Active collagen peptide with immunoregulatory function and preparation method and application thereof

Abstract

The application belongs to the technical field of active peptide preparation, and particularly relates to an active collagen peptide with immune regulation function and a preparation method and application thereof. The active collagen peptide with immune regulation function is prepared by using a method of composite enzymolysis and chromatography column separation and collection of specified collection liquid, and a new idea is provided for improving immunity. The prepared active collagen peptide can significantly improve the proliferation activity, phagocytic capacity and NO secretion amount of RAW264.7 cells, change the cell morphology, has an important regulation effect on the recovery of immune balance of immunosuppressed model mice, can regulate the immunocompromised problem of mice induced by cyclophosphamide, regulate the immune balance, and improve the immune capacity of the body.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide preparation technology, specifically relating to a bioactive collagen peptide with immunomodulatory function, its preparation method, and its application. Background Technology

[0002] Immunity is the body's defense mechanism against pathogenic microorganisms and other foreign invaders, executed by the body's immune system. Obesity, aging, unhealthy lifestyle habits, mental stress, and drug side effects can all lead to a decline in immunity, resulting in various diseases. Therefore, various health products and medicines that enhance immunity have appeared on the market.

[0003] The main mechanism of action of existing immune enhancers is to induce the maturation of dendritic cells, enabling them to 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 clear pathogens. However, immune enhancers may over-activate the immune system, leading to a series of adverse reactions and complications, such as allergic reactions, exacerbation of autoimmune diseases, and potential liver and kidney damage, cardiovascular abnormalities, and gastrointestinal discomfort.

[0004] Animal skin has a diverse chemical composition, mainly consisting of amino acids, vitamins, and peptides. Among these, peptides have attracted widespread attention due to their easy absorption and high activity. However, there are few literature reports on the relationship between animal skin peptides and immune function. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an active collagen peptide with immunomodulatory function, its preparation method, and its application. The active collagen peptide is prepared by using a method of compound enzymatic hydrolysis and chromatography column separation to collect a specified collection solution. The obtained active collagen peptide has immunomodulatory function and can be used in the preparation of immune-enhancing products.

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

[0007] This invention also provides a method for preparing the above-mentioned active collagen peptides with immunomodulatory functions, comprising the following steps:

[0008] S1. Cut the animal skin into pieces, boil it in boiling water to remove the fishy smell and impurities;

[0009] S2, add a compound enzyme for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzyme in a water bath to obtain the enzymatic hydrolysate for later use.

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

[0011] S4, the concentrate is separated by column chromatography, and the collected solution containing immunomodulatory components is retained;

[0012] S5. The collected liquid obtained in S4 is treated with an ultrafiltration membrane and concentrated 10 to 15 times. The membrane retentate is then collected.

[0013] S6, vacuum freeze-drying, yields active collagen peptides.

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

[0015] Preferably, 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 to 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, and collagenase; and 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.

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

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

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

[0020] Preferably, in S4, the collection solution containing the immunomodulatory component is the second to third column volume of the collection solution; more preferably, it is the 2.0 to 2.5 column volume of the collection solution.

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

[0022] Preferably, in S6, the parameters for vacuum freeze drying are: pre-freezing at -80℃ for 4 hours, primary drying at -40℃ / 50 mTorr for 24 hours, secondary drying at 25℃ / 20 mTorr for 8 hours, with 5% mannitol added for protection.

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

[0024] Preferably, the active collagen peptides are used in the preparation of immune-enhancing products, which include, but are not limited to, solid beverages, candies, pasta, and other foods; the daily intake of active collagen peptides is 0.1-1g.

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

[0026] (1) The present invention uses a method of compound enzymatic hydrolysis and chromatography column separation to collect a specified collection liquid to prepare active collagen peptides with immunomodulatory function, which provides a new approach to enhance immunity.

[0027] (2) The active collagen peptides of the present invention can significantly improve the proliferation activity, phagocytic capacity and NO secretion of RAW264.7 cells, change cell morphology, play an important regulatory role in the restoration of immune balance in immunosuppressed model mice, regulate the problem of cyclophosphamide-induced low immune function in mice, regulate immune balance, and improve the body's immune capacity. Attached Figure Description

[0028] Figure 1 The column graph shows the effect of the buffalo hide active peptides obtained in Example 1 on the proliferation ability of RAW264.7 cells.

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

[0030] Figure 3 The effect of the buffalo hide active peptides obtained in Example 1 on the phagocytic ability of RAW264.7 cells;

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

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

[0033] Figure 6 The value represents the number of peripheral blood leukocytes in each group of mice in this invention. Detailed Implementation

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

[0035] Example 1

[0036] A method for preparing buffalo hide bioactive peptides with immunomodulatory functions includes the following steps:

[0037] S1. Take 50g of raw buffalo hide and cut it into small pieces about 2cm long. Add 5 times the weight of the raw buffalo hide to the cut buffalo hide and pre-boil for 10 minutes.

[0038] S2, add 0.5g of trypsin and gastric lipase (m 胰蛋白酶 :m 脂肪酶 The mixture of 5:1 was adjusted to pH 5.5 with citric acid, placed in an electric stirrer, and enzymatically hydrolyzed at 50°C for 4 hours. After the enzymatic hydrolysis was completed, the enzyme was inactivated in a water bath at 90°C for 15 minutes.

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

[0040] S4. The concentrate was separated by Sephadex G25 column chromatography, and the collected liquid was retained at a volume of 2.5 times the column volume.

[0041] S5 is treated with an ultrafiltration membrane with a molecular weight cutoff of ≤5000Da and concentrated 10 times to obtain the membrane retentate.

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

[0043] The yield of buffalo hide active peptides in this embodiment was measured to be 33.61%.

[0044] Example 2

[0045] The difference from Example 1 is that in S2, neutral protease and intestinal lipase (m 中性蛋白酶 :m 肠脂肪酶 The mixture of 5:1 was enzymatically hydrolyzed, and the pH was adjusted to 7 with citric acid. All other steps were the same as in Example 1.

[0046] The yield of buffalo hide active peptides in this embodiment was measured to be 32.75%.

[0047] Example 3

[0048] The difference from Example 2 is that in S2, the enzyme is hydrolyzed at 40°C for 3 hours in an electric stirrer, while the other steps are the same as in Example 2.

[0049] The yield of buffalo hide active peptides in this embodiment 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. 胶原蛋白酶 :m 肠脂肪酶 The amount of the mixture (5:1) added was 0.3g, and the other steps were the same as in Example 3.

[0052] The yield of buffalo hide active peptides in this embodiment 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, while the other steps are the same as in Example 1.

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

[0056] Example 6

[0057] The difference from Example 1 is that in S4, the collection liquid of the second column volume is retained, while the other steps are the same as in Example 1.

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

[0059] Comparative Example 1

[0060] The difference from Example 1 is that in S4, the collection liquid of the third column volume is retained, while the other steps are the same as in Example 1.

[0061] Comparative Example 2

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

[0063] Comparative Example 3

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

[0065] Experimental 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 buffalo hide active peptides in each experimental group was determined by the Kjeldahl method, and the survival rate of RAW264.7 cells was detected by the MTT assay. The results are shown in Table 1 below.

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

[0068]

[0069]

[0070] As shown in Table 1, the buffalo hide active peptides obtained in Example 1 had the highest protein content, reaching 68.2%, and the cell viability of RAW264.7 cells reached 131.55%, demonstrating the best performance among all examples. The protein concentration and cell viability of Examples 1-6 were significantly higher than those of Comparative Examples 1-3. This indicates that retaining different collection solutions, changing the type of enzyme, or changing the packing material all affect the properties of the peptides. Furthermore, different extraction methods resulted in varying purity of the buffalo hide active peptides. Clearly, the method of this invention can more effectively increase the concentration of buffalo hide active peptides, reduce impurities, and improve the survival rate of RAW264.7 cells.

[0071] Experimental Example 2: In vitro immunomodulatory effect test of buffalo hide active peptides (all buffalo hide active peptides used were 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 the cells reached nearly 80% growth, they were passaged by repeated pipetting, and cells in the logarithmic growth phase were selected for experiments.

[0074] 2. Detection indicators

[0075] The following test indicators were analyzed using Prism 9.5 software to process the experimental data and perform statistical analysis.

[0076] 2.1 Effect of buffalo hide active peptides on the proliferation of RAW264.7 cells

[0077] Macrophages are an important component of the innate immune system. They play a key role in recognizing and engulfing pathogens and cellular debris, as well as in participating in immune and inflammatory responses. Macrophage proliferation helps enhance the immune system's surveillance and defense capabilities.

[0078] The method for detecting the proliferation effect of buffalo hide active peptides on RAW264.7 cells was as follows: RAW264.7 cells in the exponential growth phase were taken, and the cell density was adjusted. 100 μL of cell suspension was added to each well of a 96-well plate. The blank control group was added with complete DMEM culture medium, and the drug-treated groups were 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, respectively. The cells were cultured at 37℃ in an incubator containing 5% CO2 for 24 h, with 6 replicates per group. After 24 h, the supernatant was discarded, and 20 μL of MTT staining solution was added for incubation in the dark. After 4 h in the dark, MTT was discarded, and 100 μL of DMSO was added. The mixture was shaken for 10 min to dissolve the crystals, and the absorbance at 570 nm was measured immediately to calculate the cell proliferation index.

[0079] The effects of different concentrations of buffalo hide active peptides on macrophage proliferation activity, such as Figure 1 As shown, by Figure 1 It was found that buffalo hide active peptides could promote the proliferation of RAW264.7 cells. Within the concentration range of 6.25 μg / mL to 50 μg / mL, the proliferation index of RAW264.7 cells showed a trend of first increasing and then decreasing. After culturing with buffalo hide active peptides at a concentration of 25 μg / mL for 24 h, the proliferation rate of RAW264.7 cells was significantly increased compared with the control group, and there was no cytotoxic effect on RAW264.7 cells.

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

[0081] Changes in macrophage morphology are an important characteristic of macrophage activation and the realization of cellular functions when stimulated.

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

[0083] The effects of different concentrations of buffalo hide active peptides on the morphology of RAW 264.7 cells, such as... Figure 2 As shown. By Figure 2It was observed that cells in the normal group were round without obvious angles. Different concentrations of buffalo hide active peptides altered the morphology of RAW 264.7 cells (indicated by red arrows). Compared with the normal control group, the low-dose group (6.25 μg / mL) showed differentiation, with some cells developing pseudopodia. In the medium-dose groups (12.5 μg / mL and 25 μg / mL), some cells developed pseudopodia. In the high-dose group (50 μg / mL), cells became larger, showed significant differentiation, and had an increased number of pseudopodia. After LPS activation, the number of differentiated cells significantly increased, and the cell shapes were mostly irregular with pseudopodia (indicated by red arrows). These results indicate that the morphology of RAW 264.7 cells changes to varying degrees under stimulation by different concentrations of buffalo hide active peptides, suggesting that buffalo hide active peptides have a certain activating effect on RAW 264.7 cells.

[0084] 2.3 Effects of buffalo hide active peptides on the phagocytic capacity of RAW264.7 cells

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

[0086] The method for detecting the phagocytic ability of buffalo hide active peptides on RAW264.7 cells was as follows: RAW264.7 cells in the logarithmic growth phase were taken and the density was adjusted to approximately 1×10⁻⁶. 4 Cells were seeded at a density of 200 μL / mL into 96-well plates. After 24 h of incubation, the supernatant was discarded, and 200 μL of the sample solutions obtained in Example 1 (6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, and 50 μg / mL, respectively) were added to each well. The blank group received only 200 μL of complete culture medium, while the positive control group received 200 μL of culture medium containing LPS (final LPS concentration 1 μg / mL). Six replicates were set up for each group. After 24 h of incubation, the supernatant was discarded, and the cells were washed twice with PBS. 200 μL of culture medium and 20 μL of neutral red staining solution were added to each well, and the cells were incubated for 2 h. The supernatant was discarded, and 200 μL of lysis buffer was added to each well. The cells were shaken at room temperature for 10 min, and the absorbance was measured using a microplate reader to calculate the phagocytic index.

[0087] The effects of different concentrations of buffalo hide active peptides on the phagocytic capacity of RAW264.7 cells, as follows: Figure 3 As shown. By Figure 3 It was found that both lipopolysaccharide (LPS) and buffalo hide active peptides could enhance the phagocytic ability of RAW264.7 cells. Within the concentration range of 6.25 μg / mL to 50 μg / mL, the phagocytic rate of RAW264.7 cells showed a trend of first increasing and then decreasing. After culturing with buffalo hide active peptides at a concentration of 25 μg / mL for 24 h, the phagocytic rate of RAW264.7 cells showed a significant increase compared to the control group.

[0088] 2.4 Effects of buffalo hide active peptides on NO secretion in 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 has the function of killing bacteria, viruses and tumor cells.

[0090] The method for detecting NO secretion in RAW264.7 cells by buffalo hide active peptides was as follows: Logarithmic growth phase RAW264.7 cells were seeded in 96-well culture plates, with a volume of 200 μL per well and a density of 102. 5 Cells were cultured at 37°C and 5% CO2 for 24 hours. After cell attachment, the supernatant was discarded, and 200 μL of the sample solution obtained in Example 1 (6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, and 50 μg / mL) were added to each well. A blank group and a lipopolysaccharide (LPS) group were set up, with 6 replicates in each group. After culturing at 37°C and 5% CO2 for 24 hours, 200 μL of the supernatant was collected, and the NO secretion was measured according to the instructions of each kit.

[0091] The effects of different concentrations of buffalo hide active peptides on NO release from macrophages, as follows: Figure 4 As shown. By Figure 4 It was found that the stimulatory effect of buffalo hide active peptides on NO secretion in RAW264.7 cells was dose-related. At a concentration of 25 μg / mL, the amount of NO secreted by RAW264.7 cells was 6.88 μmol / mL, which was close to the amount of NO produced in the LPS-positive control group (7.01 μmol / mL). Figure 4 As shown, the horizontal axis represents the concentration of active peptides in buffalo hide, and there was no significant difference between the two groups (P>0.05).

[0092] Experimental Example 3: In vivo immunomodulatory effect of buffalo hide active peptides

[0093] 1. Animal grouping and feeding methods

[0094] After the acclimatization period, mice were randomly divided into 5 groups: a blank control group, a model control group, a positive control group, a low-dose sample group, and a high-dose sample group, with 8 mice in each group. Except for the blank control group, all other groups received intraperitoneal injections of cyclophosphamide daily at a dose of 80 mg / kg for 5 consecutive days to establish the model. On day 6, gavage treatment began. The positive control group received levamisole at a dose of 25 mg / kg, the sample treatment groups received the low-dose (100 mg / kg) and high-dose (500 mg / kg) buffalo hide active peptide samples from Example 1, respectively, and the blank control and model control groups received an equal volume of distilled water. This gavage treatment continued for 14 days. After the last gavage treatment, mice were fasted for 12 hours but allowed free access to water, and were then sacrificed for various immune marker tests. Specific experimental groups and gavage doses of the test samples are shown in Table 2. The low-dose and high-dose groups represent the low and high doses of buffalo hide active peptide converted from the recommended human dose.

[0095] Table 2. Experimental Groups and Test Samples Administered Test Substances and Doses

[0096]

[0097]

[0098] 2. Detection indicators

[0099] The following test indicators were analyzed using Prism 9.5 software to process the experimental data and perform statistical analysis.

[0100] 2.1 Effects of buffalo hide active peptides on mouse immune organs

[0101] The thymus and spleen are two important immune organs in the body. The thymus is the site of T lymphocyte differentiation, development, and maturation, while the spleen is the site of T and B lymphocyte storage and function. Therefore, their weight gain often represents lymphocyte proliferation, thus directly reflecting the strength of the immune response. This experiment investigated the effects of buffalo hide active peptides from Example 1 on the immune organs of mice. The immune organ index was determined as follows: 14 days after administration, mice were fasted but not watered for 12 hours, weighed, and then euthanized by cervical dislocation. The thymus and spleen were removed, and the peritoneal fluid on the surface of the organs was blotted dry with filter paper. The organs were then weighed, and the difference was divided by the mouse's body weight. The calculation formula is as follows:

[0102]

[0103] Figure 5 The changes in thymus and spleen indices in mice under different treatment groups are shown. Figure 5As shown, after 14 days of gavage treatment, the thymus index and spleen index of the model group mice were lower than those of the blank control group, positive control group and low- and high-dose sample groups. This indicates that buffalo hide active peptides increased the thymus index and spleen index of immunocompromised mice, which can effectively delay the degeneration of immune organs. Its effect is similar to that of the positive drug levamisole, and there is no significant dose dependence.

[0104] 2.2 Effects of buffalo hide active peptides on peripheral blood leukocyte levels in mice

[0105] Changes in peripheral blood cell levels in mice can reflect their immune status. This experiment investigated the effect of buffalo hide active peptides from Example 1 on peripheral blood leukocyte levels in mice. The procedure for measuring mouse peripheral blood leukocyte levels was as follows: after drug administration, blood was collected from the eyeballs of mice, and the peripheral blood leukocyte levels were measured using a complete blood count analyzer within 2 hours.

[0106] Figure 6 The effect of buffalo hide active peptides on peripheral blood leukocyte levels in mice was demonstrated. Figure 6 As shown, after 14 days of gavage treatment, compared with the model group, the peripheral blood leukocyte levels in the low- and high-dose groups of mice were significantly increased (p<0.05), essentially returning to the levels of the blank control group. These results indicate that buffalo hide active peptides have a regulatory effect on the immune function of immunosuppressed mice.

[0107] In summary, this invention, using a RAW264.7 cell model, verified that buffalo hide active peptides can enhance the proliferation capacity of RAW264.7 cells, alter cell morphology, and increase phagocytic capacity and NO secretion, indicating that buffalo hide active peptides have an immune-enhancing effect. An immunocompromised mouse model was established by intraperitoneal injection of cyclophosphamide. After continuous gavage administration of buffalo hide active peptides for 14 days, the spleen and thymus indices of the mice increased, effectively inhibiting the decline of immune organs induced by cyclophosphamide. The peripheral blood leukocyte level of the mice significantly increased, essentially returning to the level of the blank control group, indicating that appropriate intake of buffalo hide active peptides can effectively enhance immunity, improve immunodeficiency, and improve physical condition. Therefore, this research provides theoretical support for the application of buffalo hide active peptides in the preparation of nutritional foods, regulatory drugs, or health products with immunomodulatory effects.

Claims

1. An active collagen peptide with immunomodulatory function, characterized in that, The active collagen peptides are obtained from animal hide, specifically buffalo hide. Preparation methods include: S1. Cut the animal skin into pieces, boil it in boiling water to remove the fishy smell and impurities; S2, add a complex enzyme of protease and lipase for enzymatic hydrolysis, with a mass ratio of 8:1 to 4:1; after enzymatic hydrolysis, inactivate the enzyme in a water bath to obtain the hydrolysate for later use; the complex enzyme is selected from any one of the following: trypsin and gastric lipase, neutral protease and intestinal lipase, collagenase and intestinal lipase combination. S3. Cool the enzymatic hydrolysate to room temperature, centrifuge, and concentrate it 3-5 times to obtain the supernatant concentrate. S4, the concentrate is separated by column chromatography, and the collection solution containing immunomodulatory components is retained; the collection solution of 2 to 2.5 column volumes is retained; the packing material used for column chromatography is selected from either Sephadex G25 or Bio-Gel P-6; S5. The collected liquid obtained in S4 is treated with an ultrafiltration membrane and concentrated 10 to 15 times. The membrane retentate is collected. An ultrafiltration membrane with a molecular weight cutoff of ≤5000 Da is used. S6, vacuum freeze-drying, yields active collagen peptides.

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

3. The preparation method according to claim 1, characterized in that, In S2, the added mass of the compound enzyme is 0.05~0.2% of the mass of the liquid; the pH of the liquid is adjusted to 5.5-7.5 with citric acid; the enzymatic hydrolysis temperature is 40~60℃ and the time is 2~6h; the enzyme is inactivated by water bath at 85~95℃ for 10~20min.

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

5. The preparation method according to claim 1, characterized in that, In S6, the parameters for vacuum freeze drying are: pre-freezing at -80℃ for 4 hours, primary drying at -40℃ / 50 mTorr for 24 hours, secondary drying at 25℃ / 20 mTorr for 8 hours, with 5% mannitol added for protection.

6. The use of the active collagen peptides prepared by the preparation method according to any one of claims 1-5 in the preparation of immune-enhancing products.

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