Wheat peptide, wheat oligopeptide and preparation method and application thereof

Abstract

The invention discloses a wheat peptide, a wheat oligopeptide and a preparation method and application thereof, and belongs to the technical field of bioactive peptide preparation. The invention provides a preparation method of wheat peptide, which comprises the following steps: preparing wheat protein subjected to dry heat treatment into component dispersoid by using distilled water, and preheating in a water bath; adjusting the pH value, adding ProteAXH according to 0.5-1.0% of the mass of the substrate, and carrying out enzymolysis at 50-60 DEG C for 6-8 hours; after the enzymolysis is finished, heating for enzyme deactivation; cooling to room temperature, and centrifuging hydrolysate to obtain supernate; and freezing and drying the supernate to obtain the wheat peptide. The wheat peptide DPP-IV prepared by the preparation method disclosed by the invention is high in inhibitory activity, has alpha-glucosidase inhibitory activity and oxidation resistance, is high in safety and has an excellent hypoglycemic effect. The wheat oligopeptide screened from wheat peptides has DPP-IV inhibitory activity and alpha-glucosidase inhibitory activity at the same time, and the hypoglycemic function is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioactive peptide preparation, and particularly relates to a wheat peptide, a wheat oligopeptide, and their preparation methods and applications. Background Art

[0002] Diabetes is one of the major chronic diseases globally, with more than 90% being type II diabetes patients, characterized by insulin resistance, damaged pancreatic β-cells, and relative insulin deficiency. Diabetes usually causes serious complications, bringing a lot of pain to patients. There is also a considerable proportion of the population who, although not meeting the diabetes diagnosis criteria, have abnormal blood glucose levels. Although drugs such as liraglutide can effectively lower blood glucose, they may cause relatively serious side effects. Bioactive peptides prepared from food proteins under specific conditions have dipeptidyl peptidase IV (DPP-IV) inhibitory activity, and can promote insulin secretion by pancreatic β-cells by inhibiting the degradation of GLP-1, fundamentally controlling postprandial blood glucose levels. In addition, postprandial blood glucose levels can also be effectively controlled by inhibiting the degradation of carbohydrates. α-Glucosidase plays a key role in the digestion of polysaccharides such as starch, sucrose, and oligosaccharides, and glucose is generated during its hydrolysis process. Therefore, inhibiting the activity of α-glucosidase is also helpful for postprandial blood glucose regulation in diabetic patients. Due to impaired metabolism, the oxidative level in diabetic patients is relatively high, and a large number of free radicals generated will attack multiple tissues and organs in the body, increasing the risk of developing other metabolic diseases. Therefore, antioxidant activity is very important for repairing damaged tissue cells and promoting the health of diabetic patients.

[0003] However, in the current research on the preparation of bioactive peptides with hypoglycemic effects, most studies only focus on a specific hypoglycemic effect, such as DPP-IV inhibition or α-glucosidase inhibitory activity, so that they can only play a role in one aspect during actual application, limiting the exertion of hypoglycemic effects. Therefore, it is particularly important to provide a preparation method for bioactive peptides, so that the prepared bioactive peptides can play hypoglycemic roles from multiple aspects, thereby improving their hypoglycemic effects. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a preparation method for wheat peptides, which can improve the DPP-IV inhibitory activity of wheat peptides, and the prepared wheat peptides have DPP-IV inhibitory activity, α-glucosidase inhibitory activity, and antioxidant effects at the same time, and can play hypoglycemic roles from multiple angles, improving the hypoglycemic effect.

[0005] Another purpose of the present invention is to provide a wheat peptide prepared by the above-mentioned preparation method.

[0006] Another object of the present invention is to provide a wheat oligopeptide screened from the wheat peptide.

[0007] Another object of the present invention is to provide an application of the wheat peptide or the wheat oligopeptide in the preparation of a DPP-IV inhibitor and / or an α-glucosidase inhibitor.

[0008] Another object of the present invention is to provide an application of the wheat peptide or the wheat oligopeptide in the preparation of a hypoglycemic product.

[0009] In order to achieve the above object of the present invention, the following technical solutions are provided:

[0010] The present invention provides a preparation method of a wheat peptide, and the preparation method includes the following steps: preparing a dispersion of wheat protein after dry heat treatment with distilled water and preheating in a water bath; adjusting the pH, adding ProteAXH according to 0.5% to 1.0% of the substrate mass, and enzymatically hydrolyzing at 50 to 60 °C for 6 to 8 h; after the enzymatic hydrolysis is completed, heating to inactivate the enzyme; after cooling to room temperature, centrifuging the hydrolyzate to obtain a supernatant; and freeze-drying the supernatant to obtain the wheat peptide.

[0011] Preferably, the conditions of the dry heat treatment include: baking at 80 to 90 °C for 20 to 40 min.

[0012] Preferably, the mass-volume percentage of wheat protein in the dispersion is 6% to 11%.

[0013] Preferably, the conditions of the preheating include: preheating at 50 to 60 °C for 20 to 40 min.

[0014] Preferably, the pH is 6.5 to 8.5.

[0015] Preferably, the conditions of inactivating the enzyme include: heating in a water bath at 90 to 100 °C for 5 to 15 min.

[0016] Preferably, the centrifugation conditions include centrifuging at 8000 to 12000×g for 5 to 15 min.

[0017] The present invention also provides a wheat peptide prepared by the above preparation method.

[0018] The present invention also provides a wheat oligopeptide screened from the wheat peptide, and the amino acid sequence of the wheat oligopeptide is FPQP.

[0019] The present invention also provides an application of the wheat peptide or the wheat oligopeptide in the preparation of a DPP-IV inhibitor and / or an α-glucosidase inhibitor.

[0020] The present invention also provides an application of the wheat peptide or the wheat oligopeptide in the preparation of a hypoglycemic product.

[0021] Advantages of the present invention:

[0022] The wheat peptide prepared by the preparation method of the present invention has significantly improved DPP-IV inhibitory activity, and at the same time has α-glucosidase inhibitory activity and antioxidant activity. It has no obvious inhibitory effect on the viability of HepG2 cells, has high safety for liver cells, can promote the uptake and consumption of glucose by high-glucose-induced insulin-tolerant HepG2 cells, and has excellent hypoglycemic effects.

[0023] The wheat peptide of the present invention simultaneously exhibits DPP-IV and α-glucosidase inhibitory activities. It can promote the secretion of insulin by pancreatic islet β cells to reduce blood sugar by inhibiting the degradation of GLP-1, and can also slow down the decomposition rate of polysaccharides such as starch into glucose, reduce and delay the absorption of glucose by the small intestine to lower blood sugar. At the same time, the wheat peptide of the present invention also has antioxidant activity. Through the combined action in multiple aspects and from multiple angles, it can effectively reduce blood sugar and improve the hypoglycemic effect.

[0024] The present invention screens a wheat oligopeptide from wheat peptides. The IC 50 of the DPP-IV inhibitory activity reaches 0.68 mg / mL, which can significantly inhibit the activity of DPP-IV, effectively reduce blood sugar, and improve the hypoglycemic function; this wheat oligopeptide also has α-glucosidase inhibitory activity, and the IC 50 of the α-glucosidase inhibitory activity reaches 0.06 mg / mL, which can effectively reduce blood sugar and improve the hypoglycemic function. Description of the drawings

[0025] Figure 1 It is a micrograph of HepG2 cells under different treatments in Test Example 2 (scale bar 100 μm);

[0026] Figure 2 It is the viability of HepG2 cells treated with wheat peptides at different concentrations in Test Example 2, where ns indicates no significant difference;

[0027] Figure 3 It is the viability of HepG2 cells treated with wheat peptides at different concentrations in Test Example 2;

[0028] Figure 4 It is the relative glucose consumption of high-glucose-induced insulin-tolerant HepG2 cells under different treatments in Test Example 3, where * indicates P < 0.05;

[0029] Figure 5 It is the glucose consumption of high-glucose-induced insulin-tolerant HepG2 cells under different treatments in Test Example 3, where * indicates P < 0.05;

[0030] Figure 6 For the relative fluorescence intensity of high - glucose - induced insulin - resistant HepG2 cells in Test Example 3 under different treatments, where * indicates P < 0.05 and ** indicates P < 0.01;

[0031] Figure 7 It is the mass spectrometry detection result graph in Test Example 4. Detailed implementation mode

[0032] The present invention provides a preparation method of wheat peptides. The preparation method includes the following steps: preparing a dispersion of heat - treated wheat protein with distilled water and preheating it in a water bath; adjusting the pH, adding ProteAXH at 0.5% - 1.0% of the substrate mass, and enzymatically hydrolyzing at 50 - 60 °C for 6 - 8 h; after the enzymatic hydrolysis ends, heating to inactivate the enzyme; after cooling to room temperature, centrifuging the hydrolyzate to obtain a supernatant; and freeze - drying the supernatant to obtain wheat peptides.

[0033] In the present invention, there is no special limitation on the type and source of the wheat protein, which can be conventionally selected according to actual needs. Preferably, it is wheat gluten protein, which can be obtained through regular commercial channels. The preferred heat treatment includes: baking the wheat protein on a baking tray; the baking temperature is preferably 80 - 90 °C, more preferably 85 °C or 82.5 - 87.5 °C or 84 - 86 °C; the baking time is preferably 20 - 40 min, more preferably 30 min or 25 - 35 min or 28 - 32 min; preferably, stir - turning 1 - 2 times during baking. After the wheat protein is heat - treated, it is immediately cooled to room temperature.

[0034] In the present invention, the mass - volume (g / mL) percentage of the heat - treated wheat protein in the dispersion is preferably 6% - 11%, more preferably 10% or 8% - 10.5% or 9% - 10.25%; the preheating temperature is preferably 50 - 60 °C, more preferably 55 °C or 52.5 - 57.5 °C or 54 - 56 °C; the preheating time is preferably 20 - 40 min, more preferably 30 min or 25 - 35 min or 28 - 32 min.

[0035] In the present invention, the pH of the system can be adjusted by conventional methods as needed, and it is preferably adjusted with NaOH. The pH is preferably 6.5 - 8.5, more preferably 7 or 6.8 - 8 or 6.9 - 7.5. The ProteAXH can be obtained by purchasing through conventional channels, and the enzyme activity is preferably ≥1600 U / g. The addition amount of the ProteAXH is preferably 0.8% or 0.6% - 1.0% or 0.7% - 0.85% of the substrate mass. The temperature of the enzymatic hydrolysis is preferably 55°C or 52.5 - 57.5°C or 54 - 56°C; the time of the enzymatic hydrolysis is preferably 7 h or 6.5 - 7.5 h or 6.8 - 7.2 h.

[0036] In the present invention, the enzyme inactivation by heating is preferably carried out by heating in a water bath; the temperature of the enzyme inactivation is preferably 90 - 100°C, more preferably 95°C or 92.5 - 97.5°C or 94 - 96°C; the time of the enzyme inactivation is preferably 5 - 15 min, more preferably 10 min or 7.5 - 12.5 min or 9 - 11 min.

[0037] In the present invention, the rotation speed of the centrifugation is preferably 8000 - 12000 × g, more preferably 10000 × g or 9000 - 11000 × g or 9500 - 10500 × g; the time of the centrifugation is preferably 5 - 15 min, more preferably 10 min or 7.5 - 12.5 min or 9 - 11 min. In the present invention, the method and condition parameters of the freeze-drying can be conventionally selected according to actual needs.

[0038] By adjusting the enzymatic hydrolysis conditions, the preparation method of the present invention can release and enrich more bioactive peptides with DPP-IV inhibitory activity, expose more active sites, significantly improve the DPP-IV inhibitory activity, and the inhibitory activity is increased by 13% - 25%; meanwhile, the wheat peptides prepared by the preparation method of the present invention also have α-glucosidase inhibitory activity and antioxidant activity, have no obvious inhibitory effect on the viability of HepG2 cells, have high safety for liver cells, and can promote the uptake and consumption of glucose by high-glucose-induced insulin-tolerant HepG2 cells, thus showing excellent hypoglycemic effects.

[0039] The present invention also provides a wheat peptide prepared by the above preparation method.

[0040] The present invention also provides a wheat oligopeptide screened from the wheat peptide, and the amino acid sequence of the wheat oligopeptide is FPQP.

[0041] The IC of the DPP-IV inhibitory activity of the wheat oligopeptide screened by the present invention 50is 0.68 mg / mL, which can significantly inhibit DPP-IV activity and has excellent hypoglycemic effects. At the same time, the wheat oligopeptide also has α-glucosidase inhibitory activity, and the IC 50 reaches 0.06 mg / mL, which can effectively reduce blood sugar and improve hypoglycemic function.

[0042] The present invention also provides an application of the wheat peptide or the wheat oligopeptide in the preparation of a DPP-IV inhibitor and / or an α-glucosidase inhibitor.

[0043] The wheat peptide of the present invention has inhibitory effects on both DPP-IV and α-glucosidase. The inhibitory activity against DPP-IV reaches 62.10% - 62.61%, and the inhibitory activity against α-glucosidase reaches 95.47 ± 0.17%, which can be used to prepare a DPP-IV inhibitor and / or an α-glucosidase inhibitor. The IC 50 of the wheat oligopeptide of the present invention for DPP-IV inhibitory activity is 0.68 mg / mL, which can be used to prepare a DPP-IV inhibitor; the IC 50 of the α-glucosidase inhibitory activity reaches 0.06 mg / mL, which can be used to prepare an α-glucosidase inhibitor.

[0044] The present invention also provides an application of the wheat peptide or the wheat oligopeptide in the preparation of a hypoglycemic product.

[0045] Both the wheat peptide and the wheat oligopeptide of the present invention simultaneously exhibit significant DPP-IV inhibitory activity and α-glucosidase inhibitory activity. They can promote insulin secretion by pancreatic islet β cells to reduce blood sugar by inhibiting GLP-1 degradation, and can also slow down the decomposition rate of polysaccharides such as starch into glucose, reduce and delay the absorption of glucose by the small intestine to lower blood sugar. At the same time, the wheat peptide of the present invention also has antioxidant activity. Through the combined action in multiple aspects and from multiple angles, it can effectively reduce blood sugar and improve hypoglycemic effects. In addition, the wheat peptide of the present invention has high safety and can be used to prepare hypoglycemic products. In the present invention, the types of the products are not particularly limited, and preferably include health foods or drugs. The addition forms and addition doses can be conventionally selected according to actual needs.

[0046] The following examples are used to illustrate the technical solutions provided by the present invention in detail, but they should not be construed as limiting the protection scope of the present invention.

[0047] In the following examples, unless otherwise specified, all are conventional methods.

[0048] The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0049] In the following examples, the wheat protein is wheat gluten protein (WGP), containing 82.9% (w / w, dry basis) protein and 7.2% moisture, and is purchased from Henan Huaxin Company. ProteAXH is purchased from Amano Enzyme Co., Ltd. in Japan, and its enzyme activity is ≥1600 U / g.

[0050] Example 1

[0051] A method for preparing wheat peptides, the steps of the method are as follows:

[0052] Pretreatment: The wheat protein is subjected to dry heat treatment: The wheat protein is evenly spread on a plate and baked at 85 °C for 30 min, and it is stirred once during this period. At the end of the heat treatment, the sample is immediately cooled to ambient temperature and stored at -20 °C.

[0053] Enzymatic hydrolysis: The pretreated wheat protein is made into a 10% (w / v, g / mL) dispersion with distilled water, and it is preheated in a water bath at 55 °C for 30 min. The pH is adjusted to 7.0 with NaOH, and the addition amount of ProteAXH is 0.8% of the substrate mass. Enzymatic hydrolysis is carried out at 55 °C for 7 h. After the enzymatic hydrolysis is completed, it is heated in a water bath at 95 °C for 10 min to inactivate the protease. After cooling to room temperature, the hydrolyzate is centrifuged at 10000×g for 10 min. Subsequently, the supernatant is frozen and freeze-dried to obtain wheat peptides. The wheat peptides are stored at -20 °C for further analysis.

[0054] Example 2

[0055] A method for preparing wheat peptides, the steps of the method are as follows:

[0056] Pretreatment: The wheat protein is subjected to dry heat treatment: The wheat protein is evenly spread on a plate and baked at 85 °C for 30 min, and it is stirred once during this period. At the end of the heat treatment, the sample is immediately cooled to ambient temperature and stored at -20 °C.

[0057] Enzymatic hydrolysis: The pretreated wheat protein is made into an 8% (w / v, g / mL) dispersion with distilled water, and it is preheated in a water bath at 50 °C for 30 min. The pH is adjusted to 7.0 with NaOH, and the addition amount of ProteAXH is 1% of the substrate addition amount. Enzymatic hydrolysis is carried out at 50 °C for 6 h. After the enzymatic hydrolysis is completed, it is heated in a water bath at 95 °C for 10 min to inactivate the protease. After cooling to room temperature, the hydrolyzate is centrifuged at 10000×g for 10 min. Subsequently, the supernatant is frozen and freeze-dried to obtain wheat peptides. The wheat peptides are stored at -20 °C for further analysis.

[0058] Example 3

[0059] A method for preparing wheat peptides, the steps of the method are as follows:

[0060] Pretreatment: The wheat protein was subjected to dry heat treatment: The wheat protein was evenly spread on a plate and baked at 80 °C for 40 min, during which it was stirred twice. At the end of the heat treatment, the sample was immediately cooled to ambient temperature and stored at -20 °C.

[0061] Enzymatic hydrolysis: The pretreated wheat protein was made into a 6% (w / v, g / mL) dispersion with distilled water, and it was preheated in a water bath at 50 °C for 40 min. The pH was adjusted to 6.5 with NaOH, and the addition amount of ProteAXH was 0.5% of the substrate mass. It was enzymatically hydrolyzed at 50 °C for 8 h. After the enzymatic hydrolysis was completed, it was heated in a water bath at 90 °C for 15 min to inactivate the protease. After cooling to room temperature, the hydrolysate was centrifuged at 8000×g for 15 min. Subsequently, the supernatant was frozen and freeze-dried to obtain wheat peptides. The wheat peptides were stored at -20 °C for further analysis.

[0062] Example 4

[0063] Method for preparing wheat peptides, the steps of the method are as follows:

[0064] Pretreatment: The wheat protein was subjected to dry heat treatment: The wheat protein was evenly spread on a plate and baked at 90 °C for 20 min, during which it was stirred once. At the end of the heat treatment, the sample was immediately cooled to ambient temperature and stored at -20 °C.

[0065] Enzymatic hydrolysis: The pretreated wheat protein was made into an 11% (w / v, g / mL) dispersion with distilled water, and it was preheated in a water bath at 60 °C for 20 min. The pH was adjusted to 8.5 with NaOH, and the addition amount of ProteAXH was 1% of the substrate mass. It was enzymatically hydrolyzed at 60 °C for 6 h. After the enzymatic hydrolysis was completed, it was heated in a water bath at 100 °C for 5 min to inactivate the protease. After cooling to room temperature, the hydrolysate was centrifuged at 12000×g for 5 min. Subsequently, the supernatant was frozen and freeze-dried to obtain wheat peptides. The wheat peptides were stored at -20 °C for further analysis.

[0066] Example 5

[0067] Method for preparing wheat peptides, the steps of the method are as follows:

[0068] Pretreatment: The wheat protein was subjected to dry heat treatment: The wheat protein was evenly spread on a plate and baked at 82.5 °C for 35 min, during which it was stirred twice. At the end of the heat treatment, the sample was immediately cooled to ambient temperature and stored at -20 °C.

[0069] Enzymatic hydrolysis: The pretreated wheat protein was made into an 8% (w / v, g / mL) dispersion with distilled water, and it was preheated in a water bath at 52.5 °C for 35 min. The pH was adjusted to 6.8 with NaOH, and the addition amount of ProteAXH was 0.6% of the substrate mass. It was enzymatically hydrolyzed at 52.5 °C for 7.5 h. After the enzymatic hydrolysis was completed, it was heated in a water bath at 92.5 °C for 12.5 min to inactivate the protease. After cooling to room temperature, the hydrolyzate was centrifuged at 9000×g for 12.5 min. Subsequently, the supernatant was frozen and freeze-dried to obtain wheat peptides. The wheat peptides were stored at -20 °C for further analysis.

[0070] Example 6

[0071] Method for preparing wheat peptides, the steps of the method are as follows:

[0072] Pretreatment: The wheat protein was subjected to dry heat treatment: The wheat protein was evenly spread on a plate and baked at 87.5 °C for 25 min, and it was stirred once during this period. At the end of the heat treatment, the sample was immediately cooled to ambient temperature and stored at -20 °C.

[0073] Enzymatic hydrolysis: The pretreated wheat protein was made into a 10.5% (w / v, g / mL) dispersion with distilled water, and it was preheated in a water bath at 57.5 °C for 25 min. The pH was adjusted to 8 with NaOH, and the addition amount of ProteAXH was 0.9% of the substrate mass. It was enzymatically hydrolyzed at 57.5 °C for 6.5 h. After the enzymatic hydrolysis was completed, it was heated in a water bath at 97.5 °C for 7.5 min to inactivate the protease. After cooling to room temperature, the hydrolyzate was centrifuged at 11000×g for 7.5 min. Subsequently, the supernatant was frozen and freeze-dried to obtain wheat peptides. The wheat peptides were stored at -20 °C for further analysis.

[0074] Comparative Example 1

[0075] Method for preparing wheat peptides, the steps of the method are as follows:

[0076] Pretreatment: The wheat protein was subjected to dry heat treatment: The wheat protein was evenly spread on a plate and baked at 85 °C for 30 min, and it was stirred once during this period. At the end of the heat treatment, the sample was immediately cooled to ambient temperature and stored at -20 °C.

[0077] Enzymatic hydrolysis: The pretreated wheat protein was formulated into an 8% (w / v, g / mL) dispersion with distilled water, and it was preheated in a water bath at 55 °C for 30 min. The pH was adjusted to 7.0 with NaOH, and the addition amount of ProteAXH was 1% of the substrate mass. It was enzymatically hydrolyzed at 55 °C for 4 h. After the enzymatic hydrolysis was completed, it was heated in a water bath at 95 °C for 10 min to inactivate the protease. After cooling to room temperature, the hydrolyzate was centrifuged at 10000×g for 10 min. Subsequently, the supernatant was frozen and freeze-dried to obtain wheat peptides. The wheat peptides were stored at -20 °C for further analysis.

[0078] Comparative Example 2

[0079] Preparation method of wheat peptides, the method steps are as follows:

[0080] Pretreatment: The wheat protein was subjected to dry heat treatment: The wheat protein was evenly spread on a plate and baked at 85 °C for 30 min, and it was stirred once during this period. At the end of the heat treatment, the sample was immediately cooled to ambient temperature and stored at -20 °C.

[0081] Enzymatic hydrolysis: The pretreated wheat protein was formulated into an 8% (w / v, g / mL) dispersion with distilled water, and it was preheated in a water bath at 50 °C for 30 min. The pH was adjusted to 7.0 with NaOH, and the addition amount of ProteAXH was 0.6% of the substrate mass. It was enzymatically hydrolyzed at 50 °C for 4 h. After the enzymatic hydrolysis was completed, it was heated in a water bath at 95 °C for 10 min to inactivate the protease. After cooling to room temperature, the hydrolyzate was centrifuged at 10000×g for 10 min. Subsequently, the supernatant was frozen and freeze-dried to obtain wheat peptides. The wheat peptides were stored at -20 °C for further analysis.

[0082] Comparative Example 3

[0083] Preparation method of wheat peptides, the method steps are as follows:

[0084] Pretreatment: The wheat protein was subjected to dry heat treatment: The wheat protein was evenly spread on a plate and baked at 85 °C for 30 min, and it was stirred once during this period. At the end of the heat treatment, the sample was immediately cooled to ambient temperature and stored at -20 °C.

[0085] Enzymatic hydrolysis: The pretreated wheat protein was formulated into a 10% (w / v, g / mL) dispersion with distilled water, and it was preheated in a water bath at 45 °C for 30 min. The pH was adjusted to 7.0 with NaOH, and the addition amount of ProteAXH was 0.4% of the substrate mass. It was enzymatically hydrolyzed at 45 °C for 7 h. After the enzymatic hydrolysis was completed, it was heated in a water bath at 95 °C for 10 min to inactivate the protease. After cooling to room temperature, the hydrolysate was centrifuged at 10000×g for 10 min. Subsequently, the supernatant was frozen and freeze-dried to obtain wheat peptides. The wheat peptides were stored at -20 °C for further analysis.

[0086] Test Example 1 Determination of Biological Activities of Different Wheat Peptides

[0087] 1. Respectively take the wheat peptides prepared in Examples 1-2 and Comparative Examples 1-3, and use the colorimetric method to detect the DPP-IV inhibitory activity of different wheat peptides. Mix 25 μL of wheat peptide (final concentration 1.0 mg / mL) with 50 μL of reaction substrate (GP-pNA, final concentration 0.200 mM). The reaction was initiated by adding 50 μL of DPP-IV (final concentration 2.5 mU / mL). All reagents were diluted in 100 mmol / L Tris-HCl buffer at pH 8.0. An equal volume of Tris-HCl buffer was used instead of the sample as a negative control. After incubating at 37 °C for 30 minutes in a microplate reader, the absorbance was monitored at 405 nm. The degree of hydrolysis of different wheat peptides was detected by the OPA method, and the results are shown in Table 1.

[0088] Table 1 Biological Activities of Different Wheat Peptides

[0089] Group Degree of hydrolysis (%) DPP-IV inhibitory activity (%) Example 1 34.35±1.38 62.61±2.28 Example 2 24.11±1.12 62.10±1.61 Comparative Example 1 32.50±0.22 54.83±0.73 Comparative Example 2 14.76±0.83 50.81±4.06 Comparative Example 3 15.68±0.13 50.00±3.22

[0090] As can be seen from the results in Table 1, compared with Comparative Examples 1-3, the DPP-IV inhibitory activity of the wheat peptides prepared in Example 1 of the present invention increased by 14% - 25%; the DPP-IV inhibitory activity of the wheat peptides prepared in Example 2 of the present invention increased by 13% - 24%. As can be seen from the results of Example 1 and Comparative Example 1, by adjusting the enzymatic hydrolysis conditions in Example 1, under the condition of comparable hydrolysis degree, the preparation method of Example 1 can release and enrich more active peptides with DPP-IV inhibitory activity, expose more active sites, and significantly improve the DPP-IV inhibitory activity. After detection, the IC 50 value of the DPP-IV inhibitory activity of the wheat peptides prepared by the preparation method of Example 1 was 0.71 mg / mL. It shows that the preparation method of the present invention can significantly improve the DPP-IV inhibitory activity of wheat peptides.

[0091] 2. Take the wheat peptides prepared in Example 1 and use the colorimetric method to detect the α-glucosidase inhibitory activity, hydroxyl radical scavenging rate, and ABTS radical scavenging rate of the wheat peptides respectively. The results are shown in Table 2.

[0092] Table 2 α-Glucosidase inhibitory activity and antioxidant activity of different wheat peptides

[0093]

[0094] As can be seen from the results in Table 2, the wheat peptides of the present invention exhibit significant α-glucosidase inhibitory activity and significant antioxidant activity, indicating that the wheat peptides prepared by the preparation method of the present invention can simultaneously exhibit DPP-IV and α-glucosidase inhibitory activities, and can promote the secretion of insulin by pancreatic islet β cells to lower blood glucose by inhibiting the degradation of GLP-1. It can also slow down the rate of decomposition of polysaccharides such as starch into glucose, reduce and delay the absorption of glucose by the small intestine to lower blood glucose, and play a hypoglycemic effect through multi-faceted and multi-angle joint action; at the same time, it also shows that the wheat peptides of the present invention can promote tissue repair and protect cells from oxidative damage while controlling blood glucose levels, thus playing a positive role in diabetes management and complication prevention.

[0095] Test Example 2 Cytotoxicity experiment

[0096] Determine the safety of the wheat peptides prepared in Example 1 using the insulin-tolerant HepG2 cell line.

[0097] 1. Seed HepG2 cells into 12-well plates and divide them into 4 treatments:

[0098] High glucose + wheat peptide group: Cultured with a culture medium containing 45 mmol / L glucose and treated with 800 μg / mL of the wheat peptides prepared in Example 1;

[0099] High glucose + insulin group: Cultured with a culture medium containing 45 mmol / L glucose and treated with 1×10 -3 mmol / L insulin;

[0100] High glucose group: Cultured with a culture medium containing 45 mmol / L glucose;

[0101] Normal cell group: Cultured with a culture medium containing 25 mmol / L glucose;

[0102] Cultivation method: Cells were cultured in cell culture medium containing glucose (25 mmol / L) or high glucose (45 mmol / L) for 12 h, and cells in the high glucose + wheat peptide group and the high glucose + insulin group were treated with corresponding concentrations of wheat peptide or insulin for 24 h. CCK-8 was added to the cells and incubated in the dark for 3 h, and the absorbance at 450 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader. The results are as Figure 1 shown.

[0103] It can be Figure 1 seen that from the cell morphology, compared with the normal cell group, the cell density in the high glucose group slightly increased and the cell morphology was slightly irregular. This indicates that the high glucose environment has an impact on the cells, and it can be preliminarily judged that the induction of HepG2 cells by high glucose was successful. In addition, the cell morphology of the high glucose + wheat peptide group was similar to that of the high glucose group, and there were no obvious typical toxic manifestations such as obvious cell death, a large number of cell lyses, and cell shrinkage. This can preliminarily show that at this dose (800 μg / mL), wheat peptide has no obvious acute toxic effect on the cells, indicating that the wheat peptide prepared in Example 1 has no obvious inhibitory effect on the viability of HepG2 cells.

[0104] 2. HepG2 cells were seeded in 96-well plates and cultured in glucose culture medium containing 45 mmol / L for 12 h. The volume of the culture medium in each well was 100 μL, and the experiment was carried out when the cell confluence reached 80%. Cells were treated with different concentrations of the wheat peptide prepared in Example 1 (50, 100, 200, 400, 800, 1600 μg / mL) at 37 °C for 24 h, and the treatment without wheat peptide was used as the control group. CCK-8 was added to the cells and incubated in the dark for 3 h, and the absorbance at 450 nm was detected using an ELISA reader. The results are as Figure 2 shown (the wheat peptide concentration is the abscissa and the cell survival rate is the ordinate).

[0105] Using the same method as above, the wheat peptide prepared in Example 1 was added to HepG2 cells at final concentrations of 0.1, 1, 10, 100, 1000, 10000, and 100000 μg / mL, respectively, and cultured at 37 °C for 24 h. CCK-8 was added to the cells and incubated in the dark for 3 h, and the absorbance at 450 nm was detected using an ELISA reader. The results are as Figure 3 shown (the logarithm of the wheat peptide concentration to the base 10 is the abscissa and the cell survival rate is the ordinate).

[0106] It can be Figure 2 - Figure 3 seen from the results that the wheat peptide prepared in Example 1 has no obvious inhibitory effect on the viability of HepG2 cells at an addition amount of 0.1 - 1 × 10 5 μg / mL, indicating that it has high safety for liver cells in a wide concentration range.

[0107] Experimental Example 3 Determination of Hypoglycemic Effect of Wheat Peptide

[0108] 1. The consumption of glucose by HepG2 cells was determined by the hexokinase method

[0109] HepG2 cells were seeded in 12-well plates and divided into 5 treatments:

[0110] Control group: cultured with a culture medium containing 25 mmol / L glucose;

[0111] High-glucose group: cultured with a culture medium containing 45 mmol / L glucose;

[0112] High-glucose + insulin group: cultured with a culture medium containing 45 mmol / L glucose and treated with 1×10 -3 mmol / L insulin;

[0113] High-glucose + 200 μg / mL peptide group: cultured with a culture medium containing 45 mmol / L glucose and treated with 200 μg / mL wheat peptide prepared in Example 1;

[0114] High-glucose + 400 μg / mL peptide group: cultured with a culture medium containing 45 mmol / L glucose and treated with 400 μg / mL wheat peptide prepared in Example 1.

[0115] Culture method: Cells were cultured with cell culture medium containing normal concentration glucose (25 mmol / L) or high concentration glucose (45 mmol / L) for 12 h. The high-glucose + 200 μg / mL peptide group, high-glucose + 400 μg / mL peptide group and high-glucose + insulin group were treated with corresponding concentrations of wheat peptide or insulin for 24 h.

[0116] Detection method: Collect the cell culture medium and centrifuge at 1000 rpm / min for 10 min. Add the supernatant and standard product to a 96-well plate and operate according to the instructions of the glucose detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing). Add 200 μL of R1 reagent to the supernatant, incubate at 37°C for 3 min, and measure the absorbance A1 at 340 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Then add 50 μL of R2 reagent to the supernatant, incubate at 37°C for 5 min, and measure the absorbance A2 at 340 nm again with an ELISA reader. Calculate △A = A2 - A1. The glucose content and glucose consumption were calculated according to the following formulas:

[0117] Glucose content (mmol / L) = (△A determination - △A blank) / (△A standard product - △A blank) × standard product concentration (5.5 mmol / L)

[0118] Glucose consumption (mmol / L) = Glucose addition in the medium - Glucose content in each group

[0119] The relative glucose consumption (%) is the percentage of the glucose consumption in each group relative to that in the control group.

[0120] The results are as Figure 4 - Figure 5 shown. From Figure 4 and Figure 5 the results, it can be seen that when the addition amount of wheat peptide is 400 μg / mL, it can significantly promote the glucose consumption of high-glucose-induced HepG2 cells.

[0121] 2. Effect on glucose uptake of high-glucose-induced insulin-tolerant HepG2 cells

[0122] HepG2 cells were seeded in 12-well plates and divided into 6 treatments:

[0123] Control group: Cultured with a culture medium containing 25 mmol / L glucose;

[0124] High-glucose group: Cultured with a culture medium containing 45 mmol / L glucose;

[0125] High-glucose + insulin group: Cultured with a culture medium containing 45 mmol / L glucose and treated with 1×10 -3 mmol / L insulin;

[0126] High-glucose + 800 μg / mL wheat peptide group: Cultured with a culture medium containing 45 mmol / L glucose and treated with the wheat peptide prepared in Example 1 at 800 μg / mL;

[0127] High-glucose + 1000 μg / mL wheat peptide group: Cultured with a culture medium containing 45 mmol / L glucose and treated with the wheat peptide prepared in Example 1 at 1000 μg / mL;

[0128] High-glucose + 2000 μg / mL wheat peptide group: Cultured with a culture medium containing 45 mmol / L glucose and treated with the wheat peptide prepared in Example 1 at 2000 μg / mL.

[0129] Culture method: Cells were cultured with cell culture medium containing glucose (25 mmol / L) or high glucose (45 mmol / L) for 12 h, and the high-glucose + 800 μg / mL peptide group, high-glucose + 1000 μg / mL peptide group, high-glucose + 2000 μg / mL peptide group and high-glucose + insulin group were treated with the corresponding concentration of wheat peptide or insulin for 24 h respectively.

[0130] Detection method: Wash the cells twice with PBS, add 50 μmol / L of 2-NBDG (Beyotime Biotechnology Research Institute, Shanghai) into the cells, incubate at 37 °C for 30 min, and then wash twice with PBS to remove the residual 2-NBDG. Digest the cells with trypsin and collect them in a black 96-well plate, and detect the fluorescence intensity at the excitation / emission wavelength of 485 nm / 530 nm with a fluorescence microplate reader. The results are as Figure 6 shown.

[0131] The glucose uptake ability of cells not only reflects the energy demand of cells, but also reflects the metabolic state of cells. As Figure 6 shown by the results, wheat peptides at 800 - 1000 μg / mL significantly increased the uptake of the fluorescently labeled 2-deoxyglucose analog 2-NBDG by cells. It shows that the wheat peptides prepared in the present invention can promote the glucose uptake of high-glucose-induced insulin-tolerant HepG2 cells.

[0132] Test Example 4 Screening and Synthesis of Wheat Oligopeptides

[0133] 1. Perform reductive alkylation treatment on the wheat peptides prepared in Example 1, and then determine the identification results of the wheat peptides by liquid chromatography-tandem mass spectrometry (LC-MS / MS) and analyze them with PEAKS Studio (10.6). The results are as Figure 7 shown. Use bioinformatics tools such as PeptideRanker and UniDL4BioPep to predict the activity and analyze the physicochemical properties of oligopeptides with less than ten amino acids. For peptide segments that may have biological activity, further analyze their binding energy with DPP-IV by molecular docking. Through screening, four wheat oligopeptides were synthesized by Nanjing Peptide Biotechnology Co., Ltd. for the next step of analysis. The amino acid sequences of the four wheat oligopeptides are shown in Table 3.

[0134] Table 3 Amino Acid Sequences of Wheat Oligopeptides

[0135]

[0136]

[0137] 2. The DPP-IV inhibitory activity of the four synthesized wheat oligopeptides was determined by spectrophotometry. 25 μL of the wheat oligopeptide solution (dissolved in 0.1 M phosphate buffer, pH 6.8) was mixed with 50 μL of the reaction substrate (GP-pNA, final concentration 0.200 mM). The reaction was initiated by adding 50 μL of DPP-IV (final concentration 2.5 mU / mL). All reagents were diluted in 100 mmol / L Tris-HCl buffer, pH 8.0. An equal volume of Tris-HCl buffer was used instead of the sample as a negative control. After incubation at 37 °C for 30 minutes in a microplate reader, the absorbance was monitored at 405 nm.

[0138] The results showed that the IC 50 of wheat oligopeptide FPQP was 0.68 mg / mL (equivalent to 1.39 mM), indicating that the screened wheat oligopeptide FPQP had significant DPP-IV inhibitory activity.

[0139] 3. The α-glucosidase inhibitory activity of wheat oligopeptide FPQP was determined by spectrophotometry. 50 μL of the synthesized wheat oligopeptide FPQP solution (dissolved in 0.1 M phosphate buffer, pH 6.8) was mixed with 50 μL of α-glucosidase (0.35 U / mL, Sigma) in a microcentrifuge tube and incubated at 37 °C for 10 minutes. Subsequently, 50 μL of p-nitrophenyl α-D-glucoside (1.5 mM) was added and further incubated at 37 °C for 20 minutes. Then, 100 μL of 1 M Na2CO3 was added as a termination reagent to the reaction. After transferring the solution to a 96-well plate, the absorbance of the released p-nitrophenol was measured at 405 nm. An equal volume of 0.1 M phosphate buffer (pH 6.8) was used instead of the sample as a blank.

[0140] The results showed that the IC 50 of wheat oligopeptide FPQP was 0.06 mg / mL (equivalent to 0.12 mM), indicating that the screened wheat oligopeptide FPQP had significant α-glucosidase inhibitory activity.

[0141] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing wheat peptides, characterized in that, The preparation method comprises the following steps: preparing a dispersion of heat-dried wheat protein with distilled water and preheating it in a water bath; adjusting the pH, adding ProteAXH at 0.5% - 1.0% of the substrate mass, and enzymatically hydrolyzing for 6 - 8 h at 50 - 60 °C; after the enzymatic hydrolysis ends, heating to inactivate the enzyme; after cooling to room temperature, centrifuging the hydrolyzate to obtain a supernatant; and freeze-drying the supernatant to obtain wheat peptides.

2. The preparation method according to claim 1, characterized in that, The conditions for the heat drying treatment include: baking at 80 - 90 °C for 20 - 40 min.

3. The preparation method according to claim 1, characterized in that, The mass-volume percentage of wheat protein in the dispersion is 6% - 11%.

4. The preparation method according to claim 1, wherein The conditions for the preheating include: preheating at 50 - 60 °C for 20 - 40 min.

5. The preparation method according to claim 1, characterized in that, The pH is 6.5 - 8.

5.

6. The preparation method according to claim 1, wherein The conditions for inactivating the enzyme include: heating in a water bath at 90 - 100 °C for 5 - 15 min.

7. Wheat peptides prepared by the preparation method according to any one of claims 1 - 6.

8. Wheat oligopeptide screened from the wheat peptide according to claim 7, characterized in that, The amino acid sequence of the wheat oligopeptide is FPQP.

9. Use of the wheat peptide according to claim 7 or the wheat oligopeptide according to claim 8 in the preparation of DPP-IV inhibitors and / or α-glucosidase inhibitors.

10. Use of the wheat peptide according to claim 7 or the wheat oligopeptide according to claim 8 in the preparation of hypoglycemic products.

Images