Preparation method and application of milk-derived immunomodulatory peptide

Abstract

The invention discloses a preparation method and application of a milk-derived immunomodulatory peptide, milk-derived casein is taken as a raw material, known immunomodulatory peptide is screened, virtual enzymolysis is performed on the known immunomodulatory peptide, and the immunomodulatory peptide is separated and prepared by a verification and freeze drying method. The casein can be subjected to targeted enzymolysis, the relative content and variety of the immunomodulatory peptide are increased, the situation that the immunomodulatory effect of other immunomodulatory casein peptide products is measured after pure enzymolysis, and the efficacy of the immunomodulatory peptide is not enhanced is reduced, the better immunomodulatory effect is achieved, and the immune cell proliferation effect is effectively promoted.

Description

Technical Field

[0001] The invention relates to the field of dairy product processing, and more specifically to a method for preparing a milk-derived immunomodulatory peptide. Background Art

[0002] Under the dual background of a large number of sub-healthy people and the post-epidemic era caused by the new coronavirus, people have strengthened their awareness of actively improving their immunity. However, the related drugs of immunomodulation are not suitable for long-term regulation of the body's immunity. Therefore, people's demand for natural and non-drug immunomodulators is increasing. Immunomodulatory peptides are one of the main types of bioactive peptides. They have the effects of improving resistance, resisting viral infections and even preventing and treating diseases. Their functions are usually not accompanied by side effects of similar drugs, and they are relatively safe dietary active ingredients. For example, the immunomodulatory peptides obtained by enzymatic hydrolysis of frog meat in the invention of CN115927518 have the effect of proliferation and promoting the secretion of cytokines on mouse macrophages RAW264.7. But frog meat is not suitable as one of the raw materials for a large number of immunomodulatory peptides. Summary of the invention

[0003] The purpose of the present invention is to provide a method for preparing a milk-derived immunomodulatory peptide and its application, which mainly focuses on the preparation of immunomodulatory peptides. The method uses milk-derived casein as a raw material, screens known immunomodulatory peptides, performs virtual enzymolysis on them, and separates and prepares immunomodulatory peptides by a verified freeze-drying method. The purpose is to perform targeted enzymolysis according to the existing immunomodulatory casein peptide sequence, increase the relative content and type of immunomodulatory peptides, reduce the immunomodulatory effect of other immunomodulatory casein peptide products after simple enzymolysis, and lose the enhancement of the efficacy of immunomodulatory peptides, so as to achieve better immunomodulatory effect. The present invention can provide new materials for potential immunomodulators and provide a certain basis for the development of functional foods.

[0004] The purpose of the present invention is achieved through the following technical solutions:

[0005] A method for preparing a casein immunomodulatory peptide comprises the following steps:

[0006] Enzymes are screened based on known immunomodulatory peptides in the BIOPEP database, and the milk-derived casein raw materials are targeted for hydrolysis based on the screened enzymes to obtain casein immunomodulatory peptide products.

[0007] According to the above technical solution, preferably, the casein includes α S1 -Casein, alpha S2 -One or more of casein, β-casein, and κ-casein.

[0008] According to the above technical solution, preferably, the milk-derived casein raw material includes but is not limited to one or more of cow's milk, goat's milk, skim milk, reconstituted milk, casein, sodium caseinate, etc. Taking cow's milk as an example, its casein includes various components, such as: cow α S1 -Casein (ID:1089), α S2 -Casein (ID: 1090), β-casein (ID: 1097, 1100), κ-casein (ID: 1109).

[0009] According to the above technical solution, preferably, the method for preparing the casein immunomodulatory peptide comprises the following steps:

[0010] (1) Screening: From the BIOPEP database ( https: / / biochemia.uwm.edu.pl / biopep / start_ biopep.php ) were used to screen out immunomodulatory peptides derived from casein.

[0011] (2) Simulated enzymatic hydrolysis: The screened immunomodulatory peptides were analyzed using pLogo ( https: / / plogo.uconn.edu / # ) for enzyme screening.

[0012] (3) Enzymatic hydrolysis: Based on the screened enzyme, the optimal temperature and optimal pH of the enzyme are used to enzymatically hydrolyze the milk-derived casein raw material.

[0013] (4) The enzymatic hydrolysate is centrifuged and freeze-dried to obtain the polypeptide.

[0014] According to the above technical scheme, preferably, in step (1), the peptides obtained by screening include but are not limited to YPFPGPIH, KEMPFPK, YQQPVLGPVRGPFPIIV, RELEELNVP GEIVESLSSSEESITRINK, YPFPGPI, YPFPG, PGPIPN, LLYQEPVLGPVRGP FPIIV, RPKHPIKHQGLPQEVLNENLLRF, TTMPLW, KNTMEHVSSSEESIISQ ETYKQEKNMAINPSK.

[0015] According to the above technical solution, preferably, in step (2), the peptides screened in step (1) are first converted into FASTA format of equal width (e.g., 8 amino acid sequences), and input into pLogo for enzyme cleavage preference prediction to obtain the enzyme cleavage site of the peptide; the obtained enzyme cleavage site is searched in the BI OPEP database ( https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php )Search the enzyme library to obtain the screened enzyme.

[0016] According to the above technical solution, preferably, in step (2), the cleavage site of the enzyme includes but is not limited to one or two of F and K.

[0017] According to the above technical scheme, preferably, in step (2), the screened enzyme is one or more of ficin, pepsin, and trypsin, and the optimal enzymatic hydrolysis is ficin; the cleavage sites of ficin are K, F, Y, G, S, L, R, and H; the cleavage sites of pepsin are F, L, G, Y, A, E, Q, T, N, K, V, D, and M; and the cleavage sites of trypsin are K and R.

[0018] According to the above technical solution, preferably, in step (3), single enzyme or multi-enzyme composite hydrolysis can be used, and the multi-enzyme composite hydrolysis can be a composite hydrolysis of pepsin and trypsin.

[0019] According to the above technical solution, preferably, in step (3), the temperature of enzymolysis is 25-65°C, the pH is 3-8.5; the amount of enzyme added is 3.5-30U / mg protein, and the time is 60-280min.

[0020] According to the above technical solution, preferably, in step (3), the enzymatic hydrolysis conditions of the ficin are: temperature of 60-65°C, pH of 6.5-7.5; time of 120-150 min, and the amount of enzyme added is 6.0-7.0 U / mg protein.

[0021] According to the above technical solution, preferably, in step (3), the enzymatic hydrolysis conditions of the pepsin are: temperature of 37°C, pH of 3.0; time of 60-120 min, and the amount of enzyme added is 22.75-23.75 U / mg protein.

[0022] According to the above technical solution, in a preferred case, the enzymatic hydrolysis conditions of the trypsin are: temperature of 37°C, pH of 6.5°C, time of 120-150 min, and the amount of enzyme added is 3.5 U / mg protein.

[0023] According to the above technical solution, preferably, in step (3), the milk-derived casein raw material is a milk-derived casein solution with a protein content of less than 5%. When the milk-derived casein raw material is sodium caseinate, it is a milk-derived casein solution with a protein sodium content of less than 5%.

[0024] According to the above technical solution, preferably, in step (4), the centrifugal conditions are: 4000-5000 rpm / min, 30-35 min.

[0025] According to the above technical solution, preferably, in step (4), the pH of the enzymatic hydrolysate is first adjusted to 4.5-4.6, and then centrifuged.

[0026] According to the above technical solution, preferably, in step (4), desalting is performed after centrifugation, and then freeze-drying is performed.

[0027] According to the above technical solution, preferably, the desalination includes dialysis, electrodialysis, osmotic dialysis, ultrafiltration, nanofiltration, etc. When the desalination is dialysis desalination, the molecular weight of the dialysis bag is less than or equal to 500Da, and the dialysis is not less than 30min.

[0028] According to the above technical solution, preferably, the obtained casein immunomodulatory peptide is subjected to an activity assay to measure the immunomodulatory activity of the polypeptide.

[0029] Specifically, the immunomodulatory peptide can be obtained according to the following preparation method:

[0030] (1) Screen the bovine α-protein from the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php) S1 -Casein (ID:1089), α S2 The known immunomodulatory peptides of -casein (ID: 1090), β-casein (ID: 1097, 1100), and κ-casein (ID: 1109) are YPFPGPIH, KEMPFPK, YQQPVLGPVRGPFPIIV, RELEELNVPGEIVESLSSSEESITRINK, YPFPGPI, YPFPG, PGPIPN, LLYQEPVLGPVRGPFPIIV, RPKHPIKHQGLPQEVLNENLLRF, TTMPLW, KNTMEHVSSSEESIISQETYKQEKNMAINPSK, respectively.

[0031] (2) The peptides selected in step (1) were converted into FASTA format of equal width and input into pLogo (https: / / plogo.uconn.edu / #) for enzyme cleavage preference prediction. It was found that these peptides were more likely to be produced when the cleavage sites were F and K.

[0032] (3) The enzyme cleavage site obtained in step (2) is searched for enzymes in the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php) to obtain the screened enzyme.

[0033] (4) According to the predicted results, a protein solution of less than 5% was prepared, the pH was adjusted to 6.5-7.5, the enzyme addition amount was 6.0-7.0 U / mg protein, and the solution was incubated in a 60°C water bath for 120-150 min to obtain Solution I.

[0034] (5) Adjust the pH of solution I to 4.5-4.6, centrifuge at 4000-5000 rpm / min for 30-35 min. Take the supernatant II and set aside.

[0035] (6) Desalting the supernatant II to obtain solution III.

[0036] (7) Freeze-drying solution III and storing it at -40 to -20°C for future use, thereby obtaining the casein immunomodulatory peptide.

[0037] The casein immunomodulatory peptide prepared by the above method.

[0038] The application of the above casein immunomodulatory peptide is to prepare cosmetics, medicines, foods or health products with immunomodulatory effects, including the application in preparing acceptable excipients for pharmaceutical foods or health products.

[0039] The casein immunomodulatory peptide of the present invention has a relative purity of more than 50% and has a good immunomodulatory effect.

[0040] Beneficial effects of the present invention:

[0041] The present invention can carry out targeted enzymatic hydrolysis of casein, increase the relative content and types of immunomodulatory peptides, reduce the loss of immunomodulatory peptide efficacy when other immunomodulatory casein peptide products are measured after simple enzymatic hydrolysis, achieve better immunomodulatory effects, and effectively promote the proliferation of immune cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is the pLogo diagram of the enzyme cutting site. Among them: FG-casein; BG-Cow protein indicate the source of the amino acid sequence; P4-P4' indicate the distribution of amino acids at different positions, among which P1 and P1' are defined as the enzyme cutting preference sites.

[0043] Figure 2 The proliferation effect of casein immunomodulatory peptide on mouse macrophage RAW264.7. Among them: * indicates p < 0.05, **** indicates p < 0.0001.

[0044] Figure 3 The proliferation effect of skim milk immunomodulatory peptide on mouse macrophage RAW264.7 cells. Where: **** indicates p < 0.0001.

[0045] Figure 4 The proliferation effect of raw milk immunomodulatory peptide on mouse macrophage RAW264.7. Where: **** means p < 0.0001.

[0046] Figure 5 The proliferation effect of β,κ-casein immunomodulatory peptide on mouse macrophage RAW264.7. Among them: * indicates p < 0.05, ** indicates p < 0.01. DETAILED DESCRIPTION

[0047] The technical scheme of the present invention will be described in detail below. It should be understood that the embodiments presented here are merely implementation cases for illustrative purposes and are not intended to limit the scope of the present invention. Unless otherwise specified, the reagents and instruments used in the following examples are all commercially available products.

[0048] Example 1

[0049] Enzymatic hydrolysis of casein to prepare casein immunomodulatory peptides. Sample preparation is completed according to the following steps:

[0050] (1) The known immunomodulatory peptides derived from bovine αS1-casein (ID: 1089), αS2-casein (ID: 1090), β-casein (ID: 1097, 1100), and κ-casein (ID: 1109) were screened from the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php), which were YPFPGPIH, KEMPFPK, YQQPVLGPVRGPFPIIV, RELEELNVPGEIVESLSSSEESITRINK, YPFPGPI, YPFPG, PGPIPN, LLYQEPVLGPVRGPFPIIV, RPKHPIKHQGLPQEVLNENLLRF, TTMPLW, KNTMEHVSSSEESIISQETYKQEKNMAINPSK, respectively.

[0051] (2) The peptides selected in step (1) were converted into FASTA format of 8 amino acid sequences and input into pLogo (https: / / plogo.uconn.edu / #) for enzyme cleavage preference prediction, such as Figure 1 As shown, these peptides are more likely to be produced when the cleavage sites are F and K.

[0052] (3) The enzyme cleavage site obtained in step (2) was searched in the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php) to obtain ficin (EC3.4.22.3), whose cleavage sites are K, F, Y, G, S, L, R, and H.

[0053] (4) According to the predicted results, a 1% casein solution was prepared with deionized water using casein, the pH was adjusted to 7.0, and ficin was added at an enzyme amount of 6.0 U / mg protein. The solution was incubated at 60°C in a water bath for 120 min to obtain Solution I.

[0054] (5) Adjust solution I to pH 4.6 and centrifuge at 4500 rpm / min for 30 min. Take supernatant II and set aside.

[0055] (6) The supernatant II was desalted using a 500 Da dialysis bag for 12 h to obtain solution III.

[0056] (7) The solution III was freeze-dried and stored at -40°C for later use to obtain casein immunomodulatory peptides with a purity of 80%.

[0057] (8) The peptide powder of step (7) was dissolved in DMEM basal medium to prepare a solution with a peptide content of 100 μg / mL, and incubated with mouse macrophage RAW264.7 at 37°C and 5% CO2 for 24 h. The control group (DMEM basal medium) was used as a negative control, and the LPS group (DMEM basal medium containing 1 μg / ml lipopolysaccharide) was used as a positive control. CCK-8 was used to detect the effect of casein peptide on Raw264.7 proliferation. 2×10 5 After 24 hours, 10 μL of CCK-8 reagent was added to each well and incubated at 37°C for 2 hours. The absorbance of the cells at a wavelength of 450 nm was measured. Figure 2 As shown, it has a significant effect on the proliferation of RAW264.7, indicating that it is a casein immunomodulatory peptide.

[0058] Example 2

[0059] Enzymatic hydrolysis of fresh milk to prepare raw milk immunomodulatory peptides, the sample preparation is completed according to the following steps:

[0060] (1) Screen the bovine α-protein from the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php) S1 -Casein (ID:1089), α S2The known immunomodulatory peptides of -casein (ID: 1090), β-casein (ID: 1097, 1100), and κ-casein (ID: 1109) are YPFPGPIH, KEMPFPK, YQQPVLGPVRGPFPIIV, RELEELNVPGEIVESLSSSEESITRINK, YPFPGPI, YPFPG, PGPIPN, LLYQEPVLGPVRGPFPIIV, RPKHPIKHQGLPQEVLNENLLRF, TTMPLW, KNTMEHVSSSEESIISQETYKQEKNMAINPSK, respectively.

[0061] (2) The peptides selected in step (1) were converted into FASTA format of 8 amino acid sequences and input into pLogo (https: / / plogo.uconn.edu / #) for enzyme cleavage preference prediction, such as Figure 1 As shown, these peptides are more likely to be produced when the cleavage sites are F and K.

[0062] (3) The enzyme cleavage site obtained in step (2) was searched in the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php) to obtain ficin (EC3.4.22.3), whose cleavage sites are K, F, Y, G, S, L, R, and H.

[0063] (4) Based on the predicted results, fresh milk was used, the pH was adjusted to 7.0, and ficin was added at an enzyme dosage of 6.0 U / mg protein. The mixture was incubated in a 60°C water bath for 120 min to obtain Solution I.

[0064] (5) Adjust solution I to pH 4.6 and centrifuge at 4500 rpm / min for 30 min. Take supernatant II and set aside.

[0065] (6) The supernatant II was desalted using a 500 Da dialysis bag for 12 h to obtain solution III.

[0066] (7) The solution III was freeze-dried and stored at -40°C for later use to obtain raw cow milk immunomodulatory peptides with a purity of 78%.

[0067] (8) The peptide powder of step (7) was dissolved in DMEM basal medium to prepare a solution with a peptide content of 100 μg / mL, and incubated with mouse macrophage RAW264.7 at 37°C and 5% CO2 for 24 h. The control group (DMEM basal medium) was used as a negative control, and the LPS group (DMEM basal medium containing 1 μg / ml lipopolysaccharide) was used as a positive control. CCK-8 was used to detect the effect of the peptide on the proliferation of Raw264.7. 2×10 5 After 24 hours, 10 μL of CCK-8 reagent was added to each well and incubated at 37°C for 2 hours. The absorbance of the cells at a wavelength of 450 nm was measured. Figure 4 As shown, it has a significant effect on the proliferation of RAW264.7, indicating that it is a casein immunomodulatory peptide.

[0068] Example 3

[0069] Enzymatic hydrolysis of milk to prepare skim milk immunomodulatory peptides, the sample is completed according to the following steps:

[0070] (1) Screen the bovine α-protein from the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php) S1 -Casein (ID:1089), α S2 The known immunomodulatory peptides of -casein (ID: 1090), β-casein (ID: 1097, 1100), and κ-casein (ID: 1109) are YPFPGPIH, KEMPFPK, YQQPVLGPVRGPFPIIV, RELEELNVPGEIVESLSSSEESITRINK, YPFPGPI, YPFPG, PGPIPN, LLYQEPVLGPVRGPFPIIV, RPKHPIKHQGLPQEVLNENLLRF, TTMPLW, KNTMEHVSSSEESIISQETYKQEKNMAINPSK, respectively.

[0071] (2) The peptides selected in step (1) were converted into FASTA format of 8 amino acid sequences and input into pLogo (https: / / plogo.uconn.edu / #) for enzyme cleavage preference prediction, such as Figure 1 As shown, these peptides are more likely to be produced when the cleavage sites are F and K.

[0072] (3) The enzyme cleavage site obtained in step (2) was searched in the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php) to obtain ficin (EC3.4.22.3), whose cleavage sites are K, F, Y, G, S, L, R, and H.

[0073] (4) According to the prediction results, skim milk powder was used to prepare a 1% protein solution with deionized water, the pH was adjusted to 7.0, and ficin was added at an enzyme addition amount of 6.0 U / mg protein. The solution was incubated in a water bath at 60°C for 120 min to obtain Solution I.

[0074] (5) Adjust solution I to pH 4.6 and centrifuge at 4500 rpm / min for 30 min. Take supernatant II and set aside.

[0075] (6) The supernatant II was desalted using a 500 Da dialysis bag for 12 h to obtain solution III.

[0076] (7) The solution III was freeze-dried and stored at -40°C for later use to obtain skimmed milk immunomodulatory peptides with a purity of 75%.

[0077] (8) The peptide powder of step (7) was dissolved in DMEM basal medium to prepare a solution with a peptide content of 100 μg / mL, and incubated with mouse macrophage RAW264.7 at 37°C and 5% CO2 for 24 h. The control group (DMEM basal medium) was used as a negative control, and the LPS group (DMEM basal medium containing 1 μg / ml lipopolysaccharide) was used as a positive control. CCK-8 was used to detect the effect of the peptide on the proliferation of Raw264.7. 2×10 5 After 24 hours, 10 μL of CCK-8 reagent was added to each well and incubated at 37°C for 2 hours. The absorbance of the cells at a wavelength of 450 nm was measured. Figure 3 As shown, it has a significant effect on the proliferation of RAW264.7, indicating that it is a casein immunomodulatory peptide.

[0078] Example 4

[0079] Enzymatic hydrolysis of β,κ-casein to prepare β,κ-casein immunomodulatory peptides was performed according to the following steps:

[0080] (1) Screen the bovine α-protein from the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php) S1 -Casein (ID:1089), α S2The known immunomodulatory peptides of -casein (ID: 1090), β-casein (ID: 1097, 1100), and κ-casein (ID: 1109) are YPFPGPIH, KEMPFPK, YQQPVLGPVRGPFPIIV, RELEELNVPGEIVESLSSSEESITRINK, YPFPGPI, YPFPG, PGPIPN, LLYQEPVLGPVRGPFPIIV, RPKHPIKHQGLPQEVLNENLLRF, TTMPLW, KNTMEHVSSSEESIISQETYKQEKNMAINPSK, respectively.

[0081] (2) The peptides selected in step (1) were converted into FASTA format of 8 amino acid sequences and input into pLogo (https: / / plogo.uconn.edu / #) for enzyme cleavage preference prediction, such as Figure 1 As shown, these peptides are more likely to be produced when the cleavage sites are F and K.

[0082] (3) The enzyme cleavage sites obtained in step (2) were searched in the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php) to obtain pepsin, whose cleavage sites are F, L, G, Y, A, E, Q, T, N, K, V, D, M (pH>2); and trypsin, whose cleavage sites are K and R.

[0083] (4) According to the prediction results, β, κ-casein was mixed with deionized water in a mass ratio of 68:20 to prepare a casein solution. The final concentration (mass fraction) of the casein solution was 1%. The solution temperature was adjusted to 37°C and the pH was 3.0. Pepsin was added in an amount of 22.75 U / mg protein. After 60 minutes, the pH was adjusted to 7.0 to inactivate the pepsin. Then, the solution temperature was adjusted to 37°C and the pH was 6.5. Trypsin was added in an amount of 3.5 U / mg protein. After 120 minutes, the enzyme was inactivated in a 90°C water bath for 10 minutes to obtain Solution I.

[0084] (5) Adjust solution I to pH 4.6 and centrifuge at 4500 rpm / min for 30 min. Take supernatant II and set aside.

[0085] (6) The supernatant II was treated with electrodialysis desalination under the conditions of sample solution flow rate of 1.2 L / min and DC power supply voltage of 11.5 V to obtain solution III.

[0086] (7) Solution III was freeze-dried and stored at -40°C for later use to obtain β,κ-casein immunomodulatory peptide with a purity of 70%.

[0087] (8) The peptide powder of step (7) was dissolved in DMEM basal medium to prepare a solution with a peptide content of 100 μg / mL, and incubated with mouse macrophage RAW264.7 at 37°C and 5% CO2 for 24 h. The control group (DMEM basal medium) was used as a negative control, and the LPS group (DMEM basal medium containing 1 μg / ml lipopolysaccharide) was used as a positive control. CCK-8 was used to detect the effect of the peptide on the proliferation of Raw264.7. 5×10 3 After 24 hours, 10 μL of CCK-8 reagent was added to each well and incubated at 37°C for 2 hours. The absorbance of the cells at a wavelength of 450 nm was measured. Figure 5 As shown, it has a significant effect on the proliferation of RAW264.7, indicating that it is a casein immunomodulatory peptide.

Claims

1. A method for preparing a casein immunomodulatory peptide, characterized in that: The method comprises the following steps: screening enzymes according to known immunomodulatory peptides derived from casein in the BIOPEP database, and performing targeted hydrolysis on milk-derived casein raw materials according to the screened enzymes to obtain casein immunomodulatory peptide products.

2. The preparation method according to claim 1, characterized in that: The casein includes α S1 -Casein, alpha S2 -one or more of casein, β-casein, and κ-casein; the milk-derived casein raw material includes one or more of cow's milk, goat's milk, skim milk, reconstituted milk, casein, and sodium caseinate.

3. The preparation method according to claim 1, characterized in that: The steps include: (1) Screening: Screening immunomodulatory peptides derived from casein from the BIOPEP database; (2) Simulated enzymatic hydrolysis: using pLogo to screen the peptides screened in step (1); (3) enzymatic hydrolysis: enzymatically hydrolyzing the milk-derived casein raw material according to the enzyme screened in step (2); (4) The enzymatic hydrolysate obtained in step (3) is centrifuged and freeze-dried to obtain an immunomodulatory peptide.

4. The preparation method according to claim 3, characterized in that: In step (1), the peptides obtained by screening include YPFPGPIH, KEMPFPK, YQQPVLGPVRGPFPIIV, RELEELNVP GEIVESLSSSEESITRINK, YPFPGPI, YPFPG, PGPIPN, LLYQEPVLGPVRGP FPIIV, RPKHPIKHQGLPQEVLNENLLRF, TTMPLW, KNTMEHVSSSEESIISQ ETYKQEKNMAINPSK.

5. The preparation method according to claim 3, characterized in that: In step (2), the peptides screened out in step (1) are first converted into FASTA format of amino acid sequences of equal width, and input into pLogo for enzyme cleavage preference prediction to obtain the enzyme cleavage site of the peptide; the obtained enzyme cleavage site is searched for the enzyme in the BIOPEP database to obtain the screened enzyme.

6. The preparation method according to claim 5, characterized in that: In step (2), the restriction site contains one or two of F and K; the screened enzyme is one or more of ficin, pepsin and trypsin.

7. The preparation method according to claim 3, characterized in that: In step (2), a single enzyme or multiple enzymes are used for hydrolysis; the temperature of the enzymatic hydrolysis is 25-65° C. and the pH is 3-8.5; In step (3), the milk-derived casein raw material is a milk-derived casein solution having a protein content of less than 5%; In step (4), the centrifugal conditions are: 2000-15000 rpm / min, 5-35 min.

8. The preparation method according to claim 3, characterized in that: In step (4), the pH of the enzymatic hydrolysate is first adjusted to 4.5-4.6, and then centrifuged; or after centrifugation, desalting is performed, and then freeze-drying is performed.

9. The casein immunomodulatory peptide prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the casein immunomodulatory peptide according to claim 9, characterized in that: The casein immunomodulatory peptide is used for preparing cosmetics, medicines, foods or health products with immunomodulatory effects.

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