Cream casein hydrolysate with anti-inflammatory effect as well as preparation method and application thereof

Abstract

The invention provides a cheese protein hydrolysate with an anti-inflammatory effect as well as a preparation method and application thereof. The casein hydrolysate comprises a peptide fragment FAWPQYLK, a peptide fragment LPW, a peptide fragment MPLW and a peptide fragment PLW, and can reduce the inflammatory response of cells by reducing the release of proinflammatory cytokines, such as reducing the release of IL-1beta, IL-2, IL-6 and TNF-alpha, and improving the release of anti-inflammatory cytokines, such as improving the release of IL-10, so as to achieve the anti-inflammatory effect.

Description

Technical Field

[0001] The invention relates to biotechnology, and in particular to a casein hydrolyzate with anti-inflammatory effect, a preparation method and application thereof. Background Art

[0002] In a normal body, various pro-inflammatory cytokines and anti-inflammatory cytokines maintain a relatively stable balance. When a disease occurs, the balance is broken, and the body will secrete various cytokines that regulate inflammation to stimulate, recruit and amplify immune cells, leading to an inflammatory response. Inflammation is a defense mechanism against the invasion of harmful substances into life, allowing damaged tissues to repair and heal. But at the same time, if the inflammatory response is too strong or lasts too long, it may also cause a series of injuries to the body, and in severe cases, it may even be life-threatening. For example, inflammation may damage the vascular endothelium, and the human immune mechanism will repair the damaged tissue by itself. This repeated process will cause the vascular endothelium to thicken, making it easier for lipids to deposit on the blood vessel walls, thereby inducing atherosclerosis.

[0003] Bioactive peptides are composed of natural amino acids and are usually degraded into harmless metabolites in the body, so they are less toxic and safer. Bioactive peptides are similar to natural proteins and enzymes in the human body and usually have good biocompatibility, reducing the risk of immunogenicity and allergic reactions. Bioactive peptides have a small molecular weight and a clear characteristic structure, which is conducive to direct absorption and utilization by the body, and have the advantages of strong solubility and stability. Therefore, in order to effectively inhibit inflammatory reactions, it is urgent to find a bioactive peptide with anti-inflammatory effects. Summary of the invention

[0004] The invention provides a milk casein hydrolyzate which can reduce the release of IL-1β, IL-2 and IL-6 to reduce cellular inflammatory response.

[0005] The invention provides a method for preparing a casein hydrolysate. The method is simple and fast to operate and is convenient for large-scale production.

[0006] The present invention provides an application of the above-mentioned casein hydrolysate and / or the casein hydrolysate prepared by the above-mentioned preparation method in preparing related products with anti-inflammatory effects.

[0007] The present invention provides a milk casein hydrolysate, wherein the milk casein hydrolysate comprises a peptide segment FAWPQYLK, a peptide segment LPW, a peptide segment MPLW and a peptide segment PLW.

[0008] The casein hydrolysate as described above, wherein the content of the peptide segment FAWPQYLK is ≥ 1.50% based on the mass of the casein hydrolysate; and / or,

[0009] The content of peptide LPW is ≥ 0.05% based on the mass of casein hydrolysate; and / or,

[0010] Based on the mass of casein hydrolysate, the content of peptide fragment MPLW is ≥ 0.05%; and / or,

[0011] Based on the mass of casein hydrolysate, the content of peptide PLW is ≥ 0.05%.

[0012] The casein hydrolysate as described above, wherein the degree of hydrolysis of the casein hydrolysate is 10%-15%; and / or,

[0013] The mass content of peptides with a molecular weight of ≤10,000 Da in the casein hydrolysate is ≥80%; and / or,

[0014] The mass content of peptides with a molecular weight of ≤5000 Da in the casein hydrolysate is ≥55%; and / or,

[0015] The mass content of peptides with a molecular weight of less than 1000 Da in casein hydrolysate is ≥30%.

[0016] The casein hydrolysate as described above, wherein the casein hydrolysate is obtained by enzymatically hydrolyzing the casein raw material;

[0017] The enzymatic treatment included enzymatic digestion using trypsin and pronase.

[0018] The present invention provides a method for preparing a casein hydrolysate, which comprises the following steps:

[0019] Performing enzymatic hydrolysis on the casein raw material to obtain casein hydrolyzate;

[0020] The enzymatic treatment included enzymatic digestion using trypsin and pronase.

[0021] The preparation method as described above, wherein the enzyme activity of trypsin is 180000-220000 U / g; and / or,

[0022] Based on each gram of casein raw material, the amount of trypsin added is 0.10-0.35wt%; and / or,

[0023] The enzyme activity of Streptomyces protease is 180000-220000 U / g; and / or,

[0024] Based on each gram of casein raw material, the added amount of pronase is 0.05-0.32wt%.

[0025] In the preparation method as described above, the conditions for enzymatic hydrolysis include: an enzymatic hydrolysis temperature of 40-55° C., an enzymatic hydrolysis time of 2.0-3.5 h, or an enzymatic hydrolysis pH of 7.5-8.2.

[0026] In the preparation method as described above, the casein raw material is obtained by mixing casein and water in a volume ratio of 1:(8-12).

[0027] The present invention provides an application of the above-mentioned casein hydrolysate and / or the casein hydrolysate prepared by the above-mentioned preparation method in preparing related products with anti-inflammatory effects.

[0028] The present invention provides a casein hydrolyzate, comprising peptide segments FAWPQYLK, LPW, MPLW and PLW, which can reduce the release of pro-inflammatory cytokines, such as reducing the release of IL-1β, IL-2, IL-6 and TNF-α, and increase the release of anti-inflammatory cytokines, such as increasing the release of IL-10, thereby reducing cellular inflammatory response and achieving anti-inflammatory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a graph showing the cytokine content detection results of the peptide FAWPQYLK group in Example 4;

[0030] Figure 2 This is a graph showing the cytokine content detection results of the peptide LPW group in Example 4;

[0031] Figure 3 This is a graph showing the cytokine content detection results of the peptide segment MPLW group in Example 4;

[0032] Figure 4 This is a graph showing the cytokine content detection results of the peptide PLW group in Example 4;

[0033] Figure 5 This is a graph showing the results of detecting the cytokine TNF-α content in the casein hydrolyzate group in Example 4;

[0034] Figure 6 This is a graph showing the results of detecting the cytokine IL-10 content in the casein hydrolyzate group in Example 4;

[0035] Figure 7 This is a graph showing the results of detecting the cytokine IL-6 content in the casein hydrolyzate group in Example 4;

[0036] Figure 8 This is a graph showing the results of detecting the cytokine IL-2 content in the casein hydrolyzate group in Example 4;

[0037] Fig. 9This is a graph showing the results of detecting the cytokine IL-1β content in the casein hydrolyzate group in Example 4. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the scheme of the present invention, the present invention is further described in detail below. The specific implementation methods listed below are only for describing the principles and features of the present invention. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0039] In a normal body, various pro-inflammatory cytokines and anti-inflammatory cytokines maintain a relatively stable balance. When a disease occurs, the balance is broken, and the body will secrete various cytokines that regulate inflammation to stimulate, recruit and amplify immune cells, leading to the occurrence of inflammatory reactions. Inflammation is a defense mechanism to resist the invasion of harmful substances into life, allowing damaged tissues to repair and heal. But at the same time, if the inflammatory response is too strong or lasts too long, it may also cause a series of damage to the body, and in severe cases, it may even endanger life.

[0040] Therefore, in order to regulate pro-inflammatory cytokines and anti-inflammatory cytokines and thereby effectively inhibit inflammatory responses, the first aspect of the present invention provides a casein hydrolyzate comprising peptide segments FAWPQYLK, LPW, MPLW and PLW.

[0041] Among them, the peptide FAWPQYLK is an octapeptide (Phe-Ala–Trp-Pro-Gln-Tyr-Leu-Lys) composed of phenylalanine (Phe, F), alanine (Ala, A), tryptophan (Trp, W), proline (Pro, P), glutamine (Gln, Q), tyrosine (Tyr, Y), leucine (Leu, L), and lysine (Lys, K) from N-terminus to C-terminus. The peptide LPW is a tripeptide (Leu-Pro-Trp) composed of leucine (Leu, L), proline (Pro, P), and tryptophan (Trp, W) from N-terminus to C-terminus. The peptide MPLW is a tetrapeptide (Met-Pro-Leu-Trp) composed of methionine (Met, M), proline (Pro, P), leucine (Leu, L), and tryptophan (Trp, W) from N-terminus to C-terminus. The peptide PLW is a tripeptide (Pro-Leu-Trp) consisting of proline (Pro, P), leucine (Leu, L), and tryptophan (Trp, W) from the N-terminus to the C-terminus.

[0042] Experimental verification shows that the peptide FAWPQYLK can significantly reduce the release of cytokines IL-1β, IL-2, IL-6 and TNF-α; the peptide LPW can significantly reduce the release of cytokines IL-1β, IL-6 and TNF-α, and increase the release of cytokine IL-10; the peptide MPLW can significantly reduce the release of cytokines IL-1β, IL-2, IL-6 and TNF-α, and increase the release of cytokine IL-10; the peptide PLW can significantly reduce the release of cytokines IL-1β, IL-2, IL-6 and TNF-α. The above interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor α (TNF-α) are cytokines (Cytokine, CK). Cytokines are a class of small molecule proteins with a wide range of biological activities. They are synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) after stimulation. Cytokines can be divided into anti-inflammatory cytokines and pro-inflammatory cytokines according to their functions in regulating inflammatory responses. Anti-inflammatory cytokines include IL-10, which has the biological effect of inhibiting inflammatory responses, contributing to the resolution of inflammation and the recovery of the acute phase of autoimmune diseases. Pro-inflammatory cytokines include IL-1β, IL-2, IL-6, and TNF-α, which can send signals through type I cytokine receptors (CCR1) to regulate cell-mediated immune responses, promote the growth, activation, differentiation, and homing of immune cells to the site of infection, and contribute to the occurrence and spread of autoimmune inflammation. Therefore, the peptides FAWPQYLK, LPW, MPLW, and PLW can all reduce the inflammatory response of cells and achieve anti-inflammatory effects by reducing the release of pro-inflammatory cytokines and increasing the release of anti-inflammatory cytokines.

[0043] Casein hydrolysate is the hydrolysis product of casein. It is a small molecule polypeptide substance obtained by directional enzymatic cleavage of casein and specific small peptide separation technology. Casein hydrolysate has both the original excellent properties of casein and many new advantages, such as direct absorption, strong solubility, strong stability, high safety, etc., so it has good development prospects.

[0044] At the same time, as a hydrolysis product, casein hydrolysate also has the advantages of low preparation cost and simple preparation method. It can overcome the problems of high synthesis cost or difficulty in purification and separation of active peptides, and is therefore easier to apply to large-scale industrial production.

[0045] It has been experimentally verified that the casein hydrolyzate provided by the present invention including the above-mentioned active peptide segment also has anti-inflammatory effect, which can reduce the inflammatory response of cells by significantly reducing the release of pro-inflammatory cytokines IL-1β, IL-2 and IL-6.

[0046] Furthermore, based on the mass of casein hydrolysate, the content of peptide FAWPQYLK is ≥1.50%; based on the mass of casein hydrolysate, the content of peptide LPW is ≥0.05%; based on the mass of casein hydrolysate, the content of peptide MPLW is ≥0.05%; based on the mass of casein hydrolysate, the content of peptide PLW is ≥0.05%.

[0047] The present invention can further enhance the anti-inflammatory effect of casein hydrolysate by regulating the content of active peptides in the casein hydrolysate. It is understandable that when the content of at least one of the peptides FAWPQYLK, LPW, MPLW, and PLW of the present invention meets the above range, the anti-inflammatory effect of the casein hydrolysate can be improved.

[0048] In the above technical scheme, by regulating the degree of hydrolysis of casein hydrolysate, the enrichment of active peptides can be promoted, thereby improving the anti-inflammatory effect of casein hydrolysate; specifically, the degree of hydrolysis of casein hydrolysate can be 10%-15%. In addition, by regulating the molecular weight of casein hydrolysate, the casein hydrolysate can have the advantages of small molecular weight and easy absorption, and can also promote the enrichment of active peptides, thereby improving the anti-inflammatory effect of casein hydrolysate; specifically, the mass content of peptides with a molecular weight of ≤10000 Da in casein hydrolysate is ≥80%; the mass content of peptides with a molecular weight of ≤5000 Da in casein hydrolysate is ≥55%; the mass content of peptides with a molecular weight of <1000 Da in casein hydrolysate is ≥30%. It can be understood that when at least one of the degree of hydrolysis and molecular weight distribution of the casein hydrolysate of the present invention meets the above range, the anti-inflammatory effect of the casein hydrolysate can be improved.

[0049] In a specific embodiment, the casein hydrolysate is obtained by enzymatically hydrolyzing the casein raw material, wherein the enzymatic hydrolysis includes enzymatic hydrolysis using trypsin and pronase.

[0050] Casein is the main protein in the milk of mammals (such as cows, sheep and humans). The present invention uses casein as a raw material and enzymatically hydrolyzes it to prepare casein hydrolysate, thereby promoting the conversion of large-molecule casein into small-molecule casein hydrolysate that can be directly absorbed and utilized by the body, has a clear characteristic structure and has biological activity, so as to enhance the biological activity of casein, and at the same time give it higher added value and a wider range of usage scenarios.

[0051] The present invention does not limit the specific type of the casein raw material, as long as the protein content (dry basis) in the casein is not less than 55%. It is understood that the casein of the present invention can be in a solid form or in a liquid form.

[0052] The present invention realizes enzymatic hydrolysis by using trypsin and protease, wherein trypsin is a serine protease extracted from the pancreas of cattle, sheep and pigs, and is also a kind of endopeptidase, which can break the peptide bond formed by the carboxyl group of lysine or arginine, has strong specificity, and plays a key role in determining the amino acid arrangement of protein. Protease is an enzyme preparation prepared by fermentation, purification and compounding of Aspergillus oryzae strain, which can make the enzymatic hydrolysis product form a unique flavor and reduce the bitterness caused by enzymatic hydrolysis. In the present invention, by enzymatic hydrolysis of casein raw materials and limiting the type of enzyme preparation to meet the above range, active peptides can be further enriched to improve the anti-inflammatory effect of casein hydrolysate.

[0053] The third aspect of the present invention provides a method for preparing a casein hydrolysate, comprising the following steps:

[0054] Performing enzymatic hydrolysis on the casein raw material to obtain casein hydrolyzate;

[0055] The enzymatic treatment included enzymatic digestion using trypsin and pronase.

[0056] The present invention has conducted a lot of research and exploration on how to make the enzymatic hydrolyzate of the casein raw material contain the expected active peptide segment FAWPQYLK, LPW, MPLW or PLW, and proved that the selection of enzyme preparation has a key influence on the result. The casein raw material can be enzymatically treated with two enzyme preparations, trypsin and protease, to obtain a casein hydrolyzate with the expected active peptide segment. The above-mentioned preparation method of the enzyme preparation is limited, and the casein hydrolyzate containing the expected active peptide can be obtained simply and quickly, which is convenient for large-scale production.

[0057] During the enzymatic hydrolysis process, trypsin and protease can be used simultaneously for enzymatic hydrolysis. In order to increase the contact area between the enzyme preparation and the casein raw material and ensure that the enzyme preparation and the casein raw material are evenly mixed to improve the enzymatic hydrolysis efficiency, trypsin and protease can be first mixed in a small amount of pure water to obtain an enzyme mixture. The enzyme mixture is then added to the casein for enzymatic hydrolysis.

[0058] After the enzymolysis is completed, an enzyme inactivation treatment may be performed to make the enzyme preparation lose its catalytic activity. The present invention does not limit the enzyme inactivation method, and the enzyme inactivation may be performed by conventional enzyme inactivation methods in the art, for example, the enzyme inactivation treatment may be performed at 80° C. for 30 min.

[0059] After the enzyme inactivation treatment, the temperature can be lowered to 30-40°C for concentration and drying. The concentration treatment can further enrich the active peptides, and the drying treatment can obtain a solid form of casein hydrolyzate, which is convenient for storage and transportation. The present invention does not limit the methods of concentration and drying treatment, and can be carried out by conventional technical means in the art. For example, the concentration treatment can be alcohol precipitation or evaporation concentration; the drying treatment can be freeze vacuum drying or spray drying.

[0060] In the present invention, the enzyme activity and addition amount of the enzyme preparation can be regulated to promote a good matching effect between the casein raw material and the enzyme preparation, thereby improving the enzymatic hydrolysis efficiency, better promoting the enzymatic hydrolysis reaction, and ultimately increasing the content of active peptides in the casein hydrolysate. Specifically, the enzyme activity of trypsin is 180000-220000 U / g; based on each gram of casein raw material (dry basis), the addition amount of trypsin is 0.10-0.35wt%; the enzyme activity of Streptomyces protease is 180000-220000 U / g; based on each gram of casein raw material (dry basis), the addition amount of Streptomyces protease is 0.05-0.32wt%. It can be understood that when at least one of the enzyme activity or addition amount of the enzyme preparation meets the above range, the enzymatic hydrolysis efficiency can be improved and the active peptides can be further enriched.

[0061] Further, the enzymolysis effect of the enzyme preparation can be better exerted by regulating the enzymolysis conditions. Specifically, the enzymolysis conditions include: an enzymolysis temperature of 40-55 ° C, an enzymolysis time of 2.0-3.5 h, or an enzymolysis pH of 7.5-8.2. In the present invention, the casein raw material can be first adjusted to the optimal enzymolysis pH environment of the enzyme mixture, that is, the pH value is 7.5-8.2, to obtain an enzymolysis stock solution that is conducive to enzymolysis of the enzyme mixture. Subsequently, at the appropriate enzymolysis temperature of the enzyme mixture, i.e. 40-55 ° C, after adding the enzyme mixture composed of two enzyme preparations to the enzymolysis stock solution, enzymolysis 2.0-3.5 h, so that the two enzyme preparations fully act on the protein in the enzymolysis stock solution.

[0062] In order to better enrich the active peptides, in the present invention, casein and water can be first mixed in a mass ratio of 1: (8-12) to obtain a casein solution, and then the casein solution can be used as a casein raw material for enzymolysis. Since the casein solution has a certain fluidity, it has a larger surface area than the casein powder, and is therefore more conducive to promoting the subsequent enzymolysis reaction. If too little water is added, the fluidity of the casein solution will be poor, which is not conducive to the action of the enzyme preparation and is likely to reduce the enzymolysis efficiency; if too much water is added, the reaction volume will be too large during the enzymolysis treatment, and the load of the subsequent treatment (such as separation and purification treatment, etc.) will increase, and the treatment cost will also increase accordingly.

[0063] In the technical scheme of the present invention, the preparation method of casein hydrolyzate is as follows: casein powder and pure are mixed in a volume ratio of 1: (8-12) to obtain a casein solution; trypsin with an enzyme activity of 180000-220000 U / g and an addition amount of 0.10-0.35wt% (based on the mass of the casein powder) and Streptomyces protease with an enzyme activity of 180000-220000 U / g and an addition amount of 0.05-0.32wt% (based on the mass of the casein powder) are added to the casein solution for enzymatic hydrolysis, the enzymatic hydrolysis temperature is 40-55°C, the enzymatic hydrolysis time is 2.0-3.5 h, and the enzymatic hydrolysis pH value is 7.5-8.2 to obtain a casein hydrolyzate.

[0064] In one embodiment, casein and water can be mixed in a mass ratio of 1:10, and trypsin with an enzyme activity of 200,000 U / g and an addition amount of 0.25wt% (based on the mass of casein) and Streptomyces protease with an enzyme activity of 200,000 U / g and an addition amount of 0.12wt% (based on the mass of casein) are added thereto for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 45°C, the enzymatic hydrolysis pH is 7.5, and the enzymatic hydrolysis time is 2.0 h to obtain an enzymatic hydrolyzate, which is then subjected to enzyme inactivation treatment, evaporation concentration treatment and drying treatment to obtain a casein hydrolyzate.

[0065] In one embodiment, casein and water can be mixed in a mass ratio of 1:12, and trypsin with an enzyme activity of 200,000 U / g and an addition amount of 0.17wt% (based on the mass of casein) and Streptomyces protease with an enzyme activity of 200,000 U / g and an addition amount of 0.17wt% (based on the mass of casein) are added thereto for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 45°C, the enzymatic hydrolysis pH is 7.5, and the enzymatic hydrolysis time is 2.5 h to obtain an enzymatic hydrolyzate, which is then subjected to enzyme inactivation treatment, evaporation concentration treatment and drying treatment to obtain a casein hydrolyzate.

[0066] In one embodiment, casein and water can be mixed in a mass ratio of 1:8, and trypsin with an enzyme activity of 200,000 U / g and an addition amount of 0.35wt% (based on the mass of casein) and Streptomyces protease with an enzyme activity of 200,000 U / g and an addition amount of 0.05wt% (based on the mass of casein) are added thereto for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 45°C, the enzymatic hydrolysis pH is 8.2, and the enzymatic hydrolysis time is 3.0 h to obtain an enzymatic hydrolyzate, which is then subjected to enzyme inactivation treatment, evaporation concentration treatment and drying treatment to obtain a casein hydrolyzate.

[0067] In one embodiment, casein and water can be mixed in a mass ratio of 1:10, and trypsin with an enzyme activity of 200,000 U / g and an addition amount of 0.10wt% (based on the mass of casein) and Streptomyces protease with an enzyme activity of 200,000 U / g and an addition amount of 0.32wt% (based on the mass of casein) are added thereto for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 45°C, the enzymatic hydrolysis pH is 7.5, and the enzymatic hydrolysis time is 3.5 h to obtain an enzymatic hydrolyzate, which is then subjected to enzyme inactivation treatment, evaporation concentration treatment and drying treatment to obtain a casein hydrolyzate.

[0068] In the present invention, casein and water can be mixed in a mass ratio of 1: (8-12), and trypsin with an enzyme activity of 180,000-220,000 U / g and an addition amount of 0.10wt%-0.35wt% (based on the mass of casein) and Streptomyces protease with an enzyme activity of 180,000-220,000 U / g and an addition amount of 0.05wt%-0.32wt% (based on the mass of casein) are added thereto for enzymatic hydrolysis, the enzymatic hydrolysis temperature is 40-55°C, the enzymatic hydrolysis pH value is 7.5-8.2, the enzymatic hydrolysis time is 2.0-3.5 h, and an enzymatic hydrolyzate is obtained, which is then subjected to enzyme inactivation treatment, evaporation concentration treatment and drying treatment to obtain a casein hydrolyzate.

[0069] The fourth aspect of the present invention provides the above-mentioned casein hydrolysate, and the use of the casein hydrolysate prepared by the above-mentioned preparation method in the preparation of related products with anti-inflammatory effects.

[0070] It is understandable that the above-mentioned related products include but are not limited to food, health products, skin care products, cosmetics and medicines. It is proved by a large amount of research data that the casein hydrolysate of the present invention includes peptides FAWPQYLK, peptides LPW, peptides MPLW, peptides PLW, which can reduce the release of proinflammatory cytokines and improve the release of anti-inflammatory cytokines to reduce the inflammatory response of cells and achieve anti-inflammatory efficacy. Therefore, the present invention includes the casein hydrolysate of active peptides and the casein hydrolysate including active peptides prepared by the above-mentioned preparation method, which can not only be applied to food, health products, skin care products, cosmetics in the conventional sense, such as anti-inflammatory food, anti-inflammatory health products, anti-inflammatory skin care products or anti-inflammatory cosmetics, but also can be used to prepare medicines with anti-inflammatory efficacy, thereby widening the scope of application of the above-mentioned casein hydrolysate, and also providing new raw materials for related products with anti-inflammatory efficacy.

[0071] In addition, experimental verification has shown that the casein hydrolysate provided by the present invention and the casein hydrolysate prepared by the above preparation method can be used to alleviate the inflammatory response of THP-1 M1 macrophages, or to prepare drugs related to the inflammatory response of THP-1 M1 macrophages.

[0072] Furthermore, the peptide FAWPQYLK provided by the present invention has a significant effective concentration range of 0.02325-0.19065 mg / mL for alleviating the inflammatory response of THP-1 M1 macrophages; the peptide LPW provided by the present invention has a significant effective concentration range of 0.00045-0.00369 mg / mL for alleviating the inflammatory response of THP-1 M1 macrophages; the peptide MPLW provided by the present invention has a significant effective concentration range of 0.00035-0.00287 mg / mL for alleviating the inflammatory response of THP-1 M1 macrophages; the peptide PLW provided by the present invention has a significant effective concentration range of 0.00215-0.01763 mg / mL for alleviating the inflammatory response of THP-1 M1 macrophages; and the casein hydrolyzate has a significant effective concentration range of 0.5-4.1 mg / mL for alleviating the inflammatory response of THP-1 M1 macrophages.

[0073] The technical solution of the present application will be further explained below in conjunction with specific examples. The experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The reagents used are commercially available or available from public channels unless otherwise specified.

[0074] Example 1: Preparation of casein hydrolysate

[0075] (1) Mix casein powder and pure water in a mass ratio of 1:10 to obtain a casein solution. Mix trypsin with an enzyme activity of 200,000 U / g and an addition amount of 0.25wt% (based on the mass of casein powder) and Streptomyces protease with an enzyme activity of 200,000 U / g and an addition amount of 0.12wt% (based on the mass of casein powder) in 10 mL of pure water to obtain an enzyme mixture. Add the enzyme mixture to the casein solution for enzymatic hydrolysis to obtain an enzymatic hydrolyzate. During the enzymatic hydrolysis, the enzymatic hydrolysis temperature is 45°C, the enzymatic hydrolysis pH is 7.5, and the enzymatic hydrolysis time is 2.0 h. After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is inactivated at 80°C for 30 min. After the inactivated enzymatic hydrolyzate is cooled to 30°C-40°C, it is evaporated and concentrated and vacuum freeze-dried to obtain a casein hydrolyzate powder 1.

[0076] (2) Mix casein powder and pure water in a mass ratio of 1:12 to obtain a casein solution. Mix trypsin with an enzyme activity of 200,000 U / g and an addition amount of 0.17 wt% (based on the mass of casein powder) and Streptomyces protease with an enzyme activity of 200,000 U / g and an addition amount of 0.17 wt% (based on the mass of casein powder) in 10 mL of pure water to obtain an enzyme mixture. Add the enzyme mixture to the casein solution for enzymatic hydrolysis to obtain an enzymatic hydrolyzate. During the enzymatic hydrolysis, the enzymatic hydrolysis temperature is 45°C, the enzymatic hydrolysis pH is 7.5, and the enzymatic hydrolysis time is 2.5 h. After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is inactivated at 80°C for 30 min. After the inactivated enzymatic hydrolyzate is cooled to 30°C-40°C, it is evaporated and concentrated and vacuum freeze-dried to obtain casein hydrolyzate powder 2.

[0077] (3) Mix casein powder and pure water in a mass ratio of 1:8 to obtain a casein solution. Mix trypsin with an enzyme activity of 200,000 U / g and an addition amount of 0.35wt% (based on the mass of casein powder) and Streptomyces protease with an enzyme activity of 200,000 U / g and an addition amount of 0.05wt% (based on the mass of casein powder) in 10 mL of pure water to obtain an enzyme mixture. Add the enzyme mixture to the casein solution for enzymatic hydrolysis to obtain an enzymatic hydrolyzate. During the enzymatic hydrolysis, the enzymatic hydrolysis temperature is 45°C, the enzymatic hydrolysis pH is 8.2, and the enzymatic hydrolysis time is 3.0 h. After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is inactivated at 80°C for 30 min. After the inactivated enzymatic hydrolyzate is cooled to 30°C-40°C, it is evaporated and concentrated and vacuum freeze-dried to obtain casein hydrolyzate powder 3.

[0078] (4) Mix casein powder and pure water in a mass ratio of 1:10 to obtain a casein solution. Mix trypsin with an enzyme activity of 200,000 U / g and an addition amount of 0.10wt% (based on the mass of casein powder) and Streptomyces protease with an enzyme activity of 200,000 U / g and an addition amount of 0.32wt% (based on the mass of casein powder) in 10 mL of pure water to obtain an enzyme mixture. Add the enzyme mixture to the casein solution for enzymatic hydrolysis to obtain an enzymatic hydrolyzate. During the enzymatic hydrolysis, the enzymatic hydrolysis temperature is 45°C, the enzymatic hydrolysis pH is 7.5, and the enzymatic hydrolysis time is 3.5 h. After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is inactivated at 80°C for 30 min. After the inactivated enzymatic hydrolyzate is cooled to 30°C-40°C, it is evaporated and concentrated and vacuum freeze-dried to obtain casein hydrolyzate powder 4.

[0079] Example 2: Detection of casein hydrolysate

[0080] (1) Degree of hydrolysis of casein hydrolysate

[0081] The hydrolysis degree of the casein hydrolyzate powders 1-4 in Example 1 was determined by OPA reaction. Since each peptide bond hydrolyzed releases a free amine group, and the free amine group reacts with OPA (o-phthalaldehyde) to form a yellow complex, the absorbance at 340 nm was measured by a spectrophotometer to characterize the hydrolysis degree. The calculation formula of the hydrolysis degree is as follows:

[0082]

[0083] Where h is the number of peptide bonds hydrolyzed, mmol / g; h tot is the total number of peptide bonds, mmol / g (the total number of peptide bonds in casein is 8.2 mmol / g); C serine is the millimolar number of serine amino groups in the casein hydrolysate powder 1-4, mmol / L; V is the dissolved fixed volume of the casein hydrolysate powder 1-4, L; N is the dilution multiple of the casein hydrolysate powder 1-4; m is the mass of the casein hydrolysate powder 1-4, g; w is the protein content in the casein hydrolysate powder 1-4, %; β is a constant, which is 0.383 for casein; α is a constant, which is 1.039 for casein.

[0084] After three parallel experiments, the hydrolysis degrees of casein hydrolyzate powders 1-4 were detected to be 13.05%±0.29%, 11.68%±0.34%, 13.71%±0.26% and 11.36%±0.33%, respectively.

[0085] (2) Molecular weight distribution detection of casein hydrolysate

[0086] The molecular weight distribution of the casein hydrolyzate powders 1-4 in Example 1 was determined by referring to the GPC / UV detection method in Appendix A of the national standard GB 31645-2018, and the results are shown in Table 1, where samples 1-4 correspond to casein hydrolyzate powders 1-4, respectively.

[0087] Table 1

[0088]

[0089] As shown in Table 1, the molecular weight distribution patterns of casein hydrolyzate powders 1-4 are similar, and the mass content of peptides with a molecular weight ≤10000 Da is ≥80%, the mass content of peptides with a molecular weight ≤5000 Da is ≥55%, and the mass content of small molecule peptides and amino acids with a molecular weight <1000 Da is ≥30%.

[0090] Example 3: Screening of anti-inflammatory active peptides

[0091] (1) LC-MS / MS mass spectrometry identification

[0092] The casein hydrolysate powders 1-4 in Example 1 were dissolved in ddH2O to obtain casein hydrolysate solutions, which were then transferred to 10 kD ultrafiltration tubes and centrifuged at 4°C and 12000 rcf for 10 min to collect polypeptide samples less than 10 kD. Dithiothreitol solution was added to the polypeptide samples to a final concentration of 10 mmol / L, and the samples were reduced in a 56°C water bath for 1 h. Then, iodoacetic acid solution was added to a final concentration of 50 mmol / L. After reacting in the dark for 40 min, the samples were desalted using a self-filled desalting column, and the solvent was evaporated in a 45°C vacuum centrifugal concentrator to obtain samples 1-4 to be tested.

[0093] Samples 1-4 were subjected to LC-MS / MS detection, where the capillary liquid chromatography conditions were as follows: analytical column: 150 μm id×150 mm, packed with Acclaim PepMap RPLC C18, 1.9 μm, 100Å; mobile phase A: pure water (containing 0.1% (v / v) formic acid), mobile phase B: 80% acetonitrile (containing 0.1% (v / v) formic acid), flow rate: 600 nL / min, analysis time for each component: 66 min. Mass spectrometry conditions were as follows: primary mass spectrometry parameters: Resolution: 70000, AGCtarget: 3e6, MaximumIT: 100 ms, Scanrange: 300 to 1800 m / z; secondary mass spectrometry parameters: Resolution: 75000, AGCtarget: 1e5, MaximumIT: 50 ms, TopN: 20, NCE / steppedNCE: 28.

[0094] The mass spectrum raw files obtained after LC-MS / MS detection were used to search the Uniprot protein database using Maxquant (1.6.2.10) to obtain the amino acid sequences and content ranges of the characteristic peptides in the casein hydrolysate powders 1-4.

[0095] (2) Screening of bioactive peptides

[0096] The online prediction tool Peptide Ranker was used to predict the potential biological activity of the common characteristic peptides in casein hydrolyzate powders 1-4. The Peptide Ranker software scores peptides from 0 to 1. The higher the score, the higher the possibility that the peptide has biological activity. The score is based on the fact that the specific structural features and amino acid sequences of the bioactive peptides enable the bioactive peptides to have specific biological activities. Peptides with scores > 0.8 were screened as bioactive peptides, a total of 54, as shown in Table 2.

[0097] Table 2

[0098]

[0099] (3) Screening of anti-inflammatory active peptides

[0100] From the 54 bioactive peptides in Table 2, peptides with relatively high contents in milk casein hydrolysate powders 1-4 were screened, and 4 anti-inflammatory active peptides were obtained, namely, peptide FAWPQYLK, peptide LPW, peptide MPLW, and peptide PLW, whose molecular weights, protein names, and relative contents in milk casein hydrolysate powders 1-4 can be seen in Table 3. The above 4 anti-inflammatory active peptides were synthesized by solid phase synthesis for subsequent enzyme-linked immunosorbent assay.

[0101] Table 3

[0102]

[0103] Example 4: Functional verification of casein hydrolysate and anti-inflammatory active peptides

[0104] In a normal body, various pro-inflammatory cytokines and anti-inflammatory cytokines maintain a relatively stable balance. When a disease occurs, the balance is broken, and the body will secrete various cytokines that regulate inflammation to stimulate, recruit and amplify immune cells, leading to the occurrence of inflammatory reactions. Inflammation is a defense mechanism to resist the invasion of harmful substances into life, allowing damaged tissues to repair and heal. But at the same time, if the inflammatory response is too strong or lasts too long, it may also cause a series of damage to the body, and in severe cases, it may even endanger life.

[0105] Cytokines (CK) are a class of small molecule proteins with a wide range of biological activities, such as interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor α (TNF-α); they are synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) after stimulation. Cytokines can be divided into anti-inflammatory cytokines and pro-inflammatory cytokines according to their functions in regulating inflammatory responses. Anti-inflammatory cytokines include IL-10, which has the biological effect of inhibiting inflammatory responses, and contribute to the resolution of inflammation and the recovery of the acute phase of autoimmune diseases. Proinflammatory cytokines include IL-1β, IL-2, IL-6, and TNF-α, which can send signals through type I cytokine receptor (CCR1) to regulate cell-mediated immune responses, promote the growth, activation, differentiation, and homing of immune cells to the site of infection, and contribute to the occurrence and spread of autoimmune inflammation.

[0106] Therefore, in order to verify the anti-inflammatory effect of the above-mentioned casein hydrolysate and anti-inflammatory active peptides, this experiment will detect the content of pro-inflammatory cytokines and anti-inflammatory cytokines released by the macrophage-like inflammation model under the treatment of casein hydrolysate and anti-inflammatory active peptides, so as to characterize the anti-inflammatory effect of casein hydrolysate and anti-inflammatory active peptides.

[0107] (1) Culture and induction of THP-1 cells

[0108] At 37°C and 5% CO2, human mononuclear cells (THP-1) were routinely cultured and passaged using a basal medium, which was RPMI-1640 medium containing 10% fetal bovine serum, 0.05 mM β-mercaptoethanol, and 1% penicillin-streptomycin solution. THP-1 cells, basal medium, and its components were purchased from Nanjing Senbeijia Biotechnology Co., Ltd. Before the start of the ELISA experiment, THP-1 cells were cultured for 10 4 Pieces / cm 2 The cells were seeded in 12-well plates and phorbol methyl ester (PMA) was added at a final concentration of 100 ng / mL for 24-48 h to induce THP-1 cells into macrophage-like cells.

[0109] (2) Construction of macrophage-like cell inflammation model

[0110] The macrophage-like cells in (1) were stimulated with lipopolysaccharide (LPS) at a final concentration of 20 ng / mL for 6 h, and then the basal culture medium was replaced with a serum-free culture medium without 10% fetal bovine serum and cultured for 24 h for starvation treatment to assimilate the cell state. A macrophage-like cell inflammation model was constructed to simulate the macrophage inflammatory response.

[0111] (3) Enzyme-linked immunosorbent assay (ELISA)

[0112] The old culture medium of macrophage-like cells in (1) was removed, a new basal culture medium was added and cultured for 24 h, and the culture supernatant was collected, which was set as the Blank group (blank control group). The old culture medium of the macrophage-like cell inflammation model in (2) was removed, a new basal culture medium was added and cultured for 24 h, and the culture supernatant was collected, which was set as the LPS group (experimental control group). The old culture medium of the macrophage-like cell inflammation model in (2) was removed, a test culture medium was added and cultured for 24 h, and the culture supernatant was collected. The culture supernatant was set as the peptide FAWPQYLK group, peptide LPW group, peptide MPLW group, peptide PLW group and casein hydrolyzate group (experimental group) according to the test components of the test culture medium. Among them, the test culture medium is a basal culture medium containing different final concentrations of the test components. The above test components containing different final concentrations can be seen in Table 4.

[0113] Table 4

[0114]

[0115] The culture supernatant of each group was tested using the ELISA kit produced by Wuhan Elaruite Biotechnology Co., Ltd. to obtain the content of cytokines IL-1β, IL-2, IL-6, IL-10 and TNF-α in each group. Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 and Fig. 9 The detection operation was carried out according to the instructions of the ELISA kit.

[0116] Figure 1 This is the result of cytokine content detection in the peptide FAWPQYLK group. Figure 1 The results showed that the contents of cytokines IL-1β, IL-2, IL-6, IL-10 and TNF-α in the LPS group were significantly higher than those in the Blank group, indicating that the macrophage-like cell inflammation model was successfully constructed using LPS in this experiment. After treatment with low (L), medium (M) and high (H) concentrations of the peptide FAWPQYLK, the contents of cytokines IL-1β, IL-2, IL-6 and TNF-α were significantly lower than those in the LPS group; the contents of cytokines IL-1β, IL-2, IL-6 and TNF-α were not concentration-dependent. After treatment with low concentration of peptide FAWPQYLK, the content of cytokine IL-10 was higher than that in the LPS group; after treatment with medium concentration of peptide FAWPQYLK, the content of cytokine IL-10 was not statistically different from that in the LPS group; after treatment with high concentration of peptide FAWPQYLK, the content of cytokine IL-10 was lower than that in the LPS group; the content of cytokine IL-10 was concentration-dependent, and the lower the treatment concentration of peptide FAWPQYLK, the higher the IL-10 content. Figure 1 The results showed that the peptide FAWPQYLK can reduce the inflammatory response of cells and achieve anti-inflammatory effect by significantly reducing the content of cytokines IL-1β, IL-2, IL-6 and TNF-α.

[0117] Figure 2 This is the result of cytokine content detection in the peptide LPW group. Figure 2The results showed that the contents of cytokines IL-1β, IL-2, IL-6, IL-10 and TNF-α in the LPS group were significantly higher than those in the Blank group, indicating that the macrophage-like cell inflammation model was successfully constructed using LPS in this experiment. After treatment with low (L), medium (M) and high (H) concentrations of peptide LPW, the contents of cytokines IL-1β, IL-6 and TNF-α were significantly lower than those in the LPS group; the contents of cytokines IL-1β, IL-6 and TNF-α were concentration-dependent, and the higher the concentration of peptide LPW treatment, the lower the contents of cytokines IL-1β, IL-6 and TNF-α. After treatment with low and medium concentrations of peptide LPW, the content of cytokine IL-2 was not statistically different from that in the LPS group; after treatment with high concentrations of peptide LPW, the content of cytokine IL-2 was lower than that in the LPS group; the content of cytokine IL-2 was also concentration-dependent, and the higher the concentration of peptide LPW treatment, the lower the content of cytokine IL-2. After treatment with low and medium concentrations of peptide LPW, the content of cytokine IL-10 was higher than that in the LPS group; after treatment with high concentration of peptide LPW, the content of cytokine IL-10 was not statistically different from that in the LPS group; the content of cytokine IL-10 was concentration-dependent, and the lower the concentration of peptide LPW treatment, the higher the content of cytokine IL-10. Figure 2 The results showed that the peptide LPW can reduce the inflammatory response of cells and achieve anti-inflammatory effect by significantly reducing the content of cytokines IL-1β, IL-6 and TNF-α, and increasing the content of cytokine IL-10.

[0118] Figure 3 This is the result of cytokine content detection in the peptide MPLW group. Figure 3 The results showed that the contents of cytokines IL-1β, IL-2, IL-6, IL-10 and TNF-α in the LPS group were significantly higher than those in the Blank group, indicating that the macrophage-like cell inflammation model was successfully constructed using LPS in this experiment. After treatment with low (L), medium (M), and high (H) concentrations of the peptide segment MPLW, the contents of cytokines IL-1β, IL-2, IL-6, and TNF-α were significantly lower than those in the LPS group; the contents of cytokines IL-1β, IL-2, IL-6, and TNF-α did not have typical concentration dependence. After treatment with low, medium, and high concentrations of the peptide segment MPLW, the content of cytokine IL-10 was significantly higher than that in the LPS group; the content of cytokine IL-10 was concentration-dependent, and the lower the concentration of peptide segment MPLW treatment, the higher the content of cytokine IL-10. Figure 3 The results showed that the peptide MPLW can reduce the inflammatory response of cells and achieve anti-inflammatory effect by significantly reducing the content of cytokines IL-1β, IL-2, IL-6 and TNF-α, and increasing the content of cytokine IL-10.

[0119] Figure 4 This is the result of cytokine content detection in the peptide PLW group. Figure 4 The results showed that the contents of cytokines IL-1β, IL-2, IL-6, IL-10 and TNF-α in the LPS group were significantly higher than those in the Blank group, indicating that the macrophage-like cell inflammation model was successfully constructed using LPS in this experiment. After treatment with low (L), medium (M) and high (H) concentrations of peptide PLW, the contents of cytokines IL-1β, IL-2, IL-6 and TNF-α were significantly lower than those in the LPS group; the content of cytokine IL-2 was not concentration-dependent; the contents of cytokines IL-1β, IL-6 and TNF-α were concentration-dependent, and the higher the concentration of peptide PLW treatment, the lower the contents of cytokines IL-1β, IL-6 and TNF-α. After treatment with low concentration of peptide PLW, the content of cytokine IL-10 was higher than that in the LPS group; after treatment with medium and high concentrations of peptide PLW, the content of cytokine IL-10 was not statistically different from that in the LPS group; the content of cytokine IL-10 was concentration-dependent, and the lower the concentration of peptide PLW treatment, the higher the content of cytokine IL-10. Figure 4 The results showed that the peptide PLW can reduce the inflammatory response of cells by significantly reducing the content of cytokines IL-1β, IL-2, IL-6 and TNF-α, thereby achieving anti-inflammatory efficacy.

[0120] Figure 5 This is the result of the test of cytokine TNF-α content in the casein hydrolyzate group. Figure 6 This is the result of the test of cytokine IL-10 content in the casein hydrolysate group. Figure 7 This is the result of the test of cytokine IL-6 content in the casein hydrolysate group. Figure 8 This is the result of the test of cytokine IL-2 content in the casein hydrolysate group. Fig. 9 This is the result of the test on the cytokine IL-1β content in the casein hydrolysate group. Figure 5-Figure 9The results showed that the contents of cytokines IL-1β, IL-2, IL-6, IL-10 and TNF-α in the LPS group were significantly higher than those in the Blank group, indicating that the macrophage-like cell inflammation model was successfully constructed using LPS in this experiment. In addition, after treatment with low (0.5 mg / mL), medium (2.3 mg / mL) and high (4.1 mg / mL) concentrations of casein hydrolysate powder 1, the contents of cytokines IL-1β, IL-2 and IL-6 were significantly lower than those in the LPS group (p < 0.001), indicating that casein hydrolysate powder 1 can reduce the inflammatory response of cells by significantly reducing the contents of cytokines IL-1β, IL-2 and IL-6, thereby achieving anti-inflammatory effects. After treatment with low (0.5 mg / mL), medium (2.3 mg / mL), and high (4.1 mg / mL) concentrations of casein hydrolyzate powder 2, the contents of cytokines IL-1β, IL-2, and IL-6 were significantly lower than those in the LPS group, indicating that casein hydrolyzate powder 2 can significantly reduce the contents of cytokines IL-1β, IL-2, and IL-6 to reduce the inflammatory response of cells and achieve anti-inflammatory effects. After treatment with low (0.5 mg / mL), medium (2.3 mg / mL), and high (4.1 mg / mL) concentrations of casein hydrolyzate powder 3, the contents of cytokine IL-1β were significantly lower than those in the LPS group, and at medium and high concentrations, the contents of cytokines IL-2 and IL-6 were significantly lower than those in the LPS group, indicating that casein hydrolyzate powder 3 can also reduce the contents of cytokines IL-1β, IL-2, and IL-6 to reduce the inflammatory response of cells and achieve anti-inflammatory effects. After treatment with low (0.5 mg / mL), medium (2.3 mg / mL), and high (4.1 mg / mL) concentrations of casein hydrolyzate powder 4, the contents of cytokines IL-1β, IL-2, and IL-6 were significantly lower than those in the LPS group, indicating that casein hydrolyzate powder 4 can reduce the inflammatory response of cells and achieve anti-inflammatory effects by significantly reducing the contents of cytokines IL-1β, IL-2, and IL-6.

[0121] In summary, the peptides FAWPQYLK, LPW, MPLW, and PLW can all reduce the inflammatory response of cells by reducing the content of cytokines IL-1β, IL-2, IL-6, and TNF-α, and increase the content of cytokine IL-10 at low concentrations to achieve anti-inflammatory effects. In addition, the casein hydrolysate containing the above four active peptides can also reduce the inflammatory response of cells by reducing the content of cytokines IL-1β, IL-2, and IL-6 to achieve anti-inflammatory effects.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A casein hydrolysate, characterized in that: The casein hydrolysate comprises peptide segment FAWPQYLK, peptide segment LPW, peptide segment MPLW and peptide segment PLW.

2. The casein hydrolyzate according to claim 1, characterized in that Based on the mass of the casein hydrolysate, the content of the peptide segment FAWPQYLK is ≥ 1.50%; and / or, Based on the mass of the casein hydrolysate, the content of the peptide segment LPW is ≥ 0.05%; and / or, Based on the mass of the casein hydrolysate, the content of the peptide segment MPLW is ≥ 0.05%; and / or, Based on the mass of the casein hydrolysate, the content of the peptide segment PLW is ≥0.05%.

3. The casein hydrolyzate according to claim 1 or 2, characterized in that The degree of hydrolysis of the casein hydrolysate is 10%-15%; and / or, The mass content of peptides with a molecular weight of ≤10000 Da in the casein hydrolysate is ≥80%; and / or, The mass content of peptides with a molecular weight of ≤5000 Da in the casein hydrolysate is ≥55%; and / or, The mass content of peptides with a molecular weight of less than 1000 Da in the casein hydrolysate is ≥30%.

4. The casein hydrolyzate according to claim 1 or 2, characterized in that The casein hydrolysate is obtained by enzymatically hydrolyzing the casein raw material; The enzymatic treatment includes enzymatic hydrolysis using trypsin and pronase.

5. The method for preparing the casein hydrolyzate according to any one of claims 1 to 4, characterized in that: The steps include: Performing enzymatic hydrolysis on the casein raw material to obtain the casein hydrolyzate; The enzymatic treatment includes enzymatic hydrolysis using trypsin and pronase.

6. The preparation method according to claim 5, characterized in that: The trypsin has an enzymatic activity of 180,000-220,000 U / g; and / or, Based on each gram of the casein raw material, the amount of trypsin added is 0.10-0.35wt%; and / or, The enzyme activity of the Streptomyces protease is 180000-220000 U / g; and / or, Based on each gram of the casein raw material, the added amount of the pronase is 0.05-0.32wt%.

7. The preparation method according to claim 5 or 6, characterized in that: The conditions of the enzymatic hydrolysis treatment include: an enzymatic hydrolysis temperature of 40-55° C., an enzymatic hydrolysis time of 2.0-3.5 h, or an enzymatic hydrolysis pH of 7.5-8.

2.

8. The preparation method according to claim 5 or 6, characterized in that: The casein raw material is obtained by mixing casein and water in a volume ratio of 1:(8-12).

9. Use of the casein hydrolyzate according to any one of claims 1 to 4 and / or the casein hydrolyzate prepared by the preparation method according to any one of claims 5 to 8 in preparing related products with anti-inflammatory effects.

Images