Corn hypoglycemic peptide as well as preparation method and application thereof

Abstract

The invention provides a corn hypoglycemic peptide as well as a preparation method and application thereof, and belongs to the technical field of active peptides. The amino acid sequence of the corn hypoglycemic peptide provided by the invention is as shown in SEQ ID NO. 1. According to the invention, the corn hypoglycemic peptide with remarkable DPP-IV (dipeptidyl peptidase-IV) inhibition activity and hypoglycemic effect is obtained by separation and purification from corn germ meal for the first time, and IC50 of the DPP-IV inhibition rate is 0.024 mg / mL.

Description

Technical Field

[0001] The present invention belongs to the technical field of active peptides, and in particular relates to a corn blood sugar-lowering peptide and a preparation method and application thereof. Background Art

[0002] Current drugs used to treat type 2 diabetes primarily regulate blood glucose levels by inhibiting α-glucosidase and dipeptidyl peptidase-IV (DPP-IV). DPP-IV is an enzyme that plays a crucial physiological role in the body, blocking the transport of glucagon-like peptide-1 (GLP-1) in the small intestine. GLP-1 increases insulin secretion, inhibits glucagon secretion, and significantly inhibits gastric emptying. DPP-IV inhibitors inhibit DPP-IV activity, reducing the inactivation of GLP-1 and gastric inhibitory polypeptide (GIP), increasing GLP-1 and GIP levels in the body, and prolonging the duration of GLP-1 and GIP effects. DPP-IV inhibitors are primarily indicated for the treatment of type 2 diabetes, but they are commonly associated with side effects. Their use can cause serious adverse reactions, such as upper respiratory tract infections, nasopharyngitis, and headaches. In fact, many patients with type 2 diabetes can prevent elevated blood glucose levels by improving their lifestyle and dietary habits, in addition to medication. Studies have shown that functional foods have fewer toxic side effects than synthetic drugs. Therefore, the search for a safe and non-toxic functional food to prevent the occurrence and development of diabetes has attracted widespread attention. Studies have reported that the intake of special foods or dietary ingredients is associated with the incidence of hyperglycemia. Special diets can also play a key role in the prevention of type 2 diabetes. One of the reasons is that food components have the ability to inhibit DPP-IV. Hypoglycemic peptides are a class of active oligopeptides or polypeptides that are endogenous to organisms or produced by the hydrolysis of biological proteins. They have the effect of inhibiting α-glucosidase and DPP-IV. In the research field of hypoglycemic peptides, edible plant-derived hypoglycemic peptides are more widely available and safer and more reliable than chemically synthesized α-glucosidase and DPP-IV inhibitors. Therefore, the production and application of hypoglycemic peptides have gradually expanded to become a research hotspot in the food industry.

[0003] Corn germ meal contains approximately 25% crude protein, of which soluble protein accounts for approximately 80-88% of the total corn germ protein. Compared to corn endosperm protein, which primarily consists of alcohol-soluble proteins, corn germ protein primarily consists of soluble albumins and globulins (albumin (30%), globulins (30%), alcohol-soluble proteins (5%), and glutenins (25%)). Corn germ protein has a relatively balanced amino acid profile and a bioavailability (BV) of 64-72%. Corn germ protein contains lysine, which is lacking in most cereal proteins, and is richer in lysine and tryptophan than corn endosperm protein. Therefore, corn germ protein can be used as a high-quality protein raw material for further processing. However, research on the preparation of hypoglycemic peptides using corn germ meal as a raw material is lacking. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a corn blood sugar lowering 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: The present invention provides a corn hypoglycemic peptide, wherein the amino acid sequence of the corn hypoglycemic peptide is PPAT (SEQ ID NO. 1).

[0006] The present invention also provides a corn germ protein hydrolysate, which contains the corn blood sugar-lowering peptide.

[0007] 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; ultrafiltration of the supernatant using an ultrafiltration membrane, wherein the ultrafiltration membrane has a molecular weight cutoff of 3 kDa, and collecting a permeate with a molecular weight less than 3 kDa 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 1000 U / g protein based on the protein content in the corn germ meal.

[0008] The present invention does not specifically limit the specific source of corn germ meal; any commercially available product in this field can be used. In the present invention, before enzymatic hydrolysis, the corn germ meal is preferably mixed with water to form a suspension. The mass concentration of the suspension is preferably 4% (based on protein). In the present invention, after enzymatic hydrolysis, an enzyme inactivation treatment is preferably performed, followed by centrifugation. The temperature for the enzyme inactivation treatment is preferably 100°C; the duration of the enzyme inactivation treatment is preferably 10 minutes. In the present invention, the centrifugation speed is preferably 3000 rpm, and the duration of the centrifugation is preferably 15 minutes.

[0009] The present invention also provides a method for screening corn hypoglycemic peptides, which, based on the above-mentioned preparation method, further comprises the following steps: subjecting the permeate to anion exchange chromatography to separate the fraction with the highest DPP-IV inhibitory activity, thereby obtaining an anion exchange chromatographic fluid; subjecting the anion exchange chromatographic fluid to reverse phase chromatography to separate the fraction with the highest DPP-IV inhibitory activity, thereby obtaining the corn hypoglycemic peptide. The corn hypoglycemic peptide comprises a corn hypoglycemic peptide having an amino acid sequence of PPAT.

[0010] In the present invention, the ion exchanger for anion exchange chromatography separation is preferably Q-Sepharose TM FastFlow strong anion exchanger, the eluent is preferably 20mmol / L Tris-HCl containing 0.5mol / mL NaCl, 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 as a semi-preparative reversed-phase chromatography separation, and then as an 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 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.

[0011] The present invention also provides the use of the corn hypoglycemic peptide, the corn germ protein hydrolysate, the preparation method or the screening method in preparing a product that inhibits DPP-IV activity or in preparing a product that lowers blood sugar.

[0012] In the present invention, the types of the products preferably include food or medicine. When the product is food, the above-mentioned corn blood sugar-lowering peptide, the above-mentioned corn germ protein hydrolysate, the above-mentioned preparation method or the above-mentioned screening method plays a role in helping to maintain healthy blood sugar levels.

[0013] Beneficial effects of the present invention: The present invention is the first to separate and purify corn germ meal to obtain corn hypoglycemic peptides with significant DPP-IV inhibition and blood sugar lowering effects. The DPP-IV inhibition rate is IC 50 It is 0.024mg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the elution profile of ion exchange chromatography; Figure 2 The DPP-IV inhibition results of the elution peak separated by ion exchange chromatography are shown in Table 1. 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 between 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 between the two groups (p<0.05). Figure 3 This is the elution profile of semi-preparative reverse phase chromatography; Figure 4 The DPP-IV inhibition results of the semi-preparative reverse-phase chromatography elution peaks are shown. If the superscript letters of any two components are the same, it indicates that there is no significant difference in the DPP-IV inhibition rates between 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 rates between the two groups (p<0.05). Figure 5 This is the analytical reverse phase chromatography elution profile; Figure 6 The DPP-IV inhibition results of the analytical reverse phase chromatography elution peaks, 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 rates 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 rates of the two groups, p<0.05; Figure 7 This is the mass spectrometry detection diagram of the polypeptide PPAT. DETAILED DESCRIPTION

[0015] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0016] In the following examples, unless otherwise specified, all methods are conventional.

[0017] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0018] Example 1 A corn hypoglycemic peptide, the preparation steps are as follows: 1. Preparation of supernatant: Corn germ meal (purchased from Jilin COFCO Co., Ltd.) was mixed with water to prepare a 4% (m / v) substrate suspension. The pH of the suspension was adjusted to 7.5, and alkaline protease (Alcalase 2.4 L, Novozymes (China) Biotechnology Co., Ltd.) was added at a rate of 1000 U / g protein, based on the protein content of the corn germ meal. The reaction was incubated at 60°C for 3 h. After enzymatic hydrolysis, the hydrolyzate was heated at 100°C for 10 min to inactivate the protease. The hydrolyzate was centrifuged at 3000 rpm for 15 min, and the precipitate was discarded to obtain the supernatant.

[0019] 2. Ultrafiltration of the supernatant obtained in step 1 was performed using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain permeates with molecular weights >3 kDa and <3 kDa. The DPP-IV inhibitory abilities of the two permeates were determined to evaluate their hypoglycemic activity. The DPP-IV inhibitory ability determination method was as follows: Mix 40 μL of sample solution (1 mg / mL) and 10 μL of DPP-IV (100 ng / mL) solution and react at 37°C for 10 minutes. Then, add 50 μL of the substrate Gly-Pro-PNA (Sigma-Aldrich, USA) (100 μM concentration) and continue reacting at 37°C for 1 hour. After the reaction is complete, the absorbance is measured at 405 nm. The inhibitory activity of the sample against DPP-IV is calculated using the following formula: A s : absorbance when enzyme and sample are added; A sb : absorbance of sample blank (sample, buffer, substrate); A c : absorbance of control (enzyme, substrate, buffer); A cb : absorbance when compared with blank (buffer, substrate); The results are shown in Table 1. After ultrafiltration, the inhibition rate of the fraction <3 kDa on DPP-IV was much higher than that of the fraction >3 kDa. Therefore, the permeate with a molecular weight <3 kDa, i.e., corn germ protein hydrolysate, was selected for the next step of separation and purification.

[0020] Table 1 Inhibitory activity of different fractions obtained by ultrafiltration against DPP-IV

[0021] 3. Ion exchange chromatography separation The permeate (corn germ protein hydrolysate) with a concentration of <3 kDa obtained in step 2 was passed through a 0.22 μm microporous filter membrane and the filtrate was subjected to Q-Sepharose TMFast Flow strong anion exchange chromatography (φ1.6cm×25cm). Starting buffer: 20mmol / L pH 9.0 Tris-HCl buffer, eluent: 20mmol / L pH 9.0 Tris-HCl buffer containing 0.5mol NaCl, gradient elution 170min, flow rate: 2mL / min, detection at UV214nm. Determine the inhibition rate (hypoglycemic activity) of the collected fluid in each tube on DPP-IV, the determination method is the same as step 2 above. The elution profile of ion exchange chromatography separation and the DPP-IV inhibition results of each peak are shown as follows: Figure 1 and Figure 2 shown.

[0022] 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 future use, namely component 27.

[0023] 4. Reversed-phase chromatography separation 4.1 Determination of the active region of DPP-IV inhibitory peptides using a semi-preparative reversed-phase column 5 mg of the active component (component 27) obtained in step 3 was weighed and dissolved in 5 mL of 2% acetonitrile aqueous solution, and filtered through a 0.22 μm microporous filter membrane. The chromatographic column model was XSelect CSH C18 OBD Prep Column, 130Å (φ 10 mm × 250 mm, 10 μm), the detection wavelength was 214 nm, the flow rate was 1 mL / min, the mobile phase A was 2 vol% acetonitrile aqueous solution containing 0.065 vol% TFA, and the mobile phase B was 80 vol% acetonitrile aqueous solution containing 0.05 vol% TFA. The gradient elution was performed for 53 min. The elution program is shown in Table 2 below. The elution profile of the semi-preparative reversed-phase chromatography is shown in Table 2. Figure 3 As shown, Table 2 Semi-preparative reversed-phase chromatography gradient elution program

[0024] Collect the components of the peak and determine the DPP-IV inhibitory activity of each tube 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 3.

[0025] Table 3 DPP-IV inhibition results of semi-preparative reverse phase chromatography elution peaks

[0026] according to Figure 4 As shown in Table 3, it can be determined that the active component with relatively higher hypoglycemic activity is in tube 1.

[0027] 4.2 Separation of DPP-IV Inhibitory Peptides by Analytical Reversed-Phase Chromatographic Column The active component from tube 1 obtained in step 4.1 was dissolved in 2% acetonitrile aqueous solution and further separated and purified using an analytical column. The analytical column type was an XSelect Peptide CSH C18 Column, 130Å (φ 4.6mm×150mm, 3.5μm), with a detection wavelength of 214nm, a flow rate of 1mL / min, and a sample volume of 20μL. Mobile phase A was 2vol% acetonitrile aqueous solution containing 0.065vol% TFA, and mobile phase B was 100vol% acetonitrile aqueous solution containing 0.05vol% TFA. The gradient elution program is shown in Table 4. The elution profile of the analytical reversed-phase chromatography is shown in Figure 5 shown.

[0028] Table 4 Analytical reversed-phase chromatography gradient elution program

[0029] Depend on Figure 5 It can be seen that after the components were separated by the analytical column, 6 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 2. Figure 6 and as shown in Table 5.

[0030] Table 5 DPP-IV inhibition results of analytical reverse phase chromatography elution peaks

[0031] Depend on Figure 5 and 6 The results in Table 5 indicate that Peak 3 is a corn hypoglycemic peptide with relatively higher hypoglycemic activity. Peak 3 was lyophilized and prepared for mass spectrometry sequencing.

[0032] 5. Mass spectrometry sequencing The peak 3 with high activity isolated in step 4.2 was subjected to mass spectrometry sequencing to obtain the sequence of corn hypoglycemic peptide: Pro-Pro-Ala-Thr (PPAT), with 4 amino acids and a molecular weight of 384.2009 Da. The mass spectrum of the determination is shown in FIG. Figure 7 shown.

[0033] Example 2 According to the mass spectrometry sequencing results in Example 1, the peptide PPAT was chemically synthesized (commissioned to Shanghai Qiangyao Biotechnology Co., Ltd. for synthesis), and the inhibitory ability of corn hypoglycemic peptide PPAT on DPP-IV was determined. The determination method was the same as step 2 of Example 1. The results showed that the IC of corn hypoglycemic peptide PPAT on DPP-IV inhibition was 50 The value is 0.024mg / mL.

[0034] From the above results, it can be concluded that the corn hypoglycemic peptide prepared by the present invention has a high hypoglycemic activity.

[0035] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A corn hypoglycemic peptide, characterized in that: The amino acid sequence of the corn hypoglycemic peptide is shown in SEQ ID NO.

1.

2. A corn germ protein hydrolysate, characterized in that: The corn germ protein hydrolysate contains the corn blood sugar lowering 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, and centrifuging to obtain supernatant; ultrafiltering the supernatant with an ultrafiltration membrane, wherein the cut-off molecular weight of the ultrafiltration membrane is 3 kDa, and collecting permeate with a molecular weight less than 3 kDa as the corn germ protease hydrolyzate; the enzymolysis temperature is 60 DEG C, the enzymolysis time is 3 hours, and the enzymolysis pH is 7.5; and the amount of alkaline protease used is 1000 U / g protein based on the protein mass in the corn germ meal.

4. A method for screening corn hypoglycemic peptides, characterized in that: Based on the preparation method described in claim 3, the method further comprises the following steps: subjecting the permeate to anion exchange chromatography separation, collecting the component with the highest DPP-IV inhibitory activity to obtain 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 to obtain a corn glucose-lowering peptide, wherein the corn glucose-lowering peptide comprises the corn glucose-lowering 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 Fast Flow strong anion exchanger, the eluent is 20 mmol / L Tris-HCl containing 0.5 mol / mL NaCl, the pH of the Tris-HCl is 9.0, and the flow rate is 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 mobile phase A is a 2 vol% acetonitrile aqueous solution containing 0.065 vol% TFA, and 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 corn glucose-lowering 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 corn blood sugar lowering 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 blood sugar lowering products.

Images