Tuna arterial globulin enzymolysis polypeptide as well as preparation method and application thereof
Through enzymatic optimization of tuna arterial spheres, an enzymatic polypeptide with specific amino acid sequences was prepared, which solved the problem that tuna arterial sphere resources were not effectively utilized, and its effect on preventing vascular calcification was verified through functional prediction and animal models, achieving the effect of reducing blood lipids and blood calcium levels.
Patent Information
- Application Number
- CN202510075939.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-17
- Publication Date
- 2025-03-21
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The prior art has failed to effectively utilize tuna arterial sphere resources and has failed to effectively prevent vascular calcification.
By enzymatically decomposing the tuna arterial balls, enzymatic polypeptides with specific amino acid sequences were prepared, and their effect on preventing vascular calcification was verified through functional prediction and animal models.
The prepared tuna arterial sphere enzyme polypeptide can reduce blood lipid levels, blood calcium and blood phosphorus content, and has the effect of preventing vascular calcification.
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Figure CN119661644A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of bioactive peptides, and in particular relates to a tuna glomus arteriosum enzymatic hydrolyzed polypeptide and a preparation method and application thereof. Background Art
[0002] The number of amino acids in a polypeptide is generally between 2 and 50. In recent years, many polypeptides with biologically active functions have been prepared and extracted, indicating that bioactive peptides have great application prospects in the food and pharmaceutical industries. Tuna processing produces a large number of by-products, most of which are used at a low value and cause environmental pollution. Effective enzymatic hydrolysis of tuna processing by-products to obtain bioactive peptides through enzymatic hydrolysis technology is one of the effective ways to achieve high-value utilization of aquatic resources.
[0003] The bulbus arteriosus is a tissue unique to some bony fish with well-developed arterial trunks. It is an auxiliary organ of the heart. It is rich in elastic fibers and a variety of bioactive substances. It has a high utilization value and plays an important role in maintaining skin and vascular function. However, due to its small size and small amount, the bulbus arteriosus of tuna has not attracted much attention and is often discarded as waste. The bulbus arteriosus of tuna is rich in elastin, which plays an important role in maintaining and repairing skin elasticity and preventing cardiovascular diseases. Vascular calcification is a cardiovascular disease, an active process initiated and regulated by molecular signaling pathways, and its molecular mechanism is still unclear.
[0004] Therefore, it is of great significance to develop a new enzymatic peptide from tuna arteriosclerosis and find its application. Summary of the invention
[0005] The technical problem to be solved by the present invention is to provide a tuna arteriosphere enzymatic hydrolyzed polypeptide in view of the deficiencies in the prior art. The tuna arteriosphere is enzymatically optimized to prepare the arteriosphere enzymatic hydrolyzed polypeptide, the polypeptide sequence is identified and the function is predicted using Discovery Studio to find the molecular mechanism related to the study of vascular calcification, and finally, vascular calcification is induced in mice by feeding a high-fat diet based on a large dose of vitamin D3, a vascular calcification model is established, and the effect of tuna arteriosphere enzymatic hydrolyzate on preventing vascular calcification is studied.
[0006] In order to solve the above technical problems, the present invention discloses a tuna glomus arteriosum enzymatic hydrolysis polypeptide, wherein the amino acid sequence of the tuna glomus arteriosum enzymatic hydrolysis polypeptide comprises any one or a combination of several of CGDRGFFY, MLGMGGGEEET and HAFVMIFFM.
[0007] The present invention also discloses a method for preparing the above-mentioned tuna arteriosclerosis enzymatic polypeptide, which specifically comprises the following steps:
[0008] S1. Adding tuna arterial bulb powder into distilled water to obtain tuna arterial bulb substrate solution;
[0009] S2. adding protease to the tuna arterial bulb substrate solution obtained in S1 to enzymatically hydrolyze and inactivate the enzyme, and taking the supernatant after centrifugation to obtain a tuna arterial bulb enzymatic hydrolyzate;
[0010] S3. The tuna glomus arteriosus enzymatic hydrolyzate obtained in S2 is freeze-dried into powder to obtain tuna glomus arteriosus enzymatic hydrolyzate polypeptide.
[0011] Specifically, the solid-liquid ratio of the tuna glomus arteriosus powder and distilled water is 1g:(3-5)ml; preferably, the solid-liquid ratio of the tuna glomus arteriosus powder and distilled water is 1g:4ml.
[0012] Specifically, the added amount of the protease is 0.4-0.8% of the mass of the tuna glomus arteriosus powder; preferably, the added amount of the protease is 0.6% of the mass of the tuna glomus arteriosus powder.
[0013] Specifically, the proteases are flavor protease and alkaline protease; the weight ratio of the flavor protease to the alkaline protease is 1:1.
[0014] Specifically, in S2, the enzymatic hydrolysis is carried out under the following conditions: temperature of 48 to 52° C. and time of 4 to 8 h;
[0015] Preferably, in S2, the enzymatic hydrolysis is carried out under the following conditions: temperature is 50.33° C. and time is 6.13 h.
[0016] Specifically, in S2, the enzyme inactivation is carried out under the following conditions: water bath at 80-100°C for 8-12 minutes;
[0017] Preferably, in S2, the enzyme inactivation is carried out under the following conditions: water bath at 90°C for 10 min.
[0018] Specifically, in S2, the centrifugation is carried out under the following conditions: centrifugation at a speed of 7000 to 9000 r / min for 15 to 25 min;
[0019] Preferably, in S2, the centrifugation is carried out under the condition of: centrifugation at a rotation speed of 8000 r / min for 20 min.
[0020] Furthermore, the use of the above-mentioned tuna arteriosclerotic polypeptide in the preparation of BMP-2 inhibitors is also within the protection scope of the present invention.
[0021] Specifically, in some embodiments of the present invention, reverse target searching was performed using the PharmaDB module and molecular docking of the above-mentioned polypeptide with BMP-2 was performed using the Discovery Studio software, which proved that the tuna arteriolar enzymatic polypeptide provided by the present invention has the ability to affect the activity of BMP-2 protein, thereby providing the application prospect of the above-mentioned tuna arteriolar enzymatic polypeptide in the preparation of BMP-2 inhibitors.
[0022] Furthermore, the use of the above-mentioned tuna arteriosclerotic polypeptide in the preparation of a preparation for inhibiting vascular calcification is also within the protection scope of the present invention.
[0023] On the basis of the above, the prior art records that high expression of BMP-2 protein is closely related to an increased risk of vascular calcification. In some embodiments of the present invention, vascular calcification is induced in mice by feeding them a high-fat diet on the basis of a large dose of vitamin D3, and a vascular calcification model is established. Through animal experiments, the function of tuna enzymatic hydrolyzed polypeptides in inhibiting vascular calcification is analyzed, demonstrating the application prospects of the above tuna arteriolar hydrolyzed polypeptides in the preparation of preparations for inhibiting vascular calcification.
[0024] Beneficial effects: The present invention uses tuna arterial bulbs as raw materials to prepare polypeptides capable of inhibiting vascular calcification, which fully utilizes tuna resources, increases the added value of tuna arterial bulbs as waste, and provides a new development idea for the production and processing of tuna.
[0025] The tuna arteriolar enzymatic polypeptide prepared by the invention can reduce blood lipid level, blood calcium and blood phosphorus content to a certain extent, and has a good effect of preventing vascular calcification.
[0026] The method of the invention is simple to operate, highly practical and easy to industrialize for production. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.
[0028] Figure 1 This is a mass spectrometry analysis diagram of the enzymatic hydrolysis polypeptide of tuna arteriophorum in the embodiment of the present invention;
[0029] Figure 2 This is a diagram showing the interaction between the tuna arteriolar enzymatic polypeptide and the BMP-2 protein in the embodiment of the present invention;
[0030] Figure 3 : is a graph showing changes in blood physiological and biochemical indices of mice in an embodiment of the present invention, wherein A is TC, TG, HDL-C and LDL-C indices, B is a blood calcium index, and C is a blood phosphorus index; in the graph, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001;
[0031] Figure 4 This is a hematoxylin-eosin staining analysis diagram of the mouse thoracic aorta in the embodiment of the present invention, wherein A is a control group, B is a model group, C is a positive drug group, D is a low-dose group, E is a medium-dose group, and F is a high-dose group;
[0032] Figure 5 This is a von Kossa staining analysis diagram of the mouse thoracic aorta in the example of the present invention, wherein A is a control group, B is a model group, C is a positive drug group, D is a low-dose group, E is a medium-dose group, and F is a high-dose group. DETAILED DESCRIPTION
[0033] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.
[0034] In the following examples, the flavor protease and alkaline protease were purchased from Nanning Pangbo Bioengineering Co., Ltd., wherein the flavor protease had an activity of 20,000 U / g, and the alkaline protease had an activity of 200,000 U / g.
[0035] Example 1: Preparation of Tuna Glomus Arteriosus Enzymatic Hydrolyzed Peptides
[0036] The tuna arterial bulb was thawed and crushed with a wall-breaking machine at room temperature of 20-30°C to obtain tuna arterial bulb powder. The tuna arterial bulb powder was mixed with tuna arterial bulb substrate solution at a solid-liquid ratio of 1:4, i.e. 10g powder: 40ml distilled water (pH 7), and the temperature was adjusted to 50.33°C. The solution was hydrolyzed by a combination of flavor protease and alkaline protease with a mass ratio of 1:1 for 6.13 hours. The amount of enzyme added was 0.6% of the mass of the tuna arterial bulb powder, and the degree of hydrolysis was 64.23%. After the enzymolysis was completed, the enzyme was inactivated in a boiling water bath at 90°C for 10 minutes, and the solution was centrifuged at 8000r / min for 20 minutes. The supernatant was taken, which was the tuna arterial bulb enzymatic hydrolyzate. The solution was freeze-dried into dry powder using a freeze dryer and stored at -20°C for later use.
[0037] The amino acid sequence of the enzymatic hydrolysate was determined by MALDI-TOF / TOF. Table 1 shows the identified peptides. Figure 1 For primary mass spectrometry (MS) and secondary mass spectrometry (MS / MS). Figure 1It can be seen that the signal intensity of the parent ion m / z 964.3163 is the highest, followed by the signal intensity of m / z 1142.3833, and the signal intensity of the other parent ions is relatively low. It can be considered that the content of polypeptides with molecular weights of 964.3136 and 1142.3833 in the enzymatic hydrolyzate is dominant. The parent ions of m / z 964.3163 and 1142.3833 were selected for secondary mass spectrometry analysis, and the obtained results were compared with the library to determine their amino acid sequences as CGDRGFFY (Cys-Gly-Asp-Arg-Gly-Phe-Phe-Tyr), MLGMGGGEEET (Met-Leu-Gly-Met-Gly-Gly-Gly-Glu-Glu-Glu-Thr) and HAFVMIFFM (His-Ala-Phe-Val-Met-Ile-Phe-Phe-Met).
[0038] Table 1 Identification of enzymatic peptide sequences of Glomus arteriosus
[0039]
[0040] Example 2: Functional prediction of tuna glomus arteriosus enzymatic peptides
[0041] The PharmaDB module was used for reverse target search, and the test results showed that the dominant peptides prepared in Example 1 had a potential effect on the activity of 14 protein models. The results are shown in Table 2. It can be seen that CGDRGFFY, MLGMGGGEEET and HAFVMIFFM all interact with Bone morphogenetic protein 2 (3bmp), which is a BMP-2 protein. The prior art records that this protein is related to vascular calcification. Therefore, the tuna arteriolar enzymatic polypeptide prepared in Example 1 may have the ability to inhibit vascular calcification.
[0042] Table 2 Prediction of the function of glomus arteriosum enzymatic peptides
[0043]
[0044]
[0045] Example 3: Molecular docking of tuna arteriolar enzymatic peptides and BMP-2 protein
[0046] BMP-2 is an important vascular calcification factor. In order to clarify the molecular mechanism of enzymatic peptides on vascular calcification, the dominant peptides (CGDRGFFY, MLGMGGGEEET and HAFVMIFFM) were docked with BMP-2 using Discovery Studio software. The molecular docking results of BMP-2 and each peptide are shown in Table 3 and Figure 2 The results of the CDOCKER molecular docking method mainly determine the degree of binding based on the energy (-CDOCKER_Energy, -Ec) between the receptor and the ligand, electrostatic energy (Eele) and van der Waals energy (Evdw). The value of -CDOCKER_Energy indicates the strength of affinity between the peptide and BMP-2. It can be seen from Table 3 that MLGMGGGEEET (130.17 kcal / mol) showed a higher affinity than HAFVMIFFM (112.924 kcal / mol) or CGDRGFFY (114.397 kcal / mol), indicating that MLGMGGGEEET may release a higher BMP-2 inhibitory effect than the other two peptides; the electrostatic energy (Eele) of CGDRGFFY, MLGMGGGEEET and HAFVMIFFM were -375.665 kcal / mol, -357.486 kcal / mol and -294.648 kcal / mol, respectively, indicating that the three peptides have a certain binding ability with BMP-2: Van der Waals energy (Van der Waals energy, Evdw) were -16.6728 kcal / mol, -26.4965 kcal / mol and -33.8058 kcal / mol, respectively, which also showed that the three polypeptides had a certain binding ability with BMP-2.
[0047] Table 3 Molecular docking results
[0048]
[0049] Depend on Figure 2(From left to right are the active sites of the interaction between the polypeptide molecule and BMP-2, the interacting amino acid residues and the two-dimensional diagram) It can be seen that CGDRGFFY has a total of 11 amino acid residues that interact with BMP-2, forms 4 hydrogen bonds (HBs) with Asp30, Leu92, Asp93 and ASP105, and 7 residues participate in van der Waals (VDW) interaction; MLGMGGGEEET has a total of 11 amino acid residues that interact with BMP-2, forms 5 HBs with Tyr91, Glu94, Asn95, Tyr103 and Gln104, and 6 amino acid residues participate in VDW; HAFVMIFFM has a total of 8 amino acid residues that interact with BMP-2, forms 2 HBs with Phe41 and Asp105, and 6 amino acid residues participate in VDW. From the above results, it can be seen that the polypeptide and BMP-2 protein can be docked at the active site, indicating that it has a certain ability to inhibit BMP-2 protein, that is, it has a molecular effect of inhibiting vascular calcification.
[0050] Example 4: Animal model validation of tuna arteriolar enzymatic polypeptide inhibiting vascular calcification
[0051] The mice in this example were purchased from Zhejiang Medical Experimental Animal Center (Hangzhou, China), specifically male Balb / c mice (6-8 weeks old, 18-20 g); the mice were divided into control group C, model group M, positive drug group P, low-dose polypeptide group PL, medium-dose polypeptide group PM, and high-dose polypeptide group PH, with 6 mice in each group. After one week of adaptation to the standard diet, the control group was fed with ordinary feed, and the other groups were fed with high-fat feed (15% fat, 1.25% cholesterol, 0.5% sodium cholate, 0.2% propylthiouracil, basic feed). Except for the control group, the mice in the other groups were gavaged with 300,000 IU / kg / d of vitamin D3 for 1 to 3 days at the beginning of the experiment, followed by drug intervention. The positive drug group was gavaged with 6 mg / kg / d of fluvastatin sodium; the experimental group was gavaged with low-dose 50 mg / kg / d of arterial bulb enzymatic hydrolysate, the medium-dose group was gavaged with 150 mg / kg / d of arterial bulb enzymatic hydrolysate, and the high-dose group was gavaged with 450 mg / kg / d of arterial bulb enzymatic hydrolysate. The control group and the model group were gavaged with an equal amount of normal saline. The experiment lasted for 8 weeks. During the experiment, all animals had free access to water, the room temperature was maintained at around 23-25°C, and the light was on for 12 hours every day and night.
[0052] Table 4 Body weight changes of mice in each group
[0053]
[0054] Note: Compared with the control group, # p<0.05, ## p<0.01, ###p<0.001; compared with the model group, *p<0.05; **p<0.01; ***p<0.001.
[0055] Except for the control group, mice in all groups showed acute poisoning symptoms such as mild diarrhea, decreased appetite, and rough fur color after gavage of vitamin D3, and the symptoms were relieved after about one week.
[0056] Table 4 shows that the weight of mice in each group increased over time. The weight of mice in the control group increased linearly, and the weight of mice in the other groups dropped sharply after vitamin D3 was given. The weight on the 7th day was significantly lower than that of mice in the control group. Compared with the control group, the weight of mice in the model group gradually recovered and continued to grow under the effect of high-fat diet. At the end of the experiment, the weight of the model group increased compared with the control group, but there was no significant difference. Compared with mice in the model group, the weight of the enzymatic polypeptide group was lighter at the end of the experiment, which was significantly different.
[0057] The blood of mice was tested. The specific detection method was to collect blood from the mouse eyeball, bathe in 37°C water for 10 minutes, and then centrifuge at 3000rpm / min for 10 minutes, take the upper serum, and use the kit to determine the TC, TG, LDL-C, HDL-C, blood calcium and blood phosphorus levels in the serum. Figure 3 A is the total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) indicators of mice. It can be seen from the figure that TC and LDL in the model group increased significantly, TG increased, and HDL decreased. After intragastric administration of arterial bulb enzymatic hydrolysate, TC and LDL decreased significantly compared with the model group; while TG and HDL did not change significantly.
[0058] Figure 3 B is the blood calcium index of mice in each group. Figure 3C is the blood phosphorus index of mice in each group. It can be seen from the figure that compared with the control group, the blood calcium of the model group mice increased significantly, but there was no significant difference in blood phosphorus; compared with the model group, after gavage with medium and high doses of tuna arteriolar enzymatic hydrolysate, blood calcium and blood phosphorus showed significant differences (p < 0.05). Our experiments preliminarily showed that the model group mice had abnormal blood lipids, with significantly increased TC and LDL, an upward trend in TG, and a downward trend in HDL, indicating that feeding mice with a high-fat diet increased their lipid intake, because the levels of TC, TG, LDL-C and HDL-C in serum can show the viscosity of the blood and are closely related to the formation of atherosclerosis. Higher TC, TG, LDL-C levels and lower HDL-C levels can lead to the occurrence of hyperlipidemia; at the same time, there were significant differences in blood calcium in the model mice, and blood phosphorus showed an upward trend, indicating that vascular calcification has a certain connection with serum calcium and phosphorus levels. Gavage with enzymatic polypeptides can reduce the blood lipids, blood calcium and blood phosphorus levels in mice, and the slice results showed no obvious calcification, which proves that tuna arteriolar enzymatic hydrolysate has the effect of preventing vascular calcification to a certain extent.
[0059] Morphological observation of the mouse thoracic aorta with hematoxylin-eosin (HE) showed the basic structure of the blood vessels. The specific detection method is to manually separate the aorta after killing the mouse by dislocating the neck, fix it with tissue fixative for more than 24 hours, dehydrate it, dip it in wax, embed and slice it, bake it dry with water and then take it out and store it at room temperature for later use, and use it for hematoxylin-eosin (HE) staining. The staining process uses hematoxylin staining solution for 3-5 minutes, and then washes with tap water; differentiates with differentiation solution, i.e. hydrochloric acid alcohol, washes with tap water, blues with blueing solution, and rinses with running water; dehydrate with 85% and 95% alcohol gradient for 5 minutes each, and then put it into eosin staining solution for 5 minutes, and finally seal the slide for microscopic examination. Figure 4 This is a morphological observation diagram of hematoxylin-eosin (HE). Figure 4 It can be seen that the thickness of the thoracic aorta wall of the mice in the control group was uniform, the elastic fibers were arranged in a wavy ring shape, the long axis and nuclear long axis of the vascular smooth muscle cells were arranged in a ring along the lumen, and the cell nuclei were mostly long spindle-shaped. Figure 4 A; The thoracic aorta of the model mouse is irregular in shape, with varying thickness of the wall. It can be observed that the intima has protrusions bulging into the lumen; the thickness of the media layer increases, the elastic plate breaks, and the calcification formed in the media breaks to form a small cavity. Figure 4 B. The tube walls of the positive drug group and the test group were relatively smooth, with no obvious calcification. Figure 4 C~F.
[0060] Von Kossa morphological observation was performed on the thoracic aorta of mice. The specific detection method was to manually separate the aorta after the mice were killed by dislocating the neck, fix it with tissue fixative for more than 24 hours, dehydrate it, dip it in wax, embed and slice it, bake it dry with water and then take it out and store it at room temperature for later use for Von Kossa staining. Silver nitrate solution was added to the slices, and they were continuously irradiated with ultraviolet light for 2-3 hours and washed with distilled water for several times; the slices were put into hematoxylin staining solution for 3-5 minutes and washed with tap water; differentiated with differentiation solution, washed with tap water, blued with blueing solution, and rinsed with running water; dehydrated with 85% and 95% alcohol gradient for 5 minutes each, put into eosin staining solution for 5 minutes, and finally sealed for microscopic examination. Von Kossa staining judges the vascular calcification by observing whether there is dark crystalline particle deposition (calcium salt deposition) in the vascular tunica. If there is calcium salt deposition, the area will be dark brown or brownish black. The results are as follows Figure 5 As shown, no obvious calcium salt deposition was observed in the control group mice ( Figure 5 A), a large number of dark particles were observed to aggregate between the elastic fibers of the tunica media of the blood vessels of the mice in the model group, showing a dark brown color ( Figure 5 B), indicating that calcium deposition is serious, while such dark aggregates are less common on the intima, indicating that the modeling is successful. In addition, no obvious dark particles were found in the vascular arteries of mice in the positive drug group and the experimental group ( Figure 5 C~F).
[0061] The invention prepares tuna arteriolar enzymatic polypeptides, and through MALDI-TOF / TOF identification, the dominant peptide sequences in the enzymatic hydrolyzate are CGDRGFFY, MLGMGGGEEET and HAFVMIFFM. The potential target is screened as BMP-2 protein through molecular simulation method, and its mechanism of action is simulated through molecular docking to predict its molecular mechanism of vascular calcification. Finally, by constructing a vascular calcification model for animal experiments, it is found that the enzymatic polypeptide can reduce blood lipid levels, blood calcium and blood phosphorus content to a certain extent, and no obvious calcification is found in the slice results, which further proves the effect of the enzymatic polypeptide in preventing vascular calcification.
[0062] The present invention provides a tuna arteriolar enzymatic polypeptide and its preparation method and application ideas. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.
Claims
1. A tuna arteriosclerosis enzymatic polypeptide, characterized in that: The amino acid sequence of the tuna arteriophora enzymatic polypeptide includes any one or a combination of several of CGDRGFFY, MLGMGGGEEET and HAFVMIFFM.
2. The method for preparing the tuna arteriosclerosis enzymatic polypeptide according to claim 1, characterized in that: The specific steps are: S1. Adding tuna arterial bulb powder into distilled water to obtain tuna arterial bulb substrate solution; S2. adding protease to the tuna arterial bulb substrate solution obtained in S1 to enzymatically hydrolyze and inactivate the enzyme, and taking the supernatant after centrifugation to obtain a tuna arterial bulb enzymatic hydrolyzate; S3. The tuna glomus arteriosus enzymatic hydrolyzate obtained in S2 is freeze-dried into powder to obtain tuna glomus arteriosus enzymatic hydrolyzate polypeptide.
3. The preparation method according to claim 2, characterized in that: In S1, the solid-liquid ratio of the tuna arterial bulb powder to distilled water is 1 g: (3-5) ml.
4. The preparation method according to claim 2, characterized in that: The added amount of the protease is 0.4-0.8% of the mass of the tuna glomus arteriosus powder.
5. The preparation method according to claim 4, characterized in that: The proteases are flavor protease and alkaline protease; the usage mass ratio of the flavor protease to the alkaline protease is 1:
1.
6. The preparation method according to claim 2, characterized in that: In S2, the enzymatic hydrolysis is carried out under the following conditions: temperature is 48-52° C. and time is 4-8 h.
7. The preparation method according to claim 2, characterized in that: In S2, the enzyme inactivation is carried out under the following conditions: water bath at a temperature of 80-100°C for 8-12 minutes.
8. The preparation method according to claim 2, characterized in that: In S2, the centrifugation is carried out under the conditions of: centrifugation at a rotation speed of 7000-9000 r / min for 15-25 min.
9. Use of the tuna arteriosclerosis enzymatic polypeptide according to claim 1 in the preparation of a BMP-2 inhibitor.
10. Use of the tuna arteriosclerosis enzymatic polypeptide according to claim 1 in the preparation of a preparation for inhibiting vascular calcification.