Tortoise plastron peptide with immunomodulatory activity as well as preparation method, product and application of tortoise plastron peptide

Abstract

The invention provides a tortoise plastron peptide with immunomodulatory activity, and a preparation method, a product and application thereof, and relates to the field of biological medicines. According to the method, total protein in the tortoise plastron is extracted by means of decoction, concentration, extraction, filtration and the like, meanwhile, the content of fat-soluble impurities and mineral calcium in the extract is reduced, and then the total protein is subjected to enzymolysis to obtain polypeptide; the obtained polypeptide has good immunocompetence, and can effectively improve the multiplication capacity of splenocytes, promote macrophages to generate immune response and enhance the immune regulation function.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, relates to tortoise plastron peptides, and specifically relates to tortoise plastron peptides with immunomodulatory activity, and methods for preparing the same, products and applications thereof. Background Art

[0002] Immunity is the body's defense system, which has the ability to recognize and eliminate foreign invading viruses and bacteria, as well as recognize and process mutated and infected cells in the body, and plays an important role in human health. Immunopeptides are peptide segments with immunological activity, composed of amino acids and amino acid residues, and have a variety of immunomodulatory functions. By coordinating pro-inflammatory and anti-inflammatory responses, enhancing bactericidal ability, cell differentiation, activating immunity, wound healing, autophagy, apoptosis, etc., they can improve human immunity. Therefore, it is very necessary to search for polypeptides with immunological activity for human health.

[0003] Tortoise plastron is a kind of traditional Chinese medicine. "Compendium of Materia Medica" records that tortoise plastron can treat lumbago and leg pain, tonify the heart and kidney, benefit the large intestine, stop chronic diarrhea, be mainly used for difficult labor, disperse swelling and carbuncles, and burn the ash to apply to ecthyma. Its shell can tonify the heart, kidney and blood, all of which are for nourishing yin. Previous studies have shown that tortoise plastron contains a variety of amino acids and trace minerals, which play an important role in human growth and development, immunity improvement, etc. Tortoise plastron peptides belong to animal protein immune natural peptides with both medicinal and edible properties. Their sequences are close to or complementary to endogenous peptides. They not only have good digestion and absorption characteristics, but also have more effective physiological functions. They can act on antibodies and antigens, activate T cell immunity, regulate autoimmune diseases and tumors, etc. Tortoise plastron peptides have the characteristics of low molecular weight, weak toxicity and side effects, and low cost. Therefore, screening immunologically active peptides from tortoise plastron is of great significance to human health.

[0004] The prior art CN107858392A discloses a method for preparing active polypeptides from soft-shelled turtle shell, including: degreasing, alkali dissolution, enzymatic hydrolysis, and purification. Petroleum ether is used to degrease soft-shelled turtle shell powder. After degreasing, the powder is dissolved in alkali and centrifuged to obtain soft-shelled turtle shell protein powder. A composite protease is used to enzymatically hydrolyze the soft-shelled turtle shell protein powder. After filtering the enzymatic hydrolysis solution through an acetic acid fiber microporous filter membrane, it is filtered through an ultrafiltration membrane to obtain active polypeptides from soft-shelled turtle shell. Adding (2R,4R)-pentanediol to the enzymatic hydrolysis system can reduce the energy barrier between the enzyme and the substrate, increase the reactivity between the enzyme and the substrate, and improve the reaction efficiency. The disadvantage of this technology is that there is no validation of effectiveness and safety, and (2R,4R)-pentanediol is added additionally, which has certain toxicity and may have a negative impact on the use of active polypeptides from soft-shelled turtle shell for preparing products to improve immunity.

[0005] Research shows that tortoise plastron peptide can be prepared by enzymatic hydrolysis of tortoise plastron glue. Tortoise plastron peptide not only has good digestion and absorption characteristics, but also has more effective physiological functions than tortoise plastron glue. Zhao Jinsong et al. (Zhao Jinsong, et al. Research on the Optimization Conditions of Enzymatic Hydrolysis of Tortoise Plastron Glue [J]. Modern Food Science and Technology, 2006) conducted an optimization study on the enzymatic hydrolysis conditions of tortoise plastron glue: Weigh the test amount of tortoise plastron glue respectively, add an appropriate amount of distilled water, and prepare the required concentration. Dissolve it in a 250 ml Erlenmeyer flask. The tortoise plastron glue is boiled and dissolved, and after dissolution, it is cooled to room temperature. After the solution is cooled to the corresponding enzymatic hydrolysis temperature, it is adjusted to the required pH value with hydrochloric acid or sodium hydroxide solution. The solution with the adjusted pH is placed in a constant temperature water bath at the corresponding enzymatic hydrolysis temperature and incubated. Hydrolyze under certain conditions. During the hydrolysis process, stir slowly, generally 10 - 20 times / min is appropriate, and add a standard sodium hydroxide solution in a timely manner to maintain its pH value. After the enzymatic hydrolysis is completed, stop stirring, directly heat to 85 °C, and keep it for 5 min to obtain the hydrolyzed protein solution. The optimal process conditions are: enzymatic hydrolysis temperature 50 °C, pH value 8.5, substrate concentration 8%, and enzymatic hydrolysis time 8 h. The disadvantages of this method are that it is necessary to prepare tortoise plastron glue first, secondly, the enzymatic hydrolysis time is long, and then there is a lack of verification of the effectiveness and safety of the product. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention provides a tortoise plastron peptide with immunomodulatory activity, its preparation method, product and application. The present invention extracts the total protein in tortoise plastron through means such as boiling, concentration, extraction, and filtration, while reducing the content of fat-soluble substances and mineral calcium in the extract. Then, the total protein is enzymatically hydrolyzed to obtain polypeptides. The obtained polypeptides have good immune activity, can effectively improve the proliferation ability of spleen cells, promote macrophages to produce immune responses, and enhance the immune regulation function.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] On the one hand, the present invention provides a preparation method of a tortoise plastron peptide with immunomodulatory activity, including the following steps:

[0009] 1) Wash, break and boil the tortoise plastron;

[0010] 2) Concentrate the soup obtained by boiling;

[0011] 3) After concentration, perform extraction and dialysis, and then freeze-dry;

[0012] 4) After drying, add it to an EDTA solution, stir, filter, and freeze-dry the filtrate to obtain a crude protein extract;

[0013] 5) Mix the crude protein extract with an NaOH solution, adjust the pH value, add protease, mix well, and perform enzymatic hydrolysis;

[0014] 6) Inactivate the enzyme after the enzymatic hydrolysis ends;

[0015] 7) Centrifuge and collect the centrifugate;

[0016] 8) Filter the centrifugate through an ultrafiltration membrane, freeze-dry it, and pulverize it to obtain a polypeptide powder.

[0017] Preferably, the above preparation method includes the following steps:

[0018] 1) Wash and break the turtle shell, put it into a pot and boil for 8 - 16 hours, and boil three times;

[0019] 2) Combine and filter the soups obtained from the three boilings, and rotary evaporate and concentrate at 50 - 60 °C until basically no condensate flows out to obtain a concentrate;

[0020] 3) Extract the concentrate with petroleum ether, then filter it through a dialysis bag, and then freeze-dry it;

[0021] 4) Pulverize after freeze-drying, add it to a 0.4 - 8 moL / L EDTA solution at an addition amount of 4 - 8%, stir and react for 15 - 60 min, filter, and freeze-dry the filtrate again to obtain a crude protein extract;

[0022] 5) Add the crude protein extract to a NaOH solution with a pH of 7.5 at a solid-liquid ratio of 1:25, adjust the pH value to 8.0 - 8.5, add protease to make the enzyme concentration in the solution 1.5 - 2.0%, and perform enzymatic hydrolysis in a water bath at a constant temperature of 55 °C for 2 - 6 h;

[0023] 6) Then put it into a hot water bath and continuously stir to inactivate the enzyme;

[0024] 7) Centrifuge for 10 minutes and collect the centrifugate;

[0025] 8) Filter the centrifugate through an ultrafiltration membrane with a molecular weight cut-off less than 10000 D, then put it into a freeze-drying oven to freeze-dry, and pulverize it to obtain a polypeptide powder.

[0026] Preferably, the above preparation method includes the following steps:

[0027] 1) Wash and break the turtle shell, put it into a pot and boil for 12 hours, and boil three times;

[0028] 2) Combine and filter the soups obtained from the three boilings, and rotary evaporate and concentrate at 55 °C until basically no condensate flows out to obtain a concentrate;

[0029] 3) Extract the concentrate with petroleum ether three times, and then filter it through a dialysis bag;

[0030] 4) Put it into a vacuum freeze-drying oven for freeze-drying. After pulverization, add it to a 0.6 moL / L EDTA solution at an addition amount of 6%, stir and react for 30 min, filter, and freeze-dry the filtrate again to obtain a crude protein extract;

[0031] 5) Add the crude protein extract to a NaOH solution with a pH of 7.5 at a solid-liquid ratio of 1:25, adjust the pH value to 8.0, add protease to make the enzyme concentration in the solution 1.8%, and carry out enzymatic hydrolysis in a water bath at a constant temperature of 55 °C for 3 h;

[0032] 6) Then put it into a 95 °C water bath and continuously stir for 15 minutes to inactivate the enzyme;

[0033] 7) Centrifuge at 4 °C and 10000 r / min for 10 minutes, and collect the centrifugate;

[0034] 8) Filter the centrifugate through an ultrafiltration membrane with a molecular weight cut-off less than 6000 D, then put it into a vacuum freeze-drying oven for freeze-drying, and pulverize to obtain a polypeptide powder.

[0035] Preferably, the tortoise plastron is from a tortoise of the genus Cuora.

[0036] Preferably, the protease is compound protease.

[0037] On the other hand, the present invention provides the tortoise plastron peptide prepared by the above preparation method.

[0038] Preferably, the tortoise plastron peptide contains a polypeptide with the amino acid sequence shown in SEQ ID NO.1-179.

[0039] On the other hand, the present invention provides a product containing the above tortoise plastron peptide.

[0040] Preferably, the product includes food, medicine and health products.

[0041] On the other hand, the present invention provides the application of the above preparation method, the above tortoise plastron peptide or the above product, and the application includes one or more of the following applications:

[0042] 1) Application in the preparation of a product for improving the proliferation ability of spleen cells;

[0043] 2) Application in the preparation of a product for increasing the proliferation ability of macrophages;

[0044] 3) Application in the preparation of a product for increasing the NO release ability of macrophages;

[0045] 4) Application in the preparation of a product for increasing the phagocytosis ability of macrophages;

[0046] 5) Application in the preparation of a product for alleviating the inflammatory response of macrophages.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The present invention extracts the total protein in tortoise plastron through means such as boiling, concentration, extraction, and filtration, while reducing the contents of fat-soluble substances and mineral calcium in the extract, and then enzymatically hydrolyzes the total protein to obtain polypeptides, including the polypeptides with the amino acid sequences shown in SEQ ID NO.1-179;

[0049] The polypeptides obtained by the present invention have good immunological activity, can effectively improve the proliferation ability of spleen cells, promote macrophages to produce immune responses, and enhance the immune regulation function. Description of the Drawings

[0050] Figure 1 Effect of tortoise plastron peptides on the survival rate of mouse spleen lymphocytes; among them, compared with the "0mg / mL" group,

[0051] **p<0.01, ***p<0.001, ****p<0.0001.

[0052] Figure 2 Effect of tortoise plastron peptides on the survival rate of macrophages RAW264.7; among them, compared with the "0mg / mL" group,

[0053] *p<0.05.

[0054] Figure 3 Effect of tortoise plastron peptides on the inflammatory proliferation of macrophages induced by LPS; among them, compared with the "0mg / mL" group,

[0055] ****p<0.0001; compared with the "LPS" group, #### p<0.0001.

[0056] Figure 4 Effect of tortoise plastron peptides on the phagocytosis ability of macrophages RAW264.7; among them, compared with the "0mg / mL" group,

[0057] **p<0.01, ***p<0.001, ****p<0.0001.

[0058] Figure 5 Effect of tortoise plastron peptides on the inflammatory phagocytosis ability of macrophages induced by LPS; among them, compared with the "0mg / mL" group,

[0059] ***p<0.001; compared with the "LPS" group, ## p<0.01, ### p<0.001, #### p<0.0001.

[0060] Figure 6Effect of tortoise plastron peptide on activating macrophages RAW264.7 to release NO; among them, compared with the "0 mg / mL" group,

[0061] ****p < 0.0001.

[0062] Figure 7 Effect of tortoise plastron peptide on LPS-induced inflammatory release of NO by macrophages; among them, compared with the "0 mg / mL" group,

[0063] ****p < 0.0001; compared with the "LPS" group, # p < 0.05, ### p < 0.001.

[0064] Figure 8 Effect of tortoise plastron peptide on the viability of macrophages RAW264.7.

[0065] Figure 9 Effect of tortoise plastron peptide on LPS-induced inflammatory proliferation of macrophages; among them, compared with the "0 mg / mL" group,

[0066] *p < 0.05.

[0067] Figure 10 Effect of tortoise plastron peptide on the viability of macrophages RAW264.7.

[0068] Figure 11 Effect of tortoise plastron peptide on LPS-induced inflammatory proliferation of macrophages; among them, compared with the "0 mg / mL" group,

[0069] *p < 0.05. Specific implementation manner

[0070] Unless otherwise specified, the raw materials used in the present invention are all ordinary commercially available products, and their sources are not specifically limited.

[0071] Data analysis:

[0072] SPSS 20.0 software was used to perform repeated measures analysis of variance and t-test on the data, and P < 0.05 was considered to be statistically significant.

[0073] Example 1: Preparation of tortoise plastron peptide

[0074] The total protein in tortoise plastron was extracted by means of boiling, extraction, concentration, filtration, etc., and the contents of fat-soluble and other small-molecule water-soluble components such as mineral calcium in the extract were reduced. Then, the total protein was enzymatically hydrolyzed to obtain polypeptides. The specific method is as follows:

[0075] I. Extraction of total protein

[0076] (1) The yellow pond turtle is sacrificed in hot water. After removing the internal organs, blood, and scales, the turtle shell is washed and broken up.

[0077] (2) The turtle shell is placed in a pot and boiled with pure water for 12 hours, and this is repeated 3 times.

[0078] (3) The soups obtained from the 3 boilings are combined, the residues are filtered out, and it is rotary evaporated and concentrated at 55 °C until basically no condensate flows out.

[0079] (4) The concentrate is extracted three times with petroleum ether (Shanghai Macklin Biochemical Co., Ltd., product number P816693) to remove fat-soluble impurities, and then filtered through a dialysis bag with a molecular weight cut-off of 10000 D to remove some water-soluble small molecules.

[0080] (5) It is freeze-dried in a vacuum freeze-drying oven, pulverized, and added to a 0.6 moL / L EDTA solution at an addition amount of 6%, and stirred and reacted for 30 min to remove the calcium bound in the protein. After filtration, the filtrate is freeze-dried again to obtain a crude protein extract.

[0081] II. Preparation of Polypeptides

[0082] (1) Weigh the crude protein extract and add it to a NaOH solution with a pH of 7.5 at a solid-liquid ratio of 1:25 (g / mL).

[0083] (2) Add a complex protease (Nanning Suipeptide Biotechnology Co., Ltd., product number ST2301) to make the enzyme concentration in the solution 1.8%, and enzymatically hydrolyze it in a water bath at a constant temperature of 55 °C for 3 h.

[0084] (3) Then place it in a 95 °C water bath and continuously stir for 15 minutes to inactivate the enzyme.

[0085] (4) Centrifuge at 4 °C and 10000 r / min for 10 minutes, and collect the centrifugate.

[0086] (5) Filter the centrifugate through an ultrafiltration membrane with a molecular weight cut-off less than 6000 D to remove the inactivated enzyme and unhydrolyzed macromolecular proteins, and then place it in a vacuum freeze-drying oven to be freeze-dried and pulverized to obtain a polypeptide powder.

[0087] Example 2: Identification of Peptide Fragments

[0088] Before mass spectrometry identification, the tortoise plastron peptides prepared in Example 1 were separated using nano liquid chromatography (Thermo Fisher, model Nano LC). Buffer A was an aqueous solution of 2% acetonitrile and 0.1% formic acid, and buffer B was an aqueous solution of 98% acetonitrile and 0.1% formic acid. The flow rate was 250 nL / min, and the gradient elution conditions were as follows: 0 - 5 min, 2% - 6% buffer B; 5 - 32 min, 6% - 15% buffer B; 32 - 49 min, 15% - 23% buffer B; 49 - 51 min, 23% - 98% buffer B; 51 - 56 min, 98% - 2% buffer B; 56 - 61 min, 2% buffer B.

[0089] Q-Exactive mass spectrometer from Thermo Fisher was used for mass spectrometry detection, and the mass spectrometry parameters were set as follows: the resolution of the first-level mass spectrometry was 70000; the normalized collision energy was 27; the mass spectrometry scanning range was 300 - 2000 m / z; the resolution of the second-level mass spectrometry was 17500; the collision mode was HCD (High-energy collision dissociation); the dynamic exclusion duration was 20 s.

[0090] Proteome Discoverer 2.1 software was used for mass spectrometry data analysis. The retrieval conditions were as follows: the first-level mass error was ±20 ppm, the second-level mass error was ±0.02 Da, and the maximum number of missed cleavage sites was 2. The false discovery rate (FDR) for protein and peptide identification was set to 1%. The sequences of the tortoise plastron peptides of Mauremys mutica were determined, and a total of 179 peptide segments were identified (see SEQ ID NO.1 - 179), and the specific peptide segment information is shown in Table 1.

[0091] Table 1 Description of the peptide sequences of tortoise plastron peptides

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] Example 3: Functional verification of tortoise plastron peptides I. Detection of the proliferation ability of mouse spleen cells

[0098] The mice were purchased from Guangdong Vital River Laboratory Animal Co., Ltd., C57BL / 6 mice, 8 - 10 weeks old, weighing 20 - 22 g. After the mice were euthanized, the mice were dissected, the spleen was isolated, and quickly placed into a culture dish containing RPMI 1640 complete medium. The spleen was ground with the bottom of the inner core of a 1 mL syringe. The cells were filtered through a 70 μm nylon cell strainer, and the cell strainer and the culture dish were rinsed with RPMI 1640 complete medium during the grinding process, and finally filtered into a 50 mL centrifuge tube. The cell suspension was centrifuged at 20 °C and 3000 rpm for 10 min. The supernatant was carefully discarded, 2 mL of red blood cell lysate (Solarbio, catalog number R1010) was added, the cells were resuspended, and left standing at room temperature for 2 min. Then 10 mL of RPMI 1640 complete medium was added, and the cell suspension was centrifuged at 20 °C and 3000 rpm for 10 min. The supernatant was carefully discarded, the cells were resuspended with the medium, the cells were counted and the concentration was adjusted to 5x10 4 cells / mL. 100 μL of cell suspension (about 5000 cells / well) was inoculated into each well of a 96-well plate. The plate was cultured in an incubator at 37 °C and 5% CO2 for 24 h. 10 μL of tortoise shell peptides with different concentrations (0 mg / mL, 0.15 mg / mL, 0.3 mg / mL, 0.45 mg / mL, 0.6 mg / mL, 0.75 mg / mL, 0.9 mg / mL, 1.05 mg / mL) were added to the wells, and the culture plate was incubated in the incubator for an appropriate time length of 24 h. Using a CCK-8 detection kit (APEXbio, catalog number K1018), 10 μL of CCK-8 solution was added to each well of the plate, and the culture plate was incubated in the incubator for 3 hours, and then the absorbance at 450 nm was measured using a microplate reader to detect cell proliferation. Figure 1 It shows the effects of tortoise shell peptides with different concentrations on the survival rate of mouse spleen lymphocytes. The results show that tortoise shell peptides with different concentrations can significantly improve the survival rate, and the survival rate of mouse spleen lymphocytes is the highest when 0.75 mg / mL of tortoise shell peptide is added.

[0099] II. Detection of macrophage proliferation ability

[0100] Resuscitate RAW264.7 macrophages, inoculate 100 μL of cell suspension (about 5000 cells / well) in each well of a 96-well plate, and culture in the 96-well plate for 24 h. The cells are divided into two parts. For the immunocompetence verification part: treat with tortoise plastron peptides at different mass concentrations (0 mg / mL, 0.15 mg / mL, 0.3 mg / mL, 0.45 mg / mL, 0.6 mg / mL, 0.75 mg / mL, 0.9 mg / mL, 1.05 mg / mL) for 48 h; for the inflammation alleviation verification part: co-treat with LPS at a mass concentration of 1 μg / mL and tortoise plastron peptides at different mass concentrations (0 mg / mL, 0.15 mg / mL, 0.3 mg / mL, 0.45 mg / mL, 0.6 mg / mL, 0.75 mg / mL, 0.9 mg / mL, 1.05 mg / mL) for 48 h. Use a CCK-8 detection kit, add 10 μL of CCK-8 solution to each well of the plate, incubate the culture plate in an incubator for 3 h, and then use a microplate reader to measure the absorbance at 450 nm to detect cell proliferation. Figure 2 Figure 2 shows the effects of tortoise plastron peptides at different concentrations on the survival rate of macrophages. The results show that the survival rate of macrophages increases significantly when 0.6 or 0.75 mg / mL of tortoise plastron peptides is added; Figure 3 Figure 3 shows the effects of tortoise plastron peptides at different concentrations on the inflammatory proliferation of LPS-induced macrophages. The results show that at 1.05 mg / mL, it can significantly reduce the inflammatory effect of LPS on macrophages.

[0101] III. Neutral red phagocytosis ability detection

[0102] Resuscitate RAW264.7 macrophages, inoculate 100 μL of cell suspension (about 5000 cells / well) in each well of a 96-well plate, and culture in the 96-well plate for 24 h. The cells are divided into two parts. For the immunocompetence verification part: treat with tortoise plastron peptides at different mass concentrations (0 mg / mL, 0.15 mg / mL, 0.3 mg / mL, 0.45 mg / mL, 0.6 mg / mL, 0.75 mg / mL, 0.9 mg / mL, 1.05 mg / mL) for 48 h; for the inflammation alleviation verification part: co-treat with LPS at a mass concentration of 1 μg / mL and tortoise plastron peptides at different mass concentrations (0 mg / mL, 0.15 mg / mL, 0.3 mg / mL, 0.45 mg / mL, 0.6 mg / mL, 0.75 mg / mL, 0.9 mg / mL, 1.05 mg / mL) for 48 h. Carefully discard the culture medium, add 100 μL of neutral red solution with a mass concentration of 1 mg / mL to each well, and incubate for 30 min. Wash the cells 2 times with 200 μL of PBS to remove the residual neutral red. Then add 100 μL of cell lysis buffer (volume ratio of glacial acetic acid to ethanol is 1:1) and gently shake at room temperature for 2 h, measure the absorbance at a wavelength of 540 nm, and detect the phagocytic ability of macrophages to neutral red under different conditions. Among them Figure 4 shows the effects of tortoise plastron peptides at different concentrations on the phagocytic ability of macrophages RAW264.7 to neutral red. The results show that tortoise plastron peptides at different concentrations significantly improve the phagocytic ability; Figure 5 shows the effects of tortoise plastron peptides at different concentrations on the phagocytic ability of LPS-induced macrophage inflammation. The results show that tortoise plastron peptide concentrations of 0.75 mg / mL, 0.9 mg / mL, and 1.05 mg / mL significantly reduce the effect of LPS on the phagocytic ability of macrophages.

[0103] IV. Detection of NO content

[0104] Resuscitate RAW264.7 macrophages, inoculate 100 μL of cell suspension (about 5000 cells / well) in each well of a 96-well plate, and culture in the 96-well plate for 24 h. The cells are divided into two parts. For the immunocompetence verification part: treat with tortoise plastron peptides at different mass concentrations (0 mg / mL, 0.15 mg / mL, 0.3 mg / mL, 0.45 mg / mL, 0.6 mg / mL, 0.75 mg / mL, 0.9 mg / mL, 1.05 mg / mL) for 48 h; for the inflammation alleviation verification part: co-treat with LPS at a mass concentration of 1 μg / mL and tortoise plastron peptides at different mass concentrations (0 mg / mL, 0.15 mg / mL, 0.3 mg / mL, 0.45 mg / mL, 0.6 mg / mL, 0.75 mg / mL, 0.9 mg / mL, 1.05 mg / mL) for 48 h. Using a NO detection kit (Beyotime Biotechnology, catalog number S0021S), take the supernatant of the cell culture medium, add standards and samples in the 96-well plate at 50 μL / well; add Griess Reagent I and Griess Reagent II at room temperature to each well, and measure the absorbance at 540 nm. Figure 6 Shows the effects of tortoise plastron peptides at different concentrations on activating macrophages RAW264.7 to release NO. The results show that tortoise plastron peptide concentrations greater than 0.15 mg / mL significantly increased the NO release; Figure 7 Shows the effects of tortoise plastron peptides at different concentrations on LPS-induced macrophage inflammation to release NO. The results show that tortoise plastron peptide concentrations of 0.9 and 1.05 mg / mL significantly reduced the effect of LPS on macrophage inflammation to release NO.

[0105] Comparative Example 1: Preparation and Effect Verification of Tortoise Plastron Peptide

[0106] Prepare tortoise plastron peptide according to the method of Example 1, with the difference that in step (5) of "I. Extraction of Total Protein", add it to pure water without EDTA and stir; test the tortoise plastron peptide according to the method of "II. Detection of Macrophage Proliferation Ability" in Example 3, and the results are as Figure 8 and Figure 9 shown. The tortoise plastron peptide prepared in this comparative example has no significant effect on macrophage viability and LPS-induced macrophage inflammation proliferation.

[0107] Comparative Example 2: Preparation and Effect Verification of Tortoise Plastron Peptide

[0108] According to the method of Example 1 in CN107858392A, take the enzymatic hydrolysate of the molecular segment less than 6 KD to prepare tortoise plastron peptide; test the tortoise plastron peptide according to the method of "II. Detection of Macrophage Proliferation Ability" in Example 3, and the results are as Figure 10 and Figure 11As shown, the tortoise plastron peptide prepared in this comparative example has no significant effect on the survival rate of macrophages and the inflammatory proliferation of macrophages induced by LPS.

[0109] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A preparation method of tortoise plastron peptide with immunomodulatory activity, characterized in that, It includes the following steps: 1) Wash, break and boil the turtle shell; 2) Concentrate the soup obtained by boiling; 3) After concentration, perform extraction and dialysis, and then freeze-dry; 4) After drying, add it to an EDTA solution, stir, filter, and freeze-dry the filtrate to obtain a crude protein extract; 5) Mix the crude protein extract with an NaOH solution, adjust the pH value, add protease, mix well, and perform enzymatic hydrolysis; 6) Inactivate the enzyme after enzymatic hydrolysis ends; 7) Centrifuge and collect the centrifugate; 8) Filter the centrifugate through an ultrafiltration membrane, freeze-dry, and pulverize to obtain a polypeptide powder.

2. The preparation method according to claim 1, characterized in that, It includes the following steps: 1) Wash and break the turtle shell, put it into a pot and boil for 8 - 16 hours, and boil three times; 2) Combine and filter the soups obtained from the three boilings, and perform rotary evaporation and concentration at 50 - 60 °C until basically no condensate flows out to obtain a concentrate; 3) Extract the concentrate with petroleum ether, then filter it through a dialysis bag, and then freeze-dry; 4) Pulverize after freeze-drying, add it to a 0.4 - 8 moL / L EDTA solution at an addition amount of 4 - 8%, stir and react for 15 - 60 min, filter, and freeze-dry the filtrate again to obtain a crude protein extract; 5) Add the crude protein extract to an NaOH solution with a pH of 7.5 at a solid-liquid ratio of 1:25, adjust the pH value to 8.0 - 8.5, add protease to make the enzyme concentration in the solution 1.5 - 2.0%, and perform enzymatic hydrolysis in a water bath at a constant temperature of 55 °C for 2 - 6 h; 6) Then put it into a hot water bath and continuously stir to inactivate the enzyme; 7) Centrifuge for 10 minutes and collect the centrifugate; 8) Filter the centrifugate through an ultrafiltration membrane with a molecular weight cut-off less than 10000 D, then put it into a freeze-drying oven to freeze-dry, and pulverize to obtain a polypeptide powder.

3. The preparation method according to claim 2, characterized in that, It includes the following steps: 1) Wash and break the turtle shell, put it into a pot and boil for 12 hours, and boil three times; 2) Combine and filter the soups obtained from the three boilings, and perform rotary evaporation and concentration at 55 °C until basically no condensate flows out to obtain a concentrate; 3) Extract the concentrate with petroleum ether three times, and then filter it through a dialysis bag; 4) Put it into a vacuum freeze-drying oven to freeze-dry, pulverize, add it to a 0.6 moL / L EDTA solution at an addition amount of 6%, stir and react for 30 min, filter, and freeze-dry the filtrate again to obtain a crude protein extract; 5) Add the crude protein extract to an NaOH solution with a pH of 7.5 at a solid-liquid ratio of 1:25, adjust the pH value to 8.0, add protease to make the enzyme concentration in the solution 1.8%, and perform enzymatic hydrolysis in a water bath at a constant temperature of 55 °C for 3 h; 6) Then put it into a 95 °C hot water bath and continuously stir for 15 minutes to inactivate the enzyme; 7) Centrifuge at 4 °C and 10000 r / min for 10 minutes and collect the centrifugate; 8) Filter the centrifugate through an ultrafiltration membrane with a molecular weight cut-off less than 6000 D, then put it into a vacuum freeze-drying oven to freeze-dry, and pulverize to obtain a polypeptide powder.

4. The preparation method according to any one of claims 1-3, characterized in that, The turtle shell is from a turtle of the genus Cuora.

5. The preparation method according to any one of claims 1-3, characterized in that, The protease is a compound protease.

6. The turtle shell peptide prepared by the preparation method according to any one of claims 1 - 5.

7. The tortoise plastron peptide according to claim 6, characterized in that, The turtle shell peptide contains polypeptides with the amino acid sequences shown in SEQ ID NO.1 - 179.

8. A product containing the tortoise plastron peptide described in claim 6 or 7.

9. The product according to claim 8, wherein The product includes food, medicine, and health products.

10. Use of the preparation method according to any one of claims 1 to 5, the tortoise plastron peptide according to any one of claims 6 to 7, or the product according to any one of claims 8 to 9, characterized in that The applications include one or more of the following applications: 1) Application in the preparation of a product for enhancing the proliferative ability of spleen cells; 2) Application in the preparation of a product for increasing the proliferative ability of macrophages; 3) Application in the preparation of a product for increasing the NO release ability of macrophages; 4) Application in the preparation of a product for increasing the phagocytic ability of macrophages; 5) Application in the preparation of a product for alleviating the inflammatory response of macrophages.

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