a method for screening DPP-IV activity inhibitory peptides. Based on the above preparation method, the method further comprises the following steps: performing anion exchange chromatography separation on the DPP-IV activity inhibitory peptides, collecting components with the highest DPP-IV inhibitory activity, and obtaining anion exchange chromatography fluid; performing reverse phase chromatography separation on the anion exchange chromatography fluid, collecting components with the highest DPP-IV inhibitory activity, which are DPP-IV activity inhibitory peptides, wherein the DPP-IV activity inhibitory peptides comprise at least one of the amino acid sequences of LSPY, AGFAGDPR, ENPILQPY, and IIPY.

[0011] In the present invention, the ion exchanger for the anion exchange chromatography separation is preferably Q-Sepharose TMFastFlow strong anion exchanger, the eluent is preferably 20mmol / LTris-HCl containing 1mol / mLNaCl, the pH of the Tris-HCl is preferably 9.0, and the flow rate is preferably 2mL / min. In the present invention, the reversed-phase chromatography separation is first performed by semi-preparative reversed-phase chromatography separation, and then analytical reversed-phase chromatography separation. The column type of the semi-preparative reversed-phase chromatography separation is XSelect CSH C18 OBD Prep Column; the mobile phase of the semi-preparative reversed-phase chromatography separation includes mobile phase A and mobile phase B, the mobile phase A is a 2vol% acetonitrile aqueous solution containing 0.065vol% TFA, and the mobile phase B is an 80vol% acetonitrile aqueous solution containing 0.05vol% TFA. The column type of the analytical reversed-phase chromatography separation is XSelect Peptide CSH C18 Column; the mobile phase of the analytical reversed-phase chromatography separation includes mobile phase A and mobile phase B, wherein the mobile phase A is a 2 vol% acetonitrile aqueous solution containing 0.065 vol% TFA, and the mobile phase B is a 100 vol% acetonitrile aqueous solution containing 0.05 vol% TFA.

[0012] The present invention also provides the use of the above-mentioned DPP-IV activity inhibitory peptide, the above-mentioned corn germ protein hydrolysate, the above-mentioned preparation method or the above-mentioned screening method in preparing a product for inhibiting DPP-IV activity or in preparing a product for lowering blood sugar.

[0013] In the present invention, the types of the products preferably include food or medicine. When the product is food, the role played by the above-mentioned DPP-IV activity inhibitory peptide, the above-mentioned corn germ protein hydrolysate, the above-mentioned preparation method or the above-mentioned screening method is to help maintain a healthy blood sugar level.

[0014] Beneficial effects of the present invention:

[0015] The present invention is the first to separate and purify DPP-IV activity inhibitory peptides with significant DPP-IV activity inhibition and hypoglycemic efficacy from corn germ meal, wherein LSPY inhibits DPP-IV with an IC 50 The IC of AGFAGDPR to inhibit DPP-IV is 0.9385 mg / mL. 50 The IC of ENPILQPY for inhibiting DPP-IV is 1.293 mg / mL. 50 The IC of IIPY for inhibiting DPP-IV is 3.177 mg / mL. 50 The DPP-IV activity inhibitory peptide provided by the present invention can be used as an additive for products with blood sugar lowering function including food and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1It is the elution profile of ion exchange chromatography separation;

[0017] Figure 2 It is the DPP-IV inhibition result of the elution peak separated by ion exchange chromatography, where if the superscript letters of any two components are the same, it indicates that there is no significant difference in the DPP-IV inhibition rate of the two groups, p>0.05; if the superscript letters of any two components are different, it indicates that there is a significant difference in the DPP-IV inhibition rate of the two groups, p<0.05;

[0018] Figure 3 This is the elution profile of semi-preparative reverse phase chromatography;

[0019] Figure 4 is the DPP-IV inhibition result of the semi-preparative reverse phase chromatography elution peak, wherein if the superscript letters of any two components are the same, it indicates that the DPP-IV inhibition rates of the two groups are not significantly different, p>0.05; if the superscript letters of any two components are different, it indicates that the DPP-IV inhibition rates of the two groups are significantly different, p<0.05;

[0020] Figure 5 It is the analytical reverse phase chromatography elution profile;

[0021] Figure 6 It is the DPP-IV inhibition result of analytical reverse phase chromatography elution peak, wherein if the superscript letters of any two components are the same, it indicates that there is no significant difference in the DPP-IV inhibition rate of the two groups, p>0.05; if the superscript letters of any two components are different, it indicates that there is a significant difference in the DPP-IV inhibition rate of the two groups, p<0.05. DETAILED DESCRIPTION

[0022] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0023] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0024] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0025] Example 1

[0026] A DPP-IV activity inhibitory peptide, the preparation steps are as follows:

[0027] 1. Preparation of corn germ protein hydrolysate:

[0028] Corn germ meal (purchased from Jilin COFCO Co., Ltd.) was mixed with water to prepare a suspension with a substrate concentration of 4% (m / v). The pH of the suspension was adjusted to 7.5 and then alkaline protease (Alcalase 2.4L) (Novozymes (China) Biotechnology Co., Ltd.) was added. The amount of enzyme added was 1000U / g protein based on the protein content in the corn germ meal. The reaction was carried out at 60°C for 3h. After the enzymolysis was completed, the enzymolysis solution was heated at 100°C for 10min to inactivate the protease activity. The enzymolysis product was centrifuged at 3000r / min for 15min and the precipitate was discarded. The supernatant was the corn germ protease hydrolysate.

[0029] 2. Ion exchange chromatography separation

[0030] The corn germ protease hydrolysate obtained in step 1 was centrifuged and filtered through a 0.22 μm microporous membrane. TM Fast Flow Strong Anion Exchange Chromatography Starting buffer: 20mmol / L pH 9.0 Tris-HCl buffer, eluent: 20mmol / L pH 9.0 Tris-HCl buffer containing 1mol NaCl, gradient elution 170min, flow rate: 2mL / min, UV detection at 214nm. Determine the inhibition rate (hypoglycemic activity) of the collected solution in each tube on DPP-IV. The DPP-IV inhibition ability determination method is:

[0031] 40 μL of sample solution (1 mg / mL) and 10 μL of DPP-IV (100 ng / mL) solution were mixed and reacted at 37°C for 10 min. Then 50 μL of substrate Gly-Pro-PNA (Sigma Reagent Company, USA) (concentration 100 μM) was added and the reaction continued at 37°C for 1 h. After the reaction was completed, the absorbance was measured at a wavelength of 405 nm. The inhibitory activity of the sample on DPP-IV was calculated using the following formula:

[0032] Inhibition of DPP-IV activity % = [[(A c -A cb )-(A s -A sb )] / (A c -A cb )]×100%

[0033] A s : absorbance when enzyme and sample are added;

[0034] A sb : Absorbance of sample blank (sample, buffer, substrate);

[0035] A c: Absorbance of control (enzyme, substrate, buffer);

[0036] A cb : Absorbance compared with blank (buffer, substrate);

[0037] The elution profiles of ion exchange chromatography and the DPP-IV inhibition results of each peak are shown in Figure 1 and Figure 2 shown.

[0038] according to Figure 1 and Figure 2 Based on the results in , the component with the strongest blood sugar lowering function in the elution peak was collected for use, i.e. component 38.

[0039] 3. Reversed phase chromatography separation

[0040] 3.1 Determination of the active region of DPP-IV inhibitory peptides using semi-preparative reversed-phase chromatography

[0041] Weigh 5 mg of the active component (component 38) obtained in step 2 and dissolve it in 5 mL of 2% acetonitrile aqueous solution. Filter it with a 0.22 μm microporous filter membrane. The chromatographic column model is XSelect CSH C18 OBD Prep Column. The detection wavelength was 214 nm, the flow rate was 1 mL / min, the mobile phase A was a 2 vol% acetonitrile aqueous solution containing 0.065 vol% TFA, the mobile phase B was an 80 vol% acetonitrile aqueous solution containing 0.05 vol% TFA, the gradient elution was 70 min, and the elution program is shown in Table 1 below. The elution profile of the semi-preparative reverse phase chromatography is shown in Figure 3 As shown,

[0042] Table 1 Semi-preparative reversed phase chromatography gradient elution program

[0043] Time / min A / ％ B / ％ 0 98 2 15 85 15 25 70 30 30 42 58 60 9 91 65 2 98 70 98 2

[0044] Collect each component of the peak to determine the DPP-IV inhibitory activity of each tube of sample. The determination method is the same as the DPP-IV inhibitory ability determination method described in step 2. The DPP-IV inhibition results of each peak are as follows: Figure 4 And as shown in Table 2.

[0045] Table 2 DPP-IV inhibition results of semi-preparative reverse phase chromatography elution peaks

[0046] Semi-preparative reversed phase chromatography elution peak DPP-IV inhibition rate (%) 1 19.23±2.22 2 17.95±2.22 3 26.92±2.72 4 38.46±3.85 5 30.77±0.00

[0047] according to Figure 4 As shown in Table 2, it can be determined that the active component with relatively higher hypoglycemic activity is in tube 4.

[0048] 3.2 Separation of DPP-IV inhibitory peptides using analytical reversed-phase chromatography

[0049] The active component of the fourth tube obtained in step 3.1 was dissolved in 2% acetonitrile aqueous solution and further separated and purified using an analytical column. The column type of the analytical column was XSelect Peptide CSH C18 Column. The detection wavelength was 214 nm, the flow rate was 1 mL / min, and the sample volume was 20 μL. Mobile phase A was a 2 vol% acetonitrile aqueous solution containing 0.065 vol% TFA, and mobile phase B was a 100 vol% acetonitrile aqueous solution containing 0.05 vol% TFA. The gradient elution program is shown in Table 3. The elution profile of analytical reverse phase chromatography is shown in Figure 5 shown.

[0050] Table 3 Analytical reverse phase chromatography gradient elution program

[0051] Time / min A / ％ B / ％ 0 100 2 40 75 25 45 50 50 80 0 100 85 0 100 90 100 0 95 100 0

[0052] Depend on Figure 5 It can be seen that after the components were separated by the analytical column, 5 corn germ peptides were obtained, and the peak tip of each peak was collected for DPP-IV inhibitory activity determination. The determination method was the same as step 2. The DPP-IV inhibition results of each peak are shown in Figure 6 And as shown in Table 4.

[0053] Table 4 DPP-IV inhibition results of analytical reverse phase chromatography elution peaks

[0054] Analytical reverse phase chromatography elution peak DPP-IV inhibition rate (%) 1 19.12±1.05 2 20.00±1.76 3 22.50±0.00 4 25.00±0.00 5 16.25±1.76

[0055] Depend on Figure 5 and 6 The results in Table 4 indicate that Peak 4 is a DPP-IV activity inhibitory peptide with relatively higher hypoglycemic activity. Peak 4 was lyophilized for sequencing.

[0056] 4. Sequencing

[0057] The highly active peak 4 isolated in step 3.2 was sequenced, and the sequences of the DPP-IV activity inhibitory peptides were Leu-Ser-Pro-Tyr (LSPY), Ala-Gly-Phe-Ala-Gly-Asp-Pro-Arg (AGFAGDPR), Glu-Asn-Pro-Ile-Leu-Gln-Pro-Tyr (ENPILQPY) and Ile-Ile-Pro-Tyr (IIPY), with 4, 8, 8 and 4 amino acids, respectively, and molecular weights of 478.24 Da, 789.85 Da, 972.49 Da and 504.29 Da.

[0058] Example 2

[0059] According to the mass spectrometry sequencing results in Example 1, the peptides LSPY, AGFAGDPR, ENPILQPY and IIPY were chemically synthesized (commissioned to Shanghai Qiangyao Biotechnology Co., Ltd. for synthesis), and the inhibitory ability of each peptide on DPP-IV was determined. The determination method was the same as step 2 of Example 1. The results showed that the IC values ​​of the peptides LSPY, AGFAGDPR, ENPILQPY and IIPY for inhibiting DPP-IV were 50 They are 0.9385 mg / mL, 1.293 mg / mL, 3.177 mg / mL and 3.491 mg / mL respectively.

[0060] From the above results, it can be concluded that the DPP-IV activity inhibitory peptide prepared by the present invention has a high blood sugar lowering activity.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A DPP-IV activity inhibitory peptide, characterized in that: The DPP-IV activity inhibitory peptide comprises at least one of the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.

4.

2. A corn germ protein hydrolysate, characterized in that: The corn germ protein hydrolysate contains the DPP-IV activity inhibitory peptide according to claim 1.

3. The method for preparing the corn germ protein hydrolysate according to claim 2, characterized in that: The method comprises the following steps: mixing corn germ meal with alkaline protease for enzymolysis, centrifuging and taking supernatant as the corn germ protease hydrolysate; the enzymolysis temperature is 60°C, the enzymolysis time is 3 hours, and the enzymolysis pH is 7.5; the amount of alkaline protease used is 1000U / g protein based on the protein mass in the corn germ meal.

4. A method for screening a DPP-IV activity inhibitory peptide, characterized in that: Based on the preparation method described in claim 3, the method further comprises the following steps: subjecting the corn germ protein hydrolysate to anion exchange chromatography separation, collecting the component with the highest DPP-IV inhibitory activity, and obtaining an anion exchange chromatography fluid; subjecting the anion exchange chromatography fluid to reverse phase chromatography separation, collecting the component with the highest DPP-IV inhibitory activity, which is a DPP-IV active inhibitory peptide, and the DPP-IV active inhibitory peptide comprises the DPP-IV active inhibitory peptide described in claim 1.

5. The screening method according to claim 4, characterized in that The ion exchanger for the anion exchange chromatography separation is Q-Sepharose TM FastFlow strong anion exchanger, the eluent was 20 mmol / L Tris-HCl containing 1 mol / mL NaCl, the pH of the Tris-HCl was 9.0, and the flow rate was 2 mL / min.

6. The screening method according to claim 4, characterized in that The reverse phase chromatography separation is performed by first performing semi-preparative reverse phase chromatography separation and then performing analytical reverse phase chromatography separation.

7. The screening method according to claim 6, characterized in that The column type of the semi-preparative reversed-phase chromatography separation is XSelect CSH C18 OBD Prep Column; the mobile phase of the semi-preparative reversed-phase chromatography separation includes mobile phase A and mobile phase B, wherein the mobile phase A is a 2 vol% acetonitrile aqueous solution containing 0.065 vol% TFA, and the mobile phase B is an 80 vol% acetonitrile aqueous solution containing 0.05 vol% TFA.

8. The screening method according to claim 6, characterized in that The column type of the analytical reversed-phase chromatography separation is XSelect Peptide CSH C18 Column; the mobile phase of the analytical reversed-phase chromatography separation includes mobile phase A and mobile phase B, wherein mobile phase A is a 2 vol% acetonitrile aqueous solution containing 0.065 vol% TFA, and mobile phase B is a 100 vol% acetonitrile aqueous solution containing 0.05 vol% TFA.

9. Use of the DPP-IV activity inhibitory peptide according to claim 1, the corn germ protein hydrolysate according to claim 2, the preparation method according to claim 3 or the screening method according to any one of claims 4 to 8 in the preparation of a product that inhibits DPP-IV activity.

10. Use of the DPP-IV activity inhibitory peptide according to claim 1, the corn germ protein hydrolysate according to claim 2, the preparation method according to claim 3 or the screening method according to any one of claims 4 to 8 in the preparation of a blood sugar-lowering product.

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