Dipeptidyl peptidase-IV inhibitor in sheep bone as well as preparation method and application of dipeptidyl peptidase-IV inhibitor

Abstract

The invention discloses a dipeptidyl peptidase-IV inhibitor in sheep bones as well as a preparation method and application thereof, and relates to the technical field of biology. The sheep bone dipeptidyl peptidase-IV inhibitor is obtained by the following steps: cleaning and crushing sheep bones, cooking under high pressure, drying and crushing, degreasing bone meal, carrying out ultrasonic pretreatment, carrying out enzymolysis by using protease, carrying out enzyme deactivation after enzymolysis, centrifuging, and freeze-drying supernate. The optimal process conditions are obtained by controlling the influence of the ultrasonic pretreatment power, time, enzymolysis protease type, enzyme addition amount, temperature, pH and time on the DPP-IV inhibition rate and hydrolysis degree, and the prepared DPP-IV inhibitor is high in DPP-IV inhibition rate and hydrolysis degree, has high practical value and can be used for developing common food and health care products or applied to the field of medicine.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a dipeptidyl peptidase-IV inhibitor in sheep bones and a preparation method and application thereof. Background Art

[0002] Sheep bones, as a by-product of the sheep slaughtering process, are very nutritious and contain a lot of nutrients such as protein, fat, minerals, etc. However, due to the backwardness of sheep bone deep processing technology in my country, sheep bone resources cannot be fully utilized, resulting in a huge waste of resources. Therefore, the development of high value-added products using sheep bones has become one of the mainstreams of the industry development.

[0003] Incretins are a group of metabolic hormones that promote the decrease of blood sugar levels. They are released after eating and increase the secretion of insulin released by pancreatic β cells through a blood sugar-dependent mechanism. Incretins mainly include glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Both GLP-1 and GIP can be rapidly inactivated by dipeptidyl peptidase-IV (DPP-IV), thus losing their hypoglycemic effect. Therefore, preventing the degradation of incretins by inhibiting dipeptidyl peptidase-IV has gradually become a powerful means of treating type 2 diabetes. Common oral dipeptidyl peptidase-IV inhibitors for type 2 diabetes that have been marketed include sitagliptin, vildagliptin, etc. However, due to the long-term use of drug treatment, adverse reactions such as edema, heart failure, diarrhea, and increased risk of fractures may occur. Therefore, it is crucial to develop natural, safe and side-effect-free hypoglycemic drugs. The traditional enzymatic hydrolysis method for preparing bioactive peptides is costly and the resulting polypeptides have low activity. Therefore, the present invention aims to improve the biological activity of the prepared polypeptides by using ultrasound-assisted enzymatic hydrolysis, which has application prospects in the inhibition of dipeptidyl peptidase-IV and the treatment of type II diabetes. Summary of the invention

[0004] The purpose of the present invention is to provide a dipeptidyl peptidase-IV inhibitor in sheep bones and a preparation method thereof. The extracted inhibitor can significantly inhibit dipeptidyl peptidase-IV and has application potential in the treatment of type II diabetes.

[0005] In order to achieve the above object, the present invention provides a method for preparing a dipeptidyl peptidase-IV inhibitor in sheep bones, comprising the following steps: S1: Fresh sheep bones were washed and crushed, and then steamed under a pressure of 0.1 MPa; S2: drying the steamed sheep bones and then crushing them, and passing them through an 80-mesh sieve to obtain sheep bone powder; S3: mixing the sheep bone powder and anhydrous ethanol and stirring them, washing them repeatedly with distilled water after stirring, discarding the supernatant after centrifugation, and drying the precipitate to obtain defatted sheep bone powder; S4: dissolving defatted sheep bone powder in distilled water to obtain a sheep bone powder solution, and then performing ultrasonic treatment; S5: adding protease to the sheep bone powder solution after ultrasonic treatment for enzymatic hydrolysis, and performing enzyme inactivation treatment after enzymatic hydrolysis to obtain sheep bone enzymatic hydrolyzate; S6: cooling the sheep bone enzymatic hydrolyzate to room temperature, centrifuging and collecting the supernatant to obtain a sheep bone polypeptide solution; S7: freeze-drying the sheep bone polypeptide solution to obtain a final product which is a dipeptidyl peptidase-IV inhibitor.

[0006] Preferably, the mass ratio of sheep bone powder to anhydrous ethanol in the above S3 is 1:10; the centrifugation is 8000 r / min for 15 min; and the drying temperature is 80°C.

[0007] More preferably, the sheep bone powder and anhydrous ethanol are mixed in a mass ratio of 1:10 and stirred for 24 hours, and the anhydrous ethanol is replaced every 6 hours.

[0008] Preferably, the mass percentage concentration of defatted sheep bone powder in the sheep bone powder solution in the above S4 is 8%; the ultrasonic treatment has an ultrasonic temperature of 25°C, an ultrasonic power of 360 W, and an ultrasonic time of 30 min.

[0009] Preferably, the protease in S5 is papain; and the enzyme inactivation treatment is heating at 100° C. for 15 min.

[0010] More preferably, the amount of papain is 8000 U / g, the pH of the enzymatic hydrolysis system is adjusted to 6.0, and the enzymatic hydrolysis is carried out at 55° C. for 4 h.

[0011] Preferably, the centrifugation in S6 is performed at 8000 r / min for 15 min.

[0012] Preferably, the above-mentioned freeze-drying is selected by segmented drying, the first segment: -35°C, 2h; the second segment: -25°C, 8h; the third segment: -20°C, 6h; the fourth segment: -10°C, 8h; the fifth segment: 0°C, 2h; the sixth segment: 10°C, 3h; wherein the second segment is set as a vacuum segment.

[0013] The present invention also provides a dipeptidyl peptidase-IV inhibitor prepared by the above preparation method, and the use of the inhibitor in any of the following items includes: For the inhibition of dipeptidyl peptidase-IV; Relieve insulin resistance and tolerance, stabilize blood sugar concentration; Used for the treatment of type 2 diabetes.

[0014] The present invention has the following advantages: The invention uses sheep bones as raw materials and uses ultrasound-assisted enzymatic hydrolysis technology to prepare active peptides with high DPP-IV inhibitory ability. Through a large number of studies, extraction process parameters suitable for sheep bone dipeptidyl peptidase-IV inhibitors are obtained, the power and time in the ultrasonic pretreatment process are controlled, and the effects of the type of added protease, enzyme addition amount, temperature, pH and time in the enzymatic hydrolysis process on the hydrolysis degree and the DPP-IV inhibition rate are controlled, and the optimal sheep bone dipeptidyl peptidase-IV inhibitor preparation process conditions are obtained, and the DPP-IV inhibitory activity thereof is greatly improved.

[0015] The inhibitor provided by the sheep bone of the present invention can be used for the treatment of type II diabetes, and has significant application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The results are the results of measuring the effect of the inhibitor prepared in the present invention on the fasting blood glucose level of rats.

[0017] Figure 2 The results of oral glucose tolerance test in rats after oral administration of the inhibitor prepared in the present invention for four weeks. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Note: The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0020] Example 1 This embodiment provides a method for extracting and preparing a dipeptidyl peptidase-IV inhibitor from sheep bones, which is as follows: (1) Raw material pretreatment: Select fresh sheep bones, remove the meat and fat remaining on the surface, clean them, and break them into pieces of about 5 cm; (2) High-pressure cooking: Place the broken sheep bone fragments in a high-temperature back-pressure cooking pot, add ultrapure water, cook at a pressure of 0.1 MPa for 40 min, and then rinse; (3) Preparation of sheep bone powder: The sheep bones after high-pressure steaming were dried in an oven at 75°C, crushed using a grinder (1000W, 27000r / min), and passed through an 80-mesh sieve to obtain sheep bone powder; (4) Defatting sheep bone powder: Mix sheep bone powder and anhydrous ethanol in a ratio of 1:10 (w / w), place on a magnetic stirrer and stir for 24 h, replace anhydrous ethanol every 6 h, wash repeatedly with distilled water after stirring, centrifuge at 8000 r / min for 15 min, discard the supernatant, and place in an oven at 80 °C to dry to obtain defatted sheep bone powder; (5) Enzymatic hydrolysis: Defatted sheep bone powder was prepared into a solution with a mass percentage concentration of 8% using distilled water, and alkaline protease (Beijing Solebow Technology Co., Ltd., B8360-100) was added for enzymatic hydrolysis at an amount of 8000 U / g. The pH was adjusted to 9.0 using 0.5 mol / L NaOH and 0.5 mol / L HCl. The enzymatic hydrolysis was carried out at a temperature of 50°C for 4 h. The enzymatic hydrolysis product was heated at 100°C for 15 min to inactivate the enzyme, thereby obtaining a sheep bone enzymatic hydrolyzate. (6) Centrifugation: The obtained sheep bone enzymatic hydrolysate was cooled to room temperature, and then centrifuged at 8000 r / min for 15 min in a low-temperature high-speed centrifuge to obtain the supernatant, which is the sheep bone polypeptide solution; (7) Freeze drying: The obtained sheep bone polypeptide solution was freeze dried in a freeze dryer for 29 hours. The freeze drying was performed in stages: the first stage: -35°C, 2 hours; the second stage: -25°C, 8 hours; the third stage: -20°C, 6 hours; the fourth stage: -10°C, 8 hours; the fifth stage: 0°C, 2 hours; the sixth stage: 10°C, 3 hours. The second stage was set to vacuum conditions. The final product sample was obtained after the drying was completed.

[0021] Example 2 This embodiment also provides a method for extracting and preparing a dipeptidyl peptidase-IV inhibitor from sheep bones, which is as follows: (1) Raw material pretreatment: Select fresh sheep bones, remove the meat and fat remaining on the surface, clean them, and break them into pieces of about 5 cm; (2) High-pressure cooking: Place the crushed sheep bones in a high-temperature back-pressure cooking pot, add ultrapure water, cook at a pressure of 0.1 MPa for 40 min, and then rinse; (3) Preparation of sheep bone powder: The sheep bones after high-pressure steaming were dried in an oven at 75°C, crushed using a grinder (1000W, 27000r / min), and passed through an 80-mesh sieve to obtain sheep bone powder; (4) Defatting sheep bone powder: Mix sheep bone powder and anhydrous ethanol in a ratio of 1:10 (w / w), place on a magnetic stirrer and stir for 24 h, replace anhydrous ethanol every 6 h, wash repeatedly with distilled water after stirring, centrifuge at 8000 r / min for 15 min, discard the supernatant, and place in an oven at 80°C to dry to obtain defatted sheep bone powder; (5) Enzymatic hydrolysis: Papain (CP8131-10g, Beijing Coolbo Technology Co., Ltd.) was added to the sheep bone powder solution after ultrasonic pretreatment for enzymatic hydrolysis. The enzyme amount was 8000 U / g, and the pH was adjusted to 7.0. The enzymatic hydrolysis temperature was 50°C for 4 h. After the enzymatic hydrolysis, the solution was heated at 100°C for 15 min to inactivate the enzyme, and the sheep bone enzymatic hydrolyzate was obtained. (6) Centrifugation: The obtained sheep bone enzymatic hydrolysate was cooled to room temperature, and then centrifuged at 8000 r / min for 15 min in a low-temperature high-speed centrifuge to obtain the supernatant, which is the sheep bone polypeptide solution; (7) Freeze drying: The obtained sheep bone polypeptide solution was freeze dried in a freeze dryer for 29 hours. The freeze drying was performed in stages: the first stage: -35°C, 2 hours; the second stage: -25°C, 8 hours; the third stage: -20°C, 6 hours; the fourth stage: -10°C, 8 hours; the fifth stage: 0°C, 2 hours; the sixth stage: 10°C, 3 hours. The second stage was set as a vacuum stage. The final product sample was obtained after the drying was completed.

[0022] Example 3 This embodiment also provides a method for extracting and preparing a dipeptidyl peptidase-IV inhibitor from sheep bones, which is as follows: (1) Raw material pretreatment: Select fresh sheep bones, remove the meat and fat remaining on the surface, and then wash and crush them; (2) High-pressure cooking: Place the crushed sheep bones in a high-temperature back-pressure cooking pot, add ultrapure water, cook at a pressure of 0.1 MPa for 40 min, and then rinse; (3) Preparation of sheep bone powder: The sheep bones after high-pressure steaming were dried in an oven at 75°C, crushed using a grinder (1000W, 27000r / min), and passed through an 80-mesh sieve to obtain sheep bone powder; (4) Defatting sheep bone powder: Mix sheep bone powder and anhydrous ethanol in a ratio of 1:10 (w / w), place on a magnetic stirrer and stir for 24 h, replace anhydrous ethanol every 6 h, wash repeatedly with distilled water after stirring, centrifuge at 8000 r / min for 15 min, discard the supernatant, and place in an oven at 80°C to dry to obtain defatted sheep bone powder; (5) Ultrasonic pretreatment: Use distilled water to prepare a solution with a mass percentage concentration of 8% of defatted sheep bone powder, set the ultrasonic parameters to 25 °C, ultrasonic power to 440 W, and ultrasonic time to 10 min; (6) Enzymatic hydrolysis: Add papain to the sheep bone powder solution after ultrasonic pretreatment for enzymatic hydrolysis, the enzyme amount is 8000 U / g, the pH is adjusted to 6.0, the enzymatic hydrolysis temperature is 55°C for 4 h, and after the enzymatic hydrolysis, the solution is heated at 100°C for 15 min to inactivate the enzyme, thereby obtaining a sheep bone enzymatic hydrolyzate; (7) Centrifugation: After cooling the obtained sheep bone enzymatic hydrolysate to room temperature, centrifuge it in a low-temperature high-speed centrifuge at 8000 r / min for 15 min to obtain the supernatant, which is the sheep bone polypeptide solution; (8) Freeze drying: The obtained sheep bone polypeptide solution was freeze dried in a freeze dryer for 29 hours. The freeze drying was performed in stages: the first stage: -35°C, 2 hours; the second stage: -25°C, 8 hours; the third stage: -20°C, 6 hours; the fourth stage: -10°C, 8 hours; the fifth stage: 0°C, 2 hours; the sixth stage: 10°C, 3 hours. The second stage was set as a vacuum stage. The final product sample was obtained after the drying was completed.

[0023] Example 4 This embodiment also provides a method for extracting and preparing a dipeptidyl peptidase-IV inhibitor from sheep bones, which is as follows: (1) Raw material pretreatment: Select fresh sheep bones, remove the meat and fat remaining on the surface, and then wash and crush them; (2) High-pressure cooking: Place the crushed sheep bones in a high-temperature back-pressure cooking pot, add ultrapure water, cook at a pressure of 0.1 MPa for 40 min, and then rinse; (3) Preparation of sheep bone powder: The sheep bones after high-pressure steaming were dried in an oven at 75°C, crushed using a grinder (1000W, 27000r / min), and passed through an 80-mesh sieve to obtain sheep bone powder; (4) Defatting sheep bone powder: Mix sheep bone powder and anhydrous ethanol in a ratio of 1:10 (w / w), place on a magnetic stirrer and stir for 24 h, replace anhydrous ethanol every 6 h, wash repeatedly with distilled water after stirring, centrifuge at 8000 r / min for 15 min, discard the supernatant, and place in an oven at 80°C to dry to obtain defatted sheep bone powder; (5) Ultrasonic pretreatment: Use distilled water to prepare a solution with a mass percentage concentration of 8% of defatted sheep bone powder, and set the ultrasonic parameters to 25 °C, ultrasonic power to 360 W, and ultrasonic time to 30 min; (6) Enzymatic hydrolysis: Add papain to the sheep bone powder solution after ultrasonic pretreatment for enzymatic hydrolysis, the enzyme amount is 8000 U / g, the pH is adjusted to 6.0, the enzymatic hydrolysis temperature is 55°C for 4 h, and after the enzymatic hydrolysis, the solution is heated at 100°C for 15 min to inactivate the enzyme, thereby obtaining a sheep bone enzymatic hydrolyzate; (7) Centrifugation: After cooling the obtained sheep bone enzymatic hydrolysate to room temperature, centrifuge it in a low-temperature high-speed centrifuge at 8000 r / min for 15 min to obtain the supernatant, which is the sheep bone polypeptide solution; (8) Freeze drying: The obtained sheep bone polypeptide solution was freeze dried in a freeze dryer for 29 hours. The freeze drying was performed in stages: the first stage: -35°C, 2 hours; the second stage: -25°C, 8 hours; the third stage: -20°C, 6 hours; the fourth stage: -10°C, 8 hours; the fifth stage: 0°C, 2 hours; the sixth stage: 10°C, 3 hours. The second stage was set as a vacuum stage. The final product sample was obtained after the drying was completed.

[0024] Experimental Example 1 The dipeptidyl peptidase-IV (DPP-IV) inhibitory effect of the final product prepared in Examples 1 to 4 above was determined, including the hydrolysis degree of the final product sample and the inhibition rate of DPP-IV, as follows: 1. In vitro validation Among them, the DPP-IV inhibition rate determination method is as follows: DPP-IV enzyme and glycylproline p-nitroanilide (GPP) are respectively prepared into 0.025 U / mL and 2 mmol / L solutions with 100 mol / L Tris-HCL buffer at pH=8.0, and the prepared final product samples are respectively prepared into 20 mg / mL peptide solutions with Tris-HCL buffer. 25 μL of GPP solution and 25 μL of peptide solution are transferred to a 96-well ELISA plate, mixed thoroughly, incubated at 37 °C for 10 min, and then 50 μL of DPP-IV enzyme solution is added to start the reaction, and the reaction is fully reacted at 37 °C for 60 min. The absorbance value at a wavelength of 405 nm is detected and recorded by an ELISA instrument.

[0025] Among them, the wells added with DPP-IV enzyme solution, final product sample polypeptide solution and GPP solution were recorded as sample groups, A 样品组 is the absorbance value after the reaction of this group; Tris-HCL buffer is used instead of A 样品组 The wells containing DPP-IV enzyme solution were recorded as sample control group, A 样品对照组 The wells to which Tris-HCL buffer, DPP-IV enzyme solution and GPP solution were added were recorded as the enzyme activity group, B 酶活性组 is the absorbance value after the reaction of this group; Tris-HCL buffer was used instead of B 酶活性组 The wells containing the DPP-IV enzyme solution in B were recorded as the enzyme blank group, 酶空白组 is the absorbance value after the reaction. Three parallel reactions were performed for each sample and the calculation was performed according to the following formula (1):

[0026] DPP-IV inhibition rate = ×100% Formula (1) The degree of hydrolysis is calculated as follows: 5 mL of sheep bone hydrolysate and 25 mL of distilled water are added to a beaker, and the solution is titrated to pH = 8.2 with 0.1 M NaOH standard solution. Then, 10 mL of formaldehyde solution is added, stirred evenly, and titrated with 0.1 M NaOH standard solution until the pH is 9.2. The volume of NaOH solution consumed from pH = 8.2 to pH = 9.2 is recorded as V1. At the same time, 5.0 mL of deionized water is used to replace the sheep bone hydrolysate as a blank group for a blank test, which is recorded as V0. Calculate according to the following formula (2):

[0027] D H = Formula (2) Wherein, V is the total volume of sheep bone enzymatic hydrolysate (mL), C is the actual concentration of NaOH (mol / L), 5 is the amount of sheep bone enzymatic hydrolysate used, and TPN is the protein content in sheep bone.

[0028] The hydrolysis degree and DPP-IV inhibition rate of the final product prepared in the above embodiment were determined by the above method, and the results are shown in Table 1 below. Dipeptidyl peptidase-IV inhibitory peptides were prepared from sheep bones using different preparation processes. The results show that the use of different proteases will affect the activity of the prepared dipeptidyl peptidase-IV inhibitory peptides. Papain has great potential in the preparation of DPP-IV inhibitory peptides. At the same time, the microjet generated by the cavitation of ultrasonic treatment can help open the protein structure, expose more enzyme action sites, and have a positive effect on the enzymatic reaction. Therefore, the effect of preparing DPP-IV inhibitory peptides in combination with ultrasonic pretreatment technology before enzymatic hydrolysis is better than that of single enzyme enzymatic hydrolysis. By optimizing the process of preparing polypeptides by ultrasonic-assisted enzymatic hydrolysis, the DPP-IV inhibition rate of the polypeptides finally prepared can reach 60.95±0.29%. It can be seen that the final product prepared in this embodiment has a significant inhibitory effect on DPP-IV, can be used as an inhibitor for the inhibition of DPP-IV, and has application prospects in the treatment of type II diabetes.

[0029] Table 1 Hydrolysis degree and DPP-IV inhibition rate of dipeptidyl peptidase-IV inhibitory peptides prepared by the embodiment of the present invention

[0030] 2. In vivo validation The in vivo validation experimental method for the final product prepared in the above examples is as follows: Five-week-old male SD rats were selected and divided into three groups. One group was fed with conventional diet and regarded as the control group (group C). The other two groups were fed with high-fat and high-sugar diet to induce type 2 diabetes mellitus (T2DM) model and regarded as the experimental group. After the rats in the experimental group were fed with high-fat and high-sugar diet for 4 weeks, streptozotocin (35 mg / kg) was intraperitoneally injected. Blood glucose level was measured 5 days after injection. The rats with fasting blood glucose level ≥11.1 mmol / L were used as T2DM model rats. The rats in the experimental group were divided into model group (group M) and inhibitor gavage group (group P). The inhibitor gavage group was gavaged with 800 mg / kg sheep bone polypeptide daily, and the normal group and model group were gavaged with the same amount of saline daily for four consecutive weeks. A total of 5 fasting blood glucose were collected, which were blood glucose after modeling and 1-4 weeks after gavage, respectively recorded as week0, 1, 2, 3, 4, and oral glucose tolerance test was performed on rats four weeks after gavage.

[0031] The changes of fasting blood glucose levels in each group of rats during the treatment period were as follows Figure 1 As shown, it should be noted that one asterisk * represents p<0.05, two asterisks represent p<0.01, three asterisks represent p<0.001, and four asterisks represent p<0.0001, which is a very significant difference. Figure 1 It can be seen that the fasting blood glucose of the experimental group rats after modeling was higher than that of the C group. The blood glucose of the M group was kept above 20 mmol / L during the intragastric administration. From the third week, the fasting blood glucose of the P group showed a significant downward trend, which was significantly different from the fasting blood glucose of the M group. By the fourth week after intragastric administration, the fasting blood glucose was reduced by 33.47% compared with the M group. The above results show that intragastric administration of the inhibitor prepared by the present invention to T2DM rats can effectively reduce their fasting blood glucose values.

[0032] After four weeks of oral gavage treatment, the changes in blood glucose levels of rats in each group were as follows Figure 2 As shown. Figure 2 It can be seen that the blood glucose level of group C reached the maximum value 15 minutes after gavage, then gradually decreased, and basically returned to normal levels at 120 minutes; the blood glucose levels of groups M and P both reached the maximum value at 30 minutes, and the blood glucose levels at 120 minutes were still greater than the initial blood glucose levels. Compared with group M, the blood glucose level of group P decreased faster after 30 minutes, indicating that sheep bone polypeptide can alleviate the trend of insulin resistance in rats, thereby helping to stabilize the blood glucose concentration of rats.

[0033] In summary, the present invention provides a method for preparing a dipeptidyl peptidase-IV inhibitor extracted from sheep bones. The extracted final product can be used as an inhibitor and has a significant inhibitory effect on DPP-IV. At the same time, oral administration of the inhibitor can also significantly reduce the fasting blood glucose value of type II diabetes, alleviate the trend of insulin resistance in rats, and help stabilize the blood glucose concentration of rats. It has a significant application effect in the treatment of type II diabetes.

[0034] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A dipeptidyl peptidase-IV inhibitor prepared from sheep bones, characterized in that: The preparation of the dipeptidyl peptidase-IV inhibitor comprises the following steps: S1: Fresh sheep bones were washed and crushed, and then steamed under a pressure of 0.1 MPa; S2: drying the steamed sheep bones and then crushing them, and passing them through an 80-mesh sieve to obtain sheep bone powder; S3: mixing the sheep bone powder and anhydrous ethanol and stirring them, washing them repeatedly with distilled water after the stirring, discarding the supernatant after centrifugation, and drying the precipitate to obtain defatted sheep bone powder; S4: dissolving the defatted sheep bone powder in distilled water to obtain a sheep bone powder solution, and then performing ultrasonic treatment; S5: adding protease to the sheep bone powder solution after ultrasonic treatment for enzymatic hydrolysis, and performing enzyme inactivation treatment after enzymatic hydrolysis to obtain sheep bone enzymatic hydrolyzate; S6: cooling the sheep bone enzymatic hydrolyzate to room temperature, centrifuging and taking the supernatant to obtain a sheep bone polypeptide solution; S7: freeze-drying the sheep bone polypeptide solution to obtain the final product, which is the dipeptidyl peptidase-IV inhibitor.

2. The dipeptidyl peptidase-IV inhibitor according to claim 1, characterized in that The mass ratio of sheep bone powder to anhydrous ethanol in S3 is 1:10; the centrifugation is 8000 r / min for 15 min; and the drying temperature is 80°C.

3. The dipeptidyl peptidase-IV inhibitor according to claim 2, characterized in that Sheep bone powder and anhydrous ethanol were mixed at a mass ratio of 1:10 and stirred for 24 hours, with the anhydrous ethanol replaced every 6 hours.

4. The dipeptidyl peptidase-IV inhibitor according to claim 1, characterized in that The mass percentage concentration of defatted sheep bone powder in the sheep bone powder solution in S4 is 8%; the ultrasonic treatment has an ultrasonic temperature of 25° C., an ultrasonic power of 360 W, and an ultrasonic time of 30 min.

5. The dipeptidyl peptidase-IV inhibitor according to claim 1, characterized in that The protease in S5 is papain; the enzyme inactivation treatment is heating at 100° C. for 15 min.

6. The dipeptidyl peptidase-IV inhibitor according to claim 5, characterized in that The amount of papain was 8000 U / g, the pH of the enzymatic hydrolysis system was adjusted to 6.0, and the enzymatic hydrolysis was carried out at 55° C. for 4 h.

7. The dipeptidyl peptidase-IV inhibitor according to claim 1, characterized in that The centrifugation in S6 is performed at 8000 r / min for 15 min.

8. The dipeptidyl peptidase-IV inhibitor according to claim 1, characterized in that The freeze drying method is divided into segmented drying, the first segment: -35°C, 2h; the second segment: -25°C, 8h; the third segment: -20°C, 6h; the fourth segment: -10°C, 8h; the fifth segment: 0°C, 2h; the sixth segment: 10°C, 3h; wherein the second segment is set as a vacuuming segment.

9. Use of the inhibitor according to any one of claims 1 to 8 in any one of the following, comprising: For the inhibition of dipeptidyl peptidase-IV; Relieve insulin resistance and tolerance; Used for the treatment of type 2 diabetes.

Images