Active collagen peptide with immune regulation function as well as preparation method and application of active collagen peptide

Abstract

The invention belongs to the technical field of active peptide preparation, and particularly relates to an active collagen peptide with an immunoregulation function and a preparation method and application thereof. The active collagen peptide with an immunoregulation function is prepared by using a method of performing composite enzymolysis and separating and collecting specified collection liquid by using a chromatographic column, and a new thought is provided for improving the immunity; the prepared active collagen peptide can significantly improve the proliferative activity, phagocytic ability and NO secretion amount of RAW264.7 cells and change the cell morphology, has an important regulation effect on immune balance recovery of immunosuppression model mice, and can regulate the problem of low immunologic function of mice induced by cyclophosphamide, regulate immune balance and improve the body immunity.

Description

Technical Field

[0001] The present invention belongs to the technical field of active peptide preparation, and specifically relates to an active collagen peptide with immunomodulatory function and a preparation method and application thereof. Background Art

[0002] Immunity is the body's defense against pathogenic microorganisms and other foreign invasions, and is performed by the body's immune system. Obesity, aging, bad living habits, mental stress, and drug side effects can all lead to a decline in immunity, which in turn leads to the occurrence of various diseases. Therefore, a variety of health products or medicines that enhance immunity have begun to appear on the market.

[0003] The main mechanism of action of the existing immunopotentiators is to induce the maturation of dendritic cells, so that they can present antigens more effectively, activate T cells, enhance the activity of natural killer cells and the phagocytic function of phagocytes, thereby improving the body's ability to eliminate pathogenic microorganisms. However, immunopotentiators may over-activate the immune system, leading to a series of adverse reactions and complications, such as allergic reactions, aggravation of autoimmune diseases, and may also cause liver and kidney damage, cardiovascular system abnormalities, gastrointestinal discomfort and other problems.

[0004] The chemical composition of animal skins is diverse, mainly composed of amino acids, vitamins, and peptides. Among them, peptides have the advantages of being easily absorbed and highly active, and have received widespread attention. However, there are few literature reports on the relationship between animal skin peptides and immune function. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides an active collagen peptide with immunomodulatory function and a preparation method and application thereof. The active collagen peptide is prepared by using a method of composite enzymatic hydrolysis and chromatography column separation to collect a specified collection liquid. The obtained active collagen peptide has immunomodulatory function and can be used in the preparation of immune enhancement products.

[0006] On the one hand, the present invention provides an active collagen peptide with immunomodulatory function, wherein the active collagen peptide is made from animal skin; preferably, the animal skin is cowhide, and more preferably, the animal skin is selected from at least one of buffalo skin, yak skin, cow skin, and yellow cattle skin.

[0007] The present invention also provides a method for preparing the above-mentioned active collagen peptide having immunomodulatory function, comprising the following steps:

[0008] S1, cut the animal skin into pieces, pre-boil in boiling water, and remove the fishy smell and impurities;

[0009] S2, adding compound enzyme for enzymolysis, after the enzymolysis is completed, inactivate the enzyme in a water bath, and obtain the enzymolysis solution for use;

[0010] S3. Cool the enzymatic hydrolysate to room temperature, centrifuge it, and concentrate it 3 - 5 times to obtain the supernatant concentrate.

[0011] S4. Separate the concentrate by column chromatography and retain the collected liquid containing the immunomodulatory component.

[0012] S5. Treat the collected liquid obtained in S4 with an ultrafiltration membrane, concentrate it 10 - 15 times, and collect the retentate on the membrane.

[0013] S6. Vacuum freeze - dry to obtain the active collagen peptide.

[0014] Preferably, in S1, the mass ratio of the water added during boiling pre - cooking to the mass of the animal skin is 5:1, and the boiling pre - cooking time is 10 - 30 min.

[0015] Preferably, in S2, the composite enzyme is selected from at least one of protease and lipase; the mass ratio of the protease to the lipase is 8:1 - 4:1.

[0016] Preferably, the protease is selected from at least one or a mixture of two or more of papain, pepsin, neutral protease, trypsin, subtilisin, elastase, collagenase; the lipase is selected from at least one or a mixture of two or more of pancreatic lipase, gastric lipase, intestinal lipase, lipoprotein lipase, hepatic lipase.

[0017] Preferably, in S2, the added mass of the composite enzyme is 0.05 - 0.2% of the mass of the feed liquid; the pH of the feed liquid is adjusted to 5.5 - 7.5 with citric acid; the enzymatic hydrolysis temperature is 40 - 60 °C, the enzymatic hydrolysis time is 2 - 6 h, more preferably 3 - 4 h; the temperature for inactivating the enzyme by water bath is 85 - 95 °C, and the time is 10 - 20 min.

[0018] Preferably, in S3, the centrifugal force is 8000 - 10000 g, and the time is 15 - 20 min.

[0019] Preferably, in S4, the packing material for column chromatography separation is selected from at least one of Sephadex G25, Bio - Gel P - 6, Toyopearl HW - 40, Superdex 30 Increase, Cellufine GCL - 25; the mobile phase is a PBS buffer solution with a concentration of 0.1 M and a pH of 6.5 - 7.5.

[0020] Preferably, in S4, the collected liquid containing the immunomodulatory component is the collected liquid of the 2 - 3 times of the column volume; more preferably, it is the collected liquid of 2.0 - 2.5 times of the column volume.

[0021] Preferably, in S5, an ultrafiltration membrane with a molecular weight cut - off ≤5000 Da is used.

[0022] Preferably, in S6, the parameters of vacuum freeze-drying are pre-freezing at -80°C for 4 h, primary drying at -40°C / 50 mTorr for 24 h, secondary drying at 25°C / 20 mTorr for 8 h, and adding 5% mannitol for protection.

[0023] On the other hand, the active collagen peptide prepared by the above method, and the application of the active collagen peptide in the preparation of immune-enhancing products are also within the protection scope of the present invention.

[0024] Preferably, for the application of the active collagen peptide in the preparation of immune-enhancing products, the immune-enhancing products include but are not limited to foods such as solid beverages, candies, and pasta; the daily intake of the active collagen peptide is 0.1 - 1 g.

[0025] The beneficial effects of the present invention are as follows:

[0026] (1) The present invention uses the methods of composite enzymatic hydrolysis and chromatography column separation to collect the specified collection liquid, and prepares an active collagen peptide with immune regulation function, providing a new idea for improving immunity;

[0027] (2) The active collagen peptide of the present invention can significantly improve the proliferation activity, phagocytosis ability and NO secretion of RAW264.7 cells, change the cell morphology, play an important regulatory role in restoring the immune balance of immunosuppressive model mice, can regulate the problem of immune function decline induced by cyclophosphamide in mice, regulate the immune balance, and improve the body's immune ability. Description of the Drawings

[0028] Figure 1 It is a column chart showing the effect of the buffalo hide active peptide obtained in Example 1 on the proliferation ability of RAW264.7 cells;

[0029] Figure 2 It is the effect of the buffalo hide active peptide obtained in Example 1 on the morphological changes of RAW264.7 cells;

[0030] Figure 3 It is the effect of the buffalo hide active peptide obtained in Example 1 on the phagocytosis ability of RAW264.7 cells;

[0031] Figure 4 It is the effect of the buffalo hide active peptide obtained in Example 1 on the NO secretion of RAW264.7 cells;

[0032] Figure 5 It is the effect of the buffalo hide active peptide obtained in Example 1 on the immune organ index of immunosuppressed mice;

[0033] Figure 6 It is the peripheral blood white blood cell count of each group of mice in the present invention. Detailed Embodiments

[0034] In order to enable those skilled in the art to better understand the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0035] Example 1

[0036] A preparation method of a water buffalo hide active peptide with immunomodulatory function, comprising the following steps:

[0037] S1. Take 50 g of raw water buffalo hide, cut it into small pieces about 2 cm long, add 5 times the mass of distilled water of the raw water buffalo hide, add it to the cut raw water buffalo hide, and pre-cook it in boiling water for 10 min;

[0038] S2. Add a mixture of 0.5 g of trypsin and gastric lipase (m 胰蛋白酶 :m 脂肪酶 = 5:1), adjust the pH to 5.5 with citric acid, place it in an electric stirrer, enzymatically hydrolyze at 50 °C for 4 h, and after the enzymatic hydrolysis is completed, inactivate the enzyme in a water bath at 90 °C for 15 min;

[0039] S3. Cool the enzymatic hydrolysate to room temperature, centrifuge at high speed (centrifugation speed is 10000 g, time is 20 min), and concentrate 4 times to obtain a supernatant concentrate;

[0040] S4. Separate the concentrate by Sephadex G25 column chromatography, and retain the collected liquid at 2.5 times the column volume;

[0041] S5. Treat with an ultrafiltration membrane with a molecular weight cut-off ≤ 5000 Da, concentrate 10 times to obtain a membrane retentate;

[0042] S6. Pre-freeze the membrane retentate at -80 °C for 4 h, primary drying at -40 °C / 50 mTorr for 24 h, secondary drying at 25 °C / 20 mTorr for 8 h, and perform vacuum drying under the protection of 5% mannitol to obtain the water buffalo hide active peptide.

[0043] The yield of the water buffalo hide active peptide in this example was measured to be 33.61%.

[0044] Example 2

[0045] The difference from Example 1 is that in S2, a mixture of neutral protease and intestinal lipase (m 中性蛋白酶 :m 肠脂肪酶 = 5:1) is used for enzymatic hydrolysis, and the pH is adjusted to 7 with citric acid, and other steps are the same as those in Example 1.

[0046] The yield of the water buffalo hide active peptide in this example was measured to be 32.75%.

[0047] Example 3

[0048] The difference from Example 2 is that in S2, it is enzymatically hydrolyzed at 40 °C for 3 h in an electric stirrer, and other steps are the same as those in Example 2.

[0049] The yield of buffalo hide active peptides in this example was measured to be 32.94%.

[0050] Example 4

[0051] The difference from Example 3 is that in S2, a mixture of collagenase and intestinal lipase is used for enzymatic hydrolysis, and the addition amount of the mixture of collagenase and intestinal lipase (m 胶原蛋白酶 :m 肠脂肪酶 = 5:1) is 0.3 g, and other steps are the same as those in Example 3.

[0052] The yield of buffalo hide active peptides in this example was measured to be 29.64%.

[0053] Example 5

[0054] The difference from Example 1 is that in S4, Bio-Gel P-6 is used as the filler, and other steps are the same as those in Example 1.

[0055] The yield of buffalo hide active peptides in this example was measured to be 28.11%.

[0056] Example 6

[0057] The difference from Example 1 is that in S4, the collected liquid of 2.0 times the column volume is retained, and other steps are the same as those in Example 1.

[0058] The yield of buffalo hide active peptides in this example was measured to be 30.23%.

[0059] Comparative Example 1

[0060] The difference from Example 1 is that in S4, the collected liquid of 3.0 times the column volume is retained, and other steps are the same as those in Example 1.

[0061] Comparative Example 2

[0062] The difference from Example 1 is that in S2, 0.5 g of aspergillus protease is used for enzymatic hydrolysis, and other steps are the same as those in Example 1.

[0063] Comparative Example 3

[0064] The difference from Example 1 is that in S4, Sephadex G100 is used as the filler, and other steps are the same as those in Example 1.

[0065] Test Example 1

[0066] The properties of the buffalo hide active peptides prepared in Examples 1-6 and Comparative Examples 1-3 were characterized. The protein concentration of the buffalo hide active peptides in each experimental group was determined by the Kjeldahl method, and the viability of RAW264.7 cells was detected by the MTT method. The results are shown in Table 1 below.

[0067] Table 1 Protein concentration of buffalo hide active peptides obtained in each example and comparative example and viability of RAW264.7 cells

[0068]

[0069]

[0070] According to the data in Table 1, the protein content in the buffalo hide active peptide obtained in Example 1 reached a maximum of 68.2%, and the viability of RAW264.7 cells reached 131.55%, showing the best effect among all examples. The protein concentration and cell viability in Examples 1-6 were significantly higher than those in Comparative Examples 1-3. That is, retaining different collecting solutions, changing the type of enzyme, or changing the filler would all affect the properties of the peptide. The purity of the buffalo hide active peptide obtained by different extraction methods was also different. Obviously, the method of the present invention can more effectively increase the concentration of the buffalo hide active peptide, reduce impurities, and improve the viability of RAW264.7 cells.

[0071] Test Example 2 Test on the in vitro immunomodulatory application effect of buffalo hide active peptides (all buffalo hide active peptides used were those obtained in Example 1)

[0072] 1. Cell culture

[0073] RAW264.7 cells were cultured in DMEM containing 10% FBS and incubated in an incubator at 37°C and 5% CO2. When they grew to nearly 80% or more, they were passaged by repeated pipetting, and cells in the logarithmic growth phase were selected for the experiment.

[0074] 2. Detection indexes

[0075] The following detection indexes were processed and statistically analyzed for the experimental data using Prism 9.5 software.

[0076] 2.1 Proliferation effect of buffalo hide active peptides on RAW264.7 cells

[0077] Macrophages are an important part of the innate immune system. They play a key role in recognizing and phagocytosing pathogens, cell debris, and participating in immune and inflammatory responses. The proliferation of macrophages helps to enhance the surveillance and defense capabilities of the immune system.

[0078] The method for detecting the proliferation effect of buffalo hide active peptide on RAW264.7 cells is as follows: RAW264.7 cells in the exponential growth phase are taken, the cell density is adjusted, and 100 μL of cell suspension is added to each well of a 96-well plate. The blank control group is added with DMEM complete culture medium, and the drug administration groups are added with 200 μL of the sample solution obtained in Example 1 at concentrations of 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, and 50 μg / mL. They are cultured in an incubator at 37 °C with 5% CO2 for 24 h, and each group has 6 replicate wells. After 24 h, the upper-layer culture medium is discarded, 20 μL of MTT staining solution is added, and the culture is carried out in the dark. After 4 h in the dark, the MTT is discarded, 100 μL of DMSO is added, and the mixture is shaken for 10 min to dissolve the crystals. Immediately, the absorbance at 570 nm is measured, and the cell proliferation index is calculated.

[0079] The effects of buffalo hide active peptide at different concentrations on the proliferation activity of macrophages are as Figure 1 shown. It can be Figure 1 seen that buffalo hide active peptide can promote the proliferation of RAW264.7 cells. In the range of mass concentration from 6.25 μg / mL to 50 μg / mL, the proliferation index of RAW264.7 cells shows a trend of first increasing and then decreasing. After culturing RAW264.7 cells with 25 μg / mL of buffalo hide active peptide added for 24 h, the proliferation rate of RAW264.7 cells shows a significant increase compared with the blank group, and there is no cytotoxic effect on RAW264.7 cells.

[0080] 2.2 Effects of buffalo hide active peptide on the morphological changes of RAW264.7 cells

[0081] The morphological change of macrophages is an important feature of their being stimulated, activated, and realizing cell functions.

[0082] The method for detecting the morphological changes of RAW264.7 cells by buffalo hide active peptide is as follows: When the cells are in the logarithmic growth phase, their density is adjusted to 5×10 4 cells / mL and inoculated into a 6-well plate, with 2 mL added to each well. After culturing for 12 h, the old nutrient solution is discarded. In the experimental groups, buffalo hide active peptide at different concentrations (6.25, 12.5, 25, 50 μg / mL) is added. A blank control group (DMEM complete culture medium) and a positive control group (1 μg / mL LPS) are set, and 2 mL is added to each well. After culturing for 24 h, the cell morphology is observed under an inverted microscope and photographed.

[0083] The changes in the morphology of RAW 264.7 cells by buffalo hide active peptide at different concentrations are as Figure 2 shown. It can be Figure 2It can be seen that the cells in the normal group are round without obvious edges and corners; the morphology of RAW 264.7 cells is changed by active peptides from buffalo hide at different concentrations (indicated by the red arrows). Compared with the normal control group, differentiation occurred in the low-dose group (6.25 μg / mL), with individual cells growing pseudopodia. In the medium-dose groups (12.5 μg / mL and 25 μg / mL), some cells had pseudopodia. In the high-dose group (50 μg / mL), the cell volume increased, differentiation was obvious, and the number of pseudopodia increased. After LPS activated the cells, the number of differentiated cells increased significantly, and the cell shapes were mostly irregular with pseudopodia (indicated by the red arrows). These results indicate that the morphology of RAW 264.7 cells changes to varying degrees under the stimulation of active peptides from buffalo hide at different concentrations, suggesting that active peptides from buffalo hide have a certain activating effect on RAW 264.7 cells.

[0084] 2.3 Effects of Active Peptides from Buffalo Hide on the Phagocytic Ability of RAW264.7 Cells

[0085] Macrophages can recognize and phagocytose bacteria through mechanisms such as pattern recognition receptors, thus playing an important role in the host defense against bacterial infections.

[0086] The method for detecting the phagocytic ability of active peptides from buffalo hide on RAW264.7 cells is as follows: RAW264.7 cells in the logarithmic growth phase are taken, and the density is adjusted to about 1×10 4 cells / mL. The cells are inoculated into a 96-well plate, 200 μL per well. After culturing for 24 h, the supernatant is aspirated and discarded. Then, 200 μL of the sample solution obtained in Example 1 at 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, and 50 μg / mL are added respectively. Only 200 μL of complete culture medium is added to the blank group, and 200 μL of culture medium containing LPS (the final concentration of LPS is 1 μg / mL) is added to the positive control group. Six replicates are set for each group. After incubating for 24 h, the supernatant is aspirated and discarded, and the cells are washed twice with PBS. Then, 200 μL of culture medium and 20 μL of neutral red staining solution are added to each well, and incubated for 2 h. The supernatant is aspirated and discarded, and 200 μL of lysis solution is added to each well. The mixture is shaken at room temperature for 10 min, and the absorbance is detected by an enzyme-linked immunosorbent assay (ELISA) reader to calculate the phagocytosis index.

[0087] The effects of active peptides from buffalo hide at different concentrations on the phagocytic ability of RAW264.7 cells are as Figure 3 shown. As Figure 3 can be seen, both lipopolysaccharide (LPS) and active peptides from buffalo hide can improve the phagocytic ability of RAW264.7 cells. In the range of mass concentration from 6.25 μg / mL to 50 μg / mL, the phagocytosis rate of RAW264.7 cells shows a trend of first increasing and then decreasing. After culturing for 24 h with the addition of 25 μg / mL active peptides from buffalo hide, the phagocytosis rate of RAW264.7 cells shows a significant increase compared with the blank group.

[0088] 2.4 Effect of Buffalo Hide Active Peptide on NO Secretion of RAW264.7 Cells

[0089] Nitric oxide (NO) is a key regulator of immune cell function, participating in the body's inflammatory and immune regulation processes, and having the function of killing bacteria, viruses, and tumor cells.

[0090] The method for detecting the NO secretion of buffalo hide active peptide on RAW264.7 cells is as follows: RAW264.7 cells in the logarithmic growth phase are inoculated into a 96-well culture plate, with a volume of 200 μL per well and a density of 10 5 cells / well. They are cultured in an incubator at 37°C and 5% CO2 for 24 h. After the cells adhere to the wall, the supernatant is discarded. Then, 200 μL of the sample solution obtained in Example 1 with concentrations of 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, and 50 μg / mL are added respectively. A blank group and a lipopolysaccharide LPS group are set, with 6 replicates in each group. After culturing for 24 h under the conditions of 37°C and 5% CO2, 200 μL of the supernatant is taken, and the NO secretion is measured according to the instructions of each kit.

[0091] The effects of different concentrations of buffalo hide active peptide on the NO release of macrophages are as Figure 4 shown. As Figure 4 can be seen, the stimulating effect of buffalo hide active peptide on the NO secretion of RAW264.7 cells is dose-related. When the mass concentration of buffalo hide active peptide is 25 μg / mL, the amount of NO secreted by RAW264.7 cells is 6.88 μmol / mL, which is close to the amount of NO (7.01 μmol / mL) generated by the LPS positive control group in RAW264.7, as Figure 4 shown. The abscissa is the concentration of buffalo hide active peptide, and there is no significant difference between the two groups (P>0.05).

[0092] Experimental Example 3 Test on the In Vivo Immunomodulatory Application Effect of Buffalo Hide Active Peptide

[0093] 1. Animal Grouping and Feeding Method

[0094] After the adaptation period of the mice, they were randomly divided into 5 groups, namely the blank control group, the model control group, the positive control group, the low-dose sample group, and the high-dose sample group, with 8 mice in each group. Except for the blank control group, the other groups were intraperitoneally injected with cyclophosphamide every day. The injection dose of cyclophosphamide was 80 mg / kg, and it was continuously injected for 5 days for modeling. On the 6th day, gavage treatment began. The positive control group was gavaged with 25 mg / kg levamisole, the sample administration groups were respectively gavaged with the low-dose 100 mg / kg water buffalo hide active peptide sample and the high-dose 500 mg / kg water buffalo hide active peptide sample of Example 1, and the blank control group and the model control group were gavaged with an equal amount of distilled water. The gavage treatment was continuously carried out for 14 days. After the last gavage treatment, the mice were fasted but not water-restricted for 12 h, then sacrificed, and various immune indexes were detected. The specific test grouping and the gavage doses of the test samples are shown in Table 2. Among them, the low- and high-dose groups were the low- and high-dose water buffalo hide active peptides converted from the recommended human dose by gavage.

[0095] Table 2 Test grouping and the test substances and doses administered to the test samples

[0096]

[0097]

[0098] 2. Detection indexes

[0099] The following detection indexes were processed and statistically analyzed for the test data using Prism 9.5 software.

[0100] 2.1 Effect of water buffalo hide active peptide on the immune organs of mice

[0101] The thymus and spleen are two important immune organs of the body. The thymus is the place where T lymphocytes differentiate, develop, and mature, and the spleen is the place where T and B lymphocytes are stored and play their roles. Therefore, their weight gain often represents the proliferation of lymphocytes, thus directly reflecting the strength of the immune response. This test example examines the effect of the water buffalo hide active peptide of Example 1 on the immune organs of mice. Among them, the determination process of the immune organ index is as follows: 14 days after administration, the mice were fasted but not water-restricted for 12 h, weighed, then sacrificed by cervical dislocation. The thymus and spleen of the mice were removed, and after blotting the peritoneal fluid on the surface of the organs with filter paper, they were weighed. The differences were divided by the body weight of the mice respectively. The calculation formula is as follows:

[0102]

[0103] Figure 5 Shows the changes in the thymus index and spleen index of mice in different treatment groups. As Figure 5As shown, 14 days after intragastric administration, the thymus index and spleen index of the mice in the model group were lower than those in the blank control group, positive control group, and low- and high-dose sample groups, indicating that the active peptides from buffalo hide increased the thymus index and spleen index of immunosuppressed mice, could effectively delay the degeneration of immune organs, and had a similar effect to the positive drug levamisole without significant dose dependence.

[0104] 2.2 Effects of Active Peptides from Buffalo Hide on the Level of Peripheral Blood Leukocytes in Mice

[0105] The changes in the level of peripheral blood cells in mice can reflect their immune status. In this experimental example, the effects of the active peptides from buffalo hide in Example 1 on the level of peripheral blood leukocytes in mice were investigated. Among them, the determination process of the level of peripheral blood leukocytes in mice was as follows: after the administration ended, blood was collected from the mouse eyeballs, and the level of peripheral blood leukocytes in mice was measured within 2 hours using a blood routine analyzer.

[0106] Figure 6 Shows the effects of active peptides from buffalo hide on the level of peripheral blood leukocytes in mice. As Figure 6 shown, 14 days after intragastric administration, compared with the model group, the levels of peripheral blood leukocytes in the low- and high-dose sample groups of mice were significantly increased (p < 0.05), and basically returned to the level of the blank control group. The above results indicate that the active peptides from buffalo hide have a regulatory effect on the immune function of immunosuppressed mice.

[0107] In summary, through the RAW264.7 cell model, the present invention verified that the active peptides from buffalo hide can improve the proliferation ability of RAW264.7 cells, change the cell morphology, and increase the phagocytosis ability and NO secretion of RAW264.7 cells, indicating that the active peptides from buffalo hide have the effect of enhancing immunity. An immunosuppressed mouse model was established by intraperitoneal injection of cyclophosphamide. After continuous intragastric administration of the active peptides from buffalo hide for 14 days, the spleen index and thymus index of the mice both increased, effectively curbing the attenuation of immune organs caused by cyclophosphamide. The level of peripheral blood leukocytes in the mice was significantly increased and basically returned to the level of the blank control group, indicating that appropriate intake of the active peptides from buffalo hide can effectively enhance immunity, improve the state of low immunity, and improve the physical condition. Therefore, this research of the present invention can provide certain theoretical support for the application of the active peptides from buffalo hide in the preparation of nutritional foods, regulatory drugs, or health care products with immunomodulatory effects.

Claims

1. An active collagen peptide with immunomodulatory function, characterized in that, The active collagen peptide is prepared from animal skins, and the animal skins are selected from at least one of buffalo skins, yak skins, dairy cow skins, and yellow cow skins.

2. The preparation method of the active collagen peptide according to claim 1, wherein, It includes the following steps: S1. Cut the animal skins into pieces, pre-cook them in boiling water to remove fishy smell and impurities. S2. Add a complex enzyme for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzyme in a water bath to obtain an enzymatic hydrolysate for standby. S3. Cool the enzymatic hydrolysate to room temperature, centrifuge it, and concentrate it 3 - 5 times to obtain a supernatant concentrate. S4. Separate the concentrate by column chromatography, and retain the collected liquid containing the immunomodulatory component. S5. Treat the collected liquid obtained in S4 with an ultrafiltration membrane, concentrate it 10 - 15 times, and collect the membrane retentate. S6. Vacuum freeze-dry it to obtain the active collagen peptide.

3. The preparation method according to claim 2, characterized in that, In S1, the mass ratio of the water added during pre-cooking in boiling water to the mass of the animal skins is 5:1, and the pre-cooking time in boiling water is 10 - 30 min.

4. The preparation method according to claim 2, characterized in that, In S2, the complex enzyme is selected from at least one of protease and lipase; the mass ratio of the protease to the lipase is 8:1 - 4:1; the added mass of the complex enzyme is 0.05 - 0.2% of the mass of the feed liquid; the pH of the feed liquid is adjusted to 5.5 - 7.5 with citric acid; the enzymatic hydrolysis temperature is 40 - 60 °C, and the time is 2 - 6 h; the temperature for inactivating the enzyme in the water bath is 85 - 95 °C, and the time is 10 - 20 min.

5. The preparation method according to claim 4, characterized in that, The protease is selected from at least one or a mixture of two or more of papain, pepsin, neutral protease, trypsin, subtilisin, elastase, and collagenase. The lipase is selected from at least one or a mixture of two or more of pancreatic lipase, gastric lipase, intestinal lipase, lipoprotein lipase, and hepatic lipase.

6. The preparation method according to claim 1, characterized in that, In S3, the centrifugal force is 8000 - 10000 g, and the time is 15 - 20 min.

7. The preparation method according to claim 1, characterized in that, In S4, the packing material for column chromatography separation is selected from at least one of Sephadex G25, Bio-Gel P-6, Toyopearl HW-40, Superdex 30 Increase, and Cellufine GCL-25; the mobile phase is a PBS buffer solution with a concentration of 0.1 M and a pH of 6.5 - 7.5; the collected liquid containing the immunomodulatory component is the collected liquid of the 2 - 3 times of the column volume.

8. The preparation method according to claim 1, characterized in that, In S5, an ultrafiltration membrane with a molecular weight cut-off ≤ 5000 Da is used.

9. The preparation method according to claim 1, characterized in that, In S6, the parameters for vacuum freeze-drying are pre-freezing at -80 °C for 4 h, primary drying at -40 °C / 50 mTorr for 24 h, secondary drying at 25 °C / 20 mTorr for 8 h, and protected with 5% mannitol.

10. Application of the active collagen peptide described in claim 1 or the active collagen peptide prepared by the preparation method described in any one of claims 2 - 9 in the preparation of immune-enhancing products.

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